Studies Of The Biological Activities Of The Recombinant Human Zona Pellucida-3 Protein (rhZP3)

Zona pellucida (ZP), an extracellular matrix surrounding oocytes, has received considerable attentions as its key role in reproductive processes. However, the exiguity of human ZP resource has impeded its study and application. Human zona pellucida protein and peptides have obtained for reproductive research by gene-recombination technology. In the present study, the activities of the recombinant human zona pellucida 3 protein (rhZP3) was observed by two methods, inducing acrosome reaction (AR) and sperm-egg interaction. The results will provide basic evidence for anti-fertility research and developing some reagents for detection of the fertility.The effect of rhZP3 on ZP-induced AR was determined by the AR rate of incubating sperm from the same donor with BSA medium, progestone and different concentration of rhZP3 respectively. The acrosome status of sperm was evaluated by Coomassie brilliant blue (CBB) staining. The effects of purified rhZP3 on human sperm-egg interaction were investigated by two ways and the sperm and ZP were from the same donor. Sperm-egg interaction of mouse was used to be an accessorial model due to the limitation of human oocytes. The sperm were pretreated with rhZP3 in different concentration and control medium and then co-incubated with zona-intact oocytes. After the oocytes were washed for several times, the number of sperm binding on oocytes was recorded. The effect of anti-rhZP3 antibody on sperm-egg binding was evaluated also. The oocytes pretreated with different concentration antiserum and negative serum co-incubated with sperm from same donor and the numbers of sperms binding on each group were record. Meantime, the antiserum was used in immunohistochemistry to evaluate the effect on ovrian.The result of induced AR showed that there were significant differences between the rhZP3 group and the BSA group (P<0.05) and the AR rate increased with the concentration of rhZP3. rhZP3 can induce the acrosome reaction and the AR percentage of sperm was correlated with the concentration of rhZP3 to some extent. Statistically differences between control and tested group were observed in sperm-egg binding tests(P<0.05) and the results showed that rhZP3 and anti-rhZP3 antibody could strongly interfere with sperm-egg binding both in mouse and human. This decrease of bound sperm in sperm-egg interaction depended on the dose of rhZP3 and rhZP3 antiserum employed and the inhibitory effect on human was more prominent than that on mouse. The immunoreaction of anti-rhZP3 serum to ovarian section was weakly.In conclusion, the biological activities of rhZP3 were similar to natural human ZP and the immunoreaction of anti-rhZP3 serum to ovary was low. These results implied rhZP3 has the potential to develop diagnostic kits for the AR and safe, effective contraceptive vaccine.