The impact of furan exposure on steroidogenesis in Leydig cells: cellular and molecular observations.

BACKGROUND: Furan is an organic compound that occurs as a result of heat treatment during the processing and cooking of many food products. Furthermore, the environment contains furan in tobacco smoke and vehicle exhaust gases, and it serves as an intermediate molecule in the synthesis of various pharmaceutical and chemical agents, pesticides, and stabilizers. Studies on the male reproductive system have not been able to elucidate the pathway through which furan exerts its negative effects. METHODS AND RESULTS: In this study, the TM3 Leydig cell line was exposed to various furan concentrations (0.03, 0.3, and 3 mM) for 24 h. In order to assess the cytotoxic effects of furan on Leydig cells, we examined cell viability, cell proliferation, and lactate dehydrogenase enzyme levels. To investigate the detrimental effects of furan on testosterone biosynthesis, quantitative analyses were conducted on cAMP and testosterone levels, as well as the expression levels of key genes and transcription factors implicated in the steroidogenic pathway. The results indicate that furan inhibited the viability and proliferation of Leydig cells and enhanced the activity of lactate dehydrogenase. Leydig cells administered to furan exhibited notable reductions in cAMP and testosterone levels. Additionally, while the expression levels of steroidogenic genes were downregulated, significant changes were detected in the expression levels of the transcription factors responsible for the regulation of these genes. CONCLUSIONS: Consequently, our findings suggest that furan exerts inhibitory effects on steroidogenesis in Leydig cells through multiple mechanisms, ultimately leading to infertility by inducing dysfunction in Leydig cells.

L-Cysteine Upregulates Testosterone Biosynthesis and Blood-Testis Barrier Genes in Cultured Human Leydig Cells and THP-1 Monocytes and Increases Testosterone Secretion in Human Leydig Cells.

Leydig cells are the primary source of testosterone or androgen production in male mammals. The blood-testis barrier (BTB) maintains structural integrity and safeguards germ cells from harmful substances by blocking their entry into the seminiferous tubules. L-cysteine is essential to the production of glutathione, a powerful antioxidant crucial to protecting against oxidative stress-induced damage. Animal studies have demonstrated the protective effect of L-cysteine in preventing testicular damage caused by chemicals or radiation. This study examines whether L-cysteine enhances the expression of testosterone biosynthesis and the BTB genes in human Leydig cells and THP-1 monocytes. The Leydig cells and THP-1 monocytes were treated with L-cysteine for 24 h. RNA was extracted following treatment, and the gene expression was analyzed using quantitative RT-PCR. Testosterone levels in the cell supernatant were measured using an ELISA kit. L-cysteine treatment in Leydig cells significantly upregulated the expression of CYP11A1 (p = 0.03) and the BTB genes CLDN1 (p = 0.03), CLDN11 (p = 0.02), and TJP1 (p = 0.02). Similarly, L-cysteine significantly upregulated the expression of CYP11A1 (p = 0.03) and CYP19A1 (p < 0.01), and the BTB genes CLDN1 (p = 0.04), CLDN2 (p < 0.01), CLDN4 (p < 0.01), CLDN11 (p < 0.01), and TJP1 (p = 0.03) in THP-1 monocytes. Further, L-cysteine supplementation increased the testosterone secretion levels in human Leydig cells. The findings suggest that L-cysteine supplementation could be used as an adjuvant therapy to promote the integrity of the BTB genes, testosterone biosynthesis and secretion, and the maintenance of testicular functions, which in turn mitigates the risk of male infertility.

Supplementing ageing male laying breeders with lycopene alleviates oxidative stress in testis and improves testosterone secretion.

