Strandabø Rønnaug A U, Grønlien Heidi K, Ager-Wick Eirill, Nourizadeh-Lillabadi Rasoul, Hildahl Jon P, Weltzien Finn-Arne, Haug Trude M
University of Oslo, Department of Biosciences, P.O. Box 1066 Blindern, N-0316 Oslo, Norway.
Østfold University College, Faculty of Health and Social Studies, P.O. 700, N-1757 Halden, Norway.
Gen Comp Endocrinol. 2016 Apr 1;229:19-31. doi: 10.1016/j.ygcen.2016.02.019. Epub 2016 Feb 17.
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 Ca(2+) concentration. In the current study we present the corresponding responses to Gnrh1 and Gnrh3. Ca(2+) 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 Ca(2+) signal was considerably lower for Gnrh1 and Gnrh3 than for Gnrh2, and the distribution between the two different biphasic patterns differed. The different putative Ca(2+) sources were examined by depleting intracellular Ca(2+) stores with thapsigargin, or preventing influx of extracellular Ca(2+) by either extracellular Ca(2+) depletion or the L-type Ca(2+)-channel blocker verapamil. Both Gnrh1 and 3 relied on Ca(2+) 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 Ca(2+) 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 Ca(2+) signaling pathways.
我们之前已对绿色荧光蛋白（Gfp）转基因青鳉（Oryzias latipes）中表达促黄体生成素（lhb）的细胞对促性腺激素释放激素（Gnrh）2的反应进行了表征，涉及胞质Ca(2+)浓度的变化。在本研究中，我们展示了对Gnrh1和Gnrh3的相应反应。Ca(2+)成像揭示了对Gnrh1和Gnrh3的三种反应模式，一种单相模式和两种双相模式。三种Gnrh形式之间反应模式的形状几乎没有显著差异，尽管Gnrh1和Gnrh3的Ca(2+)信号幅度明显低于Gnrh2，并且两种不同双相模式之间的分布有所不同。通过用毒胡萝卜素耗尽细胞内Ca(2+)储存，或通过细胞外Ca(2+)耗尽或L型Ca(2+)通道阻滞剂维拉帕米阻止细胞外Ca(2+)内流，研究了不同的假定Ca(2+)来源。Gnrh1和3都依赖于细胞内和细胞外来源的Ca(2+)，在相对贡献方面存在一些意外差异。此外，在从幼年到成年的发育过程中，研究了整个垂体中Gnrh受体（gnrhr）的基因表达。在已鉴定的四种青鳉受体中，只有两种在垂体中表达，即gnrhr1b和gnrhr2a，通过定量PCR分析，新发现的gnrhr2a在所有阶段都显示出最高的表达水平。虽然两种受体的表达水平根据发育阶段而有所不同，但只有gnrhr2a的表达随着性腺成熟而明显增加。对通过荧光激活细胞分选（FACS）分离的Gfp阳性lhb细胞进行RNA测序分析表明，gnrhr1b和gnrhr2a都在表达lhb的细胞中表达，并证实gnrhr2a的表达高于gnrhr1b。这些结果表明，尽管青鳉中表达lhb的促性腺细胞通过激活两种不同的受体gnrhr1b和gnrhr2a，对所有三种内源性Gnrh形式表现出相似的Ca(2+)反应模式，但Gnrh形式之间观察到的差异表明不同的Ca(2+)信号通路被激活。
