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Reprinted in the IVIS website with the permission of AAEP Close window to return to IVIS IN DEPTH: REPRODUCTION The Physiology of Early Pregnancy in the Mare Professor W. R. Allen, BVSc, PhD, ScD, DESM, MRCVS Author’s address: University of Cambridge, Department of Clinical Veterinary Medicine, Equine Fertility Unit, Mertoun Paddocks, Woodditton Road, Newmarket, Suffolk CB8 9BH, United Kingdom. © 2000 AAEP. Introduction Many features of early pregnancy in the mare appear to be unique to the genus Equus and are of considerable academic interest and practical significance. From the time of fertilization of the oocyte soon after ovulation until establishment of the mature and fully functional placenta some 150 days later, a series of morphological, immunological, and endocrinological changes take place in the oviduct and uterus which may be presumed to be important components of the establishment and maintenance of the pregnancy state, but which differ markedly from equivalent events in the other common large domestic animal species and for which it is difficult to imagine a precise evolutionary reason for their occurrence. This paper aims to highlight a few of these equine pregnancy-related reproductive oddities and discuss their significance in modern equine veterinary medicine. Oviductal Transport van Niekerk and Gerneke1 first drew attention to the differential transport of oocytes and embryos in the equine oviduct. Namely, if the freshly ovulated oocyte remains unfertilized, it passes down the oviduct only to the ampullary-isthmus junction where it remains lodged in the highly convoluted folds of oviductal mucosa and degenerates slowly over many months.2 On the other hand, if the oocyte is fertilized by spermatozoa accumulated at the sperm reservoir in the same ampullary-isthmic region of the oviduct,3 the resulting embryo continues its onward passage and passes through the very constricted and prominent uterotubal junction (UTJ) to enter the uterus between 144 and 168 hours after ovulation.4 Thus, flushing the oviducts of mares post mortem typically yields multiple flattened and degenerate oocytes accumulated from previous sterile ovulations in preceding estrous cycles,5,6 while the intervention of fertilization can result in the young embryo bypassing the still-accumulated oocytes to enter the uterus at the expected time.7 What 338 mechanism could be responsible for such an unusual differential movement of gametes in the oviduct? In early studies, Betteridge et al8 argued that the process of cleavage bestowed oviductal mobility on the equine embryo, while Onuma and Ohnami7 and others proposed that ultrastructural changes in the surface of the zona pellucida during early development of the embryo enabled its selective propulsion through the oviduct lumen by the organized beating of the cilia extruding from the apical surface of the lumenal epithelial cells. However, it was Weber and his colleagues in northwest America who eventually provided the definitive answer to the puzzle in a series of elegant experiments that involved both the culture of embryos in vitro9,10 and surgical implantation of mini-pumps to enable perfusion of hormones into the mesosalpinx, followed by embryo recovery attempts at fixed times after ovulation.11–13 In this way they demonstrated convincingly that the embryo, but not the unfertilized oocyte, begins secreting appreciable quantities of prostaglandin E2 (PGE2) when it reaches the compact morula stage of development on day 5 after ovulation. The smooth muscle relaxing properties of this hormone act locally on the circular smooth muscle fibers in the wall of the oviduct and thereby allow the embryo to move onwards, with the aid of the rhythmically beating cilia, to enter the uterus approximately 24 hours later. Thus, it is the stagedependent development of the hormone-secreting capacity of the embryo, not any subtle change in maternal recognition of size or structural changes in the outermost coat of it, which brings about its desired onward movement to the uterus (Fig. 1a). The protracted 6-day sojourn of the equine embryo in the oviduct compared to the 48-hour oviductal period of the 4-cell pig embryo14 and the 72-hour transport time of the 8-cell ruminant embryo,15 has disadvantageous practical implications for embryo transfer and related embryo technologies in equids. For example, the bisection of embryos to produce 2000 Ⲑ Vol. 46 Ⲑ AAEP PROCEEDINGS Proceedings of the Annual Convention of the AAEP 2000 Reprinted in the IVIS website with the permission of AAEP Close window to return to IVIS Fig. 1. Cartoon depicting: a) the differential rates of oviductal transport between the embryos of the pig, sheep and horse and the unique need for the latter to secrete PGE2 to relax the oviductal smooth muscle for its onward passage to the uterus; and b) the contrasting mechanisms employed by the three species to achieve maternal recognition of pregnancy and luteostasis for maintenance of the pregnancy state. AAEP PROCEEDINGS Ⲑ Vol. 46 Ⲑ 2000 Proceedings of the Annual Convention of the AAEP 2000 339 Reprinted in the IVIS website with the permission of AAEP Close window to return to IVIS IN DEPTH: REPRODUCTION monozygotic (identical) twins is successful when performed on morulae but the success rate falls dramatically if the embryo is showing even the earliest signs of blastulation when bisected.16,17 Similarly, the success of deep-freezing embryos in liquid nitrogen falls off sharply with increasing developmental age and size of the embryo,18 due probably to a combination of damage to cells of the inner cell mass (ICM) and impermeability of the equine blastocyst capsule to cryoprotectants.19 In their painstaking and elegant study, Battut et al determined that the majority of horse embryos enter the uterus (from which they can be recovered by simple non-surgical flushing methods) between 144 and 156 hours after ovulation when they are at the late morula stage of development and may already be beginning to blastulate.4 But even when the time of ovulation is known to within a few hours by repeated ultrasound scanning of the ovaries, flushing the uterus at closely timed intervals between 144 and 156 hours later yields embryos that differ markedly in their stage of development. Similarly, in a large experiment designed to recover morulae for the purposes of bisection by flushing normal, fertile mares at fixed times after a carefully estimated time of ovulation, Boyle et al obtained a lower-than-normal overall embryo recovery rate of only 43% due to flushing some mares too early when the embryo was still in the oviduct.20 And it was disappointing and illuminating to find that, from the 236 flushing attempts, only 57 (24%) produced a morula. A major improvement in this unsatisfactory situation occurred when Weber et al11 and Freeman et al10 showed accelerated passage of the embryo through the oviduct and its resulting premature entry into the uterus on day 5 after ovulation in mares in which a mini-pump giving a low-dose, slow release of PGE2 was surgically implanted into the ipsilateral mesosalpinx of the ovary containing the new corpus luteum on day 4 after ovulation. This stimulated Robinson et al21 to attempt a more practical approach to hastening oviductal transport by dripping onto the ipsilateral oviduct on day 4 after ovulation a long acting triacetin-based gel formulation of PGE2a applied with the aid of a 0.5 ml straw in a disposable plastic equine embryo transfer gun passed through the working channel of a rigid laparoscope under local anaesthetic. Non-surgical flushing of the uterus one day later (day 5) yielded 12 morulae from 20 mares treated with the PGE2impregnated gel (60%) compared to no embryos on day 5 from 19 mares treated similarly with only the gel vehicle, 12 of which (63%) did produce an expanded blastocyst when re-flushed on day 8.21 Thus, it now seems safe to conclude that the 30year riddle of delayed and differential oviductal transport in the mare, posed by the startling original discovery of van Niekerk and Gerneke,1 has been solved. The local smooth muscle relaxing properties of the stage-dependent secretion of PGE2 by the day 5 morula seems to be the key to its 340 onward passage into the uterus. But the question of whether this unusual method of oviductal transport is no more than an evolutionary quirk in the mare, or is a necessary developmental mechanism to delay entry of the embryo into the uterus until such time as the latter is biologically ready and prepared to nurture the former, remains an interesting one for future investigation. Maternal Recognition of Pregnancy Short first coined the phrase “maternal recognition of pregnancy” when he highlighted the different strategies employed by the common domestic animal species to ensure continuation of the secretory function of the corpus luteum beyond its normal cyclical lifespan and so maintain the uterus in the correct progestational state to support pregnancy and the growth of the fetus.22 Prior to this time, a series of elegant experiments in sheep, cows, and pigs had demonstrated that: 1) the luteolytic hormone which induces cyclical regression of the corpus luteum is secreted by the endometrium; 2) this uterine luteolysin reaches the ovary by means of a local utero-ovarian transfer mechanism rather than via the peripheral circulation; and 3) one or more embryos must be present in the ipsilateral uterine horn