Cumulus expansion, nuclear maturation and connexin 43, cyclooxygenase-2 and FSH receptor mRNA expression in equine cumulus-oocyte complexes cultured in vitro in the presence of FSH and precursors for hyaluronic acid synthesis

Maria Dell'Aquila,Filippo Maritato,Ghylène Goudet,Maud Caillaud,Paolo Minoia,Nadine Gérard,Giulio Aiudi,Alain Martoriati

doi:10.1186/1477-7827-2-44

Abstract

The aim of this study was to investigate cumulus expansion, nuclear maturation and expression of connexin 43, cyclooxygenase-2 and FSH receptor transcripts in equine cumuli oophori during in vivo and in vitro maturation in the presence of equine FSH (eFSH) and precursors for hyaluronic acid synthesis. Equine cumulus-oocyte complexes (COC) were cultured in a control defined medium supplemented with eFSH (0 to 5 micrograms/ml), Fetal Calf Serum (FCS), precursors for hyaluronic acid synthesis or glutamine according to the experiments. After in vitro maturation, the cumulus expansion rate was increased with 1 microgram/ml eFSH, and was the highest with 20% FCS. It was not influenced by precursors for hyaluronic acid synthesis or glutamine. The expression of transcripts related to cumulus expansion was analyzed in equine cumulus cells before maturation, and after in vivo and in vitro maturation, by using reverse transcription-polymerase chain reaction (RT-PCR) with specific primers. Connexin 43, cyclooxygenase-2 (COX-2) and FSH receptor (FSHr) mRNA were detected in equine cumulus cells before and after maturation. Their level did not vary during in vivo or in vitro maturation and was influenced neither by FSH nor by precursors for hyaluronic acid synthesis. Results indicate that previously reported regulation of connexin 43 and COX-2 proteins during equine COC maturation may involve post-transcriptional mechanisms.

Highlights

In mammals, embryos produced in vitro, in sequential steps of oocyte maturation in vitro (IVM), conventional fertilization in vitro (IVF) or intracytoplasmic sperm injection (ICSI) and embryo culture (EC), display marked differences from their in vivo counterparts with regard to morphology, timing of development, resistance to low temperature, metabolism and gene expression [1]
The aim of this study was to improve in vitro cumulus expansion in the equine cumulus-oocyte complexes (COC), since optimal expansion of the cumulus mass is essential for cytoplasmic maturation
In order to elucidate the mechanism of equine cumulus expansion, we analyzed the expression of connexin 43, COX-2 and FSH receptors mRNA during COCs maturation

Summary

Introduction

Embryos produced in vitro, in sequential steps of oocyte maturation in vitro (IVM), conventional fertilization in vitro (IVF) or intracytoplasmic sperm injection (ICSI) and embryo culture (EC), display marked differences from their in vivo counterparts with regard to morphology, timing of development, resistance to low temperature, metabolism and gene expression [1]. Their clinical applications remain sub-optimal [2,3,4]. Expanded oocytes issuing from apoptotic follicles show increased meiotic competence, but not increased activation rate after ICSI [10]

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