Seminal fluid predominantly epitomizes the reproductive process of boars, with variations in their reproductive capabilities across different age cohorts, potentially attributed to individual sperm quality, semen vitality, health status, and oxidative stress [20, 21]. Recently, proteomic analysis of semen from older adult human males has been reported, revealing a positive correlation between age and the DNA fragmentation index [22]. Furthermore, the DEPs were significantly enriched in pathways related to energy metabolism. To discern the disparities in protein levels among boars of varying ages, we conducted LC-MS-based proteomic analysis on juvenile (1-year-old) and mature (7-year-old) boars. A total of 4050 proteins were identified, including 130 proteins that were differentially expressed between the two groups.
FOLR2, an isoform of the folate receptor FR, is expressed in the placenta, hematopoietic cells, and macrophages and is equipped with a cellular glycosylphosphatidylinositol (GPI) anchor [23, 24]. Therefore, FOLR2 expression may serve as a predictive marker of male fertility. Immunostaining results revealed that sperm in semen exhibited a more robust immune response than sperm in testicular tissue [25]. The presence of FOLR in sperm enables the formation of folate complexes, thereby safeguarding the folate content within the sperm microenvironment and facilitating normal DNA replication post-fertilization through the transfer of folate carriers into the interior of the sperm. The role of energy metabolism in sperm flagellar motility has been extensively studied. Mammalian sperms require sustained motility from ejaculation to fertilization, necessitating the generation of sufficient energy to meet their locomotion demands [26]. The tricarboxylic acid cycle serves as a crucial pathway for ATP production in sperm mitochondria. Aconitate hydratase (ACO2), an enzyme that regulates the tricarboxylic acid cycle, translocates from the cytoplasm to the nucleus during somatic cell reprogramming, thereby influencing cellular totipotency [27]. It also affects ATP-dependent sperm motility in sperm [28]. The expression level of ACO2 was significantly lower in males with asthenospermia than in those with normal fertility. The addition of isocitrate results in a significant increase in sperm motility, which could be attributed to enhanced ATP production [28]. In our study, the expression level of ACO2 was higher in young boars than in old boars, suggesting that young boars exhibited greater semen motility, which is consistent with previously reported findings. Outside of ACO2, a larger proportion of the upregulated proteins was localized in the mitochondria (15%), indicating that young boars have a stronger energy source in the semen, which can enhance sperm motility.
Carboxypeptidase O (CPO) was identified as the most significant DEP, exhibiting over 11-fold higher expression in the Y group than in the O group. This protein belongs to the M14 family of metal carboxypeptidases and displays a preference for cleaving C-terminal acidic amino acids. In insects, seminal fluid proteins are synthesized in the male reproductive tract and transferred to females during mating, along with sperm, inducing physiological and behavioral changes in females. CPB, another member of the carboxypeptidase family, possesses this functionality; therefore, we hypothesized that CPO may also exhibit similar functions [29]. Mitochondria-associated cysteine-rich protein (SMCP) is a rapidly evolving protein that mainly localizes to the outer membrane of sperm mitochondria and enhances sperm motility [30]. In vitro fertilization experiments in SMCP-knockout mice showed that the fertilization success rate was three-fold lower than that in wild-type mice, indicating that sperm motility and oocyte penetration were reduced [31]. The findings of knockout experiments by Karim et al. further support this perspective [32]. Our results demonstrated a significantly higher expression of SMCP in the Y group, indicating enhanced motility in the semen of young boars.
Ribosomal proteins are the most highly expressed genes in virtually all cells, and their products play a pivotal role in ribosome biogenesis, thereby influencing protein folding [33]. Among these proteins, 40S ribosomal protein S5 (RPS5) is a key RNA-binding component that contributes significantly to translation [34]. Our findings highlight the central involvement of RPS5 in PPIs and its pronounced expression in Group Y. In a study conducted by Sandeep et al., transcriptome sequencing was performed on 60 males with known fertility (n = 20), idiopathic infertility, and asthenospermia, revealing high expression of RPS5 in the normal group and low expression in the asthenospermia group [35]. Similarly, other members of the ribosomal protein family, such as RPLP0 and RPL7, interacted with RPS5, underscoring the crucial role played by ribosomal family members in sperm function, a finding consistent with that of Laxman's transcriptome data analysis [36]. The downregulated proteins in our study were partially localized in the Golgi apparatus and were enriched in the ribosomal pathway, indicating that protein translation, processing, and folding are more important in the semen of old boars.
The AWN protein, a member of the sperm adhesin family, was initially discovered in boar seminal plasma and is found in the seminal plasma of most mammalian species [37, 38]. Electron microscopy indicated that AWN predominantly localizes on the surface of sperm cells and exhibits a range of ligand-binding capabilities to interact with receptor molecules in the anterior region of ejaculated sperm, thereby facilitating sperm capacitation [39]. This is consistent with the subcellular localization results of our proteomic analysis. Sajjad et al. conducted single-cell transcriptome sequencing of mouse spermatogonia and mesenchymal stem cells and identified KAT2A as the predominant central regulator, based on centrality analysis [40]. KAT2A, an enzyme responsible for regulating various acylation modifications, potentially modulates its function by influencing post-translational modifications of both histone and non-histone proteins [41, 42]. Histone modifications exert epigenetic effects on spermatogenesis by modulating gene transcription, either by activating or repressing transcription. Acetylation of histone H3K14 is associated with testosterone production and spermatogenesis. Exposure enhances the level of H3K14ac in rat testes by promoting the expression of KAT2A, which subsequently leads to the repression of steroidogenic-related genes, resulting in reduced testosterone production and impaired spermatogenesis [43]. Lower expression levels of KAT2A were also observed in Group Y, suggesting that Group Y may correspond to decreased acetylation and normal spermatogenesis. Protein modifications are closely associated with spermatogenesis. Protein acetylation serves as a protective mechanism against spontaneous acrosome reactions in sperm, thereby enhancing fertilization rates [44]. Additionally, it plays a crucial role in regulating sperm capacitation and influences semen quality post-thawing [45, 46]. Histone crotonylation plays a pivotal role in promoter activation and male germ cell differentiation and exerts a significant influence during the late stages of spermatogenesis [47]. In addition to exerting an impact on gene expression in the testis, CDYL-mediated knockdown of histone crotonylation results in impaired fertility in mice, as evidenced by a decrease in the number of sperms found in the epididymis and reduced motility of spermatids [48].
In conclusion, the reproductive performance of breeding boars in practical production is influenced by age, and the variation in reproductive ability among boars of different ages may be associated with the protein composition of their semen. Comparative proteomic analysis of semen proteins from 1- and 7-year-old breeding boars identified 130 differentially expressed genes, including 33 upregulated and 97 downregulated genes. Functional enrichment analysis revealed that these differentially expressed genes were associated with energy metabolism, reproduction, and fertilization. Furthermore, protein post-translational modification may affect the quality of boar semen at different ages. This study provides insights into the age-related differences in boar semen at the protein level. In future studies, we will further explore the epigenetic effects on semen through post-translational modifications.