Paternal trans fatty acid and vitamin E diet affect the expression pattern of androgen signaling pathway genes in the testis of rat offspring.

Numerous studies have shown that an improper diet in parents has a negative impact on offspring's health. Furthermore, the negative effects of trans fatty acids (TFA) in maternal diets on fertility and health and their impact on future generations have been documented. However, there is limited research on the negative effects of TFA in paternal diets on male children. The current work used qRT-PCR to investigate the effects of trans fatty acids and vitamin E in the paternal diet on the expression pattern of androgen signaling pathway genes such as STAR, CYP11a1, HSD3B, SRD5a2, and SCARB1 in offspring testes. In this experiment, parental rats were randomly separated into four groups, each with ten father rats, and were fed for eight weeks (60 days) as follows. 1: Standard diet group plus liquid sunflower oil (control). 2: Standard diet group containing trans fatty acids (CTH). 3: The regular diet group received 2.5 times the recommended quantity of vitamin E supplement. 4: Standard diet group with vitamin E and trans fatty acid supplementation (ETH). The testis tissue samples from 35 offspring were then used. Following RNA extraction from tissues and cDNA synthesis, quantitative real-time PCR was used to evaluate the expression levels of androgen signaling pathway genes such as STAR, CYP11A1, HSD3B, SCARB1, and SRD5A2. Our findings showed that the expression of CYP11A1 was considerably reduced in the progeny of paternal rats given ETH compared to the CTH group. The expression levels of the STAR gene were significantly lower in the progeny of paternal rats administered TFA, ETH, and vitamin E compared to the controls. Although the CTH group had lower SCARB1 expression than the other groups, the difference was not statistically significant. Paternal vitamin E consumption substantially affected SRD5A2 expression when compared to offspring of paternal rats fed vitamin E + trans fatty acid or those fed a conventional diet containing trans fatty acid. Furthermore, the vitamin E group showed a statistically significant increase in HSD3B expression compared to the other groups. Bioinformatics analyses, such as protein-protein interaction networks and gene ontology term enrichment, revealed that these genes play roles in lipid biosynthesis, hormone metabolism, male sex differentiation, reproductive development, and steroid biosynthesis. Our data indicate that paternal trans fatty acid consumption influences the expression of particular androgen signaling pathway genes in offspring testis, with vitamin E potentially mitigating some of these effects.

Functional characterization of the SDR42E1 reveals its role in vitamin D biosynthesis.

Vitamin D deficiency poses a widespread health challenge, shaped by environmental and genetic determinants. A recent discovery identified a genetic regulator, rs11542462, in the SDR42E1 gene, though its biological implications remain largely unexplored. Our bioinformatic assessments revealed pronounced SDR42E1 expression in skin keratinocytes and the analogous HaCaT human keratinocyte cell lines, prompting us to select the latter as an experimental model. Employing CRISPR/Cas9 gene-editing technology and multi-omics approach, we discovered that depleting SDR42E1 showed a 1.6-fold disruption in steroid biosynthesis pathway (P-value = 0.03), considerably affecting crucial vitamin D biosynthesis regulators. Notably, SERPINB2 (P-value = 2.17 × 10-103), EBP (P-value = 2.46 × 10-13), and DHCR7 (P-value = 8.03 × 10-09) elevated by ∼2-3 fold, while ALPP (P-value <2.2 × 10-308), SLC7A5 (P-value = 1.96 × 10-215), and CYP26A1 (P-value = 1.06 × 10-08) downregulated by ∼1.5-3 fold. These alterations resulted in accumulation of 7-dehydrocholesterol precursor and reduction of vitamin D3 production, as evidenced by the drug enrichment (P-value = 4.39 × 10-06) and total vitamin D quantification (R2 = 0.935, P-value = 0.0016) analyses. Our investigation unveils SDR42E1's significance in vitamin D homeostasis, emphasizing the potential of precision medicine in addressing vitamin D deficiency through understanding its genetic basis.

