Conceptus-mediated integrity of endometrial epithelial cells and maintenance of relaxin synthesis in pregnant rabbits: effects of unilateral oviduct ligation

A previous study indicated rabbit endometrial relaxin synthesis is stimulated by blastocyst (Lee VH, Fields PA, Biol Reprod 1990; 40:737-745). To evaluate this hypothesis, unilateral oviduct ligations were placed (A) at the oviduct isthmus on Day 1 post-copulation and (B), in a separate group of rabbits, at the infundibulum before copulation. Blastocysts migrate into and implant in the uterine horn contralateral to the ligated oviduct only (conceptus-bearing uterus). The uterine horn ipsilateral to the ligated oviduct will be referred to as the non-conceptus-bearing uterus. Uteri and ovaries were removed on Days 4-28 of pregnancy and were evaluated for relaxin using guinea pig anti-porcine relaxin serum and avidin-biotin light microscopy immunohistochemistry. Results were identical for both models. Blastocysts first attach to the antimesometrial uterine surface by Day 7 post-copulation. Implantation on the mesometrial surface occurs on Days 8-11. Relaxin was observed in antimesometrial endometrial glands of both conceptus and non-conceptus-bearing uteri on Days 4-7 of pregnancy. Beyond Day 7, relaxin was observed in antimesometrial and mesometrial endometrial glandular and luminal epithelial cells at implantation sites of the conceptus-bearing uterus only. Relaxin was not found between implantation sites. Endometrial epithelial cells of the non-conceptus-bearing uterus were regressing by Day 9. These data indicate a conceptus-mediated maintenance of endometrial epithelial cells. Furthermore, the data suggest a paracrine maintenance of epithelial cell integrity and relaxin synthesis since these parameters are preserved only in the conceptus-bearing uterus. Cell-cell communication between conceptus and endometrium appears to be specific since endometrium between implantation sites does not contain relaxin. Uterine tissue from pseudopregnant rabbits (Days 1-16) was evaluated. Relaxin was observed in the antimesometrial glands on Day 7 only. Like the endometrium in the ligation model, endometrial epithelial cells of the pseudopregnant rabbit uterus were regressing by Day 9. These results indicate that pregnancy is not required for, but may enhance, relaxin synthesis. In addition, endometrial epithelial cells regress in the absence of pregnancy. Regression of endometrial epithelial cells on Day 9 suggests that maternal recognition of pregnancy occurs during the preimplantation period (Days 4-8).     

Immunocytochemical localization of relaxin in the golden hamster (Mesocricetus auratus) during the last half of gestation

The objective of this study was to determine the tissue source of relaxin in pregnant hamsters by immunocytochemical techniques. Ovarian, uterine, and placental tissues were recovered from hamsters on Days 8, 10, 12, 14, and 15 of gestation and processed for light microscopy. Relaxin immunoreactivity was localized in tissue sections by the avidin-biotin-peroxidase technique using antiserum to porcine relaxin. On Day 8 of gestation, relaxin immunoreactivity was localized in primary giant trophoblast cells (GTC-1s) adjacent to the uterine decidua. On Day 10, relaxin immunoreactivity was localized in GTC-1s, secondary giant trophoblast cells (GTC-2s) adjacent to the ectoplacental cone, and endometrial granulocytes in the wall of sheathed arteries. On Day 12, relaxin immunoreactivity was observed primarily in GTC-2s interspersed among cells of the placental trophospongium but not in cells of the placental labyrinth. The intensity of staining and number of relaxin immunoreactive GTCs increased between Days 12 and 14 but was decreased by Day 15 PM. Relaxin was not localized in uterine glands or corpora lutea. These observations suggest that the placenta is the tissue source of relaxin in pregnant hamsters.     

Presence of the acute phase protein, bikunin, in the endometrium of gilts during estrous cycle and early pregnancy

Noninvasive, epitheliochorial placental attachment in the pig is regulated through endometrial production of protease inhibitors. The objective of the present study was to determine if the light-chain serine protease inhibitor of the inter-alpha-trypsin inhibitor family, bikunin, is produced by the porcine endometrium during the estrous cycle and early pregnancy. Western blot analysis revealed the presence of bikunin in uterine flushings of gilts collected during the luteal phase of the estrous cycle and early pregnancy (Days 12-18). However, bikunin unbound to the inter-alpha-trypsin heavy chains was detected only in endometrial explant culture medium obtained from estrus and pregnant (Days 12, 15, and 18) gilts. Endometrial bikunin gene expression was lowest on Day 10 of the estrous cycle and pregnancy, followed by a 30- to 77-fold increase on Day 15 of the estrous cycle and pregnancy. Bikunin gene expression decreased on Day 18 of the estrous cycle, whereas endometrial bikunin gene expression continued to increase in pregnant gilts. Bikunin mRNA was localized to the uterine glands between Days 15 and 18 of the estrous cycle and pregnancy. In addition to its role as a protease inhibitor, bikunin functions in stabilization of the extracellular matrix, which suggests that bikunin could be involved with facilitating placental attachment to the uterine epithelial surface in the pig.     

