Regulation of Oocyte mRNA Metabolism: A Key Determinant of Oocyte Developmental Competence.

The regulation of mRNA transcription and translation is uncoupled during oogenesis. The reason for this uncoupling is two-fold. Chromatin is only accessible to the transcriptional machinery during the growth phase as it condenses prior to resumption of meiosis to ensure faithful segregation of chromosomes during meiotic maturation. Thus, transcription rates are high during this time period in order to produce all of the transcripts needed for meiosis, fertilization, and embryo cleavage until the newly formed embryonic genome becomes transcriptionally active. To ensure appropriate timing of key developmental milestones including chromatin condensation, resumption of meiosis, segregation of chromosomes, and polar body extrusion, the translation of protein from transcripts synthesized during oocyte growth must be temporally regulated. This is achieved by the regulation of mRNA interaction with RNA binding proteins and shortening and lengthening of the poly(A) tail. This chapter details the essential factors that regulate the dynamic changes in mRNA synthesis, storage, translation, and degradation during oocyte growth and maturation.

Transcriptomic Profiling at the Maternal-to-Zygotic Transition in Leech, Helobdella austinensis.

The glossiphoniid leech, Helobdella austinensis, is an experimentally tractable member of the superphylum, Lophotrochozoa. Its large embryonic cells, stereotyped asymmetric cell divisions and ex vivo development capabilities makes it a favorable model for studying the molecular and cellular events of a representative spiralian. In this study, we focused on a narrow developmental time window of ~6-8 h, comprising stages just prior to and immediately following zygote deposition. Employing RNA-Seq methodology, we identified differentially expressed transcripts at this fundamental ontogenic boundary, known as the maternal-to-zygotic transition (MZT). Gene expression changes were characterized by the massive degradation of maternal RNAs (~45%) coupled with the rapid transcription of ~5000 zygotic genes (~20% of the genome) in the first mitotic cell cycle. The latter transcripts encoded a mixture of cell maintenance and regulatory proteins that predictably influence downstream developmental events.

Germ cell-specific eIF4E1b regulates maternal mRNA translation to ensure zygotic genome activation.

Translation of maternal mRNAs is detected before transcription of zygotic genes and is essential for mammalian embryo development. How certain maternal mRNAs are selected for translation instead of degradation and how this burst of translation affects zygotic genome activation remain unknown. Using gene-edited mice, we document that the oocyte-specific eukaryotic translation initiation factor 4E family member 1b (eIF4E1b) is the regulator of maternal mRNA expression that ensures subsequent reprogramming of the zygotic genome. In oocytes, eIF4E1b binds to transcripts encoding translation machinery proteins, chromatin remodelers, and reprogramming factors to promote their translation in zygotes and protect them from degradation. The protein products are thought to establish an open chromatin landscape in one-cell zygotes to enable transcription of genes required for cleavage stage development. Our results define a program for rapid resetting of the zygotic epigenome that is regulated by maternal mRNA expression and provide new insights into the mammalian maternal-to-zygotic transition.

Co-expression network analysis of environmental canalization in the ascidian Ciona.

BACKGROUND: Canalization, or buffering, is defined as developmental stability in the face of genetic and/or environmental perturbations. Understanding how canalization works is important in predicting how species survive environmental change, as well as deciphering how development can be altered in the evolutionary process. However, how developmental gene expression is linked to buffering remains unclear. We addressed this by co-expression network analysis, comparing gene expression changes caused by heat stress during development at a whole-embryonic scale in reciprocal hybrid crosses of sibling species of the ascidian Ciona that are adapted to different thermal environments. RESULTS: Since our previous work showed that developmental buffering in this group is maternally inherited, we first identified maternal developmental buffering genes (MDBGs) in which the expression level in embryos is both correlated to the level of environmental canalization and also differentially expressed depending on the species' gender roles in hybrid crosses. We found only 15 MDBGs, all of which showed high correlation coefficient values for expression with a large number of other genes, and 14 of these belonged to a single co-expression module. We then calculated correlation coefficients of expression between MDBGs and transcription factors in the central nervous system (CNS) developmental gene network that had previously been identified experimentally. We found that, compared to the correlation coefficients between MDBGs, which had an average of 0.96, the MDBGs are loosely linked to the CNS developmental genes (average correlation coefficient 0.45). Further, we investigated the correlation of each developmental to MDBGs, showing that only four out of 62 CNS developmental genes showed correlation coefficient > 0.9, comparable to the values between MDBGs, and three of these four genes were signaling molecules: BMP2/4, Wnt7, and Delta-like. CONCLUSIONS: We show that the developmental pathway is not centrally located within the buffering network. We found that out of 62 genes in the developmental gene network, only four genes showed correlation coefficients as high as between MDBGs. We propose that loose links to MDBGs stabilize spatiotemporally dynamic development.

