Lats1 and Lats2 regulate YAP and TAZ activity to control the development of mouse Sertoli cells.

Recent reports suggest that the Hippo signaling pathway regulates testis development, though its exact roles in Sertoli cell differentiation remain unknown. Here, we examined the functions of the main Hippo pathway kinases, large tumor suppressor homolog kinases 1 and 2 (Lats1 and Lats2) in developing mouse Sertoli cells. Conditional inactivation of Lats1/2 in Sertoli cells resulted in the disorganization and overgrowth of the testis cords, the induction of a testicular inflammatory response and germ cell apoptosis. Stimulated by retinoic acid 8 (STRA8) expression in germ cells additionally suggested that germ cells may have been preparing to enter meiosis prior to their loss. Gene expression analyses of the developing testes of conditional knockout animals further suggested impaired Sertoli cell differentiation, epithelial-to-mesenchymal transition, and the induction of a specific set of genes associated with Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ)-mediated integrin signaling. Finally, the involvement of YAP/TAZ in Sertoli cell differentiation was confirmed by concomitantly inactivating Yap/Taz in Lats1/2 conditional knockout model, which resulted in a partial rescue of the testicular phenotypic changes. Taken together, these results identify Hippo signaling as a crucial pathway for Sertoli cell development and provide novel insight into Sertoli cell fate maintenance.

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.

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

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

Characterization of sexual maturity-associated N6-methyladenosine in boar testes.

BACKGROUND: The health and size of the testes are crucial for boar fertility. Testicular development is tightly regulated by epigenetics. N6-methyladenosine (m6A) modification is a prevalent internal modification on mRNA and plays an important role in development. The mRNA m6A methylation in boar testicular development still needs to be investigated. RESULTS: Using the MeRIP-seq technique, we identify and profile m6A modification in boar testes between piglets and adults. The results showed 7783 distinct m6A peaks in piglets and 6590 distinct m6A peaks in adults, with 2,471 peaks shared between the two groups. Enrichment of GO and KEGG analysis reveal dynamic m6A methylation in various biological processes and signalling pathways. Meanwhile, we conjointly analyzed differentially methylated and expressed genes in boar testes before and after sexual maturity, and reproductive related genes (TLE4, TSSK3, TSSK6, C11ORF94, PATZ1, PHLPP1 and PAQR7) were identified. Functional enrichment analysis showed that differential genes are associated with important biological functions, including regulation of growth and development, regulation of metabolic processes and protein catabolic processes. CONCLUSION: The results demonstrate that m6A methylation, differential expression and the related signalling pathways are crucial for boar testicular development. These results suggest a role for m6A modification in boar testicular development and provided a resource for future studies on m6A function in boar testicular development.

Prenatal nicotine exposure leads to epigenetic alterations in peripheral nervous system signaling genes in the testis of the rat.

BACKGROUND: Prenatal nicotine exposure (PNE) has been documented to cause numerous deleterious effects on fetal development. However, the epigenetic changes promoted by nicotine exposure on germ cells are still not well understood. OBJECTIVES: In this study, we focused on elucidating the impact of prenatal nicotine exposure on regulatory epigenetic mechanisms important for germ cell development. METHODS: Sprague-Dawley rats were exposed to nicotine during pregnancy and male progeny was analyzed at 11 weeks of age. Testis morphology was analyzed using frozen testis sections and expression of germ cell markers was examined by RT-qPCR; histone modifications were assessed by Western Blot (WB). DNA methylation analysis was performed by methylation-specific PCR of bisulfite converted DNA. Genome-wide DNA methylation was analyzed using Methylated DNA immunoprecipitation (MeDIP)-seq. We also carried out transcriptomics analysis of pituitary glands by RNA-seq. RESULTS: We show that gestational exposure to nicotine reduces germ cell numbers, perturbs meiosis, affects the expression of germ line reprogramming responsive genes, and impacts the DNA methylation of nervous system genes in the testis. PNE also causes perturbation of gene expression in the pituitary gland of the brain. CONCLUSIONS: Our data demonstrate that PNE leads to perturbation of male spermatogenesis, and the observed effects are associated with changes of peripheral nervous system signaling pathways. Alterations in the expression of genes associated with diverse biological activities such as cell migration, cell adhesion and GABA signaling in the pituitary gland underscore the complexity of the effects of nicotine exposure during pregnancy.

