Aldosterone-producing nodules and CYP11B1 signaling correlate in primary aldosteronism

Autonomous cortisol secretion (ACS) could be found in some patients with unilateral primary aldosteronism (uPA). However, the histopathological patterns of uPA with concurrent ACS has not been well elucidated. The adrenal gland with the adenoma from 61 uPA patients who underwent unilateral adrenalectomy were assessed by immunohistochemistry. Bioinformatics analysis, including the Cancer Genome Atlas (TCGA) and Kyoto Encyclopedia of Genes and Genomes (KEGG), was applied. The prevalence of multiple aldosterone-producing nodules or micronodules (mAPN/mAPM) was 65.6% (40/61) among our uPA patients. Concurrent ACS was identified in 32% of this uPA cohort; they were associated with the interaction of larger tumor size (> 1.98 cm) and mAPN/mAPM (OR= 3.08, p= 0.004). Transcriptome analysis uncovered a dominant enrichment of HSD3B7 over-expression (p= 0.004) in the adenomas of the histopathologically classical adrenal uPA lesions with concomitant mAPN/mAPM, compared with those uPA adenomas without concurrent surrounding mAPN/mAPM. We identified a novel linkage of enhanced steroidogenic genes of HSD3B7 expression concurrent with downstream higher CYP11B1 expression; further relationship was confirmed by immunohistochemical staining and validated by TCGA bioinformatics. The presence of mAPN/mAPM in uPA patients had lower rate for biochemical success after adrenalectomy (p=0.047). In summary, two thirds of uPA patients had concomitant mAPN/mAPM; 1/3 of uPA patients had concurrent ACS. Steroidogenic HSD3B7/ CYP11B1 signaling was associated with uPA adenomas with surrounding mAPN/mAPM. Interaction of larger adenoma size with the presence of mAPN/mAPM was linked to co-existent ACS. Such uPA patients with concomitant mAPN/mAPM had lower rate of biochemical success.

Unexpected Interacting Effects of Physical (Radiation) and Chemical (Bisphenol A) Treatments on Male Reproductive Functions in Mice

For decades, numerous chemical pollutants have been described to interfere with endogenous hormone metabolism/signaling altering reproductive functions. Among these endocrine disrupting substances, Bisphenol A (BPA), a widely used compound, is known to negatively impact germ and somatic cells in the testis. Physical agents, such as ionizing radiation, were also described to perturb spermatogenesis. Despite the fact that we are constantly exposed to numerous environmental chemical and physical compounds, very few studies explore the impact of combined exposure to chemical and physical pollutants on reproductive health. The aim of this study was to describe the impact of fetal co-exposure to BPA and IR on testicular function in mice. We exposed pregnant mice to 10 µM BPA (corresponding to 0.5 mg/kg/day) in drinking water from 10.5 dpc until birth, and we irradiated mice with 0.2 Gy (γ-ray, RAD) at 12.5 days post-conception. Co-exposure to BPA and γ-ray induces DNA damage in fetal germ cells in an additive manner, leading to a long-lasting decrease in germ cell abundance. We also observed significant alteration of adult steroidogenesis by RAD exposure independently of the BPA exposure. This is illustrated by the downregulation of steroidogenic genes and the decrease of the number of adult Leydig cells. As a consequence, courtship behavior is modified, and male ultrasonic vocalizations associated with courtship decreased. In conclusion, this study provides evidence for the importance of broadening the concept of endocrine disruptors to include physical agents, leading to a reevaluation of risk management and regulatory decisions.

Bee bread mitigates down-regulation of steroidogenic genes, decreased spermatogenesis, and epididymal oxidative stress in male rats fed with high-fat diet

