Abstract
Mammalian spermatogenesis consists of many cell types and biological processes and serves as an excellent model for studying gene regulation at transcriptional and post-transcriptional levels. Many key proteins, miRNAs, and perhaps piRNAs have been shown to be involved in post-transcriptional regulation of spermatogenesis. However, a systematic method for assessing the relationship between protein and mRNA expression has not been available for studying mechanisms of post-transcriptional regulation. In the present study, we used the iTRAQ-based quantitative proteomic approach to identify 2008 proteins in mouse type A spermatogonia, pachytene spermatocytes, round spermatids, and elongative spermatids with high confidence. Of these proteins, 1194 made up four dynamically changing clusters, which reflect the mitotic amplification, meiosis, and post-meiotic development of germ cells. We identified five major regulatory mechanisms termed "transcript only," "transcript degradation," "translation repression," "translation de-repression," and "protein degradation" based on changes in protein level relative to changes in mRNA level at the mitosis/meiosis transition and the meiosis/post-meiotic development transition. We found that post-transcriptional regulatory mechanisms are related to the generation of piRNAs and antisense transcripts. Our results provide a valuable inventory of proteins produced during mouse spermatogenesis and contribute to elucidating the mechanisms of the post-transcriptional regulation of gene expression in mammalian spermatogenesis.
Highlights
From the ‡State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100080, P
PiRNAs are a unique set of small RNAs expressed in germ cells and associated with PIWI proteins including MIWI, MILI, and MIWI2. piRNAs generated at different stages of spermatogenesis have different features. piRNAs produced by primordial germ cells (PGCs) and spermatogonia map to repeat sequences and play important roles in retrotransposon control at the levels of RNA metabolism and DNA methylation [13]. piRNAs are processed from mRNAs mainly in spermatogonia and from long precursor RNAs transcribed from the intergenic regions in spermatocytes and spermatids [14]
It is still unclear whether these piRNAs have biological functions or if they are instead products of RNA metabolism. It is well-documented that proteins related to piRNA production—such as MILI, MIWI, and MIWI2—are essential for mouse spermatogenesis because their gene knockouts result in spermatogenesis arrest at multiple stages [15]. piRNAs and/or PIWI proteins may be involved in translational regulation because they are detected in RNP, monosomes, and polysomes [16, 17]
Summary
These data indicated discrepancy between mRNA and protein level changes, suggesting the existence of the post-transcriptional regulation of gene expression. Functional annotation analysis of the 336 genes showed that they were involved in “sexual reproduction,” “flagellum,” “spermatogenesis,” “glycolysis,” and “monosaccharide metabolic process.” Consistently, while only a very small fraction of proteins in Clusters 0ϳ3 belonged to the “translation repression” set at SG-A/pacSC, more than half in Cluster 4 (139/253) were translationally repressed in pacSC, the majority of which (83/139) were derepressed in rST.
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