Leydig Cell Testosterone Production Research Articles

Effects of empagliflozin on reproductive system in men without diabetes

Sodium-glucose cotransporter (SGLT) 2 inhibition is a well-known target for the treatment of type 2 diabetes, renal disease and chronic heart failure. The protein SGLT2 is encoded by SLC5A2 (Solute Carrier Family 5 Member 2), which is highly expressed in renal cortex, but also in the testes where glucose uptake may be essential for spermatogenesis and androgen synthesis. We postulated that in healthy males, SGLT2 inhibitor therapy may affect gonadal function. We examined the impact on gonadal and steroid hormones in a post-hoc analysis of a double-blind, randomized, placebo-controlled research including 26 healthy males who were given either placebo or empagliflozin 10 mg once daily for four weeks. After one month of empagliflozin, there were no discernible changes in androgen, pituitary gonadotropin hormones, or inhibin B. Regardless of BMI category, the administration of empagliflozin, a highly selective SGLT2 inhibitor, did not alter serum androgen levels in men without diabetes. While SGLT2 is present in the testes, its inhibition does not seem to affect testosterone production in Leydig cells nor inhibin B secretion by the Sertoli cells.

Bioactive Components in Black Currant Fruit, Red Perilla (Shiso) Leaf, and Chinese Sweet Tea That Enhance Testosterone Production in Leydig Cells and Their Combined Effect in Male Mice

Bioactive Components in Black Currant Fruit, Red Perilla (Shiso) Leaf, and Chinese Sweet Tea That Enhance Testosterone Production in Leydig Cells and Their Combined Effect in Male Mice

Effect of Curcuma longa extract on reproduction function in mice and testosterone production in Leydig cells.

Curcuma longa, best known for its culinary application as the main constituent of curry powder, has shown potential impact on the reproductive system. This study aimed to investigate the efficacy of Curcuma longa extract (CLE) on Kidney-Yang deficiency mice induced by hydrocortisone and the possible roles in testosterone secretion in Leydig cells. We evaluated male sexual behaviour, reproductive organ weight, testosterone levels, and histological tissue changes in hydrocortisone-induced mice. CLE effectively reversed hydrocortisone-induced Kidney-Yang deficiency syndrome by improving sexual behaviour, testis and epididymis weight, testosterone levels and reducing pathological damage. Our invitro study further indicated that CLE stimulated testosterone production via upregulating the mRNA and protein expression of steroidogenic enzymes in Leydig cells. It significantly improved H89-inhibited protein expression of StAR and cAMP-response element-binding (CREB), as well as melatonin-suppressed StAR protein expression. The data obtained from this study suggest that CLE could alleviate Kidney-Yang deficiency symptoms and stimulate testosterone production by upregulating the steroidogenic pathway. This research identifies CLE as a potential nutraceutical option for addressing testosterone deficiency diseases.

METTL3-m6A-SIRT1 axis affects autophagic flux contributing to PM2.5-induced inhibition of testosterone production in Leydig cells

Epidemiological studies have found that long-term inhalation of PM2.5 is closely related to spermatogenesis disorders and infertility, but the underlying molecular mechanism is still unidentified. Testosterone, an essential reproductive hormone produced by Leydig cells, whose synthesis is disrupted by multiple environmental pollutants. In the current study, we explored the role of METTL3-m6A-SIRT1 axis-mediated abnormal autophagy in PM2.5-induced inhibition of testosterone production in in vivo and in vitro models. Our in vivo findings shown that long-term inhalation of PM2.5 decreased sperm count, increased sperm deformity rates, and altered testicular interstitial morphology accompanied by reduced testosterone in serum and testes. Further, data from the in vitro model displayed that exposure to PM2.5 caused an increase in m6A modification and METTL3 levels, followed by a decrease in testosterone levels and autophagy dysfunction in Leydig cells. The knockdown of METTL3 promotes autophagy flux and testosterone production in Leydig cells. Mechanistically, PM2.5 increased METTL3-induced m6A modification of SIRT1 mRNA in Leydig cells, bringing about abnormal autophagy. Subsequently, administration of SRT1720 (a SIRT1 activator) enhanced autophagy and further promoted testosterone biosynthesis. Collectively, our discoveries indicate that METTL3-m6A-SIRT1 axis-mediated autophagic flux contributes to PM2.5-induced inhibition of testosterone biosynthesis. This research offers a novel viewpoint on the mechanism of male reproductive injury following PM2.5 exposure.