Background: The pituitary-gonadal axis plays an important role in steroidogenesis and spermatogenesis and by extension, fertility. Objectives: The aim of this study was to investigate the protective role of bee bread, which is a natural bee product, against obesity-induced decreases in steroidogenesis and spermatogenesis. Methods: Thirty-two adult male Sprague-Dawley rats weighing between 200-300 g were divided into four groups (n=8/group), namely: normal control (NC), high-fat diet (HFD), HFD plus bee bread administered concurrently for 12 weeks (HFD+B), HFD plus orlistat administered concurrently for 12 weeks (HFD+O) groups. Bee bread (0.5 g/kg) or Orlistat (10 mg/kg/day) was suspended in distilled water and given by oral gavage daily for 12 weeks. Results: Levels of follicle stimulating hormone, luteinizing hormone testosterone, and adiponectin, as well as sperm count, motility, viability, normal morphology and epididymal antioxidants decreased whereas levels of leptin and malondialdehyde, and sperm nDNA fragmentation increased significantly in HFD group relative to NC group. There were significant decreases in the testicular mRNA transcript and protein levels of androgen receptor, luteinizing hormone receptor, steroidogenic acute regulatory protein, cytochrome P450 enzyme, 3β-hydroxysteroid dehydrogenase (HSD) and 17β-HSD in the testes of the HFD group. Further, mount latency, mount frequency, intromission, ejaculation, penile iNOS and cGMP levels deceased significantly in the HFD group. Supplementation with bee bread significantly reduced leptin level and increased adiponectin level, enhanced sperm parameters and reduced sperm nDNA fragmentation, upregulated the levels of steroidogenic genes and proteins in HFD-induced obese male rats. Discussion and Conclusion: Bee bread improved spermatogenesis and steroidogenesis by upregulating steroidogenic genes. Therefore, bee bread may be considered as a potential supplementation to protect against infertility in overweight or obese men.

Modulation of Calcium Signaling by Chemogenetic Tools to Elucidate the Pathogenesis of Primary Aldosteronism

Primary aldosteronism (PA) is the most frequent form of secondary arterial hypertension. The identification of germline or somatic mutations in different genes coding for ion channels (KCNJ5, CACNA1D, CACNA1H and CLCN2) and ATPases (ATP1A1 and ATP2B3) defines PA as a channelopathy. These mutations promote increased intracellular calcium concentrations and activation of calcium signaling, the main trigger for aldosterone biosynthesis. The aim of our study was to elucidate the mechanisms underlying the development of PA by modulating calcium signaling using chemogenetic tools. We have generated two different adrenocortical H295R_S2 cell lines stably expressing different chimeric ion channels generated by fusing the mutated extracellular ligand binding domain of the α7 nicotinic acetylcholine receptor to the ion pore domain of large Cys-loop receptor ion channel family; these receptors constitute PSAM (Pharmacologically Selective Actuator Modules). The mutations introduced in the ligand-binding domain allow to use synthetic ligands, PSEM (Pharmacologically Selective Effector Molecules) to activate the PSAM. We used two different PSAM: the chimeric receptor α7-5HT3 or a mutated acetylcholine receptor nAchR, allowing respectively modulation of sodium or calcium entry into the cells in response to the specific PSEM: Varenicline for α7-5HT3 or Compound 9S for mutated nAChR. The cells lines were characterized in terms of intracellular calcium concentrations, cell proliferation, aldosterone production and steroidogenic gene expression. Cells expressing α7-5HT3 treated for 24h with increasing concentrations of Varenicline (10–9 to 10-5M) showed increased intracellular calcium concentrations and an increase in expression of steroidogenic genes such as StAR CYP17A1, CYP21A2 and CYP11B2. Cell proliferation was not affected. Calcium entry into cells expressing the mutated nAChR receptor treated for 24h with increasing concentrations of Compound 9S (10–9 to 10-5M) induced an increase in expression of steroidogenic genes such as StAR, CYP21A2 and HSD3B2, but not CYP11B2. Similarly to the results obtained in cells expressing α7-5HT3, cell proliferation was unaffected in response to Compound 9S. These cell lines, in which we can modulate the intracellular calcium concentration « on demand », are a useful tool for a better understanding of the alterations of intracellular ion balance and calcium signaling in the pathophysiology of PA.

