RESUMEN
Upon C. elegans 's oocyte fertilization, the sperm brings mitochondria and membranous organelles (MOs) which are rapidly eliminated by autophagy. Their poly-ubiquitylation is suspected to be a signal for their recognition and degradation but mitochondria poly-ubiquitylation remains debated. Using fluorescent Tandem-repeated Ubiquitin-Binding Entities (TUBEs) we confirmed the presence of K48- and K63-ubiquitin chains on MOs contrasting with the absence of signal on sperm mitochondria. This new and sensitive approach confirmed the poly-ubiquitylation of the MOs while providing additional arguments for the absence of substantial poly-ubiquitylation of sperm-derived mitochondria, suggesting that K63- and K48-poly-ubiquitylation are unlikely acting as a common targeting signal for their degradation.
RESUMEN
Caenorhabditis elegans is a model system widely used in fundamental research. Even though, nematodes are easy to maintain in the laboratory, obtaining large populations of worms require a lot of work and is time consuming. Furthermore, because C. elegans are mainly hermaphrodite it is even more complicated to obtain large amounts of males which make high-throughput experiments using C. elegans males very challenging. In order to overcome these limitations, we developed affordable and rapid methods to: (1) grow large synchronous worm populations (2) easily obtain large amounts of males We developed a culture method on plates to grow big synchronized worm populations with the standard incubators used on all worm labs. We also established an easy filtration method allowing to obtain large male populations in an hour. After filtering, the worm population contains more than 90% of adult males and no adult hermaphrodites since all the contaminants are larva and embryos. The culture and the filtering methods we developed are easy to implement and require a very limited investment in equipment and consumables beside the standard one present in worm labs. In addition, this filtering method could be applied to nematode's species similar in size to C. elegans.
RESUMEN
Loss of membrane potential of sperm mitochondria has been regarded as the first step preceding mitophagy degradation after their entry into the C. elegans oocyte at fertilization. This is in line with the classical view of mitophagy of defective or abnormal mitochondria and could serve as a recognition signal for their specific and quick autophagy degradation. Here, using TMRE (tetramethylrhodamine ethyl ester) and live imaging we show that this is not the case. Instead, sperm inherited mitochondria show a stable labeling with TMRE before and at the time of autophagosomes formation. Interestingly, this labeling remains in late-stage-embryos of autophagy-defective-mutants suggesting that the loss of membrane potential occurs upon the entry of the mitochondria into the autophagy pathway. These stabilized and still polarized sperm mitochondria remain distinct but associated with the maternal-derived mitochondrial network suggesting a mechanism that prevents their fusion and represents an efficient additional protective system against fertilization-induced heteroplasmy.
