Apoptosis Of Spermatogonial Stem Cells Research Articles

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.

The Effects of Pifithrin-µ on Spermatogonial Stem Cell Viability and Pluripotency

Introduction: Spermatogonial stem cells (SSCs) offer remarkable competencies for animal reproduction and overcoming human disease as a result of their differentiation capability. We evaluated the effect of small molecule pifithrin-mu (PFT-µ), a well-known inhibitor of P53 on SSC biological processes such as viability, apoptosis, and gene expression pattern. Methods: The SSCs were isolated from the testes of adult NMRI mice and then cultured in DMEM/F12 medium containing 10% FBS. Then, they were characterized by the immunocytochemistry technique by high PLZF and low c-Kit expressions. SSC colony formation assay was carried out and their viability was estimated by methylthiazolyldiphenyl-tetrazolium bromide (MTT, or 3-[4,5-dimethyl-2-thiazolyl]-2,5-diphenyl-2H-tetrazolium bromide) assay upon exposure to PFT-µ (0, 0.6, 1.2, 2.5, and 5 µm). The apoptosis percentages were also measured using FACS analysis, and finally, Oct4 and Stra8 expression at mRNA levels was assessed using real-time quantitative PCR. Results: The 0.6 and 1.2 µm PFT-µ improved the viability of SSC based on MTT assay results; however, 2.5 and 5 µm PFT-µ reduced SSC viability compared with the control group. Moreover, PFT-µ at lower concentrations enhanced the colony size of SSCs and diminished their apoptosis. As well, exposure to PFT-µ upregulated Oct4 expression while downregulating the meiotic entry marker, Stra8. Conclusion: Based on findings, optimized concentrations of PFT-µ can decrease SSC apoptosis, and conversely potentiate their pluripotency and self-renewal capacities in vitro.

Youjing granules ameliorate spermatogenesis in rats through regulating the prolifereation of spermatogonial stem cells.

Male infertility has evolved from a common reproductive system disease to a major social issue. Youjing granule (YG) is a Chinese medicinal material used as a therapy method for tonifying the kidneys and removing dampness due to its pathogenic characteristics. YG has been shown to regulate sperm quality in clinical trials, but the underlying mechanism is not fully understood. The present study was aimed to explore the protective effects and mechanism of action of YG on male reproductive system damage caused by methyl methane sulfonate (MMS). We first established an infertility model of rats through oral administration of MMS and then treated with YG. To determine the effect of YG, spermatogenesis, microvascular density, and secretory function of Leydig cells and Sertoli cells in rats were assessed. Spermatogonial stem cells (SSCs) were co-cultured with mouse embryo fibroblast (MEF) cells as an in vitro cell model before exposure to serum containing YG. Furthermore, the proliferation and apoptosis of SSCs were measured. Results indicated that YG increased the expression of self-renewal and proliferation-related molecules such as glial cell line derived neurotrophic factor (GDNF) and fibroblast growth factor-2 (FGF2), and improved the quality of sperm and the proliferation of SSCs. In conclusion, YG may protect spermatogenetic function of rats through regulating the proliferation and self-renewal of SSCs.

β-estradiol promotes the growth of primary human fetal spermatogonial stem cells via the induction of stem cell factor in Sertoli cells.

Mammalian spermatogenesis is responsible for male fertility and is supported by the self-renewal and differentiation of spermatogonial stem cells (SSCs). Sertoli cells provide a supportive microenvironment for SSCs, in part by the production of stem cell factor (SCF), which is a potent regulator of spermatogonia proliferation and survival. We investigated the novel role of β-estradiol in modulating the proliferation and apoptosis of fetal SSCs via the regulation of SCF secretion in Sertoli cells isolated from human fetal testes. The proliferation of SSCs in the co-culture system was determined by colony formation and BrdU incorporation assays. TUNEL assay was used to measure SSC apoptosis in co-culture in response to treatment with control, β-estradiol, or the combination of β-estradiol and the estrogen receptor inhibitor ICI 182780. In the system with purified human fetal Sertoli cells (MIS+/c-Kit-/AP-), β-estradiol upregulated the production of SCF in a dose- and time-dependent manner. In the co-culture system of primary human fetal SSCs (c-Kit+/SSEA-4+/Oct-4+/AP+) and Sertoli cells (MIS+), β-estradiol markedly increased the proliferation of SSCs. Moreover, SSC apoptosis was significantly inhibited by β-estradiol and was completely reversed by the combination of β-estradiol and ICI 182780. Here we report, for the first time, that β-estradiol can induce the increase of SCF expression in human fetal Sertoli cells and regulates the growth and survival of human fetal SSCs. These novel findings provide new perspectives on the current understanding of the role of estrogen in human spermatogenesis.

