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During the first days of development the preimplantation embryo is supplied with nutrients from the surrounding milieu. Maternal diabetes mellitus affects the uterine microenvironment, leading to a metabolic adaptation processes in the embryo. We analysed embryonic fatty acid (FA) profiles and expression of processing genes in rabbit blastocysts, separately in embryoblasts (EBs) and trophoblasts (TBs), to determine the potential consequences of maternal diabetes mellitus on intracellular FA metabolism. Insulin-dependent diabetes was induced by alloxan in female rabbits. On Day 6 post coitum, FA profiles in blastocysts (EB, TB and blastocoel fluid) and maternal blood were analysed by gas chromatography. The expression levels of molecules involved in FA elongation (fatty acid elongases, ELOVLs) and desaturation (fatty acid desaturases, FADSs) were measured in EB and TB. Maternal diabetes mellitus influenced the FA profile in maternal plasma and blastocysts. Independent from metabolic changes, rabbit blastocysts contained a higher level of saturated fatty acids (SFAs) and a lower level of polyunsaturated fatty acids (PUFAs) compared to the FA profile of the maternal plasma. Furthermore, the FA profile was altered in the EB and TB, differently. While SFAs (palmitic and stearic acid) were elevated in EB of diabetic rabbits, PUFAs, such as docosahexaenoic acid, were decreased. In contrast, in the TB, lower levels of SFAs and higher levels of oleic acid were observed. EB and TB specific alterations in gene expression were found for ELOVLs and FADSs, key enzymes for FA elongation and desaturation. In conclusion, maternal diabetes mellitus alters embryonic FA metabolism differently in EB and TB, indicating a lineage-specific metabolic adaptive response.

The neuroblastoma cell line SH-SY5Y is commonly employed to study neuronal function and disease. This includes cells grown under standard conditions or differentiated to neuron-like cells by administration of chemical reagents such as retinoic acid (RA) or phorbol-12-myristate-13-acetate (PMA). Even though SH-SY5Y cells are widely explored, a complete description of the resulting proteomes and cellular reorganisation during differentiation is still missing. Here, we relatively quantify the proteomes of cells grown under standard conditions and obtained from two differentiation protocols employing RA or a combination of RA and PMA. Relative quantification and KEGG pathway analysis of the proteins reveals the presence of early differentiating cells and provides a list of marker proteins for undifferentiated and differentiated cells. For characterisation of neuronal sub-types, we analyse expression of marker genes and find that RA-differentiated cells are acetylcholinergic and cholinergic, while RA/PMA-differentiated cells show high expression of acetylcholinergic and dopaminergic marker genes. In-cell cross-linking further allows capturing protein interactions in different cellular organelles. Specifically, we observe structural reorganisation upon differentiation involving regulating protein factors of the actin cytoskeleton.

In a diabetic pregnancy, an altered maternal metabolism led to increased formation of reactive α-dicarbonyls such as glyoxal (GO) and methylglyoxal (MGO) in the reproductive organs and embryos. The enzyme glyoxalase (GLO) 1 detoxifies reactive α-dicarbonyls thus protecting cells against malfunction or modifications of proteins by advanced glycated end products (AGEs). The aim of this study was to analyse the influence of a maternal insulin-dependent diabetes mellitus (IDD) on GLO1 expression and activity in preimplantation embryos in vivo and human trophoblast cells (Ac-1M88) in vitro. Maternal diabetes was induced in female rabbits by alloxan before conception and maintained during the preimplantation period. GLO1 expression and activity were investigated in 6-day-old blastocysts from healthy and diabetic rabbits. Furthermore, blastocysts and human trophoblast cells were exposed in vitro to hyperglycaemia, GO and MGO and analysed for GLO1 expression and activity. During gastrulation, GLO1 was expressed in all compartments of the rabbit blastocyst. Maternal diabetes decreased embryonic GLO1 protein amount by approx. 30 per cent whereas the enzymatic activity remained unchanged, indicating that the specific GLO1 activity increases along with metabolic changes. In in vitro cultured embryos, neither hyperglycaemia nor MGO and GO had an effect on GLO1 protein amount. In human trophoblast cells, a stimulating effect on the GLO1 expression was shown in the highest GO concentration, only. Our data show that maternal diabetes mellitus affects the specific activity of GLO1, indicating that GLO1 was post-translationally modified due to changes in metabolic processes in the preimplantation embryos.

Stromal mesenchymal stem cells (MSCs) have the capability to self-renew and can differentiate into multiple cell types of the mesoderm germ layer, but their properties are affected by molecular aging mechanisms. MSCs can be obtained from adipose tissue termed as adipose-derived stem/stromal cells (ASCs) representing a promising tool for studying age-related diseases in detail. ASCs from young (16 weeks) and old (>108 weeks) rabbits were successfully isolated and propagated. ASCs showed the typical morphology and stained positive for CD105, Vimentin, Collagenase 1A, and negative for CD14, CD90, and CD73, demonstrating their mesenchymal origin. ASCs expressed MSC markers, including MYC, KLF4, CHD1, REST, and KAT6A, whereas pluripotency-related genes, such as NANOG, OCT4, and SOX2, were not expressed. Aged ASCs showed altered protein and mRNA levels of APOE, ATG7, FGF2, PTEN, and SIRT1. Adipogenic differentiation of old visceral ASCs was significantly decreased compared with young visceral ASCs. We successfully established rabbit ASC cultures representing an in vitro model for the analysis of stem cell aging mechanisms. ASCs, obtained from old female rabbits, showed age- and source-specific alteration due to aging of the donor. Stem cell plasticity was altered with age as shown by reduced adipogenic differentiation capacity.

MicroRNAs are promising biological markers for prenatal diagnosis. They regulate placental development and are present in maternal plasma. Maternal metabolic diseases are major risk factors for placental deterioration. We analysed the influence of a maternal insulin-dependent diabetes mellitus on microRNA expression in maternal plasma and in blastocysts employing an in vivo rabbit diabetic pregnancy model and an in vitro embryo culture in hyperglycaemic and hypoinsulinaemic medium. Maternal diabetes led to a marked downregulation of Dicer protein in embryoblast cells and Drosha protein in trophoblast cells. MiR-27b, miR-141 and miR-191 were decreased in trophoblast cells and in maternal plasma of diabetic rabbits. In vitro studies indicate, that maternal hyperglycaemia and hypoinsulinaemia partially contribute to the downregulation of trophoblastic microRNAs. As the altered microRNA expression was detectable in maternal plasma, too, the plasma microRNA signature could serve as an early biological marker for the prediction of trophoblast function during a diabetic pregnancy.

Metabolic disorders of the mother adversely affect early embryo development, causing changes in maternal metabolism and consequent alterations in the embryo environment in the uterus. The goal of this study was to analyse the biochemical profiles of embryonic fluids and blood plasma of rabbits with and without insulin-dependent diabetes mellitus (DT1), to identify metabolic changes associated with maternal diabetes mellitus in early pregnancy. Insulin-dependent diabetes was induced by alloxan treatment in female rabbits 10 days before mating. On day 6 post-coitum, plasma and blastocoel fluid (BF) were analysed by ultrahigh performance liquid chromatography-tandem mass spectroscopy (UPLC-MS/MS) (Metabolon Inc. Durham, NC, USA). Metabolic datasets comprised a total of 284 and 597 compounds of known identity in BF and plasma, respectively. Diabetes mellitus had profound effects on maternal and embryonic metabolic profiles, with almost half of the metabolites changed. As predicted, we observed an increase in glucose and a decrease in 1,5-anhydroglucitol in diabetic plasma samples. In plasma, fructose, mannose, and sorbitol were elevated in the diabetic group, which may be a way of dealing with excess glucose. In BF, metabolites of the pentose metabolism were especially increased, indicating the need for ribose-based compounds relevant to DNA and RNA metabolism at this very early stage of embryo development. Other changes were more consistent between BF and plasma. Both displayed elevated acylcarnitines, body3-hydroxybutyrate, and multiple compounds within the branched chain amino acid metabolism pathway, suggesting that lipid beta-oxidation is occurring at elevated levels in the diabetic group. This study demonstrates that maternal and embryonic metabolism are closely related. Maternal diabetes mellitus profoundly alters the metabolic profile of the preimplantation embryo with changes in all subclasses of metabolites.

