Potential role of mitochondria and endoplasmic reticulum in the response elicited by D-aspartate in TM4 Sertoli cells.

D-Aspartic Acid (D-Asp) affects spermatogenesis by enhancing the biosynthesis of the sex steroid hormones acting either through the hypothalamus-pituitary-testis axis or directly on Leydig cells. Recently, in vitro studies have also demonstrated the direct effects of D-Asp on the proliferation and/or activity of germ cells. However, although D-Asp is present in Sertoli cells (SC), the specific role of the amino acid in these cells remains unknown. This study investigated the effects of D-Asp on the proliferation and activity of TM4 SC, focusing on the mitochondrial compartment and its association with the endoplasmic reticulum (ER). We found that D-Asp enhanced the proliferation and activity of TM4 cells as evidenced by the activation of ERK/Akt/PCNA pathway and the increase in the protein levels of the androgen receptor. Furthermore, D-Asp reduced both the oxidative stress and apoptotic process. An increase in mitochondrial functionality and dynamics, as well as a reduction in ER stress, were also found in D-Asp-treated TM4 cells. It is known that mitochondria are closely associated with ER to form the Mitochondrial-Associated Endoplasmic Reticulum Membranes (MAM), the site of calcium ions and lipid transfer from ER to the mitochondria, and vice versa. The data demonstrated that D-Asp induced stabilization of MAM in TM4 cells. In conclusion, this study is the first to demonstrate a direct effect of D-Asp on SC activity and to clarify the cellular/molecular mechanism underlying these effects, suggesting that D-Asp could stimulate spermatogenesis by improving the efficiency of SC.

Profile of key metabolites and identification of HMGCS1-DHEA pathway in porcine Sertoli cells treated by Vitamin C.

Vitamin C (Ascorbic acid, AA), as vital micro-nutrient, plays an essential role for male animal reproduction. Previously, we showed that vitamin C reprogrammed the transcriptome and proteome to change phenotypes of porcine immature Sertoli cells (iSCs). Here, we used LC-MS-based non-targeted metabolomics to further investigate the metabolic effects of vitamin C on porcine iSCs. The results identified 43 significantly differential metabolites (DMs) (16 up and 27 down) as induced by vitamin C (L-ascorbic acid 2-phosphate sesquimagnesium salt hydrate, AA2P) treatment of porcine iSCs, which were mainly enriched in steroid related and protein related metabolic pathways. ELISA (Enzyme-Linked ImmunoSorbent Assay) showed that significantly differential metabolites of Dehydroepiandrosterone (DHEA) (involved in steroid hormone biosynthesis) and Desmosterol (involved in steroid degradation) were significantly increased, which were partially consistent with metabolomic results. Further integrative analysis of metabolomics, transcriptomics and proteomics data identified the strong correlation between the key differential metabolite of Dehydroepiandrosterone and 6 differentially expressed genes (DEGs)/proteins (DEPs) (HMGCS1, P4HA1, STON2, LOXL2, EMILIN2 and CCN3). Further experiments validated that HMGCS1 could positively regulate Dehydroepiandrosterone level. These data indicate that vitamin C could modulate the metabolism profile, and HMGCS1-DHEA could be the pathway to mediate effects exerted by vitamin C on porcine iSCs.

Maternal exposure to phenanthrene induces testicular apoptosis and Sertoli cell dysfunction in F1 adult male mice: a histological and molecular study.

Objective: Phenanthrene, a polycyclic aromatic hydrocarbon, is found in abundance in environmental pollutants, food, and drinking water. This substance can accumulate in body tissues and exert harmful effects. Moreover, phenanthrene can cross the placental barrier, potentially impacting fetal development. We aimed to explore the impacts of maternal exposure to phenanthrene on testicular tissue and Sertoli cell function in F1 mice. Methods: Female rats with vaginal plugs were randomly assigned to one of three groups: control, sham, or phenanthrene. The control group received no intervention during pregnancy. In the sham and phenanthrene groups, corn oil and a phenanthrene solution, respectively, were administered via gavage once every 2 days. Offspring were separated by sex 21 days after birth. At 56 days postnatal, male F1 offspring were euthanized, and their testes were harvested for histological and molecular analyses. Results: Phenanthrene exposure was associated with a lower testicular weight and volume, a smaller diameter of the seminiferous tubules, and a relative thinning of the germinal epithelium. These changes were associated with increased cellular apoptosis, as shown by the upregulation of caspase 3 expression. Additionally, we observed an increase in vacuolization and residual bodies within the tissue. Conversely, the number of Sertoli cells and expression levels of Sox9, as well as the Ocln and Itgb1 genes, were found to be lowered. Conclusion: Maternal exposure to phenanthrene impacts both germ cells and Sertoli cells, disrupting their function and leading to fertility disorders in male F1 offspring mice.

