304. Murine HIF-1α localisation by immunohistochemistry in a mouse reproductive tissue

Abstract

Hypoxia-inducible factors (HIFs) are heterodimeric transcription factors consisting of an α and β subunit. The level of O₂ within cells regulates the stability of HIF-1α and HIF-1 is considered the primary mediator of cellular responses to hypoxia, helping restore O₂ homeostasis by promoting the expression of hypoxia-sensitive genes involved in cell survival, angiogenesis, cell proliferation and metabolism.

There are few published studies investigating the role of HIF-1 protein in mouse tissues using immunohistochemistry, due to the lack of a reliable protocol and the inability of many commercially available antibodies to detect murine HIF-1α protein. We have developed a protocol that has allowed us to analyse the presence and location of HIF-1α protein in the mouse epididymis and found that it was predominantly located in the nucleus of discrete principal cells in the epididymis of mice housed under normoxic conditions and sacrificed by cervical dislocation. Interestingly, a 2.5× increase (P < 0.05) of HIF-1α protein staining intensity in both the nucleus and cytoplasm of principal cells in the epididymis was detected in mice housed under normoxic conditions but sacrificed with CO₂ gas, compared to mice sacrificed by cervical dislocation. HIF-1α protein detection was 3-fold increased in the nucleus and cytoplasm of principal cells when mice were exposed to hypoxic conditions (6% O₂ for 1 h). Our results demonstrate that murine HIF-1α can be detected in discrete cells under normoxic conditions, suggesting local differences in O₂. Acute hypoxic responses, via deliberate exposure or even CO₂ euthanasia can significantly upregulate HIF-1α protein levels. Further studies will investigate the role of HIFs and hypoxia in male and female reproductive function.