The mammalian target of rapamycin complex 1 (mTORC1) is known to be a central cellular nutrient sensor and master regulator of protein metabolism; therefore, it is indispensable for normal embryonic development. We showed previously in a diabetic pregnancy that embryonic mTORC1 phosphorylation is increased in case of maternal hyperglycaemia and hypoinsulinaemia. Further, the preimplantation embryo is exposed to increased L-leucine levels during a diabetic pregnancy. To understand how mTOR signalling is regulated in preimplantation embryos, we examined consequences of L-leucine and glucose stimulation on mTORC1 signalling and downstream targets inin vitrocultured preimplantation rabbit blastocysts andin vivo. High levels of L-leucine and glucose lead to higher phosphorylation of mTORC1 and its downstream target ribosomal S6 kinase 1 (S6K1) in these embryos. Further, L-leucine supplementation resulted in higher embryonic expression of genes involved in cell cycle (cyclin D1;CCND1), translation initiation (eukaryotic translation initiation factor 4E;EIF4E), amino acid transport (large neutral amino acid transporter 2; Lat2: geneSLC7A8) and proliferation (proliferating cell nuclear antigen;PCNA) in a mTORC1-dependent manner. Phosphorylation of S6K1 and expression patterns ofCCND1andEIF4Ewere increased in embryos from diabetic rabbits, while the expression of proliferation markerPCNAwas decreased. In these embryos, protein synthesis was increased and autophagic activity was decreased. We conclude that mammalian preimplantation embryos sense changes in nutrient supply via mTORC1 signalling. Therefore, mTORC1 may be a decisive mediator of metabolic programming in a diabetic pregnancy.

Maternal metabolic diseases such as diabetes mellitus with diabetogenic hypoinsulinemia and hyperglycemia change periconceptional developmental conditions in utero. In preimplantation rabbit embryos, all major metabolic pathways are affected. Alterations in protein, lipid and glucose metabolism, adipokines, advanced glycation end products (AGEs) and reactive oxygen species (ROS) are described in this review. The embryonic metabolism is characterized by a high plasticity which enables survival of most preimplantation embryos under the non-physiological developmental conditions in diabetic mothers. Adiponectin, for example, compensates for the missing insulin-driven glucose supply and stimulates intracellular lipid accumulation in embryonic cells. AGEs and ROS are clear indicators of metabolic stress. The price paid for survival, however, needs to be taken into consideration. It is an increase in lipogenesis and proteinogenesis, leading to metabolic stress and with potentially negative long-term health effects.

The accumulation of advanced glycation end products (AGEs) occurs in ageing and in many degenerative diseases as a final outcome of persistent oxidative stress on cells and organs. Environmental alterations taking place during early embryonic development can also lead to oxidative damage, reactive oxygen species (ROS) production, and AGE accumulation. Whether similar mechanisms act on somatic and embryonic stem cells (ESC) exposed to oxidative stress is not known; and therefore, the modelling of oxidative stress in vitro on human ESC has been the focus of this study. We compared changes in Nε-carboxymethyl-lysine (CML) advanced glycation end products and RAGE levels in hESC versus differentiated somatic cells exposed to H2O2 within the noncytotoxic range. Our data revealed that hESC accumulates CML and RAGE under oxidative stress conditions in different ways than somatic cells, being the accumulation of CML statistically significant only in somatic cells and, conversely, the RAGE increase exclusively appreciated in hESC. Then, following cardiac and neural differentiation, we observed a progressive removal of AGEs and at the same time an elevated activity of the 20S proteasome. We conclude that human ESCs constitute a unique model to study the consequence of an oxidative environment in the pluripotent cells of the embryo during the human preimplantation period.

The accumulation of advanced glycation end products (AGEs) occurs in ageing and in many degenerative diseases as a final outcome of persistent oxidative stress on cells and organs. Environmental alterations taking place during early embryonic development can also lead to oxidative damage, reactive oxygen species (ROS) production, and AGE accumulation. Whether similar mechanisms act on somatic and embryonic stem cells (ESC) exposed to oxidative stress is not known; and therefore, the modelling of oxidative stress in vitro on human ESC has been the focus of this study. We compared changes in Nε-carboxymethyl-lysine (CML) advanced glycation end products and RAGE levels in hESC versus differentiated somatic cells exposed to H2O2 within the noncytotoxic range. Our data revealed that hESC accumulates CML and RAGE under oxidative stress conditions in different ways than somatic cells, being the accumulation of CML statistically significant only in somatic cells and, conversely, the RAGE increase exclusively appreciated in hESC. Then, following cardiac and neural differentiation, we observed a progressive removal of AGEs and at the same time an elevated activity of the 20S proteasome. We conclude that human ESCs constitute a unique model to study the consequence of an oxidative environment in the pluripotent cells of the embryo during the human preimplantation period.

Maternal metabolic diseases such as diabetes mellitus with diabetogenic hypoinsulinemia and hyperglycemia change periconceptional developmental conditions in utero. In preimplantation rabbit embryos, all major metabolic pathways are affected. Alterations in protein, lipid and glucose metabolism, adipokines, advanced glycation end products (AGEs) and reactive oxygen species (ROS) are described in this review. The embryonic metabolism is characterized by a high plasticity which enables survival of most preimplantation embryos under the non-physiological developmental conditions in diabetic mothers. Adiponectin, for example, compensates for the missing insulin-driven glucose supply and stimulates intracellular lipid accumulation in embryonic cells. AGEs and ROS are clear indicators of metabolic stress. The price paid for survival, however, needs to be taken into consideration. It is an increase in lipogenesis and proteinogenesis, leading to metabolic stress and with potentially negative long-term health effects.

Diabetes mellitus (DM) during pregnancy is one of the leading causes of perinatal morbidity and birth defects. The mechanism by which maternal hyperglycemia, the major teratogenic factor, induces embryonic malformations remains unclear. Advanced glycation end products (AGEs) are known to accumulate during the course of DM and contribute to the development of diabetic complications. Employing a diabetic rabbit model, we investigated the influence of maternal hyperglycemia during the preimplantation period on AGE formation (pentosidine, argpyrimidine, andNε-carboxymethyllysine (CML)) in the reproductive tract and the embryo itself. As a consequence of type 1 DM, the AGE levels in blood plasma increased up to 50%, correlating closely with an AGE accumulation in the endometrium of diabetic females. Embryos from diabetic mothers had increased protein-bound CML levels and showed enhanced fluorescent signals for AGE-specific fluorescence in the blastocyst cavity fluid (BCF). The quantification of CML by HPLC–mass spectrometry (MS/MS) showed a higher amount of soluble CML in the BCF of blastocysts from diabetic rabbits (0.26±0.05 μmol/l) compared with controls (0.18±0.02 μmol/l). The high amount of AGEs in blastocysts from diabetic mothers correlates positively with an increasedAGER(receptor for AGE (RAGE)) mRNA expression. Our study gives alarming insights into the consequences of poorly controlled maternal diabetes for AGE formation in the embryo. Maternal hyperglycemia during the preimplantation period is correlated with an increase in AGE formation in the uterine environment and the embryo itself. This may influence the development of the embryo through increased AGE-mediated cellular stress by RAGEs.

