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Mechanisms of maternal mRNA regulation: Implications for mammalian early embryonic development
Abstract
Mammalian oocytes accumulate a large pool of mRNA molecules that orchestrate subsequent embryonic development. The transcriptional machinery is silent during oocyte meiotic maturation and early embryogenesis, and thereby the early decisive events in embryo development prior to initiation of transcription from the embryonic genome are directed by the translation of pre-existing maternal mRNAs. Oocytes display remarkable post-transcriptional regulatory mechanisms that control mRNA stability and translation. The regulatory mechanisms are generally negative, and target mRNAs are either subjected to degradation or repressed from undergoing translation until specifically activated. Such negative regulatory mechanisms generally are mediated by transcript deadenylation, interaction of transcripts with RNA-binding proteins in a nonspecific or sequence-specific fashion, and/or potentially via actions of microRNA and repeat- associated small interfering RNA, which degrade maternal RNA transcripts. In contrast, translational activation is initiated via cytoplasmic polyadenylation of maternal transcripts facilitated via the binding of embryo-specific poly(A)-binding proteins (ePABs). In certain instances, translational regulation (positive or negative) is dictated by the balance of positive and negative trans-acting factors that compete for specific sequence motifs present in maternal transcripts. Coordinate post-transcriptional regulation of the oocyte mRNA pool is critical for normal progression of early embryonic development.
... Increased oocyte size during the growth phase is largely due to the accumulation of water, ions, and lipids within the ooplasm (Gosden and Bownes 1995). However, synthesis and storage of RNA and protein during oocyte growth is crucial for subsequent survival (reviewed by Bettegowda and Smith 2007). These stores of maternal transcripts and proteins are necessary after GV breakdown (GVBD) and fertilization until embryonic genome activation (i.e., when the embryo initiates transcription of its own genome for further survival, reviewed by Bettegowda and Smith 2007). ...
... However, synthesis and storage of RNA and protein during oocyte growth is crucial for subsequent survival (reviewed by Bettegowda and Smith 2007). These stores of maternal transcripts and proteins are necessary after GV breakdown (GVBD) and fertilization until embryonic genome activation (i.e., when the embryo initiates transcription of its own genome for further survival, reviewed by Bettegowda and Smith 2007). Transcriptional activity, as assessed by [ 3 H] uridine uptake by the nucleolus and nucleus of murine oocytes within ovarian follicles is rapid early in oocyte growth and declines in conjunction with the formation of the follicular antrum in tertiary follicles (Oakberg 1968; reviewed by Mermillod et al., 2008). ...
... Specifically, oocytes become transcriptionally-quiescent upon resumption of meiosis and GV breakdown (Rodman and Bachvarova 1976;Fair et al., 1995;Lodde et al., 2008). Thus, they must rely upon pools of RNA accumulated during the growth phase for protein synthesis (Wassarman and Letourneau 1976) during meiotic maturation, fertilization, and early embryonic cleavage divisions (reviewed by Bettegowda and Smith 2007), until embryonic genome activation. This occurs in bovine embryos at the 8 to 16-cell stage, 4 days after resumption of meiosis (Memili et al., 1998). ...
... In WT P14 oocytes transcripts corresponding to 10791 genes were detected. Importantly, many of these detected transcripts have been transcribed at earlier stages and are stored in growing oocytes 37 . As there is no Pol II transcription in Tbpl2 −/− growing oocytes 16 , RNAs detected in the Tbpl2 −/− mutant oocytes represent mRNAs transcribed by a TBP/TFIID-dependent mechanism and deposited into the growing oocytes independently of TBPL2 activity at earlier stages, i.e., at the primordial follicular stage, where TBP is still expressed. ...
... c Distribution of the best TATA box PWM matches within a −35 to −20 region upstream of the TBPL2-dependent (blue) and TBPL2-independent (orange) dominant TSS of the shifting promoters. The dashed lines indicate the median of the TATA box PWM matches for the TBPL2-dependent (blue) and the TBPL2-independent (orange) shifting TSS clusters (P value after a two-tailed Wilcoxon rankthe oocyte genome is transcriptionally active, and the newly synthesised maternal mRNAs are either translated or stored in a dormant form (reviewed in ref.37 ). The newly synthesised transcripts receive a long poly(A) tail and subsequently undergo poly(A) shortening in the oocyte cytoplasm, preventing translation. ...
During oocyte growth, transcription is required to create RNA and protein reserves to achieve maternal competence. During this period, the general transcription factor TATA binding protein (TBP) is replaced by its paralogue, TBPL2 (TBP2 or TRF3), which is essential for RNA polymerase II transcription. We show that in oocytes TBPL2 does not assemble into a canonical TFIID complex. Our transcript analyses demonstrate that TBPL2 mediates transcription of oocyte-expressed genes, including mRNA survey genes, as well as specific endogenous retroviral elements. Transcription start site (TSS) mapping indicates that TBPL2 has a strong preference for TATA-like motif in core promoters driving sharp TSS selection, in contrast with canonical TBP/TFIID-driven TATA-less promoters that have broader TSS architecture. Thus, we show a role for the TBPL2/TFIIA complex in the establishment of the oocyte transcriptome by using a specific TSS recognition code.
... Nevertheless, before becoming fully competent oocytes also need to go through molecular maturation. Transcriptional activity is supposed to be low during maturation (Bettegowda & Smith, 2007) and much of the mRNA transcribed and stored in the cytoplasm during oocyte growth is degraded, whereas some of it is protected from degradation and conferred stability by the 3' untranslated regions (Brevini et al., 2007). Polyadenylated oocyte mRNAs are required not only for meiotic resumption, but also for early embryo development (Piccioni et al., 2005;Brevini et al., 2007;Evsikov & Marín de Evsikova, 2009). ...
... The reasons behind the effects of in vitro maturation on the abundance of transcripts in the ooplasm have not been entirely elucidated, and may involve a combination of factors including a more stressful environment (Morado et al., 2009) requiring mRNAs for the synthesis of specific proteins, decreased ability of the oocyte transcription machinery (Bettegowda & Smith, 2007) to synthese news mRNAs, and degradation or deadenylation of transcripts during in vitro maturation (Thelie et al., 2009). A RNAseq study showed that some polyadenylated transcripts decreased in abundance during oocyte maturation, while others associated to cell-cycle progression, cytoskeletal organization, and macromolecule metabolism increased (Reyes et al., 2015). ...
Objective
In vitro maturation has been shown to influence gene expression in oocytes, but a common shortcoming in reports on the matter has been the use of different donors in each experimental group thus disregarding donor effects. This study aimed to investigate the abundance of mRNA in oocytes matured in vivo and in vitro obtained from the same group of donors.
Methods
A bovine model was used to assess the relative abundance of specific transcripts in in vitro-matured (IN VITRO-OPU) and in vivo-matured (IN VIVO-OPU) oocytes collected from the same donors by transvaginal ovum pick-up (OPU). Transcript abundance in oocytes from the IN VIVO-OPU group and oocytes matured in vitro but retrieved from different cows slaughtered at a commercial abattoir (IN VITRO-Abattoir group) was also compared. Total RNA was extracted from denuded oocytes and cDNA was produced via reverse transcription using an oligo(dT) primer for relative quantification of eight target transcripts by real-time PCR.
Results
Oocytes in the IN VITRO-OPU group had lower (p<0.05) abundance of peroxiredoxin 1 (Prdx1), heat shock protein 70.1 (Hsp70.1), growth and differentiation factor 9 (Gdf9), and maternal antigen that embryo requires (Mater) transcripts than the oocytes in the IN VIVO-OPU group, all obtained from the same pool of donor cows. Similar results were seen in the comparisons involving the IN VIVO-OPU and IN VITRO-Abattoir groups (p<0.05).
Conclusion
In vitro maturation affected the abundance of polyadenylated transcripts in the oocyte cytoplasm when compared to in vivo maturation induced by exogenous hormones in oocytes collected from the same donor pool.
... Therefore, any developmental events required for early embryogenesis (i.e. meiotic maturation, fertilization, initial cleavage divisions, and programming of EGA) prior to the onset of EGA are regulated by the translation of pre-existing maternal transcripts [6]. ...
... Twenty CpG sites from ~1,600 bp flanking the predicted TSS region were identified (Supplemental Fig. S2). The 3'UTR was found to contain two nuclear poly(A) signals (AAUAAA) and three cytoplasmic polyadenylation elements (U 5 A 1-2 U), which are required for translational activation of maternally derived mRNAs [6]. ...
... In mammalian oocytes, gene expression is mainly regulated at the post-transcriptional level; in particular, this regulation primarily occurs through cytoplasmic polyadenylation, a process that plays an important role in protecting mRNAs from degradation and stimulating the translation of these mRNAs [4]. During the meiotic maturation of mammalian oocytes, the mother must accumulate more energy than is required for in vitro oocyte maturation and early embryonic development. ...
... This process plays an extremely important role in preventing mRNA degradation and stimulating translation. The translation potential of maternal mRNAs primarily depends on the poly-(A) tail length, and deadenylation is typically the first sign of mRNA degradation (reviewed by Bettegowda and Smith, 2007). It can be inferred that the functions of polyadenylation/deadenylation may not only affect mRNA stability and translation efficiency during the course of oocyte maturation, but they may also affect mRNA degradation during early embryonic development, either before or after genome activation. ...
Aims:
This study aims to clarify the effects of polyadenylation status on M-phase promoting factors (MPFs) during in vitro porcine oocyte maturation.
Methods:
In this study, porcine follicular oocytes from large follicles (> 5 millimeter (mm)) and small follicles (< 3 mm) were examined at different follicular developmental stages. The polyadenylation of maternal mRNAs was inhibited by the addition of 3'-deoxyadenosine (3'-da) during the germinal vesicle (GV)(0 h), GV breakdown (GVBD)(18 h), metaphase I (MI)(28 h), and metaphase II (MII) (44 h) stages. In addition, the expression levels and poly-(A) tail lengths of the maternal mRNAs Cyclin B1 and cell division cycle 2 (Cdc2) were determined by real-time quantitative PCR. Immunofluorescence was used to assess spindle formation and chromosome alignment in the examined oocytes.
Results:
In large-follicle oocytes, the effects of inhibiting polyadenylation caused the percentage of mature to be significantly lower for the treated group than for the untreated group (p < 0.01). 3'-da can significantly improve the rate of small oocyte maturation in vitro and inhibits Cdc2 polyadenylation. Cyclin B1 plays a significant role in promoting the maturation of large-follicle oocytes. Polyadenylation contributes to the formation of dominant follicles and facilitates the selection of dominant follicles. However, the inhibition of adenylation affected spindle formation-related propulsion and chromosome alignment in both large- and small-follicle oocytes. The first polar body could not be extruded in certain large follicles.
Conclusions:
3'-da can significantly improve the rate of small oocyte maturation in vitro, but it can also affect spindle formation-related propulsion and chromosome alignment.
... Furthermore, the results of embryo culture in the presence of a-amanitin (Fig. 1, D and F) suggest that the decline in SMAD2 and SMAD3 mRNA, which also was observed previously for SMAD4 [16], during maternal-embryonic transition is itself transcription dependent. Although further investigation will be required, this regulation is possibly mediated by transcript deadenylation because adenylation and deadenylation of mRNAs are prominent mechanisms involved in the regulation of mRNA levels during the maternal-embryonic transition [30,31]. ...
... The dramatic decrease in SMAD2/3 protein abundance is in contrast with the slight change in SMAD2/3 mRNA level between pre-and postfertilization, which potentially suggests dynamic posttranslational regulation of SMAD2/3 protein. Dynamic posttranscriptional and posttranslational regulatory mechanisms are characteristic of the maternal-to-embryonic transition and critical to development [30]. Indeed, regulation of SMAD2/3 via ubiquitination is implicated in the degradation of SMAD2/3 and regulation of the TGF-b signaling pathway [32][33][34]. ...
The TGF-beta-SMAD signaling pathway is involved in regulation of various aspects of female reproduction. However, the intrinsic functional role of SMADs in early embryogenesis remains poorly understood. Previously, we demonstrated that treatment with follistatin, an activin (TGF-beta superfamily ligand) binding protein, is beneficial for bovine early embryogenesis and specific embryotropic actions of follistatin are dependent on SMAD4. Since SMAD4 is a common SMAD that can bind both SMAD2/3 and SMAD1/5, the objective of this study was to further determine the intrinsic role of SMAD2/3 in control of early embryogenesis and delineate if embryotropic actions of follistatin in early embryos are SMAD2/3 dependent. By using a combination of pharmacological and siRNA-mediated inhibition of SMAD2/3 signaling in the presence or absence of follistatin treatment, our results indicate SMAD2 and SMAD3 are both required for bovine early embryonic development and stimulatory actions of follistatin on 8-16 cell and blastocyst rates, but not early cleavage, are muted when SMAD2/3 signaling is inhibited. SMAD2 deficiency also results in reduced expression of the bovine trophectoderm cell specific gene CTGF. In conclusion, the present work provides evidence supporting a functional role of SMAD2/3 in bovine early embryogenesis and that specific stimulatory actions of follistatin are not observed in the absence of SMAD2/3 signaling.
Copyright 2015 by The Society for the Study of Reproduction.
... Many of the TPR1 transcripts are produced in the female gametocyte but remain translationally repressed by the RNA helicase DOZI; they are then supplied to the zygote as maternal mRNAs where they are translated . This process is widely observed across life kingdoms whereby quiescent female germ cells store mRNAs that are translated later in the zygote (Bettegowda and Smith, 2006;Sheth et al., 2010). In Plasmodium, products of these genes are required for zygote and ookinete development and include the ookinete surface antigens P25 and P28 as well as the Apetela 2 (AP2) transcription factor AP2-O Yuda et al., 2009). ...
... The first hypothesis is that both the paternal and maternal alleles are silenced during zygotic development, and that transcripts are inherited to the zygote by the female gametocyte as maternal mRNAs (Fig. 4A). Indeed, a defining feature of sexual development in metazoans is the detainment of a subset of transcripts in quiescent messenger ribonucleoprotein particles in the oocytes for later translation in the zygote (Bettegowda and Smith, 2006;Sheth et al., 2010). These particles involve DDX6 class DEAD box RNA helicases that directly bind maternal mRNAs and are often located in structures designated as P granules (Pitt et al., 2000;Schisa et al., 2001). ...
The malaria parasite develops sexually in the mosquito midgut upon entry with the ingested blood meal, before it can invade the midgut epithelium and embark on sporogony. Recent data have identified a number of distinct transcriptional programs operating during this critical phase of the parasite lifecycle. We aimed at characterizing the parental contribution to these transcriptional programs and establish the genetic framework that would guide further studies of Plasmodium zygotic development and ookinete-to-oocyst transition. To achieve this we used in vitro and in vivo cross-fertilization experiments of various parasite lines expressing fluorescent reporters under the control of constitutive and stage specific promoters. The results revealed that the zygote/ookinete stage exhibits a maternal phenotype with respect to constitutively expressed reporters, which is derived from either maternal mRNA inheritance or transcription of the maternal allele. The respective paternal alleles are silenced in the zygote/ookinete but reactivated after midgut invasion and transformation to oocyst. Transcripts specifically produced in the zygote/ookinete are synthesized de novo by both parental alleles. These findings highlight a putative role of epigenetic regulation of Plasmodium zygotic development and add substantially to the emerging picture of the molecular mechanisms regulating this important stage of malaria transmission.
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... Multiple mechanisms for maternal RNA degradation were reviewed by (Bettegowda and Smith 2007) and include microRNAs. MicroRNAs, short 21 -22 nucleotide non-coding sequences, downregulate gene expression by binding to known miRNA-target sites, on mRNA 92 encoding a protein, in the 3' untranslated regions (UTR; . ...
... However, the authors are unaware of any studies showing AGO2 expression in oocytes and embryos. Therefore, it is postulated that miR-181a promotes translational silencing but is not involved in the degradation of NPM2, which is likely to require another mechanism involving the 3' UTR (Bettegowda and Smith, 2007). ...
... In mammals, maternally stored messenger ribonucleic acids (mRNAs) undergo a translational control to promote timely synthesis of the required proteins during oocyte maturation and early embryo development [1,2]. RNA-binding proteins (RBPs) as a major component of translation complex play key roles in these processes. ...
Expression of the embryonic poly(A)-binding protein (EPAB) in frog, mouse, and human oocytes and early-stage embryos is maintained at high levels until embryonic genome activation (EGA) after which a significant decrease occurs in EPAB levels. Studies on the vertebrate oocytes and early embryos revealed that EPAB plays key roles in the translational regulation, stabilization, and protection of maternal mRNAs during oocyte maturation and early embryogenesis. However, it remains elusive whether EPAB interacts with other cellular proteins and undergoes phosphorylation to perform these roles. For this purpose, we identified a group of Epab-interacting proteins and its phosphorylation status in mouse germinal vesicle (GV)- and metaphase II (MII)-stage oocytes, and in 1-cell, 2-cell, and 4-cell preimplantation embryos. In the oocytes and early preimplantation embryos, Epab-interacting proteins were found to play roles in the translation and transcription processes, intracellular signaling and transport, maintenance of structural integrity, metabolism, posttranslational modifications, and chromatin remodeling. Moreover, we discovered that Epab undergoes phosphorylation on the serine, threonine, and tyrosine residues, which are localized in the RNA recognition motifs 2, 3, and 4 or C-terminal. Conclusively, these findings suggest that Epab not only functions in the translational control of maternal mRNAs through binding to their poly(A) tails but also participates in various cellular events through interacting with certain group proteins. Most likely, Epab undergoes a dynamic phosphorylation during the oocyte maturation and the early embryo development to carry out these functions.
