Development of the discoidal hemochorial placenta in the black mastiff bat, Molossus ater: evidence for a role of maternal endothelial cells in the control of trophoblastic growth

In an effort to define further the factors that can influence trophoblastic growth, development of the discoidal chorioallantoic placenta was examined histologically in laboratory-bred black mastiff bats, Molossus ater. This placenta normally forms only at the cranial end of the right uterine horn. Its positioning was found to be related to the development of an unusual vascular tuft in the endometrium at this site following ovulation. When a decidual reaction occurred, the endothelial cells of the tuft vessels hypertrophied and their basal laminae became more prominent in sections stained for glycoproteins. Cytotrophoblast then proliferated preferentially around the tuft in the right horn and its vessels became surrounded by trophoblastic cuffs. A functional placenta was formed when trophoblastic tubules grew out from these cuffs, became interconnected, developed lumina, and began to carry maternal blood. Maternal endothelial cells generally persisted only in portions of the larger vascular tubules. These observations suggest that the endothelial cells of the tuft vessels may be secreting factors that influence early trophoblastic growth and are at least partially incorporated into the basal laminae of the endothelial cells. Evidence is reviewed which raises the possibility that endometrial endothelial cells might have a similar morphogenetic role in many other mammals with invasive trophoblast. Mitotic activity in the trophoblastic tubules of M. ater ceased when the tubules became patent and began to carry blood. Further growth was then accomplished by hypertrophy of the existing cytotrophoblast cells. Electron microscopic examination of near-term placentas confirmed that the interhemal barrier was hemomonochorial and lacked a continuous layer of syncytiotrophoblast.     

Morphology of the endometrial microvasculature during early placentation in the rat

Summary The morphology of the uterine microvasculature during early placentation was investigated by light microscopy, scanning electron microscopy of microvascular corrosion casts and transmission electron microscopy in rats 26 and 50 h after initiation of implantation. Increased vascular permeability at implantation sites was observed as a positive blue-dye test, spacing of vessels, and as localized extravasations of resin from postcapillary venules in the center of the endometrium. The subepithelial capillary plexus in the primary decidual zone adjacent to the blastocyst was shut down 50 h after initiation of implantation, most probably due to swelling of the metabolically activated endothelium and volume expansion of the decidual cells. This phenomenon coincided with the mesometrial orientation of the inner cell mass of the blastocyst; it may be a uterine mechanism to direct the ectoplacental cone toward the patent vessels in the mesometrial portion of the uterus. The remaining vessels at implantation sites were generally fewer, larger in diameter, more irregular in caliber, and more uniformly oriented along the implantation axis than their counterparts at inter-implantation sites. No vascular sprouts were observed during the interval studied.     

Expression of stathmin family genes in the murine uterus during early pregnancy

Stathmin, a cytosolic phosphoprotein that regulates microtubule dynamics during cell-cycle progression, is abundantly expressed at embryo implantation sites in rats. Here, we characterized the expression of stathmin and its family genes in the murine uterus during the peri-implantation period. Stathmin protein was expressed in the glandular and luminal epithelium, blood vessels, and stromal cells on day 3 of pregnancy. On the day of implantation (day 5), stathmin was mainly localized in blood vessels in the endometrium. On day 7, intense stathmin expression was limited to capillary vessels and secondary decidual cells. Stathmin expression was higher at implantation sites than at uterine segments between implantation sites and increased during oil-induced decidualization. Although the artificially-induced deciduoma weights and number of implantation sites were similar between stathmin-knockout (KO) and wild-type (WT) mice, the stathmin-KO mice had fewer newborn pups (reduced by 30%). The expression of alkaline phosphatase, desmin, and cyclin D3 was attenuated in decidual zones of stathmin-KO mice. Messenger RNA level of the stathmin family gene, SCG10, was high at the time of decidualization in WT and stathmin-KO mice. In contrast, the others of stathmin family members, SCLIP and RB3 were highly expressed in stathmin-KO mice compared to WT mice. These results suggest that stathmin and stathmin family genes are expressed in the murine endometrium with enhanced expression in the implantation or the decidualization process.     

