Group Title: Characterization of protein secretions by the porcine uterus and their relationship to reproductive physiology /
Title: Characterization of protein secretions by the porcine uterus and their relationship to reproductive physiology
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Title: Characterization of protein secretions by the porcine uterus and their relationship to reproductive physiology
Physical Description: 63 leaves : ill. ; 28 cm.
Language: English
Creator: Murray, Finnie Ardrey, 1943-
Publication Date: 1971
Copyright Date: 1971
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Subject: Chemical embryology   ( lcsh )
Animal Science thesis Ph. D
Dissertations, Academic -- Animal Science -- UF
Genre: bibliography   ( marcgt )
non-fiction   ( marcgt )
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Thesis: Thesis (Ph. D.)--University of Florida, 1971.
Bibliography: Bibliography: leaves 54-62.
Additional Physical Form: Also available on World Wide Web
General Note: Typescript.
General Note: Vita.
Statement of Responsibility: by Finnie Ardrey Murray.
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Bibliographic ID: UF00097684
Volume ID: VID00001
Source Institution: University of Florida
Holding Location: University of Florida
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Resource Identifier: alephbibnum - 000402344
oclc - 24664080
notis - ACE8222

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CHARACTERIZATION OF PROTEIN SECRETIONS BY THE PORCINE
UTERUS AND THEIR RELATIONSHIP TO REPRODUCTIVE PHYSIOLOGY










By


Finnie Ardrey Murray, Jr.


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








University of Florida


1971











ACKNOWLEDGMENTS


For their advice and assistance in this research, the author

wishes to acknowledge sincere gratitude to the members of his super-

visory committee: Dr. A. C. Warnick, chairman; Dr. F. W. Bazer;

Dr. J. A. Himes; Dr. O. II. Rennert; and Dr. R. L. Shirley.

The author wishes to express deep appreciation to Dr. F. W. Bazer

for his constant encouragement and willingness to be of assistance.

Without his continual help and guidance this research could not have

been completed.

Dr. H. D. Wallace kindly furnished the swine used in this

research; and without this valuable assistance, the work could not have

been done. Thanks are also due D. E. Pogue for care and feeding of

animals. Also greatly appreciated was the valuable assistance which

was received from G. D. Squire, who carried out the electrophoresis

in this research.

Thanks are also due the author's fellow graduate students who

generously assisted with surgery. These are Lee E. Anderson,

Yvonne Barber, James R. Dickey, Diego M. Giminez, Albert C. Mills,

Richard H. Smith, and Gregory D. Squire.

The author deeply appreciates the sacrifices, encouragement,

and assistance so freely given by his wife, Margaret Alice.













TABLE OF CONTENTS

Page

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

LIST OF FIGURES................................................ iv

ABSTRACT....................................................... v

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

CHAPTER 1. REVIEW OF LITERATURE............................... 3

CHAPTER 2. EFFECTS ON DEVELOPMENT OF PORCINE EMBRYOS
CAUSED BY RESTRICTION TO THE OVIDUCT............... 18

Materials and Methods................................. 18

Results and Discussion.............................. 19

CHAPTER 3. QUANTITATIVE AND QUALITATIVE CHANGES IN PROTEIN
SECRETION BY THE SWINE UTERUS DURING THE ESTROUS
CYCLE............................................. ...... 24

Materials and Methods.......... ...................... 24

Results and Discussion ............................. 27

CHAPTER 4. PREGNANCY IN GILTS AFTER INTRAVENOUS INJECTIONS
OF ANTI-PORCINE UTERINE FLUID PROTEINS............. 38

Materials and Methods................................ 38

Treatment of Gilts Trial I.................. 39

Treatment of Gilts Trial II................. 39

Results and Discussion.............................. 40

CHAPTER 5. GENERAL DISCUSSION................................. 42

SUMMARY........................................................ 51

LIST OF REFERENCES............................................. 54

BIOGRAPHICAL SKETCH............................................ 63












LIST OF FIGURES

Figure Page

Plate 1.................................... .......... 22

1 A tube-locked embryo recovered from gilt 324
seven days post coitum......................... 22

2 A uterine blastocyst recovered from gilt 324
seven days post coitum........................... 22

3 A tube-locked embryo recovered from gilt 331
seven days post coitum........... ............... 22

4 Variation in the total quantity of swine uterine
fluid protein secreted per day of the
estrous cycle.................................. 29

5 Variation in gel filtration protein profiles with
days of the estrous cycle..................... 32


Plate 2............................................... 34

6a Polyacrylamide gel disc electrophoresis of swine
day 15 uterine fluid proteins, migration
toward the anode................................ 34

6b Polyacrylamide gel disc electrophoresis of
Fractions I through V, migration toward
the anode..................................... 34

6c Polyacrylamide gel disc electrophoresis of
Fractions I through V, migration toward
the cathode................................... 34

7 Variation with days of the estrous cycle in the
relative percentage of uterine fluid proteins
with molecular weight estimates of 10,000
to 25,000..................................... 35













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


CHARACTERIZATION OF PROTEIN SECRETIONS BY THE PORCINE
UTERUS AND THEIR RELATIONSHIP TO REPRODUCTIVE PHYSIOLOGY

By

Finnie Ardrey Murray, Jr.

March, 1971


Chairman: A. C. Warnick
Co-chairman: F. W. Bazer
Major Department: Animal Science


The uterus actively influences reproduction in many species

of mammals by participation in embryonic differentiation and in

determination of estrous cycle length. In this work the secretion

of proteins by the porcine uterus during the estrous cycle was

characterized and related to reproductive phenomena.

To determine if the porcine uterus provides an environment

essential for continuous development of early embryos, an experiment

which involved restriction of embryos to the oviduct was carried out.

One tubo-uterine junction was ligated in each of 10 mated gilts under

surgical anesthesia at 48 to 72 hours post coitum. The contralateral

uterine horn was ligated approximately 10 cm posterior to the tubo-

uterine junction and served as a control. The animals were slaughtered

either 7 or 8 days after mating and the embryos were recovered, examined,

and measured. The tube-locked embryos failed to develop past the

morula stage and many had begun degenerating by the time of recovery.

No tube-locked embryo had escaped from the zona pellucida. However,

v








uterine embryos in the same females were mostly blastocysts, and

84 percent had emerged from the zona pellucida and formed a distinct

inner cell mass. These data suggest that some factor secreted by

the porcine uterus is required by embryos for continued development.

Daily quantitative and qualitative variation in the protein

constituents of uterine intraluminal fluid was studied on days 2 to

18 and day 20 of the estrous cycle. Sixty-six gilts furnished one

sample of uterine fluid each. Samples were centrifuged at 3000 g,

sterilized by millipore filtration, concentrated from approximately

40 ml to 1 ml, and dialyzed against 0.05 M phosphate-citrate buffer,

pH 7.4. Gel filtration through Sephadex G-200 was used to establish

protein profiles for each sample and for estimation of molecular

weights. Polyacrylamide gel disc electrophoresis was employed to

compare the protein components among the various samples.

Gel filtration revealed three groups of proteins (Fractions I,

II, III) which did not vary with the estrous cycle and two groups of

proteins (Fractions IV and V) which were not present early or late in

the estrous cycle. Fraction IV, which appeared on day 12, contained a

lavender protein with an estimated molecular weight of 45,000, while

Fraction V had an estimated molecular weight of about 20,000, and

appeared as early as day 9. Fractions IV and V were not present in the

gel filtration protein profiles after day 16 of the cycle.

Disc electrophoresis demonstrated that Fraction IV contained at

least three basic proteins, while Fraction V contained one basic protein

and six acidic proteins at pH 8.0. The basic proteins, especially,

are interesting as possible regulators of embryonic differentiation by

control of genetic expression. The fact that these proteins appear in









the uterine flushings shortly before the corpus luteum begins to regress

and are not present after regression suggests an interrelationship with

the corpus luteum.

Attempts to determine the role of the uterine fluid proteins

in reproductive phenomena were largely unsuccessful. Anti-uterine

fluid sera did not interfere with pregnancy when injected intravenously

into mated gilts. Final conclusions cannot be drawn from these data

since it is possible that the antibodies injected did not reach the

uterine lumen in sufficient quantities to bind proteins secreted by the

uterus.

This study showed that the uterine environment is required for

embryonic development and that there are cyclic changes in the protein

secretion pattern of the uterus. These findings provide additional

support for the concept that proteins of maternal origin may regulate

genetic expression in the embryo during early differentiation.












INTRODUCTION


The mammalian uterus serves in the reproductive process to

protect the embryo, to supply nutrition to the embryo, and to par-

ticipate in the differentiation of the embryo. The latter function

is the least understood, but perhaps the most important. Evidence

has accumulated that some factor provided by the uterine environment

is required for uninterrupted embryonic development and that the

uterus is actively involved in the reproductive process.

The active role of the uterus in reproduction has been studied

in a variety of experiments, using indirect methods such as ova culture,

superovulation, embryo superinduction, and hysterectomy. Using a direct

approach Krishnan and Daniel (1967) identified, in rabbit uterine fluid,

a protein which stimulates blastulation of rabbit embryos in vitro.

This is the first uterine protein demonstrated to have an apparent

regulatory function in embryonic development. This information is

extremely important because it provides clues into the mechanism by

which the uterus exerts an active control over embryonic development.

There is also direct evidence that the uterus actively par-

ticipates in the regulation of the life span of the corpus luteum.

The evidence suggests that this regulatory factor is also proteinic in

character. If uterine proteins with regulatory functions on embryonic

development and the corpus luteum are a general rule in mammalian

reproduction, methods for either increasing fertility or decreasing it

to zero may be near at hand. Also, interesting possibilities for study









of the mechanism of differentiation of mammalian cells as influenced

by specific proteins are apparent.

Considering the available information on these roles of the

uterus, a working hypothesis can be formulated concerning the regulatory

function of the uterus in reproduction. The working hypothesis, which

served as a basis for these studies can be stated as follows: the uterus

secretes a protein(s) which is required by the embryo for uninterrupted

development; but, if embryos are not present to utilize the protein(s),

it causes regression of the corpus luteum.

This study was carried out to determine if the uterine environment

is required for embryonic development in the pig and to qualitatively

and quantitatively characterize the secretion of protein by the porcine

uterus during the estrous cycle. In addition, attempts were made to relate

the proteins secreted by the uterus to embryonic development.












CHAPTER 1. REVIEW OF LITERATURE


In recent years research has made it clear that the uterus

plays a much more profound role in mammalian reproduction than the

rather passive functions of nutrition and protection of the embryo.

There is much evidence that the uterus influences embryonic develop-

ment in a very specific way and that the life span of the corpus luteum

is under the control of the uterus. These active roles of the uterus

are suggested by several lines of evidence as will be discussed below.

One line of evidence was developed from experiments with

in vitro culture of the embryos. Lewis and Gregory (1929) found that

rabbit ova developed in rabbit blood plasma to the blastocyst stage

but no further. This failure of development past the early blastocyst

stage in vitro has been the general rule regardless of the composition

of the culture medium or the species of animal studied (Chang, 1949;

Hammond, 1949; Tarkowski, 1961; Brinster, 1963; Adams, 1965; Cole and

Paul, 1965; Onuma et al., 1968; Maurer et al., 1969; and Rundell, 1969).

Recently two groups of workers (Whitten and Biggers, 1968; and Kane and

Foote, 1970 a,b,c) have been somewhat more successful in embryo culture

and have grown embryos to the beginning of blastocyst expansion in

chemically defined media alone or in media containing bovine serum

fractions. However, continued development of preblastocyst embryos past

the early blastocyst stage in vitro has not been reported.

