Endometrial changes due primarily to sex steriod stimulation are considered very important for the maintenance of human pre-implantation embryos and for successful pregnancies. One such change in the rapid rise in tissue glycogen that occurs shortly after ovulation. Our long-term objectives are the understanding of the hormonal mechanisms involved in the glycogen increase and its potential significance in the evaluation of clinical problems of infertility and early wastage.
The specific aims are: 1) Isolation and characterization of glycogen synthase (GS) from human uterus; 2) Generation of antibodies to the purified enzyme and development of a valid immunoassay for use with very small endometrial samples; 3) Measurement of turnover and production rates of the GS in normal endometrical organ cultures; how they are affected by estrogen and progesterone; and how they relate to progesterone receptor properties and distribution; 4) Measurement of GS in endometrial specimens from normal and infertile women to establish if there are cyclic variations and if they are useful in the evaluation of infertility.
Aim #1 will rely on methods (primarily Soderling's) that have proved successful in the purification of GS from rat liver, rabbit skeletal muscle and other sources. Monoclonal antibodies will be produced by i.p. injection of purified GS to Balb/c mice; fusion of spleen cells with myeloma cells; selection of hybrids in HAT medium; cloning and screening; and pertioneal inoculation of pristine mice with selected clones. Polyclonal antibodies will be prepared by injecting rabbits with GS purified in two-dimensional PAGE gels as described by Dunbar. The purifier antibodies will be used to develop a double antibody assay for GS (Aim #2). Endometrical organ cultures and cytosolic and nuclear receptor assays are standard in our laboratories; the effects of progestins (with or without addition of estrogens) on GS amount and activity, the degree of phosphorylation and their relationship to receptor distribution and properties can therefore be assessed (Aim #3). It is hoped that a more thorough knowledge of the mechanisms of glycogen metabolism will contribute to a better understanding of the human reproduction process by determining its place in the sequence of events that take place in the endometrium preparing it for implantation, and of the disruptions of this sequence that may occur in some cases of infertility (Aim #4).