BACKGROUND: Reproductive performance is a crucial aspect of poultry production and is carefully controlled by endocrine, paracrine, and autocrine factors. This study aimed to investigate the effect of lycopene on testosterone synthesis in Leydig cells of laying breeder roosters, clarify the mechanism of lycopene improving Leydig cells function and promoting testosterone production, and explore the role of related signal transduction pathways in testosterone synthesis. RESULTS: A total of 96 healthy 55-week-old breeding roosters were randomly assigned to one of five dietary treatments. They were provided with a corn-soybean meal-based diet containing different levels of lycopene: 0 mg/kg (control), 50 mg/kg, 100 mg/kg, or 200 mg/kg. The experiment lasted for 6 weeks. With the increase in lycopene levels, the testosterone content in the plasma was significantly higher than in the control group. Testicular Leydig cells were isolated and cultured from fresh testicular tissue of 45-wk-old to 60-wk-old breeding roosters. Various doses of lycopene were administered to Leydig cells, and subsequently, cells were collected for the detection of cell viability and testosterone content. The optimal concentration of lycopene to be added was determined, and changes in mRNA expression and protein levels of key proteins involved in testosterone synthesis were investigated. The results showed that lycopene treatment significantly increased testosterone secretion, mRNA expression, and protein levels of steroid-producing enzymes. Cells were collected to measure the activity of antioxidant enzymes, the mRNA transcription level of apoptotic factors, and the protein expression of apoptotic factors after treatment with lycopene. The results showed that lycopene significantly increased the activities of antioxidant enzymes, and the ability to inhibit oxygen radicals, and decreased the content of malondialdehyde. Apoptosis was inhibited by regulating the expression of apoptosis-inducing and anti-apoptosis factors. After that, the MAPK signaling pathway and downstream SF-1, Nrf2 gene, and protein expression levels were detected. The results showed that lycopene treatment significantly increased the gene and protein expression of JNK, SF-1, and Nrf2, and significantly decreased the gene and protein expression of p38. CONCLUSIONS: Lycopene treatment could promote testosterone synthesis of testicular Leydig cells by activating MAPK-SF-1 (increasing steroid-producing enzyme level) and MAPK-Nrf2 pathways (resisting oxidative damage).

Pregnenolone 16-Alpha Carbonitrile, an Agonist of Rodent Pregnane X Receptor, Regulates Testosterone Biosynthesis in Rodent Leydig Cells.

Leydig cells (LCs) in the testes produce the male sex hormone testosterone (T). Several xenobiotics, including clinical drugs, supplements, and environmental chemicals, are known to disrupt T homeostasis. Notably, some of these xenobiotics are known to activate the pregnane X receptor (PXR), a ligand-dependent nuclear receptor. However, it is currently unknown whether PXR is expressed in LCs and whether PXR activation alters T synthesis in rodent LCs. Therefore, in this study, we sought to determine whether PXR is expressed in rodent LCs and whether pregnenolone 16-alpha carbonitrile (PCN), the prototype agonist of rodent PXR, regulates T biosynthesis in rodent LCs. Hormonal as well as protein and gene expression analyses were conducted in rat primary LCs and MA-10 mouse Leydig cells. Results showed that PXR was expressed at the mRNA and protein level in both rat primary LCs and MA-10 cells. Incubation of rat primary LCs with PCN resulted in a significant decrease in T secretion. This PCN-induced decrease in T secretion was associated with decreased protein expression of key steroidogenic enzymes such as 3ß-HSD and CYP17A1. RNA-seq results from MA-10 cells showed that PCN down-regulated the transcripts of steroidogenic enzymes and proteins involved in the T synthesis pathway. Together, these results suggest that PCN, an agonist of rodent PXR, can regulate T biosynthesis in rodent LCs by down-regulating the expression of the steroidogenic enzymes involved in T biosynthesis. Our results are significant as they provide a potential novel mechanism for disruption of testosterone homeostasis by a variety of xenobiotics.

Leydig cells pyroptosis in testis mediates deoxynivalenol-induced male reproductive toxicity in mice.