Endometrial immunoreactive beta-endorphin increases during mid-estrous cycle and early pregnancy in gilts.

Immunoreactive (ir) beta-endorphin (BEND) was recently identified in porcine uterine fluids. In the study reported here, we examined the hypothesis that porcine endometrium serves as a source of uterine fluid ir-BEND during the estrous cycle and early pregnancy. Endometrial ir-BEND was chromatographically characterized, sites of ir-BEND synthesis were immunocytochemically localized, and concentrations of endometrial ir-BEND during the estrous cycle and early pregnancy were measured. Sephadex G-50 chromatographic profiles of endometrial extracts from Day 15 of the estrous cycle revealed three distinct peaks of ir-BEND, with the first peak occurring near void volume and the second and third peaks coinciding with standard porcine beta-lipotropin and standard porcine BEND, respectively. Reverse-phase HPLC C18 chromatographic profiles indicated that endometrial ir-BEND contained both standard BEND and alpha-N-acetylated BEND. Immunocytochemical studies demonstrated ir-BEND in the surface and glandular epithelial cells of the endometrium, with immunostaining most prominent in the apical portion of epithelial cells. Concentrations of ir-BEND in endometrial tissues were higher on Days 14-15 than on Days 8-12 during the estrous cycle and pregnancy (p less than 0.05); however, values were not different in pregnant and cyclic gilts. Biochemical and immunocytochemical evidence supports our hypothesis that ir-BEND present in uterine fluids is derived from the endometrium. The increase in endometrial ir-BEND concentration during Days 14-15 in cyclic and pregnant gilts indicates that ovarian steroids may influence the synthesis of endometrial ir-BEND.     

Measurement of plasma and tissue relaxin concentrations in the pregnant hamster and fetus using a homologous radioimmunoassay

A homologous hamster relaxin RIA was developed to evaluate plasma and tissue concentrations of relaxin in the latter half of pregnancy in this species. Relaxin protein and mRNA were localized using antibodies developed to synthetic hamster relaxin and gene-specific molecular probes, respectively. Molecular weight and isoelectric point of the synthetic and native hormones were identical by electrophoretic methods, and synthetic hamster relaxin was active in the mouse interpubic ligament bioassay. Synthetic hormone was used as tracer and standard with rabbit antiserum to the synthetic hormone in the RIA. Relaxin was assayed in blood samples recovered from the retro-orbital plexus on Days 6, 8, 10, 12, 14, 15, and 16 of gestation and on Days 1 and 5 postpartum. Relaxin was first detected on Day 8 of gestation (3.7 +/- 0.6 ng/ml), increased to reach a maximum in the evening of Day 15 (826.0 +/- 124.0 ng/ml), and decreased by Day 16 (day of parturition). Relaxin concentrations were assayed in aqueous extracts of implantation sites (Days 6, 8, and 10) and chorioallantoic placentae (Days 12, 14, and 15). Concentrations were low on Day 6 (0.02 +/- 0.001 microg/g tissue), increased to Day 15 (6.96 +/- 0.86 microg/g tissue), and subsequently declined by the evening of Day 15. Relaxin protein and mRNA were localized to primary and secondary giant trophoblast cells in the chorioallantoic placental trophospongium. However, relaxin protein was not localized in ovaries of pregnant animals or oviductal tissues of cycling animals. Significant quantities of relaxin were detected in the serum of fetal hamsters recovered on Day 15.     

Light and electron microscopic radioautography of DNA synthesis in the endometria of pregnant-ovariectomized mice during activation of implantation window.

