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.

References 46

[1]	LAROSE H,SHAMI A N,ABBOTT H,et al.Gametogenesis:a journey from inception to conception[J].Curr Top Dev Biol, 2019,132:257-310.
[2]	KUBOTA H,WU X,GOODYEAR S M,et al.Glial cell line-derived neurotrophic factor and endothelial cells promote self-renewal of rabbit germ cells with spermatogonial stem cell properties[J].FASEB J,2011,25(8):2604-2614.
[3]	GONZÁLEZ R,DOBRINSKI I.Beyond the mouse monopoly:studying the male germ line in domestic animal models[J]. ILAR J, 2015,56(1):83-98.
[4]	GUO Y,LIU L H,SUN M,et al.Expansion and long-term culture of human spermatogonial stem cells via the activation of SMAD3 and AKT pathways[J].Exp Biol Med (Maywood),2015,240(8):1112-1122.
[5]	KORUJI M,SHAHVERDI A,JANAN A,et al.Proliferation of small number of human spermatogonial stem cells obtained from azoospermic patients[J].J Assist Reprod Genet,2012,29(9):957-967.
[6]	ISHⅡ K,KANATSU-SHINOHARA M,TOYOKUNI S,et al.FGF2 mediates mouse spermatogonial stem cell selfrenewal via upregulation of Etv5 and Bcl6b through MAP2K1 activation[J].Development,2012,139(10):1734-1743.
[7]	SARIOLA H,SAARMA M.Novel functions and signalling pathways for GDNF[J].J Cell Sci,2003,116(19):3855-3862.
[8]	AZIZI H,HAMIDABADI H G,SKUTELLA T.Differential proliferation effects after short-term cultivation of mouse spermatogonial stem cells on different feeder layers[J].Cell J,2019,21(2):186-193.
[9]	PORTELA J M D,MULDER C L,VAN DAALEN S K M,et al.Strains matter:success of murine in vitro spermatogenesis is dependent on genetic background[J].Dev Biol,2019,456(1):25-30.
[10]	ZHENG Y,ZHANG Y Q,QU R F,et al.Spermatogonial stem cells from domestic animals:progress and prospects[J].Reproduction, 2014,147(3):R65-R74.
[11]	KUBOTA H,BRINSTER R L.Spermatogonial stem cells[J].Biol Reprod,2018,99(1):52-74.
[12]	王烁程,陈晓丽,张林波,等.支持细胞对精原干细胞增殖、分化调控的研究进展[J].畜牧兽医学报,2019,50(2):253-260.WANG S C,CHEN X L,ZHANG L B,et al.The progress of the study on the regulation of the proliferation,differentiation of spermatogonial stem cells by the Sertoli cells[J].Acta Veterinaria et Zootechnica Sinica,2019,50(2):253-260.(in Chinese)
[13]	TSUDA M,SASAOKA Y,KISO M,et al.Conserved role of nanos proteins in germ cell development[J]. Science, 2003,301(5637):1239-1241.
[14]	LUO J P,MEGEE S,DOBRINSKI I.Asymmetric distribution of UCH-L1 in spermatogonia is associated with maintenance and differentiation of spermatogonial stem cells[J].J Cell Physiol,2009,220(2):460-468.
[15]	COSTOYA J A,HOBBS R M,BARNA M,et al.Essential role of Plzf in maintenance of spermatogonial stem cells[J].Nat Genet,2004,36(6):653-659.
[16]	BRAYDICH-STOLLE L,KOSTEREVA N,DYM M,et al.Role of Src family kinases and N-Myc in spermatogonial stem cell proliferation[J].Dev Biol,2007,304(1):34-45.
