Male infertility has become a global concern, accounting for 20-70% of infertility. Dysfunctional spermatogenesis is the most common cause of male infertility; thus, treating abnormal spermatogenesis may improve male infertility and has attracted the attention of the medical community. Mitochondria are essential organelles that maintain cell homeostasis and normal physiological functions in various ways, such as mitochondrial oxidative phosphorylation (OXPHOS). Mitochondrial OXPHOS transmits electrons through the respiratory chain, synthesizes adenosine triphosphate (ATP), and produces reactive oxygen species (ROS). These mechanisms are vital for spermatogenesis, especially to maintain the normal function of testicular Sertoli cells and germ cells. The disruption of mitochondrial OXPHOS caused by external factors can result in inadequate cellular energy supply, oxidative stress, apoptosis, or ferroptosis, all inhibiting spermatogenesis and damaging the male reproductive system, leading to male infertility. This article summarizes the latest pathological mechanism of mitochondrial OXPHOS disorder in testicular Sertoli cells and germ cells, which disrupts spermatogenesis and results in male infertility. In addition, we also briefly outline the current treatment of spermatogenic malfunction caused by mitochondrial OXPHOS disorders. However, relevant treatments have not been fully elucidated. Therefore, targeting mitochondrial OXPHOS disorders in Sertoli cells and germ cells is a research direction worthy of attention. We believe this review will provide new and more accurate ideas for treating male infertility.
Male ; Humans ; Infertility, Male/metabolism* ; Oxidative Phosphorylation ; Mitochondria/metabolism* ; Spermatogenesis/physiology* ; Sertoli Cells/metabolism* ; Oxidative Stress/physiology* ; Animals ; Reactive Oxygen Species/metabolism*

Researchers commonly use cyclization recombination enzyme/locus of X-over P1 (Cre/loxP) technology-based conditional gene knockouts of model mice to investigate the functional roles of genes of interest in Sertoli and Leydig cells within the testis. However, the shortcomings of these genetic tools include high costs, lengthy experimental periods, and limited accessibility for researchers. Therefore, exploring alternative gene silencing techniques is of great practical value. In this study, we employed adeno-associated virus (AAV) as a vector for gene silencing in Sertoli and Leydig cells. Our findings demonstrated that AAV serotypes 1, 8, and 9 exhibited high infection efficiency in both types of testis cells. Importantly, we discovered that all three AAV serotypes exhibited exquisite specificity in targeting Sertoli cells via tubular injection while demonstrating remarkable selectivity in targeting Leydig cells via interstitial injection. We achieved cell-specific knockouts of the steroidogenic acute regulatory ( Star ) and luteinizing hormone/human chorionic gonadotropin receptor (Lhcgr) genes in Leydig cells, but not in Sertoli cells, using AAV9-single guide RNA (sgRNA)-mediated gene editing in Rosa26-LSL-Cas9 mice. Knockdown of androgen receptor ( Ar ) gene expression in Sertoli cells of wild-type mice was achieved via tubular injection of AAV9-short hairpin RNA (shRNA)-mediated targeting. Our findings offer technical approaches for investigating gene function in Sertoli and Leydig cells through AAV9-mediated gene silencing.
Animals ; Male ; Leydig Cells/metabolism* ; Mice ; Dependovirus/genetics* ; Sertoli Cells/metabolism* ; Gene Silencing ; Genetic Vectors ; Testis/cytology*

The patient, assigned female at birth and aged 1 year and 7 months, presented with clinical manifestations of 46,XY disorders of sex development. The external genitalia exhibited a severely undermasculinized phenotype. Laboratory tests and gonadal biopsy indicated poor Leydig cell function and good Sertoli cell function. Genetic testing revealed compound heterozygous mutations of c.867-2A>C and c.547G>A (p.G183R) in the LHCGR gene. The patient was ultimately diagnosed with type II Leydig cell hypoplasia. Type II Leydig cell hypoplasia presents a broad spectrum of clinical phenotypes, characterized by a lack of parallel function between Leydig cells and Sertoli cells, and significant individual variability in spermatogenesis and gender assignment. This condition should be considered when there is poor Leydig cell function but good development of Wolffian duct derivatives.
Female ; Humans ; Infant ; Disorder of Sex Development, 46,XY/genetics* ; Leydig Cells/pathology* ; Mutation ; Receptors, LH/genetics* ; Testis/abnormalities*

