Single cell RNA sequencing and lineage tracing confirm mesenchyme to epithelial transformation (MET) contributes to repair of the endometrium at menstruation

The human endometrium experiences repetitive cycles of tissue wounding characterised by piecemeal shedding of the surface epithelium and rapid restoration of tissue homeostasis. In this study, we used a mouse model of endometrial repair and three transgenic lines of mice to investigate whether epithelial cells that become incorporated into the newly formed luminal epithelium have their origins in one or more of the mesenchymal cell types present in the stromal compartment of the endometrium. Using scRNAseq, we identified a novel population of PDGFRb + mesenchymal stromal cells that developed a unique transcriptomic signature in response to endometrial breakdown/repair. These cells expressed genes usually considered specific to epithelial cells and in silico trajectory analysis suggested they were stromal fibroblasts in transition to becoming epithelial cells. To confirm our hypothesis we used a lineage tracing strategy to compare the fate of stromal fibroblasts (PDGFRa+) and stromal perivascular cells (NG2/CSPG4+). We demonstrated that stromal fibroblasts can undergo a mesenchyme to epithelial transformation and become incorporated into the re-epithelialised luminal surface of the repaired tissue. This study is the first to discover a novel population of wound-responsive, plastic endometrial stromal fibroblasts that contribute to the rapid restoration of an intact luminal epithelium during endometrial repair. These findings form a platform for comparisons both to endometrial pathologies which involve a fibrotic response (Asherman’s syndrome, endometriosis) as well as other mucosal tissues which have a variable response to wounding

The antiestrogen ICI 182,780 decreases the expression of estrogen receptor-alpha but has no effect on estrogen receptor-beta and androgen receptor in rat efferent ductules

BACKGROUND: The antiestrogen ICI 182,780 has been used successfully as an alternative experimental model for the study of estrogen action in the rodent adult male reproductive tract. Although ICI 182,780 causes severe alterations in testicular and efferent ductule morphology and function, the effects on the expression of estrogen and androgen receptors in the male have not been shown. METHODS: In the present study, adult male rats were treated with ICI 182,780 for 7 to 150 days, to evaluate the time-response effects of the treatment on the pattern of ERα, ERβ and AR protein expression in the efferent ductules. The receptors were localized using immunohistochemistry. RESULTS: ERα, ERβ and AR have distinct cellular distribution in the testis and efferent ductules. Staining for ERα is nearly opposite of that for ERβ, as ERα shows an increase in staining intensity from proximal to distal efferent ductules, whereas ERβ shows the reverse. Androgen receptor follows that of ERα. ICI 182,780 caused a gradual but dramatic decrease in ERα expression in the testis and efferent ductules, but no change in ERβ and AR expression. CONCLUSIONS: The differential response of ERα and ERβ proteins to ICI 182,780 indicates that these receptors are regulated by different mechanisms in the male reproductive tract

The effect of a sertoli cell-selective knockout of the androgen receptor on testicular gene expression in prepubertal mice

To unravel the molecular mechanisms mediating the effects of androgens on spermatogenesis, testicular gene expression was compared in mice with Sertoli cell-selective androgen receptor knockout (SCARKO) and littermate controls on postnatal d 10. Microarray analysis identified 692 genes with significant differences in expression. Of these, 28 appeared to be down-regulated and 12 up-regulated at least 2-fold in SCARKOs compared with controls. For nine of the more than 2-fold down-regulated genes, androgen regulation was confirmed by treatment of wild-type mice with an antiandrogen ( flutamide). Some of them were previously described to be androgen regulated or essential for spermatogenesis. Serine-type protease inhibitors were markedly overrepresented in this down-regulated subgroup. A time study (d 8-20), followed by cluster analysis, allowed identification of distinct expression patterns of differentially expressed genes. Three genes with a pattern closely resembling that of Pem, a prototypical an-drogen-regulated gene expressed in Sertoli cells, were selected for confirmation by quantitative RTPCR and additional analysis. The data confirm that the SCARKO model allows identification of novel androgen-regulated genes in the testis. Moreover, they suggest that protease inhibitors and other proteins related to tubular restructuring and cell junction dynamics may be controlled in part by androgens

Expression of oestrogen receptors, ERα, ERβ, and ERβ variants, in endometrial cancers and evidence that prostaglandin F may play a role in regulating expression of ERα

