Production and characterization of endothelin released by human endometrial epithelial cells in culture.

This study identified and characterized endothelin (ET) produced by human endometrial epithelial cells cultured under serum-free conditions, compared the ET released by cells derived from proliferative and secretory phase endometrium, and examined the regulation of ET released by these cells. ET messenger RNA was detected in normal human endometrium with maximal expression in the mid-late secretory phase. Immunoreactive ET released into culture media by separated endometrial epithelial and stromal cells was almost entirely of epithelial cell origin, consistent with the previous immunohistochemical findings. This was identified as ET-1 by reverse phase high-pressure liquid chromatography, and the fractionated conditioned media exhibited bioactivity similar to that of standard ET-1. Mean ET production was greater from cells derived from proliferative phase endometrium cultured either in serum (P < 0.02) or serum-free conditions (P < 0.02). Fetal calf serum stimulated ET-1 production from epithelial cells in a dose-responsive manner. ET production was also stimulated by transforming growth factor-beta 1 (2, 5 & 10 ng/mL) and IL-1 alpha (10 & 100 IU/mL) under serum-free conditions but always to a lesser extent than stimulation by serum. The production of ET in human endometrium underlines a potential role for ET in endometrial function.

Matrix metalloproteinase production by cultured human endometrial stromal cells: identification of interstitial collagenase, gelatinase-A, gelatinase-B, and stromelysin-1 and their differential regulation by interleukin-1 alpha and tumor necrosis factor-alpha.

Matrix metalloproteinases (MMPs) together degrade virtually all the components of the extracellular matrix and are likely to play a role in remodeling of endometrial tissue during the normal menstrual cycle. Primary cultures of human endometrial stromal cells secreted a number of MMPs. MMP-1 (interstitial collagenase) and MMP-3 (stromelysin-1) were measured in culture medium by specific enzyme assays. Production of the enzymes did not correlate with the time of the menstrual cycle at which the tissue was collected. Identities of MMP-1 and MMP-3 were confirmed by Western blots, by comparison of mol wt with those of purified enzymes on casein zymography, and by inhibition of these activities with EDTA and 1,10-phenanthroline. Northern analysis demonstrated specific messenger ribonucleic acid for pro-MMP-1 and pro-MMP-3 in phorbol myristate acetate-stimulated stromal cells. Two gelatinases were detected by gelatin zymography: MMP-2 (gelatinase-A) was present in two forms (72 and 67 kilodaltons), and MMP-9 (gelatinase-B) was present as a homodimer with a mol wt of approximately 180 kilodaltons. MMP-9, but not MMP-2, secretion was stimulated by phorbol myristate acetate. All enzymes could be activated in vitro by (4-aminophenyl)mercuric acetate. Both interleukin-1 alpha and tumor necrosis factor-alpha stimulated the secretion of MMP-1, MMP-3, and MMP-9, but not MMP-2, from the cells in a concentration-dependent manner. MMP production by endometrial stromal cells has a potentially important role in the processes of menstruation and implantation.

Expression of messenger ribonucleic acid encoding matrix metalloproteinases and their tissue inhibitors is related to menstruation.

Matrix metalloproteinases (MMP) degrade components of the extracellular matrix and the balance between enzyme production and that of their specific tissue inhibitors (TIMPs) is likely to be crucial for menstruation. The temporal expression of messenger (m) RNA for proMMP-1 (interstitial collagenase), proMMP-3 (stromelysin 1), TIMP-1 and TIMP-2 has been examined by Northern analysis in 73 individually-dated endometrial tissue samples from normal women. Thirteen tissues expressed the mRNA for proMMP-3 and mRNA for proMMP-1 was detected in eleven of the same tissues. All of these tissues were from the menstrual (11) or perimenstrual (2) phases. No expression of mRNA for proMMP-1 or -3 was detected between cycle days 4 and 26. By contrast, mRNA for both TIMP-1 and TIMP-2 was detected in all tissue samples. The abundance of mRNA for TIMP-1 was significantly elevated in menstrual tissue compared with tissue from the rest of the cycle. TIMP-2 mRNA expression displayed considerable variability between individual tissues but mean abundance was also higher in menstrual tissue. Menstruation is therefore associated with a perturbation of the balance between the expression of MMPs and their tissue inhibitors which could lead to tissue degradation.

