NOTCH SIGNALING REGULATES GRANULOSA CELL DIFFERENTIATION

Arizona State University-Tempe Campus, Tempe AZ

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Abstract

DESCRIPTION (Adapted from the applicant's description): Each year 1.3 million women seek medical help for infertility. Understanding the molecular and cellular egulation of folliculogenesis and oocyte maturation will improve the diagnostic and treatment options for those hose infertility currently is unexplained. Development of ovarian follicles and maturation of fertilization ompetent oocytes is a complex process. Notch signaling regulates cell fate decisions between adjacent cells and otch pathway genes were found to be expressed in granulosa cells of developing follicles. Recent work has shown hat granulosa cells differentially express cell cycle regulators during proliferation and terminal differentiation. ome researchers have hypothesized that granulosa cells may differentiate into three subtypes. The goal of this pplication is to examine the role of the Notch pathway in granulosa cell differentiation. Initial work will determine he spatial and temporal pattern of expression of components of the Notch signaling pathway in the mammalian vary. Factors which affect expression of genes in the ovary include, age, pregnancy, and hormonal regulation of he ovulatory cycle. The effect of these factors on Notch genes will be tested in two ways, first, quantitative changes n the level of expression, and second, change in the stage of follicle or cell type, using semiquantitative RT-PCR and in situ hybridization on thick ovary sections. Lunatic fringe (L-fng) is an important modulator of Notch signaling. ost L-fng -I- mice die at birth, some survive to adulthood but have reduced fertility. The role of L-fng during olliculogenesis and oocyte maturation in vivo will be examined. If L-fng regulates signaling in granulosa cells, there may be alterations in the pattern of follicle maturation in the mutants. Any change in expression of Notch pathway genes in follicles will be determined by in situ hybridization of L-fng -/- ovaries. Ovary serial sections will be examined for gross abnormalities histologically and the number of follicles in each stage quantified. Alterations in granulosa populations may result in unfertilizable eggs, so superovulation will be induced and eggs collected, and maturation tested by parthenogenesis using ionophore. Several null mutants of Notch receptors and ligands have been generated, all are embryonically lethal. This approach will not enable us to examine the in vivo role of Notch signaling in granulosa cell differentiation therefore, we will use tissue- targeted transgenic analysis, a powerful method of examining Notch function in vivo. The control regions of the mouse alpha-inhibin gene have been used to target transgenes to the granulosa cells of developing follicles in a gonadotropin responsive manner. This promoter/enhancer will be used to express dominant negative and constitutively active mutants of Notch in granulosa cells.