Interference of trophoblastin in ruminant embryonic mortality. A review

Livestock Production Science, 17 (1987) 193-210 Elsevier Science Publishers B.V., Amsterdam - - Printed in The Netherlands

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Interference of Trophoblastin in Ruminant Embryonic Mortality. A R e v i e w J. MARTAL, M. CHARLIER, G. CHARPIGNY, S. CAMOUS, N. CHENE, P. REINAUD, S. SADE and M. GUILLOMOT I.N.R.A., Unitd Endocrinologie de l'Embryon, Station de Physiologie animale, 78350 Jouy-enJosas (France) (Accepted 24 March 1987)

ABSTRACT Martal, J., Charlier, M., Charpigny, G., Camous, S., Chene, N., Reinaud, P., Sade, S. and Guillomot, M., 1987. Interference of trophoblastin in ruminant embryonic mortality. A review. Livest. Prod. Sci., 17: 193-210. The endocrine factors involved in the mechanisms of pregnancy recognition, and particularly those leading to the maintenance of the progesterone environment necessary for embryonic survival, have been reviewed. An antiluteolytic protein, trophoblastin, inhibits luteolysis during early pregnancy. Some cases of embryonic mortality might result from an insufficient secretion of this embryonic signal. Trophoblastin or oTP1 (ovine trophoblastic protein 1 ) is secreted by the trophectoderm. It has only been demonstrated in sheep and cattle. Its presence is necessary during a short critical period (from day 12 to 22 in sheep, 16 to 24 in cattle). Daily intra-uterine injections of 14-16-day-old ruminant embryo homogenates into ewes or heifers from Day 12 of the oestrous cycle maintained progesterone secretion. Removal of 25-day-old embryos provoked the maintenance of luteal activity for several weeks or months. This persistence of corpus luteum can explain some erroneous pregnancy diagnoses based on progesterone determination. Injected into the uterine lumen, purified trophoblastin inhibited cyclic luteolysis. This 20 Kdal protein bound to its endometrial receptors which appeared during the oestrous cycle. In vitro, it stimulated the synthesis of endometrial proteins. The different hypotheses concerning the mechanisms of trophoblastin action, which might be involved in embryonic mortality, are thoroughly discussed.

INTRODUCTION T h e e n d o c r i n e c a u s e s of e a r l y e m b r y o n i c m o r t a l i t y are n u m e r o u s , a n d m a y v a r y c o n s i d e r a b l y f r o m o n e species to a n o t h e r ( H a w k et al., 1955; W e b e l et al., 1970). I n r u m i n a n t s , e m b r y o n i c m o r t a l i t y is m a i n l y o b s e r v e d b e f o r e i m p l a n t a t i o n ( a b o u t 30% ), a n d c o n s i d e r a b l y r e d u c e s t h e r e p r o d u c t i v e p o t e n t i a l of r u m i n a n t s . A n y i m p o r t a n t d i s f u n c t i o n i n g in t h e s e c r e t i o n of e s s e n t i a l e m b r y onic signals m a y lead to e a r l y a b o r t i o n . P r o g e s t e r o n e is a b s o l u t e l y n e c e s s a r y

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to ovo-implantation in all placental mammalian species. The control of progesterone secretion is variable according to the species concerned (Martal and Charlier, 1985). In primates, hysterectomy does not result in the maintenance of the corpus luteum, contrary to other species in which the uterine luteolytic activity is predominant. Thus, in the woman, the luteal activity is particularly stimulated by the chorionic gonadotrophin. In the other species, the main mechanism of corpus luteum maintenance is the blockade, by different ways, of the luteolytic activity of uterine prostaglandin F2~. In the cow, ovariectomy performed on Days 200-215 of pregnancy does not lead to abortion (Erb et al., 1968). In the cow, placental progesterone secretion is particularly low (Ainsworth and Ryan, 1967; Peterson et al., 1975b). In the goat, ovariectomy throughout gestation causes abortion, while in the sheep it may be performed from Day 50 of pregnancy because from this stage the placenta is capable of progesterone secretion (Martal, 1981a; Martal and Charlier, 1985). In ruminants, ovariectomy can be carried out a few days after fertilization without abortion risks provided that the animals are supplemented with progesterone ( Hawk et al., 1955; Foote et al., 1957; Ricketts and Flint, 1980). This hormone considerably increases the uterine permeability for plasma proteins. It also induces the synthesis of specific uterine proteins (Laster, 1977; Dixon and Gibbons, 1979), but to a small extent: less than 2% of total proteins (Roberts and Parker, 1974 ). The nature of progesterone-dependent uterine proteins has not been established yet in ruminants (Bazer et al., 1981; Findlay et al., 1981; Salamonsen et al., 1985). In other species, some uterine proteins have been characterized as uteroglobin (or blastokinin) in the rabbit (Beier, 1976, 1982), the inhibitor of plasminogen activator in the pig (Mullins et al., 1980) or the uteroferrin in the pig (Bazer and Thatcher, 1977; Frank et al., 1978) and the horse (Zavy et al., 1979, 1982). Secretion of rabbit uteroglobin and sow uteroferrin is modulated by oestrogens. In sheep, Salamonsen et al. (1985) have found that, in endometrial cell cultures, protein synthesis is induced either by progesterone or by oestrogens. After preliminary investigations by Rowson and Moor (1967), we demonstrated, in 1979, the existence of an antiluteolytic protein called trophoblastin (Martal et al., 1979). It is involved in the control of progesterone secretion by the ruminant corpus luteum in early pregnancy. It was also called Ovine Trophoblastic Protein 1 or oTP1 by Godkin et al. (1984a). The purpose of this paper is to study how trophoblastin might be involved in embryonic mortality. I N H I B I T I O N OF CYCLIC L U T E O L Y S I S BY T R O P H O B L A S T H O M O G E N A T E S

