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

Abstract

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 O 2 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% O 2 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% O 2 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% O 2 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 < † Co-first authors.

Repeated hyperstimulation affects the ultrastructure of mouse fallopian tube epithelium

“…COH, there was a significant increase in the frequency and density of damaged mitochondria from the oviductal epithelium, thus strenghtening the ultrastructural observations described [55][56][57]. The slight improvement observed in terms of density and frequency of damaged mitochondria at 8R, respect to 4R, could be probably due to cytoplasmic remodeling mechanisms activated by the oxidative damage, that may establish domains of autonomous regulation of mitochondrial function and activity, as previously reported in mammalian oocytes and embryos [44,64]. The mitochondrial dysfunction here observed may, probably, be connected to the absence of ciliated epithelium in the sections observed, as the fibrils' tips during ciliogenesis are laterally connected to mitochondria [65].…”

“…These adverse effects on the mitochondrial function after superovulation in oocytes, cumulus cells and embryos [44,48,49], prompted us to focus on the ultrastructure of mitochondria dispersed in the columnar epithelial cells of FTs. Interestingly, it was found the presence of numerous swelled or even disrupted elements, most of them undergoing to phenomena of mitophagy, evident already after 4 repeated cycles, in agreement to a previous study in rabbit oviducts after hCG injection [55].…”

“…The ImageJ software (http://rsbweb.nih.gov/ij/) was used to measure the numerical density and dimension of normal and damaged mitochondria from columnar epithelial cells (CECs) of the tunica mucosa on low-magnification TEM micrographs of Ctr, 4R and 8R groups. The numerical density of mitochondria was expressed as number of organelle per 100 μm 2 of epithelial area [44]. Mitochondria were classified, according to their ultrastructural appearance, in: i) normal, i.e.…”

HYPOXIA AND REPRODUCTIVE HEALTH: Hypoxic regulation of preimplantation embryos: lessons from human embryonic stem cells

“…This results in the trophectoderm being metabolically energetic, consuming more oxygen, producing more ATP and possessing more active mitochondria than the quiescent ICM (Houghton 2006). The precise mechanisms which regulate mitochondrial development in the embryo remain to be discovered but environmental oxygen has been shown to be involved (Belli et al 2019). Mouse IVF embryos cultured at 20% oxygen contained fewer normal mitochondria and more vacuoles compared to those cultured at 5% oxygen, or in vivo produced embryos (Belli et al 2019).…”

“…The precise mechanisms which regulate mitochondrial development in the embryo remain to be discovered but environmental oxygen has been shown to be involved (Belli et al 2019). Mouse IVF embryos cultured at 20% oxygen contained fewer normal mitochondria and more vacuoles compared to those cultured at 5% oxygen, or in vivo produced embryos (Belli et al 2019). Further research is required to understand the mechanisms regulating these results but the authors' suggestion that atmospheric oxygen may alter mitochondrial division, leading to an abnormal morphology and a reduction in total mitochondrial number warrants further investigation.…”