Somatic cell reprogramming technology can reverse fate determinations of the differentiated cells by regulating their epigenetic and gene expression programs. This reprogramming enables the acquisition of pluripotency to differentiate into mature and functional cells, which provides sufficient cells for regenerative and reproductive medicine. Significant progress has recently been made by peers and us in reprogramming somatic cells, e.g., Sertoli cells, fibroblasts, and peripheral blood mononuclear cells into functional stem cells. In this review, we systematically summarize the development and optimization of multiple core methods for cell reprogramming, including somatic cell nuclear transfer (SCNT), transcription factor-induced reprogramming, chemical reprogramming, and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (CRISPR/Cas)-based reprogramming. We also address the epigenetic remodeling mechanisms that are involved in somatic cell reprogramming, including the dynamic processes of chromatin accessibility regulation, DNA demethylation, histone modifications, and the regulation of non-coding RNAs (ncRNAs). Moreover, we discuss current challenges and perspectives in this field. Significantly, stem cells derived from somatic cells have great applications in regenerative medicine for treating various kinds of diseases, such as infertility, diabetes, neurodegenerative diseases, and chimeric antigen receptor T-cell (CAR-T) immunotherapy with attention to cell maturity, heterogeneity, and safety. Our in-depth understanding of approaches, mechanisms, and applications of somatic cell reprogramming into stem cells is essential for cell therapy and tissue engineering.

The fate determinations of stem cells are governed by signalling molecules and pathways, and notably, the TGF-β superfamily members play pivotal roles in mediating these decisions. Significantly, TGF-β3 participates in affecting the development of stem cells and their microenvironment. In this review, we address the functions and regulatory networks of TGF-β3 in the fate decisions of several types of stem cells, including neural stem cells, haematopoietic stem cells, odontogenic stem cells, hair-follicle stem cells, adipose-derived stem cells, and mesenchymal stem cells. Specifically, we discuss the biosynthesis, activation, the roles, and mechanisms of TGF-β in influencing the proliferation, differentiation, and cell death of these stem cells, and we further highlight the perspective in this field. TGF-β signalling in stem cells begins with the activation of either integrin-dependent or integrin-independent pathways by binding to cell surface receptors TbRII and TbRI and co-receptor Betaglycan (TR3). TGF-β acts via both classical Smad signalling pathways and a variety of non-classical pathways. Notably, the biological functions of TGF-β3 depend primarily on specific cell type and the existing conditions of stem cells, reflecting the integration of multiple factors including the concentration, duration of action, interactions with other genes and/or non-coding RNAs. This review provides in-depth analyses of the molecular mechanisms through which TGF-β3 affects the fate decisions of adult stem cells, which lays a basis for identifying potential targets and developing future interventions for treating human diseases.

Spermatogonial stem cells (SSCs) are essential for male fertility because they form the cellular foundation for normal spermatogenesis. Here we address the regulatory mechanisms governing cell deaths of SSCs, e.g., apoptosis, ferroptosis, pyroptosis, and cuproptosis, including transcriptional and post-transcriptional regulation, RNA-binding proteins, and epigenetic modifications (e.g., non-coding RNAs). We systematically elucidate testicular microenvironment and signaling pathways in controlling SSC deaths, including mitochondrial signaling, death receptor signaling, PI3K/AKT/mTOR, MAPK, and WNT/β-catenin pathways. We also discuss the translational applications of targeting key pathways or remodeling the microenvironment to intervene in SSC deaths. We highlight the prospects and requirements to develop the advanced technologies, e.g., the long-term in vitro human primary SSC culture systems, single cell multi-omics, novel gene editing approaches with high safety and efficiency, and translating efficacy from basic research to clinical applications. The present review aims to provide new and overall insights into better understanding the molecular mechanisms underlying cell deaths of SSCs and the pathogenesis of non-obstructive azoospermia (NOA), which could offer novel strategies for precise treatment of male infertility.

Male infertility has become a serious issue in reproductive health. Inflammation of male reproductive system is a prevalent factor that influences male fertility. On the one hand, inflammation serves as the innate defense mechanism against injury, infection, and abnormal stimuli; and notably, it plays crucial roles in pathogen elimination, tissue repair, and the maintenance of internal homeostasis. On the other hand, inflammation can adversely affect spermatogenesis and lead to male infertility through oxidative stress, immune dysregulation, and cellular damage. In this review, we discuss the categories and etiology of inflammation diseases in male reproductive system and the functions of testicular immune cells, e.g., testicular macrophages, T and B lymphocytes, dendritic cells (DCs), mast cells, and Leydig cells, as well as signaling molecules and pathways by inflammation in male reproduction. Understanding these mechanisms may offer targets for therapeutic strategies to improve male fertility outcomes in inflammation-related condition. We also address therapeutic strategies for male reproductive system inflammation, including the use of antibiotics and lifestyle modifications. This review would help us better understand the mechanisms underlying the influence of inflammation on the male reproductive system. More significantly, it could be beneficial for treating inflammatory-related diseases of male reproductive system.

