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

最近，我们成功地在含血清的培养液中诱导雌雄同体的三斑鱼(Halichoeres Trimaculatus)在卵巢器官培养中发生精子。该培养体系为进一步研究控制性腺性别变化的机制提供了一个很好的模型。在本研究中，我们对这种三点鱼的培养体系进行了重新检验。采用漂浮组织培养法，将性未成熟的三点鱼的卵巢组织在添加0.5%牛血清白蛋白或10%胎牛血清的莱博维茨L-15培养液中进行培养。在26℃时，组织暴露在雄激素下数周。还检测了不同浓度的雄激素和雌激素的影响。同时，采用原位末端标记法(TUNEL)检测培养过程中的细胞凋亡情况，观察到对照组(不含类固醇)、卵母细胞变性和精子发生的启动情况。雄激素促进卵巢组织通过精原细胞向生精隐窝的完全转变。补充牛血清白蛋白与补充牛血清白蛋白无差异。雄激素对精子发生的诱导作用呈剂量依赖性。雌激素处理减少了卵母细胞的退化，并且不会导致精子发生。在培养过程中观察到TUNEL阳性的卵母细胞并不断增多，这表明在无血清培养条件下，可在体外诱导鲤鱼从卵巢到睾丸的重构。这些结果还表明，由于缺乏女性性行为的内源性因素(尤其是雌激素)，基础体外培养可以触发性腺的性别变化，雄激素可以加速性腺的性别变化。此外，卵母细胞的凋亡可能是性腺变性的一个重要机制。

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.