Retinoic acid mitigates the NSC319726-induced spermatogenesis dysfunction through cuproptosis-independent mechanisms.

Copper ionophore NSC319726 has attracted researchers' attention in treating diseases, particularly cancers. However, its potential effects on male reproduction during medication are unclear. This study aimed to determine whether NSC319726 exposure affected the male reproductive system. The reproductive toxicity of NSC319726 was evaluated in male mice following a continuous exposure period of 5 weeks. The result showed that NSC319726 exposure caused testis index reduction, spermatogenesis dysfunction, and architectural damage in the testis and epididymis. The exposure interfered with spermatogonia proliferation, meiosis initiation, sperm count, and sperm morphology. The exposure also disturbed androgen synthesis and blood testis barrier integrity. NSC319726 treatment could elevate the copper ions in the testis to induce cuproptosis in the testis. Copper chelator rescued the elevated copper ions in the testis and partly restored the spermatogenesis dysfunction caused by NSC319726. NSC319726 treatment also decreased the level of retinol dehydrogenase 10 (RDH10), thereby inhibiting the conversion of retinol to retinoic acid, causing the inability to initiate meiosis. Retinoic acid treatment could rescue the meiotic initiation and spermatogenesis while not affecting the intracellular copper ion levels. The study provided an insight into the bio-safety of NSC319726. Retinoic acid could be a potential therapy for spermatogenesis impairment in patients undergoing treatment with NSC319726.

MSC-derived exosomes mitigate cadmium-induced male reproductive injury by ameliorating DNA damage and autophagic flux.

Cadmium, an environmental toxicant, severely impairs male reproductive functions and currently lacks effective clinical treatments. Mesenchymal stem cell-derived exosomes (MSC-Exos) are increasingly recognized as a potential alternative to whole-cell therapy for tissue injury and regeneration. This study aims to investigate the protective effects of MSC-Exos against cadmium toxicity on male reproduction. Our findings reveal that MSC-Exos treatment significantly promotes spermatogenesis, improves sperm quality, and reduces germ cell apoptosis in cadmium-exposed mice. Mechanistically, MSC-Exos dramatically mitigate cadmium-induced cell apoptosis in a spermatogonia cell line (GC-1 spg) in vitro by reducing DNA damage and promoting autophagic flux. These results suggest that MSC-Exos have a protective effect on cadmium-induced germ cell apoptosis by ameliorating DNA damage and autophagy flux, demonstrating the therapeutic potential of MSC-Exos for cadmium toxicity on male reproduction.

Inhibition of reactive oxygen species generation by N-Acetyl Cysteine can mitigate male germ cell toxicity induced by bisphenol analogs.

The estrogen-like effect of bisphenol A (BPA) disrupting the maintenance of functional male germ cells is associated with male sub-fertility. This study investigated toxicity of male germ cells induced by four bisphenol analogs: BPA, BPAF, BPF, and BPS. The investigation of bisphenol analogs' impact on male germ cells included assessing proliferation, apoptosis induction, and the capacity to generate reactive oxygen species (ROS) in GC-1 spermatogonia (spg) cells, specifically type B spermatogonia. Additionally, the therapeutic potential and protective effects of N-Acetyl Cysteine (NAC) and NF-κB inhibitor parthenolide was evaluated. In comparison to BPA, BPF and BPS, BPAF exhibited the most pronounced adverse effect in GC-1 spg cell proliferation. This effect was characterized by pronounced inhibition of phosphorylation of PI3K, AKT, and mTOR, along with increased release of cytochrome c and subsequent cleavages of caspase 3, caspase 7, and poly (ADP-ribose) polymerase. Both NAC and parthenolide were effective reducing cellular ROS induced by BPAF. However, only NAC demonstrated a substantial recovery in proliferation, accompanied by a significant reduction in cytochrome c release and cleaved PARP. These results suggest that NAC supplementation may play an effective therapeutic role in countering germ cell toxicity induced by environmental pollutants with robust oxidative stress-generating capacity.

Restoration of fertility in nonablated recipient mice after spermatogonial stem cell transplantation.

