Low oxygen conditions (2%) during post-compaction culture of bovine blastocysts improve embryo quality, which is associated with a small yet significant increase in the expression of glucose transporter 1 (GLUT-1), suggesting a role of oxygen in embryo development mediated through oxygen-sensitive gene expression. However, bovine embryos to at least the blastocyst stage lack a key regulator of oxygen-sensitive gene expression, hypoxia-inducible factor 1alpha (HIF1alpha). A second, less well-characterized protein (HIF2alpha) is, however, detectable from the 8-cell stage of development. Here we use differential display to determine additional gene targets in bovine embryos in response to low oxygen conditions. While development to the blastocyst stage was unaffected by the oxygen concentration used during post-compaction culture, differential display identified oxygen-regulation of myotrophin and anaphase promoting complex 1 expression, with significantly lower levels observed following culture under 20% oxygen than 2% oxygen. These results further support the hypothesis that the level of gene expression of specific transcripts by bovine embryos alters in response to changes in the oxygen environment post-compaction. Specifically, we have identified two oxygen-sensitive genes that are potentially regulated by HIF2 in the bovine blastocyst.

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.

The addition of insulin during in vitro culture has beneficial effects on rabbit preimplantation embryos leading to increased cell proliferation and reduced apoptosis. We have previously described the expression of the insulin receptor (IR) and the insulin-responsive glucose transporters (GLUT) 4 and 8 in rabbit preimplantation embryos. However, the effects of insulin on IR signaling and glucose metabolism have not been investigated in rabbit embryos. In the present study, the effects of 170 nM insulin on IR, GLUT4 and GLUT8 mRNA levels, Akt and Erk phosphorylation, GLUT4 translocation and methyl glucose transport were studied in cultured day 3 to day 6 rabbit embryos. Insulin stimulated phosphorylation of the mitogen-activated protein kinase (MAPK) Erk1/2 and levels of IR and GLUT4 mRNA, but not phosphorylation of the phosphatidylinositol 3-kinase-dependent protein kinase, Akt, GLUT8 mRNA levels, glucose uptake or GLUT4 translocation. Activation of the MAPK signaling pathway in the absence of GLUT4 translocation and of a glucose transport response suggest that in the rabbit preimplantation embryo insulin is acting as a growth factor rather than a component of glucose homeostatic control.

IL-4 receptor signaling is supposed to play a major role in anti-inflammatory polarization and proliferation of adipose tissue macrophages. In this study, we examined the metabolic and inflammatory phenotype of C57BL/6J mice (IIl4ra) with LysM-dependent knockout (IIl4raΔmyel) of the IL-4 receptor α-chain (IL-4Rα), the mandatory signaling component of IL-4 and IL-13, on chow and high-fat diet. Lean IIl4raΔmyel mice showed decreased insulin sensitivity, no divergent adipose tissue macrophage polarization, but an increased percentage of CD8+ T cells in visceral adipose tissue. After 20 wk of a high-fat diet, IIl4raΔmyel mice exhibited higher glucose tolerance, no changes in the lymphocyte compartment and fewer M1 macrophages in visceral adipose tissue. In vivo adipose tissue macrophage proliferation measured by BrdU incorporation was unaffected by Il4ra knockout. Interestingly, we show that IL-4Rα signaling directly augmented Itgax (Cd11c) gene expression in bone marrow–derived macrophages and increased the amount of CD11c+ macrophages in adipose tissue explants. Myeloid cell–specific knockout of Il4ra deteriorated insulin sensitivity in lean mice but improved parameters of glucose homeostasis and partially protected from adipose tissue inflammation in obese mice. Hence, IL-4Rα signaling probably plays a minor role in maintaining the macrophage M2 population and proliferation rates in vivo. Moreover, our data indicate that IL-4 signaling plays a proinflammatory role in adipose tissue inflammation by directly upregulating CD11c on adipose tissue macrophages.

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Oxygen-regulated gene expression in the bovine embryo contrasts markedly with that observed in the mouse. Under low (2%) oxygen moderate changes in gene expression are observed in the bovine blastocyst, compared with 3- to 4-fold increases in the mouse. We have determined that these moderate gene expression changes are most likely regulated by Hypoxia-Inducible Factor (HIF)-2 transcription factor activity in the bovine, in the absence of HIF1, although HIF2 target genes are largely unknown. The aim of this study was to screen, by differential display RT-PCR, for putative oxygen-regulated transcripts that might confer developmental competence in blastocysts cultured under varying oxygen atmospheres post compaction. In vitro-produced bovine blastocysts were generated using standard protocols. Compact morulae were randomly allocated to treatments under either 2%, 7% or 20% oxygen for 72 h from Day 5. Blastocyst RNA was isolated using TriReagent and samples were reverse transcribed using Superscript II. cDNA was amplified using 10-mer primers in reactions containing 32Pα-labelled dCTP. Resulting bands were detected by autoradiography, excised, purified and ligated into pGEMT vectors for transformation and sequencing. Seven clones were identified as having high homology with known sequences in GenBank. Real-time PCR was undertaken to confirm oxygen-regulation using Sybr green master mix. Myotrophin mRNA was significantly increased following 2% oxygen culture, compared with 20% cultured blastocysts (P�<�0.01), as was GLUT1 (P�<�0.01). The expression of anaphase-promoting complex showed a significant association with oxygen, being higher in 2% cultured blastocysts (P�<�0.05). Acetyl-coA-acetyltransferase I, chronic myelogenous leukemia tumor antigen (CML66), cyclin I, NADH dehydrogenase subunit 2 and ribonucleotide reductase M1, genes identified using differential display, were not altered by post compaction oxygen concentration. This study has identified potentially HIF2-specific regulated genes, and supports the hypothesis that reduced oxygen concentrations post-compaction may influence bovine embryo development through oxygen-regulated changes in gene expression.