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Anthony D Horlock, Thomas J R Ormsby, Martin J D Clift, José E P Santos, John J Bromfield, and I Martin Sheldon

Bovine granulosa cells are often exposed to energy stress, due to the energy demands of lactation, and exposed to lipopolysaccharide from postpartum bacterial infections. Granulosa cells mount innate immune responses to lipopolysaccharide, including the phosphorylation of mitogen-activated protein kinases and production of pro-inflammatory interleukins. Cellular energy depends on glycolysis, and energy stress activates intracellular AMPK (AMP-activated protein kinase), which in turn inhibits mTOR (mechanistic target of rapamycin). Here, we tested the hypothesis that manipulating glycolysis, AMPK or mTOR to mimic energy stress in bovine granulosa cells limits the inflammatory responses to lipopolysaccharide. We inhibited glycolysis, activated AMPK or inhibited mTOR in granulosa cells isolated from 4–8mm and from > 8.5 mm diameter ovarian follicles, and then challenged the cells with lipopolysaccharide and measured the production of interleukins IL-1α, IL-1β, and IL-8. We found that inhibiting glycolysis with 2-deoxy-d-glucose reduced lipopolysaccharide-stimulated IL-1α > 80%, IL-1β > 90%, and IL-8 > 65% in granulosa cells from 4–8 mm and from > 8.5 mm diameter ovarian follicles. Activating AMPK with AICAR also reduced lipopolysaccharide-stimulated IL-1α > 60%, IL-1β > 75%, and IL-8 > 20%, and shortened the duration of lipopolysaccharide-stimulated phosphorylation of the mitogen-activated protein kinase ERK1/2 and JNK. However, only the mTOR inhibitor Torin 1, and not rapamycin, reduced lipopolysaccharide-stimulated IL-1α and IL-1β. In conclusion, manipulating granulosa cell energy metabolism with a glycolysis inhibitor, an AMPK activator, or an mTOR inhibitor, limited inflammatory responses to lipopolysaccharide. Our findings imply that energy stress compromises ovarian follicle immune defences.

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Anthony D Horlock, Thomas J R Ormsby, Martin J D Clift, José E P Santos, John J Bromfield, and I Martin Sheldon

In brief

Bovine granulosa cells need to be cultured with serum to generate inflammation in response to bacterial lipopolysaccharide. This study shows that it is cholesterol that facilitates this lipopolysaccharide-stimulated cytokine secretion.


During bacterial infections of the bovine uterus or mammary gland, ovarian granulosa cells mount inflammatory responses to lipopolysaccharide (LPS). In vitro, LPS stimulates granulosa cell secretion of the cytokines IL-1α and IL-1β and the chemokine IL-8. These LPS-stimulated inflammatory responses depend on culturing granulosa cells with serum, but the mechanism is unclear. Here, we tested the hypothesis that cholesterol supports inflammatory responses to LPS in bovine granulosa cells. We used granulosa cells isolated from 4 to 8 mm and >8.5 mm diameter ovarian follicles and manipulated the availability of cholesterol. We found that serum or follicular fluid containing cholesterol increased LPS-stimulated secretion of IL-1α and IL-1β from granulosa cells. Conversely, depleting cholesterol using methyl-β-cyclodextrin diminished LPS-stimulated secretion of IL-1α, IL-1β and IL-8 from granulosa cells cultured in serum. Follicular fluid contained more high-density lipoprotein cholesterol than low-density lipoprotein cholesterol, and granulosa cells expressed the receptor for high-density lipoprotein, scavenger receptor class B member 1 (SCARB1). Furthermore, culturing granulosa cells with high-density lipoprotein cholesterol, but not low-density lipoprotein or very low-density lipoprotein cholesterol, increased LPS-stimulated inflammation in granulosa cells. Cholesterol biosynthesis also played a role in granulosa cell inflammation because RNAi of mevalonate pathway enzymes inhibited LPS-stimulated inflammation. Finally, treatment with follicle-stimulating hormone, but not luteinising hormone, increased LPS-stimulated granulosa cell inflammation, and follicle-stimulating hormone increased SCARB1 protein. However, changes in inflammation were not associated with changes in oestradiol or progesterone secretion. Taken together, these findings imply that cholesterol supports inflammatory responses to LPS in granulosa cells.