SN Versus NSN Oocytes: The Endless Battle for the Achievement of the Developmental Competence.

In the mouse, the antral compartment of the ovary contains two different types of oocytes (SN and the NSN) distinguishable by a different transcriptional activity and developmental competence: only the SN types maturated and fertilized in vitro are able to develop until the blastocyst stage. Since the functional meaning of SN and NSN oocytes is still highly debated, we used a multi-approach analysis employing molecular, biophysics and biochemical methods to understand the molecular events responsible for the two different chromatin organizations and thus for the two different prospective zygotic developments.Gene expression analyses suggested that the NSN-derived zygotic epigenome shows reduced levels of expression of some important genes involved in cell differentiation and transcription. For instance, the transcription factor Oct4 is detected with low levels of expression in the NSN compared to the SN type: this might explain the greater developmental capacity of the SN oocytes and its correlation with the totipotent characteristic of the future zygote. Using microarrays technology we characterized global changes in gene expression in SN versus NSN oocytes showing an over-expression of many early response genes often associated with stress and apoptosis together with many genes involved in the translation and polyadenylation process.To get new insights on the molecular composition and organization of SN and NSN oocytes at the GV stage and during the transition MI-MII stage, we recently used Fourier transformed infrared microspectroscopy, showing important differences in the protein absorption regions concerning the extent of transcription, polyadenylation and methylation. The NSN oocytes maintain, during the maturation process, a higher transcriptional activity than the SN type. At MII, SN-derived oocytes showed a significant polyA content that could reflect the storage of maternal mRNAs with polyA tails, crucial for the subsequent early embryonic development, while the NSN-derived oocytes lacking a properly polyadenylated maternal mRNAs, are kept in an unsuccessful transcriptional state. However, the most evident separation between the two types of oocytes is at the MI stage, considered a crucial checkpoint where some of the molecules and/or mechanisms rearrange to then participate in the decision of oocyte fate and destiny towards, or not, meiotic resumption.NSN oocytes reflect also a partially methylated DNA (indicated by a reduction in the CpG island methylation) and enter into a transcriptional standby state at the MI stage. The maturation of the NSN oocytes is characterized by a higher transcriptional activity at MII than at the antral stage and by a lower level of polyadenylation of mRNAs. In addition, we are currently identifying the protein profiles of the two different oocytes by means of MudPIT (Multidimensional Protein Identification Technology), a gel-free approach based on two dimensional micro-chromatography coupled to tandem mass spectrometry (2DC-MS/MS). By doing this, hundreds of proteins will be identified for each sample and differentially expressed proteins determined by comparing SN versus NSN oocytes.Taken together these results will allow us to better comprehend the mechanisms involved in the generation of developmentally fully competent oocytes in vitro and in the reliable genetic control of mammalian reproduction. (poster)