... Among these accumulated RNAs, more than 60% are ribosomal RNAs (rRNAs) and about 19% are mRNAs that likely correspond to more than half of all the protein-coding genes in the genome of mice and humans (Evsikov et al., 2006;Wang et al., 2004;Sha et al., 2020b,a;Wu & Dean, 2020;Liu et al., 2016;Yu et al., 2016). In addition to mRNAs which are translated shortly after transcription, a large class of maternal mRNAs (30-45%) are stabilized in various ribonucleoprotein (RNP) complexes in a dormant state (Bettegowda & Smith, 2007;Balagopal & Parker, 2009). ...
Vertebrate oocytes face a particular challenge concerning the regulation of gene expression during meiotic maturation. Global transcription becomes quiescent in fully grown oocytes, remains halted throughout maturation and fertilization, and only resumes upon embryonic genome activation. Hence, the oocyte meiotic maturation process is largely regulated by protein synthesis from pre‐existing maternal messenger RNAs (mRNAs) that are transcribed and stored during oocyte growth. Rapidly developing genome‐wide techniques have greatly expanded our insights into the global translation changes and possible regulatory mechanisms during oocyte maturation. The storage, translation, and processing of maternal mRNAs are thought to be regulated by factors interacting with elements in the mRNA molecules. Additionally, posttranscriptional modifications of mRNAs, such as methylation and uridylation, have recently been demonstrated to play crucial roles in maternal mRNA destabilization. However, a comprehensive understanding of the machineries that regulate maternal mRNA fate during oocyte maturation is still lacking. In particular, how the transcripts of important cell cycle components are stabilized, recruited at the appropriate time for translation, and eliminated to modulate oocyte meiotic progression remains unclear. A better understanding of these mechanisms will provide invaluable insights for the preconditions of developmental competence acquisition, with important implications for the treatment of infertility. This review discusses how the storage, localization, translation, and processing of oocyte mRNAs are regulated, and how these contribute to oocyte maturation progression.
... Uncoupling of transcription and translation is prominent during spermiogenesis (round spermatid differentiation into spermatozoa) (Braun, 1998;Bettegowda and Wilkinson, 2010;Idler and Yan, 2012), oogenesis (maternal transcript production) (Bettegowda and Smith, 2007;Sha et al., 2019), preimplantation embryonic development ( protein production before zygotic genome activation) (Cui and Kim, 2007;Gohin et al., 2014;Lim et al., 2016) and neuronal cell functions (mRNA synthesis in the cell body and translation in the axon) (Richter, 2001;Andreassi and Riccio, 2009;Norbury, 2013). Several potential mechanisms have been identified to achieve uncoupled transcription and translation, including physical sequestration of mRNAs and proteins in RNP granules (Braun, 1998;Bettegowda and Wilkinson, 2010;Zhang et al., 2017), 3′ UTR length control through alternative polyadenylation (Di Giammartino et al., 2011;Mayr, 2017) and 3′ UTR lengthdependent selective decay of transcripts by UPF proteins (Boehm et al., 2014;Bao et al., 2016). ...
As one of the post-transcriptional regulatory mechanisms, uncoupling of transcription and translation plays an essential role in development and adulthood physiology. However, it remains elusive how thousands of mRNAs get translationally silenced while stability is maintained for up to hours or even days before translation. In addition to oocytes and neurons, developing spermatids display significant uncoupling of transcription and translation for delayed translation. Therefore, spermiogenesis represents an excellent in vivo model for investigating the mechanism underlying uncoupled transcription and translation. Through full-length poly(A) deep sequencing, we discovered dynamic changes in poly(A) length through deadenylation and re-polyadenylation. Deadenylation appeared to be mediated by microRNAs (miRNAs), and transcripts with shorter poly(A) tails tend to be sequestered into ribonucleoproteins (RNPs) for translational repression and stabilization. In contrast, re-polyadenylation might allow for translocation of the translationally repressed transcripts from RNPs to polysomes. Overall, our data suggest that miRNA-dependent poly(A) length control represents a novel mechanism underlying uncoupled translation and transcription in haploid male germ cells.
... Unlike most somatic cells, the oocyte can store mRNA for days. The transcriptional machinery is silent during oocyte meiotic maturation and early embryogenesis and oocytes display remarkable post-transcriptional regulatory mechanisms that control mRNA stability and translation [28,29]. We postulate that BFARv3 may be a component of these regulatory mechanisms. ...
The mouse 3110001I22Rik gene located in the first intron of Bfar is considered as a Bfar variant coding for the BFARv3 protein. However, it differs from other BFAR isoforms and resembles periphilin 1 (PPHLN1) due to its two (Lge1 and serine-rich) conserved domains. We identified the BFARv3/EGFP-interacting proteins by co-immunoprecipitation coupled to mass spectrometry, which revealed 40S ribosomal proteins (RPS3, RPS14, RPS19, RPS25, RPS27), histones (H1.2, H1.4, H3.3C), proteins involved in RNA processing and splicing (SFPQ, SNRPA1, HNRNPA3, NONO, KHDRBS3), calcium signaling (HPCAL1, PTK2B), as well as HSD17B4, GRB14, POSTN, and MYO10. Co-immunoprecipitation revealed that both Lge1 and Ser-rich domains of BFARv3 were necessary for binding to RNA-interacting factors NONO and SFPQ, known to be components of paraspeckles. Reciprocal co-immunoprecipitation and the proximity ligation assay confirmed that both BFARv3 and PPHLN1 could interact with NONO and SFPQ, suggesting a new function for PPHLN1 as well. BFARv3 and its Lge1 or Ser-rich-deficient mutants preferentially localize in the nucleus. We found an accumulation of BFARv3/EGFP (but not its mutated forms) in the nuclear granules, which was enhanced in response to arsenite treatment and ionizing radiation. Although Bfar v3 is expressed ubiquitously in mouse tissues, its expression is the highest in metaphase II oocytes. The BFARv3 interactome suggests its role in RNA metabolism, which is critical for the transcriptionally silent MII oocyte. Mouse BFARv3 has no ortholog in the human genome, thus it may contribute to the differences between these two species observed in oocyte maturation and early embryonic development.
... However, there is limited research on RNA methylation modifications during embryonic development. RNA is widely involved in mammalian reproduction processes, such as the maturation of sperm and oocytes, embryonic development, and maintenance of the pluripotency of embryonic stem cells (Bettegowda and Smith, 2007;Sha et al., 2020). Previous reports showed that RNA was essential for embryonic and fetal development (Giraldez et al., 2005;Murchison et al., 2005;Melton et al., 2010;Stadler et al., 2010;Suh et al., 2010). ...
N6-methyl-adenosine (m6A) methylation is one of the most common and abundant modifications of RNA molecules in eukaryotes. Although various biological roles of m6A methylation have been elucidated, its role in embryonic development is still unclear. In this review, we focused on the function and expression patterns of m6A-related genes in mammalian embryonic development and the role of m6A modification in the embryonic epigenetic reprogramming process. The modification of m6A is regulated by the combined activities of methyltransferases, demethylases, and m6A-binding proteins. m6A-related genes act synergistically to form a dynamic, reversible m6A pattern, which exists in several physiological processes in various stages of embryonic development. The lack of one of these enzymes affects embryonic m6A levels, leading to abnormal embryonic development and even death. Moreover, m6A is a positive regulator of reprogramming to pluripotency and can affect embryo reprogramming by affecting activation of the maternal-to-zygotic transition. In conclusion, m6A is involved in the regulation of gene expression during embryonic development and the metabolic processes of RNA and plays an important role in the epigenetic modification of embryos.
... This phenomenon was observed in the mouse and other species, which indicates that maternal transcript degradation is required for normal embryonic development [16]. Multiple negative regulatory mechanisms including mRNA deadenylation, interaction with RNA-binding proteins and miRNA-mediated degradation are involved in post-transcriptional degradation of maternal transcripts [17]. miRNAs such as miRNA-430 in zebra sh and miRNA-427 in Xenopus were shown to be present prior to embryonic genome activation and further studies revealed more evidence to support the role of these miRNAs in degradation of hundreds of maternal transcripts [18][19][20]. ...
Background Epigenetic regulation of oocyte-specific maternal factors is essential for oocyte and early embryonic development. KPNA7 is an oocyte-specific maternal factor, which controls transportation of nuclear proteins important for early embryonic development. To elucidate the epigenetic mechanisms involved in the controlled expression of KPNA7, both DNA methylation associated transcriptional silencing and miRNA-mediated mRNA degradation of KPNA7 were examined. Results Comparison of DNA methylation profiles at the proximal promoter of KPNA7 gene between oocyte and 6 different somatic tissues identified 3 oocyte-specific differentially methylated CpG sites. Expression of KPNA7 mRNA was reintroduced in bovine kidney-derived CCL2 cells after treatment with the methylation inhibitor, 5-aza-2-deoxycytidine (5-Aza). Analysis of the promoter region of KPNA7 gene in CCL2 cells treated with 5-Aza showed a lighter methylation rate in all the CpG sites. Bioinformatic analysis predicted 4 miRNA-1296 binding sites in the coding region of KPNA7 mRNA. Ectopic co-expression of miRNA-1296 and KPNA7 in HEK293 cells led to reduced expression of KPNA7 protein. Quantitative real time PCR analysis revealed that miRNA-1296 is expressed in oocytes and early stage embryos, and the expression reaches a peak level in 8-cell stage embryos, coincident with the time of embryonic genome activation and the start of declining of KPNA7 expression. Conclusions These results suggest that DNA methylation may account for oocyte-specific expression of KPNA7, and miRNA-1296 targeting the coding region of KPNA7 is a potential mechanism for KPNA7 transcript degradation during the maternal-to-zygotic transition.
... Interestingly, numerous genes were more highly expressed in oocytes compared with the other follicular cell types (cluster 1). Because mRNA and correspondent protein abundances did not correlate in the oocyte (2,(69)(70)(71), overexpression in the oocyte suggests that these transcripts may be stored as maternal RNAs for translation during oocyte maturation and/or the first rounds of embryonic cleavage (72). An increased abundance of these transcripts in oocyte and presence of their corresponding proteins in ffEVs are of particular importance to oocyte maturation and further embryonic development. ...
Follicular fluid (FF) fills the interior portion of the ovarian antral follicle and provides a suitable microenvironment for the growth of the enclosed oocyte through molecular factors that originate from plasma and the secretions of follicular cells. FF contains extracellular nanovesicles (ffEVs), including 30–100-nm membrane-coated exosomes, which carry different types of RNA, proteins, and lipids and directly influence oocyte competence to develop embryo. In the present study, we aimed to characterize the protein cargo of EVs from the FF of 3–6-mm follicles and uncover the origins of ffEVs by assessing expression levels of corresponding mRNAs in bovine follicular cells and oocyte and cell proteomes. Isolated exosome-like ffEVs were 53.6 + 23.3 nm in size and could be internalized by cumulus-oocyte complex. Proteomes of ffEVs and granulosa cells (GC) were assessed using nanoflow liquid chromatography coupled with high-resolution tandem mass spectrometry after the gel fractionation of total proteins. In total, 460 protein isoforms corresponding to 322 unique proteins were identified in ffEVs; among them, 190 were also identified via GC. Gene Ontology terms related to the ribosome, protein and RNA folding, molecular transport, endocytosis, signal transduction, complement and coagulation cascades, apoptosis, and developmental biology pathways, including PI3K-Akt signaling, were significantly enriched features of ffEV proteins. FfEVs contain numerous ribosome and RNA-binding proteins, which may serve to compact different RNAs to regulate gene expression and RNA degradation, and might transfer ribosomal constituents to the oocyte. Majority of genes encoding ffEV proteins expressed at different levels in follicular cells and oocyte, corroborating with numerous proteins, which were reported in bovine oocyte and cumulus cells in other studies thus indicating possible origin of ffEV proteins. The limited abundance of several mRNAs within follicular cells indicated that corresponding ffEV proteins likely originated from circulating exosomes released by other tissues. Analysis of bovine ffEV transcriptome revealed that mRNAs present in ffEV accounted for only 18.3% of detected ffEV proteins. In conclusion, our study revealed numerous proteins within ffEVs, which originated from follicular and other cells. These proteins are likely involved in the maintenance of follicular homeostasis and may affect oocyte competence.
... However, before they become fully competent oocytes, they also need to undergo molecular maturation. Transcriptional activity is supposed to be low during maturation (Bettegowda & Smith, 2007), and much of the mRNA transcribed and stored in the cytoplasm during oocyte growth is degraded, while part of it is protected from degradation and imparts stability to the untranslated regions 3′ (Brevini, Cillo, Antonini, Tosetti, & Gandolfi, 2007). In this study, oocytes undergoing prematuration and IVM increased mRNA levels for GDF9, CCNB1, PARN, cMOS, eIF4E, and H1FOO, which indicate that these RNAs are stored in the oocyte throughout the oocyte growth. ...
During the last 10 to 15 years, in vitro research to predict antral follicle growth and oocyte maturation has delivered interesting advances in the knowledge of processes regulating follicle growth and developmental competence of oocytes. This review discusses the contribution of cumulus and mural granulosa cells in the process of oocyte maturation and cumulus expansion in cumulus oocyte complexes (COCs) from follicles of different sizes and shows that differences in gene expression in oocytes, granulosa and theca cells of small and large follicles impact the success of in vitro blastocyst development. In addition, the molecular mechanisms by which COC metabolism and antioxidant defense provide oocyte competence are highlighted. Furthermore, new insights and perspectives on molecular and cellular regulation of in vitro oocyte maturation are emphasized.
... This phenomenon was observed in the mouse and other species, which indicates that maternal transcript degradation is required for normal embryonic development [16]. Multiple negative regulatory mechanisms including mRNA deadenylation, interaction with RNA-binding proteins and miRNA-mediated degradation are involved in post-transcriptional degradation of maternal transcripts [17]. miRNAs such as miRNA-430 in zebra sh and miRNA-427 in Xenopus were shown to be present prior to embryonic genome activation and further studies revealed more evidence to support the role of these miRNAs in degradation of hundreds of maternal transcripts [18][19][20]. ...
Background Epigenetic regulation of oocyte-specific maternal factors is essential for oocyte and early embryonic development. KPNA7 is an oocyte-specific maternal factor, which controls transportation of nuclear proteins important for early embryonic development. To elucidate the epigenetic mechanisms involved in the controlled expression of KPNA7, both DNA methylation associated transcriptional silencing and miRNA-mediated mRNA degradation of KPNA7 were examined. Results Comparison of DNA methylation profiles at the proximal promoter of KPNA7 gene between oocyte and 6 different somatic tissues identified 3 oocyte-specific differentially methylated CpG sites. Expression of KPNA7 mRNA was reintroduced in bovine kidney-derived CCL2 cells after treatment with the methylation inhibitor, 5-aza-2-deoxycytidine (5-Aza). Analysis of the promoter region of KPNA7 gene in CCL2 cells treated with 5-Aza showed a lighter methylation rate in all the CpG sites. Bioinformatic analysis predicted 4 miRNA-1296 binding sites in the coding region of KPNA7 mRNA. Ectopic co-expression of miRNA-1296 and KPNA7 in HEK293 cells led to reduced expression of KPNA7 protein. Quantitative real time PCR analysis revealed that miRNA-1296 is expressed in oocytes and early stage embryos, and the expression reaches a peak level in 8-cell stage embryos, coincident with the time of embryonic genome activation and the start of declining of KPNA7 expression. Conclusions These results suggest that DNA methylation may account for oocyte-specific expression of KPNA7, and miRNA-1296 targeting the coding region of KPNA7 is a potential mechanism for KPNA7 transcript degradation during the maternal-to-zygotic transition.
... This phenomenon was observed in the mouse and other species, which indicates that maternal transcript degradation is required for normal embryonic development [16]. Multiple negative regulatory mechanisms including mRNA deadenylation, interaction with RNA-binding proteins and miRNA-mediated degradation are involved in post-transcriptional degradation of maternal transcripts [17]. miRNAs such as miRNA-430 in zebra sh and miRNA-427 in Xenopus were shown to be present prior to embryonic genome activation and further studies revealed more evidence to support the role of these miRNAs in degradation of hundreds of maternal transcripts [18][19][20]. ...
