Mid-blastula Stage Research Articles

Fertilization by short-term stored sperm alters DNA methylation patterns at single-base resolution in common carp (Cyprinus carpio) embryos

Sperm after short-term storage in vitro is widely used for artificial fertilization in aquaculture. It has been shown that short-term storage affects sperm motility characteristics, resulting in diminished fertility. However, the detrimental effects of short-term sperm storage on embryos development have remained unexplored in single-base methylome resolution. The main aim of the present study was to investigate DNA methylation in the offspring of common carp (Cyprinus carpio) derived from short-term stored sperm. Sperm were stored in artificial seminal plasma on ice (0–2 °C) for 0, 3 and 6 days in vitro, fertilization was performed using oocytes from a single female, and embryos were collected at the mid-blastula stage. In the DNA methylation study, DNA from both sperm and embryos was extracted and analysed using liquid chromatography with tandem mass spectrometry (LC–MS/MS). Concurrently, DNA methylation levels of embryos in single base were evaluated through whole genome bisulfite sequencing (WGBS). Sperm storage showed negative effects on sperm motility, viability, and DNA integrity, but had no effect on global DNA methylation of spermatozoa and resulting embryos. Results from the WGBS showed that methylation of 3313 differentially methylated regions (DMRs)-target genes was affected in the embryos fertilized with the 6-day-stored sperm, and the identified DMRs were mainly involved in cell adhesion, calcium, mitogen-activated protein kinase and adrenergic signalling, melanogenesis, metabolism and RNA transport. Such results suggest that prolongation of storage time may have certain impacts on embryonic development. These initial results provide valuable information for future consideration of the DNA methylome in embryos generated from short-term stored sperm, which are used for genetic management of broodstock in aquaculture.

Evaluating potential developmental toxicity of perfluoroalkyl and polyfluoroalkyl substances in Xenopus laevis embryos and larvae.

As part of the US Environmental Protection Agency's perfluoroalkyl and polyfluoroalkyl substances (PFAS) Action Plan, the agency is committed to increasing our understanding of the potential ecological effects of PFAS. The objective of these studies was to examine the developmental toxicity of PFAS using the laboratory model amphibian species Xenopus laevis. We had two primary aims: (1) to understand the developmental toxicity of a structurally diverse set of PFAS compounds in developing embryos and (2) to characterize the potential impacts of perfluorooctanesulfonic acid (PFOS), perfluorohexanesulfonic acid (PFHxS), perfluorooctanoic acid (PFOA), and hexafluoropropylene oxide-dimer acid (HFPO-DA a.k.a. GenX), on growth and thyroid hormone-controlled metamorphosis. We employed a combination of static renewal and flow-through exposure designs. Embryos were exposed to 17 structurally diverse PFAS starting at the midblastula stage through the completion of organogenesis (96 h). To investigate impacts on PFOS, PFOA, PFHxS, and HFPO-DA on development and metamorphosis, larvae were exposed from premetamorphosis (Nieuwkoop Faber stage 51 or 54) through pro metamorphosis. Of the PFAS tested in embryos, only 1H,1H,10H,10H-perfluorodecane-1,10-diol (FC10-diol) and perfluorohexanesulfonamide (FHxSA) exposure resulted in clear concentration-dependent developmental toxicity. For both of these PFAS, a significant increase in mortality was observed at 2.5 and 5mg/L. For FC10-diol, 100% of the surviving embryos were malformed at 1.25 and 2.5mg/L, while for FHxSA, a significant increase in malformations (100%) was observed at 2.5 and 5mg/L. Developmental stage achieved was the most sensitive endpoint with significant effects observed at 1.25 and 0.625 mg/L for FC10-diol and FHxSA, respectively. In larval studies, we observed impacts on growth following exposure to PFHxS and PFOS at concentrations of 100 and 2.5mg/L, respectively, while no impacts were observed in larvae when exposed to PFOA and HFPO-DA at concentration of 100 mg/L. Further, we did not observe impacts on thyroid endpoints in exposed larvae. These experiments have broadened our understanding of the impact of PFAS on anuran development.

