Date of Award

Summer 2008

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biological Sciences

Program/Concentration

Biomedical Sciences

Committee Director

Diane M. Duffy

Committee Member

Peter F. Blackmore

Committee Member

Anca D. Dobrian

Committee Member

Gustavo F. Doncel

Abstract

Elevated follicular prostaglandin E2 (PGE2) levels are necessary for follicle rupture during ovulation. As the degradation of the follicle wall requires proteolysis, it was hypothesized that regulation of proteolysis by gonadotropins is PGE2 mediated.

There are four PGE2 receptors: EP1, EP2, EP3 and EP4. To determine which PGE2 receptors are present in monkey periovulatory follicles, granulosa cells and whole ovaries were collected 0, 12, 24, and 36 hours after administration of an ovulatory dose of human chorionic gonadotropin (hCG), or 36 hours after hCG from monkeys that received the prostaglandin (PG) synthesis inhibitor celecoxib. All EP receptor mRNAs were present in granulosa cells. By immunofluorescence, EP1 was detectable only 36 hours after hCG. EP2 and EP3 proteins were present throughout the periovulatory interval. By western blotting, EP2 and EP3 proteins increased between 0 and 36 hours after hCG. EP4 protein was not detected in periovulatory granulosa cells. An increase in EP2 and EP3 receptor responsiveness was confirmed by measuring elevated cAMP levels in response to receptor selective agonists. EP1 receptor function, measured as an increase in intracellular calcium, was present only 36 hours after hCG administration.

To elucidate the role of the ovulatory gonadotropin surge and PGs in the regulation of periovulatory proteolytic events, the expression of mRNA and protein for the tissue type plasminogen activator (tPA) and urokinase type plasminogen activator (uPA) and their inhibitor PAI-1 was measured in granulosa cells. uPA and PAI-1 mRNAs peaked 12 hours and 36 hours after hCG administration respectively. tPA mRNA decreased 36 hours after hCG. Celecoxib administration had no effect on any of these mRNAs. By immunofluorescence, tPA and PAI-1 protein expression increased 12 hours after hCG and persisted until 36 hours after hCG. tPA and PAI-1 proteins were not detectable 36 hours after hCG in celecoxib treated animals. uPA was not detectable in granulosa cells before and after hCG administration. By in situ zymography, plasminogen activator (PA) activity was low before hCG administration and detectable 12, 24 and 36 hours after hCG. PA activity was attenuated by pre-incubation with an anti-tPA antibody. Animals treated for 36 hours with hCG+celecoxib had low ovarian PA activity. To determine whether PGE2 is the key PG in the regulation of PA family members, 0 hour granulosa cells were hCG treated with or without PGE2 or EP receptor selective agonists. tPA protein increased only in the presence of PGE2, the EP2 agonist or the EP3 agonist. PAI-1 protein was detected in cells cultured with PGE2, the EP1 agonist or the EP3 agonist.

These studies demonstrate that the ovulatory gonadotropin stimulates granulosa cell EP receptor expression and responsiveness. PGE2, acting through EP2 and EP3 receptors, may mediate the hCG induced increase in tPA proteolytic activity.

Taken together, this study elucidates the mechanisms by which the ovulatory gonadotropin surge and PGE2 cooperate to regulate proteolysis in the periovulatory follicle. This information may lead to improved treatments for anovulation or to the development of new contraceptive options.

DOI

10.25777/kt2y-bs69

ISBN

9780549792918

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