Date of Award

1-1-2013

Document Type

Open Access Thesis

Department

Biomedical Engineering

First Advisor

Holly A LaVoie

Abstract

After the luteinizing hormone surge of the menstrual cycle, the ovarian follicular granulosa and theca cells terminally differentiate to form the luteal cells of the corpus luteum. During this process known as luteinization, granulosa cells begin to synthesize large quantities of progesterone, a hormone essential for pregnancy. The rate limiting step for the de novo synthesis of pregnenolone (the precursor to progesterone) is the transport of cholesterol from the outer to the inner mitochondrial membrane, a process mediated by STARD1. STARD1 contains a C-terminal lipid binding domain holding one molecule of cholesterol, and an N-terminal domain targeting STARD1 to the mitochondrial membrane. STARD1 is the founding member of the mammalian START domain family, which includes 15 members. Two other members, STARD4 and STARD6, have recently been found in the ovary, and there is evidence that both can transfer cholesterol in certain settings, but unlike STARD1, STARD4 and STARD6 lack mitochondrial targeting. In this study, we aimed to determine the regulation of STARD1, STARD4, and STARD6 by protein kinase A (PKA) and Protein Kinase C (PKC) signals and sterol levels in cultured human luteinized granulosa cells. We found that both STARD1 and STARD4 mRNA, but not STARD6, were increased by PKA and PKC signaling agonists. STARD4 mRNA was sensitive to cellular sterol level, and STARD1 mRNA was responsive to cholesterol under low dose phorbol ester (PMA) stimulation. Moreover, STARD1 protein level and phosphorylation was increased by both cAMP analog and PMA. In transfected COS-1 cells, fluorescent confocal images showed that the localization of STARD6 was mostly in the cytoplasm with some nuclear presence but STARD4 was throughout the cytoplasm and nucleus. In addition, to confirm a prior result in our laboratory to test if STARD6 can facilitate de novo steroidogenesis, COS-1 cells transfected with the components of the P450 cholesterol side chain cleavage complex were co-transfected with vector alone or vectors containing either human STARD1 or STARD6. STARD6 was able to modestly increase pregnenolone production above vector, but not nearly to the extent of STARD1. These studies provide new insight into the regulation of START domain proteins in the human ovary.

Rights

© 2013, Bo Shi

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