Marco Contextual

In summary, we have made several novel discoveries in the present study. We identify potential functions for SPON1 in the ovary, namely, regulation of: a) granulosa cell steroidogenesis, b) STAR and CYP11A1 expression, c) STAR promoter activity, and d) granulosa cell viability and proliferation. We also identify PRAS40 as a kinase target that is phosphorylated in the presence of SPON1, and is potentially involved in the signalling pathways regulating these functions. In addition, we demonstrate that cAMP- induced STAR protein levels are reduced by an ECM protein, and very few ECM proteins with this ability have been identified. These results strongly suggest that in addition to its role in other tissues, SPON1 plays a role in ovarian folliculogenesis by regulating granulosa cell function. We believe that these findings may help explain why SPON1 is highly overexpressed in ovarian cancer, and provide further support for its utility as an ovarian cancer marker.

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2.5

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3

Chapter 3: Characterization of Spondin 1 function in

mouse ovarian folliculogenesis

3.1

Introduction

Folliculogenesis, followed by successful ovulation, is a complex process that transforms a primordial follicle, an oocyte surrounded by a single layer of pre-granulosa cells, into a fully-mature pre-ovulatory follicle, consisting of an oocyte surrounded by several layers of cumulus granulosa cells, multiple layers of mural granulosa cells, a fluid-filled antrum, and a layer of vascularized theca cells separated from the rest of the follicle by a basal lamina. In response to luteinizing hormone, ovulation occurs and the oocyte is released, resulting in formation of a highly-vascularized corpus luteum (CL). The CL produces progesterone to prepare the uterus for implantation and maintain pregnancy, should it occur. This process requires the action of a large number of factors, including pituitary hormones, steroid hormones produced by the ovary itself, the

extracellular matrix (ECM), glycoproteins, and growth factors. Despite extensive characterization of many of these factors, many more remain to be discovered, as indicated by the numerous genome-wide expression analyses that have been carried out using follicles at various stages of folliculogenesis. Identification of new regulatory factors may point to new directions of therapeutic intervention for women since unexplained sub-infertility or infertility contributes to a large proportion of female infertility, and defects in ovulation account for the greatest proportion of causes of infertility.

Spondin 1 (SPON1) was first characterized as a secreted (ECM) protein highly expressed in the mouse embryonic floorplate [1]. SPON1 has been primarily studied for its role in the central nervous system, where it regulates migration of neurons during early development and cell adhesion in many cell types of the central nervous system [2- 6]. In C. elegans SPON1 maintains cell-ECM adhesion in several tissues, both neural and non-neural [7]. SPON1 also increases cell viability in neural cells. Chicken ciliary

ganglion cells treated with recombinant SPON1 in culture have increased viability [8]. In a mouse neuroblastoma cell line knockdown of SPON1 resulted in decreased survival under adverse conditions that was rescued by SPON1 treatment [9].

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Several lines of evidence suggest that SPON1 regulates angiogenesis, and in fact, may inhibit it. Angiogenesis is the growth of new blood vessels from pre-existing vessels and consists of several steps. Existing blood vessels are destabilized by separation of endothelial cells from mural cells, including pericytes and vascular smooth muscle cells (vSMC). The ECM must be degraded by proteases to allow migration of endothelial and mural cells. These cells then migrate towards the angiogenic stimuli. And finally,

depending on the size of the vessel, mural pericytes (small and large vessels) and vSMC (large vessels) reattach to promote structural integrity. With the exception of tissue injury or tumour progression, angiogenesis only occurs in a few adult tissues, including the CL, the endometrium of the uterus, and the placenta. In relation to angiogenesis, SPON1 inhibits migration and tube formation by human umbilical vein endothelial (HUVEC) cells, and neovascularization in rat cornea [10]. SPON1 also promotes vSMC proliferation [11], and was isolated from bovine ovarian follicular fluid in a search for a factor that promoted vSMC proliferation in the ovary [11].

Despite the fact that SPON1 mRNA is highly expressed in the ovary in humans, as shown by Northern blot [11] there have been few reports describing SPON1 gene expression in the mammalian ovary or uterus [12]. As mentioned, SPON1 was identified in bovine follicular fluid [11]. In addition, when ovariectomized mice are treated with 17β-estradiol, Spon1 mRNA expression increases in both the uterus [13] and mammary gland [14], suggesting SPON1 may play a role in these reproductive tissues.

Interestingly, SPON1 is also highly overexpressed in ovarian cancer [15, 16]. Our previous microarray studies [17] comparing the gene expression profiles of granulosa cells isolated from gonadotropin-treated immature (PND 23-29) C57Bl/6 wildtype (WT) and estrogen receptor  (ER)-null mice identified SPON1 (Spon1) expression to be lower in ER-null granulosa cells than in WT cells (Gene Expression Omnibus accession number GSE11585). Together this information points to a potentially significant role for SPON1 in the ovary. Given that there is evidence for a role for SPON1 in cell signalling in survival, differentiation and angiogenesis in other tissues, we also hypothesized that SPON1 may play a role in these functions in the uterus and ovary. Currently, it is not known in what cells of the ovary or uterus SPON1 is expressed. It is also not known

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whether SPON1 expression is regulated by gonadotropins, or what functional roles it plays in these tissues.

Therefore, to further characterize the localization, potential function, and regulation of SPON1 by gonadotropins in the mouse, we examined SPON1 expression,