Mouse Spermatogonial Cells Research Articles

TRIM59 is required for mouse GC-1 cell maintenance through modulating the ubiquitination of AXIN1

Tripartite motif-containing protein 59 (TRIM59) is a biomarker for multiple tumors with crucial roles. However, the specific role of TRIM59 in germ cells remains largely unknown. Here, we investigated the effects and underlying regulatory mechanisms of TRIM59 on germ cells using the mouse spermatogonial cell line GC-1. Our results demonstrated that TRIM59 promoted proliferation and inhibited apoptosis of GC-1 cells. Mechanistically, TRIM59 maintained GC-1 cell behaviors through ubiquitination of AXIN1 to activate β-catenin signaling. Furthermore, activation of β-catenin signaling reversed the effects mediated by Trim59 knockdown in GC-1 cells. Collectively, our study revealed a major role and regulatory mechanism of TRIM59 in GC-1 cells, which sheds new light on the molecular pathogenesis of defects in spermatogenesis and may provide therapeutic targets for treatment of male infertility.

In vitro proliferation and differentiation of mouse spermatogonial stem cells in decellularized human placenta matrix.

Utilizing natural scaffold production derived from extracellular matrix components presents a promising strategy for advancing in vitro spermatogenesis. In this study, we employed decellularized human placental tissue as a scaffold, upon which neonatal mouse spermatogonial cells (SCs) were cultured three-dimensional (3D) configuration. To assess cellular proliferation, we examined the expression of key markers (Id4 and Gfrα1) at both 1 and 14 days into the culture. Our quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analysis revealed a notable increase in Gfrα1 gene expression, with the 3D culture group exhibiting the highest levels. Furthermore, the relative frequency of Gfrα1-positive cells significantly rose from 38.1% in isolated SCs to 46.13% and 76.93% in the two-dimensional (2D) and 3D culture systems, respectively. Moving forward to days 14 and 35 of the culture period, we evaluated the expression of differentiating markers (Sycp3, acrosin, and Protamine 1). Sycp3 and Prm1 gene expression levels were upregulated in both 2D and 3D cultures, with the 3D group displaying the highest expression. Additionally, acrosin gene expression increased notably within the 3D culture. Notably, at the 35-day mark, the percentage of Prm1-positive cells in the 3D group (36.4%) significantly surpassed that in the 2D group (10.96%). This study suggests that the utilization of placental scaffolds holds significant promise as a bio-scaffold for enhancing mouse in vitro spermatogenesis.

X-linked RBBP7 mutation causes maturation arrest and testicular tumors.

Maturation arrest (MA) is a subtype of non-obstructive azoospermia, and male infertility is a known risk factor for testicular tumors. However, the genetic basis for many affected individuals remains unknown. Here, we identified a deleterious hemizygous variant of X-linked retinoblastoma-binding protein 7 (RBBP7) as a potential key cause of MA, which was also found to be associated with the development of Leydig cell tumors. This mutation resulted in premature protein translation termination, affecting the sixth WD40 domain of the RBBP7 and the interaction of the mutated RBBP7 with histone H4. Decreased BRCA1 and increased γH2AX were observed in the proband. In mouse spermatogonial and pachytene spermatocyte-derived cells, deprivation of rbbp7 led to cell cycle arrest and apoptosis. In Drosophila, knockdown of RBBP7/Caf1-55 in germ cells resulted in complete absence of germ cells and reduced testis size, whereas knockdown of RBBP7/Caf1-55 in cyst cells resulted in hyperproliferative testicular cells. Interestingly, male infertility caused by Caf1-55 deficiency was rescued by ectopic expression of wild-type human RBBP7 but not mutant variants, suggesting the importance of RBBP7 in spermatogenesis. Our study provides insights into the mechanisms underlying the co-occurrence of MA and testicular tumors and may pave the way for innovative genetic diagnostics of these 2 diseases.

YTHDF2 promotes DNA damage repair by positively regulating the histone methyltransferase SETDB1 in spermatogonia†.

