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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, EquineFertility Unit, Mertoun Paddocks, Woodditton Road, Newmarket, Suffolk CB8 9BH,United Kingdom. © 2000 AAEP.

Introduction

Many features of early pregnancy in the mare ap-pear to be unique to the genus Equus and are ofconsiderable academic interest and practical signif-icance. From the time of fertilization of the oocytesoon after ovulation until establishment of the ma-ture and fully functional placenta some 150 dayslater, a series of morphological, immunological, andendocrinological changes take place in the oviductand uterus which may be presumed to be importantcomponents of the establishment and maintenanceof the pregnancy state, but which differ markedlyfrom equivalent events in the other common largedomestic animal species and for which it is difficultto imagine a precise evolutionary reason for theiroccurrence. This paper aims to highlight a few ofthese equine pregnancy-related reproductive oddi-ties and discuss their significance in modern equineveterinary medicine.

Oviductal Transport

van Niekerk and Gerneke1 first drew attention tothe differential transport of oocytes and embryos inthe equine oviduct. Namely, if the freshly ovulatedoocyte remains unfertilized, it passes down the ovi-duct only to the ampullary-isthmus junction whereit remains lodged in the highly convoluted folds ofoviductal mucosa and degenerates slowly over manymonths.2 On the other hand, if the oocyte is fertil-ized by spermatozoa accumulated at the sperm res-ervoir in the same ampullary-isthmic region of theoviduct,3 the resulting embryo continues its onwardpassage and passes through the very constricted andprominent uterotubal junction (UTJ) to enter theuterus between 144 and 168 hours after ovulation.4

Thus, flushing the oviducts of mares post mortemtypically yields multiple flattened and degenerateoocytes accumulated from previous sterile ovula-tions in preceding estrous cycles,5,6 while the inter-vention of fertilization can result in the youngembryo bypassing the still-accumulated oocytes toenter the uterus at the expected time.7 What

mechanism could be responsible for such an unusualdifferential movement of gametes in the oviduct?

In early studies, Betteridge et al8 argued that theprocess of cleavage bestowed oviductal mobility onthe equine embryo, while Onuma and Ohnami7 andothers proposed that ultrastructural changes in thesurface of the zona pellucida during early develop-ment of the embryo enabled its selective propulsionthrough the oviduct lumen by the organized beatingof the cilia extruding from the apical surface of thelumenal epithelial cells. However, it was Weberand his colleagues in northwest America who even-tually provided the definitive answer to the puzzle ina series of elegant experiments that involved boththe culture of embryos in vitro9,10 and surgical im-plantation of mini-pumps to enable perfusion of hor-mones into the mesosalpinx, followed by embryorecovery attempts at fixed times after ovula-tion.11–13 In this way they demonstrated convinc-ingly that the embryo, but not the unfertilizedoocyte, begins secreting appreciable quantities ofprostaglandin E2 (PGE2) when it reaches the com-pact morula stage of development on day 5 afterovulation. The smooth muscle relaxing propertiesof this hormone act locally on the circular smoothmuscle fibers in the wall of the oviduct and therebyallow the embryo to move onwards, with the aid ofthe rhythmically beating cilia, to enter the uterusapproximately 24 hours later. Thus, it is the stage-dependent development of the hormone-secretingcapacity of the embryo, not any subtle change inmaternal recognition of size or structural changes inthe outermost coat of it, which brings about its de-sired onward movement to the uterus (Fig. 1a).

The protracted 6-day sojourn of the equine embryoin the oviduct compared to the 48-hour oviductalperiod of the 4-cell pig embryo14 and the 72-hourtransport time of the 8-cell ruminant embryo,15 hasdisadvantageous practical implications for embryotransfer and related embryo technologies in equids.For example, the bisection of embryos to produce

338 2000 / Vol. 46 / AAEP PROCEEDINGS

IN DEPTH: REPRODUCTION

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Fig. 1. Cartoon depicting: a) the differential rates of oviductal transport between the embryos of the pig, sheep and horse and theunique need for the latter to secrete PGE2 to relax the oviductal smooth muscle for its onward passage to the uterus; and b) thecontrasting mechanisms employed by the three species to achieve maternal recognition of pregnancy and luteostasis for maintenanceof the pregnancy state.

AAEP PROCEEDINGS / Vol. 46 / 2000 339



monozygotic (identical) twins is successful whenperformed on morulae but the success rate falls dra-matically if the embryo is showing even the earliestsigns of blastulation when bisected.16,17 Similarly,the success of deep-freezing embryos in liquid nitro-gen falls off sharply with increasing developmentalage and size of the embryo,18 due probably to acombination of damage to cells of the inner cell mass(ICM) and impermeability of the equine blastocystcapsule to cryoprotectants.19 In their painstakingand elegant study, Battut et al determined that themajority of horse embryos enter the uterus (fromwhich they can be recovered by simple non-surgicalflushing methods) between 144 and 156 hours afterovulation when they are at the late morula stage ofdevelopment and may already be beginning to blas-tulate.4 But even when the time of ovulation isknown to within a few hours by repeated ultrasoundscanning of the ovaries, flushing the uterus atclosely timed intervals between 144 and 156 hourslater yields embryos that differ markedly in theirstage of development. Similarly, in a large experi-ment designed to recover morulae for the purposesof bisection by flushing normal, fertile mares at fixedtimes after a carefully estimated time of ovulation,Boyle et al obtained a lower-than-normal overallembryo recovery rate of only 43% due to flushingsome mares too early when the embryo was still inthe oviduct.20 And it was disappointing and illumi-nating to find that, from the 236 flushing attempts,only 57 (24%) produced a morula.

