Developmental arrest and mouse antral not-surrounded nucleolus oocytes

Abstract

The antral compartment in the ovary consists of two populations of oocytes that differ by their ability to resume meiosis and to develop to the blastocyst stage. For reasons still not entirely clear, antral oocytes termed surrounded nucleolus (SN; 70% of the population of antral oocytes) develop to the blastocyst stage, whereas those called not-surrounded nucleolus (NSN) arrest at two cells. We profiled transcriptomic, proteomic, and morphological characteristics of antral oocytes and observed that NSN oocyte arrest is associated with lack of cytoplasmic lattices coincident with reduced expression of MATER and ribosomal proteins. Cytoplasmic lattices have been shown to store maternally derived mRNA and ribosomes in mammalian oocytes and embryos, and MATER has been shown to be required for cytoplasmic lattice formation. Thus, we isolated antral oocytes from a Mater(tm/tm) mouse and we observed that 84% of oocytes are of the NSN type. Our results provide the first molecular evidence to account for inability of NSN-derived embryos to progress beyond the two-cell stage; these results may be relevant to naturally occurring preimplantation embryo demise in mammals.

FIG. 1

Microarray analysis on 50 oocytes/type shows an overexpression of ribosomal proteins in SN oocytes. A) Scatter plot of 44K features on the NIA 60-mer oligo microarray comparing gene expression in antral oocytes (SN, green dots; NSN, red dots). Microarray data were analyzed by ANOVA-false discovery rate (FDR) statistics (averaged log intensity, FDR ≤ 0.05 and P ≤ 0.01). B) Bars representing transcripts detected in antral oocytes by ANOVA statistics and grouped according to the main Gene Ontology (GO) biological process categories they belong to. C) Heat map showing hierarchical clustering of significant ribosomal genes with one-fold expression change between SN and NSN oocytes. Expression levels are represented by the color; green indicates lower expression and red indicates higher expression.

FIG. 2

Proteomic profile on 300 oocytes/type reveals the up-regulation of several proteins in the NSN oocytes together with a higher number of peptide modifications. A) Two-dimensional map of SN and NSN oocytes obtained from the related identified protein and plotted by MAProMa software. Specifically, proteins are plotted according to their theoretical isoelectric point (pI) and molecular weight (MW KDa), and the color/shape code for each identified protein is related to its identification confidence by SEQUEST score value (yellow/triangle <15, blue/square from >15 to <35, and red/circle >35). B) Differentially abundant proteins characterized through DAVE and DCI algorithms from MAProMa software (black/negative and grey/positive values correspond to up-regulated proteins in NSN and SN cells, respectively). C) Posttranslational modifications of peptides (acetylation, methylation, and deimination) in SN versus NSN oocytes.

FIG. 3

Map of the interactome networks correlating the main biological processes/functions. Identified proteins were plotted on the networks by means of Cytoscape application. Node colors correspond to specific proteins up-regulated in SN (red) or NSN (blue) oocytes: pink are proteins equally distributed in the oocytes; white are not-identified proteins. Blue, green, yellow, and red connecting lines represent protein-protein, genetic, and metabolic pathways and protein-DNA interactions, respectively. The dashed circle indicates the magnification of the proteins belonging to the subcortical maternal complex and to the proteins directly or indirectly connected to it.

FIG. 4

MATER and the CPLs are essential for meiotic acquisition. A) Comparison between the percentages of SN and NSN oocytes in Mater^+/+ and Mater^tm/tm females (3 mo old). SN and NSN nucleoli are stained with Hoechst 33342. Bars = 10 μm. B) Transmission electron microscopy images of SN and NSN oocytes. Black arrows point to CPLs. Bars = 200 nm.

FIG. 5

CPLs and lipid droplet content are good candidate markers for oocyte developmental competence. A) Model of GV SN and NSN oocytes showing the abundance or absence of CPLs (pink lines) and ribosomes (dark blue dots) freely dispersed in the cytoplasm. A magnification of a portion of the subcortical maternal complex (SCMC) is schematically represented (dotted box) as a model showing the interactions between the components (red, MATER; yellow, FILIA; green, PADI6; blue, TLE6; black, OOEP) and their overexpression (filled shape) or underexpression (empty shape) in the antral SN and NSN oocytes. In this model, the functional SN maternal complex (due to MATER and FILIA overexpression) is compared to a nonfunctional NSN maternal complex characterized by MATER and FILIA underexpression. For this reason, the SCMC disassembly of NSN oocytes is probably due to the inability of all four proteins to bind to MATER. B) Number of lipid droplets content in SN and NSN oocytes after morphometric computerized image analysis performed with ImageJ software. Data is displayed as means ± SEM. *P < 0.05 versus SN oocytes (Student t-test). C, D) Semithin sections of SN and NSN oocytes stained with toluidine blue. Black arrows point to lipid droplets. Bars = 10 μm. C', D') Binary images from C and D after ImageJ thresholding segmentation for the evaluation of oocyte lipid droplet content.