DEVELOPMENTAL EXPRESSION OF THE 84-KDA ODF SPERM PROTEIN - LOCALIZATION TO BOTH THE CORTEX AND MEDULLA OF OUTER DENSE FIBERS AND TO THE CONNECTING PIECE

Outer dense fibers (ODF) are specialized cytoskeletal elements of the mammalian sperm tail which are composed of several prominent proteins. me previously reported the isolation of a cDNA (111-450) encoding a p utative 84-kDa ODF protein Here we demonstrate by independent cDNA iso lations and by translational/immunoprecipitation of testicular mRNAs u sing anti-ODF 84 antibodies that 111-450 cDNA encodes the 84-kDa prote in. We then analyzed the testicular expression of the ODF 84 mRNA and protein. Riboprobes generated from the clones recognized four testicul ar-specific transcripts of 1.6, 2.2, 2.4, and 2.8 kb in both rat and b ull of which the immunoprecipitable product of the 2.4-kb mRNA comigra tes with ODF 84 protein. Developmental Northerns indicated that the 2. 2- and 2.4-kb mRNAs are first transcribed during meiotic prophase whil e the other two species are first expressed in round spermatids. The l evels of all the transcripts steadily increased up to elongated sperma tids. Immunocytochemistry revealed that the anti-84 reactive ODF prote ins were synthesized and assembled in the cytoplasm of elongated sperm atids (steps 9-18) with peak activity occurring in step 16 of spermiog enesis. Immunogold labeling was selective to the assembling ODF and co nnecting piece of the tail and to granulated bodies of the cytoplasmic lobe. Both the striated collar and capitulum of the connecting piece were immunolabeled as well as the bas;al plate of the implantation fos sa. A combination of pre- and postembedding immunogold labeling provid ed evidence that the 84-kDa ODF protein is localized to both the corte x and medulla of the ODF in contrast to the sole medullary localizatio n of the major 27-kDa ODF protein Thus the 84-kDa ODF protein, encoded by the 2.4 transcript, is translationally regulated, packaged after s ynthesis into granulated bodies, assembled in a proximal to distal dir ection along the axoneme and may interact by means of leucine zippers specifically with the 27-kDa ODF protein during assembly. Its localiza tion to both the cortex and medulla of the ODF, as opposed to exclusiv e medullary localization of the 27-kDa ODF protein, and the presence o f two leucine zippers, only one of which interacts with the 27-kDa ODF , suggests that it could act as a link between proteins of the two reg ions of the ODF. (C) 1998 Academic Press.