雌激素及相关因子对肾阳虚模型大鼠精子发生的影响
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摘要
目的:
观察不同剂量腺嘌呤诱导的肾阳虚不育模型大鼠血清雌二醇(Estradiol, E2)水平及相关因子芳香化酶P450 (Aromatase cytochrome, P450arom)、细胞色素P-19 (Cytochrome P-19, CYP19)、转化生长因子1 (Transforming growth facter beta1, TGF-β1)在睾丸中的定位及表达,探讨肾阳虚致精子发生障碍的病理机制。
方法:
将32只成年雄性SD大鼠随机分为4组(低、中、高剂量组,正常对照组),每组8只。分别用腺嘌呤100mg/kg·d、150 mg/kg.、200mg/kg·d,给低、中、高剂量组大鼠灌胃30天,诱导肾阳虚不育模型;正常对照组给与0.9%的生理盐水灌胃30天。观察大鼠体征变化,用放射免疫法测定血清性激素水平,生化检测方法检测血清中尿素氮(Blood urea nitrogen, BUN)、肌酐(Serum creatinine, SCr),全自动精子分析仪进行精子计数,HE染色观察大鼠睾丸、肾脏的形态学改变;免疫组织化学SABC法及Western blot方法检测P450arom、CYP19和TGF-β1在大鼠睾丸中的定位及表达。
结果:
与正常对照组相比,各模型组睾酮(Testosterone , T)、雌二醇(Estradiol, E2)显著降低(P<0.05);卵泡刺激素(Follicle-stimulating hormone, FSH )、促黄体激素(Luteotrophic hormone, LH)水平无显著性差异(P>0.05);SCr、BUN水平显著升高(P<0.05);精子数目明显减少(P<0.05);而在不同模型组之间,其变化均无显著性差异(P>0.05)。各模型组大鼠睾丸均出现了生精小管中生精细胞排列紊乱,细胞疏松,生精小管之间的结缔组织增多,间质细胞数量明显减少,管腔内生殖上皮变薄,精子数目减少,大部分管腔中可见脱落的精子细胞;肾小管内有大量结晶物沉着,且有异物肉芽肿形成,占据整个管腔,肾小管上皮肿胀、坏死、脱落,肾小管显著扩张成囊状,皮质、髓质间质纤维组织增生,大量炎性细胞浸润,可见纤维素样坏死;且随着腺嘌呤给药剂量的增加,肾脏、睾丸的损害呈进行性加重。CYP19主要在睾丸的间质细胞、长形精子细胞表达;P450arom、TGF-β1主要表达在睾丸的间质细胞。与正常对照组相比,模型组大鼠睾丸中TGF-β1的表达显著增强(P<0.05),而CYP19、P450arom的表达则明显降低(P<0.05);三个模型组之间相比,TGF-β1、CYP19、P450arom的表达均无显著性差异(P>0.05)。
结论:
不同剂量腺嘌呤诱导的大鼠肾阳虚模型均出现了血清E2水平降低及精子数目的减少,同时伴有睾丸TGF-β1、CYP19和P450arom的表达改变,初步说明雌激素及相关因子与肾阳虚引起的精子发生障碍有关。
Objective
To observe the level of estradiol (E2) in serum by different doses of adenine on infertile rats with kidney-yang deficiency and the localization and expression of P450arom、CYP19 and TGF-β1 in the testis of infertile rats.To investigate the pathological mechanism of spermatogenesis caused by kidney-yang deficiency.
Methods
32 SD male rats were averagely randomized into 4 groups: normal control group and groups treated with low (100 mg/kg daily), middle (150 mg/kg daily) and high (200 mg/kg daily) dosage of adenine. The rat model of kidney-yang deficiency infertility was induced by intragastric injection of adenine for 30 days. Symptoms, signs and serum levels of sex hormones, sperm count with automatic sperm analyzer ,blood urea nitrogen (BUN) and serum creatinine (SCr) as well as pathological changes of the testis and kidney were observed. The expression and change levels of P450arom,CYP19,TGF-β1 were measured with immunohistochemical SABC and Western blot methods.
