三倍体雌性虹鳟性腺发育阶段细胞色素相关基因的表达

Expression of cytochrome genes during the early gonadal development in triploid female rainbow trout Oncorhynchus mykiss

Cyp19a1b、Cyp11a1、Cyp11b2、Cyp17a1、Hsd3b1等细胞色素相关基因能够调节硬骨鱼类性类固醇的合成,对性腺发育和性别决定产生影响。本研究以全雌三倍体虹鳟(Oncorhynchus mykiss)为研究对象,正常雌性二倍体虹鳟为对照,选取31~68 dpf(days post fertilization)时间段的虹鳟仔鱼脑组织,采用q RT-PCR和酶联免疫的方法研究以上几种基因的表达状况和脑芳香化酶的活性变化,以期探明导致三倍体雌性虹鳟性腺发育异常的关键原因。q RTPCR结果显示,二倍体中Cyp19a1b在30~50 dpf时表达量上调并且维持在较稳定水平,但50~56 dpf时表达量逐渐下调,之后56~68 dpf表达量持续上调;三倍体中Cyp19a1b表达量在30~35 dpf开始上调,35~47 dpf逐渐下调,47~55 dpf开始第二次上调,之后维持在较稳定水平直至68 dpf,但三倍体Cyp19a1b的表达量显著(P<0.05)低于同期二倍体的。二倍体Cyp11a1表达量在34 dpf出现峰值,三倍体Cyp11a1在38 dpf时出现峰值。二倍体Hsd3b1表达量在33~42 dpf时维持在较高水平,在38 dpf时出现高峰;三倍体Hsd3b1表达量在47~59 dpf时较高,在49 dpf出现高峰。二倍体中Cyp11b2在37 dpf出现峰值,之后开始下调;三倍体在40 dpf出现峰值,之后逐渐下调,但三倍体Cyp11b2表达量显著低于同期二倍体。二倍体Cyp17a1的表达量在35~46 dpf时逐渐上升,在45 dpf时达到高峰之后直至69 dpf逐渐下降,并且维持在较为平稳的水平上;但是在相同的实验条件下未检测到同一时期三倍体Cyp17a1的表达量。酶联免疫结果显示,在40 dpf时二者的脑芳香化酶活性到达高峰,但在40~60 dpf时期,二倍体虹鳟脑芳香化酶活性显著(P<0.05)高于三倍体虹鳟,尤其在45~50 dpf时,该酶活性分别较三倍体的高1.15倍和1.12倍。以上结果表明三倍体虹鳟早期性腺发育迟缓的原因之一是Cyp19a1b、Cyp11a1、Cyp11b2、Cyp17a1、Hsd3b1等基因的表达晚于二倍体,且表达量低于二倍体,造成雌二醇不能正常合成,最终导致性腺发育迟缓。

The genes Cyp19a1b,Cyp11a1,Cyp11b2,Cyp17a1,and Hsd3b1 play an important role in the steroidogenesis and gonadal differentiation of teleosts.Therefore,in this study,we chose triploid and diploid rainbow trouts(Oncorhynchus mykiss)at 31–68 days post fertilization(dpf)cultured in the same environment as object.In this study,triploid rainbow trouts were used as object and diploid rainbow trouts were used as controls to evaluate the expression of genes and the activity of CYP19A1B enzyme in the brain of rainbow trout.The real-time polymerase chain reaction results showed that the expression of Cyp19a1b in the diploid rainbow trout was up-regulated during 30–50 dpf and maintained a stable level during this period.Subsequently,during 50–56 dpf,the expression was down-regulated;during 56–68 dpf,the expression was up-regulated again.The expression of Cyp19a1b in the triploid rainbow trout was up-regulated during 30–35 dpf,down-regulated during 35–47 dpf,and then upregulated during 47–55 dpf;thereafter,until 68 dpf,the expression was maintained at a stable level.The expression of Cyp11a1 in diploid rainbow trout peaked at 34 dpf and in triploid rainbow trout peaked at 38 dpf.The expression of Hsd3b1 in the diploid rainbow trout was maintained at a high level during 33–42 dpf and its expression peaked at 38 dpf.The expression of Hsd3b1 in the triploid rainbow trout was high during 47–59 dpf and peaked at 49 dpf.The expression of Cyp11b2 in diploid rainbow trout peaked at 37 dpf,and then the expression was down-regulated.In the triploid rainbow trout,the peak value was recorded at 40 dpf,and then the expression was down-regulated,but the expression of Cyp11b2 in the triploid rainbow trout was lower than that in the diploid rainbow at the same period.The expression of Cyp17a1 in the diploid rainbow trout was up-regulated at 35–46 dpf and the peak value was recorded at 45 dpf,and then the expression was down-regulated until 69 dpf.However,under the same experimental conditions,the expression of Cyp17a1 in the triploid rainbow trout was not detected.The results of enzyme-linked immunosorbent assay demonstrated that at 40 dpf,the activity of CYP19A1B enzyme in the diploid and triploid rainbow trout peaked,but during 40–60 dpf,the activity of CYP19A1B enzyme in the diploid rainbow trout was significantly higher than that in the triploid rainbow trout;especially at 45 dpf and 50 dpf,the activity of this enzyme in the diploid trout was 1.15 and 1.12 times higher than that in the triploid trout,respectively.The results suggest that one of the reasons for early gonadal differentiation delay in triploid rainbow trout is the expression of Cyp19a1b,Cyp11a1,Cyp11b2,Cyp17a1,and Hsd3b1 is lower and later than those in the diploid rainbow trout.Moreover,estradiol cannot be synthesized normally in triploid rainbow trout,and therefore,gonadal differentiation is delayed.