Comparative reproduction of the female horse, elephant and rhinoceros: implications for advancing Assisted Reproductive Technologies (ART).

Recent loss of rhinoceros subspecies has renewed interest in using more advanced assisted reproductive technologies (ART) in rhinoceroses and elephants. Currently, only semen collection, semen preservation and artificial insemination (AI) have been used repeatedly with success in these species. Although ovum pick-up (OPU) and intra-cytoplasmic sperm injection (ICSI) have been reported in rhinoceroses, the techniques are not yet optimised. In contrast, multiple ART applications are routinely used in the horse. Since elephant and rhinoceroses share some reproductive features with equids, we postulate that procedures such as OPU, ICSI, in vitro fertilisation (IVF) and embryo transfer (ET), which are well established in the horse, may represent a basis to develop protocols for endangered pachyderms. In this review, we summarize current knowledge on reproductive physiology relevant to ART. We discuss the current state of ART in all three families and the requirements for the successful implementation of OPU, ICSI, IVF and ET in these species.

Observations on the glycosylation of the term placenta of the Indo-Pacific Bottlenose Dolphin (Tursiops aduncus): A lectin histochemical study.

INTRODUCTION: Little is known about the glycosylation of placental villi and areolae of cetaceans. Term tissue from the delivered placenta of an Indo-Pacific Bottlenose Dolphin (Tursiops aduncus) was examined using lectin histochemistry to compare trophoblast glycosylation in these two locations. METHODS: Placental blocks fixed in 10% formalin were resin-embedded before semithin sections were stained with 24 biotinylated lectins and an avidin-biotin revealing system. RESULTS: Areolar trophoblast was composed of large, bulbous cells packed with numerous granules compared to the smaller, cuboidal cells clothing the chorionic villi, which had a sparser, mainly subapical granule population. Both were richly glycosylated; generally areolar cells were more heavily stained apart from poor binding to some N-acetylgalactosamine and N-acetylglucosamine termini. Most striking was the distribution of α1,2-linked fucosyl residues, weakly expressed in villous trophoblast but intensely stained in some areolar cells, also terminal sialic acids. Some lectins bound in a variable fashion. Staining of terminal α-d-mannose, which locates mainly to lysosomes, was heavy in areolar cells compared to scattered irregular foci in villous cells. DISCUSSION AND CONCLUSION: The many intracellular inclusions reflect ongoing lysosomal breakdown of histotroph in areolar cells which often show heterogeneous glycosylation staining unlike the uniformly stained villous cells, possibly reflecting partial breakdown of ingested sialoglycoprotein, cell turnover or regional variation in uptake of histotroph. Our results indicate that Dolphin areolae are functionally distinct from villous trophoblast, performing absorptive and phagocytic functions similar to other Artiodactyla.

The effect of embryo reduction and transfer on luteostasis in the mare.

This study investigated the effects of embryo reduction and transfer of Day 11 embryos, with or without subsequent reduction, on luteostasis in the mare. In Experiment 1, reduction of embryos at Days 10 (n = 15), 11 (n = 47), 12 (n = 36), 13 (n = 27), 14 (n = 5) and 16 (n = 2) of pregnancy resulted in luteostasis in 13%, 47%, 78%, 89%, 80% and 100% mares. Mares undergoing > 1 embryo reduction showed consistency in when luteostasis occurred. In Experiment 2, transfer of Day 11 embryos to recipient mares 10 (n = 9), 11 (n = 8), 12 (n = 9) and 13 (n = 8) days post ovulation resulted in luteostasis in 78%, 87.5%, 78% and 37.5% of mares. Only 22%, 37%, 0% and 12%, respectively, of these mares remained pregnant. In the Day 10, 11 and 12 recipients luteostasis occurred on at least one occasion when an embryo was detected at 24 h but not at 48 h post transfer. In the Day 12 recipients luteostasis occurred on three occasions (3/9;33%) when the transferred embryo was not detected at 24 h. In Experiment 3 reduction of a Day 11 embryo 24 h after transfer to a Day 10 (n = 4), 11 (n = 6), 12 (n = 6) or 13 (n = 6) recipient resulted in luteostasis in 100%, 83%, 100%, and 83% of mares. All five Day 11 recipients that had an embryo reduced 12 h post transfer became luteostatic. These results suggest there is plasticity overall, but individual rigidity, in the timing of maternal recognition of pregnancy. Furthermore, an intact embryo need only be present in the uterus for 12 h to cause luteostasis.