between days 12 and 14 after ovulation to achieve the necessary luteostasis (see Moor15). Further and equally elegant experiments during the early 1970s established that: 1) prostaglandin F2␣ (PGF2␣) is the essential component of the uterine luteolysin in mammals; 2) it is released from the endometrium in spike-like pulses late in dioestrus; and 3) it reaches the corpus luteum via direct local countercurrent transfer between the uterine vein and the ovarian artery in the ovarian pedicle (see McCracken et al23). In the pig, Kidder et al24 and others reported that injections of estradiol benzoate given to cycling gilts between days 10 and 16 after ovulation would significantly prolong the secretory lifespan of the corpora lutea and so delay a return to estrus. Subsequently, Perry et al25 associated the dramatic elongation of the trophoblast by the pig embryo between days 10 and 14 after ovulation with the onset of its capacity to synthesize and secrete appreciable quantities of estrogens (Fig. 1b) and, a few years later Bazer and Thatcher26 published their now widely accepted hypothesis that embryonic estrogens function as the maternal recognition of pregnancy signal in the pig by redirecting the flow of endometrial PGF2␣ away from the uterine vein to an exocrine secretory route into the uterine lumen instead. Vigorous experimental activity in the 1980s unravelled the interactions and complexities of the mechanism which brings about maternal recognition of pregnancy in the sheep, cow, deer, and other ruminants. Namely, the synthesis and release of large quantities of a protein hormone, interferon tau, by the elongating trophoblast between days 10 and 16 after ovulation which suppresses the normal 2000 Ⲑ Vol. 46 Ⲑ AAEP PROCEEDINGS Proceedings of the Annual Convention of the AAEP 2000 Reprinted in the IVIS website with the permission of AAEP Close window to return to IVIS IN DEPTH: REPRODUCTION cyclical development of oxytocin receptors in the endometrium (Fig. 1b).27 This, in turn, prevents oxytocin secreted by the corpus luteum28 from binding to the endometrium and driving the pulsatile releases of PGF2␣ that would normally induce luteolysis in the cycling animal (see Lamming and Mann).29 The mare provides a distinct, and apparently unique, contrast to the pig and wide range of ruminant species in the manner in which its embryo transmits the all-important maternal recognition of pregnancy signal in early gestation. Enveloped in a tough and closely fitting glycocalyx capsule between days 6.5 and 22 after ovulation,30 the equine embryo is unable to rearrange and elongate its trophectoderm between days 10 and 14 after ovulation like its porcine and ruminant counterparts so as to bring trophoblast into close contact with a sizeable area of endometrium in the gravid uterine horn.25,31 Instead, the equine conceptus remains spherical and completely unattached within the uterine lumen, where it moves continually throughout the uterine domain, propelled by strong and peristaltic contractions of the myometrium (Fig. 1b).32,33 This unusual process of conceptus mobility in the mare persists until day 17 after ovulation when a sudden and spasm-like increase in myometrial tone immobilizes and “fixes” the conceptus at the site of eventual implantation at the base of one or other of the uterine horns.34,35 It is now clear that this constant movement of the equine conceptus throughout the uterus between days 7 and 17 after ovulation is an integral part of an evolutionary adaptation to ensure that the embryonic maternal recognition of pregnancy signal reaches the endometrium in all parts of the uterus. The utero-ovarian pedicle in ruminants which enables direct countercurrent transfer of endometrial PGF2␣ from the uterine vein to the ovarian artery, and thereby creates a very effective local ipsilateral uterine control of luteal lifespan, is absent in equids.36 Thus, endometrial prostaglandin can only reach the ovaries via the peripheral circulation which removes the possibility for any ipsilateral function of the uterus in the mare. Indeed, surgical restriction of the equine conceptus to only one-third of the total uterine area is followed by luteolysis and a return to estrus at the expected time of the estrous cycle, regardless of whether the unoccupied portion of the uterus is ipsilateral or contralateral to the ovary containing the corpus luteum.37 The nature of the signal by which the equine embryo “informs” the mare biochemically of its presence in her uterus, and so achieves the necessary luteostasis for pregnancy maintenance, remains a mystery. Unlike the ruminants, the equine conceptus does not produce any interferon-like protein molecules with luteostatic properties38 but, like the pig embryo, it does begin to secrete appreciable quantities of estrogens from as early as day 10 after ovulation.39 – 41 It has frequently been speculated that, like the situation in the pig in which the embryonic estrogens achieve luteostasis by re-directing the flow of endometrial PGF2␣ away from the uterine veinous drainage,26 embryonic estrogens may similarly constitute the maternal recognition of pregnancy signal in the mare. However, the many experiments undertaken to date to prove or disprove this theory have given equivocal results. For example, Vanderwall et al42 induced prolongation of luteal lifespan in only 6 of 11 mares into the uteri of which they surgically inserted an estradiol-17-releasing minipump intended to mimic a conceptus and 4 of 11 control mares showed an equivalent prolongation. Similarly, Ginther et al prolonged luteal lifespan in 2 of the 3 diestrous mares they injected daily with 5 mg estradiol and 2 of the 5 they injected with 100 ng of estrone during days 7–18 after ovulation.43 But Woodley et al prolonged the cycle in only one of 5 mares treated with 10 mg estradiol-17 per day and in none of 5 mares at each of 3 lower doses.44 More recently, Stout achieved similarly encouraging, although still equivocal, results when he treated diestrous mares, parenterally or by the intrauterine route, with estradiol-17.45 Four of 7 mares given a daily intramuscular (IM) injection of 20 mg estradiol benzoate between days 10 and 20 after ovulation passed into prolonged diestrous, as did 3 of 7 mares given an intrauterine silastic implant impregnated with estradiol 17 on day 8 after ovulation. Thus, on the face of it, around 60% of diestrous mares to which estrogens are administered parenterally over a number of days, or placed in the uterine lumen, undergo luteal prolongation. However, there is no obvious explanation to account for the 40% or so of mares that do not respond in this way to estrogen therapy. Clearly, more experimentation is required, with emphasis perhaps being placed on local intrauterine administration regimes of the most appropriate estrogen in the correct dose to better mimic the probably pulsatile releases of estrogen directly onto the lumenal surface of the endometrium (Fig. 1b) from the as yet non-vascularized choriovitelline membrane of the day 10 –16 conceptus as the latter is “squeezed” around the uterus by the remarkably powerful myometrial contractions.33,46 Despite the continuing uncertainty about the nature of the embryonic maternal recognition of pregnancy signal in equids, recent experiments have established convincingly that, as in ruminants, suppression of the normal cyclical upregulation of oxytocin receptors in the endometrium between days 10 and 16 after ovulation is an integral part of the luteostatic mechanism in the pregnant mare. Endometrial oxytocin receptor concentrations are greatly reduced in pregnant versus cycling mares between days 10 and 1647 and the normal spike-like releases of PGF2␣ from the endometrium, measured in plasma as 13,14 dihydro 15-keto PGF2␣ (PGFM), which occur in response to an intravenous (IV) injection of oxytocin between days 10 and 16 after AAEP PROCEEDINGS Ⲑ Vol. 46 Ⲑ 2000 Proceedings of the Annual Convention of the AAEP 2000 341 Reprinted in the IVIS website with the permission of AAEP Close window to return to IVIS IN DEPTH: REPRODUCTION Fig. 2. High-power photomicrograph of a section of a day 14 horse conceptus showing the bilaminar blastocyst capsule overlying and closely investing the single layer of trophectoderm cells which are stained with an anti-equine trophoblast antibody (F102.1). Photograph kindly supplied by Dr J. C. Oriol of the Dominican Republic. ovulation in the cycling mare, are abolished during the same period in pregnancy.47,48 It is of interest that the oxytocin involved in establishing this positive feedback loop with PGF2␣ to induce luteolysis in the cycling mare is, like the pig,49 secreted by the endometrium.50,51 This is in contrast to the ruminant species in which the oxytocin involved in the luteolytic pathway is secreted by the corpus luteum itself.52 One other fascinating anomaly in the mare is the ability of the equine conceptus to secrete appreciable quantities of both PGF2␣ and PGE2 when cultured in vitro (Fig. 1b).52 It is reasonable to assume that this prostanoic synthetic capacity of the choriovitelline membrane is necessary to stimulate locally the peristaltic contractions and relaxations of the myometrium required to propel the conceptus throughout the uterine lumen during the period of release of the maternal recognition of pregnancy factor. Indeed, such a hypothesis is supported by the recent finding of Stout and Allen that conceptus mobility is virtually abolished when the