Spermatogonial stem cell (SSC) transplantation is a valuable tool for studying stem cell-niche interaction. However, the conventional approach requires the removal of endogenous SSCs, causing damage to the niche. Here we introduce WIN18,446, an ALDH1A2 inhibitor, to enhance SSC colonization in nonablated recipients. Pre-transplantation treatment with WIN18,446 induced abnormal claudin protein expression, which comprises the blood-testis barrier and impedes SSC colonization. Consequently, WIN18,446 increased colonization efficiency by 4.6-fold compared with untreated host. WIN18,446-treated testes remained small despite the cessation of WIN18,446, suggesting its irreversible effect. Offspring were born by microinsemination using donor-derived sperm. While WIN18,446 was lethal to busulfan-treated mice, cyclophosphamide- or radiation-treated animals survived after WIN18,446 treatment. Although WIN18,446 is not applicable to humans due to toxicity, similar ALDH1A2 inhibitors may be useful for SSC transplantation into nonablated testes, shedding light on the role of retinoid metabolism on SSC-niche interactions and advancing SSC research in animal models and humans.

Pubertal exposure to Microcystin-LR arrests spermatogonia proliferation by inducing DSB and inhibiting SIRT6 dependent DNA repair in vivo and in vitro.

The reproduction toxicity of pubertal exposure to Microcystin-LR (MC-LR) and the underlying mechanism needs to be further investigated. In the current study, pubertal male ICR mice were intraperitoneally injected with 2 µg/kg MC-LR for four weeks. Pubertal exposure to MC-LR decreased epididymal sperm concentration and blocked spermatogonia proliferation. In-vitro studies found MC-LR inhibited cell proliferation of GC-1 cells and arrested cell cycle in G2/M phase. Mechanistically, MC-LR exposure evoked excessive reactive oxygen species (ROS) and induced DNA double-strand break in GC-1 cells. Besides, MC-LR inhibited DNA repair by reducing PolyADP-ribosylation (PARylation) activity of PARP1. Further study found MC-LR caused proteasomal degradation of SIRT6, a monoADP-ribosylation enzyme which is essential for PARP1 PARylation activity, due to destruction of SIRT6-USP10 interaction. Additionally, MG132 pretreatment alleviated MC-LR-induced SIRT6 degradation and promoted DNA repair, leading to the restoration of cell proliferation inhibition. Correspondingly, N-Acetylcysteine (NAC) pre-treatment mitigated the disturbed SIRT6-USP10 interaction and SIRT6 degradation, causing recovered DNA repair and subsequently restoration of cell proliferation inhibition in MC-LR treated GC-1 cells. Together, pubertal exposure to MC-LR induced spermatogonia cell cycle arrest and sperm count reduction by oxidative DNA damage and simultaneous SIRT6-mediated DNA repair failing. This study reports the effect of pubertal exposure to MC-LR on spermatogenesis and complex mechanism how MC-LR induces spermatogonia cell proliferation inhibition.

Stage-specific regulation of undifferentiated spermatogonia by AKT1S1-mediated AKT-mTORC1 signaling during mouse spermatogenesis.

Undifferentiated spermatogonia are composed of a heterogeneous cell population including spermatogonial stem cells (SSCs). Molecular mechanisms underlying the regulation of various spermatogonial cohorts during their self-renewal and differentiation are largely unclear. Here we show that AKT1S1, an AKT substrate and inhibitor of mTORC1, regulates the homeostasis of undifferentiated spermatogonia. Although deletion of Akt1s1 in mouse appears not grossly affecting steady-state spermatogenesis and male mice are fertile, the subset of differentiation-primed OCT4+ spermatogonia decreased significantly, whereas self-renewing GFRα1+ and proliferating PLZF+ spermatogonia were sustained. Both neonatal prospermatogonia and the first wave spermatogenesis were greatly reduced in Akt1s1-/- mice. Further analyses suggest that OCT4+ spermatogonia in Akt1s1-/- mice possess altered PI3K/AKT-mTORC1 signaling, gene expression and carbohydrate metabolism, leading to their functionally compromised developmental potential. Collectively, these results revealed an important role of AKT1S1 in mediating the stage-specific signals that regulate the self-renewal and differentiation of spermatogonia during mouse spermatogenesis.

JMJD3 regulate H3K27me3 modification via interacting directly with TET1 to affect spermatogonia self-renewal and proliferation.

