Higgy N A, Zackson S L, van der Hoorn F A
Department of Medical Biochemistry, University of Calgary Health Sciences Centre, Alberta, Canada.
Dev Genet. 1995;16(2):190-200. doi: 10.1002/dvg.1020160211.
Possible functions of the c-mos proto-oncogene during spermatogenesis were investigated through perturbations of its expression in transgenic mice. Two promoters, one from the pre-meiotic male germ cell-specific mouse phosphoglycerate kinase 2 gene, and the other from the post-meiotic male germ cell-specific rat RT7 gene were used to direct expression of c-mos. Northern blot analysis of testis RNA from transgenic PGK-c-mos mice indicated elevated levels of c-mos RNA in spermatocytes and spermatids compared to controls. No transgene expression was detected in any other tissue examined, suggesting that the mouse PGK2 promoter, like the previously used human PGK2 promoter, confers correct cell-specific expression onto c-mos. The promoter from a newly characterized rat gene, RT7, was shown to direct expression specific to post-meiotic spermatids. Transgenic mice carrying an RT7-lacZ construct displayed immunoreactive bacterial beta-galactosidase as well as enzyme activity in round spermatids. The cellular specificity for beta-galactosidase expression observed in RT7-lacZ transgenic animals was in agreement with endogenous RT7 transcript expression. Northern blot analysis of testis RNA of RT7-c-mos transgenic mice showed elevated levels of c-mos in spermatids, but not in other cells or tissues examined. Western blot analysis demonstrated elevated levels of p43c-mos in spermatids of both PGK-c-mos and RT7-c-mos transgenic animals, but only PGK-c-mos transgenics had increased p43c-mos levels in spermatocytes. Both RT7-c-mos and PGK-c-mos transgenic mice are fertile and show no tendency toward transformation. RT7-c-mos mice have no discernible phenotype associated with the c-mos overexpression in spermatids. However, PGK-c-mos transgenic males exhibited a significant increase in germ cell number, as determined by cell counts using total germ cells and germ cells fractionated by centrifugal elutriation. Because mitotic divisions of germ cells occur prior to PGK-c-mos transgene expression, our observations suggest that c-mos overexpression in spermatocytes causes an alteration in cell-cell interactions.
通过干扰转基因小鼠中c-mos原癌基因的表达，研究了其在精子发生过程中的可能功能。使用了两个启动子，一个来自减数分裂前雄性生殖细胞特异性的小鼠磷酸甘油酸激酶2基因，另一个来自减数分裂后雄性生殖细胞特异性的大鼠RT7基因，来指导c-mos的表达。对转基因PGK-c-mos小鼠睾丸RNA的Northern印迹分析表明，与对照组相比，精母细胞和精子细胞中c-mos RNA水平升高。在检查的任何其他组织中均未检测到转基因表达，这表明小鼠PGK2启动子与先前使用的人PGK2启动子一样，赋予c-mos正确的细胞特异性表达。来自新鉴定的大鼠基因RT7的启动子被证明可指导减数分裂后精子细胞特异性表达。携带RT7-lacZ构建体的转基因小鼠在圆形精子细胞中显示出免疫反应性细菌β-半乳糖苷酶以及酶活性。在RT7-lacZ转基因动物中观察到的β-半乳糖苷酶表达的细胞特异性与内源性RT7转录本表达一致。对RT7-c-mos转基因小鼠睾丸RNA的Northern印迹分析显示，精子细胞中c-mos水平升高，但在检查的其他细胞或组织中未升高。蛋白质印迹分析表明，PGK-c-mos和RT7-c-mos转基因动物的精子细胞中p43c-mos水平均升高，但只有PGK-c-mos转基因动物的精母细胞中p43c-mos水平升高。RT7-c-mos和PGK-c-mos转基因小鼠均能生育，且无转化倾向。RT7-c-mos小鼠没有与精子细胞中c-mos过表达相关的可识别表型。然而，通过使用总生殖细胞和通过离心淘析分离的生殖细胞进行细胞计数确定，PGK-c-mos转基因雄性生殖细胞数量显著增加。由于生殖细胞的有丝分裂发生在PGK-c-mos转基因表达之前，我们的观察结果表明，精母细胞中c-mos过表达会导致细胞间相互作用的改变。