Motor proteins, spermatogenesis and testis function.

The role of motor proteins in supporting intracellular transports of vesicles and organelles in mammalian cells has been known for decades. On the other hand, the function of motor proteins that support spermatogenesis is also well established since the deletion of motor protein genes leads to subfertility and/or infertility. Furthermore, mutations and genetic variations of motor protein genes affect fertility in men, but also a wide range of developmental defects in humans including multiple organs besides the testis. In this review, we seek to provide a summary of microtubule and actin-dependent motor proteins based on earlier and recent findings in the field. Since these two cytoskeletons are polarized structures, different motor proteins are being used to transport cargoes to different ends of these cytoskeletons. However, their involvement in germ cell transport across the blood-testis barrier (BTB) and the epithelium of the seminiferous tubules remains relatively unknown. It is based on recent findings in the field, we have provided a hypothetical model by which motor proteins are being used to support germ cell transport across the BTB and the seminiferous epithelium during the epithelial cycle of spermatogenesis. In our discussion, we have highlighted the areas of research that deserve attention to bridge the gap of research in relating the function of motor proteins to spermatogenesis.

A new type of spermiogenesis in teleost fish: Formation of the aflagellate sperm in Campylomormyrus compressirostris (Osteoglossomorpha: Mormyridae).

Osteoglossomorpha, the bony tongue fishes, show great variation in morphology, behavioural strategies, reproductive biology and gamete ultrastructure. The order Osteoglossiformes is the only vertebrate taxon, in which four types of sperm (monoflagellate, biflagellate and aflagellate aquasperm and the complex introsperm) have been described. It is also the only vertebrate lineage in which aflagellate spermatozoa exist. The aim of this study was to analyse the structure of the testis and the process of spermiogenesis in the mormyrid Campylomormyrus compressirostris during the breeding season using light and electron microscopy (transmission and scanning). Males of this species have a single testis of the anastomosing tubular type. The tubules of the anterior part of the testis contain cysts with developing germ cells, and this region is much wider than the posterior part, which consists of efferent ducts filled with sperm cells. The cysts are filled with single or mitotic spermatogonia, primary and secondary spermatocytes and early spermatids. At the stage of spermatids with fine granular chromatin, the cysts rupture and successive stages of spermatid differentiation take place in the testicular lumen; we therefore characterise this process as 'extracystic spermiogenesis'. Sperm development in C. compressirostris is extremely simple and involves chromatin condensation in the central region of the nucleus, a slight decrease in nuclear volume, the appearance of numerous vesicles in the cytoplasm that form a tubular-vesicular system at the base of the nucleus. Both centrioles and mitochondria are translocated to the peripheral region of the midpiece, which forms the opposite pole to the nucleus. There are many differences between the types of spermiogenesis described so far in teleosts and that found in C. compressirostris, including the loss of flagellum formation. This unique type of spermiogenesis is restricted to species of the families Mormyridae and Gymnarchidae, all of which possess aflagellate spermatozoa. Our data demonstrate that the spermatid differentiation and existence of the aflagellate spermatozoon are a unique phenomena not only among teleosts but also in the whole vertebrate lineage.

Oxylipin profiling analyses reveal that ω-3 PUFA is more susceptible to lipid oxidation in sheep testis under oxidative stress.

