Minisymposium XV. Transcriptional Regulation of Gene Expression in the Ovary
Chair(s): Davis, John,
Location: CCQ 2000A
MS43. MOLECULAR REGULATION OF FETAL OVARY DEVELOPMENT. Koopman, Peter1, 1 The University of Queensland, Brisbane, QLD, Australia
We are investigating the early development of the vertebrate ovary – that is, the steps prior to formation of definitive follicles. To this end we have undertaken screens comparing gene expression in mouse fetal ovaries relative to testes, and different stages of ovarian development. We are particularly interested in identifying genes that may encode transcription factors involved in regulating cell type specification, and components of signalling pathways that may be involved in communication between cell types and co-ordination of the histogenesis of the early ovary. We have identified a large number of genes whose expression is upregulated in fetal ovaries shortly after sex determination, and are in the process of analysing the cell type-specificity and timing of expression of these genes which, together with information regarding the class of protein encoded, prioritizes the genes for further study. One gene resulting from these screens encodes the forkhead transcription factor Foxl2. Mutations in human FOXL2 underlie blepharophimosis ptosis epicanthus inversus syndrome (BPES), characterised by facial dysmorphology, combined in some cases with ovarian failure. We detected Foxl2 expression in embryonic ovaries of mice, chickens and red-eared slider turtles around the time of sex determination, associated with both somatic and germ cell populations. Moreover, sequence analysis of turtle and chicken FoxL2 orthologues indicated an unusually high degree of structural conservation during evolution. Our observations, combined with recent knockout analyses, are consistent with BPES resulting from early abnormalities in regulating the development of the foetal ovary, and identify Foxl2 as one of the earliest known regulators of ovarian development. Overall our results point to a surprising degree of transcriptional complexity in the fetal ovary at stages prior to overt histological differentiation.
MS44. EPIGENETIC REGULATION OF TRANSCRIPTION IN OVARIAN CANCER. Nephew, Kenneth1, 2, Huang, Tim3, Wei, Susan3, Balch, Curtis1, Brown, Robert4, 1 Indiana University School of Medicine, Bloomington, IN2 Indiana University Cancer Center, Indianapolis, IN3 The Ohio State University, Columbus, OH4 Glasgow University, Glasgow, UK, Scotland
Ovarian cancer is the most lethal gynecological malignancy and fifth leading cause of cancer death worldwide. Aberrant epigenetic regulation, such as CpG island methylation and associated transcriptional silencing of genes, has been implicated in a variety of human diseases, including ovarian cancer. Our group, utilizing a microarray-based technique known as differential methylation hybridization, examined 19 advanced ovarian cancer patients, for 7700 specific loci. Hierarchical clustering revealed the delineation of two groups of patients, corresponding to high vs. low levels of concurrent DNA methylation. Independently, it was found that the high methylation group bore a significant shorter progression-free survival (PFS less than 8 months versus greater than 12 months). Specifically, we identified 182 methylated sequences significantly associated with short PFS; we are now in the process of characterizing those sequences for patterns that could predict DNA methylation. In a separate approach, we are examining the possibility of re-expression of silenced ovarian cancer tumor suppressor genes by inhibition of DNA methylation. To this end, we are investigating a novel inhibitor, zebularine, and comparing its effect to the well-established (but relatively unstable) inhibitor 2-deoxy-5-azacytidine (DAC). In widely utilized ovarian cancer cell lines, we found that zebularine exerted impressive antigrowth effects, greater than those elicited by DAC. Further, zebularine effected impressive demethylation and induction of two tumor suppressor genes, hMLH1 and RASSF1A. To examine global methylation effects, we performed methyl acceptance and differential methylation hybridization analyses. While we found statistically identical demethylation by both zebularine and DAC, we discovered differences in demethylation of specific genes by the two agents. Finally, we found that zebularine could resensitize a platinum-resistant ovarian cancer cell line to cisplatin. As epigenetic dysregulation is responsible for the transcriptional silencing of hundreds of genes in ovarian tumors, epigenetic-based agents, both alone and in combination with conventional agents, may provide effective therapeutic approaches for this devastating disease.
