| The aim of this study was to investigate the productive damage of Diethylstilbestrol(DES) on F0 adult male hamster and F1 adult male offspring after F0 adult male hamster exposuring, and determine the protect effect of Lycium barbarum Polysaccharides(LBP) against the damage. Ninety adult male hamsters were randomly divided into 6 groups. After 24 hours of the last treatment,(1) 5 male hamsters were randomly selected in each group, the potency of Caspase-8, Caspase-9, P53, AR and ER in testicular tissue were tested through immunohistochemistry. Histopathological changes of the epididymis tissue were observed, morphology of sperm, sperm number and teratospermia were detected under the microscope, immunohistochemical technique was used to check the expression of Caspase-8、Caspase-9 and P53.(2) After treatment, each male hamster was mated with two adult females, with total 5 males in each group. After 2 estrous cycles(9 days), pregnant hamsters were fed with single cage until delivery, and F1 male offsprings were fed until sexual maturity(PND 56 d). The pregnancy rate and gestation length of F0 females were tested, total litter size and sex ratio of F1 pups were detected, the body weight of F1 hamsters on PNDs 0, 7, 14, 21, 28 and 56 were tested, the weight and coefficient of testis and epididymides were determined, testicular and epididymal histopathologic structure were observed under microscope, the sperm number and teratospermia were detected and pro-apoptotic proteins Fas, Fas L and Caspase-3 in testes of adult F1 male hamsters were tested.(3) Five F0 males in each group were raised as normal for another 3 months, then the histopathological changes of the testicular tissue were observed, the sperm number and teratospermia were detected.Results showed that:(1) Atrophy was bring out at ductus epididymis, large vacuolization could be seen on the epididymis epithelial cell after DES treatment. DES decrease the sperm counts, while increase the teratospermia of F0 male hamster(P<0.01). After supplement with different doses of LBP, the sperm number was increased, especially in the 50 mg/kg LBP group than that in the DES group(P<0.01), the teratospermia were diminished in the 10 mg/kg LBP group and 50 mg/kg LBP group compared with the DES group(P<0.01). DES induced apoptosis of spermatogenic cells in F0 hamster with dramatically increased expression of Caspase-9(P<0.01). After treated with different dosage of LBP, the expression of Caspase-9 and p53 decreased in a dose dependent manner with 50 mg/kg LBP being the best(P<0.01). There were no significant difference compared the high dose of LBP group with the control group referring to the expression of Caspase-8. DES dramatically decreased the expression of AR(P<0.01), after different dosage of LBP treatment, the expression of AR gradually increased with the increasing dose of LBP. DES dramatically increase the expression of ER gene(P<0.01), after supplement with LBP, the expression of Bax were decreased. DES can enhance the expression of apoptosis Caspase-8, Caspase-9 and P53 proteins in epididymis of F0 hamster(P<0.01), after treated with LBP, the expression of Caspase-8 was significantly decreased at all LBP groups than that in the DES group(P<0.01), the level of Caspase-9 positive cells was decreased in 10/50 mg/kg LBP groups(P<0.01), the positive expression of P53 was decreased in 50 mg/kg LBP groups(P<0.01).(2) After 2 estrous cycle’s mating, there were only four females in the DES group did become pregnant, the pregnancy rate in 1 mg/kg LBP group and 10 mg/kg LBP group were 60% and 70% respectively, all females in control group, 50 mg/kg LBP group and vitamin groups did become pregnant, and all pregnant females in all groups delivered live pups. There were no significant differences in the body weight of F1 hamsters on PND 7. The body weight of F1 pups was significantly decreased at DES group and vitamin group on PNDs 14, 21 and 28(P<0.05). No significant changes were observed in the body weight of F1 hamsters at all LBP groups and control group on PNDs 14, 21 and 28. A significantly lower body weight was observed in F1 pups at vitamin group on PND 56(P<0.05). There were no significant difference of the body weight of F1 hamsters on PND 56 among control group, DES group and all LBP groups. The weight and coefficient of epididymis and sperm number were significantly decreased, and the expression of Fas/Fas L and Caspase-3 were increased, but no changes of the testicular weight and coefficient, teratospermia and epididymis histopathology in the F1 generation after F0 male hamster exposed to DES. The weight and coefficient of epididymis in F1 adult male hamster were increased in LBP groups compared with DES group. The sperm density of F1 offspring increased in 50 mg/kg LBP group comparing to DES group. The expression of Fas was significantly decreased in 10 mg/kg and 50 mg/kg LBP group, and the positive expression of Fas L and Caspase-3 were all diminished after supplement with different dosage of LBP compared with DES group. There were no significant differences in the gestation length, total litter size and birth weight, and sex ratio of F1 pups.(3) After recovery for 3 months, the weight of testis and epididymis and the testicular tissue morphology in all groups were reach to the levels showing in normal animal, and sperm malformation rate was reduced to normal level. The number of sperm in DES group, 1/10 mg/kg LBP groups and Vc+VE group were still lowder than that in control group, but the amount of sperm in 50 mg/kg LBP group recovered to the normal level. The body weight in all groups was increased significantly(P<0.01). The organ weight of testis in all groups was higher than that pre-recovery, but no significant difference was observed. The weight of epididymis was increased 81.25% and 55.81% respectively in 1 mg/kg DES and vitamin groups after 3 months recovery, but no significant difference of the epididymal weight after recovery. The weight of epididymis in 1/10/50 mg/kg LBP groups were higher than that pre-recovery. The number of sperm in DES group, 1/10 mg/kg LBP groups and vitamin group was increased significantly after recovering 3 months. The teratospermia in all groups was decreased significantly after recover(P<0.01).Conclusion:(1) The study indicate that DES induce the apoptosis of germ cells in F0 hamster, the morphology of epididymis was obviously changed.DES also enhance the expression of pro-apoptotic Caspase-8, Caspase-9 and P53 proteins, disrupte the normal activity of AR and ER, diminishe the sperm number, and increase the sperm abnormality rate, which all leading to the reproductive damage of F0 adult male hamster. However, after supplement with LBP, the expression of apoptosis proteins in spermatogenic cells of testis and epithelial cells of epididymis decreased. Moveover, LBP significantly relieve the epididymal histomorphologic damage induced by DES, redresse the function of AR and ER, increase the sperm number and decrease the sperm abnormality rate, alleviate the reproductive damage of F0 adult male hamster induced by DES.(2) The reproductive damage of DES on F1 hamster include: decreasing the body weight on PNDs 14 d, 21 d, 28 d and 56 d, diminishing the testicular weight and coefficient, reducing sperm number, increasing the levels of Fas/Fas L and Caspase-3 in spermatogenic cells. While supplement with LBP can alleviate the reproductive damage of F1 adult male hamsters, with the best dose of LBP being 50 mg/kg.(3) The reproductive damage induced by DES was reversible after F1 males recovering for 3 months. Reproductive indicators in DES group were improved, but the number of sperm was still lower than that in the control group. The indicators for reproduction recover well after supplement with LBP, the sperm number back to normal level in 50 mg/kg LBP group, this indicate that LBP can help the recovery of reproductive damage induced by DES. |
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