Apoptosis in uterine epithelium and decidua in response to implantation: evidence for two different pathways

During the initial steps of implantation, the mouse uterine epithelium of the implantation chamber undergoes apoptosis in response to the interacting blastocyst. With progressing implantation, regression of the decidual cells allows a restricted and coordinated invasion of trophoblast cells into the maternal compartment. In order to investigate pathways of apoptosis in mouse uterine epithelium and decidua during early pregnancy (day 4.5–7.0 post coitum), we have investigated different proteins such as TNFalpha, TNF receptor1, Fas ligand, Fas receptor1, Bax and Bcl2 as well as caspase-9 and caspase-3 using immunohistochemistry. To detect cells undergoing apoptosis the Tunel assay was performed. Immunoreactivity for TNFalpha as well as for TNF receptor1 was observed exclusively in the epithelium of the implantation chamber and the adjacent luminal epithelium from day 4.5 post coitum onwards. In the developing decidua the Fas ligand, but not the Fas receptor, was expressed. Bax and Bcl2 revealed a complementary expression pattern with Bax in the primary and Bcl2 in the adjacent decidual zone. Strong immunolabelling for the initiator caspase-9 was restricted to the decidual compartment, whereas caspase-3 expression characterized the apoptotic uterine epithelium. Only some caspase-3 positive decidual cells were found around the embryo which correlated to the pattern of Tunel staining. Taken together, the apoptotic degeneration of the uterine epithelium seems to be mediated by TNF receptor1 followed by caspase-3, whereas the very moderate regression of the decidua did not show the investigated death receptor, but Bax and Blc2 instead and in addition caspase-9, which indicates a different regulation for epithelial versus decidual apoptosis.     

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 secretory protease inhibitor SPINK3 in mouse uterus during early pregnancy

Successful embryo implantation depends on intricate epithelial-stromal cross-talk. However, molecular modulators involved in this cellular communication remain poorly elucidated. Using multiple approaches, we have investigated the spatiotemporal expression and regulation of serine protease inhibitor Kazal type 3 (SPINK3) in mouse uterus during the estrous cycle and early pregnancy. In cycling mice, both SPINK3 mRNA and protein are only expressed during proestrus. In the pregnant mouse, the expression levels of both SPINK3 mRNA and protein increase on days 5-8 and then decline. Spink3 mRNA is expressed exclusively in the uterine glandular epithelium, whereas SPINK3 protein is localized on the surface of both luminal and glandular epithelium and in the decidua. Moreover, SPINK3 in the decidua has been observed in the primary decidual zone on day 6 and the secondary decidual zone on days 7-8; this is tightly associated with the progression of decidualization. SPINK3 has also been found in decidual cells of the artificially decidualized uterine horn but not control horn, whereas Spink3 mRNA localizes in the glands of both horns. The expression of endometrial Spink3 is not regulated by the blastocyst according to its expression pattern during pseudopregnancy and delayed implantation but is induced by progesterone and further augmented by a combination of progesterone and estrogen in ovariectomized mice. Thus, uterine-gland-derived SPINK3, as a new paracrine modulator, might play an important role in embryo implantation through its influence on stromal decidualization in mice.     

Norrin immunolocalization and its possible functions in rat endometrium during the estrus cycle and early pregnancy

Mutations in Norrie Disease Pseudoglioma (NDP) gene cause serious sight loss, deafness and mental retardation in Norrie disease patients via the impairment of angiogenesis. Since norrin is a Wnt pathway ligand, it could function in several tissues other than eye and nervous systems. Therefore, the aim of the present study was to determine the possible function of norrin in angiogenesis, cellular differentiation in stroma and in decidua and the survival of those cells using immunofluorescent labeling. While norrin had a uniform distribution in stroma and in blood vessels, it had a strong expression in luminal and glandular epithelia during the estrus cycle. Norrin had strong immunolocalization in the antimesometrial decidual reaction zone on day 7 of gestation, whereas it had a decreased expression in the mesometrial uterine luminal epithelium along with an increased localization in blood vessels and decidual cells of the same region on day 8 of gestation. As from day 9 of gestation, norrin demonstrated rather strong expression in the decidual cells and blood vessels of the mesometrial region in which the chorioallantoic placenta was going to develop. In all periods studied, norrin had rather weak expression in the primary decidual zone surrounding the embryo. Findings of the present study suggested that norrin might regulate the decidual reaction and the placental angiogenesis along with the survival and the differentiation of luminal and glandular epithelial and decidual cells in rats. In addition, it could play indirect important roles in the control of trophoblastic invasion and the programmed cell death.     

