Development of techniques for in vitro production of good quality eggs by elucidation of mechanisms of oocyte cytoplasmic maturation in teleost

通过阐明硬骨鱼卵母细胞胞质成熟机制，开发体外生产优质卵子的技术

• 批准号: 18580187
• 负责人: KAGAWA Hirohiko
• 金额: $ 2.49万
• 依托单位: University of Miyazaki
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2006
• 资助国家: 日本
• 起止时间: 2006 至 2007
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-18580187/
• 关键词: eel; oocyte; oocyte maturation; cytoplasmic maturation; aquaporin; in vitro; egg quality; maturation-inducing steroid; 卵細胞質成熟; 吸水; IGF-I; 卵細胞質; 吸水現象; プロトンポンプ

To elucidate mechanisms of oocyte cytoplasmic maturation (such as a hydration, liquid coalescence and clearing of the ooplasm) during oocyte maturation and to develop techniques for in vitro production of good quality eggs, the present study examined the following studies in the Japanese eel spawn buoyant eggs.1. The present study shows the process of oocyte cytoplasmic maturation during de novo oocyte maturation in the Japanese eel. Moreover, we also developed the incubation techniques for mimic the cytoplasmic maturation occurred in vivo. Using the techniques we developed, we found that cytoplasmic maturation was induced by the maturation-inducing steroid that was synthesized in the ovarian follicles by the stimulation of gonadotropin. Cytoplasmic maturation induced by maturation-inducing steroid may be mediated by protein synthesis in the oocyte. IGF-I, but not GAP junction may be involved in the cytoplasmic maturation of eel oocytes.2. The present study also indicates that oocyte h … More ydration is mediated by yolk proteolysis which creates the osmotic driving force and aquaporin possibly facilitates water uptake into oocytes. Moreover, the study indicates that the ovarian follicles in the Japanese eel may play an essential role in oocyte hydration, although the precise role must be identified in the further research.3. The present study also examined the Japanese eel for the presence and cellular distribution of eel AQP-1o in the ovarian follicles during oocyte growth, oocyte maturation and ovulation by the immunocytochemistry. Anti-AQP-1o antibody was raised in a rabbit against a synthetic peptide corresponding to a part of the C-terminal region of eel AQP-1o. Immunoreactivity was first observed in the cytoplasm around the yolk globules which were located in the peripheral region of the oocytes during the primary yolk globule stage; thereafter, the immunoreactivity was observed throughout the cytoplasm of the oocyte as vitellogenesis progressed. The immunoreactivity became localized around the large membrane-limited yolk masses which were formed by the fusion of yolk globules. It also localized below the oocyte plasma membrane in the migratory nucleus stage oocytes, mature oocytes and ovulated eggs. These results are different from those observed in the oocytes of gilthead sea bream and suggest the existence of a distinct and characteristic mechanism of oocyte hydration in the eel.4. We tried to develop the techniques for in vitro production of good quality eggs in the Japanese eel. Although we found the oocyte maturation and ovulation occurred at 12 and 18 hours after incubation with maturation-inducing steroid respectively, we could not obtained fertilized eggs. Further studies are necessary to develop the techniques for obtaining good quality eggs in vitro. Less