In sheep, uterine injections of 14-16-day-old embryo homogenates from Days 14-15 of the oestrous cycle lead to the maintenance of functional corpora lutea (Rowson and Moor, 1967; Martal et al., 1979). In contrast, uterine injections of 21-23-day-old ovine embryo homogenates did not inhibit luteolysis. Similar

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experiments were made in heifers from Day 14-15 of the oestrous cycle by uterine injections of 16-18-day-old embryo homogenates (Northey and French, 1980; Humblot and Dalla Porta, 1984). In cattle, the disappearance of embryonic antiluteolytic factor has been shown to take place on Day 24 of pregnancy (Thatcher et al., 1985 ). Thus, the presence of the embryonic signal inhibiting the uterine luteolytic activity would only be necessary in the ewe from Days 12 to 22 of pregnancy and in the cow from Days 16 to 23. T H E NATURE OF T H E ANTILUTEOLYTIC SUBSTANCE

Homogenates of 14-16-day-old ovine trophoblasts were found to lose their antiluteolytic capacity subsequent to thermal or proteolytic treatments. After extraction and gel filtration, homogenates of 14-16-day-old conceptuses remained able to maintain the cyclic corpus luteum of recipient ewes. The presence of intact trophoblastic cells is not necessary in vivo to the inhibition of luteolysis, since the antiluteolytic activity was observed in the effluents of ultrogel columns. Thus, the active factor is soluble. It is different from some known luteotrophic hormones because uterine injections of chorionic gonadotrophin ( hCG ), and/or ovine placental lactogen, do not inhibit cyclic luteolysis (Martal et al., 1979). For these reasons, this new antiluteolytic factor was called trophoblastin. It is also totally different from E P F (Early Pregnancy Factor) (Martal and Charlier, 1985). Cultured in vitro, in the presence of radioactive amino acids, ovine blastocysts (Godkin et al., 1982) and trophoblastic vesicles (blastocyst after removal of the embryonic disc ) ( Martal et al., 1984b) secrete a protein in large amounts into the media, though in trace amounts in the tissues. Its presence was demonstrated from Day 12 to 21 of pregnancy. Its apparent molecular weight is 20 000 daltons and its isoelectric point about 5.3, according to electrophoretic and isoelectrofocusing analyses. This protein is immunoprecipitable by an immuneserum against oTP1 ( M. Charlier, P. Gaye and J. Martal, unpublished data, 1985). It inhibits luteolysis when introduced in the uterine lumen of recipient ewes on Day 12 of the oestrous cycle ( Godkin et al., 1984b ). In cattle, two similar molecules were characterized: their apparent molecular weights were 20 000 and 22 000 daltons, respectively, and their isoelectric points were also acid. These bovine and ovine proteins exhibit an immunological crossreaction (Thatcher et al., 1985). oTP1 was found to prolong corpus luteum life-span when administered into the uterus of cyclic recipient ewes. Similar results were obtained with bovine conceptus proteins (Knickerbocker et al., 1984). Ovine trophoblastic vesicles injected into the uterus of recipient heifers, or bovine vesicles administered to cyclic ewes, inhibited luteolysis (Martal et al., 1984a,b). These heterologous transfers show similarities between ovine and bovine trophoblastins.