According to the “developmental origin of health and disease” hypothesis, the metabolic set points of glucose and lipid metabolism are determined prenatally. In the case of a diabetic pregnancy, the embryo is exposed to higher glucose and lipid concentrations as early as during preimplantation development. We used the rabbit to study the effect of maternal diabetes type 1 on lipid accumulation and expression of lipogenic markers in preimplantation blastocysts. Accompanied by elevated triglyceride and glucose levels in the maternal blood, embryos from diabetic rabbits showed a massive intracellular lipid accumulation and increased expression of fatty acid transporter 4, fatty acid–binding protein 4, perilipin/adipophilin, and maturation of sterol-regulated element binding protein. However, expression of fatty acid synthase, a key enzyme for de novo synthesis of fatty acids, was not altered in vivo. During a short time in vitro culture of rabbit blastocysts, the accumulation of lipid droplets and expression of lipogenic markers were directly correlated with increasing glucose concentration, indicating that hyperglycemia leads to increased lipogenesis in the preimplantation embryo. Our study shows the decisive effect of glucose as the determining factor for fatty acid metabolism and intracellular lipid accumulation in preimplantation embryos.

Improvements in health care and lifestyle have led to an elevated lifespan and increased focus on age-associated diseases, such as neurodegeneration, cardiovascular disease, frailty and arteriosclerosis. In all these chronic diseases protein, lipid or nucleic acid modifications are involved, including cross-linked and non-degradable aggregates, such as advanced glycation end products (AGEs). Formation of endogenous or uptake of dietary AGEs can lead to further protein modifications and activation of several inflammatory signaling pathways. This review will give an overview of the most prominent AGE-mediated signaling cascades, AGE receptor interactions, prevention of AGE formation and the impact of AGEs during pathophysiological processes.

STUDY QUESTION How does a maternal diabetic hyperadiponectineamia affect signal transduction and lipid metabolism in rabbit preimplantation blastocysts? SUMMARY ANSWER In a diabetic pregnancy increased levels of adiponectin led to a switch in embryonic metabolism towards a fatty acid-dependent energy metabolism, mainly affecting genes that are responsible for fatty acid uptake and turnover. WHAT IS KNOWN ALREADY Although studies in cell culture experiments have shown that adiponectin is able to regulate lipid metabolism via 5′-AMP-activated protein kinase (AMPK) and peroxisome proliferator-activated receptor α (PPARα), data on the effects of adiponectin on embryonic lipid metabolism are not available. In a diabetic pregnancy in rabbits, maternal adiponectin levels are elevated fourfold and are accompanied by an increase in intracellular lipid droplets in blastocysts, implying consequences for the embryonic hormonal and metabolic environment. STUDY DESIGN, SIZE, DURATION Rabbit blastocysts were cultured in vitro with adiponectin (1 μg/ml) and with the specific AMPK-inhibitor Compound C for 15 min, 1 h and 4 h (N ≥ 3 independent experiments: for RNA analysis, n ≥ 4 blastocysts per treatment group; for protein analysis three blastocysts pooled per sample and three samples used per experiment). Adiponectin signalling was verified in blastocysts grown in vivo from diabetic rabbits with a hyperadiponectinaemia (N ≥ 3 independent experiments, n ≥ 4 samples per treatment group, eight blastocysts pooled per sample). PARTICIPANTS/MATERIALS, SETTING, METHODS In these blastocysts, expression of molecules involved in adiponectin signalling [adaptor protein 1 (APPL1), AMPK, acetyl-CoA carboxylase (ACC), p38 mitogen-activated protein kinases (p38 MAPK)], lipid metabolism [PPARα, cluster of differentiation 36 (CD36), fatty acid transport protein 4 (FATP4), fatty acid binding protein (FABP4), carnitine palmityl transferase 1 (CPT1), hormone-senstive lipase (HSL), lipoprotein lipase (LPL)] and members of the insulin/insulin-like growth factor (IGF)-system [IGF1, IGF2, insulin receptor (InsR), IGF1 receptor (IGF1R)] were analyzed by quantitative RT-PCR and western blot. Analyses were performed in both models, i.e. adiponectin stimulated blastocysts (in vitro) and in blastocysts grown in vivo under increased adiponectin levels caused by a maternal diabetes mellitus. MAIN RESULTS AND THE ROLE OF CHANCE In both in vitro and in vivo models adiponectin increased AMPK and ACC phosphorylation, followed by an activation of the transcription factor PPARα, and CPT1, the key enzyme of β-oxidation (all P < 0.05 versus control). Moreover, mRNA levels of the fatty acid transporters CD36, FATP4 and FABP4, and HSL were upregulated by adiponectin/AMPK signalling (all P < 0.05 versus control). Under diabetic developmental conditions the amount of p38 MAPK was upregulated (P < 0.01 versus non-diabetic), which was not observed in blastocysts cultured in vitro with adiponectin, indicating that the elevated p38 MAPK was not related to adiponectin. However, a second effect of adiponectin has to be noted: its intensification of insulin sensitivity, by regulating IGF availability and InsR/IGF1R expression. LARGE SCALE DATA Not applicable. LIMITATIONS REASONS FOR CAUTION There are two main limitations for our study. First, human and rabbit embryogenesis can only be compared during blastocyst development. Therefore, the inferences from our findings are limited to the embryonic stages investigated here. Second, the increased adiponectin levels and lack of maternal insulin is only typical for a diabetes mellitus type one model. WIDER IMPLICATIONS OF THE FINDINGS This is the first mechanistic study demonstrating a direct influence of adiponectin on lipid metabolism in preimplantation embryos. The numbers of young women with a diabetes mellitus type one are increasing steadily. We have shown that preimplantation embryos are able to adapt to changes in the uterine milieu, which is mediated by the adiponectin/AMPK signalling. A tightly hormonal control during pregnancy is essential for survival and proper development. In this control process, adiponectin plays a more important role than known so far. STUDY FUNDING/COMPETING INTEREST(S) This work was supported by the German Research Council (DFG RTG ProMoAge 2155), the EU (FP7 Epihealth No. 278418, FP7-EpiHealthNet N°317146), COST Action EpiConcept FA 1201 and SALAAM BM 1308. The authors have no conflict(s) of interest to disclose.

Background: Recent studies have disclosed a close relationship between maternal obesity, fetal metabolism and pre- and postnatal development. The lipid metabolism in preimplantation embryos is a possible target of metabolic programming. Methods: 31 genes of beta-oxidation and fatty acid and cholesterol uptake, synthesis and regulation were analyzed in day 3.5 blastocysts from NZO (obese) and C57Bl/6 (normal weight) mice by RT-PCR and semiquantitative PCR. Results: The most obvious difference between both strains was the lack of the RXR gamma transcript in NZO blastocysts. In adult NZO mice, RXR gamma is detectable in most tissues. In a semiquantitative analysis, a higher transcription rate of fatty acid transport protein 4 (p = 0.004) and a reduced transcript number of fatty acid synthase (p = 0.049) was found in NZO blastocysts. Cholesterol synthesis regulation was modified in NZO blastocysts, as indicated by the ratio of sterol regulatory element-binding protein (SREBP) 2 / insulin-induced gene 1 (Insig 1) (p = 0.001). Conclusion: In mouse blastocysts enzymes and signal molecules of fatty acid and cholesterol metabolism resemble those expressed postnatally. Distinct differences in transcription rates of genes between blastocysts from obese and non-obese mothers indicate that preimplantation embryo development is an early target for metabolic programming.

