Alexandra J. Harvey

Oxygen-Regulated Gene Expression in Bovine Blastocysts

Abstract 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 HIFα 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 (HIF1α, HIF2α) was unaltered by oxygen concentration. HIF2α protein was predominantly localized to the nucleus of blastocysts. In contrast, HIF1α 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 HIF2α.

Regulation of Gene Expression in Bovine Blastocysts in Response to Oxygen and the Iron Chelator Desferrioxamine

Abstract Low (2%) oxygen conditions during postcompaction culture of bovine blastocysts improve embryo quality and are associated with small increases in the expression of glucose transporter 1 (SLC2A1), anaphase promoting complex (ANAPC1), and myotrophin (MTPN), suggesting a role for oxygen in the regulation of embryo development, mediated through oxygen-sensitive gene expression. However, bovine embryos, to at least the blastocyst stage, lack detectable levels of the key regulator of oxygen-sensitive gene expression, hypoxia-inducible 1 alpha (HIF1A), while the less well-characterized HIF2 alpha protein is readily detectable. Here we report that other key HIF1 regulated genes are not significantly altered in their expression pattern in bovine blastocysts in response to reduced oxygen concentrations postcompaction—with the exception of lactate dehydrogenase A (LDHA), which was significantly increased following 2% oxygen culture. Antioxidant enzymes have been suggested as potential HIF2 target genes, but their expression was not altered following low-oxygen culture in the bovine blastocyst. The addition of desferrioxamine (an iron chelator and inducer of HIF-regulated gene expression) during postcompaction stages significantly increased SLC2A1, LDHA, inducible nitric oxide synthase (NOS2A), and MTPN gene expression in bovine blastocysts, although development to the blastocyst stage was not significantly affected. These results further suggest that expression of genes, known to be regulated by oxygen via HIF-1 in somatic cells, is not influenced by oxygen during preimplantation postcompaction bovine embryo development. Oxygen-regulated expression of LDHA and SLC2A1 in bovine blastocysts suggests that regulation of these genes may be mediated by HIF2. Furthermore, the effect of a reduced-oxygen environment on gene expression can be mimicked in vitro through the use of desferrioxamine. These results further support our data that the bovine blastocyst stage embryo is unique in its responsiveness to oxygen compared with somatic cells, in that the lack of HIF1-mediated gene expression reduces the overall response to low (physiological) oxygen environments, which appear to favor development.

Inner Cell Mass Localization of NANOG Precedes OCT3/4 in Rhesus Monkey Blastocysts

The mechanism by which the inner cell mass (ICM) and trophectoderm (TE) become specified is poorly understood. Considerable species variation is evident in the expression of lineage-specific and embryonic stem cell (ESC) regulatory markers. We sought to investigate localization patterns of these markers in rhesus macaque compact morulae and blastocysts. NANOG protein was restricted to the ICM of blastocysts. In contrast to a previous report, the expression of CDX2 was detected in the primate blastocyst, localized specifically to the TE. Unlike the mouse embryo, OCT4 protein was detected using two different antibodies in both the ICM and TE. The ubiquitous pattern of OCT4 expression is consistent with observations in human, cow, and pig embryos. Significantly, lack of restricted OCT4 protein, and ICM localization of NANOG in primate blastocysts, suggests that NANOG may determine inner cell mass fate more specifically during primate development or may be less susceptible to culture artifacts. These results contrast markedly with current mechanistic hypotheses, although other factors may lie upstream of NANOG to constitute a complex interactive network. This difference may also underlie observations that regulatory mechanisms in ESC differ between mice and primates.

Molecular control of mitochondrial function in developing rhesus monkey oocytes and preimplantation-stage embryos.

The mitochondrion undergoes significant functional and structural changes, as well as an increase in number, during preimplantation embryonic development. The mitochondrion generates ATP and regulates a range of cellular processes, such as signal transduction and apoptosis. Therefore, mitochondria contribute to overall oocyte quality and embryo developmental competence. The present study identified, for the first time, the detailed temporal expression of mRNAs related to mitochondrial biogenesis in rhesus monkey oocytes and embryos. Persistent expression of maternally encoded mRNAs was observed, in combination with transcriptional activation and mRNA accumulation at the eight-cell stage, around the time of embryonic genome activation. The expression of these transcripts was significantly altered in oocytes and embryos with reduced developmental potential. In these embryos, most maternally encoded transcripts were precociously depleted. Embryo culture and specific culture media affected the expression of some of these transcripts, including a deficiency in the expression of key transcriptional regulators. Several genes involved in regulating mitochondrial transcription and replication are similarly affected by in vitro conditions and their downregulation may be instrumental in maintaining the mRNA profiles of mitochondrially encoded genes observed in the present study. These data support the hypothesis that the molecular control of mitochondrial biogenesis, and therefore mitochondrial function, is impaired in in vitro-cultured embryos. These results highlight the need for additional studies in human and non-human primate model species to determine how mitochondrial biogenesis can be altered by oocyte and embryo manipulation protocols and whether this affects physiological function in progeny.

