Effect Of Protecting Spermatogenesis And Regulating The Expression Of CYP17A1 On The Tests Of BPA-infected Mice By LBP Gavage

Endocrine disrupting chemicals?EDCs?are hormone-like chemical which released into the surroundings by production and daily life.As a typical environmental hormone,Bisphenol A may cause infertility due to disturb spermatogenic function.There is no effective method to prevent and treat.Lycium barbarum polysaccharide?LBP?is the based effective component of Chinese medicine fructuslycii,which can antagonize the testicular morphological damage caused by BPA and protect the spermatogenic function of mice testes through antioxidant and inhibition of apoptosis.However,there are few reports about the effect of LBP on sperm quantity and quality of male BPA-infected mice.Spermatogenic function is closely related to androgen,CYP17 A is a key enzyme for androgen synthesis,therefore,its expression in testis is closely related to spermatogenesis.Based on our previous studies,the protective effect of LBP at 200mg/kg on spermatogenic function and testicular CYP17A1 expression in mice infected with BPA were discussed,we aim not only to explore the mechanism of LBP to improve spermatogenesis,but also to provide a reliable experimental basis to research and development LBP further.Part One Modelling spermatogenic disorder in BPA-infected miceObjective: To make mode of spermatogenic disorder in mice infected BPA.Method:1)groups: take random number table method,10 kunming SPF 8-week-old adult male mice bw?37 ±2?g were divided into the normal control group?Ctrl group?and model group?BPA group?.2)Model preparation: BPA100mg/kg/d was injected subcutaneously in the BPA group and 0.2 ml coin oil in the control group,respectively,for 7 days.3)Testicular coefficient measurement: Testicular coefficient = testicular weight / body weight.4)Detecting of sperm density,sperm malformation rate and sperm viability:SQA-V semen analyzer was used to detect.5)Detecting of Serum T,E₂ and T/E₂: Serum T and E2 were detected by radioimmunoassay.6)HE staining of testis: paraffin sections of testis were dewaxed with xylene,alcohol dehydration;Hematoxylin was stained;Dyeing with 1% eosin for 1 min;alcohol solution;Xylene transparent.Results:1)Compared with control group,there was no significant difference in body weight in BPA group?P> 0.05?.testis coefficient in BPA group was also significantly lower,and the difference was statistically significant?P< 0.05?.2)Compared with control group,both sperm density and sperm motility were significantly decreased in BPA group,sperm malformation rate was significantly increased?P<0.01?.3)Compared with control group,the serum T,T/E₂ ratio of BPA group was significantly lower than control group?P<0.05?.There was no significant difference in E₂?P>0.05?.4)The spermatogenic tubules of mice in BPA group were hollow and tubular in different degrees,the wall of the functional cavity was damaged and became thinner,the continuity of basement membrane was damaged,and the reproductive cells were disorderly and disorderly arranged,the count of layers was reduced,the structure was unclear,the count of sperm in the cavity was reduced,and the distribution of interstitial cells was uneven.Conclusion:BPA 100mg/kg/d can induce spermatogenic dysfunction in mice by subcutaneous injection for 7d,and the spermatogenic dysfunction model in mice were established succesessfully.Part Two Protective effect of LBP on spermatogenic function of BPA-infected miceObjective: To explore the protective effect of LBP on spermatogenic function of model mice.Method:1)groups: taking random number table method,35 male mice were divided into 7groups,5 in each group,respectively,the control group?Ctrl group?,model group?BPA group?,LBP7 d group?7d group?,LBP14 d group?14d group?,LBP21 d group?21d group?,LBP28 d group?28d group?,and LBP35 d group?35d group?.2)spermatogenic disorder model in mice infected with BPA: the same as the first part3)LBP intervention in BPA-infected reproductive disorder mouse model: LBP group was divided into 7d,14 d,21d,28 d,35d,200mg/kg LBP gavage,respectively,the body weight was measured on 7d,14 d,21d,28 d,35d,respectively.4)Testicular coefficient determination: the same as the first part5)Sperm density,sperm malformation rate and sperm viability detection: the same as part one.6)Serum T,E₂,T/E₂: the same as the part one.7)HE staining of testicular tissue: the same as the part one.8)Testicular TUNEL immunofluorescence staining: paraffin section in xylene;Immerse in distilled water;Add 0.1% Triton X-100 50 ul.TUNEL reaction solution was added to.Section was taken out and restained with DAPI.Drop the fluorescence quenching agent to seal the tablet.Results:1)Compared with the BPA group,the testicular coefficient was significantly increased in the control group,the 7d group,the 21 d group,the 28 d group and the35 d group?P<0.05?,but there was no significant difference in the 14 d group?P>0.05?.Among the LBP groups,the 14 d group was lower than the 21 d group?P<0.05?,the 28 d group was higher than the 21 d group?P<0.05?,and there was no significant difference between the 7d group,35 d group and 21 d group?P>0.05?.2)Compared with BPA group,control group and LBP group showed significant increase in sperm density and motility,and significant decrease in deformity rate?P<0.05?.In the LBP group,the sperm density and motility were significantly lower 7d group and 14 d group than 21 d group,and the sperm malformation rate was significantly higher than 21 d group?P<0.05?,while there were significant differences between the 28 d and 35 d groups and the 21 d group?P>0.05?.3)Compared with BPA group,control group and LBP 21d?28d?35d group showed significant increase in the serum T and T/E2 ratio,there was no significant difference in other groups.There was no significant difference in E₂?P>0.05?.4)In