Tebuconazole Induces Mouse Fetal Testes Damage via ROS Generation in an Organ Culture Method.

The fungicide tebuconazole (TEB) poses risks to human and animal health via various exposure routes. It induces toxicity in multiple organs and disrupts reproductive health by affecting steroid hormone synthesis and fetal development. In this study, we investigated the impact of TEB on fetal testes using in vitro models, focusing on germ, Sertoli, and Leydig cells, and explored the mechanisms underlying cellular damage. The results revealed significant damage to germ cells and disruption of Leydig cell development. TEB exposure led to a decrease in germ cell numbers, as indicated by histological and immunostaining analyses. TEB induced the up- and down-regulation of the expression of fetal and adult Leydig cell markers, respectively. Additionally, TEB-treated fetal testes exhibited increased expression of oxidative-stress-related genes and proteins. However, co-treatment with the antioxidant N-acetylcysteine mitigated TEB-induced germ cell damage and prevented abnormal Leydig cell development. These findings suggest that administration of antioxidants can prevent the intratesticular damage typically caused by TEB exposure.

Loss of Function of Vasoactive-intestinal Peptide Alters Sex Ratio and Reduces Male Reproductive Fitness in Zebrafish.

Vasoactive-intestinal peptide (Vip) is a pleiotropic peptide with a wide range of distribution and functions. Zebrafish possess 2 isoforms of Vip (a and b), in which Vipa is most homologous to the mammalian form. In female zebrafish, Vipa can stimulate LH secretion from the pituitary but is not essential for female reproduction, as vipa-/- females display normal reproduction. In contrast, we have found that vipa-/- males are severely subfertile and sex ratio of offspring is female-biased. By analyzing all aspects of male reproduction with wild-type (WT) males, we show that the testes of vipa-/- are underdeveloped and contain ∼70% less spermatids compared to WT counterparts. The sperm of vipa-/- males displayed reduced potency in terms of fertilization (by ∼80%) and motility span and duration (by ∼50%). In addition, vipa-/- male attraction to WT females was largely nonexistent, indicating decreased sexual motivation. We show that vipa mRNA and protein is present in Leydig cells and in developing germ cells in the testis of WT, raising the possibility that endogenous Vipa contributes to testicular function. Absence of Vipa in vipa-/- males resulted in downregulation of 3 key genes in the androgen synthesis chain in the testis, 3ß-hsd, 17ß-hsd1, and cyp11c1 (11ß-hydrogenase), associated with a pronounced decrease in 11-ketotestosterone production and, in turn, compromised reproductive fitness. Altogether, this study establishes a crucial role for Vipa in the regulation of male reproduction in zebrafish, like in mammals, with the exception that Vipa is also expressed in zebrafish testis.

Apigenin improves testosterone synthesis by regulating endoplasmic reticulum stress.

Obesity is a growing epidemic among reproductive-age men, which can cause and exacerbate male infertility by means of associated comorbidities, endocrine abnormalities, and direct effects on the fidelity and throughput of spermatogenesis. A prominent consequence of male obesity is a reduction in testosterone levels. Natural products have shown tremendous potential anti-obesity effects in metabolic diseases. This study aimed to investigate the potential of apigenin (AP) to alleviate testicular dysfunction induced by a high-fat diet (HFD) and to investigate the underlying mechanisms, focusing on endoplasmic reticulum stress (ERS) and testosterone synthesis. A murine model of obesity was established using HFD-fed mice. The effects of AP on obesity, lipid metabolism, testicular dysfunction, and ERS were assessed through various physiological, histological, and molecular techniques. Administration of AP (10 mg/kg) ameliorated HFD-induced obesity and testicular dysfunction in a mouse model, as evidenced by decreased body weight, improved lipid profiles and testicular pathology, and restored protein levels related to testosterone. Furthermore, in vitro studies demonstrated that AP relieved ERS and recovered testosterone synthesis in murine Leydig cells (TM3) treated with free fatty acids (FFAs). It was also observed that AP rescued testosterone synthesis enzymes in TM3 cells, similar to that observed with the inhibitor of the PERK pathway (GSK2606414). In addition, ChIP, qPCR, and gene silencing showed that the C/EBP homologous protein (CHOP) bound directly to the promoter region of steroidogenic STAR and negatively modulated its expression. Collectively, AP has remarkable potential to alleviate HFD-induced obesity and testicular dysfunction. Its protective effects are attributable partly to mitigating ERS and restoring testosterone synthesis in Leydig cells.

