Reproduction,placentation, and embryonic development of the Atlantic sharpnose shark,Rhizoprionodon terraenovae

The Atlantic sharpnose shark Rhizoprionodon terraenovae (Richardson) is a small carcharhinid that is a common year-round resident along the southeast coast of the United States. It is viviparous and its embryos develop an epithelio-vitelline placenta. Females enter shallow water to give birth in late May and early June. Mating occurs shortly after parturition, and four to seven eggs are ovulated. Fertilized eggs attain the blastoderm stage in early June to early July. Separate compartments for each egg are formed in the uterus when the embryos reach 3–30 mm. Embryos depend on yolk for the first 8 weeks of development. When embryos reach 72 mm their yolk supply is nearly depleted and they shift to matrotrophic nutrition. When the embryos reach 40–55 mm, placental development begins with the vascularization of the yolk sac where it contacts the uterine wall. Implantation occurs at an age of 8–10 weeks by which time the embryos reach 70–85 mm. The expanding yolk sac engulfs the maternal placental villi, and its surface interdigitates with the villi to form the placenta. The rest of the lumenal surface of the uterus is covered by non-placental villi that appear shortly after implantation. Histotrophe production by the non-placental villi begins just after their formation. The placenta grows continuously during gestation. The egg envelope is present throughout gestation, separating maternal and fetal tissues. Embryos develop numerous appendiculae on the umbilical cord. Young sharks are born at 290–320 mm after a gestation period of 11 to 12 months. © 1993 Wiley-Liss, Inc.     

Developmental regulation of human cytomegalovirus receptors in cytotrophoblasts correlates with distinct replication sites in the placenta

Cytomegalovirus (CMV), the major viral cause of congenital disease, infects the uterus and developing placenta and spreads to the fetus throughout gestation. Virus replicates in invasive cytotrophoblasts in the decidua, and maternal immunoglobulin G (IgG)-CMV virion complexes, which are transcytosed by the neonatal Fc receptor across syncytiotrophoblasts, infect underlying cytotrophoblasts in chorionic villi. Immunity is central to protection of the placenta-fetal unit: infection can occur when IgG has a low neutralizing titer. Here we used immunohistochemical and function-blocking methods to correlate infection in the placenta with expression of potential CMV receptors in situ and in vitro. In placental villi, syncytiotrophoblasts express the virion receptor epidermal growth factor receptor (EGFR) but lack integrin coreceptors, and virion uptake occurs without replication. Focal infection can occur when transcytosed virions reach EGFR-expressing cytotrophoblasts that selectively initiate expression of alphaV integrin. In cell columns, proximal cytotrophoblasts lack receptors and distal cells express integrins alpha1beta1 and alphaVbeta3, enabling virion attachment. In the decidua, invasive cytotrophoblasts expressing coreceptors upregulate EGFR, thereby dramatically increasing susceptibility to infection. Our findings indicate that virion interactions with cytotrophoblasts expressing receptors in the placenta (i) change as the cells differentiate and (ii) correlate with spatially distinct sites of CMV replication in maternal and fetal compartments.     

Temporal expression profiles of organic anion transport proteins in placenta and fetal liver of the rat

Physiological cholestasis linked to immature hepatobiliary transport systems for organic anions occurs in rat and human neonates. In utero, the placenta facilitates vectorial transfer of certain fetal-derived solutes to the maternal circulation for elimination. We compared the ontogenesis of organic anion transporters in the placenta and the fetal liver of the rat to assess their relative abundance throughout gestation and to determine whether the placenta compensates for the late maturation of transporters in the developing liver. The mRNA of members of the organic anion transporting polypeptide (Oatp) superfamily, the multidrug resistance protein (Mrp) family, one organic anion transporter (OAT), and the bile acid carriers Na(+)-taurocholate cotransporting polypeptide (Ntcp) and bile salt export pump (Bsep) was quantified by real-time PCR. The most abundant placental transporters were Oatp4a1, whose mRNA increased 10-fold during gestation, and Mrp1. Mrp1 immunolocalized predominantly to epithelial cells of the endoplacental yolk sac, suggesting an excretory role that sequesters fetal-derived solutes in the yolk sac cavity, and faintly to the basal syncytiotrophoblast surface. The mRNA levels of Oatp2b1, Mrp3, and Bsep in the placenta exceeded those in the fetal liver until day 20 of gestation, suggesting that the fetus relies on placental clearance of substrates when expression in the developing liver is low. Mrp3 immunolocalized to the epithelium of the endoplacental yolk sac and less abundantly in the labyrinth zone and endothelium of the maternal arteries. The placental expression of Oatp1a1, Oatp1a4, Oatp1a5, Oatp1b2, Oat, Ntcp, Mrp2, and Mrp6 was low.     

