Formation and Integrity of paternal chromosomes in the zygote – ZygoPat

Our understanding of the molecular mechanisms involved in zygote formation is largely based on the use of model organisms. The role of MH in paternal chromosome transmission was indeed discovered in the fruit fly Drosophila by Partner #1. Our main methodology combines the powerful Drosophila genetics and the use of confocal microscopy which allows for the observation of nuclei deep into the gigantic egg cell. This strategy will be reinforced by the use of the worm C. elegans by Partner#2, another well-established animal model for genetics and in vivo observations. Finally, a large-scale genetic screen based on the RNA interference technology will be conducted in Drosophila in order to discover new maternal functions involved in the transmission of paternal chromosomes to the zygote.

The results already obtained have shown that MH is critically required for the preparation of the paternal genome for the first zygotic replication. The MH protein is a protease that could be involved in the timely elimination of sperm specific chromosomal proteins or maternal proteins involved in the topological reorganization of the male nucleus at fertilization. MH is highly conserved in animals and its human representative, named Spartan, has been recently involved in the resistance to UV radiation by several research groups. This led us to analyze this putative function in flies. Indeed, we have found that mh mutant larvae are hypersensitive to UV, thus demonstrating the functional conservation between MH and Spartan.
In addition, we have performed a genetic screen in Drosophila and identified new maternal functions required for the formation of paternal chromosomes during zygote formation. We are now currently studying these new genes.

To be completed later.

1. Delabaere L, Orsi G, Sapey-Triomphe L, Horard B, Couble P and Loppin B. The Spartan ortholog maternal haploid is required for paternal chromosome integrity in the Drosophila zygote (2014) Current Biology, in press.

2. Tirmarche S, Kimura S, Sapey-Triomphe L, Sullivan W, Landmann F, and Loppin B. Drosophila protamine-like Mst35Ba and Mst35Bb are required for proper sperm nuclear morphogenesis but are dispensable for male fertility. G3 :Genes, Genomes, Genetics, in press

Sexual reproduction implies the union of two haploid gametes to form the diploid zygote, the first cell of the embryo. In animals, the sperm nucleus is highly condensed and its DNA is packaged in a way that is not compatible with basic nuclear activities. At fertilization, its transformation into a pronucleus involves the genome-wide replacement of sperm chromosomal proteins with maternally-provided histones, the repair of DNA lesions accumulated on sperm DNA and the de novo acquisition of a DNA replication program. The correct realization of these steps during the rapid decondensation of the male pronucleus is critical for the successful transmission of paternal chromosomes to the zygote. Drosophila is a powerful model to study such poorly known processes at the functional level. In this project, we propose to study male pronucleus formation through the functional characterization of a conserved protein, Maternal Haploid (MH), which is specifically and critically required for the integration of paternal chromosomes in the zygote. By employing a diverse array of experimental approaches, such as transgenic recombinant proteins, high quality imaging techniques and the purification of interacting proteins, we (Partner#1 – B. Loppin group) will explore in a first task the molecular role of MH during male pronucleus formation. More specifically, we will address the potential implication of MH in the replication, repair and condensation of paternal chromosomes, as suggested by the detailed characterization of its mutant phenotype.
These efforts will be combined with those of another group (Partner#2 – F. Palladino group) to determine if the MH protein has a general role in male pronucleus formation, or if it has evolved different functions in other sexually reproducing animals. For this goal, we will characterize the C. elegans mh ortholog both functionally (a loss of function allele is available) and cytologically. Another major interest associated with MH is the presence of a protein domain called SprT, which is highly conserved in all MH orthologs. Interestingly, this ancient domain contains a predicted Zinc metalloprotease signature found in non-SprT proteases. Animal genomes contain a surprisingly small number of genes encoding SprT-like proteins but none of them has been studied so far. We will thus characterize its function for the first time in both model organisms.
In the third principal task, Partner#1 will take advantage of a new, germline specific RNAi resource to conduct for the first time a large-scale genetic screen aimed at identifying genes required for paternal chromosome formation. We hope more specifically to select lines associated with a mh-like phenotype to identify genes involved in the same molecular pathway.
In conclusion, our combined analysis of MH orthologs in both Drosophila and the nematode C. elegans should not only decipher the molecular role of MH in the male pronucleus but will also give us a broader perspective regarding its functional conservation in metazoans.