Cells Of Endometrium Research Articles

Endometrium from women with and without endometriosis, and peritoneal, ovarian and bowel endometriosis, show different c-kit protein expression

Background: Endometriosis is defined by the presence of endometrium outside the uterus. Changes in the expression of the proto-oncogene c- kit are associated with aggressive behaviour of both benign and malignant tumours, but there are few data on its c- kit expression in endometriosis. Here we examined c-kit expression in endometrium and endometriotic tissue. Methods: Immunohistochemistry was used for qualitative and semi-quantitative (mean ± S.D. positive cells) analysis of c-kit expression in endometrium from women with ( n = 9) and without endometriosis ( n = 18), and in peritoneal ( n = 20), ovarian ( n = 20) and colorectal endometriosis ( n = 20). Results: Semi-quantitative c-kit expression was higher in endometrial glandular cells from women with endometriosis than from women without endometriosis (15.0 ± 14.6% versus 3.9 ± 7.4%, p = 0.01). No difference in c-kit expression was found in qualitative analysis and according to the phase of the menstrual cycle. C-kit expression values in peritoneal, ovarian and colorectal endometriosis were 2.0 ± 3.8%, 2.0 ± 4.1% and 21.7 ± 18.4%, respectively. Qualitative and semi-quantitative c-kit expression was higher in colorectal endometriosis than in peritoneal and ovarian endometriosis ( p < 0.001); no difference was found between ovarian and peritoneal endometriosis. No c-kit expression was detected in stromal cells of either endometrium or endometriotic tissue. Conclusion: These results suggest that the c-kit/stem cell factor axis is involved in the pathogenesis of endometriosis. Strong c-kit protein expression was associated with invasive endometriotic lesions.

Expression of Human Prostaglandin Transporter in the Human Endometrium across the Menstrual Cycle

Prostaglandins (PGs) are important regulators of reproductive function. The mechanism by which PGs are transported across the biological membrane is a new emerging field of investigation. Prostaglandin transporter (PGT) has been identified as a functional PG carrier. The aim of our study was to outline the expression of PGT in the human endometrium across the menstrual cycle. Quantitative RT-PCR showed human PGT (hPGT) expression to be strong in the proliferative and early secretory phases and low in the middle to late secretory phase. Northern blot analysis revealed hPGT mRNA transcript of 4 kb in the human endometrium. A peptide-directed polyclonal antibody was generated in rabbits against the 22 amino acids forming the C terminus of hPGT. Antibody specificity was demonstrated by Western blot. Immunoblots of endogenous hPGT in the human endometrium revealed a 70-kDa protein in endometrial cells. Endometrial biopsies collected across the menstrual cycle were used to assess hPGT protein expression by immunohistochemistry. hPGT was immunolocalized to luminal, glandular epithelial, and stromal cells. Because it was observed at the mRNA level, semiquantitative analysis showed a higher protein expression in proliferative and early secretory phases than in the mid-late secretory phase. In conclusion, our study revealed that hPGT expression is modulated in epithelial and stromal cells of the human endometrium at both mRNA and protein levels during the menstrual cycle. These findings support a role for hPGT as an important new player in the regulation of PG action in the human endometrium.

Activity of alkaline and acidic phosphatase in glandular cells of uterine endometrium of puerperal ewes after exposure to polychlorinated biphenyls

