Preliminary Study Of Estradiol Benzoate On Cell Proliferation And Glucose Metabolism Of Testis In Mice

Objective Establishment of estradiol benzoate(EB)experimental animal models,analysis of estradiol benzoate(EB)impact on the male reproductive,the content of ATP,glycolysis products and limiting enzyme activies.Preliminary studies on the mechanism for EB impairment of spermatogenesis.Methods Ninety Kunming male mice aged 4 weeks were random Ly divided into three groups,the mice of the control group were injected with 150 ?l corn oil only by every other day intramuscular injection for 4 weeks;meantime,the mice of the treatment groups were injected with EB at the concentration of 5 or 10 mg/kg,respectively.After the experiment,the test analysis was divided into two parts.Part One :(1)After the experiment,three mice of each group were kept for fertility detection respectively,and two female were randomly set for cohabiting with each male mouse.In the end,the litter size of each female mouse was counted fot the fertility statistic.(2)The mice were killed,the testes and epididymis weight of mice were weighted and the coefficient were calculated.(3)The number of the sperm were counted from the epididymis sperm suspension.cauda epididymidis were stained with HE and observed under light microscope.(4)The paraffin sections of testis were stained with HE and PAS,at stage VII of the seminiferous cycle,including spermatogonia,spermatocytes,step 7spermatids were counted;testis transmission electron microscopy examination were executed.(5)The proportion of cell cycle phase of the testes were measured by flow cytometry.(6)The m RNA expression of Cyclin A1,Cyclin B1,Vasa and PCNA in the testes were detected by q RT-PCR.(7)The activity of SOD,GSH-Px,CAT,NOS and the contents of MDA,NO in testicular tissue were detected by chemical colorimetry.The m RNA expression of SOD,CAT,GPx4 in the testes was detected by q RT-PCR.Part Two:(1)High-performance liquid chromatography method for determination the contents of ATP in testes.(2)Preparation of testes homogenate,the content of glucose,lactate,pyruvate and the activity of HK,PK,LDH were specifically detected by chemical colorimetry.(3)The m RNA expression of MCT2 and MCT4 in the testes were detected by q RT-PCR.Results Part One: Compared with control group:(1)The fertility tests showed that EB-treated mice were infertile;however,the control mice had normal fertility.(2)The weight and coefficients of testis and epididymis were significantly decreased.(3)No sperm was found in the epididymis sperm suspension and epididymal ducts in EB-treated group(P<0.05).(4)In control group,the seminiferous tubules were normal and large numbers of sperm were observed in the lumen;no sperm were observed in the seminiferous tubules in EB-terated groups.The number of spermatogonia,spermatocyte and round spermatids were significantly decreased(P<0.05).The spermatogonia,spermatocyte,round spermatids and sertoli cells in EB-treated group,the mitochondria were transformed to vacuoles with irregular cristae.(5)The proportion of G0/G1 and S phase cells reduced significantly,the cells of G2/M phase increased significantly with the doses of EB increased(P<0.05).(6)The m RNA expression level of PCNA,Cyclin A1,Cyclin B1 were significantly downregulated in the testes of the EB-treated groups(P<0.01);The m RNA expression level of Vasa was significantly downregulated in 10 mg/kg of EB-treated group(P<0.01),5mg/kg of EB-treated group are same as control group(P>0.05).(7)The activity and m RNA expression level of SOD,CAT,GSH-Px were significantly declined in the EB-treated group(P<0.05);the activity of NOS and the contents of MDA and NO were significantly raised in the testes of the EB-treated groups(P<0.05).Part Two: Compared with control group,in testicular tissue:(1)HPLC analyse result showed ATP content in EB-treated groups were significantly decreased(P<0.05).(2)The content of glucose and pyruvate in EB-treated groups were increased(P<0.05);the lactate content in EB treatment groups were similar with control group(P>0.05).HK and PK activies were significantly decreased when compared with control group(P<0.05),LDH activity were very similar in three groups(P>0.05).(3)The m RNA expression levels of MCT2 and MCT4 were significantly decreased in the EB-treated group(P<0.05).Conclusion Part one1.EB blocked cell cycle and inhibited cell proliferation of spermatogeic cell by downregulating the m RNA expression of Cyclin A1,Cyclin B1,Vasa and PCNA,decreased the number of spermatogeic cells.2.Decreased antioxidase activities and oxidative stress in the testis of mice induced by EB,oxidative damage to mitochondria of sertoli cells and spermatogenic cells,which might be one of the causes of spermatogenic dysfunction.Part two EB decreased the levels of ATP,the activities of HK,PK and the m RNA expression of MCT4 and MCT2 in testis,inhibited glycolysis to produce pyruvate,lactic acid transport capacity declined.It has been speculated that glucose metabolism disorder and energy deficiency were the important reason for spermatogenic dysfunction induced by EB.