Abstract

Our commitment to studying the role of calcium effector pathways in ovarian differentiation will be continued by focusing on the regulatory actions of the calcium-phospholipid-diacylglycerol (PKC) second-messenger system in a well defined and established in vitro model of swine granulosa cells that retain responsivity to a variety of hormonal agonists. We will particularly investigate the manner in which FSH and angiotensin II (presumptively acting respectively through distinct cyclic AMP and PKC intracellular signaling pathways) alter intracellular calcium ion concentrations in single porcine granulosa cells (Time-resolved fluorescence video-microscopy); how hormonal agonists trigger increased secretory activity by single ovarian cells (reverse hemolytic plaque assay of relaxin secretion by pig luteal cells); employ a microperifusion system to test the temporal concordance between intracellular calcium-ion concentration changes and secretory events; probe the causal association between increased intracellular calcium-ion concentrations and resultant secretion using experimentally induced calcium transients in permeabilized cells; and begin to dissect the important interactions between the calcium effector signaling pathway and the cyclic AMP second messenger pathway on sterol-metabolizing enzymes. These emphases reflects work carried out under my parallel RCDA, in which I have has occasion to learn appropriate single-cell and reproductive biology techniques that can now be applied to this R01 renewal. The significance of the calcium second-messenger system in the ovary will therefore be explored at the single cell level (Specific Aim I), in relation to calcium's causal relevance to hormone secretion (Specific Aim II), and in relation to the interactions between the PKC and classical cyclic AMP effector pathways (interactions to be studied at the level of steroidogenesis). Overall, we anticipate that these approaches will yield significant and interesting new information regarding ovarian cellular physiology, which will enhance our physiological understanding or reproductive biology and fertility regulation in higher mammals.

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD)
Type
Research Project (R01)
Project #
5R01HD016806-10
Application #
3313966
Study Section
Reproductive Biology Study Section (REB)
Project Start
1984-04-01
Project End
1995-06-30
Budget Start
1993-07-01
Budget End
1994-06-30
Support Year
10
Fiscal Year
1993
Total Cost
Indirect Cost

  Institution

Name
University of Virginia
Department
Type
Schools of Medicine
DUNS #
001910777
City
Charlottesville
State
VA
Country
United States
Zip Code
22904

  Publications

Veldhuis, Johannes D; Zhang, George; Garmey, James C (2002) Troglitazone, an insulin-sensitizing thiazolidinedione, represses combined stimulation by LH and insulin of de novo androgen biosynthesis by thecal cells in vitro. J Clin Endocrinol Metab 87:1129-33
Schoppee, Pamela D; Garmey, James C; Veldhuis, Johannes D (2002) Putative activation of the peroxisome proliferator-activated receptor gamma impairs androgen and enhances progesterone biosynthesis in primary cultures of porcine theca cells. Biol Reprod 66:190-8
Sekar, N; Veldhuis, J D (2001) Concerted transcriptional activation of the low density lipoprotein receptor gene by insulin and luteinizing hormone in cultured porcine granulosa-luteal cells: possible convergence of protein kinase a, phosphatidylinositol 3-kinase, and mitogen-activated Endocrinology 142:2921-8
Sekar, N; Garmey, J C; Veldhuis, J D (2000) Mechanisms underlying the steroidogenic synergy of insulin and luteinizing hormone in porcine granulosa cells: joint amplification of pivotal sterol-regulatory genes encoding the low-density lipoprotein (LDL) receptor, steroidogenic acute regulatory (stAR Mol Cell Endocrinol 159:25-35
Garmey, J C; Schnorr, J A; Bruns, M E et al. (2000) Expression of parathyroid hormone-related peptide (PTH-rp) and its receptorin the porcine ovary: regulation by transforming growth factor-beta and possible paracrine effects of granulosa cell PTH-rp secretion on theca cells. Biol Reprod 62:334-9
Sekar, N; Lavoie, H A; Veldhuis, J D (2000) Concerted regulation of steroidogenic acute regulatory gene expression by luteinizing hormone and insulin (or insulin-like growth factor I) in primary cultures of porcine granulosa-luteal cells. Endocrinology 141:3983-92
Zhang, G; Garmey, J C; Veldhuis, J D (2000) Interactive stimulation by luteinizing hormone and insulin of the steroidogenic acute regulatory (StAR) protein and 17alpha-hydroxylase/17,20-lyase (CYP17) genes in porcine theca cells. Endocrinology 141:2735-42
Garmey, J C; Guthrie, H D; Garrett, W M et al. (2000) Localization and expression of low-density lipoprotein receptor, steroidogenic acute regulatory protein, cytochrome P450 side-chain cleavage and P450 17-alpha-hydroxylase/C17-20 lyase in developing swine follicles: in situ molecular hybridization and immu Mol Cell Endocrinol 170:57-65
Flores, J A; Garmey, J C; Lahav, M et al. (1999) Mechanisms underlying endothelin's inhibition of FSH-stimulated progesterone production by ovarian granulosa cells. Mol Cell Endocrinol 156:169-78
Flores, J A; Aguirre, C; Sharma, O P et al. (1998) Luteinizing hormone (LH) stimulates both intracellular calcium ion ([Ca2+]i) mobilization and transmembrane cation influx in single ovarian (granulosa) cells: recruitment as a cellular mechanism of LH-[Ca2+]i dose response. Endocrinology 139:3606-12

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