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ABSTRACT: Previously, we have shown that a maternal low protein diet, fed exclusively during the preimplantation period of mouse development (Emb-LPD), is sufficient to induce by the blastocyst stage a compensatory growth phenotype in late gestation and postnatally, correlating with increased risk of adult onset cardiovascular disease and behavioural dysfunction. Here, we examine mechanisms of induction of maternal Emb-LPD programming and early compensatory responses by the embryo. Emb-LPD induced changes in maternal serum metabolites at the time of blastocyst formation (E3.5), notably reduced insulin and increased glucose, together with reduced levels of free amino acids (AAs) including branched chain AAs leucine, isoleucine and valine. Emb-LPD also caused reduction in the branched chain AAs within uterine fluid at the blastocyst stage. These maternal changes coincided with an altered content of blastocyst AAs and reduced mTORC1 signalling within blastocysts evident in reduced phosphorylation of effector S6 ribosomal protein and its ratio to total S6 protein but no change in effector 4E-BP1 phosphorylated and total pools. These changes were accompanied by increased proliferation of blastocyst trophectoderm and total cells and subsequent increased spreading of trophoblast cells in blastocyst outgrowths. We propose that induction of metabolic programming following Emb-LPD is achieved through mTORC1signalling which acts as a sensor for preimplantation embryos to detect maternal nutrient levels via branched chain AAs and/or insulin availability. Moreover, this induction step associates with changes in extra-embryonic trophectoderm behaviour occurring as early compensatory responses leading to later nutrient recovery.
PLoS ONE 01/2012; 7(12):e52791. · 4.09 Impact Factor
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ABSTRACT: Maternal periconceptional (PC) nutrition, coupled with maternal physiological condition, can impact on reproductive performance and potential across mammalian species. Oocyte quality and embryo development are affected adversely by either nutrient restriction or excess. Moreover, the quality of maternal PC nutrition can have lasting effects through fetal development and postnatally into adulthood. Chronic disease, notably cardiovascular and metabolic disease, and abnormal behaviour have been identified in adult offspring in small and large animal models of PC nutrient restriction. These long-term effects associate with compensatory responses that begin from the time of early embryo development. This review assesses the field of PC nutrition in vivo on short- and long-term developmental consequences in rodent and ruminant models and considers the implications for human health.
Reproduction Fertility and Development 12/2011; 24(1):35-44. · 2.11 Impact Factor
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ABSTRACT: Environmental perturbations during early mammalian development can affect aspects of offspring growth and cardiovascular health. We have demonstrated previously that maternal gestational dietary protein restriction in mice significantly elevated adult offspring systolic blood pressure. Therefore, the present study investigates the key mechanisms of blood pressure regulation in these mice. Following mating, female MF-1 mice were assigned to either a normal-protein diet (NPD; 18 % casein) or an isocaloric low-protein diet throughout gestation (LPD; 9 % casein), or fed the LPD exclusively during the pre-implantation period (3.5 d) before returning to the NPD for the remainder of gestation (Emb-LPD). All offspring received standard chow. At 22 weeks, isolated mesenteric arteries from LPD and Emb-LPD males displayed significantly attenuated vasodilatation to isoprenaline (P = 0.04 and P = 0.025, respectively), when compared with NPD arteries. At 28 weeks, stereological analysis of glomerular number in female left kidneys revealed no significant difference between the groups. Real-time RT-PCR analysis of type 1a angiotensin II receptor, Na+/K+ ATPase transporter subunits and glucocorticoid receptor expression in male and female left kidneys revealed no significant differences between the groups. LPD females displayed elevated serum angiotensin-converting enzyme (ACE) activity (P = 0.044), whilst Emb-LPD males had elevated lung ACE activity (P = 0.001), when compared with NPD offspring. These data demonstrate that elevated offspring systolic blood pressure following maternal gestational protein undernutrition is associated with impaired arterial vasodilatation in male offspring, elevated serum and lung ACE activity in female and male offspring, respectively, but kidney glomerular number in females and kidney gene expression in male and female offspring appear unaffected.
