野外采集牦牛睾丸组织的冷冻保存及其生精细胞的复苏培养研究

Study on Cryopreservation of Field Sampled Yak Testicular Tissues and Culture of the Isolated Frozen-thawed Spermatogenic Cells

中文关键词: 牦牛  睾丸组织  玻璃化冷冻  细胞复苏  细胞培养

英文关键词: Yak  Testicular tissues  Vitrification Freezing  Cell thawing  Cell culture

中文摘要:

      采用玻璃化冷冻方法对野外采集的牦牛睾丸组织进行冷冻,通过HE(hematoxylin-eosin)染色分析发现玻璃化冷冻后的牦牛睾丸组织曲细精管结构保存较完好,曲细精管可见大量形态完好的各类生精细胞。采用台盼蓝染色检测细胞活率,发现冷冻复苏后细胞活率可达80.20%。分别通过生精细胞和精原干细胞标志蛋白DDX4和GFRA1免疫荧光染色发现复苏后培养14天后的生精细胞和精原干细胞的数量明显减少。通过RT-qPCR对复苏后不同实验处理组牦牛睾丸细胞标志基因的表达分析,发现培养30天的生精细胞中的精原干细胞标志基因Thy1和UCHL1的表达量显著增高。因此,玻璃化冷冻保存的牦牛睾丸组织中曲细精管及其生精细胞得到了较好的保护,该方法对于其他哺乳动物生精细胞的长久有效保存具有重要的参考价值。

英文摘要:

      Vitrification freezing was employed to preserve the field sampled testicular tissues of yak. Hematoxylin-eosin staining of the frozen testicular tissues indicated that the structure of seminiferous tubules of the testicular tissue was well preserved and a large number of spermatogenic cells in different developmental stages were morphologically intact. Trypan blue stainingSof the frozen-thawed testicular cells showed that the viability rate of the cells was 80.20 %. Immunofluorescence staining of the spermatogenic cells and spermatogenic stem cells by the cell marker DDX4 and GFRA1 respectively revealed that the number of spermatogenic stem cells was significantly reduced after culturing of the frozen-thawed testicular cells for two weeks. RT-qPCR was used to detect the marker gene expressions in the testicular cells in different experimental groups, and the expression of marker genes of spermatogenic stem cells (Thy1 and UCHL1) was significantly upregulated in the spermatogenic cells cultured for 30 days. Therefore, the structure of seminiferous tubules and spermatogenic cells of yak were well preserved in the vitrification frozen testicular tissues, which also provided a valuable freezing method to preserve spermatogenic cells of other mammals.