The study is focused on the observation of alkaline and acidic phosphatase activity in the glandular cells of uterine endometrium in puerperal ewes after exposure to polychlorinated biphenyls. Ewes of Slovak merino breed (n=25) divided into 2 groups were included in the experiment. The animals in the experimental group (n=14) and control group (n=11) were euthanised on day 17, 25 and 34 postpartum. The ewes in the experimental group were given per os capsules of the chemical preparation Delor 105 of domestic proveniance containing polychlorinated biphenyls (PCB) for a period of 30 days. This preparation is equivalent to the foreign preparation Aroclor 1254. A dose of 100 µg/kg of Delor 105 was given to the animals of the experimental group. These animals were euthanised on day 17 postpartum (n=4) i. e. 5 days from the end of a 30-day period of application; on day 25 postpartum (n=5) i.e. 17 days from the last application of PCB; on day 34 postpartum (n=5), which was equivalent to day 28 from the last application. The ewes from the control group were euthanised on day 17 (n=3), day 25 (n=4) and on day 34 (n-4) postpartum. When evaluating alkaline phosphatase (ALP) activity in the glandular cells of the endometrium in the control group, a statistically significant increase (P<0.01) was observed on day 25 and on day 34 (P<0.001) compared to day 17 postpartum. No statistically significant differences in alkaline phosphatase (ALP) activity were observed (P>0.05) in the experimental group. The mean values of its activity in the observed period were below the level of values of day 17 in the control group. Acidic phosphatase activity in the glandular cells of the ewes' endometrium showed a statistically conclusive increase between day 17 and day 25 as well as day 34 postpartum (P<0.001). Acidic phosphatase density in the experimental group of ewes showed no statistically marked change (P>0.05) at the observed intervals postpartum. The discussion is focused on PCB effect on the activity of alkaline and acidic phosphatase in the glandular cells of the endometrium of ewes in the puerperal period.

Changes in Immune Cell Distribution and IL‐10 Production are Regulated through Endometrial IP‐10 Expression in the Goat Uterus

Changes in distribution or redistribution of immune cells are required for the establishment and maintenance of pregnancy, but these changes during early pregnancy have been poorly understood in the ruminant ungulates. Expression of a chemokine, interferon-gamma (IFN-gamma)-inducible protein 10 kDa (IP-10, CXCL10), was identified in the endometrium of pregnant goats. Population and/or distribution of endometrial immune cells and their cytokine productions could be regulated by IP-10 during the period of pregnancy establishment. Using reverse transcriptase-polymerase chain reaction (RT-PCR), expression of IP-10, IFN-gamma, tumor necrosis factor-alpha, interleukin-10 (IL-10), CXCR3 mRNA and leukocyte cell surface markers, CD4, CD8, CD11b and CD45 mRNA during the caprine early pregnancy was investigated. The ability of IP-10 to stimulate peripheral blood mononuclear cells (PBMCs) migration was demonstrated using a chemotaxis assay. Changes in migration of PBMCs' immune cell population and cytokine expressions with IP-10 stimulation were investigated using flow cytometry and RT-PCR respectively. Levels of IP-10, IL-10, CD4 and CD11b mRNA, and the number of CD4 and CD11b positive cells in pregnant goat endometrium were higher than those of cyclic goat endometrium. Migration of PBMCs was stimulated by recombinant caprine IP-10, and the effect was significantly reduced by neutralization with the use of an anti-IP-10 antibody. In the flow cytometric and RT-PCR analyses, migrated cells stimulated by IP-10 increased the expression of IL-10 and CD11b mRNA. Furthermore, IP-10 could stimulate the expression of IL-10 mRNA from PBMCs. Endometrial chemokine IP-10 could regulate IL-10 production by resident and possibly migrated cells expressing CD11b, probably natural killer cells, and these changes may result in immune environments of the uterus suitable for conceptus implantation in ruminants.

286. The role of uterine natural killer cells in causing irregular bleeding in HT users

Menopausal hormone therapy (HT) causes irregular bleeding in up to 60% of user. This is extremely unpopular with patients, and commonly leads to invasive and expensive investigations to rule out underlying pelvic pathology. In most cases no cause is found. The aim of this study was to further elucidate the mechanisms of vascular fragility. Uterine NK cells are known to increase vascular fragility during the normal menstrual cycle. We hypothesise that HT is associated with an increase in uterine natural killer (uNK) cells. Eighty six endometrial biopsies were obtained from 59 postmenopausal users of continuous combined HT. Uterine NK cells were identified using immunohistochemistry as being CD56+. Image analysis was used to identify absolute number of CD56+ cells and their distribution within the stroma. Endometrial histology was classified using Noyes criteria. A statistically significant increase in endometrial uNK cell density was observed in HT users compared to postmenopausal women not using HT (P &lt; 0.001). uNK cell populations were more marked in biopsies taken during a bleeding episode compared to those HT users with amenorrhoea (P = 031). uNK cells are a major regulator of endometrial vascular integrity and are known to be disrupted in irregular bleeding with progestin only contraceptives. This is the first study to report the presence of uNK cells in postmenopausal endometrium and the first to report a significant association between bleeding patterns and uNK cell density. We postulate that HT induces an increase in endometrial uNK cell populations and that their presence stimulates endometrial vascular fragility leading to bleeding.

