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.

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.

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.

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.

Casticin inhibits stemness of hepatocellular carcinoma cells via disrupting the reciprocal negative regulation between DNMT1 and miR-148a-3p.

Casticin (CAS) is a polymethyl flavonoid from Fructus viticis and has multiple pharmacological activities, including anticancer. However, whether the molecular mechanism underlying CAS represses stemness characteristics in hepatocellular carcinoma (HCC) cells involves intervention in the reciprocal negative regulation between DNA methyltransferase 1 (DNMT1) and miR-148a-3p has not yet been reported. In this study, the effect of CAS on stemness characteristics of HCC cells and its mechanism were investigated. Results showed that CAS selectively reduced the viabilities of HCC cells but not L02 cells, as determined by CCK-8 assay. Importantly, the sub-cytotoxic concentrations of CAS could inhibit the stemness characteristics in HCC cells, as demonstrated by the expression of stemness biomarkers (CD44, EpCAM, Bmi1, Nanog, and Oct4), sphere forming assay, RT-qPCR, and Western blotting. In addition, CAS repressed DNMT1 activity and expression and increased miR-148a-3p. The effect of CAS on stemness characteristics was abolished by stable DNMT1 overexpression. MiR-148a-3p overexpression enhanced the reduction of CAS on stemness characteristics. DNMT1 overexpression promoted miR-148a-3p promoter hypermethylation as detected by methylation-specific PCR (MSP), which repressed its expression. Conversely, miR-148a-3p repressed DNMT1 expression by specific site binding to 3'-UTR of DNMT1 mRNA, as determined by luciferase assay. Moreover, the combination of CAS and agomir-148a-3p had robust effects on tumor suppression as compared to the sole activity of either molecule in nude mouse xenograft experiments in vivo. The findings suggested that CAS could inhibit stemness characteristics in HCC cells by interruption of the reciprocal negative regulation between DNMT1 and miR-148a-3p.

Chrysin Inhibits Proinflammatory Factor-Induced EMT Phenotype and Cancer Stem Cell-Like Features in HeLa Cells by Blocking the NF-κB/Twist Axis.

BACKGROUND/AIMS: TNF-α and TGF-ß associated epithelial-mesenchymal transition (EMT) occurs via NF-κB-dependent transcriptional upregulation of Twist1.Chrysin (ChR) is a major active ingredient ofpropolis, which inhibits various cancer cells and possesses anti-inflammatory activities. This study aimed to assess whether and how ChR inhibits proinflammatory cytokine-induced EMT phenotype and cancer stem-like cell (CSLC) features in the HeLa cell line. METHODS: HeLa cells were co-administered TNF-α (10.0 ng/mL) and TGF-ß (5.0 ng/mL) for 24h following TGF-ß (5.0 ng/mL) alone for 6 d in the presence or absence of ChR (5.0, 10.0 and 20.0µM). Then, the levels of EMT-related factors, multi-potential transcription factors, and stem cell markers were analyzed by immunoblot. Wound healing and tumor sphere formation assays were performed to assess the migration and self-renewal capabilities of cells, respectively. Overexpression and/or knockdown of NF-κBp65 and/or Twsit1 were used to explore the molecular mechanisms. RESULTS: The results showed that ChR inhibited EMT and CSLC properties in HeLa cells administered TNF-α after prolonged TGF-ß treatment, in a concentration-dependent fashion. NF-κBp65 knockdown and ChR(10.0µM) cooperatively enhanced the inhibition of NF-κBp65 and Twist1 expression, EMT, and CSLC properties. Conversely, overexpression of NF-κBp65 combined with ChR(10.0µM) antagonized such activities. Meanwhile, Twist1silencing or overexpression combined with ChR treatment did not affect NF-κBp65 levels, but also reduced or enhanced EMT and CSLC properties. Importantly, overexpressing Twist1 combined with ChR reversed the effects of NF-κBp65 knockdown and ChR. CONCLUSION: ChR inhibits proinflammatory cytokine-induced EMT and CSLC features in HeLa cells by blocking the NF-κB/Twist axis.

8-bromo-7-methoxychrysin suppress stemness of SMMC-7721 cells induced by co-culture of liver cancer stem-like cells with hepatic stellate cells.