BACKGROUND: In epigenetic modification, histone modification and DNA methylation coordinate the regulation of spermatogonium. Not only can methylcytosine dioxygenase 1 (TET1) function as a DNA demethylase, converting 5-methylcytosine to 5-hydroxymethylcytosine, it can also form complexes with other proteins to regulate gene expression. H3K27me3, one of the common histone modifications, is involved in the regulation of stem cell maintenance and tumorigenesis by inhibiting gene transcription. METHODS: we examined JMJD3 at both mRNA and protein levels and performed Chip-seq sequencing of H3K27me3 in TET1 overexpressing cells to search for target genes and signaling pathways of its action. RESULTS: This study has found that JMJD3 plays a leading role in spermatogonia self-renewal and proliferation: at one extreme, the expression of the self-renewal gene GFRA1 and the proliferation-promoting gene PCNA was upregulated following the overexpression of JMJD3 in spermatogonia; at the other end of the spectrum, the expression of differentiation-promoting gene DAZL was down-regulated. Furthermore, the fact that TET1 and JMJD3 can form a protein complex to interact with H3K27me3 has also been fully proven. Then, through analyzing the sequencing results of CHIP-Seq, we found that TET1 targeted Pramel3 when it interacted with H3K27me3. Besides, TET1 overexpression not only reduced H3K27me3 deposition at Pramel3, but promoted its transcriptional activation as well, and the up-regulation of Pramel3 expression was verified in JMJD3-overexpressing spermatogonia. CONCLUSION: In summary, our study identified a novel link between TET1 and H3K27me3 and established a Tet1-JMJD3-H3K27me3-Pramel3 axis to regulate spermatogonia self-renewal and proliferation. Judging from the evidence offered above, we can safely conclude that this study provides new ideas for further research regarding the mechanism of spermatogenesis and spermatogenesis disorders on an apparent spectrum.

Isolation and In Vitro Propagation of Human Spermatogonial Stem Cells (SSCs).

Preservation of human spermatogonial stem cells (SSCs) may be suitable for young male patients at risk of male infertility due to various causes, such as gonadotoxic treatment or genetic diseases. With optimal cryopreservation, cell viability can be retained to reestablish spermatogenesis in the future through autologous transplantation or in vitro differentiation of SSCs. This protocol outlines techniques to optimize the SSCs isolation and in vitro culture. With particular emphasis on the microscopic characteristics encountered, this protocol outlines a broader approach to processing tissues with varying morphologies among patients.

Transcriptome Analysis of Key Genes Involved in the Initiation of Spermatogonial Stem Cell Differentiation.

PURPOSE: The purpose of this study was to screen the genes and pathways that are involved in spermatogonia stem cell (SSC) differentiation regulation during the transition from Aundiff to A1. Methods: RNA sequencing was performed to screen differentially expressed genes at 1 d and 2 d after SSC differentiation culture. KEGG pathway enrichment and GO function analysis were performed to reveal the genes and pathways related to the initiation of early SSC differentiation. RESULTS: The GO analysis showed that Rpl21, which regulates cell differentiation initiation, significantly increased after 1 day of SSC differentiation. The expressions of Fn1, Cd9, Fgf2, Itgb1, Epha2, Ctgf, Cttn, Timp2 and Fgfr1, which are related to promoting differentiation, were up-regulated after 2 days of SSC differentiation. The analysis of the KEGG pathway revealed that RNA transport is the most enriched pathway 1 day after SSC differentiation. Hspa2, which promotes the differentiation of male reproductive cells, and Cdkn2a, which participates in the cell cycle, were significantly up-regulated. The p53 pathway and MAPK pathway were the most enriched pathways 2 days after SSC differentiation. Cdkn1a, Hmga2, Thbs1 and Cdkn2a, microRNAs that promote cell differentiation, were also significantly up-regulated. CONCLUSIONS: RNA transport, the MAPK pathway and the p53 pathway may play vital roles in early SSC differentiation, and Rpl21, Fn1, Cd9, Fgf2, Itgb1, Epha2, Ctgf, Cttn, Timp2, Fgfr1, Hspa2, Cdkn2a, Cdkn1a, Hmga2 and Thbs1 are involved in the initiation of SSC differentiation. The findings of this study provide a reference for further revelations of the regulatory mechanism of SSC differentiation.

Successful xenotransplantation of testicular cells following fractionated chemotherapy of recipient birds.

