Effects of resveratrol treatment on mitochondria and subsequent embryonic development of bovine blastocysts cryopreserved by slow freezing

Abstract

This study evaluated the effects of cryopreservation by slow freezing on the mitochondrial function, DNA integrity, and developmental ability of bovine embryos and examined whether resveratrol treatment of the frozen‐thawed blastocysts improved embryonic viability. In vitro produced bovine embryos were subjected to slow freezing. After thawing, the ATP content and mitochondrial DNA integrity (mtDNA), determined by real‐time PCR targeting short and long mitochondrial sequences, was found to be lower in frozen‐thawed embryos than in fresh embryos, and mtDNA copy number was significantly reduced during the 24‐hr incubation post warming. Furthermore, immunostaining against double‐strand DNA revealed DNA damage in frozen‐thawed embryos. When frozen‐thawed embryos were incubated in the medium containing 0.5 µM resveratrol, SIRT1 expression, and survival rate of the embryos significantly improved compared with the vehicle‐treated embryos. In addition, cell‐free mtDNA content in medium was higher in case of resveratrol‐treated embryos than of vehicle‐treated embryos. In conclusion, slow freezing affects mitochondrial integrity and function in the blastocysts. In the frozen‐thawed embryos, mitochondria were removed during post‐thawing incubation and resveratrol enhanced the process, resulting in improved survivability of the embryos.

Comparative analysis of cell-free DNA content in culture medium and mitochondrial DNA copy number in porcine parthenogenetically activated embryos

“…When granulosa cells were treated with mitochondrial uncoupler inducing mitochondrial dysfunction, the amount of cf-mtDNA in the medium increased [44]. Vitrification tends to affect the levels of cf-mtDNA in the SCM of bovine embryos, as does the treatment of vitrifiedwarmed embryos with resveratrol, which was previously reported to improve embryonic development and increases cf-mtDNA content in the medium [22,45]. In addition, it has been reported that the inhibition of autophagy increases the cf-mtDNA content and that the inhibition of either proteasome or intracellular vesicle formation decreases cf-tDNA content in the SCM of bovine granulosa cells [41].…”

Resveratrol enhanced mitochondrial recovery from cryopreservation‐induced damages in oocytes and embryos

“… 7 , 8 , 9 In our studies based on porcine oocytes, bovine eight‐cell stage and blastocyst stage embryos, ATP content, mitochondrial genome integrity and developmental ability in cryopreserved samples was low compared with those fresh counterparts. 10 , 11 , 12 A previous study showed that vitrification of ovine oocytes induced low mitochondrial function and high ROS content; however, optimal restoration period, in this case 4 hours after vitrification‐warming partly improved oocyte developmental ability. 13 However, little is known about the restoration of mitochondria after the warming of oocytes and embryos.…”

“… 64 In this context, resveratrol increased mitochondrial cell‐free DNA in the culture medium of cryopreserved‐warmed embryos and concomitantly, decreased mitochondrial DNA copy number and protein levels in the embryos. 10 , 11 …”

The current state of the problem of in vitrogene pool preservation in poultry

“…Besides, SIRT1 expression and cell-free mtDNA content in the medium were higher in the case of embryos treated with resveratrol. It should be noted that slow freezing affects mitochondrial integrity and function in blastocysts (Hayashi et al, 2019). It is important to improve in vitro maturation (IVM) conditions for immature oocytes after cryopreservation, especially if a limited number of oocytes are collected from specific donors.…”