IDENTIFICATION OF OSTEOPONTIN (OPN) MESSENGER-RNA AND PROTEIN IN THE RAT TESTIS AND EPIDIDYMIS, AND ON SPERM

We have identified a bone cell adhesion molecule, osteopontin, in the rat testis and epididymis by Northern analysis, RT-PCR, Western immuno blot analysis, and immunocytochemistry. A polyclonal antibody raised a gainst rat epididymal fluid proteins was used to detect fusion protein s produced by a testis lambda gt11 cDNA library. Sequence analysis of one of four positive cDNA clones, designated as pREP5, revealed identi ty with the rat osteopontin (OPN) cDNA. The partial cDNA clone pREP5 e ncompasses 64% of the 1,457 residues reported by Oldberg et al. (1986; Proc Natl Acad Sci USA 83:8819-8823). Immunoblot analysis with a mono clonal antibody against OPN detects the presence of immunoreactive pol ypeptides in rat testis homogenates as well as in epididymal fluid and sperm extracts. Immunocytochemical localization to the basal and adlu minal region of the seminiferous tubule suggests that OPN could be a S ertoli cell product. Indeed, Northern blot analysis of testicular cell preparations demonstrated positive hybridization to Sertoli cell-enri ched RNA, but not to RNA isolated from interstitial cell preparations or to isolated germ cell RNA preparations. OPN is also detected in the rat epididymis and on epididymal spermatozoa. This is the first repor t on the presence of OPN mRNA and protein in rat testis and epididymis and on the presence of OPN on the surface of epididymal spermatozoa. The characterization of this protein in other tissue suggests that OPN could play a role in testicular cell adhesion during spermatogenesis and/or epididymal maturation, although other potential functions in th e male reproductive tract are discussed. (C) 1995 Wiley-Liss, Inc.