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Factors affecting sperm transport in the mammalian oviduct

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Factors affecting sperm transport in the mammalian oviduct
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Demott, Robert P
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x, 138 leaves : ill. ; 29 cm.

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Animal reproduction ( jstor )
Antibodies ( jstor )
Fallopian tubes ( jstor )
Fertility ( jstor )
Fertilization ( jstor )
Hamsters ( jstor )
In vitro fertilization ( jstor )
Oviducts ( jstor )
Sperm transport ( jstor )
Spermatozoa ( jstor )
Department of Veterinary Medicine thesis Ph.D ( mesh )
Dissertations, Academic -- College of Veterinary Medicine -- Department of Veterinary Medicine -- UF ( mesh )
Fallopian Tubes ( mesh )
Ligands ( mesh )
Mucus ( mesh )
Sialic Acids -- physiology ( mesh )
Sperm Motility -- physiology ( mesh )
Sperm Transport -- immunology ( mesh )
Sperm Transport -- physiology ( mesh )
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bibliography ( marcgt )
theses ( marcgt )
non-fiction ( marcgt )

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Thesis:
Thesis (Ph. D.)--University of Florida, 1993.
Bibliography:
Includes bibliographical references (leaves 116-137).
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Also available online.
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Typescript.
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Vita.
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by Robert P. Demott.

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FACTORS AFFECTING SPERM TRANSPORT IN THE
MAMMALIAN OVIDUCT



















By

ROBERT P. DEMOTT


















A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 1993













ACKNOWLEDGMENTS


First thanks go to Dr. Susan Suarez. Her prods were inevitably in the right direction, guided by insight that has served as a good example of why it is important to know intimately the details of your research material. Her financial and professional support of my work has been most unselfish. Next thanks go to the other members of my supervisory committee, Drs. Maarten Drost, Louis Guillette, Paul Klein, William Buhi, and also Fuller Bazer for advising during the first half of my program. Their diverse approaches and interests helped lead to intriguing questions and provided a wide selection of ways to answer them. They did a good job teaching me to keep my trees and forests in proper perspective.

Next, thanks go to all the faculty members who provided generously of their knowledge, time, and equipment including Drs. Kevin Anderson, John Harvey, Roger Reep, and Chris West. The varied nature of my experiments had me knocking on many doors and their cooperation was wonderful, as was the cooperation from all the scientists who spent time training me in new methods including Idania Alvarez, Katy Gropp, Melanie Pate, Michael Sapper and Heidi Wearne. Several college administrators have done a good job providing support and helping me through the Graduate School procedures including Drs. Darryl Buss, Phillip Kosch and Tom Wronski.


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My labmates, Xiao-bing Dai, John Donald, Samir Raychoudhury, Carmen Stauss and Steve Varosi were always willing to lend a hand, or at least an opinion, and I appreciate their help. Thanks also go to Lori Dixon for her help with antibody screening and Rejean LeFebvre for his involvement in the sperm binding experiments. The ICBR core facilities are an invaluable asset at the University of Florida, and the monoclonal core and electron microscopy core staff provided expert support.

Finally thanks go to Kember DeMott; as a scientist and wife, untoppable. Having a soulmate with a detailed understanding of the work you are immersed in, able to share ideas and contribute to your career, is a blessing.



























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TABLE OF CONTENTS



ACKNOWLEDGMENTS ....................................... ii

LIST OF TABLES ............................................ vi

LIST OF FIGURES ........................................... vii

ABSTRACT ................................................ ix

CHAPTERS

1 INTRODUCTION ....................................... 1

Significance of Controlled Sperm Transport .................... 1
Features of Tubal Sperm Transport .......................... 5
Sperm Retention in the Isthmic Reservoir ................... 6
Sperm Movement out of the Isthmic Reservoir ............... 8
Changes in Sperm Cell Biology .......................... 11
Sperm Path through the Uterine Tube ..................... 22
Description of Experiments ............................... 26
Nature of Sperm Binding in the Tubal Reservoir ............. 27
Sperm Motility Patterns in the Uterine Tube ................ 27
Sperm Surface Changes ................................ 27
The Morphology and Nature of Tubal Contents .............. 28

2 MECHANISM OF SPERM BINDING IN THE
ISTHMIC RESERVOIR ................................. 29

Introduction ........................................... 29
Materials and Methods ................................... 31
Medium and Chemicals ............................... 31
Binding Inhibition in Excised Uterine Tubes ................ 32
Binding of Fetuin to Fresh and Hyperactivated Sperm ......... 37 Characterization of Fetuin Binding Proteins ................. 38
Results ......................................... 40


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Discussion ............................................ 51
Conclusions ........................................... 56

3 SPERM MOTILITY PATTERNS IN THE UTERINE TUBE ...... 58

Introduction ........................................... 58
Materials and Methods ................................... 61
Results ......................................... 66
Qualitative Observations ............................... 66
Quantitative Comparisons .............................. 69
Discussion ............................................ 72
Conclusions ........................................... 77

4 INVESTIGATION OF CHANGING SPERM ANTIGENICITY .... 78

Introduction ........................................... 78
Materials and Methods ................................... 80
Production of Monoclonal Antibodies ..................... 80
Immunogold Labelling of Sperm ......................... 84
Immunoblotting of Sperm Proteins ....................... 85
R esults ............................................... 86
Discussion ............................................ 93
Conclusions ........................................... 96

5 DEMONSTRATION OF TUBAL MUCUS IN THE PATH
OF SPERM TRANSPORT ............................... 97

Introduction ........................................... 97
Materials and Methods .................................. 100
Design and Sampling ................................. 100
Processing ........................................ 101
Sectioning ........................................ 101
Staining .......................................... 102
R esults .............................................. 103
Discussion ........................................... 104
Conclusions ........................................... 111

6 SUMMARY .......................................... 112

REFERENCE LIST ....................................... 116

BIOGRAPHICAL SKETCH ................................. 138




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LIST OF TABLES


Table page

2-1 Candidate competitive inhibitors of carbohydrate interactions
in the uterine tube .................................... 34

2-2 Carbohydrate binding treatments for blotted
sperm components ................................... 41

3-1 Analysis of factors affecting proportion of sperm swimming
freely within the uterine tube ............................ 70

3-2 Attachment status interactions for FCR ....................... 71

4-1 Modified hamster capacitation medium formulation .............. 82

4-2 Criteria for staging sperm .................................. 82






















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LIST OF FIGURES


Figure page

2-1 Photomicrographs of hamster sperm within the
tubal isthmus ....................................... 36

2-2 Bar graph showing effect of treatment on sperm ................. 43

2-3 Bar graph showing regional differences in inhibition
by fetuin and sialic acid ................................ 45

2-4 Bar graph showing that effect of treatment is due
primarily to isthmic inhibition ........................... 46

2-5 Photomicrographs of silver-enhanced labelled hamster sperm ....... 48 2-6 PVDF blots probed for carbohydrate binding. ................... 50

3-1 The method for calculating flagellar curvature ratio
(FCR) is shown ...................................... 65

3-2 Illustration of a typical pattern and timecourse for
sperm progress in the uterine tube ....................... 68

4-1 Stage-related changes observed in indirect immunofluorescent
labelling with monoclonal antibodies ...................... 88

4-2 Epifluorescent images of monoclonal antibody binding patterns ...... 89











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4-3 1-D Immunoblots probed with stage-specific
monoclonal antibodies ................................. 91

4-4 2-D Immunoblot of fresh sperm extract labelled with HL 787 ....... 92 5-1 Differential preservation of luminal contents by
processing method .................................. 105

5-2 Regional differences in luminal staining characteristics ........... 106

5-3 Sperm observed within luminal mucus ....................... 107






































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Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy

FACTORS AFFECTING SPERM TRANSPORT IN THE MAMMALIAN OVIDUCT

By

Robert P. DeMott

December 1993

Chairperson: Susan S. Suarez, Ph.D. Major Department: Veterinary Medicine

The mammalian oviduct, or uterine tube, is the site of fertilization. During passage through the uterine tube, sperm complete the physiological processes that prepare them for fertilization; they may also begin using the vigorous hyperactivated motility pattern. Sperm numbers drop several orders of magnitude between the entrance to the uterine tube and the site of fertilization approximately mid-way along it. This is primarily due to the presence of a sperm reservoir in the lower uterine tube formed relatively quickly after insemination. Sperm are retained here until fertilization is imminent. Then, a very few move out of the reservoir and on to the site of fertilization.

The experiments described here were intended to assess the effects of several factors potentially regulating sperm transport through the uterine tube. The nature of retention in the reservoir was investigated and found to involve a specific ix








carbohydrate interaction between the sperm surface and the tubal wall. A sialic acidbearing ligand apparently mediates this lectin-like binding. The use of particular motility patterns in the uterine tube was investigated and the switch to hyperactivated motility appeared prior to release from the reservoir. Thus, hyperactivation may help sperm break free from the wall. Sperm modifications coincident with transport were investigated by developing monoclonal antibodies to sperm epitopes. Antigens that serve as markers and possibly play a functional role for the transition to hyperactivation and other stages were described. Finally, a specialized histological protocol was used to characterize the nature, morphology and location of the luminal contents which sperm encounter. The presence of mucus in the path of sperm was demonstrated. Based on these experiments, it appears that there is a specific binding interaction affecting sperm retention in the reservoir, that motility changes occur when sperm can release from the reservoir, that changing antigenicity can be used to detect cellular modifications associated with functional changes, and that the tubal environment contains material which has the capability to affect sperm passage.

















x













CHAPTER 1
INTRODUCTION


Significance of Controlled Sperm Transport


There is a common perception that mammalian fertilization occurs after a large number of sperm move up the female reproductive tract and encounter the egg. The sheer number of sperm in the tract is seen as a guarantee that enough will make it up to the egg. This perception is strengthened by the images we typically see of fertilization in which the event has been contrived, either for the sake of the image, or to ensure that fertilization takes place in an artificial system. The procedure for in vitro fertilization, which requires large excesses of sperm, have been empirically determined to provide high percentages of fertilization while limiting polyspermy. It appears, however, that it is not adequate to extrapolate from these conditions to describe fertilization in vivo.

More than 40 years ago, observations were published based on timed matings and subsequent serial sectioning indicating that the ratio of sperm to eggs at the time of fertilization in the rabbit (Chang, 1951) and rat (Moricard & Bossu, 1951) is approximately 1:1. Zamboni reported similar findings in the mouse (Zamboni, 1972), noting that it was only after fertilization was completed that excess sperm reached the area. Serial sectioning, tubal flushing and microscopic observations of sperm within


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excised uterine tubes have been used to show that the number of sperm present at the time and place of fertilization is very low in the mouse (Tessler & Olds-Clarke, 1981; Suarez, 1987), hamster (Cummins & Yanagimachi, 1982; Smith et al., 1987) rabbit (Overstreet et al., 1978), rat (Bedford & Kim, 1993), sheep (Cummins, 1982) and pig (Hunter et al., 1987).

Other observations imply that the reduction of sperm numbers to these very low levels does not occur as a regular, gradual process in the female reproductive tract. There is likely a constant loss of sperm during their ascent, but there are also very restrictive barriers (Katz et al., 1989). For species that inseminate at or below the cervix, this structure appears to reduce the number of progressing sperm by orders of magnitude (Hunter, 1988). The junction between the uterus and uterine tube (the preferred anatomical term "uterine tube" will be used throughout in place of the more common "oviduct"), the uterotubal junction, also appears to serve as a restrictive filter (Gaddum-Rosse, 1981; Shalgi et al., 1992), causing another large drop in sperm numbers. Finally, within the uterine tube, there is not a regular distribution of sperm. Sperm appear to be sequestered in a reservoir located in the most proximal portion of the uterine tube until ovulation, and thus fertilization, is imminent. This phenomenon has been demonstrated in the hamster (Yanagimachi & Chang, 1963; Smith et al., 1987), mouse (Zamboni, 1972; Suarez, 1987), rabbit (Harper, 1973a, 1973b; Overstreet et al., 1978), cow (Thibault et al., 1975), guinea pig (Yanagimachi & Mahi, 1976), sheep (Hunter & Nichol, 1983), pig (Hunter, 1984; Hunter et al., 1987) and rat (Shalgi & Kraicer, 1978; Shalgi & Phillips, 1988).







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The tubal region of the reproductive tract serves to store and support sperm for a number of other taxa that have lengthy periods between insemination and fertilization, including bats (Krutzsch et al., 1982; Racey et al., 1987) and the fattailed dunnart (Breed et al., 1989). There are also birds (Bobr et al., 1964a, 1964b; Bakst, 1992) and reptiles (Halpert et al., 1982; Palmer & Guillette, 1988; Kumari et al., 1990; Gist & Fischer, 1993) that have sperm storage sites in various regions of the female reproductive tract.

The formation of the sperm reservoir occurs relatively quickly after fertilization. Using serial sectioning after timed matings, Smith and coworkers (Smith et al., 1987) showed that the isthmic reservoir in the hamster is populated with sperm from 1-3 hours after the onset of mating. Population of the reservoir in the mouse appears to be functionally limited to the first hour after mating, as the uterotubal junction begins constricting after this point (Zamboni, 1972; Suarez, 1987). Observing sperm within excised mouse uterine tubes, Suarez (1987) noted that by 1-2 hours post-coitus there are many sperm in the lower isthmus and tubal portion of the uterotubal junction, but that the intramural region, where the junction is within the muscular walls of the uterus, is constricted and sperm numbers are low. By ligating the reproductive tract at various points and various times after mating, Hunter (1981) demonstrated that the reservoir is established within 1 hour in the pig isthmus. For the cow, a vaginal inseminator where the interval between mating and ovulation may be up to 30 hours, formation of the reservoir occurs around 8 hours after insemination (Wilmut & Hunter, 1984; Hawk, 1987).







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It is also well established that the sperm that will go on to fertilize eggs are derived from the population in the isthmic reservoir. Using the timed ligation approach, Harper (1973b) demonstrated that the rabbit sperm that reach the isthmus relatively early are sufficient for fertilization and Hunter (1984) demonstrated that it was the boar sperm that had rapidly populated the isthmus that were responsible for fertilization. The closing of the uterotubal junction shortly after mating in the mouse (Zamboni, 1972; Suarez, 1987) implies that the fertilizing sperm must come from the early arriving population. In the hamster, Smith and Yanagimachi (1991) flushed out the sperm that had not bound to the epithelium after mating and later obtained fertilized eggs from these uterine tubes. In an earlier study (Smith & Yanagimachi, 1990), they had found that at 2 hours post-insemination, the number of sperm in the isthmus remains fairly constant. Since there was not yet ascent to the ampulla (Smith et al., 1987), this implies that a stable population forms early and provides the fertilizing sperm. Considering all these observations, our current model for fertilization in vivo is that the fertilizing sperm ascend rapidly through the lower portion of the female reproductive tract and are retained in a reservoir in the proximal portion of the uterine tube. Around the time of ovulation, after a species specific delay, a very few of these sperm complete passage to the ampullary-isthmic junction where they meet and fertilize the eggs. In this model, retention, maintenance, and release of sperm in the isthmus seem to serve as a key control point for fertilization. Yet, the functional details of sperm binding, release and ascent through the uterine tube remain poorly described.








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The mechanisms that control tubal sperm transport to the site of fertilization have not been given sufficient attention for several reasons. Since it is not yet possible to observe natural fertilization and to track the responsible sperm, inferential approaches are required. Thus, experimental results can be difficult to interpret definitively and conclusions must be narrowly qualified. Further, using such a complex physiological system for experimentation is laborious, and accounting for and holding potentially complicating factors constant can be difficult. Additionally, the interest in manipulating reproduction and the numerous difficulties associated with these technologies have provided a selection of research questions that can be addressed with well controlled in vitro approaches.

However, the study of fertilization in vivo will improve our understanding of intercellular interactions, sperm cell and reproductive tract biology, and allow improvements to be made to manipulated reproduction technologies. Consider that the naturally regulated system provides successful fertilization and reproduction with a gamete ratio close to unity, whereas our artificial technologies require thousands of times more sperm than eggs. It seems clear that there is much information, both mechanistic and applied, to be found studying the process of sperm transport in the uterine tube.


Features of Tubal Sperm Transport


There are several features of sperm transport through the uterine tube that need further investigation. The biochemical mechanisms of sperm retention in the







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reservoir have not been described. While several mechanisms for sperm release have been postulated, which actually operate and are pertinent to fertilization remains unclear. Observations of sperm motility patterns in the uterine tube raise the question of how motility and other aspects of sperm cell biology contribute to reaching the site of fertilization. And, the path that sperm take in the uterine tube and substances that they encounter may represent another level of regulation. Our current understanding of each of these features will be reviewed.


Sperm Retention in the Isthmic Reservoir


At least for the mouse (Suarez, 1987) and hamster (Smith & Yanagimachi, 1990), it seems that the most important method of retaining sperm in the reservoir involves adherence to the tubal mucosa. Attachment to the mucosa has also been suggested in the rabbit (Cooper et al., 1979) and pig isthmus (Hunter et al., 1987). The morphology of the uterine tube provides a series of mucosal folds and pockets that appear to serve as storage crypts in some species (Nilsson & Reinius, 1969; Suarez, 1987; Hunter et al., 1987; Smith & Yanagimachi, 1990). Smith and Yanagimachi (1987) flushed hamster uterine tubes to remove first luminal and then adherent sperm. There is also evidence that bull sperm bound to tubal cells in culture (Pollard et al., 1991; Ellington et al., 1991) maintain viability and fertilizability while unattached cells become immotile and may have disrupted acrosomes. In a species with extended sperm storage, the little brown bat, sperm interact with and appear to be maintained by the epithelium in the uterotubal junction (Racey et al.,








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1987). In reptilian sperm-storing species there appear to be some storage regions with sperm in close association to the epithelium and others where large agglutinated masses of sperm occupy the lumen of specialized tubules (Gist & Fischer, 1993). This second type of storage organ is found in fowl (Bakst, 1992).

Several other mechanisms for retaining sperm in the isthmus have been put forth and they may play an accessory role in some species. Post-coital constriction of the isthmus may hamper the ability of mouse sperm to move within the uterine tube (Suarez, 1987). Depressed motility observed in isthmic sperm has also been postulated to help prevent sperm escaping the reservoir (Overstreet & Cooper, 1975; Cooper et al., 1979; Cummins, 1982). Hunter and Nichol (1986) found a slight temperature gradient in the pig uterine tube and speculated that the cooler temperatures in the reservoir region may help subdue sperm. Another contributing factor in the pig uterine tube may be the presence of viscous secretions in the mucosal crypts that Hunter and coworkers inferred based on the degree of flagellar bending seen by scanning electron microscopy (SEM) of sperm in the crypts compared to sperm in the main lumen (Hunter et al., 1987). The presence of a mucous layer, again detected by SEM, before ovulation but not after in the rabbit (Jansen, 1978) and human (Jansen, 1980) uterine tube led to the proposal that ciliary beating, which aids in sperm transport, may be dampened by this mucus until ovulation has occurred. An actual role for any of these alternative mechanisms in controlling sperm transport has not been demonstrated. In light of the observations of tight adherence between sperm and the isthmic mucosa in situ (Suarez, 1987;








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Smith & Yanagimachi, 1990), this mechanism seems to be the most relevant, though its biochemical basis has not yet been determined.


Sperm Movement out of the Isthmic Reservoir


Several mechanisms for moving sperm from the isthmic reservoir to the site of fertilization have been proposed. First, through in situ observations of sperm in the mouse (Suarez, 1987) and hamster uterine tube (Smith & Yanagimachi, 1990) sperm have been seen to detach from the wall and move by their own flagellar beating. Another mechanism involved is the movement of sperm along with the contents of the uterine tube. The most efficient propulsion is likely due to muscular contractions of the uterine tube directed toward the ampulla. By injecting a particulate solution into the uterine tubes and observing its redistribution in anesthetized hamsters, Battalia and Yanagimachi (1979) noted that there is coordinated muscular activity directing the tubal contents toward the ovary only during the period immediately preceding ovulation. A subsequent study showed that the shifts in ovarian steroid ratios around ovulation trigger this coordinated movement (Battalia & Yanagimachi, 1980). Waves of muscular contraction in the pig uterine tube, measured in anesthetized animals, also appear to change direction around ovulation (Rodriguez-Martinez, et al., 1982).

Control of the muscular contractions may be affected by the products of ovulation as well. Ito and coworkers (1991) propose that prostaglandins in the ovulatory products have a local effect in the uterine tube regulating the contractions.








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They had observed that superovulated hamsters have enhanced sperm transport. Presumably the additional ovulations produce additional prostaglandins that enhances the contractions. The transport of extra sperm in superovulated rats has also been documented (Shalgi & Phillips, 1988). The presence of high levels of estrogens in boar semen has also been postulated to induce prostaglandin and LH releases in the female that could affect muscular activity in the reproductive tract, and the timing of ovulation (Claus, 1990)

Another possible mechanism for transporting sperm is ciliary currents. Gaddum-Rosse and Blandau (1973) observed ciliary transport of particles in longitudinally opened uterine tubes and reported that the current in the isthmus of rabbits and pigs is toward the ampulla. However, these species seem to be atypical in terms of the direction of the current when compared to rats, guinea pigs, humans, and cows (Gaddum-Rosse & Blandau, 1976) and also in terms of how much ciliation is present in the isthmus (Nilsson & Reinius, 1969; Hunter, 1988). So, while ciliary beating is important for egg transport (Norwood et al., 1978; Norwood & Anderson, 1980; Mahi-Brown & Yanagimachi, 1983), it probably plays a minimal role in isolated species for sperm movement (Gaddum-Rosse & Blandau, 1976).

While the uterine tube may play a predominant role in the movement of sperm that have released from the reservoir, it seems that it does not control whether or not adherence of sperm to the mucosa is maintained. By sequentially flushing the hamster uterine tube to remove free then loosely bound sperm at several time points, Smith and Yanagimachi (1990) found that there were no significant changes in the








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number of tubal sperm until at least 2 hours after ovulation. And, at this time, sperm were only slightly less susceptible to being flushed out. These results imply that the uterine tube does not undergo marked changes in its affinity for binding sperm at ovulation.

A significant change in sperm binding was, however, associated with the condition of the sperm (Smith & Yanagimachi, 1991). When sperm capacitated in vitro (see below "Changes in Sperm Cell Biology"), i.e., sperm capable of fertilization, were injected into the uterine tube, they remained free from the mucosa whereas non-capacitated sperm bound to the mucosa as soon as they encountered it. This implies that part of capacitation involves a loss of affinity for the tubal reservoir and it is this reduction in binding affinity that allows sperm to move along the uterine tube. Interpretation of these results is confounded, however, because the capacitated sperm were also using the vigorous hyperactivated motility pattern which could contribute to their ability to pull away from the mucosa.

In summary, movement out of the tubal reservoir appears to involve release from the mucosa due to changes in the binding affinity of sperm and/or changes in flagellar beating, as well as indirect effects of tubal physiology. The primary contribution to indirect effects is the muscular contractions of the uterine tube which serve to move the luminal contents. Ciliary beating may play a small role as well.










Changes in Sperm Cell Biology


The production of sperm capable of fertilizing an egg is a continuous process from testis to uterine tube. Sperm leave the testis immotile and undergo a series of modifications in the epididymis, collectively called sperm maturation, which result in the attainment of motility and the stabilization of the cell membrane (Yanagimachi, 1988). Nevertheless, they require further processing before becoming capable of fertilization. Chang (1951) and Austin (1951) first recognized that sperm need to spend a certain time in the female reproductive tract before they are able to fertilize an egg. This part of the process, capacitation (Austin, 1952), has been defined nebulously as a set of cellular changes that allow the sperm cell to undergo the acrosome reaction when challenged with a specific inducer (Yanagimachi, 1988). The acrosome reaction is an exocytotic event required for fertilization (Meizel, 1985). Another observable phenomenon that can not yet be explained in terms of overall sperm cell biology is the use of the hyperactivated flagellar movement pattern. First noticed for hamster sperm capacitated in vitro (Yanagimachi, 1969; Gwatkin & Anderson, 1969), this obvious alteration in frequency, amplitude, and shape of the flagellar beat yields an erratic, albeit vigorous, pattern of movement. Hyperactivation has since been observed within the hamster ampulla (Katz & Yanagimachi, 1980) and in the upper regions of the mouse reproductive tract (Suarez & Osman, 1987). While the relationship of hyperactivation to the other steps in the process of preparing a sperm for fertilization remains unclear, its occurrence in parallel with capacitation








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and use in the uterine tube mean that the change to this pattern may contribute to the regulation of sperm transport in the uterine tube.


Maturation. The maturational changes that occur primarily in the epididymis include modifications of the glycoproteins on the sperm surface, changes in the lipid composition of the membrane, and stabilization of the tail structures and condensed nucleus by disulfide bonding (Yanagimachi, 1988). Membrane glycoproteins may be modified, added, or removed and some of these changes have been associated with functional changes in sperm. A protein that appears to modify boar sperm, preventing the head-to-head agglutination seen in corpus epididymal sperm, was extracted from caudal epididymal fluid (Dacheux et al., 1983).

The proteins present on rat sperm from the caput, corpus, and caudal epididymis and changes induced by incubating these sperm with caudal epididymal fluid have been analyzed by two-dimensional polyacrylamide gel electrophoresis (Hall & Killian, 1989). They demonstrated that a variety of different glycoproteins appear and disappear from the sperm membrane as they move along the epididymis and that treatment with caudal fluid can cause some of these changes.

Extrinsic rat sperm proteins of the acrosomal region that are produced in the caudal epididymis have been identified (Rifkin & Olson, 1985). Additionally, changes in the localization of rat sperm antigens occurring during epididymal passage and capacitation have been reported (Petruszak et al., 1991; Phillips et al., 1991; Rochwerger & Cuasnicu, 1992). Hamster sperm proteins that are added in the epididymis have also been described (Moore & Hartman, 1984; Smith et al., 1986;








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Hoos & Olson, 1988; Robataille et al., 1991). A sialylated glycoprotein produced from the distal corpus to the cauda appears to be processed and is associated with the mouse sperm tail (Vernon et al., 1987; Feuchter et al., 1988; Toshimori et al., 1990) and a ram sperm sialoglycoprotein added in the cauda redistributes during the acrosome reaction (McKinnon et al., 1991).

Another group of studies relate to changes in protein glycosylation during maturation. By comparing the binding of various lectins to sperm from different parts of the epididymis, changing populations of carbohydrate moieties on sperm have been identified for the hamster (Nicolson & Yanagimachi, 1972; Koehler, 1981), rabbit (Nicolson & Yanagimachi, 1972; Nicolson et al., 1977), ram (Hammerstedt et al., 1982; Magargee et al., 1988), macaque (Fain-Maurel et al., 1984), mouse (Rankin et al., 1989), dog (Bains et al., 1993b), and goat (Bains et al., 1993a). The results of these studies imply that the exposed carbohydrates and their modifications are highly species specific. Galactose and N-acetylgalactosamine distribution on testicular and caudal rat sperm, analyzed by binding to labeled galactose oxidase, has also identified proteins modified during maturation (Brown et al., 1983). Another approach has been to demonstrate the presence of certain glycosyltransferases in epididymal fluid. Sialyltransferase has been demonstrated in rat epididymal fluid with the strongest activity in the caput (Bernal et al., 1980), and galactosyltranferase activity also appears in rat epididymal fluid (Hamilton, 1980).

Sperm are considered mature when they reach the caudal epididymis since they may become motile, capacitate, and fertilize. However, in the natural scheme








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of fertilization, sperm are exposed to seminal plasma before the environment of the female tract and this exposure seems to affect their cell biology. The effects of seminal plasma were initially considered to prevent premature capacitation of the sperm (Yanagimachi, 1988). Chang (1957) showed that capacitation could be reversed by a component of rabbit seminal plasma produced in the epididymis. Subsequently a number of specific decapacitation components were identified (Oliphant et al., 1985). A family of proteins found in bull, guinea pig, mouse and rat semen seem to inhibit calcium uptake by sperm (Coronel et al., 1993), a necessary occurrence for capacitation and hyperactivation (Yanagimachi, 1988; Fraser, 1990; Suarez et al., 1993). After insemination, these proteins, caltrins, may become enhancers of calcium uptake (San Agustin et al., 1987; Coronel & Lardy, 1992) presumably enhancing capacitation. A similar model of decapacitating activity and subsequent enhancement of capacitation by boar seminal plasma proteins has been described (Desnoyers & Manjunath, 1992). These proteins, which bind specific sperm phospholipids and presumably stabilize the membrane, are proposed to coat the sperm during early transport and then be removed, taking some membrane lipid along; the resulting leakiness of the membrane allows calcium entrance (Desnoyers & Manjunath, 1992). These proteins also bind calmodulin (Manjunath et al., 1993) which may play a role in calcium regulation during capacitation. A calmodulin-like protein from seminal plasma, that may help control calcium levels in buffalo sperm, has also been reported (Sidhu & Guraya, 1993)








15

Recently, other functions of seminal plasma components have been reported. The proposal for a direct effect of seminal plasma steroids on the endocrine regulation of the female tract has been described above (Claus, 1990). Two separate groups have identified seminal plasma proteins that have the ability to bind to the zona pellucida (Parry et al., 1992; Veselsky et al., 1992), though some of the same proteins may have been found by both groups. Though they are unlikely to represent the primary ligand for sperm/egg binding, these proteins present the interesting possibility that seminal plasma components enhance sperm/egg binding. Additionally, a marked reduction in catalase activity, one of the major oxidative damage protection systems, has been found in the seminal plasma of infertile men compared to sperm and seminal plasma from normal, fertile men (Jeulin et al., 1989). In light of the growing awareness of the importance of free radical scavenging systems, research clarifying this potential protective role of seminal plasma may intensify.


Capacitation. Determining which of the myriad events of sperm cell biology are part of capacitation is a matter of semantics. The sperm cells are constantly being affected by their environment and undergoing modifications; our distinctions are convenient but must be recognized as artificial and irrelevant in terms of the sperm's life history. And, while capacitation is by definition directed toward producing the acrosome reaction, along the way some changes in the sperm cell may play a role in sperm transport. Contributions to the control of capacitation by the female tract may also be relevant to controlling sperm transport.








16

A number of sperm surface changes associated with capacitation have been described. Following the report of lectin binding characteristics for sperm by Nicolson and Yanagimachi (1972), a number of surface carbohydrate changes were identified using lectins. In the hamster, the distribution of binding sites for the lectin Concanavalin A over the head changes as sperm are capacitated in vitro (Kinsey & Koehler, 1978). Decapacitation factors which are removed during capacitation have been described for the rabbit (Reyes et al., 1975) and mouse (Fraser, 1984). Membrane antigens that are redistributed have been identified for mouse (Okabe et al., 1986), boar (Saxena et al., 1986; Berger, 1990; Topfer-Petersen et al., 1990) and rat (Jones et al., 1990) sperm. The redistribution of intramembranous particles has been demonstrated in the guinea pig (Koehler & Gaddum-Rosse, 1975). The appearance of certain antigens with capacitation, suggesting that they were masked or have been modified, has been documented in the rat (Jones et al., 1990) and ram (Voglmayr & Sawyer, 1986) and for integrin binding proteins on human sperm (Fusi et al., 1992).

Another type of change that has been associated with capacitation involves alteration of the lipid characteristics of sperm membranes. The ratio of cholesterol to phospholipid, which affects membrane fluidity (Go & Wolf, 1983), was shown to decrease due to the gradual removal of cholesterol. This increasing fluidity of the membrane was proposed to enhance capacitation (Davis, 1981). A similar change in the ratio was found during human sperm capacitation (Hoshi et al., 1990). The enhancement of capacitation, measured by the ability of the sperm to penetrate the








17

egg, coincident with the removal of cholesterol from bovine sperm has also been demonstrated (Ehrenwald et al., 1988). Besides the change in sterol to phospholipid ratio, there is also evidence in guinea pig sperm that the relative amounts of different membrane phospholipids change during capacitation (Stojanoff et al., 1988).

Another occurrence during capacitation is the alteration of the ionic balance of the sperm cell. Before capacitation, sperm maintain typical ionic gradients relative to the medium with potassium high inside the cell and sodium low inside the cell. They also maintain a calcium gradient with lower concentrations inside than outside (Yanagimachi, 1988). Changes in the calcium gradient may be a part of capacitation. There is a clear requirement for an extracellular calcium pool to support hyperactivation and the acrosome reaction (Yanagimachi & Usui, 1974; Yanagimachi, 1982). There is also evidence that an influx of this calcium triggers the acrosome reaction (Roldan & Harrison, 1990). Fraser (1987; Fraser & McDermott, 1992) reported that mouse sperm require at least 0.09 mM for capacitation, but markedly higher fertility results at 1.8 mM. These results fit well with the proposal that there are two changes in the calcium gradient, the first associated with hyperactivation and the second, larger influx at the time of the acrosome reaction (Suarez et al., 1993).

Several mechanisms that may contribute to the calcium influx have been described. The presence of calcium channels has been inferred based on the sensitivity of the influx to channel blockers (Fraser, 1987). Chou and coworkers (1989) used a voltage sensitive dye to show that during mouse sperm capacitation there is a shift in membrane polarity from negative inside to positive inside, primarily








18

due to potassium ion redistribution, and propose that this polarity shift stimulates voltage sensitive channels. The ability of Ca-ATPase inhibitors to affect capacitation (Fraser & McDermott, 1992) and the influx of extracellular calcium (Blackmore, 1993) suggests that this enzyme may also play a role. Decreased Na/K-ATPase activity observed during guinea pig capacitation (Hang et al., 1990) fits with a model proposed by Fraser and coworkers (1993) where Na/K-ATPase activity may be contributing to early mouse capacitation events and help promote increased intracellular sodium ion levels that activate Na+/H+ exchange, resulting in reduced intracellular pH and activation of calcium channels.

This model also provides a potential role of the female tract in controlling capacitation. Differences in the ionic composition of uterine, ampullary, and bursal sac fluid (Borland et al., 1977) are proposed to allow sperm to undergo the initial stages of capacitation. However, sperm do not complete the acrosome reaction until they reach the ampulla and ovulation has occurred, which shifts the extracellular sodium and potassium levels (Fraser, 1983; Fraser et al., 1993). Killian and coworkers (1989) analyzed the lipid characteristics of bovine tubal fluid and found that the cholesterol to phospholipid ratio drops and the prevalence of lysophospholipids increases around estrus. Both of these conditions are seemingly favorable for mediating the sperm membrane lipid changes described above.

Contributions of the female tract to capacitation have long been suspected (Yanagimachi, 1981, 1988; Oliphant et al., 1985). A variety of enzymes, glycosaminoglycans, and other secretions have been proposed as effectors of








19

capacitation within the tract (Yanagimachi, 1988), but few details have been sorted out. In the rabbit, a vaginal inseminating species, capacitation was optimized by sequential exposure to the uterine, then the tubal environment (Bedford, 1969). Tubal secretions associate with ram and human sperm (Sutton et al., 1986; Wagh & Lippes, 1989), especially if the sperm are first treated with uterine fluid (Voglmayr & Sawyer, 1986). Recently, a functional role in enhancing capacitation was proposed for tubal proteins that interact with bull sperm (McNutt et al., 1993). Clearly the role of the uterine tube in controlling capacitation needs further definition.


Hyperactivation. This striking motility pattern has now been described for more than a dozen mammalian species (Yanagimachi, 1988; Katz et al., 1989). First observed in hamster sperm during capacitation in vitro (Yanagimachi, 1969; Gwatkin & Anderson, 1969), sperm were later observed using the pattern within the hamster ampulla (Katz & Yanagimachi, 1980) and in the mouse uterus and uterine tube (Suarez & Osman, 1987). Additionally, hyperactivated sperm have been flushed from the tracts of female mice (Phillips, 1972), rabbits (Cooper et al., 1979; Suarez et al., 1983), hamsters (Cummins & Yanagimachi, 1982b), and sheep (Cummins, 1982a).

The specific form of movement appears to differ from species to species and is somewhat hard to describe accurately, as evidenced by the array of anthropomorphic terms that have been used, e.g., dashing, dancing and bobbing (Yanagimachi, 1988; Katz et al., 1989). Generally it involves increased amplitude of the flagellar waveform, usually asymmetrically, producing an erratic path, and may involve changes in the 3-dimensional aspects of beat propagation (Katz et al., 1989).








20

Since hyperactivation was first described in preparations of sperm capacitated in vitro (Yanagimachi, 1969; Gwatkin & Anderson, 1969), it was originally considered to be a part of the capacitation process. Nevertheless, there is now evidence that while these processes may be complementary and parallel, they can be regulated independently. Yanagimachi (1981) pointed out that hyperactivation occurs before capacitation is complete and can be experimentally induced in the absence of capacitation (defined by the inability to undergo the acrosome reaction). He also noted that hyperactivation is primarily a tail associated phenomenon whereas capacitation involves the sperm head. While this logic does not fit well with the continual, dynamic model of capacitation developed here, it has led to clearer demonstrations of the separate regulation of the two events.

Assessing the contribution of the carrier protein bovine serum albumin (BSA) that is typically used in capacitation medium, Neill and Olds-Clarke (1987) reported that mouse sperm could develop hyperactivation, but could not be fully capacitated without protein present. Altering the sodium bicarbonate levels in capacitating medium has allowed the production of seemingly non-hyperactivated, capacitated hamster sperm (Boatman & Robbins, 1991). The finding of separate calcium influxes associated with hyperactivation and the acrosome reaction implies that there are at least two mechanisms of calcium ion regulation at work (Suarez et al., 1993) and lends further support to the idea of separable control.

There is little other information directly relating to the cellular/molecular basis of controlling hyperactivation. For unclear reasons, increased levels of cAMP seem








21

to positively affect the expression of hyperactivated motility (Fraser & Monks, 1990). Including either cAMP analogs or phosphodiesterase inhibitors in capacitating medium appears to enhance hyperactivation (Fraser, 1979, 1981; Mrsny & Meizel, 1980). White and Aitken (1989) have detected a rise in cAMP levels preceding the attainment of hyperactivation in the hamster. It has been proposed that calcium regulation and cAMP metabolism are the cellular keys to the shift to hyperactivation (Ishijima, 1990).

Several functions for hyperactivated motility have been postulated: (1) providing additional force to release sperm from the mucosa and to aid in penetration of viscous or viscoelastic substances; (2) generating a movement path that helps sperm avoid entrapment in the twists and folds of the uterine tube and covers more area within the uterine tube, improving the likelihood of finding the egg; and

(3) creating disturbances in the tubal medium to improve the exchange of signalling and metabolic components (Yanagimachi, 1981; Katz et al., 1989; Suarez & DeMott, 1991). Though actual analysis of the functional significance of hyperactivation remains limited (Drobnis et al., 1988a, 1988b; Suarez et al., 1991a; Suarez & Dai, 1992), the implications of a potential role for hyperactivation in affecting sperm transport within the uterine tube seem clear.

While the studies described above point out many changes in sperm cell surface antigens, membrane composition and intracellular metabolism, none of these changes have been interpreted in terms of sperm transport. They are generally assigned as part of the maturational process aimed at the stabilization of the sperm








22
and attainment of motility, or part of capacitation aimed at the preparation for sperm/egg interactions and the acrosome reaction. Some of these cellular changes, while observed during maturation, capacitation or hyperactivation, may play their functional role in helping control the transport of sperm though the uterine tube.


Sperm Path through the Uterine Tube


A final subject that needs to be considered for a potential role in controlling sperm transport is the features of the female tract that directly interact with sperm. The female tract has both physical and physiological features that are potentially involved. Again, there has been a great deal discovered which may be relevant to sperm transport, but little interpreted in terms of this phenomenon.

For vaginal inseminating species, the cervix is generally considered the first obstacle to sperm (Hunter, 1988). The morphology of the bovine cervix has been shown to include a series of mucosal folds and blind passages that may complicate sperm passage (Mullins & Saacke, 1989). Based on 3-dimensional reconstructions from serial sections that noted the position of the passages through the cervix and the cervical mucus, Mullins and Saacke (1989) proposed that the path of least resistance for sperm is in longitudinal grooves away from the center of the cervix. The role of cervical mucus for sequestering sperm and removing seminal plasma, especially in the human was studied intensively (Hunter, 1988; Barratt & Cooke, 1991) and there is recent evidence that human cervical mucus may enhance capacitation (Lambert et al., 1985) but prevent premature acrosome reactions (Bielfeld et al., 1992).








23
The uterotubal junction is the first major obstacle for species, such as the hamster and mouse, where large amounts of the ejaculate enter the uterus. Like the cervix, the uterotubal junction appears to help the sperm migrate out of seminal plasma and into female tract fluids (Hunter, 1988). It contains mucosal folds, but the orientation and degree of folding are highly variable among species (Hook & Hafez, 1968; Nilsson & Reinius, 1969). Histology (Zamboni, 1972) and in situ observations (Suarez, 1987) indicate that in the mouse, the uterotubal junction can serve as a valve controlled by coitus. Studies on the migration of rat and hamster sperm through the uterotubal junction indicate that sperm motility is critical for passage, yet hyperactivated sperm are not able to pass (Gaddum-Rosse, 1981; Smith & Yanagimachi, 1988; Shalgi et al., 1992). This implies that, at least in these species, the onset of hyperactivation must occur in the uterine tube for any sperm destined to fertilize (Shalgi et al., 1992). Based on observations of sperm passage following natural mating, Bedford and Yanagimachi (1992) reported that while rat sperm attain their fully active motility pattern in the uterus, hamster sperm motility is subdued until they have passed through the uterotubal junction and are exposed to tubal fluid.

Since the fertilizing sperm come from a population established rapidly in the isthmic reservoir, the uterine tube environment is the most likely, compared to the other segments of the female tract, to influence the transport of the fertilizing sperm. Again, the morphological features of the uterine tube may affect transport. In the mouse (Nilsson & Reinius, 1969; Suarez, 1987) and hamster isthmus (Smith et al., 1987; Smith & Yanagimachi, 1990) there are pockets formed by transverse folding








24

of the mucosa in which the isthmic sperm lodge. The folding pattern gradually changes to longitudinal around the ampullary/isthmic junction in these species, forming mucosal grooves oriented along the long axis of the uterine tube (Nilsson & Reinius, 1969). While the species examined have mucosal folds, the degree and orientation is species dependent (Nilsson & Reinius, 1969). For the rodents, the uterine tube is held in a tightly coiled spiral by the mesosalpinx (Suarez, 1987; Smith et al., 1987). This may also affect sperm transport, as the sperm must be able to change direction and follow this twisting path.

Another relevant factor of uterine tube morphology is the distribution of secretory and ciliated cells in the epithelium. Again, there is wide species diversity in the proportion of each cell type (Nilsson & Reinius, 1969; Hunter, 1988) and both regional and cyclical variations in their morphogenesis (Brower & Anderson, 1969; Patek, 1974; Hagiwara et al., 1992; Abe & Oikawa, 1993). Sperm appear capable of binding to both cell types, though there may be species-dependent preferences for one type or the other (Hunter et al., 1987; Smith & Yanagimachi, 1990; Suarez et al., 1991b).

The secretory products of the uterine tube may play a physiological role in the regulation of sperm transport. There are a few reports of tubal secretions interacting with sperm (Sutton et al., 1986; Voglmayr & Sawyer, 1986; Wagh & Lippes, 1989; McNutt et al., 1993), and in the case of human sperm this secretion has been identified as the sialylated glycoprotein alpha-fetoprotein (Wagh & Lippes, 1993). A functional role for these secretions remains to be clearly demonstrated, though








25

McNutt and coworkers (McNutt et al., 1993) have associated enhanced bull sperm capacitation with tubal fluid treatment.

A wide array of other tubal secretory proteins have been characterized that have been proposed to influence the egg or embryo. Some of these show regional specificity, as demonstrated with rabbit (Hyde & Black, 1986), pig (Buhi et al., 1990) and sheep (Murray, 1992) uterine tubes. Others have been demonstrated to be under hormonal control either by variation during the estrous cycles of sheep and mice (Sutton et al., 1986; Horvat et al., 1992; Vrcic et al., 1993) or following hormonal treatments of rabbits (Hyde & Black, 1986; Erickson-Lawrence et al., 1989), baboon (Verhage & Fazleabas, 1988; Verhage et al., 1990), and sheep (Murray, 1992). Though some of these proteins obviously could not affect sperm based on their secretory location and timing, clear demonstrations of association with the eggs and embryos are few (Kapur & Johnson, 1988; Minami et al., 1992; Boice et al., 1992; Buhi et al., 1993). More tubal secretions may eventually turn out to bind sperm or indirectly affect transport out of the reservoir by altering the tubal environment.

Another type of secretion that could potentially affect sperm transport is tubal mucus. Because of the difficulties of preserving mucopolysaccharides, the presence and prevalence of this material in the lumen remains unclear. In standard immersion fixation protocols followed by routine histological preparations, the luminal contents are lost (Schulte et al., 1985). The inclusion of polycationic alcian blue with aldehydes and use of perfusion fixation appears to improve the retention of luminal








26
contents and suggests that in the rabbit uterine tube there are both serous and mucous secretions present (Jansen & Bajpai, 1982). In scanning electron microscopy studies, the luminal contents appear patchy and as a matrix or honeycomb on the surface of the mucosa; sperm associated with the mucosa have been seen coated with this material (Jansen, 1978, 1980; Boyle et al., 1987; Hunter et al., 1987). Whether this material actually spans the luminal spaces, potentially affecting sperm movement as well as sperm adherence to the mucosa remains has not yet been clearly demonstrated.


Description of Experiments


The experiments described below address four aspects of the control of sperm transport to the site of fertilization. The characteristics of the binding of sperm to the tubal wall were examined to address how the sperm reservoir is formed and maintained. An analysis of sperm motility patterns in the uterine tube addresses a possible mechanism for release from the reservoir. The establishment of surface markers associated with various sperm conditions addresses how sperm changes may contribute to tubal transport. Finally, an examination and characterization of the tubal luminal contents addresses what role the luminal environment may play in sperm transport.








27

Nature of Sperm Binding in the Tubal Reservoir


The nature of the interaction between sperm and the tubal mucosa was examined by treating hamster sperm with a series of potential binding inhibitors and introducing them into excised hamster uterine tubes. Binding levels were assessed by observing and scoring the live sperm within the uterine tube by videomicroscopy. Sialic acid, especially as presented on the glycoprotein fetuin, appears to be a potent inhibitor of sperm binding in the hamster uterine tube. The binding of sperm in the tubal reservoir may rely on a carbohydrate mediated, interaction with sialic acid. Sperm Motility Patterns in the Uterine Tube


Sperm motility patterns were examined within the uterine tube by recording live mouse sperm in excised uterine tubes following natural mating. Qualitative observations about the bound and free-swimming sperm were made and quantitative measures taken from the recordings were used to characterize the hyperactivated motility pattern. All free-swimming sperm appeared to be using the hyperactivated pattern. Thus, it appears that when mouse sperm are able to release from the reservoir, they have switched to the hyperactivated pattern. Hyperactivation may contribute to their ability to release.


Sperm Surface Changes


Changes on the sperm surface were examined by producing monoclonal antibodies that recognized epitopes that were somehow changing as hamster sperm








28

went through a set of observable conditions during capacitation in vitro. The binding of antibodies to sperm in each condition was determined by indirect immunofluorescence staining patterns of fixed sperm and gold-labelled immunostaining of live sperm. The antigens were also characterized on Western blots. One antibody recognized an epitope that appeared to be unmasked when sperm had reached the hyperactivated stage. Another recognized an epitope that was lost or modified when they had reached hyperactivation. A third antibody recognized an epitope that was lost or modified after only a short period in culture. These antibodies are useful tools for isolating and identifying surface components that may play a role in binding and release from the tubal mucosa. The Morphology and Nature of Tubal Contents


In order to determine whether the tubal luminal contents could play a role in controlling sperm transport, a histological protocol was developed to optimally preserve the structure and position of the luminal contents. This protocol used celloidin-stabilized cryosections of mouse uterine tube that had been post-fixed with cetylpyridinium chloride. A substance with the staining characteristics of a mucopolysaccharide was found to occlude some of the luminal spaces, and sperm were observed in this substance. This tubal mucus seems to be in the path of sperm transport and could provide a selective advantage for the passage of hyperactivated sperm.













CHAPTER 2
MECHANISM OF SPERM BINDING IN THE ISTHMIC RESERVOIR


Introduction


The existence of a reservoir in the isthmic portion of the uterine tube in which sperm are maintained until the time of fertilization approaches has been described for a number of mammalian species (Zamboni, 1972; Thibault et al., 1975; Yanagimachi & Mahi, 1976; Overstreet et al., 1978; Shalgi & Kraicer, 1978; Hunter et al., 1987; Suarez, 1987). Sperm appear to ascend relatively quickly to this region and then are retained; the population of sperm responsible for fertilization move on to the ampulla only when fertilization is imminent (Harper, 1973b; Hunter, 1984; Suarez, 1987; Smith & Yanagimachi, 1991).

In the hamster uterine tube at least, it appears that the modulation of binding and release of sperm from the reservoir is primarily dependent on changes in the sperm cell, not the uterine tube (Smith & Yanagimachi, 1990, 1991). Noncapacitated hamster sperm samples injected into excised uterine tubes have been shown to bind almost completely to the epithelium whereas sperm from capacitated samples, that were also hyperactivated, are able to remain free (Smith & Yanagimachi, 1991). Further, following natural mating in the mouse, very few free swimming sperm were seen in the uterine tube and those that were appeared to be


29








30
hyperactivated (see Chapter 3). These results imply that there is a change in the affinity for the tubal epithelium coincident with capacitation and the switch to hyperactivated motility that allows sperm in the appropriate condition for fertilization to release from the reservoir.

The biochemical nature of sperm adherence in the reservoir and the changes leading to release have not yet been described, however. To address this question, the binding of non-capacitated sperm in excised hamster uterine tubes following treatment with potential inhibitors was analyzed. The binding of sperm in the isthmus appears to be quite strong, since repeated flushing is required to release the bound sperm (Smith & Yanagimachi, 1990). Also, various pretreatments of the uterine tube and enzymatic treatments of tubal explants with adherent sperm were unsuccessful for preventing sperm binding (Raychoudhury & Suarez, 1991; T.T. Smith, personal communication). For this study, a different approach was attempted where sperm were treated with potential inhibitors prior to exposure to the uterine tube. Carbohydrate inhibitors in the form of large glycoproteins were chosen due to the ineffectiveness of proteases (Raychoudhury & Suarez, 1991) and the importance of carbohydrate interactions for another sperm binding phenomenon, adherence to the zona pellucida (Wassarman, 1990; Cornwall et al., 1991; Noguchi & Nakano, 1992; Tulsiani et al., 1993).

In the present study, screening resulted in the detection of the glycoprotein fetuin as an inhibitor of sperm binding. Fetuin, a 43-49 kDa glycoprotein, contains 12-13 O-linked and N-linked carbohydrate chains that end in the N-acetylneuraminic








31

acid form of sialic acid (Graham, 1966; Spiro & Bhoyroo, 1974; Krusius et al., 1976). Subsequent analysis revealed that the sialic acid residues appear to be involved in the binding inhibition. Fetuin labelling of fresh and hyperactivated sperm cells and of proteins separated from sperm extracts was investigated to assess changes that might correlate with the release of sperm from the tubal reservoir.


Materials and Methods


Medium and Chemicals


For the culture of sperm cells and induction of hyperactivated motility, hamster sperm capacitation medium, similar to that shown to sustain hyperactivation and capacitation previously (Suarez et al., 1993) was used. The medium contains 105 mM NaCI, 5 mM KCI, 2.4 mM CaC2, 0.49 mM MgC2, 0.36 mM NaH2PO4, 25 mM HEPES buffer, 25mM NaHCO3, 5.00 mM glucose, 6.26 mM sodium lactate, 0.125 mM pyruvic acid, 12 mg/ml Fraction V bovine serum albumin (BSA) and 0.06 g/l penicillin G. The pH was adjusted to 7.5 prior to filter sterilization (0.22 ;m MillexGV filter, Millipore Corp., Bedford, MA). Osmolarity was 285-295 mOsm/kg. Prior to use, 1 j.m epinephrine, 100 ;km hypotaurine, and 20 M D-penicillamine were added from frozen 100X stock solutions (Bavister, 1989). Incomplete hamster capacitation medium lacked BSA and the metabolic substrates, glucose, lactate, and pyruvic acid.








32

Protease inhibitors were also prepared as a 10OX concentrate. The concentrate contained 10 mg/ml aprotinin, 1 mg/ml Na-t-boc-deacetylleupeptin, and 500 mM benzamidine HCI dissolved in incomplete medium.

Sperm extraction buffer (modified from Saling & Lakoski, 1985) contained 2% sodium dodecyl sulfate (SDS), 125 mM Tris buffer, pH 6.8, 20% glycerol, 10 g/ml Na-t-boc-deacetylleupeptin, and 2 mM phenylmethylsulfonyl fluoride (PMSF).

All chemicals were from Sigma Chemical Co. (St. Louis, MO) except those specifically noted here. Limax flavus lectin (LFA), BSA and HEPES were from Calbiochem Corp. (La Jolla, CA). Fetuin conjugated to 5 nm colloidal gold particles and chick pea lectin (CPA) were obtained from E-Y Labs, Inc. (San Mateo, CA). Tris was from Biorad Laboratories (Richmond, CA). Glycine was from ICN Biomedicals, Inc. (Cleveland, OH). SDS was from BDH Chemicals, Ltd. (Poole, UK).


Binding Inhibition in Excised Uterine Tubes


Golden Syrian hamsters (Mesocricetus auratus) from Charles Rivers Laboratories (Wilmington, MA) were maintained on a 14L:10D light cycle with lights on from 0700-2100 h and provided food and water ad libitum. Retired breeder males were used to obtain caudal epididymal sperm. Females (9-18 weeks old) were visually staged with Day 1, estrus, being determined by the presence of clear vaginal discharge (Hafez, 1970). Animals, males first, were killed by CO2 inhalation followed








33

by cervical dislocation at 1400 h on Day 1, approximately 12 hours before the females were expected to ovulate (Hafez, 1970).

From the males, the epididymides were exposed and the caudae were punctured with a 25 ga. needle. A drop of epididymal contents was placed in 1 ml of hamster capacitation medium that had been prewarmed to 37"C and equilibrated under 5% CO2. Sperm were allowed to disperse for 5 minutes at 37* C then the top 2/3 of the suspension, containing the highly motile fraction, were removed. After the motility was assessed, the suspension was centrifuged at 140 G for 5 minutes to concentrate the sperm. The bottom 100 1 of this concentrate was retained and sperm concentration was determined with a hemacytometer. Concentrations were generally 1-2 X 10s sperm/ml. From this stock suspension, dilutions were made containing 3 X 106 sperm/mi in 500 jl hamster capacitation medium plus candidate inhibitors dissolved in incomplete capacitation medium (see Table 2-1) or incomplete medium alone and incubated at 37C for 10 minutes. At each experiment, the assignment of treatments to the first and second uterine tube preparation was randomized by flipping a coin.

While the sperm samples were being prepared, females were killed in the same manner and their uterine tubes were removed and uncoiled. To accomplish this, the cranial tip of the uterine horn was bisected and lifted up to allow the ovarian mesenteries to be cut. These segments were rinsed with prewarmed medium and placed in petri dishes in a small drop of medium. One of the dishes was placed in a 37C, 5% CO2 incubator while the other uterine tube was dissected. The side to








34
Table 2-1. Candidate competitive inhibitors of carbohydrate interactions in the
uterine tube.


Candidate Concentration j Inhibitory element Fetuin 5 mg/ml sialic acid Asialofetuin 5 mg/ml galactose Fucoidan 5 mg/ml fucose Ovalbumin 5 mg/ml n-acetylglucosamine, mannose

Poly-l-lysine 5 mg/ml + charge





be dissected first was randomized by flipping a coin during each experiment. Using a dissecting microscope, the coiled uterine tube was straightened by carefully cutting the mesosalpinx. The uterine horn was pinned in wax and only the horn and ovary were used to hold the tissue. When the uterine tube had been completely straightened, the ovary and ovarian bursa were removed, the first loop of the isthmus was freed from the uterine serosa, and the uterine tube was cut through the extramural uterotubal junction.

Incubated sperm samples were gently drawn into a 1 ml syringe equipped with a blunted 30 ga. needle. The needle was introduced into the lumen at the infundibulum and at the first loop of the isthmus and 50 + 5 ul was injected into








35

each end. In several cases, the isthmus could not be successfully injected and an additional 50 pC was injected through the infundibulum. The flow of sperm suspension through the entire uterine tube was observed to ensure that the lumen was patent and had been completely flushed. The uterine tubes were rinsed in each of 5 drops of medium in a clean petri dish to remove most of the sperm on the outside and placed on a microscope slide. A coverslip, supported by 4 pillars of silicone grease, was gently pressed down to slightly flatten the uterine tube (modified from Suarez and Osman, 1987).

The slide chamber was immediately placed on the heated stage of an inverted videomicroscope. The preparations were observed under bright field illumination with a halogen light source and a 30X extra-long working distance Hoffman Modulation Contrast objective (Modulation Optics, Greenvale, NY) (Figure 2-1). Images from a solid state Dage CCD 72 camera (MTI Inc., Michigan City, IN) were recorded at 30 frames/second on a SuperVHS videocassette recorder (Panasonic AG7300, Panasonic Industrial Co., Secaucus, NJ) along with time/date information (Model VTG 33, For-A Co., Ltd., Newton, MA). Uterine tubes were taped beginning at the ampulla progressing toward the isthmus. To ensure that differences seen between the isthmus and ampulla were not an effect of the sequential taping, in several uterine tubes the isthmus was taped briefly before the usual sequence was followed. In several other uterine tubes, the ampulla was retaped after the usual progression had been completed.








