P-085 Reactive molecules drive sperm capacitation via protein posttranslational modifications

Abstract Study question Are S-sulfenylation and S-nitrosylation, two essential posttranslational modifications (PTMs), increased during sperm capacitation? Which proteins are subjected to S-sulfenylation? Summary answer S-sulfenylation and S-nitrosylation increase during sperm capacitation. S-sulfenylation affects certain proteins, including A-kinase anchor protein 13, Fibrilline-1, and Spermatogenesis-associated protein 7. What is known already There is a significant increase in reactive oxygen species (ROS) and reactive nitrogen species (RNS) production within sperm capacitation. In contrast to tyrosine phosphorylation, a well-established mark of sperm capacitation, ROS, and RNS lead to PTMs themselves and modify thiol groups of cysteines. Despite these facts, the dynamics of ROS/RNS-derived PTMs during sperm capacitation remain unknown. Therefore, we consider both S-nitrosylation and S-sulfenylation of sperm proteins as novel modifications associated with the capacitation. Study design, size, duration C57BL6 mouse sperm were isolated from the caudae epididymae of 12-20-week-old males (n = 5). The study compared non-capacitated and capacitated spermatozoa using proteomic profiling. Participants/materials, setting, methods Sperm proteins were extracted into RIPA buffer. The protein lysate was incubated with 5mM dimedone, a hapten that specifically labels S-sulfenylated proteins, and immunogenically detected with an anti-dimedone antibody (Merk, Germany). Alternatively, S-nitrosylated proteins were detected by the anti-nitrosocysteine antibody (Merk, Germany). S-sulfenylated and S-nitrosylated proteins were visualized by western blotting. S-sulfenylated proteins were simultaneously identified by mass spectrometry. Main results and the role of chance We demonstrated an increase in S-sulfenylation and S-nitrosylation along with sperm capacitation. In addition, both S-sulfenylation and S-nitrosylation are positively correlated with tyrosine phosphorylation. Using mass spectrometry, we identified S-sulfenylated proteins, mostly belonging to the cytoskeleton: A-kinase anchor protein 13, Fibrilline-1, Spermatogenesis-associated protein 7, and Rab-like protein 2A. Limitations, reasons for caution The limitation of this study is the instability of PTMs. ROS/RNS-derived PTMs hypothetically arise and cease during protein sample preparation. The experiment was limited to one animal model. Further experiments should be performed on other mammalian models. Wider implications of the findings Studying novel sperm PTMs and their mechanism of action may improve the diagnosis of male infertility. Further, it could bring a novel approach to modulate the fertilizing ability of sperm used for in vitro fertilization. Trial registration number 260 536