Deoxynivalenol (DON) is a toxic secondary metabolite produced by Fusarium spp. It is widely distributed among various cereals and has attracted much attention as a potential health threat to humans and domestic animals. However, the effects of DON on the reproductive systems of mammals are still ambiguous. In this study, the toxic effects of DON in the male reproduction of mice were investigated. The results showed that DON caused the shedding of sperm cells at all testis levels and the presence of inflammatory cells in the testicular interstitium. The rate of living sperm was significantly reduced, and the rate of sperm deformity was increased after DON exposure. The DON exposure resulted in decreased levels of testosterone (T) and increased levels of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) in the serum. Measurements of oxidative stress markers showed that DON induced oxidative stress in mice testis. Meanwhile, DON triggered the assembly of NLRP3-ASC-Caspase-1 inflammatory complex and pyroptosis in both mice testis and TM3 cells, further causing the activation of GSDMD, promoting the leakage of inflammatory cytokines, including IL-1ß and IL-18. Notably, the inhibition of oxidative stress was found to protect pyroptosis in TM3 cells exposed to DON. We identified a novel mechanism of reproductive damage induced by DON, demonstrating the activation of the canonical Caspase-1-dependent pyroptosis pathway and clarifying the protection of antioxidation against pyroptosis damage. Our discovery provided support for the risk assessment of DON and target exploration for clinical treatment related to pyroptosis.

Bisphenol H exposure disrupts Leydig cell function in adult rats via oxidative stress-mediated m6A modifications: Implications for reproductive toxicity.

Bisphenol H (BPH) has emerged as a potential alternative to bisphenol A (BPA), which has been curtailed for use due to concerns over its reproductive and endocrine toxicity. This study investigates whether BPH exerts antiandrogenic effects by impairing Leydig cell function, a critical component in testosterone production. We administered orally BPH to adult male rats at doses of 0, 1, 10, and 100 mg/kg/day for 7 days. Notably, BPH treatment resulted in a dose-dependent reduction in testicular testosterone levels, with significant decreases observed at ≥ 1 mg/kg/day. Additionally, BPH affected the expression of key genes involved in steroidogenesis and cholesterol metabolism, including Nr5a1, Nr3c4, Lhcgr, Scarb1, and Star, at higher doses (10 and/or 100 mg/kg/day). The study also revealed alterations in antioxidant gene expression (Sod2 and Cat) and modulation of m6A-related genes (Ythdf1-3 and Foxo3) and their proteins. Through MeRIP-qPCR analysis, we identified increased m6A modifications in Scarb1 and Star genes following BPH exposure. In vitro experiments with primary Leydig cells confirmed that BPH enhanced oxidative stress and diminished testosterone production, which were partially mitigated by antioxidant vitamin E supplementation and Ythdf3 knockdown. Meanwhile, simultaneous administration of BPH and vitamin E to primary Leydig cells partially counteracted BPH-induced alterations in the Ythdf3 expression. Our findings underscore a novel mechanism by which BPH disrupts Leydig cell function through the oxidative stress-m6A modification-autophagy pathway, raising concerns about its potential reproductive toxicity.

Integrated stress response mediates HSP70 to inhibit testosterone synthesis in aging testicular Leydig cells.

The integrated stress response (ISR) is implicated in age-related diseases, while the molecular chaperone heat shock protein 70 (HSP70) can facilitate proper protein folding. However, the regulatory mechanism of ISR in insufficient testosterone synthesis of aging Leydig cells (LCs) remains unclear. This study aims to elucidate the regulatory role of ISR in inadequate testosterone synthesis of aging LCs. We observed a positive correlation between testosterone and HSP70 levels, which were found to be decreased in elderly men. ISR was detected in testicular tissue from old mice. The expression of testosterone synthesis related protein and the content of testosterone decreased in testicular tissue of old mice. Conversely, inhibition of the integrated stress response in testicular tissue led to an increase in steroid synthase expression among old mice. Furthermore, inhibiting ISR specifically within aging LCs resulted in enhanced protein translation efficiency and increased expression levels of new HSP70 and steroidogenic acute regulatory protein (StAR). These findings suggest that ISR occurrence within aging LCs affects StAR protein expression through regulation of HSP70-mediated translation, consequently impairing testosterone synthesis.

Effects of Leydig cell elimination on testicular interstitial cell populations: characterization by scRNA-seq and immunocytochemical techniques.