Pregastrular Development of Amphibians: Ontogenetic Diversity and Eco-Devo

Comparative and ecological aspects of the reorganizations of early development in the class Amphibia are analyzed. We used data on the developmental diversity in a number of families belonging to the orders Anura and Caudata, in which many species had lost their connection with the aquatic environment. Model representatives of the class Amphibia (Ambystoma mexicanum, Rana temporaria, and Xenopus laevis) have small eggs (no more than 2.5 mm in diameter). In these species, the slowdown in the rate of cell divisions and the loss of synchrony occur at the midblastula stage. However, phylogenetically basal amphibian species (Ascaphus truei, Cryptobranchus alleganiensis) are characterized by the large (4–6 mm in diameter) yolky eggs and a short series of synchronous blastomere divisions (the synchrony is already lost at the 8-cell stage of cleavage). They do not have a “midblastula transition”, which is characteristic of the above model species. On the other hand, many evolutionarily advanced non-model species of caudate and anuran amphibians (for example, Desmognathus fuscus, Gastrotheca riobambae, Philoria sphagnicolus), as well as the basal species, are characterized by the large, yolk-rich eggs and the early loss of cell division synchrony. Phylogenetic analysis suggests that the cleavage pattern of the most extensively studied amphibians, the Mexican axolotl (Caudata) and the African clawed frog (Anura), represents a homoplasy. The midblastula transition, which is characteristic of these two species, might have evolved convergently in these two orders of amphibians as an embryonic adaptation to development in lentic water.

MiR-202-3p determines embryo viability during mid-blastula transition.

Developmental growth is an intricate process involving the coordinated regulation of the expression of various genes, and microRNAs (miRNAs) play crucial roles in diverse processes throughout animal development. The mid-blastula transition (MBT) is a developmental milestone when maternal RNAs are cleared and the zygotic genome programmed asynchronous cell division begins to drive embryogenesis. While mechanisms underlying MBT have been intensively revealed, factors regulating cell proliferation at the transition remain largely unknown. We report here a microRNA, miR-202-3p to be a key factor that determines embryonic fate during MBT in zebrafish. A miR-202-3p antagomir specifically terminated embryo development at the mid-blastula stage. In vivo deletion of the miR-202 locus recapitulated the fatal phenotypes, which were rescued only by miR-202-3p or its precursor. Transcriptome comparison revealed >250 RNAs including both maternal and zygotic origins were dysregulated at MBT in the miR-202−/− embryos, corresponding with arrays of homeostatic disorders leading to massive apoptosis. A trio of genes: nfkbiaa, perp and mgll, known to be intimately involved with cell proliferation and survival, were identified as direct targets of miR-202-3p. Importantly, over- or under-expression of any of the trio led to developmental delay or termination at the blastula or gastrula stages. Furthermore, nfkbiaa and perp were shown to inter-regulate each other. Thus, miR-202-3p mediates a regulatory network whose components interact closely during MBT to determine embryonic viability and development.

Control of zygotic genome activation in Xenopus.

The fertilized frog egg contains all the materials needed to initiate development of a new organism, including stored RNAs and proteins deposited during oogenesis, thus the earliest stages of development do not require transcription. The onset of transcription from the zygotic genome marks the first genetic switch activating the gene regulatory network that programs embryonic development. Zygotic genome activation occurs after an initial phase of transcriptional quiescence that continues until the midblastula stage, a period called the midblastula transition, which was first identified in Xenopus. Activation of transcription is programmed by maternally supplied factors and is regulated at multiple levels. A similar switch exists in most animals and is of great interest both to developmental biologists and to those interested in understanding nuclear reprogramming. Here we review in detail our knowledge on this major switch in transcription in Xenopus and place recent discoveries in the context of a decades old problem.

A biphasic role of non-canonical Wnt16 signaling during early anterior-posterior patterning and morphogenesis of the sea urchin embryo.

ABSTRACTA Wnt signaling network governs early anterior-posterior (AP) specification and patterning of the deuterostome sea urchin embryo. We have previously shown that non-canonical Fzl1/2/7 signaling antagonizes the progressive posterior-to-anterior downregulation of the anterior neuroectoderm (ANE) gene regulatory network (GRN) by canonical Wnt/β-catenin and non-canonical Wnt1/Wnt8-Fzl5/8-JNK signaling. This study focuses on the non-canonical function of the Wnt16 ligand during early AP specification and patterning. Maternally supplied wnt16 is expressed ubiquitously during cleavage and zygotic wnt16 expression is concentrated in the endoderm/mesoderm beginning at mid-blastula stage. Wnt16 antagonizes the ANE restriction mechanism and this activity depends on a functional Fzl1/2/7 receptor. Our results also show that zygotic wnt16 expression depends on both Fzl5/8 and Wnt/β-catenin signaling. Furthermore, Wnt16 is necessary for the activation and/or maintenance of key regulatory endoderm/mesoderm genes and is essential for gastrulation. Together, our data show that Wnt16 has two functions during early AP specification and patterning: (1) an initial role activating the Fzl1/2/7 pathway that antagonizes the ANE restriction mechanism; and (2) a subsequent function in activating key endoderm GRN factors and the morphogenetic movements of gastrulation.

