Map-1a regulates Sertoli cell BTB dynamics through the cytoskeletal organization of microtubule and F-actin.

Microtubule-associated protein 1a (Map1a) is a microtubule (MT) regulatory protein that binds to the MT protofilaments in mammalian cells to promote MT stabilization. Maps work with MT cleavage proteins and other MT catastrophe-inducing proteins to confer MT dynamics to support changes in the Sertoli cell shape to sustain spermatogenesis. However, no functional studies are found in the literature to probe its role in spermatogenesis. Using an RNAi approach, coupled with the use of toxicant-induced testis (in vivo)- and Sertoli cell (in vitro)-injury models, RNA-Seq analysis, transcriptome profiling, and relevant bioinformatics analysis, immunofluorescence analysis, and pertinent biochemical assays for cytoskeletal organization, we have delineated the functional role of Map1a in Sertoli cells and testes. Map1a was shown to support MT structural organization, and its knockdown (KD) also perturbed the structural organization of actin, vimentin, and septin cytoskeletons as these cytoskeletons are intimately related, working in concert to support spermatogenesis. More importantly, cadmium-induced Sertoli cell injury that perturbed the MT structural organization across the cell cytoplasm was associated with disruptive changes in the distribution of Map1a and a surge in p-p38-MAPK (phosphorylated p38-mitogen-activated protein kinase) expression but not total p38-MAPK. These findings thus support the notion that p-p38-MAPK activation is involved in cadmium-induced Sertoli cell injury. This conclusion was supported by studies using doramapimod, a specific p38-MAPK phosphorylation (activation) inhibitor, which was capable of restoring the cadmium-induced disruptive structural organization of MTs across the Sertoli cell cytoplasm. In summary: this study provides mechanistic insights regarding restoration of toxicant-induced Sertoli cell and testis injury and male infertility.

Development of peptides for targeting cell ablation agents concurrently to the Sertoli and Leydig cell populations of the testes: An approach to non-surgical sterilization.

The surgical sterilization of cats and dogs has been used to prevent their unwanted breeding for decades. However, this is an expensive and invasive procedure, and often impractical in wider contexts, for example the control of feral populations. A sterilization agent that could be administered in a single injection, would not only eliminate the risks imposed by surgery but also be a much more cost-effective solution to this worldwide problem. In this study, we sought to develop a targeting peptide that would selectively bind to Leydig cells of the testes. Subsequently, after covalently attaching a cell ablation agent, Auristatin, to this peptide we aimed to apply this conjugated product (LH2Auristatin) to adult male mice in vivo, both alone and together with a previously developed Sertoli cell targeting peptide (FSH2Menadione). The application of LH2Auristatin alone resulted in an increase in sperm DNA damage, reduced mean testes weights and mean seminiferous tubule size, along with extensive germ cell apoptosis and a reduction in litter sizes. Together with FSH2Menadione there was also an increase in embryo resorptions. These promising results were observed in around a third of all treated animals. Given this variability, we discuss how these reagents might be modified in order to increase target cell ablation and improve their efficacy as sterilization agents.

Hormetic effects of a cannabinoid system component, 2-arachidonoyl glycerol, on cell viability and expression profile of growth factors in cultured mouse Sertoli cells: Friend or foe of male fertility?

The generally undesired effects of exocannabinoids on male reproduction include alterations in testicular cell proliferation and function, as well as apoptosis induction. However, this paradigm has been challenged by the ability of endocannabinoids to regulate reproductive function. The present study addresses these paradoxical facts by investigating the effects of the endocannabinoid 2-arachidonoyl glycerol (2-AG) on mouse Sertoli cells' survival and apoptosis, with a mechanistic insight into Sertoli cell-based growth factors' production. The Mus musculus Sertoli cell line (TM4) was exposed to different concentrations of 2-AG, and cell viability was evaluated using MTT assay. Growth factors' gene and protein expressions were analyzed through RT-PCR and western blotting. 2-AG concentration dependently increased TM4 viability, with a slight increase starting at 0.0001 µM, a peak of 190% of the control level at 1 µM, and a decrease at 3 µM. Moreover, 2-AG paradoxically altered mRNA expression of caspase-3 and growth factors. Caspase-3 mRNA expression was down-regulated, and growth factors mRNA and protein expression were up-regulated when using a low concentration of 2-AG (1 µM). Opposite effects were observed by a higher concentration of 2-AG (3 µM). These paradoxical effects of 2-AG can be explained through the concept of hormesis. The results indicate the pivotal role of 2-AG in mediating Sertoli cell viability and apoptosis, at least in part, through altering growth factors secretion. Furthermore, they suggest the involvement of endocannabinoids in Sertoli cell-based physiological and pathological conditions and reflect the ability of abnormally elevated 2-AG to mimic the actions of exocannabinoids in reproductive dysfunction.

