Establishment and Characterization of Testis Organoids with Proliferation and Differentiation of Spermatogonial Stem Cells into Spermatocytes and Spermatids.

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.

The functions and mechanisms of piRNAs in mediating mammalian spermatogenesis and their applications in reproductive medicine.

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.

LncRNA ACVR2B-as1 interacts with ALDOA to regulate the self-renewal and apoptosis of human spermatogonial stem cells by controlling glycolysis activity.

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.

Gene regulation and signaling transduction in mediating the self-renewal, differentiation, and apoptosis of spermatogonial stem cells.

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.

USP11 regulates proliferation and apoptosis of human spermatogonial stem cells via HOXC5-mediated canonical WNT/ß-catenin signaling pathway.

Spermatogonial stem cells (SSCs) are capable of transmitting genetic information to the next generations and they are the initial cells for spermatogenesis. Nevertheless, it remains largely unknown about key genes and signaling pathways that regulate fate determinations of human SSCs and male infertility. In this study, we explored the expression, function, and mechanism of USP11 in controlling the proliferation and apoptosis of human SSCs as well as the association between its abnormality and azoospermia. We found that USP11 was predominantly expressed in human SSCs as shown by database analysis and immunohistochemistry. USP11 silencing led to decreases in proliferation and DNA synthesis and an enhancement in apoptosis of human SSCs. RNA-sequencing identified HOXC5 as a target of USP11 in human SSCs. Double immunofluorescence, Co-immunoprecipitation (Co-IP), and molecular docking demonstrated an interaction between USP11 and HOXC5 in human SSCs. HOXC5 knockdown suppressed the growth of human SSCs and increased apoptosis via the classical WNT/ß-catenin pathway. In contrast, HOXC5 overexpression reversed the effect of proliferation and apoptosis induced by USP11 silencing. Significantly, lower levels of USP11 expression were observed in the testicular tissues of patients with spermatogenic disorders. Collectively, these results implicate that USP11 regulates the fate decisions of human SSCs through the HOXC5/WNT/ß-catenin pathway. This study thus provides novel insights into understanding molecular mechanisms underlying human spermatogenesis and the etiology of azoospermia and it offers new targets for gene therapy of male infertility.

KLF2 controls proliferation and apoptosis of human spermatogonial stem cells via targeting GJA1.

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.

WNT5A regulates the proliferation, apoptosis and stemness of human stem Leydig cells via the ß-catenin signaling pathway.

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 ß-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 ß-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.

Ferritinophagy: research advance and clinical significance in cancers.

Ferritinophagy, a process involving selective autophagy of ferritin facilitated by nuclear receptor coactivator 4 (NCOA4), entails the recognition of ferritin by NCOA4 and subsequent delivery to the autophagosome. Within the autophagosome, ferritin undergoes degradation, leading to the release of iron in the lysosome. It is worth noting that excessive iron levels can trigger cell death. Recent evidence has elucidated the significant roles played by ferritinophagy and ferroptosis in regulation the initiation and progression of cancer. Given the crucial role of ferritinophagy in tumor biology, it may serve as a potential target for future anti-tumor therapeutic interventions. In this study, we have provided the distinctive features of ferritinophagy and its distinctions from ferroptosis. Moreover, we have briefly examined the fundamental regulatory mechanisms of ferritinophagy, encompassing the involvement of the specific receptor NCOA4, the Nrf2/HO-1 signaling and other pathways. Subsequently, we have synthesized the current understanding of the impact of ferritinophagy on cancer progression and its potential therapeutic applications, with a particular emphasis on the utilization of chemotherapy, nanomaterials, and immunotherapy to target the ferritinophagy pathway for anti-tumor purposes.

AZGP1P2/UBA1/RBM15 Cascade Mediates the Fate Determinations of Prostate Cancer Stem Cells and Promotes Therapeutic Effect of Docetaxel in Castration-Resistant Prostate Cancer via TPM1 m6A Modification.

