Karen L. Kind

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.

Beyond oxygen: complex regulation and activity of hypoxia inducible factors in pregnancy

In the first trimester the extravillous cytotrophoblast cells occlude the uterine spiral arterioles creating a low oxygen environment early in pregnancy, which is essential for pregnancy success. Paradoxically, shallow trophoblast invasion and defective vascular remodelling of the uterine spiral arteries in the first trimester may result in impaired placental perfusion and chronic placental ischemia and hypoxia later in gestation leading to adverse pregnancy outcomes. The hypoxia inducible factors (HIFs) are key mediators of the response to low oxygen. We aimed to elucidate mechanisms of regulation of HIFs and the role these may play in the control of placental differentiation, growth and function in both normal and pathological pregnancies. The Pubmed database was consulted for identification of the most relevant published articles. Search terms used were oxygen, placenta, trophoblast, pregnancy, HIF and hypoxia. The HIFs are able to function throughout all aspects of normal and abnormal placental differentiation, growth and function; during the first trimester (physiologically low oxygen), during mid-late gestation (where there is adequate supply of blood and oxygen to the placenta) and in pathological pregnancies complicated by placental hypoxia/ischemia. During normal pregnancy HIFs may respond to complex alterations in oxygen, hormones, cytokines and growth factors to regulate placental invasion, differentiation, transport and vascularization. In the ever-changing environment created during pregnancy, the HIFs appear to act as key mediators of placental development and function and thereby are likely to be important contributors to both normal and adverse pregnancy outcomes.

Placental control of fetal growth

The placenta exerts its effects on the growth of the fetus from the beginning of pregnancy via metabolic and endocrine mechanisms. To achieve this, the placenta exchanges a wide array of nutrients, endocrine signals, cytokines and growth factors with the mother and the fetus. These exchanges modulate or programme fetal growth and development. This review concentrates on the function and structure of the placenta in humans and in animals, and the effects of experimental perturbation of placental size and function on fetal growth. The consequences for fetal growth of varying the abundance of peptides or, by deleting genes, insulin-like growth factors or cytokines, are also described. Maternal nutritional and hormonal state from as early as the first few days after fertilization, can influence the growth rate of the placenta and the fetus and also the length of gestation. Influences on placental development and their consequences will clearly have an impact on the placental control of fetal growth. Variations in the maternal environment and consequent perturbation of the metabolic and endocrine environment of the placenta and fetus are implicated as being responsible for the associations between prenatal growth of the placenta and its fetus and the subsequent risk of adult disease. The next challenge will be to determine the dominant influences at each stage of fetal and placental growth.

Restricted fetal growth and the response to dietary cholesterol in the guinea pig

Epidemiological studies suggest that retarded growth before birth is associated with increased plasma total and low-density lipoprotein (LDL) cholesterol concentrations in adult life. Thus perturbations of prenatal growth may permanently alter cholesterol metabolism. To determine directly whether restriction of prenatal nutrition and growth alters postnatal cholesterol homeostasis, the plasma cholesterol response to cholesterol feeding (0.25% cholesterol) was examined in adult guinea pig offspring of ad libitum-fed or moderately undernourished mothers. Maternal undernutrition (85% ad libitum intake throughout pregnancy) reduced birth weight (-13%). Plasma total cholesterol was higher prior to and following 6 wk cholesterol feeding in male offspring of undernourished mothers compared with male offspring of ad libitum-fed mothers ( P< 0.05). The influence of birth weight on cholesterol metabolism was examined by dividing the offspring into those whose birth weight was above (high) or below (low) the median birth weight. Plasma total cholesterol concentrations prior to cholesterol feeding did not differ with size at birth, but plasma total and LDL cholesterol were 31 and 34% higher, respectively, following cholesterol feeding in low- compared with high-birth weight males ( P < 0.02). The response to cholesterol feeding in female offspring was not altered by variable maternal nutrition or size at birth. Covariate analysis showed that the effect of maternal undernutrition on adult cholesterol metabolism could be partly accounted for by alterations in prenatal growth. In conclusion, maternal undernutrition and small size at birth permanently alter postnatal cholesterol homeostasis in the male guinea pig.

