Periconceptional alcohol exposure causes female-specific perturbations to trophoblast differentiation and placental formation in the rat.

The development of pathologies during pregnancy, including pre-eclampsia, hypertension and fetal growth restriction (FGR), often originates from poor functioning of the placenta. In vivo models of maternal stressors, such as nutrient deficiency, and placental insufficiency often focus on inadequate growth of the fetus and placenta in late gestation. These studies rarely investigate the origins of poor placental formation in early gestation, including those affecting the pre-implantation embryo and/or the uterine environment. The current study characterises the impact on blastocyst, uterine and placental outcomes in a rat model of periconceptional alcohol exposure, in which 12.5% ethanol is administered in a liquid diet from 4 days before until 4 days after conception. We show female-specific effects on trophoblast differentiation, embryo-uterine communication, and formation of the placental vasculature, resulting in markedly reduced placental volume at embryonic day 15. Both sexes exhibited reduced trophectoderm pluripotency and global hypermethylation, suggestive of inappropriate epigenetic reprogramming. Furthermore, evidence of reduced placental nutrient exchange and reduced pre-implantation maternal plasma choline levels offers significant mechanistic insight into the origins of FGR in this model.

Effects of periconceptional maternal alcohol intake and a postnatal high-fat diet on obesity and liver disease in male and female rat offspring.

The effects of maternal alcohol consumption around the time of conception on offspring are largely unknown and difficult to determine in a human population. This study utilized a rodent model to examine if periconceptional alcohol (PC:EtOH) consumption, alone or in combination with a postnatal high-fat diet (HFD), resulted in obesity and liver dysfunction. Sprague-Dawley rats were fed a control or an ethanol-containing [12.5% (vol/vol) EtOH] liquid diet from 4 days before mating until 4 days of gestation ( n = 12/group). A subset of offspring was fed a HFD between 3 and 8 mo of age. In males, PC:EtOH and HFD increased total body fat mass ( PPC:EtOH < 0.05, PHFD < 0.0001); in females, only HFD increased fat mass ( PHFD < 0.0001). PC:EtOH increased microvesicular liver steatosis in male, but not female, offspring. Plasma triglycerides, HDL, and cholesterol were increased in PC:EtOH-exposed males ( PPC:EtOH < 0.05), and LDL, cholesterol, and leptin (Lep) were increased in PC:EtOH-exposed females ( PPC:EtOH < 0.05). mRNA levels of Tnf-α and Lep in visceral adipose tissue were increased by PC:EtOH in both sexes ( PPC:EtOH < 0.05), and Il-6 mRNA was increased in males ( PPC:EtOH < 0.05). These findings were associated with reduced expression of microRNA-26a, a known regulator of IL-6 and TNF-α. Alcohol exposure around conception increases obesity risk, alters plasma lipid and leptin profiles, and induces liver steatosis in a sex-specific manner. These programmed phenotypes were similar to those caused by a postnatal HFD, particularly in male offspring. These results have implications for the health of offspring whose mothers consumed alcohol around the time of conception.

Placental O-GlcNAc-transferase expression and interactions with the glucocorticoid receptor are sex specific and regulated by maternal corticosterone exposure in mice.

Maternal stress programs offspring disease in a sexually dimorphic manner with males often more adversely affected. Previous studies of maternal glucocorticoid exposure suggest male vulnerability may derive from placental alterations. The hexosamine signalling pathway and O-linked glycosylation (O-GlcNAcylation) are part of an essential adaptive survival response in healthy cells. The key enzyme involved is O-linked-N-acetylglucosamine transferase (OGT), a gene recently identified as a sex-specific placental biomarker of maternal stress. Using a mouse model of maternal corticosterone (Cort) exposure, we examined components of hexosamine biosynthesis/signalling and O-GlcNAcylation in whole placentae at E14.5. Our results demonstrate sex-specific differences in OGT levels and O-GlcNAcylation during Cort exposure which impacts on key mediators of cell survival, in particular AKT as well as the stress responsive OGT/GR transrepression complex. In male placentae only, Cort exposure increased Akt O-GlcNacylation which correlated with decreased phosphorylation. Female placentae had higher basal OGT and OGT/GR complex compared with male placentae. Cort exposure did not alter these levels in female placentae but increased global O-GlcNacylation. In male placentae Cort increased OGT and OGT/GR complex with no change in global O-GlcNacylation. These findings suggest that sex-specific differences in placental OGT play a key role in the sexually dimorphic responses to stress.

