Effect of holding equine oocytes in meiosis inhibitor-free medium before in vitro maturation and of holding temperature on meiotic suppression and mitochondrial energy/redox potential.

Nicola A Martino,Manuel Filioli Uranio,Lucia Rutigliano,Katrin Hinrichs,Maria E Dell’Aquila,Giovanni M Lacalandra,Michele Nicassio

doi:10.1186/1477-7827-12-99

Abstract

BackgroundEvaluation of mitochondrial function offers an alternative to evaluate embryo development for assessment of oocyte viability, but little information is available on the relationship between mitochondrial and chromatin status in equine oocytes. We evaluated these parameters in immature equine oocytes either fixed immediately (IMM) or held overnight in an Earle’s/Hank’s’ M199-based medium in the absence of meiotic inhibitors (EH treatment), and in mature oocytes. We hypothesized that EH holding may affect mitochondrial function and that holding temperature may affect the efficiency of meiotic suppression.MethodsExperiment 1 - Equine oocytes processed immediately or held in EH at uncontrolled temperature (22 to 27°C) were evaluated for initial chromatin configuration, in vitro maturation (IVM) rates and mitochondrial energy/redox potential. Experiment 2 - We then investigated the effect of holding temperature (25°C, 30°C, 38°C) on initial chromatin status of held oocytes, and subsequently repeated mitochondrial energy/redox assessment of oocytes held at 25°C vs. immediately-evaluated controls.ResultsEH holding at uncontrolled temperature was associated with advancement of germinal vesicle (GV) chromatin condensation and with meiotic resumption, as well as a lower maturation rate after IVM. Holding did not have a significant effect on mitochondrial distribution within chromatin configurations. Independent of treatment, oocytes having condensed chromatin had a significantly higher proportion of perinuclear/pericortical mitochondrial distribution than did other GV configurations. Holding did not detrimentally affect oocyte energy/redox parameters in viable GV-stage oocytes. There were no significant differences in chromatin configuration between oocytes held at 25°C and controls, whereas holding at higher temperature was associated with meiosis resumption and loss of oocytes having the condensed chromatin GV configuration. Holding at 25°C was not associated with progression of mitochondrial distribution pattern and there were no significant differences in oocyte energy/redox parameters between these oocytes and controls.ConclusionsMitochondrial distribution in equine GV-stage oocytes is correlated with chromatin configuration within the GV. Progression of chromatin configuration and mitochondrial status during holding are dependent on temperature. EH holding at 25°C maintains meiotic arrest, viability and mitochondrial potential of equine oocytes. This is the first report on the effects of EH treatment on oocyte mitochondrial energy/redox potential.

Highlights

Evaluation of mitochondrial function offers an alternative to evaluate embryo development for assessment of oocyte viability, but little information is available on the relationship between mitochondrial and chromatin status in equine oocytes
Experiment 1: effects of EH treatment on initial nuclear chromatin configuration, meiotic competence and mitochondrial energy/redox potential of equine oocytes A total of 95 non-cultured oocytes was evaluated for chromatin configuration, 45 in the IMM group and 50 in the EH group, comprising four replicates
There were no significant differences in mitochondrial distribution within chromatin configurations between EH and IMM oocytes (P >0.1), results for oocytes within the IMM and EH treatments were pooled to determine the association of chromatin configuration with mitochondrial distribution

Summary

Introduction

Evaluation of mitochondrial function offers an alternative to evaluate embryo development for assessment of oocyte viability, but little information is available on the relationship between mitochondrial and chromatin status in equine oocytes We evaluated these parameters in immature equine oocytes either fixed immediately (IMM) or held overnight in an Earle’s/Hank’s’ M199-based medium in the absence of meiotic inhibitors (EH treatment), and in mature oocytes. Use of a modified Earle’s/Hank’s M199 (EH medium, [1]) at room temperature has been shown to effectively maintain equine oocytes in meiotic arrest in the absence of meiotic inhibitors This oocyte holding technique has been utilized in studies on both intracytoplasmic sperm injection (ICSI) and nuclear transfer [2,3,4,5,6,7,8], to enable scheduling of in vitro maturation (IVM) and subsequent manipulations. The mode by which this method maintains meiotic arrest is currently unknown; from initial findings in our study, we hypothesized that holding temperature affects oocyte meiotic arrest

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Conclusion