A conserved NR5A1-responsive enhancer regulates SRY in testis-determination.

The Y-linked SRY gene initiates mammalian testis-determination. However, how the expression of SRY is regulated remains elusive. Here, we demonstrate that a conserved steroidogenic factor-1 (SF-1)/NR5A1 binding enhancer is required for appropriate SRY expression to initiate testis-determination in humans. Comparative sequence analysis of SRY 5' regions in mammals identified an evolutionary conserved SF-1/NR5A1-binding motif within a 250 bp region of open chromatin located 5 kilobases upstream of the SRY transcription start site. Genomic analysis of 46,XY individuals with disrupted testis-determination, including a large multigenerational family, identified unique single-base substitutions of highly conserved residues within the SF-1/NR5A1-binding element. In silico modelling and in vitro assays demonstrate the enhancer properties of the NR5A1 motif. Deletion of this hemizygous element by genome-editing, in a novel in vitro cellular model recapitulating human Sertoli cell formation, resulted in a significant reduction in expression of SRY. Therefore, human NR5A1 acts as a regulatory switch between testis and ovary development by upregulating SRY expression, a role that may predate the eutherian radiation. We show that disruption of an enhancer can phenocopy variants in the coding regions of SRY that cause human testis dysgenesis. Since disease causing variants in enhancers are currently rare, the regulation of gene expression in testis-determination offers a paradigm to define enhancer activity in a key developmental process.

Dissecting the dynamic cellular transcriptional atlas of adult teleost testis development throughout the annual reproductive cycle.

Teleost testis development during the annual cycle involves dramatic changes in cellular compositions and molecular events. In this study, the testicular cells derived from adult black rockfish at distinct stages - regressed, regenerating and differentiating - were meticulously dissected via single-cell transcriptome sequencing. A continuous developmental trajectory of spermatogenic cells, from spermatogonia to spermatids, was delineated, elucidating the molecular events involved in spermatogenesis. Subsequently, the dynamic regulation of gene expression associated with spermatogonia proliferation and differentiation was observed across spermatogonia subgroups and developmental stages. A bioenergetic transition from glycolysis to mitochondrial respiration of spermatogonia during the annual developmental cycle was demonstrated, and a deeper level of heterogeneity and molecular characteristics was revealed by re-clustering analysis. Additionally, the developmental trajectory of Sertoli cells was delineated, alongside the divergence of Leydig cells and macrophages. Moreover, the interaction network between testicular micro-environment somatic cells and spermatogenic cells was established. Overall, our study provides detailed information on both germ and somatic cells within teleost testes during the annual reproductive cycle, which lays the foundation for spermatogenesis regulation and germplasm preservation of endangered species.

Grafted Sertoli Cells Exert Immunomodulatory Non-Immunosuppressive Effects in Preclinical Models of Infection and Cancer.