Changes in the temporal and spatial expression of H beta 58 during formation and maturation of the chorioallantoic placenta in the Rat

Cloning and sequencing of a cDNA amplified by RNA fingerprinting at the implantation site of pregnant rats revealed 80% similarity with H beta 58, previously shown to be essential for formation of the chorioallantoic placenta in the mouse. H beta 58 mRNA was detected in the endometrium of hormonally sensitized rats stimulated to undergo decidualization and in the contralateral uterine horns lacking a decidual stimulus, indicating that uterine expression of H beta 58 mRNA did not require decidualization or the presence of a blastocyst. Immunodetection in the early postimplantation uterus (Days 6-8 of pregnancy) showed H beta 58 localized in the luminal and glandular epithelia and some stromal cells. Decidual cells at Day 6 of pregnancy expressed H beta 58, and by Day 9 of pregnancy, the protein localized throughout the maternal decidua. The temporal and spatial distribution of H beta 58 in the developing chorioallantoic placenta was assessed at Days 10, 12, and 14 of pregnancy. Immunoreactive H beta 58 localized to erythroid cells within the developing fetal vasculature of the chorioallantoic primordia at Day 10 of pregnancy. By Day 12, the fetal vasculature extended into the placental labyrinth, and the erythroid stem cells continued to strongly express H beta 58. At Day 14 of pregnancy, immunoreactivity became evident in the trophoblast giant cells and syncytiotrophoblast of the fetal placenta. As the chorioallantoic placenta matured (Day 18), H beta 58 mRNA was 3.6-fold higher in the labyrinth compared with the junctional region. Stable cell lines (HRP/LRP) isolated from the rat labyrinthine placenta expressed H beta 58 mRNA and protein. The expression pattern of H beta maternal and fetal placental tissues and its early expression in fetal erythroid stem cells during formation and maturation of the chorioallantoic placenta suggest that H beta 58 plays key roles in the regulatory networks that control hematopoietic development and placentation.     

Expression of vascular endothelial growth factor and its receptors in the rhesus monkey (Macaca mulatta) endometrium and placenta during early pregnancy

Vascular endothelial growth factor (VEGF) is fundamental for development and maintenance of endometrial and placental vascular function during pregnancy. While there are a number of studies on VEGF in the human placenta, they are mostly restricted to late pregnancy. To further understand the role of VEGF in mediating angiogenesis during human early pregnancy, we employed a rhesus monkey early pregnancy model to study the temporal and spatial expression of VEGF and its receptors, fms-like tyrosine kinase (Flt)-1, and kinase-insert domain-containing receptor (KDR) mRNAs and proteins in the uteri on day 12, 18, and 26 of pregnancy using in situ hybridization, RT-PCR, and immunohistochemistry. VEGF mRNA had been identified in the luminal epithelium on day 12, in the glandular epithelium on day 12 and 18, and the highest expression was detected in the walls of some spiral arterioles adjacent to the implantation site on day 18, in the placental villi and in the fetal-maternal border on day 18 and 26. Besides, immunostaining of VEGF was detected in the placental villi and endometrial compartments including spiral arteries walls and the glandular epithelium. The localization of VEGF in the endothelium correlates with the presence of Flt-1 and KDR receptors on vascular structure. All the results above suggest that VEGF-VEGFR pairs were involved in the process of trophoblast invasion, maternal vascular transformation, and fetoplacental vascular differentiation and development during the rhesus monkey early pregnancy. Expression of VEGF, Flt-1, and KDR in the epithelial cells also hints some additionally functional roles of VEGF during early pregnancy.     

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.     

Expression of cellCAM-105 in the apical surface of rat uterine epithelium is controlled by ovarian steroid hormones

Affinity-purified antibodies to cellCAM-105, an adhesive cell surface glycoprotein, were used in immunohistochemical investigations of rat uteri at various functional stages: (i) the oestrous, pro-oestrous, metoestrous, and dioestrous stages of the oestrous cycle, (ii) Days 1-8 of normal pregnancy, (iii) delayed implantation, (iv) 18 h after oestrogen reactivation from delay of implantation, and (v) juvenile rats, and normal ovariectomized adults, respectively, before and after experimental injection of progesterone and/or oestrogen. CellCAM-105 was present in the apical zones of the luminal and glandular epithelium cells in a stage-specific and hormone-dependent manner. The results indicate that: (1) steroid hormones are essential for the expression of cellCAM-105 in the uterine epithelial cells; (2) progesterone induces cellCAM-105 expression in the glandular epithelium, and oestrogen induces cellCAM-105 expression in the luminal epithelium; (3) progesterone induces down-regulation of cellCAM-105 from the surface of the uterine luminal epithelium of juvenile rats; (4) cellCAM-105 is absent in the luminal epithelial cells but present in the glandular epithelial cells of the rat uterus at the time of blastocyst implantation.     