The development of rat alpha 2-macroglobulin. Studies in vivo and in cultured fetal rat hepatocytes

During inflammation and tissue injury, there is an increase in the plasma concentration of several proteins, the acute-phase proteins. The levels of some acute-phase proteins have been reported to increase in pregnant and tumour-bearing animals. Rat alpha 2-macroglobulin is classified as an acute-phase protein. In this study we report the expression of alpha 2-macroglobulin in various tissues during development of the rat embryo by analysis of mRNA. The tissues studied are liver, visceral yolk sac, placental labyrinth, decidua and trophoblast. In addition, the sites of alpha 2-macroglobulin expression are localized by in situ hybridization of cDNA for alpha 2-macroglobulin to mid-sagittal cryosections of rat embryos. The level of mRNA coding for alpha 2-macroglobulin is determined in the liver of rats aged between 12 days gestation and 2 days postnatal. alpha 2-Macroglobulin mRNA is first observed in fetal liver from 12 days of gestation and increases after day 17, reaching a maximum on day 20. At this time the level is greater than that found in the liver of an adult rat suffering from acute inflammation. alpha 2-Macroglobulin mRNA is detectable in the yolk sac, placental labyrinth, trophoblast tissue and decidua. In the decidua the alpha 2-macroglobulin message is first detected at 8 days of gestation, with high levels observed from 10 to 21 days of gestation. These observations are supported by in situ hybridization studies. Experiments using cultured hepatocytes show that cells derived from rats at 15 days and 19 days of gestation are capable of synthesizing and secreting alpha 2-macroglobulin. Both synthesis and secretion can be induced by the addition of dexamethasone to the culture medium.     

Histological analysis of spontaneous abortions with trisomy 2: first description of an embryo.

Three spontaneous abortions with trisomy 2 were analyzed histologically. In one of these, beside chorionic membranes and villi, yolk sac, yolk stalk, body stalk and an embryo are described. Concerning the development stage there seems to be an order; villi and body stalk (16 days), embryo (end of 3rd week to beginning of 4th week) and yolk sac with yolk stalk (2nd half of 4th week).     

Evolution of placental specializations in viviparous African and South American lizards

Phylogenetic information offers an important resource in analyses of reproductive diversity, including interpretations of fetal membrane evolution. In this paper, we draw upon ongoing studies of South American and African lizards to consider the value of combining phylogenetic and reproductive evidence in the construction of evolutionary interpretations. South American lizards of the genus Mabuya exhibit several reproductive specializations that are convergent on those of eutherian mammals, including viviparity, long gestation periods, ovulation of tiny eggs, and placental supply of the nutrients for development. Studies of placental morphology and development indicate that New World Mabuya share several other derived features, including chorionic areolae and a "Type IV" epitheliochorial placenta with a villous, mesometrial placentome. Some characteristics of these lizards are shared by two African skinks, M. ivensii and Eumecia anchietae, including minuscule eggs, placentotrophy, an absorptive chorioallantois, and features of the yolk sac. Available evidence is consistent with two explanations: (1) placentotrophy originated in Africa, predating a trans-Atlantic colonization by Mabuya of the New World; and (2) placentotrophy arose two or three separate times among these closely related skinks. As illustrated by analysis of these animals, not only can data on fetal membrane morphology yield phylogenetic information, but phylogenetic evidence in turn provides a valuable way to reconstruct the evolution of fetal membranes in a biogeographic context. When appropriately interpreted, morphological and phylogenetic evidence can be combined to yield robust evolutionary conclusions that avoid the pitfalls of circular reasoning.     

