Cannabidiol-induced transcriptomic changes and cellular senescence in human Sertoli cells.

Cannabidiol (CBD), one of the major cannabinoids in the plant Cannabis sativa L., is the active ingredient in a drug approved for the treatment of seizures associated with certain childhood-onset epileptic disorders. CBD has been shown to induce male reproductive toxicity in multiple animal models. We previously reported that CBD inhibits cellular proliferation in the mouse Sertoli cell line TM4 and in primary human Sertoli cells. In this study, using a transcriptomic approach with mRNA-sequencing analysis, we identified molecular mechanisms underlying CBD-induced cytotoxicity in primary human Sertoli cells. Analysis of differentially expressed genes demonstrated that DNA replication, cell cycle, and DNA repair were the most significantly affected pathways. We confirmed the concentration-dependent changes in the expression of key genes in these pathways using real-time PCR. mRNA sequencing showed upregulation of a group of genes tightly associated with the senescence-associated secretory phenotype (SASP) and with the activation of the p53 signaling pathway, a key upstream event in cellular senescence. Prolonged treatment of 10 µM CBD-induced cellular senescence, as evidenced by the stable cessation of proliferation and the activation of senescence-associated ß-galactosidase (SA-ß-gal), 2 hallmarks of senescence. Additionally, using real-time PCR and Western blotting assays, we observed that CBD treatment increased the expression of p16, an important marker of cellular senescence. Taken together, our results show that CBD exposure disturbs various interrelated signaling pathways and induces cellular senescence in primary human Sertoli cells.

Fumonisin B1 promotes germ cells apoptosis associated with oxidative stress-related Nrf2 signaling in mice testes.

Fumonisins (FBs) are widespread Fusarium toxins commonly found in corn. This study aimed to establish the mechanism of oxidative stress via the Nrf2 signaling pathway associated with FB1-induced toxicity in mice testis. Male mice were fed with 5 mg/kg FB1 diet for 21 or 42 days, the expression of inflammatory related genes, apoptosis related genes and Nrf2 pathway genes were detected by RT-qPCR, Western blot and immunohistochemical. Furthermore, Sertoli cell was treatment with FB1. Cell viability was measured by CCK8 assay, ROS level and apoptosis related genes were detected by immunofluorescence staining. The results showed that FB1 had toxic effects on testis, which could increase the ROS level of Sertoli cells, affect the Keap1-Nrf2 pathway related factors, destroy the oxidative balance of testis, lead to the occurrence of inflammation and the initiation of apoptosis, and finally destroy the testicular tissue structure and affect the formation of sperm.

A comparative study of the effects of 3 testicular toxicants in cultures of seminiferous tubules of rats or of domestic cats (veterinary waste): An alternative method for reprotoxicology.

In response to the EU cosmetics directive regulation and REACH legislation which encourage cell culture methods in order to reduce or replace the use of animals in toxicology studies, we settled the culture of prepubertal domestic cat seminiferous tubules in our validated BioAlter® model, usually used with prepubertal rat, called here BioAlter®-rat, by opposition to BioAlter®-cat settled here. We carried out a comparative study on the effects of 3 testicular toxicants, 1,3-dinitrobenzene at 60 µM, 2-methoxyacetic acid at 2.5 mM and carbendazim at 50 nM or 500 nM in both BioAlter®-cat and BioAlter®-rat over a 3-week culture period. Sertoli cell or each germ cell populations as well as the levels of Sertoli cell or germ cell specific mRNAs were studied. The harmful effects of the 3 toxicants on pre-meiotic, meiotic and post-meiotic cell numbers and on Sertoli or germ cell specific mRNAs were clearly observed in the two species, even if there might be some small differences in the intensity of the effects on some of the studied parameters. Hence, BioAlter®-cat might be a solution to the requirements of the EU cosmetics directive and REACH legislation for male reproductive toxicology studies.

4-[1-Ethyl-1-methylhexy]-phenol induces apoptosis and interrupts Ca2+ homeostasis via ROS pathway in Sertoli TM4 cells.

