Immunohistochemical localization of oestrogen receptors alpha and beta, progesterone receptor and aromatase in the equine placenta.

The functions of placental oestrogens during equine pregnancy are still unclear. Yet, they may act predominantly as local regulators of growth and differentiation in the microplacentomes. Thus, expression patterns of oestrogen receptors (ERs) alpha and beta were investigated in the microcotyledonary placenta from pregnant mares at 110, 121, 179, 199 and 309 days of gestation by immunohistochemistry. In microplacentomes, both the ER isoforms were detected in trophoblast (T) cells, chorionic villous stroma (FS), microcaruncular epithelium (ME) and microcaruncular stroma (MS). Proportions of positive cells were 38-91% (T), 11-41% (FS), 55-89% (ME), 17-51% (MS) for ERalpha and 66-76% (T), 21-37% (FS), 41-68% (ME) and 24-55% (MS) for ERbeta. Between days 110 and 199, proportions of cells positive for progesterone receptor (PR) varied between 19% and 62% (T), 3% and 50% (CS), 15% and 46% (ME), and 4% and 33% (MS). At day 309, PR was virtually absent in T, CS and ME (percentages < 0.1), whereas in MS 14.3% of cells were still positive. The expression of ERs and PR in equine microplacentomes gives evidence for a role of placental steroids as regulators of placental growth, differentiation and function. The detection of ERalpha, ERbeta and PR in foetal and maternal vascular tissue suggests that placental steroids are also involved in the control of placental angiogenesis and /or vascular functions. The co-localization of ERs with aromatase in T suggests auto- or intracrine functions of oestrogens in this cell type.

Prenatal development of murine gonads with special reference to germ cell differentiation: a morphological and immunohistochemical study.

The prenatal differentiation of male and female gonads of the mouse was investigated both morphologically and immunohistochemically. Sexual dimorphism could be detected as early as 12 days post-coitum (dpc) by the appearance of the primary elements of the tunica albuginea and positive immunoreactivity for anti-Muellerian hormone in the Sertoli cells of the male gonad. Male germ cells passed two waves of mitotic activity, a first wave between 12 and 14 dpc, which is followed by apoptosis of the old germ cell generation, and a second wave between 17 and 20 dpc. Oct-4 was expressed as a juxtanuclear ring in the cytoplasm of germ cells up to 17 dpc. Subsequently, it was down-regulated and completely disappeared in 20 dpc full-term fetuses. By contrast, M2A antigen revealed only a weak immunoreaction in some germ cells of 14 dpc gonads, but exhibited strong signals in all germ cells of 20 dpc full-term fetuses. Therefore, we postulate that, in the mouse, prenatal germ cells represent two populations: the first is immunopositive for Oct-4 and disappeared in full-term fetuses, whereas the second appeared in 14 dpc and is immunopositive for M2A antigen.

Microvascular architecture of the near-term uterine caruncles in water buffaloes (Bubalus bubalis).

The present investigation was carried out on five near-term pregnant water buffaloes for studying the microvascular architecture of the uterine caruncles. The vascular casts were obtained by injection of 4:1 mixture of mercox and methylmethacrylate through the branches of the uterine arteries. After complete polymerization of the plastic, corrosion was conducted in 20% potassium hydroxide, then the vessel casts were immersed in distilled water, cut into small pieces, sputter coated with gold, and examined by using a scanning electron microscope. The buffalo uterine caruncle is highly vascularized through two slightly convoluted arteries and a single less tortuous vein. The arteries branch into several stem arteries at the base of the uterine caruncle, which follow nearly straight course in the primary septa towards the fetal side. During the courses of these stem arteries arterioles of variable diameters arise. The arterioles run in the secondary and tertiary septae and at this location arterioles and venules are connected through a voluminous capillary complex. The latter consists of capillaries of greatly variable diameters with vigorous coiling and sinusoidally dilated zones. From the capillary complexes the blood is driven through postcapillary venules back to the tertiary, secondary and primary septa, respectively, and then converge into stem veins which leave the caruncles through the branches of the uterine vein.

Structural and haemovascular aspects of placental growth throughout gestation in young and aged mares.