Polystyrene microplastics trigger testosterone decline via GPX1.

As an emerging environmental endocrine disruptor, polystyrene microplastics (PS-MPs) are considered to have the anti-androgenic feature and impair male reproductive function. To explore the adverse effects of PS-MPs on testosterone synthesis and male reproduction and further elucidate underlying mechanisms, BALB/c mice and Leydig cells were employed in the present work. The results indicated that 50 µm PS-MPs accumulated in mouse testes and were internalized into the cytoplasm. This not only damaged the testicular histomorphology and ultrastructure, but also reduced the viability of Leydig cells and the serum level of GnRH, FSH, LH, and testosterone. After PS-MPs exposure, the ubiquitination degradation and miR-425-3p-targeted modulation synergistically contributed to the suppression of GPX1, which induced oxidative stress and subsequently activated the PERK-EIF2α-ATF4-CHOP pathway of endoplasmic reticulum (ER) stress. The transcription factor CHOP positively regulated the expression of SRD5A2 by directly binding to its promoter region, thereby accelerating testosterone metabolism and ultimately lowing testosterone levels. Besides, PS-MPs compromised testosterone homeostasis via interfering with the hypothalamic-pituitary-testis (HPT) axis. Taken together, PS-MPs possess an anti-androgenic characteristic and exert male reproductive damage effects. The antioxidant enzyme GPX1 plays a crucial role in the PS-MPs-mediated testosterone decline.

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.

Chlormequat Chloride Inhibits TM3 Leydig Cell Growth via Ferroptosis-Initiated Inflammation.

Ferroptosis hallmarked by lipid peroxidation and iron homeostasis imbalance is involved in the occurrence and development of various diseases. The plant growth regulator chlormequat chloride (CCC) can contribute to the causality and exacerbation of reproductive disorders. However, the mechanism by which CCC may cause Leydig cell attenuation remains poorly understood. In this study, TM3 Leydig cells were used to investigate the inhibitory effect of CCC on cell growth and its possible mechanism. The results showed that CCC caused apoptosis, pyroptosis, ferroptosis and necroinflammation in TM3 cells. By comparing the effects of ferroptosis inhibitor Ferrostatin-1 (Fer-1) and pan-Caspase inhibitor Z-VAD-FMK (ZVF) on lipid peroxidation and Caspase-mediated regulated cell death (RCD), we found that Fer-1 was better at rescuing the growth of TM3 cells than ZVF. Although ZVF reduced mitochondrial ROS level and inhibited the activation of Caspase3 and Caspase1, it could not significantly ameliorate lipid peroxidation and the levels of IL-1ß and HMGB1 like Fer-1. Therefore, ferroptosis might be a key non apoptotic RCD mode responsible for CCC-driven inflammation, leading to weakened viability and proliferation of TM3 cells. In addition, overexpression of ferritin light chain (FTL) promoted the resistance of TM3 cells to CCC-induced ferroptosis-mediated inflammation and to some extent improved the inhibition of viability and proliferation. Altogether, ferroptosis-initiated inflammation might play a key role in CCC-impaired TM3 cell growth.

Inhibition of WNT/ß-catenin signalling during sex-specific gonadal differentiation is essential for normal human fetal testis development.