BACKGROUND: Our previous works have demonstrated that 8-bromo-7-methoxychrysin suppressed stemness of human hepatocellular carcinoma (HCC) cell line SMMC-7721 induced by condition medium from hepatic stellate cell line LX-2 that was activated by liver cancer stem-like cells (LCSCs). However, whether and whereby BrMC inhibits the stemness induced by co-culture of LCSCs and LX-2 cells remains to be investigated. METHODS: The second-generation spheres by sphere culture were identified and used as SMMC-7721-and MHCC97H-derived LCSLCs. SMMC-7721-and MHCC97-derived LCSCs/LX-2 cells transwell co-culture system was treated with BrMC and its lead compound chrysin. The concentrations of IL-6, IL-8, HGF and PDGF in condition medium from co-culture were measured by enzyme-linked immunosorbent assay (ELISA). The stemness of SMMC-7721 cells was evaluated by sphere formation assay and western blot analysis for expression levels of cancer stem cell markers (CD133 and CD44).The expression levels of cancer-associated fibroblast markers (FAP-α and α-SMA) were employed to evaluate pathologic activation of LX-2 cells. Addition of IL-6 and/or HGF or deletion of IL-6 and/or HGF was conducted to investigate the mechanisms for BrMC and chrysin treatment in SMMC-7721-derived LCSLCs co-cultured with LX-2cells. RESULTS: The co-culture of LCSLCs with LX-2 cells increased sphere formation capability as well as expression of CD133 and CD44 in SMMC-7721 cells, meanwhile, upregulated expression of FAP-α in LX-2 cells. ELISA indicated that the concentrations of IL-6 and HGF were significantly elevated in Co-CM than that of condition media from co-cultured SMMC-7721 cells/LX-2 cells. Treatment of BrMC and chrysin with co-cultures of SMMC-7721- and MHCC97H-derived LCSLCs and LX-2 cells effectively inhibited the above responses. Moreover, addition of IL-6 and/or HGF induced stemness of SMMC-7721 cells and activation of LX-2 cells, conversely, deletion of IL-6 and/or HGF suppressed those. Furthermore, the inhibitory effects of BrMC and chrysin on stemness of SMMC-7721 cells and activation of LX-2 cells were attenuated by addition of IL-6 or HGF, and enhanced by deletion of IL-6 or HGF. CONCLUSIONS: Our results suggest IL-6 and HGF may be the key communication molecules for the interaction between LCSLCs and HSCs, and BrMC and chrysin could block these effects and be the novel therapeutic candidates for HCC management.

CK2 acts as a potent negative regulator of receptor-mediated insulin release in vitro and in vivo.

G protein-coupled receptors (GPCRs) regulate virtually all physiological functions including the release of insulin from pancreatic ß-cells. ß-Cell M3 muscarinic receptors (M3Rs) are known to play an essential role in facilitating insulin release and maintaining proper whole-body glucose homeostasis. As is the case with other GPCRs, M3R activity is regulated by phosphorylation by various kinases, including GPCR kinases and casein kinase 2 (CK2). At present, it remains unknown which of these various kinases are physiologically relevant for the regulation of ß-cell activity. In the present study, we demonstrate that inhibition of CK2 in pancreatic ß-cells, knockdown of CK2α expression, or genetic deletion of CK2α in ß-cells of mutant mice selectively augmented M3R-stimulated insulin release in vitro and in vivo. In vitro studies showed that this effect was associated with an M3R-mediated increase in intracellular calcium levels. Treatment of mouse pancreatic islets with CX4945, a highly selective CK2 inhibitor, greatly reduced agonist-induced phosphorylation of ß-cell M3Rs, indicative of CK2-mediated M3R phosphorylation. We also showed that inhibition of CK2 greatly enhanced M3R-stimulated insulin secretion in human islets. Finally, CX4945 treatment protected mice against diet-induced hyperglycemia and glucose intolerance in an M3R-dependent fashion. Our data demonstrate, for the first time to our knowledge, the physiological relevance of CK2 phosphorylation of a GPCR and suggest the novel concept that kinases acting on ß-cell GPCRs may represent novel therapeutic targets.

8-Bromo-7-methoxychrysin-blocked STAT3/Twist axis inhibits the stemness of cancer stem cell-like cell originated from SMMC-7721 cells.

