Cell shape cell adhesion and regulation of ovarian folliculogenesis

Female mammals are born with all the eggs they will ever have, and this stock is irreplaceable. The majority of these eggs in the ovary are held in a resting stock of small immature eggs. Each egg is surrounded by a layer of a few flattened cells, called granulosa cells, and forms a structure known as a follicle. During reproductive life a steady trickle of follicles leave the resting stage and start to grow. This continues until the stock of eggs is exhausted and, in the human female, this results in the menopause, normally at the age of about 50 years. The progression of follicles from the resting to the growing phase has to be tightly regulated to ensure a normal reproductive lifespan. Premature depletion of oocytes, leading to an early menopause, is a common cause of infertility in women. Little is known about the factors that control the start of follicle growth. Growth factors produced locally in the ovary seem to have an important role but there are several possible candidates and it is not clear which ones are the key factors, and why some follicles start growing while others which are close by don't. In this project, using the mouse ovary as a model, we focus on the change that occurs in the shape of the granulosa cells, which is the first indication of follicle growth. This will give us a new insight into the way in which granulosa cells communicate with each other and with the oocyte. Using microscopy, one of the most dramatic changes that we see as the follicles start to grow is that the flattened granulosa cells become fatter, and more cuboidal in shape. After the granulosa cells have changed shape, they begin to divide, leading to an increase in number and at this point the oocyte begins to grow. Studies in skin cells have shown that this critical change in cell shape must involve an increase in the proteins that stick the cells together (so called adhesion molecules) along with changes in the internal protein 'scaffold', or cytoskeleton, of the cell. Adhesion molecules are remarkable in that, as well as sticking cells together, they also link tightly to the cytoskeleton and, in addition, send molecular signals to the nucleus. If the cell changes shape, the adhesion molecules can send signals to produce new proteins and other molecules needed for cell division. Surprisingly little is known about adhesion molecules and the cytoskeleton in follicles. We therefore want to map which adhesion molecules and cytoskeleton proteins are present in resting follicles and look to see if some of the signalling molecules can be seen near the nucleus. We will investigate how these molecules change as the follicle starts to grow. If we culture ovaries in dishes whilst blocking adhesion, we can see how important the adhesion molecules are in the change in granulosa cell shape, cell division and oocyte growth by blocking their ability to stick the cells together. Overall, by defining the proteins involved in the change in cell shape and showing where they are localized during the various stages of follicle growth, we will be better able to identify the most important input signals from the environment that lead to the change in shape of granulosa cells. We can also then examine the resultant output signals from the changing granulosa cells to the egg which stimulate its growth. In other words, we aim to identify the key factors that determine the reproductive lifespan of the mammal.

Female mammals are born with a finite supply of oocytes and reproductive lifespan ends with exhaustion of this supply. Each oocyte becomes enclosed in a layer of flattened somatic granulosa cells (GCs) to form a follicle. The rate at which oocytes leave the quiescent follicle pool and start growing must be tightly regulated to ensure a normal reproductive lifespan but little is known about this regulation. Several growth factors have been implicated in controlling initiation of follicle growth but these studies have treated initiation as a single event which actually encompasses several discrete cellular and morphogenetic processes including shape change of GCs from flattened to cuboidal, onset of GC cell division, oocyte growth, synthesis of zona pellucida around oocyte and development of multiple layers of GCs. We hypothesize that GC shape change is the key event in initiation of folliculogenesis, involving changes in intercellular adhesion which trigger signalling pathways resulting in downstream events of oocyte growth and GC division. We also suggest that GCs undergo an epithelial-mesenchymal transition during folliculogenesis, which is unusual in healthy adult tissue. We will characterize changes in cell adhesion (mainly cadherins), intercellular junctions, cytoskeleton and the intracellular localization of related signalling molecules (catenins) during follicle formation and early folliculogenesis in the mouse, using RT-PCR, immunohistochemistry, light, transmission and confocal microscopy. The influence of adhesion molecules on downstream events will be determined by targeted disruption using specific neutralising antibodies in organ culture. The role of the oocyte in regulating GC adhesion will be investigated using individual follicle culture following oocyte disruption. Increased understanding of the precise cellular changes during initiation of follicle growth will help identify candidate regulatory pathways, and their mechanisms of action.