The RNA ligase RNA terminal phosphate cyclase B regulates mRNA alternative splicing and is required for mouse oocyte development and maintenance.

Recent large-scale mRNA sequencing has shown that introns are retained in 5-10% of mRNA, and these events are named intron retention (IR). IR has been recognized as a key mechanism in the regulation of gene expression. However, the role of this mechanism in female reproduction in mammals remains unclear. RNA terminal phosphate cyclase B (RTCB) is a RNA ligase; we found that RTCB conditional knockout mice have premature ovarian failure and that RTCB plays a crucial role in follicular development. RTCB regulated the splicing of transcripts related to DNA methylation and DNA damage repair. In addition, it regulated the resumption of oocyte meiosis by affecting CDK1 activation. Moreover, the loss of RTCB suppressed zygotic genome activation (ZGA) and decreased translation at the global level. In addition, Rtcb deletion resulted in the accumulation of maternal mRNAs containing unspliced introns and in a decline in the overall level of transcripts. As a result, the Rtcb-/- females were sterile. Our study highlights the important role of RTCB-regulated noncanonical alternative splicing in female reproduction.

Biallelic mutations in MOS cause female infertility characterized by human early embryonic arrest and fragmentation.

Early embryonic arrest and fragmentation (EEAF) is a common phenomenon leading to female infertility, but the genetic determinants remain largely unknown. The Moloney sarcoma oncogene (MOS) encodes a serine/threonine kinase that activates the ERK signaling cascade during oocyte maturation in vertebrates. Here, we identified four rare variants of MOS in three infertile female individuals with EEAF that followed a recessive inheritance pattern. These MOS variants encoded proteins that resulted in decreased phosphorylated ERK1/2 level in cells and oocytes, and displayed attenuated rescuing effects on cortical F-actin assembly. Using oocyte-specific Erk1/2 knockout mice, we verified that MOS-ERK signal pathway inactivation in oocytes caused EEAF as human. The RNA sequencing data revealed that maternal mRNA clearance was disrupted in human mature oocytes either with MOS homozygous variant or with U0126 treatment, especially genes relative to mitochondrial function. Mitochondrial dysfunction was observed in oocytes with ERK1/2 deficiency or inactivation. In conclusion, this study not only uncovers biallelic MOS variants causes EEAF but also demonstrates that MOS-ERK signaling pathway drives human oocyte cytoplasmic maturation to prevent EEAF.

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.

[Function and molecular mechanism of mitogen-activated protein kinase (MAPK) in regulating oocyte meiotic maturation and ovulation].

The mitogen-activated protein kinase (MAPK) signaling pathway is a highly conserved signal transduction pathway from yeast to human species, and is widely distributed in various eukaryotic cells. In almost all of the species studied over the past three decades, this signaling pathway plays a crucial role in the development of female germ cells and meiotic maturation. Especially in a variety of mammalian species including primates, rodents, and domestic animals, the MAPK signaling pathway is activated during the resumption of first oocyte meiosis and plays an indispensable role in meiotic spindle assembly and cell cycle progression. In granulosa cells of fully grown ovarian follicles, the MAPK pathway also mediates the physiological action of gonadotropins, including cumulus expansion, ovulation, and corpus luteum formation. Although the MAPK signaling pathway plays a wide range of physiological functions during the female reproduction process, and these functions are highly conserved in evolution, their underlying mechanisms, especially their direct and physiological target molecules, have not been sufficiently studied for a long time. In recent years, based on some new gene-editing mouse models and theoretical findings, as well as the wide application of various omics techniques, it has been further revealed that MAPK directly phosphorylates and activates the RNA binding protein cytoplasmic polyadenylation element-binding protein-1 (CPEB1), promoting poly(A) tail extension of maternal mRNA to regulate protein translation during meiotic recovery. These findings not only constitute the current basic mechanism of mammalian oocyte maturation and ovulation, but also provide useful research ideas for other related research in this field. In this review, we summarize the research findings in our laboratory and from other groups regarding the role of MAPK cascade in regulating oocyte maturation and ovulation. We also discuss the latest research progress on MAPK regulation of mRNA translation and degradation by directly activating the translation initiation complex and mRNA poly(A) polymerase by phosphorylation in the granulosa cells.

CFP1-dependent histone H3K4 trimethylation in murine oocytes facilitates ovarian follicle recruitment and ovulation in a cell-nonautonomous manner.

