A Study Of Testis Protein Expression Profiling

As the major male gonad, the function of testis is spermatogenesis and produce androgen. Spermatogenesis is a process of development from undifferentiated spermatogonia to sperm, which include the mitosis of spermatogonia and meiosis of spermatocyte to produce haploid round spermatids and subsequent spermiogenesis from round spermatids to sperm. These strict and complex programmed processes need lots of genes and proten coordinately expressed and regulated. Because in testis many proteins are regulated in the translational level, it is of significance to study the testis proteome in the system level.We used one-dimensional SDS-PAGE and liquid chromatography to separate human testis protein and identified human testis proteome with 1430 proteins by advanced LTQ Orbitrap, in which 39 were testis specific and may be important for testis function. Domain analysis of these proteins revealed three large protein families which are RRM protein family, Rab protein family and Arf protein family. Pathway analysis of RRM domain containing proteins showed that many of them function in alternative splicing, which may also be involved in testis-specific splicing events. Additinoally, Rab and Arf protein families were annotated to vesicle events and possibly function in acrosome reaction. Compared with the studies on brain proteome, research on the testis proteome is still very limited. Study of these proteins will give a better understanding on the function of the testis.To better characterize the process of spermatogenesis, we constructed the proteomes of spermatogenic cells in advance. Due to the restriction of human sample, we used mouse, which is a frequently used mammalian model studying spermatogenesis. After purifying spermatid/spermatozoa with 1C DNA content and spermatogenic cells with 4C DNA content using flow cytometry from mouse testis, we construct a spermatid/spermatozoa proteome of 2116 proteins involving in spermiogenesis and a 4C spermatogenic cell proteome of 3507 proteins involving in the first stage of meiosis based on similar strategy used in human testis proteome construction.In the testis spermatid/spermatozoa proteome, we found 299 testis specific and 157 novel proteins. Bionformatics analysis of these proteins showed many proteins possibly functioning in unique processes of spermiogenesis. In the 4C spermatogenic cell proteome, bioinformatics analysis showed the existence of 216 proteins with orthologs in yeast essential for meiosis. They are annotated to participate in various events in cycle, including pachytene regulation and DNA recombination. Interation analysis of these proteins showed complex regulation relationship between them. Chromosome distribution analysis of these two proteomes showed hyporepresentation of proteins identified on X chromosome, which may be due to meiotic sex chromosome inactivation. Comparison between these two proteomes showed that most proteins from spermatid/spermatozoa proteome were already expressed in 4C spermatogenic cells, in which testis-specific ones were related to male gamete generation. Additionally, proteins identified only in 4C spermatogenic cells functions related to cell cycle and possibly important for meiosis regulation. And proteins identified only in spermatid/spermatozoa function mainly in dramatic structural changes during spermiogenesis.It has been known that there is translational suppression in testis, microRNA as a member of non-coding small RNAs, which have been studied a lot recently, can regulate protein translation through diverse machanisms. We studied the distribution and evolution of genome-wide microRNAs in mammals, and found higher substitution rate of X-linked testis microRNAs compared with autosomal testis ones using human and mouse orthologous microRNAs. X-linked testis mature microRNAs have an average substitution rate between mouse and human almost 25 fold as high as testis microRNAs found on autosomes, unlike microRNAs with precursors not expressed in testis, where no significant difference in the substitution rate between the X chromosome and autosomes was found. Additionally, the testis precursor microRNAs also evolved faster than testis autosomal ones, which was also observed in the branches of rodent and primates in mammals. Functional analysis of target genes of X-linked testis microRNAs with conserved seed region in mammals showed that they may function in regulation of mitosis of spermatogonia and/or meiosis of spermatocytes, and regulate cell cycle-related proteins from human testis proteome, and consequently be important for the regulation of spermatogenesis.This large-scale identification of testis and spermatogenic cell proteins could serve as a reference for future studies on the mechanisms underlying male infertility, searching for potential contraceptive targets, and developing new treatments for testis cancer. And functional analysis of testis microRNA implied their possible important function in testis function, and in-depth study could help explain the phenomenon of translational suppresion and help better study on the level of proteins.