Pregnant mice were ovariectomized at pre-implantation stage and exogenous nidatory estradiol was administered to evaluate the DNA synthesis of the endometrial cells during activation of uterine receptivity for blastocyst implantation. After 0, 3, 6, 12 and 18 hrs. of estradiol treatment, the animals received 3H-thymidine injection, sacrificed 1 hr. later, and the uteri were prepared for light and electron microscopic radioautography. At time 0, no labelled stromal or epithelial cells was found in the endometrium. According to the time-lapse after estradiol induction, a gradual increase of labelled stromal and endothelial cells was seen in the endometrium. The highest labeling index was observed at the antimesometrial side of the implantation sites and the lowest value was found at the interimplantation site. The cells found at mesometrial side of the implantation site showed an intermediate labeling index. Eighteen hrs. after estradiol treatment, the labelled stromal cells found near the implantation chamber resembled the morphology of decidual cells while those labelled cells localized at the interimplantation sites were similar to the fibroblast. The uterine luminal epithelial cells showed low DNA synthesis after estradiol treatment resulting in only a few labelled cells at the interimplantation sites and no labelled cells at the implantation sites. A similar labeling pattern was seen in the glandular epithelium. The distribution of labelled cells seen among the regions of pregnant endometrium under estradiol effect suggest that DNA synthesis related to uterine activation for blastocyst implantation is a focal reaction, where the luminal epithelium does nt proliferate while the stromal and endothelial cells around the conceptus increase the DNA synthesis to prepare the endometrial decidualization.     

Stage-specific expression of the intermediate filament protein cytokeratin 13 in luminal epithelial cells of secretory phase human endometrium and peri-implantation stage rabbit endometrium

In preparation for blastocyst implantation, uterine luminal epithelial cells express new cell adhesion molecules on their apical plasma membrane. Since one mechanism epithelial cells employ to regulate membrane polarity is the establishment of specific membrane-cytoskeletal interactions, this study was undertaken to determine if new cytokeratin (CK) intermediate filament assemblies are expressed in endometrial epithelial cells during developmental stages related to blastocyst implantation. Type-specific CK antibodies were used for immunocytochemical and immunoblot analyses of 1) intermediate filament networks of the endometrial epithelium during embryo implantation in rabbits and 2) proliferative and secretory phases of the human menstrual cycle. CK18, a type I CK found in most simple epithelia, was expressed in all luminal and glandular epithelial cells of both the human and rabbit endometrium at all developmental stages analyzed; it was also strongly expressed in trophectoderm of the implanting rabbit blastocyst. In contrast, CK13, another type I cytokeratin, exhibited a regulated expression pattern in luminal, but not glandular, epithelial cells of secretory phase human and peri-implantation stage rabbit endometrium. Furthermore, in the rabbit implantation chambers, CK13 was predominantly localized at the cell apex of luminal epithelial cells, where it assembled into a dense filamentous network. These data suggest that the stage-specific expression of CK13 and a reorganization of the apical intermediate filament cytoskeleton of uterine luminal epithelial cells may play important functions in preparation for the implantation process.     

Analysis of osteopontin at the maternal-placental interface in pigs

Noninvasive, epitheliochorial placentation in the pig follows a prolonged preimplantation period characterized by migration, spacing and elongation of conceptuses, and secretion of estrogen for maternal recognition of pregnancy. Osteopontin (OPN) is an extracellular matrix protein that binds integrins to promote cell-cell attachment and communication. OPN appears to play a key role in conceptus implantation and maintenance of pregnancy in sheep; however, a role for OPN in the porcine uterus has not been established. Therefore, this study examined OPN expression and function in the porcine uterus and conceptus (embryo/fetus and associated extraembryonic membranes). Northern and slot blot hybridization detected an increase in endometrial OPN expression between Days 25 and 30, and levels remained elevated through Day 85 of pregnancy. In situ hybridization localized OPN mRNA to discrete regions of the uterine luminal epithelium (LE) on Day 15 of pregnancy and to the entire LE thereafter. Glandular epithelial (GE) expression of OPN mRNA was first detected on Day 35 of pregnancy and increased through Day 85. Both 70- and 45-kDa forms of OPN protein were detected in cyclic and pregnant endometrium by Western blotting. OPN protein was localized to the LE and GE by immunofluorescence; however, only the 70-kDa OPN was detected in uterine flushings. OPN protein was present along the entire uterine-placental interface after Day 30 of pregnancy. In addition, OPN mRNA and protein were localized to immune-like cells within the stratum compactum of the endometrium in both Day 9 cyclic and pregnant gilts. Incubation of OPN-coated microbeads with porcine trophectoderm and uterine luminal epithelial cells induced Arg-Gly-Asp (RGD)-dependent integrin activation and transmembrane accumulation of cytoskeletal molecules at the apical cell surface as assessed by immunofluorescence detection of talin or alpha-actinin as markers for focal adhesions. These results suggest that OPN, expressed by uterine epithelium and immune cells, may interact with receptors (i.e., integrins) on conceptus and uterus to promote conceptus development and signaling between these tissues as key contributors to attachment and placentation in the pig.     

