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

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.

DNA methylation and miRNA-1296 act in concert to mediate spatiotemporal expression of KPNA7 during bovine oocyte and early embryonic development

“…Electrophoresis and transfer were performed according to a previous study with minor modifications [45]. HEK293 cell were harvested with Pierce IP Lysis Buffer (Thermo Fisher Scientific, Waltham, MA), and 10 μl of cell lysate were mixed with an equal volume of Laemmli sample buffer (Bio-Rad Laboratories, Hercules, CA).…”

SCF Ligases and Their Functions in Oogenesis and Embryogenesis—Summary of the Most Important Findings throughout the Animal Kingdom

“…Another option is that FBXW15/FBXO12J might function as a regulator of oocyte-granulosa communication [ 50 ]. Wang et al [ 60 ] reported the expression analysis of an oocyte-specific gene coding a novel F-box protein in rainbow trout. They observed the Fbxoo mRNA and protein expression profiles and found high expression in oocytes at the early pre-vitellogenesis stage.…”

“…They observed the Fbxoo mRNA and protein expression profiles and found high expression in oocytes at the early pre-vitellogenesis stage. This finding suggests an essential role of this F-box protein in early oocyte development by regulating other critical proteins [ 60 ].…”

Lhx8 interacts with a novel germ cell-specific nuclear factor containing an Nbl1 domain in rainbow trout (Oncorhynchus mykiss)

“…Understanding the functional contribution of oocyte-specific genes to egg quality will help us develop biomarkers for prediction of egg quality in rainbow trout. Our previous studies have resulted in the discovery of a number of novel oocyte-specific genes in rainbow trout, which include oorp-t [ 6 ], rtgst-1 [ 7 ], fbxoo [ 8 ] and knpa7 [ 9 ].…”