Genomic integrity is critical for sexual reproduction, ensuring correct transmission of parental genetic information to the descendant. To preserve genomic integrity, germ cells have evolved multiple DNA repair mechanisms, together termed as DNA damage response. The RNA N6-methyladenosine is the most abundant mRNA modification in eukaryotic cells, which plays important roles in DNA damage response, and YTH N6-methyladenosine RNA binding protein 2 (YTHDF2) is a well-acknowledged N6-methyladenosine reader protein regulating the mRNA decay and stress response. Despite this, the correlation between YTHDF2 and DNA damage response in germ cells, if any, remains enigmatic. Here, by employing a Ythdf2-conditional knockout mouse model as well as a Ythdf2-null GC-1 mouse spermatogonial cell line, we explored the role and the underlying mechanism for YTHDF2 in spermatogonial DNA damage response. We identified that, despite no evident testicular morphological abnormalities under the normal circumstance, conditional mutation of Ythdf2 in adult male mice sensitized germ cells, including spermatogonia, to etoposide-induced DNA damage. Consistently, Ythdf2-KO GC-1 cells displayed increased sensitivity and apoptosis in response to DNA damage, accompanied by the decreased SET domain bifurcated 1 (SETDB1, a histone methyltransferase) and H3K9me3 levels. The Setdb1 knockdown in GC-1 cells generated a similar phenotype, but its overexpression in Ythdf2-null GC-1 cells alleviated the sensitivity and apoptosis in response to DNA damage. Taken together, these results demonstrate that the N6-methyladenosine reader YTHDF2 promotes DNA damage repair by positively regulating the histone methyltransferase SETDB1 in spermatogonia, which provides novel insights into the mechanisms underlying spermatogonial genome integrity maintenance and therefore contributes to safe reproduction.

ScRNA-seq reveals that origin recognition complex subunit 6 regulates mouse spermatogonial cell proliferation and apoptosis via activation of Wnt/β-catenin signaling.

The regulation of spermatogonial proliferation and apoptosis is of great significance for maintaining spermatogenesis. The single-cell RNA sequencing (scRNA-seq) analysis of the testis was performed to identify genes upregulated in spermatogonia. Using scRNA-seq analysis, we identified the spermatogonia upregulated gene origin recognition complex subunit 6 ( Orc6 ), which is involved in DNA replication and cell cycle regulation; its protein expression in the human and mouse testis was detected by western blot and immunofluorescence. To explore the potential function of Orc6 in spermatogonia, the C18-4 cell line was transfected with control or Orc6 siRNA. Subsequently, 5-ethynyl-2-deoxyuridine (EdU) and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assays, flow cytometry, and western blot were used to evaluate its effects on proliferation and apoptosis. It was revealed that ORC6 could promote proliferation and inhibit apoptosis of C18-4 cells. Bulk RNA sequencing and bioinformatics analysis indicated that Orc6 was involved in the activation of wingless/integrated (Wnt)/β-catenin signaling. Western blot revealed that the expression of β-catenin protein and its phosphorylation (Ser675) were significantly decreased when silencing the expression of ORC6. Our findings indicated that Orc6 was upregulated in spermatogonia, whereby it regulated proliferation and apoptosis by activating Wnt/β-catenin signaling.

An N-terminal peptide of Tar DNA binding Protein 43 lacking nuclear localization signal translocates to the nucleus of GC-1 spermatogonial cells

Objectives: TAR DNA-binding protein of 43 kDa (TDP-43) is an RNA/DNA binding protein expressed in the brain and the testis. Mutations in TDP-43 lead to mislocalization and cytoplasmic aggregation of this protein causing neurodegenerative diseases such as amyotrophic lateral sclerosis and frontotemporal dementia. TDP-43 has also been implicated in maintaining spermatogenesis. While homodimerization of TDP-43 is critical for its physiological functions, higher-order aggregation of this protein impairs its functions. This study was aimed to map the critical amino acids of the N-terminus of this protein in mediating its homodimerization. Materials and Methods: We generated deletion constructs of Tdp-43 containing NRRM1 domain alone (TDP-43∆3-183) and N-terminal peptide of TDP-43 which lacks the nuclear localization signal (NLS) (TDP-43∆1-50) with fluorescent reporters having non-overlapping emission properties. These constructs were co-transfected into a mouse spermatogonial cell line to examine their dimerization and nuclear translocation capabilities in vitro. Results: We found that TDP-43∆3-183 alone was not capable of homodimerization. On the other hand, TDP-43∆1-50 when co-transfected into GC1-spg cells along with full length TDP-43 translocated to the nucleus oligomerized with the latter and translocated to the nucleus, indicating the importance of amino acids 1-50 of TDP-43 in dimerization. Conclusion: The N-terminal segment of TDP-43 spanning amino acids 1-50 is responsible for dimerization, while that spanning amino acids 51-183 directs it to the nucleus.The physiological and pathological implications of this finding need to be examined.

