甲基睾酮(mt)对雌性及未性分化斑马鱼(daniorerio)影响

摘 要 I 摘 要 斑马鱼（Danio rerio）是一种典型的小型热带观赏鱼。由于其具有个体小、产卵多、发 育快、体外受精、胚胎透明、易于观察、饲养简便等优点，被广泛应用于发育生物学、遗传 学、环境毒理学、免疫学等学科，斑马鱼也因此成为研究背景最广泛的脊椎动物模式生物之 一。本文以斑马鱼为研究对象，运用组织学、毒理学及繁殖生物学等方法比较深入研究不同 浓度乙醇和 MT 对斑马鱼成活率、生长及性腺结构的影响。 采用波恩氏液固定、常规石蜡切片的方法观察斑马鱼卵巢结构及发育情况。结果表明， 斑马鱼卵巢为封闭型， 分为左右大致对称的一对， 90 日龄左右达到性成熟。 成熟卵巢乳白色， 卵巢内充满 II-V 时相的卵母细胞，80%以上体积被 V 时相卵母细胞所占据，与斑马鱼为典型 的非同步型卵巢相适应；II 时相卵母细胞染色为深紫色，III 时相卵母细胞开始出现放射膜， IV 时相卵母细胞卵黄迅速堆积，V 时相成熟卵母细胞呈游离状态、细胞核消失。 利用乙醇浓度梯度为 0 ml/L（对照） 、0.50 ml/L、1.00 ml/L、2.00 ml/L、4.00 ml/L、8.00 ml/L 的溶液孵化受精卵及饲养稚鱼 5 d，得到受精卵孵化率分别为 61%、59.5%、55%、40%、 17.5%、10%；稚鱼成活率分别为 67.78 %、66.84 %、58.8 %、47.68 %、20.2 %、12.3 %，并 在 4ml/L 以上组观察到独眼畸形、脊柱弯曲和死亡等现象。结果表明，0.50 ml/L 的乙醇浓度 对受精卵孵化率及稚鱼成活率与对照组差异不显著， 因此小于等于 0.50 ml/L 的乙醇溶液对斑 马鱼是安全剂量。大于此剂量的乙醇则对孵化率、存活率有明显影响。 利用浓度梯度为 0mg/L （对照） 、 1.00 mg/L、 2.50 mg/L、 5.00 mg/L、 10.00 mg/L、 20.00 mg/L 的甲基睾酮（MT）溶液孵化受精卵及浸泡孵化后 10d 的稚鱼 40d，孵化和饲养期间每 3d 采 集孵化后 12d 以上稚鱼固定切片观察性腺发育。 结果显示， 受精卵组的成活率分别为 34.75%、 33%、 31%、 23%、 15.75%、 9.75%； 雄性比分别为 50.35%、 56.89%、 74.94%、 93.61%、 95.3%、 0。 稚鱼最晚于 35 d 时可以区分性别， 成活率分别为 74.92%、 66.60%、 62.15%、 41.74%、 24.39%、 12.81%，雄性比率为 49.61%、58.17%、78.23%、92.89%、98.75%、0。并随着激素浓度的增 高，各组体长、体重均有所降低。由此表明 MT 对斑马鱼的生长活力和成活率都有明显的抑 制作用；当 MT 剂量达到 10mg/L 处理鱼卵和稚鱼均会出现少量的不育个体，20 mg/L 的 MT 为可诱导群体不育，利用 MT 浸泡法对斑马鱼进行全雄控制的剂量应在 510mg/L 之间。 利用含 MT0mg/kg（对照） 、30 mg/kg、60 mg/kg、120 mg/kg、240 mg/kg、400 mg/kg 的 饲料饲喂 60 日龄及经产雌性斑马鱼 30 d，并于饲喂后进行测交。结果显示，60 日龄组成活 率分别为 95%、 90%、 75%、 55%、 35%、 70%， 成熟度分别为 0.1074、 0.0998、 0.0756、 0.0582、 0.0386、0.0278；经产组成活率分别为 100%、95%、80%、55%、40%、65%，成熟度分别为 0.2247、0.1905、0.1531、0.1210、0.0275、0.0407。随着激素剂量的增加，斑马鱼的生长受 到明显的抑制，但在 400 mg/kg 组有明显回升。MT 对雌性斑马鱼产卵量、受精卵孵化率、 生长活力、成活率及成熟度都有明显的抑制作用；400 mg/kg 处理雌性斑马鱼 30 d 可产生激 素阉割。 经产雌性斑马鱼性腺切片观察结果表明：随着 MT 剂量的增加，各剂量组性腺的发育受 到的抑制加深，卵巢结构变化也明显。30 mg/kg 组 V 时相成熟卵母细胞数量减少；60 mg/kg 组 II 时相以上卵母细胞所占面积减少为 60%，120 mg/kg 组开始观测到明显的卵母细胞退化东北农业大学农学硕士学位论文 II 过程， 240 mg/kg 组 II 时相以上卵母细胞仅占性腺内 10%左右， 400 mg/kg 组性腺内除少量退 化中的 II 时相卵母细胞外， 其他组织已难以确认为卵巢。 卵母细胞在 MT 作用下的退化过程： 抑制卵母细胞发育细胞核结构变化累及细胞器形成凋亡小体、破坏细胞膜结构 凋亡小体逸散、细胞固缩崩解。 关键词 斑马鱼(Danio rerio)；乙醇；甲基睾酮；卵巢结构；激素阉割；细胞退化 Abstract III Effects of Methyltestosterone on Adult Female and Sex Undifferentiation Fry Zebrafish (Danio rerio) Abstract Zebrafish(Danio rerio) is a kind of typical small tropical fish，because of its small individual, fast development, eggs embryos, invitro fertilization, embryos transparent, easy to observe and feed, etc. Its widely applied in developmental biology, genetics, environment toxicology, immunology etc. Zebrafish