Multi-Omics Reveals the Role of Arachidonic Acid Metabolism in the Gut-Follicle Axis for the Antral Follicular Development of Holstein Cows.

In vitro embryonic technology is crucial for improving farm animal reproduction but is hampered by the poor quality of oocytes and insufficient development potential. This study investigated the relationships among changes in the gut microbiota and metabolism, serum features, and the follicular fluid metabolome atlas. Correlation network maps were constructed to reveal how the metabolites affect follicular development by regulating gene expression in granulosa cells. The superovulation synchronization results showed that the number of follicle diameters from 4 to 8 mm, qualified oocyte number, cleavage, and blastocyst rates were improved in the dairy heifers (DH) compared with the non-lactating multiparous dairy cows (NDC) groups. The gut microbiota was decreased in Rikenellaceae_RC9_gut_group, Alistipes, and Bifidobacterium, but increased in Firmicutes, Cyanobacteria, Fibrobacterota, Desulfobacterota, and Verrucomicrobiota in the NDC group, which was highly associated with phospholipid-related metabolites of gut microbiota and serum. Metabolomic profiling of the gut microbiota, serum, and follicular fluid further demonstrated that the co-metabolites were phosphocholine and linoleic acid. Moreover, the expression of genes related to arachidonic acid metabolism in granulosa cells was significantly correlated with phosphocholine and linoleic acid. The results in granulosa cells showed that the levels of PLCB1 and COX2, participating in arachidonic acid metabolism, were increased in the DH group, which improved the concentrations of PGD2 and PGF2α in the follicular fluid. Finally, the expression levels of apoptosis-related proteins, cytokines, and steroidogenesis-related genes in granulosa cells and the concentrations of steroid hormones in follicular fluid were determinants of follicular development. According to our results, gut microbiota-related phosphocholine and linoleic acid participate in arachidonic acid metabolism in granulosa cells through the gut-follicle axis, which regulates follicular development. These findings hold promise for enhancing follicular development and optimizing oocyte quality in subfertile dairy cows.

Nicotine as a female reproductive toxicant-A review.

The preceding decades have seen an extensive emergence of the harmful effects of tobacco smoke on systemic health. Among the various compounds of tobacco, nicotine is one of the principal, potentially hazardous, and toxic components which is an oxidant agent that can affect both men's and women's fertility. Nicotine exerts its effect by modulating the expression of transmembrane ligand-gated ion channels called nicotinic acetylcholine receptors. The activities of female reproduction might be disrupted by exposure to nicotine at various sites, such as the ovary or reproductive tract. It's been demonstrated that nicotine might cause oxidative stress, apoptosis, hormonal imbalance, abnormalities in chromosomal segregation, impact oocyte development, and disruption in ovarian morphology and functions. This review paper summarizes the findings and provides an updated overview of the evidence on the harmful effects of nicotine use on women's reproductive health and the resulting detrimental impacts on the body. Additionally, it provides the detailed possible mechanisms involved in impairing reproductive processes like folliculogenesis, oocyte maturation, steroidogenesis, and pregnancy in different animal species.

Long-term nanoplastics exposure contributes to impaired steroidogenesis by disrupting the hypothalamic-testis axis: Evidence from integrated transcriptome and metabolome analysis.

Cumulative evidence suggested that nanoplastics (NPs) cause male toxicity, but the mechanisms of which are still misty. Steroidogenesis is a key biological event that responsible for maintaining reproductive health. However, whether dysregulated steroidogenesis is involved in NPs-induced impaired male reproductive function and the underlying mechanism remains unclear. In our study, Balb/c mice were continuously exposed to pristine-NPs or NH2-NPs for 12 weeks, spanning the puberty and adult stage. Upon the long-term NPs treatment, the hypothalamus and testis were subjected to transcriptome and metabolome analysis. And the results demonstrated that both primitive-NPs and NH2-NPs resulted in impaired spermatogenesis and steroidogenesis, as evidenced by a significant reduction in sperm quality, testosterone, FSH, and LH. The expression of genes involved in hypothalamic-pituitary-testis (HPT) axis, such as Kiss-1 and Cyp17a1 that encoded the key steroid hormone synthetase, was also diminished. Furthermore, the phosphatidylcholine and pantothenic acid that mainly enriched in glycerophospholipid metabolism were significantly reduced in the testis. Comprehensive analysis of the transcriptome and metabolome indicated that down-regulated Cyp17a1 was associated with decreased metabolites phosphatidylcholine and pantothenic acid. Overall, we speculate that the disturbed HPT axis induced by long-term NPs contributes to disordered glycerophospholipid metabolism and subsequently impaired steroidogenesis. Our findings deepen the understanding of the action of the mechanism responsible for NPs-induced male reproductive toxicology.

