新型促黄体素受体在斜带石斑鱼性反转中的功能研究

Sex reversal is a common phenomenon. Its regulatory mechanisms are an interesting topic in the area of fish reproduction. Evidence indicated that luteinizing hormone (LH) could effectively induce the sex reversal in hermaphrodite fish species, but how the LH delivers its signals to gonad and activate the process of sex changes remains unknown. Orange-spotted grouper (Epinephelus coioides) is protogynous hermaphrodite species undergoing sex reversal from female to male during their life history. Therefore, orange-spotted grouper becomes a good model for studying the regulatory mechanisms of sex reversal. Using orange-spotted grouperas model, we have sequenced its genome, and found a novel luteinizing hormone receptor gene (LHR), namely LHR2. During the process of sex reversal in orange-spotted grouper, LHR2 mRNA expression could not be detected in the ovary before hormone induction, but was significantly increased in the intersexual gonad, and then was reduced in the testis. This initial data suggest that LHR2 may be the main receptor mediating the LH signal in inducing the sex reversal in hermaphrodite fish species. In order to reveal the role of LHR2 in sex reversal, therefore, in this project, we will investigate its expression profiles in the process of natural and artificial sex reversal, and its interaction with the enzymes associated with steroid hormone synthesis and the genes related to sex control. Finally, we will examine whether the sex reversal was interrupted and study the change of transcriptome and proteome of gonad after the LHR2 gene knockdown and knockout. Our study will provide valuable evidence for revealing the molecular regulatory mechanisms of sex reversal in groupers.

在鱼类中，性反转是一个普遍现象，其调控机制一直是鱼类生殖生理学的研究热点。众多实验表明促性腺激素在雌雄同体鱼类性反转过程中发挥重要作用，但其信号如何传递至今仍未清楚。斜带石斑鱼为典型的雌雄同体雌性先成熟鱼类，是研究鱼类性反转调控机理的理想模型。在分析斜带石斑鱼全基因数据的过程中，我们发现了一种新型的促黄体素受体（LHR2）基因，这是首次在脊椎动物中证实了两个LHR基因的存在。初步研究发现，在性反转过程中，LHR2在卵巢中没有表达，在间性性腺中表达最高，在精巢中表达下降，提示LHR2可能是介导LH信号诱导雌雄同体鱼类性反转的主要受体。本研究将通过研究LHR2的表达调控、与性类固醇激素合成酶和性别调控因子的关系、LHR2基因受到干扰后斜带石斑鱼性反转的变化及转录组和蛋白组学分析，来深入探讨LHR2在斜带石斑鱼性反转过程中的功能。我们的研究成果将有助于更深入理解石斑鱼类性反转的调控机制。

鱼类性反转是一个普遍现象，其调控机制一直是鱼类生殖生理学的研究热点。在分析斜带石斑鱼全基因数据的过程中，我们发现了一种新型的促黄体素受体（LHR2）基因，这是首次在脊椎动物中证实了两个LHR基因的存在。通过配体受体相互作用分析，发现LH和hCG能分别与LHR1和LHR2结合，并激活cAMP/PKA信号通路，而且LHR2能引起SRF表达的上升，但LHR1不能，说明二者在信号通路的转导上有相同点但又存在差异；表明二者都具有生物学活性，但可能在功能上有所区别。在此基础上，研究GtH/GtHR信号系统在斜带石斑鱼性逆转过程中的表达模式，发现FSH和FSHR基因 mRNA水平显著提高，而LH和LHR基因表达则受到抑制，表明两种GtH在斜带石斑鱼性逆转过程中扮演不同的角色。进一步的研究发现FSH连续注射能显著促进雌性斜带石斑鱼血清11-KT的含量，并成功诱导雌性转变为雄性。不同浓度的外源FSH都能在一定程度上抑制cyp19a1a的表达，同时促进cyp11b和hsd11b2的表达。表明FSH可能通过调控性类固醇激素的合成，决定卵巢的走向，诱导性逆转的发生。同时发现 FSH在斜带石斑鱼的性别分化中有重要作用，其在天然的卵巢分化过程中呈现显著上升的表达模式。低剂量的FSH能够上调cyp19a1a的表达，从而使雌激素合成上升，雄激素合成下降，进而促进石斑鱼的卵巢分化；而过高剂量的FSH持续激活性腺中性类固醇激素合成，抑制cyp19a1a的表达，导致石斑鱼分化为雄鱼。以上的研究成果丰富了我们对鱼类性逆转和性别分化调控机制的认识，同时也为雌雄同体鱼类性别控制提供了新思路和新途径。