Identified lhb-expressing cells from medaka (Oryzias latipes) show similar Ca2+-response to all endogenous Gnrh forms, and reveal expression of a novel fourth Gnrh receptor

• Ca2+ imaging revealed three distinct response patterns to Gnrh1 and Gnrh3.
• There were few significant differences in the response between Gnrh1, 2 and 3.
• Two of four identified mGnrhRs were expressed in pituitary, gnrhr1b and gnrhr2a.
• First documented expression of gnrhr2a except in silico, dominating all stages.
• gnrhr2a showed a clear increase with gonadal maturation.

We have previously characterized the response to gonadotropin-releasing hormone (Gnrh) 2 in luteinizing hormone (lhb)-expressing cells from green fluorescent protein (Gfp)-transgenic medaka (Oryzias latipes), with regard to changes in the cytosolic Ca2+ concentration. In the current study we present the corresponding responses to Gnrh1 and Gnrh3. Ca2+ imaging revealed three response patterns to Gnrh1 and Gnrh3, one monophasic and two types of biphasic patterns. There were few significant differences in the shape of the response patterns between the three Gnrh forms, although the amplitude of the Ca2+ signal was considerably lower for Gnrh1 and Gnrh3 than for Gnrh2, and the distribution between the two different biphasic patterns differed. The different putative Ca2+ sources were examined by depleting intracellular Ca2+ stores with thapsigargin, or preventing influx of extracellular Ca2+ by either extracellular Ca2+ depletion or the L-type Ca2+-channel blocker verapamil. Both Gnrh1 and 3 relied on Ca2+ from both intracellular and extracellular sources, with some unexpected differences in the relative contribution. Furthermore, gene expression of Gnrh-receptors (gnrhr) in whole pituitaries was studied during development from juvenile to adult. Only two of the four identified medaka receptors were expressed in the pituitary, gnrhr1b and gnrhr2a, with the newly discovered gnrhr2a showing the highest expression level at all stages as analyzed by quantitative PCR. While both receptors differed in expression level according to developmental stage, only the expression of gnrhr2a showed a clear-cut increase with gonadal maturation. RNA sequencing analysis of FACS-sorted Gfp-positive lhb-cells revealed that both gnrhr1b and gnrhr2a were expressed in lhb-expressing cells, and confirmed the higher expression of gnrhr2a compared to gnrhr1b. These results show that although lhb-expressing gonadotropes in medaka show similar Ca2+ response patterns to all three endogenous Gnrh forms through the activation of two different receptors, gnrhr1b and gnrhr2a, the differences observed between the Gnrh forms indicate activation of different Ca2+ signaling pathways.