Cloning and expression of the estrogen receptor-alpha (Esr1) from the Harderian gland of the sea turtle (Lepidochelys olivacea).
The effects of estradiol on the Harderian gland (HG) are believed to be partially regulated by the transcriptional regulation of the estrogen-related genes via estrogen receptor (ER). In reptiles, however, it has not been well established whether the HG contains or expresses steroid nuclear receptors. As a first step toward investigating the molecular mechanisms of estrogen signalling in the HG, we isolated the cDNA for ERalpha in the sea turtle Lepidochelys olivacea. ERalpha was cloned using RT-PCR coupled with 5' and 3' RACE procedures. The cDNA contains a complete open reading frame encoding 588 amino acid residues. Comparative analysis of this amino acid sequence showed moderate to strong conservation of the ERalpha (Esr1) gene within divergent vertebrate groups. In transfection studies, the cloned ER displayed high affinity K(d)=0.25nM and high specificity for 17beta-estradiol. Binding assays using sucrose density gradients demonstrated a specific 7-7.5 S binding component in the HG cytosolic fractions. RT-qPCR analysis showed significant ERalpha mRNA expression in the liver, HG, lung and brain. Altogether, these results provide evidence for the expression of intracellular ERs in the HG of the sea turtle and suggest that ERalpha may be an important modulator of the estrogen-mediated response in the HG of reptiles.
Chávez
Bertha
B
Department of Reproductive Biology, Instituto Nacional de Ciencias Médicas y Nutrición S.Z., México D.F., Mexico.
Ramos
Luis
L
Merchant-Larios
Horacio
H
Vilchis
Felipe
F
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D003001:Cloning, Molecular; D047628:Estrogen Receptor alpha / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D012333:RNA, Messenger / Q000378:metabolism; D030162:Reptilian Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016415:Sequence Alignment; D017421:Sequence Analysis; D014162:Transfection; D014426:Turtles / Q000235:genetics / Q000378:metabolism*
D047628:Estrogen Receptor alpha; D012333:RNA, Messenger; D030162:Reptilian Proteins