RESUMEN
Mammalian oocyte development depends on the temporally controlled translation of maternal transcripts, particularly in the coordination of meiotic and early embryonic development when transcription has ceased. The translation of mRNA is regulated by various RNA-binding proteins. We show that the absence of cytoplasmic polyadenylation element-binding protein 3 (CPEB3) negatively affects female reproductive fitness. CPEB3-depleted oocytes undergo meiosis normally but experience early embryonic arrest due to a disrupted transcriptome, leading to aberrant protein expression and the subsequent failure of embryonic transcription initiation. We found that CPEB3 stabilizes a subset of mRNAs with a significantly longer 3'UTR that is enriched in its distal region with cytoplasmic polyadenylation elements. Overall, our results suggest that CPEB3 is an important maternal factor that regulates the stability and translation of a subclass of mRNAs that are essential for the initiation of embryonic transcription and thus for embryonic development.
Asunto(s)
Oocitos , Proteínas de Unión al ARN , Oocitos/metabolismo , Animales , Proteínas de Unión al ARN/metabolismo , Proteínas de Unión al ARN/genética , Femenino , Ratones , Meiosis/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Desarrollo Embrionario/genética , Regulación del Desarrollo de la Expresión Génica , Regiones no Traducidas 3'/genética , Poliadenilación , Estabilidad del ARN/genéticaRESUMEN
Translation is critical for development as transcription in the oocyte and early embryo is silenced. To illustrate the translational changes during meiosis and consecutive two mitoses of the oocyte and early embryo, we performed a genome-wide translatome analysis. Acquired data showed significant and uniform activation of key translational initiation and elongation axes specific to M-phases. Although global protein synthesis decreases in M-phases, translation initiation and elongation activity increases in a uniformly fluctuating manner, leading to qualitative changes in translation regulation via the mTOR1/4F/eEF2 axis. Overall, we have uncovered a highly dynamic and oscillatory pattern of translational reprogramming that contributes to the translational regulation of specific mRNAs with different modes of polysomal occupancy/translation that are important for oocyte and embryo developmental competence. Our results provide new insights into the regulation of gene expression during oocyte meiosis as well as the first two embryonic mitoses and show how temporal translation can be optimized. This study is the first step towards a comprehensive analysis of the molecular mechanisms that not only control translation during early development, but also regulate translation-related networks employed in the oocyte-to-embryo transition and embryonic genome activation.
Asunto(s)
Desarrollo Embrionario , Oocitos , Biosíntesis de Proteínas , Regulación del Desarrollo de la Expresión Génica , Meiosis , Oocitos/citología , Oocitos/crecimiento & desarrollo , Oocitos/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Animales , RatonesRESUMEN
Preimplantation mouse embryo development involves temporal-spatial specification and segregation of three blastocyst cell lineages: trophectoderm, primitive endoderm and epiblast. Spatial separation of the outer-trophectoderm lineage from the two other inner-cell-mass (ICM) lineages starts with the 8- to 16-cell transition and concludes at the 32-cell stages. Accordingly, the ICM is derived from primary and secondary contributed cells; with debated relative EPI versus PrE potencies. We report generation of primary but not secondary ICM populations is highly dependent on temporal activation of mammalian target of Rapamycin (mTOR) during 8-cell stage M-phase entry, mediated via regulation of the 7-methylguanosine-cap (m7G-cap)-binding initiation complex (EIF4F) and linked to translation of mRNAs containing 5' UTR terminal oligopyrimidine (TOP-) sequence motifs, as knockdown of identified TOP-like motif transcripts impairs generation of primary ICM founders. However, mTOR inhibition-induced ICM cell number deficits in early blastocysts can be compensated by the late blastocyst stage, after inhibitor withdrawal; compensation likely initiated at the 32-cell stage when supernumerary outer cells exhibit molecular characteristics of inner cells. These data identify a novel mechanism specifically governing initial spatial segregation of mouse embryo blastomeres, that is distinct from those directing subsequent inner cell formation, contributing to germane segregation of late blastocyst lineages.