The corrective role of superparamagnetic iron oxide nanoparticles for the genes controlling hypothalamus-pituitary-testis-axis in male obesity-associated secondary hypogonadism.

Background: Obesity is one of the most prevalent and perilous health affairs. Male obesity-associated secondary hypogonadism (MOSH) is one of many of its complexities, which is mounting in parallel with the aggravation of obesity. Magnetic nanoparticles seem to be an advanced favorable trend in multiple biomedical fields. Aim: In this study, we explore the therapeutic effects of superparamagnetic iron oxide nanoparticles (SPIONs) coated with carboxymethyl cellulose (CMC) on an obese male rat model with MOSH syndrome, comparing their impacts with a well-known anti-obesity medication (Orlistat). Methods: 42 male albino rats split into 7 equal groups: 1-negative control: nonobese, untreated; 35 rats fed the high fat-high fructose (HFHF) diet for a period of 12 weeks. Obese rats splitted into 6 equal groups; 2-positive control: obese untreated; 3-obese given Orlistat (30 mg/kg); 4-obese given CMC-SPIONs (25 mgFe/kg); 5-obese given CMC-SPIONs (50 mgFe/kg); 6-obese given CMC-SPIONs(25 mgFe/kg) + Orlistat (30 mg/kg), 7-obese given CMC-SPIONs (50 mgFe/kg) + Orlistat (30 mg/kg); all treatments given orally for 4 weeks. During sacrifice, blood serum and sectioned hypothalamic, pituitary, testicular, and adipose tissues were collected for biochemical and biomolecular assessments. Results: The HFHF diet for 12 weeks resulted in a significant upsurge in body weight, body mass index, serum fasting glucose, insulin resistance, TAG, total cholesterol, and LDL-c; HDL-c was dropped. Serum FSH, LH, and testosterone values declined. A significant disorder in expression levels of genes regulating the hypothalamic-pituitary-testicular-axis pathway. Hypothalamic GnRH, Kisspeptin-1, Kisspeptin-r1, and Adipo-R1 values declined. GnIH and Leptin-R1 values raised up. Pituitary GnRH-R values declined. Testicular tissue STAR, HSD17B3, and CYP19A1 values declined. Adipose tissue adiponectin declined, while leptin raised up. CMC-SPIONs 25-50 mg could modulate the deranged biochemical parameters and correct the deranged expression levels of all previous genes. Co-treatments revealed highly synergistic effects on all parameters. Overall, CMC-SPIONs have significant efficiency whether alone or with Orlisat in limiting obesity and consequence subfertility. Conclusion: CMC-SPIONs act as an incoming promising contender for obesity and MOSH disorders management, and need more studies on their mechanisms.

NAD+ precursors promote the restoration of spermatogenesis in busulfan-treated mice through inhibiting Sirt2-regulated ferroptosis.