E-cadherin maintains the undifferentiated state of mouse spermatogonial progenitor cells via β-catenin

BackgroundCadherins play a pivotal role in facilitating intercellular interactions between spermatogonial progenitor cells (SPCs) and their surrounding microenvironment. Specifically, E-cadherin serves as a cellular marker of SPCs in many species. Depletion of E-cadherin in mouse SPCs showed no obvious effect on SPCs homing and spermatogenesis.ResultsHere, we investigated the regulatory role of E-cadherin in regulating SPCs fate. Specific deletion of E-cadherin in germ cells was shown to promote SPCs differentiation, evidencing by reduced PLZF+ population and increased c-Kit+ population in mouse testes. E-cadherin loss down-regulated the expression level of β-catenin, leading to the reduced β-catenin in nuclear localization for transcriptional activity. Remarkably, increasing expression level of Cadherin-22 (CDH22) appeared specifically after E-cadherin deletion, indicating CDH22 played a synergistic effect with E-cadherin in SPCs. By searching for the binding partners of β-catenin, Lymphoid enhancer-binding factor 1 (LEF1), T-cell factor (TCF3), histone deacetylase 4 (HDAC4) and signal transducer and activator 3 (STAT3) were identified as suppressors of SPCs differentiation by regulating acetylation of differentiation genes with PLZF.ConclusionsTwo surface markers of SPCs, E-cadherin and Cadherin-22, synergically maintain the undifferentiation of SPCs via the pivotal intermediate molecule β-catenin. LEF1, TCF3, STAT3 and HDAC4 were identified as co-regulatory factors of β-catenin in regulation of SPC fate. These observations revealed a novel regulatory pattern of cadherins on SPCs fate.

Bisphenol A exposure causes testicular toxicity by targeting DPY30-mediated post-translational modification of PI3K/AKT signaling in mice

Bisphenol A (BPA), one of the chemicals with the highest volume of production worldwide, has been demonstrated to cause testicular toxicity via different pathways. However, there is little evidence concerning the mechanism of BPA exposure induced histone modification alterations, especially regarding the effect on the histone H3 lysine 4 (H3K4) epigenetic modification. Our results demonstrated a new epigenetic regulation of BPA exposure on testicular damage using both cell culture and mouse models. With BPA treatment, disordered and shrunken seminiferous tubules and poor sperm quality were observed in vivo, and mouse spermatogonial germ cell proliferation was inhibited in vitro. BPA attenuated PI3K expression inducing phospho-AKT inhibition in vivo and in vitro. DPY30 was the only downregulated subunit in BPA and MEK2206 (AKT inhibitor) treated cells, which contributed to reducing H3K4me3 recruitment at the PIK3CA transcriptional start site (TSS) in BPA treated cells. The toxicity caused by BPA exposure was relieved after the transduction of adenoviruses expressing DPY30 transgenes, which resulted in the stimulation of PI3K/AKT with H3K4me3 enriched at the PI3KCA TSS. DPY30 promoted cell glycolysis via AMPK and proliferation through AKT/P21. DPY30 was mainly located in the round and elongated spermatids for energy accumulation in mature sperm in AD-DPY30-treated mice which showed higher sperm quality. Overall, our results indicated that BPA exposure causes testicular toxicity through a DPY30-mediated H3K4me3 epigenetic modification, which serves to regulate the PI3K/AKT/P21 pathway.

