CORPUS LUTEUM FUNCTION AND UTERINE LUMENAL FLUID PROTEIN IN ewes DURING THE ESTROUS CYCLE AND EARLY PREGNANCY _ Dissertation for the Degree of Ph. D. MICHIGAN STATE UNIVERSITY ROGER JOHN KITTOK 1977 LIBRARY man State University This is to certify that the thesis entitled CORPUS LUTEUM FUNCTION AND UTERINE LUMENAL FLUID PROTEIN IN ENES DURING THE ESTROUS CYCLE AND EARLY PREGNANCY presented by Roger John Kittok has been accepted towards fulfillment of the “requiremefifor . ”A? degree 1n M% Date wig/76 WW 7"?" 0-7639 30,4054 ABSTRACT CORPUS LUTEUM FUNCTION AND UTERINE LUMENAL FLUID PROTEIN IN ENES DURING THE ESTROUS CYCLE AND EARLY PREGNANCY By Roger John Kittok In the ewe, luteal maintenance during the third week of preg- nancy requires maternal recognition of that pregnancy. Since the mechanism of this recognition is unclear, the objectives of the present study were to assess the antiluteolytic capacity of the con- ceptus and determine if the early maternal recognition of pregnancy involved either qualitative or quantitative changes in uterine lumenal fluid protein. Ewes given 250 ug l7B-estradiol per day on days ll and l2 or l2 and l3 following mating to a vasectomized ram had less serum pro- gesterone (ng/ml) on day l4 than did similarly mated control ewes (.4 i .1 vs l.9 i .6; P < .01). However, estradiol treatment caused a decline in serum progesterone in only 6 of 12 ewes mated to fertile rams. .Of ewes mated to fertile rams in experiment II, four of eight and one of eight given 125 or 250 pg estradiol per day, respectively, on days ll and 12 post mating were pregnant on day 16. Pregnant ewes had higher (P < .05) serum progesterone on day 15 (1.6 i .8) than non- pregnant ewes (.3 i .1). In experiment III, five of 17 ewes mated to Roger John Kittok fertile rams and given 125 pg estradiol on days l1 and 12 were preg- nant on day 20. These pregnant ewes had higher progesterone (P < .01) on day 14 than ewes similarly mated but not pregnant on day 20 (4.1 i .3 vs 1.3 i .3 ng/ml). Serum progesterone in estradiol treated ewes that were pregnant on day 20 did not differ from levels in normal pregnant ewes. In experiments IV and V, recoverable uterine lumenal protein increased (P < .05) from 1.84 t.21 mg on day 3 (five ewes/day) to 4.97 i 1.20 mg on day 9 of an estrous cycle and remained at that level until day l4. Uterine protein recovered on day 14 of pregnancy did not differ from day 14 of an estrous cycle (6.61 i .76 vs 5.1 i 1.60 mg, respectively). Up to 35 protein bands were detected after isoelectric focusing of uterine protein collected on day 14 of an estrous cycle; only two faint bands (pI 7.2 to 7.6) were not present also in blood serum. With the exception of the two bands focused between pH 7.2 and 7.6, protein collected on days 3 and 9 of an estrous cycle focused a similar number of bands. During pregnancy, uterine protein differed from that collected during an estrous cycle as follows: (1) after day 14, a pregnancy-specific protein migrated toward the cathode at pH 4.5; (2) after day 13 of pregnancy, increased staining intensity of a protein of = 9500 MN; and (3) decreased pro- portions of proteins focused between pH 5.4 and 7.0 occurred on day 14 of pregnancy. Compared to blood serum, uterine protein collected from ovari- ectomized ewes in experiment VI (five ewes/treatment) had a higher pro- portion of proteins focused at less than pH 4.7. After 10 days of Roger John Kittok progesterone replacement, the proportion of proteins that focused at less than pH 4.7 decreased while recoverable protein increased (1.20 i .26 vs 3.48 i 1.20 mg; P < .05). Twenty-one protein bands were detected after progesterone treatment and only one of these (pI 7.9) was not present also in serum. Estradiol replacement increased recoverable uterine protein (4.98 i 2.37 mg) but profiles of protein collected after estrogen treatment were not different from those observed in control ovariectomized ewes. Progesterone plus estradiol replacement increased the proportion of lumenal proteins focused between pH 4.7 and 5.8. Estradiol (125 ug/day; experiment VII) on days 11 and 12 of an estrous cycle increased the proportion of proteins focused at less than pH 4.7 in three of five ewes and decreased the proportion focused between pH 5.2 and 6.9 when compared to day 14 of a normal estrous cycle and the two uterine-specific proteins (pI 7.2 to 7.6) were not detected. Estradiol administration on days 11 and 12 of pregnancy did not alter uterine protein collected on day 14. The present study had demonstrated that (1) the conceptus had a definite antiluteolytic capacity and was able to exert that capacity between days 11 and 13 of pregnancy, (2) the majority of proteins in uterine lumenal fluid were present also in blood serum, and (3) progesterone influenced the presence of proteins in uterine fluid. CORPUS LUTEUM FUNCTION AND UTERINE LUMENAL FLUID PROTEIN IN ENES DURING THE ESTROUS CYCLE AND EARLY PREGNANCY By Roger John Kittok A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Dairy Science 1977 ACKNOWLEDGEMENTS The author wishes to express thanks and appreciation for the perceptive advice and skilled assistance so willingly given by his major professor, Dr. J.H. Britt. And he is especially indebted to Drs. E.M. Convey, H.D. Hafs and H.A. Tucker for assistance in experimental design, laboratory techniques and critical review of data. The advice and approval received from his graduate guidance committee, 0rs. R.S. Emery, P.K. Kindel and J.R. Shaver, was gratefully appreciated. The author also acknowledges a sense of obligation to the Department of Dairy Science for the provision of facilities and financial assistance necessary for his graduate study. ii TABLE OF CONTENTS Page LIST OF TABLES .......................... v LIST OF FIGURES .......................... vi INTRODUCTION ........................... 1 REVIEW OF LITERATURE ....................... 4 Uterine Influence on Corpus Luteum Function in Sheep . . . . 4 Pathway of Uterine Luteolysin to Ovary .......... 5 Prostaglandin FZa Induced luteolysis ........... 7 Prostaglandin an Secretion During the Estrous Cycle. . . 8 Prostaglandin an Response to Steroid Hormone Administration .................... 10 Estradiol-Induced Luteolysis ............... ll Inhibition of Luteolysis by Gonadotropins ........ 12 Luteal Maintenance Prolonged by Embryo ........... 13 Blood-Borne Factor of Pregnancy . . . , .......... 14 Prostaglandin Fed During Pregnancy ............ 15 Prostaglandin Fga Administration During Pregnancy . . . . 15 Proteins in Uterine Lumenal Fluid .............. 16 Uterine Lumenal Fluid Proteins in the Rabbit ....... l6 Uterine Lumenal Fluid Proteins in the Pig ........ 20 Uterine Lumenal Fluid Proteins in the Cow ........ 23 Uterine Lumenal Fluid Protein in the Ewe ......... 24 Uterine Lumenal Fluid Protein in Primates ........ 25 Uterine Lumenal Fluid Protein in Other Species ...... 26 MATERIALS AND METHODS ....................... 27 Experiment I ..................... . . . 27 Experiment 11 ........................ 28 Experiment III ....................... 28 Experiment IV ........................ 28 Experiment V ........................ 30 Experiment VI ........................ 30 Experiment VII ....................... 31 RESULTS ............................. Experiment I ........................ Experiment II ....................... Experiment III .................. . . . . . Experiment IV ....................... Experiment V ........................ Experiment VI ....................... Experiment VII ....................... Discussion ......................... SUMMARY AND CONCLUSIONS ..................... APPENDICES ............................ A. Extraction and Radioimmunoassay of Progesterone . . . . 8. Disc Gel Electrophoresis pH 4.5 ............ C. Total Protein Determination .............. 0. SDS Electrophoresis .................. E. Isoelectric Focusing in Polyacrylamide Gels ...... F. Radioreceptor Assay of Gonadotropin Activity ...... G. Extraction and Radioimmunoassay of Estradiol ...... H. Serum Progesterone in Ewes Given Estradiol During the Estrous Cycle or Early Pregnancy ......... 1. Representative Spectrophotometric Scans of Polyacrylamide Gels After Isoelectric Focusing of Uterine Lumenal Fluid Protein ........... LITERATURE CITED ......................... iv 96 101 104 109 111 LIST OF TABLES Table Page 1 Uterine lumenal fluid protein recovered from ewes during estrous cycle or early pregnancy (Experiment IV) ..... 39 2 Uterine lumenal fluid protein recovered from ewes during an estrous cycle or early pregnancy (Experiment V) ..... 48 3 Gradients of pH formed during isoelectric focusing in poly- acrylamide gels ..................... 48 4 Serum estradiol and progesterone in ovariectomized ewes with estradiol implants and/or progesterone pessaries (Experiment VI) ..................... 55 5 Uterine lumenal fluid protein recovered from ovari- ectomized ewes with estradiol implants and/or progesterone pessaries (Experiment VI) .......... 57 6 Uterine lumenal fluid protein recovered on day 14 from ewes mated to intact or vasectomized rams and given either 0 or 125 ug 17B-estradiol on days 11 and 12 ‘after mating (Experiment VII) .............. 57 7 Serum progesterone (ng/ml) in ewes mated to a vasectomized ram and given 250 pg 17B-estradiol on days 11 and 12 or days 12 and 13 and in non-injected control ewes mated to a vasectomized ram (Experiment I) ............ 105 8 Serum progesterone (ng/ml) in ewes mated to intact rams and given 250 pg l7B-estradiol on days 11 and 12 or days 12 and 13 (Experiment I) ................. 106 9 Serum progesterone (ng/ml) in ewes mated to intact or vasectomized rams and given 125 or 250 pg l7B-estradiol on days 11 and 12 after mating (Experiment II) ...... 107 10 Serum progesterone (ng/ml) in ewes mated to intact or vasectomized rams and given either 0 or 125 pg 178 estradiol on days 11 and 12 after mating (Experiment III) ........... . ......... 