BPA group,spermatogenic tubules were hollow and tubular,the continuity of basement membrane was damaged,the number of layers of germ cells were disorderly and disorderly arranged,the number of sperm in the cavity was reduced,and the distribution of interstitial cells was uneven.After LBP gavage,the wall of the lumen was damaged,thinned and the continuity of basement membrane was damaged,the arrangement of germ cells was disordered,the number of spermatogenic cell layers was reduced,and the uneven distribution of interstitial cells was alleviated to varying degrees.5)The positive signal of apoptosis in control group was mainly located in spermatogonial cells close to the spermatogenic tubule basement membrane,with obvious cytoplasmic staining.The apoptosis in the 21 d group was significantly lower than that in the BPA group,7d group and 14 d group,and there was no significant difference from that in the 28 d group and 35 d group.Conclusion:LBP can protect the spermatogenic function of BPA-infected mice,and the maximum effect and the minimum time of treatment was 21 d.Part Three Effect of LBP on testicular CYP17A1 expression in BPA-infected miceObjective: To investigate the effect of CYP17A1 expression in LBP on spermatogenic function of BPA-infected mice.Method:1)group: the same as the part two.2)spermatogenic disorder model in mice infected with BPA: the same as the part one3)Testicular CYP17A1 immunohistochemical staining: the paraffin section in xylene ?,?,immerse 15 min;Put it into anhydrous ethanol for 5min.Put in 95%,85% and75% ethanol successively.The sections were put into the antigen repair solution and heated for 10 min.,Incubate at room temperature with 3% H2O2 for 15 min.Normal goat serum was added and incubated at room temperature for 15 min.Primary anti-CYP17A1 was diluted with PBS at 1:100,and then added as biotin-labeled goat anti-rabbit Ig G secondary anti-CYP17A1.Wet with in 4 ? 8h.PBS diluted 200 times the two resistance,in 37 ? for 30 min incubation in the box,.Add HRP labeling avidin,1:200 dilution with PBS,37 ? for 30 min incubation.PBS 5min x3.added DAB reagent 100 ul.Differentiation for 3s,water 20 min.Dehydrate the ethanol and seal the slices.4)Immunofluorescence staining: paraffin sections were immersed in xylene ? and? for 15min respectively.Immerse in 95%,85% and 75% ethanol for 1min respectively.Goat serum was added and kept in a wet box at room temperature for 30 min.anti-CYP17A1 was diluted with PBS at 1:100,added as biotin-labeled goat secondary anti-CYP17A1 of anti-rabbit Ig G.4 ? 8h.add fluorescence secondary antibody.Both were diluted with PBS1:200 and incubated at room temperature for90 min.add DAPI restain core.;Cover the slide and seal it.5)Western-Blot: lysis sample was added,protein was extracted,standard curve was prepared,protein solution was prepared,BCA reaction was performed,and protein concentration was calculated.Preparation of polyacrylamide gel and protein loading solution,SDS-PAGE;The gel "sandwich" structure was prepared and transferred to the printing tank for 1.5h.Occluded residual antigen,incubat primary antibody,secondary antibody,ECL substrate luminescence,antibody stripping,antibody,internal reference antibody,secondary antibody,ECL substrate luminescence,take photos,and the optical density values of the target bands were analyzed using Gel image processing system?gel-pro-analyzer software?.6)RT-PCR: the upstream primer sequence was 5–TGGGCACTGCATCA CGATAA-3?,and the downstream primer sequence was 5?-GCTCCGAAGGGCAAATAAC T-3?.actin upstream primer: 5?CTGTGCCCATCTACGAGGGCTAT-3? downstream primer: 5?-TTTGATGTCACGCACGATTTCC-3?.1ml Trizol lysate was added,mixed,and set aside for 5min.Centrifugation after antigen repair;Add 30 l RNase-free dd H₂O,dissolve completely.Determin RNA concentrations.CYP17A1-RNA reverse transcription to c DNA,and the expression of CYP17A1 m RNA was detected.Detect the dissolution curve.Results:1)Immunohistochemical staining of CYP17A1 in testis: cytoplasm of testicular cells in each group showed brown positive reactant and negative nucleus.2)Immunofluorescence staining of CYP17A1 in testis: cytoplasm of testicular cells in each group showed red fluorescence signal,and the nucleus was negative.The red fluorescence signal of testicular stromal cells in BPA group was significantly lower than that in Ctrl group,showing a diffuse distribution with uneven distribution.After LBP treatment,CYP17A1 red fluorescence signal of testicular stromal cells at 21 d was higher than that of BPA group,7d group,and there was no significant difference from that of 28 d group and 35 d group.3)Western-Blot detection of CYP17A1: compared with the BPA group,the expression of CYP17A1 in the testicles of male mice in the control,14 d,21d,28 d and 35 d groups was significantly increased?P<0.05?,while no significant change was observed in the 7d group?P>0.05?.After LBP intervention,21 d was significantly higher than 7d?P<0.05?,while there was no significant difference with 28 d and 35d?P >0.05?.4)RT-PCR detection of testicular CYP17A1: compared with the BPA group,the expression of testicular CYP17A1 m RNA was significantly increased in the control group,the 14 d group,the 21 d group,the 28 d group and the 35 d group,with statistical difference?P<0.05?,while there was no statistical difference in the 7d group?P>0.05?.Among the treatment groups,the 21 d group was significantly higher than the 7d group,the 14 d group,the 28 d group and the 35 d group?P<0.05?.Conclusion:1)The mechanism of BPA inducing spermatogenic dysfunction in mice maybe inhibit the expression of CYP17A1 by BPA,reduce the secretion of T and the production of sperm.2)LBP may increase the expression of testicular CYP17A1 and the secretion of T,and protect the spermatogenic function of BPA-infected mice.The maximum effect and the minimum time of treatment was 21 d.