Background: The mammalian testicular interstitial cells are not well-defined. The present study characterized the interstitial cell types and their turnover dynamics in adult rats. Additionally, the heterogeneity of the mesenchymal population and the effects of Leydig cell elimination on interstitial homeostasis were further analyzed by scRNA-seq datasets and immunocytochemical techniques. Methods: Interstitial cells were defined at the transcriptomic level by scRNA-seq and then confirmed and quantified with protein markers. The dividing activity of the major cell types was determined by continuous EdU labeling of the animals for one week. Some of the rats were also treated with a dose of ethylenedimethylsulfonate (EDS) to examine how the loss of Leydig cells (LCs) could affect interstitial homeostasis for three weeks. Results: Seven interstitial cell types were identified, including cell types (percentage of the whole interstitial population) as follows: Leydig (44.6%), macrophage and dendritic (19.1%), lymphoid (6.2%), vascular endothelial (7.9%), smooth muscle (10.7%), and mesenchymal (11.5%) cells. The EdU experiment indicated that most cell types were dividing at relatively low levels (<9%) except for the mesenchymal cells (MCs, 17.1%). Further analysis of the transcriptome of MCs revealed 4 subgroups with distinct functions, including 1) glutathione metabolism and xenobiotic detoxification, 2) ROS response and AP-1 signaling, 3) extracellular matrix synthesis and binding, and 4) immune response and regulation. Stem LCs (SLCs) are primarily associated with subgroup 3, expressing ARG1 and GAP43. EDS treatment not only eliminated LCs but also increased subgroup 3 and decreased subgroups 1 and 2 of the mesenchymal population. Moreover, EDS treatment increased the division of immune cells by more than tenfold in one week. Conclusion: Seven interstitial cell types were identified and quantified for rat testis. Many may play more diversified roles than previously realized. The elimination of LCs led to significant changes in MCs and immune cells, indicating the importance of LCs in maintaining testicular interstitial homeostasis.

The hepatokine FGF21 stopped lipogenesis and reduced testosterone production in mLTC-1 Leydig Cell Line.

Beyond their link to metabolic issues like type 2 diabetes, factors like lifestyle, environment, and excess weight may also influence fertility. Fibroblast growth factor 21 (FGF21), a liver-derived hormone linked to energy balance, has recently emerged as a potential player in female mammalian reproduction. In male, only two studies have described potential effects of FGF21 on fertility. A recent study has described a negative correlation observed in obese patients presenting a low testosterone level associated with elevated FGF21 plasma levels. To investigate the role of FGF21 in steroidogenesis, we have studied the involvement of FGF21 in lipid and steroid activity by Leydig cells. Leydig cell model expressed all FGF21 receptors and ß-Klotho cofactor as determined by RT-qPCR and by western-blot. Cultured mLTC-1 Leydig cell line exposed to increasing FGF21 concentration induced phosphorylation (Ser 473) of Akt and modified the CREB factor activity, suggesting the functionality of the FGF21 pathway. FGF21 consequences on mLTC-1 Leydig cells are inhibition of the lipid synthesis, leading to a reduction in the content of lipid droplets. The drop in lipid synthesis is associated with a reduction in the amount of lipids (mainly PUFA, cholesterol esterified, and triglycerides) as measured by lipidomic approach. The main consequence is to reduce the quantity of cholesterol, the steroid precursor, in mLTC-1 Leydig cells and is associated with a low production in testosterone. The decrease in androgens was also associated with a reduction in the steroid enzyme genes expression, which are under the control of CREB activity, and present a lower activity due to low cAMP intracellular levels. In vivo, steroid production was lowering after FGF21 administration in adult male mice associated to a decrease in progressive motility and velocity of sperm. In addition, these experimental data are reinforced by a data mining analysis focused on "gonad" terms in 1,319,905 article references showing the link already described between FGF21 with the fatty acids pathways, cholesterol storage, and steroid production. In conclusion, we demonstrated that Leydig cells in the testes present a functional FGF21 pathway, which regulates lipid metabolism and steroid function. In mLTC-1 Leydig cells, FGF21 reduced cholesterol, PUFA content, and testosterone production. Finally, this work highlighted that the hepatokine FGF21 could have a negative impact on androgen synthesis and testicular activity.

The Smoky Impact of Nicotinic Acetylcholine Receptors on Testicular Function.