The British journal of nutrition 06/2010; 103(12):1762-70. · 3.45 Impact Factor
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ABSTRACT: The negative impact of obesity on reproductive success is well documented but the stages at which development of the conceptus is compromised and the mechanisms responsible for the developmental failure still remain unclear. Recent findings suggest that mitochondria may be a contributing factor. However to date no studies have directly addressed the consequences of maternal obesity on mitochondria in early embryogenesis.Using an established murine model of maternal diet induced obesity and a live cell dynamic fluorescence imaging techniques coupled with molecular biology we have investigated the underlying mechanisms of obesity-induced reduced fertility. Our study is the first to show that maternal obesity prior to conception is associated with altered mitochondria in mouse oocytes and zygotes. Specifically, maternal diet-induced obesity in mice led to an increase in mitochondrial potential, mitochondrial DNA content and biogenesis. Generation of reactive oxygen species (ROS) was raised while glutathione was depleted and the redox state became more oxidised, suggestive of oxidative stress. These altered mitochondrial properties were associated with significant developmental impairment as shown by the increased number of obese mothers who failed to support blastocyst formation compared to lean dams. We propose that compromised oocyte and early embryo mitochondrial metabolism, resulting from excessive nutrient exposure prior to and during conception, may underlie poor reproductive outcomes frequently reported in obese women.
PLoS ONE 01/2010; 5(4):e10074. · 4.09 Impact Factor
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ABSTRACT: NANOG, POU5F1, and SOX2 are required by the inner cell mass of the blastocyst and act cooperatively to maintain pluripotency in both mouse and human embryonic stem cells. Inadequacy of any one of them causes loss of the undifferentiated state. Mouse primordial germ cells (PGCs), from which pluripotent embryonic germ cells (EGCs) are derived, also express POU5F1, NANOG, and SOX2. Thus, a similar expression profile has been predicted for human PGCs. Here we show by RT-PCR, immunoblotting, and immunohistochemistry that human PGCs express POU5F1 and NANOG but not SOX2, with no evidence of redundancy within the group B family of human SOX genes. Although lacking SOX2, proliferative human germ cells can still be identified in situ during early development and are capable of culture in vitro. Surprisingly, with the exception of FGF4, many stem cell-restricted SOX2 target genes remained detected within the human SOX2-negative germ cell lineage. These studies demonstrate an unexpected difference in gene expression between human and mouse. The human PGC is the first primary cell type described to express POU5F1 and NANOG but not SOX2. The data also provide a new reference point for studies attempting to turn human stem cells into gametes by normal developmental pathways for the treatment of infertility.
Biology of Reproduction 06/2008; 78(5):852-8. · 4.01 Impact Factor
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ABSTRACT: The tight junction (TJ) is an essential component of the differentiated epithelial cell required for polarised transport and intercellular integrity and signalling. Whilst much can be learnt about how the TJ is constructed and maintained and how it functions using a wide range of cellular systems, the mechanisms of TJ biogenesis within developmental models must be studied to gain insight into this process as an integral part of epithelial differentiation. Here, we review TJ biogenesis in the early mammalian embryo, mainly considering the mouse but also including the human and other species, and, briefly, within the amphibian embryo. We relate TJ biogenesis to inherent mechanisms of cell differentiation and biosynthesis occurring during cleavage of the egg and the formation of the first epithelium. We also evaluate a wide range of exogenous cues, including cell-cell interactions, protein kinase C signalling, gap junctional communication, Na+/K+-ATPase and cellular energy status, that may contribute to TJ biogenesis in the embryo and how these may shape the pattern of early morphogenesis.
