Major Sperm Protein: life at the leading edge

Hi!

The main goal of an activated spermatid is to get in touch with an oocyte and deliver a haploid nucleus to it (fertilization). How this fertilization is done is beyond the scope of our lab’s work. The diagram below (L’Hernault, 2009) outlines well what we’re focusing on, the steps prior to fertilization.

Summary of Spermatogenesis. Click on the figure for a link to the full paper.

 

In the diagram, the little green rings in the cell are the fibrous bodies-membranous organelles (FB-MOs). In part (B), we can see the FB-MO progression with respect to the whole cellular steps (part A). The major sperm protein (MSP) filaments localize to the fibrous bodies in a sort of paracrystallized arrangement. While other nonessential components (e.g. tubulin, Golgi, ribosomes) are discarded to the residual body, it is very important that the FB-MOs segregate into the spermatids. At this point, the fibrous body filaments start to depolymerize and disperse throughout the cytoplasm. The membranous organelles dock to the cell membrane and create a fusion pore. MSP leaves the FB and needs to reach the edge of the cell to form a pseudopod; it is the polymerization and depolymerization of MSP, with the help of other components such as phosphotyrosine and ATP that help push the spermatids forward. There is no actin involve in this process but the dynamics are similar to that of actin and the leading edge.

In the rrf-3 mutant we have been studying, we first thought there was a decrease in MSP concentration just by using immunofluorescence. As we improved our preparations and microscopy techniques, and therefore reduced technical erros, we started to think this is not longer the case; both him8 (control) pictures and rrf-3 look similar in terms of MSP concentration and localization. What we did notice was a defect in the FB-MO disassembly; the membranous organelles (stained with the 1CB4 antibody) are being diffused through the cytoplasm instead of localizing to the fusion pores. It was also intriguing to see that MSP is somehow leaving the FB and making to the leading edge, even though the membranous organelle looks defective. These results are not at all conclusive though; we have to run more preps and quantify the defective spermatids. As far as MSP concentration goes, we thought that a Western blot might confirm whether or not there is an actual difference in amount of MSP.  However, a Western might not be necessary if we standarize the exposure times (same for all) for each of the control and experimental slides when we do fluorescence microscopy.

During the next few days, we will be re-doing the 1CB4 incubation to look at the membranous organelle. A look at MSP and 1CB4 in the same incubation would be ideal but there are problems with co-staining between these two antibodies. Hopefully two separate preps, with maximal standardization between them, will yield results just as well. We’re getting excited about these possible MSP defects in rrf-3 mutants but, as good scientists-in-training, we’re keeping our skeptical hats on for a bit longer.

André