Effects Of Genistein And Royal Jelly Proteins On Testist Development In Male Mice

Estrogen palys a crucial role on the development of animal reproductive system,especially in the period of neonatal state.Genistein(GEN)and Main Royal Jelly Proteins(MRJPs)have been defined as estrogen-like substances or endocrine disruptors,which attracted attentions widely.The intake of these endocrine disruptors leads to the disorder of reproductive hormone secretion and the abnormal of testis structure,which may result in infertility.GEN and other substances are reported to have their effects by binding to estrogen receptors.Meanwhile,they may affect testicular function via intervention of hypothalamus pituitary gonad axis feedback regulation,which lead to reproductive function disorders.Hematoxylin and eosin(HE)staining,immunohistochemistry(IHC),enzyme-linked immunosorbent assay(ELISA),real time-PCR,electron microscopy,apoptosis detection(TUNEL cell apoptosis detection)were used to detect the effects of different dose of endocrine disruptors(GEN and RJ)on testis of young mice.By detecting the proliferation and apoptosis of testicular germ cells and the concentration of serum reproductive hormone testosterone that may provide some regulatory mechanisms of animal reproductive function,which helpful to reproductive biology and clinic application.The main contents are as follows:1.Effects of genistein on the testis structure in miceIn order to clarify the effects of genistein(GEN)on reproductive function of puberty male mice,we detected the testis weight,morphological structure of seminiferous tubules and some reproduction related genes in experimental animals.In present experiment,GEN fedding was administrated to pregnant mice(the 7th day of pregnancy).During the pregnancy and lactation period,food which was containing different doses of GEN,was fedding to the mothers.After weaning,that diet was provided to pups directly until the pups were 35 days.The experiment was designed into low dose group(LGE:40 mg/kg GEN),high dose group(HGE:800 mg/kg GEN):and control group(CON).After cervical dislocation under CO2 anesthesia,blood was collected and testis were separated.The weight of testis,diameter of seminiferous tubules and height of reproductive epithelium were analyzed by one-way ANOVA,with multiple comparisons among groups tested by Tukey'srange test.The expression levels of GAPDH,ESR1,ESR2,CYP19A1,SOX9 and BRD7 genes in testis were detected by real-time PCR.The results showed that body weights of the mice in LGE group were higher than that of CON group at PND 35(P<0.05),while,in HGE group were lower than that in CON group(P<0.05).Similarly,testis weights of mice in LGE group was higher,while,in HGE group were lower than that in CON group(P<0.001).In addition,seminiferous epithelium height and diameter of testicular seminiferous tubule in LGE group was significantly increased,while,in HGE group significantly decreased(P<0.05)compared to CON group.Meanwhile,deformity rate of testicular germ cell in LGE group was significantly decreased,while,in HGE group was higher than that of CON group(P<0.05).Furthermore,the testicular spermatogenesis scoring in LGE group was significantly increased,while,in HGE group was significantly decreased compared to CON group(P<0.05).Finally,compared with CON group,the gene expression of ESR2 in the treatment group(LGE and HGE)were higher than that in CON group(P<0.05).The expression of CYP19A1 in LGE and HGE group were significantly increased compared to CON group(P<0.001).The expression of SOX9 and BRD7 in LGE group were significantly increased compared to CON group.However,the expression of SOX9 and BRD7 in the HGE group were significantly decreased than that in CON group(P<0.001).These results above suggested that low-dose of GEN might promote the growth of mouse testis,while,high-dose GEN might inhibit the testis development.2.Effects of MRJPs on testis structure of puberty miceIn order to investigate the effects of MRJPs on the reproductive function of young male mice,the weight of testis,seminiferous epithelium height and other indexes were detected for statistical analysis and reproductive assessment.MRJPs were fed orally to new born pups until they were 35 days old.The experiment was divided into four groups:control group(CON),low-dose MRJPs group(L-RJ:125 mg/kg L-RJ),moderate-dose MRJPs group(MRJ:250 mg/kg M-RJ)and high-dose MRJPs group(H-RJ:500 mg/kg H-RJ).Four time point(PND 7,14,21 and 35)were selected.The results showed that testis weights in M-RJ group were significantly increased compared to the CON group(P<0.05)at PND 14.Diameters of seminiferous tubules in H-RJ group were significantly decreased compared to CON group(P<0.05).At PND 21,testicular weights and testicular spermatogenesis score of mice in MRJ group were significantly increased compared to CON group(P<0.05).Height of testicular epithelium in H-RJ group was lower than that of CON group(P<0.05).At PND 35,testicular weight and testicular epithelium height of mice in M-RJ group were significantly increased,while,in H-RJ group was significantly decreased compared to CON group(P<0.05).In addition,testicular spermatogenesis score of H-RJ group was significantly lower than that of CON group(P<0.05).Results above indicated that fedding moderate dose of MRJPs could promote the testis development of mice.While,500 mg/kg MRJPs might inhibit the development of testis.Therefore,it is suggested that testes function might be affected by RJ according to its concentration.3.Effects of Genistein and MRJPs on sexual hormones and testis apoptosis in miceEnzyme-linked immunosorbent assay(ELISA)and TUNEL were employed to detect the effects of genistein(GEN)and Main Royal Jelly Proteins(MRJPs)on sexual hormones and apoptosis of germ cells.Results showed that testosterone(T)in LGE group was significantly increased compared to CON group(P<0.05).At 21 days old,the FSH level of the treatment group(L-RJ,M-RJ and H-RJ)was significantly decreased compared to CON group(P<0.05).E2 level of the treatment group(L-RJ and M-RJ)were significantly decreased compared to CON group(P<0.05).In addition,the TUNEL detection showed that the apoptosis of germ cell in HGE group was significantly increased compared to the CON group(P<0.05).Similarly,the number of germ cell apoptosis of H-RJ group was significantly decreased compared to CON group(P<0.05).In conclusion,these results suggested that GEN and MRJPs have a dose-dependent effect on testis development.Meanwhile,high-dose GEN and MRJPs could has an adverse effect on the development of germ cells of testes.Through the establishment of the experimental model of feeding new born mice with GEN and MRJPs,those treatment changed the structure of testis,some related genes expression of testes and the level of sexual hormones in the mice.The results suggested that there were dose differences in the effects of genistein and MRJPs on the testis development of mice.Therefore,teenagers should avoid excessive consumption of beans and royal jelly products in their daily lives.