低氧条件下小鼠精母细胞中热休克蛋白A2及几种热休克转录因子的表达变化

Expressions of heat shock protein A2 and several heat shock factors in mouse spermatocytes under hypoxia

目的探索低氧条件下精子发育功能障碍的可能机制。方法使用6孔细胞培养板常规传代培养小鼠精母细胞GC-2至对数生长期,选择8块细胞培养板,随机等分为低氧组和对照组。将低氧组与对照组分别培养于Inviv O_(2)工作站(参数设置:1%O_(2);5%CO_(2);94%N_(2);37℃)和温、湿度恒定的二氧化碳(5%)培养箱中,低氧处理6、12、24及48 h后,各随机取出1块细胞培养板作为对应时间梯度的低氧组及其对照组,将低氧组6孔细胞再随机等分为两个小组,其中3孔用于提取总RNA,另3孔用于提取总蛋白,对照组作相同处理。分别通过实时荧光定量PCR技术和western blot技术检测GC-2中HSPA2、HSF1及HSF2基因表达。结果HSPA2基因mRNA水平仅在低氧处理24 h后一过性升高(P<0.05),但低氧处理6、12及48 h后,均未见明显变化(P>0.05)。HSPA2蛋白在低氧处理6 h后未见明显变化(P>0.05),但低氧处理12、24及48 h后显著降低(P<0.05)。HSF1基因mRNA水平在低氧处理6及12 h后显著降低(P<0.05),低氧处理24 h后无明显变化(P>0.05),但在低氧处理48 h后显著上升(P<0.05)。HSF1蛋白在低氧处理6、12、24及48 h后均显著降低(P<0.05)。HSF2基因mRNA在低氧处理6、12、24及48 h后均未发生显著变化(P>0.05),而其对应的HSF2蛋白在低氧处理6、12及24 h后也未见明显差异(P>0.05),但在低氧处理48 h后显著降低(P<0.05)。结论低氧条件下,HSPA2、HSF1、HSF2基因表达均发生显著变化,HSPA2、HSF1、HSF2可能都是低氧致精子发育功能障碍的关键介导因子。

Objective To investigate the underlying mechanism by which hypoxia caused sperm developmental dysfunction.Methods Mouse spermatocytes GC-2 were conventionally sub-cultured in 6-well plates and grew to the logarithmic growth stage.8 cell culture plates were selected and randomly divided into the hypoxia group and the control group.The cells in the hypoxia group and the control group were cultured in a Inviv O,work station in 1%O_(2),5%CO_(2)and 94%N_(2)at 37℃and a constant temperature and humidity of 5%carbon dioxide incubator at 37℃,respectively.After hypoxia treatment for6,12,24 and 48 h,one cell culture plate was randomly taken from the Inviv O_(2) work station and the control from the carbon dioxide incubator.The cells in the hypoxia group and the control group were all randomly divided into two parts,of which one was used to extract total RNA and the other to extract total protein.The expressions of HSPA2,HSF1 and HSF2 in CC-2 were detected by real-time quantitative PCR and western blot.Results A transient increase in HSPA2 mRNA levels was found in GC-2 cells under hypoxia treatment for 24 h(P<0.05),but no significant changes were observed at the other time points(6,12 and 48 h)(P>0.05).No significant change in HSPA2 protein was found in GC-2 cells under hypoxia treatment for 6 h(P>0.05),but significant decreases at the other time points(12,24 and 48 h)were observed(P<0.05).HSF1 mRNA expressions were significantly decreased in GC-2 cells under hypoxia treatment for 6 and 12 h(P<0.05),and no obvious change at the time point of 24 h was observed(P>0.05),but a significant increase was detected at the time point of 48 h(P<0.05).HSF1 protein levels were all significantly downregulated under hypoxia treatment at four time points(6,12,24 and 48 h)(P<0.05).HSF2 mRNA expressions had no significant changes in GC-2 cells at all four time points(6,12,24 and 48 h)(P>0.05),and HSF2 protein levels also showed no significant changes under hypoxia treatment at three time points(6,12 and 24 h)(P>0.05),but a significant decrease occurred at the time point of 48 h(P<0.05).Conclusion HSPA2,HSFI and HSF2 expressions in GC-2 cells are all significantly changed under hypoxia,and they may participate in hypoxia-caused sperm developmental dysfunction.