Cysteine protease inhibitor is specifically expressed in pre‐ and early‐vitellogenic oocytes from the brook trout periovulatory ovary*

Abstract

A cDNA fragment hybridizing with a transcript abundant in the periovulatory ovary was obtained while performing subtractive cloning on brook trout ovulatory and postovulatory ovarian tissue. Using this fragment as a probe, a 478 bp full-length cDNA was obtained by screening an ovulatory ovarian cDNA library. This cDNA presumably codes for an 88 amino acid protein that is structurally related to a new family of cysteine protease inhibitors characterized by the presence of a type I thyroglobulin motif in the amino acid sequence. Therefore, the protein was tentatively named an oocyte cysteine protease inhibitor (OCPI). On Northern blots, the OCPI cDNA hybridizes with a 0.5 kb transcript present in the ovary during the periovulatory period. The OCPI transcript and protein were localized to the cytoplasm of pre- and early-vitellogenic oocytes. On Northern blots of RNA from other tissues, the OCPI transcript was detected only in the ovary. On Western blots, OCPI was detected in the ovarian tissue at all periovulatory stages tested. The specific localization of both OCPI transcript and protein to pre- and early-vitellogenic oocytes and the structural similarity to protease inhibitors, suggest that OCPI might be involved in the protection of oocytes during the periovulatory period or in the regulation of yolk formation and degradation.

Thyroglobulin type‐1 domain protease inhibitors exhibit specific expression in the cortical ooplasm of vitellogenic rainbow trout oocytes

“…The expression pattern of rainbow trout OPI-1 differs somewhat from the reported expression pattern of the brook trout homolog (OCPI). Bobe and Goetz (2001) detected brook trout OCPI mRNA and protein in what were described as previtellogenic follicles, whereas we were unable to detect OPI-1 expression in the previtellogenic ovary, using both immunocytochemistry and the more sensitive RT-PCR technique. One plausible explanation for these divergent observations relates to the specific stages of ovarian development of fish used in each study.…”

“…In this study, we report the identification and characterization of two oocyte protease inhibitor cDNAs in rainbow trout ovary. One of these cDNAs (OPI-1) is predicted to encode the rainbow trout homolog of an OPI recently identified in related teleosts (Yamashita and Konagaya, 1996;Bobe and Goetz, 2001;Davey et al, 2001). The second cDNA (OPI-2) is a novel gene that resembles a tandem duplication of OPI-1, but which has not previously been reported.…”

“…Vitellogenic ovarian follicles were punctured and samples of whole yolk were diluted 1:100 in SDS-PAGE sample buffer (1% SDS, 0.1 M Tris-HCl, 10% glycerol, 0.02% bromophenol blue, 2% 2-mercaptoethanol), heated for 10 min at 658C, electrophoretically separated on 14% polyacrylamide gels, and transferred for 30 min to Immobilon-P PVDF membranes (Millipore, Milwaukee, WI). After transfer, the membranes were incubated for 2 hr at room temperature in blotting buffer (phosphate-buffered saline, 0.1% Tween 1 20, 3% BSA, pH 7.4), and then blotted overnight at 48C with rabbit antiserum (1:1,000 in blotting buffer) raised against a recombinantly expressed maltose-binding protein:brook trout OCPI fusion protein (anti-OCPI) (Bobe and Goetz, 2001), generously provided by Dr. Frederick W. Goetz (Marine Biological Laboratories, Wood's Hole, MA). Membranes were then blotted for 2 hr at room temperature with secondary antibody (HRP-conjugated goat anti-rabbit IgG, 1:5,000 in blotting buffer) and immunoreactive proteins were visualized using the ECL detection system.…”

“…The defining feature of ECI is a cysteine-rich thyroglobulin type-1 (TY) domain, characteristic of a protein group recently termed ''thyroglobulin type-1 domain protease inhibitors'' (Molina et al, 1996;Lenarcic et al, 1997). A cDNA encoding a putatively homologous TY domain inhibitor (oocyte cysteine protease inhibitor, OCPI) was recently cloned from brook trout ovarian tissues (Bobe and Goetz, 2001), and shown to exhibit ovary-specific expression. These expression data, however, were derived almost exclusively from periovulatory ovarian tissues, thus there is no information on the expression of this gene during the prolonged vitellogenic phase of oocyte development, during which the specific regulation of oocyte proteases is required for proper oocyte development.…”

Characterization of rainbow trout gonad, brain and gill deep cDNA repertoires using a Roche 454-Titanium sequencing approach

“…In agreement with existing data on rainbow trout (Bobe et al, 2008), zygote arrest 1 (zar1) and growth and differentiation factor 9 (gdf9) exhibited a very strict ovarian-predominant profile. Similarly, oocyte cysteine protease inhibitor (ocpi) was dramatically overexpressed in the ovarian libraries, again in agreement with existing data (Bobe and Goetz, 2001). In addition to these previously documented patterns, which validate our approach, the DDD analysis has led to the identification of many genes that also putatively exhibit predominant or specific ovarian and/or oocyte expression.…”