Reactive oxygen species causes oxidative stress, which oxidizes polyunsaturated fatty acids (PUFAs) to form oxidative metabolites. Sertoli cell is an important cellular metabolism of PUFA in testicular cells, and it regulates the testis development and spermatogenesis. However, the oxylipins generated in testes with different developmental statuses are lacking. In this study, twelve 6-month-old Hu sheep were selected and divided into large testicular group (L) and the small testicular group (S) (n=6). UPLC-MS/MS was conducted to screen oxylipins in the testis, and the total oxylipin and ω-3 PUFA-derived oxylipin contents in the S group were higher. A total of 20 differential oxylipins between the two groups were screened. Among them, the contents of ω-3 PUFA, DHA-derived oxylipins were increased in the S group. The arachidonic acid-derived oxylipin was lower in the S group. The mRNA expression levels of genes related to oxylipin regulation (AKR1B1, PTGER2, and PTGDS) were higher in the S group (P < 0.05). In vitro, 200 µM α-linolenic acid alleviated oxidative stress damage to Sertoli cells and improved cell viability by increasing the superoxide dismutase contents and mRNA expression levels of GPX4 and Bcl2. These results indicate that ω-3 PUFA is more susceptible to lipid oxidation in the S group under oxidative stress, which might alleviate the damage of oxidative stress to testis. Moreover, ALA could stimulate the proliferation of Sertoli cells by increasing the capacity of antioxidants. This work may provide a theoretical basis for further studies on the antioxidant properties of the testis for Hu sheep.

Engineering of gelatin scaffold by extracellular matrix of Sertoli cells for embryonic stem cell proliferation.

Mimicking the microenvironment of seminiferous tubules plays an indispensable role in directing differentiation of stem cells toward germ cells in vitro. In this work, we fabricated electrospun gelatin (EG) mats (i.e., with diameter <500 nm) conditioned with Sertoli cells' extracellular matrix (ECM) to simulate both 3D structures and composition of normal testis tissue. Sertoli cells were isolated from mice testis and represented through immunocytochemistry (ICC) staining for expression of vimentin, a specific marker of Sertoli cells. The morphological characteristics of ECM-coated scaffold were investigated under scanning electron microscope (SEM). The efficient elimination of cells was confirmed by MTT assay. Furthermore, the cyto/biocompatibility of ECM-conditioned EG scaffold was determined for Sertoli cells and embryonic stem cells (ESCs), alone and as in co-culture. According to the results, the designed scaffold provided a mat for cell proliferation with negligible toxicity (almost 100% cell viability). SEM micrographs displayed cells with elongated shape and complete stretching morphology when compared with those cultured on scaffold without ECM. Moreover, an enhanced differentiation of ESCs toward sperm-generating cells was obtained through co-culturing of Sertoli cells and ESCs, where cell viability was found almost 100%. Our findings introduce the ECM-conditioned EG scaffold as a potentially influential engineered substrate for in vitro guidance of stem cells differentiation by mimicking the native microenvironment.

Ultrastructure of the Bovine Testis in Cattle (Bos taurus): New View.

The purpose of this study was to analyze the ultrastructure of the testes of sexually immature calves and reproductive bulls of the Polish Holstein-Friesian Black-and-White breed. Utilizing TEM, this study identified three distinct stages of seminiferous tubule development in calves, characterized by varying shapes, distributions, and arrangements of individual cells. In immature animals, early developing spermatocytes, prespermatogonia, and pre-Sertoli cells were observed within the seminiferous tubules. In sexually mature bulls, all cells of the spermatogenic series were observed, situated on a thin, multilayered basal lamina, which forms characteristic undulations. An abundant smooth endoplasmic reticulum was observed in the cytoplasm of spermatogonia in both groups of animals, forming characteristic membranous swirls. In adult bulls, spermatogonia maintain contact with each other through numerous cytoplasmic bridges and cell connections, forming small spaces with visible microvilli between them. The ultrastructural analysis facilitated the identification of morphological changes occurring during the maturation of pre-Sertoli cells, transitioning from a large euchromatic nucleus to a nucleus in which the formation of characteristic vesicles and tubules could be observed. It should also be emphasized that two types of Sertoli cells, namely dark and light electron-dense cells, can be found in cattle. These cells differ from each other, indicating that they may perform different functions. The widespread recognition of the presence of two types of Sertoli cells in cattle will undoubtedly contribute to a better understanding of the processes occurring within the testes and provide a basis for further research in this area.