Rationale: In recent years, nicotinamide adenine dinucleotide (NAD+) precursors (Npre) have been widely employed to ameliorate female reproductive problems in both humans and animal models. However, whether and how Npre plays a role in the male reproductive disorder has not been fully clarified. Methods: In the present study, a busulfan-induced non-obstructive azoospermic mouse model was used, and Npre was administered for five weeks following the drug injection, with the objective of reinstating spermatogenesis and fertility. Initially, we assessed the NAD+ level, germ cell types, semen parameters and sperm fertilization capability. Subsequently, testis tissues were examined through RNA sequencing analysis, ELISA, H&E, immunofluorescence, quantitative real-time PCR, and Western blotting techniques. Results: The results indicated that Npre restored normal level of NAD+ in blood and significantly alleviated the deleterious effects of busulfan (BU) on spermatogenesis, thereby partially reestablishing fertilization capacity. Transcriptome analysis, along with recovery of testicular Fe2+, GSH, NADPH, and MDA levels, impaired by BU, and the fact that Fer-1, an inhibitor of ferroptosis, restored spermatogenesis and semen parameters close to CTRL values, supported such possibility. Interestingly, the reduction in SIRT2 protein level by the specific inhibitor AGK2 attenuated the beneficial effects of Npre on spermatogenesis and ferroptosis by affecting PGC-1α and ACLY protein levels, thus suggesting how these compounds might confer spermatogenesis protection. Conclusion: Collectively, these findings indicate that NAD+ protects spermatogenesis against ferroptosis, probably through SIRT2 dependent mechanisms. This underscores the considerable potential of Npre supplementation as a feasible strategy for preserving or restoring spermatogenesis in specific conditions of male infertility and as adjuvant therapy to preserve male fertility in cancer patients receiving sterilizing treatments.

Apigetrin ameliorates doxorubicin prompted testicular damage: biochemical, spermatological and histological based study.

Doxorubicin (DOX) is a highly effective, commonly prescribed, potent anti-neoplastic drug that damages the testicular tissues and leads to infertility. Apigetrin (APG) is an important flavonoid that shows diverse biological activities. The present research was designed to evaluate the alleviative role of APG against DOX-induced testicular damages in rats. Forty-eight adult male albino rats were randomly distributed into 4 groups, control, DOX administered (3 mgkg-1), DOX + APG co-administered (3 mgkg-1 of DOX; 15 mgkg-1 of APG), and APG administered group (15 mgkg-1). Results of the current study indicated that DOX treatment significantly reduced the activities of superoxide dismutase (SOD), glutathione reductase (GSR), catalase (CAT) and glutathione peroxidase (GPx), while increasing the levels of malondialdehyde (MDA) and reactive oxygen species (ROS). DOX treatment also reduced the sperm count, viability, and motility. Moreover, DOX significantly increased the sperm morphological anomalies and reduced the levels of plasma testosterone, luteinizing hormone (LH) and follicle-stimulating hormone (FSH). The administration of DOX significantly increased the expressions of Bax and Caspase-3, as well as the levels of inflammatory markers. Additionally, DOX treatment significantly downregulated the expressions of steroidogenic enzymes (StAR, 3ß-HSD and 17ß-HSD) and Bcl-2. Furthermore, DOX administration provoked significant histopathological abnormalities in the testicular tissues. However, APG supplementation significantly reversed all the testicular damages due to its androgenic, anti-apoptotic, anti-oxidant and anti-inflammatory nature. Therefore, it is concluded that APG may prove a promising therapeutic agent to treat DOX-induced testicular damages.

Uncovering Novel Protein Partners of Inducible Nitric Oxide Synthase in Human Testis.

Peroxidative damage to human spermatozoa has been shown to be the primary cause of male infertility. The possible role of nitric oxide (NO) in affecting sperm motility, capacitation, and acrosome reaction has been reported, too. The overproduction of NO by the enzyme inducible nitric oxide synthase (iNOS) could be responsible as it has been implicated in the pathogenesis of many diseases. There have been many studies on regulating iNOS function in various tissues, especially by protein-protein interaction; however, no study has looked for iNOS-interacting proteins in the human testis. Here, we have reported the identification of two proteins that interact with iNOS. We initially undertook a popular yeast two-hybrid assay to screen a human testis cDNA library in yeast using an iNOS-peptide fragment (amino acids 181-335) as bait. We verified our data using the mammalian chemiluminescent co-IP method; first, employing the same peptide and, then, a full-length protein co-expressed in HEK293 cells in addition to the candidate protein. In both cases, these two protein partners of iNOS were revealed: (a) sperm acrosome-associated 7 protein and (b) retinoblastoma tumor-suppressor binding protein.