This investigation was carried out to study the equine placenta from early gestation to near term, with special reference to morphological changes associated with the development of the vasculature of the fetal component of the microcotyledons. Pregnant uteri were removed post mortem from five Thoroughbred mares between 110 and 309 days of gestation, two of which were aged, multiparous animals suffering age-related degenerative changes in their endometrium (endometrosis), while the other three were young, and had primigravid normal healthy uteri. Pieces of endometrium with placenta attached were fixed for light microscopy and fetal vascular casts were made by injecting the placental arteries with a mixture of Mercox and methylmethacrylate. The casts were examined under the scanning electron microscope. In an aged, endometrotic mare at 110 days of gestation, most of the microplacentomes were irregular in shape with a mean+/-sem diameter of 399+/-30.53 microm. Capillaries with variable diameters made up widely meshed network villi with pointed ends (Type 1 terminal villi), and narrow-meshed networks with finger-like ends (Type 2 terminal villi). In the "paired" young healthy mare at day 121 of gestation, most of the microplacentomes were globular in shape and appeared smaller in diameter than those in the 110-day "pair". The narrow-meshed capillary networks formed villi with stems that consisted of both intermediate and terminal parts, the latter of which represented more the Type 2 than the Type 1 terminal villus. In another aged endometrotic mare at 179 days of gestation, the microplacentomes were typically globular in shape and they showed a mean diameter of 534+/-36.07 microm. The villi were short and thick and they were distinctly differentiated into stem, intermediate and terminal parts. The density of the fetal capillaries had now greatly increased so that, three dimensionally, they constituted bulb-like capillary networks at the base of the stem of each villus. At 199 days in the young healthy "pair", the microplacentomes were again smaller in diameter (402+/-16.24 microm) than in the old mare at 179 days and the interhaemal distance had now reduced to 14.28+/-0.42 microm. The vascular density was lower than in the day 179 aged mare and the fetal villi were much longer and thinner. In the single late stage, healthy young mare at 309 days of gestation (term=336 days), the microplacentomes, each of around 2 mm diameter, exhibited maximal length villi. The capillaries were arranged simply, mostly in straight lines along the axis of the villus, and with communications visible at irregular intervals. Simple and slightly more complicated side capillary loops could be seen along the whole length of the villi and at the top of the terminal villi. Most of the capillaries were characterized by zones containing dilated sinusoids, which increased the surface area for materno-fetal exchange. Thus, the morphological development of the microplacentomes on the surface of the horse placenta during gestation was studied, with special reference to the growth and organisation of the fetal and maternal capillary beds within each microplacentome. The study also reinforced previous work showing the disadvantageous influence of age-related endometrial degenerative changes on microplacentome development and on both the extent and intimacy of physical and haematological contact at the fetomaternal interface, and hence upon fetal growth.

Development of the areola in the early placenta of the one-humped camel (Camelus dromedarius): a light, scanning and transmission electron microscopical study.

This study aimed to elucidate development of the areola in the early dromedary placenta in comparison with that of the pig and mare. Placental tissues from 25 pregnant camels were obtained from Cairo abattoir and prepared for light, scanning and transmission electron microscopy by routine methods. Vascular casts were made by injection of 4 : 1 liquid plastic mixture of mercox and methylmethacrylate. Areolar formation was first observed at 4.5 cm curved-crown-rump CVR length, while by 5-9 cm CVR length, the endometrial surface was uneven and studded with numerous uterine gland openings, where corresponding foetal areolae were barely detectable and the foetal areolar cells were of variable appearance and covered with long microvilli. At 10-13 cm CVR length the uterine gland openings developed irregular folds and the maternal areolar cells showed numerous apical blebs. At 14-29 cm CVR length the foetal areolae showed a great increase in height at the expense of their width. At 30-34 cm (CVR) length the maternal areolae appeared discoid and sharply demarcated from the surrounding inter-areolar tissues and the foetal areolae were rounded to irregular in shape with well-developed areolar rims. The vascular casts showed a widely meshed capillary network on the maternal areola, connecting with the pre- and post-capillary vessels, whereas the foetal side showed a relatively dense capillary meshwork. These studies indicate that the areola in the placenta of the one-humped camel is of the regular type like in the pig, and is poorly vascularized.

The blood vasculature as the forming element of the uterus of the estrous donkey (Equus asinus).