Sertoli cells play an important role in the process of spermatogenesis, and the abnormalities in spermatogenesis are closely related to disruptions in glycolipid metabolism. The metabolic environment of Sertoli cells is hypoxic, with glycolysis and fatty acid β-oxidation being the primary metabolic pathways. In Sertoli cells, glycolysis produces lactate to provide energy for spermatogenic cells, while fatty acid β-oxidation generates ATP. Currently, the relationship between glycolipid metabolism in Sertoli cells and spermatogenic cell development, as well as the interplay between glucose and lipid metabolism remain unclear. Various hormones, including sex hormones, can affect glucose metabolism in Sertoli cells by endocrine regulation. The activation or inhibition of signaling pathways such as AMPK, mTOR, and Akt can alter the expression levels of glycolysis-related transporter genes and the synthesis of fatty acids, thereby affecting glycolipid metabolism in Sertoli cells. Some transcription factors such as PPARγ can regulate downstream fatty acid metabolism-related genes by directly binding to their response elements and promoting the oxidation of fatty acids in Sertoli cells. In this article we elaborate on the key factors influencing glycolipid metabolism in Sertoli cells and their interconnections, as well as their potential clinical implications, offering new insights for precisely targeted treatments of male infertility.
Sertoli Cells/cytology* ; Male ; Glycolipids/metabolism* ; Spermatogenesis/physiology* ; Humans ; Lipid Metabolism ; Animals ; Fatty Acids/metabolism* ; Signal Transduction ; Glycolysis

OBJECTIVE: To comprehensively evaluate the effect of icariin in alleviating reproductive function damage (RFD) in male mice via in vitro and in vivo experiments. METHODS: We isolated Leydig cells from 60 KM male mice in vitro, and examined the toxic effect of icariin on the Leydig cells using Cell Counting Kit-8 (CCK-8). We equally randomized the mice into six groups: normal control, RFD model control (made by intraperitoneal injection of busulfan at 10 mg/kg combined with cyclophosphamide (CP) at 120 mg/kg), positive control, and low-, medium- and high-dose icariin. After modeling, we treated the mice in the positive control group with Wuziyanzong Pills and those in the low-, medium- and high-dose icariin groups by intragastrical administration of icariin at 20, 40 and 80 mg/kg-1, respectively, for 30 successive days. Then we obtained the weight and visceral coefficients of the reproductive organs, calculated the sperm count, observed the pathological changes in the testis tissue by HE staining, measured the serum testosterone (T) level by ELISA, determined the indexes of testicular oxidative stress and nitric oxide (NO) signaling pathway by colorimetric assay, and detected the expression levels of the pro-apoptotic genes Fas and Bax by qRT-PCR. RESULTS: CCK-8 assay confirmed that icariin had no toxic effect on the isolated Leydig cells of the mice, and could effectively reduce busulfan- and CP-induced cytotoxicity and promote the secretion of serum T. Icariin at 80 mg/kg significantly increased the visceral coefficient of the testis and promoted spermatogenesis (P<0.05), but had little effect on the visceral coefficient of the epididymis in the RFD model mice. Testicular histomorphometric observation revealed significantly improved testis structure, intact boundary membrane of seminiferous tubules and increased numbers of various types of spermatogenic cells of the model mice after treated with icariin. Compared with the mice in the model control group, those treated with high-dose icariin showed a significantly reduced content of malondialdehyde (MDA) (by 35.3%, P<0.01), elevated total antioxidant capacity (TAOC) and superoxide dismutase (T-SOD) activity (P<0.05), and decreased NO content and nitric oxide synthase (NOS) activity in the testis tissue (P<0.01). In addition, icariin exhibited an evident inhibitory effect on the expressions of the pro-apoptotic genes Bax and Fas. CONCLUSION Icariin can ameliorate oxidative stress-induced damage to the testicular function and protect spermatogenesis of male mice by elevating TAOC, decreasing NOS activity, inhibiting the NO level in the testis, and suppressing busulfan- and CP-induced apoptosis of testicular cells.
Animals ; Male ; Cyclophosphamide/adverse effects* ; Mice ; Busulfan/adverse effects* ; Flavonoids/pharmacology* ; Leydig Cells/drug effects* ; Oxidative Stress/drug effects* ; Testis/drug effects* ; Apoptosis/drug effects* ; Testosterone/blood*