Implantation, development of the fetal membranes, and placentation in the captive black mastiff bat, Molossus ater

Uterine events during pregnancy were examined histologically in laboratory-bred black mastiff bats (Molossus ater) as part of an effort to develop this species as a model for studies of the factors controlling trophoblastic growth. Embryos entered the uterus at the morula stage and in most cases shed their zonae pellucidae reasonably intact, apparently as blastocyst expansion occurred. Implantation was superficial and observed to occur only in the right uterine horn. During implantation to the endometrium by both blastocyst expansion and closure of the uterine lumen. A decidual reaction was evident at an early stage of uterine epithelial displacement and spread rapidly through the endometrium. Initial trophoblastic proliferation occurred along the uterine lumen and into the glands, while its invasion of the endometrial stroma was delayed. Although one or several primordial cavities have been observed to develop within the epiblast during implantation, these subsequently opened to a trophoepiblastic cavity, and the definitive amnion was formed by folding. A choriovitelline placenta was present briefly at thesomite stage, but disappeared as the exocoelom enlarged and the yolk sac collapsed. The latter persisted through pregnancy, however, as a glandular-appearing body. As the yolk sac retracted from the chorion, it was replaced by allantoic mesoderm, creating a diffuse labyrinthine endotheliodichorial placenta. This was prominent during mid-gestation, but was supplanted by the discoidal hemochorial placenta as the major site of feto-maternal exchange during late pregnancy.     

Immunohistochemical localization of prostaglandin synthase in the rat uterus and embryo during the peri-implantation period

Prostaglandins (PGs) appear to have a role in the appearance of the increased uterine vascular permeability and subsequent decidualization observed at implantation in many species. However, the sites of production of these PGs have not been clearly established. To clarify the PG synthetic capacity of the blastocyst and the various types of cells in the uterus at implantation, we have studied the immunohistochemical localization of PG synthase in the rat blastocyst on Days 5 to 7 and uterus on Days 1, 4, 5, 6, and 7 of pregnancy. Labeling of PG synthase was negligible in the uterus on Day 1 of pregnancy. On Day 4, there was increased labeling in the luminal and glandular epithelium, in stromal cells adjacent to the luminal epithelium, and in blood vessels and some leukocytes. PG synthase was detected in the blastocysts on Days 5 to 7, but there was a gradual loss of label in the luminal and glandular epithelial cells during this period. Early differentiating stromal cells adjacent to the luminal epithelium in the implantation site on Day 5 showed bright labeling, whereas peripheral stromal cells were only slightly labeled. By Day 7, the differentiated cells of the primary decidual zone showed little or no label, but cells in the secondary decidual zone were brightly labeled. These results indicate that PG synthase is present in the rat blastocyst and in several kinds of uterine cells, and that its localization in uterine cells changed markedly during the implantation process.     

Role of the sympathetic innervation on uterine electromyographic activity in nonpregnant ovariectomized sheep under estrogen supplementation.

The aims of the present study were to characterize the sympathetic innervation of the nonpregnant sheep uterus, to determine the catecholamine content in myometrium (MYO) and endometrium, and to study the effects of chemical sympathectomy (CHSPX) on uterine catecholamine content and on uterine electromyographic (EMG) activity recorded from the MYO and mesometrium (MESO) in the nonpregnant ovariectomized sheep. After synchronization of estrus, 9 nonpregnant sheep were anesthetized with halothane, ovariectomized, and fitted with vascular catheters and EMG electrodes. Estradiol-17 beta was administered intravascularly at a rate of 50 micrograms/24 h for 10 days. CHSPX was induced with 6-hydroxy dopamine (20 mg/kg). Uterine tissues were obtained for determination of catecholamine content by HPLC and for immunocytochemical staining using an antibody against tyrosine hydroxylase (TH). In nonpregnant ovariectomized sheep, TH immunostaining was present in nerve fibers located in endometrium and MYO. In all layers of the uterus, catecholamine fibers were found in the proximity of blood vessels as well as in defined regions of the parenchyma. Throughout the uterus, norepinephrine content and TH immunostaining were dramatically decreased after CHSPX. CHSPX decreased uterine short EMG event activity in both MYO and MESO. Contracture-type activity was not affected in MYO and was increased in MESO. We conclude that sympathetic innervation modulates the MYO and MESO EMG activity in nonpregnant ovariectomized sheep under estradiol supplementation, and that the removal of the sympathetic innervation induces a decrease in the spontaneous activity.     

