ASPECTS OF UTERINE RECEPTIVITY FOR IMPLANTATION

植入前胚胎向囊胚的同步发育 子宫进入接受状态的阶段和准备是必要的 用于植入。 胚泡着床的子宫容受性 时间和类固醇激素依赖性。 在大鼠和小鼠中，子宫 接受性仅在怀孕期间的有限时期内发生， 假孕激素，或当动物用孕激素（P4）治疗时， 雌激素（E）适当。 在这些物种中，子宫敏感性 分为中性、接受和非接受（不应）阶段。 在妊娠或假孕小鼠中，子宫仅在 第4天（植入当天），而到第5天（如在 通过胚胎移植假孕小鼠），子宫变得顽固 并且对胚泡或创伤刺激的存在没有反应 用于诱导着床或蜕膜细胞反应（DCR）。 的 当子宫仅暴露于P4时达到中性相。 下 这种情况下，虽然子宫可以经历一些DCR后， 它对胚泡的存在没有反应。 P4- 致敏的小鼠子宫可以接受胚泡植入， 在P4引发24-48小时后暴露于少量E。 子宫 在注射E后24至36小时之间变得难治，并且 只要P4治疗继续，则维持阶段。 虽然 子宫敏感性的各个阶段可以通过实验诱导， 啮齿动物，这些阶段的合适的细胞或分子标记物没有 尚未被确认。 几种生长因子的表达及其生物学活性 围着床期子宫和胚胎中的受体 表明这些生长因子在介导胚胎发育中很重要， 着床期间子宫的相互作用。 待测试的假设 本发明涉及转化生长因子-α（TGF-α）， 在接受子宫的腔上皮顶端作为膜， 结合前体（proTGF-α），作为受体子宫的标志物 并与表达的表皮生长因子受体（EGF-R）相互作用， 在胚泡滋养外胚层（Tr）的细胞表面顶端， 启动小鼠体内的植入过程。 我们的具体目标是 检测：（1）在中性、接受性和 非接受性（难治性）子宫;（2）EGF-R在 中性、接受性和非接受性（不应性）子宫，以及 延迟和激活囊胚的作用;（3）囊胚的状态， 在接受子宫中的着床活动;和（4）相互作用 在腔上皮proTGF-α和胚泡EGF-R之间。 的 这些研究的结果将确定是否管腔上皮 proTGF-α或Tr EGF-R可作为子宫容受性的标志物 或囊胚的活动状态，以及proTGF-α/EGF-R 信号传导对于植入是重要的。 原位北方印迹法 杂交和RT-PCR将用于检测TGF-α和EGF-R mRNA、免疫组织化学、Western印迹和ELISA将用于 检测proTGF-α和EGF-R蛋白。 EGF-R也将由 125 I-EGF的放射自显影结合和亲和交联。 将通过以下方法检测proTGF-α和EGF-R之间的相互作用： EGF-R和磷脂酶c-γ的磷酸化。

Synchronized development of the preimplantation embryo to the blastocyst stage and preparation of the uterus to the receptive state are essential for implantation. Uterine receptivity for blastocyst implantation is time and steroid hormone dependent. In the rat and mouse, uterine receptivity occurs only for a limited period during pregnancy, pseudopregnancy, or when the animal is treated with progesterone (P4) and estrogen (E) appropriately. In these species, uterine sensitivity is divided into neutral, receptive, and non-receptive (refractory) phases. In the pregnant or pseudopregnant mouse, the uterus is receptive only on day 4 (the day of implantation), while by day 5 (as examined in the pseudopregnant mouse by embryo transfer), the uterus becomes refractory and fails to respond to the presence of blastocysts or traumatic stimuli for the induction of implantation or decidual cell reaction (DCR). The neutral phase is achieved when the uterus is exposed to P4 only. Under this condition, although the uterus can undergo some DCR after traumatization, it does not respond to the presence of a blastocyst. P4- primed mouse uterus can be receptive for blastocyst implantation if exposed to a small amount of E after 24-48 h of P4 priming. The uterus becomes refractory between 24 and 36 h after an injection of E, and this phase is maintained as long as P4 treatment is continued. Although various phases of uterine sensitivity can be induced experimentally in rodents, suitable cellular or molecular markers for these phases have not yet been identified. Expression of several growth factors and their receptors in the uterus and embryo during the periimplantation period suggests that these growth factors are important in mediating embryo- uterine interactions during implantation. The hypothesis to be tested herein is that transforming growth factor-alpha (TGF-alpha), expressed apically in the luminal epithelium of the receptive uterus as a membrane- bound precursor (proTGF-alpha), serves as a marker for receptive uterus and interacts with the epidermal growth factor receptor (EGF-R) expressed apically on the cell surface of the blastocyst trophectoderm (Tr) to initiate the process of implantation in the mouse. Our specific aims are to examine: (1) Expression of proTGF-alpha in the neutral, receptive and non-receptive (refractory) uterus; (2) Expression of EGF-R in the neutral, receptive and non-receptive (refractory) uterus, and in the delayed and activated blastocysts; (3) Role of blastocyst's states of activity in implantation in the receptive uterus; and (4) Interaction between luminal epithelial proTGF-alpha and blastocyst EGF-R. The results of these studies will establish whether luminal epithelial proTGF-alpha or Tr EGF-R can serve as a marker for uterine receptivity or blastocyst's states of activity, and whether proTGF-alpha/EGF-R signalling is important for implantation. Northern blotting, in situ hybridization and RT-PCR will be used to detect TGF-alpha and EGF-R mRNAs, immunohistochemistry, Western blotting and ELISA will be used to detect proTGF-alpha and EGF-R proteins. EGF-R will also be studied by autoradiographic binding of 125I-EGF and affinity cross-linking. Interaction between proTGF-alpha and EGF-R will be tested by phosphorylation of EGF-R and phospholipase c-gamma.