9-cis-retinoic acid signaling in Sertoli cells regulates their immunomodulatory function to control lymphocyte physiology and Treg differentiation.

BACKGROUND: Testis is an immune privileged organ, which prevents the immune response against sperm antigens and inflammation. Testicular cells responsible for immune tolerance are mainly Sertoli cells, which form the blood-testis barrier and produce immunosuppressive factors. Sertoli cells prevent inflammation in the testis and maintain immune tolerance by inhibiting proliferation and inducing lymphocyte apoptosis. It has been shown that 9-cis-retinoic acid (9cRA) blocks ex vivo apoptosis of peripheral blood lymphocytes and promotes the differentiation of Treg cells in the gut. However, the role of retinoid signaling in regulating the immune privilege of the testes remains unknown. OBJECTIVE: The aim of this study was to determine whether 9cRA, acting via the retinoic acid receptors (RAR) and the retinoic X receptors (RXR), controls the immunomodulatory functions of Sertoli cells by influencing the secretion of anti-inflammatory/pro-inflammatory factors, lymphocyte physiology and Treg cell differentiation. METHODS: Experiments were performed using in vitro model of co-cultures of murine Sertoli cells and T lymphocytes. Agonists and antagonists of retinoic acid receptors were used to inhibit/stimulate retinoid signaling in Sertoli cells. RESULTS: Our results have demonstrated that 9cRA inhibits the expression of immunosuppressive genes and enhances the expression of pro-inflammatory factors in Sertoli cells and lymphocytes, increases lymphocyte viability and decreases apoptosis rate. Moreover, we have found that 9cRA blocks lymphocyte apoptosis acting through both RAR and RXR and inhibiting FasL/Fas/Caspase 8 and Bax/Bcl-2/Caspase 9 pathways. Finally, we have shown that 9cRA signaling in Sertoli cells inhibits Treg differentiation. CONCLUSION: Collectively, our results indicate that retinoid signaling negatively regulates immunologically privileged functions of Sertoli cells, crucial for ensuring male fertility. 9cRA inhibits lymphocyte apoptosis, which can be related to the development of autoimmunity, inflammation, and, in consequence, infertility.

Spermatogonial quantity in prepubertal boys undergoing fertility preservation is comparable between haematological and non-haematological cancers.

Fertility restoration potential of immature testicular tissue (ITT) depends on the number of spermatogonial cells in the retrieved tissue prior to cryopreservation in oncofertility programme. There are limited data on the association between type of malignancy and testicular germ cell population. Hence, this study is aimed to investigate the spermatogonial and Sertoli cell population in ITT retrieved from 14 pre-pubertal boys who opted for fertility preservation. Histopathological and immunochemical analysis of seminiferous tubules from haematological (N = 7) and non-haematological (N = 7) malignant patients revealed 3.43 ± 2.92 and 1.71 ± 1.81 spermatogonia per tubular cross section (S/T), respectively. The Sertoli cell number was comparable between haematological and non-haematological group (18.42 ± 3.78 and 22.03 ± 10.43). Spermatogonial quantity in ITT did not vary significantly between haematological and non-haematological cancers. This observation, though preliminary, would contribute to the limited literature on paediatric male oncofertility.

Combined exposure of PS-MPs with NaF induces Sertoli cell death and dysfunction via ferroptosis and apoptosis.

