Structure and Steroidogenesis of the Placenta in the Antarctic Minke Whale (Balaenoptera bonaerensis)

There are few reports describing the structure and function of the whale placenta with the advance of pregnancy. In this study, therefore, the placenta and nonpregnant uterus of the Antarctic minke whale were observed morphologically and immunohistochemically. Placentas and nonpregnant uteri were collected from the 15th, 16th and 18th Japanese Whale Research Programme with Special Permit in the Antarctic (JARPA) and 1st JARPA II organized by the Institute of Cetacean Research in Tokyo, Japan. In the macro- and microscopic observations, the placenta of the Antarctic minke whale was a diffuse and epitheliochorial placenta. The chorion was interdigitated to the endometrium by primary, secondary and tertiary villi, which contained no specialized trophoblast cells such as binucleate cells, and the interdigitation became complicated with the progress of gestation. Furthermore, fetal and maternal blood vessels indented deeply into the trophoblast cells and endometrial epithelium respectively with fetal growth. The minke whale placenta showed a fold-like shape as opposed to a finger-like shape. In both nonpregnant and pregnant uteri, many uterine glands were distributed. The uterine glands in the superficial layer of the pregnant endometrium had a wide lumen and large epithelial cells as compared with those in the deep layer. On the other hand, in the nonpregnant endometrium, the uterine glands had a narrower lumen and smaller epithelial cells than in the pregnant endometrium. In immunohistochemical detection, immunoreactivity for P450scc was detected in most trophoblast cells, but not in nonpregnant uteri, suggesting that trophoblast epithelial cells synthesized and secreted the sex steroid hormones and/or their precursors to maintain the pregnancy in the Antarctic minke whale.     

Influence of breed and oestrous cycle on endometrial gland surface density in the mare

REASON FOR PERFORMING STUDY: The diffuse noninvasive epitheliochorial equine placenta develops an intimate and complex interdigitation with the maternal endometrium throughout gestation to maximise surface contact and, consequently, optimise nutritional and gaseous maternofetal exchanges. A significant reduction occurs in the surface density of microcotyledons on the placentae of Welsh Pony vs. Thoroughbred mares that may relate to a difference in either the number or density of endometrial glands between these breeds. OBJECTIVES: To examine this hypothesis and to determine the influence of the oestrous cycle upon the development and surface density of endometrial glands. METHODS: Endometrial biopsies were taken under videoendoscopic visual control from the base of a uterine horn from young, fertile, Welsh Pony and Thoroughbred mares at defined stages of the oestrous cycle. Computer-assisted morphometric analysis then permitted the surface density of endometrial glands within the stratum spongiosum to be assessed. CONCLUSIONS: There was a statistically significant reduction in endometrial gland surface density in the Welsh Pony vs. Thoroughbred mares during both oestrus and dioestrus. POTENTIAL RELEVANCE: A substantial upregulation of epidermal growth factor (EGF) mRNA in the epithelial cells lining the apical portions of endometrial glands has been demonstrated in pregnant mares between Days 35 and 40 after ovulation, coincident with the onset of interdigitation between the allantochorion and endometrium to form the microcotyledonary placenta. The increased surface density of endometrial glands noted in the uteri of Thoroughbred mares might account for the greater surface density of placental microcotyledons in this breed.     

Tumour necrosis factor in the canine endometrium: an immunohistochemical study

Tumour necrosis factor (TNF), a pleiotropic cytokine that regulates cell growth and differentiation as well as the synthesis of other cytokines, has been identified in the uterus of several species describing a cyclic pattern, eventually under ovarian steroid regulation. Information is yet limited on the presence of TNF protein in the canine endometrium during the oestrous cycle and early pregnancy. This study depicts the temporal immunolocalization of TNF in the bitch endometrium along the oestrous cycle and changes associated with the early steps of embryo invasion. TNF immunolabelling was found in both the stromal fibroblasts and epithelial components of the canine endometrium in all stages studied. Stromal immunostaining was more intense than that of the epithelia, in all the stages of the oestrous cycle. In addition, a tendency for a decrease in the surface epithelium intensity score was found in early dioestrus. A positive glandular content was only observed in anoestrus and proestrus stages. In early pregnancy (days 13-16), TNF immunolabelling was detected at the embryo-maternal surface, in the syncytium cords and the trophoblast, as well in the endometrial stroma and the basal endometrial glands, but not in the lacunar epithelium. The overall TNF immunoreactivity was higher in early pregnancy samples in comparison with those of the early dioestrus and dioestrus stages, suggesting it plays a role during implantation.     

