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1 CANINE SPERMATOZOA: PURIFICATION, ASSESSMENT, AND PRESERVATION By TAMEKA CERISE PHILLIPS A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEG REE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2010
2 2010 Tameka C. Phillips
3 To my Lord and Savior Jesus Christ, because all that I am is because of you Faith without works is dead. (James 2:20)
4 ACKNOWLEDGMENTS First and foremost, al l praises goes to God who is the head of my life, for without him I am nothing. I would like to express my appreciation to those who contributed in any fashion for the completion of this dissertation. I am equally grateful for my Ph.D. funding provided by the Florida Education Fund McKnight Doctoral Fellowship and the College of Veterinary Medicine Office of Research and Graduate Studies. To my PhD advisor, Dr. John Verstegen, thank for the decision to stay at the UF as my advisor. To my committee, thank yo u for the support, guidance, edited materials, and the ability to spend time in your labs and clinics. To Dr. Joseph DiPietro and Dr. Louis Archbald thank you for guiding me back to Florida to pursue my Ph.D. To my wonderful family and friends, there is n ot enough space to speak the volumes of acknowledgements that I have for you. So, I say thank you for all the food, laughter, love, prayers, support, and correction. Finally, to my research dogs: Bashful, Borat, Deogee, Freckles, and Jimbo this would not h ave been possible without each of you. Thank you all. Kimmie we made it!
5 TABLE OF CONTENTS page ACKNOWLEDGMENTS ...................................................................................................... 4 LIST OF TABLES ................................................................................................................ 7 LIST OF FIGURES .............................................................................................................. 8 LIST OF ABBREVIATIONS ................................................................................................ 9 ABSTRACT ........................................................................................................................ 10 CHAPTER 1 INTRODUCTION ........................................................................................................ 12 Dissertation Objectives ............................................................................................... 13 2 LITERATURE REVIEW .............................................................................................. 16 Purification .................................................................................................................. 16 Assessment ................................................................................................................ 19 Microscopy ........................................................................................................... 19 Evaluation of Sperm Motility ................................................................................ 20 Evaluation of Sperm Morphology ........................................................................ 23 Evaluation of Sperm Functional Test and Membrane Integrity .......................... 25 Evaluation of Sperm at Molecular Level .............................................................. 28 Evaluation of Sperm Membrane Proteins (Antigens or Receptors) ................... 30 Preservation ................................................................................................................ 32 3 EFFICACY OF FOUR DIFFERENT DENSITY GRADIENT SEPARATION MEDIA TO REMOVE RED BLOOD CELLS AND NON VIABLE SPERMATOZOA FROM CANINE SEMEN ............................................................... 38 Introduction ................................................................................................................. 38 Materials and Methods ............................................................................................... 39 Materials ............................................................................................................... 39 Animals ................................................................................................................. 40 Experiments ......................................................................................................... 40 Statistical Analysis ............................................................................................... 42 Results ........................................................................................................................ 42 Discussion ................................................................................................................... 43 4 IDENTIFICATION OF SPERM PROTEINS SPAG6, SP17, SP56, AND CATSPER2 IN THE CANINE ..................................................................................... 50
6 Introduction ................................................................................................................. 50 Materials and Methods ............................................................................................... 54 Materials ............................................................................................................... 54 Animals and Sperm Preparation .......................................................................... 55 Experiments ......................................................................................................... 56 Statistical Analysis ............................................................................................... 57 Results ........................................................................................................................ 57 Discussion ................................................................................................................... 60 5 CHARACTERIZATION OF SPERM PROTEINS SPAG6, SP17, SP56, AND CATSPER2 IN CANINE BY WESTERN BLOT ANALYSIS ...................................... 70 Introduction ................................................................................................................. 70 Materials and Methods ............................................................................................... 71 Materials ............................................................................................................... 71 Animals and Sperm Preparation .......................................................................... 72 Experiments ......................................................................................................... 73 Results ........................................................................................................................ 75 Discussion ................................................................................................................... 76 6 EFFECTS OF CANINE SEMEN PRESERVATION ON SPERM PROTEIN EXPRESSION ............................................................................................................. 84 Introduction ................................................................................................................. 84 Materials and Methods ............................................................................................... 87 Materials ............................................................................................................... 87 Animals ................................................................................................................. 88 Sperm Preparation and Preservation .................................................................. 89 Experiments ......................................................................................................... 90 Effect of chilled extenders on motility and acrosomal status ....................... 90 Effect of chilling and different extenders on sperm protein expression ....... 90 Effect of acrosome reaction induction on sperm protein expression ........... 91 Stat istical Analysis ............................................................................................... 92 Results ........................................................................................................................ 92 Effect of chilled extenders on motility and acrosomal status .............................. 93 Effect of chilling and different extenders on sperm protein expression ............. 94 Effect of acrosome reaction induction on sperm protein expression ................. 94 Discussion ................................................................................................................... 95 7 GENERAL DISCUSSION ......................................................................................... 107 LIST OF REFERENCES ................................................................................................. 123 BIOGRAPHICAL SKETCH .............................................................................................. 139
7 LIST OF TABLES Table page 5 -1 Molecular weight standard densitogram data. ...................................................... 81 5 -2 SPAG6 at 1:200 dilution densitogram data. .......................................................... 82 5 -3 SP17 at 1:100 dilution densitogram data. ............................................................. 82 5 -4 CATSPER2 at 1:2 dilution densitogram data. ....................................................... 83
8 LIST OF FIGURES Figure page 3 -1 DGC media separation of sperm and RB Cs with a 45% and 90% Percoll gradient.. ................................................................................................................. 47 3 -2 Sperm separation media parameters for the collected fractions. ......................... 48 3 -3 Characteri stics of fractions (%) with optimal recovery for the different DGC: ...... 49 4 -1 Diagram of a sperm cell. ........................................................................................ 65 4 -1 SPAG6 immunocytochemi stry. .............................................................................. 66 4 -2 SPAG6 confocal immunofluorescence. ................................................................. 67 4 -3 Canine SP17 with fresh negative and positive controls.. ...................................... 68 4 -4 SP17 localization on fresh canine spermatozoa.. ................................................. 68 4 -5 SP56 localization on fresh canine spermatozoa. .................................................. 69 4 -6 CATSPER2 localization on canine spermatozoa. ................................................. 69 5 -1 Canine sperm protein western blot. ....................................................................... 81 5 -2 Molecular weight standard densitogram. ............................................................... 81 5 -3 SPAG6 at 1:200 dilution densitogram. .................................................................. 82 5 -5 CATSPER2 at 1:2 dilution densitogram. ............................................................... 83 6 -1 Overall motility (%) of sperm preserved at 4C overtime in different chilled semen extenders. ................................................................................................. 101 6 -2 Mean (%) of acroso me intact sperm overtime at 4C in different chilled semen extenders. ................................................................................................. 102 6 -3 SPAG6 chilled spermatozoa Day 3 of preservation at 4 C. ............................... 103 6 -4 SP17 chilled spermatozoa Day 10 and Day 20 of preservation at 4 C.. ............ 104 6 -5 Mean (%) of acrosome intact sperm before and after acrosome reaction induction. .............................................................................................................. 105 6 -6 Effect of acrosome reaction induction on sperm proteins. .................................. 106
9 LIST OF ABBREVIATION S BSA Bovine Serum Albumin CASA Computer Assisted Sperm Analysis CATSPER2 Cation Channe l, Sperm Associated 2 DGC Density Gradient Centrifugation ICC Immunocytochemistry IVF In vitro Fertilization PBS Phosphate Buffer Saline RBC s Red Blood Cells SP17 Sperm Protein 17 SP56 Sperm Fertilization Protein 56 SPAG6 Sperm Associated A ntigen 6
10 Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of theRequirements for the Degree of Doctor of Philosophy CANINE SPEMATOZOA: PURIFICATION, ASSESSMENT, AND PRESERVATI ON By Tameka Cerise Phillips May 2010 Chair: John Verstegen Major: Veterinary Medical Sciences A series of experiments were conducted to determine optimum methods to purify, assess canine spermatozoa, and detect the presence of sperm associated proteins and the effect of preservation on these proteins. Density gradient centrifugation media (DGC) were compared ( Isolate, Perc oll, PureCeption, and PureSperm) for separation of viable, motile sperm from nonmotile or dead sperm and red blood cells (RBCs). DGC media were effective tools for separation of RBCs from motile sperm. PureCeption more efficiently allowed for purification of motile sperm than Isolate and Percoll (P<0.01) in the optimal fraction. Using immunocytochemistry, ferti lity associated sperm pr oteins: SPAG6, SP17, SP56, and CATSPER2 were identified. SPAG6 (61.55 kDa) was strongly localized at the acrosomal region with none to weak localization at the flagellum. SP17 (15.45, 28.75, and 53.75 kDa) was strongly localized at the acrosome and princi pal piece with weak to moderate expression at the midpiece. SP56 was moderately localized at the acrosome with moderate to stron g localization at the flagellum, and was not confirmed using western blot. CATSPER2 (48-49 and 63 kDa) was weakly to moderately localized at acrosome with moderate to strong localization at flagellum.
11 Chill ed semen preservation (4 C) using semen extenders [Camelot, Synbiotics, CaniPRO Chill 5 and Chill 10] had decreased overall motility (%) overtime with a difference between the e xtenders when motility was assessed. No significant differences overtime of the acrosome reaction, with an average of 40% of sperm being acrosome reacted by day 20. Chilled sperm protein expression, no labeling was present by day 1 for CATSPER2, weak or no labe ling from day 3 for SPAG6, but SP17 remained labeled up to day 20. When acrosome reaction was artificially induced with SP-TALP, acrosome-intact sperm decreased ~40% when compared to controls. SPAG6 and CATSPER2 had post acrosomal region staining with minimal staining of the flagellum. SP17 had no labeling present. In conclusion, PureCeption more efficiency separated motile sperm from contaminants than Isolate, Percoll or PureSperm. Th e differential expressions of the sperm proteins in fresh, chill ed preserved or acrosome -induced sperm suggest a possible role in canine spermatozoal function and their variable expressions may correlate with different functional status of the spermatozoa.
12 CHAPTER 1 INTRODUCTION An owner comes into the clinic with a se ven year old male dog. The owner complains that recently bitches have not had litters after mating with this stud dog and the owner suspects that the male dog is infertile. A breeding soundness evaluation (Johnston et al. 2001) which includes a complete physical examination, semen collection and evaluation (sperm concentration, progressive motility, mor phology, color, and volume) determines that the male dog suffers from asthenozoospermia, reduced sperm motility without any other major signs. The presence of blood is also observed in the ejaculate. The owner is informed that the cause of the reduction in sperm motility is unknown, as no evidence of disease or infection was noticed. The blood that was observed in the ejaculate was linked to a little non significant trauma of the penis. The owner is told to rest the dog and follow up with rechecks at interv als which take into account the length of the canine spermatogenesis cycle that is over 2 months (Foote et al. 1972) As it has been ruled out that the different bitches mated to the stud dog were not responsible for the observed infertility, the male fact or for infertility needed to be determined. There are many possible reasons why a male dog is suddenly termed infertile. Incomplete ejaculates, obstruction of the male reproductive tract, hormonal abnormities, environme ntal toxins, trauma, or prostatic d isease can be some of the reasons reported(Harrop 1966; Larsen 1977) Also, as dogs get older there is an association between decreased semen production and quality and the development of
13 prostatic disease that has been shown to lead to infertility (England and Allen 1992; Rijsselaere et al. 2004) A proper semen evaluation is thus needed. However it is generally admitted that the classical laboratory and clinical assessment tools (Graham 2001; Henkel and Schill 2003) currently av ailable can only guess the potential fertility value of a semen sample ; and that infertility can still be observed in otherwise apparently normal samples. For these reasons, new and more detailed approaches are required. A combination of fertility tests in cluding classical evaluation, semen purification, sperm protein assessment, effects of semen preservation, in vitro and/or in vivo fertilization, and functional tests of acrosome integrity is needed in order to gain a fuller understanding of the fertilizing ability of a semen sample. Dissertation Objectives Currently, there is no single in vitro method providing an accurate fertility prognosis. This is why for many years numerous studies have been conducted to find fertility tests trustworthy enough to dete rmine the fertilization ability of sperm (Mahi and Yanagimachi 1978; Kawakami et al. 1993; Farstad 2000a) Nowadays it is generally recognized that a combination of tests, including classical semen evaluation for motility, concentrat ion, and morphology, in addition to sperm function tests including acrosome reaction induction, will increase the accuracy of the prognosis concerning the potential fertility of an evaluated semen sample. This dissertation describes a combination of techni ques for semen purification, sperm protein assessment, effects of semen preservation on spermatozoa, and the functional test of acrosome status after acrosome reaction induction as predictors of canine spermatozoa fertility. In order to accomplish these go als the following objectives were defined and tested.
14 The objective of the first experiment was to compare commercially available density gradient centrifugation media [Isolate, Percoll, PureCeption, PureSperm] in their ability to optimally separate viable, motile spermatozoa from nonviable (non motile and/or dead) spermatozoa and red blood cells (RBC) contamination. The hypothesis tested was that there would be differences between the four DGC media in terms of their efficiency on canine spermatozoa, as det ermined by the ability to separate motile sperm from non motile and RBC for optimal sperm recovery. The objective of the second experiment was to identify and characterize using immunocytochemistry the presence of sperm proteins SPAG6, SP17, SP56, and CAT SPER2 proteins in canine spermatozoa. The hypothesis was that sperm proteins SPAG6, SP17, SP56, and CATSPER2 are localized and expressed in similar patterns in canine sperm cells as in other species and may play a role in sperm function. The objective of the third experiment was to confirm the presence of sperm proteins SPAG6, SP17, SP56, and CATSPER2 by western blot analysis on canine sperm, this way validating the immunocytochemistry results presented in the previous chapter. The hypothesis to be tested was: western blot analysis will confirm by immunolabeling in the dog the presence of sperm proteins with molecular weights corresponding to the previously described SPAG6, SP17, SP56, and CATSPER2 proteins in other species spermatozoa. The objectives of th e last set of experiments were: a) to evaluate the changes in selected sperm protein expression during chill preservation over a period of time, b) to establish a possible relationship between sperm protein expression and different semen parameters includi ng motility, capacitation, and acrosome reaction. The hypotheses to
15 be tested were: a) semen preservation overtime affect protein expression, b) differences may be observed between the different evaluated extenders, c) acrosome induced functional changes m ay be detected in relation to the preservation processes, d) acrosome reaction induction is associated with changes in the expression of the different semen proteins evaluated, e) overtime changes in motility may be related to changes associated with acros ome reaction and modification in the expression of the different proteins. Male fertility is complex. In the dog, semen assessment has been limited in comparison to other species such as mice and humans (Fulton et al. 1998; Farstad 2000a) Even in these other species, in vitro clinical and laboratory semen evaluations can only partially predict the outcome of fertility: a successful pregnancy the birth of offspring. The a chievement of our objectives will produce sound evidence in support for in vitro canine semen assessment, which will improve chances of a successful pregnancy and attaining a litter. The ov erall hypothesis of this study wa s that the incorporation of semen purification protocols sperm protein assessment, and the effects of semen preservation or acrosome reaction induction techniques to canine semen will improve the ability to determine fertilizing potential of a semen sample.
