SIRT2 Is Critical for Sheep Oocyte Maturation through Regulating Function of Surrounding Granulosa Cells
Abstract
:1. Introduction
2. Results
2.1. Inhibition or Activation of SIRT2 Affects Sheep Oocytes In Vitro Maturation
2.2. Sirt2 Is Critical for Sheep Oocytes In Vitro Maturation by Affecting the Apoptosis, Expansion, and Secretion of Granulosa Cells
2.3. Sirt2 Is Required for the Dynamic Balance of Mitochondrial Fusion and Fission and the Mitochondrial Quality
2.4. Effect of Sirt2 Overexpression on Granulosa Cell Mitochondrial Function
2.5. Sirt2 Knockdown Increases the Mitophagy Level
2.6. SIRT2 Regulates Steroid Hormones Secretion through ERK1/2 Signaling Pathway in Granulosa Cell
3. Discussion
4. Materials and Methods
4.1. Ethics Statement
4.2. Chemicals and Reagents
4.3. Isolation and Culture of Granulosa Cells and Oocytes
4.4. RNA Interference
4.5. Gene Overexpression
4.6. Granulosa Cells Were Cocultured with DOs
4.7. Cell Scratch Test
4.8. Intracellular Reactive Oxygen Species (ROS) and Glutathione (GSH) Levels Assay
4.9. Detection of Mitochondrial Membrane Potential
4.10. Immunofluorescence Staining
4.11. RNA Extraction and Quantitative Real-Time Reverse-Transcription Polymerase Chain Reaction (qRT-PCR)
4.12. Western Blot
4.13. ELISA
4.14. Determination of Intracellular ATP
4.15. Statistical Analysis
5. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
- Eppig, J.J. Reproduction: Oocytes Call, Granulosa Cells Connect. Curr. Biol. 2018, 28, R354–R356. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Sanchez-Lazo, L.; Brisard, D.; Elis, S.; Maillard, V.; Uzbekov, R.; Labas, V.; Desmarchais, A.; Papillier, P.; Monget, P.; Uzbekova, S. Fatty acid synthesis and oxidation in cumulus cells support oocyte maturation in bovine. Mol. Endocrinol. 2014, 28, 1502–1521. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Li, H.J.; Sutton-McDowall, M.L.; Wang, X.; Sugimura, S.; Thompson, J.G.; Gilchrist, R.B. Extending prematuration with cAMP modulators enhances the cumulus contribution to oocyte antioxidant defence and oocyte quality via gap junctions. Hum. Reprod. 2016, 31, 810–821. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Sugiyama, M.; Sumiya, M.; Shirasuna, K.; Kuwayama, T.; Iwata, H. Addition of granulosa cell mass to the culture medium of oocytes derived from early antral follicles increases oocyte growth, ATP content, and acetylation of H4K12. Zygote 2016, 24, 848–856. [Google Scholar] [CrossRef]
- Richani, D.; Dunning, K.R.; Thompson, J.G.; Gilchrist, R.B. Metabolic co-dependence of the oocyte and cumulus cells: Essential role in determining oocyte developmental competence. Hum. Reprod. Update 2021, 27, 27–47. [Google Scholar] [CrossRef]
- Liu, H.Y.; Gale, J.R.; Reynolds, I.J.; Weiss, J.H.; Aizenman, E. The Multifaceted Roles of Zinc in Neuronal Mitochondrial Dysfunction. Biomedicines 2021, 9, 489. [Google Scholar] [CrossRef]
- Kansaku, K.; Itami, N.; Kawahara-Miki, R.; Shirasuna, K.; Kuwayama, T.; Iwata, H. Differential effects of mitochondrial inhibitors on porcine granulosa cells and oocytes. Theriogenology 2017, 103, 98–103. [Google Scholar] [CrossRef]
