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Final Report Summary - MATERNA (Somatic cells regulation of maternal mRNA translation in mammalian oocytes)

The MateRNA project aims at investigating the relationship between the oocyte competence to sustain embryonic development and the program of maternal mRNA translation in mammals.
The last hours of oocyte differentiation, before ovulation, are characterized by well-coordinated events that regulates cell cycle re-entry, chromosomes segregation, chromatin remodelling and reprogramming of the genome. Since transcription is silent, these events are regulated at a translational level. Specifically, the interaction between cis elements on the 3′UTR of mRNAs and the cognate RNA binding proteins (RBPs) activates translation. The disruption of these regulatory circuits impairs oocyte maturation. With the MateRNA project we showed that in presence of more subtle disturbances of translation, the oocytes undergo apparently normal maturation but, once fertilized, fail to develop into an embryo. This was the case when somatic follicular compartment was not activated (specific objective 1 – SO1) and in older individuals (specific objective 2 – SO2). Specific objective 3 (SO3) verified this hypothesis in a non-murine model.
SO1: define the biochemical mechanisms by which somatic cells regulate the translational program of mouse oocytes. Hypothesis: signals coming from the somatic compartment modulate maternal mRNA translation and are critical for developmental competence
SO2: investigate whether the pattern of translation is disrupted in oocyte from aging mice. Hypothesis: the program of maternal mRNA translation during maturation is impaired in aging oocytes.
SO3: investigate whether the mechanisms of regulation of maternal mRNAs are conserved in non-murine mammalian species. Hypothesis: Temporal regulation of maternal mRNA translation is required for oocyte maturation and embryo development in monovulatory mammalian species.
Experimental approach: oocytes enclosed in the cumulus cells (CEOs) were treated with hormones/growth factors/signal molecules or inhibitors of intracellular pathways. The translation was monitored using luciferase reporters. Endogenous proteins and pathway activation were monitored by western blot. Morpholino-induced knock down and genetic models were used to manipulate the identified pathways. In vitro fertilization and embryo culture were used to investigate developmental competence. Bioinformatic analyses were conducted to mine previously published datasets.

SO1.A: previous studies conducted at the Host Institution showed that EGF-like growth factors initiate a cascade in the cumulus cells that surround the oocyte. Such activation of the somatic follicular compartment promotes the translation of a subset of transcripts in the oocyte together with an increase in embryo development. We asked: 1) whether different hormonal treatment can increase the developmental competence by influencing translation; 2) which pathway is triggered by this hormonal stimulation.
SO1.B: results obtained in 1A indicated PI3K/AKT as key pathway linking somatic cells activation to induction of oocyte specific translation. We developed a genetically engineered mouse model, where AKT was constitutively activated in the oocyte to ask whether AKT activation in the oocyte induced increased translation and higher embryo rate.
SO1.C: we performed a pharmacological screening and analyzed previously deposited databases to identify pathway(s) responsible for signal transduction between cumulus cells and activation of AKT in the oocyte.

SO2.A: a published study analyzed transcript expression level in oocytes from young and old mice before (GV) and after (MII) maturation, concluding that a number of transcripts are lower in MII oocytes of aging mice. However, analysis of this dataset showed that levels of a sizable number of transcripts significantly increased from GV to MII. Given the genome-wide transcriptional silencing, mRNA expression studies in MII oocytes should only yield stable mRNA or decreased mRNA due to degradation. Hence we hypothesized that in the published dataset a bias towards polyadenylated mRNA species was introduced by priming for cDNA synthesis using oligo-dT. Preliminary experiments showed that our hypothesis was correct. GO analyses were conducted on the deposited dataset comparing young and old mice to identify the biological processes mainly affected by reproductive aging.
SO2.B: having demonstrated in 2A that oligo-dT priming may capture differences in polyA length, we surmised that if levels of a transcript decrease in aging oocytes this may be due to disrupted polyadenylation or translation rather than actual overall transcript levels. We tested this hypothesis by comparing the translation of target transcript in oocytes from old and young mice.
SO2.C: we generated an animal model for unbiased profiling of translation in oocytes from aged mice, using a technique recently developed at the Host Institution.

SO3.A: We asked whether the obtained results are representative also of mono-ovulatory mammals and conducted experiments to investigate translation in bovine oocytes. Culture and assay conditions have been developed to: 1) conduct luciferase based reporter assays; 2) prepare a construct coding for a tagged ribosomal protein allowing precipitation of ribosomal associated RNA.
SO3.B: chromatin and epigenetic remodelers are translated during oocyte maturation in the mouse for future use by the embryo. We investigated epigenetic remodelers in bovine oocytes using an integrated bioinformatics and immunohistochemistry approach.

