In vitro decondensation of mammalian sperm and subsequent formation of pronuclei-like structures for micromanipulation

Abstract

In this study, we describe an efficient protocol for the formation of in vitro developed pronuclei for micromanipulation techniques. Our approach involved incubation of demembranated or permeabilized mammalian sperm in a phosphate buffer supplemented with heparin and β-mercaptoethanol. Under the prevailing conditions, we achieved a uniform and reliable synchronous decondensation of sperm nuclear DNA. This initial decondensation facilitated the removal of mammalian protamines upon subsequent incubation in an amphibian egg extract. The interchange of protamines for histones to stabilize the DNA structure is recognized as a prerequisite for pronuclear formation. Furthermore, immunocytochemical studies have revealed that pronuclear development is accompanied by the formation of a nuclear lamina with corresponding DNA synthesis. The method described gave a high yield of nuclei during pronuclear formation. Ultimately, our aim is to transfer the in vitro-developed pronuclei into mammalian oocytes by micromanipulation. This novel procedure may prove useful in alleviating severe male factor problems especially in oligozoospermic cases in our in vitro fertilization center.