36



































Figure 2-1. Photomicrographs of hamster sperm within the tubal isthmus.
Magnification 30X.








37

The sperm on the videotapes were counted and scored as either free or bound to the mucosa based on the first two seconds in which they were in focus. Counts from three uterine tubes were duplicated by two separate observers to provide an assessment of interobserver variation. From the counts, the overall proportions of free and bound sperm were calculated as well as proportions for each region of the uterine tube. The proportions were calculated based on all the sperm that could be seen, approximately 200 per uterine tube. For testing by analysis of variance (ANOVA), the proportions were transformed by taking the arcsin of the square root (Sokal & Rohlf, 1989) and the significance level was set at p < 0.05. Values reported are not transformed.


Binding of Fetuin to Fresh and Hyperactivated Sperm


Cauda epididymal sperm were obtained and the highly motile fraction was collected as above. The concentration of this fraction was determined and dilutions were set up containing 3 X 106 sperm in 1 ml of capacitation medium. Immediately after dilution, aliquots of the non-capacitated, non-hyperactivated sperm were incubated with either 0.05 ;g/ml of 5 nm colloidal gold-conjugated fetuin or an equivalent volume of medium for 15 minutes. Hyperactivated sperm were treated similarly once they were obtained, after approximately 3 hours of culture in a 37"C, 5% CO2 incubator. Samples were checked occasionally, and when at least 70% of the sperm were using the characteristic hyperactivated motility pattern (Suarez et al., 1993), a sample was removed for labelling.







38

Sperm were pelleted in a microcentrifuge and washed 3X with incomplete medium. They were fixed in suspension with 2.5% glutaraldehyde in phosphate buffer, pH 7.3, for 5 minutes and then washed again. Samples were placed in the wells of multi-well immunofluorescence slides (Cel-line Assoc., Newfield, NJ) and allowed to dry down. They were rinsed 5X with PBS then 5X with distilled water. They were incubated with silver enhancer solution (Sigma, St. Louis, MO) for 7 minutes, rinsed again with distilled water, and the enhancement reaction was stabilized by treatment with 2.5% sodium thiosulfate for 2-3 minutes (according to the instructions of the supplier). The samples were rinsed again with distilled water and coverslipped with the aqueous mounting medium Gel/Mount (biomedia, Foster City, CA). Patterns of fetuin binding were observed using differential interference contrast (DIC) optics.


Characterization of Fetuin Binding Proteins


For the extraction of fresh sperm membrane components, cauda epididymal contents from 2 males (4 epididymides) were released into six 1.5 ml-eppendorf tubes. The highly motile fractions (top 700 Al) were pooled after 10 minutes and samples were removed to check motility and determine concentration. Protease inhibitors, 100X concentrate, were added and the sperm were pelleted at 960 G for 5 min, washed in 5 ml of incomplete medium plus protease inhibitors (960 G, 5 minutes), then suspended in 1.5-2 ml of cold extraction buffer to yield a final concentration of approximately 2 X 108/ml. The suspension was kept on ice and after








39

15 minutes, it was sonicated in three 20 second bursts separated by 30 seconds (Heat Systems Ultrasonics, model W-225R, Farmingdale, NY). The extraction was continued for another 45 minutes and then the mixture was cleared by centrifugation, 12,000 G, 10 minutes. Total protein concentration in the supernatant was determined using the Biorad protein assay (Biorad Labs, Richmond, CA). Concentrations ranged from 1-1.5 mg/ml. The supernatants were stored at 80 OC until used.

To extract hyperactivated sperm components, caudal epididymal contents were collected from 4 males and pooled after swim up. Motility and concentration were assessed and a series of 15-ml centrifuge tubes were set up containing 3 X 106 sperm/ml or 6 X 106 sperm/ml in 10 ml of capacitating medium. When the sperm were hyperactivated, determined as described above, the top 8 ml from each tube was pooled, protease inhibitors were added, and the sperm were pelleted and washed as above. Sperm concentration was determined after they were suspended in 2.25 ml of extraction buffer. Final concentrations were approximately 2 X 10/ml. Thereafter, sperm were treated as above.

Fresh and hyperactivated sperm extracts were thawed and prepared for reducing polyacrylamide gel electrophoresis (PAGE) by boiling for 3 minutes in the presence of 10% 2-mercaptoethanol (Buhi et al., 1989). Bromophenol blue was added for color. Samples (500-800 Mg) and molecular weight standards were separated with 4.5% stacking gels and 10% running gels in a Tris-glycine buffer system (Roberts et al., 1984). Stacking current was 15 mA per gel and running current was 30 mA per gel.








40

Proteins were transferred to Immobilon-P membranes (Millipore Corp., Bedford, MA) by semi-dry electrophoretic transfer at 2.5 mA/cm2 for 15 minutes (Buhi et al., 1993). Standards and a lane of sperm proteins were separated and stained with Coomassie blue in 50% methanol. Gels were also stained with Coomassie blue to ensure the transfer was successful. The blots were blocked with 3% BSA in PBS, pH 7.4, for 2 hours at room temperature. After rinsing with PBS, blot strips were incubated with the treatments listed in Table 2-2. All treatments were diluted with PBS containing 3% BSA and inhibitory substrates were included with lectins as a control for the specificity of their binding.

Slug lectin, LFA, was not available conjugated to a label and was detected using an indirect method (Roth et al., 1984, 1985). The lectin is multivalent and will bind to fetuin/gold. LFA-treated blots were incubated for 1 hour at room temperature with 0.5 Ag/ml of fetuin/gold. Fetuin/gold treatment for 1 hour at room temperature without previous lectin treatment served as a control. Labelling with fetuin/gold was detected using the silver enhancement reaction (Sigma, St. Louis, MO). Peroxidase-labelled chick pea lectin, CPA, was detected using diaminobenzidine (DAB) as substrate with nickel enhancement (Harlow & Lane, 1988).


Results


For preliminary screening, each of the inhibitors listed in Table 2-1 was tested in two uterine tubes from two different females and compared to sperm treated with








41

Table 2-2. Carbohydrate binding treatments for blotted sperm components. Treatment Conc. Conditions Detection Fetuin/gold' 0.5 p/g/ml overnight, 4*C Ag enhance Fetuin/gold 0.5 /g/ml 1 h, 22"C Ag enhance LFA2 10 pg/ml overnight, 4C fet/gold, Ag enhance

LFA + 10 /kg/ml + overnight, 40C fet/gold, Sialic acid3 10 mM Ag enhance CPA4 10 /zg/ml overnight, 4"C HRP CPA + 10 gg/ml + overnight, 4C HRP Fetuin 1 mg/ml

1. Fetuin conjugated to 5 nm colloidal gold particles.

2. Lectin from the garden slug, Limaxflavus, recognizes sialic acid (Roth et al.,
1984).

3. n-acetylneuraminic acid.

4. Lectin from the chickpea, recognizes several sialic acid containing
glycoproteins including fetuin (Kolberg et al., 1983).








42

PBS vehicle only. The motility of the sperm at the end of the ten minute treatment was also checked. During videotaping following treatment with asialofetuin, ovalbumin, fucoidan, poly-l-lysine and PBS, almost no motile sperm were seen free of the mucosa. Some immotile sperm could be seen being transported passively with the tubal contents. Further, motile sperm were not observed to pull away from the mucosa. In contrast, numerous free swimming sperm were observed following treatment with fetuin. The free sperm were observed to encounter the mucosa and not stick to it.

Having identified fetuin as the only inhibitor of sperm binding to the tubal mucosa among the candidates, a quantitative analysis was completed to more carefully study the binding inhibition by fetuin and determine whether the terminal sialic acid residues were responsible for the inhibition. Uterine tubes from a series of females were randomly assigned by flipping a coin to sperm that had been treated with 5 mg/ml of either fetuin or asialofetuin, 25 mM sialic acid (n-acetylneuraminic acid), or PBS vehicle. The pH of the sialic acid treatment was readjusted to 7.5 prior to the addition of sperm. There were significant differences between the groups in the proportion of free sperm found in the uterine tube by ANOVA (F=13.5, df=20, p=0.0001). The proportion of free sperm was significantly higher than buffer control by Fisher's test for multiple comparisons for both fetuin and sialic acid-treated sperm (Figure 2-2). The binding inhibition by fetuin was not significantly stronger than that by sialic acid. Also, asialofetuin was again ineffective as an inhibitor of sperm binding. The proportion of free sperm in the asialofetuin group was not different








43









% Free Sperm
30
n=7

a
25


20n15 15


10 n-5





Fetuin Sialic Asialo. Buffer Treatment






Figure 2-2. Bar graph showing effect of treatment on sperm. Sperm counted from
entire uterine tube. n = number of uterine tubes, error bars represent
SEM, bars marked with different letters differ significantly.







44

from buffer control and was significantly lower than that observed in the fetuin and sialic acid groups.

While analyzing the videotapes, a clear regional difference in sperm binding was observed. In the fetuin and sialic acid treatments, most of the free sperm were seen in the isthmus. As the morphology gradually changed in the region of the ampullary/isthmic junction (AIJ), numbers of free sperm decreased. In the ampulla, very few free sperm were observed. When the proportions of free sperm in each of these regions were compared, significant differences were detected by ANOVA for fetuin (F=12.9, df= 17, p=0.005) and sialic acid (F= 10.7, df= 14, p=0.002). Since the analysis was carried out a posteriori, the more stringent Scheffe's F test was used for multiple comparisons (Marks, 1990). The inhibition in the isthmus was significantly stronger than in the ampulla for both fetuin and sialic acid (Figure 2-3). In the fetuin treatment, the decrease between isthmus and AIJ was also statistically significant. In the sialic acid treatment, however, the variability was such that the proportion free in the AIJ was not statistically different from the isthmus or ampulla.

When the sperm from the isthmus and ampulla were separated and differences between the treatments analyzed by region, it was found that most of the fetuin-induced inhibition was occurring in the isthmus (Figure 2-4). In the isthmus, inhibition in the fetuin-treated group was significantly higher by Scheffe's F test than the asialofetuin or buffer groups. The sialic acid group was significantly higher than the asialofetuin and buffer groups, which remained statistically indistinguishable. In the ampulla, however, there was no significant inhibition in any group. The mean







45









% Free Sperm
50 40

30


20


10


0
Isthmus AIJ Ampulla Region


Fetuin M Sialic






Figure 2-3. Bar graph showing regional differences in inhibition by fetuin and sialic
acid. Error bars represent SEM.








46







% Free Sperm
50


40


30


20 10


0
Isthmus Ampulla Treatment


fl Fetuin E Sialic 0 Asialo M Buffer








Figure 2-4. Bar graph showing that effect of treatment is due primarily to isthmic
inhibition. Error bars represent SEM.








47

proportions were distributed in the same pattern, but they were very low and the differences were slight. There was also a significant inhibition effect of treatment in the AIJ, but only the fetuin treatment was high enough to differ statistically from control (data not shown).

The binding of fetuin to the sperm cells was demonstrated using colloidal goldlabelled fetuin. The silver-enhanced label was found over the acrosomal region of the head on fresh, acrosome intact sperm (Figure 2-5a). The staining pattern covered the dorsal and anterior surfaces of the sperm head, apparently demarcating the shape of the hamster acrosome. There also appeared to be a band of fetuin labelling at the neck of the sperm, though this was variable. On the midpiece, labelling was restricted to the distal portion and was spotty. Where acrosomes had been disrupted during processing, the acrosomal fragments labelled heavily but the regions that had been exposed underneath the acrosome did not label.

Hyperactivated sperm showed a different pattern of fetuin binding (Fig. 2-5b). Acrosome intact sperm had a characteristic clear patch on the dorsal and anterior acrosomal region. This region appeared to have lost its fetuin binding ability during incubation. The midpiece also had decreased labelling as the spottiness became very sparse. The posterior margin of the acrosome and the neck were still able to bind fetuin, however. Disrupted acrosomes stained variably.

Specific fetuin binding components were detected in Western blots of sperm extracts and differences were noted between fresh and hyperactivated sperm (Figure 2-6). Results were similar with blots from 2 separate sets of extracts. Colloidal gold








48



A
























C









Figure 2-5. Photomicrographs of silver-enhanced labelled hamster sperm. A) Fresh
sperm with fetuin-gold, B) Hyperactivated sperm with fetuin-gold, C)
Control sperm with no label.


f








49

labelled fetuin incubated with the blots overnight bound to fresh sperm components at a number of molecular weights. The intensity of labelling was generally lower for hyperactivated sperm extracts though the gels had been loaded with the same amount of total protein. Distinct bands at 27.5 kDa and >335 kDa in the fresh sperm extracts were absent in the hyperactivated extracts. A strong band at 32-33 kDa in the fresh sperm extracts was very dim at hyperactivation. Also, a strong band in the fresh sperm extracts at 49-50 kDa seemed to have shifted to an apparent molecular weight of 53 kDa in the hyperactivated extracts.

Lectin binding on the blots incubated overnight with LFA indicated that the fetuin binding proteins seem to be sialylated (Figure 2-6). The same group of proteins was recognized by the LFA, though some of the bands that labelled weakly with fetuin were not detectable in the LFA blots. LFA staining was also generally decreased in the hyperactivated extracts and the differences observed agreed with the results in the fetuin-labelled blots.

Since LFA was detected using an indirect technique including a 1 hour, room temperature incubation with colloidal gold-labelled fetuin (Roth et al., 1985; Roth et al., 1984), control blots that had been incubated overnight with diluent (3% BSA in PBS), then treated for 1 hour at room temperature with labelled fetuin were included. These strips were developed with silver enhancement reagent for the same amount of time as the LFA strips. The interference due to colloidal gold-labelled fetuin was minimal under these conditions (Figure 2-6). Also, LFA labelling was inhibited when sialic acid was included during the incubation.







50








A B








50k -32k 27k



f h ctrl f h



Figure 2-6. PVDF blots probed for carbohydrate binding. A) fetuin-gold-labelled
sperm components, B) LFA-labelled sperm components.








51

Staining with CPA was similar to the previous results though the labelling was generally dim. The stronger fetuin binding bands could be detected and one band at 42-44 kDa that had not been detected with fetuin or LFA was observed (data not shown). CPA labelling was inhibited by fetuin.




Discussion


Since fetuin and sialic acid, but not asialofetuin, are able to inhibit sperm binding to the tubal mucosa, the terminal sialic acid residues of the fetuin molecule appear to be important for blocking the interaction. This implies that the binding between sperm and the uterine tube involves a specific interaction with sialic acid. At least for hamster sperm, none of the common sugars other than sialic acid seem to be involved in sperm binding to the uterine tube. Also, it does not appear that charge interactions are the basis of this interaction, since poly-l-lysine was also ineffective as an inhibitor. Sialic acid is an effective inhibitor by itself, but as might be expected, the large fetuin molecule is significantly more effective. The glycoprotein contains 12-13 carbohydrate chains that end in sialic acid (Spiro & Bhoyroo, 1974; Krusius et al., 1976), providing multiple binding possibilities to stabilize the interaction.

The labelling of the fresh sperm over the region of the head that characteristically binds to the tubal mucosa (Suarez, 1987; Smith & Yanagimachi, 1990) and identification of specific sperm glycoproteins that label with fetuin on








52

Western blots suggests that the inhibition by fetuin is due to the presence of a fetuin binding component on the sperm. The fetuin binding component appears to be serving as a lectin-like receptor for sialic acid containing ligands. Such a role for carbohydrates in cellular interactions is well established. The most extensively studied are the selectins, glycoproteins involved in leukocyte/endothelial adhesion that bind to sialylated oligosaccharide ligands (Phillips et al., 1990; Walz et al., 1990; Gahmberg et al., 1992; Varki, 1993). CD 22, a receptor from B cells belonging to another class of glycoproteins, the immunoglobulin superfamily, also operates as a sialic acidbinding lectin (Sgroi et al., 1993; Powell et al., 1993). Additionally, pertussis toxin appears to contain a subunit that is capable of binding fetuin and other sialic acid bearing substrates (Heerze et al., 1992).

The presence of carbohydrate mediated interactions involving sperm has also been documented. Sperm/egg binding in the mouse appears to involve an interaction with the oligosaccharide portion of the mouse ZP3 glycoprotein, specifically a galactose residue (Wassarman, 1990). Sperm also seem to bind to the carbohydrate portion of the pig ZP3 molecule (Noguchi & Nakano, 1992). The involvement of mannose (Tesarik et al., 1991; Cornwall et al., 1991; Tulsiani et al., 1992) and fucose (Oehninger et al., 1991) in sperm/egg binding have also been proposed. These findings demonstrate the importance of carbohydrate mediated interactions for sperm physiology.

The fetuin binding components in sperm extracts appeared to be predominantly sialylated themselves. The similarities between the labelling with







53

fetuin gold and with the two sialic acid recognizing lectins, LFA and CPA, were striking. Sialylated molecules on sperm have previously been identified and changes during maturation have been documented (Nicolson & Yanagimachi, 1972; Hammerstedt et al., 1982; Feuchter et al., 1988; Magargee et al., 1988; Rankin et al., 1989; Bains et al., 1993b). Capacitation also seems to involve changes in the sialylation patterns of sperm. Treatment with neuraminidase accelerated the capacitation of rabbit and guinea pig sperm in vitro (Gwatkin et al., 1972; Oliphant, 1976; Srivastava et al., 1988) and sialylated molecules from sperm have been found released into capacitation medium (Focarelli et al., 1990). For hamster sperm, the binding of sialic acid lectins decreases during capacitation (Ahuja, 1984; Nicolson & Yanagimachi, 1972). Part of the decrease in sialylation may represent the loss of a fetuin binding component responsible for maintaining the adherence of sperm in the isthmic reservoir.

The regional restriction of the inhibition to the isthmus suggests that the fetuin binding component is only involved in binding in the isthmus. This specificity matches the restriction of the sperm reservoir to the isthmus. Binding of noncapacitated sperm in the ampulla apparently proceeds by another mechanism and is presumably irrelevant in vivo, since non-capacitated sperm cannot pass through the isthmus to reach the ampulla.

Alteration of the interaction between the isthmic epithelium and the fetuinbinding component as part of the sperm changes occurring during capacitation and/or hyperactivation may allow release from the reservoir. The extremely low numbers








54
of free sperm seen in these experiments, in agreement with previous observations when non-capacitated, non-hyperactivated sperm were injected into the hamster uterine tube (Smith & Yanagimachi, 1991) speak to the strength of this interaction for fresh sperm. However, once sperm have been capacitated in vitro (Smith & Yanagimachi, 1991) or begun using the hyperactivated motility pattern following natural mating (see Chapter 3), they are able to release from the tubal mucosa. Smith and Yanagimachi (1991) demonstrated that this difference was likely due to changes in the sperm, not the condition of the uterine tube. While the use of hyperactivated motility may provide additional force for sperm to break free, there is substantial support for a mechanism of release involving the loss of the fetuinbinding substance.

The binding patterns seen with colloidal gold-labelled fetuin indicate that during incubation to the hyperactivated stage, the fetuin binding affinity over the dorsal and anterior surface of the acrosomal region decreases. This is the region of the sperm head that typically binds to the tubal epithelium (Suarez, 1987; Smith & Yanagimachi, 1990). The reduction in the fetuin binding ability is thus well correlated in terms of both timing and localization with release from the tubal mucosa.

In a study on zona pellucida glycoprotein binding to sperm, fetuin was used as a control for non-specific binding (Mortillo & Wassarman, 1991). The study found very low levels of fetuin binding sites on the plasma membrane of acrosome intact, capacitated sperm and slightly higher levels on the acrosomal membranes of reacted







55
sperm using colloidal gold labelling and electron microscopy. Also, capacitated mouse sperm were observed to have little ability to bind fetuin-coated beads (Vazquez et al., 1989). These results further support the proposal that when sperm are able to release from the tubal reservoir they have reduced fetuin binding affinity.

The differences observed in fetuin/gold labelling between blots of fresh and hyperactivated sperm extracts suggest candidates for the fetuin binding component involved in adherence in the isthmus. The bands that appear to be lost or sharply reduced prior to hyperactivation, one high molecular weight band >335 kDa, and 3233 and 27.5 kDa bands, are obvious choices for further characterization. Also, the potential change in the 49-50 kDa band is interesting. Further characterization and antibodies directed against each of these fetuin binding glycoproteins would be useful for establishing the identity of the component involved in sperm/epithelial binding.

Similarities to several previously described glycoproteins can be noted. A 50 kDa extrinsic sialoglycoprotein that seems to be localized over the acrosomal region of cauda epididymal rat sperm has been described (Rifkin & Olson, 1985). Other rat sperm glycoproteins at 32 and 33 kDa have been identified (Hall & Killian, 1989) and a minor component of the sialylglycoconjugates released during human sperm capacitation appeared at 32 kDa (Focarelli et al., 1990). Additionally, a hamster sperm glycoprotein of epididymal origin that may be localized over the acrosome appears to migrate around 26 kDa, and a 26 kDa wheat germ agglutinin-binding protein was detected in mouse caudal epididymal fluid (Rankin et al., 1989).







56

Considering the current information, a possible model for the maintenance of the isthmic reservoir involves the binding of non-capacitated, non-hyperactivated sperm to the tubal mucosa by a sialoglycoprotein receptor that serves as a sialic acid lectin. Subsequent loss or modification of this receptor during the course of capacitation, potentially by desialylation, other enzymatic processing, or by absorption onto carrier molecules in the fluid, may decrease the affinity of the sperm for the mucosa and enable sperm to release. Hyperactivation may also contribute by providing extra force for pulling away. The factors responsible for the binding of non-capacitated sperm in the ampulla must also be changed during capacitation so that the sperm arriving there are not retained.


Conclusions


The binding of hamster sperm to the tubal mucosa has been characterized based on the ability of the sialylated glycoprotein fetuin and sialic acid to inhibit the binding specifically in the isthmus. Non-capacitated, non-hyperactivated sperm appear to have a sialylated component on their surface that acts as a lectin-like receptor for sialic acid containing molecules. This mechanism is proposed to maintain the adherence of sperm in the isthmic reservoir until the time of fertilization approaches. The fetuin binding characteristics of the sperm appear to change coincident with hyperactivation, the stage at which sperm can detach from the isthmic mucosa. The area over the acrosome by which sperm adhere to the mucosa shows a sharp reduction in fetuin binding, indicating a loss of the component involved in








57

adhesion. These results agree with previous findings about the ability of hyperactivated and capacitated sperm to release from the mucosa, a reduction in sialylation as part of the capacitation sequence, and the inability of capacitated sperm to bind fetuin. However, this is the first time that these phenomena have been related to a function, specifically the regulation of sperm passage from the isthmic reservoir. Western blots labelled with fetuin and sialic acid-recognizing lectins identified proteins at several molecular weights that show changes in labelling between fresh and hyperactivated sperm extracts. These proteins are good candidates for further study as the fetuin binding component responsible for sperm adherence in the isthmic reservoir.













CHAPTER 3
SPERM MOTILITY PATTERNS IN THE UTERINE TUBE Introduction


The types of cellular interactions involving sperm discussed in the previous chapter are a part of the complement of regulatory mechanisms that many cell types contain. The ability to interact with biomolecules in a specific manner and modify these interactions is a foundation of modern cell biology. Beyond these typical mechanisms, sperm have the unique characteristic, among the cells of complex organisms, of being capable of relatively rapid, independent movement. Motility provides alternative ways for sperm to interact with their environment. The propulsive force generated by the sperm tail may affect the ability of sperm to release from the epithelium and pass through the tubal environment. Also, the dynamic nature of motility suggests that different patterns may be expressed in a given environment and this then allows the assessment of functional advantages associated with particular motility patterns. In this light, sperm motility and its modification becomes a potential regulatory feature of sperm transport through the uterine tube. The aim of this study was to relate the progress of sperm in the uterine tube to active motility patterns and describe some factors that affect these patterns.




58








59

Investigations of sperm motility and progress through the uterine tube have relied on serial sectioning (Yanagimachi & Chang, 1963; Olds, 1970; Smith et al., 1987), flushes with oil and physiological medium (Cooper et al., 1979; Overstreet et al., 1980; Suarez et al., 1983; Smith & Yanagimachi, 1990), blocking passage through the uterine tube at various times (Hunter, 1984), and in situ observations in animals with small, translucent uterine tubes (Katz & Yanagimachi, 1980; Suarez, 1987; Smith & Yanagimachi, 1991). Serial sectioning allows accurate localization of sperm and, coupled with timed sampling, can provide information about distribution changes. A disadvantage is that the relative importance of sperm motility and the factors that affect it cannot be determined. Tubal flushes provide more information about the condition of the sperm since their motility, or lack of it, and capacitation status can be characterized. However, location in the uterine tube cannot be accurately described and motility must be analyzed and interpreted out of physiological context. Studies on the effects of tubal ligation at various positions and times relative to ovulation or fertilization offer an advantage in that the actual fertilizing population of sperm can be localized. Again, however, the motility and capacitation status of individual sperm cannot be assessed. Observing sperm within the uterine tube has the advantage of allowing investigators to localize sperm at different times and also to observe the motility patterns actually used in the uterine tube.

In this study, a previously described in situ preparation (Suarez, 1987) for observing mouse sperm within the uterine tube has been modified to allow longer contiguous segments of uterine tube and, thus, longer segments of sperm transport








60

to be observed. This modification lets us follow individual sperm longer and observe dynamic aspects of sperm motility. These observations contribute to our understanding of two specific phenomena: the establishment and release of sperm from isthmic reservoirs, and the function of hyperactivated motility.

Hyperactivation is a vigorous, erratic motility pattern assumed by some sperm in the uterine tube (Katz & Yanagimachi, 1980; Suarez, 1987). It is characterized by sharply curved, asymmetric tail beats and frequent changes of direction (Katz et al., 1989). Katz and coworkers (1989) have reviewed several proposed functions for hyperactivation in the confined spaces and varied substances of the uterine tube including an increased ability for sperm to free themselves from the tubal wall, an increased ability to penetrate viscous or viscoelastic fluids such as the egg vestments, and an increased probability for escaping from between epithelial folds. The possible advantage of hyperactivation for sperm release from the tubal wall could result from increased forces and/or torques generated in various directions. Similarly, these increases may help sperm penetrate the cumulus matrix and zona pellucida surrounding the egg (Katz & Yanagimachi, 1981; Katz et al., 1989). Recent evidence for such an advantage comes from experimental comparisons of hyperactivated and non-hyperactivated hamster sperm penetrating artificial viscous media (Suarez et. al., 1991a; Suarez & Dai, 1992).

To address release from the isthmic reservoir and the use of hyperactivated motility, sperm were observed in the uterine tube and the pattern of progress for individual sperm was characterized. The effects of ovulatory status, local








61

environment in the uterine tube and time since insemination on flagellar bending and sperm sticking to the tubal mucosa were analyzed.


Materials and Methods


Medium ingredients were purchased from Sigma Chemical Co. (St. Louis, MO). The medium consisted of Earle's Balanced Salts supplemented with 2.2 g/1 sodium bicarbonate, 0.06 g/l penicillin, and 0.06 g/1l streptomycin. The final pH was adjusted to 7.6 and the medium was sterilized by filtration through 0.22 gm filters (Millipore Products Div., Bedford, MA).

Outbred ICR strain mice from Harlan Sprague Dawley (Indianapolis, IN) were kept under a 14:10 hour light cycle with lights on from 0700 h to 2100 h. A delayed mating protocol (Braden & Austin, 1954) was used to reduce the time between insemination and sperm arrival at the site of fertilization. This allowed us to observe a larger portion of the period between insemination and fertilization. Virgin females, 8-16 weeks old, were placed with retired breeder males on the morning of estrus and allowed to mate. After ejaculation was observed (Wimer & Fuller, 1966), sperm were allowed to ascend through the tract for 1, 1.5, 2, or 3 hours. The females were killed by CO2 inhalation and ovulatory status was determined by observing the ovaries, ovarian bursa, and uterine tubes. Based on preliminary observations, the ovulation period for delayed mated mice of this strain extended up to approximately 4 hours after lights on (1100). To observe mating, the lights had to be on, and by starting matings between 0730-0830 and sampling between 0900-1130, mice could be








62

obtained shortly before and shortly after ovulation at all of the desired sperm incubation time points.

Uterine tubes were removed by clamping across the uterine horn at the level of the intramural uterotubal junction with a pair of forceps, cutting the horn just caudal to the forceps, and lifting the uterine tube and ovary up so that the ovarian mesenteries could be cut. Uterine tubes and ovaries were placed in a petri dish and kept moist with medium that had been prewarmed to 370 C and equilibrated under 5% CO2. The uterine tubes were uncoiled by cutting the mesosalpinx while handling only the tip of the uterine horn and the ovary. The ovarian bursa was cut away once the ampulla was uncoiled. The tip of the uterine horn was cut off at the end of the dissection leaving a straightened uterine tube from the extramural uterotubal junction to the infundibulum. Once straightened, the uterine tubes were placed on a microscope slide with a small drop of medium and covered with a coverslip supported by silicone grease (modified from Suarez and Osman (1987). They were stored in a 37"C, 5% CO2 incubator when not being observed.

The uterine tube preparations were observed, beginning about 15 minutes after killing the mouse, through a 30x Hoffman Modulation Contrast objective (Modulation Optics, Greenvale, NY) on a Zeiss Axiovert inverted microscope with a heated stage (Carl Zeiss, Inc., Thornbrook, NY). A xenon stroboscopic light source (Model 10030, Chadwick Helmuth Co., Inc., El Monte, CA) was used to reduce exposure of sperm to light and provide crisp images in individual video frames. Using a Dage CCD 72 solid-state camera (Dage MTI, Inc., Michigan City,








63
IN), sperm movement within the uterine tubes was recorded at 30 frames/second, along with time-date information to 0.01 second (Model VTG 33, For-A Co., Ltd., Newton, MA), on a Panasonic AG-7300 SuperVHS video cassette recorder (Panasonic Industrial Co., Secaucus, NJ). Recording both uterine tubes was completed in approximately 30 minutes. Preliminary experiments indicated that extending the observation period of an uterine tube up to an hour or more was associated with vigorous, uncoordinated contractions of the tissue, and the lumen would begin to fill with refractile spheres, presumably released by the epithelium. Similar stress responses were found if the uterine tube was roughly handled during straightening or compressed too much under the coverslip. Data was not collected from preparations that contained refractile spheres or exhibited these contractions.

From the videotapes, observed on a Panasonic WV-5410 monitor, sperm were counted and categorized for region of the uterine tube they were found in (isthmus or ampulla), location (in the lumen or between mucosal folds), and attachment status (stuck or free). The regions were easily distinguished by their morphology (Suarez, 1987). The location was scored as "lumen" if the sperm was swimming in the central luminal space or stuck to the epithelium where it was in direct contact with the central luminal contents. Location was scored as "fold" if the sperm was out of contact with the central lumen, either in an isthmic pocket, or an ampullar longitudinal fold. The attachment status was recorded based on the first 5 seconds that each sperm was visible. If a sperm swam freely during any portion of this period it was scored "free." It was scored "stuck" if it appeared to remain associated with








64
the epithelium throughout the first 5 seconds. Sperm in all treatment groups were scored by the same criteria. For qualitative observations, some sperm were observed for up to 20 min. The proportion of free sperm in each region was calculated for each experiment by dividing the number of free sperm by the total number of sperm observed.

The flagellar curvature ratio (FCR) was measured as an indicator of hyperactivation for those sperm in which, within a single video frame, the entire principal bend could be found in focus at the maximally bent part of the beat (Suarez et al., 1983). The restrictive criteria for choosing sperm from which to measure FCR were required to avoid parallax errors and other image artifacts. The number of sperm for which FCR was measured was approximately 15% of the counted sperm, and these appeared to be representative based on observations of the sperm at normal video speed. FCR is calculated as the straight-line distance from the headmidpiece junction to the first inflection point of the principal bend, divided by the curved path distance between these two points (Figure 3-1). As the principal bend becomes flatter, the ratio approaches one; conversely, the ratio becomes smaller as the bend becomes more curved. FCR was measured from the stopped video frames using a Graf/Bar sonic digitizer (Science Accessories Corp., Stanford, CT) connected to an Apple Macintosh 512K (Apple Computer, Inc., Cupertino, CA) running a BASIC program written by W. Gottlieb and R.P. DeMott. Each measurement was made three times and the mean was recorded for each sperm.







65



A




AB
A,= FCR
AB


B










Figure 3-1. The method for calculating flagellar curvature ratio (FCR) is shown.
The tail was traced with the digitizer pen to calculate curved-path distance as well as straight-line distance from the ends of the trace.
(Used with permission from DeMott & Suarez, 1992).




All statistical tests were carried out with StatView 512+ (BrainPower, Inc., Calabasas, CA). Proportional measurement data (proportion of free sperm and FCR) were transformed by taking the arcsine of the square root of the proportion before testing by ANOVA (Sokal & Rohlf, 1989). One-factor and multi-factor ANOVA were performed and the a priori determined significance level was p

0.05. Values reported are nontransformed.








66

Results


Qualitative Observations

Using the straightened uterine tube preparation for observing sperm motility, we noted certain typical patterns of movement. There appeared to be two distinct populations of sperm, one that showed a very regular, moderately bent tail beat and another more dynamic population defined by regular moderate tail beats interspersed with groups of erratic, highly curved beats characteristic of hyperactivation. The erratic beats were of increased amplitude, varying frequency, and were propagated in randomly changing planes. The dynamic group, those sperm which varied their beat pattern, represented a small proportion of the total, approximately 20%. Sperm of both types were typically found in the same area, but occasionally an isthmic fold would contain a large number of regular sperm and no dynamic sperm.

Sperm in the dynamic group appeared to be the ones capable of swimming freely and making progress along the uterine tube. Regularly beating sperm were observed for up to 20 minutes and none were ever seen to break free from the epithelium. The typical pattern of motility for the dynamic sperm involved a period of regular beating ranging from 10 seconds to approximately 1 minute followed by a series of erratic, high curvature beats during which the sperm might successfully break free. This series typically lasted 5-10 seconds regardless of whether the sperm broke free. Some sperm that did not break free were seen to repeat the cycle. There were occasional sperm, approximately 1%, that continually beat erratically, but they did not








67
seem able to release from the epithelium any more frequently than those that used the erratic pattern intermittently. Sperm were seen to move in a similar fashion in both the isthmus and ampulla. There appeared to be relatively more dynamic sperm in the ampulla; specifically, there were no large clusters of regularly beating attached sperm.

Those sperm which released from the epithelium and swam freely did so for a maximum of approximately 5 seconds. Within this time they would again stick to the epithelium (Figure 3-2). During free swimming, sperm used both erratic, highly curved beats, and regular, moderately curved beats. This cycle was seen to repeat up to four times. The direction that a sperm swam after release appeared to be random. Sometimes the sperm would reattach the first time it contacted the epithelium, and sometimes it would bounce off, frequently making a direction change, and continue swimming freely. Sperm swam across isthmic pockets only to stick to the other side, others swam out of pockets and across the lumen to stick again, and some swam out of one pocket and into an adjacent one without entering the main area of the lumen.

In the mouse tubal isthmus, the predominant folding feature of the mucosa is separate, relatively narrow-mouthed, occasionally branching pockets oriented transverse to the central lumen. In the ampulla, there are predominantly longitudinal folds that provide lengthy channels up the uterine tube lateral to the central lumen. Sperm were more scattered and appeared to be less sheltered from the luminal contents and flow in the ampullar folds than in the isthmic ones. For example, in







68




o .0









releases sticks releases contact
1 1 I I I 11/ 1
0 2 3 4 5 6 7 30 free free sticks
Time (sec)


Figure 3-2. Illustration of a typical pattern and timecourse for sperm progress in
the uterine tube. Made from composite tracings of a single sperm moving within an isthmic pocket. Dotted line indicates the sperm path.
(Used with permission from DeMott & Suarez, 1992).


contracting preparations, the isthmic sperm in the pockets seemed unaffected by the currents that interfered with the beat patterns of sperm in the central lumen. In the ampulla, however, all sperm seemed to be subject to reorientation as the current changed with contractions. It should be noted that the contractions in the ampulla appeared less vigorous than the isthmic ones.








69
Quantitative Comparisons


A total of 1296 sperm from 5 males randomly mated in 19 experiments were counted and categorized. The transformed proportions of free sperm were used to test for an effect of the following factors: region, ovulation status, and time in the tract. Two uterine tubes were discounted because unusually low numbers of sperm were counted, ten in one, one in the other. Sperm stuck in the cumulus mass were not included in this analysis.

There was a highly significant effect of region on the proportion of freeswimming sperm by ANOVA (Table 3-1) with the proportion of free sperm in the ampulla more than twice that in the isthmus. There was also a significantly higher proportion of free sperm in post-ovulatory compared to pre-ovulatory uterine tubes. ANOVA showed no significant difference related to the factor of time in the tract and no interactions between factors. However, since many more sperm were found in the isthmus than ampulla, most of the effect observed for ovulation status resulted from isthmic sperm.

FCR values were measured for 174 sperm and analyzed by ANOVA for differences related to region of the uterine tube, attachment status, location relative to the lumen, ovulation state, and time in tract. All single factors and two-factor combinations were tested. Attachment status, region, and location relative to the lumen were all significant factors in two-way interactions (Table 3-2). A posteriori three-factor ANOVA for the combinations of these factors showed no significant








70

Table 3-1. Analysis of factors affecting proportion of sperm swimming freely
within the uterine tube; calculated from videotapes of in situ
preparations from naturally mated mice.


Factor Treatment Mean SEM N Region" Isthmus 11.8% 1.0% 1196 Ampulla 26.3% 0.8% 100


Ovulation Pre- 10.6% 1.6% 553 Status"
Post- 16.2% 2.0% 743


Time 1 h 18.7% 3.5% 311 in
Tract 1.5 h 12.8% 3.0% 412 2 h 12.6% 2.3% 198 3 h 11.6% 1.8% 375


Treatments of this factor significant at p < 0.01 Treatments of this factor significant at p < 0.05 (Used with permission from DeMott & Suarez, 1992).








71
Table 3-2. Attachment status interactions for FCR with tubal region and sperm
location analyzed by two-factor ANOVA showing mean + SEM for
each treatment combination.


Attachment Status2

Free Stuck

Region1,2 Isthmus 0.656 + 0.034 0.777 + 0.013 n=33 n=85

Ampulla 0.786 + 0.029 0.795 + 0.034 n=28 n=28

Location' Lumen 0.750 + 0.030 0.759 + 0.024 n=31 n=44

Fold 0.680 + 0.036 0.796 + 0.014 n=30 n=69



1. Significant interaction (p < 0.05) with Attachment Status.

2. Differences between treatments (ie., free vs. stuck, and isthmus vs.
ampulla) are significant (p< 0.05). Overall treatment means and SEM
are

Free: 0.716 + 0.024 Stuck: 0.782 + 0.013 Isthmus: 0.743 + 0.014 Ampulla: 0.790 + 0.022 Lumen: 0.756 + 0.019 Fold: 0.761 + 0.015

(Used with permission from DeMott & Suarez, 1992).








72

three-way interactions. Ovulation status and time in tract were not involved in interactions, nor were they significant as individual factors.

While free sperm were significantly more sharply bent than stuck sperm, an interaction between attachment status and tubal region was detected, since the difference between free and stuck sperm was much greater in the isthmus than the ampulla. Also, while isthmic sperm were significantly more sharply bent than ampullar sperm, the difference between the isthmus and ampulla was greater for free sperm than stuck sperm. An interaction of attachment status with location relative to the lumen was also detected, because free sperm in the folds were more sharply bent than free sperm in the lumen; however, stuck sperm in the folds were LESS sharply bent than stuck sperm in the lumen. Also, free sperm were more sharply bent in both the folds and lumen than stuck sperm, but the difference between free and stuck sperm was much greater in the folds than in the lumen.


Discussion


The general pattern of mouse sperm progress up the uterine tube appears to involve periods of free swimming between periods of attachment to the tubal epithelium. This pattern was observed in both the isthmus and the ampulla but our analysis of the proportion of free swimming sperm showed that sperm were more likely to be free in the ampulla. While earlier in situ studies in the mouse (Suarez, 1987) reported the apparently decreased stickiness of ampullar sperm, this is the first








73

report of a progressive pattern involving sequential binding and release from the ampullar wall.

Analysis of the proportion of free-swimming sperm at different times relative to ovulation supports the idea that sperm are better able to stay free from the epithelium as the time of fertilization approaches. This is one mechanism for shifting sperm from the isthmic reservoir to the site of fertilization. Previous research indicates that while sperm numbers in the ampulla are low, near the time of fertilization the number of sperm reaching this region increases (Katz & Yanagimachi, 1980; Cummins & Yanagimachi, 1982; Smith et al., 1987; Suarez, 1987). These sperm appear to be moving up from the isthmic reservoir (Suarez, 1987). The increased ability to remain free near the time of fertilization may reflect changes in the sperm such as capacitation or hyperactivation, or changes in the affinity of the tubal epithelium for sperm. There is evidence that different levels of estradiol may affect the sticking of boar sperm to pig tubal explants (Suarez et al., 1991b; Raychoudhury & Suarez, 1991).

FCR has proven to be a consistent indicator of hyperactivation in vitro (Suarez et al., 1983; Suarez & Osman, 1987; Suarez et al., 1992) and has the particular advantage of being relatively insensitive to small errors in determining the inflection point. For measurements within the mouse uterine tube, decreases in FCR have been a reliable indicator of hyperactivation (Suarez & Osman, 1987) and there are distinct advantages over other indicators. Since the sperm are confined in a narrow, convoluted lumen, especially in the isthmus, it is impossible to measure their








74
undisturbed swimming trajectory or their velocity over a long enough distance to correlate it with in vitro measurements. In our laboratory, beat amplitude and wave length are more sensitive to measurement errors than FCR and this becomes significant when taking measurements from images of sperm within the uterine tube.

Based on the results of the FCR measurements, it appears that sperm which have broken free are more likely to be hyperactivated than stuck sperm. Also, based on these observations and previous descriptions of the hyperactivated pattern for mouse sperm (Fraser, 1977; Olds-Clarke, 1986; Suarez & Osman, 1987), the pool of erratically beating, dynamic sperm that intermittently released and reattached to the tubal wall appeared to be hyperactivated. All free sperm were a subset of this pool. The erratic beating pattern generated a sharply curved, hyperactivated beat while sperm were attached to the tubal wall and all sperm releases were preceded by erratic beating. This supports the possibility of hyperactivation functioning to give sperm an advantage in remaining free from the epithelium and progressing up to the site of fertilization.

Hyperactivation may provide the direction changes needed to navigate the convoluted path up the uterine tube and provide the forces or torques that allow sperm to bounce off rather than stick when they encounter the wall. Since not all of the erratically beating sperm were free, hyperactivation is not always sufficient for sperm release. Capacitation may also be required. Capacitation may decrease the stickiness of the sperm head, making it easier to release from the epithelium when they are stuck. In the hamster uterine tube, sperm have also been noted breaking








75

free and reattaching to the epithelium (Smith et al., 1987). A recent study indicates that hamster sperm incubated in capacitating conditions before being introduced into the uterine tube lose their stickiness and may encounter the epithelium and not attach (Smith & Yanagimachi, 1991).

Based on the two-factor ANOVA results, it appears that differences in FCR should not be interpreted simply in terms of the effects of each factor we examined. Although the factors tubal region, sperm attachment status, and sperm location relative to the lumen all significantly affected FCR, their major importance lies in how they vary in combination, rather than in how each singly affects FCR. The statistical interactions imply that there are physiological interactions whereby the tract features act on the sperm's potential to affect the movement pattern and progress. The large difference between free and stuck sperm in the isthmus compared to the ampulla may reflect a reduction in the proportion of the relatively high FCR, regularly beating sperm that were not seen to move up the uterine tube. In the isthmus there may be more distinct populations, one containing the sperm that have begun erratic, low FCR beating, and are able to break free and move along the uterine tube, and another one containing the stuck, regularly beating, high FCR sperm that do not move up to the ampulla. The population in the ampulla may be more homogeneous, containing only the sperm able to use erratic beating and move up from the isthmus. Separating the sperm in this manner need not imply sperm selection based on the ability to become hyperactivated. Interactions between the relative timing of hyperactivation and capacitation could produce only a small








76

population ready and able to move up the uterine tube at any time. Other sperm may become hyperactivated, but if this is not properly coupled to capacitation, and/or other unidentified factors, it may not help them reach the site of fertilization at the appropriate time.

The increase in FCR for both free and stuck sperm in the ampulla is harder to understand. If hyperactivation is indeed a factor helping sperm release from repeated bindings to the epithelium, we might assume that the most hyperactivated sperm should reach the ampulla. There may be some difference in the fluid surrounding the sperm that dampens their tail beat in the ampulla. The secretions of the ampullar epithelium may be more viscous or viscoelastic than those of the isthmus, and this may be further enhanced after ovulation by cumulus secretions and follicular fluid. Since there is a bursa surrounding the mouse ovary (Hunter, 1988), the products of ovulation cannot escape into the peritoneal cavity and must instead enter the ampulla. There is experimental evidence indicating that increases in the viscosity of the medium can dampen sperm tail beat amplitude and frequency (Drobnis et al., 1988b; Rikmenspoel, 1984; Suarez et al., 1991a; Suarez & Dai, 1992). Also, hyperactivated hamster sperm appear to have an advantage over nonhyperactivated in penetrating fluids of elevated viscosity and their tail beats do flatten out (develop higher FCR) in more viscous fluid (Suarez et al., 1991a). Measurements of the hydrodynamic properties of the fluids in which sperm swim in vivo would be valuable for our understanding of the modulation of sperm motility and its possible functions. Besides the regional differences in fluid composition that may affect








77
sperm, there may be important effects of the local environment (lumen vs. fold). Being out of the main fluid flow through the lumen, free sperm in the folds may be able to generate more sharply curved tail beats observed than sperm in the lumen. The drag on the sperm would be reduced in the relatively quiet eddy of the folds.


Conclusions


In summary, most mouse sperm are stuck to the tubal wall most of the time. As the time for fertilization approaches, however, there is an increase in the proportion of free-swimming sperm. Progress up the uterine tube to the site of fertilization occurs intermittently as sperm repeatedly release from the wall, swim a short distance and reattach. This may be the means of release from the isthmic reservoir. Those that do break free appear to be using hyperactivated tail beats to give them an advantage for releasing from the epithelium. The coincidental occurrence of hyperactivation and release from the tubal wall resulting in progress up the uterine tube suggests an association between the phenomena. Nevertheless, the two-factor interactions affecting FCR serve to remind us that the onset of hyperactivation and any advantages that it provides are likely to be regulated by a number of physiological factors.













CHAPTER 4
INVESTIGATION OF CHANGING SPERM ANTIGENICITY Introduction


There is a wide variety of evidence showing that there are modifications of the mammalian sperm cell surface between the time sperm leave epididymal storage and the time they reach the site of fertilization (Yanagimachi, 1988). Antigenic rearrangements (O'Rand, 1977; Cowan et al., 1986; Okabe et al., 1986; Jones et al., 1990), glycosylation pattern changes (Kinsey & Koehler, 1978; Ahuja, 1984; Cross & Overstreet, 1987), and alterations of the lipid structure of the sperm membranes (Scott et al., 1967; Davis, 1981; Go & Wolf, 1985; Ehrenwald et al., 1988) have all been described. Some of these changes have been associated with capacitation and the acrosome reaction (see Chapter 1), but there has been no specific investigation of a potential role in controlling sperm transport through the uterine tube. Changes appearing during the course of capacitation may not relate directly to preparing the cell for the acrosome reaction but may instead play a functional role in regulating sperm transport.

There are two observable sperm phenomena that relate to transport through the uterine tube, release from the isthmic reservoir and the onset of hyperactivated motility. Sperm release from the epithelium appears to be dependent on changes in


78







79
the sperm rather than the uterine tube (Smith & Yanagimachi, 1990; Smith & Yanagimachi, 1991) and may involve the modification of a sperm glycoprotein (see Chapter 2). Hyperactivated motility is involved in ascent from the isthmic reservoir to the site of fertilization (Suarez, 1987, see Chapter 3) and thus physiological changes affecting hyperactivation necessarily affect sperm transport as well.

It was hypothesized that some of the sperm changes associated with adherence in the tubal reservoir and the onset of hyperactivated motility would be reflected as antigenic changes on the surface of the sperm that could be detected based on differential labelling with monoclonal antibodies directed against sperm components. It was assumed that the changes occurring on sperm capacitated in vitro would be similar, allowing sperm that had reached various stages of the capacitation/hyperactivation sequence to be used to analyze antibody binding. The antibodies identified would then serve as both markers for changes in epitope associated with observable sperm changes and a means to characterize the antigen. Monoclonal antibodies have previously been used to describe antigens that change during capacitation and the acrosome reaction (O'Rand, 1977; Cowan et al., 1986; Okabe et al., 1986; Saxena et al., 1986; Topfer-Petersen et al., 1990; Berger, 1990; Jones et al., 1990).

Under capacitating conditions in vitro, hamster sperm pass through four stages before undergoing the acrosome reaction. Sequentially, sperm begin in the fresh or activated stage, then agglutinate head-to-head, then separate and hyperactivate coincidentally, and finally complete capacitation (Suarez, 1988). Monoclonal








80

antibodies were generated against the complement of epididymal hamster sperm antigens and screened for changes in activity associated with agglutination, hyperactivation, capacitation and the acrosome reaction. Identifying antigenicity changes associated with the agglutinated state and with hyperactivation were of primary interest as these molecules may be associated with the ability of sperm to bind and release the tubal epithelium. Three antibodies were identified that recognized epitopes changing during agglutination or hyperactivation.

Antibody labelling patterns were identified by indirect immunofluorescent staining of fixed sperm and were subsequently analyzed by immunogold labelling of unfixed sperm to better localize the antigenicity and by immunoblotting to provide a preliminary characterization of the antigens.


Materials and Methods


Production of Monoclonal Antibodies


Immunization. All chemicals were obtained from Sigma Chemical Co. (St. Louis, MO) unless otherwise noted. Caudae epididymides from mature golden syrian hamsters were punctured and the sperm in epididymal fluid were diluted in an equal volume of RIBI's adjuvant (RIBI Immunochemical Research, Inc., Hamilton, MT) at 37"C. This mixture was used immediately for subcutaneous injections into BALB/c mice. The mice received three injections of 7-9 X 107 sperm over two months and were tested for titer following each injection. They received a final boost of 1.2 X 10 sperm six days before fusion.







81
Fusion. The fusion was carried out in a standard manner using polyethylene glycol and selection on HAT medium (Simrell & Klein, 1979; Kao & Klein, 1986). Splenocytes were fused with SP2/0 cells. Fused cells were plated at a concentration to yield 3-10 different populations in each culture well. Supernatants from the resulting hybridoma cultures were screened for anti-sperm activity using an indirect immunofluorescence assay.

Screening. To identify antibodies against epitopes that were modified as sperm went through their functional changes, we compared indirect immunofluorescent staining patterns of fresh, agglutinated, hyperactivated, and capacitated sperm. Supernatants that showed differential patterns of labelling between sperm stages were selected for cloning.

In order to maximize the time between hyperactivation and the completion of capacitation, the concentrations of motility stimulators, BSA and metabolic substrates in the medium were optimized to provide rapid hyperactivation and slow capacitation based on previous reports of the effects of modifications of hamster capacitation medium (Dravland & Meizel, 1981; Bavister, 1989). The composition of modified hamster capacitation medium is shown in Table 4-1. Using this medium, populations of hyperactivated sperm could be obtained 60-90 minutes before capacitation was complete (assay described below).

Caudal epididymal sperm were allowed to disperse into 1 ml of modified hamster capacitation medium for 5-10 minutes at 370C, then the top 2/3 of the sperm suspension was collected. Sperm numbers were adjusted to 3 X 106 sperm/ml and








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Table 4-1. Modified hamster capacitation medium formulation


Component Cone. Component Conc. NaCI 102 mM Glucose 3.24 mM KCI 5 mM Pyruvate 3.00 mM CaCl2 2.4 mM Hypotaurine 100 tM MgCl2 0.5 mM Penacillamine 20 iM NaH2PO4 0.35 mM Epinephrine 1 iLM NaHCO3 25 mM Penicillin 0.06 g/l HEPES 25 mM Fraction V BSA 3 mg/ml Note: Formulated from Dravland and Meizel, 1981; Suarez, 1988; and
Bavister, 1989.




Table 4-2. Criteria for staging sperm



Stage Criteria

Fresh Caudal epididymal sperm after 5-10 min.
dispersal in medium.

Agglutinated 70% of sperm are agglutinated head to head. Typically after 1 h.

Hyperactivated 70% of sperm exhibit vigorous, circular hyperactivated pattern. Typically after 33.5 h.

Capacitated Acrosome reactions induced in >50% of motile sperm treated with 0.3 mg/ml lysophosphatidylcholine for 5-10 min. Typically 4.25-5 h.








83

samples were used immediately as fresh sperm. The remaining suspensions were incubated at 37*C, checked periodically, and used for the agglutinated, hyperactivated and capacitated stages when they met the criteria in Table 4-2. Capacitation was assayed by the ability of lysophosphatidylcholine (LPC) to induce acrosome reactions in motile sperm (Llanos & Meizel, 1983). Samples were tested periodically beginning at the onset of hyperactivation. When sperm were collected for the hyperactivated stage, the percent responding to LPC was 10% or less.

Sperm were dried down on multi-well microscope slides (Cel-line Assoc. Newfield, NJ), rinsed with phosphate-buffered saline (PBS), fixed for 5-10 min with 7% formaldehyde and rinsed again with PBS. All treatments were carried out at room temperature. The sperm were blocked with 3% bovine serum albumin (Calbiochem, La Jolla, CA) in PBS (PBS/BSA) for 30 minutes, then individual wells for each sperm stage were incubated with PBS/BSA alone, neat hybridoma culture supernatant, hybridoma growth media, 1:1000 normal mouse serum (non-immune) in PBS/BSA, and immune mouse serum, diluted similarly for 1.5-1.75 hours. The wells were rinsed with PBS/BSA, incubated with TRITC-labelled secondary antibody (F(ab')2 of rabbit anti-mouse IgG-F(ab')2) (Jackson Immunoresearch Labs, Inc., West Grove, PA) for 20-25 minutes, and rinsed again. Slides were examined using combined Nomarski/epifluorescence optics, 100W mercury lamp, and a rhodamine (TRITC) filter set (Carl Zeiss, Inc., Thornbrook, NY). The staining pattern for each supernatant with each type of sperm was recorded.








84
Cloning. Cultures producing supernatants that yielded interesting staining patterns were cloned (Simrell & Klein, 1979; Kao & Klein, 1986). The resulting cultures were screened, subcloned, and rescreened at least three times to obtain active monoclonal cultures. The monoclonal cell lines were expanded, isotyped and stored.

Production of Ascites. Cultures from three cell lines producing antibodies that recognized epitopes changing prior to hyperactivation, HL 772, HL 778, and HL 787, were used to induce ascites fluid production in BALB/c mice. RIBI's adjuvant was used. Ascites fluid was cleared by centrifugation at 960 G for 10 minutes, then stored frozen in aliquots at -200C.


Immunogold Labelling of Sperm


Sperm from the four stages were obtained as described above. Medium was removed following centrifugation, 5 minutes, 960 G, and the sperm were washed in PBS/BSA, 5 minutes, 960 G. Labelling was carried out based on the protocol of Jones and coworkers (1990). The sperm were then suspended in either 1:1 culture supernatant or 1:10 ascites diluted with PBS/BSA and incubated for 35 minutes at room temperature. The supernatant was removed as above and the sperm were washed by centrifugation twice with PBS/0.1% BSA. They were then incubated for 30 minutes with 5 nm colloidal gold-labelled secondary antibody, anti-mouse IgG, IgM (E-Y Labs, San Mateo CA) diluted 1:30 with PBS/0.1% BSA. Supernatant was removed and the sperm were washed with PBS by centrifugation 3X.








85

For analysis by light microscopy, the labelled, washed sperm suspensions were dried down on multi-well slides, rinsed 3X with PBS and fixed for 5 minutes with 2.5% phosphate buffered glutaraldehyde. They were rinsed with PBS and distilled water, 3X each, then the label was enhanced with silver as described in Chapter 1. Slides were analyzed following a 9-11 minute enhancement.


Immunoblotting of Sperm Proteins


1-D blots. Fresh and hyperactivated sperm extracts were prepared as described in Chapter 2. Electrophoresis and semi-dry transfer to Immobilon-P membranes were carried out under the conditions previously described as well (see Chapter 1). Blots were blocked for 2 hours with PBS/BSA then washed 4X with PBS. Strips were incubated overnight at 40C with 1:50 ascites, 1:1000 normal mouse serum, 1:1000 immune mouse serum, and PBS/BSA alone. All dilutions were made with PBS/BSA. Following 4 washes with PBS, the blot strips were incubated for 2 hours at room temperature with 1:1000 horseradish peroxidase-labelled F(ab')2 of rabbit anti-mouse IgG-F(ab')2) (Jackson Immunoresearch Labs, Inc., West Grove, PA). This product contains the same antibodies previously used for immunofluorescence conjugated to a different label. After 4 washes with PBS, the peroxidase reaction was developed as described in Chapter 2.

