Within the period of a few days, the newly-formed bovine embryo is transformed from an organism that is unable to successfully survive exposure to elevated temperature to one that is capable of continuing development after exposure to physiological heat shock. Thermal tolerance becomes acquired at about the 8–16 cell stage [2–6], which is coincident with the period of development when the embryonic genome becomes fully activated . Here we hypothesized that the morula-stage embryo is resistant to heat shock because it can undergo transcriptional changes in genes related to cellular survival that stabilize cellular function at elevated temperature.
In fact, however, the transcriptional response to heat shock was muted. Only a few genes involved in the heat shock protein response or antioxidant function were increased by heat shock. Thus, it is likely that heat shock at this stage of development does not cause a large increase in the signals leading to transcription of heat shock protein genes (denatured protein, ref. ) or antioxidant genes (free radicals, ref [40–42]). Put differently, there was not a large-scale transcriptional response of heat shock protein genes or antioxidant genes in response to heat shock because the increased need for the proteins encoded by these genes is minimal. Embryonic resistance of the morula to physiological heat shock as compared to embryos at earlier, more thermosensitive stages is more likely to be due to possession of mechanisms to prevent accumulation of denatured proteins and free radical damage rather than due to differences in transcriptional regulation of heat shock protein and antioxidant genes. Indeed, the two-cell embryo, which is very sensitive to heat shock is capable of increasing transcription of HSPA1A and synthesis of HSP70 [44, 45] in response to heat shock. In addition, the steady-state amount of mRNA for HSPA1A in the two-cell embryo not exposed to heat shock is higher than amounts in the Day 5 embryo after heat shock .
Heat shock did induce expression of some heat shock protein and antioxidant genes. As determined in the 3’DGE analysis, heat shock increased expression of HSPB11 significantly and tended to increase expression of HSPA1A and HSPB1. Also, four genes regulated by NRF2, a transcription factor activated by oxidative stress , were increased by heat shock (AKR7A2, CBR1, GSTA4, and MAP2K5) as was another gene involved in glutathione metabolism (GGH). The experiment using real-time PCR was performed to determine if effects of heat shock on expression of selected heat shock protein and antioxidant genes depended on the duration of heat shock. Results indicated that responses to heat shock were similar after 2, 4 and 8 h of exposure to elevated temperature, Moreover, the apparent increase in expression of HSPA1A detected by 3’DGE was repeatable and significant, as would be expected from previous papers [2, 43], and there was a tendency for expression of HSP90AA1 to increase slightly after heat shock. The increase was small and of a magnitude similar to that seen in the 3’DGE experiment (although the difference was non-significant). In an earlier experiment, there was no increase in steady-state amounts of HSP90AA1 after 24 h of heat shock in one-cell or Day 5 embryos . There was also no effect of heat shock of any duration on expression of CAT or SOD1, in agreement with the results of 3’DGE and an earlier experiment in which embryos were exposed to 24 h of heat shock .
However, heat shock did not alter expression of 64 other genes that encoded heat shock proteins, heat shock transcription factors or heat shock protein binding proteins or expression of 42 other genes involved in antioxidant defense. Analysis of the literature and the results from the present study provide clues as to why the induction of expression of heat shock protein and antioxidant genes was so muted. The lack of a large-scale heat shock protein response could reflect efficient clearance of denatured protein. The most striking change in response to heat shock in the current study was a change in expression of genes encoding for proteins that bind to UBC. This protein is part of the ubiquitination pathway that is critical for removal of damaged protein including in cells damaged by heat shock . Changes in expression of genes involved in the ubiquitin pathway could be reflective of perturbation of the ubiquitin-proteasome pathway by heat shock. Removal of damaged proteins by this pathway could conceivably reduce the signal for activation of HSF1, the transcription factor controlling expression of heat shock protein genes, because this protein is activated as a result of increased accumulation of denatured protein . The idea that embryos that have developed thermotolerance are less likely to accumulate denatured proteins than embryos at earlier stages is consistent with earlier results in mouse embryos, where a more severe heat shock was required to induce HSP70 synthesis in blastocysts than in eight-cell embryos .
The lack of broad increase in genes involved in antioxidant defense probably reflects the inhibition of free radical accumulation in response to heat shock for embryos at this stage of development. Previous results indicate that heat shock did not increase free radical production at Day 4 or 6 after fertilization (16-cell through morula stage of development) although it did when embryos were at Days 0–2 of development (one-cell to early eight-cell stage) . The most likely cause is increased accumulation of intracellular antioxidants. Indeed, one such antioxidant, glutathione, increases in amount by the 9–16 cell stage .
One mechanism contributing to the thermotolerance of the preimplantation embryo is the acquisition of the capacity for apoptosis at the 8–16 cell stage . At physiological temperatures, heat shock causes apoptosis in about 10-20% of the blastomeres [49–51]. Inhibition of apoptosis through administration of caspase 3 inhibitors exacerbates the effects of heat shock on development [51, 52]. In the present study, effects of heat shock on genes involved in apoptosis were inconsistent. Three genes that are associated with activation of apoptosis were increased by heat shock: BOK, NGFR and STK4, which activated apoptosis through a p53-dependent mechanism . However, another gene that inhibits apoptosis, SERINC3 was increased by heat shock and the pro-apoptotic gene, PDIA3, was decreased by heat shock. The possible involvement of the ubiquitin system in clearance of proteins damaged by heat shock, as indicated by the large number of genes regulated by heat shock that bind UBC, may also have implications for apoptosis since ubiquitination plays a role in activation of apoptosis responses .
There were few genes involved in developmental processes whose expression was affected by heat shock. This is not surprising because, as seen previously [2, 5, 6], the development of the morula-stage embryo to the blastocyst stage was unaffected by heat shock. It is likely that adjustments to cellular function that limited the heat shock protein and antioxidant gene response to heat shock also allowed development to continue normally after exposure to 40°C. It may be, however, that some changes in gene expression induced by heat shock compromise ability of the blastocyst to continue development in later embryonic or fetal life. In one study in which Day 4 embryos were exposed to a more severe heat shock than used here (41°C for 12 h), pregnancy rate of the blastocysts formed after heat shock was lower after transfer into recipient females than for blastocysts formed from control embryos . Among the pathways affected by heat shock that could potentially compromise subsequent development was the WNT signaling pathway, where heat shock increased expression of AXIN1 and LEF1 and decreased expression of CUL1 and PPP2A. WNT signaling regulates cell proliferation, cell fate decision and stem cell maintenance . LEF1 is involved in trophectoderm differentiation , and CUL1 is required for development in the mouse . Heat shock also caused changes in expression of genes regulating mucopolysaccharide synthesis. One mucopolysaccharide, hyaluronan, has been reported to increase competence of morula-stage and early blastocyst-stage bovine embryos to establish pregnancy after transfer into recipients . Heparan sulfate proteoglycans also regulate WNT availability by binding to secreted WNTs .