REGULATION OF OVIDUCT HOMEOSTASIS AND FERTILITY BY Pax2 AND Pax8 GENES

Abstract

In mouse and humans, the development regulatory genes Pax2 and Pax8 are expressed in ovarian duct surface epithelia, yet the function of these genes and proteins in adult females remains unclear. By Generating double & single KO mouse modules of Pax2 and Pax8 we investigate their roles in maintaining the integrity of the oviductal cells. Our studies directly address aspects of oviduct epithelial homeostasis and the effects on fertility. To create Pax2, Pax8, and Pax2/8 double mutants specifically in oviduct epithelia and characterize changes in gene expression, epithelial cell integrity, and fertility. We will implement detailed analyses of oviducts isolated at various times post tamoxifen administration from mice with conditional Pax2, Pax8, or both alleles, using the Ovigp1-CreER driver. Mice will also carry reporter allele (Gt(ROSA)26Sortm4(ACTB-tdTomato,-EGFP)Luo) to mark all active Cre expressing cells with cell surface EGFP. In addition to defining novel functions for Pax proteins in the oviduct, we will utilize state-of-the-art methods for single cell analyses, transcriptomics, and chromatin remodeling. Mutation of Pax2 and Pax8 in adult female oviducts results in significant changes in gene expression and morphology. Loss of both Pax2 and Pax8 resulted in infertility (Table 1). To date, most of the preliminary data was obtained from Pax2/8 double mutants since we were concerned with redundancy and expected the most significant phenotypes in the double mutants. Analyses of histology, immunohistochemistry and changes in gene expression of total RNA from whole oviducts isolated after tamoxifen administration. For a rapid first pass screen, we compared controls (Pax2f/f;Pax8f/f) to single Pax mutants and Pax2/8 double mutants (Pax2f/f;Pax8f/f; Ovigp1-CreER) by Affymetrix microarrays. These data show hundreds of changes in gene expression levels upon Pax2 or Pax8 deletion, with significant overlap between the Pax2 and Pax8 mutants but also expression changes unique to each single mutant.Tabled 1TABLE 1. Fertility of Oviduct Specific Pax MutantsGenotype # females # plugs Litters Litter sizePax2f/f, Pax8f/f, Ovi-Cre 5 15 0 0Pax2f/f, Pax8f/f, 4 12 12 6-9Pax8f/f, Ovi-Cre 3 6 2 6-7 Open table in a new tab Little is known regarding the mechanisms underlying epithelial homoeostasis, the proteins that determine cell fates, and epithelial integrity in the adult oviduct. Pax8 expressing secretory cells are thought to give rise to ciliated cells, which help move the oocyte down the duct and Pax2 is associated with cilia motility and physiology. Using a conditional KO model of Pax2 & Pax8 will allow us to understand those mechanisms and identify future novel therapeutic targets for diagnostics and treatments