Human spermatogonial stem cells (SSCs) are crucial for spermatogenesis and male reproduction. Although abnormal pyroptosis caused by inflammation impacts male fertility, the molecular mechanisms underlying the pyroptosis of human SSCs are elusive. To induce pyroptosis, lipopolysaccharide (LPS) was introduced into the vas deferens of mice. RNA sequencing (RNA-Seq) of human SSCs was employed to identify NOD-like receptor thermal protein domain associated protein 3 (NLRP3), and RNA interference (RNAi) was used to determine the function and mechanism of NLRP3 in controlling pyroptosis of human SSCs. Guanylate-binding protein 4 (GBP4) was analyzed using the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway and protein–protein interaction (PPI) analyses, and the impact of GBP4 on NLRP3 in human SSCs was subsequently assessed using GBP4 short hairpin RNA (shRNA). LPS reduced the testicular weight of mice, disrupted spermatogenesis, and increased the levels of inflammatory factors (interleukin-18 [IL-18] and interleukin-1β [IL-1β]). NLRP3 siRNAs antagonized the LPS-induced increases in IL-18 and IL-1β, proliferative inhibition, and pyroptosis (Caspase 1 and Gasdermin D) in human SSCs. An association between GBP4 and NLRP3 in human SSCs was identified by co-immunoprecipitation (Co-IP). Human SSCs with stable GBP4 shRNA decreased the expression level of NLRP3 in human SSCs under inflammatory conditions. Collectively, these results imply that LPS-induced NLRP3 activation enhances the pyroptosis of human SSCs via the regulation of GBP4. This study offers novel insights into molecular mechanisms underlying the fate determinations of human SSCs.

As key factors of cellular development, epigenetic regulation can accurately control gene expression through multiple manners, e.g., DNA methylation, histone modification, and chromatin remodeling complexes (CRCs). Epigenetic factors play pivotal roles in various kinds of cell processes, including cell proliferation, differentiation, and apoptosis, and diseases may be resulted from their dysfunction. Spermatogenesis refers to the complex process by which spermatogonial stem cells (SSCs) self-renew and differentiate into the differentiating spermatogonia that further develop to spermatocytes and mature spermatids. Significantly, epigenetic regulation has recently been shown to mediate fate determinations of SSCs to ensure normal spermatogenesis. Interestingly, much progress has recently been made in epigenetic regulation and their dysfunction in controlling spermatogenesis and male infertility, respectively. In this review, we address the dynamic expression patterns, functions and mechanisms of DNA methylation, histone modification, and CRCs in mediating the development of SSCs and spermatogenesis, and we also discuss the association between epigenetic dysfunction and male infertility. We further point out the perspectives in epigenetic regulation on human spermatogenesis. Our review on the in-depth analysis of epigenetic regulatory mechanisms in normal and abnormal spermatogenesis not only helps us better understand the etiology of male infertility but also provides novel targets for treating this disease.

It remains unknown about molecular mechanisms underlying the transition of somatic cells into male germ cells. It is observed that RAD21L1 transcript is upregulated during reprogramming of Sertoli cells into human spermatogonial stem cells (SSCs) by overexpressing DAZ family genes. Significantly, RAD21L1 overexpression transits Sertoli cells into phenotypic and functional human SSCs with high safety. RNA sequencing shows that DNMT1 is expressed at a higher level by RAD21L1 overexpression when Sertoli cells are reprogrammed to become human SSCs. Whole genome bisulfite sequencing elucidates that RAD21L1 modulates DNA methylation to reprogram Sertoli cells into human SSCs, and RAD21L1 interacts with DNMT1 in human SSCs generated from Sertoli cells. Intriguingly, RAD21L1 mutation results in the decreases in stemness maintenance of human SSCs and DNMT1 expression levels. Notably, RAD21L1 mutations are positively related to risk of non-obstructive azoospermia (NOA) and male infertility. Collectively, these results implicate that RAD21L1 is sufficient and effective for reprogramming human Sertoli cells to SSCs through modulating DNMT1 and RAD21L1 mutations leads to NOA. This study is of particular significance because it provides a novel molecular mechanism that reprograms human somatic cells into human SSCs and it could offer invaluable gametes for treating male infertility.