Expression of the chemokines, monocyte chemotactic protein (MCP)-1 and MCP-2 in endometrium of normal women and Norplant users, does not support a central role in macrophage infiltration into endometrium.

The endometrium contains many leukocytes, including macrophages, the numbers varying with the time of the menstrual cycle and being maximal peri-menstrually. The long-acting progestogenic contraceptive Norplant, has a high rate of discontinuation due to uterine bleeding; this is associated with large numbers of endometrial macrophages. Monocyte chemotactic proteins (MCP) act to recruit and activate monocytes into sites of inflammation. This study compared the cellular localization of endometrial MCP-1 and MCP-2 across the normal menstrual cycle and in users of Norplant. Both MCP-1 and MCP-2 were present in normal endometrium, but with very different patterns of cellular location and considerable variability between individuals. MCP-1 of epithelial origin was present in 77% of tissues, while stromal staining was present in 52% and vascular staining in 34% of samples. MCP-1 was also released from both epithelial and stromal cells in culture. MCP-2 staining was predominantly epithelial and was found in 52% of tissues while stromal staining was present in only 3/56 samples. Vascular staining of MCP-2 was found in 2/56 samples. The epithelial staining was mostly punctate and sometimes within uterine secretions. No correlation of staining for MCP-1 or -2 with the phase of the cycle was found in any cellular compartment. Very little immunoreactive MCP-1 or MCP-2 was detected in endometrium from Norplant users regardless of morphological subtype. These distributions do not support a role for either MCP-1 or MCP-2 in the migration of macrophages into the endometrium and suggest that these cytokines may have other functions in this tissue.

Progesterone analogues similarly modulate endometrial matrix metalloproteinase-1 and matrix metalloproteinase-3 and their inhibitor in a model for long-term contraceptive effects.

Matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) are involved in normal menstruation, while MMP-1 and MMP-3 production by human endometrial stromal cells (HESCs) is repressed in vitro by progesterone. We postulated that the repression by synthetic progestins of MMP production from HESCs may not be fully maintained in the long term, and that this may account for the disturbed uterine bleeding patterns in women using long-acting progestins. In this study, a long-term HESC culture model was established to compare the effects of natural progesterone and a number of synthetic analogues (ORG2058, medroxyprogesterone acetate, norethindrone acetate, levonorgestrel and drospirenone) on the production by these cells of MMP-1 and MMP-3 and TIMP-1. Zymographic and enzyme-linked immunosorbent analysis of culture medium after 2 weeks showed that both natural progesterone and all of the synthetic progestins tested maintained a significant inhibition of MMP-1 and MMP-3 production. Production of mRNA for MMP-1 and MMP-3 was also suppressed by all progestins, while TIMP production was increased. Thus, menstrual bleeding disturbances which occur during the use of synthetic progestins is not likely to result directly from changes in the effect of long-term progestin exposure on MMP-1 or MMP-3 or TIMP-1 production by HESCs.

Modulation of production of matrix metalloproteinases from ovine endometrial cells by ovine trophoblast interferon.