Sex-specific gonadal differentiation is directed by complex signalling promoting development in either male or female direction, while simultaneously inhibiting the opposite pathway. In mice, the WNT/ß-catenin pathway promotes ovarian development and the importance of actively inhibiting this pathway to ensure normal testis development has been recognised. However, the implications of alterations in the tightly regulated WNT/ß-catenin signalling during human fetal gonad development has not yet been examined in detail. Thus, the aim of this study was to examine the consequences of dysregulating the WNT/ß-catenin signalling pathway in the supporting cell lineage during sex-specific human fetal gonad development using an established and extensively validated ex vivo culture model. Inhibition of WNT/ß-catenin signalling in human fetal ovary cultures resulted in only minor effects, including reduced secretion of RSPO1 and reduced cell proliferation although this was not consistently found in all treatment groups. In contrast, promotion of WNT/ß-catenin signalling in testes severely affected development and function. This included disrupted seminiferous cord structures, reduced cell proliferation, reduced expression of SOX9/AMH, reduced secretion of Inhibin B and AMH as well as loss of the germ cell population. Additionally, Leydig cell function was markedly impaired with reduced secretion of testosterone, androstenedione and INSL3. Together, this study suggests that dysregulated WNT/ß-catenin signalling during human fetal gonad development severely impairs testicular development and function. Importantly, our study highlights the notion that sufficient inhibition of the opposite pathway during sex-specific gonadal differentiation is essential to ensure normal development and function also applies to human fetal gonads.

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.

Single-cell transcriptome profiling highlights the importance of telocyte, kallikrein genes, and alternative splicing in mouse testes aging.

Advancing healthcare for elderly men requires a deeper understanding of testicular aging processes. In this study, we conducted transcriptomic profiling of 43,323 testicular single cells from young and old mice, shedding light on 1032 telocytes-an underexplored testicular cell type in previous research. Our study unveiled 916 age-related differentially expressed genes (age-DEGs), with telocytes emerging as the cell type harboring the highest count of age-DEGs. Of particular interest, four genes (Klk1b21, Klk1b22, Klk1b24, Klk1b27) from the Kallikrein family, specifically expressed in Leydig cells, displayed down-regulation in aged testes. Moreover, cell-type-level splicing analyses unveiled 1838 age-related alternative splicing (AS) events. While we confirmed the presence of more age-DEGs in somatic cells compared to germ cells, unexpectedly, more age-related AS events were identified in germ cells. Further experimental validation highlighted 4930555F03Rik, a non-coding RNA gene exhibiting significant age-related AS changes. Our study represents the first age-related single-cell transcriptomic investigation of testicular telocytes and Kallikrein genes in Leydig cells, as well as the first delineation of cell-type-level AS dynamics during testicular aging in mice.

Radio frequency electromagnetic radiations interfere with the Leydig cell functions in-vitro.

A growing threat to male infertility has become a major concern for the human population due to the advent of modern technologies as a source of radiofrequency radiation (RFR). Since these technologies have become an integral part of our daily lives, thus, it becomes necessary to know the impression of such radiations on human health. In view of this, the current study aims to focus on the biological effects of radiofrequency electromagnetic radiations on mouse Leydig cell line (TM3) in a time-dependent manner. TM3 cells were exposed to RFR emitted from 4G cell phone and also exposed to a particular frequency of 1800 MHz and 2450 MHz from RFR exposure system. The cells were then evaluated for different parameters such as cell viability, cell proliferation, testosterone production, and ROS generation. A considerable reduction in the testosterone levels and proliferation rate of TM3 cells were observed at 120 min of exposure as compared to the control group in all exposure settings. Conversely, the intracellular ROS levels showed a significant rise at 60, 90 and 120 min of exposure in both mobile phone and 2450 MHz exposure groups. However, RFR treatment for different time durations (15, 30, 45, 60, 90, and 120 min) did not have significant effect on cell viability at any of the exposure condition (2450 MHz, 1800 MHz, and mobile phone radiation). Therefore, our findings concluded with the negative impact of radiofrequency electromagnetic radiations on Leydig cell's physiological functions, which could be a serious concern for male infertility. However, additional studies are required to determine the specific mechanism of RFR action as well as its long-term consequences.

Effect of IL-1ß on the Development of Spermatogenesis In Vitro in Normal and Busulfan-Treated Immature Mice.