In vitro culture of more advanced embryos has lately been

achieved by use of a device developed by New (1967) for recirculating








the culture medium. New (1967) reported that 5- to 10-somite rat embryos

grew to 30 to 35 somites, and 25-somite embryos grew to 40 to 45 somites

in recirculating homologous serun. Embryos more advanced than 45 somites

did not develop further in vitro. Using the New (1967) recirculator

or modifications of it, hamster embryos have been grown to a stage of

development at which 50 percent of the embryos had limb buds and 63 percent

had good circulation of blood (Givelber and DiPaolo, 1968), rat embryos

explanted at 7.5 days post coitum have been grown to the 15- to 20-somite

stage (New and Daniel, 1969), and rabbit embryos explanted at 6.8 days

post coitum have been cultured to the 23-somite stage (Daniel, 1970a).

Thus after achieving the blastocyst stage in utero and implanting, embryos

become at least temporarily independent of the uterine environment.

Other evidence for an active role of the uterus in embryonic

development is derived from research which indicates that the oviduct

can support development to the morula or early blastocyst stage, but no

further. Restriction of embryos to the oviducal environment by ligation

of the tubo-uterine junction does not prevent development to the early

blastocyst stage in mice (Kirby, 1962; and Orsini and McLaren, 1967),

rabbits (Westman et al., 1931; Pincus and Kirsch, 1936; and Adams, 1958),

rats (Alden, 1942), and sheep (Wintenberger-Torres, 1956); however, further

development of tube-locked embryos has not been observed. Adams (1958),

Kirby (1962), and Orsini and McLaren (1967) reported that the tube-locked

embryos not only failed to continue development, but underwent degenera-

tion within the oviduct.

Transplantation of rat and mouse tubal embryos to extrauterine

sites has been attempted, but with little apparent success. Fawcett et al.

(1947) and Runner (1947) transplanted mouse oviducal embryos to the

anterior chamber of the eye. Implantation occurred but only extraembryonic









membranes developed. Similar abnormal growth was observed in mouse

tubal embryos (Fawcett, 1950; and Kirby, 1962) and in rat tubal embryos

(Nicholas, 1942) transplanted beneath the kidney capsule. However,

developmental failure of extrauterine embryos is not a universal

phenomenon. It is common knowledge that extrauterine embryonic develop-

ment occurs in at least one species, i.e., the human, where ectopic

pregnancies in a variety of extrauterine sites have been reported.

Experiments involving superovulation and embryo superinduction

have provided still further evidence for the role of the uterus in

control of early embryonic development. Bazer et al. (1969a) reported

that litter size in swine at 90 days post coitum was similar for

unoperated controls, sham-operated controls, and for three levels

of embryo superinduction. The number of corpora lutea in each recipient

was counted and considered to represent the number of native embryos.

Embryo superinduction was achieved by transfer of additional 2- to 4-cell

embryos into the uteri of pregnant recipients. The three groups receiving

extra embryos had 16, 22, or 28 potential embryos after transfer. At

90 days post coitum average litter size in the five groups was 9.9, 8.2,

8.8, 8.5, and 9.9 for the control, sham, 16-embryo, 22-embryo, and

28-embryo groups respectively. The authors referred to this limitation

in litter size as "uterine capacity."

In the same experiment, Bazer et al. (1969a) observed that

synchrony of transferred embryos and recipient uteri is very important.

When development of foreign (transferred) embryos relative to native

embryos was equal, the survival of native embryos was 51 percent compared

to 35 percent for foreign embryos and this difference was not significant.

When the foreign embryos were 12 hours behind the native embryos, the








survival rates were 56 percent for native embryos compared to 20 percent

for foreign; and, even more strikingly, when foreign embryos were 24

hours behind the native embryos, survival rates were 72 percent for native

compared to 3 percent for foreign embryos. These differences in survival

rates were highly significant, and they indicate that embryos in synchrony

with the uterus are at a great advantage in competing for possible uterine

factors as compared to asynchronous embryos. This suggests that embryos

must reach a precise stage of development before they can utilize the

uterine factor and that the uterine factor is available only at a specific

time. Kirby (1962) found this to be the case in the mouse. When 3.5-day

tube-locked blastocysts were transferred to the uterus, development

continued normally, but if the blastocysts were forced to remain in the

oviduct until 4.5 days post coitum'further development did not occur after

transfer to the uterus. Other authors have also reported that synchroniza-

tion of donors and recipients is necessary for survival of transferred ova

in the rat (Dickmann and Noyes, 1960) and in the pig (Hunter et al., 1967).

However Webel et al. (1970) reported that asynchrony by plus or minus 1

day did not affect embryo survival in swine.

Bazer et al. (1969b) compared embryonic survival after embryo

superinduction at 25 days and at 105 days post coitum. Among gilts

slaughtered at 25 days post coitum the average number of potential embryos

corporaa lutea plus transferred embryos) was 23.9, and the average litter

size was 12.0 (50 percent survival). In the group of gilts slaughtered

at 105 days, there were 23.6 potential embryos and the average litter

size was 9.8 (42 percent survival). Unoperated control gilts had litter

sizes of 9.6 at 25 days and at 105 days post coitum, with 74 and 75 percent

survival, respectively. Thus this experiment shows clearly that the







majority of embryonic deaths following embryo superinduction occur prior

to 25 days post coitum. This is in agreement with data from untreated

gilts (Gossett and Sorenson, 1959).

Fenton et al. (1969a) reported that embryo superinduction of

gilts by transfer of 7-day blastocysts resulted in a significant increase

in average litter size at 25 days after mating, as compared to litter size

in control gilts. Therefore, it was postulated that "uterine capacity" is

established prior to day 7. To test this hypothesis, Fenton et al. (1969b)

used 2.5-day and 7-day embryos and Schwartz et al. (1970) used 7-day embryos

in embryo superinduction of gilts. Average litter sizes at 25 days post

coitum were not different from those in control gilts; therefore, contrary

to the results of Fenton et al. (1969a), it appears that "uterine capacity"

is not established prior to day 7, but instead between day 7 and day 25.

The effects of restricted uterine space on embryonic development

has also been studied. In swine unilateral hysterectomy-ovariectomy

(Dziuk, 1968; and Fenton et al., 1970) did not affect litter size at 25

days after mating. Variation of the amount of uterine space by ligation

of the uterine horns so that embryos on one side had half the uterine

space available to embryos in the contralateral horn (Dziuk, 1968) did

not affect embryonic survival in swine. In mice, rabbits, and rats

Johnson (1970) established pregnancy in one of the two uterine horns by

unilateral ovariectomy. In these animals, ovulation rate was similar to

that in intact females, and litter size was no different even though the

amount of uterine space was only half that available to embryos in intact

females. Similar results have been reported earlier by Hafez (1964) using

rabbits. Thus in mice, rabbits, and rats one uterine horn can support

as many embryos as two uterine horns. However, final litter size at








105 days after mating was decreased by surgical reduction in uterine

tissue in the pig (Fenton et al., 1970).

Superovulation is followed by increased embryonic death in

swine so that litter size at farrowing is not significantly greater

than in control gilts (Hammond, 1921; Perry, 1954; Rathnasabapathy

et al., 1957; King and Young, 1957; Hunter, 1966; Dziuk, 1968;

Longenecker and Day, 1968; Pope et al., 1968; and Bazer et al., 1969c).

Also, in rabbits superovulation does not increase litter size and even

tends to decrease the number of feti surviving to term. Adams (1960)

reported that superovulation resulted in a mean of 28.7 implantations

but only 6.5 surviving feti at term in rabbits. Hafez (1964) also found

that superovulation failed to increase litter size in rabbits and that

the maximum number of viable feti at term was 8 per uterine horn and 15

per litter. Johnson (1970) reported that in rabbits average litter size

could not be increased significantly, but that one uterine horn could

carry as many embryos as two horns. In mice the litter size is increased

following superovulation (McLaren and Michie, 1956; McLaren and Michie,

1959 a,b; and Wilson and Edwards, 1963). On the other hand, Fowler and

Edwards (1957) were unable to increase litter size by superovulation of

mice. In general, polytocous species appear to have a limit to the

number of embryos that can be maintained to term and in pigs and rabbits

this limit is apparently closely approached in the normal pregnancy.

Mice seem to be an exception to this general rule, suggesting that the

number of ova shed is less than the number required to reach the "uterine

capacity" of this species.

Further evidence for uterine control of the fate of blastocysts

is found in the research on delayed implantation of embryos. As indicated







in a review of this subject by Daniel (1970b), delayed implantation

occurs in a wide variety of species. In some species the delay is

obligative while in others it is facultative (occurring only when the

female becomes pregnant during lactation). In rats and mice implanta-

tion can be prevented without degeneration of the blastocysts by

ovariectomy prior to day 4 post coitum and daily treatment with 2 mg

progesterone (Dickmann, 1967). Implantation can be induced in such

animals by a single 1 pg dose of estrone. On the other hand, Humphrey

(1969) did not need to administer estrogen to ovariectomized, progesterone-

treated mice in order to cause implantation.

The role of the uterus in implantation and the importance of the

hormonal state of the female in this role was clearly demonstrated by

Dickmann and DeFeo (1967). In this experiment rats were ovariectomized

on the second day of pregnancy in order to provide uteri and blastocysts

in the delayed state, i.e., dormant. A group of intact female rats

served as a source of active uteri and blastocysts. When dormant embryos

were transplanted to the active uteri, they became active and implanted

within a few hours after being placed in the active uteri. When active

blastocysts were transplanted to dormant uteri, these embryos became

dormant and remained dormant until implantation was induced by administering

estrogen to the female. Thus it is the uterus which determines the condi-

tion of the embryos.

Experiments on delayed implantation in general and, more specifically,

the work of Dickmann and DeFeo (1967) indicate that progesterone and estrogen

are very important to the role of the uterus in controlling embryonic develop-

ment. The specificity of the uterus as a target organ for estrogen has been

clearly shown by Gorski and co-workers (1968). In a series of experiments,








these authors found that estrogenic compounds were specifically bound

to receptor molecules in the rat uterus. One receptor was a 9.5 S

protein component in the uterine cytosol and the other was a 5 S protein

in the nucleus. Nonestrogenic steroids had no apparent interaction

with the receptor molecules. Jensen and DeSombre (1969) showed that the

uterine uptake of estrogen is unsaturable even with hyperphysiological

levels of estrogen; however, the retention of estrogen is limited.

Estrogens have been shown to induce nuclear RNA synthesis and

uptake of RNA precursors in the uterus within two minutes after

injection (Means and Hamilton, 1967). Hamilton et al. (1967) reported

that the rate of nuclear RNA synthesis and activity of Mg -activated

RNA polymerase was greater at estrus than at diestrus; whereas, nuclear

RNA and protein content did not vary with the estrous cycle. Whole

uterine tissue content of both RNA and protein per cell was two and five

times greater, respectively, at estrus than during diestrus. Contrary

to the work of Hamilton and co-workers, Billing et al. (1969) reported

that uterine RNA synthesis, as indicated by incorporation of radioisotope-

labeled precursors, did not increase until five hours after estradiol

treatment and that the RNA synthesized was mainly r-RNA and t-RNA with

very little m-RNA. From these experiments it is evident that estrogenic

compounds profoundly influence the metabolism of the uterus, inducing

both RNA and protein synthesis. Notides and Gorski (1966) reported

that estradiol stimulated synthesis of a new protein specific to the rat

uterus both in vivo and in vitro. This protein appeared within 30 minutes

after administration of estrogen, but contributed only a minor amount

to the quantity of total protein in the uterus.