- Sakaguchi, K.; Huang, W.; Yang, Y.; Yanagawa, Y.; Nagano, M. Relationship between in vitro growth of bovine oocytes and steroidogenesis of granulosa cells cultured in medium supplemented with bone morphogenetic protein-4 and follicle stimulating hormone. Theriogenology 2017, 97, 113–123. [Google Scholar] [CrossRef]
- Singh, C.K.; Chhabra, G.; Ndiaye, M.A.; Garcia-Peterson, L.M.; Mack, N.J.; Ahmad, N. The Role of Sirtuins in Antioxidant and Redox Signaling. Antioxid. Redox Signal. 2018, 28, 643–661. [Google Scholar] [CrossRef]
- Wang, T.; Wang, Y.; Liu, L.; Jiang, Z.; Li, X.; Tong, R.; He, J.; Shi, J. Research progress on sirtuins family members and cell senescence. Eur. J. Med. Chem. 2020, 193, 112207. [Google Scholar] [CrossRef]
- Wu, B.; You, S.; Qian, H.; Wu, S.; Lu, S.; Zhang, Y.; Sun, Y.; Zhang, N. The role of SIRT2 in vascular-related and heart-related diseases: A review. J. Cell Mol. Med. 2021, 25, 6470–6478. [Google Scholar] [CrossRef] [PubMed]
- Xu, D.; Wu, L.; Jiang, X.; Yang, L.; Cheng, J.; Chen, H.; Hua, R.; Geng, G.; Yang, L.; Li, Q. SIRT2 Inhibition Results in Meiotic Arrest, Mitochondrial Dysfunction, and Disturbance of Redox Homeostasis during Bovine Oocyte Maturation. Int. J. Mol. Sci. 2019, 20, 1365. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Wang, Y.P.; Zhou, L.S.; Zhao, Y.Z.; Wang, S.W.; Chen, L.L.; Liu, L.X.; Ling, Z.Q.; Hu, F.J.; Sun, Y.P.; Zhang, J.Y.; et al. Regulation of G6PD acetylation by SIRT2 and KAT9 modulates NADPH homeostasis and cell survival during oxidative stress. EMBO J. 2014, 33, 1304–1320. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Xu, D.; He, H.; Liu, D.; Geng, G.; Li, Q. A novel role of SIRT2 in regulating gap junction communications via connexin-43 in bovine cumulus-oocyte complexes. J. Cell Physiol. 2020, 235, 7332–7343. [Google Scholar] [CrossRef]
- Gyles, S.L.; Burns, C.J.; Whitehouse, B.J.; Sugden, D.; Marsh, P.J.; Persaud, S.J.; Jones, P.M. ERKs regulate cyclic AMP-induced steroid synthesis through transcription of the steroidogenic acute regulatory (StAR) gene. J. Biol. Chem. 2001, 276, 34888–34895. [Google Scholar] [CrossRef] [Green Version]
- Dalton, C.M.; Szabadkai, G.; Carroll, J. Measurement of ATP in single oocytes: Impact of maturation and cumulus cells on levels and consumption. J. Cell Physiol. 2014, 229, 353–361. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Zhang, L.; Hou, X.; Ma, R.; Moley, K.; Schedl, T.; Wang, Q. Sirt2 functions in spindle organization and chromosome alignment in mouse oocyte meiosis. FASEB J. 2014, 28, 1435–1445. [Google Scholar] [CrossRef] [Green Version]
- Xu, D.; Jiang, X.; He, H.; Liu, D.; Yang, L.; Chen, H.; Wu, L.; Geng, G.; Li, Q. SIRT2 functions in aging, autophagy, and apoptosis in post-maturation bovine oocytes. Life Sci. 2019, 232, 116639. [Google Scholar] [CrossRef]
- Pan, Y.; Zhang, H.; Zheng, Y.; Zhou, J.; Yuan, J.; Yu, Y.; Wang, J. Resveratrol Exerts Antioxidant Effects by Activating SIRT2 To Deacetylate Prx1. Biochemistry 2017, 56, 6325–6328. [Google Scholar] [CrossRef]
- Jiang, W.; Wang, S.; Xiao, M.; Lin, Y.; Zhou, L.; Lei, Q.; Xiong, Y.; Guan, K.L.; Zhao, S. Acetylation regulates gluconeogenesis by promoting PEPCK1 degradation via recruiting the UBR5 ubiquitin ligase. Mol. Cell. 2011, 43, 33–44. [Google Scholar] [CrossRef] [Green Version]