Asunto(s)
Blastocisto , Embrión de Mamíferos , Ratones , Animales , Diferenciación Celular/fisiología , Diana Mecanicista del Complejo 1 de la Rapamicina , Linaje de la Célula , MamíferosRESUMEN
High-resolution ribosome fractionation and low-input ribosome profiling of bovine oocytes and preimplantation embryos has enabled us to define the translational landscapes of early embryo development at an unprecedented level. We analyzed the transcriptome and the polysome- and non-polysome-bound RNA profiles of bovine oocytes (germinal vesicle and metaphase II stages) and early embryos at the two-cell, eight-cell, morula and blastocyst stages, and revealed four modes of translational selectivity: (1) selective translation of non-abundant mRNAs; (2) active, but modest translation of a selection of highly expressed mRNAs; (3) translationally suppressed abundant to moderately abundant mRNAs; and (4) mRNAs associated specifically with monosomes. A strong translational selection of low-abundance transcripts involved in metabolic pathways and lysosomes was found throughout bovine embryonic development. Notably, genes involved in mitochondrial function were prioritized for translation. We found that translation largely reflected transcription in oocytes and two-cell embryos, but observed a marked shift in the translational control in eight-cell embryos that was associated with the main phase of embryonic genome activation. Subsequently, transcription and translation become more synchronized in morulae and blastocysts. Taken together, these data reveal a unique spatiotemporal translational regulation that accompanies bovine preimplantation development.
Asunto(s)
Blastocisto , Desarrollo Embrionario , Embarazo , Femenino , Bovinos , Animales , Desarrollo Embrionario/genética , Mórula/metabolismo , Blastocisto/metabolismo , Oocitos/metabolismo , Ribosomas/genética , Regulación del Desarrollo de la Expresión GénicaRESUMEN
In mammalian females, oocytes are stored in the ovary and meiosis is arrested at the diplotene stage of prophase I. When females reach puberty oocytes are selectively recruited in cycles to grow, overcome the meiotic arrest, complete the first meiotic division and become mature (ready for fertilization). At a molecular level, the master regulator of prophase I arrest and meiotic resumption is the maturation-promoting factor (MPF) complex, formed by the active form of cyclin dependent kinase 1 (CDK1) and Cyclin B1. However, we still do not have complete information regarding the factors implicated in MPF activation. In this study we document that out of three mammalian serum-glucocorticoid kinase proteins (SGK1, SGK2, SGK3), mouse oocytes express only SGK1 with a phosphorylated (active) form dominantly localized in the nucleoplasm. Further, suppression of SGK1 activity in oocytes results in decreased CDK1 activation via the phosphatase cell division cycle 25B (CDC25B), consequently delaying or inhibiting nuclear envelope breakdown. Expression of exogenous constitutively active CDK1 can rescue the phenotype induced by SGK1 inhibition. These findings bring new insights into the molecular pathways acting upstream of MPF and a better understanding of meiotic resumption control by presenting a new key player SGK1 in mammalian oocytes.
Asunto(s)
Proteínas Inmediatas-Precoces , Factor Promotor de Maduración , Animales , Puntos de Control del Ciclo Celular , Femenino , Proteínas Inmediatas-Precoces/genética , Proteínas Inmediatas-Precoces/metabolismo , Mamíferos/metabolismo , Factor Promotor de Maduración/metabolismo , Meiosis , Profase Meiótica I , Ratones , Oocitos/metabolismo , Proteínas Serina-Treonina Quinasas/genéticaRESUMEN
Successful reproduction requires an oocyte competent to sustain early embryo development. By the end of oogenesis, the oocyte has entered a transcriptionally silenced state, the mechanisms and significance of which remain poorly understood. Histone H3.3, a histone H3 variant, has unique cell cycle-independent functions in chromatin structure and gene expression. Here, we have characterised the H3.3 chaperone Hira/Cabin1/Ubn1 complex, showing that loss of function of any of these subunits causes early embryogenesis failure in mouse. Transcriptome and nascent RNA analyses revealed that transcription is aberrantly silenced in mutant oocytes. Histone marks, including H3K4me3 and H3K9me3, are reduced and chromatin accessibility is impaired in Hira/Cabin1 mutants. Misregulated genes in mutant oocytes include Zscan4d, a two-cell specific gene involved in zygote genome activation. Overexpression of Zscan4 in the oocyte partially recapitulates the phenotypes of Hira mutants and Zscan4 knockdown in Cabin1 mutant oocytes partially restored their developmental potential, illustrating that temporal and spatial expression of Zscan4 is fine-tuned at the oocyte-to-embryo transition. Thus, the H3.3 chaperone Hira complex has a maternal effect function in oocyte developmental competence and embryogenesis, through modulating chromatin condensation and transcriptional quiescence.