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Phosphorylation is a key post-translational modification regulating protein function and biological outcomes. However, the phosphorylation dynamics orchestrating mammalian oocyte development remains poorly understood. In the present study, we apply high-resolution mass spectrometry-based phosphoproteomics to obtain the first global in vivo quantification of mouse oocyte phosphorylation. Of more than 8000 phosphosites, 75% significantly oscillate and 64% exhibit marked upregulation during meiotic maturation, indicative of the dominant regulatory role. Moreover, we identify numerous novel phosphosites on oocyte proteins and a few highly conserved phosphosites in oocytes from different species. Through functional perturbations, we demonstrate that phosphorylation status of specific sites participates in modulating critical events including metabolism, translation, and RNA processing during meiosis. Finally, we combine inhibitor screening and enzyme-substrate network prediction to discover previously unexplored kinases and phosphatases that are essential for oocyte maturation. In sum, our data define landscape of the oocyte phosphoproteome, enabling in-depth mechanistic insights into developmental control of germ cells. Synopsis: Oocytes plays a central role in the success of reproduction, yet the phosphorylation dynamics during mammalian oocyte development remain poorly understood. This resource article describes the comprehensive stage-resolved phosphoproteome and proteome landscape of mouse oocytes, and reveals novel molecular mechanisms controlling meiotic maturation. The phosphorylation landscape of oocytes during in vivo maturation is delineated. Novel phosphosites in oocytes and conserved phosphosites across multiple species are identified. Key kinases orchestrating the regulation of oocyte meiosis are pinpointed Phosphatase dynamics during meiotic maturation are deciphered. This resource article provides the most comprehensive stage-resolved phosphoproteome and proteome landscape of mouse oocytes to date, revealing novel molecular control mechanisms. [ABSTRACT FROM AUTHOR]

In fully grown oocytes, the genome is considered to be globally transcriptionally silenced. However, this conclusion is primarily derived from the results obtained through immunofluorescence staining or inferred from the highly condensed state of chromosomes, lacking more direct evidence. Here, by using a kethoxal-assisted single-stranded DNA sequencing (KAS-seq) approach, we investigated the landscape of single-strand DNA (ssDNA) throughout the genome and provided a readout of the activity and dynamics of transcription during oocyte meiotic maturation. In non-surrounded nucleolus (NSN) oocytes, we observed a robust KAS-seq signal, indicating the high transcriptional activity. In surrounded nucleolus (SN) oocytes, the presence of ssDNA still persists although the KAS-seq signal was relatively weak, suggesting the presence of transcription. Accompanying with the meiotic resumption, the transcriptional activity gradually decreased, and global repression was detected in matured oocytes. Moreover, we preformed the integrative genomics analysis to dissect the transcriptional dynamics during mouse oocyte maturation. In sum, the present study delineates the detailed transcriptional activity during mammalian oocyte maturation. [ABSTRACT FROM AUTHOR]

Mitochondria are essential for female reproductive processes, yet the function of mitochondrial DNA (mtDNA) mutation in oocytes remains elusive. By employing an mtDNA mutator (Polgm) mouse model, we found the fetal growth retardation and placental dysfunction in post-implantation embryos derived from Polgm oocytes. Remarkably, Polgm oocytes displayed the global loss of DNA methylation; following fertilization, zygotic genome experienced insufficient demethylation, along with dysregulation of gene expression. Spindle–chromosome exchange experiment revealed that cytoplasmic factors in Polgm oocytes are responsible for such a deficient epigenetic remodeling. Moreover, metabolomic profiling identified a significant reduction in the α-ketoglutarate (αKG) level in oocytes from Polgm mice. Importantly, αKG supplement restored both DNA methylation state and transcriptional activity in Polgm embryos, consequently preventing the developmental defects. Our findings uncover the important role of oocyte mtDNA mutation in controlling epigenetic reprogramming and gene expression during embryogenesis. αKG deserves further evaluation as a potential drug for treating mitochondrial dysfunction-related fertility decline. [ABSTRACT FROM AUTHOR]

Maternal obesity is associated with multiple adverse reproductive outcomes, whereas the underlying molecular mechanisms are still not fully understood. Here, we found the reduced nicotinamide phosphoribosyl transferase (NAMPT) expression and lowered nicotinamide adenine dinucleotide (NAD+) content in oocytes from obese mice. Next, by performing morpholino knockdown assay and pharmacological inhibition, we revealed that NAMPT deficiency not only severely disrupts maturational progression and meiotic apparatus, but also induces the metabolic dysfunction in oocytes. Furthermore, overexpression analysis demonstrated that NAMPT insufficiency induced NAD+ loss contributes to the compromised developmental potential of oocytes and early embryos from obese mice. Importantly, in vitro supplement and in vivo administration of nicotinic acid (NA) was able to ameliorate the obesity‐associated meiotic defects and oxidative stress in oocytes. Our results indicate a role of NAMPT in modulating oocyte meiosis and metabolism, and uncover the beneficial effects of NA treatment on oocyte quality from obese mice. [ABSTRACT FROM AUTHOR]

