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

Abstract

Hypoxia-inducible factors (HIF) are transcription factors that serve essential regulatory roles in cellular and molecular responses to oxygen debt. HIFs are composed of hypoxiadependent a subunits (1a, 2a, 3a) and an oxygen-independent b 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-2a and HIF-3a are expressed in rat testis, identify testis cell types that express HIF-1a, and examine patterns of testicular HIF-1a protein expression under conditions of ischemia and hypoxia in vivo and in vitro. Reverse transcriptase polymerase chain reaction revealed that mRNA for Hif-1a, Hif-2a, and Hif-3a is expressed in the testis. The HIF-1a protein is the predominant subunit in testis. HIF-1a 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-1a 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 H 2 O 2 , were analyzed by immunoblotting. Levels of HIF1a were significantly diminished in 5% or 21% oxygen cultures compared with freshly isolated cells. Treating Leydig cells with H 2 O 2 as a source of reactive oxygen species did not affect HIF-1a levels. High levels of constitutively expressed HIF-1a in normoxic Leydig cells suggest potentially unique roles for HIF-1 in Leydig cell responsiveness to oxygen.

Myeloid cell leukemia-1 (Mc1-1) is a candidate target gene of hypoxia-inducible factor-1 (HIF-1) in the testis

“…Apoptosis of germ cells in the testes following I and I/R was evaluated via terminal deoxynucleotidyltransferase mediated dUTP-biotin nick end labelling (TUNEL) assays to validate cell damage. These data were previously reported in Supplemental Data of Palladino et al [12] and demonstrated an increase in germ cell-specific apoptosis following I and I/R, but no statistically significant changes in apoptosis of Leydig cells after I and I/R.…”

“…We previously demonstrated [12] that unlike many other tissues in which HIF-1 accumulates only after hypoxia, HIF-1 is abundant in normoxic testes. This result suggested potentially novel roles for HIF-1 in the testis.…”

“…We hypothesize that HIF-1 plays important roles in mediating antiapoptotic responses in the testis and may protect Leydig cells from apoptosis. Previously we demonstrated that HIF-1α mRNA and protein is abundantly expressed by Leydig cells in the rat testis [12,13]. These studies revealed that, unlike many tissues in which HIF-1α expression is induced by hypoxia, HIF-1 is constitutively expressed in the normoxic testis and the levels of testicular HIF-1 are unaffected following ischemia and hypoxia caused by testicular torsion and reperfusion [12].…”

Development of resistance to antiglioma agents in rat C6 cells caused collateral sensitivity to doxorubicin

“…PCR reactions were performed with primers specific for Bax, Bad, Bcl-2 [15], Bcl-xL [16], caspase 3 [17], HIF-1α [18], MnSOD [19] and iNOS [20]. β-actin [21], gapdh [22] and p1B15 [23] were used as an internal control and were coamplified with the gene of interest in all PCR reactions.…”

Erdosteine protects rat testis tissue from hypoxic injury by reducing apoptotic cell death

“…HIF-1a, the predominant subunit in the testis, may mediate proapoptotic or antiapoptotic responses in the ischaemic testis depending on the duration and extent of ischaemia and hypoxia (Powell et al, 2002;Palladino et al, 2011).…”