Developmentally regulated expression of Hsp70-2 and a Hsp70-2/lacZ transgene during spermatogenesis

• Published in: Developmental biology Vol. 174; no. 2; pp. 310 - 321
• Main Authors: Dix, D J, Rosario-Herrle, M, Gotoh, H, Mori, C, Goulding, E H, Barrett, C V, Eddy, E M
• Format: Journal Article
• Language: English
• Published: United States 15.03.1996
• Subjects: Animals; Base Sequence; beta-Galactosidase - genetics; DNA Primers; Gene Expression Regulation, Developmental; Homozygote; HSP70 Heat-Shock Proteins - genetics; Introns; Male; Mice; Mice, Transgenic; Molecular Sequence Data; Promoter Regions, Genetic; Spermatogenesis - genetics; Testis - growth & development; Testis - metabolism; Transgenes
• ISSN: 0012-1606
• DOI: 10.1006/dbio.1996.0076

Germ 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 of Hsp70-2, 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 of Hsp70-2 during spermatogenesis. Results with multiple lines of transgenic mice containing promoter-reporter transgenes with varying lengths of Hsp7-2 sequence indicate that promoter sequences up to 640 bp upstream of the start codon and 287 bp upstream of the transcription start site are required for Hsp70-2/lacZ expression in spermatocytes. Histochemical detection of transgene beta- 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 of Hsp7O-2 and Hsp7O-2/lacZ mRNAs was determined by Northern blot, in situ hybridization, and RT-PCR, and it was found that upregulation of expression of both Hsp7O-2 and Hsp7O-2/lacZ was specific to the meiotic phase of spermatogenesis.