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

为了阐明卵母细胞成熟过程中卵母细胞胞质成熟(如水化、液体结合和卵质清除)的机制，并发展体外生产高质量卵子的技术，本研究对日本鳗鱼产卵浮卵进行了以下研究。本文研究了鳗鱼卵母细胞体外成熟过程中卵母细胞胞质成熟的过程。此外，我们还开发了模拟体内发生的细胞质成熟的孵化技术。利用我们开发的技术，我们发现胞浆成熟是由促性腺激素刺激在卵泡中合成的成熟诱导类固醇诱导的。成熟诱导类固醇诱导的细胞质成熟可能是通过卵母细胞内蛋白质的合成来实现的。结论：1.鳗鱼卵母细胞胞质成熟可能与IGF-I有关，而与缝隙连接无关。本研究还表明，卵母细胞h…卵黄蛋白分解产生渗透推动力，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，鳗鱼的卵泡在卵母细胞的水合过程中可能起着重要的作用，但其确切的作用还有待进一步研究。本研究还用免疫细胞化学方法检测了日本鳗鱼卵母细胞生长、成熟和排卵过程中卵泡中鳗鱼AQP-10的存在和细胞分布。抗AQP-1o抗体是针对鳗鱼AQP-1o C-末端部分区域的合成肽而在兔体内制备的。在初级卵黄小球阶段，卵黄小球周围的细胞质中首先观察到免疫反应，之后随着卵黄发生的进行，免疫反应遍及整个卵母细胞的细胞质。免疫反应定位于卵黄小球融合形成的大的膜限制的卵黄团周围。在迁移核期的卵母细胞、成熟的卵母细胞和排卵的卵母细胞中，它也定位于卵母细胞质膜之下。这些结果与在金枪鱼卵母细胞中观察到的结果不同，表明鳗鱼卵母细胞存在一种独特的卵子水化机制。我们试图开发出日本鳗鱼体外生产优质卵的技术。虽然我们发现卵母细胞成熟和排卵分别发生在成熟诱导激素孵育后12小时和18小时，但未能获得受精卵。在体外获得高质量卵子的技术还需要进一步的研究。较少

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