【作者】 倪娜；

【导师】 柳学周； 温海深；

【作者基本信息】 中国海洋大学 ， 水产养殖， 2011， 硕士

【摘要】 本文以人工养殖雌性条斑星鲽为研究对象,运用组织学、免疫组织化学、放射免疫测定技术和透射电镜等技术方法对条斑星鲽卵巢组织学,雌、雄激素受体在卵巢组织的定位,垂体组织学和超微结构观察以及血浆性类固醇激素表达水平等方面进行了初步研究,初步查明了条斑星鲽HPG轴的生殖内分泌机能及其调控机制。研究结果如下:1.条斑星鲽卵巢周年发育组织学研究条斑星鲽属于非同步分批产卵类型。卵母细胞按其组织学特点可分为6个时相,卵巢发育依据其形态特征可划分为相应的6个时期。8月份卵巢开始发育,性腺指数（GSI）开始升高,在1月份达到峰值,此时性腺成熟进入产卵期,在产卵结束后的4月份,GSI迅速下降,并保持较低的水平至下次性腺发育开始前。11月份,肝脏指数（HSI）达到峰值,表明肝脏储能增加迅速,产卵期间保持相对较高水平,在4月份繁殖季节结束后再次下降并保持至下一次繁殖季节。肥满度（CF）在12月份达到最大值, 4月份繁殖季节结束后CF逐渐下降并在6月份达到最低值。这些结果可为亲鱼促熟培育和人工激素催产时机选择提供坚实的技术依据。2.条斑星鲽卵巢雌、雄激素受体免疫组化分析首次在条斑星鲽（Verasper moseri）性腺发育过程中利用雌激素受体（ER）和雄激素受体（AR）的多克隆抗体进行了免疫组织化学定位分析的研究。结果表明:ER和AR在条斑星鲽卵巢发育的各个时期均有表达。卵黄发生早期,ER呈强阳性表达,而在卵黄形成后期和成熟的卵母细胞中表达较弱;AR在不同时相卵母细胞的滤泡膜上均有分布,在早期卵母细胞胞质和成熟期的卵母细胞胞核中一直保持较强表达。由此总结出:性类固醇激素及其受体免疫阳性物定位在生殖细胞的胞质、胞膜和核膜等部位,在性腺不同发育时期表达强度不同,阐明了这两种受体在卵巢发育过程各阶段发挥不同的生理作用。这些结果为性类固醇激素及受体参与调节条斑星鲽卵巢发育成熟机制提供了重要组织学依据。3.条斑星鲽垂体组织学和超微结构观察条斑星鲽脑垂体呈背腹型,由神经垂体和腺垂体两部分组成。神经垂体为一束神经纤维,中间夹杂有两种颗粒垂体细胞和两种胶质。腺垂体结构复杂,分为前腺垂体（RPD）、中腺垂体（PPD）和后腺垂体（PI）三部分。腺垂体内共鉴别出6种内分泌细胞。前腺垂体内有3种:催乳激素（PROL）分泌细胞、促肾上腺皮质激素（ACTH）分泌细胞和促甲状腺激素（TSH）分泌细胞;中腺垂体含有生长激素（GH）分泌细胞、促甲状腺激素（TSH）分泌细胞和促性腺激素（GtH）分泌细胞;后腺垂体内含有一种促黑色素激素（MSH）分泌细胞。性成熟条斑星鲽脑垂体内只发现一种类型的GtH分泌细胞,其内含有大小两种分泌颗粒。4.条斑星鲽血浆性类固醇激素周年变化研究采用125I标记的放射免疫测定方法检测条斑星鲽亲鱼血浆中的雌二醇（E₂）和睾酮（T）的周年表达变化水平。研究得出:条斑星鲽亲鱼血浆中E₂含量在6月份开始升高,9月份达到年周期中的第一个小高峰,2月份E₂表达水平达到全年最大值,4月产卵结束后,E₂表达水平降低并维持较低水平至下一个生殖季节。条斑星鲽亲鱼血浆中T含量在7月份达到第一个峰值,并在1月份达到第二个峰值,于4月份产卵后降至最低水平并维持至下一个生殖季节。统计分析表明,E₂与T存在正相关关系,E₂与水温存在正相关关系,这些结果可为亲鱼促熟培育和选择人工激素催产时机提供坚实的理论依据。更多还原

