Effect of Embryos on Luteolysis and Termination of Early Pregnancy in Sheep with Cloprostenol

BOLOGY OF REPRODUTON 26, 26-269 (1982) Effect of Embryos on Luteolysis and Termination of Early Pregnancy in Sheep with loprostenol. D. NANARROW,1 B. M. EVSON and P. J. ONNELL.S..R.O. Division ofanimal Production Black town, New South Wales, Australia ABSTRAT An investigation of the control of the function of the corpus luteum of early pregnancy in sheep has been carried out by assessing the luteolytic effects of a dose of 1 of the synthetic prostaglandin, cloprostenol. loprostenol injected into 21-day pregnant sheep carrying single or multiple embryos caused luteolysis and termination of pregnancy within 2-5 days in 8 of 2 sheep. The mean numbers of corpora lutea in these ewes differed significantly from those which carried their pregnancy (1.8 ±., SEM, vs.2 ±.4; P<O.5). t appeared that the greater number of embryos, or a greater mass of embryonic tissue, afforded increased protection to the corpora lutes through unidentified antiluteolytic or luteotrophic factors. The number of corpora lutea present did not appear to be an operative factor, as the same dose of cloprostenol was luteolytic in 4 of 46 nonpregnant ewes with 1-6 corpora lutea; the refractive ewes each having only 1 corpus luteum. The progesterone concentration in the plasma of pregnant ewes which underwent luteolysis following cloprostenol treatment was.5 ±.1 ng/ml 48 h after injection, while that of the ewes remaining pregnant was significantly higher at 2.1 ±. ng/ml. Seven of these refractive ewes which were given a second injection of cloprostenol on Day 28 of pregnancy subsequently experienced luteolysis and abortion. The corpora lutea of the remaining 8 ewes were not affected by saline injections given on Day 28, but 4 of these ewes later aborted after Day 44. t is concluded that pregnancies in ewes carrying 1 embryo can be terminated by a single dose of 1 zg cloprostenol given around Day 21 of pregnancy, but 2 injections given about 7 days apart may be needed when multiple embryos are present. Hormonal control of the sensitivity of the corpus luteum to luteolytic prostaglandins appears to reside in the conceptus in such a way that a decrease in sensitivity is related to the number of embryos present. NTRODUTON Pregnancy is maintained in sheep during the first 55 days by the action of progesterone produced by the corpus luteum (asida and Warwick, 1945; Foote et al., 1957). Thereafter the placenta synthesizes sufficient progesterone to maintain the pregnancy. loprostenol, and other prostaglandin F2 analogues, will cause luteolysis in nonpregnant sheep (Acritopoulou et al., 1977; Hearnshaw et al., 1974) and by similar mechanisms should cause absorption or abortion of embryos in sheep during early pregnancy (up to 55 days). Preliminary observations at this laboratory showed Accepted November 2, 1981. Received April 27, 1981. orrespondence: Dr.. D. Nancarrow,.S..R.O., Division of Animal Production, P.. Box 29, Blacktown, N.S.W., 2148, Australia. that while some pregnancies of 19-25 days duration could be terminated with cloprostenol, luteolysis and abortion did not invariably occur if multiple embryos were present. Luteolysis induced by the release of prostaglandin F2 around the 1th to 16th days after estrus (Thorburn et al., 197; ox et al., 1972) does not occur in the presence of embryos or embryonic components (Moor and Rowson, 1966; Rowson and Moor, 1967). This effect may act through a decrease in the synthesis and release of prostaglandin F2a or an increase in the resistance of the corpus luteum to the luteolytic effects of prostaglandin. Recently, it has been shown that the conceptus affords a degree of protection to the corpus luteum against exogenous prostaglandin F2 (Pratt et al., 1977). f the occasional failure of cloprostenol to induce abortion is due to a similar protection of the corpus luteum by products of the conceptus, then the efficacy of this antiluteolytic mechanism may be related to 26