Asymmetric expression of proteins in the granules of the placentomal Binucleate cells in Giraffa camelopardalis†.

Mature granulated trophoblast binucleate cells (BNC) have been found in all ruminant placentas examined histologically so far. BNC are normally fairly evenly distributed throughout the fetal villus and all their granules contain a similar variety of hormones and pregnancy associated glycoproteins (PAGs). Only the Giraffe is reported to show a different BNC protein expression, this paper is designed to investigate that. Gold labelled Lectin histochemistry and protein immunocytochemistry were used on deplasticised 1 µm sections of a wide variety of ruminant placentomes with a wide range of antibodies and lectins. In the Giraffe placentomes, even though the lectin histochemistry shows an even distribution of BNC throughout the trophoblast of the placental villi, the protein expression in the BNC granules is limited to the BNC either in the apex or the base of the villi. Placental lactogens and Prolactin (PRL) are present only in basally situated BNC: PAGs only in the apical BNC. PRL is only found in the Giraffe BNC which react with many fewer of the wide range of antibodies used here to investigate the uniformity of protein expression in ruminant BNC. The possible relevance of these differences to ruminant function and evolution is considered to provide a further example of the versatility of the BNC system.

Placentation in Equids.

This chapter focuses on the early stages of placental development in horses and their relatives in the genus Equus and highlights unique features of equid reproductive biology. The equine placenta is classified as a noninvasive, epitheliochorial type. However, equids have evolved a minor component of invasive trophoblast, the chorionic girdle and endometrial cups, which links the equine placenta with the highly invasive hemochorial placentae of rodents and, particularly, with the primate placenta. Two types of fetus-to-mother signaling in equine pregnancy are mediated by the invasive equine trophoblast cells. First, endocrinological signaling mediated by equine chorionic gonadotrophin (eCG) drives maternal progesterone production to support the equine conceptus between days 40 and 100 of gestation. Only in primates and equids does the placenta produce a gonadotrophin, but the evolutionary paths taken by these two groups of mammals to produce this placental signal were very different. Second, florid expression of paternal major histocompatibility complex (MHC) class I molecules by invading chorionic girdle cells stimulates strong maternal anti-fetal antibody responses that may play a role in the development of immunological tolerance that protects the conceptus from destruction by the maternal immune system. In humans, invasive extravillous trophoblasts also express MHC class I molecules, but the loci involved, and their likely function, are different from those of the horse. Comparison of the cellular and molecular events in these disparate species provides outstanding examples of convergent evolution and co-option in mammalian pregnancy and highlights how studies of the equine placenta have produced new insights into reproductive strategies.

Placentation in the African Elephant (Loxodonta africana).

The female elephant shows a 3-week "follicular phase" to commence her 16-week estrous cycle at the end of which a second surge in pituitary luteinizing hormone (LH) release matures and ovulates an ovarian follicle in association with estrous behavior and mating, whereas the first LH surge at the start of the follicular phase causes luteinization of 3-5 partially developed follicles. The prolonged pregnancy of 22 months is supported by a zonary endotheliochorial placenta which secretes placental lactogen (ePL) from around 40 days of gestation in association with replacement of the lumenal epithelium of the endometrium by trophoblast and the development of large corpora lutea (CLs) in the maternal ovaries from the previously formed luteinized follicles in response to the first LH peak early in the follicular phase. The zonary placenta develops above, rather than within, the endometrium. The elephant placenta secretes neither estrogens nor progestagens throughout gestation, as pregnancy maintenance relies on 5α-dihyroprogesterone and other 5α reduced progestagens secreted by secondary CLs stimulated by ePL and the stromal tissue of the fetal gonads, which become extremely enlarged during the second half of the 22-month pregnancy. In female fetuses, this ovarian enlargement includes the development and subsequent regression of multiple primary and secondary follicles with a consequent substantial decline in primary follicle numbers at birth. During the next 8-9 years of pre-pubertal life, however, oocyte and primary follicle numbers recover to levels near those found in late gestation, which may be evidence of postnatal oogenesis occurring in the elephant.