[17]	OATLEY J M,AVARBOCK M R,TELARANTA A I,et al.Identifying genes important for spermatogonial stem cell self-renewal and survival[J].Proc Natl Acad Sci U S A,2006,103(25):9524-9529.
[18]	OATLEY M J,KAUCHER A V,RACICOT K E,et al.Inhibitor of DNA binding 4 is expressed selectively by single spermatogonia in the male germline and regulates the selfrenewal of spermatogonial stem cells in mice[J].Biol Reprod, 2011,85(2):347-356.
[19]	DE ROOIJ D G.Recent developments in the spermatogonial stem cell field[J].Anim Reprod,2017,14(1):82-88.
[20]	YOSHINAGA K,NISHIKAWA S,OGAWA M,et al.Role of c-kit in mouse spermatogenesis:identification of spermatogonia as a specific site of c-kit expression and function[J].Development,1991,113(2):689-699.
[21]	VERVER D E,VAN PELT A M,REPPING S,et al.Role for rodent Smc6 in pericentromeric heterochromatin domains during spermatogonial differentiation and meiosis[J].Cell Death Dis,2013,4(8):e749.
[22]	AZIZI H,RANJBAR M,RAHAIEE S,et al.Investigation of VASA gene and protein expression in neonate and adult testicular germ cells in mice in vivo and in vitro[J].Cell J,2020,22(2):171-177.
[23]	DE ROOIJ D G.The nature and dynamics of spermatogonial stem cells[J].Development,2017,144(17):3022-3030.
[24]	KANATSU-SHINOHARA M,SHINOHARA T.Spermatogonial stem cell self-renewal and develop-ment[J].Annu Rev Cell Dev Biol,2013,29:163-187.
[25]	余冬冬.精原干细胞体外培养和移植的探究[D].杨凌:西北农林科技大学,2013.YU D D.Exploration of the culture of spermatogonial stem cells in vitro and transplantation[D].Yangling:Northwest A&F University,2013.(in Chinese)
[26]	陈庭锋,王霄燕,李东,等.猪精原干细胞体外分离培养及诱导分化的研究[J].畜牧兽医学报,2014,45(7):1104-1112.CHEN T F,WANG X Y,LI D,et al.Study on the culture and induced differentiation of spermatogonial stem cells in porcine[J].Acta Veterinaria et Zootechnica Sinica,2014,45(7):1104-1112.(in Chinese)
[27]	何莹.猪精原干细胞分离纯化及分子标记[D].杨凌:西北农林科技大学,2012.HE Y.Molecular marker,isolation and purification of spermatogonial stem cells in pigs[D].Yangling:Northwest A&F University,2012.(in Chinese)
[28]	GROMPE M.Tissue stem cells:new tools and functional diversity[J].Cell Stem Cell,2012,10(6):685-689.
[29]	MOSCATELLI D,PRESTA M,JOSEPH-SILVERSTEIN J,et al.Both normal and tumor cells produce basic fibroblast growth factor[J].J Cell Physiol,1986,129(2):273-276.
[30]	CHEN S,CHOO A,CHIN A,et al.TGF-β2 allows pluripotent human embryonic stem cell proliferation on E6/E7 immortalized mouse embryonic fibroblasts[J].J Biotechnol,2006,122(3):341-361.
[31]	CONRAD S,AZIZI H,HATAMI M,et al.Expression of genes related to germ cell lineage and pluripotency in single cells and colonies of human adult germ stem cells[J].Stem Cells Int,2016,2016:8582526.
