Normoxic Expression of Hypoxia-Inducible Factor 1 in Rat Leydig Cells In Vivo and In Vitro

Hypoxia-inducible factors (HIF) are transcription factors that serve essential regulatory roles in cellular and molecular responses to oxygen debt. HIFs are composed of hypoxia-dependent alpha subunits (1 alpha, 2 alpha, 3 alpha) and an oxygen-independent beta subunit. Previously we demonstrated that HIF-1, the master regulator of hypoxic responses, is expressed in the adult rat testis. We hypothesized that HIF-1 is involved in regulating responses to oxygen tension in the testis. Goals of this study were to determine if HIF-2 alpha and HIF-3 alpha are expressed in rat testis, identify testis cell types that express HIF-1 alpha, and examine patterns of testicular HIF-1 alpha protein expression under conditions of ischemia and hypoxia in vivo and in vitro. Reverse transcriptase polymerase chain reaction revealed that mRNA for Hif-1 alpha, Hif-2 alpha, and Hif-3 alpha is expressed in the testis. The HIF-1 alpha protein is the predominant subunit in testis. HIF-1 alpha protein was abundant in normoxic testis, and its levels remained unchanged following ischemia created by surgically induced testicular torsion and reperfusion. Immunoblot and immunocytochemical experiments demonstrated that Leydig cells are the major source of HIF-1 alpha in normoxic and hypoxic testes. To examine potential mechanisms of testicular HIF-1 stabilization, nuclear proteins from Leydig cells cultured in 5% or 21% oxygen, or cells cultured with H2O2, were analyzed by immunoblotting. Levels of HIF-1 alpha a were significantly diminished in 5% or 21% oxygen cultures compared with freshly isolated cells. Treating Leydig cells with H2O2 as a source of reactive oxygen species did not affect HIF-1 alpha levels. High levels of constitutively expressed HIF-1 alpha in normoxic Leydig cells suggest potentially unique roles for HIF-1 in Leydig cell responsiveness to oxygen.