Maternal body condition and season influence RNA deposition in the oocytes of alfalfa leafcutting bees (Megachile rotundata).

Maternal effects are an important source of phenotypic variance, whereby females influence offspring developmental trajectory beyond direct genetic contributions, often in response to changing environmental conditions. However, relatively little is known about the mechanisms by which maternal experience is translated into molecular signals that shape offspring development. One such signal may be maternal RNA transcripts (mRNAs and miRNAs) deposited into maturing oocytes. These regulate the earliest stages of development of all animals, but are understudied in most insects. Here we investigated the effects of female internal (body condition) and external (time of season) environmental conditions on maternal RNA in the maturing oocytes and 24-h-old eggs (24-h eggs) of alfalfa leafcutting bees. Using gene expression and WGCNA analysis, we found that females adjust the quantity of mRNAs related to protein phosphorylation, transcriptional regulation, and nuclease activity deposited into maturing oocytes in response to both poor body condition and shorter day lengths that accompany the late season. However, the magnitude of these changes was higher for time of season. Females also adjusted miRNA deposition in response to seasonal changes, but not body condition. We did not observe significant changes in maternal RNAs in response to either body condition or time of season in 24-h eggs, which were past the maternal-to-zygotic transition. Our results suggest that females adjust the RNA transcripts they provide for offspring to regulate development in response to both internal and external environmental cues. Variation in maternal RNAs may, therefore, be important for regulating offspring phenotype in response to environmental change.

Maternal Transcripts of Hox Genes Are Found in Oocytes of Platynereis dumerilii (Annelida, Nereididae).

Hox genes are some of the best studied developmental control genes. In the overwhelming majority of bilateral animals, these genes are sequentially activated along the main body axis during the establishment of the ground plane, i.e., at the moment of gastrulation. Their activation is necessary for the correct differentiation of cell lines, but at the same time it reduces the level of stemness. That is why the chromatin of Hox loci in the pre-gastrulating embryo is in a bivalent state. It carries both repressive and permissive epigenetic markers at H3 histone residues, leading to transcriptional repression. There is a paradox that maternal RNAs, and in some cases the proteins of the Hox genes, are present in oocytes and preimplantation embryos in mammals. Their functions should be different from the zygotic ones and have not been studied to date. Our object is the errant annelid Platynereis dumerilii. This model is convenient for studying new functions and mechanisms of regulation of Hox genes, because it is incomparably simpler than laboratory vertebrates. Using a standard RT-PCR on cDNA template which was obtained by reverse transcription using random primers, we found that maternal transcripts of almost all Hox genes are present in unfertilized oocytes of worm. We assessed the localization of these transcripts using WMISH.

Comparisons among rainbow trout, Oncorhynchus mykiss, populations of maternal transcript profile associated with egg viability.