196 THE SYNTHESIS LOCATIONOF TROPHOBLASTIN Although deprived of its embryonic disc, the blastocyst keeps on secreting trophoblastin. This protein is therefore synthesized by the trophoblast (Heyman et al., 1984; Martal et al., 1984b). Using anti-oTP1 immunoserum, Godkin et al. (1984a) showed by immunocytology that trophoblastin was only located in the trophectoderm. The gene (s) encoding for trophoblastin is transcribed before the beginning of the critical phase of luteolysis inhibition ( Day 12 ) : cultured in the presence of radioactive methionine, 9-day-old blastocysts secreted trophoblastin while 6-day-old ones had not yet produced it {M. Charlier and J. Martal, unpublished data, 1985). Removal by micromanipulation of the embryonic discs from 9-day-old ovine blastocysts, and transfer of these vesicles into recipient ewes on Day 8 of the oestrous cycle (J. Martal, M. Charlier and P. Chesnd, unpublished data, 1985 ), inhibited luteolysis. Although trophoblastin synthesis starts before the critical phase of corpus luteum maintenance (Days 12-22 of pregnancy), it has not been established yet whether blastomeres which will differentiate into an embryonic disc are involved in the induction of the expression of this protein. The role of endoderm in the control of this secretion has not been determined either. THE PERSISTENCE OF THE TROPHOBLASTINEFFECT ON CYCLICLUTEOLYSIS Uterine injections of 14-16-day-old trophoblast homogenates into cyclic ewes from Day 12 for only a few days led to the maintenance of a functional corpus luteum for more than one month (Martal et al., 1979). Similar results were obtained after transfer of ovine and bovine trophoblastic vesicles ( Heyman et al., 1984). Removal of ovine embryos on Days 14-16 of pregnancy did not prevent return to oestrous, but slightly delayed the cycle (Moor and Rowson, 1966a; Edey, 1967). A similar observation was made in the cow on Day 16 (Humblot and Dalla Porta, 1984). Surgical abortion on Day 25 of pregnancy, i.e. after the critical period of luteolysis inhibition, also caused the persistence of the pregnancy corpus luteum ( Martal et al., 1979). These animals exhibited an authentic pseudopregnancy, characterized by a secretory corpus luteum and an often abundant amount of sterile liquid within the uterus. MECHANISMS OF TROPHOBLASTINACTION The present hypothesis on the mechanisms of trophoblastin action are shown in Fig. 1.

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PRESENT

H Y P O T H E S I S ON THE M E C H A N I S M S OF T R O P H O B L A S T I N ACTION

Trophectoderm I I-'*TROPHOBLASTIN ( ~ Sheep:Ol2- 022

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Cow :O,i~-D2,

ENDOMETRIUM epithelium + stroma neosynthesized proteins | ~ ~ ~ . ~ "* "..~,$~.9 ('(PGi.) ~

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PROGESTERONE i A CORPUS ~ LUTEUM ,IF*, ., PGE>PGF21ower :(peaks)

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~_,_:., ~ IV21 9 + [/

EMBRYO

PGsynthetase I 1 ?-'~t Phospholipase I Ovarian i + ~ ro__] A2 / artery Utero-ovarian vein OT ROT, oxytocin R: receptor : [R]:number of R

Fig. 1. Present hypothesis on the mechanisms of trophoblastin action. During early pregnancy in ruminants, the conceptus is constituted by an embryonic disc (ED) and trophoblastic membranes composed of trophectoderm and endoderm (Day 14, for instance). The trophectoderm secretes an antiluteolytic protein, trophoblastin (T), at least from Day 12 to 22 in sheep and from Day 16 to 24 in cattle. Trophoblastin binds to its own endometrial receptors (R). Oxytocin (OT) binds to its endometrial receptors (ROT). The corpus luteum (CL) secretes progesterone which allows the uterus and embryo development. Prostaglandin E (E2 and El) favours progesterone secretion, while PGFe~ inhibits it. Serum PGF2~ peaks are lower and less numerous in early pregnancy than during oestrous. Endometrial PGF2~ and PGE cross the utero-ovarmn vein wall then that of the ovarian artery to bind to their specific luteal receptors. In the endometrium (constituted by an epithelium and a stroma) several proteins (6) are neosynthesized after trophoblastin stimulation but their nature and function are unknown yet. They might be involved in prostaglandin (PG) regulation, some small peptides might reach the ovary and intervene in the pregnancy corpus luteum regulation. They might also participate in the control of the oxytocin receptors the number of which is particularly decreased in early pregnancy. Trophoblastin might inhibit (--), by other ways, oxytocin and/or oestradiol receptors (RE2). Trophoblastin might also be implicated in the regulation of prostaglandin synthesis, and for instance, favour the transformation of PGF2~ into PGE2 by an oxydoreductase, the PGF2~-9dehydrogenase.

