Compartmental Adrenomedullin Signaling in the Uterus during Implantation

着床期间子宫内的隔室肾上腺髓质素信号传导

• 批准号: 9521405
• 负责人: Brooke Matson
• 金额: $ 3.58万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-07-01 至 2020-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9521405
• 关键词: Affect; Animal Model; Animals; Binding; Biological Assay; Birth; Cells; Clinical; Contraceptive methods; Cues; Cultured Cells; Data; Defect; Disease; Embryo; Endocrine; Endometrial; Endometrial Stromal Cell; Endometrium; Enzyme-Linked Immunosorbent Assay; Epithelial; Epithelial Cells; Epithelium; Equation; F Factor; Female; Fertility; Functional disorder; Genetic; Genetically Engineered Mouse; Genotype; Gestational Age; Glean; Human; Implant; In Vitro; Individual; Infertility; Intercellular Junctions; Invaded; Investigation; LIF gene; Lactoferrin; Light; Litter Size; LoxP-flanked allele; Luciferases; Maintenance; Messenger RNA; Methods; Molecular; Mus; Outcome; Pathway interactions; Phenotype; Pregnancy; Pregnancy Complications; Process; Proteins; Quantitative Reverse Transcriptase PCR; Signal Transduction; Site; Small Interfering RNA; Stat3 protein; Stromal Cells; Testing; Therapeutic; Tight Junctions; United States; Uterus; Woman; adrenomedullin; blastocyst; calcitonin receptor-like receptor; dosage; experimental study; fetal; healthy pregnancy; implantation; in utero; in vivo; infertility treatment; inhaled nitric oxide; innovation; insight; loss of function; mouse model; natural Blastocyst Implantation; novel; prevent; promoter; protein expression; public health relevance; pup; receptor; subfertility; uterine receptivity

 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.

 描述（由申请人提供）：胚泡植入缺陷可阻止妊娠或为妊娠晚期出现的临床并发症奠定基础。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够附着和侵入子宫内膜。然而，子宫容受性的细胞和分子机制还不清楚。我们的实验室已经确定肾上腺髓质素（Adm，AM）作为母体和胎儿来源的内分泌因子，对建立和维持健康的妊娠非常重要。使用遗传小鼠模型，我们发现了Adm+/-雌性中的生育力低下表型，其在子宫内出生异常间隔和过度拥挤的较小窝仔。重要的是，我们已经表明，母体基因型是这种表型的原因，强调了母体来源的AM剂量在子宫着床期间的重要性。然而，进一步的研究受到Adm-/-幼仔胚胎致死性的限制，因此Adm+/-雌性的生育力低下表型仍然无法解释。信号转导子和转录激活子3（STAT 3）是Adm表达的候选调节因子，其已被证明通过干扰子宫腔上皮中的连接重构来消除子宫容受性和生育力。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形成。因此，我建议测试这一假设，即STAT 3-Adm轴有助于生育提供组织线索连接蛋白在子宫durig着床期。目的1将使用标准ChIP、荧光素酶、qRT-PCR和ELISA测定法在体外确定STAT 3和Adm之间的关系。在目标1中，我还将测试AM是否直接促进了子宫容受性的标志物--棘突的形成，或新植入胚胎周围保护屏障的形成。目的2将使用功能丧失小鼠模型来表征AM信号在不同子宫隔室中对生育力和着床期间子宫的连接完整性的贡献。具体地，将floxed降钙素受体样受体（Calcrl，Calcr 2）小鼠与乳铁蛋白-iCre和Amhr 2-Cre系杂交，以分别在子宫上皮和间质区室中缺失AM受体（Calcrl，Calcr 2）。然后，我将对Cre+和Cre-动物进行全面的生育力表型分析。我还将评估是否在上皮和间质的AM信号的损失影响交界处的完整性，在这些车厢在围着床期。来自这些实验的结果将提供对子宫中Adm表达的控制及其对生育力的隔室特异性贡献的深入了解，这可能是通过组织细胞-细胞连接实现的。最终，这些研究将促进我们对子宫容受性的理解，并为开发不孕症治疗方法、妊娠并发症预防策略和新型避孕方法提供信息。