The Sertoli cells (SeCs) of the seminiferous tubules secrete a multitude of immunoregulatory and trophic factors to provide immune protection and assist in the orderly development of germ cells. Grafts of naked or encapsulated SeCs have been proved to represent an interesting therapeutic option in a plethora of experimental models of diseases. However, whether SeCs have immunosuppressive or immunomodulatory effects, which is imperative for their clinical translatability, has not been demonstrated. We directly assessed the immunopotential of intraperitoneally grafted microencapsulated porcine SeCs (MC-SeCs) in murine models of fungal infection (Aspergillus fumigatus or Candida albicans) or cancer (Lewis lung carcinoma/LLC or B16 melanoma cells). We found that MC-SeCs (i) provide antifungal resistance with minimum inflammatory pathology through the activation of the tolerogenic aryl hydrocarbon receptor/indoleamine 2,3-dioxygenase pathway; (ii) do not affect tumor growth in vivo; and (iii) reduce the LLC cell metastatic cancer spread associated with restricted Vegfr2 expression in primary tumors. Our results point to the fine immunoregulation of SeCs in the relative absence of overt immunosuppression in both infection and cancer conditions, providing additional support for the potential therapeutic use of SeC grafts in human patients.

Reprograming skin fibroblasts into Sertoli cells: a patient-specific tool to understand effects of genetic variants on gonadal development.

BACKGROUND: Disorders/differences of sex development (DSD) are congenital conditions in which the development of chromosomal, gonadal, or anatomical sex is atypical. With overlapping phenotypes and multiple genes involved, poor diagnostic yields are achieved for many of these conditions. The current DSD diagnostic regimen can be augmented by investigating transcriptome/proteome in vivo, but it is hampered by the unavailability of affected gonadal tissue at the relevant developmental stage. We try to mitigate this limitation by reprogramming readily available skin tissue-derived dermal fibroblasts into Sertoli cells (SC), which could then be deployed for different diagnostic strategies. SCs form the target cell type of choice because they act like an organizing center of embryonic gonadal development and many DSD arise when these developmental processes go awry. METHODS: We employed a computational predictive algorithm for cell conversions called Mogrify to predict the transcription factors (TFs) required for direct reprogramming of human dermal fibroblasts into SCs. We established trans-differentiation culture conditions where stable transgenic expression of these TFs was achieved in 46, XY adult dermal fibroblasts using lentiviral vectors. The resulting Sertoli like cells (SLCs) were validated for SC phenotype using several approaches. RESULTS: SLCs exhibited Sertoli-like morphological and cellular properties as revealed by morphometry and xCelligence cell behavior assays. They also showed Sertoli-specific expression of molecular markers such as SOX9, PTGDS, BMP4, or DMRT1 as revealed by IF imaging, RNAseq and qPCR. The SLC transcriptome shared about two thirds of its differentially expressed genes with a human adult SC transcriptome and expressed markers typical of embryonic SCs. Notably, SLCs lacked expression of most markers of other gonadal cell types such as Leydig, germ, peritubular myoid or granulosa cells. CONCLUSIONS: The trans-differentiation method was applied to a variety of commercially available 46, XY fibroblasts derived from patients with DSD and to a 46, XX cell line. The DSD SLCs displayed altered levels of trans-differentiation in comparison to normal 46, XY-derived SLCs, thus showcasing the robustness of this new trans-differentiation model. Future applications could include using the SLCs to improve definitive diagnosis of DSD in patients with variants of unknown significance.

Individuals with disorders/differences of sex development (DSD) frequently do not get a specific genetic diagnostic. A limitation in the field is that the relevant cell types that would be needed to study the molecular events occurring at the time of onset of many DSD are found in the embryonic gonad. This, of course, is not accessible for research or diagnostic purposes. We set out to develop a method for directly transforming more accessible cells, from adult skin, into the cells known to organize the male gonad in the embryo, Sertoli cells. A combination of unique transcription factors was stably integrated into skin fibroblasts, and culture under appropriate conditions allowed differentiation into Sertoli-like cells (SLC), but not other gonadal cell types. The SLCs recapitulated known patterns of gene expression, shape, and behavior of Sertoli cells. The method was also tested on commercially available fibroblasts from a variety of DSD genetic backgrounds. The resulting cells exhibited condition-specific behavior (gene expression, adhesion to substrate, division rate…). This method provides a new tool to study molecular events occurring at the time of onset of DSD in the embryonic gonad, and the impact of patient-specific mutations on those. It could allow identification of new developmental pathways (and, thus, new candidate genes for DSD), as well as a provide models to validate the impact of variants of unknown significance, or to test approaches to correct the genetic anomaly in patient cells.

