C. elegans provides an excellent model system in which to explore pathways involved in fertilization and early embryogenesis. Copious genetic resources, the transparency of the germline and embryo, and the rapid developmental time course are all features that support developmental studies in this system. The worm has already proven to be a rich source of information about egg activation and zygote polarity.
Like other animals, mitochondrial inheritance in C. elegans is strictly maternal. The fate of paternal mitochondria can be followed using males labeled with Mitotracker prior to mating. (C. elegans normally reproduce hermaprhoditically. However, males arise at a low frequency and can be used to perform matings.) Labeled sperm mitochondria are detected in the fertilized oocyte after matings with labeled males . When labeled sperm are used to fertilize oocytes expressing GFP::Ub (tagged Ubiquitin protein), we observe that MOs (Membranous Organelles, found in sperm cells) are heavily ubiquitinated, but the mitochondria themselves are not. The mitochondria remain closely associated with the MOs up until the start of the first mitosis. Our model for mitochondrial elimination is that the mitochondria become autophagocytized via their close proximity to the ubiquitinated MOs. We have quantified the numbers of mitochondria and ubiquitinated MOs as well as their clustering during early embryogenesis. Those counts demonstrated that mitochondria and MOs are removed from the embryo very early in development.
Our published studies show that ubiquitination of the sperm MOs precedes the elimination of sperm mitochondria through autophagy. We have also demonstrated that both K48 and K63 linked ubiquitin chains as well as proteasomes colocalize to MOs in meiotic embryos. Follow up studies have shown that the different ubiquitin chains exhibit a different temporal profile in their localization.
An interesting finding from our studies is that proteasomes are present at significant levels in sperm. The presence of proteasomes in sperm seems a conundrum as sperm are transcriptionally and translationally inactive and, thus, are incapable of new protein synthesis. Therefore, if sperm are engaged in protein degradation, they would experience an overall decrease in protein levels over time. Although, proteasomes are present in sperm, their activity has not been tested. Another interesting finding from our studies is that C. elegans sperm contain very low levels of ubiquitin, but relatively high levels of proteasomes.
In order to further characterize the pathway that leads to ubiquitination of the sperm MOs, we ware in the process of performing a whole genome RNAi screen using commercially available RNAi libraries. The two RNAi collections account for about 17,500 genes (out of a total of 20,470 genes in C. elegans).