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染料木黄酮对青春期前雄性大鼠生殖系统影响的机制研究

Mechanism of Effects of Genistein on the Reproductive System of Prepubertal Male Rats

    摘要
  • 摘要:
      目的  研究染料木黄酮（genistein，GEN）对青春期前雄性大鼠生殖系统影响的机制。
      方法  选用30只初断乳（21日龄）SPF级雄性SD大鼠，随机分为对照组（Con组）、GEN低剂量组（G1组）、GEN高剂量组（G2组），每组10只。根据大鼠体质量，分别以5 mL/kg玉米油、150 mg/kg和300 mg/kg用玉米油溶解的GEN灌胃6周，隔天称取体质量1次。6周后处死大鼠，解剖睾丸、附睾、前列腺组织，计算脏器系数；观察睾丸组织病理结构变化；计数精子数量并计算精子畸形率；放射免疫法检测血清中睾酮和雌二醇的质量浓度；免疫荧光技术检测睾丸组织中蛋白磷酸酶2调节亚基2C（PPP2R2C）蛋白的表达定位；分别采用实时荧光定量PCR（RT-qPCR）和蛋白质印迹法（Western blot）检测大鼠睾丸组织中PPP2R2C和细胞周期依赖性蛋白激酶（CDK2）的mRNA和蛋白表达水平；免疫共沉淀法检测睾丸组织中蛋白磷酸酶2A（PP2A）活性。
      结果  各组大鼠体质量、精子数量、血清雌二醇、PP2A酶活性差异均无统计学意义（P>0.05）；G2组睾丸组织的病理结构紊乱；G1和G2组的精子畸形率高于Con组（P<0.05）；G2组的血清睾酮质量浓度低于Con组（P<0.05）；G2组PPP2R2C和CDK2的mRNA水平高于Con组（P<0.05），但蛋白水平低于Con组（P<0.05）；PPP2R2C蛋白在各组睾丸组织中均有表达。
      结论  青春期前长期暴露高剂量（300 mg/kg）GEN会对雄性大鼠成年后的生殖功能造成不良影响。由于在PP2A酶活性没有改变的情况下，磷酸化CDK2（p-CDK2）蛋白的表达量出现下降，PPP2R2C-PP2A-CDK2磷酸化路径可否影响大鼠的生殖系统仍需进一步研究。

     

    Abstract:
      Objective  To study the effects of genistein (GEN) on reproductive system in prepubertal male rats.
      Methods  Thirty SPF-rated male SD rats were randomly divided into control group (Con group), low-dose group (G1 group) and high-dose group (G2 group), with 10 rats in each group. Corn oil, 150 mg/kg and 300 mg/kg GEN dissolved in corn oil of equal volume were respectively administered every day and weighed the next day. After 6 weeks, the rats were sacrificed, and the testis, epididymis and prostate were dissected, and organ coefficients were calculated. Histopathological changes of testis was observed. The number of sperm was counted and the rate of sperm malformation was calculated. The concentrations of serum testosterone and estradiol were detected by radioimmunoassay. The protein phosphatase 2, regulatory subunit B, gamma (PPP2R2C) protein expression in testicular tissue was detected by immunofluorescence assay. The mRNA and protein expression levels of PPP2R2C and cyclin dependent protein kinases 2 (CDK2) in rat testis were detected by real-time quantitative fluorescence PCR (RT-qPCR) and Western blot, respectively. The protein phosphatase 2A (PP2A) activity in testicular tissue was detected by immunoprecipitation.
      Results  There were no statistically significant differences in body mass, sperm number, serum estradiol and PP2A enzyme activity among the groups (P>0.05). The pathological structure of testicular in G2 group was disordered. Sperm abnormality rate in G1 and G2 groups was higher than that in Con group (P<0.05). Serum testosterone concentration in G2 group was lower than that in Con group (P<0.05). The expression of PPP2R2C and CDK2 in G2 group was higher than that in Con group (P<0.05), but the protein level was lower than that in Con group (P<0.05). PPP2R2C protein was expressed in testicular tissue in each group.
      Conclusion  Long-term exposure to high dose (300 mg/kg) GEN during prepuberty may cause adverse effects on reproductive function in adult male rats. Further investigation is needed to determine whether PPP2R2C-PP2A-CDK2 phosphorylation pathway affects reproductive system in rats.

     

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