2-D blots. Fresh sperm extract prepared as described above containing 650 A.g of total protein was diluted to 5 ml and dialyzed against three 2-liter changes of distilled water over 24 hours. Dialysis tubing (Spectra/Por, mW cutoff 3500,








86

Spectrum Medical Industries Inc., Los Angeles, CA) had been boiled in the presence of EDTA to reduce protein binding (Harlow & Lane, 1988). The sample was then lyophilized. Two-dimensional polyacrylamide gel electrophoresis was carried out according to Roberts and coworkers (1984). For isoelectric focussing (IEF), the pH gradient ranged from 4-10. The IEF gels were then loaded on 10% polyacrylamide slab gels and separated as previously described (see Chapter 2). Semi-dry transfer to Immobilon-P membranes and blocking was carried out as described above. Blots were then incubated overnight at 40C with either 1:2000 normal mouse serum or 1:100 HL 787 ascites diluted with PBS/BSA. The blots were washed, incubated with 1:1000 peroxidase-labelled secondary antibody for 1 hour, and developed as described above.


Results


From one fusion yielding 316 cultures, we identified 6 antibodies that showed stage specific differences in immunofluorescent labelling of hamster sperm. Four of these were cloned; three that showed labelling changes associated with agglutination or hyperactivation that could be relevant to sperm transport in the uterine tube, and one that was an exceptionally strong acrosome-specific antibody that is useful for determining acrosome reaction status.

Staining patterns show that the epitope associated with antibody HL 787 changes at agglutination while those associated with HL 772 and HL 778 change at hyperactivation (Figures 4-1, 4-2). Between the fresh stage and agglutination, the








87

labelling with HL 787, an IgM class antibody, decreased over the head and midpiece. Labelling at hyperactivation and capacitation remained low. Labelling with HL 772, an IgG1, decreased over the head and midpiece between the agglutinated and hyperactivated stages. Labelling of fresh sperm was similar to agglutinated, and capacitated continued to show the decrease seen at hyperactivation. HL 778, another IgM, showed the opposite reaction at hyperactivation, increasing in intensity and redistributing from a spotty pattern with isolated areas at the rostral tip of the head and the neck region to an evenly distributed pattern over the entire head. Again, the fresh stage resembled agglutination and the capacitated stage resembled hyperactivation. HL 784, an IgG, relevant to the acrosome reaction rather than sperm transport, stained the heads of the acrosome-reacted, capacitated sperm brightly and evenly. It did not stain the heads of non-capacitated sperm that had lost their acrosomes, presumably precociously or during processing, however. It also brightly stained the acrosome itself whether intact or in fragments (data not shown). This antibody would be useful for studies of the acrosome reaction but was not further characterized here.

Controls labelled with normal serum, hybridoma culture medium and secondary antibody alone characteristically showed very low signal. The detectable fluorescence was predominately on the midpiece. The positive control, serum obtained at the time of fusion, however, strongly labelled the entire sperm cell. The silver-enhanced colloidal gold labelling assessed by light microscopy indicated a positive reaction for HL 772, 778, and 787 on the surface of the sperm. Patterns,







A B 787









772









778








Figure 4-1. Stage-related Changes Observed in Indirect Immunofluorescent
Labelling with Monoclonal Antibodies. Column A shows labelling of
fresh sperm, column B shows Agglutinated labelling for HL 787,
Hyperactivated labelling for HL 772, 778.







89
HL 787 Fresh Agglutinated












II1, 772 Frcsh I I peracti ated












111, 778 Fresh II lhpc'ractivated











Figure 4-2. Epifluorescent Images of Monoclonal Antibody Binding Patterns.
Fixed, dried down sperm treated with culture supernatant.
Photographed at 100X with Zeiss Plan-neofluar objective on Ektar 1000 film. Exposures 75-90 sec. Images for a given antibody exposed
the same duration and all negatives printed similarly.








90
however, were very patchy and variable. The stage specific characteristics identified by immunofluorescence could be seen, but not consistently.

Immunostaining of blots allowed the identification of potential antigens for two of the antibodies (Figure 4-3). HL 787 clearly identified a specific antigen that migrated at 22-23 kDa under reducing conditions. This band was seen in blots stained with immune serum but not with normal serum or secondary antibody alone. It also showed decreased labelling on blots of hyperactivated sperm extracts compared to fresh. A strong band at approximately 42-44 kDa was also detected with the other monoclonals and controls, though it was clearly enhanced by binding to the monoclonals. This band did not label with immune serum. A weak band at 46 kDa was also unique to the fresh blots stained with HL 787. Staining with HL 772 seemed to identify 2 separate bands at approximately 44 and 42 kDa, and the lower molecular weight band appeared to be distinct from the band labelling in the other treatments. It also showed decreased labelling of hyperactivated sperm extracts. Labelling with HL778 did not suggest any strong candidates for the antigen associated with this antibody. Results were similar on blots from 2 separate sets of extracts representing eight hamsters.

Analysis of HL 787 labelling by 2-D PAGE again detected a protein of approximately 22 kDa (Figure 4-4). The protein is acidic with an approximate pI range of 5.1-5.2. This spot was again absent in the control blot stained with normal mouse serum.




Full Text
77
sperm, there may be important effects of the local environment (lumen vs. fold).
Being out of the main fluid flow through the lumen, free sperm in the folds may be
able to generate more sharply curved tail beats observed than sperm in the lumen.
The drag on the sperm would be reduced in the relatively quiet eddy of the folds.
Conclusions
In summary, most mouse sperm are stuck to the tubal wall most of the time.
As the time for fertilization approaches, however, there is an increase in the
proportion of free-swimming sperm. Progress up the uterine tube to the site of
fertilization occurs intermittently as sperm repeatedly release from the wall, swim a
short distance and reattach. This may be the means of release from the isthmic
reservoir. Those that do break free appear to be using hyperactivated tail beats to
give them an advantage for releasing from the epithelium. The coincidental
occurrence of hyperactivation and release from the tubal wall resulting in progress
up the uterine tube suggests an association between the phenomena. Nevertheless,
the two-factor interactions affecting FCR serve to remind us that the onset of
hyperactivation and any advantages that it provides are likely to be regulated by a
number of physiological factors.


136
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105
Figure 5-1. Differential Preservation of Luminal Contents by Processing Method.
(A) Photomicrograph of oviductal cryosection coated with celloidin and
post-fixed with CPC, stained with PAS, hematoxylin. (B) Untreated
oviductal cryosection, stained with alcian blue/PAS, hematoxylin. (C)
Oviductal paraffin section fixed with CPC and stained with H&E. m =
mucus, magnification 40X.


32
Protease inhibitors were also prepared as a 100X concentrate. The
concentrate contained 10 mg/ml aprotinin, 1 mg/ml Na-t-boc-deacetylleupeptin, and
500 mM benzamidine HC1 dissolved in incomplete medium.
Sperm extraction buffer (modified from Saling & Lakoski, 1985) contained 2%
sodium dodecyl sulfate (SDS), 125 mM Tris buffer, pH 6.8, 20% glycerol, 10 /ig/ml
Na-t-boc-deacetylleupeptin, and 2 mM phenylmethylsulfonyl fluoride (PMSF).
All chemicals were from Sigma Chemical Co. (St. Louis, MO) except those
specifically noted here. Limax flavus lectin (LFA), BSA and HEPES were from
Calbiochem Corp. (La Jolla, CA). Fetuin conjugated to 5 nm colloidal gold particles
and chick pea lectin (CPA) were obtained from E-Y Labs, Inc. (San Mateo, CA).
Tris was from Biorad Laboratories (Richmond, CA). Glycine was from ICN
Biomedicals, Inc. (Cleveland, OH). SDS was from BDH Chemicals, Ltd. (Poole,
UK).
Binding Inhibition in Excised Uterine Tubes
Golden Syrian hamsters (Mesocricetus auratus) from Charles Rivers
Laboratories (Wilmington, MA) were maintained on a 14L:10D light cycle with lights
on from 0700-2100 h and provided food and water ad libitum. Retired breeder males
were used to obtain caudal epididymal sperm. Females (9-18 weeks old) were
visually staged with Day 1, estrus, being determined by the presence of clear vaginal
discharge (Hafez, 1970). Animals, males first, were killed by C02 inhalation followed


My labmates, Xiao-bing Dai, John Donald, Samir Raychoudhury, Carmen
Stauss and Steve Varosi were always willing to lend a hand, or at least an opinion,
and I appreciate their help. Thanks also go to Lori Dixon for her help with antibody
screening and Rejean LeFebvre for his involvement in the sperm binding
experiments. The ICBR core facilities are an invaluable asset at the University of
Florida, and the monoclonal core and electron microscopy core staff provided expert
support.
Finally thanks go to Kember DeMott; as a scientist and wife, untoppable.
Having a soulmate with a detailed understanding of the work you are immersed in,
able to share ideas and contribute to your career, is a blessing.
in


133
Smith TT & Yanagimachi R (1988) Capacitation status of hamster spermatozoa at
various times after mating. Biology of Reproduction, 38,Suppl 1, 33-a.
Smith TT & Yanagimachi R (1990) The viability of hamster spermatozoa stored in
the isthmus of the oviduct: the importance of sperm-epithelium contact for
sperm survival. Biology of Reproduction, 42, 450-457.
Smith TT & Yanagimachi R (1991) Attachment and release of spermatozoa from the
caudal isthmus of the hamster oviduct. Journal of Reproduction and Fertility,
91, 567-573.
Sokal RR & Rohlf FJ (1989) Biometry. San Francisco: W.H.Freeman.
Spiro RG & Bhoyroo VD (1974) Structure of the O-glycosidically linked
carbohydrate units of fetuin. Journal of Biological Chemistry, 249, 5704-5717.
Srivastava PN, Kumar VM & Arbtan KD (1988) Neuraminidase induces capacitation
and acrosome reaction in mammalian spermatozoa. Journal of Experimental
Zoology, 245, 106-110.
Stojanoff A, Bourne H, Andrews AG & Hyne RV (1988) Phospholipid composition
of isolated guinea pig sperm outer acrosomal membrane and plasma
membrane during capacitation in vitro. Gamete Research, 21, 297-311.
Suarez SS (1987) Sperm transport and motility in the mouse oviduct: observations in
situ. Biology of Reproduction, 36, 203-210.
Suarez SS (1988) Hamster sperm motility transformation during development of
hyperactivation in vitro and epididymal maturation. Gamete Research, 19,
51-65.
Suarez SS & Dai XB (1992) Hyperactivation enhances mouse sperm capacity for
penetrating viscoelastic media. Biology of Reproduction, 46, 686-691.
Suarez SS, Dai XB, DeMott RP, Redfern K & Mirando MA (1992) Movement
characteristics of boar sperm obtained from the oviduct or hyperactivated in
vitro. Journal of Andrology, 13, 75-80.
Suarez SS & DeMott RP (1991) Functions of hyperactivated motility of sperm in the
oviduct. Archivos de Biologa y Medicina Experimentales, 24, 331-337.
Suarez SS, Katz DF & Overstreet JW (1983) Movement characteristics and
acrosomal status of rabbit spermatozoa recovered at the site and time of
fertilization. Biology of Reproduction, 29, 1277-1287.


49
labelled fetuin incubated with the blots overnight bound to fresh sperm components
at a number of molecular weights. The intensity of labelling was generally lower for
hyperactivated sperm extracts though the gels had been loaded with the same amount
of total protein. Distinct bands at 27.5 kDa and >335 kDa in the fresh sperm
extracts were absent in the hyperactivated extracts. A strong band at 32-33 kDa in
the fresh sperm extracts was very dim at hyperactivation. Also, a strong band in the
fresh sperm extracts at 49-50 kDa seemed to have shifted to an apparent molecular
weight of 53 kDa in the hyperactivated extracts.
Lectin binding on the blots incubated overnight with LFA indicated that the
fetuin binding proteins seem to be sialylated (Figure 2-6). The same group of
proteins was recognized by the LFA, though some of the bands that labelled weakly
with fetuin were not detectable in the LFA blots. LFA staining was also generally
decreased in the hyperactivated extracts and the differences observed agreed with the
results in the fetuin-labelled blots.
Since LFA was detected using an indirect technique including a 1 hour, room
temperature incubation with colloidal gold-labelled fetuin (Roth et al., 1985; Roth et
al., 1984), control blots that had been incubated overnight with diluent (3% BSA in
PBS), then treated for 1 hour at room temperature with labelled fetuin were
included. These strips were developed with silver enhancement reagent for the same
amount of time as the LFA strips. The interference due to colloidal gold-labelled
fetuin was minimal under these conditions (Figure 2-6). Also, LFA labelling was
inhibited when sialic acid was included during the incubation.


117
Battalia DE & Yanagimachi R (1980) The change in oestrogen and progesterone
levels triggers adovarian propulsive movement of the hamster oviduct. Journal
of Reproduction and Fertility, 59, 243-247.
Bavister BD (1989) A consistently successful procedure for in vitro fertilization of
golden hamster eggs. Gamete Research, 23, 139-158.
Bedford JM (1969) Limitations of the uterus in the development of the fertilizing
ability (capacitation) of spermatozoa. Journal of Reproduction & Fertility, suppl.
8, 19-26.
Bedford JM & Kim HH (1993) Cumulus oophorus as a sperm sequestering device,
in vivo. Journal of Experimental Zoology, 256, 321-328.
Bedford JM & Yanagimachi R (1992) Initiation of sperm motility after mating in the
rat and hamster. Journal of Andrology, 13, 444-449.
Berger T (1990) Changes in exposed membrane proteins during in vitro capacitation
of boar sperm. Molecular Reproduction and Development, 27, 249-253.
Bernal A, Torres J, Reyes A & Rosado A (1980) Presence and regional distribution
of sialyl transferase in the epididymis of the rat. Biology of Reproduction, 23,
290-293.
Bielfeld P, Graf M, Jeyendran RS & Zaneveld LJD (1992) No change in acrosome
reaction of human spermatozoa during storage in cervical mucus. Andrologia,
24, 83-86.
Blackmore PF (1993) Thapsigargin elevates and potentiates the ability of
progesterone to increase intracellular free calcium in human sperm: possible
role of perinuclear calcium. Cell Calcium, 14, 53-60.
Boatman DE & Robbins RS (1991) Bicarbonate: carbon-dioxide regulation of sperm
capacitation, hyperactivated motility, and acrosome reactions. Biology of
Reproduction, 44, 806-813.
Bobr LW, Ogasawara FX & Lorenz FW (1964a) Distribution of spermatozoa in the
oviduct and fertility in domestic birds II. Transport of spermatozoa in the
fowl oviduct. Journal of Reproduction and Fertility, 8, 48-58.
Bobr LW, Lorenz FW & Ogasawara FX (1964b) Distribution of spermatozoa in the
oviduct and fertility in domestic birds I. Residence sites of spermatozoa in
fowl oviducts. Journal of Reproduction and Fertility, 8, 39-47.


59
Investigations of sperm motility and progress through the uterine tube have
relied on serial sectioning (Yanagimachi & Chang, 1963; Olds, 1970; Smith et al.,
1987), flushes with oil and physiological medium (Cooper et al., 1979; Overstreet et
al., 1980; Suarez et al., 1983; Smith & Yanagimachi, 1990), blocking passage through
the uterine tube at various times (Hunter, 1984), and in situ observations in animals
with small, translucent uterine tubes (Katz & Yanagimachi, 1980; Suarez, 1987; Smith
& Yanagimachi, 1991). Serial sectioning allows accurate localization of sperm and,
coupled with timed sampling, can provide information about distribution changes.
A disadvantage is that the relative importance of sperm motility and the factors that
affect it cannot be determined. Tubal flushes provide more information about the
condition of the sperm since their motility, or lack of it, and capacitation status can
be characterized. However, location in the uterine tube cannot be accurately
described and motility must be analyzed and interpreted out of physiological context.
Studies on the effects of tubal ligation at various positions and times relative to
ovulation or fertilization offer an advantage in that the actual fertilizing population
of sperm can be localized. Again, however, the motility and capacitation status of
individual sperm cannot be assessed. Observing sperm within the uterine tube has
the advantage of allowing investigators to localize sperm at different times and also
to observe the motility patterns actually used in the uterine tube.
In this study, a previously described in situ preparation (Suarez, 1987) for
observing mouse sperm within the uterine tube has been modified to allow longer
contiguous segments of uterine tube and, thus, longer segments of sperm transport


110
The production of the mucous portion of the isthmic secretions may remain fairly
constant during the cycle. Cyclical variation in the ampullar cells have been noted
(Nilsson & Reinius, 1969; Abe & Oikawa, 1993), but the connection between such
changes and mucus staining is not well established. The cryosectioning method
described here is excellent for demonstrating the presence of luminal mucous
secretions, however, the nature of this material is probably better studied using other
methods.
Finding more sperm in the uterotubal junction and isthmus of late mated mice
compared to those mated early in estrus was in agreement with results previously
reported (Smith et al., 1987). It appears that the tubal reservoir may be populated
more rapidly when ovulation is imminent. Also, sampling only 1 hour after mating,
sperm were not expected to have reached the ampulla yet. For the live sperm that
will potentially go on to fertilize, passage to the ampulla appears to require somewhat
more than 1 hour (Suarez, 1987; Smith et al., 1987).
Several of the technical details noted while experimenting with the
preservation technique deserve mention. Rapid freezing was important for the
morphological preservation of both mucus and tissue. Placing the uterine tube
directly in isopentane gave noticeably better results than either freezing it in a tube
of O.C.T. or using liquid nitrogen alone. Both of these alternatives led to more
freeze artifact due, presumably, to slower freezing of the tissue. Also, in sections that
had not been treated with celloidin and CPC, the mucus strands did not remain
consistently apposed to the epithelium after the slower freezing protocols. The


ACKNOWLEDGMENTS
First thanks go to Dr. Susan Suarez. Her prods were inevitably in the right
direction, guided by insight that has served as a good example of why it is important
to know intimately the details of your research material. Her financial and
professional support of my work has been most unselfish. Next thanks go to the
other members of my supervisory committee, Drs. Maarten Drost, Louis Guillette,
Paul Klein, William Buhi, and also Fuller Bazer for advising during the first half of
my program. Their diverse approaches and interests helped lead to intriguing
questions and provided a wide selection of ways to answer them. They did a good
job teaching me to keep my trees and forests in proper perspective.
Next, thanks go to all the faculty members who provided generously of their
knowledge, time, and equipment including Drs. Kevin Anderson, John Harvey, Roger
Reep, and Chris West. The varied nature of my experiments had me knocking on
many doors and their cooperation was wonderful, as was the cooperation from all the
scientists who spent time training me in new methods including Idania Alvarez, Katy
Gropp, Melanie Pate, Michael Sapper and Heidi Wearne. Several college
administrators have done a good job providing support and helping me through the
Graduate School procedures including Drs. Darryl Buss, Phillip Kosch and Tom
Wronski.
ii


128
Mrsny RJ & Meizel S (1980) Evidence suggesting a role for cyclic nucleotides in
acrosome reactions of hamster sperm in vitro. Journal of Experimental Zoology,
211, 153-157.
Mullins KJ & Saacke RG (1989) Study of the functional anatomy of bovine cervical
mucosa with special reference to mucus secretion and sperm transport.
Anatomical Record, 225, 106-117.
Murray MK (1992) Biosynthesis and immunocytochemical localization of an
estrogen-dependent glycoprotein and associated morphological alterations in
the sheep ampulla oviduct. Biology of Reproduction, 47, 889-902.
Neill JM & Olds-Clarke P (1987) A computer-assisted assay for mouse sperm
hyperactivation demonstrates that bicarbonate but not bovine serum albumin
is required. Gamete Research, 18, 121-140.
Nicolson GL, Usui N, Yanagimachi R, Yanagimachi H & Smith JR (1977)
Lectin-binding sites on the plasma membranes of rabbit spermatozoa. Journal
of Cell Biology, 74, 950-962.
Nicolson GL & Yanagimachi R (1972) Terminal saccharides on sperm plasma
membranes: identification by specific agglutinins. Science, 177, 276-279.
Nilsson O & Reinius S (1969) Light and electron microscopic structure of the oviduct
in The Mammalian Oviduct, edited by ESE Hafez & RJ Blandau. Chicago:
University of Chicago Press, pp. 57-82.
Noguchi S fe Nakano M (1992) Structure of the acidic N-linked carbohydrate chains
of the 55-kDa glycoprotein family (PZP3) from porcine zona pellucida.
European Journal of Biochemistry, 209, 883-894.
Norwood JT oviduct cilia membranes are required for ovum pickup in situ. Biology of
Reproduction, 23, 788-791.
Norwood JT, Hein CE, Halbert SA macromolecules inhibit cilia-mediated ovum transport in the rabbit oviduct.
Proceedings of the National Academy of Sciences, USA, 75, 4413-4416.
ORand MG (1977) Restriction of a sperm surface antigens mobility during
capacitation. Developmental Biology, 55, 260-270.


LIST OF TABLES
Table page
2-1 Candidate competitive inhibitors of carbohydrate interactions
in the uterine tube 34
2-2 Carbohydrate binding treatments for blotted
sperm components 41
3-1 Analysis of factors affecting proportion of sperm swimming
freely within the uterine tube 70
3-2 Attachment status interactions for FCR 71
4-1 Modified hamster capacitation medium formulation 82
4-2 Criteria for staging sperm 82
vi


10
number of tubal sperm until at least 2 hours after ovulation. And, at this time, sperm
were only slightly less susceptible to being flushed out. These results imply that the
uterine tube does not undergo marked changes in its affinity for binding sperm at
ovulation.
A significant change in sperm binding was, however, associated with the
condition of the sperm (Smith & Yanagimachi, 1991). When sperm capacitated in
vitro (see below "Changes in Sperm Cell Biology"), i.e., sperm capable of fertilization,
were injected into the uterine tube, they remained free from the mucosa whereas
non-capacitated sperm bound to the mucosa as soon as they encountered it. This
implies that part of capacitation involves a loss of affinity for the tubal reservoir and
it is this reduction in binding affinity that allows sperm to move along the uterine
tube. Interpretation of these results is confounded, however, because the capacitated
sperm were also using the vigorous hyperactivated motility pattern which could
contribute to their ability to pull away from the mucosa.
In summary, movement out of the tubal reservoir appears to involve release
from the mucosa due to changes in the binding affinity of sperm and/or changes in
flagellar beating, as well as indirect effects of tubal physiology. The primary
contribution to indirect effects is the muscular contractions of the uterine tube which
serve to move the luminal contents. Ciliary beating may play a small role as well.


47
proportions were distributed in the same pattern, but they were very low and the
differences were slight. There was also a significant inhibition effect of treatment in
the AIJ, but only the fetuin treatment was high enough to differ statistically from
control (data not shown).
The binding of fetuin to the sperm cells was demonstrated using colloidal gold-
labelled fetuin. The silver-enhanced label was found over the acrosomal region of
the head on fresh, acrosome intact sperm (Figure 2-5a). The staining pattern
covered the dorsal and anterior surfaces of the sperm head, apparently demarcating
the shape of the hamster acrosome. There also appeared to be a band of fetuin
labelling at the neck of the sperm, though this was variable. On the midpiece,
labelling was restricted to the distal portion and was spotty. Where acrosomes had
been disrupted during processing, the acrosomal fragments labelled heavily but the
regions that had been exposed underneath the acrosome did not label.
Hyperactivated sperm showed a different pattern of fetuin binding (Fig. 2-5b).
Acrosome intact sperm had a characteristic clear patch on the dorsal and anterior
acrosomal region. This region appeared to have lost its fetuin binding ability during
incubation. The midpiece also had decreased labelling as the spottiness became very
sparse. The posterior margin of the acrosome and the neck were still able to bind
fetuin, however. Disrupted acrosomes stained variably.
Specific fetuin binding components were detected in Western blots of sperm
extracts and differences were noted between fresh and hyperactivated sperm (Figure
2-6). Results were similar with blots from 2 separate sets of extracts. Colloidal gold


38
Sperm were pelleted in a microcentrifuge and washed 3X with incomplete
medium. They were fixed in suspension with 2.5% glutaraldehyde in phosphate
buffer, pH 7.3, for 5 minutes and then washed again. Samples were placed in the
wells of multi-well immunofluorescence slides (Cel-line Assoc., Newfield, NJ) and
allowed to dry down. They were rinsed 5X with PBS then 5X with distilled water.
They were incubated with silver enhancer solution (Sigma, St. Louis, MO) for 7
minutes, rinsed again with distilled water, and the enhancement reaction was
stabilized by treatment with 2.5% sodium thiosulfate for 2-3 minutes (according to
the instructions of the supplier). The samples were rinsed again with distilled water
and coverslipped with the aqueous mounting medium Gel/Mount (biomedia, Foster
City, CA). Patterns of fetuin binding were observed using differential interference
contrast (DIC) optics.
Characterization of Fetuin Binding Proteins
For the extraction of fresh sperm membrane components, cauda epididymal
contents from 2 males (4 epididymides) were released into six 1.5 ml-eppendorf
tubes. The highly motile fractions (top 700 /l) were pooled after 10 minutes and
samples were removed to check motility and determine concentration. Protease
inhibitors, 100X concentrate, were added and the sperm were pelleted at 960 G for
5 min, washed in 5 ml of incomplete medium plus protease inhibitors (960 G, 5
minutes), then suspended in 1.5-2 ml of cold extraction buffer to yield a final
concentration of approximately 2 X 108/ml. The suspension was kept on ice and after


27
Nature of Sperm Binding in the Tubal Reservoir
The nature of the interaction between sperm and the tubal mucosa was
examined by treating hamster sperm with a series of potential binding inhibitors and
introducing them into excised hamster uterine tubes. Binding levels were assessed
by observing and scoring the live sperm within the uterine tube by videomicroscopy.
Sialic acid, especially as presented on the glycoprotein fetuin, appears to be a potent
inhibitor of sperm binding in the hamster uterine tube. The binding of sperm in the
tubal reservoir may rely on a carbohydrate mediated, interaction with sialic acid.
Sperm Motility Patterns in the Uterine Tube
Sperm motility patterns were examined within the uterine tube by recording
live mouse sperm in excised uterine tubes following natural mating. Qualitative
observations about the bound and free-swimming sperm were made and quantitative
measures taken from the recordings were used to characterize the hyperactivated
motility pattern. All free-swimming sperm appeared to be using the hyperactivated
pattern. Thus, it appears that when mouse sperm are able to release from the
reservoir, they have switched to the hyperactivated pattern. Hyperactivation may
contribute to their ability to release.
Sperm Surface Changes
Changes on the sperm surface were examined by producing monoclonal
antibodies that recognized epitopes that were somehow changing as hamster sperm


94
from caudal epididymal sperm and also from fluid of the distal cauda (Robataille et
al., 1991). A monoclonal antibody was produced which recognized a 23 kDa protein,
pi 3.8-4.2, from extracted cauda sperm separated under reducing conditions (Ellis et
al., 1985). However, immunofluorescent labelling with this antibody was restricted
to the sperm tail. Neither of these studies included potential changes associated with
in vitro capacitation.
The epitopes for HL 772 and HL 778 appear to be changed by the time sperm
reach the hyperactivated stage. A loss or modification of epitope seems to occur for
antibody HL 772. Since the staining with HL 778 increases in intensity and becomes
evenly distributed at hyperactivation, the epitope may be unmasked during culture.
A change in glycosylation could expose an epitope that had been previously
inaccessible to the antibody. Increased labelling of sperm surface components during
in vitro capacitation has been previously described (Okabe et al., 1986; Berger, 1990;
Fusi et al., 1992). Additionally, the antigen containing the recognized epitope may
be redistributed during capacitation. There are also documented examples of this
phenomenon (Saxena et al., 1986; Topfer-Petersen et al., 1990; Jones et al., 1990).
Since the antigenic changes for these two antibodies are coincident with
hyperactivation and this may be the first stage at which sperm can break free from
the isthmic reservoir (see Chapter 3), they may play a role in either the switch of
motility patterns or release from the mucosa.
The antigen associated with HL 772 may be approximately 42 kDa under
reducing conditions. This band seems to be reduced in the hyperactivated sperm


40
Proteins were transferred to Immobilon-P membranes (Millipore Corp.,
Bedford, MA) by semi-dry electrophoretic transfer at 2.5 mA/cm2 for 15 minutes
(Buhi et al., 1993). Standards and a lane of sperm proteins were separated and
stained with Coomassie blue in 50% methanol. Gels were also stained with
Coomassie blue to ensure the transfer was successful. The blots were blocked with
3% BSA in PBS, pH 7.4, for 2 hours at room temperature. After rinsing with PBS,
blot strips were incubated with the treatments listed in Table 2-2. All treatments
were diluted with PBS containing 3% BSA and inhibitory substrates were included
with lectins as a control for the specificity of their binding.
Slug lectin, LFA, was not available conjugated to a label and was detected
using an indirect method (Roth et al., 1984,1985). The lectin is multivalent and will
bind to fetuin/gold. LFA-treated blots were incubated for 1 hour at room
temperature with 0.5 jug/ml of fetuin/gold. Fetuin/gold treatment for 1 hour at room
temperature without previous lectin treatment served as a control. Labelling with
fetuin/gold was detected using the silver enhancement reaction (Sigma, St. Louis,
MO). Peroxidase-labelled chick pea lectin, CPA, was detected using
diaminobenzidine (DAB) as substrate with nickel enhancement (Harlow & Lane,
1988).
Results
For preliminary screening, each of the inhibitors listed in Table 2-1 was tested
in two uterine tubes from two different females and compared to sperm treated with


31
acid form of sialic acid (Graham, 1966; Spiro & Bhoyroo, 1974; Krusius et al., 1976).
Subsequent analysis revealed that the sialic acid residues appear to be involved in the
binding inhibition. Fetuin labelling of fresh and hyperactivated sperm cells and of
proteins separated from sperm extracts was investigated to assess changes that might
correlate with the release of sperm from the tubal reservoir.
Materials and Methods
Medium and Chemicals
For the culture of sperm cells and induction of hyperactivated motility,
hamster sperm capacitation medium, similar to that shown to sustain hyperactivation
and capacitation previously (Suarez et al., 1993) was used. The medium contains 105
mM NaCl, 5 mM KC1, 2.4 mM CaCl2, 0.49 mM MgCl2, 0.36 mM NaH2P04, 25 mM
HEPES buffer, 25mM NaHC03, 5.00 mM glucose, 6.26 mM sodium lactate, 0.125
mM pyruvic acid, 12 mg/ml Fraction V bovine serum albumin (BSA) and 0.06 g/1
penicillin G. The pH was adjusted to 7.5 prior to filter sterilization (0.22 cm Millex-
GV filter, Millipore Corp., Bedford, MA). Osmolarity was 285-295 mOsm/kg. Prior
to use, 1 fim epinephrine, 100 on hypotaurine, and 20 iM D-penicillamine were
added from frozen 100X stock solutions (Bavister, 1989). Incomplete hamster
capacitation medium lacked BSA and the metabolic substrates, glucose, lactate, and
pyruvic acid.


86
Spectrum Medical Industries Inc., Los Angeles, CA) had been boiled in the presence
of EDTA to reduce protein binding (Harlow & Lane, 1988). The sample was then
lyophilized. Two-dimensional polyacrylamide gel electrophoresis was carried out
according to Roberts and coworkers (1984). For isoelectric focussing (IEF), the pH
gradient ranged from 4-10. The IEF gels were then loaded on 10% polyacrylamide
slab gels and separated as previously described (see Chapter 2). Semi-dry transfer
to Immobilon-P membranes and blocking was carried out as described above. Blots
were then incubated overnight at 4C with either 1:2000 normal mouse serum or
1:100 HL 787 ascites diluted with PBS/BSA. The blots were washed, incubated with
1:1000 peroxidase-labelled secondary antibody for 1 hour, and developed as described
above.
Results
From one fusion yielding 316 cultures, we identified 6 antibodies that showed
stage specific differences in immunofluorescent labelling of hamster sperm. Four of
these were cloned; three that showed labelling changes associated with agglutination
or hyperactivation that could be relevant to sperm transport in the uterine tube, and
one that was an exceptionally strong acrosome-specific antibody that is useful for
determining acrosome reaction status.
Staining patterns show that the epitope associated with antibody HL 787
changes at agglutination while those associated with HL 772 and HL 778 change at
hyperactivation (Figures 4-1, 4-2). Between the fresh stage and agglutination, the


65
41 = FCR
AB
Figure 3-1. The method for calculating flagellar curvature ratio (FCR) is shown.
The tail was traced with the digitizer pen to calculate curved-path
distance as well as straight-line distance from the ends of the trace.
(Used with permission from DeMott & Suarez, 1992).
All statistical tests were carried out with StatView 512+ (Brainpower, Inc.,
Calabasas, CA). Proportional measurement data (proportion of free sperm and
FCR) were transformed by taking the arcsine of the square root of the proportion
before testing by ANOVA (Sokal & Rohlf, 1989). One-factor and multi-factor
ANOVA were performed and the a priori determined significance level was p <.
0.05. Values reported are nontransformed.


93
Discussion
The decline in immunofluorescent staining seen at agglutination with antibody
HL 787 implies that the epitope is lost, either through modification of the antigen or
release from the cell surface. The results on both 1-D and 2-D immunoblots suggest
that this antibody recognizes a 22-23 kDa acidic antigen. Also, the blots of
hyperactivated and fresh sperm extracts showed changes in staining parallel to the
immunofluorescent results. Gels had been loaded with similar amounts of total
protein. Taken together, these results imply that before agglutination occurs, a sperm
protein migrating at approximately 22 kDa in reducing conditions is altered or lost
from the sperm surface. The loss and alteration of sperm components during in vitro
culture has been well documented and proposed to be a part of the capacitation
process (reviewed by Yanagimachi, 1988).
Modification of this antigen prior to agglutination in vitro implies that there
may be an analogous early change after sperm have been deposited in the female
tract. The timing, approximately 1 hour in culture, suggests that this modification
could be associated with the binding of sperm to the isthmic epithelium in the tubal
reservoir, occurring during the first 2 hours after insemination in the hamster (Smith
& Yanagimachi, 1991). It is possible that this change plays a functional role in the
ability of sperm to bind as well. The antibody will be a useful tool for investigating
these possibilities.
Similar hamster sperm antigens have previously been described. Antiserum
produced against sperm extracts recognized a 22.5 kDa protein (reducing conditions)


132
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Schulte BA, Rao KPP, Kreutner A, Thomopoulos GN & Spicer SS (1985)
Histochemical examination of glycoconjugates of epithelial cells in the human
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Shalgi R & Kraicer PF (1978) Timing of sperm transport, sperm penetration and
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passage of capacitated and uncapacitated hamster spermatozoa through the
uterotubal junction. Biology of Reproduction, 46, 419-424.
Sidhu KS & Guraya SS (1993) Effect of calmodulin-like protein from buffalo
(Bubalus bubalis) seminal plasma on Ca2+, Mg2+-ATPase of purified plasma
membrane of buffalo spermatozoa. Andrologia, 25, 25-28.
Simrell CR & Klein PA (1979) Antibody responses of tumor-bearing mice to their
own tumors captured and perpetuated as hybridomas. Journal of Immunology,
123, 2386-2394.
Smith CA, Hartman TD & Moore HDM (1986) A determinant of Mr 34000
expressed by hamster epididymal epithelium binds specifically to spermatozoa
in co-culture. Journal of Reproduction and Fertility, 78, 337-345.
Smith TT, Koyanagi F & Yanagimachi R (1987) Distribution and number of
spermatozoa in the oviduct of the golden hamster after mating and artificial
insemination. Biology of Reproduction, 37, 225-234.


107
Figure 5-3. Sperm Observed Within Luminal Mucus. Photomicrographs of the
uterotubal junction (A) and isthmus (B) containing sperm in the
luminal mucus, s = sperm heads, magnification 40X.


100
Materials and Methods
Design and Sampling
Mice were obtained and housed as described in Chapter 4. Uterine tubes
from three females in each of six different groups (proestrus, early estrus-mated and
unmated, late estrus-mated and unmated, and diestrus) were examined. Mice were
considered in proestrus on the fourth day following a standing estrus. This was
backed up by visual staging using the criteria of Champlin and coworkers (1973).
Mice were considered early estrus as soon as they allowed a male to mount, usually
around 3-5 hours after lights out, approximately 12:00 to 2:00 A.M. For mated mice,
the female was left with the male until one hour after ejaculation had been observed,
then the uterine tubes were removed. Uterine tubes were taken from unmated mice
60-90 minutes after the male was allowed to mount. For late estrus, females were
placed with males shortly after lights on, around 7:30 A.M. Again, mated animals
were taken 1 hour after ejaculation, unmated were taken 60-90 minutes after allowing
mounts. Diestrus was considered the third day following a standing estrus. This was
also backed up by visual staging (Champlin et al., 1973).
The uterine tubes were processed to preserve the morphology of the mucus,
and sections were examined for differences in the staining and morphology of the
tissue and the luminal contents that related to tubal region, stage of cycle, or mating
status. The location of sperm in the mated animals was also noted.


CHAPTER 5
DEMONSTRATION OF TUBAL MUCUS IN THE PATH
OF SPERM TRANSPORT
Introduction
The tubal environment may present another level of regulation for sperm
passage to the site of fertilization. When sperm are swimming freely within the
uterine tube they encounter morphological barriers such as mucosal folds and pockets
(Nilsson & Reinius, 1969; Suarez, 1987; Smith & Yanagimachi, 1990) that may make
progress toward the egg difficult (Suarez & DeMott, 1991). They may also encounter
mucous epithelial secretions that may affect their movement (Hunter et al., 1987;
Suarez et al., 1991b). There are also cycles of epithelial secretion correlated with the
estrous cycle that may be relevant to the ability of sperm and eggs to move along the
uterine tube (Jansen, 1980; Schulte et al., 1985).
One of the potential functions of hyperactivated motility is proposed to be the
generation of additional force and torque which provides an advantage for passage
through viscoelastic materials (Katz et al., 1989). Analyses of sperm motility and
passage through the cumulus matrix and zona pellucida (Drobnis et al., 1988b;
Drobnis et al., 1988a), and also through artificial viscous and viscoelastic media
(Suarez et al., 1991a; Suarez fe Dai, 1992) indicate that there are changes in flagellar
97


104
In the uterotubal junction and isthmus of the mated mice, sperm were seen
within pools of the luminal material (Figure 5-3). Sperm in the isthmic pockets and
the central lumen were seen completely surrounded by the material. There appeared
to be more sperm in the uterine tubes of the animals mated late in estrus compared
to the early estrous matings even though the same amount of time had passed since
ejaculation. No sperm were found in the ampulla.
Discussion
Treatment of frozen sections with both celloidin and CPC results in the
retention of the luminal contents as a relatively homogeneous material that is clearly
in the path of sperm moving along the uterine tube. The staining characteristics of
the luminal material implies the presence of some sulfated acidic muco
polysaccharides. It stained with PAS, Alcian blue, pH 2.5 and preferentially with
Alcian blue when treated with combined PAS/Alcian blue. This material thus
appears to be mucous secretions maintained in position and structure as in vivo. The
diffuse nature of the PAS reactivity in the coated, post-fixed sections suggests that
when the material is hydrated, the mucopolysaccharides are somewhat dispersed.
Additional characterization of the material is required to determine the proportions
of the various mucous elements. This method has since been used to examine
uterine tubes from rabbits, cows, and pigs and yields similar preservation but species-
dependent staining differences (Suarez, S.S. and DeMott, K.R., unpublished results).


23
The uterotubal junction is the first major obstacle for species, such as the
hamster and mouse, where large amounts of the ejaculate enter the uterus. Like the
cervix, the uterotubal junction appears to help the sperm migrate out of seminal
plasma and into female tract fluids (Hunter, 1988). It contains mucosal folds, but the
orientation and degree of folding are highly variable among species (Hook & Hafez,
1968; Nilsson & Reinius, 1969). Histology (Zamboni, 1972) and in situ observations
(Suarez, 1987) indicate that in the mouse, the uterotubal junction can serve as a valve
controlled by coitus. Studies on the migration of rat and hamster sperm through the
uterotubal junction indicate that sperm motility is critical for passage, yet
hyperactivated sperm are not able to pass (Gaddum-Rosse, 1981; Smith &
Yanagimachi, 1988; Shalgi et al., 1992). This implies that, at least in these species,
the onset of hyperactivation must occur in the uterine tube for any sperm destined
to fertilize (Shalgi et al., 1992). Based on observations of sperm passage following
natural mating, Bedford and Yanagimachi (1992) reported that while rat sperm attain
their fully active motility pattern in the uterus, hamster sperm motility is subdued
until they have passed through the uterotubal junction and are exposed to tubal fluid.
Since the fertilizing sperm come from a population established rapidly in the
isthmic reservoir, the uterine tube environment is the most likely, compared to the
other segments of the female tract, to influence the transport of the fertilizing sperm.
Again, the morphological features of the uterine tube may affect transport. In the
mouse (Nilsson & Reinius, 1969; Suarez, 1987) and hamster isthmus (Smith et al.,
1987; Smith & Yanagimachi, 1990) there are pockets formed by transverse folding


22
and attainment of motility, or part of capacitation aimed at the preparation for
sperm/egg interactions and the acrosome reaction. Some of these cellular changes,
while observed during maturation, capacitation or hyperactivation, may play their
functional role in helping control the transport of sperm though the uterine tube.
Sperm Path through the Uterine Tube
A final subject that needs to be considered for a potential role in controlling
sperm transport is the features of the female tract that directly interact with sperm.
The female tract has both physical and physiological features that are potentially
involved. Again, there has been a great deal discovered which may be relevant to
sperm transport, but little interpreted in terms of this phenomenon.
For vaginal inseminating species, the cervix is generally considered the first
obstacle to sperm (Hunter, 1988). The morphology of the bovine cervix has been
shown to include a series of mucosal folds and blind passages that may complicate
sperm passage (Mullins & Saacke, 1989). Based on 3-dimensional reconstructions
from serial sections that noted the position of the passages through the cervix and the
cervical mucus, Mullins and Saacke (1989) proposed that the path of least resistance
for sperm is in longitudinal grooves away from the center of the cervix. The role of
cervical mucus for sequestering sperm and removing seminal plasma, especially in the
human was studied intensively (Hunter, 1988; Barratt & Cooke, 1991) and there is
recent evidence that human cervical mucus may enhance capacitation (Lambert et
al., 1985) but prevent premature acrosome reactions (Bielfeld et al., 1992).


119
Chang MC (1951) Fertilizing capacity of spermatozoa deposited in the fallopian
tubes. Nature, 168, 697-698.
Chang MC (1957) A detrimental effect of seminal plasma on the fertilizing capacity
of sperm. Nature, 184, 466-467.
Chou K, Chen J, Yuan S & Haug A (1989) The membrane potential changes polarity
during capacitation of murine epididymal sperm. Biochemical and Biophysical
Research Communications, 165, 58-64.
Claus R (1990) Physiological role of seminal components in the reproductive tract of
the female pig. Journal of Reproduction & Fertility, suppl. 40, 117-131.
Cook HC (1977) Carbohydrates in Theory and Practice of Histological Techniques,
edited by JD Bancroft & A Stevens. New York: Churchill Livingstone, pp.
141-167.
Cooper GW, Overstreet JW & Katz DF (1979) The motility of rabbit spermatozoa
recovered from the female reproductive tract. Gamete Research, 2, 35-42.
Cornwall GA, Tulsiani DRP & Orgebin-Crist M-C (1991) Inhibition of the mouse
sperm surface a-D-mannosidase inhibits sperm-egg binding in vivo. Biology of
Reproduction, 44, 913-921.
Coronel CE & Lardy HA (1992) Functional properties of caltrin proteins from
seminal vesicle of the guinea pig. Molecular Reproduction and Development, 33,
74-80.
Coronel CE, Novella ML, Winnica DE & Lardy HA (1993) Isolation and
characterization of a 54-kilodalton precursor of caltrin, the calciium transport
inhibitor protein from seminal vesicles of the rat. Biology of Reproduction, 48,
1326-1333.
Cowan AE, Primakoff P & Myles DG (1986) Sperm exocytosis increases the amount
of PH-20 antigen on the surface of guies pig sperm. J.Cell Biol., 103,
1289-1297.
Cross NL & Overstreet JW (1987) Glycoconjugates of the human sperm surface:
Distribution and alterations that accompany capacitation in vitro. Gamete
Research, 16, 23-35.
Cummins JM (1982) Hyperactivated motility patterns of ram spermatozoa recovered
from oviducts of mated ewes. Gamete Research, 6, 53-64.


Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy
FACTORS AFFECTING SPERM TRANSPORT IN THE
MAMMALIAN OVIDUCT
By
Robert P. DeMott
December 1993
Chairperson: Susan S. Suarez, Ph.D.
Major Department: Veterinary Medicine
The mammalian oviduct, or uterine tube, is the site of fertilization. During
passage through the uterine tube, sperm complete the physiological processes that
prepare them for fertilization; they may also begin using the vigorous hyperactivated
motility pattern. Sperm numbers drop several orders of magnitude between the
entrance to the uterine tube and the site of fertilization approximately mid-way along
it. This is primarily due to the presence of a sperm reservoir in the lower uterine
tube formed relatively quickly after insemination. Sperm are retained here until
fertilization is imminent. Then, a very few move out of the reservoir and on to the
site of fertilization.
The experiments described here were intended to assess the effects of several
factors potentially regulating sperm transport through the uterine tube. The nature
of retention in the reservoir was investigated and found to involve a specific
IX


TABLE OF CONTENTS
ACKNOWLEDGMENTS
LIST OF TABLES vi
LIST OF FIGURES vii
ABSTRACT ix
CHAPTERS
1 INTRODUCTION 1
Significance of Controlled Sperm Transport 1
Features of Tubal Sperm Transport 5
Sperm Retention in the Isthmic Reservoir 6
Sperm Movement out of the Isthmic Reservoir 8
Changes in Sperm Cell Biology 11
Sperm Path through the Uterine Tube 22
Description of Experiments 26
Nature of Sperm Binding in the Tubal Reservoir 27
Sperm Motility Patterns in the Uterine Tube 27
Sperm Surface Changes 27
The Morphology and Nature of Tubal Contents 28
2 MECHANISM OF SPERM BINDING IN THE
ISTHMIC RESERVOIR 29
Introduction 29
Materials and Methods 31
Medium and Chemicals 31
Binding Inhibition in Excised Uterine Tubes 32
Binding of Fetuin to Fresh and Hyperactivated Sperm 37
Characterization of Fetuin Binding Proteins 38
Results 40
IV


126
Koehler JK (1981) Lectins as probes of the spermatozoon surface. Archives of
Andrology, 6, 197-217.
Koehler JK & Gaddum-Rosse P (1975) Media induced alterations of the membrane
associated particles of the guinea pig sperm tail. Journal of Ultrastructure
Research, 51, 106-118.
Kolberg J, Michaelsen TE & Sletten K (1983) Properties of a lectin purified from the
seeds of Cicer arietinum. Hoppe-Seylers Zeitschrift fur Physiologische Chemie,
364, 655-664.
Krusius T, Finne J & Rauvala H (1976) The structural basis of the different affinities
of two types of acidic N-glycosidic glycopetides for concanavalin A-sepharose.
FEBS Letters, 71, 117-120.
Krutzsch PH, Crichton EG & Nagle RB (1982) Studies on prolonged spermatozoa
survival in Chiroptera: A morphological examination of storage and clearance
of intrauterine and cauda epididymal spermatozoa in the bats Myotis lucifugus
and M. velifer. American Journal of Anatomy, 165, 421-434.
Kumari TRS, Sarkar HBD & Shivanandappa T (1990) Histology and histochemistry
of the oviductal sperm storage pockets of the agamid lizard Calotes versicolor.
Journal of Morphology, 203, 97-106.
Lambert H, Overstreet JW, Morales P, Hanson FW & Yanagimachi R (1985) Sperm
capacitation in the human female reproductive tract. Fertility and Sterility, 43,
325-327.
Landemore G, Quillec M, Oulhaj N & Izard J (1993) Kurloff cell ultrastructure after
combined formaldehyde-cetylpyridinium chloride fixation and high-iron
diamine staining. Histochemical Journal, 25, 64-76.
Lillie RD & Fullmar HM (1976) Histopathologic Technic and Practical
Histochemistry. New York: McGraw Hill.
Llanos MN & Meizel S (1983) Phospholipid methylation increases during
capacitation of golden hamster sperm in vitro. Biology of Reproduction, 28,
1043-1051.
Luna LG (1968) Manual of Histological Staining Methods of the Armed Forces
Institute of Pathology. New York: McGraw Hill.


98
beat shape associated with exposure to these media and that hyperactivated sperm
appear to have a functional advantage.
Prior to encountering the cumulus or zona, however, sperm may be influenced
by increased viscosity or viscoelasticity in the tubal lumen due to the presence of
mucous secretions. Sperm observed within excised uterine tubes (see Chapters 2, 3)
seemed to be moving in a thick, viscous fluid. Hyperactivated sperm in the tubal
ampulla had relatively reduced flagellar bending compared to those in the isthmus
(see Chapter 2). While regional differences in the fluid were not visible, the changed
flagellar beating patterns resembled those observed in experiments where sperm were
added to viscous solutions (Suarez et al., 1991a). It was hypothesized for the present
study that sperm pass through mucous secretions of the uterine tube and that there
may be regional differences in the secretions that affect sperm motility.
The presence of cervical mucus, its regulation and effects on sperm transport
have been extensively studied (Iacobelli et al., 1971; Barratt & Cooke, 1991; Katz et
al., 1989). The penetrability of cervical mucus appears to differ at various stages of
the cycle (Barratt & Cooke, 1991; Katz et al., 1989) and exposure to cervical mucus
appears to alter motility parameters (Zhu et al., 1992). The distribution of the mucus
within the cervix may also affect sperm transport by creating certain paths of reduced
resistance (Mullins & Saacke, 1989).
Though tubal mucus has been described (Jansen, 1978,1980; Jansen & Bajpai,
1982; Hunter et al., 1987), the distribution of the mucus in the lumen and its relation
to the path of sperm ascent remains unclear. There is little information as to


66
Results
Qualitative Observations
Using the straightened uterine tube preparation for observing sperm motility,
we noted certain typical patterns of movement. There appeared to be two distinct
populations of sperm, one that showed a very regular, moderately bent tail beat and
another more dynamic population defined by regular moderate tail beats
interspersed with groups of erratic, highly curved beats characteristic of
hyperactivation. The erratic beats were of increased amplitude, varying frequency,
and were propagated in randomly changing planes. The dynamic group, those sperm
which varied their beat pattern, represented a small proportion of the total,
approximately 20%. Sperm of both types were typically found in the same area, but
occasionally an isthmic fold would contain a large number of regular sperm and no
dynamic sperm.
Sperm in the dynamic group appeared to be the ones capable of swimming
freely and making progress along the uterine tube. Regularly beating sperm were
observed for up to 20 minutes and none were ever seen to break free from the
epithelium. The typical pattern of motility for the dynamic sperm involved a period
of regular beating ranging from 10 seconds to approximately 1 minute followed by a
series of erratic, high curvature beats during which the sperm might successfully break
free. This series typically lasted 5-10 seconds regardless of whether the sperm broke
free. Some sperm that did not break free were seen to repeat the cycle. There were
occasional sperm, approximately 1%, that continually beat erratically, but they did not


76
population ready and able to move up the uterine tube at any time. Other sperm
may become hyperactivated, but if this is not properly coupled to capacitation, and/or
other unidentified factors, it may not help them reach the site of fertilization at the
appropriate time.
The increase in FCR for both free and stuck sperm in the ampulla is harder
to understand. If hyperactivation is indeed a factor helping sperm release from
repeated bindings to the epithelium, we might assume that the most hyperactivated
sperm should reach the ampulla. There may be some difference in the fluid
surrounding the sperm that dampens their tail beat in the ampulla. The secretions
of the ampullar epithelium may be more viscous or viscoelastic than those of the
isthmus, and this may be further enhanced after ovulation by cumulus secretions and
follicular fluid. Since there is a bursa surrounding the mouse ovary (Hunter, 1988),
the products of ovulation cannot escape into the peritoneal cavity and must instead
enter the ampulla. There is experimental evidence indicating that increases in the
viscosity of the medium can dampen sperm tail beat amplitude and frequency
(Drobnis et al., 1988b; Rikmenspoel, 1984; Suarez et al., 1991a; Suarez & Dai, 1992).
Also, hyperactivated hamster sperm appear to have an advantage over non-
hyperactivated in penetrating fluids of elevated viscosity and their tail beats do flatten
out (develop higher FCR) in more viscous fluid (Suarez et al., 1991a). Measurements
of the hydrodynamic properties of the fluids in which sperm swim in vivo would be
valuable for our understanding of the modulation of sperm motility and its possible
functions. Besides the regional differences in fluid composition that may affect


135
Tulsiani DRP, Nagdas SK, Cornwall GA & Orgebin-Crist M-C (1992) Evidence for
the presence of high-mannose/hybrid oligosaccharide chain(s) on the mouse
ZP2 and ZP3. Biology of Reproduction, 46, 93-100.
Tulsiani DRP, Skudlarek MD, Holland MK & Orgebin-Crist M-C (1993)
Glycosylation of rat sperm plasma membrane during epididymal maturation.
Biology of Reproduction, 48, 417-428.
Varki A (1993) Biological roles of oligosaccharides: all of the theories are correct.
Gtycobiology, 3, 97-130.
Vazquez MH, Phillips DM & Wasserman PM (1989) Interaction of mouse sperm
with purified sperm receptors covalently linked to silica beads. Journal of Cell
Science, 92, 713-722.
Verhage H & Fazleabas AT (1988) The in vitro synthesis of estrogen-dependent
proteins by the baboon (Papio anubis) oviduct. Endocrinology, 123, 552-558.
Verhage HG, Mavrogianis PA, Boice ML, Li W & Fazleabas AT (1990) Oviductal
epithelium of the baboon: hormonal control and the immuno-gold localization
of oviduct-specific glycoproteins. American Journal of Anatomy, 187, 81-90.
Vernon RB, Muller CH & Eddy EM (1987) Further characterization of a secreted
epididymal glycoprotein in mice that binds to sperm tails. Journal ofAndrology,
8, 123-128.
Veselsky L, Jonakova V, Sanz ML, Topfer-Petersen E & Cechova D (1992) Binding
of a 15kDa glycoprotein from spermatozoa of boars to surface of zona
pellucida and cumulus oophorus cells. Journal of Reproduction and Fertility, 96,
593-602.
Voglmayr JK & Sawyer RF Jr (1986) Surface transformation of ram spermatozoa in
uterine, oviduct, and cauda epididymal fluid in vitro. Journal of Reproduction
and Fertility, 78, 315-325.
Vrcic H, Horvat B & Damjanov I (1993) Estrous-cycle-related changes in the
expression of mouse endometrial and oviductal glycoproteins. Gynecological
and Obstetric Investigations, 35, 44-48.
Wagh PV & Lippes J (1989) Human oviductal fluid proteins. III. Identification and
partial purification. Fertility and Sterility, 51, 81-89.
Wagh PV & Lippes J (1993) Human oviductal fluid proteins. V. identification of
human oviductin-1 as alpha-fetoprotein. Fertility and Sterility, 59, 148-156.


