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Connexin 43 Knockdown Induces Mitochondrial Dysfunction and Affects Early Developmental Competence in Porcine Embryos

Published online by Cambridge University Press:  10 February 2020

Kyung-Tae Shin
Affiliation:
Department of Animal Sciences, Chungbuk National University, Chungbuk, Cheongju361-763, Republic of Korea
Zheng-Wen Nie
Affiliation:
Department of Animal Sciences, Chungbuk National University, Chungbuk, Cheongju361-763, Republic of Korea
Wenjun Zhou
Affiliation:
Department of Animal Sciences, Chungbuk National University, Chungbuk, Cheongju361-763, Republic of Korea
Dongjie Zhou
Affiliation:
Department of Animal Sciences, Chungbuk National University, Chungbuk, Cheongju361-763, Republic of Korea
Ju-Yeon Kim
Affiliation:
Department of Animal Sciences, Chungbuk National University, Chungbuk, Cheongju361-763, Republic of Korea
Sun A. Ock
Affiliation:
Animal Biotechnology Division, National Institute of Animal Science, Rural Development Administration, Jeonju55365, Republic of Korea
Ying-Jie Niu
Affiliation:
Department of Animal Sciences, Chungbuk National University, Chungbuk, Cheongju361-763, Republic of Korea
Xiang-Shun Cui*
Affiliation:
Department of Animal Sciences, Chungbuk National University, Chungbuk, Cheongju361-763, Republic of Korea
*
*Author for correspondence: Xiang-Shun Cui, E-mail: xscui@cbnu.ac.kr
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Abstract

Connexin 43 (CX43) is a component of gap junctions. The lack of functional CX43 induces oxidative stress, autophagy, and apoptosis in somatic cells. However, the role of CX43 in the early development of porcine embryos is still unknown. Thus, the aim of this study was to investigate the role of CX43, and its underlying molecular mechanisms, on the developmental competence of early porcine embryos. We performed CX43 knockdown by microinjecting dsRNA into parthenogenetically activated porcine parthenotes. The blastocyst development rate and the total number of cells in the blastocysts were significantly reduced by CX43 knockdown. Results from FITC-dextran assays showed that CX43 knockdown significantly increased membrane permeability. ZO-1 protein was obliterated in CX43 knockdown blastocysts. Mitochondrial membrane potential and ATP production were significantly reduced following CX43 knockdown. Reactive oxygen species (ROS) levels were significantly increased in the CX43 knockdown group compared to those in control embryos. Moreover, CX43 knockdown induced autophagy and apoptosis. Our findings indicate that CX43 is essential for the development and preimplantation of porcine embryos and maintains mitochondrial function, cell junction structure, and cell homeostasis by regulating membrane permeability, ROS generation, autophagy, and apoptosis in early embryos.