Sertoli cell-only syndrome (SCOS) is one of the most severe non-obstructive azoospermia (NOA) types, since only Sertoli cells with not any male germ cells exist with the seminiferous tubules. As such, it is of particular significance to elucidate molecular mechanisms underlying SCOS for improving the diagnosis and treatment strategies for this disease. Due to the difficulties in obtaining sufficient human testicular tissues and the limited availability of human cells, the traditional proteomics is inadequate for comparing the differences in large scale of protein expression patterns of human Sertoli cells between SCOS and normal men. To solve this issue on the requirement of large amount of cell numbers, we employed micro-proteomics to reveal distinct global protein expression profiles of human Sertoli cells between SCOS and obstructive azoospermia (OA) with normal spermatogenesis utilizing single human Sertoli cells. We found a significant downregulation of proteins involved in cell adhesion pathways in SCOS Sertoli cells, whereas proteins related to apoptosis were markedly upregulated. Interestingly, we identified the lower expression of SPARC (secreted protein acidic and rich in cysteine) and the higher expression of FGF2 (fibroblast growth factor 2) in human Sertoli cells of the SCOS compared to normal men. SPARC silencing led to upregulation of FGF2 in human Sertoli cells, and SPARC may be associated with the occurrence of SCOS and serves as a reliable marker for the diagnosis of this disease. This study thus comprehensively offers the proteomic landscape of human Sertoli cells in the testes of SCOS patients and it sheds a novel insight into the pathogenesis of SCOS.

Stem cells have important applications in both regenerative and reproductive medicine. The cohesin complex comprises 4 core subunits, namely, SMC1, SMC3, RAD21, and STAG, and notably, it plays pivotal roles in controlling the fate determinations of stem cells by facilitating the dynamic regulation of the 3-dimensional genome architecture. We have recently reported that RAD21 forms a complex with YAP1 and NEDD4 to promote the self-renewal of human spermatogonial stem cells and inhibit their apoptosis. In this review, we address the molecular properties of the cohesin complex and its multiple regulatory mechanisms in mediating the fate decisions of various kinds of stem cells, including hematopoietic stem cells, embryonic stem cells, spermatogonial stem cells, neural stem cells, and other types of stem cells. By maintaining the chromatin loop structure, the cohesin complex is involved in DNA repair and gene transcription, which in turn controls the pluripotency, self-renewal, and differentiation of stem cells. In addition, the cohesin complex ensures faithful DNA replication and sister chromatid cohesion, which indirectly supports genetic and epigenetic programs. Variants in the subunit components of the cohesin complex and proteins’ modifications further confer functional plasticity, and its mutations can lead to abnormal stem cell functions and are correlated with diseases including cancers. Future studies need to integrate multidisciplinary approaches including single-cell multi-omics and cryo-electronic microscopy to resolve the dynamic regulatory networks of the cohesin complex in stem cell fate regulation and further explore its potential applications in regenerative and reproductive medicine.

Icariin is a pure compound derived from Epimedium brevicornu Maxim, and it helps the regulation of male reproduction. Nevertheless, the role and underlying mechanisms of Icariin in mediating male germ cell development remain to be clarified. Here, we have demonstrated that Icariin promoted proliferation and DNA synthesis of mouse spermatogonial stem cells (SSCs). Furthermore, surface plasmon resonance iron (SPRi) and molecular docking (MOE) assays revealed that phosphodiesterase 5A (PDE5A) was an important target of Icariin in mouse SSCs. Mechanically, Icariin decreased the expression level of PDE5A. Interestingly, hydrogen peroxides (H2O2) enhanced the expression level of phosphorylation H2A.X (p-H2A.X), whereas Icariin diminished the expression level of p-H2A.X and DNA damage caused by H2O2 in mouse SSCs. Finally, our in vivo animal study indicated that Icariin protected male reproduction. Collectively, these results implicate that Icariin targets PDE5A to regulate mouse SSC viability and DNA damage and improves male reproductive capacity. This study thus sheds new insights into molecular mechanisms underlying the fate decisions of mammalian SSCs and offers a scientific basis for the clinical application of Icariin in male reproduction.

