Mammalian oocyte maturation depends on the translational activation of stored maternal mRNAs upon meiotic resumption. Cytoplasmic polyadenylation element binding protein 1 (CPEB1) is a key oocyte factor that regulates maternal mRNA translation. However, the signal that triggers CPEB1 activation at the onset of mammalian oocyte maturation is not known. We provide evidence that a mitogen-activated protein kinase (MAPK) cascade couples maternal mRNA translation to meiotic cell cycle progression in mouse oocytes by triggering CPEB1 phosphorylation and degradation. Mutations of the phosphorylation sites or ubiquitin E3 ligase binding sites in CPEB1 have a dominant-negative effect in oocytes, and mimic the phenotype of ERK1/2 knockout, by impairing spindle assembly and mRNA translation. Overexpression of the CPEB1 downstream translation activator DAZL in ERK1/2-deficient oocytes partially rescued the meiotic defects, indicating that ERK1/2 is essential for spindle assembly, metaphase II arrest and maternal-zygotic transition (MZT) primarily by triggering the translation of key maternal mRNAs. Taken together, ERK1/2-mediated CPEB1 phosphorylation/degradation is a major mechanism of maternal mRNA translational activation, and is crucial for mouse oocyte maturation and MZT.
In vertebrates, fully-grown oocytes are arrested at the diplotene stage of meiosis I, and contain a large amount of maternal mRNAs that are translationally dormant (Chen et al., 2011). Upon meiotic maturation, oocytes undergo two consecutive M phases and arrest again at metaphase of meiosis II (MII). Many maternal mRNAs are translationally activated during this process (Chen et al., 2013; Piccioni et al., 2005). These temporally translated proteins play key roles in meiotic spindle assembly, MII arrest maintenance and mRNA clearance during maternal-zygotic transition (MZT).
The mechanisms of temporal and selective activation of maternal mRNAs are complicated and diverse among species (Komrskova et al., 2014). Cytoplasmic polyadenylation of the 3′-untranslated region (3′-UTR) is largely correlated with mRNA stability and translational activation of mRNA, and plays an essential role in oocyte maturation. Cytoplasmic polyadenylation of maternal mRNAs requires a cytoplasmic polyadenylation element (CPE) that binds specific trans-acting proteins (Pique et al., 2008). Studies in Xenopus and mouse oocytes found that the CPE-binding protein 1 (CPEB1) mediates cytoplasmic polyadenylation of many CPE-containing mRNAs (Ivshina et al., 2014). During Xenopus oocyte maturation, phosphorylation of CPEB on several serine/threonine residues is required for early activation of a class of maternal mRNAs, while a large fraction (70-90%) of CPEB1 proteins undergo a polyubiquitylation-dependent degradation in meiosis I (Mendez et al., 2002; Setoyama et al., 2007). This degradation causes a change in the CPEB/CPE ratio and results in activation of another class of mRNAs. However, the precise mechanism of CPEB1 activation and degradation in mammalian species is not known.
Recently, we reported that meiotic cell cycle-coupled CPEB1 activation in mouse oocyte is essential for translation of maternal mRNAs encoding B-cell translocation gene 4 (Btg4), a MZT licensing factor (Yu et al., 2016b). Our studies also found that extracellular signal-regulated kinases 1 and 2 (ERK1/2), two of the best-studied mitogen-activated protein kinase (MAPK) family members, are indispensable for BTG4 protein accumulation after meiotic resumption (Yu et al., 2016b). However, it remains unclear whether ERK1/2 is also required for translational activation of other important maternal mRNAs, and whether ERK1/2 accomplishes this function by regulating CPEB1 phosphorylation and degradation.
The ERK1/2 cascade plays pivotal roles in regulating oocyte meiotic cell cycle progression, but the identity of the phosphorylation substrates of ERK1/2 in mammalian oocytes remain elusive. ERK1/2 inhibition or knockout in mouse oocytes severely impairs microtubule organization and meiotic spindle assembly (Fan and Sun, 2004; Zhang et al., 2015). Deletion of ERK1/2 or their upstream kinase MOS (c-mos Moloney murine sarcoma viral oncogene homolog) leads to precocious sister chromatid separation and oocyte parthenogenetic activation (Verlhac et al., 1996; Zhang et al., 2015). Efforts have been made to identify the ERK1/2 phosphorylation substrates in regulating these processes. Verlhac and colleagues have reported MISS (MAPK interacting and spindle stabilizing) and DOC1R (deleted in oral cancer 1 related) as ERK1/2 substrates that regulate microtubule organization of mouse MII oocytes (Lefebvre et al., 2002; Terret et al., 2003). However, considering the profound influence of ERK1/2 knockout on oocyte meiotic division, the physiological function of MAPK cascade in oocytes could not be sufficiently explained by these mechanisms.