HDAC3 promotes Sertoli cell maturation and maintains the blood-testis barrier dynamics.

Germ cell development depends on the capacity of somatic Sertoli cells to undergo differentiation into a mature state and establish a germ cell-specific blood-testis barrier (BTB). The BTB structure confers an immunological barrier for meiotic and postmeiotic germ cells, and its dynamic permeability facilitates a transient movement of preleptotene spermatocytes through BTB to enter meiosis. However, the regulatory factors involved in Sertoli cell maturation and how BTB dynamics coordinate germ cell development remain unclear. Here, we found a histone deacetylase HDAC3 abundantly expresses in Sertoli cells and localizes in both cytoplasm and nucleus. Sertoli cell-specific Hdac3 knockout in mice causes infertility with compromised integrity of blood-testis barrier, leading to germ cells unable to traverse through BTB and an accumulation of preleptotene spermatocytes in juvenile testis. Mechanistically, nuclear HDAC3 regulates the expression program of Sertoli cell maturation genes, and cytoplasmic HDAC3 forms a complex with the gap junction protein Connexin 43 to modulate the BTB integrity and dynamics through regulating the distribution of tight junction proteins. Our findings identify HDAC3 as a critical regulator in promoting Sertoli cell maturation and maintaining the homeostasis of the blood-testis barrier.

Heme oxygenase 1 (HO1) regulates autophagy and apoptosis via the PI3K/AKT/mTOR signaling pathway of yak Sertoli cells.

Successful male reproduction depends on healthy testes. Autophagy has been confirmed to be active during many cellular events associated with the testes. It is not only crucial for testicular spermatogenesis but is also an essential regulatory mechanism for Sertoli cell (SCs) ectoplasmic specialization integrity and normal function of the blood-testis-barrier. Hypoxic stress induces oxidative damage, apoptosis, and autophagy, negatively affecting the male reproductive system. Cryptorchidism is a common condition associated with infertility. Recent studies have demonstrated that hypoxia-induced miRNAs and their transcription factors are highly expressed in the testicular tissue of infertile patients. Heme oxygenase 1 (HO1) is a heat-shock protein family member associated with cellular antioxidant defense and anti-apoptotic functions. The present study found that the HO1 mRNA and protein are up-regulated in yak cryptorchidism compared to normal testes. Next, we investigated the expression of HO1 in the SCs exposed to hypoxic stress and characterized the expression of key molecules involved in autophagy and apoptosis. The results showed that hypoxic stress induced the upregulation of autophagy of SCs. The down-regulation of HO1 using siRNA increases autophagy and decreases apoptosis, while the over-expression of HO1 attenuates autophagy and increases apoptosis. Furthermore, HO1 regulates autophagy and apoptosis via the PI3K/AKT/mTOR signaling pathway. These results will be helpful for further understanding the regulatory mechanisms of HO1 in yak cryptorchidism.

The role of Sertoli cells-secreted factors in different stages of germ cells development in mice exposed to BDE-209.