There are few reports in the literature which suggest induction

of RNA and protein synthesis by progesterone. However there is ample

evidence for induction of at least one specific protein by progesterone.

Hertz et al. (1943) showed that progesterone induced thebiosynthesis of

avidin in the estrogen pretreated oviduct of the immature chick. O'Malley

(1967) found that progesterone could induce avidin synthesis in minced

chick oviduct tissue in vitro. Dingman et al. (1969) reported that

progesterone stimulated biosynthesis of both nuclear and cytoplasmic

t-RNA in the chick oviduct. It has been shown that progesterone induces

synthesis of new species of m-RNA by the chick oviduct prior to the

initiation of avidin biosynthesis (O'Malley and McGuire, 1969). Aside

from its effect on avidin biosynthesis, progesterone reportedly has a

detrimental influence on estrogen-induced RNA synthesis in the rat uterus

(Trams and Maass, 1969) and in rat mammary tissue (Eddington, 1969).

The meager amount of information available suggests that any role of

progesterone in RNA and protein biosynthesis is likely to be highly

specific.

The protein constituents of uterine luminal fluids have been

investigated in a number of studies in recent years. Junge and Blandau

(1958) found four major electrophoretic components present at low levels

in rat uterine fluids. Albumin was usually not present in the uterine

fluid obtained by these authors. Ringler (1961) reported that rat

uterine fluid contained proteins with five different electrophoretic

mobilities. Of these five components, a prealbumin fraction was specific

to uterine fluid. Ringler proposed that uterine luminal fluid is

essentially an ultrafiltrate of plasma but includes specific uterine

secretions. Utilizing the techniques of buchterlony gel diffusion and








immunoelectrophoresis, Albers and Castro (1961) showed that pooled

and concentrated rat uterine fluid contains at least five protein

components. Only one of the five components was specific to uterine

fluid, and it migrated to a position occupied by B-globulins in serum.

The proteins of rabbit uterine fluid were first studied by

Stevens et al. (1964). These workers used diffusion-in-gel, moving

boundary electrophoresis,and immunoelectrophoresis to characterize

the proteins in fluids from the ligated uteri of estrous rabbits.

Moving boundary electrophoresis separated eight components, among which

a prealbumin and an a-globulin were specific to uterine fluid and not

found in serum. Diffusion of uterine fluid in gel revealed 13 antigenic

components of which three were unique to uterine fluid. Five antigens

specific to uterine fluid were made evident by immunoelectrophoresis.

Two of the five antigens had mobilities in the prealbumin region and

three were similar to B-globulins.

In 1967, Krishnan and Daniel studied the uterine fluid protein

constituents of rabbits in early pregnancy. The fluid was subjected to

gel filtration through Sephadex G-200 and five major protein fractions

were obtained. The fourth fraction was not present in the chromatographic

profiles until day 3 post coitum. The maximum concentration of this

fraction occurred on day 5 post coitum and afterward appeared in pro-

gressively smaller proportions of the total protein until day 9. The

variable fraction was also observed in uterine fluid of pseudopregnant

rabbits on day 7 post coitum and blastocoelic fluid, but not in uterine

fluid accumulated by ligation of the rabbit uterus from day 3 to day 10

of pregnancy, maternal serum, fetal serum, or fetal amniotic fluid. This

fraction was used as a supplement to Ham's F 10 culture medium in rabbit








embryo culture and it promoted the development of embryos to the

expanding blastocyst stage. This property prompted the authors to

call this fraction "blastokinin." The molecular weight of "blastokinin"

was estimated by gel filtration to be 27,000, and it was found to be

a glycoprotein. In a later paper (Krishnan and Daniel, 1968) it was

reported that amino acids constituted about 74 percent of the protein

and 6 percent was contributed by carbohydrates. No sialic acid was

found in the glycoprotein.

Beier (1968) reported that, based on immunologic and electro-

phoretic techniques, flushed uterine fluid and blastocoelic fluid

obtained on the sixth day of pregnancy contained plasma proteins as

well as specific uterine fluid proteins. One of the proteins specific

to the uterus was reported to be involved in blastocyst development

and was designated "uteroglobin." "Uteroglobin" was shown to be a

glycoprotein with a molecular weight of about 30,000 as determined by

ultracentrifugation. Steroid hormones were shown to have a profound

effect on the level of "uteroglobin" in 6-day post coitum uterine

fluid. Treatment of rabbits with estradiol for three days (100, 100,

and 50 pg) plus 5 mg progesterone per day for five days caused the

relative percent of "uteroglobin" to be double that in the normal 6-day

pregnant rabbit. Administration of excessive amounts of estrogen caused

a reduction in the amount of "uteroglobin" produced. Kirchner (1969)

also reported a specific glycoprotein in rabbit uterine secretion.

Hamana and Hafez (1970) demonstrated "uteroglobin" in rabbit blastocoelic

fluid between days 3 and 9 of pregnancy. Both Kirchner (1969) and Beier

(1970) believed "uteroglobin" and "blastokinin" to be the same protein.









As was discussed earlier in this literature review, it is a

general rule that embryos can develop no further than the morula or

early blastocyst stage in synthetic media in vitro. There have been

exceptions to this rule (Whitten and Biggers, 1968; and Kane and Foote,

1970 a,b,c); however, this does not diminish the significance of

"blastokinin" since the effect of the presence and absence of "blastokinin"

were observed under the same experimental conditions. The stimulatory

effect of "blastokinin" on rabbit embryos in vitro suggests that it may be

involved in the process of implantation (Beier, 1970).

Daniel (1968 and 1970 b) and Daniel and Krishnan (1969) have

studied the relationship between uterine fluid proteins and embryonic

diapause in a number of species of mammals having delayed implantation.

Daniel (1968) obtained fluid from the rat, during delayed implantation

and in normal pregnancy; the fur seal; the armadillo; and the mink.

Polyacrylamide gel disc electrophoresis revealed no protein with similar

electrophoretic mobility to "blastokinin" except possibly in low con-

centration in uterine fluid of mink.

Daniel and Krishnan (1969) found that the protein content of

uterine flushings of animals undergoing delayed implantation was much

lower than was the case in other species. These authors found a small

"blastokinin"-like fraction in the Sephadex gel filtration profiles of

mink uterine fluid obtained on day 20 post coitum, but not prior to

this time. This is just before the time at which mink embryos implant

(Baevsky, 1963). Diapausing embryos of animals with obligative delayed

implantation (mink, fur seal, and armadillo) were stimulated to grow

in vitro in the presence of rabbit "blastokinin" as indicated by mitotic

activity but not in media containing serum. Diapausing rat blastocysts








(i.e., species with facultative delayed implantation) expanded in vitro

in Ham's F 10 medium alone, in medium containing maternal serum, or in

medium containing macromolecular components of uterine secretion, but

not rabbit blastokinin. Thus, in facultative delayed implantation

there is apparently not only a protein deficiency but also some other

uterine condition which inhibits growth of the blastocysts.

The role of the uterus is not limited solely to control of

embryonic development but it also affects life span of the corpus luteum

in at least some animals (see reviews by Anderson et al., 1963; Bland

and Donovan, 1966; Anderson et al., 1969; Schomberg, 1969; and Rowson,

1970). In these reviews it was pointed out that hysterectomy in cattle,

pigs, sheep, and other species results in maintenance of the corpus

luteum for approximately the length of normal gestation. Goding et al.

(1967) showed that if the uterus of a sheep was transplanted to the

neck, while leaving the ovaries intact, estrous cycle lengths were

increased. Harrison et al. (1968) showed that if both the ovary and the

uterus were transplanted together to the neck the cycle lengths were

normal. These experiments suggest that the luteolytic factor is not

mediated via the pituitary gland or nervous connections.

Caldwell et al. (1967) reported that homologous uterine trans-

plantation into the cheek pouches of the Syrian hamster caused regression

of the corpora lutea in hysterectomized hamsters. Transplantation of

uteri from estrous rats, uteri from 14-day pseudopregnant rabbits, and

homologous endometrium into the cheek pouch of the hysterectomized

Syrian hamster caused regression of corpora lutea. Similar trans-

plantations of human endometrium and homologous myometrium were

ineffective. Caldwell et al. (1968) reported that when transplanted









to the neck of hysterectomized pseudopregnant hamsters, sheep endometrium

obtained toward the end of the cycle caused regression of the corpora

lutea. This suggests that an endometrial secretion that is not species

specific is responsible for luteolysis.

Several early attempts to demonstrate luteolytic effects of

endometrial extracts were largely unsuccessful (see Schomberg, 1969)

until Williams et al. (1967 a,b) reported that acetone-dried extracts

of bovine uteri caused luteal regression in the rabbit. Schomberg

(1967) applied swine uterine flushings to luteinized granulosa cells

in vitro. Uterine flushings on days 1 to 10 and day 20 of the estrous

cycle produced no effect on the growth of granulosa cells or their

progesterone production. Flushings obtained on days 14 to 18 of the

estrous cycle were very luteolytic and had completely destroyed the

cells within 6 to 8 hours. The luteolytic effect was also sometimes

present in uterine flushings obtained on day 12 of the estrous cycle.

Schomberg (1969) demonstrated that the uterine fluid component with

luteolytic activity was thermolabile and nondialyzable, and the

molecular weight was estimated to be about 200,000.

Mazer and Wright (1968) found a nondialyzable luteolytic factor

in the uterus of the hamster on days 6 and 7 of pseudopregnancy.

Lukaszewaska and Hansel (1970) found that extracts from bovine endo-

metrium reduced hamster corpus luteum weight and progesterone content.

The endometrial extracts were water soluble and precipitable in 55

percent ammonium sulfate. The 55 percent ammonium sulfate fraction had

a partition coefficient of 0.271 when filtered through Sephadex G-100.

The active factor was concluded to be either a large molecular weight

protein or a smaller molecule bound to protein. Thus, the available








data relating to the endometrial luteolytic factor strongly suggest

that it is proteinaceous.

Data from studies on embryo culture, tube-locked embryos, and

delayed implantation suggest that embryos require some uterine factor

in order to reach and surpass the expanding blastocyst stage of develop-

ment. Experiments on superovulation and embryo superinduction have shown

that in general (except in mice) polytocous species lack the ability to

maintain litter sizes larger than those now characteristic of the species

and that most of the embryonic death occurs early in gestation. Estrogens

have been shown to stimulate DNA, RNA, and protein synthesis in the uterus

and specific estrogen-induced proteins have been described. Also, a

specific protein which stimulates expansion of blastocysts in vitro has

been demonstrated in the uterine intraluminal fluid of rabbits during

early pregnancy. These data seem adequate for formulation of the hypothesis

that the uterus secretes a specific protein(s) which is necessary for

blastocysts to expand and to implant.

Data are available to indicate that a protein(s) secreted by the

endometrium is involved in the regression of the corpus luteum. In

considering all these data together it is possible to hypothesize that

the embryotrophic and luteolytic factors are the same protein(s), and in

the absence of embryos to metabolize this protein(s), it exerts a lytic

effect on the corpus luteum. This hypothesis would provide an explana-

tion for estrous cycles in the nonpregnant female and absence of estrous

cycles during pregnancy.













