Reversible Disassembly of the Actin Cytoskeleton Improves the Survival Rate and Developmental Competence of Cryopreserved Mouse Oocytes

Basarab G Hosu,John K Critser,Steven F Mullen,Gabor Forgacs

doi:10.1371/journal.pone.0002787

Basarab G Hosu, John K Critser + Show 2 more

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https://doi.org/10.1371/journal.pone.0002787

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Abstract

Effective cryopreservation of oocytes is critically needed in many areas of human reproductive medicine and basic science, such as stem cell research. Currently, oocyte cryopreservation has a low success rate. The goal of this study was to understand the mechanisms associated with oocyte cryopreservation through biophysical means using a mouse model. Specifically, we experimentally investigated the biomechanical properties of the ooplasm prior and after cryopreservation as well as the consequences of reversible dismantling of the F-actin network in mouse oocytes prior to freezing. The study was complemented with the evaluation of post-thaw developmental competence of oocytes after in vitro fertilization. Our results show that the freezing-thawing process markedly alters the physiological viscoelastic properties of the actin cytoskeleton. The reversible depolymerization of the F-actin network prior to freezing preserves normal ooplasm viscoelastic properties, results in high post-thaw survival and significantly improves developmental competence. These findings provide new information on the biophysical characteristics of mammalian oocytes, identify a pathophysiological mechanism underlying cryodamage and suggest a novel cryopreservation method.

Highlights

Assisted reproductive technologies have become a solution for millions of infertile couples worldwide, and an essential tool for transgenic animal production [1], embryonic stem cell research and reproductive or therapeutic cloning [2]
Since microfilaments control a number of cellular functions [18] and postfertilization events in oocytes [19], in the present work we investigated the relationship between the survival rate and developmental competence of cryopreserved oocytes and those biophysical properties of the cytoplasm that strongly depend on the integrity of the actin cytoskeleton
The aim of this study was to investigate the mechanisms associated with cryodamage and apply this new knowledge to improve on the survival rate and developmental competence of cryopreserved oocytes

Summary

Introduction

Assisted reproductive technologies have become a solution for millions of infertile couples worldwide, and an essential tool for transgenic animal production [1], embryonic stem cell research and reproductive or therapeutic cloning [2]. Mature oocytes are arrested in metaphase of the second meiotic reduction (metaphase II or MII oocytes) and are not capable of normal cell division until fertilization. In this regard, MII oocytes are unlike cell lines which can ‘‘survive’’ cryopreservation by compensating for low cryopreservation efficiencies via rapid cell division either in vitro (e.g., many immortalized cell lines) or in vivo (e.g., hematopoietic stem cells). For most mammalian species studied to date, the overall success of the method (taking into account the cryosurvival rate, the fertilization and the developmental competence of thawed oocytes) is very low; recent publications using human oocytes report developmental competence less than 10% (1%, [4]; 5.6% [5]). Understanding the mechanism of cryodamage would facilitate the development of improved cryopreservation techniques that yield higher survival rates and better developmental competence

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