Background: MicroRNAs (miRNAs), which are small, non-coding RNAs approximately 21-nucleotides in length, have become a major focus of research in molecular biology. Mammalian miRNAs are proposed to regulate approximately 30% of all protein-coding genes. Previous studies have focused on highly conserved miRNAs, but nonconserved miRNAs represent a potentially important source of novel functionalities during evolution. Results: An analysis of the chromosome distribution of miRNAs showed higher densities of miRNAs on the X chromosome compared to the average densities on autosomes in all eight mammalian species analyzed. The distribution pattern did not, however, apply well to species beyond mammals. In addition, by comparing orthologous human and mouse miRNAs, we found that X-linked miRNAs had higher substitution rates than autosomal miRNAs. Since the highest proportion of X-linked miRNAs were found in mouse testis, we tested the hypothesis that testis miRNAs are evolving faster on the X chromosome than on autosomes. Mature X-linked testis miRNAs had an average substitution rate between mouse and human that was almost 25-fold higher than mature testis miRNAs on autosomes. In contrast, for mature miRNAs with precursors not expressed in testis, no significant difference in the substitution rate between the X chromosome and autosomes was found. Among mammals, the rapid evolution of X-linked testis miRNAs was also observed in rodents and primates. Conclusion: The rapid evolution of X-linked testis miRNAs implies possible important male reproductive functions and may contribute to speciation in mammals.

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.

Background: The mature mouse oocyte contains the full complement of maternal proteins required for fertilization, reprogramming, zygotic gene activation (ZGA), and the early stages of embryogenesis. However, due to limitations of traditional proteomics strategies, only a few abundantly expressed proteins have yet been identified. Our laboratory applied a more effective strategy: one-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (1D SDS-PAGE) and reverse-phase liquid chromatography tandem mass spectrometry (RP-LC-MS/MS) were employed to analyze the mature oocyte proteome in depth. Results: Using this high-performance proteomic approach, we successfully identified 625 different proteins from 2700 mature mouse oocytes lacking zona pellucidae. This is the largest catalog of mature mouse oocyte proteins compiled to date. According to their pattern of expression, we screened 76 maternal proteins with high levels of mRNA expression both in oocytes and fertilized eggs. Many well-known maternal effect proteins were included in this subset, including MATER and NPM2. In addition, our mouse oocyte proteome was compared with a recently published mouse embryonic stem cell (ESC) proteome and 371 overlapping proteins were identified. Conclusion: This proteomics analysis will be a valuable resource to aid in the characterization of important maternal proteins involved in oogenesis, fertilization, early embryonic development and in revealing their mechanisms of action.

AJA publishes new findings in basic and clinical (including modern, traditional and epidemiological) research on Andrology.