为了阐明卵母细胞成熟过程中卵母细胞胞质成熟（如水化、液体聚结和卵质透明化）的机制，并开发体外生产优质卵的技术，本研究对日本鳗鲡产卵浮卵进行了以下研究.本研究揭示了日本鳗鲡卵母细胞从头成熟过程中卵母细胞胞质的成熟过程。此外，我们还发展了模拟细胞质在体内成熟的孵育技术。使用我们开发的技术，我们发现细胞质成熟是由促性腺激素刺激卵泡合成的成熟诱导类固醇诱导的。成熟诱导激素诱导的细胞质成熟可能是通过卵母细胞中的蛋白质合成来介导的。IGF-I可能参与了鳗鱼卵母细胞的胞质成熟，而GAP连接可能不参与.本研究还表明，卵母细胞h ...更多信息 水化是由卵黄蛋白水解介导的，卵黄蛋白水解产生渗透驱动力，水通道蛋白可能促进卵母细胞对水的吸收。此外，本研究还表明，日本鳗鲡卵泡在卵母细胞水化过程中可能发挥着重要作用，但具体作用有待于进一步研究.本研究还通过免疫细胞化学方法检测了鳗鱼AQP-1 o在卵母细胞生长、成熟和排卵过程中卵泡中的存在和细胞分布。抗AQP-1 o抗体在兔中针对对应于鳗鱼AQP-1 o的C-末端区域的一部分的合成肽产生。免疫反应性首先观察到周围的卵黄球，这是位于在初级卵黄球阶段的卵母细胞的外周区域的细胞质中;此后，免疫反应性观察到整个细胞质的卵母细胞的卵黄发生进展。免疫反应性变得局限于由卵黄球融合形成的大的膜限制性卵黄团周围。在移行核期卵母细胞、成熟卵母细胞和排卵卵中，它也定位于卵母细胞质膜下。这些结果与在金头鲷卵母细胞中观察到的结果不同，并且表明鳗鱼卵母细胞水化存在独特的和特征性的机制。本研究旨在探索日本鳗鲡体外生产优质卵子的技术。虽然我们发现卵母细胞分别在促成熟激素孵育后12和18小时成熟和排卵，但我们未能获得受精卵。进一步的研究是必要的，以发展技术，获得良好的质量卵子在体外。少

项目成果

The first success of glass eel production in the world: basic biology on fish reproduction advances new applied technology in aquaculture.

• DOI: 10.1007/s10695-006-0024-3
• 发表时间: 2005-04
• 期刊: FISH PHYSIOLOGY AND BIOCHEMISTRY
• 影响因子: 2.9
• 作者: Kagawa, Hirohiko;Tanaka, Hideki;Ohta, Hiromi;Unuma, Tatsuya;Nomura, Kazuharu
• 通讯作者: Nomura, Kazuharu

オスモティックポンプを用いたサケ脳下垂体抽出液投与が雌ウナギの成熟に及ぼす影響

使用渗透泵施用鲑鱼垂体提取物对雌性鳗鱼成熟的影响

• 发表时间: 2007
• 作者: Fujie;N.;Adachi;J.;Nishi;A.;Hashimoto;H.;Kaji;S.;Kishi;T.;Kagawa;H.;堀内 泰宏;香川 浩彦
• 通讯作者: 香川 浩彦

Expression of growth hormone family and growth hormone receptor during early development in the Japanese eel (Anguilla japonica)

日本鳗鱼（Anguilla japonica）早期发育过程中生长激素家族和生长激素受体的表达

• 发表时间: 2006
• 期刊: Comparative Biochemistry and Physiology, Part B 145
• 作者: Amano;H.;香川 浩彦;Ozaki Y.
• 通讯作者: Ozaki Y.

Molecular characterization of three forms of vitellogenin and their yolk protein products during oocyte growth and maturation in red seabream (Pagrus majar). a marine teleost spawning pelagic eggs

红鲷（Pagrus majar）卵母细胞生长和成熟过程中三种形式卵黄蛋白原及其卵黄蛋白产物的分子特征。

• 发表时间: 2006
• 期刊: Molecular Reproduction and Development 73
• 作者: Sawaguchi;S.;Kagawa;H.;Ohkubo;N.;Hiramatsu;N.;Sullivan;C.V.;Matsubara;T.;香川 浩彦;Ozaki Y.;Sawaguchi S.
• 通讯作者: Sawaguchi S.

Aquaporin and proteolysis of yolk and ovarian follicles play essential roles in the regulation of oocyte hydration during oocyte maturation in the Japanese eel

水通道蛋白以及卵黄和卵泡的蛋白水解在日本鳗鱼卵母细胞成熟过程中卵母细胞水合的调节中发挥重要作用

• 发表时间: 2007
• 作者: Kagawa;H.
• 通讯作者: H.