The long-term goal of this research is to determine the role of an oocyte catecholaminergic system in ovarian function. Dopamine (DA) enters cells in one of three manners; intracellular production, through membrane bound receptors or an active dopamine transporter (DAT). Intracellular DA can be converted to norepinephrine (NE) by the enzyme dopamine-beta-hydroxylase (DBH). In the monkey oocyte, the presence of an adrenergic pathway utilizing active DBH has recently been demonstrated. Monkey oocytes also contain a dopamine transporter system enabling oocytes to take-up dopamine and produce NE. Experiments have shown that this de novo synthesized NE is capable of regulating intrafollicular cyclic adenosine monophosphate (cAMP). Because of the expense and physical limitations of performing this research in the monkey, continued research demands a shift to a rodent model. Therefore, the specific aim of this project is to demonstrate the presence of a functional catecholaminergic system in the mouse oocyte. In addition, comparisons will be made between oocytes of functional divergence, those characterized as germinal vesicle breakdown-incompetent (GVB-I) and GVB-competent (GVB-C). Experiments are proposed utilizing molecular, biochemical and cell physiology approaches investigating presence, location and function of the enzyme DBH and the transporter DAT which appear to control de novo biosynthesis of intrafollicular NE. GVB-I, GVB-C oocytes and ovaries (prepubertal and PMSG-stimulated) will be collected from mice. In Experiment 1 transcripts for DBH and DAT will be identified in GVB-I and GVB-C oocytes using RT-PCR, restriction enzyme digest, and PAGE. RT-PCR products for DBH and DAT will be sequenced by automated cycle sequencing. Ovarian tissues will be subjected to in situ hybridization using cRNA probes for DBH and DAT to confirm transcript presence and to provide insights as to oocyte expression in relation to folliculogenesis. Experiment 2 will focus on identification and characterization of the DBH and DAT proteins using Western blotting, immunohistochemistry and confocal immunocytochemistry. Experiment 3 deals with functionality of the mouse catecholamine system. Oocytes will be incubated in the presence or absence of DA followed by measurement of NE in culture media by HPLC. These experiments are preliminary in nature, although absolutely necessary to establish the catecholaminergic system in the mouse oocyte. Such experiments will form the building blocks for future experiments elucidating the functional importance of a catecholaminergic paracrine communication system between the oocyte and its follicular environment. Understanding such cross-talk systems between the oocyte and the follicle may be pivotal in addressing the pathophysiology of polycystic ovarian syndrome, aberrant responses of follicles to exogenous gonadotropins in assisted reproductive procedures and short-comings in embryonic development following oocyte in vitro maturation.
| Smith, G D (2001) In vitro maturation of oocytes. Curr Womens Health Rep 1:143-51 |