Melatonin Protects Goat Spermatogonial Stem Cells against Oxidative Damage during Cryopreservation by Improving Antioxidant Capacity and Inhibiting Mitochondrial Apoptosis Pathway

Spermatogonial stem cells (SSCs) are the only adult stem cells that pass genes to the next generation and can be used in assisted reproductive technology and stem cell therapy. SSC cryopreservation is an important method for the preservation of immature male fertility. However, freezing increases the production of intracellular reactive oxygen species (ROS) and causes oxidative damage to SSCs. The aim of this study was to investigate the effect of melatonin on goat SSCs during cryopreservation and to explore its protective mechanism. We obtained SSCs from dairy goat testes by two-step enzymatic digestion and differential plating. The SSCs were cryopreserved with freezing media containing different melatonin concentrations. The results showed that 10−6 M of melatonin increased significantly the viability, total antioxidant capacity (T-AOC), and mitochondrial membrane potential of frozen-thawed SSCs, while it reduced significantly the ROS level and malondialdehyde (MDA) content (P < 0.05). Further analysis was performed by western blotting, flow cytometry, and transmission electron microscopy (TEM). Melatonin improved significantly the enzyme activity and protein expression of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px) (P < 0.05), thereby activating the antioxidant defense system of SSCs. Furthermore, melatonin inhibited significantly the expression of proapoptotic protein (Bax) and increased the expression of antiapoptotic proteins (Bcl-2 and Bcl-XL) (P < 0.05). The mitochondrial apoptosis pathway analysis showed that the addition of melatonin reduced significantly the mitochondrial swelling and vacuolation, and inhibited the release of cytochrome C from mitochondria into the cytoplasm, thereby preventing the activation of caspase-3 (P < 0.05) and inhibiting SSC apoptosis. In addition, melatonin reduced significantly the autophagosome formation and regulated the expression of autophagy-related proteins (LC3-I, LC3-II, P62, Beclin1, and ATG7) (P < 0.05), thereby reversing the freeze-induced excessive autophagy. In summary, melatonin protected goat SSCs during cryopreservation via antioxidant, antiapoptotic, and autophagic regulation.

The Histone Methyltransferase SETDB1 Modulates Survival of Spermatogonial Stem/Progenitor Cells Through NADPH Oxidase.

SETDB1, a histone H3 lysine 9 (H3K9) methyltransferase, is crucial in meiosis and embryo development. This study aimed to investigate whether SETDB1 was associated with spermatogonial stem cells (SSC) homeostasis. We found that knockdown of Setdb1 impaired cell proliferation, led to an increase in reactive oxygen species (ROS) level through NADPH oxidase, and Setdb1 deficiency activated ROS downstream signaling pathways, including JNK and p38 MAPK, which possibly contributed to SSC apoptosis. Melatonin scavenged ROS and rescued the phenotype of Setdb1 KD. In addition, we demonstrated that SETDB1 regulated NADPH oxidase 4 (Nox4) and E2F1. Therefore, this study uncovers the new roles of SETDB1 in mediating intracellular ROS homeostasis for the survival of SSC.

Cytotoxicity of nonylphenol on spermatogonial stem cells via phosphatidylinositol-3-kinase/protein kinase B/mammalian target of rapamycin pathway.