Morphological Characteristics of the Placenta and Umbilical Cord of Arabian Mares Foaling in the United Arab Emirates.

A total of 127 normal placentas from Arabian mares resident in the United Arab Emirates were examined. The mean linear dimensions of the placenta were, on average, 84% of those previously recorded for the placentas of the Thoroughbred. Significant differences in the size of the allantochorion between primigravid and multiparous mares were seen only in the linear dimensions of the body portion. The pregnant horn was more commonly on the right than left side of the uterus (P = .01; 74/127; 58%). Cord attachment was primarily at the base of the two placental horns (112/127; 88%), with the remainder showing anomalies from this position. The mean (±SEM) total length of the umbilical cords was 62.2 ± 1.2 cm, being composed of the allantoic portion (29.7 ± 0.9 cm) and amniotic portion (32.5 ± 0.6 cm), which averaged 53 ± 0.01% of the total length. The amniotic portion was usually, but not always, longer than the allantoic portion (79/127; 62%). Longer cords were associated with a greater proportion of allantoic length. An enlarged yolk sac remnant (YSR) was present in 16/127 (13%) placentas. Cords with YSRs displayed a significantly longer allantoic portion than those without (P = .02). The total cord length was not correlated with the weight or area of the allantochorion or amnion, any linear measurement of the allantochorion, gestation length, the month of foaling, parity of the dam, or birthweight or sex of the foal. The purpose for which the mare was bred (i.e., racing or showing) influenced the cord length, those of show mares being significantly longer.

Historical Aspects of Equine Embryo Transfer.

Early embryo transfer in equids was undertaken simultaneously in the early 1970s in Cambridge, England, and Kyoto, Japan. Both groups achieved limited success when flushing the uterine horn ipsilateral to the side of ovulation but the rates improved markedly when the whole uterus was flushed on realization of the continued movement of the embryo throughout the uterine lumen after day 6. Initial transfers of embryos to recipient mares were carried out surgically, but nonsurgical transfer via the cervix has been used subsequently with increasing success, culminating in pregnancy rates of 75%-90% today. Experimental use of embryo transfer in horses and donkeys demonstrated the unique ability of equids to carry to term a full range of interspecies hybrid conceptuses and extraspecies pregnancies created by embryo transfer. Furthermore, splitting of day 4-8 cell embryos and day 6 compact morulae allowed the creation of genetically identical twin foals. But despite these and other significant advances over the past 45 years, a persisting limitation is the relatively low embryo recovery rates from donor mares treated with exogenous gonadotropins in attempts to induce them to superovulate. This is due to the toughness of the ovarian tunica albuginea which forces ovulation through the ventrally situated ovulation fossa where multiple follicles compete with each other and luteinize before they can ovulate properly.

The influences of cycle stage and pregnancy upon cell glycosylation in the endometrium of the mare.

From Day 6.5-7 post-conception until its loss around Day 22, the equine embryo is enclosed in a mucinous capsule that prevents direct intercellular interaction between the trophectoderm and uterine epithelium. The embryo is, however, bathed in glycoprotein-rich secretions. In this study, lectin histochemistry was used to characterise the distribution and glycan composition of uterine glycoproteins destined for secretion, and to ascertain the local effect of an embryo on glycosylation in the endometrium. Endometrial biopsies were taken from mares in estrus, on Days 5, 8, 12 and 15 of diestrus, and on Days 12 and 15 of pregnancy and processed for lectin histochemistry. During estrus, lumenal epithelial cells were as truncated pyramids and mainly non-ciliated with glycosylated granules in the cytoplasm. Occasional ciliated cells contained few granules. Five days post-ovulation, non-ciliated cells of the lumenal epithelium were taller, and had accumulated many highly glycosylated apical granules. By Days 12 and 15 post-ovulation these cells were more cuboidal and some showed fewer secretory granules. In marked contrast, by Days 12 and 15 of pregnancy, the ciliated cells were distended, with numerous granules but non-ciliated cells had only a few in the apical cytoplasm. Glycosylation changed dramatically in pregnancy in the luminal and superficial gland epithelium, with fewer fucosylated termini, more N-acetyl galactosamine residues, together with an overall reduction in sialic acid and several other sugar structures. Glycosylation in ciliated cells on Days 12 and 15 of pregnancy showed a striking similarity to that of the blastocyst capsule. The data strongly suggests that glycoprotein production by luminal epithelial cells is influenced by the presence of a conceptus. We speculate that, as well as providing nourishment for the developing embryo, epithelial secretory glycoproteins may contribute components to the capsule, which develops only partially in embryos cultured in vitro.

