Role of STARD6 in ovarian steroidogenesis

描述（由申请人提供）：在女性中，黄体酮用于正常月经周期和维持妊娠。除了为排卵准备卵子外，卵巢的另一项重要功能是从胆固醇中重新合成类固醇。在黄体生成素（LH）激增之前，卵巢卵泡的卵膜细胞层以及黄体细胞在LH激增之后，类固醇的重新合成主要发生在黄体细胞中。与卵泡相比，黄体的新生甾体生成显著增加。START结构域蛋白STARD1（甾体生成急性调节蛋白）介导甾体生成的限速步骤，即胆固醇从外部线粒体转运到内部线粒体，在那里发生了新生甾体生成的第一个酶促步骤。有体外证据表明，一种新的START结构域蛋白STARD6的功能类似于STARD1。其他实验室先前进行的两项初步研究未能在卵巢中检测到STARD6。利用芯片技术，我们在黄体生成素猪颗粒细胞中偶然发现了STARD6 mRNA，并进一步发现它在黄体中期高水平表达，黄体是一个高水平的STARD1和高黄体酮合成的组织。我们的长期目标是更好地了解胆固醇在卵巢中的运输，因为它关系到卵巢正常功能和卵巢疾病的治疗。本提案的主要目标是确定STARD6对人类卵巢细胞卵巢新生类固醇生成的贡献。在Aim 1中，我们将确定STARD6是否定位于人类卵巢的类固醇生成细胞。人类卵巢切片将被探测以确定表达STARD6和mRNA的细胞群，并在结构中评估蛋白质水平。目的2将测试两个子假设，即在人黄体化颗粒细胞中，STARD6以stard1独立或stard1依赖的方式介导甾体生成。这些研究将利用shRNA过表达STARD6和敲低STARD6来评估其对类固醇生成的影响。Aim 3将验证STARD6与黄体化颗粒细胞中的胆固醇区室相关的假设，并在类固醇合成活跃的条件下改变其在区室之间的分布。这些研究将确定STARD6在细胞内的定位，并确定在基础和camp刺激的条件下，当细胞补充低密度脂蛋白（LDL）时，是否会发生STARD6的动员。目的4将验证在黄体化颗粒细胞中，STARD6 mRNA和蛋白受特异性非促性腺激素或生长因子调控的假设。人黄体化颗粒细胞的原代培养将用胰岛素样生长因子和其他候选调节因子处理，以确定STARD6 mRNA和蛋白是否发生改变。该项目的成功完成将为卵巢甾体生成的胆固醇转运提供新的见解，并将为今后的研究奠定基础

DESCRIPTION (provided by applicant): 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.