Effect of maternal feed restriction on blood pressure in the adult guinea pig

Small size at birth has been associated with increased blood pressure in adult men and women. In rats, isocaloric protein restriction reduces fetal growth and increases systolic blood pressure in adult offspring. Balanced maternal undernutrition in the rat also increases adult blood pressure, but not consistently. The aim of this study was to determine the effect of moderate balanced maternal undernutrition (85% of ad libitum intake from 4 weeks before, and throughout pregnancy) on blood pressure of adult offspring in the guinea pig, a species that is relatively mature at birth. Blood pressure was measured in chronically catheterised offspring of ad libitum fed or feed‐restricted mothers, at 3 months of age (young adult). Maternal feed restriction reduced birth weight (‐17%) and increased systolic blood pressure (+9%, P < 0.03) in young adult male offspring. In offspring of ad libitum fed and feed‐restricted mothers, combined data showed that systolic blood pressure and mean arterial pressure correlated negatively with head width at birth (P = 0.02 and P = 0.04, respectively, n = 28). Systolic blood pressure also correlated negatively with birth weight and the ratio birth weight/birth length, but only in offspring of ad libitum fed mothers (P = 0.04 and P = 0.03, respectively, n = 22). The effect of maternal feed restriction on systolic blood pressure in male offspring was not significant when adjusted for these measures of size at birth. Thus, moderate balanced undernutrition in the guinea pig increases systolic blood pressure in young adult male offspring; however, these effects may be mediated, at least in part, through effects on fetal growth.

Embryo culture and long-term consequences

The development ofpre-elongation (ruminants) and preimplantation (e.g. mouse and humans) embryos ex vivo has evolved over the past four decades into a reliable technology that is used as a research tool in developmental biology, as well as other embryo technologies, for application in infertility treatment, species conservation and selective breeding. It is clear from a variety of embryo culture studies that adaptive responses by embryos during culture can lead to significant alterations in subsequent developmental profiles, the mechanisms of which are not entirely clear but are unlikely to be limited to a single mechanism because this does not account for the variability seen in responses and the emerging list of specific cellular stressors that cause long-term deviations in fetal development. Epigenetic mechanisms, especially deviation of methylation patterns, and adaptation via causal pathways linking gene expression signalling with critical developmental time points, especially of placental development, are two candidates. Observational studies on post-transfer consequences must now be designed so that specific candidate pathways are followed to elucidate their role in perturbed development following transfer.

Effect of culturing mouse embryos under different oxygen concentrations on subsequent fetal and placental development.

The oxygen concentration used during embryo culture can influence embryo development and quality. Reducing the oxygen concentration in the atmosphere to 2% during post‐compaction culture of mouse embryos perturbs embryonic gene expression. This study examined the effect of culturing mouse embryos under different oxygen concentrations on subsequent fetal and placental development. Embryos were cultured from the zygote to morula stage under 7% oxygen, followed by 20, 7 or 2% oxygen to the blastocyst stage. Cultured and in vivo developed blastocysts were transferred into pseudopregnant recipients. Fetal and placental outcomes were analysed at day 18 of pregnancy. Implantation rate was not influenced by embryo culture conditions, but resorption rates were increased in embryos cultured under 2% oxygen, compared with 7% oxygen. Day 18 fetal weights were reduced following culture under 2%, compared with 7 or 20% oxygen, or in vivo development. Placental weight was not influenced by culture conditions. No differences in the proportion of junctional or labyrinthine exchange regions within the placenta or the morphometry of the labyrinthine region were detected. Surface density (surface area/gram labyrinth) of trophoblast available for exchange was reduced in placentas developed from embryos cultured under 2% oxygen, compared with 7% oxygen. Placental gene expression of Slc2a1, Slc2a3, Igf2, Igf2r and H19 was not influenced by oxygen conditions during embryo culture. Thus, exposure to 2% oxygen during post‐compaction pre‐implantation embryo development has adverse consequences for fetal development in the mouse. Oxygen is a significant component of the embryonic environment and reductions in oxygen availability can influence both embryonic gene expression and subsequent fetal development.