MS45. FoxO3 AND PRIMORDIAL FOLLICLE ACTIVATION. Castrillon, Diego1, Gallardo, Teresa1, John, George1, 1 University of Texas Southwestern Medical Center, Dallas, TX
In mammals, oocytes arrested in the diplotene stage of meiosis become invested soon after birth in a single layer of pregranulosa cells to form primordial follicles, the reserve precursor pool for maturing follicles during reproductive life. Primordial follicle activation (PFA) is the metered process by which small numbers of primordial follicles are selected from this reserve pool into the growing follicle pool. This process is irreversible, in that follicles that have initiated growth undergo atresia if not selected for subsequent stages of maturation. PFA is a delicately balanced and tightly regulated process ensuring that some number of growing follicles is available during each estrus/menstrual cycle while preserving the majority of follicles for later in life, thereby forestalling depletion of oocytes and reproductive senescence. Despite advances in our understanding of many aspects of ovarian function, the mechanisms that regulate PFA remain largely obscure. We have shown that the forkhead transcription factor FoxO3, which functions downstream of the PI3K/AKT pathway, is a master regulator (suppressor) of PFA. FoxO3-null females undergo global PFA within a few days of birth, leading secondarily to increased atresia, premature depletion of ovarian follicles, and consequent female infertility. Our model, which we are currently validating by several approaches, is that FoxO3 functions within the oocyte itself to suppress follicle activation via the transcriptional regulation of currently unknown targets. To begin identifying and validating physiologically relevant transcriptional targets of FoxO3, we have compared 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 has permitted a unique view of ovarian genes that are induced during PFA and early follicle growth.
MS46. REGULATION OF TRANSFORMING GROWTH FACTOR-BETA1 (TGF) TRANSCRIPTION BY THE EARLY GROWTH RESPONSE FACTOR 1 (Egr1) TRANSCRIPTION FACTOR IN THE CORPUS LUTEUM (CL). Hou, Xiaoying1, 2, Arvisais, Edward1, 2, Davis, John1, 2, 1 University of Nebraska Medical Center, Omaha, NE2 VA Medical Center, Omaha, NE
In mammals, including primates and humans, prostaglandin F2 (PGF2) is believed to be the trigger that induces the regression of the CL, whereby progesterone synthesis is inhibited, the luteal structure involutes, and the menstrual or estrus cycle resumes. However, the cellular and molecular mechanisms of PGF2-induced CL regression remain poorly understood. As it is well established that PGF2 activates the PKC/Raf/MEK/ERK signaling pathway, our objectives were to identify genes responsive to PGF2 and activators of this pathway. Our data demonstrate that the expression of Egr1 mRNA and protein is up-regulated in the CL during PGF2-induced luteolysis in vivo and in PGF2-treated luteal cells in vitro. Studies have shown that Egr1 protein can induce the activation of various proapoptotic proteins, suggesting that Egr1 may play a role in luteal regression. Our hypothesis is that Egr1 mediates the actions of PGF2 by inducing the expression of key proapoptotic proteins. Egr1 protein was elevated in nuclear extracts prepared from PGF2-treated cells, and nuclear extracts bound a GC-rich Egr1 consensus motif, indicating that the newly synthesized Egr1 transcription factor was functional. Using a variety of chemical and genetic approaches, the PKC/Raf/MEK/ERK pathway was identified as the proximal signaling event required for the induction of Egr1 in PGF2-treated cells. TGF is believed to exert proapoptotic effects in many cell types, and is expressed in the CL during regression. Treatment with PGF2 increased the expression of TGF mRNA and protein in bovine luteal cells. The effect of PGF2 on TGF expression was mimicked by PKC activators or overexpression of Egr1. The stimulatory effect of PGF2 on TGF mRNA and protein secretion was inhibited by blockade of PKC/Raf/MEK/ERK signaling and overexpression of NAB2, a co-repressor that binds to and inhibits Egr-1 transcriptional activity. Treatment of luteal cells with TGF reduced progesterone secretion and DNA synthesis, implicating TGF in luteal regression. These studies demonstrate that PGF2 via the PKC/Raf/MEK/ERK signaling pathway regulates the expression of Egr1 and TGF in the CL. We suggest that Egr1 may play a role the induction of genes that coordinate the regression of the CL.