Delayed Implantation

这项研究计划的长期目标是确定一种被称为胚胎滞育的哺乳动物生殖策略的特征和调节。滞育也被称为延迟着床，是在130种或更多种类的哺乳动物中发现的一种特征，在这种特征下，胚胎在囊胚期经历可逆的发育停滞，随后重新激活并植入子宫。这一策略将交配从分娩中分离出来，并允许两者在最有利于繁殖成功和后代存活的条件下发生。我们建议继续通过比较食肉动物和啮齿动物模型来比较专性滞育和兼性滞育，重点放在胚胎的变化和子宫内膜对发育停滞的调节上。第一个短期目标是确定基因表达模式，这些基因表达模式既描述了桑拿期胚胎进入滞育的囊胚，也描述了发育复兴期间囊胚退出滞育的过程。这将通过对囊胚的两个部分--内细胞团和滋养外胚层的单细胞转录组分析来实现，然后通过转录本和蛋白质分析进行验证。第二个目标是确定调节滞育进出的子宫(子宫内膜)机制和途径，同样是通过转录组测序，然后是蛋白质组学验证。第三个目标是在体外用胚胎测试来自目标1和目标2的候选分泌因子，以确定它们作为滞育调节因子的真实性。在这个目标中，第一个目标将通过药物抑制细胞内通路来探索啮齿动物胚胎的体外靶点，第二个目标将通过使用抑制RNA和过度表达策略来控制基因表达。胚胎将与蛋白质和其他子宫来源因素一起孵化。然后，将探索这些因素，以及被证明在诱导或维持啮齿动物胚胎滞育方面有效的药理学试剂和基因组工具，以确定它们诱导牛滞育的潜力，牛是一种不会自然产生这种特征的物种。将使用体外培养的牛囊胚，终点将是发育停滞和随后重新激活的能力。在这项研究的多重影响中，将进一步发现调节滞育的机制，可能允许干预以增加表现滞育的濒危物种的繁殖。如果可以在牛胚胎中诱导滞育，就可以在不冷冻的情况下保存胚胎及其相关的细胞损伤。

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.