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

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...

Isolation,Culture and Comparison of Biological Characteristics of Sertoli Cells from Yak and Cattle-yak

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.