16 CHAPTER 2 LITERATURE REVIEW Purification Purification of semen prior to artificial insemination and semen preservation freezing is vital to the success of canine fertility. There are many techniques for semen purification, which can be classified in several families: the motility bas ed separation techniques (swim-up and migration sedimentation (Henkel and Schill 2003) filtration based techniques (gel, wool, or glass bead filtrations (Henkel and Schill 2003) and the density based (Percoll) techniques (Parrish et al. 1995) More recently, more complex techniques have also been presented, one of which is c entrifugal counter -current distribution analysis (CCCD) an aqueous two part partition system. This technique has been proven to show sperm heterogeneity in semen samples (Rodriguez Martinez 2003) Transmembrane migration has also been presented. It is a migration/filtration s ystem where the spermatozoa have straight channels to swim through the membrane. Unfortunately, the membrane has a very low ratio of the total cross -sectional area of the pores to the overall membrane area and the yield is extremely low (Henkel and Schill 2003) M any of these techniques are expensive, diffi cult to apply in clinical conditions and most often still at the development phase. For these reason, we will only focus on the technique of direct potential clinical application. Swim -up is an aqueous two-phase sperm separation system that reveals sperm h eterogeneity in semen samples. The swim up technique requires a short amount of time (incubation 1 hour) and is indirectly correlated with fertility as it al lows for the separation of the swimming spermatozoa from dead spermatozoa or inactive contaminant s. However, it is difficult to initially achieve high levels of repeatability and
17 the recovery rate being low; it is difficult to recover adequate numbers of spermatozoa for artificial insemination (Parrish and Foote 1987; Parrish et al. 1995; Chen et al. 1995b; Henkel and Schill 2003) Migration-sedimentation is a very gentle sperm separation system that works as a swim up method combined with a sedimentation step. Sperm separated using m igration-sedimentation represent usually a very clean fraction of highly motile spermatozoa. Nonetheless, special glass or plastic tubes are required and tubes are expensive and sensitive (Henkel and Schill 2003) This technique has not yet been applied or validated in dogs. Glass Bea ds is a column separation technique of motile spermatozoa across glass bead columns. Glass beads have a high yield selection of motile human spermatozoa. However, with glass beads there is the risk of a possible spill over of beads into the insemination medium (Henkel and Schill 2003) This technique has no t yet been applied or validated in dogs. Glass wool filtration is a variation of the previous technique in which motile spermatozoa are separated from nonmotile spermatozoa by means of densely packed glass wool fibers. Glass wool filtration is simple to pe rform, a good yield with recovery of spermatozoa with good motility, and contaminants are eliminated to a large extent. However, the technique is expensive, the filtrate is not as clean as it is with other sperm separation methods, and remnants of debris are still present (Henkel and Schill 2003) Sephadex beads can also be used in a separation column also sold commercial as SpermPrep ( http://www.zdlinc.biz/ ) a sperm separation kit to purify good quality sperm from poorly motile or dead sperm and c ontaminants. The Sephadex based
18 semen separation technique (SpermPrep) is similar to the density gradient separation methods using Percoll. However, i t has significantly lower recovery and separation abilities for sperm count and morphology (Ruiz -Romero et al. 1995; Henkel and Schill 2003) than density gradient separation methods The d ensity gradient centrifugation method is well known in the literature for separation of viable, motile spermatozoa from contaminants in several species i.e. : bull (Parrish et al. 1995; Samardzi ja et al. 2006) dog (Hishinuma and Sekine 2004) human (Miller et al. 1996; Centola et al. 1998; Hammadeh e t al. 2001a) There are different types of substrate available. The most commercially known substrate is Percoll which is composed of a silicabased colloidal/silica particles coated with polyvinylpyrrolidone (PVP). Isolate, PureCeption and PureSperm are silane -coated silica-based substrates that are also currently available and often used. Percoll is continuous or discontinuous density centrifugation media. Separation with Percoll is based upon differences in densities (live and dead sperm cell have different densities). Percoll is simple, repeatable, and faster, antibiotics are not needed and has six -fold greater recovery rate of motile spermatozoa than swim -up procedure as shown in human (Chen et al. 1995b) and in bovine (Parrish et al. 1995) However, Percoll is less effective for the recovery rate of progressively motile spe rmatozoa than swim up in c anine (Hishinuma and Sekine 2004) I t allow s for the separ ation of motile from dead spermatozoa without high diff er entiation between motile cells. It has also been suggested that contaminated cells often will be trapped at the wrong layer (Parrish and Foote 1987; Parrish et al. 1995; Chen et al. 1995b; Miller et al. 1996; Hishinuma and Sekine 2004)
19 Isolate, PureCeption, and PureSperm are more recently developed density centrifugation media that have primarily been used in human studies (Perez et al. 1997; Hammadeh et al. 2001b; Hirabayashi et al. 2006) These silane -coated silica-based substrates have become more widely used since the discontinuation of Percol l from clinical human usage (Scott and Smith 1997) In some comparative studies (Perez et al. 1997; Polit ch et al. 2004; Mousset -Simeon et al. 2004) these substrates are listed as very similar to Percoll in their ability to separate live from dead spermatozoa. However, t here are conflicting results as to whether PureSperm and PureCeption are as effective as Percoll for spermatozoa separation (Centola et al. 1998; Chen and Bongso 1999; Samardzija et al. 2006) i.e. for HIV -1 remove from motile spermatozoa (Politch et al. 2004; Kuji et al. 2008) Isolate, PureC eption, and PureSperm have not been evaluated in dogs. Assessment Regardless, of whether a semen sample is purified or not the semen sample should be analy zed for several factors such as motility, morphology, concentration, acrosomal status, and membrane i ntegrity. T here are numerous techniques for assessment of semen quality. In order to limit confusion, these techniques will be placed into categories. Microscopy Brightfield microscopy was developed in 1590 by Z and H Janssen and improved by Antonie van L eeuwenhoek in the late 1600s. Microscopy allows for the direct evaluation of sperm concentration and motility on non fixed, non stained samples. However, viability, except on fixed stained samples, can poorly be analyzed The use of fluorescence microscop y, using a fluorescent dye which emits a specific color when
20 excited, allow s for the identification of either specific cells (dead or alive) or specific location on the cell (Becker et al. 2003) Transmission electron microscopy (TEM) and scanning electron mic roscopy (SEM) allow for the observation at the subcellular level of details not visualized with light microscopy. Transmission electron microscopy functions by transmitting elections through the specimen and producing two dimensional images. S canning electron mic roscopy functions by electron beams scanning the surface of the specimen and producing three-dimensional images. Both TEM and SEM require ex pensive equipment and specialized training (Rijsselaere et al. 2005; Pesch et al. 2006) Evaluation of Sperm Motility In vivo fertilization relies on the ability of the propelling force of the flagell um to push the spermatozoa towards the oviduct to fertilize the ovulated oocyte. A disruption of flagella movement can be detrimental to fertility. For this reason, assessing the motility of the semen sample is a main component for a breeding soundness examination. In most normal dog semen samples, motile spermatozoa are more than 70%. It is beneficial if the spermatozoa are not only motile but linear, progressively motile (forward movement), so that the sperm can be transported to the oviduct for capacitat ion and fertilization to occur. Capacitation causes a change in the pattern of spermatozoa movement which becomes hyperactivated. Hyperactivated sperm have increased flagella movement with decrease linear, forward progressive movement, and increased curvil inearity. Capacitation and hyperactivation of sperm cells are prerequisite s for the acrosome reaction to occur, which is essential penetration of the zona pellucida and fertilization (Ho and Suarez 2001b; Suarez 2008)
21 Studies have shown (Gunzel Apel et al. 1993; Tak agi et al. 2001; Iguer -Ouada and Verstegen 2001a) that information regarding sperm motion characteristics is advantageous for assessment of male fertility. An example, the utilization of these motility parameters have demonstrated that canine semen freezi ng extender induced changes of sperm motion characteristics bear a resemblance to hyperactive motion dynamics of spermatozoa associated with capacitation (Nizanski et al. 2009) Furthermore, the combination of various motility parameters and other sperm function tests were strongly correlated and produced predictive values for bovine fertility (Januskauskas et al. 2001; Rodriguez Martinez 2003) Assessment of sperm motility has been subjectively performed for many years with individual visual observation using light microscopy. These methods, though cheap in cost, are variable in results due to individual interpretation and investigator level of experience Therefore, w ith the advent of semen analyzers and the standardization of sperm parameters, objective analysis was developed. Objective analysis elimina ted most of the bias es that w ere present with subjective analysis. In addition, with objective analysis, multiple factors can be assessed simultaneously such as: motility differential analysis of motion based on velocity (progressive, local, immotile, hype ractive) and path (linear, curvilinear), concentration or morphology (Verstegen et al. 2002) Motility parameters such as the following are generated by CASA systems: the percentage of motile spermatozoa; velocity average pathway (VAP); the average velocity of the smoothed cell path in rn /sec; the velocity straight line (VSL); the average velocity measured in a straight line from the beginning to the end of track in rn/sec; the curvilinear velocity (VCL); the aver age velocity measured over the actual point to point
22 track followed by the cell in rn/sec; the amplitude lateral head (ALH); amplitude of lateral head displacement in lam; the beat cross frequency (BCF); frequency of sperm head crossing the sperm average path in Hertz; the straightness (STR); the average value of the ratio VSL/VAP in percentage (straightness estimates the proximity of the cell path to a straight line, with 100% corresponding to the optimal straightness); and the linearity (LIN); the averag e value of the ratio VSL/VCL in percentage (linearity estimates the proximity of the cell track to a straight line). The Computer Assisted Semen Analyzer ( CASA) system, allow for large amounts of spermatozoa to be analyzed in a short amount of time. A CASA can be used on fresh, chilled, frozenthawed, and capacitated sperm. However, appropriate technical training to use the equipment is required. The CASA system s most often use phase contrast microscopy for image analysis However fluorescence is also sometime used particularly to eva luate viability of samples over time. Phase contrast microscopy is used for the examination of unstained specimens, especially living cells. Phase contrast microscopy has the major advantage of quickly providing better discrimin ation of moving cells from the background contaminants Disadvantages to CASA usage are its expensive equipment including phase contrast rings and objectives, variable results depending on user experience, and potential variability in parameter settings in different laboratories (Iguer -Ouada and V erstegen 2001a; Iguer -Ouada and Verstegen 2001b; Iguer -Ouada and Verstegen 2001c; Verstegen et al. 2002; Martinez 2004; Verstegen et al. 2005) .The correlation between CASA results and fertility has also been questioned (Rota et al. 19 99a; Martinez 2004)
23 If CASA systems appear optimal, intermediate systems, less expensive, easier to use but providing less objective information are also available. The Spermacue provides objective automated numeration of sperm cells. Similarly, t he Sper m Quality Analyzer (SQA) is a simple, less expensive system that can be used to assess fresh or cryopreserved sperm. It functions by detection of the variations in optical density included by sperm concentration and movement. The sperm motility index (SMI) generated by the SQA is an accurate estimation of sperm concentration and motility. The value is repeatable enough for canine sperm analysis (Iguer -Ouada and Verstegen 2001c; Rijsselaere et al. 2002b) and is of clinical interest even if not providing as thorough and informative results as the CASA systems. Evaluation of Sperm Morphology Another main component for a breeding soundness examination is assessment of sperm morphology. Morphological evaluation is important because a high number of abnormal sperm can be associated with infertility. In most normal dogs, morphological abnormal spermatozoa are less than 20%. Sperm with primary and secondary defects such as: double head, proximal or distal droplets, coiled or kinked tails are to be less than 20%. Morphology assessment, with the appropriate stain also allows for the determination of acrosomal status. I f sperm are already acrosome reacted, they may not function correctly, due to the fact that proteins and enzymes that are a part of the acrosome are no longer present and thus can not perform their specific function. Staining : To identify sperm cell struc ture and viability, numerous stains have been routinely used in clini c. Eosin nigrosin staining is commonly used to discriminate live from dead spermatozoa (sometime called the livedead stain). It is s imple and quick to use, assessing membrane integrity (live sperm do not allow the stain to penetrate the
24 cytoplasm while the modified membrane in dead sperm will not prevent the stain to penetrate). Eosin -nigrosin stain has been shown to be hypotonic and possibly cause sperm defects and incomplete stain ing of spermatozoa (Rijsselaere et al. 2002a; Tsutsui et al. 2003; Rijsselaere et al. 2005) Diff -Quik, a modification of the Wright Giemsa stain, is a basic commonly used in practice stain that differentiate s cells and cellular components based on eosinphilic (acidic) or basophilic properties. This stain is easy to use and provides fast results. In a comparative study (Root Kustritz et al. 1998) Diff -Quik was shown to significantly stain a larger percentage of morphological normal spermatozoa when compared to eosin nigosin stains. Spermac stain has a two -fold function. It differentiates normal from abnormal sperm morphology and determines the acrosomal status of spermatozoa. The stain provides quick results, easy to use, and can be used on fresh, chilled, or frozen semen (Oettle 1986; Bencharif et al. 2010) Coomassie Blue stain is an effective and inexpensive acrosomal staining method that provides fast results. The acrosome is defined as intact (un iform dark -blue staining overlaying the entire acrosome region), damaged (patchy staining over the acrosome region), or non intact (total absence of staining or staining only in the equatorial segment) (Brum et al. 2006) Immunostaining is a different more specialized and specific type of staining where the reaction can either induce a colorimetric (i mmunocytochemistry and i mmunohistochemistry ), a fluorescent (IF) or a luminescent reaction (chemiluminescence). Immunocytochemistry (ICC ) allows for the specific detection of
25 the expression of molecules (antigens) at the cellular and/or tissue level depending on the specific interaction of an antibody with the antigen of interest. The immune reaction, specific to the antibody used, is detected either by an enzyme mediated reaction associated with the local precipitation of a stain (i.e. 3, 3 diami nobenzidine tetrahydrochloride (DAB) and 5bromo-4 -chloro 3 ind olylphosphate p -toluidine salt (BCIP ) or the production of a chemiluminescent or fluorescent signal such as the green one produced by fluorescein isothiocynate (FITC). There are several advantages to using fluorescent staining: it has less interference due to cryopreservation media; it can be used in solution to analyze fluorescent living stained sperm in culture or by means of flow cyt ometry. The immunostaining approach not only is specific but allows for the detection of several parameters at the same time (membrane integrity and acrosomal status); and stains can be combined. Flow cytometry also called cell sorting, when used with sem en analysis has the advantages of allowing simultaneous evaluation of different and numerous characteristics (i.e. viability, acrosomal integrity, DNA fragmentation and integrity or mitochondrial function (Graham 2001; Evenson and Wixon 2006) However, to date its clinical applications are limited by cost, time, and the number of cells that can be analyzed in a minimal amount of time (Brewis et al. 2001; Graham 2001; Petrunkina et al. 2004) Evaluation of Sperm Functional Test and Membrane Integrity Assessment of sperm motility and morphology alone are not strong predictors of fertilization ability (Amann 1989) The incorporation in the panel of evaluation of a sperm functional test should provide a n improved and more detailed evaluation of sperm ch aracteristics. Acrosomal status, membrane integrity, and fertilization ability can be
26 tested using functional tests (Amann 1989; Farstad 2000b; Rijsselaere et al. 2005) The ultimate goal would be to establish a correlation between subjective and objective tests (functional tests) to increase the probability that in vitro evaluation will result in favorable fertility prognosis Outside the previously described basic stains to evaluate acrosomal status, more complex and expensive technologies have been descr ibed. The Chlortetracycline Assay (CTC) is used with a fluorescent antibody probe for detection of the acrosomal status and advancement of spermatozoa capacitation. In canine, there are three fluorescent patterns: uncapacitated and intact acrosome is repre sented with F -pattern, capacitated and intact acrosome is represented with B -pattern, and capacitated and acrosome reacted is represented with AR -pattern (Rota et al. 1999b; Iguer -Ouada and Verstegen 2001b; Rodriguez Martinez 2003) Fluorescent SYBR14PI ( PI propidium iodide, which binds to DNA in membrane defective cells ) is a sensitive method for separation of live, dead, and dying state spermatozoa (Kawakami et al. 2004; Rijsselaere et al. 2005; Samardzija et al. 2006) Triple staining (SNARF -1/YO -PRO 1/ethidium homodimer) permits viability and acrosomal integrity to be evaluated at the same time in fresh or frozen-thawed semen utilizing fluoresce nt microscopy or flow cytometry without removal of cryopreservation extender (Tsutsui et al. 2003; Pena et al. 2006) .Epifluorescence microscopy (CFDA/PI fluorophores) is used for the recognition of ruptured or damaged acrosomal membranes and can be used with flow cytometry. E pifluorescence with CFDA -carboxyfluorescein and PI propidium iodide fluorophores are not adequate for evaluation of the existence of
27 acrosomal defects (Graham 2001; Rijsselaere et al. 2005) Moreover, availability of equipment limits clinical application. Fluoresceinated lectins combined with propidium iodide are regularly used to detect the acros ome reaction in fresh or frozen-thawed dog sperm diluted in various ext enders. Fluoresceinated lectins, Pisum sativum agglutinin (PSA) and peanut agglutinin (PNA) are harmful due to the fact that they are extremely toxic when added to sperm incubating in c apacitating environments (Graham 2001; Kawakami et al. 2004; Rijsselaere et al. 2005) Moreover, availability of equipment li mits clinical application. Functional tests permit in vitro experimentation to test the potential fertilization ability of sperm cells. Hypo osmotic swelling test (HOST) evaluates the permeability of the sperm membrane. The HOST is advantageous due to the fact that there is a high correlation between assessment of membrane integrity and the ability of sperm to penetrate the oocyte (Kumi -Diaka 1993; Hishinuma and Sekine 2003; Samardzija et al. 2006; Bencharif et al. 2 010) If in vivo fertilization is the definitive studies answer for the fertilization ability of spermatozoa, the cost and the laborious nature of in vivo studies strongly and negatively influence the use of such in vivo technologies to assess semen qual ity through fertilization studies. To alleviate the difficulties in performing in vivo studies, additional in vitro works have been described. In Vitro Fertilization (IVF) is a technique that involves in vitro spermatozoa and oocyte interaction, which results in fertilization outside of the body. The procedure is well established and commonly used in many species: bull (Parrish et al. 1995; Samardzija et al. 2006) mice (Songsasen and Leibo 1997) human (Bungum et al. 2008) Unfortunately, this is not the case in the canine,
28 due to the low percentage of oocytes fertilized ( low percentage passing from ooc yte maturation to morula and blast ocyst stage) difficulties with in vitro development, and is largely dependency on laboratory expertise (Verstegen et al. 2005; Rodrigues and Rodrigues 2006; Songsasen and Wildt 2007) Intracytoplasmic sperm injection (ICSI) involves the injection of one spermatozoon directly into the ooplasm of the oocyte. This procedure is very useful for cases where a small number of sperm is available (Evenson et al. 2002; Henkel and Schill 2003; Rijsselaere et al. 2005) Intracytoplasmic sperm injection was performed using chilled canine semen for treatment of canine infertility (Fulton et al. 1998) They reported that the results were not as successful as in other species treated with by ICSI and that further studies are warranted. Currently, this technique is not yet available in canine. Zona pellucida binding assay functions to determine the ability of spermatozoa to bin d to and penetrate intact zona pellucida. The zona pellucida binding assay is able to detect sperm damage at the molecular level. Unfortunately, canine salt -stored oocytes and frozen -thawed oocytes do not do as well as fresh oocytes (Strom et al. 2000b) ; and canine spermatozoa chilled for 4 days have the tendency to reduce zona-binding (Strom et al. 2000a) Hemi -zona assay is performed by bisecting an oocyte by means of microdissection. The two halves of the zona are th en incubated with a semen sample. Both the hemi -zona assay and the zona pellucida assays are time-consuming and difficult procedures to perform that not yet readily and easily available in canine (Mayenco Aguirre and Perez Cortes 1998; Ivanova e t al. 1999) Evaluation of Sperm at Molecular Level Upon visual observation, sperm may have normal motility and morphology. The sperm may successfully pass functional tests. However, the male is still deemed