Biochimica et Biophysica Acta 04/2008; 1778(3):717-28. · 4.66 Impact Factor
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Adam J Watkins,
Adrian Wilkins,
Colm Cunningham,
V Hugh Perry,
Meei J Seet,
Clive Osmond, Judith J Eckert,
Christopher Torrens,
Felino R A Cagampang,
Jane Cleal,
William P Gray,
Mark A Hanson,
Tom P Fleming
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ABSTRACT: Early embryonic development is known to be susceptible to maternal undernutrition, leading to a disease-related postnatal phenotype. To determine whether this sensitivity extended into oocyte development, we examined the effect of maternal normal protein diet (18% casein; NPD) or isocaloric low protein diet (9% casein; LPD) restricted to one ovulatory cycle (3.5 days) prior to natural mating in female MF-1 mice. After mating, all females received NPD for the remainder of gestation and all offspring were litter size adjusted and fed standard chow. No difference in gestation length, litter size, sex ratio or postnatal growth was observed between treatments. Maternal LPD did, however, induce abnormal anxiety-related behaviour in open field activities in male and female offspring (P < 0.05). Maternal LPD offspring also exhibited elevated systolic blood pressure (SBP) in males at 9 and 15 weeks and in both sexes at 21 weeks (P < 0.05). Male LPD offspring hypertension was accompanied by attenuated arterial responsiveness in vitro to vasodilators acetylcholine and isoprenaline (P < 0.05). LPD female offspring adult kidneys were also smaller, but had increased nephron numbers (P < 0.05). Moreover, the relationship between SBP and kidney or heart size or nephron number was altered by diet treatment (P < 0.05). These data demonstrate the sensitivity of mouse maturing oocytes in vivo to maternal protein undernutrition and identify both behavioural and cardiovascular postnatal outcomes, indicative of adult disease. These outcomes probably derive from a direct effect of protein restriction, although indirect stress mechanisms may also be contributory. Similar and distinct postnatal outcomes were observed here compared with maternal LPD treatment during post-fertilization preimplantation development which may reflect the relative contribution of the paternal genome.
The Journal of Physiology 04/2008; 586(8):2231-44. · 4.72 Impact Factor
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Adam J Watkins,
Elizabeth Ursell,
Rose Panton,
Thomas Papenbrock,
Lisa Hollis,
Colm Cunningham,
Adrian Wilkins,
V Hugh Perry,
Bhavwanti Sheth,
Wing Yee Kwong, Judith J Eckert,
Arthur E Wild,
Mark A Hanson,
Clive Osmond,
Tom P Fleming
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ABSTRACT: Poor maternal nutrition during pregnancy can alter postnatal phenotype and increase susceptibility to adult cardiovascular and metabolic diseases. However, underlying mechanisms are largely unknown. Here, we show that maternal low protein diet (LPD), fed exclusively during mouse preimplantation development, leads to offspring with increased weight from birth, sustained hypertension, and abnormal anxiety-related behavior, especially in females. These adverse outcomes were interrelated with increased perinatal weight being predictive of later adult overweight and hypertension. Embryo transfer experiments revealed that the increase in perinatal weight was induced within blastocysts responding to preimplantation LPD, independent of subsequent maternal environment during later pregnancy. We further identified the embryo-derived visceral yolk sac endoderm (VYSE) as one mediator of this response. VYSE contributes to fetal growth through endocytosis of maternal proteins, mainly via the multiligand megalin (LRP2) receptor and supply of liberated amino acids. Thus, LPD maintained throughout gestation stimulated VYSE nutrient transport capacity and megalin expression in late pregnancy, with enhanced megalin expression evident even when LPD was limited to the preimplantation period. Our results demonstrate that in a nutrient-restricted environment, the preimplantation embryo activates physiological mechanisms of developmental plasticity to stablize conceptus growth and enhance postnatal fitness. However, activation of such responses may also lead to adult excess growth and cardiovascular and behavioral diseases.
Biology of Reproduction 03/2008; 78(2):299-306. · 4.01 Impact Factor
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ABSTRACT: Blastocyst biogenesis occurs over several cell cycles during the preimplantation period comprising the gradual expression and membrane assembly of junctional protein complexes which distinguish the outer epithelial trophectoderm (TE) cells from the inner cell mass (ICM). In the human, TE integrity and the formation of a junctional seal can often be impaired. Embryos likely to result in a successful pregnancy after transfer are mostly selected according to morphological criteria. Recent data suggest that non-invasive measurement of amino acid turnover may be useful to complement such morphological scores. Whether morphological and metabolic criteria can be linked to poor TE differentiation thereby underpinning developmental predictions mechanistically remains unknown.
We examined TE intercellular junction formation in human embryos by immunofluorescence and confocal microscopy and correlated this process with morphological criteria and amino acid turnover during late cleavage.
Our results show that TE differentiation may be compromised by failure of membrane assembly of specific junction constituents. This abnormality relates more closely to metabolic profiles than morphological criteria.
