【作者】 徐永江；

【导师】 王清印；

【作者基本信息】 中国海洋大学 ， 海洋生物学， 2011， 博士

【摘要】 本文以我国重要海水养殖经济鱼种—星鲽为研究对象,以建立稳定的星鲽人工繁育技术体系为研究目的,以脑垂体、性腺发育规律、性类固醇激素表达、配子发育成熟、GnRH克隆表达为研究核心和主线,综合运用组织学、电镜技术、组织化学、放射免疫技术、分子克隆技术、基因定量表达和激素诱导技术等方法,全面认识了星鲽生殖生理和内分泌特征。以此为参考,在生产实践中成功诱导星鲽亲鱼性腺成熟产卵,为建立稳定的星鲽人工繁育技术提供了参考依据和技术支持。本文各部分研究结果简述如下:首次系统研究和认识了星鲽亲鱼卵巢发育规律、性类固醇激素表达与温光调控的关系。星鲽卵巢发育属非同步分批发育模式,一年一次成熟分批产卵类型。卵巢年周期发育过程中可见5个发育时相的卵母细胞,卵巢发育相应划分为5个发育时期。性腺指数(GSI)、肝脏指数(HSI)和肥满度(CF)在亲鱼的年周期发育中呈现规律性的变化。血浆性类固醇激素水平呈现周期性变化,并与性腺成熟周期具有密切的藕联关系。水温和光周期对卵巢发育具有明显的影响并显著影响性类固醇激素的表达。圆斑星鲽和条斑星鲽卵巢发育的有效积温分别为1050日·度和1180日·度,研究结果可为亲鱼促熟和激素催产提供技术依据。首次研究了外源激素sGnRH、LHRHa对2龄雌性圆斑星鲽后备亲鱼性腺发育的诱导效果:2龄亲鱼对外源激素的诱导应答效应较性成熟亲鱼弱,卵母细胞发育可达到5时相早中期但未能达到水合成熟,性类固醇激素表达水平显著升高,但未能引发排卵。探明了星鲽精巢年周期发育的生理和内分泌特征:精巢年周期发育过程中可见5个时相的生殖细胞,精巢发育相应分为5期。研究中首次发现圆斑星鲽雄鱼存在雌雄同体现象,但机制不明。认识了星鲽精巢年周期发育过程的性类固醇激素表达变化规律。圆斑星鲽和条斑星鲽精子具有相似的共同结构,都具有头部、中段和鞭毛3部分,其结构与其它高等鱼类精子结构相似。精子不具顶体,但具有开口于核前端稍偏处呈不规则状的核前部凹陷及核内核泡。圆斑星鲽精子活力最适宜温度8 - 12℃,最适宜盐度25 - 35,最适宜pH 7 - 8。超低温冷冻保存了圆斑星鲽精子,以12%GLY+10%YK及12%MeOH+10%YK保存精子的效果较好,解冻后受精率和孵化率较高,与新鲜精液无显著差异。外源激素GnRH和HCG处理都可以显著提高条斑星鲽精液质量,精液流动性和液化能力增强、精子活动率和快速活动率显著提高、快速活动时间和精子寿命延长,性类固醇激素表达水平明显升高,表明缓释激素诱导可有效提升条斑星鲽雄鱼的精液质量。首次观察了条斑星鲽脑垂体组织和超微结构:脑垂体呈背腹型,包括神经垂体和腺垂体两部分。神经垂体为一束神经纤维,中间夹杂两种垂体细胞和有两种不同的胶质。腺垂体分为前腺垂体(RPD)、中腺垂体(PPD)和后腺垂体(PI)。腺垂体内鉴别出6种内分泌细胞。前腺垂体表达3种,中腺垂体表达3种,后腺垂体表达1种。采用分子克隆技术,首次系统研究了圆斑星鲽GnRH信号系统及其可能的生理功能。在圆斑星鲽脑中克隆到3种GnRH基因,分别是:cGnRH-Ⅱ、sGnRH和sbGnRH。每一种GnRH都包括一个信号肽、一个Gly-Lys-Arg连接序列和一个GnRH相关肽。分析了3种GnRH基因的氨基酸序列与其它脊椎动物的同源性。系统进化分析表明圆斑星鲽3种不同分子形式的GnRH分别归属于3个不同分支:cGnRH-Ⅱ分支、sGnRH分支和鱼类特异性GnRH分支(如sbGnRH、cfGnRH等)。GnRH基因都在脑中表现出最高表达水平且具有性别特异性表达模式:雌性中不同组织的GnRH mRNA表达水平都相应高于雄性。cGnRH-Ⅱ的mRNA仅在脑中表达,而sbGnRH在各个组织都有表达,sGnRH仅在脑、垂体和性腺中表达。sbGnRH mRNA的表达水平变化与卵巢年周期发育过程具有最密切的关系,表明sbGnRH可能是性腺成熟调控的关键GnRH基因。综合采用温度、光照、激素诱导等技术,成功诱导条斑星鲽亲鱼性腺发育成熟和排卵,获得了良好的产卵效果,筛选了一种有效地性腺促熟剂,利用该技术可在繁殖季节稳定获取受精卵,为星鲽养殖业发展提供技术支持。更多还原

【Abstract】 The physiological and endocrine characteristics of reproduction of farmed Spotted halibut and Barfin flounder were investigated and elucidated in this thesis. This thesis aimed at construction of stable technology for artificial breeding of farmed Spotted halibut and Barfin flounder. The contents mainly focused on pituitary structure, gonadal maturation cycle, sex steroids expression pattern, gametes characteristics, structure and function of GnRH system. Multiple and combined methods including histological observation, electron microscopy technique, immunohistochemistry, radioimmunoassay (RIA), molecular cloning, quantitative real time PCR and exogenous hormone induction etc. were employed. Based on results from this study, successful control of gonadal maturation, ovulation and batches of embryos with high quality from barfin flounder were obtained. Results from this study would be helpful to better understand the physiological and endocrine mechanisms underlying the reproductive cycle of spotted halibut and barfin flounder, also would facilitate the artificial control of gonadal maturation and obtain spawnings from broodstocks under farming conditions.The annual ovarian maturation cycle and related serum sex steroids levels variation, the relationships between gonadosomatic index (GSI), condition factor (CF), hepatosomatic index (HSI), sex steroids expression levels and photothermal regulation were investigated and elucidated for farmed Spotted halibut and Barfin flounder. These two species feature asynchronous oocyte development and batch spawnings. In sexually matured females, the oocyte growth could be divided into five phases and ovary maturation cycle was