pregnant mare is treated with the prostaglandin synthetase inhibitor, flumixin meglumine.53 b The situation seems ironic, and no doubt reflects a finely balanced mechanism of action and interaction, that, in order to distribute its all important recognition message throughout the uterus, the equine conceptus must secrete the very hormone, PGF2␣, which is striving to prevent the neighboring maternal endometrium from releasing to ensure its survival in a progesterone-dominated uterus. One cannot help the suspicion that at least some of the relatively high proportion of the total pregnancy losses in the mare which occur between days 12 and 30 after ovulation (32%)54 stem not from any failure of release of sufficient maternal recognition of pregnancy factor from the conceptus to suppress the normal cyclical luteolytic pathway, but more from the secretion of too much PGF2␣ by the wandering conceptus (Fig. 1b) which then gains untoward access to the peripheral circulation and thereby accidentally induces luteolysis of the ultrasensitive corpus luteum. The resulting ultrasound scanning image, which is en342 countered occasionally by the stud farm veterinary clinician when scanning mares for pregnancy between days 14 and 18 after ovulation, is of a well developed and apparently normal conceptus surrounded by a clearly edematous endometrium that is heralding the imminent onset of true estrus and the resulting relaxation of the cervix, and leading to expulsion of the conceptus from the uterus. Development of the Fetal Membranes In addition to providing strength and elasticity to the expanding blastocyst to enable it to withstand the rigours of the myometrial contractions which propel it through the uterus,30 the equine blastocyst capsule is clearly also important in accumulating and regulating the supply of nutrients to the young, free-living conceptus.55 The capsular material is secreted initially by the trophectoderm cells from around day 6.5 and is molded into shape as it coagulates by the zona pellucida to create an intact envelope that completely surrounds the embryo.56 It would be reasonable to suppose that this process of molding within the zona would create an outer investment that would be snug and close-fitting from the outset (Fig. 2). Curiously, however, this is not the case and physical removal of the zona pellucida with the aid of the micromanipulator some hours before hatching would occur naturally, reveals a capsule that is creased and folded upon itself and which unfolds and expands rapidly, rather like a coiled spring being freed from restraint, as soon as the zona is removed.c This unusual process is presumably necessary to accommodate the rapid expansion of the blastocyst that does occur over the 2 or 3 days after it hatches from the zona pellucida57 but the physico-chemical mechanisms which enable an exocrine secretion to coagulate and harden in this manner in a series of “pleats,” yet at the same time creates a contiguous layer that can completely envelop the embryo within it, remains a fascinating area for future investigation. Due to its negative electrostatic charge and its unusual glycocalyx configuration,58 the outer surface of the capsule is very “sticky” to other proteins. 2000 Ⲑ Vol. 46 Ⲑ AAEP PROCEEDINGS Proceedings of the Annual Convention of the AAEP 2000 Reprinted in the IVIS website with the permission of AAEP Close window to return to IVIS IN DEPTH: REPRODUCTION Fig. 3. Videoendoscopic view of a 14-day horse conceptus bathed in endometrial gland secretions (“uterine milk”) as it moves freely about the uterine lumen to broadcast its maternal recognition of pregnancy luteostatic signal to the maternal endometrium. The capsule therefore functions to accumulate proteins and other components of the endometrial gland secretions (the “uterine milk”)59 onto its surface as the conceptus moves throughout the uterus between days 7 and 17 after ovulation. This accumulation process, of what is in effect the only source of nutrients for the rapidly growing embryo, is attested to both by a doubling in weight of the capsule between hatching of the blastocyst around day 7.5 after ovulation and immobilization of the conceptus at day 17,56 and by the very large quantities found adhered to, and almost incorporated into the structure of, the capsule of one component of uterine milk, the 19KDa progesterone-dependent protein called P19. This was first isolated, sequenced, and identified as a member of the lipocalin family of carrier proteins by Stewart et al60 and Crossett et al61 and it no doubt transports vital minerals and/or vitamins through the capsule to the underlying embryonic membranes and the primitive embryo itself.55 Thus, a free-living, fully encapsulated equine embryo that rattles around the maternal uterus for 10 days liberating significant quantities of estrogens and prostaglandins through the capsule in an outward direction to maintain progesterone-dominance of the uterus for its very existence, while at the same time imbibing quantities of protein-rich uterine milk through its capsule in the opposite or inward direction to sustain the growth and development it must undergo during this period (Fig. 3). Movement stops abruptly around day 17 with the sudden increase in myometrial tone, the precise underlying cause of which has yet to be determined although it is, quite reasonably, considered widely to be the result of an interaction between the longer than normal period of progesterone dominance from the now-prolonged maternal corpus luteum and the increasing quantity of estrogens secreted by the enlarging conceptus.41,43 The enveloping capsule also begins to disintegrate and melt away from around day 20 –21, presumably as a consequence of enzymes secreted by the trophoblast and/or lumenal epithelium of the endometrium.62 This once gain exposes the trophectoderm to the external environment which enables the rapid, although temporary, development of fingerlike tufts of trophoblast cells on the external surface of the non-vascularized bilaminar choriovitelline membrane. Termed aerolae by Amoroso,59 due to their structural similarity to the absorptive aerolae that cover the external surface of the similarly noninvasive allantochorion of the porcine placenta, these tufts protrude into the mouths of endometrial glands to provide important physical adherence of the conceptus to the endometrium and increase the extent and efficiency of imbibition of endometrial gland secretions. Their nutritional importance is shown by the high rate (i.e., 70 – 80%) of spontaneous death and resorbtion, between days 15 and 25 after ovulation, of one of twin conceptuses in mares in which both conceptuses become fixed together at the base of the same uterine horn (unilateral twins) in such a manner that the absorptive bilaminar choriovitelline portion of one conceptus abuts up against its co-twin conceptus rather than to the nutritionally provident endometrium (Fig. 4).33 Around day 20 –21 after ovulation the embryo itself becomes more clearly visible at one pole of the still spherical, but now increasingly capsule-free conceptus.63 Organogenesis is proceeding rapidly and the primitive embryonic heart is already pumping blood through the vitelline artery to the sinus terminalis, and through the myriad of tiny blood vessels developing within the advancing mesodermal tissue between the outer chorionic and inner yolk sac membranes (Fig. 5). The allantoic membrane first appears as an out-pouching of the embryonic hind gut around day 2164 and it grows rapidly to surround the embryo and fuse with the outer chorion to form the allantochorion that will eventually become the definitive placenta (Fig. 5). By day 25 the allantochorion constitutes about one-quarter of the total volume of the conceptus (Fig. 6a) and, over the next 15–20 days, it continues to enlarge rapidly to eventually replace the yolk sac completely by about day 45.63 The vascularized mesoderm continues to expand until, by day 33–35, it encompasses the whole conceptus apart from one small circle of bilaminar omphalopleure which persists within the sinus terminals at the abembryonic pole (Fig. 6b). This concomitant enlargement of the allantois above the embryo, and regression of the yolk sac beneath it, gives the optical illusion that, between about day 23 and 40, the embryo migrates from one pole of the conceptus to the other (Fig. 6a). In fact it is the pole that moves, not the embryo, and when serially scanning a mare over the same interval, the embryo appears to lift off the ventral floor of the uterus and rise steadily towards roof, apparently bisected all the while by the echogenic line created by the abutAAEP PROCEEDINGS Ⲑ Vol. 46 Ⲑ 2000 Proceedings of the Annual Convention of the AAEP 2000 343 Reprinted in the IVIS website with the permission of AAEP Close window to return to IVIS IN DEPTH: REPRODUCTION Fig. 5. Diagrammatic representation of the development and differentiation of the equine embryonic membranes between days 14 and 35 after ovulation. Fig. 4. Diagrammatic representations of two possible arrangements of day 18 unilateral twin conceptuses in the uterine horn of a mare. In the upper panel the non-vascularized highly absorptive bilaminar choriovitelline membrane of the anterior conceptus is abutted up against its posterior co-twin and is therefore prevented from imbibing uterine milk for its sustenance and growth. In the lower panel the absorptive bilaminar membranes of both conceptuses have the potential