Nodal expression in the uterus of the mouse is regulated by the embryo and correlates with implantation

Nodal, a transforming growth factor beta (TGFB) superfamily member, plays a critical role during early embryonic development. Recently, components of the Nodal signaling pathway were characterized in the human uterus and implicated in the tissue remodeling events during menstruation. Furthermore, the Nodal inhibitor, Lefty, was identified in the mouse endometrium during pregnancy, and its overexpression led to implantation failure. Nonetheless, the precise function and mechanism of Nodal signaling during pregnancy remains unclear. In order to elucidate the potential roles Nodal plays in these processes, we have generated a detailed profile of maternal Nodal expression in the mouse uterus throughout pregnancy. NODAL, although undetectable during the nonpregnant estrus cycle, was localized throughout the glandular epithelium of the endometrium during the peri-implantation period. Interestingly, Nodal expression generated a banding pattern along the proximal-distal axis of the uterine horn on Day 4.5 that directly correlated with blastocyst implantation. Embryo transfer experiments indicate the embryo regulates Nodal expression in the uterus and directs its expression at the time of implantation, restricting NODAL to the sites between implantation crypts. During the later stages of pregnancy, Nodal exhibits a dynamic expression profile that suggests a role in regulating the endometrial response to decidualization and associated trophoblast invasion.     

New insights into human endometrial aminopeptidases in both implantation and menstruation

The endometrium cycle involves proliferation of endometrial epithelial cells in preparation for implantation of fertilized ovum. With ovulation, the endometrium secretes nutrients such as peptides and amino acids into the endometrial cavity. The histological evidence of ovulation in normal menstrual cycle includes subnuclear glycogen vacuoles surrounded by placental leucine aminopeptidase (P-LAP) in endometrial epithelial cells. P-LAP is an essentially involved in intracellular trafficking of glucose transporter (GLUT) 4 which is primarily important for glucose uptake in skeletal muscles and fat tissues. On the other hand, glucose influx from blood into endometrial epithelial cells is not mainly mediated by GLUTs, but by coincident appearing progesterone just after ovulation. Progesterone increases permeability of not only plasma membranes, but also lysosomal membranes, and this may be primarily involved in glucose influx. Progesterone also expands the exocytosis in the endometrium after ovulation, and endometrial secretion after ovulation is possibly apocrine and holocrine, which is augmented and exaggerated exocytosis of the lysosomal contents. The endometrial spiral arteries/arterioles are surrounded by endometrial stromal cells which are differentiated into decidual/pre-decidual cells. Decidual cells are devoid of aminopeptidase A (APA), possibly leading to enhancement of Angiotensin-II action in decidual cell area due to loss of its degradation by APA. Angiotensin-II is thought to exert growth-factor-like effects in post-implantation embryos in decidual cells, thereby contributing to implantation. Without implantation, angiotensin-II constricts the endometrial spiral arteries/arterioles to promote menstruation. Thus, P-LAP and APA may be involved in homeostasis in uterus via regulating glucose transport and vasoconstrictive peptides.     

Development of the implantation chamber in the pregnant bitch.

Uteri taken from 25 bitches at various times during the early stages of pregnancy were studies cytologically to determine how the implantation chamber developed and how fetal-maternal relations were established. On day 13 after the end of estrus, knobs of trophoblastic syncytium formed and became wedged between cells of the uterine luminal epithelium. The syncytium quickly spread along the uterine lumen and into the mouths of the glands, dislodging and surrounding maternal cells. As invasion continued trophoblastic villi, consisting of cores of cytotrophoblast covered by a continuous layer of syncytium, penetrated deeper into the endometrium. The syncytium spread to surround maternal vessels and decidual cells. By day 26 the trophoblast had extended down to the large lacunae. Here syncytial trophoblast covering tips of the villi degenerated, leaving cytotrophoblast exposed to the necrotic zone. These cells possessed characteristics of absorbing cells. Hematomas were formed by focal necrosis of fetal and endometrial tissue at the poles of the implantation sites. Large pools of extravasated blood accumulated and red blood cells were phagocytized by surrounding trophoblastic cells. Therefore, the endotheliochorial relationship in the canine placenta appeared to be established by syncytial trophoblast invading a cellular endometrium. In the necrotic zone and hematomas, cellular trophoblast may have lost its syncytial covering, but elsewhere maternal vessels and decidual cells in the placenta were in direct contact only with syncytial trophoblast.     