The role of hexosamine biosynthesis and signaling in early development.

Although the culture requirements and the metabolic profile of the preimplantation embryo have been thoroughly investigated since their first successful culture in a defined medium, now more than 50 years ago (Whitten, Nature 177:96, 1956), it is only recently that we have begun to appreciate the impact of the environment on life-course trajectory. The mechanisms involved in how nutrient availability may potentially modulate developmental potential are consequently not well defined. Originally thought of as simple energy substrates and biosynthetic precursors, the currently emerging paradigm suggests that nutrients may act in non-classical roles to impact on developmental potential. This is now an area of considerable activity thanks to pioneering epidemiological studies (Barker et al., BMJ 298:564-7, 1989) that have led to the establishment of the Developmental Origins of Health and Disease (DoHAD) hypothesis and a whole new field of research activity. The period prior to implantation is of particular interest as this has been identified as a critical window of developmental sensitivity to environmental or nutrient stress (Fleming et al., Biol Reprod 71:1046-54, 2004a). This review seeks specifically to explore the pivotal role of glucose in early mouse development and the mechanisms by which it may impact on the cellular functions of the developing embryo. The emerging paradigm suggests that this humble hexose sugar may be at the heart of a rather sophisticated mechanism of cellular control that not only impacts on cellular proliferation and viability in the short term but on cellular memory through to the next generation.

Maternal alcohol intake around the time of conception causes glucose intolerance and insulin insensitivity in rat offspring, which is exacerbated by a postnatal high-fat diet.

Alcohol consumption throughout pregnancy can cause metabolic dysregulation, including glucose intolerance in progeny. This study determined if periconceptional (PC) alcohol (12% v/v in a liquid diet) (PC:EtOH) consumed exclusively around conception results in similar outcomes in Sprague-Dawley rats. Control (C) rats were given a liquid diet containing no alcohol but matched to ensure equal caloric intake. PC maternal alcohol intake (from 4 days before conception until day 4 of gestation), resulted in offspring with elevated fasting plasma glucose (∼10-25%, P < 0.05), impaired glucose tolerance (P < 0.05), and decreased insulin sensitivity (P < 0.01) at 6 months of age. This was associated with increased hepatic gluconeogenesis and sex-specific alterations in peripheral protein kinase B (AKT) signaling. These changes were accompanied by increased mRNA expression of DNA methyltransferases (DNMTs) 1, 3a, and 3b (1.5- to 1.9-fold, P < 0.05) in fetal liver in late gestation, suggesting PC:EtOH may cause epigenetic changes that predispose offspring to metabolic dysfunction. Exposure to a postnatal (PN) high-fat and cholesterol diet (HFD) from 3 months of age caused hyperinsulinemia (∼2-fold increase, P < 0.001) and exacerbated the metabolic dysfunction in male offspring exposed to PC:EtOH but had no additive effects in females. Given many women may drink alcohol while planning a pregnancy, it is crucial to increase public awareness regarding the effects of alcohol consumption around conception on offspring health.

Adverse prenatal environment and kidney development: implications for programing of adult disease.

The 'developmental origins of health and disease' hypothesis suggests that many adult-onset diseases can be attributed to altered growth and development during early life. Perturbations during gestation can be detrimental and lead to an increased risk of developing renal, cardiovascular, metabolic, and neurocognitive dysfunction in adulthood. The kidney has emerged as being especially vulnerable to insult at almost any stage of development resulting in a reduction in nephron endowment. In both humans and animal models, a reduction in nephron endowment is strongly associated with an increased risk of hypertension. The focus of this review is twofold: i) to determine the importance of specific periods during development on long-term programing and ii) to examine the effects of maternal perturbations on the developing kidney and how this may program adult-onset disease. Recent evidence has suggested that insults occurring around the time of conception also have the capacity to influence long-term health. Although epigenetic mechanisms are implicated in mediating these outcomes, it is unclear as to how these may impact on kidney development. This presents exciting new challenges and areas for research.

