Valentin M, Balvers M, Pusch W, Weinbauer G F, Knudsen J, Ivell R
Institute for Hormone and Fertility Research, University of Hamburg, Germany.
Eur J Biochem. 2000 Sep;267(17):5438-49. doi: 10.1046/j.1432-1327.2000.01603.x.
The endozepine-like peptide (ELP) represents a testis-specific isoform of the ubiquitous acyl-CoA binding protein (ACBP) and is highly expressed in late haploid stages of male germ cell development. The genomic sequence of the functional ELP gene as well as that of a pseudogene were analysed from independent bacteriophage clones of a 129sv mouse genomic library. Unlike the ACBP gene, which comprises four exons, the ELP gene has only a single intron within the region of the 5' untranslated region, suggesting that, like some other haploid expressed genes, the ELP gene might have evolved by retroposon-mediated gene duplication. Primer extension analysis was used to define the start site for transcription and hence the 5' promoter region. Electrophoretic mobility shift analysis was carried out on this region comparing nuclear extracts from adult mouse testis with those from mouse liver. Several testis-specific DNA-protein complexes were observed throughout 700 bp upstream of the transcription start site. One of these could be identified as corresponding to a steroidogenic factor-1 (SF-1) binding element. Further analysis using pure transcription factors showed that this element at position -340 was able to bind specifically to both SF-1 and to the germ cell nuclear factor (GCNF). Immunohistochemical analysis using an ELP-specific antibody showed that expression was very restricted within the testis to the postmeiotic germ cells, and in the ovary to interstitial/luteal cells, cell-types known to express GCNF and SF-1, respectively. Testes of CREM-tau knockout mice, lacking all spermatogenic stages later than round spermatids, were devoid of ELP immunoreactivity, whereas in RAD6 knockout mice the few remaining elongated spermatids were clearly defined by this excellent late haploid marker product. The ELP gene and its product thus offer an ideal system with which to investigate the differentiation of late haploid stages of spermatogenesis.
内啡肽样肽（ELP）是普遍存在的酰基辅酶A结合蛋白（ACBP）的睾丸特异性异构体，在雄性生殖细胞发育的单倍体晚期高度表达。从129sv小鼠基因组文库的独立噬菌体克隆中分析了功能性ELP基因以及假基因的基因组序列。与包含四个外显子的ACBP基因不同，ELP基因在5'非翻译区区域内只有一个内含子，这表明，与其他一些单倍体表达基因一样，ELP基因可能是通过逆转座子介导的基因复制进化而来的。引物延伸分析用于确定转录起始位点，从而确定5'启动子区域。对该区域进行了电泳迁移率变动分析，比较了成年小鼠睾丸和小鼠肝脏的核提取物。在转录起始位点上游700 bp范围内观察到几种睾丸特异性DNA-蛋白质复合物。其中之一可被鉴定为对应于类固醇生成因子-1（SF-1）结合元件。使用纯转录因子的进一步分析表明，位于-340位置的该元件能够特异性结合SF-1和生殖细胞核因子（GCNF）。使用ELP特异性抗体的免疫组织化学分析表明，ELP在睾丸内的表达非常局限于减数分裂后的生殖细胞，并在卵巢中局限于间质/黄体细胞，已知这两种细胞类型分别表达GCNF和SF-1。CREM-tau基因敲除小鼠的睾丸缺乏圆形精子细胞之后的所有生精阶段，没有ELP免疫反应性，而在RAD-基因敲除小鼠中，少数剩余的细长精子细胞被这种出色的单倍体晚期标记产物清楚地界定。因此，ELP基因及其产物为研究精子发生单倍体晚期的分化提供了一个理想的系统。