The indirect effect of trophoblastin on the corpus luteum I n sheep, t r a n s f e r of a n e m b r y o into t h e u t e r i n e h o r n c o n t r a l a t e r a l to t h e c o r p u s l u t e u m , t h i s h o r n b e i n g i s o l a t e d f r o m t h e o t h e r b y a ligature, did n o t i n h i b i t cyclic luteolysis. O n t h e o t h e r h a n d , t r a n s f e r o f a c o n c e p t u s into t h e o t h e r h o r n m a i n t a i n e d p r o g e s t e r o n e s e c r e t i o n ( M o o r a n d R o w s o n , 1966b).

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These results indicate that the antiluteolytic signal does not reach the corpus luteum via the blood circulation. The size of the trophoblastin molecule (20 000 daltons) prevents it from using the prostaglandin pathway to reach the ovary: McCracken et al. (1972) have demonstrated by means of radioactive PGF2~ that this hormone crossed the utero-ovarian vein wall, then that of the ovarian artery to bind to its specific luteal receptors (Rao, 1974). These results were confirmed in cattle and sheep by Mapletoft et al. (1976a,b) with cross anastomoses experiments. Trophoblastin does not seem to reach the ovary by the lymphatic pathway either: only negligible amounts of radioactivity were detected in the ovary after uterine injections of labelled oTP1 into cyclic ewes (Godkin et al., 1984a). Radioactive trophoblastin bound to specific endometrial receptors. In the presence of endometrial cells obtained for ewes on Day 12 of the oestrous cycle, oTP1 increased the rate of synthesis of the proteins in the culture medium, and induced the synthesis of at least 6 proteins whose functions are still unknown ( Godkin et al., 1984a ). It seems, therefore, that trophoblastin inhibits luteolysis by direct action on the endometrium. The action o[ trophoblastin on the endometrium secretion of PGF2~ Plasma PGF2~ concentrations During early pregnancy in sheep, despite the maintenance of corpus luteum, the basic plasma-PGF2~ concentrations in the utero-ovarian vein are similar or higher than those of cyclic ewes (Wilson et al., 1972; Pexton et al., 1975a,b; Peterson et al., 1975a; Cerini et al., 1976; Lewis et al., 1977, 1978; Ellinwood et al., 1979; Nett et al., 1976). The endometrium of pregnant ewes (Wilson et al., 1972; Inskeep et al., 1975; Roberts et al., 1976; Reynolds et al., 1981 ) and cows ( Shemesh et al., 1979; Lewis et al., 1982 ) contains and secretes PGF2~ in similar or even higher amounts than that found in cyclic animals. The ruminant blastocyst also produces very large amounts of PGF2~ during the critical phase of luteolysis inhibition. It even secretes more PGF2~ than PGE2 in sheep (Lacroix and Kann, 1982) and cattle (Shemesh et al., 1979; Lewis et al., 1982). The higher plasma PGF2~ concentrations in early pregnancy may be explained by the intense secretion from the endometrial and trophoblastic tissues. Thus, trophoblastin does not inhibit cyclic luteolysis by reducing the basic plasma PGF2~ secretion. PGF2~ secretion in the uterine lumen It was assumed that trophoblastin might affect the exocrine secretion of PGF2~ ( Martal et al., 1979). Bazer and Thatcher (1977) explained the luteotrophic action of oestrogens in the pig by the exocrine secretion of endometrial

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PGF2~ towards the uterine lumen. Although oestrogens exhibit a luteolytic effect in late oestrous cycle in ruminants (Martal, 1981a,b), this hypothesis might be applied to other factors of corpus luteum maintenance, such as trophoblastin. In pseudopregnant ewes after uterine injections of trophoblast homogenates, embryo removal or transfer of trophoblastic vesicles, the uterine fluid formed in most cases contained a much higher proportion of PGF2~ than that of PGE2 (J. Martal and M.C. Lacroix, unpublished data, 1982 ). Although this experimental model was rather different from the natural conditions of pregnancy, it might indicate a possible bi-directional secretion by the endometrium. However, the higher proportion of PGF2~ might result from the instability of PGE2 ( GranstrSm et al., 1980). Nevertheless, ovine (Ellinwood et al., 1979 ) and bovine ( Lewis et al., 1982 ) uterine fluids from pregnant animals exhibited a higher proportion of PGF2~ than those from cyclic animals. These results could be confirmed as the manipulated endometrium and the blastocyst may secrete large amounts of PGF2~. In addition, the uterine fluid of pregnant animals contains a higher proportion of proteins, among which some carrier proteins could specifically bind to PGF2~. None of them have been identified yet, however. In conclusion, the present state of our knowledge does not permit us to transpose to ruminants the hypothesis of change in the PGF2~ direction.