Epigenetic regulation of oocyte-specific maternal factors is essential for oocyte and early embryonic development. KPNA7 is an oocyte-specific maternal factor, which controls transportation of nuclear proteins important for early embryonic development. To elucidate the epigenetic mechanisms involved in the controlled expression of KPNA7, both DNA methylation associated transcriptional silencing and miRNA-mediated mRNA degradation of KPNA7 were examined. Results Comparison of DNA methylation profiles at the proximal promoter of KPNA7 gene between oocyte and 6 different somatic tissues identified 3 oocyte-specific differentially methylated CpG sites. Expression of KPNA7 mRNA was reintroduced in bovine kidney-derived CCL2 cells after treatment with the methylation inhibitor, 5-aza-2-deoxycytidine (5-Aza). Analysis of the promoter region of KPNA7 gene in CCL2 cells treated with 5-Aza showed a lighter methylation rate in all the CpG sites. Bioinformatic analysis predicted 4 miRNA-1296 binding sites in the coding region of KPNA7 mRNA. Ectopic co-expression of miRNA-1296 and KPNA7 in HEK293 cells led to reduced expression of KPNA7 protein. Quantitative real time PCR analysis revealed that miRNA-1296 is expressed in oocytes and early stage embryos, and the expression reaches a peak level in 8-cell stage embryos, coincident with the time of embryonic genome activation and the start of declining of KPNA7 expression. Conclusions These results suggest that DNA methylation may account for oocyte-specific expression of KPNA7, and miRNA-1296 targeting the coding region of KPNA7 is a potential mechanism for KPNA7 transcript degradation during the maternal-to-zygotic transition.
... This phenomenon was observed in the mouse and other species, which indicates that maternal transcript degradation is required for normal embryonic development [16]. Multiple negative regulatory mechanisms including mRNA deadenylation, interaction with RNA-binding proteins and miRNA-mediated degradation are involved in post-transcriptional degradation of maternal transcripts [17]. miRNAs such as miRNA-430 in zebra sh and miRNA-427 in Xenopus were shown to be present prior to embryonic genome activation and further studies revealed more evidence to support the role of these miRNAs in degradation of hundreds of maternal transcripts [18][19][20]. ...
Background Epigenetic regulation of oocyte-specific maternal factors is essential for oocyte and early embryonic development. KPNA7 is an oocyte-specific maternal factor, which controls transportation of nuclear proteins important for early embryonic development. To elucidate the epigenetic mechanisms involved in the controlled expression of KPNA7, both DNA methylation associated transcriptional silencing and miRNA-mediated mRNA degradation of KPNA7 were examined. Results Comparison of DNA methylation profiles at the proximal promoter of KPNA7 gene between oocyte and 6 different somatic tissues identified 3 oocyte-specific differentially methylated CpG sites. Expression of KPNA7 mRNA was reintroduced in bovine kidney-derived CCL2 cells after treatment with the methylation inhibitor, 5-aza-2-deoxycytidine (5-Aza). Analysis of the promoter region of KPNA7 gene in CCL2 cells treated with 5-Aza showed a lighter methylation rate in all the CpG sites. Bioinformatic analysis predicted 4 miRNA-1296 binding sites in the coding region of KPNA7 mRNA. Ectopic co-expression of miRNA-1296 and KPNA7 in HEK293 cells led to reduced expression of KPNA7 protein. Quantitative real time PCR analysis revealed that miRNA-1296 is expressed in oocytes and early stage embryos, and the expression reaches a peak level in 8-cell stage embryos, coincident with the time of embryonic genome activation and the start of declining of KPNA7 expression. Conclusions These results suggest that DNA methylation may account for oocyte-specific expression of KPNA7, and miRNA-1296 targeting the coding region of KPNA7 is a potential mechanism for KPNA7 transcript degradation during the maternal-to-zygotic transition.
... This phenomenon was observed in the mouse and other species, which indicates that maternal transcript degradation is required for normal embryonic development [16]. Multiple negative regulatory mechanisms including mRNA deadenylation, interaction with RNA-binding proteins and miRNA-mediated degradation are involved in post-transcriptional degradation of maternal transcripts [17]. miRNAs such as miRNA-430 in zebra sh and miRNA-427 in Xenopus were shown to be present prior to embryonic genome activation and further studies revealed more evidence to support the role of these miRNAs in degradation of hundreds of maternal transcripts [18][19][20]. ...
Background Epigenetic regulation of oocyte-specific maternal factors is essential for oocyte and early embryonic development. KPNA7 is an oocyte-specific maternal factor, which controls transportation of nuclear proteins important for early embryonic development. To elucidate the epigenetic mechanisms involved in the controlled expression of KPNA7, both DNA methylation associated transcriptional silencing and miRNA-mediated mRNA degradation of KPNA7 were examined. Results Comparison of DNA methylation profiles at the proximal promoter of KPNA7 gene between oocyte and 6 different somatic tissues identified 3 oocyte-specific differentially methylated CpG sites. Expression of KPNA7 mRNA was reintroduced in bovine kidney-derived CCL2 cells after treatment with the methylation inhibitor, 5-aza-2-deoxycytidine (5-Aza). Analysis of the promoter region of KPNA7 gene in CCL2 cells treated with 5-Aza showed a lighter methylation rate in all the CpG sites. Bioinformatic analysis predicted 4 miRNA-1296 binding sites in the coding region of KPNA7 mRNA. Ectopic co-expression of miRNA-1296 and KPNA7 in HEK293 cells led to reduced expression of KPNA7 protein. Quantitative real time PCR analysis revealed that miRNA-1296 is expressed in oocytes and early stage embryos, and the expression reaches a peak level in 8-cell stage embryos, coincident with the time of embryonic genome activation and the start of declining of KPNA7 expression. Conclusions These results suggest that DNA methylation may account for oocyte-specific expression of KPNA7, and miRNA-1296 targeting the coding region of KPNA7 is a potential mechanism for KPNA7 transcript degradation during the maternal-to-zygotic transition.
... This phenomenon was observed in the mouse and other species, which indicates that maternal transcript degradation is required for normal embryonic development [16]. Multiple negative regulatory mechanisms including mRNA deadenylation, interaction with RNA-binding proteins and miRNA-mediated degradation are involved in post-transcriptional degradation of maternal transcripts [17]. miRNAs such as miRNA-430 in zebrafish and miRNA-427 in Xenopus were shown to be present prior to embryonic genome activation and further studies revealed more evidence to support the role of these miRNAs in degradation of hundreds of maternal transcripts [18][19][20]. ...
Background:
Epigenetic regulation of oocyte-specific maternal factors is essential for oocyte and early embryonic development. KPNA7 is an oocyte-specific maternal factor, which controls transportation of nuclear proteins important for early embryonic development. To elucidate the epigenetic mechanisms involved in the controlled expression of KPNA7, both DNA methylation associated transcriptional silencing and microRNA (miRNA)-mediated mRNA degradation of KPNA7 were examined.
Results:
Comparison of DNA methylation profiles at the proximal promoter of KPNA7 gene between oocyte and 6 different somatic tissues identified 3 oocyte-specific differentially methylated CpG sites. Expression of KPNA7 mRNA was reintroduced in bovine kidney-derived CCL2 cells after treatment with the methylation inhibitor, 5-aza-2'-deoxycytidine (5-Aza-CdR). Analysis of the promoter region of KPNA7 gene in CCL2 cells treated with 5-Aza-CdR showed a lighter methylation rate in all the CpG sites. Bioinformatic analysis predicted 4 miRNA-1296 binding sites in the coding region of KPNA7 mRNA. Ectopic co-expression of miRNA-1296 and KPNA7 in HEK293 cells led to reduced expression of KPNA7 protein. Quantitative real time PCR (RT-qPCR) analysis revealed that miRNA-1296 is expressed in oocytes and early stage embryos, and the expression reaches a peak level in 8-cell stage embryos, coincident with the time of embryonic genome activation and the start of declining of KPNA7 expression.
Conclusions:
These results suggest that DNA methylation may account for oocyte-specific expression of KPNA7, and miRNA-1296 targeting the coding region of KPNA7 is a potential mechanism for KPNA7 transcript degradation during the maternal-to-zygotic transition.
... However, before they become fully competent oocytes, they also need to undergo molecular maturation. Transcriptional activity is supposed to be low during maturation (Bettegowda & Smith, 2007), and much of the mRNA transcribed and stored in the cytoplasm during oocyte growth is degraded, while part of it is protected from degradation and imparts stability to the untranslated regions 3′ (Brevini, Cillo, Antonini, Tosetti, & Gandolfi, 2007). In this study, oocytes undergoing prematuration and IVM increased mRNA levels for GDF9, CCNB1, PARN, cMOS, eIF4E, and H1FOO, which indicate that these RNAs are stored in the oocyte throughout the oocyte growth. ...
This study evaluates the levels of messenger RNA (mRNA) for eIF4E, PARN, H1FOO, cMOS, GDF9, and CCNB1 in oocytes from secondary and antral follicles at different stages of development. The effects of in vitro culture, in vitro prematuration, and in vitro maturation on the expression of these genes on oocytes were also analyzed. The results showed that mRNA levels for H1FOO, GDF9, and PARN were higher in oocytes from small, medium, and large antral follicles, respectively, than those seen in secondary follicles. Oocytes from small, medium, and large antral follicles had higher levels of CCNB1 than oocytes from secondary follicles. Oocytes from cultured secondary follicles had higher levels of GDF9, CMOS, PARN, eIF4E, CCNB1, and H1FOO than before culture. Prematured oocytes from small antral follicles had higher levels of mRNA for GDF9, PARN, and eIF4E than before culture. In addition, higher levels of cMOS and H1FOO were identified in prematured oocytes from medium antral follicles. In conclusion, follicular growth is associated with an increase in the expression of H1FOO, GDF9, CCNB1, and PARN. The culture of secondary follicles, prematuration, and maturation of oocytes from antral follicles increase the expression of eIF4E, PARN, H1FOO, cMOS, GDF9, and CCNB1. Follicular growth increases expression of maturation‐related genes in vitro culture of preantral follicles increase gene expression prematuration and maturation of oocytes increases gene expression.
... Consistent with those findings, GO analysis of the 2853 genes specifically expressed in the control group showed preferential enrichment in oxidative phosphorylation, metabolic pathways, and RNA transport (Fig. 5b), showing the dysfunction of these pathways after light exposure. The clearance of maternal mRNAs is another important event during this maternal-to-zygotic transition (MZT) [9]. Compared to the control group, highintensity light caused the embryos to fail to downregulate (Fig. 5c), which might also have contributed to the low blastulation rate after light exposure. ...
Background:
Light exposure is a common stress factor in in vitro manipulation of embryos in the reproductive center. Many studies have shown the deleterious effects of high-intensity light exposure in different animal embryos. However, no transcriptomic studies have explored the light-induced injury and response in preimplantation embryos.
Results:
Here, we adopt different time-courses and illumination intensities to treat mouse embryos at the 2-cell stage and evaluate their effects on blastulation. Meanwhile, single-cell transcriptomes from the 2-cell to blastocyst stage were analyzed after high-intensity light exposure. These data show that cells at each embryonic stage can be categorized into different light conditions. Further analyses of differentially expressed genes and GO terms revealed the light-induced injury as well as the potential repair response after high-intensity lighting. Maternal-to-zygote transition is also affected by the failure to remove maternal RNAs and deactivate zygotic genome expression.
Conclusion:
Our work revealed an integrated response to high-intensity lighting, involving morphological changes, long-lasting injury effects, and intracellular damage repair mechanisms.
... Within an OCC, a bovine full-grown oocyte exercises low transcriptional activity or is transcriptionally silent [89]. Transcripts are accumulated during oocyte growth and then stored as long as protein synthesis is required. ...
Ovarian follicle provides a favorable environment for enclosed oocytes, which acquire their competence in supporting embryo development in tight communications with somatic follicular cells and follicular fluid (FF). Although steroidogenesis in theca (TH) and granulosa cells (GC) is largely studied, and the molecular mechanisms of fatty acid (FA) metabolism in cumulus cells (CC) and oocytes are emerging, little data is available regarding lipid metabolism regulation within ovarian follicles. In this study, we investigated lipid composition and the transcriptional regulation of FA metabolism in 3–8 mm ovarian follicles in bovine. Using liquid chromatography and mass spectrometry (MS), 438 and 439 lipids were identified in FF and follicular cells, respectively. From the MALDI-TOF MS lipid fingerprints of FF, TH, GC, CC, and oocytes, and the MS imaging of ovarian
sections, we identified 197 peaks and determined more abundant lipids in each compartment. Transcriptomics revealed lipid metabolism-related genes, which were expressed constitutively or more specifically in TH, GC, CC, or oocytes. Coupled with differential lipid composition, these data suggest that the ovarian follicle contains the metabolic machinery that is potentially capable of metabolizing FA from nutrient uptake, degrading and producing lipoproteins, performing de novo lipogenesis, and accumulating lipid reserves, thus assuring oocyte energy supply, membrane synthesis, and lipid-mediated signaling to maintain follicular homeostasis.
... In this study, we investigated both the temporal and spatial expression of the newly identified Pl-Fra mRNA throughout the sea urchin embryo development. The Pl-Fra transcripts are already present in the unfertilized eggs, in agreement with a well-known attitude of the sea urchins as well as of other organisms including vertebrates, i.e. the synthesis of large pools of mRNA and proteins during oogenesis and their storage in the cytoplasm for later use throughout embryogenesis (Bettegowda and Smith, 2007;Flytzanis et al., 1982). A similar behavior has been observed for the expression of Pl-jun in unfertilized eggs (Russo et al., 2014b). ...
Lithium (Li), Nickel (Ni), and Zinc (Zn) are metals normally present in the seawater, although they can have adverse effects on the marine ecosystem at high concentrations by interfering with many biological processes. These metals are toxic for sea urchin embryos, affecting their morphology and developmental pathways. In particular, they perturb differently the correct organization of the embryonic axes (animal-vegetal, dorso-ventral): Li is a vegetalizing agent and Ni disrupts the dorso-ventral axis, while Zn has an animalizing effect. To deeply address the response of Paracentrotus lividus embryos to these metals, we studied the expression profiling of Pl-Fra transcription factor (TF), relating it to Pl-jun, a potential partner for AP-1 complex formation, and to Pl-MT, known to be an AP-1 target and to have a protective role against heavy metals. The AP-1 TFs are found throughout the animal kingdom and are involved in many cellular events, i.e. cell proliferation and differentiation, immune and stress responses, cancer growth. Here, we isolated the complete Pl-Fra cDNA and showed that Pl-Fra transcript, already present in the unfertilized eggs, was newly synthesized from the blastula stage, while its spatial distribution was mainly observed in skeletogenic cells, similarly to Pl-jun. Interestingly, Pl-Fra expression was induced by the different metals and the induction kinetics revealed its persistent expression during treatments. Moreover, its temporal and spatial behavior in response to the three metals was comparable to that of Pl-jun and Pl-MT. The understanding of AP-1 functions in invertebrates may provide new knowledge about the mechanisms of response to metal injuries, as well as it might lead to acknowledge the TFs as new type of biomarkers for the evaluation of hazards in polluted environment.
... In this study, we investigated both the temporal and spatial expression of the newly identified Pl-Fra mRNA throughout the sea urchin embryo development. The Pl-Fra transcripts are already present in the unfertilized eggs, in agreement with a well-known attitude of the sea urchins as well as of other organisms including vertebrates, i.e. the synthesis of large pools of mRNA and proteins during oogenesis and their storage in the cytoplasm for later use throughout embryogenesis (Bettegowda and Smith, 2007;Flytzanis et al., 1982). A similar behavior has been observed for the expression of Pl-jun in unfertilized eggs (Russo et al., 2014b). ...
Many industrial activities release Nickel (Ni) in the environment with harmful effects for terrestrial and marine organisms. Despite many studies on the mechanisms of Ni toxicity are available, the understanding about its toxic effects on marine organisms is more limited. We used Paracentrotus lividus as a model to analyze the effects on the stress pathways in embryos continuously exposed to different Ni doses, ranging from 0.03 to 0.5 mM. We deeply examined the altered embryonic morphologies at 24 and 48 h after Ni exposure. Some different phenotypes have been classified, showing alterations at the expenses of the dorso-ventral axis as well as the skeleton and/or the pigment cells. At the lowest dose used, Ni mainly induced a multi-spicule phenotype observed at 24 h after treatment. On the contrary, at the highest dose of Ni (0.5 mM), 90% of embryos showed no skeleton and no pigment cells. Therefore, we focused on this dose to study protein and gene expression patterns at 24 and 48 h after exposure. Among the proteins analyzed, i.e. p38MAPK, Grp78 and Mn-SOD, only p38MAPK was induced by Ni treatment. Moreover, we analyzed the mRNA profiles of a pool of genes that are involved in stress response and in development mechanisms, i.e. the transcription factors Pl-NFkB and Pl-FOXO; a marker of DNA repair, Pl-XPB/ERCC3; a mitogen-activated protein kinase (MAPK), Pl-p38; an ER stress gene, Pl-grp78; an adapter protein, Pl-14-3-3ε; two markers of pigment cells, Pl-PKS1 and Pl-gcm. The spatial expression of mesenchymal marker genes has been evaluated in Ni-treated embryos at both 24 and 48 h after exposure. Our results indicated that Ni acts at several levels in P. lividus sea urchin, by affecting embryo development, influencing the embryonic immune response and activating stress response pathways to counteract the suffered injury and to promote embryos surviving.