Association of -3826 G Variant in uncoupling protein-1 with increased BMI in overweight Australian women

Aims/hypothesis. To determine whether genetic variation in uncoupling protein-1 (UCP-1) is associated with obesity or obesity-related risk factors in overweight women.¶Methods. We genotyped 526 overweight/obese women (mean body mass index 34.1 kg/m2, range 25.0 to 47.5 kg/m2) for the –3826 A→G uncoupling protein-1 polymorphism. Of the 526 women genotyped 144 had fasting blood samples analysed for glucose and lipid measurements.¶Results. The –3826 G allele was found with a frequency of 0.23 and was associated with higher BMI (p = 0.02). A higher frequency of this polymorphism (0.33) was found in subjects with Type II (non-insulin-dependent) diabetes mellitus (p = 0.02), though adjustment for BMI weakened this significance (p = 0.06). The –3826 G variant was associated with increased fasting glucose (p = 0.01). This was, however, a result of a greater proportion of women with Type II diabetes also having the G variant (p = 0.10, adjusted for Type II diabetes). The –3826 G variant of uncoupling protein-1 did not have an effect on other metabolic variables associated with obesity.¶Conclusion/interpretation. In overweight Australian women the –3826 G variant of UCP-1 increased the susceptibility to obesity indicating that UCP-1 could be involved in weight regulation. [Diabetologia (2000) 43: 242–244]

Food restriction alters pregnancy-associated changes in IGF and IGFBP in the guinea pig

The effect of moderate food restriction on pregnancy-associated changes in weight gain, body composition, and circulating insulin-like growth factors (IGF) I and II and IGF-binding proteins (IGFBP)-1 through-4 and their relationship was determined in the guinea pig. Pregnancy did not stimulate weight gain but reduced fat deposition in ad libitum-fed animals and increased weight gain and fat deposition in food-restricted animals relative to their respective virginal group. Pregnancy increased the abundance of circulating IGF-I regardless of food intake and increased that of IGF-II in food-restricted animals only. Pregnancy also increased circulating IGFBP-1 and -2 in ad libitum-fed and food-restricted animals and IGFBP-4 in ad libitum-fed animals. Multiple regression analysis showed that maternal weight gain was negatively associated with circulating IGF-II and IGFBP-2. Fetal weight was positively associated with maternal circulating IGF-II and negatively associated with maternal circulating IGFBP-1 and -2. Significant interactions indicate, however, that the role of IGF-II and IGFBP-1 on fetal growth is dependent on the nutritional status of the mother.The effect of moderate food restriction on pregnancy-associated changes in weight gain, body composition, and circulating insulin-like growth factors (IGF) I and II and IGF-binding proteins (IGFBP)-1 through -4 and their relationship was determined in the guinea pig. Pregnancy did not stimulate weight gain but reduced fat deposition in ad libitum-fed animals and increased weight gain and fat deposition in food-restricted animals relative to their respective virginal group. Pregnancy increased the abundance of circulating IGF-I regardless of food intake and increased that of IGF-II in food-restricted animals only. Pregnancy also increased circulating IGFBP-1 and -2 in ad libitum-fed and food-restricted animals and IGFBP-4 in ad libitum-fed animals. Multiple regression analysis showed that maternal weight gain was negatively associated with circulating IGF-II and IGFBP-2. Fetal weight was positively associated with maternal circulating IGF-II and negatively associated with maternal circulating IGFBP-1 and -2. Significant interactions indicate, however, that the role of IGF-II and IGFBP-1 on fetal growth is dependent on the nutritional status of the mother.

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.