Abstract
To elucidate the molecular mechanisms involved in the delayed induction of PGHS-2 in species with a long ovulatory process, a 1. 6-kilobase fragment of the bovine PGHS-2 promoter was isolated, and its activity was characterized in primary cultures of bovine granulosa cells. Promoter activity assays performed with a series of deletion mutants revealed that the promoter region from -149 to -2 (+1 = transcription start site) confers full-length promoter activity in response to forskolin (10 microM). Four consensus cis-elements were identified within this region, including an E-box, ATF/CRE, C/EBP, and AP2 site. Site-directed mutagenesis showed that the E-box was required for PGHS-2 promoter activity, that disruption of the C/EBP element decreased forskolin inducible activity by 29%, whereas point mutation within the ATF/CRE and AP2 element had no inhibitory effect. Electrophoretic mobility shift assays (EMSAs) performed with the -149/-2 fragment and granulosa cell nuclear extracts obtained before (0 h) and after (18 and 20 h) human chorionic gonadotropin (hCG) revealed the regulation of multiple DNA-protein complexes. The 0-h extract generated four complexes at the E-box, whereas only one complex was produced at this site with the 18-h extract. Supershift EMSAs identified that upstream stimulatory factor-1 and -2 (USF-1 and -2) were part of these complexes. Interestingly, the presence of the amino-terminal truncated USF-2, which lacks the transcription activation domain, was detected in the 0-h extract, but not in extracts prepared post-hCG. Supershift EMSAs also indicated high levels of C/EBPbeta binding to its cis-element in the 0-h extract, which contrasts with results previously reported in rats. Thus, high levels of amino-terminal truncated USF-2 and C/EBPbeta in bovine granulosa cells prior to hCG treatment could repress gene expression, and be involved in the delayed induction of PGHS-2 in species with a long ovulatory process.
Highlights
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AF031699
Induction of PGHS-2 in Primary Cultures of Bovine Granulosa Cells—To establish a model in vitro for the study of delayed PGHS-2 induction, the regulation of PGHS-2 was characterized in primary cultures of bovine granulosa cells stimulated with forskolin
To determine if the induction of PGHS-2 mRNA and protein was associated with changes in prostaglandin synthetic activities, PGE2 concentrations were measured in culture media
Summary
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AF031699. Induction of PGHS-2 in species with a long ovulatory process like cows (28 h post-hCG) and mares (39 – 42 h post-hCG) is remarkably delayed and occurs only at 18- and 30 h post-hCG, respectively [21, 23, 26]. This marked difference among species in the control of PGHS-2 gene expression could. Delayed Induction of PGHS-2 in Bovine Granulosa Cells be involved in the control of the mammalian ovulatory clock [27]; its molecular basis remains unknown
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