264 NANAR ROW ET AL. the total mass of embryonic tissue or the number of embryos present. Thus, we have investigated the sensitivity of the ovine corpus luteum to cloprostenol administered during the first trimester of pregnancy by relating it to the number of corpora lutea present. The data so obtained will aid our investigations of the factors which control the maintenance of early pregnancy in sheep and of the causes and extent of early embryonic mortality in livestock. MATERALS AND METHODS Experiment 1. Nonpregnant Ewes Fifty-three cyclic, nonsynchronized, merino ewes, 4-5 years old, were identified and used toward the end of their breeding season (July-August, 1978). The animals were prepared by giving 1 of 4 i.m. treatments with pregnant mare s serum gonadotropin (PMSG) (Folligon, ntervet (Australia) Pry. Ltd.) during the luteal phase of their estrous cycles in order to obtain a range of individual ovulation rates. These treatments were: 1).5 ml.9% saline (11 ewes); 2)1 t) PMSG in.5 ml water (15 ewes); ) 4 t) PMSG in.5 ml water (1 ewes); or 4) 7 U PMSG in 1. ml water (14 ewes), and were followed 24 h later by an i.m. injection of a luteolytic dose of 1 g cloprostenol (, Australia Ltd.) in.5 ml of.9% saline. The ewes were not individually examined following this preparatory treatment but daily records of markings made by vasectomized rams wearing raddle harnesses were taken. Five ml blood samples were taken into heparinized syringes from all ewes at 8, 6 and 4 days before and, 2, and 9 days after the cloprostenol injection. Plasma was immediately separated by centrifugation and stored at -2#{176} until assayed for progesterone to confirm those animals which had responded to the prostaglandin treatment. The ewes were laparotomized under sodium thiopentone anesthesia 9 days after cloprostenol treatment to determine the number and site of the corpora lutea produced in response to the PMSG treatment. Four days later (Days 1-11 of the induced estrous cycle) the luteolytic response to a second injection of 1 g cloprostenol was tested. Blood samples were taken at, 1, 2,, 4 and 7 days after this treatment to determine the changes in plasma progesterone concentrations. On this last day, the ewes were killed and the ovaries were reexamined for confirmation of luteolysis. Experiment 2. Pregnant Ewes Thirty-three cyclic merino ewes were given progestogen for 1 days via intravaginal sponges (Repromap, Upjohn Pty. Ltd.) during September, 1978. On the day of sponge removal, either 2 (Group 1, 16 ewes) or 7 (Group 2, 17 ewes) lu PMSG were injected i.m. and intact raddled rams were placed in the flock for 4 days. All fertile matings occurred over the latter 2 days, designated as Days and 1, and further experimental details and responses relate to these days for convenience. Then the rams were replaced with vasectomized rams and any returns to estrus by the ewes were noted. On Day 21, all ewes mated on Days and 1 were injected i.m. with 1 g cloprostenol, including 6 that had returned to service, and observed for induced estrus. All ewes were laparotomized on Days 2 or 26 to determine the response of the corpora lutea to cloprostenol, the number of corpora lutes which had been present during pregnancy and which ewes remained pregnant (fluid-filled uterine horns). The number of embryos could not be determined because either they had been resorbed or aborted or, if the ewes were still pregnant, further treatment was to be given as indicated below. Blood samples for progesterone assays were collected into 1 ml heparinized syringes by jugular venipuncture on Days 14, 16 and 19 for confirmation of pregnancy or cyclicity, and on Days 21, 22, 2, 26, 28 and 29 to estimate the extent of luteolysis and to monitor subsequent events. Prior to being assayed these samples were treated as previously described. A total of 15 ewes remained pregnant following the first injection of cloprostenol. Possible changes in the sensitivity of the corpora lutea of these were assessed by giving 7 of these a second injection of 1 g on Day 28 while the remainder received a control treatment of.5 ml saline i.m. All 15 animals were bled on Days 28, 29. and, but those given saline continued to be bled at intervals up to Day 44 (Fig. 1). Vasectomized rams bearing raddles were used to detect estrous ewes. The ewes were again laparotomized on Day 6 to determine the continuence of pregnancy. A second injection of cloprostenol was administered on Day 28 to the 6 mated animals which returned to estrus (Days 16-19, Fig. ic) and to 2 previously pregnant animals which showed estrus after the first treatment on Day 21 (Fig. is). These ewes acted as an additional control to test the efficacy of cloprostenol action in the nonpregnant sheep. Rodioimmunoassay of Progesterone The method of assay described by Thorneycroft and