- Qu, Z.A.; Ma, X.J.; Huang, S.B.; Hao, X.R.; Li, D.M.; Feng, K.Y.; Wang, W.M. SIRT2 inhibits oxidative stress and inflammatory response in diabetic osteoarthritis. Eur. Rev. Med. Pharmacol. Sci. 2020, 24, 2855–2864. [Google Scholar] [PubMed]
- Ciesiółka, S.; Budna, J.; Bryja, A.; Kranc, W.; Chachuła, A.; Dyszkiewicz-Konwińska, M.; Piotrowska, H.; Bukowska, D.; Antosik, P.; Bruska, M.; et al. Association between expression of cumulus expansion markers and real-time proliferation of porcine follicular granulosa cells in a primary cell culture model. J. Biol. Regul. Homeost Agents 2016, 30, 971–984. [Google Scholar] [PubMed]
- Jin, J.; Ma, Y.; Tong, X.; Yang, W.; Dai, Y.; Pan, Y.; Ren, P.; Liu, L.; Fan, H.Y.; Zhang, Y.; et al. Metformin inhibits testosterone-induced endoplasmic reticulum stress in ovarian granulosa cells via inactivation of p38 MAPK. Hum. Reprod. 2020, 35, 1145–1158. [Google Scholar] [CrossRef] [PubMed]
- Adebayo, M.; Singh, S.; Singh, A.P.; Dasgupta, S. Mitochondrial fusion and fission: The fine-tune balance for cellular homeostasis. FASEB J. 2021, 35, e21620. [Google Scholar] [CrossRef] [PubMed]
- Kandimalla, R.; Manczak, M.; Yin, X.; Wang, R.; Reddy, P.H. Hippocampal phosphorylated tau induced cognitive decline, dendritic spine loss and mitochondrial abnormalities in a mouse model of Alzheimer’s disease. Hum. Mol. Genet. 2018, 27, 30–40. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Youle, R.J.; van der Bliek, A.M. Mitochondrial fission, fusion, and stress. Science 2012, 337, 1062–1065. [Google Scholar] [CrossRef] [Green Version]
- Gunaydin Akyildiz, A.; Boran, T.; Jannuzzi, A.T.; Alpertunga, B. Mitochondrial dynamics imbalance and mitochondrial dysfunction contribute to the molecular cardiotoxic effects of Lenvatinib. Toxicol. Appl. Pharmacol. 2021, 423, 115577. [Google Scholar] [CrossRef]
- Westermann, B. Mitochondrial fusion and fission in cell life and death. Nat. Rev. Mol. Cell Biol. 2010, 11, 872–884. [Google Scholar] [CrossRef]
- Song, Z.Q.; Li, X.; Wang, Y.K.; Du, Z.Q.; Yang, C.X. DMBA acts on cumulus cells to desynchronize nuclear and cytoplasmic maturation of pig oocytes. Sci. Rep. 2017, 7, 1687. [Google Scholar] [CrossRef] [Green Version]
- Merry, B.J. Molecular mechanisms linking calorie restriction and longevity. Int. J. Biochem. Cell Biol. 2002, 34, 1340–1354. [Google Scholar] [CrossRef]
- Ashrafi, G.; Schwarz, T.L. The pathways of mitophagy for quality control and clearance of mitochondria. Cell Death Differ. 2013, 20, 31–42. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Wu, H.; Chen, Q. Hypoxia activation of mitophagy and its role in disease pathogenesis. Antioxid. Redox Signal. 2015, 22, 1032–1046. [Google Scholar] [CrossRef] [PubMed]
- Tsubouchi, K.; Araya, J.; Kuwano, K. PINK1-PARK2-mediated mitophagy in COPD and IPF pathogeneses. Inflamm. Regen. 2018, 38, 18. [Google Scholar] [CrossRef] [PubMed]
- Xu, D.; He, H.; Jiang, X.; Hua, R.; Chen, H.; Yang, L.; Cheng, J.; Duan, J.; Li, Q. SIRT2 plays a novel role on progesterone, estradiol and testosterone synthesis via PPARs/LXRalpha pathways in bovine ovarian granular cells. J. Steroid. Biochem. Mol. Biol. 2019, 185, 27–38. [Google Scholar] [CrossRef]