<p>Abstract</p> <p>Background</p> <p>Endometrial cancer is the most common gynaecological malignancy; risk factors include exposure to oestrogens and high body mass index. Expression of enzymes involved in biosynthesis of oestrogens and prostaglandins (PG) is often higher in endometrial cancers when compared with levels detected in normal endometrium. Oestrogens bind one of two receptors (ERα and ERβ) encoded by separate genes. The full-length receptors function as ligand-activated transcription factors; splice variant isoforms of ERβ lacking a ligand-binding domain have also been described. PGs act in an autocrine or paracrine manner by binding to specific G-protein coupled receptors.</p> <p>Methods</p> <p>We compared expression of ERs, progesterone receptor (PR) and cyclooxygenase-2 (COX-2) in stage 1 endometrial adenocarcinomas graded as well (G1), moderately (G2) or poorly (G3) differentiated (n ≥ 10 each group) using qRTPCR, single and double immunohistochemistry. We used endometrial adenocarcinoma cell lines to investigate the impact of PGF2α on expression of ERs and PR.</p> <p>Results</p> <p>Full length ERβ (ERβ1) and two ERβ variants (ERβ2, ERβ5) were expressed in endometrial cancers regardless of grade and the proteins were immunolocalised to the nuclei of cells in both epithelial and stromal compartments. Immunoexpression of COX-2 was most intense in cells that were ERα^neg/low. Expression of PR in endometrial adenocarcinoma (Ishikawa) cell lines and tissues broadly paralleled that of ERα. Treatment of adenocarcinoma cells with PGF2α reduced expression of ERα but had no impact on ERβ1. Cells incubated with PGF2α were unable to increase expression of PR mRNA when they were incubated with E2.</p> <p>Conclusion</p> <p>We have demonstrated that ERβ5 protein is expressed in stage 1 endometrial adenocarcinomas. Expression of three ERβ variants, including the full-length protein is not grade-dependent and most cells in poorly differentiated cancers are ERβ^pos/ERα^neg. We found evidence of a link between COX-2, its product PGF2α, and expression of ERα and PR that sheds new light on the cross talk between steroid and PG signalling pathways in this disease.</p

Intratubular germ cell neoplasia of the human testis:heterogeneous protein expression and relation to invasive potential

Testicular germ cell cancer develops from premalignant intratubular germ cell neoplasia, unclassified cells that are believed to arise from failure of normal maturation of fetal germ cells from gonocytes (OCT4(+)/MAGEA4(-)) into pre-spermatogonia (OCT4(-)/MAGEA4(+)). Intratubular germ cell neoplasia cell subpopulations based on stage of germ cell differentiation have been described, however the importance of these subpopulations in terms of invasive potential has not been reported. We hypothesized that cells expressing an immature (OCT4(+)/MAGEA4(-)) germ cell profile would exhibit an increased proliferation rate compared with those with a mature profile (OCT4(+)/MAGEA4(+)). Therefore, we performed triple immunofluorescence and stereology to quantify the different intratubular germ cell neoplasia cell subpopulations, based on expression of germ cell (OCT4, PLAP, AP2γ, MAGEA4, VASA) and proliferation (Ki67) markers, in testis sections from patients with preinvasive disease, seminoma, and non-seminoma. We compared these subpopulations with normal human fetal testis and with seminoma cells. Heterogeneity of protein expression was demonstrated in intratubular germ cell neoplasia cells with respect to gonocyte and spermatogonial markers. It included an embryonic/fetal germ cell subpopulation lacking expression of the definitive intratubular germ cell neoplasia marker OCT4, that did not correspond to a physiological (fetal) germ cell subpopulation. OCT4(+)/MAGEA4(-) cells showed a significantly increased rate of proliferation compared with the OCT4(+)/MAGEA4(+) population (12.8 versus 3.4%, P<0.0001) irrespective of histological tumor type, reflected in the predominance of OCT4(+)/MAGEA4(-) cells in the invasive tumor component. Surprisingly, OCT4(+)/MAGEA4(-) cells in patients with preinvasive disease showed significantly higher proliferation compared to those with seminoma or non-seminoma (18.1 versus 10.2 versus 7.2%, P<0.05, respectively). In conclusion, this study has demonstrated that OCT4(+)/MAGEA4(-) cells are the most frequent and most proliferative cell population in tubules containing intratubular germ cell neoplasia, which appears to be an important factor in determining invasive potential of intratubular germ cell neoplasia to seminomas

