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大菱鲆性腺分化以及Mtnrs,kiss/kissr和GnRH在性腺成熟过程中的表达特征
赵春彦1,2
学位类型博士
导师李军
2017-05-25
学位授予单位中国科学院大学
学位授予地点北京
学位专业水产养殖
关键词大菱鲆 性腺分化 褪黑激素 促性腺激素释放激素 Kisspeptin
摘要大菱鲆(Scophthalmus maximus)属于鲆科(Bothidae),菱鲆属( Scophthalmus),俗称“多宝鱼”,是原产于欧洲沿海的、具有较高经济价值的重要海水养殖良种之一。因肉质鲜美,生长速度快,适应低水温生活和养殖效益高等优点,大菱鲆于1992年首次引入我国。然而,在世代养殖中,大菱鲆出现了种质资源退化现象,养殖户养殖效益逐年下降,因此本文从性腺发育的角度,研究了大菱鲆早期性腺分化及生殖周期性腺发育过程中Mtnrskiss/kissrGnRH的表达特征,为促进大菱鲆性腺成熟及排卵,提高大菱鲆精卵质量提供科学依据。
(1)大菱鲆鱼苗在18-19℃水温条件下养成,通过0-90dph组织学观察发现,大菱鲆原始生殖细胞经历胚后迁移,于15dph迁移至生殖嵴,此后体细胞也开始聚集;孵化后20天原始性腺形成,由迁移至生殖嵴的原始生殖细胞和体细胞共同组成;至孵化后36天原始性腺出现形态学及细胞学的分化,一类性腺表现为含有生殖细胞数量较少的细长的柳叶型性腺,另一类性腺为含有生殖细胞数量较多的短粗的球棒状性腺,依据性腺形态学及生殖细胞的增殖规律,我们推测柳叶型性腺将来发育成精巢,而球棒状性腺将来发育成卵巢;孵化后60天左右,卵巢腔出现,雌性性腺分化完成,孵化后90天左右,精小叶出现,雄性性腺分化完成。
(2)克隆获得大菱鲆lymphocyte antigen 75smly75)基因全长,通过原位杂交的方法对smly75在大菱鲆精巢的定位分析发现,smly75在A型精原细胞检测到表达,在B型精原细胞、精母细胞及精子细胞均未检测到表达,结果表明smly75可以作为大菱鲆A型精原细胞的标记基因;同时,通过组织学观察发现,8-14月龄大龄鲆精巢含有比例较高的A型精原细胞,此时期smly75的表达量相对较高,因此,8-14月龄大龄鲆精巢可以用于后续的A型精原细胞的分离和富集实验。
(3)克隆获得大菱鲆三种褪黑激素受体分子,分别为Mtnr1Mtnr2Mtnr1c,通过荧光定量发现,Mtnrs mRNA均在脑中有高表达,且Mtnr1Mtnr2的表达量要显著性高于Mtnr1c,同时下丘脑中Mtnr1的表达显著性高于Mtnr2,并且随着性腺的发育Mtnr1的表达呈现规律性波动,即性腺发育早期表达量较高(II-IV),而在性腺成熟季节达到最低(V),推测Mtnr1在生殖调控轴上发挥重要作用。
(4)克隆获得大菱鲆kiss1kiss2kiss receptorkissr)cDNA序列,多重序列比对发现kissr与kissr4同源,属于kiss2受体家族,荧光定量分析发现,kiss/kissr均高表达于脑,并且kiss2仅表达于下丘脑,同时下丘脑中kiss2kissr的表达量要显著性高于kiss1。生殖周期性腺发育过程中,kiss2kissr呈现先降低后升高的表达趋势,即性腺成熟期(V)的表达量最低,而性腺发育期(II-IV)与退化期(VI)的表达量升高,我们推测kiss2kissr参与到了大菱鲆生殖活动。
(5)促性腺激素释放激素(Gonadotrophin-releasing hormone, GnRH)在脊椎动物性腺发育的调节和控制性成熟中起着至关重要的作用,本研究克隆获得三种GnRH cDNA序列:sbGnRHcGnRH-IIsGnRH,荧光定量分析显示:三种GnRH mRNAs在脑中均有高表达,在一个生殖周期内,sbGnRH和sGnRH都呈现出成熟期(V)表达量最高而退化期(VI)表达量下降的趋势,并且sbGnRH的表达量远远高于sGnRH,表明参与生殖内分泌的主要GnRH分子类型为sbGnRH,sGnRH辅助sbGnRH发挥作用。通过生殖周期中Mtnr1kiss2/kissrsbGnRH的表达变化趋势分析显示,Mtnr1kiss2/kissr均在繁殖期(V)的表达量最低,并与sbGnRH的表达趋势相反;同时随着性腺的发育血清中褪黑激素的含量则呈现先升高后降低的趋势,并结合GnRH与FSH、LH的血清含量比较来看,褪黑激素具有促进大菱鲆性腺成熟的作用,且通过下调Mtnr1kiss2/kissr的表达来调控sbGnRH表达进而参与大菱鲆的生殖活动。
其他摘要As a flatfish, the turbot (Scophthalmus maximus) is one of the most important farmed fish species with great commercial value. The turbot was firstly introduced to China at the year of 1992. However, in recent years, the efficiency of aquaculture has been brought down for the reccession of genetic resource for the rapid development of the industry. In this study, we investigated the formation and differentiation of turbot gonad and evaluated the localizations of expressions and the temporal transcription patterns of melatonin receptors, kiss/kissr and GnRH throughout their reproductive cycle. This study will provide basic knowledge in the regulation of reproductive endocrine and artificial reproduction.