Infertility has become one of the most serious diseases worldwide, and 50% of this disease can be attributed to male-related factors. Spermatogenesis, by definition, is a complex process by which spermatogonial stem cells (SSCs) self-renew to maintain stem cell population within the testes and differentiate into mature spermatids. It is of great significance to uncover gene regulation and signaling pathways that are involved in the fate determinations of SSCs with aims to better understand molecular mechanisms underlying human spermatogenesis and identify novel targets for gene therapy of male infertility. Significant achievement has recently been made in demonstrating the signaling molecules and pathways mediating the fate decisions of mammalian SSCs. In this review, we address key gene regulation and crucial signaling transduction pathways in controlling the self-renewal, differentiation, and apoptosis of SSCs, and we illustrate the networks of genes and signaling pathways in SSC fate determinations. We also highlight perspectives and future directions in SSC regulation by genes and their signaling pathways. This review could provide novel insights into the genetic regulation of normal and abnormal spermatogenesis and offer molecular targets to develop new approaches for gene therapy of male infertility.

The normal development of spermatogonial stem cells (SSCs) is essential for maintaining male fertility. Nevertheless, signaling molecules and pathways regulating the fate decisions of human SSCs remain elusive. We have reported that Opa interacting protein 5 (OIP5) is involved in controlling human SSC self-renewal and apoptosis. Notably, we found that insulin-like growth factor 2 mRNA-binding protein 1 (IGF2BP1) interacted with OIP5 in human SSCs. Our RNA sequencing (RNA-seq) and immunofluorescence–fluorescence in situ hybridization (IF-FISH) identified heme oxygenase 1 (HMOX1) as a downstream target of IGF2BP1 in human SSCs. Methylated RNA immunoprecipitation-binding qPCR (MeRIP-qPCR) and RNA stability assays revealed that IGF2BP1 could bind to HMOX1 mRNA and enhance its stability and HMOX1 expression level. Functional assays demonstrated that IGF2BP1 or HMOX1 silencing resulted in the decreased expression levels of ferroptosis-associated genes (e.g., SLC7A11 and SLC3A2), the increased intracellular reactive oxygen species (ROS) level and ferrous ion (Fe2+) content, and the impaired stemness maintenance of human SSCs. Additionally, we observed that IGF2BP1-mediated HMOX1 stabilization activated system Xc−, thereby inhibiting ferroptosis of human SSCs. Furthermore, IGF2BP1 gene variants were positively correlated with the occurrence of non-obstructive azoospermia (NOA). Collectively, these results imply a critical role of IGF2BP1/HMOX1 mRNA/system Xc− axis in regulating human SSC ferroptosis, autophagy, and stemness maintenance. This study thus provides novel insights into the regulatory mechanisms of human SSC fate determinations and offers new targets for treating male infertility.

Organoids refer to three-dimensional (3D) multicellular tissues derived from stem cells or single cells through their self-assembly capacity, and significantly, they mimic structural and functional characteristics of the organ from which they are derived. Organoids can maintain the gene expression profiles and mutational features of parental cells during long-term culture. This makes organoids more relevant to the human bodies than gene knockout or overexpression animal models. Consequently, organoids have been widely used in various kinds of fields, including studies on organ developmental mechanisms, regenerative medicine, organ repair, the construction of disease models, high-throughput drug screening, and personalized medicine. Notably, significant progress has recently been made in organoid construction methodologies and regulatory mechanisms. These include the selections of starting cell sources, optimizing matrix materials, and the related cell signaling pathways. The rapid development of organoid technologies has provided new opportunities for their applications in organ transplantation, drug and toxicity screening, and molecular mechanisms for cell and tissue development. In this review, we discuss organoid construction methods involving the starting cell selection and spatiotemporal mediation, regulatory mechanisms with signaling molecules and pathways, and their applications in unveiling organogenesis mechanisms and disease etiology, drug screening, toxicity testing, personalized medicine, regenerative medicine, and alternatives to animal experiments. We also address the perspectives and challenges in this field with an aim to promote the development of organoids in basic research and translational medicine.