In vitro effects of uncarboxylated osteocalcin on buffalo Leydig cell steroidogenesis.

Uncarboxylated osteocalcin (UcOCN), a bone derived circulating protein, has been demonstrated to influence steroidogenesis in testicular Leydig cells of murine and human species. However, the role of UcOCN in testosterone biosynthesis remains unexplored in domestic animals. The present study aimed to investigate the impact of UcOCN on the expressions of steroidogenic genes (HSD3β1, HSD3β6, CYP17A1, CYP11A1), testosterone production and GPRC6A receptor localization in buffalo Leydig cells. Leydig cells from the testes of adult Murrah buffalo were isolated, with an average cell count and viability after digestion and Percoll enrichment of 1.43 × 106 cells/g of testes and 78.5%, respectively. Immunophenotyping of Percoll-enriched cell suspension by flow cytometry showed populations of Leydig cells ranging between 69 and 73.9%. Immunostaining confirmed the presence of GPRC6A receptors and CYP11A1 positive Leydig cells. When these cells were cultured and incubated with varying levels of UcOCN (6, 12, 24, and 48 ng/ml) and LH, there was a significant (P < 0.01) increase in testosterone production and up-regulation (P < 0.05) of CYP11A1, CYP17A1, HSD3β1 and HSD3β6 gene expression. In summary, the present study underscored the effects of UcOCN on testosterone biosynthesis, expression of crucial steroidogenic genes and interaction with GPRC6A receptors in buffalo Leydig cells, emphasizing its potential implications in andrology.

Protective effect of gomisin N on benzyl butyl phthalate-induced dysfunction of testosterone production in TM3 Leydig cells.

Exposure to benzyl butyl phthalate (BBP) may induce disorders in the male reproductive system. However, the molecular mechanisms remain unknown. Here we investigated the effect of BBP on testosterone production and its molecular mechanisms. Furthermore, we also investigated the role of gomisin N (GN) from Schisandra chinensis (S. chinensis) in testosterone synthesis in TM3 Leydig cells. First, we examined the effects of BBP on expression levels of testosterone biosynthesis-related genes (StAR, CYP11α1, CYP17α1, 3βHSD, and 17βHSD) and attenuation-related genes (CYP1β1, CYP19α1, and Srd5α1-3). Although testosterone biosynthesis-related genes did not change, attenuation-related genes such as CYP1β1 and CYP19α1 were upregulated with ROS generation and testosterone level attenuation in the presence of 50 µM of BBP. However, the compound with the highest ROS and ONOO- scavenging activity from S. chinensis, GN, significantly reversed the expression of BBP-induced testosterone attenuation-related gene to normal levels. Subsequently, GN improved the testosterone production levels in TM3 Leydig cells. These events may be regulated by the antioxidant effect of GN. On conclusion, our study suggests, for the first time, that BBP impairs testosterone synthesis by the modulation of CYP1β1 and CYP19α1 expression in TM3 cells; GN could potentially minimize the BBP-induced dysfunction of TM3 cells to produce testosterone by suppressing CYP19α1 expression.

Protective effect of TNFAIP3 on testosterone production in Leydig cells under an aging inflammatory microenvironment