29 infertile. Indeed, molecular DNA defects may b e present but not detected by classical approaches. The next step is then to assess the sperm at a molecular level. At the molecular level, DNA, RNA, protein, and/or subcellular structures such as chromatin can be evaluated for defects. The detection of sp erm molecular defects may be the determinate for fertile or infertile status. Assessment of sperm at a molecular level, also allows for identification of genes and proteins of interest. Many modern techniques have been described to evaluate DNA integrity In situ Nick Translation assay allows for assessment of DNA integrity by quantifying the incorporation of biotinylated dUTP at single-stranded DNA breaks in a reaction catalyzed by the template dependent enzyme DNA polymerase I. Flow cytometry can also b e used to evaluate DNA integrity. Single -cell electrophoresis assay (COMET) is a technique that is used to assess DNA integrity. The COMET assay detects damage at the level of single and double strand breaks. Terminal transferase dUTP nick end labeling (TU NEL) assay is a common method used to assess DNA integrity by quantifying the incorporation of dUTP at single and double stranded DNA breaks in a reaction catalyzed by the template-independent enzyme, terminal transferase, TdT (Evenson et al. 2002; Rodriguez Martinez 2003) These techniques have not been evaluated and validated in canine. Sperm chromatin structure assay (SCSA) evaluates the vulnerability of chromatin to denaturation when sperm are incubated under denaturing conditions. The sperm chromatin structure assay is an objective assay that uses flow cytometry to detect abnormalities. This assay, which was first described by (Evenson et al. 1980) can be used on fresh as well as frozen -thawed semen. The assay also has the advantage of
30 repeatability and of having data supporting it as a strong predictor of semen quality (Evenson et al. 1980; Evenson et al. 2002; Nunez Martinez et al. 2005) Justified data have been presented in dogs (Nunez -Martinez et al. 2005; Shahiduzzaman and LindeForsberg 2007) Among the newer technique s developed, Fluorescent in situ hybridization (FISH) is an interesting and sensitive technique with a promising future. It permits the analysis of sperm by using chromosome-specific probes labeled with fluorochromes. Fluorescent in situ hybridization is used to map out chromosomes that contain microsatellite markers and to identify specific genes. FISH like ICC is specific and when an opt imal probe is available providing reliable results. However, FISH can not yet been applied on live sperm cells and is missing sensibility due to the low amount of DNA available into the sperm cells (Breen et al. 1999; Olivier et al. 1999; Breen et al. 2001; Gianaroli et al. 2005) Eva luation of Sperm Membrane Proteins (Antigens or Receptors) At the level of the spermatozoa, there are many proteins that can provide vital information as it relates to fertility. These proteins can be receptors localized in the membrane (proteins and pepti des receptors) or within the cytoplasm (steroid receptors) and their activation may be related to specific important functional events. They can also be antigens localized on the membrane or within the cytoplasm and be important for the overall spermatozoa activity and metabolism Sperm proteins localized on the flagellum may be associated with motility. Sperm proteins localized at the acrosomal region may be associated with capacitation and/or acrosome reaction. Knockout mice that are infertile or have reduced fertility as a result
31 of gene or protein disruption, might give clues as which sperm proteins are required for fertilization (Ren et al. 2001; Sapiro et al. 2002) Heparin -binding proteins are largely located on the surface of ejaculated spermatozoa and less on the plasma membrane of epididymal spermatozoa. Heparin is described as the most potent enhancer of capacitation of bovine and rabbit spermatozoa. Thus, heparin binding proteins function to increase affinity for heparin and enhance capacitation (Miller et al. 1990; Braundmeier and Miller 2001) Fertilization associated antigen (FAA) is a heparin-binding protein tha t binds to spermatozoa (Braundmeier and Miller 2001; Gatti et a l. 2004) and is secreted by the seminal vesicles and the prostate gland. A study has been published (de Souza et al. 2006) stating that there are heparin-binding proteins in canine seminal plasma. The report does not mention however, which fraction the seminal plasma was taken from; as the only seminal fluid in the canine would come from the prostate gland, its only sexually accessory gland. Estrogen receptor alpha (ER sent in human ejaculated spermatozoa (Aquila et al. 2004; Lambard et al. 2004; Carreau et al. 2006) Estrogen receptor beta (ER ) is present in rodent ejaculated spermatozoa (Saunders et al. 1998; Carreau et al. 2006) In the dog, ER spermatocytes, but is not expressed in the spermatozoa (Nie et al. 2002) A major olfactory receptor transcript (DTMT) and its corresponding protein have been identified on canine spermatids and the midpiece of mature spermatozoa (Vanderhaeghen et al. 1993) Due to the pattern of expression, it is hypothesized that
32 the function of the olfactory receptor and protein is associated with sperm maturation, motility, or chemota xis (Vanderhaeghen et al. 1997) Odorant receptors hOR17-4 in human and mOR23 in mouse mediate calcium signals in mature spermatozoa. However, the specific physiological function o f hOR17-4 and mOR23 is unknown (Spehr et al. 2006) The presence o f s perm associated antigen 6 (SPAG6) has been linked with sperm flagella motility and structural integrity of the axoneme central apparatus of mature sperm in mice and humans (Neilson et al. 1999; Sapiro et al. 2000; Horowitz et al. 2005) S perm protein 17 (SP17), initial ly was rep orted as a rabbit sperm protein that was highly expressed in spermatozoa and had a primary function of zona pellucida binding (Richardson et al. 1994) Sperm fertilization protein 56 (SP56), is a sperm oocyte recognition sperm protein that has an affinity for ZP3 an oocyte gly coprotein associated with sperm oocyte interaction (Cheng et al. 1994) Cation channel, sperm associated 2 (CATSPER2), is a voltage-gated protein that is present in spermatozoa (Quill et al. 2001) Nothing is known of SPAG6, SP17, SP56, or CATSPER2 spermatozoa expression in dogs. Preservation The first reportedly successful canine artificial insemination (AI) using fresh semen was done by an Italian physiologist and priest, Abbe Lazzaro Spallanzani in 1784. Dr. AE Harrop, using heat -treated pasteurized milk performed the first successful canine chilled semen insemination in 1954 (Harrop 1954) In 1969, Seager reportedly performed the first successful canine insemination with frozen -thawed semen (Seager and Fletcher 1972; Platz Jr. 1990; Farstad 2000a)
33 Preservation of semen in volves providing an optimal in vitro environment for preserving sperm viability. Semen preservation consists of fresh, chilled, and frozenthawed semen. Short -term preservation (days) includes fresh or chilled semen. Long term preservation (months and year s) includes frozen semen. Normally, canine semen is shipped chilled or frozen in canine semen extender. Semen, in chilled extender, is chilled to 45 C and placed in a shipping box with ice packaging for transport. Frozen semen, in freezing semen extender, is shipped in a l iquid nitrogen container at a temperature around 196C (Linde-F orsberg 1991) Since the first successful artificial insemination in canine species using chilled semen in 1954, several studies have been conducted in order to evaluate the effects of temperature over freezing point on semen conservation; the range of 4 5 C was determined as the best temperature conditions for sustaining semen motility by reducing gamete metabolism (Province et al. 1984; Bouchard et al. 1990; Rota et al. 1995) This temperature was demonstrated initially to allow semen conservation for up to 4 5 d, as opposed to a few hours when s emen was conserved either at room temperature or at 35 C. Different cooling techniques and semen extenders were proposed during the last 30 years (Foote and Leonard 1964; Province et al. 1984; Bouchard et al. 1990; Brown 1992; Goodman and Cain 1993; Rota et al. 1995) There are several essential components for semen extender: it must be isotonic with the semen of interest, a good buffer to avoid significant pH changes overtime, minimize cold shock and damage during chilling, provide appropriate nutrients, allowing sperm to survive, prevent microbial growth, and be relatively low in cost (Wales and White 1958; Foote 1964a; Linde Forsberg 1995) Buffers such as Tris, sodium citrate,
34 and sodium phosphate are commonly used (Rota et al. 2001; Iguer Ouada and Verstegen 2001b; Verstegen et al. 2005) The main nutrients required for sperm metabolism are glucose and/or fructose (Ponglowhapan et al. 2004) Du ring preservation when semen is chilled or frozen the semen extender must provide protection against cold shock and damage. Egg yolk or low density lipoproteins (LDL) are used to provide energy and prevent cold shock which effects cell membrane integrity (Foote and Leonard 1964; Songsasen et al. 2002; Okano et al. 2004) Last, antibiotics such as penicillin and streptomycin, or gentamycin will help prevent microbial growth. In a canine study (Rota et al. 1995) demonstrated that Tris egg yolk extender appears to be better than other extenders, such as, egg yolk milk or egg yolk cream in preservation of do g semen at 4 C. In another canine study (Iguer -Ouada and Verstegen 2001b) comparing different commercial and laboratory made extenders; it was demonstrated that a Tris -glucose egg yolk based extender had the highest quality motility and percentages of motile sperm. All extenders showed a significant improvement in motility parameters with the addition of egg yolk. Among commercial extenders that were examined Biladyl had the best motility parameters when egg yolk was added. While the Tris -glucose egg yolk extender, a laboratory created extender had the best overall motility par ameters and allowed for the longest viability after extension (more than 80% of the initial motility up to 14 days after collection). Egg yolk had an obvious effect for protecting the membranes and preventing acrosome reaction. Glucose and fructose provid e nourishment for metabolism and support for motility in sperm. There is still significant controversy in the canine as to which of the two
35 sugars has the highest activity. If Iguer -Ouada and Verstegen (Iguer Ouada and Verstegen 2001b) did consider glucose as having the most activity, it was described later that egg yolk Tris ex tender supplemented with fructose at a concentration of 70mM was superior for longterm preservation of chilled canine semen (Ponglowhapan et al. 2004) On the other hand, Tris -glucose egg yolk extender was found, after several changes of extender, to extend chilled semen motility up to 3 weeks with preservation of good quality sperm for at least 15 days. When the extender was changed, sperm cells that were thought to be nonmotile or of poor m otility were reactivated. To verify fertilization ability of this prolonged, chilled extended semen, 20 bitches were inseminated with 711 day chilled semen; 12 became pregnant proving that motility can be preserved while chilling semen in the dog and that as semen motility can be reactivated, motility without further evaluation cannot always be a reliable predictor of fertility (Verstegen et al. 2005) The Iguer -Ouada and Verstegen study (Iguer -Ouada and Verstegen 2001b) was recently confirmed (Shahiduzzaman and Linde-Forsberg 2007) to maintain preservation of good quality semen up to 14 days at 5 C. Other sugars added to a Tris -citric acid e xtender had variable effects. Galatose, lactose, trehalose, maltose, and sucrose reduced damaged acrosome percentages in equilibrated sperm. Monosacchrides (fructose and xylose) improved motility along with post -thaw viability and intact acrosome percentag es. Trehalose, xylose, and fructose significant increased total active sperm rates in frozen-thawed semen samples (Yildiz et al. 2000) The inclusion of Equex STM paste (in cluding sodium dodecyl sulfate ( SDS) demonstrated to prevent protein agglutination and coagulation) to semen extenders has
36 been confirmed to be beneficial for semen preservation (Rota et al. 1997; Pena and Linde -Forsberg 2000; Petrunkina et al. 2005; Ponglowhapan and Chatdarong 2008) In a study involving Equex (Petrunkina et al. 2005) before freezing, canine semen wa s diluted with or without Equex STM paste. The extender containing Equex STM paste had protective effect on isotonic cell volume, on regulatory function under hypertonic conditions, and on the proportion of live acrosome reacted cells. The use of egg yolk Tris -citrateglucose extender with the addition of 0.5% Equex STM paste significantly increased the proportion of spermatozoa having an intact plasmalemma after thawing compared to without Equex STM paste (Rota et al. 1997) Furthermore, there is increased long evity of the thawed spermatozoa which prolongs the maintenance of both motility and plasma membrane integrity. During freezing, a cryoprotectant such as glycerol, dimethyl sulfoxide (DMSO), methanol, or methyl glycol will protect against damage due to int racellular ice formation. Nevertheless, egg yolk has been used for many years as a membrane protectant and energy substrate for the prevention of cold shock during semen chilling and freezing. However, with the insurgence of bioterrorism and avian bird flu the usage of egg yolk in semen extenders for international shipping has decreased. Pace and Graham, reported that the low density fraction or low density lipoproteins (LDL) was the component of egg yolk that in the absence of glycerol protects motility of sperm cells during the freezing process (Pace and Graham 1974) They also suggested the presence of unkn own substances in purified egg yolk [without LDL] that diminish sperm motility. Low density lipoproteins which adheres to cell membranes during chilling, freezing, and thawing were shown to improve bull sperm motility when compared to an egg yolk extender
37 (Moussa et al. 2002) The specific protective effect of LDL on canine semen has never been analyzed. Nevertheless, it has been demonstrated that canine semen frozen wit h LDL instead of egg yolk had better post -thaw characteristics (Bencharif et al. 2008; Bencharif et al. 2010)
38 CHAPTER 3 EFFICACY OF FOUR DIFFERENT DENSITY GRADIENT SEPARATION MEDIA TO REMOVE RED BLOOD CELLS AND NON VIABLE SPERMATOZOA FROM CANINE SEMEN Introduction Man y factors can affect seminal and prostatic fluid and consequently have an impact on male fertility. Blood, urine, or other products (bacteria, debris, and pus) have been demonstrated (England and Allen 1992; Chen et al. 1995a; Johnston et al. 2001) to contaminate semen and prevent its use for artificial insemination (AI) and/or semen processing including freezing. It is well known that in dogs, blood of different origins is negatively affect ing semen preservation (Rijsselaere et al. 2004) Moreover, the aging dog is prone to prostatic disease. Prostatic disease commonly contributes to cellu lar contamination (blood, bacteria, and neutrophils) that can compromise semen quality and reduce its suitability for AI and preservation procedures. Therefore, at the time of AI or prior to application of assisted reproductive technology (ART), it is crit ical to remove contaminants prior to further processing. Removal of liquid contaminants, such as urine, can easily be achieved by centrifugation and multiple washes with semen extender. However, cellular contaminants are more of a concern as they cannot easily be spundown and excluded from the viable, motile sperm cells. The density gradient centrifugation (DGC) method has been demonstrated in many species (Miller et al. 1996; Centola et al. 1998; Samardzija et al. 2006) including dogs as being not only simple and relatively inexpensive, but also resulting in favorable sperm cell recovery. However, the fir st published studies demonstrated a poor efficiency in separation of cellular contaminants.
39 Percoll is a commonly used DGC media for sperm separation. Unfortunately, Percoll was removed from human clinical usage in 1996 and has been reported to have delet erious effect on sperm membranes (Strehler et al. 1998) However, in many non human clinics and laboratories Percoll is still used as the DGC media of choice. The objective of the study was to compare commercially available prepared density gradient centrifugation media ( Isolate, Percoll, PureCeption, PureSperm) in their ability to optimally sepa rate viable, motile spermatozoa from nonviable (nonmotile and/or dead) spermatozoa and contamination of red blood cells (RBC). The hypothesis to be tested was that there would be differences between the four DGC media in terms of their efficiency on canin e spermatozoa, as determined by the ability to separate motile from nonmotile sperm and RBC for optimal sperm recovery. Materials and Methods Materials SpermVision SAR was purchased from M initube of America (Verona, WI) CaniPRO C hill 5, canine semen exten der was purchased from Minitube of America (Verona, WI). Isolate, Percoll, and PureCeption DGC media were purchased from Irv ine Scientific (Santa Ana, CA), Pharmacia (Uppsala, Sweden), and SA GE In Vitro Fertilization, Inc. ( Trumbull, CT), respectively Pur eSperm in 40% and 80% ready -to use -solutions were purchased from Nidacon International AB ( Molndal, Sweden) The sp erm separation media gradients (Isolate, Percoll, and PureSperm) were prepared by combining the undiluted sperm separation media with CaniPRO Chill 5 semen extender to reach desired working dilutions. For all media, the pH was adjusted to be within the range of 6.8 to 7.2 and the osmolarity adjusted to be within the range of 280 to 325
40 mOsm. For RBC and spermatozoa staining Diff -Quik was purcha sed from Webster Veterinary Supply (Sterling, MA). Animals Four healthy male dogs of unknown fertility, ranging between 2 to 5 years old were used in this study. Semen was collected a maximum of three times each week with significant periods of rest (more than a week) occurring between collection cycles as collections were performed continuously during the year but only as needed. To reduce variability in the different studies concerning semen, semen was pooled from the four dogs Using a pool of semen al lowed for a reduction of variability due to animals and allowed for a reduction of the number of animals needed for every study The pools of semen submitted to the different treatments will indeed vary only depending on the effect of the treatment studied and not depending on the animals. The number of dogs is then justified by the need to have access to sample volumes large enough to allow the submission of the pools of semen to the different treatments (extenders sperm protein, etc. ). Five animals have been demonstrated to be optimum for this purpose during CASA validations and semen extenders studies (Verstegen et al. 2005) However, one of our animals had to be removed from the study just after the start of this research for health reasons. Anim al care and research was conducted under the approved protocol (# E434) by the University of Florida Institutional Animal Use and Care Committee. Experiments Semen was collected by digital manipulation (Foote 1964b; Linde-Forsberg 1991) and blood was sampled by jugular venipuncture by means of a vacutainer containing K2 EDTA from BD (Franklin Lakes, NJ). The sperm -rich fraction s of semen from the fo ur
41 dogs were pooled to reduce individual variability between trials as previously described by Silva & Verstegen (Silva and Verstegen 1995) The pooled semen was objectively analyzed with a computer assisted semen analyzer (CASA), SpermVision 1.0 Minitube of America (Ver ona, WI) for concentration, motility and patterns of motility. The semen was then washed with CaniPRO Chill 5 canine semen extender at 700 x g for 15 minutes at room temperature. After centrifugation, the supernatant was removed and the semen was re extend ed if needed with CaniPRO Chill 5 canine semen extender to reach a final volume of 4 ml and a sperm concentration around 100 x106/ml. When needed, whole blood was then added to the washed sperm at concentrations of 1 to 4% v/v to mimic a natural contaminat ion. Then a volume of 1 ml of the blood/sperm admixture was gently pipetted over 4 ml of a double layered-column ( the higher density mi xture was layered on the bottom) (Fig. 3 1) of the following working gradients: 50/90% Isolate, 45/90% Percoll, 40/80% Pu reCeption, and 40/80% PureSperm as recommended by the manufacturers. The columns were then centrifuged at 400 x g for 20 min at room temperature and the different phases of the column gradients were removed layer by layer into 1 ml fractions (A, B, C, D, E ,) and when present an additional layer or a pellet (F). The different fractions were finally analyzed using the Spermvision SAR system for sperm concentration and motility parameters. Slides were also prepared for the evaluation of the RBC/spermatozoa rat io as well as the sperm cell morphology in the different fractions. The smears were stained using Diff -Quik (Root Kustritz et al. 1998) and examined under light microscopy (200 400x). A hundred spermatozoa were counted in three fields of each slide and an average was obt ained. To evaluate morphology, ( norm al, double
42 tail, distal droplet, coiled tail, kinked tail, abnormal head shape, and detac hed head) smears of each collected fraction were stained and examined under light microscopy under oil immersion (1000x). A hundred spermatozoa were counted for each s lide with a laboratory counter Fisher Scientific (Pittsburg, PA). A minimum of 3 slides were analyzed per fraction. The study was repeated three times. Statistical Analysis One way analysis of variance (ANOVA) was used to compare spermatozoa parameters and efficiency of separation of contaminants of the different sperm separation media. Two by two comparisons were performed when P was significant (P<0.05) using the Bonferroni multiple comparisons test. Two way ANOVA was used to compare the different sperm s eparation media and fractions for morphology. Data were expressed as mean SD. Differences were considered to be significant when P<0.05 Results All media except Isolate presented a pellet which generally included the maximum number of motile spermatozoa and the lowest concentration of dead or immotile spermatozoa and RBC. The overall percent recovery of total and motile spermatozoa, the percent motility of spermatozoa in the specific fractions and RBC/ spermatozoa ratio (RBC/S) per fraction for each separ ation media are given in (Fig s 3 2 A-D ). All DGC media allowed for separation of RBC from sperm cells and the highest ratio RBC/S for each DGC media were: Isolate fraction A (29.4 29.7), Percoll fraction A (28.2 20.8), PureCeption fractions A and B (37.0 22.8, 39. 6 24.3, respectively), and PureSperm fractions A and B (25.2 5.9, 23.0 3.9, respectively). For all DGC media, the fractions with the highest RBC/S were significantly different than all the remaining fractions (P<0.05). The optimal fract ion (highest sperm
43 cell recovery, motile sperm cell recovery and overall motility respectively) in each of the media were: Isolate fraction D (33.9 29.4%; 40.99 27.9%; 71.2 21.8%), Percoll fraction D (36.4 14.5%; 39.3 15.8%; 88.6 2.3%), PureCeption fraction F (78.8 28.3%; 88.0 17.4%; 70.2 11.1%), and PureSperm fraction F (73.1 21.0%; 75.4 24.6%; 80.6 17.1%). The fraction F for PureCeption and the fractions E and F for PureSperm were significantly different than all the other fractions (P<0.05). The optimal fraction of PureCeption (Fig. 3 -3 ) had a significantly higher recovery of total and motile sperm cells compared to the optimal one for Percoll and Isolate (P<0.0163). PureCeption and PureSperm fractions F were not significantly dif ferent; however the maximum number of total and motile spermatozoa was concentrated in only one fraction (F) in PureCeption while for PureSperm they were observed in either fraction E or F as demonstrated by the high standard deviations observed (39 and 21, 44 and 24 for total and motile sperm cell recoveries respectively). DGC separation does not improve morphology (data not shown). Discussion In the canine, it has been shown that the presence of increased concentrations of blood in semen stored for a prolonged period of time has detrimental effects on sperm motility parameters, membrane integrity, and acrosome status. The detrimental effects are partially due to the high concentration of hemoglobin originating from RBC hemolysis (England and Allen 1992; Rijsselaere et al. 2004) Using bovine sperm, it was found that with an increase in blood and serum contamination, there is a decrease of in vitro fertilizing capacity (Verberckmoes et al. 2004) These studies provide the relevance for the removal of blood from canine semen.
