Melatonin accelerates the developmental competence and telomere elongation in ovine SCNT embryos

PLOS ONE RESEARCH ARTICLE Melatonin accelerates the developmental competence and telomere elongation in ovine SCNT embryos Parisa Nadri1, Saeid Ansari-Mahyari1*, Farnoosh Jafarpour2, Amir Hossein Mahdavi1, Nima Tanhaei Vash2, Liana Lachinani3, Kianoush Dormiani3, Mohammad Hossein NasrEsfahani ID2* a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 1 Department of Animal Science, College of Agriculture, Isfahan University of Technology, Isfahan, Iran, 2 Department of Animal Biotechnology, Reproductive Biomedicine Research Center, Royan Institute for Biotechnology, ACECR, Isfahan, Iran, 3 Department of Animal Biotechnology, Cell Science Research Center, Royan Institute for Biotechnology, ACECR, Isfahan, Iran * (SAM); , (MHNE) Abstract OPEN ACCESS Citation: Nadri P, Ansari-Mahyari S, Jafarpour F, Mahdavi AH, Tanhaei Vash N, Lachinani L, et al. (2022) Melatonin accelerates the developmental competence and telomere elongation in ovine SCNT embryos. PLoS ONE 17(7): e0267598. https://doi.org/10.1371/journal.pone.0267598 Editor: Christine Wrenzycki, Justus Liebig Universitat Giessen, GERMANY Received: October 23, 2021 Accepted: April 11, 2022 Published: July 21, 2022 Copyright: © 2022 Nadri 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 manuscript files. There is no additional relevant data for supporting information files. SCNT embryos suffer from poor developmental competence (both in vitro and in vivo) due to various defects such as oxidative stress, incomplete epigenetic reprogramming, and flaws in telomere rejuvenation. It is very promising to ameliorate all these defects in SCNT embryos by supplementing the culture medium with a single compound. It has been demonstrated that melatonin, as a multitasking molecule, can improve the development of SCNT embryos, but its function during ovine SCNT embryos is unclear. We observed that supplementation of embryonic culture medium with 10 nM melatonin for 7 days accelerated the rate of blastocyst formation in ovine SCNT embryos. In addition, the quality of blastocysts increased in the melatonin-treated group compared with the SCNT control groups in terms of ICM, TE, total cell number, and mRNA expression of NANOG. Mechanistic studies in this study revealed that the melatonin-treated group had significantly lower ROS level, apoptotic cell ratio, and mRNA expression of CASPASE-3 and BAX/BCL2 ratio. In addition, melatonin promotes mitochondrial membrane potential and autophagy status (higher number of LC3B dots). Our results indicate that melatonin decreased the global level of 5mC and increased the level of H3K9ac in the treated blastocyst group compared with the blastocysts in the control group. More importantly, we demonstrated for the first time that melatonin treatment promoted telomere elongation in ovine SCNT embryos. This result offers the possibility of better development of ovine SCNT embryos after implantation. We concluded that melatonin can accelerate the reprogramming of telomere length in sheep SCNT embryos, in addition to its various beneficial effects such as increasing antioxidant capacity, reducing DNA damage, and improving the quality of derived blastocysts, all of which led to a higher in vitro development rate. Funding: All the authors have declared that the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist. PLOS ONE | https://doi.org/10.1371/journal.pone.0267598 July 21, 2022 1 / 19 PLOS ONE Abbreviations: DNMTis, DNA methyltransferases inhibitors; GSH, Glutathione; HMTis, histone methyltransferase inhibitors; ICM, inner cell mass; IVF, in vitro fertilization; IVM, in vitro maturation; ROS, reactive oxygen species; SCNT, Somatic cell nuclear transfer; TCN, total cells number; TE, trophectoderm. Melatonin improves efficiency of ovine SCNT embryos Introduction The oocyte, as a unique cell in both structure and function, is capable of supporting the full development of embryos from somatic cell nuclear transfer (SCNT). After the birth of Dolly in 1997, the SCNT technique has been applied to more than 20 animal species for the purpose of reproductive cloning [1]. Despite the successful results of SCNT technique, the low efficiency (1–2%) of this technique is no longer a matter of debate, which limits its application [2]. Several factors such as suboptimal culture conditions, oxidative stress, autophagy status, telomere length, epigenetic barriers in donor somatic cells, etc. are responsible for the low success rate of the SCNT technique [3–5]. The physiological level of intracellular reactive oxygen species (ROS) is an essential factor for maintaining the proper function of male and female reproductive systems [6, 7]. Supraphysiological level of ROS lead to oxidative stress, which impairs the ability of sperm, oocytes, and resulting embryos to develop [8–10]. Several studies have shown that elevated levels of ROS under in vitro conditions can contribute to embryonic apoptosis, mitochondrial dysfunction, and subsequent developmental arrest [10–12]. Moreover, oxidative stress and DNA damage have been shown to be higher in SCNT embryos than in in vitro fertilized (IVF) embryos due to some specific procedures such as enucleation, cell attachment, electrical fusion and chemical activation [4, 13]. One of the most effective strategies to improve cellular antioxidant capacity to overcome the excessive production of ROS is the addition of exogenous antioxidants to the culture media. In this regard, numerous studies have reported that supplementing the maturation or/and culture medium with exogenous antioxidants accelerates embryonic development [14]. Scientists have demonstrated the role of autophagy in several developmental features, including pre- and post-embryonic development and placental and fetal growth. Tsukamoto and colleagues have shown that autophagy can be used as a valuable marker for selecting highquality embryos for further development [15, 16]. Limited studies have shown that autophagy is not induced in SCNT embryos compared to IVF embryos. Tsukamoto and colleagues have also shown that pharmacological activation of autophagy in SCNT-reconstituted oocytes increases the blastocyst rate [15, 16]. Several studies have shown that certain epigenetic markers such as DNA and histone methylation (H3K9 and H3K27) in donor somatic cells are crucial barriers in the epigenetic reprogramming process in SCNT [17]. In this regard, there are several evidences in the literature that the removal of these barriers using DNA methyltransferase inhibitors (DNMTis) and histone methyltransferase inhibit (...truncated)