... However, the post-transcriptional regulation of oocyte and CC transcripts needs to be elucidated. This is particularly important also because the stability and translation of the maternal mRNAs, that are accumulated during oocyte maturation (Niakan et al., 2012) and that drive human preimplantation development, are controlled by post-transcriptional regulatory mechanisms (Bettegowda and Smith, 2007). ...
STUDY QUESTION:
What is the expression pattern of microRNAs (miRNAs) in human cumulus-oocyte complexes (COCs)?
SUMMARY ANSWER:
Several miRNAs are enriched in cumulus cells (CCs) or oocytes, and are predicted to target genes involved in biological functions of the COC.
WHAT IS KNOWN ALREADY:
The transcriptional profiles of human MII oocytes and the surrounding CCs are known. However, very limited data are available about post-transcriptional regulators, such as miRNAs. This is the first study focussing on the identification and quantification of small RNAs, including miRNAs, in human oocytes and CCs using a deep-sequencing approach.
STUDY DESIGN, SIZE, DURATION:
MII oocytes and CCs were collected from women who underwent IVF.
PARTICIPANTS/MATERIALS, SETTING, METHODS:
Using the Illumina/deep-sequencing technology, we analyzed the small RNAome of pooled MII oocytes (n = 24) and CC samples (n = 20). The mRNA targets of CC and MII oocyte miRNAs were identified using in silico prediction algorithms. Using oligonucleotide microarrays, genome-wide gene expression was studied in oocytes (10 pools of 19 ± 3 oocytes/each) and 10 individual CC samples. TaqMan miRNA assays were used to confirm the sequencing results in independent pools of MII oocytes (3 pools of 8 ± 3 oocytes/each) and CC samples (3 pools of 7 ± 3 CCs/each). The functional role of one miRNA, MIR23a, was assessed in primary cultures of human CCs.
MAIN RESULTS AND THE ROLE OF CHANCE:
Deep sequencing of small RNAs yielded more than 1 million raw reads. By mapping reads with a single location to the human genome, known miRNAs that were abundant in MII oocytes (MIR184, MIR100 and MIR10A) or CCs (MIR29a, MIR30d, MIR21, MIR93, MIR320a, MIR125a and the LET7 family) were identified. Predicted target genes of the oocyte miRNAs were associated with the regulation of transcription and cell cycle, whereas genes targeted by CC miRNAs were involved in extracellular matrix and apoptosis. Comparison of the predicted miRNA target genes and mRNA microarray data resulted in a list of 224 target genes that were differentially expressed in MII oocytes and CCs, including PTGS2, CTGF and BMPR1B that are important for cumulus-oocyte communication. Functional analysis using primary CC cultures revealed that BCL2 and CYP19A1 mRNA levels were decreased upon MIR23a overexpression.
LIMITATIONS, REASONS FOR CAUTION:
Only known miRNAs were investigated in the present study on COCs. Moreover, the source of the material is MII oocytes that failed to fertilize.
WIDER IMPLICATIONS OF THE FINDINGS:
The present findings suggest that miRNA could play a role in the regulation of the oocyte and CC crosstalk.
STUDY FUNDING/COMPETING INTEREST(S):
This work was partially supported by a grant from Ferring Pharmaceuticals. The authors of the study have no conflict of interest to report.
... During oocyte maturation, maternal mRNAs are accumulated in the cytoplasm [18]. Most of these maternal mRNAs are in a masked state, and the translation of these masked mRNAs in mature oocytes is regulated at the post-transcriptional level [19]. Since miRNAs function by causing mRNA translational inhibition or degradation [20,21], it is reasonable to assume that miRNAs may take part in the regulation of maternal mRNA translation in aging oocytes. ...
In this study, microRNA (miRNA) profiles in postovulatory aging mouse oocytes were analyzed by microarray screening and RT-qPCR. Hierarchical cluster analysis on the microarray data and KEGG pathway enrichment analysis on the mRNAs targeted by differentially expressed (DE) miRNAs between two adjacent egg-ages suggest that while only a mild alteration in miRNA expression occurred from 13 to 18 h, a great change took place from 18 to 24 h post hCG injection. Theoretical exploration on functions of the predicted target genes suggest that KEGG pathways enriched by 13-18 h DE miRNAs are correlated with early events of oocyte aging while pathways most enriched by 18-24 h or 24-30 h DE miRNAs are correlated with the late symptoms of aged oocytes. Experimental verification on functions of the key proteins predicted by the KEGG analysis and injection of miR-98 mimics or inhibitors further confirmed that miRNAs played stimulatory/inhibitory roles in postovulatory oocyte aging. In conclusion, marked changes in miRNA expression are associated with significant alterations in function and morphology of postovulatory aging oocytes.
... How maternal mRNA is regulated at the levels of translation and stability is the subject of intense investigation. Cytoplasmic polyadenylation represents a conserved mechanism for translational enhancement of dormant mRNAs with short poly(A) tails (Bettegowda and Smith 2007). Strong coupling between poly(A) tail length and translation efficiency (TE) has been observed in the cleavage embryos of many species (Subtelny et al. 2014;Eichhorn et al. 2016;Lim et al. 2016), although the molecular mechanisms that enable this coupling remain unclear. ...
During the maternal-to-zygotic transition (MZT), transcriptionally silent embryos rely on post-transcriptional regulation of maternal mRNAs until zygotic genome activation (ZGA). RNA-binding proteins (RBPs) are important regulators of post-transcriptional RNA processing events, yet their identities and functions during developmental transitions in vertebrates remain largely unexplored. Using mRNA interactome capture, we identified 227 RBPs in zebrafish embryos before and during ZGA, hereby named the zebrafish MZT mRNA-bound proteome. This protein constellation consists of many conserved RBPs, some of which are potential stage-specific mRNA interactors that likely reflect the dynamics of RNA-protein interactions during MZT. The enrichment of numerous splicing factors like hnRNP proteins before ZGA was surprising, because maternal mRNAs were found to be fully spliced. To address potentially unique roles of RBPs in embryogenesis, we focused on Hnrnpa1. iCLIP and subsequent mRNA reporter assays revealed a function for Hnrnpa1 in the regulation of poly(A) tail length and translation of maternal mRNAs through sequence-specific association with 3'UTRs before ZGA. Comparison of iCLIP data from two developmental stages revealed that Hnrnpa1 dissociates from maternal mRNAs at ZGA and instead regulates the nuclear processing of pri-mir-430 transcripts, which we validated experimentally. The shift from cytoplasmic to nuclear RNA targets was accompanied by a dramatic translocation of Hnrnpa1 and other pre-mRNA splicing factors to the nucleus in a transcription-dependent manner. Thus, our study identifies global changes in RNA-protein interactions during vertebrate MZT and shows that Hnrnpa1 RNA-binding activities are spatially and temporally coordinated to regulate RNA metabolism during early development.
... Although the exact identity of most of these factors remains a mystery, they are nevertheless instrumental in preparing the oocyte for the egg to embryo transition. [1][2][3][4][5] The presence and correct storage of these maternal factors is crucial for embryonic genome activation (EGA) which needs to occur for proper embryonic development. 6,7 During this transition, mRNAs are released, translated, and the resulting proteins, along with other stored maternal factors, are targeted to the nucleus in a regulated fashion where they then orchestrate the nuclear reprogramming events leading to the EGA. ...
The oocyte cytoplasmic lattices (CPLs) have long been predicted to function as a storage form for the maternal contribution of ribosomes to the early embryo. Our previous studies have demonstrated that ribosomal component S6 is stored in the oocyte CPLs and peptidylarginine deiminase 6 (PADI6) is critical for CPLs formation. Additionally, we found that depletion of PADI6 reduced de novo protein synthesis prior to the maternal-to-embryonic transition, therefore causing embryos to arrest at the two-cell stage. Here, we present evidence further supporting the association of ribosomes with the CPLs by demonstrating that rRNAs are dramatically decreased in Padi6 KO oocytes. We also show that the abundance and localization of mRNAs is affected upon PADI6 depletion, suggesting that mRNAs are very possibly associated with CPLs. Consistent with this observation, the amount of the major RNA binding protein, MSY2, that is associated with the insoluble fraction of the oocytes after Triton X-100 extraction is also markedly decreased in the Padi6 KO oocytes. Furthermore, treatment of the oocytes with RNase A followed by Triton X-100 extraction severely impairs the localization of PADI6 and MSY2 in oocytes. These results indicate that mRNAs, possibly in a complex with MSY2 and PADI6, are bound in the CPLs and may play a role in securing the mRNA-MSY2 complex to the CPLs.
... Embryo implantation is a developmental biological event, which requires a mature embryo and a receptive endometrium [1,2]. The mature embryo invades and penetrates the endometrium in a process called placentation, which is crucial for successful establishment of pregnancy. ...
Placental trophoblast cells differentiate into invasive trophoblasts or syncytiotrophoblasts. Abnormal trophoblast invasion results in pregnancy-associated disease and abortion. uPAR is a cell membrane-bound glycosylated protein, involved in physiological and pathological processes. However, uPAR expression in villi during threatened abortion and its role in trophoblast differentiation are unclear. We determined that, uPAR expression in the villi was reduced in threatened abortion patients than that in normal pregnancy. uPARsiRNA inhibited the potential for trophoblast migration and invasion in explants culture and HTR8/SVneo cells. It also enhanced forskolin-induced fusion of HTR8/SVneo cells. Overall, this study provides a possible reason for abortion.
... However, in the presence of both concentrations of BSA, 10 mM control MO, and 6 mM EP, the development of all zygotes tested was arrested before the morula stage (data not shown). In mice, the embryonic genome is activated during the development from zygotes to the 2cell embryos [15,16]. We considered that the application of MOs and EP to this critical period is unsuitable. ...
Knockdown of gene expression by antisense morpholino oligos (MOs) is a simple and effective method for analyzing the roles of genes in mammalian cells. Here, we demonstrate the efficient delivery of MOs by Endo-Porter (EP), a special transfection reagent for MOs, into preimplantation mouse embryos cultured in vitro. A fluorescein-labeled control MO was applied for monitoring the incorporation of MOs into developing 2-cell embryos in the presence of varying amounts of EP and bovine serum albumin. In optimized conditions, fluorescence was detected in 2-cell embryos within a 3-h incubation period. In order to analyze the validity of the optimized conditions, an antisense Oct4 MO was applied for knockdown of the synthesis of OCT4 protein in developing embryos from the 2-cell stage. In blastocysts, the antisense Oct4 MO induced a decrease in the amount in OCT4 protein to less than half. An almost complete absence of OCT4-positive cells and nearly complete disappearance of the inner cell mass in the outgrowths of blastocysts were also noted. These phenotypes corresponded with those of Oct4-deficient mouse embryos. Overall, we suggest that the delivery of MOs using EP is useful for the knockdown of gene expression in preimplantation mouse embryos cultured in vitro.
... An increased understanding of the maternal transcriptome is needed to elucidate the underlying molecular mechanism of normal oocyte maturation, which depends on a precise sequence of changes in maternal genes expression. The translational potential of a maternal mRNA is generally determined by the length of the poly(A) tail, and deadenylation is usually Effect of maternal gene expression on porcine oocytes in vitro maturation the first sign of mRNA degradation [1]. The in vivo overexpression of poly(A) polymerase (PAP), which further intensified the level of cellular polyadenylation, resulted in embryonic lethality in Drosophila [2]. ...
Understanding of the maternal transcriptome increased to elucidate the underlying molecular mechanism of normal oocyte maturation, which depends on a precise sequence of changes in maternal genes expression. Previous reports that the translational potential of a maternal mRNA is generally determined by the length of the poly(A) tail, and deadenylation is usually the first sign of mRNA degradation. However, in vitro cultured system has the underlying molecular mechanisms remain unclear. We determined whether the role of molecular basis, four important maternal genes, C-mos, cyclin-B1 (regulatory subunit of MPF), BMP15 and GDF9, were selected for detection of their precise mRNA expression patterns by real-time PCR and for determination of their polyadenylation status by poly(A) tail PCR during oocyte maturation. In the present study. the abnormal expression of maternal mRNAs prior to zygotic genome activation, which results in suppression of the corresponding protein level, may be responsible for, at least in part, a profound defect in further embryonic development. Reasonable expression of maternal gene is crucial for proper oocyte maturation and further embryonic development.
... Several theories have been proposed to explain this unaccounted heritability, such as part of heritability being 'hidden' in numerous weakly contributing genetic risk factor patterns of linkage disequilibrium (Visscher et al., 2012), or heritability estimates being inflated by epistatic gene interactions (Zuk et al., 2012). Given the large unaccounted heritability of major psychosis (collective term for SCZ and BPD), other molecular mechanisms may be involved, including the passage of epigenetic marks through the germ line (Chong and Whitelaw, 2004), the passage of maternal RNA molecules into the embryo (Bettegowda and Smith, 2007;Rassoulzadegan et al., 2006), the potential passage of prion proteins from parent to offspring (Shorter and Lindquist, 2005), the biochemical state of the gametes at the time of conception, and the transmission of nutrients, bacteria, or antibodies from maternal circulation to that of the offspring (Boulinier and Staszewski, 2008;Grindstaff et al., 2003;Hasselquist and Nilsson, 2009). All of these factors can, and do, influence phenotypic variability during development. ...
Development of psychiatric disorders such as major depressive disorder, bipolar disorder, and schizophrenia involve - as with most complex diseases - both inherited and nonheritable components, which traditionally have been attributed to alterations in DNA and hazardous environment, respectively. Molecular genetic studies, however, have been able to explain only a very small fraction (1-2%) of heritability, a sharp contrast with population estimates of 30-80% heritability in the three diseases. Similarly, epidemiological studies have demonstrated correlations between life experiences (pre- and/or perinatal events, socioeconomic status, etc.) and psychiatric disease risk, but are unable to detect any specific causal environmental hazards. It has become evident that the efforts to identify the primary causes of complex psychiatric disease may significantly benefit from epigenetic studies. Inherited and/or acquired epigenetic factors are relatively stable and they have regulatory roles in numerous genomic activities, which translate into phenotypic outcomes. In this article, we outline the characteristics of psychiatric diseases and survey recent developments revealing epigenetic aspects of mental illness. We also discuss some rare psychiatric syndromes in light of recent discoveries of epigenetic mechanisms in learning and memory. Finally, we review some of the challenges of epigenetic approaches to psychiatric disorders and describe some future directions in the field of behavioral and psychiatric epigenetics.
... In transcriptionally quiescent maturing oocytes and early embryos, stored maternal factors like mRNAs and proteins become recruited, modified and expressed in a stage-specific and timed manner to ensure normal OET and zygotic gene activation (ZGA) (Bettegowda and Smith, 2007;Fair, 2010;Li et al., 2010;Zuccotti et al., 2011;Clift and Schuh, 2013;Norbury, 2013;Ma et al., 2015). Long-term storage of mRNAs during oocyte growth requires mRNA-binding proteins like the intracellular germ cell-specific mRNA binding protein MSY2 (also termed YBX2, Y-box-binding protein 2 in human, and FRGY2 in Xenopus), a conserved protein that confers stability to mRNAs as well as translational repression (Medvedev et al., 2011;Ma et al., 2015). ...
Study question:
Is the postovulatory aging-dependent differential decrease of mRNAs and polyadenylation of mRNAs coded by maternal effect genes associated with altered abundance and distribution of maternal effect and RNA-binding proteins (MSY2)?
Summary answer:
Postovulatory aging results in differential reduction in abundance of maternal effect proteins, loss of RNA-binding proteins from specific cytoplasmic domains and critical alterations of pericentromeric proteins without globally affecting protein abundance.
What is known already:
Oocyte postovulatory aging is associated with differential alteration in polyadenylation and reduction in abundance of mRNAs coded by selected maternal effect genes. RNA-binding and -processing proteins are involved in storage, polyadenylation and degradation of mRNAs thus regulating stage-specific recruitment of maternal mRNAs, while chromosomal proteins that are stage-specifically expressed at pericentromeres, contribute to control of chromosome segregation and regulation of gene expression in the zygote.
