Molecular Biology of Mammalian Germ Cells

斑点玻璃微阵列和基因表达系列分析（SAGE）已经成功地结合起来，以确定下游的FIGLA目标。利用Figla基因缺失小鼠和NIA 22 K微阵列（其含有在卵母细胞和早期发育中富集表达的元件），在从E12.5到新生儿的四个胚胎时间点分析卵巢基因表达。然而，微阵列在制造过程中受到玻璃上斑点的元素的限制。因此，为了拓宽对潜在的直接或间接下游基因靶标的搜索，从分离自新生儿正常卵巢和Figla无效卵巢的poly（A）+ RNA构建SAGE文库。紧邻3个引物的Sau 3A 1限制酶切割位点的10个碱基标签用于鉴定上调和下调的转录物。FIGLA差异调节的基因的进一步表征应证明在定义影响出生后雌性生殖细胞的发育途径中是有用的。这些目标不仅代表影响卵泡发生和受精的基因，而且还代表成功完成小鼠早期发育所需的母体效应基因。 在鉴定FIGLA的另外下游靶标的替代方法中，使用二维凝胶电泳来比较从正常小鼠和FIGLA缺失小鼠分离的新生卵巢的蛋白质表达谱。差异表达的蛋白质进行了鉴定，通过微量质谱和差异进行了确认，在转录水平上的定量RT-PCR。出乎意料的是，二十个睾丸特异性蛋白质在Figla null新生儿卵巢中过表达，表明FIGLA不仅是卵母细胞特异性基因的激活剂，而且是睾丸特异性基因的阻遏物，在雌性性腺发育过程中。为了进一步研究这一假设，建立了在雄性生殖细胞中异位表达FIGLA的转基因小鼠系。尽管表达FIGLA的可育转基因雄性小鼠的生殖力降低，但随着小鼠年龄的增长，在生精小管内偶尔观察到空泡，这与生殖细胞凋亡的斑块一致。总的来说，这些数据表明，FIGLA作为卵母细胞特异性基因和睾丸特异性基因的抑制物的转录激活起着关键作用，这些基因是雌性性腺成功发育所必需的。

Spotted-glass microarrays and Serial Analysis of Gene Expression (SAGE) have been combined successfully to identify downstream targets of FIGLA. Taking advantage of Figla null mice and the NIA 22K microarray that contains elements enriched for expression in oocyte and early development, ovarian gene expression was profiled at four embryonic time points from E12.5 to newborn. However, microarrays are limited by the elements spotted on glass during their fabrication. Therefore, to broaden the search for potential direct or indirect downstream gene targets, SAGE libraries were constructed from poly(A)+ RNA isolated from newborn normal and Figla null ovaries. A 10 base tag immediately adjacent to the 3 prime most Sau3A1 restriction enzyme cleavage site was used to identify both up-regulated and down-regulated transcripts. The further characterization of the genes that are differentially regulated by FIGLA should prove useful in defining developmental pathways that affect the postnatal female germ cell. These targets represent not only genes that affect folliculogenesis and fertilization, but also maternal effect genes required for successful completion of early mouse development. In an alternative approach to identify additional downstream targets of FIGLA, two-dimensional gel electrophoresis was used to compare the protein expression profiles of newborn ovaries isolated from normal and Figla null mice. Differentially expressed proteins were identified by microscale mass spectrometry and differences were confirmed at a transcriptional level by quantitative RT-PCR. Unexpectedly, twenty testis-specific proteins were over-expressed in Figla null newborn ovaries suggesting that FIGLA is not only an activator of oocyte-specific genes, but also a repressor of testis-specific genes, during female gonadogenesis. To further investigate this hypothesis, transgenic mouse lines ectopically expressing FIGLA in male germ cells were established. Although fertile, transgenic male mice expressing FIGLA had decreased fecundity and as mice aged, vacuoles were sporadically observed within seminiferous tubules consistent with patches of germ cell apoptosis. Collectively, these data indicate that FIGLA plays a critical role as the transcriptional activating of oocyte-specific and repressor of testis-specific genes necessary for successful development of the female gonad.