thus becomes one of the most extensive research background mode vertebrate.We taked zebrafish as the research object, using histology and toxicology and reproductive biology research methods to compare the effece of different concentration of ethanol and methyltestosterone to the survival rate,growth vitality and gonads structure of Zebrafish. We observe the structure and developmental capacity of ovaries by fixing with Bouins and paraffin section.The result showed that zebrafish ovaries is close type, roughly symmetrical couple,grows to sexual maturation about 90 days after born. Mature ovaries were milk-white，full of II-V phases oocytes，more than 80% volume was occupied by V phase oocytes，its fit for zebrafish was typical asynchronic ovarian. II phase oocytes dyeing were modena,phase III oocyte began to appear zona radiata, phase IV oocyte yolk produce quickly, phase V oocyte was maturation in the free state, nuclei disappeaered. Using the concentration gradient of 0 ml/L (control group), 0.50 ml/L, 1.00 ml/L, 2.00 ml/L, 4.00 ml/L, 8.00 ml/L ethanol feeding fertilized eggs and breeding juvenile fishes 5 d. Got fertilized eggs hatch rates was 61%, 59.5%, 55%, 40%, 17.5%, 10%, juvenile fishes survival rate were 67.78%, 66.84% ,58.8%, 47.68 %, 20.2%, 12.3%. In 4ml/L above groups observed one-eyed deformity, spinal bending and die. Results showed that 0.50 mg/L group of fertilized eggs hatch rate and the survival rate of juvenile fishes with no significant difference, not deformities. So less than 0.50ml/L ethanol solution of zebrafish was safe doses. Above 0.50 mg/L could obviously effect to hatch rate and surviva rate. Using the concentration gradient of 0mg/L (control group), 1.00 mg/L, 2.50 mg/L, 5.00 mg/L, 10 .00mg/L, 20.00 mg/L of methyltestosterone (MT) hatch fertilized eggs and breeding fishes after hatched 10 d for 40 d, and fixed juvenile after hatched above 12 d observation development of gonad per 3 d. Experimental results showed that the group can distinguish sex latest in 35d. The survival rate respectively fertilized group were 34.75%、33%、31%、23%、15.75%、9.75%, Male rate 东北农业大学农学硕士学位论文 IV was50.35%, 56.89%, 74.94%, 93.61%, 95.3%, 0%. Survival rate of juvenile fishes were 74.92%、66.60%、62.15%、41.74%、24.39%、12.48%;male rate were 49.60%、58.17%、78.23%、92.89%、98.75%、0% in each group.And with the increase of the concentration of MT, fishes length and weight were reduced. Results show that the MT has obvious inhibition to growth vitality and surviva