Cloning, Regulation of Messenger Ribonucleic Acid Expression, and Function of a New Isoform of Pituitary Adenylate Cyclase-Activating Polypeptide in the Zebrafish Ovary

Abstract

Increasing evidence suggests that pituitary adenylate cyclase-activating polypeptide (PACAP) acts as a local factor in the ovary of mammals. In nonmammalian vertebrates, although the expression of PACAP has also been demonstrated in the ovary, the information on its functions and regulation is limited. In the present study, we identified a new type of PACAP, zebrafish (zf)PACAP38-2, from the zebrafish ovary. The precursor of GHRH-zfPACAP38-2 consists of 175 amino acids with only 64% homology with another type of zebrafish PACAP, ZfPACAP38-1. RT-PCR analysis detected two messengers of zfPACAP38-2 in the zebrafish ovary. The short product was more abundant, and it encodes zfPACAP38-2 only, whereas the long form codes for both zfPACAP38-2 and GHRH. Using a primary culture of zebrafish follicle cells, we demonstrated that gonadotropin (human chorionic gonadotropin and goldfish pituitary extract) significantly stimulated zfPACAP38-2 expression within 2 h; however, the effect decreased to the control level after 8 h of treatment. The stimulation of zfPACAP38-2 expression by gonadotropin could be mimicked by cAMP analogs and forskolin but suppressed by H89 (10 μM), suggesting the involvement of the cAMP-protein kinase A signaling pathway. We also examined the expression of PACAP receptor VPAC2-R in the zebrafish ovary. Unlike zfPACAP38-2, which showed a trend of increase during follicle development, the expression of VPAC2-R mRNA in the follicles showed no significant stage-dependent variation, and its expression in the follicle cells did not respond to gonadotropin treatment. Our studies further demonstrated that synthetic zfPACAP38-2 stimulated oocyte maturation and increased the expression of follistatin in zebrafish ovarian follicle cells. These results suggest that zfPACAP38-2 is a potential ovarian factor that mediates gonadotropin actions in paracrine/autocrine manners, and its functional roles are likely, to some extent, related to the ovarian activin/follistatin system.