Oxygen concentration alters mitochondrial structure and function in in vitro fertilized preimplantation mouse embryos

Abstract

Study question: Does the oxygen concentration in the culture medium [either physiologic (5%) or atmospheric (20%)] affect mitochondrial ultrastructure and function in preimplantation mouse embryos generated by IVF?

Summary answer: Embryos cultured in 20% oxygen show increased mitochondrial abnormalities compared to embryos cultured in 5% oxygen.

What is known already: ART are widely used and have resulted in the birth of more than 8 million children. A variety of media and oxygen concentrations are used to culture embryos. Embryos cultured under physiological O2 tension (5%) reach the blastocyst stage faster and have fewer alterations in gene expression when compared with embryos cultured under atmospheric oxygen conditions (20%). The mechanisms by which oxygen tension affects preimplantation development remain unclear, but mitochondria are believed to play an important role. The aim of this study was to evaluate how mitochondrial ultrastructure and function in IVF embryos were affected by culture under physiologic (5%) or atmospheric (20%) oxygen concentrations.

Study design, size, duration: Zygotes, 2-cell, 4-cell, morula and blastocyst were flushed out of the uterus after natural fertilization and used as controls. IVF was performed in CF1 x B6D2F1 mice and embryos were cultured in Potassium simplex optimized medium (KSOM) with amino acids (KAA) under 5% and 20% O2 until the blastocyst stage. Embryo development with the addition of antioxidants was also tested.

Participants/materials, setting, methods: Mitochondrial function was assessed by measuring mitochondrial membrane potential, reactive oxygen species (ROS) production, ATP levels, and the expression of selected genes involved in mitochondrial function. Mitochondria ultrastructure was evaluated by transmission electron microscopy (TEM).

Main results and the role of chance: Embryos cultured under 20% O2 had fewer mitochondria and more vacuoles and hooded (abnormal) mitochondria compared to the other groups (P < 0.05). At the blastocyst stage the mitochondria of IVF embryos cultured in 20% O2 had lower mtDNA copy number, a denser matrix and more lamellar cristae than controls. Overall IVF-generated blastocysts had lower mitochondrial membrane potential, higher ROS levels, together with changes in the expression of selected mitochondrial genes (P < 0.05). ATP levels were significantly lower than controls only under 5% O2, with the 20% O2 IVF group having intermediate levels. Unexpectedly, adding antioxidant to the culture medium did not improve development.

Large scale data: N/A.

Limitations, reasons for caution: Findings in mice embryos might be different from human embryos.

Wider implications of the findings: This study suggests that changes in the mitochondria may be part of the mechanism by which lower oxygen concentration leads to better embryo development and further emphasize the importance of mitochondria as a locus of reprogramming.

Study funding/competing interest(s): This study was funded by R01 HD 082039 to PFR, the Department of Life, Health and Environmental Sciences, University of L'Aquila, Italy (RIA 2016-2018) and the Department of Anatomy, Histology, Forensic Medicine and Orthopaedics, La Sapienza University of Rome, Italy (University grants 2016-2017). The authors declare no competing interests.

Keywords: IVF; ROS; embryo culture; mitochondria; oxygen levels.

Figure 1

Mitochondrial density by developmental stage. The numerical density (mitochondria per 3800 μm²) of total (A), abnormal (B) and normal (C) mitochondria, as well as vacuoles (D) varies with developmental stage. IVF embryos have more abnormal mitochondria and vacuoles and fewer normal mitochondria. Bars with a different letter superscript (within a stage) are statistically different. The absence of superscript letter indicates that there were no statistically significant differences between any of the treatment groups. TE, trophectoderm; ICM, inner cell mass; FB, in vivo fertilized embryos flushed from the uterus; IVF 5%/20%, IVF embryos cultured under 5 / 20% oxygen.

Figure 2

Numerical density of mitochondria and mtDNA content. The numerical density (mitochondria per 3800 μm²) of total mitochondria in blastocysts tended to decline from in vivo fertilized embryos flushed from the uterus (FB) to IVF embryos cultured under-5% group and again to those cultured under 20% oxygen (IVF5%/20% groups) (A). The mtDNA copy number (B) reflects the pattern seen morphologically. Bars with a different letter superscript are statistically different. The absence of superscript letter indicates that there were no statistically significant differences between any of the treatment groups.

