Objetivos De acuerdo a los resultados obtenidos se analizan los objetivos que

granulosa cells

SPON1 has been shown to affect cell survival and cell differentiation in neural and non-neural cells. We show that treatment of wildtype mouse granulosa cells with recombinant SPON1 led to increased viability. We could not determine if there was increased proliferation as primary granulosa cells that are not treated with hormones or cultured with fetal bovine serum do not proliferate. As mentioned above, SPON1

promotes cell viability of chicken ciliary ganglion[8] and mouse neuroblastoma cells [9]. In chicken ciliary ganglion this was achieved through increased active TGFβ levels [8] while in neuroblastoma cells loss of viability following SPON1 knockdown was attributed to lower IL-6 levels [9]. With current knowledge a likely candidate for the effect of SPON1 on cell viability in granulosa cells is alteration of TGFβ signalling, as TGFβ is an important protein in folliculogenesis and also regulates IL-6.

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Differentiation of early granulosa and theca cells to steroid producing cells is an essential step in folliculogenesis. Differentiation of cells due to SPON1 has been observed in nerve cells [5], cementoblasts [31], and chondrocytes [32]. The ability of SPON1 to alter gene expression has been seen in chondrocytes where expression of proteins involved in ECM remodelling is increased by SPON1 [32, 33]. Again, these changes in gene expression were brought about by increased levels of TGFβ [32, 33]. Granulosa cells treated with SPON1 and either dbcAMP or FSH and testosterone resulted in increased production of progesterone compared to cells that were not treated with SPON1. Because SPON1 affects both viability and steroid production of wildtype granulosa cells we hypothesize that it may play an important role in vivo in these functions.

3.4.6

Conclusion

In summary, we have shown for the first time that SPON1 is expressed in both the ovary and uterus and that expression changes in response to gonadotropins. We have also shown that SPON1 localizes to the theca cell layer and corpus luteum in the mouse ovary, and to the endometrial stroma and the myometrial stratum vasculare of the mouse uterus. Through co-localization studies with vascular cell markers we have shown that SPON1 is present at small and, to a lesser extent, large blood vessels. This is the first in vivo

evidence showing that SPON1 is associated with blood vessels and therefore may have a role in angiogenesis. We have also found possible functional roles for SPON1 within ovarian folliculogenesis as a mediator of FSH-induced granulosa cell proliferation and differentiation. These studies suggest the enticing possibility that SPON1 may be a new regulator of angiogenesis and folliculogenesis with potential therapeutic implications.

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3.5

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22. Madekurozwa MC. An immunohistochemical study of the distribution of intermediate filaments in the ovary of the emu (dromaius novaehollandiae). Anatomia, histologia, embryologia 2007; 36:336-342.

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28. Shynlova O, Tsui P, Dorogin A, Chow M and Lye SJ. Expression and localization of alpha-smooth muscle and gamma-actins in the pregnant rat myometrium. Biology of Reproduction 2005; 73:773-780.

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32. Palmer GD, Piton AH, Thant LM, Oliveira SM, D'Angelo M, Attur MG, Abramson SB and Teixeira CC. F-spondin regulates chondrocyte terminal differentiation and endochondral bone formation. Journal of Orthopaedic Research 2010; 28:1323-1329.

33. Attur MG, Palmer GD, Al-Mussawir HE, Dave M, Teixeira CC, Rifkin DB, Appleton CTG, Beier F and Abramson SB. F-spondin, a neuroregulatory protein, is up-regulated in osteoarthritis and regulates cartilage metabolism via TGF-b activation. The FASEB Journal 2009; 23:79-89.

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4

Chapter 4 – Spondin 1 is required for normal ovarian

function in Mus musculus

This chapter is based on a manuscript that has been prepared but not yet submitted.

