Kappa Opioid Receptors Are Internalized in Mbh GnRH Cells during GnRH Pulse Termination in the Ewe

Presentation Number: SUN 472
Date of Presentation: April 2nd, 2017

Peyton Wood Weems*1, Lique M Coolen1, Stanley M. Hileman2, Steven L. Hardy2, Richard B. McCosh2, Robert L. Goodman2 and Michael N Lehman1
1University of Mississippi Medical Center, Jackson, MS, 2West Virginia University School of Medicine, Morgantown, WV


Dynorphin (Dyn), an endogenous opioid peptide, regulates the negative feedback influence of progesterone on pulsatile GnRH secretion in sheep. Dyn action is mediated via its high affinity receptor, kappa opioid receptor (KOR), present on kisspeptin/neurokinin B/dynorphin (KNDy) and GnRH neurons in the sheep medial basal hypothalamus (MBH). The Dyn/ KOR interaction is believed to play a critical role within the KNDy model of GnRH pulse generation, acting as a “stop signal” terminating each pulse. KOR is an inhibitory G-protein coupled receptor and after repeated or sustained exposure to agonists, KORs are desensitized by receptor phosphorylation and endocytosis. Recently we have shown that KOR is internalized in KNDy cells shortly after GnRH pulse onset and this internalization increases at the time of pulse termination, reflecting the release of Dyn from presynaptic afferents. However, it is unknown if Dyn also acts directly upon GnRH neurons during pulse onset and/or termination. To test this, GnRH pulses were artificially induced in unanesthetized, gonad-intact anestrous ewes via icv injection of 0.2 nmole neurokinin B (NKB) into the third ventricle. Portal blood samples were taken every two minutes prior to and following injection of NKB or vehicle to measure GnRH and LH concentrations. Animals were sacrificed at times corresponding to either GnRH pulse onset or termination and brain tissue was collected. Hypothalamic sections (45µm) were processed for immunofluorescent labeling of KOR in GnRH cells and counterstained with Fluoro-Nissl to aid in the assessment of membrane-bound vs internalized KOR immunoreactivity using confocal microscopy. Numbers of KOR-immunoreactive endosome-like particles within the cytosol and not associated with the cell membrane were counted in 1 µm optical sections through the middle of each GnRH cell body (n = 10/animal). No differences in the number of internalized particles were observed between control (n=2) and NKB (n=4) injected ewes during pulse onset. Furthermore, control animals showed no differences between pulse onset and termination. However, animals that received NKB injection showed significantly higher numbers of internalized particles during pulse termination (n=2) compared to onset (n=2). Thus it appears that, unlike KNDy cells, Dyn is released onto GnRH neurons only during pulse termination and likely acts at this time directly on both KNDy and GnRH neurons to end each GnRH pulse.


Nothing to Disclose: PWW, LMC, SMH, SLH, RBM, RLG, MNL