Pre-Implantation Mouse Embryos Cultured In Vitro under Different Oxygen Concentrations Show Altered Ultrastructures

Abstract

Assisted Reproductive Technologies routinely utilize different culture media and oxygen (O2) concentrations to culture human embryos. Overall, embryos cultured under physiological O2 tension (5%) have improved development compared to embryos cultured under atmospheric O2 conditions (20%). The mechanisms responsible for this remain unclear. This study aimed to evaluate the effect of physiologic (5%) or atmospheric O2 (20%) tension on the microscopic ultrastructure of pre-implantation mouse embryos using Transmission Electron Microscopy (TEM). Embryos flushed out of the uterus after natural mating were used as the control. For use as the control, 2-cells, 4-cells, morulae, and blastocysts were flushed out of the uterus after natural fertilization. In vitro fertilization (IVF) was performed using potassium simplex optimized medium (KSOM) under different O2 tensions (5% and 20%) until the blastocyst stage. After collection, embryos were subjected to the standard preparative for light microscopy (LM) and TEM. We found that culture in vitro under 5% and 20% O2 results in an increase of vacuolated shaped mitochondria, cytoplasmic vacuolization and presence of multi-vesicular bodies at every embryonic stage. In addition, blastocysts generated by IVF under 5% and 20% O2 showed a lower content of heterochromatin, an interruption of the trophectodermal and inner cell mass cell membranes, an increased density of residual bodies, and high levels of glycogen granules in the cytoplasm. In conclusion, this study suggests that in vitro culture, particularly under atmospheric O2 tension, causes stage-specific changes in preimplantation embryo ultrastructure. In addition, atmospheric (20%) O2 is associated with increased alterations in embryonic ultrastructure; these changes may explain the reduced embryonic development of embryos cultured with 20% O2.

Mitochondrial function and intracellular distribution is severely affected in in vitro cultured mouse embryos

“…In the present study, we have demonstrated that in vitro culture of mouse embryos significantly affects the functionality of the mitochondria and might be responsible for the impaired embryonic development observed here and by others 7 , 15 , 16 …”

“…A number of reports have shown the influence of culture medium (oxygen concentration, medium components etc.) at the in vitro culture stage on embryonic development, fetal growth, and the postnatal health of the offspring 15 , 16 , 18 .…”

Ultrastructural Analysis of Large Japanese Field Mouse (Apodemus speciosus) Testes Exposed to Low-Dose-Rate (LDR) Radiation after the Fukushima Nuclear Power Plant Accident

“…After collection, testicular samples, divided into 4-5 pieces from each group (control, low-, intermediate-, and high-level LDR radiation), were sectioned and immediately fixed in 2.5% glutaraldehyde (Sigma-Aldrich, St. Louis, MO, USA) in PBS, at 4 • C for at least 48 h before processing for LM and TEM as previously described [31,32]. Semithin sections (1 µm thick) were stained with methylene blue and images were taken using a digital camera (Leica DFC230) mounted on a LM (Zeiss Axioscope, Carl Zeiss Microscopy GmbH, Jena, Germany).…”

“…ImageJ 1.53 software (http://rsbweb.nih.gov/ij/) (accessed on 30 January 2020) was used to measure the mean intercellular space diameter and the numerical density of cytoplasmic vacuolization, vacuolated mitochondria, lipid droplets, acrosomal vesicles, and mitochondrial cristae on low-magnification TEM micrographs of control and LDR radiation groups [32,35]. More specifically, the numerical density of cytoplasmic vacuolization, vacuolated mitochondria, lipid droplets, acrosomal vesicles, and mitochondria cristae and the width of intercellular spaces were determined on at least five equatorial sections (distance between the sections: 3 µm) and was performed by analyzing TEM micrographs at 2600×.…”

Ultrastructural Changes of Neuroendocrine Pheochromocytoma Cell Line PC-12 Exposed In Vitro to Rotenone

“…PC12, after diverse treatments, were collected and fixed overnight in 2.5% glutaraldehyde with 0.1 M sodium hydroxide, 0.1 M, pH 7.3 [25]. Samples were washed 6 times in the sodium hydroxide buffer, and then post-fixed in 2% osmium tetroxide in the same buffer for 2 h at room temperature and treated following a standard protocol for embedding in EPON resin [26]. Next, polymerization procedure overnight at 65 • C was performed, ultrathin sections of 80 nm of thickness, were cut on a Leica Ultracut E Ultramicrotome (Leica Microsystems, Wetzlar, Germany) and placed on copper grids, contrasted with UranyLess stain and lead hydroxide, and, lastly, examined in a JEOL-1400 Plus TEM (Jeol Ltd., Tokyo, Japan).…”