CHAPTER 2. EFFECTS ON DEVELOPMENT OF PORCINE EMBRYOS
CAUSED BY RESTRICTION TO THE OVIDUCT


As indicated previously, many lines of evidence suggest that the

uterus supplies the necessary factors) which allows continued develop-

ment of the early embryo. Restriction of embryos to the oviducal

environment allows development only to the early blastocyst stage in mice

(Kirby, 1962 and Orsini and McLaren, 1967), rabbits (Westman et al., 1931;

Pincus and Kirsch, 1936; and Adams, 1958), rats (Alden, 1942), and sheep

(Wintenberger-Torres, 1956).

This study was initiated for the purpose of establishing whether

or not the uterus plays a similar essential role in early embryonic

development in swine.



Materials and Methods


Ten crossbred gilts (nulliparous females, Sus scrofa) were used in

this experiment. Intact boars were used at 12-hour intervals to check

for estrous behavior in the gilts. The females were bred at 12 and 24

hours after onset of estrus, and surgery was performed 48 to 72 hours after

onset of estrus. Anesthesia was induced with 5 percent sodium thiopental

and maintained with methoxyflurane. The reproductive tracts were exposed

by mid-ventral laporotomies, and one oviduct in each gilt was ligated at

the tubo-uterine junction while the contralateral uterine horn was ligated

about 10 cm posterior to the tubo-uterine junction and served as a con-

trol. The gilts were slaughtered either seven or eight days post coitum,








and the embryos present were recovered by flushing the oviducts and

uteri with a physiological saline solution. Recovered embryos were

observed under a stereomicroscope at a magnification of 10 X or 45 X

to determine stage of development, and the diameter of the embryos

was measured with an ocular micrometer. Gilts from which no embryos

were recovered were considered nonpregnant and eliminated from the

experiment. Data were analyzed by chi-square and the Student's

t-Test (Steel and Torrie, 1960).



Results and Discussion


A total of 53 embryos were recovered from seven of the ten gilts

and the data are summarized in Table 1. The difference between uterine

and tube-locked embryos was striking. The tube-locked embryos were

mostly fragmented and all remained within the zona pellucida while 83.8

percent of the recovered uterine blastocysts had emerged from the zona

pellucida, formed an inner cell mass, and expanded to about three times

the diameter of the zona pellucida (Plate 1). This difference in size

of the embryos was highly significant (P < 0.001).

Recovery rates were based on the number of embryos recovered

compared to the number of corpora lutea. The overall recovery rate from

pregnant gilts was 58.9 percent. The rate of recovery of tube-locked

embryos (45.7 percent) tended to be less than the recovery rate of

uterine embryos (65.0 percent), but this difference was not statistically

significant. The nonsignificant difference suggests that many of the

tube-locked embryos may have already disintegrated by the time of

recovery.

















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PLATE 1


A tube-locked embryo recovered from gilt 324 seven
days post coitum X 150.

A uterine blastocyst recovered from gilt 324 seven
days post coitum X 150.

A tube-locked embryo recovered from gilt 331 seven
days post coitum X 150.


Figure 1.


Figure 2.


Figure 3.







22






















..
:.ii. ..iii.:..




23



Since no blastocysts were recovered from ligated oviducts,

it is apparent that pig embryos must reach the uterine environment

in order for blastulation to occur as is true for mice, rabbits,

rats, and sheep. This provides an even broader basis for the hypo-

thesis that the uterus of the gilt may secrete a protein(s) required

for continued embryonic development.













CHAPTER 3. QUANTITATIVE AND QUALITATIVE CHANGES IN PROTEIN SECRETION
BY THE SWINE UTERUS DURING THE ESTROUS CYCLE


In the preceding chapter data are presented that demonstrate

that pig embryos fail to reach the blastocyst stage in vivo if they are

prevented from reaching the uterus. This, along with data presented by

other workers on embryo culture, superovulation, and embryo superinduction,

strongly suggests that some uterine factor is necessary for continued

development of swine embryos through the blastocyst stage. In the rabbit,

a protein component of uterine fluid has already been shown to stimulate

blastocyst growth in vitro (Krishnan and Daniel, 1967). Therefore, the

protein components of swine uterine fluid were studied to determine

whether there are proteins which are specific to the uterus, and whether

there is variation in uterine specific protein which might be related to

embryonic development.



Materials and Methods


Sixty-six crossbred gilts served as uterine fluid donors. The

females were checked for estrous activity twice daily at 12-hour intervals

with mature boars. Gilts were subjected to cervical stimulation with

an insemination rod at 12 and 24 hours after the onset of estrus. The day

of the estrous cycle on which the uterus of a given gilt was to be flushed

was randomly assigned prior to the experimental period. Surgery dates

were assigned as multiples of 24 6 hours after onset of estrus. The

first day of estrus was designated as day 0. Animals were held off feed

and water for 24 to 36 hours before surgery. At surgery anesthesia was

24








induced with 5 percent sodium thiopental and maintained with methoxy-

flurane. The reproductive tract was exposed by mid-ventral laporotomy.

A small incision was made in an avascular area of the oviduct approx-

imately 1 cm from the tubo-uterine junction and a polyvinyl catheter

(I.D. = 1.25 mm) was inserted through this incision and the tubo-uterine

junction into the lumen of the uterus. While the external uterine

bifurcation was being restricted with hand pressure, 20 ml of 0.33 M NaC1

was injected into each uterine horn via the cannula. The saline was

forced to the external bifurcation of the uterus by pulling the uterus

between the thumb and forefinger; then it was forced back and out through

the cannula into a sterile serum bottle. Flushings from the two horns

were pooled and kept in an ice bath until centrifugation and sterilization

could be carried out. The flushings were centrifuged at 3000 g and

sterilized by filtration through 0.2 p millipore filters. The sterilized

flushings were then stored at -700C until they could be analyzed.

The uterine flushings of approximately 40 ml were concentrated

to about 1 ml by pressure dialysis. The fluid was then dialyzed against

0.05 M phosphate-citrate buffer, pH 7.4, for 24 to 36 hours. The protein

concentration of each sample was determined by the method of Lowry et al.

(1951), and the total quantity of protein in each sample was then cal-

culated. These data were used to compare the relative amounts of total

protein per uterus for each day of the cycle. This comparison depends

upon the assumption that the same proportion of intraluminal fluid was

recovered and the same proportion of the protein in the uterine flushings

was lost during processing,regardless of the day of the estrous cycle.

Whole dialyzed samples, remaining after the protein determination

and polyacrylamide disc electrophoresis, or aliquots of samples were









loaded on columns of Sephadex G-200. For most samples 1.5 x 90 cm

columns were used and the bed heights varied from 75 to 85 cm. Other

samples were loaded onto a 2.5 x 100 cm column. The eluent used was

0.05 M phosphate-citrate buffer, pH 7.4, and the flow rate was

approximately 4 ml per hour for the small columns and 8 ml per hour

for the large column. Gel filtration was carried out at 30C. The

fractions were approximately 2 and 4 ml in the small and large columns,

respectively. Aliquots of each fraction were used for protein deter-

mination. The optical density of each fraction was measured and plotted

against the elution volume at which the fraction occurred, so that a

protein profile was constructed.

It was impossible to load a standard quantity of protein from

each sample onto the column; therefore, the optical densities obtained

from the fractions of one sample were not directly comparable to those

from other samples. In order to standardize all gel filtration data,

the optical density of each fraction was converted to a percentage of

the total protein eluted from the column for each sample. This allowed

the construction of protein profiles which were directly comparable to

each other. Thus, protein profiles of samples within and among days

of the estrous cycle were compared.

Polyacrylamide gel disc electrophoresis was employed for further

characterization of the proteins in the uterine flushings during the

various days of the estrous cycle. The electrophoresis was carried out

in 7 percent acrylamide gel; however, neither sample gel nor spacer gel

was used. Instead, the samples were applied in 1 M sucrose. Electro-

phoresis was carried out at 2.2 mA. per gel in 0.05 M Tris-0.38 M glycine








buffer, adjusted to pH 8.0 by addition of HC1. Gels were stained with

amido black or periodic acid-Schiff (PAS) and then destined. The gels

were then compared directly or indirectly from densitometer tracings.



Results and Discussion


One of the samples of uterine flushings obtained on day 3 and one

obtained on day 6 were excluded from the results due to the presence of

excessive amounts of blood and hemoglobin. A sample of day 4 and one of

day 7 flushings were lost during dialysis due to defective dialysis tubing,

and a sample of day 18 flushings was not used since the animal from which

it was obtained exhibited one estrous cycle that was abnormally long

(24 days).

The mean values for total protein flushed from uteri on each day

of the estrous cycle and the standard error of each mean are presented in

Figure 4. This shows clearly that the protein content of uterine secre-

tion remained fairly stable from day 2 through day 9 of the cycle and then

it began to increase. The increase became sharper on day 13 and the

maximum value was reached on day 15, after which the total protein content

of uterine flushings decreased sharply so that on days 18 and 20, the

amount was comparable to that on days 6 to 9 of the cycle. The peak in

protein secretion occurred while the reproductive system was under the

influence of progesterone, suggesting that progesterone, possibly in

synergism with estrogen, is responsible for the increased production of

protein. Also the abrupt decrease in the protein content of uterine

flushings coincided with the regression of the corpora lutea (Masuda et al.,

1967) and reduced progesterone production in the pig (Gomes et al., 1965;



















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Schomberg et al., 1966; and Masuda et al., 1967). This further suggests

that progesterone plays a role in the secretion of protein by the uterus

of the pig.

Protein profiles of uterine flushings obtained on days 2 through

18 and day 20 are presented in Figure 5. From these profiles it can be

seen that all profiles contain three protein fractions which do not appear

to vary with days of the estrous cycle. These three fractions (Fractions I,

II, and III) have estimated average molecular weights of 400,000 and greater,

200,000 and 90,000, respectively. Beginning on day 9 and continuing through

day 16, an additional peak (Fraction V) with an average molecular weight of

20,000 occurred in the protein profile. Fraction V constituted greater

than 20 percent of the total protein on days 12 through 16, although it is

quantitatively greatest on day 15.

Polyacrylamide gel disc electrophoresis revealed that Fraction V

consists of six acidic proteins (Plate 2, Figures 6a and b). Although

most, if not all, of these six proteins were absent from flushings obtained

prior to day 9 and after day 16, it is possible that they may have been

present in undetectable quantities prior to day 9. When the portion of

the gel filtration eluent which contained Fraction V was compared across

all the days of the cycle, a slight increase occurred on days 5 and 6

(see Figure 7). It is not known whether this transitory increase is

physiologically significant; however, it would seem that any uterine

secretion critical for the development of the blastocyst must be present

at the time when this stage is reached.

On days 12 through 16, the region between Fraction III and Fraction

V of the gel filtration protein profile was occupied by a lavender protein

with a molecular weight of about 45,000, as determined by gel filtration.





























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PLATE 2


Figure 6a.



Figure 6b.




Figure 6c.


Polyacrylamide gel disc electrophoresis of swine
day 15 uterine fluid proteins, migration toward
the anode (from left to right).

Polyacrylamide gel disc electrophoresis of
Fractions I (top of figure) through V (bottom
of figure), migration toward the anode (from
left to right).

Polyacrylamide gel disc electrophoresis of
Fractions I (top of figure) through V (bottom
of figure), migration toward the cathode (from
left to right).

















