DEVELOPMENTALLY-REGULATED EXPRESSION OF HSP70-2 AND A HSP70-2 LACZ TRANSGENE DURING SPERMATOGENESIS/

Germ cells synthesize large amounts of HSP70-2 protein during the meio tic phase of spermatogenesis. This developmentally regulated expressio n of HSP70-2 contrasts with the constitutive or inducible expression o f other 70-kDa heat shock proteins (HSP70s). To better understand the genetic regulation of Hsp70-2, we used mRNA primer-extension, reverse transcriptase PCR (RT-PCR), and cDNA sequencing to determine that tran scription began as far as 353 bp upstream of the start codon. We also identified a previously unrecognized 239-bp intron which is spliced ou t 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 of Hsp70-2 during sperma togenesis. Results with multiple lines of transgenic mice containing p romoter-reporter transgenes with varying lengths of Hsp70-2 sequence i ndicate that promoter sequences up to 640 bp upstream of the start cod on and 287 bp upstream of the transcription start site are required fo r Hsp70-2/lacZ expression in spermatocytes. Histochemical detection of transgene p-galactosidase activity was coincident with immunohistoche mical detection of HSP70-2 protein, both in the fist wave of spermatog enesis in juvenile mice and in ongoing spermatogenesis of adult mice. The distribution of Hsp70-2 and Hsp70-2/lacZ mRNAs was determined by N orthern blot, in situ hybridization, and RT-PCR, and it was found that upregulation of expression of both Hsp70-2 and Hsp70-2/lacZ was speci fic to the meiotic phase of spermatogenesis. (C) 1996 Academic Press, Inc.