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Analysis of mechanisms controlling gonadal sex change in fish using an in vitro culture system

利用体外培养系统分析控制鱼类性腺性别变化的机制

基本信息

批准号：
18580172
负责人：
TODO Takashi
金额：
$ 2.49万
依托单位：
Hokkaido University
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-18580172/
关键词：
sex change organ culture sex steroid ovary testis germ cell wrasse アポトーシス

项目摘要

Recently, we succeeded to induce in vitro spermatogenesis in a protogynous hermaphrodite fish, the three-spotted wrasse (Halichoeres trimaculatus) during organ culture of ovary with a serum-supplemented medium. This culture system appears to be a good model for further study of the mechanisms controlling gonadal sex change. In the present study, we re-examined this culture system for the three-spotted wrasse.Ovarian tissue from sexually immature three-spotted wrasse was cultured in Leibovitz L-15 medium supplemented with 0.5% bovine serum albumin (BSA) or 10% fetal bovine serum (FBS) using a floating tissue culture method. Tissue was exposed to androgens for a few, weeks at 26℃. The effects of various concentrations of androgens and estrogen were also examined. Furthermore, the detection of apoptotic cells during the culture was performed using the TUNEL method.In all experimental groups, including the control (no steroid), degeneration of oocytes and initiation of spermatogenesis was observed. Androgens enhanced the complete transition of ovarian tissue via spermatogonia to formation of spermatogenic crypts. There was no difference between supplementation with BSA and FBS. The effects of androgens on inducing spermatogenesis showed a dose dependent manner. Estrogen treatments reduced the degeneration of oocytes, and did not lead the occurrence of spermatogenesis. The TUNEL-positive oocytes were observed and increased during culture period.These results show that the restructuring from ovary to testis in the wrasse could be induced in vitro with a serum-free medium. The results also suggest that the gonadal sex change can be triggered by basic in vitro culture due to a lack of endogenous factors (especially, estrogens) for female sexuality, and accelerated by androgens. Furthermore, oocyte apoptosis is might be an important mechanism of the gonadal sex change.

最近，我们成功地在体外诱导精子发生的原生雌雄同体的鱼类，三斑海猪鱼（Halichoeres trimaculatus）在卵巢器官培养与血清补充培养基。该培养体系为进一步研究性腺性别转变的调控机制提供了良好的模型。在本研究中，我们重新审视了这种培养系统的三点濑鱼，卵巢组织从性未成熟的三点濑鱼培养在Leibovitz L-15培养基添加0.5%的牛血清白蛋白（BSA）或10%的胎牛血清（FBS）使用浮动组织培养法。将组织在26℃下暴露于雄激素几周。还检查了不同浓度的雄激素和雌激素的作用。同时，用TUNEL法检测培养过程中凋亡细胞的变化，结果表明，所有实验组（包括对照组）的卵母细胞均发生变性，精子发生开始。雄激素促进卵巢组织经精原细胞向生精窝的完全转化。补充BSA和FBS之间没有差异。雄激素诱导精子发生的作用呈剂量依赖性。雌激素处理可减轻卵母细胞的变性，不导致精子发生。在体外培养过程中，可观察到TUNEL阳性卵母细胞的出现，并呈上升趋势，表明在无血清培养液中可以诱导隆头鱼卵巢向精巢的重构。结果还表明，由于缺乏雌性性行为的内源性因素（特别是雌激素），基本的体外培养可以触发性腺的性别变化，并被雄激素加速。此外，卵母细胞凋亡可能是性腺性别改变的重要机制。