A major improvement in this unsatisfactory situ-ation occurred when Weber et al11 and Freeman etal10 showed accelerated passage of the embryothrough the oviduct and its resulting premature en-try into the uterus on day 5 after ovulation in maresin which a mini-pump giving a low-dose, slow re-lease of PGE2 was surgically implanted into theipsilateral mesosalpinx of the ovary containing thenew corpus luteum on day 4 after ovulation. Thisstimulated Robinson et al21 to attempt a more prac-tical approach to hastening oviductal transport bydripping onto the ipsilateral oviduct on day 4 afterovulation a long acting triacetin-based gel formula-tion of PGE2

a applied with the aid of a 0.5 ml strawin a disposable plastic equine embryo transfer gunpassed through the working channel of a rigid lap-aroscope under local anaesthetic. Non-surgicalflushing of the uterus one day later (day 5) yielded12 morulae from 20 mares treated with the PGE2-impregnated gel (60%) compared to no embryos onday 5 from 19 mares treated similarly with only thegel vehicle, 12 of which (63%) did produce an ex-panded blastocyst when re-flushed on day 8.21

Thus, it now seems safe to conclude that the 30-year riddle of delayed and differential oviductaltransport in the mare, posed by the startling origi-nal discovery of van Niekerk and Gerneke,1 hasbeen solved. The local smooth muscle relaxingproperties of the stage-dependent secretion of PGE2by the day 5 morula seems to be the key to its

onward passage into the uterus. But the questionof whether this unusual method of oviductal trans-port is no more than an evolutionary quirk in themare, or is a necessary developmental mechanism todelay entry of the embryo into the uterus until suchtime as the latter is biologically ready and preparedto nurture the former, remains an interesting onefor future investigation.

Maternal Recognition of Pregnancy

Short first coined the phrase “maternal recognitionof pregnancy” when he highlighted the differentstrategies employed by the common domestic animalspecies to ensure continuation of the secretory func-tion of the corpus luteum beyond its normal cyclicallifespan and so maintain the uterus in the correctprogestational state to support pregnancy and thegrowth of the fetus.22 Prior to this time, a series ofelegant experiments in sheep, cows, and pigs haddemonstrated that: 1) the luteolytic hormonewhich induces cyclical regression of the corpus lu-teum is secreted by the endometrium; 2) this uterineluteolysin reaches the ovary by means of a localutero-ovarian transfer mechanism rather than viathe peripheral circulation; and 3) one or more em-bryos must be present in the ipsilateral uterine hornbetween days 12 and 14 after ovulation to achievethe necessary luteostasis (see Moor15). Furtherand equally elegant experiments during the early1970s established that: 1) prostaglandin F2a

(PGF2a) is the essential component of the uterineluteolysin in mammals; 2) it is released from theendometrium in spike-like pulses late in dioestrus;and 3) it reaches the corpus luteum via direct localcountercurrent transfer between the uterine veinand the ovarian artery in the ovarian pedicle (seeMcCracken et al23).

In the pig, Kidder et al24 and others reported thatinjections of estradiol benzoate given to cycling giltsbetween days 10 and 16 after ovulation would sig-nificantly prolong the secretory lifespan of thecorpora lutea and so delay a return to estrus. Sub-sequently, Perry et al25 associated the dramaticelongation of the trophoblast by the pig embryo be-tween days 10 and 14 after ovulation with the onsetof its capacity to synthesize and secrete appreciablequantities of estrogens (Fig. 1b) and, a few yearslater Bazer and Thatcher26 published their nowwidely accepted hypothesis that embryonic estro-gens function as the maternal recognition of preg-nancy signal in the pig by redirecting the flow ofendometrial PGF2a away from the uterine vein to anexocrine secretory route into the uterine lumen in-stead. Vigorous experimental activity in the 1980sunravelled the interactions and complexities of themechanism which brings about maternal recogni-tion of pregnancy in the sheep, cow, deer, and otherruminants. Namely, the synthesis and release oflarge quantities of a protein hormone, interferontau, by the elongating trophoblast between days 10and 16 after ovulation which suppresses the normal





cyclical development of oxytocin receptors in theendometrium (Fig. 1b).27 This, in turn, preventsoxytocin secreted by the corpus luteum28 from bind-ing to the endometrium and driving the pulsatilereleases of PGF2a that would normally induce lute-olysis in the cycling animal (see Lamming andMann).29

The mare provides a distinct, and apparentlyunique, contrast to the pig and wide range of rumi-nant species in the manner in which its embryotransmits the all-important maternal recognition ofpregnancy signal in early gestation. Enveloped ina tough and closely fitting glycocalyx capsule be-tween days 6.5 and 22 after ovulation,30 the equineembryo is unable to rearrange and elongate itstrophectoderm between days 10 and 14 after ovula-tion like its porcine and ruminant counterparts so asto bring trophoblast into close contact with a size-able area of endometrium in the gravid uterinehorn.25,31 Instead, the equine conceptus remainsspherical and completely unattached within theuterine lumen, where it moves continually through-out the uterine domain, propelled by strong andperistaltic contractions of the myometrium (Fig.1b).32,33 This unusual process of conceptus mobil-ity in the mare persists until day 17 after ovulationwhen a sudden and spasm-like increase in myome-trial tone immobilizes and “fixes” the conceptus atthe site of eventual implantation at the base of oneor other of the uterine horns.34,35

It is now clear that this constant movement of theequine conceptus throughout the uterus betweendays 7 and 17 after ovulation is an integral part ofan evolutionary adaptation to ensure that the em-bryonic maternal recognition of pregnancy signalreaches the endometrium in all parts of the uterus.The utero-ovarian pedicle in ruminants which en-ables direct countercurrent transfer of endometrialPGF2a from the uterine vein to the ovarian artery,and thereby creates a very effective local ipsilateraluterine control of luteal lifespan, is absent inequids.36 Thus, endometrial prostaglandin canonly reach the ovaries via the peripheral circulationwhich removes the possibility for any ipsilateralfunction of the uterus in the mare. Indeed, surgicalrestriction of the equine conceptus to only one-thirdof the total uterine area is followed by luteolysis anda return to estrus at the expected time of the estrouscycle, regardless of whether the unoccupied portionof the uterus is ipsilateral or contralateral to theovary containing the corpus luteum.37