The expression profiles of cyp19a1, sf-1, esrs and gths in the brain-pituitary during gonadal sex differentiation in juvenile Japanese eels.

Eels are gonochoristic species whose gonadal differentiation initiates at the yellow eel stage and is influenced by environmental factors. We revealed some sex-related genes were sex dimorphically expressed in gonads during gonadal sex differentiation of Japanese eel (Anguilla japonica); however, the expression of sex-related genes in the brain-pituitary during gonadal sex differentiation in eels is still unclear. This study aimed to investigate the sex-related gene expressions in the brain-pituitary and tried to clarify their roles in the brain and gonads during gonadal sex differentiation. Based on our previous histological study, the control eels developed as males, and estradiol-17ß (E2) was used for feminization. Our results showed that during testicular differentiation, the brain cyp19a1 transcripts and aromatase proteins were increased significantly; moreover, the cyp19a1, sf-1, foxl2s, and esrs (except gperb) transcripts in the midbrain/pituitary also were increased significantly. Forebrain gnrh1 transcripts increased slightly during gonadal differentiation of both sexes, but the gnrhr1b and gnrhr2 transcripts in the midbrain/pituitary were stable during gonadal differentiation. The expression levels of gths and gh in the midbrain/pituitary were significantly increased during testicular differentiation and were much higher in males than in E2-feminized females. These results implied that endogenous estrogens might play essential roles in the brain/pituitary during testicular differentiation, sf-1, foxl2s, and esrs may have roles in cyp19a1 regulation in the midbrain/pituitary of Japanese eels. For the GnRH-GTH axis, gths, especially fshb, may be regulated by esrs and involved in regulating testicular differentiation and development in Japanese eels.

Semaglutide mitigates testicular damage in diabetes by inhibiting ferroptosis.

Diabetes is linked to male infertility, but the mechanisms and therapeutic options remain unclear. This study investigates the effects of semaglutide on testicular function in a diabetes mouse model. Clinical data shows that diabetes affects blood glucose, lipid levels, and sperm quality. Single-cell and transcriptome analyses reveal changes in testicular tissue cell proportions and activation of ferroptosis pathways in diabetic patients/rats. In the diabetes mouse model, sperm quality decreases significantly. Treatment with semaglutide (Sem) and the ferroptosis inhibitor ferrostatin-1 (Fer-1) alleviates testicular damage, as evidenced by improved lipid peroxidation and ferroptosis markers. Moreover, the diabetes-induced decrease in the TM-3 cell line's vitality, increased lipid peroxidation, ROS, ferrous ions, and mitochondrial membrane potential damage are all improved by semaglutide and ferrostatin-1 intervention. Overall, these findings highlight semaglutide's potential as a therapeutic approach for mitigating diabetes-induced testicular damage through modulation of the ferroptosis pathway.

Fetal Leydig cells: What we know and what we don't.

During male fetal development, testosterone plays an essential role in the differentiation and maturation of the male reproductive system. Deficient fetal testosterone production can result in variations of sex differentiation that may cause infertility and even increased tumor incidence later in life. Fetal Leydig cells in the fetal testis are the major androgen source in mammals. Although fetal and adult Leydig cells are similar in their functions, they are two distinct cell types, and therefore, the knowledge of adult Leydig cells cannot be directly applied to understanding fetal Leydig cells. This review summarizes our current knowledge of fetal Leydig cells regarding their cell biology, developmental biology, and androgen production regulation in rodents and human. Fetal Leydig cells are present in basement membrane-enclosed clusters in between testis cords. They originate from the mesonephros mesenchyme and the coelomic epithelium and start to differentiate upon receiving a Desert Hedgehog signal from Sertoli cells or being released from a NOTCH signal from endothelial cells. Mature fetal Leydig cells produce androgens. Human fetal Leydig cell steroidogenesis is LHCGR (Luteinizing Hormone Chronic Gonadotropin Receptor) dependent, while rodents are not, although other Gαs -protein coupled receptors might be involved in rodent steroidogenesis regulation. Fetal steroidogenesis ceases after sex differentiation is completed, and some fetal Leydig cells dedifferentiate to serve as stem cells for adult testicular cell types. Significant gaps are acknowledged: (1) Why are adult and fetal Leydig cells different? (2) What are bona fide progenitor and fetal Leydig cell markers? (3) Which signaling pathways and transcription factors regulate fetal Leydig cell steroidogenesis? It is critical to discover answers to these questions so that we can understand vulnerable targets in fetal Leydig cells and the mechanisms for androgen production that when disrupted, leads to variations in sex differentiation that range from subtle to complete sex reversal.