In the rabbit reproductive model, maternal experimentally induced insulin-dependent diabetes mellitus (expIDD) leads to accumulation of lipid droplets in blastocysts. Cholesterol metabolism is a likely candidate to explain such metabolic changes. Therefore, in the present study we analysed maternal and embryonic cholesterol concentrations and expression of related genes in vivo (diabetic model) and in vitro (embryo culture in hyperglycaemic medium). In pregnant expIDD rabbits, the serum composition of lipoprotein subfractions was changed, with a decrease in high-density lipoprotein cholesterol and an increase in very low-density lipoprotein cholesterol; in uterine fluid, total cholesterol concentrations were elevated. Expression of 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR), very low-density lipoprotein receptor (VLDLR), sterol regulatory element binding transcription factor 2 (SREBF2), insulin-induced gene-1 (INSIG1) and cholesterol 7 alpha-hydroxylase (CYP7A1) mRNA was decreased in the liver and low-density lipoprotein receptor (LDLR) mRNA expression was decreased in the adipose tissue of diabetic rabbits. In embryos from diabetic rabbits, the mean (+/- s.e.m.) ratio of cholesterol concentrations in trophoblasts to embryoblasts was changed from 1.27 +/- 2.34 (control) to 0.88 +/- 3.85 (expIDD). Rabbit blastocysts expressed HMGCR, LDLR, VLDLR, SREBF2 and INSIG1 but not CYP7A1, without any impairment of expression as a result of maternal diabetes. In vitro hyperglycaemia decreased embryonic HMGCR and SREBF2 transcription in rabbit blastocysts. The findings of the present study show that a diabetic pregnancy leads to distinct changes in maternal cholesterol metabolism with a minor effect on embryo cholesterol metabolism.

Pluripotent embryonic stem cells (ESC) have the potential to differentiate into any cell type of the three germ layers. Differentiation processes depend on genetic and epigenetic factors. The guidance of cell fate determination by microRNAs (miRs) seems important for embryonic development and cell lineage decisions. MiRs are short, single-stranded, noncoding RNA molecules that regulate through posttranscriptional modulation, a subset of target genes involved in cell differentiation and specific cell function. We have used microarray profiling of miRs in the mouse embryonic stem cell line CGR8. Comparison of the miR profiles of undifferentiated stem cells with mesodermal progenitors cells ( day 5), preadipocytes ( day 10), and adipocytes ( day 21) showed that the expression level of 129 miRs changed (twofold) during adipogenic differentiation. We identified 10 clusters of differentially expressed miRs, which contain putative markers and regulators of mesodermal differentiation and cell fate determination into adipocytes. Notably, the adipocyte-specific miRs 143 and 103 were upregulated from day 10 onward. We have therefore demonstrated and characterized the dynamic profile of miR expression during murine adipogenic differentiation in vitro, including the initial differentiation from ESC via mesenchymal progenitors up to adipocytes. Our findings and experimental approach provide a suitable system to directly interrogate the role of miRs during adipogenic differentiation of embryonic stem cells.

Since the discovery of adipokines, the adipose tissue is no longer considered to be an inactive fat storage. It secretes a variety of bioactive molecules, which regulate body metabolism and energy homeostasis. One of these molecules is the adipokine adiponectin. In different tissues, adiponectin triggers metabolic effects through the adenosine monophosphate-activated protein kinase (PRKA), which is a master regulator in glucose and lipid metabolism. Recent studies point to a role for adiponectin in reproduction. Adiponectin and its receptors are present in female reproductive tract during pregnancy, and the preimplantation embryo is fully equipped with adiponectin. Here, we show that both receptor isoforms, ADIPOR1 and ADIPOR2, are expressed in 6-day-old rabbit blastocysts. To investigate the signaling pathway of adiponectin in preimplantation embryos, rabbit blastocysts were cultured in vitro and stimulated with adiponectin. Supplementation of adiponectin (1 μg/ml) enhanced PRKA alpha 1/2 (PRKAA1/2) phosphorylation and decreased expression of phosphoenolpyruvate carboxykinase 2 (PCK2), a key regulator of gluconeogenesis. Inhibition of PRKAA1/2 by Compound C (10 μM) restored PCK2 transcription. Adiponectin enhanced embryonic glucose uptake and led to a translocation of solute carrier family 2 (facilitated glucose transporter), member 4 (SLC2A4), previously known as GLUT4. We conclude that adiponectin influences the glucose metabolism of rabbit blastocysts via the phosphorylation of PRKAA1/2, which in turn results in a decrease of gluconeogenesis and an increase in glycolysis. The regulatory influence of adiponectin on glucose metabolism of blastocysts may be of specific interest in pathophysiological situations, such as obesity during pregnancy.

Because of the increasing incidence of worldwide obesity, cell culture models which enable the study of adipose tissue development are of particular importance. The murine embryonic stem cell (ESC) line CGR8 differentiates into adipocytes with a differentiation efficiency of up to 15%. A critical step for the analysis of stem cell-derived adipogenesis is the reliable separation of adipocytes. Here we report on how to (i) gently separate the cells of embryoid bodies (EBs) and (ii) identify and sort adipocytes from the rest of the heterogeneous cell mixture. Up to the present, no adipocyte specific surface marker is known for fluorescence activated cell sorting (FACS). After separation we employed two independently existing FACS methods for adipocyte cell sorting. These methods are based on Nile red staining and granularity. For stem cell-derived adipocytes only the combination of both methods led to a reliable, efficient, and highly reproducible FACS analysis, as shown by the presence and absence of adipocyte specific markers in positively and negatively sorted cells. ' 2010 International Society for Advancement of Cytometry

During preimplantation development, embryos of many species are known to express up to five isoforms of the facilitative glucose transporter proteins (GLUT). Development of hamster blastocysts is inhibited by glucose. We therefore investigated GLUT isoform and insulin receptor (IR) expression in hamster preimplantation embryos cultured in glucose-free medium from the 8-cell stage onwards. We show that GLUT1, 3 and 8 mRNA are constitutively expressed from the 8-cell to the blastocyst stage. The IR is expressed from the morula stage onwards. Messenger RNA of the insulin-responsive GLUT4 was not detected at any stage. GLUT1 and 3 were localised by immunocytochemistry. GLUT1 was expressed in both embryoblast and trophoblast, in the latter, mainly in basal and lateral membranes directed towards the blastocoel. and embryoblast. GLUT3 was exclusively localised in the apical. membrane of trophoblast cells. We show that hamster preimplantation embryos express several GLUT isoforms thus closely resembling embryos of other mammalian species. Despite endogenous IR expression, the insulin-sensitive isoform GLUT4 was not expressed, indicating that the insulin-mediated glucose uptake known from classical insulin target cells may not be relevant for hamster blastocysts.