Our data identify that amino acid turnover can predict TE differentiation. These findings are the first to link two mechanisms, metabolism and junction membrane assembly, which contribute to early embryo development.
Human Reproduction 09/2007; 22(8):2214-24. · 4.47 Impact Factor
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ABSTRACT: A key factor in the use of assisted reproductive technologies (ART) for diverse species is the safety of procedures for long-term health. By using a mouse model, we have investigated the effect of in vitro culture and embryo transfer (ET) of superovulated embryos on postnatal growth and physiological activity compared with that of embryos developing in vivo. Embryo culture from two-cell to blastocyst stages in T6 medium either with or without a protein source reduced blastocyst trophectoderm and inner cell mass cell number compared with that of embryos developing in vivo. Embryo culture and ET had minimal effects on postnatal growth when compared with in vivo development with an equivalent litter size. However, embryo culture, and to a lesser extent ET, led to an enhanced systolic blood pressure at 21 weeks compared with in vivo development independent of litter size, maternal origin, or body weight. Moreover, activity of enzymatic regulators of cardiovascular and metabolic physiology, namely, serum angiotensin-converting enzyme and the gluconeogenesis controller, hepatic phosphoenolpyruvate carboxykinase, were significantly elevated in response to embryo culture and/or ET in female offspring at 27 weeks, independent of maternal factors and postnatal growth. These animal data indicate that postnatal physiological criteria important in cardiovascular and metabolic health may be more sensitive to routine ART procedures than growth.
Proceedings of the National Academy of Sciences 04/2007; 104(13):5449-54. · 9.68 Impact Factor
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ABSTRACT: In mouse early development, cell contact patterns regulate the spatial organization and segregation of inner cell mass (ICM) and trophectoderm epithelium (TE) during blastocyst morphogenesis. Progressive membrane assembly of tight junctional (TJ) proteins in the differentiating TE during cleavage is upregulated by cell contact asymmetry (outside position) and suppressed within the ICM by cell contact symmetry (inside position). This is reversible, and immunosurgical isolation of the ICM induces upregulation of TJ assembly in a sequence that broadly mimics that occurring during blastocyst formation. The mechanism relating cell contact pattern and TJ assembly was investigated in the ICM model with respect to PKC-mediated signaling and gap junctional communication. Our results indicate that complete cell contact asymmetry is required for TJ biogenesis and acts upstream of PKC-mediated signaling. Specific inhibition of two PKC isoforms, PKCdelta and zeta, revealed that both PKC activities are required for membrane assembly of ZO-2 TJ protein, while only PKCzeta activity is involved in regulating ZO-1alpha+ membrane assembly, suggesting different mechanisms for individual TJ proteins. Gap junctional communication had no apparent influence on either TJ formation or PKC signaling but was itself affected by changes of cell contact patterns. Our data suggest that the dynamics of cell contact patterns coordinate the spatial organization of TJ formation via specific PKC signaling pathways during blastocyst biogenesis.
Developmental Biology 01/2006; 288(1):234-47. · 4.07 Impact Factor
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ABSTRACT: We have investigated the contribution of the tight junction (TJ) transmembrane protein junction-adhesion-molecule 1 (JAM-1) to trophectoderm epithelial differentiation in the mouse embryo. JAM-1-encoding mRNA is expressed early from the embryonic genome and is detectable as protein from the eight-cell stage. Immunofluorescence confocal analysis of staged embryos and synchronized cell clusters revealed JAM-1 recruitment to cell contact sites occurred predominantly during the first hour after division to the eight-cell stage, earlier than any other TJ protein analysed to date in this model and before E-cadherin adhesion and cell polarization. During embryo compaction later in the fourth cell cycle, JAM-1 localized transiently yet precisely to the apical microvillous pole, where protein kinase Czeta (PKCzeta) and PKCdelta are also found, indicating a role in cell surface reorganization and polarization. Subsequently, in morulae and blastocysts, JAM-1 is distributed ubiquitously at cell contact sites within the embryo but is concentrated within the trophectoderm apicolateral junctional complex, a pattern resembling that of E-cadherin and nectin-2. However, treatment of embryos with anti-JAM-1-neutralizing antibodies indicated that JAM-1 did not contribute to global embryo compaction and adhesion but rather regulated the timing of blastocoel cavity formation dependent upon establishment of the trophectoderm TJ paracellular seal.
