MicroRNA-202 maintains spermatogonial stem cells by inhibiting cell cycle regulators and RNA binding proteins.
ABSTRACT: miRNAs play important roles during mammalian spermatogenesis. However, the function of most miRNAs in spermatogenesis and the underlying mechanisms remain unknown. Here, we report that miR-202 is highly expressed in mouse spermatogonial stem cells (SSCs), and is oppositely regulated by Glial cell-Derived Neurotrophic Factor (GDNF) and retinoic acid (RA), two key factors for SSC self-renewal and differentiation. We used inducible CRISPR-Cas9 to knockout miR-202 in cultured SSCs, and found that the knockout SSCs initiated premature differentiation accompanied by reduced stem cell activity and increased mitosis and apoptosis. Target genes were identified with iTRAQ-based proteomic analysis and RNA sequencing, and are enriched with cell cycle regulators and RNA-binding proteins. Rbfox2 and Cpeb1 were found to be direct targets of miR-202 and Rbfox2 but not Cpeb1, is essential for the differentiation of SSCs into meiotic cells. Accordingly, an SSC fate-regulatory network composed of signaling molecules of GDNF and RA, miR-202 and diverse downstream effectors has been identified.
Project description:Spermatogonial stem cells (SSCs) are required for spermatogenesis. Earlier studies showed that glial cell line-derived neurotrophic factor (GDNF) was indispensable for SSC self-renewal by binding to the GFRA1/RET receptor. Mice with mutations in these molecules showed impaired spermatogenesis, which was attributed to SSC depletion. Here we show that SSCs undergo GDNF-independent self-renewal. A small number of spermatogonia formed colonies when testis fragments from a Ret mutant mouse strain were transplanted into heterologous recipients. Moreover, fibroblast growth factor 2 (FGF2) supplementation enabled in vitro SSC expansion without GDNF. Although GDNF-mediated self-renewal signaling required both AKT and MAP2K1/2, the latter was dispensable in FGF2-mediated self-renewal. FGF2-depleted testes exhibited increased levels of GDNF and were enriched for SSCs, suggesting that the balance between FGF2 and GDNF levels influences SSC self-renewal in vivo. Our results show that SSCs exhibit at least two modes of self-renewal and suggest complexity of SSC regulation in vivo.
Project description:Self-renewal and differentiation by spermatogonial stem cells (SSCs) is the foundation for continual spermatogenesis. SSC self-renewal is dependent on glial cell line-derived neurotrophic factor (GDNF); however, intracellular mechanisms stimulated by GDNF in SSCs are unknown. To investigate these mechanisms we utilized a culture system that maintains a mouse undifferentiated germ cell population enriched for self-renewing SSCs. In these cultures mRNA for the transcription factors Bcl6b, Erm, and Lhx1 are up-regulated by GDNF and decreased in its absence. The expression of all three molecules was further identified in undifferentiated spermatogonia in vivo. Using small interfering RNA to reduce expression and transplantation to quantify stem cell numbers, Bcl6b, Erm, and Lhx1 were shown to be important for SSC maintenance in vitro. Next, GDNF was shown to activate both Akt and Src family kinase (SFK) signaling in SSCs, and culture of SSCs with inhibitors to Akt or SFKs followed by transplantation analysis showed significant impairment of SSC maintenance in vitro. Apoptosis analysis revealed a significant increase in the percentage of apoptotic cells when Akt, but not SFK, signaling was impaired, indicating that multiple signaling pathways are responsible for SSC self-renewal and survival. Biochemical and gene expression experiments revealed that GDNF up-regulated expression of Bcl6b, Erm, and Lhx1 transcripts is dependent on SFK signaling. Overall, these data demonstrate that GDNF up-regulation of Bcl6b, Erm, and Lhx1 expression through SFK signaling is a key component of the intracellular mechanism for SSC self-renewal.
