Glyoxalase 1 expression is downregulated in preimplantation blastocysts of diabetic rabbits

Abstract

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.

Elimination of apoptotic cells by non-professional embryonic phagocytes can be stimulated or inhibited by external stimuli

“…The cell line was produced by the fusion of human normal invasive extravillous cytotrophoblasts from Chorion laeve with AC1-1, an HGPRT-negative mutant of the choriocarcinoma cell line JEG-3 50 . The Ac-1M88 cells were cultured in vitro under standard conditions (humidi ed atmosphere with 5% CO 2 and 37°C) in DMEM (DMEM Glutamax F12, Gibco) with 10% FBS to 70 % con uency 51 . Then they were cultured in fresh media with/without the presence of actinomycin D at 4 nM concentration or ba lomycin A1 at 0.01 nM, 0.1 nM, 0.2 nM, 0.5 nM and 2.0 nM concentrations for 24 h. Analysis of internalisation of apoptotic cells in mouse blastocysts and the human Ac-1M88 cell line…”

Rabbit as an Aging Model in Reproduction: Advanced Maternal Age Alters GLO1 Expression in the Endometrium at the Time of Implantation

“…Furthermore, the GLO1 enzyme is active in rabbit blastocysts as early as during preimplantation. The GLO1 amount decreases under pathophysiological conditions of maternal diabetes mellitus in embryos [29].…”

“…For GLO1 the amplification and quantification forward (5 -ACCCCAGCACCAAGGATTTT-3 ) and reverse primer (5 -GTGCCCCAATTGTGTGTCAG-3 ) were designed based on the rabbit GLO1 gene sequence (Gene ID: 100,354,439 NCBI, accession number: XM_002714686) by Primer-BLAST. PCR products were sequenced and checked for specificity as described previously by Seeling et al [29].…”

“…The activity of GLO1 was measured spectrophotometrically in accordance with Bélanger et al [35]. A total amount of 10 µg isolated protein per sample were loaded onto an UV microplate (Corning Inc., Corning, NY, USA) and 200 µL of reaction mix was added, as previously described in Seeling et al [29]. The change in absorbance (∆E) at 240 nm was measured every minute for one hour at 37 • C, to allow the reaction to reach saturation.…”

Post-Translational Modifications and Diabetes

“…The enzyme glyoxalase (GLO) 1 is known to detoxify the reactive α-dicarbonyls and safeguard against modifications of proteins by advanced glycated end products (AGEs). It was shown that maternal diabetes affects the activity of GLO1 by post-translational modification due to changes in the metabolic processes in the embryos [ 15 ]. Better understanding of the potential roles of PTMs in diabetes is providing insights into the molecular mechanisms that can be targeted to develop antidiabetic drugs [ 16 ].…”