Prostate cancer (PCa) is a common malignant tumor with high morbidity and mortality worldwide. The prostate cancer stem cell (PCSC) model provides novel insights into the pathogenesis of PCa and its therapeutic response. However, the roles and molecular mechanisms of specific genes in mediating fate decisions of PCSCs and carcinogenesis of PCa remain to be elusive. In this study, we have explored the expression, function, and mechanism of AZGP1P2, a pseudogene of AZGP1, in regulating the stemness and apoptosis of PCSCs and treatment resistance of docetaxel in castration-resistant prostate cancer (CRPC). We revealed that AZGP1P2 was downregulated in CRPC cell lines and PCSCs, while it was positively associated with progression-free interval. Upregulation of the AZGP1P2 enhanced the sensitivity of docetaxel treatment in CRPCs via inhibiting their stemness. RNA pull-down associated with mass spectrometry analysis, co-immunoprecipitation assay, and RNA immunoprecipitation assay demonstrated that AZGP1P2 could bind to UBA1 and RBM15 as a "writer" of methyltransferase to form a compound. UBA1, an E1 ubiquitin-activating enzyme, contributed to RBM15 protein degradation via ubiquitination modification. Methylated RNA immunoprecipitation assay displayed that RBM15 controlled the mRNA decay of TPM1 in m6A methylation. Furthermore, a xenograft mouse model and patient-derived organoids showed that the therapeutic effect of docetaxel in CRPC was increased by AZGP1P2 in vivo. Collectively, these results imply that AZGP1P2 mediates the stemness and apoptosis of PCSCs and promotes docetaxel therapeutic effect by suppressing tumor growth and metastasis via UBA1/RBM15-mediated TPM1 mRNA decay in CRPC.

ZBTB40 is a telomere-associated protein and protects telomeres in human ALT cells.

Alternative lengthening of telomeres (ALTs) mechanism is activated in some somatic, germ cells, and human cancer cells. However, the key regulators and mechanisms of the ALT pathway remain elusive. Here we demonstrated that ZBTB40 is a novel telomere-associated protein and binds to telomeric dsDNA through its N-terminal BTB (BR-C, ttk and bab) or POZ (Pox virus and Zinc finger) domain in ALT cells. Notably, the knockout or knockdown of ZBTB40 resulted in the telomere dysfunction-induced foci and telomere lengthening in the ALT cells. The results also show that ZBTB40 is associated with ALT-associated promyelocytic leukemia nuclear bodies, and the loss of ZBTB40 induces the accumulation of the ALT-associated promyelocytic leukemia nuclear bodies in U2OS cells. Taken together, our results implicate that ZBTB40 is a key player of telomere protection and telomere lengthening regulation in human ALT cells.

GPx3 knockdown inhibits the proliferation and DNA synthesis and enhances the early apoptosis of human spermatogonial stem cells via mediating CXCL10 and cyclin B1.

Spermatogenesis is regulated by genetic and epigenetic factors. However, the genes and signaling pathways mediating human spermatogenesis remain largely unknown. Here, we have for the first time explored the expression, function, and mechanism of glutathione peroxidase 3 (GPx3) in controlling the proliferation and apoptosis of human spermatogonial stem cells (SSCs). We found that GPx3 was expressed in human SSCs. Notably, we revealed that GPx3 knockdown resulted in the decrease in the proliferation, DNA synthesis, and cyclin B1 level in human SSC lines, which possessed the phenotypic features of human primary SSCs. Flow cytometry and TUNEL assays showed that GPx3 silencing led to enhancement of early apoptosis of human SSC line. RNA sequencing was utilized to identify CXCL10 as a target of GPx3 in human SSCs, and notably, both double immunostaining and co-immunoprecipitation (co-IP) demonstrated that there was an association between GPx3 and CXCL10 in these cells. CXCL10-shRNA resulted in the reduction in the proliferation and DNA synthesis of human SSC line and an increase in apoptosis of these cells. Taken together, these results implicate that GPx3 regulates the proliferation, DNA synthesis, and early apoptosis of human SSC line via mediating CXCL10 and cyclin B1. This study, thus, offers a novel insight into the molecular mechanism regulating the fate determinations of human SSCs and human spermatogenesis.

