Cloning and characterization of a novel oocyte-specific gene encoding an F-Box protein in rainbow trout (Oncorhynchus mykiss)

Lei Wang,Warapond Wanna,Jianbo Yao,Caird E Rexroad ,Swamy K Tripurani

doi:10.1186/1477-7827-11-86

Abstract

BackgroundOocyte-specific genes play critical roles in oogenesis, folliculogenesis and early embryonic development. The objectives of this study were to characterize the expression of a novel oocyte-specific gene encoding an F-box protein during ovarian development in rainbow trout, and identify its potential interacting partners in rainbow trout oocytes.MethodsThrough analysis of expressed sequence tags (ESTs) from a rainbow trout oocyte cDNA library, a novel transcript represented by ESTs only from the oocyte library was identified. The complete cDNA sequence for the novel gene (named fbxoo) was obtained by assembling sequences from an EST clone and a 5′RACE product. The expression and localization of fbxoo mRNA and protein in ovaries of different developmental stages were analyzed by quantitative real time PCR, immunoblotting, in situ hybridization and immunohistochemistry. Identification of Fbxoo binding proteins was performed by yeast two-hybrid screening.Resultsfbxoo mRNA is specifically expressed in mature oocytes as revealed by tissue distribution analysis. The fbxoo cDNA sequence is 1,996 bp in length containing an open reading frame, which encodes a predicted protein of 514 amino acids. The novel protein sequence does not match any known protein sequences in the NCBI database. However, a search of the Pfam protein database revealed that the protein contains an F-box motif at the N-terminus, indicating that Fbxoo is a new member of the F-box protein family. The expression of fbxoo mRNA and protein is high in ovaries at early pre-vitellogenesis stage, and both fbxoo mRNA and protein are predominantly expressed in early pre-vitellogenic oocytes. Several proteins including tissue inhibitor of metalloproteinase 2 (Timp2) were identified as potential Fbxoo protein binding partners.ConclusionsResults suggest that the novel oocyte-specific F-box protein may play an important role in early oocyte development by regulating other critical proteins involved in oogenesis in rainbow trout.

Highlights

Oocyte-specific genes play critical roles in oogenesis, folliculogenesis and early embryonic development
Based on analysis of expressed sequence tags (ESTs) sequences from a rainbow trout oocyte cDNA library, we have previously identified two novel oocyte-specific genes in rainbow trout. oorp-t encodes a protein with a conserved oxysterol binding protein (OSBP) domain, suggesting its role in the synthesis, transportation and metabolism of lipids during oogenesis [8]. rtgst-1 is a noncoding mRNA-like transcript and its predominant expression in early previtellogenic oocytes suggests a crucial role of this transcript in the differentiation and/or early development of oocytes [9]
Cloning and sequence analysis of fbxoo cDNA Through analysis of expressed sequence tags (ESTs) from a rainbow trout oocyte cDNA library, we identified a novel transcript, which is represented by multiple ESTs derived from the oocyte cDNA library

Summary

Introduction

Oocyte-specific genes play critical roles in oogenesis, folliculogenesis and early embryonic development. The finely orchestrated development and maturation of the oocyte have been the focus of many studies in which essential oocyte-specific genes have been identified [1,2] Examples of such genes include factor in germline alpha (Figla), growth differentiation factor 9 (Gdf9) and newborn ovary homeobox (Nobox). An oocyte-specific gene, zorg (zebrafish oogenesis related gene), has been reported and its expression profiles in different stages of oocytes suggests a role of this gene in the formation of female germ cells [7] Another zebrafish gene, zvep (zebrafish vitelline envelope protein), was shown to be expressed in zebrafish ovary and brain [6]. Based on analysis of EST sequences from a rainbow trout oocyte cDNA library, we have previously identified two novel oocyte-specific genes (oorp-t and rtgst-1) in rainbow trout. oorp-t encodes a protein with a conserved oxysterol binding protein (OSBP) domain, suggesting its role in the synthesis, transportation and metabolism of lipids during oogenesis [8]. rtgst-1 is a noncoding mRNA-like transcript and its predominant expression in early previtellogenic oocytes suggests a crucial role of this transcript in the differentiation and/or early development of oocytes [9]

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