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Chaperonas de Histonas/metabolismo , Histonas/metabolismo , Oocitos/crecimiento & desarrollo , Oocitos/metabolismo , Transducción de Señal/genética , Factores de Transcripción/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Proteínas de Ciclo Celular/genética , Cromatina/metabolismo , Desarrollo Embrionario/genética , Femenino , Técnicas de Silenciamiento del Gen , Chaperonas de Histonas/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Oogénesis/genética , Factores de Transcripción/genética , Cigoto/metabolismoRESUMEN
Human multipotent neural stem cells could effectively be used for the treatment of a variety of neurological disorders. However, a defining signature of neural stem cell lines that would be expandable, non-tumorigenic, and differentiate into desirable neuronal/glial phenotype after in vivo grafting is not yet defined. Employing a mass spectrometry approach, based on selected reaction monitoring, we tested a panel of well-described culture conditions, and measured levels of protein markers routinely used to probe neural differentiation, i.e. POU5F1 (OCT4), SOX2, NES, DCX, TUBB3, MAP2, S100B, GFAP, GALC, and OLIG1. Our multiplexed assay enabled us to simultaneously identify the presence of pluripotent, multipotent, and lineage-committed neural cells, thus representing a powerful tool to optimize novel and highly specific propagation and differentiation protocols. The multiplexing capacity of this method permits the addition of other newly identified cell type-specific markers to further increase the specificity and quantitative accuracy in detecting targeted cell populations. Such an expandable assay may gain the advantage over traditional antibody-based assays, and represents a method of choice for quality control of neural stem cell lines intended for clinical use.
Asunto(s)
Diferenciación Celular , Células-Madre Neurales/citología , Células-Madre Neurales/metabolismo , Biomarcadores , Línea Celular , Linaje de la Célula/genética , Perfilación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Humanos , Inmunohistoquímica , Espectrometría de Masas , Neuroglía , NeuronasRESUMEN
Fully grown mammalian oocytes store a large amount of RNA synthesized during the transcriptionally active growth stage. A large part of the stored RNA belongs to the long non-coding class which contain either transcriptional noise or important contributors to cellular physiology. Despite the expanding number of studies related to lncRNAs, their influence on oocyte physiology remains enigmatic. We found an oocyte specific antisense, long non-coding RNA, "Rose" (lncRNA in Oocyte Specifically Expressed) expressed in two variants containing two and three non-coding exons, respectively. Rose is localized in the nucleus of transcriptionally active oocyte and in embryo with polysomal occupancy in the cytoplasm. Experimental overexpression of Rose in fully grown oocyte did not show any differences in meiotic maturation. However, knocking down Rose resulted in abnormalities in oocyte cytokinesis and impaired preimplantation embryo development. In conclusion, we have identified an oocyte-specific maternal lncRNA that is essential for successful mammalian oocyte and embryo development.
RESUMEN
During oocyte growth the cell accumulates RNAs to contribute to oocyte and embryo development which progresses with ceased transcription. To investigate the subcellular distribution of specific RNAs and their translation we developed a technique revealing several instances of localized translation with distinctive regulatory implications. We analyzed the localization and expression of candidate non-coding and mRNAs in the mouse oocyte and embryo. Furthermore, we established simultaneous visualization of mRNA and in situ translation events validated with polysomal occupancy. We discovered that translationally dormant and abundant mRNAs CyclinB1 and Mos are localized in the cytoplasm of the fully grown GV oocyte forming cloud-like structures with consequent abundant translation at the center of the MII oocyte. Coupling detection of the localization of specific single mRNA molecules with their translation at the subcellular context is a valuable tool to quantitatively study temporal and spatial translation of specific target mRNAs to understand molecular processes in the developing cell.