New insights into the all-testis differentiation in zebrafish with compromised endogenous androgen and estrogen synthesis.

The regulatory mechanism of gonadal sex differentiation, which is complex and regulated by multiple factors, remains poorly understood in teleosts. Recently, we have shown that compromised androgen and estrogen synthesis with increased progestin leads to all-male differentiation with proper testis development and spermatogenesis in cytochrome P450 17a1 (cyp17a1)-/- zebrafish. In the present study, the phenotypes of female-biased sex ratio were positively correlated with higher Fanconi anemia complementation group L (fancl) expression in the gonads of doublesex and mab-3 related transcription factor 1 (dmrt1)-/- and cyp17a1-/-;dmrt1-/- fish. The additional depletion of fancl in cyp17a1-/-;dmrt1-/- zebrafish reversed the gonadal sex differentiation from all-ovary to all-testis (in cyp17a1-/-;dmrt1-/-;fancl-/- fish). Luciferase assay revealed a synergistic inhibitory effect of Dmrt1 and androgen signaling on fancl transcription. Furthermore, an interaction between Fancl and the apoptotic factor Tumour protein p53 (Tp53) was found in vitro. The interaction between Fancl and Tp53 was observed via the WD repeat domain (WDR) and C-terminal domain (CTD) of Fancl and the DNA binding domain (DBD) of Tp53, leading to the K48-linked polyubiquitination degradation of Tp53 activated by the ubiquitin ligase, Fancl. Our results show that testis fate in cyp17a1-/- fish is determined by Dmrt1, which is thought to stabilize Tp53 by inhibiting fancl transcription during the critical stage of sexual fate determination in zebrafish.

Liraglutide improved the reproductive function of obese mice by upregulating the testicular AC3/cAMP/PKA pathway.

BACKGROUND: The incidence of male reproductive dysfunction is increasing annually, and many studies have shown that obesity can cause severe harm to male reproductive function. The mechanism of male reproductive dysfunction caused by obesity is unclear, and there is no ideal treatment. Identification of effective therapeutic drugs and elucidation of the molecular mechanism involved in male reproductive health are meaningful. In this study, we investigated the effects of the GLP-1 receptor agonist liraglutide on sex hormones, semen quality, and testicular AC3/cAMP/PKA levels in high-fat-diet-induced obese mice. METHODS: Obese mice and their lean littermates were treated with liraglutide or saline for 12 weeks. Body weight was measured weekly. Fasting blood glucose (FBG) was measured using a blood glucose test strip. The serum levels of insulin (INS), luteinizing hormone (LH), follicle stimulating hormone (FSH), testosterone (T), free testosterone (F-TESTO), estradiol (E2), and sex hormone binding globulin (SHBG) were detected using ELISA. The sperm morphology and sperm count were observed after Pap staining. The mRNA and protein expression levels of testicular GLP-1R and AC3 were measured by RT-qPCR and Western blot, respectively. Testicular cAMP levels and PKA activity were detected using ELISA. RESULTS: Liraglutide treatment can decrease body weight, FBG, INS, HOMA-IR, E2 and SHBG levels; increase LH, FSH, T, and F-TESTO levels; increase sperm count; decrease the sperm abnormality rate; and increase GLP-1R and AC3 expression levels and cAMP levels and PKA activity in testicular tissue. CONCLUSIONS: Liraglutide can improve the sex hormone levels and semen quality of obese male mice. In addition to its weight loss effect, liraglutide can improve the reproductive function of obese male mice, which may also be related to the upregulation of AC3/cAMP/PKA pathway in the testis. This work lays the groundwork for future clinical studies.