OIP5 Interacts with NCK2 to Mediate Human Spermatogonial Stem Cell Self-Renewal and Apoptosis through Cell Cyclins and Cycle Progression and Its Abnormality Is Correlated with Male Infertility.

Spermatogonial stem cells (SSCs) have important applications in both reproduction and regenerative medicine. Nevertheless, specific genes and signaling transduction pathways in mediating fate decisions of human SSCs remain elusive. Here, we have demonstrated for the first time that OIP5 (Opa interacting protein 5) controlled the self-renewal and apoptosis of human SSCs. RNA sequencing identified that NCK2 was a target for OIP5 in human SSCs, and interestingly, OIP5 could interact with NCK2 as shown by Co-IP (co-immunoprecipitation), IP-MS (mass spectrometry), and GST pulldown assays. NCK2 silencing decreased human SSC proliferation and DNA synthesis but enhanced their apoptosis. Notably, NCK2 knockdown reversed the influence of OIP5 overexpression on human SSCs. Moreover, OIP5 inhibition decreased the numbers of human SSCs at S and G2/M phases, while the levels of numerous cell cycle proteins, including cyclins A2, B1, D1, E1 and H, especially cyclin D1, were remarkably reduced. Significantly, whole-exome sequencing of 777 patients with nonobstructive azoospermia (NOA) revealed 54 single-nucleotide polymorphism mutations of the OIP5 gene (6.95%), while the level of OIP5 protein was obviously lower in testes of NOA patients compared to fertile men. Collectively, these results implicate that OIP5 interacts with NCK2 to modulate human SSC self-renewal and apoptosis via cell cyclins and cell cycle progression and that its mutation and/or lower expression is correlated with azoospermia. As such, this study offers novel insights into molecular mechanisms underlying the fate determinations of human SSCs and the pathogenesis of NOA, and it provides new targets for treating male infertility.

Alpha-tocopherol enhances spermatogonial stem cell proliferation and restores mouse spermatogenesis by up-regulating BMI1.

Purpose: Spermatogonial stem cells (SSCs) are essential for maintaining reproductive function in males. B-lymphoma Mo-MLV insertion region 1 (BMI1) is a vital transcription repressor that regulates cell proliferation and differentiation. However, little is known about the role of BMI1 in mediating the fate of mammalian SSCs and in male reproduction. This study investigated whether BMI1 is essential for male reproduction and the role of alpha-tocopherol (α-tocopherol), a protective agent for male fertility, as a modulator of BMI1 both in vitro and in vivo. Methods: Methyl thiazolyl tetrazolium (MTT) and 5-ethynyl-2'-deoxyuridine (EDU) assays were used to assess the effect of BMI1 on the proliferative ability of the mouse SSC line C18-4. Real-time polymerase chain reaction (PCR), western blotting, and immunofluorescence were applied to investigate changes in the mRNA and protein expression levels of BMI1. Male mice were used to investigate the effect of α-tocopherol and a BMI1 inhibitor on reproduction-associated functionality in vivo. Results: Analysis revealed that BMI1 was expressed at high levels in testicular tissues and spermatogonia in mice. The silencing of BMI1 inhibited the proliferation of SSCs and DNA synthesis and enhanced the levels of γ-H2AX. α-tocopherol enhanced the proliferation and DNA synthesis of C18-4 cells, and increased the levels of BMI1. Notably, α-tocopherol rescued the inhibition of cell proliferation and DNA damage in C18-4 cells caused by the silencing of BMI1. Furthermore, α-tocopherol restored sperm count (Ctrl vs. PTC-209, p = 0.0034; Ctrl vs. PTC-209 + α-tocopherol, p = 0.7293) and normalized sperm malformation such as broken heads, irregular heads, lost and curled tails in vivo, as demonstrated by its antagonism with the BMI1 inhibitor PTC-209. Conclusion: Analysis demonstrated that α-tocopherol is a potent in vitro and in vivo modulator of BMI1, a transcription factor that plays an important role in in SSC proliferation and spermatogenesis. Our findings identify a new target and strategy for treating male infertility that deserves further pre-clinical investigation.

