Data_Sheet_8_Characterization of Gonadotropin-Releasing Hormone (GnRH) Genes From Cartilaginous Fish: Evolutionary Perspectives.DOCX

The neuropeptide gonadotropin-releasing hormone (GnRH) plays an important role in the control of reproductive functions. Vertebrates possess multiple GnRH forms that are classified into three main groups, namely GnRH1, GnRH2, and GnRH3. In order to gain more insights into the GnRH gene family in vertebrates, we sought to identify which paralogs of this family are present in cartilaginous fish. For this purpose, we searched the genomes and/or transcriptomes of three representative species of this group, the small-spotted catshark, Scyliorhinus canicula, the whale shark, Rhincodon typus and the elephant shark Callorhinchus milii. In each species, we report the identification of three GnRH genes. In catshark and whale shark, phylogenetic and synteny analysis showed that these three genes correspond to GnRH1, GnRH2, and GnRH3. In both species, GnRH1 was found to encode a novel form of GnRH whose primary structure was determined as follows: QHWSFDLRPG. In elephant shark, the three genes correspond to GnRH1a and GnRH1b, two copies of the GnRH1 gene, plus GnRH2. 3D structure prediction of the chondrichthyan GnRH-associated peptides (GAPs) revealed that catshark GAP1, GAP2, and elephant shark GAP2 peptides exhibit a helix-loop-helix (HLH) structure. This structure observed for many osteichthyan GAP1 and GAP2, may convey GAP biological activity. This HLH structure could not be observed for elephant shark GAP1a and GAP1b. As for all other GAP3 described so far, no typical 3D HLH structure was observed for catshark nor whale shark GAP3. RT-PCR analysis revealed that GnRH1, GnRH2, and GnRH3 genes are differentially expressed in the catshark brain. GnRH1 mRNA appeared predominant in the diencephalon while GnRH2 and GnRH3 mRNAs seemed to be most abundant in the mesencephalon and telencephalon, respectively. Taken together, our results show that the GnRH gene repertoire of the vertebrate ancestor was entirely conserved in the chondrichthyan lineage but that the GnRH3 gene was probably lost in holocephali. They also suggest that the three GnRH neuronal systems previously described in the brain of bony vertebrates are also present in cartilaginous fish.

神经肽促性腺激素释放激素（gonadotropin-releasing hormone, GnRH）在生殖功能的调控中发挥着重要作用。脊椎动物拥有多种GnRH亚型，可划分为三大主要类群，即GnRH1、GnRH2与GnRH3。为深入解析脊椎动物的GnRH基因家族，我们旨在明确软骨鱼中存在该家族的哪些旁系同源基因。为此，我们对该类群的三个代表性物种的基因组和/或转录组进行了检索：小斑猫鲨（Scyliorhinus canicula）、鲸鲨（Rhincodon typus）以及象鲨（Callorhinchus milii）。
本研究在每个物种中均鉴定出3个GnRH基因。对小斑猫鲨和鲸鲨的系统发育与共线性分析显示，这三个基因分别对应GnRH1、GnRH2和GnRH3。在两个物种中，GnRH1均编码一种新型GnRH，其一级结构经测定为：QHWSFDLRPG。
在象鲨中，这三个基因分别为GnRH1a、GnRH1b（两份GnRH1基因拷贝）以及GnRH2。对软骨鱼GnRH相关肽（GnRH-associated peptides, GAPs）的三维结构预测结果显示，小斑猫鲨的GAP1、GAP2以及象鲨的GAP2肽段均呈现螺旋-环-螺旋（helix-loop-helix, HLH）结构。该结构在众多硬骨鱼的GAP1与GAP2中均有发现，可能与GAP的生物学活性相关。但象鲨的GAP1a与GAP1b未观察到此类HLH结构。与迄今已报道的所有GAP3一致，小斑猫鲨和鲸鲨的GAP3均未呈现典型的三维HLH结构。
逆转录聚合酶链式反应（Reverse Transcription-Polymerase Chain Reaction, RT-PCR）分析显示，小斑猫鲨脑中的GnRH1、GnRH2和GnRH3基因呈现差异化表达：GnRH1 mRNA在间脑中表达量最高，而GnRH2与GnRH3 mRNA分别在中脑和端脑中丰度最高。
综上，本研究结果表明，脊椎动物祖先的GnRH基因库在软骨鱼谱系中得到了完整保留，但全头亚纲类群可能丢失了GnRH3基因。研究结果还提示，此前在硬骨脊椎动物脑中发现的三类GnRH神经元系统，在软骨鱼中同样存在。