【Abstract】 In this thesis, the ovary maturation cycle,pituitary structure, sexual steroid hormone and steroid hormone recepters of barfin flounder （Verasper moseri） were studied by histological staining methods, electron microscopy technique, immunohistochemical and radioimmunoassay methods. The main results are as follows:1. Histological study on the development and annual change in the ovary of female barfin flounderThe annual ovarian maturation cycle and correlated plasma sex steroids levels variation were studied using light microscopy and morphometric methods combined radioimmunoassay. In addition, the relationships between GSI, CF, HSI, sex steroids and temperature, photoperiod were investigated using statistical analysis. The results showed that Barfin flounder features asynchronous oocyte development and batch spawnings. The oocyte growth could be divided into six phases and ovary maturation cycle was divided into six stages accordingly. Gonadosomatic index （GSI）, hepatosomatic index （HSI） and condition factor （CF） were all found to increase prior to, or during the peak phase of vitellogenic growth corresponding to the gonad development. These results could highlight the reproductive cycle of captive female barfin flounder.2. Immunohistochemical study on estrogen receptor and androgen receptor in the ovary of female barfin flounderImmunohistochemical localization of estrogen receptor （ER） and androgen receptor （AR） was investigated for the first time in the ovary during development of barfin flounder using polyclonal antibodies. The results showed that both ER and AR presented in the ovary of each development period. ER showed strong immunologically positive expression at early stage of development during vitellogenesis, while expressed weakly in mature oocytes. AR existed in follicular cell during all developmental stages of the ovary, and strongly expressed in early oocytes cytoplasm and mature oocytes nuclei. The sex steroid hormone and their receptors were expressed in cytomembrane, cytoplasm, nuclear membrane or nuclear plasm at different developmental stages with different intensity respectively, which showed their special functional characters. These results provided new morphological proofs that ER and AR participate in the regulation of physiological function in ovary development of barfin flounder.3. Histological and ultrastructure study of the pituitary gland in barfin flounderPituitary gland in sexually matured barfin flounder was of dorsoventral style, which consists of neurohypophysis and adenohysis. There were neurosecretory fibres in neurohypophysis. Two of pituicytes were recognized in the neurohypophysial tissue, except for two of neuroepithelial cells. The adenohysis which was complicated in structure was divided into rostral pars distails （RPD）, proximal pars distails （PPD） and pars intermedia （PI）. Six types of hormone secretory cells were found in adenohypophysis. There were prolactin hormone （PROL） cell, adrenocorticotopic hormone （ACTH） cell and thyrotropic hormone （TSH） cell formed in RPD. The PPD have three types of hormone secretory cells, named as growth hormone （GH） cell, gonadotropin hormone （GtH） cell and thyrotropic （TSH） cell. The PI was conposed of melanotropic stimulating hormone （MSH） cell. One type of gonadotropin hormone （GtH） cell was found in the pituitary of matured female barfin flounder, where the two types of secreted granules existed.4. Study on the changes of plasma sex steroid hormone levels in barfin flounder in the seasonal and reproductive cyclePlasma 17β-Estradiol （E₂） and testosterone （T） in female barfin flounder were detected with radio immunoassay （RIA）. E₂ level began to increase in June （stageⅢ） and reached the first relatively lower peak in September and peaked in February, during spawning. After the spawning, it dropped down to a relative low level and maintained until the next reproductive cycle.T level achieved the first relatively higher peak in July and peaked in January, which was a month earlier than that of E₂. A positive relationship was found between E₂ and T level. Statistical analysis revealed that a positive relationship existed between temperature and E₂.更多还原