Pulsatile exocytosis is functionally associated with GnRH gene expression in immortalized GnRH-expressing cells.
Pulsatile release of GnRH is essential for proper reproductive function, but little information is available on the molecular processes underlying this intermittent activity. Recently, GnRH gene expression (GnRH-GE) episodes and exocytotic pulses have been identified separately in individual GnRH-expressing cells, raising the exciting possibility that both activities are linked functionally and are fundamental to the pulsatile process. To explore this, we monitored GnRH-GE (using a GnRH promoter-driven luciferase reporter) and exocytosis (by FM1-43 fluorescence) in the same, living GT1-7 cells. Our results revealed a strong temporal association between exocytotic pulses and GnRH-GE episodes. To determine whether a functional link existed, we blocked one process and evaluated the other. Transcriptional inhibition with actinomycin D had only a modest influence on exocytosis, suggesting that exocytotic pulse activity was not dictated acutely by episodes of gene expression. In contrast, blockage of exocytosis with anti-SNAP-25 (which obstructs secretory granule fusion) abolished GnRH-GE pulse activity, indicating that part of the exocytotic process is responsible for triggering episodes of GnRH-GE. When taken together, our findings suggest that a careful balance is maintained between release and biosynthesis in GT1-7 cells. Such a property may be important in the hypothalamus to ensure that GnRH neurons are in a constant state of readiness to respond to changes in reproductive function.
Vazquez-Martinez
R
R
Laboratory of Molecular Dynamics, Department of Cell Biology and Anatomy, Medical University of South Carolina, Charleston, South Carolina 29425, USA.
Shorte
S L
SL
Faught
W J
WJ
Leaumont
D C
DC
Frawley
L S
LS
Boockfor
F R
FR
D000818:Animals; D000911:Antibodies, Monoclonal / Q000494:pharmacology; D002461:Cell Line, Transformed; D005089:Exocytosis / Q000187:drug effects / Q000502:physiology*; D005456:Fluorescent Dyes / Q000493:pharmacokinetics; D015870:Gene Expression / Q000187:drug effects / Q000502:physiology*; D017930:Genes, Reporter / Q000502:physiology; D007987:Gonadotropin-Releasing Hormone / Q000235:genetics*; D008156:Luciferases / Q000235:genetics; D008565:Membrane Proteins / Q000276:immunology; D009419:Nerve Tissue Proteins / Q000276:immunology; D011673:Pulsatile Flow; D011726:Pyridinium Compounds / Q000493:pharmacokinetics; D000644:Quaternary Ammonium Compounds / Q000493:pharmacokinetics; D050825:Synaptosomal-Associated Protein 25
D000911:Antibodies, Monoclonal; C073804:FM1 43; D005456:Fluorescent Dyes; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D011726:Pyridinium Compounds; D000644:Quaternary Ammonium Compounds; D050825:Synaptosomal-Associated Protein 25; D007987:Gonadotropin-Releasing Hormone; D008156:Luciferases