Placentation and hormonal maintenance of pregnancy in the impala (Aepyceros melampus).

INTRODUCTION: The impala is a widely distributed African ungulate. Detailed studies of the placenta and ovaries in impala undertaken in the 1970s did not address the endocrine functions of the placenta. METHODS: The uteri of 25 pregnant impala estimated to be between 49 and 113 days of the 190 day gestation were examined grossly, histologically and immunohistochemically. RESULTS: A single corpus luteum was present in either maternal ovary but the conceptus was always situated in the right uterine horn. The fetal membranes extended to the tips of both uterine horns. The amnion was in intimate contact with, but not fused to, the allantochorion. Placentation was typically ruminant with fetal macrocotyledons attached to the rows of maternal caruncles. The fetal villi were highly branched, especially in the centre of each placentome where the attenuated maternal epithelium lining the placental crypts was absent in some places. Both the corpus luteum and the uninucleate trophoblast cells of the interplacentomal allantochorion stained strongly for 3-ß hydroxysteroid dehydrogenase, and progestagen concentrations in allantoic and amniotic fluids increased significantly as gestation progressed, with a tendency to do likewise in maternal serum. Binucleate trophoblast cells stained positively for bovine placental lactogen, but neither the placenta nor the maternal corpus luteum showed evidence of oestrogen synthesis. DISCUSSION: Despite exhibiting the same basic type of placentation, both the gross and histological structure of the impala placenta, along with its immunohistochemical properties, demonstrates that great variation exists across ruminant placentas.

Managed wildlife breeding-an undervalued conservation tool?

Knowledge of and the technologies and resources applied to the ex situ care for wildlife have improved greatly in recent years. This has resulted in numerous successes bringing back populations from the brink of extinction by the reintroduction or restoration of animals from conservation breeding programmes. Controlled breeding of wildlife by humans is discussed controversially in society and in scientific circles and it faces a number of significant challenges. When natural breeding fails, Assisted Reproduction Technologies (ART) have been postulated to increase reproductive output and maintain genetic diversity. Furthermore, technical advances have improved the potential for successful collection and cryopreservation of gametes and embryos in many wildlife species. With the aim of creating a better understanding of why ex situ and in situ conservation of threatened species must complement each other, and under which circumstances ART provide additional tools in the rescue of a threatened population, we elucidate the current situation here by using as examples three different megavertebrate families: elephantidae, rhinoceridae and giraffidae. These mammal families consist of charismatic species, and most of their members are currently facing dramatic declines in population numbers. On the basis of these and other examples, we highlight the importance of captive zoo and other managed wildlife populations for species survival in a human dominated world. Without the possibility to study reproductive physiology in trained or habituated captive individuals, major advances made in wildlife ART during the past 20 years would not have been possible. This paper reviews the benefits and future challenges of large mammal conservation breeding and examines the role of assisted reproduction in such efforts.

The immunolocalization of Galectin-1 and Progesterone-Induced Blocking Factor (PIBF) in equine trophoblast: Possible roles in trophoblast invasion and the immunological protection of pregnancy.

