Ž .
Animal Reproduction Science 60–61 2000 691–702 www.elsevier.comrlocateranireprosci
Comparative aspects of equine embryonic development
Keith J. Betteridge
Animal Biotechnology – Embryo Laboratory, Department of Biomedical Sciences, Ontario Veterinary College, UniÕersity of Guelph, Building 165-ABEL, Guelph Ontario, Canada, N1G 2W1
Abstract
The developmental changes in the equine conceptus, its maternal environment and their interaction during the first 4 weeks following fertilization are reviewed. Attention is drawn to
species-specific events to show why the horse is such a valuable model in which to study early pregnancy. q 2000 Elsevier Science B.V. All rights reserved.
Keywords: Horse; Embryo; Pregnancy; Capsule; Blastocyst; Endometrium; Oviduct
1. Introduction
Ž . The three purposes of this paper are to: 1 review briefly our knowledge of the
embryonic development in the horse during the 4 weeks following fertilization, paying special attention to features that distinguish early equine ontogeny from analogous
Ž . events in other mammals; 2 argue that many reproductive characteristics of horses
provide us with unusual, and under-used, opportunities to contribute to comparative Ž .
studies of early mammalian pregnancy; 3 draw attention to the practical importance of understanding early pregnancy in the mare.
The advocacy of the horse as a model in studying early pregnancy is by no means Ž
. new. More than a century ago, Ewart 1897 beautifully illustrated how the early equine
Ž .
conceptus resembles that of the Virginian opossum and reasoned probably wrongly that pregnancy failure at about 42 days in horses could reflect an evolutionary call for
the conceptus to be ‘‘born’’ at the time appropriate to such a marsupial. More recently, Ž
. Renfree 1982 has drawn attention to the similarity of blastocyst expansion in the horse
Tel.: q1-519-824-4120; fax: q1-519-824-1643. Ž
. E-mail address: kbetteruguelph.ca K.J. Betteridge .
0378-4320r00r - see front matter q 2000 Elsevier Science B.V. All rights reserved. Ž
. PII: S 0 3 7 8 - 4 3 2 0 0 0 0 0 0 7 5 - 0
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and another marsupial, the tammar wallaby. Betteridge 1989,1995a,b and Denker Ž
. 2000 have reviewed how horse studies have taught us much about comparative aspects
of early development, and of blastocyst coverings in particular.
2. Developmental events
2.1. General Ž
It should be emphasized at the outset that the conceptus i.e., the embryo proper and .
its associated membranes and fluid needs always to be considered as only one partner in the physiological dialogue between it and its environment. Focusing on the conceptus
is both useful and convenient but can be misleading if this interaction is forgotten. Clinically, such a focus is much easier in horses than in other domestic animals because
the whole conceptus, and even the embryo itself, are so easy to monitor by ultrasonogra-
Ž phy Palmer and Driancourt, 1980; Chevalier and Palmer, 1982; Simpson et al., 1982;
. Ginther, 1986 . On the other hand, developmental studies in horses have suffered in
comparison with those of other species in which embryo production in vitro has progressed so very much further. These considerations should be kept in mind in the
following commentary on the future foal’s acquisition of steadily more complex
Ž .
conformation and activity in the mare’s reproductive tract Fig. 1 . 2.2. DeÕelopment in the oÕiduct
2.2.1. The oÕiductal enÕironment Released as it is from a follicle containing in excess of 20 ml of rich liquor, it might
be expected that the equine oocyte’s final preparation for fertilization would be strongly influenced by follicular fluid. Strangely, this may not be the case; Townson and Ginther
Ž .
1989 found no corresponding distension of the oviduct immediately after ovulation, Ž
. and Palmer et al. 1997 demonstrated that follicular fluid, while probably helpful, is not
absolutely essential to convey the oocyte to the ampulla. Nevertheless, fibroblasts apparently do enter the oviduct at ovulation and produce collagen masses in its lumen,
Ž .
which may affect early development Lantz et al., 1998 . Considering that fertilized and Ž
. unfertilized equine eggs are transported differentially in the mare see below , it seems
unlikely that follicular contents influence embryo transport in the mare, as has been Ž
. proposed to occur in cattle Wijayagunawardane et al., 1999 .
The reciprocal nature of the dialogue between the embryo and the mare’s reproduc- tive tract almost certainly begins immediately in the oviduct. The histochemical charac-
teristics of the oviductal epithelium vary with reproductive status and from region to Ž
. region Ball et al., 1997 . Consequently, it should be remembered that the equine
embryo spends most of its 6-day sojourn in the oviduct near the ampullary–isthmic Ž
. junction of that organ
Weber et al., 1996 . The oviductal secretory products are plentiful but, paradoxically — in view of the histochemical findings, show no clear
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variation with region or reproductive status of the mare McDowell et al., 1993 .