Spermatogonial stem cells (SSCs) are essential for initiating and maintaining normal spermatogenesis, and notably, they have important applications in both reproduction and regenerative medicine. Nevertheless, the molecular mechanisms controlling the fate determinations of human SSCs remain elusive. In this study, we identified a selective expression of APBB1 in dormant human SSCs. We demonstrated for the first time that APBB1 interacted with KAT5, which led to the suppression of GDF15 expression and consequent inhibition of human SSC proliferation. Intriguingly, Apbb1-/- mice assumed the disrupted spermatogenesis and markedly reduced fertility. SSC transplantation assays revealed that Apbb1 silencing enhanced SSC colonization and impeded their differentiation, which resulted in the impaired spermatogenesis. Notably, 4 deleterious APBB1 mutation sites were identified in 2,047 patients with non-obstructive azoospermia (NOA), and patients with the c.1940C>G mutation had a similar testicular phenotype with Apbb1-/- mice. Additionally, we observed lower expression levels of APBB1 in NOA patients with spermatogenic arrest than in obstructive azoospermia patients with normal spermatogenesis. Collectively, our findings highlight an essential role of APBB1/KAT5/GDF15 in governing human SSC fate decisions and maintaining normal spermatogenesis and underscore them as therapeutic targets for treating male infertility.

Spermatogenesis is a sophisticated biological process by which spermatogonial stem cells (SSCs) undergo self-renewal and differentiation into spermatozoa. Molecular mechanisms underlying fate determinations of human SSCs by key genes and signaling pathways remain elusive. Here, we report for the first time that Yes1-associated transcriptional regulator (YAP1) is required for fate determinations of SSCs and male fertility by interacting with RAD21 and targeting NEDD4 in humans and mice. YAP1 was mainly located at cell nuclei of human SSCs. YAP1 silencing resulted in the decreases in proliferation and DNA synthesis as well as an enhancement in apoptosis of human SSCs both in vivo and in vitro. RNA sequencing and real-time polymerase chain reaction assays identified NEDD4 as a target of YAP1, and NEDD4 knockdown inhibited the proliferation of human SSCs and increased their apoptosis. Furthermore, YAP1 interacted with RAD21 to regulate NEDD4 transcription in human SSCs. Importantly, YAP1 abnormalities were found to be associated with non-obstructive azoospermia (NOA) as manifested as lower expression level of YAP1 in testicular tissues of NOA patients and YAP1 single-nucleotide variants (SNVs) in 777 NOA patients. Finally, Yap1 germline conditional knockout (cKO) mice assumed mitotic arrest, low sperm count, and motility. Collectively, these results highlight a critical role of YAP1 in determining the fate determinations of human SSCs and male infertility through the YAP1/RAD21/NEDD4 pathway. This study provides new insights into the genetic regulatory mechanisms underlying human spermatogenesis and the pathogenesis of NOA, and it offers new targets for gene therapy of male infertility.

As the most abundant small RNAs, piwi-interacting RNAs (piRNAs) have been identified as a new class of non-coding RNAs with 24–32 nucleotides in length, and they are expressed at high levels in male germ cells. PiRNAs have been implicated in the regulation of several biological processes, including cell differentiation, development, and male reproduction. In this review, we focused on the functions and molecular mechanisms of piRNAs in controlling spermatogenesis, including genome stability, regulation of gene expression, and male germ cell development. The piRNA pathways include two major pathways, namely the pre-pachytene piRNA pathway and the pachytene piRNA pathway. In the pre-pachytene stage, piRNAs are involved in chromosome remodeling and gene expression regulation to maintain genome stability by inhibiting transposon activity. In the pachytene stage, piRNAs mediate the development of male germ cells via regulating gene expression by binding to mRNA and RNA cleavage. We further discussed the correlations between the abnormalities of piRNAs and male infertility and the prospective of piRNAs’ applications in reproductive medicine and future studies. This review provides novel insights into mechanisms underlying mammalian spermatogenesis and offers new targets for diagnosing and treating male infertility.

Human spermatogonial stem cells (SSCs) have significant applications in reproductive medicine and regenerative medicine because of their great plasticity. Nevertheless, it remains unknown about the functions and mechanisms of long non-coding RNA (LncRNA) in regulating the fate determinations of human SSCs. Here we have demonstrated that LncRNA ACVR2B-as1 (activin A receptor type 2B antisense RNA 1) controls the self-renewal and apoptosis of human SSCs by interaction with ALDOA via glycolysis activity. LncRNA ACVR2B-as1 is highly expressed in human SSCs. LncRNA ACVR2B-as1 silencing suppresses the proliferation and DNA synthesis and enhances the apoptosis of human SSCs. Mechanistically, our ChIRP-MS and RIP assays revealed that ACVR2B-as1 interacted with ALDOA in human SSCs. High expression of ACVR2B-as1 enhanced the proliferation, DNA synthesis, and glycolysis of human SSCs but inhibited their apoptosis through up-regulation of ALDOA. Importantly, overexpression of ALDOA counteracted the effect of ACVR2B-as1 knockdown on the aforementioned biological processes. Collectively, these results indicate that ACVR2B-as1 interacts with ALDOA to control the self-renewal and apoptosis of human SSCs by enhancing glycolysis activity. This study is of great significance because it sheds a novel insight into molecular mechanisms underlying the fate decisions of human SSCs and it may offer innovative approaches to address the etiology of male infertility.

