In the present study, we report the cloning and expression analysis of an oocyte-specific gene encoding a novel F-box protein in rainbow trout. We show that the expression of both fbxoo mRNA and protein is high in oocytes at early pre-vitellogenesis stage, suggesting an important role for this protein in early oocyte development. The novel protein does not contain any of the common secondary motifs and therefore it belongs to the family of FBXO proteins. It is the first oocyte-specific F-box protein identified in a fish species.
In mouse, an oocyte-specific F-box protein gene, Fbxw15/Fbxo12J, has been reported . The gene was identified by microarray analysis as the top increaser of 24 genes showing increased expression in neonatal mouse ovaries at 48 h and 96 h relative to < 24 h after birth. The oocyte-specific F-box protein is believed to play a role in the regulation of oocyte development based on its specific expression in oocytes and its expression pattern during follicular development . Despite the fact that FBXW15/FBXO12J is also an F-box only protein  and is exclusively expressed in oocytes, it is not an orthlogue of Fbxoo as the two proteins share only 20% protein sequence identity. The fact that Fbxoo only shows high sequence similarity to an uncharacterized protein in zebrafish and Xenopus indicates that Fbxoo is likely fish/amphibian specific. This speculation is supported by synteny analysis showing that the corresponding loci of the zebrafish gene on the syntenic regions of human chromosome 6 and mouse chromosome 10 do not code for any genes (data not shown).
In growing oocytes, some transcripts undergo deadenylation in the cytoplasm where they are packaged into messenger ribonucleoprotein (mRNP) particles [18, 19]. During oocyte maturation, cytoplasmic polyadenylation extends the poly (A) tail of these transcripts, a process associated with their timely translation [20, 21]. The presence of a typical U-rich cytoplasmic polyadenylation element (UUUUUAA) in the 3′UTR of fbxoo mRNA indicates that this gene may have controlled translation during oocyte maturation. In addition, Fbxoo is predicted to have a SUMO site located on lysine residue 190, indicating that the function of Fbxoo protein may be regulated by sumoylation, a post-translational protein modification process known to change and regulate the function of a protein . Sumoylation of oocyte-specific proteins have been reported previously. For example, POU5F1, an oocyte-specific transcription factor, is sumoylated  and sumoylation of POU5F1 leads to increased stability, DNA binding, and transactivation function of the protein .
Many F-box proteins are located in both cytoplasm and nucleus . Our results also show that the expression of fbxoo mRNA and protein is detected in both cytoplasm and nucleus of oocyte at early developmental stages. While many F-box proteins are ubiquitously expressed , tissue specific F-box proteins with specific functions have been reported [26–28]. In particular, muscle-specific F-box proteins such as ATROGIN-1 and MURF1 are known to have unique functions in the degradation of important regulatory proteins during muscle atrophy [27, 29]. As an oocyte-specific F-box protein predominantly expressed in early pre-vitellogenic and early vitellogenic oocytes, it is reasonable to believe that Fbxoo plays a crucial role during the early oocyte growth in rainbow trout.
F-box proteins bind to their specific substrates for ubiquitin-mediated proteolysis. They often have additional carboxy-terminal motifs (e.g. WD repeats and leucine-rich repeats) capable of protein-protein interaction. As an FBXO protein, Fbxoo does not have the common secondary motifs but may contain potential protein-protein interaction domains not yet identified. As an attempt to understand the function of the novel protein, we screened for Fbxoo-interacting proteins using the yeast two-hybrid system and identified three proteins including Timp2 as potential substrates of Fxboo protein. TIMP2 is a member of the TIMP family proteins known to play an important role in regulating the activity of matrix metalloproteinases (MMPs), which are the key metal-dependent enzymes in the extracellular matrix remodeling of follicular tissue. TIMPs are highly abundant in reproductive tissues and are well known to be involved in follicular development and early embryogenesis in a number of mammalian and fish species [30–32]. The expression of Timps has been detected in unfertilized oocytes, zygotes, cleavage stage embryos and blastocysts as well as in granulosa and theca cells . The functional interactions between TIMPs and MMPs have been thoroughly investigated during folliculogenesis, whereas much less is known about the post-secretory mechanisms regulating the activity of TIMPs . As a TIMP2 binding partner involved in the ubiquitin proteosome system, Fbxoo may regulate the post-secretory activity of Timp2 to modulate oocyte development in rainbow trout.