Molecular and functional characterization of a testis-specific TRS4 gene in spermatogenesis

Abstract

INTRODUCTION: Spermatogenesis is a process in which diploid spermatogonia undergo mitotic, meiotic divisions and cellular differentiation to produce haploid spermatozoa that are capable to fertilize an egg. To understand the molecular mechanisms on how spermatogenesis in rodents is regulated, we investigated the gene expression of heat-treated testis in a rat model and identified TRS4 as a heat-sensitive gene in testis. Mouse TRS4 gene is located at chromosome 6A 3.1 with 13 exons, which encodes a protein with a predicted molecular weight, 90 kDa. Bioinformatic analysis showed that TRS4 has a high sequence homology among rat, mouse and human. TRS4 protein possesses an ubiquitin domain near N-terminus and an IQ-calmodulin binding motif inside exon 4-6 region of the gene. METHODS: In this study, we aimed: (i) to study the spatiotemporal expression of TRS4 mRNA in mouse tissues and post-natal mouse testes by quantitative PCR; (ii) to study interacting partners of TRS4 protein in the testis; and (iii) to generate TRS4 conditionally knockout mice by genetargeting approach. RESULTS AND DISCUSSION: Quantitative PCR studies showed that TRS4 mRNA was specifically expressed in the testis and post-natally on Day 20 onward. TRS4 mRNA was also localized at the spermatids stage of seminiferous tubules of adult mouse testes by in-situ hybridization. By co-immunoprecipitation and Western blotting, TRS4 was found to interact with α-actin, but not VAD1.2 and VAD1.3 (acrosome-expressing proteins), or syntaxin 1. TRS4 conditional gene targeting vector was constructed by flanking the exon 4-6 region of TRS4 gene with two LoxP sites. The Cre/Flp recombination of this completed vector was characterized in vivo by 293-Cre and 293-Flp E.Coli cells respectively. Putative TRS4 targeted mouse ES clones were being screened by Southern Blotting. Results from the present study should shed light to understand the role of TRS4 in spermatogenesis.