This work was supported by the Business for Cooperative R&D between Industry, Academy, and Research Institute, funded by the Korea Small and Medium Business Administration in 2018 (Grants No. 17-05).
We aimed to evaluate the effects of different oxygen conditions (20% [high O2], 5% [low O2] and 5% decreased to 2% [dynamic O2]) on mouse pre- and peri-implantation development using a novel double-channel gas supply (DCGS) incubator (CNC Biotech Inc.) to alter the oxygen concentration during
The high-O2 and low-O2 groups were cultured from the one-cell to the blastocyst stage under 20% and 5% oxygen concentrations, respectively. In the dynamic-O2 group, mouse embryos were cultured from the one-cell to the morula stage under 5% O2 for 3 days, followed by culture under 2% O2 to the blastocyst stage. To evaluate peri-implantation development, the blastocysts from the three groups were individually transferred to a fibronectin-coated dish and cultured to the outgrowth stage in droplets.
The blastocyst formation rate was significantly higher in the low-O2 and dynamic-O2 groups than in the high-O2 group. The total cell number was significantly higher in the dynamic-O2 group than in the low-O2 and high-O2 groups. Additionally, the apoptotic index was significantly lower in the low-O2 and dynamic-O2 groups than in the high-O2 group. The trophoblast outgrowth rate and spread area were significantly higher in the low-O2 and dynamic-O2 groups than in the high-O2 group.
Our results showed that a dynamic oxygen concentration (decreasing from 5% to 2%) had beneficial effects on mouse pre- and peri-implantation development. Optimized, dynamic changing of oxygen concentrations using the novel DCGS incubator could improve the developmental competence of
Oxygen is an essential physiological component for regulating embryonic development in the environments of the oviduct and the uterus. Oxygen concentration impacts the rate of embryonic development and the quality of
The physiological oxygen concentration in the mammalian oviduct is around 5% [
Recently, new suggestions have emerged regarding whether a further reduction of oxygen concentration on day 3 after fertilization represents a physiological condition that is more similar to
In the present study, we hypothesized that sequential exposure to a 5% oxygen concentration from day 1 to day 3 (the pre-compaction stage), and then to a 2% oxygen concentration from day 3 to day 5 (the post-compaction stage), may improve developmental competence compared with continuous exposure to either a 5% or a 20% oxygen concentration. This study was performed to evaluate the effects of dynamic conditions using a novel double-channel gas supply (DCGS) incubator (CNC Biotech Inc., Suwon, Korea) on mouse preand peri-implantation development.
This study was approved by the Eulji University Institutional Animal Care and Use Committee (No. EUIACUC 17-14). The protocol for superovulated mice was described by Park et al. [
Once 19 hours had passed after mating, female mice with a confirmed vaginal plug were sacrificed by cervical dislocation, and cumulus-enclosed one-cell embryos (zygotes) were retrieved from the oviductal ampullae. Then, the zygotes were denuded by incubation for 1 minute with 0.1% hyaluronidase (Sigma-Aldrich, St. Louis, MO, USA) in phosphate-buffered saline. The zygotes were pooled and washed three times in Continuous Single Culture-NX (CSCM-NX; FUJIFILM Irvine Scientific, Santa Ana, CA, USA) with 10% human serum albumin (HSA; Irvine). The five healthy zygotes were cultured in 10 μL of CSCM medium with 10% HSA covered with mineral oil for 4 days under three different oxygen conditions.
