Production of prostaglandin f2alpha and its metabolite by endometrium and yolk sac placenta in late gestation in the tammar wallaby, Macropus Eugenii.
In this study, we investigated production of prostaglandin (PG) F2alpha and its metabolite, PGFM, by uterine tissues from tammar wallabies in late pregnancy. Endometrial explants were prepared from gravid and nongravid uteri of tammars between Day 18 of gestation (primitive streak) and Day 26.5 (term) and were incubated in Ham's F-10 medium supplemented with glutamine and antibiotics for 20 h. PGF2alpha and PGFM in the medium were assayed by specific, validated RIAs. Control tissues (leg muscle) did not produce detectable amounts of either PG. Both gravid and nongravid endometria secreted PGF2alpha, and production increased significantly in both gravid and nongravid uteri towards term. PGFM was produced in small amounts by both gravid and nongravid uteri, and the rate of production did not increase. Neither oxytocin nor dexamethasone stimulated PG production in vitro in any tissue at any stage. Thus, the surge in peripheral plasma PGFM levels seen at parturition may arise from increased uterine PG production, but further study is needed to define what triggers this release. (+info)
Expression of trophinin, tastin, and bystin by trophoblast and endometrial cells in human placenta.
Trophinin, tastin, and bystin comprise a complex mediating a unique homophilic cell adhesion between trophoblast and endometrial epithelial cells at their respective apical cell surfaces. In this study, we prepared mouse monoclonal antibodies specific to each of these molecules. The expression of these molecules in the human placenta was examined immunohistochemically using the antibodies. In placenta from the 6th week of pregnancy, trophinin and bystin were found in the cytoplasm of the syncytiotrophoblast in the chorionic villi, and in endometrial decidual cells at the utero placental interface. Tastin was exclusively present on the apical side of the syncytiotrophoblast. Tissue sections were also examined by in situ hybridization using RNA probes specific to each of these molecules. This analysis showed that trophoblast and endometrial epithelial cells at the utero placental interface express trophinin, tastin, and bystin. In wk 10 placenta, trophinin and bystin were found in the intravillous cytotrophoblast, while tastin was not found in the villi. After wk 10, levels of all three proteins decreased and then disappeared from placental villi. (+info)
Down-regulation of oxytocin-induced cyclooxygenase-2 and prostaglandin F synthase expression by interferon-tau in bovine endometrial cells.
Oxytocin (OT) is responsible for the episodic release of luteolytic prostaglandin (PG) F2alpha from the uterus in ruminants. The attenuation of OT-stimulated uterine PGF2alpha secretion by interferon-tau (IFN-tau) is essential for prevention of luteolysis during pregnancy in cows. To better understand the mechanisms involved, the effect of recombinant bovine IFN-tau (rbIFN-tau) on OT-induced PG production and cyclooxygenase-2 (COX-2) and PGF synthase (PGFS) expression in cultured endometrial epithelial cells was investigated. Cells were obtained from cows at Days 1-3 of the estrous cycle and cultured to confluence in RPMI medium supplemented with 5% steroid-free fetal calf serum. The cells were then incubated in the presence or absence of either 100 ng/ml OT or OT+100 ng/ml rbIFN-tau for 3, 6, 12, and 24 h. OT significantly increased PGF2alpha and PGE2 secretion at all time points (p < 0.01), while rbIFN-tau inhibited the OT-induced PG production and reduced OT receptor binding in a time-dependent manner. OT increased the steady-state level of COX-2 mRNA, measured by Northern blot, which was maximal at 3 h (9-fold increase) and then decreased with time (p < 0.01). OT also caused an increase in COX-2 protein, which peaked at 12 h (11-fold increase), as measured by Western blot. Addition of rbIFN-tau suppressed the induction of COX-2 mRNA (89%, p < 0.01) and COX-2 protein (50%, p < 0.01) by OT. OT also increased PGFS mRNA, and this stimulation was attenuated by rbIFN-tau (p < 0.01). To ensure that the decrease in COX-2 was not solely due to down-regulation of the OT receptor, cells were stimulated with a phorbol ester (phorbol 12-myristate 13-acetate; PMA) in the presence and absence of rbIFN-tau. The results showed that rbIFN-tau also decreased PMA-stimulated PG production and COX-2 protein. It can be concluded that rbIFN-tau inhibition of OT-stimulated PG production is due to down-regulation of OT receptor, COX-2, and PGFS. (+info)
Molecular and cellular aspects of endometrial receptivity.