DNA methylation dynamics during epigenetic reprogramming of medaka embryo

ABSTRACTPost-fertilization epigenome reprogramming erases epigenetic marks transmitted through gametes and establishes new marks during mid-blastula stages. The mouse embryo undergoes dynamic DNA methylation reprogramming after fertilization, while in zebrafish, the paternal DNA methylation pattern is maintained throughout the early embryogenesis and the maternal genome is reprogrammed in a pattern similar to that of sperm during the mid-blastula transition. Here, we show DNA methylation dynamics in medaka embryos, the biomedical model fish, during epigenetic reprogramming of embryonic genome. The sperm genome was hypermethylated and the oocyte genome hypomethylated prior to fertilization. After fertilization, the methylation marks of sperm genome were erased within the first cell cycle and embryonic genome remained hypomethylated from the zygote until 16-cell stage. The DNA methylation level gradually increased from 16-cell stage through the gastrula. The 5-hydroxymethylation (5hmC) levels showed an opposite pattern to DNA methylation (5-mC). The mRNA levels for DNA methyltransferase (DNMT) 1 remained high in oocytes and maintained the same level through late blastula stage and was reduced thereafter. DNMT3BB.1 mRNA levels increased prior to remethylation. The mRNA levels for ten-eleven translocation methylcytosine dioxygenases (TET2 & TET3) were detected in sperm and embryos at cleavage stages, whereas TET1 and TET3 mRNAs decreased during gastrulation. The pattern of genome methylation in medaka was identical to mammalian genome methylation but not to zebrafish. The present study suggests that a medaka embryo resets its DNA methylation pattern by active demethylation and by a gradual remethylation similar to mammals.

Transcriptome analysis of regeneration during Xenopus laevis experimental twinning

Animal embryos have the remarkable property of self-organization. Over 125 years ago, Hans Driesch separated the two blastomeres of sea urchin embryos and obtained twins, in what was the foundation of experimental embryology. Since then, embryonic twinning has been obtained experimentally in many animals. In a recent study, we developed bisection methods that generate identical twins reliably from Xenopus blastula embryos. In the present study, we have investigated the transcriptome of regenerating half-embryos after sagittal and dorsal-ventral (D-V) bisections. Individual embryos were operated at midblastula (stage 8) with an eyelash hair and cultured until early gastrula (stage 10.5) or late gastrula (stage 12) and the transcriptome of both halves were analyzed by RNA-seq. Since many genes are activated by wound healing in Xenopus embryos, we resorted to stringent sequence analyses and identified genes up-regulated in identical twins but not in either dorsal or ventral fragments. At early gastrula, cell division-related transcripts such as histones were elevated, whereas at late gastrula, pluripotency genes (such as sox2) and germ layer determination genes (such as eomesodermin, ripply2 and activin receptor ACVRI) were identified. Among the down-regulated transcripts, sizzled, a regulator of Chordin stability, was prominent. These findings are consistent with a model in which cell division is required to heal damage, while maintaining pluripotency to allow formation of the organizer with a displacement of 90 0 from its original site. The extensive transcriptomic data presented here provides a valuable resource for data mining of gene expression during early vertebrate development.

Characterization and localization of primordial germ cells in Totoaba macdonaldi.