Regulation of Interleukin-8 Expression in Human Endometrial Endothelial Cells: A Potential Mechanism for the Pathogenesis of Endometriosis

The elevation of the proinflammatory chemoattractant cytokine levels in ectopic and eutopic endometrium of endometriosis implies an inflammatory basis for this disease. The relationship between endothelial cells and leukocytes is likely to be important in the regulation of inflammatory mediators of endometriosis. The aim of this study was to describe the temporal and spatial expression of IL-8 in human endometrial endothelial cells (HEEC) in vivo and to compare the in vitro regulation of IL-8 expression by sex steroids in HEEC from women with or without endometriosis. Eutopic endometrial tissues and endometriosis implants were grouped according to menstrual cycle phase and examined by immunohistochemistry for IL-8 expression. Endothelial cells of endometriotic implants expressed higher IL-8 immunoreactivity compared with endothelial cells of eutopic endometrium from women with or without endometriosis (P < 0.02). For in vitro studies, HEEC were isolated from women with or without endometriosis and grown to preconfluence. The purity of cultured HEEC (90-95%) was confirmed by immunocytochemistry using endothelium-specific markers, CD31 and CD146. The effects of estradiol (5 x 10(-8) m), progesterone (10(-7) m), or both on IL-8 mRNA and protein levels were analyzed by RT-PCR and ELISA, respectively. Sex steroids reduced the expression of IL-8 mRNA and protein in HEEC from women without endometriosis. In contrast, both estradiol and progesterone stimulated IL-8 mRNA and protein expression in HEEC from women with endometriosis. We postulate that the stimulation of chemokine expression by sex steroids in HEEC of women with endometriosis may play a role in the inflammatory aspect of this disease.

Cyclooxygenase-2 expression in deep endometriosis and matched eutopic endometrium

To identify any relationship between cyclooxygenase-2 expression and the intensity of severe, endometriosis-related dysmenorrhea. Prospective study. University hospital. Patients with deep endometriosis. During surgery, paired samples of tissue representing deep endometriosis and eutopic endometrium were obtained from 46 patients. Control endometrial tissue samples were obtained from 34 fertile women who underwent laparoscopic tubal ligation or reversal of tubal sterilization. Pain assessment for dysmenorrhea was done with a 10-point linear analogue scale. The percentage of surface immunostained for Cox-2 was determined by an immunohistochemical technique. Relationships between pain score for dysmenorrhea and Cox-2 expression were analyzed. Cox-2 expression was significantly higher in eutopic endometrial stromal cells from patients with deep endometriosis than in stroma from controls during the early, mid, and late secretory phases. In endometriosis patients, Cox-2 expression in eutopic endometrial stromal cells was significantly higher in women with more severe dysmenorrhea (pain score > or =7 vs. <7) during early and mid secretory phases. Elevated Cox-2 expression in stromal cells in eutopic endometrium from patients with deep endometriosis may play a role in severe, endometriosis-related dysmenorrhea.

Expression of interleukin-11 receptor α and interleukin-11 protein in the endometrium of normal fertile women and women with recurrent miscarriage

Interleukin-11 (IL-11) is a member of the IL-6 family of cytokines. Previous studies have suggested that IL-11 may play a role in human endometrial function. In this study, we have used immunocytochemistry to compare endometrial IL-11Rα and IL-11 expression in precisely timed peri-implantation biopsies from 9 normal fertile women and 16 recurrent miscarriage (RM) women. Immunocytochemistry was semi-quantified by obtaining an H-score value, which showed increased expression of both IL-11 and IL-11Rα in epithelial cells compared to stromal cells in all biopsies. There was a significant ( P < 0.01) reduction in epithelial cell IL-11, but not stromal cell IL-11, expression in endometrium from RM women compared to normal fertile women. There were no significant differences in expression of IL-11Rα protein in both stromal and epithelial cells in endometrium from the two groups of women. This work shows the presence of IL-11 and IL-11Rα within the endometrium of RM women during the peri-implantation period. The decreased expression of IL-11 in epithelial endometrium in RM women suggests that this cytokine may play a role in preventing miscarriage.

