Effect Of Gibberellin On Motility And Apoptosis Of Human Sperm And Its Mechanism

Objectives:Gibberellic acid（GA3）,is a one of the most commonly plant growth regulator which was be used in the agricultural aim to promote stem elongation,improve fruit rate,produce parthenocarpic fruits and increase harvest index.Although GA3 are usually accepted as non-toxic especially in proper doses and times,but the high dose or inappropriate use may be lead to adverse effects on humans and animals.Several studies were carried out in animals,however,its effects on human sperm in vitro haven’t been suggested.In the current study the effect of gibberellin on sperm motility and the mechanism of oxidative stress and apoptosis were investigated by adding different concentrations of gibberellin into the culture medium of human spermatozoa.Methods:Ejaculated human sperm samples were collected in sterile containers by masturbation after 3-7 days of sexual abstinence from some healthy normozoospermic subjects.（1）Fractions of highly motile sperm were separated using a discontinuous Percoll gradient（45-90%）,sperm samples cultured in the G-IVF PLUS medium with different concentration of gibberellins.After Gibberellin exposure 1、5、9 and19 hour,the sperm motility parameters were analyzed using the CASA system.The minimum concentration of gibberellin,which affects the motility of sperm,is screened as far as possible.（2）Oxidative stress reaction detection:the ROS level was detected by Reactive oxygen detection kit,the protein levels of CAT、SOD were detected by immunofluorescence and western-bloting.（3）Apoptosis detection:the apoptosis level was detected by Annexin-V-FITC detection kit,the protein levels of BAX、P53 were detected by immunofluorescence and western-bloting.（4）ATP detection:The activity of Na⁺-K⁺-ATP synthesis enzymes（Na⁺-K⁺-ATPase）and Ca²⁺-ATP synthesis enzymes(Ca²⁺-ATPase)were determined by the kit from Nanjing Jiancheng Biochemistry Co.Results:1)Gibberellin exposure resulted in dose-dependent and time-dependent declines in sperm motility;2)The level of reactive oxygen species（ROS）was significantly increased than the control group（F=21.4,P<0.05）,the level of peroxidase（SOD）protein was significantly decreased than the control group（F=28.13,P<0.05）;3)the expressions of apoptotic proteins P53and BAX were significantly increased than the control group（F=104.54,59.29,P<0.05）;4)The activity of Na⁺-K⁺-ATP enzyme and Ca²⁺-ATP enzyme were significantly decreased than the control group（F=24.48,18.81,P<0.05）.Conclusion:GA3 can reduce sperm motility and promote the occurrence of oxidative stress and apoptosis.