Fig 1.
Effects of vitrification on the kinetics of ATP content in vitrified-warmed embryos.
Fresh and vitrified-warmed embryos were incubated in IVC medium for 24 h. The ATP content of the post-warming embryos was measured at 0, 3, 6, and 24 h. Bars with different letters represent difference between treatments (P < 0.05). Data are presented as mean ± SEM.
Fig 2.
Effect of vitrification on the mitochondrial DNA integrity of embryos immediately after warming.
The integrity of the mitochondrial genome was assessed according by the ratio of the Mt-number (long sequence/short sequence) predicted by real-time PCR targeting short and long mitochondrial sequences. A) Relative Mt-number predicted by PCR targeting the short and long mitochondrial sequences; the relative value for fresh embryos was taken as 1.0. B) Ratio of the Mt-numbers (Mt-number predicted by PCR targeting long sequence/Mt-number predicted by PCR targeting short sequence PCR). Value for fresh embryos was taken as 1.0. Bars with different letters represent differences between treatments (P < 0.05). Data are presented as mean ± SEM.
Fig 3.
Effect of vitrification on mitochondrial number and function in blastocysts.
A) ATP content, B) Mt-number per embryo, C) total cell number, and D) Mt-number per blastomere. Fresh and vitrified-warmed embryos were cultured for 5 days, and the randomly selected blastocysts were assessed. Bars with different letters represent differences between treatments (P < 0.05). Data are presented as mean ± SEM.
Table 1.
Effect of vitrification on the embryonic developmental rate till the blastocyst stage.
Fig 4.
Effect of resveratrol on the characteristics of blastocysts derived from vitrified-warmed embryos.
Vitrified-warmed embryos were divided into two groups and incubated in a medium containing either vehicle (control) or resveratrol (0.5 μM) for 5 days. A) ATP content, B) Mt-number per embryo, C) total cell number, D) Mt-number per blastomere. Bars with different letters represent difference between treatments (P < 0.05). Data are presented as mean ± SEM.
Fig 5.
Effect of resveratrol on the ROS content in blastocysts and blastomeres.
Vitrified-warmed embryos were cultured with either vehicle (control) or resveratrol (0.5μM), and the blastocysts were stained with CellROX. A) ROS content per blastocyst, B) ROS content per blastomere, C) Representative images of CellROX-stained blastocysts. The average fluorescence intensity of vitrified-warmed embryos cultured with vehicle (control) was defined as 1.0. Bars with different letters represent difference between treatments (P < 0.05). Data are presented as mean ± SEM.
Table 2.
Effect of resveratrol on the developmental ability of vitrified-warmed embryos during early developmental stage.
Fig 6.
Effect of resveratrol on the cell-free mitochondrial DNA copy number in the spent culture medium of blastocysts.
The spent culture medium of the blastocysts derived from fresh or vitrified-warmed embryos cultured with or without resveratrol (0.5 μM) for 5 days was examined. Bars with different letters represent differences between treatments (P < 0.05). Data are presented as mean ± SEM.
Fig 7.
Effect of resveratrol supplementation of the culture medium on TOMM20 and double-stranded DNA (dsDNA) levels in vitrified-warmed embryos.
Vitrified-warmed embryos were cultured with or without (0 or 0.5 μM) resveratrol for 24 h, and the embryos were stained for TOMM20 (an outer mitochondrial membrane protein) and dsDNA. A) and C): Representative images of embryos stained with antibodies against TOMM20 and dsDNA, respectively, B) and D): Levels of TOMM20 (n = 27) and dsDNA (n = 37), respectively. Levels of TOMM20 and dsDNA in vitrified-warmed embryos cultured without resveratrol (vehicle) were defined as 1.0. E) A representative image of embryos stained for TOMM20 (red), dsDNA (green), and nucleus (blue); ten stacked images of 0.03 μm thickness captured in the equatorial region of the embryo. Bars with different letters represent differences between treatments (P < 0.05). Data are presented as mean ± SEM.
Fig 8.
Schematic diagram of the effect of resveratrol on mitochondria in blastocysts derived from post-warming embryos.
Before vitrification, the blastomeres of embryos (left side) contained healthy mitochondria (green dots), but vitrification induced mitochondrial damage (brown dots). The blastocysts derived from the vitrified-warmed embryos had low numbers of blastomeres; however, these blastocysts still contained similar numbers of mitochondria per blastomere as compared to the blastocysts derived from fresh embryos (non-vitrified, upper right). Supplementation of the culture medium with resveratrol improved the process of embryonic development up to the blastocyst stage, and the resultant blastocysts had similar numbers of blastomeres but lower numbers of mitochondria per blastomere (lower right). This reduced mitochondrial number corresponded to an increase in cell-free mitochondrial DNA in the spent culture medium and a decrease in dsDNA in the embryo.