divided into five stages accordingly. GSI, HSI and CF values were all found to increase prior to, or during the peak phase of vitellogenic growth corresponding to the gonad development. Sex steroids levels fluctuated with the gonadal maturation cycle and closely related with photothermal regimes based on expression results and statistical analysis. The effective accumulated temperatures for Spotted halibut and Barfin flounder broodstocks were 1050 and 1180 d·℃respectively. The effects of exogenous hormone (sGnRH and LHRHa) on gonadal development of immatured females (2 year old) were studied. The results showed that the immatured females exhibited weak endocrine response compared with sexually matured broodstocks, which was manifested by the fact that the oocyte could attain the late vitellogenic stage but could not experience hydration and final maturation. Although the serum sex steroids levels increased significantly, the ovulation failed. These results would highlight the physiological and endocrine mechanism of annual reproductive cycle of captive female broodstocks of spotted halibut and barfin flounder, also could serve as useful tools for broodstocks breeding and hormonal induction of spawning of broodstocks.The physiological and endocrine characteristics during annual maturation cycle of male spotted halibut and barfin flounder were elucidated. The spermatogenesis could be divided into five phases and testis maturation cycle was divided into five stages. The hermaphroditism phenomenon was found in one male spotted halibut, but the mechanism remained unclear. The serum steroids expression patterns during testis maturation cycle were also determined. Spotted halibut sperm shared similar structure with that of barfin flounder, and exhibited similar general structure including spherical head, the reduced mid piece with numerous round unmodified small mitochondria, and the typical‘9+2’pattern of the flagellum with quite simple structure. These common structure features meant that the spermatozoon of spotted halibut and barfin flounder can be considered to be of primitive type, which is found in most of the advanced fish taxa. There is no acrosome in the sperm. Anterolateral to the nucleus, there is a chromatin-free, electron-lucent area, that we named the anterior pit of the nucleus. Within and in front of the pit, there is a triangular shaped structure and some vesicles of different size that are encapsulated in a layer of membrane were found. With regarding to acrosome and acrosome vestige present in some other euteleosts, we presume the vesicles may represent acrosome vestiges though they may also function to release nuclear material, whilst the riangular shaped structure in front the nucleus may be caused by the improper manipulation. Study on relationship between sperm and environmental factors indicated that when salinity at 25-35, temperature at 8-12 and pH at 7-8, the spotted halibut spermatozoa showed best motility and longest life span. The sperm of spotted halibut was cryopreserved by a simple and convenient protocol. The sperm was cryopreserved in the different extenders. The fertilization rates of this sperm ranged from 59±2.4% to 70±3.0% with the sperm cryopreserved in the extender containing Gly + egg yolk giving the highest fertility, while the hatching rates of eggs fertilized with this sperm were from 11±3.0% to 18±2.6% with the eggs fertilized by the sperm cryopreserved in Gly + egg yolk producing the highest