Enhancing in vitro fertilization of mouse oocytes by partial zona pellucida digestion

Abstract

This work was undertaken in order to evaluate the effect of partial zona digestion on fertilization in vitro of mouse oocytes and assess zona surface changes induced by the procedure. Three hundred forty-six oocytes allocated for treatment were exposed to Ham's F-10 medium supplemented with 0.5% Pronase for either 3 min (188 oocytes) or 5 min (158 oocytes); 324 oocytes served as controls. Oocyte losses incurred as a result of the procedure were small (15 oocytes; 4.3%). Control and Pronase-treated oocytes were each divided into four subgroups and inseminated with 5 ×10 ⁵,5 ×10 ⁴,5 ×10 ³,or 5 ×10 ² sperm cells/ml. Fertilization was assessed 8 hr following insemination by the appearance of two pronuclei and development to the two- to four-cell stage the following day. The morphology of the zona pellucida following Pronase treatment was assessed by phase-contrast and scanning electron (SEM) microscopies performed immediately after treatment. Fertilization rate of control oocytes was 80% at a sperm concentration of 500,000/ml and gradually declined to ~30% at 500 cells/ml. In contrast, treated oocytes inseminated with 500 sperm cells/ml demonstrated a normal rate of fertilization. At this low sperm concentration the longer Pronase treatment was significantly (P < 0.05) more efficient in enhancing fertilization (69 and 88% for 3 and 5 min of Pronase treatment, respectively). Polyspermic fertilization was not observed in any of the subgroups. Phase-contrast microscopic examination of oocytes at the time of Pronase treatment showed an initial swelling of the zona pellucida for 30–60 sec with a time-dependent increase in its transparency. SEM demonstrated that the fine meshlike structure of the outer surface of zona pellucida digested away, leaving a smoother surface. These morphologic changes were not associated with a diminution in sperm binding or penetration. This work demonstrates that partial zona digestion, which causes uniform dissolution of the zona pellucida and reduction of its thickness, is simple and safe. The procedure significantly increased fertilization efficiency at very low sperm concentrations and could, by itself or in conjunction with other methodologies, improve the reproductive capacity of men producing sperm with a reduced penetrating ability.