Study design, size, duration:
Germinal vesicle (GV) and metaphase II (MII) oocytes from sexually mature C57B1/6J female mice were investigated. Denuded in vivo or in vitro matured MII oocytes were postovulatory aged and analyzed by semiquantitative confocal microscopy for abundance and localization of polyadenylated RNAs, proteins of maternal effect genes (transcription activator BRG1 also known as ATP-dependent helicase SWI/SNF related, matrix associated, actin dependent regulator of chromatin, subfamily a, member 4 (SMARCA4) and NOD-like receptor family pyrin domain containing 5 (NLRP5) also known as MATER), RNA-binding proteins (MSY2 also known as germ cell-specific Y-box-binding protein, YBX2), and post-transcriptionally modified histones (trimethylated histone H3K9 and acetylated histone H4K12), as well as pericentromeric ATRX (alpha thalassemia/mental retardation syndrome X-linked, also termed ATP-dependent helicase ATRX or X-linked nuclear protein (XNP)). For proteome analysis five replicates of 30 mouse oocytes were analyzed by selected reaction monitoring (SRM).
Material and methods:
GV and MII oocytes were obtained from large antral follicles or ampullae of sexually mature mice, respectively. Denuded MII oocytes were aged for 24 h post ovulation. For analysis of distribution and abundance of polyadenylated RNAs fixed oocytes were in situ hybridized to Cy5 labeled oligo(dT)20 nucleotides. Absolute quantification of protein concentration per oocyte of selected proteins was done by SRM proteome analysis. Relative abundance of ATRX was assessed by confocal laser scanning microscopy (CLSM) of whole mount formaldehyde fixed oocytes or after removal of zona and spreading. MSY2 protein distribution and abundance was studied in MII oocytes prior to, during and after exposure to nocodazole, or after aging for 2 h in presence of H2O2 or for 24 h in presence of a glutathione donor, glutathione ethylester (GEE).
Main results and role of chance:
The significant reduction in abundance of proteins (P < 0.001) translated from maternal mRNAs was independent of polyadenylation status, while their protein localization was not significantly changed by aging. Most of other proteins quantified by SRM analysis did not significantly change in abundance upon aging except MSY2 and GTSF1. MSY2 was enriched in the subcortical RNP domain (SCRD) and in the spindle chromosome complex (SCC) in a distinct pattern, right and left to the chromosomes. There was a significant loss of MSY2 from the SCRD (P < 0.001) and the spindle after postovulatory aging. Microtubule de- and repolymerization caused reversible loss of MSY2 spindle-association whereas H2O2 stress did not significantly decrease MSY2 abundance. Aging in presence of GEE decreased significantly (P < 0.05) the aging-related overall and cytoplasmic loss of MSY2. Postovulatory aging increased significantly spindle abnormalities, unaligned chromosomes, and abundance of acetylated histone H4K12, and decreased pericentromeric trimethylated histone H3K9 (all P < 0.001). Spreading revealed a highly significant increase in pericentromeric ATRX (P < 0.001) upon ageing. Thus, the significantly reduced abundance of MSY2 protein, especially at the SCRD and the spindle may disturb the spatial control and timely recruitment, deadenylation and degradation of developmentally important RNAs. An autonomous program of degradation appears to exist which transiently and specifically induces the loss and displacement of transcripts and specific maternal proteins independent of fertilization in aging oocytes and thereby can critically affect chromosome segregation and gene expression in the embryo after fertilization.
Limitation, reasons for caution:
We used the mouse oocyte to study processes associated with postovulatory aging, which may not entirely reflect processes in aging human oocytes. However, increases in spindle abnormalities, unaligned chromosomes and H4K12 acetylated histones, as well as in mRNA abundance and polyadenylation have been observed also in aged human oocytes suggesting conserved processes in aging.
Wider implications of the findings:
Postovulatory aging precociously induces alterations in expression and epigenetic modifications of chromatin by ATRX and in histone pattern in MII oocytes that normally occur after fertilization, possibly contributing to disturbances in the oocyte-to-embryo transition (OET) and the zygotic gene activation (ZGA). These observations in mouse oocytes are also relevant to explain disturbances and reduced developmental potential of aged human oocytes and caution to prevent oocyte aging in vivo and in vitro.
Study funding/competing interests:
The study has been supported by the German Research Foundation (DFG) (EI 199/7-1 | GR 1138/12-1 | HO 949/21-1 and FOR 1041). There is no competing interest.
... Some of these maternal RNAs are required for oocyte maturation while others are stored in the form of mRNPs and are translated and/or degraded in an orchestrated manner during early phases of embryonic development. Hence, maternally deposited RNAs are under tight post-transcriptional regulation that includes regulated processing, localization and translation [59][60][61][62][63]. It is widely believed that the major biological function of localization and translational control of RNAs in oocytes and embryos is spatial and temporal regulation of the corresponding protein product. ...
For many decades, the major function of mRNA was thought to be to provide protein-coding information embedded in the genome. The advent of high-throughput sequencing has led to the discovery of pervasive transcription of eukaryotic genomes and opened the world of RNA-mediated gene regulation. Many regulatory RNAs have been found to be incapable of protein coding and are hence termed as non-coding RNAs (ncRNAs). However, studies in recent years have shown that several previously annotated non-coding RNAs have the potential to encode proteins, and conversely, some coding RNAs have regulatory functions independent of the protein they encode. Such bi-functional RNAs, with both protein coding and non-coding functions, which we term as 'cncRNAs', have emerged as new players in cellular systems. Here, we describe the functions of some cncRNAs identified from bacteria to humans. Because the functions of many RNAs across genomes remains unclear, we propose that RNAs be classified as coding, non-coding or both only after careful analysis of their functions.
... In addition to mRNAs which are translated shortly after synthesis, the oocyte maternal transcriptome contains stable dormant maternal mRNAs, which are recruited later during meiotic maturation and/or during early embryogenesis. Selective polyadenylation and decapping are the two major mechanisms regulating the recruitment and stability of dormant maternal mRNAs (Bettegowda and Smith 2007;Ma et al. 2013). RNA molecules are rarely dispersed in cells, as their function usually requires the formation of various ribonucleoprotein (RNP) complexes (Balagopal and Parker 2009). ...
A hallmark of oocyte development in mammals is the dependence on the translation and utilization of stored RNA and proteins rather than the de novo transcription of genes in order to sustain meiotic progression and early embryo development. In the absence of transcription, the completion of meiosis and early embryo development in mammals relies significantly on maternally synthesized RNAs. Post-transcriptional control of gene expression at the translational level has emerged as an important cellular function in normal development. Therefore, the regulation of gene expression in oocytes is controlled almost exclusively at the level of mRNA and protein stabilization and protein synthesis. This current review is focused on the recently emerged findings on RNA distribution related to the temporal and spatial translational control of the meiotic progression of the mammalian oocyte.
... However, the post-transcriptional regulation of oocyte and CC transcripts needs to be elucidated. This is particularly important also because the stability and translation of the maternal mRNAs, that are accumulated during oocyte maturation (Niakan et al., 2012) and that drive human preimplantation development, are controlled by post-transcriptional regulatory mechanisms (Bettegowda and Smith, 2007). ...
... The fertilized egg divides into a 2-, 4-, and 8-cell embryo before developing into the morula and then into the mature blastocyst. 1,2 In the mature blastocysts, the inner cell mass (ICM), which comprises embryonic stem cells, differentiates into various tissues in the fetus. The trophoblast is the outer layer of embryonic tissue (also called villi) that facilitates the adhesion and penetration of the blastocysts into the endometrium during implantation. ...
Trophoblast cell migration and invasion are crucial for the establishment of a successful pregnancy. Protein O-fucosyltransferases, such as poFUT1 and poFUT2, catalyze the O-fucosylation of proteins and have important roles in embryonic development. Leukemia inhibitory factor (LIF) is a critical cytokine in the regulation of embryonic development and implantation. However, the exact roles of poFUTs in embryo migration and invasion and the effects of LIF on the expression of poFUTs have not been studied in detail. In the current study, we showed that poFUT1 and LIF were highly expressed in human trophoblast cells and in the serum of women during the first trimester of a normal pregnancy. However, in patients with threatened abortion, poFUT1 and LIF levels were found to be reduced. There were no significant differences in the expression levels of poFUT2 between the two groups. The migration and invasion potential of trophoblasts in an explant culture and in an in vitro implantation model was decreased or increased upon altering poFUT1 expression levels by siRNA or cDNA transfection. Our results also revealed that LIF upregulated the expression of poFUT1. The upregulation of poFUT1 by LIF promoted trophoblast cell migration and invasion at the fetal-maternal interface by activating the PI3K/Akt signaling pathway. Taken together, these study findings suggest that poFUT1 may be used as a marker of embryo implantation.
... It has been shown previously that in vivo matured oocytes have lower abundance of GDF9 transcripts than their in vitro matured counterparts (Lonergan et al., 2003), despite their higher developmental competence (Humblot et al., 2005). As the transcription activity in oocytes during meiotic maturation is low (Bettegowda & Smith, 2007) and transcripts may be degraded during maturation (Thélie et al., 2007), the decrease of GDF9 transcripts in oocytes with high competence may be due to their translation, favoring further development, coupled to RNA degradation. ...
Summary The aim of this study was to evaluate the dose-response effect of insulin, plus follicle-simulating hormone (FSH) at a fixed concentration, in a serum-free defined culture medium (DCM) on the in vitro maturation of bovine cumulus-oocyte complexes (COCs). For oocyte nuclear maturation, the expression levels of GDF9, GLUT1, PRDX1 and HSP70.1 transcripts related to oocyte and embryo developmental competence were analysed. For in vitro maturation (IVM), cumulus-oocyte complexes from slaughterhouse ovaries were distributed into four groups based on insulin concentration added to serum-free DCM, which was composed of alpha minimum essential medium (α-MEM), as basal medium: (1) DCM control: 0 ng/ml; (2) DCM1: 1 ng/ml; (3) DCM10: 10 ng/ml; and (4) DCM100: 100 ng/ml. After IVM, the nuclear status of a sample of oocytes was analysed and the other oocytes were submitted for in vitro fertilization (IVF) and in vitro culture (IVC). Different concentrations of insulin did not affect significantly the nuclear maturation and cleavage rate (72 h post-insemination) across all groups. Blastocyst rate (192 h post-insemination) did not differ in DCM control (24.3%), DCM1 (27.0%) and DCM10 (26.3%) groups, but the DCM100 (36.1%) group showed a greater blastocyst rate (P < 0.05) than the DCM control. Insulin concentrations of 1, 10, or 100 ng/ml decreased the relative levels of GDF9 and HSP70-1 transcripts in oocytes at the end of IVM (P < 0.05). The transcripts levels of PRDX1 decreased (P < 0.05) only when 10 or 100 ng/ml insulin was added to the DCM medium. No difference in levels of GLUT1 transcripts (P > 0.05) was observed at the different insulin concentrations. The results indicated that insulin added to DCM influenced levels of transcripts related to cellular stress (HSP70-1 and PRDX1) and oocyte competence (GDF9) in bovine oocytes and at higher concentrations enhanced blastocyst production.
... Oocyte maturation and early embryo development until embryonic genome activation (EGA) largely rely on translation of maternally stored specific mRNAs with a short poly(A) tail (Oh et al. 2000;Bettegowda and Smith 2007). The RNA-binding proteins embryonic poly(A)-binding protein (EPAB; also known as ePABP and PABPc1-like) and poly(A)-binding protein, cytoplasmic 1 (PABPC1; also known as PABP1) are essential members of the poly(A)-binding protein (PABP) family, and they function primarily in translational regulation and stability of maternal mRNAs. ...
Embryonic poly(A)-binding protein (EPAB) and poly(A)-binding protein, cytoplasmic 1 (PABPC1) play critical roles in translational regulation of stored maternal mRNAs required for proper oocyte maturation and early embryo development in mammals. Superovulation is a commonly used technique to obtain a great number of oocytes in the same developmental stages in assisted reproductive technology (ART) and in clinical or experimental animal studies. Previous studies have convincingly indicated that superovulation alone can cause impaired oocyte maturation, delayed embryo development, decreased implantation rate and increased postimplantation loss. Although how superovulation results in these disturbances has not been clearly addressed yet, putative changes in genes related to oocyte and early embryo development seem to be potential risk factors. Thus, the aim of the present study was to determine the effect of superovulation on Epab and Pabpc1 gene expression. To this end, low- (5IU) and high-dose (10IU) pregnant mare's serum gonadotropin (PMSG) and human chorionic gonadotrophin (hCG) were administered to female mice to induce superovulation, with naturally cycling female mice serving as controls. Epab and Pabpc1 gene expression in germinal vesicle (GV) stage oocytes, MII oocytes and 1- and 2-cell embryos collected from each group were quantified using quantitative reverse transcription-polymerase chain reaction. Superovulation with low or high doses of gonadotropins significantly altered Epab and Pabpc1 mRNA levels in GV oocytes, MII oocytes and 1- and 2-cell embryos compared with their respective controls (P<0.05). These changes most likely lead to variations in expression of EPAB- and PABPC1-regulated genes, which may adversely influence the quality of oocytes and early embryos retrieved using superovulation.
Heat shock protein 90 (Hsp90) is critical for cell homeostasis but its role on bovine oocyte maturation is not well known. We investigated the importance of Hsp90 for competence of bovine oocyte using 17-(allylamino)-17-demethoxygeldanamycin (17AAG), an inhibitor of Hsp90, during in vitro maturation (IVM). Three experiments evaluated the effect of 17AAG on developmental competence of oocytes matured in vitro under thermoneutral (38.5ºC) or heat shock (HS; 41.5ºC) temperatures. The first experiment found that the blastocyst rates were lower ( P < 0.05) with 2 µM 17AAG compared with the untreated control (0 µM). The abundance of HSF1 transcripts was higher in oocytes matured with 2 µM than with 0 and 1 µM 17AAG, whereas the abundance of HSP90AA1 and HSPA1A transcripts was lower ( P < 0.05) with 1 and 2 µM than with 0 µM. The second experiment found that 2 µM 17AAG for 12 or 24 h during IVM decreased ( P < 0.05) the blastocysts rates. In the third experiment, the association of 2 μM 17AAG with HS for 12 h during IVM resulted in lower ( P < 0.05) blastocysts rates than 17AAG, HS or untreated control. In conclusion, inhibition of Hsp90 during in vitro maturation compromises further embryo development; the association of Hsp90 inhibition with HS aggravates the deleterious effect of both on oocyte developmental competence.
As one of the post-transcriptional regulatory mechanisms, transcription and translation’s uncoupling plays an essential role in development and adulthood physiology. However, it remains elusive how thousands of mRNAs get translationally silenced while stability is maintained for up to hours or even days before translation. In addition to oocytes and neurons, developing spermatids have significant uncoupling of transcription and translation for delayed translation. Therefore, spermiogenesis represents an excellent in vivo model for investigating the mechanism underlying uncoupled transcription and translation. Through full-length poly(A) deep sequencing, we discovered dynamic changes in poly(A) length through deadenylation and re-polyadenylation. Deadenylation appeared to be mediated by microRNAs (miRNAs), and transcripts with shorter poly(A) tails tend to be sequestered into ribonucleoproteins (RNPs) for translational repression and stabilization. In contrast, re-polyadenylation allows for translocation of the translationally repressed transcripts from RNPs to polysomes for translation. Overall, our data suggest that miRNA-dependent poly(A) length control represents a novel mechanism underlying uncoupled translation and transcription in haploid male germ cells.
During the maternal-to-zygotic transition (MZT), mRNAs and proteins stored in oocytes are degraded, and zygotic genes are activated. We have previously shown that the ubiquitin-proteasome system (UPS)-mediated degradation of maternal proteins plays a role in the onset of zygotic transcription. However, it is still unclear which maternal proteins should be degraded for zygotic genome activation and ensuring subsequent embryonic development. In this study, we screen for these maternal factors that are degraded via the UPS. We thus identified a maternal protein PIASy (Protein inhibitor of activated STAT y), which is an E3 SUMO ligase. The overexpression of PIASy in fertilized embryos causes developmental arrest at the 2-cell stage due to severe abnormal chromosome segregation and impaired zygotic transcription. We find that this developmental role of PIASy is related to its SUMOylation activity. Moreover, PIASy overexpression leads to increased trimethylation of histone H3 lysine 9 (H3K9me3) in 2-cell nuclei and enhanced translocation of H3K9me3 methyltransferase to the pronucleus. Hence, PIASy is a maternal factor that is degraded after fertilization and may be important for the proper induction of zygotic genome activation and embryonic development.
Wingless-int (WNT) signaling pathway is vital to modulate life processes, including cell fate determination, cell differentiation, cell proliferation, cell apoptosis and embryogenesis. To demonstrate the uncertain effect of the canonical WNT signaling pathway on oocyte maturation, immature porcine oocytes were collected and cultured in vitro with the WNT/β-catenin inhibitor FH535. The concentrations of FH535 were selected as 0.00, 0.01, 0.10, 1.00 and 10.00 μmol/L. The results showed that the optimum concentration of FH535 on oocyte maturation was 1.00 μmol/L. In this concentration, the proportion of MII oocytes increased (P < 0.05). The rate of cleavage was the same with the control (P > 0.05), while the rate of blastocysts in the 1.00 μmol/L FH535 treated group was higher than that of control (P < 0.01). Additionally, the average number of nuclei in blastocysts raised significantly (P < 0.05). The inhibition of WNT could regulate expression of maturation-related genes, including Cdc-2, Bmp-15, Gdf-9 and Mos. In the 1.00 μmol/L FH535 treated group, the messenger RNA level of β-catenin showed no significant change compared to the control (P > 0.05), but the protein abundance was decreased (P < 0.05). This study revealed that the inhibition of FH535 on the WNT signaling pathway could promote the maturation of porcine oocytes and altered gene expressions in vitro.