Zbtb40 Deficiency Leads to Morphological and Phenotypic Abnormalities of Spermatocytes and Spermatozoa and Causes Male Infertility.

Studies on the gene regulation of spermatogenesis are of unusual significance for maintaining male reproduction and treating male infertility. Here, we have demonstrated, for the first time, that a loss of ZBTB40 function leads to abnormalities in the morphological and phenotypic characteristics of mouse spermatocytes and spermatids as well as male infertility. We revealed that Zbtb40 was expressed in spermatocytes of mouse testes, and it was co-localized with γH2AX in mouse secondary spermatocytes. Interestingly, spermatocytes of Zbtb40 knockout mice had longer telomeres, compromised double-strand break (DSB) repair in the sex chromosome, and a higher apoptosis ratio compared to wild-type (WT) mice. The testis weight, testicular volume, and cauda epididymis body weight of the Zbtb40+/- male mice were significantly lower than in WT mice. Mating tests indicated that Zbtb40+/- male mice were able to mate normally, but they failed to produce any pups. Notably, sperm of Zbtb40+/- mice showed flagellum deformities and abnormal acrosome biogenesis. Furthermore, a ZBTB40 mutation was associated with non-obstructive azoospermia. Our results implicate that ZBTB40 deficiency leads to morphological and phenotypic abnormalities of spermatocytes and spermatids and causes male infertility. This study thus offers a new genetic mechanism regulating mammalian spermatogenesis and provides a novel target for gene therapy in male infertility.

Generation of male germ cells in vitro from the stem cells.

Infertility has become a serious disease since it affects 10%-15% of couples worldwide, and male infertility contributes to about 50% of the cases. Notably, a significant decrease occurs in the newborn population by 7.82 million in 2020 compared to 2016 in China. As such, it is essential to explore the effective methods of obtaining functional male gametes for restoring male fertility. Stem cells, including embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs), spermatogonial stem cells (SSCs), and mesenchymal stem cells (MSCs), possess the abilities of both self-renewal and differentiation into germ cells. Significantly, much progress has recently been achieved in the generation of male germ cells in vitro from various kinds of stem cells under the specified conditions, e.g., the coculturing with Sertoli cells, three-dimensional culture system, the addition of growth factors and cytokines, and/or the overexpression of germ cell-related genes. In this review, we address the current advance in the derivation of male germ cells in vitro from stem cells based on the studies of the peers and us, and we highlight the perspectives and potential application of stem cell-derived male gametes in reproductive medicine.

MAP4K4/JNK Signaling Pathway Stimulates Proliferation and Suppresses Apoptosis of Human Spermatogonial Stem Cells and Lower Level of MAP4K4 Is Associated with Male Infertility.

Spermatogonial stem cells (SSCs) serve as a foundation for spermatogenesis and they are essential for male fertility. The fate of SSC is determined by genetic and epigenetic regulatory networks. Many molecules that regulate SSC fate determinations have been identified in mice. However, the molecules and signaling pathways underlying human SSCs remain largely unclear. In this study, we have demonstrated that MAP4K4 was predominantly expressed in human UCHL1-positive spermatogonia by double immunocytochemical staining. MAP4K4 knockdown inhibited proliferation of human SSCs and induced their apoptosis. Moreover, MAP4K4 silencing led to inhibition of JNK phosphorylation and MAP4K4 phosphorylation at Ser801. RNA sequencing indicated that MAP4K4 affected the transcription of SPARC, ADAM19, GPX7, GNG2, and COLA1. Interestingly, the phenotype of inhibiting JNK phosphorylation by SP600125 was similar to MAP4K4 knockdown. Notably, MAP4K4 protein was lower in the testes of patients with non-obstructive azoospermia than those with normal spermatogenesis as shown by Western blots and immunohistochemistry. Considered together, our data implicate that MAP4K4/JNK signaling pathway mediates proliferation and apoptosis of human SSCs, which provides a novel insight into molecular mechanisms governing human spermatogenesis and might offer new targets for gene therapy of male infertility.