108 LIST OF FIGURES Figure Serum progesterone in ewes mated to a vasectomized ram and given 250 pg 17B-estradiol on days 11 and 12 or days 12 and 13 and in non-injected control ewes mated to a vasectomized ram (Experiment I) ........... Serum progesterone in ewes mated to intact rams and given 250 pg 17B-estradiol on days 11 and 12 or days 12 and 13 (Experiment I) ............. . . . . Serum progesterone in ewes mated to intact rams and given 125 or 250 pg l7B-estradiol on days 11 and 12 after mating (Experiment II) ............... Serum progesterone in ewes mated to intact rams and given 125 pg 17B-estradiol on days 11 and 12 after mating (Experiment III) ..................... Serum progesterone in ewes mated to a vasectomized ram and given either 0 or 125 pg 17B-estradiol on days 11 and 12 after mating (Experiment III) ......... Serum progesterone in ewes mated to intact rams and iven 125 pg l7B-estradiol on days 11 and 12 after mating IExperiment III) ..................... Electrophoresis in polyacrylamide gel at pH 4.5 of uterine lumenal fluid protein collected on day 14 of an estrous cycle and pregnancy (Experiment IV) ...... SDS electrophoresis of uterine lumenal fluid proteins collected on days 12, 13, 14 and 15 of an estrous cycle and pregnancy (Experiment IV) ........... Determination of molecular weight by $05 electrophoresis of uterine lumenal fluid protein collected on day 14 of an estrous cycle and pregnancy (Experiment IV) . . . . vi Page 33 34 36 37 38 4O 42 44 47 Figure Page 10 Isoelectric focusing in polyacrylamide gel of uterine lumenal fluid protein collected on days 3, 9 and 14 of an estrous cycle (Experiment V) ............ 49 11 Isoelectric focusing in polyacrylamide gel of uterine lumenal fluid protein collected on day 14 of an estrous cycle and pregnancy (Experiment V) ...... . . 52 12 Isoelectric focusing in polyacrylamide gel of uterine lumenal fluid protein collected from ovariectomized ewes after replacement of estradiol and/or progesterone (Experiment IV) .............. . 58 13 Isoelectric focusing in polyacrylamide gel of uterine lumenal fluid protein collected on day 14 from ewes mated to intact or vasectomized rams and given 125 pg 17B-estradiol on days 11 and 12 after mating. . . . . . . 61 14 Spectrophotometric scans of polyacrylamide gels after isoelectric focusing of uterine lumenal fluid protein collected on day 14 of an estrous cycle and pregnancy (Experiment V) ...................... 110 vii INTRODUCTION In the ewe, normal regression of the corpus luteum during the late luteal phase of an estrous cycle requires the presence of the uterus. Evidence accumulated from many studies indicates that a factor(s) emanates from the uterus, transverses a local venoarterial pathway to the ovary and causes the demise of the corpus luteum. Much of this evidence would suggest that prostaglandin F2a (PGan) is the uterine luteolysin in the sheep (Goding, 1974). Presence of an embryo in the ovine uterus on day 12 after estrus results in maintenance rather than regression of the corpus luteum. The mechanism by which the conceptus prolongs luteal life may be through support of the corpus luteum (luteotropic effect) and/or through protection of the corpus luteum (antiluteolytic effect). In the ewe, premature luteal regression can be induced by daily injection of estradiol after day 8 of an estrous cycle or by administration of PGF2 Estradiol's luteolytic effect can be a. blocked by administration of gonadotropins and hysterectomy. The luteolytic effect of exogenous PGFZa can be blocked by gonadotropins and pregnancy. Estradiol-induced luteolysis is probably mediated through release of PGFZa from the uterus. Estradiol has been implicated in a similar role in luteolysis during the late luteal phase of an estrous cycle. The luteolytic effect of exogenous PGFZa can be blocked by pregnancy, however, assessment of the antiluteolytic capacity of the conceptus during an estrogen mediated luteolytic challenge has not been reported. The first objective of the present study was to assess the antiluteolytic action of the conceptus when pregnant ewes were given doses of estradiol capable of causing premature regression of the corpus luteum in cycling ewes. The present study also involved investigation of the means by which the conceptus was capable of preventing luteolysis. Liter- ature reports had indicated the factor involved was a protein and was active prior to day 13 after mating. This factor may be produced by the conceptus or by the uterus in response to the presence of the conceptus. It was assumed that this protein factor would be in the fluid secreted into the lumen of the uterus because: (1) if the conceptus was flushed from the uterus, the factor was also removed and was contained in a homogenate of the embryo; and, (2) if the factor was produced by the embryo, it must pass through the lumenal fluid to leave the uterine lumen since placental attachment does not begin until day 15 in the sheep. Since adequate preliminary information was not available, the present study was to describe the uterine lumenal fluid protein in the ewe. Specifically, the objectives were to determine if proteins in uterine lumenal fluid change qualitatively or quantitatively during the estrous cycle, and to determine the contribution of the conceptus to uterine lumenal fluid protein. REVIEW OF LITERATURE Uterine Influence on Corpus Luteum Function inTSheep Complete hysterectomy between days 3 and 8 of an estrous cycle in ewes prolonged the life-span of the corpus luteum to the termination of the experiment at 100 days after pre-surgery estrus (Wiltbank and Casida, 1956). Kiracofe and Spies (1966) observed luteal maintenance in hysterectomized ewes for at least 160 days; however, corpora lutea regressed within the next 40 days and new ovulations were observed at 200 days after the pre-operation estrus. Hysterectomy on day 15 of an estrous cycle interrupted luteal regression provided the luteal tissue was still capable of progesterone secretion (Moor et al., 1970). The corpus luteum was prolonged to at least 34 days in five of six ewes after removal of the uterine horn ipsilateral to the ovary containing the corpus luteum on day 6 of an estrous cycle; the sixth ewe was observed in estrus on day 24 (Inskeep and Butcher, 1966). Moor and Rowson (1966b) observed that on day 8 of a cycle unilateral hysterectomy ipsilateral to the ovary containing the corpus luteum lengthened 22 of 31 observed estrous cycles. Removal of the uterine horn contralateral to the corpus luteum did not effect cycle length (Inskeep and Butcher, 1966; Moor and Rowson, 1966b). Pathwaygof Uterine Luteolysin to Ovary Bilateral ligation of uterine arteries and veins prior to day 7 of an estrous cycle prolonged luteal maintenance to at least 25 days after pretreatment estrus in seven of eight ewes (Kiracofe et al., 1966). However, ligation of uterine arteries alone did not affect cycle length. On days 7 to 9 of an estrous cycle Ginther et a1. (1973) anastomosed the uterine vein from the intact side of unilaterally hysterectomized ewes to the uterine vein on the operated side and observed luteal regression on the hysterectomized side in three ewes. Also, anastomosis of the ovarian artery on the intact side to the ovarian artery on the hysterectomized side decreased corpus luteum weight in six ewes. These data indicated that the uterine vein and ovarian artery were the proximal and distal components, respectively, of a local venoarterial pathway between the uterine horn and its adjacent ovary. Reconstituted uterine venous plasma from day 14 of an estrous cycle decreased plasma progesterone 50 percent within six to eight hours when infused into the ovarian artery of seven recipient ewes (Caldwell and Moor, 1971); jugular venous plasma from day 14 or uterine venous plasma from day 8 had no effect. In cross circulation experiments, McCracken et a1. (1972) reported that uterine venous blood from donors on day 15 of an estrous cycle decreased plasma progesterone more than 50 percent in five ewes with ovarian trans- plants on the neck. Progesterone decreased less than 20 percent when recipients received uterine venous blood from donors on days 2, 6, 10 or 13 of an estrous cycle. Baird et a1. (1973) observed a 33 percent decrease in progesterone secretion from seven ovarian transplants upon infusion of uterine venous plasma collected on days 14, 15 and 16 of an estrous cycle, whereas, plasma collected on days 9, 10 and 11 decreased progesterone secretion only 10 percent and infusion of jugular venous plasma from day 14 had no effect on progesterone secretion. The ovarian artery lies adjacent to the uterine vein and this contact area is increased by the tortuous path of the artery over the surface of the vein (Del Campo and Ginther, 1973a; 1973b). Coudert at al. (1974a) found that this contact between the ovarian artery and uterine vein allowed diffusion of xenon from the vein to the artery, but there was no direct shunt to allow transfer of proteins (human serum albumen). When the ovarian artery and uterine vein were dissected on day 2 or 3 of an estrous cycle and folds of peritoneum interposed between them, the corpus luteum was maintained until day 25 (Barrett et al., 1971). As further evidence of counter-current exchange, 3 McCracken et a1. (1972) reported increased H-PGan in the ovarian artery within 20 minutes after the start of a one hour infusion of 3H-PGF2a into the uterine vein. Radioactive PGan peaked in the ovarian artery after 80 to 90 minutes and this transfer accounted for approximately two percent of the total 3H-PGF2a infused. Land et a1. (1976) also observed increased levels of 3 H-PGF2 in the OI ovarian artery within 40 minutes of the start of a uterine vein infusion. However, Coudert et al. (1974b) were unable to detect 3 transfer of H-PGFZa from the uterine vein to ovarian artery within 80 minutes after 11 ewes were infused for 40 to 60 minutes with 3H-PGF2a into a uterine vein. Prostaglandin an Induced Luteolysis