Intoxication by dioxins such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) leads, among other damages, to early embryo loss, fetal malformations, and cardiovascular toxicity. Apart from binding to the arylhydrocarbon receptor (AhR), the mechanism of TCDD-mediated embryo toxicity is still unclear. We investigated possible modes of a TCDD-mediated toxicity, particularly in glucose metabolism, in pluripotent P19 mouse embryonic carcinoma cells. Undifferentiated P19 cells were exposed to 1–100 nm TCDD and characterized for AhR signaling. For studying cell differentiation, P19 cells were exposed to 10 nm TCDD at stage of embryoid body formation, and analyzed on glucose metabolism and cardiac differentiation during the next 3 wk. TCDD treatment activated the AhR-signaling cascade within 1 h, confirmed by AhR translocation, induction of cytochrome P450 1A1 expression, and activation of the xenobiotic response element. Although cell viability and transcription of the cardiac marker protein α-myosin heavy chain were affected, TCDD did not inhibit the differentiation of P19 cells to pulsating cardiomyocytes. TCDD significantly down-regulated the expression levels of the glucose transporter (GLUT) isoforms 1 and 3. After 24-h TCDD treatment, GLUT1 was no longer localized in the plasma membrane of P19 cells. The impaired GLUT expression correlated with a lower glucose uptake in 5-d-old embryoid bodies. The TCDD effects were mediated by AhR, as shown by preculture with the AhR antagonist α-naphthoflavone. Our data demonstrate that an AhR-mediated disturbance in GLUT expression and insufficient glucose uptake may be major mechanisms in TCDD embryo toxicity.

The insulin/IGF system plays a critical role in embryo growth and development. We have investigated the expression of insulin receptor (IR) and IGF-I receptor (IGF-IR) and the activation of their downstream pathways in rabbit 6-d-old blastocysts. IR was expressed in embryoblast (Em, inner cell mass) and trophoblast (Tr) cells, whereas IGF-IR was localized mainly in Em. Isoform A (IR-A) represents the main insulin isoform in blastocysts and was found in Em and Tr cells. IR-B was detectable only in Tr. IR/IGF-IR signaling pathways were analyzed after stimulation with insulin (17 nm) or IGF-I (1.3 nm) in cultured blastocysts. Insulin stimulated Erk1/2 in Em and Tr and Akt in Tr but not in Em. IGF-I activated both kinases exclusively in Em. The target genes c-fos (for MAPK kinase-1/Erk signaling) and phosphoenolpyruvate carboxykinase (PEPCK, for PI3K/Akt signaling) were also specifically regulated. Insulin down-regulated PEPCK RNA amounts in Tr by activation of the phosphatidylinositol 3-kinase/Akt pathway. Expression of c-fos by insulin and IGF-I was different with respect to time and fortitude of expression, mirroring again the specific IR and IGF-IR expression patterns in Em and Tr. Taken together, we show that IGF-I acts primarily mitogenic, an effect that is cell lineage-specifically restricted to the Em. By contrast, insulin is the growth factor of the Tr stimulating mitogenesis and down-regulating metabolic responses. As soon as blastocyst differentiation in Em and Tr has been accomplished, insulin and IGF-I signaling is different in both cell lineages, implying a different developmental impact of both growth factors.

The peptidyl-prolyl cis/trans isomerase (PPIase) activity and the expression of cyclophilins were studied in 6-day-old rabbit preimplantation embryos cultured under physiological and increased oxygen concentrations of 5% and 20% O(2), respectively. The PPIase activity was completely inhibited by cyclosporin A (CsA). The inhibitor of FK506-binding proteins, rapamycin, had no effect on the PPIase activity, indicating that the PPIase activity in rabbit blastocysts originates from cyclophilins. Using CsA affinity chromatography, only one cyclophilin with a molecular mass of about 17.8 kDa was separated. The cDNA of rabbit cyclophilin was cloned and sequenced. Analysis of the 682-base pair cDNA revealed an open reading frame coding for a polypeptide of 164 amino acid residues with a molecular weight of 17.83 kDa. Homologies of 90% and 96% for the cDNA and amino acid sequence, respectively, to the human CyP18 were found, suggesting that the novel rabbit cyclophilin is a member of the CyP18 family (rabCyP18). The transcription level of rabCyP18 mRNA was 8.3 +/- 0.6 pg in 100 ng total RNA in noncultured blastocysts. In vitro culture with moderate oxygen stress (20% O(2)) resulted in a 1.5-fold increase in rabCyP18 transcription and an increased PPIase activity compared to that of blastocysts cultured with 5% O(2). Increase in transcription rate and PPIase activity by oxygen stress suggests an involvement of CyP18 in oxygen defense in rabbit preimplantation embryos.

The food contaminant tributyltin (TBT) is an endocrine disrupting compound (EDC) promoting adipogenic differentiation in vitro and in vivo. Although prenatal TBT exposure has been shown to induce obesity, the underlying mechanisms and the role of the transcription factor PPARγ are not clarified yet. At different stages of adipogenesis, multipotent murine mesenchymal stem cells (MSC), C3H10T1/2, were exposed to TBT and analyzed for adipogenic differentiation, PPARγ promoter activation and PPARγ1, PPARγ2, Pref-1 and SOX9 expression. Depending on the exposure window, TBT promoted subsequent adipogenesis independently and dependently from PPARγ. In undifferentiated MSC, TBT exposure induced a transcriptional PPARγ-independent repression of Pref-1 and SOX9, which are both suppressors of adipogenic cell fate commitment. During hormonal induction TBT additionally enhanced adipogenic differentiation by PPARγ signaling. The impact of TBT on early cell fate development documents a novel mechanistic insight in the development of adipocytes derived from MSC and its susceptibility to EDC.

The transcription factor cAMP responsive element-binding protein (CREB) and activating transcription factors (ATFs) are downstream components of the insulin/IGF cascade, playing crucial roles in maintaining cell viability and embryo survival. One of the CREB target genes is adiponectin, which acts synergistically with insulin. We have studied the CREB-ATF-adiponectin network in rabbit preimplantation development in vivo and in vitro. From the blastocyst stage onwards, CREB and ATF1, ATF3, and ATF4 are present with increasing expression for CREB, ATF1, and ATF3 during gastrulation and with a dominant expression in the embryoblast (EB). In vitro stimulation with insulin and IGF-I reduced CREB and ATF1 transcripts by approximately 50%, whereas CREB phosphorylation was increased. Activation of CREB was accompanied by subsequent reduction in adiponectin and adiponectin receptor (adipoR)1 expression. Under in vivo conditions of diabetes type 1, maternal adiponectin levels were up-regulated in serum and endometrium. Embryonic CREB expression was altered in a cell lineage-specific pattern. Although in EB cells CREB localization did not change, it was translocated from the nucleus into the cytosol in trophoblast (TB) cells. In TB, adiponectin expression was increased (diabetic 427.8 ± 59.3 pg/mL vs normoinsulinaemic 143.9 ± 26.5 pg/mL), whereas it was no longer measureable in the EB. Analysis of embryonic adipoRs showed an increased expression of adipoR1 and no changes in adipoR2 transcription. We conclude that the transcription factors CREB and ATFs vitally participate in embryo-maternal cross talk before implantation in a cell lineage-specific manner. Embryonic CREB/ATFs act as insulin/IGF sensors. Lack of insulin is compensated by a CREB-mediated adiponectin expression, which may maintain glucose uptake in blastocysts grown in diabetic mothers.

