稀有鮈鲫脑中KISS/GPR54系统介导MT对性腺发育影响研究
Effect of 17α-methyltestosterone on Gonad Development Mediated through KISS/GPR54 System in Brain of Gobiocypris rarus
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摘要: 17α-甲基睾酮（17α-methyltestosterone，MT）是一种人工合成的具有雄激素效应的环境内分泌干扰物。为了探究MT通过KISS/GPR54系统及其相关miRNAs干扰稀有鮈鲫性腺成熟作用机制，采用0、25、50和100 ng·L-1 MT暴露稀有鮈鲫7、14和21 d。石蜡组织切片（H-E染色）观察MT对稀有鮈鲫性腺组织学的影响；实时荧光定量PCR （qRT-PCR）检测雌、雄鱼脑中kiss1、kiss2、GPR54α和GPR54β基因mRNA的表达量以及kiss1相关miRNAs （miR-25-3p、miR-92a-3p、miR-137-3p、miR-199-3p和miR-324-3p）的表达量。结果显示，随着暴露时间的延长和暴露浓度的升高，性腺退化的程度逐渐严重，成熟卵细胞和成熟精子比例逐渐降低。MT处理雌鱼7 d，各处理组GPR54β基因表达量均显著降低。处理14 d，高浓度组kiss1、kiss2、GPR54α和GPR54β基因mRNA表达量均显著低于对照组。暴露雌鱼21 d，各处理组kiss1和GPR54α表达量均显著降低。MT处理雄鱼7 d，中浓度组kiss1基因表达量显著降低，高浓度组kiss1基因表达量显著升高，MT （25、50和100 ng·L-1）处理组kiss2基因表达量均显著升高。MT处理雄鱼14 d，kiss2基因表达量均显著降低。MT处理21 d，3个处理组雄鱼kiss1表达量均显著升高，GPR54β表达量则显著降低。低浓度MT处理雌鱼7 d，脑中miR-25-3p、miR-92a-3p、miR-137-3p和miR-199-3p表达显著升高，kiss1基因表达显著降低，呈负相关。MT处理雄鱼21 d，各处理组miR-92a-3p和miR-137-3p表达量均降低，与kiss1表达量呈负相关。MT通过干扰稀有鮈鲫脑中miR-25-3p、miR-92a-3p、miR-137-3p、miR-199-3p和miR-324-3p表达量，调控kiss1基因mRNA表达，进而干扰下丘脑-垂体-性腺轴上游刺激因子KISS/GPR54系统，导致类固醇激素分泌异常，性腺发育受阻。
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关键词:
- 17α-甲基睾酮 /
- 稀有鮈鲫 /
- KISS/GPR54系统 /
- miRNAs
Abstract: 17α-methyltestosterone (MT) is an artificially synthesized environmental endocrine disruptor with androgen effect. To explore the interference of MT through the KISS/GPR54 system and its related miRNAs of Gobiocypris rarus, G. rarus were exposed to 0, 25, 50 and 100 ng·L-1 of MT for 7, 14 and 21 d. The tissue paraffin slice (H-E staining) was used to study the effect of MT on the gonads tissues. Real-time fluorescent quantitative PCR (qRT-PCR) was used to detect the expression of kiss1, kiss2, GPR54α, GPR54β gene mRNA and miRNAs (miR-25-3p, miR-92a-3p, miR-137-3p, miR-199-3p and miR-324-3p) in the brain of female and male fish. The results show that MT can lead to the involution of the gonads and the reduction of mature eggs and sperm in the G. rarus in a time- and dose-dependent manner. For female G. rarus, the expression of GPR54β gene was decreased significantly of MT treatment for 7 d. The kiss1, kiss2, GPR54α and GPR54β expressions in MT (100 ng·L-1) group were decreased significantly for 14 d. MT (25, 50 and 100 ng·L-1) exposure for 21 d, the mRNA expressions of kiss1 and GPR54α were decreased significantly of female fish. For male G. rarus, the expression of kiss1 gene in MT (50 ng·L-1) group was decreased significantly, but its expression was increased significantly in 100 ng·L-1 group for 7 d. The mRNA expression of kiss2 was increased significantly in groups of MT (25, 50 and 100 ng·L-1) for 7 d. In all the three MT exposure groups, kiss2 gene expression was decreased significantly for 14 d. After MT treatment for 21 d, the expression of kiss1 in male fish in the three treatment groups was significantly increased, while the expression of GPR54β was significantly decreased. The miR-25-3p, miR-92a-3p, miR-137-3p and miR-199-3p expressions were increased in females and they were negatively correlated with the expression of kiss1 in 25 ng·L-1 MT group following 7 d exposure. The miR-92a-3p and miR-137-3p expression were decreased in males following 21 d MT exposure, and they were negatively correlated with the kiss1 expression. The present study suggests that MT regulates the expression of the kiss1 by interfering with the expression of miR-25-3p, miR-92a-3p, miR-137-3p, miR-199-3p and miR-324-3p in the brain of G. rarus. In turn, it interferes with the KISS/GPR54 system, an upstream stimulator of the hypothalamic-pituitary-gonad axis, resulting in abnormal secretion of steroid hormones and gonad development.-
Key words:
- 17α-methyltestosterone /
- Gobiocypris rarus /
- KISS/GPR54 system /
- miRNAs
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