The Effects of Organophosphate Esters Used as Flame Retardants and Plasticizers on Granulosa, Leydig, and Spermatogonial Cells Analyzed Using High-Content Imaging.

The replacement of regulated brominated flame retardants and plasticizers with organophosphate esters (OPEs) has led to their pervasive presence in the environment and in biological matrices. Further, there is evidence that exposure to some of these chemicals is associated with reproductive toxicity. Using a high-content imaging approach, we assessed the effects of exposure to 9 OPEs on cells related to reproductive function: KGN human granulosa cells, MA-10 mouse Leydig cells, and C18-4 mouse spermatogonial cells. The effects of OPEs were compared with those of 2,2',4,4'-tetrabromodiphenyl ether (BDE-47), a legacy brominated flame retardant. Alterations in several important cell features, including cell survival, mitochondrial dynamics, oxidative stress, lysosomes, and lipid droplets, were analyzed. Most of the OPEs tested displayed higher cytotoxicity than BDE-47 in all 3 cell lines. Effects on phenotypic parameters were specific for each cell type. Several OPEs increased total mitochondria, decreased lysosomes, increased the total area of lipid droplets, and induced oxidative stress in KGN cells; these endpoints were differentially affected in MA-10 and C18-4 cells. Alterations in cell phenotypes were highly correlated in the 2 steroidogenic cell lines for a few triaryl OPEs. Potency ranking using 2 complementary approaches, Toxicological Prioritization Index analyses and the lowest benchmark concentration/administered equivalent dose method, revealed that while most of the OPEs tested were more potent than BDE-47, others showed little to no effect. We propose that these approaches serve as lines of evidence in a screening strategy to identify the potential for reproductive and endocrine effects of emerging chemicals and assist in regulatory decision-making.

Novel expression of zona pellucida 3 protein in normal testis; potential functional implications

The expression of the zona pellucida glycoprotein 3 (ZP3), originally thought to be specific for oocytes, was recently extended to ovarian, prostate, colorectal and lung cancers. Earlier successful ZP3 immunization of a transgenic mouse model carrying a ZP3 positive ovarian tumor emphasized the suitability of ZP3 for cancer immunotherapy. This study was carried out to determine whether any other normal tissues besides the ovary in healthy human and mouse tissues may express ZP3, considered important to exclude off-target effects of ZP3 cancer immunotherapy. Strong ZP3 expression was found in normal human and mouse testis. ZP3 protein and mRNA transcripts were localized in spermatogonia, spermatocytes and round and elongated spermatids of both human and mouse testis, as well as in a mouse spermatogonial cell line, but absent in testicular Sertoli, Leydig, spermatogonial stem and progenitor cells. All other normal human and mouse tissues were ZP3 negative. This surprising testicular ZP3 expression has implications for the development of ZP3 cancer immunotherapies, and it also alludes to the potential of using ZP3 as a target for the development of a male immunocontraceptive.

Involvement of Cytokines and Hormones in the Development of Spermatogenesis In Vitro from Spermatogonial Cells of Cyclophosphamide-Treated Immature Mice.

Aggressive chemotherapy treatment may lead to male infertility. Prepubertal boys do not produce sperm at this age, however, they have spermatogonial stem cells in their testes. Here, we examined the effect of intraperitoneal injection of cyclophosphamide (CP) on the capacity of immature mice (IM) to develop spermatogenesis in vivo and in vitro [using methylcellulose culture system (MCS)]. Our results show a significant decrease in testicular weight, total number of testicular cells, and the number of Sertoli, peritubular, premeiotic, and meiotic/post-meiotic cells, but an increase in the percentages of damaged seminiferous tubules in CP-treated IM compared to control. The functionality of Sertoli cells was significantly affected. The addition of testosterone to isolated cells from seminiferous tubules of CP-treated IM significantly increased the percentages of premeiotic (CD9-positive cells) and meiotic/post-meiotic cells (ACROSIN-positive cells) developed in MCS compared to control. The addition of FSH did not affect developed cells in MCS compared to control, but in combination with testosterone, it significantly decreased the percentages of CD9-positive cells and ACROSIN-positive cells. The addition of IL-1 did not affect developed cells in MCS compared to control, but in combination with testosterone, it significantly increased the percentages of VASA-positive cells and BOULE-positive cells compared to IL-1 or testosterone. Addition of TNF significantly increased only CD9-positive cells in MCS compared to control, but in combination with testosterone, it significantly decreased ACROSIN-positive cells compared to testosterone. Our results show a significant impairment of spermatogenesis in the testes of CP-treated IM, and that spermatogonial cells from these mice proliferate and differentiate to meiotic/post-meiotic cells under in vitro culture conditions.

