A Dominant-Negative Isoform of Hypoxia-Inducible Factor-1α Specifically Expressed in Human Testis1

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Abstract
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)-1α in the mouse, termed mHIF-1αI.1. Here, we demonstrate that expression of mHIF-1αI.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-1α isoform in the human testis, called hHIF-1αTe. Like mHIF-1αI.1, hHIF-1αTe 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-1αTe is exclusively expressed in the testis. As determined by immunofluorescence of ejaculated sperm cells, HIF-1α 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-1αTe nor hHIF-1α associated with mitochondria. In contrast with the ubiquitously expressed HIF-1α protein and the mouse testis-specific mHIF-1αI.1 isoform, the hHIF-1αTe mRNA sequence predicts a protein with an N-terminal truncation of the DNA-binding domain. As shown by yeast two-hybrid assays, hHIF-1αTe still formed heterodimeric complexes with HIF-1β. However, hHIF-1αTe was incapable of forming a DNA-binding HIF-1 complex. Overexpression of exogenous hHIF-1αTe resulted in the inhibition of the endogenous HIF-1 transcriptional activity, demonstrating that the testis-specific hHIF-1αTe isoform is a dominant-negative regulator of normal HIF-1 activity.