Glycine ameliorates MBP-induced meiotic abnormalities and apoptosis by regulating mitochondrial-endoplasmic reticulum interactions in porcine oocytes

Monobutyl phthalate (MBP) is the main metabolite of dibutyl phthalate (DBP) in vivo. MBP has a stable structure, can continuously accumulate in living organisms, and has the potentially to harm animal and human reproductive function. In the ovarian follicle microenvironment, MBP may lead to defects in follicular development and steroid production, abnormal meiotic maturation, impaired ovarian function and other reproductive deficits. In this study, SMART-seq was used to investigate the effects of MBP exposure on the in vitro maturation (IVM) and development of porcine oocytes. The results showed that differentially expressed genes after MBP exposure were enriched in the biological processes cytoskeleton, cell apoptosis, endoplasmic reticulum (ER) and mitochondria. Glycine (Gly) improved the developmental potential of porcine oocytes by regulating mitochondrial and ER function. The effect of Gly in protecting oocytes against MBP-induced damage was studied. The results showed that the addition of Gly significantly decreased the rate of MBP-induced spindle abnormalities, decreased the frequency of MBP-induced mitochondria-associated ER membrane (MAM) interactions, and downregulated the protein and gene expression of the linkage molecules Mitofusin 1 (MFN1) and Mitofusin 2 (MFN2) in the MAM. Additionally, treatment with Gly restored the distribution of the 1,4,5-triphosphate receptor 1 (IP₃R1) and voltage-dependent anion channel 1 (VDAC1), further decreasing the intracellular free calcium concentration ([Ca²⁺]ᵢ) levels and mitochondrial Ca²⁺ ([Ca²⁺]ₘ) , increasing the ER Ca²⁺ ([Ca²⁺]ER) levels, and thus significantly increasing the ER levels and mitochondrial membrane potential (ΔΨ m). Gly also decreased the levels of reactive oxygen species (ROS) and increased the levels of Glutathione (GSH), oocyte apoptosis-related indicators (Caspase-3 activity and Annexin V) and oocyte apoptosis-related genes (BAX, Caspase 3 and AIFM1). Our results suggest that Gly can ameliorate microtubule cytoskeleton abnormalities and improve oocyte maturation by reducing the defective mitochondrial–ER interactions caused by MBP exposure in vitro.