Lipid content, active mitochondria and brilliant cresyl blue staining in bovine oocytes.

Bovine oocytes that stain with brilliant cresyl blue (BCB) have a relatively higher developmental competence. The aim of the present study was to investigate the relationships among BCB staining, lipid content, and active mitochondria. Bovine oocytes (N = 133) with at least three layers of cumulus cells were segregated as BCB retained (BCB+) or metabolized (BCB-) and then stained for active mitochondria (Mitotracker Red) and lipid (Bodipy), with analysis by confocal microscopy. The BCB+ oocytes (N = 45) contained approximately 26% more cytoplasmic lipid than BCB- oocytes (N = 26-27; P < 0.05). Staining for active mitochondria did not differ between the groups. In BCB- oocytes but not BCB+ oocytes, lipid content correlated with active mitochondrial staining (r = 0.48; P < 0.05). Diameter correlated with lipid content for BCB+ oocytes (r = 0.46; P < 0.05), but not for BCB- oocytes (r = 0.16; P > 0.05). Irrespective of BCB staining, both lipid and active mitochondrial content correlated with diameter. In conclusion, the higher lipid content of BCB+ bovine oocytes might provide a cellular and functional basis for their greater developmental competence.

In vitro manipulation of mammalian preimplantation embryos can alter transcript abundance of histone variants and associated factors.

Manipulation of mammalian embryos and gametes in vitro reduces viability. Specific causes for these reductions are still largely undetermined. Accumulating evidence suggests that survival rates and developmental competency may be reduced following disruptions in the epigenetic regulation of gene expression. Chromatin-based epigenetics can regulate the transcriptome through the establishment of different transcriptionally permissive and repressive chromatin environments. Recently, support has been gathering for the hypothesis that the in vitro embryo displays reduced viability due to abnormal remodelling of the paternal chromatin, which is hypothesized to result in global transcriptional repression. In this study, we have used quantitative real-time PCR to document the effect of in vitro culture on the transcription of genes that code for proteins that are directly involved in the establishment of chromatin environments. We compare in vitro embryos to embryos generated through parthenogenetic activation to determine how the absence of paternal chromatin remodeling affects transcriptional activity. Through these studies, we show that the expression of many genes encoding for histone proteins and other modifiers involved in chromatin-based epigenetic regulation are perturbed by in vitro culture. In addition, we show that the expression of many candidate genes was reduced in in vitro embryos but not in parthenogenetic embryos. These results support the hypothesis that events linked to remodeling of paternal chromatin may influence transcriptional activity in the in vitro embryo and that chromatin-based reprogramming events in developing embryos are dynamically responsive to prevailing conditions.

Expression of genes coding for histone variants and histone-associated proteins in pluripotent stem cells and mouse preimplantation embryos.

The histone code is an epigenetic regulatory system thought to play a crucial role in cellular events such as development, differentiation and in the maintenance of pluripotency. In order to gain an insight into the role variant histones may play during mammalian development; we studied gene expression of histone variants and remodelling enzymes in mouse embryonic stem (ES) cells and during mouse preimplantation development. Using quantitative reverse-transcription PCR (qRT-PCR) we document the gene expression pattern of 12 histone variants and 2 of their associated remodelling enzymes in undifferentiated ES cells and during preimplantation embryo development. All histone variants were detected in undifferentiated ES cells, with H2AZ showing the highest expression levels of all the histone variants tested. The results also show that H2A variant levels tend to increase later in embryo development whilst H3 variant levels are elevated in early preimplantation stages. In addition, the expression of SWI/SNF, a remodeler protein involved in specifically remodelling H2A-H2B dimers, mirrors the expression of H2B and H2A variants, and the H3-H4 specific chaperone CAF-1 expression mirrors H3 variant expression. These results provide a foundation for further studies on the functions of histone variants during development, differentiation and in pluripotency.

Toxic effects of hyperglycemia are mediated by the hexosamine signaling pathway and o-linked glycosylation in early mouse embryos.