to absorb the endometrial gland secretions. ment within the conceptus of the enlarging allantois and the regressing yolk sac. The Endometrial Cup Reaction A unique and puzzling feature of equine embryogenesis is the development of the so-called chorionic girdle65 on the outer surface of the chorion between days 25 and 35 after ovulation (Fig. 6b)66 and its subsequent invasion of the maternal endometrium between days 36 and 38 to form the endometrial cups.67 The girdle is first seen around day 25 as a series of shallow undulations in the chorion which deepen markedly over the next 10 days to become elongated finger-like villous ridges due to the very rapid hyperplasia of the trophoblast cells at the tops of each fold (Fig. 7a). The resulting ridges become bent over and flattened due to the compressive effects of uterine tone and conceptus expansion and the clefts between adjacent ridges become gland-like in appearance and function.66 They begin to release increasing quantities of an alcian blue-positive exocrine secretion which adheres the outer surface of the girdle to the lumenal surface of the overlying 344 endometrium. Then, at around day 36, but with some temporal variation between individual mares, the entire girdle peels off the fetal membranes and the now binucleate girdle cells begin invading the maternal tissue (Figure 7b).67 In searching for an underlying mechanism to explain the rapid development of this discrete annulate band of highly invasive trophoblast cells situated adjacent to the otherwise non-invasive trophoblast of the allantochorion, Stewart et al observed that the girdle is thickest and best developed at its end next to the allantochorion but shows a definite thinning and general tapering off at the other end adjacent to the choriovitelline membrane.68 Furthermore, a series of small blood vessels extend from the highly vascularized mesoderm associated with the allantois into the space beneath the girdle to about halfway across the width of the latter. In the light of her previous in situ, hybridization studies of growth factor synthetic capabilities of the component membranes of the horse conceptus that allantoic mesenchyme is a major source of the highly mitogenic and motogenic growth factor, hepatocyte growth factor:scatter factor (HGF:SF) at this early stage of gestation,68 Stewart hypothesized that HGF:SF secreted by the allantoic mesenchyme acts as the principal mitogen to stimulate the rapid multiplication of both the trophoblast and the allantoic cells. Since these two membranes are fused together by the mesodermal tissue secreting the mitogen, and since the trophoblast cells are firmly attached to an underlying basement membrane, growth occurs as rapid and simple expansion of the allantochorion. But in the region of the chorionic girdle, which is not sited above allantoic mesoderm 2000 Ⲑ Vol. 46 Ⲑ AAEP PROCEEDINGS Proceedings of the Annual Convention of the AAEP 2000 Reprinted in the IVIS website with the permission of AAEP Close window to return to IVIS IN DEPTH: REPRODUCTION Fig. 6. Intact horse conceptuses at: a) 28 days and b) 35 days after ovulation. cg ⫽ chorionic girdle; e ⫽ embryo; ys ⫽ yolk sac. but is nonetheless still exposed to the mitogenic effects of the HGF:SF secreted by the extending mesenchymal blood vessels, the multiplying trophoblast cells can only pile up on each other, rather than expand in a linear manner. Thus, the discrete and thickened chorionic girdle develops (Fig. 7a).68 This growth factor-driven development of the chorionic girdle could also explain the striking and interacting effects of fetal genotype and uterine environment on both the development of the girdle and its subsequent hormone secreting capacity in the form of the endometrial cups it turns into. Namely, the girdle that develops on the conceptus of the donkey (Equus asinus ⫻ E. asinus, 2n ⫽ 62) and the hybrid mule (E. asinus 么 ⫻ E. caballus 乆, 2n ⫽ 63), both of which have a donkey as the sire, is very much narrower and less well developed at the time of invasion of the maternal endometrium around day 36 than its counterpart which develops on the conceptus of the horse (E. caballus ⫻ E. caballus, 2n ⫽ 64) and the reciprocal hybrid, the hinny (E. caballus 么 ⫻ E. asinus 乆, 2n ⫽ 63), both of which have a horse as the sire.69 While this difference might initially appear to be likely to be caused by maternal imprinting of genes associated with development of the chorionic girdle portion of the placenta, the dominant role of uterine environment on the whole process was illustrated dramatically by using embryo transfer to place one half of a bisected mule morula in the uterus of a recipient mare and the other half in the uterus of a recipient donkey.70 The mule conceptus in the mare developed a typically narrow chorionic girdle which gave rise to small endometrial cups with low hormone output ac ⫽ allantochorion; bo ⫽ bilaminar omphalopleure; whereas its other half in the donkey produced a very wide, thick and productive chorionic girdle, typical of that which develops on a hinny conceptus sired by a horse (Fig. 8). Thus, uterine environment was able to completely override any genetic effects which may have been operating.70 Returning to the invasion of the endometrium by the chorionic girdle at around day 36 –38 after ovulation, the now binucleate trophoblast cells pass both between, and occasionally straight through, the lumenal epithelial cells of the endometrium to reach the basement membrane below. They track down the endometrial glands (Fig. 7b), dislodging the lining epithelial cells as they go, before breaking through the basement membranes and streaming out into the endometrial stroma during day 38 – 40. Then, as though triggered by a developmental time switch, all the invading cells suddenly become sensile, round up, and enlarge greatly so as to become tightly packed together within the endometrial stroma. This gives rise to the protuberances, originally called endometrial cups by Schauder,71 that first become visible to the naked eye around day 40 as a series of pale, slightly raised plaques on the endometrial surface, arranged in a horseshoe or circle at the base of the gravid uterine horn and thereby mimicking the annulate chorionic girdle of the conceptus from which they originated.72,73 They vary in size and shape, from small circular structures of only 1–2 mm in diameter to long, unbroken ribbons of tissue that may be 3–5 cm in width and up to 30 cm in length (Fig. 9a). This range in dimensions stems from differences in the configuration of the endometrium at the time of invasion of AAEP PROCEEDINGS Ⲑ Vol. 46 Ⲑ 2000 Proceedings of the Annual Convention of the AAEP 2000 345 Reprinted in the IVIS website with the permission of AAEP Close window to return to IVIS IN DEPTH: REPRODUCTION Fig. 7. Low-power photomicrograph of: a) A day 35 horse chorionic girdle showing the folded back finger-like projections of rapidly multiplying trophoblast cells (⫻100); and b) The endometrium of a mare overlain by the invading chorionic girdle on day 38 after ovulation. The mass of girdle cells have eroded and ablated the lumenal epithelium and they can be seen traversing down the mouths of the endometrial glands, lifting the glandular epithelium of its basement membrane as they proceed (⫻156). the chorionic girdle, with the longer ribbons of cup tissue forming in areas where the endometrium opposing the chorionic girdle is flattened and nonundulating, while the smaller, isolated cups form on the tops of folds or ridges in a more undulating region of the endometrium which later become flattened out as the uterus expands with the growth of the conceptus. The cups reach their maximum size and productivity around day 60 –70 of gestation when they are elevated above the surface of the endometrium and appear saucer shaped and ulcer-like due to overgrowth at the edges and commencing cell degeneration in the central region (Fig. 9b). Histologically, each cup now consists of a densely packed mass of 346 the large binucleate epitheliod-type cells interspersed with occasional blood vessels and the dilated fundic portions of the endometrial glands, the apical regions and outlets of which were obliterated during the original invasion of the chorionic girdle around day 38.72,74 A collection of large lymph sinuses forms in the stroma beneath each cup and an increasing number of maternal leucocytes, consisting of CD4⫹ and CD8⫹ lymphocytes, plasma cells, macrophages, and eosinophils accumulate in the stroma at the periphery.74,75 Beyond day 70 the cups become increasingly pale and cheesy in appearance due to commencing degeneration and death of the large cup cells, especially in the central depression at the lumenal surface of the cup (Fig. 9c). Slough- 2000 Ⲑ Vol. 46 Ⲑ AAEP PROCEEDINGS Proceedings of the Annual Convention of the AAEP 2000 Reprinted in the IVIS website with the permission of AAEP Close window to return to IVIS IN DEPTH: REPRODUCTION Fig. 8. Comparison of the endometrial cups at day 60 of gestation produced by the chorionic girdles which developed on two halves of the same mule (乆 horse ⫻ 么 donkey) morula bisected on day 6 after ovulation. One demi embryo was transferred to the uterus of a horse recipient mare (a) and the other was transferred to the uterus of a recipient donkey (b). Note the small, narrow and already necrosing cups in the horse uterus (a) compared to the much larger and still active cups in the donkey uterus (b). ing of this necrotic