Immunohistochemical assessment of intrinsic and extrinsic markers of hypoxia in reproductive tissue: differential expression of HIF1α and HIF2α in rat oviduct and endometrium

Abstract

Hypoxia is thought to be critical in regulating physiological processes within the female reproductive system, including ovulation, composition of the fluid in the oviductal/uterine lumens and ovarian follicle development. This study examined the localisation of exogenous (pimonidazole) and endogenous [hypoxia inducible factor 1α and 2α (HIF1α, -2α), glucose transporter type 1 (GLUT1) and carbonic anhydrase 9 (CAIX)] hypoxia-related antigens within the oviduct and uterus of the rat reproductive tract. The extent to which each endogenous antigen co-compartmentalised with pimonidazole was also assessed. Female Wistar Furth rats (n = 10) were injected intraperitoneally with pimonidazole (60 mg/kg) 1 h prior to death. Reproductive tissues were removed immediately following death and fixed in 4% paraformaldehyde before being embedded in paraffin. Serial sections were cut (6-7 μm thick) and antigens of interest identified using standard immunohistochemical procedures. The mucosal epithelia of the ampulla, isthmus and uterus were immunopositive for pimonidazole in most sections. Co-compartmentalisation of pimonidazole with HIF1α was only expressed in the mucosa of the uterus whilst co-compartmentalisation with HIF2α was observed in the mucosa of the ampulla, isthmus and uterus. Both GLUT1 and CAIX were co-compartmentalised with pimonidazole in mucosa of the isthmus and uterus. This study confirms that mucosal regions of the rat oviduct and uterus frequently experience severe hypoxia and there are compartment specific variations in expression of endogenous hypoxia-related antigens, including the HIF isoforms. The latter observation may relate to target gene specificity of HIF isoforms or perhaps HIF2α's responsiveness to non-hypoxic stimuli such as hypoglycaemia independently of HIF1α.

Fig. 1

a–f Sections taken from a representative rat showing the results for immunohistochemical staining in the ampulla region of the oviduct for pimonidazole, HIF1α, HIF2α, GLUT1, GLUT4 and CAIX, respectively. The boxed diagrams in a–f show a higher magnification detail for each stain. Scale bars 100 μm (a–f) or 30 μm (detail pictures in a–f)

Fig. 2

a–f Sections taken from a representative rat showing the results for immunohistochemical staining in the isthmus region of the oviduct for pimonidazole, HIF1α, HIF2α, GLUT1, GLUT4 and CAIX, respectively. The boxed diagrams in a–f show a higher magnification detail for each stain. Scale bars 100 μm (a–f) or 30 μm (detail pictures in a–f)

Fig. 3

a–f Sections taken from a representative rat showing the results for immunohistochemical staining in the uterus for pimonidazole, HIF1α, HIF2α, GLUT1, GLUT4 and CAIX, respectively. The boxed diagrams in a–f show a higher magnification detail for each stain. Scale bars 100 μm (a–f) or 30 μm (detail in a–f)

Fig. 4

a–f Sections taken from a representative rat showing examples of immunohistochemical staining in glandular epithelia for pimonidazole, HIF1α, HIF2α, GLUT1, GLUT4 and CAIX, respectively. Scale bar 50 μm

Fig. 5

Charts showing the staining intensity of the 6 hypoxia-related antigens in the mucosa of a the ampulla, b the isthmus and c the endometrium as a percentage of the total number of sections examined (n = 20). (+) denotes strong-very strong positive staining, (±) denotes weak/moderate or sporadic staining and (−) denotes an absence of staining. Charts d–f show the proportions of co-compartmentalisation of the 5 endogenous hypoxia-related antigens with pimonidazole in the mucosa of the ampulla, isthmus and endometrium, respectively. Percentages are relative to the total number of sections examined (n = 20). (P+/A+) denotes the presence of both pimonidazole and the respective endogenous antigen within the same compartment in a section: (P+/A−) is as before, but with the absence of the respective antigen; (P−/A+) is as for the 1st criterion, but with the absence of pimonidazole and (P−/A−) denotes that both pimonidazole and the respective antigen were absent from the same compartment. X axis labels: P pimonidazole, H1α HIF1α, H2α HIF2α, G1 GLUT1 and G4 GLUT4

Fig. 6

Charts showing the staining intensity of the 6 hypoxia-related antigens in the lamina propria of a the ampulla, b the isthmus and c the endometrium as a percentage of the total number of sections examined (n = 20). (+) denotes strong-very strong positive staining, (±) denotes weak/moderate or sporadic staining and (−) denotes an absence of staining. Charts d–f show the proportions of co-compartmentalisation of the 5 endogenous hypoxia-related antigens with pimonidazole in the lamina propria of the ampulla, isthmus and endometrium, respectively. Percentages are relative to the total number of sections examined (n = 20). (P+/A+) denotes the presence of both pimonidazole and the respective endogenous antigen within the same compartment in a section: (P+/A−) is as before, but with the absence of the respective antigen; (P−/A+) is as for the 1st criterion, but with the absence of pimonidazole and (P−/A−) denotes that both pimonidazole and the respective antigen were absent from the same compartment. X axis labels: P pimonidazole, H1α HIF1α, H2α HIF2α, G1 GLUT1 and G4 GLUT4

Fig. 7

Charts showing the staining intensity of the 6 hypoxia-related antigens in the muscularis/myometrium of a the ampulla, b the isthmus and c the endometrium as a percentage of the total number of sections examined (n = 20). (+) denotes strong-very strong positive staining, (±) denotes weak/moderate or sporadic staining and (−) denotes an absence of staining. Charts d–f show the proportions of co-compartmentalisation of the 5 endogenous hypoxia-related antigens with pimonidazole in the muscularis/myometrium of the ampulla, isthmus and endometrium, respectively. Percentages are relative to the total number of sections examined (n = 20). (P+/A+) denotes the presence of both pimonidazole and the respective endogenous antigen within the same compartment in a section: (P+/A−) is as before, but with the absence of the respective antigen; (P−/A+) is as for the 1st criterion, but with the absence of pimonidazole and (P−/A−) denotes that both pimonidazole and the respective antigen were absent from the same compartment. X axis labels: P pimonidazole, H1α HIF1α, H2α HIF2α, G1 GLUT1 and G4 GLUT4