The nature of the signal by which the equine em-bryo “informs” the mare biochemically of its pres-ence in her uterus, and so achieves the necessaryluteostasis for pregnancy maintenance, remains amystery. Unlike the ruminants, the equine concep-tus does not produce any interferon-like protein mol-ecules with luteostatic properties38 but, like the pigembryo, it does begin to secrete appreciable quanti-ties of estrogens from as early as day 10 after ovu-lation.39–41 It has frequently been speculated that,

like the situation in the pig in which the embryonicestrogens achieve luteostasis by re-directing theflow of endometrial PGF2a away from the uterineveinous drainage,26 embryonic estrogens may simi-larly constitute the maternal recognition of preg-nancy signal in the mare. However, the manyexperiments undertaken to date to prove or disprovethis theory have given equivocal results. For exam-ple, Vanderwall et al42 induced prolongation of lu-teal lifespan in only 6 of 11 mares into the uteri ofwhich they surgically inserted an estradiol-17b-re-leasing minipump intended to mimic a conceptusand 4 of 11 control mares showed an equivalentprolongation. Similarly, Ginther et al prolongedluteal lifespan in 2 of the 3 diestrous mares theyinjected daily with 5 mg estradiol and 2 of the 5 theyinjected with 100 ng of estrone during days 7–18after ovulation.43 But Woodley et al prolonged thecycle in only one of 5 mares treated with 10 mgestradiol-17b per day and in none of 5 mares at eachof 3 lower doses.44 More recently, Stout achievedsimilarly encouraging, although still equivocal, re-sults when he treated diestrous mares, parenterallyor by the intrauterine route, with estradiol-17b.45

Four of 7 mares given a daily intramuscular (IM)injection of 20 mg estradiol benzoate between days10 and 20 after ovulation passed into prolongeddiestrous, as did 3 of 7 mares given an intrauterinesilastic implant impregnated with estradiol 17b onday 8 after ovulation. Thus, on the face of it,around 60% of diestrous mares to which estrogensare administered parenterally over a number ofdays, or placed in the uterine lumen, undergo lutealprolongation. However, there is no obvious expla-nation to account for the 40% or so of mares that donot respond in this way to estrogen therapy.Clearly, more experimentation is required, with em-phasis perhaps being placed on local intrauterineadministration regimes of the most appropriate es-trogen in the correct dose to better mimic the prob-ably pulsatile releases of estrogen directly onto thelumenal surface of the endometrium (Fig. 1b) fromthe as yet non-vascularized choriovitelline mem-brane of the day 10–16 conceptus as the latter is“squeezed” around the uterus by the remarkablypowerful myometrial contractions.33,46

Despite the continuing uncertainty about the na-ture of the embryonic maternal recognition of preg-nancy signal in equids, recent experiments haveestablished convincingly that, as in ruminants, sup-pression of the normal cyclical upregulation of oxy-tocin receptors in the endometrium between days 10and 16 after ovulation is an integral part of theluteostatic mechanism in the pregnant mare. En-dometrial oxytocin receptor concentrations aregreatly reduced in pregnant versus cycling maresbetween days 10 and 1647 and the normal spike-likereleases of PGF2a from the endometrium, measuredin plasma as 13,14 dihydro 15-keto PGF2a (PGFM),which occur in response to an intravenous (IV) in-jection of oxytocin between days 10 and 16 after





ovulation in the cycling mare, are abolished duringthe same period in pregnancy.47,48 It is of interestthat the oxytocin involved in establishing this posi-tive feedback loop with PGF2a to induce luteolysis inthe cycling mare is, like the pig,49 secreted by theendometrium.50,51 This is in contrast to the rumi-nant species in which the oxytocin involved in theluteolytic pathway is secreted by the corpus luteumitself.52

One other fascinating anomaly in the mare is theability of the equine conceptus to secrete appreciablequantities of both PGF2a and PGE2 when cultured invitro (Fig. 1b).52 It is reasonable to assume thatthis prostanoic synthetic capacity of the chorio-vitelline membrane is necessary to stimulate locallythe peristaltic contractions and relaxations of themyometrium required to propel the conceptusthroughout the uterine lumen during the period ofrelease of the maternal recognition of pregnancyfactor. Indeed, such a hypothesis is supported bythe recent finding of Stout and Allen that conceptusmobility is virtually abolished when the pregnantmare is treated with the prostaglandin synthetaseinhibitor, flumixin meglumine.53 b The situationseems ironic, and no doubt reflects a finely balancedmechanism of action and interaction, that, in orderto distribute its all important recognition messagethroughout the uterus, the equine conceptus mustsecrete the very hormone, PGF2a, which is strivingto prevent the neighboring maternal endometriumfrom releasing to ensure its survival in a progester-one-dominated uterus. One cannot help the suspi-cion that at least some of the relatively highproportion of the total pregnancy losses in the marewhich occur between days 12 and 30 after ovulation(32%)54 stem not from any failure of release of suf-ficient maternal recognition of pregnancy factorfrom the conceptus to suppress the normal cyclicalluteolytic pathway, but more from the secretion oftoo much PGF2a by the wandering conceptus (Fig.1b) which then gains untoward access to the periph-eral circulation and thereby accidentally induces lu-teolysis of the ultrasensitive corpus luteum. Theresulting ultrasound scanning image, which is en-

countered occasionally by the stud farm veterinaryclinician when scanning mares for pregnancy be-tween days 14 and 18 after ovulation, is of a welldeveloped and apparently normal conceptus sur-rounded by a clearly edematous endometrium thatis heralding the imminent onset of true estrus andthe resulting relaxation of the cervix, and leading toexpulsion of the conceptus from the uterus.

Development of the Fetal Membranes

In addition to providing strength and elasticity tothe expanding blastocyst to enable it to withstandthe rigours of the myometrial contractions whichpropel it through the uterus,30 the equine blastocystcapsule is clearly also important in accumulatingand regulating the supply of nutrients to the young,free-living conceptus.55 The capsular material issecreted initially by the trophectoderm cells fromaround day 6.5 and is molded into shape as it coag-ulates by the zona pellucida to create an intact en-velope that completely surrounds the embryo.56

It would be reasonable to suppose that this processof molding within the zona would create an outerinvestment that would be snug and close-fittingfrom the outset (Fig. 2). Curiously, however, this isnot the case and physical removal of the zona pellu-cida with the aid of the micromanipulator somehours before hatching would occur naturally, revealsa capsule that is creased and folded upon itself andwhich unfolds and expands rapidly, rather like acoiled spring being freed from restraint, as soon asthe zona is removed.c This unusual process is pre-sumably necessary to accommodate the rapid expan-sion of the blastocyst that does occur over the 2 or 3days after it hatches from the zona pellucida57 butthe physico-chemical mechanisms which enable anexocrine secretion to coagulate and harden in thismanner in a series of “pleats,” yet at the same timecreates a contiguous layer that can completely en-velop the embryo within it, remains a fascinatingarea for future investigation.