Gastrin-releasing peptide (GRP) in the ovine uterus: regulation by interferon tau and progesterone

Gastrin-releasing peptide (GRP) is abundantly expressed by endometrial glands of the ovine uterus and processed into different bioactive peptides, including GRP1-27, GRP18-27, and a C-terminus, that affect cell proliferation and migration. However, little information is available concerning the hormonal regulation of endometrial GRP and expression of GRP receptors in the ovine endometrium and conceptus. These studies determined the effects of pregnancy, progesterone (P4), interferon tau (IFNT), placental lactogen (CSH1), and growth hormone (GH) on expression of GRP in the endometrium and GRP receptors (GRPR, NMBR, BRS3) in the endometrium, conceptus, and placenta. In pregnant ewes, GRP mRNA and protein were first detected predominantly in endometrial glands after Day 10 and were abundant from Days 18 through 120 of gestation. Treatment with IFNT and progesterone but not CSH1 or GH stimulated GRP expression in the endometrial glands. Western blot analyses identified proGRP in uterine luminal fluid and allantoic fluid from Day 80 unilateral pregnant ewes but not in uterine luminal fluid of either cyclic or early pregnant ewes. GRPR mRNA was very low in the Day 18 conceptus and undetectable in the endometrium and placenta; NMBR and BRS3 mRNAs were undetectable in ovine uteroplacental tissues. Collectively, the present studies validate GRP as a novel IFNT-stimulated gene in the glands of the ovine uterus, revealed that IFNT induction of GRP is dependent on P4, and found that exposure of the ovine uterus to P4 for 20 days induces GRP expression in endometrial glands.     

Immunocytochemical localization and messenger ribonucleic acid levels of a progesterone-dependent endometrial secretory protein (cathepsin L) in the pregnant cat uterus.

The purpose of this study was to localize immunocytochemically a progesterone-dependent protein (PDP) and to determine PDP mRNA levels during the initial stage of the implantation period. Uterine tissue was collected from Day 0-18 postcoital animals. The tissue was processed for immunocytochemical localization of PDP, and the endometrial RNA was isolated and analyzed for PDP gene expression by slot-blot hybridization. PDP was detected immunocytochemically as early as Day 5 postcoitus in the epithelial cells of the deep uterine glands, and the intensity of immunostaining appeared to peak by Day 12 postcoitus. PDP was absent in the endometrium obtained from implantation sites after Day 16 postcoitus, but the synthesis of PDP was maintained in the endometrium obtained from nonimplantation sites. Immunogold electron microscopy demonstrated that PDP was present in electron-dense granules of the glandular epithelial cells. PDP mRNA was detectable in the endometrium at Day 5 postcoitus and peaked around Day 10 postcoitus. PDP mRNA was absent in the endometrium from implantation sites after Day 16 postcoitus, but was maintained in the endometrium from nonimplantation sites. In summary, the results of this study illustrate that PDP is synthesized within the epithelial cells of the deep uterine glands, packaged within membrane-bound secretory granules, and released into the uterine lumen. Also, the process of implantation alters the gene expression in a very localized way since PDP mRNA and PDP-positive granules were absent in the endometrial glands obtained from the implantation site within 1-2 days of the onset of implantation, whereas both PDP mRNA and PDP-positive granules were maintained in the endometrial glands from nonimplantation-site regions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Immunocytochemical localization of relaxin in corpora lutea of sows throughout the estrous cycle

Porcine relaxin has been sought by localization in the corpus luteum of sows on Days 3, 7, 9, 11, 12, 15, 18, 19, and 21 of the estrous cycle, using the avidin-biotin immunoperoxidase method and an antiserum to purified porcine relaxin. Simultaneous localization of relaxin in corpora lutea from sows on Days 108 and 113 of pregnancy was used to compare the intensity of immunostaining with that of corpora lutea of cyclic animals. However, the antiserum dilution necessary for optimal localization differed considerably in these two states (1:10,000 in pregnancy and 1:750 in the cycle), suggesting that lower levels of antigen are present in the luteal cells of the cycle. Relaxin immunostaining was undetectable on Day 3 of the cycle but became evident by Days 7 and 9. At Day 11 staining intensity increased and persisted through Day 15. On Day 18 some stain was still evident, but by Days 19, 20, and 21 there was complete absence of immunostain. Relaxin immunostaining appeared to be located throughout the cytoplasm of the luteal cell, as clear areas in the nuclear region were often observed. The results suggest that relaxin is produced in low amounts by the luteal cells of the cyclic sow and that the levels fluctuate with stage of the cycle. Lack of evidence from radioimmunoassay for a surge of relaxin secretion into the systemic circulation prior to luteolysis in the pig estrous cycle suggests that the relaxin localized in the luteal cells of the cycle may have an intraovarian function.     

