【作者】 孙鹏；

【导师】 张培军； 尤锋；

【作者基本信息】 中国科学院研究生院（海洋研究所） ， 海洋生物学， 2009， 博士

【摘要】 本文采用组织学手段研究了牙鲆性腺在分化、发育和成熟过程中的变化。然后,通过放射性免疫方法(RIA)测定了牙鲆仔稚幼鱼全组织匀浆液中的性类固醇激素—睾酮(T)和雌二醇(E2)的含量,并结合牙鲆血清中T和E2含量的年周期测定,从内分泌学水平探讨了T和E2在其性腺分化、发育和成熟过程中水平的变化规律。同时,采用高温和雌性激素对性腺未分化的普通和雌核发育牙鲆仔稚鱼进行诱导处理,获得了较高比例的雄性鱼/假雄鱼或100%雌性鱼;并研究了这些外界环境因子对牙鲆性腺分化、性别比率及体内T和E2水平的影响,藉此探讨了牙鲆性别决定与性腺分化的细胞学和内分泌学机制。对牙鲆仔稚幼鱼性腺的组织切片观察发现,培育水温18~20℃下,孵化后第45天、平均全长<22.0±2.8 mm的牙鲆,其性腺分化尚未开始,属于原始性腺;在孵化后70日龄、平均全长为38.0±1.7 mm左右,部分个体中观察到卵巢的雏形,其余个体的性腺在此阶段以及之后的一段时间内变化并不明显;到了第110天、平均全长达到86.5±5.9 mm时,雌性个体卵巢出现了卵原细胞向卵母细胞的转变,标志着卵巢分化的结束。在90日龄、平均全长为63.5±3.4 mm的雄性牙鲆中,精原细胞快速增殖,并观察到了输精管结构;进一步的细胞学分化则出现在100日龄、平均全长为76.0±8.6 mm的个体中,此时可以看到精小叶的形成;在平均全长为140.0±15.2 mm时,精巢中出现初级精母细胞,标志着性腺分化的基本完成。对牙鲆仔稚幼鱼全组织匀浆和成鱼血清中的T和E2水平的比较发现,在全长为6 mm左右的仔鱼中T和E2含量均较高。随后,在性腺分化过程中T含量大大降低,E2的含量急剧增高,而性腺分化后期E2含量又降到较低的水平。在雄性牙鲆成鱼中,从精巢第Ⅲ期开始,T含量随着精巢的发育而增加,到了精巢第Ⅴ期性腺发育成熟并排精后,又降低到较低的水平;E2含量在从精巢第Ⅲ期发育至精巢第Ⅴ期过程中略呈降低的趋势,但是总体上来说没有明显的差异。在雌性牙鲆成鱼中,卵巢从第Ⅱ期到第Ⅳ期的过程中,T水平逐渐升高,在第Ⅴ期时则明显降低;而E2含量在卵巢第Ⅱ期时保持较低的水平,随着卵巢的发育,E2含量逐渐增高,在卵巢第Ⅳ期时达到最高水平,在第Ⅴ期产卵后又有所降低。在雌雄个体中T和E2均呈现周期性的变化。5月份随着水温的升高,雄性个体T和E2含量显著上升;到了9月份又逐渐下降至最低值。雌性个体E2含量自3月份开始增高,在5月份急剧升高,并在6月份达到最高值;在7月份的时候,E2突然降低,而到了8月份又有所回升;9月份之后E2逐渐降低并在1月份左右降到最低;而T的含量分别在2月份和6月份出现两次高峰。温度诱导牙鲆幼鱼性腺分化的结果表明,牙鲆中存在明显的TSD机制,即其性腺分化因饲育水温的不同而变化:在一定温度范围内,随着饲育温度的增加,牙鲆的雄性比例逐渐增高,常温对照组和21℃组中的雄性比例分别为51.62%、60.00%,而在24℃和28℃高温组中,雄性比例显著高于对照组,分别达到73.33%和87.27%。T和E2含量测定显示,在性腺分化时期,高温和对照组中T含量没有明显的变化,而温度处理组中的E2水平则低于对照组,特别是在28℃高温组,其E2水平显著低于对照组(P<0.05)。外源E2处理性腺未分化的牙鲆幼鱼的结果也表明,牙鲆的死亡率与雌性化比率均为雌性激素剂量依赖型的。随着外源E2剂量的增加,雌性比率增加,但同时死亡率也增高。此期间T和E2水平比较发现,在性腺分化时期,对照组中的T含量稍高于雌激素处理组;而对照组中的E2含量高于0.2 ppm和2 ppm两个低剂量组,却低于20 ppm和100 ppm两个高剂量组。同时,还对人工诱导培育的雌核发育牙鲆和性反转牙鲆进行了性腺发育观察,在所观察的雌核发育牙鲆个体中,其雌性比例为83.33%,而高温28℃饲育群体中的雄性比例(即假雄鱼比例)为91.67%;在雌性个体中,也有一定比例的个体性腺发育不正常,有的性腺发育较小,有的则缺少部分性腺。进一步对雌核发育成体的血清中T和E2含量进行测量,发现在普通牙鲆个体中T含量显著低于雌核发育个体,而E2含量则高于雌核发育牙鲆;在雌核发育牙鲆中,性腺发育不正常的个体比性腺发育正常的个体中的T含量稍高,而E2含量则显著低于正常雌核发育牙鲆个体和普通牙鲆个体(P< 0.05)。更多还原