An essential step in the success of germ cell transplantation is the preparation of the recipient's testicular environment to increase the availability of stem cell niches. However, most methods for this purpose in birds face serious limitations such as partial germ cell depletion, high toxicity and mortality, or the need to use expensive technologies. Here, we validated a simple and practical technique of transferring quail testicular cells into chicken testes depleted of endogenous spermatozoa by fractioned chemotherapy (20 mg/kg/week busulfan for 5 weeks). This protocol resulted in a very low mortality of the treated day-old chicks and, despite maintenance of androgenic activity, sperm production was decreased by 84.3% at 25 weeks of age. NANOG immunostaining revealed that very few to no germ cells were present following treatment with 20 and 40 mg/kg, respectively. RT-qPCR data also showed that c-MYC and NANOG expression declined in these treatments, but GRFα1 and BID expressions remained unaltered among groups. After xenotransplantation, quail germ cells were immunodetected in chicken testes using a species-specific antibody (QCPN), and quail ovalbumin DNA was found in seminal samples collected from chicken recipients. Together, these data confirm that fractionated administration of busulfan in hatchlings is a practical, effective, and safe protocol to prepare recipient male birds capable of supporting xenogeneic spermatogenesis.

Postnatal development of mouse spermatogonial stem cells as determined by immunophenotype, regenerative capacity, and long-term culture-initiating ability: a model for practical applications.

Spermatogonial stem cells (SSCs) are the foundation of life-long spermatogenesis. While SSC research has advanced greatly over the past two decades, characterization of SSCs during postnatal development has not been well documented. Using the mouse as a model, in this study, we defined the immunophenotypic profiles of testis cells during the course of postnatal development using multi-parameter flow cytometry with up to five cell-surface antigens. We found that the profiles progress over time in a manner specific to developmental stages. We then isolated multiple cell fractions at different developmental stages using fluorescent-activated cell sorting (FACS) and identified specific cell populations with prominent capacities to regenerate spermatogenesis upon transplantation and to initiate long-term SSC culture. The data indicated that the cell fraction with the highest level of regeneration capacity exhibited the most prominent potential to initiate SSC culture, regardless of age. Interestingly, refinement of cell fractionation using GFRA1 and KIT did not lead to further enrichment of regenerative and culture-initiating stem cells, suggesting that when a high degree of SSC enrichment is achieved, standard markers of SSC self-renewal or commitment may lose their effectiveness to distinguish cells at the stem cell state from committed progenitors. This study provides a significant information resource for future studies and practical applications of mammalian SSCs.

Evaluating testicular tissue for future autotransplantation: focus on cancer cell contamination and presence of spermatogonia in tissue cryobanked for boys diagnosed with a hematological malignancy.