Decabromodiphenyl ether (BDE-209), a frequently used brominated flame retardant, readily enters the environment and is difficult to degrade with bioaccumulation. BDE-209 could cause male reproductive toxicity, but the regulatory functions of Sertoli cells-secreted factors remain uncertain. In present study, male mice were treated with 75 mg/kg BDE-209 and then stopped exposure for 50 days. Exogenous Glial cell line-derived neurotrophic factor (GDNF), a Sertoli cell-secreted factor, was injected into testes of mice treated with BDE-209 for 50 days to explore the role of GDNF in BDE-209-induced reproductive toxicity. The mouse spermatogonia cell line GC-1 spg was used in vitro to further verify regulatory effects of Sertoli cells-secreted factors on meiotic initiation. The results showed that BDE-209 inhibited expressions of the self-renewal pathway GFRα-1/RAS/ERK1/2 in spermatogonial stem cells (SSCs), and reduced expressions of spermatogonia proliferation-related pathway NRG3/ERBB4 and meiosis initiation factor Stra8. Furthermore, BDE-209 decreased the levels of both GDNF and retinoic acid (RA) secreted by Sertoli cells in testes. Importantly, the alterations of above indicators induced by BDE-209 did not recover after 50-day recovery period. After exogenous GDNF injection, the decreased expression of GFRα-1/RAS/ERK in SSCs was reversed. However, the level of RA and expressions of NRG3/ERBB4/Stra8 were not restored. The in vitro experimental results showed that exogenous RA reversed the reductions in NRG3/ERBB4/Stra8 and ameliorated inhibition of GC-1 spg cells proliferation induced by BDE-209. These results suggested that Sertoli cells-secreted factors play roles in regulating various stages of germ cell development. Specifically, BDE-209 affected the self-renewal of SSCs by decreasing GDNF secretion resulting in the inhibition of GFRα-1/RAS/ERK pathway; BDE-209 hindered the proliferation of spermatogonia and initiation of meiosis by inhibiting the secretion of RA and preventing RA from binding to RARα, resulting in the suppression of NRG3/ERBB4/Stra8 pathway. As a consequence, spermatogenesis was compromised, leading to persistent male reproductive toxicity.

Isolation and In Vitro Culture of Germ Cells and Sertoli Cells from Human Fetal Testis.

In the human fetal testis, fetal germ cells (FGCs) are progressively surrounded by supporting Sertoli cells inside seminiferous cords. During the second trimester, the FGCs develop asynchronously and can be observed in several stages of development. However, the mechanism that regulates the transition between the different developmental stages as well as the formation of spermatogonia is currently not well understood. For this, it is necessary to develop suitable isolation protocols and a platform for in vitro culture of FGCs of different stages. Here, we report a method to isolate distinct populations of FGCs and Sertoli cells from second trimester human testis using a panel of conjugated antibodies for THY1, PDPN, ALPL, KIT, and SUSD2 for fluorescence-activated cell sorting (FACS) followed by in vitro culture up to 7 days. This platform provides the base for cellular and molecular characterization of the different testicular cell populations to investigate the transition between FGCs and spermatogonia and shed some light on crucial processes of early human gametogenesis unknown until now.

Defective autophagic flux aggravates cadmium-induced Sertoli cell apoptosis.

The male reproductive dysfunction accounts for 50% of infertile couples in the world. Cadmium (Cd) is one of the most harmful heavy metals to both the environment and inhabitants. Accumulating data suggest that Cd could cause male infertility. Sertoli cell (SC) is a somatic cell of testis and a key regulator of spermatogenesis by providing physical and nutritional support for developing sperm. Many studies showed that Cd induced dysfunction of SCs was directly related to male reproductive damage. However, the mechanism of SCs injury caused by Cd remains to be clarified. We found that Cd treatment caused a significant increase of apoptosis in SCs cells, accompanied by a marked increase in the production of ROS. These results were associated with the formation of mitochondria-containing autophagosomes and increased expression of LC3-II in vitro. Interestingly, our results showed that Cd did not promote but inhibited the fusion of mitochondria-containing autophagosomes with lysosomes by reducing the function of lysosomes. Together, this study provides insight into the negative effects of Cd, which interferes with autophagic flux and induces the apoptosis of SCs.

Rapamycin protects Sertoli cells against BPA-induced autophagy disorders.

Bisphenol A (BPA) is a well-known environmental contaminant that can negatively impact reproductive function. Disruption of autophagy is implicated in BPA-induced cell injury, the specific molecular mechanisms through which BPA affects autophagy in Sertoli cells are still unknown. In the present study, TM4 cells were exposed to various doses of BPA (10, 100, and 200 µM), and the results indicated that BPA exposure led to the accumulation of autophagosomes, this change was accompanied by increased expression of p-mTOR and decreased expression of Atg12, a protein involved in regulating autophagy initiation. Additionally, BPA exposure upregulated the expression levels of p62, a protein involved in autophagic degradation. The inhibition of autophagy initiation and autophagic degradation contributes to the accumulation of autophagosomes. Further studies showed that BPA exposure didn't affect the expression of the lysosome protein LAMP1; however, decreased cytoplasmic retention of acridine orange in TM4 cells may explain the disruption of autophagy. The role of rapamycin and chloroquine (CQ), an autophagy inhibitor that impairs lysosomal degradation also confirmed the effect of BPA on autophagy regulation. Specifically, rapamycin can protect Sertoli cells against BPA-induced cell injury by promoting autophagy. These findings contribute to our understanding of the mechanisms underlying reproductive toxicity caused by BPA.

