As oocytes experience prolonged aging, reactive oxygen species (ROS) typically accumulate, the mitochondrial electrochemical potential gradient dissipates, and cumulus cells undergo expansion. Trichostatin A (TSA), used to stimulate aging, delays oocyte maturation by inhibiting germinal vesicle breakdown during meiosis. The objective of this study was to study the effects of aging during in vitro oocyte maturation in pigs. Oocytes (n=881) were matured with or without TSA (100 ng/mL) for 24 or 48 h followed by an additional 16 h of maturation without TSA. At the end of maturation, oocytes (n=446) were evaluated for cumulus cell expansion (CCE). A portion of the oocytes were stained to determine the relative levels of ROS (n=476) or mitochondrial electrochemical potential gradient dissipation (n=405). Fluorescent images of the oocytes were acquired, images were analyzed using ImageJ, and statistical analysis of the data was performed using ANOVA and Tukey’s test. Oocytes matured with or without TSA for 48 h had significantly less (P < 0.05) CCE compared to oocytes matured without TSA for 24 h. Oocytes matured without TSA for 24 h generated significantly different (P < 0.05) levels of ROS compared to oocytes matured with TSA for 48 h. Oocytes matured without TSA for 48 h had significantly higher (P < 0.05) mitochondrial membrane potential compared to the all other treatments. Results indicate that oocytes experiencing prolonged aging have less CCE and a decrease in mitochondrial membrane potential but no consistent or predictable trends in ROS formation. The use of TSA to stimulate aging in pig oocytes remains a valid and a reliable option.
L-α-amino butyrate is a low-molecular weight thiol compound that acts to increase the levels of glutathione in the oocyte. Glutathione acts as an antioxidant during oocyte maturation and promotes male pronuclear formation during fertilization. Supplementing the L-α-amino butyrate helps to decrease polyspermic penetration rates and improve early embryonic development in swine. However, it is unknown if L-α-amino butyrate supplementation affects the environment of the oocyte or the oocyte directly. Therefore, the objective of this study was to determine if L-α-amino butyrate supplementation to the maturation media acted on the oocyte or had alternative beneficial effects in the surrounding environment. Oocytes were randomly assigned to a maturation media containing an amino acid transport inhibitor, quisqualic acid (QA) (0 or 1 mM) and then supplemented with L-α-amino butyrate (0 or 3.3 mM). Oocytes were evaluated for stage of meiosis (n=380) and cumulus cell expansion (n=411) at the end of maturation. The remaining oocytes were fertilized and evaluated for cortical granule exocytosis (n=400) and IVF kinetics (n=456). Supplementation of L-α-amino butyrate with or without QA significantly increased (P < 0.05) cumulus cell expansion, cortical granule exocytosis and male pronuclear formation compared to no supplementation or QA supplementation. There was no difference in meiotic progression, fertilization or polyspermic penetration rates between the treatment groups. Results suggest that when L-α-amino butyrate is supplemented during maturation, it improves the maturation of the oocyte by acting directly on the oocyte and not through the surrounding environment of the oocyte.
Oocytes of older animals are less likely to be fertilized during the optimal time window post ovulation, resulting in the potential diminished fertilization and embryonic development success. The activity of the epigenetic modifications during this period is a possible target to reverse these damaging effects of aging. The objective of this study was to study the effects of aging during in vitro oocyte maturation in pigs on epigenetic modifications. Oocytes (n = 54) were matured with or without Trichostatin A (TSA; 100 ng/mL), a known meiotic inhibitor, for 24 h, then for an additional 16 h without TSA or hormones for a total of 40 h. At the end of maturation, oocytes were denuded and their zona pellucida’s removed. Oocytes were stained with anti-5-methylcytosine (5mC, 1:500). Fluorescent images of the oocytes were acquired, images were analyzed using ImageJ, and data analysis was performed using ANOVA and Tukey’s test. Oocytes matured with TSA had significantly greater (P < 0.05) levels of DNA methylation by the end of in vitro maturation compared to those not supplemented with TSA These results suggest that TSA can be used to develop an in vitro model to study the effects of epigenetic modifications in oocytes from aged livestock.
Quisqualic acid is a known inhibitor of sodium-dependent amino acid transporters. However, it is unknown if quisqualic acid has similar effects in in vitro mature oocytes. Therefore, the objective of this study was to determine the optimal dose and effects of quisqualic acid supplemented during maturation. Oocytes (n=362) were supplemented during maturation with quisqualic acid (0, 0.5, 0.75, 1.0, 2.5 mM) to determine the minimum concentration of quisqualic acid that had no effect on oocyte maturation but significantly decreased the intracellular glutathione concentration. The addition of 1.0 mM quisqualic acid was the lowest concentration observed to cause intracellular glutathione levels to be significantly less (P < 0.05) without affecting maturation compared to no quisqualic acid. Based on those results, oocytes were supplemented with or without 1.0 mM quisqualic acid then evaluated for cumulus cell expansion (n=410) and stage of meiosis (n=380) at the end of maturation. Additional oocytes were fertilized and assessed for cortical granule exocytosis (n=400) and kinetics at 12 h after IVF (n=420). Supplementing quisqualic acid to the media did not have an effect on stage of meiosis, fertilization, polyspermic penetration, or cortical granule exocytosis. Supplementing 1.0 mM quisqualic acid significantly decreased (P < 0.05) cumulus cell expansion by the end of maturation and male pronuclear formation by 12 h after IVF. These results suggest that quisqualic acid supplementation during maturation in pigs inhibits sodium-dependent amino acid transporters.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.