The upregulation of messenger ribonucleic acids during 17 alpha,20 beta-dihydroxy-4-pregnen-3-one-induced ovulation in the perch ovary

While progestins appear to be involved in the local ovarian regulation of v ertebrate ovulation, their specific role is unclear. In yellow perch (Perca flavescens) the progestin, 17 alpha,20 beta-dihydroxy-4-pregnen-3-one (17, 20-P), stimulates ovulation in vitro and this induction requires gene activ ation. Therefore, the perch model was used to isolate progestin-upregulated mRNAs. Perch ovaries were incubated for 32 h with or without 17,20-P (0.1 mu g/ml). Messenger ribonucleic acids were isolated from the tissue and use d for differential display PCR (DDPCR). From DDPCR, 5 bands were eventually obtained that were verified by Northern analysis to be consistently upregu lated by 17,20-P at 32 h. Using these bands, full-length cDNAs were obtaine d by library screening and completely sequenced. Based on similarity to kno wn sequences, four of the cDNAs presumably encode for perch forms of (1) ne prilysin (PNEP-1; 63% identical); (2) a lysyl oxidase-type protein (PLO-2; 43.2% identical); (3) calmodulin (PCAL-1; 100% identical); and (4) a microt ubule aggregate-like protein (PMAP-1; 29.6% identical). The fifth cDNA obta ined from DDPCR most likely encodes for an egg protein and will be reported separately. Each of the cDNAs was used to probe Northern blots of ovarian mRNA taken at 0, 12, 24, 32 and 42 h of incubation with 17,20-P. This tempo ral Northern analysis verified that all four were upregulated by 32 h. In a ddition, PNEP and PMAP transcripts began to increase by 12 h, while PCAL an d PLO transcripts remained elevated through 42 h. On Northern blots of RNA from other perch tissues, calmodulin was found in all tissues, PLO mRNA was ovarian specific, and PMAP mRNA was also present in the gills and liver. M ultiple transcripts were observed for PNEP, but the ovarian form induced by 17,20-P was only found in high abundance in the heart. To our knowledge, t his is the first report that specifically characterizes progestin upregulat ed mRNAs in the vertebrate ovary at ovulation.