Trophoblastin and PGF2~pulsatility Demonstration of PGF2~ pulsatility is difficult to perform, and requires collection of frequent blood samples in the utero-ovarian vein. Some authors (Thorburn et al., 1973; Barcikowski et al., 1974; Fairclough et al., 1980, 1984) have observed a reduction in the frequency and magnitude of PGF2~ secretion peaks in pregnant ewes as compared to cyclic ones. PGF2~ secretion peaks occur simultaneously with those of oxytocin (Fairclough et al., 1980, 1984; Flint and Sheldrick, 1982b). In vitro, when endometrial pieces were collected between Days 13 and 17 of the oestrous cycle, large releases of PGF2~ were induced by oxytocin, but they were much lower between Days 3 and 13 (Roberts et al., 1976). Luteolytic activity of oxytocin in the late oestrous cycle has been well established for a long time in sheep and cattle. Its effect disappears after hysterectomy (Armstrong and Hansel, 1959; Milne, 1963; Ginther et al., 1967; Sharma and Fitzpatrick, 1974; Mitchell et al., 1975 ). Active immunization against oxytocin delays luteolysis ( Sheldrick et al., 1980; Schams et al., 1982 ). The number of high-affinity oxytocin receptors increases during oestrous. The binding capacity of the endometrium is twice as high as that of the myometrium (Roberts et al., 1976). Oestrogens could modulate PGF2~ synthesis by regulating that of endometrial oxytocin receptors (Roberts et al., 1976). At the end of the oestrous cycle oestrogens exert, like oxytocin, a luteolytic effect in sheep (Denamur and Kann, 1973; Barcikowski et al., 1974) and cattle (Greenstein et al., 1958; Wiltbank

200 et al., 1961 ). Hysterectomy suppresses this activity, which normally takes place via the uterine PGF2, in both species (Wiltbank and Casida, 1956; Malven and Hansel, 1964; Brunner et al., 1969; Bolt and Hawk, 1975). X-Ray treatment of ovarian follicles considerably delays cyclic luteolysis by blocking oestrogen synthesis (Karsh et al., 1970; Denamur, 1973). Progesterone administration between Days 3-10 also reduces the length of the oestrous cycle in heifers, but this effect is not observed after hysterectomy (Woody and Ginther, 1968). McCracken {1980) and McCracken et al. (1984) have investigated the progesterone-oestrogen-oxytocin interactions involved in the PGF2~ pulsatile secretion. In intact sheep, oxytocin injections produce serum PGF2, releases, which are only significant in late oestrous cycle (Day 13-15): for example, on Day 8 they are not yet observed in the uterine vein. In contrast, from Day 13 the magnitude of PGF2~ secretion peaks after oxytocin administration is larger. Nevertheless, from Day 6, oestrogen infusions can intensify the effect of oxytocin injections and lead to small serum PGF2, peaks. On the other hand, in intact sheep magnitude of PGF2, is increased if progesterone is infused for 10 days, from Day 3, in addition to oestrogen infusion and oxytocin injections. This shows that progesterone exerts an antagonistic effect of oestrogens on the pulsatility of PGF2~ release induced by oxytocin at the beginning of the oestrous cycle, and a synergic one at the end. The respective mechanisms of action of progesterone, oestrogens and oxytocin on cyclic luteolysis can be explained as shown below. During the luteal phase, progesterone favours phospholipid synthesis and storage in the epithelial cells of the endometrium ( Brinsfield and Hawk, 1973 ) to constitute the stock necessary for prostaglandin synthesis at the end of the oestrous cycle. In addition, progesterone would decrease the number of endometrial oestrogen receptors (Koligian and Stormshak, 1977a,b; Henricks and Harris, 1978). They are probably restricted in number at the beginning of the cycle, which strengthens the progesterone action (McCracken et al., 1972). Oestrogens could act upon luteolysis in several ways: they could considerably enhance the phospholipase A activity (this enzyme is able to cleave arachidonic acid from phospholipids ) ( Pakrasi et al., 1983 ). It has already been demonstrated that free arachidonic acid is a limiting factor in prostaglandin synthesis. Oestrogens also stimulate the enzyme activity of the prostaglandin synthetase complex (Naylor and Poyser, 1975; Ham et al., 1975; Wlodawer et al., 1976). They could also increase the number of endometrial oxytocin receptors (Roberts et al., 1976). Thus, oestrogens could stimulate the PGF2~ pulsatile secretion. Oxytocin could play a major role in the PGF2, releases. It would be secreted both by the posterior hypophysis and by the corpus luteum. Uterine handling provokes the reflex PGF2~ release (McCracken et al., 1984). Besides, it was recently demonstrated in sheep that luteal structures have a high oxytocin