The totoaba, Totoaba macdonaldi, is an endangered fish of the Gulf of California with high economic and ecological potential. Therefore, our purpose was to characterize the Primordial Germ Cells (PGCs) of this Sciaenid with two objectives: (1) to provide the basis for PGCs cryopreservation to preserve the genetic resources and (2) to take the first step to know the gonadal genesis and sex differentiation of totoaba. Immunofluorescence analysis performed from 2-cell stage to 8-day after hatch (DAH) shows that Vasa protein is specific for PGCs. These cells were first observed in the peripheral and dorsal regions of the blastodisc at approximately the 50%-epiboly stage and migrated to both sides of embryo body during the development. Finally, at 7 DAH the PGCs of the hatching embryo reached the place where the gonad will be developed. Histology analysis of larvae showed a genital ridge with enclosed PGCs on the dorsal side of the peritoneum at 9 DAH, gonadal primordium growth was observed at 11 DAH as a result of the interaction between PGCs and somatic cells derived from the peritoneum. Results of qPCR showed that vasa expression was restricted to the embryonic and early larval development, highest values were observed in 2-cell and mid-blastula stage suggesting the maternal inheritance of vasa mRNA. These findings support the hypothesis of preformation in T. macdonaldi PGCs. The migration pattern of PGCs allow us to recommend the isolation and subsequent cryopreservation of these cells before 7 DAH when the embryonic and larval development is given at 21 °C.

Tolerance to paternal genotoxic damage promotes survival during embryo development in zebrafish (Danio rerio).

ABSTRACTSpermatozoa carry DNA damage that must be repaired by the oocyte machinery upon fertilization. Different strategies could be adopted by different vertebrates to face the paternal genotoxic damage. Mammals have strong sperm selection mechanisms and activate a zygotic DNA damage response (DDR) (including cell cycle arrest, DNA repair and alternative apoptosis) in order to guarantee the genomic conformity of the reduced progeny. However, external fertilizers, with different reproductive strategies, seem to proceed distinctively. Previous results from our group showed a downregulation of apoptotic activity in trout embryos with a defective DNA repairing ability, suggesting that mechanisms of tolerance to damaged DNA could be activated in fish to maintain cell survival and to progress with development. In this work, zebrafish embryos were obtained from control or UV-irradiated sperm (carrying more than 10% of fragmented DNA but still preserving fertilization ability). DNA repair (γH2AX and 53BP1 foci), apoptotic activity, expression of genes related to DDR and malformation rates were analyzed throughout development. Results showed in the progeny from damaged sperm, an enhanced repairing activity at the mid-blastula transition stage that returned to its basal level at later stages, rendering at hatching a very high rate of multimalformed larvae. The study of transcriptional and post-translational activity of tp53 (ZDF-GENE-990415-270) revealed the activation of an intense DDR in those progenies. However, the downstream pro-apoptotic factor noxa (ZDF-GENE-070119-3) showed a significant downregulation, whereas the anti-apoptotic gene bcl2 (ZDF-GENE-051015-1) was upregulated, triggering a repressive apoptotic scenario in spite of a clear genomic instability. This repression can be explained by the observed upregulation of p53 isoform Δ113p53, which is known to enhance bcl2 transcription. Our results showed that tp53 is involved in DNA damage tolerance (DDT) pathways, allowing the embryo survival regardless of the paternal DNA damage. DDT could be an evolutionary mechanism in fish: tolerance to unrepaired sperm DNA could introduce new mutations, some of them potentially advantageous to face a changing environment.

Relation of Nodal expression to the specification of the dorsal-ventral axis and tissue patterning in the starfish Patiria pectinifera.

In this study, we attempted to reveal fundamental aspects of starfish embryogenesis, particularly embryonic axis specification or determination, in Patiria pectinifera. We first cloned PpNodal, which is known to play an important role in the specification of the embryonic axis in a wide range of animals, and studied its expression profile. PpNodal expression was first detected at the mid-blastula stage and showed a single peak around the onset of gastrulation. These features of Nodal expression were shifted to later stages by several hours, compared with those of sea urchin embryos. After the gastrulation started, the expression level became gradually lowered up to the early bipinnaria stage, while the expression level became drastically lowered in sea urchin embryos during gastrulation. The localized Nodal expression in the presumptive oral region was not observed in starfish embryos, unlike in sea urchin embryos. Furthermore, SB431542, an inhibitor of Nodal receptor, did not affect the formation of the DV axis, although it caused the loss of left-right asymmetry. In contrast to this, SB525334, a specific inhibitor of TGF-beta receptor, caused the complete loss of the DV axis. Thus, the usage of signaling molecules during early embryogenesis likely varies among echinoderm classes.

