Continuous self-renewal and differentiation of spermatogonial stem cells (SSCs) is vital for maintenance of adult spermatogenesis. Although several spermatogonial stem cell regulators have been extensively investigated in rodents, regulatory mechanisms of human SSC self-renewal and differentiation have not been fully established. We analyzed single-cell sequencing data from the human testis and found that forkhead box P4 (FOXP4) expression gradually increased with development of SSCs. Further analysis of its expression patterns in human testicular tissues revealed that FOXP4 specifically marks a subset of spermatogonia with stem cell potential. Conditional inactivation of FOXP4 in human SSC lines suppressed SSC proliferation and significantly activated apoptosis. FOXP4 expressions were markedly suppressed in tissues with dysregulated spermatogenesis. These findings imply that FOXP4 is involved in human SSC proliferation, which will help elucidate on the mechanisms controlling the fate decisions in human SSCs.
Adult ; Humans ; Male ; Cell Differentiation ; Cell Proliferation ; Forkhead Transcription Factors/metabolism* ; Spermatogenesis/genetics* ; Spermatogonia/metabolism* ; Stem Cells/metabolism* ; Testis/metabolism*

Mammalian testis exhibits remarkably high transcriptome complexity, and spermatogenesis undergoes two periods of transcriptional cessation. These make the RNA-binding proteins (RBPs) the utmost importance during male germ cell development. Heterogeneous nuclear ribonucleoproteins (hnRNPs) are a large family of RBPs implicated in many steps of RNA processing; however, their roles in spermatogenesis are largely unknown. Here, we investigated the expression pattern of 12 hnRNP family members in mouse testes and found that most detected members are highly expressed in the testis. Furthermore, we found that most of the detected hnRNP proteins (hnRNPD, hnRNPK, hnRNPQ, hnRNPU, and hnRNPUL1) display the highest signals in the nuclei of pachytene spermatocytes, round spermatids, and Sertoli cells, whereas hnRNPE1 exclusively concentrates in the manchette of elongating spermatids. The expression of these hnRNP proteins showed both similarities and specificity, suggesting their diverse roles in spermatogenesis.
Mice ; Male ; Animals ; Heterogeneous-Nuclear Ribonucleoproteins/metabolism* ; Spermatogenesis/genetics* ; Testis/metabolism* ; Spermatids/metabolism* ; Sertoli Cells ; Spermatocytes/metabolism* ; RNA-Binding Proteins/metabolism* ; Mammals

Bisphenol A is a common environmental factor and endocrine disruptor that exerts a negative impact on male reproductive ability. By exploring bisphenol A-induced testicular cell death using the Institute of Cancer Research (ICR) mouse model, we found that a ferroptosis phenomenon may exist. Mice were divided into six groups and administered different doses of bisphenol A via intragastric gavage once daily for 45 consecutive days. Serum was then collected to determine the levels of superoxide dismutase and malondialdehyde. Epididymal sperm was also collected for semen analysis, and testicular tissue was collected for ferritin content determination, electron microscope observation of mitochondrial morphology, immunohistochemistry, real-time quantitative polymerase chain reaction, and western blot analysis. Exposure to bisphenol A was found to decrease sperm quality and cause oxidative damage, iron accumulation, and mitochondrial damage in the testes of mice. In addition, bisphenol A was confirmed to affect the expression of the ferroptosis-related genes, glutathione peroxidase 4 (GPX4), ferritin heavy chain 1 (FTH1), cyclooxygenase 2 (COX2), and acyl-CoA synthetase 4 (ACSL4) in mouse testicular tissues. Accordingly, we speculate that bisphenol A induces oxidative stress, which leads to the ferroptosis of testicular cells. Overall, the inhibition of ferroptosis may be a potential strategy to reduce male reproductive toxicity caused by bisphenol A.
Male ; Mice ; Animals ; Testis/metabolism* ; Ferroptosis ; Semen ; Oxidative Stress

