Egg-Induced Modifications of the Murine Zona Pellucida

Abstract

We are interested in the biochemical mechanisms involved in the early events of mammalian egg activation, with special reference to the mechanism(s) by which the block to polyspermy at the level of the zona pellucida (ZP) is established. Since some of the initial events of sperm-induced egg activation in lower species are accompanied by the hydrolysis of polyphosphoinositides, we examined the effects of protein kinase C (PKC) activators (phorbol diesters and diacylglycerols) and microinjected inositol 1,4,5-trisphosphate (InsP₃) on sperm-ZP binding, the ZP-induced acrosome reaction, fertilization, and egg induced modifications of the ZP.

Sperm penetration of the ZP and fertilization are inhibited in mouse eggs treated with biologically active phorbol diesters and the diacylglycerol, sn-I ,2,-dioctanoyl glycerol (diC₈). The effect is mediated by the ZP, since ZP-free eggs treated with these compounds are fertilized to the same extent as untreated eggs; these compounds also exert no direct inhibitory effect on the sperm’s ability to fertilize an egg. The inhibitory effect of these compounds on fertilization is due to a step subsequent to sperm binding, since the ability of untreated and treated eggs to bind sperm is similar. Two-dimensional reduction gel electrophoresis of iodinated ZP obtained from phorbol diester or diC₈-treated eggs demonstrated that ZP2 is modified to ZP2_f; such a modification is similar to that observed in ZP isolated from 2-cell embryos. Analysis of the sperm receptor and acrosome reaction-inducing activities of the ZP (i.e., properties of ZP3) from these treated eggs demonstrated that receptor activity remained but that the acrosome reaction-inducing activity was altered; sperm could initiate but not complete the acrosome reaction. Similarly, purified ZP3 prepared from phorbol diester-treated eggs possesses full sperm receptor activity, and can initiate but not complete the acrosome reaction. This is in contrast to ZP3 from 2-cell embryos, which cannot even initiate an acrosome reaction. Thus, we have been able to elicit an egg-induced dissociation of the receptor and acrosome reaction-inducing activities of the ZP, which should provide a means to study the components of ZP3 involved in the acrosome reaction. Moreover, ZP obtained from phorbol diester-treated eggs may serve as a probe to delineate the sequence of events comprising the acrosome reaction.

Since the previous experiments utilizing PKC activators represent the examination of one-half of a second messenger cascade generated upon the hydrolysis of phosphatidylinositol-1,4,-bisphosphate (PIP₂) by a PIP₂-specific phospholipase C, we investigated the effects of microinjecting InsP₃ into mouse eggs on egg activation and egg-induced modifications of the ZP.

A concentration-dependent increase in the percentage of eggs displaying an egg-induced modification of ZP2 to ZP2_f was observed in cells microinjected with increasing concentrations of InsP3 (0.1 nM to 4 μM final concentration). InsP₃ concentrations greater than 1 nM elicited an egg-induced modification of ZP2 to ZP2_f ; EC₅₀=5 nM. These InsP₃ effects also occurred in the absence of extracellular Ca²⁺. Microinjection of inositol 1,3,4,5-tetrakisphosphate, which has been implicated in activating plasma membrane associated Ca^2- channels in eggs of lower species, did not result in an egg-induced modification of ZP2. Similarly, microinjection of I(1,4)P₂’ I(2,4,5)P₃, or I(1,3,4)P₃, which do not release intracellular Ca²⁺, failed to cause an egg-induced modification of ZP2. Analysis of sperm binding to the ZP of InsP₃ microinjected eggs demonstrated that binding was decreased when compared to vehicle injected eggs. These binding studies suggest that at least one of the biological activities of the ZP (i.e., sperm receptor activity) may be modified by InsP₃ microinjection. It remains to be determined whether the acrosome reaction-inducing properties of the ZP are modified by such treatments. Fluorograms of ³⁵S-methionine labeled mouse eggs thlt had been injected with InsP₃ revealed labeling patterns similar to that of vehicle injected eggs; these patterns contrasted with those seen with I-cell embryos. This is consistent with a low level of egg activation (17%), as demonstrated by emission of a second pollr body. InsP₃ injection, therefore, did not generate those global changes in protein synthesis normally associated with fertilization.