Gonadotoxic agents could impair spermatogenesis and may lead to male infertility. The present study aimed to evaluate the effect of IL-1ß on the development of spermatogenesis from cells isolated from seminiferous tubules (STs) of normal and busulfan-treated immature mice in vitro. Cells were cultured in a 3D in vitro culture system for 5 weeks. We examined the development of cells from the different stages of spermatogenesis by immunofluorescence staining or qPCR analyses. Factors of Sertoli and Leydig cells were examined by qPCR analysis. We showed that busulfan (BU) treatment significantly reduced the expression of testicular IL-1ß in the treated mice compared to the control group (CT). Cultures of cells from normal and busulfan-treated immature mice induced the development of pre-meiotic (Vasa), meiotic (Boule), and post-meiotic (acrosin) cells. However, the percentage of developed Boule and acrosin cells was significantly lower in cultures of busulfan-treated mice compared to normal mice. Adding IL-1ß to both cultures significantly increased the percentages of Vasa, Boule, and acrosin cells compared to their controls. However, the percentage of Boule and acrosin cells was significantly lower from cultures of busulfan-treated mice that were treated with IL-1ß compared to cultures treated with IL-1ß from normal mice. Furthermore, addition of IL-1ß to cultures from normal mice significantly increased only the expression of androgen receptor and transferrin but no other factors of Sertoli cells compared to their CT. However, the addition of IL-1ß to cultures from busulfan-treated mice significantly increased only the expression of androgen-binding protein and the FSH receptor compared to their CT. Adding IL-1ß to cultures of normal mice did not affect the expression of 3ßHSD compared to the CT, but it significantly reduced its expression in cultures from busulfan-treated mice compared to the CT. Our findings demonstrate the development of different stages of spermatogenesis in vitro from busulfan-treated mice and that IL-1ß could potentiate this development in vitro.

Human benign Leydig cell tumor - Biochemical evaluation.

Recently we reported expressional alterations in 219 genes and their transcripts in Leydig cell tumors but nowadays there is still a lack of full basic biochemical characteristics of these tumors. The discovery of potential biochemical markers for tumor management from early detection, treatments, and control of therapy results may markedly supplement genetic data. Leydig cell micronodules were obtained from patients with azoospermia who were qualified for testicular biopsy. The biochemistry of Leydig cell tumors was analyzed using histological staining and spectrophotometric measurements of total proteins, carbohydrates, lipids, and nucleic acids. In addition, the levels of calcium (Ca2 +), copper (Cu2 +), zinc (Zn2 +), and selenium (Se2 +) ions were measured. When compared to healthy testis we revealed, for the first time, that in the interstitial tissue with Leydig cell tumors, great amounts of proteins, carbohydrates, lipids, and acids were dislocated from the seminiferous tubules. Measurements of organic compounds showed a decrease (P < 0.05) only in the Cu2 + content in Leydig cell tumors which may be related to their altered biochemical structure. This specific result may be promising for designing further approaches to manage this tumor based on combining morphological and molecular data.

VD3/VDR attenuates bisphenol A-induced impairment in mouse Leydig cells via regulation of autophagy.

Bisphenol A (BPA) is an endocrine disruptor with reproductive toxicity. Further, 1,25-dihydroxyvitamin D3 (VD3) plays an important role in male reproduction by binding vitamin D receptor (VDR) and mediating the pleiotropic biological actions that include spermatogenesis. However, whether VD3/VDR regulates the effect of BPA on Leydig cells (LCs) injury remains unknown. This study aimed to explore the effects of VD on BPA-induced cytotoxicity in mouse LCs. Hereby, LCs treated with BPA, VD3, or both were subjected to the assays of cell apoptosis, proliferation, autophagy, and levels of target proteins. This study unveiled that cell viability was dose-dependently reduced after exposure to BPA. BPA treatment significantly inhibited LC proliferation, induced apoptosis, and also downregulated VDR expression. By jointly analyzing transcriptome data and Comparative Toxicogenomics Database (CTD) data, autophagy signaling pathways related to testicular development and male reproduction were screened out. Therefore, the autophagy phenomenon of cells was further detected. The results showed that BPA treatment could activate cell autophagy, Vdr-/- inhibits cell autophagy, and active VD3 does not have a significant effect on the autophagy of normal LCs. After VD3 and BPA were used in combination, the autophagy of cells was further enhanced, and VD3 could alleviate BPA-induced damage of LCs. In conclusion, this study found that supplementing VD3 could eliminate the inhibition of BPA on VDR expression, further enhance LCs autophagy effect, and alleviate the inhibition of LCs proliferation and induction of apoptosis by BPA, playing a protective role in cells. The research results will provide valuable strategies to alleviate BPA-induced reproductive toxicity.