Ovine trophoblast interferon modulates the secretion of a number of proteins by ovine endometrium, but only one of these proteins has so far been identified. We examined the effects of trophoblast interferon on the secretion of matrix metalloproteinase-1, -2 and -3 by cultured ovine endometrial cells and determined whether they are mediated via effects on prostaglandin synthesis. Both ovine trophoblast interferon (30 ng ml-1) and human recombinant interferon alpha (50 U ml-1) inhibited the production of latent matrix metalloproteinase-1 and -3 (P < 0.05), as measured by enzyme assays, but had no effect on the secretion of latent matrix metalloproteinase-2. These inhibitory effects were not overcome by PGE2 or PGF2 alpha (each 10 mumol l-1) either alone or in combination. Indomethacin (12 mumol l-1) similarly inhibited the production of latent matrix metalloproteinase-1 and -3, but production was partially restored by adding the prostaglandins either singly or in combination. PGE2 and PGF2 alpha together had no effect on enzyme production. These data were confirmed by gelatin and casein zymography. Northern analysis showed a 4.5-fold increase in the abundance of specific mRNA for latent matrix metalloproteinase-1 following treatment of cells with phorbol myristate acetate, but a marked decrease following interferon treatment. Thus, ovine trophoblast interferon inhibits the production of the latent forms of matrix metalloproteinase-1 and -3 by ovine endometrial cells, and this is independent of its effect on prostaglandin production.

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.

Progesterone inhibits activation of latent matrix metalloproteinase (MMP)-2 by membrane-type 1 MMP: enzymes coordinately expressed in human endometrium.

Matrix metalloproteinases (MMP) have specific spatial and temporal expression patterns in human endometrium and are critical for menstruation. Expression and activation mechanisms for proMMP-2 differ from other MMPs; in many cells proMMP-2 is specifically activated by membrane-type (MT)-MMPs. We examined the expression and localization of proMMP-2, MT1-MMP, and MT2-MMP in human endometrium across the menstrual cycle; and we examined the expression of MT1-MMP and activation of proMMP-2 in cultured endometrial stromal cells and their regulation by progesterone. MMP-2 was immunolocalized in 25 of 32 endometrial samples in all cellular compartments but with greatest intensity in degrading menstrual tissue. MT1-MMP mRNA was present throughout the cycle, and immunoreactive protein was detected in 24 of 32 samples, with the strongest staining in subsets of macrophages, neutrophils, and granular lymphocytes (but not mast cells or eosinophils) during the menstrual, mid-proliferative and mid-secretory phases. Patchy epithelial staining and staining of decidual cells, often periglandular in menstrual tissue, were also seen. MT2-MMP was more widespread than MT1-MMP without apparent cyclical variation and with maximal intensity in glandular epithelium. Cultured endometrial stromal cells released proMMP-2, and progesterone treatment significantly reduced the percentage level of its active (62 kDa) form (22.5 +/- 1.8% vs. 3.0 +/- 1.3%, without and with treatment, respectively, mean +/- SEM, P < 0.0001). This activation was blocked by a specific MMP inhibitor and restored following inhibitor removal. Progesterone also attenuated cell expression of MT1-MMP mRNA. We postulate that MT1-MMP activates proMMP-2 in endometrium, this activity being increased at the end of the cycle when progesterone levels fall, thus contributing to menstruation.

Tissue inhibitors of metalloproteinases in endometrium of ovariectomized steroid-treated ewes and during the estrous cycle and early pregnancy.

Tissue inhibitors of metalloproteinases (TIMPs) have an important role in remodeling of tissues and are likely to be implicated in uterine function, including embryo implantation and placentation. Expression of mRNA for TIMP-1 and TIMP-2 was examined by Northern analysis of endometrial RNA derived from steroid-treated ovariectomized ewes and from intact ewes during the estrous cycle and early pregnancy. Expression of mRNA for TIMP-1 (transcript size 0.9 kb), high in ovariectomized ewes, was substantially reduced by estrogen and to a lesser extent by progesterone. In cyclic and pregnant animals, abundance remained low until Day 10 and then increased, with high abundance continuing to Day 20 in the pregnant animals. Two transcripts for TIMP-2 were detected in ovine tissues--the 3.5-kb transcript and, in greater abundance, the 1.0-kb transcript. In ovariectomized ewes, endometrial abundance of both transcripts was low, and it decreased following estrogen treatment but was stimulated by progesterone alone or progesterone in the presence of estrogen. Abundance of TIMP-2 mRNA increased from Day 4 to Day 14 of the cycle. During early pregnancy, expression of the 1.0-kb transcript increased from Day 4 to Days 12-14 and was maintained at a high level to Day 20, whereas the 3.5-kb transcript decreased after Day 14 to very low levels by Day 20. In contrast with this pattern of regulated expression of TIMP, mRNA for proMMP-1 and for proMMP-3 was not detectable in any of the same tissues by Northern analysis. TIMP-1 protein was immunolocalized to both epithelium and stroma of intact endometrium, and the intensity of immunostaining was correlated with mRNA levels. TIMP-1 was secreted by both epithelial and stromal cells in primary culture, and its identity was confirmed by Western analysis, while reverse zymography demonstrated TIMP-1 and TIMP-2 along with a putative ovine TIMP-3 in the culture medium from both cell types. The precise role of TIMP in the endometrium remains to be established.

