Separation of spermatogenic cells from adult transgenic mouse testes using unit-gravity sedimentation

During the final stage of spermatogenesis (i.e., spermiogenesis), round spermatids differentiate into mature spermatozoa. This transformation is mediated by a suite of nuclear packaging proteins. These include the transition proteins and the protamines. The two human protamines PRM1 and PRM2, and transition protein TNP2, are encoded by a single chromatin domain bounded by two regions of matrix attachment. Previous transgenic studies in our laboratory have shown that mice harboring a 40-kb segment of human chromosome 16p13.13 containing the PRM1--> PRM2-->TNP2 domain express the transgene in a haploid-specific, copy number-dependent, and position-independent manner. While these results indicate that this segment of the genome is a complete structural and functional regulatory unit, the elements governing the haploid expression of this suite of genes remain to be clearly defined. The preparation of spermatogenic cells is required to begin to address this mechanism. The CELSEP (Wescor/Dupont Inc. Wilmington, DE) unit-gravity sedimentation apparatus provides a simple, efficient, and reproducible means to separate testicular germ cells at all stages along this differentiative pathway. The high quality and integrity of germ cells obtained by this means provides a valuable resource for characterizing the molecular mechanisms governing the regulation of the PRM1-->PRM2-->TNP2 domain during spermatogenesis. A discussion of the CELSEP apparatus and the application of this methodology in our laboratory are presented.