The Aryl Hydrocarbon Receptor May Regulate Genes Involved in Cell Cycle and X Chromosome Inactivation in the Neonatal Mouse Ovary.

Abstract

The aryl hydrocarbon receptor (AHR) is a ligand activated transcription factor known for its role in mediating the toxic effects of numerous environmental contaminants. Recent data also indicate that the AHR plays a role in normal ovarian physiology. Specifically, previous studies have shown that Ahr deletion results in a decrease in the number of antral follicles, slow follicular growth, decreased estradiol synthesis, and reduced fertility in adult female mice. However, the role of the AHR in early neonatal reproductive function is relatively unknown. Normally, just after birth, the mouse ovary contains a finite number of germ cells available for follicle formation. These germ cells are organized in clusters called oocyte nests that will break apart and form primordial follicles. During this period more than 70% of the germ cells die by apoptosis and the surviving germs cells are then surrounded by a single layer of flattened granulosa cells, forming primordial follicles. Very little is understood about what determines the fate of the germ cells and what signals the development of the neonatal ovarian cell types. Previous studies in our laboratory have shown that the AHR may play a role in regulating the timing of follicle formation. Thus, this study tested the hypothesis that on postnatal day (PND) 3, when oocyte nests are breaking apart to form primordial follicles, there are changes in expression of genes involved in regulating apoptosis and cell survival between aryl hydrocarbon receptor null (AhRKO) and wild type (WT) mouse ovaries. To test this hypothesis, ovaries were collected from AhRKO and WT mice on PND3 and subjected to Affymetrix GeneChip® Mouse Genome 430 2.0 arrays to compare the expression of more than 20,000 genes. A comparative analysis revealed differential expression of 147 genes: 123 genes were up-regulated, whereas 24 genes were down-regulated in the AhRKO ovaries compared to WT ovaries. Several of these are known to play essential roles in cell cycle regulation and apoptosis. For example, a gene necessary for maintaining spindle structure and proper movement of chromosomes during the cell cycle, kinesin family member 11 (Kif11), was significantly increased by 2 fold in the AhRKO ovaries compared to the WT ovaries. This result was confirmed by quantitative RT-PCR (AhRKO=3.32±0.55, WT=1.67±0.15, relative expression level, n=4, p≤0.05). Interestingly, a gene essential for initiation of X chromosome inactivation, inactive X specific transcripts (Xist), was decreased by 2 fold in the AhRKO ovary compared to the WT ovary. This finding was also confirmed by quantitative RT-PCR (AhRKO=0.68±0.04, WT=1.10±0.12 relative expression level, n=5, p≤0.05). We further screened both Kif11 and Xist as candidate target genes for the ligand activated AHR by searching for potential aryl hydrocarbon receptor response elements (AHREs) within the promoter regions of these genes. We found AHREs within the promoter regions of both Kif11 and Xist genes. Collectively, these data suggest that the AHR regulates genes involved in cell cycle and the X chromosome inactivation during early follicle formation. (platform)