Dysregulation of mitochondrial dynamics and mitophagy are involved in high-fat diet-induced steroidogenesis inhibition.

Male obesity is a pandemic health issue and can disrupt testicular steroidogenesis. Here, we explored the mechanism by which a high-fat diet (HFD) induced steroidogenic inhibition. As expected, HFD induced lipid droplet accumulation and reduced the expression of StAR, P450scc, and 3ß-HSD, three steroidogenic enzymes, in mouse testes. Palmitic acid (PA), a saturated fatty acid usually used to trigger lipotoxicity in vitro, induced greater accumulation of lipid droplets and the downregulation of steroidogenic enzymes in TM3 cells. Mechanistically, both HFD and PA disturbed mitochondrial fusion/fission dynamics and then induced mitochondrial dysfunction and mitophagy inhibition in mouse Leydig cells. Additionally, mitochondrial fusion promoter M1 attenuated PA-induced imbalance of mitochondrial dynamics, mitophagy inhibition, mitochondrial reactive oxygen species (ROS) production, and mitochondrial dysfunction in TM3 cells. Mitofusin 2 (Mfn2) knock-down further aggravated the PA-induced imbalance of mitochondrial dynamics, mitochondrial ROS production, and mitochondrial dysfunction in TM3 cells. Importantly, M1 rescued PA-induced downregulation of steroidogenic enzymes, whereas Mfn2 knock-down further aggravated PA-induced downregulation of steroidogenic enzymes in TM3 cells. Overall, our results provide laboratory evidence that mitochondrial dysfunction and mitophagy inhibition caused by dysregulation of mitochondrial fusion may be involved in HFD-induced steroidogenesis inhibition in mouse Leydig cells.

Personalized mixture toxicity testing: A proof-of-principle in vitro study evaluating the steroidogenic effects of reconstructed contaminant mixtures measured in blood of individual adults.

Chemical risk assessments typically focus on single substances, often overlooking real-world co-exposures to chemical mixtures. Mixture toxicology studies using representative mixtures can reveal potential chemical interactions, but these do not account for the unique chemical profiles that occur in the blood of diverse individuals. Here we used the H295R steroidogenesis assay to screen personalized mixtures of 24 persistent organic pollutants (POPs) for cytotoxicity and endocrine disruption. Each mixture was reconstructed at a human exposure relevant concentration (1×), as well as at 10- and 100-fold higher concentration (10×, 100×) by acoustic liquid handling based on measured blood concentrations in a Swedish cohort. Among the twelve mixtures tested, nine mixtures decreased the cell viability by 4-18%, primarily at the highest concentration. While the median and maximum mixtures based on the whole study population induced no measurable effects on steroidogenesis at any concentration, the personalized mixture from an individual with the lowest total POPs concentration was the only mixture that affected estradiol synthesis (35% increase at the 100× concentration). Mixtures reconstructed from blood levels of three different individuals stimulated testosterone synthesis at the 1× (11-15%) and 10× concentrations (12-16%), but not at the 100× concentration. This proof-of-principle personalized toxicity study illustrates that population-based representative chemical mixtures may not adequately account for the toxicological risks posed to individuals. It highlights the importance of testing a range of real-world mixtures at relevant concentrations to explore potential interactions and non-monotonic effects. Further toxicological studies of personalized contaminant mixtures could improve chemical risk assessment and advance the understanding of human health, as chemical exposome data become increasingly available.