Perfluorooctanoic acid induces cytotoxicity in spermatogonial GC-1 cells

Perfluorooctane acid (PFOA), a typical perfluorinated chemical, has been suggested to interfere with male reproductive function. In this study, mouse spermatogonial GC-1 cells were in vitro treated with PFOA (250, 500 or 750 μM) for 24 h to investigate the cytotoxicity of PFOA and its underlying mechanisms. Our results indicated that exposure to intermediate and high doses of PFOA suppressed the viability of GC-1 cells in a concentration-dependent manner. Furthermore, PFOA treatment markedly enhanced the generation of reactive oxygen species and malondialdehyde, with diminished activity of superoxide dismutase. Particularly, PFOA exposure evoked a decline in mitochondrial membrane potential and ATP production. Furthermore, the apoptotic index and caspase-3 activity were significantly elevated after treatment with PFOA. In addition, PFOA incubation caused an increase in LC3B-II/LC3B–I ratio. Meanwhile, PFOA resulted in an excessive accumulation of autophagosomes in the cytoplasm. Taken together, exposure to PFOA can elicit cytotoxicity to spermatogonial GC-1 cells in vitro, which may be link to the mitochondrial oxidative damage and induction of apoptosis and autophagy.

MicroRNA-382 inhibits the proliferation of mouse spermatogonia by targeting Kmt5a

Spermatogenesis is a highly efficient and intricate process in the testis by which mature spermatozoa are produced daily to maintain lifelong male fertility. Essential to this process are spermatogonia capable of both proliferation and differentiation. Nevertheless, the underlying mechanisms for spermatogonial proliferation and differentiation remain poorly understood. MicroRNAs (miRNAs) are a category of non-coding small RNAs with regulatory functions by binding to the 3’ untranslated region (UTR) of the target mRNA. Previous studies have demonstrated that miRNAs are capable of modulating cell proliferation, differentiation and apoptosis, but the roles of individual miRNAs in spermatogonial fate determination remain largely elusive. Here, by using a mouse spermatogonial cell line (GC-1), we investigated the role for miRNA-382 in spermatogonial proliferation. We found that pre-miRNA-382 was expressed in spermatogonia. The luciferase reporter assay demonstrated Kmt5a but not Top1 as a target gene of miRNA-382. Overexpression of miRNA-382 by transfecting a miRNA mimic downregulated Kmt5a at both RNA and protein levels, and further reduced the proliferation and viability of spermatogonia. Knockdown of Kmt5a by RNA interference (RNAi) resulted in a uniform phenotype in spermatogonia. We therefore conclude that miRNA-382 inhibits the proliferation of mouse spermatogonia by targeting Kmt5a. Our finding extends the knowledge about the regulatory roles of miRNAs in spermatogonia and lays the groundwork for diagnosis and treatment of male infertility.

PLZF suppresses differentiation of mouse spermatogonial progenitor cells via binding of differentiation associated genes.

Promyelocytic leukaemia zinc finger (PLZF) is a key factor in inhibiting differentiation of spermatogonial progenitor cells (SPCs), but the underlying mechanisms are still largely unknown. In this study, the regulation of PLZF on Kit, Stra8, Sohlh2, and Dmrt1 (SPCs differentiation related genes) was investigated. We found some PLZF potential binding sites existed in the promoters of Kit, Stra8, Sohlh2, and Dmrt1. Additionally, the expressions of KIT, STRA8, SOHLH2, and DMRT1 were upregulated when PLZF was knockdown in SPCs. Furthermore, chromatin immunoprecipitation quantitative polymerase chain reaction revealed PLZF directly bound to the promoters of Kit, Stra8, Sohlh2, and Dmrt1. Besides, dual luciferase assay verified PLZF repressed those gene expressions. Collectively, our finding indicate that PLZF binds to the promoter regions of Kit, Stra8, Sohlh2, and Dmrt1 to regulate SPCs differentiation, which facilitate us to further understand the regulatory mechanism of PLZF in SPCs fates.

