Sperm capacitation triggers transcriptomic changes linked to the proteasome-mediated ubiquitin-dependent catabolic pathway.

In brief: Molecular changes during human sperm in vitro capacitation were investigated through RNA sequencing and bioinformatic analysis. The ubiquitin-dependent protein catabolic process emerged as a key pathway, and proteomic analysis supported that the ubiquitin-proteasome system plays a regulatory role in human sperm capacitation.

Abstract: Capacitation involves a series of biochemical and physiological changes that spermatozoa undergo during their transit along the female reproductive tract, which are essential for fertilizing the oocyte. While several processes associated with capacitation, such as increased tyrosine phosphorylation, have been extensively studied, the molecular mechanisms regulating this process remain unclear. This study aimed to identify biological processes associated with human sperm in vitro capacitation using next-generation RNA sequencing. Our findings revealed that sperm capacitation is associated with transcriptomic changes, characterized by 337 differential gene transcripts. Notably, one of the primary biological processes associated with capacitation was the ubiquitin-dependent protein catabolic process. To explore this further, we compared the ubiquitinated protein profiles of non-capacitated and capacitated spermatozoa using Western blot analysis after protein separation by denaturing gel electrophoresis and two-dimensional electrophoresis. The results showed increased protein ubiquitination during capacitation, which paralleled the expected increase in tyrosine phosphorylation. Interestingly, inhibition of proteasome activity with 50 μM MG132 avoided the degradation of ubiquitin conjugates, whereas tyrosine phosphorylation levels remained constant. These findings suggest that ubiquitin-conjugated sperm proteins and their subsequent degradation by the proteasome may play a role in sperm capacitation. Further investigation of ubiquitin-mediated mechanisms during capacitation could provide valuable insights into the signaling pathways essential for fertilization.