Expression of nidogens in rat uterus and embryo during decidualization and implantation

The purpose of this study was to demonstrate the expression of nidogen-1 and nidogen-2 and their possible role in decidualization and implantation events during early pregnancy in rats. The tissue samples were examined from pregnant animals between gestational days 1-8 using immunocytochemistry. The uterine luminal epithelium, the glandular epithelium, and the myometrial smooth muscle cells stained strongly from gestational days 1-8 with both nidogen antibodies. At day 4 the decidual reaction areas began to appear in the stromal matrix and immunostaining of both nidogens revealed that the basement membrane of the surface epithelium was discontinuous. The differentiation of stromal cells into decidual cells was seen at gestational day 5 and both nidogens were weakly expressed in the decidualizing cells. At day 6, nidogen-2 immunoreactivity was higher in the primary decidual cells close to the embryo than nidogen-1, and during development of the decidual tissue both nidogens appeared in the endometrial stromal cells. At day 7, while expression of both nidogens declined in the primary decidual cells, their expression was markedly observed in the secondary decidual cells close to the myometrium. At day 8, expression of both nidogens was also observed to increase in the primary decidual cells. While nidogen-2 expression was seen in the parietal endoderm and primary ectoderm of the rat embryos at this developmental stage, nidogen-1 expression was only detected in the parietal endoderm. These results indicate that nidogen-1 and nidogen-2 could play important roles during embryogenesis, decidualization, and implantation in the endometrium of rat uterus.     

Distribution of laminin,vimentin and desmin in the rat uterus during initial stages of implantation

The aim of this study was to investigate the immunohistochemical distribution of laminin, vimentin and desmin during the implantation period in the rat since ECM remodelling and the expression of intermediate filaments (Ifs) is essential for successful decidualization and implantation. On day 4 of pregnancy, laminin was found in a few endometrial stromal cells (ESC), the basement membrane of the numerous endometrial blood vessels, in endometrial glands and as well as in the uterine epithelium. The localization of vimentin on day 4 of pregnancy was widespread in the ESC. However, desmin immunoreactivity was low in ESC on this day of pregnancy. On day 6 of pregnancy, laminin and vimentin were localized in the decidual area underlying luminal epithelium and around the implanting embryo. Additionally, desmin was found to be present densely in decidual cells of the anti-mesometrial region where implantation takes place. Finally, on day 8 of pregnancy, laminin was present in decidual and parietal endodermal cells, whereas vimentin was immunolocalized in primary and secondary decidual regions in the endometrium. In contrast, desmin was detected in some parts of the secondary decidual zone. In conclusion, these proteins could have crucial roles in decidualization and implantation.     

Fibronectin and cell shape in vivo: studies on the endometrium during pregnancy

The rat endometrium during pregnancy was used as a model system to study fibronectin in vivo. Fibronectin distribution on stromal fibroblasts, as determined by indirect immunofluorescence staining, was studied in relationship to cell shape during decidual transformation. Fibroblasts of the estrus endometrial stroma were elongated cells with a fibrillar pattern of fibronectin on their surfaces. During days 1-6 of pregnancy, as these elongated cells acquired a round morphology, fibronectin changed first to a patched distribution on the cells'a surfaces and then disappeared. The change in fibronectin was specific for the fibroblasts since over the same time period there was no decrease in fibronectin found associated with blood vessels or in the epithelial-stromal basement membrane. These results support the proposed relationship between cell surface fibronectin and cell shape that has been inferred from in vitro experiments. After implantation, fibronectin distribution was studied in relationship to the position of the conceptus. In the stroma proximal to the implanting conceptus, fibronectin was absent except around blood vessels, which may help explain how decidual tissue could act as a barrier to trophoblast invasion. Finally, fibronectin distribution was studied in the uterus after parturition. Debris in the uterine lumen was coated with fibronectin, which may be important in the rapid removal of this material by phagocytic cells. Also, fibronectin associated with the epithelial-stromal basement membrane was reorganized after reepithelialization had occurred.     