Objectives: It has been widely reported that maternal diabetes impairs oocyte quality. However, the responsible mechanisms remain to be explored. In the present study, we focused on whether SIRT3‐GSK3β pathway mediates the meiotic defects in oocytes from diabetic mice. Materials and methods: GSK3β functions in mouse oocyte meiosis were first detected by targeted siRNA knockdown. Spindle assembly and chromosome alignment were visualized by immunostaining and analysed under the confocal microscope. PCR‐based site mutation of specific GSK3β lysine residues was used to confirm which lysine residues function in oocyte meiosis. siRNA knockdown coupled with cRNA overexpression was performed to detect SIRT3‐GSK3β pathway functions in oocyte meiosis. Immunofluorescence was performed to detect ROS levels. T1DM mouse models were induced by a single intraperitoneal injection of streptozotocin. Results: In the present study, we found that specific depletion of GSK3β disrupts maturational progression and meiotic apparatus in mouse oocytes. By constructing site‐specific mutants, we further revealed that acetylation state of lysine (K) 15 on GSK3β is essential for spindle assembly and chromosome alignment during oocyte meiosis. Moreover, non–acetylation‐mimetic mutant GSK3β‐K15R is capable of partly preventing the spindle/chromosome anomalies in oocytes with SIRT3 knockdown. A significant reduction in SIRT3 protein was detected in oocytes from diabetic mice. Of note, forced expression of GSK3β‐K15R ameliorates maternal diabetes‐associated meiotic defects in mouse oocytes, with no evident effects on oxidative stress. Conclusion: Our data identify GSK3β as a cytoskeletal regulator that is required for the assembly of meiotic apparatus, and discover a beneficial effect of SIRT3‐dependent GSK3β deacetylation on oocyte quality from diabetic mice. [ABSTRACT FROM AUTHOR]

Abstract: SIRT4 modulates energy homeostasis in multiple cell types and tissues. However, its role in meiotic oocytes remains unknown. Here, we report that mouse oocytes overexpressing SIRT4 are unable to completely progress through meiosis, showing the inadequate mitochondrial redistribution, lowered ATP content, elevated reactive oxygen species (ROS) level, with the severely disrupted spindle/chromosome organization. Moreover, we find that phosphorylation of Ser293‐PDHE1α mediates the effects of SIRT4 overexpression on metabolic activity and meiotic events in oocytes by performing functional rescue experiments. By chance, we discover the SIRT4 upregulation in oocytes from aged mice; and importantly, the maternal age‐associated deficient phenotypes in oocytes can be partly rescued through the knockdown of SIRT4. These findings reveal the critical role for SIRT4 in the control of energy metabolism and meiotic apparatus during oocyte maturation and indicate that SIRT4 is an essential factor determining oocyte quality. [ABSTRACT FROM AUTHOR]

Summary: The level of Sirt2 protein is reduced in oocytes from aged mice, while exogenous expression of Sirt2 could ameliorate the maternal age‐associated meiotic defects. To date, the underlying mechanism remains unclear. Here, we confirmed that specific depletion of Sirt2 disrupts maturational progression and spindle/chromosome organization in mouse oocytes, with compromised kinetochore–microtubule attachments. Candidate screening revealed that acetylation state of lysine 243 on BubR1 (BubR1‐K243, an integral part of the spindle assembly checkpoint complex) functions during oocyte meiosis, and acetylation‐mimetic mutant BubR1‐K243Q results in the very similar phenotypes as Sirt2‐knockdown oocytes. Furthermore, we found that nonacetylatable‐mimetic mutant BubR1‐K243R partly prevents the meiotic deficits in oocytes depleted of Sirt2. Importantly, BubR1‐K243R overexpression in oocytes derived from aged mice markedly suppresses spindle/chromosome anomalies and thereupon lowers the incidence of aneuploid eggs. In sum, our data suggest that Sirt2‐dependent BubR1 deacetylation involves in the regulation of meiotic apparatus in normal oocytes and mediates the effects of advanced maternal age on oocyte quality. [ABSTRACT FROM AUTHOR]

Maternal obesity in humans is associated with poor outcomes across the reproductive spectrum. Emerging evidence indicates that these defects are likely attributed to factors within the oocyte. Although various molecules and pathways may contribute to impaired oocyte quality, prevention of fertility issues associated with maternal obesity is a challenge. Using mice fed a high-fat diet ( HFD) as an obesity model, we document spindle disorganization, chromosome misalignment, and elevated reactive oxygen species ( ROS) levels in oocytes from obese mice. Oral administration of melatonin to HFD mice not only reduces ROS generation, but also prevents spindle/chromosome anomalies in oocytes, consequently promoting the developmental potential of early embryos. Consistent with this finding, we find that melatonin supplement during in vitro maturation also markedly attenuates oxidative stress and meiotic defects in HFD oocytes. Finally, by performing morpholino knockdown and acetylation-mimetic mutant overexpression assays, we reveal that melatonin ameliorates maternal obesity-induced defective phenotypes in oocytes through the SIRT3- SOD2-dependent mechanism. In sum, our data uncover the marked beneficial effects of melatonin on oocyte quality from obese females; this opens a new area for optimizing culture system as well as fertility management. [ABSTRACT FROM AUTHOR]