Due to its negative electrostatic charge and itsunusual glycocalyx configuration,58 the outer sur-face of the capsule is very “sticky” to other proteins.

Fig. 2. High-power photomicrograph of a section of a day 14 horse conceptus showing the bilaminar blastocyst capsule overlying andclosely 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.





The capsule therefore functions to accumulate pro-teins and other components of the endometrial glandsecretions (the “uterine milk”)59 onto its surface asthe conceptus moves throughout the uterus betweendays 7 and 17 after ovulation. This accumulationprocess, of what is in effect the only source of nutri-ents for the rapidly growing embryo, is attested toboth by a doubling in weight of the capsule betweenhatching of the blastocyst around day 7.5 after ovu-lation and immobilization of the conceptus at day17,56 and by the very large quantities found adheredto, and almost incorporated into the structure of, thecapsule of one component of uterine milk, the 19KDaprogesterone-dependent protein called P19. Thiswas first isolated, sequenced, and identified as amember of the lipocalin family of carrier proteins byStewart et al60 and Crossett et al61 and it no doubttransports vital minerals and/or vitamins throughthe capsule to the underlying embryonic membranesand the primitive embryo itself.55

Thus, a free-living, fully encapsulated equine em-bryo that rattles around the maternal uterus for 10days liberating significant quantities of estrogensand prostaglandins through the capsule in an out-ward direction to maintain progesterone-dominanceof the uterus for its very existence, while at the sametime imbibing quantities of protein-rich uterine milkthrough its capsule in the opposite or inward direc-tion to sustain the growth and development it mustundergo during this period (Fig. 3). Movementstops abruptly around day 17 with the sudden in-crease in myometrial tone, the precise underlyingcause of which has yet to be determined although itis, quite reasonably, considered widely to be theresult of an interaction between the longer thannormal period of progesterone dominance from thenow-prolonged maternal corpus luteum and the in-creasing quantity of estrogens secreted by the en-larging conceptus.41,43

The enveloping capsule also begins to disintegrateand melt away from around day 20–21, presumablyas a consequence of enzymes secreted by the tropho-blast and/or lumenal epithelium of the endome-trium.62 This once gain exposes the trophectodermto the external environment which enables therapid, although temporary, development of finger-like tufts of trophoblast cells on the external surfaceof the non-vascularized bilaminar choriovitellinemembrane. Termed aerolae by Amoroso,59 due totheir structural similarity to the absorptive aerolaethat cover the external surface of the similarly non-invasive allantochorion of the porcine placenta,these tufts protrude into the mouths of endometrialglands to provide important physical adherence ofthe conceptus to the endometrium and increase theextent and efficiency of imbibition of endometrialgland secretions. Their nutritional importance isshown by the high rate (i.e., 70–80%) of spontane-ous death and resorbtion, between days 15 and 25after ovulation, of one of twin conceptuses in maresin which both conceptuses become fixed together atthe base of the same uterine horn (unilateral twins)in such a manner that the absorptive bilaminarchoriovitelline portion of one conceptus abuts upagainst its co-twin conceptus rather than to the nu-tritionally provident endometrium (Fig. 4).33

Around day 20–21 after ovulation the embryo it-self becomes more clearly visible at one pole of thestill spherical, but now increasingly capsule-freeconceptus.63 Organogenesis is proceeding rapidlyand the primitive embryonic heart is already pump-ing blood through the vitelline artery to the sinusterminalis, and through the myriad of tiny bloodvessels developing within the advancing mesoder-mal tissue between the outer chorionic and inneryolk sac membranes (Fig. 5). The allantoic mem-brane first appears as an out-pouching of the embry-onic hind gut around day 2164 and it grows rapidly tosurround the embryo and fuse with the outer cho-rion to form the allantochorion that will eventuallybecome the definitive placenta (Fig. 5). By day 25the allantochorion constitutes about one-quarter ofthe total volume of the conceptus (Fig. 6a) and, overthe next 15–20 days, it continues to enlarge rapidlyto eventually replace the yolk sac completely byabout day 45.63 The vascularized mesoderm con-tinues to expand until, by day 33–35, it encompassesthe whole conceptus apart from one small circle ofbilaminar omphalopleure which persists within thesinus terminals at the abembryonic pole (Fig. 6b).This concomitant enlargement of the allantois abovethe embryo, and regression of the yolk sac beneathit, gives the optical illusion that, between about day23 and 40, the embryo migrates from one pole of theconceptus to the other (Fig. 6a). In fact it is thepole that moves, not the embryo, and when seriallyscanning a mare over the same interval, the embryoappears to lift off the ventral floor of the uterus andrise steadily towards roof, apparently bisected allthe while by the echogenic line created by the abut-

Fig. 3. Videoendoscopic view of a 14-day horse conceptus bathedin endometrial gland secretions (“uterine milk”) as it moves freelyabout the uterine lumen to broadcast its maternal recognition ofpregnancy luteostatic signal to the maternal endometrium.





ment within the conceptus of the enlarging allantoisand the regressing yolk sac.

