[Expression of rat annexin 5 and its effect on human sperm motility in vitro].
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).


CONCLUSIONS
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.
Tao
Xiao-qian
XQ
PLA Research Institute of Clinical Laboratory Medicine, Nanjing General Hospital of Nanjing Military Region, Nanjing, Jiangsu 210002, China.
Liu
Hai-yan
HY
Shi
Shan-shan
SS
Han
Xue-feng
XF
Lin
Chai-ying
CY
Yao
Bing
B
D000818:Animals; D017304:Annexin A5 / Q000235:genetics / Q000494:pharmacology*; D005822:Genetic Vectors; D006801:Humans; D008297:Male; D010957:Plasmids; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000494:pharmacology; D013081:Sperm Motility
D017304:Annexin A5; D011993:Recombinant Fusion Proteins