施氏鲟脑的性别分化及其调控机制初探

Gonadal sex differentiation is the most important event of fish, which is regulated by the brain via the HPG axis. As a crucial organ of sexual differentiation and reproductive regulation, brain is essential for normal gonadal development in structural differentiation and physiological functions. A number of neuropeptides and neurotransmitters brain synthesizes involves in the regulation of Gonadotropin-releasing hormone (GnRH) pulse release to regulate gonadal development and maturation via the HPG axis. GnRH are the key signal molecules of HPG axis , in which the communication between the brain and the pituitary is carried out through the pituitary portal system in GnRH neurons which innervates the anterior pituitary and forms a physical and functional connection between the brain and the pituitary. Amur sturgeon is an important freshwater aquaculture species in China. Our recent study found that some individuals are abnormal in gonadal differentiation and with a higher proportion, especially for female fish, which has a significant impact on the Amur sturgeon caviar industry. This phenomenon indicates that the HPG axis during gonadal sex differentiation is affected by endocrine or environmental factor.Now, it is confirmed that Kiss-1/GPR54 signaling is central to the regulation of GnRH, which leads to the secretion of LH and FSH in fish. Meanwhile, it's also thought that Kiss is the link mediating the response between environmental cues and metabolic signals to the reproductive axis. Therefore, we supposed that Kiss system involves in brain sex differentiation of Amur sturgeon, and relates to abnormal gonad sex differentiation. In this project, we use the methods of histology, endocrinology and molecular biology to investigate the characteristics of brain sex differentiation, the expression of kiss, GPR54, GnRH and GtH genes during brain sex differentiation. We also detect the expression and regulation of kisspeptin on brain GnRH via intraventricular injection, and the main differential protein for Kisspeptin metabolism. This study aims at exploring the relationships between gene Kiss and brain sex differentiation, and attempts to reveal the molecular mechanism of brain sex differentiation, and to provide a theoretical basis for reproductive regulation of fish in intensive culture system.

性别分化是鱼类生活史最重要的事件，脑是发生性别分化和调控性腺发育的重要器官之一。鱼类性别分化和性腺发育多受下丘脑-垂体-性腺轴(HPG)的调控，脑通过神经内分泌因子将外部环境信号或内分泌信号转换为GnRH的脉冲释放，参与性腺轴的生殖调控。施氏鲟是我国北方重要淡水养殖种类，新近研究发现，同龄施氏鲟中存在性腺分化异常个体，且比例较高，严重影响了作为鲟鱼子酱生产的施氏鲟种业发展。据此，我们提出了本项申请，以来自同一亲本的施氏鲟为研究对象，以脑这一生殖调控中枢为切入点，采用组织学、内分泌学及分子生物学方法，阐明施氏鲟脑的性别分化特性、Kiss等基因在脑性别分化过程中的表达、Kisspeptin对GnRH的调控作用及参与其代谢的主要差异蛋白质，从表型、内分泌代谢和基因表达三个层面探讨Kiss基因与脑性别分化的关系，认识施氏鲟脑性别分化的分子调控机制，为实现这一名贵养殖鱼类的生殖调控提供理论依据。

施氏鲟是我国北方重要淡水养殖种类，同龄施氏鲟中存在性腺分化异常个体，且比例较高，严重影响了作为鲟鱼子酱生产的施氏鲟种业发展。本项目以来自同一亲本的施氏鲟为研究对象，以脑这一生殖调控中枢为切入点，采用组织学、内分泌学及分子生物学方法，研究施氏鲟脑的性别分化特性、Kiss 等基因在脑性别分化过程中的表达、Kisspeptin 对GnRH 的调控作用及参与其代谢的主要差异蛋白质，拟揭示Kiss 基因与其脑性别分化的关系。 结果如下：（1）对整个性别分化期间施氏鲟的脑组织结构研究表明（0-199pdf），性腺早期发育过程中，雌雄鱼脑组织结构无明显差异，且幼鲟体内的17ß-E2和T含量与其发育时期显著相关；（2）GnRH、kiss、GPR54、ER2、AR、cyp19a1b等基因参与了施氏鲟早期的性腺发育及分化（0-199pdf），且随着施氏鲟性腺发育时期的不同，其相关基因的表达规律不同；ER2和kiss2等基因不参与施氏鲟早期的性腺发育及性别分化（0-49 pdf）；（3）Kisspeptin 脑室注射后，施氏鲟脑组织和性腺组织内的GnRH、kiss、GPR54、cyp19a1b、ERα、ERβ与AR mRNA的表达量均发生改变，此外脑和性腺内的17β-E2含量和T含量也随着注射次数的增加而变化；（4）Kisspeptin 脑室注射后，共鉴定4 720种脑组织蛋白，其中3 720种蛋白质有定量信息，发现354种差异蛋白质，其中177种上调，177种下调。GO分析显著富集到生物学过程中的细胞代谢过程、细胞组成成分的细胞部分和分子功能的黏附部分。经Pathway 富集分析，注射组与未注射组之间的差异蛋白显著富集到繁殖功能的GnRH信号通路、卵母细胞减数分裂通路 和P53信号通路上，其中MAPK信号通路、钙离子信号存在于以上3个pathway 中。发现了CALU、CaMK 、Jnk 、53BP1、SSR3为候选蛋白，与kisspeptin调控性腺早期发育与分化相关的候选蛋白。综上，施氏鲟早期性腺发育过程中，kiss1基因激活GnRH信号通路，抑制CaMK、ERK等蛋白的合成和JNK的磷酸化水平，从而参与施氏鲟早期性别分化期间的性轴（HPG轴）的生殖调控，kiss1基因在施氏鲟的早期性别分化过程中发挥着重要作用。