Developmental competence of IVF and SCNT goat embryos is improved by inhibition of canonical WNT signaling

PLOS ONE RESEARCH ARTICLE Developmental competence of IVF and SCNT goat embryos is improved by inhibition of canonical WNT signaling Marjan Sadeghi1,2, Mohsen Rahimi Andani2, Mehdi Hajian2, Nafiseh Sanei2, Reza MoradiHajidavaloo2, Nasrin Mahvash1,2, Farnoosh Jafarpour2*, Mohammad Hossein NasrEsfahani ID2* 1 Department of Biology, Faculty of Science and Technology, ACECR Institute of Higher Education (Isfahan), Isfahan, Iran, 2 Department of Animal Biotechnology, Reproductive Biomedicine Research Center, Royan Institute for Biotechnology, ACECR, Isfahan, Iran a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 OPEN ACCESS Citation: Sadeghi M, Andani MR, Hajian M, Sanei N, Moradi-Hajidavaloo R, Mahvash N, et al. (2023) Developmental competence of IVF and SCNT goat embryos is improved by inhibition of canonical WNT signaling. PLoS ONE 18(4): e0281331. https://doi.org/10.1371/journal.pone.0281331 Editor: Carlos E. Ambrósio, USP FZEA: Universidade de Sao Paulo Faculdade de Zootecnia e Engenharia de Alimentos, BRAZIL Received: August 6, 2022 Accepted: January 20, 2023 * (FJ); (MHNE) Abstract The specific role of the canonical WNT/β-catenin signaling pathway during the preimplantation development of goat remains unclear. Our objective was to investigate the expression of β-CATENIN, one of the critical components of Wnt signaling pathway, in IVF embryos and compare it with SCNT embryos in goat. In addition, we evaluated the consequence of inhibition of β-catenin using IWR1. Initially, we observed cytoplasmic expression of β-CATENIN in 2 and 8–16 cell stage embryos and membranous expression of β-CATENIN in compact morula and blastocyst stages. Furthermore, while we observed exclusively membranous localization of β-catenin in IVF blastocysts, we observed both membranous and cytoplasmic localization in SCNT blastocysts. We observed that Inhibition of WNT signaling by IWR1 during compact morula to blastocyst transition (from day 4 till day 7 of in vitro culture) increased blastocyst formation rate in both IVF and SCNT embryos. In conclusion, it seems that WNT signaling system has functional role in the preimplantation goat embryos, and inhibition of this pathway during the period of compact morula to blastocyst transition (D4-D7) can improve preimplantation embryonic development. Published: April 19, 2023 Copyright: © 2023 Sadeghi et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting information files. Funding: The author(s) received no specific funding for this work. Competing interests: The authors have declared that no competing interests exist. Introduction Successful development of newly formed embryos [either IVF (in vitro fertilization) or SCNT embryos] is a complex process which requires precise and synchronized regulation of signaling pathways [1–6]. Any abnormal regulations of these mechanisms in embryos during early and late development results in an embryonic arrest or numerous phenotypically defects in the live offspring [4, 7, 8]. Even though the results of assisted reproductive techniques (ARTs) indicate that maternal signals are not essential during the early embryo development [9, 10], the importance of maternal signaling is revealed by higher defects in ART-derived embryos in terms of the transcriptome [10], proteome [11], metabolome [12] and epigenome [13] as compared to PLOS ONE | https://doi.org/10.1371/journal.pone.0281331 April 19, 2023 1 / 19 PLOS ONE IWR1 improves blastocyst rate of goat IVF and SCNT embryos their in vivo counterparts. Furthermore, the higher proportion of defects in ART-derived embryos manifests itself through a lower pregnancy rate than that for in vivo-produced embryos [1, 14]. The highly orchestrated WNT signaling pathways were first identified for their critical role in the development of cancer [15, 16]. Then, later their function was recognized in a wide range of embryonic development processes, including body axis formation [17], maintenance of pluripotency [18, 19], the commitment of differentiation and the proliferation of cells [20, 21], migration of cells [22], and maintenance of hemostasis in adult tissues. Wnt signaling pathway is a highly regulated and complex signaling pathway which is regulated by various WNT ligands, and these ligands interact and bind with a variety of receptors, including frizzled (FZD), receptor tyrosine kinase-like orphan receptor (ROR), receptor tyrosine kinase-related tyrosine kinase (RYK) and protein tyrosine kinase-7 (PTK7) and also coreceptors including low-density lipoprotein receptor-related proteins 5 and 6 (LRP5 and LRP6) [23–26]. Currently, three different WNT pathways are introduced to be activated upon WNT-receptor interaction [25, 27–30]. Canonical WNT/β-catenin signaling pathway is the well-identified downstream pathway and is strongly dependent on β-catenin [23]. The two other pathways are non-canonical pathways and are independent of β-catenin. A typical example of a non-canonical pathway is the planar cell polarity pathway (PCP). The other non-canonical pathway is the Ca2+ signaling pathway. Each pathway is activated by specific ligands and receptors that can be varied among different cell types or different physiological conditions, even in a defined cell type. These differences are related to multifaceted interactions between various ligands and receptors, and regulatory molecules in intercellular and intracellular microenvironments. Thus, specific ligand-receptor interactions can have various consequences in different cell types [26, 27, 29]. The canonical WNT/ß-Catenin pathway is initiated by the interacting WNT ligands with FRZ/LRP5 and LRP6 co-receptors. This complex recruits the dishevelled (Dvl), and subsequently Dvl traps the destruction complex containing Axin/APC/CK1/GSK-3β. This event prevents phosphorylation and degradation of β-catenin and then leads to the accumulation of β-catenin in the cytoplasm and translocation to the nucleus, where it activates transcription factors in the nucleus [31–33]. Canonical WNT/ß-Catenin is a maternally-derived pathway that seems to be essential for the preimplantation development of embryos [34]. Studies in the mouse indicate that this pathway is active as early as the two-cell stage around embryonic genome activation (EGA) [35]. In bovines, it has been shown that stimulation of WNT signaling using AMBMP (activator of WNT signaling pathway) during days 5 to 7 post-fertilization decreased the blastocyst formation rate. Supplementation of bovine embryo culture medium with DKK1 reversed the effect of AMPMP on blastocyst formation rate and CTNNB1 accumulation [36, 37]. In addition, some studies demonstrated that supplementation of bovine embryo c (...truncated)