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.
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