Developmentally Regulated Expression ofHsp70-2and aHsp70-2/lacZTransgene during Spermatogenesis

AbstractGerm cells synthesize large amounts of HSP70-2 protein during the meiotic phase of spermatogenesis. This developmentally regulated expression of HSP70-2 contrasts with the constitutive or inducible expression of other 70-kDa heat shock proteins (HSP70s). To better understand the genetic regulation ofHsp70-2,we used mRNA primer-extension, reverse transcriptase PCR (RT–PCR), and cDNA sequencing to determine that transcription began as far as 353 bp upstream of the start codon. We also identified a previously unrecognized 239-bp intron which is spliced out of the pre-mRNA transcript to leave a 114 nt 5′-untranslated region. Transgenic mice were then produced to delimit the upstream regulatory region required for developmental expression ofHsp70-2during spermatogenesis. Results with multiple lines of transgenic mice containing promoter–reporter transgenes with varying lengths ofHsp70-2sequence indicate that promoter sequences up to 640 bp upstream of the start codon and 287 bp upstream of the transcription start site are required forHsp70-2/lacZexpression in spermatocytes. Histochemical detection of transgene β-galactosidase activity was coincident with immunohistochemical detection of HSP70-2 protein, both in the first wave of spermatogenesis in juvenile mice and in ongoing spermatogenesis of adult mice. The distribution ofHsp70-2andHsp70-2/lacZmRNAs was determined by Northern blot,in situhybridization, and RT–PCR, and it was found that upregulation of expression of bothHsp70-2andHsp70-2/lacZwas specific to the meiotic phase of spermatogenesis