Isolation and preliminary characterization of the synaptonemal complex from rat pachytene spermatocytes.

A method for preparation of the morphologically intact synaptonemal complex from rat pachytene spermatocytes is described. Pachytene spermatocytes were fractionated from rat testicular cells by centrifugal elutriation. Nuclei from fractionated pachytene cells were prepared and extensively digested with micrococcal nuclease. The digested nuclei were sedimented through 20% (w/v) sucrose containing 2 M NaCl by centrifugation. About 10% of total nuclear proteins and 1-2% of total genomic DNA was found to be associated with the residual structure. The residual structure, which contains mainly the synaptonemal complex, but may be still contaminated with other nuclear components including membrane and matrix, was stained with silver and examined under light microscopy. It was found that a silver-staining component of the synaptonemal complex is not grossly different from that in pachytene nuclei not subjected to digestion and extraction. Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis revealed that virtually all the proteins in the residual structure are non-histones. The DNA isolated from the residual structure was about 135 base pairs (bp), long. The DNA was end-labeled and hybridized with a large excess of sonicated rat genomic DNA. The hybridization displayed a kinetics virtually identical to that of total nuclear DNA. We also prepared restricted DNA fragments associated with the residual structure. Southern blot analyses using a probe made from a recombinant DNA clone containing the albumin gene revealed that the DNA associated with the residual structure was not enriched (or depleted) in this gene sequence. Our results strongly suggest that (1) the synaptomenal complex may play a structural role to support the chromatin domains inside pachytene nucleus; and (2) a simple common DNA sequence in the chromatin domain is not required for association with the residual structure which contains morphologically intact synaptonemal complex in rat spermatocytes.