INTRODUCTION: The proteins galectin-1 and Progesterone Induced Blocking Factor (PIBF) are present on human and murine trophoblast and are thought to influence both immunomodulation and trophoblast invasion. In equids, the invasive component of the placenta, the endometrial cups, stimulate maternal cell-mediated and humoral immune responses. It was therefore of interest to know if galectin-1 or PIBF could be immunolocalised to the invasive and/or non-invasive components of the equine placenta. MATERIALS: Horse and mule (♀ horse X ♂ donkey) embryos and placental tissues between Days 12 and 124 of gestation were stained immunohistochemically with antibodies raised against galectin-1 and PIBF. RESULTS: Galectin-1 stained the non-invasive trophoblast between Days 15 and 20 but thereafter stained only the invasive trophoblast cells of the chorionic girdle, both before and after they invaded the endometrium to form the endometrial cups. PIBF, on the other hand, stained both the invasive and non-invasive trophoblast throughout the period of gestation studied. Of particular interest was the relative lack of staining of the endometrial cup cells in mule compared to horse pregnancies for galectin-1 and PIBF prior to the earlier and more rapid death and desquamation of the mule cup cells. DISCUSSION: The expression of galectin-1 and PIBF proteins in equine trophoblast and the marked difference in lifespan between the endometrial cups in intraspecies horse versus interspecies mule pregnancies support a likely role for these two proteins protecting the fetal trophoblast from maternal immune attack and/or modulation of the invasiveness of endometrial cup cells.

A preliminary study of the heterogeneity in endometrial morphology and glycosylation in the uterine horns of the non-pregnant impala (Aepyceros melampus).

Placentation commences only in the right uterine horn in impala (Aepyceros melampus). To investigate possible differences in morphology or glycosylation between the two horns, right and left uterine horns from six non-pregnant, wild impala were examined morphometrically and histochemically using a panel of 23 lectins and an avidin-biotin revealing system. The presence of ovarian 3ß hydroxysteroid dehydrogenase (3ßHSD) and aromatase was also investigated using immunocytochemistry. There were few detectable differences in morphology and glycosylation between right and left uterine horns in five of the specimens, but the sixth had deep clefts and plentiful exocrine secretions in the right horn, and not the left. Heavily glycosylated clusters of supranuclear granules were present in the epithelial cells, which had many classes of O-linked glycans. The serum progestagen was not markedly different, however, from that of the other specimens. In five of the six specimens, the height of luminal epithelium was greater on the right than that on the left, and the height of the gland epithelium was also greater on the right side in four of these. The 3ßHSD and aromatase activities were present in the ovaries and were similar in impala with or without progestagen concentrations >1 ng/ml in peripheral blood. No corpus haemorrhagicum or corpus luteum could be discerned. These findings indicate there are morphological and biochemical differences between right and left uterine horns in the impala and further studies are needed on both impala and other species in which placentation commences only in one uterine horn, to establish the cyclical hormone changes which induce them.

Vitrification of equine expanded blastocysts following puncture with or without aspiration of the blastocoele fluid.

BACKGROUND: Historically, cryopreservation of equine embryos >300 µm gave poor pregnancy rates until researchers collapsed the blastocoele cavity and aspirated the blastocoele fluid prior to vitrification. OBJECTIVE: To determine if aspiration of the blastocoele fluid prior to vitrification is essential for post warming survival. STUDY DESIGN: In vivo experiments. METHODS: Fifty embryos were recovered on day 7-8 and washed in holding medium (HM; M-199HEPES + 20% FBS + antibiotics). Embryos were punctured using a micromanipulator mounted 30 µm biopsy needle; following this 28 had >90% of their blastocoelic fluid actively aspirated while the remaining 22 were not-aspirated. Embryos were then vitrified using a two-step process with increasing concentrations of DMSO and ethylene glycol (7.5-15% v:v), and 0.5 mol/L sucrose in the final solution before being loaded onto a Cryolock device and plunged into liquid nitrogen. The embryos were warmed by plunging the Cryolock tip into HM with 1 mol/L sucrose at 37°C. After 1 min, the embryos were transferred to HM + 0.5 mol/L sucrose at RT for 4 min before transfer into HM for a further 4 min prior to transfer to a recipient mare. RESULTS: Mean (±s.e.) embryo diameter was not significantly different between the punctured and punctured plus aspirated group (646.4 ± 61.7 vs. 754.8 ± 59.1 µm, respectively; P = 0.215). Nonaspirated and aspirated embryos gave pregnancy rates of 10/22 (45%) and 21/28 (75%) respectively (P = 0.061). Sub-dividing embryos on the basis of size showed that vitrification of larger embryos (>550 µm) yielded a significantly higher pregnancy rate when they were aspirated vs. not-aspirated (13/18 [72%] vs. 1/10 [10%], respectively; P = 0.006), whereas there was no difference for smaller embryos (8/10 [80%] vs. 9/12 [75%], respectively; P = 0.8). MAIN LIMITATIONS: Group sizes are limited. CONCLUSION: Aspiration of blastocoele fluid from embryos ≤550 µm is not a pre-requisite for successful vitrification. The Summary is available in Spanish - see Supporting Information.