Placentation in watersnakes II: Placental ultrastructure in Nerodia erythrogaster (Colubridae: Natricinae)

Ultrastructure of the placental tissues from redbelly watersnakes (Nerodia erythrogaster ) was analyzed during late pregnancy to provide insight into placental development and function. Examination of the chorioallantoic placenta with transmission electron microscopy reveals that chorionic and uterine epithelia are extremely attenuated but intact and that the eggshell membrane is vestigial and lacks a calcareous layer. These features minimize the interhemal diffusion distance across the placenta. Scanning electron microscopy reveals that fetal and maternal components of the placentas are richly vascularized by dense networks of capillaries. Although the yolk sac omphalopleure has largely been replaced by chorioallantois by late gestation, it retains patches of yolk droplets and regions of absorptive cells with microvilli and abundant mitochondria. Transmission electron microscopy reveals that yolk material is taken up for digestion by endodermal cells. As yolk is removed, allantoic capillaries invade to occupy positions just beneath the epithelium, forming regions of chorioallantoic placentation. Ultrastructural features indicate that the chorioallantoic placenta is specialized for gas exchange, while the omphalallantoic (“yolk sac”) placenta shows evidence of functions in yolk digestion and maternal‐fetal nutrient transfer. Placental features of this species are consistent with those of other thamnophines, and are evolutionarily convergent on snakes of other viviparous clades.     

The ontogeny of expression of murine metallothionein: comparison with the alpha-fetoprotein gene

The ontogeny of expression of mouse metallothionein was studied by RNA dot and Northern blot hybridization using a cloned cDNA probe. In some instances the synthesis of metallothionein was analyzed by cell-free translation of RNA as well as pulse-labeling of proteins in short-term organ cultures followed by polyacrylamide gel electrophoresis. Interesting parallels between metallothionein and alpha-fetoprotein gene expression during development were noted. Like alpha-fetoprotein mRNA ( Dziadek and Andrews, 1983), metallothionein mRNA was found to be abundant in developing liver as well as in visceral yolk sac endoderm. In addition, metallothionein mRNA was abundant in parietal yolk sac. During liver development metallothionein and alpha-fetoprotein mRNAs were abundant by Day 12 of gestation, increasing to maximal levels on Day 16 and decreasing during late fetal and neonatal life to basal levels in adult. Metallothionein mRNA increased in maternal liver and was also abundant in certain hepatomas. Synthesis of metallothionein and levels of metallothionein mRNA in visceral yolk sac increased from Day 9 of gestation to maximal levels on Days 11-12 and then decreased abruptly after Day 15. RNA from differentiated teratocarcinoma cells with primitive, parietal or visceral endoderm characteristics each contained high levels of metallothionein mRNA, whereas, levels of this mRNA varied widely among embryonal carcinoma stem cell lines. alpha-Fetoprotein mRNA was not detected in embryonal carcinoma cells but was expressed in visceral endoderm-like differentiated cells. These results indicate that parietal and visceral endoderm cells actively express the metallothionein gene and further suggest that expression may be initiated at the earlier stage of primitive endoderm.     

Immunohistochemical localization of laminin and fibronectin isoforms in human placental villi.

We studied the localization of laminin alpha1, alpha2, alpha3, alpha5, beta1, beta2, and gamma1 chains and extradomain A- (EDA), EDB-, and oncofetal fibronectin by immunohistochemistry in human placental villi during placental development. The laminin alpha2, alpha5, beta1, beta2, and gamma1 chains were detected in the trophoblastic basement membrane (BM) at all stages of gestation, suggesting the presence of laminin-2, -4, -10, and -11 trimers. The laminin alpha1 chain was selectively found at sites where the villous BM was in contact with proliferating cells in trophoblastic islands or columns. EDA-Fn, but not other Fn isoforms, was found in the trophoblastic BM during the first trimester. The laminin alpha2, beta1, beta2, and gamma1 chains were detected in the villous stroma and capillaries throughout placental development, while the laminin alpha5 chain emerged distinctly during development. Extensive EDA-Fn immunoreactivity was found in first-trimester villous stroma, but distinctly fewer Fn isoforms were seen in the villous stroma during the later stages of gestation. Our results also suggest that, during the formation of new villi, laminins are not found in trophoblastic sprouts before the ingrowth of the villous mesenchyme. Rather, laminins may be deposited at the villous epithelial-mesenchymal interface. Furthermore, the results show that distinct changes occur in the localization of various laminin and Fn isoforms during the maturation of villous trophoblastic and capillary BMs.     

Pattern of serum protein gene expression in mouse visceral yolk sac and foetal liver.