Type
Biological Applications
Copyright
Copyright © Microscopy Society of America 2020

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References

Akanuma, SI, Higashi, H, Maruyama, S, Murakami, K, Tachikawa, M, Kubo, Y & Hosoya, KI (2018). Expression and function of connexin 43 protein in mouse and human retinal pigment epithelial cells as hemichannels and gap junction proteins. Exp Eye Res 168, 128137.CrossRefGoogle ScholarPubMed
Alexander, DB & Goldberg, GS (2003). Transfer of biologically important molecules between cells through gap junction channels. Curr Med Chem 10(19), 20452058.CrossRefGoogle ScholarPubMed
Ali, I, Shah, SZ, Jin, Y, Li, ZS, Ullah, O & Fang, NZ (2017). Reactive oxygen species-mediated unfolded protein response pathways in preimplantation embryos. J Vet Sci 18(1), 19.CrossRefGoogle ScholarPubMed
Antelman, J, Manandhar, G, Yi, YJ, Li, R, Whitworth, KM, Sutovsky, M, Agca, C, Prather, RS & Sutovsky, P (2008). Expression of mitochondrial transcription factor A (TFAM) during porcine gametogenesis and preimplantation embryo development. J Cell Physiol 217(2), 529543.CrossRefGoogle ScholarPubMed
Bloor, DJ, Wilson, Y, Kibschull, M, Traub, O, Leese, HJ, Winterhager, E & Kimber, SJ (2004). Expression of connexins in human preimplantation embryos in vitro. Reprod Biol Endocrinol 2, 25.CrossRefGoogle ScholarPubMed
Chen, Y, Azad, M & Gibson, S (2009). Superoxide is the major reactive oxygen species regulating autophagy. Cell Death Differ 16(7), 1040.CrossRefGoogle ScholarPubMed
Chen, YN, Dai, JJ, Wu, CF, Zhang, SS, Sun, LW & Zhang, DF (2018). Apoptosis and developmental capacity of vitrified parthenogenetic pig blastocysts. Anim Reprod Sci 198, 137144.CrossRefGoogle ScholarPubMed
Choi, I, Carey, TS, Wilson, CA & Knott, JG (2012). Transcription factor AP-2gamma is a core regulator of tight junction biogenesis and cavity formation during mouse early embryogenesis. Development 139(24), 46234632.CrossRefGoogle ScholarPubMed
Davies, TC, Barr, KJ, Jones, DH, Zhu, D & Kidder, GM (1996). Multiple members of the connexin gene family participate in preimplantation development of the mouse. Dev Genet 18(3), 234243.3.0.CO;2-A>CrossRefGoogle ScholarPubMed
Davis, HM, Pacheco-Costa, R, Atkinson, EG, Brun, LR, Gortazar, AR, Harris, J, Hiasa, M, Bolarinwa, SA, Yoneda, T, Ivan, M, Bruzzaniti, A, Bellido, T & Plotkin, LI (2017). Disruption of the CX43/miR21 pathway leads to osteocyte apoptosis and increased osteoclastogenesis with aging. Aging Cell 16(3), 551563.CrossRefGoogle ScholarPubMed
Day, BN (2000). Reproductive biotechnologies: Current status in porcine reproduction. Anim Reprod Sci 60–61, 161172.CrossRefGoogle ScholarPubMed
De Sousa, PA, Valdimarsson, G, Nicholson, BJ & Kidder, GM (1993). Connexin trafficking and the control of gap junction assembly in mouse preimplantation embryos. Development 117(4), 13551367.Google ScholarPubMed
Devasagayam, T, Tilak, J, Boloor, K, Sane, KS, Ghaskadbi, SS & Lele, R (2004). Free radicals and antioxidants in human health: Current status and future prospects. J Assoc Physicians India 52, 794804.Google ScholarPubMed
Eckert, JJ & Fleming, TP (2008). Tight junction biogenesis during early development. Biochim Biophys Acta 1778(3), 717728.CrossRefGoogle ScholarPubMed
Ferris, J, Mahboubi, K, MacLusky, N, King, WA & Favetta, LA (2016). BPA exposure during in vitro oocyte maturation results in dose-dependent alterations to embryo development rates, apoptosis rate, sex ratio and gene expression. Reprod Toxicol 59, 128138.CrossRefGoogle ScholarPubMed
