# Proteomic dynamics of bull sperm during post-testicular maturation

## TL;DR

This study maps changes in bull sperm proteins during maturation, revealing shifts in function and metabolism as sperm move through the epididymis.

## Contribution

The study provides a detailed comparative proteomic profile of bull sperm at different maturation stages, revealing novel insights into sperm function and histone dynamics.

## Key findings

- Sperm proteomes show distinct biological processes at each maturation stage, including post-transcriptional regulation in testicular sperm and OXPHOS in cauda epididymal sperm.
- Histone replacement by protamines is not completed in the testis but continues through epididymal transit, with histone modifying enzymes detected in caput sperm.
- The ubiquitin–proteasome system is significantly enriched in conserved sperm proteomes across species, suggesting a role in proteomic remodeling.

## Abstract

Mammalian sperm maturation during epididymal transit is driven by sequential interactions with the epididymal environment allowing the acquisition, loss and modification of sperm protein content. We report a comparative proteomic profile of testicular, caput and cauda epididymal spermatozoa using shotgun proteomics. Analysis rendered 2,305 proteins in testicular sperm (TestSperm), 2,554 in caput epididymal sperm (CaputSperm) and 2,038 in cauda epididymal sperm (CaudaSperm), including 702, 483 and 314 unique proteins, respectively. Gene Ontology (GO) enrichment analysis of total proteomes and unique proteins from each population allowed us to map biological processes (BP) to spermatozoa with sequential degrees of maturation. TestSperm was mostly enriched in processes related to post-transcriptional regulation, that may be reminiscent mechanisms to regulate spermatogenesis and sperm differentiation. GO enrichment analysis of BP in CaputSperm proteome identified a significant enrichment of integrin-mediated and non-classical endocytic pathways may be aiding in sperm-epididymosome interaction and vesicle fusion events. CaudaSperm was significantly enriched in BP related to specific sperm functions (motility, fertilization) and associated to oxidative phosphorylation (OXPHOS) reflecting that sperm maturation involves a transition from a glycolytic metabolism (prevalent in testicular sperm) to being more dependent on OXPHOS, as ejaculated sperm. An in silico analysis further revealed how proteins are associated with specific sperm functions/events (motility, acrosome reaction/capacitation, fertilization and embryo development). Most proteins related to these functions seem to be present since the testicular phase and maintained through maturation. Interestingly, ubiquitin–proteasome system (UPS) catabolic processes appear to be significantly enriched in the conserved epididymal sperm proteome across species. Finally, we identified several sperm histones and variants and, together with an in silico analysis, we also identified histone modifying enzymes in each sperm population. Contrarily to common believe, our results suggest that histone replacement by protamines is not completed in the testis and continues through epididymal transit. Detection of histone modifying enzymes in CaputSperm suggests that histone PTMs might be more prevalent in caput epididymis where they may play a role in achieving the necessary balance between histone eviction and retainment. Our study emphasizes the pivotal role that sperm proteomic remodeling plays in post-testicular maturation, in the sequential acquisition of competence.

The online version contains supplementary material available at 10.1186/s12864-026-12614-0.