低剂量棉酚与甾体激素联合用药对大鼠生精细胞凋亡的影响

Effect of low dose gossypol combined with use of steroid hormone on the apoptosis of rat spermatogenic cells

采用检测细胞凋亡的微核 (MN)、原位末端标记检测 (ISEL)及单细胞凝胶电泳 (SCGE)等方法 ,研究低剂量棉酚与激素组合用药对大鼠生精细胞凋亡的影响 ,分析不同剂量棉酚 (高剂量 10 0mg/(kg·d)、中剂量 6 0mg/(kg·d)及低剂量 12mg/(kg·d) )及维持量棉酚对生精细胞凋亡及DNA的作用。结果表明不同剂量的棉酚G与单剂量激素H(甲基睾丸素 2 0mg/(kg·d)、炔雌醇 10 0 (g/(kg·d) )合并用药 6周后 ,生精细胞的凋亡系数和微核率随棉酚剂量的增大而增加。高和中剂量棉酚组的凋亡系数分别为 3 5 2 0± 0 4 6 5和 2 0 2 0± 0 0 17,显著高于对照组 (0 2 33± 0 0 88)(P <0 0 5 )。微核率在高剂量组为 2 7 6 33± 3 75 5 ,显著高于对照组 (5 6 0 0± 1 137) (P <0 0 5 ) ;而低剂量组 (3 5 6 7±0 86 9)反而显著低于对照组。服低剂量棉酚G +H 6周后继续服单独低剂量棉酚 (12mg/(kg·d) ) 12周 ,单细胞凝胶电泳未见生精细胞出现DNA单链断裂的彗星状细胞核 ,与高、中剂量组较多的出现率形成明显对照。结果提示低剂量棉酚与甾体激素组合用药在睾丸生精过程中互为调节 。

To determine the effects of combined use of low dose gossypol and steroid hormone as male contraceptive upon apoptosis of adult Wistar rat spermatogenic cells. We applied the techniques of micronuclei (MN), In situ end labeling (ISEL) and single cell gel electrophoresis (SCGE) to detect apoptosis and DNA damage of rat spermatogenic cells following high [100mg/(kg·d)], medium [60mg/(kg·d)] and low [12mg/(kg·d)] doses of gossypol respectively combined with a single dose steroid hormone [methytestosterone 20mg/(kg·d) and ethinyl oestradiol 100μg/(kg·d)] and as well after a periods of maintenance dosage of gossypol. The apoptosic coefficients (A) and the rate of MN of spermatogenic cells after 6 weeks treatment of different groups of gossypol combined with steroid hormone were increased steadily with the increase of gossypol dosage. Both of the high and medium groups showing remarkable increment of apoptosic coefficient compared with the control group ( P <0.05, table 1); the index of low dose group is also higher than that of control group, but the difference is not significant statistically. The rate micronuclei in high and medium groups were 27.633±3.755 and 7.900±1.674 respectively, which were notably higher than that of the control group (5.600±1.137) ( P <0.05), whereas that of the low dose group (3.567±0.869) was revealed even lower than the control ( P <0.05). Analysis of single cell gel electrophoresis demonstrated that both the high and medium groups had a percentage (15%～27%) of spermatogenic cells exhibited breakage of single strain DNA in nucleus, but neither the low dose group treated for 6 weeks nor the maintenance group dosage for further 12 weeks showed changes in nuclear DNA damage. Results indicated that that the low dosage regimen of gossypol combined with steroid hormones could affect spermatogenesis and negate each other in action between the combined components. The apoptosic effect of gossypol could be reduced by regulation of steroid hormones then to protect spermatogenesis.