Patrick E. Chappell, Ph.D. - US grants

The pulsatile secretion of gonadotropin-releasing hormone (GnRH) from the hypothalamus is crucial for normal reproductive function in mammals, including humans. Although much past research has concentrated on what neuronal and hormonal factors may modulate this pulsatile release, the cellular and molecular mechanisms regulating the timing of these events has yet to be elucidated. Recent studies suggest that a transcriptional feedback loop may be responsive for conferring circadian oscillations of gene expression found in multiple cell types. It is as yet unclear, however, whether circadian genes may also influence GnRH gene expression or secretion. Given this, we will investigate the role of the circadian genes mper1, mper2 and clock on GnRH gene expression and secretion in vitro using GT1 cells and in vivo using transgenic mice, as outlined in the following specific aims: Specific Aim 1 Determine whether GT1 cells can be induced to express a circadian pattern of mper1 expression, and whether any changes in transcription of mper1 are associated with the synchronization of GnRH gene expression and/or peptide secretion. Specific Aim 2 Determine whether disruption of normal CLOCK signaling in Vitro in GT1 cells, and in vivo in transgenic mice, affects patterns and timing of GnRH gene expression and/or GnRH release. Results from these studies will allow for a greater understanding of possible molecular and cellular mechanisms underlying the pulsatile secretion of GnRH required for normal reproductive function.

DESCRIPTION (provided by applicant): Whereas it has long been known that normal mammalian reproduction depends upon the pulsatile secretion of gonadotropin-releasing hormone (GnRH) from the hypothalamus, it is yet unclear what cellular and molecular mechanisms lie at the heart of this exceptionally timed pulse release. Additionally, while previous studies have implicated the 24 hour biological clock in the control of reproductive hormone secretion, it remains unknown how the circadian clock might modulate the amplitude or frequency of synchronous GnRH secretory release in order to regulate reproduction. Preliminary work in this laboratory, recently submitted for publication, indicates that not only are all molecular clock components, such as mPer1, mPer2, and clock present in the GnRH-secreting GT1-7 cell line, but that transcripts of mPer1 and mPer2 oscillate in these cultured cells with a circadian period. Strikingly, perturbation of the clock in perifused GT1-7 cells, via transient transfection of a dominant negative clock, disrupts normal secretory pulse patterns, suggesting that an intracellular circadian clock within GnRH neurons may function to modulate secretion. To investigate this further, the following proposal will attempt to 1) determine the extent of regulation of GnRH by the molecular clock, by examining both transcriptional and secretory effects; 2) investigate how cell-specific disruption of clock function only in GnRH neurons in vivo impacts fertility in transgenic mice; 3) dissect inter- and intracellular mechanisms linking clock oscillation to timed GnRH pulsatile secretion, by examining real-time changes in both clock gene oscillations and membrane kinetics. Results from this proposal have the potential to answer many fundamental questions regarding the nature of the GnRH "pulse generator," provide insight into broad mechanisms of endocrine neurosecretion, and allow for the candidate to fully develop into a principal investigator capable of eventually becoming a leader in reproductive neurobiology. Additionally, these studies could also advance the field of circadian biology, by ultimately demonstrating how oscillation of transcripts at the molecular level can control synchronous events at the multi-cellular and tissue level, in order to regulate numerous biological processes, from cellular metabolism to exocytosis, and even to orchestrate complex series of behaviors. Potential applications could lead to new directions in treating a range of physiological disorders that result from malfunction of hypothalamic neurosecretion, such as polycystic ovarian syndrome and primary ideopathic hypogonadism.

DESCRIPTION (provided by applicant): Whereas it has long been known that normal mammalian reproduction depends upon the pulsatile secretion of gonadotropin-releasing hormone (GnRH) from the hypothalamus, it is yet unclear what cellular and molecular mechanisms lie at the heart of this exceptionally timed pulse release. Additionally, while previous studies have implicated the 24-hour biological clock in the control of reproductive hormone secretion, it remains unknown how the circadian clock might modulate the amplitude or frequency of synchronous GnRH secretory release to regulate reproduction. Preliminary work in this laboratory demonstrates that not only are all molecular clock components expressed in the GnRH-secreting GT1-7 cell line, but that transcripts oscillate in these cultured cells with a circadian period. Strikingly, perturbation of the clock in perifused GT1-7 cells, via transient transfection of a dominant-negative Clock, disrupts normal secretory pulse patterns, showing that an intracellular circadian clock within GriRH neurons can modulate secretion. To investigate this further, the following proposal will 1) use in vitro and in vivo models to determine the necessity of a GnRH-specific molecular clock on patterns of release required for proper reproductive status, 2) examine potential intracellular mechanisms, including transcriptional regulation and properties of cell excitability, underlying circadian clock modulation of GnRH, and 3) explore the role of the suprachiasmatic nucleus (SCN), the brain's master clock, in influencing GnRH secretion, and the involvement of cell-specific molecular oscillators in this regulation. Results from this proposal have the potential to answer many fundamental questions regarding the nature of the GnRH "pulse generator", and how patterns of secretion may be altered by cell-cell communication to produce preovulatory surges. Additionally, these studies could provide insight into broader mechanisms of endocrine neurosecretion, and advance the field of circadian biology by demonstrating how transcriptional oscillations can control synchronous events at the multi-cellular and tissue level, in order to regulate numerous biological processes, from cellular metabolism to exocytosis, and even to orchestrate complex series of behaviors. Potential applications could lead to new directions in treating a range of physiological disorders that result from malfunction of hypothalamic reproductive neurosecretion, including polycystic ovarian syndrome and primary ideopathic hypogonadism.

EAGER: Building Networks and Study Systems to Advance Research on the Biology of Pacific Corals
This EAGER award provides funding to develop an international and multidisciplinary network of researchers to study the biology of coral reefs and their resilience relative to global climate change. This study will also undertake pilot and proof-of-concept experiments, both in the laboratory and in the field, to provide better understanding of the vulnerability of coral reefs to changing environment and measures for their protection. A team of researchers from Oregon State University, the University of Hawaii, and Vassar College will make trips to research sites both in Taiwan, at their National Museum of Marine Biology and Aquarium, and in China, at their Tropical Biological Research Station in Sanya, to engage with coral-reef researchers and to lay the groundwork for on-going collaborations. The outcomes of this project, including the testing of research hypotheses and sharing of views, promise to lead to transformative results, both in biology and ecology.
This proposed activity addresses an important research area that has important implications for global environmental health. The potential for fruitful collaborations and a broader international network of coral-reef researchers should produce valuable benefits. In addition to the scientific outcomes, benefits will derive from the engagement of a number of young researchers, both post-doctoral fellows and undergraduates, who will travel to Asia and participate in this groundbreaking research. In a broader sense, better understanding of reef resilience and local ecological phenomena should lead to methods for remediation and control of reef destruction, a serious environmental problem. The establishment of an East Asia Coral Reef Alliance could be instrumental in saving critical reef populations.