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The lavender material was stained by both amido black and PAS. In

addition to the lavender protein, polyacrylamide gel disc electro-

phoresis demonstrated that two other proteins may be included in

Fraction IV (Plate 2, Figure 6c). All three of the proteins migrated

toward the cathode at pH 8.0, and Fraction IV contained no protein which

migrated toward the anode at this pH. The secretion pattern of Fraction

IV proteins, as is the case with Fraction V proteins, suggests that if

any role in embryonic development is fulfilled by these molecules, it

must relate to the growth of the expanding blastocyst rather than the

process of blastulation.

The pattern of secretion of both Fractions IV and V proteins

implies that the presence of a functional corpus luteum is necessary for

their secretion. There was a rather gradual increase in the amounts of

these proteins until their peak was reached on day 15. At this point the

corpus luteum in the pig has reached an advanced state of regression and

lost much of its secretary function (Gomes et al., 1965; Schomberg et al.,

1966; and Masuda et al., 1967), and the secretion of Fractions IV and V

decreased abruptly. The data also support the hypothesis that uterine

proteins are responsible for regression of the corpus luteum, as is

suggested by the data of Schomberg (1967), Mazer and Wright (1968), and

Lukaszewaska and Hansel (1970). However, the secretion patterns of

Fractions IV and V do not suggest a role for them in the process of blastu-

lation and blastocyst expansion as is true of the "blastokinin" secretion

pattern in the rabbit (Krishnan and Daniel, 1967 and 1968; Beier, 1968;

and Kirchner, 1969). It is possible that the amount of stimulatory

proteins needed for blastulation may be very small and therefore undetect-

able by the methods used in this study. If this is true, the transitory




37



increase in the low molecular weight fraction on days 5 and 6 may be

related to early embryonic development. On the other hand, Fractions IV

and V may be related to the rapid growth and expansion of the more

advanced embryo.












CHAPTER 4. PREGNANCY IN GILTS AFTER INTRAVENOUS INJECTIONS
OF ANTI-PORCINE UTERINE FLUID PROTEINS


Evidence was presented in the preceding chapter that two groups

of proteins specific to the uterus are secreted from day 9 to day 16 of

the estrous cycle of the pig. Some possible roles for these proteins

in reproduction were also discussed. This experiment was designed to

determine the effect of passive immunization of gilts against uterine

fluid proteins.



Materials and Methods


Day 14 is approximately midway through the phase of the estrous

cycle in which major quantitative and qualitative changes occur in the

swine uterine fluid protein secretion pattern. Therefore, uterine proteins

obtained on day 14 of the estrous cycle were used in this experiment.

Uterine fluid was obtained, prepared, and chromatographed through Sephadex

G-200 as described in Chapter 3.

Sheep were used to produce antibodies against Fractions I through

V (anti-total) of swine uterine proteins, Fractions I, II, and III (anti-

high) of swine uterine proteins, Fractions IV and V (anti-low) of swine

uterine proteins, and Freund's complete adjuvant (anti-Freund). Uterine

proteins in doses of 2 16 mg in suspensions of Freund's complete

adjuvant were injected intradermally into the sheep, and these doses were

administered twice for Fractions I to V and for Fractions I, II, and III,

but three times for Fractions IV and V. In each case all of the antigens








available at the time of the injections were used. Totals of 5.7 mg

of Fractions I to V; 32.0 mg of Fractions I, II, and III; and 6.9 mg

of Fractions IV and V were administered to the appropriate sheep.

The sheep were challenged with the respective antigens at intervals

of two weeks and harvesting of antisera was begun four weeks after

the initial challenge. A commercial preparation of goat anti-porcine

serum was used for comparison with the sheep anti-porcine uterine

protein sera prepared for this experiment. Specificities of the

antisera were determined by comparing the anti-porcine serum with

the anti-porcine uterine protein sera using diffusion-in-gel and

immunoelectrophoresis. The antisera were stored at -200C.


Treatment of Gilts Trial I

Five mated gilts received injections of antisera via an ear

vein. Two gilts were injected with anti-total sera, two with anti-low

sera, and one with anti-Freund sera. Each gilt received 5 ml of the

appropriate antiserum on days 8 and 10 post coitum and 10 ml of the

proper antiserum on days 12 and 14. The gilts were laporotomized on

day 30 after mating and their reproductive tracts were examined for

pregnancy and condition of corpora lutea.


Treatment of Gilts Trial II

Eleven gilts were used in Trial II. Six gilts were mated to

fertile boars and five gilts were nonmated. One nonmated and two

mated gilts received 10, 15, and 20 ml of anti-high serum on days

5, 7, and 9 after the onset of estrus, respectively. Two mated and

nonmated gilts received the same amounts of anti-low on days 5, 7,

and 9. Two mated and two nonmated gilts were treated with 50, 100,







and 200 ml of anti-low on days 10, 12, and 14 after the onset of estrus.

Using techniques described in Chapter 3, uterine fluid was obtained from

all nonmated gilts on day 15 of the estrous cycle. The fluids were

processed in the manner previously described and each sample was sub-

jected to gel filtration. Mated gilts were laporotomized on day 30

post coitum and their reproductive tracts were examined for pregnancy

and condition of corpora lutea.



Results and Discussion


Immunoelectrophoresis revealed four antibodies in anti-low serum

which cross-reacted with basic proteins in rechromatographed Fraction IV.

Two diffuse precipitation crescents were evident after immunoelectrophoresis

of rechromatographed Fraction V. None of these six antibodies was present

in anti-porcine serum, although at least three antigens in total uterine

fluid cross-reacted with anti-total, anti-high, and anti-porcine sera.

Anti-high serum contained at least six antibodies not found in anti-porcine

serum and anti-total contained a combination of the antibodies found in

anti-high and anti-low sera. Thus, at least 12 antigenic substances,

apparently unique to the secretions of the uterus, were identified using

immunoelectrophoresis. Anti-Freund serum did not cross-react with any

antigen in pig uterine flushings.

After intravenous injections of sheep anti-swine uterine protein

sera into mated pigs, no effects were apparent at laporotomy 30 days after

mating in either Trial I or Trial II. Possible reasons for the lack of

effect include lack of antibodies against specific proteins required by

the embryos, failure of antibodies to reach the uterine lumen, inadequate

quantities of antibodies, and lack of a requirement by swine embryos for

specific uterine fluid protein.







There was also no apparent effect of antisera on the total

protein content or gel filtration protein profile of uterine flushings

obtained from the nonmated gilts. The total quantity of protein

obtained from the uterine fluid of these animals was well within the

range of values for total protein in other samples of day 15 uterine

fluid described in Chapter 3. The failure of these antisera to affect

the protein constituents of uterine flushings suggests that the anti-

bodies did not reach the uterine lumen. Also, estrous cycle lengths

were normal following antisera treatments of nonmated gilts. Therefore,

there was evidently no effect on the corpora lutea.

Although this experiment did not provide data on the relation-

ship of uterine fluid proteins to embryonic development or to corpus

luteum regression, it did provide valuable information. In this study

it was learned that at least 15 different antigenic substances constitute

the macromolecular fraction of swine uterine secretions, and that there

are at least 12 antigens of uterine fluid which do not cross-react with

anti-porcine serum.












CHAPTER 5. GENERAL DISCUSSION


A variety of studies has provided information regarding the

influence of uterine factors on the early development of the mammalian

embryo and, equally important, on the function of the uterus in the

regression of the corpus luteum. The data obtained in this research

provide additional information and support the concept that secretions

of the uterus control embryonic development and the status of the

corpus luteum.

In this experiment pig embryos failed to reach the blastocyst

stage of development when prevented from reaching the uterus by ligation

of the oviduct. In the mouse, rabbit, rat, and sheep a similar develop-

mental failure of tube-locked embryos has been reported (Westman et al.,

1931; Pincus and Kirsch, 1936; Alden, 1942; Wintenberger-Torres, 1956;

Adams, 1958; Kirby, 1962; and Orsini and McLaren, 1967); however, in

these species development to the early blastocyst stage in the oviduct

was observed. These data lead to the unavoidable conclusion that the

uterus provides some factor which is absolutely required for continuous

development of the early embryo.

It does not appear likely that simple nutrients constitute the

uterine factors which enable the uterine embryos to develop continuously.

A wide variety of culture media have been used for in vitro cultivation

of ova from a number of species of mammals to the blastocyst stage

(Chang, 1949; Hammond, 1949; Whitten, 1956 and 1957; McLaren and Biggers,

1958; Tarkowski, 1961; Brinster, 1963; Adams, 1965; Cole and Paul, 1965;

Onuma et al., 1968; Maurer et al., 1969; and Rundell, 1969). In these

42








studies the embryos underwent cleavage in vitro in synthetic media

containing simple carbohydrates, inorganic salts, and sources of

fixed nitrogen or in heated sera; yet development did not continue

past the early blastocyst stage. These data indicate that simple

nutrients are not adequate to support development through this

critical phase.

An opposing view is held by two groups of workers. Mouse

zygotes (Whitten and Biggers, 1968) and 2- to 4-cell rabbit embryos

were grown to the expanding blastocyst stage in vitro. Possible

reconciliation between these results and data indicating that embryos

fail to continue development in the absence of the uterine environment

is found in the work of Nicholas (1942), Fawcett et al. (1947), Runner

(1947), Fawcett (1950), and Kirby (1962), which indicates that tropho-

blasts of transplanted oviducal embryos expand in a morphologically

normal manner, but the embryonic disc cells failed to develop.

Once the embryo has surpassed the critical period of development,

which appears to require the uterine environment, growth and development

can again take place in vitro in the New (1967) culture medium re-

circulator. Hamster embryos (Givelber and DiPaolo, 1968), rabbit embryos

(Daniel, 1970a), and rat embryos (New, 1967; and New and Daniel, 1969)

have been so cultured. This information is important because it indicates

that, if the embryo is allowed access to the uterine environment during

the critical phase of differentiation, the embryo can be cultured in vitro

during the period of organogenesis.

Evidence that the uterus exerts control over the embryo has been

obtained from studies involving embryo superinduction, superovulation,

asynchrony of embryonic and uterine development, and delayed implantation.








In a series of experiments it was shown that embryo superinduction

of gilts with 2.5-day embryos did not affect litter size at 105 days,

90 days, or 25 days after mating (Bazer et al., 1969 a,b). Fenton

et al. (1969a) reported that 7-day embryos used for superinduction

of gilts increased litter size at 25 days post coitum. This led to

the hypothesis that the uterine control of embryonic development is

exerted prior to day 7 post coitum. To test this hypothesis 2.5-day

and 7-day embryos were used for embryo superinduction (Fenton et al.,

1969 b), but in this experiment 7-day embryos did not result in an

increase in litter size as compared to unoperated controls or gilts

superinducted with 2.5-day embryos. Schwartz et al. (1970) obtained

similar results after embryo superinduction with 7-day embryos. These

data suggest that the uterine control of embryonic survival is exerted

after day 7 but before day 25 post coitum.

The effect of reduced uterine space on embryonic development

was studied by Dziuk (1968), Fenton et al. (1970), and Johnson (1970).

In gilts (Dziuk, 1968; and Fenton et al., 1970) the reduction of uterine

space had no effect on litter size at 25 days after mating although

105-day litter size was reduced. In mice, rabbits, and rats (Johnson,

1970) one uterine horn was found to carry as many embryos to term as

two uterine horns. These data suggest that some factor other than

uterine space limits litter size since the majority of the embryonic

deaths occur prior to the time when uterine space becomes limiting.