Luteolysis has been reported after administration of PGan in various doses and via several routes. Plasma progesterone gradually decreased in four ewes infused (ovarian artery) with PGan at the rate of 2 pg/hr for 9.5 to 18 hours, however only one ewe exhibited estrus (Chamley et al., 1972). Increased rate of infusion (10 to 110 pg PGFZa/hr for three to seven hours) decreased pro- gesterone secretion to at least 30 percent of control values within 24 hours or less from the start of infusion and induced estrus behavior within 48 to 72 hours (McCracken et al., 1970; Barrett et al., 1971; Thorburn and Nicol, 1971; Chamley et al., 1972; McCracken et al., 1972). V Uterine vein PGan infusion required doses of 20 pg/hr for nine hours (Goding et al., 1972) or 40 pg/hr for six hours (Thorburn and Nicol, 1971) before a consistent decrease in progesterone secretion and estrus were observed. Infusion of 200 pg/hr PGan for three hours into the uterine horn ipsilateral to the corpus luteum caused luteolysis, estrus and ovulation in one ewe, while 50 pg/hr for nine hours resulted in luteolysis in one of two ewes with no signs of estrus 03 (Goding et al., 1972). Douglas and Ginther (1973) reported reduced corpus luteum weight (96 vs 725 mg) after a 2 mg intrauterine bolus of PGan' On day 8 of an estrous cycle, the minimum intramuscular dose of PGan to shorten the interval from treatment to estrus was 6 mg (Douglas and Ginther, 1973). Prostaglandin an Secretion During the Estrous Cycle Many reports describe the pattern of production and/or release of PGFZQ from the uterus during an estrous cycle in the ewe. Different methods of quantitation do not allow comparison of abso- lute values, however, most investigators have observed increased PGFZa during the late luteal phase of the cycle compared to the early luteal phase. Also, some investigators have not eliminated the possible contribution of PGFla to reported values and have expressed their results in terms of prostaglandins of the F series (PGF). Wilson et a1. (1972) observed both increased content and concentration of PGan in endometrium on day 14 of an estrous cycle compared to values found on days 3, 5 and 11. Vena cava plasma PGF was less than 100 pg/ml until four days prior to estrus in five ewes and peaked (1260 pg/ml) 72 hours prior to estrus in four of five ewes; 2705 pg/ml was observed 84 hours prior to estrus in the fifth animal (Fitzpatrick and Sharma, 1973). In uterine venous plasma, Bland et a1. (1971) estimated 3.4 to 8.0 ng/ml PGan on days 14, 15 and 16 of an estrous cycle, but concentrations were undetectable (<2.9 ng/ml) between days 2 and 13. Thorburn et a1. (1972) reported basal concentrations of less than 1.0 ng/ml PGF in uterine venous plasma between days 13 and 17 of an estrous cycle but during this period, a series of peaks of PGF (4 to 22 ng/ml) were observed with individual peak duration of less than two to three hours. Nett et a1. (1976) measured uterine venous plasma between days 11 and 17 of an estrous cycle and observed 32 peaks averaging 41.3125.l ng/ml PGFZa per peak with basal levels of 9.6 i.6 ng/ml. Although variation in uterine venous plasma estradiol occurred throughout an estrous cycle, it was not until day 13 or 14 that increases in estrogen (12 to 132 pg/ml) were associated with peaks of PGFZa (3 to 8.5 ng/ml; Barcikowski et al., 1974). Luteal regrese sion was associated with 9 to 23 ng/ml PGan peaks lasting two hours or less on day 15. Beginning on days 12 to 14 of five estrous cycles, Baird et a1. (1976) observed transient increases in uterine venous plasma PGan (peak height 2 to 10 ng/ml) and maximum PGFZa (10 to 16 ng/ml) released on the day prior to onset of estrus. In most cases, estradiol increased (50 to 180 pg/ml) within six hours before each increase of PGFZa' Evidence that endogenous PGan causes luteolysis was provided by Scaramuzzi and Baird (1976). Four of six ewes failed to exhibit regular estrous cycles after active immunization against PGFZa protein conjugates. 10 Prostaglandin F20 Response to Steroid Hormone Administra- tion Estradiol injections (500 ug/day) on days 9 and 10 of an estrous cycle increased uterine venous plasma PGF on day 11 over control values (8.4::2.4 vs 1.31:.4 ng/ml; Ford et al., 1975). However when 500 ug/day estradiol was given on days 4 and 5, PGF did not increase on day 6 unless progesterone was given (10 mg/day) on days 1 to 5 (2.82:1.3 vs .5 t.2 ng/ml, progesterone/estradiol treatment vs control, respectively). Infusion of estradiol (1.0 ng/min) for six hours into the arterial blood supply of an autotransplanted uterus increased PGFZa in the uterine vein from 1.4 to 102.2 ng/hr within 90 minutes (Barcikowski et al., 1974). Systemic infusion of a similar amount of estradiol had no effect on uterine venous PGFZa in two ewes. After 11 days of progesterone replacement (20 mg/day on alternate days) in ovariectomized ewes, 50 pg estradiol increased jugular venous plasma PGF from .3 to 1.0 ng/ml within 24 hours (Caldwell et al., 1972). However, if ovariectomized ewes were immunized against estradiol, the above replacement of steroid hormones had no effect on PGF; less than .05 ng/ml PGF was detected. In ovariectomized ewes, uterine venous plasma PGF did not respond to an intra-arterial infusion of sufficient estradiol to increase plasma estradiol to 5 pg/ml (Scaramuzzi et al., 1974). After 14 days of progesterone replacement (10 mg/day), estradiol infusion increased PGF over control levels (22 to 110 ng/ml vs .1 to 3.0 ng/ml, respectively). However, Ford et a1. (1975) 11 reported increased uterine venous plasma PGF in ovariectomized ewes within 12 hours of 12.5 pg estradiol, whether or not (10.51:2.2 and 9.91:3.3, respectively, vs .lrt.l ng/ml) the ewes were given five days of progesterone replacement (3 mg/day). Progesterone replacement (3 mg/day) in ovariectomized ewes for five days (Ford et al., 1975) or 11 days (20 mg/day on alternate days; Caldwell et al., 1972) had no effect on uterine or jugular venous PGF, respectively. However, 10 mg/day progesterone for 14 days increased uterine venous plasma PGF from basal secretion (.1 to 3.0 ng/ml to 1.0 to 25.0 ng/ml; Scaramuzzi et al., 1974). Estradiol-Induced Luteglysis Daily injection of estradiol beginning after day 8 of an estrous cycle results in premature luteolysis in intact ewes. Many reports have demonstrated premature luteal regression from various doses given on different days of an estrous cycle: 500 and 750 ug/day on days 11 and 12 (Stormshak et al., 1969); 500 ug/day on days 8 to 11 (Ginther, 1970); 250 and 750 pg/day on days 9 and 10 or days 11 and 12 (Hawk and Bolt, 1970); 500 and 1000 ug/day on days 11 and 12 (Akbar et al., 1971); 100 to 1000 pg/day on days 10 and 11 (Bolt and Hawk, 1971), and 1000 pg on day 10 (Bolt et al., 1971). Luteal regression was not observed in ewes hysterectomized five days after estrus and given 750 ug/day estradiol on days 11 and 12 (Stormshak et al., 1969). Bolt and Hawk (1975) also observed no luteal regression in ewes hysterectomized on day 9 after estrus and given 1 or 10 mg/day estradiol on days 9 and 10, 1 mg/day estradiol 12 on days 9 to 13, or 1 mg/day estradiol on days 9 to 24. In six unilaterally hysterectomized ewes, Akbar et a1. (1971) reported premature luteal regression from 1 mg/day estradiol given on days 11 and 12 regardless of whether the corpus luteum was adjacent or opposite the removed side (4461:50 vs 6881:61 mg, treated vs control, respectively). However, in ten unilaterally hysterec- tomized ewes with bilateral ovulations, estradiol reduced corpus luteum weight to a greater extent on the ovary adjacent to the intact horn. Inhibition of Luteolysis by_ Gonadotropins Daily injection of 2 mg/day ovine luteinizing hormone on days 9 to 13 blocked the luteolytic effect of 500 ug/day estradiol on days 10 and 11 (Akbar et al., 1971). Ginther (1970) observed 1500 I.U./day human chorionic gonadotropin (HCG) given with 500 ug/ ‘ day estradiol from days 8 to 11 prevented premature luteal regression. Bolt et a1. (1971) demonstrated HCG (750 I.U.) blocked estrogen- induced (1 mg on day 10) luteolysis if given with estradiol on day 10 or seven days earlier; a commercial pituitary preparation, Vetrophin (equivalent to 1 mg FSH and 1 mg LH; Abbott Lab., North Chicago, 111.), prevented luteolysis if given seven days prior to estradiol but not if given at the same time as estradiol.~ Neither HCG nor the commercial preparation altered corpus luteum weight when administered alone. 