- Smith, D.M.; Tenney, D.Y. Effects of steroids on mouse oocyte maturation in vitro. J. Reprod. Fertil. 1980, 60, 331–338. [Google Scholar] [CrossRef]
- Tang, S.; Li, X.; Wu, X.; Gong, Y. WT1 suppresses follicle-stimulating hormone-induced progesterone secretion by regulating ERK1/2 pathway in chicken preovulatory granulosa cells. Gene 2022, 812, 146097. [Google Scholar] [CrossRef]
- Meng, K.; Wang, X.; He, Y.; Yang, J.; Wang, H.; Zhang, Y.; Quan, F. The Wilms tumor gene (WT1) (+/−KTS) isoforms regulate steroidogenesis by modulating the PI3K/AKT and ERK1/2 pathways in bovine granulosa cells. Biol. Reprod. 2019, 100, 1344–1355. [Google Scholar] [CrossRef]
Primers/siRNA Name | Primer Sequences (5’-3’) | Fragment Size (bp) |
---|---|---|
β-actin | F: CTCTTCCAGCCTTCCTTCCT R: GGGCAGTGATCTCTTTCTGC | 178 |
sirt2 | F: CGCCAACCTGGAGAAATA R: ATGGTGGGCTTGAACTGC | 129 |
gclm | F: CCTATTGAAGATGGAGTGAATC R: GCAGGAGGCAAGATTAACT | 189 |
prdx1 | F: CAGATGGTCAGTTCAAGGAT R: CAGGTGACAGAAGTGAGAAT | 191 |
nfe2l2 | F: CATCACCAGACCACTCAG R: GGACTTACAGGCACTTCTT | 241 |
bcl-2 | F: GATGACCGAGTACCTGAACCG R: GACAGCCAGGAGAAATCAAACA | 120 |
bax | F: CCGACGGCAACTTCAACTGG R: GATCAACTCGGGCACCTTGG | 98 |
c-myc | F: TTGATGTTGTCTCTGTGGAA R: AATTGTGCTGATGCGTAGA | 141 |
has2 | F: CCTCATCATCCAAAGCCTG R: ACATTTCCGCAAATAGTCTG | 139 |
ptx3 | F: GCTATCGGTCCATAATGCTTG R: TTTCTTTGAATCCCAGGTGC | 113 |
ptgs2 | F: AGGAGGTCTTTGGTCTGGTG R: TCTGGAACAACTGCTCATCG | 126 |
tnfaip6 | F: CTACTGGCACATTAGACTCA R: AGCATCACTTAGGAACTTCA | 217 |
mfn1 | F: AAGCACATAGAAGACGGAAT R: ACGATGGACAAGAGAAGAC | 264 |
opa1 | F: ATGAAATAGAACTCCGAATG R: GTCAACAAGCACCATCCT | 112 |
mff | F: GTGCTTACGCTGAGTGAA R: ACGAGTGGAAGACTGGATA | 354 |
fis1 | F: GGGGAACTACAGGCTCAAG R: GACACAGCAAGTCCGATGA | 207 |
drp1 | F: GGAGTTGAAGCAGAAGAATG R: AGTGACAGCGAGGATAATG | 306 |
tomm20 | F: GGACCATCTGACGAATGC R: GAGCACTTACAATTCTCTGAC | 140 |
pink1 | F: CCTGAAGTCCGACAACAT R: AGCCAATCATCTCGTCTG | 266 |
parkin | F: CTTCATCATCAACCAGTTCTC R: CTTCAGCACAGGAATCAGT | 371 |
lc3 | F: TATCCGAGAGCAGCATCC R: GATCAGGCACCAAGAACTT | 104 |
si-Sirt2 | Sense: UCUUGAAGUAGCUGAUUUCAA Antisense: GAAAUCAGCUACUUCAAGAAG | |
NC | Sense: UUCUCCGAACGUGUCACGUTT Antisense: ACGUGACACGUUCGGAGAATT |
Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. |
© 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
Share and Cite
Fang, X.; Xia, W.; Li, S.; Qi, Y.; Liu, M.; Yu, Y.; Li, H.; Li, M.; Tao, C.; Wang, Z.; et al. SIRT2 Is Critical for Sheep Oocyte Maturation through Regulating Function of Surrounding Granulosa Cells. Int. J. Mol. Sci. 2022, 23, 5013. https://doi.org/10.3390/ijms23095013
Fang X, Xia W, Li S, Qi Y, Liu M, Yu Y, Li H, Li M, Tao C, Wang Z, et al. SIRT2 Is Critical for Sheep Oocyte Maturation through Regulating Function of Surrounding Granulosa Cells. International Journal of Molecular Sciences. 2022; 23(9):5013. https://doi.org/10.3390/ijms23095013
Chicago/Turabian StyleFang, Xiaohuan, Wei Xia, Sa Li, Yatian Qi, Mingzhi Liu, Yang Yu, Hanxing Li, Mengqi Li, Chenyu Tao, Zhigang Wang, and et al. 2022. "SIRT2 Is Critical for Sheep Oocyte Maturation through Regulating Function of Surrounding Granulosa Cells" International Journal of Molecular Sciences 23, no. 9: 5013. https://doi.org/10.3390/ijms23095013