MOLECULAR CHARACTERIZATION OF A RIBONUCLEIC-ACID TRANSCRIPT THAT IS HIGHLY UP-REGULATED AT THE TIME OF OVULATION IN THE BROOK TROUT (SALVELINUS-FONTINALIS) OVARY

Using stage-specific subtractive cDNA cloning, a 0.8-kilobase (kb) cDN A (TOP-1; trout ovulatory protein-1) was previously obtained that hybr idized only with ovarian RNA and was highly up-regulated at the time o f ovulation. In the present study, a 1.4-kb cDNA (TOP-2; trout ovulato ry protein-9 was obtained after rescreening a brook trout ovulatory cD NA library using TOP-1 as a probe. The levels of ovarian transcripts h ybridizing on Northern blots probed with TOP-2 begin to increase after the breakdown of the germinal vesicle in the oocyte and appear to pea k approximately 24 h postovulation. Antibodies produced to a recombina nt maltose binding protein-TOP-2 fusion protein recognize 3-4 protein bands (53, 39, 29, and 24 kDa) on Western blots of ovarian tissue and/ or fluid. Levels of the two largest proteins increase immediately afte r ovulation and decrease significantly by 4-8 days postovulation. TOP- 2 has an open reading frame of 262 amino acids. The presumed amino aci d sequence of TOP-2 is rich in cysteine and is arranged in 5 polypepti de domains consisting of two types, one of which is homologous to the domains of TOP-1. Both domain types also share homology with a group o f mammalian leukocytic protease inhibitors called anti leukoproteinase s.