Structure of Caspase9 gene in Qianhua Mutton Merino and its association with spermatogenic function analysis

Semen quality influences mating efficiency and is a key trait for evaluating reproductive performance. Sperm concentration and motility are important indicators of semen quality and are closely associated with the microenvironment and dynamic regulation of apoptosis during spermatogenesis. Cysteinyl aspartate-specific proteinase 9 (Caspase9) is a key component of the intrinsic mitochondrial apoptotic pathway and contributes to the balance between spermatogenesis and apoptosis. However, studies on Caspase9 gene polymorphisms, protein characteristics, and expression in male sheep reproduction remain limited. The cloned Caspase9 coding sequence encoded a protein of 426 amino acids. Bioinformatic analyses predicted Caspase9 to be an unstable protein, with an instability index of 50.11 and an average hydropathy score of − 0.350, and predicted the absence of a signal peptide and transmembrane regions. Two conserved domains, CARD and CASc, were identified. Nine SNPs were identified in the Caspase9 gene. The g.252 locus showed low polymorphism, whereas the other eight loci showed moderate polymorphism. Three SNPs, g.252 C > T, g.345 G > A, and g.397 T > C, were synonymous mutations. After correction for multiple comparisons, the ejaculate volume of rams with the CC genotype at the g.129 C > T locus was significantly greater than that of rams with the CT and TT genotypes ( P < 0.01). Caspase9 mRNA expression in the testis was significantly upregulated after sexual maturity, accompanied by a significant increase in Caspase9 protein expression ( P < 0.01). NFATc4 mRNA expression in the testis was significantly downregulated after sexual maturity, whereas its protein expression was significantly increased ( P < 0.01). VEGF mRNA expression was significantly higher in the testis than in the other reproductive tissues ( P < 0.05), whereas testicular VEGF protein expression did not differ significantly between reproductive stages. This study characterized the Caspase9 gene and protein in Qianhua Mutton Merino rams and identified nine SNPs. The g.129 C > T polymorphism was significantly associated with ejaculate volume after multiple-comparison correction. Caspase9 showed tissue- and reproductive-stage-dependent expression, with both its mRNA and protein expression in the testis significantly increased after sexual maturity. NFATc4 and VEGF also exhibited distinct expression patterns among reproductive tissues and developmental stages. These findings provide a basis for further investigation of Caspase9 as a candidate molecular marker associated with semen-quality traits and its potential role in male reproductive function in sheep.