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.     

Estrogen regulation of aquaporins in the mouse uterus: potential roles in uterine water movement

Estrogen stimulates water imbibition in the uterine endometrium. This water then crosses the epithelial cells into the lumen, leading to a decrease in viscosity of uterine luminal fluid. To gain insight into the mechanisms underlying this estrogen-stimulated water transport, we have explored the expression profile and functionality of water channels termed aquaporins (AQPs) in the ovariectomized mouse uterus treated with ovarian steroid hormones. Using immunocytochemical analysis and immunoprecipitation techniques, we have found that AQP-1, -3, and -8 were constitutively expressed. AQP-1 expression was restricted to the myometrium and may be slightly regulated by ovarian steroid hormones. AQP-3 was expressed at low levels in the epithelial cells and myometrium, whereas AQP-8 was found in both the stromal cells and myometrium. AQP-2 was absent in vehicle controls but strongly up-regulated by estrogen in the epithelial cells and myometrium of the uterus. This localization implicates all four isotypes in movement of water during uterine imbibition and, based on their localization to the luminal epithelial cells, AQP-2 and -3 in facilitating water movement into the lumen of the uterus. The analysis of the plasma membrane permeability of luminal epithelial cells by two separate cell swelling assays confirmed a highly increased water permeability of these cells in response to estrogen treatment. This finding suggests that estrogen decreases the luminal fluid viscosity, in part, by enhancing the water permeability of the epithelial layer, most likely by increasing the expression of AQP-2 and/or the availability of AQP-3. Together these results provide novel information concerning the mechanism by which estrogen controls water imbibition and luminal fluid viscosity in the mouse uterus.     

Deregulation of the serum- and glucocorticoid-inducible kinase SGK1 in the endometrium causes reproductive failure

Infertility and recurrent pregnancy loss (RPL) are prevalent but distinct causes of reproductive failure that often remain unexplained despite extensive investigations. Analysis of midsecretory endometrial samples revealed that SGK1, a kinase involved in epithelial ion transport and cell survival, is upregulated in unexplained infertility, most prominently in the luminal epithelium, but downregulated in the endometrium of women suffering from RPL. To determine the functional importance of these observations, we first expressed a constitutively active SGK1 mutant in the luminal epithelium of the mouse uterus. This prevented expression of certain endometrial receptivity genes, perturbed uterine fluid handling and abolished embryo implantation. By contrast, implantation was unhindered in Sgk1-/- mice, but pregnancy was often complicated by bleeding at the decidual-placental interface and fetal growth retardation and subsequent demise. Compared to wild-type mice, Sgk1-/- mice had gross impairment of pregnancy-dependent induction of genes involved in oxidative stress defenses. Relative SGK1 deficiency was also a hallmark of decidualizing stromal cells from human subjects with RPL and sensitized these cells to oxidative cell death. Thus, depending on the cellular compartment, deregulated SGK1 activity in cycling endometrium interferes with embryo implantation, leading to infertility, or predisposes to pregnancy complications by rendering the feto-maternal interface vulnerable to oxidative damage.     

Interleukin-18 (IL-18) mRNA expression and localization of IL-18 mRNA-expressing cells in the mouse uterus

Interleukin-18 (IL-18) belongs to the interleukin-1 family and was identified as an interferon-gamma inducing factor. We investigated IL-18 mRNA-expressing cells in the mouse uterus. By RNase protection assay, IL-18 mRNA and alpha subunit of IL-18 receptor mRNA were detected in the uterus. In the uterus, IL-18 mRNA levels increased during sexual maturation. In situ hybridization analysis demonstrated IL-18 mRNA-expressing cells in the mouse uterus of different ages. At 21 days of age, IL-18 mRNA-expressing cells were detected in the luminal epithelial cells and stromal cells although the IL-18 mRNA signal was weak. At 42 days of age, IL-18 mRNA signal was mainly detected in the stromal cells located near the myometrium, and in some of the luminal and glandular epithelial cells. In the uterus of 63-day-old adult mice, a strong hybridization signal for IL-18 mRNA was detected at estrus, but was weak at diestrus. IL-18 mRNA was mainly detected in the glandular epithelial cells and stromal cells. The effect of estradiol-17beta (E(2)) on IL-18 mRNA-expressing cells in the uterus was examined in ovariectomized mice. In oil-treated mice IL-18 mRNA signal was localized in luminal epithelial cells and stromal cells, while in E(2)-treated mice IL-18 mRNA signal was localized in stromal cells alone. These results suggest that the mouse uterus has an IL-18 system, and IL-18 exerts a physiological role within the uterus in a paracrine manner, and that IL-18 gene expression is regulated by estrogen.     

