Regulation of prolactin gene expression in goldfish Carassius auratus

Prolactin (PRL) is present in all vertebrates and is mainly secreted by lactotrophic cells of the anterior pituitary. PRL has over 300 separate biological activities, ranging from growth and development, water and electrolyte balance, endocrinology and metabolism, brain and behavior, reproduction, to immunoregulation and protection. In fish, the role of PRL is particularly important in water and electrolyte balance as well as in growth and development. In view of the important biological role of PRL in fish, we have therefore used the goldfish as a model in this project to investigate the regulation of PRL gene expression.; In our study, a 1.1 kb 5'-flanking region of the goldfish PRL gene has been cloned. To understand the molecular mechanism that regulate the expression of the goldfish PRL gene, deletion mutation analysis was performed. The results revealed that there are three stimulatory elements and an inhibitory element in this goldfish PRL gene promoter region. The effects of thyrotropin releasing hormone (TRH), dopamine (DA) and estrogen on the goldfish PRL gene expression in vitro were also investigated.; To investigate the effects of the DA, TRH and estrogen on goldfish PRL gene expression under mere physiological conditions, goldfish pituitary primary cell culture and fish intraperitoneal injection experiments were performed. These in vitro and in vivo studies indicated that DA and TRH exert inhibitory effects on PRL gene expression but estrogen stimulates the expression of PRL in goldfish. The transcription factor Pit-1 plays an important role in regulating the expression of the PRL gene. In addition, the seasonal expression level of PRL/growth hormone (GH)/somatolactin (SL) and Pit-1 in goldfish pituitary was investigated by real-time PCR. The data indicated that the expression of PRL, GH, and SL genes was closely associated with sexual maturity and there were obvious increases before the spawning period. A remarkable rise of pituitary PRL and GH mRNA in the summer was associated with enhanced growth rates. These studies revealed that Pit-1 is crucial for the expression of the GH/PRL/SL family genes. These results would help us to understand the molecular mechanism that controls the regulation of goldfish PRL gene expression.; The cDNA encoding the goldfish Pit-1 transcription factor was cloned and sequenced. Tissue distribution study shows that the Pit-1 expression is pituitary-specific in goldfish. Cotransfection of Pit-1 with the goldfish 1.1 kb proximal PRL promoter was performed in HeLa cells. It was found that Pit-1 stimulates the goldfish PRL promoter activity in HeLa cells. In order to study the biological function, especially the molecular mechanism of its regulation on the expression of PRL, the recombinant protein of goldfish Pit-1 was expressed and purified.