The 5'-flanking region of the ovarian promoter of the bovine CYP19 gene contains a deletion in a cyclic adenosine 3',5'-monophosphate-like responsive sequence.
Conversion of C19 steroids to estrogens is catalyzed by aromatase P450 (P450arom; the product of the CYP19 gene). In the ovary, P450arom is expressed in granulosa cells of both human (h) and bovine (b) follicles. After the ovulatory surge of gonadotropins, however, P450arom expression is maintained only in the luteinized granulosa cells of the human ovary and is absent from the bovine corpus luteum. We compared the regulation of expression of the ovary-specific human CYP19 (hCYP19ov) and the bovine CYP19 (bCYP19ov) gene by cAMP (forskolin) and sought to determine whether the divergence in the expression of P450arom with the onset of luteinization could be explained by specific cis-acting elements present uniquely in the 5'-flanking DNA of the hCYP19ov or bCYP19ov gene. We, therefore, subcloned DNA encompassing the promoters and 5'-flanking regions of the hCYP19ov or bCYP19ov gene into a promoterless luciferase vector. These constructs were transfected into luteinized bovine granulosa cells or bovine luteal cells in primary culture. Neither cell type exhibits endogenous expression of bovine P450arom. After transfection, cells were treated with either vehicle or 25 microM forskolin. There was little or no increase in luciferase activity after forskolin treatment in cells transfected with any of the bCYP19ov constructs, whereas all of the corresponding hCYP19ov constructs (-693/-16 to -214/-16 bp) expressed reporter activity in the presence of forskolin. This dramatic difference between the activities of the constructs of the two species occurred despite the fact that there is an 88% sequence identity between the bovine and human promoters in the region between -214 to -16 bp. One possible explanation for this variability may be that the bCYP19ov gene has a 1-bp deletion in a cAMP-response element-like sequence (CLS) present at -208 to -201 bp in the hCYP19ov gene that we have shown to be critical for cAMP-stimulated transcription of hCYP19ov in the ovary. When this region of the bCYP19ov promoter was mutated to the hCLS, a partial restoration in luciferase activity was observed after forskolin treatment. Therefore, these results suggest that another sequence in this -214 bp region of the bCYP19ov gene is also contributing to the lack of expression of P450arom after luteinization in the bovine ovary. This lack of expression of the bCYP19ov gene may be due to the presence of a repressive trans-acting factor expressed with the onset of luteinization of the bovine granulosa cell. These results further suggest that in the cow, elements upstream of those employed by the hCYP19ov gene may have been recruited to facilitate regulated expression of the bCYP19ov gene in the absence of a functional CLS.
Hinshelwood
M M
MM
Cecil H. and Ida Green Center for Reproductive Biology Sciences and the Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas 75235-9051, USA.
Michael
M D
MD
Simpson
E R
ER
D000818:Animals; D001141:Aromatase / Q000235:genetics*; D001483:Base Sequence; D002417:Cattle; D000242:Cyclic AMP / Q000494:pharmacology*; D017362:Cyclic AMP Response Element-Binding Protein / Q000378:metabolism; D004247:DNA / Q000737:chemistry; D004268:DNA-Binding Proteins / Q000378:metabolism; D005260:Female; D017353:Gene Deletion; D006107:Granulosa Cells / Q000201:enzymology; D006801:Humans; D008184:Luteal Cells / Q000201:enzymology; D008969:Molecular Sequence Data; D010053:Ovary / Q000201:enzymology*; D011401:Promoter Regions, Genetic; D017385:Sequence Homology; D016385:TATA Box; D014162:Transfection
D017362:Cyclic AMP Response Element-Binding Protein; D004268:DNA-Binding Proteins; D004247:DNA; D000242:Cyclic AMP; D001141:Aromatase