44 In the present study, four density gradient centrifugation media were compared taking into account not only the highest recovery of total and motile spermatozoa, but also separation of red blood cells from spermatozoa and morphology preservation. The ideal media should separate the ejaculate into different fractions allowing for the highest recovery of total an d motile spermatozoa and the lowest contamination with other cells including RBC, immotile or dead spermatozoa. On a practical point of view to ease separation, the location of spermatozoa would be best in the bottom of the column, away from other cellular contaminants such as blood, which ideally would be located in upper phases. Among the evaluated media, Percoll is the most extensively used. However, it was removed from human clinical usage because of a risk of endotoxin contamination (Scott and Smith 1997) It has also been reported that Percoll has a deleterious effect on sperm membranes (Strehler et al. 1998) In a recent study, in cats it was reported that Percoll centrifugation was associated with a low final yield of sperm cells (Filliers et al. 2008) Since Percoll has been shown to provide poor results and negative effects on sperm, there is a further need for comparison of other DGC media. In the present study, Percoll centrifugation resulted in poor separation. To get a good overall recovery we would have had to pool the fractions C to E making the process more complicated. Although Isolate resulted in recovery of motile spermatozoa, it had percentages of recovery which were significantly lower than PureSperm and PureCeption. PureSperm had better percent recovery of total and motile spermatozoa than Isolate and Percoll. However, the separation of motile spermatozoa was not optimal as it was split with high variations bet ween 2 fractions (E and F). Furthermore,
45 the percentages of recovery were lower than the one observed when PureCeption was used (P<0.05 for fraction E). PureCeption resulted in the highest percent recovery of total and motile spermatozoa concentrated in on ly one fraction (fraction F), which also was characterized by a very low RBC/S ratio corresponding to our initial objective. T he centrifugation settings selected for this study were within the range that had been shown to result in the least amount of damage to spermatozoa (Parrish et al. 1995; Hishinuma and Sekine 2003; Politch et al. 2004; Samardzija et al. 2006) Our centrifugation speed being at the upper limit, made increasing the speed not an option without inducing significant sperm cell damage while decreasing the speed has been shown to reduce t he separation efficacy. If the use of centrifugation has not been shown to be optimal for the sperm cell (Aitken and Clarkson 1988; Alvarez et al. 1993; Mortimer 1994) the only other alternative presently available (swim up) has also been associated with a poor recovery in the canine Furthermore, several studies have illustrated, that using Percoll at the present centrifugation speed and time, centrifugation provided better recovery and is not associated with more sperm damage than swim up (Tanphaichitr et al. 1988; Moohan and Lindsay 1995; Chen et al. 1995b) PureCeption usage ha s up to now only been reported in human studies (Politch et al. 2004; Kuji et al. 2008) for separation of human immunodeficiency virus type 1 (HIV 1) from spermatozoa or for sperm separation. Isolate and PureSperm have been use d in human (Politch et al. 2004; Mousset -Simeon et al. 2004) and animal studies (Hernandez Lopez et al. 2005; Underwood et al. 2009) In conclusion, we have demonstrated in this study a significant difference in the percent recovery of total and motile sperm between PureCeption and Percoll and
46 difference in their ability to separation sperm between different DGC media and their ability to separation sperm from RBC. On a practical and clinical point of view, PureCeption more efficiently allowed separation of the motile sperm cells in different fractions with the highest recovery concentrated in the bottom of the column and better recovery than Isolat e, Percoll, and PureSperm.
47 Fig ure 31. DGC media separation of sperm and RBCs with a 45% and 90% Percoll gradient. One ml of sperm mixed with 1-4% whole blood for contamination was added to the layered aliquots of 45% and 90% phases. Centrifugation wa s for 20 minutes and 100 x 106 sperm cells/ml were layered. One ml fractions (A -F) were pipetted from the column and number of motile sperm, nonmotile sperm, and RBCs were determined per fraction. Diagram created by Tameka C. Phillips.
48 Fig ure 32 Sperm separation media parameters for the collected fractions. A) Isolate, B) Percoll, C) PureCeption, D) PureSperm
49 Figure 3 3 Characteristics of fractions (%) with optimal recovery for the different DGC: Isolate D, Percoll D, PureCeption F, and PureSperm F.
50 CHAPTER 4 IDENTIFICATION OF SPERM PROTEINS SPAG6, SP17, SP56, AND CATSPER2 IN THE CANINE Introduction For many species, when semen characteristics are poor, procedures such as in vitro fertilization and intracytoplasmic spe rm injection provide successful outcomes (Hammadeh et al. 2001a; Henkel and Schill 2003) In the dog, these in vitro approaches are up to now disappointing and the results poor (Fulton et al. 1998; Songsasen and Wildt 2007; de Avila Rodrigues et al. 2007) : fertilization results are low, the embryos do not develop, the oocytes availability (in number, maturation and quality) or accessibility (high lipid content and dark aspect) are mediocre. To date, only in vivo approaches remain available. Achi eving optimal pregnancy rates requires good breeding management, a fertile male and fertile female. The semen should be optimally concentrated, devoid of contaminants and of optimal motility and morphology. However, even when these parameters are present, there are still numerous cases where pregnancy is not obtained. It is believed that more than 70% of the infertile cycles are related to female causes while the male seems to be responsible for the other 30% In the previous chapter, we saw that semen contamination could be an issue and that we presently have approach es to purify contaminated semen thereby improving the quality of the ejaculate (Hishinuma and Sekine 2004; Mousset Simeon et al. 2004) L ow c oncentration, abnormal motility or abnormal morphology can be re sponsible for the infertility However, infertility can still be observed in classically evaluated normal samples. For these reasons, there is a real need for other complementary approaches to evaluate semen characteristics and function Recently sever al sperm proteins have
51 been identified some of which are directly related to sperm function and motility (Bookbinder et al. 1995; Sapiro et al. 2000; Wen et al. 2001; Quill et al. 2001) The Basic Local Alignment Search Tool (BLAST) for bioinformatics (Altschul et al. 1990) provided the technology to perform a search to scan the c anine genome for genes associated with fertility previously described in mice or other species. At the initiation of the present work, a Protein-Protein BLAST (blastp) was completed to determine in the dog if the equivalent sequences of proteins associated with sperm function and identified in mice. With confirmed similarities between the two species of specific sequences for sperm proteins, the expectation was that these proteins would be conserved and potentially identified in canine sperm. Four proteins were identified: SPAG6, SP17, SP56, and CATSPER2 with SPAG6: 11 blast hits, SP17: 2 blast hits, SP56: 6 blast hits, and CATSPER2: 1 blast hit ( www.ncbi.nlm.nih.gov/ BLAST / ). For these reasons we considered that there was a high probability to be able to identify the proteins also in the dog and developed the second p art of our work. It was expected that SPAG6 and SP56 with 11 and 6 blast hits, respectively, would have a high probability to be identified in canine sperm cells. SP17 and CATSPER2 with 2 and 1 b last hits, respectively, were more questionable but worth investigating as they have never been studied in the dog. Th e presence of sperm associated antigen 6 (SPAG6), a sperm protein, which is localized on the flagellum in mice and human, has not been inv estigated in canine. The presence of SPAG6 has been linked with sperm flagella motility and structural integrity of the axoneme central apparatus of mature sperm in mice and humans (Neilson et al. 1999; Sapiro et al. 2000) Specifically, SPAG6 is expressed on the principal piece of
52 epididymal sperm, with weaker expression located on the midpiece and head regi ons (Sapiro et al. 2000) Mature SPAG6 knock out mice are infertile and produce sperm with low concentration, abnormal motility and structural defects (fragmentation of midpiece, shortened flagella, unstable central apparatus, missing axonemes, or decapitat ions) (Sapiro et al. 2002) These mice have hydrocephalus and 50% die within 8 weeks of life T he presence of SPAG6 as observed in human and mice appear to be highly conserved among mammals (Neilson et al. 1999; Sapiro et al. 2000; Horowitz et al. 2005) However, t his sperm protein has yet to be identified in canine sperm. Sperm protein 17 (SP17) was initial ly described as a rabbit sperm autoantigen protein (RSA) that was highly expressed in spermatozoa and had a primary function of zona pellucida binding (Richardson et al. 1994) Additional studies stated that SP17 though strongly expressed in the testes, was also p resent in somatic tissues. It also functions in somatic and germ cell migration and/or cell adhesion, and is vital for heparin binding (Wen et al. 2001; Frayne and Hall 2002) SP17 appears to be a highly conse rved mammalian protein and has been observed in mouse sperm where it is expressed on the principal piece, weakly on the midpiece, and over the acrosomal region of the head (Kong et al. 1995) On human sperm, SP17 appears to be located on the principal piece, midpiece, and in scattered patches throughout the head region (Lea et al. 2004) While another group reported that SP17 was detected throughout the principal piece, but not intermediate, head, acrosome vesicle in human ejaculated sperm, and that spermatogonia, Sertoli and Leydig cells were immunonegat ive for SP17 (Grizzi et al. 2003) SP17 has yet to be identified in canine sperm.
53 Sperm fertilization protein 56 (SP56), is a sperm oocyte recognition protein that has an affinity for ZP3 an oocyte glycoprotein associated with sperm oocyte interaction (Cheng et al. 1994) Initially, SP56 was reported in mice as localized on the peripheral sperm membrane and on the outer surface of the sperm head (Cheng et al. 1994) In later reports, SP56 was demonstrated as a part of the acrosomal matrix and n ot a component of the plasma membrane (Kim et al. 2001; Buffone et al. 2008) However, in mice with an abnormal spermatozoon head shape mutation (Azh ) t hat produce d 100% abnormal sperm SP56 was localized on the fl agellum as well as the acrosome (Moreno et al. 2006) SP56 is presently accepted as an intraacrosomal component, part of the acrosomal matrix (Kim et al. 2001) expressed primarily in the acrosomal vesicle (Cohen and Wassarman 2001) In the rat and hamster, SP56 is expressed on the sperm head (He et al. 2003) SP56 has not been detected in human sperm (Bookbinder et al. 1995) nor has it been ident ified in canine sperm. Cation channel, sperm associated 2 protein (CATSPER2), is a voltage -gated protein identified in mice spermatozoa (Quill et al. 2001) Catsper2 kn ockout mice are infertile. Disruption of the CATSPE R2 gene through gene mutation does not significantly a ffect sperm production or overall motility; however, It was demonstrated in these knockout mice an inability to achieve hyperactive sperm motility at the time of activation, which is vital for in vivo fertilization (Quill et al. 2003) Mouse CATSPER2 is located on the plasma membrane o f the principal piece of the sperm tail (Quill et al. 2001; R en et al. 2001; Xia et al. 2007) while human CATSPER2 protein is located on the flagellum (Avidan et al. 2003) CATSPER2 has yet to be identified in canine sperm.
54 These proteins have critical role s in sp erm motility and in the process of fertilization. The objective of this study was to use immunocytochemistry to identify and characterize the presence of sperm proteins SPAG6, SP17, SP56, and CATSPER2 proteins in canine spermat ozoa. The hypothesis was that SPAG6, SP17, SP56, and CATSPER2 are localized and expressed in similar patterns in canine sperm cells as in other species and may have a role in sperm motility, capacitation, and/or acrosome reaction. Materials and Methods Materials SPAG6 mouse monoclon al SP17 rabbit polyclonal SP56 mouse monoclonal and CATSPER2 rabbit polyclonal antibodies were purchased from Abnova Corporation (Taipei, Taiwan) Delta Biolabs (Gilroy, CA) GeneTex, Inc. (Irvine, CA) and Aviva Systems Biology (San Diego, CA) respective ly Zamboni fixative was purchased from Newcomer Supply (Middleton, WI). SpermVision SAR and Spermac stain were purchased from Minitube of America (Verona, WI). Methanol (HPLC) was purchased from Fisher Scientific (Fair Lawn, NJ). DakoCytomation LSAB + Sys tem -HRP Kit with swine anti mouse/rabbit/goat biotinylated secondary antibody were purchased from DakoCytomation, Inc. (Carpinteria, CA). BX51 Olympus microscope was purchased from Olympus Industrial America, Inc. (Orangeburg, NY). Alexa Fluor 647 IgG goat anti mouse antibody and Alexa Fluor 594 IgG goat anti -rabbit antibody were purchased from Invitrogen (Eugene, OR). Leica TCS SP5 Laser Scanning Confocal microscope was purchased from Leica Microsystems, Inc. (Bannockburn, IL).
55 Animals and Sperm Preparati on Semen was collected from four sexually mature dogs ranging from 26 years of age according to IACUC authorization protocols # E434 and #200903106. Semen was collected a maximum of three times each week with significant periods of rest (more than a week) occurring between collection cycles as collections were performed continuously during the year but only as needed. The sperm rich fraction of the ejaculate was collected by digital manipulation in a calibrated plastic vial (Foote 1964b; Linde -Forsberg 1991) To reduce variability between trials concerning semen, the semen was pooled to have access to sample volumes large enough to allow the submission of the pools of semen t o the different antibodies tested. Using a pool of semen allowed for a reduction of varia bility due to animals and allowed for a reduction of the number of animals needed for every study. After pooling, the semen was evaluated for concentration, sperm moti lity using SpermVision SAR, and morphology using Spermac staining (Silva and Verstegen 1995; Verstegen et al. 2002) Five animals have been demonstrated to be optimum for this purpose during CASA validations and semen extenders studies (Verstegen et al. 2005) However, one of our animals had to be removed for health reasons from the program just after the start of this research. Mature BALB/c male mice were euthanized by CO2 gas and cervical dislocation, according to IACUC authorization protocol #200903106. Directly after euthanasia, the cauda epididymides were excised and washed twice in PBS at room temperature. Each cauda epididymis was punctured with a 23guage needle and sperm were released f rom the epididymis into the petri dish.
56 Experiments Brightfield immunocytochemistry : Twenty l of semen was deposited on a Superfrost/Plus microscope slide, a smear was prepared and allowed to air dry overnight. The air dried slides were fixed with Zamboni fixative for one hour at 4 C, permal ized with methanol for 10 minutes at -20 C, and processed following Dako standardized immunocytochemistry protocol (www. dako.com) using a primary mouse monoclonal antibody against SPAG6 or SP56, or primary rabbit polyclonal antibody against SP17 or CATSPER2. Primary antibody concentrations were optimized prior to experimentation and were diluted as follows: SPAG6 at 1/200, SP17 at 1/200, SP56 at 1/100, and CATSPER2 at 1/50 with PBS, incubated overnight at 4 C, rinsed in distilled water washed 3 times in PBS, then incubated 1 hour at room temperature with a swine anti mouse/rabbit/goat biotinylated secondary antibody. Chromogenic DAB + Substrate buffer were used to visualize the binding sites. In the negative controls (canine and mice) the primary antibody was omitted. Mouse sperm was used for the positive control. Visualizations were ascertained using a BX51 Olympus microscope at 200600X magnifications. This study was repeated five times. Confocal immunofluorescence : One ml of sperm was incubated with one of the following primary antibodies after dilution as follows: SPAG6 at 1/200, SP17 at 1/200, SP56 at 1/100, and CATSPER2 at 1/50 with PBS The diluted semen was then incubated at room temperature for 30 minutes in the dark, centrifuged at 2000 x g for 5 minutes in 2 ml of 2% fetal bovine serum in PBS, the supernatant was removed, incubated at room temperature for 30 minutes in the dark with a secondary Alexa Fluor 647 IgG goat anti mouse antibody or Alexa Fluor 594 IgG goat anti -rabbit antibody, and centr ifuged at 2000 x g for 5 minutes in 2 ml of 2% fetal bovine serum in PBS, the
57 supernatant was removed. The negative controls were as before with the addition of a tissue negative control consisting of immortalized cat T cells. Visualizations were ascertain ed using a Leica TCS SP5 Laser Scanning Confocal microscope at 200600X magnifications. This study was repeated three times. Assessment of immunostaining: Points of interest for localization are: acrosome, equatorial band, post acrosomal region, midpiece, principal piece, or flagella (both midpiece and principal piece (Fig, 4 -1 ). Scoring for spermatozoa staining intensity was classified: negative (no staining), weak (+), moderate (++), or strong (+++) expression. Spermatozoa were reported negative when the stain did not differ from the negative control. Statistical Analysis For each trial, three slides were labeled and 100 sperm cells were evaluated per slide ; thus = 5 with 3 replicates One way ANOVA was used to compare sperm protein expression of the indi vidual sperm protein with the three other sperm proteins. Tukey Kramer multiple comparison test was performed when the P value was significant (<0.05). Data were expressed as mean SD. Differences were considered significant when P<0.05. Results The mean values for concentration, overall motility, and forward progression of sperm samples were 596.14 373.54 x 106 sperm /ml, 98.49 1.1%, 94.19 0.80% respectively. For sperm morphology, the mean percentage of normal spermatozoa was 93 5.70% sperm. Second ary s perm abnormalities were tail defects (5.8%). The mean percentage of acrosome-intact spermatozoa was 95 2.94%.
58 SPAG6 labeling was identified on 97.6 2.88% spermatozoa counted per slide. Brightfield and confocal allowed both for the detection of a similar pattern of expression of SPAG6 immunostaining on fresh canine sperm. SPAG6 was mainly and strongly expressed in the head of the spermatozoa (Fig. 4 2C ) with a high incidence of panacrosome labeling but variation of the expression was also noticed with punctate and equatorial (Fig. 43) SPAG6 labeling. In addition, SPAG6 had a variable (n egative to moderate) expression at the level of the midpiece section of the flagellum (Fig. 4 -2C) Midpiece expression was found in less than 20% of canine spermat ozoa. The canine SPAG6 negative control (Fig. 4 -2A ) mouse positive (Fig. 4 2D), and negative (Fig. 42B) controls were as expected. No sperm were labeled in the canine or mouse sperm negative controls. In agreement with previous reports, the mouse positiv e control had stronger expression of the principal piece, with weaker expression of midpiece and acrosome. SP17 labeling was identified on 93.6 4.0 % spermatozoa counted per slide. Brightfield and confocal allowed both for the detection of a similar patt ern of expression of SP17 immunostaining on fresh canine sperm. SP17 was strongly expressed at the acrosome and principal piece, with weaker expression at the midpiece (Fig s 4 4 and 45 ). Acrosome and principal piece expression were observed in more than 85% of canine spermatozoa. The SP17 negative and positive (Fig. 4 -4 ) controls were as expected. No sperm were labeled in the canine or mouse sperm negative controls. In agreement with previous reports, the mouse positive control had strong flagella and acr osomal labeling. SP56 labeling was identified on 97.3 2.5 % spermatozoa counted per slide. SP56 was strongly expressed and localized mostly (>95%) at the principal piece. Weak to
59 moderate acrosomal expression (Fig. 4 6 ) was observed in more than 25% of th e sperm. Confocal results were negative for canine SP56 expression. The SP56 negative and positive (Fig. 4 -6 ) controls were as expected. No sperm were labeled in the canine or mouse sperm negative controls. The mouse positive control had labeling at the level of the acrosome as previously reported. CATSPER2 labeling was identified on 75.0 5 % spermatozoa counted per slide. CATSPER2 was expressed and localized at the flagella region. There was also weak expression localized at the acrosomal region (Fig. 4 7 ) and this was seen in more than 70% of the sperm. Confocal results were not consistent for canine CATSPER2 expression. No sperm were labeled in the canine or mouse sperm negative controls. The mouse positive control had labeling at the level of the flagell um as previously reported. CATSPER2 labeling was identified on 75.0 5 % spermatozoa counted per slide. CATSPER2 was expressed and localized at the flagella region. There was also weak expression localized at the acrosomal region (Fig. 4 7 ) and this was seen in more than 70% of the sperm. Confocal results were not consistent for canine CATSPER2 expression. No sperm were labeled in the canine or mouse sperm negative controls. The mouse positive control had labeling at the level of the flagellum as previously reported. The percentages for the number of spermatozoa that were labeled of the four proteins were compared. There was not a statistical difference in the labeling of SPAG6, SP17, and SP56. However, CATSPER2 was significantly labeled in fewer spermatozoa than the other three studied proteins (p<0.05).