Gao, J, Cheng, TS, Qin, A, Pavlos, NJ, Wang, T, Song, K, Wang, Y, Chen, L, Zhou, L, Jiang, Q, Takayanagi, H, Yan, S & Zheng, M (2016). Glucocorticoid impairs cell-cell communication by autophagy-mediated degradation of connexin 43 in osteocytes. Oncotarget 7(19), 2696626978.CrossRefGoogle ScholarPubMed
Giepmans, BN & Moolenaar, WH (1998). The gap junction protein connexin43 interacts with the second PDZ domain of the zona occludens-1 protein. Curr Biol 8(16), 931934.CrossRefGoogle ScholarPubMed
Guerin, P, El Mouatassim, S & Menezo, Y (2001). Oxidative stress and protection against reactive oxygen species in the pre-implantation embryo and its surroundings. Hum Reprod Update 7(2), 175189.CrossRefGoogle ScholarPubMed
Guo, J, Kim, NH & Cui, XS (2017 a). Inhibition of fatty acid synthase reduces blastocyst hatching through regulation of the AKT pathway in pigs. PLoS One 12(1), e0170624.CrossRefGoogle ScholarPubMed
Guo, J, Zhao, MH, Shin, KT, Niu, YJ, Ahn, YD, Kim, NH & Cui, XS (2017 b). The possible molecular mechanisms of bisphenol A action on porcine early embryonic development. Sci Rep 7(1), 8632.CrossRefGoogle ScholarPubMed
Hardy, K, Warner, A, Winston, RM & Becker, DL (1996). Expression of intercellular junctions during preimplantation development of the human embryo. Mol Hum Reprod 2(8), 621632.CrossRefGoogle ScholarPubMed
Houghton, FD (2005). Role of gap junctions during early embryo development. Reproduction 129(2), 129135.CrossRefGoogle ScholarPubMed
Houghton, FD, Barr, KJ, Walter, G, Gabriel, HD, Grummer, R, Traub, O, Leese, HJ, Winterhager, E & Kidder, GM (2002). Functional significance of gap junctional coupling in preimplantation development. Biol Reprod 66(5), 14031412.CrossRefGoogle ScholarPubMed
Juraver-Geslin, HA & Durand, BC (2015). Early development of the neural plate: New roles for apoptosis and for one of its main effectors caspase-3. Genesis 53(2), 203224.CrossRefGoogle ScholarPubMed
Kim, SN, Kwon, HJ, Im, SW, Son, YH, Akindehin, S, Jung, YS, Lee, SJ, Rhyu, IJ, Kim, IY, Seong, JK, Lee, J, Yoo, HC, Granneman, JG & Lee, YH (2017). Connexin 43 is required for the maintenance of mitochondrial integrity in brown adipose tissue. Sci Rep 7(1), 7159.CrossRefGoogle ScholarPubMed
Kowluru, RA & Mishra, M (2015). Oxidative stress, mitochondrial damage and diabetic retinopathy. Biochim Biophys Acta 1852(11), 24742483.CrossRefGoogle ScholarPubMed
Kwon, J, Kim, NH & Choi, I (2016 a). ROCK activity regulates functional tight junction assembly during blastocyst formation in porcine parthenogenetic embryos. PeerJ 4, e1914.CrossRefGoogle ScholarPubMed
Kwon, JW, Kim, NH & Choi, I (2016 b). CXADR is required for AJ and TJ assembly during porcine blastocyst formation. Reproduction 151(4), 297304.CrossRefGoogle ScholarPubMed
Laird, DW (2005). Connexin phosphorylation as a regulatory event linked to gap junction internalization and degradation. Biochim Biophys Acta 1711(2), 172182.CrossRefGoogle ScholarPubMed
Lim, KT, Gupta, MK, Lee, SH, Jung, YH, Han, DW & Lee, HT (2013). Possible involvement of Wnt/beta-catenin signaling pathway in hatching and trophectoderm differentiation of pig blastocysts. Theriogenology 79(2), 284290.e1–2CrossRefGoogle ScholarPubMed
Mizushima, N, Yoshimori, T & Levine, B (2010). Methods in mammalian autophagy research. Cell 140(3), 313326.CrossRefGoogle ScholarPubMed