Portulaca oleracea seeds’ extract alleviates acrylamide-induced testicular dysfunction by promoting oxidative status and steroidogenic pathway in rats

Background Acrylamide (ACR) is a widespread industrial and food contaminant that garnered considerable attention for its carcinogenic, neurotoxic, and reproductive toxic effects. The antioxidant effects of Portulaca oleracea seeds extract (POS) and its fertility-enhancing effects were inspiring to evaluate the protective potential and pinpoint the mechanisms and molecular targets of the UPLC-MS fingerprinted POS extract on ACR-induced testicular toxicity in rats. Methods Male Wistar rats were divided into 6 equal groups of negative control, ACR model (10 mg/kg b.wt.), POS at doses of (200 and 400 mg/kg b.wt.) and POS-treated ACR groups. All treatments were given by oral dosing every day for 60 days. Results Administration of POS extract reversed the ACR-induced epididymides weight loss with improved semen quality and count, ameliorated the ACR-decreased testicular lesion scoring, testicular oxidative stress, testicular degeneration, Leydig cell apoptosis and the dysregulated PCNA and Caspase-3 expression in a dose-dependent manner. It upregulated the declined level of serum testosterone and the expression of steroidogenic genes such as CYP11A1 and 17β3-HSD with an obvious histologic improvement of the testes with re-establishment of the normal spermatogenic series, Sertoli and Leydig cells. Conclusions The supplementation with POS extract may provide a potential protective effect for ACR-induced testicular dysfunction which is mediated by its antioxidant, antiapoptotic and steroidogenic modulatory effects.

A Testis-Specific Long Noncoding RNA, Start, Is a Regulator of Steroidogenesis in Mouse Leydig Cells

The testis expresses many long noncoding RNAs (lncRNAs), but their functions and overview of lncRNA variety are not well understood. The mouse Prss/Tessp locus contains six serine protease genes and two lncRNAs that have been suggested to play important roles in spermatogenesis. Here, we found a novel testis-specific lncRNA, Start (Steroidogenesis activating lncRNA in testis), in this locus. Start is 1822 nucleotides in length and was found to be localized mostly in the cytosol of germ cells and Leydig cells, although nuclear localization was also observed. Start-knockout (KO) mice generated by the CRISPR/Cas9 system were fertile and showed no morphological abnormality in adults. However, in adult Start-KO testes, RNA-seq and qRT-PCR analyses revealed an increase in the expression of steroidogenic genes such as Star and Hsd3b1, while ELISA analysis revealed that the testosterone levels in serum and testis were significantly low. Interestingly, at 8 days postpartum, both steroidogenic gene expression and testosterone level were decreased in Start-KO mice. Since overexpression of Start in two Leydig-derived cell lines resulted in elevation of the expression of steroidogenic genes including Star and Hsd3b1, Start is likely to be involved in their upregulation. The increase in expression of steroidogenic genes in adult Start-KO testes might be caused by a secondary effect via the androgen receptor autocrine pathway or the hypothalamus-pituitary-gonadal axis. Additionally, we observed a reduced number of Leydig cells at 8 days postpartum. Collectively, our results strongly suggest that Start is a regulator of steroidogenesis in Leydig cells. The current study provides an insight into the overall picture of the function of testis lncRNAs.

FOXA3, a Negative Regulator of Nur77 Expression and Activity in Testicular Steroidogenesis

Biosynthesis of testosterone occurs mainly in the testicular Leydig cells. Nur77, an orphan nuclear receptor that is expressed in response to the luteinizing hormone/cyclic adenosine monophosphate (LH/cAMP) signaling pathway, is one of the key factors that regulate steroidogenesis in Leydig cells. The function of Nur77 is modulated through interaction with other proteins. FOXA3, a transcription factor that is crucial for male fertility, is also expressed in Leydig cells. Here, we sought to elucidate the role of FOXA3 in testicular steroidogenesis by focusing on its interaction with Nur77. LH/cAMP signaling induces the onset of steroidogenesis in Leydig cells but has a repressive effect on the expression of FOXA3. Overexpression of FOXA3 in MA-10 Leydig cells repressed cAMP-induced expression of Nur77 and its target steroidogenic genes (StAR, P450c17, and Hsd3β). Furthermore, FOXA3 suppressed Nur77 transactivation of the promoter of steroidogenic genes. In mouse primary Leydig cells, adenovirus-mediated overexpression of FOXA3 had similar effects and resulted in decreased production of testosterone. Taken together, these results suggest the role of FOXA3 in the regulation of steroidogenic genes in Leydig cells and fine-tuning steroidogenesis in the testis.