The translational functions of embryonic poly(A)‐binding protein during gametogenesis and early embryo development

Embryonic poly(A)‐binding protein (EPAB) is an RNA‐binding protein that binds to the poly(A) tails and AU‐rich element at the 3′ ends of messenger RNA (mRNAs). The main functions of EPAB are to protect stored mRNAs from undergoing deadenylation and subsequent degradation and to be involved in their translational regulation during spermatogenesis, oogenesis, and early embryogenesis. Following the first characterization of Epab in the Xenopus oocytes and early embryos, spatial and temporal expression and potential roles of the Epab gene have been determined in the vertebrate germ cells and early embryos. In this review, we have comprehensively evaluated all studies in this field and discussed the particular functions of EPAB in the spermatogenic cells, oocytes, early embryos, and somatic cells in vertebrates.     

Embryonic poly(A)-binding protein (EPAB) is required for oocyte maturation and female fertility in mice

Gene expression during oocyte maturation and early embryogenesis up to zygotic genome activation requires translational activation of maternally-derived mRNAs. EPAB [embryonic poly(A)-binding protein] is the predominant poly(A)-binding protein during this period in Xenopus, mouse and human. In Xenopus oocytes, ePAB stabilizes maternal mRNAs and promotes their translation. To assess the role of EPAB in mammalian reproduction, we generated Epab-knockout mice. Although Epab(-/-) males and Epab(+/-) of both sexes were fertile, Epab(-/-) female mice were infertile, and could not generate embryos or mature oocytes in vivo or in vitro. Epab(-/-) oocytes failed to achieve translational activation of maternally-stored mRNAs upon stimulation of oocyte maturation, including Ccnb1 (cyclin B1) and Dazl (deleted in azoospermia-like) mRNAs. Microinjection of Epab mRNA into Epab(-/-) germinal vesicle stage oocytes did not rescue maturation, suggesting that EPAB is also required for earlier stages of oogenesis. In addition, late antral follicles in the ovaries of Epab(-/-) mice exhibited impaired cumulus expansion, and a 8-fold decrease in ovulation, associated with a significant down-regulation of mRNAs encoding the EGF (epidermal growth factor)-like growth factors Areg (amphiregulin), Ereg (epiregulin) and Btc (betacellulin), and their downstream regulators, Ptgs2 (prostaglandin synthase 2), Has2 (hyaluronan synthase 2) and Tnfaip6 (tumour necrosis factor α-induced protein 6). The findings from the present study indicate that EPAB is necessary for oogenesis, folliculogenesis and female fertility in mice.     

Induction of DNA replication in the germinal vesicle of the growing mouse oocyte

Growing mouse oocytes are physiologically arrested in the G2 phase of prophase of the first meiotic division. Growing oocytes were isolated from ovaries of 9- to 12-day-old mice and fused with parthenogenetic one-cell eggs or two-cell embryos derived from fertilized eggs. Resulting hybrids were injected with Dig-11-dUTP and examined for DNA replication using immunofluorescence. Parthenogenetic one-cell eggs fused at telophase II, G1, and middle-to-late S phase, and also S-phase two-cell blastomeres, were able to trigger DNA synthesis in oocyte germinal vesicle (GV) in the majority of hybrids cultured to the end of the first cell cycle. Activation of replication in the GV occurred within 2-3 h after fusion of growing oocytes with S-phase eggs. We show indirectly that the reactivation of replication in GVs was not dependent on the breakdown of the GV envelope. Although GVs had the ability to renew DNA replication after fusion, the G2 blastomere nuclei were incapable of reinitiating DNA replication under the influence of S-phase one-cell eggs. We hypothesize that the nuclei of growing oocytes arrested in meiotic prophase are in a physiological state that is equivalent to replication-competent G1, and not G2, nuclei.     