Journal of Cell Science 12/2004; 117(Pt 23):5599-608. · 6.11 Impact Factor
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ABSTRACT: The preimplantation mammalian embryo from different species appears sensitive to the environment in which it develops, either in vitro or in vivo, for example, in response to culture conditions or maternal diet. This sensitivity may lead to long-term alterations in the characteristics of fetal and/or postnatal growth and phenotype, which have implications for clinical health and biotechnological applications. We review the breadth of environmental influences that may affect early embryos and their responses to such conditions along epigenetic, metabolic, cellular, and physiological directions. In addition, we evaluate how embryo environmental responses may influence developmental potential and phenotype during later gestation. We conclude that a complex of different mechanisms may operate to associate early embryo environment with future health.
Biology of Reproduction 11/2004; 71(4):1046-54. · 4.01 Impact Factor
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ABSTRACT: During early mammalian development, blastocyst morphogenesis is achieved by epithelial differentiation of trophectoderm (TE) and its segregation from the inner cell mass (ICM). Two major interrelated features of TE differentiation required for blastocoel formation include intercellular junction biogenesis and a directed ion transport system, mediated by Na+/K+ ATPase. We have examined the relative contribution of intercellular signalling mediated by protein kinase C (PKC) and gap junctional communication in TE differentiation and blastocyst cavitation. The distribution pattern of four (delta, theta, iota/lambda, zeta) PKC isoforms and PKCmicro/PKD1 showed partial colocalisation with the tight junction marker ZO-1alpha+ in TE and all four PKCs (delta, theta, iota/lambda, zeta) showed distinct TE/ICM staining patterns (predominantly at the cell membrane within the TE and cytoplasmic within the ICM), indicating their potential contribution to TE differentiation and blastocyst morphogenesis. Specific inhibition of PKCdelta and zeta activity significantly delayed blastocyst formation. Although modulation of these PKC isoforms failed to influence the already established programme of epithelial junctional differentiation within the TE, Na+/K+ ATPase alpha1 subunit was internalised from membrane to cytoplasm. Inhibition of gap junctional communication, in contrast, had no influence on any of these processes. Our results demonstrate for the first time that distinct PKC isotypes contribute to the regulation of cavitation in preimplantation embryos via target proteins including Na+/K+ ATPase.
Developmental Biology 11/2004; 274(2):384-401. · 4.07 Impact Factor
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ABSTRACT: Epithelial differentiation including tight junction (TJ) formation occurs exclusively within the trophectoderm (TE) lineage of the mouse blastocyst. Here we examine mechanisms by which TJ protein membrane assembly might be regulated by protein kinase C (PKC) in the embryo. To overcome the inherent staging asynchrony of individual blastomeres within intact embryos, we have used isolated inner cell masses (ICMs) from early blastocysts to induce epithelial differentiation in their outer cells responding to their new cell contact pattern. Two TJ proteins examined retain their order of membrane assembly in isolated ICMs in culture as during normal development (early-assembling ZO-2 and late-assembling ZO-1alpha(+)), but this process is highly accelerated. Using six chemical modulators of PKC activity, we show here that PKC signalling is involved in the regulation of TJ membrane assembly. While indolactam-mediated PKC activation stimulates membrane assembly of both TJ proteins, TPA-mediated PKC activation stimulates only that of ZO-1alpha(+). The PKC inhibitors Ro-31-8220, Ro-31-8425 and Gö 6983 suppress the stimulatory effect of both PKC activators on membrane assembly to varying extents according to inhibitor and TJ protein examined. Gö 6983 similarly inhibits ZO-2 and ZO-1alpha(+) membrane assembly. PKC inhibition by Gö 6976 appeared to stimulate TJ membrane assembly. Despite the broad PKC isotype specificity of the inhibitors used, these data suggest that the two TJ proteins are differently regulated by PKC isotypes or subfamilies. As Gö 6983 uniquely affects aPKC (particularly PKCzeta) and we find that both PKCdelta and zeta relocate upon activator treatment to colocalise partially with the TJ proteins in isolated ICMs, we suggest that at least PKCdelta and zeta may play a central role in regulating TJ membrane assembly.
