Identification, cloning and functional characterization of novel sperm associated antigen II (SPAG II) isoforms in the rat

Abstract

Background: Sperm binding proteins and their C-terminal peptides of the Sperm Associated Antigen II (SPAG II) family were found to play an important role in epididymal innate immunity in addition to their role in sperm maturation. However, the expression of SpagII transcripts in rodents is not well documented. Methods: Computational analysis was employed to identify novel SpagII isoforms in the rat. RTPCR analyses were carried out on RNAs isolated from the male reproductive tract tissues of rat using gene specific primers for SpagIIc and SpagIIt. The identities of PCR products were confirmed by sequencing. Tissue distribution, developmental expression and androgen regulation of SpagIIt and SpagIIc were studied using RT-PCR. The antimicrobial activities of recombinant SpagIIt and SpagIIc were tested against E coli in a colony forming unit assay. Results: In this study, we identified two novel SpagIIt transcripts, namely, SpagIIt and SpagIIc derived from the long arm of chromosome 16 in the rat (Rattus norvegicus), using both in silico and molecular biology approaches. SpagIIc is expressed in all three regions of the epididymis, in testis and in ovary but is absent from the seminal vesicle. SpagIIt expression is confined to the caput and it is not expressed in the testis, seminal vesicle or ovary. Age dependent expression of SpagIIt and SpagIIc was observed in the epididymides of rats (10-60 day old). Their expression was found to be most abundant in the adult rat (60 day) suggesting roles in mature reproductive function. Further, both SpagIIt and SpagIIc expression was down regulated in castrated rat epididymides and the expression was maintained in the testosterone replaced castrated rats. SPAGIIC is a potent antibacterial agent. SPAGIIT also displayed bactericidal capacity although weaker than SPAGIIC and SPAGIIE. Conclusion: The abundant expression of SpagIIt and SpagIIc in the male reproductive tract suggests an important role in male reproductive tract immunity. Their expression is developmentally regulated and androgen dependent. Characterization of novel SPAGII isoforms will contribute to our understanding of the role of epididymal proteins in sperm maturation and innate immunity. © 2006 Yenugu et al; licensee BioMed Central Ltd.