BackgroundThe aging inflammatory microenvironment surrounding Leydig cells is linked to reduced testosterone levels in males. Tumor necrosis factor alpha-induced protein 3 (TNFAIP3) acts as a critical anti-inflammatory factor in various aging-related diseases. This study aims to investigate the protective effect of TNFAIP3 on testosterone production in Leydig cells under an aging inflammatory microenvironment. MethodsBioinformatics analysis examined TNFAIP3 expression differences in aging rat testes and validated the findings in aging mouse testes. In vitro models of inflammation were established using two Leydig cell lines, with tumor necrosis factor alpha (TNF-α) as the inflammatory factor. Lentiviral transduction was utilized to manipulate TNFAIP3 expression in these cell lines. Transcriptomic sequencing identified differentially expressed genes in TNFAIP3-overexpressing cells. ResultsBioinformatics analysis and validation experiments revealed increased inflammatory signaling and elevated TNFAIP3 expression in aging rat and mouse testes. TNFAIP3 knockdown worsened testosterone synthesis inhibition and apoptosis in cells, while TNFAIP3 overexpression reversed these effects. Transcriptome analysis identified alterations in the P38MAPK pathway following TNFAIP3 overexpression. TNFAIP3 knockdown enhanced TNF-induced P38MAPK signaling, whereas its overexpression attenuated this effect. TNFAIP3 was found to regulate testosterone synthesis by upregulating CEBPB expression. ConclusionsTNFAIP3 exhibits inhibitory effects on apoptosis and promotes testosterone production in Leydig cells. The protective influence of TNFAIP3 on Leydig cells within an inflammatory microenvironment is likely mediated through by inhibiting the P38MAPK pathway and upregulating CEBPB expression.

Betaine regulates steroidogenesis, endoplasmic reticulum stress response and Nrf2/HO-1 antioxidant pathways in mouse Leydig cells under hyperglycaemia condition

We studied the effects of betaine on steroidogenesis, endoplasmic reticulum stress and Nrf2 antioxidant pathways of mice Leydig cells under hyperglycaemia conditions. Leydig cells were grown in low and high glucose concentrations (5 mM and 30 mM) in the presence of 5 mM of betaine for 24 h. Gene expression was determined using a real-time PCR method. The protein levels were determined by Western blot analysis. The testosterone production was evaluated by the ELISA method. Cellular contents of reduced and oxidised glutathione were measured by colorimetric method. Hyperglycaemia caused impaired steroidogenesis and ERS in Leydig cells associated with the down-regulation of 3β-HSD, StAR, P450scc, LH receptor and increased expression of GRP78, CHOP, ATF6 and IRE1. Betaine could improve cell viability, attenuate the ERS, and restore testosterone production in Leydig cells under hyperglycaemia conditions. Betaine can protect Leydig cells against the adverse effects of hyperglycaemia by regulating steroidogenesis, antioxidants, and ERS.

SARS-CoV-2 replicates in the human testis with slow kinetics and has no major deleterious effects ex vivo.

SARS-CoV-2 is a new virus responsible for the Covid-19 pandemic. Although SARS-CoV-2 primarily affects the lungs, other organs are infected. Alterations of testosteronemia and spermatozoa motility in infected men have raised questions about testicular infection, along with high level in the testis of ACE2, the main receptor used by SARS-CoV-2 to enter host cells. Using an organotypic culture of human testis, we found that SARS-CoV-2 replicated with slow kinetics in the testis. The virus first targeted testosterone-producing Leydig cells and then germ-cell nursing Sertoli cells. After a peak followed by the upregulation of antiviral effectors, viral replication in the testis decreased and did not induce any major damage to the tissue. Altogether, our data show that SARS-CoV-2 replicates in the human testis to a limited extent and suggest that testicular damages in infected patients are more likely to result from systemic infection and inflammation than from viral replication in the testis.

Yangjing Capsule Increases Testosterone Production through SET/PI3K/Akt Pathway in Leydig Cell

Background. Our previous works revealed Yangjing capsule (YC) could inhibit cell apoptosis and promote testosterone production in Leydig cells. SET protein has been reported to be important in regulating testosterone synthesis and apoptosis. The purpose of this study was to investigate whether the steroidogenic and antiapoptotic effects of YC are mediated in part by the SET/PI3K/Akt pathway in Leydig cells. Methods. MLTC-1 cells were treated with YC-medicated serum. si-RNA was used to knockdown SET expression in MLTC-1 cells. The elderly BALB/c mice were treated with different doses of YC extract. Flow cytometry and TUNEL staining were used to assess apoptosis in MLTC-1 cells and mouse testes. HE staining was conducted to detect the morphological changes in mouse testes. Testosterone levels in cell culture medium and serum were measured by ELISA kit. Quantitative real-time reverse-transcription polymerase chain reaction (qRT-PCR) and Western blot were used to detect the expression levels of key molecules. Results. YC-medicated serum could significantly increase the expression of SET and StAR, P450scc, HSD17B, and testosterone production in MLTC-1 cells. YC-medicated serum could increase Akt phosphorylation and Bcl-2/Bax ratio to suppress apoptosis in MLTC-1 cells. SET knockdown significantly inhibited YC-medicated serum-induced steroidogenic and antiapoptotic effects in MLTC-1 cells. In vivo experiments revealed the YC extract could enhance SET expression, trigger Akt phosphorylation, and suppress apoptosis in mouse testis to raise serum testosterone levels. Conclusions. A possible mechanism of promoting testosterone production induced by YC in Leydig cell was by triggering SET/PI3K/Akt pathway.