Betaine postpones hyperglycemia-related senescence in ovarian and testicular cells: Involvement of RAGE and ß-galactosidase.

The structural and functional disorders of the testis and ovary are one of the main complications of hyperglycemia. Betaine is a trimethyl glycine with antioxidant, antidiabetic, and anti-inflammatory potential. The aim of this study is to investigate the potential of betaine on the expression of aging and oxidative stress markers in ovarian and testicular cells under hyperglycemic conditions. Testicular and ovarian cells were subjected to four different conditions, including normal glucose and hyperglycemia, with or without betaine (5 mM). The cells with hyperglycemia saw an increase in malondialdehyde (MDA), methylglyoxal (MGO), expression of a receptor for AGE, and aging-related genes (ß-GAL), and a decrease in the activity of antioxidant enzymes including catalase, glutathione peroxidase, and superoxide dismutase. The treatment with betaine, in contrast, decreased the amount of MGO and MDA, and also downregulated aging-related signaling. Although hyperglycemia induces senescence in testicular and ovarian cells, the use of betaine may have a protective effect against the cell senescence, which may be useful in the management of infertility.

Camel milk or silymarin could improve the negative effects that experimentally produced by aflatoxin B1 on rat's male reproductive system.

BACKGROUND: Camel milk and silymarin have many different beneficial effects on several animal species. Meanwhile, Aflatoxins are mycotoxins with extraordinary potency that pose major health risks to several animal species. Additionally, it has been documented that aflatoxins harm the reproductive systems of a variety of domestic animals. The present design aimed to investigate the impact of aflatoxin B1 (AFB1) on rat body weight and reproductive organs and the ameliorative effects of camel milk and silymarin through measured serum testosterone, testes pathology, and gene expression of tumor necrosis factor (TNF-α), luteinizing hormone receptor (LHR), and steroidogenic acute regulatory protein (StAR) in the testes. A total of sixty mature male Wister white rats, each weighing an average of 83.67 ± 0.21 g, were used. There were six groups created from the rats. Each division had ten rats. The groups were the control (without any treatment), CM (1 ml of camel milk/kg body weight orally), S (20 mg silymarin/kg b. wt. suspension, orally), A (1.4 mg aflatoxin/kg diet), ACM (aflatoxin plus camel milk), and AS (aflatoxin plus silymarin). RESULTS: The results indicated the positive effects of camel milk and silymarin on growth, reproductive organs, and gene expression of TNF-α, LHR, and StAR with normal testicular architecture. Also, the negative effect of AFB1 on the rat's body weight and reproductive organs, as indicated by low body weight and testosterone concentration, was confirmed by the results of histopathology and gene expression. However, these negative effects were ameliorated by the ingestion of camel milk and silymarin. CONCLUSION: In conclusion, camel milk and silymarin could mitigate the negative effect of AFB1 on rat body weight and reproductive organs.

Effects of intratesticular injection of hypertonic mannitol and saline on the quality of donkey sperm, indicators of oxidative stress and testicular tissue pathology.

