The embryoid body as a model system to study the effect of heat stress on post-implantation development of mammalian embryos

101

The odjectives of this study were to establish an in vitro system to determine the effect of heat shock on germ layer differentiation, heat shock protein (hsp70) expression and apoptosis markers of mouse and rabbit embryoid bodies (EB). In Exp. 1, 5.5-8.5 day-old mouse fetuses and 1-7 day-old mouse EBs were collected to examine their gene expressions of the three germ layers. Results showed that Days 3 and 6 EBs are retrospectively associated with the Days 6.5 and 7.5 fetuses, respectively, in terms of the germ layer gene expressions. In Exp. 2.1, mouse EBs were subjected to heat shock and randomly allocated to one of the eight treatment groups, i.e., incubation at 37℃ for 12 h or 24 h, heat-shock at 39℃ for 12 h or 24 h, heat-shock at 41℃ for 12 h or 24 h, incubation at 37℃ for 12 h following 39℃ or 41℃ (12 h) of heat-shock treatment. TUNEL and LIVE/DEAD assays were used to inspect apoptosis of EB and western blotting for the expressions of heat shock proteins (Hsc70 and Hsp72). Our observations showed that the peripheral cell layer of EBs started to degenerate and significantly increased the apoptotic cells after heat-shock at 41℃ for 12 h or 24 h (P ＜ 0.05). When resumed to a normal culture temperature (37℃), EBs morphologically recovered from the damages progressively during further culture. The changes of gene expression in the germ layers after various treatments, namely, timing for the expressions of mesoderm marker Brachyury and the endoderm markers, α-fetoprotein (AFP) and Transthyretin (TTR), were set back for 1-2 days in the heat-shocked groups compared to the control group. In another experiment (Exp. 2.2), we determinated whether a mild heat shock could enhance thermotolerance of mEB when followed by another more severe heat shock. Mouse EBs were randomly allocated to one of the eight treatment groups, i.e., heat-shock at 39℃ for 12 h (M12) or 24 h (M24), heat-shock at 41℃ for 12 h (H12) or 24 h (H24), incubation for 12 h at 39℃ and heat-shock at 41℃for 12 h (M12H12) or 24 h (M12H24), and incubation for 24 h at 39 ℃ and heat-shock at 41 ℃ for 12 h (M24H12) or 24 h (M24H24). Results showed the expression of Casepase3 and Hsp70 were significantly decreased in the M24H24 group (P＜0.05). In Exp. 3., we tested the effect of heat shock on the germ layer differentiation of rabbit EB,apoptosis cells and Hsp72 expression. Similar results were also observed as that in mouse EB, where the peripheral cellular layer started to degenerate and significant increased apoptosis cells after heat shock (P＜0.05). Hsp72 expression significantly increased with the increases of temperatures and treatment duration. Timing for the expressions of mesoderm marker Desmin and BMP4 were also set back for 1-2 days in the heat-shocked groups (41℃) compared to the control group, but ectoderm marker Pax6 expressed 1-2 days earlier in the 39℃ heated group than the control group. Based on our current observation, we conclude that EB are similar to in vivo developing embryos in terms of their germ layer marker expression. They can be a potential model system for the study of various environmental insults to post-implantation embryos. In general, in vitro heat shock delays the expressions of germ layer marker genes of EBs, but enhances Hsp72 expressions in both mouse and rabbit systems. Further in vivo studies are required to confirm the physiologic alterations of embryo/fetus in response to the elevation of ambient temperatures.