Fig. 1. Approximate timing of some key developmental events during the first 4 weeks of gestation of the equine conceptus.
Functional characteristics of the oviduct, manifest as effects on embryo survival, Ž
. evidently vary with age Carnevale et al., 1993 . The clearest evidence, however, that
the oviductal epithelium affects embryo development comes from in vitro culture studies, which have demonstrated the beneficial effects of co-culture of cleaving
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embryos with oviductal epithelial cells Ball and Miller, 1992 . On the other hand, the equine endosalpinx cannot be absolutely essential for normal development, given that
Ž viable horse embryos can develop in the oviduct of nonpregnant ewes Fehilly and
. Willadsen, 1986 . In cattle, at least, the oviductal epithelium produces endothelin, which
Ž may affect gamete transport either directly, by local effects on contractility Rosselli et
. Ž
. al., 1994 , or indirectly, by affecting local blood flow Garcia-Pascual et al., 1996 .
2.2.2. Embryonic deÕelopment in the oÕiduct The most tangible evidence of all that the embryo–oviduct interaction is reciprocal
has arisen from the investigation of differential transport of fertilized and unfertilized eggs into the uterus; unless fertilized, eggs are usually retained in the oviduct and can be
‘overtaken’ by developing embryos resulting from subsequent ovulations. Stemming Ž
. from the original observation by Van Niekerk and Gerneke 1966 , experiments have
shown that this is likely to involve the embryonic production of prostaglandin E2 in Ž
. horses Weber and Woods, 1993; Weber et al., 1991a,b, 1995; Freeman et al., 1992 .
Acceptance of the reality of this phenomenon in horses led to analogous observations in Ž
. other species bat, Rasweiler, 1979; hamster, Ortiz et al., 1986; rats, Ortiz et al., 1989 .
Ž Completing the circle, it is interesting to note that hamster studies Velasquez et al.,
. 1995, 1997 now suggest that other embryonic products, such as platelet activating
factor, may also deserve investigation in the horse. In this context, it should be noted that activation of the embryonic genome occurs while the embryo is still in the oviduct
Ž and is a gradual process at the four- to eight-cell stage Ball et al., 1993; Grøndahl et al.,
. 1993; Brinsko et al., 1995 .
In the mouse at least, the structure of the oviduct wall is affected locally by the Ž .
Ž .
presence of the oocyte s in the cumulus mass soon after ovulation Sato et al., 1995 . The mouse has also provided us with fascinating indications that the interaction between
developing embryos and the oviduct affects the subsequent interactions between em- Ž
. bryos and the uterus, and thereby pregnancy rates Wakuda et al., 1999 .
Several comprehensive descriptions of fertilization and embryo cleavage in the horse have been summarized and compared with the timing of successive divisions in other
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domestic species Betteridge, 1995a . One unusual feature of early equine embryogene- sis is the polarity and opacity of the oocyte and zygote, a topic of increasing interest and
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practical significance in other mammals, including humans Fulka et al., 1998 . Others include the contents of the perivitelline space, the often ellipsoidal shape of the cleaving
egg, and the fact that the cleaving embryo acquires additional coats on the outside of its zona pellucida. However, these have received relatively little attention, largely because
of the logistical difficulties and expense of obtaining embryos from the oviduct at surgery or slaughter. Attempts to gain access to cleaving embryos by hastening their
transport through the oviduct by application of a gel preparation of prostaglandin E2 to the parietal surface of the oviduct through an endoscope have met with some success
Ž
. Robinson et al., 1998 . Unfortunately, however, it is suspected that an irritant substance
Ž in the gel vehicle may cause inflammation and lead to scarring of the mesosalpinx W.R.
. Allen, personal communication .
The time taken for the cleaving embryo to traverse the oviduct, previously estimated Ž
. to be 5.5–6.0 days, has recently been shown to be 6.0–6.5 days Battut et al., 1997 .
The last part of the journey, through the isthmus, is accomplished relatively rapidly Ž
. Weber et al., 1996 . By the time the embryo enters the uterus, development has
progressed to the late morula or early blastocyst stage. Much more is known about development from this point onwards because of the ease with which pre-attachment
conceptuses can be recovered from the uterus by transcervical lavage.
