Fu Chun, Begum Khurshida, Jordan Philip W, He Yan, Overbeek Paul A
Department of Obstetrics and Gynecology, Second Xiangya Hospital, Central South University, Changsha, 410011, China.
Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, TX, 77030, United States of America.
PLoS One. 2016 Aug 3;11(8):e0159800. doi: 10.1371/journal.pone.0159800. eCollection 2016.
After using a self-inactivating lentivirus for non-targeted insertional mutagenesis in mice, we identified a transgenic family with a recessive mutation that resulted in reduced fertility in homozygous transgenic mice. The lentiviral integration site was amplified by inverse PCR. Sequencing revealed that integration had occurred in intron 8 of the mouse Fance gene, which encodes the Fanconi anemia E (Fance) protein. Fanconi anemia (FA) proteins play pivotal roles in cellular responses to DNA damage and Fance acts as a molecular bridge between the FA core complex and Fancd2. To investigate the reduced fertility in the mutant males, we analyzed postnatal development of testicular germ cells. At one week after birth, most tubules in the mutant testes contained few or no germ cells. Over the next 2-3 weeks, germ cells accumulated in a limited number of tubules, so that some tubules contained germ cells around the full periphery of the tubule. Once sufficient numbers of germ cells had accumulated, they began to undergo the later stages of spermatogenesis. Immunoassays revealed that the Fancd2 protein accumulated around the periphery of the nucleus in normal developing spermatocytes, but we did not detect a similar localization of Fancd2 in the Fance mutant testes. Our assays indicate that although Fance mutant males are germ cell deficient at birth, the extant germ cells can proliferate and, if they reach a threshold density, can differentiate into mature sperm. Analogous to previous studies of FA genes in mice, our results show that the Fance protein plays an important, but not absolutely essential, role in the initial developmental expansion of the male germ line.
在使用一种自我失活的慢病毒对小鼠进行非靶向插入诱变后，我们鉴定出一个具有隐性突变的转基因家族，该突变导致纯合转基因小鼠的生育力降低。通过反向PCR扩增慢病毒整合位点。测序显示整合发生在小鼠Fance基因的第8内含子中，该基因编码范可尼贫血E（Fance）蛋白。范可尼贫血（FA）蛋白在细胞对DNA损伤的反应中起关键作用，而Fance作为FA核心复合物与Fancd2之间的分子桥梁。为了研究突变雄性小鼠生育力降低的原因，我们分析了睾丸生殖细胞的出生后发育。出生后一周，突变小鼠睾丸中的大多数曲细精管几乎没有或根本没有生殖细胞。在接下来的2至3周内，生殖细胞在有限数量的曲细精管中积累，因此一些曲细精管的整个周边都含有生殖细胞。一旦积累了足够数量的生殖细胞，它们就开始进入精子发生的后期阶段。免疫分析显示，在正常发育的精母细胞中，Fancd2蛋白在细胞核周边积累，但在Fance突变小鼠的睾丸中我们未检测到Fancd2的类似定位。我们的分析表明，尽管Fance突变雄性小鼠出生时生殖细胞不足，但现存的生殖细胞可以增殖，如果它们达到阈值密度，就可以分化为成熟精子。与先前对小鼠FA基因的研究类似，我们的结果表明，Fance蛋白在雄性生殖系的初始发育扩展中起重要但非绝对必要的作用。