44
from buffer control and was significantly lower than that observed in the fetuin and
sialic acid groups.
While analyzing the videotapes, a clear regional difference in sperm binding
was observed. In the fetuin and sialic acid treatments, most of the free sperm were
seen in the isthmus. As the morphology gradually changed in the region of the
ampullary/isthmic junction (AIJ), numbers of free sperm decreased. In the ampulla,
very few free sperm were observed. When the proportions of free sperm in each of
these regions were compared, significant differences were detected by ANOVA for
fetuin (F=12.9, df=17, p=0.005) and sialic acid (F=10.7, df=14, p=0.002). Since the
analysis was carried out a posteriori, the more stringent Scheffes F test was used for
multiple comparisons (Marks, 1990). The inhibition in the isthmus was significantly
stronger than in the ampulla for both fetuin and sialic acid (Figure 2-3). In the fetuin
treatment, the decrease between isthmus and AIJ was also statistically significant. In
the sialic acid treatment, however, the variability was such that the proportion free
in the AIJ was not statistically different from the isthmus or ampulla.
When the sperm from the isthmus and ampulla were separated and
differences between the treatments analyzed by region, it was found that most of the
fetuin-induced inhibition was occurring in the isthmus (Figure 2-4). In the isthmus,
inhibition in the fetuin-treated group was significantly higher by Scheffes F test than
the asialofetuin or buffer groups. The sialic acid group was significantly higher than
the asialofetuin and buffer groups, which remained statistically indistinguishable. In
the ampulla, however, there was no significant inhibition in any group. The mean


FACTORS AFFECTING SPERM TRANSPORT IN THE
MAMMALIAN OVIDUCT
By
ROBERT P. DEMOTT
A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
1993

ACKNOWLEDGMENTS
First thanks go to Dr. Susan Suarez. Her prods were inevitably in the right
direction, guided by insight that has served as a good example of why it is important
to know intimately the details of your research material. Her financial and
professional support of my work has been most unselfish. Next thanks go to the
other members of my supervisory committee, Drs. Maarten Drost, Louis Guillette,
Paul Klein, William Buhi, and also Fuller Bazer for advising during the first half of
my program. Their diverse approaches and interests helped lead to intriguing
questions and provided a wide selection of ways to answer them. They did a good
job teaching me to keep my trees and forests in proper perspective.
Next, thanks go to all the faculty members who provided generously of their
knowledge, time, and equipment including Drs. Kevin Anderson, John Harvey, Roger
Reep, and Chris West. The varied nature of my experiments had me knocking on
many doors and their cooperation was wonderful, as was the cooperation from all the
scientists who spent time training me in new methods including Idania Alvarez, Katy
Gropp, Melanie Pate, Michael Sapper and Heidi Wearne. Several college
administrators have done a good job providing support and helping me through the
Graduate School procedures including Drs. Darryl Buss, Phillip Kosch and Tom
Wronski.
ii

My labmates, Xiao-bing Dai, John Donald, Samir Raychoudhury, Carmen
Stauss and Steve Varosi were always willing to lend a hand, or at least an opinion,
and I appreciate their help. Thanks also go to Lori Dixon for her help with antibody
screening and Rejean LeFebvre for his involvement in the sperm binding
experiments. The ICBR core facilities are an invaluable asset at the University of
Florida, and the monoclonal core and electron microscopy core staff provided expert
support.
Finally thanks go to Kember DeMott; as a scientist and wife, untoppable.
Having a soulmate with a detailed understanding of the work you are immersed in,
able to share ideas and contribute to your career, is a blessing.
in

TABLE OF CONTENTS
ACKNOWLEDGMENTS
LIST OF TABLES vi
LIST OF FIGURES vii
ABSTRACT ix
CHAPTERS
1 INTRODUCTION 1
Significance of Controlled Sperm Transport 1
Features of Tubal Sperm Transport 5
Sperm Retention in the Isthmic Reservoir 6
Sperm Movement out of the Isthmic Reservoir 8
Changes in Sperm Cell Biology 11
Sperm Path through the Uterine Tube 22
Description of Experiments 26
Nature of Sperm Binding in the Tubal Reservoir 27
Sperm Motility Patterns in the Uterine Tube 27
Sperm Surface Changes 27
The Morphology and Nature of Tubal Contents 28
2 MECHANISM OF SPERM BINDING IN THE
ISTHMIC RESERVOIR 29
Introduction 29
Materials and Methods 31
Medium and Chemicals 31
Binding Inhibition in Excised Uterine Tubes 32
Binding of Fetuin to Fresh and Hyperactivated Sperm 37
Characterization of Fetuin Binding Proteins 38
Results 40
IV

Discussion 51
Conclusions 56
3 SPERM MOTILITY PATTERNS IN THE UTERINE TUBE 58
Introduction 58
Materials and Methods 61
Results 66
Qualitative Observations 66
Quantitative Comparisons 69
Discussion 72
Conclusions 77
4 INVESTIGATION OF CHANGING SPERM ANTIGENICITY .... 78
Introduction 78
Materials and Methods 80
Production of Monoclonal Antibodies 80
Immunogold Labelling of Sperm 84
Immunoblotting of Sperm Proteins 85
Results 86
Discussion 93
Conclusions 96
5 DEMONSTRATION OF TUBAL MUCUS IN THE PATH
OF SPERM TRANSPORT 97
Introduction 97
Materials and Methods 100
Design and Sampling 100
Processing 101
Sectioning 101
Staining 102
Results 103
Discussion 104
Conclusions Ill
6 SUMMARY 112
REFERENCE LIST 116
BIOGRAPHICAL SKETCH 138
v

LIST OF TABLES
Table page
2-1 Candidate competitive inhibitors of carbohydrate interactions
in the uterine tube 34
2-2 Carbohydrate binding treatments for blotted
sperm components 41
3-1 Analysis of factors affecting proportion of sperm swimming
freely within the uterine tube 70
3-2 Attachment status interactions for FCR 71
4-1 Modified hamster capacitation medium formulation 82
4-2 Criteria for staging sperm 82
vi

LIST OF FIGURES
Figure page
2-1 Photomicrographs of hamster sperm within the
tubal isthmus 36
2-2 Bar graph showing effect of treatment on sperm 43
2-3 Bar graph showing regional differences in inhibition
by fetuin and sialic acid 45
2-4 Bar graph showing that effect of treatment is due
primarily to isthmic inhibition 46
2-5 Photomicrographs of silver-enhanced labelled hamster sperm 48
2-6 PVDF blots probed for carbohydrate binding 50
3-1 The method for calculating flagellar curvature ratio
(FCR) is shown 65
3-2 Illustration of a typical pattern and timecourse for
sperm progress in the uterine tube 68
4-1 Stage-related changes observed in indirect immunofluorescent
labelling with monoclonal antibodies 88
4-2 Epifluorescent images of monoclonal antibody binding patterns 89
vn

4-3 1-D Immunoblots probed with stage-specific
monoclonal antibodies 91
4-4 2-D Immunoblot of fresh sperm extract labelled with HL 787 92
5-1 Differential preservation of luminal contents by
processing method 105
5-2 Regional differences in luminal staining characteristics 106
5-3 Sperm observed within luminal mucus 107
vm

Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy
FACTORS AFFECTING SPERM TRANSPORT IN THE
MAMMALIAN OVIDUCT
By
Robert P. DeMott
December 1993
Chairperson: Susan S. Suarez, Ph.D.
Major Department: Veterinary Medicine
The mammalian oviduct, or uterine tube, is the site of fertilization. During
passage through the uterine tube, sperm complete the physiological processes that
prepare them for fertilization; they may also begin using the vigorous hyperactivated
motility pattern. Sperm numbers drop several orders of magnitude between the
entrance to the uterine tube and the site of fertilization approximately mid-way along
it. This is primarily due to the presence of a sperm reservoir in the lower uterine
tube formed relatively quickly after insemination. Sperm are retained here until
fertilization is imminent. Then, a very few move out of the reservoir and on to the
site of fertilization.
The experiments described here were intended to assess the effects of several
factors potentially regulating sperm transport through the uterine tube. The nature
of retention in the reservoir was investigated and found to involve a specific
IX

carbohydrate interaction between the sperm surface and the tubal wall. A sialic acid
bearing ligand apparently mediates this lectin-like binding. The use of particular
motility patterns in the uterine tube was investigated and the switch to hyperactivated
motility appeared prior to release from the reservoir. Thus, hyperactivation may help
sperm break free from the wall. Sperm modifications coincident with transport were
investigated by developing monoclonal antibodies to sperm epitopes. Antigens that
serve as markers and possibly play a functional role for the transition to
hyperactivation and other stages were described. Finally, a specialized histological
protocol was used to characterize the nature, morphology and location of the luminal
contents which sperm encounter. The presence of mucus in the path of sperm was
demonstrated. Based on these experiments, it appears that there is a specific binding
interaction affecting sperm retention in the reservoir, that motility changes occur
when sperm can release from the reservoir, that changing antigenicity can be used to
detect cellular modifications associated with functional changes, and that the tubal
environment contains material which has the capability to affect sperm passage.
x

CHAPTER 1
INTRODUCTION
Significance of Controlled Sperm Transport
There is a common perception that mammalian fertilization occurs after a
large number of sperm move up the female reproductive tract and encounter the egg.
The sheer number of sperm in the tract is seen as a guarantee that enough will make
it up to the egg. This perception is strengthened by the images we typically see of
fertilization in which the event has been contrived, either for the sake of the image,
or to ensure that fertilization takes place in an artificial system. The procedure for
in vitro fertilization, which requires large excesses of sperm, have been empirically
determined to provide high percentages of fertilization while limiting polyspermy. It
appears, however, that it is not adequate to extrapolate from these conditions to
describe fertilization in vivo.
More than 40 years ago, observations were published based on timed matings
and subsequent serial sectioning indicating that the ratio of sperm to eggs at the time
of fertilization in the rabbit (Chang, 1951) and rat (Moricard & Bossu, 1951) is
approximately 1:1. Zamboni reported similar findings in the mouse (Zamboni, 1972),
noting that it was only after fertilization was completed that excess sperm reached the
area. Serial sectioning, tubal flushing and microscopic observations of sperm within
1

2
excised uterine tubes have been used to show that the number of sperm present at
the time and place of fertilization is very low in the mouse (Tessler & Olds-Clarke,
1981; Suarez, 1987), hamster (Cummins & Yanagimachi, 1982; Smith et al., 1987)
rabbit (Overstreet et al, 1978), rat (Bedford & Kim, 1993), sheep (Cummins, 1982)
and pig (Hunter et al., 1987).
Other observations imply that the reduction of sperm numbers to these very
low levels does not occur as a regular, gradual process in the female reproductive
tract. There is likely a constant loss of sperm during their ascent, but there are also
very restrictive barriers (Katz et al., 1989). For species that inseminate at or below
the cervix, this structure appears to reduce the number of progressing sperm by
orders of magnitude (Hunter, 1988). The junction between the uterus and uterine
tube (the preferred anatomical term "uterine tube" will be used throughout in place
of the more common "oviduct"), the uterotubal junction, also appears to serve as a
restrictive filter (Gaddum-Rosse, 1981; Shalgi et al., 1992), causing another large drop
in sperm numbers. Finally, within the uterine tube, there is not a regular distribution
of sperm. Sperm appear to be sequestered in a reservoir located in the most
proximal portion of the uterine tube until ovulation, and thus fertilization, is
imminent. This phenomenon has been demonstrated in the hamster (Yanagimachi
& Chang, 1963; Smith et al., 1987), mouse (Zamboni, 1972; Suarez, 1987), rabbit
(Harper, 1973a, 1973b; Overstreet et al., 1978), cow (Thibault et al., 1975), guinea
pig (Yanagimachi & Mahi, 1976), sheep (Hunter & Nichol, 1983), pig (Hunter, 1984;
Hunter et al., 1987) and rat (Shalgi & Kraicer, 1978; Shalgi & Phillips, 1988).

3
The tubal region of the reproductive tract serves to store and support sperm
for a number of other taxa that have lengthy periods between insemination and
fertilization, including bats (Krutzsch et al., 1982; Racey et al., 1987) and the fat
tailed dunnart (Breed et al., 1989). There are also birds (Bobr et al., 1964a, 1964b;
Bakst, 1992) and reptiles (Halpert et al., 1982; Palmer & Guillette, 1988; Kumari et
al., 1990; Gist & Fischer, 1993) that have sperm storage sites in various regions of the
female reproductive tract.
The formation of the sperm reservoir occurs relatively quickly after
fertilization. Using serial sectioning after timed matings, Smith and coworkers (Smith
et al., 1987) showed that the isthmic reservoir in the hamster is populated with sperm
from 1-3 hours after the onset of mating. Population of the reservoir in the mouse
appears to be functionally limited to the first hour after mating, as the uterotubal
junction begins constricting after this point (Zamboni, 1972; Suarez, 1987). Observing
sperm within excised mouse uterine tubes, Suarez (1987) noted that by 1-2 hours
post-coitus there are many sperm in the lower isthmus and tubal portion of the
uterotubal junction, but that the intramural region, where the junction is within the
muscular walls of the uterus, is constricted and sperm numbers are low. By ligating
the reproductive tract at various points and various times after mating, Hunter (1981)
demonstrated that the reservoir is established within 1 hour in the pig isthmus. For
the cow, a vaginal inseminator where the interval between mating and ovulation may
be up to 30 hours, formation of the reservoir occurs around 8 hours after
insemination (Wilmut & Hunter, 1984; Hawk, 1987).

4
It is also well established that the sperm that will go on to fertilize eggs are
derived from the population in the isthmic reservoir. Using the timed ligation
approach, Harper (1973b) demonstrated that the rabbit sperm that reach the isthmus
relatively early are sufficient for fertilization and Hunter (1984) demonstrated that
it was the boar sperm that had rapidly populated the isthmus that were responsible
for fertilization. The closing of the uterotubal junction shortly after mating in the
mouse (Zamboni, 1972; Suarez, 1987) implies that the fertilizing sperm must come
from the early arriving population. In the hamster, Smith and Yanagimachi (1991)
flushed out the sperm that had not bound to the epithelium after mating and later
obtained fertilized eggs from these uterine tubes. In an earlier study (Smith &
Yanagimachi, 1990), they had found that at 2 hours post-insemination, the number
of sperm in the isthmus remains fairly constant. Since there was not yet ascent to the
ampulla (Smith et al., 1987), this implies that a stable population forms early and
provides the fertilizing sperm. Considering all these observations, our current model
for fertilization in vivo is that the fertilizing sperm ascend rapidly through the lower
portion of the female reproductive tract and are retained in a reservoir in the
proximal portion of the uterine tube. Around the time of ovulation, after a species
specific delay, a very few of these sperm complete passage to the ampullary-isthmic
junction where they meet and fertilize the eggs. In this model, retention,
maintenance, and release of sperm in the isthmus seem to serve as a key control
point for fertilization. Yet, the functional details of sperm binding, release and ascent
through the uterine tube remain poorly described.

5
The mechanisms that control tubal sperm transport to the site of fertilization
have not been given sufficient attention for several reasons. Since it is not yet
possible to observe natural fertilization and to track the responsible sperm, inferential
approaches are required. Thus, experimental results can be difficult to interpret
definitively and conclusions must be narrowly qualified. Further, using such a
complex physiological system for experimentation is laborious, and accounting for and
holding potentially complicating factors constant can be difficult. Additionally, the
interest in manipulating reproduction and the numerous difficulties associated with
these technologies have provided a selection of research questions that can be
addressed with well controlled in vitro approaches.
However, the study of fertilization in vivo will improve our understanding of
intercellular interactions, sperm cell and reproductive tract biology, and allow
improvements to be made to manipulated reproduction technologies. Consider that
the naturally regulated system provides successful fertilization and reproduction with
a gamete ratio close to unity, whereas our artificial technologies require thousands
of times more sperm than eggs. It seems clear that there is much information, both
mechanistic and applied, to be found studying the process of sperm transport in the
uterine tube.
Features of Tubal Sperm Transport
There are several features of sperm transport through the uterine tube that
need further investigation. The biochemical mechanisms of sperm retention in the

6
reservoir have not been described. While several mechanisms for sperm release have
been postulated, which actually operate and are pertinent to fertilization remains
unclear. Observations of sperm motility patterns in the uterine tube raise the
question of how motility and other aspects of sperm cell biology contribute to
reaching the site of fertilization. And, the path that sperm take in the uterine tube
and substances that they encounter may represent another level of regulation. Our
current understanding of each of these features will be reviewed.
Sperm Retention in the Isthmic Reservoir
At least for the mouse (Suarez, 1987) and hamster (Smith & Yanagimachi,
1990), it seems that the most important method of retaining sperm in the reservoir
involves adherence to the tubal mucosa. Attachment to the mucosa has also been
suggested in the rabbit (Cooper et al., 1979) and pig isthmus (Hunter et al., 1987).
The morphology of the uterine tube provides a series of mucosal folds and pockets
that appear to serve as storage crypts in some species (Nilsson & Reinius, 1969;
Suarez, 1987; Hunter et al., 1987; Smith & Yanagimachi, 1990). Smith and
Yanagimachi (1987) flushed hamster uterine tubes to remove first luminal and then
adherent sperm. There is also evidence that bull sperm bound to tubal cells in
culture (Pollard et al., 1991; Ellington et al., 1991) maintain viability and fertilizability
while unattached cells become immotile and may have disrupted acrosomes. In a
species with extended sperm storage, the little brown bat, sperm interact with and
appear to be maintained by the epithelium in the uterotubal junction (Racey et al.,

7
1987). In reptilian sperm-storing species there appear to be some storage regions
with sperm in close association to the epithelium and others where large agglutinated
masses of sperm occupy the lumen of specialized tubules (Gist & Fischer, 1993).
This second type of storage organ is found in fowl (Bakst, 1992).
Several other mechanisms for retaining sperm in the isthmus have been put
forth and they may play an accessory role in some species. Post-coital constriction
of the isthmus may hamper the ability of mouse sperm to move within the uterine
tube (Suarez, 1987). Depressed motility observed in isthmic sperm has also been
postulated to help prevent sperm escaping the reservoir (Overstreet fe Cooper, 1975;
Cooper et al., 1979; Cummins, 1982). Hunter and Nichol (1986) found a slight
temperature gradient in the pig uterine tube and speculated that the cooler
temperatures in the reservoir region may help subdue sperm. Another contributing
factor in the pig uterine tube may be the presence of viscous secretions in the
mucosal crypts that Hunter and coworkers inferred based on the degree of flagellar
bending seen by scanning electron microscopy (SEM) of sperm in the crypts
compared to sperm in the main lumen (Hunter et al., 1987). The presence of a
mucous layer, again detected by SEM, before ovulation but not after in the rabbit
(Jansen, 1978) and human (Jansen, 1980) uterine tube led to the proposal that ciliary
beating, which aids in sperm transport, may be dampened by this mucus until
ovulation has occurred. An actual role for any of these alternative mechanisms in
controlling sperm transport has not been demonstrated. In light of the observations
of tight adherence between sperm and the isthmic mucosa in situ (Suarez, 1987;

8
Smith & Yanagimachi, 1990), this mechanism seems to be the most relevant, though
its biochemical basis has not yet been determined.
Sperm Movement out of the Isthmic Reservoir
Several mechanisms for moving sperm from the isthmic reservoir to the site
of fertilization have been proposed. First, through in situ observations of sperm in
the mouse (Suarez, 1987) and hamster uterine tube (Smith & Yanagimachi, 1990)
sperm have been seen to detach from the wall and move by their own flagellar
beating. Another mechanism involved is the movement of sperm along with the
contents of the uterine tube. The most efficient propulsion is likely due to muscular
contractions of the uterine tube directed toward the ampulla. By injecting a
particulate solution into the uterine tubes and observing its redistribution in
anesthetized hamsters, Battalia and Yanagimachi (1979) noted that there is
coordinated muscular activity directing the tubal contents toward the ovary only
during the period immediately preceding ovulation. A subsequent study showed that
the shifts in ovarian steroid ratios around ovulation trigger this coordinated
movement (Battalia & Yanagimachi, 1980). Waves of muscular contraction in the
pig uterine tube, measured in anesthetized animals, also appear to change direction
around ovulation (Rodriguez-Martinez, et al., 1982).
Control of the muscular contractions may be affected by the products of
ovulation as well. Ito and coworkers (1991) propose that prostaglandins in the
ovulatory products have a local effect in the uterine tube regulating the contractions.

9
They had observed that superovulated hamsters have enhanced sperm transport.
Presumably the additional ovulations produce additional prostaglandins that enhances
the contractions. The transport of extra sperm in superovulated rats has also been
documented (Shalgi & Phillips, 1988). The presence of high levels of estrogens in
boar semen has also been postulated to induce prostaglandin and LH releases in the
female that could affect muscular activity in the reproductive tract, and the timing of
ovulation (Claus, 1990)
Another possible mechanism for transporting sperm is ciliary currents.
Gaddum-Rosse and Blandau (1973) observed ciliary transport of particles in
longitudinally opened uterine tubes and reported that the current in the isthmus of
rabbits and pigs is toward the ampulla. However, these species seem to be atypical
in terms of the direction of the current when compared to rats, guinea pigs, humans,
and cows (Gaddum-Rosse & Blandau, 1976) and also in terms of how much ciliation
is present in the isthmus (Nilsson & Reinius, 1969; Hunter, 1988). So, while ciliary
beating is important for egg transport (Norwood et al., 1978; Norwood & Anderson,
1980; Mahi-Brown & Yanagimachi, 1983), it probably plays a minimal role in isolated
species for sperm movement (Gaddum-Rosse & Blandau, 1976).
While the uterine tube may play a predominant role in the movement of
sperm that have released from the reservoir, it seems that it does not control whether
or not adherence of sperm to the mucosa is maintained. By sequentially flushing the
hamster uterine tube to remove free then loosely bound sperm at several time points,
Smith and Yanagimachi (1990) found that there were no significant changes in the

10
number of tubal sperm until at least 2 hours after ovulation. And, at this time, sperm
were only slightly less susceptible to being flushed out. These results imply that the
uterine tube does not undergo marked changes in its affinity for binding sperm at
ovulation.
A significant change in sperm binding was, however, associated with the
condition of the sperm (Smith & Yanagimachi, 1991). When sperm capacitated in
vitro (see below "Changes in Sperm Cell Biology"), i.e., sperm capable of fertilization,
were injected into the uterine tube, they remained free from the mucosa whereas
non-capacitated sperm bound to the mucosa as soon as they encountered it. This
implies that part of capacitation involves a loss of affinity for the tubal reservoir and
it is this reduction in binding affinity that allows sperm to move along the uterine
tube. Interpretation of these results is confounded, however, because the capacitated
sperm were also using the vigorous hyperactivated motility pattern which could
contribute to their ability to pull away from the mucosa.
In summary, movement out of the tubal reservoir appears to involve release
from the mucosa due to changes in the binding affinity of sperm and/or changes in
flagellar beating, as well as indirect effects of tubal physiology. The primary
contribution to indirect effects is the muscular contractions of the uterine tube which
serve to move the luminal contents. Ciliary beating may play a small role as well.

11
Changes in Sperm Cell Biology
The production of sperm capable of fertilizing an egg is a continuous process
from testis to uterine tube. Sperm leave the testis immotile and undergo a series of
modifications in the epididymis, collectively called sperm maturation, which result in
the attainment of motility and the stabilization of the cell membrane (Yanagimachi,
1988). Nevertheless, they require further processing before becoming capable of
fertilization. Chang (1951) and Austin (1951) first recognized that sperm need to
spend a certain time in the female reproductive tract before they are able to fertilize
an egg. This part of the process, capacitation (Austin, 1952), has been defined
nebulously as a set of cellular changes that allow the sperm cell to undergo the
acrosome reaction when challenged with a specific inducer (Yanagimachi, 1988). The
acrosome reaction is an exocytotic event required for fertilization (Meizel, 1985).
Another observable phenomenon that can not yet be explained in terms of overall
sperm cell biology is the use of the hyperactivated flagellar movement pattern. First
noticed for hamster sperm capacitated in vitro (Yanagimachi, 1969; Gwatkin &
Anderson, 1969), this obvious alteration in frequency, amplitude, and shape of the
flagellar beat yields an erratic, albeit vigorous, pattern of movement. Hyperactivation
has since been observed within the hamster ampulla (Katz & Yanagimachi, 1980) and
in the upper regions of the mouse reproductive tract (Suarez & Osman, 1987). While
the relationship of hyperactivation to the other steps in the process of preparing a
sperm for fertilization remains unclear, its occurrence in parallel with capacitation

12
and use in the uterine tube mean that the change to this pattern may contribute to
the regulation of sperm transport in the uterine tube.
Maturation. The maturational changes that occur primarily in the
epididymis include modifications of the glycoproteins on the sperm surface, changes
in the lipid composition of the membrane, and stabilization of the tail structures and
condensed nucleus by disulfide bonding (Yanagimachi, 1988). Membrane
glycoproteins may be modified, added, or removed and some of these changes have
been associated with functional changes in sperm. A protein that appears to modify
boar sperm, preventing the head-to-head agglutination seen in corpus epididymal
sperm, was extracted from caudal epididymal fluid (Dacheux et al., 1983).
The proteins present on rat sperm from the caput, corpus, and caudal
epididymis and changes induced by incubating these sperm with caudal epididymal
fluid have been analyzed by two-dimensional polyacrylamide gel electrophoresis (Hall
& Killian, 1989). They demonstrated that a variety of different glycoproteins appear
and disappear from the sperm membrane as they move along the epididymis and that
treatment with caudal fluid can cause some of these changes.
Extrinsic rat sperm proteins of the acrosomal region that are produced in the
caudal epididymis have been identified (Rifkin & Olson, 1985). Additionally, changes
in the localization of rat sperm antigens occurring during epididymal passage and
capacitation have been reported (Petruszak et al., 1991; Phillips et al., 1991;
Rochwerger & Cuasnicu, 1992). Hamster sperm proteins that are added in the
epididymis have also been described (Moore & Hartman, 1984; Smith et al., 1986;

13
Hoos & Olson, 1988; Robataille et al., 1991). A sialylated glycoprotein produced
from the distal corpus to the cauda appears to be processed and is associated with
the mouse sperm tail (Vernon et al., 1987; Feuchter et al., 1988; Toshimori et al.,
1990) and a ram sperm sialoglycoprotein added in the cauda redistributes during the
acrosome reaction (McKinnon et al., 1991).
Another group of studies relate to changes in protein glycosylation during
maturation. By comparing the binding of various lectins to sperm from different
parts of the epididymis, changing populations of carbohydrate moieties on sperm have
been identified for the hamster (Nicolson & Yanagimachi, 1972; Koehler, 1981),
rabbit (Nicolson & Yanagimachi, 1972; Nicolson et al., 1977), ram (Hammerstedt et
al., 1982; Magargee et al., 1988), macaque (Fain-Maurel et al., 1984), mouse (Rankin
et al., 1989), dog (Bains et al., 1993b), and goat (Bains et al., 1993a). The results of
these studies imply that the exposed carbohydrates and their modifications are highly
species specific. Galactose and N-acetylgalactosamine distribution on testicular and
caudal rat sperm, analyzed by binding to labeled galactose oxidase, has also identified
proteins modified during maturation (Brown et al., 1983). Another approach has
been to demonstrate the presence of certain glycosyltransferases in epididymal fluid.
Sialyltransferase has been demonstrated in rat epididymal fluid with the strongest
activity in the caput (Bernal et al., 1980), and galactosyltranferase activity also
appears in rat epididymal fluid (Hamilton, 1980).
Sperm are considered mature when they reach the caudal epididymis since
they may become motile, capacitate, and fertilize. However, in the natural scheme

14
of fertilization, sperm are exposed to seminal plasma before the environment of the
female tract and this exposure seems to affect their cell biology. The effects of
seminal plasma were initially considered to prevent premature capacitation of the
sperm (Yanagimachi, 1988). Chang (1957) showed that capacitation could be
reversed by a component of rabbit seminal plasma produced in the epididymis.
Subsequently a number of specific decapacitation components were identified
(Oliphant et al., 1985). A family of proteins found in bull, guinea pig, mouse and rat
semen seem to inhibit calcium uptake by sperm (Coronel et al., 1993), a necessary
occurrence for capacitation and hyperactivation (Yanagimachi, 1988; Fraser, 1990;
Suarez et al., 1993). After insemination, these proteins, caltrins, may become
enhancers of calcium uptake (San Agustn et al., 1987; Coronel & Lardy, 1992)
presumably enhancing capacitation. A similar model of decapacitating activity and
subsequent enhancement of capacitation by boar seminal plasma proteins has been
described (Desnoyers & Manjunath, 1992). These proteins, which bind specific sperm
phospholipids and presumably stabilize the membrane, are proposed to coat the
sperm during early transport and then be removed, taking some membrane lipid
along; the resulting leakiness of the membrane allows calcium entrance (Desnoyers
& Manjunath, 1992). These proteins also bind calmodulin (Manjunath et al., 1993)
which may play a role in calcium regulation during capacitation. A calmodulin-like
protein from seminal plasma, that may help control calcium levels in buffalo sperm,
has also been reported (Sidhu & Guraya, 1993)

15
Recently, other functions of seminal plasma components have been reported.
The proposal for a direct effect of seminal plasma steroids on the endocrine
regulation of the female tract has been described above (Claus, 1990). Two separate
groups have identified seminal plasma proteins that have the ability to bind to the
zona pellucida (Parry et al., 1992; Veselsky et al., 1992), though some of the same
proteins may have been found by both groups. Though they are unlikely to represent
the primary ligand for sperm/egg binding, these proteins present the interesting
possibility that seminal plasma components enhance sperm/egg binding. Additionally,
a marked reduction in catalase activity, one of the major oxidative damage protection
systems, has been found in the seminal plasma of infertile men compared to sperm
and seminal plasma from normal, fertile men (Jeulin et al., 1989). In light of the
growing awareness of the importance of free radical scavenging systems, research
clarifying this potential protective role of seminal plasma may intensify.
Capacitation. Determining which of the myriad events of sperm cell biology
are part of capacitation is a matter of semantics. The sperm cells are constantly
being affected by their environment and undergoing modifications; our distinctions
are convenient but must be recognized as artificial and irrelevant in terms of the
sperms life history. And, while capacitation is by definition directed toward
producing the acrosome reaction, along the way some changes in the sperm cell may
play a role in sperm transport. Contributions to the control of capacitation by the
female tract may also be relevant to controlling sperm transport.

16
A number of sperm surface changes associated with capacitation have been
described. Following the report of lectin binding characteristics for sperm by
Nicolson and Yanagimachi (1972), a number of surface carbohydrate changes were
identified using lectins. In the hamster, the distribution of binding sites for the lectin
Concanavalin A over the head changes as sperm are capacitated in vitro (Kinsey &
Koehler, 1978). Decapacitation factors which are removed during capacitation have
been described for the rabbit (Reyes et al., 1975) and mouse (Fraser, 1984).
Membrane antigens that are redistributed have been identified for mouse (Okabe et
al., 1986), boar (Saxena et al., 1986; Berger, 1990; Topfer-Petersen et al., 1990) and
rat (Jones et al., 1990) sperm. The redistribution of intramembranous particles has
been demonstrated in the guinea pig (Koehler & Gaddum-Rosse, 1975). The
appearance of certain antigens with capacitation, suggesting that they were masked
or have been modified, has been documented in the rat (Jones et al., 1990) and ram
(Voglmayr fe Sawyer, 1986) and for integrin binding proteins on human sperm (Fusi
et al., 1992).
Another type of change that has been associated with capacitation involves
alteration of the lipid characteristics of sperm membranes. The ratio of cholesterol
to phospholipid, which affects membrane fluidity (Go decrease due to the gradual removal of cholesterol. This increasing fluidity of the
membrane was proposed to enhance capacitation (Davis, 1981). A similar change
in the ratio was found during human sperm capacitation (Hoshi et al., 1990). The
enhancement of capacitation, measured by the ability of the sperm to penetrate the

17
egg, coincident with the removal of cholesterol from bovine sperm has also been
demonstrated (Ehrenwald et al., 1988). Besides the change in sterol to phospholipid
ratio, there is also evidence in guinea pig sperm that the relative amounts of different
membrane phospholipids change during capacitation (Stojanoff et al., 1988).
Another occurrence during capacitation is the alteration of the ionic balance
of the sperm cell. Before capacitation, sperm maintain typical ionic gradients relative
to the medium with potassium high inside the cell and sodium low inside the cell.
They also maintain a calcium gradient with lower concentrations inside than outside
(Yanagimachi, 1988). Changes in the calcium gradient may be a part of capacitation.
There is a clear requirement for an extracellular calcium pool to support
hyperactivation and the acrosome reaction (Yanagimachi & Usui, 1974; Yanagimachi,
1982). There is also evidence that an influx of this calcium triggers the acrosome
reaction (Roldan & Harrison, 1990). Fraser (1987; Fraser & McDermott, 1992)
reported that mouse sperm require at least 0.09 mM for capacitation, but markedly
higher fertility results at 1.8 mM. These results fit well with the proposal that there
are two changes in the calcium gradient, the first associated with hyperactivation and
the second, larger influx at the time of the acrosome reaction (Suarez et al., 1993).
Several mechanisms that may contribute to the calcium influx have been
described. The presence of calcium channels has been inferred based on the
sensitivity of the influx to channel blockers (Fraser, 1987). Chou and coworkers
(1989) used a voltage sensitive dye to show that during mouse sperm capacitation
there is a shift in membrane polarity from negative inside to positive inside, primarily

18
due to potassium ion redistribution, and propose that this polarity shift stimulates
voltage sensitive channels. The ability of Ca-ATPase inhibitors to affect capacitation
(Fraser & McDermott, 1992) and the influx of extracellular calcium (Blackmore,
1993) suggests that this enzyme may also play a role. Decreased Na/K-ATPase
activity observed during guinea pig capacitation (Hang et al., 1990) fits with a model
proposed by Fraser and coworkers (1993) where Na/K-ATPase activity may be
contributing to early mouse capacitation events and help promote increased
intracellular sodium ion levels that activate Na+/H+ exchange, resulting in reduced
intracellular pH and activation of calcium channels.
This model also provides a potential role of the female tract in controlling
capacitation. Differences in the ionic composition of uterine, ampullary, and bursal
sac fluid (Borland et al., 1977) are proposed to allow sperm to undergo the initial
stages of capacitation. However, sperm do not complete the acrosome reaction until
they reach the ampulla and ovulation has occurred, which shifts the extracellular
sodium and potassium levels (Fraser, 1983; Fraser et al., 1993). Killian and
coworkers (1989) analyzed the lipid characteristics of bovine tubal fluid and found
that the cholesterol to phospholipid ratio drops and the prevalence of
lysophospholipids increases around estrus. Both of these conditions are seemingly
favorable for mediating the sperm membrane lipid changes described above.
Contributions of the female tract to capacitation have long been suspected
(Yanagimachi, 1981, 1988; Oliphant et al., 1985). A variety of enzymes,
glycosaminoglycans, and other secretions have been proposed as effectors of

19
capacitation within the tract (Yanagimachi, 1988), but few details have been sorted
out. In the rabbit, a vaginal inseminating species, capacitation was optimized by
sequential exposure to the uterine, then the tubal environment (Bedford, 1969).
Tubal secretions associate with ram and human sperm (Sutton et al., 1986; Wagh &
Lippes, 1989), especially if the sperm are first treated with uterine fluid (Voglmayr
& Sawyer, 1986). Recently, a functional role in enhancing capacitation was proposed
for tubal proteins that interact with bull sperm (McNutt et al., 1993). Clearly the role
of the uterine tube in controlling capacitation needs further definition.
Hvperactivation. This striking motility pattern has now been described for
more than a dozen mammalian species (Yanagimachi, 1988; Katz et al., 1989). First
observed in hamster sperm during capacitation in vitro (Yanagimachi, 1969; Gwatkin
& Anderson, 1969), sperm were later observed using the pattern within the hamster
ampulla (Katz & Yanagimachi, 1980) and in the mouse uterus and uterine tube
(Suarez & Osman, 1987). Additionally, hyperactivated sperm have been flushed from
the tracts of female mice (Phillips, 1972), rabbits (Cooper et al., 1979; Suarez et al.,
1983), hamsters (Cummins & Yanagimachi, 1982b), and sheep (Cummins, 1982a).
The specific form of movement appears to differ from species to species and
is somewhat hard to describe accurately, as evidenced by the array of
anthropomorphic terms that have been used, e.g., dashing, dancing and bobbing
(Yanagimachi, 1988; Katz et al., 1989). Generally it involves increased amplitude of
the flagellar waveform, usually asymmetrically, producing an erratic path, and may
involve changes in the 3-dimensional aspects of beat propagation (Katz et al., 1989).

20
Since hyperactivation was first described in preparations of sperm capacitated
in vitro (Yanagimachi, 1969; Gwatkin & Anderson, 1969), it was originally considered
to be a part of the capacitation process. Nevertheless, there is now evidence that
while these processes may be complementary and parallel, they can be regulated
independently. Yanagimachi (1981) pointed out that hyperactivation occurs before
capacitation is complete and can be experimentally induced in the absence of
capacitation (defined by the inability to undergo the acrosome reaction). He also
noted that hyperactivation is primarily a tail associated phenomenon whereas
capacitation involves the sperm head. While this logic does not fit well with the
continual, dynamic model of capacitation developed here, it has led to clearer
demonstrations of the separate regulation of the two events.
Assessing the contribution of the carrier protein bovine serum albumin (BSA)
that is typically used in capacitation medium, Neill and Olds-Clarke (1987) reported
that mouse sperm could develop hyperactivation, but could not be fully capacitated
without protein present. Altering the sodium bicarbonate levels in capacitating
medium has allowed the production of seemingly non-hyperactivated, capacitated
hamster sperm (Boatman & Robbins, 1991). The finding of separate calcium influxes
associated with hyperactivation and the acrosome reaction implies that there are at
least two mechanisms of calcium ion regulation at work (Suarez et al., 1993) and
lends further support to the idea of separable control.
There is little other information directly relating to the cellular/molecular basis
of controlling hyperactivation. For unclear reasons, increased levels of cAMP seem

21
to positively affect the expression of hyperactivated motility (Fraser & Monks, 1990).
Including either cAMP analogs or phosphodiesterase inhibitors in capacitating
medium appears to enhance hyperactivation (Fraser, 1979, 1981; Mrsny & Meizel,
1980). White and Aitken (1989) have detected a rise in cAMP levels preceding the
attainment of hyperactivation in the hamster. It has been proposed that calcium
regulation and cAMP metabolism are the cellular keys to the shift to hyperactivation
(Ishijima, 1990).
Several functions for hyperactivated motility have been postulated: (1)
providing additional force to release sperm from the mucosa and to aid in
penetration of viscous or viscoelastic substances; (2) generating a movement path that
helps sperm avoid entrapment in the twists and folds of the uterine tube and covers
more area within the uterine tube, improving the likelihood of finding the egg; and
(3) creating disturbances in the tubal medium to improve the exchange of signalling
and metabolic components (Yanagimachi, 1981; Katz et al., 1989; Suarez & DeMott,
1991). Though actual analysis of the functional significance of hyperactivation
remains limited (Drobnis et al., 1988a, 1988b; Suarez et al., 1991a; Suarez & Dai,
1992), the implications of a potential role for hyperactivation in affecting sperm
transport within the uterine tube seem clear.
While the studies described above point out many changes in sperm cell
surface antigens, membrane composition and intracellular metabolism, none of these
changes have been interpreted in terms of sperm transport. They are generally
assigned as part of the maturational process aimed at the stabilization of the sperm

22
and attainment of motility, or part of capacitation aimed at the preparation for
sperm/egg interactions and the acrosome reaction. Some of these cellular changes,
while observed during maturation, capacitation or hyperactivation, may play their
functional role in helping control the transport of sperm though the uterine tube.
Sperm Path through the Uterine Tube
A final subject that needs to be considered for a potential role in controlling
sperm transport is the features of the female tract that directly interact with sperm.
The female tract has both physical and physiological features that are potentially
involved. Again, there has been a great deal discovered which may be relevant to
sperm transport, but little interpreted in terms of this phenomenon.
For vaginal inseminating species, the cervix is generally considered the first
obstacle to sperm (Hunter, 1988). The morphology of the bovine cervix has been
shown to include a series of mucosal folds and blind passages that may complicate
sperm passage (Mullins & Saacke, 1989). Based on 3-dimensional reconstructions
from serial sections that noted the position of the passages through the cervix and the
cervical mucus, Mullins and Saacke (1989) proposed that the path of least resistance
for sperm is in longitudinal grooves away from the center of the cervix. The role of
cervical mucus for sequestering sperm and removing seminal plasma, especially in the
human was studied intensively (Hunter, 1988; Barratt & Cooke, 1991) and there is
recent evidence that human cervical mucus may enhance capacitation (Lambert et
al., 1985) but prevent premature acrosome reactions (Bielfeld et al., 1992).

23
The uterotubal junction is the first major obstacle for species, such as the
hamster and mouse, where large amounts of the ejaculate enter the uterus. Like the
cervix, the uterotubal junction appears to help the sperm migrate out of seminal
plasma and into female tract fluids (Hunter, 1988). It contains mucosal folds, but the
orientation and degree of folding are highly variable among species (Hook & Hafez,
1968; Nilsson & Reinius, 1969). Histology (Zamboni, 1972) and in situ observations
(Suarez, 1987) indicate that in the mouse, the uterotubal junction can serve as a valve
controlled by coitus. Studies on the migration of rat and hamster sperm through the
uterotubal junction indicate that sperm motility is critical for passage, yet
hyperactivated sperm are not able to pass (Gaddum-Rosse, 1981; Smith &
Yanagimachi, 1988; Shalgi et al., 1992). This implies that, at least in these species,
the onset of hyperactivation must occur in the uterine tube for any sperm destined
to fertilize (Shalgi et al., 1992). Based on observations of sperm passage following
natural mating, Bedford and Yanagimachi (1992) reported that while rat sperm attain
their fully active motility pattern in the uterus, hamster sperm motility is subdued
until they have passed through the uterotubal junction and are exposed to tubal fluid.
Since the fertilizing sperm come from a population established rapidly in the
isthmic reservoir, the uterine tube environment is the most likely, compared to the
other segments of the female tract, to influence the transport of the fertilizing sperm.
Again, the morphological features of the uterine tube may affect transport. In the
mouse (Nilsson & Reinius, 1969; Suarez, 1987) and hamster isthmus (Smith et al.,
1987; Smith & Yanagimachi, 1990) there are pockets formed by transverse folding

24
of the mucosa in which the isthmic sperm lodge. The folding pattern gradually
changes to longitudinal around the ampullary/isthmic junction in these species,
forming mucosal grooves oriented along the long axis of the uterine tube (Nilsson &
Reinius, 1969). While the species examined have mucosal folds, the degree and
orientation is species dependent (Nilsson & Reinius, 1969). For the rodents, the
uterine tube is held in a tightly coiled spiral by the mesosalpinx (Suarez, 1987; Smith
et al., 1987). This may also affect sperm transport, as the sperm must be able to
change direction and follow this twisting path.
Another relevant factor of uterine tube morphology is the distribution of
secretory and ciliated cells in the epithelium. Again, there is wide species diversity
in the proportion of each cell type (Nilsson & Reinius, 1969; Hunter, 1988) and both
regional and cyclical variations in their morphogenesis (Brower & Anderson, 1969;
Patek, 1974; Hagiwara et al., 1992; Abe & Oikawa, 1993). Sperm appear capable of
binding to both cell types, though there may be species-dependent preferences for
one type or the other (Hunter et al., 1987; Smith & Yanagimachi, 1990; Suarez et al.,
1991b).
The secretory products of the uterine tube may play a physiological role in the
regulation of sperm transport. There are a few reports of tubal secretions interacting
with sperm (Sutton et al., 1986; Voglmayr & Sawyer, 1986; Wagh & Lippes, 1989;
McNutt et al., 1993), and in the case of human sperm this secretion has been
identified as the sialylated glycoprotein alpha-fetoprotein (Wagh & Lippes, 1993).
A functional role for these secretions remains to be clearly demonstrated, though

25
McNutt and coworkers (McNutt et al., 1993) have associated enhanced bull sperm
capacitation with tubal fluid treatment.
A wide array of other tubal secretory proteins have been characterized that
have been proposed to influence the egg or embryo. Some of these show regional
specificity, as demonstrated with rabbit (Hyde & Black, 1986), pig (Buhi et al., 1990)
and sheep (Murray, 1992) uterine tubes. Others have been demonstrated to be
under hormonal control either by variation during the estrous cycles of sheep and
mice (Sutton et al., 1986; Horvat et al., 1992; Vrcic et al., 1993) or following
hormonal treatments of rabbits (Hyde & Black, 1986; Erickson-Lawrence et al.,
1989), baboon (Verhage & Fazleabas, 1988; Verhage et al., 1990), and sheep
(Murray, 1992). Though some of these proteins obviously could not affect sperm
based on their secretory location and timing, clear demonstrations of association with
the eggs and embryos are few (Kapur & Johnson, 1988; Minami et al., 1992; Boice
et al., 1992; Buhi et al., 1993). More tubal secretions may eventually turn out to bind
sperm or indirectly affect transport out of the reservoir by altering the tubal
environment.
Another type of secretion that could potentially affect sperm transport is tubal
mucus. Because of the difficulties of preserving mucopolysaccharides, the presence
and prevalence of this material in the lumen remains unclear. In standard immersion
fixation protocols followed by routine histological preparations, the luminal contents
are lost (Schulte et al., 1985). The inclusion of polycationic alcian blue with
aldehydes and use of perfusion fixation appears to improve the retention of luminal

26
contents and suggests that in the rabbit uterine tube there are both serous and
mucous secretions present (Jansen & Bajpai, 1982). In scanning electron microscopy
studies, the luminal contents appear patchy and as a matrix or honeycomb on the
surface of the mucosa; sperm associated with the mucosa have been seen coated with
this material (Jansen, 1978, 1980; Boyle et al., 1987; Hunter et al., 1987). Whether
this material actually spans the luminal spaces, potentially affecting sperm movement
as well as sperm adherence to the mucosa remains has not yet been clearly
demonstrated.
Description of Experiments
The experiments described below address four aspects of the control of sperm
transport to the site of fertilization. The characteristics of the binding of sperm to
the tubal wall were examined to address how the sperm reservoir is formed and
maintained. An analysis of sperm motility patterns in the uterine tube addresses a
possible mechanism for release from the reservoir. The establishment of surface
markers associated with various sperm conditions addresses how sperm changes may
contribute to tubal transport. Finally, an examination and characterization of the
tubal luminal contents addresses what role the luminal environment may play in
sperm transport.

27
Nature of Sperm Binding in the Tubal Reservoir
The nature of the interaction between sperm and the tubal mucosa was
examined by treating hamster sperm with a series of potential binding inhibitors and
introducing them into excised hamster uterine tubes. Binding levels were assessed
by observing and scoring the live sperm within the uterine tube by videomicroscopy.
Sialic acid, especially as presented on the glycoprotein fetuin, appears to be a potent
inhibitor of sperm binding in the hamster uterine tube. The binding of sperm in the
tubal reservoir may rely on a carbohydrate mediated, interaction with sialic acid.
Sperm Motility Patterns in the Uterine Tube
Sperm motility patterns were examined within the uterine tube by recording
live mouse sperm in excised uterine tubes following natural mating. Qualitative
observations about the bound and free-swimming sperm were made and quantitative
measures taken from the recordings were used to characterize the hyperactivated
motility pattern. All free-swimming sperm appeared to be using the hyperactivated
pattern. Thus, it appears that when mouse sperm are able to release from the
reservoir, they have switched to the hyperactivated pattern. Hyperactivation may
contribute to their ability to release.
Sperm Surface Changes
Changes on the sperm surface were examined by producing monoclonal
antibodies that recognized epitopes that were somehow changing as hamster sperm

28
went through a set of observable conditions during capacitation in vitro. The binding
of antibodies to sperm in each condition was determined by indirect
immunofluorescence staining patterns of fixed sperm and gold-labelled
immunostaining of live sperm. The antigens were also characterized on Western
blots. One antibody recognized an epitope that appeared to be unmasked when
sperm had reached the hyperactivated stage. Another recognized an epitope that was
lost or modified when they had reached hyperactivation. A third antibody recognized
an epitope that was lost or modified after only a short period in culture. These
antibodies are useful tools for isolating and identifying surface components that may
play a role in binding and release from the tubal mucosa.
The Morphology and Nature of Tubal Contents
In order to determine whether the tubal luminal contents could play a role in
controlling sperm transport, a histological protocol was developed to optimally
preserve the structure and position of the luminal contents. This protocol used
celloidin-stabilized cryosections of mouse uterine tube that had been post-fixed with
cetylpyridinium chloride. A substance with the staining characteristics of a
mucopolysaccharide was found to occlude some of the luminal spaces, and sperm
were observed in this substance. This tubal mucus seems to be in the path of sperm
transport and could provide a selective advantage for the passage of hyperactivated
sperm.

CHAPTER 2
MECHANISM OF SPERM BINDING IN THE ISTHMIC RESERVOIR
Introduction
The existence of a reservoir in the isthmic portion of the uterine tube in which
sperm are maintained until the time of fertilization approaches has been described
for a number of mammalian species (Zamboni, 1972; Thibault et al., 1975;
Yanagimachi & Mahi, 1976; Overstreet et al., 1978; Shalgi & Kraicer, 1978; Hunter
et al., 1987; Suarez, 1987). Sperm appear to ascend relatively quickly to this region
and then are retained; the population of sperm responsible for fertilization move on
to the ampulla only when fertilization is imminent (Harper, 1973b; Hunter, 1984;
Suarez, 1987; Smith & Yanagimachi, 1991).
In the hamster uterine tube at least, it appears that the modulation of binding
and release of sperm from the reservoir is primarily dependent on changes in the
sperm cell, not the uterine tube (Smith & Yanagimachi, 1990, 1991). Non-
capacitated hamster sperm samples injected into excised uterine tubes have been
shown to bind almost completely to the epithelium whereas sperm from capacitated
samples, that were also hyperactivated, are able to remain free (Smith &
Yanagimachi, 1991). Further, following natural mating in the mouse, very few free
swimming sperm were seen in the uterine tube and those that were appeared to be
29

30
hyperactivated (see Chapter 3). These results imply that there is a change in the
affinity for the tubal epithelium coincident with capacitation and the switch to
hyperactivated motility that allows sperm in the appropriate condition for fertilization
to release from the reservoir.
The biochemical nature of sperm adherence in the reservoir and the changes
leading to release have not yet been described, however. To address this question,
the binding of non-capacitated sperm in excised hamster uterine tubes following
treatment with potential inhibitors was analyzed. The binding of sperm in the
isthmus appears to be quite strong, since repeated flushing is required to release the
bound sperm (Smith & Yanagimachi, 1990). Also, various pretreatments of the
uterine tube and enzymatic treatments of tubal explants with adherent sperm were
unsuccessful for preventing sperm binding (Raychoudhury fe Suarez, 1991; T.T.
Smith, personal communication). For this study, a different approach was attempted
where sperm were treated with potential inhibitors prior to exposure to the uterine
tube. Carbohydrate inhibitors in the form of large glycoproteins were chosen due to
the ineffectiveness of proteases (Raychoudhury of carbohydrate interactions for another sperm binding phenomenon, adherence to
the zona pellucida (Wassarman, 1990; Cornwall et al., 1991; Noguchi & Nakano,
1992; Tulsiani et al., 1993).
In the present study, screening resulted in the detection of the glycoprotein
fetuin as an inhibitor of sperm binding. Fetuin, a 43-49 kDa glycoprotein, contains
12-13 O-linked and N-linked carbohydrate chains that end in the N-acetylneuraminic

31
acid form of sialic acid (Graham, 1966; Spiro & Bhoyroo, 1974; Krusius et al., 1976).
Subsequent analysis revealed that the sialic acid residues appear to be involved in the
binding inhibition. Fetuin labelling of fresh and hyperactivated sperm cells and of
proteins separated from sperm extracts was investigated to assess changes that might
correlate with the release of sperm from the tubal reservoir.
Materials and Methods
Medium and Chemicals
For the culture of sperm cells and induction of hyperactivated motility,
hamster sperm capacitation medium, similar to that shown to sustain hyperactivation
and capacitation previously (Suarez et al., 1993) was used. The medium contains 105
mM NaCl, 5 mM KC1, 2.4 mM CaCl2, 0.49 mM MgCl2, 0.36 mM NaH2P04, 25 mM
HEPES buffer, 25mM NaHC03, 5.00 mM glucose, 6.26 mM sodium lactate, 0.125
mM pyruvic acid, 12 mg/ml Fraction V bovine serum albumin (BSA) and 0.06 g/1
penicillin G. The pH was adjusted to 7.5 prior to filter sterilization (0.22 cm Millex-
GV filter, Millipore Corp., Bedford, MA). Osmolarity was 285-295 mOsm/kg. Prior
to use, 1 fim epinephrine, 100 on hypotaurine, and 20 iM D-penicillamine were
added from frozen 100X stock solutions (Bavister, 1989). Incomplete hamster
capacitation medium lacked BSA and the metabolic substrates, glucose, lactate, and
pyruvic acid.

32
Protease inhibitors were also prepared as a 100X concentrate. The
concentrate contained 10 mg/ml aprotinin, 1 mg/ml Na-t-boc-deacetylleupeptin, and
500 mM benzamidine HC1 dissolved in incomplete medium.
Sperm extraction buffer (modified from Saling & Lakoski, 1985) contained 2%
sodium dodecyl sulfate (SDS), 125 mM Tris buffer, pH 6.8, 20% glycerol, 10 /ig/ml
Na-t-boc-deacetylleupeptin, and 2 mM phenylmethylsulfonyl fluoride (PMSF).
All chemicals were from Sigma Chemical Co. (St. Louis, MO) except those
specifically noted here. Limax flavus lectin (LFA), BSA and HEPES were from
Calbiochem Corp. (La Jolla, CA). Fetuin conjugated to 5 nm colloidal gold particles
and chick pea lectin (CPA) were obtained from E-Y Labs, Inc. (San Mateo, CA).
Tris was from Biorad Laboratories (Richmond, CA). Glycine was from ICN
Biomedicals, Inc. (Cleveland, OH). SDS was from BDH Chemicals, Ltd. (Poole,
UK).
Binding Inhibition in Excised Uterine Tubes
Golden Syrian hamsters (Mesocricetus auratus) from Charles Rivers
Laboratories (Wilmington, MA) were maintained on a 14L:10D light cycle with lights
on from 0700-2100 h and provided food and water ad libitum. Retired breeder males
were used to obtain caudal epididymal sperm. Females (9-18 weeks old) were
visually staged with Day 1, estrus, being determined by the presence of clear vaginal
discharge (Hafez, 1970). Animals, males first, were killed by C02 inhalation followed

33
by cervical dislocation at 1400 h on Day 1, approximately 12 hours before the females
were expected to ovulate (Hafez, 1970).
From the males, the epididymides were exposed and the caudae were
punctured with a 25 ga. needle. A drop of epididymal contents was placed in 1 ml
of hamster capacitation medium that had been prewarmed to 37C and equilibrated
under 5% C02. Sperm were allowed to disperse for 5 minutes at 37 C then the top
2/3 of the suspension, containing the highly motile fraction, were removed. After the
motility was assessed, the suspension was centrifuged at 140 G for 5 minutes to
concentrate the sperm. The bottom 100 /nl of this concentrate was retained and
sperm concentration was determined with a hemacytometer. Concentrations were
generally 1-2 X 108 sperm/ml. From this stock suspension, dilutions were made
containing 3 X 106 sperm/ml in 500 ¡A hamster capacitation medium plus candidate
inhibitors dissolved in incomplete capacitation medium (see Table 2-1) or incomplete
medium alone and incubated at 37C for 10 minutes. At each experiment, the
assignment of treatments to the first and second uterine tube preparation was
randomized by flipping a coin.
While the sperm samples were being prepared, females were killed in the
same manner and their uterine tubes were removed and uncoiled. To accomplish
this, the cranial tip of the uterine horn was bisected and lifted up to allow the ovarian
mesenteries to be cut. These segments were rinsed with prewarmed medium and
placed in petri dishes in a small drop of medium. One of the dishes was placed in
a 37C, 5% C02 incubator while the other uterine tube was dissected. The side to

34
Table 2-1. Candidate competitive inhibitors of carbohydrate interactions in the
uterine tube.
Candidate
Concentration
Inhibitory element
Fetuin
5 mg/ml
sialic acid
Asialofetuin
5 mg/ml
galactose
Fucoidan
5 mg/ml
fucose
Ovalbumin
5 mg/ml
n-acetylglucosamine,
mannose
Poly-l-lysine
5 mg/ml
+ charge
be dissected first was randomized by flipping a coin during each experiment. Using
a dissecting microscope, the coiled uterine tube was straightened by carefully cutting
the mesosalpinx. The uterine horn was pinned in wax and only the horn and ovary
were used to hold the tissue. When the uterine tube had been completely
straightened, the ovary and ovarian bursa were removed, the first loop of the isthmus
was freed from the uterine serosa, and the uterine tube was cut through the
extramural uterotubal junction.
Incubated sperm samples were gently drawn into a 1 ml syringe equipped with
a blunted 30 ga. needle. The needle was introduced into the lumen at the
infundibulum and at the first loop of the isthmus and 50 + 5 /I was injected into

35
each end. In several cases, the isthmus could not be successfully injected and an
additional 50 ¡ was injected through the infundibulum. The flow of sperm
suspension through the entire uterine tube was observed to ensure that the lumen
was patent and had been completely flushed. The uterine tubes were rinsed in each
of 5 drops of medium in a clean petri dish to remove most of the sperm on the
outside and placed on a microscope slide. A coverslip, supported by 4 pillars of
silicone grease, was gently pressed down to slightly flatten the uterine tube (modified
from Suarez and Osman, 1987).
The slide chamber was immediately placed on the heated stage of an inverted
videomicroscope. The preparations were observed under bright field illumination
with a halogen light source and a 30X extra-long working distance Hoffman
Modulation Contrast objective (Modulation Optics, Greenvale, NY) (Figure 2-1).
Images from a solid state Dage CCD 72 camera (MTI Inc., Michigan City, IN) were
recorded at 30 frames/second on a SuperVHS videocassette recorder (Panasonic AG-
7300, Panasonic Industrial Co., Secaucus, NJ) along with time/date information
(Model VTG 33, For-A Co., Ltd., Newton, MA). Uterine tubes were taped
beginning at the ampulla progressing toward the isthmus. To ensure that differences
seen between the isthmus and ampulla were not an effect of the sequential taping,
in several uterine tubes the isthmus was taped briefly before the usual sequence was
followed. In several other uterine tubes, the ampulla was retaped after the usual
progression had been completed.

