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 struc-ture, 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 devel-opment 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 mitochon-drial 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 re-ceptor 1 (IP(3)R1) 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 (delta Psi 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, Cas-pase 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.

Automated summary

This study investigated the effects of mono-n-butyl phthalate (MBP) on the in vitro maturation (IVM) and development of porcine oocytes using SMART-seq technology. The results showed that differentially expressed genes were enriched in cytoskeleton, cell apoptosis, endoplasmic reticulum (ER), and mitochondria. Glycine (Gly) was found to improve oocyte developmental potential by regulating mitochondrial and ER function. Gly supplementation significantly reduced MBP-induced spindle abnormalities and mitochondria-associated ER membrane (MAM) interactions. Moreover, Gly treatment restored the distribution of certain proteins and genes, leading to decreased intracellular free calcium concentration, increased ER levels and mitochondrial membrane potential, reduced levels of reactive oxygen species, and increased levels of Glutathione.