Biological effect of an individual nonylphenol (NP) isomer extremely relies upon the side chain structure. This research was designed to evaluate the impact of NP isomer, 4-[1-ethyl-1-methylhexy]-phenol (NP65), on Sertoli cells in vitro. Sertoli TM4 cells were exposed to various concentration (0, 0.1, 1, 10, or 20 µM) of NP65 for 24 h, and the outcomes indicated that treatment of NP65 induced reactive oxygen species (ROS) generation, oxidative stress, and apoptosis for Sertoli TM4 cells. In addition, it was found that NP65 exposure affected homeostasis of Ca2+ in Sertoli TM4 cells by increasing cytoplasm [Ca2+]i, inhibiting Ca2+-ATPase activity and decreasing cyclic adenosine monophosphate (cAMP) concentration. Pretreatment with ROS scavenger, N-acetylcysteine (NAC), attenuated NP65-induced oxidative stress as well as apoptosis for TM4 cells. Furthermore, NAC blocked NP65-induced disorders of Ca2+ homeostasis by attenuating the growth of intracellular [Ca2+]i and the inhibition of Ca2+-ATPase and cAMP activities. Thus, we have demonstrated that NP65 induced apoptosis as well as acted as a potent inhibitor of Ca2+-ATPase activity and resulted in disorder of Ca2+ homeostasis in Sertoli TM4 cells; ROS participated in the process. Our results supported the view that oxidative stress acted an essential role within the development of apoptosis and Ca2+ overload in TM4 cells as a consequence of NP65 stimulation.

Follicle-stimulating hormone-mediated decline in miR-92a-3p expression in pubertal mice Sertoli cells is crucial for germ cell differentiation and fertility.

Sertoli cells (Sc) are the sole target of follicle-stimulating hormone (FSH) in the testis and attain functional maturation post-birth to significantly augment germ cell (Gc) division and differentiation at puberty. Despite having an operational microRNA (miRNA) machinery, limited information is available on miRNA-mediated regulation of Sc maturation and male fertility. We have shown before that miR-92a-3p levels decline in pubertal rat Sc. In response to FSH treatment, the expressions of FSH Receptor, Claudin11 and Klf4 were found to be elevated in pubertal rat Sc coinciding with our finding of FSH-induced decline in miR-92a-3p levels. To investigate the association of miR-92a-3p and spermatogenesis, we generated transgenic mice where such pubertal decline of miR-92a-3p was prevented by its overexpression in pubertal Sc under proximal Rhox5 promoter, which is known to be activated specifically at puberty, in Sc. Our in vivo observations provided substantial evidence that FSH-induced decline in miR-92a-3p expression during Sc maturation acts as an essential prerequisite for the pubertal onset of spermatogenesis. Elevated expression of miR-92a-3p in post-pubertal testes results into functionally compromised Sc, leading to impairment of the blood-testis barrier formation and apoptosis of pre-meiotic Gc, ultimately culminating into infertility. Collectively, our data suggest that regulation of miR-92a-3p expression is crucial for Sc-mediated induction of active spermatogenesis at puberty and regulation of male fertility.

Assessment of Zearalenone-Induced Cell Survival and of Global Gene Regulation in Mouse TM4 Sertoli Cells.

Zearalenone (ZEA) is a non-steroidal xenoestrogen mycotoxin produced by many Fusarium fungal species, which are common contaminants of cereal crops destined for worldwide human and animal consumption. ZEA has been reported in various male reproduction dysfonctions, including decreased fertility potential. In this report, the direct effect of ZEA on the immature Sertoli TM4 cell line was evaluated. The results show that high concentrations of ZEA increase reactive oxygen species via the activation of MAPK signaling. Transcriptome analysis was performed on the TM4 cell line treated with ZEA, and genes involved in sex differentiation (Fgfr2, Igf1, Notch1, Sox9) and extracellular matrix (ECM) formation (Ctgf, Fam20a, Fbn1, Mmp9, Postn, Sparcl1, Spp1) were identified at the center of the functional protein association network, suggesting that ZEA could be detrimental to the early steps of Sertoli cell differentiation.

3-Monochloropropane-1,2-diol causes spermatogenesis failure in male rats via Sertoli cell dysfunction but not testosterone reduction.