In this study, we provide evidence that ERK1/2 triggers the phosphorylation and degradation of CPEB1 at an early stage of oocyte meiotic resumption. By activating CPEB1, ERK1/2 couples the translation of a series of maternal mRNAs, including Dazl, Tpx2 and Btg4, to meiotic maturation. The translation products of these maternal mRNAs are required for meiotic divisions and MZT. CPEB1 mutants that fail to be phosphorylated and degraded in response to ERK1/2 signaling have dominant-negative effects, and cause oocyte maturation defects similar to ERK1/2 inhibition. The meiosis defects caused by ERK1/2 deficiency were partially rescued by exogenous expression of Dazl, a CPEB1 downstream mRNA translation activator. Taken together, we elucidated the fundamental role of ERK1/2 in regulating oocyte meiotic maturation and MZT: ERK1/2 trigger meiosis-dependent maternal mRNA translation by activating CPEB1.
ERK1/2 promotes Btg4 mRNA translation as a ubiquitous regulation mechanism in mammals
We recently reported that ERK1/2 activity is required to activate translation of Btg4mRNA, by targeting the three CPEs in its 3′-UTR (Yu et al., 2016b). This important mechanism couples maternal mRNA decay to mouse oocyte maturation. Therefore, we investigated whether it is conserved in other mammals such as pigs. In isolated pig oocytes, BTG4 protein was undetectable in germinal vesicle (GV) oocytes, but was highly expressed after germinal vesicle breakdown (GVBD), indicating that its expression was also coupled with oocyte maturation (Fig. 1A). The MEK1/2 inhibitor U0126 blocked ERK1/2 activation as well as BTG4 accumulation (Fig. 1B). CNOT7, the BTG4-targeted RNA deadenylase, showed a similar meiotic maturation-coupled and ERK1/2-dependent expression pattern to BTG4 (Fig. 1A,B). The crucial CPE sites in the 3′-UTR of mouse Btg4 were conserved in pigs (Fig. 1C). Similar to our observations in mouse, the 3′-UTR of pig Btg4 was activated only after meiotic resumption, and in an ERK1/2-dependent manner (Fig. 1D), suggesting that this is a ubiquitous regulation mechanism in mammals.
ERK1/2 deletion in mouse oocyte causes female infertility associated with maternal-zygotic transition defects
To investigate whether maternal ERK1/2 in mouse oocytes is required for MZT, we selectively deleted Erk2 in oocytes with an Erk1−/− background using transgenic mice expressing Gdf9 promoter-mediated Cre recombinase (Erk1−/−;Erk2fl/fl;Gdf9-Cre, later referred to Erk1/2oo−/−). Efficient ERK1/2 deletion was confirmed by western blotting using isolated oocytes from wild-type and Erk1/2oo−/− females, and oocyte-specific ERK1 deletion was also demonstrated using immunohistochemistry (Fig. 2A,B). The Erk1/2oo−/− females were sterile, whereas the males exhibited normal fertility. Erk1/2-deleted oocytes resume meiotic maturation (characterized by GVBD) at a similar rate to wild-type oocytes (Fig. 2C). However, the polar body 1 (PB1) emission was delayed after ERK1/2 deletion (Fig. 2D). Spindles with distorted shapes were observed in these oocytes (Fig. 2E). We then determined whether ERK1/2 was also required for MZT. Erk1/2oo−/− females were superovulated and then mated with wild-type males. When most control zygotes developed to the 2-cell stage at 1.5 days post-coitus (1.5 dpc), a remarkable proportion of zygotes from Erk1/2oo−/− mice were arrested at the zygote stage (76%, n=162) and quickly degenerated (Fig. 2F,G). Only 24% (n=46) of maternal ERK1/2-deleted embryos developed to the 2-cell stage, and 12% (n=56) developed to the 4-cell stage (2.5 dpc), but none developed into blastocysts at 4 dpc (Fig. 2F,G).
Maternal transcript degradation during GV-to-MII transition is blocked in Erk1/2oo−/− oocytes
Because ERK1/2 activity is required for accumulation of BTG4 protein and MZT, we compared the global mRNA levels between Erk1/2oo−/− and wild-type oocytes at GV and MII stages using RNA-seq analyses. Differential expression of transcripts was analyzed by comparing the fragments per kb of exon per million fragments mapped (FPKM) values between wild-type and Erk1/2oo−/− oocytes. We found a significant difference in transcriptomes between wild-type and Erk1/2oo−/− oocytes at the GV/MII stages (Fig. 3A). Compared with the GV oocytes of wild type, we detected 2572 upregulated and 3276 downregulated transcripts in Erk1/2oo−/− GV oocytes. Similarly, compared with the MII oocytes of wild type, 5157 significantly upregulated and 1606 downregulated transcripts were detected in Erk1/2oo−/− MII oocytes (Fig. 3A). Among the top 1000 most abundant transcripts (10%) in wild-type GV oocytes, 998 transcripts decrease more than 10-fold during GV to MII transition. At GV stage, only 16 and 75 of these transcripts were upregulated and downregulated in Erk1/2oo−/− oocytes, respectively (Fig. 3B). However, among the top 10% of abundant transcripts found in GV oocytes of wild-type mice, 455 were upregulated and 23 were downregulated in MII oocytes of Erk1/2oo−/− mice, when compared with MII oocytes of wild-type mice (Fig. 3B). These results indicate that half of the top 10% abundant transcripts that should have been degraded during the transition from GV to MII were stabilized in Erk1/2oo−/− oocytes.
