Probes for INS

Endocrine disrupting chemicals (EDCs) are raising concerns about adverse effects on fertility in women as they have been shown to disrupt steroidogenesis and ovarian function in animal studies, and they associate to reduced fertility in human cohort studies. However, there is a lack of information regarding mechanisms of action and effects in humans. Our study aims to identify molecular mechanisms of endocrine disruption using two well-known human EDCs, diethylstilbestrol (DES) and ketoconazole (KTZ), via controlled exposure studies in ovarian cell lines and human ovarian tissue culture in vitro. Ovarian cortical tissue slices obtained from tissue collected from Caesarean section (c-section) patients at Karolinska University Hospital was exposed to 10-9 M to 10-5 M KTZ and 10-10 M to 10-6 M DES in vitro for 6 days. Follicle survival and growth were studied using histology, steroid production by liquid-chromatography-mass spectrometry (LC-MS/MS), and tissue viability by cytotoxicity and fibrosis assays. RNA sequencing was performed on primary ovarian cells and ovarian granulosa cell cancer cell lines COV434 and KGN that were exposed for 24 hours to the same concentrations of DES and KTZ as the tissue culture. Selected potential biomarkers were validated using real-time quantitative polymerase chain reaction (RT-qPCR) in the cells, and by in situ RNA hybridization in exposed tissue. Significantly lower non-growing follicle densities (i.e. primordial, intermediary, and primary follicles) were observed in DES 10-10 M group compared to vehicle control. A decrease trend was also observed in DES high dose group and low level KTZ exposed group. On the other hand, slightly higher growing follicle density was shown in high level KTZ exposed group. Levels of pregnenolone and progesterone were significantly reduced in KTZ 10-5 M exposed group. RNA sequencing showed that 445 and 233 differential expressed genes (DEGs) (FDR< 0.1) were affected in DES and KTZ exposed group, respectively, in the cell culture. Gene set variation analysis (GSVA) showed that both DES and KTZ modulated MTORC1 signaling, which was critical for primordial follicle activation and steroidogenesis. We selected stear-oyl-CoA desaturase (SCD), a gene that was shown to involved in cholesterol homeostasis, oocyte maturation and steroidogenesis, for validation as a potential biomarker. Up-regulation of was confirmed in response to KTZ by PCR and RNAscope. In conclusion, DES and KTZ affected folliculogenesis and steroidogenesis in human adult ovarian cortex and SCD may serve as a potential biomarker in response to exposure. Further validation of this potential biomarker may help improve the existing testing guideline and subsequently, contributing to better regulation of chemical in our global market.

Opioid peptides are well-known modulators of the central control of reproduction. Among them, dynorphin coexpressed in kisspeptin (KP) neurons of the arcuate nucleus (ARC) has been thoroughly studied for its autocrine effect on KP release through κ opioid receptors. Other studies have suggested a role for β-endorphin (BEND), a peptide cleaved from the pro-opiomelanocortin precursor, on food intake and central control of reproduction. Similar to KP, BEND content in the ARC of sheep is modulated by day length and BEND modulates food intake in a dose-dependent manner. Because KP levels in the ARC vary with photoperiodic and metabolic status, a photoperiod-driven influence of BEND neurons on neighboring KP neurons is plausible. The present study aimed to investigate a possible modulatory action of BEND on KP neurons located in the ovine ARC. Using confocal microscopy, numerous KP appositions on BEND neurons were found but there was no photoperiodic variation of the number of these interactions in ovariectomized, estradiol-replaced ewes. By contrast, BEND terminals on KP neurons were twice as numerous under short days, in ewes having an activated gonadotropic axis, compared to anestrus ewes under long days. Injection of 5 μg BEND into the third ventricle of short-day ewes induced a significant and specific increase of activated KP neurons (16% vs. 9% in controls), whereas the percentage of overall activated (c-Fos positive) neurons, was similar between both groups. These data suggest a photoperiod-dependent influence of BEND on KP neurons of the ARC, which may influence gonadotropin-releasing hormone pulsatile secretion and inform KP neurons about metabolic status.