We have shown by molecular hybridisation that the mRNAs for albumin, transferrin, apolipoprotein-A1, and alpha 1-antitrypsin are expressed at high levels in mouse visceral yolk sac. In contrast, the mRNAs for contrapsin (a plasma protease inhibitor) and the major urinary proteins (MUPs) are not detected in the visceral yolk sac at any stage of embryonic development. Contrapsin and MUP mRNAs both appear late in liver development. These differences in expression suggest that the visceral yolk sac is more similar to the foetal than adult mouse liver in its pattern of gene expression. However, the developmental time course of expression of these mRNAs is different between the foetal liver and the yolk sac. Evidence is also presented that the visceral yolk sac synthesises and secretes other apolipoproteins in addition to apolipoprotein-A1. These results suggest that the visceral yolk sac and foetal liver, two tissues with different embryological lineages, perform similar functions but are independently programmed for expression of the same set of serum protein genes.     

Development of trophoblast and placenta of the mouse. A reinvestigation with regard to the in vitro culture of mouse trophoblast and placenta

At 5 days post conceptionem (p.c.) shortly after implantation, giant cell transformation starts at the abembryonic pole of the blastocyst, spreading over the mural trophoblast; 1 day later, the first ectoplacental giant cells appear at the base of the fast growing ectoplacental cone (derived from the polar trophoblast). Giant cell transformation expands over it periphery. Thus, by the 8th day p.c., the conceptus is separated from the maternal tissue by a continuous layer of giant cells, variable in thickness. Giant cells reach their greatest size by 10 days p.c. in the mural tophoblast and by 12 days p.c. in the chorioallantoic placenta. They are probably no longer formed after that stage. Around the 8th day p.c., the allantois reaches contact with the ectoplacental cone, which develops into the chorioallantoic (definitive) placenta. At 9 days p.c., its four zones can already be discriminated: chorionic plate, labyrinth, junctional zone (trophospongium), and zone of giant cells, respectively. Within the next day, the chorioallantoic placental circulation is established. The yolk sac placental circulation is established by the 9th day p.c. The villi of the proximal layer of the yolk sac increase in size and number, and their capillary network becomes more dense until the 12th to 14th day p.c. This provides evidence that the yolk sac placenta exerts its function--to a certain extent--beyond the establishment of the definitive placenta. Around the 14th day p.c., the placental labyrinth reaches its definitive features. Fetal capillaries in the labyrinth, branching from unbilical blood vessels within the septa of connective tissue are surrounded by trophoblast cells. They form a dense vascular network bathing in maternal blood. The structures of the placental zones remain almost the same during further development, the borders becoming sometimes little blurred. Adjacent to the chorionic plate, subchorionic clefts appear at the 14th day p.c. These clefts become confluent to form the intraplacental space, regularly communicating with the yolk sac cavity. At the end of gestation (19th day p.c.) there is a considerable amount of eosinophilic material ('fibrinoid') between the zone of giant cells and the decidua, probably produced by the giant cells.     

Retinol-binding protein synthesis and secretion by the rat visceral yolk sac. Effect of retinol status

Studies were conducted to explore in rats the role of retinol in the regulation of the synthesis and secretion of retinol-binding protein (RBP) by the visceral yolk sac compared to the liver. Previous studies have shown that in retinol deficiency, hepatic RBP secretion is specifically inhibited, whereas hepatic RBP synthesis rate is unchanged. Retinol-depleted, retinoic acid-supplemented female rats were mated, and maternal liver, fetal liver, and visceral yolk sac were obtained at 14 days of gestation (retinol-depleted group). A group of identically treated, retinol-depleted rats were repleted with retinol on the 14th day of gestation, and the same tissues were collected 6 h later (retinol-repleted group). Normal female rats were used as controls. RBP was assayed by radioimmunoassay and RBP mRNA levels by RNase protection assay using a rat RBP cDNA clone. RBP levels in the visceral yolk sac were elevated 10-fold in the retinol-depleted as compared to the control rats and had declined to near normal values in the retinol-repleted animals. The relative levels of RBP mRNA in the visceral yolk sac were very similar in all three groups of rats. Thus, as in the liver, in the visceral yolk sac retinol deficiency inhibits RBP secretion without altering RBP mRNA levels. In the visceral yolk sac, as in the liver, retinol status appears to regulate RBP secretion specifically, without affecting the rate of RBP biosynthesis.     