【Abstract】 Histological changes during gonadal differentiation, development and maturation were observed in Paralichthys olivaceus. Combined with RIA analysis of T and E2 levels in whole body extract of larvae and juvenile, and also in serum, we discussed changes of T and E2 concentrations during gonadal differentiation, development and maturation in endocrine level. Then, by treating undifferentiated normal diploid and gynogenetic fish with high temperature and E2, respectively, we obtained flounder stock with high percentage of male or femal, and studied effects of temperature and exougenous sex steroids estradio-17β(E2) on growth, gonadal differentiation, sex ratios and levels of T and E2. Mechanisms of sex determination and gonadal differentiation in viewpoint of endocrine were also discussed.Histological observation found that when fish were reared at temperature of 18-20℃, gonad remained undifferentiated till the 45th day post hatching (D45). When larvae reached about 38.0±1.7 mm of mean total length (TL) on the D70, ovarian cavity was observed in presumptive ovary. On the D110, when mean total length reached about 86.5±5.9 mm, ovarian cavity grew bigger, and oocytes were firstly detected. Differentiation of testis was firstly observed in juvenile flounder with mean total length of 63.5±3.4 mm on the D90 when spermatogenous cell proliferated rapidly and spermaduct formed. Seminal lobule appeared on the D100 when juveniles reached about 76.0 mm of total length. And when flounders reached total length about 140.0 mm, appearance of spermatocyte indicated the completion of gonadal sex differentiation.Analysis of T and E2 levels in whole body extract from larvae and juvenile, and also in serum in Paralichthys olivaceus, showed that concentrations of T and E2 were higher than those in larvae with the total length of 6 mm. Then, T levels decreased with the process of gonadal differentiation, but E2 levels increased sharply during period of gonadal differentiation and decreased to a lower level thereafter. In male, from tesits stageⅢ, T levels in serum increased with the development of gonad, and finally, it dropped again at the stageⅤafter maturation of testis and spermiation. E2 levels decreased with the development of male gonad from the stageⅡto the stageⅤ, but had no significant difference in general. In female, both T and E2 levels increased gradually with the development of ovary and peaked at the stageⅣ, and then dropped after ovulation. Both levels of T and E2 have an annual cycle. In male, levels of T and E2 increase sharply with the increase of temperature from May, and then it begin to drop in September. In female, E2 increased from March and peaked in June and August, and then dropped, too. As to levels of T, it also had two peaks, one in Feberary and the other in June.Result of temperature treatment showed that there is mechanism of TSD in Paralichthys olivaceus, and ratios of male increased with the increase of temperature. The percentage of male in control, 21℃and 24℃were 51.62%, 60.00% and 73.33%, respectively. And in the groups with high temperature of 28℃, significantly high ratios of male compared to control were observed (87.27%). Analysis of T and E2 levels suggested that there were no obvious difference among control and different temperature treated groups in T levels, but E2 levels in temperature treated groups were lower than that of in control. Especially, E2 levels in high temperature groups of 28℃were significantly lower than that in control groups (P<0.05). By treating gonadal undifferentiated fish with different dose of E2, we found that both mortality and ratios of female were doze-depended. With the increase of dose of E2 in feed, percentage of female was increased in Paralichthys olivaceus. Analysis of T and E2 levels showed that level of T were lower than that in E2 treated group. But as to E2, levels were lower than those in the treatment groups with doze of 0.2 ppm and 2 ppm, but higher than those in the treatment groups with dose of 20 ppm and 100 ppm.In the meantime, we compared gonadal development conditions of gynogenetic flounder. The ratio of female in gynogenetic flounder was 83.33%, and the ratio of XX male in gynogenetic flounder treated with high temperature of 28℃was 91.67%. In the gynogenetic flounders, a certain proportion of individuals had abnormal gonad, some of the gonad grew small and some even lost part of it. Further studies on T and E2 levels in serum showed that gynogenetic individuals had higher T levels and lower E2 levels than those normal diploid groups. And also, higher T level and lower E2 level was observed in flounder with abnormal gonad when compared with control and gynogenetic flounder.更多还原