Deep freezing of mouse one-cell embryos and oocytes using different cryoprotectants

The objective of this study was to compare iso-osmolar concentrations (1.5 M) of 1,2-propanediol, glycerol, dimethylsulphoxide and a combination of 1 M propanediol + 0.5M glycerol (PDGLY) as cryoprotectants for murine ovulated oocytes and one-cell embryos. A higher (P < 0.01) percentage of one-cell embryos developed to the two-cell stage when frozen-thawed with 1,2-propanediol (83%) as compared with glycerol (43%), dimethylsulfoxide (51%) or PDGLY (7%). Data recalculated on the basis of two-cell embryos/number of normal one-cell embryos after thawing indicated no differences among single cryoprotectant groups. More (P < 0.01) frozen-thawed, in-vitro fertilized oocytes developed to the two-cell stage when 1,2-propanediol (35%) was used as cryoprotectant as compared with glycerol (15%). Freezing-thawing resulted in a reduced number of two-cell embryos after oocytes were fertilized in-vitro as compared with fresh oocytes. 1,2-propanediol was a better cryoprotectant than glycerol, dimethylsulphoxide or PDGLY for deep freezing of murine oocytes or one-cell embryos.     

Proximity of the poly(A)-binding protein to a premature termination codon inhibits mammalian nonsense-mediated mRNA decay

mRNA surveillance pathways selectively clear defective mRNAs from the cell. As such, these pathways serve as important modifiers of genetic disorders. Nonsense-mediated decay (NMD), the most intensively studied surveillance pathway, recognizes mRNAs with premature termination codons (PTCs). In mammalian systems the location of a PTC more than 50 nucleotides 5' to the terminal exon-exon junction is a critical determinant of NMD. However, mRNAs with nonsense codons that fulfill this requirement but are located very early in the open reading frame can effectively evade NMD. The unexpected resistance of such mRNAs with AUG-proximal PTCs to accelerated decay suggests that important determinants of NMD remain to be identified. Here, we report that an NMD-sensitive mRNA can be stabilized by artificially tethering the cytoplasmic poly(A) binding protein 1, PABPC1, at a PTC-proximal position. Remarkably, the data further suggest that NMD of an mRNA with an AUG-proximal PTC can also be repressed by PABPC1, which might be brought into proximity with the PTC during cap-dependent translation and 43S scanning. These results reveal a novel parameter of NMD in mammalian cells that can account for the stability of mRNAs with AUG-proximal PTCs. These findings serve to expand current mechanistic models of NMD and mRNA translation.     

Protein kinase Akt2/PKBβ is involved in blastomere proliferation of preimplantation mouse embryos

Activation of Akt/Protein Kinase B (PKB) by phosphatidylinositol-3-kinase (PI3K) controls several cellular functions largely studied in mammalian cells, including preimplantation embryos. We previously showed that early mouse embryos inherit active Akt from oocytes and that the intracellular localization of this enzyme at the two-cell stage depends on the T-cell leukemia/lymphoma 1 oncogenic protein, Tcl1. We have now investigated whether Akt isoforms, namely Akt1, Akt2 and Akt3, exert a specific role in blastomere proliferation during preimplantation embryo development. We show that, in contrast to other Akt family members, Akt2 enters male and female pronuclei of mouse preimplantation embryos at the late one-cell stage and thereafter maintains a nuclear localization during later embryo cleavage stages. Depleting one-cell embryos of single Akt family members by microinjecting Akt isoform-specific antibodies into wild-type zygotes, we observed that: (a) Akt2 is necessary for normal embryo progression through cleavage stages; and (b) the specific nuclear targeting of Akt2 in two-cell embryos depends on Tcl1. Our results indicate that preimplantation mouse embryos have a peculiar regulation of blastomere proliferation based on the activity of the Akt/PKB family member Akt2, which is mediated by the oncogenic protein Tcl1. Both Akt2 and Tcl1 are essential for early blastomere proliferation and embryo development.     