Effect of Temperature on the Development of Stages of Spermatogenesis and the Functionality of Sertoli Cells In Vitro.

Spermatogenesis is the process of proliferation and differentiation of spermatogonial cells to meiotic and post-meiotic stages and sperm generation. Normal spermatogenesis occurs in vivo at 34 °C to 35 °C, and high temperatures are known to cause male infertility. The aim of the present study was to examine the effect of temperature (35 °C compared to 37 °C) on the viability/apoptosis of developed cells, on the development of different stages of spermatogenesis in 3D in vitro culture conditions, and the functionality of Sertoli cells under these conditions. We used isolated cells from seminiferous tubules of sexually immature mice. The cells were cultured in methylcellulose (as a three-dimensional (3D) in vitro culture system) and incubated in a CO2 incubator at 35 °C or 37 °C. After two to six weeks, the developed cells and organoids were collected and examined for cell viability and apoptosis markers. The development of different stages of spermatogenesis was evaluated by immunofluorescence staining or qPCR analysis using specific antibodies or primers, respectively, for cells at each stage. Factors that indicate the functionality of Sertoli cells were assessed by qPCR analysis. The developed organoids were examined by a confocal microscope. Our results show that the percentages and/or the expression levels of the developed pre-meiotic, meiotic, and post-meiotic cells were significantly higher at 35 °C compared to those at 37 °C, including the expression levels of the androgen receptor, the FSH receptor, transferrin, the androgen-binding protein (ABP), and the glial-derived nerve growth factor (GDNF) which were similarly significantly higher at 35 °C than at 37 °C. The percentages of apoptotic cells (according to acridine orange staining) and the expression levels of BAX, FAS, and CASPAS 3 were significantly higher in cultures incubated at 37 °C compared to those incubated at 35 °C. These findings support the in vivo results regarding the negative effect of high temperatures on the process of spermatogenesis and suggest a possible effect of high temperatures on the viability/apoptosis of spermatogenic cells. In addition, increasing the temperature in vitro also impaired the functionality of Sertoli cells. These findings may deepen our understanding of the mechanisms behind optimal conditions for normal spermatogenesis in vivo and in vitro.

Inhibition of miR-143-3p Restores Blood-Testis Barrier Function and Ameliorates Sertoli Cell Senescence.

Due to the increasing trend of delayed childbirth, the age-related decline in male reproductive function has become a widely recognized issue. Sertoli cells (SCs) play a vital role in creating the necessary microenvironment for spermatogenesis in the testis. However, the mechanism underlying Sertoli cell aging is still unclear. In this study, senescent Sertoli cells showed a substantial upregulation of miR-143-3p expression. miR-143-3p was found to limit Sertoli cell proliferation, promote cellular senescence, and cause blood-testis barrier (BTB) dysfunction by targeting ubiquitin-conjugating enzyme E2 E3 (UBE2E3). Additionally, the TGF-ß receptor inhibitor SB431542 showed potential in alleviating age-related BTB dysfunction, rescuing testicular atrophy, and reversing the reduction in germ cell numbers by negatively regulating miR-143-3p. These findings clarified the regulatory pathways underlying Sertoli cell senescence and suggested a promising therapeutic approach to restore BTB function, alleviate Sertoli cell senescence, and improve reproductive outcomes for individuals facing fertility challenges.

Regulation of spermatogonial stem cell differentiation by Sertoli cells-derived exosomes through paracrine and autocrine signaling.

