2023년 한국동물생명공학회 학술 대회 [Young-Seo Jo] > 학회발표

Role of GRP75 knockdown on the porcine embryonic developmental ability in vitro 

​Young-Seo Jo^1,2, Hyo-Jin Park^1,2, Seul-Gi Yang^1,2, Dong-woo kim^1,2, Min-Hyung Lee^1,2, Deog-Bon Koo^1,2,3

¹ Department of Biotechnology, College of Engineering, Daegu University, 201 Daegudae-ro, Jillyang, Gyeongsan, Gyeonbuk 38453, Korea, ² Institute of Intfertility, Daegu University, 201 Daegudae-ro, Jillyang, Gyeongsan, Gyeonbuk 38453, Republic of Korea, ³ Department of Companion Animal Industry, College of Natural and Life Sciences, Daegu University, Gyeongbuk 38453, Republic of Korea

 The glucose-regulated protein 75 (GRP75), known as the mitochondria-associated membrane (MAM) key factor, plays an important role in physical connection between the endoplasmic reticulum (ER) and mitochondria in mammalian cells. The effects of the GRP75 of MAM on mitochondria and ER functions were reported in mammalian cells, however the relation between GRP75 expression and preimplantation embryo development in pigs has not been clearly defined. In present study, we investigated the role of GRP75 using porcine specific GRP75 siRNA during in vitro culture (IVC) and confirmed the morphological changes in MAM on porcine early embryonic developmental progression. First, we observed the effects of knockdown of GRP75 (a MAM main factor) using porcine-specific siRNAs (Gene no. XM_005661695) in porcine embryos. We performed the microinjection using GRP75 siRNA in porcine zygotes after in vitro fertilization (IVF). We observed that the protein levels of GPR75 were reduced in developed blastocysts from GRP75 siRNA microinjected embryos by using Western blot and immunofluorescence analysis. In addition, we also investigated that the reduction of GRP75 gene expression by siRNA significantly inhibited the blastocyst development rate (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) and expanded blastocyst formation (Control: 30%, N.C siRNA (25 nM): 29% vs GRP75-siRNA (25 nM): 15%) in porcine embryos. The porcine blastocysts produced by GRP75 siRNA microinjection showed a dramatic reduction of GRP75 expression present in the mitochondria and ER, respectively. Additionally, the co-localization between ER and mitochondria indicative of MAM region in GRP75 siRNA microinjected blastocysts were significantly lower (1.9 fold; Control: 29.3%; N.C siRNA (25 nM): 29.1% vs GRP75-siRNA (25 nM): 15.7%, p < 0.001) than in other groups. Finally, we confirmed the increase (p < 0.01) in the numbers of mitochondrial and ER aggregation (diameter; > 5 μm) in porcine blastocysts after GRP75 siRNA microinjection. Therefore, these findings demonstrated that GRP75 knockdown disrupted the early embryos developmental competence until blastocyst stage through reduction of MAM mechanisms.

*This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF-2021R1C1C2009469, NRF-2022R1A2C1002800, and NRF-2021R1A6A3A01087623) funded by and the Ministry of Science and ICT, Republic of Korea.

Key words: GRP75, MAM, Knockdown, Porcine embryo development