Apoptosis in endometrium of mouse during estrous cycle

The present study was carried out to evaluate apoptosis in endometrium and to correlate these changes with the circulating levels of estradiol and progesterone in the mouse. Apoptosis was observed in various compartments of mouse uterus i.e. stroma, glandular epithelium and luminal epithelium depending on the stage of cycle. Stromal cell apoptosis was observed during various stages of cyclicity except on estrus day. Luminal epithelial cells showed apoptotic changes during all stages of cyclicity except on diestrus day. During metestrus, apoptosis was observed in glandular and luminal epithelia as well as stromal cells. Steroid antagonists such as tamoxifen and onapristone altered the apoptotic changes in the uterus. The results suggest that epithelial cell apoptosis is regulated by estrogen while stromal cell apoptosis is under the control of progesterone.     

The resistance of the mouse uterine lumen to flushing and possible contamination of samples by plasma and interstitial fluid

The hydrostatic pressures generated during controlled flushing of the mouse uterus increased at implantation and under conditions of uterine closure. These pressures may be responsible for inducing tissue damage during flushing. The possibility that samples collected by flushing might be contaminated with interstitial fluid or plasma was studied using intravenously administered 51Cr-labelled EDTA and 125I-labelled human serum albumin as markers. The presence of both tracers was detected in all flushings and was greatest in flushings from uteri with luminal closure and early implantation sites. These observations raise serious doubts about the validity of the flushing technique for analysing uterine luminal constituents in mice.     

Monoclonal antibodies recognize a cell surface marker of epithelial differentiation in the rabbit reproductive tract

Monoclonal antibodies against the cell surface were produced by immunizing mice with endometrial scrapings prepared from 6-day pregnant rabbits. Spleen cells from an immune mouse were fused with myeloma cells and cultured by standard hybridoma technology methods. Hybridoma supernatants were screened for reaction with the apical epithelial surface by immunohistochemistry on frozen sections of uterus from 6-day pregnant rabbits, and positive colonies were cloned by limiting dilution. Ascites fluid was produced in mice from hybridoma clones that gave a consistent pattern of apical epithelial surface staining through 6 sub-clonings. Antibodies in the ascites fluid were tested by immunohistochemistry on frozen sections of uterus, oviduct, lung, liver and kidney from nonpregnant or 6-day pregnant rabbits. At a dilution of 1:5000, the antibodies recognized an antigen that was specific to the apical surface of luminal but not glandular epithelium of the 6-day pregnant uterus and could not be detected in the nonpregnant uterine epithelium. At higher concentrations of antibody (1:100 to 1:1000), crossreaction was seen with antigens in stromal and myometrial cells of pregnant and nonpregnant uterus. At a dilution of 1:5000, the antibody also crossreacted with some components of lung, liver and kidney but without discriminating between the two reproductive states. In the oviduct, staining of the surface epithelium was specific to the pregnant state. We conclude that this monoclonal antibody has a high affinity for a luminal epithelial cell surface antigen in the reproductive tract of the pregnant rabbit and shows multiple organ reactivity with other tissues that is not affected by pregnancy. This antigen will provide a useful cell surface marker of epithelial differentiation in the progestational reproductive tract.     

Expression of Ly-6A/E in the mouse uterus during implantation period

To investigate the molecular mechanisms of implantation, we constructed a complementary DNA library of mouse uterus enriched with pregnancy-induced genes by subtractive hybridization and polymerase chain reaction. One of the isolated clones was a part of complementary DNA for the Ly-6A/E. Ly-6A/E is reported to be differentially expressed on hematopoietic stem cells and some lymphoid and nonlymphoid tissues, mediate cell-cell adhesion on lymphoid cells, and associate with cell proliferation and angiogenesis of tumor cells. Northern blot, in situ hybridization, and immunohistochemical analyses demonstrated that the Ly-6A/E mRNA and protein were expressed in the endometrial epithelial cells as well as myometrial cells and vascular endothelial cells in the uterus of nonpregnant mouse. The expression was downregulated in luminal epithelial cells during pregnancy days 1-5, while it was upregulated in decidualized stromal cells around the implanted embryo at the time of implantation. The signals were primarily localized in stromal cells at the mesometrial pole on day 9. The increased expression was also observed in stromal cells of the embryo-transferred uterus and artificially-induced deciduoma, indicating that the expression of Ly-6A/E in the endometrial cells is concurrent with decidualization. These findings suggest that Ly-6A/E plays a role in embryo implantation.     