Light, scanning electron microscopy of endometrial surface and vascular casts were used to study the vascular architecture of the donkey uterus during estrous. The arterial blood supply of the uterus comes from three arteries: the uterine branch of the ovarian artery, the uterine artery of the external iliac artery, and the uterine branch of the urogenital artery. All arteries enter the uterus at its mesometrial border and divide into smaller ones. Segmentally constricted arteries are seen to circumscribe large veins at the perimetrium which become highly convoluted in the intermuscular vascular layer of the myometrium. Small arteries and arterioles originate at the borderline between the myometrium and the endometrium and radiate to the surface of the endometrium to constitute a system of numerous ridges and grooves by a widely meshed plexus of subepithelial capillary network. The post-capillary venules of the endometrium arise from the subepithelial capillary plexus to form slightly larger veins than the concurrent arteries which join up to the large tortuous veins in the intermuscular vascular layer of the uterus. This arrangement of blood vessels in the donkey uterus and particularly in the endometrium provides the requirement for instant blood flow on the arterial side and for the slow flow rate on the venous side to ameliorate the process of substances exchange.

Glycosylation in the near-term epitheliochorial placenta of the horse, donkey and camel: a comparative study of interbreeding and non-interbreeding species.

Studies from this laboratory have shown great diversity in the glycosylation of tissues comprising the interhaemal barrier of species with different placental types. This diversity may be one of the factors preventing interbreeding between species. Glycan expression within the uterine epithelium and trophoblast of the interhaemal barrier was examined to test this proposition in three species with similar diffuse, microcotyledonary, epitheliochorial allantochorionic types of placenta: the horse (Equus caballus) and donkey (Equus asinus), which can interbreed with each other, and the camel (Camelus dromedarius), which cannot interbreed with either of the other two species. A panel of 14 lectins was used and it was found that glycosylation patterns were generally similar between placental tissues of the horse and donkey, except for the expression of non-bisected complex N-glycan and some sialic acids, whereas those of the camel showed striking differences in the binding of lectins to many structures carrying terminal residues of fucose, N-acetyl galactosamine and beta-galactose, as well as to complex N-glycans and sialic acids. These results are consistent with the proposition that interbreeding species carry similar glycans in tissues forming the interhaemal barrier whereas glycodiversity is one of the factors preventing implantation and subsequent placental development in interspecific hybrids.

Fetomaternal attachment and anchorage in the early diffuse epitheliochorial placenta of the camel (Camelus dromedarius). Light, transmission, and scanning electron microscopic study.

Placentae of 22 one-humped camel concepti with crown-rump lengths (CRL) ranging from 2.5 to 26 cm were studied. The placentae were processed for light, transmission and scanning electron microscopy of exposed surfaces and microvascular corrosion casts. In very early stages of pregnancy (2.5-4.5 cm CRL) three froms of fetomaternal interrelationship are described. (1) Precontact, where the mononuclear trophoblast cells are still separated from the uterine epithelium by a gap containing interareolar histotroph. Both fetal and maternal epithelia develop apical ectoplasmic pads in this location. (2) Apposition, where microvilli of the apical cell membrane of the trophoblast contact the uterine epithelium focally. Multinuclear trophoblast giant cells develop beside the population of already present mononuclear trophoblast cells. Uterine ectoplasmic pads can be observed. (3) Adhesion occurs when apical cell membranes of fetal and maternal epithelia adhere to each other closely, thus forming a 'normal' intercellular space of 20 nm width, without any intervening uterine luminal space. Microvillous interdigitation in this location varies from a non-microvillous 'smooth adhesion', to a distinctly villiform 'rough adhesion', and a 'semismooth adhesion' is achieved when trophoblastic microvilli make intimate contact with the non-microvillous uterine apical cell membranes of ectoplasmic pads. This fetomaternal attachment process is sufficient until the conceptus reaches approximately 9 cm CRL. Then, from 10 to 13 cm CRL, additional anchorage of the placenta to the endometrium is accomplished by the growth of temporary grooves and ridges of the allantochorion and the endometrium, which indent each other in a complementary fashion. The height of these groove-ridge structures increases gradually in 14 to 18 cm CRL fetuses, and they also widen at about 25 cm CRL, thus forming globular fetal troughs and irregular, thick maternal ridges. These together create units responsible for improved fetomaternal anchorage and metabolic exchange for the increasing needs of the growing fetus.