The individual toxicity of sodium fluoride (NaF) and microplastics (MPs) has been extensively documented. Owing to their high specific surface area, widespread presence and durability, MPs can adsorb a broad spectrum of environmental contaminants into the organism. However, the combined toxicity of NaF and MPs has not been investigated. This study aimed to assess the effects of combined exposure to NaF and MPs on the function of testicular Sertoli cells (SCs) in male mice, and to investigate the underlying molecular mechanisms. The study revealed that combined exposure to NaF and MPs resulted in a decrease in the negative surface charge of MPs, along with an increase in the number of MPs entering the SCs. Through in vivo observation of the testicular pathological structure, spermatogenesis, and cell apoptosis in 180-day-old male mice, we discovered that combined exposure to NaF (80 mg/L) and MPs (10 mg/L) heightened reproductive toxicity compared to the individual exposure groups. This was evidenced by testicular structural defects, impaired spermatogenesis, and increased testicular cell apoptosis. Our in vitro studies showed that NaF (21 µg/mL) and MPs (100 µg/mL) synergistically induced SCs apoptosis and ferroptosis, leading to a reduction in SCs number and dysfunction. This ultimately resulted in structural and functional damage to the testes. Our findings demonstrate, for the first time, the synergistic effects of NaF and MPs on reproductive toxicity in mammals. These insights may provide valuable contributions to co-toxicity studies involving MPs and other environmental pollutants.

Assessing testicular morphofunctionality under Roundup WG® herbicide exposure in zebrafish.

Glyphosate-based herbicides, like Roundup WG® (RWG) used for a range of crops, such as corn, soybean, coffee, sugarcane, rice, apple, and citrus, can reach aquatic ecosystems and impact non-target organisms like fish. Thus, the fish were exposed to three RWG concentrations plus one negative control, which represents the concentration allowed for inland Brazilian waters and concentrations found in surface water worldwide (0.0, 0.065, 0.65, and 6.5 mg a.i./L) for 7 and 15 days. Morphological analysis revealed significant alterations in the testicular structure, particularly in Sertoli cell extensions and cytoplasmic bridges between germ cells. Subcellular compartments also displayed alterations, including dilated mitochondria and the loss of electron density and autophagic vesicles. Gene transcript levels related to autophagy and steroidogenic regulation were upregulated in exposed fish. Germ cell quality was also affected, increasing ROS (reactive oxygen species) production and DNA fragmentation. The study highlighted the RWG reproductive toxicity, providing valuable insights into understanding the morphofunctional alterations in somatic and germ cells of Danio rerio. In conclusion, the environmental relevant concentrations used in this study were toxic to male somatic and germ cells, which raises a concern about the concentrations considered safe for human and animal use.

Retinoic acid tiers mitochondrial metabolism to Sertoli Cell-Mediated efferocytosis via a non-RAR-dependent mechanism.

Efferocytosis of massive non-viable germ cells by Sertoli cells (SCs), the specialized phagocytes, is essential for maintaining testis homeostasis. What elusive is the contribution of mitochondrial metabolism to this energy-consuming process, as SC has a preference of aerobic glycolysis. All-trans retinoic acid (ATRA, hereafter referred to as RA) is a well-known morphogen that primarily acts through the nuclear RA receptor (RAR). It sustains SC blood-testisbarrier integrity, and it's SC-derived RA sets the timing of meiotic commitment. In this study, we revisited RA in SC biology, from the perspective of SC-mediated efferocytosis. We provide evidence that RA induces transcriptional programming of multiple regulators involved in efferocytosis, which thereby represses SC-mediated efferocytosis, via a RAR-independent mechanism, as blocking pan-RAR activity fails to rescue RA-induced defective efferocytosis. RA-treated SCs exhibit alternations in mitochondrial dynamics and metabolism, and the hindered efferocytosis can be rescued by stimulating mitochondrial OXPHOS via pharmacological targeting of AMPK and PDK. We thus prefer to propose a signaling axis of RA-mitochondrial metabolism-efferocytosis. Our study uncovers a hitherto unappreciated role of RA in SC biology and tiers mitochondria metabolism to SC-mediated efferocytosis, contributing a deeper understanding of SC in male reproduction.

Cryptorchidism and testicular cancer in the dog: unresolved questions and challenges in translating insights from human studies.

