P-282 The role of activin A in the development of embryo: a preliminary insight using a mouse in vitro model

Abstract

Abstract Study question Is activin A involved in the preimplantation development of mouse embryo? Do maternal-derived and zygotic protein have a different impact on embryogenesis? Summary answer Deletion of zygotic activin A does not impair the preimplantation development of mouse embryos. However, reduced level of maternal activin A affects female fertility. What is known already Activin A is a protein secreted by the reproductive system of females, both in mouse and human. Disruption of its signalling pathway is correlated with the occurrence of ectopic pregnancies and miscarriages. Activin A is also a known regulator of the hormone FSH, affecting the growth of ovarian follicles and, consequently, the formation of functional gametes. Furthermore, expression of this protein is detected in embryos themselves and is imperative during postimplantation development. Although activin has been reported to be present from the preimplantation stages, due to the multiple sources of secretion, its role during this period of embryogenesis remains elusive. Study design, size, duration In the first part of our project, we compared the phenotype of zygotic knockout mouse embryos (Inhba−/−, n = 43) with stage-matched heterozygotes (Inhba+/−, n = 113) and wild-type (Inhba+/+, n = 42) embryos. In addition, the blastocyst outgrowth assay has been used as an in vitro implantation model. The second part of our studies focuses on the protein provided by the mother. Using hypomorphic mutants, we investigated the impact of reduced level of activin A on mouse female fertility. Participants/materials, setting, methods We analysed the morphokinetic parameters of Inhba−/−, Inhba+/− and Inhba+/+ embryos by recording their development using time-lapse microscopy. Then blastocysts were immunostained and the percentage of each cell lineage was quantified using Imaris software. To assess the fertility of hypomorphic mice, we mated them with wild-type individuals. We evaluated the time needed to receive the offspring and the size of the litter. Furthermore, we examined the functionality of oocytes of hypomorphs by in vitro fertilisation. Main results and the role of chance We revealed that the deletion of zygotic activin A does not affect the rate and course of development of embryos from the zygote to the blastocyst stage. Inhba−/− embryos have similar timing of cleavage divisions, compaction, cavitation and synchrony of the cleavage rounds to the stage-matched Inhba+/− and Inhba+/+ embryos. Moreover, Inhba−/− embryos do not significantly differ in the number and percentage of cells contributing to the cell lineages of a blastocyst. Despite the reports on the possible involvement of activin A in the embryo implantation process, the obtained Inhba−/− outgrowths have normal morphology and a similar surface area to the corresponding control embryos. Therefore, our results indicate that zygotic activin A is indispensable for preimplantation development and embryo implantation. To study the effect of activin A secreted by the maternal reproductive system on embryogenesis, we used mice with a hypomorphic mutation of the activin A gene. We revealed that homozygotic mice have a characteristic phenotype and significantly reduced fertility. Crossing hypomorphic homozygous females with wild-type males resulted in the pregnancy of only one of the seven females. These results, combined with successful oocyte in vitro fertilisation experiments, point to the role of maternal-derived activin A in mouse reproduction. Limitations, reasons for caution Literature data indicate that oocytes have a pool of protein that is a product of the maternal genome. This maternal activin A can partially compensate for the lack of zygotic protein. Therefore, our further research will address the consequences of maternal and maternal-zygotic activin A knockout on embryo development. Wider implications of the findings Separate examination of zygotic and maternal-derived activin A allowed a comprehensive investigation of the contribution of this protein to mouse reproduction and preimplantation embryo development. Despite using a mouse as a model organism, the obtained results may become an argument for discussion of the impact of activin on human pregnancy. Trial registration number not applicable