Human endometrium is a dynamic tissue whose components undergo cyclic changes under the influence of steroid hormones. Blastocyst nidation occurs in secretory endometrium, and its growth and development are believed to be directly influenced by local endometrial conditions. Proteins that bind with high affinity the insulin-like growth factors (IGF- I and -II), peptides with broad mitogenic effects, have been identified in secretory endometrium and human decidua. IGF receptors and mRNAs encoding the IGFs have also been identified in endometrium, and this IGF autocrine/paracrine system is believed to play a role in cyclic endometrial differentiation and trophoblastic growth. The ultimate goals of this laboratory are to determine optimal endometrial conditions for successful embryo implantation and to examine endometrial gene expression in response to steroid hormones. The specific goals of this project are to determine the ontogeny of the IGFs, their binding proteins (bps) and their receptors in the changing hormonal milieu of the endometrium and to determine in which cell types they are synthesized. Endometrial tissue from biopsy and hysterectomy specimens will be used as a source of RNA as well as of stromal cells for culture. Tissue from different cycle stages have been exposed to a changing hormonal environment in vivo, and cultured cells will be exposed to a variety of hormonal stimuli in vitro. Effects on cellular gene expression subsequently will be examined. Induction and regulation of IGF-bp, growth factor, and receptor mRNA will be determined by RNA hybridization analysis using cDNA probes encoding these proteins and RNA isolated from tissue and cells. A ribonuclease protection assay will be used to detect IGF-I mRNAs. Expression of IGF-bp mRNAs relative to each other will be examined by in vitro translation of poly A+ RNA and immunoprecipitation of translated bps using specific antisera. To verify protein synthesis of the IGF-bps in tissues and cells, cross-linking studies, Western immunoblots and ligand blots of tissue extracts and conditioned media will be performed. A sensitive radioimmunoassay will be used to detect IGF-I and -II peptide synthesis. In situ hybridization and immunohistochemical studies will confirm mRNA expression and protein synthesis, respectively, of the IGFs, their binding proteins and receptors as well as define their cells of origin.