Half a century of equine reproduction research and application: A veterinary tour de force.

Over the past 50 years, per season pregnancy rate in Thoroughbred mares have risen from 70 to >90% and foaling rates from 55 to >80%. Some of the significant pharmacological treatments and diagnostic methods which have driven this marked improvement in breeding efficiency are reviewed. They include the application of artificial lighting to hasten the onset of ovulatory oestrous cyclicity in early Spring, rapid steroid hormone assays to aid in determining the stage and normality of the mare's cycle, prostaglandin analogues, synthetic progestagens and Gonadotrophin-releasing Hormone (GnRH) analogues to better control and manipulate her cycle to good effect, transrectal ultrasound scanning to monitor follicle growth, endometrial architecture and ovulation and to allow accurate, early pregnancy diagnosis thereby enabling successful ablation of one of twin conceptuses. Also, flexible videoendoscopy to monitor physiological and pathological changes in the uterine endometrium and rigid laparoscopy to apply prostaglandin to the oviducts to dislodge and clear suspected blockages of them to restore fertility. The outbreak of Contagious Equine Metritis in Newmarket in the spring of 1977 and the swabbing-related changes to mare and stallion management, plus the improved veterinary hygiene methods, which followed are also recounted. The past half century has witnessed many technical and therapeutic advances that have enhanced tremendously the diagnostic and treatment capabilities of studfarm veterinary surgeons. They, in turn, have improved greatly the efficiency of breeding Thoroughbreds and other types of horses.

A lectin histochemical study to detect variation in glycosylation at the feto-maternal interface in three interbreeding equine species.

INTRODUCTION: In this study, we compare glycosylation at the fetomaternal interface in 3 equine species: horse, donkey and zebra, all of which can interbreed to produce hybrids, to assess their glycan similarities and differences. METHODS: Sections cut from 3 specimens of horse (Equus caballus) placenta (50, 200 and 280 days gestation), one donkey (Equus asinus) placenta (65 cm crown-rump length) and 5 specimens of zebra (Equus quagga) placentae (81-239 days gestation) were stained with a panel of 24 biotinylated lectins using an avidin-peroxidase revealing system. RESULTS: There were only slight quantitative differences in the lectin histochemistry at the feto-maternal interface between all three specimens; zebra placentae expressed more α2,6-linked sialic acid, with α1,2fucosyl residues at the microvillous interface. However, zebra trophoblast showed histological differences from the other two species, with polarised cells, prominent supranuclear Golgi bodies, and fewer intracellular granules. DISCUSSION: Our findings appear to confirm the hypothesis that closely related, interbreeding species with epitheliochorial placentae express similar glycans at the feto-maternal interface, thereby supporting the existence of a placental glycocode. We also observed intraspecies evolutionary diversion to be associated with a different histological architecture and the absence of significant intracellular granules.

Placentation in the plains zebra (Equus quagga).