Regional differences in expression of progesterone receptor in oviduct and uterus of rabbit during early pregnancy

We characterized the expression pattern of progesterone receptor (PR) in two regions of the oviduct (ampullae and isthmus), and the uterus (epithelium and stroma) of the rabbit (Oryctolagus cuniculus) during early pregnancy (1-4 days) by RT-PCR and immunohistochemistry. We observed a significant increase in the expression of PR at mRNA level in the uterus on days 1 and 2 of pregnancy, followed by a decrease on days 3 and 4. These changes were also observed at protein level in the uterine epithelium. Interestingly, PR immunoreactivity decreased in stromal cells in all days of pregnancy as compared with non-pregnant rabbits (NG). In the isthmus PR mRNA expression significantly increased on day 2 of pregnancy and diminished on days 3 and 4, whereas no significant changes were observed in the ampullae. In epithelial and stromal cells of the isthmus, PR immunostaining was reduced through pregnancy as compared with NG group. In contrast, a reduction in PR immunostaining was observed on days 1-3 with an increase on day 4 in epithelial and stromal cells of the ampullae. The overall results suggest that PR exhibit a differential expression pattern in the oviduct and the uterus during early pregnancy of the rabbit, and that these differences are related to different functions of PR in the reproductive tract during early pregnancy.     

Conditional gene recombination by adenovirus-driven Cre in the mouse uterus

Cre-mediated conditional gene targeting has been shown to be successful in many cell and tissue types. However, gene recombination in the uterus with heterogeneous cell types by Cre activation is not yet well established. Using recombinant adenoviruses expressing a functional Cre (ADV-Cre) and ROSA26 reporter mice, we show here that ADV-Cre infused intraluminally in a small volume (10 microl) conditionally excises the loxP site, resulting in lacZ expression in uterine luminal epithelial cells without significantly affecting pregnancy. In contrast, a similar intraluminal infusion of ADV-Cre in a larger volume (50 microl) damages the normal architecture and integrity of the luminal epithelium, inducing gene recombination in the underneath stromal cells, with disruption of pregnancy. Further, decidualizing stromal cells at the implantation sites can be targeted by ADV-Cre after intravenous administration on days 5-6. This route of administration also elicits Cre activity in other tissues, including the liver, spleen, ovary, and, more remarkably, in the adrenal cortex. These findings demonstrate the feasibility of achieving conditional expression or deletion of specific genes in uterine cells at desired times and physiological states.     

A simple method for the preparation of luminal epithelial and stromal cells from rat uterus.

An apparatus and procedure is described permitting the ready separation of luminal epithelial and stromal cells (together with the glands) from the rat uterus.     

Dynamic changes to claudins in the uterine epithelial cells of the marsupial Sminthopsis crassicaudata (Dasyuridae) during pregnancy

The fluid that surrounds the embryo in the uterus contains important nourishing factors and secretions. To maintain the distinct microenvironment in the uterine lumen, the tight junctions between uterine epithelial cells are remodeled to decrease paracellular movement of molecules and solutes. Modifications to tight junctions between uterine epithelial cells is a common feature of pregnancy in eutherian mammals, regardless of placental type. Here we used immunofluorescence microscopy and western blot analysis to describe distributional changes to tight junctional proteins, claudin‐1, ‐3, ‐4, and ‐5, in the uterine epithelial cells of a marsupial species, Sminthopsis crassicaudata. Immunofluorescence microscopy revealed claudin‐1, ‐3, and ‐5 in the tight junctions of the uterine epithelium of S. crassicaudata during pregnancy. These specific claudins are associated with restricting passive movement of fluid between epithelial cells in eutherians. Hence, their function during pregnancy in S. crassicaudata may be to maintain the uterine luminal content surrounding developing embryos. Claudin‐4 disappears from all uterine regions of S. crassicaudata at the time of implantation, in contrast with the distribution of this claudin in some eutherian mammals. We conclude that like eutherian mammals, distributional changes to claudins in the uterine epithelial cells of S. crassicaudata are necessary to support pregnancy. However, the combination of individual claudin isoforms in the tight junctions of the uterine epithelium of S. crassicaudata differs from that of eutherian mammals. Our findings suggest that the precise permeability of the paracellular pathway of the uterine epithelium is species‐specific.     