Hyperhomocysteinemia lowers serum testosterone concentration via impairing testosterone production in Leydig cells.

Hyperhomocysteinemia (HHcy) plays a salient role in male infertility. However, whether HHcy interferes with testosterone production remains inconclusive. Here, we reported a lower serum testosterone level in HHcy mice. Single-cell RNA sequencing revealed that genes related to testosterone biosynthesis, together with nuclear receptor subfamily 5 group A member 1 (Nr5a1), a key transcription factor for steroidogenic genes, were downregulated in the Leydig cells (LCs) of HHcy mice. Mechanistically, Hcy lowered trimethylation of histone H3 on lysine 4 (H3K4me3), which was bound on the promoter region of Nr5a1, resulting in downregulation of Nr5a1. Intriguingly, we identified an unknown cell cluster annotated as Macrophage-like Leydig cells (McLCs), expressing both LCs and macrophages markers. In HHcy mice, McLCs were shifted toward pro-inflammatory phenotype and thus promoted inflammatory response in LC. Betaine supplementation rescued the downregulation of NR5A1 and restored the serum testosterone level in HHcy mice. Overall, our study highlights an etiological role of HHcy in LCs dysfunction.

Selenium nanoparticles improve nickel-induced testosterone synthesis disturbance by down-regulating miR-708-5p/p38 MAPK pathway in Leydig cells.

The present study was designed to investigate the role of miR-708-5p/p38 mitogen-activated protein kinase (MAPK) pathway during the mechanism of selenium nanoparticles (Nano-Se) against nickel (Ni)-induced testosterone synthesis disorder in rat Leydig cells. We conducted all procedures based on in vitro culture of rat primary Leydig cells. After treating Leydig cells with Nano-Se and NiSO4 alone or in combination for 24 h, we determined the cell viability, reactive oxygen species (ROS) levels, testosterone production, and the protein expression of key enzymes involved in testosterone biosynthesis: steroidogenic acute regulatory (StAR) and cytochrome P450 cholesterol side chain cleavage enzyme (CYP11A1). The results indicated that Nano-Se antagonized cytotoxicity and eliminated ROS generation induced by NiSO4 , suppressed p38 MAPK protein phosphorylation and reduced miR-708-5p expression. Importantly, we found that Nano-Se upregulated the expression of testosterone synthase and increased testosterone production in Leydig cells. Furthermore, we investigated the effects of p38 MAPK and miR-708-5p using their specific inhibitor during Nano-Se against Ni-induced testosterone synthesis disorder. The results showed that Ni-inhibited testosterone secretion was alleviated by Nano-Se co-treatment with p38 MAPK specific inhibitor SB203580 and miR-708-5p inhibitor, respectively. In conclusion, these findings suggested Nano-Se could inhibit miR-708-5p/p38 MAPK pathway, and up-regulate the key enzymes protein expression for testosterone synthesis, thereby antagonizing Ni-induced disorder of testosterone synthesis in Leydig cells.

Exposure to acrylamide inhibits testosterone production in mice testes and Leydig cells by activating ERK1/2 phosphorylation.

