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 α subunits (1α, 2α, 3α) and an oxygen-independent β 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-2α and HIF-3α are expressed in rat testis, identify testis cell types that express HIF-1α, and examine patterns of testicular HIF-1α protein expression under conditions of ischemia and hypoxia in vivo and in vitro. Reverse transcriptase polymerase chain reaction revealed that mRNA for Hif-1α, Hif-2α, and Hif-3α is expressed in the testis. The HIF-1α protein is the predominant subunit in testis. HIF-1α 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-1α 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-1α 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-1α levels. High levels of constitutively expressed HIF-1α in normoxic Leydig cells suggest potentially unique roles for HIF-1 in Leydig cell responsiveness to oxygen.
School of Science, Department of Biology, Monmouth University, 400 Cedar Avenue, West Long Branch, NJ 07764, USA. mpalladi@monmouth.edu
M A
MA
Palladino
P R
PR
Pirlamarla
J
J
McNamara
C M
CM
Sottas
N
N
Korah
M P
MP
Hardy
D B
DB
Hales
L
L
Hermo
D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D051793:Hypoxia-Inducible Factor 1 / Q000235:genetics / Q000378:metabolism*; D047468:Immunoprecipitation; D066298:In Vitro Techniques; D007985:Leydig Cells / Q000378:metabolism*; D008297:Male; D012333:RNA, Messenger / Q000235:genetics; D051381:Rats; D017207:Rats, Sprague-Dawley; D020133:Reverse Transcriptase Polymerase Chain Reaction
D017931:DNA Primers; D051793:Hypoxia-Inducible Factor 1; D012333:RNA, Messenger