Smoking habits (from classic cigarettes to e-cigarettes and heated tobacco) are a relatively common finding in the medical histories of couples referred to fertility centers. Tobacco smoke and e-cigarettes may deliver many substances with known harmful effects on both general and reproductive health, including nicotine. Nicotinic Acetylcholine receptors (nAChRs) form a heterogeneous family of ion channels that are differently expressed in different tissues. According to the homomeric or heteromeric combination of at least five different subunits (named from α to ε), they have peculiar pharmacological and biophysical properties. nAChRs respond to the neurotransmitter acetylcholine, which influences a number of physiological functions not restricted to neurons and plays an important role in the structure and function of non-neuronal tissues such as the testis. nAChRs are also the target of Nicotine, the active element responsible for tobacco addiction. This review summarizes recent findings on the involvement of nAChRs in testicular physiology, highlighting the effects of nicotine exposure observed in animal studies and clinical settings. We will discuss the latest data on fertility outcomes and the implications for understanding nAChR functions in reproductive health.

Rotenone-induced cell apoptosis via endoplasmic reticulum stress and PERK-eIF2α-CHOP signalling pathways in TM3 cells.

Rotenone (ROT), a widely used natural pesticide, has an uncertain effect on reproductive toxicity. In this study, we used 20 mice distributed randomly into four groups, with each group receiving ROT doses of 0, 2, 4, and 8 mg/kg/day for 28 days. The results demonstrated that ROT induced significant testicular damage, including impaired spermatogenesis, inhibition of testosterone synthesis, and apoptosis of Leydig cells. Additionally, ROT disrupted the normal ultrastructure of the endoplasmic reticulum (ER) in testicular tissue, leading to ER stress in Leydig cells. To further explore whether ROT-induced apoptosis in Leydig cells is related to ER stress, the mouse Leydig cell line (TM3 cells) was treated with ROT at 0, 250, 500, and 1000 nM. ROT inhibited TM3 cell viability, induced cytotoxicity, and reduced testosterone content in the culture supernatants. Furthermore, ROT treatment triggered apoptosis in TM3 cells by activating ER stress and the PERK-eIF2α-CHOP signalling pathway. Pre-treatment of TM3 cells exposed to ROT with the ER stress inhibitor 4-phenylbutyric acid (4-PBA) alleviated these effects, decreasing apoptosis and preserving testosterone levels. Further intervention with the PERK inhibitor GSK2606414 reduced ROT-induced apoptosis and testosterone reduction by inhibiting PERK activity. In summary, ROT-induced male reproductive toxicity is specifically driven by apoptosis, with the PERK-eIF2α-CHOP signalling pathway activated by ER stress playing a crucial role in the apoptosis of Leydig cells triggered by ROT.

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.

Single-cell RNA sequencing reveals transcriptomic landscape and potential targets for human testicular ageing.