Degeneration and apoptosis of endometrial cells in the bitch

The relationships between changes in plasma progesterone concentrations, degeneration of the luminal epithelium, the occurrence of apoptosis of endometrial cells and endometrial leucocyte populations in the bitch were determined. Mature bitches (n = 15) were euthanized and necropsied when in diestrus (Days 7-75, n = 12) or in anestrus (Days 10, 32 and 53). Degeneration of the luminal epithelium was observed in bitches in late diestrus (Days 38-75, n = 5) when plasma progesterone concentrations were decreasing and in anestrus (Days 10 and 32, n = 2) when plasma progesterone concentrations were < 0.5 ng/mL. Endometrial leucocyte populations increased after degeneration of the luminal epithelium (around Day 42 of diestrus). Apoptosis was mainly observed in the basal glandular epithelial cells and endothelial cells of blood capillaries in all except anestrous bitches. Very few apoptotic cells were found in the superficial glandular epithelial cells and stromal cells. Higher apoptotic indices were detected in the basal glandular epithelium on Days 12-42 of diestrus than at other stages. Therefore, apoptosis of glandular basal epithelial cells occurred mainly in early diestrus, degeneration of cells of the luminal epithelium occurred from mid-diestrus to early anestrus, and the increase in leucocyte numbers may have been a consequence and not a cause of luminal epithelial degeneration.     

Spatiotemporal expression of three gap junction gene products involved in fetomaternal communication during rat pregnancy.

The expression of three different members of the gap junction multigene family, alpha 1 (Cx43), beta 1 (Cx32), and beta 2 (Cx26), was analysed in the rat implantation chamber (a structural unit containing fetal, extraembryonic and maternal components within the pregnant uterus) during mid- and late stages of gestation as well as in the delivering, post-partum and non-pregnant uterus. A differential, spatiotemporal and cell-type-specific regulation of gap junctional coexpression was observed for beta 1 and beta 2 in all epithelia examined (visceral, luminal and glandular), as well as for alpha 1 and beta 2 in decidual cells and keratinocytes of the fetal epidermis. alpha 1 antigen was detected in the mesometrial stroma, mesometrial myometrium, connective tissue, mesothelia of the amnion and visceral yolk sac and in the allantoic mesodermal layer throughout gestation. In addition, expression of alpha 1 in the placental basal zone and trophoblast giant cells coincided with the differentiation of these cells. beta 2 expression was observed prominently in the chorionic villi of the placental labyrinth. The presence of beta 1 and beta 2 in the visceral epithelium (visceral yolk sac = the primary route for embryonic nourishment prior to the formation of the chorioallantoic placenta) and beta 2 in the chorionic villi (placental barrier = the major fetomaternal exchange route) suggests that gap junctions have an important role in fetomaternal communication.     

Cell-specific metallothionein gene expression in mouse decidua and placentae

Oligodeoxyribonucleotide excess solution hybridization, Northern blot and in situ hybridization were used to analyze metallothionein gene expression in mouse decidua and placentae during gestation. Metallothionein (MT) -I and -II mRNA levels were constitutively elevated, 11- and 13-fold, respectively, relative to the adult liver, in the deciduum (D8), and decreased coordinately about 6-fold during the period of development when the deciduum is replaced by the developing placenta (D10-16). Coincident with this decline, levels of MT mRNA increased dramatically in the visceral yolk sac endoderm. In situ hybridization established that MT-I mRNA was present at low levels in the uterine luminal epithelium (D4), but was elevated at the site of embryo implantation exclusively in the primary decidual zone by D5, and then in the secondary decidual zone (D6-8). Although low levels of MT mRNA were detected in total placental RNA, in situ hybridization revealed constitutively high levels in the outer placental spongiotrophoblasts. Analysis of pulse-labeled proteins from decidua and placentae established that these tissues are active in the synthesis of MT. The constitutively high levels of MT mRNA in decidua were only slightly elevated following injection of cadmium (Cd) and/or zinc (Zn), whereas in placentae they increased several-fold. MT mRNA levels were equally high in decidua and experimentally induced deciduomata (D8) which establishes that decidual MT gene expression is not dependent on the presence of the embryo or some embryo-derived factor. Although the functional role of MT during development is speculative, these results establish the concept that, from the time of implantation to late in gestation, the mouse embryo is surrounded by cells, interposed between the maternal and embryonic environments, which actively express the MT genes. This suggests that MT plays an important role in the establishment and maintenance of normal pregnancy.     