13 Human chorionic gonadotropin (HCG), given on days 9 and 10 of an estrous cycle, prevented the luteolytic effect of 10 but not of 20 mg PGFZa given on day 10 of that cycle (Bolt, 1973). The activity of endogenous PGFZa may be blocked by continuous infusion of ovine luteinizing hormone starting prior to day 13 of an estrous cycle, since this treatment was capable of prolonging the life-span of the corpus luteum to day 30 after estrus (Karsch et al., 1971). Luteal Maintenance Prolonged by Embryo Histologically, Deane et a1. (1966) observed early signs of luteal regression in luteal cells on day 12 or 13 of an estrous cycle. However, corpus luteum maintenance is prolonged if an embryo is present in the uterus between days 12 and 13. Transfer of day 12, 13 and 14 embryos to recipients on respective days of an estrous cycle resulted in 67, 22 and zero percent of the recipients becoming pregnant, respectively (Moor and Rowson, 1964; 1966d). Failure to maintain pregnancy when transfers were on days 13 and 14 of a cycle was attributed to recipient ewes since the transfer of day 13 and 14 blastocysts to day 12 recipients results in pregnancy. Surgical removal of embryos on day 5, 7, 9 or 12 did not affect return to estrus (cycle length of 18.01:.3 days); removal of embryos on day 13, 14 or 15 of pregnancy extended the estrous cycle to 24.5 t.8 days (Moor and Rowson, 1966c). Rowson and Moor (1967) prolonged the length of an estrous cycle with daily intra-uterine infusion of day 14 and 15 embryo homogenates; infusion began on day 12 and ewes received the equivalent of one embryo per day. 14 Following synchronous transfer of day 5 or 9 embryos to the uterine horn either ipsilateral (12 ewes) or contralateral (eight ewes) to the corpus luteum, pregnancy rate did not differ (75 and 63 percent, respectively) provided the uterus was intact (Moor and Rowson, 1966a). Luteal regression occurred in all 15 ewes after transfer of day 5 embryos to an isolated horn contralateral to the corpus luteum, while the transfer to an isolated horn ipsilateral to the corpus luteum resulted in luteal maintenance in eight of ten ewes (Moor and Rowson, 1966a). Blood-Borne Factor of Pregnancy Mapletoft et a1. (1975) observed corpus luteum regression in all five ewes on day 20 when the uterine vein ipsilateral to the corpus luteum contained blood from only the nongravid uterine horn, whereas the corpus luteum was maintained when the ipsilateral uterine vein contained blood from a gravid horn whether or not it also con- tained blood from a nongravid horn (three ewes/treatment). Mapletoft et al. (1976b) demonstrated the transfer of this blood-borne factor from uterine vein to ovarian artery via a venoarterial pathway. In order to maintain the corpus luteum, arterial blood to the ovary containing the corpus luteum was required to pass through the segment of the ovarian artery in contact with the uterine vein draining the gravid horn. In unilaterally pregnant ewes with bilateral ovula- tions, anastomosis of the ovarian artery of the gravid horn to the ovarian artery supplying the ovary of the nongravid side prior to day 7 after estrus resulted in the prolonged maintenance of the 15 corpus luteum ipsilateral to the isolated nongravid horn until day 20 in four ewes (Mapletoft et al., 1976b). Prostaglandin F2a During Pregnancy Barcikowski et a1. (1974), in one pregnant ewe, observed suppression of peaks of PGFZa normally occurring on days 15 and 16 of an estrous cycle but small peaks of PGFZa were still observed on days 13 and 14. However, Pexton et a1. (1975) did not observe any differ- ences in uterine venous PGF in pregnant and non-pregnant ewes sampled once on day 15 after estrus (5.2 i.9 vs 7.12:3.4 ng/ml, respectively). Nett et a1. (1976) observed no differences in uterine venous plasma PGFZa.from pregnant and non-pregnant ewes sampled at three hour intervals between days 11 and 17 after estrus (8.11:.1 vs 9.11:.6 ng/ml, respectively). Non-pregnant ewes had more variation than pregnant ewes as reflected by more peaks of PGan (32 vs 12). Uptake by ovarian and luteal tissues did not differ between pregnant and non-pregnant ewes on day 15 after estrus, although increased blood flow (4.5-fold) exposed the pregnant ovary to more PGF (2.1 i.5 vs .5 t.2 ng/min.) than the ovary of the non-pregnant animal (Pexton et al., 1975). Prostaglandin F20L Administration During Pregnangy Serum progesterone declined similarly in mated and non-mated ewes after PGan was injected on day 12 after estrus into a follicle on the same ovary as the corpus luteum; however, within 24 hours 16 progesterone recovered to pretreatment concentrations in mated ewes carrying embryos on day 17 after estrus (Inskeep et al., 1975). Infusion of 200 pg PGFZa into the ovarian artery on day 13 after mating was luteolytic in six of seven ewes hysterectomized before day 7, however, this dose caused luteal regression in only two of seven pregnant ewes (Mapletoft et al., 1976a). Proteins in Uterine Lumenal Fluid A functional relationship is required between embryonic and maternal systems after fertilization. Each of these systems is composed of many compartments and are significant in several ways. One area of importance is uterine environment and its provision of nourishment and shelter for the conceptus. Prior to implantation, fluid in the uterine lumen may provide not only nutrients, but also means of regulation of embryonic development and/or maternal premonition of pregnancy. Several early reports documented electrophoretically and immunologically the presence of proteins in uterine lumenal fluid of estrous rabbits (Stevens et al., 1964) and rats (Junge and Blandau, 1958; Albers and Castro, 1961; Ringler, 1961) that were undetectable in blood serum of the respective species. Uterine Lumenal Fluid Proteins In The Rabbit In the pregnant rabbit, Beier (1968b) observed uterine lumenal fluid proteins to differ from serum proteins in the prealbumen, postalbumen, B-globulin and macroglobulin electrophoretic regions. Independently, Krishnan and Daniel (1967) and Beier (1968a; 1968b) 17 reported the time related occurrence of a uterine-specific protein that migrated as a postalbumen. The protein was named "blastokinin" by Krishnan and Daniel (1967) and "uteroglobin" by Beier (1968a; 1968b), however, in this review, blastokinin will be used exclusively. Blastulation and onset of implantation were associated with the initial detection and diminution, respectively, of blastokinin. Effect of Uterine Proteins on Embryonic Development.-- Krishnan and Daniel (1967) demonstrated addition of uterine lumenal fluid protein to synthetic culture media increased the incidence of initiation of morulae blastulation (78 vs 4 percent). In a subse- quent experiment, similar results were observed after supplementation of media with a partially purified blastokinin preparation. Although uterine proteins may be essential for blastocyst formation in utero, the in vitro requirement was challenged by Kane and Foot (1970). Chemically defined synthetic media was sufficient for 42 percent of two- and four-cell rabbit ova to develop to the expanded blastocyst stage. Thymidine incorporation was not affected by blastokinin in day 3, 4, or 5 rabbit embryos, but uridine incorporation was stimu- lated by blastokinin in day 5 blastocysts (Gulyas et al., 1969). El-Banna and Daniel (1972a) also observed increased uridine incor- poration (40+ percent) in day 5 blastocysts cultured in the presence of uterine proteins. Steroid Binding by Blastokinin.--Uterine proteins may be steroid hormone carriers. El-Banna and Daniel (1972b) reported 18 increased growth (150 percent) of day 5 blastocysts cultured in progesterone-containing media after addition of uterine proteins. Urzua et a1. (1970) observed uterine lumenal protein to bind more progesterone on day 5 post-coitum (p.c.) than on day l p.c. (74 vs 5 percent). Progesterone binding on day 5 p.c. was limited to the blastokinin containing fraction and exhibited a binding constant of 5.95 x 108 (Arthur et al., 1972). Arthur et a1. (1972) reported estradiol also binds to blastokinin, but to a lesser extent than progesterone (binding constant 2.72 x 107). Sites of Blastokinin Production.--Johnson (1972) suggested on the basis of immunofluorescence experiments that blastokinin was synthesized in the cytoplasm of uterine crypt epithelial cells. Maximum intensity of the fluorescence was observed between days 3 and 7 of pregnancy. During that period, immunofluorescence was associated with the surface villous endometrial epithelium as well as the supranuclear and paranuclear cytoplasm of crypt epithelial cells and crypt lumens (Johnson, 1972; Kirchner, 1972). In estrous rabbits, a faint immunofluorescence was confined to the uterine crypt areas (Johnson, 1972). Passive Immunity to Blastokinin.--Requirement of blastokinin during early pregnancy was affirmed by passive immunity. Krishnan (1971) observed a reduction of implantation sites on day 11 (8.4 vs 1.4 sites per rabbit) after three alternate day injections (days 2 to 6) of IgG fraction from antiserum against blastokinin. Anti- blastokinin injections reduced offspring from 6.7 to .7 per female. 19 Occurrence of Blastokinin.-7Refined techniques allowed Bullock and Connell (1973) and Mayol and Longenecker (1974) to observe a trace of blastokinin in uterine lumenal fluid of estrous rabbits. Originally, Krishnan and Daniel (1967) could not detect the protein until day 3 p.c. Maximum amounts of blastokinin were recovered from the uterine lumen on day 5 p.c. and by day 9 p.c., the protein was undetectable (Krishnan and Daniel, 1967; Urzua et al., 1970; Bullock and Connell, 1973; Mayol and Longenecker, 1974) except by the sensitive radioimmunoassay technique of Mayol and Longenecker (1974). By this technique, blastokinin was observed in very low amounts in uterine lumenal fluid until at least day 12 p.c. Urzua et a1. (1970) reported a secretion pattern in pseudo- pregnant rabbits similar to that observed in pregnant animals. Krishnan and Daniel (1967) and Beier (1968b) detected blastokinin in pseudopregnant rabbits on days 6 and 7, respectively, the only days investigated in those studies. On day 6 of pregnancy and pseudopregnancy, 8.0 and 3.2 mg of total protein, respectively, was recovered from the uterine lumen. Blastokinin made up 22 and 32 percent, respectively, of the protein (Beier, 1968b). Arthur and Daniel (1972) observed uterine lumenal fluid protein to be 40 percent blastokinin in pregnant animals on day 5 p.c. Steroid Control of Blastokinin.