Semi-quantitative measurement of specific proteins in human cumulus cells using reverse phase protein array

Vincent Puard,Veronique Cadoret,Dominique Royere,Christophe Gauthier,Fabrice Guerif,Eric Reiter,Thibaud Tranchant

doi:10.1186/1477-7827-11-100

Abstract

BackgroundThe ability to predict the developmental and implantation ability of embryos remains a major goal in human assisted-reproductive technology (ART) and most ART laboratories use morphological criteria to evaluate the oocyte competence despite the poor predictive value of this analysis. Transcriptomic and proteomic approaches on somatic cells surrounding the oocyte (granulosa cells, cumulus cells [CCs]) have been proposed for the identification of biomarkers of oocyte competence. We propose to use a Reverse Phase Protein Array (RPPA) approach to investigate new potential biomarkers of oocyte competence in human CCs at the protein level, an approach that is already used in cancer research to identify biomarkers in clinical diagnostics.MethodsAntibodies targeting proteins of interest were validated for their utilisation in RPPA by measuring siRNA-mediated knockdown efficiency in HEK293 cells in parallel with Western blotting (WB) and RPPA from the same lysates. The proteins of interests were measured by RPPA across 13 individual human CCs from four patients undergoing intracytoplasmic sperm injection procedure.ResultsThe knockdown efficiency of VCL, RGS2 and SRC were measured in HEK293 cells by WB and by RPPA and were acceptable for VCL and SRC proteins. The antibodies targeting these proteins were used for their detection in human CCs by RPPA. The detection of protein VCL, SRC and ERK2 (by using an antibody already validated for RPPA) was then carried out on individual CCs and signals were detected for each individual sample. After normalisation by VCL, we showed that the level of expression of ERK2 was almost the same across the 13 individual CCs while the level of expression of SRC was different between the 13 individual CCs of the four patients and between the CCs from one individual patient.ConclusionsThe exquisite sensitivity of RPPA allowed detection of specific proteins in individual CCs. Although the validation of antibodies for RPPA is labour intensive, RRPA is a sensitive and quantitative technique allowing the detection of specific proteins from very small quantities of biological samples. RPPA may be of great interest in clinical diagnostics to predict the oocyte competence prior to transfer of the embryo using robust protein biomarkers expressed by CCs.

Highlights

The ability to predict the developmental and implantation ability of embryos remains a major goal in human assisted-reproductive technology (ART) and most ART laboratories use morphological criteria to evaluate the oocyte competence despite the poor predictive value of this analysis
Pearson correlations were calculated for each antibody in all dilutions of HEK293 cells transfected by small interfering RNA (siRNA) targeting the four proteins (VCL, SRC, regulator of G-protein signalling 2 (RGS2) and GAPDH)
The extinction level of GAPDH, VCL and SRC were acceptable in Western blotting (WB) and in Reverse Phase Protein Array (RPPA) with differences in knockdown efficiencies measured by WB and RPPA for SRC and GAPDH (Figure 3C), which validated these antibodies for RPPA

Summary

Introduction

The ability to predict the developmental and implantation ability of embryos remains a major goal in human assisted-reproductive technology (ART) and most ART laboratories use morphological criteria to evaluate the oocyte competence despite the poor predictive value of this analysis. Transcriptomic and proteomic approaches on somatic cells surrounding the oocyte (granulosa cells, cumulus cells [CCs]) have been proposed for the identification of biomarkers of oocyte competence. Such morphological criteria (zygote scoring, early cleavage and embryo morphology at day 2 or 3) remain poorly predictive of developmental or implantation ability [1] Direct studies, such as genomic or proteomic analyses, are difficult in human embryos, since such approaches remains invasive and might alter the embryo integrity [2]. To date no study investigating individual CC proteomes and specific proteins according to oocyte competence has been reported

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