Our results demonstrate that mammalian embryo development strongly depends on the fulfilment of the oocyte translational program before ovulation and that disruptions of such program contribute to decreased female fertility. The innovative contribution of these studies to the field of medical assisted reproduction is detailed in an invited review (Conti M & Franciosi F, 2017 Hum Reprod Updates, in press)
Proposed models of our main findings are given in Figures 1.I, 1.II and 1.III (attached documents).
SO1: we demonstrate that 1) FSH stimulation increases the translation of specific transcripts; 2) activation of the oocyte AKT pathway is a common downstream to EGF-like growth factors and FSH stimulation; 3) oocyte AKT activation triggers the increase in translation; 4) oocyte AKT activation improves the developmental competence; 5) the pattern of oocyte secretion (OSFs) is controlled at the translational level and changes according to the meiotic stages. These results were published: Franciosi F et al. Endocrinology 2016, 157(2):872-82); Cakmak H, Franciosi F et al. Proc Natl Acad Sci U S A 2016, 113(9):2424-9. Preliminary results will allow dissecting the signaling pathway responsible for inducing AKT activation and translation. Specifically we observed that de novo transcription in the cumulus cells is required to propagate the EGF-like growth factors-induced cascade and we identified putative ligands responsible for oocyte AKT activation. As additional outcome, Dr Franciosi was invited to several meetings to present these results.
SO2: oligo-dT primed retrotranscription and subsequent qPCR may detect differences in the polyA tail length of mRNAs during oocyte maturation. We confirmed this hypothesis also by meta-analysis of published databases. By conducting GO analysis we observed that in oocytes from old mice the increase in oligo-dT primed transcript levels of several regulators of sister chromatid cohesion and epigenetic remodelers is significantly impaired. This finding is consistent with the higher incidence of aneuploidy typically observed in old oocyte and in line with the idea that differences observed between young and old oocytes originate from differences in polyadenylation and/or translation rather than absolute transcripts levels. We substantiated this hypothesis by measuring the translation activity of luciferase reporters of target transcripts in oocytes from young and old mice. These results constituted the core preliminary data for grant application (RAP2016: financed, ERC Consolidator 2017: under evaluation), to support Dr Franciosi in achieving scientific independence.
SO3: Our results suggest that approaches similar to the ones adopted in mice are applicable in bovine oocytes, opening the possibility that these mechanisms are conserved in mono-ovulatory species, comprising humans. These preliminary results sustained the proposal of a 2-years post-doc fellowship application successfully financed to Dr Franciosi to continue these studies. The epigenetic remodeling part of the study paved the way to a collaboration with the EmbryoGENE Network that led to the publication of a manuscript (Lodde V, Luciano AM, Franciosi F, Labreque R, Sirard MA. In: Kloc M (ed.) Oocytes - Maternal information and functions, vol. in press: Springer; 2017, Part of series: Results and Problems in Cell Differentiation).

1.4 THE EXPECTED FINAL RESULTS AND THEIR POTENTIAL IMPACT AND USE (including the socio-economic impact and the wider societal implications of the project so far)
Overall our results provide evidence that the regulation of translation and protein accumulation during oocyte maturation is associated with the acquisition of developmental competence, with important repercussion in the field of assisted reproduction. Currently approx. 10% of the couples in Western countries seek for infertility treatment to achieve conception. Considering that social pressure is delaying the time at which women decide to become pregnant, this rate will definitely increase in the next future. Consequently the development of more accurate stimulation and culture treatments, as well as non-invasive tests to predict oocyte fitness, will be useful to tackle the social impact of declining fertility with early aging.
In this view, our published results provide a molecular rationale for the use of FSH during oocyte in vitro maturation (IVM) procedures and suggest the possibility of a cooperative function of the FSH and LH mid-cycle surges in promoting the acquisition of oocyte developmental competence. This finding supports the use of combined FSH/hCG administration in patients undergoing oocyte retrieval for fertility treatment. Moreover we propose to monitor the translation pattern non-invasively by measuring the accumulation of secreted proteins, in order to identify the oocyte with the best chances to sustain embryo development. Also our results indicate that manipulation of the PI3K/AKT pathway may represent a new line of intervention to activate translation in oocytes and promote their quality. Finally we included in our project a specific objective to investigate the regulation of the translation program in oocyte during early aging. We believe that the results coming from this part of the study will shed light on molecular aspects that can be targeted to improve the developmental outcome in aging oocytes.

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