Functional characterization of porcine septin12 and its role in male reproduction.

The objective is to identify and functionally characterize the pig septin12 gene, focusing on its expression pattern, subcellular localization, and interacting proteins, in order to explore its potential roles in male reproduction. Full-length septin12 cDNA was obtained from the testis of the Banna mini-pig inbred line (BMI) using rapid amplification of cDNA ends. Quantitative real-time polymerase chain reaction and western blot were employed to assess tissue-specific expression in BMI pigs. Subcellular localization was determined by fluorescence microscopy following transfection of EGFP-tagged septin12. A yeast two-hybrid (Y2H) screen using an adult BMI testis cDNA library identified potential interacting proteins, which were then validated by Co-immunoprecipitation (Co-IP) and immunofluorescence (IF). Gene Ontology (GO) analysis was performed to ascertain the functional categories of the interactors. The cloned septin12 cDNA from the BMI testis was 1,289 base pairs (bp) in length. septin12 mRNA and protein expression was predominantly observed in the BMI testis, suggesting its critical role in male reproductive function. septin12 localized to both fibrous filamentous and punctate cytoplasmic structures. Thirteen interacting proteins were identified by Y2H, including ATAD5, ATP1B3, CENPL, ENO1, and septin5, with GO analysis indicating enrichment in mitotic nuclear division, cell growth, and G2/M phase transition. Furthermore, Co-IP and IF confirmed interactions between septin12 and electron transfer flavoprotein subunit alpha (ETFA) as well as lactate dehydrogenase B (LDHB), supporting the results of the Y2H screen. This study identified and characterized the septin12 gene in BMI pig, highlighting its predominant expression in the testis and implicating its role in male reproductive function. Protein interaction analysis uncovered partners involved in cell division and metabolism, suggesting potential mechanisms by which septin12 may influence spermatogenesis. Co-IP and IF further validated interactions with ETFA and LDHB. These findings offer a groundwork for future investigations on the role of septin12 in male fertility.