Dynamic regulation of mitochondrial function in preimplantation embryos and embryonic stem cells

Mitochondrial function is dependent upon regulation of biogenesis and dynamics. A number of studies have documented the importance of these organelles in both preimplantation embryos and embryonic stem cells (ESCs), however it remains unclear how mitochondria respond to their immediate microenvironment through modulation of morphology and movement, or whether perturbations in these processes will have a significant impact following differentiation/implantation. Here we review existing literature on two key aspects of nuclear-mitochondrial cross-talk and the dynamic processes involved in mediating mitochondrial function through regulation of mitochondrial biogenesis, morphology and movement, with particular emphasis on embryos and ESCs.

Metaboloepigenetic Regulation of Pluripotent Stem Cells

The differentiation of pluripotent stem cells is associated with extensive changes in metabolism, as well as widespread remodeling of the epigenetic landscape. Epigenetic regulation is essential for the modulation of differentiation, being responsible for cell type specific gene expression patterns through the modification of DNA and histones, thereby establishing cell identity. Each cell type has its own idiosyncratic pattern regarding the use of specific metabolic pathways. Rather than simply being perceived as a means of generating ATP and building blocks for cell growth and division, cellular metabolism can directly influence cellular regulation and the epigenome. Consequently, the significance of nutrients and metabolites as regulators of differentiation is central to understanding how cells interact with their immediate environment. This review serves to integrate studies on pluripotent stem cell metabolism, and the regulation of DNA methylation and acetylation and identifies areas in which current knowledge is limited.

Effect of the oxidative phosphorylation uncoupler 2,4-dinitrophenol on hypoxia-inducible factor-regulated gene expression in bovine blastocysts.

In cattle embryos, development to the blastocyst stage is improved in the presence of 10 micro;m 2,4-dinitrophenol (DNP), an uncoupler of oxidative phosphorylation, coincident with an increase in glycolytic activity following embryonic genome activation. The present study examined redox-sensitive gene expression and embryo development in response to the addition of DNP post-compaction. 2,4-Dinitrophenol increased the expression of hypoxia-inducible factor 1alpha and 2alpha (HIF1alpha, HIF2alpha) mRNA. Although HIF1alpha protein remained undetectable in bovine blastocysts, HIF2alpha protein was localised within the nucleus of trophectoderm and inner cell mass (ICM) cells of blastocysts cultured in the presence or absence of DNP, with a slight increase in staining evident within the ICM in blastocysts cultured in the presence of DNP. However, the expression of GLUT1 and VEGF mRNA, genes known to be regulated by HIFs, was unaffected by the addition of DNP to the culture. Although the development of Grade 1 and 2 blastocysts was unaltered by the addition of DNP post compaction in the present study, a significant increase in the proportion of ICM cells was observed. Results indicate that 10 microm DNP improves the quality of bovine embryos, coincident with increased HIF2alpha protein localisation within ICM cells and increased HIFalpha mRNA levels. Therefore, the results demonstrate redox-regulated expression of HIF2.

Oxygen modulates human embryonic stem cell metabolism in the absence of changes in self-renewal

Human embryonic stem (ES) cells are routinely cultured under atmospheric oxygen (~20%), a concentration that is known to impair embryo development in vitro and is likely to be suboptimal for maintaining human ES cells compared with physiological (~5%) oxygen conditions. Conflicting reports exist on the effect of oxygen during human ES cell culture and studies have been largely limited to characterisation of typical stem cell markers or analysis of global expression changes. This study aimed to identify physiological markers that could be used to evaluate the metabolic impact of oxygen on the MEL-2 human ES cell line after adaptation to either 5% or 20% oxygen in extended culture. ES cells cultured under atmospheric oxygen displayed decreased glucose consumption and lactate production when compared with those cultured under 5% oxygen, indicating an overall higher flux of glucose through glycolysis under physiological conditions. Higher glucose utilisation at 5% oxygen was accompanied by significantly increased expression of all glycolytic genes analysed. Analysis of amino acid turnover highlighted differences in the consumption of glutamine and threonine and in the production of proline. The expression of pluripotency and differentiation markers was, however, unaltered by oxygen and no observable difference in proliferation between cells cultured in 5% and 20% oxygen was seen. Apoptosis was elevated under 5% oxygen conditions. Collectively these data suggest that culture conditions, including oxygen concentration, can significantly alter human ES cell physiology with coordinated changes in gene expression, in the absence of detectable alterations in undifferentiated marker expression.

