SAK-57, AN INTERMEDIATE FILAMENT KERATIN PRESENT IN INTERCELLULAR BRIDGES OF RAT PRIMARY SPERMATOCYTES

We have previously reported the purification of Sak 57(for spermatogen ic cell/sperm-associated keratin of molecular mass 57 kDa) from outer dense fibers of rat sperm tails, Internal protein sequence analysis of Sak 57 revealed 70-100% homology to the 1A and 2A regions of the alph a-helical rod domain of human, mouse, and rat keratins. A multiple ant igen peptide was synthesized using the KQYEDIAQK sequence correspondin g to the 2A region and a polyclonal antibody was produced in rabbit to detect Sak 57, During spermiogenesis, Sak 57 associates with the micr otubular manchette before becoming a component of para-axonemal kerati n structures of the developing tail. We now report that during late me iotic prophase, intercellular bridges linking late pachytene-diplotene spermatocytes display a distinct ribbon containing a Sak 57/beta-tubu lin complex, separated by a nonimmunoreactive midzone, Indirect immuno fluorescence demonstrates that the ribbon is the final stage of a thre e-step developmental sequence: (1) a spindlelike arrangement radiating from equidistant spherical centers in early pachytene spermatocytes, (2) an ectoplasmic shell-like framework in mid-to-late pachytene sperm atocytes, and (3) a Sak 57/beta-tubulin-containing ribbon found in int ercellular bridges linking adjacent late pachytene-diplotene spermatoc ytes. Shear forces causing a breakdown of one of the conjoined spermat ocytes do not disrupt the cytoskeletal ribbon. Results of this work, t ogether with previous observations during spermiogenesis, show that Sa k 57 associates with cytoplasmic microtubules in a timely fashion. Upo n completion of late meiotic prophase, the Sak 57/microtubule complex behaves as an intercellular ligament and contributes to both the stren gth of intercellular bridges and the cohesiveness of members of a sper matocyte lineage. (C) 1996 Wiley-Liss, Inc.