Fine particulate matter (PM2.5) induces testosterone disruption by triggering ferroptosis through SIRT1/HIF-1α signaling pathway in male mice.

Fine particulate matter (PM2.5), a significant component of air pollution particulate matter, is inevitable and closely associated with increasing male reproductive disorder. However, the testicular targets of PM2.5 and its toxicity related molecular mechanisms are still not fully understood. In this study, the conditional knockout (cKO) mice and primary Leydig cells were used to explore the testicular targets of PM2.5 and the related underlying mechanisms. First, apparent the structure impairment of seminiferous tubules, Leydig cells vacuolization, decline of serum testosterone and sperm quality reduction were found in male wild-type (WT) and Sirt1 knockout mice after exposure to PM2.5. Enrichment analyses revealed that differentially expressed genes (DEGs) were enriched in steroid hormone biosynthesis, ferroptosis, and HIF-1 signaling pathway in the mice testes after exposure to PM2.5, which were subsequently verified by the molecular biological analyses. Notably, similar enrichment analyses results were also observed in primary Leydig cells after treatment with PM2.5. In addition, Knockdown of Sirt1 significantly increased PM2.5-induced expression and activation of HIF-1α, which was in parallel to the changes of cellular iron levels, oxidative stress indicators and the ferroptosis markers. In conclusion, this highlights that PM2.5 triggers ferroptosis via SIRT1/HIF-1α signaling pathway to inhibit testosterone synthesis in males. These findings provide a novel research support for the study that PM2.5 causes male reproductive injury.

Mechanistic screening of reproductive toxicity in a novel 3D testicular co-culture model shows significant impairments following exposure to low-dibutyl phthalate concentrations.

To improve the mechanistic screening of reproductive toxicants in  chemical-risk assessment and drug development, we have developed a three-dimensional (3D) heterogenous testicular co-culture model from neonatal mice. Di-n-butyl phthalate (DBP), an environmental contaminant that can affect reproductive health negatively, was used as a model compound to illustrate the utility of the in vitro model. The cells were treated with DBP (1 nM to 100 µM) for 7 days. Automated high-content imaging confirmed the presence of cell-specific markers of Leydig cells (CYP11A1 +), Sertoli cells (SOX9 +), and germ cells (DAZL +). Steroidogenic activity of Leydig cells was demonstrated by analyzing testosterone levels in the culture medium. DBP induced a concentration-dependent reduction in testosterone levels and decreased the number of Leydig cells compared to vehicle control. The levels of steroidogenic regulator StAR and the steroidogenic enzyme CYP11A1 were decreased already at the lowest DBP concentration (1 nM), demonstrating upstream effects in the testosterone biosynthesis pathway. Furthermore, exposure to 10 nM DBP decreased the levels of the germ cell-specific RNA binding protein DAZL, central for the spermatogenesis. The 3D model also captured the development of the Sertoli cell junction proteins, N-cadherin and Zonula occludens protein 1 (ZO-1), critical for the blood-testis barrier. However, DBP exposure did not significantly alter the cadherin and ZO-1 levels. Altogether, this 3D in vitro system models testicular cellular signaling and function, making it a powerful tool for mechanistic screening of developmental testicular toxicity. This can open a new avenue for high throughput screening of chemically-induced reproductive toxicity during sensitive developmental phases.

Differentiation of bone marrow mesenchymal stem cells into Leydig-like cells with testicular extract liquid in vitro.

