2022년 한국동물생명공학회 학술 대회 [Seul-Gi Yang] > 학회발표

Inhibition of DRP1 induces meiotic maturation failure through decreasing spindle formation genes expressions during in vitro maturation in pigs

Seul-Gi Yang1,2, Hyo-Jin Park1,2, Ji-Hyun Shin1,2, Jae-Hun Choi1,2, Young-Seo Jo1,2, Ye-Won Kim1,2 and Deog-Bon Koo1,2

1Department of Biotechnology, College of Engineering, Daegu University,

201 Daegudae-ro, Jillyang, Gyeongsan, Gyeongbuk 38453, Republic of Korea

2Institute of Infertility, Daegu University, 201 Daegudae-ro, Jillyang, Gyeongsan, Gyeongbuk 38453, Republic of Korea

   The dynamin-related protein (DRP1) plays an essential role in mitochondrial fission during porcine oocyte maturation. However, the effect of reduction of DRP1 expression by siRNA transfection on spindle formation during porcine oocyte maturation is unknown. Here, we investigated the DRP1 siRNA knockdown efficiency by transfection (Lipofectamine) during porcine meiotic maturation using orcein staining. Oocyte maturation rate in the DRP1 knockdown group was significantly decreased compared to the other control and negative control groups (p ＜ 0.05, control group: 84.5 ± 7.9%; negative control group (100 nM): 84.2 ± 7.7% vs DRP1 knockdown group (100 nM): 66.7 ± 5.3%). To confirm the reduction of DRP1 protein expression, we examined mitochondrial fission-related protein expressions (pDRP1-Ser616, DRP1, and FIS1) using Western blot analysis. All mitochondrial fission-related protein expressions in the DRP1 knockdown group were significantly lower (p ＜ 0.05) than those of control group. Based on these results, we investigated correlations between mitochondrial dynamics and spindle formation in the total RNA level of the DRP1 knockdown group by using transcriptome analysis. Ontology analysis showed that changes in cell cycle and mitosis-related genes expressions were highlighted most distinct represented biological phenomena in DRP1 knockdown group. In particular, the expressions of spindle assembly-related genes from scatter plot and heatmap cluster analysis through RNA-seq were 10 genes revealed ≥1.2-fold increased abundance, however, 6 genes indicated a decrease in abundance (≤1.2-fold) by silencing DRP1. According to RNA-seq results, we confirmed the cell cycle and spindle formation-related genes (Rad51, H2ax, Tuba1a, Atat1, and Map2) using qRT-PCR analysis. The cell cycle-related genes expressions were significantly increased (p ＜ 0.05) in DRP1 knockdown group compared with control group. Additionally, the spindle assembly-related genes expressions in DRP1 knockdown group were reduced (p ＜ 0.05) than those of control group. In summary, our results suggest that the knockdown of DRP1 is closely related to a decrease in the expression of cell cycle and spindle formation-related genes related, which leads to a meiotic maturation reduction of pig oocytes.

*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) F-actin, microtubule, spindle assembly, oocyte aging, in vitro maturation

E-mail : foreverday37@naver.com (Seul-Gi, Yang).