129 Activation of membrane progesterone receptors induces glycogenolysis in uterine epithelial cells

Glycogen metabolism in the uterine epithelium is critical for embryo survival. Abnormal glucose concentrations in uterine fluid is a major cause of embryonic death, and glycogen serves as a buffer to regulate glucose concentrations. The demand for glucose increases dramatically once the embryo enters the uterus at the morula stage. In a previous publication, we showed the glycogen content of the bovine uterine epithelium was lower during the luteal phase (Day 11) than near oestrus (Day 1). In the current study, our objective was to elucidate the role of progesterone in glycogen breakdown in bovine uterine epithelial (BUTE) cells. The BUTE cells were treated with insulin-like growth factor 1 (IGF1) to stimulate glycogenesis for 24 h, followed by experimental treatment for 48 h. Statistical analysis was performed with a t-test or a one-way ANOVA for experiments with more than two treatments. Progesterone decreased glycogen levels in BUTE cells by 99% (P = 0.0002). RU486, which antagonizes the nuclear progesterone receptor (nPR), did not block progesterone’s effect. Therefore, we hypothesised that progesterone was acting through the membrane progesterone receptors (mPRs). The reverse-transcription polymerase chain reaction confirmed that BUTE cells expressed all five mPRs (α, β, γ, δ, and ε). Like progesterone, a specific mPR agonist (Org OD 02–0) reduced glycogen levels in BUTE cells by 94% (P < 0.0001). Indicating a potential for mPRs to regulate glycogen in vivo, immunohistochemistry showed that the bovine uterine epithelium expressed high levels of mPRα during oestrus and the luteal phase. These results suggest that progesterone acts through mPRs to stimulate glycogenolysis in BUTE cells. Next, we searched current literature and determined that mPRs can mediate effects via cAMP. However, we found that neither progesterone nor Org OD 02–0 changed intracellular cAMP concentrations. In agreement, the adenylyl cyclase activator forskolin increased cAMP concentration (P = 0.0005) but did not significantly decrease glycogen levels. The mPRs can also activate AMPK, a master regulator of metabolism. We found that progesterone increased in phospho-AMPK levels by 18% at 24 h compared to the control (P = 0.0001). Supporting these results in vivo, phospho-AMPK levels in the uterine epithelium were high in the bovine uterine epithelium during the luteal phase when glycogen levels were low. However, the AMPK activator (A-769662) did not reduce glycogen in BUTE cells. And dorsomorphin, an AMPK inhibitor, did not block the effect of progesterone on glycogen breakdown. While BUTE cells treated with D942, which increases intracellular AMP concentrations, had a 92% decrease in glycogen levels compared to the control (P = 0.0051). AMP allosterically activates glycogen phosphorylase, which would directly decrease glycogen levels. Human Ishikawa cells express mPRs but not nPRs. In these cells, progesterone and Org OD 02–0 also decreased glycogen levels (P = 0.0027 and P = 0.0076, respectively). In conclusion, progesterone acts through the mPR to stimulate glycogenolysis, likely through direct effects of AMP on glycogen phosphorylase, which plays a crucial role in providing glucose to endometrial tissue or the growing embryo.