Reproduction (Cambridge, England) 07/2004; 127(6):653-67. · 3.09 Impact Factor
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ABSTRACT: During early development, the eutherian mammalian embryo forms a blastocyst comprising an outer trophectoderm epithelium and enclosed inner cell mass (ICM). The short-term goal of blastocyst morphogenesis, including epithelial differentiation and segregation of the ICM, is mainly regulated autonomously and comprises a combination of temporally controlled gene expression, cell polarisation, differentiative cell divisions and cell-cell interactions. This aspect of blastocyst biogenesis is reviewed, focusing, in particular, on the maturation and role of cell adhesion systems. Early embryos are also sensitive to their environment, which can affect their developmental potential in diverse ways and may lead to long-term consequences relating to fetal or postnatal growth and physiology. Some current concepts of embryo-environment interactions, which may impact on future health, are also reviewed.
Reproduction Fertility and Development 02/2004; 16(3):325-37. · 2.11 Impact Factor
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ABSTRACT: We investigated gene expression associated with trophectoderm epithelial intercellular junction formation in single human embryos at different stages of cleavage using RT-PCR methods based upon magnetic bead separation of polyA+ RNA. Trophectoderm tight junction (TJ) and desmosome biogenesis contribute to intercellular sealing and tissue integrity, critical for vectorial transport and blastocoel cavity formation. Expression of the various genes throughout human preimplantation development showed differing levels of sensitivity of detection; these genes included claudin-1, occludin (TM4+ and TM4 isoforms), ZO-1 (ZO-1alpha+ and ZO-1alpha- isoforms), ZO-2 and JAM (junction adhesion molecule), and the desmosome junction gene, DSC2 (desmocollin 2). Some transcripts appeared to be expressed throughout preimplantation development (claudin-1, JAM, occludin TM4+ and TM4, ZO-1alpha- isoform) while others tended to be expressed preferentially in later cleavage and associated with blastocyst formation (ZO-2, ZO-1alpha+ isoform, DSC-2), illustrating an expression pattern broadly similar to mouse cleavage stages. Human embryo transcript detection was significantly decreased when reverse transcription was performed in solid phase to generate a bead/cDNA transient library rather than after mRNA elution from beads. Transcript detection tended to be positively correlated with embryo morphological grade using the solid phase method. In blastocysts, occludin TM4-, ZO-1alpha+ and DSC2 transcripts were the most susceptible to failure of detection, indicative of low levels of expression which may impact on trophectoderm differentiation competence. Immunoconfocal microscopy analysis of selected adhesion and TJ proteins in human embryos indicated poor membrane assembly compared with mouse blastocysts, which may further affect embryo viability.
Molecular Human Reproduction 06/2003; 9(5):245-52. · 3.85 Impact Factor
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ABSTRACT: We have established a transcription map of individual bovine embryos using semiquantitative reverse transcriptase-polymerase chain reaction to detect the levels of six marker genes involved in early embryo differentiation. The critical step of compaction during preimplantation development is often not accomplished or it takes place for only a short period in in vitro generated embryos, which may result in reduced viability. Compaction is accompanied by the assembly of intercellular tight junctions (TJs) as a barrier against the extraembryonic environment and as a prerequisite for blastocele formation. In the present study, we have related the expression of TJ gene mRNA in individual bovine embryos to their developmental stage, their competence to undergo a clear period of compaction before blastocyst formation, and their in vitro or in vivo origin. Our results indicate that embryos that showed a detectable and well-formed compaction period in vitro are of similar quality to their in vivo counterparts. Starting from the same amount of maternal message, in vivo and in vitro development differ most during the critical period of the major switch from maternal to embryonic genomic control before a dramatic increase of TJ mRNAs occurs upon blastocyst formation. Failure to compact in vitro results in significant reduction of specific transcript levels, in a manner that depends on culture conditions, which may contribute to reduced viability. We conclude that TJ mRNA expression levels are sensitive to environmental conditions that may influence the developmental potential of bovine blastocysts.
Biology of Reproduction 05/2003; 68(4):1394-402. · 4.01 Impact Factor