With continuous advancement of industrial society, environmental pollution has become more and more serious. There has been an increase in infertility caused by environmental factors. Nonylphenol (NP) is a stable degradation product widely used in daily life and production and has been proven to affect male fertility. However, the underlying mechanisms therein are unclear. Thus, it is necessary to study the effect and mechanism of NP on spermatogonial stem cells (SSCs). To investigate the cytotoxic effect of NP on SSCs via the phosphatidylinositol-3-kinase/protein kinase B/mammalian target of rapamycin (PI3K/AKT/mTOR) pathway. SSCs were treated with NP at 0, 10, 20 or 30 µmol. MTT assay was performed to evaluate the effect of NP on the proliferation of SSCs. Flow cytometry was conducted to measure SSC apoptosis. The expression of Bad, Bcl-2, cytochrome-c, pro-Caspase 9, SOX-2, OCT-4, Nanog, Nanos3, Stra8, Scp3, GFRα1, CD90, VASA, Nanos2, KIT, PLZF and PI3K/AKT/mTOR-related proteins was observed by western blot, and the mRNA expression of SOX-2, OCT-4 and Nanog was detected by quantitative reverse transcription polymerase chain reaction. Compared with untreated cells (0 μmol NP), SSCs treated with NP at all concentrations showed a decrease in cell proliferation and expression of Bcl-2, Nanog, OCT-4, SOX-2, Nanos3, Stra8, Scp3, GFRα1, CD90, VASA, Nanos2, KIT, and PLZF (P < 0.05), whereas the expression of Bad, cytochrome-c, and pro-Caspase 9 increased significantly (P < 0.05). We further examined the PI3K/AKT/mTOR pathway and found that the phosphorylation of PI3K, AKT, mTORC1, and S6K was significantly decreased by NP at all concentrations compared to that in untreated SSCs (P < 0.05). NP exerted the greatest effect at 30 μmol among all NP concentrations. NP attenuated the proliferation, differentiation and stemness maintenance of SSCs while promoting apoptosis and oxidative stress. The associated mechanism may be related to the PI3K/AKT/mTOR pathway.

Molecular characterization of AwSox2 from bivalve Anodonta woodiana: Elucidating its player in the immune response.

Sox2 is an embryonal stem cell Ag essential for early embryonic development, tissue homeostasis and immune regulation. In the current study, one complete Sox2 cDNA sequence was cloned from freshwater bivalve Anodonta woodiana and named AwSox2. Histological changes of testis derived from Bisphenol A (BPA) treatment were analyzed by hematoxylin and eosin staining. Expressions of AwSox2 derived from BPA, LPS and polyinosinic:polycytidylic (Poly I:C) challenge were measured by quantitative real-time PCR. The full-length cDNA of AwSox2 contained an open reading frame of 927 nucleotides bearing the typical structural features of Sox2 family. Obvious degeneration, irregular arrangement of spermatids, and clotted dead and intertwined spermatids were observed in BPA-treated groups. Administration of BPA could result in a dose-dependent up-regulation of AwSox2 expression in the male gonadal tissue of A. woodiana. In addition, expression of AwSox2 was significantly induced by LPS and Poly I:C treatment in the hepatopancreas, gill and hemocytes, compared with that of control group. These results indicated that up-regulations of AwSOx2 are closely related to apoptosis of spermatogonial stem cells derived from BPA treatment as well as enhancement of immune defense against LPS and Poly I:C challenge in A. woodiana.

Histone methyltransferase SETDB1 maintains survival of mouse spermatogonial stem/progenitor cells via PTEN/AKT/FOXO1 pathway

Spermatogonial stem cells (SSCs) possess the capacity of self-renewal and differentiation, which are the basis of spermatogenesis. In maintenance of SSC homeostasis, intrinsic/extrinsic factors and various signaling pathways tightly control the fate of SSCs. Methyltransferase SETDB1 (Set domain, bifurcated 1) catalyzes histone H3 lysine 9 (H3K9) trimethylation and represses gene expression. SETDB1 is required for maintaining the survival of spermatogonial stem cells in mice. However, the underlying molecular mechanism remains unclear. In the present study, we found that Setdb1 regulates PTEN/AKT/FOXO1 pathway to inhibit SSC apoptosis. Co-immunoprecipitation and reporter gene assay revealed that SETDB1 interacted and coordinated with AKT to regulate FOXO1 activity and expression of the downstream target genes Bim and Puma. Among the SETDB1-bound genes, the H3K9me3 levels on the promoter regions of Bim and Pten decreased in Setdb1-KD group; in contrast, H3K9me3 status on promoters of Bax and Puma remained unchanged. Therefore, SETDB1 was responsible for regulating the transcription activity of genes in the apoptotic pathway at least in part through modulating H3K9me3. This study replenishes the research on the epigenetic regulation of SSC survival, and provides a new insight for the future study of epigenetic regulation of spermatogenesis.

Bisphenol A induced apoptosis and transcriptome differences of spermatogonial stem cells in vitro.

