Objective: Gonadotropin releasing hormones (GnRH) regulate the expression of annexin 5 in Leydig cells, and annexin 5 is supposed to be a signal molecule in regulating testosterone secretion. This study aimed to investigate the function of annexin 5 in male reproduction by observing its effect on human sperm motility in vitro.
Methods: The encoding sequence of rat annexin 5 was chemically synthesized and inserted into the HIS fusion expression vector pET28a. The expression of the fusion protein HIS-annexin 5 was induced by isopropyl-beta-D-thiogalactoside (IPTG) under the control of the T7 promoter, and the products were purified by affinity chromatography. The anticoagulant activity of annexin 5 was determined by the modified activated partial thromboplastin time (APTT) test. Semen samples from 15 donors were assigned to a control and an annexin 5 group, the latter treated with recombinant annexin 5 at the concentration of 10(-8) mol/L. Sperm motility and the percentage of grade a + b sperm were measured by computer-assisted semen analysis (CASA) after 20 and 60 min exposure, and the sperm ascending experiment was done after 20 min treatment.
Results: The product of the synthesized target gene was 947 bp in length, and the inserted sequence corresponded to the published encoding sequence of rat annexin 5. The plasmid pET28a-annexin 5 was transformed into E. coli BL21(DE3) and IPTG induced a fusion protein with a relative molecular weight of about 36,000, a purity of 95% and a high anticoagulant activity. Compared with the control group, sperm motility and the percentage of grade a + b sperm were increased by 40% (P < 0.01) and 21% (P < 0.01), respectively, after 20 min treatment with annexin 5, but neither showed any significant improvement after 60 min. The sperm ascending altitude was remarkably elevated after annexin 5 treatment, with extremely significant difference from the control group (37.84 +/- 6.35 vs. 49.5 +/- 12.27, P < 0.01).
Conclusion: An annexin 5 recombinant expression vector was successfully constructed. The protein annexin 5 can be efficiently expressed in E. coli and effectively improve human sperm motility in vitro.