--After progesterone replace- ment, blastokinin and other uterine-specific proteins could be detected in considerable amounts in lumenal fluid of ovariectomized 20 rabbits; and after sufficient replacement, protein profiles were essentially identical to intact day 5 p.c. rabbits. Arthur and Daniel (1972) observed .5 mg/kg/day progesterone for four days was the minimum dose required to elicit a blastokinin response. A dose of 1.0 mg/kg/day progesterone doubled (as a percentage of total uterine protein) blastokinin response and appeared to effect a maximum dbse response. In ovariectomized rabbits, total uterine lumenal protein was 32 percent blastokinin after five days of progesterone (1 mg/kg/day) replacement (Bullock and Willen, 1974). Prior exposure to estradiol was not required to achieve a blastokinin response to progesterone replacement (Urzua et al., 1970; Arthur and Daniel, 1972; Bullock and Willen, 1974). Blastokinin could not be detected in ovariectomized rabbits after estradiol replacement (Urzua et al., 1970; Arthur and Daniel, 1972). Uterine Lumenal Fluid Proteins in the Pig Murray et a1. (1972) observed no difference in amount (<6 mg) of uterine lumenal fluid protein recovered between days 2 and 9 of an estrous cycle. Total uterine protein increased to 141:3 mg by day 12 and to 452:8 mg by day 15. Thereafter, secretion decreased rapidly and values on day 18 were similar to those prior to day 9 of an estrous cycle. On day 15, five fractions were eluted by gel filtration chromatography of uterine lumenal fluid protein (Murray et al., 1972). Three of these protein fractions (1, MW<200,000; 21 11, MW 3 200,000; and, III, MW 3 90,000) were observed throughout the estrous cycle. Fraction IV (MW 3 45,000) was observed in uterine fluid only on days 12 to 16; and fraction V (MW 3 20,000) was detected on days 9 to 16. The primary component of fraction IV was demonstrated to be a basic purple-colored protein which migrated toward the cathode (-) at pH 8.0 (Squire et al., 1972). On days 14 to 16, fraction V was composed of six proteins which migrated in the postalbumen and posttransferrin electrophoretic regions. Progesterone Control.--Qualitative and quantitative changes in uterine lumenal fluid proteins are influenced by progesterone. Knight et a1. (1973) reported an increase in total uterine protein (13.7 vs 77.5 mg) from ovariectomized gilts after 12 days of progesterone replacement (2.2 mg/kg/day). Estradiol (1.1 Ug/kg/day) in addition to progesterone resulted in 167 mg uterine protein; estradiol alone had no effect. After 12 days of progesterone replacement, fractions I, II, III, IV and V were present in uterine lumenal protein from ovariectomized gilts, whether or not estradiol was also given (Knight et al., 1973). Uterine flushing from non- treated ovariectomized gilts consisted of fractions I, II and III; and estradiol replacement had no effect of the protein profile. Knight et a1. (1973) observed only fractions 1, II and 111 after four days of progesterone (2.2 mg/kg/day) in ovariectomized gilts; after six days of progesterone, fraction IV was detected and after ten days of replacement, all five fractions were observed. Knight et a1. (1974) reported a correlation of .88 between total 22 uterine lumenal fluid protein and amount of progesterone replacement (0 to 3000 mg/45.4 kg/day) in ovariectomized gilts. Purple-Colored Protein.--Uterine lumenal fluid collected between days 12 and 16 of an estrous cycle had a purple color (Murray et al., 1972; Squire et al., 1972). This color was attributed to a basic protein (Squire et al., 1972) eluted as fraction IV during gel filtration chromatography (Murray et al., 1972) which comprised approximately 15 percent of the total uterine protein on day 15 of an estrous cycle and was not detected in serum (Chen et al., 1973). Chen et al. (1973) further characterized the protein as a 32,000 MW glycoprotein (12.5 percent carbohydrate) with an isoelectric point of 9.7. Schlosnagle et a1. (1974) associated acid phosphatase activity with this protein. Sites of Purple-Colored Protein Production.--Based on immunofluorescence experiments, Chen et a1. (1975) suggested the purple-colored protein was synthesized and secreted by uterine sur- face epithelial and glandular epithelial cells. Although activity was observed throughout the estrous cycle, fluorescence increased after day 9. Between days 12 and 15, fluorescence was also present in the lumen of uterine glands. During pregnancy, fluorescence was detected in placental arealae, as well as the above tissues. The purple-colored protein was immunologically detected in allantoic fluid only after day 30 of gestation (Chen et al., 1973). Bazer et a1. (1975) demonstrated the presence of a protein of 23 identical physical and chemical properties as the purple-colored protein in allantoic fluid collected on days 30 to 100 of gestation. Passive Immunity to Purple Protein.-—Chen and Bazer (1973) passively immunized gilts with repeated injections of antiserum against the purple—colored protein on days 7, 11, 13 and 15 of gestation and observed decreased placental length and allantoic fluid protein concentrations on day 30. Injection of antiserum on days 34, 36, 38, 40 and 42 of gestation resulted in decreased placental length and weight, fetal wet weight and crown-rump length on day 50. Uterine Lumenal Fluid Proteins in the Cow In uterine lumenal fluid, Palubicki and Hunter (1974) detected three antigens not present in serum; these proteins were observed throughout the estrous cycle. One of these proteins migrated electrophoretically as an aZ—globulin and the other two as B-globulins. Mills et al. (1973) also detected a uterine-specific protein which migrated as a B-globulin on days 15 and 16 of an estrous cycle. Utilizing antiserum against bovine serum, immuno- electrophoresis of uterine lumenal protein formed ten precipitant lines (Roberts and Parker, 1974; serum and anti-uterine lumenal pro- teins yielded only seven precipitant arcs. Roberts and Parker (1974) used two dimensional immunoelectrophoresis to detect differences between uterine protein and serum in the a- and B-globulin regions. Uterine lumenal fluid and serum proteins reacted with antiserum against bovine serum to yield 14 and 16 precipitant arcs, respectively. 24 Immunoelectrophoresis provided no detectable differences in uterine lumenal fluid protein from pregnant and non-pregnant cows (Roberts and Parker, 1974). I Electrophoresis of uterine lumenal proteins at pH 4.5 revealed the transient appearance of three proteins which migrated more rapidly than albumen (Roberts and Parker, 1974). The slowest migrating of the three was detected on day 7 of pregnancy but not on day 14. The two other bands were observed on day 14, but not on day 20 of pregnancy. On day 20, a protein was observed with mobility similar to the protein on day 7. These proteins were not detected on day 14 of an estrous cycle. 0f the 35 protein bands detected by isoelectric focusing of’ uterine lumenal fluid from pregnant cows on day 14, all but two (pI 7.5 to 8.5) were also present in serum (Roberts and Parker, 1974). Uterine Lumenal Fluid Protein in the Ewe Lumenal fluid protein collected via cannulation did not differ in electrophoretic profiles during estrus, diestrous or anestrous periods (Wales, 1973). Although comparable proteins were detected in both uterine lumenal fluid and serum, the al-globulin of lumenal protein migrated slightly ahead of serum al-globulin. Wales (1973) observed albumen to make up a smaller proportion of the total protein in uterine lumenal fluid than in serum (522:.7 vs 602:1.8 percent); the opposite was true of B-globulin (31:t1.4 vs 22 t1.7 percent; uterine protein vs serum, respectively). 25 Uterine Lumenal Fluid Protein in Primates Hgmgg,--Bernstein et a1. (1971) detected two uterine-specific proteins in lumenal fluid by immunoelectrophoresis; however, the samples had been collected at random throughout the menstrual cycle. Shirai et a1. (1972) observed a uterine-specific protein migrating between albumen and transferrin in uterine lumenal protein collected six to ten days after the rise in basal body temperature. Also during that time period, protein bands in the a-globulin region were more prominent than those in samples collected either imme— diately before or after that period. Daniel (1973) reported a component of uterine lumenal fluid collected eight to nine days after ovulation had the immunological and electrophoretic characteristics of blastokinin. Babggg,--Peplow et a1. (1973) detected 18 protein bands by electrophoresis of uterine lumenal fluid protein; all but one which migrated as a prealbumen were also present in plasma. Uterine fluid collected during the proliferative or secretory phases of the men- strual cycle did not differ in number of protein bands detected; how- ever, band intensity did differ with cycle phases as well as between uterine protein and serum. Two dimensional immunoelectrophoresis of uterine lumenal proteins yielded nine precipitant arcs with antiserum against uterine lumenal protein (Peplow et al., 1974). 26 Uterine Lumenal Fluid Protein in Other Species Northern Fur Seal.--Daniel (1971) reported total uterine lumenal protein increased from less than 2 mg during the period of delayed implantation to 10 mg during the time associated with expansion of the blastocyst. An additional increase (22 mg) was observed at the time of implantation. Uterine lumenal proteins migrated electrophoretically similar to albumen and blastokinin as well as several other less well-defined protein components; however, blastokinin could not be detected immunologically (Daniel, 1972). Golden Hamster.