36
Figure 2-1. Photomicrographs of hamster sperm within the tubal isthmus.
Magnification 30X.

37
The sperm on the videotapes were counted and scored as either free or bound
to the mucosa based on the first two seconds in which they were in focus. Counts
from three uterine tubes were duplicated by two separate observers to provide an
assessment of interobserver variation. From the counts, the overall proportions of
free and bound sperm were calculated as well as proportions for each region of the
uterine tube. The proportions were calculated based on all the sperm that could be
seen, approximately 200 per uterine tube. For testing by analysis of variance
(ANOVA), the proportions were transformed by taking the arcsin of the square root
(Sokal & Rohlf, 1989) and the significance level was set at p < 0.05. Values reported
are not transformed.
Binding of Fetuin to Fresh and Hvperactivated Sperm
Cauda epididymal sperm were obtained and the highly motile fraction was
collected as above. The concentration of this fraction was determined and dilutions
were set up containing 3 X 106 sperm in 1 ml of capacitation medium. Immediately
after dilution, aliquots of the non-capacitated, non-hyperactivated sperm were
incubated with either 0.05 ¡xg/ml of 5 nm colloidal gold-conjugated fetuin or an
equivalent volume of medium for 15 minutes. Hyperactivated sperm were treated
similarly once they were obtained, after approximately 3 hours of culture in a 37C,
5% CO2 incubator. Samples were checked occasionally, and when at least 70% of
the sperm were using the characteristic hyperactivated motility pattern (Suarez et al.,
1993), a sample was removed for labelling.

38
Sperm were pelleted in a microcentrifuge and washed 3X with incomplete
medium. They were fixed in suspension with 2.5% glutaraldehyde in phosphate
buffer, pH 7.3, for 5 minutes and then washed again. Samples were placed in the
wells of multi-well immunofluorescence slides (Cel-line Assoc., Newfield, NJ) and
allowed to dry down. They were rinsed 5X with PBS then 5X with distilled water.
They were incubated with silver enhancer solution (Sigma, St. Louis, MO) for 7
minutes, rinsed again with distilled water, and the enhancement reaction was
stabilized by treatment with 2.5% sodium thiosulfate for 2-3 minutes (according to
the instructions of the supplier). The samples were rinsed again with distilled water
and coverslipped with the aqueous mounting medium Gel/Mount (biomedia, Foster
City, CA). Patterns of fetuin binding were observed using differential interference
contrast (DIC) optics.
Characterization of Fetuin Binding Proteins
For the extraction of fresh sperm membrane components, cauda epididymal
contents from 2 males (4 epididymides) were released into six 1.5 ml-eppendorf
tubes. The highly motile fractions (top 700 /l) were pooled after 10 minutes and
samples were removed to check motility and determine concentration. Protease
inhibitors, 100X concentrate, were added and the sperm were pelleted at 960 G for
5 min, washed in 5 ml of incomplete medium plus protease inhibitors (960 G, 5
minutes), then suspended in 1.5-2 ml of cold extraction buffer to yield a final
concentration of approximately 2 X 108/ml. The suspension was kept on ice and after

39
15 minutes, it was sonicated in three 20 second bursts separated by 30 seconds (Heat
Systems Ultrasonics, model W-225R, Farmingdale, NY). The extraction was
continued for another 45 minutes and then the mixture was cleared by centrifugation,
12,000 G, 10 minutes. Total protein concentration in the supernatant was determined
using the Biorad protein assay (Biorad Labs, Richmond, CA). Concentrations ranged
from 1-1.5 mg/ml. The supernatants were stored at 80 C until used.
To extract hyperactivated sperm components, caudal epididymal contents were
collected from 4 males and pooled after swim up. Motility and concentration were
assessed and a series of 15-ml centrifuge tubes were set up containing 3 X 106
sperm/ml or 6 X 106 sperm/ml in 10 ml of capacitating medium. When the sperm
were hyperactivated, determined as described above, the top 8 ml from each tube
was pooled, protease inhibitors were added, and the sperm were pelleted and washed
as above. Sperm concentration was determined after they were suspended in 2.25
ml of extraction buffer. Final concentrations were approximately 2 X 108/ml.
Thereafter, sperm were treated as above.
Fresh and hyperactivated sperm extracts were thawed and prepared for
reducing polyacrylamide gel electrophoresis (PAGE) by boiling for 3 minutes in the
presence of 10% 2-mercaptoethanol (Buhi et al., 1989). Bromophenol blue was
added for color. Samples (500-800 /xg) and molecular weight standards were
separated with 4.5% stacking gels and 10% running gels in a Tris-glycine buffer
system (Roberts et al., 1984). Stacking current was 15 mA per gel and running
current was 30 mA per gel.

40
Proteins were transferred to Immobilon-P membranes (Millipore Corp.,
Bedford, MA) by semi-dry electrophoretic transfer at 2.5 mA/cm2 for 15 minutes
(Buhi et al., 1993). Standards and a lane of sperm proteins were separated and
stained with Coomassie blue in 50% methanol. Gels were also stained with
Coomassie blue to ensure the transfer was successful. The blots were blocked with
3% BSA in PBS, pH 7.4, for 2 hours at room temperature. After rinsing with PBS,
blot strips were incubated with the treatments listed in Table 2-2. All treatments
were diluted with PBS containing 3% BSA and inhibitory substrates were included
with lectins as a control for the specificity of their binding.
Slug lectin, LFA, was not available conjugated to a label and was detected
using an indirect method (Roth et al., 1984,1985). The lectin is multivalent and will
bind to fetuin/gold. LFA-treated blots were incubated for 1 hour at room
temperature with 0.5 jug/ml of fetuin/gold. Fetuin/gold treatment for 1 hour at room
temperature without previous lectin treatment served as a control. Labelling with
fetuin/gold was detected using the silver enhancement reaction (Sigma, St. Louis,
MO). Peroxidase-labelled chick pea lectin, CPA, was detected using
diaminobenzidine (DAB) as substrate with nickel enhancement (Harlow & Lane,
1988).
Results
For preliminary screening, each of the inhibitors listed in Table 2-1 was tested
in two uterine tubes from two different females and compared to sperm treated with

41
Table 2-2. Carbohydrate binding treatments for blotted sperm components.
Treatment
Cone.
Conditions
Detection
Fetuin/gold1
0.5 ¡iglml
overnight, 4C
Ag enhance
Fetuin/gold
0.5 fxg/ml
1 h, 22C
Ag enhance
LFA2
10 fig/ml
overnight, 4C
fet/gold,
Ag enhance
LFA +
10 ig/ml +
overnight, 4C
fet/gold,
Sialic acid3
10 mM
Ag enhance
CPA4
10 /xg/ml
overnight, 4C
HRP
CPA +
10 ig/ml +
overnight, 4C
HRP
Fetuin
1 mg/ml
T Fetuin conjugated to 5 nm colloidal gold particles.
2. Lectin from the garden slug, Umax flavus, recognizes sialic acid (Roth et al.,
1984).
3. n-acetylneuraminic acid.
4. Lectin from the chickpea, recognizes several sialic acid containing
glycoproteins including fetuin (Kolberg et al., 1983).

42
PBS vehicle only. The motility of the sperm at the end of the ten minute treatment
was also checked. During videotaping following treatment with asialofetuin,
ovalbumin, fucoidan, poly-l-lysine and PBS, almost no motile sperm were seen free
of the mucosa. Some immotile sperm could be seen being transported passively with
the tubal contents. Further, motile sperm were not observed to pull away from the
mucosa. In contrast, numerous free swimming sperm were observed following
treatment with fetuin. The free sperm were observed to encounter the mucosa and
not stick to it.
Having identified fetuin as the only inhibitor of sperm binding to the tubal
mucosa among the candidates, a quantitative analysis was completed to more
carefully study the binding inhibition by fetuin and determine whether the terminal
sialic acid residues were responsible for the inhibition. Uterine tubes from a series
of females were randomly assigned by flipping a coin to sperm that had been treated
with 5 mg/ml of either fetuin or asialofetuin, 25 mM sialic acid (n-acetylneuraminic
acid), or PBS vehicle. The pH of the sialic acid treatment was readjusted to 7.5 prior
to the addition of sperm. There were significant differences between the groups in
the proportion of free sperm found in the uterine tube by ANOVA (F=13.5, df=20,
p=0.0001). The proportion of free sperm was significantly higher than buffer control
by Fishers test for multiple comparisons for both fetuin and sialic acid-treated sperm
(Figure 2-2). The binding inhibition by fetuin was not significantly stronger than that
by sialic acid. Also, asialofetuin was again ineffective as an inhibitor of sperm
binding. The proportion of free sperm in the asialofetuin group was not different

43
% Free Sperm
30 =
25
Fetuin Sialic Asalo. Buffer
Treatment
Figure 2-2. Bar graph showing effect of treatment on sperm. Sperm counted from
entire uterine tube, n = number of uterine tubes, error bars represent
SEM, bars marked with different letters differ significantly.

44
from buffer control and was significantly lower than that observed in the fetuin and
sialic acid groups.
While analyzing the videotapes, a clear regional difference in sperm binding
was observed. In the fetuin and sialic acid treatments, most of the free sperm were
seen in the isthmus. As the morphology gradually changed in the region of the
ampullary/isthmic junction (AIJ), numbers of free sperm decreased. In the ampulla,
very few free sperm were observed. When the proportions of free sperm in each of
these regions were compared, significant differences were detected by ANOVA for
fetuin (F=12.9, df=17, p=0.005) and sialic acid (F=10.7, df=14, p=0.002). Since the
analysis was carried out a posteriori, the more stringent Scheffes F test was used for
multiple comparisons (Marks, 1990). The inhibition in the isthmus was significantly
stronger than in the ampulla for both fetuin and sialic acid (Figure 2-3). In the fetuin
treatment, the decrease between isthmus and AIJ was also statistically significant. In
the sialic acid treatment, however, the variability was such that the proportion free
in the AIJ was not statistically different from the isthmus or ampulla.
When the sperm from the isthmus and ampulla were separated and
differences between the treatments analyzed by region, it was found that most of the
fetuin-induced inhibition was occurring in the isthmus (Figure 2-4). In the isthmus,
inhibition in the fetuin-treated group was significantly higher by Scheffes F test than
the asialofetuin or buffer groups. The sialic acid group was significantly higher than
the asialofetuin and buffer groups, which remained statistically indistinguishable. In
the ampulla, however, there was no significant inhibition in any group. The mean

45
% Free Sperm
50
40
30
20
10
0
Isthmus
AIJ
Region
Ampulla
Fetuin
Sialic
Figure 2-3. Bar graph showing regional differences in inhibition by fetuin and sialic
acid. Error bars represent SEM.

46
% Free Sperm
50
Treatment
Fetuin
Sialic
H Asalo
Buffer
Figure 2-4. Bar graph showing that effect of treatment is due primarily to isthmic
inhibition. Error bars represent SEM.

47
proportions were distributed in the same pattern, but they were very low and the
differences were slight. There was also a significant inhibition effect of treatment in
the AIJ, but only the fetuin treatment was high enough to differ statistically from
control (data not shown).
The binding of fetuin to the sperm cells was demonstrated using colloidal gold-
labelled fetuin. The silver-enhanced label was found over the acrosomal region of
the head on fresh, acrosome intact sperm (Figure 2-5a). The staining pattern
covered the dorsal and anterior surfaces of the sperm head, apparently demarcating
the shape of the hamster acrosome. There also appeared to be a band of fetuin
labelling at the neck of the sperm, though this was variable. On the midpiece,
labelling was restricted to the distal portion and was spotty. Where acrosomes had
been disrupted during processing, the acrosomal fragments labelled heavily but the
regions that had been exposed underneath the acrosome did not label.
Hyperactivated sperm showed a different pattern of fetuin binding (Fig. 2-5b).
Acrosome intact sperm had a characteristic clear patch on the dorsal and anterior
acrosomal region. This region appeared to have lost its fetuin binding ability during
incubation. The midpiece also had decreased labelling as the spottiness became very
sparse. The posterior margin of the acrosome and the neck were still able to bind
fetuin, however. Disrupted acrosomes stained variably.
Specific fetuin binding components were detected in Western blots of sperm
extracts and differences were noted between fresh and hyperactivated sperm (Figure
2-6). Results were similar with blots from 2 separate sets of extracts. Colloidal gold

48
Figure 2-5. Photomicrographs of silver-enhanced labelled hamster sperm. A) Fresh
sperm with fetuin-gold, B) Hyperactivated sperm with fetuin-gold, C)
Control sperm with no label.
/

49
labelled fetuin incubated with the blots overnight bound to fresh sperm components
at a number of molecular weights. The intensity of labelling was generally lower for
hyperactivated sperm extracts though the gels had been loaded with the same amount
of total protein. Distinct bands at 27.5 kDa and >335 kDa in the fresh sperm
extracts were absent in the hyperactivated extracts. A strong band at 32-33 kDa in
the fresh sperm extracts was very dim at hyperactivation. Also, a strong band in the
fresh sperm extracts at 49-50 kDa seemed to have shifted to an apparent molecular
weight of 53 kDa in the hyperactivated extracts.
Lectin binding on the blots incubated overnight with LFA indicated that the
fetuin binding proteins seem to be sialylated (Figure 2-6). The same group of
proteins was recognized by the LFA, though some of the bands that labelled weakly
with fetuin were not detectable in the LFA blots. LFA staining was also generally
decreased in the hyperactivated extracts and the differences observed agreed with the
results in the fetuin-labelled blots.
Since LFA was detected using an indirect technique including a 1 hour, room
temperature incubation with colloidal gold-labelled fetuin (Roth et al., 1985; Roth et
al., 1984), control blots that had been incubated overnight with diluent (3% BSA in
PBS), then treated for 1 hour at room temperature with labelled fetuin were
included. These strips were developed with silver enhancement reagent for the same
amount of time as the LFA strips. The interference due to colloidal gold-labelled
fetuin was minimal under these conditions (Figure 2-6). Also, LFA labelling was
inhibited when sialic acid was included during the incubation.

50
Figure 2-6. PVDF blots probed for carbohydrate binding. A) fetuin-gold-labelled
sperm components, B) LFA-labelled sperm components.

51
Staining with CPA was similar to the previous results though the labelling was
generally dim. The stronger fetuin binding bands could be detected and one band
at 42-44 kDa that had not been detected with fetuin or LFA was observed (data not
shown). CPA labelling was inhibited by fetuin.
Discussion
Since fetuin and sialic acid, but not asialofetuin, are able to inhibit sperm
binding to the tubal mucosa, the terminal sialic acid residues of the fetuin molecule
appear to be important for blocking the interaction. This implies that the binding
between sperm and the uterine tube involves a specific interaction with sialic acid.
At least for hamster sperm, none of the common sugars other than sialic acid seem
to be involved in sperm binding to the uterine tube. Also, it does not appear that
charge interactions are the basis of this interaction, since poly-l-lysine was also
ineffective as an inhibitor. Sialic acid is an effective inhibitor by itself, but as might
be expected, the large fetuin molecule is significantly more effective. The
glycoprotein contains 12-13 carbohydrate chains that end in sialic acid (Spiro &
Bhoyroo, 1974; Krusius et al., 1976), providing multiple binding possibilities to
stabilize the interaction.
The labelling of the fresh sperm over the region of the head that
characteristically binds to the tubal mucosa (Suarez, 1987; Smith & Yanagimachi,
1990) and identification of specific sperm glycoproteins that label with fetuin on

52
Western blots suggests that the inhibition by fetuin is due to the presence of a fetuin
binding component on the sperm. The fetuin binding component appears to be
serving as a lectin-like receptor for sialic acid containing ligands. Such a role for
carbohydrates in cellular interactions is well established. The most extensively studied
are the selectins, glycoproteins involved in leukocyte/endothelial adhesion that bind
to sialylated oligosaccharide ligands (Phillips et al., 1990; Walz et al., 1990; Gahmberg
et al., 1992; Varki, 1993). CD 22, a receptor from B cells belonging to another class
of glycoproteins, the immunoglobulin superfamily, also operates as a sialic acid
binding lectin (Sgroi et al., 1993; Powell et al., 1993). Additionally, pertussis toxin
appears to contain a subunit that is capable of binding fetuin and other sialic acid
bearing substrates (Heerze et al., 1992).
The presence of carbohydrate mediated interactions involving sperm has also
been documented. Sperm/egg binding in the mouse appears to involve an interaction
with the oligosaccharide portion of the mouse ZP3 glycoprotein, specifically a
galactose residue (Wassarman, 1990). Sperm also seem to bind to the carbohydrate
portion of the pig ZP3 molecule (Noguchi & Nakano, 1992). The involvement of
mannose (Tesarik et al., 1991; Cornwall et al., 1991; Tulsiani et al., 1992) and fucose
(Oehninger et al., 1991) in sperm/egg binding have also been proposed. These
findings demonstrate the importance of carbohydrate mediated interactions for sperm
physiology.
The fetuin binding components in sperm extracts appeared to be
predominantly sialylated themselves. The similarities between the labelling with

53
fetuin gold and with the two sialic acid recognizing lectins, LFA and CPA, were
striking. Sialylated molecules on sperm have previously been identified and changes
during maturation have been documented (Nicolson & Yanagimachi, 1972;
Hammerstedt et al., 1982; Feuchter et al., 1988; Magargee et al., 1988; Rankin et al.,
1989; Bains et al., 1993b). Capacitation also seems to involve changes in the
sialylation patterns of sperm. Treatment with neuraminidase accelerated the
capacitation of rabbit and guinea pig sperm in vitro (Gwatkin et al., 1972; Oliphant,
1976; Srivastava et al., 1988) and sialylated molecules from sperm have been found
released into capacitation medium (Focarelli et al., 1990). For hamster sperm, the
binding of sialic acid lectins decreases during capacitation (Ahuja, 1984; Nicolson &
Yanagimachi, 1972). Part of the decrease in sialylation may represent the loss of a
fetuin binding component responsible for maintaining the adherence of sperm in the
isthmic reservoir.
The regional restriction of the inhibition to the isthmus suggests that the fetuin
binding component is only involved in binding in the isthmus. This specificity
matches the restriction of the sperm reservoir to the isthmus. Binding of non-
capacitated sperm in the ampulla apparently proceeds by another mechanism and is
presumably irrelevant in vivo, since non-capacitated sperm cannot pass through the
isthmus to reach the ampulla.
Alteration of the interaction between the isthmic epithelium and the fetuin-
binding component as part of the sperm changes occurring during capacitation and/or
hyperactivation may allow release from the reservoir. The extremely low numbers

54
of free sperm seen in these experiments, in agreement with previous observations
when non-capacitated, non-hyperactivated sperm were injected into the hamster
uterine tube (Smith & Yanagimachi, 1991) speak to the strength of this interaction
for fresh sperm. However, once sperm have been capacitated in vitro (Smith &
Yanagimachi, 1991) or begun using the hyperactivated motility pattern following
natural mating (see Chapter 3), they are able to release from the tubal mucosa.
Smith and Yanagimachi (1991) demonstrated that this difference was likely due to
changes in the sperm, not the condition of the uterine tube. While the use of
hyperactivated motility may provide additional force for sperm to break free, there
is substantial support for a mechanism of release involving the loss of the fetuin-
binding substance.
The binding patterns seen with colloidal gold-labelled fetuin indicate that
during incubation to the hyperactivated stage, the fetuin binding affinity over the
dorsal and anterior surface of the acrosomal region decreases. This is the region of
the sperm head that typically binds to the tubal epithelium (Suarez, 1987; Smith &
Yanagimachi, 1990). The reduction in the fetuin binding ability is thus well
correlated in terms of both timing and localization with release from the tubal
mucosa.
In a study on zona pellucida glycoprotein binding to sperm, fetuin was used
as a control for non-specific binding (Mortillo & Wassarman, 1991). The study found
very low levels of fetuin binding sites on the plasma membrane of acrosome intact,
capacitated sperm and slightly higher levels on the acrosomal membranes of reacted

55
sperm using colloidal gold labelling and electron microscopy. Also, capacitated
mouse sperm were observed to have little ability to bind fetuin-coated beads
(Vazquez et al., 1989). These results further support the proposal that when sperm
are able to release from the tubal reservoir they have reduced fetuin binding affinity.
The differences observed in fetuin/gold labelling between blots of fresh and
hyperactivated sperm extracts suggest candidates for the fetuin binding component
involved in adherence in the isthmus. The bands that appear to be lost or sharply
reduced prior to hyperactivation, one high molecular weight band >335 kDa, and 32-
33 and 27.5 kDa bands, are obvious choices for further characterization. Also, the
potential change in the 49-50 kDa band is interesting. Further characterization and
antibodies directed against each of these fetuin binding glycoproteins would be useful
for establishing the identity of the component involved in sperm/epithelial binding.
Similarities to several previously described glycoproteins can be noted. A 50
kDa extrinsic sialoglycoprotein that seems to be localized over the acrosomal region
of cauda epididymal rat sperm has been described (Rifkin & Olson, 1985). Other rat
sperm glycoproteins at 32 and 33 kDa have been identified (Hall & Killian, 1989) and
a minor component of the sialylglycoconjugates released during human sperm
capacitation appeared at 32 kDa (Focarelli et al., 1990). Additionally, a hamster
sperm glycoprotein of epididymal origin that may be localized over the acrosome
appears to migrate around 26 kDa, and a 26 kDa wheat germ agglutinin-binding
protein was detected in mouse caudal epididymal fluid (Rankin et al., 1989).

56
Considering the current information, a possible model for the maintenance of
the isthmic reservoir involves the binding of non-capacitated, non-hyperactivated
sperm to the tubal mucosa by a sialoglycoprotein receptor that serves as a sialic acid
lectin. Subsequent loss or modification of this receptor during the course of
capacitation, potentially by desialylation, other enzymatic processing, or by absorption
onto carrier molecules in the fluid, may decrease the affinity of the sperm for the
mucosa and enable sperm to release. Hyperactivation may also contribute by
providing extra force for pulling away. The factors responsible for the binding of
non-capacitated sperm in the ampulla must also be changed during capacitation so
that the sperm arriving there are not retained.
Conclusions
The binding of hamster sperm to the tubal mucosa has been characterized
based on the ability of the sialylated glycoprotein fetuin and sialic acid to inhibit the
binding specifically in the isthmus. Non-capacitated, non-hyperactivated sperm
appear to have a sialylated component on their surface that acts as a lectin-like
receptor for sialic acid containing molecules. This mechanism is proposed to
maintain the adherence of sperm in the isthmic reservoir until the time of fertilization
approaches. The fetuin binding characteristics of the sperm appear to change
coincident with hyperactivation, the stage at which sperm can detach from the isthmic
mucosa. The area over the acrosome by which sperm adhere to the mucosa shows
a sharp reduction in fetuin binding, indicating a loss of the component involved in

57
adhesion. These results agree with previous findings about the ability of
hyperactivated and capacitated sperm to release from the mucosa, a reduction in
sialylation as part of the capacitation sequence, and the inability of capacitated sperm
to bind fetuin. However, this is the first time that these phenomena have been
related to a function, specifically the regulation of sperm passage from the isthmic
reservoir. Western blots labelled with fetuin and sialic acid-recognizing lectins
identified proteins at several molecular weights that show changes in labelling
between fresh and hyperactivated sperm extracts. These proteins are good
candidates for further study as the fetuin binding component responsible for sperm
adherence in the isthmic reservoir.

CHAPTER 3
SPERM MOTILITY PATTERNS IN THE UTERINE TUBE
Introduction
The types of cellular interactions involving sperm discussed in the previous
chapter are a part of the complement of regulatory mechanisms that many cell types
contain. The ability to interact with biomolecules in a specific manner and modify
these interactions is a foundation of modem cell biology. Beyond these typical
mechanisms, sperm have the unique characteristic, among the cells of complex
organisms, of being capable of relatively rapid, independent movement. Motility
provides alternative ways for sperm to interact with their environment. The
propulsive force generated by the sperm tail may affect the ability of sperm to release
from the epithelium and pass through the tubal environment. Also, the dynamic
nature of motility suggests that different patterns may be expressed in a given
environment and this then allows the assessment of functional advantages associated
with particular motility patterns. In this light, sperm motility and its modification
becomes a potential regulatory feature of sperm transport through the uterine tube.
The aim of this study was to relate the progress of sperm in the uterine tube to active
motility patterns and describe some factors that affect these patterns.
58

59
Investigations of sperm motility and progress through the uterine tube have
relied on serial sectioning (Yanagimachi & Chang, 1963; Olds, 1970; Smith et al.,
1987), flushes with oil and physiological medium (Cooper et al., 1979; Overstreet et
al., 1980; Suarez et al., 1983; Smith & Yanagimachi, 1990), blocking passage through
the uterine tube at various times (Hunter, 1984), and in situ observations in animals
with small, translucent uterine tubes (Katz & Yanagimachi, 1980; Suarez, 1987; Smith
& Yanagimachi, 1991). Serial sectioning allows accurate localization of sperm and,
coupled with timed sampling, can provide information about distribution changes.
A disadvantage is that the relative importance of sperm motility and the factors that
affect it cannot be determined. Tubal flushes provide more information about the
condition of the sperm since their motility, or lack of it, and capacitation status can
be characterized. However, location in the uterine tube cannot be accurately
described and motility must be analyzed and interpreted out of physiological context.
Studies on the effects of tubal ligation at various positions and times relative to
ovulation or fertilization offer an advantage in that the actual fertilizing population
of sperm can be localized. Again, however, the motility and capacitation status of
individual sperm cannot be assessed. Observing sperm within the uterine tube has
the advantage of allowing investigators to localize sperm at different times and also
to observe the motility patterns actually used in the uterine tube.
In this study, a previously described in situ preparation (Suarez, 1987) for
observing mouse sperm within the uterine tube has been modified to allow longer
contiguous segments of uterine tube and, thus, longer segments of sperm transport

60
to be observed. This modification lets us follow individual sperm longer and observe
dynamic aspects of sperm motility. These observations contribute to our
understanding of two specific phenomena: the establishment and release of sperm
from isthmic reservoirs, and the function of hyperactivated motility.
Hyperactivation is a vigorous, erratic motility pattern assumed by some sperm
in the uterine tube (Katz & Yanagimachi, 1980; Suarez, 1987). It is characterized by
sharply curved, asymmetric tail beats and frequent changes of direction (Katz et al.,
1989). Katz and coworkers (1989) have reviewed several proposed functions for
hyperactivation in the confined spaces and varied substances of the uterine tube
including an increased ability for sperm to free themselves from the tubal wall, an
increased ability to penetrate viscous or viscoelastic fluids such as the egg vestments,
and an increased probability for escaping from between epithelial folds. The possible
advantage of hyperactivation for sperm release from the tubal wall could result from
increased forces and/or torques generated in various directions. Similarly, these
increases may help sperm penetrate the cumulus matrix and zona pellucida
surrounding the egg (Katz & Yanagimachi, 1981; Katz et al., 1989). Recent evidence
for such an advantage comes from experimental comparisons of hyperactivated and
non-hyperactivated hamster sperm penetrating artificial viscous media (Suarez et. al.,
1991a; Suarez & Dai, 1992).
To address release from the isthmic reservoir and the use of hyperactivated
motility, sperm were observed in the uterine tube and the pattern of progress for
individual sperm was characterized. The effects of ovulatory status, local

61
environment in the uterine tube and time since insemination on flagellar bending and
sperm sticking to the tubal mucosa were analyzed.
Materials and Methods
Medium ingredients were purchased from Sigma Chemical Co. (St. Louis,
MO). The medium consisted of Earles Balanced Salts supplemented with 2.2 g/1
sodium bicarbonate, 0.06 g/1 penicillin, and 0.06 g/1 streptomycin. The final pH was
adjusted to 7.6 and the medium was sterilized by filtration through 0.22 /xm filters
(Millipore Products Div., Bedford, MA).
Outbred ICR strain mice from Harlan Sprague Dawley (Indianapolis, IN) were
kept under a 14:10 hour light cycle with lights on from 0700 h to 2100 h. A delayed
mating protocol (Braden & Austin, 1954) was used to reduce the time between
insemination and sperm arrival at the site of fertilization. This allowed us to observe
a larger portion of the period between insemination and fertilization. Virgin females,
8-16 weeks old, were placed with retired breeder males on the morning of estrus and
allowed to mate. After ejaculation was observed (Wimer & Fuller, 1966), sperm
were allowed to ascend through the tract for 1,1.5, 2, or 3 hours. The females were
killed by C02 inhalation and ovulatory status was determined by observing the
ovaries, ovarian bursa, and uterine tubes. Based on preliminary observations, the
ovulation period for delayed mated mice of this strain extended up to approximately
4 hours after lights on (1100). To observe mating, the lights had to be on, and by
starting matings between 0730-0830 and sampling between 0900-1130, mice could be

62
obtained shortly before and shortly after ovulation at all of the desired sperm
incubation time points.
Uterine tubes were removed by clamping across the uterine horn at the level
of the intramural uterotubal junction with a pair of forceps, cutting the horn just
caudal to the forceps, and lifting the uterine tube and ovary up so that the ovarian
mesenteries could be cut. Uterine tubes and ovaries were placed in a petri dish and
kept moist with medium that had been prewarmed to 37 C and equilibrated under
5% C02. The uterine tubes were uncoiled by cutting the mesosalpinx while handling
only the tip of the uterine horn and the ovary. The ovarian bursa was cut away once
the ampulla was uncoiled. The tip of the uterine horn was cut off at the end of the
dissection leaving a straightened uterine tube from the extramural uterotubal junction
to the infundibulum. Once straightened, the uterine tubes were placed on a
microscope slide with a small drop of medium and covered with a coverslip supported
by silicone grease (modified from Suarez and Osman (1987). They were stored in
a 37C, 5% C02 incubator when not being observed.
The uterine tube preparations were observed, beginning about 15 minutes
after killing the mouse, through a 30x Hoffman Modulation Contrast objective
(Modulation Optics, Greenvale, NY) on a Zeiss Axiovert inverted microscope with
a heated stage (Carl Zeiss, Inc., Thornbrook, NY). A xenon stroboscopic light
source (Model 10030, Chadwick Helmuth Co., Inc., El Monte, CA) was used to
reduce exposure of sperm to light and provide crisp images in individual video
frames. Using a Dage CCD 72 solid-state camera (Dage MTI, Inc., Michigan City,

63
IN), sperm movement within the uterine tubes was recorded at 30 frames/second,
along with time-date information to 0.01 second (Model VTG 33, For-A Co., Ltd.,
Newton, MA), on a Panasonic AG-7300 SuperVHS video cassette recorder
(Panasonic Industrial Co., Secaucus, NJ). Recording both uterine tubes was
completed in approximately 30 minutes. Preliminary experiments indicated that
extending the observation period of an uterine tube up to an hour or more was
associated with vigorous, uncoordinated contractions of the tissue, and the lumen
would begin to fill with refractile spheres, presumably released by the epithelium.
Similar stress responses were found if the uterine tube was roughly handled during
straightening or compressed too much under the coverslip. Data was not collected
from preparations that contained refractile spheres or exhibited these contractions.
From the videotapes, observed on a Panasonic WV-5410 monitor, sperm were
counted and categorized for region of the uterine tube they were found in (isthmus
or ampulla), location (in the lumen or between mucosal folds), and attachment status
(stuck or free). The regions were easily distinguished by their morphology (Suarez,
1987). The location was scored as "lumen" if the sperm was swimming in the central
luminal space or stuck to the epithelium where it was in direct contact with the
central luminal contents. Location was scored as "fold" if the sperm was out of
contact with the central lumen, either in an isthmic pocket, or an ampullar
longitudinal fold. The attachment status was recorded based on the first 5 seconds
that each sperm was visible. If a sperm swam freely during any portion of this period
it was scored "free." It was scored "stuck" if it appeared to remain associated with

64
the epithelium throughout the first 5 seconds. Sperm in all treatment groups were
scored by the same criteria. For qualitative observations, some sperm were observed
for up to 20 min. The proportion of free sperm in each region was calculated for
each experiment by dividing the number of free sperm by the total number of sperm
observed.
The flagellar curvature ratio (FCR) was measured as an indicator of
hyperactivation for those sperm in which, within a single video frame, the entire
principal bend could be found in focus at the maximally bent part of the beat (Suarez
et al., 1983). The restrictive criteria for choosing sperm from which to measure FCR
were required to avoid parallax errors and other image artifacts. The number of
sperm for which FCR was measured was approximately 15% of the counted sperm,
and these appeared to be representative based on observations of the sperm at
normal video speed. FCR is calculated as the straight-line distance from the head-
midpiece junction to the first inflection point of the principal bend, divided by the
curved path distance between these two points (Figure 3-1). As the principal bend
becomes flatter, the ratio approaches one; conversely, the ratio becomes smaller as
the bend becomes more curved. FCR was measured from the stopped video frames
using a Graf/Bar sonic digitizer (Science Accessories Corp., Stanford, CT) connected
to an Apple Macintosh 512K (Apple Computer, Inc., Cupertino, CA) running a
BASIC program written by W. Gottlieb and R.P. DeMott. Each measurement was
made three times and the mean was recorded for each sperm.

65
41 = FCR
AB
Figure 3-1. The method for calculating flagellar curvature ratio (FCR) is shown.
The tail was traced with the digitizer pen to calculate curved-path
distance as well as straight-line distance from the ends of the trace.
(Used with permission from DeMott & Suarez, 1992).
All statistical tests were carried out with StatView 512+ (Brainpower, Inc.,
Calabasas, CA). Proportional measurement data (proportion of free sperm and
FCR) were transformed by taking the arcsine of the square root of the proportion
before testing by ANOVA (Sokal & Rohlf, 1989). One-factor and multi-factor
ANOVA were performed and the a priori determined significance level was p <.
0.05. Values reported are nontransformed.

66
Results
Qualitative Observations
Using the straightened uterine tube preparation for observing sperm motility,
we noted certain typical patterns of movement. There appeared to be two distinct
populations of sperm, one that showed a very regular, moderately bent tail beat and
another more dynamic population defined by regular moderate tail beats
interspersed with groups of erratic, highly curved beats characteristic of
hyperactivation. The erratic beats were of increased amplitude, varying frequency,
and were propagated in randomly changing planes. The dynamic group, those sperm
which varied their beat pattern, represented a small proportion of the total,
approximately 20%. Sperm of both types were typically found in the same area, but
occasionally an isthmic fold would contain a large number of regular sperm and no
dynamic sperm.
Sperm in the dynamic group appeared to be the ones capable of swimming
freely and making progress along the uterine tube. Regularly beating sperm were
observed for up to 20 minutes and none were ever seen to break free from the
epithelium. The typical pattern of motility for the dynamic sperm involved a period
of regular beating ranging from 10 seconds to approximately 1 minute followed by a
series of erratic, high curvature beats during which the sperm might successfully break
free. This series typically lasted 5-10 seconds regardless of whether the sperm broke
free. Some sperm that did not break free were seen to repeat the cycle. There were
occasional sperm, approximately 1%, that continually beat erratically, but they did not

67
seem able to release from the epithelium any more frequently than those that used
the erratic pattern intermittently. Sperm were seen to move in a similar fashion in
both the isthmus and ampulla. There appeared to be relatively more dynamic sperm
in the ampulla; specifically, there were no large clusters of regularly beating attached
sperm.
Those sperm which released from the epithelium and swam freely did so for
a maximum of approximately 5 seconds. Within this time they would again stick to
the epithelium (Figure 3-2). During free swimming, sperm used both erratic, highly
curved beats, and regular, moderately curved beats. This cycle was seen to repeat
up to four times. The direction that a sperm swam after release appeared to be
random. Sometimes the sperm would reattach the first time it contacted the
epithelium, and sometimes it would bounce off, frequently making a direction change,
and continue swimming freely. Sperm swam across isthmic pockets only to stick to
the other side, others swam out of pockets and across the lumen to stick again, and
some swam out of one pocket and into an adjacent one without entering the main
area of the lumen.
In the mouse tubal isthmus, the predominant folding feature of the mucosa is
separate, relatively narrow-mouthed, occasionally branching pockets oriented
transverse to the central lumen. In the ampulla, there are predominantly longitudinal
folds that provide lengthy channels up the uterine tube lateral to the central lumen.
Sperm were more scattered and appeared to be less sheltered from the luminal
contents and flow in the ampullar folds than in the isthmic ones. For example, in

68

releases
t
I

sticks
releases
a
contact
,t ,
0 J
| '
1 2
I '
3 4 I 5
6 I
free
free
sticks
0


Time (sec)
Figure 3-2. Illustration of a typical pattern and timecourse for sperm progress in
the uterine tube. Made from composite tracings of a single sperm
moving within an isthmic pocket. Dotted line indicates the sperm path.
(Used with permission from DeMott & Suarez, 1992).
contracting preparations, the isthmic sperm in the pockets seemed unaffected by the
currents that interfered with the beat patterns of sperm in the central lumen. In the
ampulla, however, all sperm seemed to be subject to reorientation as the current
changed with contractions. It should be noted that the contractions in the ampulla
appeared less vigorous than the isthmic ones.

69
Quantitative Comparisons
A total of 1296 sperm from 5 males randomly mated in 19 experiments were counted
and categorized. The transformed proportions of free sperm were used to test for
an effect of the following factors: region, ovulation status, and time in the tract. Two
uterine tubes were discounted because unusually low numbers of sperm were
counted, ten in one, one in the other. Sperm stuck in the cumulus mass were not
included in this analysis.
There was a highly significant effect of region on the proportion of free-
swimming sperm by ANOVA (Table 3-1) with the proportion of free sperm in the
ampulla more than twice that in the isthmus. There was also a significantly higher
proportion of free sperm in post-ovulatory compared to pre-ovulatory uterine tubes.
ANOVA showed no significant difference related to the factor of time in the tract
and no interactions between factors. However, since many more sperm were found
in the isthmus than ampulla, most of the effect observed for ovulation status resulted
from isthmic sperm.
FCR values were measured for 174 sperm and analyzed by ANOVA for
differences related to region of the uterine tube, attachment status, location relative
to the lumen, ovulation state, and time in tract. All single factors and two-factor
combinations were tested. Attachment status, region, and location relative to the
lumen were all significant factors in two-way interactions (Table 3-2). A posteriori
three-factor ANOVA for the combinations of these factors showed no significant

70
Table 3-1.
Analysis of factors affecting proportion of sperm swimming freely
within the uterine tube; calculated from videotapes of in situ
preparations from naturally mated mice.
Factor
Treatment
Mean
SEM
N
Region*
Isthmus
11.8%
1.0%
1196
Ampulla
26.3%
0.8%
100
Ovulation
Pre-
10.6%
1.6%
553
Status**
Post-
16.2%
2.0%
743
Time
1 h
18.7%
3.5%
311
in
Tract
1.5 h
12.8%
3.0%
412
2 h
12.6%
2.3%
198
3 h
11.6%
1.8%
375
Treatments of this factor significant at p < 0.01
Treatments of this factor significant at p < 0.05
(Used with permission from DeMott & Suarez, 1992).

71
Table 3-2. Attachment status interactions for FCR with tubal region and sperm
location analyzed by two-factor ANOVA showing mean SEM for
each treatment combination.
Attachment Status2
Free Stuck
Region1,2
Isthmus
0.656 0.034
n=33
0.777 + 0.013
n=85
Ampulla
0.786 0.029
n=28
0.795 0.034
n=28
Location1
Lumen
0.750 0.030
n=31
0.759 0.024
n=44
Fold
0.680 0.036
n=30
0.796 0.014
n=69
1. Significant interaction (p < 0.05) with Attachment Status.
2. Differences between treatments (ie., free vs. stuck, and isthmus vs.
ampulla) are significant (p< 0.05). Overall treatment means and SEM
are
Free: 0.716 + 0.024
Isthmus: 0.743 0.014
Lumen: 0.756 + 0.019
Stuck: 0.782 0.013
Ampulla: 0.790 0.022
Fold: 0.761 + 0.015
(Used with permission from DeMott & Suarez, 1992).

72
three-way interactions. Ovulation status and time in tract were not involved in
interactions, nor were they significant as individual factors.
While free sperm were significantly more sharply bent than stuck sperm, an
interaction between attachment status and tubal region was detected, since the
difference between free and stuck sperm was much greater in the isthmus than the
ampulla. Also, while isthmic sperm were significantly more sharply bent than
ampullar sperm, the difference between the isthmus and ampulla was greater for free
sperm than stuck sperm. An interaction of attachment status with location relative
to the lumen was also detected, because free sperm in the folds were more sharply
bent than free sperm in the lumen; however, stuck sperm in the folds were LESS
sharply bent than stuck sperm in the lumen. Also, free sperm were more sharply
bent in both the folds and lumen than stuck sperm, but the difference between free
and stuck sperm was much greater in the folds than in the lumen.
Discussion
The general pattern of mouse sperm progress up the uterine tube appears to
involve periods of free swimming between periods of attachment to the tubal
epithelium. This pattern was observed in both the isthmus and the ampulla but our
analysis of the proportion of free swimming sperm showed that sperm were more
likely to be free in the ampulla. While earlier in situ studies in the mouse (Suarez,
1987) reported the apparently decreased stickiness of ampullar sperm, this is the first

73
report of a progressive pattern involving sequential binding and release from the
ampullar wall.
Analysis of the proportion of free-swimming sperm at different times relative
to ovulation supports the idea that sperm are better able to stay free from the
epithelium as the time of fertilization approaches. This is one mechanism for shifting
sperm from the isthmic reservoir to the site of fertilization. Previous research
indicates that while sperm numbers in the ampulla are low, near the time of
fertilization the number of sperm reaching this region increases (Katz &
Yanagimachi, 1980; Cummins & Yanagimachi, 1982; Smith et al., 1987; Suarez, 1987).
These sperm appear to be moving up from the isthmic reservoir (Suarez, 1987). The
increased ability to remain free near the time of fertilization may reflect changes in
the sperm such as capacitation or hyperactivation, or changes in the affinity of the
tubal epithelium for sperm. There is evidence that different levels of estradiol may
affect the sticking of boar sperm to pig tubal explants (Suarez et al., 1991b;
Raychoudhury & Suarez, 1991).
FCR has proven to be a consistent indicator of hyperactivation in vitro (Suarez
et al., 1983; Suarez & Osman, 1987; Suarez et al., 1992) and has the particular
advantage of being relatively insensitive to small errors in determining the inflection
point. For measurements within the mouse uterine tube, decreases in FCR have
been a reliable indicator of hyperactivation (Suarez & Osman, 1987) and there are
distinct advantages over other indicators. Since the sperm are confined in a narrow,
convoluted lumen, especially in the isthmus, it is impossible to measure their

74
undisturbed swimming trajectory or their velocity over a long enough distance to
correlate it with in vitro measurements. In our laboratory, beat amplitude and wave
length are more sensitive to measurement errors than FCR and this becomes
significant when taking measurements from images of sperm within the uterine tube.
Based on the results of the FCR measurements, it appears that sperm which
have broken free are more likely to be hyperactivated than stuck sperm. Also, based
on these observations and previous descriptions of the hyperactivated pattern for
mouse sperm (Fraser, 1977; Olds-Clarke, 1986; Suarez & Osman, 1987), the pool of
erratically beating, dynamic sperm that intermittently released and reattached to the
tubal wall appeared to be hyperactivated. All free sperm were a subset of this pool.
The erratic beating pattern generated a sharply curved, hyperactivated beat while
sperm were attached to the tubal wall and all sperm releases were preceded by
erratic beating. This supports the possibility of hyperactivation functioning to give
sperm an advantage in remaining free from the epithelium and progressing up to the
site of fertilization.
Hyperactivation may provide the direction changes needed to navigate the
convoluted path up the uterine tube and provide the forces or torques that allow
sperm to bounce off rather than stick when they encounter the wall. Since not all of
the erratically beating sperm were free, hyperactivation is not always sufficient for
sperm release. Capacitation may also be required. Capacitation may decrease the
stickiness of the sperm head, making it easier to release from the epithelium when
they are stuck. In the hamster uterine tube, sperm have also been noted breaking

75
free and reattaching to the epithelium (Smith et al., 1987). A recent study indicates
that hamster sperm incubated in capacitating conditions before being introduced into
the uterine tube lose their stickiness and may encounter the epithelium and not
attach (Smith & Yanagimachi, 1991).
Based on the two-factor ANOVA results, it appears that differences in FCR
should not be interpreted simply in terms of the effects of each factor we examined.
Although the factors tubal region, sperm attachment status, and sperm location
relative to the lumen all significantly affected FCR, their major importance lies in
how they vary in combination, rather than in how each singly affects FCR. The
statistical interactions imply that there are physiological interactions whereby the tract
features act on the sperms potential to affect the movement pattern and progress.
The large difference between free and stuck sperm in the isthmus compared to the
ampulla may reflect a reduction in the proportion of the relatively high FCR,
regularly beating sperm that were not seen to move up the uterine tube. In the
isthmus there may be more distinct populations, one containing the sperm that have
begun erratic, low FCR beating, and are able to break free and move along the
uterine tube, and another one containing the stuck, regularly beating, high FCR
sperm that do not move up to the ampulla. The population in the ampulla may be
more homogeneous, containing only the sperm able to use erratic beating and move
up from the isthmus. Separating the sperm in this manner need not imply sperm
selection based on the ability to become hyperactivated. Interactions between the
relative timing of hyperactivation and capacitation could produce only a small

76
population ready and able to move up the uterine tube at any time. Other sperm
may become hyperactivated, but if this is not properly coupled to capacitation, and/or
other unidentified factors, it may not help them reach the site of fertilization at the
appropriate time.
The increase in FCR for both free and stuck sperm in the ampulla is harder
to understand. If hyperactivation is indeed a factor helping sperm release from
repeated bindings to the epithelium, we might assume that the most hyperactivated
sperm should reach the ampulla. There may be some difference in the fluid
surrounding the sperm that dampens their tail beat in the ampulla. The secretions
of the ampullar epithelium may be more viscous or viscoelastic than those of the
isthmus, and this may be further enhanced after ovulation by cumulus secretions and
follicular fluid. Since there is a bursa surrounding the mouse ovary (Hunter, 1988),
the products of ovulation cannot escape into the peritoneal cavity and must instead
enter the ampulla. There is experimental evidence indicating that increases in the
viscosity of the medium can dampen sperm tail beat amplitude and frequency
(Drobnis et al., 1988b; Rikmenspoel, 1984; Suarez et al., 1991a; Suarez & Dai, 1992).
Also, hyperactivated hamster sperm appear to have an advantage over non-
hyperactivated in penetrating fluids of elevated viscosity and their tail beats do flatten
out (develop higher FCR) in more viscous fluid (Suarez et al., 1991a). Measurements
of the hydrodynamic properties of the fluids in which sperm swim in vivo would be
valuable for our understanding of the modulation of sperm motility and its possible
functions. Besides the regional differences in fluid composition that may affect

77
sperm, there may be important effects of the local environment (lumen vs. fold).
Being out of the main fluid flow through the lumen, free sperm in the folds may be
able to generate more sharply curved tail beats observed than sperm in the lumen.
The drag on the sperm would be reduced in the relatively quiet eddy of the folds.
Conclusions
In summary, most mouse sperm are stuck to the tubal wall most of the time.
As the time for fertilization approaches, however, there is an increase in the
proportion of free-swimming sperm. Progress up the uterine tube to the site of
fertilization occurs intermittently as sperm repeatedly release from the wall, swim a
short distance and reattach. This may be the means of release from the isthmic
reservoir. Those that do break free appear to be using hyperactivated tail beats to
give them an advantage for releasing from the epithelium. The coincidental
occurrence of hyperactivation and release from the tubal wall resulting in progress
up the uterine tube suggests an association between the phenomena. Nevertheless,
the two-factor interactions affecting FCR serve to remind us that the onset of
hyperactivation and any advantages that it provides are likely to be regulated by a
number of physiological factors.

CHAPTER 4
INVESTIGATION OF CHANGING SPERM ANTIGENICITY
Introduction
There is a wide variety of evidence showing that there are modifications of the
mammalian sperm cell surface between the time sperm leave epididymal storage and
the time they reach the site of fertilization (Yanagimachi, 1988). Antigenic
rearrangements (ORand, 1977; Cowan et al., 1986; Okabe et al., 1986; Jones et al.,
1990), glycosylation pattern changes (Kinsey & Koehler, 1978; Ahuja, 1984; Cross &
Overstreet, 1987), and alterations of the lipid structure of the sperm membranes
(Scott et al., 1967; Davis, 1981; Go & Wolf, 1985; Ehrenwald et al., 1988) have all
been described. Some of these changes have been associated with capacitation and
the acrosome reaction (see Chapter 1), but there has been no specific investigation
of a potential role in controlling sperm transport through the uterine tube. Changes
appearing during the course of capacitation may not relate directly to preparing the
cell for the acrosome reaction but may instead play a functional role in regulating
sperm transport.
There are two observable sperm phenomena that relate to transport through
the uterine tube, release from the isthmic reservoir and the onset of hyperactivated
motility. Sperm release from the epithelium appears to be dependent on changes in
78

79
the sperm rather than the uterine tube (Smith & Yanagimachi, 1990; Smith &
Yanagimachi, 1991) and may involve the modification of a sperm glycoprotein (see
Chapter 2). Hyperactivated motility is involved in ascent from the isthmic reservoir
to the site of fertilization (Suarez, 1987, see Chapter 3) and thus physiological
changes affecting hyperactivation necessarily affect sperm transport as well.
It was hypothesized that some of the sperm changes associated with adherence
in the tubal reservoir and the onset of hyperactivated motility would be reflected as
antigenic changes on the surface of the sperm that could be detected based on
differential labelling with monoclonal antibodies directed against sperm components.
It was assumed that the changes occurring on sperm capacitated in vitro would be
similar, allowing sperm that had reached various stages of the
capacitation/hyperactivation sequence to be used to analyze antibody binding. The
antibodies identified would then serve as both markers for changes in epitope
associated with observable sperm changes and a means to characterize the antigen.
Monoclonal antibodies have previously been used to describe antigens that change
during capacitation and the acrosome reaction (ORand, 1977; Cowan et al., 1986;
Okabe et al., 1986; Saxena et al., 1986; Topfer-Petersen et al., 1990; Berger, 1990;
Jones et al., 1990).
Under capacitating conditions in vitro, hamster sperm pass through four stages
before undergoing the acrosome reaction. Sequentially, sperm begin in the fresh or
activated stage, then agglutinate head-to-head, then separate and hyperactivate
coincidentally, and finally complete capacitation (Suarez, 1988). Monoclonal

80
antibodies were generated against the complement of epididymal hamster sperm
antigens and screened for changes in activity associated with agglutination,
hyperactivation, capacitation and the acrosome reaction. Identifying antigenicity
changes associated with the agglutinated state and with hyperactivation were of
primary interest as these molecules may be associated with the ability of sperm to
bind and release the tubal epithelium. Three antibodies were identified that
recognized epitopes changing during agglutination or hyperactivation.
Antibody labelling patterns were identified by indirect immunofluorescent
staining of fixed sperm and were subsequently analyzed by immunogold labelling of
unfixed sperm to better localize the antigenicity and by immunoblotting to provide
a preliminary characterization of the antigens.
Materials and Methods
Production of Monoclonal Antibodies
Immunization. All chemicals were obtained from Sigma Chemical Co. (St.
Louis, MO) unless otherwise noted. Caudae epididymides from mature golden Syrian
hamsters were punctured and the sperm in epididymal fluid were diluted in an equal
volume of RIBFs adjuvant (RIBI Immunochemical Research, Inc., Hamilton, MT)
at 37C. This mixture was used immediately for subcutaneous injections into BALB/c
mice. The mice received three injections of 7-9 X 107 sperm over two months and
were tested for titer following each injection. They received a final boost of 1.2 X
108 sperm six days before fusion.

81
Fusion. The fusion was carried out in a standard manner using polyethylene
glycol and selection on HAT medium (Simrell & Klein, 1979; Kao & Klein, 1986).
Splenocytes were fused with SP2/0 cells. Fused cells were plated at a concentration
to yield 3-10 different populations in each culture well. Supernatants from the
resulting hybridoma cultures were screened for anti-sperm activity using an indirect
immunofluorescence assay.
Screening. To identify antibodies against epitopes that were modified as
sperm went through their functional changes, we compared indirect
immunofluorescent staining patterns of fresh, agglutinated, hyperactivated, and
capacitated sperm. Supernatants that showed differential patterns of labelling
between sperm stages were selected for cloning.
In order to maximize the time between hyperactivation and the completion of
capacitation, the concentrations of motility stimulators, BSA and metabolic substrates
in the medium were optimized to provide rapid hyperactivation and slow capacitation
based on previous reports of the effects of modifications of hamster capacitation
medium (Dravland & Meizel, 1981; Bavister, 1989). The composition of modified
hamster capacitation medium is shown in Table 4-1. Using this medium, populations
of hyperactivated sperm could be obtained 60-90 minutes before capacitation was
complete (assay described below).
Caudal epididymal sperm were allowed to disperse into 1 ml of modified
hamster capacitation medium for 5-10 minutes at 37C, then the top 2/3 of the sperm
suspension was collected. Sperm numbers were adjusted to 3 X 106 sperm/ml and

82
Table 4-1. Modified hamster capacitation medium formulation
Component
Cone.
Component
Cone.
NaCl
102 mM
Glucose
3.24 mM
KC1
5 mM
Pyruvate
3.00 mM
CaCl2
2.4 mM
Hypotaurine
100/M
MgCl2
0.5 mM
Penacillamine
20 fiM
NaH2P04
0.35 mM
Epinephrine
1 fiM
NaHC03
25 mM
Penicillin
0.06 g/l
HEPES
25 mM
Fraction V BSA
3 mg/ml
Note: Formulated from Dravland and Meizel, 1981; Suarez, 1988; and
Bavister, 1989.
Table 4-2. Criteria for staging sperm
Stage
Criteria
Fresh
Caudal epididymal sperm after 5-10 min.
dispersal in medium.
Agglutinated
70% of sperm are agglutinated head to
head. Typically after 1 h.
Hyperactivated
70% of sperm exhibit vigorous, circular
hyperactivated pattern. Typically after 3-
3.5 h.
Capacitated
Acrosome reactions induced in >50% of
motile sperm treated with 0.3 mg/ml
lysophosphatidylcholine for 5-10 min.
Typically 4.25-5 h.

83
samples were used immediately as fresh sperm. The remaining suspensions were
incubated at 37C, checked periodically, and used for the agglutinated, hyperactivated
and capacitated stages when they met the criteria in Table 4-2. Capacitation was
assayed by the ability of lysophosphatidylcholine (LPC) to induce acrosome reactions
in motile sperm (Llanos & Meizel, 1983). Samples were tested periodically beginning
at the onset of hyperactivation. When sperm were collected for the hyperactivated
stage, the percent responding to LPC was 10% or less.
Sperm were dried down on multi-well microscope slides (Cel-line Assoc.
Newfield, NJ), rinsed with phosphate-buffered saline (PBS), fixed for 5-10 min with
7% formaldehyde and rinsed again with PBS. All treatments were carried out at
room temperature. The sperm were blocked with 3% bovine serum albumin
(Calbiochem, La Jolla, CA) in PBS (PBS/BSA) for 30 minutes, then individual wells
for each sperm stage were incubated with PBS/BSA alone, neat hybridoma culture
supernatant, hybridoma growth media, 1:1000 normal mouse serum (non-immune)
in PBS/BSA, and immune mouse serum, diluted similarly for 1.5-1.75 hours. The
wells were rinsed with PBS/BSA, incubated with TRITC-labelled secondary antibody
(F(ab)2 of rabbit anti-mouse IgG-F(ab)2) (Jackson Immunoresearch Labs, Inc., West
Grove, PA) for 20-25 minutes, and rinsed again. Slides were examined using
combined Nomarski/epifluorescence optics, 100W mercury lamp, and a rhodamine
(TRITC) filter set (Carl Zeiss, Inc., Thornbrook, NY). The staining pattern for each
supernatant with each type of sperm was recorded.

84
Cloning. Cultures producing supernatants that yielded interesting staining
patterns were cloned (Simrell & Klein, 1979; Kao & Klein, 1986). The resulting
cultures were screened, subcloned, and rescreened at least three times to obtain
active monoclonal cultures. The monoclonal cell lines were expanded, isotyped and
stored.
Production of Ascites. Cultures from three cell lines producing antibodies
that recognized epitopes changing prior to hyperactivation, HL 772, HL 778, and HL
787, were used to induce ascites fluid production in BALB/c mice. RIBIs adjuvant
was used. Ascites fluid was cleared by centrifugation at 960 G for 10 minutes, then
stored frozen in aliquots at -20C.
Immunogold Labelling of Sperm
Sperm from the four stages were obtained as described above. Medium was
removed following centrifugation, 5 minutes, 960 G, and the sperm were washed in
PBS/BSA, 5 minutes, 960 G. Labelling was carried out based on the protocol of
Jones and coworkers (1990). The sperm were then suspended in either 1:1 culture
supernatant or 1:10 ascites diluted with PBS/BSA and incubated for 35 minutes at
room temperature. The supernatant was removed as above and the sperm were
washed by centrifugation twice with PBS/0.1% BSA. They were then incubated for
30 minutes with 5 nm colloidal gold-labelled secondary antibody, anti-mouse IgG, IgM
(E-Y Labs, San Mateo CA) diluted 1:30 with PBS/0.1% BSA. Supernatant was
removed and the sperm were washed with PBS by centrifugation 3X.