Acrylamide (ACR) is formed during the cooking of starchy foods at high temperatures. Accumulating evidence has shown that ACR has toxic effects, but the mechanism of its potential reproductive toxicity remains unclear. In this study, we observed that ACR caused weight loss in mice. There was no significant difference in the weight of testis and epididymis between the low/medium-dose ACR group and the control group. And the number of epididymal sperms, testicular Leydig cells, serum testosterone level, testicular steroidogenic genes and enzymes, including cytochrome P450 family 11 subfamily A member 1 (CYP11A1) and cytochrome P450 family 17 subfamily A member 1 (CYP17A1), were decreased in the medium/high-dose ACR group. Additional cell experiments showed that the apoptosis rate and the level of reactive oxygen species (ROS) were increased, and testosterone levels and CYP17A1 protein expression were reduced in Leydig cells with treated ACR. Furthermore, the phosphorylation levels of extracellular signal-regulated kinases (ERK1/2) increased significantly; however, there was no significant difference in the levels of serine-threonine protein kinase (AKT) phosphorylation in the testis of mice and Leydig cells treated with ACR. These results suggest that ACR exposure leads to the damage of testicular structure and function and a decline in testosterone synthesis in Leydig cells and mouse testis, which may be related to the activated phosphorylation of ERK1/2.

Effect of Organic Zinc Supplementation on Semen Production and Quality in Kankrej Bulls

Background: Zinc is found in very high concentration in semen and male reproductive tract and acts as a hormone receptor modulator for testosterone production in Leydig cells. Thus, it plays a very important role in male reproduction. Moreover, Indian soils are very deficient in zinc as per earlier reports and recent studies have shown that supplementation of zinc has improved qualitative as well as the quantitative aspects of semen quality in bulls. So, a study has been planned to study the effect of organic zinc supplementation on semen production and quality in kankrej bulls. Methods: A total of 8 healthy Kankrej bulls under semen collection were selected for the study. Basal diet (concentrate mixture, green and dry fodder) was fed during pre-supplementation and zinc propionate @ 100 ppm/bull was supplemented to the basal diet during post-supplementation for a period of 60 days to complete one spermatogenic cycle. A total of 128 ejaculates (64 pre-supplementation and 64 post-supplementation) were collected. Result: Mean semen volume was non-significant (P greater than 0.05) between the treatment groups. Mean sperm concentration was 1325.68±80.08 and 2162.36±112.0 million/ml pre- and post-supplementation, respectively. Feeding of zinc propionate significantly (P less than 0.01) increased semen sperm concentration. The average mass motility of sperms was significantly (P less than 0.01) higher during post-supplementation (3.92%) than the pre-supplementation (3.78%). The overall live sperms were significantly (P less than 0.01) higher during post-supplementation (73.33±0.91%) than the pre-supplementation (63.53±1.03%). It may be concluded that supplementation of 100 ppm of zinc propionate in the diet of Kankrej bulls improved semen quality in terms of quantitative and qualitative characteristics of semen.

Comprehensive and Quantitative Analysis of the Changes in Proteomic and Phosphoproteomic Profiles during Stimulation and Repression of Steroidogenesis in MA-10 Leydig Cells.

Leydig cells produce testosterone, a hormone essential for male sex differentiation and spermatogenesis. The pituitary hormone, LH, stimulates testosterone production in Leydig cells by increasing the intracellular cAMP levels, which leads to the activation of various kinases and transcription factors, ultimately stimulating the expression of the genes involved in steroidogenesis. The second messenger, cAMP, is subsequently degraded to AMP, and the increase in the intracellular AMP levels activates AMP-dependent protein kinase (AMPK). Activated AMPK potently represses steroidogenesis. Despite the key roles played by the various stimulatory and inhibitory kinases, the proteins phosphorylated by these kinases during steroidogenesis remain poorly characterized. In the present study, we have used a quantitative LC-MS/MS approach, using total and phosphopeptide-enriched proteins to identify the global changes that occur in the proteome and phosphoproteome of MA-10 Leydig cells during both the stimulatory phase (Fsk/cAMP treatment) and inhibitory phase (AICAR-mediated activation of AMPK) of steroidogenesis. The phosphorylation levels of several proteins, including some never before described in Leydig cells, were significantly altered during the stimulation and inhibition of steroidogenesis. Our data also provide new key insights into the finely tuned and dynamic processes that ensure adequate steroid hormone production.