The genetic regulation of female fertility (follicular development, oocyte maturation, and early preimplantation embryo development) involves the spatio-temporal regulation of those genes that play key roles in various stages of the female reproductive axis. MicroRNAs (miRNAs), a class of small non-coding RNA, are known to regulate the expression of a large proportion of such genes. In recent decades, multiple studies have aimed to determine the roles of these non-coding RNAs in mammalian follicular development, oocyte growth, and embryo development. These studies have applied a variety of approaches, including conditional knockout (KO) of miRNA biogenesis genes, high-throughput sequencing technologies for pattern recognition in miRNA expression, and loss- and gain-of-function of miRNAs in various animal models. In addition to the cellular miRNAs, a large variety of RNAs are found in circulation, being coupled with extracellular vesicles, proteins, and lipids. Because of their potential as diagnostic markers for abnormal physiologies, there is increasing interest in the identification of extracellular miRNAs in various biological fluids and spent in vitro culture media. This review focuses on studies addressing the expression and potential role of cellular and extracellular miRNAs in mammalian follicular cell physiology and subsequent ovarian functionality and oocyte maturation.
Temperature is a crucial factor in mammalian spermatogenesis. The scrotum, pampiniform plexus, and cremaster and dartos muscles in mammals are specific adaptations to ensure sperm production in a regulated environment 4-6°C below internal body temperature. However, the limited endogenous antioxidant systems inherent in mammalian spermatozoa compounded by the loss of cytosolic repair mechanisms during spermatogenesis, make the DNA in these cells particularly vulnerable to oxidative damage. Boar sperm is likely to be more susceptible to the effects of heat stress and thus oxidative damage due to the relatively high unsaturated fatty acids in the plasma membrane, low antioxidant capacity in boar seminal plasma, and the boar's non-pendulous scrotum. Heat stress has a significant negative impact on reproductive performance in piggeries, which manifests as summer infertility and results in productivity losses that amount to millions of dollars. This problem is particularly prevalent in tropical and subtropical regions where ambient temperatures rise beyond the animal's zone of thermal comfort. Based on preliminary studies in the pig and other species, this article discusses whether heat stress could induce sufficient DNA damage in boar sperm to significantly contribute to the high rates of embryo loss and pregnancy failure observed in the sow during summer infertility. Heat stress-induced damage to sperm DNA can lead to disrupted expression of key developmental genes essential for the differentiation of early cell lineages, such as the trophectoderm, and can distort the timely formation of the blastocyst; resulting in a failure of implantation and ultimately pregnancy loss. Confirming such a link would prompt greater emphasis on boar management and strategies to mitigate summer infertility during periods of heat stress.
Zygotic genome activation (ZGA) is essential for normal development of mammalian preimplantation embryos. Estrogen receptor alpha (ERα) has been implicated in early embryogenesis, and controls the expression of genes associated with proliferation, differentiation and development of cell and target organs via a genomic effect. The objective of this study was to determine whether ERα plays a role in early embryo development and affects ZGA gene expression. Toward this objective, 1-cell embryos from B6C3F1 mouse were cultured with the antiestrogen ICI182780, ERα-specific antagonist MPP, ERα-specific antibody and ERβ-specific antagonist PHTPP. Development of 2-cell to 4-cell in vitro was significantly blocked by ICI182780, MPP and ERα-antibody treatment in a dose-dependent manner but not affected by PHTPP exposure. MPP decreased nuclear ERα protein levels and reduced mRNA expression levels of MuERV-L, one of the ZGA related genes. The results indicate that ERα has a functional role in early embryo development by regulation of ZGA-related genes.
To address the biological function of RNA interference (RNAi)-related pathways in mammals, we disrupted the gene Dicer1 in mice. Loss of Dicer1 lead to lethality early in development, with Dicer1-null embryos depleted of stem cells. Coupled with our inability to generate viable Dicer1-null embryonic stem (ES) cells, this suggests a role for Dicer, and, by implication, the RNAi machinery, in maintaining the stem cell population during early mouse development.
Genes whose expression is restricted to oogenesis and early development may have important functions in these processes. Northern analysis showed that Xenopus B4 mRNA is expressed in oogenesis and embryogenesis through to the neurula stage. Immunocytochemistry with anti-B4 antibodies showed that B4 protein is only detectable in preneurula stages; it is localized to nuclei and is associated with metaphase chromosomes. Immunoblotting revealed approximately constant levels of B4 protein per embryo for the first 2 days of development. Thus, as the number of nuclei increases during early development, the amount of B4 protein per nucleus is diluted out. Sequencing of two B4 cDNA clones revealed that the predicted B4 translation product is a 29-kD protein with 29% identity with histone H1, distributed over the entire length of its sequence. The B4 protein also has certain other H1 protein characteristics--a tripartite structure consisting of a mainly hydrophobic central domain flanked by an amino-terminal segment and a long hydrophilic carboxyterminal tail containing a tandemly repeated amino acid motif. However, in contrast to histone H1 mRNA, B4 mRNA has a classic polyadenylation signal, is polyadenylated, and lacks the histone H1 3' noncoding consensus sequence involved in RNA processing.
As the oocytes of Xenopus laevis grow and develop they accumulate vast stores of mRNA for use during early embryogenesis. The stored mRNA is stabilized and may be prevented from being translated in oocytes by the binding of a defined set of oocyte-specific proteins to form messenger RNP (mRNP) particles. A key event in the interaction of protein with mRNA is the phosphorylation of those few polypeptides that bind directly to all classes of polyadenylated mRNA. In this study we show that the phosphorylating enzyme (protein kinase), in addition to its target phosphoproteins, is an integral component of the mRNP particles. This association extends through various stages in the formation and use of the mRNP particles. Examination of material from oocytes of an early developmental stage (early stage 1), when the level of accumulated mRNA is low, reveals an excess of protein particles free of RNA, sedimenting at 6-18 S, and containing protein kinase activity and mRNA-binding phosphoproteins. At stages of maximum rate of mRNA accumulation (stages 1 and 2), the phosphoproteins and kinase are found primarily in individual mRNP particles that sediment at 40-80 S. As ribosomes become abundant (stages 2 and 3), the mRNP particles tend to interact with ribosomal subunits, at least in vitro, to form blocked translation initiation complexes that sediment at 80-110 S. These results are compared with observation on stored mRNP in other developmental systems.
The mammalian zona pellucida is an extracellular matrix that surrounds growing oocytes, ovulated eggs and early embryos. The mouse zona is composed of three sulfated glycoproteins: ZP1, ZP2 and ZP3. Each is critically involved in fertilization, the postfertilization block to polyspermy and protection of the preimplantation embryo. We have previously isolated cDNAs encoding mouse ZP2 and ZP3 and now report the isolation of a full-length cDNA encoding ZP1. Mouse ZP1 is composed of a 623 amino acid polypeptide chain with a signal peptide and a carboxyl terminal transmembrane domain, typical of all zona proteins. Sequence comparison demonstrate that mouse ZP1 is an orthologue of a rabbit zona protein, R55. The expression of R55 has been reported previously in both oocytes and granulosa cells. However, by northern analysis and in situ hybridization with 33P-labelled antisense probes to each of the three mouse zona mRNAs, we have determined that the expression of each mouse zona gene is restricted to the oocyte. ZP2 transcripts, but not ZP1 or ZP3, are detected in resting (15 microns diameter) oocytes, and all three zona transcripts coordinately accumulate as oocytes begin to grow. Together they represent approximately 1.5% of the total poly(A)+ RNA in 50-60 microns oocytes. In the latter stages of oogenesis, their abundance declines and each zona transcript is present in ovulated eggs at less than 5% of its maximal level. No zona transcripts were detected above background signal in granulosa cells. We conclude that, in mice, the three zona pellucida genes are expressed in a coordinate, oocyte-specific manner during the growth phase of oogenesis. Our data support the hypothesis that the transcription of the zona genes is controlled, in part, by shared regulatory element(s).
Sperm-induced activation of mammalian eggs is associated with a transient increase in the concentration of intracellular Ca2+. The role of inositol 1,4,5-trisphosphate (IP3)-mediated release of Ca2+ from intracellular stores during mouse egg activation was examined in the present study by determining the effects of microinjected monoclonal antibody (mAb) 18A10, which binds to the IP3 receptor and inhibits IP3-induced Ca2+ release, on endpoints of egg activation following insemination. The antibody inhibited in a concentration-dependent manner the ZP2 to ZP2f conversion that is involved in the zona pellucida block to polyspermy, as well as the ZP2 to ZP2f conversion promoted by microinjected IP3 in non-inseminated eggs. As anticipated, inseminated eggs that had been microinjected with the antibody were polyspermic. In addition, the antibody inhibited the fertilization-associated decrease in H1 kinase activity and pronucleus formation, and the concentration dependence for inhibition of these events was similar to that observed for inhibiting the ZP2 to ZP2f conversion. Last, the antibody inhibited the fertilization-induced recruitment of maternal mRNAs and post-translational modifications of proteins. In each case, eggs microinjected with the mAb 4C11, which also binds to the IP3 receptor but does not inhibit IP3-induced Ca2+ release, had no inhibitory effect on fertilization and egg activation. Results of these studies suggest that IP3-mediated Ca2+ release is essential for both early and late events of mouse egg activation.
The Y-box proteins are defined by their ability to bind to Y-box promoter elements and to help regulate transcription of a wide variety of genes. However, Y-box proteins are also identified as abundant proteins in the cytoplasm of germ cells, where they are found bound to stored mRNA molecules. Binding of Y-box proteins to mRNA sequences, both in vitro and in vivo, has been shown to effect their translational repression ("masking"). Here we discuss the ability of Y box proteins to recognize different nucleic acid structures and to become involved in regulation of both transcription and translation.
The transition from a late 1-cell mouse embryo to a 4-cell embryo, the period when zygotic gene expression begins, is accompanied by an increasing ability to repress the activities of promoters and replication origins. Since this repression can be relieved by either butyrate or enhancers, it appears to be mediated through chromatin structure. Here we identify changes in the synthesis and modification of chromatin bound histones that are consistent with this hypothesis. Oocytes, which can repress promoter activity, synthesized a full complement of histones, and histone synthesis up to the early 2-cell stage originated from mRNA inherited from the oocyte. However, while histones H3 and H4 continued to be synthesized in early 1-cell embryos, synthesis of histones H2A, H2B and H1 (proteins required for chromatin condensation) was delayed until the late 1-cell stage, reaching their maximum rate in early 2-cell embryos. Moreover, histone H4 in both 1-cell and 2-cell embryos was predominantly diacetylated (a modification that facilitates transcription). Deacetylation towards the unacetylated and monoacetylated H4 population in fibroblasts began at the late 2-cell to 4-cell stage. Arresting development at the beginning of S-phase in 1-cell embryos prevented both the appearance of chromatin-mediated repression of transcription in paternal pronuclei and synthesis of new histones. These changes correlated with the establishment of chromatin-mediated repression during formation of a 2-cell embryo, and the increase in repression from the 2-cell to 4-cell stage as linker histone H1 accumulates and core histones are deacetylated.
RBM and DAZ/SPGY are two families of genes located on the Y chromosome that encode proteins containing RNA-binding motifs, and both have been described as candidate human spermatogenesis genes. Transmission of deletions from father to son has been observed in the case of DAZ, but neither gene family has been shown to be essential for spermatogenesis in human males. The DAZ/SPGY genes are particularly amenable to a knockout approach, as they are found on the Y chromosome in Old World primates and apes, but in other mammals, they are represented only by an autosomal gene, DAZLA, which is also present in Old World primates and apes. It has also been shown that a Dazla homologue is essential for spermatogenesis in Drosophila. Here we show that Dazla protein is cytoplasmic in male and female germ cells, unlike the nuclear RBM protein. Disruption of the Dazla gene leads to loss of germ cells and complete absence of gamete production, demonstrating that Dazla is essential for the differentiation of germ cells.
Exonucleolytic degradation of the poly(A) tail is often the first step in the decay of eukaryotic mRNAs and is also used to silence certain maternal mRNAs translationally during oocyte maturation and early embryonic development. We previously described the purification of a poly(A)-specific 3'-exoribonuclease (deadenylating nuclease, DAN) from mammalian tissue. Here, the isolation and functional characterization of cDNA clones encoding human DAN is reported. Recombinant DAN overexpressed in Escherichia coli has properties similar to those of the authentic protein. The amino acid sequence of DAN shows homology to the RNase D family of 3'-exonucleases. DAN appears to be localized in both the nucleus and the cytoplasm. It is not stably associated with polysomes or ribosomal subunits. Xenopus oocytes contain nuclear and cytoplasmic DAN isoforms, both of which are closely related to the human DAN. Anti-DAN antibody microinjected into oocytes inhibits default deadenylation during progesterone-induced maturation. Ectopic expression of human DAN in enucleated oocytes rescues maturation-specific deadenylation, indicating that amphibian and mammalian DANs are functionally equivalent.
Regulation of translation initiation is a central control point in animal cells. We review our current understanding of the mechanisms of regulation, drawing particularly on examples in which the biological consequences of the regulation are clear. Specific mRNAs can be controlled via sequences in their 5' and 3' untranslated regions (UTRs) and by alterations in the translation machinery. The 5'UTR sequence can determine which initiation pathway is used to bring the ribosome to the initiation codon, how efficiently initiation occurs, and which initiation site is selected. 5'UTR-mediated control can also be accomplished via sequence-specific mRNA-binding proteins. Sequences in the 3' untranslated region and the poly(A) tail can have dramatic effects on initiation frequency, with particularly profound effects in oogenesis and early development. The mechanism by which 3'UTRs and poly(A) regulate initiation may involve contacts between proteins bound to these regions and the basal translation apparatus. mRNA localization signals in the 3'UTR can also dramatically influence translational activation and repression. Modulations of the initiation machinery, including phosphorylation of initiation factors and their regulated association with other proteins, can regulate both specific mRNAs and overall translation rates and thereby affect cell growth and phenotype.
Stage VI Xenopus oocytes are suspended at the G2/M transition of meiosis I, and represent an excellent system for the identification and examination of cell cycle regulatory proteins. Essential cell cycle regulators such as MAPK, cyclins and mos have the ability to induce oocyte maturation, causing the resumption of the cell cycle from its arrested state. We have identified the product of a novel Xenopus gene, Speedy or Spy1, which is able to induce rapid maturation of Xenopus oocytes, resulting in the induction of germinal vesicle breakdown (GVBD) and activation of M-phasepromoting factor (MPF). Spy1 activates the MAPK pathway in oocytes, and its ability to induce maturation is dependent upon this pathway. Spy1-induced maturation occurs much more rapidly than maturation induced by other cell cycle regulators including progesterone, mos or Ras, and does not require any of these proteins or hormones, indicating that Spy1-induced maturation proceeds through a novel regulatory pathway. In addition, we have shown that Spy1 physically interacts with cdk2, and prematurely activates cdk2 kinase activity. Spy1 therefore represents a novel cell cycle regulatory protein, inducing maturation through the activation of MAPK and MPF, and also leading to the premature activation of cdk2.
The activation of maturation-promoting factor (MPF) is required for G(2)/M progression in eukaryotic cells. Xenopus oocytes are arrested in G(2) and are induced to enter M phase of meiosis by progesterone stimulation. This process is known as meiotic maturation and requires the translation of specific maternal mRNAs stored in the oocytes. We have used an expression cloning strategy to functionally identify proteins involved in G(2)/M progression in Xenopus oocytes. Here we report the cloning of two novel cDNAs that when expressed in oocytes induce meiotic maturation efficiently. The two cDNAs encode proteins of 33 kD that are 88% identical and have no significant homologies to other sequences in databases. These proteins, which we refer to as p33(ringo) (rapid inducer of G(2)/M progression in oocytes), induce very rapid MPF activation in cycloheximide-treated oocytes. Conversely, ablation of endogenous p33(ringo) mRNAs using antisense oligonucleotides inhibits progesterone-induced maturation, suggesting that synthesis of p33(ringo) is required for this process. We also show that p33(ringo) binds to and activates the kinase activity of p34(cdc2) but does not associate with p34(cdc2)/cyclin B complexes. Our results identify a novel p34(cdc2) binding and activating protein that regulates the G(2)/M transition during oocyte maturation.