3-Monochloropane-1,2-diol (3-MCPD), a common food contaminant, has been confirmed to impair male fertility, but the mechanism has not been fully clarified. This study systematically explored the spermatogenesis impairment induced by 3-MCPD in vivo and in vitro with a focus on Sertoli cells (SCs) and spermatogonial stem cells (SSCs). After adult male Sprague-Dawley rats were administered 36 and 72 mg/kg b.w./day 3-MCPD daily for 4 weeks, the total sperm concentration dramatically decreased by 28.9 % and 57.7 %, respectively, and obvious testicular seminiferous tubule atrophy was observed. 3-MPCD exposure decreased serum testosterone levels but not intratesticular testosterone levels and upregulated the expression of steroidogenesis enzymes in both rat testes and primary Leydig cells. 3-MCPD did not reduce the number and self-renewal marker PLZF+ of SSCs; however, it downregulated the key meiotic genes Stra8 and Rec8 in the rat testis but not in primary germ cells. Although SC counts were not affected, 3-MCPD downregulated androgen receptor (AR) in rat testes and primary SCs. In addition, 3-MCPD downregulated p-CREB (transcription factor of AR), paracrine meiosis regulators Nrg1 and Nrg3 and retinoic acid synthetase Aldh1a1 in primary SCs. In summary, 3-MCPD caused impairment of spermatogenesis by inhibiting secretion of meiosis regulators and disturbing testosterone signalling in SCs.

In vitro effects of cannabidiol and its main metabolites in mouse and human Sertoli cells.

Cannabidiol (CBD) is a major cannabinoid present in extracts of the plant Cannabis sativa (marijuana). While the therapeutic effects of CBD on epilepsy have been demonstrated, less is understood regarding its potential adverse effects. Recent studies revealed that CBD induced toxicity in the male reproductive system of animal models. In this study, we used TM4, an immortalized mouse Sertoli cell line, and primary human Sertoli cells to evaluate the toxicities of CBD and its main metabolites, 7-carboxy-CBD and 7-hydroxy-CBD. CBD induced concentration- and time-dependent cytotoxicity in mouse and human Sertoli cells, which mainly resulted from the inhibition of the G1/S-phase cell cycle transition. CBD also inhibited DNA synthesis and downregulated key cell cycle proteins. Moreover, CBD reduced the mRNA and protein levels of a functional marker, Wilms' tumor 1. Similar to CBD, 7-carboxy-CBD and 7-hydroxy-CBD inhibited cellular proliferation and decreased DNA synthesis. 7-Carboxy-CBD was less cytotoxic than CBD, while 7-hydroxy-CBD showed comparable cytotoxicity to CBD in both mouse and human Sertoli cells. Compared to mouse Sertoli cells, CBD, 7-hydroxy-CBD, and 7-carboxy-CBD were more cytotoxic in human Sertoli cells. Our results indicate that CBD and its main metabolites can inhibit cell proliferation in mouse and human Sertoli cells.

Dihydrotestosterone through blockade of TGF-ß/Smad signaling mediates the anti-fibrosis effect under hypoxia in canine Sertoli cells.

The hypoxic microenvironment of cryptorchidism is an important factor to induce the impairment of the structure and function of Sertoli cells and thus lead to spermatogenesis loss or tumorigenesis. Dihydrotestosterone (DHT), as a potent nonaromatizable 5α-reduced androgen, has both positive and negative effect on pathological fibrosis process. However, it is still unknown whether DHT can regulate hypoxia-induced fibrosis of Sertoli cells. Herein, in this study, we evaluate the DHT level, two 5α-reductase isoforms, 5α-red1 and 5α-red2, as well as HIF-1α expression pattern in canine cryptorchidism and contralateral normal testis. Results showed that the abdominal testes presented low DHT levels and 5α-red1 and 5α-red2 expression, while significantly higher HIF-1α expression and ECM production compared with the scrotum. Moreover, we established a hypoxia-induced fibrosis model in canine Sertoli cells induced by cobalt chloride (CoCl2), and found that DHT inhibited the fibrosis of Sertoli cells in a dose-dependent manner. Meanwhile, DHT interfered with the TGF-ß signaling by reducing the expression of TGF-ßRI and TGF-ßRII and inhibiting the expression and phosphorylation of Smad2 and Smad3, while flutamide (androgen receptor inhibitor) inhibited these effects of DHT. Furthermore, use of LY2109761 (TGF-ß receptor type I/II inhibitor) to interfere with the TGF-ß/Smad pathway showed a similar effect with DHT suppression of the fibrosis in Sertoli cells. Our research data demonstrated that cryptorchidism is located in a hypoxic and DHT deficiency microenvironment. Moreover, supplementing DHT can alleviate the fibrosis process of Sertoli cells caused by hypoxia, which is associated with AR regulating the inhibition of TGF-ß/Smad signaling.

