Mutation of a putative sperm membrane protein in Caenorhabditis elegans prevents sperm differentiation but not its associated meiotic divisions.

Spermatogenesis in the nematode Caenorhabditis elegans uses unusual organelles, called the fibrous body-membranous organelle (FB-MO) complexes, to prepackage and deliver macromolecules to spermatids during cytokinesis that accompanies the second meiotic division. Mutations in the spe-4 (spermatogenesis-defective) gene disrupt these organelles and prevent cytokinesis during spermatogenesis, but do not prevent completion of the meiotic nuclear divisions that normally accompany spermatid formation. We report an ultrastructural analysis of spe-4 mutant sperm where the normally close association of the FB's with the MO's and the double layered membrane surrounding the FB's are both defective. The internal membrane structure of the MO's is also disrupted in spe-4 mutant sperm. Although sperm morphogenesis in spe-4 mutants arrests prior to the formation of spermatids, meiosis can apparently be completed so that haploid nuclei reside in an arrested spermatocyte. We have cloned the spe-4 gene in order to understand its role during spermatogenesis and the molecular basis of how mutation of this gene disrupts this process. The spe-4 gene encodes an approximately 1.5-kb mRNA that is expressed during spermatogenesis, and the sequence of this gene suggests that it encodes an integral membrane protein. These data suggest that mutation of an integral membrane protein within FB-MO complexes disrupts morphogenesis and prevents formation of spermatids but does not affect completion of the meiotic nuclear divisions in C. elegans sperm.

A technique for freeze fracturing minute amounts of isolated cardiac membrane

Electron microscopy (EM) of freeze-fractured membranes provides more information about internal membrane structure than EM of thin-sectioned or negatively stained material. However, it has heretofore been impractical to use freeze fracture routinely for analysis of highly purified membrane fractions obtainable in small (micrograms) amounts, because the technique, when conventionally applied to minute pellets, yields only one fracture of unpredictable quality; it also precludes in parallel biochemical studies by using up the entire preparation. To solve this problem, we have developed a method for freeze fracturing tiny droplets of suspended membranes containing 1-10 micrograms membrane protein, thereby allowing both multiple fractures and biochemical studies. Before fracture, the final membrane fractions can be concentrated, subjected to experimental manipulations, cross-linked, and glycerinated in a dialysis bag. The technique is illustrated on isolated gap junctions from rabbit hearts, which were chosen because their unique internal membrane structure allows unequivocal identification of membrane type based on structural criteria.