In the orchestrated environment of the testicular niche, the equilibrium between self-renewal and differentiation of spermatogonial stem cells (SSCs) is meticulously maintained, ensuring a stable stem cell reserve and robust spermatogenesis. Within this milieu, extracellular vesicles, specifically exosomes, have emerged as critical conveyors of intercellular communication. Despite their recognized significance, the implications of testicular exosomes in modulating SSC fate remain incompletely characterized. Given the fundamental support and regulatory influence of Sertoli cells (SCs) on SSCs, we were compelled to explore the role of SC-derived exosomes (SC-EXOs) in the SSC-testicular niche. Our investigation hinged on the hypothesis that SC-EXOs, secreted by SCs from the testes of 5-day-old mice-a developmental juncture marking the onset of SSC differentiation-participate in the regulation of this process. We discovered that exposure to SC-EXOs resulted in an upsurge of PLZF, MVH, and STRA8 expression in SSC cultures, concomitant with a diminution of ID4 and GFRA1 levels. Intriguingly, obstructing exosomal communication in a SC-SSC coculture system with the exosome inhibitor GW4869 attenuated SSC differentiation, suggesting that SC-EXOs may modulate this process via paracrine signaling. Further scrutiny revealed the presence of miR-493-5p within SC-EXOs, which suppresses Gdnf mRNA in SCs to indirectly restrain SSC differentiation through the modulation of GDNF expression-an indication of autocrine regulation. Collectively, our findings illuminate the complex regulatory schema by which SC-EXOs affect SSC differentiation, offering novel perspectives and laying the groundwork for future preclinical and clinical investigations.

Low androgen signaling rescues genome integrity with innate immune response by reducing fertility in humans.

Development of the gonads under complex androgen regulation is critical for germ cells specification. In this work we addressed the relationship between androgens and genomic integrity determining human fertility. We used different study groups: individuals with Differences of Sex Development (DSD), including Complete Androgen Insensitivity Syndrome (CAIS) due to mutated androgen receptor (AR), and men with idiopathic nonobstructive azoospermia. Both showed genome integrity status influenced by androgen signaling via innate immune response activation in blood and gonads. Whole proteome analysis connected low AR to interleukin-specific gene expression, while compromised genome stability and tumorigenesis were also supported by interferons. AR expression was associated with predominant DNA damage phenotype, that eliminated AR-positive Sertoli cells as the degeneration of gonads increased. Low AR contributed to resistance from the inhibition of DNA repair in primary leukocytes. Downregulation of androgen promoted apoptosis and specific innate immune response with higher susceptibility in cells carrying genomic instability.

Cypermethrin induces Sertoli cell apoptosis through endoplasmic reticulum-mitochondrial coupling involving IP3R1-GRP75-VDAC1.

A widely used type II pyrethroid pesticide cypermethrin (CYP) is one of endocrine disrupting chemicals (EDCs) with anti-androgenic activity to induce male reproductive toxicology. However, the mechanisms have not been fully elucidated. This study was to explore the effects of CYP on apoptosis of mouse Sertoli cells (TM4) and the roles of endoplasmic reticulum (ER)-mitochondria coupling involving 1,4,5-trisphosphate receptor type1-glucose-regulated protein 75-voltage-dependent anion channel 1 (IP3R1-GRP75-VDAC1). TM4 were cultured with different concentrations of CYP. Flow cytometry, calcium (Ca2+) fluorescent probe, transmission electron microscopy and confocal microscopy, and western blot were to examine apoptosis of TM4, mitochondrial Ca2+, ER-mitochondria coupling, and expressions of related proteins. CYP was found to increase apoptotic rates of TM4 significantly. CYP was shown to significantly increase expressions of cleaved caspase-3, cleaved poly ADP-ribose polymerase (PARP). Concentration of mitochondrial Ca2+ was increased by CYP treatment significantly. CYP significantly enhanced ER-mitochondria coupling. CYP was shown to increase expressions of IP3R, Grp75 and VDAC1 significantly. We suggest that CYP induces apoptosis in TM4 cells by facilitating mitochondrial Ca2+ overload regulated by ER-mitochondria coupling involving IP3R1-GRP75-VDAC1. This study identifies a novel mechanism of CYP-induced apoptosis in Sertoli cells.