A potential molecular mechanism for regulating pre-mRNA splicing of implantation-related genes through unique uterine expression of splicing factor SC35 in women and rhesus monkeys.

Splicing factor SC35 is an essential component of the spliceosome, the cellular apparatus that removes introns from pre-mRNA to provide alternatively spliced isoforms. Many proteins associated with development of uterine receptivity and embryo implantation are present as isoforms, the tissue-specific expression of which may be regulated through alternative splicing. SC35 was identified as being increased at implantation sites during early pregnancy in mice. However, the present study has demonstrated that SC35 is present in human and rhesus monkey endometrium, that the protein is increased during the secretory phase of the oestrous cycle compared with the proliferative phase in both these primates and that it is present in a distinct pattern within the nucleus of both epithelial and stromal cells, as well as in cells of the vasculature. Both the intensity of immunoreactive protein and the proportion of cells that stain for SC35 alter with the phase of the oestrous cycle. A very precise expression pattern of SC35 (both protein and mRNA) was seen during early placentation in rhesus monkeys. At implantation sites between day 24 and day 35 of early pregnancy, SC35 was expressed strongly in cytotrophoblasts within the trophoblastic shell, in syncytiotrophoblast at the periphery of the cell column and in both cytotrophoblast and syncytiotrophoblast in the floating villi. In the adjacent maternal decidua, expression of SC35 was weak. These results indicate a role for SC35 in preparation of a receptive uterus, in the provision of secreted proteins to support blastocyst development and in trophoblast invasion.

Identification of monoclonal nonspecific suppressor factor beta (mNSFbeta) as one of the genes differentially expressed at implantation sites compared to interimplantation sites in the mouse uterus.

Successful implantation requires synchronous development of and active dialogue between the maternal endometrium and the implanting blastocyst. While it is well established that appropriate maternal steroid hormones are essential for endometrial preparation for implantation, the molecular events at the actual site of implantation are still little understood. The aims of our studies were to identify genes explicitly expressed or repressed at the sites of implantation by utilising RNA differential display (DDPCR), and to establish the roles of these genes in the implantation process in a mouse model. Ten bands unique in implantation sites compared to interimplantation sites were identified by DDPCR and subsequently confirmed by Northern blotting. One of these bands contained a cDNA fragment that was highly homologous to mouse monoclonal nonspecific suppressor factor beta (MNSFbeta) or Fau. The full cDNA sequence of this gene, obtained by screening a lambdagt11 cDNA library, was essentially the same as MNSFbeta, except that it had much longer 5' untranslated region. Interestingly, both Northern and immunohistochemical analysis showed that the expression of this gene was much lower in implantation sites compared to interimplantation sites on day 4.5 of pregnancy, when embryos first attach to the uterus and initiate implantation, and on day 5.5, when implantation has advanced. These results suggest a role for MNSF during implantation and early pregnancy, possibly through regulating the proliferation and/or differentiation of uterine stromal cells. It may also be involved in the selective production of TH2-type cytokines in implantation sites to regulate the immune system at the maternal-fetal interface.