Objective: To investigate the clinicopathological characteristics of testicular biopsies from Klinefelter syndrome (KS) patients. Methods: The testicular biopsy specimens of 87 patients with KS (a total of 107 biopsy specimens) were collected from the Department of Pathology, Peking University Third Hospital, Beijing, China from January 2017 to July 2022. All patients were diagnosed as KS by peripheral blood karyotyping analysis. The testicular histopathologic features, testicular volume and hormone levels were evaluated retrospectively. The histopathologic analysis was used to assess the quantity and morphology of Leydig cells, the spermatogenic state of seminiferous tubules, the thickening of the basement membrane of seminiferous tubules and the changes of stroma. Results: Leydig cell proliferative nodules were seen in 95.3% (102/107) of KS testicular biopsy tissues. The eosinophilic inclusion bodies and lipofuscin in Leydig cells were found in 52.3% (56/107) and 57.9% (62/107) of specimens, respectively. The Sertoli cell only seminiferous tubules and the hyalinized tubules were found in 66.4% (71/107) and 76.6% (82/107) of the examined tissues, respectively. The tubules with complete spermatogenic arrest were found in 15.9% (17/107) of specimens, and 5.6% (6/107) of the specimens showed low spermatogenesis or incomplete spermatogenic arrest. In 85.0% (91/107) of the specimens, increased thick-walled small vessels with hyaline degeneration were identified. Conclusions: The most common features of KS testicular specimens are Leydig cell proliferative nodules, hyaline degeneration of seminiferous tubules and proliferation of thick-walled blood vessels. Testicular biopsy specimens of KS are rare. The pathologists can make a tentative diagnosis of KS based on the histological findings, combined with the ultrasound and laboratory results, which is helpful for further diagnosis and treatment of KS.
Male ; Humans ; Testis/pathology* ; Klinefelter Syndrome/pathology* ; Retrospective Studies ; Seminiferous Tubules/pathology* ; Biopsy

Objective: To study the effects of cadmium chloride (CdCl(2)) exposure on testicular autophagy levels and blood-testis barrier integrity in prepubertal male SD rats and testicular sertoli (TM4) cells. Methods: In July 2021, 9 4-week-old male SD rats were randomly divided into 3 groups: control group (normal saline), low dose group (1 mg/kg·bw CdCl(2)) and high dose group (2 mg/kg·bw CdCl(2)), and were exposed with CdCl(2) by intrabitoneal injection. 24 h later, HE staining was used to observe the morphological changes of testis of rats, biological tracer was used to observe the integrity of blood-testis barrier, and the expression levels of microtubule-associated protein light chain 3 (LC3) -Ⅰ and LC3-Ⅱ in testicular tissue were detected. TM4 cells were treated with 0, 2.5, 5.0 and 10.0 μmol/L CdCl(2) for 24 h to detect the toxic effect of cadmium. The cells were divided into blank group (no exposure), exposure group (10.0 μmol/L CdCl(2)), experimental group[10.0 μmol/L CdCl(2)+60.0 μmol/L 3-methyladenine (3-MA) ] and inhibitor group (60.0 μmol/L 3-MA). After 24 h of treatment, Western blot analysis was used to detect the expression levels of LC3-Ⅱ, ubiquitin binding protein p62, tight junction protein ZO-1 and adhesion junction protein N-cadherin. Results: The morphology and structure of testicular tissue in the high dose group were obvious changed, including uneven distribution of seminiferous tubules, irregular shape, thinning of seminiferous epithelium, loose structure, disordered arrangement of cells, abnormal deep staining of nuclei and vacuoles of Sertoli cells. The results of biological tracer method showed that the integrity of blood-testis barrier was damaged in the low and high dose group. Western blot results showed that compared with control group, the expression levels of LC3-Ⅱ in testicular tissue of rats in low and high dose groups were increased, the differences were statistically significant (P<0.05). Compared with the 0 μmol/L, after exposure to 5.0, 10.0 μmol/L CdCl(2), the expression levels of ZO-1 and N-cadherin in TM4 cells were significantly decreased, and the expression level of p62 and LC3-Ⅱ/LC3-Ⅰ were significantly increased, the differences were statistically significant (P<0.05). Compared with the exposure group, the relative expression level of p62 and LC3-Ⅱ/LC3-Ⅰ in TM4 cells of the experimental group were significantly decreased, while the relative expression levels of ZO-1 and N-cadherin were significantly increased, the differences were statistically significant (P<0.05) . Conclusion: The mechanism of the toxic effect of cadmium on the reproductive system of male SD rats may be related to the effect of the autophagy level of testicular tissue and the destruction of the blood-testis barrier integrity.
Rats ; Male ; Animals ; Testis ; Cadmium Chloride/metabolism* ; Cadmium ; Blood-Testis Barrier/metabolism* ; Rats, Sprague-Dawley ; Cadherins/metabolism* ; Autophagy