BACKGROUND: Transcription is arrested in the late stage oocyte and therefore the maternal transcriptome stored in the oocyte provides nearly all the mRNA required for oocyte maturation, fertilization, and early cleavage of the embryo. The transcriptome of the unfertilized egg, therefore, has potential to provide markers for predictors of egg quality and diagnosing problems with embryo production encountered by fish hatcheries. Although levels of specific transcripts have been shown to associate with measures of egg quality, these differentially expressed genes (DEGs) have not been consistent among studies. The present study compares differences in select transcripts among unfertilized rainbow trout eggs of different quality based on eyeing rate, among 2 year classes of the same line (A1, A2) and a population from a different hatchery (B). The study compared 65 transcripts previously reported to be differentially expressed with egg quality in rainbow trout. RESULTS: There were 32 transcripts identified as DEGs among the three groups by regression analysis. Group A1 had the most DEGs, 26; A2 had 15, 14 of which were shared with A1; and B had 12, 7 of which overlapped with A1 or A2. Six transcripts were found in all three groups, dcaf11, impa2, mrpl39_like, senp7, tfip11 and uchl1. CONCLUSIONS: Our results confirmed maternal transcripts found to be differentially expressed between low- and high-quality eggs in one population of rainbow trout can often be found to overlap with DEGs in other populations. The transcripts differentially expressed with egg quality remain consistent among year classes of the same line. Greater similarity in dysregulated transcripts within year classes of the same line than among lines suggests patterns of transcriptome dysregulation may provide insight into causes of decreased viability within a hatchery population. Although many DEGs were identified, for each of the genes there is considerable variability in transcript abundance among eggs of similar quality and low correlations between transcript abundance and eyeing rate, making it highly improbable to predict the quality of a single batch of eggs based on transcript abundance of just a few genes.

Exploring Translational Control of Maternal mRNAs in Zebrafish.

The study of translational regulation requires reliable measurement of both mRNA levels and protein synthesis. Cytoplasmic polyadenylation is a prevalent mode of translational regulation during oogenesis and early embryogenesis. Here the length of the poly(A) tail of an mRNA is coupled to its translatability. We describe a protocol to identify translationally regulated genes and measure their translation rate in the early zebrafish embryo using genome-wide polysome profiling. This protocol relies on the isolation of mRNA by means of an rRNA depletion strategy, which avoids capture bias due to short poly(A) tail that can occur when using conventional oligo(dT)-based methods. We also present a simple PCR-based method to measure the poly(A) tail length of selected mRNAs.

Preliminary Identification of Candidate Genes Related to Survival of Gynogenetic Rainbow Trout (Oncorhynchus mykiss) Based on Comparative Transcriptome Analysis.

In the present research, the eggs from four rainbow trout females were used to provide four groups of gynogenetic doubled haploids (DHs). The quality of the eggs from different clutches was comparable, however, interclutch differences were observed in the gynogenetic variants of the experiment and the survival of DH specimens from different groups varied from 3% to 57% during embryogenesis. Transcriptome analysis of the eggs from different females exhibited inter-individual differences in the maternal genes' expression. Eggs originating from females whose gynogenetic offspring had the highest survival showed an increased expression of 46 genes when compared to the eggs from three other females. Eggs with the highest survival of gynogenetic embryos showed an up-regulation of genes that are associated with cell survival, migration and differentiation (tyrosine-protein kinase receptor TYRO3-like gene), triglyceride metabolism (carnitine O-palmitoyltransferase 1 gene), biosynthesis of polyunsaturated fat (3-oxoacyl-acyl-carrier-protein reductase gene), early embryogenic development (protein argonaute-3 gene, leucine-rich repeat-containing protein 3-like gene), 5S RNA binding (ribosome biogenesis regulatory protein homolog) as well as senescence and aging (telomerase reverse transcriptase, TERT gene), among others. Positive correlation between the genotypic efficiency and egg transcriptome profiles indicated that at least some of the differentially expressed genes should be considered as potential candidate genes for the efficiency of gynogenesis in rainbow trout.

Maternal factors regulating preimplantation development in mice.

Mammalian embryogenesis depends on maternal factors accumulated in eggs prior to fertilization and on placental transfers later in gestation. In this review, we focus on initial events when the organism has insufficient newly synthesized embryonic factors to sustain development. These maternal factors regulate preimplantation embryogenesis both uniquely in pronuclear formation, genome reprogramming and cell fate determination and more universally in regulating cell division, transcription and RNA metabolism. Depletion, disruption or inappropriate persistence of maternal factors can result in developmental defects in early embryos. To better understand the origins of these maternal effects, we include oocyte maturation processes that are responsible for their production. We focus on recent publications and reference comprehensive reviews that include earlier scientific literature of early mouse development.

