Human recombinant ZPC: evaluation of its biological activity

MARIN BRIGGILER, CLARA I; CAPECE, LORENA; GONZALEZ ECHEVERRIA, FERNANDA; FURLONG, LAURA; HARRIS, JEFF; VAZQUEZ LEVIN, MÓNICA

Buenos Aires

Congreso; Reunión Anual de la Sociedad Argentina de Biología; 2003

HUMAN RECOMBINANT ZPC: EVALUATION OF ITS BIOLOGICAL ACTIVITY. 1Marín-Briggiler CI, 1Capece L, 2Gonzalez-Echeverria F, 1Furlong  L, 3Harris J, 1Vazquez-Levin MH. 1IBYME-CONICET; 2Fertilab; Buenos Aires, Argentina. 3Zonagen, The Woodlands, Tx, EEUU.   The egg´s extracellular matrix (zona pellucida; ZP) is composed of three glycoproteins: ZPA, B and C. Native ZPC has been shown to interact with mouse sperm receptors and to induce the acrosome reaction (AR). Similar studies can not be done in the human due to scarcity of ZP. To overcome this limitation, ZP proteins have been produced in vitro using recombinant DNA technology. Objective: To evaluate the biological activity of human recombinant ZPC (rZPC) produced in CHO cells. Material and Methods: rZPC ability to recognize surface sperm antigens was evaluated after sperm incubation with rZPC, followed by its immunolocalization with an specific antibody. rZPC ability to interfere with sperm-ZP interaction was assessed using the hemizona assay, and calculating the HZ index (HZI=HZrZPC/HZcontrol). rZPC ability to induce the AR was determined, expressing the results as AR inducibility (I=% ARinducer – % ARspontaneous). Results: When incubated with rZPC (100 mg/ml), 28 % of the 4-h capacitated sperm showed specific staining over the acrosome. Sperm preincubation with rZPC reduced their ability to interact with the ZP (HZI=0.8±0.1 (100 mg/ml rZPC), 0.5±0.1 (10 mg/ml) vs 1.5±0.4 (0.1 mg/ml), p<0.05). Such reduction could not be attributed to alterations in sperm motility or to premature AR (data not shown). rZPC was able to induce the AR in 18-h capacitated sperm: I=19±6 % (200 mg/ml), 22±9 % (400 mg/ml) vs 26±7 % (10 % follicular fluid, positive control). Conclusions: rZPC was able to associate to human sperm, to compete with the native protein during gamete interaction and to induce the AR. The results indicate that rZPC could be used in the development of functional assays to assess human sperm fertilizing ability.