Modulation of vascular endothelial growth factor and thymidine phosphorylase in normal human endometrial stromal cells

To study the regulation of vascular endothelial growth factor (VEGF) and thymidine phosphorylase (TP), two angiogenic factors, in cultured endometrial stromal cells. The effects of steroids, E2, and P, the gonadotropin, hCG, and hypoxia were investigated. Quantitative experimental study. Academic medical department. Women undergoing hysterectomy for benign causes. Endometrial cells were collected from subjects and cultured. The secretion of VEGF in supernatant media and the numbers of cells expressing VEGF or TP. Estradiol increased VEGF secretion and the number of cells that contained VEGF and TP, and those effects were inhibited by hCG. Human chorionic gonadotropin alone could increase number of cells that expressed VEGF and TP. Hypoxia increased both VEGF secretion and number of cells containing VEGF and TP. Progesterone had no observed effect on VEGF secretion or number of VEGF- or TP-containing cells. Vascular endothelial growth factor and TP are present in stromal cells of normal endometrium, and E2 may interact with gonadotropins to regulate angiogenic compounds to modulate stromal functioning.

Pregnancy and Bovine Somatotropin in Nonlactating Dairy Cows: II. Endometrial Gene Expression Related to Maintenance of Pregnancy

The objective was to evaluate the effects of pregnancy and bovine somatotropin (bST) on endometrial gene and protein expression related to maintenance of pregnancy in nonlactating dairy cows at d 17. In endometrial tissues, treatment with bST increased the steady state concentration of oxytocin receptor (OTR) mRNA; bST-treated cyclic (bST-C) cows had greater OTR mRNA than bST-treated pregnant (bST-P) cows. Estradiol receptor α (ERα) mRNA was reduced in bST-P cows compared with control P and C (no bST) cows. Western blotting revealed that pregnancy decreased the abundance of ERα protein, and bST stimulated an increase in ERα protein in C and P cows. Treatment with bST increased steady state concentrations of progesterone receptor (PR) mRNA. No differences were detected in steady state mRNA concentrations of prostaglandin H synthase-2 (PGHS-2), prostaglandin E synthase, and prostaglandin F synthase due to pregnancy or bST treatment. However, PGHS-2 protein was increased in response to pregnancy and bST treatment. Immunostaining indicated that P decreased ERα protein in luminal epithelium and increased PR protein in epithelial cells of the uterine glands. The PR protein response in the glands was less in bST-P cows than in P cows. In the stromal layer of the endometrium, bST decreased PR protein abundance in C and P cows. The PGHS-2 protein was localized exclusively in the luminal epithelium cells of endometrium and was increased in P cows. In conclusion, distinctly different mRNA and protein responses were detected between C and P cows related to prostaglandin biosynthesis, and bST-induced changes may potentially impact mechanisms associated with maintenance of pregnancy in nonlactating cows.

Interaction of stress-activated protein kinase-interacting protein-1 with the interferon receptor subunit IFNAR2 in uterine endometrium.

During early pregnancy in ruminants, a type I interferon (IFN-tau) signals from the conceptus to the mother to ensure the functional survival of the corpus luteum. IFN-tau operates through binding to the type I IFN receptor (IFNR). Here we have explored the possibility that IFNAR2, one of the two subunits of the receptor, might interact with hitherto unknown signal transduction factors in the uterus that link IFN action to pathways other than the well established Janus kinase-signal transducer and activator of transcription pathways. A yeast two-hybrid screen of an ovine (ov) endometrial cDNA library with the carboxyl-terminal 185 amino acids of ovIFNAR2 as bait identified stress-activated protein kinase-interacting protein 1 (ovSin1) as a protein that bound constitutively through its own carboxyl terminus to the receptor. ovSin1 is a little studied, 522-amino acid-long polypeptide (molecular weight, 59,200) that is highly conserved across vertebrates, but has identifiable orthologs in Drosophila and yeast. It appears to be expressed ubiquitously in mammals, although in low abundance, in a wide range of mammalian tissues in addition to endometrium. Sin1 mRNA occurs in at least two alternatively spliced forms, the smaller of which lacks a 108-bp internal exon. ovSin1, although not exhibiting features of a membrane-spanning protein, such as IFNAR2, is concentrated predominantly in luminal and glandular epithelial cells of the uterine endometrium. When ovSin1 and ovIFNAR2 are coexpressed, the two proteins can be coimmunoprecipitated and colocalized to the plasma membrane and to perinuclear structures. Sin1 provides a possible link among type I IFN action, stress-activated signaling pathways, and control of prostaglandin production.