The Effects of Human Chorionic Gonadotropin and Gonadotropin-Releasing Hormone Receptor Antagonist on Testicular Steroidogenesis in Normal and Obese Rats.

We studied the effect of a high-fat, high-carbohydrate diet (HFHCD) on basal testosterone levels in the blood and testosterone, its precursors, and expression of steroidogenic genes in the testes of rats treated with human chorionic gonadotropin (hCG, 10 IU/rat, subcutaneously, once), gonadotropin-releasing hormone receptor antagonist cetrorelix (75 µg/kg, subcutaneously, 3 days), and their combination. In HFHCD rats, no obvious signs of androgen deficiency were observed and the response of the testes to hCG stimulation was preserved. Unlike control rats (normal diet), the expression of the luteinizing hormone receptor gene in these rats did not change in response to hCG stimulation and cetrorelix administration; they also showed a paradoxical, more pronounced response to hCG administration under conditions of suppression of the gonadotropin secretion by cetrorelix. This suggests that the etiology and pathogenesis of obesity may have different effects on the hormonal status of the male reproductive system.

Effects of polystyrene micro- and nanoplastics on androgen- and estrogen receptor activity and steroidogenesis in vitro.

While many plastic additives show endocrine disrupting properties, this has not been studied for micro- and nanoplastics (MNPs) particles despite their ubiquitous presence in humans. The objective of this study was to determine the effects of various sizes and concentrations of polystyrene (PS)-MNPs (50-10,000 nm, 0.01-100 µg/mL) on estrogen- and androgen receptor (ER and AR) activity and steroidogenesis in vitro. Fluorescent (F)PS-MNPs of ≤1000 nm were internalized in VM7 and H295R cells and FPS-MNPs ≤200 nm in AR-ecoscreen cells. H295R cells displayed the highest uptake and particles were closer to the nucleus than other cell types. None of the sizes and concentrations PS-MNPs tested affected ER or AR activity. In H295R cells, PS-MNPs caused some statistically significant changes in hormone levels, though these showed no apparent concentration or size-dependent patterns. Additionally, PS-MNPs caused a decrease in estriol (E3) with a maximum of 37.5 % (100 µg/mL, 50 nm) and an increase in gene expression of oxidative stress markers GPX1 (1.26-fold) and SOD1 (1.23-fold). Taken together, our data show limited endocrine-disrupting properties of PS-MNPs in vitro. Nevertheless the importance of E3 in the placenta warrants further studies in the potential effects of MNPs during pregnancy.

High concentrations of progesterone inhibit the expression of genes related to steroid metabolism in MA-10 Leydig cells.

Leydig cells are the main testosterone-producing cells in males. During androgen synthesis, cholesterol enters the mitochondria via the STAR protein and is converted into pregnenolone by the CYP11A1 enzyme. This steroid is then exported from the mitochondria to be metabolized to progesterone by the HSD3B1 enzyme in the endoplasmic reticulum. In this study, we used 3'Tag-RNA-Seq to identify progesterone-regulated genes in MA-10 Leydig cells. Our results indicate that high concentrations of progesterone (30 µM) are involved in a negative feedback loop that inhibits cAMP/PKA-dependent activation of Star and Cyp11a1 expression and participate in cAMP/PKA-dependent down-regulation of genes related to the metabolism of steroid hormones. Linked to activation of the MAPK signaling pathway, endoplasmic reticulum stress and apoptosis, most of the genes encoding bZIP transcription factors are upregulated by progesterone in MA-10 Leydig cells. However, only DDIT3 protein levels are increased in response to progesterone in MA-10 Leydig cells. Like normal Leydig cells, MA-10 cells very weakly express the classical nuclear receptor for progesterone, suggesting that gene regulation by progesterone is rather mediated by one of the non-classical membrane receptors for progesterone However, current findings suggest that the inhibitory effect of progesterone on STAR protein increase in response to forskolin is not dependent on PGRMC1/2 or PAQR9. Furthermore, the increase in progesterone synthesis in response to activation of the cAMP/PKA pathway is rather inhibited by siRNA-mediated knockdown of PAQR9. Overall, this study shows that progesterone produced by Leydig cells participates in the regulation of steroidogenesis through autocrine action involving negative feedback upon activation of the cAMP/PKA pathway.