Moreover, the RNA-seq data were verified using quantitative RT-PCR (qRT-PCR). Fig. 3C,D show the RNA-seq and qRT-PCR results of representative genes that were differentially expressed in wild-type and Erk1/2oo−/− oocytes. These transcripts were degraded during MZT of wild-type oocytes, but were stabilized in Erk1/2oo−/− oocytes. Significantly upregulated abundant transcripts (n=380, FPKM>100) were selected for gene ontology (GO) analysis. We found transcripts of 45 genes that were related to translation (Fig. 3E). It has been reported that the abundant ribosomal transcripts are significantly degraded during oocyte maturation (Su et al., 2007). Our RNA-seq data of wild-type GV/MII oocytes are inconsistent with this observation. The genes that encode the 60S and 40S ribosome subunits are listed in Fig. 3F. Among the 84 transcripts detected in wild-type oocytes, we found that 45 transcripts were upregulated after Erk1/2 deletion, whereas only eight were downregulated. This finding suggests that ERK1/2 promoted degradation of the ribosomal transcripts during the transition from GV to MII.
ERK1/2 is required for translational activation of multiple CPEB1-targeted maternal mRNAs during oocyte meiotic maturation
As translational activation of Btg4 mRNA is triggered by ERK1/2 in a CPE-dependent manner, we investigated whether the oocyte maturation-coupled translation of other CPE-containing maternal mRNAs also relied on ERK1/2 activity. Maternally accumulated cyclin B1 and microtubule nucleation factor (TPX2) are responsible for meiotic cell cycle progression and spindle assembly, respectively. However, in oocytes that underwent meiotic maturation in the presence of U0126, the levels of both proteins were lower than normal (Fig. 4A). Immunofluorescence results also showed that TPX2 protein is significantly accumulated onto the spindle of normal MII oocytes, but was absent or considerably downregulated on the distorted spindle of ERK1/2-inhibited oocytes (Fig. 4B). Poly(A) tail assay demonstrated that the poly(A) tails of multiple CPE-containing mRNAs, including Tpx2, Ccnb1 and Tex19.1, were elongated during the GV-to-MI progression. However, this effect was blocked by U0126 (Fig. 4C). In addition, we conducted qRT-PCR for CPE-containing transcripts that were reverse transcribed using oligo-dT or random primers. Oligo-dT favorably mediates the RT reaction of transcripts containing long poly(A) tails, but random primers do not have this preference. Therefore, the ratio changes of qRT-PCR results obtained from oligo-dT- versus random primer-mediated RT reactions also reflect the poly(A) tail length changes of given maternal transcripts (Moore et al., 1996). As shown in Fig. 4D, oligo-dT mediated more favorable reverse transcription of CPE-containing transcripts (Dazl, Tpx2, Ccnb1, Btg4 and Cnot7), but not of Gapdh, in MI oocytes when compared with GV oocytes (wild type). However, U0126 inhibited the oligo-dT-favored reverse transcriptions, indicating that ERK1/2 regulates the poly(A) tail elongation of these mRNAs.
Deleted in azoospermia-like (DAZL) is a CPEB1-downstream factor that mediates maternal mRNA translation in mouse oocytes (Chen et al., 2011). The 3′-UTR of Dazl mRNA contains four putative CPEs that bind with CPEB1. Dazl mRNA exhibited high levels of polyadenylation at the MI stage (Fig. 4C,D). To test whether ERK1/2 activity is also required for the translational activation of Dazl mRNA, we cloned the Dazl 3′-UTR from mouse, fused it with Flag-GFP, in vitro transcribed it into mRNAs [FLAG-GFP-(Dazl)3′-UTR], and injected them into GV oocytes. The experimental strategy is similar to that of the Btg4 3′-UTR reporter (Fig. 2C). After an overnight culture, GFP signals were detected in mature oocytes, but not in oocytes that were arrested at the GV stage using milrinone (Fig. 4E). U0126 blocked the translation of injected FLAG-GFP-(Dazl)3′-UTR mRNA (Fig. 4E). These observations were confirmed by western blotting of the translated Flag-GFP (Fig. 4F), indicating that the accumulation of DAZL protein in maturing mouse oocytes relies on ERK1/2 activity. Collectively, these results indicate that ERK1/2 targets the 3′-UTR of multiple CPE-containing maternal mRNAs and triggers their translational activation during oocyte meiotic maturation.