Follistatin-related gene expression, but not follistatin expression, is decreased in human endometrial adenocarcinoma.

Activin A is a multifunctional growth and cell differentiation factor produced by normal endometrium, and secreted in high amounts by endometrial adenocarcinoma. In the present study we evaluated the expression of two inhibitory activin A ligands, follistatin and follistatin-related gene (FLRG), in endometrial adenocarcinoma and in age-matched healthy human endometrium. Atropic menopausal (n=13) and tumoral (n=9 adenocarcinoma) tissues were processed to evaluate mRNA expression levels (by semiquantitative RT-PCR) and peptide localization (by immunohistochemistry). Differences were evaluated by the unpaired t-test and assumed to be statistically significant when P<0.05. Both control and tumoral endometrial samples express and localize follistatin and FLRG. However, whereas follistatin mRNA expression did not differ significantly, FLRG was significantly lower in endometrial adenocarcinoma than in healthy endometrial specimens (P<0.0001). With respect to the localization of proteins, follistatin was immunolocalized in endometrial epithelial and vascular cells both in tumoral and healthy endometrium without any significant difference in intensity. Nuclear and cytoplasmic FLRG immunolocalization was seen in glands, and only nuclear immunolocalization was found in stroma and vessels of healthy endometrium. FLRG was weakly immunostained in endometrial adenocarcinoma. Whilst follistatin expression is unchanged, FLRG is down-regulated in endometrial carcinoma. As activin A is a differentiation factor of human endometrium, the present findings support an imbalance between increased activin A and decreased FLRG expression in endometrial cancer, so that the failure of the activin A pathway through FLRG may be pivotal in endometrial tumorigenesis.

Expression of nuclear factor-kappaB in mouse uterus during peri-implantation.

To investigate the expression of the subunit p65 of NF-kappaB and inhibitor kappa B alpha (IkappaBalpha) in mouse uterus during peri-implantation, thereby investigating whether transient activation of nuclear factor-kappaB (NF-kappaB) takes place during embryo implantation in mice. Immunohistochemical technique was used to examine the expression and localization of p65 in endometrium or deciduas, and Western blot analysis was employed to detect the levels of IkappaBalpha protein in mouse endometrium or deciduas. P65 protein was detected in stromal cells, epithelial cells of endometrium as well as in myometrium. Staining was predominately seen in the cytoplasm of the cells. Staining intensity for p65 was stronger in the epithelial compartment than the stromal compartment and myometrium. Staining intensity increased slightly during pregnancy, and it reached a high level on pregnancy day 5 and day 8. In contrast to p65, the level of IkappaBalpha protein was lowest on pregnancy day 5 in all groups. Our results suggested that NF-kappaB may regulate embryo implantation by its transient activation in mice.

Decreased expression of killer cell inhibitory receptors on natural killer cells in eutopic endometrium in women with adenomyosis.

Increased expression of killer cell inhibitory receptors (KIRs) has been found on natural killer (NK) cells in peritoneal fluid in women with endometriosis. In this study, we tried to measure the expression of KIRs on NK and T cells in women with adenomyosis, in an attempt to find the possible role of KIRs in the development of adenomyosis. A total of 10 women with adenomyosis (study group) and 12 women with uterine myoma (control group) were included in this study. The expression of KIRs, including NKB1, GL183, EB6 and CD94, on NK and T cells in myometrium and endometrium was examined by flow cytometry. There was a decreased expression of NKB1 and GL183 on NK cells in the endometrium, but not in the myometrium, in women with adenomyosis. However, the expression of KIRs on T cells, either CD4+ or CD8+, was not different in either myometrium or endometrium between women with and without adenomyosis. The expression of KIRs on NK cells was decreased in eutopic endometrium in women with adenomyosis. It may be a compensatory effect in which the NK cytotoxicity is activated in order to eradicate the abnormal endometrial cells that might exit of the eutopic site of the endometrium.

