Turner B Elizabeth, Basecke Sophia M, Bazan Grace C, Dodge Eric S, Haire Cassy M, Heussman Dylan J, Johnson Chelsey L, Mukai Chelsea K, Naccarati Adrianna M, Norton Sunny-June, Sato Jennifer R, Talavera Chihara O, Wade Michael V, Hillers Kenneth J
Department of Chemistry and Biochemistry; California Polytechnic State University ; San Luis Obispo, CA USA ; ; Department of Biological Sciences; California Polytechnic State University ; San Luis Obispo, CA USA.
Department of Biological Sciences; California Polytechnic State University ; San Luis Obispo, CA USA.
Worm. 2015 Feb 9;4(1):e1008903. doi: 10.1080/21624054.2015.1008903. eCollection 2015 Jan-Mar.
Sexual reproduction involves fusion of 2 haploid gametes to form diploid offspring with genetic contributions from both parents. Gamete formation represents a unique developmental program involving the action of numerous germline-specific proteins. In an attempt to identify novel proteins involved in reproduction and embryonic development, we have carried out a proteomic characterization of the process in Caenorhabditis elegans. To identify candidate proteins, we used 2D gel electrophoresis (2DGE) to compare protein abundance in nucleus-enriched extracts from wild-type C. elegans, and in extracts from mutant worms with greatly reduced gonads (glp-4(bn2) worms reared at 25°C); 84 proteins whose abundance correlated with germline presence were identified. To validate candidates, we used feeding RNAi to deplete candidate proteins, and looked for reduction in fertility and/or germline cytological defects. Of 20 candidates so screened for involvement in fertility, depletion of 13 (65%) caused a significant reduction in fertility, and 6 (30%) resulted in sterility (<5 % of wild-type fertility). Five of the 13 proteins with demonstrated roles in fertility have not previously been implicated in germline function. The high frequency of defects observed after RNAi depletion of candidate proteins suggests that this approach is effective at identifying germline proteins, thus contributing to our understanding of this complex organ.
有性生殖涉及两个单倍体配子的融合，形成具有来自双亲遗传贡献的二倍体后代。配子形成代表了一个独特的发育程序，涉及众多生殖系特异性蛋白质的作用。为了鉴定参与生殖和胚胎发育的新蛋白质，我们对线虫（Caenorhabditis elegans）这一过程进行了蛋白质组学表征。为了鉴定候选蛋白质，我们使用二维凝胶电泳（2DGE）比较野生型线虫富含细胞核提取物中的蛋白质丰度，以及性腺大大减少的突变体线虫（在25°C饲养的glp-4(bn2)线虫）提取物中的蛋白质丰度；鉴定出了84种其丰度与生殖系存在相关的蛋白质。为了验证候选蛋白质，我们使用喂食RNA干扰技术来耗尽候选蛋白质，并观察生育力的降低和/或生殖系细胞学缺陷。在如此筛选的20个参与生育力的候选蛋白质中，13个（65%）的耗尽导致生育力显著降低，6个（30%）导致不育（<野生型生育力的5%）。在13个已证明在生育力中起作用的蛋白质中，有5个以前未被认为与生殖系功能有关。在RNA干扰耗尽候选蛋白质后观察到的高缺陷频率表明，这种方法在鉴定生殖系蛋白质方面是有效的，从而有助于我们对这个复杂器官的理解。