The Endometrial Cup Reaction

A unique and puzzling feature of equine embryo-genesis is the development of the so-called chorionicgirdle65 on the outer surface of the chorion betweendays 25 and 35 after ovulation (Fig. 6b)66 and itssubsequent invasion of the maternal endometriumbetween days 36 and 38 to form the endometrialcups.67 The girdle is first seen around day 25 as aseries of shallow undulations in the chorion whichdeepen markedly over the next 10 days to becomeelongated finger-like villous ridges due to the veryrapid hyperplasia of the trophoblast cells at the topsof each fold (Fig. 7a). The resulting ridges becomebent over and flattened due to the compressive ef-fects of uterine tone and conceptus expansion andthe clefts between adjacent ridges become gland-likein appearance and function.66 They begin to re-lease increasing quantities of an alcian blue-positiveexocrine secretion which adheres the outer surfaceof the girdle to the lumenal surface of the overlying

endometrium. Then, at around day 36, but withsome temporal variation between individual mares,the entire girdle peels off the fetal membranes andthe now binucleate girdle cells begin invading thematernal tissue (Figure 7b).67

In searching for an underlying mechanism to ex-plain the rapid development of this discrete annu-late band of highly invasive trophoblast cellssituated adjacent to the otherwise non-invasive tro-phoblast of the allantochorion, Stewart et al ob-served that the girdle is thickest and best developedat its end next to the allantochorion but shows a def-inite thinning and general tapering off at the otherend adjacent to the choriovitelline membrane.68

Furthermore, a series of small blood vessels ex-tend from the highly vascularized mesoderm associ-ated with the allantois into the space beneath thegirdle to about halfway across the width of the lat-ter. In the light of her previous in situ, hybridiza-tion studies of growth factor synthetic capabilities ofthe component membranes of the horse conceptusthat allantoic mesenchyme is a major source of thehighly mitogenic and motogenic growth factor, he-patocyte growth factor:scatter factor (HGF:SF) atthis early stage of gestation,68 Stewart hypothesizedthat HGF:SF secreted by the allantoic mesenchymeacts as the principal mitogen to stimulate the rapidmultiplication of both the trophoblast and the allan-toic cells. Since these two membranes are fusedtogether by the mesodermal tissue secreting the mi-togen, and since the trophoblast cells are firmly at-tached to an underlying basement membrane,growth occurs as rapid and simple expansion of theallantochorion. But in the region of the chorionicgirdle, which is not sited above allantoic mesoderm

Fig. 4. Diagrammatic representations of two possible arrange-ments of day 18 unilateral twin conceptuses in the uterine horn ofa mare. In the upper panel the non-vascularized highly absorp-tive bilaminar choriovitelline membrane of the anterior concep-tus is abutted up against its posterior co-twin and is thereforeprevented from imbibing uterine milk for its sustenance andgrowth. In the lower panel the absorptive bilaminar membranesof both conceptuses have the potential to absorb the endometrialgland secretions.

Fig. 5. Diagrammatic representation of the development anddifferentiation of the equine embryonic membranes between days14 and 35 after ovulation.





but is nonetheless still exposed to the mitogeniceffects of the HGF:SF secreted by the extendingmesenchymal blood vessels, the multiplying tropho-blast cells can only pile up on each other, ratherthan expand in a linear manner. Thus, the discreteand thickened chorionic girdle develops (Fig. 7a).68

This growth factor-driven development of the cho-rionic girdle could also explain the striking and in-teracting effects of fetal genotype and uterineenvironment on both the development of the girdleand its subsequent hormone secreting capacity inthe form of the endometrial cups it turns into.Namely, the girdle that develops on the conceptus ofthe donkey (Equus asinus 3 E. asinus, 2n 5 62) andthe hybrid mule (E. asinus ? 3 E. caballus /, 2n 563), both of which have a donkey as the sire, is verymuch narrower and less well developed at the timeof invasion of the maternal endometrium aroundday 36 than its counterpart which develops on theconceptus of the horse (E. caballus 3 E. caballus,2n 5 64) and the reciprocal hybrid, the hinny (E.caballus ? 3 E. asinus /, 2n 5 63), both of whichhave a horse as the sire.69 While this differencemight initially appear to be likely to be caused bymaternal imprinting of genes associated with devel-opment of the chorionic girdle portion of the pla-centa, the dominant role of uterine environment onthe whole process was illustrated dramatically byusing embryo transfer to place one half of a bisectedmule morula in the uterus of a recipient mare andthe other half in the uterus of a recipient donkey.70

The mule conceptus in the mare developed a typi-cally narrow chorionic girdle which gave rise tosmall endometrial cups with low hormone output

whereas its other half in the donkey produced a verywide, thick and productive chorionic girdle, typicalof that which develops on a hinny conceptus sired bya horse (Fig. 8). Thus, uterine environment wasable to completely override any genetic effects whichmay have been operating.70

Returning to the invasion of the endometrium bythe chorionic girdle at around day 36–38 after ovu-lation, the now binucleate trophoblast cells passboth between, and occasionally straight through, thelumenal epithelial cells of the endometrium to reachthe basement membrane below. They track downthe endometrial glands (Fig. 7b), dislodging the lin-ing epithelial cells as they go, before breakingthrough the basement membranes and streamingout into the endometrial stroma during day 38–40.Then, as though triggered by a developmental timeswitch, all the invading cells suddenly become sen-sile, round up, and enlarge greatly so as to becometightly packed together within the endometrialstroma. This gives rise to the protuberances, orig-inally called endometrial cups by Schauder,71 thatfirst become visible to the naked eye around day 40as a series of pale, slightly raised plaques on theendometrial surface, arranged in a horseshoe orcircle at the base of the gravid uterine hornand thereby mimicking the annulate chorionic gir-dle of the conceptus from which they originated.72,73

They vary in size and shape, from small circularstructures of only 1–2 mm in diameter to long, un-broken ribbons of tissue that may be 3–5 cm in widthand up to 30 cm in length (Fig. 9a). This range indimensions stems from differences in the configura-tion of the endometrium at the time of invasion of

Fig. 6. Intact horse conceptuses at: a) 28 days and b) 35 days after ovulation. ac 5 allantochorion; bo 5 bilaminar omphalopleure;cg 5 chorionic girdle; e 5 embryo; ys 5 yolk sac.





the chorionic girdle, with the longer ribbons of cuptissue forming in areas where the endometrium op-posing the chorionic girdle is flattened and non-undulating, while the smaller, isolated cups form onthe tops of folds or ridges in a more undulatingregion of the endometrium which later become flat-tened out as the uterus expands with the growth ofthe conceptus.