Metabolomic and Transcriptomic Analyses Reveal the Potential Mechanisms of Dynamic Ovarian Development in Goats during Sexual Maturation.

The ovary is a crucial reproductive organ in mammals, and its development directly influences an individual's sexual maturity and reproductive capacity. To comprehensively describe ovarian sexual maturation in goats, we integrated phenotypic, hormonal, metabolomic, and transcriptomic data from four specific time points: after birth (D1), at 2 months old (M2), at 4 months old (M4), and at 6 month old (M6). The study showed that during the early stage (D1-M2), ovarian growth was the most rapid, with weight and morphology increasing by 284% and 65%, respectively, and hormone levels rose significantly, with estradiol increasing by 57%. Metabolomic analysis identified 1231 metabolites, primarily lipids, lipid molecules, and organic acids, which can support hormone balance and follicle development by providing energy and participating in signaling transduction. Transcriptomic analysis identified 543 stage-specific differentially expressed genes, mainly enriched in steroid biosynthesis, amino acid metabolism, and the PI3K/AKT pathway, which are key factors influencing ovarian cell proliferation, apoptosis, hormone secretion, and metabolism. The integrated analysis revealed the key processes in the ovarian steroid hormone biosynthesis pathway and gene/metabolite networks associated with ovarian phenotypes and hormone levels, ultimately highlighting scavenger receptor class B type 1 (SCARB1), Cytochrome P450 Family 1 Subfamily A Member 1 (CYP11A1), 3beta-hydroxysteroid dehydrogenase (3BHSD), progesterone, estradiol, and L-phenylalanine as key regulators of ovarian morphological and functional changes at different developmental stages. This study is the first to reveal the metabolic changes and molecular regulatory mechanisms during ovarian sexual maturation in goats, providing valuable insights for understanding reproductive system development and optimizing reproductive performance and breeding efficiency.

Unraveling the impact of hyperleptinemia on female reproduction: insights from transgenic pig model.

BACKGROUND: Infertility is a growing global health concern affecting millions of couples worldwide. Among several factors, an extreme body weight adversely affects reproductive functions. Leptin is a well-known adipokine that serves as an endocrine signal between adiposity and fertility. However, the exact mechanisms underlying the effects of high leptin level on female reproduction remain unclear. METHODS: Transgenic pigs overexpressing leptin (♀) were produced by backcrossing and screened for leptin overexpression. The growth curve, fat deposition, reproductive performance, apoptosis, serum hormones and cholesterol production, RNA sequencing, and single-nucleus RNA sequencing (snRNA-seq) of the leptin-overexpressing pigs and wild-type group were evaluated. RESULTS: Transgenic pigs overexpressing leptin (♀) were obtained, which exhibited significantly reduced body weight, body size, and back fat thickness. These pigs manifested a late onset of puberty (330 ± 54.3 vs. 155 ± 14.7 days), irregular estrous behavior characterized by increased inter-estrous interval (29.2 ± 0 vs. 21.3 ± 0.7 days), and more number of matings until pregnancy (at least 3 times). This reproductive impairment in leptin pigs was related to hormonal imbalances characterized by increased levels of FSH, LH, prolactin, E2, P4, and TSH, altered steroidogenesis such as increased levels of serum cholesterol esters along with steroidogenic markers (StAR, CYP19A), and ovarian dysfunctions manifested by neutrophilic infiltration and low expression of caspase-3 positive cells in the ovaries. Moreover, bulk RNA sequencing of the ovaries also revealed neutrophilic infiltration followed by upregulation of inflammation-related genes. Furthermore, snRNA-seq reflected that leptin overexpression triggered immune response, suppressed follicle development and luteinization, resulting in metabolic dysfunction and hormone imbalance in the ovary. CONCLUSIONS: Low body weight in leptin overexpressing pigs adversely affects the reproductive performance, causing delayed puberty, irregular estrous cycles, and reduced breeding efficiency. This is linked to metabolic imbalances, an increased immune response, and altered ovarian functions. This study provides a theoretical basis for the complex mechanisms underlying leptin, and infertility by employing leptin-overexpressing female pigs.

