ATF3在糖皮质激素与邻苯二甲酸二丁酯联合作用致大鼠尿道下裂发生中的表达变化
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摘要
目的:利用糖皮质激素与邻苯二甲酸二丁酯(DBP)联合作用致大鼠尿道下裂,验证转录激活因子3(ATF3)在正常大鼠生殖结节(GT)与不同严重程度尿道下裂大鼠生殖结节中的差异表达,以强调组合因素在尿道下裂病因中的重要性,并探讨ATF3在尿道下裂发生中的作用机制。
方法:SD孕鼠80只,随机分为4组,每组20只,在妊娠14～18天(GD14～18)分别给予:A组:大豆油2ml/d灌胃;B组:地塞米松(DXM)0.1mg/(kg·d)皮下注射;C组:DBP700mg/(kg·d)灌胃;D组:地塞米松0.1mg/(kg·d)皮下注射加DBP700mg/(kg·d)灌胃。于GD19将各组半数孕鼠处死剖腹统计仔鼠数、雄性仔鼠出生体重(BW)及肛门生殖器间距离(AGD),取雄性仔鼠生殖结节,运用免疫印迹法和免疫组织化学方法分析ATF3的表达情况,剩余孕鼠自然分娩后,于出生后第70天(PND70)观察统计尿道下裂和隐睾的发生情况。
结果:地塞米松单独作用于孕鼠仅表现为后代仔鼠出生体重的下降,但与DBP组合作用却能显著增强DBP的致畸作用,导致后代雄性仔鼠出生体重及肛门生殖器间距离/出生体重(AGD/BW)明显减少,且尿道下裂发生率、严重程度以及隐睾发生率较DBP单因素作用下更为显著,统计显示隐睾在C组和D组的发生率分别为:58%和88%,尿道下裂的在C组和D组的发生率分别为:37%和60%,而尿道下裂严重程度则根据出生后70天(PND70)大鼠外生殖器观察统计。蛋白质组学结果显示,ATF3在A组的蛋白相对表达量为0.351±0.012(n=10),B组为0.336±0.015(n=10),C组为0.603±0.014(n=10),D组为0.851±0.016(n=10),C组或D组与A组,以及C组与D组之间的差异均有统计学意义(P<0.05);免疫组化结果显示ATF3主要定位于生殖结节的尿道上皮和侧翼间质,C组和D组免疫组化染色强度明显强于A组,而D组染色强于C组。
结论:地塞米松联合DBP进一步增强了DBP的致畸作用,表现为后代雄性仔鼠出生体重及肛门生殖器间距离/出生体重(AGD/BW)明显减少,且尿道下裂发生率、严重程度以及隐睾发生率较DBP单因素作用下更为显著,提示我们尿道下裂的发生是环境中多因素协同作用下的结果;ATF3在对照组与实验组组生殖结节中的表达有明显变化,且随着尿道下裂程度的加重而表达增高,ATF3的高表达影响了生殖结节的发育及尿生殖褶的融合,这可能是尿道下裂发生的机制之一。
Objective To verify the differential expression of ATF3 in the genital tubercle(GT) of hypospadiac rats induced by maternal exposure to DBP or DBP+DXMamethasone (DXM) in order to stress the importance of combination of factors rather than single angents and to further explore the mechanism of hypospadias.
Methods Eighty pregnant SD rats were randomly divided into four groups which were given: Group A: soybean oil oral gavage 2ml/d; Group B: DXM sc-injection 0.1mg/(kg·d); Group C: DBP oral gavage 700 mg/(kg·d); Group D: DBP(700mg/kg gavage)+DXM(0.1mg/kg sc-injection) during GD14-18. Half of the pregnant rats were killed at GD19, we recorded fetal rats’number, birth weight (BW), anogenital distance (AGD) and harvested GT to verify the expression of ATF3 by Western blotting and immunohistochemistry. The rest spontaneous labor rats were used to record the incidence of hypospadias and cryptorchidism at PND70.
Results DXM alone had no effect except to reduce BW but can increase the teratogenic effect of DBP when combined with it. The BW and AGD/BW of group D was significantly lower than group C, and the incidence of hypospadias and cryptor- chidism vice versa. The incidence of cryptorchidism in group C was 58%,in group D was 88%. The incidence of hypospadias in group C was 37%,in group D was 60%. And the severity of hypospadias is according to PND70 rats genitals observation statistics. The expression of ATF3 in group A was:0.351±0.012(n=10), in group B was: 0.336±0.015(n=10), in group C was: 0.603±0.014(n=10), in group D was: 0.851±0.016(n=10), it had statistical difference between group C or D with A, as well as C with D (P<0.05). ATF3 was mainly located in the urethral epithelium and mesenchy- me, the result of staining intensity was in common with western blotting.
