MON-229 IRX3 and IRX5 Regulate Downstream Targets that Promote Ovarian Follicle Integrity in Mice

Appropriate cell-cell interactions and communication within ovarian follicles are required for their healthy development and maturation and are critical for maintaining female reproductive life quality. Our lab previously showed that two Iroquois transcription factor family genes, Irx3 and Irx5, were important for ovarian follicle integrity. Deletion of Irx3 and Irx5 (Irx3(-)Irx5(EGFP)/Irx3(-)Irx5(EGFP), Irx3/5 DKO) resulted in disrupted granulosa cell-oocyte contacts, ectopic gap junction protein expression, and abnormal basement membrane morphology. The mechanisms by which Irx3 and Irx5 promote follicle integrity, however, are unknown. Therefore, we performed RNA-seq experiments on control and Irx3/5 DKO ovarian samples at two critical phases of ovarian development: embryonic day 13.5 when germline nests are established and just after birth when germline nests break down and primordial follicles form. RNA-seq analyses revealed that Inhbb, a TGF-β superfamily gene, was significantly upregulated in Irx3/5 DKO samples at both time points. It was previously reported that Inhbb was also upregulated in Wnt4 and β-catenin mutant ovaries. Notably, similar to the Irx3/5 DKO ovary, cell-cell interaction failure and germ cell death are prominent features of these models and removal of the excessive Inhbb by breeding to Inhbb knockout animals rescued the defects. These data suggest that Inhbb must be blocked to maintain germ cell and follicle survival. Based on the similarities in ovarian phenotype and Inhbb upregulation resulting from both Wnt4/β-catenin and Irx3/5 mutant models, we hypothesized that Irx3 and Irx5 mediate the Wnt4/β-catenin repression of Inhbb while they also regulate the expression of other downstream target genes required for cell contacts and germ cell health within follicles. First, we used gain- and loss-of-function gonad culture paradigms to manipulate β-catenin activity in ovaries and testes, respectively. Results showed that Irx3 and Irx5 expression levels were positively regulated by β-catenin signaling within the canonical Wnt4 pathway. Next, additional analysis of the RNA-seq data using the Database for Annotation, Visualization and Integrated Discovery (DAVID) showed that the differentially expressed genes (DEGs) in Irx3/5 DKO samples included factors critical to extracellular matrix, cell projections, actin-binding and cell adhesion. We are currently validating the DEGs relevant to our Irx3/5 DKO follicle defects and investigating their relationship to Inhbb in ovaries. Results from this study will help us to discover the means by which initial cell contacts are formed between support cells and oocytes in germline nests and new primordial follicles. These interactions provide the foundation for follicle integrity within the ovarian reserve and ultimately ensure that healthy follicles are available for recruitment and maturation.