Serine protease inhibitor Kazal-type 2 is expressed in the male reproductive tract of carp with a possible role in antimicrobial protection

The presence of the low-molecular-mass serine protease inhibitor Kazal-type (Spink) is a characteristic feature of vertebrate semen. Its main function is control of the serine protease in the acrosome, acrosin. Here we showed for the first time that Spink is present in the seminal plasma of carp, which have anacrosomal spermatozoa. Using a three-step isolation procedure that consisted in gel filtration and RP-HPLC and re-RP-HPLC, we isolated this inhibitor and identified it as serine protease inhibitor Kazal-type 2 (Spink2), a reproductive-derived member of the Spink family. The cDNA sequence of this inhibitor obtained from carp testis encoded 77 amino acids, including a 17 amino acids signal peptide; this sequence was distinct from fish Kazal-type inhibitors. The mRNA expression analysis showed that Spink2 is expressed predominantly in carp testis and spermatic duct. Immunohistochemical analysis demonstrated its localization in testis in Sertoli, Leydig and germ cells at all developmental stages (with the exception of spermatozoa) and in the epithelium of the spermatic duct. Aside from strong inhibition of trypsin, this inhibitor acts strongly against subtilisin and possesses bacteriostatic activities against Lactobacillus subtilis, Escherichia coli and Aeromonas hydrophila. The localization of Spink2 in carp reproductive tract suggests an important function in spermatogenesis and in maintenance of the microenvironment in which sperm maturation occurs and sperm are stored. Our results suggest that Spink2 from carp seminal plasma may play a role in antibacterial semen defense, protecting semen against unwanted proteolysis within the reproductive tract.