Differentiation of Leydig cells plays a key role in male reproductive function. Bone marrow mesenchymal stem cells (BMSCs) have emerged as a potential cell source for generating Leydig-like cells due to their multipotent differentiation capacity and accessibility. This study aimed to investigate the morphological and genetic expression changes of BMSCs during differentiation into Leydig-like cells. Testicular extract liquid, which simulates the microenvironment in vivo, induced the third passage BMSCs differentiated into Leydig-like cells. Changes in cell morphology were observed by microscopy, the formation of lipid droplets of androgen precursor was identified by Oil Red Staining, and the expression of testicular specific genes 3ß-HSD and SF-1 in testicular stromal cells was detected by RT-qPCR. BMSCs isolated from the bone marrow of Sprague-Dawley (SD) rats were cultured for 3 generations and identified as qualified BMSCs in terms of morphology and cell surface markers. After 14 days of induction with testicular tissue lysate, lipid droplets appeared in the cytoplasm of P3 BMSCs by Oil Red O staining. RT-qPCR detection was performed on BMSCs on the 3rd, 7th, 14th, and 21st day after induction. Relative expression levels of 3ß-HSD mRNA significantly increased after 14 days of induction, while the relative expression of SF-1 mRNA increased after 14 days of induction but was not significant. BMSCs can differentiate into testicular interstitial cells with reserve androgen precursor lipid droplets after induction by testicular tissue lysate. The differentiation ability of BMSCs provides the potential to reconstruct the testicular microenvironment and is expected to fundamentally improve testicular function and provide new treatment options for abnormal spermatogenesis diseases.

Single-cell insights into mouse testicular toxicity under peripubertal exposure to di(2-ethylhexyl) phthalate.

Male fertility depends on normal pubertal development. Di-(2-ethylhexyl) phthalate (DEHP) is a potent antiandrogen chemical, and exposure to DEHP during peripuberty can damage the developing male reproductive system, especially the testis. However, the specific cellular targets and differentiation processes affected by DEHP, which lead to testicular toxicity, remain poorly defined. Herein, we presented the first single-cell transcriptomic profile of the pubertal mouse testis following DEHP exposure. To carry out the experiment, 2 groups (n = 8 each) of 3-week-old male mice were orally administered 0.5% carboxymethylcellulose sodium salt or 100 mg/kg body weight DEHP daily from postnatal day 21-48, respectively. Using single-cell RNA sequencing, a total of 31 distinct cell populations were identified, notably, Sertoli and Leydig cells emerged as important targets of DEHP. DEHP exposure significantly decreased the proportions of Sertoli cell clusters expressing mature Sertoli markers (Sox9 and Ar), and selectively reduced the expression of testosterone synthesis genes in fetal Leydig cells. Through cell-cell interaction analyses, we observed changed numbers of interactions in Sertoli cells 1 (SCs1), Leydig cells 1 (LCs1), and interstitial macrophages, and we also identified cell-specific ligand gene expressions in these clusters, such as Inha, Fyn, Vcam1, and Apoe. Complementary in vitro assays confirmed that DEHP directly reduced the expression of genes related to Sertoli cell adhesion and intercellular communication. In conclusion, peripubertal DEHP exposure reduced the number of mature Sertoli cells and may disrupt testicular steroidogenesis by affecting the testosterone synthesis genes in fetal Leydig cells rather than adult Leydig cells.

Isolation, culture, characterization and the functional study of testicular Leydig cells from Hezuo pig.

