The glycogenolytic enzyme acid α-glucosidase is expressed in the bovine uterine endometrium.

Progesterone has been shown to stimulate glycogen catabolism in uterine epithelial cells. Acid α-glucosidase (GAA) is an enzyme that breaks down glycogen within lysosomes. We hypothesized that progesterone may stimulate glycogenolysis in the uterine epithelium via GAA. We found that GAA was more highly expressed in the stroma on Day 1 than on Day 11. However, GAA did not appear to differ in the epithelium on Days 1 and 11. Progesterone (0-10 µM) had no effect on the levels of the full-length inactive protein (110 kDa) or the cleaved (active) peptides present inside the lysosome (70 and 76 kDa) in immortalized bovine uterine epithelial (BUTE) cells. Furthermore, the activity of GAA did not differ between the BUTE cells treated with 10 µM progesterone or control. Overall, we confirmed that GAA is present in the cow endometrium and BUTE cells. However, progesterone did not affect protein levels or enzyme activity.

Effect of estradiol and IGF1 on glycogen synthesis in bovine uterine epithelial cells.

In brief: Glucose is an important nutrient for the endometrium and embryo during pregnancy. This study shows that estradiol (E2)/IGF1 signaling stimulates glycogen synthesis in the uterine epithelium of cows, which could provide glucose when needed. Abstract: Glycogen storage in the uterine epithelium peaks near estrus and is a potential source of glucose for the endometrium and embryos. However, the hormonal regulation of glycogen synthesis in the uterine epithelium is poorly understood. Our objective was to evaluate the effect of E2 and insulin-like growth factor 1 (IGF1) on glycogenesis in immortalized bovine uterine epithelial (BUTE) cells. Treatment of BUTE cells with E2 (0.1-10 nM) did not increase glycogen levels. However, treatment of BUTE cells with IGF1 (50 or 100 ng/mL) resulted in a >2-fold increase in glycogen. To determine if the uterine stroma produced IGF1 in response to E2, bovine uterine fibroblasts were treated with E2, which increased IGF1 levels. Immunohistochemistry showed higher levels of IGF1 in the stroma on day 1 than on day 11, which coincides with higher glycogen levels in the uterine epithelium. Western blots revealed that IGF1 treatment increased the levels of phospho-AKT, phospho-GSKß, hexokinase 1, and glycogen synthase in BUTE cells. Metabolomic (GC-MS) analysis showed that IGF1 increased 3-phosphoglycerate and lactate, potentially indicative of increased flux through glycolysis. We also found higher levels of N-acetyl-glucosamine and protein glycosylation after IGF1 treatment, indicating increased hexosamine biosynthetic pathway activity. In conclusion, IGF1 is produced by uterine fibroblasts due to E2, and IGF1 increases glucose metabolism and glycogenesis in uterine epithelial cells. Glycogen stored in the uterine epithelium due to E2/IGF1 signaling at estrus could provide glucose to the endometrium or be secreted into the uterine lumen as a component of histotroph.

Endometrial glycogen metabolism on days 1 and 11 of the reproductive cycle in dairy cows.

Embryos need glucose or fructose to remain viable; however, it is not well understood how secretion of these carbohydrates is regulated. This study was conducted to evaluate endometrial glycogen and associated enzymes on Days 1 and 11 of the estrous cycle (Day 0 = behavioral estrus) in cattle. Diastase-liable periodic acid-Schiff (PAS) staining of luminal epithelia decreased 81 % between Days 1 and 11. Similarly, glycogen content of glandular epithelia was 66 % less on Day 11 than Day 1. There was dense PAS staining in the lumen of some glands, and this staining was removed when there was pretreatment with diastase. Based on western blot results, there was no difference in glycogen metabolizing enzymes between Days 1 and 11. Results from conducting immunohistochemistry procedures indicated hexokinase 1 was more abundant in the epithelial cells than stroma, but immunostaining was not different between Day 1 and 11. In contrast, phospho-glycogen synthase was undetectable on Day 1 but was present in glandular epithelia on Day 11. Glycogen synthase was localized to the epithelia, and was in larger abundance on Day 1. The abundance of glycogen phosphorylase was greater in the epithelium than stroma and on Day 11 than 1. Furthermore, glucose-6-phosphatase 3 was more abundant in the epithelium on both Days 1 and 11. In conclusion, in the uterus of cattle glycogen is stored in a reproductive cycle-dependent manner. Glucose released from endometrial glycogen stores could potentially be utilized by the endometrium or secreted into the uterine lumen.

Paraben exposure alters cell cycle progression and survival of spontaneously immortalized secretory murine oviductal epithelial (MOE) cells.

The mammalian oviduct is a central organ for female reproduction as it is the site of fertilization and it actively transports the embryo to the uterus. The oviduct is responsive to ovarian steroids and thus, it is a potential target of endocrine disrupting chemicals. Parabens are antimicrobial compounds that are prevalently found in daily-used products. However, recent studies suggest that some parabens can impact female reproductive health. Yet, their effects on the oviduct are unknown. Here, we hypothesized that in vitro exposure of immortalized murine oviductal secretory epithelial (MOE) cells to methylparaben or propylparaben will result in disrupted cell cycle progression and increased cell death by dysregulation of molecular mechanisms that involve the cell cycle and apoptosis. Thus, we examined the effects of exposure to parabens on cell proliferation, cell cycle progression by flow cytometry, and mRNA levels of major cell cycle regulators and apoptotic factors, in MOE cells. Protein levels of estrogen and progesterone receptors were also quantified. Differences between treatments and controls were analyzed by linear mixed model followed by Dunnett post-hoc tests. The results indicate that methylparaben and propylparaben selectively reduce MOE cellular proliferation and colony numbers, compared to controls. Additionally, paraben exposure selectively dysregulates the progression through the cell cycle and decreases the levels of cell cycle regulators, compared to controls. Last, paraben selectively alters the levels of progesterone receptor. Overall, these findings suggest that parabens can affect mouse oviductal secretory epithelial cell proliferation and survival.