马胚胎附植及其分子调控研究进展

胚胎附植是处于活化状态的囊胚滋养层细胞与处于接受态的母体子宫上皮细胞之间逐步建立组织和生理上联系的过程。马（马属动物）在该过程中先后存在侵入性的绒毛膜带（子宫内膜杯）和非侵入性的尿囊绒毛膜（微子叶型上皮绒毛膜胎盘）,是一种集侵入性与非侵入性胚胎滋养层于一体的哺乳动物。多种分子共同调节妊娠母马的胚胎附植过程。作者综述了马胚胎附植特点、过程与相关分子调控的研究进展,对减少母马妊娠早期胚胎丢失与流产具有重要参考价值。     

Rechallenge of previously-infected pregnant ewes with Chlamydophila abortus

In an attempt to ascertain the means whereby previous exposure to Chlamydophila (C.) abortus can protect against the re-occurrence of enzootic abortion of ewes (EAE), ten previously-exposed ewes were intravenously rechallenged with a large infective dose of C. abortus during pregnancy. The patterns of development of chlamydial placentitis and its sequelae closely resembled that observed following first-time challenge of previously-na?ve ewes, although placentitis appeared to develop more slowly following rechallenge infection and none of the rechallenged ewes aborted. Chorioallantoic and foetal pathology and foetal immune responses were qualitatively similar whilst the local maternal response to C. abortus infection of the endometrium did not appear to differ in rechallenged and first-time challenged sheep. This demonstrates that if C. abortus reaches the foetal side of the placenta, a stereotypical response is elicited, regardless of the status of maternal immunity. Therefore it appears that in natural circumstances, acquired immunity of the dam protects against the re-occurrence of EAE by preventing the causative agent from reaching the susceptible foetal trophoblast.     

Oxidative Stress During Pregnancy In The Sheep

During physiological pregnancy, all tissues and, mostly, placenta and foetus require high amounts of oxygen. Reactive oxygen species (ROS), generated both by mother and foetus, are implicated in foetal growth because they promote replication, differentiation and maturation of cells and organs. Nevertheless, ROS excess, if not properly counterbalanced, may lead to an alteration in cell constituents, with harmful effects both on mother and foetus.ROS exert a biphasic effect because adequate ROS concentration is essential for embryo development, implant, foetal defence against uterine infections, steroidogenesis, pregnancy maintainance and partum. On the other hand, an uncontrolled ROS generation, beyond physiological antioxidant defences, may lead to embryo resorption, placental degeneration with subsequent alteration in maternal‐foetal exchanges, delay in foetal growth, pregnancy interruption, stillbirths. This review investigates the mechanisms underlying ROS generation and effects, throughout physiological and pathological pregnancy in sheep, with a look to antioxidants and their importance in such a critical phase of the reproductive cycle of the sheep.     

VEGF system expression in different stages of estrous cycle in the corpus luteum of non-treated and superovulated water buffalo

Water buffaloes are easily adaptable animals, whose raising and economical exploitation have been growing in the last three decades all over the world. Hyperstimulation of ovarian function in this species is a common technique aiming to improve reproductive performance. Superovulatory treatment affects corpus luteum (CL) function, which is highly correlated to angiogenic process. The aim of this study was therefore to assess the temporal protein and mRNA expression of VEGF and its receptors in the CL of non-treated and superovulated buffaloes. For that purpose blood samples and CL from 36 healthy (30 untreated, groups 1–5, and 6 superovulated, group 6) non-pregnant buffaloes were collected and the samples were divided into 6 groups according to the age of CL. Plasma samples were submitted to RIA to measure progesterone concentration and CL were subjected to immunohistochemistry and real time PCR for VEGF (vascular endothelial growth factor), Flt-1 (fms-like tyrosine kinase receptor 1) and KDR (kinase insert domain containing region). The VEGF system protein and mRNA expression during CL life span of untreated animals showed a specific time-dependent profile, although protein did not always reflect mRNA concentrations. VEGF expression in luteal cells was high correlated to plasma progesterone levels. Superovulated CL showed a significant increase of the VEGF-system protein and a significant decrease of mRNA expression compared to untreated animals in the same stage of the oestrous cycle. We conclude that VEGF, Flt-1 and KDR protein and mRNA expression in buffalo CL is dependent of estrous cycle stage and superovulatory treatment is able to increase the translation rate of this system.     