85
For analysis by light microscopy, the labelled, washed sperm suspensions were
dried down on multi-well slides, rinsed 3X with PBS and fixed for 5 minutes with
2.5% phosphate buffered glutaraldehyde. They were rinsed with PBS and distilled
water, 3X each, then the label was enhanced with silver as described in Chapter 1.
Slides were analyzed following a 9-11 minute enhancement.
Immunoblotting of Sperm Proteins
1-D blots. Fresh and hyperactivated sperm extracts were prepared as
described in Chapter 2. Electrophoresis and semi-dry transfer to Immobilon-P
membranes were carried out under the conditions previously described as well (see
Chapter 1). Blots were blocked for 2 hours with PBS/BSA then washed 4X with PBS.
Strips were incubated overnight at 4C with 1:50 ascites, 1:1000 normal mouse serum,
1:1000 immune mouse serum, and PBS/BSA alone. All dilutions were made with
PBS/BSA. Following 4 washes with PBS, the blot strips were incubated for 2 hours
at room temperature with 1:1000 horseradish peroxidase-labelled F(ab)2 of rabbit
anti-mouse IgG-F(ab)2) (Jackson Immunoresearch Labs, Inc., West Grove, PA).
This product contains the same antibodies previously used for immunofluorescence
conjugated to a different label. After 4 washes with PBS, the peroxidase reaction was
developed as described in Chapter 2.
2-D blots. Fresh sperm extract prepared as described above containing 650
¡ig of total protein was diluted to 5 ml and dialyzed against three 2-liter changes of
distilled water over 24 hours. Dialysis tubing (Spectra/Por, mW cutoff 3500,

86
Spectrum Medical Industries Inc., Los Angeles, CA) had been boiled in the presence
of EDTA to reduce protein binding (Harlow & Lane, 1988). The sample was then
lyophilized. Two-dimensional polyacrylamide gel electrophoresis was carried out
according to Roberts and coworkers (1984). For isoelectric focussing (IEF), the pH
gradient ranged from 4-10. The IEF gels were then loaded on 10% polyacrylamide
slab gels and separated as previously described (see Chapter 2). Semi-dry transfer
to Immobilon-P membranes and blocking was carried out as described above. Blots
were then incubated overnight at 4C with either 1:2000 normal mouse serum or
1:100 HL 787 ascites diluted with PBS/BSA. The blots were washed, incubated with
1:1000 peroxidase-labelled secondary antibody for 1 hour, and developed as described
above.
Results
From one fusion yielding 316 cultures, we identified 6 antibodies that showed
stage specific differences in immunofluorescent labelling of hamster sperm. Four of
these were cloned; three that showed labelling changes associated with agglutination
or hyperactivation that could be relevant to sperm transport in the uterine tube, and
one that was an exceptionally strong acrosome-specific antibody that is useful for
determining acrosome reaction status.
Staining patterns show that the epitope associated with antibody HL 787
changes at agglutination while those associated with HL 772 and HL 778 change at
hyperactivation (Figures 4-1, 4-2). Between the fresh stage and agglutination, the

87
labelling with HL 787, an IgM class antibody, decreased over the head and midpiece.
Labelling at hyperactivation and capacitation remained low. Labelling with HL 772,
an IgG1? decreased over the head and midpiece between the agglutinated and
hyperactivated stages. Labelling of fresh sperm was similar to agglutinated, and
capacitated continued to show the decrease seen at hyperactivation. HL 778, another
IgM, showed the opposite reaction at hyperactivation, increasing in intensity and
redistributing from a spotty pattern with isolated areas at the rostral tip of the head
and the neck region to an evenly distributed pattern over the entire head. Again, the
fresh stage resembled agglutination and the capacitated stage resembled
hyperactivation. HL 784, an IgG1 relevant to the acrosome reaction rather than
sperm transport, stained the heads of the acrosome-reacted, capacitated sperm
brightly and evenly. It did not stain the heads of non-capacitated sperm that had lost
their acrosomes, presumably precociously or during processing, however. It also
brightly stained the acrosome itself whether intact or in fragments (data not shown).
This antibody would be useful for studies of the acrosome reaction but was not
further characterized here.
Controls labelled with normal serum, hybridoma culture medium and
secondary antibody alone characteristically showed very low signal. The detectable
fluorescence was predominately on the midpiece. The positive control, serum
obtained at the time of fusion, however, strongly labelled the entire sperm cell.
The silver-enhanced colloidal gold labelling assessed by light microscopy indicated a
positive reaction for HL 772, 778, and 787 on the surface of the sperm. Patterns,

88
Figure 4-1. Stage-related Changes Observed in Indirect Immunofluorescent
Labelling with Monoclonal Antibodies. Column A shows labelling of
- fresh sperm, column B shows Agglutinated labelling for HL 787,
Hyperactivated labelling for HL 772, 778.

89
HL 787 Fresh Agglutinated
Figure 4-2. Epifluorescent Images of Monoclonal Antibody Binding Patterns.
Fixed, dried down sperm treated with culture supernatant.
Photographed at 100X with Zeiss Plan-neofluar objective on Ektar
1000 film. Exposures 75-90 sec. Images for a given antibody exposed
the same duration and all negatives printed similarly.

90
however, were very patchy and variable. The stage specific characteristics identified
by immunofluorescence could be seen, but not consistently.
Immunostaining of blots allowed the identification of potential antigens for two
of the antibodies (Figure 4-3). HL 787 clearly identified a specific antigen that
migrated at 22-23 kDa under reducing conditions. This band was seen in blots
stained with immune serum but not with normal serum or secondary antibody alone.
It also showed decreased labelling on blots of hyperactivated sperm extracts
compared to fresh. A strong band at approximately 42-44 kDa was also detected
with the other monoclonals and controls, though it was clearly enhanced by binding
to the monoclonals. This band did not label with immune serum. A weak band at
46 kDa was also unique to the fresh blots stained with HL 787. Staining with HL 772
seemed to identify 2 separate bands at approximately 44 and 42 kDa, and the lower
molecular weight band appeared to be distinct from the band labelling in the other
treatments. It also showed decreased labelling of hyperactivated sperm extracts.
Labelling with HL778 did not suggest any strong candidates for the antigen associated
with this antibody. Results were similar on blots from 2 separate sets of extracts
representing eight hamsters.
Analysis of HL 787 labelling by 2-D PAGE again detected a protein of
approximately 22 kDa (Figure 4-4). The protein is acidic with an approximate pi
range of 5.1-5.2. This spot was again absent in the control blot stained with normal
mouse serum.

91
A B C D
| ( 44 k
- 22k
f h f h f h
Figure 4-3. 1-D immunoblots probed with stage-specific monoclonal antibodies. A)
control normal mouse serum, B) HL 772 lanes of fresh (left) and
hyperactivated sperm extract, C) HL 778 lanes of fresh (left) and
hyperactivated sperm extract, D) HL 787 lanes of fresh (left) and
hyperactivated sperm extract.

92
mW
Figure 4-4. 2-D Immunoblot of fresh sperm extract labelled with HL 787.

93
Discussion
The decline in immunofluorescent staining seen at agglutination with antibody
HL 787 implies that the epitope is lost, either through modification of the antigen or
release from the cell surface. The results on both 1-D and 2-D immunoblots suggest
that this antibody recognizes a 22-23 kDa acidic antigen. Also, the blots of
hyperactivated and fresh sperm extracts showed changes in staining parallel to the
immunofluorescent results. Gels had been loaded with similar amounts of total
protein. Taken together, these results imply that before agglutination occurs, a sperm
protein migrating at approximately 22 kDa in reducing conditions is altered or lost
from the sperm surface. The loss and alteration of sperm components during in vitro
culture has been well documented and proposed to be a part of the capacitation
process (reviewed by Yanagimachi, 1988).
Modification of this antigen prior to agglutination in vitro implies that there
may be an analogous early change after sperm have been deposited in the female
tract. The timing, approximately 1 hour in culture, suggests that this modification
could be associated with the binding of sperm to the isthmic epithelium in the tubal
reservoir, occurring during the first 2 hours after insemination in the hamster (Smith
& Yanagimachi, 1991). It is possible that this change plays a functional role in the
ability of sperm to bind as well. The antibody will be a useful tool for investigating
these possibilities.
Similar hamster sperm antigens have previously been described. Antiserum
produced against sperm extracts recognized a 22.5 kDa protein (reducing conditions)

94
from caudal epididymal sperm and also from fluid of the distal cauda (Robataille et
al., 1991). A monoclonal antibody was produced which recognized a 23 kDa protein,
pi 3.8-4.2, from extracted cauda sperm separated under reducing conditions (Ellis et
al., 1985). However, immunofluorescent labelling with this antibody was restricted
to the sperm tail. Neither of these studies included potential changes associated with
in vitro capacitation.
The epitopes for HL 772 and HL 778 appear to be changed by the time sperm
reach the hyperactivated stage. A loss or modification of epitope seems to occur for
antibody HL 772. Since the staining with HL 778 increases in intensity and becomes
evenly distributed at hyperactivation, the epitope may be unmasked during culture.
A change in glycosylation could expose an epitope that had been previously
inaccessible to the antibody. Increased labelling of sperm surface components during
in vitro capacitation has been previously described (Okabe et al., 1986; Berger, 1990;
Fusi et al., 1992). Additionally, the antigen containing the recognized epitope may
be redistributed during capacitation. There are also documented examples of this
phenomenon (Saxena et al., 1986; Topfer-Petersen et al., 1990; Jones et al., 1990).
Since the antigenic changes for these two antibodies are coincident with
hyperactivation and this may be the first stage at which sperm can break free from
the isthmic reservoir (see Chapter 3), they may play a role in either the switch of
motility patterns or release from the mucosa.
The antigen associated with HL 772 may be approximately 42 kDa under
reducing conditions. This band seems to be reduced in the hyperactivated sperm

95
extract. However, the presence of a large nearby band, apparently non-specific,
complicates interpretation. Though the separate bands appeared in two different
samples, both bands fall within the range of molecular weights calculated for the non
specific band in other treatments. Alternative methods for characterizing the antigen
might provide clearer information.
HL 778 did not appear to react with a specific antigen on Western blots. The
epitope recognized by this antibody may have been destroyed during the extraction,
denaturation and reduction of disulfide bonds. Although screening was based on
fixed sperm to specifically identify antibodies that would be effective for denatured
protein antigens, the changes in proteins during PAGE could be sufficiently different
to disrupt the affinity of the antibody. Also, the protein may not have been
transferred successfully to the Immobilon-P membrane. Alternatively, the antibody
may recognize a non-protein component that was retained on fixed sperm but lost
during electrophoresis and blotting.
The inconsistent results obtained by labelling unfixed sperm are most likely
due to the multivalent nature of the antibodies used for these experiments. The
primary antibodies and the colloidal gold-labelled secondary antibody were all intact
immunoglobin molecules with multiple binding sites that could cause artificial shifts
of antigens in unfixed cell membranes.
The antigen associated with antibody HL 784 was not characterized, since its
relevance to sperm transport appeared minimal. However, this antibody could prove
very useful for studies involving the acrosome reaction. First, it provides a good

96
marker for the acrosome, or acrosomal fragments. Second, the selective labelling
over the heads of capacitated, acrosome-reacted sperm, but not sperm that have
prematurely lost their acrosomes suggests that the epitope is not present on the inner
acrosomal membrane until the sperm have completed, or nearly completed the
capacitation process. Alternatively, however, premature loss of the acrosome in non-
capacitated sperm may just disrupt the membrane in a manner that destroys the
epitope.
Conclusions
We have used a stage-specific screening technique to identify monoclonal
antibodies against antigens that are somehow changed during in vitro capacitation of
hamster sperm. An antigenic change associated with agglutination involving a 22-23
kDa acidic protein was found. Two such changes associated with hyperactivation, one
possibly involving a 42 kDa protein, were also identified. These changes are
coincident with phenomena related to sperm transport in the uterine tube, specifically
the formation and subsequent release from the isthmic reservoir. Also, an antibody
that may be useful for examining the acrosome reaction was produced.
At the least these antibodies serve as markers for the respective changes with
which they are associated. In addition, their antigens are potentially involved in the
functional changes the sperm goes through as it prepares for fertilization. The
antibodies will serve as powerful tools for investigating this potential.

CHAPTER 5
DEMONSTRATION OF TUBAL MUCUS IN THE PATH
OF SPERM TRANSPORT
Introduction
The tubal environment may present another level of regulation for sperm
passage to the site of fertilization. When sperm are swimming freely within the
uterine tube they encounter morphological barriers such as mucosal folds and pockets
(Nilsson & Reinius, 1969; Suarez, 1987; Smith & Yanagimachi, 1990) that may make
progress toward the egg difficult (Suarez & DeMott, 1991). They may also encounter
mucous epithelial secretions that may affect their movement (Hunter et al., 1987;
Suarez et al., 1991b). There are also cycles of epithelial secretion correlated with the
estrous cycle that may be relevant to the ability of sperm and eggs to move along the
uterine tube (Jansen, 1980; Schulte et al., 1985).
One of the potential functions of hyperactivated motility is proposed to be the
generation of additional force and torque which provides an advantage for passage
through viscoelastic materials (Katz et al., 1989). Analyses of sperm motility and
passage through the cumulus matrix and zona pellucida (Drobnis et al., 1988b;
Drobnis et al., 1988a), and also through artificial viscous and viscoelastic media
(Suarez et al., 1991a; Suarez fe Dai, 1992) indicate that there are changes in flagellar
97

98
beat shape associated with exposure to these media and that hyperactivated sperm
appear to have a functional advantage.
Prior to encountering the cumulus or zona, however, sperm may be influenced
by increased viscosity or viscoelasticity in the tubal lumen due to the presence of
mucous secretions. Sperm observed within excised uterine tubes (see Chapters 2, 3)
seemed to be moving in a thick, viscous fluid. Hyperactivated sperm in the tubal
ampulla had relatively reduced flagellar bending compared to those in the isthmus
(see Chapter 2). While regional differences in the fluid were not visible, the changed
flagellar beating patterns resembled those observed in experiments where sperm were
added to viscous solutions (Suarez et al., 1991a). It was hypothesized for the present
study that sperm pass through mucous secretions of the uterine tube and that there
may be regional differences in the secretions that affect sperm motility.
The presence of cervical mucus, its regulation and effects on sperm transport
have been extensively studied (Iacobelli et al., 1971; Barratt & Cooke, 1991; Katz et
al., 1989). The penetrability of cervical mucus appears to differ at various stages of
the cycle (Barratt & Cooke, 1991; Katz et al., 1989) and exposure to cervical mucus
appears to alter motility parameters (Zhu et al., 1992). The distribution of the mucus
within the cervix may also affect sperm transport by creating certain paths of reduced
resistance (Mullins & Saacke, 1989).
Though tubal mucus has been described (Jansen, 1978,1980; Jansen & Bajpai,
1982; Hunter et al., 1987), the distribution of the mucus in the lumen and its relation
to the path of sperm ascent remains unclear. There is little information as to

99
whether the mucus is an effective trap or a selective barrier for sperm. Conventional
studies of the uterine tube using immersion or perfusion fixation followed by
dehydration and preparation for light and scanning electron microscopy (Jansen,
1978,1980; Jansen & Bajpai, 1982; Schulte et al., 1985; Hunter et al., 1987; Smith et
al., 1987; Boyle et al., 1987; Abe & Oikawa, 1992) suggest the presence of a mucous
substance in the tubal lumen. The most detailed descriptions of tubal mucus come
from scanning electron microscopy studies, and in these preparations, the mucus
typically appears as a lattice overlaying the mucosa (Jansen, 1978, 1980; Hunter et
al., 1987).
The presence of mucus that completely fills the tubal lumen is suggested but,
due to the difficulties in preserving and retaining luminal mucopolysaccharides during
processing, has not been clearly shown. It was hypothesized for the present study
that, when hydrated, this mucus fills the luminal spaces, especially within the mucosal
folds and pockets, and potentially affects sperm movement.
A technique for preserving tubal mucus and maintaining its position in the
lumen during processing was developed to test this hypothesis. From frozen sections
of mouse uterine tubes treated with a combination of celloidin coating and
cetylpyridinium chloride (CPC) post-fixation, a homogeneous substance that fills many
of the luminal spaces was detected. There appeared to be regional variations and
sperm were seen within the material.

100
Materials and Methods
Design and Sampling
Mice were obtained and housed as described in Chapter 4. Uterine tubes
from three females in each of six different groups (proestrus, early estrus-mated and
unmated, late estrus-mated and unmated, and diestrus) were examined. Mice were
considered in proestrus on the fourth day following a standing estrus. This was
backed up by visual staging using the criteria of Champlin and coworkers (1973).
Mice were considered early estrus as soon as they allowed a male to mount, usually
around 3-5 hours after lights out, approximately 12:00 to 2:00 A.M. For mated mice,
the female was left with the male until one hour after ejaculation had been observed,
then the uterine tubes were removed. Uterine tubes were taken from unmated mice
60-90 minutes after the male was allowed to mount. For late estrus, females were
placed with males shortly after lights on, around 7:30 A.M. Again, mated animals
were taken 1 hour after ejaculation, unmated were taken 60-90 minutes after allowing
mounts. Diestrus was considered the third day following a standing estrus. This was
also backed up by visual staging (Champlin et al., 1973).
The uterine tubes were processed to preserve the morphology of the mucus,
and sections were examined for differences in the staining and morphology of the
tissue and the luminal contents that related to tubal region, stage of cycle, or mating
status. The location of sperm in the mated animals was also noted.

101
Processing
Mice were killed by cervical dislocation and the uterine tubes were dissected
free by cutting across the uterotubal junction and through the ovarian bursa. Most
of the uterine tubes were placed in a copper mesh basket and snap frozen for 30
seconds in isopentane (2-methylbutane, Fisher Chemical Co., Fair Lawn, NJ) cooled
with liquid nitrogen. The basket was placed on dry ice. A drop of O.C.T. frozen
sectioning medium (Ames, Elkhart, IN) was added to a Beem tube (Polysciences,
Warrington, PA) that had been cooled on dry ice. As the O.C.T. hardened, the
uterine tube was introduced, followed by O.C.T. to fill the tube. The tube was
immediately placed in isopentane for an additional 30-45 sec. Then, the frozen
O.C.T. block was pressed out of the tube, wrapped in Parafilm and stored at -80C.
For comparison, one uterine tube from each group was fixed for 24 hours at
4C in 1% glutaraldehyde/4% formaldehyde in phosphate buffer (0.176 M) with 0.5%
CPC (Sigma, St. Louis, MO) added to help preserve the negatively charged mucus.
The fixed uterine tubes were then processed for routine paraffin histology.
Sectioning
The blocks containing frozen tissue were cut at -18C to -21C in a Reichert
Histostat cryostat (Scientific Instruments, Buffalo, NY) and 10-12 gm sections were
mounted on acid-washed slides. Some slides had been coated with 0.05% Elmers
Glue-all (Borden Industries, Columbus, OH) in distilled water (M. Baccala, personal
communication). The sections were dried for 15-45 min on a 40C warming plate.

102
They were then coated with celloidin according to a method used to stabilize colonic
mucus (Szentkuti & Eggers, 1990). Slides were incubated for 3 minutes with 0.2%
celloidin (Fisher Chemical, Co.) dissolved in a 1:1 mixture of absolute ethanol and
ethyl ether. They were dried for 5-10 minutes and hardened in 70% ethanol for 3
minutes. To further stabilize the mucus, the slides were post-fixed for 10 minutes in
10% phosphate buffered formalin containing 0.5% CPC. For controls, alternate
serial slides were prepared omitting one or both of the celloidin and CPC steps.
Staining
Chemicals and stains were from Sigma except where noted. Slides were
primarily stained with the periodic acid Schiff reaction (PAS), as described below,
because it was compatible with the processing technique and identifies a broad array
of carbohydrates. They were treated with 1% periodic acid in distilled water (5
minutes), rinsed and treated with Schiffs base (12-15 minutes, cold Schiff method
(Lillie & Fullmar, 1976)). Slides were rinsed and counterstained with Harris
hematoxylin (Luna, 1968) for 20-40 seconds. They were rinsed in distilled water, acid
alcohol and ammonia water and dehydrated through a graded ethanol series, cleared
in xylene and mounted with Eukitt (Calibrated Instruments, Hawthorne, NY).
Some of the frozen sections were stained with 1% alcian blue, pH 1 & 2.5,
combined alcian blue (pH 2.5)/PAS and 1% toluidine blue. The paraffin sections
were stained with hematoxylin/eosin (H&E), PAS, alcian blue (pH 2.5) and combined
alcian blue/PAS. These slides were dehydrated, cleared and mounted as above.

103
Results
The combination of celloidin coating and CPC post-fixation for frozen sections
resulted in the detection of a fairly homogeneous substance completely filling many
of the luminal spaces between mucosal folds and in the central lumen (Figure 5-la).
The substance showed a light, or diffuse, PAS reaction. This luminal substance was
found throughout the segments of the uterine tube, but between the mucosal folds
there were scattered regions that did not stain for this material.
In frozen sections where both treatments had been left out, the luminal
contents were relatively sparse and the remnants appeared as strands (Figure 5-lb).
These strands stained darkly with PAS, alcian blue and toluidine blue. In combined
alcian blue/PAS, much of the material preferentially stained with alcian blue.
Immersion fixation in aldehydes, even with CPC present, followed by standard
paraffin processing led to the loss of almost all the luminal contents though the tissue
morphology was well preserved and the cilia are visible (Figure 5-lc).
Slight regional and cyclical differences in the luminal contents were observed.
The contents in the ampulla of the early and late estrous animals appeared as an
even pool of light pink material (Figure 5-2a). The isthmic loops, compared on the
same section, showed a more granular material that appeared to take up more of the
hematoxylin counterstain, since there was a gray cast to the material (Figure 5-2b).
The latter staining characteristics were found in all regions at proestrus and diestrus.
At proestrus, however, there was a band of strong PAS staining in the apical portions
of the isthmic epithelial cells.

104
In the uterotubal junction and isthmus of the mated mice, sperm were seen
within pools of the luminal material (Figure 5-3). Sperm in the isthmic pockets and
the central lumen were seen completely surrounded by the material. There appeared
to be more sperm in the uterine tubes of the animals mated late in estrus compared
to the early estrous matings even though the same amount of time had passed since
ejaculation. No sperm were found in the ampulla.
Discussion
Treatment of frozen sections with both celloidin and CPC results in the
retention of the luminal contents as a relatively homogeneous material that is clearly
in the path of sperm moving along the uterine tube. The staining characteristics of
the luminal material implies the presence of some sulfated acidic muco
polysaccharides. It stained with PAS, Alcian blue, pH 2.5 and preferentially with
Alcian blue when treated with combined PAS/Alcian blue. This material thus
appears to be mucous secretions maintained in position and structure as in vivo. The
diffuse nature of the PAS reactivity in the coated, post-fixed sections suggests that
when the material is hydrated, the mucopolysaccharides are somewhat dispersed.
Additional characterization of the material is required to determine the proportions
of the various mucous elements. This method has since been used to examine
uterine tubes from rabbits, cows, and pigs and yields similar preservation but species-
dependent staining differences (Suarez, S.S. and DeMott, K.R., unpublished results).

105
Figure 5-1. Differential Preservation of Luminal Contents by Processing Method.
(A) Photomicrograph of oviductal cryosection coated with celloidin and
post-fixed with CPC, stained with PAS, hematoxylin. (B) Untreated
oviductal cryosection, stained with alcian blue/PAS, hematoxylin. (C)
Oviductal paraffin section fixed with CPC and stained with H&E. m =
mucus, magnification 40X.

106
Figure 5-2. Regional Differences in Luminal Staining Characteristics.
Photomicrographs of the oviductal ampulla (A) and isthmus (B) from
the same cryosection from a late estrous mouse. Section treated with
combined celloidin/CPC, stained with PAS and hematoxylin.
Magnification 40X.

107
Figure 5-3. Sperm Observed Within Luminal Mucus. Photomicrographs of the
uterotubal junction (A) and isthmus (B) containing sperm in the
luminal mucus, s = sperm heads, magnification 40X.

108
The sperm seem to be forced to penetrate through this mucus as they move
along the uterine tube, not just trapped in it when they are on or near the epithelial
surface (Hunter et al., 1987; Suarez et al., 1991b). These results support the
observations of sperm appearing to be enmeshed in a viscous material while
swimming in the uterine tube. Also, since an advantage for hyperactivated sperm in
penetrating viscous and viscoelastic media has been demonstrated (Suarez et al.,
1991a; Suarez & Dai, 1992), hyperactivation may be functional not only for
detachment from the isthmic reservoir and egg penetration, but may also be
advantageous for movement in luminal mucus.
The strand-like morphology of the luminal contents in untreated frozen
sections is reminiscent of the results seen with standard scanning electron microscopy.
Previously published micrographs show luminal secretions as a layer adherent to the
epithelium, or as strands or a honeycomb (Jansen & Bajpai, 1982; Jansen, 1980;
Jansen, 1978). While some of these sections leave the impression that there is a
matrix which may fill the lumen, they do not clearly illustrate the extent or nature of
the luminal contents.
Sections prepared using either celloidin coating or CPC fixation alone yielded
intermediate results. In both cases, preservation was better than untreated sections,
but stronger staining characteristics and a more homogeneous appearance of the
contents was achieved by combining the treatments. Celloidin coating has recently
been used successfully to stabilize colonic mucus (Szentkuti et al., 1992; Szentkuti &
Eggers, 1990). The use of polycationic additives such as CPC to aldehyde fixatives

109
has been widely used to enhance mucopolysaccharide preservation (Landemore et
al., 1993; Sames & Hoyer, 1992; Cook, 1977). The combination of these treatments
appeared beneficial for preserving the contents of the tubal lumen, however, there
are limitations on the stains that can be subsequently used.
While the celloidin coating and CPC fixation were optimal for morphological
preservation, the non-treated control sections were important for verifying staining
characteristics. Celloidin, as previously reported (Cook, 1977), and CPC both
interfered with Alcian blue staining. Toluidine blue also gave anomalous results when
used with CPC or celloidin, though the stain was still picked up to some degree.
Therefore, care must be taken in drawing conclusions about staining characteristics
without using appropriate controls.
The regional variations in staining, and cyclical changes in the ampulla, difficult
to see clearly in black-and-white photographs, may indicate differences in the
composition or amount of the mucus present. Since the uterine tube was sectioned
as a coiled mass, loops from all the regions were visible in the same section and
stained together. This provided an added measure of confidence that the differences
observed were not due to variations in staining times or handling.
However, the conclusions that can be drawn from this histological
characterization are limited. The secretory cells in the mouse uterine tube are
located predominantly in the isthmus (Nilsson & Reinius, 1969) and relatively little
cyclical variation of cell ultrastructure, and presumably secretory activity, has been
observed in the murine isthmus (Nilsson & Reinius, 1969; Abe & Oikawa, 1993).

110
The production of the mucous portion of the isthmic secretions may remain fairly
constant during the cycle. Cyclical variation in the ampullar cells have been noted
(Nilsson & Reinius, 1969; Abe & Oikawa, 1993), but the connection between such
changes and mucus staining is not well established. The cryosectioning method
described here is excellent for demonstrating the presence of luminal mucous
secretions, however, the nature of this material is probably better studied using other
methods.
Finding more sperm in the uterotubal junction and isthmus of late mated mice
compared to those mated early in estrus was in agreement with results previously
reported (Smith et al., 1987). It appears that the tubal reservoir may be populated
more rapidly when ovulation is imminent. Also, sampling only 1 hour after mating,
sperm were not expected to have reached the ampulla yet. For the live sperm that
will potentially go on to fertilize, passage to the ampulla appears to require somewhat
more than 1 hour (Suarez, 1987; Smith et al., 1987).
Several of the technical details noted while experimenting with the
preservation technique deserve mention. Rapid freezing was important for the
morphological preservation of both mucus and tissue. Placing the uterine tube
directly in isopentane gave noticeably better results than either freezing it in a tube
of O.C.T. or using liquid nitrogen alone. Both of these alternatives led to more
freeze artifact due, presumably, to slower freezing of the tissue. Also, in sections that
had not been treated with celloidin and CPC, the mucus strands did not remain
consistently apposed to the epithelium after the slower freezing protocols. The

Ill
frozen uterine tubes were embedded in O.C.T. blocks because their size made them
difficult to handle and section without a supporting matrix. For any celloidin-treated
sections, clean, uncoated slides were sufficient for adhesion; however a strong subbing
solution such as Elmers glue was required when CPC was used alone for post
fixation. Background staining of the subbing solution was minimal.
Conclusions
Mucous secretions do fill much of the luminal space in the mouse uterine tube,
requiring that sperm pass through this viscous media as they move through the
uterine tube to reach the site of fertilization. Thus, tubal mucus is not only likely to
affect sperm when they are bound to the epithelium, but also as they attempt to
move up the uterine tube. Having to swim through this type of medium may provide
a selective advantage for hyperactivated sperm. Also, there appear to be regional
and cyclical changes in the concentration or composition of the mucus. The regional
changes could cause differential affects on sperm motility, however, other methods
are needed to assess the nature of these secretory differences.
Coating rapidly frozen sections with celloidin and post-fixing them with CPC
is an effective method for retaining the morphology and position of the tubal luminal
contents. This improved method for examination will allow us to better study the
role and nature of tubal mucus and to develop a more detailed picture of the its
interactions with sperm.

CHAPTER 6
SUMMARY
Controlled sperm transport through the uterine tube is an important
phenomenon for providing successful fertilization. The results described here pertain
to four aspects of the control of sperm transport to the site of fertilization. The
nature of the binding of sperm to the tubal mucosa in the isthmic reservoir was
investigated using inhibition experiments and showing the association of the inhibitor
to sperm. Potential differences in transport based on the motility pattern of the
sperm were analyzed by qualitatively and quantitatively characterizing the movement
of sperm within the uterine tube. Markers or possibly controlling features for the
physiological changes of the sperm coincident with binding and release from the
isthmic reservoir and their changing motility were developed by generating stage-
specific monoclonal antibodies. Finally, a specialized histological method was
developed to investigate the nature and localization of tubal mucus that may
potentially regulate the type of sperm able to progress in the tubal environment.
The interaction between sperm and mucosa in the isthmus was shown to
involve a lectin-like association between a sialylated glycoprotein on the sperm and
a sialic acid-bearing ligand. The binding capabilities of sperm for both the
competetive inhibitor and the mucosa itself seem to change as the sperm goes
through the capacitation sequence, specifically, by the time they switch to
112

113
hyperactivated motility. This suggests that adherence in the isthmic reservoir may be
influenced by biochemical changes of the sperm surface that aid in their release when
they are becoming ready to fertilize an egg.
The antibodies generated provide a useful tool for identifying the cellular
components changing on the sperm as it undergoes functional changes in the uterine
tube. Antigens associated with sperm agglutination and hyperactivation were
detected, and, while none of them appear to be the binding site detected in the
inhibition experiments, their characterization could provide clues about the signalling
or metabolic processes involved in the changing binding and motility characteristics.
The switch to hyperactivated motility is closely tied to release from the tubal
reservoir and the additional force and torque presumably provided by this vigorous
pattern may help sperm break free. The relative contributions of reduced affinity for
the mucosa and the changed motility pattern remain unclear, but there is clearly
room for both to be operating. Since all free sperm appeared to be hyperactivated
but not all hyperactivated sperm were able to break free, the reduction in binding
affinity may be a permissive regulatory component. Such a fail-safe system, requiring
that sperm can both develop hyperactivated motility and undergo the membrane
transformations that occur during capacitation, could play an important selective role
in providing sperm in the appropriate condition to reach the site of fertilization.
Given the observations that so few sperm actually have a chance to participate in
fertilization, there are clear benefits to a stringent selection process helping ensure
that those few are appropriate for fertilization.

114
Finally, the demonstration of tubal mucus occupying the entire luminal cross-
section provides yet another potentially selective regulatory feature. Sperm have little
chance of avoiding this material, whether close to the mucosa or in the central lumen
and, there do not appear to be paths of decreased resistance. Edge effects and other
hydrodynamic properties may still be relevant to the ability of sperm to navigate
some areas more easily than others, however. With the only path appearing to be
straight through the tubal mucus, the differential abilities of sperm using different
motility patterns to penetrate becomes important for determining the population of
sperm that ascend the uterine tube. Hyperactivated sperm have an advantage in
material of this nature; therefore, the mucus potentially serves as another selective
barrier to sperm.
The picture arising out of this investigation of the transport of sperm through
the uterine tube shows the sperm as a dynamic cell, responding to a variety of factors
that may help optimize fertilization. Their viability may be supported by adherence
in the isthmic reservoir, until the sperm reaches a certain point in the physiological
process preparing it for fertilization. This appears to be somehow tied to ovulation
and the impending time of fertilization. Following desialylation or some other
modification reducing the affinity of the receptor for mucosal binding, the recently
acquired hyperactivated motility pattern can release sperm from the reservoir and
enable them to penetrate the luminal mucous secretions. Sperm that undergo the
surface modification and manage to release without hyperactivating are constrained

115
by the mucous secretions. In the end, only sperm that have capacitated at the
appropriate rate and hyperactivated make it to the eggs.

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137
Zhu JJ, Barratt CLR & Cooke ID (1992) Effect of human cervical mucus on human
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BIOGRAPHICAL SKETCH
Robert P. DeMott was born in Bellrose, New York, and raised throughout the
U.S.A. His biological curiosity was piqued at high school in Valley Stream, New
York, primarily by a biology teacher/soccer coach whose lectures bore remarkable
resemblance to the nonstop, all-over-the-field action of a soccer match. Riding this
wave to a biology major at Williams College, where he received a B.A. in 1987, his
interests in developmental and reproductive biology coalesced into ambition partly
thanks to the timely birth of a sister. After working as a technical editor for two
years he entered the Ph.D. program in animal biology in the College of Veterinary
Medicine at the University of Florida to study reproductive physiology. Apparently
still motivated by practical exposure to reproductive biology, he completed graduate
school as the father of three. Immediately following graduate school, he will shift
fields, much to the relief of his wife, to gain experience in toxicology, then aim for a
research career combining this specialty with reproductive physiology.
138

I certify that I have read this study and that in my opinion it conforms to
acceptable standards of scholarly presentation and is fully adequate, in scope and
quality, as a dissertation for the degree of Doctor of Philosophy.
4
V
Susan Suarez, Chair
Associate Professor of
Veterinary Medicine
I certify that I have read this study and that in my opinion it conforms to
acceptable standards of scholarly presentation and is fully adequate, in scope and
quality, as a dissertation for the degree of Doctor of Philosophy^_
William Buhi
Associate Professor of Biochemistry
and Molecular Biology
I certify that I have read this study and that in my opinion it conforms to
acceptable standards of scholarly presentation and is fully adequate, in scope and
quality, as a dissertation for the degree of Doctor of Philosophy.
yi\ Osf,\rJ^
Maarten Drost
Professor of Veterinary Medicine
I certify that I have read this study and that in my opinion it conforms to
acceptable standards of scholarly presentation and is fully adequate, in scope and
quality, as a dissertation for the degree of Doctor of Philosophy.
Professor of Zoology

I certify that I have read this study and that in my opinion it conforms to
acceptable standards of scholarly presentation and is fully adequate, in scope and
quality, as a dissertation for the degree of Doctor of Philosophy.
Paul Klein
Professor of Pathology
and Laboratory Medicine
This dissertation was submitted to the Graduate Faculty of the College of
Veterinary Medicine and to the Graduate School and was accepted as partial
fulfillment of the requirements for the degree of Doctor of Philosophy.
December, 1993
4
Dean, College of Veterinary
Medicine
Dean, Graduate School

3 1262 08554 8120



69
Quantitative Comparisons
A total of 1296 sperm from 5 males randomly mated in 19 experiments were counted
and categorized. The transformed proportions of free sperm were used to test for
an effect of the following factors: region, ovulation status, and time in the tract. Two
uterine tubes were discounted because unusually low numbers of sperm were
counted, ten in one, one in the other. Sperm stuck in the cumulus mass were not
included in this analysis.
There was a highly significant effect of region on the proportion of free-
swimming sperm by ANOVA (Table 3-1) with the proportion of free sperm in the
ampulla more than twice that in the isthmus. There was also a significantly higher
proportion of free sperm in post-ovulatory compared to pre-ovulatory uterine tubes.
ANOVA showed no significant difference related to the factor of time in the tract
and no interactions between factors. However, since many more sperm were found
in the isthmus than ampulla, most of the effect observed for ovulation status resulted
from isthmic sperm.
FCR values were measured for 174 sperm and analyzed by ANOVA for
differences related to region of the uterine tube, attachment status, location relative
to the lumen, ovulation state, and time in tract. All single factors and two-factor
combinations were tested. Attachment status, region, and location relative to the
lumen were all significant factors in two-way interactions (Table 3-2). A posteriori
three-factor ANOVA for the combinations of these factors showed no significant


82
Table 4-1. Modified hamster capacitation medium formulation
Component
Cone.
Component
Cone.
NaCl
102 mM
Glucose
3.24 mM
KC1
5 mM
Pyruvate
3.00 mM
CaCl2
2.4 mM
Hypotaurine
100/M
MgCl2
0.5 mM
Penacillamine
20 fiM
NaH2P04
0.35 mM
Epinephrine
1 fiM
NaHC03
25 mM
Penicillin
0.06 g/l
HEPES
25 mM
Fraction V BSA
3 mg/ml
Note: Formulated from Dravland and Meizel, 1981; Suarez, 1988; and
Bavister, 1989.
Table 4-2. Criteria for staging sperm
Stage
Criteria
Fresh
Caudal epididymal sperm after 5-10 min.
dispersal in medium.
Agglutinated
70% of sperm are agglutinated head to
head. Typically after 1 h.
Hyperactivated
70% of sperm exhibit vigorous, circular
hyperactivated pattern. Typically after 3-
3.5 h.
Capacitated
Acrosome reactions induced in >50% of
motile sperm treated with 0.3 mg/ml
lysophosphatidylcholine for 5-10 min.
Typically 4.25-5 h.


Ill
frozen uterine tubes were embedded in O.C.T. blocks because their size made them
difficult to handle and section without a supporting matrix. For any celloidin-treated
sections, clean, uncoated slides were sufficient for adhesion; however a strong subbing
solution such as Elmers glue was required when CPC was used alone for post
fixation. Background staining of the subbing solution was minimal.
Conclusions
Mucous secretions do fill much of the luminal space in the mouse uterine tube,
requiring that sperm pass through this viscous media as they move through the
uterine tube to reach the site of fertilization. Thus, tubal mucus is not only likely to
affect sperm when they are bound to the epithelium, but also as they attempt to
move up the uterine tube. Having to swim through this type of medium may provide
a selective advantage for hyperactivated sperm. Also, there appear to be regional
and cyclical changes in the concentration or composition of the mucus. The regional
changes could cause differential affects on sperm motility, however, other methods
are needed to assess the nature of these secretory differences.
Coating rapidly frozen sections with celloidin and post-fixing them with CPC
is an effective method for retaining the morphology and position of the tubal luminal
contents. This improved method for examination will allow us to better study the
role and nature of tubal mucus and to develop a more detailed picture of the its
interactions with sperm.


129
Oehninger S, Clark GF, Acosta AA & Hodgen GD (1991) Nature of the inhibitory
effect of complex saccharide moieties on the tight binding of human
spermatozoa to the human zona pellucida. Fertility and Sterility, 55, 165-169.
Okabe M, Takada K, Adachi T, Kohama Y & Mimura T (1986) Inconsistent
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Olds PJ (1970) Effect of the T locus on sperm distribution in the house mouse.
Biology of Reproduction, 2, 91-97.
Olds-Clarke P (1986) Motility characteristics of sperm from the uterus and oviducts
of female mice after mating to congenie males differing in sperm transport
and fertility. Biology of Reproduction, 34, 453-467.
Oliphant G (1976) Removal of sperm-bound seminal plasma components as a
prerequisite to induction of the rabbit acrosome reaction. Fertility and Sterility,
27, 28-38.
Oliphant G, Reynolds AB & Thomas TS (1985) Sperm surface components involved
in the control of the acrosome reaction. American Journal of Anatomy, 174,
269-283.
Overstreet JW & Cooper GW (1975) Reduced sperm motility in the isthmus of the
rabbit oviduct. Nature, 258, 718-719.
Overstreet JW, Cooper GW & Katz DF (1978) Sperm transport in the reproductive
tract of the female rabbit: II. The sustained phase of transport. Biology of
Reproduction, 19, 115-132.
Overstreet JW, Katz DF & Johnson LL (1980) Motility of rabbit spermatozoa in the
secretions of the oviduct. Biology of Reproduction, 22, 1083-1088.
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Anatomy, 183, 200-211.
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1-28.


88
Figure 4-1. Stage-related Changes Observed in Indirect Immunofluorescent
Labelling with Monoclonal Antibodies. Column A shows labelling of
- fresh sperm, column B shows Agglutinated labelling for HL 787,
Hyperactivated labelling for HL 772, 778.


3 1262 08554 8120


7
1987). In reptilian sperm-storing species there appear to be some storage regions
with sperm in close association to the epithelium and others where large agglutinated
masses of sperm occupy the lumen of specialized tubules (Gist & Fischer, 1993).
This second type of storage organ is found in fowl (Bakst, 1992).
Several other mechanisms for retaining sperm in the isthmus have been put
forth and they may play an accessory role in some species. Post-coital constriction
of the isthmus may hamper the ability of mouse sperm to move within the uterine
tube (Suarez, 1987). Depressed motility observed in isthmic sperm has also been
postulated to help prevent sperm escaping the reservoir (Overstreet fe Cooper, 1975;
Cooper et al., 1979; Cummins, 1982). Hunter and Nichol (1986) found a slight
temperature gradient in the pig uterine tube and speculated that the cooler
temperatures in the reservoir region may help subdue sperm. Another contributing
factor in the pig uterine tube may be the presence of viscous secretions in the
mucosal crypts that Hunter and coworkers inferred based on the degree of flagellar
bending seen by scanning electron microscopy (SEM) of sperm in the crypts
compared to sperm in the main lumen (Hunter et al., 1987). The presence of a
mucous layer, again detected by SEM, before ovulation but not after in the rabbit
(Jansen, 1978) and human (Jansen, 1980) uterine tube led to the proposal that ciliary
beating, which aids in sperm transport, may be dampened by this mucus until
ovulation has occurred. An actual role for any of these alternative mechanisms in
controlling sperm transport has not been demonstrated. In light of the observations
of tight adherence between sperm and the isthmic mucosa in situ (Suarez, 1987;


LIST OF FIGURES
Figure page
2-1 Photomicrographs of hamster sperm within the
tubal isthmus 36
2-2 Bar graph showing effect of treatment on sperm 43
2-3 Bar graph showing regional differences in inhibition
by fetuin and sialic acid 45
2-4 Bar graph showing that effect of treatment is due
primarily to isthmic inhibition 46
2-5 Photomicrographs of silver-enhanced labelled hamster sperm 48
2-6 PVDF blots probed for carbohydrate binding 50
3-1 The method for calculating flagellar curvature ratio
(FCR) is shown 65
3-2 Illustration of a typical pattern and timecourse for
sperm progress in the uterine tube 68
4-1 Stage-related changes observed in indirect immunofluorescent
labelling with monoclonal antibodies 88
4-2 Epifluorescent images of monoclonal antibody binding patterns 89
vn


carbohydrate interaction between the sperm surface and the tubal wall. A sialic acid
bearing ligand apparently mediates this lectin-like binding. The use of particular
motility patterns in the uterine tube was investigated and the switch to hyperactivated
motility appeared prior to release from the reservoir. Thus, hyperactivation may help
sperm break free from the wall. Sperm modifications coincident with transport were
investigated by developing monoclonal antibodies to sperm epitopes. Antigens that
serve as markers and possibly play a functional role for the transition to
hyperactivation and other stages were described. Finally, a specialized histological
protocol was used to characterize the nature, morphology and location of the luminal
contents which sperm encounter. The presence of mucus in the path of sperm was
demonstrated. Based on these experiments, it appears that there is a specific binding
interaction affecting sperm retention in the reservoir, that motility changes occur
when sperm can release from the reservoir, that changing antigenicity can be used to
detect cellular modifications associated with functional changes, and that the tubal
environment contains material which has the capability to affect sperm passage.
x


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Ahuja KK (1984) Lectin-coated agarose beads in the investigation of sperm
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Austin CR (1952) The capacitation of mammalian sperm. Nature, 170, 326-326.
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Bains HK, Pabst MA & Bawa SR (1993b) Changes in the lectin binding sites on the
testicular, epididymal, vas, and ejaculated spermatozoon surface of dog.
Andrologia, 25, 19-24.
Bakst MR (1992) Observations on the turkey oviductal sperm-storage tubule using
differential interference contrast microscopy. Journal of Reproduction and
Fertility, 95, 877-883.
Barratt CLR & Cooke 10 (1991) Sperm transport in the human female reproductive
tract-a dynamic interaction. International Journal of Andrology, 14, 394-411.
Battalia DE & Yanagimachi R (1979) Enhanced and coordinated movement of the
hamster oviduct during the periovulatory period. Journal of Reproduction and
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116


96
marker for the acrosome, or acrosomal fragments. Second, the selective labelling
over the heads of capacitated, acrosome-reacted sperm, but not sperm that have
prematurely lost their acrosomes suggests that the epitope is not present on the inner
acrosomal membrane until the sperm have completed, or nearly completed the
capacitation process. Alternatively, however, premature loss of the acrosome in non-
capacitated sperm may just disrupt the membrane in a manner that destroys the
epitope.
Conclusions
We have used a stage-specific screening technique to identify monoclonal
antibodies against antigens that are somehow changed during in vitro capacitation of
hamster sperm. An antigenic change associated with agglutination involving a 22-23
kDa acidic protein was found. Two such changes associated with hyperactivation, one
possibly involving a 42 kDa protein, were also identified. These changes are
coincident with phenomena related to sperm transport in the uterine tube, specifically
the formation and subsequent release from the isthmic reservoir. Also, an antibody
that may be useful for examining the acrosome reaction was produced.
At the least these antibodies serve as markers for the respective changes with
which they are associated. In addition, their antigens are potentially involved in the
functional changes the sperm goes through as it prepares for fertilization. The
antibodies will serve as powerful tools for investigating this potential.


BIOGRAPHICAL SKETCH
Robert P. DeMott was born in Bellrose, New York, and raised throughout the
U.S.A. His biological curiosity was piqued at high school in Valley Stream, New
York, primarily by a biology teacher/soccer coach whose lectures bore remarkable
resemblance to the nonstop, all-over-the-field action of a soccer match. Riding this
wave to a biology major at Williams College, where he received a B.A. in 1987, his
interests in developmental and reproductive biology coalesced into ambition partly
thanks to the timely birth of a sister. After working as a technical editor for two
years he entered the Ph.D. program in animal biology in the College of Veterinary
Medicine at the University of Florida to study reproductive physiology. Apparently
still motivated by practical exposure to reproductive biology, he completed graduate
school as the father of three. Immediately following graduate school, he will shift
fields, much to the relief of his wife, to gain experience in toxicology, then aim for a
research career combining this specialty with reproductive physiology.
138


17
egg, coincident with the removal of cholesterol from bovine sperm has also been
demonstrated (Ehrenwald et al., 1988). Besides the change in sterol to phospholipid
ratio, there is also evidence in guinea pig sperm that the relative amounts of different
membrane phospholipids change during capacitation (Stojanoff et al., 1988).
Another occurrence during capacitation is the alteration of the ionic balance
of the sperm cell. Before capacitation, sperm maintain typical ionic gradients relative
to the medium with potassium high inside the cell and sodium low inside the cell.
They also maintain a calcium gradient with lower concentrations inside than outside
(Yanagimachi, 1988). Changes in the calcium gradient may be a part of capacitation.
There is a clear requirement for an extracellular calcium pool to support
hyperactivation and the acrosome reaction (Yanagimachi & Usui, 1974; Yanagimachi,
1982). There is also evidence that an influx of this calcium triggers the acrosome
reaction (Roldan & Harrison, 1990). Fraser (1987; Fraser & McDermott, 1992)
reported that mouse sperm require at least 0.09 mM for capacitation, but markedly
higher fertility results at 1.8 mM. These results fit well with the proposal that there
are two changes in the calcium gradient, the first associated with hyperactivation and
the second, larger influx at the time of the acrosome reaction (Suarez et al., 1993).
Several mechanisms that may contribute to the calcium influx have been
described. The presence of calcium channels has been inferred based on the
sensitivity of the influx to channel blockers (Fraser, 1987). Chou and coworkers
(1989) used a voltage sensitive dye to show that during mouse sperm capacitation
there is a shift in membrane polarity from negative inside to positive inside, primarily


30
hyperactivated (see Chapter 3). These results imply that there is a change in the
affinity for the tubal epithelium coincident with capacitation and the switch to
hyperactivated motility that allows sperm in the appropriate condition for fertilization
to release from the reservoir.
The biochemical nature of sperm adherence in the reservoir and the changes
leading to release have not yet been described, however. To address this question,
the binding of non-capacitated sperm in excised hamster uterine tubes following
treatment with potential inhibitors was analyzed. The binding of sperm in the
isthmus appears to be quite strong, since repeated flushing is required to release the
bound sperm (Smith & Yanagimachi, 1990). Also, various pretreatments of the
uterine tube and enzymatic treatments of tubal explants with adherent sperm were
unsuccessful for preventing sperm binding (Raychoudhury fe Suarez, 1991; T.T.
Smith, personal communication). For this study, a different approach was attempted
where sperm were treated with potential inhibitors prior to exposure to the uterine
tube. Carbohydrate inhibitors in the form of large glycoproteins were chosen due to
the ineffectiveness of proteases (Raychoudhury of carbohydrate interactions for another sperm binding phenomenon, adherence to
the zona pellucida (Wassarman, 1990; Cornwall et al., 1991; Noguchi & Nakano,
1992; Tulsiani et al., 1993).
In the present study, screening resulted in the detection of the glycoprotein
fetuin as an inhibitor of sperm binding. Fetuin, a 43-49 kDa glycoprotein, contains
12-13 O-linked and N-linked carbohydrate chains that end in the N-acetylneuraminic


16
A number of sperm surface changes associated with capacitation have been
described. Following the report of lectin binding characteristics for sperm by
Nicolson and Yanagimachi (1972), a number of surface carbohydrate changes were
identified using lectins. In the hamster, the distribution of binding sites for the lectin
Concanavalin A over the head changes as sperm are capacitated in vitro (Kinsey &
Koehler, 1978). Decapacitation factors which are removed during capacitation have
been described for the rabbit (Reyes et al., 1975) and mouse (Fraser, 1984).
Membrane antigens that are redistributed have been identified for mouse (Okabe et
al., 1986), boar (Saxena et al., 1986; Berger, 1990; Topfer-Petersen et al., 1990) and
rat (Jones et al., 1990) sperm. The redistribution of intramembranous particles has
been demonstrated in the guinea pig (Koehler & Gaddum-Rosse, 1975). The
appearance of certain antigens with capacitation, suggesting that they were masked
or have been modified, has been documented in the rat (Jones et al., 1990) and ram
(Voglmayr fe Sawyer, 1986) and for integrin binding proteins on human sperm (Fusi
et al., 1992).
Another type of change that has been associated with capacitation involves
alteration of the lipid characteristics of sperm membranes. The ratio of cholesterol
to phospholipid, which affects membrane fluidity (Go decrease due to the gradual removal of cholesterol. This increasing fluidity of the
membrane was proposed to enhance capacitation (Davis, 1981). A similar change
in the ratio was found during human sperm capacitation (Hoshi et al., 1990). The
enhancement of capacitation, measured by the ability of the sperm to penetrate the


18
due to potassium ion redistribution, and propose that this polarity shift stimulates
voltage sensitive channels. The ability of Ca-ATPase inhibitors to affect capacitation
(Fraser & McDermott, 1992) and the influx of extracellular calcium (Blackmore,
1993) suggests that this enzyme may also play a role. Decreased Na/K-ATPase
activity observed during guinea pig capacitation (Hang et al., 1990) fits with a model
proposed by Fraser and coworkers (1993) where Na/K-ATPase activity may be
contributing to early mouse capacitation events and help promote increased
intracellular sodium ion levels that activate Na+/H+ exchange, resulting in reduced
intracellular pH and activation of calcium channels.
This model also provides a potential role of the female tract in controlling
capacitation. Differences in the ionic composition of uterine, ampullary, and bursal
sac fluid (Borland et al., 1977) are proposed to allow sperm to undergo the initial
stages of capacitation. However, sperm do not complete the acrosome reaction until
they reach the ampulla and ovulation has occurred, which shifts the extracellular
sodium and potassium levels (Fraser, 1983; Fraser et al., 1993). Killian and
coworkers (1989) analyzed the lipid characteristics of bovine tubal fluid and found
that the cholesterol to phospholipid ratio drops and the prevalence of
lysophospholipids increases around estrus. Both of these conditions are seemingly
favorable for mediating the sperm membrane lipid changes described above.
Contributions of the female tract to capacitation have long been suspected
(Yanagimachi, 1981, 1988; Oliphant et al., 1985). A variety of enzymes,
glycosaminoglycans, and other secretions have been proposed as effectors of


106
Figure 5-2. Regional Differences in Luminal Staining Characteristics.
Photomicrographs of the oviductal ampulla (A) and isthmus (B) from
the same cryosection from a late estrous mouse. Section treated with
combined celloidin/CPC, stained with PAS and hematoxylin.
Magnification 40X.


52
Western blots suggests that the inhibition by fetuin is due to the presence of a fetuin
binding component on the sperm. The fetuin binding component appears to be
serving as a lectin-like receptor for sialic acid containing ligands. Such a role for
carbohydrates in cellular interactions is well established. The most extensively studied
are the selectins, glycoproteins involved in leukocyte/endothelial adhesion that bind
to sialylated oligosaccharide ligands (Phillips et al., 1990; Walz et al., 1990; Gahmberg
et al., 1992; Varki, 1993). CD 22, a receptor from B cells belonging to another class
of glycoproteins, the immunoglobulin superfamily, also operates as a sialic acid
binding lectin (Sgroi et al., 1993; Powell et al., 1993). Additionally, pertussis toxin
appears to contain a subunit that is capable of binding fetuin and other sialic acid
bearing substrates (Heerze et al., 1992).
The presence of carbohydrate mediated interactions involving sperm has also
been documented. Sperm/egg binding in the mouse appears to involve an interaction
with the oligosaccharide portion of the mouse ZP3 glycoprotein, specifically a
galactose residue (Wassarman, 1990). Sperm also seem to bind to the carbohydrate
portion of the pig ZP3 molecule (Noguchi & Nakano, 1992). The involvement of
mannose (Tesarik et al., 1991; Cornwall et al., 1991; Tulsiani et al., 1992) and fucose
(Oehninger et al., 1991) in sperm/egg binding have also been proposed. These
findings demonstrate the importance of carbohydrate mediated interactions for sperm
physiology.
The fetuin binding components in sperm extracts appeared to be
predominantly sialylated themselves. The similarities between the labelling with


21
to positively affect the expression of hyperactivated motility (Fraser & Monks, 1990).
Including either cAMP analogs or phosphodiesterase inhibitors in capacitating
medium appears to enhance hyperactivation (Fraser, 1979, 1981; Mrsny & Meizel,
1980). White and Aitken (1989) have detected a rise in cAMP levels preceding the
attainment of hyperactivation in the hamster. It has been proposed that calcium
regulation and cAMP metabolism are the cellular keys to the shift to hyperactivation
(Ishijima, 1990).
Several functions for hyperactivated motility have been postulated: (1)
providing additional force to release sperm from the mucosa and to aid in
penetration of viscous or viscoelastic substances; (2) generating a movement path that
helps sperm avoid entrapment in the twists and folds of the uterine tube and covers
more area within the uterine tube, improving the likelihood of finding the egg; and
(3) creating disturbances in the tubal medium to improve the exchange of signalling
and metabolic components (Yanagimachi, 1981; Katz et al., 1989; Suarez & DeMott,
1991). Though actual analysis of the functional significance of hyperactivation
remains limited (Drobnis et al., 1988a, 1988b; Suarez et al., 1991a; Suarez & Dai,
1992), the implications of a potential role for hyperactivation in affecting sperm
transport within the uterine tube seem clear.
While the studies described above point out many changes in sperm cell
surface antigens, membrane composition and intracellular metabolism, none of these
changes have been interpreted in terms of sperm transport. They are generally
assigned as part of the maturational process aimed at the stabilization of the sperm


130
Petruszak JAM, Nehme CL & Bartles JR (1991) Endoproteolytic cleavage in the
extracellular domain of the integral plasma membrane protein CE9 precedes
its redistribution from the posterior to the anterior tail of the rat
spermatozoon during epididymal maturation. Journal of Cell Biology, 114,
917-927.
Phillips DM (1972) Comparative analysis of mammalian sperm motility. Journal of
Cell Biology, 53, 561-573.
Phillips DM, Jones R & Shalgi R (1991) Alterations in distribution of surface and
intracellular antigens during epididymal maturation of rat spermatozoa.
Molecular Reproduction and Development, 29, 347-356.
Phillips ML, Nudelman E, Gaeta FCA, Perez M, Singhal AK, Hakomori S-I &
Paulson JC (1990) ELAM-1 mediates cell adhesion by recognition of a
carbohydrate ligand, sialyl-le(x). Science, 250, 1130-1132.
Pollard JW, Plante C, King WA, Hansen PJ, Betteridge KJ & Suarez SS (1991)
Fertilizing capacity of bovine sperm may be maintained by binding to oviductal
epithelial cells. Biology of Reproduction, 44, 102-107.
Powell LD, Sgroi D, Sjoberg ER, Stamenkovic I & Varki A (1993) Natural ligands
of the B cell adhesion molecule CD22b carry N-linked oligosaccharides with
a-2,6-linked sialic acids that are required for recognition. Journal of Biological
Chemistry,
Racey PA, Uchida TA, Mori T, Avery MI & Fenton MB (1987) Sperm-epithelium
relationships in relation to the time of insemination in little brown bats
(Myotis lucifugus). Journal of Reproduction and Fertility, 80, 445-454.
Rankin RL, Holland MK & Orgebin-Crist M-C (1989) Lectin binding characteristics
of mouse epididymal fluid and sperm extracts. Gamete Research, 24, 439-451.
Raychoudhury SS & Suarez SS (1991) Porcine sperm binding to oviductal explants
in culture. Theriogenology, 36, 1059-1070.
Reyes A, Oliphant G & Brackett BG (1975) Partial purification and identification of
a reversible decapacitation factor from rabbit seminal plasma. Fertility and
Sterility, 26, 148-157.
Rifkin JM & Olson GE (1985) Characterization of maturation-dependent extrinsic
proteins of the rat sperm surface. Journal of Cell Biology, 100, 1582-1591.