Biological role of the two overlapping poly(A)-binding protein interacting motifs 2 (PAM2) of eukaryotic releasing factor eRF3 in mRNA decay

Eukaryotic releasing factor GSPT/eRF3 mediates translation termination-coupled mRNA decay via interaction with a cytosolic poly(A)-binding protein (PABPC1). A region of eRF3 containing two overlapping PAM2 (PABPC1-interacting motif 2) motifs is assumed to bind to the PABC domain of PABPC1, on the poly(A) tail of mRNA. PAM2 motifs are also found in the major deadenylases Caf1–Ccr4 and Pan2–Pan3, whose activities are enhanced upon PABPC1 binding to these motifs. Their deadenylase activities are regulated by eRF3, in which two overlapping PAM2 motifs competitively prevent interaction with PABPC1. However, it is unclear how these overlapping motifs recognize PABC and regulate deadenylase activity in a translation termination-coupled manner. We used a dominant-negative approach to demonstrate that the N-terminal PAM2 motif is critical for eRF3 binding to PABPC1 and that both motifs are required for function. Isothermal titration calorimetry (ITC) and NMR analyses revealed that the interaction is in equilibrium between the two PAM2–PABC complexes, where only one of the two overlapping PAM2 motifs is PABC-bound and the other is PABC-unbound and partially accessible to the other PABC. Based on these results, we proposed a biological role for the overlapping PAM2 motifs in the regulation of deadenylase accessibility to PABPC1 at the 3′ end of poly(A).     

The effects of U.V.-light, ionizing radiation and the carcinogen N-acetoxy-2-fluorenylacetamide on the development in vitro of one- and two-cell mouse embryos.

The development of one- and two-cell mouse embryos to morula-blastula stages was followed in vitro after treatment with low doses of U.V.-light, ionizing radiation or N-acetoxy-2-fluorenylacetamide. Exposure of one-cell embryos to either radiation source 18 and 24 hours after human chorionic gonadotropin injections prevented maturation, most embryos being arrested at the one-cell stage and a few at the two-cell stage. Two-cell embryos, however, were not sensitive to low doses of either U.V. or X-irradiation and developed normally. Treatment of early one-cell embryos with the carcinogen, N-acetoxy-2-fluorenyl-acetamide (0-7 muM), also arrested development, whereas exposure of late one-cell embryos did not completely prevent maturation to morula-blastula stages. Exposure of two-cell embryos to the same concentration of carcinogen had no effect on their development to blastulas. Results with all three agents showed that mouse embryos at the one-cell stage are more sensitive than those at the two-cell stage, as judged by their ability to develop in vitro.     

Differential expression of alloantigens of the major histocompatibility complex on unfertilized and fertilized mouse eggs

Mouse oocytes at the dictyate and metaphase II stages as well as fertilized eggs have been studied by indirect immunofluorescence for the expression of H-2 histocompatibility antigens on surface membranes. Serologically specific reactivity to H-2 antibody was observed as patchy fluorescence distributed over the surface of the oocyte membrane. In contrast, one-cell zygotes exhibited variable reactivity, and early two-cell stages were negative. Absorption studies confirmed the serologic specificity of the reactivity on oocytes, which could be shown to be due to H-2 antibody. The results suggest that fertilization results in altered expression of major histocompatibility complex surface antigens, and confirms earlier studies that cleavage stage mouse embryos are not reactive with H-2 antibody.     

A new method to efficiently produce transgenic embryos and mice from low-titer lentiviral vectors

Vector injection into the perivitelline space has emerged as the standard delivery method to transduce lentivirus to mammalian oocytes or one-cell embryos, but its application is limited by the need for high titers of lentivirus. Herein we developed a new method by using a Piezo impact micro-manipulator for injecting low titer of lentivirus into the subzonal space of two-cell embryos or the perivitelline space of one-cell embryos that were shrunk with a highly concentrated sucrose solution. The survival rate of embryos was greater than 98% using this micromanipulation strategy, which was increased compared to the normal one-cell embryo injection method. More than 90% of injected embryos were GFP positive after subzonal injection of a lentivirus vector carrying the GFP gene with titers of 2 × 10⁸ I.U./ml. Even when a low titer of lentivirus (2 × 10⁶ I.U./ml) was used, 53.26% and 40.85% transgenic embryos were obtained after two-cell embryonic injection and one-cell sucrose treated embryonic injection, respectively. The GFP-positive rates were also greater than in the conventional method of injecting one-cell embryos (25.39%). In addition, blastocysts from the two-cell embryo injection group displayed stronger GFP fluorescence than the one-cell embryo injection groups treated with or without the sucrose solution. Increased expression of GFP suggests that the embryos obtained from this injection method have higher exogenous gene expression levels compared to previous methods. Therefore, in contrast with the traditional injection method, we have demonstrated a simplified and efficient lentivirus-mediated gene transfer method based on a low-titer virus preparation.     