60 Discussion Brightfield immunocytochemistry and confocal fluorescent imaging allowed us to identify and localize the four tested proteins in canine s perm To the best of our knowledge, this is th e first repor t of these proteins in canine sperm Their pattern of distribution and intensity of staining were clear. In the present study immunolabeling was observed on almost all sperm cells from neat fresh ejaculates from adult mature male dogs for SPAG6, SP17 and SP56. The percentage of sperm cells labeled by these antibodies (97.6 2.88, 93.6 4, 97.3 2.5 for SPAG6, SP17 and SP56, respectively) was not different from the percentage of acrosome normal sperm cells (95 2.4). CATSPER2 labeling (75 5.0) was the only one to be significantly lower than the other labeled proteins and lower than the percentage of acrosome-intact spermatozoa (P<0.05). In the present study it was shown that SPAG6 had strong expression localized mainly at the acrosomal region of the sp erm head. In addition, SPAG6 had negative to moderate expression localized at the midpiece of the sperm tail. The strong expression of SPAG6 localized to the canine acrosomal region of the sperm head has not been shown in mice and humans (Neilson et al. 1999; Sapiro et al. 2000) In a mouse model, SPAG6 was essentially localized to the principal piece of the flagellum and a weaker signal in the sperm tail midpiece and head region (Sapiro et al. 2000) Flagella local ization of SPAG6 in the canine was less consistent than that seen in mice and humans (Neilson et al. 1999; Sapiro et al. 2002) However it may still indicate a similar role in flagella movement and motility as found in other species. As illustrated in mice and humans, it may be proposed that SPAG6 in the canine has a function in sperm motility However, the essential involvement of SPAG6 in fertility is still
61 controversial as SPAG6 -deficent mice have several disruptions of the spermatozoa particularly at the midpiece (Sapiro et al. 2002) However, in humans with a heterozygous mutation that affects SPAG16L, which interacts with SPAG6, male infertility is not observed (Zhang et al. 2007) The exact role of SPAG6 in the canine needs to be investigated and warrants further studies. In the canine, we have shown that SP17 is localized with strong and similar intensity at the acrosomal region and the principal piece of the flagellum These findings are in agreement with those previously observed in the mouse and human (Kong et al. 1995; Grizzi et al. 2003) SP17 h as yet to be clearly demonstrated to be involved in the fertilization process. In fact, due to its l ocalization within the tail region, it has been postulated by (Frayne and Hall 2002) that the role of SP17 in oocyte binding through the zona pellucida is unlikely to be its principal function. However, direct involvement in sperm cell motility has not been demonstrated. SP17 seems to be nonspecific to sperm cells as it is also expressed in tumors and normal somatic tissues (Wen et al. 2001; Straughn, Jr. et al. 2004) In agreement with what has been shown in these species, canine SP17 function can not yet be proposed except that it may be involved in both fertilization and motility as seen with the other sperm proteins w ith flagella protein expression. In the present study, c anine SP56 was consistently expressed predominantly at the principal piece of the flagellum with some moderate acrosomal expression. The observed primary localization of SP56 in the canine was differ ent than that reported for the mouse. In the normal mouse, SP56 is reported as essentially localized at the acrosomal matrix (Kim et al. 2001; Buffone et al. 2008) However, it has been
62 demonstrated (Moreno et al. 2006) that A zh mutated mice have SP56 localized on the flagellum as well as the acrosome. The reason f or this discrepancy between normal and mutated animals is currently unknown When SP56 was first identified it was reported to be localized on the peripheral sperm membrane (Cheng et al. 1994) T his type of localization could account for the strong expression found mostly at the principal piece of the sperm tail in the current study CATSPER2 is part of a cation-channel family of four similar, non identical (CATSPER1, CATSPER2, CATSPER3, CATSPER4) proteins. Cats per 2 knockout mice are void of Cats per1 protein expression and the inverse. Hence, both proteins are required in order for the other to function (Carlson et al. 2005) The family of four proteins is essential for male fertility and hyperactive sperm motility (Qi et al. 2007) In the present study, c anine CATSPER2 flagellar localization was shown to be in agreement with that sh own in other species (Ren et al. 2001; Xia et al. 2007) However, t here was also some weak to moderate expres sion of CATSPER2 at the level of the acrosomal region. The localization at the level of the flagellum may indicate a role in spermatozoa l movement and calcium exchanges. The flagella localization of the four proteins (SPAG6, SP17, SP56, CATSPER2), comparab le to that seen in mice and humans, may suggest a similar role in motility as found in other species. For fertilization to occur, the sperm must be motile and have the ability to capacitate and undergo an acrosome reaction (Kawakami et al. 1993; Brewis et al. 2001; Witte et al. 2009) The changes to the acrosome will occur as part o f sperm activation. Capacitation is a stepwise process. During the progression through the female reproductive tract, sperm change their pattern of motility from linear, forward
63 progression to a vigorous, nonprogressive, curvi linear, hyperactivated movemen t. This change in motility pattern is a result of capacitation (Mahi and Yanagimachi 1978) It is understood that hyperactivated sperm assist with transport through oviductal secretions and binding of sperm to the zona pell uci da. Then the hyperactivated sperm will penetrate the zona pell uci da (Mahi and Yanagimachi 1978; Kawakami et al. 1993; Suarez 2 008) Only a capacitated sperm cell can fertilize an oocyte, and the inability of spermatozoa to fertilize could be the result of failure in all motility and acrosome associated changes. The localization and expression patterns of SPAG6, SP17, SP56, and C ATSPER2, to the acrosomal region of canine spermatozoa that were observed in the present study suggest that these proteins ha ve functions associated with capacitation, acrosome reaction and fertilization in the canine Lastly, as reported (Ho and Suarez 2001b) it was demonstrated that an increase in cAMP and calcium stimulate sperm motility and hyperactivation. This is illustrated in CatSper knockout mice that have defects in the cAMP -induced increase in calcium, and where defective hyperactivated motility is observed (Carlson et al. 2003) The confirmation of CATSPER2 pro tein presence in canine spermatozoa will warrant investigation for its function. Similarly it would be interesting to evaluate changes in the expression of these proteins with acrosome reaction, capacitation and hyperactivation in the dog like in other sp ecies. In conclusion, we have demonstrated in this study the presence of sperm proteins SPAG6, SP17, SP56, and CATSPER2 in canine spermatozoa using immunocytochemistry and confocal immunofluorescence. The objective to identify and characterize the presence of sperm proteins SPAG6, SP17, SP56, and CATSPER2 in
64 canine spermatozoa was achieved. The sperm proteins SPAG6, SP17, SP56, and CATSPER2 were localized and expressed similar pattern with minor differences to that found in other species.
65 Figure 41 Diagram of a sperm cell. The head includes the acrosome, equatorial band, and post acrosomal regions. The principal piece is the tail according to this diagram. The picture is taken from Dr. Danton ODay website (www.erin.utoronto.edu).
66 Figure 4 2 SPAG6 immunocytochemistry. A) canine negative control, B) SPAG6 mouse negative control C) canine SPAG6, D) mouse positive control. Magnification at 200600x. A B C D
67 Figure 43 SPAG6 confocal immunofluorescence. A) negative tissue control (immortalized cat T cel ls), B) canine SPAG6 at 200x C) canine SPAG6 at 400x, D) canine SPAG6 at 600x. Top) fluorescence, Center) phase contrast, Bottom: fluorescence/phase contrast. A B Top B A Center Bottom C D
68 Fig ure 44 Canine SP17 with fresh neg ative and positive controls. A) mouse sperm positive control, B) canine sperm negative control. Black arrow: normal SP17 expression. White arrow: acrosome reacted sperm. Magnification at 400x. Figure 45 SP17 localization on fresh canine spermatozoa. C onfocal immunofluorescence red labeling at acrosome, midpiece, and principal piece of the canine spermatozoa. Magnification at 400x.
69 Figure 46 SP56 localization on fresh canine spermatozoa. Canine SP56 expressed mainly at the principal piece, with so me expression at the acrosome. The insert is a mouse positive control. Magnification at 600x. Figure 47 CATSPER2 localization on canine spermatozoa. Canine CATSPER2 expressed at the flagellum, with some expression at the acrosome. Magnification at 60 0x.
70 CHAPTER 5 CHARACTERIZATION OF SPERM PROTEINS SPAG6, SP17, SP56, AND CATSPER2 IN CANINE BY WESTERN BLOT ANALYSIS Introduction While immunocytochemistry can identify the presence of immunoreactivity it does not demonstrate the presence of the protein itself since similar labeling can be observed in case of nonspecific reaction In order t o validate and confirm the identity of the observed labeling, the uses of protein electrophoresis and protein characterization are absolutely required. For these reas ons, identification and characterization of protein (including molecular weight) using western blot analysis is warranted. Western blot is a powerful protein characterization tool in which small amounts of protein can be detected, protein molecular weight can be determined, and antibody antigen interaction can be visualized as seen with specific band development. Western blotting, also known as immunoblotting of proteins was first described as a method for detection and identification of proteins transfe rred from a gel to a membrane (nitrocellulose or PDVF or other membrane s) and then detected with a substrate (Towbin et al. 1979) Prior to transfer and detection the protein sample of interest along with molecular weight markers are separated using electrophoresis either native without denaturation and separation based on protein mass: charge ratio or SDS-PAG E (with denaturation and separation of subunits and neutralization of charges) based on molecular mass only After electrophoretic separation and transfer of a membrane, the actual proce ss of western blotting involves several blocking steps to neutralize the nonspecific binding sites, washing between steps with or without detergents to reduce nonspecific binding, incubation with primary and then secondary antibodies of interest and finally the
71 addition of substrate reagent for chromogenic visualization (Towbin et al. 1979; Tash et al. 1988; Sabeur et al. 2002) The disadvantages of using western blot are extensive optimization, presence or absence of bands is subject to interpretation, and nonspecific interactions may result in variability of results. In the present study, w estern blot analyses were performed for each of the four proteins of interest o n canine spermatozoa. According to the literature, SPAG6 is listed as a 55.5 kDa molecular weight protein (Neilson et al. 1999; Zhang et al. 2005) SP17 is less homogenous and the following molecular weights have been presented: 14 -15 kDa, 17 -19 kDa, 2124 kDa, 26 kDa, 29 kDa (Richardson et al. 1994; Wen et al. 1999; Lea et al. 2002; Grizzi et al. 2003) SP56 has the following recorded molecular weights: 31 kDa, 40 kDa, 42 kDa, 43 kDa, and 67 kDa (Cheng et al. 1994; He et al. 2003) CATSPER2 according to the manufacturing company has the band sizes of 48kDa and 61 kDa. However, Carsons group has for CATSPER2 a band size of ~72 kDa (Carlson et al. 2005) The objective of this study was to confirm the presence of sperm proteins SPAG 6, SP17, SP56, and CATS PER2 by western blot analysis in the canine. The hypothesis tested was that western blot analysis will confirm in the dog the presence of sperm proteins with molecular weights corresponding to the previously described SPAG6, SP17, SP 56, and CATSPER2 proteins in other species spermatozoa. Materials and Methods Materials SpermVision SAR was purchased from Minitube of America (Verona, WI). BX51 Olympus microscope was purchased from Olympus Industrial America, Inc. (Orangeburg, NY). Tris, EDTA, and Triton X 100 were purchased from Fisher Scientific (Fair Lawn, NJ). PMSF was purchased from MP Biomedical, LLC (Solon, Ohio). N -
72 ethylmaleimide was purchased from Acros Organics (Morris Plaines, NJ). Pierce 660 nm Protein Assay and the Fast Blot Developer were purchased from Thermo Scientific (Rockford, IL). Spectra Max Plus 384 plate reader was purchased from Molecular Devices (Sunnydale, CA). NuPage 10% Bis -Tris Precast Gels, NuPage MOPS SDS Running Buffer (20X), Sample Reducing Agent (10X), NuP age LDS Sample Buffer (4X), NuPage Antioxidant, SeeBlue Plus 2 Prestained Standard (1X), iBlot Transfer Stack, Mini (Nitrocellulose), iBlot Gel Transfer Device were purchased from (Invitrogen, Carlsbad, CA). PowerPac Basic was purchased by Bio-Rad (Hercu les, CA). Carnation instant nonfat dried milk was purchased from a local store. Alkaline phosphatase -g oat anti mouse IgG Fc specific -, alkaline p hosphatase goat anti -rabbit IgG whole molecule, NBT/BCIP substrate tablet, and Tween20 were purchased from Si gma Aldrich (St. Louis, MO). PBS was purchased from Fisher Scientific (Fair Lawn, NJ). BSA was purchased from Roche (Mannheim, Germany). SPAG6 mouse monoclon al, SP17 rabbit polyclonal SP56 mouse monoclonal and CATSPER2 rabbit polyclonal antibodies were purchased from Abnova Corporation (Taipei, Taiwan) Delta Biolabs (Gilroy, CA) GeneTex, Inc. (Irvine, CA) and Aviva Systems Biology (San Diego, CA) respectively SoftmaxPro version 5.3 (Molecular Devices, Sunnydale, CA) software was used to determine tota l sperm protein concentration of the semen sample s. A serial dilution BSA standard was used as a reference. This study was repeated five times. Animals and Sperm Preparation Semen was collected from four sexually mature dogs ranging from 26 years of age, according to IACUC authorization protocols # E434 and #200903106. The sperm rich fraction of the ejaculate was collected by digital manipulation in a 15 ml calibrated plastic vial (Foote 1964b; Linde Forsberg 1991) To reduce variability between trials
73 concerning semen, the semen was pooled to have access to sample volumes large enough to allow the submission of the pools of semen to the different antibodies tested. Using a p ool of semen allowed for a reduction of variability due to animals and allowed for a reduction of the number of animals needed for every study. Five animals have been demonstrated to be optimum for this purpose during CASA validations and semen extenders s tudies (Verstegen et al. 2005) However, one of our animals had to be removed for health reasons from the program just after the start of this research. After pooling, the semen was evaluated for concentration, sperm moti lity using SpermVision SAR, and morphology using Spermac staining (Silva and Verstegen 1995; Verstegen et al. 2002) The fresh semen was then centrifuged twice at 700 x g for 10 minutes at room temperature in PBS and the supernatant was removed after each wash. The spe rm extract was serial diluted with 1% BSA from undiluted to 1:128 dilutions and analyzed for total protein concentration. Mature BALB/c male mice were euthanized by CO2 gas and cervical dislocation, according to IACUC authorization protocol #200903106. Di rectly after euthanasia, the cauda epididymides were excised and washed twice in PBS at room temperature. Each cauda epididymis was punctured with a 23guage needle and sperm was released from the epididymis into the petri dish. The sperm cells were then c entrifuged twice at 700 x g for 10 minutes at room temperature in PBS and the supernatant was removed after each wash. The sperm extract was serial diluted with 1% BSA from undiluted to 1:128 dilutions and analyzed for total protein concentration Experiments During preliminary trials (data not shown), p rimary and secondary antibody dilutions were optimized. Primary antibodies diluted in 1% BSA were used as follows:
74 SPAG6 at 1/100 and 1/200, SP17 at 1/50 and 1/100, SP56 at 1/100 and 1/200, and CATSPER2 at and 1/50. Secondary antibodies diluted in 1% BSA were used as follows: alkaline p hosphatase goat anti mouse and goat anti -rabbit at 1:2000. Canine and mouse sperm samples were analyzed on separate membranes to allow for concurrent evaluation of sperm prot ein expressions. Total protein concentration was determined with Pierce 660 nm protein assay Sperm samples were diluted with a NuPAGE sample reducing agent that contains dithiothreitol, which denatures the protein, NuPAGE LDS sample buffer which neutraliz es the pH of the environment to minimize protein modifications, and PBS Samples were then heated at 70 C for 10 minutes to denature the proteins prior to electrophoresis. The NuPage 10% Bis -Tris precast 2 lane gel casse tte was prepared along with the ele ctrophoresis tank (MOPS SDS running buffer with antioxidant). In the first lane, 200 l of canine sperm sample (109 g/per lane of total protein) and in the second lane 7 l of molecular weight standards (191, 97, 64, 51, 39, 28, 19, and 14 kDa molecular w eights used as recommended) were added to the gel cassette within the electrophoresis tank. The voltage on the tank was set at 200V, constant for 40 minutes at room temperature. After electrophoresis the gel was removed from the cassette and placed in an IBlot Gel Transfer Device (Invitrogen, Carlsbad, CA) for 8 minutes. After the transfer of proteins from the gel to the membrane, the membrane was blocked in 5% non-fat dried milk in PBS for 1 hour at room temp erature on a rocker. After blocking, the membr ane was briefly (510 seconds) rinsed twice in PBS -Tween (0.1%) The membrane was then placed in a Fast Blot Developer (a 10-lane template that allows for the detection of different antibodies or dilutions at the same time (Thermo Scientific,
75 Rockford, IL ) and 1000 l of each primary antibody were loaded into the 10 -lane template in the following order: lane 1 -CatSper2 undiluted, lane 2-CatSper2 1:2, lane 3BSA only, lane-4 goat anti rabbit secondary antibody only, lane 5-SP17 1:50, lane 6SP17 1:100, lane 7 -goat anti -mouse secondary antibody only, lane 8SPAG6 1:100, lane 9 -SPAG6 1:200, lane 10-BSA only. The primary antibodies were allowed to i ncubate overnight at 4 C in a cold room on a rocker. T he following day, the liquid was discarded, the membrane rin sed in PBS -Tween (0.1%) aspirated again, and washed three times for 5 minutes with PBS -Tween (0.1%) at room temperature. Secondary antibodies diluted to 1:2000 in 1% BSA were added to the appropriate lanes and incubated for 1 hour at room temperature on a rocker. Following completion of secondary antibody incubation, the liquid was discarded, the membrane rinsed in PBS Tween (0.1%) aspirated again, and washed twice for 5 minutes with PBS -Tween (0.1%). The membrane was then removed from the template and pl aced in a tray on a rocker for a final rinse with PBS -Tween (0.1%) The wash buffer was removed and the colormetric NBT -BCIP substrate tablet was then added to the tray for 10 minutes. If color development did not occur after 10 minutes the substrate was d iscarded and new NBT -BCIP tablet was added for an additional 10-20 minutes. To stop the reaction the membrane was washed in distilled wat er and then allowed to air dry. Results The mean value for concentration, overall motility, and forward progression of sperm samples were 596.14 373.54 x 106 sperm /ml, 98.49 1.1%, 94.19 0.80% respectively. For sperm morphology, the mean percentage of normal spermatozoa was 93 5.70% sperm. The protein concentration of the sperm samples were canine (9.3 mg/ml) and m ouse sperm ( 0.365 mg/ml ). The canine negative controls and molecular
76 weight standards (Figs 5 1 and 5-2; Table 5 1 ) were as expected. The molecular weight standards lane had all associated bands accounted for and the calculated standard curves were linear as expected. Based upon five trials, the coefficient of variation for the measurement of molecular weight standards was estimated to be 1020%. The canine negative control lanes which contained one of the following: BSA only, goat anti mouse secondary ant ibody only, or goat anti -rabbit secondary antibody only, had no visible bands present. Under reduced conditions, (SDS -PAGE) western blot analysis of canine sperm demonstrated SPAG6 was detected in a band at 61.57 kDa (Figs. 5 1 and 53; Table 52 ). SP17 a ntibodies allowed us to localize different bands at 15.45 and 28.75 kDa (Figs. 5 -1 and 5-4; Table 5 3 ). For SP56 there were no bands immunodetected in the canine protein extracted sperm (data not shown). CATSPER2 antibodies allowed us to localize different bands corresponding to molecular weights of 48.48, 49.18 and 63 kDa (Figs 5 .1 and 5 5; Table 5 4). Mouse sperm was used as a positive control and results were as followed: SPAG6 immunoreactivity was detected in a band at 49.75 kDa. SP17 immunoreactivity was detected in associated bands at 12.83 and 21.99 kDa. The SP56 immunoreactivity was detected a t 5253 kDa. CATSPER2 immunoreactivity was detected in a band at 48.16 kDa. Discussion The results of this study confirmed the presence of SPAG6, SP17, and C ATSPER2 proteins and allowed us to define their molecular weight in canine spermatozoa extracted samples For sperm cell extraction, s ome authors (Sabeur et al. 2002; De Los et al. 2009) added protease inhibitors to their samples O ther authors do
77 not add protease inhibitors (Baxendale and Fraser 2003; de Souza et al. 2007) simply wash ing the sperm sample after heat ing and SDS denaturation. In the present study, sample preparation was initially done (data not shown) using the addition of protease inhibitors (50 mM Tris, 20mM EDTA, 1mM PMSF, 5mM N ethylmaleimide) and solubilization using with 0.5 1%Triton X 100. Unfortunately, after several attempts, western blot protein identification was unsuccessful. It was postulated th at the addition of the protease inhibitor cocktail was associated with protein degradation preventing their detection by immunoblotting Canine SPAG6 immunoreactivity was detected with a molecular weight of 61.57 kDa while in the control mouse samples; SPAG 6 was detected in a band at 49.75 kDa. Based on the published results, the expected molecular weight for mouse SPAG6 is 55.5 kDa (Neilson et al. 1999; Zhang et al. 2005). If we accept 10-20% variation in molecular weight evaluation as estimated by western blot, the molecular weight calculated here can be considered as correct. Canine SPAG6 is expressed on canine spermatozoa and this was verified with the presence of a 61.57 kDa band on the western blot. The difference between the 61.57 kDa molecular weight observed here for the canine SPAG6 and the predicted 55.5 kDa band observed in mouse may again just be related to the variability in molecular weight evaluation between western blot or may be due to a different isoform of SPAG6 in the dog. To confirm the p rotein identity, an amino acid composition will have to be performed. However, the existence of different isoforms between species or within species has been postulated (personal communication with Dr. Jerome Strauss) and needs to be evaluated in dogs.
