Vitrification preserves chromatin integrity, bioenergy potential and oxidative parameters in mouse embryos

Abstract

Background: The aim of this study was to evaluate the effects of vitrification on morpho-functional parameters (blastomere/chromatin integrity and bioenergy/oxidative potential) of mouse preimplantation embryos.

Methods: In vivo produced mouse (4/16-cell, morulae and blastocyst-stage) embryos were randomly divided into vitrification and control groups. For vitrification, embryos were exposed to a 2-step loading of ethylene glycol and propylene glycol, before being placed in a small nylon loop and submerged into liquid nitrogen. After warming, the cryoprotectants were diluted by a 3-step procedure. Embryo morphology, chromatin integrity and energy/oxidative status were compared between groups.

Results: Vitrification induced low grade blastomere cytofragmentation (P < 0.05) and low chromatin damage only in embryos at the morula stage (P < 0.001). Mitochondrial (mt) distribution pattern was affected by vitrification only in early embryos (P < 0.001). Mitochondrial activity did not change upon vitrification in morula-stage embryos but it was reduced in blastocyst-stage embryos (P < 0.05). Intracellular ROS levels significantly increased in embryos at the morula and blastocyst stages (P < 0.001). Colocalization of active mitochondria and ROS increased only in vitrified blastocysts.

Conclusions: In conclusion, this study elucidates the developmentally-related and mild effects of vitrification on morphology, nuclear and bioenergy/oxidative parameters of mouse embryos and demonstrates that vitrification is a suitable method for preserving predictive parameters of embryo ability to induce a full-term pregnancy.

Figure 1

Effects of vitrification on chromatin integrity and mitochondrial distribution pattern of mouse 4/16-cell, morula and blastocyst stage embryos. Panel A: overall percentages of embryos (grade B + grade C) with damaged chromatin observed after vitrification (black bars), grouped according to their developmental stage, and compared with controls (white bars). In detail, in embryos at the morula stage, vitrification increased the rate of embryos showing chromatin damage whereas it had no effect in early embryos and in blastocysts. Chi square test: within each stage: a,b P<0.001; c,d P<0.05. Panel B:Percentages of embryos showing the P/P mt pattern observed after vitrification (black bars), grouped according to examined developmental stages, and compared with controls (white bars). In detail, in embryos at the 4/16-cell stage, vitrification reduced the rate of embryos showing P/P mt pattern whereas it had no effect in embryos at the morula and blastocyst stages. Chi square test: a,b P<0.001; c,d P<0.05; * P<0.001. Numbers of analyzed oocytes per group are indicated on the top of each histogram.

Figure 2

Photomicrographs of fresh and vitrified/warmed mouse embryos at early (4/16-cell), morula and blastocyst stages of development as assessed for their nuclear chromatin and bioenergy/oxidative potential. For each embryo, corresponding bright-field (phase contrast; lane 1), UV light (lane 2) and confocal images showing mt distribution pattern (lane 3), ROS localization (lane 4) and mt/ROS merge (lane 5), are shown. Representative photomicrographs of control non-vitrified and vitrified/warmed embryos at the 4- (row A) and 8-cell (row B), morula (rows C and D) and blastocyst stages (rows E and F), are shown. Nuclear chromatin was stained with Hoechst 33258.MitoTracker Orange CMTM Rosand DCDH FDA were used to label mitochondria and ROS, respectively. In control fresh early embryos and in morulae, in all blastomeres, there were detectable MitoTracker signals in the form of continuous rings around the nuclei and clusters of mitochondria at the cortex, namely perinuclear/pericorticalmt pattern (heterogeneous, healthy P/P mt pattern) (A3, C3). Vitrified early embryos(B3) showed a uniform/diffused (homogeneous) mt distribution pattern throughout the blastomere cytoplasm. In embryos at the morula and blastocyst stage, the mt pattern was apparently not affected by vitrification (D3 vs C3 and F3 vs E3). A higher number of red fluorescent spots is evident on the trophoectoderma (white arrows) compared with the inner cell mass, indicating differences in mt number/cell between these two embryo lineages and higher mt/number and aggregate formation in the trophoectoderma compared with ICM. This feature can be observed in embryos of both groups, thus it was not influenced by vitrification. ROS intracellular localization (lane 4) corresponded to the distribution pattern of mitochondria. In fresh control embryos, apart areas/sites of mt/ROS overlapping (merge, lane 5), intracellular ROS appeared diffused throughout the cytoplasm (A4, C4, E4). In vitrified (B4, D4 and F4) embryos, diffused MitoTracker and DCDH FDA labelling were evident throughout the cytoplasm. Scale bar represents 20 μm.

Figure 3

Effects of vitrification on mitochondrial activity, intracellular ROS levels and mt/ROS colocalization in single mouse morulae and blastocysts. In each group, energy status and ROS intracellular levels are expressed as mean±SD of Mitotracker Orange CMTM Ros (panel a) and DCF (panel b) fluorescence intensity of individual embryos in arbitrary densitometric units (ADU). In each group, mt/ROS colocalization is expressed as mean±SD of Pearson’s correlation coefficient of individual embryos (panel c). Representative mt/ROS colocalization scatterplots graph of a fresh (panel d) and a vitrified (panel e) blastocyst are shown. In embryos at the morula stage, mt activity did not change after vitrification whereas in embryos at the blastocyst stage, mt activity was significantly reduced (P<0.05). ROS levels significantly increased in vitrified embryos at morulaand blastocyst stages (P<0.001). Mt/ROS colocalizationsignificantly increased in vitrifiedmorulae. Numbers of analyzed embryos per group are indicated on the top of each histogram. Student’s t-Test: a,b P<0.05; c,d P<0.001.