大鼠睾丸前促甲状腺激素释放激素原及其受体的表达与发育变化

摘要

为研究促甲状腺激素释放激素(thyrotrophin-releasing hormone,TRH)及其受体 (TRH receptor, TRH-R)在大鼠睾丸组织中的表达规律和在生殖发育调节中的作用，依据大鼠下丘脑中的前TRH原(preproTRH, ppTRH)和垂体中的TRH-R cDNA设计引物，采用RT-PCR法从大鼠睾丸组织中获得了ppTRH和TRH-R的cDNA克隆，测序后构建表达载体，在大肠杆菌中表达了可溶性的ppTRH和TRH-R融合蛋白。利用实时动态定量RT-PCR (real time quantitative RT-PCR)法观察了ppTRH和TRH-R在不同发育阶段大鼠睾丸中的表达变化，发现在睾丸间质细胞发育的初期阶段(第8天)，没有ppTRH和TRH-R的表达，但从第15天起能观察到ppTRH和TRH-R的表达，并且表达量在20天、35天、60天和90天逐渐增加。这些结果表明：大鼠睾丸组织能特异性表达ppTRH和TRH-R，并且表达量与发育过程相关。ppTRH和TRH-R体外表达产物的获得为后续研究其功能奠定了基础。%Recently, testis has been known as a source of numerous hypothalamic neuropeptid es, including thyrotrophinreleasing hormone (TRH). We have found that TRH rece p tor (TRHR) mRNA was identified in leydig cells of mouse testis usingin situ hy bridization. In order to investigate the expression regulation of thyrotrophin releasing hormone (TRH) and TRH receptor (TRHR) in rat testis, and to study thei r function in reproduction and development, we designed oligonucleotide primers rnf rom the sequences of rat hypothalamus prepro(pp)TRH and pituitary TRHR cDNA fo r 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 fr om 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 p rotein band of 60 kD and that of PGEXTRHR demonstrated 40 kD, which were of th e same as the expected size of fusional proteins respectively. The quantitative analyses demonstrated that no ppTRH and TRH mRNA could be detected at the earlie st stage (day 8). PpTRH and TRH mRNA signals were detected on day 15 and increas ed progressively on day 20, 35, 60 and 90. These Results suggest that rat tes tis 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 ex pr essed products for ppTRH and TRHR can be used for further research on physiolo gical significance of TRH and TRHR expression in rat testis.