Performing Department
BIOMEDICAL SCIENCES
Non Technical Summary
Estradiol is a major regulator of the reproductive system in females. During periods of greater sensitivity, estradiol prevents normal estrous cycles from occurring. Increased sensitivity to estradiol occurs prior to puberty, postpartum, and in some species, during certain seasons of the year. These periods during which females cannot become pregnant can result in great economic losses to the livestock industry. This project is designed to test how estradiol inhibits the ability of the reproductive hormone, gonadotropin-releasing hormone (GnRH) to induce secretion of luteinizing hormone (LH). Luteinizing hormone is required for normal ovarian function, specifically ovulation. Our hypothesis is estradiol signaling via cell membrane receptors inhibits the ability of GnRH to stimulate secretion of LH. In our first aim we propose to identify estrogen receptors at the plasma membrane of gonadotropes, cells which secrete LH. In our second aim we propose to determine which
component of the GnRH cell signaling pathway is inhibited by estradiol, to disrupt secretion of LH. Currently, no data are available pertaining to membrane estrogen receptors in domestic livestock. It is anticipated that this research can be used to develop analogs of estradiol that could be used in management of domestic livestock to both shorten the periods of greater sensitivity to estradiol and hasten the onset of reproductive cycles.
Animal Health Component
(N/A)
Research Effort Categories
Basic
100%
Applied
(N/A)
Developmental
(N/A)
Goals / Objectives
The long-term goal of the proposed research is to determine the role of membrane estrogen receptors in regulation of luteinizing hormone (LH) secretion. That estradiol (E2) has both positive and negative effects on LH secretion has been known for decades. In general positive effects of E2 in target tissues usually occurs over hours, involves E2 crossing the plasma membrane of the cell to bind to the estrogen receptor in the cytoplasm and translocate to the nucleus to activate the genome. The negative effects of E2 on LH secretion occur very rapidly, usually less than 15 min. The rapidity which the time to response to E2 occurs is too short to be a genomic effect. Recent results from our laboratory demonstrated that membrane impermeable forms of E2 mimic the rapid inhibitory effects of E2 on LH secretion both in vitro and in vivo. These effects are presumably via interactions with estrogen receptors located at the plasma membrane of gonadotropes. The specific aims of
the research are to: 1) Determine if E2 receptors exist in the plasma membrane and if so, if they couple to G-proteins in gonadotropes; 2) Examine the effects of E2 and membrane impermeable E2 on GnRH-induced signal transduction pathways and subsequent LH release.
Project Methods
The first objective is to determine if estrogen receptors exist in the plasma membrane within lipid rafts and if so, if they couple to G-proteins in gonadotropes. Two separate approaches will be used to determine if E2 receptors are localized to lipid rafts: 1) membrane homogenates will be subjected to sucrose gradient and SDS-polyacrylamide gel electrophoresis; 2) cells will be labeled with anti-estrogen receptor antibodies and a lipid raft marker and visualized by confocal microscopy. To examine possible interactions of the E2 receptors with G-proteins coimmunoprecipitation studies will be conducted to determine if E2 or membrane impermeable E2, E2-BSA, stimulates an interaction between E2 receptors and Galphai, Galphas, or Galphaq proteins. Association of G-proteins with E2 receptors will be determined in quiescent and E2-stimulated cells in a dose- and time-dependent manner. For the second portion of this objective mouse gonadotrope cell lines, alphaT3-1 and Lbeta
T2, will be used because the pituitary consists of multiple endocrine cell types able to respond to E2 and use of the gonadotrope-derived cell lines will provide a homogenous cell model. The second objective is to determine the effects of E2 and E2-BSA on GnRH-induced signal transduction pathways. Binding of GnRH to the GnRH receptor leads to production of inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG), as well as activation of protein kinase C and mobilization of calcium. Further, GnRH signaling elevates cytosolic calcium by opening voltage-gated calcium channels. Activation of PKC and increases in intracellular calcium by GnRH stimulate release of LH. Therefore, we will determine if E2 or E2-BSA inhibits production of IP3 and PKC activation. Time-dependent effects (15, 30 and 60 min) of 10 nM GnRH in the presence or absence of 0.1 nM E2 or E2-BSA on IP3 production and PKC activity will be measured in alphaT3-1 and LbetaT2 cells. To evaluate the effect E2 has on
GnRH-induced calcium mobilization, cells will be loaded with Fura2-AM and pre-incubated with various concentrations of E2 or E2-BSA (0 - 100 nM) for 15 min prior to calcium measurements. With the previous treatments remaining in the media, 10nM GnRH will be added to the cells and changes in intracellular calcium will be recorded. Additional experiments will be conducted to determine if the changes in intracellular calcium are due to an effect of E2 on intracellular calcium stores or the influx of calcium through voltage-gated calcium channels. We are currently validating the use of an adenovirus containing the bovine LHbeta subunit promoter which expresses green fluorescent protein (GFP) to infect and identify gonadotropes. Upon validation of the specificity of this adenovirus, we will be able to sort gonadotropes from the remaining cell populations of the pituitary by flow cytometry and conduct calcium measurements using mature ovine gonadotropes.