Apolipoprotein A1 Is Located In Human Sperm And Relates With Sperm Activity

To discover the mechanism of immune infertility,we apply co-immunoprecipitation combined with liquid chromatogram mass/mass(LC-MS/MS)to screen the human sperm membrane protein involved in sterility.Through the software analysis,15 different proteins are found in human spermatozoa membrane related with fertility.The apolipoprotein A1(APOA1)is selected to analyze its expression and function in human spermatozoa.Semen samples(n=26)were obtained from healthy males by masturbation after 3 to 5 days of abstinence.APOA1 expression was detected by reverse transcription–polymerase chain reaction(RT-PCR),Western blot and immunofluorescence in sperm cells.Set up a blank control group,rabbit polyclonal IgG group,anti-APOA1 antibody group which contains 10μg/ml,20μg/ml and 40μg/ml APOA1 antibody,to treat normal semen samples and incubated at 37℃for 1h,2h and 3h.The progressive motility of spermatozoa was determined by a computer-assisted motion analyzer and apoptosis rate and mitochondrial membrane potential(MMP)were analyzed by flow cytometry.The mouse sperm was incubated with 40μg/ml IgG antibody or APOA1 antibody for 1 h,and then subjected to in vitro fertilization experiments to observe the difference in fertilization rate.The percentage of APOA1-active sperm cells were detected by flow cytometry.Further the correlation of APOA1-active sperm cells with sperm motility in 83 semen samples were calculated by statistical software.We identified APOA1 mRNA was presented in sperm and the APOA1,the molecular weight of 31kD,was located at the post-acrosomal region and necks of human sperm.A significant decline in sperm progressive motility(PR)was observed after 1,2 and 3 hours of incubation with APOA1 antibody.There was a statistically significant difference between the blank control group and the APOA1 antibody concentration of 20μg/ml and 40μg/ml groups(1 hour after incubation,blank control group(68.65±15.7)%with 20μg/ml group(48.45±5.2)%,40μg/ml group(39.25±7.89)%,t=2.442,3.345,both P<0.05;2 hours after incubation,blank control group(55.33±10.12)%with 20μg/ml group(28.68±11.7)%,40μg/ml group(18.13±10.52)%,t=3.445,5.097,both P<0.05;3 hours after incubation,blank control group(35.73±14.08)%with 20μg/ml group(15.53±8.42)%,40μg/ml group(9.98±7.08)%,t=2.462,3.268,both P<0.05).Moreover,we analyzed the changes of sperm immobility after 2h incubation,the blank control group and the APOA1 antibody concentration of 20μg/ml and 40μg/ml groups were significant statistically,blank control group(32.15±8.67)%with 20μg/ml group(57.85±11.93)%,40μg/ml group(68.15±13.92)%,t=3.485,4.390,both P<0.05After incubation 2 hours,the percentage of apoptotic cells increased from(16.02±4.28)%in the blank control group to(21.72±2.67)%in the20μg/ml APOA1 antibody group and(28.01±3.93)%in the 40μg/ml APOA1 antibody group,respectively(t=3.177,5.834,both P<0.01).After incubation 2 hours,the percentage of lowΔψspermatozoa increases from(21.53±2.47)%in the blank control group to(33.13±5.48)%and(36.36±5.35)%,respectively in 20 and 40μg/ml concentrations of APOA1antibody treated group(t=3.34 and 4.355,both P<0.05).The oocyte fertilization level of inhibition by APOA1 antibody(35%)was statistically significant(χ~2=48.664,p<0.001)as compared to control IgG(81%).The percentage of APOA1-positive cells showed a strong positive correlation with the percentage of PR spermatozoa(r=0.427,P<0.01).A negative correlation between the percentage of immotile sperm and APOA1 levels were observed(r=-0.504,p<0.01).The percentage of APOA1-positive sperm cells was significantly lower in asthenozoospermic patients(57.74±11.43)%than in normozoospermia(66.54±12.96)%(t=2.812,P<0.01).The APOA1 mRNA and protein are expressed in human spermatozoa.The APOA1 antibody causes the deduce of PR sperm,the increase of apoptosis rate,decrease of the sperm MMP and the mouse fertilization in vitro.Therefore,APOA1 plays an important role in maintaining sperm motility and vitality.The levels of APOA1 in human sperm have a strong positive correlation with the percentage of PR spermatozoa and a negative correlation with the immotile spermatozoa.The percentage of APOA1-positive sperm cells is significantly lower in asthenozoospermic samples,therefore it could be the potential marker of sperm motility to assess the sperm quality.