ERK1/2 is required for CPEB1 phosphorylation and degradation after oocyte meiotic resumption
In the following experiment, we investigated whether ERK1/2 triggers maternal mRNA translation by modulating CPEB1 in mouse oocytes. CPEB1 protein was present in GV oocytes, and was degraded between 4 and 6 h at meiosis resumption (Fig. 5A). An upshift band of CPEB1 was detected 4 h after meiotic resumption, coinciding with the phosphorylation activation of ERK1/2 at the same time point (Fig. 5A). When protein degradation was blocked, using the 26S proteosome MG132, the CPEB1 upshift was more clearly demonstrated (Fig. 5B). Western blots following phosphate affinity SDS-PAGE (Phos-tag-WB) further demonstrated that CPEB1 was phosphorylated before its degradation (Fig. 5C). The MEK1/2 inhibitor U0126 blocked both upshift and degradation of CPEB1 in maturing oocytes (Fig. 5A,B). Phos-tag-WB results also showed that GVBD-coupled CPEB1 phosphorylation was significantly inhibited by U0126 (Fig. 5C). CPEB1 phosphorylation and degradation were also impaired in Erk1/2oo−/− oocytes (Fig. 5D). These data demonstrate that ERK1/2 activity was required for triggering CPEB1 phosphorylation and degradation after oocyte meiotic resumption.
CPEB1 is phosphorylated and partially degraded during mouse oocyte meiotic maturation
To determine the CPEB1 phosphorylation sites in maturing oocytes, we focused on the two conserved SP motifs in mammalian CPEB1 (serine 181 and 207, both putative ERK1/2 phosphorylation sites) (Fig. 5E) and mutated the two serine residues to alanine either separately (CPEB1-S181/A or CPEB1-S207/A) or together (CPEB1-2A). We injected mRNAs encoding wild-type and mutated CPEB1 in oocytes arrested at the GV stage using milrinone. Following an overnight culture, oocytes were released from GV arrest by being transferred to milrinone-free medium. At 6 h following meiotic resumption, CPEB1 phosphorylation was decreased by either the S181/A or S207/A mutations, and was more significantly inhibited in CPEB1-2A mutants (Fig. 5F). Furthermore, we tested whether phosphorylation at S181 and S207 also regulates CPEB1 stability during meiotic maturation. Wild-type and mutated CPEB1 were expressed at a comparable level at GV stage (Fig. 5G). At 6 h after release from GV arrest, most wild-type CPEB1 proteins were degraded, whereas the CPEB1 mutant forms were stabilized, with CPEB1-2A being the most stable form (Fig. 5G).
The ubiquitin-dependent degradation of CPEB in maturing Xenopus oocytes requires a conserved PEST (Pro/Glu/Ser/Thr) sequence that binds with CRL1-βTrCP ubiquitin E3 ligase (Fig. 5E). Deleting the conserved PEST sequence in mouse CPEB1 (CPEB1-ΔPEST) prevented its binding with βTrCP, the substrate adaptor of CRL1 (Fig. 5H). Overexpression of βTrCP induced polyubiquitylation of CPEB1-WT, but not CPEB1-ΔPEST (Fig. 5I). The PEST sequence is spanned by S181 and S207 (Fig. 5E). CPEB1-2A neither bound with βTrCP, nor showed increased polyubiquitylation after βTrCP overexpression (Fig. 5H,I). Collectively, these results indicate that CRL1βTrCP mediates CPEB1 degradation in maturing mouse oocytes, and phosphorylation of S181 and S207 facilitates CPEB1 degradation by increasing its binding with βTrCP.
The activation and partial degradation of CPEB1 through phosphorylation is essential for meiotic maturation
In the following experiments, we investigated whether ERK1/2-triggered CPEB1 phosphorylation and degradation play a key role in oocyte maturation. GV-arrested oocytes were injected with mRNAs encoding CPEB1-WT or mutant proteins, and then allowed to undergo meiotic maturation 10-12 h after injection. Overexpression of CPEB1-WT in GV oocytes was quickly degraded after meiotic resumption, and did not cause any abnormalities (Fig. 5G and Fig. 6A-D). Overexpression of CPEB1-2A or CPEB1-ΔPEST did not affect GVBD (4 h after release from GV arrest), but resulted in decreased PB1 emission rates (12 h after release from GV-arrest) (Fig. 6A). It is noteworthy that the PB1 emission rate of CPEB1-2A-overexpressing oocytes was lower than that of Erk1/2oo−/− oocytes, indicating a strong dominant-negative effect (Fig. 6B). Distorted spindles were observed in most CPEB1-2A-overexpressing oocytes (Fig. 6C). TPX2, a microtubule-associated protein that is important for spindle assembly and chromosome alignment, accumulated at the MII spindle of normal oocytes (Fig. 6C). However, the TPX2 signal was undetectable on spindles of CPEB1-2A-overexpressing oocytes (Fig. 6C). Chromosome spreading and immunofluorescence showed that control MII-arrested oocytes contained 20 pairs of sister chromatids, but the CPEB1-2A-overexpressing oocytes still have paired homologous chromosomes (tetrads), indicating impaired chromosome separation in anaphase I (Fig. 6D). Deletion of the PEST sequence stabilized CPEB1 in maturing oocytes (Fig. 6E). Although spindle shape was distorted in CPEB1-ΔPEST-overexpressing oocytes, it was not as severe as that found in CPEB1-2A-overexpressing oocytes; however, the decrease of spindle-localized TPX2 was remarkable (Fig. 6C). In addition, compact chromosomes did not properly align at the equator of the spindles in these oocytes, and homologous chromosome separation was blocked (Fig. 6C,D). At the molecular level, overexpression of CPEB1-2A or PEST deletion blocked the accumulation of cyclin B1, BTG4 and CNOT7 (Fig. 6F). Taken together, the phosphorylation site- or degradation site-mutated CPEB1 has a dominant-negative effect in preventing bipolar spindle formation and homologous chromosome separation during oocyte maturation, both of which are meiotic events controlled by ERK1/2 in mammalian oocytes.