The cups reach their maximum size and produc-tivity around day 60–70 of gestation when they areelevated above the surface of the endometrium andappear saucer shaped and ulcer-like due to over-growth at the edges and commencing cell degenera-tion in the central region (Fig. 9b). Histologically,each cup now consists of a densely packed mass of

the large binucleate epitheliod-type cells inter-spersed with occasional blood vessels and the dilatedfundic portions of the endometrial glands, the apicalregions and outlets of which were obliterated duringthe original invasion of the chorionic girdle aroundday 38.72,74 A collection of large lymph sinusesforms in the stroma beneath each cup and an in-creasing number of maternal leucocytes, consistingof CD41 and CD81 lymphocytes, plasma cells, mac-rophages, and eosinophils accumulate in the stromaat the periphery.74,75 Beyond day 70 the cups be-come increasingly pale and cheesy in appearancedue to commencing degeneration and death of thelarge cup cells, especially in the central depressionat the lumenal surface of the cup (Fig. 9c). Slough-

Fig. 7. Low-power photomicrograph of: a) A day 35 horse chorionic girdle showing the folded back finger-like projections of rapidlymultiplying trophoblast cells (3100); and b) The endometrium of a mare overlain by the invading chorionic girdle on day 38 afterovulation. The mass of girdle cells have eroded and ablated the lumenal epithelium and they can be seen traversing down the mouthsof the endometrial glands, lifting the glandular epithelium of its basement membrane as they proceed (3156).





ing of this necrotic surface tissue re-establishes out-lets for the distended endometrial glands which thendisgorge their accumulated secretory material ontothe surface of the cup. It mixes with the necrosingcup cells to form a thick, honey-colored coagulum,termed endometrial cup secretion, which is ex-ceedingly rich in eCG activity76 and adheres to thesurface of the overlying allantochorion (Fig. 9d).Coincidentally, the lymphocytes accumulated at theperiphery of the cup begin to actively invade the cuptissue and destroy the foreign fetal cup cells (Fig.10). Eventually, between days 100 and 120 of ges-tation in most mares, but with considerable individ-ual variation, the whole necrotic cup and itsadmixed, inspisated pabulum of exocrine secretionis sloughed off the surface of the endometriumwhere it will sometimes indent into the surface ofthe allantochorion to form a pendulous sac, termedan allantochorionic pouch72 which hangs into the

allantoic cavity and is still readily visible in the termplacenta some 200 days later.

Two aspects of this unusual, short-lived, and bio-logically bizarre injection of specialized fetal tropho-blast cells into the maternal endometrium are ofsignificance in terms of the maintenance of equinepregnancy. Endocrinologically, the gonadotrophin(eCG) which is secreted in large quantities by thefetal cup cells is a high molecular weight glycopro-tein78 which expresses both Follicle StimulatingHormone (FSH)-like and Luteinzing Hormone (LH)-like biological activities in roughly equal propor-tions.79 Concentrations of eCG in maternal serumrise rapidly from day 38–40 to reach a variable peak(20–300 iu/ml) at around day 60–70 and then de-cline again steadily in parallel with the steady de-generation and death of the endometrial cups.80,81

The hormone shows low binding affinity for gonado-trophin receptors in horse gonadal tissues82 but itsLH-like component nonetheless ovulates, orlutenizes without ovulation, the dominant follicle insuccessive waves of follicles which are stimulated todevelop during the first half of pregnancy by contin-uation of the 10–12 day surge-like releases of pitu-itary FSH that control follicular development duringthe estrous cycle.83,84 Thus, secondary corporalutea begin to accumulate in the maternal ovariesfrom the time of the very first appearance of eCG inmaternal blood at around day 38 after ovulationwith a consequential rise in maternal serum proges-terone concentrations each time one of these acces-sory luteal structures develops (Fig. 11).85–87

In addition to the rise in progesterone, the com-mencement of eCG secretion by the newly developedendometrial cups stimulates a sharp and pro-nounced rise in peripheral serum conjugated estro-gen concentrations in the pregnant mare.84,88

These conjugated estrogens are ovarian in origin88

and the experiments of Daels et al87 have revealedthey are secreted by the primary and/or secondarycorpora lutea, rather than the Graffian follicles, indirect response to the gonadotrophic action of eCG(Fig. 11). Once risen in this manner, the serumestrogen levels tend to plateau, or even decline againslightly, until around day 70–80 when they begin afurther and more prolonged rise that culminates in arelatively enormous peak in conjugated estrogenconcentrations in both the blood and the urine of themare around day 200–240 of gestation.89,90 Thistime the estrogens are placental in origin and theyinclude both the common phenolic estrogens, es-trone and estradiol-17b, and the unusual and equine-specific ring B unsaturated estrogen, equilin andequilenin,91 which are synthesized by placental aro-matization of the large quantities of dihydroandro-sterone (DHA) and dihydroepiandosterone (DHEA),and the rare 3b-hydroxy-5,7-prenandien-20-one and3b-hydroxy-5,7 androstadien-17-one forms of theseC-19 precursors, secreted by the dramatically en-larged gonads of the fetus.91–94 The gonads, boththe ovaries in the female fetus and the testes in the

Fig. 8. Comparison of the endometrial cups at day 60 of gesta-tion produced by the chorionic girdles which developed on twohalves of the same mule (/ horse 3 ? donkey) morula bisected onday 6 after ovulation. One demi embryo was transferred to theuterus of a horse recipient mare (a) and the other was transferredto the uterus of a recipient donkey (b). Note the small, narrowand already necrosing cups in the horse uterus (a) compared tothe much larger and still active cups in the donkey uterus (b).





male fetus, begin to enlarge from around day 80 ofgestation to reach a maximum size around day 240,

when they occupy almost half the total volume of theabdomen of the fetus and are usually bigger than thenow-inactive ovaries of the mare.95 Their growth isoccasioned by a massive hypertrophy and hyperpla-sia of the interstitial cells of both types of gonad96

and they decline again steadily during the last quar-ter of pregnancy to more normal proportions andmorphological configurations by the time the foal isborn at around day 336–340.77

Immunologically, the equine endometrial cup re-action is a huge puzzle. The invasive chorionic gir-dle trophoblast cells, but not the non-invasivetrophoblast of the adjacent allantochorion, expresshigh concentrations of paternally inherited Class IMajor Histocompatibility Complex (MHC) antigenson their cell surface before, and for a few days after,they invade the maternal endometrium to form theendometrial cups.97,98 This blatant display of for-eign antigenic molecules stimulates a strong hu-moral immune response in the mother such that allmares, including primigravid maidens, carrying fe-tuses which differ paternally at the Class I MHCbarrier, develop high titres of specific anti-paternallymphocytotoxic antibody in their serum within

Fig. 10. Photomicrograph of the base of an endometrial cup atday 87 of gestation. The accumulated maternal lymphocytes areseen migrating into the cup tissue and destroying the large,binucleate fetal cup cells (3100).

