Reelin/Thrombospondin signaling in granulosa cells

The Reelin signaling pathway is indispensable for the development of laminated structures in the brain where it orchestrates correct positioning of newly generated neurons in the neocortex, hippocampus, and cerebellum. Key players of the Reelin signaling pathway are apolipoprotein E receptor 2 (ApoER2) and very low density lipoprotein receptor (VLDLR) that relay the Reelin signal into radially migrating neurons. Loss of any of these components results in neuroanatomical defects resulting in the characteristic "reeler-phenotype". In addition, Thrombospondin plays an important role as alternative ligand for both receptors in the development of the rostral migratory stream which guides neurons from the sub-ventricular zone to the olfactory bulb. Surprisingly, the components of this signaling pathway are also expressed in ovarian follicles of egg laying species. Development of the oocyte in egg laying species has to obey to other physiological aspects than in mammals. Here, the oocyte has to carry all nutrients necessary for the embryo to develop. This is achieved by massive amounts of yolk deposited in the oocyte proper during development resulting in enormous sizes of the oocytes in these species. In the chicken for example, the mature oocyte weighs about 15 grams and corresponds to the egg yolk of a laid egg. Our effort over the last decade has resulted in delineating the molecular basis of the rapid oocyte growth by delineating the endocytosis machinery supporting the accumulation of yolk precursors into the growing oocyte. Our knowledge about the function(s) of the surrounding cells constituting the follicle which supports oocyte growth is still scarce. The recent finding that ApoER2 becomes up-regulated during bovine folliculogenesis opens a door for a novel approach to unravel mechanisms responsible for regulating the growth of this organ. This projects aims at defining the signaling pathways in the ovarian follicle supported by ApoER2 and VLDLR and their cognate ligands Reelin and Thrombospondin. We propose that a pathway similar to the Reelin-pathway in the brain is operable within ovarian follicles regulating cell survival and proliferation of granulosa cells and thus determining the growth and the architecture of the follicle. The results of this project are expected to shed light on grossly missing molecular and mechanistic aspects of follicular growth in egg laying species and to have a significant impact on the field of reproduction and poultry science in general.

ApoER2 and VLDL receptor are members of the LDL receptor family and are part of a signaling cascade which orchestrates correct development of laminated structures of the central nervous system. This signaling pathway is elicited by Reelin an extracellular matrix protein which is secreted from specialized neurons during embryonic development of the brain. Reelin binds to ApoER2 and VLDL receptor which induces phosphorylation of Dab1 an intracellular adapter protein bound to the intracellular domains of both receptors. Both receptors however, are also expressed in the chicken follicle. VLDL receptor is expressed on the surface of growing oocytes and mediates endocytosis of yolk components which are synthesized in the liver of laying hens. A variant of VLDL receptor is expressed in granulosa cells which surround and support the growing oocyte. In these cells however, VLDL receptor does not take up yolk components although these molecules come in close contact with the granulosa cells.During the first phase of the project we could demonstrate that, in addition to VLDL receptor, granulosa cells also express ApoER2 and Dab1. In addition, it became evident, that Theca cells, which are part of the outermost layer of the follicle, secrete Reelin. Thus, in the chicken follicle exist the major components of the Reelin signaling cascade, which might orchestrate the development of the laminated structure of the follicle. Indeed, we could demonstrate that such a cascade is functional in the follicle and that Reelin induces Dab1 phosphorylation in granulosa cells.During our studies on Dab1 phosphorylation in granulosa cells we discovered a shorter splice variant of Dab1 to be expressed in granulosa cells. In the second part of the project we could demonstrate that the shorter Dab1 variant acts as a dominant inhibitor of the Reelin signaling pathway. Thus, in the chicken follicle exists a complex Reelin signaling network with intrinsic inhibitors which orchestrates the development of this organ and the development of the oocyte.