Delayed Implantation

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The long-term objective of this research program is to determine the characteristics and regulation of a mammalian reproductive strategy known as embryonic diapause. Also known as delayed implantation, diapause is a trait found in 130 or more species of mammals, whereby which the embryo undergoes reversible developmental arrest at the blastocyst stage, to be later reactivated to implant in the uterus. This strategy uncouples mating from parturition and allows both to occur when conditions are most favorable for reproductive success and offspring survival. We propose to continue studies comparing obligate and facultative diapause by comparing carnivore and rodent models, with focus on changes in the embryo and on regulation of developmental arrest by the endometrium. The first short-term objective is to determine the gene expression patterns that characterize both the entry of the morula stage embryo into the blastocyst in diapause, and the exit of the blastocyst from diapause during the renaissance of development. This will be achieved by single cell transcriptome analysis of the two compartments of the blastocyst, the inner cell mass and the trophectoderm, followed by validation by transcript and protein analysis. The second objective is to determine the uterine (endometrial) mechanisms and pathways that regulate the entry and exit from diapause, again by transcriptome sequencing, followed by proteomic validation. The third objective is to test candidate secretory factors derived from Objectives 1 and 2 with embryos in vitro to establish their authenticity as regulators of diapause. In this objective, targets from the first objective will be explored in rodent embryos in vitro by pharmacological inhibition of intracellular pathways, and secondly, by using inhibitory RNA and overexpression strategies to manipulate gene expression. Embryos will be incubated with protein and other factors of uterine provenance. These factors, along with pharmacological agents and genomic tools that prove effective in inducing or maintaining diapause in rodent embryos will then be explored to determine their potential for inducing diapause in cattle, a species where the trait does not naturally occur. Bovine blastocysts in vitro will be employed and the endpoint will be the capacity for developmental arrest and consequent reactivation. Among multiple impacts of the study will be further discovery of the mechanisms that regulate diapause, potentially allowing for intervention to increase reproduction in endangered species that display diapause. If diapause can be induced in the bovine embryo, it opens the door to embryo preservation without freezing and its associated cellular damage.