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To better define the tissue- and sex-specific roles of aromatase in fishes, we have isolated a CYP19A1 cDNA sequence from a well-developed model of teleost reproduction, the Atlantic croaker (Micropogonias undulatus). This cDNA encodes a protein which has high identity (57-90%) to known CYP19A1 proteins and segregates with teleost CYP19A1 proteins in molecular phylogenetic analysis. In both sexes, the gene encoding Atlantic croaker CYP19A1 is expressed primarily in gonadal tissue, but also in the brain and other tissues at much lower levels, as determined relative to ribosomal 18S RNA expression by real-time quantitative RT-PCR. In females, the highest levels of CYP19A1 mRNA are found in the developing ovary compared to spawning, regressing and resting ovaries. In contrast, testicular CYP19A1 expression is lowest in developing testes and increases in spawning and regressing testes, although there were no statistically significant differences between stages. Brain CYP19A1 mRNA levels are lower in animals with developing gonads compared to spawning fish. In vitro treatment with human chorionic gonadotropin (10 IU/ml) for 6 or 24h increases CYP19A1 mRNA approximately 16- and 43-fold, respectively, in isolated Atlantic croaker ovarian follicles, but has no effect on CYP19A1 mRNA in testicular or brain minces. Six hour in vitro treatment with sex steroids (estradiol, testosterone or 17,20 beta,21-trihydroxy-4-pregnen-3-one; 290 nM) does not alter CYP19A1 mRNA in ovary, testis or brain. The regulation of CYP19A1 in the Atlantic croaker therefore differs in a tissue- and sex-specific manner.

Abstract: The steroidogenic acute regulatory protein (StAR), a member of the StAR-related lipid transfer domain (START) family, is critical to regulated steroidogenesis in vertebrates. We have isolated a cDNA encoding StAR from a well-studied model of teleost physiology, the Atlantic croaker Micropogonias undulatus. This cDNA (1204 nucleotides total length) contains an open reading frame of 858 nucleotides encoding a protein of 286 amino acids. Molecular phylogenetic analysis indicates the putative Atlantic croaker StAR protein is more closely related to StAR proteins (62–85% identity) than to the related START protein MLN-64 (28–31% identity). Green monkey kidney cells (COS-1) cotransfected with Atlantic croaker StAR and human cholesterol side chain cleavage (SCC) expression constructs are able to produce significantly more pregnenolone than cells transfected with SCC alone. StAR mRNA is detected in the Atlantic croaker head kidney by reverse transcriptase-polymerase chain reaction (RT-PCR) and in the kidney and hypothalamus in some individuals. Gonadal StAR gene expression is below the level of detection by RT-PCR in most individuals, but can be detected using fluorescent probes in quantitative RT-PCR. StAR mRNA is not detected in the Atlantic croaker brain. Six hour in vitro treatment of Atlantic croaker ovarian follicles with human chorionic gonadotropin (hCG) is insufficient to significantly alter StAR mRNA levels; however, 24h hCG treatment induces StAR mRNA levels 17-fold over untreated controls. Neither 6 nor 24h treatment with hCG significantly alters StAR mRNA levels in Atlantic croaker testicular minces. Likewise, 6h in vitro treatment with estradiol, testosterone or the maturation-inducing steroid 17,20β,21-trihydroxy-4-pregnen-3-one is without effect on gonadal StAR mRNA levels. [Copyright &y& Elsevier]