OBJECTIVES: The aim of the present study was to examine donkey sperm quality after intratesticular injection of hypertonic mannitol (HM) and saline (HS). METHODS: Randomly assigned to five treatment groups were 15 adult male donkeys: (1) Control group (no treatment), (2) Surgery group (surgical castration for testosterone control), (3) NS group (normal saline intratesticular injection), (4) HS group (hypertonic saline), and (5) HM group. We injected 20 mL per testicle. We took 5 mL blood from all donkeys before injection. Castration was performed under general anesthesia 60 days later. Samples included blood and testicular tissue. Total motility (TM), progressive motility (PM), movementy features, DNA damage, morphology, viability, and plasma membrane functionality were evaluated. Hormone analyses, histomorphometric studies and oxidative stress indices including total antioxidant capacity (TAC), glutathione peroxidase (GPx), glutathione (GSH), superoxide dismutase (SOD), malondialdehyde (MDA), and NADP+/NADPH were evaluated. Apoptosis, pyroptosis-related Bax, Caspase-1, GSDMD, and Bcl-2 expression were also assessed. RESULTS: In HS and HM groups, testosterone, epididymal sperm count, motility, viability, and plasma membrane functionality dropped while sperm DNA damage increased. HS and HM groups had significantly lower histomorphometric parameters, TAC, GPx, SOD, GSH, and Bcl-2 gene expression. MDA, NADP+/NADPH, Bax, Caspase-1, and GSDMD gene expression were substantially higher in the HS and HM groups than in the control group. CONCLUSIONS: Toxic effects of hypertonic saline and mannitol on reproductive parameters were seen following, hence, they might be considered as a good chemical sterilizing treatment in donkeys.

Piperine mitigates oxidative stress, inflammation, and apoptosis in the testicular damage induced by cyclophosphamide in mice.

Although cyclophosphamide (CP) has been approved as an anticancer drug, its toxic effect on most organs, especially the testis, has been established. Piperine (PIP) is an alkaloid that has antioxidant, antiapoptotic, and anti-inflammatory activities. This study was investigated the protective effects of PIP on CP-induced testicular toxicity in the mice. In this experimental study, 48 adult male BALB/c mice (30-35 g) were divided into six groups (n = 8), receiving normal saline (C), 5 mg/kg of PIP (PIP5), 10 mg/kg of PIP (PIP10), 200 mg/kg of CP, 200 mg/kg of CP + PIP5, and 200 mg/kg of CP + PIP10. On the eighth day of the study, blood and testis samples were prepared for serum testosterone hormone quantification, sperm analysis, histological, and immunohistochemical assays. The results of this study showed that CP induced testicular toxicity with the decrease of sperm count, motility, and viability. Also, CP treatment caused histological structure alterations in the testis, including exfoliation, degeneration, vacuolation of spermatogenic cells, and reducing the thickness of the epithelium and the diameter of the seminiferous tubule. In addition, CP decreased glutathione (GSH) levels, increased malondialdehyde (MDA) levels, Caspase-3, and NF-κB. At the same time, PIP treatment reduced testicular histopathological abnormalities, oxidative stress, and apoptosis that were induced by CP. These results showed that PIP improved CP-induced testicular toxicity in mice, which can be related to its antioxidant, antiapoptotic, and anti-inflammatory activities.

mTORC1 is required for differentiation of germline stem cells in the Drosophila melanogaster testis.

Metabolism participates in the control of stem cell function and subsequent maintenance of tissue homeostasis. How this is achieved in the context of adult stem cell niches in coordination with other local and intrinsic signaling cues is not completely understood. The Target of Rapamycin (TOR) pathway is a master regulator of metabolism and plays essential roles in stem cell maintenance and differentiation. In the Drosophila male germline, mTORC1 is active in germline stem cells (GSCs) and early germ cells. Targeted RNAi-mediated downregulation of mTor in early germ cells causes a block and/or a delay in differentiation, resulting in an accumulation of germ cells with GSC-like features. These early germ cells also contain unusually large and dysfunctional autolysosomes. In addition, downregulation of mTor in adult male GSCs and early germ cells causes non-autonomous activation of mTORC1 in neighboring cyst cells, which correlates with a disruption in the coordination of germline and somatic differentiation. Our study identifies a previously uncharacterized role of the TOR pathway in regulating male germline differentiation.

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.