Objective： Varicocele (VC) induces male infertility by mediating changes in the testicular microenvironment, in which testicular hypoxia and high-pressure are important pathological conditions. This study aims to compare the mouse spermatogenesis (GC-2spd) cells and Sertoli (TM4) cells of mouse testis after hypoxic modeling and hypoxic and high-pressure combined modeling, and to explore the feasibility of establishing a hypoxic and high-pressure combined cell model. Methods： On the basis of cell hypoxia induced by CoCl2, the complex model of testicular cell hypoxia and high pressure was constructed by changing the osmotic pressure of GC-2 and TM4 cell medium with a high concentration of NaCl solution. After selecting the intervention concentration of CoCl2 by MTT test and detecting the expression level of HIF-1α for the determination of the optimal osmotic pressure conditions of the cell model, the cells were divided into normal group, hypoxia model group and composite model group. And the levels of OS, programmed cell death, inflammatory factors, and the expression levels of pyroptosis-related proteins were compared between the normal group and the groups with different modeling methods. Results： The optimal intervention concentration of CoCl2 in GC-2 and TM4 cells was 150 and 250μmol/L, respectively, and the expression of HIF-1α was the highest in both cells under osmotic pressure of 500 mOsmol/kg (P<0.05). Compared with the normal group, the SOD levels of GC-2 and TM4 cells decreased (all P<0.05), CAT level decreased (all P<0.05), and MDA level increased (all P<0.01), and the OS level of GC-2 and TM4 cells was more obvious than that of the hypoxia model group (all P<0.05). Compared with the normal group, apoptosis occurred in GC-2 and TM4 cells after composite modeling (all P<0.05). Compared with the normal group, the mRNA expressions of IL-1β, IL-18, TNF-α and COX-2 in GC-2 and TM4 cells significantly increased (P<0.01) and higher than those in hypoxia model group (P<0.05) and induced pyroptosis (P<0.01). The expression level of GSDMD increased (P<0.05). Conclusion： The cell model with hypoxia and high pressure combined modeling can not only induce oxidative stress and apoptosis of cells better than that with hypoxia alone, but also further cause inflammatory response damage and pyroptosis, which simulates the changes of testis microenvironment mediated by hypoxia and high pressure combined conditions in VC. This cell model can be used for studying the pathogenesis of VC-associated male infertility, evaluating drug efficacy, and exploring pharmacological mechanisms.
Male ; Animals ; Varicocele/pathology* ; Mice ; Testis/metabolism* ; Hypoxia-Inducible Factor 1, alpha Subunit/metabolism* ; Cell Hypoxia ; Cobalt ; Sertoli Cells/metabolism* ; Osmotic Pressure ; Spermatogenesis ; Cellular Microenvironment ; Infertility, Male ; Disease Models, Animal

Trophoblast cells serve as the foundation for placental development. We analyzed published multiomics sequencing data and found that trophoblast cells highly expressed RRS1 compared to primitive endoderm and epiblast. We used HTR-8/SVneo cells for further investigation, and Western blot and immunofluorescence staining confirmed that HTR-8/SVneo cells highly expressed RRS1. RRS1 was successfully knocked down in HTR-8/SVneo cells using siRNA. Using IncuCyte S3 live-cell analysis system based on continuous live-cell imaging and real-time data, we observed that proliferation, migration, and invasion abilities were all significantly decreased in RRS1-knockdown cells. RNA-seq revealed that knockdown of RRS1 affected the gene transcription, and upregulated pathways in extracellular matrix organization, DNA damage response, and intrinsic apoptotic signaling, downregulated pathways in embryo implantation, trophoblast cell migration, and wound healing. Differentially expressed genes were enriched in diseases related to placental development. Consistent with these findings, human chorionic villus samples collected from spontaneous abortion cases exhibited significantly reduced RRS1 expression compared to normal controls. Our results highlight the functional importance of RRS1 in human trophoblasts and suggest that its deficiency contributes to early pregnancy loss.
Humans ; Trophoblasts/physiology* ; Cell Movement/genetics* ; Cell Proliferation/genetics* ; Female ; Pregnancy ; Abortion, Spontaneous/metabolism* ; Cell Line ; Placentation/genetics*

The testis is pivotal for male reproduction, and its progressive functional decline in aging is associated with infertility. However, the regulatory mechanism underlying primate testicular aging remains largely elusive. Here, we resolve the aging-related cellular and molecular alterations of primate testicular aging by establishing a single-nucleus transcriptomic atlas. Gene-expression patterns along the spermatogenesis trajectory revealed molecular programs associated with attrition of spermatogonial stem cell reservoir, disturbed meiosis and impaired spermiogenesis along the sequential continuum. Remarkably, Sertoli cell was identified as the cell type most susceptible to aging, given its deeply perturbed age-associated transcriptional profiles. Concomitantly, downregulation of the transcription factor Wilms' Tumor 1 (WT1), essential for Sertoli cell homeostasis, was associated with accelerated cellular senescence, disrupted tight junctions, and a compromised cell identity signature, which altogether may help create a hostile microenvironment for spermatogenesis. Collectively, our study depicts in-depth transcriptomic traits of non-human primate (NHP) testicular aging at single-cell resolution, providing potential diagnostic biomarkers and targets for therapeutic interventions against testicular aging and age-related male reproductive diseases.
Animals ; Male ; Testis ; Sertoli Cells/metabolism* ; Transcriptome ; Spermatogenesis/genetics* ; Primates ; Aging/genetics* ; Stem Cells

