Quantitative Phosphoproteomics Analysis Reveals a Key Role of Insulin Growth Factor 1 Receptor (IGF1R) Tyrosine Kinase in Human Sperm Capacitation*

Abstract

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. Molecular & Cellular Proteomics 14: 10.1074/mcp.M114.045468, 1104-1112, 2015.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 Ca 2ϩ 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). Tyrosinephosphorylated 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...

Phosphoproteomic and Functional Analyses Reveal Sperm-specific Protein Changes Downstream of Kappa Opioid Receptor in Human Spermatozoa

“…According to this, Wang et al in 2015 (14), previously identified 3303 phosphosites belonging to 965 proteins. Comparing our data with the largest description of the sperm phosphoproteome reported so far (14), we found 2692 novel phosphosites being 835 common in both studies. From these 2692 phosphosites, 535 were previously reported in humans in the PhosphoSitePlus database.…”

Unravelling the genomic architecture of bull fertility in Holstein cattle

“…This gene belongs to a family of insulin-like growth factors that has important roles in sex determination, testis development, spermatogenesis and steroidogenesis [46]. Interestingly, IGF1R has been implicated in regulating Sertoli cell proliferation and maturation, testis size, and sperm capacitation [47, 48]. Therefore, our findings provide more evidence of the association between IGF1R and male fertility.…”

A role for KIF9 in male fertility

“…We confirmed the presence of KIF9 at the protein level in the spermatocytes using an antipeptide antibody. Proteomic studies have revealed the presence of KIF9 in human sperm [12][13][14] as well as sperm of several mouse species [15]. Thus, based on studies in lower organisms and localization in mammalian sperm, we anticipated that there would be a strong motility defect.…”