|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Mtnrs，kiss/kissr和GnRH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。|
（2）克隆获得大菱鲆lymphocyte antigen 75 （smly75）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。
（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：sbGnRH，cGnRH-II和sGnRH，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Mtnr1与kiss2/kissr及sbGnRH的表达变化趋势分析显示，Mtnr1与kiss2/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Mtnr1与kiss2/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.
|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.|