Insulin-like growth factor-I expression during early conceptus development in the pig

Porcine conceptuses (embryo and associated membranes) in utero undergo developmental morphological transformations coincident with structural and biochemical changes in the uterine endometrium during early gestation. To elucidate a possible role for insulin-like growth factor-I (IGF-I) in these events, porcine endometrial (Days 8, 10, 11, 12, 14, and 30) and conceptus (Days 12, 14, and 16) tissues were characterized for the presence of IGF-I peptide and mRNAs. The corresponding uterine luminal fluids (ULF) at these stages of pregnancy were also analyzed for immunoreactive IGF-I concentration. ULF IGF-I was lowest on Day 8, highest on Day 12, and declined by Day 14. In contrast, endometrial tissue IGF-I content remained constant during this period. Conceptus tissues contained less IGF-I than endometrial tissues; however, conceptus IGF-I values were maximum on Day 12 coincident with peak values for ULF IGF-I. Dot-blot hybridization analyses revealed temporal variation in steady-state levels of IGF-I mRNAs in endometrium. Highest levels of endometrial IGF-I mRNA were detected on Day 12 and were about 4-fold greater than on Day 30 of pregnancy. IGF-I mRNA expression in conceptus tissues on Days 12, 14, and 16 was the same and was significantly less than that in endometrium on Day 12. These results demonstrate the temporal variation of IGF-I mRNA abundance in uterine endometrium and of immunoreactive IGF-I in ULF and in conceptus tissues, with the developmental processes occurring in the conceptuses at early pregnancy.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Ovarian hormones regulate expression of the focal adhesion proteins,talin and paxillin,in rat uterine luminal but not glandular epithelial cells

During early pregnancy in the rat, focal adhesions disassemble in uterine luminal epithelial cells at the time of implantation to facilitate their removal so that the implanting blastocyst can invade into the underlying endometrial decidual cells. This study investigated the effect of ovarian hormones on the distribution and protein expression of two focal adhesion proteins, talin and paxillin, in rat uterine luminal and glandular epithelial cells under various hormone regimes. Talin and paxillin showed a major distributional change between different hormone regimes. Talin and paxillin were highly concentrated along the basal cell surface of uterine luminal epithelial cells in response to oestrogen treatment. However, this prominent staining of talin and paxillin was absent and also a corresponding reduction of paxillin expression was demonstrated in response to progesterone alone or progesterone in combination with oestrogen, which is also observed at the time of implantation. In contrast, the distribution of talin and paxillin in uterine glandular epithelial cells was localised on the basal cell surface and remained unchanged in all hormone regimes. Thus, not all focal adhesions are hormonally dependent in the rat uterus; however, the dynamics of focal adhesion in uterine luminal epithelial cells is tightly regulated by ovarian hormones. In particular, focal adhesion disassembly in uterine luminal epithelial cells, a key component to establish successful implantation, is predominantly under the influence of progesterone.     

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.     

Immunohistological localization of human endometrial secretory protein, 'pregnancy-associated endometrial alpha 2-globulin' (alpha 2-PEG), during the menstrual cycle

The distribution of alpha 2-PEG, a human analogue of beta-lactoglobulin, in endometrium at different phases of the cycle was determined using immunohistochemistry with monoclonal and polyclonal antibodies. In the epithelial cells of glands in the functional zone of the endometrium, alpha 2-PEG was first detectable from Days 19 to 21 during the mid-luteal phase and maximal immunostaining was observed during the end of the late luteal phase. Intense staining in the glandular secretions and weaker staining in surface luminal epithelial cells during this period were observed. A minor population of basal glands contained alpha 2-PEG during the follicular phase. These results suggest that alpha 2-PEG synthesis by the glandular epithelium of the regenerated endometrium is hormonally regulated. Maximal staining occurring during the late luteal phase suggests that regulation may be related to the hormonal requirement for pre-decidualization rather than that required for histologically defined glandular epithelial secretion.     

