cAMP-dependent and tissue-specific expression of genes encoding steroidogenic enzymes in bovine luteal and granulosa cells in primary culture.
Steroidogenic enzymes are differentially expressed throughout the ovarian cycle. The complex pattern of cell-specific up- and down-regulation accounts, at least in part, for the cyclic production of estrogens, androgens and progesterone. The gonadotropins follicle-stimulating hormone and luteinizing hormone are the main regulators of ovarian steroid hormone production and act primarily via the cAMP second-messenger system. Previous studies have identified cAMP-responsive sequences (CRS) in a number of genes encoding steroidogenic enzymes. In the present study we attempted to compare the cAMP responsiveness of some of these sequences with each other and with the classical cAMP-response element (CRE), as identified in the somatostatin gene. In addition, we were interested to determine whether or not the information for tissue-specific expression is contained by these sequences. Using transient transfection of reporter gene constructs, comprising the CRS of bCYP11A, bCYP17, hCYP21B and bovine adrenodoxin, we investigated cAMP-dependent and tissue-specific expression in primary cultures of bovine luteal and granulosa cells. Treatment of transfected luteal cells with forskolin markedly increased the expression of all but the CYP17-specific reporter gene constructs. A similar pattern of forskolin responsiveness was observed when these reporter gene constructs were transfected in bovine granulosa cells in primary culture. Furthermore, when a reporter gene construct containing the classical CRE genomic was transfected in bovine luteal cells, its expression was also highly stimulated upon treatment with forskolin. Thus, the classical cAMP/CRE system appears to be functional in these cells. Northern blot analysis of primary cultures of bovine luteal and granulosa cells revealed that bCYP17 and bCYP21B are not expressed in control and forskolin-treated cultures.(ABSTRACT TRUNCATED AT 250 WORDS)
Lauber
M E
ME
Cecil H. and Ida Green Center for Reproductive Biology Sciences, University of Texas Southwestern Medical Center, Dallas 75235-9051.
Kagawa
N
N
Waterman
M R
MR
Simpson
E R
ER
D000325:Adrenodoxin / Q000096:biosynthesis* / Q000235:genetics; D000446:Aldehyde-Lyases / Q000096:biosynthesis / Q000235:genetics; D000818:Animals; D001141:Aromatase / Q000096:biosynthesis / Q000235:genetics; D001483:Base Sequence; D002417:Cattle; D002478:Cells, Cultured; D002786:Cholesterol Side-Chain Cleavage Enzyme / Q000096:biosynthesis / Q000235:genetics; D005576:Colforsin / Q000494:pharmacology; D003338:Corpus Luteum / Q000166:cytology / Q000187:drug effects* / Q000201:enzymology; D000242:Cyclic AMP / Q000502:physiology*; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis* / Q000235:genetics; D004790:Enzyme Induction / Q000187:drug effects; D005260:Female; D012739:Gonadal Steroid Hormones / Q000096:biosynthesis*; D006107:Granulosa Cells / Q000187:drug effects* / Q000201:enzymology; D008969:Molecular Sequence Data; D009928:Organ Specificity; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D015290:Second Messenger Systems / Q000502:physiology*; D013254:Steroid 17-alpha-Hydroxylase / Q000096:biosynthesis / Q000235:genetics; D013256:Steroids / Q000096:biosynthesis*; D014162:Transfection
D012739:Gonadal Steroid Hormones; D011993:Recombinant Fusion Proteins; D013256:Steroids; D000325:Adrenodoxin; D005576:Colforsin; D003577:Cytochrome P-450 Enzyme System; D000242:Cyclic AMP; D001141:Aromatase; D013254:Steroid 17-alpha-Hydroxylase; D002786:Cholesterol Side-Chain Cleavage Enzyme; D000446:Aldehyde-Lyases