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The first differentiative event in mammalian development is segregation of the inner cell mass and trophectoderm (TE) lineages. The epithelial TE cells pump f luid into the spherical blastocyst to form the blastocyst cavity. This activity is fuelled by glucose supplied through facilitative glucose transporters. However, the reported kinetic characteristics of blastocyst glucose transport are inconsistent with those of the previously identified transporters and suggested the presence of a high-affinity glucose carrier. We identified and localized the primary transporter in TE cells. It is glucose transporter GLUT3, previously described in the mouse as neuron-specific. In the differentiating embryo, GLUT3 is targeted to the apical membranes of the polarized cells of the compacted morula and then to the apical membranes of TE cells where it has access to maternal glucose. In contrast, GLUT1 was restricted to basolateral membranes of the outer TE cells in both compacted morulae and blastocysts. Using antisense oligodeoxynucleotides to specifically block protein expression, we confirmed that GLUT3 and not GLUT1 is the functional transporter for maternal glucose on the apical TE. More importantly, however, GLUT3 expression is required for blastocyst formation and hence this primary differentiation in mammalian development. This requirement is independent of its function as a glucose transporter because blastocysts will form in the absence of glucose. Thus the vectorial expression of GLUT3 into the apical membrane domains of the outer cells of the morula, which in turn form the TE cells of the blastocyst, is required for blastocyst formation.

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.

The results of this study challenge the widely held view that growth hormone (GH) acts only during the postnatal period. RNA phenotyping shows transcripts for the GH receptor and GH-binding protein in mouse preimplantation embryos of all stages from fertilized eggs (day 1) to blastocysts (day 4). An antibody specific to the cytoplasmic region of the GH receptor revealed receptor protein expression, first in two-cell embryos, the stage of activation of the embryonic genome (day 2), and in all subsequent stages. In cleavage-stage embryos this immunoreactivity was localized mainly to the nucleus, but clear evidence of membrane labeling was apparent in blastocysts. GH receptor immunoreactivity was also observed in cumulus cells associated with unfertilized oocytes but not in the unfertilized oocytes. The blastocyst receptor was demonstrated to be functional, exhibiting the classic bell-shaped dose-response curves for GH stimulation of both 3-O-methyl glucose transport and protein synthesis. Maximal stimulation of 40-50% was seen for both responses at less than 1 ng͞ml recombinant GH, suggesting a role for maternal GH. However mRNA transcripts for GH were also detected from the morula stage (day 3) by using reverse transcription-PCR, and GH immunoreactivity was seen in blastocysts. These observations raise the possibility of a paracrine͞autocrine GH loop regulating embryonic development in its earliest stages.Embryonic and fetal growth have long been considered to be independent of pituitary growth hormone (GH). However, this view is challenged by a growing body of evidence which demonstrates a role for GH in the development of the fetus. Newborn Laron dwarfs, lacking a functional GH receptor, are more than 2 SD shorter than normal (1). Exogenous GH has been shown to restore embryonic growth in rats after transplantation of parts of embryos into hypophysectomized hosts (2). A number of fetal tissues has been shown to respond to GH in vitro (3-6). GH receptor transcripts have been demonstrated in day 12 rat embryos and placentae (7), day 51 sheep embryos (8), and mouse placenta (9). Immunoreactive GH receptor was observed in the human fetus from the second trimester (10, 11). Recently GH receptor transcripts and immunoreactivity have been demonstrated in germ-line competent mouse embryonic stem cells, and GH receptor transcripts were also demonstrated in mouse blastocysts (12).Preimplantation stages earlier than the blastocyst, however, were not examined by Ohlsson et al. (12). Furthermore, there was no evidence that these very early embryos were capable of receptor synthesis or of signal transduction after ligand binding to expressed receptor. In this study we demonstrate the presence of GH receptors in the early embryo from fertilization to the blastocyst stage and the ability of GH to influence the metabolism of blastocyst. Moreover, we report the expression of GH by preimplantation blastocysts, raising the possibility of paracrine͞autocrine regulation of embryonic growth by GH. MATERIALS AND METHODSGene Ex...

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.

Oxygen concentrations used during in vitro embryo culture can influence embryo development, cell numbers, and gene expression. Here we propose that the preimplantation bovine embryo possesses a molecular mechanism for the detection of, and response to, oxygen, mediated by a family of basic helix-loop-helix transcription factors, the hypoxia-inducible factors (HIFs). Day 5 compacting bovine embryos were cultured under different oxygen tensions (2%, 7%, 20%) and the effect on the expression of oxygen-regulated genes, development, and cell number allocation and HIFalpha protein localization were examined. Bovine in vitro-produced embryos responded to variations in oxygen concentration by altering gene expression. GLUT1 expression was higher following 2% oxygen culture compared with 7% and 20% cultured blastocysts. HIF mRNA expression (HIF1alpha, HIF2alpha) was unaltered by oxygen concentration. HIF2alpha protein was predominantly localized to the nucleus of blastocysts. In contrast, HIF1alpha protein was undetectable at any oxygen concentration or in the presence of the HIF protein stabilizer desferrioxamine (DFO), despite being detectable in cumulus cells following normal maturation conditions, acute anoxic culture, or in the presence of DFO. Oxygen concentration also significantly altered inner cell mass cell proportions at the blastocyst stage. These results suggest that oxygen can influence gene expression in the bovine embryo during postcompaction development and that these effects may be mediated by HIF2alpha.

FAM is a developmentally regulated substrate-specific deubiquitylating enzyme. It binds the cell adhesion and signalling molecules beta-catenin and AF-6 in vitro, and stabilises both in mammalian cell culture. To determine if FAM is required at the earliest stages of mouse development we examined its expression and function in preimplantation mouse embryos. FAM is expressed at all stages of preimplantation development from ovulation to implantation. Exposure of two-cell embryos to FAM-specific antisense, but not sense, oligodeoxynucleotides resulted in depletion of the FAM protein and failure of the embryos to develop to blastocysts. Loss of FAM had two physiological effects, namely, a decrease in cleavage rate and an inhibition of cell adhesive events. Depletion of FAM protein was mirrored by a loss of beta-catenin such that very little of either protein remained following 72h culture. The residual beta-catenin was localised to sites of cell-cell contact suggesting that the cytoplasmic pool of beta-catenin is stabilised by FAM. Although AF-6 levels initially decreased they returned to normal. However, the nascent protein was mislocalised at the apical surface of blastomeres. Therefore FAM is required for preimplantation mouse embryo development and regulates beta-catenin and AF-6 in vivo.

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