Superovulation of the pig (Hammond, 1921; Perry, 1954; King

and Young, 1957; Rathnasabapathy et al., 1957; Hunter, 1966; Longenecker

and Day, 1968; Pope et al., 1968; and Bazer et al., 1969 c) and the

rabbit (Adams, 1960; Hafez, 1964; and Johnson, 1970) did not result in








increased litter size. On the other hand, litter size was increased

by superovulation of mice (McLaren and Michie, 1956; McLaren and

Michie, 1959 a,b; and Wilson and Edwards, 1963), except in the

experiment of Fowler and Edwards (1957). In general, litter size

cannot be increased by embryo superinduction or by superovulation in

polytocous species. This suggests that there is a limit to the number

of embryos that can be maintained ("uterine capacity"), and that this

limit is closely approached during normal pregnancy in pigs and rabbits.

Embryonic survival is greatest when the transferred embryos

and recipient uteri are closely synchronized (Bazer et al., 1969a;

Dickmann and Noyes, 1960; and Hunter et al., 1967). Furthermore,

Bazer et al. (1969a) showed that when transferred embryos and recipient

uteri were in asynchrony, the embryos native to the recipient uteri

had significantly higher survival rates than transferred embryos; whereas,

there was no statistical difference in survival rates if the transfer

was made in synchrony. From this it can be inferred that the uterus

secretes some substance or a group of substances which are necessary for

embryonic development, that the substances) is produced at a specific

time, and that the embryo must be present in the uterus at the proper

time in order to utilize the secretion.

Further information on the relationship between the uterus and

embryonic development is available from experiments with animals

exhibiting delayed implantation (Daniel, 1970b). Dickmann and DeFeo

(1967) demonstrated that the uterine development rather than the embryo

determines whether or not implantation in the rat is to be delayed.

This also supports the idea that uterine secretions are necessary for

continuous embryonic development and that the secretion of the required









material is not constant but instead is produced at a specific time.

Because of their interesting biological functions as enzymes

and regulators of genetic material, proteins are worthy of study as

possible uterine specific substances required by embryos in order

for differentiation to proceed in a normal manner. Studies based on

electrophoresis (Junge and Blandau, 1958; Ringler, 1961; Albers and

Castro, 1961; and Stevens et al., 1964) led to the conclusion that

intraluminal fluids from rat and rabbit uteri contain proteins

similar to serum proteins as well as proteins specific to the uterus.

Krishnan and Daniel (1967) were the first investigators to

show that a specific uterine protein is secreted in large quantities

at a precise time in early pregnancy in the rabbit, and that this

protein can influence a definite phase in the development of the

embryo in vitro. The protein, designated as "blastokinin," was

shown to appear on day 3 post coitum, reach a maximum concentration

on day 5, and then decline. "Blastokinin" was found to stimulate

blastulation in vitro. Other workers (Beier, 1968; Kirchner, 1969;

and Hamana and Hafez, 1970) have also demonstrated the same protein

or a very similar protein in rabbits.

In the present experiment the proteins in swine uterine

flushings were studied on days 2 through 18 and day 20 of the estrous

cycle. Gel filtration of the proteins in these flushings demonstrated

that at least two groups of proteins were not secreted constantly

throughout the estrous cycle, but instead appeared at mid-cycle and

disappeared before the next estrus. The fraction (Fraction IV) having

the higher molecular weight (45,000) contained at least three proteins

as revealed by polyacrylamide gel disc electrophoresis. All three








proteins are basic at pH 8.0. The remaining protein fraction

(Fraction V) had an estimated molecular weight of approximately

20,000. Polyacrylamide gel disc electrophoresis demonstrated

six acidic proteins and one slightly basic protein in Fraction V

at pH 8.0. Thus a total of at least 10 proteins with molecular

weights between 10,000 and 50,000 are secreted in a cyclic pattern

by the uterus. Proteins with higher molecular weights may also be

secreted at specific periods during the estrous cycle; however,

the methods used in this study were not adequate to permit drawing

such a conclusion.

Since all the proteins in Fraction IV and one in Fraction V

of gilt uterine fluid are basic at pH 8.0, they are well suited as

repressors or inducers, and therefore they are possibly regulatory

in function. Induction of regulatory protein synthesis by the

uterus may be an important function of estrogens and progesterone.

Estrogen (Hamilton, 1963; Ui and Mueller, 1963; Hamilton, 1964;

Gorski and Nelson, 1965; Notides and Gorski, 1966; Hamilton et al.,

1967; and Billing et al., 1969) and progesterone (Dingman et al.,

1969; and O'Malley and McGuire, 1969) have been shown to stimulate

RNA and protein synthesis in mammalian uterine and chick oviduct

tissues. Proteins induced by these hormones may possibly serve

regulatory functions in the uterus and/or in embryos.

Prasad et al. (1968) found that only very low rates of DNA,

RNA, and protein synthesis occur in rat uteri and blastocysts

undergoing delayed implantation; however, estrogen stimulated DNA,

RNA, and protein synthesis in the inner cell mass of the blastocysts

and RNA and protein synthesis in the uterus. These data indicate









that estrogen can initiate a resumption of growth and metabolic

activity in diapausing rat embryos, apparently by induction of

protein synthesis. If the basic proteins observed in the present

study serve as repressors or inducers of specific regions of the

DNA of swine embryos, they should offer excellent opportunities

for the study of basic mechanisms of embryonic differentiation.

An attempt to determine biological functions of the

protein fractions obtained in the present study has been carried

out. Immunization of gilts against uterine fluid proteins did not

affect the embryos, corpora lutea, or uterine protein secretions

in any apparent way. The gilts may have received insufficient

doses of the antisera, or the antibodies may not have reached the

antigens. Use of larger quantities of antisera and early administra-

tion directly into the uterine lumen might be advantageous in a

future experiment. Injections of antisera into gilts did not provide

additional data on the relationship between uterine proteins and

embryonic development or corpus luteum regression. However, valuable

information on the specificity of the uterine protein secretion was

obtained.

By use of the techniques of diffusion-in-gel and immuno-

electrophoresis, it was learned that at least 12 proteins, not

found in pig serum, are secreted by the uterus of the pig and of

these, six occurred in Fractions IV and V. Three additional

uterine proteins are also present in pig serum. These data indicate

that the uterine fluids used in this research represent true secre-

tions of the uterus.








Recently the effects of uterine fluid proteins on mouse

embryonic development were tested (unpublished data, Ulberg et al.,

1970). In this experiment 4- to 8-cell mouse embryos were cultured

in Brinster's medium for ova culture (Brinster, 1963), supplemented

with total uterine fluid protein or fractions of the total. The

fractions used were those described in Chapter 3. The supplements

were added in concentrations of 1 mg/ml or 0.1 mg/ml. The embryos

were cultured in these media for up to 96 hours. During this time

most embryos (82.0 percent) reached the blastocyst stage and 27.2

percent hatched. As evaluated by percentage of embryos which

hatched and embryonic size, no fraction was as stimulatory to mouse

embryonic development in vitro as was the total of all uterine

proteins. However, the percent of embryos hatching in media contain-

ing Fractions I, II, IV, and V in concentrations of 0.1 mg/ml was

superior to that in concentrations of 1 mg/ml. The percent of

embryos hatching in media containing Fraction III was about the same

at 0.1 mg/ml and 1 mg/ml. On the other hand, embryonic diameter

tended to be smaller when embryos were grown in media containing

uterine protein at 0.1 mg/ml than at concentrations of 1 mg/ml.

The data suggest that more than a single protein may be required

for optimum embryonic development, since no single fraction was

as stimulatory as the total of all proteins. For this reason, it

would be informative to test the effects of all possible combinations

of the five fractions on swine embryos in vitro.

Possible luteolytic effects of the proteins in swine uterine

flushings were not tested in the present study; however, there is

ample evidence that uterine proteins are luteolytic (Schomberg, 1967,








1969; Williams et al., 1967 a,b; Mazer and Wright, 1968; and

Lukaszewaska and Hansel, 1970). Therefore it would be useful to

know which fraction(s) of the swine uterine flushings is luteolytic

in order to gain more information concerning interrelationships

among the uterus, corpus luteum, and embryo. It is very interesting

that Fraction IV proteins and Fraction V proteins were observed to

be present in highest concentrations in swine uterine flushings at

the same time in the estrous cycle that Schomberg (1967) found swine

uterine flushings to be luteolytic.

After consideration of the several lines of evidence which

suggest that the uterus plays an active role in mammalian reproduc-

tion, the implications were integrated to form a hypothesis concerning

the active role of the uterus. The hypothesis assumes that the uterus

secretes a protein(s) which is required by the embryo for development;

and if no embryos are present to utilize the protein(s), it causes

regression of the corpus luteum and the estrous cycle begins anew.

The information gained in the present study is inadequate to allow

conclusions to be drawn concerning the validity of this hypothesis;

however, the data obtained provide a basis for further experiments to

test the hypothesis.













SUMMARY


The uterus actively influences mammalian reproduction by

participation in embryonic differentiation and in determination of

estrous cycle length. In these experiments the secretion of proteins

by the porcine uterus during the estrous cycle was characterized and

related to reproductive phenomena.

A preliminary experiment was designed to determine if the

porcine uterus provides an environment essential for continued develop-

ment of the early embryo. One tubo-uterine junction was ligated in

each of 10 mated gilts under surgical anesthesia at 48 to 72 hours post

coitum. The contralateral uterine horn was ligated approximately 10 cm

posterior to the tubo-uterine junction and served as the control side.

The animals were slaughtered at either 7 or 8 days after mating and

the embryos were recovered. The tube-locked embryos failed to develop

past the morula stage and many had begun degenerating by the time of

recovery. No tube-locked embryo had escaped from the zona pellucida.

On the other hand, uterine embryos in the same females were mostly

blastocysts, and 84 percent had emerged from the zona pellucida, formed

a distinct inner cell mass, and expanded to approximately three times

the diameter of the zona pellucida. Thus it was shown that developmental

failure occurred in embryos prevented from reaching the environment

provided by the uterus.

The daily variation in the protein content of porcine uterine

intraluminal fluid was studied on days 2 to 18 and day 20 of the estrous

cycle. Sixty-six gilts furnished one sample of uterine fluid each. The

51








samples were centrifuged at 3000 g, sterilized by filtration, con-

centrated from approximately 40 ml to 1 ml, and dialyzed against

0.05 M phosphate-citrate buffer, pH 7.4. A portion of each sample

was chromatographed through Sephadex G-200 and another portion was

subjected to polyacrylamide gel disc electrophoresis. Gel filtra-

tion through Sephadex revealed three groups of proteins (Fractions

I, II, and III) which did not vary with the estrous cycle and two

groups of proteins (Fractions IV and V) which were not present

early in the estrous cycle. Fraction IV, which appeared on day 12,

contained a lavender protein with an estimated molecular weight

of 45,000. Fraction V had an estimated molecular weight of about

20,000 and appeared as early as day 9. Fractions IV and V were not

present in the gel filtration protein profiles after day 16 of the

cycle.

Polyacrylamide gel disc electrophoresis demonstrated that

Fraction IV contained at least three basic proteins, while Fraction V

contained one basic protein and six acidic proteins at pH 8.0. The

basic proteins, especially, are interesting as possible regulators of

the differentiation of embryos by control of genetic expression. Also,

these proteins appear in the uterine flushings shortly before the

corpus luteum begins to regress and are not present after regression.

This coincidence suggests an interrelationship between the low molec-

ular weight proteins and the corpus luteum.