The proliferation inhibitory proteins p27(Kip1) and retinoblastoma are involved in the control of equine lymphocyte proliferation

Observations in early equine pregnancy clearly reveal maternal immune recognition of and response to the presence of the conceptus. Nevertheless, both maternal cellular and humoral responses appear ineffective in destroying the developing placenta and fetus in early pregnancy. Our previous studies had shown that the pre-conditioned medium generated from the culture of equine invasive trophoblast inhibited mitogen-induced lymphocyte proliferation and the expression of cytokine messenger RNA in vitro. Those findings also suggested that lymphocytes might have been halted in the G0/G1 phase of the cell cycle. To characterize the cell cycle and the intracellular mechanisms involved in the inhibition of lymphocyte proliferation, equine peripheral blood lymphocytes were cultured in the presence or absence of pokeweed mitogen (PWM) in fresh medium, or in medium pre-conditioned through cell culture of invasive trophoblast cells or fetal fibroblasts. Two-color flow cytometric analysis for bromodeoxyuridine (BrdU) incorporation by stimulated lymphocytes, and concomitant DNA staining with 7-amino-actinomycin D (7-AAD), indicated that a greater proportion of lymphocytes were found in the G0/G1 phase of the cell cycle when cultured in the invasive trophoblast cell pre-conditioned medium compared to controls. Analysis using carboxyfluorescein diacetate succinimidyl ester (CFSE) fluorescence intensity demonstrated that lymphocytes cultured in the presence of invasive trophoblast cell pre-conditioned medium had fewer cells going through division, but that those fewer cells sustained similar numbers of cell divisions as in control cultures. Hypophosphorylated retinoblastoma (Rb) protein expression was increased and p27Kip1 expression was maintained at higher levels in lymphocytes cultured in invasive trophoblast pre-conditioned medium compared to fresh medium. In agreement with these data, flow cytometric measurement of the Ki-67 protein expression in lymphocytes cultured in invasive trophoblast pre-conditioned medium was lower in comparison to controls. These findings suggest that the equine lymphocyte proliferation is at least partially regulated by the expression of proliferation inhibitory proteins such as p27Kip1 and hypophosphorylated Rb. These proteins seem to be important regulators of cell cycle transition between G1 and S phase in equine lymphocytes.     

T-cell tolerance to the developing equine conceptus

One of the most intriguing and dramatic examples of immunological tolerance is displayed by the mammalian foetal-placental unit, which thrives as a semi-allograft in the mother's uterus during pregnancy. The success of the so-called foetal allograft stands in stark contrast to the failure of most tissue and organ grafts to survive without genetic matching of donor and recipient or drastic immunosuppression of the recipient's immune system. Experiments conducted over the past 60 years have revealed multiple mechanisms that enable the conceptus to avoid immunological detection or destruction. Many of these mechanisms are directed towards evading immune-mediated damage by maternal T lymphocytes, and they can be grouped into three classes: (i) downregulation of major histocompatibility complex (MHC) gene expression in placental trophoblast cells; (ii) local and systemic alterations in maternal immune reactivity; and (iii) innate defence mechanisms of the trophoblast cells that comprise the barrier between foetal and maternal tissues. The redundancy in these protective mechanisms helps ensure the transmission of life from generation to generation and provides a rich field of study of ways in which functional immunological tolerance can be manifest. The variation in placental forms and function among mammalian species present opportunities to discover and understand novel tolerogenic mechanisms that may have broad application in biology, medicine and animal husbandry. This review focuses on the evidence obtained from studies of pregnancy in the mare that support the case for selective T-cell tolerance to the mammalian conceptus.     

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.     

Streptozotocin-induced diabetes decreases placenta growth factor (PlGF) levels in rat placenta

The placenta produces several growth factors, including placenta growth factor (PlGF), which are essential for placenta growth and fetal growth. Diabetic pregnancy induces the abnormal placental growth and fetal development. This study investigated whether diabetes in pregnant rats induces changes in PlGF expression in the placenta. Diabetes was induced by a single intravenous injection of streptozotocin (35 mg/kg body weight) on day 0 of pregnancy, blood and tissue samples were collected on day 20 of pregnancy. In the diabetic group, maternal body weight and fetal weight significantly decreased compared to controls. RT-PCR and Western blot analyses showed that expression of PlGF was significantly decreased in placenta by streptozotocin treatment. Immunohistochemical study showed that the positive signal of PlGF in trophoblast cells was decreased in the diabetic group compared to controls. These findings demonstrate the decline of PlGF in the placenta in diabetic pregnancy.     

