Abstract
This study aimed to examine whether the addition of glutathione ethyl ester (GSH-OEt) to the in vitro maturation (IVM) medium would improve the resilience of bovine oocytes to withstand vitrification. The effects of GSH-OEt on spindle morphology, levels of reactive oxygen species (ROS), mitochondrial activity and distribution, and embryo developmental potential were assessed together with the expression of genes with a role in apoptosis (BAX, BCL2), oxidative-stress pathways (GPX1, SOD1), water channels (AQP3), implantation (IFN-τ) and gap junctions (CX43) in oocytes and their derived blastocysts. Vitrification gave rise to abnormal spindle microtubule configurations and elevated ROS levels. Supplementation of IVM medium with GSH-OEt before vitrification preserved mitochondrial distribution pattern and diminished both cytoplasmic and mitochondrial ROS contents and percentages of embryos developing beyond the 8-cell stage were similar to those recorded in fresh non-vitrified oocytes. Although not significantly different from control vitrified oocytes, vitrified oocytes after GSH-OEt treatment gave rise to similar day 8-blastocyst and hatching rates to fresh non-vitrified oocytes. No effects of GSH-OEt supplementation were noted on the targeted gene expression of oocytes and derived blastocysts, with the exception of GPX1, AQP3 and CX43 in derived blastocysts. The addition of GSH-OEt to the IVM medium before vitrification may be beneficial for embryo development presumably as the consequence of additional anti-oxidant protection during IVM.
Highlights
The successful cryopreservation of mammalian oocytes is useful for assisted reproductive technologies including animal breeding programs and somatic cell nuclear transfer as this method of storage resolves the temporal and spatial limitations of oocyte supplies
Rates of oocytes showing an abnormal microtubule configuration were similar in fresh control oocytes and VIT GSH-OEt, no differences were observed among VIT GSH-OEt, VIT control and GSH-OEt groups
GSH-OEt was added as a supplement to the medium during the in vitro maturation (IVM) of bovine oocytes to examine its protective role against damage induced by the vitrification/warming protocol
Summary
The successful cryopreservation of mammalian oocytes is useful for assisted reproductive technologies including animal breeding programs and somatic cell nuclear transfer as this method of storage resolves the temporal and spatial limitations of oocyte supplies (for a review, see [1,2]). Vitrification has proved more efficient and reliable than slow freezing for the cryopreservation of oocytes of many species including cow [3,4,5], mouse [6], and human [7,8], among others. The impaired embryo developmental potential of vitrified oocytes has been attributed to abnormal meiotic spindle assembly, destabilization of the microfilament and microtubular elements of the cytoskeleton, plasma membrane rupture, premature cortical granule exocytosis, and zona pellucida hardening, among other factors (reviewed by [1]). Vitrification severely affects the morphofunctional integrity of the oocyte’s mitochondria and endogenous antioxidant systems, with the consequence of increased levels of reactive oxygen species (ROS) [10,11]. ROS can compromise the developmental capacity of embryos and promote apoptosis in oocytes and early embryos [14,15] by activating the caspase cascade that executes the apoptotic program [16,17]
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