Rucker E B, Dierisseau P, Wagner K U, Garrett L, Wynshaw-Boris A, Flaws J A, Hennighausen L
Laboratory of Genetics and Physiology, National Institute of Diabetes, Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892, USA.
Mol Endocrinol. 2000 Jul;14(7):1038-52. doi: 10.1210/mend.14.7.0465.
Restricted germ cell loss through apoptosis is initiated in the fetal gonad around embryonic day 13.5 (E13.5) as part of normal germ cell development. The mechanism of this germ cell attrition is unknown. We show that Bcl-x plays a crucial role in maintaining the survival of mouse germ cells during gonadogenesis. A bcl-x hypomorphic mouse was generated through the introduction of a neomycin (neo) gene into the promoter of the bcl-x gene by homologous recombination. Mice that contained two copies of the hypomorphic allele had severe reproductive defects attributed to compromised germ cell development. Males with two mutant alleles lacked spermatogonia and were sterile; females showed a severely reduced population of primordial and primary follicles and exhibited greatly impaired fertility. Primordial germ cells (PGCs) in bcl-x hypomorph mice migrated to the genital ridge by E12.5 but were depleted by E15.5, a time when Bcl-x and Bax were present. Two additional bcl-x transcripts were identified in fetal germ cells more than 300 bp upstream of previously reported start sites. Insertion of a neo cassette led to a down-regulation of the bcl-x gene at E12.5 in the hypomorph. Bax was detected by immunohistochemistry in germ cells from bcl-x hypomorph and control testes at E12.5 and E13.5. Bcl-x function was restored, and animals of both genders were fertile after removal of the neo selection cassette using Cre-mediated recombination. Alternatively, the loss of Bcl-x function in the hypomorph was corrected by the deletion of both copies of the bax gene, resulting in a restoration of germ cell survival. These findings demonstrate that the balance of Bcl-x and Bax control PGC survival and apoptosis.
作为正常生殖细胞发育的一部分，通过凋亡导致的生殖细胞损失受限在胚胎第13.5天（E13.5）左右的胎儿性腺中开始。这种生殖细胞损耗的机制尚不清楚。我们发现，Bcl-x在性腺发生过程中维持小鼠生殖细胞的存活方面起着关键作用。通过同源重组将新霉素（neo）基因引入bcl-x基因的启动子中，产生了bcl-x低表达小鼠。含有两个低表达等位基因拷贝的小鼠由于生殖细胞发育受损而出现严重的生殖缺陷。具有两个突变等位基因的雄性小鼠缺乏精原细胞且不育；雌性小鼠的原始卵泡和初级卵泡数量大幅减少，生育能力严重受损。bcl-x低表达小鼠中的原始生殖细胞（PGCs）在E12.5时迁移至生殖嵴，但在E15.5时耗尽，此时Bcl-x和Bax均存在。在胎儿生殖细胞中，在先前报道的起始位点上游300多个碱基对处鉴定出另外两种bcl-x转录本。neo盒的插入导致低表达小鼠在E12.5时bcl-x基因下调。在E12.5和E13.5时，通过免疫组织化学在bcl-x低表达和对照睾丸的生殖细胞中检测到Bax。使用Cre介导的重组去除neo选择盒后，Bcl-x功能得以恢复，雌雄动物均具有生育能力。或者，通过缺失bax基因的两个拷贝来纠正低表达小鼠中Bcl-x功能的丧失，从而恢复生殖细胞的存活。这些发现表明，Bcl-x和Bax的平衡控制着原始生殖细胞的存活和凋亡。