Development of Spermatogenesis In Vitro in Three-Dimensional Culture from Spermatogonial Cells of Busulfan-Treated Immature Mice.

Aggressive chemotherapy may lead to permanent male infertility. Prepubertal males do not generate sperm, but their testes do contain spermatogonial cells (SPGCs) that could be used for fertility preservation. In the present study, we examined the effect of busulfan (BU) on the SPGCs of immature mice, and the possible induction of the survivor SPGCs to develop spermatogenesis in 3D in-vitro culture. Immature mice were injected with BU, and after 0.5–12 weeks, their testes were weighed and evaluated histologically compared to the control mice. The spermatogonial cells [Sal-like protein 4 (SALL4) and VASA (a member of the DEAD box protein family) in the testicular tissue were counted/seminiferous tubule (ST). The cells from the STs were enzymatically isolated and cultured in vitro. Our results showed a significant decrease in the testicular weight of the BU-treated mice compared to the control. This was in parallel to a significant increase in the number of severely damaged STs, and a decrease in the number of SALL4 and VASA/STs compared to the control. The cultures of the isolated cells from the STs of the BU-treated mice showed a development of colonies and meiotic and post-meiotic cells after four weeks of culture. The addition of homogenates from adult GFP mice to those cultures induced the development of sperm-like cells after four weeks of culture. This is the first study demonstrating the presence of biologically active spermatogonial cells in the testicular tissue of BU-treated immature mice, and their capacity to develop sperm-like cells in vitro.

Perturbation of microRNA signalling by doxorubicin in spermatogonial, Leydig and Sertoli cell lines in vitro.

We have previously shown that in addition to its widely recognised cardiotoxicity, the chemotherapeutic doxorubicin (DOX) is able to induce transcriptional, microRNA (miRNA) and DNA methylation changes in the mouse testis. These changes perturb pathways involved in stress/cell death and survival and testicular function and lead to germ cell loss and reproductive organ damage. Here, we further investigated the differential miRNA expression induced by DOX in mouse spermatogonial (GC1), Leydig (TM3) and Sertoli (TM4) cell lines in vitro. We began by performing cell cycle analysis of the three mouse testicular cell lines to evaluate their sensitivity to DOX and thus select suitable doses for miRNA profiling. In keeping with our in vivo data, the spermatogonial cell line was the most sensitive, and the Sertoli cell line the most resistant to DOX-induced cell cycle arrest. We then further demonstrated that each cell line has a distinct miRNA profile, which is perturbed upon treatment with DOX. Pathway analysis identified changes in the miRNA-mediated regulation of specialised signalling at germ-Sertoli and Sertoli-Sertoli cell junctions following treatment with DOX. Amongst the most significant disease categories associated with DOX-induced miRNA expression were organismal injury and abnormalities, and reproductive system disease. This suggests that miRNAs play significant roles in both normal testicular function and DOX-induced testicular toxicity. Comparison of our in vitro and in vivo data highlights that in vitro cell models can provide valuable mechanistic information, which may also help facilitate the development of biomarkers of testicular toxicity and high-throughput in vitro screening methods to identify potential testicular toxicants.

