Transcriptome based identification of mouse cumulus cell markers that predict the developmental competence of their enclosed antral oocytes

Giulia Vigone,Maurizio Zuccotti,Riccardo Bellazzi,Carlo Alberto Redi,Valeria Merico,James Adjaye,Matteo Gabetta,Giuliano Mazzini,Alessandro Prigione,Francesca Mulas,Silvia Garagna,Lucia Sacchi

doi:10.1186/1471-2164-14-380

Abstract

BackgroundThe cumulus cells (CCs) enveloping antral and ovulated oocytes have been regarded as putative source of non-invasive markers of the oocyte developmental competence. A number of studies have indeed observed a correlation between CCs gene expression, embryo quality, and final pregnancy outcome. Here, we isolated CCs from antral mouse oocytes of known developmental incompetence (NSN-CCs) or competence (SN-CCs) and compared their transcriptomes with the aim of identifying distinct marker transcripts.ResultsGlobal gene expression analysis highlighted that both types of CCs share similar transcriptomes, with the exception of 422 genes, 97.6% of which were down-regulated in NSN-CCs vs. SN-CCs. This transcriptional down-regulation in NSN-CCs was confirmed by qRT-PCR analysis of CC-related genes (Has2, Ptx3, Tnfaip6 and Ptgs2). Only ten of the 422 genes were up-regulated with Amh being the most up-regulated in NSN-CCs, with an average 4-fold higher expression when analysed by qRT-PCR.ConclusionsThe developmental incompetence (NSN) or competence (SN) of antral oocytes can be predicted using transcript markers expressed by their surrounding CCs (i.e., Has2, Ptx3, Tnfaip6, Ptgs2 and Amh). Overall, the regulated nature of the group of genes brought out by whole transcriptome analysis constitutes the molecular signature of CCs associated either with developmentally incompetent or competent oocytes and may represent a valuable resource for developing new molecular tools for the assessment of oocyte quality and to further investigate the complex bi-directional interaction occurring between CCs and oocyte.

Highlights

The cumulus cells (CCs) enveloping antral and ovulated oocytes have been regarded as putative source of non-invasive markers of the oocyte developmental competence
To confirm our microarrays results, we analysed by quantitative RealTime PCR (qRT-PCR) the profile of expression of Amh and Has2; we extended this analysis to three more CC-related genes previously shown to play a critical role during the latest phases of oocyte maturation and whose expression pattern was correlated to the developmental outcome: Ptx3 (Pentraxin 3), Tnfaip6 (Tumor necrosis factor alpha-induced protein 6) and Ptgs2 (Prostaglandin-endoperoxide synthase 2), associated with the stabilisation of the matrix at the time of cumulus expansion [17,18,19,21,25,26]
The microarray-based evaluation of their transcriptome highlighted a similar transcriptional activity, with the exception of 422 genes, 97.6% of which are down-regulated in non-surrounded nucleolus (NSN) oocytes

Summary

Introduction

The cumulus cells (CCs) enveloping antral and ovulated oocytes have been regarded as putative source of non-invasive markers of the oocyte developmental competence. We isolated CCs from antral mouse oocytes of known developmental incompetence (NSN-CCs) or competence (SN-CCs) and compared their transcriptomes with the aim of identifying distinct marker transcripts Based on their chromatin morphology, fully-grown antral oocytes may be classified into two distinct types: surrounded nucleolus (SN) oocytes, that present a ring of Hoechst-positive chromatin around the nucleolus and non-surrounded nucleolus (NSN) oocytes, that lack this ring and have a more widespread chromatin [1,2,3,4,5,6]. When matured in vitro to metaphase II (MII) and fertilised, SN oocytes may reach the blastocyst stage [4,7] and complete development to the birth of new individuals [13], whereas NSN oocytes cease development at the 2-cell stage [4,13,14] This protocol of antral oocyte classification, though allowing the recognition of gametes that are potentially developmentally competent or incompetent, has several limitations. The use of the supravital fluorochrome Hoechst 33342 and the subsequent steps of classification under UV light do not alter significantly the oocytes developmental competence [4], for obvious reasons the employment of this DNAbinding molecule is a strong limitation and is not advisable, outside the laboratory routine with model animals, for the selection of oocytes of domestic species or of humans

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