Molecular cloning and expression of Octamer-binding transcription factor (Oct4) in the large yellow croaker, Larimichthys crocea

Octamer-binding transcription factor 4 (Oct4) is a crucial pluripotent transcription factor in controlling pluripotency of embryonic stem cells (ESCs), formation of primordial germ cells (PGCs), early embryonic development, and gonadal germ cells. To understand the roles of Oct4 in the large yellow croaker (Larimichthys crocea) (Lc-Oct4), the full-length cDNA of Oct4 was cloned and analyzed. Lc-Oct4 includes a 104-bp 5′ untranslated region (UTR), a 567-bp 3′ UTR and a 1431-bp coding region encoding a protein of 476 amino acids (aa) with a predicted molecular mass (Mm) of 52.40 kDa and an isoelectric point (PI) of 6.34. Quantitative real time PCR (qRT-PCR) in tissues showed that Lc-Oct4 was only expressed in ovary. During ovarian development, the expression level in 635 days post hatching (635-dph) ovary was about 6.3-fold higher than in 270-dph and 1000-dph ovary. The in situ hybridization analysis showed that Lc-Oct4 had a high expression in the different developmental stages of oocytes with the highest level in stages II (later) and III oocytes. Lc-Oct4 was also expressed in spermatogonia and primary spermatocytes. Furthermore, the qRT-PCR analysis of Lc-Oct4 in different developmental embryos revealed that the expression from high to low was multiple-cell stage > mid-blastula stage = mid-gastrula stage, and the expression in multiple-cell stage was at less 1.6-fold higher than in other stages. Accordingly, the whole mount in situ hybridization (WISH) in embryos demonstrated that Lc-Oct4 was expressed only in early embryonic development from 2-cell stage to early-gastrula stage with the peak at 16-cell stage, but not detected in PGCs area. In conclusion, Lc-Oct4 participated in the oogenesis and early embryonic development in large yellow croaker. Overall, this study provided better understanding of Lc-Oct4 gene and lay the foundation for its function research in the large yellow croaker.

넙치(Paralichthys olivaceus)와 강도다리(Platichthys stellatus)간 유도된 잡종 3배체의 난발생

We investigated the characteristics and rate of development of allotriploid embryos derived from a cross between female olive flounder Paralichthys olivaceus and male starry flounder Platichthys stellatus. The allotriploidy was induced by cold shocking fertilized eggs three minutes post-fertilization at 3°C for 45 minutes. The average cellular DNA content of the allotriploid embryos was 2.06±0.03 pg/cell, which is equal to the sum of the cellular DNA content of a diploid olive flounder (1.42 pg/cell) and a haploid starry flounder (0.66 pg/haploid cell). The first cleavage, midblastula, gastrula and Kupffer's vesicle appearance stages of the allotriploid eggs began at 1.5, 8, 13 and 26 hours after cold shocking at 18°C, respectively. The developmental rate of allotriploid eggs was equivalent to that of diploid and triploid olive flounder eggs at 10, 14 and 18°C. However, the hatching times of allotriploid eggs, 7 h at 10°C, 5 h at 14°C and 4 h at 18°C, were earlier than those of diploid and triploid olive flounder.

N6-Methyladenosine Sequencing Highlights the Involvement of mRNA Methylation in Oocyte Meiotic Maturation and Embryo Development by Regulating Translation in Xenopus laevis

During the oogenesis of Xenopus laevis, oocytes accumulate maternal materials for early embryo development. As the transcription activity of the oocyte is silenced at the fully grown stage and the global genome is reactivated only by the mid-blastula embryo stage, the translation of maternal mRNAs accumulated during oocyte growth should be accurately regulated. Previous evidence has illustrated that the poly(A) tail length and RNA binding elements mediate RNA translation regulation in the oocyte. Recently, RNA methylation has been found to exist in various systems. In this study, we sequenced the N6-methyladenosine (m6A) modified mRNAs in fully grown germinal vesicle-stage and metaphase II-stage oocytes. As a result, we identified 4207 mRNAs with m6A peaks in germinal vesicle-stage or metaphase II-stage oocytes. When we integrated the mRNA methylation data with transcriptome and proteome data, we found that the highly methylated mRNAs showed significantly lower protein levels than those of the hypomethylated mRNAs, although the RNA levels showed no significant difference. We also found that the hypomethylated mRNAs were mainly enriched in the cell cycle and translation pathways, whereas the highly methylated mRNAs were mainly associated with protein phosphorylation. Our results suggest that oocyte mRNA methylation can regulate cellular translation and cell division during oocyte meiotic maturation and early embryo development.

