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Implantation Failure of Blastocysts Derived from Oocyte-directed Connexin 43 depleted Mice is Associated with Impaired Ribosomal and Translational Machinery Gene Expression

ABSTRACT: Oocyte quality is a well- established determinant of embryonic fate. However, the molecular participants and biological markers that affect and predict adequate embryonic development are largely elusive. We have previously reported that oocyte- directed Connexin 43 (Cx43) depletion leads to embryo implantation defects, although both the morphology of the oocyte and processes presiding embryo implantation appear to undergo normally. In the context of previous data determining Cx43 indispensability to oocyte and embryonic development, we show here that the timing of Cx43 depletion from the oocyte and the ovarian follicle is crucial in determining the severity of subsequent embryonic defects. Specifically, we show that the implantation defects of blastocysts resulting from oocyte- directed Cx43- depleted follicles (depletion occurs at day 3 postnatal), is not due to maternal luteal insufficiency but rather depends solely on the defective blastocysts. Gene expression microarray analysis revealed global defects in the expression of ribosomal proteins, translation initiation factors and other genes associated with cellular biosynthetic and metabolic processes in these defective oocytes and specifically blastocysts. We therefore propose that timely expression of Cx43 in the oocyte and ovarian follicles is a major determinant of oocyte developmental competence, by determining the ability of the resulting blastocyst to facilitate biomass expansion and undergo adequate embryo implantation

ORGANISM(S): Mus musculus

PROVIDER: GSE35299 | GEO | 2013/01/01

SECONDARY ACCESSION(S): PRJNA151607

REPOSITORIES: GEO

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Project description:Objective: To study the uptake by human embryos of extracellular vesicles secreted by the maternal endometrium, and to investigate their miRNA cargo, in order to describe their role in implantation and early embryo development. Design: Prospective descriptive study. Subjects: Healthy women oocyte donors with confirmed fertility and day 5 human blastocysts. Intervention: Endometrial biopsies were collected from healthy oocyte donors undergoing transvaginal ultrasound-guided cyst aspiration for oocyte retrieval. Main Outcome Measures: Extracellular vesicle were isolated from culture media of primary human endometrial epithelial cells by ultracentrifugation. Concentration and size were analyzed by nanoparticle tracking analysis, their morphology visualized by transmission electron microscopy and extracellular vesicle protein markers expression was determined by western blotting. Vesicles were fluorescently labelled with Bodipy-TR ceramide, and their uptake by human blastocysts was analyzed using confocal microscopy. Analysis of the miRNA cargo of extracellular vesicles was performed using miRNAseq, target genes of the most expressed miRNAs were annotated and functional enrichment analysis was performed. Results: Extracellular vesicle characterization revealed a size within 100-300 nm, and expression of extracellular vesicle protein markers HSP70, TSG101, CD9 and CD81. Fluorescent microscopy showed an efficient extracellular vesicle internalization by human blastocysts within 1-2h, being the fluorescent signal stronger in the hatched area of the embryo. miRNAseq described 149 annotated miRNAs and top 37 most expressed miRNAs targeted 6,592 genes. Functional enrichment analysis of these targeted genes indicated that they participate in several processes related to embryo development, oxygen metabolism, cell cycle, cell differentiation, apoptosis, metabolism, cellular organization or gene expression. Among miRNAs contained in these EVs, hsa-miR-92a-3p, hsa-let-7b-5p, hsa-miR-30a-5p, hsa-miR-24-3p, hsa-miR-21-5p and hsa-let-7a-5p were highly implicated in all these biological processes. Conclusion: Data suggest that extracellular vesicles secreted by human endometrial epithelial cells are internalized by human blastocysts, and transport miRNAs to modulate biological processes related to implantation events and early embryo development. Knowledge of the communication system between human endometrium and embryo via miRNA cargo of these vesicles could describe new biomarkers of implantation success and embryo competence.

Project description:Somatic cell nuclear transfer (SCNT) is the most efficient cell reprogramming technique available, especially when working with bovine species. Although SCNT blastocysts performed equally well or better than controls in the weeks following embryo transfer at Day 7, elongation and gastrulation defects were observed prior to implantation. To understand the developmental implications of embryonic/extra-embryonic interactions, the morphological and molecular features of elongating and gastrulating tissues were analysed. At Day 18, 30 SCNT conceptuses were compared to 20 controls (AI and IVP: 10 conceptuses each); one-half of the SCNT conceptuses appeared normal while the other half showed signs of atypical elongation and gastrulation. SCNT was also associated with a high incidence of discordance in embryonic and extraembryonic patterns, as evidenced by morphological and molecular M-bM-^@M-^XM-bM-^@M-^XuncouplingM-bM-^@M-^YM-bM-^@M-^Y. Elongation appeared to be secondarily affected; only 3 of 30 conceptuses had abnormally elongated shapes and there were very few differences in gene expression when they were compared to the controls. However, some of these differences could be linked to defects in microvilli formation or extracellular matrix composition and could thus impact extra-embryonic functions. In contrast to elongation, gastrulation stages included embryonic defects that likely affected the hypoblast, the epiblast, or the early stages of their differentiation. When taking into account SCNT conceptus somatic origin, i.e. the reprogramming efficiency of each bovine ear fibroblast (Low: 0029, Med: 7711, High: 5538), we found that embryonic abnormalities or severe embryonic/extra-embryonic uncoupling were more tightly correlated to embryo loss at implantation than were elongation defects. Alternatively, extra-embryonic differences between SCNT and control conceptuses at Day 18 were related to molecular plasticity (high efficiency/high plasticity) and subsequent pregnancy loss. Finally, because it alters redifferentiation processes in vivo, SCNT reprogramming highlights temporally and spatially restricted interactions among cells and tissues in a unique way. Amplified material from each extra-embryonic tissue was indirectly labelled using "random" hexamers. One independent target per tissue was generated and hybridised onto one array. 10 measurements per reproduction mode were generated.

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Implantation Failure of Blastocysts Derived from Oocyte-directed Connexin 43 depleted Mice is Associated with Impaired Ribosomal and Translational Machinery Gene Expression

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