The Studies On Apoptosis Of Rat Sperm Induced By Antisperin Antibody And Its Mechanism Of Action

Objective:1 Summarize and explore the methods of seting up AsAb positive rat model for accumulating the data and empirical methods of the experiment study on rat about antisperm antibody;2 Investigate the differences of the effects about sperm apoptosis acted by serum AsAb and semen AsAb, in the application of staining of PI and FCM analysis the relationship of anti-sperm antibodies and sperm apoptosis, and accumulate the information about studing on the etiological factor of autoimmune infertility;3 In application of staining of JC-1 and FCM, electron microscopy, western blot, and spectrophotometric analysis on apoptosis sperms about MMP , ultrastructure of mitochondria, release of cytochrome c and caspase-3 activity for the documentation of the study on apoptosis pathway AsAb induced by.Method: 1 Set up SD rat animal model of AsAb positive : The 40 male SD rats, animals Level SPF, body weight 220 ~ 250g, rear under constant temperature conditions, free water and eating.Rats were randomly divided into 2 groups: experimental group 30, control group 10. Using the sperm of homogeneity and Freund's adjuvant on the rats of experiment for immune. The control group was injected with normal saline.Repeat every 7 day.Modeling cycle for 1-1.5 months;2 The electric technologies was used to take rats semen: provide electric ejaculation instrument. Fix rat by tubular cage, cut the hair of the back waist region to the Department of Bioglass,wipe and wet the skin by normal saline.Fix the copper electrodes(3×3cm) at theb shearing region, abut the auxiliary electrode of electric ejaculation instrument. Rod-shaped electrode inserted gently to anus of rat about 2 ~ 3cm, abut the exciting electrode of electric ejaculation instrument. Connected to power.Every rat shoud be ejaculated by stimulated 3-7 times on average.3 Detect serum, semen AsAb of two groups of rats by ELISA: collect semen and blood samples of rats abd steps below elisa kit instructions in accordance. AsAb in serum and semen are positive proves the success of the local model, it's the semen group. Serum AsAb positive proves success of cycle model, it's the serum group.4 Annexin V/PI staining combined with FCM detect the rate of sperm apoptosis of three groups of rats: Prepare sperm samples of three groups of rats. sperm samples were made with incubated sperm buffer into suspension of (3~6)×106/ml.Take Annexin V(0.2mg/ml)5ul into suspension of sperms 195 ul. Incubated 10min in room temperature and away from light. centrifugalization(200×g,5min). Scoured were the samples by PBS 3 times and taked into PBS 190ul. Take PI(0.1mg/ml)10ul into it. Incubated 20min in room temperature and away from light.Detect it by FCM. Excitation wavelength was 488nm.Cell Quest software used to collect 10000 cell, analysis the percentage of eupyrene sperms,earlier period or advanced stage apoptosis sperms and death sperms.5 Detection of Sperm MMP of three groups of rats by JC-1 Fluorescent Staining and FCM. Semen samples will be washed with PBS 2 times and centrifugated. Re-suspended it by PBS and adjusted the density of sperm 1×106/ml. Adding JC-1 of final concentration was 5μg/ml, incubated in incubator under 37℃, dark 10min, washed by PBS to remove excess free dye, re-suspended by PBS and detected on flow cytometry immediately. Excitation wavelength was 488nm, In application of prior to the scattered light (FSC) and side scatter (SSC) gating, through fluorescence channel FL1 (green) and FL2 (red) to detect fluorescence intensity. Each sperm suspension samples to obtain 10000 cells. CellQuest software was used to access and analyze data, using the rate of fluorescent orange-red sperm express the percentage of MMP in normal of sperms.6 Detect the release of cytochrome c of sperm of three groups of rats by Western blot: sperm samples was homogenated in the ice bath in the glass, separated nucleus and mitochondria by taking graded differential centrifugation, collected for Samples of cytoplasmic proteins and mitochondrial protein. Determination the content of protein respectively, add in the buffer, heating denaturation, separate the proteins by SDS-polyacrylamide gel electrophoresis, semi-dry trarsmembrane, closure. Add in Rabbit Anty-Cytochrome C orβ-Actin separately, incubated overnight. Add the secondary antibody of horseradish peroxidase labeled, incubated 1h, ECL detection, to take photographs. Use Image-Pro Plus 6.0 software to analysis the gray value.7 Spectrophotometry Detect the activity of caspase-3 : Collect the sperm of three groups of rats and follow with the directions of kit to analysis the activity of caspase-3, reading by microplate reader.8 By TEM observed the mitochondrial ultrastructural of sperms of 3 group of rats. After collecting sperm samples, centrifuged and fixed. Treatment and cross-section was finished in electron microscopy room, the mitochondrial ultrastructure was observed by transmission electron microscope.9 By optical microscopy observed the organizational structure of the epididymis of three groups of rats. Get the epididymis of three groups of rats, Treatment and cross-section was finished in patho-department, the organizational structurewas observed by the optical microscope the organizational structure.10 Methods of statistical analysis: All statistical analysis were finished by SAS statistical software. 