Maternal hyperglycemia is believed to be the metabolic derangement associated with both early pregnancy loss and congenital malformations in a diabetic pregnancy. Using an in vitro model of embryo exposure to hyperglycemia, this study questioned if increased flux through the hexosamine signaling pathway (HSP), which results in increased embryonic O-linked glycosylation (O-GlcNAcylation), underlies the glucotoxic effects of hyperglycemia during early embryogenesis. Mouse zygotes were randomly allocated to culture treatment groups that included no glucose (no flux through HSP), hyperglycemia (27 mM glucose, excess flux), 0.2 mM glucosamine (GlcN) in the absence of glucose (HSP flux alone), and O-GlcNAcylation levels monitored immunohistochemically. The impact of HSP manipulation on the first differentiation in development, blastocyst formation, was assessed, as were apoptosis and cell number in individual embryos. The enzymes regulating O-GlcNAcylation, and therefore hexosamine signaling, are the beta-linked-O-GlcNAc transferase (OGT) and an O-GlcNAc-selective beta-N-acetylglucosaminidase (O-GlcNAcase). Inhibition of these enzymes has a negative impact on blastocyst formation, demonstrating the importance of this signaling system to developmental potential. The ability of the OGT inhibitor benzyl-2-acetamido-2-deoxy-alpha-D-galactopyranoside (BADGP) to reverse the glucotoxic effects of hyperglycemia on these parameters was also sought. Excess HSP flux arising from a hyperglycemic environment or glucosamine supplementation reduced cell proliferation and blastocyst formation, confirming the criticality of this signaling pathway during early embryogenesis. Inhibition of OGT using BADGP blocked the negative impact of hyperglycemia on blastocyst formation, cell number, and apoptosis. Our results suggest that dysregulation of HSP and O-GlcNAcylation is the mechanism by which the embryotoxic effects of hyperglycemia are manifested during preimplantation development.

Glucose deprivation, oxidative stress and peroxisome proliferator-activated receptor-alpha (PPARA) cause peroxisome proliferation in preimplantation mouse embryos.

Ex vivo two-cell mouse embryos deprived of glucose in vitro can develop to blastocysts by increasing their pyruvate consumption; however, zygotes when glucose-deprived cannot adapt this metabolic profile and degenerate as morulae. Prior to their death, these glucose-deprived morulae exhibit upregulation of the H+-monocarboxylate co-transporter SLC16A7 and catalase, which partly co-localize in peroxisomes. SLC16A7 has been linked to redox shuttling for peroxisomal beta-oxidation. Peroxisomal function is unclear during preimplantation development, but as a peroxisomal transporter in embryos, SLC16A7 may be involved and influenced by peroxisome proliferators such as peroxisome proliferator-activated receptor-alpha (PPARA). PCR confirmed Ppara mRNA expression in mouse embryos. Zygotes were cultured with or without glucose and with the PPARA-selective agonist WY14643 and the developing embryos assessed for expression of PPARA and phospho-PPARA in relation to the upregulation of SLC16A7 and catalase driven by glucose deprivation, indicative of peroxisomal proliferation. Reactive oxygen species (ROS) production and relationship to PPARA expression were also analysed. In glucose-deprived zygotes, ROS was elevated within 2 h, as were PPARA expression within 8 h and catalase and SLC16A7 after 12-24 h compared with glucose-supplied embryos. Inhibition of ROS production prevented this induction of PPARA and SLC16A7. Selective PPARA agonism with WY14643 also induced SLC16A7 and catalase expression in the presence of glucose. These data suggest that glucose-deprived cleavage stage embryos, although supplied with sufficient monocarboxylate-derived energy, undergo oxidative stress and exhibit elevated ROS, which in turn upregulates PPARA, catalase and SLC16A7 in a classical peroxisomal proliferation response.

Characterization and regulation of monocarboxylate cotransporters Slc16a7 and Slc16a3 in preimplantation mouse embryos.

