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Gonadotropin releasing hormone (GnRH) is the central hormone of reproduction in vertebrates. This hormone is secreted from the hypothalamus in response to environmental, steroid hormone feedback and other stimuli in a pulsatile manner and travels via the hypophyseal portal vasculature to the anterior pituitary. GnRH binding to its receptor on the surface of pituitary gonadotropes stimulates the release of the gonadotropin hormones: luteinizing hormone (LH) and follicle stimulating hormone (FSH), heterodimers of the common [alpha] subunit with the hormone-specific [beta] subunits. In addition to secretion of gonadotropin hormones, GnRH stimulates the transcription of the gonadotropin subunit genes and that of its own receptor (GnRHR). The GnRHR has been shown in recent years to be a constitutive occupant of membrane raft microdomains within the plasma membrane. GnRHR association with these microdomains appears to be required for the initiation of downstream signaling processes within the GnRH signaling network including activation of the mitogenactivated protein kinase, extracellular signal regulated kinase (ERK). GnRHR-induced ERK activation is absolutely required for gonadotropin subunit gene expression and fertility in mice. In this dissertation the components of the GnRHR-associated membrane raft microdomain are explored providing insight into how the receptor might be connected to membrane microdomains, the actin cytoskeletal network, and the initiation of downstream transcriptional events. These studies introduce the flotillin/reggie proteins and [beta] catenin as novel members of the GnRHR-associated membrane raft proteome in addition to identifying a list of proteins for future studies.

In somatic cells, membrane rafts are dynamic, existing in various time and space scales. The transient nature of these membrane microdomains and the use of detergents or multivalent probes/cross-linkers to isolate or visualize them have incited controversy in this field. In the following studies, I demonstrate for the first time in live spermatozoa, the presence of a micrometer-scale, stable domain in the plasma membrane enriched in sterols and the glycosphingolipid GM1 at physiological temperature. This sterol-enriched membrane platform is positioned to regulate the acquisition of sperm fertilization competence. I examine the properties of this domain and the mechanism behind its segregation and maintenance. I also conduct in-depth functional studies on FABP9, which was a strong candidate for tethering membrane lipids to underlying cytoskeletal elements in this region. My studies showed that the maintenance of this stable segregation in the sperm head was at least in part due to a specialized diffusion barrier at a region called the sub-acrosomal ring. I found that FABP9 did not play a role in this membrane segregation. Interesting dynamics of GM1 bound to cholera toxin subunit B (CTB) revealed that complex membrane interactions occur in this region. This led to the finding that the acrosomal vesicle that immediately underlies this domain is a GM1-enriched organelle. The dynamics of CTB-bound GM1 also resulted in our ability to distinguish different functional changes in sperm membrane properties occurring in response to capacitating stimuli. Based on these stimulus specific changes seen with CTB-bound GM1 distribution, we found that sperm could respond to bicarbonate ions or mediators of sterol efflux independently, thereby refining existing models of capacitation. The applicability of CTB-bound GM1 dynamics as a diagnostic tool to assay sperm response to stimuli for capacitation is potentially of significant clinical importance. These studies set the stage for exploring both capacitation-related changes in the microheterogeneities within this domain and ensuing signaling events in response to stimuli for capacitation. Furthermore, my data have led to a complex model that involves several complementary mechanisms of lipid segregation acting at different spatial scales. Testing this model will be the subject of continuing investigations.