Poly(A) and synthesis of polyadenylated RNA in the preimplantation mouse embryo

Investigations were conducted to quantitate polyadenylic acid and estimate the synthesis of polyadenylated RNA in mouse embryos at several stages of preimplantation development. Poly(A) was assayed by molecular hybridization of total embryonic RNA with [³H]polyuridylic acid. The mean values of poly(A) in the ovulated oocytes and in the one-cell, two-cell, and blastocyst stages of the embryo were 1.9, 1.6, 0.68, and 3.8 pg, respectively. Synthesis of polyadenylated RNA was estimated by affinity chromatography of [³H]uridine-labeled embryo RNA on oligo(dT)-cellulose. The proportions of newly synthesized RNA bound by oligo(dT)-cellulose at the 2-cell, 8- to 16-cell, and blastocyst stages were 6.7, 3.5, and 3.3%, respectively. These results suggest that significant quantities of maternal mRNA are present during early development of the mouse, but that polyadenylation of RNA transcribed from the embryonic genome occurs as early as the two-cell stage.     

The Saccharomyces cerevisiae poly(A)-binding protein is subject to multiple post-translational modifications,including the methylation of glutamic acid

Poly(A)-binding protein in mouse and man was recently found to be highly post-translationally modified. Here we analysed an ortholog of this protein, Pab1 from Saccharomyces cerevisiae, to assess the conservation and thus likely importance of these modifications. Pab1 showed the presence of six sites of methylated glutamate, five sites of lysine acetylation, and one phosphorylation of serine. Many modifications on Pab1 showed either complete conservation with those on human or mouse PABPC1, were present on nearby residues and/or were present in the same domain(s). The conservation of methylated glutamate, an unusual modification, was of particular note and suggests a conserved function. Comparison of methylated glutamate sites in human, mouse and yeast poly(A)-binding protein, along with methylation sites catalysed by CheR l-glutamyl protein methyltransferase from Salmonella typhimurium, revealed that the methylation of glutamate preferentially occurs in EE and DE motifs or other small regions of acidic amino acids. The conservation of methylated glutamate in the same protein between mouse, man and yeast suggests the presence of a eukaryotic l-glutamyl protein methyltransferase and that the modification is of functional significance.     

Inhibition of endoplasmic reticulum stress improves mouse embryo development

X-box binding protein-1 (XBP-1) is an important regulator of a subset of genes during endoplasmic reticulum (ER) stress. In the current study, we analyzed endogenous XBP-1 expression and localization, with a view to determining the effects of ER stress on the developmental competency of preimplantation embryos in mice. Fluorescence staining revealed that functional XBP-1 is localized on mature oocyte spindles and abundant in the nucleus at the germinal vesicle (GV) stage. However, in preimplantation embryos, XBP-1 was solely detected in the cytoplasm at the one-cell stage. The density of XBP-1 was higher in the nucleus than the cytoplasm at the two-cell, four-cell, eight-cell, morula, and blastocyst stages. Furthermore, RT-PCR analysis confirmed active XBP-1 mRNA splicing at all preimplantation embryo stages, except the one-cell stage. Tunicamycin (TM), an ER stress inducer used as a positive control, promoted an increase in the density of nuclear XBP-1 at the one-cell and two-cell stages. Similarly, culture medium supplemented with 25 mM sorbitol displayed a remarkable increase active XBP-1 expression in the nuclei of 1-cell and 2-cell embryos. Conversely, high concentrations of TM or sorbitol led to reduced nuclear XBP-1 density and significant ER stress-induced apoptosis. Tauroursodeoxycholic acid (TUDCA), a known inhibitor of ER stress, improved the rate of two-cell embryo development to blastocysts by attenuating the expression of active XBP-1 protein in the nucleus at the two-cell stage. Our data collectively suggest that endogenous XBP-1 plays a role in normal preimplantation embryonic development. Moreover, XBP-1 splicing is activated to generate a functional form in mouse preimplantation embryos during culture stress. TUDCA inhibits hyperosmolar-induced ER stress as well as ER stress-induced apoptosis during mouse preimplantation embryo development.     