Enhanced endocytotic and transcytotic activity in the rat endometrium prior to embryo implantation

Background Understanding the mechanisms by which fluid absorption and secretion occur in the endometrium is clinically important since conditions that deregulate this process reduce fertility. It has been suggested that luminal epithelial cells induce a crucial step in the process of embryo implantation called uterine closure via endocytotic fluid uptake. Uterine lumen closure is a key step in the process of embryo implantation and is absent in some infertile strains of mice. Methods To investigate the process of uterine closure a ferritin-based tracer, used as a marker of endocytosis, was injected into the uterine lumen on day 5 of pregnancy when closure occurs. Results Unexpectedly, luminal epithelial uptake of tracer was minimal on day 5 of pregnancy discrediting endocytosis as the induction method of uterine closure. In contrast, ferritin was found deep in the stromal portion of the endometrium in pre-pregnant animals. Conclusions We have shown for the first time that uterine closure is not induced by luminal epithelial cell driven endocytosis. Another novel finding of this study was the passage of the tracer ferritin up to 15 cells deep into the endometrium suggesting an as yet unstudied mechanism by which information can be transported from the uterine lumen to the underlying stroma.     

Progesterone and estrogen receptor distribution in the endometrium of the mare

An immunoperoxidase staining technique was used to localize receptors for progesterone and estrogen in the uterus of the mare. Specific staining for receptors was limited to cell nuclei. During estrus, stromal cells tended to stain more intensely for both receptor types than myometrial cells or luminal and glandular epithelial cells. During diestrus, staining intensities in stromal and myometrial cells tended to decrease. Staining intensities of epithelial cells were not affected by the cycle stage. Early pregnancy did not markedly affect the staining intensities of pregnant mares compared with the nonpregnant mares on Day 14 of diestrus. In mares susceptible to endometritis from which samples were taken during diestrus, stromal and myometrial staining for estrogen receptors was more intense than in endometrium from genitally-normal mares.     

Characterization of the uterine phenotype during the peri-implantation period for LIF-null, MF1 strain mice

Leukemia inhibitory factor plays a major role in the uterus and in its absence embryos fail to implant. Our knowledge of the targets for LIF and the consequences of its absence is still very incomplete. In this study, we have examined the ultrastructure of the potential implantation site in LIF-null MF1 female mice compared to that of wild type animals. We also compared expression of proteins associated with implantation in luminal epithelium and stroma. Luminal epithelial cells (LE) of null animals failed to develop apical pinopods, had increased glycocalyx, and retained a columnar shape during the peri-implantation period. Stromal cells of LIF-null animals showed no evidence of decidual giant cell formation even by day 6 of pregnancy. A number of proteins normally expressed in decidualizing stroma did not increase in abundance in the LIF-null animals including desmin, tenascin, Cox-2, bone morphogenetic protein (BMP)-2 and -7, and Hoxa-10. In wild type animals, the IL-6 family member Oncostatin M (OSM) was found to be transiently expressed in the luminal epithelium on late day 4 and then in the stroma at the attachment site on days 5-6 of pregnancy, with a similar but not identical pattern to that of Cox-2. In the LIF-null animals, no OSM protein was detected in either LE or stroma adjacent to the embryo, indicating that expression requires uterine LIF in addition to a blastocyst signal. Fucosylated epitopes: the H-type-1 antigen and those recognized by lectins from Ulex europaeus-1 and Tetragonolobus purpureus were enhanced on apical LE on day 4 of pregnancy. H-type-1 antigen remained higher on day 5, and was not reduced even by day 6 in contrast to wild type uterus. These data point to a profound disturbance of normal luminal epithelial and stromal differentiation during early pregnancy in LIF-nulls. On this background, we also obtained less than a Mendelian ratio of null offspring suggesting developmental failure.     

