Proteomic landscape of crotonylation and acetylation in pig testes across male reproduction.

Lysine crotonylation (Kcr) and acetylation (Kac) are evolutionarily conserved post-translational modifications that alter protein's charge, polarity, and structure. To explore the potential roles and critical modified proteins in male reproduction, we employed the affinity enrichment of pan-anti-Kcr and pan-anti-Kac antibodies with high-resolution LC-MS/MS from testes tissues of six boars (three at 5 months and three at 24 months of age), respectively. As a result, a large number of Kcr and Kac proteins were identified in testicular tissue between two groups. Differential analysis further revealed that Kcr proteins were significantly enriched to Gene Ontology biological process terms concerning reproductive system development, response to steroid hormone and response to hydroxy isoflavone, whereas Kac proteins primarily regulated mitochondria-localized proteins involved in testicular metabolism. RPS5, RPS6, CYP17A1, CYP11A1, and CYP19A1, which may act as important regulators of testicular development through crotonylation modification. In addition, we observed indications of crosstalk between the two modifications, involving shared enzymes (including HDAC1-3), overlapping substrates, and CoA donor metabolism. This study performed a large-scale analysis of Kcr and Kac proteins in porcine testis, providing a post-translational modification perspective to testis development. SIGNIFICANCE: The testis is a key organ determining male reproductive capacity, and pigs of different ages exhibit distinct reproductive performance. Most previous studies have focused on transcriptomic and proteomic differences in testes across developmental stages, while post-translational modifications (PTMs) remain poorly explored. Here, using affinity enrichment with pan-anti-Kcr and pan-anti-Kac antibodies combined with high-resolution LC-MS/MS, we profiled lysine crotonylation (Kcr) and acetylation (Kac) in pig testes. Bioinformatic analyses identified CYP17A1, CYP19A1, CYP11A1, RPS5, and RPS6 as potential regulators of testicular maturation via crotonylation, while acetylation was found to regulate metabolic activity by modifying mitochondrial proteins. Furthermore, crosstalk between crotonylation and acetylation at the metabolic level was revealed. These findings advance our understanding of PTMs in porcine testis development and provide novel data resources for future studies.