STUDY QUESTION: What is the contamination rate by cancer cells and spermatogonia numbers in immature testicular tissue (ITT) harvested before the start of gonadotoxic therapy in boys with a hematological malignancy? SUMMARY ANSWER: Among our cohort of boys diagnosed with acute lymphoblastic leukemia (ALL) and lymphomas, 39% (n = 11/28) had cancer cells identified in their tissues at the time of diagnosis and all patients appeared to have reduced spermatogonia numbers compared to healthy reference cohorts. WHAT IS KNOWN ALREADY: Young boys affected by a hematological cancer are at risk of contamination of their testes by cancer cells but histological examination is unable to detect the presence of only a few cancer cells, which would preclude autotransplantation of cryobanked ITT for fertility restoration, and more sensitive detection techniques are thus required. Reduced numbers of spermatogonia in ITT in hematological cancer patients have been suggested based on results in a limited number of patients. STUDY DESIGN, SIZE, DURATION: This retrospective cohort study included 54 pre- and peri-pubertal boys who were diagnosed with a hematological malignancy and who underwent a testicular biopsy for fertility preservation at the time of diagnosis before any gonadotoxic therapy between 2005 and 2021. PARTICIPANTS/MATERIALS, SETTING, METHODS: Among the 54 patients eligible in our database, formalin-fixed paraffin-embedded (FFPE) testicular tissue was available for 28 boys diagnosed either with ALL (n = 14) or lymphoma (n = 14) and was used to evaluate malignant cell contamination. Hematoxylin and eosin (H&E) staining was performed for each patient to search for cancer cells in the tissue. Markers specific to each patient's disease were identified at the time of diagnosis on the biopsy of the primary tumor or bone marrow aspiration and an immunohistochemistry (IHC) was performed on the FFPE ITT for each patient to evidence his disease markers. PCR analyses on the FFPE tissue were also conducted when a specific gene rearrangement was available. MAIN RESULTS AND THE ROLE OF CHANCE: The mean age at diagnosis and ITT biopsy of the 28 boys was 7.5 years (age range: 19 months-16 years old). Examination of ITT of the 28 boys on H&E stained sections did not detect malignant cells. Using IHC, we found contamination by cancerous cells using markers specific to the patient's disease in 10 of 28 boys, with a higher rate in patients diagnosed with ALL (57%, n = 8/14) compared with lymphoma (14%, n = 2/14) (P-value < 0.05). PCR showed contamination in three of 15 patients who had specific rearrangements identified on their bone marrow at the time of diagnosis; one of these patients had negative results from the IHC. Compared to age-related reference values of the number of spermatogonia per ST (seminiferous tubule) (Spg/ST) throughout prepuberty of healthy patients from a simulated control cohort, mean spermatogonial numbers appeared to be decreased in all age groups (0-4 years: 1.49 ± 0.54, 4-7 years: 1.08 ± 0.43, 7-11 years: 1.56 ± 0.65, 11-14 years: 3.37, 14-16 years: 5.44 ± 3.14). However, using a cohort independent method based on the Z-score, a decrease in spermatogonia numbers was not confirmed. LIMITATIONS, REASONS FOR CAUTION: The results obtained from the biopsy fragments that were evaluated for contamination by cancer cells may not be representative of the entire cryostored ITT and tumor foci may still be present outside of the biopsy range. WIDER IMPLICATIONS OF THE FINDINGS: ITT from boys diagnosed with a hematological malignancy could bear the risk for cancer cell reseeding in case of autotransplantation of the tissue. Such a high level of cancer cell contamination opens the debate of harvesting the tissue after one or two rounds of chemotherapy. However, as the safety of germ cells can be compromised by gonadotoxic treatments, this strategy warrants for the development of adapted fertility restoration protocols. Finally, the impact of the hematological cancer on spermatogonia numbers should be further explored. STUDY FUNDING/COMPETING INTEREST(S): The project was funded by a grant from the FNRS-Télévie (grant n°. 7.4533.20) and Fondation Contre le Cancer/Foundation Against Cancer (2020-121) for the research project on fertility restoration with testicular tissue from hemato-oncological boys. The authors declare that they have no conflict of interest. TRIAL REGISTRATION NUMBER: N/A.

PBA alleviates cadmium-induced mouse spermatogonia apoptosis by suppressing endoplasmic reticulum stress.

OBJECTIVE: Endoplasmic reticulum (ER) stress mediates Cd-caused germ cell apoptosis in testis. The effects of 4-phenylbutyric acid (PBA), a classical chaperone, were investigated on Cd-induced apoptosis in mouse GC-1 spermatogonia cells. METHODS: The cells were pretreated with PBA before Cd exposure. TUNEL and flow cytometry assays were applied to determine apoptosis. Some key biomarkers of ER stress were analyzed using RT-PCR and western blot. RESULTS: as expected, the apoptotic cells exposed to Cd apparently increased. The mRNA and protein expression levels of GRP78 and ATF6α, were elevated in the Cd groups. Additional experiments displayed that Cd notably increased IRE1α and JNK phosphorylation, and upregulated XBP-1 mRNA and protein expression. Moreover, p-eIF2α and CHOP expressions were clearly elevated in the Cd groups. Interestingly, PBA almost completely inhibited ER stress and protected spermatogonia against apoptosis induced by Cd. CONCLUSION: PBA alleviated Cd-induced ER stress and spermatogonia apoptosis, and may have the therapeutic role in Cd-induced male reproductive toxicity.

Splicing factor SRSF1 is essential for homing of precursor spermatogonial stem cells in mice.

