Liu Na-Chuan, Wang Yi-Zhou, Yao Chen-Cheng, Zhao Liang-Yu, Zhu Zi-Jue, Huang Yu-Hua, Zhi Er-Lei, Chen Hui-Xing, Tian Ru-Hui, Li Zheng
Department of Andrology, Center of Urology / Center for Men's Health, Shanghai Jiao Tong University Institute of Urology / Shanghai Key Lab of Reproductive Medicine, Shanghai General Hospital of Shanghai Jiao Tong University, Shanghai 200080, China.
Zhonghua Nan Ke Xue. 2018 Sep;24(9):811-818.
To investigate the effect of Qilin Pills (QP) in facilitating the recovery of spermatogenic function in azoospermia (AS) mice and to explore its mechanism of regulating testicular spermatogenesis.
Fifteen 4-week-old male mice were equally randomized into an AS model control, a low-dose QP and a high-dose QP group. The AS model was established in the mice by intraperitoneal injection of busulfan at 35 mg/kg. After modeling, the animals in the low- and high-dose QP groups were treated with Qilin Pills intragastrically at 2 000 and 8 000 mg/kg/d respectively while those in the model control group fed on a normal diet, all for 28 days. Then, all the mice were sacrificed for examination of the ultrastructures of the epididymis and testis by HE staining, detection of the specific markers of spermatogenic, Sertoli and Leydig cells by Western blot, and determination of the expressions of these markers in the testis tissue by immunofluorescence assay.
The number of spermatogenic cells in the testis tissue was significantly decreased in the AS model controls, with no spermatozoa in most of the seminiferous tubules in the epididymis (Johnsen's score: 5.2 ± 0.5). In the high-dose QP group, spermatogenic cells were tightly arranged with distinct layers in the seminiferous tubules, with a large number of spermatozoa but no non-sperm cells in the lumens of the epididymis (Johnsen's score: 9.4 ± 0.6). The number of spermatogenic cells in the testis was increased in the low-dose QP group with some spermatozoa in the seminiferous tubules as compared with that in the model control, but lower than in the high-dose group (Johnsen's score: 7.6 ± 0.6). The Johnsen's score was significantly lower in the model control than in the high- and low-dose QP groups (P < 0.01), and higher in the high-dose than in the low-dose QP group (P < 0.05). The expressions of the specific markers of Sertoli cells SCF, BMP4, SYCP3, DMC1 and Ki67 were also remarkably lower in the model control than in the high- and low-dose QP groups (P < 0.01), and higher in the high-dose than in the low-dose QP group (P < 0.05 or P < 0.01). No statistically significant differences were observed among the three groups of mice in the markers of spermatogonial stem cells (SSC) and undifferentiated SSCs UCHL1, STRA8, NGN3 and PLZF3 (P > 0.05). The expressions of the spermatocyte markers DMC1 and SYCP3 were markedly lower in the model control than in the high- and low-dose QP groups (P < 0.05 or P < 0.01), and higher in the high-dose than in the low-dose QP group (P < 0.05 or P < 0.01). The Ki67 fluorescence signals were distributed in the spermatogonia, with a higher intensity in the model control than in the high- and low-dose QP groups. The acrosome marker PNA was found mainly in the seminiferous tubules, with abundant fluorescence signals in the high- and low-dose QP groups but no obvious dot signals in the model controls.
Qilin Pills may contribute to the meiosis of spermatogonia and promote spermatogenesis by improving the function of Sertoli cells in the testis.
研究麒麟丸（QP）对无精子症（AS）小鼠生精功能恢复的影响，并探讨其调节睾丸生精的机制。
将15只4周龄雄性小鼠随机分为AS模型对照组、低剂量QP组和高剂量QP组，每组5只。采用腹腔注射白消安35mg/kg建立小鼠AS模型。造模后，低剂量QP组和高剂量QP组小鼠分别按2000mg/kg/d和8000mg/kg/d灌胃给予麒麟丸，模型对照组小鼠给予正常饮食，连续给药28天。然后，处死所有小鼠，通过HE染色观察附睾和睾丸的超微结构，采用蛋白质免疫印迹法检测生精细胞、支持细胞和间质细胞的特异性标志物，通过免疫荧光法测定这些标志物在睾丸组织中的表达。
AS模型对照组睾丸组织中生精细胞数量显著减少，附睾大部分生精小管内无精子（Johnsen评分：5.2±0.5）。高剂量QP组生精小管中生精细胞排列紧密，层次分明，附睾管腔内有大量精子，无非精子细胞（Johnsen评分：9.4±0.6）。低剂量QP组睾丸中生精细胞数量较模型对照组增多，生精小管内有少量精子，Johnsen评分高于模型对照组，但低于高剂量QP组（Johnsen评分：7.6±0.6）。模型对照组Johnsen评分显著低于高剂量和低剂量QP组（P<0.01），高剂量QP组高于低剂量QP组（P<0.05）。支持细胞特异性标志物SCF、BMP4、SYCP3、DMC1和Ki67在模型对照组中的表达也显著低于高剂量和低剂量QP组（P<0.01），高剂量QP组高于低剂量QP组（P<0.05或P<0.01）。三组小鼠精原干细胞标志物（SSC）和未分化精原干细胞UCHL1、STRA8、NGN3和PLZF3的表达差异无统计学意义（P>0.05）。精母细胞标志物DMC1和SYCP3在模型对照组中的表达显著低于高剂量和低剂量QP组（P<0.05或P<0.01），高剂量QP组高于低剂量QP组（P<0.05或P<0.01）。Ki67荧光信号分布于精原细胞，模型对照组荧光强度高于高剂量和低剂量QP组。顶体标志物PNA主要分布于生精小管，高剂量和低剂量QP组荧光信号丰富，模型对照组无明显点状信号。
麒麟丸可能通过改善睾丸支持细胞功能促进精原细胞减数分裂，从而促进生精。