Hormonal and Allosteric Regulation of the Luteinizing Hormone/Chorionic Gonadotropin Receptor.

Luteinizing hormone (LH) and human chorionic gonadotropin (CG), like follicle-stimulating hormone, are the most important regulators of the reproductive system. They exert their effect on the cell through the LH/CG receptor (LHCGR), which belongs to the family of G protein-coupled receptors. Binding to gonadotropin induces the interaction of LHCGR with various types of heterotrimeric G proteins (Gs, Gq/11, Gi) and ß-arrestins, which leads to stimulation (Gs) or inhibition (Gi) of cyclic adenosine monophosphate-dependent cascades, activation of the phospholipase pathway (Gq/11), and also to the formation of signalosomes that mediate the stimulation of mitogen-activated protein kinases (ß-arrestins). The efficiency and selectivity of activation of intracellular cascades by different gonadotropins varies, which is due to differences in their interaction with the ligand-binding site of LHCGR. Gonadotropin signaling largely depends on the status of N- and O-glycosylation of LH and CG, on the formation of homo- and heterodimeric receptor complexes, on the cell-specific microenvironment of LHCGR and the presence of autoantibodies to it, and allosteric mechanisms are important in the implementation of these influences, which is due to the multiplicity of allosteric sites in different loci of the LHCGR. The development of low-molecular-weight allosteric regulators of LHCGR with different profiles of pharmacological activity, which can be used in medicine for the correction of reproductive disorders and in assisted reproductive technologies, is promising. These and other issues regarding the hormonal and allosteric regulation of LHCGR are summarized and discussed in this review.

Exploring the Link between Premature Ovarian Insufficiency, Insomnia and Circadian Pathways.

OBJECTIVE: To establish an interaction network for genes related to premature ovarian insufficiency (POI) and insomnia, and to identify biological processes that connect POI to the physiological clock. METHODS: Previously reported lists of genes associated to POI and insomnia were contrasted and their intersection was used as input on protein-protein interaction analyses. POI-associated genes were contrasted with gene expression markers for neural circadian control and enriched pathways among their shared content were dissected. RESULTS: The functional network generated from the intersection between POI and insomnia gene lists pointed to the central nervous system as the most relevant cellular context for this connection. After identifying POI-associated genes that play a role in neural circadian patterns, we observed the disruption of pathways related to the hypothalamic-pituitary-gonadal axis as the major genetic link between ovarian function and circadian neural circuits. CONCLUSIONS: These findings highlight neurological mechanisms that support the POI-insomnia interplay.

Efficacy of propolis and royal jelly in attenuating cadmium-induced spermatogenesis and steroidogenesis dysregulation, causing infertility in rats.

