Pablo Ross's Research


Our laboratory specializes in animal reproduction with emphasis on gametes, early embryos and embryonic stem cells. The focus within this area is on epigenetic mechanisms that regulate the acquisition of pluripotency and the impact of assisted reproductive technologies (ART) on these mechanisms. We use a comparative approach which includes different species (cattle, sheep, pig, horses, mice, monkeys, humans), different models of ART (IVF, ICSI, SCNT) and varied models of nuclear reprogramming (early embryo development, cloning, induced pluripotency, and embryonic stem cells). Also, we make extensive use of next generation sequencing approaches to determine transcriptome and epigenetic profiles. We are currently generating epigenetic data from multiple tissues from large animals to aid in efforts to annotate the functional regions of animla genomes as part of FAANG. For mechanistic studies we routinely microinject siRNA and CRISPR/Cas9 into oocytes and zygotes to then study consequences of gene knockdown/knockout on embryonic development. We further use these techniques to develop genetically engineered large animal models and blastocyst complementation approaches.

Transcriptome studies in oocytes and preimplantation embryos

Transcriptome analysis of oocytes and preimplantation embryos provide important insight into the mechanisms of early development and alterations to normal development induced by Assisted Reproductive Technologies (ART), such as IVF and SCNT. We have generated important databasets on transcript expression of oocytes and embryos of different species and under different conditions. We developed state-of-the-art RNA-Seq methodologies to analyze the transcriptome of single oocytes and preimplantation embryos. The application of this methodology has provided a comprehensive insight into the gene expression program during the first differentiation event in early embryonic development, as well as clues as to the establishment and maintenance of pluripotent cells.

Pluripotent stem cells in regenerative medicine applications

Pluripotent stem cells, such as embryonic stem cells (ESC) and induced pluripotent stem cells (iPSC) represent a great promise for regenerative medicine. We work on different aspects of derivation and characterization of large animal stem cells. We are interested in developing large animal models for regenerative medicine including the possibility of using blastocyst complementation to generate human tissues/organs in animals. Along this line, we have established new ESC culture conditions that allow inter-specific chimera formation and determined the chimeric potential of different types of human iPSC in porcine and ovine embryos.

In Vitro Breeding

Genetic improvement has contributed to a substantial progress in livestock production efficiency. However, it is limited by the generational interval, which is quite long for some mammalian species. Recent advancements such as the in vitro generation of gametes from murine embryonic stem cells[1, 2] and the derivation of embryonic stem cells (ESCs) from bovine embryos[3], allow us to propose new strategies to accelerate genetic improvement. By applying genomic selection on ESCs and subjecting them to germ cell differentiation and in vitro fertilization, the generational interval could be reduced to only a few months, thereby increasing selection intensity and accelerating genetic improvement. We are working on developing methods for the in vitro differentiation of gametes from ESCs to allow IVB approaches.

Epigenetic remodeling during preimplantation development

Embryo epigenetics research is focused on the remodeling of tri-methylation at lysine 27 of histone H3 (H3K27me3). H3K27me3 is well characterized as a negative regulator of transcription, is considered rather stable, and has significant developmental ramifications in a number of species. During our initial analysis of H3K27me3 reprogramming in preimplantation bovine embryos, we demonstrated that this epigenetic modification is dynamically remodeled, with a strong initial decrease in its abundance near the time of embryonic genome activation (8-cell stage in cattle). The decrease in H3K27me3 levels indicates a potential role for histone demethylases during the initial reprogramming of the embryonic genome. We have now established that among known H3K27me3 demethylases, only JMJD3 is expressed in oocytes and early embryos. Moreover, knockdown of JMJD3 by siRNA injection in bovine embryos resulted in abrogation of H3K27me3 reduction and impaired development to the blastocyst stage, indicating that JMJD3 has an important role during early embryonic development. Using the same siRNA knock-down model, we are expanding our research to study mechanisms of remodeling for other epigenetic marks.