Concurrent with compaction, preimplantation mouse embryos switch from the high pyruvate consumption that prevailed during cleavage stages to glucose consumption against a constant background of pyruvate uptake. However, zygotes exposed to and subsequently deprived of glucose can form blastocysts by increasing pyruvate uptake. This metabolic switch requires cleavage-stage exposure to glucose and is one aspect of metabolic differentiation that normally occurs in vivo. Monocarboxylates, such as pyruvate and lactate, are transported across membranes via the SLC16 family of H(+)-monocarboxylate cotransporter (MCT) proteins. Thus, the increase in pyruvate uptake in embryos developing without glucose must involve changes in activity and localization of MCT. In mouse embryos, continued expression of Slc16a1 (MCT1) requires glucose supply. Messenger RNA for Slc17a7 (MCT2) and Slc16a3 (MCT4) has been detected in mouse preimplantation embryos; however, protein function, localization, and regulation of expression at the basis of these net pyruvate uptake changes remain unclear. The expression and localization of SLC16A7 and SLC16A3 have therefore been examined to clarify their respective roles in embryos derived from the reproductive tract and cultured under varied conditions. SLC16A3 appears localized to the plasma membrane until the morula stage and also maintains a nuclear distribution throughout preimplantation development. However, continued Slc16a3 mRNA expression is dependent on prior exposure to glucose. SLC16A7 localizes to apical cortical regions with punctate, vesicular expression throughout blastomeres, partially colocalizing in peroxisomes with peroxisomal catalase (CAT). In contrast to SLC16A3 and SLC16A1, SLC16A7 and CAT demonstrate upregulation in the absence of glucose. These striking differences between the two isoforms in expression localization and regulation suggest unique roles for each in monocarboxylate transport and pH regulation during preimplantation development, and implicate peroxisomal SLC16A7 as an important redox regulator in the absence of glucose.

Nutrient sensing by the early mouse embryo: hexosamine biosynthesis and glucose signaling during preimplantation development.

Although mouse oocytes and cleavage-stage embryos are unable to utilize glucose as a metabolic fuel, they have a specific requirement for a short exposure to glucose prior to compaction. The reason for this requirement has been unclear. In this study we confirm that cleavage-stage exposure to glucose is required for blastocyst formation and show that the absence of glucose between 18-64 h after hCG causes an irreversible decrease in cellular proliferation and an increase in apoptosis. More importantly, this glucose signals to activate expression of Slc2a3 transcript and SLC2A3 protein, a facilitative glucose transporter (previously known as GLUT3) associated with developmental competence and increased glucose uptake used to fuel blastocyst formation. Glucosamine could substitute for glucose in these roles, suggesting that hexosamine biosynthesis may be a nutrient-sensing mechanism involved in metabolic differentiation. Inhibition of the rate-limiting enzyme in this pathway, glutamine-fructose-6-phosphate amidotransferase (GFPT), inhibited expression of the SLC2A3 transporter protein and blastocyst formation. Glucosamine, a substrate that enters this pathway downstream of GFPT, was able to overcome this inhibition and support SLC2A3 expression. These data suggest that early embryos rely on hexosamine biosynthesis as a glucose-sensing pathway to initiate metabolic differentiation.

Glucose affects monocarboxylate cotransporter (MCT) 1 expression during mouse preimplantation development.

Cleavage-stage embryos have an absolute requirement for pyruvate and lactate, but as the morula compacts, it switches to glucose as the preferred energy source to fuel glycolysis. Substrates such as glucose, amino acids, and lactate are moved into and out of cells by facilitated diffusion. In the case of lactate and pyruvate, this occurs via H+-monocarboxylate cotransporter (MCT) proteins. To clarify the role of MCT in development, transport characteristics for DL-lactate were examined, as were mRNA expression and protein localisation for MCT1 and MCT3, using confocal laser scanning immunofluorescence in freshly collected and cultured embryos. Blastocysts demonstrated significantly higher affinity for DL-lactate than zygotes (Km 20 +/- 10 vs 87 +/- 35 mmol lactate/l; P = 0.03 by linear regression) but was similar for all stages. For embryos derived in vivo and those cultured with glucose, MCT1 mRNA was present throughout preimplantation development, protein immunoreactivity appearing diffuse throughout the cytoplasm with brightest intensity in the outer cortical region of blastomeres. In expanding blastocysts, MCT1 became more prominent in the cytoplasmic cortex of blastomeres, with brightest intensity in the polar trophectoderm. Without glucose, MCT1 mRNA was not expressed, and immunoreactivity dramatically reduced in intensity as morulae died. MCT3 mRNA and immunoreactivity were not detected in early embryos. The differential expression of MCT1 in the presence or absence of glucose demonstrates that it is important in the critical regulation of pH and monocarboxylate transport during preimplantation development, and implies a role for glucose in the control of MCT1, but not MCT3, expression.

