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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。
OBJECTIVE: To study the pathogenesis of cryptorchidism in male and female offspring of middle and late pregnancy induced by androgen receptor Flutamide (Flu), and to explore its pathological basis for infertility and clinical treatment of cryptorchidism As a result of non-obstructive infertility provided basis. Methods: Twenty pregnant rats with sprague dawley were randomly divided into three groups: Flu cryptorchidism group (n = 10), normal control group (n = 10), Flu cryptorchidism group Flu 25 mg / kg · d) were injected subcutaneously, without any treatment in the normal control group. Male SD rats testis (Flu cryptorchidism test only testicular cryptococcal testis) were collected on the 60th postnatal day 60 (PND60) The morphology of testes was observed by transmission electron microscopy. The tight junctions between testicular supporting cells were observed by transmission electron microscopy. Apoptosis of spermatogenic cells was detected by TUNEL staining. Sperm count and morphology were observed by epididymal tail. Immunohistochemistry and Western blot The expression of germinal cell proliferation and differentiation testis in testis tissue was determined by Q-PCR. The expression of Stra8, Stra8 and synaptonemal complex protein 3 (SCP3) . Results: Twenty normal testes and 22 cryptorchid testis were collected from normal testes. Hematoxylin and eosin (HE) staining showed that the lumen of cryptorchid testis was significantly narrowed, the spermatogenic cells were delayed and disordered, and there was no spermatogenesis in central lumen. TUNEL Apoptosis test showed that a large number of spermatogenic cells in cryptorchid group were apoptosis, the sperm counting results were cryptorchidism group (1.99 ± 0.13) × 108 / m L, much lower than the normal control group (5.53 ± 0.17) × 108 / m L, P = 0.000]. Transmission electron microscopy (TEM) showed that the tight junctions between cryptorchid cells in SD rats were loose. Immunohistochemistry showed that the expression of Stra8 in cryptorchidism tissues was significantly higher than that in normal control group , SCP 3 was expressed in nucleus of spermatogenic cells at all levels of testicular seminiferous tubules in normal control group and weakly in testis of cryptorchid testis group. Western blot results showed that the expression of Stra8 protein in Flu cryptorchidism group ( 0.34 ± 0.05) was significantly lower than that of the normal control group (0.96 ± 0.09), P = 0.002. The expression of SCP3 protein in Flu cryptorchidism group was also significantly lower than that of the normal control group (0.97 ± 0.07, P = 0.001). The results of Q-PCR showed that the expression of Stra8 m RNA in testis of Flu rats was 0.765 ± 0.015, which was lower than that of control group (1.00 ± 0.01), P = 0.01. CONCLUSIONS: In the rat model of cryptorchidism induced by Flu, the pathological changes of the testes showed obvious morphological changes in the testes, which supported the destruction of the tight junctions between the cells and the down-regulation of the expression of Stra8 and SCP3, increased apoptosis of spermatogenic cells and decreased sperm count and quality May lead to spermatogenic cell dysplasia is an important reason.