In females, progesterone is needed for normal menstrual cyclicity and for the maintenance of pregnancy. In addition to preparation of the ovum for ovulation, another critical function of the ovary is de novo steroid synthesis from cholesterol De novo steroidogenesis occurs predominantly in the theca cell layer of the follicle prior to the luteinizing hormone (LH) surge and in the cells of the corpus luteum following the LH surge. The corpus luteum demonstrates a dramatic increase in de novo steroidogenesis compared to the follicle. A START domain protein STARD1 (steroidogenic acute regulatory protein) mediates the rate-limiting step in steroidogenesis, the transport of cholesterol from the outer to the inner mitochondrion, where the first enzymatic steps in de novo steroidogenesis occur. There is in vitro evidence that a novel START domain protein, STARD6, can function similarly to STARD1. Two preliminary studies by other laboratories previously failed to detect STARD6 in the ovary. Using microarray, we serendipitously found STARD6 mRNA in luteinizing pig granulosa cells and further found it to be highly expressed in midluteal phase corpora lutea, a tissue with high STARD1 levels and high progesterone synthesis. Our long-term goal is to better understand cholesterol trafficking in the ovary as it relates to normal ovarian function and the treatment of ovarian disorders. The main goal of this proposal is to define the contribution of STARD6 to ovarian de novo steroidogenesis in human ovarian cells. In Aim 1, we will determine if STARD6 is localized to steroidogenic cells of human ovaries. Sections of human ovaries will be probed to determine the cell populations expressing STARD6 and mRNA and protein levels will be assessed in structures. Aim 2 will test two sub-hypotheses that STARD6 mediates steroidogenesis in either a STARD1-independent or STARD1-dependent manner in human luteinized granulosa cells. These studies will overexpress STARD6 and knockdown STARD6 using shRNA to evaluate effects on steroidogenesis. Aim 3 will test the hypothesis that STARD6 is associated with cholesterol compartments in luteinized granulosa cells and changes its distribution between compartments under conditions of active steroid synthesis. These studies will determine the localization of STARD6 to intracellular compartments and determine if mobilization of STARD6 occurs when cells are supplemented with low density lipoproteins (LDL) under basal and cAMP-stimulated conditions. Aim 4 will test the hypothesis that STARD6 mRNA and protein are regulated by specific non-gonadotropin hormones or growth factors in luteinized granulosa cells. Primary cultures of human luteinized granulosa cells will be treated with insulin-like growth factors and other candidate regulators to determine if STARD6 mRNA and protein are altered. Successful completion of this project will provide new insight into cholesterol trafficking for ovarian steroidogenesis and will serve as a basis for future studies of disorders of aberrant steroidogenesis by the ovary. PUBLIC HEALTH RELEVANCE: Ovarian progesterone production is required by the uterus for pregnancy. Progesterone flux also contributes to normal menstrual cycles. Cholesterol serves as a substrate for de novo steroidogenesis and its intracellular movement into the mitochondria is tightly controlled. We propose studies to determine if a novel START domain protein, STARD6, contributes to ovarian progesterone production. The successful completion of this study will identify a new regulator of cholesterol trafficking in ovarian cells and modulator of progesterone production.