Single-cell transcriptome analysis of Bisphenol A exposure reveals the key roles of the testicular microenvironment in male reproduction.

Testicular development during juvenile is crucial for subsequent male reproductive function. However, it remains poorly understood about the contribution of the testis microenvironment to human germ cell maturation. Therefore, we systematically analyzed scRNA-seq transcriptome and found the dramatic changes in cell-type composition in human testis during puberty. Then we constructed cell-cell communication networks between germ cells and somatic cells in the juvenile testis, which may be achieved via immune-related pathways. Our results showed that maturation-promoting factors are the switches of the Sertoli cells that drive sperm maturation. Furthermore, we found that Bisphenol A(BPA) enhanced the maturation and growth of germ cells through the Sertoli cell's secretory protein. Finally, our results indicate Bisphenol A would lead to the dysregulation of secreted protein expression in Sertoli cells during spermatogenesis, which in turn has direct cytotoxicity to Sertoli cells. Bisphenol A is one of the underlying causes of non-obstructive azoospermia (NOA). In summary, our results reveal the reproductive toxicity and molecular mechanism of Bisphenol A in Sertoli cells and male reproduction. Provide a reference for the toxicity of Bisphenol A to human reproduction.

FSH inhibits autophagy and lysosomal biogenesis to regulate protein degradation in cultured goat Sertoli cells.

Although the follicle-stimulating hormone (FSH) plays a vital role in male reproduction, the molecular relationships among FSH, autophagy, and the secretory function of Sertoli cells remain largely undetermined. In this study, we sought to investigate the effects of FSH on dairy goat Sertoli cell autophagy and the role of autophagy in protein clearance. FSH treatment of primary Sertoli cells was found to enhance the expression level of LC3-II, reduce p62 degradation and the number of lysosomes, and downregulate the levels of LAMP2 protein and lysosomal gene mRNAs. Further analyses revealed that starvation-induced autophagy promotes the translocation of transcription factor EB (TFEB) from the cytoplasm to nucleus and its binding to the promoter region of LAMP2, whereas FSH suppresses the nuclear translocation of TFEB. Moreover, we found that the FSH-mediated inhibition of autophagy extends the biological half-lives of androgen-binding protein (ABP), glial-derived neurotrophic factor (GDNF), and stem cell factor (SCF) and promotes the secretion of these proteins. Collectively, these observations indicate that FSH inhibits autophagy by reducing lysosomal biogenesis, which is associated with the suppression of TFEB nuclear translocation via activation of the PI3K/Akt/mTOR pathway, thereby extending the biological half-lives and enhancing the expression of ABP, GDNF, and SCF in dairy goat Sertoli cells.

Multifaceted epigenetic regulation of porcine testicular aromatase.

Testicular aromatase catalyzes the synthesis of estradiol, which contributes to regulation of porcine Sertoli cell proliferation and postpubertal maintenance of Sertoli cell numbers. Although aromatase enzymatic activity decreases with age and is persistently reprogrammed by prepubertal treatment with the aromatase inhibitor letrozole, the molecular bases for regulation have not been identified. DNA methylation was examined as a potential regulatory mechanism using DNA from Leydig cells isolated from 16-, 40-, and 68-week-old boars and from 68- week-old littermates treated with the aromatase inhibitor, letrozole. Methylation levels of individual CpG dinucleotides located in the distal untranslated exon 1 of the relevant aromatase encoding gene, CYP19A3, were quite high in Leydig cell DNA, and increased further with maturity of boar (P < 0.05), while aromatase activity and transcript abundance decreased more than two-fold. However, reduced aromatase activity following letrozole treatment was not accompanied by altered DNA methylation. Testicular expression of miR378 was altered by prepubertal treatment with letrozole. The data provide evidence for two different epigenetic mechanisms that regulate aromatase expression and enzymatic activity in the boar testis.

