氟他胺致大鼠隐睾模型的睾丸组织形态改变及其病理基础

发布时间：2018-10-29 09:07

【摘要】：目的:研究抗雄激素受体氟他胺(Flutamide,Flu)孕中、晚期诱导子代雄鼠发生隐睾,及其病理组织学损害,探讨其不育的病理学基础,为临床治疗隐睾所致非梗阻性不育提供依据。方法:将20只SD(sprague dawley)孕鼠随机分为Flu隐睾组(n=10)、正常对照组(n=10),Flu隐睾组于妊娠12~21 d给予Flu 25 mg/(kg·d)皮下注射,正常对照组不予任何处理,取各组出生后第60天(postnatal day 60,PND60)的雄性SD大鼠睾丸组织(Flu隐睾组只取隐睾睾丸),HE染色观察睾丸组织形态学的差异,透射电镜观察睾丸支持细胞间的紧密连接结构,TUNEL凋亡染色检测生精细胞凋亡情况,取附睾尾进行精子计数及形态观测,免疫组织化学和Western blot检测睾丸组织中生殖细胞增殖分化指标视黄酸刺激因子8(stimulated by retinoic acid gene 8,Stra8)、联会复合体蛋白3(synaptonemal complex protein 3,SCP3)的表达,采用Q-PCR检测Stra8基因水平。结果:收集正常对照组正常睾丸30只与Flu隐睾组隐睾睾丸22只,HE染色显示隐睾睾丸管腔明显缩窄、生精细胞发育迟缓且排列紊乱、管腔中央无精子形成;TUNEL凋亡检测证实隐睾组有大量生精细胞发生凋亡,精子计数结果为隐睾组(1.99±0.13)×108个/m L,远低于正常对照组[(5.53±0.17)×108个/m L,P=0.000];透射电镜(transmission electron microscope,TEM)可观察到SD大鼠隐睾支持细胞间的紧密连接结构疏松;免疫组织化学显示Stra8在隐睾组织中表达较正常对照组少,SCP 3在正常对照组睾丸生精小管的各级生精细胞胞核中均有表达,而在隐睾组睾丸中表达很弱;Western blot结果显示,Flu隐睾组Stra8蛋白表达量(0.34±0.05)明显低于正常对照组(0.96±0.09),P=0.002;Flu隐睾组SCP3蛋白表达量(0.39±0.03)也明显低于正常对照组(0.97±0.07),P=0.001。Q-PCR结果显示,Flu组SD大鼠睾丸组织内Stra8 m RNA的表达(0.765±0.015)较正常对照组(1.00±0.01)低,P=0.01。结论:Flu诱导的SD大鼠隐睾模型中,睾丸呈明显病理形态学改变,支持细胞间紧密连接结构破坏,Stra8及SCP3表达明显下调,生精细胞凋亡增多,精子数量及质量下降,这可能是导致生精细胞发育障碍的重要原因。
[Abstract]:Objective: to study the histopathological damage of cryptorchidism in male offspring induced by antiandrogen receptor flutamide (Flutamide,Flu) in pregnancy and to explore the pathological basis of sterility in order to provide evidence for clinical treatment of non-obstructive sterility induced by cryptorchidism. Methods: twenty pregnant rats with SD (sprague dawley) were randomly divided into Flu cryptorchidism group (n = 10). The normal control group (n = 10), Flu) received subcutaneous injection of Flu 25 mg/ (kg d) on the 21st day of gestation, while the normal control group did not receive any treatment. The testicular tissues of male SD rats (Flu cryptorchidism group) were collected on the 60th day after birth (Flu cryptorchidism group) to observe the difference of testicular morphology with), HE staining, and the tight junctional structure of testicular Sertoli cells was observed by transmission electron microscope. The apoptosis of spermatogenic cells was detected by TUNEL apoptosis staining, sperm count and morphology were observed from epididymal tail, and Retinoic acid stimulating factor (8 (stimulated by retinoic acid gene 8 Stra8) was detected by immunohistochemistry and Western blot. The expression of synaptophysin 3 (synaptonemal complex protein 3 was detected by Q-PCR. Results: 30 normal testis in normal control group and 22 cryptorchidism testis in Flu cryptorchidism group were collected. HE staining showed that testicular lumen of cryptorchidism was obviously constricted, spermatogenic cells were slow and disordered, and azoospermia was formed in the central lumen. TUNEL apoptosis assay confirmed that a large number of spermatogenic cells were apoptotic in cryptorchidism group. The sperm count in cryptorchidism group was (1.99 卤0.13) 脳 108 / mL, which was much lower than that in normal control group [(5.53 卤0.17) 脳 108 / mL]. Transmission electron microscopy (transmission electron microscope,TEM) showed that the tight junctional structure of cryptorchidism Sertoli cells in SD rats was loose. Immunohistochemistry showed that the expression of Stra8 in cryptorchidism was less than that in control group. The expression of SCP 3 was found in the nucleus of spermatogenic cells of testicular seminiferous tubule in normal control group, but was weak in cryptorchidism testis. Western blot results showed that the expression of Stra8 protein in Flu cryptorchidism group (0.34 卤0. 05) was significantly lower than that in normal control group (0. 96 卤0. 09). The expression of SCP3 protein in Flu cryptorchidism group (0.39 卤0.03) was also significantly lower than that in normal control group (0.97 卤0.07). The expression of Stra8 m RNA in testis of SD rats in Flu group (0.765 卤0.015) was lower than that in normal control group (1.00 卤0.01). Conclusion: in the SD rat model of cryptorchidism induced by Flu, the testis showed obvious pathomorphological changes, the structure of supporting cell tight junction was destroyed, the expression of Stra8 and SCP3 was down-regulated, the apoptosis of spermatogenic cells was increased, and the quantity and quality of spermatozoa were decreased. This may be an important factor in the development of spermatogenic cells.
【作者单位】：重庆医科大学附属儿童医院泌尿外科儿童发育疾病研究教育部重点实验室儿科学重庆市重点实验室重庆市儿童发育重大疾病诊治与预防国际科技合作基地;
【基金】：国家自然科学基金资助项目(编号:81100415) 教育部博士点基金资助项目(编号:20115503120009)
【分类号】：R-332;R726.9

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