牦牛与犏牛睾丸支持细胞分离培养及生物学特性比较分析

doi:10.11843/j.issn.0366-6964.2021.06.015

摘要/Abstract

摘要： 旨在建立牦牛与犏牛睾丸支持细胞分离培养及鉴定方案，比较两种牛睾丸支持细胞的生物学特性。本研究分别采集24月龄的3头健康公牦牛与3头F1代公犏牛睾丸组织作为2个样品组，其中每组3个生物学重复，分别通过混合酶消化、差速贴壁和饥饿处理分离得到两种牛的睾丸支持细胞，采用DMEM高糖及DMEM/F12培养基培养睾丸支持细胞，筛选更佳的培养体系。采用碱性磷酸酶染色、油红O染色和免疫荧光染色鉴定细胞表型特征，利用CCK8和RT-qPCR法分别检测细胞的增殖活性和标志功能基因的表达，进一步通过不同浓度丝裂霉素C处理两类支持细胞来评价两牛种支持细胞的耐受性及其作为饲养层细胞的潜能。结果，经形态、特殊染色及标志基因表达鉴定，成功分离到牦牛与犏牛睾丸支持细胞，建立了牦牛与犏牛睾丸支持细胞体外长期培养方案。发现DMEM高糖培养基更适用于睾丸支持细胞的增殖，两牛种支持细胞形态相似、轮廓清晰、呈现多边形或长梭形，牦牛睾丸支持细胞的体外增殖速率及活性远高于犏牛。调节精原细胞增殖分化相关基因（胶质细胞源性神经营养因子（glial cell line derived neurotrophic factor，GDNF）和转化生长因子β1基因（transforming growth factor-β1，TGF-β1））的表达在犏牛支持细胞中分别下调了3.4与2.9倍（P<0.05），基质细胞源性因子12基因（stromal cell-derived factor 12，CXCL12）的表达在犏牛上调了3.6倍（P<0.05）；调节睾丸发育的SRY-盒包含蛋白9基因（sex determining region Y-box9，SOX9）和睾丸支持细胞特异表达的Wilm肿瘤基因1（Wilms tumor gene1，WT1）在犏牛支持细胞中的表达分别下调了25.9（P<0.01）与38.7倍（P<0.01）。与牦牛相比，犏牛睾丸支持细胞对于丝裂霉素C具有较差的耐受性，表现为细胞核质分界不清、胞质空泡化严重和悬浮死细胞增多。本研究成功建立了牦牛与犏牛睾丸支持细胞分离纯化与体外培养方案；与牦牛相比，犏牛睾丸支持细胞在增殖活性和睾丸生精细胞分化关键功能基因表达等方面均存在缺陷，这可能也是导致犏牛雄性不育的原因之一。

关键词: 牦牛, 犏牛, 睾丸支持细胞, 体外培养

Abstract: The purpose of this study was to establish the protocol of isolation, culture and identification of sertoli cells from yak and cattle-yak, and to compare the biological characteristics of sertoli cells between yak and cattle-yak. Testis tissues were collected from 3 healthy male yaks and 3 F1 cattle-yaks aged 24 months to constitute 2 sample groups with 3 biological replicates in each group. The sertoli cells from yak and cattle-yak were separated by mixed enzyme digestion, differential adhesion and starvation treatment. DMEM high glucose and DMEM/F12 medium were used to culture sertoli cells to select an optimum culture system. Alkaline phosphatase staining, Oil red O staining and immunofluorescence staining were employed to identify the phenotypic characteristics of sertoli cells. CCK8 and RT-qPCR methods were used to detect proliferative activity and functional gene expression of sertoli cells. The sertoli cells from yak and cattle-yak were further treated with different concentrations of mitomycin C to evaluate the tolerance of sertoli cells and their potential as feeder cells. The sertoli cells from yak and cattle-yak were successfully isolated, and a long-term in vitro culture program for testicular sertoli cells was successfully established. DMEM high glucose medium was more suitable for the proliferation of sertoli cells. There were no significant differences between two types of sertoli cells, which presented the clear cellular profile and a polygonal or long spindle morphology. The proliferation capacity and viability of sertoli cells of yak were better than that of cattle-yak. The expressions of GDNF, CXCL12, TGF-β1 genes related to spermatogonial proliferation and differentiation were significantly changed in sertoli cells of yak and cattle-yak. The expression of GDNF and TGF-β1 were down-regulated with 3.4 and 2.9 folds in the cattle-yak sertoli cells (P<0.05), the expression of and CXCL12 was up-regulated by 3.6 folds (P<0.05). The expressions of SOX9 and WT1 genes related to testicular development and sertoli cell markers were down-regulated by 25.9 (P<0.01) and 38.7 folds (P<0.01) in the cattle-yak sertoli cells, respectively. Compared with yak, cattle-yak sertoli cells possessed poorer tolerance to mitomycin C treatment and the cultured cells presented unclear boundaries between cytoplasm and nucleus, severe cytoplasmic vacuolation and more dead cells. This study successfully established the protocol of isolation, culture and identification of sertoli cells from yak and cattle-yak. Compared with yak, the cattle-yak sertoli cells have defects in proliferation activity, testicular development and functional gene expressions for spermatogonial differentiation, which may be one of the reasons for sterility of cattle-yak.

Key words: yak, cattle-yak, sertoli cells, culture in vitro

中图分类号:

陈雪梅, 易川平, 罗辉, 张鹏, 王明秀, 蔡欣, 钟金城. 牦牛与犏牛睾丸支持细胞分离培养及生物学特性比较分析[J]. 畜牧兽医学报, 2021, 52(6): 1603-1615.

CHEN Xuemei, YI Chuanping, LUO Hui, ZHANG Peng, WANG Mingxiu, CAI Xin, ZHONG Jincheng. Isolation, Culture and Comparison of Biological Characteristics of Sertoli Cells from Yak and Cattle-yak[J]. Acta Veterinaria et Zootechnica Sinica, 2021, 52(6): 1603-1615.

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