Bee-derived pharmaceutical products, including propolis (PRO) and royal jelly (ROJ), possess outstanding pharmacological properties. However, their efficiency in counteracting the deleterious influences of cadmium (Cd) in testes and the relevant mechanisms entail further investigations. Therefore, this study sheds light on the therapeutic efficacy of PRO and ROJ against testicular dysfunction and infertility induced by Cd. Toward this end, 30 mature male Wistar albino rats were randomly divided into six groups (5 animals/group), including (I) control, (II) Cd, (III) PRO, (IV) ROJ, (V) PRO + Cd, and (VI) ROJ + Cd groups. Furthermore, antioxidant factors, semen quality, hormonal levels, steroidogenic enzymes, and genotoxicity were assessed. Moreover, histopathological and ultrastructural attributes and offspring rates were investigated. The Cd-treated group revealed marked reductions in reduced glutathione (GSH), total antioxidant capacity (TAC), and superoxide dismutase (SOD) with an amplification of lipid peroxidation in testes, indicating disruption of the antioxidant defense system. Furthermore, myeloperoxidase (MPO) activity and DNA damage were significantly heightened, implying inflammation and genotoxicity, respectively. Moreover, steroidogenic enzymes, including 17ß-Hydroxy Steroid Dehydrogenase 3 (HSD17b3), 3ß-Hydroxy Steroid Dehydrogenase 2 (HSD3b2), 17α-hydroxylase/17,20-lyase (CYP17A1), and steroid 5α-reductase 2 (SRD5A2) were markedly diminished accompanied with disorders in luteinizing hormone (LH), follicle-stimulating hormone (FSH), and testosterone. Besides, spermatozoa quality was reduced, associated with a diminution in the diameter of seminiferous tubules. By contrast, PRO or ROJ significantly protected and/or counteracted the Cd-induced pathophysiological consequences, ameliorating antioxidant and inflammatory biomarkers, steroidogenic enzymes, hormonal levels, and sperm properties, along with lessening DNA impairments. Critically, histological and ultrastructural analyses manifested several anomalies in the testicular tissues of the Cd-administered group, including the Leydig and Sertoli cells and spermatozoa. Conversely, PRO or ROJ sustained testicular tissues' structure, enhancing spermatozoa integrity and productivity. Interestingly, treatment with PRO or ROJ improved fertility indices through offspring rates compared to the Cd-animal group. Our data suggest that PRO is a more effective countermeasure than ROJ against Cd toxicity for securing the delicate testicular microenvironment for spermatogenesis and steroidogenesis.

Vitamin E alleviates glyphosate-based herbicide-induced progesterone secretion inhibition and oxidative stress increase in chicken primary granulosa cells.

Glyphosate-based herbicides (GBH) are the most extensively used herbicides worldwide. Despite a presumed nondangerousness for animals, several studies reported negative effects after a GBH exposure in several animal models including birds, notably on reproductive functions. Several studies concerning the advantages of Vitamin E (VE) for antioxidant activity but also growth and reproduction have been reported in birds. However, it remains unclear whether VE could alleviate the negative effect of GBHs on chicken ovarian cells. Here we exposed chicken primary granulosa cells (GCs) from F1 and F3/4 follicles to growing doses of GBH (0.036, 0.36, 3.6, and 36 gly eq/L), with or without VE supplementation (1 mg/L) and investigated cell viability, proliferation, oxidative stress and steroidogenesis. GBH exposure did not affect F1 and F3 GCs viability but it increased cell proliferation only in F1 GCs and this effect was not altered by VE. In both F1 and F3/4 GCs, GBH exposure increased total oxidant status (TOS), reduced total antioxidant status (TAS) and consequently increased index of oxidative stress (OSI) in dose dependent manner. This latter effect for GBH 36 mg eq gly/L was totally abolished in response to VE. In both F1 and F3/4 GCs, GBH exposure reduced progesterone secretion in a dose dependent manner and this effect with GBH 0.36 and 1.8 mg eq glyphosate/L was alleviated by VE. However, we did not observe any effect of GBH and VE on the gene expression of several components of the steroidogenesis process. Taken together, these results show that GBH may have endocrine disruptor effects, and that these effects might be alleviated by antioxidant VE supplementation.

New Insights into D-Aspartate Signaling in Testicular Activity.

