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) cDNA (TOP-1; trout ovulatory protein-1) was previously obtained that hybridized only with ovarian RNA and was highly up-regulated at the time of ovulation. In the present study, a 1.4-kb cDNA (TOP-2; trout ovulatory protein-9 was obtained after rescreening a brook trout ovulatory cDNA library using TOP-1 as a probe. The levels of ovarian transcripts hybridizing on Northern blots probed with TOP-2 begin to increase after the breakdown of the germinal vesicle in the oocyte and appear to peak approximately 24 h postovulation. Antibodies produced to a recombinant 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 after ovulation and decrease significantly by 4-8 days postovulation. TOP-2 has an open reading frame of 262 amino acids. The presumed amino acid sequence of TOP-2 is rich in cysteine and is arranged in 5 polypeptide 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 of mammalian leukocytic protease inhibitors called anti leukoproteinases.