Endocrine and paracrine controls regulate the endometrium during the luteal phase of the cycle to permit implantation. Part of this differentiation process is the production of a specific secretion which fills the intrauterine cavity and glandular lumen. Its molecular composition originates from the gland secretion, from transudations from stroma, from the endometrial blood vessels, and last, but not least, from cellular components of apoptotic and exfoliated cells. We have studied the secretions of all phases during the menstrual cycle using patterns evaluated by SDS-PAGE, by laser densitometry or Western blots. Uterine secretion electrophoresis (USE) permits detailed analyses of the intrauterine micromilieu and allows clinical assessment of the receptive stage of endometrium during the luteal phase. Several individual protein bands have been defined as characteristic markers for such receptive pattern. We have isolated and identified the molecular structure of several of these proteins, e.g. histones, cyclophilin, transthyretin, haptoglobin and uteroglobin. Investigations on the endocrine regulation of these proteins, were carried out on the uterine secretions of patients treated with progesterone antagonists (mifepristone and onapristone). The results demonstrate how progesterone-dependent components produce a receptive pattern, which can serve as a useful and precise marker in the clinical diagnosis of the luteal phase. Essential progesterone-dependent components differentiating during the luteal phase may provide new targets for contraceptive interventions by preventing the physiological changes typical of receptivity. (+info)
Molecular control of the implantation window.
Human endometrium is the end organ of the hypothalamic-pituitary-ovarian axis. Therefore, endometrium is susceptible to changes in the cases of infertility that originate from disturbances in the normal functioning of this axis. In addition, some cases of unexplained infertility may be due to altered endometrial function. This disturbed endometrial function may originate from lesions in the molecular repertoire that are crucial to implantation. Human endometrium becomes receptive to implantation by the blastocyst within a defined period during the menstrual cycle. The duration of this so-called 'endometrial receptivity' or 'implantation' period seems to span from few days after ovulation to several days prior to menstruation. Successful implantation results from a co-ordinated series of events that would allow establishment of a timely dialogue between a receptive endometrium and an intrusive blastocyst. The members of the molecular repertoire that make endometrium receptive to implantation are gradually being recognized. Among these are the cytokines, integrins, heat shock proteins, tastin and trophinin. In addition, the expression of a second set of genes including tumour necrosis factor alpha (TNF-alpha) and ebaf, may be the appropriate signal for the closure of the 'implantation window', for making the endometrium refractory to implantation and for preparing it for the menstrual shedding. (+info)
Marker genes of decidualization: activation of the decidual prolactin gene.
Decidualization of human endometrial stromal (ES) cells in vitro is induced by cAMP analogues and ligands that elevate cellular cAMP levels in a manner resembling the gonadotrophins, prostaglandin E2 and relaxin (RLX). This differentiation process is marked by the onset of decidual prolactin (PRL) production in the late luteal phase of the cycle. Using transfection assays and a primary ES cell culture system, we have demonstrated that decidual PRL gene transcription is driven by an alternative upstream promoter (dPRL), approximately 6 kb upstream of the pituitary transcription start site. In primary cell cultures, RLX not only acutely but also permanently elevated cellular cAMP levels and induced PRL secretion after 6 days. Northern and Western blot analyses revealed all regulatory subunit isoforms (RIalpha, RIbeta, RIIalpha, RIIbeta) and catalytic subunits Calpha and Cbeta of protein kinase A (PKA) in ES cells. Transcript levels of PKA subunit isoforms are not altered during decidualization, but in decidualized ES cells exposed to elevated cellular cAMP levels by stimulation with RLX for >6 days, RIalpha protein levels were significantly reduced, whereas levels of all other forms remained unchanged. Reducing the availability of R subunits changed the R:C subunit ratio in favour of C and increased kinase activity. In transient transfections of undifferentiated ES cells, the dPRL promoter was activated by 8-Br-cAMP and by C subunit (Cbeta) of PKA. This induction, and the differentiation-dependent activity of the dPRL promoter in transfected decidualized cells, was effectively abolished by the co-expression of protein kinase inhibitor (PKI). A fragment of 332 bp of 5'-flanking region of the dPRL transcription start site was sufficient to mediate full inducibility by cAMP. cAMP activation of the dPRL promoter in ES cells was biphasic as an initial weak induction within 12 hours was followed by a subsequent, much more intense induction after 12 hours. The secondary induction was not seen with a control construct driven by a consensus cAMP response element (CRE) linked to a minimal promoter. The early response of the dPRL promoter depended upon a non-palindromic CRE at position -12 and mutation of this sequence led to omission of the early, but not of the delayed, induction. The major activation of the dPRL promoter depended upon a different region between position -332 and -270 since its deletion significantly reduced inducibility by cAMP. Its action was probably indirect as its kinetics differed from classic CRE-mediated responses, and it was specific to ES cells. (+info)
Changes in basement membrane thickness in the human endometrium during the luteal phase of the menstrual cycle.