surface tissue re-establishes outlets for the distended endometrial glands which then disgorge their accumulated secretory material onto the surface of the cup. It mixes with the necrosing cup cells to form a thick, honey-colored coagulum, termed endometrial cup secretion, which is exceedingly rich in eCG activity76 and adheres to the surface of the overlying allantochorion (Fig. 9d). Coincidentally, the lymphocytes accumulated at the periphery of the cup begin to actively invade the cup tissue and destroy the foreign fetal cup cells (Fig. 10). Eventually, between days 100 and 120 of gestation in most mares, but with considerable individual variation, the whole necrotic cup and its admixed, inspisated pabulum of exocrine secretion is sloughed off the surface of the endometrium where it will sometimes indent into the surface of the allantochorion to form a pendulous sac, termed an allantochorionic pouch72 which hangs into the allantoic cavity and is still readily visible in the term placenta some 200 days later. Two aspects of this unusual, short-lived, and biologically bizarre injection of specialized fetal trophoblast cells into the maternal endometrium are of significance in terms of the maintenance of equine pregnancy. Endocrinologically, the gonadotrophin (eCG) which is secreted in large quantities by the fetal cup cells is a high molecular weight glycoprotein78 which expresses both Follicle Stimulating Hormone (FSH)-like and Luteinzing Hormone (LH)like biological activities in roughly equal proportions.79 Concentrations of eCG in maternal serum rise rapidly from day 38 – 40 to reach a variable peak (20 –300 iu/ml) at around day 60 –70 and then decline again steadily in parallel with the steady degeneration and death of the endometrial cups.80,81 The hormone shows low binding affinity for gonadotrophin receptors in horse gonadal tissues82 but its LH-like component nonetheless ovulates, or lutenizes without ovulation, the dominant follicle in successive waves of follicles which are stimulated to develop during the first half of pregnancy by continuation of the 10 –12 day surge-like releases of pituitary FSH that control follicular development during the estrous cycle.83,84 Thus, secondary corpora lutea begin to accumulate in the maternal ovaries from the time of the very first appearance of eCG in maternal blood at around day 38 after ovulation with a consequential rise in maternal serum progesterone concentrations each time one of these accessory luteal structures develops (Fig. 11).85– 87 In addition to the rise in progesterone, the commencement of eCG secretion by the newly developed endometrial cups stimulates a sharp and pronounced rise in peripheral serum conjugated estrogen concentrations in the pregnant mare.84,88 These conjugated estrogens are ovarian in origin88 and the experiments of Daels et al87 have revealed they are secreted by the primary and/or secondary corpora lutea, rather than the Graffian follicles, in direct response to the gonadotrophic action of eCG (Fig. 11). Once risen in this manner, the serum estrogen levels tend to plateau, or even decline again slightly, until around day 70 – 80 when they begin a further and more prolonged rise that culminates in a relatively enormous peak in conjugated estrogen concentrations in both the blood and the urine of the mare around day 200 –240 of gestation.89,90 This time the estrogens are placental in origin and they include both the common phenolic estrogens, estrone and estradiol-17, and the unusual and equinespecific ring B unsaturated estrogen, equilin and equilenin,91 which are synthesized by placental aromatization of the large quantities of dihydroandrosterone (DHA) and dihydroepiandosterone (DHEA), and the rare 3-hydroxy-5,7-prenandien-20-one and 3-hydroxy-5,7 androstadien-17-one forms of these C-19 precursors, secreted by the dramatically enlarged gonads of the fetus.91–94 The gonads, both the ovaries in the female fetus and the testes in the AAEP PROCEEDINGS Ⲑ Vol. 46 Ⲑ 2000 Proceedings of the Annual Convention of the AAEP 2000 347 Reprinted in the IVIS website with the permission of AAEP Close window to return to IVIS IN DEPTH: REPRODUCTION Fig. 9. Endometrial cups (ec) in mares at different stages of pregnancy. a) A long unbroken ribbon of cup tissue seen at hysterotomy operation at day 45 after ovulation; b) Individual cups at day 60 of gestation; c) Aging cups exposed by retracting the allantochorion (ac) at day 83; the cups are now saucer-shaped and ulcer-like in appearance; d) Degenerating cups at day 98 showing the yellow, treacle-like endometrial cup secretion (ecs) adhered to the overlying allantochorion. Fig. 10. Photomicrograph of the base of an endometrial cup at day 87 of gestation. The accumulated maternal lymphocytes are seen migrating into the cup tissue and destroying the large, binucleate fetal cup cells (⫻100). male fetus, begin to enlarge from around day 80 of gestation to reach a maximum size around day 240, 348 when they occupy almost half the total volume of the abdomen of the fetus and are usually bigger than the now-inactive ovaries of the mare.95 Their growth is occasioned by a massive hypertrophy and hyperplasia of the interstitial cells of both types of gonad96 and they decline again steadily during the last quarter of pregnancy to more normal proportions and morphological configurations by the time the foal is born at around day 336 –340.77 Immunologically, the equine endometrial cup reaction is a huge puzzle. The invasive chorionic girdle trophoblast cells, but not the non-invasive trophoblast of the adjacent allantochorion, express high concentrations of paternally inherited Class I Major Histocompatibility Complex (MHC) antigens on their cell surface before, and for a few days after, they invade the maternal endometrium to form the endometrial cups.97,98 This blatant display of foreign antigenic molecules stimulates a strong humoral immune response in the mother such that all mares, including primigravid maidens, carrying fetuses which differ paternally at the Class I MHC barrier, develop high titres of specific anti-paternal lymphocytotoxic antibody in their serum within 2000 Ⲑ Vol. 46 Ⲑ AAEP PROCEEDINGS Proceedings of the Annual Convention of the AAEP 2000 Reprinted in the IVIS website with the permission of AAEP Close window to return to IVIS IN DEPTH: REPRODUCTION Fig. 11. Endocrinological functions of the equine endometrial cups. The dominant member of waves of ovarian follicles stimulated by continuing surge-like releases of pituitary FSH are ovulated and/or luteinized by the LH-like component of the equine Chorionic Gonadotrophin (eCG) secreted by the fetal chorionic girdle cells after they invade the maternal endometrium around day 38 to form the endometrial cups. The corpora lutea, both primary and secondary, secrete progesterone and conjugated estrogens in response to the gonadotrophic stimulus of eCG. 10 –14 days after initial invasion of the endometrium by the chorionic girdle at around day 36 –38.99,100 The antibody persists throughout pregnancy and it reappears anamnestically at earlier stages of gestation, and at even higher concentrations, in mares mated to the same MHCincompatible stallion in successive years and in mares transplanted with biopsies of skin from the stallion prior to mating.101 In addition, a very strong maternal cell-mediated reaction is mounted against the invading chorionic girdle cells. Lymphocytes appear in the endometrial stroma within hours after initial invasion by the chorionic girdle cells and their numbers increase dramatically from around day 60 –70, when they are joined by other mononuclear immune cells such as plasma cells, macrophages and eosinophils.73,74 Collectively these accumulated maternal immune cells form a definite barrier that separates fetal and maternal tissues and is reminiscent of the interface between grafted and host tissues during rejection of an allograft of skin. Initially, the accumulating leucocytes seem content to merely wall off the foreign fetal cells, but beyond day 60 –70 of gestation when the cells in the central region of the cup start to degenerate and die, the lymphocytes at the periphery begin to actively attack and destroy the fetal cup cells and they thereby hasten the death and eventual desquamation of the whole cup around day 100 –120.73 It is apparent that the paternally inherited Class I MHC antigens expressed by the invading chorionic girdle cells98,102 are the stimulus for the strong humoral maternal immune response to the fetus in equine pregnancy, but the nature of the foreign an- tigens that stimulate the equally strong cellular response is far less clear. The cups live as long, and secrete equivalent amounts of eCG, in mares carrying MHC-incompatible as MHC-compatible fetuses119 and the leucocytic response mounted against the cups is far more intense and destructive in mares carrying interspecific mule fetuses than it is in mares carrying normal intraspecific horse fetuses.73,103 Thus, it appears that species-specific non-MHC antigens, and possibly also tissue-specific trophoblast antigens, are involved in the cell-mediated response to the endometrial cups.104 The biological raison d’être for the endometrial cup reaction in equine pregnancy remains a mystery. Endocrinologically, the additional progesterone generated by the secondary corpora that are stimulated by the ovulatory and/or luteinizing properties of the relatively vast quantities of gonadotrophic hormone (eCG) secreted by the cups during their short lifespan, certainly supports the maintenance of the pregnancy state until day 100 of gestation or thereabouts when, as shown by the ovariectomy studies of Holtan et al,105 the placenta is now sufficiently well developed to take over completely the supply of enough progesterone to maintain the pregnancy state without any further contribution from the maternal ovaries. But, as demonstrated firstly by the survival to term of around 30% of extraspecific donkey-in-horse pregnancies, created by embryo transfer, in the complete absence of any detected endometrial cup formation and eCG secretion,106 and resulting failure of development of any secondary corpora lutea,103 and secondly by the marked reduction, or complete absence, of accessory ovulations in mares mated in late auAAEP PROCEEDINGS Ⲑ Vol. 46 Ⲑ 2000 Proceedings of the Annual Convention of the AAEP 2000 349 Reprinted in the IVIS website with the permission of AAEP Close window to return to IVIS IN DEPTH: REPRODUCTION Fig. 12. The “panic reaction” of the still unattached equine fetus around day 36 after ovulation to inject its specialized trophoblast of the chorionic girdle into the maternal endometrium to increase the supply of steroid hormones and suppress the potential immunological hostility of the endometrium. tumn so that they are seasonally deficient in pituitary FSH release during the first 100 days of pregnancy,73 secondary luteal progesterone is by no means obligatory to the maintenance of early pregnancy in the mare provided the primary corpus luteum does not undergo luteolysis for any untoward reason such as endotoxin production.87 Immunologically, it seems a very risky stratagem for an allotypic fetus, or worse still, a xenotypic fetus in the case of a mule,73 to deliberately immunize the dam against its paternally-derived histocompatibility antigens, merely for the sake of generating some extra, temporary luteal progesterone which is not absolutely necessary. Yet, curiously, it is in the one type of xenogeneic pregnancy, the extraspecific donkey-in-horse pregnancy created by embryo transfer, which does not have an endometrial cup reaction due to inadequate development and failure of the donkey chorionic girdle to invade the horse endometrium around day 36 after ovulation,107 that the majority (i.e., ⯝70%) of fetuses die and are aborted around day 80 –100 of gestation in conjunction with delayed and/or inadequate interdigitation of the allantochorion with the endometrium and a generalized and intense maternal leucocytic response throughout the endometrium in that is in contact with the xenogeneic donkey trophoblast.106 And, in these at-risk pregnancies, administration of either or both exogenous eCG and progesterone fails to reduce the high rate of pregnancy loss,106 whereas active immunization against donkey peripheral blood lymphocytes results in a marked increase in fetal survival above that in untreated control animals.103 350 Perhaps its injection of specialized hormone secreting and foreign antigen presenting trophoblast cells into the maternal endometrium represents something of developmental panic reaction on the part of the fetus to re-announce antigenically and endocrinologically its presence to the maternal organism after such a prolonged period of nonattachment and immunological indifference to the potentially hostile endometrium (Fig. 12). Certainly, a lack of normal interdigitation between the allantochorion and endometrium is the most striking abnormality of the unsuccessful donkey-in-horse pregnancy model in which endometrial cups do not develop and the associated maternal anti-paternal MHC humoral response is absent. This raises the possibility that some hitherto unknown influence of the whole endometrial cup reaction in equids is essential to stimulate the close and stable microvillous interaction between fetal and maternal epithelial layers which underpins and characterizes the whole process of placentation in the pregnant mare. Placentation Only as late as day 40 after ovulation, some 2 or 3 days after invasion of the endometrium by the chorionic girdle cells to start the endometrial cup reaction, does the non-invasive trophoblast of the now rapidly expanding and slowly elongating allantochorion begin to make a stable, microvillous attachment to the lumenal epithelial cells of the endometrium (Fig. 13a). During the next 20 days, blunt, fingerlike villi of allantochorion form a close fitting interdigitation with thinner, frond-like villi that develop on the endometrium, much like fingers being in- 2000 Ⲑ Vol. 46 Ⲑ AAEP PROCEEDINGS Proceedings of the Annual Convention of the AAEP 2000 Reprinted in the IVIS website with the permission of AAEP Close window to return to IVIS IN DEPTH: REPRODUCTION Fig. 14. Twin horse conceptuses in the excised uterus of a mare at an estimated 250 days of gestation. Note the much larger size of the fetus with the bigger area of placenta that occupies the body of the uterus, the non-gravid horn and the base of the gravid horn. The smaller co-twin has been pushed up to the tip of the gravid horn and is now beginning to suffer severe nutritional deprivation due to an inadequate area of placenta to meet its growth requirements. Fig. 13. Sections of the placental interface in pregnant mares. a) At day 43 showing close microvillous attachment of the trophoblast of the allantochorion to the lumenal epithelium of the endometrium and blunt villi of allantochorion beginning to indent into the surface of the allantochorion; b) At day 83 of gestation showing thickened and branched villi of allantochorion interdigitating with thinner, finger-like sulci of endometrium (⫻100). serted into a tight-fitting glove.107 Beyond day 60 the allantochorionic villi and accommodating endometrial sulci begin to branch extensively while at the same time becoming longer and deeper (Fig. 13b). This process of branching and lengthening of each primary villous and its opposing endometrium eventually creates, by about day 120 of gestation,108 the primary hemotrophic exchange unit of the noninvasive allantochorionic placenta known as the microcotyledon.109 The process maximizes the microscopic area of contact between the fetal and maternal epithelial layers for hemotrophic exchange of nutrients and waste products and it is aided by the close apposition to, and indentation into, the base of these epithelial layers by numerous blood capillaries, on both the fetal and maternal sides of the interface.110 Each microcotyledon is supplied with a sizeable artery on the maternal side and an equiv- alent placental vein on the fetal side to maximize the exchange process.111 In addition, the endometrial glands remain functional throughout gestation and they liberate their protein-rich exocrine secretions into well defined spaces between the microcotyledons. Here, the trophoblast cells become pseudostratified and are specially adapted to take up and absorb the exocrine material to establish a second, histotrophic form of nutrition for the rapidly growing fetus.112 Thus, by mid-gestation, and after an abnormally slow start, the diffuse non-invasive epitheliochorial equine placenta is established over the entire available area of endometrium and is providing both hemotrophic and histotrophic nutritional exchange for the fetal foal. Both the total gross area of the placenta, and the microscopic area of fetomaternal contact at the placental interface, continue to increase throughout the remainder of pregnancy to meet the fetal growth needs, and any diminution of this vast area of functional placenta, such as would occur in the case of twin conceptuses competing for the same limited area of endometrium (Fig. 14)113 or in older mares exhibiting age-related degenerative changes (endometrosis) in the endometrium,107,114,115 will lead at best to a degree of runting and weakness in the newborn foal, and at worst embryonic death and resorbtion early in gestation, or abortion in late pregnancy.108 An extensive and fully functional microcotyledonary placenta attached to a healthy and fully functional endometrium is an essential AAEP PROCEEDINGS Ⲑ Vol. 46 Ⲑ 2000 Proceedings of the Annual Convention of the AAEP 2000 351 Reprinted in the IVIS website with the permission of AAEP Close window to return to IVIS IN DEPTH: REPRODUCTION pre-requisite of normal pregnancy in the mare and the production of a healthy, well developed foal at term. The nature of the stimulus which both initiates placental interdigitation with the endometrium at day 40 after ovulation, and which drives the whole process of amazing growth and architectural modification of the endometrium and allantochorion throughout the remainder of gestation, is of great interest and remains something of a mystery. Locally produced growth factors almost certainly provide the main mitogenic impetus to placentation and insulin-like growth factor II (IGF-II) secreted by the trophoblast of the allantochorion and other fetal tissues throughout pregnancy116 and epidermal growth factor (EGF), secreted by the epithelium lining the apical portions of the endometrial glands, the genetic message (mRNA) for which is dramatically upregulated in these cells between days 35 and 40 of gestation,117 or after 40 days of exogenous progesterone administration in the non-pregnant mare,118 are the two most likely candidates. Conclusions So many aspects of embryonic survival, fetal development, and placentation remain puzzling in the mare. From the slow, PGE2-driven passage of the embryo down the oviduct, through the free-wheeling encapsulated movement of the embryo throughout the uterus to bring about maternal recognition of pregnancy, to the tenuous, myometrial tonecontrolled choriovitelline first attachment of the conceptus, and on through the bizarre and immunologically perilous process of endometrial cup development just prior to the final fetal utopia of stable and nutritious placentation, pregnancy in equids remains a mysterious and fascinating process that is well worthy of much further investigation. References and Notes 1. Van Niekerk CH, Gerneke WH. Persistence and parthenogenetic cleavage of tubal ova in the mare. Onderstepoort J Vet Res 1966;33:195–231. 