Effect of inhibition of matrix metalloproteinases on endometrial decidualization and implantation in mated rats

Implantation of the embryo into the endometrium is a highly regulated event that is critical for establishment of pregnancy. Molecules involved in this process provide potential targets for post-coital contraception. The aims of this study were to determine whether matrix metalloproteinases (MMPs) are present at implantation sites in rats and whether administration of a broad-based inhibitor of MMPs could inhibit embryo implantation. Uterine extracts from non-pregnant rats and from rats on days 3-9 of pregnancy were examined for the presence of MMPs. Doxycycline (5 or 15 mg day-1) was administered by gavage to rats from the day of mating (day 0) to day 7 of pregnancy and the uterus was examined for implantation sites. A number of MMPs were present in all uterine samples. MMP-2 reached a peak on day 3, whereas the highest expression of MMP-7 occurred on day 7. MMP-13 and MMP-3 were present in smaller amounts. MMP-9 was detectable only on day 9. Treatment of rats with doxycycline had no effect on the number of implantation sites or on the total uterine mass. However, in treated rats, the process of decidualization was impaired and both the width and length of the decidual zone was reduced, resulting in a decrease in total decidual area from 1.20 +/- 0.07 to 0.91 +/- 0.07 mm2 (mean +/- SEM, controls versus doxycycline treated, P < 0.02). It is concluded that administration of MMP inhibitors during early pregnancy retards decidual development, but does not block implantation.     

Temporal relationships among uterine pituitary adenylate cyclase-activating polypeptide, decidual prolactin-related protein and progesterone receptor mRNAs expressions during decidualization and gestation in rats

Pituitary adenylate cyclase-activating peptide (PACAP), a novel compound with vasoactive intestinal polypeptide-like activity, was recently shown to be localized in the neuronal endings of the human uterus. The purpose of the present study was to assess the functional presence of PACAP mRNA in the decidual endometrium and its relationship to the expression levels of decidual prolactin-related protein (dPRP) and the progesterone receptor mRNAs during decidualization and pregnancy in Sprague-Dawley rats. PACAP was constitutively and temporally expressed in the decidual endometrium and gravid uterus. The time-dependent correlated expression levels of PACAP, dPRP and the progesterone receptor were induced by the neurogenic reproductive signals, i.e. the vagino-cervical/deciduogenic stimuli of decidualization and by the normal equivalent stimuli of mating/blastocyst implantation of gestation. Correlation among the mRNA expression levels of PACAP, dPRP and the progesterone receptor and the coordinated inhibitory actions of the anti-progesterone (RU-486) suggest that there is also correlated time-dependent steroid regulation of the mRNA levels of PACAP, dPRP and the progesterone receptor in the decidual and pregnant uteri. One possible functional meaning for the time-related localization of endometrial/uterine PACAP could be to facilitate endometrial blood flow and increase the availability of metabolic substrates to the developing deciduoma or embryo. The study demonstrates the potential importance of PACAP expression in the regulation of the maternal feto-placental component and suggests a prominent reproductive role for the neuropeptide in mammalian pregnancy.     

Role of conceptus secretory products in establishment of pregnancy

Conceptuses produce steroids, prostaglandins, proteins and possibly other unidentified agents which may play a role in the establishment and maintenance of pregnancy. A key event in this process is protection of the corpus luteum (CL) from the luteolytic activity of prostaglandin (PG) F-2 alpha of uterine origin. Oestrogens produced by the pig conceptuses between Days 11 and 16 appear to exert an antiluteolytic effect resulting in the sequestering of PGF-2 alpha within the uterine lumen. Failure of the pregnant uterus to release PGF-2 alpha in an endocrine fashion, therefore, allows for maintenance of CL function. Conceptuses of sheep and cattle produce proteins which, when introduced into the uterine lumen of nonpregnant ewes and cows, suppress the ability of oestradiol and oxytocin to stimulate uterine production of PGF-2 alpha. These conceptus secretory proteins appear to exert an antiluteolytic effect by inhibiting uterine production of luteolytic amounts of PGF-2 alpha. The horse conceptus produces both oestrogens and proteins during early pregnancy when uterine production of PGF-2 alpha is suppressed. Co-culture of horse endometrium and conceptus inhibits endometrial production of PGF-2 alpha. Conceptuses of pigs, sheep and cattle undergo elongation to achieve apposition between trophectoderm and endometrium but the horse embryo migrates rapidly and consistently throughout the uterus to achieve endometrial contact.     

