The gonadotrope natriuretic peptide system is differentially sensitive to chronic and pulsatile GnRH stimulation

Program: Abstracts - Orals, Featured Poster Presentations, and Posters
Session: SAT 134-163-GnRH & Gonadotroph Biology & Signaling
Bench to Bedside
Saturday, June 15, 2013: 1:45 PM-3:45 PM
Expo Halls ABC (Moscone Center)

Poster Board SAT-155
Samantha Mary Mirczuk*1, Alice Catterick1, Andrew James Lessey1, Rebecca Mary Perrett2, Craig Alexander McArdle2, Imelda Mary McGonnell1 and Robert C Fowkes1
1The Royal Veterinary College, London, United Kingdom, 2University of Bristol, Bristol, United Kingdom
Recent studies have implicated C-type natriuretic peptide (CNP) as a component of gonadotrope function. However, the precise mechanism by which CNP is regulated, particularly by GnRH, or the consequence of CNP-mediated signaling via its receptor guanylyl cyclase-B (GC-B) within gonadotropes, remains to be elucidated.  To establish whether the gonadotrope naturietic peptide system (Nppc, Npr1, Nrp2, Npr3, and Furin) is affected by GnRH signaling, we developed a novel multiplex RT-PCR assay to examine simultaneous expression of these genes, as well as transcriptional regulators of gonadotropes (Nr5a1, Nr0b1, cFos, Egr1, cJun) and dual-specificity phosphatases (Mkp1 (Dusp1) and Mkp2 (Dusp4), in a single PCR reaction. LβT2 cells were either treated for 24hrs, or for 5 mins every hour over 4hrs with 100nM GnRH, after which RNA was extracted, multiplex RT-PCR performed and expression quantified using the GenomeLab GeXP analysis system. Exposure of GnRH for 24hrs failed to increase Nppc, or Npr3, whereas Npr1, Npr2, and Furin were up regulated (1.24± 0.04, 1.35±0.07 and 1.10±0.02 respectively), in addition to, Nr5a1 and Nr0b1, cFos, and Egr1 (1.24±0.06, 1.30±0.06, 1.39±0.03 and 1.41±0.04 respectively). However, recent studies have established that different GnRH treatment paradigms in vitro result in altered signaling and transcriptional events; therefore a more physiologically relevant, ‘pulsatile’ administration of GnRH was applied. Interestingly, pulsatile exposure of GnRH led to an increase in Nppc (1.68±0.17), in addition to Npr2, Npr3, Furin, Nr5a1, Nr0b1, cFos, Egr1 and Mkp2 (1.65±0.16, 1.89±0.13, 1.38±0.12, 1.30±0.10, 1.52±0.11, 1.28±0.08 and 1.81±0.08, and 1.23±0.09, respectively). To investigate the downstream affects of CNP-mediated signaling, LβT2 cells were treated with 100nM CNP for 0, 4, 8 and 24hrs, and expression of transcriptional regulators, described were examined. Interestingly, CNP/GC-B signaling, up-regulated Nr5a1, Nr0b1 and Mkp1 after 8hrs (1.69±0.10, 1.41±0.08, and 1.65±0.13, respectively), whereas, cJun and Mkp2 were up regulated after 4hrs (1.32±0.03, and 1.37±0.13, respectively). In summary, these data suggest that the gonadotrope natriuretic peptide system is sensitive to pulsatile GnRH signaling, and that gonadotrope transcriptional regulators are sensitive to CNP/GC-B-mediated signaling, thus providing further insight into CNP function in gonadotropes, and its potential involvement as a component of the GnRH signaling pathway.

Nothing to Disclose: SMM, AC, AJL, RMP, CAM, IMM, RCF

*Please take note of The Endocrine Society's News Embargo Policy at http://www.endo-society.org/endo2013/media.cfm

Sources of Research Support: Wellcome Trust Project Grant 093257