97 Interleukin-6 and leukemia inhibitory factor modify embryonic disc composition in post-hatching bovine blastocysts

Understanding developmental milestones during the second week of pregnancy is needed for developing novel schemes to reduce early embryonic loss in cattle. This work used an extended bovine embryo culture system to explore how recombinant human interleukin-6 (IL6) and leukemia inhibitory factor (LIF) influence the development and organization of the epiblast (EPI) and hypoblast (HYPO) lineages within the embryonic disc (ED). In vitro-produced bovine blastocysts were cultured in N2B27 medium or N2B27 containing IL6, LIF, or both (20 ng mL⁻¹ each) from Day 7 to Day 12 post-fertilization (n = 19–41 blastocysts/treatment; 4–5 replicates/study). Immunofluorescent detection of SRY-Box 2 (SOX2; EPI marker), SRY-Box 17 (SOX17; HYPO marker), and nuclear DNA (DAPI) was used to visualize EPI and HYPO cells. Analyses were performed by least-squares ANOVA using the general linear model in SAS. Pair-wise comparisons were analyzed using the probability of difference test in SAS. Supplementing either IL6 or IL6+LIF increased EPI cell number (P ≤ 0.01) and reduced HYPO migration (P ≤ 0.05), but neither treatment affected embryo survival or the presence of an ED or HYPO. Supplementing LIF alone did not affect any parameter. A second study explored including a cocktail of five pharmacological signaling inhibitors and activin A (20 ng mL⁻¹) (5iA) compared with N2B27. This cocktail reduced HYPO presence and migration (P ≤ 0.01) but did not alter the percentage of embryos containing an ED or the number of EPI cells. A follow-up study tested the effects of adding IL6 or LIF to 5iA compared with 5iA alone. Adding IL6 to 5iA increased EPI cell number (P ≤ 0.05) but reduced the percentage of embryos containing a HYPO (P ≤ 0.05); whereas, supplementing LIF to 5iA did not affect EPI cell number or the presence of a HYPO, but reduced HYPO migration (P ≤ 0.05). Neither IL6 nor LIF altered embryo survival. A final study examined how IL6+LIF co-supplementation with 5iA influenced ED development. This scheme resembled the 5iA+IL6 outcomes, with increased rates of embryos containing an ED (P ≤ 0.05) and EPI cell number (P ≤ 0.01), and reduced percentage of embryos containing a HYPO (P ≤ 0.05). No effects on HYPO migration and embryo survival were detected. In summary, supplementing IL6 primarily affected EPI cell number; whereas, supplementing LIF primarily affected HYPO presence and migration. Co-supplementing IL6+LIF in 5iA increased the percentage of embryos containing an ED and increased EPI cell number. These observations indicate that IL6 and LIF each exhibit activities that influence ED development in Day 12 blastocysts, but IL6 primarily affects the EPI cell lineage and LIF primarily affects the HYPO lineage. In addition, the 5iA cocktail could alter the EPI and HYPO lineages in the embryo. This work shows that IL6 and LIF modify ED development and organization in Day 12 embryos in different ways, both of which ultimately improve pre-gastrulation ED composition.

Funding for this work was provided by USDA-NIFA (2021-67015-34485) and by a NIFA Predoctoral Fellowship (2023-67011-40341).