Expression of the Vascular Endothelial Growth Factor (VEGF‐A) and its Receptors in the Umbilical Cord in the Course of Pregnancy in the Pig

The umbilical cord (UC) and the placenta are important organs through which respiratory gases, nutrients, wastes and biologically active substances are exchanged between the maternal and the foetal system. A rapid placental vascularization observed in the second half of pig pregnancy is positively correlated with the mRNA expression of the vascular endothelial growth factor (VEGF). Based on these findings, we hypothesized that VEGF may have a stimulatory effect in the dynamically growing UC. To further understand the role of the VEGF–VEGFR system during UC development, mRNA and protein expression as well as the cellular localization of VEGF‐A, VEGFR‐1 and VEGFR‐2 in UC were examined on days 40, 60, 75 and 90 of pregnancy and after physiological delivery in the pig (day 114 of pregnancy). Real Time RT‐PCR analysis showed an increase in the mRNA levels of VEGF120 and VEGF164 from day 90 of pregnancy. VEGFR‐1 mRNA expression was significantly increased on day 75 of pregnancy. No significant changes in VEGFR‐2 mRNA expression were detected. In turn, western blot analysis revealed an increase in VEGF‐A protein expression on day 40, compared to the later days of pregnancy. A rapid increase in the VEGFR‐1 protein level was noted on day 75 and 90 of gestation. No significant changes in VEGFR‐2 protein expression were detected on any of the analysed days of pregnancy. Immunohistochemical staining enabled detection of VEGF–VEGFR system, in endothelial and tunica media cells of the umbilical vessels and in allantoic duct and amniotic epithelium on all analysed days of pregnancy. Positive reactions for VEGF‐A and VEGFR‐1, but not VEGFR‐2, were also observed in myofibroblasts. In conclusion, this data shows that members of the VEGF–VEGFR system are temporally and spatially well localized for playing key roles during umbilical cord formation and its intensive growth observed after day 75 of pregnancy.     

Expression and Localization of Vascular Endothelial Growth Factor and its Receptors in the Corpus Luteum During Oestrous Cycle in Water Buffaloes (Bubalus bubalis)

The aim of this study was to document the expression and localization of VEGF system comprising of VEGF isoforms (VEGF 120, VEGF 164 and VEGF 188) and their receptors (VEGFR1 and VEGFR2) in buffalo corpus luteum (CL) obtained from different stages of the oestrous cycle. Real‐time RT‐PCR (qPCR), Western blot and immunohistochemistry were applied to investigate mRNA expression, protein expression and localization of examined factors. In general, all the components of VEGF system (the VEGF isoforms and their receptors) were found in the water buffalo CL during the oestrous cycle. The mRNA as well as protein expression of VEGF system was highest during the early and mid‐luteal phase, which later steadily decreased (p < 0.05) after day 10 to reach the lowest level in regressed CL. As demonstrated by immunohistochemistry, VEGF protein was localized predominantly in luteal cells; however, VEGFR1 and VEGFR2 were localized in luteal cells as well as in endothelial cells. In conclusion, the dynamics of expression and localization of VEGF system in buffalo corpora lutea during the luteal phase were demonstrated in this study, indicating the possible role of VEGF system in the regulation of luteal angiogenesis and proliferation of luteal as well as endothelial cells through their non‐angiogenic function.     

Two concepts on the immunological aspect of blastocyst implantation

The process of blastocyst implantation is a series of interactions between the blastocyst and maternal tissues. The purpose of this process is (1) to provide nourishment to the embryo for developmental growth in appropriate physiological and endocrinological environment until a placenta is established, and (2) to protect the (semi-)allogeneic embryo from any attacks from the maternal immune system. To facilitate successful implantation, therefore, these two aspects of the embryonic demand must be satisfied in the embryo-maternal interface throughout the entire process of implantation. The first concept I present in this paper is that blastocyst implantation essential factors (BIEFs) have dual functions: one, for structural and functional modification of the endometrium to accommodate the developing embryo and provide nourishment and suitable environment for its development, and the other, for modulation, directly or indirectly, of the maternal immune system to prevent attacks by the maternal immune system. The second concept is that BIEFs convert the endometrium (or uterus) from an immunologically non-privileged site to a privileged site. This endometrial (uterine) conversion is the immunological aspect of the blastocyst implantation process. When the endometrium has become receptive for blastocyst implantation, it signifies that the immunological conversion of the endometrium by BIEFs has been sufficiently attained to let the embryo start contacting maternal tissues. During the early stages of placentation, as the trophoblast cells differentiate and make their way to the maternal blood vessels to establish the placenta, BIEFs continuously provide nourishment and immunological protection to the developing embryo. The immunological protection of the embryo/fetus from potential attacks by the maternal immune system appears to reach a peak at the time of establishment of the placenta. Thus, clarification of the roles of BIEFs in both the physiological/endocrinological aspect as well as the immunological aspect is essential for understanding the biological process of implantation.     

