Hypoxia-Inducible Factor-1α Is Constitutively Expressed in Murine Leydig Cells and Regulates 3β-Hydroxysteroid Dehydrogenase Type 1 Promoter Activity

Hypoxia-inducible factor-1 alpha (HIF-1 alpha) is a transcription factor that plays an essential role in oxygen homeostasis. HIF-1 alpha is constitutively made in cells; however, it is ubiquitinated and degraded under normoxic conditions. Hypoxia prevents the ubiquitination of HIF-1 alpha, resulting in stabilization of the protein and activation of target genes. Because of its vascular arrangement and the high metabolic demand of spermatogenesis, the testis has been described previously as functioning on the brink of hypoxia; thus, we have hypothesized that HIF-1 alpha is constitutively expressed and stabilized in the testis, where it could play a role in testicular homeostasis. Western blot analysis using nuclear proteins from liver, kidney, and testis revealed the presence of HIF-1 alpha only in the testis. Immunohistochemistry confirmed this result and revealed that HIF-1 alpha was specifically located in interstitial Leydig cells. Electromobility shift assays employing nuclear extracts from the TM3 Leydig cell line revealed that these cells express HIF-1 alpha that is capable of binding DNA under normoxic conditions. Furthermore, we found that protein levels can be increased further when the TM3 cells are cultured under hypoxic conditions. Finally, transient transfections of TM3 Leydig cells revealed that the promoter of the mouse 3 beta-hydroxysteroid dehydrogenase type 1 (Hsd3b1) gene, which encodes a key enzyme in testosterone production, is a potential target of HIF-1 alpha. In conclusion, HIF-1 alpha is constitutively present in the Leydig cells of the murine testis, where it potentially regulates Hsd3b1 transcription, and thus male reproductive function.