Disruption of Leydig cell steroidogenic function by sodium arsenite and/or sodium fluoride

Arsenite (As) and fluoride (F), both of which are linked to a variety of human ailments, are regularly found in underground drinking water. Numerous studies have shown that As and/or F have negative impacts on testicular function and fertility. For this purpose, mouse Leydig cells, the main cells responsible for the generation and regulation of steroid hormones such as testosterone, were used to reveal the effects of individual and combined exposure of As and F on the steroidogenic pathway in the male reproductive system. Leydig cells were treated with 0.39 μM (50 ppb) As and 0.0476 mM (2 ppm) F alone and in combination for 24 h. The findings revealed that As and/or F exposure induced oxidative stress and apoptosis in Leydig cells and altered antioxidant equilibrium of the cells by reducing superoxide dismutase, catalase, glutathione peroxidase. Additionally, individual and combined administration of As and/or F significantly supressed the expression of both steroidogenic enzymes and the genes encoding these enzymes. In conclusion, this study showed that exposure to As and F at environmentally relevant concentrations dispersed by water decreased testosterone production in Leydig cells, an important cell of the male reproductive system. The deleterious effects of even the lowest concentrations of As and F elements that can reach humans from the environment on the Leydig cell, and therefore on male infertility, emphasize necessity new safe limits for these elements.

Local adrenomedullin gene delivery inhibits Leydig cell dysfunction by rescuing steroidogenic enzymes in vivo.

Adrenomedullin (ADM) has beneficial effects on Leydig cells under pathological conditions, including lipopolysaccharide (LPS)-induced orchitis. Our previous studies demonstrated that ADM exerts a restorative effect on steroidogenesis in LPS-treated primary rat Leydig cells by attenuating oxidative stress, inflammation and apoptosis. In this study, we aim to investigate whether ADM inhibits Leydig cell dysfunction by rescuing steroidogenic enzymes in vivo. Rats were administered with LPS and injected with Ad-ADM, an adeno-associated virus vector that expressed ADM. Then, rat testes were collected for 3β-hydroxysteroid dehydrogenase (3β-HSD) immunofluorescence staining. Steroidogenic enzymes or steroidogenic regulatory factors or protein, including steroidogenic factor-1 (SF-1), liver receptor homologue-1 (LRH1), Nur77, steroidogenic acute regulatory protein (StAR), cytochrome P450 cholesterol side chain cleavage enzyme (P450scc), 3β-HSD, cytochrome P450 17α-hydroxylase/17, 20 lyase (CYP17) and 17β-hydroxysteroid dehydrogenase (17β-HSD), were detected via gene expression profiling and western blot analysis. Plasma testosterone concentrations were measured. Results showed that ADM may inhibit Leydig cell dysfunction by rescuing steroidogenic enzymes and steroidogenic regulatory factors in vivo. The reduction in the number of Leydig cells after LPS exposure was reversed by ADM. ADM rescued the gene or protein levels of SF-1, LRH1, Nur77, StAR, P450scc, 3β-HSD, CYP17 and 17β-HSD and plasma testosterone concentrations. To summarize ADM could rescue some important steroidogenic enzymes, steroidogenic regulatory factors and testosterone production in Leydig cells in vivo.

Single-cell analysis of human testis aging and correlation with elevated body mass index.

Aging men display reduced reproductive health; however, testis aging is poorly understood at the molecular and genomic levels. Here, we utilized single-cell RNA-seq to profile over 44,000 cells from both young and older men and examined age-related changes in germline development and in the testicular somatic cells. Age-related changes in spermatogonial stem cells appeared modest, whereas age-related dysregulation of spermatogenesis and somatic cells ranged from moderate to severe. Altered pathways included signaling and inflammation in multiple cell types, metabolic signaling in Sertoli cells, hedgehog signaling and testosterone production in Leydig cells, cell death and growth in testicular peritubular cells, and possible developmental regression in both Leydig and peritubular cells. Remarkably, the extent of dysregulation correlated with body mass index in older but not in younger men. Collectively, we reveal candidate molecular mechanisms underlying the complex testicular changes conferred by aging and their possible exacerbation by concurrent chronic conditions such as obesity.

