Interplay of spermatogonial subpopulations during initial stages of spermatogenesis in adult primates.

The spermatogonial compartment maintains spermatogenesis throughout the reproductive lifespan. Single-cell RNA sequencing (scRNA-seq) has revealed the presence of several spermatogonial clusters characterized by specific molecular signatures. However, it is unknown whether the presence of such clusters can be confirmed in terms of protein expression and whether protein expression in the subsets overlaps. To investigate this, we analyzed the expression profile of spermatogonial markers during the seminiferous epithelial cycle in cynomolgus monkeys and compared the results with human data. We found that in cynomolgus monkeys, as in humans, undifferentiated spermatogonia are largely quiescent, and the few engaged in the cell cycle were immunoreactive to GFRA1 antibodies. Moreover, we showed that PIWIL4+ spermatogonia, considered the most primitive undifferentiated spermatogonia in scRNA-seq studies, are quiescent in primates. We also described a novel subset of early differentiating spermatogonia, detectable from stage III to stage VII of the seminiferous epithelial cycle, that were transitioning from undifferentiated to differentiating spermatogonia, suggesting that the first generation of differentiating spermatogonia arises early during the epithelial cycle. Our study makes key advances in the current understanding of male germline premeiotic expansion in primates.

The activation of M2 muscarinic receptor inhibits cell growth and survival in human epithelial ovarian carcinoma.

Ovarian cancer is the fifth leading cause of cancer-related deaths in females. Many ovarian tumor cell lines express muscarinic receptors (mAChRs), and their expression is correlated with reduced survival of patients. We have characterized the expression of mAChRs in two human ovarian carcinoma cell lines (SKOV-3, TOV-21G) and two immortalized ovarian surface epithelium cell lines (iOSE-120, iOSE-398). Among the five subtypes of mAChRs (M1-M5 receptors), we focused our attention on the M2 receptor, which is involved in the inhibition of tumor cell proliferation. Western blot analysis and real-time PCR analyses indicated that the levels of M2 are statistically downregulated in cancer cells. Therefore, we investigated the effect of arecaidine propargyl ester hydrobromide (APE), a preferential M2 agonist, on cell growth and survival. APE treatment decreased cell number in a dose and time-dependent manner by decreasing cell proliferation and increasing cell death. FACS and immunocytochemistry analysis have also demonstrated the ability of APE to accumulate the cells in G2/M phase of the cell cycle and to increase the percentage of abnormal mitosis. The higher level of M2 receptors in the iOSE cells rendered these cells more sensitive to APE treatment than cancer cells. The data here reported suggest that M2 has a negative role in cell growth/survival of ovarian cell lines, and its downregulation may favor tumor progression.

Regulation of Gdnf expression by retinoic acid in Sertoli cells.

Glial cell line-derived neurotrophic factor (GDNF) and retinoic acid (RA) are two molecules crucial for the regulation of the spermatogonial compartment of the testis. During the cycle of the seminiferous epithelium, their relative concentration oscillates with lower GDNF levels in stages where RA levels are high. It has been recently shown that RA negatively regulates Gdnf expression but the mechanisms behind are so far unknown. Here, we show that RA directly downregulates Gdnf mRNA levels in primary murine Sertoli cells through binding of RARα to a novel DR5-RARE on Gdnf promoter. Pharmacological inhibition and chromatin immunoprecipitation-quantitative polymerase chain reaction analysis suggested that the underlying mechanism involved histone deacetylase activity and epigenetic repression of Gdnf promoter upon RA treatment.

Benzo[a]pyrene disrupts LH/hCG-dependent mouse Leydig cell steroidogenesis through receptor/Gαs protein targeting.

Steroidogenesis of gonadal cells is tightly regulated by gonadotropins. However, certain polycyclic aromatic hydrocarbons, including Benzo[a]pyrene (BaP), induce reproductive toxicity. Several existing studies have considered higher than environmentally relevant concentrations of BaP on male and female steroidogenesis following long-term exposure. Also, the impact of short-term exposure to BaP on gonadotropin-stimulated cells is understudied. Therefore, we evaluated the effect of 1 nM and 1 µM BaP on luteinizing hormone/choriogonadotropin (LH/hCG)-mediated signalling in two steroidogenic cell models, i.e. the mouse tumor Leydig cell line mLTC1, and the human primary granulosa lutein cells (hGLC) post 8- and 24-h exposure. Cell signalling studies were performed by homogeneous time-resolved fluorescence (HTRF) assay, bioluminescence energy transfer (BRET) and Western blotting, while immunostainings and immunoassays were used for intracellular protein expression and steroidogenesis analyses, respectively. BaP decreased cAMP production in gonadotropin-stimulated mLTC1 interfering with Gαs activation. Therefore, decrease in gonadotropin-mediated CREB phosphorylation in mLTC1 treated with 1 µM BaP was observed, while StAR protein levels in gonadotropin-stimulated mLTC1 cells were unaffected by BaP. Further, BaP decreased LH- and hCG-mediated progesterone production in mLTC1. Contrastingly, BaP failed to mediate any change in cAMP, genes and proteins of steroidogenic machinery and steroidogenesis of gonadotropin-treated hGLC. Our results indicate that short-term exposure to BaP significantly impairs steroidogenic signalling in mLTC1 interfering with Gαs. These findings could have a significant impact on our understanding of the mechanism of reproductive toxicity by endocrine disruptors.

