PDF(Pubmed)
Abstract：
BACKGROUND: Testis is the only organ supporting sperm production and with the largest number of proteins and tissue-specific proteins in animals. In our previous studies, we have found that knockdown of ocnus (ocn), a testis-specific gene, resulted in much smaller testis with no germ cells in Drosophila melanogaster. However, the molecular consequences of ocn knockdown in fly testes are unknown.
RESULTS: In this study, through iTRAQ quantitative proteomics sequencing, 606 proteins were identified from fly abdomens as having a significant and at least a 1.5-fold change in expression after ocn knockdown in fly testes, of which 85 were up-regulated and 521 were down-regulated. Among the differential expressed proteins (DEPs), apart from those proteins involved in spermatogenesis, the others extensively affected biological processes of generation of precursor metabolites and energy, metabolic process, and mitochondrial transport. Protein-protein interaction (PPI) analyses of DEPs showed that several kinases and/or phosphatases interacted with Ocn. Re-analyses of the transcriptome revealed 150 differential expressed genes (DEGs) appeared in the DEPs, and their changing trends in expressions after ocn knockdown were consistent. Many common down-regulated DEGs and DEPs were testis-specific or highly expressed in the testis of D. melanogaster. Quantitative RT-PCR (qRT-PCR) confirmed 12 genes appeared in both DEGs and DEPs were significantly down-regulated after ocn knockdown in fly testes. Furthermore, 153 differentially expressed phosphoproteins (DEPPs), including 72 up-regulated and 94 down-regulated phosphorylated proteins were also identified (13 phosphoproteins appeared in both up- and down-regulated groups due to having multiple phosphorylation sites). In addition to those DEPPs associated with spermatogenesis, the other DEPPs were enriched in actin filament-based process, protein folding, and mesoderm development. Some DEPs and DEPPs were involved in Notch, JAK/STAT, and cell death pathways.
CONCLUSIONS: Given the drastic effect of the ocn knockdown on tissue development and testis cells composition, the differences in protein abundance in the ocn knockdown flies might not necessarily be the direct result of differential gene regulation due to the inactivation of ocn. Nevertheless, our results suggest that the expression of ocn is essential for Drosophila testis development and that its down-regulation disturbs key signaling pathways related to cell survival and differentiation. These DEPs and DEPPs identified may provide significant candidate set for future studies on the mechanism of male reproduction of animals, including humans.
RESULTS: In this study, through iTRAQ quantitative proteomics sequencing, 606 proteins were identified from fly abdomens as having a significant and at least a 1.5-fold change in expression after ocn knockdown in fly testes, of which 85 were up-regulated and 521 were down-regulated. Among the differential expressed proteins (DEPs), apart from those proteins involved in spermatogenesis, the others extensively affected biological processes of generation of precursor metabolites and energy, metabolic process, and mitochondrial transport. Protein-protein interaction (PPI) analyses of DEPs showed that several kinases and/or phosphatases interacted with Ocn. Re-analyses of the transcriptome revealed 150 differential expressed genes (DEGs) appeared in the DEPs, and their changing trends in expressions after ocn knockdown were consistent. Many common down-regulated DEGs and DEPs were testis-specific or highly expressed in the testis of D. melanogaster. Quantitative RT-PCR (qRT-PCR) confirmed 12 genes appeared in both DEGs and DEPs were significantly down-regulated after ocn knockdown in fly testes. Furthermore, 153 differentially expressed phosphoproteins (DEPPs), including 72 up-regulated and 94 down-regulated phosphorylated proteins were also identified (13 phosphoproteins appeared in both up- and down-regulated groups due to having multiple phosphorylation sites). In addition to those DEPPs associated with spermatogenesis, the other DEPPs were enriched in actin filament-based process, protein folding, and mesoderm development. Some DEPs and DEPPs were involved in Notch, JAK/STAT, and cell death pathways.
CONCLUSIONS: Given the drastic effect of the ocn knockdown on tissue development and testis cells composition, the differences in protein abundance in the ocn knockdown flies might not necessarily be the direct result of differential gene regulation due to the inactivation of ocn. Nevertheless, our results suggest that the expression of ocn is essential for Drosophila testis development and that its down-regulation disturbs key signaling pathways related to cell survival and differentiation. These DEPs and DEPPs identified may provide significant candidate set for future studies on the mechanism of male reproduction of animals, including humans.