Stone (1972) was carried out on single.1 ml aliquots of plasma diluted with.5 ml distilled water before extraction. Antiserum #61 (larke, 1976) was raised in sheep against progesterone-i lo-hemisuccinate-bovine serum albumin and used at a final dilution of 1/18. This antiserum displayed >1% cross-reaction; defined as amount of progesterone yielding 5% inhibition of bound ligand divided by amount of test steroid also yielding 5% inhibition, expressed as a percentage; with the following steroids: 5-dihydropregnane-,2-dione (5.1%), 1-hydroxy- 5-pregnane-2-one (6.6%), and the derivatives of progesterone hydroxylated at ila-(4.6%), 11-(6.6%), 1 7oc-(7.1%), 21-(21.2%) and 1 1f,21-(9.7%). Pregnancy plasma obtained from 2 ewes diluted out from.1 ml was in parallel with the standard curve. The sensitivity of this assay, defined as the zero standard minus 2 standard deviations of the zero standard (Scaramuzzi et al., 198), varied from 1-4 pg/tube. The intraand inter-assay coefficients of variation (standard deviation divided by the mean, multiplied by 1%) were <14% and <18%, respectively. Recoveries of

EFFET OF MULTPLE EMBRYOS ON LUTEOLYSS N SHEEP 265 internal standards were 85-15% over the range 2-512 pg. Experiment 2. Pregnant Ewes RESULTS Experiment 1. Nonpregnant Ewes Forty-six of the 5 ewes were detected in estrus following the preparatory PMSG-cloprostenol treatment and were available for the experiment. Direct examination of the ovaries and the pattern of plasma progesterone concentrations obtained during this treatment period (data not shown) confirmed the response and indicated that luteolysis and reovulation had occurred in these animals. The groups were then reformed in relation to the number of corpora lutea present at the first laparotomy (Table ). Following the second cloprostenol injection, ewes failed to undergo luteolysis, while an estrous response was detected in 7 of the 46 ewes. The mean progesterone concentrations (± SEM) of plasma taken at the time of the second cloprostenol treatment and 48 h later are shown for each group of responding ewes (Table 1). The equivalent values for the ewes which underwent partial luteolysis were 4. ±.8 ng/ml and 1.7 ±.4 ng/ml respectively. These ewes each had only 1 corpus luteum at the time of treatment. There was a trend for the mean progesterone concentration to increase with the number of corpora lutea present, but as the second doprostenol treatment was not given on the same day of the cycle (Days 1 or 11) strict comparisons were not made. Twenty-nine of the ewes were mated by the fertile rams over a 2-day period, 1 from Group 1 and 16 from Group 2. Of these 29, 6 returned to estrus on Days 16-19 and were found to have low progesterone concentrations in the plasma at the same time (Fig. lc). Two of these ewes responded to cloprostenol treatment on Day 21 (Day 5 of their new cycle) and these were included in the nonpregnant, cyclic group (Table 2). Laparotomies on Days 2-26 showed the 6 animals to be nonpregnant. The remaining 2 ewes were assumed to be pregnant because they did not return to estrus. This assessment was confirmed subsequently when plasma progesterone concentrations were found to remain elevated until at least Day 21 (Figs. la,b,d,e). Luteolysis occurred in only 8 of these 2 ewes following a single injection of cloprostenol on Day 21 as judged by the rapid decline in concentration of plasma progesterone (Fig. la, Table 2). All corpora lutea were found to have regressed. Although the uteri of 5 of the ewes appeared nongravid (pale and small) those of the other were still distended at laparotomy 2 days after treatment. Luteolysis occurred in all of the 8 nonpregnant ewes which were injected with doprostenol on Day 28 for, at laparotomy, a newly formed corpus luteum was present in each animal. The mean progesterone concentrations for this group, plus the 2 nonpregnant animals responding to treatment on Day 21, declined to low levels by 24 h and were barely detectable at 48 h (Table 2). This response is TABLE 1. Response of ewes with differing numbers of corpora lutea to an injection of 1 pg cloprostenol. of corpora lutes 1 2 4 of 1 2 ewes of ewes in estrus 25 7 1 with induced luteolysis 27 1 2 Progesteron e concentrationa h 48 h. ±..7 ±.5 5.4 ±. 7.5 ± 2.. ±..25 ±.5. ±..45 ±.4 6 1 1 1 1.5.2 Total 46 7 4 amean ng/m ± SEM, where n = number of animals in which the corpora lutes underwent complete luteolysis.

