POLARIZED-LIGHT MICROSCOPY AND DIGITAL IMAGE-PROCESSING IDENTIFY A MULTILAMINAR STRUCTURE OF THE HAMSTER ZONA-PELLUCIDA

The zona pellucida (zona) is a glycoprotein coat that envelopes the oo cyte and embryo, binds sperm during fertilization and facilitates tran sfer of the embryo through the Fallopian tube. Before implantation can occur, the blastocyst must hatch from the zona. Several lines of evid ence suggest that the zona is multilaminar. We hypothesized that the m ultilaminar structure of the zona filaments could be imaged nondestruc tively with the polarized light microscope. A recent modification of t he polarized light microscope (pol-scope), which combines innovations in polarization optics with novel image processing software, allows me asurement of birefringence at all points of the image. Hamster metapha se II oocytes were placed on glass coverslips which replaced the botto m of culture dishes, imaged under differential interference contrast ( DIG) and pol-scope optics, then digitized and processed to measure bir efringence magnitude and orientation. The pol-scope revealed the zona to be divided into outer and inner layers separated by a zone of low r etardance. This finding is consistent with filaments in the outer laye r oriented tangentially and in the inner layer oriented radially. The multilaminar structure of the mammalian zona suggested by differential lectin binding and by scanning electron microscopy could be imaged no n-destructively with the pol-scope. Because the pol-scope provides a n on-destructive method to identify macro-molecular organization of the zona, it may prove useful in developmental studies of hatching and to direct resection of the zona.