2.3. DeÕelopment in the uterus 2.3.1. The uterine enÕironment
There is an understandable tendency to divide the discussion of the maternal contributions to the uterine environment during early pregnancy in the mare into two
categories: one relates to the contrasts with the non-pregnant uterus which permit the
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corpus luteum CL to be maintained; the other is associated with the nutrition of the developing conceptus. Physiologically, however, these two functions of the pregnant
uterus are inextricably mixed and dependent on interaction with the conceptus, as has already been stressed. Indeed, the uterine environment, as reflected in uterine flushings
Ž during pregnancy, often includes materials produced by the conceptus Zavy et al., 1982,
. 1984 .
The mechanisms by which luteolysis is abrogated in the pregnant mare are still far Ž
from being completely understood. It is best viewed as being a gradual process Sharp et .
al., 1989 and clearly involves down-regulation of receptors for oxytocin in the
endometrium so that luteolytic releases of endometrial prostaglandin F2 a are prevented Ž
. see Stout et al., 1999 for references . Oxytocin itself, neurophysin, and the messages for
both have been demonstrated in the endometrium by immunohistochemistry and RT-PCR, Ž
. respectively Watson et al., 1998; Behrendt-Adam et al., 1999 . Both oxytocin and
arginine vasopressin have also been detected in the yolk sac fluid, their concentrations Ž
. increasing after day 12 of gestation Waelchli et al., 2000a .
Numerous progesterone-dependent proteins, presumably vital to the nutrition of the conceptus, have been shown to be produced by the uterus. Of these, by far the best
Ž .
characterized is the lipocalin P19 Stewart et al., 1995; Crossett et al., 1996, 1998 , Ž
which probably helps transport small, hydrophobic molecules e.g., steroids and
. Ž
. eicosanoids into the yolk sac of the conceptus see below . A distinct endometrial
Ž .
retinol-binding protein has also been cloned in the horse McDowell et al., 1995 and can be presumed to play a vital role in embryonic development by transporting
morphogenic retinoids. Ž
. Numerous proteins found in the embryonic capsule see below are under investiga-
tion to determine which are of maternal origin and which come from the conceptus. Ž
Several of these are growth factors and their binding proteins Herrler et al., 1998, 1999; .
Herrler and Beier, 2000 . 2.3.2. Conceptus deÕelopment in the uterus
It is in the uterus that developmental characteristics of the conceptus in the horse become especially distinct from those of other domestic species. First of all, the equine
embryo, its fetal membranes and its circulatory system develop to an extraordinary Ž
. extent before even the first signs of a functional yolk sac choriovitelline placenta at
Ž .
day 22 Enders et al., 1993 and allantochorionic placenta at about 40 days after ovulation. Second, the endodermal wall of the yolk sac is completed from cells that are
unusually broadly distributed around the interior of the blastocyst, and which seem to Ž
. have unusual functional characteristics Enders et al., 1993 . Third, although as in the
other species the zona pellucida is shed from the blastocyst, it is replaced in the horse by a glycoprotein capsule of considerable functional importance between approximately
days 7 and 21. Fourth, the encapsulated conceptus interacts with its environment during two distinct phases; it is extremely mobile until about days 16–17 and then becomes
Ž .
immobilized ‘‘fixed’’ at the site of eventual placentation. Fifth, the capsule maintains a spherical form for the conceptus, particularly before fixation. Sixth, it is relatively easy
to recover the intact conceptuses transcervically from the standing mare at least until about day 35 — a feature important to experimental investigation.
There are numerous structural changes associated with conceptus development during Ž
the second and third weeks of pregnancy Betteridge et al., 1982; Flood et al., 1982; .
Enders and Liu, 1991; Enders et al., 1988, 1993 . They become of major importance in interpreting the corresponding functional changes that are undergone during this period,
Ž .
as is especially well explained for the cellular tissues by Enders et al. 1993 . The formation and dissolution of the acellular capsule have also been the subject of much
attention. This interest is likely to increase, given the importance of the capsule as a Ž
. ‘‘mailbox’’ for proteins and perhaps other molecules? being exchanged between the
Ž conceptus and the endometrium Crossett et al., 1998; Herrler et al., 1998, 1999; Herrler
. and Beier, 2000 . Furthermore, the development of the capsule soon after the blastocyst
enters the uterus is of practical significance. It radically changes the ease with which equine blastocysts can be cryopreserved, presumably because of an effect on permeation
Ž .
by cryoprotective agents Bruyas, 1998; Hochi, 1998 and also makes the micromanipu- Ž
. lation of the embryo very difficult Huhtinen et al., 1997 .