-vNoske and Daniel (1974) observed total uterine lumenal protein to peak on day 4 of pregnancy, the day of implantation (.117 t.046 vs .0551:.031 mg day 4 vs day 3, respec- tively). During the first six days of pregnancy, albumen, trans- ferrin and two B-globulins were detected in uterine lumenal fluid. On day 3 of pregnancy, an a-globulin band was first detected and by day 6, that region resembled serum with four bands. A prealbumen was detected after day 3 of pregnancy that was not present in serum. Egrrgt,--Daniel (1970) observed total uterine lumenal protein to increase at the time of blastocyst expansion (<100 pg vs 225 pg, day 0 vs day 9, respectively) and increase again at the time of implantation (2150 pg, day 16). By electrophoretic tech- niques, albumen was obvious in all samples but resolution of the other proteins was poor. MATERIALS AND METHODS Multiparous crossbred ewes confined to a dry-lot management system were used throughout the study. A mixture of grease and paint was applied to the brisket of a vasectomized ram which was allowed continuous access to the ewes. Paint markings on the rump of a ewe was considered a sign of estrus; the flock was observed twice daily (0700 and 1700 hours) and fresh markings recorded. Ewes destined to be mated to intact rams were separated from the flock when marked and pen-mated to two intact rams, once at the first observance of the paint mark and again, if still receptive, at the next estrous check. The ewe was then returned to the flock. In the present study, sample collection was extended over a three year period (Experiment I, Fall 1973; Experiments II and IV, Fall 1974; and Experiments III, V, VI and VII, Fall 1975). Experiment I Ewes mated (estrus = day 0) to intact rams were given (im) 250 pg l7B-estradiol in oil per day on days 11 and 12 (six ewes) or days 12 and 13 (six ewes). Control ewes mated to a vasectomized ram were given either zero (four ewes) or 250 pg estradiol per day on days 11 and 12 (five ewes) or days 12 and 13 (six ewes). Blood was collected from each ewe via jugular puncture on days 11 to 18 27 28 to monitor corpus luteum function by radioimmunoassay of serum progesterone (Louis et al., 1973; Appendix A). On days of estradiol injections, blood was collected immediately before estrogen was given. Experiment II Sixteen ewes mated to intact rams and eight ewes mated to a vasectomized ram were given either 125 or 250 pg estradiol on days 11 and 12. Blood samples were collected on days 10 to 16 for assay of serum progesterone. On day 16, all ewes were sacrificed, and their uteri excised and flushed with saline to ascertain pregnancy by the presence of embryonic tissue. Experiment III Twenty-two ewes were mated to intact rams; then 17 of these were given 125 pg estradiol (im) on days 11 and 12. Ten control ewes were mated to a vasectomized ram and five of these ewes received 125 pg estradiol on days 11 and 12. Blood was collected on days 10 to 20 for determination of serum progesterone. Ewes mated to intact rams were sacrificed on day 20 and their uteri excised and flushed with saline. Pregnancy was diagnosed by the presence of embryonic tissue. Experiment IV Fourteen ewes mated to intact rams and 13 ewes mated to a vasectomized ram were sacrificed on days 12, 13, 14 and 15 after mating (two to five ewes/day/mating group). The uterus of each ewe was excised within two to three minutes of death. The cervix and 29 tubo-uterine junctions of the uterus were clamped (Allis's forceps and hemostats, respectively) and the uterine lumen was filled with 20 ml of chilled .33 M NaCl (4 C). The tip of one uterine horn was removed, any incident blood sponged off and after the saline was exposed to the uterine lumen for five minutes, the flushing was drained through the opening. In ewes mated to intact rams, pregnancy was ascertained by the presence of embryonic tissue. Cellular debris was removed from the uterine lumenal flushing by centrifugation (2500 g; 20 minutes; 4 C). An aliquant of the supernatant was removed for estimation of total recoverable protein from the uterus. The supernatant was dialyzed (Spectrapor Membrane #3; 3500 MW cutoff; Spectrum Med. Ind. Inc.) against distilled H20 for 36 hours (sample: H20 was 1 1:100; H20 was changed at 12 hour intervals) and then lyophilized. The lumenal protein was then stored at -20 C until analysis, at which time the lyophilized protein was suspended in the appropriate buffer required for the technique utilized. Uterine lumenal fluid protein (200 pg) from individual ewes was subjected to polyacrylamide disc gel electrophoresis at pH 4.5 (Reisfeld et al., 1962; Appendix B). The amount of protein recovered from the uterine lumen and later subjected to electrophoresis was determined as described by Miller (1959; Appendix C). Lumenal protein (300 pg) was also subjected to sodium dodecyl sulfate (SDS) electro- phoresis (Weber and Osborn, 1969; Appendix D). 30 Experiment V Fifteen ewes mated to a vasectomized ram were sacrificed on days 3, 9 and 14 of an estrous cycle (five ewes/day). Five ewes mated to intact rams were sacrificed on day 14 of pregnancy. Their uteri were excised, flushed with .33 M NaCl and centrifuged as in the previous experiment. After lyophilization, uterine lumenal protein (500 pg) from each ewe was subjected to isoelectric focusing on polyacrylamide columns (Bio-Rad Lab., Tech. Bull. 1030, 1975; Appendix E). In pregnant ewes, embryonic tissue was aspirated from the flushing before centrifugation and homogenized. The homogenate was centrifuged (10,000 g; 30 minutes) and the supernatant was dialyzed, lyophilized and subjected to a radio-receptor assay for gonadotropic activity (Saxena et al., 1974; Appendix F). Experiment VI Twenty ewes were ovariectomized. At least 14 days elapsed between surgery and random assignment to one of the following groups (five ewes/treatment): (1) control; (2) subcutaneous estradiol implant (sealed polydimethylsiloxane tubing, 4.65 0.0. 3.34 1.0. X 55 mm, packed with crystalline l7B-estradiol); (3) pessary impregnated with progesterone (polyurethane foam, 3.4 dia. X 2.5 cm; 1 gm progesterone); or (4) both estradiol implant and progesterone pessary. Blood was collected immediately before treatment (day 0) and during treatment on days 3, 6 and 9 for determination of serum estradiol (Britt et al., 1974; Appendix G) and progesterone (Louis et al., 1973; Appendix A). After ten days of treatment, all ewes 31 were sacrificed and uterine lumenal fluid protein obtained and processed as previously described. Lumenal protein was subjected to isoelectric focusing. Experiment VII Fifteen ewes were mated to intact rams; then ten of these were given 125 pg estradiol (im) on days 11 and 12. Ten ewes were mated to a vasectomized ram and five of these ewes received 125 pg estradiol on days 11 and 12. On day 14, all ewes were sacrificed, their uteri excised and flushed with .33 M NaCl. Preg- nancy in the ewes mated to intact rams was ascertained by the presence of embryonic tissue. The flushings were centrifuged, dialyzed, lyophilized and subjected to isoelectric focusing. Significant differences in serum progesterone concentrations and total protein recovered from the uterine lumen were determined by t-test if two means had homogenous variances or approximate t-test if variances were heterogenous (Sokal and Rohlf, 1969). RESULTS Experiment I Serum progesterone concentrations in ewes given 250 ug estradiol per day on days 11 and 12 tended to decline more rapidly than in ewes treated on days 12 and 13; however, on day 14 serum progesterone did not differ between the two groups (Appendix H, Table 7; P >.05) and data were pooled (Figure 1). Injection of estradiol on days 11 and 12 or days 12 and 13 caused a decline in serum progesterone to .4-:.1 ng/ml on day 14 of an estrous cycle (Figure 1), lower (P <.01) than non-injected controls (1.92:.6 ng/ml). Nine of 11 ewes given estradiol had;g.4 ng/ml progesterone on day 14.1 However, estradiol treatment caused serum progesterone to decrease in only six of 12 ewes mated to intact rams (day 14, .8 t.2 vs 2.7 t.7 ng/ml; Figure 2 and Appendix H, Table 8). The six ewes with elevated progesterone Q:2.0 ng/ml) on day 14 after mating were assumed to be pregnant, but this was not confirmed. After day 14, serum progesterone in estrogen treated ewes which were assumed to be pregnant was higher (P‘<.01) than in ewes mated to a vasectomized ram and treated with estrogen (2.7 i.7 vs .4:t.l ng/ml, respectively). Progesterone concentrations in ewes assumed to be non-pregnant were not different from those in estradiol injected ewes mated to a vasectomized ram. 32 33 NON" INJECTED /CONTROLS ESTRADIOL l c: c; <3 :2 ID Q' '0 N (pp/bu) aumatsabmd tumas m _ INJECTED I2 13 I4 15 16 I7 Days After Mating to Vasectomized Rom ted control ewes mated to a 1n non-injec on days 11 and 12 or days 12 and 13 and Figure l.-Serum progesterone in ewes mated to a vasectomized ram and given 250 pg l7B-estradiol vasectomized ram (Experiment I). (iClb 34 PROGESTERONE g 0.0 o _ 0.0.. .1 E o 0.... o z :-:-: g 4 .0.0.0. .o:o.o 2 0.0.0. o o o. I (NJ/5U) euoaatsaboad umaas LCDF B:>7 PROGESTERONE ::i3;7 13 l4. l5» IS» 17 18 Days After Mating to Intact Rams l2 Figure 2.-Serum progesterone in ewes mated to intact rams and given 250 pg l7B-estradiol on days 11 and 12 or days 12 and 13 (Experiment I). 35 Experiment 11 0f 16 ewes mated to intact rams and given either 125 or 250 pg estradiol per day on days 11 and 12, four of eight and one of eight, respectively, were pregnant on day 16. Six embryos were recovered from the five pregnant ewes; one ewe had two normal embryos. Two of the six recovered embryos were retarded in develop- ment. Pregnant ewes has higher (P <.05) serum progesterone on day 15 than non-pregnant ewes (1.6:t.8 vs .3:i.1 ng/ml; Figure 3). In the two ewes with retarded embryos, serum progesterone averaged .2 ng/ml on day 15. Serum progesterone concentrations in non- pregnant ewes did not differ from values in estrogen treated ewes mated to a vasectomized ram (Appendix H, Table 9; p>»,os), Concomitant with this experiment, 18 other ewes from this flock were mated to the same intact rams and all were confirmed pregnant prior to day 16, attesting to the fertility of the flock. Experiment III Five of 17 ewes mated to intact rams and given 125 pg estradiol on days 11 and 12 were pregnant on day 20. Pregnant ewes had higher (P <.01) serum progesterone on day 14 than non-pregnant ewes (4.12:.4 vs 1.4 i.3 ng/ml; Figure 4 and Appendix H, Table 10). Estradiol injectiOns on days 11 and 12 into ewes previously mated to a vasectomized ram caused serum progesterone to decline to 1.9 i .4 ng/ml by day 13, lower than in non-injected controls (3.2:t.5 ng/ml; P‘<.05; Figure 5 and Appendix H, Table 10). On day 14, four of five ewes given estradiol had 1.4 ng/ml of serum progesterone. Serum progesterone concentrations in estradiol injected ewes that 36 /PREGNANT NON'PREGNANT l‘ I ‘ - - I C 0 O .0 IO- (pu/DU) euomtsaboad wnaas I2 I3 I4 I5 16 Days After Mating to Intact Rams 10 Figure 3.-Serum progesterone in ewes mated to intact rams and given 125 or 250 pg l7B-estradiol on days 11 and 12 after mating (Experiment 11). 