Table_4_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关键作用。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（cartilaginous fish）中存在该家族的哪些旁系同源基因（paralogs）。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（transcriptomes）进行了检索：小斑猫鲨（Scyliorhinus canicula）、鲸鲨（Rhincodon typus）以及银鲛（Callorhinchus milii）。本研究在每个物种中均成功鉴定出3个GnRH基因。在小斑猫鲨与鲸鲨中，系统发育与共线性分析（phylogenetic and synteny analysis）显示，这三个基因分别对应GnRH1、GnRH2与GnRH3。在两个物种中，GnRH1均编码一种新型GnRH亚型，其一级结构被确定为：QHWSFDLRPG。在银鲛中，三个基因分别对应GnRH1a、GnRH1b这两份GnRH1基因拷贝，以及GnRH2。软骨鱼GnRH相关肽（GnRH-associated peptides，GAPs）的三维结构预测结果表明，小斑猫鲨的GAP1、GAP2以及银鲛的GAP2肽段呈现螺旋-环-螺旋（helix-loop-helix，HLH）结构。该结构在众多硬骨鱼（osteichthyan）的GAP1与GAP2中均有观测到，可能与GAP的生物学活性密切相关。但银鲛的GAP1a与GAP1b未观测到此类HLH结构。正如目前已报道的所有GAP3一样，小斑猫鲨与鲸鲨的GAP3均未观测到典型的三维HLH结构。逆转录聚合酶链反应（reverse transcription-polymerase chain reaction，RT-PCR）分析显示，小斑猫鲨脑内的GnRH1、GnRH2与GnRH3基因呈现差异化表达模式。GnRH1信使RNA（mRNA）在间脑（diencephalon）中表达量最高，而GnRH2与GnRH3 mRNA分别在中脑（mesencephalon）与端脑（telencephalon）中丰度最高。综上，本研究结果表明，脊椎动物祖先的GnRH基因库在软骨鱼支系中得到了完整保留，但在全头亚类（holocephali）中可能丢失了GnRH3基因。研究结果同时提示，此前在硬骨脊椎动物脑内发现的三种GnRH神经元系统，在软骨鱼中同样存在。