Male germ cell-specific protein Trs4 binds to multiple proteins

Temperature-related sequence 4 (Trs4) has been identified as a testis-specific gene with expression sensitive to the abdominal temperature changes induced by artificial cryptorchidism. In murine testes, Trs4 mRNA was detected in round spermatids and its protein was localized mainly in the elongating spermatids as well as in the acrosomes and tails of mature spermatozoa. Using a yeast two-hybrid screening system, we identified Rshl-2, Gstmu1, and Ddc8 as putative binding partners of the Trs4 protein in mouse testes. Their interactions were confirmed by in vivo and in vitro binding assays. Further studies demonstrated that Ddc8, a newly identified gene with unknown functions, displayed a similar expression pattern with Trs4 in mouse testes. In particular, Trs4, Ddc8, and Rshl-2 proteins were co-localized to the tails of mature spermatozoa. These results suggested that Trs4 might be involved in diverse processes of spermiogenesis and/or fertilization through interactions with its multiple binding partners. © 2009 Elsevier Inc.postprin

Identification of a genetic modifier of CHD in Type IIA procollagen deficient mutant mice suggests a dynamic regulatory system in cardiac development

Poster no. BD0

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

Poster Presentations - Theme 4: Reproduction & Development: no. 4.11The 13th Research Postgraduate Symposium, the University of Hong Kong, Hong Kong, China, 10-11 December 2008

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

SRF Student Prize: O23INTRODUCTION: 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.The 2009 Annual Conference of the Society for Reproduction and Fertility (SRF), St Catherine’s College, Oxford, U.K., 12-14 July 2009

Syntaxin and b-actin interact with acrosome-expressing protein VAD1.2/AEP2 in spermatogenesis

Spermatogenesis involves meiosis of the diploid spermatogonia to form the haploid spermatids and spermiogenesis to transform the round spermatids into mature spermatozoa. Normal fertilization requires the sperm to come into contact with the egg and release its enzymes from the head (acrosome). Acrosomal defects are known to cause infertility in human. To understand the molecular regulation of spermatogenesis in vivo, we used differential display RT-PCR to identify testis-specific genes in a retinol-supplemented vitamin A deficiency (VAD) rat model and identified the VAD1.2 (acrosome-expressing protein 2, AEP2) gene, which was expressed strongly in the rat testis from postnatal day 32 to adult stage. However, the functional roles of this protein on acrosome formation during spermatogenesis remain elusive. The aims of this study were to (1) study the spatiotemporal expression of VAD1.2 during spermatogenesis and (2) study the interaction of this protein with their binding protein(s) in the mouse, rat and human testis. The mouse VAD1.2 mRNA shared 85% and 67% sequence homology, and 74% and 38% amino acid homology, respectively with the rat and human counterparts. VAD1.2 transcript was abundantly expressed in the rat seminiferous tubules at stage VIII-XII and the protein was detected in the acrosome region of the round and elongated spermatids of mouse, human, monkey and pig. VAD1.2 colocalized with lectin-PNA to the acrosome region of spermatids. Interestingly, the expression of VAD1.2 protein in human testis diminished in patients with defective spermatogenesis. Co-immunoprecipitation using polyclonal VAD1.2 antibody followed by Western blotting and mass spectrometry (MS-MS) identified syntaxin-1, b-actin and myosin heavy chain (MHC) but not VAD1.3/AEP1, TRS4, SEC23, synaptotagmin and cyclophilin B as putative interacting partners. Taken together, the stage-specific expression of VAD1.2 in the acrosome of spermatids and the binding of VAD1.2 protein with vesicle forming (syntaxin) and structural (b-actin and MHC) proteins suggest that VAD1.2 is involved in acrosome formation during spermiogenesis. Research supported by CERG HKU7537/05M and NSFC/RGC N_HKU712/06 to KFL

Molecular and functional approaches to study testis-repressed sequence 4 (TRS4) in spermatogenesis

Poster Presentation - Theme 1: Reproduction and Development: P.1.19The 15th Research Postgraduate Symposium, the University of Hong Kong, Hong Kong, China, 1-2 December 2010