Cytoplasmic aggregation of DDX1 in developing embryos: Early embryonic lethality associated with Ddx1 knockout.

Temporally-regulated maternal RNA translation is essential for embryonic development, with defective degradation resulting in stalled 2-cell embryos. We show that DDX1, a DEAD box protein implicated in RNA transport, may be a key regulator of maternal RNA utilization. DDX1 protein localizes exclusively to cytoplasmic granules in both oocytes and early stage mouse embryos, with DDX1 requiring RNA for retention at these sites. Homozygous knockout of Ddx1 causes stalling of mouse embryos at the 2-4 cell stages. These results suggest a maternal RNA-dependent role for DDX1 in the progression of embryos past the 2-4 cell stage. The change in appearance of DDX1-containing granules in developing embryos further supports a role in temporally-regulated degradation of RNAs. We carried out RNA-immunoprecipitations (RNA-IPs) to identify mRNAs bound to DDX1 in 2-cell embryos, focusing on 16 maternal genes previously shown to be essential for embryonic development past the 1- to 2-cell stages. Five of these RNAs were preferentially bound by DDX1: Ago2, Zar1, Tle6, Floped and Tif1α. We propose that DDX1 controls access to subsets of key maternal RNAs required for early embryonic development.

Translational repression of the Drosophila nanos mRNA involves the RNA helicase Belle and RNA coating by Me31B and Trailer hitch.

Translational repression of maternal mRNAs is an essential regulatory mechanism during early embryonic development. Repression of the Drosophila nanos mRNA, required for the formation of the anterior-posterior body axis, depends on the protein Smaug binding to two Smaug recognition elements (SREs) in the nanos 3' UTR. In a comprehensive mass spectrometric analysis of the SRE-dependent repressor complex, we identified Smaug, Cup, Me31B, Trailer hitch, eIF4E, and PABPC, in agreement with earlier data. As a novel component, the RNA-dependent ATPase Belle (DDX3) was found, and its involvement in deadenylation and repression of nanos was confirmed in vivo. Smaug, Cup, and Belle bound stoichiometrically to the SREs, independently of RNA length. Binding of Me31B and Tral was also SRE-dependent, but their amounts were proportional to the length of the RNA and equimolar to each other. We suggest that "coating" of the RNA by a Me31B•Tral complex may be at the core of repression.

Maternal RNA regulates Aurora C kinase during mouse oocyte maturation in a translation-independent fashion.

During oocyte meiotic maturation, Aurora kinase C (AURKC) is required to accomplish many critical functions including destabilizing erroneous kinetochore-microtubule (K-MT)attachments and regulating bipolar spindle assembly. How localized activity of AURKC is regulated in mammalian oocytes, however, is not fully understood. Female gametes from many species, including mouse, contain stores of maternal transcripts that are required for downstream developmental events. We show here that depletion of maternal RNA in mouse oocytes resulted in impaired meiotic progression, increased incidence of chromosome misalignment and abnormal spindle formation at metaphase I (Met I), and cytokinesis defects. Importantly, depletion of maternal RNA perturbed the localization and activity of AURKC within the chromosomal passenger complex (CPC). These perturbations were not observed when translation was inhibited by cycloheximide (CHX) treatment. These results demonstrate a translation-independent function of maternal RNA to regulate AURKC-CPC function in mouse oocytes.

Transcriptome dynamics in early zebrafish embryogenesis determined by high-resolution time course analysis of 180 successive, individual zebrafish embryos.

