Dynamic Oxygen Conditions Promote the Translocation of HIF-1 α to the Nucleus in Mouse Blastocysts

Abstract

Oxygen tension is one of the most critical factors for mammalian embryo development and its survival. The HIF protein is an essential transcription factor that activated under hypoxic conditions. In this study, we evaluated the effect of dynamic oxygen conditions on the expression of embryonic genes and translocation of hypoxia-inducible factor-1α (HIF-1α) in cultured mouse blastocysts. Two-pronuclear (2PN) zygotes harvested from ICR mice were subjected to either high oxygen (HO; 20%), low oxygen (LO; 5%), or dynamic oxygen (DO; 5% to 2%) conditions. In the DO group, PN zygotes were cultured in 5% O₂ from days 1 to 3 and then in 2% O₂ till day 5 after hCG injection. On day 5, the percentage of blastocysts in the cultured embryos from each group was estimated, and the embryos were also subjected to immunocytochemical and gene expression analysis. We found that the percentage of blastocysts was similar among the experimental groups; however, the percentage of hatching blastocysts in the DO and LO groups was significantly higher than that in the HO group. The total cell number of blastocysts in the DO group was significantly higher than that of both the HO and LO groups. Further, gene expression analysis revealed that the expression of genes related to the embryonic development was significantly higher in the DO group than that in the HO and LO groups. Interestingly, HIF-1α mRNA expression did not significantly differ; however, HIF-1α protein translocation from the cytoplasm to the nucleus was significantly higher in the DO group than in the HO and LO groups. Our study suggests that dynamic oxygen concentrations increase the developmental capacity in mouse preimplantation embryos through activation of the potent transcription factor HIF-1α.

Figure 1

Immunocytochemistry for Oct-4 and DAPI staining for nuclei in blastocysts of in vitro culture with HO (20%), LO (5%), and DO (5% to 2%) conditions. Representative fluorescent microscopy images of nucleus with (a, d, g) DAPI (blue), (b, e, h) FITC-labeled Oct-4 (green), and (c, f, i) merged images. Scale bars indicate 50 μm.

Figure 2

The mRNA expressions of embryonic development- and oxygen-related genes in blastocysts of in vitro culture with HO (20%), LO (5%), and DO (5% to 2%) conditions. The qRT-PCR analysis of (a) caudal-type homeobox 2 (Cdx-2), (b) octamer-binding transcription factor 4 (Oct-4), (c) H2A.Z variant histone 1 (H2az1), (d) hypoxia-inducible factor-1α (HIF-1α), (e) manganese superoxide dismutase (MnSOD), and (f) 16s ribosomal RNA (16s rRNA) in blastocysts of in vitro culture with HO, LO, and DO conditions. Values are represented as mean ± SEM for each group and analyzed by one-way ANOVA using Tukey's multiple comparison test. Different letters (a, b, c) indicate statistically significant differences (p < 0.05).

Figure 3

Translocation of HIF-1α from the cytoplasm into the nucleus in mouse blastocysts of in vitro culture with HO (20%), LO (5%), and DO (5% to 2%) conditions. (a) Quantitative analysis of HIF-1α translocation ratio in epi-mounted immunofluorescence images by nuclear to total cell intensity. Values are represented as mean ± SEM for each group and analyzed by one-way ANOVA using Tukey's multiple comparison test. Different letters indicate statistically significant differences (p < 0.05). (b) Representative confocal microscopy images of the nucleus with DAPI (A, D, G, J), Alexa 488-labeled HIF-1α (B, E, H, K), and merged images (C, F, I, L). Scale bars indicated 50 μm.