78 Ca nine SP17 immunoreactivity was detected for proteins of 15 and 28-29 kDa. These molecular weights are in agreement with the previously described values. The presence of multiple bands for SP17 protein is a generalized observation has been reported in sever al publications (Kong et al. 1995; Wen et al. 1999; Grizzi et al. 2003) with variation in molecular weights as follows:14 -15, 1719 2124, 26, and 29 kDa However, in species were SP17 was observed, even if large variations in molecular weights were noticed, two isoforms were always detected and were also detected in the present study. In m ouse SP17 was detected in associated bands at 12.83 and 21.99 kDa. In addition, some studies have also observed higher molecular weight bands ranging from 54 193 kDa. In rabbits, it has been reported that t hese higher molecular weight bands may be are aggregates of rabbit sperm auto antigen protein now known as SP17 (or non-RSA) cross -reactive antigens (Richardson et al. 1994) Moreover, recombinant mouse SP17 has higher bands present in western blots. Like for many proteins, pr oteolytic product and proteolytic cleavage of pre-pro-proteins were the explanation for some of the observations (Kong et al. 1995) It has also been mentioned (Adoyo et al. 1997; Grizzi et al. 2003; Grizzi et al. 2004) that a detected 54 kDa band is consistent with a multimeric form of SP17. In accordance with these findings canine SP17 was also detected in a visible band located at the 53.27 kDa. Sperm protein 56 was not detected in canine spermatozoa, w hile mouse SP56 was detected (data not show n) in a band at 52 -53 kDa (Cheng et al. 1994; He et al. 2003) Canine CATSPER2 was detected at 48-49 and 63 kDa. Mouse CATSPER2 was detected in a band at 48.16 kDa. The bands were in agreement with the predicted molecular weight at 48 and 63 kDa as reported by the manufacturing company. Canine
79 and mouse CATSPER2 immunoreactivity of higher molecular weights were also present, which could be due to the presence of pre or pre-pro proteins and their cleavage products. The present results confirm the presence of SPAG6, SP17 and CATSPER2 proteins in canine spermatozoa. The presence of SP56 was not confirmed as immunodetection was not observed in western blot. As the sperm protein SP56 is important for mammalian fertilization by functioning in the sperm ability to bind the zona pell uci da (Cheng et al. 1994; Cohen and Wassarman 2001) it was of interest to see if this sp erm protein would also be expressed and function in canine sperm. In chapter 3, according to ICC results, SP56 was reported strongly expressed and localized at the principal piece with weak to moderate acrosomal expression. The SP56 expression that was observed in ICC could be the result of denaturation of the sperm protein or inaccessibility of isoforms. The fact that SP56 is present in mouse sperm and not canine sperm indicates that further investigation is warranted to determine if the sperm protein SP5 6 is present or not in canine sperm and to identify the immunoreactivity observed in the present study The objective of this study was to confirm the presence of sperm proteins SPAG6, SP17, SP56, and CATSPER2 by western blot analysis on canine sperm The objective to identify and characterize the presence of sperm proteins SPAG6, SP17, and CATSPER2 in canine spermatozoa was achieved. Canine SPAG6 is expressed on canine spermatozoa and this was verified with the presence of a 61.57 kDa band on the western b lot. Canine SP17 is expressed on canine spermatozoa and this was verified with the presence of several SP17 associated proteins 15, 2829, and 53 kDa. Canine
80 CATSPER2 is expressed on canine spermatozoa and was verified with the presence of 4849 and 63 kDa bands which are in agreement with what has been reported by the manufacturing company. In conclusion, the present study demonstrated t he presence of SPAG6, SP17, and CATSPER2 on canine spermatozoa using w estern blot analysis and immunostaining.
81 Figur e 51. Canine sperm protein western blot. Altered and original 1) CatSper2 undiluted, 2) CatSper2 1:2, 3) BSA only, 4) goat anti -rabbit secondary antibody only, 5) SP17 1:50, 6) SP17 1:100, 7) goat anti mouse secondary antibody only, 8) SPAG6 1:100, 9) SPAG6 1:200, 10) molecular weight marker. 1 2 3 4 5 6 7 8 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 Rf distance down track 0 10000 5000 15000 20000 Profile height 0 806 403 Distance travelled (pixels) 1 20 5 100 500 Log molecular weight A B Figur e 52 Molecular weight standard densitogram A) densitogram, B) standard curve Table 5 1 Molecular weight standard densitogram data. Track 10 Number Mol. weigh t Height Raw vol. 1 191.00 7646.113 5290350.50 2 97.00 5139.493 12072106.00 3 64.00 9080.901 7600069.50 4 51.00 9199.746 13004836.00 5 39.00 9430.046 14444542.00 6 28.00 9136.664 17888004.00 7 19.00 5913.502 12070192.00 8 14.00 10629.005 11489455.0 0 1 2 3 4 5 6 7 8 9 10 1 2 3 4 5 6 7 8 9 10 A B
82 1 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 Rf distance down track 0 10000 5000 15000 20000 Profile height Figur e 53 SPAG6 at 1:200 dilution densitogram Table 5 2 SPAG6 at 1:200 dilution densitogram data. Track 9: SPAG6 Number Mol. weight Height Raw vol. 1(m) 61.57 4252.524 1351688.25 1 2 3 4 5 6 7 8 9 10 11 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 Rf distance down track 0 10000 5000 15000 20000 Profile height Figur e 54 SP17 at 1:100 dilution densitogram. Table 5 3 SP17 at 1:100 dilution densitogram data. Track 6: SP17 Number Mol. weight Height Raw vol. 1 193.51 1166.964 528417.38 2(m) 163.35 1505.759 433985.44 3 59.24 2822.645 758897.25 4(m) 53.27 1466.241 380468.91 5 42.70 6552.807 2549770.50 6 39.28 1662 2.148 6909526.50 7 36.34 6148.989 3302094.00 8 35.08 8239.422 5784035.00 9 33.56 8251.163 6719155.00 10 28.75 3008.873 2853290.50 11 15.45 1847.011 1364073.88
83 1 2 3 4 5 6 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 Rf distance down track 0 10000 5000 15000 20000 Profile height Figur e 55 CATSPER2 at 1:2 dilution densitogram. Table 5 4 CATSPER2 at 1:2 dilution d ensitogram data. Track 2 : CATSPER 2 Number Mol. Weight Height Raw vol. 1(m) 289.81 7721.347 5181371.50 2 183.68 20769.453 15434651.00 3(m) 147.18 5741.048 3569350.00 4(m) 122.64 3517.732 1653897.50 5(m) 63.69 1856.448 573341.75 6 49.18 3065.417 5707022.00
84 CHAPTER 6 EFFECTS OF CANINE SEMEN PRESERVATION ON SPERM PROTEIN EXPRESSION Introduction Protein detection by i mmunocytochemistry or western blotting does not automatically imply that these proteins have any biological activity or function. However, if functions in motility and/or fertilization are expected, any induced changes in their motility and/or morphology should be associated with changes in their expression. If involved in sperm motility, changes in their flagellar expression may be expected and correlated with changes in motility Similarly, if involved in fertilization, artificial induction of acrosome reaction and hyperactivation should possibly be associated with changes in their acrosome related expression. Canine semen extende rs protect spermatozoa by conserving motility and fertility over time by stabilizing the sperm membrane, providing energy substrates and preventing deleterious effects of changes in pH and osmolarity (Foote and Leonard 1964; Province et al. 1984; Bouchard et al. 1990; Brown 1992; Goodman and Cain 1993; Rota et al. 1995) Cold shock is prevented in chilled semen by slowly cool ing (Bouchard et al. 1990; Ponglowhapan et al. 2004) in the presence of a defined buffer a nd preserved at 4 C. There are commercially available and laboratory prepared canine chilled semen extenders. Until recently, commercially and laboratory prepared extenders allowed for the preservation of chilled semen at 4 C for only 4 -5 days. However, some of the more recently described and produced extenders allow for the preservation of canine semen for more than 1015 days. U sing a laboratory prepared extender (Verstegen et al. 2005) chilled semen was conserved up to 3 weeks.
85 Among the laboratory -prepared buffers, egg yolk Tris fructose was tested as an extender (Rota et al. 1995) and shown to possess the best properties to allow long -term chilled semen preservation. Similarly, results from (Iguer -Ouada and Verstegen 2001b) have confirmed the interest of the egg yolk Tris based extenders (using glucose instead of fructose) in terms of motility conservation assessed objectively using computer assisted analyzer (CASA Hamilton-Thorn IVOS 10) (Iguer -Ouada and Verstegen 2001a; Iguer -Ouada and Verstegen 2001b) The glucosebased extender was also evaluated by the ability to limit the acrosome reaction, as determined using chlortetracycline staining. In this exten der, good semen quality, characterized by more than >7 0% motile spermatozoa and less than <10% acrosome -reacted cells, were observed after conservation for 10 13 d ays at 4 C (Iguer -Ouada and Verstegen 2001b) At the end of the storage period ( day 17), no semen motility was observed. Overall, quality of stored semen using gluco se instead of fructose was improved, suggesting a major role of the energy substrate glucose in maintaining c anine semen motility. S tudies in other species horse (Love et al. 2002) or boar (Boe -Hansen et al. 2005) questioned the possibility to preserve chilled semen for such a long per iod without reducing fertility. However, the preserved fertility after long-term pres ervation in canine was demonstrated with significant pregnancies obtained after insemination with chilled semen preserved for more than 10 days (Verstegen et al. 2005) In fresh canine sperm, SPAG6 has been demonstrated previously to have strong expression localized mainly at the acrosomal region of the sperm hea d. In addition, SPAG6 had negative to moderate expression localized at the midpiece of the sperm tail SP17 is localized with strong and similar intensity at the a crosomal region and the
86 principal piece of the flagellum CATSPER2 had flagella localization and t here was also some weak to moderate expression of CATSPER2 at the level of the acrosomal region. The localization of all three of these proteins either at the level of the head or at the level of the flagella may be indicative of a role of these protein in either or both of the fertilization and motility processes. Since the optimal chilling extenders were developed for the preservation of semen (i.e. by stabil izing the membrane), there should be little to no change in the plasma membrane and in acrosome integrity during chilled semen preservation in these extenders. Only at the appropriate time can capacitated sperm fertilize oocytes. It is then logical to post ulate that a sperm cells unable to undergo an acrosome reaction at the optimal time or opposite, developing premature acrosome reaction can result in fertilization failure. Acrosome reaction is associated with head permeabilization and significant motilit y changes. In the dog acrosome reaction is associated with significant changes in the motility pattern of the sperm cells and/or hyperactivation both needed for oocyte penetration. If these events do not occur (cell damage) or do occur to early (inappropri ate early acrosome reaction and hyperactivation) fertilization will be impaired. Disruption of plasma and acrosomal membranes induced by t he preservation process inhibit the ability of sperm cells to fertilize. These modifications can be artificially induc ed and assessed by the in vitro acrosome reaction protocols and staining of the sperm cells. Laboratory prepared capacitation media SP -TALP has been demonstrated to induce an acrosome reaction. In a comparison study (Sirivaidyapong et al. 2 000) of sperm capacitation media it was concluded that SP -TALP induces a significantly higher percentage of acrosome reacted sperm c ells than other capacitation media tested.
87 Different commercially available chilling semen extenders were compared for thei r semen preservation capabilities and their effects on the different membrane proteins evaluated to analyze the changes in selected sperm protein expression during preservation over a period of time and establish a possible relationship between this expres sion and different semen parameters including motility, capacitation, and acrosome reaction. The objectives of the experiments were: a) evaluate the changes in selected sperm protein expression during preservation over a period of time, b) establish a poss ible relationship between sperm protein expression and different semen parameters including motility, capacitation, and acrosome reaction. The hypotheses to be tested were: a) semen preservation over time affect protein expression, b) differences may be observed between the different evaluated extenders, c) acrosome induced functional changes may be detected in relation to the preservation processes, d) acrosome reaction induction is associated with changes in the expression of the different semen proteins evaluated e) overtime changes in motility may be related to changes associated to acrosome reaction and modification in the expression of the different proteins. Materials and Methods Materials SpermVision SAR, CaniPRO Chill 5, and CaniPRO Chill 10 and Spermac stain were purchased from Minitube of America (Verona, WI). Camelot was purchased from Camelot Farms (College Station, TX). Synbiotics was purchased from Synbiotics Corporation (San Diego, CA). SP -TALP sperm capacitation media was laboratory prepar ed according to published instructions (Sirivaidyapong et al. 2000) Briefly, the ingredients were: 100 mMol s odium chloride, 3.1 mMol p otassium chloride, 2.0 mMol
88 c alcium chloride, 0.4 mMol m agnesium chloride, 25 mMol s odium bicarbonate, 1 mMol s odium pyruvate, 3.38 ml s odium lactate 60% syrup 0.3 mMol s odium phosphate, 10 gm/L b ovine serum albumin 2.28 ml Hepes and 0.25 mMol gentamycin S. At the start of the experiment, all ingredients were dissolved in ultrapure water. Coomassie Blue stain was purchased from Fisher Scientific (Fair Lawn, NJ) and laboratory prepared according to published instructions (Larson and Miller 1999; Brum et al. 2006) SPAG6 mouse monoclon al, SP17 rabbit polyclona l CATSPER2 rabbit polyclonal were purchased from Abnova Corporation (Taipei, Taiwan), Delta Biolabs (Gilroy, CA ), Aviva Systems Biology (San Diego, CA). Zamboni fixative was purchased from Newcomer Supply (Middleton, WI). DakoCytomation LSAB + System -HRP Kit with swine anti mouse/rabbit/goat biotinylated secondary antibody were purchased from DakoCytomation, Inc. (Carpinteria, CA). BX51 Olympus microscope was purchased from Olympus Industrial America, Inc. (Orangeburg, NY). Animals Semen was collected f rom four healthy dogs ranging from 26 years of age, according to IACUC authorization protocols # E434 and #200903106. The sperm rich fraction of the ejaculate was collected by digital manipulation in a calibrated plastic vial (Foote 1964b; Linde Forsberg 1991) To reduce variability between trials concerning semen, the semen was pooled to have access to sample volumes large enough to allow the submission of the pools of semen to the different antibodies tested. Using a pool of semen allowed for a reduction of variability due to animals and allowed for a reduction of the number of animals needed for every study. Five animals have been demonstrated to be optimum for this purpose during CASA validations and semen extenders studies
89 (Verstegen et al. 2005) However, one of our animals had to be removed for health reasons from the program just after the start of this research. Mature BALB/c male mic e were euthanized by CO2 gas and cervical dislocation, according to IACUC authorization protocol #200903106. Directly after euthanasia, the cauda epididymides were excised and washed twice in PBS at room temperature. Each cauda epididymis was punctured wit h a 23guage needle and sperm were released from the epididymis into the petri dish. Sperm Preparation and Preservation After pooling, the semen was evaluated for concentration, sperm motility using SpermVision SAR, and morphology using Spermac staining (Silva and Vers tegen 1995; Verstegen et al. 2002) The fresh semen was then centrifuged twice at 700 x g for 10 minutes at room temperature in PBS and the supernatant was removed after each wash. The final concentration of the PBS extended semen was 300 x 106 sperm cell /ml. To extend and process the semen samples, each extender was warmed to room temperature before dilution of the samples. The different samples were extended according t o manufactures recommendations. Briefly, for the Camelot, and CaniPRO Chill 5, and C aniPRO Chill 10 groups, 1 part of semen was diluted with 3 parts of extender. For the Synbiotics group, 0.5 part of semen was diluted with 2 parts extender CaniPRO Chill 10 extender requires the addition of 20% egg yolk before being used to extend the sem en. After extension t en l of each semen samples were used for assessment of motility after dilution using the CASA analysis as previously described Twenty l of each of the samples were also deposited on a Superfrost/Plus microscope slide, a smear was performed and allowed to air dry overnight before fixation with the Zamboni
90 fixative. Another t en l of s ample was last deposited on a microscope slide, dried, and stained using a Spermac s tain to assess morphology and acrosomal status following the manuf actures recommendations The rest of the extended chilled semen (1.52.5 ml) was placed in 15 ml conical tubes and stored in the refrigerator at 4C in alcohol filled containers up to day 20 post collection (evaluation was stopped when all sperm were dead as demonstrated by CASA analysis). The alcohol -filled storage containers prevented both cold shock during the chilling process and temperature variations (Verstegen et al. 2005) Each CASA analysis included evaluation of sperm concentration, motility, and forward progression parameters. Experiments Effect of chilled extenders on motility and acrosomal status Sperm motility was assessed at different time point s (Days 0 (before dilution with the different extenders ) and 1 ,3 ,5,7,10,13,15,18, and when needed day 20 after dilution) ] The sperm were no longer assessed when sperm cells were no longer motile. Acrosomal status was assessed at Days 0, 5, 10, 15, 20. This study was repeated three times. Effect of chilling and differ ent extenders on sperm protein expression At each time point listed above, a ten l sperm sample from the different extenders was overnight air dried on a S uperfrost plus microscope slide and fixed with Zamboni fixative for one hour at 4C and processed f ollowing Dako standardized immunocytochemistry protocol using a primary mouse monoclonal antibody against SPAG6, or primary rabbit polyclonal antibody against SP17 or CATSPER2. Primary antibodies were diluted as follows: SPAG6 at 1/200, SP17 at 1/200, and CATSPER2 at 1/50 with PBS, incubated overnight at 4 C, rinsed in distilled water and washed 3 times
91 in PBS, then incubated 1 hour at room temperature with a swine anti mouse/rabbit/goat biotinylated secondary antibody. DAB + Substrate buffer was used to v isualize the binding sites. In the negative controls (canine and mice) the primary antibody was omitted. Mouse sperm was used for the positive control. Visualizations were ascertained using a BX51 Olympus microscope at 200600X magnifications. Effect of a crosome reaction induction on sperm protein expression SP-TALP sperm capacitation media was laboratory prepared according to published instructions (Sirivaidyapong et al. 2000) Briefly, the ingredients were: 100 mMol s odium chloride, 3.1 mMol p otassium chloride, 2.0 mMol c alcium chloride, 0.4 mMol m agnesium chloride, 25 mMol s odium bicarbonate, 1 mMol s odium pyruvate 3.38 ml s odium lactate 60% syrup 0.3 mMol s odium phosphate, 10 gm/L b ovine serum albumin 2.28 ml Hepes and 0.25 mMol gentamycin S. All ingredients were dissolved in ultrapure water when needed The pH and osmolarity (7.26 and 311 nmol/L respectively) of SP-TALP were recorded before the addition of the semen. The pooled semen was divided into two groups: sperm with SP -TALP (1 part semen and 3 parts SP-TALP) and sperm without SP -TALP but extended in PBS (1 part semen and 3 parts SP-TALP). Prior to extension and incubation, semen was analyzed for motility and evaluation of acrosome status (before incubation samples) Then, the extended samples were covered in mineral oil and incubated for 5 hours at 37 C in 5% C02 before being analyzed again as stated below (after incubation samples) After the incubation period, 250 l of each sample was fixed separately using 0.8 ml of 4% p araformaldehyde for 10 minutes at room temperature. The samples were then centrifuged at 500 x g for 6 minutes, the supernatant was removed, and the pellet was re suspended in 500 l of ammonium acetate (0.1M) then centrifuged at 500 x g for
92 5 minutes and t he clear supernatant was removed. Finally, a 50 l smear of sperm suspension was placed on a microscope slide and allowed to air dry overnight. The following day, the air dried slides were incubated at room temperature for 2 minutes in freshly made Coomass ie Blue stain, rinsed 3 -4X in distilled water, and allowed to air dry before analysis under brightfield microscopy. Statistical Analysis For the effect of extenders on motility and acrosomal status, three slides were labeled and 100 sperm were evaluated p er slide per group. Two way ANOVA was used to compare the different chilled extenders with percent motility and percent acrosomal status overtime. Tukey Kramer multiple comparison test was performed when the P value was significant (<0.05). Data were expre ssed as mean SD. Differences were considered significant when P<0.05. These studies were repeated three times. For the acrosome reaction induction study, three slides were labeled and 100 sperm were evaluated per slide per group, thus =3 per group. One way ANOVA was used to compare the results Tukey -Kramer multiple comparison test was performed when the P value was significant (<0.05). Data were expressed as mean SD. Differences were considered significant when P<0.05. These studies were repeated thr ee times. Results The mean values for concentration, overall motility, and forward progression of sperm samples were 391 80.9 x 106 sperm/ml, 94.12% 8.0, and 78.4% 5.4, respectively. For sperm morphology, mean percentage of normal spermatozoa was 95 4.8% sperm. Data for normal sperm morphology was assessed prior to experimentation.