(1) Turbot larvae were reared at the temputer of 18-19oC. The Histology showed that the primordial germ cells (PGCs) underwent a migration after embryo development, and PGCs arrived at genital ridge at 15dph. The primary gonad of turbot formed by the PGCs and the aggregation of somatic cells at 20 dph. The cytological and morphological gonadal differentiation was observed at 36dph. One type of gonad showed lance-shape with fewer germ cells, while the other type showed club-like shape with larger amount of germ cells. According to the morphology of gonad and the proliferation of germ cells, we speculated the gonad with lance-shape could develop into testes, and the gonad with club-like shape could develop into ovary. At about 60 dph, with the presentation of ovarian cavity, the differentiation of ovary finished. About 90 dph, spermatogonial cysts appeared and the differentiation of testes finished.
 (2) The full-length cDNA of turbot lymphocyte antigen 75 (smly75) homologue were cloned. In situ hybridization, smly75 mRNA were exclusively detected in A-SG. Thus, the smly75 could be a new and convincing molecular marker on identification of turbot A-SG. Through histology, we found that testes from 8 to 14 month old males contained high rate of A-SG. And the smly75 expressed highly at these gonadal development stages. Thus, testes of 8 to 14 month old males could be used for further isolation and purification of type A-SG.
(3) Three forms turbot Melatonin receptor (Mtnr) genes: Mtnr1, Mtnr2 and Mtnr1c were cloned and sequenced by isolating the cDNAs. The expression patterns by qPCR indicated Mtnr mRNAs were all highly expressed in brain and the expression of Mtnr1 and Mtnr2 were relatively higher than Mtnr1c. Meanwhile, in hypothalamus, Mtnr1 mRNA concentrations were significantly higher than Mtnr2. With the gonadal development throughout the reproductive cycle (II-VI), the expression of Mtnr1 gradually dreased and reached the lowest level at mature stage (V). Therefore, Mtnr1 could be play an importment role in the regulation of reproduction.
(4) Turbot kiss1, kiss2 and kiss receptor (kissr) cDNA were obtained. Multiple alignments of deduced amino acid indicated that turbot kissr and Japanese flounder (Paralichthys olivaceus) kissr4 are homologous. The results of qPCR showed that kiss1, kiss2 and kissr were all strongly expressed in brain, and kiss2 mRNA was restricted to hypothalamus. Meanwhile, kiss2 and kissr mRNA were significantly higher than kiss1 in hypothalamus. Through the reproductive cycle, the expression of kiss2 and kissr in brain showed a variation of decreasing firstly and then increasing. Therefore, turbot kiss2 and kissr could be involved in the reproductive activity and gonadal development.  
 (5) Three forms turbot GnRH genes: sbGnRH, cGnRH-II, and sGnRH were cloned and sequenced by isolating the cDNAs. The expression of three mRNAs by qPCR showed that all the three GnRH mRNAs were highly expressed in brain, and sbGnRH and sGnRH mRNAs increased during the immature stage and reached the highest level at breading stage (V), then decreased at the degradation stage. Moreover, the expression of sbGnRH was significant higher than sGnRH. All the results indicated the critical role of sbGnRH on the regulation of GtH and sGnRH functionally compensates for sbGnRH. In addition, the evaluation of Mtnr1, kiss2/kissr and sbGnRH throughout the reproductive cycle showed that, at breading stage (V), the expression of Mtnr1 and kiss2/kissr reached to the lowest level, which was opposite with the expression of sbGnRH. Meanwhile, in consideration of the variation of serum hormones concentrations of melatonin, GnRH, FSH and LH in male and female turbot, it is suggested that melatonin could induce the mature of gonad and regulate the reproduction by driving the expression of Mtnr1, kiss2/kissr down to elevate the sbGnRH mRNA.
学科领域水产养殖学
语种中文
文献类型学位论文
条目标识符http://ir.qdio.ac.cn/handle/337002/136631
专题实验海洋生物学重点实验室
作者单位1.中国科学院海洋研究所
2.中国科学院大学
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赵春彦. 大菱鲆性腺分化以及Mtnrs,kiss/kissr和GnRH在性腺成熟过程中的表达特征[D]. 北京. 中国科学院大学,2017.
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