STUDY QUESTION: What is the molecular landscape underlying the functional decline of human testicular ageing? SUMMARY ANSWER: The present study provides a comprehensive single-cell transcriptomic atlas of testes from young and old humans and offers insights into the molecular mechanisms and potential targets for human testicular ageing. WHAT IS KNOWN ALREADY: Testicular ageing is known to cause male age-related fertility decline and hypogonadism. Dysfunction of testicular cells has been considered as a key factor for testicular ageing. STUDY DESIGN, SIZE, DURATION: Human testicular biopsies were collected from three young individuals and three old individuals to perform single-cell RNA sequencing (scRNA-seq). The key results were validated in a larger cohort containing human testicular samples from 10 young donors and 10 old donors. PARTICIPANTS/MATERIALS, SETTING, METHODS: scRNA-seq was used to identify gene expression signatures for human testicular cells during ageing. Ageing-associated changes of gene expression in spermatogonial stem cells (SSCs) and Leydig cells (LCs) were analysed by gene set enrichment analysis and validated by immunofluorescent and functional assays. Cell-cell communication analysis was performed using CellChat. MAIN RESULTS AND THE ROLE OF CHANCE: The single-cell transcriptomic landscape of testes from young and old men was surveyed, revealing age-related changes in germline and somatic niche cells. In-depth evaluation of the gene expression dynamics in germ cells revealed that the disruption of the base-excision repair pathway is a prominent characteristic of old SSCs, suggesting that defective DNA repair in SSCs may serve as a potential driver for increased de novo germline mutations with age. Further analysis of ageing-associated transcriptional changes demonstrated that stress-related changes and cytokine pathways accumulate in old somatic cells. Age-related impairment of redox homeostasis in old LCs was identified and pharmacological treatment with antioxidants alleviated this cellular dysfunction of LCs and promoted testosterone production. Lastly, our results revealed that decreased pleiotrophin signalling was a contributing factor for impaired spermatogenesis in testicular ageing. LARGE SCALE DATA: The scRNA-seq sequencing and processed data reported in this paper were deposited at the Genome Sequence Archive (https://ngdc.cncb.ac.cn/), under the accession number HRA002349. LIMITATIONS, REASONS FOR CAUTION: Owing to the difficulty in collecting human testis tissue, the sample size was limited. Further in-depth functional and mechanistic studies are warranted in future. WIDER IMPLICATIONS OF THE FINDINGS: These findings provide a comprehensive understanding of the cell type-specific mechanisms underlying human testicular ageing at a single-cell resolution, and suggest potential therapeutic targets that may be leveraged to address age-related male fertility decline and hypogonadism. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by the National Key Research and Development Program of China (2022YFA1104100), the National Natural Science Foundation of China (32130046, 82171564, 82101669, 82371611, 82371609, 82301796), the Natural Science Foundation of Guangdong Province, China (2022A1515010371), the Major Project of Medical Science and Technology Development Research Center of National Health Planning Commission, China (HDSL202001000), the Open Project of NHC Key Laboratory of Male Reproduction and Genetics (KF202001), the Guangdong Province Regional Joint Fund-Youth Fund Project (2021A1515110921, 2022A1515111201), and the China Postdoctoral Science Foundation (2021M703736). The authors declare no conflict of interest.

[Effect of Xiongcan Yishen Formula on ferroptosis in mouse TM3 Leydig cells after oxidative stress injury].

OBJECTIVE: To investigate the effects of Xiongcan Yishen Formula (XYF) on ferroptosis in mouse TM3 Leydig cells after oxidative stress injury (OSI) induced by H2O2.　Methods: An oxidative stress injury model was established in mouse TM3 Leydig cells using H2O2 induction. The modeled TM3 cells were randomly divided into OSI group, XYF group, the ferroptosis inhibitor Ferrostatin-1 (F-1) group, and F-1+XYF group, which were respectively intervened with blank serum, 20% drug-containing serum, 2µmol/L F-1, and 2µmol/L F-1+ 20% drug-containing serum. A control group (normal TM3 cells + blank serum) was also set up. The morphology of cells in each group was observed, and the levels of testosterone, superoxide dismutase (SOD), reactive oxygen species (ROS), malondialdehyde (MDA), ferritin heavy chain 1 (FTH1), solute carrier family 7 member 11 (SLC7A11), glutathione (GSH), glutathione peroxidase 4 (GPX4), fatty acid CoA ligase 4 (FACL4), total iron ions, and ferrous ions were detected. RESULTS: Compared with the model group, the control group showed significantly decreased expression of ROS, MDA, FACL4, total iron, and ferrous ions (P<0.05), and significantly increased levels of testosterone, SOD, GSH, FTH1, SLC7A11, and GPX4 (P<0.05). The male silkworm kidney-tonifying formula group significantly promoted testosterone secretion by TM3 cells and upregulated the expression of FTH1, SLC7A11, GPX4, GSH, and SOD in TM3 cells (P<0.05), while significantly downregulating ROS, MDA, FACL4, total iron ions, and ferrous ions (P<0.05). CONCLUSION: Following H2O2 exposure, oxidative stress can induce ferroptosis in mouse TM3 Leydig cells. XYF can antagonize OSI and ferroptosis in TM3 cells by activating the SLC7A11/GSH/GPX4 axis, which may underlie the mechanism of XYF in the treatment of male late-onset hypogonadism.

Expression of SARS-CoV-2 entry molecules ACE2, NRP1, TMPRSS2, and FURIN in the reproductive tissues of male macaques.