Decreased level of the cell cycle regulator p27 and increased level of its ubiquitin ligase Skp2 in endometrial carcinoma but not in normal secretory or in hyperstimulated endometrium.

p27 is a cyclin-dependent kinase (CDK) inhibitor whose specific late G(1) destruction allows progression of the cell across the G(1)/S boundary. The protein is ubiquitinated by S-phase kinase-interacting protein-2 (Skp2) following its specific phosphorylation, and is subsequently degraded by the 26s proteasome. There is a direct relationship between low level of p27 and rapid proliferation occurring in several benign states and in many malignancies. In the glandular cells of the normal endometrium, the level of p27 is exceedingly low during the proliferative phase, whereas it is markedly increased during the secretory phase. The expression of p27 in endometrial carcinoma is very low but has been found to increase following treatment with progesterone. However, estrogen exposure is considered as a major risk factor in developing endometrial cancer. The implications of the high dose of estrogen and progesterone induced during IVF treatment are still unknown. We have examined the expression of p27 and Skp2 as well as of Ki67 proliferation marker by using endometrial extracts and cells from normal endometrium, from ovarian hyperstimulated patients, and from endometrial carcinoma patients. The expression of p27, Skp2 and Ki67 was found to be similar in both normal secretory endometrium and endometrium from ovarian hyperstimulated patients. In striking contrast, p27 is significantly lower while Skp2 and Ki67 are significantly higher in the endometrial carcinoma and in endometrium from the proliferative phase compared with their normal secretory counterpart tissue.

The differential expression of intercellular adhesion molecule-1 (ICAM-1) and regulation by interferon-gamma during the pathogenesis of endometriosis.

To establish an in vitro culture model of the endometrium and endometriotic lesions, and demonstrate the different expressions of intercellular adhesion molecule-1 (ICAM-1) in these lesions. Eight women with moderate to severe stages of endometriosis were enrolled. The specimens were collected from their eutopic endometrium, visually normal peritoneum, ovarian endometrioma and peritoneal endometriotic spots during the follicular phase of the menstrual cycle. ICAM-1 mRNA and protein were expressed by using reverse transcription-polymerase chain reaction, enzyme-linked immunosorbent assay, immunoblot, and immunocytochemistry. The results demonstrate that visually normal peritoneal cells and ovarian endometriomas of endometriotic patients can express high ICAM-1 mRNA. Normal peritoneal cells further expressed significant soluble ICAM-1 protein levels without cytokine stimulation. The eutopic endometrium expressed less soluble ICAM-1 protein, and ICAM-1 expressions increased in cultured stromal cells of eutopic endometrium, ovarian endometrioma, and peritoneal endometriotic spots under interferon-gamma (INF-gamma) stimulation. The ICAM-1 expression in visually normal peritoneal cells from women with endometriosis may play a role in the early implantation of peritoneal endometriosis. Peritoneal INF-gamma stimulation is significantly associated with ICAM-1 expression in endometriosis. Therefore, the differential expression and changes of ICAM-1 may be involved in the mechanism that can escape immunosurveillance and allow refluxed endometrial cells to spread and invade other location.

Cell type-specific expression of beta-carotene 15,15'-mono-oxygenase in human tissues.

We studied the cell type-specific expression of human beta-carotene 15,15'-mono-oxygenase (BCO1), an enzyme that catalyzes the first step in the conversion of dietary provitamin A carotenoids to vitamin A. Immunohistochemical analysis using two monoclonal antibodies against different epitopes of the protein revealed that BCO1 is expressed in epithelial cells in a variety of human tissues, including mucosa and glandular cells of stomach, small intestine, and colon, parenchymal cells in liver, cells that make up the exocrine glands in pancreas, glandular cells in prostate, endometrium, and mammary tissue, kidney tubules, and in keratinocytes of the squamous epithelium of skin. Furthermore, BCO1 is detected in steroidogenic cells in testis, ovary, and adrenal gland, as well as skeletal muscle cells. Epithelia in general are structures that are very sensitive to vitamin A deficiency, and although the extraintestinal function of BCO1 is unclear, the finding that the enzyme is expressed in all epithelia examined thus far leads us to suggest that BCO1 may be important for local synthesis of vitamin A, constituting a back-up pathway of vitamin A synthesis during times of insufficient dietary intake of vitamin A.

