Zona drilling increases the penetrability of rat oocytes matured in vitro

Immature rat follicular oocytes were cultured either with cumulus cells intact (CI) or cumulus-free (CF) in bovine serum albumin (BSA)- or serum-supplemented medium under conditions in which meiotic maturation occurs spontaneously. After 12 h of culture to permit in vitro maturation (IVM), the cumulus cells were stripped from the CI group. Control oocytes recovered 2-4 h after ovulation from oviducts of pregnant mare's serum gonadotropin (PMSG)-treated rats were similarly stripped of cumulus cells. Half the oocytes in each group had holes "drilled" in their zonae pellucidae by topical application of acid Tyrode's solution with a micropipette to enable bypass of the zona barrier to penetration. They were cultured for a further 14-16 h with epididymal sperm and then were assessed for sperm penetration and pronuclear formation. In a preliminary study using various concentration of sperm, 50,000 sperm/ml was identified as an appropriate concentration and was used in all subsequent experiments. For oocytes matured in serum-supplemented medium, penetration rates of non-drilled oocytes-expressed as a percentage of oocytes exposed to sperm for CF, CI, and ovulated oocytes were 10%, 34%, and 80%, respectively (p less than 0.01). Drilling significantly increased the penetration rates of both IVM groups (CF: 40%, CI: 77%) but not of ovulated oocytes (78%). Forty-one percent of non-drilled CF oocytes failed to form normal pronuclei after penetration. This was significantly higher than either the CI (0%) or ovulated (1%) groups (p less than 0.001). Drilling increased the incidence of failure to form normal pronuclei in penetrated oocytes of the CF group (64%) but not of the CI or ovulated groups.2z=