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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., (© 2020 The Authors. Aging Cell published by Anatomical Society and John Wiley & Sons Ltd.)

Lamin C2 (LMN C2) is a short product of the lamin a gene. It is a germ cell-specific lamin and has been extensively studied in male germ cells. In this study, we focussed on the expression and localization of LMN C2 in fully-grown germinal vesicle (GV) oocytes. We detected LMN C2 in the fully-grown germinal vesicle oocytes of various mammalian species with confirmation done by immunoblotting the wild type and Lmnc2 gene deleted testes. Expression of LMN C2 tagged with GFP showed localization of LMN C2 to the nuclear membrane of the oocyte. Moreover, the LMN C2 protein notably disappeared after nuclear envelope breakdown (NEBD) and the expression of LMN C2 was significantly reduced in the oocytes from aged females and ceased altogether during meiotic maturation. These results provide new insights regarding LMN C2 expression in the oocytes of various mammalian species., Competing Interests: The authors have declared that no competing interests exist.

Cell division cycle ( C dc) k inase s ubunit (CKS) proteins bind cyclin-dependent kinases (CDKs) and play important roles in cell division control and development, though their precise molecular functions are not fully understood. Mammals express two closely related paralogs called CKS1 and CKS2, but only CKS2 is expressed in the germ line, indicating that it is solely responsible for regulating CDK functions in meiosis. Using cks2 -/- knockout mice, we show that CKS2 is a crucial regulator of maturation-promoting factor (MPF; CDK1-cyclin A/B) activity in meiosis. cks2 -/- oocytes display reduced and delayed MPF activity during meiotic progression, leading to defects in germinal vesicle breakdown (GVBD), anaphase-promoting complex/cyclosome (APC/C) activation, and meiotic spindle assembly. cks2 -/- germ cells express significantly reduced levels of the MPF components CDK1 and cyclins A1/B1. Additionally, injection of MPF plus CKS2, but not MPF alone, restored normal GVBD in cks2 -/- oocytes, demonstrating that GVBD is driven by a CKS2-dependent function of MPF. Moreover, we generated cks2 cks1/cks1 knock-in mice and found that CKS1 can compensate for CKS2 in meiosis in vivo , but homozygous embryos arrested development at the 2- to 5-cell stage. Collectively, our results show that CKS2 is a crucial regulator of MPF functions in meiosis and that its paralog, CKS1, must be excluded from the germ line for proper embryonic development., (Copyright © 2019 American Society for Microbiology.)

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.

The tight correlation between mRNA distribution and subsequent protein localization and function indicate a major role for mRNA localization within the cell. RNA localization, followed by local translation, presents a mechanism for spatial and temporal gene expression regulation utilized by various cell types. However, little is known about mRNA localization and translation in the mammalian oocyte and early embryo. Importantly, fully-grown oocyte becomes transcriptionally inactive and only utilizes transcripts previously synthesized and stored during earlier development. We discovered an abundant RNA population in the oocyte and early embryo nucleus together with RNA binding proteins. We also characterized specific ribosomal proteins, which contribute to translation in the oocyte and embryo. By applying selected markers to mouse and human oocytes, we found that there might be a similar mechanism of RNA metabolism in both species. In conclusion, we visualized the localization of RNAs and translation machinery in the oocyte, that could shed light on this terra incognita of these unique cell types in mouse and human.

Fully grown mammalian oocytes utilize transcripts synthetized and stored during earlier development. RNA localization followed by a local translation is a mechanism responsible for the regulation of spatial and temporal gene expression. Here we show that the mouse oocyte contains 3 forms of cap-dependent translational repressor expressed on the mRNA level: 4E-BP1, 4E-BP2 and 4E-BP3. However, only 4E-BP1 is present as a protein in oocytes, it becomes inactivated by phosphorylation after nuclear envelope breakdown and as such it promotes cap-dependent translation after NEBD. Phosphorylation of 4E-BP1 can be seen in the oocytes after resumption of meiosis but it is not detected in the surrounding cumulus cells, indicating that 4E-BP1 promotes translation at a specific cell cycle stage. Our immunofluorescence analyses of 4E-BP1 in oocytes during meiosis I showed an even localization of global 4E-BP1, as well as of its 4E-BP1 (Thr37/46) phosphorylated form. On the other hand, 4E-BP1 phosphorylated on Ser65 is localized at the spindle poles, and 4E-BP1 phosphorylated on Thr70 localizes on the spindle. We further show that the main positive regulators of 4E-BP1 phosphorylation after NEBD are mTOR and CDK1 kinases, but not PLK1 kinase. CDK1 exerts its activity toward 4E-BP1 phosphorylation via phosphorylation and activation of mTOR. Moreover, both CDK1 and phosphorylated mTOR co-localize with 4E-BP1 phosphorylated on Thr70 on the spindle at the onset of meiotic resumption. Expression of the dominant negative 4E-BP1 mutant adversely affects translation and results in spindle abnormality. Taken together, our results show that the phosphorylation of 4E-BP1 promotes translation at the onset of meiosis to support the spindle assembly and suggest an important role of CDK1 and mTOR kinases in this process. We also show that the mTOR regulatory pathway is present in human oocytes and is likely to function in a similar way as in mouse oocytes.