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

最近，我们成功地用血清补充培养基在卵巢器官培养过程中诱导了原性雌雄同体的三斑隆头鱼（Halichoeres trimaculatus）的体外精子发生。这一文化体系为进一步研究性腺性变化的控制机制提供了良好的模型。在本研究中，我们重新研究了这种三斑濑鱼的培养体系。采用漂浮组织培养法，在添加0.5%牛血清白蛋白（BSA）或10%胎牛血清（FBS）的Leibovitz L-15培养基中培养性未成熟的三斑隆鱼卵巢组织。组织在26℃下暴露于雄激素中几周。还研究了不同浓度雄激素和雌激素的影响。此外，采用TUNEL法检测培养过程中的凋亡细胞。在所有实验组中，包括对照组（不使用类固醇），观察到卵母细胞的退化和精子发生的开始。雄激素促进卵巢组织通过精原细胞完全过渡到生精隐窝的形成。补充BSA和FBS之间没有差异。雄激素对精子发生的诱导作用呈剂量依赖性。雌激素治疗减少了卵母细胞的退化，并没有导致精子发生。tunel阳性卵母细胞在培养期间增多。上述结果表明，无血清培养基可以在体外诱导濑鱼从卵巢向睾丸转变。结果还表明，由于缺乏女性性征的内源性因素（尤其是雌激素），体外基本培养可触发性腺性别变化，雄激素可加速性腺性别变化。此外，卵母细胞凋亡可能是性腺性别改变的重要机制。

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.