OBJECTIVE: To explore the mechanism underlying the inhibitory effect of quercetin against testicular oxidative damage induced by a mixture of 3 commonly used phthalates (MPEs) in rats. METHODS: Forty male Sprague-Dawley rats were randomly divided into control group, MPEs exposure group, and MPEs with low-, median- and high-dose quercetin treatment groups. For MPEs exposure, the rats were subjected to intragastric administration of MPEs at the daily dose of 900 mg/kg for 30 consecutive days; Quercetin treatments were administered in the same manner at the daily dose of 10, 30, and 90 mg/kg. After the treatments, serum levels of testosterone, luteinizing hormone (LH), follicle stimulating hormone (FSH), and testicular malondialdeyhde (MDA), catalase (CAT) and superoxide dismutase (SOD) were detected, and testicular pathologies of the rats were observed with HE staining. The expressions of nuclear factor-E2-related factor 2 (Nrf2), Kelch-like ECH2 associated protein 1 (Keap1) and heme oxygenase 1 (HO-1) in the testis were detected using immunofluorescence assay and Western blotting. RESULTS: Compared with the control group, the rats with MPEs exposure showed significant reductions of the anogenital distance, weight of the testis and epididymis, and the coefficients of the testis and epididymis with lowered serum testosterone, LH and FSH levels (P < 0.05). Testicular histological examination revealed atrophy of the seminiferous tubules, spermatogenic arrest, and hyperplasia of the Leydig cells in MPEs-exposed rats. MPEs exposure also caused significant increments of testicular Nrf2, MDA, SOD, CAT and HO-1 expressions and lowered testicular Keap1 expression (P < 0.05). Treatment with quercetin at the median and high doses significantly ameliorated the pathological changes induced by MPEs exposure (P < 0.05). CONCLUSION Quercetin treatment inhibits MPEs-induced oxidative testicular damage in rats possibly by direct scavenging of free radicals to lower testicular oxidative stress and restore the regulation of the Nrf2 signaling pathway.
Rats ; Male ; Animals ; Testis ; Quercetin/pharmacology* ; Rats, Sprague-Dawley ; NF-E2-Related Factor 2/metabolism* ; Kelch-Like ECH-Associated Protein 1/metabolism* ; Oxidative Stress ; Testosterone/pharmacology* ; Superoxide Dismutase/metabolism* ; Follicle Stimulating Hormone ; Luteinizing Hormone

OBJECTIVE: To investigate the protective effects of total saponins from Panax japonicus (TSPJ) against high-fat dietinduced testicular Sertoli cell junction damage in mice. METHODS: Forty male C57BL/6J mice were randomized into normal diet group, high-fat diet group, and low-dose (25 mg/kg) and high-dose (75 mg/kg) TSPJ treatment groups (n=10). The mice in the normal diet group were fed a normal diet, while the mice in the other groups were fed a high-fat diet. After TSPJ treatment via intragastric administration for 5 months, the testes and epididymis of the mice were collected for measurement of weight, testicular and epididymal indices and sperm parameters. HE staining was used for histological evaluation of the testicular tissues and measurement of seminiferous tubule diameter and seminiferous epithelium height. The expression levels of ZO-1, occludin, claudin11, N-cadherin, E-cadherin and β-catenin in Sertoli cells were detected with Western blot, and the localization and expression levels of ZO-1 and β-catenin in the testicular tissues were detected with immunofluorescence assay. The protein expressions of LC3B, p-AKT and p-mTOR in testicular Sertoli cells were detected using double immunofluorescence assay. RESULTS: Treatment with TSPJ significantly improved high-fat diet-induced testicular dysfunction by reducing body weight (P < 0.001), increasing testicular and epididymal indices (P < 0.05), and improving sperm concentration and sperm viability (P < 0.05). TSPJ ameliorated testicular pathologies and increased seminiferous epithelium height of the mice with high-fat diet feeding (P < 0.05) without affecting the seminiferous tubule diameter. TSPJ significantly increased the expression levels of ZO-1, occludin, N-cadherin, E-cadherin and β-catenin (P < 0.05) but did not affect claudin11 expression in the testicular tissues. Immunofluorescence assay showed that TSPJ significantly increased ZO-1 and β-catenin expression in the testicular tissues (P < 0.001), downregulated LC3B expression and upregulated p-AKT and p-mTOR expressions in testicular Sertoli cells. CONCLUSION TSPJ alleviates high-fat diet-induced damages of testicular Sertoli cell junctions and spermatogenesis possibly by activating the AKT/mTOR signaling pathway and inhibiting autophagy of testicular Sertoli cells.
Male ; Animals ; Mice ; Mice, Inbred C57BL ; Testis ; Sertoli Cells ; beta Catenin ; Diet, High-Fat ; Occludin ; Proto-Oncogene Proteins c-akt ; Seeds ; Cadherins ; Intercellular Junctions

