Endogenously produced FGF2 is essential for the survival and proliferation of cultured mouse spermatogonial stem cells

Zhang, Yan; Wang, Si; Wang, Xiuxia; Liao, Shanhui; Wu, Yujian; Han, Chunsheng

doi:10.1038/cr.2012.17

Cited by 58 publications

(54 citation statements)

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“…A subsequent study indicated that FGF2 may regulate mouse SSC proliferation and stem cell activity in vitro via autocrine phosphorylation of AKT and ERK1/2 pathway (Zhang et al 2012). Conditional knockout of the Fgf2 gene in postnatal Sertoli cells and SSCs could help to elucidate the essential role of FGF2 in SSC self-renewal in vivo.…”

Section: Gdnf Signalingmentioning

confidence: 99%

Regulation of spermatogonial stem cell self-renewal and spermatocyte meiosis by Sertoli cell signaling

Chen¹,

Liu²

2015

Reproduction

239

166

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Spermatogenesis is a continuous and productive process supported by the self-renewal and differentiation of spermatogonial stem cells (SSCs), which arise from undifferentiated precursors known as gonocytes and are strictly controlled in a special 'niche' microenvironment in the seminiferous tubules. Sertoli cells, the only somatic cell type in the tubules, directly interact with SSCs to control their proliferation and differentiation through the secretion of specific factors. Spermatocyte meiosis is another key step of spermatogenesis, which is regulated by Sertoli cells on the luminal side of the blood-testis barrier through paracrine signaling. In this review, we mainly focus on the role of Sertoli cells in the regulation of SSC self-renewal and spermatocyte meiosis, with particular emphasis on paracrine and endocrine-mediated signaling pathways. Sertoli cell growth factors, such as glial cell line-derived neurotrophic factor (GDNF) and fibroblast growth factor 2 (FGF2), as well as Sertoli cell transcription factors, such as ETS variant 5 (ERM; also known as ETV5), nociceptin, neuregulin 1 (NRG1), and androgen receptor (AR), have been identified as the most important upstream factors that regulate SSC self-renewal and spermatocyte meiosis. Other transcription factors and signaling pathways (GDNF-RET-GFRA1 signaling, FGF2-MAP2K1 signaling, CXCL12-CXCR4 signaling, CCL9-CCR1 signaling, FSH-nociceptin/OPRL1, retinoic acid/FSH-NRG/ERBB4, and AR/RB-ARID4A/ARID4B) are also addressed.Reproduction (2015) 149 R159-R167

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Section: Gdnf Signalingmentioning

confidence: 99%

Regulation of spermatogonial stem cell self-renewal and spermatocyte meiosis by Sertoli cell signaling

Chen¹,

Liu²

2015

Reproduction

239

166

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“…Cell-extrinsic factor WNT5A supports self-renewal of mSSCs, while WNT3A might promote the proliferation of spermatogonial progenitor cells [5,12]. Work from our laboratory shows that endogenously produced FGF2 is essential for the proliferation and survival of mSSCs [13]. Most of these growth factors are involved in G 1 /S regulation by facilitating DNA synthesis [11,13,14].…”

Section: Introductionmentioning

confidence: 99%

“…Briefly, testes from the 5-7 days postpartum (dpp) mice were digested with 1 mg/mL collagenase I (GIBCO; 17100) and 1 mg/mL DNase I (AppliChem; A3778). The small seminiferous tubule fragments were plated on dishes in mouse embryonic fibroblast (MEF) medium containing 10% fetal bovine serum (FBS), and the undifferentiated spermatogonia were collected with several pipetting after 20 h. Then, the cells were cultured on mitomycin-inactivated MEF feeder cells in MEMa supplemented with other components such as BSA, insulin, transferrin, and free fatty acids [9,13]. Next, 1% FBS, 20 ng/ mL of GDNF, and 5 ng/mL of FGF2 were freshly added to the culture each time the medium was changed.…”

Section: Mssc Culturementioning

confidence: 99%

“…F1 pups of DBA/2 and C57BL6 mice were used for mSSC culture. Isolation, enrichment, and culture of mSSCs were performed following protocols developed by KanatsuShinohara et al and Kubota et al with some modifications [13,29,30]. Briefly, testes from the 5-7 days postpartum (dpp) mice were digested with 1 mg/mL collagenase I (GIBCO; 17100) and 1 mg/mL DNase I (AppliChem; A3778).…”

Section: Mssc Culturementioning

confidence: 99%

“…Work from our laboratory shows that endogenously produced FGF2 is essential for the proliferation and survival of mSSCs [13]. Most of these growth factors are involved in G 1 /S regulation by facilitating DNA synthesis [11,13,14].…”

Section: Introductionmentioning

confidence: 99%

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IGF-1R Signaling Is Essential for the Proliferation of Cultured Mouse Spermatogonial Stem Cells by Promoting the G₂/M Progression of the Cell Cycle

