Molecular cloning and developmental expression of an mRNA encoding the 27 kDa outer dense fiber protein of rat spermatozoa

Abstract

We have isolated a cDNA (ODF27), encoding the major 27 kDa protein of rat sperm outer dense fibers (ODF), by screening a testicular lambda-gt11 phage cDNA library with an affinity-purified anti-27 kDa ODF polyclonal antibody. A cyanogen bromide derived internal amino acid (a.a.) sequence of the 27 kDa ODF protein was identical to an internal region of the deduced a.a. sequence of this cDNA. The cDNA encodes a protein with a high proportion of a repetitive motif, Cys-Gly-Pro, at the carboxy-terminal end, reminiscent of the testis-specific Mst(3)CGP proteins of Drosophila melanogaster (Schäfer et al., 1993. Mol Cell Biol 13:1708-1718). Nick translation probes of the ODF27 cDNA recognized two complementary mRNAs of 1.2 and 1.5 kb in the rat testis. Developmental Northern blot analysis revealed that these mRNAs are first transcribed in round spermatids. In situ hybridization confirmed the haploid expression of these transcripts and demonstrated that they are found in the cytoplasm of spermatids throughout most of the duration of spermiogenesis. They reach a peak in steps 8-10 of spermiogenesis at the time transcription ceases, remain at high levels from steps 11 to 15, and diminish in steps 16-18 at the time ODF protein synthesis and assembly are shown to be maximum. The translation of these transcripts, therefore, appears to be post-transcriptionally controlled. A literature and NCBl database search revealed that the nucleotide sequence of the 027 cDNA is homologous to the rat gene RT7 (Van Der Hoorn et al., 1990. Dev Biol 142:147-154) and to the rat testis-specific cDNA rts 5/1 (Burfeind and Hoyer-Fender, 1991. Dev Biol 148:195-204), which encodes a 27 kDa polypeptide.