4.1

Introduction

The ovary functions to prepare oocytes for ovulation and fertilization via folliculogenesis and as an endocrine gland producing steroid hormones to act on itself and other organs. These processes require involvement of the extracellular matrix (ECM). The ovary is constantly undergoing tissue remodelling as follicles grow and follicular cells differentiate, and the ECM modulates these changes [1]. ECM proteins affect follicle growth and development [2] and are involved in granulosa cell proliferation, survival, morphology and steroidogenesis [3-6]. Numerous studies have found that granulosa cells or ovarian tissue cultured with ECM proteins, such as laminin, fibronectin, and collagen IV, or Matrigel (a gelatinous protein mixture rich in ECM proteins) are more viable than those grown with other growth media [2, 3, 7].

As folliculogenesis progresses, the composition of the ECM changes and several different types of ECM proteins are required for normal ovarian cell differentiation and proliferation. These include the above mentioned laminins, fibronectins, and collagens, as well as proteoglycans with heparan sulfate or chondroitin sulfate side chains.

Proteoglycans are involved in several cellular processes including cell adhesion, ligand- receptor signalling, and basement membrane organization [8]. Matrix metalloproteinases (MMPs) degrade ECM proteins to remodel the ECM [1, 5]. In addition to supporting cells physically, ECM proteins are also important for signal transduction [9]. Integrins are often responsible for signal transduction in cell-ECM interactions. There are several examples of impaired fertility in knockout mouse models where ECM genes are

disrupted, including MMP9 [10], integrin-β1 [11], Tnfaip6 [12] and ADAMTS-1[13-15].

However, because the ECM is important for normal function in all tissues, mutations or deletions of specific ECM proteins are often lethal and effects on fertility cannot be evaluated, or the deletion results in no phenotypic changes due to functional redundancy.

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Spondin 1 (SPON1) is a secreted ECM glycoprotein encoded by the gene Spon1

[16]. It was initially studied for its role in neuron outgrowth during embryonic

development [17-22]; however, Spon1 is also expressed in other tissues such as ovary, kidney, bone, and small intestine, where several other functions for SPON1 have been uncovered. In C. elegans Spondin 1 interacts with integrins to maintain cell adhesion [23]. In bone, chondrocyte differentiation and endochondral bone formation are regulated by SPON1 in mice [24] and expression of SPON1 is high in human osteoarthritic

cartilage [25]. In chondrocytes and cartilage, treatment with SPON1 increased levels of MMP-13 [24, 25] and in cartilage, SPON1 increased the active levels of TGFβ [25].

There is compelling evidence that SPON1 plays a role in the ovary. Spon1

expression increases in response to 17β-estradiol in the mouse mammary gland [26] and the uterus [27]. SPON1 isolated from bovine ovarian follicular fluid increases

proliferation of rat vascular smooth muscle cells activity, and inhibits tube formation of human umbilical vein endothelial cells, suggesting a role in angiogenesis [28, 29]. SPON1 has also been identified as an ovarian cancer marker [30-32].

The SPON1 protein is comprised of: a) an N-terminal reeler domain, homologous to a domain in the protein, Reelin, b) a spondin domain homologous to domains in

Spondin 2 (Mindin/M-spondin), and c) six thrombospondin type 1 repeats (TSRs).

Several members of the thrombospondin superfamily are known to be involved in normal ovarian function. Thrombospondin -1 and -2 are both expressed in the ovary and regulate angiogenesis [33, 34]. R-spondin is expressed in the developing ovary and female

knockout mice have masculinized gonads and impaired ovulation [35-37]. A disintegrin and metalloproteinase with thrombospondin repeats 1 (ADAMTS-1) knockout mice have defective follicular growth, abnormal steroid gene mRNA expression, and are subfertile [13, 15].

The SPON1 knockout mouse (Spon1-/-) is viable and so far has been studied for its potential role in osteoarthritis [38] but its fertility has not been rigorously examined.

Spon1-/- mice have increased bone mass compared to controls, possibly brought about due to reduced levels of TGFβ-1 resulting in increased Smad1/5 phosphorylation. In the

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present study we sought to elucidate the role of SPON1 in female fertility and the ovary using Spon1-null (Spon1-/-) mouse as a model.

4.2

Materials and Methods