Cloning, expression analysis and localization of DAZL gene implicated in germ cell development of male Hezuo pig

Deleted in azoospermia-like (DAZL) is essential for mammalian testicular function and spermatogenesis. To explore the molecular characterization, expression patterns, and cellular localization of the DAZL in Hezuo pig testes, testicular tissue was isolated from Hezuo pig at five development stages including 30 days old (30 d), 90 days old (90 d), 120 days old (120 d), 180 days old (180 d), and 240 days old (240 d). DAZL cDNA was first cloned using the RT-PCR method, and its molecular characterization was analyzed using relevant bioinformatics software. Subsequently, the expression patterns and cellular localization of DAZL were evaluated using quantitative real-time PCR (qRT-PCR), Western blot, and immunohistochemistry. The cloning and sequence analysis showed that the Hezuo pig DAZL cDNA fragment contained 888 bp open reading frame (ORF) capable of encoding 295 amino acid residues and exhibited high identities with some other mammals. The qRT-PCR and Western blot results indicated that DAZL was specifically expressed in Hezuo pig testes, and DAZL levels of both mRNA and protein were expressed at all five reproductive stages of Hezuo pig testes, with extremely significant higher expression levels in 90 d, 120 d, 180 d, and 240 d than those in 30 d (p < 0.01). Additionally, immunohistochemistry results revealed that DAZL protein was mainly localized in gonocytes at 30 d testes, primary spermatocytes, and spermatozoon at other developmental stages, and Leydig cells throughout five development stages. Together, these results suggested that DAZL may play an important role by regulating the proliferation or differentiation of gonocytes, development of primary spermatocytes and spermatozoon, and functional maintenance of Leydig cells in testicular development and spermatogenesis of Hezuo pig. Nevertheless, the specific regulatory mechanisms underlying these phenomena still requires further investigated and verified.