2. Flood PF, Jong A, Betteridge KJ. The location of eggs retained in the oviducts of mares. J Reprod Fert 1979a;57: 291–294. 3. Hunter RF. The fallopian tubes: Their role in fertility and infertility. Berlin: Springer-Verlag, 1988. 4. Battut I, Colchen S, Fieni F, et al. Success rates when attempting to nonsurgically collect equine embryos at 144, 156 or 168 hours after ovulation. Equine Vet J 1998; 25(Suppl):60 – 62. 5. Betteridge KJ, Mitchell D. Direct evidence of retention of unfertilised ova in the oviduct of the mare. J Reprod Fert 1974;39:145–148. 6. David JSE. A survey of eggs in the oviducts of mares. J Reprod Fert 1975;23(Suppl):513–517. 7. Onuma H, Ohnami Y. Retention of tubal eggs in mares. J Reprod Fert 1975;23(Suppl):507–511. 8. Betteridge KJ, Eaglesome MD, Flood PF. Embryo transport through the mare’s oviduct depends upon cleavage and is independent of the ipsilateral corpus luteum. J Reprod Fert 1979;27(Suppl):387–394. 9. Weber JA, Freeman DA, Vanderwall DK, et al. Prostaglandin E2 secretion by oviductal transport-stage equine embryos. Biol Reprod 1991a;45:540 –543. 352 10. Freeman DA, Woods GL, Vanderwall DK, et al. Embryoinitiated oviductal transport in the mare. J Reprod Fert 1992;95:535–538. 11. Weber JA, Freeman DA, Vanderwall DK, et al. Prostaglandin E2 hastens oviductal transport of equine embryos. Biol Reprod 1991b;45:544 –546. 12. Weber JA, Woods GL, Freeman DA, et al. Prostaglandin E2-specific binding to the equine oviduct. Prostaglandins 1992;43:61– 65. 13. Weber JA, Woods GL, Lichtenwalner AB. Relaxatory effect of prostaglandin E2 on circular smooth muscle isolated from the equine oviductal isthmus. Biol Reprod 1995;1: 125–130. 14. Dzuik PJ, Polge EJC, Rowson LFA. Intrauterine migration and mixing of embryos in swine following egg transfer. J Anim Sci 1964;23:37– 40. 15. Moor RM. Effect of embryos on corpus luteum function. J Anim Sci 1968;1(Suppl):97–118. 16. Skidmore JA, Boyle MS, Cran D, et al. Micromanipulation of equine embryos to produce monozygotic twins. Equine Vet J 1989;8(Suppl):126 –128. 17. McKinnon AO, Carnevale EM, Squires EL, et al. Bisection of equine embryos. Equine Vet J 1989;8(Suppl):129 –133. 18. Skidmore JA, Boyle MS, Allen WR. A comparison of two different methods of freezing horse embryos. J Reprod Fert 1991;44(Suppl):714 –716. 19. Seidel GE. Cryopreservation of equine embryos. Vet Clin North Am [Equine Practice] 1997;12:85–99. 20. Boyle MS, Sanderson MW, Skidmore JA, et al. Use of serial progesterone measurements to assess cycle length, time of ovulation and timing of uterine flushes in order to recover equine morulae. Equine Vet J 1989;8(Suppl): 10 –13. 21. Robinson SJ, Neal H, Allen WR. Modulation of oviductal transport in the mare by local application of prostaglandin E2. J Reprod Fert 2000;56(Suppl):(in press). 22. Short RV. Implantation and the maternal recognition of pregnancy. In: Wolstenholme GEW, O’Connor M, eds. Ciba Foundation Symposium on Foetal Autonomy. London: J and A Churchill, 1969;2–26. 23. McCracken JA, Schramm W, Okulicz WC. Hormone receptor control of pulsatile secretion of PGF2␣ from the ovine uterus during luteolysis and its abrogation in early pregnancy. Anim Reprod Sci 1984;7:31–55. 24. Kidder HE, Casida LE, Grummer RH. Some effects of estrogen injections on the estrual cycle of gilts. J Anim Sci 1955;14:470 – 474. 25. Perry JS, Heap RB, Amoroso EC. Steroid hormone production by pig blastocysts. Nature 1973;245:45– 47. 26. Bazer FW, Thatcher WW. Theory of maternal recognition of pregnancy in swine based on estrogen controlled endocrine versus exocrine secretion of prostaglandin F2␣ by the uterine endometrium. Prostaglandins 1977;14:397– 401. 27. Lamming GE, Wathes DC, Flint APF, et al. Local action of trophoblast interferons in suppression of the development of oxytocin and estradiol receptors in ovine endometrium. J Reprod Fert 1995;105:165–175. 28. Flint APF, Sheldrick EL. Ovarian secretion of oxytocin is stimulated by prostaglandins. Nature 1982;297:587–588. 29. Lamming GE, Mann GE. Control of endometrial oxytocin receptors and prostaglandin F2␣ production in cows by progesterone and estradiol. J Reprod Fert 1995;103:69 –73. 30. Betteridge KJ. The structure and function of the equine capsule in relation to embryo manipulation and transfer. Equine Vet J 1989;8(Suppl):92–100. 31. Wooding FBP. The role of the binucleate cell in ruminant placental structure. J Reprod Fert 1982;31(Suppl):31–39. 32. Ginther OJ. Mobility of the early equine conceptus. Theriogenology 1983a;19:603– 611. 33. Ginther OJ. Dynamic physical interactions between the equine embryo and uterus. Equine Vet J 1985;3(Suppl): 41– 47. 2000 Ⲑ Vol. 46 Ⲑ AAEP PROCEEDINGS Proceedings of the Annual Convention of the AAEP 2000 Reprinted in the IVIS website with the permission of AAEP Close window to return to IVIS IN DEPTH: REPRODUCTION 34. Van Niekerk CH. The early diagnosis of pregnancy, the development of foetal membranes and nidation in the mare. J S Afr Vet Med Ass 1965;36:483– 488. 35. Ginther OJ. Fixation and orientation of the early equine conceptus. Theriogenology 1983b;19:613– 623. 36. Ginther OJ, Garcia MC, Squires EL, et al. Anatomy of vasculature of uterus and ovaries in the mare. Am J Vet Res 1972;33:1561–1568. 37. McDowell KJ, Sharp DC, Peck LJ, et al. Effect of restricted conceptus mobility on maternal recognition of pregnancy in mares. Equine Vet J 1985;3(Suppl):23–24. 38. Baker CB, Adams MH, McDowell KJ. Lack of expression of alpha or omega interferons by the horse conceptus. J Reprod Fert 1991;44(Suppl):439 – 443. 39. Zavy MT, Mayer R, Vernon MW, et al. An investigation of the uterine luminal environment of non-pregnant and pregnant pony mares. J Reprod Fert 1979;27(Suppl):403– 411. 40. Flood PF, Betteridge KJ, Irvine DS. Estrogens and androgens in blastocoelic fluid and cultures of cells from equine conceptuses of 10 –22 days gestation. J Reprod Fert 1979b; 27(Suppl):413– 420. 41. Heap RB, Hamon M, Allen WR. Studies on estrogen synthesis by the preimplantation equine conceptus. J Reprod Fert 1982;32(Suppl):343–352. 42. Vanderwall DK, Woods GL, Weber JA, et al. Corpus luteal function in non-pregnant mares following intrauterine administration of prostaglandin E2 or estradiol-17. Theriogenology 1994;42:1069 –1083. 43. Berg SL, Ginther OJ. Effect of estrogens on uterine tone and lifespan of the corpus luteum in mares. J Anim Sci 1978;47:203–208. 44. Woodley SL, Burns PJ, Douglas RH, et al. Prolonged interovulatory interval after estradiol treatment in mares. J Reprod Fert 1979;27(Suppl):205–209. 45. Stout TAE. Maternal recognition of pregnancy in the mare. PhD thesis, University of Cambridge, 1997. 46. Allen WR, Bracher V. Videoendoscopic evaluation of the mare’s uterus. III. Findings in the pregnant mare. Equine Vet J 1992;24:285–291. 47. Starbuck GR, Stout TAE, Lamming GE, et al. Endometrial oxytocin receptor and uterine prostaglandin secretion in mares during the estrous cycle and early pregnancy. J Reprod Fert 1998;113:173–179. 48. Goff AK, Pontbriand D, Sirois J. Oxytocin stimulation of plasma 15-keto-13, 14-dihydro prostaglandin F2␣ during the estrous cycle and early pregnancy in the mare. J Reprod Fert 1987;35(Suppl):253–260. 49. Boulton MI, McGrath TJ, Goode JA, et al. Changes in content of mRNA encoding oxytocin in the pig uterus during the estrous cycle, pregnancy, at parturition and in lactational anoestrus. J Reprod Fert 1996;108:219 –227. 50. Watson ED, Bjorkstein TS, Buckingham J, et al. Immunolocalization of oxytocin in the uterus of the mare. J Reprod Fert 1997;20:31. 51. Stout TAE, Lamming GE, Allen WR. Oxytocin and its endometrial receptor are integral to luteolysis in the cycling mare. J Reprod Fert 2000;56(Suppl):(in press). 52. Stout TAE, Allen WR. Conceptus factors involved in the maternal recognition of pregnancy in the mare. J Reprod Fert 1996;17:53. 53. Stout TAE, Allen WR. Prostaglandins drive intrauterine migration of the equine conceptus. J Reprod Fert 2000;(in press). 54. Morris LH-A, Allen WR. Reproductive efficiency in the Thoroughbred. Equine Vet J 2000;(in press). 55. Crossett B, Suire S, Herrler A, et al. Transfer of uterine lipocalin from the endometrium of the mare to the developing equine conceptus. Biol Reprod 1998;59:483– 490. 56. Oriol JG, Sharom FJ, Betteridge KJ. Developmentally regulated changes in the glycoproteins of the equine embryonic capsule. J Reprod Fert 1993a;99:653– 664. 57. McKinnon AO, Squires EL. Morphological assessment of equine embryos. J Am Vet Med Assoc 1988;192:401– 406. 58. Oriol JG, Betteridge KJ, Clarke AJ, et al. Mucin-like glycoproteins in the equine embryonic capsule. Mol Reprod Dev 1993b;34:255–265. 59. Amoroso EC. Placentation. In: Parkes AS, ed. Marshall’s Physiology of Reproduction. London: Longmans, Green and Co Ltd, 1952;127–311. 