Spermatogonial stem cells (SSCs) are essential for continuous spermatogenesis and male fertility. The underlying mechanisms of alternative splicing (AS) in mouse SSCs are still largely unclear. We demonstrated that SRSF1 is essential for gene expression and splicing in mouse SSCs. Crosslinking immunoprecipitation and sequencing data revealed that spermatogonia-related genes (e.g. Plzf, Id4, Setdb1, Stra8, Tial1/Tiar, Bcas2, Ddx5, Srsf10, Uhrf1, and Bud31) were bound by SRSF1 in the mouse testes. Specific deletion of Srsf1 in mouse germ cells impairs homing of precursor SSCs leading to male infertility. Whole-mount staining data showed the absence of germ cells in the testes of adult conditional knockout (cKO) mice, which indicates Sertoli cell-only syndrome in cKO mice. The expression of spermatogonia-related genes (e.g. Gfra1, Pou5f1, Plzf, Dnd1, Stra8, and Taf4b) was significantly reduced in the testes of cKO mice. Moreover, multiomics analysis suggests that SRSF1 may affect survival of spermatogonia by directly binding and regulating Tial1/Tiar expression through AS. In addition, immunoprecipitation mass spectrometry and co-immunoprecipitation data showed that SRSF1 interacts with RNA splicing-related proteins (e.g. SART1, RBM15, and SRSF10). Collectively, our data reveal the critical role of SRSF1 in spermatogonia survival, which may provide a framework to elucidate the molecular mechanisms of the posttranscriptional network underlying homing of precursor SSCs.

Leptin promotes proliferation of human undifferentiated spermatogonia by activating the PI3K/AKT/mTOR pathway.

BACKGROUND: Male infertility is a common disease affecting male reproductive health. Leptin is an important hormone that regulates various physiological processes, including reproductive function. However, few experimental studies have been carried out to elucidate the mechanism of leptin's effects on male reproductive function. OBJECTIVE: The purpose of this study was to investigate the effects of leptin on testicular spermatogenesis and its mechanism, so as to provide potential targets for the treatment of patients with spermatogenic dysfunction. METHODS: Testicular tissues were collected from eight prostate cancer patients undergoing surgical castration. GPR125-positive spermatogonia were isolated by two consecutive magnetic activated cell sorting (MACS), followed by incubation with conditioned medium. To identify the signaling pathway(s) involved in the effects of leptin, undifferentiated spermatogonia were treated with different concentrations of leptin and antagonists of leptin-related pathways. The proliferative effect of leptin was evaluated by cell counting using a hemocytometer. Expressions of p-AKT, p-ERK, p-STAT, and p-S6K were determined by western blotting analysis. RESULTS: Leptin promoted the growth of human GPR125-positive spermatogonia in a concentration-dependent manner. The most significant proliferative effect was observed using 100 ng/mL leptin after 6 days of culture. Leptin significantly increased the phosphorylation of STAT3, AKT, and ERK in undifferentiated spermatogonia. Phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 inhibited the leptin-induced activation of AKT, ERK, and downstream S6K. Treatment with the mammalian target of rapamycin (mTOR) inhibitor rapamycin also inhibited S6K phosphorylation. Moreover, both LY294002 and rapamycin were found to inhibit the leptin-induced proliferation of undifferentiated spermatogonia. These results suggested that the leptin-induced proliferation of GPR125-positive spermatogonia was dependent on the PI3K/AKT/mTOR pathway. Further exploration of proliferation and apoptotic markers suggested that leptin may alleviate cell apoptosis by regulating the expression of Bax and FasL. CONCLUSIONS: A certain concentration of leptin (25∼100 ng/mL) could promote proliferation of undifferentiated spermatogonia, which was mediated by PI3K/AKT/mTOR pathway.

BNC1 deficiency induces mitochondrial dysfunction-triggered spermatogonia apoptosis through the CREB/SIRT1/FOXO3 pathway: the therapeutic potential of nicotinamide riboside and metformin†.

Male infertility is a global health problem that disturbs numerous couples worldwide. Basonuclin 1 (BNC1) is a transcription factor mainly expressed in proliferative keratinocytes and germ cells. A frameshift mutation of BNC1 was identified in a large Chinese primary ovarian insufficiency pedigree. The expression of BNC1 was significantly decreased in the testis biopsies of infertile patients with nonobstructive azoospermia. Previous studies have revealed that mice with BNC1 deficiency are generally subfertile and undergo gradual spermatogenic failure. We observed that apoptosis of spermatogonia is tightly related to spermatogenic failure in mice with a Bnc1 truncation mutation. Such impairment is related to mitochondrial dysfunction causing lower mitochondrial membrane potential and higher reactive oxygen species. We showed that downregulation of CREB/SIRT1/FOXO3 signaling participates in the above impairment. Administration of nicotinamide riboside or metformin reversed mitochondrial dysfunction and inhibited apoptosis in Bnc1-knockdown spermatogonia by stimulating CREB/SIRT1/FOXO3 signaling. Dietary supplementation with nicotinamide riboside or metformin in mutated mice increased SIRT1 signaling, improved the architecture of spermatogenic tubules, inhibited apoptosis of the testis, and improved the fertility of mice with a Bnc1 truncation mutation. Our data establish that oral nicotinamide riboside or metformin can be useful for the treatment of spermatogenic failure induced by Bnc1 mutation.