201 content (Flint and Sheldrick, 1982a; Wathes and Swann, 1982). Release of luteal oxytocin can be stimulated by PGF2~ (Flint and Sheldrick, 1982b, 1983 ). Blood oxytocin and progesterone levels vary simultaneously during the oestrous cycle and after ovariectomy (Sheldrick and Flint, 1981, 1984). The existence of a positive feed-back between the corpus luteum and the endometrium has been suggested. During cyclic luteolysis, uterine PGF2~ could stimulate luteal oxytocin secretion, which, in turn, would activate the pulsatile secretion of the endometrial PGF2~. It may be assumed that in early pregnancy the embryo plays a major role in the blockage of this luteal oxytocin-endometrial PGF2~ interaction. Thus, trophoblastin could be particularly implicated in the oxytocin-PGF2~ feed-back. During the critical phase of luteolysis inhibition, the concentration of oxytocin receptors in the endometrium of pregnant ewes is markedly lower than that of cyclic ewes: 33 fmoles mg -1 versus more than 250 fmoles mg -1, on Day 16 of pregnancy and 16-17 of the oestrous cycle, respectively (McCracken et al., 1984). It may be supposed that trophoblastin is capable of inhibiting, directly or indirectly, the synthesis of oxytocin receptors in the endometrium. For instance, it could be involved in their transcription, or in the stabilization of their mRNAs. It would reduce the synthesis of oestrogen receptors which would normally stimulate that of oxytocin receptors. It could intervene through one or several endometrial polypeptides, whose synthesis it induces. Although probable, this interpretation, derived from that of McCracken et al. (1984) remains a working hypothesis: the effect of trophoblastin upon endometrium oxytocin receptors has not been demonstrated yet. Nevertheless, if it were verified, it would explain the persistence of its action beyond its disappearance: progesterone secretion would persist because of the decrease in the oestrogen and PGF2~ secretion. The variability from one individual to another in the persistence of the corpus luteum in pseudopregnant animals would depend on numerous factors controlling PGF2~ and on luteotrophic factors (LH, prolactin, PGE, PGI, receptors, etc. ). Finally, trophoblastin might still exert an indirect action upon luteal oxytocin and PGF2~ receptors. Ways in which trophoblastin could reduce the ovarian receptivity to PGF2~ Resistance to luteolysis of the pregnancy corpus luteum Several experiments have shown that the corpus luteum of gestation does not exhibit the same sensitivity to luteolytic factors as the corpus luteum of the oestrous cycle. Intrafollicular or vascular PGF2~ injections into cyclic ewes, in sufficient amounts to produce luteolysis, had less effect than in pregnant ewes (Inskeep et al., 1975; Mapletoft et al., 1976a,b; Nancarrow et al., 1982; Silvia et al., 1984a). The presence of the embryo seems to be responsible for

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the luteal resistance at the beginning of the critical phase of luteolysis (Days 12-15 of pregnancy). Oestradiol also has less effect upon luteolysis of the pregnancy corpus luteum (Kittok and Britt, 1977).

Direct action of trophoblastin As already mentioned, trophoblastin is not able to reach the corpus luteum, but it is liable to exert an effect on it either by the small peptides that it has induced or by the endometrial luteotrophic prostaglandins.