Moriwaki, K, Tsukita, S & Furuse, M (2007). Tight junctions containing claudin 4 and 6 are essential for blastocyst formation in preimplantation mouse embryos. Dev Biol 312(2), 509522.CrossRefGoogle ScholarPubMed
Niu, Y-J, Nie, Z-W, Shin, K-T, Zhou, W & Cui, X-S (2019). PINK1 regulates mitochondrial morphology via promoting mitochondrial fission in porcine preimplantation embryos. FASEB J 33(7), 78827895. doi: 10.1096/fj.201802473rCrossRefGoogle ScholarPubMed
Ortiz-Escribano, N, Szymanska, KJ, Bol, M, Vandenberghe, L, Decrock, E, Van Poucke, M, Peelman, L, Van den Abbeel, E, Van Soom, A & Leybaert, L (2017). Blocking connexin channels improves embryo development of vitrified bovine blastocysts. Biol Reprod 96(2), 288301.CrossRefGoogle ScholarPubMed
Paul, DL, Ebihara, L, Takemoto, LJ, Swenson, KI & Goodenough, DA (1991). Connexin46, a novel lens gap junction protein, induces voltage-gated currents in nonjunctional plasma membrane of Xenopus oocytes. J Cell Biol 115(4), 10771089.CrossRefGoogle ScholarPubMed
Peters, A (2001). There is nothing more practical than a good theory: An overview of contemporary approaches to International Law. German YB Int'l L 44, 25.Google Scholar
Rizos, D, Lonergan, P, Boland, MP, Arroyo-Garcia, R, Pintado, B, de la Fuente, J & Gutierrez-Adan, A (2002). Analysis of differential messenger RNA expression between bovine blastocysts produced in different culture systems: Implications for blastocyst quality. Biol Reprod 66(3), 589595.CrossRefGoogle ScholarPubMed
Shin, KT, Guo, J, Niu, YJ & Cui, XS (2018). The toxic effect of aflatoxin B1 on early porcine embryonic development. Theriogenology 118, 157163.CrossRefGoogle ScholarPubMed
Sorgen, PL, Trease, AJ, Spagnol, G, Delmar, M & Nielsen, MS (2018). Protein–protein interactions with connexin 43: Regulation and function. Int J Mol Sci 19, 5.CrossRefGoogle ScholarPubMed
Thomas, FC, Sheth, B, Eckert, JJ, Bazzoni, G, Dejana, E & Fleming, TP (2004). Contribution of JAM-1 to epithelial differentiation and tight-junction biogenesis in the mouse preimplantation embryo. J Cell Sci 117(Pt 23), 55995608.CrossRefGoogle ScholarPubMed
Tien, T, Muto, T, Barrette, K, Challyandra, L & Roy, S (2014). Downregulation of connexin 43 promotes vascular cell loss and excess permeability associated with the development of vascular lesions in the diabetic retina. Mol Vision 20, 732741.Google ScholarPubMed
Van Blerkom, J, Cox, H & Davis, P (2006). Regulatory roles for mitochondria in the peri-implantation mouse blastocyst: Possible origins and developmental significance of differential Δψm. Reproduction 131(5), 961976.CrossRefGoogle ScholarPubMed
Wang, N, De Bock, M, Decrock, E, Bol, M, Gadicherla, A, Bultynck, G & Leybaert, L (2013). Connexin targeting peptides as inhibitors of voltage- and intracellular Ca2+-triggered CX43 hemichannel opening. Neuropharmacology 75, 506516.CrossRefGoogle ScholarPubMed
Wrenzycki, C, Herrmann, D, Carnwath, JW & Niemann, H (1996). Expression of the gap junction gene connexin43 (CX43) in preimplantation bovine embryos derived in vitro or in vivo. J Reprod Fertil 108(1), 1724.CrossRefGoogle ScholarPubMed
Zhao, C, Fang, J, Li, C & Zhang, M (2017). Connexin43 and AMPK have essential role in resistance to oxidative stress induced necrosis. BioMed Res Int 2017, 3962173.CrossRefGoogle ScholarPubMed
Zhao, MH, Liang, S, Kim, SH, Cui, XS & Kim, NH (2015). Fe(III) is essential for porcine embryonic development via mitochondrial function maintenance. PLoS One 10(7), e0130791.CrossRefGoogle ScholarPubMed