We used a newly developed incubator with the DCGS system (
Apoptotic cells were detected using a fluorescein isothiocyanateconjugated
The blastocysts were transferred to a fibronectin-coated dish and outgrown to examine the effects of different oxygen concentrations on peri-implantation embryonic development. The morphological changes during peri-implantation development were observed every 24 hours, and the captured images were analyzed. The area of trophoblastic outgrowth was measured after 72 hours using ImageJ software (National Institutes of Health, Bethesda, MD, USA) as described previously [
All experiments were performed at least in triplicate. All comparisons between groups were determined by one-way analysis of variance. Tukey honest significant difference post hoc test was used for all comparisons between groups. A
Mouse embryonic development rates were assessed following culture for 24 hours (constituting development to the two-cell stage) and 96 hours (constituting development to the blastocyst stage), and are presented in
The numbers of total cells and apoptotic nuclei in the blastocysts from each group were determined using Hoechst and TUNEL staining (
The developmental competence of peri-implantation embryos cultured under different oxygen concentrations was assessed using an
This study demonstrated that the
Oxygen concentration affects the rate of
In a previous report, bovine embryos were cultured at various oxygen concentrations (0%, 1%, 2%, 4%, and 7%), and these embryos displayed the highest blastocyst development rate under the 2% oxygen concentration condition [
Oxygen concentrations during the
Our study, unlike most previous studies, developed and applied a novel DCGS incubator to automatically change the oxygen concentration. This experiment incorporated sequential exposure to a 5% oxygen concentration from day 1 to day 3 (the pre-compaction stage), and then to a 2% oxygen concentration from day 3 to day 5 (the post-compaction stage), which was determined in consideration of the physiological oxygen conditions of the female reproductive tract. This DCGS incubator system may be advantageous in that it can automatically change the oxygen concentration from 5% to 2% without any interference or adaptation time. In this study, the total cell number of blastocysts was significantly higher in the dynamic-O2 (5% to 2%) group compared with the low-O2 (5%) and high-O2 (20%) groups. The apoptotic index of the dynamic-O2 (5% to 2%) group was also the lowest of the three groups. In contrast, Yang et al. [
Blastocyst outgrowth has been used as an
In conclusion, the results of this study demonstrate the beneficial effects of dynamic oxygen concentrations, shifting from 5% to 2% on day 3. This may be related to the physiological conditions experienced by preimplantation embryos in the uterus. We recommend that future studies include more samples, and we posit that embryo transfer
No potential conflict of interest relevant to this article was reported.
Conceptualization: JHJ, KWC. Data curation: SCL, JL, HCS. Method ology: SCL, JL, HCS, JHJ. Project administration: JHJ, KWC. Writing - original draft: SCL, JHJ. Writing - review & editing: JL, HCS, KWC.
Newly developed incubator with a double-channel gas supply (DCGS) system. (A) Image of a DCGS incubator with eight chambers and a touch-panel screen. (B) Main screen of the DCGS incubator that displays the temperature, incubation time, and gas concentration of each chamber. (C) Screen for controlling the first gas supply of each chamber. (D) Screen for controlling the second gas supply of each chamber.
Assessment of apoptotic cells in blastocysts using terminal deoxynucleotidyl transferase dUTP nick end labeling assay. Fluorescence analysis for the number of total and apoptotic cells of the blastocyst. The fragmented nuclei (denoted by green fluorescence) were apoptotic (denoted by white arrows) and DNA was counterstained with the Hoechst stain (denoted by blue). Scale bar, 100 μm. DAPI, 4’,6-diamidino2-phenylindole.
Effects of different oxygen concentrations on outgrowth embryos after 3-day culture of blastocysts from different groups. Morphologic assessment of outgrowth embryos under high (20%), low (5%), and dynamic (5% decreasing to 2%) oxygen concentrations. Scale bar, 100 μm.
Effects of different oxygen concentrations on the development of preimplantation embryos
Group | No. of zygotes | No. of cleaved embryos (%) | No. of developed blastocysts (%) |
---|---|---|---|
High O2 (20%) | 151 | 131 (86.9 ± 5.8) | 86 (54.0 ± 2.3) |
Low O2 (5%) | 154 | 131 (87.9 ± 3.5) | 115 (75.7 ± 2.2) |
Dynamic O2 (5% to 2%) | 159 | 148 (90.1 ± 2.0) | 122 (75.6 ± 4.6) |
Values in the parentheses are presented as mean±standard error of the mean.
Different superscripts indicate values that differ significantly at
Effects of different oxygen concentrations on the number of total cells and apoptotic index in blastocysts
Group | Total cells in blastocysts | Apoptotic index in blastocysts (%) |
---|---|---|
High O2 (20%) | 87.5 ± 2.5 |
8.67 ± 0.82 |
Low O2 (5%) | 115.4 ± 3.6 |
4.47 ± 0.49 |
Dynamic O2 (5% to 2%) | 128.9 ± 3.3 |
4.15 ± 0.35 |
Values are presented as mean±standard error of the mean.
Different superscripts indicate values that differ significantly at
Effects of different oxygen concentrations on development of peri-implantation embryos
Group | No. of outgrowth embryos from zygotes (%) | Mean spread area of trophoblast cells (mm2) |
---|---|---|
High O2 (20%) | 34/80 (38.4 ± 0.5) |
0.70 ± 0.05 |
Low O2 (5%) | 44/73 (58.3 ± 2.3) |
1.05 ± 1.11 |
Dynamic O2 (5% to 2%) | 55/83 (71.3 ± 6.5) |
1.11 ± 0.07 |
Values in the parentheses are presented as mean±standard error of the mean.
Different superscripts indicate values that differ significantly at