Cryptorchidism, the failure of one or both testes to descend into the scrotum, and testicular cancer show a strong correlation in both dogs and humans. Yet, long-standing medical debates persist about whether the location of undescended testes directly causes testicular cancer in humans or if both conditions stem from a common origin. Although testicular cancer is a prevalent disease in dogs, even less is known about its cause and correlation with testicular descent in this species. This review investigates the relation between these two disorders in dogs, drawing insights from human studies, and examines key biomarkers identified thus far. In addition, it explores potential causal links, including the impact of temperature on maturing testicular cells and a potential shared genetic origin. Notably, this literature review reveals significant differences between men and dogs in reproductive development, histological and molecular features of testicular tumors, and the prevalence of specific tumor types, such as Sertoli cell tumors (SCTs) in cryptorchid dogs and germ cell tumors (GCTs) in humans. These disparities caution against using dogs as models for human testicular cancer research and underscore the limitations when drawing comparisons between species. The paper concludes by suggesting specific research initiatives to enhance our understanding of the complex interplay between cryptorchidism and testicular cancer in dogs.

Interaction between Chromodomain Y-like Protein and Androgen Receptor Signaling in Sertoli Cells Accounts for Spermatogenesis.

Spermatogenesis is a highly regulated process dependent on androgen receptor (AR) signaling in Sertoli cells. However, the pathogenic mechanisms of spermatogenic failure, by which loss of AR impairs downstream target genes to affect Sertoli cell function, remain incompletely understood. By using microarray analysis, we identified several AR-regulated genes involved in the maturation of spermatogenesis, including chromodomain Y-like protein (CDYL) and transition proteins 1 (TNP-1), that were significantly decreased in ARKO mouse testes. AR and CDYL were found to co-localize and interact in Sertoli cells. The AR-CDYL complex bound to the promoter regions of TNP1 and modulated their transcriptional activity. CDYL acts as a co-regulator of AR transactivation, and its expression is decreased in the Sertoli cells of human testes from patients with azoospermia. The androgen receptor-chromodomain Y-like protein axis plays a crucial role in regulating a network of genes essential for spermatogenesis in Sertoli cells. Disruption of this AR-CDYL regulatory axis may contribute to spermatogenic failure. These findings provide insights into novel molecular mechanisms targeting the AR-CDYL signaling pathway, which may have implications for developing new therapeutic strategies for male infertility.

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.

Sertoli cells require hnRNPC to support normal spermatogenesis and male fertility in mice†.

Sertoli cells act as highly polarized testicular cells that nutritionally support multiple stages of germ cell development. However, the gene regulation network in Sertoli cells for modulating germ cell development has yet to be fully understood. In this study, we report that heterogeneous nuclear ribonucleoproteins C in Sertoli cells are essential for germ cell development and male fertility. Conditional knockout of heterogeneous nuclear ribonucleoprotein C in mouse Sertoli cells leads to aberrant Sertoli cells proliferation, disrupted cytoskeleton of Sertoli cells, and compromised blood-testis barrier function, resulting in loss of supportive cell function and, ultimately, defective spermiogenesis in mice. Further ribonucleic acid-sequencing analyses revealed these phenotypes are likely caused by the dysregulated genes in heterogeneous nuclear ribonucleoprotein C-deficient Sertoli cells related to cell adhesion, cell proliferation, and apoptotic process. In conclusion, this study demonstrates that heterogeneous nuclear ribonucleoprotein C plays a critical role in Sertoli cells for maintaining the function of Sertoli cells and sustaining steady-state spermatogenesis in mice.

Therapeutic potential of exosomes in spermatogenesis regulation and male infertility.

BACKGROUND: Spermatogenesis is a fundamental process crucial for male reproductive health and fertility. Exosomes, small membranous vesicles released by various cell types, have recently garnered attention for their role in intercellular communication. OBJECTIVE: This review aims to comprehensively explore the role of exosomes in regulating spermatogenesis, focusing on their involvement in testicular development and cell-to-cell communication. METHODS: A systematic examination of literature was conducted to gather relevant studies elucidating the biogenesis, composition, and functions of exosomes in the context of spermatogenesis. RESULTS: Exosomes play a pivotal role in orchestrating the complex signaling networks required for proper spermatogenesis. They facilitate the transfer of key regulatory molecules between different cell populations within the testes, including Sertoli cells, Leydig cells, and germ cells. CONCLUSION: The emerging understanding of exosome-mediated communication sheds light on novel mechanisms underlying spermatogenesis regulation. Further research in this area holds promise for insights into male reproductive health and potential therapeutic interventions.

