Secretion of zona pellucida glycoprotein mZP2 by growing oocytes from mZP3(+/+) and mZP3(-/-) mice

The mouse egg extracellular coat, or zona pellucida (ZP), is composed of three glycoproteins, called mZP1-3, which are synthesized and secreted concomitantly by growing oocytes. Disruption of the mZP3 gene by targeted mutagenesis yields mice that are homozygous nulls (mZP3(-/-)). Growing oocytes from mZP3(-/-) mice do not synthesize mZP3 mRNA or protein and, as a result, do not assemble a ZP. Here, we examined secretion of mZP2 by growing oocytes and eggs from mZP3(-/-) mice, as well as incorporation of mZP2 into the ZP of oocytes from mZP3(+/+) mice. Laser scanning confocal microscopy (LSCM) of antibody-labeled samples showed that, indeed, mZP2 was synthesized and secreted by oocytes isolated from mZP3(-/-) mice and cultured in vitro. Nascent mZP2 was found in the culture medium, associated with the surface of the plasma membrane of growing oocytes, and in the oocyte cytoplasm. By contrast, mZP2 was barely detectable at any of these sites when ovulated eggs from mZP3(-/-) mice were examined. Examination of oocytes from wild-type [mZP3(+/+)) mice showed that, while a portion of nascent mZP2 was assembled into the ZP (approximately 40%), here too a significant fraction was secreted into the culture medium (approximately 60%). Similar results also were obtained when intact pre-antral follicles were isolated from mZP3(+/+) mice and cultured in vitro. Several of these observations are consistent with previous results obtained with oocytes from heterozygous null mice (mZP3(+/- )). Furthermore, the results suggest that ZP assembly from nascent glycoproteins may be a stochastic process that requires the presence of both mZP2 and mZP3 and occurs completely outside the growing oocyte.