Boosting effect of testosterone on GDNF expression in Sertoli cell line (TM4); comparison between TM3 cells-produced and exogenous testosterone.

The Glial cell-derived neurotrophic factor (Gdnf) and testosterone induce the spermatogonial stem cells (SSCs) self-renewal and spermatogenesis, respectively. In present study the stimulating role of testosterone on Sertoli cells to produce Gdnf, and the possible effect of Gdnf on Gfrα1 and c-RET expressions were investigated. The TM4 cells (line Sertoli cells) were co-cultured with [0.1, 0.2 and 0.4 (ng/ml)] of exogenous and TM3 (line Leydig cells)-produced testosterones, and consequently the TM4-produced Gdnf concentration was evaluated. Next, the SSCs were co-cultured with the TM-4 derived media (endogenous Gdnf) and exogenous Gdnf [0.1, 0.2, and 0.4 ng/ml)]. The 0.1 and 0.2 ng/ml endogenous and 3 concentrations of exogenous testosterone up-regulated the Gdnf expression versus non-treated Sertoli cells. The TM4-produced and exogenous Gdnfs, in all concentrations, up-regulated the receptors expression. In conclusion, the testosterone, solely, stimulates the Gdnf synthesis and the Gdnf, individually, amplifies its receptor's expression at mRNA and protein levels.

Zearalenone Exposure Disrupts Blood-Testis Barrier Integrity through Excessive Ca2+-Mediated Autophagy.

Zearalenone (ZEA), a common mycotoxin in grains and animal feeds, has been associated with male reproductive disorders. However, the potential toxicity mechanism of ZEA is not fully understood. In this study, in vivo and in vitro models were used to explore the effects of ZEA on the blood-testis barrier (BTB) and related molecular mechanisms. First, male BALB/C mice were administered ZEA orally (40 mg/kg·bw) for 5-7 d. Sperm motility, testicular morphology, and expressions of BTB junction proteins and autophagy-related proteins were evaluated. In addition, TM4 cells (mouse Sertoli cells line) were used to delineate the molecular mechanisms that mediate the effects of ZEA on BTB. Our results demonstrated that ZEA exposure induced severe testicular damage in histomorphology and an ultrastructural, time-dependent decrease in the expression of blood-testis barrier junction-related proteins, accompanied by an increase in the expression of autophagy-related proteins. Additionally, similar to the in vitro results, the dose-dependent treatment of ZEA increased the level of cytoplasmic Ca2+ and the levels of the autophagy markers LC3-II and p62, in conjunction with a decrease in the BTB junction proteins occludin, claudin-11, and Cx43, with the dislocation of the gap junction protein Cx43. Meanwhile, inhibition of autophagy by CQ and 3-MA or inhibition of cytoplasmic Ca2+ by BAPTA-AM was sufficient to reduce the effects of ZEA on the TM4 cell BTB. To summarize, this study emphasizes the role of Ca2+-mediated autophagy in ZEA-induced BTB destruction, which deepens our understanding of the molecular mechanism of ZEA-induced male reproductive disorders.

Titanium dioxide nanoparticles perturb the blood-testis barrier via disruption of actin-based cell adhesive function.

As one of the most commonly used nanoparticles, titanium dioxide nanoparticles (TiO2-NPs) are widely used as coating reagents in cosmetics, medicine and other industries. The increasing risk of exposure to TiO2-NPs raises concerns about their safety. In this study, we investigated the mechanism by which TiO2-NPs cross the blood-testis barrier (BTB). TM-4 cells were selected as an in vitro Sertoli cell model of BTB. Cell viability, cell morphological changes, apoptosis, oxidative damage, and the expression levels of actin regulatory and tight junction (TJ) proteins were assessed in TM-4 cells treated with 3-nm and 24-nm TiO2-NPs. Cells treated with 3-nm TiO2-NPs exhibited increased cytotoxicity and decreased Annexin II expression, whereas cells treated with 24-nm TiO2-NPs exhibited increased Arp 3 and c-Src expression. Both TiO2-NPs induced significant oxidative stress, decreased the expression of TJ proteins (occludin, ZO-1 and claudin 5), damaged the TJ structure, and exhibited enlarged gaps between TM-4 cells. Our results indicated that both TiO2-NPs crossed the BTB by disrupting actin-based adhesive junctions of TM-4 cells; however, apoptosis was not observed. Our results provide new insights into how TiO2-NPs cross the BTB.