Fibroblast growth factor-10: a stromal mediator of epithelial function in the ovine uterus

Fibroblast growth factor-10 (FGF-10) is a stromal-derived paracrine growth factor considered to be important during embryogenesis; however, its expression by cells in the female reproductive tract has not been investigated. Therefore, an ovine FGF-10 cDNA was cloned from an ovine endometrial cDNA library to investigate expression and potential paracrine characteristics of FGF-10 in the ovine uterus. The ovine FGF-10 cDNA encodes a protein of 213 amino acids and possesses an unusually long 5' untranslated region (UTR). In situ hybridization demonstrated that ovine FGF-10 mRNA was expressed by endometrial stromal cells and by mesenchymal cells of the chorioallantoic placenta. The mRNA for FGF-7, a homologue of FGF-10, was localized in the tunica muscularis of blood vessels in endometrium and myometrium. In contrast, FGF receptor 2IIIb, the high-affinity receptor for both FGF-10 and FGF-7, was expressed exclusively in luminal epithelium, glandular epithelium, and placental trophectoderm. The in vivo spatial expression pattern suggests that FGF-10 is a novel endometrial stromal cell-derived mediator of uterine epithelial and conceptus trophectodermal functions. The nonoverlapping spatial patterns of expression for FGF-10 and FGF-7 in ovine uterus and conceptus suggest independent roles in uterine function and conceptus development.     

Leukemia inhibitory factor, leukemia inhibitory factor receptor, and glycoprotein 130 in rhesus monkey uterus during menstrual cycle and early pregnancy

This goal of this study was to examine immunohistochemical distribution of leukemia inhibitory factor (LIF), LIF receptor (LIFR), and glycoprotein (gp) 130 in rhesus monkey uterus during the menstrual cycle and early pregnancy. Pregnancy rate was significantly reduced in the control group from 66.7% (12 of 18) to 22.2% (4 of 18) with an injection of goat anti-human recombinant LIF immunoglobulin G into the uterine lumen on Day 8 of pregnancy. LIF was mainly localized in glandular and luminal epithelium. LIF immunostaining during the luteal phase was stronger than it was during the proliferative phase. LIF staining gradually increased from Day 3 of pregnancy and reached its highest level on Day 9. LIFR was mainly localized in the glandular and luminal epithelium. LIFR staining during the luteal phase was stronger than it was during the proliferative phase. LIFR staining began to increase from Day 3 of pregnancy and reached a high level on Days 9 and 11. Gp130, a signal-transducing receptor component of LIF, was mainly localized in the glandular epithelium. A high level of gp130 was found on Days 16 and 20 of menstrual cycle, and from Days 5 to 11 of pregnancy. These results suggest that LIF may play an important role in monkey implantation, as it does in mice.     

Synthesis of 'decidualization-associated protein' in tissues of the rat uterus and placenta during pregnancy.

The synthesis of a soluble protein (referred to as 'decidualization-associated protein', DAP), has been examined in uterine and placental tissues of rats during pregnancy by means of polyacrylamide gel electrophoretic analysis of [3H]leucine-labelled soluble proteins. No synthesis of the protein was detected in non-implantation regions of the uterus. In implantation site tissue, no synthesis was detected on Days 6 or 7 of pregnancy. Only slight synthesis was present in the endometrium on Day 8, but synthesis rose rapidly from Days 9 to 12 in both the endometrium and myometrium although differences in the rates of increase were observed. Synthesis fell from Day 12 to 14 in both tissues. Synthesis by the myometrium was entirely localized in the mesometrial region, which contains the metrial gland. After Day 12, when the endometrium is represented by the chorioallantoic placenta, synthesis was examined in the labyrinthine and the decidua basalis/basal zone placenta tissues. No synthesis of 'DAP' was detected in the labyrinthine placenta from Day 16 of pregnancy. Synthesis observed in the decidua basalis/basal zone placenta fell dramatically from Day 14 to 20. The pattern of synthesis of 'DAP' during pregnancy suggests a role in the establishment of the chorioallantoic placenta and metrial gland in the rat.     

Cell-type-specific expression of transforming growth factor-alpha in the mouse uterus during the peri-implantation period.

Immunohistochemistry as well as in situ and Northern blot hybridization were employed to determine temporal and cell-type-specific expression of transforming growth factor-alpha (TGF-alpha) in the mouse uterus during the peri-implantation period. The co-localization of TGF-alpha (by immunohistochemistry) with its mRNA (by in situ hybridization) in the luminal and glandular epithelia on Days 1-4 of pregnancy (Day 1 = vaginal plug) and also in many stromal cells on Days 3 and 4 indicates that these cells are the primary sites of TGF-alpha synthesis during the preimplantation period. The higher levels of TGF-alpha mRNA in total uterine RNA on Day 4, as shown by Northern blotting, is consistent with the recruitment of stromal cells expressing this gene. During the post-implantation period (Days 5-8), the co-localization of the mRNA and protein in the decidua at the implantation sites suggests that the decidualizing stromal cells synthesize TGF-alpha. Although in situ hybridization showed the presence of mRNA in embryos on Days 5-8, immunostaining was noted in the embryo only on Days 5 and 6. These results suggest that uterine and embryonic expression of TGF-alpha during the peri-implantation period could be involved in embryonic development, preparation of the uterus for implantation, and decidualization.