Greater than 25% of all men develop an inguinal hernia in their lifetime, and more than 20 million inguinal hernia repair surgeries are performed worldwide each year. The mechanisms causing abdominal muscle weakness, the formation of inguinal hernias, or their recurrence are largely unknown. We previously reported that excessively produced estrogen in the lower abdominal muscles (LAMs) triggers extensive LAM fibrosis, leading to hernia formation in a transgenic male mouse model expressing the human aromatase gene (Aromhum). To understand the cellular basis of estrogen-driven muscle fibrosis, we performed single-cell RNA sequencing on LAM tissue from Aromhum and wild-type littermates. We found a fibroblast-like cell group composed of 6 clusters, 2 of which were validated for their enrichment in Aromhum LAM tissue. One of the potentially novel hernia-associated fibroblast clusters in Aromhum was enriched for the estrogen receptor-α gene (Esr1hi). Esr1hi fibroblasts maximally expressed estrogen target genes and seemed to serve as the progenitors of another cluster expressing ECM-altering enzymes (Mmp3hi) and to upregulate expression of proinflammatory, profibrotic genes. The discovery of these 2 potentially novel and unique hernia-associated fibroblasts may lead to the development of novel treatments that can nonsurgically prevent or reverse inguinal hernias.

Perivascular mesenchymal cells (PMCs), which include pericytes, give rise to myofibroblasts that contribute to chronic kidney disease progression. Several PMC markers have been identified; however, PMC heterogeneity and functions are not fully understood. Here, we describe a novel subset of renal PMCs that express Meflin, a glycosylphosphatidylinositol-anchored protein that was recently identified as a marker of fibroblasts essential for cardiac tissue repair. Tracing the lineage of Meflin+ PMCs, which are found in perivascular and periglomerular areas and exhibit renin-producing potential, showed that they detach from the vasculature and proliferate under disease conditions. Although the contribution of Meflin+ PMCs to conventional α-SMA+ myofibroblasts is low, they give rise to fibroblasts with heterogeneous α-SMA expression patterns. Genetic ablation of Meflin+ PMCs in a renal fibrosis mouse model revealed their essential role in collagen production. Consistent with this, human biopsy samples showed that progressive renal diseases exhibit high Meflin expression. Furthermore, Meflin overexpression in kidney fibroblasts promoted bone morphogenetic protein 7 signals and suppressed myofibroblastic differentiation, implicating the roles of Meflin in suppressing tissue fibrosis. These findings demonstrate that Meflin marks a PMC subset that is functionally distinct from classic pericytes and myofibroblasts, highlighting the importance of elucidating PMC heterogeneity.

The glucocorticoid stress hormones affect brain function via high-affinity mineralocorticoid receptors (MRs) and lower-affinity glucocorticoid receptors (GRs). MR and GR not only differ in affinity for ligands, but also have distinct, sometimes opposite, actions on neuronal excitability and other cellular and higher-order parameters related to cerebral function. GR and MR messenger RNA (mRNA) levels are often used as a proxy for the responsiveness to glucocorticoids, assuming proportionality between mRNA and protein levels. This may be especially relevant for the MR, which because of its high affinity is already largely occupied at low basal (trough) hormone levels. Here we explore how GR and MR mRNA levels are associated with the expression of a shared target gene, glucocorticoid-induced leucine zipper (GILZ, coded by Tsc22d3) with basal and elevated levels of corticosterone in male mice, using in situ hybridization. Depending on the hippocampal subfield and the corticosterone levels, mRNA levels of MR rather than GR mostly correlated with GILZ mRNA in the hippocampus and hypothalamus at the bulk tissue level. At the individual cell level, these correlations were much weaker. Using publicly available single-cell RNA sequencing data, we again observed that MR and GR mRNA levels were only weakly correlated with target gene expression in glutamatergic and GABAergic neurons. We conclude that MR mRNA levels can be limiting for receptor action, but many other cell-specific and region-specific factors ultimately determine corticosteroid receptor action. Altogether, our results argue for caution while interpreting the consequences of changed receptor expression for the response to glucocorticoids.