Omega-3 polyunsaturated fatty acids and its metabolite 12-HEPE rescue busulfan disrupted spermatogenesis via target to GPR120.

Busulfan is an antineoplastic, which is always accompanied with the abnormal of spermatogonia self-renewal and differentiation. It has been demonstrated that the omega-3 polyunsaturated fatty acids (PUFAs) benefits mature spermatozoa. However, whether omega-3 can protect endogenous spermatogonia and the detailed mechanisms are still unclear. Evaluate of spermatogenesis function (in vivo) were examined by histopathological analysis, immunofluorescence staining, and western blotting. The levels of lipid metabolites in testicular tissue were determined via liquid chromatography. We investigated the effect of lipid metabolites on Sertoli cells provided paracrine factors to regulate spermatogonia proliferation and differentiation using co-culture system. In our study, we showed that omega-3 PUFAs significantly improved the process of sperm production and elevated the quantity of both undifferentiated Lin28+ spermatogonia and differentiated c-kit+ spermatogonia in a mouse model where spermatogenic function was disrupted by busulfan. Mass spectrometry revealed an increase in the levels of several omega-3 metabolites in the testes of mice fed with omega-3 PUFAs. The eicosapentaenoic acid metabolite 12-hydroxyeicosapentaenoic acid (12-HEPE) up-regulated bone morphogenic protein 4 (BMP4) expression through GPR120-ERK1/2 pathway activation in Sertoli cells and restored spermatogonia proliferation and differentiation. Our study provides evidence that omega-3 PUFAs metabolite 12-HEPE effectively protects spermatogonia and reveals that GPR120 might be a tractable pharmacological target for fertility in men received chemotherapy or severe spermatogenesis dysfunction.

Differential roles of cyclooxygenase enzymes in the regulation of murine juvenile undifferentiated spermatogonia.

BACKGROUND: Acetaminophen and ibuprofen are widely administered to babies due to their presumed safety as over-the-counter drugs. However, no reports exist on the effects of cyclooxygenase inhibitors on undifferentiated spermatogonia and spermatogonial stem cells. Infancy represents a critical period for spermatogonial stem cell formation and disrupting spermatogonial stem cells or their precursors may be associated with infertility and testicular cancer formation. OBJECTIVES: The goal of this study was to examine the molecular and functional impact of cyclooxygenase inhibition and silencing on early steps of undifferentiated spermatogonia (u spg) and spermatogonial stem cell development, to assess the potential reproductive risk of pharmaceutical cyclooxygenase inhibitors. METHODS: The effects of cyclooxygenase inhibition were assessed using the mouse C18-4 undifferentiated juvenile spermatogonial cell line model, previously shown to include cells with spermatogonial stem cell features, by measuring prostaglandins, cell proliferation, and differentiation, using cyclooxygenase 1- and cyclooxygenase 2-selective inhibitors NS398, celecoxib, and FR122047, acetaminophen, and ibuprofen. Cyclooxygenase 1 gene silencing was achieved using a stable short-hairpin RNA approach and clone selection, then assessing gene and protein expression in RNA sequencing, quantitative real-time polymerase chain reaction, and immunofluorescence studies. RESULTS: Cyclooxygenase 2 inhibitors NS398 and celecoxib, as well as acetaminophen, but not ibuprofen, dose-dependently decreased retinoic acid-induced expression of the spg differentiation gene Stra8, while NS398 decreased the spg differentiation marker Kit, suggesting that cyclooxygenase 2 is positively associated with spg differentiation. In contrast, short-hairpin RNA-based cyclooxygenase 1 silencing in C18-4 cells altered cellular morphology and upregulated Stra8 and Kit, implying that cyclooxygenase 1 prevented spg differentiation. Furthermore, RNA sequencing analysis of cyclooxygenase 1 knockdown cells indicated the activation of several signaling pathways including the TGFb, Wnt, and Notch pathways, compared to control C18-4 cells. Notch pathway genes were upregulated by selective cyclooxygenase inhibitors, acetaminophen and ibuprofen. CONCLUSION: We report that cyclooxygenase 1 and 2 differentially regulate undifferentiated spermatogonia/spermatogonial stem cell differentiation. Cyclooxygenases regulate Notch3 expression, with the Notch pathway targeted by PGD2. These data suggest an interaction between the eicosanoid and Notch signaling pathways that may be critical for the development of spermatogonial stem cells and subsequent spermatogenesis, cautioning about using cyclooxygenase inhibitors in infants.

