Expression and in vitro regulation of pituitary adenylate cyclase-activating polypeptide (pacap38) and its type I receptor (pac1-r) in the gonads of tilapia (Oreochromis mossambicus).
Pituitary adenylate cyclase-activating polypeptide (PACAP), a pleiotropic neuropeptide, has diverse functions in mammals. However, studies of the expression and function of PACAP and its receptor in fish, particularly in the reproductive system, are still limited. In this report, semi-quantitative RT-PCR and immunohistochemical staining were performed to identify expression domains of commercially important tilapia (Oreochromis mossambicus). PACAP (tpacap(38)) and its type I receptor (tpac(1)-r). Transcripts were detected in the brain, gallbladder, gill, heart, intestine, kidney, muscles, pancreas, spleen, stomach, testes, and ovaries, but not in the liver. Expression of tpacap(38) and tpac(1)-r mRNA in brain tissue was significantly higher in both sexes compared with other tissues. Addition of exogenous ovine PACAP(38) (0.25-5 nM), cAMP analog (dibutyryl-cAMP, 0.25-1.5 mM) or forskolin (adenylate cyclase activator, 1-10 microM) significantly upregulated tpacap(38) in the gonads via a dose- and time-dependent fashion. This effect reached a maximal level at 2 h after induction, and then decreased with prolonged culture for up to 4 or 8 h. Additionally, the expression levels of tpac(1)-r were not significantly affected by ovine PACAP(38) or dibutyryl-cAMP in either sex. Forskolin had a slightly inductive effect and its function could be suppressed with the addition of protein kinase A (PKA) inhibitor, H89 (10 microM), indicating involvement of the cAMP-PKA signaling pathway in the regulation of tpacap(38). Expression of tpacap(38) and tpac(1)-r in the gonads of tilapia suggests that PACAP may mediate gonadotropin action via paracrine/autocrine mechanisms in this bony fish.
Huang
Wei-Tung
WT
Department of Molecular Biotechnology, Da-Yeh University, Chang-Hua 515, Taiwan, ROC. kater@mail.dyu.edu.tw
Li
Chia-Jung
CJ
Wu
Po-Jui
PJ
Chang
Yun-Shiang
YS
Lee
Tai-Lin
TL
Weng
Ching-Feng
CF
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003994:Bucladesine / Q000494:pharmacology; D005576:Colforsin / Q000494:pharmacology; D005260:Female; D005399:Fishes / Q000378:metabolism; D015870:Gene Expression / Q000187:drug effects; D006066:Gonads / Q000378:metabolism*; D007150:Immunohistochemistry; D007546:Isoquinolines / Q000494:pharmacology; D008297:Male; D008969:Molecular Sequence Data; D051219:Pituitary Adenylate Cyclase-Activating Polypeptide / Q000032:analysis* / Q000378:metabolism / Q000494:pharmacology; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D012333:RNA, Messenger / Q000032:analysis; D051237:Receptors, Pituitary Adenylate Cyclase-Activating Polypeptide, Type I / Q000032:analysis* / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016415:Sequence Alignment; D013449:Sulfonamides / Q000494:pharmacology; D017210:Tilapia / Q000378:metabolism*; D046509:Tissue Culture Techniques; D015854:Up-Regulation
D007546:Isoquinolines; D051219:Pituitary Adenylate Cyclase-Activating Polypeptide; D047428:Protein Kinase Inhibitors; D012333:RNA, Messenger; D051237:Receptors, Pituitary Adenylate Cyclase-Activating Polypeptide, Type I; D013449:Sulfonamides; D005576:Colforsin; D003994:Bucladesine; C063509:N-(2-(4-bromocinnamylamino)ethyl)-5-isoquinolinesulfonamide