Abstract
BackgroundThe Foxl2 transcription factor is required for ovarian function during follicular development. The mechanism of Foxl2 regulation of this process has not been elucidated. Our approach to begin to understand Foxl2 function is through the identification of Foxl2 regulated genes in the ovary.MethodsTransiently transfected KK1 mouse granulosa cells were used to identify genes that are potentially regulated by Foxl2. KK1 cells were transfected in three groups (mock, activated, and repressed) and twenty-four hours later RNA was isolated and submitted for Affymetrix microarray analysis. Genesifter software was used to carry out analysis of microarray data. One identified target, the gonadotropin releasing hormone receptor (GnRHR) gene, was chosen for further study and validation of Foxl2 responsiveness. Transient transfection analyses were carried out to study the effect of Foxl2 over-expression on GnRHR gene promoter-luciferase fusion activity. Data generated was analyzed with GraphPad Prism software.ResultsMicroarray analysis identified 996 genes of known function that are potentially regulated by Foxl2 in mouse KK1 granulosa cells. The steroidogenic acute regulatory protein (StAR) gene that has been identified as Foxl2 responsive by others was identified in this study also, thereby supporting the effectiveness of our strategy. The GnRHR gene was chosen for further study because it is known to be expressed in the ovary and the results of previous work has indicated that Foxl2 may regulate GnRHR gene expression. Cellular levels of Foxl2 were increased via transient co-transfection of KK1 cells using a Foxl2 expression vector and a GnRHR promoter-luciferase fusion reporter vector. The results of these analyses indicate that over-expression of Foxl2 resulted in a significant increase in GnRHR promoter activity. Therefore, these transfection data validate the microarray data which suggest that Foxl2 regulates GnRHR and demonstrate that Foxl2 acts as an activator of the GnRHR gene.ConclusionsPotential Foxl2 regulated ovarian genes have been identified through microarray analysis and comparison of these data to other microarray studies. The Foxl2 responsiveness of the GnRHR gene has been validated and provided evidence of Foxl2 transcriptional activation of the GnRHR gene promoter in the mouse ovary derived KK1 granulosa cell line.
Highlights
The Foxl2 transcription factor is required for ovarian function during follicular development
The transcription factor Foxl2 is vital to ovarian function as evidenced by the identification of mutations in the gene encoding FoxL2 that result in the condition known as blepharophimosis/ptosis/epicanthus inversus syndrome (BPES) and in some cases, premature ovarian failure (POF) [1]
In order to increase the potential of Foxl2 to alter gene expression levels of putative target genes, two fusions were constructed consisting of Foxl2 fused to the activation domain of the Herpes simplex virus VP16 transcription factor (Foxl2-VP16) and Foxl2 fused to the repression domain of the murine MAD transcription factor (Foxl2-MAD)
Summary
The Foxl transcription factor is required for ovarian function during follicular development. In a study in which a portion of the Foxl coding region (amino acids N-61) was fused to the bgalactosidase gene (LacZ), homozygous Foxl2-lacZ mice exhibited ovarian failure resulting from the absence of granulosa cell differentiation at an early stage of follicular development [4]. These investigators observed that follicles were activated and underwent apoptosis, leading to progressive follicular depletion and ovarian atresia. A second study in which both copies of Foxl were completely knocked out determined that the development of granulosa cells was blocked at the point of primordial follicle formation [5]
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