Attempts to determine the role of the uterine fluid proteins

in reproductive phenomena have been largely unsuccessful. Intravenous

injections of anti-uterine fluid sera did not interfere with pregnancy

in mated gilts or cause any quantitative or qualitative change in the








protein content of uterine fluid from nonmated gilts. However, it is

not possible to draw final conclusions from these data, since there

is a possibility that the antibodies injected did not reach the

uterine lumen in sufficient quantities to bind the proteins secreted

by the uterus.

This study showed that the uterine environment is required for

embryonic development and that there are cyclic changes in the protein

secretion pattern of the uterus. These findings provide additional

support for the concept that the proteins of maternal origin may

regulate genetic expression in the embryo during early differentiation.













LIST OF REFERENCES


Adams, C. E. 1958. Egg development in the rabbit: the influence
of post-coital ligation of the uterine tube and of ovariectomy.
J. Endocr. 16:283.

Adams, C. E. 1960. Prenatal mortality in the rabbit, Oryctolagus
cuniculus. J. Reprod. Fert. 1:36.

Adams, C. E. 1965. The influence of maternal environment on pre-
implantation stages of pregnancy in the rabbit, p. 345.
In: G. E. W. Wolstenholme and M. O'Connor (ed.), Pre-
implantation Stages of Pregnancy. Little, Brown and
Company; Boston.

Albers, H. J. and M. Neves e Castro. 1961. The protein components
of rat uterine fluid. An analysis of its antigens by
immunoelectrophoresis and duchterlony gel diffusion
technic. Fert. Steril. 12:142.

Alden, R. H. 1942. Aspects of the egg-ovary-oviduct relationship
in the albino rat. II. Egg development within the oviduct.
J. Exp. Zool. 90:171.

Anderson, L. L., K. P. Bland, and R. M. Melampy. 1969. Comparative
aspects of uterine-luteal relationships. Recent Progress in
Hormone Research. 25:57.

Anderson, L. L., A. M. Bowerman, and R. M. Melampy. 1963. Neuro-
utero-ovarian relationships, p. 345. In: A. Nalbandov (ed.),
Advances in Neuroendocrinology. University of Illinois Press,
Urbana.

Baevsky, U. B. 1963. The effects of embryonic diapause on the nuclei
and mitotic activity of mink and rat blastocysts, p. 141.
In: A. C. Enders (ed.), Delayed Implantation. University of
Chicago Press.

Bazer, F. W., A. J. Clawson, 0. W. Robison, and L. C. Ulberg. 1969a.
Uterine capacity in gilts. J. Reprod. Fert. 18:121.

Bazer, F. W., 0. W. Robison, A. J. Clawson, and L. C. Ulberg. 1969b.
Uterine capacity at two stages of gestation in gilts following
embryo superinduction. J. Animal Sci. 29:30.

Bazer, F. W., 0. W. Robison, A. J. Clawson, and L. C. Ulberg. 1969c.
Effect of dichlorvos and PMS on reproduction in swine.
J. Animal Sci. 28:145 (Abstract).








Beier, H. M. 1968. Uteroglobin: a hormone-sensitive endometrial
protein involved in blastocyst development. Biochim.
Biophys. Acta 160:289.

Beier, H. M. 1970. Protein patterns of endometrial secretion in
the rabbit, p. 157. In: Ovo-Implantation. Human Gonado-
tropins and Prolactin. S. Karger, New York.

Billing, R. J., B. Barbiroli, and R. M. S. Smellie. 1969. The mode
of action of oestradiol. II. The synthesis of RNA. Biochim.
Biophys. Acta 190:60.

Bland, K. P., and B. T. Donovan. 1966. Uterus and control of ovarian
function, p. 179. In: McLaren, A. (ed.), Advances in
Reproductive Physiology. Vol. 1. Academic Press, New York.

Brinster, R. L. 1963. A method for in vitro cultivation of mouse
ova from two-cell to blastocyst. Exp. Cell Res. 32:205.

Caldwell, B. V., R. S. Mazer, and P. A. Wright. 1967. Luteolysis
as affected by uterine transplantation in the Syrian hamster.
Endocrinology 80:477.

Caldwell, B. V., R. M. Moor, and R. A. S. Lawson. 1968. Effects of
sheep endometrial grafts and extracts on the length of
pseudopregnancy in the hysterectomized hamster. J. Reprod.
Fert. 17:567.

Chang, M. C. 1949. Effects of heterologous sera on fertilized rabbit
ova. J. Gen. Physiol. 32:291.

Cole, R. J. and J. Paul. 1965. Properties of cultured preimplanta-
tion mouse and rabbit embryos, and cell strains derived
from them, p. 82. In: G. E. W. Wolstenholme and
Maeve O'Connor (ed.), Preimplantation Stages of Pregnancy.
Little, Brown and Co., Boston.

Daniel, J. C., Jr. 1968. Comparison of electrophoretic patterns
of uterine fluid from rabbits and mammals having delayed
implantation. Comp. Biochem. Physiol. 24:297.

Daniel, J. C., Jr. 1970a. Culture of rabbit embryo in chemically
defined medium. Nature, Lond. 225:193.

Daniel, J. C., Jr. 1970b. Dormant embryos of mammals. BioScience
20:411.

Daniel, J. C., Jr. and R. S. Krishnan. 1969. Studies on the
relationship between uterine fluid components and the
diapausing state of blastocysts from mammals having
delayed implantation. J. Exp. Zool. 172:267.









Dickmann, Z. 1967. Hormonal requirements for the survival of
blastocysts in the uterus of the rat. J. Endocr. 37:455.

Dickmann, Z. and V. J. DeFeo. 1967. The rat blastocyst during
normal pregnancy and during delayed implantation, including
an observation on the shedding of the zona pellucida.
J. Reprod. Fert. 13:3.

Dickmann, Z. and R. W. Noyes. 1960. The fate of ova transferred into
the uterus of the rat. J. Reprod. Fert. 1:197.

Dingman, C. W., A. Aronow, S. L. Bunting, A. C. Peacock, and
B. W. O'Malley. 1969. Changes in chick oviduct ribonucleic
acid following hormonal stimulation. Biochemistry 8:489.

Dziuk, P. J. 1968. Effect of number of embryos and uterine space
on embryo survival in the pig. J. Animal Sci. 27:673.

Eddington, C. L. 1969. Hormonal control of RNA biosynthesis in
mammary tissue of rats. Diss. Abst. 29:2735B.

Fawcett, D. W. 1950. The development of mouse ova under the capsule
of the kidney. Anat. Rec. 108:71.

Fawcett, D. W., G. B. Wislocki, and C. M. Waldo. 1947. The develop-
ment of mouse ova in the anterior chamber of the eye and in
the abdominal cavity. Amer. J. Anat. 81:413.

Fenton, F. R., F. W. Bazer, 0. W. Robison, and L. C. Ulberg. 1969a.
Superinduction of gilts with 7-day pig embryos. J. Animal
Sci. 28:144 (Abstract).

Fenton, F. R., 0. W. Robison, and L. C. Ulberg. 1969b. Superinduction
of gilts with 2-1/2 or 7-day embryos. J. Animal Sci. 29:189.

Fenton, F. R., F. W. Bazer, 0. W. Robison, and L. C. Ulberg. 1970.
Effect of quantity of uterus on uterine capacity in gilts.
J. Animal Sci. 31:104.

Fowler, R. E. and R. G. Edwards. 1957. Induction of superovulation
and pregnancy in mature mice by gonadotrophins. J. Endocr.
15:374.

Givelber, H. M. and J. A. DiPaolo. 1968. Growth of explanted eight
day hamster embryos in circulating medium. Nature, Lond.
220:1131.

Goding, J. R., F. A. Harrison, R. B. Heap, and J. L. Linzell. 1967.
Ovarian activity in the ewe after autotransplantation of the
ovary or uterus to the neck. J. Physiol., Lond. 191:129P.

Gomes, W. R., R. C. Herschler, and R. E. Erb. 1965. Progesterone
levels in ovarian venous effluent of the non-pregnant
sow. J. Animal Sci. 24:722.








Gorski, J. and N. J. Nelson. 1965. Ribonucleic acid synthesis in
the rat uterus and its early response to estrogen. Archs.
Biochem. Biophys. 110:284.

Gorski, J., D. Toft, G. Shyamola, D. Smith, and A. Notides. 1968.
Hormone Receptors: Studies on the interaction of estrogen
with the uterus. Recent Progress in Hormone Research
24:45.

Gossett, J. W. and A. M. Sorensen, Jr. 1959. A comparison of embryo
survival in gilts slaughtered twenty-five versus forty days
after breeding. J. Animal Sci. 18:48.

Hafez, E. S. E. 1964. Effects of over-crowding in utero on implanta-
tion and fetal development in the rabbit. J. Exp. Zool.
156:269.

Hamana, K. and E. S. E. Hafez. 1970. Disc electrophoretic patterns
of uteroglobin and serum proteins in rabbit blastocoelic
fluid. J. Reprod. Fert. 21:555.

Hamilton, T. H. 1963. Isotopic studies on estrogen-induced
acceleration of ribonucleic acid and protein synthesis.
Proc. Natn. Acad. Sci., U.S.A. 49:373.

Hamilton, T. H. 1964. Sequences of RNA and protein synthesis during
early estrogen action. Proc. Natn. Acad. Sci., U.S.A. 51:83.

Hamilton, T. H., C. C. Widnell, and J. R. Tata. 1967. Metabolism
of ribonucleic acid during the oestrous cycle. Nature,
Lond. 213:992.

Hammond, J. 1921. Further observations on the factors controlling
fertility and fetal atrophy. J. Agr. Sci. 11:337.

Hammond, J., Jr. 1949. Recovery and culture of tubal mouse ova.
Nature, Lond. 163:28.

Harrison, F. A., R. B. Heap, and J. L. Linzell. 1968. Ovarian
function in the sheep after autotransplantation of the
ovary and uterus to the neck. J. Endocr. 40:xiii.

Hertz, R., R. M. Fraps, and W. H. Sebrell. 1943. Induction of
avidin formation in the avian oviduct by stilbestrol plus
progesterone. Proc. Soc. Exptl. Biol. Med. 52:140.

Humphrey, K. W. 1969. Induction of implantation of blastocysts
transferred to ovariectomized mice. J. Endocr. 44:299.

Hunter, R. H. F. 1966. Effect of superovulation on fertilization
and embryonic survival in the pig. Anim. Prod. 8:457.








Hunter, R. H. F., C. Polge, and L. E. A. Rowson. 1967. The recovery
transfer and survival of blastocysts in pigs. J. Reprod.
Fert. 14:501.

Jensen, E. V. and E. R. DeSombre. 1969. Oestrogen-receptor inter-
action in target tissues. Biochem. J. 115:28P (Abstract).

Johnson, A. D. 1970. Limitation of fetus number in the rat, mouse,
and rabbit. J. Animal Sci. 30:978.

Junge, J. M. and R. J. Blandau. 1958. Studies on the electrophoretic
properties of cornual fluids of rats in heat. Fert. Steril.
9:353.

Kane, M. T. and R. H. Foote. 1970a. Culture of two- and four-cell
rabbit embryos to the blastocyst stage in serum and serum
extracts. Biol. Reprod. 2:245.

Kane, M. T. and R. H. Foote. 1970b. Culture of 2- and 4-cell rabbit
embryos to the expanding blastocyst stage in synthetic media.
Proc. Soc. Exp. Biol. Med. 133:921.

Kane, M. T. and R. H. Foote. 1970c. Fractionated serum dialysate
and synthetic media for culturing 2- and 4-cell rabbit
embryos. Biol. Reprod. 2:356.