60. Stewart F, Charleston B, Crossett B, et al. A novel uterine protein that associates with the blastocyst capsule in equids. J Reprod Fert 1995a;105:65–70. 61. Crossett B, Allen WR, Stewart F. A 19 kDa protein secreted by the endometrium of the mare is a novel member of the lipocalin family. Biochem J 1996;320:137–143. 62. Denker HW, Betteridge KJ, Sirois J. Shedding of the capsule and proteinase activity in the horse embryo. J Reprod Fert 1987;35(Suppl):703. 63. Van Niekerk CH, Allen WR. Early embryonic development in the horse. J Reprod Fert 1975;23(Suppl):495– 498. 64. Ewart JC. Studies on the development of the horse. I. The development during the third week. Trans R Soc Edin 1915;51(2):287–329. 65. Ewart JC. A critical period in the development of the horse. London: Adam and Charles Black, 1897. 66. Allen WR, Moor RM. The origin of the equine endometrial cups. I. Production of PMSG by fetal trophoblast cells. J Reprod Fert 1972;29:313–316. 67. Allen WR, Hamilton DW, Moor RM. The origin of equine endometrial cups. II. Invasion of the endometrium by trophoblast. Anat Rec 1973;117:475–501. 68. Stewart F, Lennard SN, Allen WR. Mechanisms controlling formation of the equine chorionic girdle. Biol Reprod 1995b;1:151–159. 69. Allen WE. Ovarian changes during early pregnancy in Pony mares in relation to PMSG production. J Reprod Fert 1975;23:425– 428. 70. Allen WR, Skidmore JA, Stewart F, et al. Effects of fetal genotype and uterine environment on placental development in equids. J Reprod Fert 1993;97:55– 60. 71. Schauder W. Untersuchungen über die eithäute und embryotrophe des pferdes. Arch Anat Physiol 1912; 259 –302. 72. Clegg MT, Boda JM, Cole HH. The endometrial cups and allanto-chorionic pouches in the mare with emphasis on the source of equine gonadotrophin. Endocrinology 1954;54: 448 – 463. 73. Allen WR. The influence of fetal genotype upon endometrial cup development and PMSG and progestagen production in equids. J Reprod Fert 1975;23(Suppl):405– 413. 74. Amoroso EC. Endocrinology of pregnancy. Br Med Bull 1955;11:117–125. 75. Grünig GG, Triplett L, Canady LK, et al. The maternal leucocyte response to the endometrial cups in horses is correlated with the developmental stages of the invasive trophoblast cells. Placenta 1995;16:539 –559. 76. Rowlands IW. Levels of gonadotrophin in tissues and fluids with emphasis on domestic animals. In: Cole HH, ed. Gonadotrophins; their chemical and biological properties and secretory control. San Francisco: WH Freeman and Co, 1963;74 –112. 77. Allen WR. Equine gonadotrophins. PhD thesis, University of Cambridge, 1970. 78. Gospodorowicz D. Purification and physiochemical properties of the Pregnant Mare Serum Gonadotrophin (PMSG). Endocrinology 1972;1:101–106. 79. Stewart F, Allen WR, Moor RM. Pregnant mare serum gonadotrophin: Ratio of follicle-stimulating hormone and luteinizing hormone activities measured by radioreceptor assay. J Endocr 1976;71:371–382. 80. Cole HH, Hart GH. The potency of blood serum of mares in progressive stages of pregnancy in effecting the sexual maturity of the immature rat. Am J Physiol 1930;93:57– 68. 81. Allen WR. The immunological measurement of pregnant mare serum gonadotrophin. J Endocr 1969;32:593–598. 82. Stewart F, Allen WR. The binding of FSH, LH and PMSG to equine gonadal tissues. J Reprod Fert 1979;27:431– 440. AAEP PROCEEDINGS Ⲑ Vol. 46 Ⲑ 2000 Proceedings of the Annual Convention of the AAEP 2000 353 Reprinted in the IVIS website with the permission of AAEP Close window to return to IVIS IN DEPTH: REPRODUCTION 83. Evans MJ, Irvine CHG. Serum concentrations of FSH, LH and progesterone during the estrous cycle and early pregnancy in the mare. J Reprod Fert 1975;23(Suppl):193–200. 84. Urwin VE, Allen WR. Pituitary and chorionic gonadotrophin control of ovarian function during early pregnancy in equids. J Reprod Fert 1982;32(Suppl):371–382. 85. Amoroso EC, Hancock JL, Rowlands IW. Ovarian activity in the pregnant mare. Nature 1948;161:355–356. 86. Bain AM. The ovaries of the mare during early pregnancy. Vet Rec 1957;80:229 –231. 87. Daels PF, DeMoraes JJ, Stabenfeldt GH, et al. The corpus luteum: source of estrogen during early pregnancy in the mare. J Reprod Fert 1991;35(Suppl):501–508. 88. Terqui M, Palmer E. Estrogen pattern during early pregnancy in the mare. J Reprod Fert 1979;27(Suppl):441– 446. 89. Cox JE. Oestrone and equilin in the plasma of the pregnant mare. J Reprod Fert 1975;23(Suppl):463– 468. 90. Raeside JI, Liptrap RM. Patterns of urinary estrogen excretion in individual pregnant mares. J Reprod Fert 1975; 23(Suppl):469 – 475. 91. Bhavnani BR, Short RV, Solomon S. Formation of estrogens by the pregnant mare. I. Metabolism of 7-3H-dehydroisoandrosterone and 4-14C-androstenedione injected into the umbilical vein. Endocrinology 1969;85:1172–1179. 92. Pashen RL, Allen WR. The role of the fetal gonads and placenta in steroid production, maintenance of pregnancy and parturition in the mare. J Reprod Fert 1979;27(Suppl): 499 –509. 93. Tait AD, Hodge LC, Allen WR. Production of an equilin precursor by the fetal horse gonad. ICRS Med Sci 1982;10: 346 –347. 94. Tait AD, Santikarn LC, Allen WR. Identification of 3hydroxy-5,7 pregnandien-20-one and 3-hydroxy-5,7 androstadien-17-one as endogenous steroids in the foetal horse gonad. J Endocr 1983;99:87–92. 95. Cole HH, Hart GH, Lyons WR, et al. The development and hormonal content of fetal horse gonads. Anat Rec 1933;56: 275–293. 96. Hay MF, Allen WR. An ultrastructural and histochemical study of the interstitial cells in the gonads of the fetal horse. J Reprod Fert 1975;23(Suppl):557–561. 97. Crump A, Donaldson WL, Miller JM, et al. Expression of major histocompatibility complex (MHC) antigens on horse trophoblast. J Reprod Fert 1987;35(Suppl):379 –388. 98. Donaldson WL, Zhang CH, Oriol JG, et al. Invasive equine trophoblast expresses conventional class I major histocompatibility complex antigens. Development 1990;110:63–71. 99. Antczak DF, Bright SM, Remick LH, et al. Lymphocyte alloantigens of the horse. I. Serologic and genetic studies. Tissue Antigens 1982;20:172–187. 100. Kydd J, Miller J, Antczak DF, et al. Maternal anti-fetal cytotoxic antibody responses of equids during pregnancy. J Reprod Fert 1982;32(Suppl):361–369. 101. Antczak DF, Miller JM, Remick LH. Lymphocyte alloantigens of the horse. II. Antibodies to ELA antigens produced during equine pregnancy. J Reprod Immunol 1984; 6:283–297. 102. Kydd J, Butcher GW, Antczak DF, et al. Expression of Major Histocompatibility Complex (MHC) Class I molecules 354 103. 104. 105. 106. 107. 108. 109. 110. 111. 112. 113. 114. 115. 116. 117. 118. 119. on early equine trophoblast. J Reprod Fert 1991;44(Suppl): 463– 477. Allen WR, Kydd JH, Boyle MS, et al. Extra-specific donkey-in-horse pregnancy as a model of early fetal death. J Reprod Fert 1987;35(Suppl):197–209. Antczak DF, Allen WR. Invasive trophoblast in the genus Equus. In: Chaouat G, ed. The Riddle of the Foetal Allograft. Ann Immunol 1984;135D:301–351. Holtan DW, Squires EL, Lapin DR, et al. Effect of ovariectomy on pregnancy in mares. J Reprod Fert 1979; 27(Suppl):457– 463. Allen WR. Immunological aspects of the equine endometrial cup reaction and the effect of xenogeneic pregnancy in horses and donkeys. J Reprod Fert 1982;31(Suppl):57–94. Samuel CA, Allen WR, Steven DH. Studies on the equine placenta. I. Development of the micro-cotyledons. J Reprod Fert 1974;41:441– 445. Bracher V, Mathias S, Allen WR. Influence of chronic degenerative endometritis (endometrosis) on placental development in the mare. Equine Vet J 1996;28:180 –188. Samuel CA, Allen WR, Steven DH. Ultra-structural development of the equine placenta. J Reprod Fert 1975; 23(Suppl):575–578. Samuel CA, Allen WR, Steven DH. Studies on the equine placenta. II. Ultrastructure of the placental barrier. J Reprod Fert 1976;48:257–264. Steven DH, Samuel CA. Anatomy of the placental barrier in the mare. J Reprod Fert 1975;23(Suppl):579 –582. Samuel CA, Allen WR, Steven DH. Studies on the equine placenta. III. Ultrastructure of the uterine glands and the overlying trophoblast. J Reprod Fert 1977;51:433– 437. Jeffcott LB, Whitwell KE. Twinning as a cause of foetal and neonatal loss in Thoroughbred mares. J Comp Path 1973;83:91–106. Kenney RM. Cyclic and pathologic changes of the mare endometrium as detected by biopsy, with a note on early embryonic death. J Am Vet Med Assoc 1978;172:241–262. Bracher V, Mathias S, Allen WR. Videoendoscopic examination of the mare’s uterus. II. Findings in sub-fertile mares. Equine Vet J 1992;24:279 –284. Lennard SN, Stewart F, Allen WR. Insulin-like growth factor II gene expression in the fetus and placenta of the horse during the first half of gestation. J Reprod Fert 1995; 103:169 –179. Lennard SN, Gerstenberg C, Allen WR, et al. Expression of epidermal growth factor and its receptor in equine placental tissues. J Reprod Fert 1998;112:49 –57. Gerstenberg C, Allen WR, Stewart F. Factors controlling epidermal growth factor (EFG) gene expression in the endometrium of the mare. Mol Reprod Devel 1999;53:255–265. Allen WR, Kydd JH, Miller J, et al. Immunological studies on feto-maternal relationships in equine pregnancy. In: Crighton DB, ed. Immunological Aspects of Reproduction in Mammals. London: Butterworth, 1983;183–194. a Dinoprostin; Pharmacia-Upjohn, Crawley, Sussex, UK. Finadyne; Schering Plough, Middlesex, UK. c TAE Stout, personal communication. b 2000 Ⲑ Vol. 46 Ⲑ AAEP PROCEEDINGS Proceedings of the Annual Convention of the AAEP 2000