| Kuning (KM) mice were superovulated and exposed to mild heat stress (37℃for 4 hours), moderate heat stress (39℃for 4 hours), severe heat stress (41℃for 4 hours) and hyperthermia (43℃for 4 hours) during 16-20hr, 28-32hr and 40-44h post-administering of human chorionic gonadotropin(hCG)respectively. Morphological observation was used to examine the affects of heat stress on subsequent embryonic development of 1-2 cell preimplantation mouse embryos both in vitro and in vivo. Immunohistochemistry method was performed to determine HSP70 expression of embryos in different stages (from zygote to blastocysts) induced by heat stress as stated above both in vitro and in vivo, in the purpose of revealing and verifing the molecular nature of the embryonic thermoprotective mechanisms and to extend the understanding for tissue speciality. Immunohistochemistry method was performed to determine HSP70 induced expression of mouse ovarian section during the recovery after exposure of mice to heat stress. The results are as follows. 1. Maternal heat stress affects 1-2 cell early embryonic development in vivo severely. Maternal heat stress reduced the percentage of 4-cell embryos at early zygote stage, late zygote stage and mid-2-cell stage. Moreover, the more degree of reduction associated with the earlier embryonic stage and the higher temperature. The adverse effects at temperature level were more obvious than that at embryos stage level. At temperature level, the adverse effects at 37℃were significantly lower than that at 41℃(p<0.01) and 43℃(p<0.01), and that at 39℃were significantly lower than that at 41℃.(p<0.01) and 43℃(p<0.01) too. But at early zygote it was significantly lower than that at mid-2-cell (p<0.05). The results indicated that the resistant to elevated temperature was increased as it progresses through embryonic development. The inhibition of subsequent embryonic development of mid-2-cell embryos was elevated significantly after exposure of pregnant mice to 37℃at late zygote stage (p<0.05). This suggested that zygote acquired no mechanisms of resistant to subsequent elevated temperature. The percentage of 4-cell embryos that developed to blastocysts was reduced at 37℃in vitro after exposure of pregnant mice to 39℃at early zygote or late zygote stage compared with control (57.9%,58.8%,59.1%), however, elevated result was found after exposure of pregnant mice to 39℃at mid-2-cell stage (67.6%). Moreover, the effect of maternal heat stress at 39℃on subsequent development of 4-cell embryos to blastocysts were significantly difference (p<0.05, p<0.01, p<0.05). Similarly, The percentage of 4-cell embryos that developed to blastocysts was reduced at 43℃in vitro after exposure of pregnant mice to 39℃at early zygote, late zygote and mid-2-cell stage stage compared with control (0.8%,2.8%,7.0%,5.0%). The results indicated that the adverse effects of moderate maternal heat stress (39℃) at early zygote and late zygote stage affected subsequent embryonic development in vitro, but it may be beneficial to mid-two cell embryos. 2. The adverse effects of direct heat stress on developmental ability of mouse embryos. After exposure of early zygote, late zygote and mid-two-cell1 embryos to heat stress at different temperature, the percentage of embryos that developed to blastcyst in vitro increased with the time of embryos development (10.5%,13.1%,20.0%) and decreased significantly with elevated temperature (33.6%,17.6%,5.6%,1.7%).These results demonstrated that the resistant to elevated temperature was significantly increased as it progresses through embryonic developmen(tp<0.05 or p<0.01). 3. The difference of adverse effects of direct heat stress on developmental ability of mouse embryos in vitro and in vivo. The percentage of embryos that developed to blastcyst in vitro (10.2%,15.4%,27.3%) was lower than that of in vivo (21.6%,23.5%,32.4%). This result indicated that maternal environment played a role to protect embryos by alleviating the damage of embryos resulted from heat stress or repairing micro-defect of embryos. 4. Expression of HSP70 was induced in early preimplantation mouse embryos by heat stress. The quantity of expression of HSP70 increased with the elevated temperature of heat stress and embryonic development of mouse embryos. The expression of HSP70 induced by heat stress was earlier at embryos stage in vivo than that in vitro (four-cell versus eight-sixteen-cell) with the lower temperature (37℃versus 41℃). The result showed there were differences of induced expression of HSP70 between in vitro and in vivo. 5. Expression of HSP70 was induced in mouse ovary by heat stress. The induced expression in mouse ovary occurred at zero hour after heat stress at 39℃(+).The quantity of expression of HSP70 increased and the duration was prolonged with the elevated temperature of heat stress, moreover, especially in the two-heat-stressed mice ovary. These results suggested that the quantity and duration of induced expression of HSP70 correlates with temperature of heat stress within certain range of heat stress, in particular, in the pre-heat-stressed mouse ovary.
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