266 NANARROW ET AL. indicative of complete luteolysis of the corpus luteum. N E Ui Ui - () Ui oncentrations of plasma progesterone in the 15 sheep remaining pregnant had decreased by 24 h (P<.1, t test) but recovered within 2- days (Table 2, Figs. lb,d,e), to sometimes exceed the preexisting levels (Figs. lb,d). At 48 h fter treatment, the mean progesterone concentration was significantly higher (P<.1, t test) than the equivalent concentrations of the other groups (Table 2). When 7 of these ewes were retreated with 1 pg cloprostenol, complete luteolysis occurred (Fig. ib, Table 2) although a significant concentration of proges- 8-4! (b) (ci terone remained in the plasma after 24 h. Estrus was detected in 5 of these ewes within 2- days of treatment while all ewes were found to have pale, empty uteri and regressed corpora lutea at laparotomy 5-7 days after treatment. The remaining 8 saline-treated ewes were unaffected (Figs. ld,e). However, it was found at laparotomy on Day 6 that 4 of these had lost their fetuses and the progesterone values suggested that the abortions occurred about Day 44 in 2 (Fig. id) and possibly later in 1 other. No values were available for the fourth ewe. At the time of the first treatment, the mean d) (e} $ p $ 1 2 6 F, - 1 2 4 DAYS AFTER MATNG FG. 1. Examples of patterns of progesterone concentrations in plasma of sheep after mating and following 1 or 2 injections of 1 pg cloprostenol (4). Saline controls (4). a) Ewe aborted to first injection. b) Ewe aborted only to second injection. c) Ewe never became pregnant. d) Ewe aborted >2 days after a single injection. e) Ewe remained pregnant after a single injection.

EFFET OF MULTPLE EMBRYOS ON LUTEOLYSS N SHEEP 267 u progesterone concentration of those pregnant s ewes which did not abort was higher than those - o which did, but the difference was not signifi- 5 cant. There was, however, a significantly greater. number of corpora lutea per ewe (P<.5, t 4- test) in the former group (Table 2). All ewes n which - had single ovulations following the -.- - PMSG treatments (single corpus albicans iden- 4- ( tified at laparotomy) were found to be not be. pregnant following the first cloprostenol +J+++t U U s v. 2 V - lrtefl O treatment. DSUSSON - 2 n the 2 experiments reported here, a dose be o of 1 pg cloprostenol was sufficient to induce 5, luteolysis in 94.6% of ewes in the luteal phase V of their estrous cycle. Because all 16 ewes with multiple corpora lutea (2-6) responded to the * +1 +1 +1 + treatment, it can be concluded that the re- F,,-SO U r-i r- sponse of individual animals to this dose of s > s cloprostenol is independent of the number of u be -. corpora lutea present, at least up to 6 per ewe. be, loprostenol, when given to ewes on Days 4- V 2-2 1 of pregnancy, induced luteolysis and V interrupted pregnancy in only 4.8% of the B E ewes including all those with only 1 corpus 5 luteum. Therefore the corpora lutea of the - U.2 r-lr-ln be : o remaining ewes were more resistant to the ef- +1 +1 *1 +1 >. 8. 5,-, fects of luteolytic prostaglandins than were V E those of nonpregnant ewes in either experi- U --.5 ment. We have defined this phenomenon as the 551 v 5 antiluteolytic Although it effect was ofnotembryos. possible to assess the V number of embryos in ewes with multiple cor-. U pora lutea, it is likely that the number of V. embryos was related to the number of eggs o --- o shed (Rhind et al., 198). n all ewes with more s than corpora lutea, luteolysis was incomplete 25, + + + +1 and the ewes failed to abort within a week. The F,O. s V > same response was obtained with some ewes - bearing 2 or corpora lutea. Therefore, the s o. ewes which did not abort (.2 ±.4 corpora s - lutea) may have borne more embryos than v 2. those which aborted (1.8 ±. corpora lutea)... Possibly, more antiluteolytic material was V secreted by the conceptus of these animals. The V 5.. corpora lutea of these ewes underwent partial.. - S 2 luteolysis and then recovered in a manner simi- - S U V tar to when prostaglandin F2a was injected into be - O - - 5. the largest follicle on each ovary bearing a Oo V v s -.. corpus luteum on Day 1 of pregnancy (n- -.2 2 Ls ooo 5>..5 skeep et al., 1975; Pratt et al., 1977). A second 5 o s o 5. U. injection given 1 or 2 days after the first may - L.. Z <Z -< complete the luteolysis and result in abortion.

