Two-Cell Embryo Developmental Block Is Due to Lack of Cytoplasmic Lattices in Mouse Antral NSN Oocytes.

Abstract

In most mammals, the antral compartment of the ovary contains two different kinds of oocytes, SN and NSN (Surrounded Nucleolus and Not Surrounded Nucleolus), whose main differentiating characteristic lies in their acquisition of developmental competence: still inexplicably only the SN (70% of the antral oocytes population) are able to develop to the blastocyst stage while the NSN arrest at the two-cell stage. Taking advantage of proteomics and microscopy techniques, we analyzed both SN and NSN oocytes, showing for the first time that NSN-derived embryo inability to develop to the blastocyst stage is due to the under-regulation of some maternal proteins together with a lack of cytoplasmic lattices (CPLs). Additionally, we analyzed the lipid droplets content, showing that NSN oocyte cytoplasm contains a significantly higher amount of lipid droplets than SN. The proteomic profile of 600 antral oocytes (300 SN and 300 NSN) showed up-regulation of 6 proteins in SN and 23 in NSN; in particular, when compared to SN oocytes, NSN showed a significant down-regulation of the proteins MATER and FILIA. MATER is the main constituent of the Subcortical Maternal Complex and it is considered one of the factors responsible for the acquisition of meiotic competence. In fact, it is already known that Matertm/tm oocytes, without any distinction based on chromatin organization, do not have cytoplasmic lattices and are not able to progress beyond the two-cell stage. Based on our proteomic results, we speculated that the Matertm/tm antral population should be composed mostly of NSN oocytes. To test this hypothesis, we analyzed Matertm/tm antral oocytes showing that 84% of these oocytes are of the NSN type. To confirm this data, we performed Transmission Electron Microscopy analysis on SN and NSN oocytes, revealing that NSN contain no or very few cytoplasmic lattices as compared to SN. For this reason, the absence of CPLs can be considered a NSN-specific molecular morphology feature. MATER has also been correlated with the anti-apoptotic pathway, so the accumulation of lipid droplets in Mater null oocytes could be related to apoptotic activity. Through the morphometric analysis of lipid droplet content, we demonstrated that NSN oocytes contain a significantly higher amount of lipid droplets than SN. This is further evidence of the role played by CPLs in the resumption of meiosis and of lipid droplets being good candidate markers for oocyte developmental competence. Our results provide the first molecular evidence that accounts for NSN-derived embryo's inability to progress beyond the two-cell stage and, thus, for some of the naturally occurring pre-implantation losses in mammals. These data will also give basic knowledge about the function of the miniature, wonderfully organized laboratory of molecular biology that is the oocyte.