Results
Compared with those of normal control group, levels of testosterone (T) and estradiol (E2) were significantly decreased in groups treated with low, middle and high dosage of adenine (P<0.05), levels of SCr and BUN were significantly increased (P<0.05), and the number of sperm was significantly reduced (P<0.05), while levels of follicle-stimulating hormone (FSH) and luteotrophic hormone (LH) did not show significant changes (P>0.05), but there are no significant changes in three model groups. Some pathological changes were found in the testis and the kidney of rats treated with different dosage of adenine. The array of spermatogenic cells in the testis was disorder, and spermatogenic cells became sparse. There was more connective tissue between contorted seminiferous tubules. The number of interstitial cells was obviously decreased. Germinal epithelium within the lumen became thinner. The number of sperms was decreased. Caducous spermatoblasts were found in bulk of lumens. Crystal object deposits were found in renal tubule. Foreign-body granuloma formed and occupied integral lumens. The endepidermis of renal tubule became swollen, necrotic and caducous, and renal tubule was capsularly broadened. Fibroplasia in the mesenchymal of the cortex and medulla as well as bulk of inflammatory cell infiltrate and fibrinoid necrosis were found.With the increase of adenine dose, the damage of kidney and testis was progressively increased. CYP19 was expressed in Leydig cells, elongating spermatids in rats’testis. P450arom and TGF-β1 were positive in Leydig’s cells. Compared with the normal control group, the expression of TGF-β1 was significantly higher in testis of model groups (P<0.05); the expression of CYP19 and P450arom was significantly lower (P<0.05). There was no significant difference (P>0.05) in the expression of TGF-β1, P450arom and CYP19 among three model groups.
Conclusion
The level of E2 in serum and sperm counts were decreased in different doses of adenine on infertile rats with kidney-yang deficiency, which accompanied the abnormal changes of P450arom, CYP19 and TGF-β1 in the model groups. It explained that estrogen and related factors may be related to the spermatogenesis with kidney-yang deficiency.
观察不同剂量腺嘌呤诱导的肾阳虚不育模型大鼠血清雌二醇(Estradiol, E2)水平及相关因子芳香化酶P450 (Aromatase cytochrome, P450arom)、细胞色素P-19 (Cytochrome P-19, CYP19)、转化生长因子1 (Transforming growth facter beta1, TGF-β1)在睾丸中的定位及表达,探讨肾阳虚致精子发生障碍的病理机制。
方法:
将32只成年雄性SD大鼠随机分为4组(低、中、高剂量组,正常对照组),每组8只。分别用腺嘌呤100mg/kg·d、150 mg/kg.、200mg/kg·d,给低、中、高剂量组大鼠灌胃30天,诱导肾阳虚不育模型;正常对照组给与0.9%的生理盐水灌胃30天。观察大鼠体征变化,用放射免疫法测定血清性激素水平,生化检测方法检测血清中尿素氮(Blood urea nitrogen, BUN)、肌酐(Serum creatinine, SCr),全自动精子分析仪进行精子计数,HE染色观察大鼠睾丸、肾脏的形态学改变;免疫组织化学SABC法及Western blot方法检测P450arom、CYP19和TGF-β1在大鼠睾丸中的定位及表达。
结果:
与正常对照组相比,各模型组睾酮(Testosterone , T)、雌二醇(Estradiol, E2)显著降低(P<0.05);卵泡刺激素(Follicle-stimulating hormone, FSH )、促黄体激素(Luteotrophic hormone, LH)水平无显著性差异(P>0.05);SCr、BUN水平显著升高(P<0.05);精子数目明显减少(P<0.05);而在不同模型组之间,其变化均无显著性差异(P>0.05)。各模型组大鼠睾丸均出现了生精小管中生精细胞排列紊乱,细胞疏松,生精小管之间的结缔组织增多,间质细胞数量明显减少,管腔内生殖上皮变薄,精子数目减少,大部分管腔中可见脱落的精子细胞;肾小管内有大量结晶物沉着,且有异物肉芽肿形成,占据整个管腔,肾小管上皮肿胀、坏死、脱落,肾小管显著扩张成囊状,皮质、髓质间质纤维组织增生,大量炎性细胞浸润,可见纤维素样坏死;且随着腺嘌呤给药剂量的增加,肾脏、睾丸的损害呈进行性加重。CYP19主要在睾丸的间质细胞、长形精子细胞表达;P450arom、TGF-β1主要表达在睾丸的间质细胞。与正常对照组相比,模型组大鼠睾丸中TGF-β1的表达显著增强(P<0.05),而CYP19、P450arom的表达则明显降低(P<0.05);三个模型组之间相比,TGF-β1、CYP19、P450arom的表达均无显著性差异(P>0.05)。
结论:
不同剂量腺嘌呤诱导的大鼠肾阳虚模型均出现了血清E2水平降低及精子数目的减少,同时伴有睾丸TGF-β1、CYP19和P450arom的表达改变,初步说明雌激素及相关因子与肾阳虚引起的精子发生障碍有关。
Objective
To observe the level of estradiol (E2) in serum by different doses of adenine on infertile rats with kidney-yang deficiency and the localization and expression of P450arom、CYP19 and TGF-β1 in the testis of infertile rats.To investigate the pathological mechanism of spermatogenesis caused by kidney-yang deficiency.
Methods
32 SD male rats were averagely randomized into 4 groups: normal control group and groups treated with low (100 mg/kg daily), middle (150 mg/kg daily) and high (200 mg/kg daily) dosage of adenine. The rat model of kidney-yang deficiency infertility was induced by intragastric injection of adenine for 30 days. Symptoms, signs and serum levels of sex hormones, sperm count with automatic sperm analyzer ,blood urea nitrogen (BUN) and serum creatinine (SCr) as well as pathological changes of the testis and kidney were observed. The expression and change levels of P450arom,CYP19,TGF-β1 were measured with immunohistochemical SABC and Western blot methods.
Results
Compared with those of normal control group, levels of testosterone (T) and estradiol (E2) were significantly decreased in groups treated with low, middle and high dosage of adenine (P<0.05), levels of SCr and BUN were significantly increased (P<0.05), and the number of sperm was significantly reduced (P<0.05), while levels of follicle-stimulating hormone (FSH) and luteotrophic hormone (LH) did not show significant changes (P>0.05), but there are no significant changes in three model groups. Some pathological changes were found in the testis and the kidney of rats treated with different dosage of adenine. The array of spermatogenic cells in the testis was disorder, and spermatogenic cells became sparse. There was more connective tissue between contorted seminiferous tubules. The number of interstitial cells was obviously decreased. Germinal epithelium within the lumen became thinner. The number of sperms was decreased. Caducous spermatoblasts were found in bulk of lumens. Crystal object deposits were found in renal tubule. Foreign-body granuloma formed and occupied integral lumens. The endepidermis of renal tubule became swollen, necrotic and caducous, and renal tubule was capsularly broadened. Fibroplasia in the mesenchymal of the cortex and medulla as well as bulk of inflammatory cell infiltrate and fibrinoid necrosis were found.With the increase of adenine dose, the damage of kidney and testis was progressively increased. CYP19 was expressed in Leydig cells, elongating spermatids in rats’testis. P450arom and TGF-β1 were positive in Leydig’s cells. Compared with the normal control group, the expression of TGF-β1 was significantly higher in testis of model groups (P<0.05); the expression of CYP19 and P450arom was significantly lower (P<0.05). There was no significant difference (P>0.05) in the expression of TGF-β1, P450arom and CYP19 among three model groups.
Conclusion
The level of E2 in serum and sperm counts were decreased in different doses of adenine on infertile rats with kidney-yang deficiency, which accompanied the abnormal changes of P450arom, CYP19 and TGF-β1 in the model groups. It explained that estrogen and related factors may be related to the spermatogenesis with kidney-yang deficiency.
引文
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