Bisphenol A (BPA) is widely used as an industrial plasticizer, which is also an endocrine disruptor and considered to have adverse effects on reproduction. In male mammals, the long-term production of billions of spermatozoa relies on the regulated proliferation and differentiation of spermatogonial stem cells (SSCs). However, little is known about the effects of BPA on the viability of SSCs. To investigate the influence of BPA exposure on SSCs in vitro, we isolated SSCs from mouse and successfully established in vitro propagation of SSCs. After BPA treatment, we found that BPA reduced the viability of SSCs and induced SSC apoptosis. For revealing the transcriptome differences of the BPA-treated SSCs, we performed high-throughput RNA sequencing and found that 860 genes were differentially expressed among 18,272 observed genes. The gene ontology (GO) terms, regulation of programmed cell death and apoptotic process, were enriched in the differentially expressed genes (DEGs). Among the cluster of DEGs associated with the kyoto encyclopedia of genes and genomes (KEGG) apoptosis pathway, activating transcription factor 4 (Atf4) and DNA damage inducible transcript 3 (Ddit3) genes were significantly up-regulated in BPA-treated SSCs, which were proved by qPCR. Taken together, these findings suggest that BPA can increase the mRNA expression of pro-apoptosis genes and reduce the viability of SSCs by inducing apoptosis.

MicroRNA-10b regulates the renewal of spermatogonial stem cells through Kruppel-like factor 4.

MicroRNAs (miRs) are functionally important in spermatogenesis, which is the self-renewal or differentiation of spermatogonial stem cells (SSCs). Here, we report a novel role for miR-10b in regulating the self-renewal of mouse SSCs. We showed that miR-10b was highly expressed in mouse SSCs in vitro and enhanced SSC proliferation. Knockdown of miR-10b significantly increased the apoptosis of SSCs compared with controls. Kruppel-like factor 4 was found to be a target gene of miR-10b in the enhancement of SSC proliferation. These findings further our understanding of the self-renewal and differentiation of SSCs and provide a basis for the diagnosis, treatment, and prevention of male infertility.

Diethylstilbestrol induces oxidative DNA damage, resulting in apoptosis of spermatogonial stem cells in vitro.

The spermatogonial stem cells (SSCs) are the only germline stem cells in adults that are responsible for the transmission of genetic information from mammals to the next generation. SSCs play a very important role in the maintenance of progression of spermatogenesis and help provide an understanding of the reproductive biology of future gametes and a strategy for diagnosis and treatment of infertility and male reproductive toxicity. Androgens/oestrogens are very important for the suitable maintenance of male germ cells. There is also evidence confirming the damaging effects of oestrogen-like compounds on male reproductive health. We investigated the effects in vitro, of diethylstilbestrol (DES) on mouse spermatogonial stem cells separated using Staput unit-gravity velocity sedimentation, evaluating any DNA damage using the Comet assay and apoptotic cells in the TUNEL assay. Immunocytochemistry assays showed that the purity of isolated mouse spermatogonial cells was 90%, and the viability of these isolated cells was over 96%. Intracellular superoxide anion production (O2-) in SSCs was detected using p-Nitro Blue Tetrazolium (NBT) assay. The viability of cells after DES treatment was examined in the CCK8 (cell counting kit-8) cytotoxicity assay. The results showed that DES-induced DNA damage causes an increase in intracellular superoxide anions which are reduced by the flavonoid, quercetin. Investigating the molecular mechanisms and biology of SSCs provides a better understanding of spermatogonial stem cell regulation in the testis.

Melatonin Ameliorates Busulfan-Induced Spermatogonial Stem Cell Oxidative Apoptosis in Mouse Testes.

Many men endure immunosuppressive or anticancer treatments that contain alkylating agents before the age of sexual maturity, especially the increasing number of preadolescent males who undergo busulfan treatment for myeloablative conditioning before hematopoietic stem cell transplantation. Before sperm production, there are no sperm available for cryopreservation. Thus, it is necessary to identify a solution to ameliorate the busulfan-induced damage of spermatogonial stem cells (SSCs). In this study, we demonstrated that melatonin relieved the previously described SSC loss and apoptosis in mouse testes. Melatonin increased the expression of manganese superoxide dismutase (MnSOD), which regulated the production of busulfan-induced reactive oxygen species (ROS). Moreover, melatonin promoted sirtuin type 1 (SIRT1) expression. SIRT1 participated in the deacetylation of p53, which promotes p53 ubiquitin degradation. Decreased concentrations of deacetylated p53 resulted in spermatogonial cell resistance to apoptosis. Acute T cell leukemia cell assay demonstrated that melatonin does not affect busulfan-induced cancer cell apoptosis and ROS. The current evidence suggests that melatonin may alleviate the side effects of alkylating drugs, such as busulfan. Melatonin promoted MnSOD and SIRT1 expression, which successfully ameliorated busulfan-induced SSC apoptosis caused by high concentrations of ROS and p53. Antioxid. Redox Signal. 28, 385-400.

