Daniel P B, Habener J F
Laboratory of Molecular Endocrinology, Massachusetts General Hospital, Howard Hughes Medical Institute, and Harvard Medical School, Boston 02114, USA.
Endocrinology. 2000 Mar;141(3):1218-27. doi: 10.1210/endo.141.3.7347.
Pituitary adenylate cyclase-activating polypeptide (PACAP) is a member of the glucagon-related family of hormones that is widely expressed in various tissues. The PACAP messenger RNA (mRNA) and protein is expressed at high levels in the germ cells of the testis, where it locally activates cAMP-coupled receptors located in the somatic Sertoli cells. The PACAP mRNA expressed specifically in the testis is shorter than the mRNA expressed in hypothalamus and includes 127 nucleotides of novel sequence at the 5'-end, suggesting a different start site of transcription in the testes and the utilization of a tissue-specific promoter. Here we present evidence that a single PACAP gene uses a testis-specific promoter to express a mRNA containing a unique exon located 13.5 kb upstream from the first coding exon. As determined by RT-PCR analysis of testis mRNA, the expression of the first testis-specific exon is relatively specific for the testis, as no PACAP mRNA containing the testis-specific first exon was detected in hypothalamic mRNAs. The promoter for the testis-specific PACAP gene was cloned, and a start site for transcription was mapped by primer extension. The testis-specific promoter sequence directs germ cell-specific expression upon transfection of promoter-transcriptional reporter plasmids to populations of testicular cells in vitro and upon expression of a promoter-reporter transgene in mice. Analyses of PACAP gene expression during the spermatogenic cycle, accomplished by RT-PCR of segments of isolated seminiferous tubules, identified intense expression in the postmeiotic round spermatids during developmental stages I-VIII. These observations establish the existence of a specialized PACAP gene promoter whose activity is highly regulated during the spermatogenic cycle.
垂体腺苷酸环化酶激活多肽（PACAP）是胰高血糖素相关激素家族的成员，在多种组织中广泛表达。PACAP信使核糖核酸（mRNA）和蛋白质在睾丸生殖细胞中高水平表达，在那里它局部激活位于体细胞支持细胞中的环磷酸腺苷（cAMP）偶联受体。睾丸中特异性表达的PACAP mRNA比下丘脑表达的mRNA短，并且在5'端包含127个核苷酸的新序列，这表明睾丸中转录起始位点不同，且利用了组织特异性启动子。在此我们提供证据表明，单个PACAP基因使用睾丸特异性启动子来表达一种mRNA，该mRNA含有一个位于第一个编码外显子上游13.5 kb处的独特外显子。通过对睾丸mRNA进行逆转录聚合酶链反应（RT-PCR）分析确定，第一个睾丸特异性外显子的表达相对特异于睾丸，因为在下丘脑mRNA中未检测到含有睾丸特异性第一个外显子的PACAP mRNA。克隆了睾丸特异性PACAP基因的启动子，并通过引物延伸确定了转录起始位点。当将启动子-转录报告质粒转染到体外睾丸细胞群体中以及在小鼠中表达启动子-报告转基因时，睾丸特异性启动子序列指导生殖细胞特异性表达。通过对分离的生精小管片段进行RT-PCR来分析生精周期中的PACAP基因表达，结果发现在发育阶段I-VIII的减数分裂后圆形精子细胞中有强烈表达。这些观察结果证实了存在一种特殊的PACAP基因启动子，其活性在生精周期中受到高度调控。