In the mouse, completion of oocyte maturation and the initiation of preimplantation development occur during transcriptional silence and depend on the presence and translation of stored mRNAs transcribed in the growing oocyte. The Spin gene has three transcripts, each with an identical open reading frame and a different 3' untranslated region (UTR). (Beta)-galactosidase-tagged reporter transcripts containing each of the different Spin 3'UTRs were injected into oocytes and zygotes and (beta)-galactosidase activity was monitored. Results from these experiments suggest that differential polyadenylation and translation occurs at two critical points in the oocyte-to-embryo transition - upon oocyte maturation and fertilization - and is dependent on sequences in the 3'UTR. The stability and mobility shifts of ten other maternal transcripts were monitored by reprobing a northern blot of oocytes and embryos collected at 12 hour intervals after fertilization. Some are more stable than others and the upward mobility shift associated with polyadenylation correlates with the presence of cytoplasmic polyadenylation elements (CPEs) within about 120 nucleotides of the nuclear polyadenylation signal. A survey of the 3' UTRs of expressed sequence tag clusters from a mouse 2-cell stage cDNA library indicates that about one third contain CPEs. We suggest that differential transcript stability and a translational control program can supply the diversity of protein products necessary for oocyte maturation and the initiation of development.
Translational control plays a central role during oocyte maturation and early embryogenesis, as these processes occur in the absence of transcription. MSY2, a member of a multifunctional Y-box protein family, is implicated in repressing the translation of paternal mRNAs. Here, we characterize MSY2 expression in mouse oocytes and preimplantation embryos. Northern blot analysis indicates that MSY2 expression is highly restricted and essentially confined to the oocyte in the female mouse. MSY2 transcript and protein, as assessed by reverse transcription-polymerase chain reaction and immunoblotting, respectively, are expressed in growing oocytes, metaphase II-arrested eggs, and 1-cell embryos, but then are degraded by the late 2-cell stage; no expression is detectable in the blastocysts. During oocyte maturation, MSY2 is phosphorylated and following fertilization it is dephosphorylated. Quantification of the mass amount of MSY2 reveals that it represents 2% of the total protein in the fully grown oocyte, i.e., it is a very abundant protein. Both endogenous MSY2 and MSY2-enhanced green fluorescent protein (EGFP), which is synthesized following microinjection of an mRNA encoding MSY2-EGFP, are primarily localized in the cytoplasm, and about 75% of the MSY2 remains associated with oocyte cytoskeletal preparations. Results of these studies are consistent with the proposal that MSY2 functions by stabilizing and/or repressing the translation of maternal mRNAs.
Gametogenesis is directed by various specialized genetic mechanisms which, to a considerable extent, apply to the production of both eggs and sperm and have been conserved across a wide spectrum of eukaryotic organisms. Two key aspects which are discussed here are: germ-cell-specific gene transcription; and translational repression (masking) of mRNA accumulated in oocytes and spermatocytes/spermatids. Together, these two processes conspire to deliver often large amounts of essential proteins at the appropriate stages of development. It is perhaps not surprising that recent evidence points to a functional link between transcription activation and translation repression, both processes being determined in the nucleus and involving common components. One set of components which has been studied recently are members of the Y-box family of regulatory proteins. Most information of the involvement of Y-box proteins in germ cell development comes from studies on amphibian oocytes and mammalian spermatids. In these cells, Y-box proteins have been detected as major components of both maternal and paternal mRNP particles and have been shown to be instrumental in the masking process. Y-box proteins are also implicated in the regulation of several germ-cell-specific genes. Possible connections between these processes are discussed.
With sperm-egg fusion at the time of fertilization the gamete nuclei are remodeled from genetically quiescent structures into pronuclei capable of DNA synthesis. Features of this process that are critical to insure the genetic integrity of the zygote and the success of subsequent embryonic development include: oocyte responses that prevent polyspermy; completion of the 2nd meiotic division by the oocyte; exchange of proteins in the sperm nucleus; and, remodelling of the oocyte chromosomes and sperm nucleus into functional pronuclei. Elucidation of the biological and molecular mechanisms underlying zygote formation and chromatin remodeling should enhance our understanding of the potential vulnerability of the zygote to toxicant-induced damage.
It has been shown previously that maternal mRNA, synthesized and stored in growing oocytes, is stabilized and blocked from translation through various mechanisms including restricted polyadenylation and the binding of proteins to 3' regulatory elements. In addition to binding sequence-specific proteins, the bulk of stored mRNA is packaged with a set of 'masking' proteins, the most abundant of which are the phosphoproteins pp56 and pp60. In this report these proteins are shown to be bound to heterogeneous mRNA sequences and not to the 3' poly(A) tract. Crosslinking studies demonstrate that all of the pp56/60 present makes direct contact with the RNA. In vitro binding studies confirm that pp56/60 interact with single-stranded RNA of heterogeneous sequence, such as occurring in the maternal mRNA encoding cyclin B1. However, binding is equally effective to capped and polyadenylated cyclin mRNA, to truncated mRNA lacking 5' and 3' non-coding regions and even to the antisense sequence. Lengths of 70-80 nucleotides are protected from ribonuclease digestion after protein binding. Although no extended binding motif could be detected, binding does appear to have some specificity in that it is not competed out by 100-fold excess of double-stranded RNA, transfer RNA, poly(A) and various other homopolymers and heteropolymers. The sequence which competes most efficiently is the mixed polypyrimidine, poly(C,U). Crosslinking of RNA-protein complexes, followed by ribonuclease digestion, suggests that the arrangement of proteins on RNA is as dimers. Dimerization appears to be stabilized by phosphorylation of pp56/60. These results are discussed in terms of the known structures of pp56/60.
Tissue-type plasminogen activator (tPA) mRNA is stored, stable and untranslated, in the cytoplasm of fully grown primary mouse oocytes. Dormancy is associated with an unusually short poly(A) tail, and poly(A) tail elongation controls tPA mRNA translational activation during meiotic maturation. Here we show that the nuclear transcript of this mRNA is extensively polyadenylated and that primary oocytes contain a deadenylating activity capable of silencing the cytoplasmic message. The sequence determinants that control deadenylation and polyadenylation overlap; this AU-rich region thus serves as an adenylation control element (ACE). The translation of a reporter mRNA in primary oocytes is prevented upon inclusion of an ACE in its 3' untranslated region. Therefore, the stage-specific regulation of poly(A) tail length accounts for the regulated synthesis of tPA in oocytes, and reversible deadenylation provides a mechanism for the translational control of dormant mRNAs.
Unfertilized eggs of many species contain large amounts of maternal mRNA that are used to support protein synthesis during the first few hours of development, before the onset of embryonic transcription. We have examined the accumulation of nonpolysomal maternal RNAs in polysomes after fertilization in Xenopus laevis by measuring the distributions of specific sequences in nonpolysomal and polysomal fractions. In an arbitrary selection of 18 maternal sequences that are largely nonpolysomal in the full-grown oocyte, 13 became enriched in polysomes by the 16-cell cleavage stage. One sequence accumulated only 50% in polysomes at this time, while four sequences became polysomal later than the 16-cell stage. Several RNA sequences decreased in titer during early embryogenesis and were rare during organogenesis. Sequences that are mobilized rapidly and efficiently into polysomes shortly after fertilization and whose cellular concentrations are highest in embryos before organogenesis may provide genetic information for developmental functions restricted to very early embryogenesis. These experiments serve to identify such sequences in Xenopus.
The total RNA content of mouse oocytes, as measured by ethidium bromide fluorescence, was found to decrease by 19% during meiotic maturation (ovulated eggs contain 19% less RNA than full-grown oocytes). Consistent with these results, prelabeled stable RNA of full-grown oocytes decreased by about 20% during in vitro maturation. Polyadenylated RNA represented 19% of total prelabeled RNA in full-grown oocytes and 10% in oocytes matured in vitro, confirming previous results on in vivo prepared material. To distinguish between deadenylation and degradation for one mRNA, the amount and state of adenylation of actin mRNA was examined using Northern blots of oocyte RNA probed with a nick-translated beta-actin cloned chicken cDNA. The results showed that the amount of actin mRNA remained similar during maturation, but its molecular weight decreased slightly. Experiments in which RNA was treated with oligo(dT) and RNase H demonstrated that the actin mRNA was deadenylated during maturation, when actin synthesis is known to decline. These results indicate that the previously defined loss of bulk RNA and changes in the state of adenylation of mRNA during the first 11/2 days of embryogenesis actually begin during the 12 hr of meiotic maturation preceding fertilization.
Previous work has shown that more than 50% or about 50 pg of polyadenylated RNA found in the full-grown mouse oocyte is deadenylated or degraded during meiotic maturation. Here we show that rRNA declines by 60 pg during this period, accounting for most of the 80-pg decline in total RNA and indicating that a significant amount of mRNA is deadenylated but not degraded during maturation. Actin mRNA is deadenylated at about 7 hr of in vitro maturation, following the decline in its translation. The poly(A) tail on hypoxanthine phosphoribosyltransferase (HPRT) mRNA is elongated at 7 hr of maturation, preceding an increase in HPRT activity. Actin mRNA is partially degraded in the one-cell embryo and falls to near the limit of detection in the late two-cell stage, while HPRT mRNA shows no change in early two-cell embryos, but is deadenylated and declines greatly during the two-cell stage. In aging unfertilized eggs, most of these changes occur on a delayed schedule. The various species of alpha-tubulin mRNA are largely deadenylated and more than half are degraded during maturation. Taken together with other published results, we conclude that each mRNA has its own pattern of changes in the length of the poly(A) tail (correlated with translation) and degradation during the period of maternal control of protein synthesis, and, for those examined, the maternal mRNAs remaining in the early two-cell embryo are degraded to low levels by the late two-cell stage.
The c-mos proto-oncogene product, pp39mos, is present in unfertilized Xenopus eggs, and disappears on fertilization. Microinjection of synthetic mos RNA into two-cell embryos induces cleavage arrest at metaphase. By contrast, egg cytosol extracts, when immunodepleted of endogenous pp39mos, lose their cleavage-arresting activity in injected embryos. These results demonstrate that Mos protein is the cytostatic factor CSF, long known as an endogenous meiotic inhibitor in vertebrate eggs.
The stored mRNP particles of Xenopus oocytes contain protein kinase activity and two major phosphoproteins of 60 kDa (pp60) and 56 kDa (pp56). These proteins
can be phospholabelled in the particles either in vivo or in vitro and then isolated by SDS-PAGE. On renaturing pp60 in the presence of globin mRNA, a stable RNA-protein complex is formed.
The complex has a uniform density in Cs salt gradients, corresponding to the binding of about 10 protein molecules to each
mRNA, probably at the poly(A) sequence. Compared with uncomplexed mRNA, the RNP complex is translated poorly both in vitro and in vivo. Translation of the complex can be regained after treatment with protein phosphatase. It is shown that dephosphorylation
destabilizes the binding of protein to RNA, making the mRNA accessible for translation. Studies with native mRNP particles
show that their translation also can be enhanced by dephosphorylation.
Much mRNA in Xenopus oocytes is translationally dormant for months until meiotic maturation and fertilization. This masking has been found to be coupled to transcription and mediated by the Y-box protein FRGY2.
We previously showed that, during mouse oocyte maturation, specific maternal mRNAs (actins) are deadenylated, while others (hypoxanthine phosphoribosyltransferase:HPRT) are adenylated. As in other systems, these changes can be correlated with changes in translational activities. Maturation-specific polyadenylation in Xenopus depends on the presence of a U-rich cytoplasmic polyadenylation element (CPE) close to the 3' end of the RNA. RNAs that lack CPEs appear to be deadenylated by default when meiosis resumes. We show here that this default program also applies to maturing mouse oocytes. Microinjected beta- and gamma-actin 3' UTR (untranslated region) transcripts lacking CPEs but including polyA tails (100-200 N) behave as endogenous maternal actin mRNAs and are deadenylated by maturing oocytes. "Nonsense" transcripts that do not include CPEs, but that do contain polyA tails, are also deadenylated. beta- and gamma-Actin 3' UTRs with short polyA tails (50-80 N) are stable and exhibit no detectable change in adenylation when injected into growing, full-grown, or maturing oocytes. In contrast, HPRT 3' UTRs, which include the CPE UUUUAAAU and a short polyA tail (50 N), are polyadenylated during maturation. HPRT 3' UTR transcripts with long polyA tails (100-200 N) are more extensively deadenylated by growing and full-grown oocytes that retain germinal vesicles than by maturing oocytes. The presence of CPEs may be required for polyA tail shortening and translational inactivation of stable mRNAs during oocyte growth and subsequent selective readenylation and translation during meiotic maturation.(ABSTRACT TRUNCATED AT 250 WORDS)
Messenger RNA can be stored in the cytoplasm of higher Eukaryotes in the form of masked messenger ribonucleoprotein particles (masked mRNPs, or informosomes). The typical example is the storage of mRNPs in germ cells (oocytes and spermatocytes). The masked mRNPs are inactive in translation, stable, i.e., protected against degradation, and unavailable for poly(A) tail processing, such as cytoplasmic polyadenylation and deadenylation. The major nonspecific mRNA-binding protein forming mRNPs and belonging to a special p50 family of basic, glycine-rich, phosphorylatable proteins seems to be necessary, but not sufficient for the masking. In some cases, mRNA-specific repressor proteins bound to the 5'-untranslated regions (5'-UTR) of mRNAs may be involved. Interactions of the 3'-untranslated regions (3'-UTR) with sequence-specific proteins seem to be of decisive importance for the masking of mRNPs. The hypothesis is proposed that the masking is achieved through a 3'-UTR-induced conformational rearrangement of mRNP; closing into a circle and condensation of mRNP are considered plausible.
The c-mos proto-oncogene product is a key element in the cascade of events leading to meiotic maturation of vertebrate oocytes. We have investigated the role of cytoplasmic polyadenylation in the translational control of mouse c-mos mRNA and its contribution to meiosis. Using an RNase protection assay we show that optimal cytoplasmic polyadenylation of c-mos mRNA requires three cis elements in the 3' UTR: the polyadenylation hexanucleotide AAUAAA and two U-rich cytoplasmic polyadenylation elements (CPEs) located 4 and 51 nucleotides upstream of the hexanucleotide. When fused to CAT coding sequences, the wild-type 3' UTR of c-mos mRNA, but not a 3' UTR containing mutations in both CPEs, confers translational recruitment during maturation. This recruitment coincides with maximum polyadenylation. To assess whether c-mos mRNA polyadenylation is necessary for maturation of mouse oocytes, we have ablated endogenous c-mos mRNA by injecting an antisense oligonucleotide, which results in a failure to progress to meiosis II after emission of the first polar body. Such antisense oligonucleotide-injected oocytes could be efficiently rescued by co-injection of a c-mos mRNA carrying a wild-type 3' UTR. However, co-injection of a c-mos mRNA lacking functional CPEs substantially lowered the rescue activity. These results demonstrate that translational control of c-mos mRNA by cytoplasmic polyadenylation is necessary for normal development.
The Y-box proteins are the most evolutionarily conserved nucleic acid binding proteins yet defined in bacteria, plants and animals. The central nucleic acid binding domain of the vertebrate proteins is 43% identical to a 70-amino-acid-long protein (CS7.4) from E. coli. The structure of this domain consists of an antiparallel five-stranded beta-barrel that recognizes both DNA and RNA. The diverse biological roles of these Y-box proteins range from the control of the E. coli cold-shock stress response to the translational masking of messenger RNA in vertebrate gametes. This review discusses the organization of the prokaryotic and eukaryotic Y-box proteins, how they interact with nucleic acids, and their biological roles, both proven and potential.
The poly(A)-dependent translational regulation of maternal mRNAs is an important mechanism to execute stage-specific programs of protein synthesis during early development. This control underlies many crucial developmental events including the meiotic maturation of oocytes and activation of the mitotic cell cycle at fertilization. A recent report demonstrates that the 3' untranslated region of the cyclin A1, B1, B2 and c-mos mRNAs determines the timing and extent of their cytoplasmic polyadenylation and translational activation during Xenopus oocyte maturation. These studies further establish that protein synthesis can be temporally and quantitatively controlled by developmentally regulated changes in the polyadenylation of maternal mRNAs.
Zygotic gene activation (ZGA) is the critical event that governs the transition from maternal to embryonic control of development. In the mouse, ZGA occurs during the 2-cell stage and appears to be regulated by the time following fertilization, i.e. a zygotic clock, rather than by progression through the first cell cycle. The onset of ZGA must depend on maternally inherited proteins, and post-translational modification of these maternally derived proteins is likely to play a role in ZGA. Consistent with this prediction is that protein phosphorylation catalyzed by the cAMP-dependent protein kinase is involved in ZGA and that protein synthesis is not required for ZGA. Recent results suggest that ZGA may occur earlier than previously thought, i.e. not during the 2-cell stage, but rather in G2 of the 1-cell embryo. Thus ZGA may comprise a period of minor gene activation in the 1-cell embryo that is followed by a period of major gene activation in the 2-cell embryo. Following ZGA, the expression of constitutively activated genes may require an enhancer.