Identification of CHD1L as an Important Regulator for Spermatogonial Stem Cell Survival and Self‐Renewal

Chromodomain helicase/ATPase DNA binding protein 1-like gene (Chd1l) participates in chromatin-dependent processes, including transcriptional activation and DNA repair. In this study, we have found for the first time that Chd1l is mainly expressed in the testicular tissues of prepubertal and adult mice and colocalized with PLZF, OCT4, and GFRα1 in the neonatal mouse testis and THY1+ undifferentiated spermatogonia or spermatogonial stem cells (SSCs). Knockdown of endogenous Chd1l in cultured mouse undifferentiated SSCs inhibited the expression levels of Oct4, Plzf, Gfrα1, and Pcna genes, suppressed SSC colony formation, and reduced BrdU incorporation, while increasing SSC apoptosis. Moreover, the Chd1l gene expression is activated by GDNF in the cultured mouse SSCs, and the GDNF signaling pathway was modulated by endogenous levels of Chd1l; as demonstrated by the gene expression levels of GDNF, inducible transcripts Etv5, Bcl6b, Pou3f, and Lhx1, but not that of GDNF-independent gene, Taf4b, were significantly downregulated by Chd1l knockdown in mouse SSCs. Taken together, this study provides the first evidence to support the notion that Chd1l is an intrinsic and novel regulator for SSC survival and self-renewal, and it exerts such regulation at least partially through a GDNF signaling pathway.

Activation of Toll like receptor 3 induces spermatogonial stem cell apoptosis.

Germ cell apoptosis may be associated with the male infertility. The pathogenesis is to be further understood. Viral infection is one of the causative factors of apoptosis of the body cells. This study aims to investigate the role of activation of Toll like receptor (TLR)3 in the induction of germ cell apoptosis. In this study, spermatogonial stem cells (SSC) were isolated from C57BL/6 mouse testes. The expression of TLR3 on SSC was by RT-qPCR and Western blotting. polyinosinic-polycytidylic acid (Poly I:C) was employed to activate TLR3 on SSCs. The results showed that re-activation by PolyI:C induced SSC apoptosis. Exposure to PolyI:C induced interferon regulatory factor 3 (IRF3) phosphorylation in SSCs. TLR3 and IRF3 formed a complex in the SSCs. The complex of TLR3/IRF3 bound to the promoter of Fas ligand and promoted Fas ligand expression in SSC, and thus induced SSC apoptosis. In conclusion, the results of the present study indicate that activation of TLR3 by PolyI:C induces the SSC apoptosis, which implies that viral infection may interfere with the male germ cell development.

The Trp53-Trp53inp1-Tnfrsf10b Pathway Regulates the Radiation Response of Mouse Spermatogonial Stem Cells

SummaryGerm cells are thought to exhibit a unique DNA damage response that differs from that of somatic stem cells, and previous studies suggested that Trp53 is not involved in the survival of spermatogonial stem cells (SSCs) after irradiation. Here, we report a critical role for the Trp53-Trp53inp1-Tnfrsf10b pathway during radiation-induced SSC apoptosis. Spermatogonial transplantation revealed that Trp53 deficiency increased the survival of SSCs after irradiation. Although Bbc3, a member of the intrinsic apoptotic pathway, was implicated in apoptosis of germ and somatic stem cells, Bbc3 depletion inhibited apoptosis in committed spermatogonia, but not in SSCs. In contrast, inhibition of Tnfrsf10b, an extrinsic apoptosis regulator, rescued SSCs. Tnfrsf10b, whose deficiency protected SSCs, was upregulated by Trp53inp1 upon irradiation. These results suggest that the Trp53-Trp53inp1-Tnfrsf10b pathway responds to genotoxic damage in SSCs and that stem and progenitor cells exhibit distinct DNA damage responses in self-renewing tissue.

