Cholesterol added prior to vitrification on the cryotolerance of immature and in vitro matured bovine oocytes

Núria Arcarons,Manel López-Bejar,Kosala Rajapaksha,Meritxell Vendrell,Muhammad Anzar,Marc Yeste,Roser Morató,Teresa Mogas,Roger Sturmey

doi:10.1371/journal.pone.0184714

Abstract

This study examines whether incorporating cholesterol-loaded methyl-β-cyclodextrin (CLC) in the bovine oocyte plasma membrane improves oocyte tolerance to vitrification. In vitro matured oocytes were incubated with 2 mg/ml BODIPY-labeled CLC for different time intervals in FCS or PVA supplemented medium or exposed to different CLC concentrations to examine the subcellular localization of cholesterol by confocal microscopy live-cell imaging. Subsequently, the effects of optimized CLC concentrations and incubation times prior to vitrification on early embryo development were assessed. Then, we evaluated the effects of pretreatment with 2 mg/ml CLC for 30 min before the vitrification of immature (GV) and in vitro matured (MII) oocytes on developmental competence and gene expression. Our results indicate a high plasma membrane labeling intensity after 30 min of incubation with 2 mg/ml CLC for 30 min, regardless of the holding medium used. When oocytes were incubated with 1 mg/ml, 2 mg/ml and 3 mg/ml of CLC, intense labeling was observed at the plasma membrane after 40, 30 and 20 min, respectively. CLC pre-treatment before the vitrification of bovine oocytes did not affect subsequent cleavage and embryo development rates irrespective of CLC concentrations, incubation times or meiotic stage. However, pretreatment seems to improve the quality of embryos derived from vitrified oocytes, mainly when oocytes were vitrified at the GV stage.

Highlights

Widespread use of animal oocytes for procedures such as in vitro embryo production, nuclear transfer or gene banking has dramatically increased interest in oocyte cryopreservation in the agricultural and scientific communities [1]
In prior work in oocytes it was observed that the co-incubation of bovine immature [7] or in vitro-matured [8] oocytes with βCDs loaded with cholesterol (CLC) improved the nuclear maturation of oocytes after vitrification, but did not benefit embryo development to the blastocyst stage. These authors attributed the lack of effect of cholesterol-loaded methyl-β-cyclodextrin (CLC) treatment in improving cryotolerance that cholesterol was preferentially transferred to lipids or proteins present in the fetal calf serum (FCS) avoiding its incorporation into the oocyte
To examine whether cholesterol in a medium supplemented with FCS was transported into the oocyte, cholesterol internalization at different chase times was imaged in living bovine oocytes incubated with 2 mg/ml CLC in a medium containing FCS (Fig 1A) or PVA (Fig 1B)

Summary

Introduction

Widespread use of animal oocytes for procedures such as in vitro embryo production, nuclear transfer or gene banking has dramatically increased interest in oocyte cryopreservation in the agricultural and scientific communities [1]. The practical benefits of vitrification to preserve bovine oocytes are limited since vitrified oocytes show impaired in vitro maturation and early embryo development. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Methods

Results

Conclusion