Maturation‐associated Dbf4 expression is essential for mouse zygotic DNA replication

Cdc7 is an S‐phase‐promoting kinase (SPK) that is required for the activation of replication initiation complex assembly because it phosphorylates the MCM protein complex serving as the replicative helicase in eukaryotic organisms. Cdc7 activity is undetectable in immature mouse GV oocytes, although Cdc7 protein is already expressed at the same level as in mature oocytes or early one‐cell embryos at zygotic S‐phase, in which Cdc7 kinase activity is clearly detectable. Dbf4 is a regulatory subunit of Cdc7 and is required for Cdc7 kinase activity. Dbf4 is not readily detectable in immature GV oocytes but accumulates to a level similar to that in one‐cell embryos during oocyte maturation, suggesting that Cdc7 is already activated in unfertilized eggs (metaphase II). RNAi‐mediated knockdown of maternal Dbf4 expression prevents the maturation‐associated increase in Dbf4 protein, abolishes the activation of Cdc7, and leads to the failure of DNA replication in one‐cell embryos, demonstrating that Dbf4 expression is the key regulator of Cdc7 activity in mouse oocytes. Dormant Dbf4 mRNA in immature GV oocytes is recruited by cytoplasmic polyadenylation during oocyte maturation and is dependent on MPF activity via its cytoplasmic polyadenylation element (CPE) upstream of the hexanucleotide (HEX) in the 3′ untranslated region (3′UTR). Our results suggest that Cdc7 is inactivated in immature oocytes, preventing it from the unwanted phosphorylation of MCM proteins, and the oocyte is qualified by proper maturation to proceed following embryogenesis after fertilization through zygotic DNA replication.     

Zygotic gene activation in the mouse embryo: involvement of cyclic adenosine monophosphate-dependent protein kinase and appearance of an AP-1-like activity.

Protein phosphorylation catalyzed by the cyclic adenosine monophosphate (cAMP)-dependent protein kinase (PKA) is implicated in regulating zygotic gene activation in the two-cell mouse embryo (Poueymirou and Schultz; Dev Biol 133:588-599, 1989). We now provide evidence that H8, which is a PKA inhibitor, inhibits expression of an hsp70-driven beta-galactosidase reporter gene and that the concentration-dependence of this inhibition is similar to that for inhibiting expression of a stage-specific gene(s) that is a product of zygotic gene activation. We also demonstrate that neither cAMP nor serum can stimulate the expression, as detected by a histochemical assay, of a cAMP response element (CRE)- or serum response element (SRE)-driven beta-galactosidase reporter gene, respectively, in either germinal vesicle-intact oocytes or aphidicolin-arrested one-cell embryos that are chronologically at the tw-cell stage. In contrast, although 12-O-tetradecanoyl phorbol-13-acetate (TPA) does not stimulate expression of a TPA response element (TRE)-driven beta-galactosidase reporter gene in germinal vesicle-intact oocytes, it stimulates such expression in aphidicolin-arrested one-cell embryos. Moreover, TPA can stimulate the expression of either a CRE- or an SRE-driven beta-galactosidase reporter gene in such embryos. Results of these studies further implicate protein phosphorylation in regulating zygotic gene activation, along with its role in modulating enhancer function in the early mouse embryo.