Wang

et al. 2015

Stem Cells and Development

Self Cite

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Culture of mouse spermatogonial stem cells (mSSCs) contributes to understanding the mechanisms of mammalian spermatogenesis. Several key growth factors such as GDNF and FGF2 have been known to be essential for the proliferation of cultured mSSCs. However, additional factors regulating SSC proliferation remain to be identified. In this study, we report that IGF-1R signaling is required for the proliferation of cultured mSSCs by promoting the G 2 /M progression of the cell cycle. IGF-1 and its receptor IGF-1R are expressed in cultured mSSCs as well as in isolated Sertoli cells and interstitial cells. Blockage of IGF-1R signaling either by knockdown of IGF-1R or by the IGF-1R-specific inhibitor picropodophyllin (PPP) significantly reduced the proliferation of mSSCs, increased their apoptosis, and impaired their stem cell activity in an insulinindependent manner. PPP treatment of mSSCs blocked the G 2 /M progression. In contrast, both GDNF withdrawal and FGF2 signaling blockade decreased the entry of mSSCs into their S phases. Consistently, IGF-1 promoted the G 2 /M progression of thymidine-treated mSSCs, which were arrested at G 1 /S boundary synchronously; while GDNF and/or FGF2 stimulated their entry into the S phase. Moreover, IGF-1 activated the phosphorylation of AKT but not that of ERK1/2 in mSSCs. These results indicate that IGF-1R signaling stimulates the proliferation of mSSCs using a distinct mechanism from those by GDNF and FGF2, and will contribute to the establishment of a chemically defined culture system.

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A Simple and Efficient Procedure for Developing Mouse Germline Stem Cell Lines with Gene Knock‐in via CRISPR/Cas9 Technology

Wang,

Hu,

Han

2024

Current Protocols

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Cultured mammalian spermatogonial stem cells (SSCs), also known as germline stem cells (GSCs), hold great promise for applications such as fertility preservation, gene therapy, and animal breeding, particularly in conjunction with accurate gene editing. Although the in vitro development of mouse GSC (mGSC) lines, and gene‐targeting procedures for such lines, were initially established about two decades ago, it remains challenging for beginners to efficiently accomplish these tasks, partly because mGSCs proliferate more slowly and are more resistant to lipid‐mediated gene transfection than pluripotent stem cells (PSCs). Meanwhile, methods for mGSC culture and gene editing have been evolving constantly to become simpler and more efficient. Here, we describe how to develop mGSC lines from small mouse testis samples and how to carry out gene knock‐in in these cells using CRISPR/Cas9 technology, detailing three basic protocols that constitute a streamlined procedure. Using these simple and efficient procedures, site‐specific knock‐in mGSC lines can be obtained in 3 months. We hope that these protocols will help researchers use genetically modified GSCs to explore scientific questions of interest and to accumulate experience for application to GSC research in other mammalian species. © 2024 Wiley Periodicals LLC.Basic Protocol 1: Establishment of mouse GSCs lines from small testicular samplesBasic Protocol 2: Preparation of plasmids for gene knock‐in using the CRISPR/Cas9 systemBasic Protocol 3: Establishment of gene knock‐in mGSC lines by electroporation gene delivery

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Endogenously produced FGF2 is essential for the survival and proliferation of cultured mouse spermatogonial stem cells

Cited by 58 publications

References 10 publications

Regulation of spermatogonial stem cell self-renewal and spermatocyte meiosis by Sertoli cell signaling

Regulation of spermatogonial stem cell self-renewal and spermatocyte meiosis by Sertoli cell signaling

IGF-1R Signaling Is Essential for the Proliferation of Cultured Mouse Spermatogonial Stem Cells by Promoting the G₂/M Progression of the Cell Cycle

A Simple and Efficient Procedure for Developing Mouse Germline Stem Cell Lines with Gene Knock‐in via CRISPR/Cas9 Technology

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Endogenously produced FGF2 is essential for the survival and proliferation of cultured mouse spermatogonial stem cells

Cited by 58 publications

References 10 publications

Regulation of spermatogonial stem cell self-renewal and spermatocyte meiosis by Sertoli cell signaling

Regulation of spermatogonial stem cell self-renewal and spermatocyte meiosis by Sertoli cell signaling

IGF-1R Signaling Is Essential for the Proliferation of Cultured Mouse Spermatogonial Stem Cells by Promoting the G2/M Progression of the Cell Cycle

A Simple and Efficient Procedure for Developing Mouse Germline Stem Cell Lines with Gene Knock‐in via CRISPR/Cas9 Technology

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Product

Resources

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IGF-1R Signaling Is Essential for the Proliferation of Cultured Mouse Spermatogonial Stem Cells by Promoting the G₂/M Progression of the Cell Cycle