Age-related differences in the translational landscape of mammalian oocytes.
ABSTRACT: Increasing maternal age in mammals is associated with poorer oocyte quality, involving higher aneuploidy rates and decreased developmental competence. Prior to resumption of meiosis, fully developed mammalian oocytes become transcriptionally silent until the onset of zygotic genome activation. Therefore, meiotic progression and early embryogenesis are driven largely by translational utilization of previously synthesized mRNAs. We report that genome-wide translatome profiling reveals considerable numbers of transcripts that are differentially translated in oocytes obtained from aged compared to young females. Additionally, we show that a number of aberrantly translated mRNAs in oocytes from aged females are associated with cell cycle. Indeed, we demonstrate that four specific maternal age-related transcripts (Sgk1, Castor1, Aire and Eg5) with differential translation rates encode factors that are associated with the newly forming meiotic spindle. Moreover, we report substantial defects in chromosome alignment and cytokinesis in the oocytes of young females, in which candidate CASTOR1 and SGK1 protein levels or activity are experimentally altered. Our findings indicate that improper translation of specific proteins at the onset of meiosis contributes to increased chromosome segregation problems associated with female ageing.
Project description:The rate of chromosome segregation errors that emerge during meiosis I in the mammalian female germ line are known to increase with maternal age; however, little is known about the underlying molecular mechanism. The objective of this study was to analyze meiotic progression of mouse oocytes in relation to maternal age. Using the mouse as a model system, we analyzed the timing of nuclear envelope breakdown and the morphology of the nuclear lamina of oocytes obtained from young (2 months old) and aged females (12 months old). Oocytes obtained from older females display a significantly faster progression through meiosis I compared to the ones obtained from younger females. Furthermore, in oocytes from aged females, lamin A/C structures exhibit rapid phosphorylation and dissociation. Additionally, we also found an increased abundance of MPF components and increased translation of factors controlling translational activity in the oocytes of aged females. In conclusion, the elevated MPF activity observed in aged female oocytes affects precocious meiotic processes that can multifactorially contribute to chromosomal errors in meiosis I.
Project description:Mammalian oocytes are arrested at prophase I of meiosis, and resume meiosis prior to ovulation. Coordination of meiotic arrest and resumption is partly dependent on the post-transcriptional regulation of maternal transcripts. Here, we report that, SPINDLIN1 (SPIN1), a maternal protein containing Tudor-like domains, interacts with a known mRNA-binding protein SERBP1, and is involved in regulating maternal transcripts to control meiotic resumption. Mouse oocytes deficient for Spin1 undergo normal folliculogenesis, but are defective in resuming meiosis. SPIN1, via its Tudor-like domain, forms a ribonucleoprotein complex with SERBP1, and regulating mRNA stability and/or translation. The mRNA for the cAMP-degrading enzyme, PDE3A, is reduced in Spin1 mutant oocytes, possibly contributing to meiotic arrest. Our study demonstrates that Spin1 regulates maternal transcripts post-transcriptionally and is involved in meiotic resumption.
Project description:During oocyte maturation, changes in gene expression depend exclusively on translation and degradation of maternal mRNAs rather than transcription. Execution of this translation program is essential for assembling the molecular machinery required for meiotic progression, fertilization, and embryo development. With the present study, we used a RiboTag/RNA-Seq approach to explore the timing of maternal mRNA translation in quiescent oocytes as well as in oocytes progressing through the first meiotic division. This genome-wide analysis reveals a global switch in maternal mRNA translation coinciding with oocyte re-entry into the meiotic cell cycle. Messenger RNAs whose translation is highly active in quiescent oocytes invariably become repressed during meiotic re-entry, whereas transcripts repressed in quiescent oocytes become activated. Experimentally, we have defined the exact timing of the switch and the repressive function of CPE elements, and identified a novel role for CPEB1 in maintaining constitutive translation of a large group of maternal mRNAs during maturation.
Project description:Germ cells divide and differentiate in a unique local microenvironment under the control of somatic cells. Signals released in this niche instruct oocyte reentry into the meiotic cell cycle. Once initiated, the progression through meiosis and the associated programme of maternal messenger RNA translation are thought to be cell autonomous. Here we show that translation of a subset of maternal mRNAs critical for embryo development is under the control of somatic cell inputs. Translation of specific maternal transcripts increases in oocytes cultured in association with somatic cells and is sensitive to EGF-like growth factors that act only on the somatic compartment. In mice deficient in amphiregulin, decreased fecundity and oocyte developmental competence is associated with defective translation of a subset of maternal mRNAs. These somatic cell signals that affect translation require activation of the PI(3)K-AKT-mTOR pathway. Thus, mRNA translation depends on somatic cell cues that are essential to reprogramme the oocyte for embryo development.
Project description:The activation of maturation-promoting factor (MPF) is required for G(2)/M progression in eukaryotic cells. Xenopus oocytes are arrested in G(2) and are induced to enter M phase of meiosis by progesterone stimulation. This process is known as meiotic maturation and requires the translation of specific maternal mRNAs stored in the oocytes. We have used an expression cloning strategy to functionally identify proteins involved in G(2)/M progression in Xenopus oocytes. Here we report the cloning of two novel cDNAs that when expressed in oocytes induce meiotic maturation efficiently. The two cDNAs encode proteins of 33 kD that are 88% identical and have no significant homologies to other sequences in databases. These proteins, which we refer to as p33(ringo) (rapid inducer of G(2)/M progression in oocytes), induce very rapid MPF activation in cycloheximide-treated oocytes. Conversely, ablation of endogenous p33(ringo) mRNAs using antisense oligonucleotides inhibits progesterone-induced maturation, suggesting that synthesis of p33(ringo) is required for this process. We also show that p33(ringo) binds to and activates the kinase activity of p34(cdc2) but does not associate with p34(cdc2)/cyclin B complexes. Our results identify a novel p34(cdc2) binding and activating protein that regulates the G(2)/M transition during oocyte maturation.
