One-step collagenase method for zona pellucida removal in unfertilized eggs: easy and gentle method for large-scale preparation.

Purpose: Zona pellucida (ZP)-free eggs are often used for studies such as evaluating the interaction of sperm-oolemma. To acquire ZP-free eggs, the most commonly used methods employ acidified Tyrode's solution, enzymatic digestion with a trypsin-like enzyme, or mechanical methods using micropipettes. However, acidified Tyrode's solution and trypsin-like enzymes often damage the oolemma, especially when many eggs are treated at once for mass sample analyses. The mechanical method requires skill, and it is time-consuming to prepare many ZP-free eggs. Therefore, in this study, to establish an easy, reliable method for preparing ZP-free eggs, we examined the ZP digestion method originally reported by Zuccotti et al. (J Reprod Fertil 93:515-520, 1991) that uses collagenase.
Methods: Mouse unfertilized eggs were treated with collagenase and acidified Tyrode's solution to compare the ZP-free rates, the effect on the oolemma, and the two-cell development rates of ZP-free eggs by in vitro fertilization. The effects on the oolemma were gauged by observing the polarity of the transmembrane protein localization of enhanced green fluorescence protein tagged CD9 protein (CD9-EGFP) and using differential interference contrast microscopy.
Results: Collagenase removed the ZP and the cumulus cells from the cumulus oocyte complex. The collagenase method had no influence on the localization of CD9-EGFP, resulting in a high two-cell development rate. Additionally, the collagenase method could exclude low quality eggs with hardened ZP, since collagenase could not digest the hardened ZP. Conclusions: The one-step collagenase method is an easy preparation method for large numbers of high-quality ZP-free eggs.