Thiocyanate Assay for Sublytic Change in the Zona Pellucida of the Mouse Egg1

Abstract

A new quantitative method is described for analyzing the state of the zona pellucida of mouse eggs and for ascertaining the extent to which the zona may have been previously exposed to sublytic levels of zonalytic factors. Thus, in vivo changes in activity of the uterine zonalytic factor with time may be indirectly titrated. The method consists of determining the time required to lyse the zona under specified controlled conditions in a solution of the chaotropic anion, thiocyanate (SCN⁻). Zona lysis in SCN⁻ (as the sodium salt) is concentration and temperature dependent. From the kinetics of the reaction, it is hypothesized that lysis results from a multihit phenomenon, and that adsorption of zonalytic agents on the zona is not a rate-limiting factor. Neither incubation of the eggs in concentrated buffers nor fertilization significantly affects the zona lysis time in SCN⁻. Preincubating the zona in pH extremes, however, does reduce the lysis time. Application of the SCN⁻ assay to zonas of eggs obtained at progressive times after the start of pseudopregnancy confirms that zonalytic factor is absent or undetectable in the oviducts but is present in the uterus. After egg transport from the oviducts in the ICR strain, eggs are in the uterus for at least 9 hr before any change in the zona can be detected with SCN⁻. By the afternoon of Day 3 in this strain (counting the plug date as Day 0), the uterine factor has caused marked changes in the zona, before in vivo lysis is evident. Our previous observations on strain differences in the time course of zona change are supported. The results are consistent with the working hypothesis (Mintz, 1970) that the uterine zonalytic agent may be an implantation-initiating factor (IIF).