Fig. 9. Endometrial cups (ec) in mares at different stages of pregnancy. a) A long unbroken ribbon of cup tissue seen at hysterotomyoperation 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.





10–14 days after initial invasion of the endome-trium by the chorionic girdle at around day36–38.99,100 The antibody persists throughoutpregnancy and it reappears anamnestically at ear-lier stages of gestation, and at even higher concen-trations, in mares mated to the same MHC-incompatible stallion in successive years and inmares transplanted with biopsies of skin from thestallion prior to mating.101

In addition, a very strong maternal cell-mediatedreaction is mounted against the invading chorionicgirdle cells. Lymphocytes appear in the endome-trial stroma within hours after initial invasion bythe chorionic girdle cells and their numbers increasedramatically from around day 60–70, when they arejoined by other mononuclear immune cells such asplasma cells, macrophages and eosinophils.73,74

Collectively these accumulated maternal immunecells form a definite barrier that separates fetal andmaternal tissues and is reminiscent of the interfacebetween grafted and host tissues during rejection ofan allograft of skin. Initially, the accumulatingleucocytes seem content to merely wall off the for-eign fetal cells, but beyond day 60–70 of gestationwhen the cells in the central region of the cup startto degenerate and die, the lymphocytes at the pe-riphery begin to actively attack and destroy the fetalcup cells and they thereby hasten the death andeventual desquamation of the whole cup around day100–120.73

It is apparent that the paternally inherited ClassI MHC antigens expressed by the invading chorionicgirdle cells98,102 are the stimulus for the strong hu-moral maternal immune response to the fetus inequine pregnancy, but the nature of the foreign an-

tigens that stimulate the equally strong cellular re-sponse is far less clear. The cups live as long, andsecrete equivalent amounts of eCG, in mares carry-ing MHC-incompatible as MHC-compatible fe-tuses119 and the leucocytic response mountedagainst the cups is far more intense and destructivein mares carrying interspecific mule fetuses than itis in mares carrying normal intraspecific horse fe-tuses.73,103 Thus, it appears that species-specificnon-MHC antigens, and possibly also tissue-specifictrophoblast antigens, are involved in the cell-medi-ated response to the endometrial cups.104

The biological raison d’etre for the endometrialcup reaction in equine pregnancy remains a mys-tery. Endocrinologically, the additional progester-one generated by the secondary corpora that arestimulated by the ovulatory and/or luteinizing prop-erties of the relatively vast quantities of gonadotro-phic hormone (eCG) secreted by the cups duringtheir short lifespan, certainly supports the mainte-nance of the pregnancy state until day 100 of gest-ation or thereabouts when, as shown by theovariectomy studies of Holtan et al,105 the placentais now sufficiently well developed to take over com-pletely the supply of enough progesterone to main-tain the pregnancy state without any furthercontribution from the maternal ovaries. But, asdemonstrated firstly by the survival to term ofaround 30% of extraspecific donkey-in-horse preg-nancies, created by embryo transfer, in the completeabsence of any detected endometrial cup formationand eCG secretion,106 and resulting failure of devel-opment of any secondary corpora lutea,103 and sec-ondly by the marked reduction, or complete absence,of accessory ovulations in mares mated in late au-

Fig. 11. Endocrinological functions of the equine endometrial cups. The dominant member of waves of ovarian follicles stimulatedby continuing surge-like releases of pituitary FSH are ovulated and/or luteinized by the LH-like component of the equine ChorionicGonadotrophin (eCG) secreted by the fetal chorionic girdle cells after they invade the maternal endometrium around day 38 to formthe endometrial cups. The corpora lutea, both primary and secondary, secrete progesterone and conjugated estrogens in response tothe gonadotrophic stimulus of eCG.





tumn so that they are seasonally deficient in pitu-itary FSH release during the first 100 days ofpregnancy,73 secondary luteal progesterone is by nomeans obligatory to the maintenance of early preg-nancy in the mare provided the primary corpus lu-teum does not undergo luteolysis for any untowardreason such as endotoxin production.87

Immunologically, it seems a very risky stratagemfor an allotypic fetus, or worse still, a xenotypic fetusin the case of a mule,73 to deliberately immunize thedam against its paternally-derived histocompatibil-ity antigens, merely for the sake of generating someextra, temporary luteal progesterone which is notabsolutely necessary. Yet, curiously, it is in the onetype of xenogeneic pregnancy, the extraspecific don-key-in-horse pregnancy created by embryo transfer,which does not have an endometrial cup reactiondue to inadequate development and failure of thedonkey chorionic girdle to invade the horse endome-trium around day 36 after ovulation,107 that themajority (i.e., .70%) of fetuses die and are abortedaround day 80–100 of gestation in conjunction withdelayed and/or inadequate interdigitation of the al-lantochorion with the endometrium and a general-ized and intense maternal leucocytic responsethroughout the endometrium in that is in contactwith the xenogeneic donkey trophoblast.106 And, inthese at-risk pregnancies, administration of eitheror both exogenous eCG and progesterone fails toreduce the high rate of pregnancy loss,106 whereasactive immunization against donkey peripheralblood lymphocytes results in a marked increasein fetal survival above that in untreated controlanimals.103

Perhaps its injection of specialized hormone se-creting and foreign antigen presenting trophoblastcells into the maternal endometrium representssomething of developmental panic reaction on thepart of the fetus to re-announce antigenically andendocrinologically its presence to the maternalorganism after such a prolonged period of non-attachment and immunological indifference to thepotentially hostile endometrium (Fig. 12). Certainly,a lack of normal interdigitation between the allan-tochorion and endometrium is the most striking ab-normality of the unsuccessful donkey-in-horsepregnancy model in which endometrial cups do notdevelop and the associated maternal anti-paternalMHC humoral response is absent. This raises thepossibility that some hitherto unknown influence ofthe whole endometrial cup reaction in equids is es-sential to stimulate the close and stable microvillousinteraction between fetal and maternal epitheliallayers which underpins and characterizes the wholeprocess of placentation in the pregnant mare.

