Maternal metabolic status and in-vitro culture conditions during embryonic genome activation deregulate the expression of energy-related genes in the bovine 16-cells embryo

PLOS ONE RESEARCH ARTICLE Maternal metabolic status and in-vitro culture conditions during embryonic genome activation deregulate the expression of energy-related genes in the bovine 16-cells embryo a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 Maria B. Rabaglino1, Niamh Forde2, Urban Besenfelder3, Vitezslav Havlicek3, Helmut Blum4, Alexander Graf4, Eckhard Wolf4, Patrick Lonergan1* 1 School of Agriculture and Food Science, University College Dublin, Dublin, Ireland, 2 Division of Reproduction and Early Development, School of Medicine, University of Leeds, Leeds, United Kingdom, 3 Reproduction Centre Wieselburg RCW, Institute for Animal Breeding and Genetics, University of Veterinary Medicine, Vienna, Austria, 4 Laboratory for Functional Genome Analysis, Gene Center, LMU, Munich, Germany * OPEN ACCESS Citation: Rabaglino MB, Forde N, Besenfelder U, Havlicek V, Blum H, Graf A, et al. (2023) Maternal metabolic status and in-vitro culture conditions during embryonic genome activation deregulate the expression of energy-related genes in the bovine 16-cells embryo. PLoS ONE 18(8): e0290689. https://doi.org/10.1371/journal. pone.0290689 Editor: Birendra Mishra, University of Hawai’i at Manoa, UNITED STATES Received: May 23, 2023 Accepted: August 14, 2023 Published: August 25, 2023 Peer Review History: PLOS recognizes the benefits of transparency in the peer review process; therefore, we enable the publication of all of the content of peer review and author responses alongside final, published articles. The editorial history of this article is available here: https://doi.org/10.1371/journal.pone.0290689 Copyright: © 2023 Rabaglino 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. Abstract The molecular consequences of the metabolic stress caused by milk production of dairy cows in the early embryo are largely unknown. The objective was to determine the impact of dam metabolic status or in vitro culture during embryonic genome activation (EGA) on the transcriptomic profiles of bovine 16-cell stage embryos. Two days after synchronized oestrus, in vitro produced 2- to 4-cell stage embryos were endoscopically transferred in pools of 50 into the oviduct ipsilateral to the corpus luteum of lactating (LACT, n = 3) or nonlactating (i.e. dried off immediately at calving; DRY, n = 3) dairy cows. On Day 4, the oviducts were flushed to recover the embryos. Pools of five Day-2 embryos (n = 5) and Day-4 16-cell stage embryos obtained in vitro (n = 3) or from LACT or DRY cows were subjected to RNAseq. Temporally differentially expressed genes (DEG; FDR<0.05) between Day-2 and Day-4 embryos were determined considering the differences between the three conditions under which EGA occurred. Also, DEG between Day-4 embryos derived from the three conditions were identified. Functional analysis of the temporal DEG demonstrated that genes involved in ribosome, translation and oxidative phosphorylation in the mitochondria were strongly more expressed in Day-4 than Day-2 embryos. Comparison of Day-4 embryos that underwent EGA in vitro, or in LACT or DRY cows, identified DEG enriching for mitochondrial respiration and protein translation, including the mTOR pathway. In conclusion, exposure of the embryo to an unfavourable maternal metabolic status during EGA influences its transcriptome and potentially the competence for pregnancy establishment. Data Availability Statement: Data were deposited in NCBI’s Gene Expression Omnibus and are PLOS ONE | https://doi.org/10.1371/journal.pone.0290689 August 25, 2023 1 / 17 PLOS ONE accessible through GEO accession number GSE226844. Funding: This research has received funding from the European Union Seventh Framework Programme FP7/2007-2013 under grant agreement n˚ 312097 (“FECUND”). MBR was funded by an H2020-MSCA-Individual Fellowship (Proposal 101021311). 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. Environmental conditions impact embryonic genome activation in the bovine embryo Introduction In mammals, several major developmental events occur during the first week of development following fertilization. These events include the first mitotic division, the timing of which has consequences for subsequent developmental competence [1], embryonic genome activation (EGA) [2], morula formation through the establishment of tight junctions between adjacent blastomeres in the developing embryo [3], blastocyst formation, involving the differentiation of inner cell mass and trophectoderm cells, and the onset of X-chromosome inactivation [4, 5]. Amongst these events, the switching on of the embryonic genome, which occurs at a speciesspecific stage, is arguably the most crucial for subsequent viability during development [6]. The EGA process occurs in two waves: a minor wave in the initial stage of active transcription followed by a second, major, wave, when widespread transcriptional activity of zygotic genes increases dramatically. This occurs between the 8- to 16-cell stage in bovine embryos [7, 8] and involves depletion of maternal transcripts stored in oocytes by degradation and translation, and their replacement by new embryo-specific transcripts. Indeed, many embryos fail to develop beyond this stage, due, at least in part, to insufficient expression of EGA-associated genes [9]. The environment in which the embryo develops in the first week after fertilization significantly affects blastocyst quality [10, 11]. In cattle, although the in vitro production (IVP) of embryos is now routine practice, the competence of the resulting embryos is often compromised [12]. Using alternative in vivo or in vitro culture (IVC) conditions before and during EGA, Gad, Hoelker [13] highlighted the critical influence of the environment in which EGA occurred on the blastocyst transcriptome. Culture conditions also affected the embryonic proteome around the time of EGA [14]. The physiological changes associated with milk production impact on circulating metabolites during the early postpartum period and have been implicated in poor reproductive efficiency in high-producing dairy cows [15, 16]. For example, the oviducts of postpartum lactating (LACT) cows, exposed to elevated concentrations of non-esterified fatty acids, betahydroxybutyrate (BHB), and reduced concentrations of insulin, IGF-I and glucose, were less capable of supporting development of early embryos to the blastocyst stage following endoscopic transfer of 2- to 4-cell embryos and recovery at Day 7, compared to the oviducts of heifers [17] or nonlactating (DRY) cows [16]. Thus, even if the embryo undergoes EGA in a more ‘optimal’ condition i.e., in (...truncated)