Estudio de las implicaciones funcionales de la proteína oviductina en la fertilidad y el desarrollo embrionario en hámster dorado y conejo utilizando la tecnología CRISPR-Cas9

Résumé

Infertility is a widespread clinical problem affecting 8-12% of couples worldwide. Of these, about 30% are diagnosed as idiopathic infertility, as no causal factor is found. Female factor-related infertility accounts for about 40-50% of all infertility cases. Among the possible reasons for idiopathic infertility, probable alterations in genes involved in fertilisation and early embryonic development are worth mentioning. Genetic causes account for 5-10% in all infertile women. The oviduct, known as the fallopian tube in humans, and in particular the oviductal fluid (OF) creates a microenvironment that is involved in key processes for fertilisation and the correct physiology of the gamete and embryo. Oviductin or oviductal glycoprotein 1, encoded by the OVGP1 gene, is the major non-serum protein in the OF, having been identified in different mammalian species, including rabbits, hamsters and humans. Based on these premises, the main objective of this Thesis has been to test the role played by the oviductin protein in the modulation of fertilisation and early embryonic development, using the golden hamster (Mesocricetus auratus) and common rabbit (Oryctolagus cuniculus) species as KO animal models. In addition, this study has allowed us to assess whether the presence of oviductin is essential for correct reproductive function in these species. The second objective, also related to the OVGP1 gene, was its phylogenetic analysis in the Murinae subfamily. Within this subfamily we find the mouse, which has a functional oviductin protein, and the rat, where OVGP1 is a pseudogene. This analysis has made it possible to date the pseudogenisation of OVGP1, as well as determine which other species within this subfamily are also affected by this process. The third and last objective of this thesis is related to the block to polyspermy. Polyploidy is an undesirable condition that results in the failure of embryonic development. To prevent it, blocking polyspermy prevents fertilisation of the egg by more than one spermatozoa. Depending on the mammalian species, block to polyspermy can occurs at different levels: the oolemma, the zona pellucida (ZP) or both. By block to polyspermy at the level of the ZP, the proteins ovastacin and ZP2 play a crucial role. In the rabbit, block to polyspermy occurs at the level of the oolemma. Therefore, the scientific aim was to determine by genomic analysis whether there is pseudogenisation of the ASTL gene, the gene coding for the ovastacin protein, in lagomorphs. For the first objective, we began by characterising both KO OVGP1 animal models by molecular biology and proteomics experiments. Next, in vivo fertility was analysed by determining whether the absence of the oviductin protein triggers fertility alterations in these species. To assess the causal reasons for these alterations, a histological study of the oviduct, an analysis of pre implantation embryo development, as well as a comparative transcriptomic analysis of the oviduct and embryos were carried out. For the second objective, the genomic sequence of the Ovgp1 gene in 22 species of the Murinae subfamily was analysed by PCR, obtaining the phylogenetic tree that allows us to date the pseudogenisation of Ovgp1 in this subfamily. For the third objective, the genomic sequence of ASTL in 9 species of lagomorphs was analysed by PCR to determine the pseudogenisation of ASTL. In conclusion, the OVGP1 KO animal models presented in this Thesis have shown that the oviductin protein is essential for correct embryonic development in hamsters, but not in rabbits. The phylogenetic analysis of the Ovgp1 gene in the Murinae subfamily has allowed us to date its pseudogenisation to approximately 12.5 million years ago, affecting, apart from the rat, other species belonging to the Rattini tribe. Finally, genomic analysis of ASTL in lagomorphs has confirmed its pseudogenisation in these species, which may be the reason why the block to polyspermy does not occur at the level of the ZP in lagomorphs.