Investigating the Actions of Interleukin-6 Family Members on Post-Hatching Blastocyst Development in Cattle

Abstract

The goal of this work was to determine if supplementing selective members of the interleukin 6 (IL6) cytokine family influences post-hatching in vitro produced (IVP) bovine blastocyst development. The first series of studies determined if adding human recombinant IL6 improved IVP blastocyst quality before and after cryopreservation. Adding IL6 at 25, 50, and 100 ng/ml increased inner cell mass (ICM) cell number but did not affect trophectoderm (TE) cell numbers. IL6 also increased the ICM:TE ratio before and after cryopreservation and ICM cell number after freezing. All IL6 doses reduced the number of cells undergoing apoptosis in the TE, but not in the ICM. In the second study, we found that both human recombinant IL6 and leukemia inhibitory factor (LIF) increases ICM cell number before cryopreservation. Supplementing interleukin 11 (IL11) had an intermediate effect between the controls and the IL6 and LIF treatments. Supplementing IL6 also increased the ICM:TE ratio. We also observed that supplementing IL11 before freezing increased hatching rate of frozen and thawed blastocysts, but no cryotolerance effects were observed in thawed blastocyst cell number or percentage of apoptotic cells within the blastocyst. A separate series of studies tested if supplementing IL6 and/or LIF altered the morphology of blastocysts during extended culture from d 7 to 12 post-fertilization. Specifically, cell allocations within the bovine embryonic disc (ED) were investigated. Supplementing IL6 from d 5-12 increased epiblast (EPI) cell number, reduced hypoblast (HYPO) presence, and increased the EPI:HYPO ratio in d 12 blastocysts. These IL6 effects could not be replicated when IL6 was provided prior to the blastocyst stage (d 5-7), but they could be replicated when IL6 was supplemented after blastocyst formation (d 7-12). Supplementing IL6 from d 7-12 also resulted in EPI cell numbers similar to IVP blastocysts that were exposed to the uterus from d 7-12. In all studies we observed that LIF has minimal effects on EPI or HYPO allocation. These results indicate that LIF has minimal function in the bovine blastocyst after d 12, but IL6 can benefit EPI development. In conclusion, supplementing IL6 and LIF before freezing increases ICM cell number, but only IL6 has cryoprotective effects on IVP bovine blastocysts. Supplementing IL6 during IVP and extended blastocyst culture improves EPI cell number, but this is at the effect of the HYPO. Overall, results from this work indicates that IL6 plays an important role in post-hatching blastocyst survival.