We have examined aspects of the fine structure of the basal laminae associated with the luminal and glandular epithelium and small blood vessels in the human endometrium. Four short studies are presented and reviewed. Study 1 examined biopsies from 20 fertile women taken on days after the luteinizing hormone surge (LH): LH +2, 4, 6, 8 and 10. The basal lamina (both lamina densa and lucida) increased in thickness over the period studied. Study 2 again studied the glandular epithelium and examined the effect of RU486 (a progesterone receptor blocker) administered on day LH +3 and biopsied on day LH +6. The basal laminae were found to be the same as LH +2 control group but thinner than LH +6 control. Study 3 documented increased thickness of the basal laminae between LH +6, 8 and 13 in the luminal epithelium. The within-group coefficient of variation was 16% and 27% for LH +6 and LH +13 groups but only 2 % for LH +8. Study 4 demonstrated an increase in basal lamina thickness associated with small blood vessels between LH +6 and LH +10 in normal fertile women. The basal lamina provides the interface between epithelial and mesenchymal environments; changes in its structure can alter the phenotypic expression of the epithelia. It is one of the maternal barriers that must be transgressed by the trophoblast during implantation. Together, these combined studies provide quantitative baseline structural information on the electron microscopical appearance of the basal lamina during the luteal phase of the menstrual cycle. (+info)
Endometrial microvascular growth in normal and dysfunctional states.
As a tissue that exhibits rapid cyclical growth and shedding throughout the reproductive life of the female, human endometrium provides a good model for the study of normal physiological angiogenesis. The objective of this paper is to summarize recent data on endometrial vascular growth, present new data on regional variability in endothelial cell proliferation within the endometrium, and interpret this information in light of current knowledge of the mechanisms by which angiogenesis occurs. Conventional angiogenesis normally involves a series of steps which include endothelial cell activation, breakdown of the basement membrane, migration and proliferation of the endothelial cell, fusion of sprouts, and tube formation. Other mechanisms by which angiogenesis occurs include intussusception and vessel elongation. Using immunohistochemical techniques we have shown repeatedly that levels of endothelial cell proliferation within human endometrium do not show any consistent pattern across the different stages of the menstrual cycle, which is unexpected since significant vascular growth must occur during the proliferative phase, when the endometrium increases in thickness by up to 4-fold. There are two possible explanations for this; either there is no obligatory link between endometrial endothelial cell proliferation and new vessel formation, or there is significant variation in endothelial cell proliferation within different regions of the same uterus. Multiple samples from hysterectomy specimens subsequently demonstrated that the variability is due to real differences between individuals, as well as showing that the endothelial cell proliferation index is significantly elevated in functionalis compared with basalis. During these studies we observed that endothelial cell proliferation nearly always appeared inside existing endometrial vessels, rather than be associated with structures that could be identified as vascular sprouts. To explore further whether sprout formation occurs during endometrial angiogenesis, we investigated the immunohistochemical distribution of integrin alphavbeta3 on endometrial endothelial cells. As for endothelial cell proliferation, integrin alphavbeta3 immunostaining was seen only on endothelial cells that appeared within existing blood vessels. The results from these studies have major implications for our understanding of the mechanisms that control endometrial angiogenesis. The lack of correlation between menstrual cycle stage and endothelial cell proliferation index, or endothelial cell expression of integrin alphavbeta3, suggests that vascular growth is not under the overall control of oestrogen and progesterone. (+info)