•We established the acetylproteome of capacitated human sperm.•Western blot was used to analyze the expression of lysine-acetylated proteins.•Synthetic peptides were used to verify proteomics data.•Experimental evidences show essential roles of acetylation in sperm functions.Protein lysine acetylation is a dynamic and reversible post-modification that is known to play diverse functions in eukaryotes. Nevertheless, the composition and function of non-histone lysine acetylation in gametes remain unknown. In humans, only capacitated sperm have the capacity to fertilize an egg. In the present study, we found complex composition of lysine acetylated proteins in capacitated human sperm. In vitro fertilization inhibition assay by anti-acetyllysine antibody showed essential roles of lysine acetylation in fertilization. And inhibition of lysine deacetylases, the histone deacetylases, by trichostatin A and nicotinamide, could significantly suppress sperm motility. After immunopurification enrichment of acetylpeptides with anti-acetyllysine antibody and high-throughput liquid chromatography–tandem mass spectrometry identification, we characterized 1206 lysine acetylated sites, corresponding to 576 lysine acetylated proteins in human capacitated sperm. Bioinformatics analysis showed that these proteins are associated with sperm functions, including motility, capacitation, acrosome reaction and sperm–egg interaction. Thus, lysine acetylation is expected to be an important regulatory mechanism for sperm functions. And our characterization of lysine acetylproteome could be a rich resource for the study of male fertility.Biological significanceMature sperm are almost transcriptionally and translationally silent, thus post-translational modifications play important roles in sperm functions. Till now, only two types of PTMs, phosphorylation and glycosylation, are well studied in normal human sperm based on large scale proteomics. In the present study, we established the acetylproteome of capacitated human sperm. Over 1000 lysine acetylated sites were identified. Bioinformatics analysis shows that lysine acetylated proteins participate in many biological events of sperm functions. We further provided functional data that the lysine acetylation is essential for sperm motility and fertilization using histone acetylase inhibitors and anti-acetyllysine antibody. These data can be strong evidences for the important function of lysine acetylation in human sperm.

Ovarian physiology and pathology are important areas of scientific research. Efforts have been made to identify the ovary-related transcriptomes in different species. However, the proteomic studies are limited. The rhesus monkey is very similar to humans, and it is widely used in the study of reproductive biology and medicine. In this study, using an optimized proteomics platform, we successfully identified 5723 rhesus ovarian proteins, of which 4325 proteins were consistently identified in all three replicates and with at least 2 unique peptides. The 4325 proteins were chosen for further analysis. Through gene ontology and pathway analyses, we obtained a preliminary understanding of the function of these proteins. A random immunohistochemistry analysis was used to determine the expression of proteins in various cell types. By comparing the genes identified in this study with genes that were reported to have relatively high levels of expression in human oocytes, we obtained genes that were predicted to play roles in maintenance of normal ovarian physiology. Searching the identified genes from this study against the MGI database gave us a list of proteins those exist in the rhesus monkey ovary and are important for female mouse reproduction as well. The overlap of genes in this study and the genes whose abnormal expression or dysfunction were reported to be associated with human polycystic ovary syndrome (PCOS) and premature ovarian failure (POF) prompted us to use the rhesus monkey to study these two common causes of female infertility. This study may provide a basis for future studies of human reproductive disorders using the rhesus monkey as a model.

N-Linked glycosylation, a type of post-translational modification, plays important roles in cell–cell recognition, adhesion, and interactions. Although N-linked glycosylated proteins in sperm are known to be important for gamete binding, little is known about the composition of these proteins, particularly glycosylation sites, in humans. In the present study, the use of glyco-FASP, coupled with the tandem mass spectrometry (MS/MS) method, led to the identification of 554 N-glycosylation sites and 297 N-glycosylated proteins in human sperm. Bioinformatics analysis revealed enrichment of proteins with functions in cell recognition and fertilization. Overall, about 91% of the human sperm N-glycosylated proteins were classified into “membrane”, “extracellular region”, and “lysosome” groups, based on subcellular localization annotation. Furthermore, glutathione peroxidase 4 (GPX4), a membrane glycoprotein identified in our glycoproteome, was shown to play a significant role in gamete interactions using the in vitro fertilization assay. Accordingly, we propose that characterization of the human sperm glycoproteome should effectively aid in clarifying the mechanisms of fertilization and provide a valuable resource for the future development of male contraceptives and diagnosis of male infertility.

The male gamete (sperm) can fertilize an egg, and pass the male genetic information to the offspring. It has long been thought that sperm had a simple protein composition. Efforts have been made to identify the sperm proteome in different species, and only about 1000 proteins were reported. However, with advanced mass spectrometry and an optimized proteomics platform, we successfully identified 4675 human sperm proteins, of which 227 were testis-specific. This large number of identified proteins indicates the complex composition and function of human sperm. Comparison with the sperm transcriptome reveals little overlap, which shows the importance of future studies of sperm at the protein level. Interestingly, many signaling pathways, such as the IL-6, insulin and TGF-beta receptor signaling pathways, were found to be overrepresented. In addition, we found that 500 proteins were annotated as targets of known drugs. Three of four drugs studied were found to affect sperm movement. This in-depth human sperm proteome will be a rich resource for further studies of sperm function, and will provide candidate targets for the development of male contraceptive drugs.Figure optionsDownload full-size imageDownload high-quality image (68 K)Download as PowerPoint slideHighlights► Proteomic analysis of human sperm indicates unexpected complex protein compositions. ► There is little overlap between sperm proteome and transcriptome. ► Three of four drugs targeting cilia proteins were found to affect sperm movement.

The genome sequence of rhesus macaque is a draft version with many errors and is lack of Y chromosome annotation. In the present dataset, we reanalyzed the previously published macaque testis proteome. We searched for refined protein sequences, potential Y chromosome proteins and transcripts predicted proteins in addition to the latest Ensembl protein sequences of macaque. A total of 74,433 peptides corresponding to 9247 protein groups were identified, and the data are supplied in this paper. The updated version of macaque testis proteome provided evidences for predicted genes or transcripts at the peptide level. It can be used for further in-depth proteogenomic annotation of macaque genome and is useful for studying the mechanisms of macaque spermatogenesis.