The placenta and fetal gonads of 12 pregnant plains zebra (Equus quagga), estimated to be between 81 and 239 days of gestation, were examined. The diffuse, microcotyledonary zebra placenta appeared, developmentally, to be 3-4 weeks behind its counterpart in horse pregnancy and this, together with the presence of small and long-lived endometrial cups, low levels of zebra chorionic gonadotrophin in maternal serum and few accessory corpora lutea in the maternal ovaries during the first half of gestation, made zebra pregnancy more similar to donkey than horse pregnancy. Zebra fetal gonads enlarged after 80 days of gestation and their interstitial cells stained positively for 3ß hydroxysteroid dehydrogenase and 17,20 lyase steroid enzymes while the trophoblast stained for aromatase. This confirmed that zebra fetal gonads, like those of the horse and donkey, can synthesise C19 androgens, which can then be aromatised by the placenta to C18 oestrogens. It is remarkable that such unusual feto-placental mechanisms of production of gonadotrophic and steroid hormones has persisted unchanged within the genus Equus despite the many physical adaptations and the considerable loss of chromosomes that have occurred during the evolution of its member species.

Localisation of epidermal growth factor (EGF), its specific receptor (EGF-R) and aromatase at the materno-fetal interface during placentation in the pregnant mare.

INTRODUCTION: Implantation and placentation in the mare does not commence until as late as day 40 after ovulation. The reasons for this and the growth factors and/or hormones which drive placentation when it does finally occur are of considerable academic and practical interest. METHOD: Placental interface tissues recovered from 11 accurately aged and perfused-fixed horse uteri between 20 and 68 days of gestation were stained immunocytochemically for Epidermal Growth Factor (EGF), its specific receptor (EGF-R) and for the steroid hormone enzyme, aromatase. RESULTS: EGF was present in endometrial gland and lumenal epithelia from day 20 but staining intensity increased noticeably for the protein between days 30 and 40, coincidentally with the commencing secretion of equine Chorionic Gonadotrophin (eCG) from the endometrial cups and immediately prior to attachment and commencing interdigitation between the allantochorion and endometrium. EGF-R, on the other hand, was expressed strongly on the cell surface membrane of both non-invasive and invasive trophoblast and it similarly increased in staining intensity between days 30 and 40. Aromatase, the enzyme necessary for conversion of C-19 androgens to C-18 oestrogens, was expressed strongly and constantly from as early as day 12 in the non-invasive trophoblast of the allantochorion, but not the invasive trophoblast of the chorionic girdle, the progenitor tissue of the endometrial cups. DISCUSSION: The findings support the hypothesis that, in equine pregnancy, the maternal growth factor EGF synergises with maternally and fetally secreted oestrogens to drive the rapid growth and extensive vascularisation of the non-invasive, epitheliochorial, microcotyledonary placenta which results in the birth of the precocious foal after only 11 months gestation.

Morphology, histochemistry and glycosylation of the placenta and associated tissues in the European hedgehog (Erinaceus europaeus).

INTRODUCTION: There are few descriptions of the placenta and associated tissues of the European hedgehog (Erinaceus europaeus) and here we present findings on a near-term pregnant specimen. METHODS: Tissues were examined grossly and then formalin fixed and wax-embedded for histology and immunocytochemistry (cytokeratin) and resin embedded for lectin histochemistry. RESULTS: Each of four well-developed and near term hoglets displayed a discoid, haemochorial placenta with typical labyrinth and spongy zones. In addition there was a paraplacenta incorporating Reichert's membrane and a largely detached yolk sac. The trophoblast of the placenta contained diverse populations of granule which expressed most classes of glycan. Intercellular membranes were also glycosylated and this tended to be heavier in the labyrinth zone. Fetal capillary endothelium had glycosylated apical surfaces expressing sialic acid and various other glycans. Glycogen was present in large cells situated between the spongy zone and the endometrium. Trophoblast cells in the placental disc and under Reichert's membrane, as well as yolk sac endoderm and mesothelium, were cytokeratin positive. Reichert's membrane was heavily glycosylated. Yolk sac inner and outer endoderm expressed similar glycans except for N-acetylgalactosamine residues in endodermal acini. DISCUSSION: New features of near-term hedgehog placenta and associated tissues are presented, including their glycosylation, and novel yolk sac acinar structures are described. The trophoblast of the placental disc showed significant differences from that underlying Reichert's membrane while the glycan composition of the membrane itself showed some similarity to that of rat thereby implying a degree of biochemical conservation of this structure.