RNF144B stimulates the proliferation and inhibits the apoptosis of human spermatogonial stem cells via the FCER2/NOTCH2/HES1 pathway and its abnormality is associated with azoospermia.

Studies on gene regulation and signaling transduction pathways of human spermatogonial stem cells (SSCs) are of the utmost significance for unveiling molecular mechanisms underlying human spermatogenesis and gene therapy of male infertility. We have demonstrated, for the first time, that RNF144B stimulated cell proliferation and inhibited the apoptosis of human SSCs. The target of RNF144B was identified as FCER2 by RNA sequencing. We revealed that RNF144B interacted with FCER2 by immunoprecipitation. Consistently, overexpression of FCER2 reversed the phenotype of proliferation and apoptosis of human SSCs caused by RNF144B knockdown. Interestingly, FCER2 pulled down N2ICD (NOTCH2 intracellular domain), while N2ICD could bind to FCER2 in human SSCs. The levels of NOTCH2, FCER2, HES1, and HEY1 were reduced by RNF144B siRNA in human SSCs. Significantly, RNF144B was expressed at a lower level in nonobstructive azoospermia (NOA) patients than in the obstructive azoospermia (OA) patients with normal spermatogenesis, and 52 patients with heterozygous mutations of RNF144B were detected in 1,000 NOA patients. These results implicate that RNF144B promotes the proliferation of human SSCs and suppresses their apoptosis via the FCER2/NOTCH2/HES1 pathway and that the abnormality of RNF144B is associated with spermatogenesis failure. This study thus provides novel molecular mechanisms regulating the fate determinations of human SSCs, and it offers new biomarkers for the diagnosis and treatment of male infertility.

The mechanisms and functions of microRNAs in mediating the fate determinations of human spermatogonial stem cells and Sertoli cells.

Human spermatogonial stem cells (SSCs) and Sertoli cells might have the applications in reproduction and regenerative medicine. Abnormal spermatogenesis results in male infertility, which seriously affects human reproduction and health. Spermatogenesis depends on the epigenetic and genetic regulation of male germ cells and somatic cells. A number of microRNAs (miRNAs) have been identified in human testicular tissues, and they are closely related to male fertility. Significantly, we and peers have recently demonstrated that numerous miRNAs are essential for regulating the self-renewal and apoptosis of human SSCs and Sertoli cells through controlling their mRNA and lncRNA targets. In this review, we critically discuss these findings regarding the important functions and mechanisms of miRNAs in mediating the fate determinations of human SSCs and Sertoli cells. Meanwhile, we illustrate the regulatory networks for miRNAs by forming the upstream and downstream regulators of mRNAs and lncRNAs in human SSCs, and we address that miRNAs regulate the decisions of Sertoli cells by targeting genes and via N6-methyladenosine (m6A). We also point out the future directions for further studies on this field. This review could offer an update on novel molecular targets for treating male infertility and new insights into epigenetic regulation of human spermatogenesis.

Direct reprogramming of human Sertoli cells into male germline stem cells with the self-renewal and differentiation potentials via overexpressing DAZL/DAZ2/BOULE genes.

We propose a new concept that human somatic cells can be converted to become male germline stem cells by the defined factors. Here, we demonstrated that the overexpression of DAZL, DAZ2, and BOULE could directly reprogram human Sertoli cells into cells with the characteristics of human spermatogonial stem cells (SSCs), as shown by their similar transcriptomes and proteomics with human SSCs. Significantly, human SSCs derived from human Sertoli cells colonized and proliferated in vivo, and they could differentiate into spermatocytes and haploid spermatids in vitro. Human Sertoli cell-derived SSCs excluded Y chromosome microdeletions and assumed normal chromosomes. Collectively, human somatic cells could be converted directly to human SSCs with the self-renewal and differentiation potentials and high safety. This study is of unusual significance, because it provides an effective approach for reprogramming human somatic cells into male germ cells and offers invaluable male gametes for treating male infertility.