Detection of heat shock element-binding activities by gel shift assay during mouse preimplantation development.

Heat shock gene expression is regulated by highly conserved sequence elements (HSE for "heat shock elements"). Some of heat shock genes display an atypical expression during preimplantation mouse development. We have examined the profile of HSE-binding activities (HSE-BA) in matured ovulated oocytes and during the preimplantation development by gel shift assay and quantified the data by PhosphorImager. In each of our experiments, the F9 embryonal carcinoma cell line that contains both constitutive and heat-induced activity has been used as a control. We determine the number of oocytes or embryos required to get reproducible signals and accurate quantification by PhosphorImager. Oocytes, one-cell, and two-cell embryos respond to heat shock by inducing a strong HSE-BA. At the four-cell stage, no HSE-BA can be induced by heat shock, which suggests that noninducibility of heat shock genes at this stage (when the general mechanism of transcription is well established) might result from a defect in HSF or in the mechanism of HSF activation. A progressive reappearance of the ability to induce HSE-BA by stress is observed between the eight-cell stage and the blastocyst stage, and this parallels the appearance of heat shock gene inducibility. Matured ovulated oocytes and the first cleavage stages of embryos do not contain any HSE-BA at normal temperature but we observed a HSE-BA at normal temperature at the morula stage, which is increased at the blastocyst stage. These data, which, to our knowledge, for the first time describe the profile of a DNA-binding activity during the mouse preimplantation development, could serve as a basis for the study of other transcription factors during early embryogenesis.

Mezger V ，Renard JP ，Christians E ，Morange M ... -  《DEVELOPMENTAL BIOLOGY》

Distinct stress-inducible and developmentally regulated heat shock transcription factors in Xenopus oocytes.

Gordon S ，Bharadwaj S ，Hnatov A ，Ali A ，Ovsenek N ... -  《DEVELOPMENTAL BIOLOGY》

Expression pattern of HSP25 in mouse preimplantation embryo: heat shock responses during oocyte maturation.

Heat shock proteins (HSPs) are known to play an important role not only in various stress conditions such as exposure to heat shock, but also in normal development and/or differentiation. The role of small heat shock proteins such as HSP25 in early embryo development remains largely unknown. In the present study, we examined temporal and spatial expression patterns of HSP25 during mouse preimplantation embryo development. Reverse transcription-polymerase chain reaction (RT-PCR) showed that hsp25 mRNA was detected in unfertilized eggs. Hsp25 mRNA was induced by zygotic gene activation at 2-cell stage, decreased slightly at 4-cell, and re-increased at morula, with the highest level at blastocyst stage. Interestingly, another form of hsp25 variant of which 156 bp (52 a.a.) was truncated within the exon1 region was observed in all stages of preimplantation embryos. We also investigated the sub-cellular localization of HSP25 by fluorescence immunocytochemistry. HSP25 was detected in the cytoplasm under normal developmental condition. While acute heat shock (at 43 degrees C for 30 min) caused no significant changes in the sub-cellular localization of HSP25 in the developing mouse embryos, chronic heat shock (at 43 degrees C for 3 hr) resulted in a denser immunostaining of HSP25 in the nucleus than in the cytoplasm, indicating a nuclear translocation of HSP25 by heat shock. As hsp25 mRNA was detected in the unfertilized egg as a maternal transcript, we examined the expression of hsp25 mRNA with RT-PCR during oocyte maturation under normal and heat shock conditions. Hsp25 mRNA was detected at GV (germinal vesicle)-, GVBD (germinal vesicle breakdown)-, and MII (metaphase II)-oocytes. The expression of hsp25 mRNA was increased markedly by both acute (for 30 min and 1 hr) and chronic (for 4 hr) heat shock, but returned to the basal level during recovery from heat shock in a time-dependent manner, suggesting a thermo-protective role of HSP25. In contrast to preimplantation embryos, HSP25 was detected both in the cytoplasm and the nucleus except for the nucleolus, and the cellular localization was not altered by heat shock. Finally, we investigated the effect of heat shock on oocyte maturation. When GV-oocytes were exposed to acute heat shock (at 43 degrees C for 15 min to 1 hr), they underwent the GVBD and the PB (polar body) emission successfully. However, under more stringent heat shock conditions (at 43 degrees C for 2-4 hr), most oocytes were arrested at the GV-stage, and the first PB was not developed, indicating that chronic heat shock might be inhibitory to the mouse oocyte maturation. Taken together, these findings suggest that HSP25 is important for mouse preimplantation embryo development and oocyte maturation.

Kim M ，Geum D ，Khang I ，Park YM ，Kang BM ，Lee KA ，Kim K ... -  《MOLECULAR REPRODUCTION AND DEVELOPMENT》

Activity of a microinjected inducible murine hsp68 gene promoter depends on plasmid configuration and the presence of heat shock elements in mouse dictyate oocytes but not in two-cell embryos.

Bevilacqua A ，Mangia F 《developmental genetics》

Heat shock factor 2-like activity in mouse blastocysts.

Mezger V ，Rallu M ，Morimoto RI ，Morange M ，Renard JP ... -  《DEVELOPMENTAL BIOLOGY》