Considerable advances continue to be made in our understanding of how pituitary and ovarian factors coordinate both early and late stages of ovarian follicle maturation. Yet, the molecular mechanisms that control the initial step of ovarian follicle growth and trigger the growth of reserve primordial follicles (primordial follicle activation) remain obscure. I propose to systematically dissect the key signalling pathway regulating this delicate balance between primordial follicle preservation and activation. An improved understanding of this pathway and its downstream targets will lead to insights into the biological and genetic basis of female infertility, create significant opportunities to develop predictive genetic tests for hereditary forms of female infertility, and may some day lead to the development of improved contraceptives that preserve primordial follicles. Our Hypotheses, based on prior biochemical studies of the PI3K / Akt / Pten pathway, genetic studies of Kit signalling, and our studies of the forkhead transcription factor FoxO3, are: 1) Two-way communication between the oocyte and surrounding cells is necessary for primordial follicle activation, but the trigger mechanism functions within the oocyte itself. The key paracrine factor is the Kit Ligand which activates the Kit receptor at the oocyte membrane, initiating a signalling cascade within the oocyte that acts via the PI3K and AKT kinases. A corollary of this hypothesis is that the tumor suppressor Pten (which inhibits AKT) is, like FoxO3, a key regulator (suppressor) of primordial follicle activation. 2) Within the oocyte, AKT directly phosphorylates and inhibits FoxO3, which acts as a nexus and master component of the trigger mechanism. In this capacity, FoxO3 functions to suppress follicle activation via the transcriptional activation of currently unknown targets that restrict the activation of ovarian follicles. 3) This finely balanced pathway influences the rate of primordial follicle activation over a woman?s entire lifetime. Hereditary factors including mutations or sequence variants of key components like FoxO3 can result, in some women, in an increased rate of primordial follicle activation. Even slight changes in the dynamics of primordial follicle activation can result in premature depletion of the primordial follicle reserve pool resulting in infertility/premature ovarian failure. To test these hypotheses, I propose the following Specific Aims: 1) Exploit genetic approaches and available conditional (floxed) alleles of key genes in this pathway including FoxO3 itself to further explore roles in follicle activation and their function within the oocyte itself. We will also generate compound mutant mice to verify that FoxO3 functions downstream of Kit and KL. 2) Identify and validate physiologically relevant transcriptional targets of FoxO3 by comparing mRNA profiles of FoxO3-deficient and wild-type control ovaries soon after birth and prior to the onset of follicle activation. Analyses of additional time points during and immediately following the onset of global primordial follicle activation in FoxO3-deficient ovaries will aid in the identification of direct FoxO targets and provide a unique view of ovarian genes that are induced during PFA and early follicle growth. 3) Employ the mouse ovary as a system to characterize changing expression patterns, subcellular localization, and activation status of pathway components in primordial and early growing follicles, using a combination of antibody probes, pharmacologic agents, and in vitro culture. These efforts will reveal essential details of how this pathway functions in primordial follicle activation.
RECENT PUBLICATIONS
George B John, Diego H Castrillon, "Role of Kit signaling in primordial follicle activation" Manuscript in preparation
George B John, Teresa D Gallardo, lane J Shirley, Diego H Castrillon, "Pten protects the egg supply and prevents ovarian aging through Foxo3" Dev Biol, (1):197-204, September 2008
Teresa Gallardo, George B John, Karen Bradshaw, Corrine Welt, Renee Reijo-Pera, Peter H. Vogt, Philippe Touraine, Silvia Bione, Daniela Toniolo, Lawrence M. Nelson, Andrew R. Zinn, Diego H Castrillon, "Sequence variation at the human FOXO3 locus: A study of premature ovarian failure and primary amenorrhea" Human Reproduction, 23(1):216-21, January 2008
Teresa Gallardo, Lane Shirley, George B John, Diego H Castrillon, "Generation of a germ cell specific mouse transgenic cre line" Genesis, 45(6):413-7, June 2007
George B John, Li Li, Christian Renken, Carmen Mannella, Jeanne M. L. Selker, Michael J Bennett, Jiping Zha, "Regulation of mitochondrial cristae morphology by the mitochondrial inner membrane protein mitofilin." Molecular Biology of the Cell, 16 (3):1543-1544, March 2005
SIGNIFICANT PUBLICATIONS
George B John, Rana Anjum, Ashok Khar, Ramakrishnan Nagaraj., "Subcellular localization and physiological consequences of introducing a mitochondrial matrix targeting signal sequence in bax and its mutants." Experimental Cell Research, 15; 278(2):198-208, August 2002
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