Testosterone, an important sex hormone, regulates sexual maturation, testicular development, spermatogenesis and the maintenance of secondary sexual characteristics in males. Testicular Leydig cells are the primary source of testosterone production in the body. Hezuo pigs, native to the southern part of Gansu, China, are characterized by early sexual maturity, strong disease resistance and roughage tolerance. This study employed type IV collagenase digestion combined with cell sieve filtration to isolate and purify Leydig cells from the testicular tissue of 1-month-old Hezuo pigs. We also preliminarily investigated the functions of these cells. The results indicated that the purity of the isolated and purified Leydig cells was as high as 95%. Immunofluorescence analysis demonstrated that the isolated cells specifically expressed the 3ß-hydroxysteroid dehydrogenase antibody. Enzyme-linked immunosorbent assay results showed that the testosterone secretion of the Leydig cells cultured in vitro (generations 5-9) ranged between 1.29-1.67 ng/mL. Additionally, the content of the cellular autophagy signature protein microtubule-associated protein 1 light chain 3 was measured at 230-280 pg/mL. Through this study, we established an in vitro system for the isolation, purification and characterization of testicular Leydig cells from 1-month-old Hezuo pigs, providing a reference for exploring the molecular mechanism behind precocious puberty in Hezuo pigs.

Loss of PBX1 function in Leydig cells causes testicular dysgenesis and male sterility.

Leydig cells are essential components of testicular interstitial tissue and serve as a primary source of androgen in males. A functional deficiency in Leydig cells often causes severe reproductive disorders; however, the transcriptional programs underlying the fate decisions and steroidogenesis of these cells have not been fully defined. In this study, we report that the homeodomain transcription factor PBX1 is a master regulator of Leydig cell differentiation and testosterone production in mice. PBX1 was highly expressed in Leydig cells and peritubular myoid cells in the adult testis. Conditional deletion of Pbx1 in Leydig cells caused spermatogenic defects and complete sterility. Histological examinations revealed that Pbx1 deletion impaired testicular structure and led to disorganization of the seminiferous tubules. Single-cell RNA-seq analysis revealed that loss of Pbx1 function affected the fate decisions of progenitor Leydig cells and altered the transcription of genes associated with testosterone synthesis in the adult testis. Pbx1 directly regulates the transcription of genes that play important roles in steroidogenesis (Prlr, Nr2f2 and Nedd4). Further analysis demonstrated that deletion of Pbx1 leads to a significant decrease in testosterone levels, accompanied by increases in pregnenolone, androstenedione and luteinizing hormone. Collectively, our data revealed that PBX1 is indispensable for maintaining Leydig cell function. These findings provide insights into testicular dysgenesis and the regulation of hormone secretion in Leydig cells.

Androgen synthesis cell-specific CREBZF deficiency alters adrenal cortex steroid secretion and develops behavioral abnormalities in adult male mice.

The global challenge of male infertility is escalating, notably due to the decreased testosterone (T) synthesis in testicular Leydig cells under stress, underscoring the critical need for a more profound understanding of its regulatory mechanisms. CREBZF, a novel basic region-leucine zipper transcription factor, regulates testosterone synthesis in mouse Leydig cells in vitro; however, further validation through in vivo experiments is essential. Our study utilized Cyp17a1-Cre to knock out CREBZF in androgen-synthesis cells and explored the physiological roles of CREBZF in fertility, steroid hormone synthesis, and behaviors in adult male mice. Conditional knockout (cKO) CREBZF did not affect fertility and serum testosterone level in male mice. Primary Leydig cells isolated from CREBZF-cKO mice showed impaired testosterone secretion and decreased mRNA levels of Star, Cyp17a1, and Hsd3b1. Loss of CREBZF resulted in thickening of the adrenal cortex, especially X-zone, with elevated serum corticosterone and dehydroepiandrosterone levels and decreased serum dehydroepiandrosterone sulfate levels. Immunohistochemical staining revealed increased expression of StAR, Cyp11a1, and 17ß-Hsd3 in the adrenal cortex of CREBZF-cKO mice, while the expression of AR was significantly reduced. Along with the histological changes and abnormal steroid levels in the adrenal gland, CREBZF-cKO mice showed higher anxiety-like behavior and impaired memory in the elevated plus maze and Barnes maze, respectively. In summary, CREBZF is dispensable for fertility, and CREBZF deficiency in Leydig cells promotes adrenal function in adult male mice. These results shed light on the requirement of CREBZF for fertility, adrenal steroid synthesis, and stress response in adult male mice, and contribute to understanding the crosstalk between testes and adrenal glands.