摘要：
背景：睾丸是唯一支持精子产生的器官，并且在动物中具有最多数量的蛋白质和组织特异性蛋白质。在我们之前的研究中,我们已经发现了ocnus(ocn)的击倒，睾丸特异性基因,导致果蝇的睾丸小得多，没有生殖细胞。然而,飞睾丸中ocn击倒的分子后果是未知的。
结果：在这项研究中，通过iTRAQ定量蛋白质组学测序，从蝇腹部鉴定出606种蛋白质，在蝇睾丸中的ocn击倒后，表达发生了显着且至少1.5倍的变化，其中85个上调，521个下调。在差异表达蛋白（DEP）中，除了那些参与精子发生的蛋白质，其他广泛影响前体代谢物和能量产生的生物过程，代谢过程，和线粒体运输。DEP的蛋白质-蛋白质相互作用（PPI）分析表明，几种激酶和/或磷酸酶与Ocn相互作用。转录组的重新分析显示，DEP中出现了150个差异表达基因（DEGs），它们在ocn击倒后的表达变化趋势是一致的。许多常见的下调的DEGs和DEP是睾丸特异性的或在黑腹D.melanogaster的睾丸中高度表达。定量RT-PCR（qRT-PCR）证实，在蝇睾丸中的ocn敲低后，DEGs和DEP中出现的12个基因显着下调。此外，153种差异表达磷蛋白（DEPPs），还鉴定出包括72个上调和94个下调的磷酸化蛋白(13个磷蛋白由于具有多个磷酸化位点而出现在上调和下调组中)。除了那些与精子发生相关的DEPPs，其他DEPP富含基于肌动蛋白丝的过程，蛋白质折叠，和中胚层发育。一些DEP和DEPP参与了Notch，JAK/STAT,和细胞死亡途径。
结论：鉴于ocn敲低对组织发育和睾丸细胞组成的巨大影响，由于ocn失活，ocn击倒果蝇中蛋白质丰度的差异不一定是差异基因调控的直接结果。然而，我们的研究结果表明，ocn的表达对果蝇睾丸发育至关重要，其下调会干扰与细胞存活和分化相关的关键信号通路。确定的这些DEP和DEPP可能为未来研究动物雄性繁殖机制提供重要的候选集，包括人类。
结果：在这项研究中，通过iTRAQ定量蛋白质组学测序，从蝇腹部鉴定出606种蛋白质，在蝇睾丸中的ocn击倒后，表达发生了显着且至少1.5倍的变化，其中85个上调，521个下调。在差异表达蛋白（DEP）中，除了那些参与精子发生的蛋白质，其他广泛影响前体代谢物和能量产生的生物过程，代谢过程，和线粒体运输。DEP的蛋白质-蛋白质相互作用（PPI）分析表明，几种激酶和/或磷酸酶与Ocn相互作用。转录组的重新分析显示，DEP中出现了150个差异表达基因（DEGs），它们在ocn击倒后的表达变化趋势是一致的。许多常见的下调的DEGs和DEP是睾丸特异性的或在黑腹D.melanogaster的睾丸中高度表达。定量RT-PCR（qRT-PCR）证实，在蝇睾丸中的ocn敲低后，DEGs和DEP中出现的12个基因显着下调。此外，153种差异表达磷蛋白（DEPPs），还鉴定出包括72个上调和94个下调的磷酸化蛋白(13个磷蛋白由于具有多个磷酸化位点而出现在上调和下调组中)。除了那些与精子发生相关的DEPPs，其他DEPP富含基于肌动蛋白丝的过程，蛋白质折叠，和中胚层发育。一些DEP和DEPP参与了Notch，JAK/STAT,和细胞死亡途径。
结论：鉴于ocn敲低对组织发育和睾丸细胞组成的巨大影响，由于ocn失活，ocn击倒果蝇中蛋白质丰度的差异不一定是差异基因调控的直接结果。然而，我们的研究结果表明，ocn的表达对果蝇睾丸发育至关重要，其下调会干扰与细胞存活和分化相关的关键信号通路。确定的这些DEP和DEPP可能为未来研究动物雄性繁殖机制提供重要的候选集，包括人类。