37 .AHHH acmewgmaxmv mcwpme Coven up new PP when co Powumgpmwrmxp a: mu? cm>wm new mac; «cape? on nouns mmzm cw mcogmamwmocn Esgmmun.¢ mcamwd «sum 88:. ch 9522 3:4 goo cu m. m. c. u. o. ......u.\..\.\.§\\ sis pzqzommaIzoz . S >< é? ..nv . $$§ omhumqi. m \ . n hz_m vcm Em; umeEopummm> m o» nouns mmzm cw mcogmummmosa Eaemmuu.m mesmwu Eom 326203.54 ob 05.22 .624 .380 cu m. o. S N. o. 3532.6; 38 ON / mpompzoo oupou..z...zoz\\ x. (gut/bu) euOJetsebmd dimes 39 were pregnant at autopsy on day 20 were not different from levels in pregnant ewes which had not received estradiol (Figure 6). Fourteen ewes mated to the intact rams used in this experi- ment were all confirmed pregnant on or prior to day 20; these pregnant ewes were either controls in this experiment or involved in a concomitant experiment. Experiment IV There was no difference (P>».05) in amount of uterine lumenal protein recovered on each day between days 12 and 15 of an estrous cycle (Table l). the amount of lumenal protein recovered (P:>.05). Furthermore, pregnancy had no effect on Table l.--Uterine lumenal fluid protein recovered from ewes during an estrous cycle or early pregnancy (Experiment IV). ‘Day Estrous Cycle Pregnancy Total Protein (mg) 12 2.90:.673 (3)b 3.64: .77 (4) 13 3.06:.75 (3) 4.29: .64 (3) l4 3.36:.46 (5) 4.76: .99 (4) 15 2.85 (2) 5.891:2.18 (3) aMean: S.E. bNumber of Observations Beginning on day 14 of pregnancy (14P), three protein bands were detected in uterine lumenal protein that were not detected in 4O .AHHH acmewcmnxmv mcwume emuwm up van FF mzmu co powumgpmmimmp m: mm— 20 o cmcupm cm>wm new man; pumucw op copes mwzm cw mcogmpmmmosa Eagwmru.m mcammd mEom 82:. o... 9522 3:4 goo . ON 2 o. w. m. o. 1 owhowaz. Jo.o<¢._.mm 1 o. N \\\\ \ .\s\\ . I" .x\;,,\ \S $3... \\. E»: SE 3 . x. .s \ «xx. \sst‘ \.. sss.\ ss . \Vs.\:s\\s. \\\,s\‘ .\. \\s.\ x. o W\\ .Rxskkk. , x v arms 2. l 5 C) :6 (Nil bu) euomtsebmd mmes mJOmhzoo awhomaz. I202 41 serum (Figure 7; 5). One of these protein bands (top arrow) migrated slower than albumen (A) and the other two bands (middle and bottom arrows), both of low intensity, migrated faster than albumen. 0f the two bands which migrated faster than albumen, the protein band of lesser mobility was not present in lumenal fluid collected on day 14 of an estrous cycle (14C). On day 15 after estrus, a similar difference was detected between pregnant and non-pregnant ewes. After SOS electrophoresis of uterine lumenal fluid protein collected between days 12 and 15 of an estrous cycle, the only con- sistent and progressive change in protein profiles was the decrease in stain intensity of a protein below 10,000 MW (Figure 8; 12C, arrow). Lower intensity in that region was observed on days 14 (14C) and 15 (15C) as compared with days 12 (12C) and 13 (13C) of an estrous cycle. Although intensities of several other protein bands varied throughout the period, the differences were related to indi- vidual animals rather than day of the cycle. Comparison of protein profiles obtained from $05 electro- phoresis of uterine fluid from pregnant and non-pregnant ewes also emphasized the small molecular weight (‘<10,000 MW) protein bands (Figure 8). On day 12, a protein in that region of the gel was more intensely stained during an estrous cycle (120; arrow) than during pregnancy (12P). Although band intensity did not differ on day 13, differences were again detected in that region on days 14 and 15; on those days, band intensity was greater in profiles of uterine fluid from pregnant ewes (14P and 15P; arrows). 42 .A>H “cmepcmaxmv xucmcmmca new m_uxu maocumm cm 40 . . «P zen co mpum ou :wmpoca uwapw chmszp mcwcmp: mo m.¢ In pm Fem mqumP>cum>Foa cw mwmmcowaogpwwrmru.m mean”; 43 1". UV. 42 .A>H ucmsPLmaxmv xucmcmmca use m—oxu maocwmm cm . mo ep xmu co ovum oo cwmuoca nvzpw chmEJF mcwgmp: we m.¢ In pm Pom mvwEszLomx—oa cw mwmmeomaocuww—MII.N mezmpm |4P |4C 44 .A>H pcmewcmaxmv xucmcmmca new mpuzu mzogpmm cm we m_ use «P .mp .N_ mxmu co vapour—cu :wmpoca cwapw Pmcmss_ mcwcmp: mo mwmmgocgocpumpm mom--.m mc:m_d am. On. I _. av.. 0... an. 2'! ‘ On. an. 'A _..__;_‘t —4e fl 0N. 44 .A>H acmewgmaxmv xocwcmmca ucm mpuxo mzogpmm cm Co mp can ep .m— .N_ mxmu :o umpumrpou Cwmuogq vwspm chm53F mcwgmuz Co mmmwcogaocuuwpm mom--.m mczmwd 45 an. ‘3‘: . ‘ II 00. I ‘al'. 1...... AN. ON. 46 The molecular weights of proteins in lumenal fluid ranged from approximately 66,000 to 9,500 (Figure 9). As detected by spectrophotometric scanning, each of the 13 bands of protein observed on day 14 of pregnancy migrated similarly to a comparable protein band detected on day 14 of an estrous cycle. There were few excep- tions to this and those that occurred were related to individual ewes rather than reproductive status. Experiment V On day 3 of an estrous cycle, 1.84 t.21 mg total protein was recovered from the uterine lumen (Table 2). Recoverable uterine protein increased to 4.97 t1.20 mg on day 9 (P‘<.05) and remained at that level (day 14, 5.11:1.60 mg). Fourteen days of pregnancy did not increase (P=>.05) the total amount of uterine protein recov- ered (6.6li:.76 mg) as compared with day 14 of an estrous cycle. During the course of isoelectric focusing of uterine lumenal proteins, it was necessary to replenish certain reagents. This change in reagents affected the pH gradient formed in the polyacrylamide gel (Table 3). The pH gradients used for comparison in the present study were averages of all analyses completed with a particular set of reagents. . After isoelectric focusing of uterine protein collected on day 14 (14C) of an estrous cycle, up to 35 bands of protein were detected by Spectrophotometric scanning of the gels (Appendix I, Figure 14) and all but two bands (pI 7.2 to 7.6; arrow) of low intensity were also observed in serum (Figure 10; S; the left pH gradient was representative of gels 3C and 14C, the right pH 47 " ‘ "a w'"" i ‘ "'; ' I a ‘I‘ ‘— —————-—--——--—--—J>-———.—»-— ___ _. a -7 — .,,___-. I»——---— --—.. —+ ——-— —— ——. v—f————— I I i I I Em”, ___._- ..-SLI5 flaurnnhnreeLsT‘llox -1'1 --:-*'~-~ ;. Lmfihami--- ..-__.. .. I l ' _,_;.... ' xx & =,¢- Stan umrd Proteins I . . A.-.-5W._.____- air — __, - f"~*——i ”" “Ir-=13“ ff firignflfl“ I é¢ i I ‘ ' I---W p-..__...__.- .I n.-..___-i.. _-..__._-_I___ -_.-.___._.ch._ -9..y.-_74..-. "cgc'. --. E Q I I I .2 ‘ i I r o q‘ ' I s W: WWW“ “Mae-*- I 7'" - ~ “7*“ WWW L‘ .‘o I I a I . a . G x .5 I O 107900w _._.__._-. -~- -i- — -2 9- "I- 4% -4 - ~— 2 . I , : I I . I i ' o I X I O o f I . x I~1munww--fle- I"““"F”“”‘T”"'””T ~-~4-- : a ()4 sin ()8 a ‘ F’Tfi" I ._ i #flllllg I I Figure 9.--Determination of molecular weight by SDS electrophoresis of uterine lumenal fluid protein collected on da 14 of an estrous cycle and pregnancy (Experiment IV 48 Table 2.--Uterine lumenal fluid recovered from ewes during an estrous cycle or early pregnancy (Experiment V). Total Protein -mg- Day of Cycle 3 1.84 t .21“:1 (5)b 9 ' 4.97 DELZOC (3) 14 5.10 il.60c (5) Day of Pregnancy 14 6.6l : .76 (5) alvlean : S.E. bNumber of Observations cSignificantly different (P<:.05) from levels recovered on day 3 of an estrous cycle. Table 3.—-Gradients of pH formed during isoelectric focusing in polyacrylamide gel. Reagent Set #l Reagent Set #2 Centimeter of gel length pH Units pH Units l 3.82 : .063 4.08 .+. .06b 2 4.32 t.ll 4.69 $.03 3 4.87i:.21 5.47 1.‘05 4 5.62 t.05 6.30 i.ll 5 6.l5i .03 6.9l i.12 6 7.26i=.06 8.09 i.l4 7 8.551“ .07 8.55 $.04 8 9.351=.lo 8.84 $.06 aMean S.E., n = 3 bn = 5 49 .A> pcmewemaxmv mpuxu mzogumm cm mo «P can a .m when so umpumrpou c»muoca n»:_w chmE:_ mcwcmp: do Pom mu»Empxgum»_oa c» mcwmauow uwgpumeomH--.OF mesa»; 50 0 o v v n o I. n a o I I l 90 I... I..- IL 9" 3. 3 -43 56 62 -13 -I.6 -94 3C 5] gradient was representative of gels 9C and S). The number of protein bands in uterine fluid collected on days 3 (3C) and 9 (9C) of an estrous cycle did not differ from day 14 with the exception of the two basic bands (pI 7.2 to 7.6). Band intensity of proteins focused at pH 6.3 to 7.3 was less on day 9 than on day 3. On day 14, protein bands.in that region were of the intensity observed on day 3 of an estrous cycle. Isoelectric focusing of uterine lumenal protein collected on day l4 of pregnancy produced two distinct profiles (Figure ll and Appendix I, Figure 14). 0f the five pregnant ewes, gel 2 was repre- sentative of two animals and gel 3 was representative of the other three. All five of these ewes had an embryo of normal size flushed from each of their respective uteri. Stain intensity of bands focused at pH 5.4 to 7.0 was greater in gel 3 than gel 2. In profiles repre- sented by gel 3, one extra protein band was detected (middle arrow; pI 6.9) which was not present in gel 2. A protein focused at pH 6.4 (top arrow) was detected in both gels, but that area was more heavily stained in gel 3 than gel 2. The two uterine-specific protein bands (pl 7.2 to 7.6) detected on day 14 of an estrous cycle were also observed on day 14 of pregnancy in profiles represented by both gels 2 and 3 (bottom arrow), although the bands were much fainter in gel 2. Uterine lumenal proteins collected on day 14 of an estrous cycle (gel l) differed from those collected on day l4 of pregnancy in stain intensity of proteins focused between pH 5.4 to 7.0. It was not dis- cernible whether the extra band detected in gel 3 (Figure ll; middle arrow) was present on day 14 of an estrous cycle due to lack of 52 .A> acmewcmaxmv aucmcmmca ucm wpuxo msocumw cm mo ep xmu co cmpum__ou :wmpoca uw:_w Foam53— mewcmu: mo pmm mnwsmfixcumzpoa cw mcwmaoom uvcpum_momH--.P_ mczmwu - ‘0’ -‘o, 53 -7.3 ”MP W (7"? V '49. . of C. V \‘T P . ’ '1') fl . S U V - Q 54 resolution of proteins in that region. If that difference was dis- regarded, uterine lumenal fluid from pregnant and non-pregnant ewes contained an equal number of detectable proteins. Uterine lumenal fluid proteins had little gonadotropic activity when assayed with a radioreceptor assay system. 