Replication-dependent histone mRNAs are not polyadenylated but end in a conserved 26-nucleotide structure that contains a stem-loop. Much of the cell cycle regulation of histone mRNA is post-transcriptional and is mediated by the 3' end of histone mRNA. The stem-loop binding protein (SLBP) that binds the 3' end of histone mRNA is a candidate for the factor that participates in most, if not all, of the post-transcriptional regulatory events. We have cloned the cDNA for the SLBP from humans, mice, and frogs, using the recently developed yeast three-hybrid system. The human SLBP is a 31-kD protein and contains a novel RNA-binding domain, which has been mapped to a 73-amino-acid region of the protein. The cloned SLBP is the protein bound to the 3' end of histone mRNA as antibodies specific for the SLBP remove all specific binding activity from nuclear and polyribosomal extracts. These depleted extracts do not cleave histone pre-mRNA efficiently, demonstrating that the SLBP is required for efficient histone pre-mRNA processing.
Cytoplasmic polyadenylylation is an essential process that controls the translation of maternal mRNAs during early development and depends on two cis elements in the 3' untranslated region: the polyadenylylation hexanucleotide AAUAAA and a U-rich cytoplasmic polyadenylylation element (CPE). In searching for factors that could mediate cytoplasmic polyadenylylation of mouse c-mos mRNA, which encodes a serine/threonine kinase necessary for oocyte maturation, we have isolated the mouse homolog of CPEB, a protein that binds to the CPEs of a number of mRNAs in Xenopus oocytes and is required for their polyadenylylation. Mouse CPEB (mCPEB) is a 62-kDa protein that binds to the CPEs of c-mos mRNA. mCPEB mRNA is present in the ovary, testis, and kidney; within the ovary, this RNA is restricted to oocytes. mCPEB shows 80% overall identity with its Xenopus counterpart, with a higher homology in the carboxyl-terminal portion, which contains two RNA recognition motifs and a cysteine/histidine repeat. Proteins from arthropods and nematodes are also similar to this region, suggesting an ancient and widely used mechanism to control polyadenylylation and translation.
We examined the distribution of the somatic subtypes of histone H1 and the variant subtype, H1(0), and their encoding mRNAs during oogenesis and early embryogenesis in the mouse. As detected using immunocytochemistry, somatic H1 was present in the nuclei of oocytes of 18-day embryos. Following birth, however, somatic H1 became less abundant in both growing and non-growing oocytes, beginning as early as 4 days of age in the growing oocytes, and was scarcely detectable by 19 days. Together with previous results, this defines a period of time when somatic H1 is depleted in oocytes, namely, from shortly after birth when the oocytes are at prophase I until the 4-cell stage following fertilization. At the stages when somatic H1 was undetectable, oocyte nuclei could be stained using an antibody raised against histone H1(0), which suggests that this may be a major linker histone in these cells. In contrast to the post-natal loss of somatic H1 protein, mRNAs encoding four (H1a, H1b, H1d, H1e) of the five somatic subtypes were present, as detected using RT-PCR in growing oocytes of 9-day pups, and all five subtypes including H1c were present in fully grown oocytes of adults. All five subtypes were also present in embryos, both before and after activation of the embryonic genome. mRNA encoding H1(0) was also detected in oocytes and early embryos. Whole-mount in situ hybridization using cloned H1c and H1e cDNAs revealed that the mRNAs were present in the cytoplasm of oocytes and 1-cell embryos, in contrast to the sea urchin early embryo where they are sequestered in the cell nucleus. We suggest that, as in many somatic cell types, the chromatin of mouse oocytes becomes depleted of somatic H1 and relatively enriched in histone H1(0) postnatally, and that somatic H1 is reassembled onto chromatin in cleavage-stage embryos. The post-natal loss of somatic H1 appears to be regulated post-transcriptionally by a mechanism not involving nuclear localization.
Embryonic genome activation (EGA) occurs by the 2-cell stage in mouse embryos. To understand the molecular basis of EGA, it is important to determine whether EGA can be supported by maternally inherited factors or if it requires the synthesis of additional transcription factors. We used a quantitative reverse transcription-polymerase chain reaction (RT-PCR) method to test whether protein synthesis is required for the transcriptional activation of six housekeeping genes (U2afbp-rs, Hprt, Pdha1, Prps1, Odc, and Cox7c). Cycloheximide treatment reduced the expression of these mRNAs in 2-cell embryos to the same degree as alpha-amanitin treatment. Cycloheximide treatment did not reduce the expression of maternally inherited mRNAs, indicating that its effect is specific for transcription-dependent gene expression. These results contrast with earlier results reported for the Hsp70 gene. This difference may reflect differences in promoter requirements. We conclude that protein synthesis is required for the activation of most, if not all, housekeeping genes in the mouse embryo, and that the time of EGA may be controlled, in part, by the regulated recruitment of maternal mRNAs encoding key transcription factors.
Here we report the isolation and characterization of mouse testicular cDNAs encoding the mammalian homologue of the Xenopus germ cell-specific nucleic acid-binding protein FRGY2 (mRNP3+4), hereafter designated MSY2. MSY2 is a member of the Y box multigene family of proteins; it contains the cold shock domain that is highly conserved among all Y box proteins and four basic/aromatic islands that are closely related to the other known germline Y box proteins from Xenopus, FRGY2, and goldfish, GFYP2. Msy2 undergoes alternative splicing to yield alternate N-terminal regions upstream of the cold shock domain. Although MSY2 is a member of a large family of nucleic acid-binding proteins, Southern blotting detects only a limited number of genomic DNA fragments, suggesting that Msy2 is a single copy gene. By Northern blotting and immunoblotting, MSY2 appears to be a germ cell-specific protein in the testis. Analysis of Msy2 mRNA expression in prepubertal and adult mouse testes, and in isolated populations of germ cells, reveals maximal expression in postmeiotic round spermatids, a cell type with abundant amounts of stored messenger ribonucleoproteins. In the ovary, MSY2 is present exclusively in diplotene-stage and mature oocytes. MSY2 is maternally inherited in the one-cell-stage embryo but is not detected in the late two-cell-stage embryo. This loss of MSY2 is coincident with the bulk degradation of maternal mRNAs in the two-cell embryo.
Translationally inactive histone mRNA is stored in frog oocytes, and translation is activated at oocyte maturation. The replication-dependent
histone mRNAs are not polyadenylated and end in a conserved stem-loop structure. There are two proteins (SLBPs) which bind
the 3′ end of histone mRNA in frog oocytes. SLBP1 participates in pre-mRNA processing in the nucleus. SLBP2 is oocyte specific,
is present in the cytoplasm, and does not support pre-mRNA processing in vivo or in vitro. The stored histone mRNA is bound
to SLBP2. As oocytes mature, SLBP2 is degraded and a larger fraction of the histone mRNA is bound to SLBP1. The mechanism
of activation of translation of histone mRNAs may involve exchange of SLBPs associated with the 3′ end of histone mRNA.
Growth and differentiation of early embryos depends almost entirely on information which is maternally inherited in the form of macromolecules accumulated by the female gamete during its growth phase. Most of the maternal mRNAs synthesized by growing oocytes are not immediately recruited onto polysomes but are stored as translationally dormant messenger ribonucleoprotein (mRNP) particles. mRNA binding proteins which have been associated with masked mRNP complexes in Xenopus oocytes fall into two main categories, those having affinity for a variety of RNA sequences (members of the Y box and DEAD box RNA helicase families) and those which interact more specifically with 3' polyA tails (the polyA binding proteins or PABPs). The objective of this study was to determine whether mouse oocytes and embryos express sequences encoding a Y box protein, (MSY1); on RNA helicase, (RCK/p54); and a universally expressed PABP and testis specific isoform (PABP1 and PABPt, respectively). RNAs were amplified by RT/PCR and the identities of targeted cDNAs were confirmed by restriction analysis and/or direct sequencing. Relative steady state levels and time courses of accumulation/decay were compared by Northern hybridization. All of the sequences are transcribed as maternal mRNAs. MSY1 transcripts accumulated during the growth phase appear to be degraded in parallel with the bulk of maternal mRNAs by the mid-late two-cell stage. RCK/p54 mRNAs are most abundant in growing oocytes; steady state levels decline in primary and secondary oocytes, and degradation appears to be complete by the mid-late two-cell stage. Zygotic transcription of MSY1 and RCK/p54 is evident in four-cell stage embryos. Most of the PABP1 message accumulated by growing oocytes decays during meiotic maturation with transcription resuming in two-cell embryos. PABPt is expressed at very low levels in oocytes and embryos. Based on the temporal patterns of expression and the reported activities of homologous sequences in other systems, we suggest that these RNA binding proteins may participate in the post-transcriptional regulation of gene expression during the period of maternal control of development in the mouse.
Maternal mRNA translation is regulated in large part by cytoplasmic polyadenylation. This process, which occurs in both vertebrates and invertebrates, is essential for meiosis and body patterning. In spite of the evolutionary conservation of cytoplasmic polyadenylation, many of the cis elements and trans-acting factors appear to have some species specificity. With the recent isolation and cloning of factors involved in both poly(A) elongation and deadenylation, the underlying biochemistry of these reactions is beginning to be elucidated. In addition to early development, cytoplasmic polyadenylation is now known to occur in the adult brain, and there is circumstantial evidence that this process occurs at synapses, where it could mediate the long-lasting phase of long-term potentiation, which is probably the basis of learning and memory. Finally, there may be multiple mechanisms by which polyadenylation promotes translation. Important questions yet to be answered in the field of cytoplasmic polyadenylation are addressed.
Formation of mRNA 3' ends in eukaryotes requires the interaction of transacting factors with cis-acting signal elements on the RNA precursor by two distinct mechanisms, one for the cleavage of most replication-dependent histone transcripts and the other for cleavage and polyadenylation of the majority of eukaryotic mRNAs. Most of the basic factors have now been identified, as well as some of the key protein-protein and RNA-protein interactions. This processing can be regulated by changing the levels or activity of basic factors or by using activators and repressors, many of which are components of the splicing machinery. These regulatory mechanisms act during differentiation, progression through the cell cycle, or viral infections. Recent findings suggest that the association of cleavage/polyadenylation factors with the transcriptional complex via the carboxyl-terminal domain of the RNA polymerase II (Pol II) large subunit is the means by which the cell restricts polyadenylation to Pol II transcripts. The processing of 3' ends is also important for transcription termination downstream of cleavage sites and for assembly of an export-competent mRNA. The progress of the last few years points to a remarkable coordination and cooperativity in the steps leading to the appearance of translatable mRNA in the cytoplasm.
In Xenopus, the CPE is a bifunctional 3' UTR sequence that maintains maternal mRNA in a dormant state in oocytes and activates polyadenylation-induced translation during oocyte maturation. Here, we report that CPEB, which binds the CPE and stimulates polyadenylation, interacts with a new factor we term maskin. Maskin contains a peptide sequence that is conserved among elF-4E-binding proteins. Affinity chromatography demonstrates that CPEB, maskin, and elF-4E reside in a complex in oocytes, and yeast two-hybrid analyses indicate that CPEB and maskin bind directly, as do maskin and elF-4E. While CPEB and maskin remain together during oocyte maturation, the maskin-elF-4E interaction is substantially reduced. The dissolution of this complex may result in the binding of elF-4E to elF-4G and the translational activation of CPE-containing mRNAs.
Embryonic genome activation (EGA) in mice is sensitive to treatment with cycloheximide, indicating that protein synthesis plays an important role in mediating EGA. We hypothesized that regulated maternal mRNA recruitment may control the time of EGA by controlling the time of appearance of certain transcription factors (TFs). We also hypothesized that synthesis of other TFs may contribute to EGA independently of controlling the timing of EGA. To test these hypotheses, we used sucrose density gradient fractionation coupled to a quantitative reverse transcription-polymerase chain reaction method to compare polysomal mRNA abundances of specific TF mRNAs between metaphase II oocytes, 1-cell-stage embryos, and 2-cell-stage embryos. We observed a 2-cell-stage-specific increase in polysomal abundance of mouse TEA DNA binding domain 2 (mTEAD-2) mRNA, coincident with the first appearance of mTEAD activity in the early embryo. The mRNAs encoding Sp1, TATA binding protein, and cyclic AMP response element binding protein did not undergo translational recruitment, but exhibited differences in polysomal abundance. We also observed a continuous, high proportion in the polysomal fraction for the mRNA encoding ribosomal protein L23 mRNA, which contrasted with the patterns observed for other maternal transcripts. These observations are consistent with the hypothesis that regulated recruitment of maternal TF mRNAs may control the time of activation of some genes during EGA, and that continuous synthesis of other TFs, like Sp1, may facilitate EGA.
Full-grown Xenopus oocytes arrest at the G2/M border of meiosis I. Progesterone breaks this arrest, leading to the resumption of the meiotic cell cycles and maturation of the oocyte into a fertilizable egg. In these oocytes, progesterone interacts with an unidentified surface-associated receptor, which induces a non-transcriptional signalling pathway that stimulates the translation of dormant c-mos messenger RNA. Mos, a mitogen-activated protein (MAP) kinase kinase kinase, indirectly activates MAP kinase, which in turn leads to oocyte maturation. The translational recruitment of c-mos and several other mRNAs is regulated by cytoplasmic polyadenylation, a process that requires two 3' untranslated regions, the cytoplasmic polyadenylation element (CPE) and the polyadenylation hexanucleotide AAUAAA. Although the signalling events that trigger c-mos mRNA polyadenylation and translation are unclear, they probably involve the activation of CPEB, the CPE binding factor. Here we show that an early site-specific phosphorylation of CPEB is essential for the polyadenylation of c-mos mRNA and its subsequent translation, and for oocyte maturation. In addition, we show that this selective, early phosphorylation of CPEB is catalysed by Eg2, a member of the Aurora family of serine/threonine protein kinases.
The protamine mRNAs are stored for up to 8 days as translationally repressed ribonucleoprotein particles during murine spermatogenesis. Translational repression of the protamine 1, Prm1, mRNA is controlled by sequences in its 3'-untranslated region (UTR). In this study we used the yeast three-hybrid system to clone Msy4, which encodes a novel member of the Y box family of nucleic acid binding proteins. MSY4 specifically binds to a site within the 5' most 37 nucleotides in the Prm1 3' UTR. Msy4 is highly expressed in the testis, and the protein is detected in the cytoplasm of germ cells in both the testis and the ovary, where repressed messages are stored. Analysis of a previously described 48/50-kDa binding activity in testis extracts by electrophoretic mobility shift assays and immunoprecipitation indicates the activity is composed of MSY4 and MSY2, another mouse Y box protein. Polysome analysis demonstrates MSY4 is associated with mRNPs, consistent with MSY4 having a role in storing repressed messages.
The release of Xenopus oocytes from prophase I arrest is largely driven by the cytoplasmic polyadenylation-induced translation of dormant maternal mRNAs. Two cis elements, the CPE and the hexanucleotide AAUAAA, and their respective binding factors, CPEB and a cytoplasmic form of CPSF, control polyadenylation. The most proximal stimulus for polyadenylation is Eg2-catalyzed phosphorylation of CPEB serine 174. Here, we show that this phosphorylation event stimulates an interaction between CPEB and CPSF. This interaction is direct, does not require RNA tethering, and occurs through the 160 kDa subunit of CPSF. Eg2-stimulated and CPE-dependent polyadenylation is reconstituted in vitro using purified components. These results demonstrate that the molecular function of Eg2-phosphorylated CPEB is to recruit CPSF into an active cytoplasmic polyadenylation complex.
Translational control plays a large role in vertebrate oocyte maturation and contributes to the induction of the germ layers. Translational regulation is also observed in the regulation of cell proliferation and differentiation. The features of an mRNA that mediate translational control are found both in the 5' and in the 3' untranslated regions (UTRs). In the 5' UTR, secondary structure, the binding of proteins, and the presence of upstream open reading frames can interfere with the association of initiation factors with the cap, or with scanning of the initiation complex. The 3' UTR can mediate translational activation by directing cytoplasmic polyadenylation and can confer translational repression by interference with the assembly of initiation complexes. Besides mRNA-specific translational control elements, the nonspecific RNA-binding proteins contribute to the modulation of translation in development. This review discusses examples of translational control and their relevance for developmental regulation.
An in vitro system that recapitulates the in vivo effect of AU-rich elements (AREs) on mRNA deadenylation has been developed from Xenopus activated egg extracts. ARE-mediated deadenylation is uncoupled from mRNA body decay, and the rate of deadenylation increases with the number of tandem AUUUAs. A novel ARE-binding protein called ePAB (for embryonic poly(A)-binding protein) has been purified from this extract by ARE affinity selection. ePAB exhibits 72% identity to mammalian and Xenopus PABP1 and is the predominant poly(A)-binding protein expressed in the stage VI oocyte and during Xenopus early development. Immunodepletion of ePAB increases the rate of both ARE-mediated and default deadenylation in vitro. In contrast, addition of even a small excess of ePAB inhibits deadenylation, demonstrating that the ePAB concentration is critical for determining the rate of ARE-mediated deadenylation. These data argue that ePAB is the poly(A)-binding protein responsible for stabilization of poly(A) tails and is thus a potential regulator of mRNA deadenylation and translation during early development.