Cytotoxicity of mancozeb on Sertoli-germ cell co-culture system: Role of MAPK signaling pathway.

Mancozeb (MZB) is a worldwide fungicide for the management of fungal diseases in agriculture and industrial contexts. Human exposure occurs by consuming contaminated plants, drinking water, and occupational exposure. There are reports on MZB's reprotoxicity such as testicular structure damage, sperm abnormalities, and decrease in sperm parameters (number, viability, and motility), but its molecular mechanism on apoptosis in testis remains limited. To investigate the molecular mechanisms involved in male reprotoxicity induced by MZB, we used primary cultures of mouse Sertoli-germ cells. Cells were exposed to MZB (1.5, 2.5, and 3.5 µM) for 3 h to evaluate viability by 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) assay, reactive oxygen species (ROS) generation, and oxidative stress parameters (lipid peroxidation). Cell death and mitogen-activated protein kinase (MAPK) signaling were measured in these cells using flow cytometry and western blotting. In addition, some groups were exposed to N-acetylcysteine (NAC, 5 mM) in the form of co-treatment with MZB. Mancozeb reduced viability and increased the level of intracellular ROS, p38 and c-Jun N-terminal kinases (JNK) MAPK proteins phosphorylation, and apoptotic cell death, which could be blocked by NAC as an inhibitor of oxidative stress. The present study indicated for the first time the toxic manifestations of MZB on the Sertoli-germ cell co-culture. Redox imbalance and p38 and JNK signaling pathway activation might play critical roles in MZB-induced apoptosis in the male reproductive system.

Gene expression profiles of two testicular somatic cell lines respond differently to 4-nitrophenol mediating vary reproductive toxicity.

4-Nitrophenol (PNP) has been extensively used in manufacturing for several decades. Its toxic effects on the male reproductive system have been reported, but the underlying mechanisms remain unclear. In this study, we utilized two testicular somatic cell lines (TM3 and TM4 cells) to explore the possible toxic effects of PNP on the male reproductive system. The activity of the cells after exposure to different doses of PNP (0.01, 0.1, 1, 10 and 100 µM) was evaluated. PNP treatment at 10 µM significantly inhibited cell viability, and 10 µM PNP was thus selected for subsequent experiments. Although PNP (10 µM) inhibited cell proliferation, promoted cell apoptosis, and changed the cell cycle distribution and ultrastructure in both types of cells, these effects were more significant in the TM4 cells. In addition, an Agilent mouse mRNA array was used to identify the gene expression differences between the control and PNP (10 µM) exposed TM3 and TM4 cells. The microarray analysis identified 67 and 1372 differentially expressed genes mainly concentrated in endothelial cell morphogenesis and anatomical structure development in TM3 cells and associated with cardiovascular system development and circulatory system development in TM4 cells. Moreover, a pathway analysis revealed that PNP not only predominately affected meiotic recombination and meiosis in TM3 cells, but also influenced axon guidance and developmental biology in TM4 cells. These results suggest that TM3 and TM4 cells exhibit different responses to PNP, which might mediate different toxic mechanisms.

Valproic acid during pregnancy decrease the number of spermatogenic cells and testicular volume in the offspring of mice: Stereological quantification.

