| In recent years，with the quickly moving forward of industrialization and urbanization andincreased environmental pollution，human reproductive is facing an unprecedented challenge. Incidence ofinfertility is increasing and the overall level of the male sperm quality decreasing. According to somestatistics, male sperm count in the whole world has fallen by half in the past50years, on average reducesby1percent each year. By that analogy, mankind may even occur “no sperm crisis” in a couple of decades,some researchers called it the scope of the global sperm landslide. But so far there has no exact effect drugcan promote both quality and quantity of sperm, this becomes a problem that needed to be solved in thefield of reproductive medicine.Active ingredient of ginseng include saponins，small peptide，polypeptide, polysaccharide etc.,ginsenoside is the most studied one. Our research team has found the following aspects in recent years.First, Rg1can significantly improve gossypol induced sperm count fell, enhance the vitality of damagedsperm. Second, after Rg1treated, not only damaged spermatogenesis capacity has be improved, but also theeffect is better than control group rats. Third, Rg1can improve sperm count in elderly rats, although Spermvitality had no obvious improvement. Fourth, Rg1can improve cold stress damaged sperm motility. And inthe experiment we also found that the Rg1metabolites Rh1,Ppt has the same effect too, and has nosignificant difference with Rg1treatment group. This means that acting time of Rg1in body could begreatly prolonged. Meanwhile, glycosyl remove made it possible to send Ppt to chemical synthesis, thiswill greatly reduce the application cost of Rg1. The relevant results have apply for a patent and obtain thesubsidize National Science Foundation. This research will make further study on the mechanism of action.In this paper, we have made two animal model of spermatogenesis obstacle by two differentdrugs, gossypol and zearalenone, to study the spermatogenesis effect of Rg1and its metabolites Rh1andPpt on different model. The experiment use male wistar rat for the object of study, all drugs and solventswere given by oral ad ministration, then detect the related indicators of the reproductive system of adultmale rats, to study the medicine function on cAMP （cyclic adenosine monophosphate，cAMP）/CREM（cAMP-responsive element modulator，CREM） signaling pathway, on energy metabolism system in rat testis tissue, and on mitochondria metabolism system in rat sperm. This experiment is mainly through thestudy of the impact of Rg1and it’s metabolites Rh1and Ppt on reproductive system of adult male rats, toresearch the spermatogenesis molecular mechanism of the three drugs, and explain part of themechanization.The result of experiment by the animal model created by ZEA show that, treated gossypol everyother day50mg/kg for two weeks, the sperm count and sperm motile were droped, proved that thespermatogenesis is available. And the body weight of gossypol group has significantly droped, meanwhile,Rg1and it’s metabolites can recover the body weight, although can’t recover it to normal level.The result of experiment by the animal model created by ZEA show that, there are no significantdifference between each group on sperm viability and sperm motility （P>0.05）. These results promptingthat the animal model is probable not created successfully. There must be more studies to verify the validityof this animal model’s function and the impact of Rg1, Rh1, Ppt on sperm viability and sperm motility. Sowe use gossypol for the model drugs in following research.The results of experiment by the animal model created by gossypol show that, afteradministration, the content of cAMP in sperms of control group is higher than medication ad ministrationgroup （P<0.05）, gossypol has no significant effect on the level of cAMP in sperms. But in esticular tissue,gossypol can remarkable induce the content of cAMP, while Rg1, Rh1, Ppt can rise the induce level ofcAMP and followed a remarkable increase of the CREM expression. So that the regulating action of Rg1,Rh1, Ppt on cAMP/CREM signaling pathway is embody localization function to different position, thisfunction embodies the diversity effect of Rg1.In addition, the content of calcium (Ca2+) in sperms also haschanged, model group content is160.07±11.29（umol g-1protein）, control group content is248.79±23.21（umol g-1protein）, the gossypol model group is significantly lower than control group （P<0.05）, and theRg1, Rh1, Ppt groups are significantly higher than model group （P<0.05）. Hence that the gossypol canreduce the calcium in sperms, and Rg1, Rh1, Ppt can confront the reduce effect.In the study of energy metabolism-related enzyme activities in rat testis tissue, Rg1etc. showsgreat impact on lactate dehydrongenase （LDH）, the model group is significantly higher than Rg1, Rh1andPpt group （P<0.05）, and the polyacrylamide gel electrophoresis result shows that the LDH isozymes ofmodel group was significantly difference from any other groups （P<0.01）. The results suggest that the ginsenoside Rg1and the metabolite Rh1and Ppt have effects on glycolysis pathway in rat testis tissue, theglycolysis of Rg1etc. groups has been reduced and increased in aerobic metabolism, indicate that the Rg1etc. can promote the product of spermatid in rat testis. The enzymes which participate in tricarboxylic acidcycle, β-fatty acid oxidation and mitochondrial respiratory chain such as isocitrate dehydrongenase（ICDH）, β-hydroxyacyl-CoA dehydrongenase （HAD） and cytochrome c oxiase （COX） were observed nosignificantly activities changes （P>0.05）. So the function of fatty acid metabolism and mitochondrialrespiratory chain in rat testis tissue is not known and the effect of Rg1and the metabolite Rh1and Ppt onmetabolism-related enzyme activities is not yet determined.It is shown by the detection result of mitochondrial membrane potential （MMP） that Rg1etc. hassome impacts on the MMP. Rg1can increase the MMP （P<0.05） indicate that it has the function of promotethe sperm capacitation. The detection result of reactive oxygen species （ROS） shows that the Rg1etc.group’s level of ROS emerge a reducing trend （P<0.05）, prompting that they have the protect effect forsperm so that it can avoid the damage from high level of ROS.The results of HE stain indicated that theRg1etc. groups can rehabilitation the damage such as morphological structure changes which caused bygossypol,this prompt that Rg1, Rh1, Ppt have reparative therapy effect on the dyszoospermia of rat.The result of HE stain shows that Rg1,Rh1,Ppt can recover the structure damage which coused bygossypol, indicate that they have spermatogenic protection effect.So far, domestic and overseas have no report on the spermatogenic protection effect of Rg1andit’s metabolites Rh1and Ppt, and the research of the molecular mechanisms. Our research hasmonopolization protect, and for the first time expound the in vivo effect basis of Rg1, make contributionfor the in vivoetabolize dynamics. Our study will make a solid basis for the clinicaladhibition of Rg1, andaffordexperiment basis for the development of spermatogenic medicine. |
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