Expression and signalling characteristics of the corticotrophin-releasing hormone receptors during the implantation phase in the human endometrium.

Corticotrophin-releasing hormone (CRH) has been identified in several peripheral tissues, including the female reproductive organs. CRH is expressed in the placenta, myometrium, epithelial endometrium and the endometrial stromal cells at all phases of the menstrual cycle. Similarly, CRH receptors are present in pregnant and non-pregnant myometrium, placenta and endometrium. Putative roles of CRH in the endometrium include involvement in implantation, decidualisation and maintenance of pregnancy. In this study we sought to investigate in detail the CRH receptor repertoire expressed in the human endometrium and their signalling characteristics. Using RT-PCR we were able to demonstrate the expression of CRH receptor 1alpha (CRH-R1alpha) and CRH-R2alpha in the human endometrium. CRH-R1beta was present in 40% of endometrial cDNAs examined. No apparent expression of CRH-R2beta, CRH-R2gamma or any other CRH-R1 splice variants was detected. Chemical cross-linking studies with 125I-ovine CRH revealed that the endometrial CRH receptor has a molecular weight of 45 kDa. Using the non-hydrolysable photoreactive analogue [alpha-32P]GTP-azidoanilide and peptide antisera raised against G-protein alpha-subunits, we then studied coupling of endometrial CRH receptors to G proteins. Treatment of endometrial membranes with human CRH (100 nM) increased the labelling of Gq and Gs, but not Gi or Go. These results were supported by experiments in epithelial cells of the non-pregnant human endometrium in the secretory phase which showed that CRH induced increases in both cAMP and inositol trisphosphate levels. These results suggested that CRH may exert multiple effects in the human endometrium via distinct signalling cascades. These events are possibly mediated via different receptor subtypes.

Cell-specific expression of estrogen-responsive genes in the uteri of cyclic, early pregnant and ovariectomized ewes

A single physiological dose of estradiol up-regulates estrogen receptor-α(ER), progesterone receptor (PR), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), c-fos, cyclophilin, and actin mRNAs in the endometrium of ovariectomized ewes. Therefore, we hypothesized that these genes would be up-regulated by the preovulatory surge of estrogen which occurs on the evening of Day 15 in the estrous cycle of sheep. ER and PR mRNA concentrations increased between Day 15 and Day 1 in cyclic ewes in most endometrial epithelial cells, while GAPDH mRNA increased in epithelial and stromal cells in the deep endometrium. Day 15 pregnant ewes had lower expression of ER, PR, GAPDH, cyclophilin and actin genes. For ER and GAPDH mRNAs, the greatest reduction occurred in the superficial endometrium. Ovariectomized ewes demonstrated concentrations of ER, PR, and GAPDH mRNAs that were similar to those in the cyclic ewes. While concentrations of c-fos mRNA did not differ between groups, those of cyclophilin and actin mRNAs were lower in the pregnant and ovariectomized ewes. In conclusion, ER, PR and GAPDH gene expression rose during estrus in endometrial cells with the highest ER gene expression and were repressed in pregnant ewes in superficial endometrial cells with the greatest PR gene expression.

Immunohistochemical localization of the growth hormone in human endometrium and decidua.

Recent evidence of growth hormone (GH) receptor expression in rat endometrium and human myometrium have focused our attention on the role of the GH in endometrial development. We tested the expression of GH in the human endometrium throughout the menstrual cycle and during pregnancy. Immunohistochemical study was performed on endometrial specimens of fertile women in different periods of the menstrual cycle and in decidua of pregnant women. Glandular cells of the human endometrium were positive for GH in the mid and late luteal phase. Furthermore, the glandular cells of decidua showed intense staining for GH, while the stromal cells were negative. No immunostaining was expressed in the proliferative or early luteal phase. The intensity levels of staining for GH in decidual specimens were significantly higher than in glandular cells of secretory endometrium specimens (P < 0.01). The glandular cells of the human endometrium express GH from the late luteal phase throughout pregnancy in the decidual tissue. We suppose that GH plays an important role in blastocyst implantation.