Stimulation of the luteotrophic prostaglandin synthesis Prostaglandins E2 and E1 are characterized by the ability to neutralize the luteolytic action of PGF2~ when they are administered concomitantly with the latter by intravenous injection (Henderson et al., 1977; Reynolds et al., 1981). Uterine injections of PGE1 or PGE2 into cyclic ewes (Pratt et al., 1977; Colcord et al., 1978; Huie et al., 1981; Magness et al., 1981) or heifers (Gimenez and Henricks, 1983; Reynolds et al., 1983 ) inhibit luteolysis for a short period. Nevertheless, uterine infusions of conceptus homogenates, transfer of trophoblastic vesicles or embryo removal after the critical phase of luteolysis inhibition markedly prolong the secretory activity of the corpus luteum ( Martal et al., 1979; Heyman et al., 1984; McCracken et al., 1984). Ovine (Marcus, 1981; Hyland et al., 1982; Lacroix and Kann, 1982; Silvia et al., 1984a; Silvia and Niswender, 1984) and bovine ( Shemesh et al., 1979; Lewis et al., 1982) blastocysts secrete PGE2~, though in lower amounts than PGF2~. The ruminant endometrium also produces PGE2, which would reach the corpus luteum by a mechanism of transfer similar to that observed with PGF2~ ( Wilson et al., 1972; Ellinwood et al., 1979; Shemesh et al., 1979; Marcus, 1981; Hyland et al., 1982; Lacroix and Kann, 1982; Lewis et al., 1982). Mean PGE2 concentrations in the utero-ovarian vein adjacent to the corpus luteum are higher in pregnant than in cyclic ewes (Ellinwood et al., 1979; Ottobre et al., 1984; Silvia et al., 1984b). The ruminant blastocyst and endometrium also secrete prostacyclin (Jones et al., 1977; Marcus, 1981) which, in vitro, stimulates, as does PGE, the secretion of luteal progesterone (Milvae and Hansel, 1980, 1983). However, the serum concentrations of the stable metabolite of prostaglandin PGI2 (6-keto-PGFl~) are not significantly different in pregnant and cyclic animals (Silvia et al., 1984b). It is, therefore, possible that trophoblastin stimulates the endometrial PGE2 synthesis, particularly by increasing the activity of prostaglandin F2c~-9-hydroxydehydrogenase, which converts PGF2~ into PGE2 ( Lee and Levine, 1974; Dunn and Hood, 1977; Watson et al., 1979). PGE2 would enhance steroidogenesis in the small luteal cells by activating adenylate cyclase (Marsh, 1970). Moreover, it would inhibit luteal regression by reducing the secretion of the

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luteolytic factor (s) produced by the large luteal cells and/or would provoke a vasodilatation in the ovarian system (Silvia et al., 1984a; Silvia and Niswender, 1984). Bergstrom et al. (1968) demonstrated the vasodilator properties of PGE2 and the vasoconstrictive ones of PGF2,. Let us mention that during luteolysis, blood flow decreases in parallel with progesterone (Niswender et al., 1976; Ford and Chenault, 1981; Ford, 1982; Reynolds et al., 1984). In brief, trophoblastin may exert its antiluteolytic effect in one or several ways: by stimulating the synthesis of luteotrophic prostaglandins, which would behave like antiluteolysins in the ovarian system; - - by inducing the synthesis of endometrial peptides, which would be involved in numerous biochemical processes and in various vessels and tissues; by inhibiting directly or indirectly the synthesis of endometrial oxytocin receptors, and thus by reducing the PGF2, pulsatile secretion. These researches on the mechanism of trophoblastin action showed the complexity of the dam-embryo dialogue. Further studies should be made to improve our knowledge of the embryonic signals, their role and their relationships and, thus, to control problems relative to embryonic mortality.

