GnRH pulses are tightly regulated for the maintenance of reproductive cycles. Pulsatile GnRH release is an intrinsic property of GT1 GnRH cells and endogenous GnRH neurons. Based on findings in GT1 cells, we hypothesize that the cAMP signaling pathway participates in the stimulation of GnRH secretion and the timing of GnRH pulses. Our findings show that increases in cAMP stimulate GnRH secretion by opening cAMP-gated cation (CNG) channels leading to increased excitability and depolarization of the neuron. Increased neuron excitability is reflected in increased action potentials, Ca2+ oscillations and GnRH secretion. Increased cAMP levels also activate PKA that appears to initiate negative feedback pathways. We will study the role of these signaling molecules on the regulation of GnRH secretion in vitro in the GT1 GnRH cell lines and in vivo in transgenic rats. We will decrease neuron excitability by lowering cAMP levels by expressing the constitutively active phosphodiesterase, PDE4D1, or inhibiting CNG channel activity by expressing a dominant/negative (D/N) mutant of the CNG2 channel subunit (DMCNG2). We will increase neuron excitability by inhibiting the PKA negative feedback pathway by expression of the D/N mutant of the regulatory subunit of PKA mRAB and by increasing cAMP levels by expressing a constitutively active soluble adenylate cyclase (sAC). In GT1 cells we will use adenovirus vectors to target expression of the genetic probes. We will study changes in GT1 neuron excitability (Ca2+ oscillations) and the frequency and amplitude of GnRH pulses. We have now shown that expression of PDE4D1 in GT1 cells inhibits Ca2+ oscillations and pulsatile GnRH release. Genetic probes shown to be effective in experiments with GT1 cells will be cell specifically targeted to GnRH neurons in transgenic rats using the rat GnRH gene promoter/enhancer. We have now shown that targeted expression of PDE4D1 in a line of transgenic rats decreased the frequency of LH pulses in castrated males and females. Females were infertile and had blunted LH ovulatory surges or polycystic ovaries. In addition to advancing our knowledge of the signaling pathways involved in timing pulsatile GnRH secretion these animals will provide important models for studying the effects of alterations in GnRH pulsatility on reproductive function. Potentailly these findings may be relevant to the understanding of human disorders.

Agency
National Institute of Health (NIH)
Institute
Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD)
Type
Research Project (R01)
Project #
5R01HD041996-05
Application #
7149186
Study Section
Reproductive Endocrinology Study Section (REN)
Program Officer
Lamar, Charisee A
Project Start
2003-02-01
Project End
2008-11-30
Budget Start
2006-12-01
Budget End
2008-11-30
Support Year
5
Fiscal Year
2007
Total Cost
$290,891
Indirect Cost
Name
University of California San Francisco
Department
Obstetrics & Gynecology
Type
Schools of Medicine
DUNS #
094878337
City
San Francisco
State
CA
Country
United States
Zip Code
94143
Chen, Qiumei; Weiner, Richard I; Blackman, Brigitte E (2010) Decreased expression of A-kinase anchoring protein 150 in GT1 neurons decreases neuron excitability and frequency of intrinsic gonadotropin-releasing hormone pulses. Endocrinology 151:281-90
Blackman, B E; Yoshida, H; Paruthiyil, S et al. (2007) Frequency of intrinsic pulsatile gonadotropin-releasing hormone secretion is regulated by the expression of cyclic nucleotide-gated channels in GT1 cells. Endocrinology 148:3299-306
Gomez, Francisca; la Fleur, Susanne E; Weiner, Richard I et al. (2005) Decreased gonadotropin-releasing hormone neuronal activity is associated with decreased fertility and dysregulation of food intake in the female GPR-4 transgenic rat. Endocrinology 146:3800-8
Yoshida, Hiroshi; Paruthiyil, Sreenivasan; Butler, Paul et al. (2004) Role of cAMP signaling in the mediation of dopamine-induced stimulation of GnRH secretion via D1 dopamine receptors in GT1-7 cells. Neuroendocrinology 80:2-10
El Majdoubi, Mohammed; Paruthiyil, Sreenivasan; Weiner, Richard I (2003) Pulsatile luteinizing hormone and follicle-stimulating hormone secretion and gonadotropin subunit mRNA levels in the ovariectomized GPR-4 transgenic rat. Neuroendocrinology 78:287-93
Yoshida, Hiroshi; Beltran-Parrazal, Luis; Butler, Paul et al. (2003) Lowering cyclic adenosine-3',5'-monophosphate (cAMP) levels by expression of a cAMP-specific phosphodiesterase decreases intrinsic pulsatile gonadotropin-releasing hormone secretion from GT1 cells. Mol Endocrinol 17:1982-90