Background: The 75-kDa glucose-regulated protein (GRP75) plays a crucial role in regulating the formation of mitochondria-associated endoplasmic reticulum (ER) membranes (MAMs), facilitating the transfer of Ca2+ ions, and is essential for lipid metabolism, mitochondrial function, calcium homeostasis, and apoptosis in mammalian cells. However, the relationship between GRP75 expression and preimplantation embryonic development in pigs remains unknown. Methods: In this study, we investigated whether GRP75 influences ER–mitochondrial junctions and mitochondrial Ca2+ levels in porcine embryos in vitro . We examined the expression of GRP75 at the zygote, cleavage, and blastocyst stages using immunofluorescence staining and western blot analysis. Results: GRP75 fluorescence and mitochondrial Ca2+ levels were significantly lower (p < 0.01) in the blastocysts than in the zygotes. Western blot analysis revealed a decline in the expression of mitochondrial fusion factors mitofusin 2, GRP75, and the mitochondrial calcium uniporter complex MICU1 protein at the blastocyst stage. To investigate the effects of GRP75 on blastocyst developmental competence, porcinespecific GRP75-siRNA (25 nM) was microinjected at the zygote stage. The results showed a significant decrease in the development capacity until the blastocyst stage (Control: 31.2 ± 2.0%, N.C. siRNA (25 nM): 29.8 ± 3.1%, vs. GRP75-siRNA (25 nM): 24.1 ± 1.6%; p < 0.05). GRP75 in the mitochondria and ER-localized GRP75 were both significantly reduced in blastocysts of pigs microinjected with GRP75 siRNA. Along with ER–mitochondrial colocalization, the MAM formation ratio was significantly reduced in the GRP75-siRNA group compared with that in the control (Control: 29.3% vs. GRP75- siRNA (25 nM): 15.7%, p < 0.001). Conclusions: These findings demonstrate that the GRP75-derived MAM region is involved in the development of early embryos in porcine blastocysts.

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
    Chemicals and animals
    In vitro maturation (IVM) of oocyte
    In vitro fertilization (IVF) and IVC
    Transfection of siRNA for porcine BIP/GRP75
    Immunofluorescence and Mito-Tracker andER-TrackerTM staining
    Measurement of the mitochondrial Ca2+ level
    Protein extraction and western blot analysis
    Statistical analysis
RESULTS
    Expression of GRP75 and mitochondrial Ca2+ in porcineembryos during IVC
    Effects of GRP75 on blastocyst developmentalcapacity through MAM in porcine embryos
DISCUSSION
CONCLUSION
REFERENCES

• Hyo-Jin Park(Department of Biotechnology, Daegu University, Gyeongsan 38453, Korea, DU Center for Infertility, Daegu University, Gyeongsan 38453, Korea)
• Young-Seo Jo(Department of Biotechnology, Daegu University, Gyeongsan 38453, Korea, DU Center for Infertility, Daegu University, Gyeongsan 38453, Korea)
• Deog-Bon Koo(Department of Biotechnology, Daegu University, Gyeongsan 38453, Korea, DU Center for Infertility, Daegu University, Gyeongsan 38453, Korea, Department of Companion Animal Industry, Daegu University, Gyeongsan 38453, Korea) Corresponding author
• Seul-Gi Yang(DU Center for Infertility, Daegu University, Gyeongsan 38453, Korea, Department of Companion Animal Industry, Daegu University, Gyeongsan 38453, Korea)