Control and manipulation of angiogenesis in the primate ovarian follicle

Abstract

The ovary is one of the most plastic tissues in the body undergoing constant serial remodelling throughout its reproductive lifetime, during both folliculogenesis and the formation and regression of the corpus luteum. The process of follicle growth and selection is intimately associated with the de novo establishment of vasculature supporting the developing follicles. Blood vessels are recruited from the ovarian stroma to form vascular sheaths surrounding each developing follicle supplying steroid hormone precursors, oxygen and nutrients to the expanding follicle. During folliculogenesis only the theca of the developing follicle becomes vascularised, the granulosa cells remaining avascular until ovulation at which point the basement membrane that has been separating the granulosa and theca breaks down. After ovulation the granulosa cells become heavily vascularised during the process of luteinisation and the formation of the corpus luteum. Angiogenesis, the growth of new blood vessels from the pre-existing vasculature, requires the degradation of the established vessels followed by endothelial cell proliferation and finally stabilisation of the new vessels. Recently, techniques to quantify angiogenesis, identify putative molecular regulators, and inhibit them in vivo have become available. The work in this thesis applies these advances to the following questions:

1) What is the effect of the inhibition of the gonadotrophins, using a gonadotrophin releasing hormone (GnRH) antagonist, on follicular angiogenesis? The hypothesis being tested was that follicular angiogenesis would be dependent on follicle stimulating hormone (FSH) / luteinising hormone (LH) and be severely inhibited by GnRH antagonist treatment. In vivo follicular angiogenesis was assessed by quantitative immunocytochemistry of bromodeoxyuridine and the endothelial cell marker CD31. The effect of treatment on follicular development and angiogenesis at the molecular level was assessed by in situ hybridisation of mRNA for vascular endothelial growth factor (VEGF) and aromatase. The results suggest that while VEGF expression in the preovulatory follicle is under gonadotrophic control, it is not dependent on normal gonadotrophin secretion in tertiary follicles, indicating that there are other paracrine factors regulating VEGF expression in the developing ovarian follicle. The second chapter extends the findings by determining granulosa cell response to FSH stimulation with respect to induction of the VEGF and aromatase genes.

2) What is the effect of inhibition of vascular endothelial growth factor, using the antagonist, VEGF trap R1R2, on follicular angiogenesis, follicular development, ovulation and the establishment of the corpus luteum (CL)? The hypothesis being tested was that VEGF is essential for increasing permeability and the growth of the selected follicles. The immunocytochemical techniques used in the first study were again employed. The effect of treatment on the molecular regulation of angiogenesis was assessed by in situ hybridisation of mRNA for VEGF and its two receptors. In vivo inhibition of VEGF caused dramatic reductions in angiogenesis and in VEGF receptor expression but did not reliably prevent dominant follicle growth or ovulation once dominant follicle selection had occurred.

3) Is a novel factor, endocrine gland vascular endothelial growth factor (EGVEGF) expressed in our animal model? The hypothesis being tested was that EGVEGF is an additional angiogenic factor that is expressed in the marmoset and the human ovary. This was assessed by in situ hybridisation in various marmoset tissues as well as in the human corpus luteum. Findings demonstrated that EG-VEGF is expressed in the granulosa lutein cells in the human corpus luteum while the marmoset ovary does not appear to express EG-VEGF.

This thesis has improved our understanding of the gonadotrophic control of follicular angiogenesis and the role VEGF plays in the latter stages of folliculogenesis.