小鼠精原干细胞体外培养体系建立及功能鉴定

doi:10.11843/j.issn.0366-6964.2021.02.013

Abstract

Abstract: The objective of the present study was to provide a reference for in vitro culture of spermatogonial stem cells (SSCs) for other strain mice and megafauna by exploring the SSCs stable culture from ICR strain mice. Testes were harvested from 6-8 days old postpartum ICR male pups and digested using a two-step enzymatic (collagenase and trypsin) digestion protocol, then SSCs were purified by differential adherent method. The growth of SSCs in vitro culture was detected by using different feeder layers (mouse embryonic fibroblasts as feeder layer or laminin combined with polylysine), different media (StemPro-34 medium or DMEM medium) and different growth factors(recombinant rat glial-cell-line-derived neurotrophic factor (GDNF), recombinant mouse leukemia inhibitory factor (LIF), epidermal growth factor (EGF), recombinant human basic fibroblast growth factor (bFGF), insulin-like growth factors-1 (IGF1)). Proliferation of SSCs in the F6 was evaluated by immunostaining and molecular detection. Finally, stable proliferative SSCs in vitro were transplanted into recipient testes for functional identification. The results showed that SSC cells with purity higher than 79% could be achieved by two-step enzymatic digestion and differential adherent method; Using mouse embryonic fibroblasts as feeder layer, StemPro-34 as base medium, and adding GDNF, LIF, EGF, bFGF, IGF1 composite growth factors, the proli-feration and long-term culture of SSCs in vitro were observed. SSC colonies showed positive signals by alkaline phosphatase (AKP) immunostaining, and promyelocyte leukemia zinc-finger factor (PLZF) and ubiquitin C-terminal hydrolase L1 (UCHL1) immunofluorescence detection. Moreover, the high expression of pluripotent and self-renewal genes in colonies indicated the stable proliferation of SSCs by reverse transcription-polymerase chain reaction (RT-PCR) and quantitative real-time PCR (qRT-PCR) testing. After transplanting SSCs (with the enhanced green fluorescent protein (EGFP) reporter gene) into recipient testes, the heads of sperm in the epididymides emitted green light, indicating the sperm originated from the donor-SSCs. The results illustrated that the culture system obtained in this study was suitable for SSCs in vitro culture of ICR strain mice, and cells cultured in vitro had normal biological function. This study provided a reference for in vitro culture of spermatogonial stem cells of other strain mice and megafauna.

Key words: SSCs, culture in vitro, cell proliferation, functional identification

CLC Number:

S814.1

ZHANG Xianyu, ZHAO Xin, LI Guoling, XING Pingping, LI Zicong, YANG Huaqiang, WU Zhenfang, CHEN Bin. Establishment of the Culture System in Vitro and Functional Identification of Mouse Spermatogonial Stem Cells[J]. Acta Veterinaria et Zootechnica Sinica, 2021, 52(2): 408-419.

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Establishment of the Culture System in Vitro and Functional Identification of Mouse Spermatogonial Stem Cells

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