G-蛋白偶联雌激素受体在肾阴虚和肾阳虚小鼠睾丸中的表达及其与生殖功能的关系

Expression of G-protein coupled estrogen receptor in the testis of the male mouse with kidney yin or kidney yang deficiency and its impact on the reproductive function of the mouse

目的:探讨肾阴虚与肾阳虚雄性小鼠睾丸G-蛋白偶联雌激素受体(GPER)的定位与表达,及其对肾阴虚与肾阳虚雄性小鼠生殖功能的影响。方法:将6周龄雄性昆明小鼠随机分为正常组、肾阳虚组和肾阴虚组。采用矿场实验、高架十字迷宫、游泳力竭等评价小鼠精神萎靡程度和自主活动次数等行为学改变;电化学发光法检测睾酮(T)和雌二醇(E2),并计算T/E2;全自动精子分析仪检测精液质量;与雌鼠合笼后记录雌鼠产仔数,计算雄鼠平均育仔数;免疫组织化学和免疫荧光染色检测GPER在小鼠睾丸中的定位和表达。结果:与肾阴虚组比较,肾阳虚组小鼠开臂进入次数百分率和中央区进入次数明显减少,同时,游泳力竭时间缩短更为明显(P<0.05),但开臂滞留和中央区滞留时间百分率无明显差异(P>0.05)。与肾阴虚组比较,肾阳虚组小鼠附睾精子计数和活动精子百分率明显减少,且育仔数显著下降(P<0.05);精子畸形率略有升高(P>0.05);血清T水平明显降低(P<0.05),E2水平略有下降(P>0.05),T/E2明显下降(P<0.05)。肾阴虚和肾阳虚小鼠睾丸GPER均表达于睾丸间质细胞的细胞质内,细胞核和细胞膜表达为阴性,肾阳虚小鼠睾丸GPER的表达显著高于肾阴虚组(P<0.05)。结论:肾阳虚与肾阴虚小鼠均出现精子数量与育仔数下降,精子畸形率增加,但以肾阳虚小鼠更为明显,这种改变可能与肾阳虚小鼠睾丸GPER表达增加有关,从而造成血清T水平及T/E2比值下降。

Objective： To investigate the expression of the G-protein coupled estrogen receptor （GPER） in the testis of the male mouse with kidney yin or kidney yang deficiency and its influence on the reproductive function of the mouse. Methods ： We ran- domized 30 six-week-old male Kunming mice into three groups of equal number： kidney yang deficiency, kidney yin deficiency, and normal control, and established the models of kidney yang deficiency and kidney yin deficiency by peritoneal injection of hydrocortisone at 50 mg/kg for 5 days and 25 mg/kg for 10 days, respectively. We observed the behavioral changes of the mice using the elevated plus-maze, exhaustive swimming and field experiment, examined the semen quality with the automatic sperm quality analyzer, calculat- ed the average number of the offspring, measured the serum testosterone （T） and estradiol （E2） levels and T/E2 ratio by Roche elec- trochemiluminescence assay, and determined the localization and expression of GPER in the testis by immunohistochemistry and immu- no fluorescence staining. Results ： Compared with the mice with kidney yin deficiency, those with kidney yang deficiency showed re- markably fewer entries into the open arm and central area （ P 〈 0.05 ） and shorter time of exhaustive swimming （ P 〈 0.05 ） , but no statistically significant difference in the time spent in the open arm or the central area （ P 〉 0.05 ） ; the latter group also exhibited sig- nificant decreases in the epididymal sperm count （ [ 7.27± 1.30 ] vs [ 3.05 ±1.06 ] × 108/g, P 〈 0.01 ）, sperm motility （[54.15±13.52] vs [51.57 ± 8.75] %, P 〈0.01） and average number of the offspring （6.46vs4.33, P 〈0.05）, a slight increase in the rate of morphologically abnormal sperm （ [ 13.42± 2.321 vs [ 15.39 ±2.48] %, P 〉0.05）, and markedly re- duced serum T （[24.96±6.18] vs [16.72 ± 5.921 ng/dl,P 〈0.05）, E2 （[19.81 ±4.01] vs [15.24 ±1.111 pg/ml,P 〈 0.05） and T/E2 ratio （ 1.41 vs 1.25, P 〈 0.05）. The expression of GPER was found in the cytoplasm of the Leydig cells, negative in the nuclei and cell membrane, significantly higher in the kidney yang than in the kidney yin deficiency group （ P 〈 0.05 ）. Conclusion ： The numbers of sperm and offspring decreased while the percentage of morphologically abnormal sperm increased in both the kidney yang and kidney yin deficiency mice, even more significantly in the former, which might be associated with the up-regulated ex- pression of GPER in the testis of the mouse with kidney yang deficiency and consequently the reduced serum T level and T/E2 ratio.