0f the uterine protein samples collected on day l4 from five pregnant ewes, gonadotropic activity was undetectable in three and the other two had activity equivalent to 2.5 and .5 ng HCG/mg protein (.0116 I.U. HCG/ng HCG). The homogenates of three of the five embryos collected on day l4 exhibited activity equivalent to .4, 4.1 and 7.l ng HCG per embryo (3.91:1.9 ng HCG/embryo). The other two embryo homogenates were lost during processing. Experiment VI In ovariectomized ewes, a subcutaneous estradiol implant increased serum estradiol from 4.32:.6 to ll.0:t.9 pg/ml (P<=.0l) within three days and maintained increased estradiol throughout the treatment period (Table 4). Serum estradiol after hormone replace- ment was approximately 75 percent of values observed on the day of estrus (Yuthasastrakosol et al., 1975). A pessary impregnated with progesterone increased (P<:.Ol) serum progesterone from .4i:.05 to 2.21:.2 ng/ml (days 0 and 3, respectively). Serum progesterone was maintained above control values for the duration of the treat— ment. Concentrations of serum progesterone achieved were comparable to those expected during the luteal phase of an estrous cycle (Figure 5; non-injected controls). Similar increases over control values were observed for each hormone in the group of ewes given both 55 mmZm Laow u : u .m.chmmzn mmZm m>wm u an .-. .-. .-. .-. .-. .-. .-. .-. 28388: m + F N a +.o N a + m P 00 +.m m _+ o w m _+ m m m _+.w w o .+o a oucm Powumcpmm N.H o._ m.Hnw.P ~.H N.N mo.H e. o. H ~.m o. H m.¢ N. H m.¢ n.HHm.¢ mmcoemummmoca Po.H a. Fo.H m. mo.“ m. mo.H m. n. H m.m n. H _.m m. H o._F o. Hm.¢ mFowummem mo.H.¢. Po.“ a. mo.H m. Fo.H m. n. H ~.¢ N. H ¢.¢ o. H n.¢ na.an.m apocpcoo m o m o m m m o xmo zoo “cmEpmmch A~E\mcv mcogmummmoga Ape\mav powumgumm Lo\u:m mu:m_aew Fowumgpmm ;H_3 .AH> Hamswgmaxmv mmwgmmmma mcogmummmoga mmzm umNHEoHomHLm>o cm mcocmummmoga can Fownmgamm Escmm--.¢ mpnmp 56 estradiol implant and progesterone pessary (Table 4). One ewe in the estradiol-progesterone treatment group was excluded from the experiment because of expulsion of a pessary. In ovariectomized ewes, replacement of progesterone and estradiol increased (P<=.05) total recoverable uterine lumenal protein from l.20:t.25 to 3.481:l.20 and 4.98::2.37 mg, respectively (Table 5). A combination of the two hormones produced neither an additive or synergistic protein increase (6.0212l.62 mg). One ewe in the estradiol treatment group produced 14 mg of lumenal protein, nearly six-fold more than the other four ewes in the group (2.62::.36 mg). After isoelectric focusing, uterine lumenal protein from control ovariectomized ewes (C) focused to a greater extent in the region less than pH 4.7 when compared to serum (Figure l2; 5). After ten days of estradiol replacement (E), the profile of proteins in uterine lumenal fluid did not differ from profiles in control ewes. Progesterone replacement (P) decreased the proportion of proteins focused at less than pH 4.7. Of the 21 protein bands detected after isoelectric focusing of lumenal protein collected after progesterone treatment, all but one band of low intensity (pI 7.9) were present in serum (S). Upon replacement of both progesterone and estradiol (PE), the majority of proteins in lumenal fluid focused between pH 4.7 and 5.8. Experiment VII The amount of uterine lumenal fluid protein recovered on day l4 was not affected (P>'.05) by administration of 125 ug estradiol per day on days ll and 12 in ewes mated to a vasectomized ram (Table 6). 57 Table 5.--Uterine lumenal fluid protein recovered from ovariectomized ewes with estradiol implants and/or progesterone pessaries (Experiment VI). Total Protein Treatment - mg _ Control 1.20 i .26a (5)b Progesterone . 3.48 11.20C (5) Estradiol 4.98 12.37c (5) (2.62 i .36) (4) Progesterone and Estradiol 6.02 «21.62c (4) aMeaniS.E. bNumber of observations cSignificantly different (P<:.05) from levels in control ewes. Table 6.--Uterine lumenal fluid protein recovered on day 14 from ewes mated to intact or vasectomized rams and given either 0 or 125 ug l7B-estradiol on days ll and 12 after mating (Experiment VII). ' Total Protein Treatment _ mg _ Mated to Vasectomized Ram Control 5.102tl.60a (5)b Estradiol 4.32:: .31 (5) Mated to Intact Rams Control 6.61:: .76 (5) Estradiol (conceptus present) 5.84:: .50 (7) Estradiol (no conceptus) 4.19:: .56c (3) a‘Meani S.E. bNumber of observations cSignificantly different (P <.05) than values recovered from control ewes mated to intact rams. 58 .AH> pcmewcqumv mcocmummmocq L0\u:m Fowumcpmm mo acmEmumPch cmpmm mmZm umNHEouumwcm>o Eocw vaomFFou cmmHoca uwspm Foamszp mcwcmpa mo .mm mqumrxgomxpoa cw mcwmauow uHLHoumOWH--.NF mesmwu 59 4 —47 -55 -6.3 -69 I -86 -II 60 Similar lack of response was observed in estradiol treated ewes mated to intact rams provided a conceptus was present on day 14. Less uterine protein (P <.05) was recovered in the absence of an embryo compared with control ewes mated to intact rams (4.19 t.56 vs 6.612i.76 mg). After estradiol administration to ewes mated to a vasectomized ram, two distinctly different protein profiles were obtained from isoelectric focusing of lumenal fluid protein collected on day 14 (Figure 13; gel 1, CE and gel 2, CE). Gel 2 (representative of three ewes) differed from gel 1 (representative of two ewes) by increased band intensity of proteins focused at less than pH 4.7 and decreased intensity of bands focused between pH 5.2 and 6.9. The two uterine-specific protein bands (pI 7.2 to 7.6) detected on day 14 of an estrous cycle (gel 3, C) were not observed after estradiol administration. The presence of a conceptus was associated with the fact that profiles of uterine lumenal proteins collected from ewes mated to intact rams and given estradiol were divided into two distinct types. If the conceptus was present on day 14, the protein profile (gel 5, PE) was similar to one of the two representative profiles obtained on day 14 of pregnancy (gel 7, P) with the exception that proteins focused between pH 5.4 and 6.3 were in higher proportion in the estrogen treated ewes. The absence of a conceptus (gel 4, PE-nc) resulted in profiles similar to uterine protein from ewes mated to a vasectomized ram and given estradiol (gel 1, CE). (In Figure 13, the pH gradient presented was not representative of gels 3. 6 and 7, consult Figure 11 for proper pH gradient.) 61 .AHH> pcmewcmaxmv mcwume cmpwm my van PF mxmu co Fowumcpmm-mmp on mm, cm>wm ucm mam; umNHEOHummm> Lo pumH:_ op umpme mmzm Eogw «F xmu co umgumppou :Hmpoca trap» Pmcm53F mcwcmus we Pmm mvHEmpacumxpoa cw mcwmsu0$ uwcpumFmOHH--.m_ mesmwu 62 0.0I 9.0 I h.¢l —.'I In «a "'1 E v ma tr >2.0 ng/ml serum progesterone on day 14 after mating. bData were pooled from ewes treated on days 11 and 12 or 12 and 13. CMean: S.E. dSignificantly different (P<:.01) from ewes which progesterone levels declined on respective days. 107 Table 9.-—Serum progesterone (ng/ml) in ewes mated to intact or vasectomized rams and given 125 or 250 pg 17B-estradiol on days 11 and 12 after mating (Experiment 11).; 1122:: 19:23:51.? 11:25:.23222. .. 93330424 Mating (n=5) (n=ll) 1 10 2.4: .5” 2.6:.2 2.4:.5 11 3.1 : .4 2.5:.4 2.8:.5 12 2.8 i .6 2.4::.2 2.6:t.5 13 3.3 :1.oC 1.6:.1 1.7:.5 14 1.6: .4c .8:.2 1.1:.4 15 1.6: .7C .3:.1 ,3:,1 16 .9: .4d .2:.01 .2:.05 aData were pooled from ewes given 125 or 250 pg 17B-estradiol. bMean:S.E. CSignificantly different (P <.01) from levels in non-pregnant ewes on respective days. dSignificantly different (P<:.05) from levels in non-pregnant ewes on respective days. 108 .35 umuumnfiuco: .2833 5 29,2 EEC Co. VA: 228:? bucmuwtcmwmu .m.m Hcmmzo .mamu m>wpumammc co so; um~_souummm> m on vmume mmzm umpumncwuco: cw mpm>mp soc» Amo.u.av ucmcmwmwu xpucmuwmwcmwmn N. Hm. F. H . up. H m. H¢.m m. Ho.m ON F.H¢. P.H . uF.H N.—H9é v. HEN or F. HN. mo. H . uS. H m. H Tm m. H m.m 9 P. HN. mo. H . uS. H m. H~.m o. HNé 2 mo. HN. P. H . UN. Hm. m. HWm P. Hmé 3 nmo. HN. m. Hm; um. H m. H56 N. H «6 mp nN. Hm.o m. HmN om. He; 3. H Te N. H Te 3 ac. Hm; m. HN.m e. Hm.N m. Hm.m e. H m.m 9 m. Hm.N m. HN.m a. Hm.m m. Ho.m e. H m.m NF ¢.Hm.m N.HN.N m.H_.¢ m. Hmé m. H73 2 m. HWN m. HNN N. Hm.m m. H mé mm. H eé o— HMMWMWCH Amucv mewmuwmm _OMWMHWHH umHuwwmwwcoz mcwumz Powumcpmm cmpowncHucoz Hamcmmcaucoz pcmcmmpm pcmcmmea mem< 5mm umeEouummm> op topaz mama Humucm op umumz .AHHH acmewcmaxmv mcwpme LmHNH NF vcm Pp what :0 Powvmcpmmumup a: mNP go o cmcuwm cm>wm new man; vaHEouommm> :0 pumps? op umgms mwzm cw A~E\mcv mcogmummmoga Ezcwmuu.o~ myam» APPENDIX I REPRESENTATIVE SPECTROPHOTOMETRIC SCANS 0F POLYACRYLAMIDE GELS AFTER ISOELECTRIC FOCUSING OF UTERINE LUMENAL FLUID PROTEIN 109 110 S 146 W 3.6 4.3 4.9 5.6 6.2 7.3 6.6 9.4 3.6 4.3 4.9 5.6 6.2 7.3 6.6 9.4 14p j— 3.6 4.3 4.9 3.6 6.2 7.3 6.6 9.4 pH Units pH Units Lg 6.6 9.4 3.6 4.3 4.9 6.6 6.2 7.3 Figure l4.--Spectrophotometric scans of polyacrylamide gels after isoelectric focusing of uterine lumenal fluid protein collected on day 14 of an estrous cycle and pregnancy (Experiment V). LITERATURE CITED 111 LITERATURE CITED Akbar, A.M.; Rowe, K.E.; and Stormshak, F. 1971. 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Sci. 33:249. (Abstr.). 1975. Prevention of estrogen-induced regression of corpora lutea in ewes by hysterectomy. J. Anim. Sci. 40:687. Bolt, D.J.; Kelley, H.E.; and Hawk, H.H. 1971. Influence of gonadotropic hormones on estradiol-induced corpus luteum regression in the ewe. J. Anim. Sci. 32:977. Boshier, D.P. 1969. A histological and histochemical examination of implantation and early placentome formation in sheep. J. Reprod. Fert. 19:51. Britt, J.H.; Kittok, R.K.; and Harrison, 0.5. 1974. Ovulation, estrus and endocrine response after GnRH in early postpartum cows. J. Anim. Sci. 39:915. Bullock, D.H. and Connell, K.M. 1973. Occurrence and molecular weight of rabbit uterine "blastokinin." Biol. Reprod. 9:125. Bullock, 0.w. and Willen,(3.F. 1974. Regulation of a specific uterine protein by estrogen and progesterone in ovariectomized rabbits. Proc. Soc. Exp. Biol. Med. 146:294. 114 Caldwell, B.V., and Moor, R.M. 1971. 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