Developmental regulation of heat shock proteins : 70.1 in murine embryos and 70.1 and 70.4 in bovine embryos

Abstract

Heat stress causes embryonic mortality in the bovine and in the mouse. A group of proteins known as heat shock proteins (HSPs) are synthesized in response to heat stress and are thought to enhance cellular thermotolerance. Because gene expression begins at variable developmental stages in post-fertilization embryos, early stage embryos may not synthesize heat shock proteins. The hypothesis states that heat shock proteins are not present in the early embryo, thus embryos are susceptible to hyperthermic conditions. In order to detect heat shock gene transcription in a limited number of embryos at early embryonic stages, we developed a 1-tube polymerase chain reaction (PCR) based protocol that minimized RNA loss and enhanced sensitivity of transcription detection. In this protocol, embryonic gDNA was cleaved between primer sites with DNAase; endogenous RNAases were inhibited with a RNAase inhibitor; and RNA integrity was maintained with E. coli rRNA. Samples were reverse transcribed to form cDNA which was then amplified with PCR. The 1-tube protocol was used to synthesize cDNA from mRNA transcripts which were transcribed from a heat inducible heat shock protein gene from murine embryos. Transcripts from HSP 70.1 were detected during the first four days of gestation in murine embryos. The 1-tube protocol was also used to identify the presence of mRNA transcripts from two bovine genes, HSP 70.1 and 70.4. In these studies, two developmental stages were evaluated in the bovine: prior to the 8 to 16-cell stage (3-5 cells) and at 12-16 cells. Heat shock protein 70.1 transcripts appeared during the 12 to 16-cell stage in both control and heat shocked embryos. Transcription was detected from HSP 70.4 before the 8 to 16-cell stage in both control and heat shocked embryos. However, by the 12 to 16-cell stage, only the control embryos synthesized HSP 70.4 mRNA transcripts. We concluded that the 1-tube protocol was sensitive and allowed detection of both murine and bovine heat shock transcripts during early, thermolabile developmental stages. Detection of HSP 70 transcripts at the earliest stages of development suggests that HSP 70 gene expression may be necessary but was not sufficient to protect embryos from thermal stress.