Inhibitory effects of E2F transcription factor 1 small interfering RNA on apoptosis of mouse spermatogonial stem cells

Objective To observe inhibitory effects of E2F transcription factor 1 (E2F-1) small interfering RNA (siRNA) on apoptosis of mouse spermatogonial stem cells (SSCs).Methods Percoll density gradient centrifugation and flow cytometry was used to isolate,enrich and purify SSCs.Real-time reverse transcriptase-polymerase chain reaction (RT-qPCR) and Western blotting were carried out to investigate the expression of E2F-1,and flow cytometry was ued to analyze the apoptosis rate.Results Compared to non-transfected group and control group,the expression of E2F-1 in experimental group was remarkably down-regulated at mRNA [2-(△△ct),0.32 vs.0.10 and 1.04,P ＜ 0.05] and protein levels (P ＜0.05).Tthe apoptosis rate in experimental group was reduced remarkably (7.69％ vs 23.07％ and 24.19％ ;P＜0.05).Conclusion E2F-1 siRNA could remarkably suppress the apoptosis of SSCs in vitro. Key words: RNA interference; Spermatogonial stem cells; E2F transcription factor 1 ; Apoptosis

The histone methyltransferase ESET is required for the survival of spermatogonial stem/progenitor cells in mice.

Self-renewal and differentiation of spermatogonial stem cells (SSCs) are the foundation of spermatogenesis throughout a male's life. SSC transplantation will be a valuable solution for young male patients to preserve their fertility. As SSCs in the collected testis tissue from the patients are very limited, it is necessary to expansion the SSCs in vitro. Previous studies suggested that histone methyltransferase ERG-associated protein with SET domain (ESET) represses gene expression and is essential for the maintenance of the pool of embryonic stem cells and neurons. The objective of this study was to determine the role of ESET in SSCs using in vitrocell culture and germ cell transplantation. Cell transplantation assay showed that knockdown of ESET reduced the number of seminiferous tubules with spermatogenesis when compared with that of the control. Knockdown of ESET also upregulated the expression of apoptosis-associated genes (such as P53, Caspase9, Apaf1), whereas inhibited the expression of apoptosis-suppressing genes (such as Bcl2l1, X-linked inhibitor of apoptosis protein). In addition, suppression of ESET led to increase in expression of Caspase9 and activation of Caspase3 (P17) as well as cleavage of poly (ADP-ribose) polymerase. Among the five ESET-targeting genes (Cox4i2, spermatogenesis and oogenesis Specific Basic Helix-Loop-Helix 2, Nobox, Foxn1 and Dazl) examined by ChIP assay, Cox4i2 was found to regulate SSC apoptosis by the rescue experiment. BSP analyses further showed that DNA methylation in the promoter loci of Cox4i2was influenced by ESET, indicating that ESET also regulated gene expression through DNA methylation in addition to histone methylation. In conclusion, we found that ESET regulated SSC apoptosis by suppressing of Cox4i2 expression through histone H3 lysine 9 tri-methylation and DNA methylation. The results obtained will provide unique insights that would broaden the research on SSC biology and contribute to the treatment of male infertility.

Mechanism of glial cell line-derived neurotrophic factor in proliferation of spermatogonial stem cells

Objectives: The aim was to investigate the mechanism of glial cell line-derived neurotrophic factor (GDNF) in the proliferation of spermatogonial stem cells (SSCs) through detecting the expression of GDNF, receptor tyrosine kinases (RTKs), fyn and focal adhesion kinase (FAK) messenger ribonucleic acids (mRNAs) after gene interference. Materials and Methods: Multiple small interfering RNAs (siRNA) of GDNF were designed and constructed to transfect SSCs. The siRNA of GDNF with the highest efficiency was applied and expressions of GDNF, RTKs, fyn and FAK mRNA and the protein were measured with reverse transcription polymerase chain reaction and western blot, respectively. The proliferation and apoptosis of SSCs were measured with enzyme-labeled meter and flow cytometry, respectively. Results: The expression intensity of GDNF mRNA in SSCs of the experimental group and control group was 12.32 ± 1.22% and 54.25 ± 1.34%, respectively; the apoptosis rate was 25.43 ± 1.91% and 5.61 ± 0.16%, respectively; and there was a significant difference between the two groups (P < 0.01). The expression intensity of RTKs, fyn and FAK in the experimental group was 16.24 ± 1.35%, 18.32 ± 1.34%, and 20.04 ± 1.65%, respectively; the expression intensity of RTKs, fyn and FAK in the control group was 45.35 ± 1.37%, 38.37 ± 1.55% and 43.27 ± 1.28%, respectively; there was a significant difference between the two groups (P < 0.01). Conclusion: These results suggest that the constructed positive vector effectively inhibits the expression of GDNF, RTKs, fyn and FAK mRNAs, and the proliferation of SSCs. GDNF plays an important role in the proliferation of SSCs.