40 SD rats were divided into semen groups, serum groups, control group, their rate of sperm apoptosis, sperm mitochondrial transmembrane potential, caspase-3 activity values, cytochrome c protein gray value firms separately statistical analysis, using the organizational structure, one-way ANOVA respectively, with measurement data ( x±s), inter-group comparison method SNK and LSD.Results:1 AsAb test results. In accordance with the Kit's standards the value of AsAb higher than 60 U/ml was positive. Experimental group, 30 SD rats ,were positive for serum AsAb, antibody value was (79.8±7.1)U/ml, 12 of them were serum, semen AsAb-positive, semen antibody value was (76.7±6.6) U/ml, serum antibody value was (81.5±7.7) U/ml, the remaining 18 were semen antisperm antibody negative. The serum AsAb-positive, antibody values was (78.1±7.5) U/ml. The control group were all negative.2 Annexin V/PI staining combined with FCM detection of apoptosis results: The rates of sperm apoptosis of semen group, serum group and control group were( 35.88±6.49)%,(14.30±2.30)%and(12.38±2.56)%。In the group comparison of serum group and control group ,the difference has no significant(P>0.05).In the group comparison of semen group and control group, the difference has statistical significance (P<0.001)。In the group comparison of semen group and serum group ,the difference has statistically significant (P<0.001)。3 JC-1 staining combined with FCM detection of sperm MMP results. In semen group, the percentage of sperm of fluorescent orange-red was ( 40.25±9.50 ) % ,Serum group was(80.87±14.15)%,The control group was(81.56±12.78)%.In the group comparison of semen and control groups the difference has significant (P<0.001); In the group comparison of semen group and serum group the difference has significant (P<0.001); In the group comparison of serum group and control group the difference has not statistically significant(P>0.05).4 The results of the detection of cytochrome c released from mitochondrial by Western blot was: The ratio of gray-scale of Cytochrome c in cytosolic and the reference was: The differences in semen group and serum group, semen group and control group were significant (P<0.05); The differences in serum group and control group was not statistically significant(P>0.05). The ratio of gray-scale of The Cytochrome c in Mitochondrial and the reference was: The differences in semen group and serum group, semen group and control group were significant (P<0.05); The differences in serum group and control group the was not statistically significant(P>0.05). Under the role of AsAb in semen, sperm's cytochrome c in mitochondrial of the semen group transfer the location, released from the mitochondria to the cytoplasm. AsAb in serum does not have affect on the release of cytochrome c.5 The results of Spectrophotometric detection of the of the caspase-3 were: The difference in semen group and serum group was significant(P<0.05); The difference in semen group and control group was significan(tP<0.05); The difference in serum group and control group was not statistically significant(P>0.05).Under the role of AsAb in semen, activity of Caspase 3 was increased, but did not have affect with AsAb in serum.6 The results of TEM observation mitochondrial ultrastructure were: In the sperms of serum group, mitochondria has various sizes, uneven distribution, obvious swelling, deformity or the giant mitochondria in part of them, shaped as vacuoles, fuzzy and even loss of mitochondrial cristae. Ultrastructure of serum and control group were normal.7 Observed epididymal organizational of three groups under the optical microscope shows that the organizational structure of rats' epididymal in semen group, epithelial cell was hyperplasia, Leydig cell was edema, luminal was stenosis, and the number of sperms in intraluminal decreased significantly. Serum and control group were normal.Conclusion:1 This experiment used allogeneic sperm with Najaf's immune adjuvants on immunity in rats modeling has high rate of success, shorter-cycle and it can be set up semen AsAb-positive model .It's the simple reliable method of establishment of AsAb in rat model.2 Semen AsAb can induce apoptosis in sperm, serum AsAb had no effect on apoptosis of sperm. It provides an important reference for the cause for the study of anti-sperm antibody-mediated immune infertility .3 Semen AsAb can lead to decrease sperms' MMP, release mitochondrials' cytochrome c, increase caspase-3 activity, pathological changes in mitochondrials' ultrastructure, histopathological change in epididymal.It provides some important evidences for the study of the mitochondria way of AsAb induced apoptosis in sperm .