The effect of cryoprotectant agents on DNA methylation patterns and progeny development in the spermatozoa of Colossoma macropomum

DNA methylation patterns are inherited from parents and are imperative for proper embryonic development; however, alterations in these patterns can compromise fertilization and development into a fully functioning adult animal because DNA methylation is part of a complex program of gene transcription. In this study, we investigated the impact of cryoprotectant agents (CPAs) on DNA methylation patterns in spermatozoa and the consequences on embryonic development and the survival rate of progeny. Global methylation was assessed by enzymatic reactions in Colossoma macropomum spermatozoa that were cryopreserved using dimethylsulfoxide, dimethylformamide, methanol, ethyl glycol and glycerol as CPAs. Fertilization was carried out to evaluate survival rates and abnormalities in embryonic development upon treatment with each of the CPAs. Fresh semen served as the control. Our results indicated that, compared to the control group, spermatozoa cryopreservation decreased the fertilization rate and delayed embryonic development from the midblastula stage. Furthermore, spermatozoa cryopreserved in all CPAs had lower methylation levels and exhibited more delays and abnormalities during embryonic development than did fresh semen. Methanol resulted in fertilization, hatching rates and embryonic development that were closer to the control but had lower methylation levels. In conclusion, ours results show significant alterations on spermatozoa DNA methylation patterns caused by CPAs that are used in the semen cryopreservation process. DNA methylation pattern alterations affected the viability of progeny (r=0.48); however, these effects can be minimized by choosing the CPA that will compose the freezing solution.

Dnd Is a Critical Specifier of Primordial Germ Cells in the Medaka Fish.

SummaryPrimordial germ cell (PGC) specification occurs early in development. PGC specifiers have been identified in Drosophila, mouse, and human but remained elusive in most animals. Here we identify the RNA-binding protein Dnd as a critical PGC specifier in the medaka fish (Oryzias latipes). Dnd depletion specifically abolished PGCs, and its overexpression boosted PGCs. We established a single-cell culture procedure enabling lineage tracing in vitro. We show that individual blastomeres from cleavage embryos at the 32- and 64-cell stages are capable of PGC production in culture. Importantly, Dnd overexpression increases PGCs via increasing PGC precursors. Strikingly, dnd RNA forms prominent particles that segregate asymmetrically. Dnd concentrates in germ plasm and stabilizes germ plasm RNA. Therefore, Dnd is a critical specifier of fish PGCs and utilizes particle partition as a previously unidentified mechanism for asymmetric segregation. These findings offer insights into PGC specification and manipulation in medaka as a lower vertebrate model.

Temperature Sensitivity of Neural Tube Defects in Zoep Mutants.

Neural tube defects (NTD) occur when the flat neural plate epithelium fails to fold into the neural tube, the precursor to the brain and spinal cord. Squint (Sqt/Ndr1), a Nodal ligand, and One-eyed pinhead (Oep), a component of the Nodal receptor, are required for anterior neural tube closure in zebrafish. The NTD in sqt and Zoep mutants are incompletely penetrant. The penetrance of several defects in sqt mutants increases upon heat or cold shock. In this project, undergraduate students tested whether temperature influences the Zoep open neural tube phenotype. Single pairs of adults were spawned at 28.5°C, the normal temperature for zebrafish, and one half of the resulting embryos were moved to 34°C at different developmental time points. Analysis of variance indicated temperature and clutch/genetic background significantly contributed to the penetrance of the open neural tube phenotype. Heat shock affected the embryos only at or before the midblastula stage. Many factors, including temperature changes in the mother, nutrition, and genetic background, contribute to NTD in humans. Thus, sqt and Zoep mutants may serve as valuable models for studying the interactions between genetics and the environment during neurulation.

Molecular characterization and mRNA expression of HIF-prolyl hydroxylase-2 (phd2) in hypoxia-sensing pathways from Megalobrama amblycephala.