Zonula occludens-1 and E-cadherin are coordinately expressed in the mouse uterus with the initiation of implantation and decidualization

Two-way interactions between the blastocyst trophectoderm and the uterine luminal epithelium are essential for implantation. The key events of this process are cell-cell contact of trophectoderm cells with uterine luminal epithelial cells, controlled invasion of trophoblast cells through the luminal epithelium and the basement membrane, transformation of uterine stromal cells surrounding the blastocyst into decidual cells, and protection of the "semiallogenic" embryo from the mother's immunological responses. Because cell-cell contact between the trophectoderm epithelium and the luminal epithelium is essential for implantation, we investigated the expression of zonula occludens-1 (ZO-1) and E-cadherin, two molecules associated with epithelial cell junctions, in the mouse uterus during the periimplantation period. Preimplantation uterine epithelial cells express both ZO-1 and E-cadherin. With the initiation and progression of implantation, ZO-1 and E-cadherin are expressed in stromal cells of the primary decidual zone (PDZ). As trophoblast invasion progresses, these two molecules are expressed in stroma in advance of the invading trophoblast cells. These results suggest that expression of these adherence and tight junctions molecules in the PDZ serves to function as a permeability barrier to regulate access of immunologically competent maternal cells and/or molecules to the embryo and provide homotypic guidance of trophoblast cells in the endometrium.     

Expression of vascular endothelial growth factor receptors and their ligands in rat uterus during the postpartum involution period

Abstract

Vascular endothelial growth factor (VEGF) and its specific receptors, FLt1/fms, Flk1/KDR and FLt4, play important roles in vasculogenesis, and physiological and pathological angiogenesis. Whether angiogenic growth factors are involved in regulating angiogenic processes during the postpartum involution period (PP) of the rat uterus is unknown. We used immunohistochemistry to analyze the expression levels of VEGF, the fms-like tyrosine kinase 1 (FLt1/fms), the kinase insert domain-containing region 1 (Flk1/KDR), Fms-related tyrosine kinase 4 (FLt4) and vascular endothelial growth inhibitor (VEGI) in the rat uterus during the days 1, 3, 5, 10 and 15 of the PP to determine the temporal and spatial expressions of VEGF and its receptors during the PP. Throughout the PP, cytoplasmic and membrane staining of VEGI, VEGF and their receptors were observed in the lumens, crypts and glandular epithelial cells as well as in connective tissue and vascular endothelial and smooth muscle cells in the endometrium. We found that the intensity of the immunoreactions in the endometrium varied with the morphological changes that occurred during involution. Immunoreactions for VEGI, VEGF and their receptor, Flk1/KDR, in the luminal epithelial cells were stronger than those in the glandular epithelial and stromal cells, particularly during PP 1, 3 and 5, which suggests that these peptides may contribute to re-epithelialization of the endometrium. On the other hand, Flt1/fms immunoreactivity was strong mainly in the stromal cells during the PP. The presence of VEGF and its receptors (FLt1/fms, Flk1/KDR, FLt4) in the stromal cells and blood vessels during the PP suggests that they may contribute to regulating stromal repair and angiogenesis in the involuting uterus of the rat.     

Uterocalin: A mouse acute phase protein expressed in the uterus around birth

Mouse SIP24/24p3 is a 24 kDa lipocalin expressed in the liver and secreted into the bloodstream during the acute phase response (APR). In this report we show that SIP24/24p3 mRNA and protein are expressed in the uterus around parturition at levels higher than are found in the liver during the APR. Because of the unique expression of this lipocalin in the uterus, we have named this protein uterocalin. Contrary to its expression pattern during the APR, there is little or no expression of uterocalin in the liver during or after pregnancy. Also, unlike the APR, and despite its high level of expression in the uterus, uterocalin was not detected in the blood or amniotic fluid. Day 19 and postpartum uterine samples were examined by immunocytochemistry. Uterocalin was found in the luminal epithelium at day 19 and in the glandular epithelium in postpartum samples. Although some uterocalin remained in the luminal epithelium, most of the uterocalin was found deposited on its luminal surface. The uterus undergoes extensive tissue remodeling during pregnancy and suffers stress and tissue damage around parturition. Uterocalin could be part of the local inflammatory response associated with parturition. Mol. Reprod. Dev. 46:507–514, 1997. © 1997 Wiley-Liss, Inc.