93 Effect of chilled extenders on motility and acrosomal status There were no statistical differences between the CaniPRO 5, CaniPRO 10 and the Camelot groups of chilled se men immediately after dilution. However, Synbiotics extender induced a significant decline of sperm cell motility immediately after dilution. One day later, all the chilled diluted samples were similar without statistical differences H owever a trend for Synbiotics to be lower was already observed Overtime the motility observed decrease progressively for all groups. However significant differences in the dynamics of the decrease were observed between groups. An overall motility of 50% was obtained for S ynbiotics after 3 to 4 days, 7 days for the Camelot group while only observed after 11 days for both the CaniPRO Chill 5 and CaniPRO Chill 10 extenders (P<0.05). The overall motility was lower than 30 % for Synbiotics at day 5, nearly all sperm cells being immotile at day 10, the same 30% were noticed at day 10 for Camelot with nearly all sperm cell s being dead at day 15 while 30 % of motility was only observed at days 1415 with and nearly all sperm cells dead at days 18 for CaniPRO Chill 5 and CaniPRO Chill 10 respectively. These di fferences were statistically significant (P<0.05). Overtime, the percentage of sperm undergoing the acrosome reaction was not significant ly different (P>0.5) between the different extenders ( Fig. 6 2). Large variations between samples and repetitions were noticed explaining the se high standard deviations. The fact that the number of living sperm cells was changing significantly overtime for some extenders also dramatically influence the standard deviation. At 50% motility (days 34, 7, and 11 for Synbiotics, Camelot, and CaniPRO Chill 5 or 10 respectively), 20 to 30 % of the sperm cells were acrosome reacted. At the end of the preservation period when motility was lower than 10%, the percentage of acrosome
94 reacted sp erm cells was betw een 30 and 50 % with variations between groups however without statistical differences. Effect of chilling and different extenders on sperm protein expression SPAG6 and CATSPER2 proteins were poorly preserved during the chilling process. Indeed, if the la beling was considered as normal before dilution and incubation at 4 C, none or only few cells were still identified after extension and incubation whatever group considered for both proteins. For all extenders, SPAG6 labeling was very weak or absent by day 3 and when observed only the acrosome or equatorial band were weakly marked for SPAG6 (Fig. 6 -3 ). No or weak labeling were observed for CATSPER2 at the same period For SP 17, the typical labeling of the acrosome changed with time and acrosome integrity In acrosome -intact sperm cells the acrosom e is labeled, but this typical labeling disappears in all acrosome modified or reacted sperm cells that become detected with preservation. This observation was consistent with the four chilled semen extenders (F ig. 64 ). The flagella labeling sometime s observed in control fresh non extended sperm cells was also observed in preserved sperm cells but with an increased intensity The intensity of the labeling was negatively correlated with the labeling of the acro some. In the non acrosome reacted sperm cells, the principal piece was weakly labeled while in the acrosome r eacted sperm cells, the flagellum was more clearly recognized by the antibody as shown by an increased labeling intensity Effect of acrosome reaction induction on sperm protein expression Acrosome reaction induction with SP -TALP significantly (P<0.0001) caused a decrease in the number of acrosome -intact sperm when compared to sperm not treated with SP -TALP (Fig. 65) Less than 50% of acrosome intac t sperm cells were observed
95 after 5 hour incubation and 20% of the sperm cells remain ed alive. Similarly, s perm protein expression was dramatically changed with acr osome reaction induction (Fig. 66). For sperm proteins SPAG6 and CATSPER2 there was moderat e expression localized at the post acrosomal region and none to weak expr ession localized at the flagellum For sperm protein SP17, no labeling was observed on the sperm cells. The negative controls had no labeling present. For each sperm protein, the mous e positive controls were normal (data not shown). Discussion W hen sperm is not immediately used for artificial insemination, semen extenders are needed and are important for sperm conservation. This present study compared commercially available canine chil led semen extenders for their ability to maintain sperm motility and acrosome integrity. Considering previously published studies on canine chilled semen preservation, it was decided to follow some semen characteristics (motility and acrosome reaction) unt il motility decreased below 10% for some extenders up to a period of 20 days at 4 C. Significant differences were observed between the chilled semen. These differences were not statistically significant when semen were compared at fixed days because of the large individual variations observed between samples. Indeed, for an extender like Synbiotics, at day 5 in some samples the semen was already dead while in some other samples at the same day 30 or 40 % of the sperm cells were still motile. However, a signi ficant difference was noticed for the dynamic of motility over time. If the samples were compared for percent of motility, we noticed that the extenders can be classified with the Symbiotic one preserving motility over the shorter period while the CaniPRO extenders allowed for a significantly extended
96 duration of preservation. These results confirm those already published with similar extenders: huge variation and differences in the overall ability of the extenders to maintain motility. In the present study the percentage of overall motility decreased significantly overtime. On day 0, the extended sperm cells were motile in the range 7898% (with motility before extension being 94%) and all the samples were immotile after 11 days for Synbiotics, 15 days for Camelot and more than 18 days for the CaniPRO extenders. The initial motility of 70% was preserved 3 days for the Synbiotics extender, 5 days for the Camelot and 10 days for the CaniPRO extenders. During the first 5 days, no significant changes in the ove rall motility were noticed for the two CaniPRO extenders. These two last observations are of significant interest in clinical conditions where most often the preserved chilled semen is used within 3 to 5 days after collection. Similarly 70% motility is mos t often considered as being the optimal for the success rate after artificial insemination in the dog. No significant differences were observed between the chilled extenders in regards to acrosome integrity. The percentage of acrosome-intact spermatozoa w as 100% on day 0 and ranged from 45-70% on day 20. The absence of difference for this parameter can be due to the limited number of samples used in this study. Though not significant, Camelot appeared to have more damaged and acrosome reacted sperm compared to the other semen extenders. Interestingly, no differences were noticed as far as acrosome reaction is concerned when the two CaniPRO products were compared. While the exact composition of the two extenders is not known and based on previous studies dem onstrating a beneficial effect of the egg yolk on semen preservation, we were expecting not only a difference in term of motility preservation but also as far as
97 acrosome reaction is concerned for the CaniPRO 10 that is added with 20% egg yolk The absence of difference can be due to the limited number of samples used in this study or to possible differences in the composition or origin of the egg yolk used in this study versus the previous published reports. It is however interesting to note that in the best present extenders, acrosome reaction after long term preservation does not seem to be a major issue. On fresh sperm, SPAG6 was strongly expressed and localized mainly at the acrosomal region of the sperm head. In addition, SPAG6 had negative to modera te expression localized at the midpiece of the sperm tail. On chilled sperm and with all extenders, SPAG6 had weak or absent expression by day 3. When present, the labeling was located at the acrosome or equatorial band either similarly to what was observed in fresh samples (acrosome) or with a new different pattern when the equatorial bands were labeled. The presence of the labeling is not directly related to the acrosome reaction as in nearly all samples at day 3 after collection, acrosome reaction is in general of less than 30% and the labeling is gone in nearly all sperm cells The fading of the labeling for SPAG6 is probably not directly correlated to changes in motility as during the first few days for all the extenders there no significant changes in motility. A similar observation was made for CATSPER2. On fresh sperm, CATSPER2 was expressed and localized at the flagellum and also had weak to moderate expression at the level of the acrosomal region. On chilled sperm, CATSPER2 localization was absent. In other species, t he CATSPER family of proteins is known to play a role in sperm motility They are also proposed to have a role in sperm calcium regulation. The absence (or weak) of labeling after 3 days of preservation without significant changes in
98 bot h acrosome reaction and motility, either indicate s that these proteins are not involved in these mechanisms or that the proteins are involved in process associated with p rolonged motility conservation or cold shock protection. A last possibility is that the extension with complex media including egg yolk made the availability of the protein to the antibodies impossible. On fresh sperm, we demonstrated earlier that SP17 is expressed and localized with strong and similar intensity at the a crosomal region and the principal piece of the flagellum On chilled sperm and with all extenders, no significant changes in the expression of SP17 were observed during the first week of preservation. Then it was noticed that SP17 had weak to moderate expression by day 10 and weaker staining present by day 20. The labeling that was present was located at the acrosome with varying degrees of acrosomal damage and with variable expression at the level of the principal piece. It was also observed that the principal piece was l abe led more when the acrosome was reacted than when the acrosome was intact and vise versa. No labeling of the head was noticed in acrosome reacted sperm cells. This observation was confirmed but the acrosome induction study was actively inducing the acrosome reaction associated with a total inhibition of the expression of the protein. This observation was of ma jor interest clearly indicating that based on the changes in absence of acrosome reaction induction, that SP17 may have a function in the preservatio n of sperm cells but not directly associated with the acrosome reaction. Indeed, the dynamic change in the expression and pattern of expression of this protein may be indicative of a functional role of SP17 which certainly should be further investigated.
99 The results demonstrated that chilling does cause a change in expression of SP17 T his was shown with the diminishing expression from day 10 to day 20. The justification for the observ ation that the principal piece wa s labeled more when the acrosome wa s r eacted can best be explained by fact that a sperm cell that is acrosome reacted no longer has plasma or acrosomal membranes in the head region of the spermatozoa. Thus, expression of SP17 at the level of the acrosome should be absent. An acrosome reacted s perm should not have disruption of the plasma membrane covering the principal piece of the flagellum. The justification for the acrosome-intact being expressed and the principal piece being no longer labeled can best be described by the semen extender envi ronment inducing the breakdown of the plasma membrane, but not enough to cause an acrosome reaction ; t h ereby preserving SP17 expression at the level of the acrosome. To verify that an acrosome reaction does cause changes in sperm protein expression, an acr osome induction study was conducted. With the use of SP -TALP a capacitation media, to first determine how many spermatozoa will acrosome react and secondly to determine sperm protein expression following acrosome reaction induction. It was believed that a significant change in acrosome reacted sperm cells will occur with a decrease in protein expression. The study confirmed the belief that acrosome reaction induction significantly decreased the amount of acrosome-intact sperm (60%) when compared to acrosome-intact sperm not treated (98%) with SP -TALP capacitation media. Sperm protein expression was dramatically changed with acrosome reaction induction. For sperm proteins SPAG6 and CATSPER2 there was moderate expression localized at the post acrosomal region and none to weak expr ession localized at the
100 flagellum For sperm protein SP17, no labeling was observed on the sperm cells. The first observation was t hat SP17 was more so affected by semen preservation and acrosome reaction induction when compared to the other sperm proteins. Under these conditions, SP17 was either weakly expressed or absent. SPAG6 and CATSPER2 both show ed evidence that an acrosome reaction occurred by the presence of post acrosomal region staining. This is the first study to characterize the expression of SPAG6, SP17 and CATSPER2 in canine spermatozoa. Furthermore, this study demonstrated that motility overtime significantly decreased when sperm were preserved in chilled semen extender. This study also showed that acrosome status overtime does not significantly change regardless of the four canine semen extender used. Finally, that acrosome reactio n induction does significantly a ffect sperm protein expression and potential sperm protein function. In conclusion, we have demonstrated in this study differences in the ability of different extenders to preserve motility. The present results however did not reveal significant effects of the extenders and chill preservation on the induction of acrosome reaction in the dog. Semen preservation was associated with significant changes in s perm membrane protein expression without obvious correlation with changes in the overall motility and acrosome status, except for SP17 which showed changes correlated to modifications of the acrosome. The in vitro induced acrosome reaction induction surprisingly was associated with changes in sperm cell labeling. These observations seem to indicate possible changes in the protein expression patterns with preservation and acrosome reaction and warrant further invest igations.
101 0 10 20 30 40 50 60 70 80 90 100 0 1 3 5 7 10 13 15 18 20 Days % overall motility Chill 5 Chill 10 Camelot Synbiotics Figure 61. Overall m otility (%) of sperm preserved at 4C overtime in different chilled semen extenders.
102 0 10 20 30 40 50 60 70 80 90 100 0 5 10 15 20 Days % Chill 5 Chill 10 Camelot Synbiotics Figure 62. Mean (%) of acrosome -intact sperm overtime at 4C in different chilled semen extenders.
103 Figure 6 3 SPAG6 chilled spermatozoa Day 3 of preservation at 4 C A) Chill 5, B) Chill 10, C) Camelot, D) Synbiotics. Poor labeling is observed with non labeled sperm. S kinny black arrow: damaged, acrosome orange arrow: no labeling even if intact acroso me Magnification at 600x. 10 m A B C D
104 Figure 6 4 SP17 chilled spermatozoa Day 10 and Day 20 of preservation at 4 C A) Chill 5 D10, B) Chill 5 D20, C) Chill 10 D10, D) Chill 10 D20, E) Camelot D10, F) Camelot D20, G) Synbiotics D10, H) Synbiotics D20. Black arrow: acrosome intact, dashed arrow: flagella labeled, skinny black arrow: acrosome damaged orange arrow: no labeling. Magnification at 400x. 10 A B C D E F G H
1 05 Figure 65. Mean (%) of acrosome -intact sperm before and after acrosome reaction induc tion.
106 Figure 6 6 Effect of acrosome reaction induction on sperm proteins A, C, E, G) before acrosome reaction induction, B, D, F, H) after acrosome reaction induction. Magnifications at 400600x. Stain SPAG6 SP 17 CATSPER2 A B C D E F G H
107 CHAPTER 7 GENERAL DISCUS SION The canine industry in the United States is a multimillion dollar business. Using a valuable male dog that has a successful career for breeding can be a major investment for the owner. Indeed dog breeders want to breed their bitches to well known and successful males with excellent fertility. Therefore, s ome breeding stud dogs are expected to breed large numbers of bitches each year. In order to satisfy these expectations, the testes must continuously produce a large number of good quality spermatozoa and the ejaculates should be devoid of any factors possibly leading to a reduced fertility. Any factor that affects semen quality and spermatozoa function may have a great impact on a dogs reproductive career and cause great financial and emotional losse s to the breeder. There is a large body of evidence that semen and prostatic fluid qualities are important factors involved in reproductive function and fertility (Foote 1964b; Strzezek and Fraser 2009) W ith age male dogs often develop prostatic disease which may lead to the production of poor quality semen that often is contaminated with b lood, urine, or other products such as bacteria, debris, and pus (England and Allen 1992; Rijsselaere et al. 2004; Sampson et al. 2007) C ontaminated semen can not be used for artificial insemination (AI) and semen freezing, the main reasons being risk of infectious contamination of the female or poor freezability (England and Allen 1992; Chen et al. 1995a; Verberckmoes et al. 2004) Furthermore, various abnormal conditions of the reproductive genital tract including infection by herpes virus or Brucella canis bacteria, balanoposthitis, prost atitis, abscesses, and hemospermia c an affect the quality of the ejaculate that can result in reduced fertility through direct effects on the semen or
108 indirectly affecting female fertility. Moreover, in older dogs, the number of good quality and highly motile sperm cells may decrease inversely proportionally to the increase number of dead or abnormal cells. For all these reasons, there is a need for the development of technique s which will successfully allow the separation of the contaminants and/or poor quality sperm from the progressively moving, highly viable cells. The discontinuous Percoll gradient centrifugation technique has been developed with this objective to separate viable, progressively motile sperm cells from dead/poor quality sperm cells in several species, including bovine (Parrish et al. 1995) human (Chen et al. 1995b; Miller et al. 1996) and more recently canine (Strom et al. 2000a; Hishinuma and Sekine 2004) If new techniques are needed to improve semen quality by purification, sperm cells need normal membrane integrity and functionality to be able to penetrate and fertilize the oocytes Recently several membrane proteins have been demonstrated to play a significant role in sperm cell motility and function. Up to now none of these sperm membran e proteins have been analyzed in the dog. Among the proteins of interest, the presence of sperm associated antigen 6 (SPAG6), a sperm protein, which is localized on the flagellum in mice and human sperm has not been investigated in canine. The presence o f SPAG6 has been linked with sperm flagellar motility and structural integrity of the axoneme central apparatus of mature sperm in mice and humans (Neilson et al. 1999; Sapiro et al. 2000) Mature SPAG6 knock out mice are infertile and produce sperm with abnormal motility and structural defects (Sapiro et al. 2002) Knowing the localization and levels of SPAG6
109 and other sperm proteins in the dog will allow for better identifi cation of their potential role in that species and evaluation of the effects of canine semen preservation. For many years, in vitro assessment of sperm has been done in attempt to accur ately predict the fertility of the sample. This has not been without di fficulty as fertilization is a complex process. There are many in vitro techniques to assess sperm, including: evaluation of sperm motility, concentration, morphology, membrane integrity, and acrosome r eaction. Unfortunately, data have shown that these sa me parameters alone are not always accurate predictors of fertility (Graham 2001; Henkel and Schill 2003; Rodriguez -Martinez 2003; Martinez 2004; Rijsselaere et al. 2005) According to the literature a combination of these parameters in addition to others such as: oocytes penetration, hemi -zona, and zona pellucida binding as says, will be the most useful and accurate approach to evaluate the semen potential to be fertile (Henkel and Schill 2003; Rijsselaere et al. 2005) However, even if t hese approaches are easily achievable in many species, d ue to various reasons including ovulation occurring only once or twice per year, oocyte maturation occurring after ovulation, or the dark lipid content in the oocytes, there are many difficult ies with conducting successful in vitro studies in the dog (Rodrigues and Rodrigues 2006; Songsasen and Wildt 2007) The American Kennel Club has over 150 registered breeds. These breeds are placed throughout the nation and the world and in many cases to avoid displacement of the dog; semen must be shipped for artificial insemination. The semen must be extended and chilled or cryopreserved prior to shippi ng, in hopes that the sperm will be viable and fertile upon arrival and insemination.