Transplacental Transfer of Iron in the Water Buffalo (Bubalus bubalis): Uteroferrin and Erythrophagocytosis

The objectives of this investigation were to understand transplacental transport of iron by secreted uteroferrin (UF) and haemophagous areas of water buffalo placenta and clarify the role(s) of blood extravasation at the placental‐maternal interface. Placentomes and interplacentomal region of 51 placentae at various stages of gestation were fixed, processed for light and transmission electron microscopy, histochemistry and immunohistochemistry. Haemophagous areas were present in placentomes collected between 4 and 10 months of pregnancy. Perl’s reaction for ferric iron was negative in placentomes, but positive in endometrial glands. Positive staining for UF indicated areas in which it was being taken up by phagocytosis and/or fluid phase pinocytosis in areolae of the interplacentomal mesenchyme, with little staining in endometrial stroma. Imunohistochemistry detected UF in trophectoderm of haemophagous regions of placentomes and in other parts of the foetal villous tree, but the strongest immunostaining was in the epithelial cells and lumen of uterine glands. Ultrastructural analyses indicated that erythrophagocytosis was occurring and that erythrocytes were present inside cells of the chorion that also contained endocytic vesicles and caveolae. Results of this study indicate that both the haemophagous areas of placentomes and the areolae at the interface between chorion and endometrial glands are important sites for iron transfer from mother to foetal‐placental tissues in buffalo throughout pregnancy.     

Immunohistochemical Localization of the Progesterone and Oestrogen α Receptors in the Uterine Horns of the African Giant Rat (Cricetomys gambianus)

The present study investigated the immunolocalization of the progesterone and oestrogen α receptors in the uterine horns of the African giant rat during the oestrous cycle. The progesterone and oestrogen α receptors were demonstrated in various cellular constituents of the endometrium, myometrium and perimetrium. The intensity of progesterone and oestrogen α receptor immunostaining in the endometrial and myometrial layers of the uterine horns varied during the oestrous cycle. The intensity of oestrogen α receptor immunoreactivity in the luminal epithelium was high during pro‐oestrus, oestrus and dioestrus. Progesterone and oestrogen α receptor immunoreactivity in the endometrial epithelia was absent during metoestrus. Moderate to strong immunostaining for the progesterone and oestrogen α receptors was demonstrated in the myometrial smooth muscle cells during pro‐oestrus, oestrus and dioestrus. The intensity of progesterone and oestrogen α receptor immunostaining in the myometrial smooth muscle cells was low during metoestrus. Stromal cells in the perimetrium consistently expressed progesterone and oestrogen α receptor immunoreactivity throughout the oestrous cycle. The findings of the study indicate that in the giant rat the immunolocalization of the progesterone and oestrogen α receptors, in endometrial and myometrial regions of the uterine horns, varies during the oestrous cycle.     

小鼠子宫内膜凝集素结合特性的生殖周期改变

用４种酶标植物凝集素（ＣｏｎＡ,ＷＧＡ,ＳＢＡ,ＲＣＡ）作探针,研究了小鼠发情周期、妊娠期和产后子宫内膜上皮、固有膜细胞和腺上皮凝集素结合特性的变化。子宫内膜与这４种凝集素的结合,随发情周期而波动。与间情期相比,妊娠期子宫内膜上皮与ＣｏｎＡ的结合,由阴性转为阳性,并随妊娠的持续逐渐加强,产后又突然转为阴性。子宫内膜上皮与ＷＧＡ的结合,在间情期和妊娠１ｄ均为阳性,妊娠７ｄ减为弱阳性,继之又加强,产后达最强。无论妊娠期还是产后,子宫内膜上皮与ＲＣＡ的结合均为强阳性,与ＳＢＡ的结合均为阴性。结果提示,生殖周期子宫内膜与凝集素结合特性的规律性变化,可能与激素调节、胚泡的着床和分娩有关