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Webel, S.A., Peters, J.B. and Anderson, L.L., 1970. Synchronous and asynchronous transfer of embryos in the pig. J. Anim. Sci., 30: 565-568. Wilson Jr., L., Butcher, R.L. and Inskeep, E.K., 1972. Prostaglandin F-2~ in the uterine of ewes during early pregnancy. Prostaglandins, 1: 479-482. Wiltbank, J.N. and Casida, L.E., 1956. Alteration of ovarian activity by hysterectomy. J. Anim. Sci., 15: 134-140. Wiltbank, J.N., Ingalls, J.E. and Rowden, W.W., 1961. Effects of various forms and levels of estrogen alone or in combinations with gonadotropins on the estrous cycle of beef heifers. J. Anim. Sci., 20: 341-346. Wlodawer, P., Kindahl, H. and Hamberg, M., 1976. Biosynthesis of prostaglandin F2~ from arachidonic acid and prostaglandin endoperoxides in the uterus. Biochem. Biophys. Acta, 431: 603-614. Woody, C.O. and Ginther, O.J., 1968. Effect of exogenous progesterone on corpora lutea in unilaterally hysterectomized heifers. J. Anim. Sci., 27: 1387-1390. Zavy, M.T., Bazer, F.W., Sharp, D.C., and Wilcox, C.J., 1979. Uterine luminal proteins in the cycling mare. Biol. Reprod., 20: 689-698. Zavy, M.T., Sharp, D.C. and Bazer, F.W., 1982. Identification of stage-specific and hormonallyinduced polypeptides in the uterine protein secretions of the mare during the oestous cycle and pregnancy. J. Reprod. Fertil., 64" 199-207. RESUME Martal, J., Charlier, M., Charpigny, G., Camous, S., Chene, M., Reinaud, P. et Guillomot, M., 1987. Interfdrence de la trophoblastine dans la mortalit~ embryonnaire chez les ruminants. Une revue bibliographique. Livest. Prod. Sci., 17:193-210 (en anglais). Les facteurs endocriniens qui conditionnent les m~canismes de reconnaissance de la gestation et principalement ceux qui permettent le maintien de renvironnement progest~ronique n$cessaire la survie de rembryon ont ~t~ gtudi~s. Une prot~ine antilut~lytique, la trophoblastine, inhibe la lut$olyselorsde l'~tablissementde la gestation.Certains cas de mortalitd embryonnaire peuvent r~sulterd'une s~cr~tion insuffisantede ce signal embryonnaire. La trophoblastine ou oTP1 (prot~ine trophoblastine ovine 1) est s$cr~tSe par le trophectoderme. Elle a seulement $t6 raise en ~vidence chez la brebis et la vache. Sa prSsence est n~cessaire pendant une courte pSriode critique (du 12e au 22e jour chez la brebis, du 16e au 24e jour chez la vache). Des injectionsintraut~rines d'homog~nats d'embryons de ruminant, ~g~s de 14-16 jours, dans des brebis ou des gdnisses, au 12e jour du cycle oestral,prolonge la s~cr~tion de progesterone. L'ablation d'embryons, ~g~s de 25 jours, entra~ne le maintien de l'activit~lut~ale pendant plusieurs semaines ou m ~ m e plusieurs mois. La persistence du corps jaune peut expliquer certains cas d'erreurs de diagnostics de gestation par dosage de la progestdrone. Administr~e dans la lumibre utdrine,la trophoblastine purifi~e inhibe la lut~olyse cyclique. Cette prot~ine de 20 Kdal se lie ~ ses r$cepteurs endomdtriaux qui apparaissent au cours du cycle oestral.In vitro,ellestimule la synth~se de prot~ines endomStriales. Les diffSrenteshypotheses concernant les mdcanismes d'action de la trophoblastine susceptibles d'etre impliqu~s dans la mortalit~ embryonnaire sont abondamment discut~s. KURZFASSUNG Martal, J., Charlier, M., Charpigny, G., Camous, S., Chene, M., Reinaud, P. und Guillomot, M., 1987. Interferenz des Trophoblastins in der embryonalen Mortalitiit der Wiederk~uer. Ein Ubersichtsreferat. Livest. Prod. Sci.,17:193-210 (auf englisch).

210 Die endokrinen Faktoren, die an den Mechanismen der Tr~chtigkeitserkennung beteiligt sind, und vor allem die Faktoren, die ffir die Aufrechterhaltung der Progesteronumwelt und damit f[ir das Uberleben des Embryos unerliisslich sind, wurden untersucht. Ein antiluteolytisches Protein, das Trophoblastin, hemmt die RUckbildung des zyklischen GelbkSrpers beim Eintritt der Tr~ichtigkeit. Gewisse F~ille embryonaler Mortalit~it sind auf eine unausreichende Trophoblastinsekretion zurfickzufiihren. Das Trophoblastin oder oTP1 (ovine trophoblastic protein 1 ) wird durch das Trophektoderm sezerniert. Es wurde bisher nut beim Schaf und Rind nachgewiesen. Sein Vorkommen ist w~ihrend einer kurzen kritischen Periode (vom 12. bis zum 22. Tag beim Schaf und vom 16. bis zum 24. Tag beim Rind) notwendig. Die ta'gliche intra-uterine Verabreichung von Homogenaten 4-16-tiigiger Schafembryonen am 12. Tag des Zyklus verl~ngert beim Mutterschaf sowie bei der Kuh die Progesteronsekretion. Nach Entfernen 25-t~giger Embryonen bleibt die Progesteronausschiittung fiber mehrere Wochen oder sogar Monate erhalten. Diese Corpus-luteum-Persistenz kann einige fehlerhafte Triichtigkeitsdiagnosen, die auf Progesteronbestimmungen beruhen, erkl~iren. Die Verabreichung von gereinigtem Trophoblastin in das Uteruslumen hemmt die zyklische Luteolyse. Dieses Protein (M.G. 20 Kdal) bindet sich auf spezifischen endometriaten Rezeptoren, die im Laufe des Sexualzyklus auftreten. In vitro f6rdert es die Proteinsynthese durch die Endometriumzellen. Die einzelnen Hypothesen fiber die Wirkungsmechanismen des Trophoblastins, die in der embryonalen Mortali~t eine Rolle spielen kSnnten, werden diskutiert.