Impaired Spermatogenesis in Infertile Patients with Orchitis and Experimental Autoimmune Orchitis in Rats.

Experimental autoimmune orchitis (EAO) is a well-established rodent model of organ-specific autoimmunity associated with infertility in which the testis immunohistopathology has been extensively studied. In contrast, analysis of testis biopsies from infertile patients associated with inflammation has been more limited. In this work, testicular biopsies from patients with idiopathic non-obstructive azoospermia diagnosed with hypospermatogenesis (HypoSp) [mild: n = 9, and severe: n = 11], with obstructive azoospermia and complete Sp (spermatogenesis) (control group, C, n = 9), and from Sertoli cell-only syndrome (SCOS, n = 9) were analyzed for the presence of immune cells, spermatogonia and Sertoli cell (SCs) alterations, and reproductive hormones levels. These parameters were compared with those obtained in rats with EAO. The presence of increased CD45+ cells in the seminiferous tubules (STs) wall and lumen in severe HypoSp is associated with increased numbers of apoptotic meiotic germ cells and decreased populations of undifferentiated and differentiated spermatogonia. The SCs showed an immature profile with the highest expression of AMH in patients with SCOS and severe HypoSp. In SCOS patients, the amount of SCs/ST and Ki67+ SCs/ST increased and correlated with high serum FSH levels and CD45+ cells. In the severe phase of EAO, immune cell infiltration and apoptosis of meiotic germ cells increased and the number of undifferentiated and differentiated spermatogonia was lowest, as previously reported. Here, we found that orchitis leads to reduced sperm number, viability, and motility. SCs were mature (AMH-) but increased in number, with Ki67+ observed in severely damaged STs and associated with the highest levels of FSH and inflammatory cells. Our findings demonstrate that in a scenario where a chronic inflammatory process is underway, FSH levels, immune cell infiltration, and immature phenotypes of SCs are associated with severe changes in spermatogenesis, leading to azoospermia. Furthermore, AMH and Ki67 expression in SCs is a distinctive marker of severe alterations of STs in human orchitis.

Ferroptosis Is Crucial for Cisplatin Induced Sertoli Cell Injury via N6-Methyladenosine Dependent Manner.

PURPOSE: This study aimed to investigate the effect of the N6-methyladenosine (m6A) dependent ferroptosis on cisplatininduced Sertoli cell injury. MATERIALS AND METHODS: A cisplatin exposure mouse model was established by intraperitoneal injection of cisplatin in our study. TM4 cell lines was used for in vitro study. Ferroptosis was detected according to metabolomic analysis and a series of assays, including malondialdehyde, glutathione, and glutathione disulfide concentration detection, 2',7'-dichlorodihydrofluorescein diacetate and BODIPY 581/591 C11 probe detection, and transmission electron microscope imaging. Key ferroptosis-related genes were identified via transcriptomic analysis, western blot and immunohistochemistry. The m6A modification was demonstrated via m6A RNA immunoprecipitation and luciferase reporter assays. Immune cell infiltration was detected by mass cytometry, and verified by flow cytometry and immunofluorescence. RESULTS: Ferroptosis, but not other types of programmed cell death, is a significant phenomenon in cisplatin-induced testis damage and Sertoli cell loss. Ferroptosis induced by cisplatin in Sertoli cell/TM4 cell is GPX4 independent but is regulated by SLC7A11 and ALOX12. Both SLC7A11 and ALOX12 are regulated via m6A dependent manner by METTL3. Furthermore, overexpressed ALOX12-12HETE pathway may result in macrophage polarization and inflammatory response in cisplatin exposure testis. CONCLUSIONS: Cisplatin-induced Sertoli cell injury via ferroptosis and promoted ferroptosis in an m6A dependent manner. m6A modification of both SLC7A11 and ALOX12 mRNA could result in ferroptosis in our in vitro model. Further, overexpressed ALOX12 can cause more production of 12-HETE, which may be responsible for testis inflammation caused by cisplatin.