A study on the role of temperature repressed sequence 4 (Trs4) in spermatogenesis

Abstract

Heat stress inhibits spermatogenesis partly by inducing apoptosis in the testicular germ cells. Using a cryptochid rat model, we identified a temperature-related ESTs 4 (TRS4) transcript from rat testis. Trs4 mRNA is specifically expressed in the mouse and rat testis from postnatal day 21 and 28 days onwards, respectively. Trs4 protein is located mainly in the elongating spermatids and mature spermatozoa at the acrosome and tail regions. Using a yeat-2-hybrid screening, Trs4 was found to bind Gstmu1, Rslh-2 and Ddc8 proteins. To further characterize the functional role of Trs4 in spermatogenesis, and study how Trs4 interacts its binding proteins for cellular functions, we aimed (1) to screen putative ES cells with Trs4 floxed allele for knockout mice generation, (2) to generate Trs4 deletion constructs and study the cellular localization of Trs4 and its putative binding partners in transfected spermatocyte GC-2spd(s) cell line, (3) to study how heat-treatment regulates the expression of Trs4 and apoptotic molecules. The Trs4 conditional targeting vector was constructed by flanking exons 4-6 with two LoxP sites and electroporated into ES cells. After screening of 480 clones, positive ES cell clones were identified by Southern blotting using 5’- and 3’- probes. Three putative positive clones were identified carrying the floxed allele. Trs4 protein contains putative ubiquitin-like motif (a.a. 119-224), IQ-calmodulin binding motif (a.a. 334-362) and a overlapping bipartite nuclear localization signal (BNL) (a.a. 346-362). Transfection of EGFP fused Trs4 truncated protein demonstrated that the IQ-calmodulin binding motif and BNL signal was important for localization of Trs4 protein in the cytoplasmic/Golgi regions; while the N-terminal contains ubiquitin-like motif and the C-terminal regions direct the expression of the EGFP-fusion protein mainly to the nucleus. The full-length sequence of Trs4 binding partners: Gstmu1, Rshl-2 and Ddc8 were cloned into the pDsRedmonomer-C1 vector, giving red fluorescence protein in the transfected cells. They were colocalized with EGFP-Trs4 in the cytoplasm of the cells, confirming that Trs4 and its interacting protein is likely interact with each other in vivo. As Trs4 colocalize with Gstmu1, a modulator of mitochondrial-dependent pathway in apoptosis, it is suggested that Trs4 is an upstream regulator of apoptosis under heat treatment in germ cells. The functional roles of Trs4 protein Trs4 and apoptotic molecules. The Trs4 conditional targeting vector was constructed by flanking exons 4-6 with two LoxP sites and electroporated into ES cells. After screening of 480 clones, positive ES cell clones were identified by Southern blotting using 5’- and 3’- probes. Three putative positive clones were identified carrying the floxed allele. Trs4 protein contains putative ubiquitin-like motif (a.a. 119-224), IQ-calmodulin binding motif (a.a. 334-362) and a overlapping bipartite nuclear localization signal (BNL) (a.a. 346-362). Transfection of EGFP fused Trs4 truncated protein demonstrated that the IQ-calmodulin binding motif and BNL signal was important for localization of Trs4 protein in the cytoplasmic/Golgi regions; while the N-terminal contains ubiquitin-like motif and the C-terminal regions direct the expression of the EGFP-fusion protein mainly to the nucleus. The full-length sequence of Trs4 binding partners: Gstmu1, Rshl-2 and Ddc8 were cloned into the pDsRedmonomer-C1 vector, giving red fluorescence protein in the transfected cells. They were colocalized with EGFP-Trs4 in the cytoplasm of the cells, confirming that Trs4 and its interacting protein is likely interact with each other in vivo. As Trs4 colocalize with Gstmu1, a modulator of mitochondrial-dependent pathway in apoptosis, it is suggested that Trs4 is an upstream regulator of apoptosis under heat treatment in germ cells. The functional roles of Trs4 protein