Objective: To investigate the expression change of the Mas1 receptor in the placenta of healthy pregnant women during different gestation periods, analyze the expression level of the Mas1 receptor in the placenta of pre-eclampsia (PE) patients, and its biological function in trophoblast cells. Methods: Placental villous tissues were collected from normal pregnant women in early, mid and late pregnancy. Human trophoblast stem cells were isolated and cultured from early pregnancy villous tissues. The expression of the Mas1 receptor was detected by fluorescence immunoassay and real-time fluorescence quantitative PCR. In a case-control study, patients with full-term PE were selected as the case group and healthy women with full-term pregnancy were selected as the control group. Placental villus tissues were collected from both groups. Immunofluorescence chemistry and immunoprotein blotting were used to study the changes in Mas1 receptor expression in PE. Mas1 receptor agonists and blockers induced HTR8/Svneo cells and BeWo cells, and the effects of the Mas1 receptor on the proliferation and migration of trophoblast cells were detected by the CCK8 proliferation test and scratch test. Results: Eight cases were included in early pregnancy, seven cases in mid-pregnancy and six cases in late pregnancy. Mas1 receptors in normal placental villi tissue were mainly expressed in human trophoblast stem cell membranes and cytoplasm, and the expression of Mas1 receptor mRNA in villi tissue was significantly higher in late pregnancy than in mid-pregnancy. There were 24 cases included in the case group and 12 cases in the control group. Mas1 receptor expression in placental villi was significantly lower in the case group compared to the control group; Activation/inhibition of the Mas1 receptor had no significant effect on the proliferation of HTR8/Svneo cells and BeWo cells. Activated Mas1 receptor had no significant effect on the migration ability of HTR8/Svneo cells. Conclusion: Mas1 receptors are expressed in placental villous tissue and their expression varies with gestation. Mas1 receptor expression is reduced in PE patients, but it does not affect the value-added or migratory function of trophoblast cells.
Humans ; Female ; Pregnancy ; Placenta ; Trophoblasts ; Case-Control Studies ; Pre-Eclampsia ; Gene Expression

Objective: To investigate the expression change of the Mas1 receptor in the placenta of healthy pregnant women during different gestation periods, analyze the expression level of the Mas1 receptor in the placenta of pre-eclampsia (PE) patients, and its biological function in trophoblast cells. Methods: Placental villous tissues were collected from normal pregnant women in early, mid and late pregnancy. Human trophoblast stem cells were isolated and cultured from early pregnancy villous tissues. The expression of the Mas1 receptor was detected by fluorescence immunoassay and real-time fluorescence quantitative PCR. In a case-control study, patients with full-term PE were selected as the case group and healthy women with full-term pregnancy were selected as the control group. Placental villus tissues were collected from both groups. Immunofluorescence chemistry and immunoprotein blotting were used to study the changes in Mas1 receptor expression in PE. Mas1 receptor agonists and blockers induced HTR8/Svneo cells and BeWo cells, and the effects of the Mas1 receptor on the proliferation and migration of trophoblast cells were detected by the CCK8 proliferation test and scratch test. Results: Eight cases were included in early pregnancy, seven cases in mid-pregnancy and six cases in late pregnancy. Mas1 receptors in normal placental villi tissue were mainly expressed in human trophoblast stem cell membranes and cytoplasm, and the expression of Mas1 receptor mRNA in villi tissue was significantly higher in late pregnancy than in mid-pregnancy. There were 24 cases included in the case group and 12 cases in the control group. Mas1 receptor expression in placental villi was significantly lower in the case group compared to the control group; Activation/inhibition of the Mas1 receptor had no significant effect on the proliferation of HTR8/Svneo cells and BeWo cells. Activated Mas1 receptor had no significant effect on the migration ability of HTR8/Svneo cells. Conclusion: Mas1 receptors are expressed in placental villous tissue and their expression varies with gestation. Mas1 receptor expression is reduced in PE patients, but it does not affect the value-added or migratory function of trophoblast cells.
Humans ; Female ; Pregnancy ; Placenta ; Trophoblasts ; Case-Control Studies ; Pre-Eclampsia ; Gene Expression