85 Impact of nobiletin supplementation of culture medium on gene expression patterns of in vitro-produced pig embryos

The presence of nobiletin (NOB) during in vitro culture (IVC) improves embryo development in several species potentially due to its antioxidant ability. We have previously observed that IVC medium supplementation with NOB during porcine in vitro early embryo development positively impacts embryo outcomes. To gain insight into the mechanisms of action of NOB during in vitro early embryo development, we investigated the expression patterns of genes related to antioxidant and developmental responses. Immature oocytes collected from abattoir-derived ovaries were cultured in 500 µL of maturation medium supplemented with 10 IU/mL equine chorionic gonadotrophin and 10 IU/mL human chorionic gonadotrophin for 22 h and for an additional 22 h without hormonal supplements. Mature oocytes were co-cultured with thawed sperm (3000 spermatozoa/oocyte) in 100-µL drops of fertilization medium for 5 h. Presumptive zygotes were cultured in 500 µL of IVC medium supplemented with pyruvate and lactate (control) with 2.5 or 5 µM NOB (N2.5 and N5, respectively), or with 0.03% dimethyl sulfoxide (CDMSO; vehicle for nobiletin dilution), for 2 days, and then changed to fresh IVC medium containing glucose for an additional 5 days. All steps were performed at 39°C, 5% of CO_2, and 95% humidity. On Day 7, pools of 10 blastocysts were snap-frozen in liquid N₂ and stored at −80°C until further analyses. Then, RNA was isolated using the RNeasy micro kit (Qiagen); RNA was reverse-transcribed to cDNA using the high-capacity cDNA Reverse Transcription Kit (Applied Biosystem). The relative mRNA expression was quantified by real-time quantitative PCR (CFX9; Bio-Rad). Primers were designed with the software primer3 and Blast. The relative gene expression was calculated using the ^ΔΔCT method. The statistical significance was determined using Student’s t-test. Target gene expression was normalized with the housekeeping reference gene glyceraldehyde-3-phosphate dehydrogenase, GAPDH). Here, changes in embryonic gene expression were triggered by NOB supplementation. Leukemia inhibitory factor (LIF), oestrogen receptor (ER), and superoxide dismutase 1 (SOD1) were up-regulated (P < 0.05) in N5, and glucose 6-phosphate dehydrogenase (G6PDH) was up-regulated (P < 0.05) in N2.5 compared to the control groups. Oestrogen and LIF play key roles in embryo development and implantation. Embryonic LIF is indispensable for initiating embryo-endometrial interaction, while ERs are crucial for attracting estrogens to regulate embryo growth and development. The overexpression of ER gene in embryos exposed to N5 suggests that NOB may induce changes in the embryo that would lead to successful implantation. Additionally, the up-regulation of antioxidant genes SOD1 and G6PDH in N5 and N2.5, respectively could be helping to counteract the oxidative stress generated during in vitro culture to avoid apoptotic signaling. In conclusion, the addition of NOB during in vitro embryo culture induces changes in embryonic gene expression potentially related to positive IVP outcomes in the porcine species.

This study was supported by Spanish MINECO (project PID2019–111641RB-100), Swedish Research Council FORMAS (Project 2019–00288).