Project description:In the murine testis, self-renewal of spermatogonial stem cells (SSCs) requires glial cell line-derived neurotrophic factor (GDNF) secreted from neighboring somatic cells. However, it not clear how GDNF promotes self-renewal in vivo or what downstream signaling pathways are required for SSC maintenance. We found that GDNF is normally expressed cyclically during spermatogenesis. Stage-specific ectopic expression of GDNF caused the accumulation of a GFRA1+ LIN28- Asingle population, which has enhanced SSC activity compared with wild type, suggesting that GDNF normally limits self-renewal to specific stages. Despite the increase in SSC cell number, EdU labeling during steady-stage spermatogenesis, and during recovery after busulfan-mediated spermatogonial depletion, indicated that GDNF promotes self-renewal by blocking differentiation and not by promoting proliferation. Increased GDNF signaling led to increased phosphorylation of AKT3 in undifferentiated spermatogonia, but not of AKT1 or AKT2, and was independent of RPS6 phosphorylation, suggesting that AKT3 functions in SSC self-renewal or progenitor cell expansion.
Project description:The development of a stem cell culture system would expedite our understanding of the biology of tissue regeneration. Spermatogonial stem cell (SSC) is the foundation for lifelong male spermatogenesis and the SSC culture has been optimized continuously in recent years. However, there have been many inconveniences to reconstruct SSC self-renewal and proliferation in vitro, such as the frequent refreshment of recombinant cytokines, including GDNF, the essential growth factor for SSC maintenance. In the present study, we observed that both STO and MEF cells, which were previously used as feeders for SSC growth, did not express GDNF, but a GDNF-expressing STO feeder could support undifferentiated mouse spermatogonia propagation in vitro for three months without the refreshment of recombinant growth factor GDNF. The cell morphology, growth rate and SSC-associated gene expression remained identical to the SSCs cultured using previous methods. The transplantation of SSCs growing on these GDNF-expressing STO feeders could generate extensive colonies of spermatogenesis in recipient testes, functionally validating the stemness of these cells. Collectively, our data indicated that the further modification of feeder cells might facilitate the self-renewal and propagation of SSCs in vitro.
Project description:Spermatogonial stem cells (SSCs) provide the foundation for spermatogenesis throughout the life of a male. Because SSCs of many species can colonize the mouse testis, and glial cell line-derived neurotrophic factor (GDNF) is responsible for stimulating SSC self-renewal in rodents, we reasoned that molecular mechanisms of SSC self-renewal are similar across species. GDNF-regulated genes have been identified in mouse SSCs; however, downstream targets of GDNF are unknown in other species. The objective of this work was to identify GDNF-regulated genes in rat SSCs and to define the biological significance of these genes for rat SSC self-renewal. We conducted microarray analysis on cultured rat germ cells enriched for SSCs in the presence and absence of GDNF. Many GDNF-regulated genes were identified, most notably, Bcl6b and Etv5, which are important for mouse SSC self-renewal. Bcl6b was the most highly regulated gene in both the rat and mouse. Additionally, we identified three novel GDNF-regulated genes in rat SSCs: Bhlhe40, Hoxc4, and Tec. Small interfering RNA treatment for Bcl6b, Etv5, Bhlhe40, Hoxc4, and Tec resulted in a decrease in SSC number, as determined by transplantation, without a change in total cell number within the culture. These data indicate that, like in the mouse SSC, Bcl6b and Etv5 are important for rat SSC self-renewal, suggesting that these genes may be important for SSCs in all mammals. Furthermore, identification of three novel GDNF-regulated genes in the rat SSC extends our knowledge of SSC activity and broadens the foundation for understanding this process in higher species, including humans.
Project description:Maintenance of adult tissues depends on stem cell self-renewal in local niches. Spermatogonial stem cells (SSC) are germline adult stem cells necessary for spermatogenesis and fertility. We show that testicular endothelial cells (TECs) are part of the SSC niche producing glial cell line-derived neurotrophic factor (GDNF) and other factors to support human and mouse SSCs in long-term culture. We demonstrate that FGF-2 binding to FGFR1 on TECs activates the calcineurin pathway to produce GDNF. Comparison of the TEC secretome to lung and liver endothelial cells identified 5 factors sufficient for long-term maintenance of human and mouse SSC colonies in feeder-free cultures. Male cancer survivors after chemotherapy are often infertile since SSCs are highly susceptible to cytotoxic injury. Transplantation of TECs alone restores spermatogenesis in mice after chemotherapy-induced depletion of SSCs. Identifying TECs as a niche population necessary for SSC self-renewal may facilitate fertility preservation for prepubertal boys diagnosed with cancer.