57
adhesion. These results agree with previous findings about the ability of
hyperactivated and capacitated sperm to release from the mucosa, a reduction in
sialylation as part of the capacitation sequence, and the inability of capacitated sperm
to bind fetuin. However, this is the first time that these phenomena have been
related to a function, specifically the regulation of sperm passage from the isthmic
reservoir. Western blots labelled with fetuin and sialic acid-recognizing lectins
identified proteins at several molecular weights that show changes in labelling
between fresh and hyperactivated sperm extracts. These proteins are good
candidates for further study as the fetuin binding component responsible for sperm
adherence in the isthmic reservoir.


103
Results
The combination of celloidin coating and CPC post-fixation for frozen sections
resulted in the detection of a fairly homogeneous substance completely filling many
of the luminal spaces between mucosal folds and in the central lumen (Figure 5-la).
The substance showed a light, or diffuse, PAS reaction. This luminal substance was
found throughout the segments of the uterine tube, but between the mucosal folds
there were scattered regions that did not stain for this material.
In frozen sections where both treatments had been left out, the luminal
contents were relatively sparse and the remnants appeared as strands (Figure 5-lb).
These strands stained darkly with PAS, alcian blue and toluidine blue. In combined
alcian blue/PAS, much of the material preferentially stained with alcian blue.
Immersion fixation in aldehydes, even with CPC present, followed by standard
paraffin processing led to the loss of almost all the luminal contents though the tissue
morphology was well preserved and the cilia are visible (Figure 5-lc).
Slight regional and cyclical differences in the luminal contents were observed.
The contents in the ampulla of the early and late estrous animals appeared as an
even pool of light pink material (Figure 5-2a). The isthmic loops, compared on the
same section, showed a more granular material that appeared to take up more of the
hematoxylin counterstain, since there was a gray cast to the material (Figure 5-2b).
The latter staining characteristics were found in all regions at proestrus and diestrus.
At proestrus, however, there was a band of strong PAS staining in the apical portions
of the isthmic epithelial cells.


13
Hoos & Olson, 1988; Robataille et al., 1991). A sialylated glycoprotein produced
from the distal corpus to the cauda appears to be processed and is associated with
the mouse sperm tail (Vernon et al., 1987; Feuchter et al., 1988; Toshimori et al.,
1990) and a ram sperm sialoglycoprotein added in the cauda redistributes during the
acrosome reaction (McKinnon et al., 1991).
Another group of studies relate to changes in protein glycosylation during
maturation. By comparing the binding of various lectins to sperm from different
parts of the epididymis, changing populations of carbohydrate moieties on sperm have
been identified for the hamster (Nicolson & Yanagimachi, 1972; Koehler, 1981),
rabbit (Nicolson & Yanagimachi, 1972; Nicolson et al., 1977), ram (Hammerstedt et
al., 1982; Magargee et al., 1988), macaque (Fain-Maurel et al., 1984), mouse (Rankin
et al., 1989), dog (Bains et al., 1993b), and goat (Bains et al., 1993a). The results of
these studies imply that the exposed carbohydrates and their modifications are highly
species specific. Galactose and N-acetylgalactosamine distribution on testicular and
caudal rat sperm, analyzed by binding to labeled galactose oxidase, has also identified
proteins modified during maturation (Brown et al., 1983). Another approach has
been to demonstrate the presence of certain glycosyltransferases in epididymal fluid.
Sialyltransferase has been demonstrated in rat epididymal fluid with the strongest
activity in the caput (Bernal et al., 1980), and galactosyltranferase activity also
appears in rat epididymal fluid (Hamilton, 1980).
Sperm are considered mature when they reach the caudal epididymis since
they may become motile, capacitate, and fertilize. However, in the natural scheme


2
excised uterine tubes have been used to show that the number of sperm present at
the time and place of fertilization is very low in the mouse (Tessler & Olds-Clarke,
1981; Suarez, 1987), hamster (Cummins & Yanagimachi, 1982; Smith et al., 1987)
rabbit (Overstreet et al, 1978), rat (Bedford & Kim, 1993), sheep (Cummins, 1982)
and pig (Hunter et al., 1987).
Other observations imply that the reduction of sperm numbers to these very
low levels does not occur as a regular, gradual process in the female reproductive
tract. There is likely a constant loss of sperm during their ascent, but there are also
very restrictive barriers (Katz et al., 1989). For species that inseminate at or below
the cervix, this structure appears to reduce the number of progressing sperm by
orders of magnitude (Hunter, 1988). The junction between the uterus and uterine
tube (the preferred anatomical term "uterine tube" will be used throughout in place
of the more common "oviduct"), the uterotubal junction, also appears to serve as a
restrictive filter (Gaddum-Rosse, 1981; Shalgi et al., 1992), causing another large drop
in sperm numbers. Finally, within the uterine tube, there is not a regular distribution
of sperm. Sperm appear to be sequestered in a reservoir located in the most
proximal portion of the uterine tube until ovulation, and thus fertilization, is
imminent. This phenomenon has been demonstrated in the hamster (Yanagimachi
& Chang, 1963; Smith et al., 1987), mouse (Zamboni, 1972; Suarez, 1987), rabbit
(Harper, 1973a, 1973b; Overstreet et al., 1978), cow (Thibault et al., 1975), guinea
pig (Yanagimachi & Mahi, 1976), sheep (Hunter & Nichol, 1983), pig (Hunter, 1984;
Hunter et al., 1987) and rat (Shalgi & Kraicer, 1978; Shalgi & Phillips, 1988).


113
hyperactivated motility. This suggests that adherence in the isthmic reservoir may be
influenced by biochemical changes of the sperm surface that aid in their release when
they are becoming ready to fertilize an egg.
The antibodies generated provide a useful tool for identifying the cellular
components changing on the sperm as it undergoes functional changes in the uterine
tube. Antigens associated with sperm agglutination and hyperactivation were
detected, and, while none of them appear to be the binding site detected in the
inhibition experiments, their characterization could provide clues about the signalling
or metabolic processes involved in the changing binding and motility characteristics.
The switch to hyperactivated motility is closely tied to release from the tubal
reservoir and the additional force and torque presumably provided by this vigorous
pattern may help sperm break free. The relative contributions of reduced affinity for
the mucosa and the changed motility pattern remain unclear, but there is clearly
room for both to be operating. Since all free sperm appeared to be hyperactivated
but not all hyperactivated sperm were able to break free, the reduction in binding
affinity may be a permissive regulatory component. Such a fail-safe system, requiring
that sperm can both develop hyperactivated motility and undergo the membrane
transformations that occur during capacitation, could play an important selective role
in providing sperm in the appropriate condition to reach the site of fertilization.
Given the observations that so few sperm actually have a chance to participate in
fertilization, there are clear benefits to a stringent selection process helping ensure
that those few are appropriate for fertilization.


89
HL 787 Fresh Agglutinated
Figure 4-2. Epifluorescent Images of Monoclonal Antibody Binding Patterns.
Fixed, dried down sperm treated with culture supernatant.
Photographed at 100X with Zeiss Plan-neofluar objective on Ektar
1000 film. Exposures 75-90 sec. Images for a given antibody exposed
the same duration and all negatives printed similarly.


118
Boice BL, Mavrogianis PA, Murphy CN, Prather RS & Day BN (1992)
Immunocytochemical analysis of the association of bovine oviduct-specific
glycoproteins with early embryos. Journal of Experimental Zoology, 263,
225-229.
Borland RM, Hazra S, Biggers JD & Lechene CP (1977) The elemental composition
of the environments of the gametes and preimplantation embryo during the
initiation of pregnancy. Biology of Reproduction, 16, 147-157.
Boyle MS, Cran DG, Allen WR & Hunter RHF (1987) Distribution of spermatozoa
in the mares oviduct. Journal of Reproduction and Fertility, Suppl.35, 79-86.
Braden AWH & Austin CR (1954) Fertilization of the mouse egg and the effect of
delayed coitus and of hot-shock treatment. Australian Journal of Biological
Sciences, 7, 552-565.
Breed WG, Leigh CM & Bennett JH (1989) Sperm morphology and storage in the
female reproductive tract of the fat-tailed dunnart, Sminthopsis crassicaudata
(Marsupialia: Dasyuridae). Gamete Research, 23, 61-75.
Brower LK & Anderson E (1969) Cytological events associated with the secretory
process in the rabbit oviduct. Biology of Reproduction, 1, 130-148.
Brown CR, von Glos KI & Jones R (1983) Changes in plasma membrane
glycoproteins of rat spermatozoa during maturation in the epididymis. Journal
of Cell Biology, 96, 256-264.
Buhi WC, Alvarez IM, Sudhipong V & Dones-Smith MM (1990) Identification and
characterization of de novo-synthesized porcine oviductal secretory proteins.
Biology of Reproduction, 43, 929-938.
Buhi WC, OBrien B, Alvarez IM, Erdos G & Dubois D (1993) Immunogold
localization of porcine oviductal secretory proteins within the zona pellucida,
perivitelline space, and plasma membrane of oviductal and uterine oocytes
and early embryos. Biology of Reproduction, 48, 1274-1283.
Buhi WC, Vallet JL & Bazer FW (1989) Denovo Synthesis and Release of
Polypeptides from Cyclic and Early Pregnant Porcine Oviductal Tissue in
Explant Culture. Journal of Experimental Zoology, 252, 79-88.
Champlin AK, Dorr DL & Gates AH (1973) Determining the stage of the estrous
cycle in the mouse by the appearance of the vagina. Biology of Reproduction,
8, 491-494.


70
Table 3-1.
Analysis of factors affecting proportion of sperm swimming freely
within the uterine tube; calculated from videotapes of in situ
preparations from naturally mated mice.
Factor
Treatment
Mean
SEM
N
Region*
Isthmus
11.8%
1.0%
1196
Ampulla
26.3%
0.8%
100
Ovulation
Pre-
10.6%
1.6%
553
Status**
Post-
16.2%
2.0%
743
Time
1 h
18.7%
3.5%
311
in
Tract
1.5 h
12.8%
3.0%
412
2 h
12.6%
2.3%
198
3 h
11.6%
1.8%
375
Treatments of this factor significant at p < 0.01
Treatments of this factor significant at p < 0.05
(Used with permission from DeMott & Suarez, 1992).


50
Figure 2-6. PVDF blots probed for carbohydrate binding. A) fetuin-gold-labelled
sperm components, B) LFA-labelled sperm components.


114
Finally, the demonstration of tubal mucus occupying the entire luminal cross-
section provides yet another potentially selective regulatory feature. Sperm have little
chance of avoiding this material, whether close to the mucosa or in the central lumen
and, there do not appear to be paths of decreased resistance. Edge effects and other
hydrodynamic properties may still be relevant to the ability of sperm to navigate
some areas more easily than others, however. With the only path appearing to be
straight through the tubal mucus, the differential abilities of sperm using different
motility patterns to penetrate becomes important for determining the population of
sperm that ascend the uterine tube. Hyperactivated sperm have an advantage in
material of this nature; therefore, the mucus potentially serves as another selective
barrier to sperm.
The picture arising out of this investigation of the transport of sperm through
the uterine tube shows the sperm as a dynamic cell, responding to a variety of factors
that may help optimize fertilization. Their viability may be supported by adherence
in the isthmic reservoir, until the sperm reaches a certain point in the physiological
process preparing it for fertilization. This appears to be somehow tied to ovulation
and the impending time of fertilization. Following desialylation or some other
modification reducing the affinity of the receptor for mucosal binding, the recently
acquired hyperactivated motility pattern can release sperm from the reservoir and
enable them to penetrate the luminal mucous secretions. Sperm that undergo the
surface modification and manage to release without hyperactivating are constrained


42
PBS vehicle only. The motility of the sperm at the end of the ten minute treatment
was also checked. During videotaping following treatment with asialofetuin,
ovalbumin, fucoidan, poly-l-lysine and PBS, almost no motile sperm were seen free
of the mucosa. Some immotile sperm could be seen being transported passively with
the tubal contents. Further, motile sperm were not observed to pull away from the
mucosa. In contrast, numerous free swimming sperm were observed following
treatment with fetuin. The free sperm were observed to encounter the mucosa and
not stick to it.
Having identified fetuin as the only inhibitor of sperm binding to the tubal
mucosa among the candidates, a quantitative analysis was completed to more
carefully study the binding inhibition by fetuin and determine whether the terminal
sialic acid residues were responsible for the inhibition. Uterine tubes from a series
of females were randomly assigned by flipping a coin to sperm that had been treated
with 5 mg/ml of either fetuin or asialofetuin, 25 mM sialic acid (n-acetylneuraminic
acid), or PBS vehicle. The pH of the sialic acid treatment was readjusted to 7.5 prior
to the addition of sperm. There were significant differences between the groups in
the proportion of free sperm found in the uterine tube by ANOVA (F=13.5, df=20,
p=0.0001). The proportion of free sperm was significantly higher than buffer control
by Fishers test for multiple comparisons for both fetuin and sialic acid-treated sperm
(Figure 2-2). The binding inhibition by fetuin was not significantly stronger than that
by sialic acid. Also, asialofetuin was again ineffective as an inhibitor of sperm
binding. The proportion of free sperm in the asialofetuin group was not different


26
contents and suggests that in the rabbit uterine tube there are both serous and
mucous secretions present (Jansen & Bajpai, 1982). In scanning electron microscopy
studies, the luminal contents appear patchy and as a matrix or honeycomb on the
surface of the mucosa; sperm associated with the mucosa have been seen coated with
this material (Jansen, 1978, 1980; Boyle et al., 1987; Hunter et al., 1987). Whether
this material actually spans the luminal spaces, potentially affecting sperm movement
as well as sperm adherence to the mucosa remains has not yet been clearly
demonstrated.
Description of Experiments
The experiments described below address four aspects of the control of sperm
transport to the site of fertilization. The characteristics of the binding of sperm to
the tubal wall were examined to address how the sperm reservoir is formed and
maintained. An analysis of sperm motility patterns in the uterine tube addresses a
possible mechanism for release from the reservoir. The establishment of surface
markers associated with various sperm conditions addresses how sperm changes may
contribute to tubal transport. Finally, an examination and characterization of the
tubal luminal contents addresses what role the luminal environment may play in
sperm transport.


48
Figure 2-5. Photomicrographs of silver-enhanced labelled hamster sperm. A) Fresh
sperm with fetuin-gold, B) Hyperactivated sperm with fetuin-gold, C)
Control sperm with no label.
/


85
For analysis by light microscopy, the labelled, washed sperm suspensions were
dried down on multi-well slides, rinsed 3X with PBS and fixed for 5 minutes with
2.5% phosphate buffered glutaraldehyde. They were rinsed with PBS and distilled
water, 3X each, then the label was enhanced with silver as described in Chapter 1.
Slides were analyzed following a 9-11 minute enhancement.
Immunoblotting of Sperm Proteins
1-D blots. Fresh and hyperactivated sperm extracts were prepared as
described in Chapter 2. Electrophoresis and semi-dry transfer to Immobilon-P
membranes were carried out under the conditions previously described as well (see
Chapter 1). Blots were blocked for 2 hours with PBS/BSA then washed 4X with PBS.
Strips were incubated overnight at 4C with 1:50 ascites, 1:1000 normal mouse serum,
1:1000 immune mouse serum, and PBS/BSA alone. All dilutions were made with
PBS/BSA. Following 4 washes with PBS, the blot strips were incubated for 2 hours
at room temperature with 1:1000 horseradish peroxidase-labelled F(ab)2 of rabbit
anti-mouse IgG-F(ab)2) (Jackson Immunoresearch Labs, Inc., West Grove, PA).
This product contains the same antibodies previously used for immunofluorescence
conjugated to a different label. After 4 washes with PBS, the peroxidase reaction was
developed as described in Chapter 2.
2-D blots. Fresh sperm extract prepared as described above containing 650
¡ig of total protein was diluted to 5 ml and dialyzed against three 2-liter changes of
distilled water over 24 hours. Dialysis tubing (Spectra/Por, mW cutoff 3500,


72
three-way interactions. Ovulation status and time in tract were not involved in
interactions, nor were they significant as individual factors.
While free sperm were significantly more sharply bent than stuck sperm, an
interaction between attachment status and tubal region was detected, since the
difference between free and stuck sperm was much greater in the isthmus than the
ampulla. Also, while isthmic sperm were significantly more sharply bent than
ampullar sperm, the difference between the isthmus and ampulla was greater for free
sperm than stuck sperm. An interaction of attachment status with location relative
to the lumen was also detected, because free sperm in the folds were more sharply
bent than free sperm in the lumen; however, stuck sperm in the folds were LESS
sharply bent than stuck sperm in the lumen. Also, free sperm were more sharply
bent in both the folds and lumen than stuck sperm, but the difference between free
and stuck sperm was much greater in the folds than in the lumen.
Discussion
The general pattern of mouse sperm progress up the uterine tube appears to
involve periods of free swimming between periods of attachment to the tubal
epithelium. This pattern was observed in both the isthmus and the ampulla but our
analysis of the proportion of free swimming sperm showed that sperm were more
likely to be free in the ampulla. While earlier in situ studies in the mouse (Suarez,
1987) reported the apparently decreased stickiness of ampullar sperm, this is the first


9
They had observed that superovulated hamsters have enhanced sperm transport.
Presumably the additional ovulations produce additional prostaglandins that enhances
the contractions. The transport of extra sperm in superovulated rats has also been
documented (Shalgi & Phillips, 1988). The presence of high levels of estrogens in
boar semen has also been postulated to induce prostaglandin and LH releases in the
female that could affect muscular activity in the reproductive tract, and the timing of
ovulation (Claus, 1990)
Another possible mechanism for transporting sperm is ciliary currents.
Gaddum-Rosse and Blandau (1973) observed ciliary transport of particles in
longitudinally opened uterine tubes and reported that the current in the isthmus of
rabbits and pigs is toward the ampulla. However, these species seem to be atypical
in terms of the direction of the current when compared to rats, guinea pigs, humans,
and cows (Gaddum-Rosse & Blandau, 1976) and also in terms of how much ciliation
is present in the isthmus (Nilsson & Reinius, 1969; Hunter, 1988). So, while ciliary
beating is important for egg transport (Norwood et al., 1978; Norwood & Anderson,
1980; Mahi-Brown & Yanagimachi, 1983), it probably plays a minimal role in isolated
species for sperm movement (Gaddum-Rosse & Blandau, 1976).
While the uterine tube may play a predominant role in the movement of
sperm that have released from the reservoir, it seems that it does not control whether
or not adherence of sperm to the mucosa is maintained. By sequentially flushing the
hamster uterine tube to remove free then loosely bound sperm at several time points,
Smith and Yanagimachi (1990) found that there were no significant changes in the


CHAPTER 2
MECHANISM OF SPERM BINDING IN THE ISTHMIC RESERVOIR
Introduction
The existence of a reservoir in the isthmic portion of the uterine tube in which
sperm are maintained until the time of fertilization approaches has been described
for a number of mammalian species (Zamboni, 1972; Thibault et al., 1975;
Yanagimachi & Mahi, 1976; Overstreet et al., 1978; Shalgi & Kraicer, 1978; Hunter
et al., 1987; Suarez, 1987). Sperm appear to ascend relatively quickly to this region
and then are retained; the population of sperm responsible for fertilization move on
to the ampulla only when fertilization is imminent (Harper, 1973b; Hunter, 1984;
Suarez, 1987; Smith & Yanagimachi, 1991).
In the hamster uterine tube at least, it appears that the modulation of binding
and release of sperm from the reservoir is primarily dependent on changes in the
sperm cell, not the uterine tube (Smith & Yanagimachi, 1990, 1991). Non-
capacitated hamster sperm samples injected into excised uterine tubes have been
shown to bind almost completely to the epithelium whereas sperm from capacitated
samples, that were also hyperactivated, are able to remain free (Smith &
Yanagimachi, 1991). Further, following natural mating in the mouse, very few free
swimming sperm were seen in the uterine tube and those that were appeared to be
29


84
Cloning. Cultures producing supernatants that yielded interesting staining
patterns were cloned (Simrell & Klein, 1979; Kao & Klein, 1986). The resulting
cultures were screened, subcloned, and rescreened at least three times to obtain
active monoclonal cultures. The monoclonal cell lines were expanded, isotyped and
stored.
Production of Ascites. Cultures from three cell lines producing antibodies
that recognized epitopes changing prior to hyperactivation, HL 772, HL 778, and HL
787, were used to induce ascites fluid production in BALB/c mice. RIBIs adjuvant
was used. Ascites fluid was cleared by centrifugation at 960 G for 10 minutes, then
stored frozen in aliquots at -20C.
Immunogold Labelling of Sperm
Sperm from the four stages were obtained as described above. Medium was
removed following centrifugation, 5 minutes, 960 G, and the sperm were washed in
PBS/BSA, 5 minutes, 960 G. Labelling was carried out based on the protocol of
Jones and coworkers (1990). The sperm were then suspended in either 1:1 culture
supernatant or 1:10 ascites diluted with PBS/BSA and incubated for 35 minutes at
room temperature. The supernatant was removed as above and the sperm were
washed by centrifugation twice with PBS/0.1% BSA. They were then incubated for
30 minutes with 5 nm colloidal gold-labelled secondary antibody, anti-mouse IgG, IgM
(E-Y Labs, San Mateo CA) diluted 1:30 with PBS/0.1% BSA. Supernatant was
removed and the sperm were washed with PBS by centrifugation 3X.


90
however, were very patchy and variable. The stage specific characteristics identified
by immunofluorescence could be seen, but not consistently.
Immunostaining of blots allowed the identification of potential antigens for two
of the antibodies (Figure 4-3). HL 787 clearly identified a specific antigen that
migrated at 22-23 kDa under reducing conditions. This band was seen in blots
stained with immune serum but not with normal serum or secondary antibody alone.
It also showed decreased labelling on blots of hyperactivated sperm extracts
compared to fresh. A strong band at approximately 42-44 kDa was also detected
with the other monoclonals and controls, though it was clearly enhanced by binding
to the monoclonals. This band did not label with immune serum. A weak band at
46 kDa was also unique to the fresh blots stained with HL 787. Staining with HL 772
seemed to identify 2 separate bands at approximately 44 and 42 kDa, and the lower
molecular weight band appeared to be distinct from the band labelling in the other
treatments. It also showed decreased labelling of hyperactivated sperm extracts.
Labelling with HL778 did not suggest any strong candidates for the antigen associated
with this antibody. Results were similar on blots from 2 separate sets of extracts
representing eight hamsters.
Analysis of HL 787 labelling by 2-D PAGE again detected a protein of
approximately 22 kDa (Figure 4-4). The protein is acidic with an approximate pi
range of 5.1-5.2. This spot was again absent in the control blot stained with normal
mouse serum.


121
Erickson-Lawrence MF, Turner TT, Thomas TS steroid hormones on sulfated oviductal glycoprotein secretion by oviductal
explants in vitro. Biology of Reproduction, 40, 1311-1320.
Fain-Maurel MA, Dadoune JP & Reger JF (1984) A cytochemical study on surface
charges and lectin-binding sites in epididymal and ejaculated spermatozoa of
Macaca fascicularis. Anatomical Record, 208, 375-382.
Feuchter FA, Tabet AJ fe Green MF (1988) Maturation antigen of the mouse sperm
flagellum. 1. Analysis of its secretion, association with sperm, and function.
American Journal of Anatomy, 181, 67-76.
Focarelli R, Rosati F fe Terrana B (1990) Sialyglycoconjugates Release During In
Vitro Capacitation of Human Spermatozoa. Journal of Andrology, 11, 97-104.
Fraser LR (1977) Motility patterns in mouse spermatozoa before and after
capacitation. Journal of Experimental Zoology, 202, 439-444.
Fraser LR (1979) Accelerated mouse sperm penetration in vitro in the presence of
caffeine. Journal of Reproduction and Fertility, 57, 377-384.
Fraser LR (1981) Dibutyryl cyclic AMP decreases capacitation time in vitro in mouse
spermatozoa. Journal of Reproduction and Fertility, 62, 63-72.
Fraser LR (1983) Potassium ions modulate expression of mouse sperm fertilizing
ability, acrosome reaction and hyperactivated motility in vitro. Journal of
Reproduction and Fertility, 69, 539-553.
Fraser LR (1984) Mouse sperm capacitation in vitro involves loss of a
surface-associated inhibitory component. Journal of Reproduction and Fertility,
72, 373-384.
Fraser LR (1987) Minimum and maximum extracellular Ca2+ requirements during
mouse sperm capacitation and fertilization in vitro. Journal of Reproduction
and Fertility, 81, 77-89.
Fraser LR (1990) Sperm capacitation and its modulation in Fertilization in Mammals,
edited by BD Bavister, J Cummins fe ERS Roldan. Norwell MA: Serono
Symposia USA, pp. 141-153.
Fraser LR <& McDermott CA (1992) Ca2+ -related changes in the mouse sperm
capacitation state: a possible role for Ca2+ -ATPase. Journal of Reproduction
and Fertility, 96, 363-377.


74
undisturbed swimming trajectory or their velocity over a long enough distance to
correlate it with in vitro measurements. In our laboratory, beat amplitude and wave
length are more sensitive to measurement errors than FCR and this becomes
significant when taking measurements from images of sperm within the uterine tube.
Based on the results of the FCR measurements, it appears that sperm which
have broken free are more likely to be hyperactivated than stuck sperm. Also, based
on these observations and previous descriptions of the hyperactivated pattern for
mouse sperm (Fraser, 1977; Olds-Clarke, 1986; Suarez & Osman, 1987), the pool of
erratically beating, dynamic sperm that intermittently released and reattached to the
tubal wall appeared to be hyperactivated. All free sperm were a subset of this pool.
The erratic beating pattern generated a sharply curved, hyperactivated beat while
sperm were attached to the tubal wall and all sperm releases were preceded by
erratic beating. This supports the possibility of hyperactivation functioning to give
sperm an advantage in remaining free from the epithelium and progressing up to the
site of fertilization.
Hyperactivation may provide the direction changes needed to navigate the
convoluted path up the uterine tube and provide the forces or torques that allow
sperm to bounce off rather than stick when they encounter the wall. Since not all of
the erratically beating sperm were free, hyperactivation is not always sufficient for
sperm release. Capacitation may also be required. Capacitation may decrease the
stickiness of the sperm head, making it easier to release from the epithelium when
they are stuck. In the hamster uterine tube, sperm have also been noted breaking


95
extract. However, the presence of a large nearby band, apparently non-specific,
complicates interpretation. Though the separate bands appeared in two different
samples, both bands fall within the range of molecular weights calculated for the non
specific band in other treatments. Alternative methods for characterizing the antigen
might provide clearer information.
HL 778 did not appear to react with a specific antigen on Western blots. The
epitope recognized by this antibody may have been destroyed during the extraction,
denaturation and reduction of disulfide bonds. Although screening was based on
fixed sperm to specifically identify antibodies that would be effective for denatured
protein antigens, the changes in proteins during PAGE could be sufficiently different
to disrupt the affinity of the antibody. Also, the protein may not have been
transferred successfully to the Immobilon-P membrane. Alternatively, the antibody
may recognize a non-protein component that was retained on fixed sperm but lost
during electrophoresis and blotting.
The inconsistent results obtained by labelling unfixed sperm are most likely
due to the multivalent nature of the antibodies used for these experiments. The
primary antibodies and the colloidal gold-labelled secondary antibody were all intact
immunoglobin molecules with multiple binding sites that could cause artificial shifts
of antigens in unfixed cell membranes.
The antigen associated with antibody HL 784 was not characterized, since its
relevance to sperm transport appeared minimal. However, this antibody could prove
very useful for studies involving the acrosome reaction. First, it provides a good


102
They were then coated with celloidin according to a method used to stabilize colonic
mucus (Szentkuti & Eggers, 1990). Slides were incubated for 3 minutes with 0.2%
celloidin (Fisher Chemical, Co.) dissolved in a 1:1 mixture of absolute ethanol and
ethyl ether. They were dried for 5-10 minutes and hardened in 70% ethanol for 3
minutes. To further stabilize the mucus, the slides were post-fixed for 10 minutes in
10% phosphate buffered formalin containing 0.5% CPC. For controls, alternate
serial slides were prepared omitting one or both of the celloidin and CPC steps.
Staining
Chemicals and stains were from Sigma except where noted. Slides were
primarily stained with the periodic acid Schiff reaction (PAS), as described below,
because it was compatible with the processing technique and identifies a broad array
of carbohydrates. They were treated with 1% periodic acid in distilled water (5
minutes), rinsed and treated with Schiffs base (12-15 minutes, cold Schiff method
(Lillie & Fullmar, 1976)). Slides were rinsed and counterstained with Harris
hematoxylin (Luna, 1968) for 20-40 seconds. They were rinsed in distilled water, acid
alcohol and ammonia water and dehydrated through a graded ethanol series, cleared
in xylene and mounted with Eukitt (Calibrated Instruments, Hawthorne, NY).
Some of the frozen sections were stained with 1% alcian blue, pH 1 & 2.5,
combined alcian blue (pH 2.5)/PAS and 1% toluidine blue. The paraffin sections
were stained with hematoxylin/eosin (H&E), PAS, alcian blue (pH 2.5) and combined
alcian blue/PAS. These slides were dehydrated, cleared and mounted as above.


24
of the mucosa in which the isthmic sperm lodge. The folding pattern gradually
changes to longitudinal around the ampullary/isthmic junction in these species,
forming mucosal grooves oriented along the long axis of the uterine tube (Nilsson &
Reinius, 1969). While the species examined have mucosal folds, the degree and
orientation is species dependent (Nilsson & Reinius, 1969). For the rodents, the
uterine tube is held in a tightly coiled spiral by the mesosalpinx (Suarez, 1987; Smith
et al., 1987). This may also affect sperm transport, as the sperm must be able to
change direction and follow this twisting path.
Another relevant factor of uterine tube morphology is the distribution of
secretory and ciliated cells in the epithelium. Again, there is wide species diversity
in the proportion of each cell type (Nilsson & Reinius, 1969; Hunter, 1988) and both
regional and cyclical variations in their morphogenesis (Brower & Anderson, 1969;
Patek, 1974; Hagiwara et al., 1992; Abe & Oikawa, 1993). Sperm appear capable of
binding to both cell types, though there may be species-dependent preferences for
one type or the other (Hunter et al., 1987; Smith & Yanagimachi, 1990; Suarez et al.,
1991b).
The secretory products of the uterine tube may play a physiological role in the
regulation of sperm transport. There are a few reports of tubal secretions interacting
with sperm (Sutton et al., 1986; Voglmayr & Sawyer, 1986; Wagh & Lippes, 1989;
McNutt et al., 1993), and in the case of human sperm this secretion has been
identified as the sialylated glycoprotein alpha-fetoprotein (Wagh & Lippes, 1993).
A functional role for these secretions remains to be clearly demonstrated, though


83
samples were used immediately as fresh sperm. The remaining suspensions were
incubated at 37C, checked periodically, and used for the agglutinated, hyperactivated
and capacitated stages when they met the criteria in Table 4-2. Capacitation was
assayed by the ability of lysophosphatidylcholine (LPC) to induce acrosome reactions
in motile sperm (Llanos & Meizel, 1983). Samples were tested periodically beginning
at the onset of hyperactivation. When sperm were collected for the hyperactivated
stage, the percent responding to LPC was 10% or less.
Sperm were dried down on multi-well microscope slides (Cel-line Assoc.
Newfield, NJ), rinsed with phosphate-buffered saline (PBS), fixed for 5-10 min with
7% formaldehyde and rinsed again with PBS. All treatments were carried out at
room temperature. The sperm were blocked with 3% bovine serum albumin
(Calbiochem, La Jolla, CA) in PBS (PBS/BSA) for 30 minutes, then individual wells
for each sperm stage were incubated with PBS/BSA alone, neat hybridoma culture
supernatant, hybridoma growth media, 1:1000 normal mouse serum (non-immune)
in PBS/BSA, and immune mouse serum, diluted similarly for 1.5-1.75 hours. The
wells were rinsed with PBS/BSA, incubated with TRITC-labelled secondary antibody
(F(ab)2 of rabbit anti-mouse IgG-F(ab)2) (Jackson Immunoresearch Labs, Inc., West
Grove, PA) for 20-25 minutes, and rinsed again. Slides were examined using
combined Nomarski/epifluorescence optics, 100W mercury lamp, and a rhodamine
(TRITC) filter set (Carl Zeiss, Inc., Thornbrook, NY). The staining pattern for each
supernatant with each type of sperm was recorded.


61
environment in the uterine tube and time since insemination on flagellar bending and
sperm sticking to the tubal mucosa were analyzed.
Materials and Methods
Medium ingredients were purchased from Sigma Chemical Co. (St. Louis,
MO). The medium consisted of Earles Balanced Salts supplemented with 2.2 g/1
sodium bicarbonate, 0.06 g/1 penicillin, and 0.06 g/1 streptomycin. The final pH was
adjusted to 7.6 and the medium was sterilized by filtration through 0.22 /xm filters
(Millipore Products Div., Bedford, MA).
Outbred ICR strain mice from Harlan Sprague Dawley (Indianapolis, IN) were
kept under a 14:10 hour light cycle with lights on from 0700 h to 2100 h. A delayed
mating protocol (Braden & Austin, 1954) was used to reduce the time between
insemination and sperm arrival at the site of fertilization. This allowed us to observe
a larger portion of the period between insemination and fertilization. Virgin females,
8-16 weeks old, were placed with retired breeder males on the morning of estrus and
allowed to mate. After ejaculation was observed (Wimer & Fuller, 1966), sperm
were allowed to ascend through the tract for 1,1.5, 2, or 3 hours. The females were
killed by C02 inhalation and ovulatory status was determined by observing the
ovaries, ovarian bursa, and uterine tubes. Based on preliminary observations, the
ovulation period for delayed mated mice of this strain extended up to approximately
4 hours after lights on (1100). To observe mating, the lights had to be on, and by
starting matings between 0730-0830 and sampling between 0900-1130, mice could be


4-3 1-D Immunoblots probed with stage-specific
monoclonal antibodies 91
4-4 2-D Immunoblot of fresh sperm extract labelled with HL 787 92
5-1 Differential preservation of luminal contents by
processing method 105
5-2 Regional differences in luminal staining characteristics 106
5-3 Sperm observed within luminal mucus 107
vm


91
A B C D
| ( 44 k
- 22k
f h f h f h
Figure 4-3. 1-D immunoblots probed with stage-specific monoclonal antibodies. A)
control normal mouse serum, B) HL 772 lanes of fresh (left) and
hyperactivated sperm extract, C) HL 778 lanes of fresh (left) and
hyperactivated sperm extract, D) HL 787 lanes of fresh (left) and
hyperactivated sperm extract.


123
Hagiwara H, Shibasaki S & Ohwada N (1992) Ciliogenesis in the human oviduct
epithelium during the normal menstral cycle. Journal of Electron Microscopy,
41, 321-329.
Hall JC & Killian GJ (1989) Two-dimensional gel electrophoretic analysis of rat
sperm membrane interaction with cauda epididymal fluid. Journal of
Andrology, 10, 64-76.
Halpert AP, Garstka WR & Crews D (1982) Sperm transport and storage and its
relation to the annual sexual cycle of the female red-sided garter snake,
Thamnophis sirtalis parietalis. Journal of Morphology, 174, 149-159.
Hamilton DW (1980) UDP-galactose: N-acetylglucosamine galctosyltransferase in
fluids from rat rete testis and epididymis. Biology of Reproduction, 23,377-385.
Hammerstedt RH, Hay SR & Amann RP (1982) Modification of ram sperm
membranes during epididymal transit. Biology of Reproduction, 27, 745-754.
Hang H-Y, Feng B-Y & Zhang Z-Y (1990) Studies on relationship between
Na,K-ATPase activity and sperm capacitation in guinea pig. Science in China,
33, 1304-1310.
Harlow E & Lane D (1988) Antibodies a laboratory manual. Cold Spring Harbor,
NY: Cold Spring Harbor Laboratory.
Harper MJK (1973a) Stimulation of sperm movement from the isthmus to the site
of fertilization in the rabbit oviduct. Biology of Reproduction, 8, 369-377.
Harper MJK (1973b) Relationship between sperm transport and penetration of eggs
in the rabbit oviduct. Biology of Reproduction, 8, 441-450.
Hawk HW (1987) Transport and fate of spermatozoa after insemination of cattle.
Journal of Dairy Science, 70, 1487-1503.
Heerze LD, Chong PCS & Armstrong GD (1992) Investigation of the lectin-like
binding domains in pertussis toxin using synthetic peptide sequences. Journal
of Biological Chemistry, 267, 25810-25815.
Hook SJ & Hafez ESE (1968) A comparative study of the mammalian uterotubal
junction. Journal of Morphology, 125, 159-184.
Hoos PC & Olson GE (1988) Characterization of a 23kDa sperm-binding
polypeptide of the golden hamster epididymis. Biology of Reproduction, 39,
131-140.


15
Recently, other functions of seminal plasma components have been reported.
The proposal for a direct effect of seminal plasma steroids on the endocrine
regulation of the female tract has been described above (Claus, 1990). Two separate
groups have identified seminal plasma proteins that have the ability to bind to the
zona pellucida (Parry et al., 1992; Veselsky et al., 1992), though some of the same
proteins may have been found by both groups. Though they are unlikely to represent
the primary ligand for sperm/egg binding, these proteins present the interesting
possibility that seminal plasma components enhance sperm/egg binding. Additionally,
a marked reduction in catalase activity, one of the major oxidative damage protection
systems, has been found in the seminal plasma of infertile men compared to sperm
and seminal plasma from normal, fertile men (Jeulin et al., 1989). In light of the
growing awareness of the importance of free radical scavenging systems, research
clarifying this potential protective role of seminal plasma may intensify.
Capacitation. Determining which of the myriad events of sperm cell biology
are part of capacitation is a matter of semantics. The sperm cells are constantly
being affected by their environment and undergoing modifications; our distinctions
are convenient but must be recognized as artificial and irrelevant in terms of the
sperms life history. And, while capacitation is by definition directed toward
producing the acrosome reaction, along the way some changes in the sperm cell may
play a role in sperm transport. Contributions to the control of capacitation by the
female tract may also be relevant to controlling sperm transport.


99
whether the mucus is an effective trap or a selective barrier for sperm. Conventional
studies of the uterine tube using immersion or perfusion fixation followed by
dehydration and preparation for light and scanning electron microscopy (Jansen,
1978,1980; Jansen & Bajpai, 1982; Schulte et al., 1985; Hunter et al., 1987; Smith et
al., 1987; Boyle et al., 1987; Abe & Oikawa, 1992) suggest the presence of a mucous
substance in the tubal lumen. The most detailed descriptions of tubal mucus come
from scanning electron microscopy studies, and in these preparations, the mucus
typically appears as a lattice overlaying the mucosa (Jansen, 1978, 1980; Hunter et
al., 1987).
The presence of mucus that completely fills the tubal lumen is suggested but,
due to the difficulties in preserving and retaining luminal mucopolysaccharides during
processing, has not been clearly shown. It was hypothesized for the present study
that, when hydrated, this mucus fills the luminal spaces, especially within the mucosal
folds and pockets, and potentially affects sperm movement.
A technique for preserving tubal mucus and maintaining its position in the
lumen during processing was developed to test this hypothesis. From frozen sections
of mouse uterine tubes treated with a combination of celloidin coating and
cetylpyridinium chloride (CPC) post-fixation, a homogeneous substance that fills many
of the luminal spaces was detected. There appeared to be regional variations and
sperm were seen within the material.


6
reservoir have not been described. While several mechanisms for sperm release have
been postulated, which actually operate and are pertinent to fertilization remains
unclear. Observations of sperm motility patterns in the uterine tube raise the
question of how motility and other aspects of sperm cell biology contribute to
reaching the site of fertilization. And, the path that sperm take in the uterine tube
and substances that they encounter may represent another level of regulation. Our
current understanding of each of these features will be reviewed.
Sperm Retention in the Isthmic Reservoir
At least for the mouse (Suarez, 1987) and hamster (Smith & Yanagimachi,
1990), it seems that the most important method of retaining sperm in the reservoir
involves adherence to the tubal mucosa. Attachment to the mucosa has also been
suggested in the rabbit (Cooper et al., 1979) and pig isthmus (Hunter et al., 1987).
The morphology of the uterine tube provides a series of mucosal folds and pockets
that appear to serve as storage crypts in some species (Nilsson & Reinius, 1969;
Suarez, 1987; Hunter et al., 1987; Smith & Yanagimachi, 1990). Smith and
Yanagimachi (1987) flushed hamster uterine tubes to remove first luminal and then
adherent sperm. There is also evidence that bull sperm bound to tubal cells in
culture (Pollard et al., 1991; Ellington et al., 1991) maintain viability and fertilizability
while unattached cells become immotile and may have disrupted acrosomes. In a
species with extended sperm storage, the little brown bat, sperm interact with and
appear to be maintained by the epithelium in the uterotubal junction (Racey et al.,


109
has been widely used to enhance mucopolysaccharide preservation (Landemore et
al., 1993; Sames & Hoyer, 1992; Cook, 1977). The combination of these treatments
appeared beneficial for preserving the contents of the tubal lumen, however, there
are limitations on the stains that can be subsequently used.
While the celloidin coating and CPC fixation were optimal for morphological
preservation, the non-treated control sections were important for verifying staining
characteristics. Celloidin, as previously reported (Cook, 1977), and CPC both
interfered with Alcian blue staining. Toluidine blue also gave anomalous results when
used with CPC or celloidin, though the stain was still picked up to some degree.
Therefore, care must be taken in drawing conclusions about staining characteristics
without using appropriate controls.
The regional variations in staining, and cyclical changes in the ampulla, difficult
to see clearly in black-and-white photographs, may indicate differences in the
composition or amount of the mucus present. Since the uterine tube was sectioned
as a coiled mass, loops from all the regions were visible in the same section and
stained together. This provided an added measure of confidence that the differences
observed were not due to variations in staining times or handling.
However, the conclusions that can be drawn from this histological
characterization are limited. The secretory cells in the mouse uterine tube are
located predominantly in the isthmus (Nilsson & Reinius, 1969) and relatively little
cyclical variation of cell ultrastructure, and presumably secretory activity, has been
observed in the murine isthmus (Nilsson & Reinius, 1969; Abe & Oikawa, 1993).


120
Cummins JM & Yanagimachi R (1982) Sperm-egg ratios and the site of the
acrosome reaction during in vivo fertilization in the hamster. Gamete Research,
5, 239-256.
Dacheux JL, Paquignon M & Combarnous Y (1983) Head-to-head agglutination of
ram and boar epididymal spermatozoa and evidence for and epididymal
antagglutinin. Journal of Reproduction & Fertility, 67, 181-189.
Davis BK (1981) Timing of fertilization in mammals: sperm cholesterol/phospholipid
ratio as a determinant of the capacitation interval. Proceedings of the National
Academy of Sciences, USA, 78, 7560-7564.
Desnoyers L & Manjunath P (1992) Major proteins of bovine seminal plasma exhibit
novel interactions with phospholipid. Journal of Biological Chemistry, 267,
10149-10155.
DeMott RP & Suarez SS (1992) Hyperactivated sperm progress in the mouse
oviduct. Biology of Reproduction, 46, 779-785.
Dravland E & Meizel S (1981) Stimulation of hamster sperm capacitation and
acrosome reaction in vitro by glucose and lactate and inhibition by the
glycolytic inhibitor a-chlorohydrin. Gamete Research, 4, 515-523.
Drobnis EZ, Yudin AI, Cherr GN & Katz DF (1988a) Hamster sperm penetration
of the zona pellucida: kinematic analysis and mechanical implications.
Developmental Biology, 130, 311-323.
Drobnis EZ, Yudin AI, Cherr GN & Katz DF (1988b) Kinematics of hamster sperm
during penetration of the cumulus cell matrix. Gamete Research, 21, 367-383.
Ehrenwald E, Parks JE & Foote RH (1988) Cholesterol efflux from bovine sperm:
II. Effect of reducing sperm cholesterol on penetration of zona-free hamster
and in vitro matured bovine ova. Gamete Research, 20, 413-420.
Ellington JE, Padilla AW, Vredenburgh WL, Dougherty EP & Foote RH (1991)
Behavior of bull spermatozoa in bovine uterine tube epithelial cell co-culture:
an in vitro model for studying the cell interactions of reproduction.
Theriogenology, 35, 977.
Ellis DH, Hartman TD & Moore HDM (1985) Maturation and function of the
hamster spermatozoon probed with monoclonal antibodies. Journal of
Reproductive Immunology, 7, 299-314.


64
the epithelium throughout the first 5 seconds. Sperm in all treatment groups were
scored by the same criteria. For qualitative observations, some sperm were observed
for up to 20 min. The proportion of free sperm in each region was calculated for
each experiment by dividing the number of free sperm by the total number of sperm
observed.
The flagellar curvature ratio (FCR) was measured as an indicator of
hyperactivation for those sperm in which, within a single video frame, the entire
principal bend could be found in focus at the maximally bent part of the beat (Suarez
et al., 1983). The restrictive criteria for choosing sperm from which to measure FCR
were required to avoid parallax errors and other image artifacts. The number of
sperm for which FCR was measured was approximately 15% of the counted sperm,
and these appeared to be representative based on observations of the sperm at
normal video speed. FCR is calculated as the straight-line distance from the head-
midpiece junction to the first inflection point of the principal bend, divided by the
curved path distance between these two points (Figure 3-1). As the principal bend
becomes flatter, the ratio approaches one; conversely, the ratio becomes smaller as
the bend becomes more curved. FCR was measured from the stopped video frames
using a Graf/Bar sonic digitizer (Science Accessories Corp., Stanford, CT) connected
to an Apple Macintosh 512K (Apple Computer, Inc., Cupertino, CA) running a
BASIC program written by W. Gottlieb and R.P. DeMott. Each measurement was
made three times and the mean was recorded for each sperm.


14
of fertilization, sperm are exposed to seminal plasma before the environment of the
female tract and this exposure seems to affect their cell biology. The effects of
seminal plasma were initially considered to prevent premature capacitation of the
sperm (Yanagimachi, 1988). Chang (1957) showed that capacitation could be
reversed by a component of rabbit seminal plasma produced in the epididymis.
Subsequently a number of specific decapacitation components were identified
(Oliphant et al., 1985). A family of proteins found in bull, guinea pig, mouse and rat
semen seem to inhibit calcium uptake by sperm (Coronel et al., 1993), a necessary
occurrence for capacitation and hyperactivation (Yanagimachi, 1988; Fraser, 1990;
Suarez et al., 1993). After insemination, these proteins, caltrins, may become
enhancers of calcium uptake (San Agustn et al., 1987; Coronel & Lardy, 1992)
presumably enhancing capacitation. A similar model of decapacitating activity and
subsequent enhancement of capacitation by boar seminal plasma proteins has been
described (Desnoyers & Manjunath, 1992). These proteins, which bind specific sperm
phospholipids and presumably stabilize the membrane, are proposed to coat the
sperm during early transport and then be removed, taking some membrane lipid
along; the resulting leakiness of the membrane allows calcium entrance (Desnoyers
& Manjunath, 1992). These proteins also bind calmodulin (Manjunath et al., 1993)
which may play a role in calcium regulation during capacitation. A calmodulin-like
protein from seminal plasma, that may help control calcium levels in buffalo sperm,
has also been reported (Sidhu & Guraya, 1993)


73
report of a progressive pattern involving sequential binding and release from the
ampullar wall.
Analysis of the proportion of free-swimming sperm at different times relative
to ovulation supports the idea that sperm are better able to stay free from the
epithelium as the time of fertilization approaches. This is one mechanism for shifting
sperm from the isthmic reservoir to the site of fertilization. Previous research
indicates that while sperm numbers in the ampulla are low, near the time of
fertilization the number of sperm reaching this region increases (Katz &
Yanagimachi, 1980; Cummins & Yanagimachi, 1982; Smith et al., 1987; Suarez, 1987).
These sperm appear to be moving up from the isthmic reservoir (Suarez, 1987). The
increased ability to remain free near the time of fertilization may reflect changes in
the sperm such as capacitation or hyperactivation, or changes in the affinity of the
tubal epithelium for sperm. There is evidence that different levels of estradiol may
affect the sticking of boar sperm to pig tubal explants (Suarez et al., 1991b;
Raychoudhury & Suarez, 1991).
FCR has proven to be a consistent indicator of hyperactivation in vitro (Suarez
et al., 1983; Suarez & Osman, 1987; Suarez et al., 1992) and has the particular
advantage of being relatively insensitive to small errors in determining the inflection
point. For measurements within the mouse uterine tube, decreases in FCR have
been a reliable indicator of hyperactivation (Suarez & Osman, 1987) and there are
distinct advantages over other indicators. Since the sperm are confined in a narrow,
convoluted lumen, especially in the isthmus, it is impossible to measure their


5
The mechanisms that control tubal sperm transport to the site of fertilization
have not been given sufficient attention for several reasons. Since it is not yet
possible to observe natural fertilization and to track the responsible sperm, inferential
approaches are required. Thus, experimental results can be difficult to interpret
definitively and conclusions must be narrowly qualified. Further, using such a
complex physiological system for experimentation is laborious, and accounting for and
holding potentially complicating factors constant can be difficult. Additionally, the
interest in manipulating reproduction and the numerous difficulties associated with
these technologies have provided a selection of research questions that can be
addressed with well controlled in vitro approaches.
However, the study of fertilization in vivo will improve our understanding of
intercellular interactions, sperm cell and reproductive tract biology, and allow
improvements to be made to manipulated reproduction technologies. Consider that
the naturally regulated system provides successful fertilization and reproduction with
a gamete ratio close to unity, whereas our artificial technologies require thousands
of times more sperm than eggs. It seems clear that there is much information, both
mechanistic and applied, to be found studying the process of sperm transport in the
uterine tube.
Features of Tubal Sperm Transport
There are several features of sperm transport through the uterine tube that
need further investigation. The biochemical mechanisms of sperm retention in the


56
Considering the current information, a possible model for the maintenance of
the isthmic reservoir involves the binding of non-capacitated, non-hyperactivated
sperm to the tubal mucosa by a sialoglycoprotein receptor that serves as a sialic acid
lectin. Subsequent loss or modification of this receptor during the course of
capacitation, potentially by desialylation, other enzymatic processing, or by absorption
onto carrier molecules in the fluid, may decrease the affinity of the sperm for the
mucosa and enable sperm to release. Hyperactivation may also contribute by
providing extra force for pulling away. The factors responsible for the binding of
non-capacitated sperm in the ampulla must also be changed during capacitation so
that the sperm arriving there are not retained.
Conclusions
The binding of hamster sperm to the tubal mucosa has been characterized
based on the ability of the sialylated glycoprotein fetuin and sialic acid to inhibit the
binding specifically in the isthmus. Non-capacitated, non-hyperactivated sperm
appear to have a sialylated component on their surface that acts as a lectin-like
receptor for sialic acid containing molecules. This mechanism is proposed to
maintain the adherence of sperm in the isthmic reservoir until the time of fertilization
approaches. The fetuin binding characteristics of the sperm appear to change
coincident with hyperactivation, the stage at which sperm can detach from the isthmic
mucosa. The area over the acrosome by which sperm adhere to the mucosa shows
a sharp reduction in fetuin binding, indicating a loss of the component involved in


28
went through a set of observable conditions during capacitation in vitro. The binding
of antibodies to sperm in each condition was determined by indirect
immunofluorescence staining patterns of fixed sperm and gold-labelled
immunostaining of live sperm. The antigens were also characterized on Western
blots. One antibody recognized an epitope that appeared to be unmasked when
sperm had reached the hyperactivated stage. Another recognized an epitope that was
lost or modified when they had reached hyperactivation. A third antibody recognized
an epitope that was lost or modified after only a short period in culture. These
antibodies are useful tools for isolating and identifying surface components that may
play a role in binding and release from the tubal mucosa.
The Morphology and Nature of Tubal Contents
In order to determine whether the tubal luminal contents could play a role in
controlling sperm transport, a histological protocol was developed to optimally
preserve the structure and position of the luminal contents. This protocol used
celloidin-stabilized cryosections of mouse uterine tube that had been post-fixed with
cetylpyridinium chloride. A substance with the staining characteristics of a
mucopolysaccharide was found to occlude some of the luminal spaces, and sperm
were observed in this substance. This tubal mucus seems to be in the path of sperm
transport and could provide a selective advantage for the passage of hyperactivated
sperm.


20
Since hyperactivation was first described in preparations of sperm capacitated
in vitro (Yanagimachi, 1969; Gwatkin & Anderson, 1969), it was originally considered
to be a part of the capacitation process. Nevertheless, there is now evidence that
while these processes may be complementary and parallel, they can be regulated
independently. Yanagimachi (1981) pointed out that hyperactivation occurs before
capacitation is complete and can be experimentally induced in the absence of
capacitation (defined by the inability to undergo the acrosome reaction). He also
noted that hyperactivation is primarily a tail associated phenomenon whereas
capacitation involves the sperm head. While this logic does not fit well with the
continual, dynamic model of capacitation developed here, it has led to clearer
demonstrations of the separate regulation of the two events.
Assessing the contribution of the carrier protein bovine serum albumin (BSA)
that is typically used in capacitation medium, Neill and Olds-Clarke (1987) reported
that mouse sperm could develop hyperactivation, but could not be fully capacitated
without protein present. Altering the sodium bicarbonate levels in capacitating
medium has allowed the production of seemingly non-hyperactivated, capacitated
hamster sperm (Boatman & Robbins, 1991). The finding of separate calcium influxes
associated with hyperactivation and the acrosome reaction implies that there are at
least two mechanisms of calcium ion regulation at work (Suarez et al., 1993) and
lends further support to the idea of separable control.
There is little other information directly relating to the cellular/molecular basis
of controlling hyperactivation. For unclear reasons, increased levels of cAMP seem


CHAPTER 6
SUMMARY
Controlled sperm transport through the uterine tube is an important
phenomenon for providing successful fertilization. The results described here pertain
to four aspects of the control of sperm transport to the site of fertilization. The
nature of the binding of sperm to the tubal mucosa in the isthmic reservoir was
investigated using inhibition experiments and showing the association of the inhibitor
to sperm. Potential differences in transport based on the motility pattern of the
sperm were analyzed by qualitatively and quantitatively characterizing the movement
of sperm within the uterine tube. Markers or possibly controlling features for the
physiological changes of the sperm coincident with binding and release from the
isthmic reservoir and their changing motility were developed by generating stage-
specific monoclonal antibodies. Finally, a specialized histological method was
developed to investigate the nature and localization of tubal mucus that may
potentially regulate the type of sperm able to progress in the tubal environment.
The interaction between sperm and mucosa in the isthmus was shown to
involve a lectin-like association between a sialylated glycoprotein on the sperm and
a sialic acid-bearing ligand. The binding capabilities of sperm for both the
competetive inhibitor and the mucosa itself seem to change as the sperm goes
through the capacitation sequence, specifically, by the time they switch to
112


115
by the mucous secretions. In the end, only sperm that have capacitated at the
appropriate rate and hyperactivated make it to the eggs.


45
% Free Sperm
50
40
30
20
10
0
Isthmus
AIJ
Region
Ampulla
Fetuin
Sialic
Figure 2-3. Bar graph showing regional differences in inhibition by fetuin and sialic
acid. Error bars represent SEM.


125
Jansen RPS (1978) Fallopian tube isthmic mucus and ovum transport. Science, 201,
349-351.
Jansen RPS (1980) Cyclical changes in the human fallopian tube isthmus and their
functional importance. American Journal of Obstetrics and Gynecology, 136,
292-308.
Jansen RPS & Bajpai VK (1982) Oviduct acid mucus glycoproteins in the estrous
rabbit: ultrastructure and histochemistry. Biology of Reproduction, 26,155-168.
Jeulin C, Soufir P, Weber P, Laval-Martin D & Calvayrac R (1989) Catalase activity
in human spermatozoa and seminal plasma. Gamete Research, 24, 185-196.
Jones R, Shalgi R, Hoyland J & Phillips DM (1990) Topographical rearrangement
of a plasma membrane antigen during capacitation of rat spermatozoa in vitro.
Developmental Biology, 139, 349-362.
Kao K-J & Klein PA (1986) A monoclonal antibody-based enzyme-linked
immunosorbent assay for quantitation of plasma thrombospondin. Journal of
Clinical Pathology, 86, 317-323.
Kapur RP & Johnson LV (1988) Ultrastructural evidence that specialized regions of
the murine oviduct contribute a glycoprotein to the extracellular matrix of
mouse oocytes. Anatomical Record, 221, 720-729.
Katz DF, Overstreet JW & Drobnis EZ (1989) Factors regulating mammalian sperm
migration through the female reproductive tract and oocyte vestments. Gamete
Research, 22, 443-469.
Katz DF & Yanagimachi R (1980) Movement characteristics of hamster spermatozoa
within the oviduct. Biology of Reproduction, 22, 759-764.
Katz DF & Yanagimachi R (1981) Movement characteristics of hamster and guinea
pig spermatozoa upon attachment to the zona pellucida. Biology of
Reproduction, 25, 785-791.
Killian GJ, Chapman DA, Kavanaugh JF, Deaver DR & Wiggin HB (1989) Changes
in phospholipids, cholesterol and protein content of oviduct fluid of cows
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capacitation of hamster sperm. Journal of Ultrastructure Research, 64, 1-13.