The CPEB1 downstream translation activator DAZL partially rescues meiosis defects in Erk1/2oo−/− oocytes
To further demonstrate that ERK1/2 regulates oocyte maturation by activating CPEB1 and triggering translation of essential maternal mRNAs, we tested whether the CPEB1 downstream protein DAZL could bypass ERK1/2 function, and rescue the meiosis defects seen in ERK1/2-inhibited oocytes. The 3′-UTR of mouse Btg4 contains four putative DAZL-binding elements (Fig. 7A). When the CPEs in the Dazl 3′-UTR were mutated, the translation levels of Flag-GFP-(Btg4)3′-UTR were significantly decreased (Fig. 7A). This result indicates that DAZL is required to maintain high levels of BTG4 expression after CPEB1 degradation. Furthermore, when we overexpressed mCherry-DAZL in maturing oocytes cultured with U0126 (Fig. 7B), expression of endogenous BTG4 and CNOT7 was partially rescued at the MII stage (Fig. 7C), confirming that DAZL functions downstream of ERK1/2 and CPEB1. In oocytes that underwent meiotic maturation in the presence of U0126, spindle assembly and TPX2 recruitment were impaired (Fig. 7D,F), and sister chromatids were prematurely segregated (Fig. 7E,G). The expression of exogenous DAZL partially rescued these phenotypes caused by ERK1/2 inhibition (Fig. 7D-G). Taken together, these results highlight the role of CPEB1 and DAZL in sequentially mediating the function of ERK1/2 in maturing oocytes.
CPEB1-mediated mRNA cytoplasmic polyadenylation is crucial to maternal mRNA translation during oocyte maturation. Its targeting sequence CPEs are associated with regulation of the poly(A) tail length, and they are present in the 3′-UTR of many key maternal mRNAs, suggesting that polyadenylation is a regulatory mechanism for the translation of these mRNAs. Both CPEB1 proteins and their target mRNAs were abundantly accumulated in GV-arrested oocytes, yet the polyadenylation and translation of these maternal mRNAs were only activated after meiotic resumption. Until this work, how CPEB1 activity is regulated in mammalian oocytes was unclear.
During Xenopus oocyte maturation, two different types of CPEB modifications contribute to translational activation. Phosphorylation of CPEB on Ser174 that occurs at an early stage of maturation is required for early activation of a class of mRNAs such as those encoding Mos (Martinez et al., 2005). However, a large fraction (70-90%) of CPEB proteins undergoes a CRL1-βTrCP-dependent degradation in meiosis I (Reverte et al., 2001). This degradation causes a change in the CPEB/CPE ratio and results in activation of another class of mRNAs, thereby driving entry into meiosis II.
Whereas the phosphorylation of mouse CPEB1 has been poorly studied, multiple MPAK and CDK1 phosphorylation sites have been identified in Xenopus CPEB. Among these, S210 phosphorylation by CDK1 is sufficient to target CPEB1 for destruction; however, one or more other proline-directed serine phosphorylations may also be involved (Mendez et al., 2002). Another study showed that MAPK activated CPEB (measured by Mos mRNA polyadenylation and translation) by directly phosphorylating it on four residues (T22, T164, S184 and S248) (Keady et al., 2007). Among these, S184 and S210 span the PEST sequence and are highly conserved in mammalian species (corresponding to S181 and S207 in mouse CPEB1). In this study, we provide evidence that ERK1/2-triggered phosphorylation at these two sites is crucial for CPEB1 degradation and oocyte maturation. The phosphorylation of these sites also facilitated the CPEB1-βTrCP binding. However, because preventing CPEB1 phosphorylation also blocked its degradation, we could not distinguish the specific roles of these two tightly associated biochemical events (CPEB1 phosphorylation and degradation). In addition, CPEB1-2A overexpression has a stronger effect on the PB emission than Erk1/2 deletion, suggesting that protein kinase(s) other than ERK1/2 may also regulate the phosphorylation of CPEB1 at S181 and S207.