Leydig cell tumor is a rare testicular neoplasm that can present as a non-palpable small testicular nodule. Here we present a case of a 36-year-old Filipino male who initially came in for fertility work-up. Semen analysis showed azoospermia. However, an incidental finding on ultrasound showed a well-circumscribed round tumor. The patient underwent radical orchiectomy. On histopathologic examination, a Leydig cell tumor was identified and supported by immunohistochemical staining. We discuss the clinical features pathogenesis, treatment, diagnosis and prognosis of this uncommon entity.
Leydig Cells ; Testis ; Orchiectomy ; Infertility

Puberty is a pivotal biological process that completes sexual maturation to achieve full reproductive capability. It is a major transformational period of life, whose timing is strongly affected by genetic makeup of the individual, along with various internal and external factors. Although the exact mechanism for initiation of the cascade of molecular events that culminate in puberty is not yet known, the process of pubertal onset involves interaction of numerous complex signaling pathways of hypothalamo-pituitary-testicular (HPT) axis. We developed a classification of the mechanisms involved in male puberty that allowed placing many genes into physiological context. These include (i) hypothalamic development during embryogenesis, (ii) synaptogenesis where gonadotropin releasing hormone (GnRH) neurons form neuronal connections with suprahypothalamic neurons, (iii) maintenance of neuron homeostasis, (iv) regulation of synthesis and secretion of GnRH, (v) appropriate receptors/proteins on neurons governing GnRH production and release, (vi) signaling molecules activated by the receptors, (vii) the synthesis and release of GnRH, (viii) the production and release of gonadotropins, (ix) testicular development, (x) synthesis and release of steroid hormones from testes, and (xi)the action of steroid hormones in downstream effector tissues. Defects in components of this system during embryonic development, childhood/adolescence, or adulthood may disrupt/nullify puberty, leading to long-term male infertility and/or hypogonadism. This review provides a list of 598 genes involved in the development of HPT axis and classified according to this schema. Furthermore, this review identifies a subset of 75 genes for which genetic mutations are reported to delay or disrupt male puberty.
Adolescent ; Male ; Humans ; Adult ; Child ; Gonadotropin-Releasing Hormone ; Gonadotropins/metabolism* ; Hypogonadism ; Testis/metabolism* ; Puberty/physiology* ; Sexual Maturation

The authors performed a comprehensive review of current literature to create a model comparing commonly evaluated variables in male factor infertility, for example, follicle-stimulating hormone (FSH), testicular volume (TV), and testosterone (T), to better predict sperm retrieval rate (SRR). Twenty-nine studies were included, 9 with data on conventional testicular sperm extraction (cTESE) for a total of 1227 patients and 20 studies including data on microdissection testicular sperm extraction (mTESE) for a total of 4760 patients. A weighted-means value of SRR, FSH, T, and TV was created, and a weighted linear regression was then used to describe associations among SRR, type of procedure, FSH, T, and TV. In this study, weighted-means values demonstrated mTESE to be superior to cTESE with an SRR of 51.9% vs 40.1%. Multiple weighted linear regressions were created to describe associations among SRR, procedure type, FSH, T, and TV. The models showed that for every 1.19 mIU ml^-1 increase in FSH, there would be a significant decrease in SRR by 1.0%. Seeking to create a more clinically relevant model, FSH values were then divided into normal, moderate elevation, and significant elevation categories (FSH <10 mIU ml^-1, 10-19 mIU ml^-1, and >20 mIU ml^-1, respectively). For an index patient undergoing cTESE, the retrieval rates would be 57.1%, 44.3%, and 31.2% for values normal, moderately elevated, and significantly elevated, respectively. In conclusion, in a large meta-analysis, mTESE was shown to be more successful than cTESE for sperm retrievals. FSH has an inverse relationship to SRR in retrieval techniques and can alone be predictive of cTESE SRR.
Humans ; Male ; Follicle Stimulating Hormone ; Follicle Stimulating Hormone, Human ; Infertility, Male ; Linear Models ; Semen ; Sperm Retrieval ; Spermatozoa ; Testis/surgery*