Glycine ameliorates mitochondrial dysfunction caused by ABT-199 in porcine oocytes

Abstract

Mitochondria play an important role in controlling oocyte developmental competence. Our previous studies showed that glycine can regulate mitochondrial function and improve oocyte maturation in vitro. However, the mechanisms by which glycine affects mitochondrial function during oocyte maturation in vitro have not been fully investigated. In this study, we induced a mitochondrial damage model in oocytes with the Bcl-2-specific antagonist ABT-199. We investigated whether glycine could reverse the mitochondrial dysfunction induced by ABT-199 exposure and whether it is related to calcium regulation. Our results showed that ABT-199 inhibited cumulus expansion, decreased the oocyte maturation rate and the intracellular glutathione (GSH) level, caused mitochondrial dysfunction, induced oxidative stress, which was confirmed by decreased mitochondrial membrane potential (Δ⍦m) and the expression of mitochondrial function-related genes (PGC-1α), and increased reactive oxygen species (ROS) levels and the expression of apoptosis-associated genes (Bax, caspase-3, CytC). More importantly, ABT-199-treated oocytes showed an increase in the intracellular free calcium concentration ([Ca 2+]i) and had impaired cortical type 1 inositol 1,4,5-trisphosphate receptors (IP3R1) distribution. Nevertheless, treatment with glycine significantly ameliorated mitochondrial dysfunction, oxidative stress and apoptosis, glycine also regulated [Ca 2+]i levels and IP3R1 cellular distribution, which further protects oocyte maturation in ABT-199-induced porcine oocytes. Taken together, our results indicate that glycine has a protective action against ABT-199-induced mitochondrial dysfunction in porcine oocytes.

Glycine Ameliorates Endoplasmic Reticulum Stress Induced by Thapsigargin in Porcine Oocytes

“…Gly is the main component of GSH. Our previous study also verified that Gly treatment had a beneficial effect on in vitro oocyte maturation and blastocyst development by decreasing ROS levels to reduce apoptosis (Qu et al, 2002;Yu et al, 2021). In this study, Gly treatment reversed the decrease in GSH levels and the increase in ROS levels induced by TG.…”

“…Our previous studies showed that 6 mM Gly treatment has a beneficial effect on oocyte IVM and subsequent blastocyst development after parthenogenetic activation (PA) by decreasing ROS levels and increasing mitochondrial function (mitochondrial ΔΨ m, ATP concentration) to reduce apoptosis and regulate gene expression related to development (FGFR2 and Hsfl) and apoptosis (Bax and Bcl2). Furthermore, we also proved that Gly could improve oocyte IVM by regulating the [Ca 2+ ] i to enhance mitochondrial function ( Yu et al, 2021 ).…”

“…The intracellular GSH and ROS levels of oocytes at the metaphase II (MII) stage were measured by CellTracker Blue CMF2HC (4-chloromethy-6.8-difluoro-7-hydroxycoumarin; Invitrogen) and H 2 DCFDA (2′,7′-dichlorodihydrofluorescein diacetate) fluorescence assays. Fluorescence staining was performed as described in a previous report ( Yu et al, 2021 ). The fluorescence was observed under a fluorescence microscope (Olympus Tokyo, Japan) and imaged with ImageJ software.…”

“…The [Ca 2+ ] i levels were detected as described in a previous report ( Yu et al, 2021 ). Briefly, oocytes were stained with 5 μM Fluo-3/AM (Beyotime) for 40 min at 38.5°C and then washed three times with PBS-PVA (without Ca 2+ ).…”

“…The PrimeScript™ RT reagent Kit with gDNA Eraser (Perfect Real Time) was utilized to synthesize complementary DNA (cDNA). The CFX96 Real-Time PCR Detection System was utilized for real-time PCR ( Yu et al, 2021 ). Specific primer sequences ( Table 1 ) were devised utilizing the NCBI database.…”