268 NANARROW ET AL. ertainly more cloprostenol given to some of these ewes 7 days later did cause luteolysis and abortion. The previous treatment may have increased the sensitivity of the corpora lutea to cloprostenol or decreased the antiluteolytic protective role of the conceptus. Alternatively, this antiluteolytic protection may decrease with progression of the pregnancy. The present observations are in agreement with the work of Kittok and Britt (1977) and nskeep et al. (1975) in which luteolysis in response to estradiol treatment or ipsilateral intrafollicular injections of prostaglandin F2a, respectively, was suppressed during pregnancy at Days 11-1. n addition, Mapletoft et al. (1975) anastomosed the uterine vein draining the nongravid horn with that draining the gravid horn on Day 5 of pregnancy and demonstrated that the luteolytic component of the former was neutralized by a component of the latter. Also, when 2 pg prostaglandin F2 was infused into the ovarian artery on Day 1 in pregnant or hysterectomized (on Days 5-7 of pregnancy) ewes, regression of the corpora lutea was significantly increased in the latter group (Mapletoft et al., 1976). Our observations extend this work by showing a quantitative effect of embryos in inhibiting artificiallyinduced luteolysis. However, we present no evidence as to the nature of possible antiluteolytic factors produced by the conceptus. Pratt et al. (1977) have prolonged the estrous cycle of ewes by a maximum of 2 days with intrauterine infusions of prostaglandin E2, while Henderson et al. (1977) interfered with the luteolytic action of prostaglandin F2a by simultaneous infusion of prostaglandin E2. Alternatively Martal et al. (1979) have obtained a protein fraction from Days 14-16 ovine trophoblasts which extended the lifespan of the corpora lutea in nonpregnant animals up to 58 days. Although this factor could not be found in 21 to 2-day-old conceptuses, their evidence for a prolonged effect is compelling, which would suggest that the effect that we have noted on Days 2-2 1 is due to trophoblastin-like factors rather than to prostaglandin E2. The amount of cloprostenol used in these experiments was probably more than necessary to achieve luteolysis in cyclic ewes (Baird and Scaramuzzi, 1975). Hence, we may have overcome the antiluteolytic protection mechanism of those sheep bearing 1, possibly 2, embryos. Further experiments using a graded series of doses of cloprostenol could uncover differences in sensitivity of the corpus luteum between cyclic ewes and ewes bearing single embryos. Perhaps either an excessive secretion of prostaglandin F2a or a failure of the embryonic antiluteolytic mechanism in the early pregnant ewe may be causative of early embryo mortality. n 4 of the 8 animals that failed to abort when given only one cloprostenol treatment, pregnancy did not persist past 6 days, the stage at which the placenta can normally synthesize sufficient progesterone to maintain pregnancy. Apparently progesterone synthesis by the corpus luteum in these animals declined from about Days 5-4 resulting in peripheral plasma concentrations becoming too low to support pregnancy (Fig. id). n untreated pregnant sheep, a decline in progesterone concentrations does not occur over this period (Bassett et al., 1969; McNatty et al., 1972; Sarda et al., 197) and the present observations may possibly reflect a long-term effect of the original cloprostenol treatment on the integrity of the corpus luteum of pregnancy. These experiments have shown that early pregnancy in ewes carrying single embryos can be terminated with an injection of 1 pg cloprostenol and this has been the subject of a recent study by Tyrrell et al. (1981). t is suggested that termination of pregnancy in ewes with multiple embryos would be more effective if the animals were given 2 injections some 2-7 days apart. The resistance of the corpora lutea of pregnant ewes to luteolysis appears to be influenced by the number of embryos rather than by the number of corpora lutea present. The most likely explanation for this observation is that the greater embryonic mass of multiple embryos contributes to an increased synthesis of 1 or more antiluteolytic factors. t cannot be determined from these experiments whether a change in sensitivity of the corpus luteum to injected cloprostenol occurs with advancing pregnancy. This subject awaits a more accurate titration of the luteolytic effects of cloprostenol in sheep. AKNOWLEDGMENTS The authors thank Dr. E. Shepherd (..l. Aust. Ltd.) for the gift of cloprostenol and Messrs. P. Hamilton and P. Stockwell for the animal handling. We were also most appreciative of the comments about the manuscript by Dr. P. E. Mattner and the help of Mr. R. N. Tyrrell with the laparotomies.

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