The renaissance of the laboratory rabbit as a reproductive model for human health is closely related to the growing evidence of periconceptional metabolic programming and its determining effects on offspring and adult health. Advantages of rabbit reproduction are the exact timing of fertilization and pregnancy stages, high cell numbers and yield in blastocysts, relatively late implantation at a time when gastrulation is already proceeding, detailed morphologic and molecular knowledge on gastrulation stages, and a hemochorial placenta structured similarly to the human placenta. To understand, for example, the mechanisms of periconceptional programming and its effects on metabolic health in adulthood, these advantages help to elucidate even subtle changes in metabolism and development during the pre- and peri-implantation period and during gastrulation in individual embryos. Gastrulation represents a central turning point in ontogenesis in which a limited number of cells program the development of the three germ layers and, hence, the embryo proper. Newly developed transgenic and molecular tools offer promising chances for further scientific progress to be attained with this reproductive model species.

Insulin-like growth factors (IGFs) are well-known regulators of embryonic growth and differentiation. IGF function is closely related to insulin action. IGFs are available to the preimplantation embryo through maternal blood (endocrine action), uterine secretions (paracrine action) and by the embryo itself (autocrine action). In rabbit blastocysts, embryonic IGF1 and IGF2 are specifically strong in the embryoblast (ICM). Signalling of IGFs and insulin in blastocysts follows the classical pathway with Erk1/2 and Akt kinase activation. The aim of this study was to analyse signalling of IGFs in experimental insulin dependent diabetes (exp IDD) in pregnancy, employing a diabetic rabbit model with uterine hypoinsulinemia and hyperglycaemia. Exp IDD was induced in female rabbits by alloxan treatment prior to mating. At 6 days p.c., the maternal and embryonic IGFs were quantified by RT-PCR and ELISA. In pregnant females, hepatic IGF1 expression and IGF1 serum levels were decreased while IGF1 and IGF2 were increased in endometrium. In blastocysts, IGF1 RNA and protein was approx. 7.5-fold and 2-fold higher, respectively, than in controls from normoglycemic females. In cultured control blastocysts supplemented with IGF1 or insulin in vitro for 1 or 12 h, IGF1 and insulin receptors as well as IGF1 and IGF2 were downregulated. In cultured T1D blastocysts activation of Akt and Erk1/2 was impaired with lower amounts of total Akt and Erk1/2 protein and a reduced phosphorylation capacity after IGF1 supplementation. Our data show that the IGF axis is severely altered in embryo-maternal interactions in exp IDD pregnancy. Both, the endometrium and the blastocyst produce more IGF1 and IGF2. The increased endogenous IGF1 and IGF2 expression by the blastocyst compensates for the loss of systemic insulin and IGF. However, this counterbalance does not fill the gap of the reduced insulin/IGF sensitivity, leading to a developmental delay of blastocysts in exp IDD pregnancy.

Insulin and insulin-like-growth-factor 1 (IGF1) are components of the uterine secretions. As potent growth factors they influence early embryo development. The underlying molecular mechanisms are largely unknown. Here we report on the effects of insulin and IGF1 on early gastrulation in rabbit blastocysts. We have studied blastocysts grown in vivo in metabolically healthy rabbits, in rabbits with type 1 diabetes and in vitro in the presence or absence of insulin or IGF1. Embryonic disc morphology and expression of Brachyury , Wnt3a and Wnt4 were analysed by qPCR and IHC. Pre-gastrulated blastocysts (stage 0/1) cultured with insulin or IGF1 showed a significantly higher capacity to form the posterior mesoderm and primitive streak (stage 2 and 3) than blastocysts cultured without growth factors. In gastrulating blastocysts the levels of the mesoderm-specific transcription factor Brachyury and the Wnt signalling molecules Wnt3a and Wnt4 showed a stage-specific expression pattern with Brachyury transcripts increasing from stage 0/1 to 3. Wnt4 protein was found spread over the whole embryoblast. Insulin induced Wnt3a, Wnt4 and Brachyury expression in a temporal- and stage-specific pattern. Only blastocysts cultured with insulin reached the Wnt3a, Wnt4 and Brachyury expression levels of stage 2 in vivo blastocysts, indicating that insulin is required for Wnt3a, Wnt4 and Brachyury expression during gastrulation. Insulin-induced Wnt3a and Wnt4 expression preceded Brachyury . Wnt3a-induced expression could be depleted by MEK1 inhibition (PD98059). Involvement of insulin in embryonic Wnt3a expression was further shown in vivo with Wnt3a expression being notably down regulated in stage 2 blastocysts from rabbits with type 1 diabetes. Blastocysts grown in diabetic rabbits are retarded in development, a finding which supports our current results that insulin is highly likely required for early mesoderm formation in rabbit blastocysts by inducing a distinct spatiotemporal expression profile of Wnt3a, Wnt4 and Brachyury .

A well-balanced activity of the aryl hydrocarbon receptor (AhR) is necessary for normal ovarian function. As known from murine AhR knock-out (KO) models, the AhR is involved in folliculogenesis, gonadotrophin receptor expression, proliferation of granulosa cells and intraovarian estrogen signalling. Highly potent, non-physiological ligands such as the dioxin 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) lead to a blockade of ovulation, estrogen receptor degradation and reduction of estrogen levels. Estrogen synthesis is a typical function of granulosa cells and essential for normal cyclicity and fertility. We employed the human granulosa cell line KGN to further characterize AhR signalling and AhR function in granulosa cell physiology. Real-time PCR quantification of the target genes Cyp1a1 and Cyp1b1 and reporter gene assays after stimulation with TCDD or beta-naphthoflavone (BNF) or inhibition with alpha-naphthoflavone (ANF) or 3'-methoxy-4'-nitroflavone (3,4-MNF) of the AhR demonstrated constitutive activity and functionality of AhR pathway in KGN granulosa cells. In untreated KGN cells, AhR protein was exclusively detected in the nuclear fraction. TCDD stimulation affected the gonadotrophin receptor but not estrogen receptor β (ERβ) protein expression. Additionally, the constitutively activated AhR suppressed aromatase expression and estrogen synthesis (enzyme-linked immunoassay, ELISA) and enhanced proliferation [Bromodeoxyuridine (BrdU) ELISA] of KGN cells. Activation of the AhR by BNF did not override this inhibitory effect on estrogen synthesis or proliferation. In conclusion, the AhR pathway is constitutively activated and functional in human KGN granulosa cells. It is a potential target for endocrine disruption by exogenous ligands and subsequent dysfunction of granulosa cells.

Since the discovery of adipokines, the adipose tissue is no longer considered to be an inactive fat storage. It secretes a variety of bioactive molecules, which regulate body metabolism and energy homeostasis. One of these molecules is the adipokine adiponectin. In different tissues, adiponectin triggers metabolic effects through the adenosine monophosphate-activated protein kinase (PRKA), which is a master regulator in glucose and lipid metabolism. Recent studies point to a role for adiponectin in reproduction. Adiponectin and its receptors are present in female reproductive tract during pregnancy, and the preimplantation embryo is fully equipped with adiponectin. Here, we show that both receptor isoforms, ADIPOR1 and ADIPOR2, are expressed in 6-day-old rabbit blastocysts. To investigate the signaling pathway of adiponectin in preimplantation embryos, rabbit blastocysts were cultured in vitro and stimulated with adiponectin. Supplementation of adiponectin (1 μg/ml) enhanced PRKA alpha 1/2 (PRKAA1/2) phosphorylation and decreased expression of phosphoenolpyruvate carboxykinase 2 (PCK2), a key regulator of gluconeogenesis. Inhibition of PRKAA1/2 by Compound C (10 μM) restored PCK2 transcription. Adiponectin enhanced embryonic glucose uptake and led to a translocation of solute carrier family 2 (facilitated glucose transporter), member 4 (SLC2A4), previously known as GLUT4. We conclude that adiponectin influences the glucose metabolism of rabbit blastocysts via the phosphorylation of PRKAA1/2, which in turn results in a decrease of gluconeogenesis and an increase in glycolysis. The regulatory influence of adiponectin on glucose metabolism of blastocysts may be of specific interest in pathophysiological situations, such as obesity during pregnancy.