By activating CPEB1, ERK1/2 triggers translational activation of multiple CPE-containing maternal mRNAs, including those encoding BTG4, TPX2 and DAZL, which are known to be essential for oocyte maturation and MZT (Brunet et al., 2008; Chen et al., 2011; Yu et al., 2016b). We provide evidence that the insufficient accumulation of these maternal factors is the primary reason for the developmental defects seen in ERK1/2-inhibited oocytes. Studies on mouse oocytes indicate that DAZL functions as a CPEB1-downstream translational activator, and several putative targets, including Tex19.1, Tpx2 and Dazl itself, have been identified (Sousa Martins et al., 2016). In this study, we add Btg4 to the list of DAZL-regulated maternal transcripts. DAZL maintains the translation of Btg4 mRNAs in oocytes during late maturation by targeting four DAZL-binding elements in its 3′-UTR, when CPEB1 has been degraded. Using a 3′-UTR reporter assay, we also demonstrated that the translational activation of Dazl mRNA was oocyte maturation coupled and depended on ERK1/2 activity.
DAZL function in oocyte maturation has previously been studied. Injection of Dazl morpholinos in GV oocytes reduced the number of oocytes that complete meiosis I (measured as polar body extrusion) and disrupted the spindle assembly at MII. Chromosome condensation and separation are also disrupted (Chen et al., 2011). These phenotypes mimicked several aspects of ERK1/2 knockout or inhibition in oocytes, suggesting that DAZL may be a key mediator of ERK1/2 function. Indeed, overexpression of DAZL in ERK1/2-inhibited oocytes partially rescued the oocyte maturation defects, including spindle assembly, MII arrest maintenance (measured as sister chromatid cohesion) and translational activation (measured as TPX2 and BTG4 accumulation). These results highlighted that the major function of ERK1/2 in oocytes was mediated by sequentially translated maternal factors, including DAZL, TPX2 and BTG4. These factors function in parallel with the directly phosphorylated targets of ERK1/2, such as the microtubule-associated target proteins MISS and DOC1R.
TPX2 is localized on spindle microtubules and activates Aurora A, an indispensable regulator of centrosome and spindle pole assembly (Eckerdt et al., 2009). In addition, TPX2 induces microtubule nucleation near chromosomes and facilitates spindle pole organization (Helmke and Heald, 2014). Our current study provides evidence that TPX2 accumulation on the meiotic spindle is severely impaired in ERK1/2-deficient oocytes, as its protein translation requires DAZL. Tpx2 mRNA depletion causes phenotypic changes very similar to those we observed with Erk1/2 knockout, including disruption of spindle assembly and failure of chromosome condensation. Therefore, defective spindle function assembly caused by inadequate levels of TPX2 protein is likely to contribute to the defects we observed after ERK1/2 inhibition or knockout.
We report for the first time that ERK1/2 is essential for MZT, in addition to its function in oocyte meiotic maturation. Although Erk1/2oo−/− oocytes have defects in spindle assembly and chromosome alignment, they are still able to develop to MII and be fertilized. However, the zygotes derived from these oocytes fail to develop beyond the 2-cell stage, and mimicked the phenotype of the Btg4 knockout. During meiotic maturation, activated ERK1/2 and CPEB1 trigger polyadenylation and translation of many maternal mRNAs, including those encoding Btg4 and Cnot7. The accumulated BTG4 and CNOT7 proteins in turn, mediate maternal mRNA deadenylation and degradation (Liu et al., 2016; Yu et al., 2016b). Therefore, a negative-feedback regulation mechanism is formed to ensure transient, but not prolonged, translation of proteins that are crucial for oocytes and zygotes. The function of ERK1/2 in regulating maternal mRNA translation and degradation during oocyte maturation and MZT is summarized in Fig. 8.
Our oocyte RNA sequencing and GO analyses indicated that ERK1/2-targeted transcripts were remarkably enriched for transcripts of genes associated with protein translation. Interestingly, this result coincides with previous reports. By microarray analyses, Su and colleagues demonstrated that the destruction of transcripts during the GV-to-MII transition is selective rather than promiscuous in mouse oocytes (Su et al., 2007). In particular, transcripts associated with meiotic arrest and the progression of oocyte maturation, such as oxidative phosphorylation, energy production and protein synthesis, were dramatically degraded. Selective degradation of these maternal transcripts is a prerequisite for transition from oocyte meiosis to blastomere mitosis, as well as transcriptional activation of the zygotic genome (Yu et al., 2016a,b). Aberrant degradation or maintenance of some classes of transcripts during oocyte maturation could be deleterious to oocyte quality and affect developmental competence, as we demonstrated in ERK1/2- and CPEB1-inhibited oocytes in this study.
MATERIALS AND METHODS
Erk1−/−, Erk2flox/flox and Gdf9-Cre mice have been previously reported (Fan et al., 2009; Zhang et al., 2015). All mouse strains had a C57B6 background. Wild-type C57B6 mice were obtained from the Zhejiang Academy of Medical Science, China. Mice were maintained under SPF conditions in a controlled environment of 20-22°C, with a 12/12 h light/dark cycle, 50-70% humidity and food and water provided ad libitum. Animal care and experimental procedures were conducted in accordance with the Animal Research Committee guidelines of Zhejiang University.
