A dominant-negative isoform of hypoxia-inducible factor-lot specifically expressed in human testis
A dominant-negative isoform of hypoxia-inducible factor-lot specifically expressed in human testis
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DOI：
10.1095/biolreprod.104.027797
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发表时间：
2004-07-01
影响因子：
3.6
通讯作者：
Katschinski, DM
中科院分区：
文献类型：
--
作者：
Depping, R;Hägele, S;Katschinski, DM
睾丸曲细精管中的精子发生在高增殖率下发生，表明相当大的耗氧量。由于缺乏血管，这些小管管腔中的氧分压非常低。我们先前在小鼠中鉴定了低氧诱导因子（HIF）-1 α的睾丸亚型，称为mHIF-1alphaI.1。在这里，我们证明了mHIF-1 α I.1的表达在青春期增加，进一步证明了其在减数分裂后生殖细胞中的基因诱导。利用cDNA末端的5 '-快速扩增，我们在人睾丸中鉴定了一种新的HIF-1 α亚型，称为hHIF-1alphaTe。与mHIF-1alphaI.1一样，hHIF-1alphaTe mRNA来源于另一种启动子-第一外显子组合，但具有不同的基因组结构和不同的核苷酸序列。逆转录-聚合酶链反应分析证实hHIF-1 α Te仅在睾丸中表达。通过免疫荧光法测定，HIF-1 α蛋白主要定位于精子顶体后头部和中段。虽然与人和小鼠精子中的线粒体定位重叠，但hHIF-1 α Te和hHIF-1 α均不与线粒体相关。与普遍表达的HIF-1 α蛋白和小鼠睾丸特异性mHIF-1alphaI.1亚型相比，hHIF-1alphaTe mRNA序列预测了一种具有DNA结合结构域N端截短的蛋白。酵母双杂交实验表明，hHIF-1 α Te仍能与HIF-1 β形成异二聚体复合物。然而，hHIF-1 α Te不能形成DNA结合HIF-1复合物。外源性hHIF-1alphaTe的过表达导致内源性HIF-1转录活性的抑制，表明睾丸特异性hHIF-1alphaTe同种型是正常HIF-1活性的显性负调控因子。
Spermatogenesis in the seminiferous tubuli of the testis occurs under a high proliferation rate, suggesting considerable oxygen consumption. Because of the lack of blood vessels, the oxygen partial pressure in the lumen of these tubuli is very low. We previously identified a testis isoform of the hypoxia-inducible factor (HIF)-1alpha in the mouse, termed mHIF-1alphaI.1. Here, we demonstrate that expression of mHIF-1alphaI.1 increases during puberty, further demonstrating its gene induction in postmeiotic germ cells. Using 5'-rapid amplification of cDNA ends, we identified a novel HIF-1alpha isoform in the human testis, called hHIF-1alphaTe. Like mHIF-1alphaI.1, hHIF-1alphaTe mRNA is derived from an alternative promoter-first exon combination, but with a different genomic organization and a different nucleotide sequence. Reverse transcription-polymerase chain reaction analysis confirmed that hHIF-1alphaTe is exclusively expressed in the testis. As determined by immunofluorescence of ejaculated sperm cells, HIF-1alpha protein is mainly localized in the postacrosomal head and in the midpiece of spermatozoa. Though overlapping with mitochondrial localization in human and mouse spermatozoa, neither hHIF-1alphaTe nor hHIF-1alpha associated with mitochondria. In contrast with the ubiquitously expressed HIF-1alpha protein and the mouse testis-specific mHIF-1alphaI.1 isoform, the hHIF-1alphaTe mRNA sequence predicts a protein with an N-terminal truncation of the DNA-binding domain. As shown by yeast two-hybrid assays, hHIF-1alphaTe still formed heterodimeric complexes with HIF-1beta. However, hHIF-1alphaTe was incapable of forming a DNAbinding HIF-1 complex. Overexpression of exogenous hHIF-1alphaTe resulted in the inhibition of the endogenous HIF-1 transcriptional activity, demonstrating that the testis-specific hHIF-1alphaTe isoform is a dominant-negative regulator of normal HIF-1 activity.