Ovarian angiogenesis: new hypotheses

Angiogenesis, the formation of new blood vessels, is critical for the normal functioning of the ovaries. The blood vessels, which are formed from endothelial cells, develop during follicular growth in the theca layer, but the inner granulosa cell layer remains avascular. After ovulation, the follicle collapses inwards followed by extensive tissue remodelling. Underpinning these changes, there is a period of intense angiogenesis with 85% of the proliferating cells being of endothelial origin. This enables the corpus luteum (CL) to grow at a rapid rate (to >5g in 5 days) that is only surpassed by the fastest growing tumours. This is essential for sufficient progesterone to be produced to support the developing embryo and studies in a variety of species have shown that blocking angiogenesis inhibits progesterone production. In cattle for example, if sufficient progesterone is not produced rapidly enough, then the embryo will die. Hence luteal angiogenesis is critical for maintaining pregnancy and the aim of this project is to understand how this process is regulated. The factors thought to regulate angiogenesis in the CL include vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF-2) and other growth factors, but their precise roles are unclear. Unexpectedly, we found that FGF-2 was more dynamic than VEGF during the follicle-luteal transition period. Levels of FGF-2 increased dramatically around ovulation, before declining to basal levels for the remainder of the luteal phase. This led us to hypothesise 1) that this FGF-2 surge is the major initiator of luteal angiogenesis. We also propose 2) that the subsequent basal levels of FGF-2 and VEGF are obligatory at specific times for the development and maintenance of blood vessels. To test these hypotheses, we have developed a new culture system using follicular granulosa and theca cells or enzymatically dispersed cells from the corpus luteum in which endothelial tubule-like structures develop in the culture dish. We can quantify the number and area of these tubules by staining them and using microscopy and image analysis. This culture system uses a mixed population of follicular or luteal cells (including steroidogenic, endothelial and pericyte cells) and hence is a more physiologically relevant model system than using cell lines. Furthermore, it offers the opportunity to elucidate the underlying mechanisms regulating ovarian angiogenesis at a level of detail that would not be possible in the whole animal. Finally, we hypothesise 3) that tubule formation involves communication between pericytes and endothelial cells via specific pathways and also 4) that the steroidogenic luteal cells influence the migration of the endothelial cells. We will test these hypotheses by 1) mimicking the dynamic changes in FGF-2 in our mixed luteinised follicular cell culture system and 2) blocking basal levels of FGF-2 and/or VEGF using receptor blockers at different times during the culture of dispersed luteal cells and determining the effect on luteal endothelial cell migration, tubule initiation and network establishment. To test hypothesis 3) we will determine the effect of removing pericytes from the mixed cell population. Then we will block two important pathways of communication between pericytes and endothelial cells and see what happens to tubule formation. Finally, for hypothesis 4) we will culture isolated steroidogenic cells and find out if endothelial cells in culture will move towards them and which specific compounds can block this effect. All these experiments will advance our understanding of how angiogenesis in the ovary is regulated, particularly during the follicle-luteal transition period and the early luteal phase. They will provide new insights into progesterone production during the critical period of early embryo development and may lead ultimately to the development of new strategies to improve fertility in cattle and other species including man.

Progesterone production by the developing corpus luteum (CL) is dependent on intense angiogenesis and the formation of new blood vessels. Adequate progesterone is critical to support the developing embryo and consequently it is important to understand how ovarian angiogenesis is regulated. We have recently developed a physiologically relevant culture system for bovine follicular or luteal cells in which networks of endothelial cell tubule-like structures develop in the presence of other ovarian cells. These are quantified by staining for von Willebrand Factor followed by image analysis. We will use this culture system to test several new hypotheses on ovarian angiogenesis generated by our previous BBSRC project on luteal development in the cow. Firstly, we will test the hypothesis that it is the surge of FGF-2 that occurs around the time of ovulation is the major initiator of angiogenesis, and secondly that subsequent basal FGF-2 and VEGF are required at specific times for luteal endothelial cell migration and clustering, tubule initiation and network establishment. We will do these experiments by either adding these factors, or using receptor blockers to inhibit their action at specific times. We also propose that there is communication between the luteal pericytes and endothelial cells that is important in angiogenesis, and is mediated by the PDGF-B and eNOS signalling pathways. We will test this by separating out these two cell types using an automated cell sorter and then using siRNA to silence gene expression of NO in the endothelial cells and the receptor for PDGF-B in the pericytes. We will also use a PDGF-B antagonist. Finally, we hypothesise that steroidogenic luteinised granulosa cells secrete signals that attract endothelial cells and this will be tested using cell migration assays. Collectively these experiments will provide new insights into angiogenesis and may lead ultimately to new strategies to overcome luteal inadequacy.