110 Semen preservation offers many advantages to the canine industry particularly in conjunction with genetic evaluation and selection programs However, the biggest obstacl e to using preserved semen is that cooling, conservation at 4 C, freezing conservation at 196 C, and thawing generally damage sperm membrane structures which lead to fewer viable and motile cells. Consequently, fertility following artificial insemination with preserved semen might be decreased compared to that with fresh semen. Due to increased demand for preservation, many laboratories have been working to better understand the properties of preserved semen in order to find more efficient methods for sp erm storage (at either 4 or 196 C) recovery, viability, and fertility. The most commonly described side effect of semen preservation is the dramatic and sharp decrease in sperm viability, motility, and plasma membrane integrity observed in some animals. Prior to preservation of semen, in vitro assessment of the semen sample must be performed. However, t here are no guarantees that sperm that are a live and potential fertile prior to chilling or cryopreservation will be viable and fertile at the time of ins emination. This is the reason why another in vitro assessment of the semen sample must be performed prior to insemination. To get the optimal results after preservation and before artificial insemination, the preservation techniques have to be optimized so that an optimal semen extender will be chosen to provide a high quality environment for sperm to remain fertile upon artificial insemination. T her e are several steps to analyze a semen sample. Initially, aft er the semen has been collected, the semen shoul d be evaluated for sperm concentration, motility, and morphology; then the semen may be purified. Purification may be important because it
111 is necessary that non-contaminated, viable, and potentially fertile sperm are used whether inseminating or freezing s perm Before and after purification, the semen needs to be assessed and finally processed for preservation. In order to improve the predictabilit y of a semen sample and gain a better understanding of canine male fertility the following objectives and hypot heses were to be tested at the beginning of this work : to compare commercially available density gradient centrifugation media (Isolate, Percoll, PureCeption, PureSperm) in their ability to optimally separate viable, motile spermatozoa from nonviable (non m otile and/or dead) spermatozoa and red blood cells (RBC) contamination. The hypothesis tested was that there would be differences between the four DGC media in terms of their efficiency on canine sperm atozoa separation and purification, as determined by the ability to separate motile sperm from nonmotile and RBC for optimal sperm recovery. After collection of a semen sample, purification to remove contaminants (blood, urine, pus) (England and Allen 1992; Chen et al. 1995a) should improve semen quality and improve the overall pregnancy success rates. Density gradient centrifugation is a valuable and practical tool for sperm separation. Based upon the principle that due to centrifugal forces sperm ar e arranged with heads downward and flagella upward, that motile sperm cells move while non motile sperm and contaminants stay static (Kuji et al. 2008) Density gradient centrifugation has been described in several species including dogs (Parrish et al. 1995; Miller et al. 1996; Centola et al. 1998; Hishinuma and Sekine 2004) as a semen purification technique that can with little difficulty be done in a clinical or laboratory setting. There are several density gradient centri fugation media available, but the selected media Isolate, Percoll, PureCeption, and PureSperm have been
112 predominantly used in animal and human studies (Politch et al. 2004; Mousset Simeon et al. 2004) for their ability to separate sperm from contaminants. In this present study, significant differences were observed between the four density gradient centrifugation media in their ability to separate viable and motile sperm from non motile sperm and red blood cells. PureCeption and PureSperm abilities to separate blood from sperm in the optimal fraction were significantly different than Isolate and Percoll. Indeed, the optimal fraction of PureCeption had significantly higher recovery of total and m otile sperm cell compared to the optimal fraction for Isolate and Percoll; though PureCeption and PureSperm optimal fractions did not significantly differ. The maximum number of total and motile spermatozoa were located in one fraction in PureCeption and l ocated in two fractions in PureSperm. The practicality of density gradient centrifugation is so that all the sperm cells of interest reside at the bottom of a tube and the upper layers of fluid which contain contaminants can be decanted off leaving the des ired semen sample purified. The direct clinical application for sperm separation using density gradient centrifugation media is that it allows for efficient separation of motile sperm cells from contaminants. The efficacy of viable sperm cell separation f rom non motile sperm cells or contaminants is essential in the canine as it has been shown that detrimental effects can occur when semen is preserved in the presence of blood (Rijsselaere et al. 2004) Cushion centrifugation is a complementary process that has be en reported recently in equine (Volkmann 1987; Loomis 2006; Waite et al. 2008) and in porcine (Matas et al. 2007) to protect sperm cells during the centrifugation process. It has been associated with less denaturation of sperm cells than during centrifugation only. The combination of
113 our gradient centrifugation approach with the cushion centrifugation using iodoxynate may be a further step that would be of interest in the dog allowing separation of sperm cells without denaturation po ssibly with the centrifugation For practical use in a clinical setting, DGC is the method of choice. The ease of the technique is obvious, does not require sophisticated equipment as all that is needed is the DGC media and a centrifuge. The results are a vailable within minutes and when using DGC media the upper layers of fluid can be poured off leaving the viable, motile sperm cells available for insemination or semen preservation. While Percoll is the most often used DGC media of choice for many non huma n studies, Isolate, PureSperm, and PureCeption are increasing in popularity with the beneficial characteristic that Isolate, PureSperm, and PureCeption are all bioassay and endotoxin-tested. In other species such as mice and humans (Sapiro et al. 2000; Ren et al. 2001; He et al. 2003; Quill et al. 2003) sperm proteins have been reported to have a role in sperm function. Our objective was to identify and characterize, using immunocyt ochemistry the presence of sperm proteins SPAG6, SP17, SP56, and CATSPER2 proteins in c anine spermatozoa. The hypothesi s was that SPAG6, SP17, SP56, and CATSPER2 are localized and expressed in similar patterns in canine sperm cells as in other species and may play a role in sperm function, such as sperm motility, capacitation and/or acrosome reaction. While immunocytochemistry is a protein characterization tool that localizes protein expression it does not allow for confirmation of the presence of the proteins of interest Indeed, even if specific the antibody may easily cr oss -react with similar sequences of other proteins. Western blot analysis was used to confirm th e presence of the sperm
114 proteins of interest The hypothesis tested was: western blot analysis will confirm in the dog the presence of sperm proteins with molecular weights corresponding to the previously described SPAG6, SP17, SP56, and CATSPER2 proteins in other species spermatozoa (Cheng et al. 1994; Richardson et al. 1994; Neilson et al. 1999) In the present study in canine spermatozoa, SPAG6 was demonstrated to be strongly expressed and localized in the head reg ion with high incidence of panacrosomal labeling and variable punctate expression and equatorial localization. SPAG6 was also variable (negativemoderate) in expression (less than 20%) labeling at the midpiece of the flagellum At the level of the midpie ce, SP17 was demonstrated to be strongly expressed (more than 85%) and localized at the acrosome and principle piece, with weaker expression at the midpiece. SP56 was demonstrated to be strongly expressed and localized predominately (more than 95%) at the principle piece, with weak to moderate acrosomal expression (more than 25%). CATSPER2 was demonstrated to be expressed and localized at the flagella region, with weak expression (75%) localized at the acrosomal region. According to what has been shown in m ouse studies, SPAG6 is localized at the principal piece with weaker expression at the midpiece and head region (Sapiro et al. 2000) Even though the level of expression is not the same, the localization is similar and may still point to a role in sperm motility and/or capacitation and acrosome reaction functions. Canine SPAG6 was detected in a band at 61.57 kDa The predicted value for SPAG6 was 55.5 kDa and based upon the coefficient of variation which was estimated to be 10 -20% canine SPAG6 is within that range.
115 The exact role of SP17 for fertilization is still unknown. It has been proposed that based on flagel la localization that the function of binding to the zona pellucida is not the sperm proteins main function (Frayne and Hall 2002) Furthermore, this protein is also found in other tissues including so matic and tumor cells (Wen et al. 2001; Straughn, Jr. et al. 2004) In the present study, SP17 immunoreactivity was detected as proteins of different molecular weights: 15.45 28 .75 and 29.01 kDa which was in agreement with predicted values. The presence of multiple bands for SP17 protein is a generalized observation that has been reported in several publications (Kong et al. 1995; Wen et al. 1999; Grizzi et al. 2003) with variation in molecular weights as follows:1415, 17-19 21 24, 26 and 29 kDa. It was reported that (Lea et al. 2004) SP17 protein is found predominately in spermatozoa, cilia, and human neoplastic cell lines. In the present study, SP17 was present in the head and tail of spermatozoa. SP17 has been shown to bind Akinase anchoring protein 3 (AKAP3) b ut not to AKAP4. However, Akap4 knock out mice had a reduction in the amount of SP17 expressed when compared to wildtype sperm (Lea et al. 2004) Akap4 knockout mice have fibrous sheath that forms incompletely, that the principal piece is shorter and they are not progr essive moti le. From this information, it is a reasonable assumption that SP17 has a function for sperm motility and due to the SP17 association with anchoring proteins, that SP17 may have a role in sperm and zona pellucida binding. As SP17 was detected wit h the same overall localization and characteristics than SP17 in mice, we can probably suggest a similar role for SP17 in the canine: involvement in sperm cell motility and oocyte binding.
116 In the canine SP56 was consistently expressed at the principal pi ece with moderate expression at the acrosome. However, western blot analysis was unable to detect sperm protein SP56. Our results in ICC for fresh canine sperm were different than the one found in the mouse. I n the mouse, SP56 was characterized as sperm me mbrane protein (Cheng et al. 1994) and then later as an acrosomal matri x protein (Kim et al. 2001) Yet, mice with an Azh mutation that produce 100% abnormal sperm have SP56 localized on both acrosome and flagella (Moreno et al. 2006) In fact, the studies concerning sperm protein SP56 have provided conflicting and controversial results also in other studies. Different results have been reported for normal and abnormal mice for SP56 expression and sperm f unction (Cheng et al. 1994 ; Kim et al. 2001; Moreno et al. 2006) Moreover, the sperm protein SP56 was reported (Bookbinder et al. 1995) as a mouse sperm fertilization protein and detectabl e in mouse and hamster sperm and not detectable in guinea pig or human sperm. Several years later, another study (He et al. 2003) described SP56 localization in rat sperm Based on blast hits it was expected that SP56 with 6 blast hits for similar mouse and canine sequence to be identified in canine sperm cells. However, SP56 was not detectable by western blot analysis in canine sperm cells. This lack of detection by wes tern blot analysis could be a possible result of denaturation of the protein. Due to the conflicting published results in mice and contradictory results observed in the canine, the sperm protein SP56 warrants further studies in all species to determine loc alization, expression, and specific function. CATSPER2 was demonstrated in agreement with mouse sperm, to be localized to the flagellum in canine spermatozoa. There was also a weak expression at level of the
117 acrosome. The flagellar localization and the p revious reports (Quill et al. 2003; Qi et al. 2007) of function ality with hyperactivated sperm imply that CATSPER2 has a role in sperm motility and the calcium fluctuations which occur during capacitation and result in sperm hyperactivation. CATSPER2 was detecte d in different bands a t 48.48, 49.18 and 63 kDa. The predicted molecular weights are 48 and 61kDa. Of the four sperm proteins selected for this dissertation, CATSPER2, with a single blast hit, had the lowest probability to be detected in the dog. However, this study was able not only to identify CATSPER2 in canine sperm cells by ICC, but also to confirm its presence with the western blot analysis CATSPER2 had weak expression at the level of the acrosome This presence at the level of the acrosome could be a result of cross reactivity of CATSPER2 with CATSPER3 or CATSPER4, which are present and localized at the acrosome (Jin et al. 2005) It has been suggested that this family of four proteins form a functional heterotetram eric channel in sperm (Quill et al 2003). Semen preservation is a major part of canine reproduction management. Increasingly, owners are shipping the semen to the female for artificial insemination instead of allowing the dogs mate naturally. To improve th e chances that the semen sample is viable when it reaches its destination, the semen is either chilled or frozen extended in canine semen extender for transport (Linde-Forsberg 1991) In our quest to improve semen sample potential, our objectives were to evaluate the changes in selected sperm protein expression during preservation over a period of time, and establish a possible relationship between this expression and different semen parameters including motility, capacitation, and acrosome reaction. The hypotheses tested were: a) semen preservation over time affect protein expression, b) differences
118 may be observed between the different evaluated extenders, c) acrosome induced functional changes may be detected in relation to the preservation processes, d) acrosome reaction induction is associated with changes in the expression of the d ifferent semen proteins evaluated e) overtime changes in motility may be related to changes assoc iated with acrosome reaction and modification in the expre ssion of the different proteins. Preservation of semen involves providing an optimal in vitro envir onment for preserving sperm viability (Linde-Forsberg 1991; Iguer -Ouada and Verstegen 2001b) It is expected that semen extender will stabilize and protect the sperm cells from cold shock and th e plasma membrane from damage. In the current study, on day 0 no statistical differences in motility were observed between CaniPRO Chill 5, CaniPRO Chill 10, and Camelot groups directly after semen dilution. Also on day 0, the Synbiotics group had a signif icant decline in motility directly after semen dilution. At day 1, all diluted samples were not significantly different, though Synbiotics tended to be lower than the other groups. Over a period of time, motility progressively decreased for all groups. Synbiotics was below 50% motility on after days 3 -4. Camelot was at 50% motility on day 7. CaniPRO Chill 5 and CaniPRO Chill10 were below 50% motility after days 12 -13. Sperm classified as immotile or dead and were significant different for Synbiotics day 10, Camelot day 15, and CaniPRO Chill 5 and CaniPRO Chill day 18. There was not a significant difference between chilled semen extenders with percentage of acrosome-intact sperm to transition to acrosome reacted sperm. Semen preservation by chilling a ffected SPAG6 and CATSPER2 protein expression. For all chilled extenders, SPAG6 expression was weak or absent by day 3
119 and if weakly present only at acrosome or equatorial band. CATSPER2 had negative expression at the same time period for all chilled extenders. F or all chilled extenders, SP17 expression varied with the time period and acrosome integrity. Meaning acrosome intact sperm had acrosomal expression of SP17 present. While, acrosome damaged or acrosome reacted sperm, SP17 expression was diminished or absent. An interesting occurrence was observed in normal acrosome-intact sperm, the principal piece was weakly expressed. However, in acrosome reacted sperm the principal piece was obviously expressed. It has been reported (Zhang et al. 2005) that SPAG6 decoration of microtubules increases their stability and renders them resistant to depolymerization by cold (incubation at 4C). Based on this observation, SPAG6 should be resistant and stable in cold temperatures (4C). However, the study did not mention how long they were maintained at this temperature. In the canine, we found that cold temperature ( 4C ) causes SPAG6 expression to be weak or absent by day 3 Furthermore, it was mentioned (Zhang et al. 2007) that freezing and boiling of sperm result in loss of SPAG6 also lost in mic e with mutated sperm Humans with heterozygous mutation are fe rtile without sperm impairment and m ice with heterozygous mutation have impaired spermatogenesis (Zhang et al. 2007) Based on these observations, it appears that changes in temperature result in loss of SPAG6 expression similar to that observed in the present study in the canine where an obvious decrease or absence of expression of SPAG6 during preservation at 4 C. T his protein warrants further investigation. For the functional test of acrosome status, SP -TALP a sperm capacitation media, was significant in producing an acrosome reaction and caused a decrease (50%) in
120 number of acrosome -intact sperm when compared to non SP -TALP treated sperm. An induced acrosome reaction substantially transformed sperm protein expression. SPAG6 and CATSPER2 were moderately expressed and localized to the post acrosomal region T here was also negative to weak expression localized at the flagellum. SP17 labeling was absent from sperm cells. In a functional test, (Kong et al. 1995) live, capacitated mouse sperm do not stain with anti SP17. H owever, live capacitated, ionophore-treated sperm do stain, indicating that initially the polypeptide of SP17 is not available to bind to the surface of live sperm but becomes available as the acrosome reaction begins. In the canine, SP -TALP treatment resulted in an absence of SP17 staining. This result was in agreement with the live, capacitation mouse sperm not expressing SP17. The Kong study, mentioned that live, capaci t ated ionophore treated mouse sperm did stain. This raises the following question: were the canine sperm in our study capaci t ated? Based on light microscopy observations after the acrosome reaction induction study, the few remaining active sperm cells were traveling in a forward progressive movement, most were spinning around in a pin wheel circular motion and the flagella had a high beat frequency typical of hyperactivation. Based on the above results, it appears that during sperm conservation in semen extender at 4 C, the sperm cells undergo physiol ogical changes which result in the slow progressive breakdown of plasma membranes overtime. The breakdown of plasma membranes and the potent ial increase in acrosome damage could justifiable be the explanation for changes of sperm protein expression observed. In addition, the slow progression of the breakdown of the membranes could explain why overtime there was
121 not a significant decrease in acrosome-intact sperm. The slowness of the deterioration may have been enough to cause some damage to the acrosome, bu t not adequate to initiate an acrosome reaction. On the other hand, induction of the acrosome reaction caused a rapid change in th e plasma membrane, which resulted in the deterioration of the plasma membrane and the releas e of acrosomal enzymes. The sperm proteins that were released were no longer present or were diminished in their availability. This was a justifiable reason for the decrease or absence in sperm protein expression after acrosome reaction induction. The supportive evidence was shown with po st acrosomal region staining present for SPAG6 and CATSPER2 proteins. In conclusion, a complementary approach to the evaluation of canine semen and potentially of semen fertility in the male dog was suggested based on the results of this study. Based on the dissertation introductory analogy, the development of more efficient readily available, and quicker ways to assess fertilization potential of a semen sample must be developed. Classical semen evaluation parameters alone are not enough to predict ferti lit y outcome. A different approach should be to evaluate sperm characteristics and functions. Mouse research models and e specially knockout models (Sapiro et al. 2002; Moreno et al. 2006) have been instrumental in characterizing sperm functionality as it relates to fer tilization ability. Research needs to be continued for the development of additional knockout mouse models, as well as, sperm studies in other species, that will help us further understand the roles that sperm proteins portray in the fertilization process and to what degree the specific sperm protein functions.
122 Taken together, t he results of this study demonstrated that the use of PureCeption for canine semen purification is of interest to separate motile sperm cells from contaminants including RBC. However, further work needs to be don e to confirm these results and its clinical implications This is the first showing that sperm proteins SPAG6, SP17, and CATSPER2 are localized in canine sperm and that they can potentially be biochemical markers for canine f ertility to predetermine if the sperm from an individual canine will be a potentially good semen sample to undergo density gradient centrifugation, functional tests, and semen preservation. With these techniques combined, the chances of accurately predicti ng fertility of fresh and preserved sperm. Above all these techniques must provide an accurate prediction of fertility, be objective, repeatable, inexpensive, fast, and easy to do in a clinical or laboratory setting (Amann 1989; Rodriguez -Martinez 2003)
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139 BIOGRAPHICAL SKETCH Tameka Cerise Phillips was born in Urbana, Illinois. She is the second of two children that were born ten years apart. She has always had a fascination with animals and the sciences. She was accepted into the University of Illinois at Urbana -Champaign in 1994. She graduated with a bachelors degree in animal sciences in 1999. While an undergraduate student, she developed a strong interest in reproductive physiology. In 1999, she was offered a masters degr ee program on fellowship from the University of Florida, College of Veterinary Medicine. In the spring of 2000, she began her masters degree program, in which she completed in 2 001. She moved back to Illinois for a few years before the decision was made for her to return to the University of Florida, College of Veterinary Medicine to start a Ph.D. program. Upon the complete of Ph.D. degree, Tameka plans to begin her career in sci ence and government.