Date of Graduation

2015

Document Type

Dissertation

Degree Type

PhD

College

Davis College of Agriculture, Natural Resources and Design

Department

Animal and Nutritional Sciences

Committee Chair

Jorge A Flores

Committee Co-Chair

Melanie J Clemmer

Committee Member

Robert A Dailey

Committee Member

Emmett K Inskeep

Committee Member

Michael W Vernon

Committee Member

Jianbo Yao

Abstract

Prostaglandin F2 alpha (PGF2&agr;) has in vivo luteolytic actions on the bovine corpus luteum (CL). Although PGF2&agr; is utilized extensively in livestock to synchronize estrus, its actions in vitro are controversial and do not always result in a reduction in progesterone (P4) secretion. The mechanism of action of PGF2&agr; is thought to involve activation of the phospholipase C-Ca2+ pathway. The luteolytic actions of PGF2&agr; are mediated through an elevation of cytosolic Ca2+ involving activation of protein kinase C (PKC). Expression of PKC isozymes and calcium/calmodulin-dependent protein kinase kinase 2 (CAMKK2) change as the CL develops to a mature stage. Either CAMKK2 directly affects steroidogenesis or works indirectly through an intermediary step, such as adenosine- monophosphate kinase (AMPK), which is activated through CAMKK2 targeted phosphorylation. Specifically, the hypothesis that activation of PGF2&agr; receptor (FP) in the mature CL involves AMPK was examined. Expression of mRNA encoding a potential target AMPK for the action of CAMKK2, was increased in the mature versus developing CL. Furthermore, activation of FP induced rapid phosphorylation of AMPK. An AMPK-specific inhibitor, dorsomorphin dichloride (DM), eliminated effects of PGF2&agr; on secretion of P4, supporting the hypothesis that activation of the FP receptor in mature CL involves participation of AMPK. Furthermore, in vitro two AMPK activators, 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) and metformin, decreased basal P4 secretion in mature but not in developing CL. Effect of FP activation on cholesterol transport in the mature CL was investigated. Both serum and luteal P4 declined as soon as 2 hours after PGF 2&agr; administration, which was paralleled by a decrease in protein concentration of low density lipoprotein receptor (LDLR). Human mural granulosal cells are collected routinely when a patient is undergoing in vitro fertilization (IVF) and are useful for studying granulosal lutein cells of the CL. Altering intracellular calcium in these cells through an ionophore, A23187, increased basal P4 production. However, at high concentrations a calcium chelator, BAPTA, decreased P4 from mural granulosal cells. Infertility is a problem that affects more than 6.1 million women and their partners across the United States. Causes of infertility range from female related tubal factors such as blockage, endometriosis, diminished ovarian reserve, to male related infertility due to abnormal sperm motility or morphology to unexplained infertility. Primary infertility types, age, BMI, and pregnancy outcome influenced ability of granulosal cells to increase P4 production when stimulated with human chorionic gonadotropin (hCG). AICAR decreased basal P4 production in these cells, which is similar to earlier observations in the mature bovine CL. Thus, AMPK appears to be a distal target in the pathway responsible for mediating the actions of PGF2&agr;. Directly activating AMPK could lead to better estrous cycle manipulation in the cow, or overcoming luteal insufficiency in women undergoing IVF.

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