Signal transducer and activator of transcription 3 (STAT3) is a member of the family of latent cytoplasmic transcriptional factors that could regulate cell proliferation, survival, and development. It has been reported that Twist is a target gene of STAT3, and STAT3/Twist signaling plays an important role in regulating cancer progress. Here, to explore whether 8-bromo-7-methoxychrysin (BrMC) inhibits liver cancer stem-like cell (LCSLC) properties via disrupting STAT3/Twist signaling, we cultured SMMC-7721 cells in vitro, and evaluated the effects of BrMC on the stemness of spheroids by determining the sphere-forming capability and migration. The sphere formation assay results showed a concentration-dependent decrease of sphere-forming capacity in LCSLCs (P < 0.05) treated with different concentrations of BrMC. Wound-healing assays results demonstrated a concentration-dependent decline in cell migration of LCSLCs treated with different concentrations of BrMC. In addition, CD133, CD44, and ALDH1 levels were decreased in LCSLCs treated with BrMC. Treatment with different concentrations of BrMC also reduced the expressions of p-STAT3 and Twist1 proteins. The effect of BrMC was substantially enhanced by co-treatment with JSI-124, a specific inhibitor of STAT3. Ectopic expression of Twist1 attenuated the inhibitory effects of BrMC on sphere formation, migration, and expression of the markers in LCSLCs. However, it had no affect on p-STAT3 expression in LCSLCs. These results demonstrated that BrMC inhibits the stemness of LCSLCs originated from SMMC-7721 cell line by inhibiting STAT3/Twist signal axis.

A candidate Chinese medicine preparation-Fructus Viticis Total Flavonoids inhibits stem-like characteristics of lung cancer stem-like cells.

BACKGROUND: Cancer stem cells (CSCs) are considered as the origin of tumor relapse. Here, we investigated the effects of Fructus Viticis total flavonoids (FVTF) on the characteristics of lung cancer stem-like cells (LCSLCs) derived from human small cell lung cancer NCI-H446 cell line and its potential mechanism. METHODS: Human small cell lung cancer NCI-H446 cell line was cultured in vitro. The CD133(+) cells were sorted from NCI-H446 cell line by magnetic separation. The suspended culture with stem cell-conditioned medium was used to amplify CD133(+) sphere-forming cells (SFCs). The stem cell characteristics of CD133(+) SFCs were evaluated using cell self-renewal capacity by tumor sphere formation assay, migration and invasion capacity by Transwell assay, tumorigenicity by xenograft model in nude mouse and cancer stem cell markers expression levels by western blot. The effects of FVTF on the properties of LCSLCs were examined by tumorsphere formation assay and transwell chamber assay. The expression level of p-Akt was determined by western blot analysis. RESULT: CD133(+) SFCs derived from human small cell lung cancer NCI-H446 cells exhibited stemness properties of tumorsphere formation and tumorigenesis capacity comparing to the parental cells. FVTF relative selectively inhibited the proliferation of LCSLCs, suppressed tumor sphere forming capacity and migration and invasion of LCSLCs, and down-regulated the protein expression of stem cell markers (CD133, CD44 and ALDH1), self-renewal associated transcription factors (Bmi1, Nanog and OCT4) and invasion associated transcription factors (Twist1 and Snail1) in a dose-dependent manner. Moreover, we found that FVTF treatment could significantly decrease the phosphorylation level of Akt in LCSLCs. Meanwhile, LY294002 and FVTF synergistically inhibited the characteristics of LCSLCs. CONCLUSION: FVTF inhibits the characteristics of LCSLCs through down-regulating expression of p-Akt.

[Effects of apigenin on self-renewal and uPAR expression in NCI-H446 cell line].

OBJECTIVE: To investigate the effect of apigenin on self-renewal for sphere-forming cells in human small cell lung cancer cell line NCI-H446 and the underlying mechanisms.
 Methods: Sphere-forming cells from NCI-H446 cell line were cultured in stem cell-conditioned culture medium with ultra-low attachment surface plates. The rate of sphere-forming cells in the second passage sphere-forming cells was used to evaluate the inhibitory effects of apigenin on the self-renewal for sphere-forming cells. The protein level of urokinase-type plasminogen activator receptor (uPAR) in spheroids was analyzed by Western blot.
 Results: Apigenin signifcantly inhibited the self-renewal of the second passage sphere-forming cells [0, 5.0, 10.0, 20.0 µmol/L apigenin: (18.2±1.9)%, (13.6±1.7)%, (10.6±1.6)%, (6.9±1.3)%, respectively] and down-regulated uPAR expression in a concentration-dependent manner (P<0.05).
 Conclusion: Apigenin inhibits the self-renewal capacity of sphere-forming cells in NCI-H446 cells, which may be associated with down-regulation of uPAR expression.

Enhanced proliferation and defective activation-induced cell death of CD4+ T cells in childhood asthma.