Project description:Since spermatogonial stem cells (SSCs) are capable of both self-renewal and differentiation to daughter cells for subsequent spermatogenesis, the development of an efficient in vitro culture system is essential for studies related to spermatogenesis. Although the currently available system is serum-free and contains only chemically-defined components, it highly relies upon bovine serum albumin (BSA), a component with batch-to-batch quality variations similar to those of fetal bovine serum. Thus, we searched for an alternative BSA-free culture system that preserved the properties of SSCs. In this study, we utilized Knockout Serum Replacement (KSR) in the SSC culture medium, as a substitute for BSA. The results demonstrated that KSR supported the continuous growth of SSCs in vitro and the SSC activity in vivo without BSA, in a feeder-cell combination with mouse embryonic fibroblasts. The addition of BSA to KSR further facilitated cell cycle progression, whereas a transplantation assay revealed that the addition of BSA did not affect the number of SSCs in vivo. The combination of KSR with BSA also allowed the elimination of GFRA1 and FGF2, and the reduction of the GDNF concentration from 20 ng/ml to 5 ng/ml, while maintaining the growth rate and the expression of SSC markers. Furthermore, KSR was also useful with SSCs from non-DBA/2 strains, such as C57BL/6 and ICR. These results suggested that KSR is an effective substitute for BSA for long-term in vitro cultures of SSCs. Therefore, this method is practical for various studies related to SSCs, including spermatogenesis and germ stem cell biology.
Project description:Spermatogenesis originates from self-renewal of spermatogonial stem cells (SSCs). Previous studies have reported conflicting roles of gonadotropic pituitary hormones in SSC self-renewal. Here, we explored the role of hormonal regulation of SSCs using Fshb and Lhcgr knockout (KO) mice. Although follicle-stimulating hormone (FSH) is thought to promote self-renewal by glial cell line-derived neurotrophic factor (GDNF), no abnormalities were found in SSCs and their microenvironment. In contrast, SSCs were enriched in Lhcgr-deficient mice. Moreover, wild-type SSCs transplanted into Lhcgr-deficient mice showed enhanced self-renewal. Microarray analysis revealed that Lhcgr-deficient testes have enhanced WNT5A expression in Sertoli cells, which showed an immature phenotype. Since WNT5A was upregulated by anti-androgen treatment, testosterone produced by luteinizing hormone (LH) is required for Sertoli cell maturation. WNT5A promoted SSC activity both in vitro and in vivo. Therefore, FSH is not responsible for GDNF regulation, while LH negatively regulates SSC self-renewal by suppressing WNT5A via testosterone.
Project description:Spermatogenesis requires retinoic acid (RA) induction of the undifferentiated to differentiating transition in transit amplifying (TA) progenitor spermatogonia, whereas continuity of the spermatogenic lineage relies on the RA response being suppressed in spermatogonial stem cells (SSCs). Here, we discovered that, in mouse testes, both spermatogonial populations possess intrinsic RA-response machinery and exhibit hallmarks of the differentiating transition following direct exposure to RA, including loss of SSC regenerative capacity. We determined that SSCs are only resistant to RA-driven differentiation when situated in the normal topological organization of the testis. Furthermore, we show that the soma is instrumental in "priming" TA progenitors for RA-induced differentiation through elevated RA receptor expression. Collectively, these findings indicate that SSCs and TA progenitor spermatogonia inhabit disparate niche microenvironments within seminiferous tubules that are critical for mediating extrinsic cues that drive fate decisions.
Project description:Spermatogonial stem cells (SSCs) are unique male germline stem cells that support spermatogenesis and male fertility. Long non-coding RNAs (lncRNA) have been identified as key regulators of stem cell fate; however, their role in SSCs has not been explored. Here, we report that a novel spermatogonia-specific lncRNA (lncRNA033862) is essential for the survival of murine SSCs. LncRNA033862 is expressed in early spermatogonia including SSC and was among 805 lncRNAs identified by global expression profiling as responsive to glial cell-derived neurotrophic factor (GDNF), a growth factor required for SSC self-renewal and survival. LncRNA033862 is an antisense transcript of the GDNF receptor alpha1 (Gfra1) that lacks protein coding potential and regulates Gfra1 expression levels by interacting with Gfra1 chromatin. Importantly, lncRNA033862 knockdown severely impairs SSC survival and their capacity to repopulate recipient testes in a transplantation assay. Collectively, our data provide the first evidence that long non-coding RNAs (lncRNAs) regulate SSC fate.