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(510) THE ROLE(S) OF TYPE II GnRH RECEPTOR IN VERTEBRATE REPRODUCTION. Alok, Deoraj1,2, Zohar, Yonathan1, 1 Center of Marine Biotechnology, Baltimore, MD2 Sygen International, Berkeley, CA ABSTRACT- Like gonadotropin releasing hormones (GnRH), multiple forms of GnRH-receptors have recently been found in vertebrates, including primates and humans. Several novel GnRH-receptor forms have been cloned and characterized. We have also cloned pituitary GnRH-receptor from the highly evolved perciform fish models, striped bass (Morone saxatilis) and gilthead seabream (Sparus aurata). Similar GnRH-receptors containing a carboxy-terminal tail have been recently identified in primates. Based on their structural similarity, these GnRH-receptors were classified as the type II GnRH-receptor that exhibits the highest selectivity for the ubiquitous form of chicken (c) GnRH-II (type II GnRH) in heterologus expression systems. In our study, type II GnRH-receptor is highly expressed in the pituitary and shows a single transcript on a northern blot analysis. However, real time reverse-transcription (RT)-PCR and in situ hybridization demonstrate a wide distribution of type II GnRH-receptor in a number of extra-pituitary tissues. In order to delineate functional activity in response to endogenous GnRHs and their agonists, recombinant type II GnRH-receptor was expressed in Cos-7 mammalian and CHSE-214 fish cell lines. Differential c-fos and cAMP mediated signaling pathways in response to cGnRH-II suggests the ability of type II GnRH-receptor to couple to multiple G-proteins. A primary pituitary cell culture has been established to examine the activation of signaling pathways in response to endogenous GnRHs and their combinations. To understand type II GnRH-receptor role(s) in the control of LH and FSH beta synthesis, pituitary cell preparations were treated separately, or in combinations with endogenous GnRHs and their agonists, protein kinase A and C inhibitor and calcium chelators (EGTA and D600). We are currently evaluating the effects on the LH and FSH beta subunits and c-fos expression levels by real-time RT-PCR analyses. Understanding type II GnRH receptor interactions with its ligands and the distinct signaling mechanism in a highly evolved fish pituitary cell, offers potential solutions to the functional significance of multiple GnRHs in the control of vertebrate reproduction. KEY WORDS: GnRH , Receptor, Signaling, Pituitary |