MP38-11 GENERATION OF LEYDIG CELLS DERIVED FROM HUMAN iPS CELLS

You have accessJournal of UrologyCME1 May 2022MP38-11 GENERATION OF LEYDIG CELLS DERIVED FROM HUMAN iPS CELLS Katsuya Sato, Masato Fujisawa, and Takashi Aoi Katsuya SatoKatsuya Sato More articles by this author , Masato FujisawaMasato Fujisawa More articles by this author , and Takashi AoiTakashi Aoi More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000002592.11AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: We have successfully induced human iPS cells (hiPSCs) to differentiate into testosterone-producing Leydig cells by forcibly expressing NR5A1 and forming embryoid bodies (Ishida et al., 2021). However, there were several problems with the conventional method. The amount of testosterone secreted by the generated cells was low, and the life span of the cells was short. Furthermore, the efficiency of differentiation into Leydig cells was unknown. To solve these problems, it was necessary to improve the method of differentiation induction. OBJECTIVE: 1) To improve the method of differentiation of iPS cells into Leydig cells. 2) To increase the amount of testosterone secreted by the cells. 3) To generate longer-lived Leydig cells. 4) To clarify the efficiency of differentiation into Leydig cells. METHODS: The duration of embryoid body formation and culture conditions for induction of differentiation were optimized by evaluating testosterone levels in the culture supernatant and expression of several known Leydig cell marker genes. Changes in cell morphology during differentiation were visualized using a microscope equipped with a time-lapse function. HSD17B3 immunostaining was performed and its positivity was used to measure the efficiency of differentiation into Leydig cells. RESULTS: Morphological changes and gene expression during differentiation induction were confirmed. With differentiation, Leydig markers were upregulated. The amount of testosterone secreted by the generated cells increased about three times compared to the conventional method. The generated cells were able to survive for more than 120 days. Evaluation of HSD17B3 immunostaining showed that the efficiency of differentiation into Leydig cells was more than 99%. CONCLUSIONS: We have succeeded in improving the differentiation method of Leydig cells from iPS cells.In the future, we are planning to evaluate the in vivo function and safety of hiPSC-derived Leydig cells in transplantation experiments using animals. In the future, this technology can be applied to regenerative medicine for patients with LOH syndrome. Source of Funding: No corporate funding © 2022 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 207Issue Supplement 5May 2022Page: e622 Advertisement Copyright & Permissions© 2022 by American Urological Association Education and Research, Inc.MetricsAuthor Information Katsuya Sato More articles by this author Masato Fujisawa More articles by this author Takashi Aoi More articles by this author Expand All Advertisement PDF DownloadLoading ...

Activation of Autophagy by Low-Dose Silica Nanoparticles Enhances Testosterone Secretion in Leydig Cells.

Silica nanoparticles (SNPs) can cause abnormal spermatogenesis in male reproductive toxicity. However, the toxicity and toxicological mechanisms of SNPs in testosterone synthesis and secretion in Leydig cells are not well known. Therefore, this study aimed to determine the effect and molecular mechanism of low doses of SNPs in testosterone production in Leydig cells. For this, mouse primary Leydig cells (PLCs) were exposed to 100 nm Stöber nonporous spherical SNPs. We observed significant accumulation of SNPs in the cytoplasm of PLCs via transmission electron microscopy (TEM). CCK-8 and flow cytometry assays confirmed that low doses (50 and 100 μg/mL) of SNPs had no significant effect on cell viability and apoptosis, whereas high doses (more than 200 μg/mL) decreased cell viability and increased cell apoptosis in PLCs. Monodansylcadaverine (MDC) staining showed that SNPs caused the significant accumulation of autophagosomes in the cytoplasm of PLCs. SNPs activated autophagy by upregulating microtubule-associated protein light chain 3 (LC3-II) and BCL-2-interacting protein (BECLIN-1) levels, in addition to downregulating sequestosome 1 (SQSTM1/P62) level at low doses. In addition, low doses of SNPs enhanced testosterone secretion and increased steroidogenic acute regulatory protein (StAR) expression. SNPs combined with rapamycin (RAP), an autophagy activator, enhanced testosterone production and increased StAR expression, whereas SNPs combined with 3-methyladenine (3-MA) and chloroquine (CQ), autophagy inhibitors, had an opposite effect. Furthermore, BECLIN-1 depletion inhibited testosterone production and StAR expression. Altogether, our results demonstrate that low doses of SNPs enhanced testosterone secretion via the activation of autophagy in PLCs.