The long-term objective of this proposal is to explore the molecular basis of the regulation of embryonic implantation by progesterone and estrogen. Progesterone induces the expression of calcitonin, a peptide hormone that regulates calcium homeostasis, in the glandular and luminal epithelium of the rat uterus in the preimplantation phase of gestation. Calcitonin expression is switched off once implantation is complete, suggesting a critical regulatory role for this hormone during implantation.
The specific aims of this proposal are: (1) to determine the uterine and/or embryonic site(s) of expression of calcitonin receptor during implantation. Calcitonin acts on target cells through specific cell surface receptors. The uterine expression of the calcitonin receptor, like its ligand, is markedly elevated prior to the onset of implantation. The potential targets of calcitonin action will be identified by localizing the cells that synthesize calcitonin receptor mRNA by in situ hybridization. (2) To define the functional role of calcitonin during implantation. Administration of antisense oligodeoxynucleotide (ODN), targeted specifically against calcitonin mRNA, into the preimplantation phase uterus results in a dramatic reduction in the number of implanted embryos. This intervention also markedly suppresses the steady-state level of the calcitonin mRNA in the uterus, suggesting that the impairment of implantation is a direct phenotypic consequence of the attenuation of calcitonin gene expression by the antisense ODN. The events during implantation that are potentially regulated by calcitonin will be identified by investigating the effect of the antisense ODN on: (a) transport of the embryo into the uterus, (b) attachment of the blastocyst at the implantation sites, and (c) development of the embryo. (3) To analyze the molecular basis of steroid hormone regulation of calcitonin gene expression. Whereas progesterone stimulates calcitonin mRNA and protein expression in the uterus, estrogen antagonizes the progesterone-mediated enhancement of calcitonin expression. Transfection studies indicate that progesterone acting through its receptor induces calcitonin gene transcription. The progesterone response element in the calcitonin promoter will be mapped and the interplay between the progesterone receptor isoforms A and B, and the estrogen receptor in regulating the promoter will be examined by transfection experiments. The proposed study will provide valuable insights into the molecular mechanisms underlying the chain of events that link the transient expression of calcitonin triggered by steroid hormone receptors in the nuclei of uterine endometrial cells, to the control of embryo-endometrial interactions during implantation.
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