Amh transcript: a non-invasive cumulus cells marker of the oocyte developmental competence

In the field of assisted reproductive technologies (ARTs) there is a growing need to identify non-invasive markers of the oocyte quality. To this end, a number of studies have screened various molecules and morphological characteristics, but reached contrasting and yet not widely accepted results. This study aims at identifying cumulus cell (CC) markers of the oocyte development competence. To this purpose, mouse fully-grown antral follicles were isolated and the CCs separated from the oocytes enclosed. Then, based on their chromatin organization, oocytes were classified as surrounded nucleolus (SN, with a ring of Hoechst-positive heterochromatin surrounding the nucleolus) and non-surrounded nucleolus (NSN, when they displayed spots of heterochromatin widespread within the nucleus). SN oocytes, when matured in vitro to the MII stage, fertilized and the preimplantation embryo transferred in the uterus of a pseudo-pregnant female may reach full term; on the contrary, NSN oocytes arrest development at the 2-cell stage. CCs belonging to SN (SN-CCs) or NSN (NSN-CCs) oocytes were collected separately and analysed. We first made a microarray analysis of the whole transcriptome profile that brought up a list of 422 differentially regulated genes, most of which (97.6%) were down-regulated in NSN-CCs surrounding developmentally incompetent oocytes. A bioinformatics analysis associated these genes to 11 major biological processes, including development and reproduction. In addition, a bibliographic analysis of the literature based on a list of MeSH terms brought out a group of 26 differentially regulated genes involved in ovarian functions. Then, by Real Time PCR (qRT-PCR), we studied the expression of a group of CC-related genes: Has2, Ptx3, Ptgs2 and Tnfaip6, involved in cumulus expansion prior to ovulation and Amh involved in primordial follicles recruitment and dominant follicles selection. In the comparison between NSN-CCs vs. SN-CCs, Has2, Ptx3, Ptgs2 and Tnfaip6 resulted down regulated, whereas Amh showed a strong 4-fold up-regulation, confirming the microarray data. Among the gene sequences highlighted in our study, Amh was clearly the most differentially regulated in the comparison between NSN-CCs vs. SN-CCs, a difference that was confirmed also at the protein level (immunofluorescence). In conclusion, with the use of a model study that allows the identification, a priori, of the developmental capacity of antral mouse oocytes, for the first time, we identified a number of transcripts that could be used as markers of the oocyte developmental competence.