Gonadotropin-dependent regulation of bovine pituitary adenylate cyclase-activating polypeptide in ovarian follicles prior to ovulation.
To study the regulation of bovine pituitary adenylate cyclase-activating polypeptide (PACAP) in preovulatory follicles prior to ovulation, PACAP cDNA was isolated by RT-PCR. Its open reading frame (ORF) is composed of 531 bp, and encodes for a 176-amino acid protein that bears 76-90% identity with other PACAP homologs. Using bovine preovulatory follicles obtained between 0 and 24 h after human chorionic gonadotropin (hCG) and semiquantitative RT-PCR/Southern blot, we demonstrate that levels of PACAP mRNA were low at 0 h, markedly increased at 6 and 12 h (P<0.05), and declined 18 and 24 h after hCG. Levels of PACAP mRNA were high in the bovine pituitary, testis, intestine and uterus, but moderate to low in other tissues. Analyses performed on isolated preparations of granulosa and theca cells showed a significant increase of PACAP transcripts in both cell types after hCG, whereas primary granulosa cell cultures revealed high levels of PACAP as well as its receptors PAC-1 and VPAC-2 mRNA after forskolin treatment. Overexpression of the catalytic subunit of protein kinase A (PKA) in granulosa cells stimulated, but treatment with H89 or PKA inhibitor protein inhibited PACAP mRNA expression, whereas PACAP overexpression stimulated an increase in abundance of transcripts for PGHS-2, PGES, EP2 receptor, progesterone receptor, and ADAMTS-1, but not for P450-side chain cleavage and P450 aromatase. Thus, this study demonstrates the gonadotropin-dependent regulation of PACAP mRNA in bovine preovulatory follicles, the importance of PKA activation in the expression of PACAP in granulosa cells, and stimulating effect of PACAP on gene expression during the ovulatory process.
Sayasith
Khampoune
K
Centre de recherche en reproduction animale et Département de biomédecine vétérinaire, Faculté de médecine vétérinaire, Université de Montréal, CP 5000, Saint-Hyacinthe, Québec, Canada J2S 7C6. k.sayasith@umontreal.ca
Brown
Kristy A
KA
Sirois
Jean
J
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015139:Blotting, Southern; D002417:Cattle; D002478:Cells, Cultured; D006063:Chorionic Gonadotropin / Q000494:pharmacology; D003001:Cloning, Molecular; D005576:Colforsin / Q000494:pharmacology; D054746:Cyclic AMP-Dependent Protein Kinase Type II; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D051546:Cyclooxygenase 2 / Q000378:metabolism; D017931:DNA Primers / Q000235:genetics; D004789:Enzyme Activation; D005260:Female; D005498:Follicular Phase / Q000502:physiology; D005786:Gene Expression Regulation; D006062:Gonadotropins / Q000502:physiology*; D006107:Granulosa Cells / Q000378:metabolism; D006801:Humans; D007422:Intestines / Q000737:chemistry; D007546:Isoquinolines / Q000494:pharmacology; D008297:Male; D008969:Molecular Sequence Data; D006080:Ovarian Follicle / Q000378:metabolism*; D010060:Ovulation / Q000502:physiology*; D051219:Pituitary Adenylate Cyclase-Activating Polypeptide / Q000235:genetics / Q000378:metabolism*; D010902:Pituitary Gland / Q000737:chemistry; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats; D051237:Receptors, Pituitary Adenylate Cyclase-Activating Polypeptide, Type I / Q000378:metabolism; D051239:Receptors, Vasoactive Intestinal Peptide, Type II / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D016415:Sequence Alignment; D013449:Sulfonamides / Q000494:pharmacology; D013737:Testis / Q000737:chemistry; D013799:Theca Cells / Q000378:metabolism; D014162:Transfection / Q000379:methods; D014599:Uterus / Q000737:chemistry
D006063:Chorionic Gonadotropin; D017931:DNA Primers; D006062:Gonadotropins; D007546:Isoquinolines; D051219:Pituitary Adenylate Cyclase-Activating Polypeptide; D012333:RNA, Messenger; D051237:Receptors, Pituitary Adenylate Cyclase-Activating Polypeptide, Type I; D051239:Receptors, Vasoactive Intestinal Peptide, Type II; D013449:Sulfonamides; D005576:Colforsin; D051546:Cyclooxygenase 2; D054746:Cyclic AMP-Dependent Protein Kinase Type II; D017868:Cyclic AMP-Dependent Protein Kinases; C063509:N-(2-(4-bromocinnamylamino)ethyl)-5-isoquinolinesulfonamide