[32]	PROWSE A B J,MCQUADE L R,BRYANT K J,et al.Identification of potential pluripotency determinants for human embryonic stem cells following proteomic analysis of human and mouse fibroblast conditioned media[J].J Proteome Res,2007,6(9):3796-3807.
[33]	HE B R,LU F,ZHANG L L,et al.An alternative long-term culture system for highly-pure mouse spermatogonial stem cells[J].J Cell Physiol,2015,230(6):1365-1375.
[34]	BAHADORANI M,HOSSEINI S M,ABEDI P,et al.Short-term in-vitro culture of goat enriched spermatogonial stem cells using different serum concentrations[J].J Assist Reprod Genet,2012,29(1):39-46.
[35]	ZHU H J,LIU C,LI M Z,et al.Optimization of the conditions of isolation and culture of dairy goat male germline stem cells (mGSC)[J].Anim Reprod Sci,2013,137(1-2):45-52.
[36]	KANATSU-SHINOHARA M,OGONUKI N,INOUE K,et al.Long-term proliferation in culture and germline transmission of mouse male germline stem cells[J].Biol Reprod,2003,69(2):612-616.
[37]	SHARMA A,SHAH S M,TIWARI M,et al.Propagation of goat putative spermatogonial stem cells under growth factors defined serum-free culture conditions[J].Cytotechnology,2020,72(3):489-497.
[38]	SHARMA A,SHAH S M,SAINI N,et al.Optimization of serum-free culture conditions for propagation of putative buffalo (Bubalus bubalis) spermatogonial stem cells[J].Cell Reprogram,2019,21(1):1-10.
[39]	LI T T,GENG S S,XU H Y,et al.Effects of different culture systems on the culture of prepuberal buffalo (Bubalus bubalis) spermatogonial stem cell-like cells in vitro[J].J Vet Sci,2020,21(1):e13.
[40]	SUYATNO,KITAMURA Y,IKEDA S,et al.Long-term culture of undifferentiated spermatogonia isolated from immature and adult bovine testes[J].Mol Reprod Dev,2018,85(3):236-249.
[41]	WILLIAMS R L,HILTON D J,PEASE S,et al.Myeloid leukaemia inhibitory factor maintains the developmental potential of embryonic stem cells[J].Nature,1988,336(6200):684-687.
[42]	PRASETYANINGTYAS W E,KARJA N W K,AGUNGPRIYONO S,et al.Characteristics of testicular cell development of 5-day-old mice in culture in vitro[J].Anim Sci J,2020,91(1):e13332.
[43]	KÖSE S,YERSAL N,ÖNEN S,et al.Comparison of hematopoietic and spermatogonial stem cell niches from the regenerative medicine aspect[J].Adv Exp Med Biol,2018,1107:15-40.
[44]	FU H Y,ZHANG W H,YUAN Q Q,et al.PAK1 promotes the proliferation and inhibits apoptosis of human spermatogonial stem cells via PDK1/KDR/ZNF367 and ERK1/2 and AKT pathways[J].Mol Ther Nucleic Acids,2018,12:769-786.
[45]	RASOULI-GHAREHSAGHAL K,SHAKERI M,ZHANDI M,et al.Improvement of in vitro proliferation of cockerel spermatogonial stem cells using different combinations of growth factors[J].Br Poult Sci,2020:1-9.
[46]	BINSILA B K,SELVARAJU S,GHOSH S K,et al.EGF,GDNF,and IGF-1 influence the proliferation and stemness of ovine spermatogonial stem cells in vitro[J].J Assist Reprod Genet,2020,37(10):2615-2630.

