Fscb/Cabyr双基因敲除对雄鼠繁殖能力的影响

Effects of Fscb/Cabyr double gene knockout on reproductive capacity in male mice

目的观察Fscb/Cabyr双基因和单基因敲除雄鼠繁殖能力,探讨CABYR蛋白及与FSCB的复合物对于维持精子授精能力的价值及其发挥生理功能的可能机制。方法取Fscb/Cabyr(--/--)双基因敲除纯合子雄鼠、Fscb/Cabyr(++/--)单基因敲除雄鼠及野生型雄鼠分别与同代野生型雌鼠交配,观察各组雌鼠怀孕、产仔情况;应用Fluo-4 AM钙离子荧光探针对不同基因型雄鼠精子进行标记,荧光显微镜下观察并应用酶标仪检测各基因型小鼠精子中钙离子水平;Western blot检测各基因型小鼠精子中各种CATSPER通道蛋白的表达情况。结果所有Fscb/Cabyr(--/--)双基因敲除与Cabyr单基因敲除纯合子雄鼠繁殖能力完全丧失,均未能使任何雌鼠怀孕和产仔。与野生型小鼠相比,Fscb/Cabyr(--/--)及Fscb/Cabyr(++/--)基因型雄鼠精子内钙离子浓度显著降低(P<0.0001);Fscb/Cabyr(--/--)型小鼠精子中的CATSPER-1蛋白表达显著减少。Fscb/Cabyr(--/--)、Fscb/Cabyr(++/--)型小鼠精子中的CATSPER-4蛋白表达显著减低。结论雄鼠精子CABYR及FSCB/CABYR蛋白复合物的缺失导致雄鼠繁殖功能的丧失,可能是通过影响精子内的钙通道蛋白CATSPER-1和CATSPER-4的表达和钙离子浓度水平,从而影响精子的运动功能而实现的。

Objective To evaluate the value of CABYR protein and its complex with FSCB in maintaining sperm fertilization ability and investigate the possible mechanism of physiological function of the coruplex by observing the natural fertility of Fscb/Cabyr double gene knockout and Cabyr single gene knockout male mice.Methods Fscb/Cabyr(--/--)double gene knockout male mice,Fscb/Cabyr(++/--)single gene knockout male mice and wild background male mice were mated with wild-type female mice respectively,and the pregnancy and litter production of female mice in each group were observed.The sperm of male mice with different genotypes were labeled with Fluo-4 AM calcium fluorescent probe in vitro and observed under fluorescence microscope,and the level of calcium ion in the sperm were detected by ELISA;The expression of CATSPER channel proteins in sperm of mice with different genotypes was measured by Western blotting.Results All the male mice with Fscb/Cabyr(--/--)double gene knockout and Fscb/Cabyr(++/--)single gene knockout lost their reproductive capacity completely,and failed to make any female mice pregnant and give birth.Compared with the wild-type male mice,the sperm calcium ion concentration of Fscb/Cabyr(--/--)and Fscb/Cabyr(++/--)male mice was decreased significantly(P<0.0001);The expression of CATSPER-1 protein in the sperm of Fscb/Cabyr(--/--)mice was decreased obviously.The expression of CATSPER-4 in the sperm of Fscb/Cabyr(--/--)and Fscb/Cabyr(++/--)mice were notably decreased.Conclusion The deletion of either CABYR or FSCB/CABYR protein complex in male sperm leads to the loss of reproductive function in the male mice,which may be realized by affecting the expression of calcium channel proteins CATSPER-1 and CATSPER-4,concentration of calcium ions in sperm,and then motility function of sperm.