Virendra B. Mahesh - US grants

Only a limited amount of information is currently available on the control of follicle growth and follicle atresia. Many clinical situations in the human that result in excessive androgen secretion are associated with ovulatory failure. Recent work in our laboratory has used an animal model for the study of follicular atresia. The model consists of injecting 32 IU of pregnant mare serum gonadotropin (PMSG) on Day 30 to female rats hypophysectomized on Day 24 and studying either ovarian histology for various healthy and atretic follicular counts or ovulation induced by hCG 54 hrs after the PMSG. This model which gives near normal ovulation and in which the histological findings can be confirmed by a functional response, was used to demonstrate that the non-aromatizable androgen DHT caused follicular atresia and that estradiol was protective against the DHT effects. The renewal proposal will study the time course of the effect of DHT in altering blood and ovarian steroids, granulosa cell and whole ovary steroidogenesis, particularly, 5 Alpha-reductase and aromatase and granulosa cell FSH and LH receptors. Attempts will be made to separate healthy and atretic granulosa cells using Percoll and metrizamide gradients. Since a single injection of DHT results in elevation of blood steroids for only short periods of time, the above will also be studied in the presence of a stable blood level of DHT achieved by implanting a Silastic capsule of DHT. The effects of DHT on altering estrogen effects in increasing FSH sensitivity and development of granulosa cel LH receptors will be studied by using increasing quantities of DHT in the presence of a constant amount of estradiol in the hypox rat. The role of androgens and estrogens in the regulation of follicular growth and atresia will be further studied in the hypox rat and in the young adult cycling rat by the use of anti-androgens ad antiestrogens and examination of granulosa androgen and estrogen receptor dynamics. The results will not only provide new information on the control of mammalian folliculogenesis but may also be of value in understanding human polycystic ovarian disease.

The steroidal modulation of FSH and LH secretion is an important area of study for a full understanding of the control of ovulation. The immature female rat castrated on Day 26 of age and primed with 0.1 Mug/kg BW of estradiol will be used to study the effects of 20 Alpha; 5 Alpha; 20 Alpha; 3 Alpha; 5 Alpha; 20 Alpha; and 3 Beta, 5 Alpha-reduced metabolites of progesterone on FSH and LH secretion. The selective release of FSH by 5 Alpha-dihydroprogesterone and selective release of LH by 3 Alpha-hydroxy-5 Alpha-pregnant-20-one will be confirmed in the 21-day-old rat injected with pregnant mare's serum gonadotropin. The requirement for 5 Alpha-reduction of progesterone for FSH release will be tested by using a 5 Alpha-reductase inhibitor. The effect of progesterone metabolites on hypothalamic LHRH secretion will be evaluated by measuring medial basal hypothalamic, preoptic area, and plasma LHRH levels along with serum LH and FSH levels at 15 min intervals after injecting the progestin in estrogen-primed castrated rats. The direct effect of progesterone and its metabolites on the pituitary will be determined by studying the effect of the steroids on cellular content and basal and LHRH stimulated release of LH and FSH in dispersed pituitary monolayer cell cultures. The separation of various gonadotroph populations will be attempted by using unit gravity and Percoll gradient techniques. The time course of progesterone effects on anterior pituitary nuclear estrogen receptors will be studied at 1, 2, 3, 4, 8 and 12 hrs after the progesterone injection in vivo and the effects of progesterone metabolites on estrogen receptor dynamics will be evaluated. The specificity of the effect of progesterone treatment of the nuclei or cytosol in depleting nuclear estradiol receptors in vitro will be studied by examining the effects of cholesterol, corticosterone and 5 Alpha-dihydrotestosterone. Whether transcription and/or protein synthesis is involved in the action of progesterone in the reduction of occupied nuclear estrogen receptors will be determined by using actinomycin-D and cycloheximide. Experiments will be done to determine the interactive effects of progesterone on estrogen receptors and estrogens on progesterone receptors and to determine if progesterone modulates its own receptors. In these studies, cytoplasmic and nuclear estradiol and progesterone receptors will be measured after treatment of castrated rats with various estrogen and progesterone regimens.

These studies are designed to examine the significance within prostatic tissue of androgen receptor species which, by virtue of differences in their avidity for androgenic ligands, may function as regulators of the responsiveness of androgen-sensitive cells. In pursuit of our observation that two such cytoplasmic forms of this receptor do co-exist, we propose to define various physicochemical properties of these forms, as well as any others which may occur, including especially nuclear and heat-transformed cytoplasmic moieties. The emphasis will be on comparative aspects of interaction with androgens, and susceptibility to interconversion by a cytoplasmic factor which has been discovered and which will be purified and characterized further. The relative levels of the different receptor forms will be assessed under a variety of experimentally-induced changes in endogenous hormonal milieu, including castration-replacement, aging, and administration of assorted types of steroid hormones and prolactin. We will exploit the ability to separate the cytoplasmic receptor forms by ammonium sulfate fractionation in experiments wherein the dynamics of nuclear uptake and mechanisms of nuclear retention of receptor will be explored. Patterned induction of specific protein synthesis in response to androgen will be investigated as a function of the relative receptor-dictated responsiveness of the cells; these analyses are an outgrowth of preliminary observations which suggest a potentially important correlation. While not within the scope of this initial stage of the project, the implications of these studies with respect to defining a relationship between functional androgen receptor concentration and prediction of responsiveness of human prostatic cancer to endocrine therapy will certainly constitute the next major phase of this work.