Women with type 1 diabetes are subfertile. Diabetes negatively affects pregnancy by causing early miscarriage and poor prenatal outcomes. In this study we examine consequences of maternal type 1 diabetes on early embryo development, metabolic gene expression, and the pattern of insulin receptor (IR) and IGF-I receptor (IGF-IR) distribution in rabbit blastocysts. In female rabbits, type 1 diabetes was induced by alloxan treatment. Six-day-old blastocysts were recovered and assessed for receptor distribution and metabolic gene expression. In vitro culture of blastocysts was performed in medium containing 1 mm, 10 mm, or 25 mm glucose, simulating normo- and hyperglycemic developmental condition in vitro. The fertility rate of the diabetic rabbits clearly mirrored subfertility with a drop in blastocyst numbers by 40% (13.3 blastocysts in diabetic vs. 21.9 in control females). In blastocysts onset and progression of gastrulation was delayed and expression of IR and IGF-IR and their metabolic target genes (hexokinase, phosphoenolpyruvate carboxykinase), both in vivo and in vitro, was down-regulated. The amount of apoptotic cells in the embryonic disc was increased, correlating closely with the reduced transcription of the bcl-x(L) gene. Blastocyst development is clearly impaired by type 1 diabetes during early pregnancy. Insulin-stimulated metabolic genes and IR and IGF-IR are down-regulated, resulting in reduced insulin and IGF sensitivity and a delay in development. Dysregulation of the IGF system and embryonic glucose metabolism are potential reasons for diabetogenous subfertility and embryopathies and start as soon as during the first days of life.

TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin) is known to promote cancer initiation and progression and accumulates in mammary fat tissue. Effects of TCDD are mediated by the aryl hydrocarbon receptor (AhR). Physiological conditions of moderate hypoxia in breast cancer also activate another transcription factor, hypoxia-inducible factor-1 alpha (HIF-1alpha). In addition, the transcription factors p53 and the estrogen receptor alpha (ERalpha) are important key players in breast cancer progression. Here, human breast cancer cells cultured under mild hypoxic conditions were exposed to TCDD and analyzed for regulation of p53 signaling and ERalpha transactivation. Simultaneous exposure to TCDD and hypoxia resulted in a moderate but reproducible inhibition of p53 expression. Both the direct activation of the ERalpha and the transcriptional regulation of Hdm2 mediated this inhibition. As consequence the p53-mediated target gene expression (Dusp5) was reduced. Silencing of Dusp5 by simultaneous exposure of TCDD and hypoxia or by RNAi led to increased phosphorylation of ERK1/2. This increase resulted in transactivation of ERalpha and induction of ERalpha-mediated transcription of Hdm2 and SOCS3. Specificity of ERalpha-transactivation by ERK1/2 was confirmed by treatment with MAPKK-inhibitor PD98059. The combination of inhibition of functional p53 protein and induction of ERalpha signaling could serve as a model for the operational sequence of TCDD effects to prevent cell death and promote breast tumor progression.

TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin) is known to promote cancer initiation and progression and accumulates in mammary fat tissue. Effects of TCDD are mediated by the aryl hydrocarbon receptor (AhR). Physiological conditions of moderate hypoxia in breast cancer also activate another transcription factor, hypoxia-inducible factor-1 alpha (HIF-1alpha). In addition, the transcription factors p53 and the estrogen receptor alpha (ERalpha) are important key players in breast cancer progression. Here, human breast cancer cells cultured under mild hypoxic conditions were exposed to TCDD and analyzed for regulation of p53 signaling and ERalpha transactivation. Simultaneous exposure to TCDD and hypoxia resulted in a moderate but reproducible inhibition of p53 expression. Both the direct activation of the ERalpha and the transcriptional regulation of Hdm2 mediated this inhibition. As consequence the p53-mediated target gene expression (Dusp5) was reduced. Silencing of Dusp5 by simultaneous exposure of TCDD and hypoxia or by RNAi led to increased phosphorylation of ERK1/2. This increase resulted in transactivation of ERalpha and induction of ERalpha-mediated transcription of Hdm2 and SOCS3. Specificity of ERalpha-transactivation by ERK1/2 was confirmed by treatment with MAPKK-inhibitor PD98059. The combination of inhibition of functional p53 protein and induction of ERalpha signaling could serve as a model for the operational sequence of TCDD effects to prevent cell death and promote breast tumor progression.

Breast cancer is characterized, among others, by the concurrence of lipophilic xenobiotica such as 2,3,7,8-tetrachlorodibenzo-para-dioxin (TCDD) with hypoxic tissue conditions. This condition activates the transcription factors hypoxia inducible factor-1alpha (HIF-1alpha) and aryl hydrocarbon receptor (AhR) that are known to promote tumor progression. An interrelation between these transcription factors and nuclear factor of activated T-cells (NFAT) was implied by gene array analysis. In the present study, the interplay of the three transcription factors was studied and correlated with the migration of MCF-7 cells in response to TCDD and/or hypoxia. An AhR-activation by 10nM TCDD and HIF-1alpha activation by 5% oxygen induced activation of NFATc1. The effects were inhibited by cyclosporine A (CsA), suggesting that the activation of NFAT by AhR or HIF-1alpha signaling is calcineurin-dependent. The expression/activity of the NFAT target gene autotaxin (ATX) was increased. ATX is known to stimulate migration of tumor cells. The hydrolysis product of ATX, lysophosphatidic acid (LPA), increased the migration of MCF-7 cells under normoxia but not under hypoxia. This effect correlated with increased migration observed after TCDD treatment. Hypoxia did not promote migration of MCF-7 cells, suggesting that ATX down-stream signaling was inhibited by hypoxia. In conclusion, the TCDD-mediated activation of NFATc1 is suggested to promote cell migration via ATX/LPA-signaling.

Recent studies point to a role for adipokines in reproduction. Leptin is involved in embryo metabolism and may participate in embryo-maternal crosstalk. Little is known about potential roles of other adipokines in reproduction. We therefore studied the expression of adiponectin and pathway members during the pre- and periimplantation period in rabbits and mice. Adiponectin protein is localized in glandular epithelium of the rabbit endometrium on day 6 and 8 p.c. and in mouse endometrium on day 3.5 and 5 p.c. Rabbit, but not mice blastocysts express adiponectin mRNA. Adiponectin receptors one and two, adiponectin paralogues and PPARs were found in both species. Both, trophoblast and embryoblast were adiponectin positive. Real time PCR for adipoR1 and adipoR2 in rabbit blastocysts of different gastrulation stages at day 6 p.c. revealed a specific switch in expression: Expression was high in the trophoblast in early stages and in the embryoblast shortly prior to implantation. In conclusion, during the pre- and periimplantation period, members of the adiponectin pathway are expressed in endometrium and blastocysts, with a specific expression pattern in the embryonic disk of the gastrulating rabbit blastocyst, giving support to a role of the adipokine network in blastocyst differentiation and embryo-maternal interactions.

