Apolipoprotein A1 localization in sperms and its antibody′s effect on human sperm function

Abstract

Objective To investigate the presence of apolipoprotein A1 (APOA1) and its function in human spermatozoa. Methods The expression assays for APOA1 were carried out by immunofluorescence and Western blotting in human sperm cells. Set up a blank control group, rabbit polyclonal IgG group, which contain anti-APOA1 antibody 10 μg/ml, 20 μg/ml and 40 μg/ml, to treat normal semen samples and incubated at 37 ℃ for 1 h, 2 h and 3 h. The progressive motility of spermatozoa was determined bya computer-assisted motion analyzer(CASA), apoptosis rate by flow cytometry and ultrastructural changes by electron microscopy. Comparisons of sperm progressive motility and apoptosis rate were performed by independent sample t tests. Results The study showed APOA1 protein mainly located in the sperm head, the molecular size was 31 000. A significant decline in sperm progressive motility 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 20 μg/ml and 40 μg/ml groups [1 hour after incubation, blank control group(68.65%±15.70%) with 20 μg/ml group(48.45%±5.20%), 40 μg/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 20 μg/ml group(28.68%±11.70%), 40 μg/ml group(18.13%±10.52%), t=3.445, 5.097, bothP<0.05; 3 hours after incubation, blank control group(35.73%±14.08%) with 20 μg/ml group(15.53%±8.42%), 40 μg/ml group(9.98%±7.08%), t=2.462, 3.268, both P<0.05]. After incubation 2 hours, the percentage of apoptotic cells increased from 16.02%±4.28% in the blank control group to 21.72%±2.67% (20 μg/ml APOA1 antibody)and 28.01%±3.93% (40 μg/ml APOA1 antibody), respectively (t=3.177, 5.834, bothP<0.01). Treatment with 40 μg/ml APOA1 antibody for 4 hours resulted in major morphological changes to sperm cells observed by electron microscope, including membrane distension, vacuole formation and different periods of apoptosis cells. Conclusion APOA1 plays an important role in maintaining sperm motility and survival rate, however the mechanism needs further study.(Chin J Lab Med, 2018, 41: 664-668) Key words: Apolipoprotein A-I; Sperm motility; Apoptosis; Flow cytometry; Microscopy, electron, transmission