Asunto(s)
Ciclina B1/genética , Embrión de Mamíferos/química , Oocitos/crecimiento & desarrollo , Proteínas Proto-Oncogénicas c-mos/genética , Imagen Individual de Molécula/métodos , Animales , Citoplasma/genética , Femenino , Regulación del Desarrollo de la Expresión Génica , Hibridación Fluorescente in Situ , Ratones , Oocitos/química , Polirribosomas/genética , Biosíntesis de Proteínas , ARN Mensajero/genética , ARN no Traducido/genéticaRESUMEN
Successful specification of the two mouse blastocyst inner cell mass (ICM) lineages (the primitive endoderm (PrE) and epiblast) is a prerequisite for continued development and requires active fibroblast growth factor 4 (FGF4) signaling. Previously, we identified a role for p38 mitogen-activated protein kinases (p38-MAPKs) during PrE differentiation, but the underlying mechanisms have remained unresolved. Here, we report an early blastocyst window of p38-MAPK activity that is required to regulate ribosome-related gene expression, rRNA precursor processing, polysome formation and protein translation. We show that p38-MAPK inhibition-induced PrE phenotypes can be partially rescued by activating the translational regulator mTOR. However, similar PrE phenotypes associated with extracellular signal-regulated kinase (ERK) pathway inhibition targeting active FGF4 signaling are not affected by mTOR activation. These data indicate a specific role for p38-MAPKs in providing a permissive translational environment during mouse blastocyst PrE differentiation that is distinct from classically reported FGF4-based mechanisms.
Asunto(s)
Blastocisto/fisiología , Endodermo/citología , Biosíntesis de Proteínas , Proteínas Quinasas p38 Activadas por Mitógenos/fisiología , Animales , Diferenciación Celular , Linaje de la Célula , Proteínas de Unión al ADN/fisiología , Desarrollo Embrionario , Ratones , Proteínas de Unión al ARN/fisiología , Serina-Treonina Quinasas TOR/fisiología , Factores de Transcripción/fisiología , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidoresRESUMEN
Formation of the hatching mouse blastocyst marks the end of preimplantation development, whereby previous cell cleavages culminate in the formation of three distinct cell lineages (trophectoderm, primitive endoderm and epiblast). We report that dysregulated expression of Wwc2, a genetic paralog of Kibra/Wwc1 (a known activator of Hippo-signaling, a key pathway during preimplantation development), is specifically associated with cell autonomous deficits in embryo cell number and cell division abnormalities. Division phenotypes are also observed during mouse oocyte meiotic maturation, as Wwc2 dysregulation blocks progression to the stage of meiosis II metaphase (MII) arrest and is associated with spindle defects and failed Aurora-A kinase (AURKA) activation. Oocyte and embryo cell division defects, each occurring in the absence of centrosomes, are fully reversible by expression of recombinant HA-epitope tagged WWC2, restoring activated oocyte AURKA levels. Additionally, clonal embryonic dysregulation implicates Wwc2 in maintaining the pluripotent epiblast lineage. Thus, Wwc2 is a novel regulator of meiotic and early mitotic cell divisions, and mouse blastocyst cell fate.
RESUMEN
Increasing maternal age in mammals is associated with poorer oocyte quality, involving higher aneuploidy rates and decreased developmental competence. Prior to resumption of meiosis, fully developed mammalian oocytes become transcriptionally silent until the onset of zygotic genome activation. Therefore, meiotic progression and early embryogenesis are driven largely by translational utilization of previously synthesized mRNAs. We report that genome-wide translatome profiling reveals considerable numbers of transcripts that are differentially translated in oocytes obtained from aged compared to young females. Additionally, we show that a number of aberrantly translated mRNAs in oocytes from aged females are associated with cell cycle. Indeed, we demonstrate that four specific maternal age-related transcripts (Sgk1, Castor1, Aire and Eg5) with differential translation rates encode factors that are associated with the newly forming meiotic spindle. Moreover, we report substantial defects in chromosome alignment and cytokinesis in the oocytes of young females, in which candidate CASTOR1 and SGK1 protein levels or activity are experimentally altered. Our findings indicate that improper translation of specific proteins at the onset of meiosis contributes to increased chromosome segregation problems associated with female ageing.
Asunto(s)
Oocitos/metabolismo , Factores de Edad , Animales , Femenino , Humanos , MamíferosRESUMEN
Protein syntheses at appropriate timings are important for promoting diverse biological processes and are controlled at the levels of transcription and translation. Pou5f1/Oct4 is a transcription factor that is essential for vertebrate embryonic development. However, the precise timings when the mRNA and protein of Pou5f1/Oct4 are expressed during oogenesis and early stages of embryogenesis remain unclear. We analyzed the expression patterns of mRNA and protein of Pou5f1/Oct4 in mouse oocytes and embryos by using a highly sensitive in situ hybridization method and a monoclonal antibody specific to Pou5f1/Oct4, respectively. Pou5f1/Oct4 mRNA was detected in growing oocytes from the primary follicle stage to the fully grown GV stage during oogenesis. In contrast, Pou5f1/Oct4 protein was undetectable during oogenesis, oocyte maturation and the first cleavage stage but subsequently became detectable in the nuclei of early 2-cell-stage embryos. Pou5f1/Oct4 protein at this stage was synthesized from maternal mRNAs stored in oocytes. The amount of Pou5f1/Oct4 mRNA in the polysomal fraction was small in GV-stage oocytes but was significantly increased in fertilized eggs. Taken together, our results indicate that the synthesis of Pou5f1/Oct4 protein during oogenesis and early stages of embryogenesis is controlled at the level of translation and suggest that precise control of the amount of this protein by translational regulation is important for oocyte development and early embryonic development.