71
Table 3-2. Attachment status interactions for FCR with tubal region and sperm
location analyzed by two-factor ANOVA showing mean SEM for
each treatment combination.
Attachment Status2
Free Stuck
Region1,2
Isthmus
0.656 0.034
n=33
0.777 + 0.013
n=85
Ampulla
0.786 0.029
n=28
0.795 0.034
n=28
Location1
Lumen
0.750 0.030
n=31
0.759 0.024
n=44
Fold
0.680 0.036
n=30
0.796 0.014
n=69
1. Significant interaction (p < 0.05) with Attachment Status.
2. Differences between treatments (ie., free vs. stuck, and isthmus vs.
ampulla) are significant (p< 0.05). Overall treatment means and SEM
are
Free: 0.716 + 0.024
Isthmus: 0.743 0.014
Lumen: 0.756 + 0.019
Stuck: 0.782 0.013
Ampulla: 0.790 0.022
Fold: 0.761 + 0.015
(Used with permission from DeMott & Suarez, 1992).


43
% Free Sperm
30 =
25
Fetuin Sialic Asalo. Buffer
Treatment
Figure 2-2. Bar graph showing effect of treatment on sperm. Sperm counted from
entire uterine tube, n = number of uterine tubes, error bars represent
SEM, bars marked with different letters differ significantly.


33
by cervical dislocation at 1400 h on Day 1, approximately 12 hours before the females
were expected to ovulate (Hafez, 1970).
From the males, the epididymides were exposed and the caudae were
punctured with a 25 ga. needle. A drop of epididymal contents was placed in 1 ml
of hamster capacitation medium that had been prewarmed to 37C and equilibrated
under 5% C02. Sperm were allowed to disperse for 5 minutes at 37 C then the top
2/3 of the suspension, containing the highly motile fraction, were removed. After the
motility was assessed, the suspension was centrifuged at 140 G for 5 minutes to
concentrate the sperm. The bottom 100 /nl of this concentrate was retained and
sperm concentration was determined with a hemacytometer. Concentrations were
generally 1-2 X 108 sperm/ml. From this stock suspension, dilutions were made
containing 3 X 106 sperm/ml in 500 ¡A hamster capacitation medium plus candidate
inhibitors dissolved in incomplete capacitation medium (see Table 2-1) or incomplete
medium alone and incubated at 37C for 10 minutes. At each experiment, the
assignment of treatments to the first and second uterine tube preparation was
randomized by flipping a coin.
While the sperm samples were being prepared, females were killed in the
same manner and their uterine tubes were removed and uncoiled. To accomplish
this, the cranial tip of the uterine horn was bisected and lifted up to allow the ovarian
mesenteries to be cut. These segments were rinsed with prewarmed medium and
placed in petri dishes in a small drop of medium. One of the dishes was placed in
a 37C, 5% C02 incubator while the other uterine tube was dissected. The side to


54
of free sperm seen in these experiments, in agreement with previous observations
when non-capacitated, non-hyperactivated sperm were injected into the hamster
uterine tube (Smith & Yanagimachi, 1991) speak to the strength of this interaction
for fresh sperm. However, once sperm have been capacitated in vitro (Smith &
Yanagimachi, 1991) or begun using the hyperactivated motility pattern following
natural mating (see Chapter 3), they are able to release from the tubal mucosa.
Smith and Yanagimachi (1991) demonstrated that this difference was likely due to
changes in the sperm, not the condition of the uterine tube. While the use of
hyperactivated motility may provide additional force for sperm to break free, there
is substantial support for a mechanism of release involving the loss of the fetuin-
binding substance.
The binding patterns seen with colloidal gold-labelled fetuin indicate that
during incubation to the hyperactivated stage, the fetuin binding affinity over the
dorsal and anterior surface of the acrosomal region decreases. This is the region of
the sperm head that typically binds to the tubal epithelium (Suarez, 1987; Smith &
Yanagimachi, 1990). The reduction in the fetuin binding ability is thus well
correlated in terms of both timing and localization with release from the tubal
mucosa.
In a study on zona pellucida glycoprotein binding to sperm, fetuin was used
as a control for non-specific binding (Mortillo & Wassarman, 1991). The study found
very low levels of fetuin binding sites on the plasma membrane of acrosome intact,
capacitated sperm and slightly higher levels on the acrosomal membranes of reacted


131
Rikmenspoel R (1984) Movements and active moments of bull sperm flagella as a
function of temperature and viscosity. Journal of Experimental Biology, 108,
205-230.
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associated with hamster sperm. Journal of Experimental Zoology, 258, 69-74.
Roberts RM, Baumbach GA, Buhi WC, Denny JB, Fitzgerald LA, Babelyn SF &
Horst MN (1984) Analysis of membrane polypeptides by two-dimensional
polyacrylamide gel electrophoresis in Molecular and Chemical
Characterization of Membrane Receptors, edited by JC Venter & LC
Harrison. New York: Alan R. Liss, Inc., pp. 61-113.
Rochwerger L & Cuasnicu PS (1992) Redistribution of a rat sperm epididymal
glycoprotein after in vitro and in vivo capacitation. Molecular Reproduction and
Development, 31, 34-41.
Rodriguez-Martinez H, Einarsson S & Larsson B (1992) Spontaneous motility of the
oviduct in the anesthetized pig. Journal of Reproduction and Fertility, 66,
615-624.
Roldan ERS & Harrison RAP (1990) Molecular mechanisms leading to exocytosis
during the sperm acrosome reaction in Fertilization in Mammals, edited by
BD Bavister, J Cummins & ERS Roldan. Norwell: Serono Symposia USA, pp.
179-196.
Roth J, Lucocq JM & Charest PM (1984) Light and electron microscopic
demonstration of sialic acid residues with the lectin iron Limax flavus: a
cytochemical affinity technique with the use of fetuin-gold conplexes. Journal
of Histochemistry and Cytochemistry, 32, 1167-1176.
Roth J, Taatjes DJ, Lucocq JM, Weinstein J & Paulson JC (1985) Demonstraton of
an extensive trans-tubular network continuous with the golgi apparatus stack
that may function in glycosylation. Cell, 43, 287-295.
Saling PM & Lakoski KA (1985) Mouse sperm antigens that participate in
fertilization. II. Inhibition of sperm penetration through the zona pellucida
using monoclonal antibodies. Biology of Reproduction, 33, 527-536.
Sames K & Hoyer S (1992) Age-related histochemical staining patterns of
glycosaminoglycans in cell nuclei of different regions of the rat brain: A pilot
study. Archives of Gerontology and Geriatrics, 14, 75-84.


41
Table 2-2. Carbohydrate binding treatments for blotted sperm components.
Treatment
Cone.
Conditions
Detection
Fetuin/gold1
0.5 ¡iglml
overnight, 4C
Ag enhance
Fetuin/gold
0.5 fxg/ml
1 h, 22C
Ag enhance
LFA2
10 fig/ml
overnight, 4C
fet/gold,
Ag enhance
LFA +
10 ig/ml +
overnight, 4C
fet/gold,
Sialic acid3
10 mM
Ag enhance
CPA4
10 /xg/ml
overnight, 4C
HRP
CPA +
10 ig/ml +
overnight, 4C
HRP
Fetuin
1 mg/ml
T Fetuin conjugated to 5 nm colloidal gold particles.
2. Lectin from the garden slug, Umax flavus, recognizes sialic acid (Roth et al.,
1984).
3. n-acetylneuraminic acid.
4. Lectin from the chickpea, recognizes several sialic acid containing
glycoproteins including fetuin (Kolberg et al., 1983).


75
free and reattaching to the epithelium (Smith et al., 1987). A recent study indicates
that hamster sperm incubated in capacitating conditions before being introduced into
the uterine tube lose their stickiness and may encounter the epithelium and not
attach (Smith & Yanagimachi, 1991).
Based on the two-factor ANOVA results, it appears that differences in FCR
should not be interpreted simply in terms of the effects of each factor we examined.
Although the factors tubal region, sperm attachment status, and sperm location
relative to the lumen all significantly affected FCR, their major importance lies in
how they vary in combination, rather than in how each singly affects FCR. The
statistical interactions imply that there are physiological interactions whereby the tract
features act on the sperms potential to affect the movement pattern and progress.
The large difference between free and stuck sperm in the isthmus compared to the
ampulla may reflect a reduction in the proportion of the relatively high FCR,
regularly beating sperm that were not seen to move up the uterine tube. In the
isthmus there may be more distinct populations, one containing the sperm that have
begun erratic, low FCR beating, and are able to break free and move along the
uterine tube, and another one containing the stuck, regularly beating, high FCR
sperm that do not move up to the ampulla. The population in the ampulla may be
more homogeneous, containing only the sperm able to use erratic beating and move
up from the isthmus. Separating the sperm in this manner need not imply sperm
selection based on the ability to become hyperactivated. Interactions between the
relative timing of hyperactivation and capacitation could produce only a small


FACTORS AFFECTING SPERM TRANSPORT IN THE
MAMMALIAN OVIDUCT
By
ROBERT P. DEMOTT
A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
1993


63
IN), sperm movement within the uterine tubes was recorded at 30 frames/second,
along with time-date information to 0.01 second (Model VTG 33, For-A Co., Ltd.,
Newton, MA), on a Panasonic AG-7300 SuperVHS video cassette recorder
(Panasonic Industrial Co., Secaucus, NJ). Recording both uterine tubes was
completed in approximately 30 minutes. Preliminary experiments indicated that
extending the observation period of an uterine tube up to an hour or more was
associated with vigorous, uncoordinated contractions of the tissue, and the lumen
would begin to fill with refractile spheres, presumably released by the epithelium.
Similar stress responses were found if the uterine tube was roughly handled during
straightening or compressed too much under the coverslip. Data was not collected
from preparations that contained refractile spheres or exhibited these contractions.
From the videotapes, observed on a Panasonic WV-5410 monitor, sperm were
counted and categorized for region of the uterine tube they were found in (isthmus
or ampulla), location (in the lumen or between mucosal folds), and attachment status
(stuck or free). The regions were easily distinguished by their morphology (Suarez,
1987). The location was scored as "lumen" if the sperm was swimming in the central
luminal space or stuck to the epithelium where it was in direct contact with the
central luminal contents. Location was scored as "fold" if the sperm was out of
contact with the central lumen, either in an isthmic pocket, or an ampullar
longitudinal fold. The attachment status was recorded based on the first 5 seconds
that each sperm was visible. If a sperm swam freely during any portion of this period
it was scored "free." It was scored "stuck" if it appeared to remain associated with


53
fetuin gold and with the two sialic acid recognizing lectins, LFA and CPA, were
striking. Sialylated molecules on sperm have previously been identified and changes
during maturation have been documented (Nicolson & Yanagimachi, 1972;
Hammerstedt et al., 1982; Feuchter et al., 1988; Magargee et al., 1988; Rankin et al.,
1989; Bains et al., 1993b). Capacitation also seems to involve changes in the
sialylation patterns of sperm. Treatment with neuraminidase accelerated the
capacitation of rabbit and guinea pig sperm in vitro (Gwatkin et al., 1972; Oliphant,
1976; Srivastava et al., 1988) and sialylated molecules from sperm have been found
released into capacitation medium (Focarelli et al., 1990). For hamster sperm, the
binding of sialic acid lectins decreases during capacitation (Ahuja, 1984; Nicolson &
Yanagimachi, 1972). Part of the decrease in sialylation may represent the loss of a
fetuin binding component responsible for maintaining the adherence of sperm in the
isthmic reservoir.
The regional restriction of the inhibition to the isthmus suggests that the fetuin
binding component is only involved in binding in the isthmus. This specificity
matches the restriction of the sperm reservoir to the isthmus. Binding of non-
capacitated sperm in the ampulla apparently proceeds by another mechanism and is
presumably irrelevant in vivo, since non-capacitated sperm cannot pass through the
isthmus to reach the ampulla.
Alteration of the interaction between the isthmic epithelium and the fetuin-
binding component as part of the sperm changes occurring during capacitation and/or
hyperactivation may allow release from the reservoir. The extremely low numbers


39
15 minutes, it was sonicated in three 20 second bursts separated by 30 seconds (Heat
Systems Ultrasonics, model W-225R, Farmingdale, NY). The extraction was
continued for another 45 minutes and then the mixture was cleared by centrifugation,
12,000 G, 10 minutes. Total protein concentration in the supernatant was determined
using the Biorad protein assay (Biorad Labs, Richmond, CA). Concentrations ranged
from 1-1.5 mg/ml. The supernatants were stored at 80 C until used.
To extract hyperactivated sperm components, caudal epididymal contents were
collected from 4 males and pooled after swim up. Motility and concentration were
assessed and a series of 15-ml centrifuge tubes were set up containing 3 X 106
sperm/ml or 6 X 106 sperm/ml in 10 ml of capacitating medium. When the sperm
were hyperactivated, determined as described above, the top 8 ml from each tube
was pooled, protease inhibitors were added, and the sperm were pelleted and washed
as above. Sperm concentration was determined after they were suspended in 2.25
ml of extraction buffer. Final concentrations were approximately 2 X 108/ml.
Thereafter, sperm were treated as above.
Fresh and hyperactivated sperm extracts were thawed and prepared for
reducing polyacrylamide gel electrophoresis (PAGE) by boiling for 3 minutes in the
presence of 10% 2-mercaptoethanol (Buhi et al., 1989). Bromophenol blue was
added for color. Samples (500-800 /xg) and molecular weight standards were
separated with 4.5% stacking gels and 10% running gels in a Tris-glycine buffer
system (Roberts et al., 1984). Stacking current was 15 mA per gel and running
current was 30 mA per gel.


81
Fusion. The fusion was carried out in a standard manner using polyethylene
glycol and selection on HAT medium (Simrell & Klein, 1979; Kao & Klein, 1986).
Splenocytes were fused with SP2/0 cells. Fused cells were plated at a concentration
to yield 3-10 different populations in each culture well. Supernatants from the
resulting hybridoma cultures were screened for anti-sperm activity using an indirect
immunofluorescence assay.
Screening. To identify antibodies against epitopes that were modified as
sperm went through their functional changes, we compared indirect
immunofluorescent staining patterns of fresh, agglutinated, hyperactivated, and
capacitated sperm. Supernatants that showed differential patterns of labelling
between sperm stages were selected for cloning.
In order to maximize the time between hyperactivation and the completion of
capacitation, the concentrations of motility stimulators, BSA and metabolic substrates
in the medium were optimized to provide rapid hyperactivation and slow capacitation
based on previous reports of the effects of modifications of hamster capacitation
medium (Dravland & Meizel, 1981; Bavister, 1989). The composition of modified
hamster capacitation medium is shown in Table 4-1. Using this medium, populations
of hyperactivated sperm could be obtained 60-90 minutes before capacitation was
complete (assay described below).
Caudal epididymal sperm were allowed to disperse into 1 ml of modified
hamster capacitation medium for 5-10 minutes at 37C, then the top 2/3 of the sperm
suspension was collected. Sperm numbers were adjusted to 3 X 106 sperm/ml and


51
Staining with CPA was similar to the previous results though the labelling was
generally dim. The stronger fetuin binding bands could be detected and one band
at 42-44 kDa that had not been detected with fetuin or LFA was observed (data not
shown). CPA labelling was inhibited by fetuin.
Discussion
Since fetuin and sialic acid, but not asialofetuin, are able to inhibit sperm
binding to the tubal mucosa, the terminal sialic acid residues of the fetuin molecule
appear to be important for blocking the interaction. This implies that the binding
between sperm and the uterine tube involves a specific interaction with sialic acid.
At least for hamster sperm, none of the common sugars other than sialic acid seem
to be involved in sperm binding to the uterine tube. Also, it does not appear that
charge interactions are the basis of this interaction, since poly-l-lysine was also
ineffective as an inhibitor. Sialic acid is an effective inhibitor by itself, but as might
be expected, the large fetuin molecule is significantly more effective. The
glycoprotein contains 12-13 carbohydrate chains that end in sialic acid (Spiro &
Bhoyroo, 1974; Krusius et al., 1976), providing multiple binding possibilities to
stabilize the interaction.
The labelling of the fresh sperm over the region of the head that
characteristically binds to the tubal mucosa (Suarez, 1987; Smith & Yanagimachi,
1990) and identification of specific sperm glycoproteins that label with fetuin on


127
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capacitation. Human Reproduction, 5, 71-74.
Hunter RHF (1981) Sperm transport and reservoirs in the pig oviduct in relation to
the time of ovulation. Journal of Reproduction and Fertility, 63, 109-117.
Hunter RHF (1984) Pre-ovulatory arrest and peri-ovulatory redistribution of
competent spermatozoa in the isthmus of the pig oviduct. Journal of
Reproduction and Fertility, 72, 203-211.
Hunter RHF (1988) The Fallopian Tubes. Their Role in Fertility and Infertility. New
York: Springer-Verlag.
Hunter RHF, Flechon B fe Flechon J-E (1987) Pre- and peri-ovulatory distribution
of viable spermatozoa in the pig oviduct: a scanning electron microscope
study. Tissue and Cell, 19, 423-436.
Hunter RHF fe Nichol R (1983) Transport of spermatozoa in the sheep oviduct:
preovulatory sequestering of cells in the caudal isthmus. Journal of
Experimental Zoology, 228, 121-128.
Hunter RHF isthmus and ampulla of pig oviducts during the phase of sperm storage.
Journal of Reproduction and Fertility, 77, 599-606.
Hyde BA rabbit oviduct explants in vitro. Journal of Reproduction and Fertility, 78, 83-91.
Iacobelli S, Garcea N fe Angeloni C (1971) Biochemistry of cervical mucus: a
comparative analysis of the secretion from the preovulatory, postovulatory,
and pregnancy periods. Fertility and Sterility, 22, 727-734.
Ishijima S (1990) Changes in the beating pattern of spermatozoa during maturation
and capacitation in Fertilization in mammals. Edited by BD Bavister, J
Cummins fe ERS Roldan. Norwell,MA: Serono Symposia,USA, pp. 101-110.
Ito M, Smith TT fe Yanagimachi R (1991) Effect of ovulation on sperm transport in
the hamster oviduct. Journal of Reproduction and Fertility, 93, 157-163.


87
labelling with HL 787, an IgM class antibody, decreased over the head and midpiece.
Labelling at hyperactivation and capacitation remained low. Labelling with HL 772,
an IgG1? decreased over the head and midpiece between the agglutinated and
hyperactivated stages. Labelling of fresh sperm was similar to agglutinated, and
capacitated continued to show the decrease seen at hyperactivation. HL 778, another
IgM, showed the opposite reaction at hyperactivation, increasing in intensity and
redistributing from a spotty pattern with isolated areas at the rostral tip of the head
and the neck region to an evenly distributed pattern over the entire head. Again, the
fresh stage resembled agglutination and the capacitated stage resembled
hyperactivation. HL 784, an IgG1 relevant to the acrosome reaction rather than
sperm transport, stained the heads of the acrosome-reacted, capacitated sperm
brightly and evenly. It did not stain the heads of non-capacitated sperm that had lost
their acrosomes, presumably precociously or during processing, however. It also
brightly stained the acrosome itself whether intact or in fragments (data not shown).
This antibody would be useful for studies of the acrosome reaction but was not
further characterized here.
Controls labelled with normal serum, hybridoma culture medium and
secondary antibody alone characteristically showed very low signal. The detectable
fluorescence was predominately on the midpiece. The positive control, serum
obtained at the time of fusion, however, strongly labelled the entire sperm cell.
The silver-enhanced colloidal gold labelling assessed by light microscopy indicated a
positive reaction for HL 772, 778, and 787 on the surface of the sperm. Patterns,


CHAPTER 1
INTRODUCTION
Significance of Controlled Sperm Transport
There is a common perception that mammalian fertilization occurs after a
large number of sperm move up the female reproductive tract and encounter the egg.
The sheer number of sperm in the tract is seen as a guarantee that enough will make
it up to the egg. This perception is strengthened by the images we typically see of
fertilization in which the event has been contrived, either for the sake of the image,
or to ensure that fertilization takes place in an artificial system. The procedure for
in vitro fertilization, which requires large excesses of sperm, have been empirically
determined to provide high percentages of fertilization while limiting polyspermy. It
appears, however, that it is not adequate to extrapolate from these conditions to
describe fertilization in vivo.
More than 40 years ago, observations were published based on timed matings
and subsequent serial sectioning indicating that the ratio of sperm to eggs at the time
of fertilization in the rabbit (Chang, 1951) and rat (Moricard & Bossu, 1951) is
approximately 1:1. Zamboni reported similar findings in the mouse (Zamboni, 1972),
noting that it was only after fertilization was completed that excess sperm reached the
area. Serial sectioning, tubal flushing and microscopic observations of sperm within
1


60
to be observed. This modification lets us follow individual sperm longer and observe
dynamic aspects of sperm motility. These observations contribute to our
understanding of two specific phenomena: the establishment and release of sperm
from isthmic reservoirs, and the function of hyperactivated motility.
Hyperactivation is a vigorous, erratic motility pattern assumed by some sperm
in the uterine tube (Katz & Yanagimachi, 1980; Suarez, 1987). It is characterized by
sharply curved, asymmetric tail beats and frequent changes of direction (Katz et al.,
1989). Katz and coworkers (1989) have reviewed several proposed functions for
hyperactivation in the confined spaces and varied substances of the uterine tube
including an increased ability for sperm to free themselves from the tubal wall, an
increased ability to penetrate viscous or viscoelastic fluids such as the egg vestments,
and an increased probability for escaping from between epithelial folds. The possible
advantage of hyperactivation for sperm release from the tubal wall could result from
increased forces and/or torques generated in various directions. Similarly, these
increases may help sperm penetrate the cumulus matrix and zona pellucida
surrounding the egg (Katz & Yanagimachi, 1981; Katz et al., 1989). Recent evidence
for such an advantage comes from experimental comparisons of hyperactivated and
non-hyperactivated hamster sperm penetrating artificial viscous media (Suarez et. al.,
1991a; Suarez & Dai, 1992).
To address release from the isthmic reservoir and the use of hyperactivated
motility, sperm were observed in the uterine tube and the pattern of progress for
individual sperm was characterized. The effects of ovulatory status, local


19
capacitation within the tract (Yanagimachi, 1988), but few details have been sorted
out. In the rabbit, a vaginal inseminating species, capacitation was optimized by
sequential exposure to the uterine, then the tubal environment (Bedford, 1969).
Tubal secretions associate with ram and human sperm (Sutton et al., 1986; Wagh &
Lippes, 1989), especially if the sperm are first treated with uterine fluid (Voglmayr
& Sawyer, 1986). Recently, a functional role in enhancing capacitation was proposed
for tubal proteins that interact with bull sperm (McNutt et al., 1993). Clearly the role
of the uterine tube in controlling capacitation needs further definition.
Hvperactivation. This striking motility pattern has now been described for
more than a dozen mammalian species (Yanagimachi, 1988; Katz et al., 1989). First
observed in hamster sperm during capacitation in vitro (Yanagimachi, 1969; Gwatkin
& Anderson, 1969), sperm were later observed using the pattern within the hamster
ampulla (Katz & Yanagimachi, 1980) and in the mouse uterus and uterine tube
(Suarez & Osman, 1987). Additionally, hyperactivated sperm have been flushed from
the tracts of female mice (Phillips, 1972), rabbits (Cooper et al., 1979; Suarez et al.,
1983), hamsters (Cummins & Yanagimachi, 1982b), and sheep (Cummins, 1982a).
The specific form of movement appears to differ from species to species and
is somewhat hard to describe accurately, as evidenced by the array of
anthropomorphic terms that have been used, e.g., dashing, dancing and bobbing
(Yanagimachi, 1988; Katz et al., 1989). Generally it involves increased amplitude of
the flagellar waveform, usually asymmetrically, producing an erratic path, and may
involve changes in the 3-dimensional aspects of beat propagation (Katz et al., 1989).


I certify that I have read this study and that in my opinion it conforms to
acceptable standards of scholarly presentation and is fully adequate, in scope and
quality, as a dissertation for the degree of Doctor of Philosophy.
4
V
Susan Suarez, Chair
Associate Professor of
Veterinary Medicine
I certify that I have read this study and that in my opinion it conforms to
acceptable standards of scholarly presentation and is fully adequate, in scope and
quality, as a dissertation for the degree of Doctor of Philosophy^_
William Buhi
Associate Professor of Biochemistry
and Molecular Biology
I certify that I have read this study and that in my opinion it conforms to
acceptable standards of scholarly presentation and is fully adequate, in scope and
quality, as a dissertation for the degree of Doctor of Philosophy.
yi\ Osf,\rJ^
Maarten Drost
Professor of Veterinary Medicine
I certify that I have read this study and that in my opinion it conforms to
acceptable standards of scholarly presentation and is fully adequate, in scope and
quality, as a dissertation for the degree of Doctor of Philosophy.
Professor of Zoology


34
Table 2-1. Candidate competitive inhibitors of carbohydrate interactions in the
uterine tube.
Candidate
Concentration
Inhibitory element
Fetuin
5 mg/ml
sialic acid
Asialofetuin
5 mg/ml
galactose
Fucoidan
5 mg/ml
fucose
Ovalbumin
5 mg/ml
n-acetylglucosamine,
mannose
Poly-l-lysine
5 mg/ml
+ charge
be dissected first was randomized by flipping a coin during each experiment. Using
a dissecting microscope, the coiled uterine tube was straightened by carefully cutting
the mesosalpinx. The uterine horn was pinned in wax and only the horn and ovary
were used to hold the tissue. When the uterine tube had been completely
straightened, the ovary and ovarian bursa were removed, the first loop of the isthmus
was freed from the uterine serosa, and the uterine tube was cut through the
extramural uterotubal junction.
Incubated sperm samples were gently drawn into a 1 ml syringe equipped with
a blunted 30 ga. needle. The needle was introduced into the lumen at the
infundibulum and at the first loop of the isthmus and 50 + 5 /I was injected into


Discussion 51
Conclusions 56
3 SPERM MOTILITY PATTERNS IN THE UTERINE TUBE 58
Introduction 58
Materials and Methods 61
Results 66
Qualitative Observations 66
Quantitative Comparisons 69
Discussion 72
Conclusions 77
4 INVESTIGATION OF CHANGING SPERM ANTIGENICITY .... 78
Introduction 78
Materials and Methods 80
Production of Monoclonal Antibodies 80
Immunogold Labelling of Sperm 84
Immunoblotting of Sperm Proteins 85
Results 86
Discussion 93
Conclusions 96
5 DEMONSTRATION OF TUBAL MUCUS IN THE PATH
OF SPERM TRANSPORT 97
Introduction 97
Materials and Methods 100
Design and Sampling 100
Processing 101
Sectioning 101
Staining 102
Results 103
Discussion 104
Conclusions Ill
6 SUMMARY 112
REFERENCE LIST 116
BIOGRAPHICAL SKETCH 138
v


35
each end. In several cases, the isthmus could not be successfully injected and an
additional 50 ¡ was injected through the infundibulum. The flow of sperm
suspension through the entire uterine tube was observed to ensure that the lumen
was patent and had been completely flushed. The uterine tubes were rinsed in each
of 5 drops of medium in a clean petri dish to remove most of the sperm on the
outside and placed on a microscope slide. A coverslip, supported by 4 pillars of
silicone grease, was gently pressed down to slightly flatten the uterine tube (modified
from Suarez and Osman, 1987).
The slide chamber was immediately placed on the heated stage of an inverted
videomicroscope. The preparations were observed under bright field illumination
with a halogen light source and a 30X extra-long working distance Hoffman
Modulation Contrast objective (Modulation Optics, Greenvale, NY) (Figure 2-1).
Images from a solid state Dage CCD 72 camera (MTI Inc., Michigan City, IN) were
recorded at 30 frames/second on a SuperVHS videocassette recorder (Panasonic AG-
7300, Panasonic Industrial Co., Secaucus, NJ) along with time/date information
(Model VTG 33, For-A Co., Ltd., Newton, MA). Uterine tubes were taped
beginning at the ampulla progressing toward the isthmus. To ensure that differences
seen between the isthmus and ampulla were not an effect of the sequential taping,
in several uterine tubes the isthmus was taped briefly before the usual sequence was
followed. In several other uterine tubes, the ampulla was retaped after the usual
progression had been completed.


92
mW
Figure 4-4. 2-D Immunoblot of fresh sperm extract labelled with HL 787.


CHAPTER 3
SPERM MOTILITY PATTERNS IN THE UTERINE TUBE
Introduction
The types of cellular interactions involving sperm discussed in the previous
chapter are a part of the complement of regulatory mechanisms that many cell types
contain. The ability to interact with biomolecules in a specific manner and modify
these interactions is a foundation of modem cell biology. Beyond these typical
mechanisms, sperm have the unique characteristic, among the cells of complex
organisms, of being capable of relatively rapid, independent movement. Motility
provides alternative ways for sperm to interact with their environment. The
propulsive force generated by the sperm tail may affect the ability of sperm to release
from the epithelium and pass through the tubal environment. Also, the dynamic
nature of motility suggests that different patterns may be expressed in a given
environment and this then allows the assessment of functional advantages associated
with particular motility patterns. In this light, sperm motility and its modification
becomes a potential regulatory feature of sperm transport through the uterine tube.
The aim of this study was to relate the progress of sperm in the uterine tube to active
motility patterns and describe some factors that affect these patterns.
58


80
antibodies were generated against the complement of epididymal hamster sperm
antigens and screened for changes in activity associated with agglutination,
hyperactivation, capacitation and the acrosome reaction. Identifying antigenicity
changes associated with the agglutinated state and with hyperactivation were of
primary interest as these molecules may be associated with the ability of sperm to
bind and release the tubal epithelium. Three antibodies were identified that
recognized epitopes changing during agglutination or hyperactivation.
Antibody labelling patterns were identified by indirect immunofluorescent
staining of fixed sperm and were subsequently analyzed by immunogold labelling of
unfixed sperm to better localize the antigenicity and by immunoblotting to provide
a preliminary characterization of the antigens.
Materials and Methods
Production of Monoclonal Antibodies
Immunization. All chemicals were obtained from Sigma Chemical Co. (St.
Louis, MO) unless otherwise noted. Caudae epididymides from mature golden Syrian
hamsters were punctured and the sperm in epididymal fluid were diluted in an equal
volume of RIBFs adjuvant (RIBI Immunochemical Research, Inc., Hamilton, MT)
at 37C. This mixture was used immediately for subcutaneous injections into BALB/c
mice. The mice received three injections of 7-9 X 107 sperm over two months and
were tested for titer following each injection. They received a final boost of 1.2 X
108 sperm six days before fusion.


55
sperm using colloidal gold labelling and electron microscopy. Also, capacitated
mouse sperm were observed to have little ability to bind fetuin-coated beads
(Vazquez et al., 1989). These results further support the proposal that when sperm
are able to release from the tubal reservoir they have reduced fetuin binding affinity.
The differences observed in fetuin/gold labelling between blots of fresh and
hyperactivated sperm extracts suggest candidates for the fetuin binding component
involved in adherence in the isthmus. The bands that appear to be lost or sharply
reduced prior to hyperactivation, one high molecular weight band >335 kDa, and 32-
33 and 27.5 kDa bands, are obvious choices for further characterization. Also, the
potential change in the 49-50 kDa band is interesting. Further characterization and
antibodies directed against each of these fetuin binding glycoproteins would be useful
for establishing the identity of the component involved in sperm/epithelial binding.
Similarities to several previously described glycoproteins can be noted. A 50
kDa extrinsic sialoglycoprotein that seems to be localized over the acrosomal region
of cauda epididymal rat sperm has been described (Rifkin & Olson, 1985). Other rat
sperm glycoproteins at 32 and 33 kDa have been identified (Hall & Killian, 1989) and
a minor component of the sialylglycoconjugates released during human sperm
capacitation appeared at 32 kDa (Focarelli et al., 1990). Additionally, a hamster
sperm glycoprotein of epididymal origin that may be localized over the acrosome
appears to migrate around 26 kDa, and a 26 kDa wheat germ agglutinin-binding
protein was detected in mouse caudal epididymal fluid (Rankin et al., 1989).


37
The sperm on the videotapes were counted and scored as either free or bound
to the mucosa based on the first two seconds in which they were in focus. Counts
from three uterine tubes were duplicated by two separate observers to provide an
assessment of interobserver variation. From the counts, the overall proportions of
free and bound sperm were calculated as well as proportions for each region of the
uterine tube. The proportions were calculated based on all the sperm that could be
seen, approximately 200 per uterine tube. For testing by analysis of variance
(ANOVA), the proportions were transformed by taking the arcsin of the square root
(Sokal & Rohlf, 1989) and the significance level was set at p < 0.05. Values reported
are not transformed.
Binding of Fetuin to Fresh and Hvperactivated Sperm
Cauda epididymal sperm were obtained and the highly motile fraction was
collected as above. The concentration of this fraction was determined and dilutions
were set up containing 3 X 106 sperm in 1 ml of capacitation medium. Immediately
after dilution, aliquots of the non-capacitated, non-hyperactivated sperm were
incubated with either 0.05 ¡xg/ml of 5 nm colloidal gold-conjugated fetuin or an
equivalent volume of medium for 15 minutes. Hyperactivated sperm were treated
similarly once they were obtained, after approximately 3 hours of culture in a 37C,
5% CO2 incubator. Samples were checked occasionally, and when at least 70% of
the sperm were using the characteristic hyperactivated motility pattern (Suarez et al.,
1993), a sample was removed for labelling.


25
McNutt and coworkers (McNutt et al., 1993) have associated enhanced bull sperm
capacitation with tubal fluid treatment.
A wide array of other tubal secretory proteins have been characterized that
have been proposed to influence the egg or embryo. Some of these show regional
specificity, as demonstrated with rabbit (Hyde & Black, 1986), pig (Buhi et al., 1990)
and sheep (Murray, 1992) uterine tubes. Others have been demonstrated to be
under hormonal control either by variation during the estrous cycles of sheep and
mice (Sutton et al., 1986; Horvat et al., 1992; Vrcic et al., 1993) or following
hormonal treatments of rabbits (Hyde & Black, 1986; Erickson-Lawrence et al.,
1989), baboon (Verhage & Fazleabas, 1988; Verhage et al., 1990), and sheep
(Murray, 1992). Though some of these proteins obviously could not affect sperm
based on their secretory location and timing, clear demonstrations of association with
the eggs and embryos are few (Kapur & Johnson, 1988; Minami et al., 1992; Boice
et al., 1992; Buhi et al., 1993). More tubal secretions may eventually turn out to bind
sperm or indirectly affect transport out of the reservoir by altering the tubal
environment.
Another type of secretion that could potentially affect sperm transport is tubal
mucus. Because of the difficulties of preserving mucopolysaccharides, the presence
and prevalence of this material in the lumen remains unclear. In standard immersion
fixation protocols followed by routine histological preparations, the luminal contents
are lost (Schulte et al., 1985). The inclusion of polycationic alcian blue with
aldehydes and use of perfusion fixation appears to improve the retention of luminal


79
the sperm rather than the uterine tube (Smith & Yanagimachi, 1990; Smith &
Yanagimachi, 1991) and may involve the modification of a sperm glycoprotein (see
Chapter 2). Hyperactivated motility is involved in ascent from the isthmic reservoir
to the site of fertilization (Suarez, 1987, see Chapter 3) and thus physiological
changes affecting hyperactivation necessarily affect sperm transport as well.
It was hypothesized that some of the sperm changes associated with adherence
in the tubal reservoir and the onset of hyperactivated motility would be reflected as
antigenic changes on the surface of the sperm that could be detected based on
differential labelling with monoclonal antibodies directed against sperm components.
It was assumed that the changes occurring on sperm capacitated in vitro would be
similar, allowing sperm that had reached various stages of the
capacitation/hyperactivation sequence to be used to analyze antibody binding. The
antibodies identified would then serve as both markers for changes in epitope
associated with observable sperm changes and a means to characterize the antigen.
Monoclonal antibodies have previously been used to describe antigens that change
during capacitation and the acrosome reaction (ORand, 1977; Cowan et al., 1986;
Okabe et al., 1986; Saxena et al., 1986; Topfer-Petersen et al., 1990; Berger, 1990;
Jones et al., 1990).
Under capacitating conditions in vitro, hamster sperm pass through four stages
before undergoing the acrosome reaction. Sequentially, sperm begin in the fresh or
activated stage, then agglutinate head-to-head, then separate and hyperactivate
coincidentally, and finally complete capacitation (Suarez, 1988). Monoclonal


8
Smith & Yanagimachi, 1990), this mechanism seems to be the most relevant, though
its biochemical basis has not yet been determined.
Sperm Movement out of the Isthmic Reservoir
Several mechanisms for moving sperm from the isthmic reservoir to the site
of fertilization have been proposed. First, through in situ observations of sperm in
the mouse (Suarez, 1987) and hamster uterine tube (Smith & Yanagimachi, 1990)
sperm have been seen to detach from the wall and move by their own flagellar
beating. Another mechanism involved is the movement of sperm along with the
contents of the uterine tube. The most efficient propulsion is likely due to muscular
contractions of the uterine tube directed toward the ampulla. By injecting a
particulate solution into the uterine tubes and observing its redistribution in
anesthetized hamsters, Battalia and Yanagimachi (1979) noted that there is
coordinated muscular activity directing the tubal contents toward the ovary only
during the period immediately preceding ovulation. A subsequent study showed that
the shifts in ovarian steroid ratios around ovulation trigger this coordinated
movement (Battalia & Yanagimachi, 1980). Waves of muscular contraction in the
pig uterine tube, measured in anesthetized animals, also appear to change direction
around ovulation (Rodriguez-Martinez, et al., 1982).
Control of the muscular contractions may be affected by the products of
ovulation as well. Ito and coworkers (1991) propose that prostaglandins in the
ovulatory products have a local effect in the uterine tube regulating the contractions.


68

releases
t
I

sticks
releases
a
contact
,t ,
0 J
| '
1 2
I '
3 4 I 5
6 I
free
free
sticks
0


Time (sec)
Figure 3-2. Illustration of a typical pattern and timecourse for sperm progress in
the uterine tube. Made from composite tracings of a single sperm
moving within an isthmic pocket. Dotted line indicates the sperm path.
(Used with permission from DeMott & Suarez, 1992).
contracting preparations, the isthmic sperm in the pockets seemed unaffected by the
currents that interfered with the beat patterns of sperm in the central lumen. In the
ampulla, however, all sperm seemed to be subject to reorientation as the current
changed with contractions. It should be noted that the contractions in the ampulla
appeared less vigorous than the isthmic ones.


I certify that I have read this study and that in my opinion it conforms to
acceptable standards of scholarly presentation and is fully adequate, in scope and
quality, as a dissertation for the degree of Doctor of Philosophy.
Paul Klein
Professor of Pathology
and Laboratory Medicine
This dissertation was submitted to the Graduate Faculty of the College of
Veterinary Medicine and to the Graduate School and was accepted as partial
fulfillment of the requirements for the degree of Doctor of Philosophy.
December, 1993
4
Dean, College of Veterinary
Medicine
Dean, Graduate School


46
% Free Sperm
50
Treatment
Fetuin
Sialic
H Asalo
Buffer
Figure 2-4. Bar graph showing that effect of treatment is due primarily to isthmic
inhibition. Error bars represent SEM.


108
The sperm seem to be forced to penetrate through this mucus as they move
along the uterine tube, not just trapped in it when they are on or near the epithelial
surface (Hunter et al., 1987; Suarez et al., 1991b). These results support the
observations of sperm appearing to be enmeshed in a viscous material while
swimming in the uterine tube. Also, since an advantage for hyperactivated sperm in
penetrating viscous and viscoelastic media has been demonstrated (Suarez et al.,
1991a; Suarez & Dai, 1992), hyperactivation may be functional not only for
detachment from the isthmic reservoir and egg penetration, but may also be
advantageous for movement in luminal mucus.
The strand-like morphology of the luminal contents in untreated frozen
sections is reminiscent of the results seen with standard scanning electron microscopy.
Previously published micrographs show luminal secretions as a layer adherent to the
epithelium, or as strands or a honeycomb (Jansen & Bajpai, 1982; Jansen, 1980;
Jansen, 1978). While some of these sections leave the impression that there is a
matrix which may fill the lumen, they do not clearly illustrate the extent or nature of
the luminal contents.
Sections prepared using either celloidin coating or CPC fixation alone yielded
intermediate results. In both cases, preservation was better than untreated sections,
but stronger staining characteristics and a more homogeneous appearance of the
contents was achieved by combining the treatments. Celloidin coating has recently
been used successfully to stabilize colonic mucus (Szentkuti et al., 1992; Szentkuti &
Eggers, 1990). The use of polycationic additives such as CPC to aldehyde fixatives


4
It is also well established that the sperm that will go on to fertilize eggs are
derived from the population in the isthmic reservoir. Using the timed ligation
approach, Harper (1973b) demonstrated that the rabbit sperm that reach the isthmus
relatively early are sufficient for fertilization and Hunter (1984) demonstrated that
it was the boar sperm that had rapidly populated the isthmus that were responsible
for fertilization. The closing of the uterotubal junction shortly after mating in the
mouse (Zamboni, 1972; Suarez, 1987) implies that the fertilizing sperm must come
from the early arriving population. In the hamster, Smith and Yanagimachi (1991)
flushed out the sperm that had not bound to the epithelium after mating and later
obtained fertilized eggs from these uterine tubes. In an earlier study (Smith &
Yanagimachi, 1990), they had found that at 2 hours post-insemination, the number
of sperm in the isthmus remains fairly constant. Since there was not yet ascent to the
ampulla (Smith et al., 1987), this implies that a stable population forms early and
provides the fertilizing sperm. Considering all these observations, our current model
for fertilization in vivo is that the fertilizing sperm ascend rapidly through the lower
portion of the female reproductive tract and are retained in a reservoir in the
proximal portion of the uterine tube. Around the time of ovulation, after a species
specific delay, a very few of these sperm complete passage to the ampullary-isthmic
junction where they meet and fertilize the eggs. In this model, retention,
maintenance, and release of sperm in the isthmus seem to serve as a key control
point for fertilization. Yet, the functional details of sperm binding, release and ascent
through the uterine tube remain poorly described.


134
Suarez SS, Katz DF, Owen DH, Andrew JB & Powell RL (1991a) Evidence for the
function of hyperactivated motility in sperm. Biology of Reproduction, 44,
375-381.
Suarez SS & Osman RA (1987) Initiation of hyperactivated flagellar bending in
mouse sperm within the female reproductive tract. Biology of Reproduction, 36,
1191-1198.
Suarez SS, Redfern K, Raynor P, Martin F & Phillips DM (1991b) Attachment of
boar sperm to mucosal explants of oviduct in vitro: possible role in formation
of a sperm reservoir. Biology of Reproduction, 44, 998-1004.
Suarez SS, Varosi SM & Dai X (1993) Intracellular calcium increases with
hyperactivation in intact, moving hamster sperm and oscillates with the
flagellar beat cycle. Proceedings of the National Academy of Sciences, USA, 90,
4660-4664.
Sutton R, Nancarrow CD & Wallace ALC (1986) Oestrogen and seasonal effects on
the production of an oestrus-associated glycoprotein in oviducal fluid of sheep.
Journal of Reproduction and Fertility, 11, 645-653.
Szentkuti L & Eggers A (1990) Stabilization of pre-epithelial mucus gel in cryostat
sections from rat colon with celloidin. Stain Technology, 65, 179-181.
Szentkuti L, Staacke S & Busche R (1992) Light microscopic lectin histohemisty on
celloidin stabilized cryostat sections of rat colon. Biotechnic & Histochemistry,
6, 360-362.
Tesarik J, Mendoza C & Carreras A (1991) Expression of D-mannose binding sites
on human spermatozoa: comparison of fertile donors and infertile patients.
Fertility and Sterility, 56:1, 113-118.
Tessler S & Olds-Clarke P (1981) Male genotype influences sperm transport in
female mice. Biology of Reproduction, 24, 806-813.
Thibault C, Gerard M & Heyman Y (1975) Transport and survival of spermatozoa
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12
and use in the uterine tube mean that the change to this pattern may contribute to
the regulation of sperm transport in the uterine tube.
Maturation. The maturational changes that occur primarily in the
epididymis include modifications of the glycoproteins on the sperm surface, changes
in the lipid composition of the membrane, and stabilization of the tail structures and
condensed nucleus by disulfide bonding (Yanagimachi, 1988). Membrane
glycoproteins may be modified, added, or removed and some of these changes have
been associated with functional changes in sperm. A protein that appears to modify
boar sperm, preventing the head-to-head agglutination seen in corpus epididymal
sperm, was extracted from caudal epididymal fluid (Dacheux et al., 1983).
The proteins present on rat sperm from the caput, corpus, and caudal
epididymis and changes induced by incubating these sperm with caudal epididymal
fluid have been analyzed by two-dimensional polyacrylamide gel electrophoresis (Hall
& Killian, 1989). They demonstrated that a variety of different glycoproteins appear
and disappear from the sperm membrane as they move along the epididymis and that
treatment with caudal fluid can cause some of these changes.
Extrinsic rat sperm proteins of the acrosomal region that are produced in the
caudal epididymis have been identified (Rifkin & Olson, 1985). Additionally, changes
in the localization of rat sperm antigens occurring during epididymal passage and
capacitation have been reported (Petruszak et al., 1991; Phillips et al., 1991;
Rochwerger & Cuasnicu, 1992). Hamster sperm proteins that are added in the
epididymis have also been described (Moore & Hartman, 1984; Smith et al., 1986;


62
obtained shortly before and shortly after ovulation at all of the desired sperm
incubation time points.
Uterine tubes were removed by clamping across the uterine horn at the level
of the intramural uterotubal junction with a pair of forceps, cutting the horn just
caudal to the forceps, and lifting the uterine tube and ovary up so that the ovarian
mesenteries could be cut. Uterine tubes and ovaries were placed in a petri dish and
kept moist with medium that had been prewarmed to 37 C and equilibrated under
5% C02. The uterine tubes were uncoiled by cutting the mesosalpinx while handling
only the tip of the uterine horn and the ovary. The ovarian bursa was cut away once
the ampulla was uncoiled. The tip of the uterine horn was cut off at the end of the
dissection leaving a straightened uterine tube from the extramural uterotubal junction
to the infundibulum. Once straightened, the uterine tubes were placed on a
microscope slide with a small drop of medium and covered with a coverslip supported
by silicone grease (modified from Suarez and Osman (1987). They were stored in
a 37C, 5% C02 incubator when not being observed.
The uterine tube preparations were observed, beginning about 15 minutes
after killing the mouse, through a 30x Hoffman Modulation Contrast objective
(Modulation Optics, Greenvale, NY) on a Zeiss Axiovert inverted microscope with
a heated stage (Carl Zeiss, Inc., Thornbrook, NY). A xenon stroboscopic light
source (Model 10030, Chadwick Helmuth Co., Inc., El Monte, CA) was used to
reduce exposure of sperm to light and provide crisp images in individual video
frames. Using a Dage CCD 72 solid-state camera (Dage MTI, Inc., Michigan City,


137
Zhu JJ, Barratt CLR & Cooke ID (1992) Effect of human cervical mucus on human
sperm motion and hyperactivation in vitro. Human Reproduction, 7,1402-1406.


36
Figure 2-1. Photomicrographs of hamster sperm within the tubal isthmus.
Magnification 30X.


101
Processing
Mice were killed by cervical dislocation and the uterine tubes were dissected
free by cutting across the uterotubal junction and through the ovarian bursa. Most
of the uterine tubes were placed in a copper mesh basket and snap frozen for 30
seconds in isopentane (2-methylbutane, Fisher Chemical Co., Fair Lawn, NJ) cooled
with liquid nitrogen. The basket was placed on dry ice. A drop of O.C.T. frozen
sectioning medium (Ames, Elkhart, IN) was added to a Beem tube (Polysciences,
Warrington, PA) that had been cooled on dry ice. As the O.C.T. hardened, the
uterine tube was introduced, followed by O.C.T. to fill the tube. The tube was
immediately placed in isopentane for an additional 30-45 sec. Then, the frozen
O.C.T. block was pressed out of the tube, wrapped in Parafilm and stored at -80C.
For comparison, one uterine tube from each group was fixed for 24 hours at
4C in 1% glutaraldehyde/4% formaldehyde in phosphate buffer (0.176 M) with 0.5%
CPC (Sigma, St. Louis, MO) added to help preserve the negatively charged mucus.
The fixed uterine tubes were then processed for routine paraffin histology.
Sectioning
The blocks containing frozen tissue were cut at -18C to -21C in a Reichert
Histostat cryostat (Scientific Instruments, Buffalo, NY) and 10-12 gm sections were
mounted on acid-washed slides. Some slides had been coated with 0.05% Elmers
Glue-all (Borden Industries, Columbus, OH) in distilled water (M. Baccala, personal
communication). The sections were dried for 15-45 min on a 40C warming plate.


CHAPTER 4
INVESTIGATION OF CHANGING SPERM ANTIGENICITY
Introduction
There is a wide variety of evidence showing that there are modifications of the
mammalian sperm cell surface between the time sperm leave epididymal storage and
the time they reach the site of fertilization (Yanagimachi, 1988). Antigenic
rearrangements (ORand, 1977; Cowan et al., 1986; Okabe et al., 1986; Jones et al.,
1990), glycosylation pattern changes (Kinsey & Koehler, 1978; Ahuja, 1984; Cross &
Overstreet, 1987), and alterations of the lipid structure of the sperm membranes
(Scott et al., 1967; Davis, 1981; Go & Wolf, 1985; Ehrenwald et al., 1988) have all
been described. Some of these changes have been associated with capacitation and
the acrosome reaction (see Chapter 1), but there has been no specific investigation
of a potential role in controlling sperm transport through the uterine tube. Changes
appearing during the course of capacitation may not relate directly to preparing the
cell for the acrosome reaction but may instead play a functional role in regulating
sperm transport.
There are two observable sperm phenomena that relate to transport through
the uterine tube, release from the isthmic reservoir and the onset of hyperactivated
motility. Sperm release from the epithelium appears to be dependent on changes in
78


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11
Changes in Sperm Cell Biology
The production of sperm capable of fertilizing an egg is a continuous process
from testis to uterine tube. Sperm leave the testis immotile and undergo a series of
modifications in the epididymis, collectively called sperm maturation, which result in
the attainment of motility and the stabilization of the cell membrane (Yanagimachi,
1988). Nevertheless, they require further processing before becoming capable of
fertilization. Chang (1951) and Austin (1951) first recognized that sperm need to
spend a certain time in the female reproductive tract before they are able to fertilize
an egg. This part of the process, capacitation (Austin, 1952), has been defined
nebulously as a set of cellular changes that allow the sperm cell to undergo the
acrosome reaction when challenged with a specific inducer (Yanagimachi, 1988). The
acrosome reaction is an exocytotic event required for fertilization (Meizel, 1985).
Another observable phenomenon that can not yet be explained in terms of overall
sperm cell biology is the use of the hyperactivated flagellar movement pattern. First
noticed for hamster sperm capacitated in vitro (Yanagimachi, 1969; Gwatkin &
Anderson, 1969), this obvious alteration in frequency, amplitude, and shape of the
flagellar beat yields an erratic, albeit vigorous, pattern of movement. Hyperactivation
has since been observed within the hamster ampulla (Katz & Yanagimachi, 1980) and
in the upper regions of the mouse reproductive tract (Suarez & Osman, 1987). While
the relationship of hyperactivation to the other steps in the process of preparing a
sperm for fertilization remains unclear, its occurrence in parallel with capacitation


122
Fraser LR & Monks NJ (1990) Cyclic nucleotides and mammalian sperm
capacitation. Journal of Reproduction & Fertility, 42, 9-21.
Fraser LR, Umar G & Sayed S (1993) Na+-requiring mechanisms modulate
capacitation and acrosomal exocytosis in mouse spermatozoa. Journal of
Reproduction and Fertility, 97, 539-549.
Fusi FM, Lorenzetti I, Vignali M & Bronson RA (1992) Sperm surface proteins after
capacitation Expression of vitronectin on the spermatozoan head and laminin
on the sperm tail. Journal of Andrology, 13, 488-497.
Gaddum-Rosse P (1981) Some observations on sperm transport through the
uterotubal junction. American Journal of Anatomy, 160, 333-341.
Gaddum-Rosse P & Blandau RJ (1973) In vitro studies of ciliary activity within the
oviducts of the rabbit and pig. American Journal of Anatomy, 136, 91-104.
Gaddum-Rosse P & Blandau RJ (1976) Comparative observations on ciliary currents
in mammalian oviducts. Biology of Reproduction, 14, 605-609.
Gahmberg CG, Kotovuori P & Tontti E (1992) Cell surface carbohydrate in cell
adhesion. Sperm cells and leukocytes bind to their target cells through specific
oligosaccharide ligands. APMIS Supplement, 100, 39-52.
Gist DH & Fischer EN (1993) Fine structure of the sperm storage tubules in the box
turtle oviduct. Journal of Reproduction and Fertility, 97, 463-468.
Go KJ & Wolf DP (1983) The role of sterols in sperm capacitation. Advances in
Lipid Research, 20, 317-330.
Go KJ & Wolf DP (1985) Albumin-mediated changes in sperm sterol content during
capacitation. Biology of Reproduction, 32, 145-153.
Graham ERB (1966) Fetuin in Glycoproteins; Their Composition, Structure and
Function, edited by A Gottschalk. New York: Elsevier, pp. 353-361.
Gwatkin RBL & Anderson OF (1969) Capacitation of hamster spermatozoa by
bovine follicular fluid. Nature, 224, 1111-1112.
Gwatkin RBL, Anderson OF & Hutchinson CF (1972) Capacitation of hamster
spermatozoa in vitro: the role of cumulus components. Journal of Reproduction
and Fertility, 30, 389-394.
Hafez ESE (1970) Reproduction and Breeding Techniques for Laboratory Animals.
Philadelphia: Lea & Febiger.


3
The tubal region of the reproductive tract serves to store and support sperm
for a number of other taxa that have lengthy periods between insemination and
fertilization, including bats (Krutzsch et al., 1982; Racey et al., 1987) and the fat
tailed dunnart (Breed et al., 1989). There are also birds (Bobr et al., 1964a, 1964b;
Bakst, 1992) and reptiles (Halpert et al., 1982; Palmer & Guillette, 1988; Kumari et
al., 1990; Gist & Fischer, 1993) that have sperm storage sites in various regions of the
female reproductive tract.
The formation of the sperm reservoir occurs relatively quickly after
fertilization. Using serial sectioning after timed matings, Smith and coworkers (Smith
et al., 1987) showed that the isthmic reservoir in the hamster is populated with sperm
from 1-3 hours after the onset of mating. Population of the reservoir in the mouse
appears to be functionally limited to the first hour after mating, as the uterotubal
junction begins constricting after this point (Zamboni, 1972; Suarez, 1987). Observing
sperm within excised mouse uterine tubes, Suarez (1987) noted that by 1-2 hours
post-coitus there are many sperm in the lower isthmus and tubal portion of the
uterotubal junction, but that the intramural region, where the junction is within the
muscular walls of the uterus, is constricted and sperm numbers are low. By ligating
the reproductive tract at various points and various times after mating, Hunter (1981)
demonstrated that the reservoir is established within 1 hour in the pig isthmus. For
the cow, a vaginal inseminator where the interval between mating and ovulation may
be up to 30 hours, formation of the reservoir occurs around 8 hours after
insemination (Wilmut & Hunter, 1984; Hawk, 1987).


67
seem able to release from the epithelium any more frequently than those that used
the erratic pattern intermittently. Sperm were seen to move in a similar fashion in
both the isthmus and ampulla. There appeared to be relatively more dynamic sperm
in the ampulla; specifically, there were no large clusters of regularly beating attached
sperm.
Those sperm which released from the epithelium and swam freely did so for
a maximum of approximately 5 seconds. Within this time they would again stick to
the epithelium (Figure 3-2). During free swimming, sperm used both erratic, highly
curved beats, and regular, moderately curved beats. This cycle was seen to repeat
up to four times. The direction that a sperm swam after release appeared to be
random. Sometimes the sperm would reattach the first time it contacted the
epithelium, and sometimes it would bounce off, frequently making a direction change,
and continue swimming freely. Sperm swam across isthmic pockets only to stick to
the other side, others swam out of pockets and across the lumen to stick again, and
some swam out of one pocket and into an adjacent one without entering the main
area of the lumen.
In the mouse tubal isthmus, the predominant folding feature of the mucosa is
separate, relatively narrow-mouthed, occasionally branching pockets oriented
transverse to the central lumen. In the ampulla, there are predominantly longitudinal
folds that provide lengthy channels up the uterine tube lateral to the central lumen.
Sperm were more scattered and appeared to be less sheltered from the luminal
contents and flow in the ampullar folds than in the isthmic ones. For example, in