HIF-prolyl-hydroxylase-2 (Phd2), amember of the iron (II) and 2-oxoglutarate-dependent dioxygenase family, is one of the key enzymes in hypoxia-sensing pathways. In this study, thephd2cDNA sequence (1231bp), including an open reading frame (ORF) and encoding 358 amino acid residues was identified inMegalobrama amblycephala(Wuchang bream). The predicted Phd2 protein contained three conserved domains, MYND type zinc finger domain with critical regulatory activity, Fe(2+)-dependent 2OG-Fe (II) oxygenase superfamilydomain with prolyl hydroxylase function, and P4Hc (prolyl 4-hydroxylase alphasubunit homologues) domain for catalyzing proline hydroxylation. The real-time PCR results showed thatphd2mRNA was ubiquitously expressed in all detected tissues with higher levels in the peripheral blood, heart and brain, and all embryogenesis stages, especially in mid-blastula stage. In larvaeM. amblycephala, the expression trend of thephd2and hypoxia-inducible factor 1 alpha (hif-1α) mRNA was opposite during hypoxia with an increase (hypoxia for 4h) and then decrease (hypoxia for 12h) forphd2. Whereas in adult fish, thephd2mRNA appeared a transient increase under hypoxia for 4h (DO: 3.46±0.59mg/L), and dramatically reduced with further hypoxia exposure to 12h in the peripheral blood, muscle, head kidney, liver and brain, but showed an opposite expression trend in the heart and gill. The hif-1αexpression was contrary withphd2in the peripheral blood, while it gradually decreased in the heart, but increased in the liver with continuous hypoxia treatment. Additionally,hif-1αalso showed lower mRNA levels thanphd2in all detected tissues under normoxia and hypoxia conditions.

Highly efficient gene knockout by injection of TALEN mRNAs into oocytes and host transfer in Xenopus laevis.

ABSTRACTZinc-finger nucleases, transcription activator-like effector nucleases (TALENs) and the CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated proteins) system are potentially powerful tools for producing tailor-made knockout animals. However, their mutagenic activity is not high enough to induce mutations at all loci of a target gene throughout an entire tadpole. In this study, we present a highly efficient method for introducing gene modifications at almost all target sequences in randomly selected embryos. The gene modification activity of TALEN is enhanced by adopting the host-transfer technique. In our method, the efficiency is further improved by injecting TALEN mRNAs fused to the 3′UTR of the Xenopus DEADSouth gene into oocytes, which are then transferred into a host female frog, where they are ovulated and fertilized. The addition of the 3′UTR of the DEADSouth gene promotes mRNA translation in the oocytes and increases the expression of TALEN proteins to near-maximal levels three hours post fertilization (hpf). In contrast, TALEN mRNAs without this 3′UTR are translated infrequently in oocytes. Our data suggest that genomic DNA is more sensitive to TALEN proteins from fertilization to the midblastula (MBT) stage. Our method works by increasing the levels of TALEN proteins during the pre-MBT stages.

Ovary transcriptome profiling via artificial intelligence reveals a transcriptomic fingerprint predicting egg quality in striped bass, Morone saxatilis.

Inherited gene transcripts deposited in oocytes direct early embryonic development in all vertebrates, but transcript profiles indicative of embryo developmental competence have not previously been identified. We employed artificial intelligence to model profiles of maternal ovary gene expression and their relationship to egg quality, evaluated as production of viable mid-blastula stage embryos, in the striped bass (Morone saxatilis), a farmed species with serious egg quality problems. In models developed using artificial neural networks (ANNs) and supervised machine learning, collective changes in the expression of a limited suite of genes (233) representing <2% of the queried ovary transcriptome explained >90% of the eventual variance in embryo survival. Egg quality related to minor changes in gene expression (<0.2-fold), with most individual transcripts making a small contribution (<1%) to the overall prediction of egg quality. These findings indicate that the predictive power of the transcriptome as regards egg quality resides not in levels of individual genes, but rather in the collective, coordinated expression of a suite of transcripts constituting a transcriptomic “fingerprint”. Correlation analyses of the corresponding candidate genes indicated that dysfunction of the ubiquitin-26S proteasome, COP9 signalosome, and subsequent control of the cell cycle engenders embryonic developmental incompetence. The affected gene networks are centrally involved in regulation of early development in all vertebrates, including humans. By assessing collective levels of the relevant ovarian transcripts via ANNs we were able, for the first time in any vertebrate, to accurately predict the subsequent embryo developmental potential of eggs from individual females. Our results show that the transcriptomic fingerprint evidencing developmental dysfunction is highly predictive of, and therefore likely to regulate, egg quality, a biologically complex trait crucial to reproductive fitness.