Identifying the role of androgens in endometrial function

The endometrium is a complex multicellular tissue that undergoes dynamic alterations under the control of ovarian-derived sex steroid hormones. During the proliferative phase of the human menstrual cycle, oestrogen induces proliferation of the endometrial epithelium while during the progesterone-dominated secretory phase, the endometrial stromal compartment differentiates in preparation for pregnancy. This differentiation event is termed decidualisation and it is accompanied by immune cell infiltration, vascular remodelling and secretion of cytokines and growth factors, as well as a newfound capacity of active steroid synthesis in the endometrium. Defective decidualisation has been described in several endometrial-associated disorders such as endometriosis, a pathology of ectopic endometrial tissue in the peritoneal cavity, often associated with infertility. Rodent models have been used for the investigation of endometrial physiology and pathology due to the similarity in uterine tissue architecture, appropriate endometrial responses to steroid hormones and the opportunity to inform cellular mechanisms using genetic manipulation. While the impact of 17β-oestradiol and progesterone on endometrial function have been extensively studied, androgens have only recently emerged as potent potential regulators of the endometrium, however, their impact on cell function has not been fully elucidated. The aims of this study were to: Identify the impact of androgens on endometrial function using a mouse model of steroid depletion (ovariectomy) followed by administration of the potent androgen dihydrotestosterone (DHT). Investigate the capacity of endometrial stromal cells to synthesise androgens during decidualisation using human primary endometrial stromal cells (hESCs) decidualised in vitro. Elucidate the decidualisation response of hESCs from women with endometriosis after modulation of androgen receptor (AR) function during decidualisation. Novel results obtained provided evidence of a role for androgens in inducing a trophic effect in the mouse uterus characterised by: pronounced endometrial epithelial proliferation, altered expression pattern of AR, changes in the expression of genes involved in cell-cycle progression and stromal-epithelial cross-talk. In addition, androgen treatment resulted in a striking and unexpected increase in the number of endometrial glands. Decidualisation of hESCs resulted in time-dependent changes in expression of the androgen synthesising enzymes AKR1C3 and 5α-reductase (accompanied by biosynthesis of both testosterone and DHT in a dynamic time-dependent manner). Notably, blocking of AR action arising from local androgen signalling during decidualisation of hESCs culminates in sub-optimal decidualisation as detected by the expression of the classical decidualisation markers IGFBP1 and PRL. Women with endometriosis are reported to exhibit defective decidualisation, which may be accompanied with infertility. Treatment of hESCs from women with endometriosis with an AR agonist (DHT) or antagonist (flutamide) during decidualisation resulted in striking differences in decidualisation response as demonstrated in a case-study approach. Taken together, these findings highlight novel roles of androgens in regulating endometrial function by impacting on cell proliferation, gland formation and decidualisation. These striking new findings have implications for endometrial disorders such as endometriosis. Future studies will focus on the use of selective androgen receptor modulators, a novel class of compounds, with tissue-selective actions and without the undesired side-effects of potent androgens. The use of AR modulators would benefit from a personalised medicine approach, instructed by patient profiling to direct therapeutic targeting of endometrial disorders

Conserved and divergent patterns of expression of DAZL, VASA and OCT4 in the germ cells of the human fetal ovary and testis-2

<p><b>Copyright information:</b></p><p>Taken from "Conserved and divergent patterns of expression of DAZL, VASA and OCT4 in the germ cells of the human fetal ovary and testis"</p><p>http://www.biomedcentral.com/1471-213X/7/136</p><p>BMC Developmental Biology 2007;7():136-136.</p><p>Published online 18 Dec 2007</p><p>PMCID:PMC2211489.</p><p></p>64 d; d, 16 wk). DAZL positive germ cells were rare in the 1st trimester (e, ovary 61 d; g, testis 64 d) but groups of cells ('nests', labelled N) with cytoplasmic staining were present in the 2nd trimester ovaries (f, 20 wk). During the 2nd trimester VASA protein was detected in the cytoplasm of female germ cells (i, 14 wk; j, 18 wk) throughout the ovary with the exception of the sub-epithelial layer. In the testes (k, 15 wk; l, 16 wk) VASA-positive germ cells were found in all cords

Conserved and divergent patterns of expression of DAZL, VASA and OCT4 in the germ cells of the human fetal ovary and testis-1

<p><b>Copyright information:</b></p><p>Taken from "Conserved and divergent patterns of expression of DAZL, VASA and OCT4 in the germ cells of the human fetal ovary and testis"</p><p>http://www.biomedcentral.com/1471-213X/7/136</p><p>BMC Developmental Biology 2007;7():136-136.</p><p>Published online 18 Dec 2007</p><p>PMCID:PMC2211489.</p><p></p>in those from the 2nd trimester. DAZL (33 Kd) was low/undetectable in 1st trimester ovaries (C) whereas it was detectable in ovarian samples from 2nd trimester and testicular samples from both 1st and 2nd trimester (D). OCT4 (42 Kd) was present in both ovaries (E) and testes (F) during both the 1st and 2nd trimester. The loading control in all cases was β-tubulin (51 Kd)

Expression of mRNAs for , and in 1st and 2nd trimester ovary and testis

<p><b>Copyright information:</b></p><p>Taken from "Conserved and divergent patterns of expression of DAZL, VASA and OCT4 in the germ cells of the human fetal ovary and testis"</p><p>http://www.biomedcentral.com/1471-213X/7/136</p><p>BMC Developmental Biology 2007;7():136-136.</p><p>Published online 18 Dec 2007</p><p>PMCID:PMC2211489.</p><p></p> Open columns, 1st trimester, shaded columns 2nd trimester. Concentrations of mRNA are all relative to that of in the same samples. * P < 0.001 vs 1st trimester. Mean ± sem, n = 5–18 per group