hatching rate. The effects of exogenous hormones including human chorionic gonadotropin (HCG) and salmon gonadotropin releasing hormone analogue (sGnRHa) on milt quality in male barfin flounder were investigated in the present study. Results showed that exogenous hormone can greatly improve the milt quality of males with milt of high viscosity, including improved milt fluidity, sperm motility and sperm life span. Meanwhile, the sex steroids elevated and remained at a relatively higher level after the exogenous hormone treatment, which was in accordance with the sperm quality elevation. The GnRH induction showed better results although there was no significant difference was observed between GnRH and HCG treatment. This study can serve as useful tool in acquiring milt with high quality and be beneficial to artificial insemination in barfin flounder culture. The histological structure and ultrastructure of barfin flounder pituitary was observed. The pituitary belongs to dorsoventral style, consists of two parts: neurohypophysis and adenohysis. Six types of secretory cells were identified in pituitary: Prolactin hormone (PROL) cell, adrenocorticotopic hormone (ACTH) cell, thyrotropic hormone (TSH) cell, growth hormone (GH) cell, gonadotropin hormone (GtH) cell, melanotropic stimulating hormone (MSH) cell.The present study cloned and sequenced the gonadotropin-releasing hormone (GnRH) genes from spotted halibut by isolating their cDNAs. This species expressed three molecular forms of GnRH in the brain: chicken type GnRH-Ⅱ(cGnRH-Ⅱ), seabream type GnRH (sbGnRH) and salmon type GnRH (sGnRH). Each GnRH cDNA encoded a signal peptide (SP), GnRH and a GnRH-associated peptide (GAP), which was connected to GnRH by a Gly–Lys–Arg sequence. Phylogenetic analysis divided multiple molecular forms of GnRHs into three branches: cGnRH-Ⅱbranch, sGnRH branch and the fish specific GnRH branch. The tissue and sex specific expression of these three genes were determined using real time PCR, they all showed the highest expression levels in brain for both sexes. cGnRH-Ⅱwas exclusively detected in brain while sbGnRH had a global expression pattern in all examined tissues for both sexes. sGnRH was detected in pituitary, gonad besides brain tissue in females but not in gonad of males. All these three GnRH genes exhibited sex-specific expression pattern with females expressed higher mRNA levels than that of males. The seasonal changes of GnRH mRNA expression levels in brains of females during ovary maturation cycle were investigated. During ovary maturation cycle, sbGnRH mRNA level increased significantly (P<0.05) since August (vitellogenic stage), peaked in December (prespawning stage) 2009, and then decreased in April 2010 (postspawning stage). On the contrary, neither sGnRH nor cGnRH-ⅡmRNA levels showed significant changes (P>0.05) correlated with ovary maturation cycle in this study. These results will be helpful to better understand the reproductive endocrine mechanism of spotted halibut.The successful control of gonadal maturation, ovulation and batches of embryos with high quality were obtained in barfin flounder by using photothermal control in combination with hormonal induction during the spawning season in 2008. A stable technique for inducing maturation and ovulation of barfin flounder was constructed and applied to practical production of seedlings, which is beneficial for sustainable and healthy development of culture industry of barfin flounder.更多还原