In vitro transcription and preparation of mRNAs for microinjections
To prepare mRNAs for microinjection, expression vectors were linearized and subjected to phenol/chloroform extraction and ethanol precipitation. The linearized DNAs were in vitro transcribed using the SP6 mMESSAGEmMACHINE Kit (Invitrogen, AM1450) following the manufacturer's instructions. mRNAs were recovered by lithium chloride precipitation and resuspended in nuclease-free water.
Microinjection of oocytes
For microinjection, fully grown GV oocytes were harvested in M2 medium with 2 μM milrinone to inhibit spontaneous GVBD. All injections were performed using an Eppendorf Transferman NK2 micromanipulator. Denuded oocytes were injected with 5 to 10 pl samples per oocyte. After injection, oocytes were washed and cultured in M16 medium plus 2 μM milrinone at 37°C with 5% CO2.
Superovulation and fertilization
Female mice (21-23 days old) were intraperitoneally injected with 5 IU of PMSG (Ningbo Sansheng Pharmaceutical, China). After 44 h, mice were injected with 5 IU of hCG (Ningbo Sansheng Pharmaceutical, China). After an additional 16 h, oocyte/cumulus masses were surgically removed from the oviducts and the numbers of oocytes were counted after digestion with 0.3% hyaluronidase (Sigma-Aldrich). Oocyte images were acquired using a Nikon SMZ1500 stereoscope. To obtain early embryos, female mice were mated with 10- to 12-week-old wild-type males. Successful mating was confirmed by the presence of vaginal plugs. Embryos were harvested from oviducts at the indicated times post-hCG injection.
Oocyte and embryo culture
Mice at 21 days of age were injected with 5 IU of PMSG and humanely euthanized 44 h later. Oocytes at the GV stage were harvested in M2 medium (M7167; Sigma-Aldrich) and cultured in mini-drops of M16 medium (M7292; Sigma-Aldrich) covered with mineral oil (M5310; Sigma-Aldrich) at 37°C in a 5% CO2 atmosphere. In some experiments, U0126 (20 μM) was added to the culture media to inhibit ERK1/2 activation. Zygotes were harvested from fertilized females 24 h after the hCG injection and cultured in KSOM medium (Millipore).
PBS-buffered formalin-fixed, paraffin wax-embedded ovary samples were sectioned (5 μm) for immunohistochemistry. Sections were deparaffinized and rehydrated. Primary antibodies were applied at suitable dilutions (Table S1) at room temperature for 1 h, and then incubated with biotinylated secondary antibodies for 30 min. Sections were then stained using Vectastain ABC and DAB peroxidase substrate kits (Vector Laboratories). Antibodies used in this study are listed in Table S1.
Confocal microscopy of mouse oocytes and embryos
Oocytes or embryos were fixed in PBS-buffered 4% paraformaldehyde (PFA) for 30 min at room temperature, followed by permeabilization with 0.2% Triton X-100. After blocking with 1% BSA in PBS, oocytes were incubated with primary antibodies diluted in blocking solution at room temperature for 1 h. After three washes with PBS, oocytes were labeled with secondary antibodies for 45 min, and then counterstained with 5 μg/ml of 4′,6-diamidino-2-phenylindole (DAPI) for 10 min. Oocytes were mounted on glass slides using SlowFade Gold Antifade Reagent (Life Technologies) and examined under a confocal laser scanning microscope (LSM 710, Carl Zeiss).
Chromosome spreading and immunofluorescence
ZP-free oocytes were fixed in a solution containing 1% paraformaldehyde, 0.15% Triton X-100 and 3 mmol/l DTT (Sigma-Aldrich) on glass slides for 30 min and air dried. Immunofluorescent staining was performed as in oocytes described above.
Western blot analysis
Oocytes were lysed with SDS sample buffer and heated for 5 min at 95°C. Total oocyte proteins were separated by SDS-PAGE and electrophoretically transferred to PVDF membranes (Millipore), followed by blocking in TBST containing 5% defatted milk (Becton Dickinson) for 30 min. After probing with primary antibodies, the membranes were washed in TBST, incubated with an HRP-linked secondary antibody (Jackson ImmunoResearch Laboratories) for 1 h, followed by three washes with TBST. Bound antibodies were detected using the Super Signal West Femto maximum sensitivity substrate (Thermo Fisher Scientific). The primary antibodies and dilution factors used are listed in Table S1.
Phos-tag SDS-PAGE analysis
Oocytes were lysed with SDS sample buffer and heated for 5 min at 95°C. Gels for Phos-tag SDS-PAGE consisted of 50 μM Phos-tag acrylamide (Wako, AAL-107) and 100 μM MnCl2 other than SDS-PAGE. Total oocyte proteins were separated by Phos-tag SDS-PAGE. Gels were washed in the transfer buffer containing 10 mM EDTA and then electrophoretically transferred to PVDF membranes (Millipore). The following steps were performed as described in the western blot analysis section.
