Role of STARD6 in ovarian steroidogenesis

描述（由申请人提供）：在女性中，正常的月经周期和维持妊娠需要黄体酮。除了为排卵准备卵子外，卵巢的另一个关键功能是从胆固醇重新合成类固醇。类固醇重新合成主要发生在促黄体激素（LH）激增之前的卵泡膜细胞层和LH激增之后的黄体细胞中。与卵泡相比，黄体的类固醇从头合成显著增加。START结构域蛋白STARD1（类固醇生成急性调节蛋白）介导类固醇生成中的限速步骤，即胆固醇从外膜转运到内膜，在那里发生从头类固醇生成中的第一个酶促步骤。有体外证据表明，一种新的START结构域蛋白STARD6可以与STARD1类似地起作用。其他实验室先前的两项初步研究未能在卵巢中检测到STARD 6。使用微阵列，我们偶然发现STARD6 mRNA在促黄体化的猪颗粒细胞中，并进一步发现它在黄体中期高表达，黄体是一种具有高STARD1水平和高孕酮合成的组织。我们的长期目标是更好地了解卵巢中的胆固醇运输，因为它与正常卵巢功能和卵巢疾病的治疗有关。该建议的主要目标是确定STARD 6对人卵巢细胞中卵巢从头类固醇生成的贡献。在目标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。将探测人卵巢切片以确定表达STARD 6的细胞群，并将在结构中评估mRNA和蛋白质水平。目的2将测试两个子假设，STARD6介导的类固醇激素合成在一个STARD1独立或STARD1依赖的方式在人类黄素化颗粒细胞。这些研究将使用shRNA过表达STARD 6并敲低STARD 6，以评估对类固醇生成的影响。目的3将检验以下假设：STARD 6与黄素化颗粒细胞中的胆固醇隔室相关，并在活性类固醇合成的条件下改变其在隔室之间的分布。这些研究将确定STARD6在细胞内区室的定位，并确定在基础和cAMP刺激条件下，当细胞补充低密度脂蛋白（LDL）时，是否发生STARD6的动员。目的4将检验STARD 6 mRNA和蛋白质受黄素化颗粒细胞中特定非促性腺激素激素或生长因子调节的假设。将用胰岛素样生长因子和其他候选调节剂处理人黄素化颗粒细胞的原代培养物，以确定STARD6 mRNA和蛋白质是否改变。该项目的成功完成将为卵巢类固醇生成的胆固醇运输提供新的见解，并将作为未来研究的基础。 卵巢异常类固醇生成的疾病。

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.