Valproic acid (VPA) is a drug used to treat epilepsy, bipolar disorders and headaches. As a secondary effect, this antiepileptic drug can cause a decrease in androgens and gonadotropins, and dose-dependent testicular defects, such as reduction of testicular weights, sperm motility and degeneration of the seminiferous tubules. In offspring exposed to VPA, its effects have not been evaluated, so the study aimed to determine the morphological effects of the use of VPA along testicular development in mice. 30 adult female BALB/c mice were crossed and divided by age, with embryos of 12.5 days post coitum (dpc), fetuses of 17.5 dpc and male mice 6 weeks postnatal. In each case, the pregnant mouse received 600 mg/kg of VPA, making up the VPA groups, or 0.3 mL of 0.9% physiological solution for the control groups, from the beginning to the end of the pregnancy, orally.t. A morpho-quantitative analysis was carried out on the gonadal development of the male offspring. In the groups treated with VPA, at all ages studied they had lower testicular volume. At 12.5 dpc, they showed less testicular development in the form of sex cords, with fewer gonocytes and somatic cells. At 17.5 dpc, they presented greater interstitial space, fewer spermatogonial, sustentacular Sertoli, peritubular and interstitial Leydig cells. At 6 weeks postnatal, they presented fewer spermatogonia, pachytene spermatocytes, elongated spermatids, sustentacular Sertoli and interstitial Leydig cells, with statistically significant differences. In conclusion, prenatal exposure to VPA causes histopathological alterations in the offspring of mice in testicular development, from the embryonic stage to 6 weeks postnatal.

Long-Term Maintenance of Viable Adult Rat Sertoli Cells Able to Establish Testis Barrier Components and Function in Response to Androgens.

A protocol for the isolation and long-term propagation of adult rat Sertoli cells (SCs) using conditional reprogramming (CR) was developed and the formation of tight junctions as an in vitro model for the blood testis barrier (BTB) was studied. Three pure primary SC lines were isolated successfully and maintained for several months without significant changes in expression levels of SC-typical markers such as SRY-box transcription factor 9 (SOX9), transferrin, clusterin, androgen receptor (AR), and GATA binding protein 1 (GATA1). In addition to AR expression, the tight junction proteins, zonula occludens-1 (ZO-1) and the junctional adhesion molecule-3 (JAM-3), were upregulated and the SC barrier integrity was enhanced by testosterone. Peritubular/myoid cells did not increase the tightness of the SC. The cytokines, interleukin-6 (IL-6), bone morphogenetic protein-2 (BMP2), and transforming growth factor beta-3 (TGF-ß3), negatively affected the tightness of the SC barrier. We have established a protocol for the isolation and long-term propagation of highly pure primary adult rat SCs, which are able to respond to androgen treatments, to form tight junctions and to maintain the mRNA expression of SC-specific genes. By applying this new method, adult SCs can now be analyzed in more detail and might serve as an in vitro model for the study of many SC functions.

Wuzi-Yanzong prescription alleviates spermatogenesis disorder induced by heat stress dependent on Akt, NF-κB signaling pathway.

Akt and nuclear factor kappa B (NF-κB) signaling pathways are involved in germ cell apoptosis and inflammation after testicular heat stress (THS). We observed that after THS induced by the exposure of rat testes to 43 °C for 20 min, their weight decreased, the fraction of apoptotic testicular germ cells significantly increased, and the proliferation of germ cells was inhibited. In addition, THS lowered serum testosterone (T) level, whereas the levels of follicle stimulating hormone and luteinizing hormone were not significantly changed. The ultrastructure of the seminiferous tubules became abnormal after THS, the structure of the blood-testis barrier (BTB) became loose, and the Sertoli cells showed a trend of differentiation. The level of phosphorylated Akt was reduced, whereas the amount of phosphorylated NF-κB p65 was augmented by THS. Wuzi-Yanzong (WZYZ), a classic Chinese medicine prescription for the treatment of male reproductive dysfunctions, alleviated the changes induced by THS. In order to determine the mechanism of action of WZYZ, we investigated how this preparation modulated the levels of T, androgen receptor (AR), erythropoietin (EPO), EPO receptor, and Tyro-3, Axl, and Mer (TAM) family of tyrosine kinase receptors. We found that WZYZ activated the Akt pathway, inhibited the Toll-like receptor/MyD88/NF-κB pathway, and repaired the structure of BTB by regulating the levels of T, AR, TAM receptors, and EPO. In conclusion, these results suggest that WZYZ activates the Akt pathway and inhibits the NF-κB pathway by acting on the upstream regulators, thereby improving spermatogenesis deficit induced by THS.