Conclusion These results suggests that exposure to common environmental chemicals in combination with environmental endocrine disruptors, may increase the risk of male reproductive abnormalities, such as hypospadias. Up-regulation of ATF3 may affect urethral fold fusion, then lead to the occurrence of hypospadias.
方法:SD孕鼠80只,随机分为4组,每组20只,在妊娠14～18天(GD14～18)分别给予:A组:大豆油2ml/d灌胃;B组:地塞米松(DXM)0.1mg/(kg·d)皮下注射;C组:DBP700mg/(kg·d)灌胃;D组:地塞米松0.1mg/(kg·d)皮下注射加DBP700mg/(kg·d)灌胃。于GD19将各组半数孕鼠处死剖腹统计仔鼠数、雄性仔鼠出生体重(BW)及肛门生殖器间距离(AGD),取雄性仔鼠生殖结节,运用免疫印迹法和免疫组织化学方法分析ATF3的表达情况,剩余孕鼠自然分娩后,于出生后第70天(PND70)观察统计尿道下裂和隐睾的发生情况。
结果:地塞米松单独作用于孕鼠仅表现为后代仔鼠出生体重的下降,但与DBP组合作用却能显著增强DBP的致畸作用,导致后代雄性仔鼠出生体重及肛门生殖器间距离/出生体重(AGD/BW)明显减少,且尿道下裂发生率、严重程度以及隐睾发生率较DBP单因素作用下更为显著,统计显示隐睾在C组和D组的发生率分别为:58%和88%,尿道下裂的在C组和D组的发生率分别为:37%和60%,而尿道下裂严重程度则根据出生后70天(PND70)大鼠外生殖器观察统计。蛋白质组学结果显示,ATF3在A组的蛋白相对表达量为0.351±0.012(n=10),B组为0.336±0.015(n=10),C组为0.603±0.014(n=10),D组为0.851±0.016(n=10),C组或D组与A组,以及C组与D组之间的差异均有统计学意义(P<0.05);免疫组化结果显示ATF3主要定位于生殖结节的尿道上皮和侧翼间质,C组和D组免疫组化染色强度明显强于A组,而D组染色强于C组。
结论:地塞米松联合DBP进一步增强了DBP的致畸作用,表现为后代雄性仔鼠出生体重及肛门生殖器间距离/出生体重(AGD/BW)明显减少,且尿道下裂发生率、严重程度以及隐睾发生率较DBP单因素作用下更为显著,提示我们尿道下裂的发生是环境中多因素协同作用下的结果;ATF3在对照组与实验组组生殖结节中的表达有明显变化,且随着尿道下裂程度的加重而表达增高,ATF3的高表达影响了生殖结节的发育及尿生殖褶的融合,这可能是尿道下裂发生的机制之一。
Objective To verify the differential expression of ATF3 in the genital tubercle(GT) of hypospadiac rats induced by maternal exposure to DBP or DBP+DXMamethasone (DXM) in order to stress the importance of combination of factors rather than single angents and to further explore the mechanism of hypospadias.
Methods Eighty pregnant SD rats were randomly divided into four groups which were given: Group A: soybean oil oral gavage 2ml/d; Group B: DXM sc-injection 0.1mg/(kg·d); Group C: DBP oral gavage 700 mg/(kg·d); Group D: DBP(700mg/kg gavage)+DXM(0.1mg/kg sc-injection) during GD14-18. Half of the pregnant rats were killed at GD19, we recorded fetal rats’number, birth weight (BW), anogenital distance (AGD) and harvested GT to verify the expression of ATF3 by Western blotting and immunohistochemistry. The rest spontaneous labor rats were used to record the incidence of hypospadias and cryptorchidism at PND70.
Results DXM alone had no effect except to reduce BW but can increase the teratogenic effect of DBP when combined with it. The BW and AGD/BW of group D was significantly lower than group C, and the incidence of hypospadias and cryptor- chidism vice versa. The incidence of cryptorchidism in group C was 58%,in group D was 88%. The incidence of hypospadias in group C was 37%,in group D was 60%. And the severity of hypospadias is according to PND70 rats genitals observation statistics. The expression of ATF3 in group A was:0.351±0.012(n=10), in group B was: 0.336±0.015(n=10), in group C was: 0.603±0.014(n=10), in group D was: 0.851±0.016(n=10), it had statistical difference between group C or D with A, as well as C with D (P<0.05). ATF3 was mainly located in the urethral epithelium and mesenchy- me, the result of staining intensity was in common with western blotting.
Conclusion These results suggests that exposure to common environmental chemicals in combination with environmental endocrine disruptors, may increase the risk of male reproductive abnormalities, such as hypospadias. Up-regulation of ATF3 may affect urethral fold fusion, then lead to the occurrence of hypospadias.
引文
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