BACKGROUND: Recently, much progress has been made in the field of gene-expression in early embryogenesis. However, the dynamic behaviour of transcriptomes in individual embryos has hardly been studied yet and the time points at which pools of embryos are collected are usually still quite far apart. Here, we present a high-resolution gene-expression time series with 180 individual zebrafish embryos, obtained from nine different spawns, developmentally ordered and profiled from late blastula to mid-gastrula stage. On average one embryo per minute was analysed. The focus was on identification and description of the transcriptome dynamics of the expressed genes in this embryonic stage, rather than to biologically interpret profiles in cellular processes and pathways. RESULTS: In the late blastula to mid-gastrula stage, we found 6,734 genes being expressed with low variability and rather gradual changes. Ten types of dynamic behaviour were defined, such as genes with continuously increasing or decreasing expression, and all expressed genes were grouped into these types. Also, the exact expression starting and stopping points of several hundred genes during this developmental period could be pinpointed. Although the resolution of the experiment was so high, that we were able to clearly identify four known oscillating genes, no genes were observed with a peaking expression. Additionally, several genes showed expression at two or three distinct levels that strongly related to the spawn an embryo originated from. CONCLUSION: Our unique experimental set-up of whole-transcriptome analysis of 180 individual embryos, provided an unparalleled in-depth insight into the dynamics of early zebrafish embryogenesis. The existence of a tightly regulated embryonic transcriptome program, even between individuals from different spawns is shown. We have made the expression profile of all genes available for domain experts. The fact that we were able to separate the different spawns by their gene-expression variance over all expressed genes, underlines the importance of spawn specificity, as well as the unexpectedly tight gene-expression regulation in early zebrafish embryogenesis.

Transcription of highly repetitive tandemly organized DNA in amphibians and birds: A historical overview and modern concepts.

Tandemly organized highly repetitive DNA sequences are crucial structural and functional elements of eukaryotic genomes. Despite extensive evidence, satellite DNA remains an enigmatic part of the eukaryotic genome, with biological role and significance of tandem repeat transcripts remaining rather obscure. Data on tandem repeats transcription in amphibian and avian model organisms is fragmentary despite their genomes being thoroughly characterized. Review systematically covers historical and modern data on transcription of amphibian and avian satellite DNA in somatic cells and during meiosis when chromosomes acquire special lampbrush form. We highlight how transcription of tandemly repetitive DNA sequences is organized in interphase nucleus and on lampbrush chromosomes. We offer LTR-activation hypotheses of widespread satellite DNA transcription initiation during oogenesis. Recent explanations are provided for the significance of high-yield production of non-coding RNA derived from tandemly organized highly repetitive DNA. In many cases the data on the transcription of satellite DNA can be extrapolated from lampbrush chromosomes to interphase chromosomes. Lampbrush chromosomes with applied novel technical approaches such as superresolution imaging, chromosome microdissection followed by high-throughput sequencing, dynamic observation in life-like conditions provide amazing opportunities for investigation mechanisms of the satellite DNA transcription.

Trans-splicing in metazoans: A link to translational control?

The trans-splicing of a spliced-leader RNA to a subset of mRNAs is a phenomenon that occurs in many species, including Caenorhabditis elegans, and yet the driving force for its evolution in disparate groups of animals remains unclear. Polycistronic mRNA resulting from the transcription of operons is resolved via trans-splicing, but operons comprise only a sub-set of trans-spliced genes. Using the marine chordate, Oikopleura dioica, we recently tested the hypothesis that metazoan operons accelerate recovery from growth arrest. We found no supporting evidence for this in O. dioica. Instead we found a striking relationship between trans-splicing and maternal mRNA in O. dioica, C. elegans and the ascidian, Ciona intestinalis. Furthermore, in O. dioica and C. elegans, we found evidence to suggest a role for mTOR signaling in the translational control of growth-related, trans-spliced maternal mRNAs. We propose that this may be a mechanism for adjusting egg number in response to nutrient levels in these species.

Localization of polyadenylated RNAs during teloplasm formation and cleavage in leech embryos.

