Comparative and Phospho Proteomics: Identification of Protein Changes During Sperm Maturation.

Abstract

New advances in proteomic technologies will have a major impact on our understanding of how the reproductive system works. Following their production in the testis, the spermatozoa rely solely on post-translational modifications (PTM) for further maturation. This includes both epididymal passage and capacitation in the female reproductive tract. A failure of these processing events can lead to infertility, a condition that now affects 1 in every 20 males worldwide and appears to be increasing. To further understand sperm maturation, we have used a new, innovative technology that compares peptides from spermatozoa driven into two different functional states. For these studies spermatozoa were tryptically digested, and the peptides were run into IPG strips for pre-fractionation. The strip was then cut into 1-cm sections, and the peptides were excised and analysed by nano-flow, mass spectrometry. The MS-survey scan was used in silico to create virtual "2D-maps" by plotting the elution profile of individual peptides against their corresponding mass:charge ratios, together with the ion count. Using appropriate software, DeCyder MS, we were then able to show statistically relevant PTMs in spermatozoa during maturation. For example, 32 peptides statistically changed during sperm capacitation. Of these, 23 have now been sequenced and manually validated. Peptides previously documented to undergo PTM, including some derived from AKAP4, were found. Furthermore, many novel proteins, with previously undiscovered roles for capacitation, have now been mapped. For example, a peptide derived from 'Sperm equatorial segment protein 1' was completely absent in the non-capacitated sample. Several RIKENS were found to undergo phosphorylation, leading to a ~2.00 fold increase from the non-capacitated to the capacitated state. A peptide derived from dynein heavy chain 3 underwent a 2.15-fold increase, although the nature of the PTM is not yet known. To confirm the biological significance of our data, we measured the enzyme activity of soribitol dehydrogenase (SDH). A peptide derived from this enzyme was shown to decrease 0.55-fold during capacitation, however, again the nature of the PTM is unknown. We could clearly show that the rate of SDH activity was ~2-fold higher in lysates derived from capacitated spermatozoa compared to non-capacitated spermatozoa, clearly suggesting the PTM occurring on this enzyme increases its activity. To extend our analysis of the PTMs occurring during sperm maturation, we have also used TiO2-enriched peptides (phosphopeptides), to compare spermatozoa derived from either the caput or caudal epididymides. In all, 52 phospho-specific peptide changes were mapped during capacitation, including sperm-specific proteins AKAP4 and Rhophillin. These studies demonstrate the power of comparative proteomics in resolving the molecular mechanisms that drive sperm maturation. Spermatozoa are ideal candidates for this form of analysis since they can be obtained in large, pure numbers, can be robustly driven into different physiological states in vitro and are entirely dependent on PTMs for the acquisition of biological competence.