The rodent GnRH receptor was characterized in Xenopus oocytes injected with RNA isolated from rat pituitary and from a gonadotrope cell line, αT3, derived from a transgenic mouse. Three to 4 days after 150-200 ng RNA injection, 93% of the oocytes, which were recorded by voltage clamp, responded to 10-7 m GnRH. The mean inward currents obtained after RNA injection were 620 ± 88 nA (n = 22) with pituitary RNA and 1415 ± 598 (n = 4) with αT3 RNA. The threshold GnRH concentration able to evoke the dose dependent current after pituitary RNA injection was 3 x 10-9 m GnRH. The GnRH receptor response of the oocyte was antagonized by [D-Phe2, 6, Pro3] GnRH and [N-Ac-D-Na](2)1, D-αD-Me, pCI-Phe2, D-Arg6, D-Ala10-NH2]GnRH and could be elicited by D-Ser(But)6, Pro9-N-ethylamide GnRH (buserelin). The reversal potential of the GnRH generated current as determined by voltage-ramp was -22.5 ± 1.0 mV (n = 7) and -25.6 ± 3.3 mV (n = 3) in pituitary and cell line RNA-injected oocytes respectively, consistent with the chloride reversal potential. The GnRH receptor response was virtually eliminated by intracellular EGTA injection but was unaffected by ligand application in calcium-free perfusate. The GnRH-evoked response is mimicked by intracellular injection of inositol 1, 4, 5-trisphosphate. To determine the size of the GnRH receptor mRNA, αT3 RNA was size fractionated through a sucrose gradient. The maximal GnRH response was induced by a fraction larger than the 28S ribosomal peak. Thus we find that oocytes injected with RNA from an appropriate source develop an electrophysiological response to GnRH which is dependent on intracellular calcium mobilization, is independent of extracellular calcium, and may be mediated by inositol 1, 4, 5-trisphosphate.
ASJC Scopus subject areas
- Molecular Biology