RESUMEN
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.
Asunto(s)
Blastocisto/metabolismo , Diabetes Mellitus Experimental/metabolismo , Lactoilglutatión Liasa/genética , Lactoilglutatión Liasa/metabolismo , Animales , Blastocisto/enzimología , Línea Celular , Diabetes Mellitus Experimental/enzimología , Diabetes Mellitus Experimental/genética , Femenino , Glioxal/farmacología , Humanos , Hiperglucemia/metabolismo , Embarazo , Piruvaldehído/farmacología , Conejos , TrofoblastosRESUMEN
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 in in vitro cultured preimplantation rabbit blastocysts and in 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: gene SLC7A8) and proliferation (proliferating cell nuclear antigen; PCNA) in a mTORC1-dependent manner. Phosphorylation of S6K1 and expression patterns of CCND1 and EIF4E were increased in embryos from diabetic rabbits, while the expression of proliferation marker PCNA was 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.
Asunto(s)
Blastocisto/patología , Diabetes Mellitus Experimental/complicaciones , Diabetes Mellitus Tipo 1/complicaciones , Hiperamonemia/etiología , Hiperglucemia/etiología , Complejos Multiproteicos/metabolismo , Serina-Treonina Quinasas TOR/metabolismo , Animales , Blastocisto/metabolismo , Western Blotting , Células Cultivadas , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/patología , Diabetes Mellitus Tipo 1/metabolismo , Diabetes Mellitus Tipo 1/patología , Femenino , Hiperamonemia/metabolismo , Hiperamonemia/patología , Hiperglucemia/metabolismo , Hiperglucemia/patología , Diana Mecanicista del Complejo 1 de la Rapamicina , Complejos Multiproteicos/genética , Fosforilación , Embarazo , ARN Mensajero/genética , Conejos , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal , Serina-Treonina Quinasas TOR/genéticaRESUMEN
Our understanding of the signalling pathways regulating early human development is limited, despite their fundamental biological importance. Here, we mine transcriptomics datasets to investigate signalling in the human embryo and identify expression for the insulin and insulin growth factor 1 (IGF1) receptors, along with IGF1 ligand. Consequently, we generate a minimal chemically-defined culture medium in which IGF1 together with Activin maintain self-renewal in the absence of fibroblast growth factor (FGF) signalling. Under these conditions, we derive several pluripotent stem cell lines that express pluripotency-associated genes, retain high viability and a normal karyotype, and can be genetically modified or differentiated into multiple cell lineages. We also identify active phosphoinositide 3-kinase (PI3K)/AKT/mTOR signalling in early human embryos, and in both primed and naïve pluripotent culture conditions. This demonstrates that signalling insights from human blastocysts can be used to define culture conditions that more closely recapitulate the embryonic niche.
Asunto(s)
Autorrenovación de las Células/fisiología , Células Madre Embrionarias Humanas/metabolismo , Factor I del Crecimiento Similar a la Insulina/metabolismo , Activinas/metabolismo , Animales , Blastocisto/metabolismo , Diferenciación Celular/efectos de los fármacos , Células Cultivadas , Técnicas de Cocultivo , Medios de Cultivo/química , Medios de Cultivo/metabolismo , Medios de Cultivo/farmacología , Endodermo/citología , Endodermo/metabolismo , Membranas Extraembrionarias/citología , Membranas Extraembrionarias/metabolismo , Fibroblastos/citología , Regulación del Desarrollo de la Expresión Génica , Células Madre Embrionarias Humanas/citología , Humanos , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Células Madre Pluripotentes Inducidas/fisiología , Ratones , Fosfatidilinositol 3-Quinasas/metabolismo , Receptor IGF Tipo 1/metabolismo , Transducción de Señal , Serina-Treonina Quinasas TOR/metabolismo , Transcriptoma , Inactivación del Cromosoma X/fisiologíaRESUMEN
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.
Asunto(s)
Diabetes Mellitus Experimental/genética , Regulación hacia Abajo/genética , MicroARNs/genética , Ribonucleasa III/antagonistas & inhibidores , Trofoblastos/metabolismo , Animales , Blastocisto/efectos de los fármacos , Blastocisto/metabolismo , Células Cultivadas , Diabetes Mellitus Experimental/sangre , Regulación hacia Abajo/efectos de los fármacos , Embrión de Mamíferos/efectos de los fármacos , Embrión de Mamíferos/metabolismo , Endometrio/efectos de los fármacos , Endometrio/metabolismo , Femenino , Glucosa/farmacología , Insulina/farmacología , MicroARNs/sangre , Placenta/efectos de los fármacos , Placenta/metabolismo , Embarazo , Precursores del ARN/genética , Precursores del ARN/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Conejos , Ribonucleasa III/genética , Ribonucleasa III/metabolismo , Análisis de Secuencia de ARN , Trofoblastos/efectos de los fármacosRESUMEN
The incidence of diabetes mellitus for young people rises since years. A preconceptional diabetes mellitus leads to subfertility. Most of the causes for a diabetic subfertility are still unknown. Stress can significantly deteriorate glycemic control in diabetes. Several mechanisms by which "stress hormones", like adrenaline and cortisol or corticosterone, can contribute to the regulation of glucose homeostasis have been identified. Using reverse transcription-polymerase chain reaction (RT-PCR) and quantitative real-time RT-PCR, we examined the expression of adrenergic receptors and the glucocorticoid receptor transcripts in the female rabbit reproductive tract and in gastrulating blastocysts developed in normoinsulinemic mothers and in mothers with experimentally induced diabetes mellitus type 1. The glucocorticoid receptor expression was detected in the reproductive tract as well as in gastrulating blastocysts at a high level. In maternal endometrium, α1D-, α2A-, ß1-, and ß2-adrenergic receptors were expressed, whereby ß1 transcript was not detectable in the endometrium from diabetic mothers. In preimplantation embryos, all 9 adrenergic receptors were expressed, most of them predominantly in the embryoblast. A maternal diabetes mellitus altered α2A-adrenergic receptor expression in the blastocyst and reversed the ratio of α2A transcript quantity between embryoblast and trophoblast. Our results show that the maternal reproductive tract and the preimplantation embryo express a distinct pattern of the stress response system. Alterations in the pattern and/or in functionality are likely linked to subfertility in diabetes mellitus.