D-aspartate (D-Asp) is an amino acid found in high concentrations in the testis and pituitary gland. Increasing evidence suggests that D-Asp promotes spermatogenesis by activating testosterone production in the Leydig cells via LH release from the pituitary gland. In vitro studies indicate that D-Asp may also influence steroidogenesis and spermatogenesis through autocrine and paracrine signals. D-Asp enhances StAR and steroidogenic enzyme expressions, facilitating testicular cell proliferation via the GluR/ERK1/2 pathway. Moreover, it supports spermatogenesis by enhancing the mitochondrial function in spermatocytes, aiding in the metabolic shift during meiosis. Enhanced mitochondrial function, along with improved MAM stability and reduced ER stress, has been observed in Leydig and Sertoli cells treated with D-Asp, indicating potential benefits in steroidogenesis and spermatogenesis efficiency. Conversely, D-Asp exerts a notable anti-apoptotic effect in the testis via the AMPAR/AKT pathway, potentially mediated by antioxidant enzyme modulation to mitigate testicular oxidative stress. This review lays the groundwork for future investigations into the molecules promoting spermatogenesis by stimulating endogenous testosterone biosynthesis, with D-amino acids emerging as promising candidates.

Assessment of the potential risk of oteseconazole and two other tetrazole antifungals to inhibit adrenal steroidogenesis and peripheral metabolism of corticosteroids.

The triazole antifungals posaconazole and itraconazole can cause pseudohyperaldosteronism with hypertension and hypokalemia, edema, and gynecomastia by inhibiting steroid synthesis and metabolism. Mechanisms underlying pseudohyperaldosteronism include inhibition of adrenal 11ß-hydroxylase cytochrome-P450 (CYP) 11B1 and 17α-hydroxylase (CYP17A1) as well as peripherally expressed 11ß-hydroxysteroid dehydrogenase type 2 (11ß-HSD2). To enhance specificity for fungal CYP51, tetrazoles have been developed. This study employed H295R adrenocortical cells and enzyme activity assays to assess the potential risk of oteseconazole and two other tetrazoles, VT-1598 and quilseconazole, to inhibit adrenal steroidogenesis or 11ß-HSD2. Steroidomic footprint analyses of H295R cell supernatants using untargeted liquid-chromatography-high-resolution mass-spectrometry (LC-HRMS) indicated overall patterns common to oteseconazole, quilseconazole and itraconazole, as well as similarities between VT-1598 and isavuconazole. Additionally, more specific features of the steroid signatures were observed. Targeted quantification of nine adrenal steroids in supernatants from treated H295R cells revealed an overall inhibition of adrenal steroidogenesis by the three tetrazoles, itraconazole and isavuconazole, providing an explanation for their similar steroidomic pattern. Applying recombinant enzymes indicated that this effect is not due to direct inhibition of steroidogenic enzymes because no or only weak inhibition could be observed. Moreover, oteseconazole and the two other tetrazoles did not inhibit 11ß-HSD2, suggesting that they do not pose a risk of pseudohyperaldosteronism. Furthermore, oteseconazole did not alter steroid concentrations in a recent clinical study. Nevertheless, follow-up studies should assess the mechanism underlying the observed overall steroidogenesis inhibition by tetrazoles, itraconazole and isavuconazole, and whether concentrations achievable in a subgroup of susceptible patients might cause adrenal insufficiency and hyperplasia.

In vitro differentiation of mouse pluripotent stem cells into corticosteroid-producing adrenocortical cells.

Directed differentiation of pluripotent stem cells into specialized cell types represents an invaluable tool for a wide range of applications. Here, we have exploited single-cell transcriptomic data to develop a stepwise in vitro differentiation system from mouse embryonic stem cells into adrenocortical cells. We show that during development, the adrenal primordium is embedded in an extracellular matrix containing tenascin and fibronectin. Culturing cells on fibronectin during differentiation increased the expression of the steroidogenic marker NR5A1. Furthermore, 3D cultures in the presence of protein kinase A (PKA)-pathway activators led to the formation of aggregates composed of different cell types expressing adrenal progenitor or steroidogenic markers, including the adrenocortical-specific enzyme CYP21A1. Importantly, in-vitro-differentiated cells responded to adrenocorticotropic hormone (ACTH) and angiotensin II with the production of glucocorticoids and mineralocorticoids, respectively, thus confirming the specificity of differentiation toward the adrenal lineage.