Regulation of prodynorphin gene expression in the ovary: distal DNA regulatory elements confer gonadotropin regulation of promoter activity.
Examination of the regulation of prodynorphin (pro-DYN) promoter activity is limited by the absence of a good cell model. The discovery of pro-DYN mRNA and derived peptides in the reproductive tract led us to examine the cellular localization and hormonal regulation of ovarian pro-DYN expression and to evaluate normal granulosa cells as a model for studying pro-DYN gene regulation. Ovarian pro-DYN mRNA levels were significantly elevated in PMSG-primed immature rats 12-24 h after receiving an ovulatory dose of hCG. Levels peaked 2 days after hCG, remained elevated throughout the ensuing pseudopregnancy, and rose again at the end of pseudopregnancy. In situ hybridization localized pro-DYN expression predominantly to granulosa and luteal cells. Transfection of primary cultures of granulosa cells revealed that the activity of the rat pro-DYN promoter [-1858 to 133 base pairs (bp)] was increased 18- to 19-fold by hCG and human FSH treatments and 7-fold by cAMP analog treatment. Deletion analysis identified a 358 bp fragment as the primary hormone-responsive sequence (-1858 to -1500 bp; containing three potential cAMP-responsive elements); its deletion resulted in severely reduced FSH responsiveness, and its ligation to hormone-unresponsive basal promoter sequences completely restored FSH responsiveness. This is an unusually distal position for cAMP-responsive elements compared to other cAMP-regulated genes. These data demonstrate specific expression of pro-DYN in granulosa and luteal cells, which is under sensitive gonadotropin regulation, and identify a distal hormone-responsive sequence within the promoter.
Kaynard
A H
AH
Division of Reproductive Biology, Oregon Regional Primate Research Center, Beaverton 97006.
McMurray
C T
CT
Douglass
J
J
Curry
T E
TE
Melner
M H
MH
D000818:Animals; D001483:Base Sequence; D002478:Cells, Cultured; D006063:Chorionic Gonadotropin / Q000494:pharmacology*; D000242:Cyclic AMP / Q000494:pharmacology; D004247:DNA / Q000235:genetics; D004745:Enkephalins / Q000096:biosynthesis* / Q000235:genetics; D005260:Female; D005640:Follicle Stimulating Hormone / Q000494:pharmacology*; D005786:Gene Expression Regulation / Q000187:drug effects*; D006064:Gonadotropins, Equine / Q000494:pharmacology; D006107:Granulosa Cells / Q000187:drug effects*; D017403:In Situ Hybridization; D008969:Molecular Sequence Data; D010053:Ovary / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D010062:Ovulation Induction; D011401:Promoter Regions, Genetic / Q000187:drug effects*; D011498:Protein Precursors / Q000096:biosynthesis* / Q000235:genetics; D051381:Rats; D017207:Rats, Sprague-Dawley; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D012045:Regulatory Sequences, Nucleic Acid; D012689:Sequence Homology, Nucleic Acid; D014162:Transfection
D006063:Chorionic Gonadotropin; D004745:Enkephalins; D006064:Gonadotropins, Equine; D011498:Protein Precursors; D011993:Recombinant Fusion Proteins; D005640:Follicle Stimulating Hormone; D004247:DNA; C033660:preproenkephalin; D000242:Cyclic AMP