125 Presence of melatonin receptors in ovine blastocysts

Abstract

Melatonin increases in vivo embryo viability in sheep and improves the blastocyst rate on in vitro sheep embryo culture. To determine whether this melatonin effect is receptor-mediated, we evaluated the presence of melatonin receptors MT1 and MT2 on in vitro-obtained sheep embryos by means of RT-PCR and indirect immunofluorescence (IIF). For in vitro embryo production, oocytes were collected from ovaries of adult ewes and matured with TCM-199; 10% oestrus sheep serum; FSH and LH, 10μg mL−1 each; cysteamine, 100 μM; sodium pyruvate, 0.3 mM; penicillin G, 100IU mL−1; and streptomycin sulfate, 100μg mL−1 for 24h at 39°C and 5% CO2. Matured oocytes were transferred to a fertilization medium (SOF without glucose, with 2% oestrus sheep serum; heparin, 10μg mL−1; and hypotaurine, 1μg mL−1) and fertilized with swim-up selected spermatozoa at a final concentration of 106 cells mL−1. After 36h, the cleaved embryos were incubated in culture medium (SOF with amino acids, 0.4% BSA; l-glutamine, 1 mM; penicillin G, 100IU mL−1; and streptomycin sulfate, 100μg mL−1) for 8 days at 39°C with 5% CO2, 5% O2, and 90% N2. In vitro fertilization was repeated 3 times. Day 8 hatched blastocysts were selected for RT-PCR or IIF. For RT-PCR assays, RNA from 5 to 10 blastocysts was extracted and reverse transcribed with SuperScript™ III CellsDirect™ cDNA Synthesis Kit (Thermo Fisher Scientific Inc., Waltham, MA, USA). The PCR amplification was carried out in a Step One Plus Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). The PCR mix contained 2μL of embryo cDNA, iTaq Universal SYBR Green Supermix (Bio-Rad Laboratories Inc., Waltham, MA, USA) and 200nM of the MT1 (forward: 5′-CTCCATCCTCATCTTCACCATC-3′, reverse: 5′-GGCTCACCACAAACACATTC-3′, 113bp) or MT2 (forward: 5′-GCTGAGAGAATGGAGCGATATG-3′, reverse: 5′-GTCCACAGTGAGAAGCCATC-3′, 81bp) primers. The RT-PCR products were visualised under ultraviolet light on 2% agarose gel in a TBE buffer (Tris, 0.9 M; boric acid, 0.9 M; and EDTA, 20mM) with 0.5μL mL−1 ethidium bromide. Ovine testis was used as positive control. For IIF, 5 blastocysts were fixed with 4% paraformaldehyde, permeabilized with 0.5% Triton X-100 and blocked with 5% BSA in PBS for 2h at RT. Embryos were incubated overnight at 4°C with MTNR1A mouse polyclonal antibody (Abnova, Taipei City, Taiwan) for MT1, and rabbit melatonin receptor 1B antibody (Acris Antibodies, Atlanta, GA, USA) for MT2, both diluted 1/50 in PBS with 1% BSA. Secondary antibodies were Alexa Fluor 594 chicken anti-mouse (Invitrogen, Carlsbad, CA, USA) for MT1 and Alexa Fluor 488 chicken anti-rabbit for MT2, diluted 1/800 in PBS containing 1% BSA. Embryos were mounted in slides and visualised on a Nikon Eclipse E-400 microscope (Nikon, Tokyo, Japan). The RT-PCR revealed a single band of 113bp for MT1 and another one of 81bp for MT2 in both the embryo samples and the positive control, which confirmed the gene expression of melatonin receptors in the ovine embryo. The IIF located the MT1 receptor around the nucleus of the trophoblastic cells, whereas MT2 was over the nucleus in the same cells. These results indicate the presence of melatonin receptors in sheep blastocyst, which could mediate the positive effects of this hormone on embryo viability.