Project description:In many cell types, the length of the poly(A) tail of an mRNA is closely linked to its fate - a long tail is associated with active translation, a short tail with silencing and degradation. During mammalian oocyte development, two contrasting patterns of polyadenylation have been identified. Some mRNAs carry a long poly(A) tail during the growth stage and are actively translated, then become deadenylated and down-regulated during the subsequent stage, termed meiotic maturation. Other mRNAs carry a short tail poly(A) tail and are translationally repressed during growth, and their poly(A) tail lengthens and they become translationally activated during maturation. As well, a program of elimination of this 'maternal' mRNA is initiated during oocyte maturation. Here we describe a third pattern of polyadenylation: mRNAs are deadenylated in growing oocytes, become polyadenylated during early maturation and then deadenylated during late maturation. We show that the deadenylase, CNOT6, is present in cortical foci of oocytes and regulates deadenylation of these mRNAs, and that PUF-binding elements (PBEs) regulate deadenylation in mature oocytes. Unexpectedly, maintaining a long poly(A) tail neither enhances translation nor inhibits degradation of these mRNAs. Our findings implicate multiple machineries, more complex than previously thought, in regulating mRNA activity in oocytes.
Project description:Errors in meiotic chromosome segregation are the leading cause of spontaneous abortions and birth defects. Almost all such aneuploidy derives from meiotic errors in females, with increasing maternal age representing a major risk factor. It was recently reported that histones are globally deacetylated in mammalian oocytes during meiosis but not mitosis. In the present study, inhibition of meiotic histone deacetylation was found to induce aneuploidy in fertilized mouse oocytes, which resulted in embryonic death in utero at an early stage of development. In addition, a histone remained acetylated in the oocytes of older (10-month-old) female mice, suggesting that the function for histone deacetylation is decreased in the oocytes of such mice. Thus, histone deacetylation may be involved in the fair distribution of chromosomes during meiotic division. The high incidence of aneuploidy in the embryos of older females may be due to inadequate meiotic histone deacetylation.
Project description:Regulation of mRNA translation by cytoplasmic polyadenylation is known to be important for oocyte maturation and further development. This process is generally controlled by phosphorylation of cytoplasmic polyadenylation element binding protein 1 (CPEB1). The aim of this study is to determine the role of Aurora kinase A in CPEB1 phosphorylation and the consequent CPEB1-dependent polyadenylation of maternal mRNAs during mammalian oocyte meiosis. For this purpose, we specifically inhibited Aurora kinase A with MLN8237 during meiotic maturation of porcine oocytes. Using poly(A)-test PCR method, we monitored the effect of Aurora kinase A inhibition on poly(A)-tail extension of long and short cyclin B1 encoding mRNAs as markers of CPEB1-dependent cytoplasmic polyadenylation. Our results show that inhibition of Aurora kinase A activity impairs neither cyclin B1 mRNA polyadenylation nor its translation and that Aurora kinase A is unlikely to be involved in CPEB1 activating phosphorylation.
Project description:Deleted in azoospermia-like (DAZL) is an RNA-binding protein critical for gamete development. In full-grown oocytes, the DAZL protein increases 4-fold during reentry into the meiotic cell cycle. Here, we have investigated the functional significance of this accumulation at a genome-wide level. Depletion of DAZL causes a block in maturation and widespread disruption in the pattern of ribosome loading on maternal transcripts. In addition to decreased translation, DAZL depletion also causes translational activation of a distinct subset of mRNAs both in quiescent and maturing oocytes, a function recapitulated with YFP-3'UTR reporters. DAZL binds to mRNAs whose translation is both repressed and activated during maturation. Injection of recombinant DAZL protein in DAZL-depleted oocytes rescues the translation and maturation to MII. Mutagenesis of putative DAZL-binding sites in these mRNAs mimics the effect of DAZL depletion. These findings demonstrate that DAZL regulates translation of maternal mRNAs, functioning both as the translational repressor and activator during oocyte maturation.
Project description:In the absence of transcription, the regulation of gene expression in oocytes is controlled almost exclusively at the level of transcriptome and proteome stabilization, and translation. A subset of maternal transcripts is stored in a translationally dormant state in the oocyte, and temporally driven translation of specific mRNAs propel meiotic progression, oocyte-to-embryo transition and early embryo development. We identified Ank2.3 as the only transcript variant present in the mouse oocyte and discovered that it is translated after nuclear envelope breakdown. Here we show that Ank2.3 mRNA is localized in higher concentration in the oocyte nucleoplasm and, after nuclear envelope breakdown, in the newly forming spindle where its translation occurs. Furthermore, we reveal that Ank2.3 mRNA contains an oligo-pyrimidine motif at 5'UTR that predetermines its translation through a cap-dependent pathway. Lastly, we show that prevention of ANK2 translation leads to abnormalities in oocyte cytokinesis.