Hyaluronic acid/silicon nanoparticle scaffold induces proliferation and differentiation of mouse spermatogonial stem cells transplanted to epididymal adipose tissue.

Spermatogonia stem cells (SSCs) are a unique cell population maintaining male spermatogenesis during life, through their potential for proliferation and differentiation. The application of silicon nanoparticles (SNs) and hyaluronic acid (HA) to induce the differentiation of SSCs seems promising. Herein, we investigate the effect of SN and HA scaffolds on the progression of SSCs spermatogenesis in mice. Initially SSCs were isolated from healthy immature mice and cultured on prepared scaffolds (HA, SN, and HA/SN) in a 3D culture system. Then viability of SSCs cultured on scaffolds was examined using MTT assay and Acridine Orange staining. Then SSCs cultured on scaffolds were transplanted into epididymal adipose tissue (EAT) in mature mice and the result was studied by H&E and IHC staining 8 weeks after transplantation. MTT and Acridine Orange analysis revealed that among three different scaffolds HA/SN based scaffold causes considerable toxicity on SSCs (P < 0.05) while H&E staining showed that culture of SSCs on HA, SN, and HA/SN scaffolds has a positive effect on the progression of SSCs spermatogenesis after transplantation into EAT. IHC staining identified TP1, TEKT1, and PLZF as crucial biomarkers in the spermatogenesis development of SSCs transplanted to EAT. According to the presence of these biomarkers in different experimental groups, we found the most spermatogenesis development in SSCs cultured on HA/SN scaffold (PLZF, P < 0.01) (TEKT1, P < 0.01) (TP1, P < 0.001). Our study showed that, although the cytotoxic effect of the HA/SN scaffold decreases the viability rate of SSCs; however, SSCs that survive on HA/SN scaffold showed more ability to progress in spermatogenesis after transplantation into EAT.

Recovering Spermatogenesis By Protected Cryopreservation Using Metformin and Transplanting Spermatogonial Stem Cells Into Testis in an Azoospermia Mouse Model.

Cryopreservation and transplantation of spermatogonial stem cells (SSCs) may serve as a new method to restore male fertility in patients undergoing chemotherapy or radiotherapy. However, SSCs may be damaged during cryopreservation due to the production of reactive oxygen species (ROS). Therefore, different antioxidants have been used as protective agents. Studies have shown that metformin (MET) has antioxidant activity. The aim of this study was to assess the antioxidant and antiapoptotic effects of MET in frozen-thawed SSCs. In addition, the effect of MET on the proliferation and differentiation of SSCs was evaluated. To this end, SSCs were isolated from mouse pups aged 3-6 days old, cultured, identified by flow cytometry (ID4, INTEGRIN ß1+), and finally evaluated for survival and ROS rate. SSCs were transplanted after busulfan and cadmium treatment. Cryopreserved SSCs with and without MET were transplanted after 1 month of cryopreservation. Eight weeks after transplantation, the recipient testes were evaluated for the expression of apoptosis (BAX, BCL2), proliferation (PLZF), and differentiation (SCP3, TP1, TP2, PRM1) markers using immunohistochemistry, Western blot, and quantitative real-time polymerase chain reaction. The findings revealed that the survival rate of SSCs was higher in the 500 µm/mL MET group compared to the other groups (50 and 5000 µm/mL). MET significantly decreased the intracellular ROS production. Transplantation of SSCs increased the expression level of proliferation (PLZF) and differentiation (SCP3, TP1, TP2, PRM1) markers compared to azoospermia group, and their levels were significantly higher in the MET group compared to the cryopreservation group containing basic freezing medium (p < 0.05). MET increased the survival rate of SSCs, proliferation, and differentiation and decreased the ROS production and the apoptosis rate. Cryopreservation by MET seems to be effective in treating infertility.