Simultaneous deep transcriptome and proteome profiling in a single mouse oocyte.

Although single-cell multi-omics technologies are undergoing rapid development, simultaneous transcriptome and proteome analysis of a single-cell individual still faces great challenges. Here, we developed a single-cell simultaneous transcriptome and proteome (scSTAP) analysis platform based on microfluidics, high-throughput sequencing, and mass spectrometry technology to achieve deep and joint quantitative analysis of transcriptome and proteome at the single-cell level, providing an important resource for understanding the relationship between transcription and translation in cells. This platform was applied to analyze single mouse oocytes at different meiotic maturation stages, reaching an average quantification depth of 19,948 genes and 2,663 protein groups in single mouse oocytes. In particular, we analyzed the correlation of individual RNA and protein pairs, as well as the meiosis regulatory network with unprecedented depth, and identified 30 transcript-protein pairs as specific oocyte maturational signatures, which could be productive for exploring transcriptional and translational regulatory features during oocyte meiosis.

Oocyte meiosis-coupled poly(A) polymerase α phosphorylation and activation trigger maternal mRNA translation in mice.

Mammalian oocyte maturation is driven by strictly regulated polyadenylation and translational activation of maternal mRNA stored in the cytoplasm. However, the poly(A) polymerase (PAP) that directly mediates cytoplasmic polyadenylation in mammalian oocytes has not been determined. In this study, we identified PAPα as the elusive enzyme that catalyzes cytoplasmic mRNA polyadenylation implicated in mouse oocyte maturation. PAPα was mainly localized in the germinal vesicle (GV) of fully grown oocytes but was distributed to the ooplasm after GV breakdown. Inhibition of PAPα activity impaired cytoplasmic polyadenylation and translation of maternal transcripts, thus blocking meiotic cell cycle progression. Once an oocyte resumes meiosis, activated CDK1 and ERK1/2 cooperatively mediate the phosphorylation of three serine residues of PAPα, 537, 545 and 558, thereby leading to increased activity. This mechanism is responsible for translational activation of transcripts lacking cytoplasmic polyadenylation elements in their 3'-untranslated region (3'-UTR). In turn, activated PAPα stimulated polyadenylation and translation of the mRNA encoding its own (Papola) through a positive feedback circuit. ERK1/2 promoted Papola mRNA translation in a 3'-UTR polyadenylation signal-dependent manner. Through these mechanisms, PAPα activity and levels were significantly amplified, improving the levels of global mRNA polyadenylation and translation, thus, benefiting meiotic cell cycle progression.

Positive Feedback Stimulation of Ccnb1 and Mos mRNA Translation by MAPK Cascade During Mouse Oocyte Maturation.

In mammalian species, both the maturation promoting factor (MPF) and the mitogen-activated protein kinase (MAPK) cascade play critical roles in modulating oocyte meiotic cell-cycle progression. MPF is a critical heterodimer composed of CDK1 and cyclin B1. Activation of MPF and ERK1/2 requires the activation of maternal Ccnb1 and Mos mRNAs translation, respectively. The phosphorylation and degradation of CPEB1 that triggered by ERK1/2 is a principal mechanism of activating maternal mRNA translation. However, the interplay of these two key kinases in mediating mammalian translational activation of cytoplasmic mRNAs during oocyte maturation is unclear. We prove evidence that the translational activation of Ccnb1 transcripts containing a long 3'-UTR during meiotic resumption works in an ERK1/2-dependent way. A low level of ERK1/2 activation was detected prior to meiotic resumption. Precocious activation of MAPK signaling in germinal vesicle stage oocytes promotes the translation of Ccnb1 mRNA and meiotic maturation. Inhibition or precocious activation of CDK1 activity has an appreciable effect on the translation of Ccnb1 mRNA, suggesting that both kinases are required for Ccnb1 mRNA translational activation. CDK1 triggers phosphorylation, but not degradation, of CPEB1 in oocytes; the degradation of CPEB1 was only triggered by ERK1/2. Moreover, the translational activation of Mos mRNA is regulated by ERK1/2 and cytoplasmic polyadenylation elements too. Taken together, the cooperation and positive feedback activation of ERK1/2 and CDK1 lead to the fine-tuning of mRNA translation and cell-cycle progression during mouse oocyte maturation.

Mammalian nucleolar protein DCAF13 is essential for ovarian follicle maintenance and oocyte growth by mediating rRNA processing.

During mammalian oocyte growth, chromatin configuration transition from the nonsurrounded nucleolus (NSN) to surrounded nucleolus (SN) type plays a key role in the regulation of gene expression and acquisition of meiotic and developmental competence by the oocyte. Nonetheless, the mechanism underlying chromatin configuration maturation in oocytes is poorly understood. Here we show that nucleolar protein DCAF13 is an important component of the ribosomal RNA (rRNA)-processing complex and is essential for oocyte NSN-SN transition in mice. A conditional knockout of Dcaf13 in oocytes led to the arrest of oocyte development in the NSN configuration, follicular atresia, premature ovarian failure, and female sterility. The DCAF13 deficiency resulted in pre-rRNA accumulation in oocytes, whereas the total mRNA level was not altered. Further exploration showed that DCAF13 participated in the 18S rRNA processing in growing oocytes. The lack of 18S rRNA because of DCAF13 deletion caused a ribosome assembly disorder and then reduced global protein synthesis. DCAF13 interacted with a protein of the core box C/D ribonucleoprotein, fibrillarin, i.e., a factor of early pre-rRNA processing. When fibrillarin was knocked down in the oocytes from primary follicles, follicle development was inhibited as well, indicating that an rRNA processing defect in the oocyte indeed stunts chromatin configuration transition and follicle development. Taken together, these results elucidated the in vivo function of novel nucleolar protein DCAF13 in maintaining mammalian oogenesis.

Maternal DCAF2 is crucial for maintenance of genome stability during the first cell cycle in mice.

Precise regulation of DNA replication and genome integrity is crucial for gametogenesis and early embryogenesis. Cullin ring-finger ubiquitin ligase 4 (CRL4) has multiple functions in the maintenance of germ cell survival, oocyte meiotic maturation, and maternal-zygotic transition in mammals. DDB1-cullin-4-associated factor-2 (DCAF2, also known as DTL or CDT2) is an evolutionarily conserved substrate receptor of CRL4. To determine whether DCAF2 is a key CRL4 substrate adaptor in mammalian oocytes, we generated a novel mouse strain that carries a Dcaf2 allele flanked by loxP sequences, and specifically deleted Dcaf2 in oocytes. Dcaf2 knockout in mouse oocytes leads to female infertility. Although Dcaf2-null oocytes were able to develop and mature normally, the embryos derived from them were arrested at one- to two-cell stage, owing to prolonged DNA replication and accumulation of massive DNA damage. These results indicate that DCAF2 is a previously unrecognized maternal factor that safeguards zygotic genome stability. Maternal DCAF2 protein is crucial for prevention of DNA re-replication in the first and unique mitotic cell cycle of the zygote.This article has an associated First Person interview with the first author of the paper.