The permeability of the primary decidual zone in the rat uterus: an ultrastructural tracer and freeze-fracture study

The rat primary decidual zone (PDZ) is a transitory avascular region of transformed fibroblasts surrounding the implanting embryo. Studies using fluorescein-labeled tracers have shown that the PDZ is selectively permeable to macromolecules, permeability decreasing with increasing molecular weight. In the present study we investigated the morphologic basis of the permeability barrier. Horseradish peroxidase (HRP) or HRP-labeled immunoglobulin G (IgG-HRP) was administered i.v. to rats on Day 7 of pregnancy, and the animals were killed 30 min to 2 h later. The reaction product of HRP was the same density in uterine blood vessels as in the intercellular spaces of the endometrium and PDZ at 30 min and 1 h after administration. Two hours after administration, the reaction product of IgG-HRP was dense in uterine blood vessels, much less dense in the interstitial spaces of the endometrium, and was not detected in the PDZ. There was an abrupt change in the density of the IgG-HRP reaction product at the intercellular clefts between endothelial cells, where cellular junctions were observed in control tissue. This suggests that the passage of large macromolecules from blood to the implantation chamber is limited initially by cellular junctions between capillary endothelial cells. The exclusion of IgG-HRP from the PDZ indicates that an additional barrier(s) to macromolecules in this region. Lanthanum nitrate tracer was uniformly present throughout the intercellular spaces of the PDZ except at tight junctions between decidual cells. Freeze-fracture replicas of the PDZ showed tight junctions that varied from single strands to interconnected networks of strands oriented mainly parallel to the long axis of the PDZ. Some strands were discontinuous. The tight junctions between decidual cells appear to be functionally discontinuous because HRP readily penetrated the PDZ, but such junctions may retard larger macromolecules such as IgG-HRP. The biological significance of the permeability barrier of the PDZ is discussed.     

Uterine sensitization-associated gene-1: a novel gene induced within the rat endometrium at the time of uterine receptivity/sensitization for the decidual cell reaction

For successful implantation, the embryo must develop to the blastocyst stage and the endometrium must attain a state that is receptive to the implanting blastocyst. In rodents, the timing, duration, and hormonal regulation of this receptive state has been well defined. However, the molecular cascade of events involved in the onset of the receptive phase remains unclear. In the present study, we sought to identify genes involved in the onset of the receptivity using the technique of suppressive subtraction hybridization. Herein we report the isolation, cloning, and characterization of a novel gene, uterine sensitization-associated gene-1 (UASG-1), that is preferentially expressed within the maximally sensitized/receptive rat endometrium. USAG-1 mRNA encodes a putative protein of 206 amino acids that contains a possible N-terminal secretion signal and a C-terminal cystine knotlike motif. Northern blot analysis revealed that induction of USAG-1 mRNA was restricted to the Day 5 pregnant or pseudopregnant uterus. In situ hybridization experiments demonstrated that this induction was restricted to the uterine glandular epithelial cells. Given the remarkably tight restriction of its expression, USAG-1 may be involved in the onset of endometrial receptivity for implantation/sensitization for the decidual cell reaction.     

Transforming growth factor beta isoforms regulation of Akt activity and XIAP levels in rat endometrium during estrous cycle, in a model of pseudopregnancy and in cultured decidual cells

Background  

During the estrous cycle, the rat uterine endometrium undergoes many changes such as cell proliferation and apoptosis. If implantation occurs, stromal cells differentiate into decidual cells and near the end of pregnancy, a second wave of apoptosis occurs. This process called decidual regression, is tightly regulated as is it crucial for successful pregnancy. We have previously shown that TGF-beta1, TGF-beta2 and TGF-beta3 are expressed in the endometrium during decidual basalis regression, but although we had demonstrated that TGF- beta1 was involved in the regulation of apoptosis in decidual cells, the ability of TGF- beta2 and TGF-beta3 isoforms to trigger apoptotic mechanisms in these cells remains unknown. Moreover, we hypothesized that the TGF-betas were also present and regulated in the non-pregnant endometrium during the estrous cycle. The aim of the present study was to determine and compare the specific effect of each TGF-β isoform in the regulation of apoptosis in sensitized endometrial stromal cells in vitro, and to investigate the regulation of TGF-beta isoforms in the endometrium during the estrous cycle in vivo.