Stem Leydig cells (SLCs) are essential for maintaining normal spermatogenesis as the significant component of testis microenvironment and gonadal aging. Although progress has been achieved in the regulation of male germ cells in mammals and humans, it remains unknown about the genes and signaling pathways of human SLCs. Here we have demonstrated, for the first time, that WNT5A (Wnt family member 5a) mediates the proliferation, apoptosis, and stemness of human SLCs, namely NGFR + Leydig cells. We revealed that NGFR + Leydig cells expressed NGFR, PDGFRA, NES, NR2F2, and THY1, hallmarks for SLCs. RNA-sequencing showed that WNT5A was expressed at a higher level in human SLCs than non-SLCs, while immunohistochemistry and Western blots further illustrated that WNT5A was predominantly expressed in human SLCs. Notably, CCK-8, EdU and Western blots displayed that WNT5A enhanced the proliferation and DNA synthesis and retained stemness of human SLCs, whereas flow cytometry and TUNEL analyses demonstrated that WNT5A inhibited the apoptosis of these cells. WNT5A knockdown caused an increase in LC lineage differentiation of human SLCs and reversed the effect of WNT5A overexpression on fate decisions of human SLCs. In addition, WNT5A silencing resulted in the decreases in nuclear translocation of β-catenin and expression levels of c-Myc , CD44 , and Cyclin D1 . Collectively, these results implicate that WNT5A regulates the proliferation, apoptosis and stemness of human SLCs through the activation of the β-catenin signaling pathway. This study thus provides a novel molecular mechanism underlying the fate determinations of human SLCs, and it offers a new insight into the niche regulation of human testis.

Organoids play pivotal roles in uncovering the molecular mechanisms underlying organogenesis, intercellular communication, and high-throughput drug screening. Testicular organoids are essential for exploring the genetic and epigenetic regulation of spermatogenesis in vivo and the treatment of male infertility. However, the formation of testicular organoids with full spermatogenesis has not yet been achieved. In this study, neonatal mouse testicular cells were isolated by two-step enzymatic digestion, and they were combined with Matrigel and transplanted subcutaneously into nude mice. Histological examination (H&E) staining and immunohistochemistry revealed that cell grafts assembled to form seminiferous tubules that contained spermatogonial stem cells (SSCs) and Sertoli cells, as illustrated by the co-expression of PLZF (a hallmark for SSCs) and SOX9 (a marker for Sertoli cells) as well as the co-expression of UCHL1 (a hallmark for SSCs) and SOX9, after 8 weeks of transplantation. At 10 weeks of transplantation, SSCs could proliferate and differentiate into spermatocytes as evidenced by the expression of PCNA, Ki67, c-Kit, SYCP3, γ-HA2X, and MLH1. Notably, testicular organoids were seen, and spermatids were observed within the lumen of testicular organoids after 16 weeks of transplantation, as shown by the presence of TNP1 and ACROSIN (hallmarks for spermatids). Collectively, these results implicate that we successfully established testicular organoids with spermatogenesis in vivo. This study thus provides an excellent platform for unveiling the mechanisms underlying mammalian spermatogenesis, and it might offer valuable male gametes for treating male infertility.

Human spermatogonial stem cells (SSCs) are essential for spermatogenesis and male fertility. However, molecular mechanisms regulating fate determinations of human SSCs remain elusive. In this study, we revealed that KLF2 decreased the proliferation, DNA synthesis and colonization of human SSCs as well as increased apoptosis of these cells. We identified and demonstrated that GJA1 was a target gene for KLF2 in human SSCs. Notably, KLF2 overexpression rescued the reduction of proliferation of human SSCs caused by GJA1 silencing as well as the enhancement of apoptosis of human SSCs. Abnormalities in the higher level of KLF2 and/or KIF2 mutations might lead to male infertility. Collectively, these results implicate that KLF2 inhibits proliferation of human SSCs and enhances their apoptosis by targeting GJA1. This study thus provides novel genetic mechanisms underlying human spermatogenesis and azoospermia, and it offers new endogenous targets for treating male infertility.