Coronavirus disease 2019 (COVID-19) reportedly affects male reproductive function by causing spermatogenesis dysfunction and suppressing testosterone secretion. However, the relationship between COVID-19 and impaired reproductive function, such as whether these effects on reproductive function are a direct effect of severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) infection in male reproductive organs or an indirect effect of high fever, is not known. Here, we examined whether the cell entry molecules of SARS-CoV-2, namely, ACE2, NRP1, TMPRSS2, and FURIN, are expressed in the male reproductive organs using the testes and accessory gonads of macaques during the breeding season. RT-PCR expression analysis showed that the testes alone expressed all four molecules. Immunohistochemical staining of testis tissue sections revealed that ACE2 is expressed in Leydig cells and the apical region of Sertoli cells, whereas NRP1 is expressed in the cell bodies surrounding the Leydig and Sertoli cell nuclei. FURIN is mainly expressed in Leydig cells, secondary spermatocytes, and spermatids. However, TMPRSS2 immunopositive cells were not observed. Therefore, it was not possible to observe cells expressing all four molecules in the gonads and accessory gonads of male primates. These results suggest that SARS-CoV-2 is unlikely to directly affect spermatogenesis in primates or proliferate in cells of the seminiferous tubules and undergo release into the semen through the previously known ACE2-mediated infection route. However, the expression of three molecules, including ACE2, was observed in Leydig cells, suggesting that testosterone synthesis and secretion may be affected when primates, including humans, are infected with SARS-CoV-2.

Diaph1 knockout inhibits mouse primordial germ cell proliferation and affects gonadal development.

BACKGROUND: Exploring the molecular mechanisms of primordial germ cell (PGC) migration and the involvement of gonadal somatic cells in gonad development is valuable for comprehending the origins and potential treatments of reproductive-related diseases. METHODS: Diaphanous related formin 1 (Diaph1, also known as mDia1) was screened by analyzing publicly available datasets (ATAC-seq, DNase-seq, and RNA-seq). Subsequently, the CRISPR-Cas9 technology was used to construct Diaph1 knockout mice to investigate the role of Diaph1 in gonad development. RESULTS: Based on data from public databases, a differentially expressed gene Diaph1, was identified in the migration of mouse PGC. Additionally, the number of PGCs was significantly reduced in Diaph1 knockout mice compared to wild type mice, and the expression levels of genes related to proliferation (Dicer1, Mcm9), adhesion (E-cadherin, Cdh1), and migration (Cxcr4, Hmgcr, Dazl) were significantly decreased. Diaph1 knockout also inhibited Leydig cell proliferation and induced apoptosis in the testis, as well as granulosa cell apoptosis in the ovary. Moreover, the sperm count in the epididymal region and the count of ovarian follicles were significantly reduced in Diaph1 knockout mice, resulting in decreased fertility, concomitant with lowered levels of serum testosterone and estradiol. Further research found that in Diaph1 knockout mice, the key enzymes involved in testosterone synthesis (CYP11A1, 3ß-HSD) were decreased in Leydig cells, and the estradiol-associated factor (FSH receptor, AMH) in granulosa cells were also downregulated. CONCLUSIONS: Overall, our findings indicate that the knockout of Diaph1 can disrupt the expression of factors that regulate sex hormone production, leading to impaired secretion of sex hormones, ultimately resulting in damage to reproductive function. These results provide a new perspective on the molecular mechanisms underlying PGC migration and gonadal development, and offer valuable insights for further research on the causes, diagnosis, and treatment of related diseases.

The origin of ovarian Leydig cells: a possibly solved enigma?

Over the years, the origin of ovarian Leydig cells has been, and still is, a topic subject to deep debate. Seven years ago, we proposed that this origin resided in intraneural elements that came from a possible reservoir of neural crest cells, a reservoir that may be located in the ganglia of the celiac plexus. We believe we have found the evidence necessary to prove this hypothesis.

Improvement in Testosterone Production by Acorus gramineus for the Alleviation of Andropause Symptoms.