Placentation

Only as late as day 40 after ovulation, some 2 or 3days after invasion of the endometrium by the cho-rionic girdle cells to start the endometrial cup reac-tion, does the non-invasive trophoblast of the nowrapidly expanding and slowly elongating allantocho-rion begin to make a stable, microvillous attachmentto the lumenal epithelial cells of the endometrium(Fig. 13a). During the next 20 days, blunt, finger-like villi of allantochorion form a close fitting inter-digitation with thinner, frond-like villi that developon the endometrium, much like fingers being in-

Fig. 12. The “panic reaction” of the still unattached equine fetus around day 36 after ovulation to inject its specialized trophoblastof the chorionic girdle into the maternal endometrium to increase the supply of steroid hormones and suppress the potentialimmunological hostility of the endometrium.





serted into a tight-fitting glove.107 Beyond day 60the allantochorionic villi and accommodating endo-metrial sulci begin to branch extensively while atthe same time becoming longer and deeper (Fig.13b). This process of branching and lengthening ofeach primary villous and its opposing endometriumeventually creates, by about day 120 of gestation,108

the primary hemotrophic exchange unit of the non-invasive allantochorionic placenta known as themicrocotyledon.109 The process maximizes the mi-croscopic area of contact between the fetal and ma-ternal epithelial layers for hemotrophic exchange ofnutrients and waste products and it is aided by theclose apposition to, and indentation into, the base ofthese epithelial layers by numerous blood capillar-ies, on both the fetal and maternal sides of theinterface.110 Each microcotyledon is supplied witha sizeable artery on the maternal side and an equiv-

alent placental vein on the fetal side to maximize theexchange process.111 In addition, the endometrialglands remain functional throughout gestation andthey liberate their protein-rich exocrine secretionsinto well defined spaces between the microcotyle-dons. Here, the trophoblast cells become pseudo-stratified and are specially adapted to take up andabsorb the exocrine material to establish a second,histotrophic form of nutrition for the rapidly grow-ing fetus.112

Thus, by mid-gestation, and after an abnormallyslow start, the diffuse non-invasive epitheliochorialequine placenta is established over the entire avail-able area of endometrium and is providing both he-motrophic and histotrophic nutritional exchange forthe fetal foal. Both the total gross area of the pla-centa, and the microscopic area of fetomaternal con-tact at the placental interface, continue to increasethroughout the remainder of pregnancy to meet thefetal growth needs, and any diminution of this vastarea of functional placenta, such as would occur inthe case of twin conceptuses competing for the samelimited area of endometrium (Fig. 14)113 or in oldermares exhibiting age-related degenerative changes(endometrosis) in the endometrium,107,114,115 willlead at best to a degree of runting and weakness inthe newborn foal, and at worst embryonic death andresorbtion early in gestation, or abortion in latepregnancy.108 An extensive and fully functionalmicrocotyledonary placenta attached to a healthyand fully functional endometrium is an essential

Fig. 13. Sections of the placental interface in pregnantmares. a) At day 43 showing close microvillous attachment ofthe trophoblast of the allantochorion to the lumenal epithelium ofthe endometrium and blunt villi of allantochorion beginning toindent into the surface of the allantochorion; b) At day 83 ofgestation showing thickened and branched villi of allantochorioninterdigitating with thinner, finger-like sulci of endometrium(3100).

Fig. 14. Twin horse conceptuses in the excised uterus of a mareat an estimated 250 days of gestation. Note the much larger sizeof the fetus with the bigger area of placenta that occupies thebody of the uterus, the non-gravid horn and the base of the gravidhorn. The smaller co-twin has been pushed up to the tip of thegravid horn and is now beginning to suffer severe nutritionaldeprivation due to an inadequate area of placenta to meet itsgrowth requirements.





pre-requisite of normal pregnancy in the mare andthe production of a healthy, well developed foal atterm.

The nature of the stimulus which both initiatesplacental interdigitation with the endometrium atday 40 after ovulation, and which drives the wholeprocess of amazing growth and architectural modi-fication of the endometrium and allantochorionthroughout the remainder of gestation, is of greatinterest and remains something of a mystery. Lo-cally produced growth factors almost certainly pro-vide the main mitogenic impetus to placentation andinsulin-like growth factor II (IGF-II) secreted by thetrophoblast of the allantochorion and other fetaltissues throughout pregnancy116 and epidermalgrowth factor (EGF), secreted by the epithelium lin-ing the apical portions of the endometrial glands,the genetic message (mRNA) for which is dramati-cally upregulated in these cells between days 35 and40 of gestation,117 or after 40 days of exogenousprogesterone administration in the non-pregnantmare,118 are the two most likely candidates.

Conclusions

So many aspects of embryonic survival, fetal devel-opment, and placentation remain puzzling in themare. From the slow, PGE2-driven passage of theembryo down the oviduct, through the free-wheelingencapsulated movement of the embryo throughoutthe uterus to bring about maternal recognition ofpregnancy, to the tenuous, myometrial tone-controlled choriovitelline first attachment of theconceptus, and on through the bizarre and immuno-logically perilous process of endometrial cup devel-opment just prior to the final fetal utopia of stableand nutritious placentation, pregnancy in equidsremains a mysterious and fascinating process that iswell worthy of much further investigation.

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aDinoprostin; Pharmacia-Upjohn, Crawley, Sussex, UK.bFinadyne; Schering Plough, Middlesex, UK.cTAE Stout, personal communication.





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