(67) Interleukin-6 and Leukemia Inhibitory Factor Influence Epiblast Cell Number and Hypoblast Migration in Cultured, Post-Hatched Bovine Blastocysts

Mary A. Oliver¹ and Alan D. Ealy¹

¹School of Animal Sciences, Virginia Polytechnic Institute and State University, Blacksburg, VA, USA


Abstract Text: Approximately one half of pregnancies are lost during the first month of gestation in cattle. Complications in embryonic disk (ED) development likely cause some of these losses. The pre-gastrulation ED is comprised of the epiblast (EPI) and hypoblast (HYPO). The EPI eventually gives rise to the three germ layers and the HYPO contains cells that migrate to the base of the blastocyst, eventually forming the yolk sac. Understanding how the EPI and HYPO develop during the first two weeks of development could provide us with insights needed to reduce pregnancy loss in cattle. This work utilized an extended blastocyst culture system to test the hypothesis that interleukin-6 (IL6) and leukemia inhibitory factor (LIF) improve EPI cell number and HYPO migration in bovine blastocysts. In vitro produced bovine blastocysts were generated and moved to a serum-free medium formulation that supports post-hatching development after blastocyst formation (DMEM/F12:Neurobasal medium [1:1], N2 and B27 supplements, non-essential amino acids, 0.55 mM 2-Mercaptoethanol, and antibiotics). Blastocysts remained in extended culture medium from days 7-12 post-fertilization. Supplementation of either IL6 (20 ng/mL), LIF (20 ng/mL), or IL6+LIF (20 ng/mL each) were performed along with a vehicle-only control group over three independent studies, where cytokines were provided on days 5-7 (prior to extended culture), days 7-12, and days 5-12. Blastocyst survival was monitored on day 12. Non-degenerate blastocysts were fixed and processed for immunofluorescent detection of SRY-Box 2 (SOX2; EPI marker), SRY-Box 17 (SOX17; HYPO marker), and either nuclear DNA (DAPI; studies 1 and 2) or CDX2 (trophectoderm marker; study 3) (n=26-44 embryos/treatment across 5 replicates for each study). Blastocysts were imaged and processed for diameter, EPI presence, HYPO presence, EPI cell number, and HYPO migration. All statistical analyses were completed using the PROC GLIMMIX procedure in SAS (Cary, NC). None of the treatments affected survival to day 12, diameter, or presence of an ED in any of the three studies. No treatment differences in HYPO presence, EPI presence, EPI cell number, or HYPO migration were observed after supplementing IL6, LIF, or IL6+LIF from days 5-7. Supplementing IL6 on days 7-12 or days 5-12 increased EPI cell numbers (P≤0.001) and reduced HYPO presence (P≤0.05) and migration (P≤0.05) when compared to the controls and LIF supplementation. Supplementing LIF on days 7-12 or days 5-12 did not influence EPI cell numbers or the presence and migration of HYPO when compared with the control. Supplementing IL6+LIF on days 7-12 or 5-12 produced outcomes similar to IL6-only supplementation, with increases in EPI cell number (P≤0.05) and reductions in HYPO presence and migration when compared to controls (P≤0.05) and LIF (P≤0.05) but not when compared with the IL6 only treatment. This work offers evidence that IL6 and LIF have different functions in post-hatching bovine blastocysts, with IL6 increasing EPI cell numbers and LIF controlling HYPO presence and migration. Supplementing then removing IL6 and LIF at day 7 did not produce any subsequent effects, indicating that these cytokines must be present as the EPI and HYPO are developing. We have not resolved why LIF actions on the HYPO are not occurring when IL6 is co-supplemented. Perhaps IL6 limits HYPO development and this activity cannot be modified by LIF supplementation. Funding for this work was provided by USDA-NIFA (2021-67015-34485) and by a NIFA Predoctoral Fellowship (2023-67011-40341).