The capsule is composed of a mucin-like glycoprotein, produced largely by the Ž
. trophoblast Oriol et al., 1993a,b . However, the presence of mRNA from the mucin
Ž .
gene MUC1 in both the trophoblast and endometrium Gillies et al., 1999 suggests that an endometrial contribution to its formation should not yet be ruled out. It is interesting
that sialic acid is lost from the capsule at the time of conceptus fixation, raising the Ž .
possibility of changes in charge being involved in the mechanism s of attachment to the endometrium. The loss of sialic acid does not occur if the mare is treated experimentally
Ž .
with PGF2 a at day 14 Chu et al., 1996 . The capsule is a strong and resilient structure and it is thought to play a protective role and to facilitate migration throughout the
Ž .
uterine lumen before day 16 Betteridge, 1989 . This migration is essential to the Ž
. maintenance of pregnancy
Sharp et al., 1989 . After fixation, capsule production appears to decline until it ruptures at about day 21 through mechanisms, perhaps
Ž .
enzymatic, that remain unclear Denker, 2000 . Blastocyst expansion mechanisms in horses seem to differ markedly from those
observed in other species; the fluid within the expanding yolk sac is markedly hypotonic Ž
. ; 120 mosMrkg until, after day 16, it begins to rise slowly to ; 250 mosMrkg at
Ž .
about day 22 Waelchli and Betteridge, 1996; Waelchli et al., 1997 . How fluid can be Ž
. imbibed from a uterine fluid environment of higher osmolality Waelchli et al., 2000b
is still under investigation. The gradual rise in osmolality after day 16 is reflected in a Ž
parallel increase in the concentration of fructose within the yolk sac Ruddock et al., .
2000; Fig. 2 . There are two basic approaches to investigating secretory products of the conceptus
that are likely to be important to its interaction with the mare. The first is to hypothesize the existence of a substance and then to search for it either in the blastocyst fluid or in
medium used to culture conceptus tissues. The second is to use various analytical techniques to detect an array of products, then to identify members of the array and
hypothesize about their functions. The approaches are complementary but the advent of
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gene banks is making the second particularly powerful see, e.g., Simpson et al., 1999 and our knowledge of conceptusrendometrium interrelationships should be about to
advance very rapidly. The major classes of secretory products that have so far been identified include steroids, eicosanoids, proteins and peptides. Any of these found in
Fig. 2. Changes in the osmolality and fructose concentrations of equine yolk sac fluid during the second to fourth weeks of pregnancy.
blastocyst fluid could have been conveyed there by transport proteins such as P19 Ž
. Crossett et al., 1998 . Identifying the origin of a putative product must, therefore,
involve culture in vitro andror tissue analysis by immunological or molecular probes. Since the first demonstrations of estrogen and androgen production by the equine
Ž .
conceptus in the 1970s Flood et al., 1979; Zavy et al., 1979 , the most intensive study of steroidogenesis in these tissues has been by Goff and his colleagues. They have
shown that 17a-hydroxyprogesterone, and not oestradiol as had been expected, is the major steroid synthesized by the equine conceptus between days 7 and 14 of pregnancy.
They also showed that this steroid is further metabolized to an unidentified steroid by the endometrium and reasoned that these steroids could play a role in conceptus
Ž .
development or the prevention of luteolysis Goff et al., 1993 . Earlier, the same group had shown that endoderm and trophectoderm cells from the conceptus exhibit different
steroidogenic profiles in vitro, leading them to suggest that the two tissues need to act in Ž
. concert if pregnancy is to proceed Marsan et al., 1987 .
With regard to the eicosanoids, the equine conceptus continues to produce PGE2 following its passage into the uterus, production increasing between days 11 and 15
Ž .
Ž Vanderwall et al., 1993 . The conceptus also produces PGF2 a by day 14 Watson and
. Sertich, 1989 .
The production of proteins and peptides by the conceptus has been the subject of Ž
. many investigations. McDowell et al. 1990 demonstrated that the array of proteins
produced by conceptuses before day 14 differed from the array produced by older conceptuses in which a pattern characteristic of yolk sac tissue emerged. Searching for a
conceptus secretory product capable of suppressing PGF2 a production from en-
Ž .
dometrium in vitro, Sharp et al. 1989 used dialysis membranes with different molecu- lar exclusion properties to conclude that a molecule of M 1000 and - 6000 is
r
Ž .
responsible. Simpson et al. 1999 have used suppression subtractive hybridization to isolate transcripts that are more abundantly expressed in day 15 than in day 12
Ž conceptuses. They have identified cDNA clones for calcyclin
a calcium binding .
Ž .
protein and phospholipase A2 involved in eicosanoid metabolism to be differentially Ž
. expressed at these times. Green et al. 1999 have identified a new aspartic proteinase
that is produced by the equine trophoblast as early as day 15 and which would seem to be related to the pregnancy-associated glycoproteins of the order Artiodactyla.
3. The value of studies of equine embryology