Asunto(s)
Desarrollo Embrionario/genética , Regulación del Desarrollo de la Expresión Génica/genética , Factor 3 de Transcripción de Unión a Octámeros/genética , Oogénesis/genética , Animales , Femenino , Ratones , Ratones Endogámicos ICR , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , EmbarazoRESUMEN
Cyclin dependent kinase 1 (CDK1) has been primarily identified as a key cell cycle regulator in both mitosis and meiosis. Recently, an extramitotic function of CDK1 emerged when evidence was found that CDK1 is involved in many cellular events that are essential for cell proliferation and survival. In this review we summarize the involvement of CDK1 in the initiation and elongation steps of protein synthesis in the cell. During its activation, CDK1 influences the initiation of protein synthesis, promotes the activity of specific translational initiation factors and affects the functioning of a subset of elongation factors. Our review provides insights into gene expression regulation during the transcriptionally silent M-phase and describes quantitative and qualitative translational changes based on the extramitotic role of the cell cycle master regulator CDK1 to optimize temporal synthesis of proteins to sustain the division-related processes: mitosis and cytokinesis.
Asunto(s)
Proteína Quinasa CDC2/metabolismo , Animales , Proteína Quinasa CDC2/genética , Ciclo Celular/genética , Ciclo Celular/fisiología , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Humanos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Serina-Treonina Quinasas TOR/genética , Serina-Treonina Quinasas TOR/metabolismoRESUMEN
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMEN
Lamin C2 (LMN C2) is a short product of the lamin a gene. It is a germ cell-specific lamin and has been extensively studied in male germ cells. In this study, we focussed on the expression and localization of LMN C2 in fully-grown germinal vesicle (GV) oocytes. We detected LMN C2 in the fully-grown germinal vesicle oocytes of various mammalian species with confirmation done by immunoblotting the wild type and Lmnc2 gene deleted testes. Expression of LMN C2 tagged with GFP showed localization of LMN C2 to the nuclear membrane of the oocyte. Moreover, the LMN C2 protein notably disappeared after nuclear envelope breakdown (NEBD) and the expression of LMN C2 was significantly reduced in the oocytes from aged females and ceased altogether during meiotic maturation. These results provide new insights regarding LMN C2 expression in the oocytes of various mammalian species.
Asunto(s)
Laminina/genética , Oocitos/crecimiento & desarrollo , Oogénesis/genética , Ovario/crecimiento & desarrollo , Animales , Femenino , Regulación del Desarrollo de la Expresión Génica/genética , Células Germinativas/crecimiento & desarrollo , Masculino , Meiosis/genética , Ratones , Ratones Noqueados , Membrana Nuclear/genética , Oocitos/metabolismo , ARN Mensajero/genética , Espermatocitos/crecimiento & desarrollo , Testículo/crecimiento & desarrolloRESUMEN
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMEN
Meiotic maturation of oocyte relies on pre-synthesised maternal mRNA, the translation of which is highly coordinated in space and time. Here, we provide a detailed polysome profiling protocol that demonstrates a combination of the sucrose gradient ultracentrifugation in small SW55Ti tubes with the qRT-PCR-based quantification of 18S and 28S rRNAs in fractionated polysome profile. This newly optimised method, named Scarce Sample Polysome Profiling (SSP-profiling), is suitable for both scarce and conventional sample sizes and is compatible with downstream RNA-seq to identify polysome associated transcripts. Utilising SSP-profiling we have assayed the translatome of mouse oocytes at the onset of nuclear envelope breakdown (NEBD)-a developmental point, the study of which is important for furthering our understanding of the molecular mechanisms leading to oocyte aneuploidy. Our analyses identified 1847 transcripts with moderate to strong polysome occupancy, including abundantly represented mRNAs encoding mitochondrial and ribosomal proteins, proteasomal components, glycolytic and amino acids synthetic enzymes, proteins involved in cytoskeleton organization plus RNA-binding and translation initiation factors. In addition to transcripts encoding known players of meiotic progression, we also identified several mRNAs encoding proteins of unknown function. Polysome profiles generated using SSP-profiling were more than comparable to those developed using existing conventional approaches, being demonstrably superior in their resolution, reproducibility, versatility, speed of derivation and downstream protocol applicability.