Cell culture, plasmid transfection and immunoprecipitation
HeLa cells were obtained from ATCC and were recently authenticated and tested for contamination. Cells were grown in DMEM (Invitrogen) supplemented with 10% fetal bovine serum (FBS; Hyclone) and 1% penicillin-streptomycin solution (Gibco) at 37°C in a humidified 5% CO2 incubator. Mouse Cpeb1 and βTrCP cDNAs were PCR amplified from a mouse ovarian cDNA pool and cloned into pCS2- or pcDNA-based eukaryote expression vectors. Transient plasmid transfection was carried out using Lipofectamine 2000 (Invitrogen). After a 48 h transfection, cells were lysed in lysis buffer [50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 10% glycerol, and 0.5% NP-40]. After centrifugation, the supernatant was subjected to immunoprecipitation with different affinity gels (Sigma). After incubation at 4°C for 4 h, beads were washed with lysis buffer. SDS sample buffer was added to the beads and the eluted proteins were used for western blot analysis.
Poly(A) tail assay
Total RNA was isolated from oocytes at indicated stages using the RNeasy Mini Kit (Qiagen). The RNA was then hybridized with or without oligodT(20) before RNaseH treatment. RNA was purified and reacted with 5 mM GTP and ITP in RNase-free water for 1 h at 37°C using yPoly(A) polymerase (Affymetrix). Reverse transcription was performed using the SuperScript III Kit (Invitrogen) with C10T2 DNA primer (5′ CCC CCCCCC CTT 3′). The products were used in a PCR reaction with gene-specific primers (Table S2) and PAT2 (5′ CA GGA AAC AGC TAT GAC CCC CCCCCC CTT 3′) under the following conditions: 30 s at 94°C, 60 s at 56°C and 60 s at 72°C. PCR products were analyzed on a 2.5% agarose gel.
RNA isolation and library construction
GV and MII oocytes were collected from wild-type and Erk1/2oo−/− mice (300 per sample). Total RNA was extracted from each sample using the RNeasy Mini Kit (Qiagen) according to the manufacturer's protocol. In the initial step, we spiked 2×106 mRNA-GFP and 2×106 mRNA-RFP, transcribed in vitro, into each lysed sample for RNA isolation. Extracted total RNA was used to prepare a sequencing library using the TruSeq RNA Sample Prep Kit V2 (Illumina). Briefly, mRNA was purified with Oligo d(T)-25 beads, fragmented, and then reverse transcribed. The second strand was then synthesized with random primers in succession. Afterwards, double-stranded cDNA was subjected to end repair, A-tailing, adaptor ligation and PCR amplification of 10-12 cycles in order to obtain the library.
RNA-Seq data analysis
We aligned RNA-seq reads, sequenced by IlluminaHiseq 2500, to Mus musculus UCSC mm9 references with the Tophat software (http://tophat.cbcb.umd.edu/) and calculated the FPKM of each gene using Cufflinks (http://cufflinks.cbcb.umd.edu). The amount of total mRNA was calculated based on the FPKM of exogenous GFP and RFP. The expression level of each gene was calculated as its FPKM normalized to the FPKM of GFP/RFP. The top 10% transcripts at GV stage in wild type were selected, and the number of upregulated (FPKM+0.1, Erk1/2oo−/−/WT>2) and downregulated (FPKM+0.1, Erk1/2oo−/−/WT<0.5) transcripts were compared at GV and MII stage, respectively. DEGs (differentially expressed genes) were assessed using cuffdiff at a P value of <0.05 and FC (fold change) of >2 or <0.5. GO analysis for enrichment of DEGs was determined using the Database for Annotation, Visualization and Integrated Discovery (DAVID).
RNA isolation and real-time RT-PCR
Total RNA was extracted using the RNeasy Mini Kit (Qiagen) according to the manufacturer's instructions. Real-time RT-PCR analysis was performed using a Power SYBR Green PCR Master Mix (Applied Biosystems, Life Technologies) and an Applied Biosystems 7500 Real-Time PCR System. Relative mRNA levels were calculated by normalizing to the levels of endogenous β-actin mRNA (internal control) using Microsoft Excel. The relative transcript levels of samples were compared with the control, and the fold changes are demonstrated. For each experiment, qPCR reactions were carried out in triplicate. Primer sequences are listed in Table S2.
Results are given as mean±s.e.m. Each experiment included at least three independent samples and was repeated at least three times. Results for two experimental groups were compared using two-tailed unpaired Student's t-tests. Statistically significant values are indicated as follows: *P<0.05, **P<0.01 and ***P<0.001.
H.-Y.F. and Y.-L.Z. conceived the project and designed the experiments. Q.-Q.S. and X.-X.D. performed and analyzed the bulk of the experiments. Y.D., Y.-L.Z. and F.T. performed and analyzed the RNA-seq and WBGS. J.P.L. helped to perform the experiments. Q.-Q.S. wrote the manuscript and H.-Y.F. and Y.-L.Z. revised it.
This study is funded by the National Key Research and Development Program of China (2016YFC1000600) and by the National Natural Science Foundation of China (31528016, 91519313, 31371449, 31671558).
The RNA-seq data have been deposited at Gene Expression Omnibus under series accession GSE92317 (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE92317).
The authors declare no competing or financial interests.