Purification, partial characterization, and localization of Sak57, an acidic 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-beta-mercaptoethanol (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 mouse 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 (corresponding to the 2A region) and used as antigen for the production of polyclonal antibodies in rabbit. Anti-MAP sera were used for further analysis of the biochemical characteristics of Sak57 in testis and sperm tails using chromatofocusing, immunoblotting, and [32P] orthophosphate-labeling. We have found that rat testis displays two immunoreactive proteins: a soluble 83 kDa protein with pl range 5.9-6.3, regarded as a precursor, and both detergent-insoluble and soluble 57 kDa protein with pl range 5.0-5.9, corresponding to the mature form Sak57. The testicular soluble form was phosphorylated. Rat sperm tail samples displayed only the Sak57 detergent-insoluble form and its pl was more acidic (4.7-4.8). Whole-mount electron microscopy of negatively stained preparations of sperm-derived Sak57 resuspended in SDS-beta ME revealed a rod-shaped pattern. A decrease in the concentration of SDS-beta ME resulted in the side-by-side aggregation of rod-shaped Sak57 forming thick bundles. Indirect immunofluorescence was used to determine the localization of Sak57 in isolated outer dense fibers, epididymal sperm, spermatids, and pachytene spermatocytes. Confocal laser scanning microscopy was used to analyze the three-dimensional arrangement of Sak57 in pachytene spermatocytes. Isolated outer dense fiber and sperm tails displayed an immunoreactive product in the form of linear clusters. In elongating spermatids (steps 10-11), Sak57 immunoreactivity was predominant in the head region whereas pachytene spermatocytes displayed a cortical cytoplasmic distribution. Results of this study demonstrate that Sak57 has the characteristics of a keratin intermediate filament and is present during meiotic and postmeiotic stages of spermatogenesis.
Kierszenbaum
A L
AL
Department of Cell Biology and Anatomical Sciences, City University of New York Medical School, New York 10031, USA.
Rivkin
E
E
Fefer-Sadler
S
S
Mertz
J R
JR
Tres
L L
LL
D000595:Amino Acid Sequence; D000818:Animals; D019084:Fluorescent Antibody Technique, Indirect; D006801:Humans; D007633:Keratins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D008297:Male; D051379:Mice; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D011498:Protein Precursors / Q000378:metabolism; D011817:Rabbits; D051381:Rats; D017207:Rats, Sprague-Dawley; D017386:Sequence Homology, Amino Acid; D013087:Spermatids / Q000378:metabolism*; D013090:Spermatocytes / Q000378:metabolism*; D013094:Spermatozoa / Q000378:metabolism*; D013737:Testis / Q000378:metabolism
D011498:Protein Precursors; D007633:Keratins