Figure 3

Mitochondrial ultrastructure. (A and B): Ultrastructure of mitochondria in in vivo fertilized embryos flushed from the uterus (FB) 2-cell and 4-8 cell embryos. A—2-cell embryo mitochondria with cristae (m) and vacuoles (V). *: granules of mono-particulate form of glycogen (Transmission electron microscopy [TEM] Bar: 0.6 μm). B—ultrastructure of 4-8 cell embryo hooded mitochondria (hm) and normal mitochondria (m) with cristae. *: granules of glycogen (TEM. Bar: 0.8 μm). (C and D): ultrastructure of round/ovoid mitochondria in a 2-cell and a 4-8 cell IVF embryo cultured under 20% oxygen (IVF20%). C—IVF 20% 2-cell embryo mitochondria (m) and hooded mitochondria (hm). V: vacuoles; ZP: zona pellucida. (TEM. Bar: 1 μm). D—IVF 20% 4-8 cell embryo mitochondria (m), hooded mitochondria (hm) and vacuoles (V) (TEM. Bar: 1 μm). E—FB morula with large round nucleus (N). Numerous blastomeres are visible. Arrows show the intercellular contacts. m: mitochondria; V: vacuole; nm: nuclear membrane; mv: microvilli (TEM. Bar: 2 μm). F—ultrastructure of IVF-20% morula. TEM micrograph shows blastomeres in a compaction stage. Arrows indicate the intercellular contacts. m: mitochondria; V: vacuoles; ZP: zona pellucida; mv: microvilli (TEM. Bar: 2 μm). (G–I): FB morula mitochondria. G—FB morula mitochondria (m) with visible cristae. Arrow shows intercellular contacts (TEM. Bar: 0.8 μm). H—high magnification of FB morula isolated mitochondria (m) and hooded mitochondria (hm) (TEM. Bar: 0.8 μm). I—high magnification of group of mitochondria (m), hooded mitochondria (hm) and vacuoles (V) (TEM. Bar: 0.6 μm). (J and K): IVF 20% morula. J—ultrastructure of high electron-density mitochondria (m) with round shape. hm: hooded mitochondria (TEM. Bar: 0.8 μm). K—high magnification of a multivesicular body (mb) (TEM. Bar: 0.6 μm).(L and M): ultrastructure of FB blastocyst. L—trophoblast cell with large nucleus. The blastomeres shows nuclear heterochromatin (He) and euchromatin (Eu). nm: nuclear membrane (TEM. Bar: 1 μm). M—elongated mitochondria with visible cristae. *: monoparticulate form of glycogen (TEM. Bar: 0.6 μm). (N–P:) ultrastructure of IVF blastocyst cultured under 20% oxygen (IVF-20%). N—trophoblast cell with large nucleus with pacth of heterochromatin (He) and euchromatin (Eu). Large vacuole (V) is visible. O—IVF 20% Trophectoderm (TE) branching mitochondria (m). (TEM. Bar: 0.4 μm). P—representative TEM micrograph of IVF TE mitochondria (m) and monoparticulate form of glycogen (*) (TEM. Bar: 0.2 μm).

Figure 4

Mitochondrial functional tests. Mitochondrial membrane potential, as measured by JC-1 staining was significantly less in embryos cultured under 20% oxygen (IVF-20%) (A) and ROS levels were greater in IVF-20% embryos (B). ATP levels were lower in IVF embryos than FB, but the group of embryos cultured under 5% oxygen (IVF-5%) was the only group for which the difference reached statistical significance (C).

Figure 5

Expression of ten selected genes involved in mitochondrial function and cellular redox state. Top row genes involved in mitochondrial protein quality control, middle row genes involved in glutathione antioxidant pathway, bottom row genes involved in mitochondrial DNA synthesis and (mtND2) electron transport chain. Bars with no letter designation show no difference among the treatment groups. Bars with different letter designations are statistically different. FB, in vivo fertilized embryos flushed from the uterus; IVF 5%/20%, IVF embryos cultured under 5 / 20% oxygen.