不同温度对体外培养精原细胞精子发生相关基因的影响

Influence of temperature on spermatogenesis-related genes in spermatogonia cultured in vitro

目的:在32℃、37℃温度下体外培养生精细胞,探讨两种温度对精原细胞增殖相关基因的影响,为进一步探讨体腔温度(37℃)导致精子发生障碍的机制提供依据。方法:将分离的大鼠生精细胞分别置于不同温度(32℃、37℃)条件下与支持细胞进行体外共培养,观察其贴壁、增殖、形态学变化;培养第8天采用非连续Percoll密度梯度离心、差异性贴壁分离富集出精原细胞,以实时定量PCR和Western印迹分别检测不同温度下精原细胞c-kit、PI3K的mRNA和蛋白表达水平的变化;对体外培养第8天的精原细胞c-kit基因进行测序检测,了解其突变情况。结果:32℃组、37℃组均可见细胞贴壁、分裂、增殖;32℃组中,精原细胞c-kit和PI3K的mRNA及蛋白表达量均显著高于37℃组(P<0.05);基因测序结果显示32℃组c-kit基因第9、11、13号外显子均未见突变,37℃组第11、13号外显子未见突变,在9号外显子附近区域可能有缺失或插入突变。结论:在体外37℃温度下培养生精细胞会抑制精原细胞增殖分化相关基因的表达,同时可能导致精原细胞c-kit基因发生突变。

Objective： To investigate the influence of temperature on the expressions of c-kit and PI3K in spermatogonia cultured in vitro at 32 ℃ and 37 ℃,and provide basic scientific data for the mechanism of spermatogenic impairment due to body temperature（37 ℃）. Methods： Isolated spermatogenic cells were cultured in vitro at 32 ℃ and 37 ℃,and their adherence,proliferation and morphologic changes were observed and recorded under the inverted phase contrast microscope.At 8 days,the spermatogonia were separated by Percoll density gradient centrifugation and the differential adhesion method.The expressions of c-kit and PI3K mRNA and proteins in the separated cells were detected by real time polymerase chain reaction and Western blot,respectively.The c-kit gene was sequenced to identify the occurrence of mutations.Results： Adherence,division and proliferation of the cells were observed in both the 32 ℃ and 37 ℃ groups.The expressions of c-kit and PI3K mRNA and proteins in the spermatogonia were significantly higher in the 32 ℃ group than in the 37 ℃ group（P0.05）.The 32 ℃ group showed no mutation of c-kit in exon 9,11 and 13;the 37 ℃ group exhibited no mutation in exon 11 and 13,but possible insertion or deletion mutations in exon 9.Conclusion： Culturing in vitro at 37 ℃ could inhibit the expression of proliferation-and differentiation-related genes in spermatogenic cells and lead to the mutation of the c-kit gene.