(77) Melatonin is Dispensable for Ovine Development up to Post-Blastocyst Stages in Vitro

P. Ramos-Ibeas¹; M. Carvajal-Serna^1,2; N. Martínez de los Reyes¹; P. Marigorta¹; P. Bermejo-Álvarez¹

¹ Animal Reproduction Departament, INIA, CSIC, Madrid, Spain

²BIOFITER - Department of Biochemistry and Molecular and Cellular Biology, Veterinary Faculty, Instituto Universitario de Investigación en Ciencias Ambientales de Aragón (IUCA), Universidad de Zaragoza, Zaragoza, Spain

Abstract Text:

Melatonin regulates reproductive seasonality in sheep. Canonical melatonin receptors MT1 and MT2 are not present in ovine embryos after the blastocyst stage, which express the nuclear RAR-Related Orphan Receptor alpha (RORA) that may mediate melatonin signalling. Our aim was to analyze the potential melatonin-mediated effects on sheep embryo development, by assessing the developmental ability of RORA knock-out (KO) embryos and testing the effects of melatonin supplementation during in vitro culture. In the first experiment, in vitro matured oocytes were microinjected with base editor BE3 mRNA and a single guide RNA (sgRNA) to introduce a stop codon in the RORA sequence (BE+G) or with BE3 alone as control (BE). Microinjected oocytes were fertilized and cultured in vitro in conventional media up to Day (D) 8 (n = 3), or until D12 in N2B27 medium (n = 3). Embryos were then imaged, fixed and immunostained for SOX2 (epiblast), SOX17 (hypoblast), and CDX2 (trophoectoderm). Embryos were genotyped by Sanger sequencing. In the second experiment, the effects of melatonin supplementation (10 nM or 1 µM) during in vitro maturation (IVM), in vitro fertilization (IVF) and in vitro culture (IVC) were assessed. Cleavage and blastocyst rates were recorded at D2 and D8, respectively. Blastocysts produced with melatonin supplementation from IVM, as well as those produced in conventional media, were subsequently cultured in N2B27 medium supplemented with either 10 nM or 1 µM melatonin. At D12, embryo survival was recorded, and embryos were imaged, fixed and immunostained for SOX2, SOX17, and CDX2. In the first experiment, blastocyst rates at D8 (43.6±7.9% vs. 40.4±2.8%) and embryo survival at D12 (89.5±3.2% vs. 93.7±4.2%; mean ± s.e.m; t-test, p >0.05) were similar between the BE+G (containing RORA KO embryos) and BE groups. No significant differences were found between KO, heterozygous (Hz) and wildtype (WT) D8 blastocysts in the number of SOX2+ (6.9±2 vs. 8.8±1.6 vs. 10.3±2.2), SOX17+ (39.4±17.5 vs. 28.3±6.3 vs. 36.1±11), CDX2+ (182.3±57.6 vs. 111.8±29.4 vs. 150.8±36.6) or total cells (261.8±66.8 vs. 199.9±32.5 vs. 225.1±43.8), nor in embryo area (0.33±0.05 vs. 0.37±0.04 vs. 0.46±0.04 mm²) and SOX2+ cell number (11.5±5.6 vs. 9.9±4 vs. 6±1.5) at D12 (mean±s.e.m; ANOVA, p >0.05). However, the percentage of hypoblast migration along the inner trophectoderm surface was significantly lower in KO and Hz embryos (35.8±8.5 vs. 36.7±5.6 vs. 57.9±4.8; ANOVA, p< 0.05). In the second experiment, no significant differences were detected in cleavage or blastocyst rates when 10 nM or 1µM melatonin was added during IVM, IVF, and IVC. Similarly, no significant differences were found in embryo survival, embryo area, or the percentage of hypoblast migration at D12, regardless of whether melatonin (10 nM or 1µM) was added from IVM or from the blastocyst stage (ANOVA, p >0.05). The percentage of embryos containing epiblast cells was significantly lower in D12 embryos treated with 1µM melatonin from the blastocyst stage. However, no significant differences were detected in the percentage of D12 embryos forming an embryonic disc. In conclusion, melatonin supplementation does not enhance ovine embryo development in vitro, and disruption of its receptor (RORA) does not impair development to D12 but may influence post-blastocyst hypoblast migration.

Work supported by PID2021-122153NA-I00 and Margarita Salas program.