Catsper1 promoter is bidirectional and regulates the expression of a novel lncRNA

The Catsper1 gene, whose expression is restricted to male germ cells, has great importance in reproductive biology because of its function in sperm motility and fertilization. We previously reported that the promoter of this gene has transcriptional activity in either direction in a heterologous system. In the present study, we found that the Catsper1 promoter has in vitro transcriptional activity in either orientation in GC-1 spg mouse spermatogonial cells. The results also showed that this promoter regulates the expression of a new divergent Catsper1 gene named Catsper1au (Catsper1 antisense upstream transcript). Catsper1au is expressed in adult male mouse testis and liver tissues but not in female mouse liver or ovary tissues. In the testis, Catsper1au is expressed in embryos at 11.5 days post-coitum and from newborns to adults. This gene is also expressed in 1- to 3-week postnatal hearts and in 1-week to adult stage livers. The analysis of the 1402 bp whole genome sequence revealed that Catsper1au is an intronless and polyadenylated lncRNA, located in the nuclei of Sertoli and spermatogenic cells from adult testis. These data indicate that Catsper1au is divergently expressed from the Catsper1 promoter and could regulate gene expression during spermatogenesis.

Mrhl Long Noncoding RNA Mediates Meiotic Commitment of Mouse Spermatogonial Cells by Regulating Sox8 Expression.

Long noncoding RNAs (lncRNAs) are important regulators of various biological processes, including spermatogenesis. Our previous studies have revealed the regulatory loop of mrhl RNA and Wnt signaling, where mrhl RNA negatively regulates Wnt signaling and gets downregulated upon Wnt signaling activation. This downregulation of mrhl RNA is important for the meiotic progression of spermatogonial cells. In our present study, we identified the transcription factor Sox8 as the regulatory link between mrhl RNA expression, Wnt signaling activation, and meiotic progression. In contrast to reports from other groups, we report the expression of Sox8 in germ cells and describe the molecular mechanism of Sox8 regulation by mrhl RNA during differentiation of spermatogonial cells. Binding of mrhl RNA to the Sox8 promoter is accompanied by the assembly of other regulatory factors involving Myc-Max-Mad transcription factors, corepressor Sin3a, and coactivator Pcaf. In the context of Wnt signaling, Sox8 directly regulates the expression of premeiotic and meiotic markers. Prolonged Wnt signaling activation in spermatogonial cells leads to changes in global chromatin architecture and a decrease in levels of stem cell markers.

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.

Generation of Mouse Spermatogonial Stem-Cell-Colonies in A Non-Adherent Culture.

Objective The properties of self-renewal and division in spermatogonial stem cells (SSCs) support spermatogenesis. There is a number of reported methods for in vitro SSC culture systems. The development of a culture system that effectively supports isolation and selfrenewal of germline stem cells (GSCs) is of tremendous benefit for clinical trials, experimental research, and as potential treatment for male infertility. The current study aims to consider the cultivation and behavior of GSCs in a non-adherent culture system. Materials and Methods In this experimental study, we cultured testicular cells from neonatal mice in agarose coated plates in the presence of Dulbecco’s modified Eagle’s medium (DMEM) medium (CTRL group), 10% fetal bovine serum (FBS)+DMEM (10% group), and growth factor (G group) that contained 2% FBS, glial cell-derived neurotrophic factor (GDNF), epidermal growth factor (EGF), and fibroblast growth factor (FGF). Mouse spermatogonial stem-like colonies were isolated approximately 3 weeks after digestion of the testis tissue. After passages 2-3, the identity of the mouse spermatogonial stem-like cells was confirmed by immunocytochemistry, reverse transcription-polymerase chain reaction (RT-PCR), and flow cytometry against the germ cell markers α6, β1, c-Kit, Thy-1, c-Ret, Plzf, and Oct4. The statistical significance between mean values in different groups was determined by one-way analysis of variance (ANOVA).Results We observed spermatogonial stem-like colonies in the G and 10% groups, but not the CTRL group. Immunocytochemistry, flow cytometry, and RT-PCR confirmed expressions of germ cell markers in these cells. In the spermatogonial stem-like cells, we observed a significant expression (P<0.05) of germ cell markers in the G and 10% groups versus the testis cells (T). Their proliferative and apoptotic activities were examined by Ki67 and PI/annexin V-FITC. Alkaline phosphatase assay showed that mouse spermato- gonial stem-like colonies were partially positive. ConclusionA non-adherent culture system could provide a favorable method for in vitro short-term culture of spermatogonial stem-like cell colonies.