CxxC-finger protein 1 (CFP1)-mediated trimethylated histone H3 at lysine-4 (H3K4me3) during oocyte development enables the oocyte genome to establish the competence to generate a new organism. Nevertheless, it remains unclear to which extent this epigenetic modification forms an instructive component of ovarian follicle development. We investigated the ovarian functions using an oocyte-specific Cxxc1 knockout mouse model, in which the H3K4me3 accumulation is downregulated in oocytes of developing follicles. CFP1-dependent H3K4 trimethylation in oocytes was necessary to maintain the expression of key paracrine factors and to facilitate the communication between an oocyte and the surrounding granulosa cells. The distinct gene expression patterns in cumulus cells within preovulatory follicles were disrupted by the Cxxc1 deletion in oocytes. Both follicle growth and ovulation were compromised after CFP1 deletion, because Cxxc1 deletion in oocytes indirectly impaired essential signaling pathways in granulosa cells that mediate the functions of follicle-stimulating hormone and luteinizing hormone. Therefore, CFP1-regulated epigenetic modification of the oocyte genome influences the responses of ovarian follicles to gonadotropin in a cell-nonautonomous manner.

A combinatorial code for mRNA 3'-UTR-mediated translational control in the mouse oocyte.

Meiotic maturation of mammalian oocytes depends on the temporally and spatially regulated cytoplasmic polyadenylation and translational activation of maternal mRNAs. Cytoplasmic polyadenylation is controlled by cis-elements in the 3'-UTRs of mRNAs including the polyadenylation signal (PAS), which is bound by the cleavage and polyadenylation specificity factor (CPSF) and the cytoplasmic polyadenylation element (CPE), which recruits CPE binding proteins. Using the 3'-UTRs of mouse Cpeb1, Btg4 and Cnot6l mRNAs, we deciphered the combinatorial code that controls developmental stage-specific translation during meiotic maturation: (i) translation of a maternal transcript at the germinal vesicle (GV) stage requires one or more PASs that locate far away from CPEs; (ii) PASs distal and proximal to the 3'-end of the transcripts are equally effective in mediating translation at the GV stage, as long as they are not close to the CPEs; (iii) Both translational repression at the GV stage and activation after germinal vesicle breakdown require at least one CPE adjacent to the PAS; (iv) The numbers and positions of CPEs in relation to PASs within the 3'-UTR of a given transcript determines its repression efficiency in GV oocytes. This study reveals a previously unrecognized non-canonical mechanism by which the proximal PASs mediate 3'-terminal polyadenylation and translation of maternal transcripts.

CNOT6L couples the selective degradation of maternal transcripts to meiotic cell cycle progression in mouse oocyte.

Meiotic resumption-coupled degradation of maternal transcripts occurs during oocyte maturation in the absence of mRNA transcription. The CCR4-NOT complex has been identified as the main eukaryotic mRNA deadenylase. In vivo functional and mechanistic information regarding its multiple subunits remains insufficient. Cnot6l, one of four genes encoding CCR4-NOT catalytic subunits, is preferentially expressed in mouse oocytes. Genetic deletion of Cnot6l impaired deadenylation and degradation of a subset of maternal mRNAs during oocyte maturation. Overtranslation of these undegraded mRNAs caused microtubule-chromosome organization defects, which led to activation of spindle assembly checkpoint and meiotic cell cycle arrest at prometaphase. Consequently, Cnot6l-/- female mice were severely subfertile. The function of CNOT6L in maturing oocytes is mediated by RNA-binding protein ZFP36L2, not maternal-to-zygotic transition licensing factor BTG4, which interacts with catalytic subunits CNOT7 and CNOT8 of CCR4-NOT Thus, recruitment of different adaptors by different catalytic subunits ensures stage-specific degradation of maternal mRNAs by CCR4-NOT This study provides the first direct genetic evidence that CCR4-NOT-dependent and particularly CNOT6L-dependent decay of selective maternal mRNAs is a prerequisite for meiotic maturation of oocytes.

CFP1 coordinates histone H3 lysine-4 trimethylation and meiotic cell cycle progression in mouse oocytes.

Trimethylation of histone H3 on lysine-4 (H3K4me3) is associated with gene-regulatory elements, but its transcription-independent function in cell division is unclear. CxxC-finger protein-1 (CFP1) is a major mediator of H3K4 trimethylation in mouse oocytes. Here we report that oocyte-specific knockout of Cxxc1, inhibition of CFP1 function, or abrogation of H3K4 methylation in oocytes each causes a delay of meiotic resumption as well as metaphase I arrest owing to defective spindle assembly and chromosome misalignment. These phenomena are partially attributed to insufficient phosphorylation of histone H3 at threonine-3. CDK1 triggers cell division-coupled degradation and inhibitory phosphorylation of CFP1. Preventing CFP1 degradation and phosphorylation causes CFP1 accumulation on chromosomes and impairs meiotic maturation and preimplantation embryo development. Therefore, CFP1-mediated H3K4 trimethylation provides 3a permission signal for the G2-M transition. Dual inhibition of CFP1 removes the SETD1-CFP1 complex from chromatin and ensures appropriate chromosome configuration changes during meiosis and mitosis.