Glucosamine supplementation during in vitro maturation inhibits subsequent embryo development: possible role of the hexosamine pathway as a regulator of developmental competence.

Glucose concentration during cumulus-oocyte complex (COC) maturation influences several functions, including progression of oocyte meiosis, oocyte developmental competence, and cumulus mucification. Glucosamine (GlcN) is an alternative hexose substrate, specifically metabolized through the hexosamine biosynthesis pathway, which provides the intermediates for extracellular matrix formation during cumulus cell mucification. The aim of this study was to determine the influence of GlcN on meiotic progression and oocyte developmental competence following in vitro maturation (IVM). The presence of GlcN during bovine IVM did not affect the completion of nuclear maturation and early cleavage, but severely perturbed blastocyst development. This effect was subsequently shown to be dose-dependent and was also observed for porcine oocytes matured in vitro. Hexosamine biosynthesis upregulation using GlcN supplementation is well known to increase O-linked glycosylation of many intracellular signaling molecules, the best-characterized being the phosphoinositol-3-kinase (PI3K) signaling pathway. We observed extensive O-linked glycosylation in bovine cumulus cells, but not oocytes, following IVM in either the presence or the absence of GlcN. Inhibition of O-linked glycosylation significantly reversed the effect of GlcN-induced reduction in developmental competence, but inhibition of PI3K signaling had no effect. Our data are the first to link hexosamine biosynthesis, involved in cumulus cell mucification, to oocyte developmental competence during in vitro maturation.

Insulin acts via mitogen-activated protein kinase phosphorylation in rabbit blastocysts.

The addition of insulin during in vitro culture has beneficial effects on rabbit preimplantation embryos leading to increased cell proliferation and reduced apoptosis. We have previously described the expression of the insulin receptor (IR) and the insulin-responsive glucose transporters (GLUT) 4 and 8 in rabbit preimplantation embryos. However, the effects of insulin on IR signaling and glucose metabolism have not been investigated in rabbit embryos. In the present study, the effects of 170 nM insulin on IR, GLUT4 and GLUT8 mRNA levels, Akt and Erk phosphorylation, GLUT4 translocation and methyl glucose transport were studied in cultured day 3 to day 6 rabbit embryos. Insulin stimulated phosphorylation of the mitogen-activated protein kinase (MAPK) Erk1/2 and levels of IR and GLUT4 mRNA, but not phosphorylation of the phosphatidylinositol 3-kinase-dependent protein kinase, Akt, GLUT8 mRNA levels, glucose uptake or GLUT4 translocation. Activation of the MAPK signaling pathway in the absence of GLUT4 translocation and of a glucose transport response suggest that in the rabbit preimplantation embryo insulin is acting as a growth factor rather than a component of glucose homeostatic control.

Identification of the facilitative glucose transporter 12 gene Glut12 in mouse preimplantation embryos.

In this study we report the cloning and characterisation of the mouse Glut12 gene and examine for the first time its expression pattern in the earliest stages of development. Mouse Glut12 (mGlut12) was cloned from preimplantation embryos by 5'RACE RT-PCR using primers designed from an EST clone corresponding to a human GLUT12 antigenic sequence after positive immunoreactivity was observed in mouse two-cell embryos by western immunoblotting. The mGlut12 gene contains an open reading frame of 1869 base pairs, potentially encoding a polypeptide of 622 amino acids. The predicted mGLUT12 protein bears all the hallmarks of the SLC2A family of hexose transporters and shares an 83% sequence homology to human GLUT12. Consistent with its human homolog mGlut12 mRNA is found highly expressed in skeletal and cardiac muscle and fat. Additionally, it was also found in the uterus and during early embryogenesis. During early development in the mouse, Glut12 expression is clearly apparent in ovulated oocytes and two-cell embryos but declines in day 3 morulae. With the exception of some Glut12 expression apparent in blastocysts, Glut12 mRNA remains at low to undetectable levels until E11.