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 significance Mature 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.

Spermiogenesis is a complex process of terminal differentiation that is necessary to produce mature sperm. Using protein expression profiles of mouse and human testes generated from our previous studies, we chose to examine the actions of lamin A/C in the current investigation. Lamin A and lamin C are isoforms of the A-type lamins that are encoded by the LMNA gene. Our results showed that lamin A/C was expressed in the mouse testis throughout the different stages of spermatogenesis and in mature sperm. Lamin A/C was also expressed in mouse haploid germ cells and was found to be localized to the acroplaxome in spermiogenesis, from round spermatids until mature spermatozoa. The decreased expression of lamin A/C following injections of siRNA against Lmna caused a significant increase in caudal sperm head abnormalities when compared with negative controls. These abnormalities were characterized by increased fragmentation of the acrosome and abnormal vesicles, which failed to fuse to the developing acrosome. This fragmentation also caused significant alterations in nuclear elongation and acrosome formation. Furthermore, we found that lamin A/C interacted with the microtubule plus-end-tracking protein CLIP170. These results suggest that lamin A/C is critical for proper structural and functional development of the sperm acrosome and head shape.

Spermatogenesis is a complex process closely associated with the phosphorylation-orchestrated cell cycle. Elucidating the phosphorylation-based regulations should advance our understanding of the underlying molecular mechanisms. Here we present an integrative study of phosphorylation events in the testis. Large-scale phosphoproteome profiling in the adult mouse testis identified 17,829 phosphorylation sites in 3955 phosphoproteins. Although only approximately half of the phosphorylation sites enriched by IMAC were also captured by TiO2, both the phosphoprotein data sets identified by the two methods significantly enriched the functional annotation of spermatogenesis. Thus, the phosphoproteome profiled in this study is a highly useful snapshot of the phosphorylation events in spermatogenesis. To further understand phosphoregulation in the testis, the site-specific kinase-substrate relations were computationally predicted for reconstructing kinase-substrate phosphorylation networks. A core sub-kinase-substrate phosphorylation networks among the spermatogenesis-related proteins was retrieved and analyzed to explore the phosphoregulation during spermatogenesis. Moreover, network-based analyses demonstrated that a number of protein kinases such as MAPKs, CDK2, and CDC2 with statistically more site-specific kinase-substrate relations might have significantly higher activities and play an essential role in spermatogenesis, and the predictions were consistent with previous studies on the regulatory roles of these kinases. In particular, the analyses proposed that the activities of POLO-like kinases (PLKs) might be dramatically higher, while the prediction was experimentally validated by detecting and comparing the phosphorylation levels of pT210, an indicator of PLK1 activation, in testis and other tissues. Further experiments showed that the inhibition of POLO-like kinases decreases cell proliferation by inducing G2/M cell cycle arrest. Taken together, this systematic study provides a global landscape of phosphoregulation in the testis, and should prove to be of value in future studies of spermatogenesis. Spermatogenesis is a complex sperm-generating process involving the mitosis of spermatogonia, meiosis of spermatocytes, and spermiogenesis of spermatids. Sperms are produced in the male testis at the speed of ∼1000 sperm per heart beat (1), which indicate that spermatogenesis is an extremely dynamic process in the testis. The protein expression levels during spermatogenesis have been well studied by high-throughput proteomic studies, and over 7000 proteins have been identified in the mammalian testis (2⇓–4). However, the dynamic regulatory events that orchestrate this complex process have yet to be elucidated. Because phosphorylation, an important and ubiquitous post-translational modification (PTM)1, is one of the most critical regulatory mechanisms of the cell cycle (5), which is particularly active during spermatogenesis, a number of pioneering studies have contributed to our understanding of phosphoregulation in spermatogenesis. For example, mitogen-activated protein kinases (MAPKs) such as ERK1/2, were found to play an important role in ectoplasmic specialization dynamics during spermatogenesis (6). As important regulators of the cell cycle (7, 8), the POLO-like kinases (PLKs) especially PLK1, were found to be required at multiple stages of spermatogenesis (9⇓⇓–12). Thus, a systematic analysis of phosphorylation in the testis is of great importance for advancing the current understanding of the molecular mechanisms of spermatogenesis. In order to elucidate the phosphorylation-mediated regulation of spermatogenesis, the characterization of

