睾丸高表达蛋白质HSD-14对小鼠精子发生的影响

Study the Effect of a Testis Highly Expressed New Gene HSD-14 on Mouse Spermatogenesis

目的研究睾丸高表达基因HSD-14及其编码蛋白质对小鼠精子发生的影响。方法利用本实验室构建的人睾丸特异ESTs文库,通过电子克隆方法获得一新基因命名为HSD-14基因。纯化原核表达的HSD-14蛋白,并制备兔多克隆抗体。通过HE染色观察腹腔注射HSD-14纯化蛋白或睾丸曲细精管显微注射HSD-14纯化抗体后小鼠睾丸组织的生精变化,并进行TUNEL凋亡检测。结果电子克隆获得HSD-14基因,在原核表达系统中表达成功。小鼠腹腔注射HSD-14纯化蛋白或睾丸曲细精管显微注射HSD-14纯化抗体后,均发现曲细精管中生精细胞大量脱落,生精细胞的层次紊乱,管腔内有时可见成团脱落的生精细胞,附睾中几乎观察不到成熟精子。TUNEL凋亡检测结果表明,抗体注射组小鼠曲细精管中生精细胞(以精母细胞为主)凋亡程度较正常小鼠睾丸和免疫前血清注射组明显,且多数细胞停留在精母细胞甚至精原细胞的阶段。结论HSD-14通过诱导精母细胞凋亡抑制小鼠精子发生。

Objective A testis highly expressed new gene, designated as HSD - 14 and the effect of its encoding protein involved in mouse spermatogenesis were investigated. Methods HSD - 14 was acquired by electronic cloning method. A specific rabbit polyclonal antibody against HSD - 14 was prepared. BALB/c mice were immunized with purified His6 - HSD - 14 or anti - HSD - 14 antibody was injected into mice seminiferous tubules. The pathological change of spermatogenesis was observed by HE staining and apoptosis of germ cells was detected by TUNEL assay. Results The sequence of HSD - 14 was obtained by electronic cloning method. His6 - HSD - 14 protein was expressed in E. Coli BL21 （DE3） and purified by Ni - NTA purification system. Polyclonal antibodies to HSD -14 protein were raised in rabbits. Both of seminiferous tubules of mice immunized with His6 - HSD - 14 protein and mice injected with anti - HSD - 14 antibody through testicular efferent duct were revealed severe pathological disorders. Most of spermatogenic cells were degenerated and exfoliated from seminiferous epithelium into their lumen and few mature sperms were found in epididymis. Degeneration and apoptosis were observed in many germ cells, especially in spermatocytes by TUNEL assay. Conclusion HSD - 14 protein inhibited mouse spermatogenesis by inducing spermatocyte apoptosis.