Manipulation of the in vitro environment aiming at the acquisition of totipotency in vitro by means of bovine induced pluripotent stem cells

Abstract

After fertilization and during the early phases of embryogeneisis, germ cells have the essential function of achieving totipotency so that genetic information is correctly passed on to the next generation, thereby ensuring genetic diversity. In this period, totipotent are subjected to epigenetic reprogramming and converted towards the germinal lineage. Much has been investigated about the mechanisms and systems of development of such cells and, in the last decade, many efforts have been made to reconstitute all these processes in vitro (in vitro gametogenesis). The in vitro reconstitution of the entire gametogenesis process through the differentiation of pluripotent stem cells has been successfully reported only in humans and mice, and such studies open the possibility of producing viable oocytes or spermatozoa in other species, such as pigs and cattle. In this context, our main unprecedented goal is to provide a model of in vitro generation of germ cells in cattle by studying the effect of the environment on gene and epigenetic expression of such cells, thus evaluating their potential to generate competent gametes. In vitro gemetogenesis would greatly contribute to the growth and development of the reproductive biology of production animals in Brazil. With this purpose, bovine induced pluripotent stem cells (biPSCs) previously produced and characterized by our research team will be initially differentiated into primordial germ cell-like cells (PGCLCs) and then subjected to aggregation with follicular fluid using a reconstituted ovary culture system (rOvary), previously described in mice. Follicular fluid has been shown in species such as cattle and pigs, to provide a favorable environment for the process of differentiation of ovarian stem cells or fibroblasts into oocyte-like cells (OLCs), mainly by helping in the maturation process, reducing production levels of reactive oxygen species (ROS), and increasing responsiveness to gonadotropin. Cell samples will be analyzed for morphology, gene expression, protein, and epigenetic changes during different periods of in vitro culture. The results generated in this project will enable novel perspectives for applications in assisted reproductive technologies, including transgenesis, cloning, genetic improvement and precision farming, as well as contributing to a better understanding of reproductive and developmental biology. (AU)