Short-Term Exposure to Bisphenol A Does Not Impact Gonadal Cell Steroidogenesis In Vitro.

Bisphenol A (BPA) is a ubiquitous, synthetic chemical proven to induce reproductive disorders in both men and women. The available studies investigated the effects of BPA on male and female steroidogenesis following long-term exposure to the compound at relatively high environmental concentrations. However, the impact of short-term exposure to BPA on reproduction is poorly studied. We evaluated if 8 and 24 h exposure to 1 nM and 1 µM BPA perturbs luteinizing hormone/choriogonadotropin (LH/hCG)-mediated signalling in two steroidogenic cell models, i.e., the mouse tumour Leydig cell line mLTC1, and human primary granulosa lutein cells (hGLC). Cell signalling studies were performed using a homogeneous time-resolved fluorescence (HTRF) assay and Western blotting, while gene expression analysis was carried out using real-time PCR. Immunostainings and an immunoassay were used for intracellular protein expression and steroidogenesis analyses, respectively. The presence of BPA leads to no significant changes in gonadotropin-induced cAMP accumulation, alongside phosphorylation of downstream molecules, such as ERK1/2, CREB and p38 MAPK, in both the cell models. BPA did not impact STARD1, CYP11A1 and CYP19A1 gene expression in hGLC, nor Stard1 and Cyp17a1 expression in mLTC1 treated with LH/hCG. Additionally, the StAR protein expression was unchanged upon exposure to BPA. Progesterone and oestradiol levels in the culture medium, measured by hGLC, as well as the testosterone and progesterone levels in the culture medium, measured by mLTC1, did not change in the presence of BPA combined with LH/hCG. These data suggest that short-term exposure to environmental concentrations of BPA does not compromise the LH/hCG-induced steroidogenic potential of either human granulosa or mouse Leydig cells.

cAMP-specific phosphodiesterase 8A and 8B isoforms are differentially expressed in human testis and Leydig cell tumor.

Cyclic adenosine monophosphate/Protein kinase A (cAMP/PKA) signaling pathway is the master regulator of endocrine tissue function. The level, compartmentalization and amplitude of cAMP response are finely regulated by phosphodiesterases (PDEs). PDE8 is responsible of cAMP hydrolysis and its expression has been characterized in all steroidogenic cell types in rodents including adrenal and Leydig cells in rodents however scarce data are currently available in humans. Here we demonstrate that human Leydig cells express both PDE8A and PDE8B isoforms. Interestingly, we found that the expression of PDE8B but not of PDE8A is increased in transformed Leydig cells (Leydig cell tumors-LCTs) compared to non-tumoral cells. Immunofluorescence analyses further reveals that PDE8A is also highly expressed in specific spermatogenic stages. While the protein is not detected in spermatogonia it accumulates nearby the forming acrosome, in the trans-Golgi apparatus of spermatocytes and spermatids and it follows the fate of this organelle in the later stages translocating to the caudal part of the cell. Taken together our findings suggest that 1) a specific pool(s) of cAMP is/are regulated by PDE8A during spermiogenesis pointing out a possible new role of this PDE8 isoform in key events governing the differentiation and maturation of human sperm and 2) PDE8B can be involved in Leydig cell transformation.

TNF-α inhibits GDNF levels in Sertoli cells, through a NF-κB-dependent, HES1-dependent mechanism.

BACKGROUND: Glial cell line-derived neurotrophic factor (GDNF) is a soluble molecule crucial for the regulation of the spermatogonial stem cells (SSC) of the testis. The effects of GDNF on target cells have been extensively described, but mechanisms underlying GDNF regulation are currently under investigation. In the nervous system, GDNF expression is regulated by pro-inflammatory cytokines including lipopolysaccharide (LPS), interleukin 1 beta (IL-1ß), and tumor necrosis factor alpha (TNF-α) but the effect of these cytokines on GDNF expression in the testis is unclear. OBJECTIVES: The aim of the present study was to investigate the impact of TNF-α on GDNF expression levels using primary murine Sertoli cells as experimental model. MATERIAL AND METHODS: The expression of TNF-α-regulated genes including Gdnf in different culture conditions was determined by real-time PCR. GDNF protein levels were determined by ELISA. The activation of the NF-κb pathway and HES1 levels were assessed by Western Blot analysis and immunofluorescence. HES1 expression was downregulated by RNAi. RESULTS: In primary Sertoli cells, TNF-α downregulates GDNF levels through a nuclear factor-κB (NF-κB)-dependent mechanism. Mechanistically, TNF-α induces the transcriptional repressor HES1 by a NF-Κb-dependent mechanism, which in turn downregulates GDNF. DISCUSSION: Under physiological conditions, TNF-α is secreted by germ cells suggesting that this cytokine plays a role in the paracrine control of SSC niche by modulating GDNF levels. HES1, a well-known target of the Notch pathway, is implicated in the regulation of GDNF expression. In Sertoli cells, TNF-α and Notch signaling may converge at molecular level, to regulate the expression of HES1 and HES1- target genes, including GDNF. CONCLUSIONS: Because of the importance of GDNF for spermatogonial stem cell self-renewal and proliferation, this data may give important insights on how cytokine signals in the testis modulate the expression of niche-derived factors.