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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
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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 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.
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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
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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
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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.
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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
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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
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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
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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-
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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
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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
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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
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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-
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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
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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.
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a
Dinoprostin; Pharmacia-Upjohn, Crawley, Sussex, UK.
Finadyne; Schering Plough, Middlesex, UK.
c
TAE Stout, personal communication.
b
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