King, J. W. B. and G. B. Young. 1957. Maternal influences on litter
size in pigs. J. Agr. Sci. 48:457.

Kirby, D. R. S. 1962. The influence of the uterine environment on
the development of mouse eggs. J. Embryol. Exp. Morphol.
10:496.

Kirchner, C. 1969. Untersuchungen an uterusspezifischen glyko-
proteinen wahrend der fruhen Graviditat des Kaninchens
Oryctolagus cuniculus. Wilh. Roux' Arch. Entwicklungsmech.
Organ. 164:97.

Krishnan, R. S. and J. C. Daniel, Jr. 1967. "Blastokinin" inducer
and regulator of blastocyst development in the rabbit
uterus. Science 158:490.

Krishnan, R. S. and J. C. Daniel, Jr. 1968. Composition of
"blastokinin" from rabbit uterus. Biochim. Biophys.
Acta 168:579.

Lewis, W. H. and P. W. Gregory. 1929. Cinematographs of living
developing rabbit-eggs. Science 69:226.

Longenecker, D. E. and B. N. Day. 1968. Fertility level of sows
superovulated at post weaning estrus. J. Animal Sci.
27:709.








Lowry, O. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall. 1951.
Protein measurement with the folin phenol reagent. J. Biol.
Chem. 193:265.

Lukaszewaska, J. H. and W. Hansel. 1970. Extraction and partial
purification of luteolytic activity from bovine endometrial
tissue. Endocrinology 86:261.

Masuda, M., L. L. Anderson, D. M. Henricks, and R. M. Melampy. 1967.
Progesterone in ovarian venous plasma and corpora lutea of the
pig. Endocrinology 80:240.

Maurer, R. R., R. H. Whitener, and R. H. Foote. 1969. Relationship of
in vivo gamete aging and exogenous hormones to early embryo
development in rabbits. Proc. Soc. Exp. Biol. Med. 131:882.

Mazer, R. S. and P. A. Wright. 1968. A hamster uterine luteolytic
extract. Endocrinology 83:1065.

McLaren, A. and J. D. Biggers. 1958. Successful development and birth
of mice cultivated in vitro as early embryos. Nature, Lond.
182:877.

McLaren, A. and D. Michie. 1956. Studies on the transfer of fertilized
mouse eggs to uterine foster mothers I. Factors affecting
the implantation and survival of native and transferred eggs.
J. Exp. Biol. 33:394.

McLaren, A. and D. Michie. 1959a. Superpregnancy in the mouse I.
Implantation and foetal mortality after induced superovulation
in females of various ages. J. Exp. Biol. 36:281.

McLaren, A. and D. Michie. 1959b. Studies on the transfer of fertilized
mouse eggs to uterine foster mothers II. The effect of transferring
large numbers of eggs. J. Exp. Biol. 36:40.

Means, A. R. and T. H. Hamilton. 1967. Early estrogen action: concomitant
stimulations within two minutes of nuclear RNA synthesis and uptake
of RNA precursor by the uterus. Proc. Natn. Acad. Sci., U.S.A.
56:1594.

New, D. A. T. 1967. Development of explanted rat embryos in circulating
medium. J. Embryol. Exp. Morphol. 17:513.

New, D. A. T. and J. C. Daniel, Jr. 1969. Cultivation of rat embryos
explanted at 7.5 to 8.5 days of gestation. Nature, Lond. 223:515.

Nicholas, J. S. 1942. Experiments on developing rats IV. The growth and
differentiation of eggs and egg-cylinders when transplanted under
the kidney capsule. J. Exp. Zool. 90:41.

Notides, A. and J. Gorski. 1966. Estrogen-induced synthesis of a specific
uterine protein. Proc. Natn. Acad. Sci., U.S.A. 56:230.









O'Malley, B. W. and W. L. McGuire. 1969. Progesterone-induced synthesis
of a new species of nuclear RNA. Endocrinology 84:63.

Onuma, H., R. R. Maurer, and R. H. Foote. 1968. In vitro culture of
rabbit ova from early cleavage stages to the blastocyst stage.
J. Reprod. Fert. 16:491.

Orsini, M. W. and A. McLaren. 1967. Loss of the zona pellucida in mice
and the effect of tubal ligation and ovariectomy. J. Reprod.
Fert. 13:485.

Perry, J. S. 1954. Fecundity and embryonic mortality in pigs. J. Embryol.
Exp. Morph. 2:308.

Pincus, G. and R. E. Kirsch. 1936. The sterility in rabbits produced by
injection of oestrone and related compounds. Amer. J. Physiol.
115:219.

Pincus, G. and N. T. Werthessen. 1938. The comparative behavior of
mammalian eggs in vivo and in vitro III. Factors controlling
the growth of the rabbit blastocyst. J. Exp. Zool. 78:1.

Pope, C. E., C. K. Vincent, and D. M. Thrasher. 1968. Effect of I. C. I.
33,828 and PMS on reproduction in gilts. J. Animal Sci. 27:303
(Abstract).

Prasad, M. R. N., C. M. S. Dass, and S. Mohla. 1968. Action of oestrogen
on the blastocyst and uterus in delayed implantation--an auto-
radiographic study. J. Reprod. Fert. 16:97.

Rathnasabapathy, V., J. F. Lasley, and D. T. Mayer. 1957. Some genetic
and environmental factors affecting litter size in swine. Mo.
Agr. Expt. Sta. Res. Bull. 615, Columbia, Mo.

Ringler, I. 1961. The composition of rat uterine luminal fluid.
Fed. Proc. 15:281.

Rowson, L. E. A. 1970. Evidence for luteolysin. Br. Med. Bull. 26:14.

Rundell, J. W. 1969. In vitro culture of swine ova. M. S. Thesis
Louisiana State University, Baton Rouge, La.

Runner, M. N. 1947. Development of mouse eggs in the anterior chamber
of the eye. Anat. Rec. 98:1.

Schomberg, D. W. 1967. A demonstration in vitro of luteolytic activity
in pig uterine flushings. J. Endocr. 38:359.

Schomberg, D. W. 1969. The concept of a uterine luteolytic hormone,
p. 383. In: K. W. McKerns (ed.), The Gonads. Appleton-
Century-Crofts, New York.








Schomberg, D. W., P. H. Jones, R. E. Erb, and W. R. Gomes. 1966.
Metabolites of progesterone in urine compared with progesterone
in ovarian venous plasma of the cycling domestic sow. J. Animal
Sci. 25:1181.

Schwartz, F. L., F. R. Fenton, O. W. Robison, and L. C. Ulberg. 1970.
Capacity of the uterus to support extra embryos. J. Animal
Sci. 30:327 (Abstract).

Steel, R. G. D. and J. H. Torrie. 1960. Principles and procedures of
statistics. McGraw-Hill Co., New York.

Stevens, K. R., H. D. Hafs, and A. G. Hunter. 1964. Immunochemical and
electrophoretic properties of oestrous rabbit uterine fluid
proteins obtained by uterine ligation. J. Reprod. Fert. 8:319.

Tarkowski, A. K. 1961. Mouse chimaeras developed from fused eggs.
Nature, Lond. 190:857.

Trams, G. and H. Maass. 1969. The effects of progesterone on the
ribonucleic acid metabolism in the rat uterus. Acta Endocr.
Copenhagen. Suppl. 138:39 (Abstract).

Ui, H. and G. C. Mueller. 1963. The role of RNA synthesis in early
estrogen action. Proc. Natn. Acad. Sci., U.S.A. 50:256.

Ulberg, L. C., F. W. Bazer, D. W. Schomberg, C. Rowland, and
F. A. Murray, Jr. 1970. Unpublished data.

Webel, S. K., J. B. Peters, and L. L. Anderson. 1970. Synchronous
and asynchronous transfer of embryos in the pig. J. Animal
Sci. 30:565.

Westman, A., E. Jorpes, and G. Widstram. 1931. Untersuchungen uber
den Schleimhautzyklus in der Tuba Uterina, seine hormonale
Regulierung und die Bedeutung des Tubensekrets fur die
Vitalitat der befruchteten Eier. Acta Obstet. et Gynecol.
Scand. 11:279.

Whitten, W. K. 1956. Culture of tubal mouse ova. Nature, Lond.
177:96.

Whitten, W. K. 1957. Culture of tubal ova. Nature, Lond. 179:1081.

Whitten, W. K. and J. D. Biggers. 1968. Complete development in vitro
of the pre-implantation stages of the mouse in a chemically
defined medium. J. Reprod. Fert. 17:399.

Williams, W. F., J. O. Johnston, M. Lauterbach, B. Fagan. 1967a.
Luteolytic effect of a bovine uterine powder on the corpora
lutea, follicular development, and progesterone synthesis of
the pseudopregnant rabbit ovary. J. Dairy Sci. 50:555.




62




Williams, W. F., J. O. Johnston, and M. Lauterbach. 1967b. Uterine
luteolytic hormone effect on ovarian progesterone content.
J. Dairy Sci. 50:1515.

Wilson, E. D. and R. G. Edwards. 1963. Parturition and increased
litter size in mice after superovulation. J. Reprod. Fert.
5:179.

Wintenberger-Torres, S. 1956. Les rapports entire l'oeuf en segmentation
et le tractus maternel chez la brebis. Third Int. Congr. Animal
Reprod., Cambridge 1:62.













BIOGRAPHICAL SKETCH


Finnie Ardrey Murray, Jr. was born May 30, 1943, at Burgaw,

North Carolina. In May, 1961, he was graduated from Burgaw High School.

During the summer of 1963, he served in Operation Crossroads, Africa

and worked in Serowe, Botswana. From 1964 until 1965 he worked as a

laboratory technician in the Dairy Science Department of North Carolina

State University. In June, 1966, he received the degree of Bachelor

of Science with majors in Animal Science and Zoology from North Carolina

State University. In 1966 he enrolled in the Graduate School of North

Carolina State University. He worked as a graduate research assistant

in the Department of Animal Science until September, 1968, when he

received the degree of Master of Science with a major in Physiology.

In 1968 he enrolled in the Graduate School of the University of Florida

and worked as a graduate research assistant until October, 1970. From

September, 1968, until the present time he has pursued his work toward

the degree of Doctor of Philosophy.

Finnie Ardrey Murray, Jr. is married to the former

Margaret Alice Kelly and is the father of a son, Michael Edward. He

is a member of Alpha Zeta, the American Society of Animal Science,

Phi Sigma, Sigma Xi, and the Society for the Study of Reproduction.








I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully
adequate, in scope and quality, as a dissertation for the degree of
Doctor of Philosophy.




Alvin C. Warnick, Cairman
Professor of Animal Science


I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully
adequate, in scope and quality, as a dissertation for the degree of
Doctor of Philosophy.


Fuller W. Bazer
Assistant Professor of


Animal Science


I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully
adequate, in scope and quality, as a dissertation for the degree of
Doctor of Philosophy.




Jaims A. Himes
Assistant Professor of Veterinary
Science


I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully
adequate, in scope and quality, as a dissertation for the degree of
Doctor of Philosophy.



t,- '. V .^ i>,,-__
Owen M. Rennert
Assistant Professor of Biochemistry









I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully
adequate, in scope and quality, as a dissertation for the degree of
Doctor of Philosophy.




Ray S Shirley
Professor of Animal Science



This dissertation was submitted to the Dean of the College of Agriculture
and to the Graduate Council, and was accepted as partial fulfillment of
the requirements for the degree of Doctor of Philosophy.

March, 1971




Dean, Colle e/of Agriculture


Dean, Graduate School




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