Mechanisms by which steroid hormone receptor levels and functionality are controlled represent the focus of these studies. Model systems will be used in which we can examine receptor regulation by steroid hormones themselves, by peptide hormones, and by a class of compounds that may directly interfere with nuclear interactions of steroid-receptor complexes. The first system utilizes estrogen- and androgen-responsive tissues of the rat (primarily uterus, ventral prostate and anterior pituitary) to probe the dynamics of estrogen and androgen receptor turnover as influenced by the respective favored classes of steroid hormones themselves. A key feature of these studies at their present stage of development is the painstaking and systematic analysis of fluctuations in all populations of intracellular receptor. An off- shoot of this work has been the description of a discrete set of high affinity microsomal binding sites that are specific for steroid hormones and appear to mask microsomal acceptor sites for classical steroid-receptor complexes, thus being capable of regulating intranuclear levels of these complexes. In the next phase of these investigations, we will define acceptor specificity, explore the function of the microsomal binders as receptors for elicitation of extra-genomic actions of steroids, and, using monoclonal antibodies to the estrogen receptor, probe the origin and turnover of these proteins. Localization within specific organelles and initial attempts at purification will also be undertaken. The second major system, receptor control by peptide hormones, will continue to be addressed by the prolactin-mammary gland estrogen receptor interplay. We will use viral infection studies to investigate a correlation between levels of MMTV expression and sensitivity of mammary tissue to prolactin as a regulator of estrogen receptor activity. We will continue to study the mechanism by which prolactin regulates these receptors, concentration on differential nuclear binding affinities of prolactin-induced vs direct-estrogen-induced receptors, under conditions where graded responses can be engendered. We will begin to look for other factors involved in receptor regulation in this tissue. The third system involves modulation of steroid-receptor complex interactions at the nuclear level by agents which may interact directly with DNA to alter receptor binding to its effector sites. We have at hand a natural product that prevents formation of estrogen-responsive populations of mammary tumors by regulating receptor activity without direct interaction with the receptor. This may represent a novel form of receptor activity regulation.

biomedical equipment resource; biomedical equipment purchase;

The growth and development of ovarian follicles is dependent upon the population of granulosa cells present in the follicle. A systematic study of the growth and differentiation of granulosa cells has been difficult because of the differences in granulosa cells within the same follicle. Functionally different populations of granulosa cells can be separated using Percoll gradients from the ovaries of immature rats treated with diethyl stilbestrol (DES) to induce follicular growth and pregnant mare's serum gonadotropins (PMSG) to induce follicular growth and differentiation. Two populations of granulosa cells gave been shown to exist by flow cytometry and EM studies. Granulosa cells from DES and PMSG treated rats will be separated by the use of an EPICS 5 flow cytometer and Percoll gradients. The cells will be characterized by light and electron microscopy, presence of FSH and LH receptors and FSH induced estradiol and progesterone production. A combination of the enrichment of ovarian follicles by size and granulosa cell separation is expected to yield populations of granulosa cells in various stages of growth and differentiation. The use of the DES and PMSG primed immature rats is being made to work out experimental procedures in a cost effective manner with minimal animal use and the results obtained will be verified in the adult cycling rat. In order to obtain sufficient numbers of granulosa cells for further studies in different stages of growth and differentiation with use of a minimal number of animals, immortalization by transfection with the large T antigen of the SV40 virus will be attempted to generate stable cell lines. Such transfection studies have been successful in our hands using bovine adrenal cells in which the cells retained their pretransfection characteristics. With purified granulosa cell population with different growth and differentiation characteristics, detailed studies will then be done on the regulation of growth and differentiation by agents such as growth factors, estradiol, cAMP analogues, and FSH singly or in combination in a serum free defined medium. The effect of androgens on the stage dependent stimulation or inhibition of aromatase will also be studied. These studies are expected to provide new insights into the factors that lead to healthy ovulatory follicles or atretic follicles during the process of folliculogenesis.

The major objectives of this proposal are to obtain training and experience in molecular biology techniques so that they can be used in the Principal Investigator's laboratory in his research. The training will be provided by Drs. Frank French and Elizabeth Wilson at the University of North Carolina at Chapel Hill. The designated trainee will be Jaime Steinsapir, M.D., Ph.D. who will work for at least one year after completion of training in the P.I.'s laboratory. The research to be conducted is as follows: Prostate cells are responsive to androgens by virtue of the presence of functional androgen receptors (AR). The goal of these studies is to determine mechanisms by which androgens and antiandrogens control AR activity and turnover in normal and neoplastic prostate cells. The effects of androgen withdrawal on androgen and antiandrogen control of mRNA-AR levels and initial transcription rate of the AR gene will be examined in rat ventral prostate and human prostate cancer cells (LNCaP). The assessment of the effects of androgen and antiandrogens on the transcriptional activity of the AR gene upstream enhancer/promoter coupled with studies on AR binding to promoter sequences should determine specific response elements involved in AR autoregulation of the AR gene. The results will further help to understand the molecular mechanisms of regulation of AR gene transcription by androgens and antiandrogens. This project will be used as a basis for training in the use of molecular biology techniques in hormone receptor synthesis and turnover.