Placentation in watersnakes I: Placental histology and development in North American Nerodia (Colubridae: Natricinae)

As part of a broad survey of placental structure, function, and evolution in reptilian sauropsids paraffin‐section histology was used to study microscopic anatomy of the uterus and fetal membranes of three species of North American watersnakes (Nerodia : Colubridae). The pre‐ovulatory uterus is poorly vascularized with inactive shell glands. These shell glands are activated during vitellogenesis but regress during pregnancy. Two placentas develop through apposition of the uterine lining to the chorioallantois and the yolk sac omphalopleure. Fetal and maternal components of the chorioallantoic placenta are progressively vascularized during development. Their epithelia are attenuated, but (contrary to a previous report), epithelia of neither the uterus nor the chorion are eroded. The fetal portion of the yolk sac placenta is an omphalallantois, formed of avascular omphalopleure, isolated yolk mass, and allantois. This placenta is progressively replaced by chorioallantoic placenta during mid‐ to late‐development through depletion of the isolated yolk mass. The chorioallantoic placenta is anatomically specialized for maternal–fetal gas exchange, and its expansion during development reflects the growing needs of the fetus for gas exchange. The yolk sac placenta is morphologically unsuited for gas exchange, but may serve other functions in maternal‐fetal exchange.     

Implantation and placental development in somatic cell clone recipient cows

Successful somatic cloned animal production has been reported in various domesticated species, including cattle; however, it is associated with a high rate of pregnancy failure. The low cloning yield could possibly arise from either an abnormal and/or poorly developed placenta. In comparison to control cows, fewer placentomes were found in somatic cell nuclear recipient (NT) cows at day 60 of gestation, suggesting a retardation of fetal/placental growth in these animals. NT cows not only had fewer numbers of chorionic villi but also had poorly developed caruncles. Macroscopic examination revealed atypical development of the placentome in terms of shape and size. Histological disruption of chorionic villi and caruncular septum was found in NT cows. Of particular interest was that the expression of genes, as well as proteins in the placentome, was disparate between NT and artificially inseminated cows, especially placental lactogen (PL) and pregnancy-associated glycoprotein (PAG). In contrast, prolactin-related protein-1 (PRP-1) signals were comparable across cows, including NT cows carrying immotile fetuses. The expression of extracellular matrix degrading molecule, heparanase (HPA), in NT cows was divergent from that of control cows. Microarray data suggest that gene expression was disorientated in early stages of implantation in NT cows, but this was eliminated with progression of gestation. These findings strongly support a delay in trophoblast development during early stages of placentation in NT cows, and suggest that placental specific proteins, including PLs, PAGs, and HPA, are key indicators for the aberration of gestation and placental function in cows.     

Origin and differentiation of the yolk sac and extraembryonic mesoderm in presomite human and rhesus monkey embryos.

Reexamination of presomite human and rhesus monkey embryos in the Carnegie Collection provides no evidence to corroborate the hypothesis that the trophoblast is the source of all extraembryonic tissues in these embryos. Instead, the present study indicates that the developmental pattern of the yolk sac and extraembryonic mesoderm is homologous to that in other eutharian mammals. The primary yolk sac of 10- to 11-day human blastocysts is partially filled with a meshwork of extraembryonic endoderm, whereas such a meshwork is absent in the rhesus monkey. It is suggested that this endodermal meshwork develops as the result of interstitial implantation in the human embryo. A small secondary yolk sac develops in 12- to 13-day human and macaque embryos as the result of pinching off of a portion of the larger primary yolk sac. Development of a secondary yolk sac in higher primates appears to be related causally to differential rates of expansion of the blastocyst and primary yolk sac within the simplex uterus. The caudal margin of the primitive streak develops precociously in 12- to 14-day human and macaque embryos, and this appears to be the source of all the extraembryonic mesoderm of the chorion, chorionic villi, and body stalk. It is suggested that the peripheral spread of extraembryonic mesoderm plays in inductive role in the development of chorionic villi, similar to other types of epithelial-mesenchymal inductive interactions. In contrast to previous hypotheses, the human and macaque trophoblasts appear to give rise only to additional trophoblast.     