In the embryos of glossiphoniid leeches, as in many annelids, cytoplasmic reorganization prior to first cleavage generates domains of yolk-deficient cytoplasm (called teloplasm) that are sequestered during the first three cell divisions to the D' macromere. Subsequently, the D' macromere generates a set of embryonic stem cells (teloblasts) that are the progenitors of the definitive segmental tissues. The hypothesis that fate-determining substances are localized within the teloplasm and segregated to the D' macromere during cleavage is supported by experiments in which a redistribution of yolk-defcient cytoplasm changes the fate of blastomeres that inherit it (Astrow et al. 1987; Devries 1973; Nelson and Weisblat 1992). As a step toward identifying fate-determining factors in teloplasm, we describe the distribution of polyadenylated RNAs (polyA+ RNA) in the early embryo of the leech, Helobdella triserialis, as inferred from in situ hybridization using tritiated polyuridylic acid (3H-polyU). Our results indicate that polyA+ RNA colocalizes with teloplasm during cytoplasmic rearrangements resulting in teloplasm formation, and that it remains concentrated in the teloplasm during the cell divisions and a second cytoplasmic rearrangement during early embryogenesis. Lesser amounts of polyA+ RNA appear to be localized in cortical cytoplasm at most stages.

Electrophoretic analysis of newly synthesized and stored maternal RNA during oogenesis ofCalliphora erythrocephala (Dipt.).

Ovaries ofC. erythrocephala synthesize large amounts of poly(A)+ and poly(A)- RNA during early and middle stages of oogenesis as shown by labelling with3H-uridine in vivo. After incubation for 1 h, a striking difference in the electrophoretic pattern of newly synthesized labelled poly(A)+ RNA and the poly(A)+ RNA present in sufficient amounts for optical density measurements (steady state poly(A)+ RNA) was observed. During early and mid-oogenesis, in the poly(A)- RNA fraction, ≧4S predominantly mature rRNA, 5S RNA and tRNA were labelled. These fractions were no longer synthesized during late oogenesis, whereas poly(A)+ RNA was labelled continously During oogenesis stage specific differences in the size distribution of newly synthesized and steady state poly(A)+ RNA were not obvious. However, different sizes of labelled poly(A)+ RNA species were detected in 0-2h old preblastoderm embryos, after injection of3H-uridine into females either 3-4 days (stage 3-4 of oogenesis) or 24 h before oviposition (stage 5-6 of oogenesis). This difference in RNA synthesis was related to the presence of active nurse cell nuclei. The poly(A)+ RNA fraction represents about 2-3% of the total RNA in both ovaries and freshly laid eggs as judged by measurements of optical density and radioactivity bound to oligo(dT). The length of poly(A)-segments in ovarian poly(A)+ RNA varied from about 30 to 200 nucleotides.

Insect egg cortex isolated by microsurgery: Specific protein pattern and uridine incorporation.

Egg cortex material (ECM) containing the oolemma and adherent periplasm has been isolated from an insect egg by means of micromanipulation. Analysis of proteins by SDS-PAAG-electrophoresis demonstrated striking differences between the protein banding patterns of ECM and of whole eggs. ECM and whole eggs also differ clearly with respect to3H-uridine uptake; the ECM contains RNA labeled during the previtellogenetic period of oogenesis (probably in the germarium) but, in contrast to the central parts of the oocyte, is largely void of label taken up during the last stages of oogenesis.

Degradation of maternal poly(A)-containing RNA during early embryogenesis of an insect (Smittia spec., chironomidae, diptera).

Eggs of the chironomid midgeSmittia spec. were shown to contain maternal rRNA, tRNA and poly(A)-containing RNA. The ribonucleoprotein spectrum consisted of monosomes, ribosomal subunits, and subribosomal particles, whereas polysomes could be detected only in small amounts. Poly(A)-containing RNA was found in different regions of the RNP spectrum, mainly between 15 S and 60 S. After labelling maternal RNA by feeding tritiated uridine to the larvae, the radioactivity associated with poly(A)-containing RNA accounted for about 4% of the label in the total RNA extracted from newly deposited eggs. About half of the radioactivity in the poly(A)-containing RNA was lost between egg deposition and an advanced blastoderm stage. The loss was accompanied by both a decrease in the size of the poly(A)-containing RNA molecules and a shift of poly(A)-containing RNP particles to less dense regions in sucrose gradients. Comparison with poly(A)-containing RNA synthesized by the embryo indicates that the reduction in size of maternal poly(A)-containing RNA is not artifactual but reflects its degradation after the formation of blastoderm.