PURIFICATION, PARTIAL CHARACTERIZATION, AND LOCALIZATION OF SAK57, ANACIDIC INTERMEDIATE FILAMENT KERATIN PRESENT IN RAT SPERMATOCYTES, SPERMATIDS, AND SPERM

We have purified a 57 kDa protein (designated Sak57, for spermatogenic cell/sperm-associated keratin) from sodium dodecyl sulfate-p-mercapto ethanol (SDS-beta ME)-dissociated outer dense fibers isolated from rat sperm tails. Internal protein sequence analysis of Sak57 yielded two 15-mer and 10-mer fragments with 70-100% homology to human, rat, and m ouse keratins and corresponding to the 1A and 2A regions of the alpha- helical rod domain of keratins. A multiple antigenic peptide (MAP) was constructed using the 10-mer amino acid sequence KAQYEDIAQK (correspo nding to the 2A region) and used as antigen for the production of poly clonal antibodies in rabbit. Anti-MAP sera were used for further analy sis of the biochemical characteristics of Sak57 in testis and sperm ta ils using chromatofocusing, immunobloting, and [P-32]orthophosphate-la beling. We have found that rat testis displays two immunoreactive prot eins: a soluble 83 kDa protein with pI range 5.9-6.3, regarded as a pr ecursor, and both detergent-insoluble and soluble 57 kDa protein with pi range 5.0-5.9, corresponding to the mature form Sak57. The testicul ar soluble form was phosphorylated. Rat sperm tail samples displayed o nly the Sak57 detergent-insoluble form and its pI was more acidic (4.7 -4.8). Whole-mount electron microscopy of negatively stained preparati ons of sperm-derived Sak57 resuspended in SDS-beta ME revealed a rod-s haped pattern. A decrease in the concentration of SDS-beta ME resulted in the side-by-side aggregation of rod-shaped Sak57 forming thick bun dles. Indirect immunofluorescence was used to determine the localizati on of Sak57 in isolated outer dense fibers, epididymal sperm, spermati ds, and pachytene spermatocytes, Confocal laser scanning microscopy wa s used to analyze the three-dimensional arrangement of Sak57 in pachyt ene spermatocytes. Isolated outer dense fiber and sperm tails displaye d an immunoreactive product in the form of linear clusters. In elongat ing spermatids (steps 10-11), Sak57 immunoreactivity was predominant i n the head region whereas pachytene spermatocytes displayed a cortical cytoplasmic distribution. Results of this study demonstrate that Sak5 7 has the characteristics of a keratin intermediate filament and is pr esent during meiotic and postmeiotic stages of spermatogenesis. (C) 19 96 Wiley-Liss, Inc.