Uterine epithelial changes during placentation in the viviparous skink Eulamprus tympanum

We used scanning electron microscopy (SEM) and transmission electron microscopy (TEM) to describe the complete ontogeny of simple placentation and the development of both the yolk sac placentae and chorioallantoic placentae from nonreproductive through postparturition phases in the maternal uterine epithelium of the Australian skink, Eulamprus tympanum. We chose E. tympanum, a species with a simple, noninvasive placenta, and which we know, has little net nutrient uptake during gestation to develop hypotheses about placental function and to identify any difference between the oviparous and viviparous conditions. Placental differentiation into the chorioallantoic placenta and yolk sac placenta occurs from embryonic Stage 29; both placentae are simple structures without specialized features for materno/fetal connection. The uterine epithelial cells are not squamous as previously described by Claire Weekes, but are columnar, becoming increasingly attenuated because of the pressure of the impinging underlying capillaries as gestation progresses. When the females are nonreproductive, the luminal uterine surface is flat and the microvillous cells that contain electron-dense vesicles partly obscure the ciliated cells. As vitellogenesis progresses, the microvillous cells are less hypertrophied than in nonreproductive females. After ovulation and fertilization, there is no regional differentiation of the uterine epithelium around the circumference of the egg. The first differentiation, associated with the chorioallantoic placentae and yolk sac placentae, occurs at embryonic Stage 29 and continues through to Stage 39. As gestation proceeds, the uterine chorioallantoic placenta forms ridges, the microvillous cells become less hypertrophied, ciliated cells are less abundant, the underlying blood vessels increase in size, and the gland openings at the uterine surface are more apparent. In contrast, the yolk sac placenta has no particular folding with cells having a random orientation and where the microvillous cells remain hypertrophied throughout gestation. However, the ciliated cells become less abundant as gestation proceeds, as also seen in the chorioallantoic placenta. Secretory vesicles are visible in the uterine lumen. All placental differentiation and cell detail is lost at Stage 40, and the uterine structure has returned to the nonreproductive condition within 2 weeks. Circulating progesterone concentrations begin to rise during late vitellogenesis, peak at embryonic Stages 28-30, and decline after Stage 35 in the later stages of gestation. The coincidence between the time of oviposition and placental differentiation demonstrates a similarity during gestation in the uterus between oviparous and simple placental viviparous squamates.     

Defective vascular development in connexin 45-deficient mice

In order to reveal the biological function(s) of the gap-junction protein connexin 45 (Cx45), we generated Cx45-deficient mice with targeted replacement of the Cx45-coding region with the lacZ reporter gene. Heterozygous Cx45(+/)(-) mice showed strong expression of the reporter gene in vascular and visceral smooth muscle cells. Cx45-deficient embryos exhibited striking abnormalities in vascular development and died between embryonic day (E) 9.5 and 10.5. Differentiation and positioning of endothelial cells appeared to be normal, but subsequent development of blood vessels revealed impaired formation of vascular trees in the yolk sac, impaired allantoic mesenchymal ingrowth and capillary formation in the labyrinthine part of the placenta, and arrest of arterial growth, including a failure to develop a smooth muscle layer surrounding the major arteries of the embryo proper. As a consequence, the hearts of most Cx45-deficient embryos were dilated. The abnormal development of the vasculature in the yolk sac of Cx45(-)(/)(-) embryos could be caused by defective TGFbeta signalling, as the amount of TGF beta1 protein in the epithelial layer of the yolk sac was largely decreased in the E9.5 Cx45(-)(/)(-) embryo, compared with the wild-type embryo. The defective vascular development was accompanied by massive apoptosis, which began in some embryos at E8.5 and was abundant in virtually all tissues of the embryos at E9.5. We conclude that in Cx45(-)(/)(-) embryos, vasculogenesis was normal, but subsequent transformation into mature vessels was interrupted. Development of different types of vessels was impaired to a varying extent, which possibly reflects the complementation by other connexin(s).     

Oestrogen production by the preimplantation donkey conceptus compared with that of the horse and the effect of between-species embryo transfer

Aromatase distribution in membranes of preimplantation horse and donkey conceptuses was compared by measuring the incorporation of [3H]androstenedione into oestrone and oestradiol-17 beta. In the donkey conceptus, aromatase activity was similar in all the tissues examined (yolk sac, chorionic girdle and allantochorion), whereas in the horse it was generally lower and showed the relationship chorionic girdle greater than yolk sac greater than allantochorion. A higher proportion of labelled precursor was incorporated into oestradiol-17 beta by extra-embryonic tissues of the donkey compared with those of the horse. In contrast to previous results, aromatase in the chorionic girdle did not decline progressively before its migration into the endometrium on Day 36 to form the endometrial cups. The chorionic girdle of a donkey conceptus carried in the uterus of a mare failed to invade the surrogate horse endometrium and aromatase activity was still high in this tissue at Day 42. Aromatase distribution in 2 transferred donkey-in-horse conceptuses resembled that of the fetal, rather than the maternal, genotype indicating a lack of effect of the maternal environment.