EXPRESSION AND DEVELOPMENT OF RAT PREPROTHYROTROPIN-RELEASING HORMONE AND ITS RECEPTORS IN THE TESTI OF RAT

摘要

Recently, testis has been known as a source of numerous hypothalamic neuropeptides, including thyrotrophin-releasing hormone (TRH). We have found that TRH receptor (TRH-R) mRNA was identified in leydig cells of mouse testis using in situ hybridization. In order to investigate the expression regulation of thyrotrophin-releasing hormone (TRH) and TRH receptor (TRH-R) in rat testis, and to study their function in reproduction and development, we designed oligonucleotide primers from the sequences of rat hypothalamus prepro(pp)TRH and pituitary TRH-R cDNA for reverse transcription-polymerase chain reaction (RT-PCR). Specific fragments of ppTRH and TRH-R cDNA were cloned and sequenced. Expression plasmids containing ppTRH and TRH-R genes were then constructed and expression carried out in E.coli DH5α cells. Based on the developmental pattern of sexual maturation of the rat, ppTRH and TRH-R mRNA in the testis was quantitated in RNA samples prepared from rats of 8, 15, 20, 35, 60 and 90 days of age by real time quantitative RT-PCR. SDS-PAGE showed that the supernatant of PGEX-ppTRH demonstrated a new obvious protein band of 60 kD and that of PGEX-TRH-R demonstrated 40 kD, which were of the same as the expected size of fusional proteins respectively. The quantitative analyses demonstrated that no ppTRH and TRH mRNA could be detected at the earliest stage (day 8). PpTRH and TRH mRNA signals were detected on day 15 and increased progressively on day 20, 35, 60 and 90. These Results suggest that rat testis specifically express TRH and TRH-R, and the transcription of ppTRH and TRH-R gene in the rat testis is development-dependent. The acquirement of expressed products for ppTRH and TRH-R can be used for further research on physiological significance of TRH and TRH-R expression in rat testis.