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

PDF (PC)

Knowledge

Abstract

Cite this article

share this article

References 46

Related Articles 15

Recommended Articles

Metrics

Comments

[1]	LI Qiuyun, TIAN Xinyuan, LIAO Wensheng, ZHANG Huanrong, REN Yupeng, YANG Falong, ZHU Jiangjiang, XIANG Hua. Effects of SOCS2 on Proliferation, Cycle and Apoptosis of Turbinate Bone Cells in Goats [J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(5): 2226-2240.
[2]	ZHANG Wanfeng, ZHAO Tianzhi, LI Jiao, YOU Ziwei, YANG Yang, CAI Chunbo, GAO Pengfei, CAO Guoqing, GUO Xiaohong, LI Bugao. Study on NR2F2 Gene Regulating Proliferation and Apoptosis of Porcine PK15 Cells [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(8): 3242-3251.
[3]	WANG Wanjie, CHEN Nanzhu, ZOU Huiying, ZHOU Xinyi, HAO Haisheng, PANG Yunwei, ZHU Huabin, ZHAO Xueming, YU Dawei, DU Weihua. Effects of Histone Methyltransferase ASH1L Overexpression on Proliferation and Apoptosis of Bovine Cumulus Cells [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(8): 3358-3368.
[4]	ZHANG Peng, WANG Mingxiu, JING Kemin, PENG Wei, TIAN Yuan, LI Yuqian, FU Changqi, SHU Shi, ZHONG Jincheng, CAI Xin. Abnormal Expression of FGFs/FGFRs and Their Mediated Signaling Pathway Genes Affect the Proliferative Activity of Undifferentiated Spermatogonia in Cattleyak [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(7): 2886-2897.
[5]	XU Tiantian, ZHANG Tongtong, WANG Meng, WANG Xin. Transcription Factor Foxq1 Affects the Proliferation of Hair Follicle Stem Cells in Cashmere Goats via WNT/β-catenin Signaling Pathway [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(6): 2653-2661.
[6]	REN Xiaoli, FAN Yuying, HUANGFU Heping, LIU Yun, SHI Dongmei. Effect of GSK126 on Epithelial-mesenchymal Transition of Canine Mammary Tumor Cells [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(4): 1721-1729.
[7]	YANG Guang, XU Jing, LI Xin, HU Debao, GUO Yiwen, DING Xiangbin, GUO Hong, ZHANG Linlin. Effect of Interfering lncbMD on Proliferation and Differentiation of Bovine Skeletal Muscle Satellite Cells [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(3): 1015-1025.
[8]	SUN Jinkui, XU Houqiang, SHI Pengfei, RUAN Yong. Construction of MEF2A Gene Interference Vector and Effect of Its Transfection on Myoblasts in Guanling Cattle [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(2): 584-595.
[9]	MA Tianwen, YU Yue, LÜ Liangyu, JIA Lina, RUAN Hongri, WANG Haoran, WANG Xinyu, ZHANG Yuxin, ZHANG Jiantao, GAO Li. Effects of Bilobalide on Autophagy, Proliferation and Apoptosis of IL-1β-induced ATDC5 Chondrocytes [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(2): 837-846.
[10]	REN Xiaoli, FAN Yuying, HUANGFU Heping, WANG Jun, JIN Shuangxing, LIU Yun, SHI Dongmei. Mechanism of miR-502 Regulating Proliferation, Migration, Invasion and EMT of Canine Mammary Tumor Cells [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(10): 4379-4388.
[11]	WEI Jiayuan, ZHU Qian, YANG Yaxing, SHEN Ming. Inhibition of Porcine Follicular Granulosa Cell Proliferation by Cobalt Chloride Induced DNA Oxidative Damage [J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(9): 2982-2992.
[12]	CHEN Lan, ZHANG Tao, DING Hao, XIE Kaizhou, ZHANG Genxi, WANG Jinyu. Effects of Kruppel-like Factor 15 Gene on Proliferation and Differentiation of Preadipocytes of Heying Black Chickens [J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(7): 2118-2129.
[13]	ZHANG Zhenzhen, LI Jianzeng, JIANG Ruirui, MA Yanchao, CAI Chunxia, ZHANG Lujie, GUO Yujie, JI Jinqing, HAN Lu, TIAN Yadong, KANG Xiangtao, HAN Ruili. Isolation, Culture and Identification of Chicken Chondrocytes and the Effect of Overexpression of miR-15a on Chondrocytes [J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(6): 1749-1758.
[14]	LIU Junhong, ZHAO Yue, LI Hongmei, WANG Ying, WANG Moyu, CHENG Ziqiang, QIU Jianhua, HOU Qiuling, GUO Huijun. Comparison of Tissue Hyperplasia Lesions and Cytokines' Expressions Induced by Two Different Avian Leukosis Virus Subgroup J Strains [J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(6): 1895-1904.
[15]	YE Zhuang, WANG Lele, WANG Feiyan, LIU Yue, PENG Yuemei, SU Shijie, HOU Zhaofeng, XU Jinjun, TAO Jianping, LIU Dandan. Procaryotic Expression and Immunolocalization Analysis of the EnOXIO1 of Eimeria necatrix [J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(5): 1553-1561.