基于PI3K/Akt通路探究蒙古羊ADAMTS1基因的作用机制

doi:10.16431/j.cnki.1671-7236.2023.09.002

摘要/Abstract

摘要： 【目的】基于PI3K/Akt通路研究含凝血酶敏感素基序的去解联金属蛋白酶1(a disintegrin and metalloproteinase with thrombospondin motifs 1,ADAMTS1)在产双胎和单胎蒙古羊发情周期不同阶段卵巢内的表达规律,探究ADAMTS1基因影响蒙古羊繁殖性状的作用机制。【方法】选取经产2次的双胎和单胎蒙古羊共40只,自然发情,确定母羊发情期和间情期,分为双胎发情期、双胎间情期、单胎发情期和单胎间情期组,每组各10只羊。利用注射器抽取2~3岁蒙古羊颗粒细胞,分为对照组和试验组,对照组不添加药物,试验组添加15 μmol/L LY294002处理颗粒细胞,分别在24、48和72 h测定细胞活力。通过实时荧光定量PCR技术检测母羊卵巢组织和颗粒细胞内ADAMTS1、PI3K、PTEN、Akt、RPS6、Bcl-2和BAX基因的相对表达量;通过Western blotting技术检测卵巢和颗粒细胞内ADAMTS1、PI3K和Akt蛋白的表达丰度。【结果】实时荧光定量PCR结果显示,双胎发情期组ADAMTS1、Bcl-2基因表达量均显著高于其余各组(P＜0.05);双胎发情期组和双胎间情期组PI3K、Akt和PTEN基因表达量均显著高于单胎发情期组和单胎间情期组(P＜0.05);单胎间情期组RPS6基因表达量显著低于其余各组(P＜0.05);间情期与发情期相比,BAX基因表达显著上调(P＜0.05)。Western blotting 结果显示,双胎发情期ADAMST1蛋白表达量显著高于其余各组(P＜0.05);双胎组与单胎组相比,PI3K和Akt蛋白均表达上调,其中,双胎发情期和双胎间情期PI3K蛋白表达量均显著高于单胎发情期组和单胎间情期组(P＜0.05),双胎间情期Akt蛋白表达量最高,显著高于其余各组(P＜0.05)。与对照组相比,试验组蒙古羊颗粒细胞形态异常,细胞增殖缓慢,且ADAMTS1、PI3K和Akt基因和蛋白的相对表达量均显著降低(P＜0.05)。【结论】ADAMTS1、PI3K和Akt基因和蛋白在蒙古羊卵巢和颗粒细胞内的表达趋势一致,呈正向相关关系,证明ADAMTS1基因PI3K/Akt轴在蒙古羊产双胎性状中发挥重要作用。

关键词: 蒙古羊; ADAMTS1基因; PI3K/Akt通路; 双胎; 单胎; 发情周期

Abstract: 【Objective】 This study was aimed to analyze the expression pattern of a disintegrin and metalloproteinase with thrombospondin motifs 1 (ADAMTS1) on PI3K/Akt pathway in ovary of twin and singleton Mongolian sheep at different stages of estrous cycle,and explore the mechanism of ADAMTS1 gene influencing reproductive traits in Mongolian sheep.【Method】 A total of 40 Mongolian sheep with twin and single births were selected for natural estrus,ewe estrus and diestrus periods were determined.Groups of 10 Mongolian sheep each in twin estrus,twin diestrus,single estrus and single diestrus groups. The granulosa cells of 2-3 years old Mongolian sheep were extracted by syringe,which were divided into control and experiment groups.No drug added to granulosa cells in control group,and 15 μmol/L LY294002 added to granulosa cells in experiment group,the cell viability were measured at 24,48 and 72 h.The relative expression of ADAMTS1,PI3K,PTEN,Akt,RPS6,Bcl-2 and BAX genes in ovary and granulosa cells of ewe were detected by Real-time quantitative PCR.The expression of ADAMTS1,PI3K and Akt proteins in ovary and granulosa cells were detected by Western blotting.【Result】 The results of Real-time quantitative PCR showed that the expression of ADAMTS1 and Bcl-2 genes in twin estrus group were significantly higher than that in other groups (P＜0.05).The expression of PI3K,Akt and PTEN genes in twin estrus and twin diestrus groups were significantly higher than that in single estrus and single diestrus groups (P＜0.05).The expression of RPS6 gene in single diestrus group was significantly lower than that in other groups (P＜0.05).Compared with estrus,the expression of BAX gene was significantly upregulated during diestrus (P<0.05).Western blotting results showed that the expression of ADAMST1 protein in twin estrus group was significantly higher than that in other groups (P＜0.05).Compared with single group,the expression of PI3K and Akt proteins in twin group were upregulated,the expression of PI3K protein in twin estrus and twin diestrus were significantly higher than that in single estrus and single diestrus groups (P＜0.05),and the expression of Akt protein in twin estrous was the highest,which were significantly higher than that in other groups (P＜0.05).Compared with control group,the granulosa cells in experiment group of Mongolian sheep had abnormal morphology and slow proliferation,and the relative expression of ADAMTS1, PI3K and Akt genes and proteins were significantly decreased (P＜0.05).【Conclusion】 The expression trends of ADAMTS1,PI3K and Akt genes and proteins were consistent and showed a positive correlation in ovary and granulosa cells of Mongolian sheep,suggested that ADAMTS1 gene played an important role in twin birth of Mongolian sheep mediated by PI3K/Akt signaling pathway.

Key words: Mongolian sheep; ADAMTS1 gene; PI3K/Akt pathway; twin; single; estrus cycle

中图分类号:

S814.8

陈晓良, 赵梦瑶, 李秀男, 苏乐德, 田瑛, 菅瑞珍, 巴音吉日嘎拉, 杨燕燕. 基于PI3K/Akt通路探究蒙古羊ADAMTS1基因的作用机制[J]. 中国畜牧兽医, 2023, 50(9): 3459-3468.

CHEN Xiaoliang, ZHAO Mengyao, LI Xiunan, SU Lede, TIAN Ying, JIAN Ruizhen, BAYIN Jiri Gala, YANG Yanyan. Study on the Mechanism of ADAMTS1 Gene in Mongolian Sheep Based on PI3K/Akt Pathway[J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(9): 3459-3468.

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