Abstract
The present study investigated the vitrification-induced deterioration of mitochondrial functions that may reduce the developmental ability of post-warming bovine embryos. In addition, the effect of supplementation of the culture medium with resveratrol on the mitochondrial functions and post-warming embryonic development was examined. Two days after in vitro fertilization, embryos with 8–12 cells (referred to hereafter as 8-cell embryos) were vitrified and warmed, followed by in vitro incubation for 5 days in a culture medium containing either the vehicle or 0.5 μM resveratrol. Vitrification reduced embryonic development until the blastocyst stage, reduced the ATP content of embryos, and impaired the mitochondrial genome integrity, as determined by real-time polymerase chain reaction. Although the total cell number and mitochondrial DNA copy number (Mt-number) of blastocysts were low in the vitrified embryos, the Mt-number per blastomere was similar among the blastocysts derived from fresh (non-vitrified) and vitrified-warmed embryos. Supplementation of the culture medium with resveratrol enhanced the post-warming embryonic development and reduced the Mt-number and reactive oxygen species level in blastocysts and blastomeres without affecting the ATP content. An increase in the content of cell-free mitochondrial DNA in the spent culture medium was observed following cultivation of embryos with resveratrol. These results suggested that vitrification induces mitochondrial damages and that resveratrol may enhance the development of post-warming embryos and activates the degeneration of damaged mitochondria, as indicated by the increase in the cell-free mitochondrial DNA content in the spent culture medium and the decrease in the Mt-number of blastocysts and blastomeres.
Highlights
We examined the mitochondrial DNA integrity by real-time polymerase chain reaction (PCR) and found that the vitrification-induced damage of mitochondrial DNA was higher for long mitochondrial sequence than for short mitochondrial genome sequence
The present study demonstrates that the vitrification at the early developmental stage of embryos results in the decrease in the mitochondrial functions and mitochondrial DNA integrity
We showed that resveratrol improved the development of post-warming embryos possibly through mitochondrial clearance, which corresponded to a decrease in Mt-number, mitochondrial protein, and dsDNA of the embryos and an increase in cell-free mitochondrial DNA in the spent culture medium of the resultant blastocysts
Summary
Resveratrol (trans-3,5,40-trihydroxystilbene), is a phytoalexin contained in many plant species such as grapes, peanuts, and berries, has received special attention worldwide for its potential. Treatment of porcine oocytes with resveratrol increased mitochondrial biogenesis and degradation in the oocytes, through SIRT1 activation and improved their developmental ability [10]. CCCP-induced mitochondrial dysfunction has been found to differentially influence oocyte developmental ability and mitochondrial synthesis between young and aged cows; the differential mitochondrial response may be attributed to the differential activation of SIRT1 between the two age groups following CCCP treatment [11]. Based on these studies, it may be suggested that the up-regulation of SIRT1 expression induced by resveratrol may attenuate mitochondrial dysfunction in oocytes and embryos. We investigated if the incubation of vitrified-warmed embryos in a resveratrol-containing culture medium may support the recovery of embryos from cryoinjury and enhanced their developmental ability through the degeneration of damaged mitochondria
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