Regulation of gene expression and cellular localization of prostaglandin synthase by oestrogen and progesterone in the ovine uterus

Expression of the gene for prostaglandin synthase (PGS) was examined in whole endometrial tissue derived from ewes during the oestrous cycle (Days 4-14), on Day 15 of pregnancy and following ovariectomy and treatment with ovarian steroid hormones. Whilst no significant differences were seen in PGS mRNA concentrations analysed by Northern blot analysis in endometrial tissue during the oestrous cycle or in early pregnancy, treatment of ovariectomized (OVX) ewes with oestradiol-17 beta markedly reduced endometrial PGS mRNA concentration. There was no difference in PGS mRNA concentration in ewes treated with progesterone, either alone or in conjunction with oestrogen, from that in OVX controls. In contrast, differences in immunolocalization of PGS observed in uterine tissue from OVX-steroid-treated ewes were much more marked and reflected similar changes seen previously in the immunocytochemical distribution of endometrial PGS during the oestrous cycle. In OVX ewes and those treated with oestrogen, immunocytochemical staining for PGS was seen in stromal cells, but little immunoreactive PGS was located in the endometrial epithelial cells. However, in ewes treated with progesterone alone or with oestrogen plus progesterone, PGS was found in luminal and glandular epithelial cells and in stromal cells. Intensity of immunostaining for PGS in endothelial cells and myometrium did not differ between the treatments. Thus, whilst oestrogen lowers PGS mRNA in the endometrium, presumably in stroma, it may also increase the stability of the enzyme itself in the stromal cells. Although oestradiol-17 beta has no effect on PGS in endometrial epithelium, progesterone stimulates the production of PGS in endometrial epithelial cells without altering the overall abundance of PGS mRNA in the endometrium as a whole. Conceptus-induced changes in PGF-2 alpha release by ovine endometrium would not appear to be mediated via effects on PGS gene expression or protein synthesis.     

A light microscopic and ultrastructural study on the presence and location of oxytocin in the equine endometrium

It has been reported that oxytocin is produced not only in the hypothalamus and posterior pituitary but also in outside the classical hypothalamo-neurohypophyseal axis such as the ovary, testis, placenta and in some nonreproductive sites. In the mare, oxytocin-mRNA has been identified in the endometrium, and oxytocin and its neurophysin have been identified in the uterus. In the present study, oxytocin was localised in the endometrium of the mare at the light microscopic and ultrastructural level by immunostaining and immunogold labelling of endometrial biopsy specimens collected during estrus.Strong positive immunostaining for oxytocin was found in the secretory vesicles of the secretory (nonciliated) epithelial cells of the uterine lumen and of the superficial glands. Using immunogold labelling, oxytocin was detected in the secretory vesicles of secretory epithelial cells. The vesicles containing immunoreactive oxytocin were present on the luminal surface suggesting that oxytocin is secreted into the uterine lumen by apical exocytosis. There was no positive immunostaining in ciliated epithelial cells of the uterine lumen and endometrial glands, in the stromal cells, or in the basal endometrial glands. To our knowledge, this is the first report of the location of oxytocin in specific secretory cells in the endometrium of any domestic species. This locally synthesised uterine oxytocin may have an important role in the autocrine/paracrine control of uterine contractility and luteolysis in the mare.     

Spatiotemporal expression of cyclooxygenase 1 and cyclooxygenase 2 during delayed implantation and the periimplantation period in the Western spotted skunk

Embryonic development in the western spotted skunk is arrested after blastocyst formation for about 200 days. This developmental arrest is believed to be due to insufficiency of uterine conditions to support continuous development. Implantation and decidualization are defective in cyclooxygenase 2 (Cox2)-, but not Cox1-, deficient mice. We therefore used Northern and in situ hybridization to investigate changes in uterine expression of Cox1 and Cox2 genes during various stages of pregnancy in the spotted skunk. Cox1 was constitutively expressed at all stages of pregnancy examined, but it did exhibit localized up-regulation in the trophoblast and necks of uterine glands at early implantation sites. Cox2 expression was highly regulated with little or no expression during delayed implantation. Cox2 expression was first detected in the uterus and trophoblast prior to blastocyst attachment and remained detectable for 5-6 days after blastocyst attachment. Cox2 expression was also localized in the luminal and glandular epithelia of uterine segments located between implantation chambers. Changes in Cox expression were not correlated with the abrupt increase in uterine weight that occurs simultaneously with renewed embryonic development but was correlated with an influx of serum proteins into the uterus observed in a previous study.