Testicular expression of heat SHOCK proteins 60, 70, and 90 in cryptorchid horses.

Heat shock proteins are the most evolutionarily conserved protein families induced by stressors including hyperthermia. In the context of pathologies of the male reproductive tract, cryptorchidism is the most common genital defect that compromises the reproductive potential of the male because it induces an increase in intratesticular temperature. In equine species, cryptorchidism affects almost 9 % of newborns and few studies have been carried out on the molecular aspects of the retained testis. In this study, the expression pattern of HSP60, 70, and 90 in abdominal and inguinal testes, in their contralateral descended normally testes, and in testes of normal horses were investigated by Western blot and immunohistochemistry. The histomorphological investigation of retained and scrotal testes was also investigated. The seminiferous epithelium of the retained testes showed a vacuolized appearance and displayed a completely blocked spermatogenesis for lacking meiotic and spermiogenetic cells. On the contrary, the contralateral scrotal testes did not show morphological damage and the seminiferous epithelium displayed all phases of the spermatogenetic cycle as in the normal testes. The morphology of Leydig cells was not affected by the cryptorchid state. Western blot and immunohistochemistry evidenced that equine testis (both scrotal and retained) expresses the three investigated HSPs. More in detail, the Western blot evidenced that HSP70 is the more expressed chaperone and that together with HSP90 it is highly expressed in the retained gonad (P < 0.05). The immunohistochemistry revealed the presence of the three HSPs in the spermatogonia of normal and cryptorchid testes. Spermatogonia of retained testes showed the lowest expression of HSP60 and the highest expression of HSP90. Spermatocytes, spermatids of scrotal testes, and the Sertoli cells of retained and scrotal testes did not display HSP60 whereas expressed HSP70 and HSP90. These two proteins were also localized in the nucleus of the premeiotic cells. The Leydig cells displayed the three HSPs with the higher immunostaining of HSP70 and 90 in the cryptorchid testes. The results indicate that the heat stress condition occurring in the cryptorchid testis influences the expression of HSPs.

Dysregulation of MTFR2, ATP5IF1 and BAK1 in Sertoli cells relates to idiopathic non-obstructive azoospermia via inhibiting mitochondrial fission and inducing mitochondrial dysfunction†.

Non-obstructive azoospermia affects more than 10% of infertile men with over 70% patients are idiopathic with uncharacterized molecular mechanisms, which is referred as idiopathic non-obstructive azoospermia. In this study, we checked the morphology of Sertoli cell mitochondria in testis biopsies from patients with idiopathic non-obstructive azoospermia and patients with obstructive azoospermia who have normal spermiogenesis. The expression of 104 genes controlling mitochondria fission and fusion were analyzed in three gene expression datasets including a total of 60 patients with non-obstructive azoospermia. The levels of 7 candidate genes were detected in testis biopsies from 38 patients with idiopathic non-obstructive azoospermia and 24 patients with obstructive azoospermia who have normal spermatogenesis by RT-qPCR. Cell viability, apoptosis, mitochondria membrane potential, adenosine triphosphate production, oxygen consumption, and mitochondria morphology were examined in primary human Sertoli cells. Mouse spermatogonial stem cells were used to detect the cell supporting capacity of Sertoli cells. We observed that patients with idiopathic non-obstructive azoospermia had elongated mitochondria. MTFR2 and ATP5IF1 were downregulated, whereas BAK1 was upregulated in idiopathic non-obstructive azoospermia testis and Sertoli cells. Sertoli cells from patients with idiopathic non-obstructive azoospermia had reduced viability, mitochondria membrane potential, adenosine triphosphate production, oxygen consumption rate, glycolysis and increased apoptosis. Knockdown MTFR2 in Sertoli cells increased the mitochondria size. Knockdown ATP5IF1 did not change mitochondrial morphology but increased adenosine triphosphate hydrolysis. Overexpression of BAK1 reduced membrane potential and upregulated cell apoptosis. The dysregulation of all these three genes contributed to the dysfunction of Sertoli cells, which provides a clue for idiopathic non-obstructive azoospermia treatment.