502
EVALUATION OF HUMAN SPERM BINDING AND ACROSOME REACTION USING RECOMBINANT HUMAN ZONA PELLUCIDA PROTEINS.
Prasad, Sarvamangala1, Skinner, Sheri1, Son, Jung-Ho2, Meizel, Stanley2, Lamb, Dolores1, Dunbar, Bonnie1, 1 2
ABSTRACT- We have established a new method to rapidly and efficiently evaluate human sperm adherence to human zona pellucida (ZP) proteins using a modified whole cell enzyme linked assay procedure (ELISA). We have used recombinant ZP proteins expressed in Sf9 insect cells (baculovirus expression system) and native human and porcine ZP proteins to evaluate human sperm-ZP interactions. Initially we demonstrated the binding of native and recombinant human ZP proteins to ELISA plates using rabbit antibodies against porcine ZP since these antibodies are cross-reactive with most mammalian ZP proteins. In the sperm adherence assay, human ZP protein, native or recombinant was coated onto microtiter plates at various dilutions and incubated with capacitated human sperm for 1 hr at 37oC. Plates were gently rinsed in BWW media and bound sperm were fixed with glutarladehyde and incubated with anti-human sperm antisera followed by peroxidase labeled secondary antibody and the signal was developed using ABTS as chromophore. These studies demonstrate that capacitated human sperm adhere to all three baculovirus expressed ZP proteins and to heat solubilized native human ZP proteins in a dose dependent manner. Sperm adherence to recombinant ZP3 protein was the greatest and there was no sperm adherence to porcine ZP that is used as a non- specific control for this assay. We have also investigated the ZP induced acrosome reaction (AR) with recombinant human ZP3 protein expressed in CHO cells that was partially purified by DEAE chromatography. Human sperm capacitated for 24hr were incubated with recombinant ZP3 for various time points and AR was detected using FITC labeled ConA. These results demonstrate that ZP3 triggers acrosomal loss as compared to the vector protein control with maximal AR occurring at 30min (31.2% ± 1.0, n=3) although significant AR (17.5% ± 1.8, n=3) was observed even at 5min after incubation with ZP3. These studies therefore demonstrate the feasibility of utilizing human recombinant ZP proteins in studies to evaluate human sperm-ZP interactions and should provide a major source of ZP protein for use in clinical assays to evaluate male fertility. This project was funded by grants from the NIH to BSD (HD-17543 and HD-07495) and S.M. (HD-33368).
KEY WORDS: sperm-adherence assay, acrosome reaction, fertility assay, recombinant human zona pellucida