Compartmental Adrenomedullin Signaling in the Uterus during Implantation

 描述(由申请人提供)：胚泡植入的缺陷可以阻止怀孕或为妊娠的临床并发症奠定基础，这些并发症出现在较高的孕龄。着床方程的一个关键变量是子宫容受性，它决定了胚泡是否能附着并侵入子宫内膜。然而，子宫容受性的细胞和分子机制还不是很清楚。我们的实验室已经确定肾上腺髓质素(Adm，AM)是一种母体和胎儿来源的内分泌因子，对于建立和维持健康的妊娠非常重要。利用遗传小鼠模型，我们在Adm+/-雌性中发现了一种亚生育表型，它们产下的小鼠在子宫中异常间隔和过度拥挤。重要的是，我们已经证明了母体基因是导致这种表型的原因，强调了在植入过程中母体来源的AM剂量在子宫中的重要性。然而，由于Adm-/-幼鼠的胚胎致死性限制了进一步的研究，因此Adm+/-雌性幼鼠的亚育表型仍然不清楚。信号转导和转录激活因子3(STAT3)是Adm表达的一个候选调节因子，已被证明通过干扰子宫腔上皮细胞的连接重塑来取消子宫的容受性和生育能力。我的初步体外和体内研究表明，AM有助于子宫内适当的紧密连接的形成。因此，我建议检验这一假设，即STAT3-Adm轴通过在围着床期为子宫中的连接蛋白提供组织线索来促进生育。目的1利用标准芯片、荧光素酶、qRT-PCR和ELISA法在体外确定STAT3与Adm的关系。在目标1中，我还将测试AM是否直接促进小齿足的形成，子宫容受性的标志，或新植入的胚胎周围保护性屏障的形成。目的2将使用功能丧失的小鼠模型来表征不同子宫间隔中AM信号对着床期间生育力和子宫连接完整性的贡献。具体地说，克隆的降钙素受体样受体(Calcr1，CLR)小鼠将与Lactoferrin-iCre和Amhr2-Cre品系杂交，以分别删除子宫上皮和间质中的AM受体CLR。然后，我将对Cre+和Cre-动物进行全面的生育表型分析。我还将评估在种植围术期，上皮和间质中AM信号的丢失是否会影响这些脑室的连接完整性。这些实验的结果将有助于深入了解Adm在子宫中的表达控制及其对生育能力的特殊贡献，可能是通过细胞-细胞连接的组织。最终，这些研究将促进我们对子宫容受性的理解，并为开发不孕不育的治疗方法、妊娠并发症的预防策略和新的避孕方法提供信息。

 DESCRIPTION (provided by applicant): Defects in blastocyst implantation can prevent pregnancy or set the stage for clinical complications of pregnancy that present at advanced gestational ages. A critical variable of the implantation equation is uterine receptivity, which determines whether a blastocyst can attach to and invade the endometrium. However, the cellular and molecular mechanisms underlying uterine receptivity are not well understood. Our lab has identified adrenomedullin (Adm, AM) as a maternal- and fetal-derived endocrine factor that is important for the establishment and maintenance of a healthy pregnancy. Using genetic mouse models, we have uncovered a subfertility phenotype in Adm+/- females, which birth smaller litters that are abnormally spaced and overcrowded in utero. Importantly, we have shown that the maternal genotype is causative of this phenotype, underscoring the importance of maternal-derived AM dosage in the uterus during implantation. However, further investigation has been limited by the embryonic lethality of Adm-/- pups, so the subfertility phenotype of Adm+/- females remains unexplained. Signal transducer and activator of transcription 3 (STAT3) is a candidate regulator of Adm expression that has been shown to abolish uterine receptivity and fertility by interfering with junctional remodeling in the uterine luminal epithelim. My preliminary in vitro and in vivo studies suggest that AM contributes to proper tight junction formation in the uterus. Therefore, I propose to test the hypothesis that the STAT3-Adm axis contributes to fertility by providing organizational cues to junctional proteins in the uterus durig the peri- implantation period. Aim 1 will define the relationship between STAT3 and Adm in vitro using standard ChIP, luciferase, qRT-PCR, and ELISA assays. In Aim 1, I will also test whether AM directly promotes the formation of pinipodes, markers of uterine receptivity, or the formation of a protective barrier surrounding the newly implanted embryo. Aim 2 will use loss-of-function mouse models to characterize the contributions of AM signaling in different uterine compartments to fertility and the junctional integrity of the uterus during implantation. Specifically, floxed calcitonin receptor-like receptor (Calcrl, CLR) mice will be crossed to Lactoferrin-iCre and Amhr2-Cre lines to delete CLR, the AM receptor, in the uterine epithelial and stromal compartments, respectively. I will then subject Cre+ and Cre- animals to a comprehensive fertility phenotyping analysis. I will also assess whether loss of AM signaling in the epithelium and stroma affects junctional integrity in these compartments during the peri-implantation period. Results from these experiments will provide insight into the control of Adm expression in the uterus and its compartment-specific contributions to fertility, potentially via organization of cell- cell junctions. Ultimately, these studies will advance our understanding of uterine receptivity and inform efforts to develop therapeutics for infertility; preventative strateies for complications of pregnancy; and novel contraception methods.