Acorus gramineus has a number of beneficial effects, including protective effects against age-related disorders. In this study, the effects of A. gramineus on testosterone production and andropause symptoms were evaluated. We first treated TM3 mouse Leydig cells, responsible for testosterone production, with A. gramineus aqueous extract at different concentrations. In TM3 cells, the testosterone concentration increased in a concentration-dependent manner compared with those in the control. In addition, at 400 µg/mL extract, the mRNA expression level of the steroidogenic enzyme CYP11A1 was increased. Subsequently, 23-week-old Sprague-Dawley (SD) rats exhibiting an age-related reduction in serum testosterone (approximately 80% lower than that in 7-week-old SD rats) were administered A. gramineus aqueous extract for 8 weeks. Serum total testosterone and free testosterone levels were higher and serum estradiol, prostate-specific antigen levels, and total cholesterol levels were lower in the AG50 group (A. gramineus aqueous extract 50 mg/kg of body weight/day) than in the OLD (control group). The AG50 group also showed significant elevations in sperm count, grip strength, and mRNA expression of StAR, CYP11A1, 17ß-HSD, and CYP17A1 compared with those in the OLD group. In conclusion, A. gramineus aqueous extract facilitated steroidogenesis in Leydig cells, elevated testosterone levels, lowered serum estradiol and total cholesterol levels, and increased muscle strength and sperm count, thus alleviating the symptoms of andropause. These findings suggest that A. gramineus aqueous extract is a potentially effective therapeutic agent against various symptoms associated with andropause.

Prevention of Male Late-Onset Hypogonadism by Natural Polyphenolic Antioxidants.

Androgen production primarily occurs in Leydig cells located in the interstitial compartment of the testis. In aging males, testosterone is crucial for maintaining muscle mass and strength, bone density, sexual function, metabolic health, energy levels, cognitive function, as well as overall well-being. As men age, testosterone production by Leydig cells of the testes begins to decline at a rate of approximately 1% per year starting from their 30s. This review highlights recent findings concerning the use of natural polyphenolics compounds, such as flavonoids, resveratrol, and phenolic acids, to enhance testosterone production, thereby preventing age-related degenerative conditions associated with testosterone insufficiency. Interestingly, most of the natural polyphenolic antioxidants having beneficial effects on testosterone production tend to enhance the expression of the steroidogenic acute regulatory protein (Star) gene in Leydig cells. The STAR protein facilitates the entry of the steroid precursor cholesterol inside mitochondria, a rate-limiting step for androgen biosynthesis. Natural polyphenolic compounds can also improve the activities of steroidogenic enzymes, hypothalamus-pituitary gland axis signaling, and testosterone bioavailability. Thus, many polyphenolic compounds such as luteolin, quercetin, resveratrol, ferulic acid phenethyl ester or gigantol may be promising in delaying the initiation of late-onset hypogonadism accompanying aging in males.

MicroRNA-361-5p Alleviates Leydig Cell Apoptosis and Promotes Cell Growth by Targeting PIAS1 in Late-Onset Hypogonadism.

Late-onset hypogonadism (LOH) is an age-related syndrome characterized by deficiency of serum testosterone produced by Leydig cells. Previous evidence suggested that microRNA (miR)-361-3p can serve as a promising biomarker for LOH. Nonetheless, its detailed function and molecular mechanism in LOH remain unclarified. The 24-month-old male mice were selected as an animal LOH model, and mouse Leydig cell line TM3 was stimulated with H2O2. ELISA was employed for testosterone level evaluation. Hematoxylin-eosin staining was implemented for histologic analysis of mouse testicular tissues. Western blotting and RT-qPCR were utilized for evaluating molecular protein and RNA expression, respectively. Functional experiments were conducted to test miR-361-5p roles. Luciferase reporter assay was for verifying the interaction between miR-361-5p and protein inhibitor of activated STAT 1 (PIAS1). miR-361-5p displayed a decreased level in the testes of LOH mice. Overexpressing miR-361-5p attenuated Leydig cell loss in the testis and elevated serum and intratesticular testosterone levels in LOH mice. H2O2 stimulation impaired TM3 cell viability, proliferation and intracellular testosterone production and enhanced cell apoptosis. miR-361-5p targeted PIAS1 in TM3 cell. PIAS1 upregulation counteracted miR-361-5p overexpression-mediated alleviation of cell apoptosis and elevation of testosterone synthesis in H2O2-stimualetd TM3 cells. miR-361-5p ameliorates LOH progression by increasing testosterone production and alleviate Leydig cell apoptosis via downregulation of PIAS1.