Hypoxia increases glucose transporter 1 expression in bovine corpus luteum at the early luteal stage

Abstract

A major role of the corpus luteum (CL) is to produce progesterone (P4). The CL has immature vasculature shortly after ovulation, suggesting it exists under hypoxic conditions. Hypoxia-inducible factor-1 (HIF1) induces the expression of glucose transporter 1 (GLUT1). To clarify the physiological roles of GLUT1 in bovine CL, we examined GLUT1 mRNA expression in the CL under hypoxic conditions by quantitative RT-PCR. We also measured the effects of glucose (0-25 mM) and GLUT1 inhibitors (cytochalasin B, STF-31) on P4 production in bovine luteal cells. GLUT1 mRNA expression in bovine CL was higher at the early luteal stage compared to the other later stages. Hypoxia (3% O₂) increased GLUT1 mRNA expression in early luteal cells, but not in mid luteal cells. Glucose (0-25 mM) increased P4 production in early luteal cells, but not in mid luteal cells. Both GLUT1 inhibitors decreased P4 production in early and mid luteal cells. Overall, the results suggest that GLUT1 (possibly induced by hypoxic conditions in the early CL) plays a role in the establishment and development of bovine CL, especially in supporting luteal P4 synthesis at the early luteal stage.

Keywords: HIF1; corpus luteum; glucose transporter-1; hypoxia; progesterone.

Fig. 1.

Changes in the relative amounts of GLUT1 mRNA in the bovine CL throughout the estrous cycle (E: early, Days 2–3; D: developing, Days 5–6; M: mid, Days 8–12; L: late, Days 15–17; R: regressed luteal stages, Days 19–21). Data are the means ± SEM for 4 samples/stage and are expressed as relative ratios of GLUT1 mRNA to 18S rRNA. Different letters indicate significant differences (P<0.05), as determined by ANOVA followed by a multiple comparison with Bonferroni correction.

Fig. 2.

Effect of hypoxia on GLUT1 mRNA expression in cultured bovine early luteal cells (A) and mid luteal cells (B). Cells were cultured under 20% O₂ (normoxia) or 3% O₂ (hypoxia) for 24 hr. The amount of GLUT1 mRNA is expressed relative to the amount of 18S rRNA (early: n=5, mid: n=5). An asterisk indicates a significant difference between oxygen tension (P<0.05), as determined by the Student’s t-test.

Fig. 3.

Dose-dependent effects of glucose on P4 production by cultured bovine early luteal cells (A) and mid luteal cells (B). The cells were cultured in glucose-free medium without or with glucose (0.25, 2.5 and 25 mM) for 24 hr (early: n=3, mid: n=3). All values are represented as a percentage of the control (none) value (mean ± SEM). The concentration of P4 in the early and mid luteal control cells was 193 ± 25.7 ng/ml and 613 ± 36.7 ng/ml, respectively. Asterisks indicate significant differences compared with none (*P<0.05, **P<0.01), as determined by ANOVA followed by a multiple comparison with Bonferroni correction.

Fig. 4.

Effect of GLUT1 inhibitors (cytochalasin B [CytoB], STF-31 [STF]) on P4 production by cultured bovine early luteal cells (A) and mid luteal cells (B). Cells were cultured in medium containing 5% calf serum without or with GLUT1 inhibitor (cytochalasin B; 10 µM, STF-31; 10 µM) in combination without or with hCG stimulation (1 U/ml) for 24 hr (early: n=6, mid: n=5). All values are represented as a percentage of the control (none without hCG) value (mean ± SEM). The concentration of P4 in the controls for early and mid luteal cells was 238 ± 19.5 ng/ml and 674 ± 58.0 ng/ml, respectively. Asterisks indicate significant differences (P<0.05), as determined by ANOVA followed by a multiple comparison with Bonferroni correction.