BACKGROUND: Hyperactivation of CD4+ T cells in peripheral blood and airway tissues has been suggested to play a key role in the development and maintenance of chronic inflammation in childhood asthma. However, the underlying mechanisms are not yet clear. OBJECTIVE: To investigate alterations in serum levels of T helper cell-related cytokines, mitogen-stimulated CD4+ T cell proliferation and activation-induced cell death (AICD) in childhood asthma. METHODS: 21 children with untreated asthma and 21 healthy volunteers (age and gender matched) participated in this study. Th1/Th2/Th17 cytokines in serum were analyzed by flow cytometry. CD4+ T cells were isolated from participants by using immuno-magnetic beads and were stimulated by phytohemagglutinin (PHA). Cell proliferation was evaluated with a Cell Counting Kit-8 (CCK-8). Activation induced cell death (AICD) of CD4+ T cells was also induced by PHA and apoptosis was assayed by annexin V/PI staining. Quantitative RT-PCR was carried out to analyze Fas and FasL mRNA expression. FLIPL, caspase-8 and Bcl-2 were detected by western blot analysis. RESULTS: In children with asthma, the proliferative capacity of CD4+ T cells was enhanced and AICD was inhibited significantly, while serum IL-4, IL-10 and TNF were markedly higher compared with the control group. Fas mRNA expression in the asthma group was obviously lower than that in the control group, while no change was detected in FasL mRNA expression. Western blot analysis showed that the levels of the anti-apoptotic proteins, FLIPL and Bcl-2 in CD4+ T cells of the asthma group were significantly higher than in the control group. Spearman's correlation tests showed that only IL-4 correlated positively with FLIPL and Bcl-2 expression, while IL-10 and TNF were unrelated to FLIPL and Bcl-2 expression. CONCLUSIONS: These results indicate that enhanced proliferation and defective AICD of CD4+ T cells influence the T cell-mediated inflammatory reaction in childhood asthma and that increased IL-4, FLIPL and Bcl-2 expression and decreased Fas expression jointly participate in these changes in cell proliferation and apoptosis.ression and decreased Fas expression jointly participate in these changes in cell proliferation and apoptosis.

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.

Intra-islet α-cell Gs signaling promotes glucagon release.

Glucagon, a hormone released from pancreatic α-cells, is critical for maintaining euglycemia and plays a key role in the pathophysiology of diabetes. To stimulate the development of new classes of therapeutic agents targeting glucagon release, key α-cell signaling pathways that regulate glucagon secretion need to be identified. Here, we focused on the potential importance of α-cell Gs signaling on modulating α-cell function. Studies with α-cell-specific mouse models showed that activation of α-cell Gs signaling causes a marked increase in glucagon secretion. We also found that intra-islet adenosine plays an unexpected autocrine/paracrine role in promoting glucagon release via activation of α-cell Gs-coupled A2A adenosine receptors. Studies with α-cell-specific Gαs knockout mice showed that α-cell Gs also plays an essential role in stimulating the activity of the Gcg gene, thus ensuring proper islet glucagon content. Our data suggest that α-cell enriched Gs-coupled receptors represent potential targets for modulating α-cell function for therapeutic purposes.

Spinophilin as a novel regulator of M3 muscarinic receptor-mediated insulin release in vitro and in vivo.

Spinophilin (SPL), a multidomain scaffolding protein known to modulate the activity of different G-protein-coupled receptors, regulates various central nervous system (CNS) functions. However, little is known about the role of SPL expressed in peripheral cell types including pancreatic ß cells. In this study, we examined the ability of SPL to modulate the activity of ß-cell M(3) muscarinic acetylcholine receptors (M3Rs), which play an important role in facilitating insulin release and maintaining normal blood glucose levels. We demonstrated, by using both in vitro and in vivo approaches (mouse insulinoma cells and SPL-deficient mice), that SPL is a potent negative regulator of M3R-mediated signaling and insulin release. Additional biochemical and biophysical studies, including the use of bioluminescence resonance energy transfer technology, suggested that SPL is able to recruit regulator of G-protein signaling 4 (RGS4) to the M3R signaling complex in an agonist-dependent fashion. Since RGS4 is a member of the RGS family of proteins that act to reduce the lifetime of activated G proteins, these findings support the concept that the inhibitory effects of SPL on M3R activity are mediated by RGS4. These data suggest that SPL or other G-protein-coupled receptor-associated proteins may serve as novel targets for drug therapy aimed at improving ß-cell function for the treatment of type 2 diabetes.

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.

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.

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.