Regulation and functional studies of the testis-specific temperature-related sequence 4 (TRS4) in spermatogenesis

Temperature-related sequence 4 (Trs4) is a transcript identified from the testis of a cryptorchid rat model. TRS4 is firstly detected in round spermatids on postnatal day 21 mice, and is localized on the acrosome and tails of elongating spermatids and mature spermatozoa, suggesting a role in spermatogenesis. The present study aimed to investigate the regulatory and functional roles of Trs4 in spermatogenesis. To investigate the hormonal regulation of Trs4 in vitro, mouse germ cell were co-cultured with Sertoli (TM4) cell-line for 24 hours with 1μM or 10nM testosterone and the expression of Trs4 was quantitated. It was found that testosterone treatment at physiological level (10nM) significantly down-regulated Trs4 mRNA expression. However, TRS4 protein level did not change significantly. To investigate the functional roles of Trs4 and its interacting proteins (Rshl2, Gstmu1 and Ddc8) in spermatogenesis, the scrotal heat-treated rat model was used. The expression of Trs4, Rshl2, Gstmu1 and Ddc8 transcript were investigated on day 1, 9, 25, 37, 46, 56 and 79 post-heat treatment. Trs4, Rshl2 and Ddc8 transcripts were down-regulated on day 9 after heat-treatment, and recovered from day 37 onward. Immunohistochemical staining of TRS4 and RSHL-2 proteins showed they specifically localized in the cytoplasm of elongating spermatids in rat seminiferous tubules, suggesting that they might interact in regulating the development of elongating spermatids. Ten sessions of electroacupuncture (EA) was applied to the rats on day 9-36 to study the effect of EA in spermatogenesis. It was found that EA did not have any effect on the expression of Trs4, Rshl-2 and Ddc8 during recovery from hyperthermia when compared to the heat-treated only group. However, qPCR analysis on Gstmu1 mRNA expression showed a significant decrease in expression on day 46, and consistent with our TUNEL assay indicating a decrease in apoptosis in the EA group. Therefore, it is possible that Trs4 interacts with Gstmu1 and acts as anti-apoptotic factor for cell survival. To understand functional roles of Trs4 in mouse spermatogenesis, five Trs4 chimeric mice were generated in our laboratory and germline transmissible 〖Trs4〗^(flox/+) mice were identified. Sycp1-Cre mice were used to breed with Trs4 floxed mice to generate Trs4 floxed mice expressing Cre recombinase in spermatocytes (〖Trs4〗^(flox/+);〖Sycp-Cre〗^(+/0)) and aiming to generate Trs4 heterozygous KO mouse. However, after screening 74 offspring, no Trs4 heterozygous KO mouse was identified in this study and was likely to be caused by the low efficiency of recombination in Sycp1-Cre mice. Then, Zp3-Cre mice that expressing Cre-recombinase in oocyte were employed. Crossing of 〖Trs4〗^(flox/+):Zp3-〖Cre〗^(+/0) mice and Trs4 floxed mice could not produce heterozygous or homozygous Trs4 KO mouse, but suggested an aberrant recombination process happened in vivo. PCR assay confirmed DNA rearrangement occurred in regions flanking the exon 4 to the second LoxP site in Trs4 floxed mice. Furthermore, the absence of 〖Trs4〗^(flox/flox) homozygous transgene suggested a possibility of insertional mutagenesis in the transgene. Future study is needed to confirm and characterize the insertion site of the floxed allele in order to generate Trs4 knockout mice for fertility study.published_or_final_versionObstetrics and GynaecologyMasterMaster of Philosoph