Combined parental obesity negatively impacts preimplantation mouse embryo development, kinetics, morphology and metabolism

STUDY QUESTION Does combined parental obesity, both an obese mother and father, have a greater effect on mouse preimplantation embryo development and quality than single-parent obesity? SUMMARY ANSWER Combined parental obesity causes a greater reduction in the blastocyst rate and a greater delay to the timing of key embryonic developmental events than single-parental obesity, as well as altering embryonic characteristics, such as zona pellucida width. WHAT IS KNOWN ALREADY Maternal or paternal obesity alone are known to have significant and detrimental impacts on preimplantation embryo development. Furthermore, these early embryonic perturbations can have long-term impacts on both offspring health and further generations. This is one of the first studies to examine the effects of having both an obese mother and an obese father. STUDY DESIGN, SIZE, DURATION A cross-sectional control versus treatment mouse study of diet-induced obesity was employed, in which 300 embryos per group were generated and studied from reciprocal matings: (i) control female and control male (Lean Parented Embryos); (ii) control female and obese male (Paternal Obese Parented Embryos); (iii) obese female and control male (Maternal Obese Parented Embryos) and (iv) obese female and obese male (Combined Obese Parented embryos). Assessments of the embryonic development rate, timing of development, morphological characteristics, metabolic gene expression, metabolism and cell lineage allocation were made at selected time points and analysed in relation to parental obesity status. PARTICIPANTS/MATERIALS, SETTING, METHODS Three-week-old C57BL6 male and female mice were fed control (7% total fat) or high fat (21% total fat) diets for a minimum of 8 weeks. Females were superovulated, mated, fertilized zygotes recovered and standard mouse in vitro embryo culture performed. Time-lapse monitoring was undertaken to compare developmental timings and morphological characteristics (embryonic area and zona pellucida width) for embryos from all four reciprocal matings. Differential staining identified cell lineage allocation. Real-time quantitative RT-PCR (qRT-PCR) and microfluorescence were used to measure gene expression and metabolism (glucose consumption and lactate production), respectively, in embryos from Lean Parented and Combined Obese Parented matings. This research was completed in a University research laboratory. MAIN RESULTS AND THE ROLE OF CHANCE Blastocyst rate was reduced in Combined Obese Parented embryos when compared with both Single Obese (11% decrease for Maternal Obese Parented, P < 0.05; 15% for Paternal Obese Parented, P < 0.05) and Lean Parented embryos (25% decrease, P < 0.01). Time-lapse analysis of developmental kinetics highlighted a delay of 1 h at the 2-3 cell division, extending to 6 h delay by the blastocyst stage for Combined Obese Parented embryos (P < 0.05). A reduction in the total cell number of Combined Obese Parented blastocysts was a further manifestation of this developmental delay (P < 0.05). Zona pellucida width was reduced in Combined Obese Parented embryos (P < 0.05). Glucose consumption was increased in Combined Obese Parented embryos (P < 0.05), which was associated with the up-regulation of Glucose transporter 1 expression (P < 0.05). LIMITATIONS AND REASON FOR CAUTION This study was completed in fertile C57BL/6 mice using a well-defined model of diet-induced obesity in which embryos were fertilized in vivo. Human obesity is complex, with many causes and co-morbidities, and therefore, the impact of combined obesity would require further investigation in human settings. WIDER IMPLICATIONS OF THE FINDINGS This study demonstrates that combined parental obesity has a detrimental impact on mouse embryo development, a finding consistent with previous studies on individual parent obesity. Of note, the effect of combined parental obesity upon embryo development markers was greater than that of individual parental obesity. Plausibly, human embryos will be similarly impacted. The reduction in the blastocyst rate and delayed time to developmental events confirms that embryos of obese parents differ from those of lean parents. Allowance for this should therefore be incorporated into clinical practice when selecting the best embryo for the transfer of an obese couple. STUDY FUNDING/COMPETING INTERESTS Funding was provided by University of Melbourne research monies. M.P.G. currently holds the position of Merck Serono Lecturer of Reproductive Biology. D.K.G. received research funds from Vitrolife AB Sweden. The other authors of this manuscript have nothing to declare and no conflicts of interest.

The effects of 2,4-dinitrophenol and d-glucose concentration on the development, sex ratio, and interferon-tau (IFNT) production of bovine blastocysts

The preimplantation bovine embryo displays sexual dimorphism in glucose sensitivity and interferon‐tau (IFNT) secretion that are negated by inhibition of the pentose phosphate pathway, suggesting that the association between glucose metabolism and IFNT likely underpins the selective loss of female embryos. The aim of this study was to determine if altered glucose metabolism, through glucose supplementation and/or uncoupling oxidative phosphorylation with 2,4‐dinitrophenol (DNP), affected embryo development. Bovine blastocyst development, sex, and IFNT production were examined in embryos cultured in the presence or absence of glucose (0, 1.5, 4 mM) with or without exposure to DNP (0, 10, 100 μM) between Days 5 and 8 post‐fertilization. The absence or presence of high (4 mM) glucose reduced blastocyst development and favored the development of male embryos (P < 0.001). DNP at 10 μM had no effect, whereas 100 μM had a negative impact on blastocyst development. Notably, in the presence or even absence of glucose, supplementation with 10 μM DNP further skewed the sex ratio toward males (P < 0.05). Sexually dimorphic IFNT production was maintained in these conditions, although total production was reduced in the presence of high glucose and DNP, irrespective of embryo sex. These data suggest that the pentose phosphate pathway can modulate embryonic sex ratio and development. Therefore, bovine embryo culture should be undertaken in a low glucose (<2.5 mM) medium to minimize potential embryonic stress, as higher concentrations have sexually dimorphic effects on development and an embryos ability to signal to the maternal reproductive tract. Mol. Reprod. Dev. 83: 50–60, 2016.