Asunto(s)
Membrana Nuclear/genética , Oocitos/crecimiento & desarrollo , Polirribosomas/genética , Proteínas de Unión al ARN/genética , Animales , Femenino , Regulación del Desarrollo de la Expresión Génica/genética , Meiosis/genética , Ratones , Membrana Nuclear/metabolismo , Oocitos/metabolismo , ARN Mensajero Almacenado/genética , ARN Ribosómico 18S/genética , ARN Ribosómico 28S/genética , RNA-SeqRESUMEN
Tens of thousands of rapidly evolving long non-coding RNA (lncRNA) genes have been identified, but functions were assigned to relatively few of them. The lncRNA contribution to the mouse oocyte physiology remains unknown. We report the evolutionary history and functional analysis of Sirena1, the most expressed lncRNA and the 10th most abundant poly(A) transcript in mouse oocytes. Sirena1 appeared in the common ancestor of mouse and rat and became engaged in two different post-transcriptional regulations. First, antisense oriented Elob pseudogene insertion into Sirena1 exon 1 is a source of small RNAs targeting Elob mRNA via RNA interference. Second, Sirena1 evolved functional cytoplasmic polyadenylation elements, an unexpected feature borrowed from translation control of specific maternal mRNAs. Sirena1 knock-out does not affect fertility, but causes minor dysregulation of the maternal transcriptome. This includes increased levels of Elob and mitochondrial mRNAs. Mitochondria in Sirena1-/- oocytes disperse from the perinuclear compartment, but do not change in number or ultrastructure. Taken together, Sirena1 contributes to RNA interference and mitochondrial aggregation in mouse oocytes. Sirena1 exemplifies how lncRNAs stochastically engage or even repurpose molecular mechanisms during evolution. Simultaneously, Sirena1 expression levels and unique functional features contrast with the lack of functional importance assessed under laboratory conditions.
Asunto(s)
Mitocondrias/genética , Oocitos/metabolismo , ARN Largo no Codificante/genética , ARN Mensajero/genética , ARN Mitocondrial/genética , Animales , Técnicas de Inactivación de Genes , Ratones , Mitocondrias/ultraestructura , Oocitos/crecimiento & desarrollo , Oocitos/ultraestructura , Poliadenilación/genética , Ratas , Transcriptoma/genéticaRESUMEN
Maternal starvation coincident with preimplantation development has profound consequences for placental-fetal development, with various identified pathologies persisting/manifest in adulthood; the 'Developmental Origin of Health and Disease' (DOHaD) hypothesis/model. Despite evidence describing DOHaD-related incidence, supporting mechanistic and molecular data relating to preimplantation embryos themselves are comparatively meager. We recently identified the classically recognized stress-related p38-mitogen activated kinases (p38-MAPK) as regulating formation of the extraembryonic primitive endoderm (PrE) lineage within mouse blastocyst inner cell mass (ICM). Thus, we wanted to assay if PrE differentiation is sensitive to amino acid availability, in a manner regulated by p38-MAPK. Although blastocysts appropriately mature, without developmental/morphological or cell fate defects, irrespective of amino acid supplementation status, we found the extent of p38-MAPK inhibition induced phenotypes was more severe in the absence of amino acid supplementation. Specifically, both PrE and epiblast (EPI) ICM progenitor populations remained unspecified and there were fewer cells and smaller blastocyst cavities. Such phenotypes could be ameliorated, to resemble those observed in groups supplemented with amino acids, by addition of the anti-oxidant NAC (N-acetyl-cysteine), although PrE differentiation deficits remained. Therefore, p38-MAPK performs a hitherto unrecognized homeostatic early developmental regulatory role (in addition to direct specification of PrE), by buffering blastocyst cell number and ICM cell lineage specification (relating to EPI) in response to amino acid availability, partly by counteracting induced oxidative stress; with clear implications for the DOHaD model.