Hypoxia-inducible factor-1 alpha (HIF-1 alpha) and the aryl hydrocarbon receptor (AhR) work as environmental sensors in human tissues. These proteins are members of the helix-loop-helix/Per-ARNT-SIM transcription factor family and form heterodimers with the aryl hydrocarbon receptor nuclear translocator. HIF-1 alpha can be activated by low oxygen concentrations and hypoxia-inducing agents. The AhR is activated by xenobiotica such as dioxins. Here, we analyze the interference between the AhR signaling, activated by 10 nM 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD), and the HIF-1 alpha pathway, induced by hypoxia (5% O2), in two human cell lines, the breast carcinoma cell line MCF-7 and the hepatocyte cell line HepG2. In both cell lines, treatment with TCDD and hypoxia clearly reduced the stabilization of HIF-1 alpha and HRE-mediated promoter activity when compared to the induction under hypoxia alone. Because these effects were not observed after alpha-naphthoflavone treatment and HIF-1 alpha mRNA was not down-regulated, HIF-1 alpha stabilization was revealed to be the target by TCDD in an AhR-depended mechanism. Under exposure to TCDD or hypoxia, the main regulator of HIF-1 alpha stability, the prolyl hydroxylase domain containing protein 2 (PHD2) showed an increase in promoter activity, transcript numbers, and protein amount. Therefore, PHD2 expression is regulated in an AhR-dependent manner under normoxia. The AhR-dependent regulation of PHD2 under normoxia, however, is overwritten by the TCDD-mediated destabilization of HIF-1 alpha. The destabilization of HIF-1 alpha is the dominant effect causing the reduced PHD2 expression after simultaneous exposure to TCDD and hypoxia. We conclude that PHD2 does not mediate the TCDD-mediated HIF-1 alpha destabilization and does not control the interference of AhR and HIF-1 alpha pathways.

During pregnancy an adequate amino acid supply is essential for embryo development and fetal growth. We have studied amino acid composition and branched chain amino acid (BCAA) metabolism at day 6 p.c. in diabetic rabbits and blastocysts. In the plasma of diabetic rabbits the concentrations of 12 amino acids were altered in comparison to the controls. Notably, the concentrations of the BCAA leucine, isoleucine and valine were approximately three-fold higher in diabetic rabbits than in the control. In the cavity fluid of blastocysts from diabetic rabbits BCAA concentrations were twice as high as those from controls, indicating a close link between maternal diabetes and embryonic BCAA metabolism. The expression of BCAA oxidizing enzymes and BCAA transporter was analysed in maternal tissues and in blastocysts. The RNA amounts of three oxidizing enzymes, i.e. branched chain aminotransferase 2 (Bcat2), branched chain ketoacid dehydrogenase (Bckdha) and dehydrolipoyl dehydrogenase (Dld), were markedly increased in maternal adipose tissue and decreased in liver and skeletal muscle of diabetic rabbits than in those of controls. Blastocysts of diabetic rabbits revealed a higher Bcat2 mRNA and protein abundance in comparison to control blastocysts. The expression of BCAA transporter LAT1 and LAT2 were unaltered in endometrium of diabetic and healthy rabbits, whereas LAT2 transcripts were increased in blastocysts of diabetic rabbits. In correlation to high embryonic BCAA levels the phosphorylation amount of the nutrient sensor mammalian target of rapamycin (mTOR) was enhanced in blastocysts caused by maternal diabetes. These results demonstrate a direct impact of maternal diabetes on BCAA concentrations and degradation in mammalian blastocysts with influence on embryonic mTOR signalling.

In mouse blastocysts six facilitative glucose transporter isoforms (GLUT)1–4, 8 and 9 are expressed. We have used the mouse embryonic stem (ES) cell line D3 and spontaneously differentiating embryoid bodies (EB) to investigate GLUT expression and the influence of glucose during differentiation of early embryonic cells. Both ES cells and EBs (2d–20d) expressed GLUT1, 3, and 8, whereas the isoforms 2 and 4 were detectable exclusively in EBs. Differentiation-associated expression of GLUT was analyzed by double staining with stage-specific embryonic antigen (SSEA-1), cytokeratins (CK18, 19), nestin, and desmin. Similar to trophoblast cells in mouse blastocysts the outer cell layer of endoderm-like cells showed a high GLUT3 expression in early EBs. In 20-day-old EBs no GLUT3 protein and only minor GLUT3 mRNA amounts could be detected. A minimal glucose concentration of 5 mM applied during 2 and 8 days of EB culture resulted in up-regulated GLUT4, Oct-4 and SSEA-1 levels and a delay in EB differentiation. We conclude that GLUT expression depends on cellular differentiation and that the expression is modulated by glucose concentration. The developmental and glucose-dependent regulation of GLUT strongly suggests a functional role of glucose and glucose transporters in ES cell differentiation and embryonic development.

The intestinal epithelium has one of the greatest regenerative capacities in the body; however, neither stem nor progenitor cells have been successfully cultivated from the intestine. In this study, we applied an "artificial niche" of mouse embryonic fibroblasts to derive multipotent cells from the intestinal epithelium. Cocultivation of adult mouse and human intestinal epithelium with fibroblast feeder cells led to the generation of a novel type of nestin-positive cells (intestinal epithelium-derived nestin-positive cells [INPs]). Transcriptome analyses demonstrated that mouse embryonic fibroblasts expressed relatively high levels of Wnt/bone morphogenetic protein (BMP) transcripts, and the formation of INPs was specifically associated with an increase in Lef1, Wnt4, Wnt5a, and Wnt/BMP-responsive factors, but a decrease of BMP4 transcript abundance. In vitro, INPs showed a high but finite proliferative capacity and readily differentiated into cells expressing neural, pancreatic, and hepatic transcripts and proteins; however, these derivatives did not show functional properties. In vivo, INPs failed to form chimeras following injection into mouse blastocysts but integrated into hippocampal brain slice cultures in situ. We conclude that the use of embryonic fibroblasts seems to reprogram adult intestinal epithelial cells by modulation of Wnt/BMP signaling to a cell type with a more primitive embryonic-like stage of development that has a high degree of flexibility and plasticity.

Glucose is the most important energy substrate for mammalian blastocysts. Its uptake is mediated by glucose transporters (GLUT). In muscle and adipocyte cells insulin stimulates glucose uptake by activation of the insulin receptor (IR) pathway and translocation of GLUT4. GLUT4 is expressed in bovine preimplantation embryos. A new insulin-responsive isoform, GLUT8, was recently described in mouse blastocysts. Thus, potentially, two insulin-responsive isoforms are expressed in early embryos. The mechanism of insulin action on embryonic cells, however, is still not clear. In the present study expression of IR, GLUT1, 2, 3, 4, 5 and 8 was studied in rabbit preimplantation embryos using RT-PCR, Western blotting and immunohistochemistry. The rabbit mRNA sequences for the complete coding region of IR, GLUT4 and a partial GLUT8 sequence were determined by RACE-PCR and sequencing. GLUT4 was expressed in 3-day-old morulae and in 4- and 6-day-old blastocysts. IR and GLUT8 transcripts were detectable only in blastocysts. Blastocysts also expressed GLUT1 and 3, but not GLUT2 and 5. Transcript numbers of GLUT4 and 8 were higher in trophoblast than in embryoblast cells. Translation of IR, GLUT4 and 8 proteins in blastocysts was confirmed by Western blotting. GLUT4 was localized mainly in the membrane and in the perinuclear region in trophoblast cells while in embryoblast cells its localization was predominantly in the perinuclear cytoplasm. The possible function(s) of two insulin-responsive isoforms, GLUT4 and GLUT8, in rabbit preimplantation embryos needs further investigation. It may not necessarily be linked to insulin-stimulated glucose transport.