Male macaques produce faster sperm than male humans due to a higher pressure of sperm competition in macaques. To explore the molecular basis of this biological difference, we firstly constructed macaque and human sperm proteomes using LC−MS/MS. We then detected the positively selected genes specifically on the branch of macaque based on branch‐site likelihood method. We identified 197 positively selected genes specifically on the branch of macaque that are unselected in corresponding human orthologs. These genes are highly associated with mitochondria and axoneme that directly drive sperm motility. We further compared the ultrastructural differences of the midpiece between macaque and human sperms to provide evidence for our findings using transmission electron microscopy. In conclusion, our results provide potential molecular targets for explaining the different phenotypes under sperm competition between macaques and humans, and also provide resources for the analysis of male fertility.

One of the most important changes during sperm capacitation is the enhancement of tyrosine phosphorylation. However, the mechanisms of protein tyrosine phosphorylation during sperm capacitation are not well studied. We used label-free quantitative phosphoproteomics to investigate the overall phosphorylation events during sperm capacitation in humans and identified 231 sites with increased phosphorylation levels. Motif analysis using the NetworKIN algorithm revealed that the activity of tyrosine phosphorylation kinases insulin growth factor 1 receptor (IGF1R)/insulin receptor is significantly enriched among the up-regulated phosphorylation substrates during capacitation. Western blotting further confirmed inhibition of IGF1R with inhibitors GSK1904529A and NVP-AEW541, which inhibited the increase in tyrosine phosphorylation levels during sperm capacitation. Additionally, sperm hyperactivated motility was also inhibited by GSK1904529A and NVP-AEW541 but could be up-regulated by insulin growth factor 1, the ligand of IGF1R. Thus, the IGF1R-mediated tyrosine phosphorylation pathway may play important roles in the regulation of sperm capacitation in humans and could be a target for improvement in sperm functions in infertile men. Austin (1) and Chang (2) discovered that sperm must reside in the female genital tract for a specific period of time to acquire the ability to fertilize an egg and named this process “capacitation.” During capacitation, several biochemical changes occur, including enhancement of tyrosine phosphorylation (3), increased intracellular Ca2+ and cAMP levels (4), hyperactivated motility (5), and increased membrane plasma permeability (6). Mature sperm are highly differentiated and specialized cells, there is almost no transcription, and the genomic ribosome is inactive (5). Therefore, regulation of proteins at the level of post-translational modification is expected to play important roles in sperm functions. In mammalian sperm, phosphorylated proteins, protein kinases, and phosphatases are reported to function in sperm motility, capacitation, and acrosome reaction (7, 8). Tyrosine phosphorylation and dephosphorylation are required for sperm to reach, bind, penetrate, and fuse with the oocyte (5). Tyrosine-phosphorylated proteins have been found in human (9), monkey (10), rat (11), and mouse (12) sperm. The sperm tail is the main location of protein tyrosine phosphorylation, and tyrosine phosphorylation of the sperm tail is related to hyperactivated motility (13). However, the mechanism of protein tyrosine phosphorylation regulation in sperm capacitation is not well studied. With high throughput ability, proteomics has been used to characterize phosphorylation in sperm. For the human sperm, Ficarro et al. (14) used two-dimensional polyacrylamide gel electrophoresis (PAGE), anti-phosphotyrosine antibody labeling, and tandem mass spectrometry (MS/MS) to identify tyrosine phosphoproteins during capacitation. They identified a total of five tyrosine phosphorylation sites, 56 serine phosphorylation sites, and two threonine phosphorylation sites. Because of the low abundance of phosphorylation, recent studies used an enrichment approach to identify phosphorylation sites. In rodents, Platt et al. (15) labeled uncapacitated and capacitated mouse sperm protein using an isotope labeling reagent based on Fisher esterification and quantified 55 phosphorylation sites during sperm capacitation. Baker et al. (16), using a rat model, quantified 288 phosphorylated peptides during sperm capacitation. However,