Women and other mammalian females are born with a finite supply of oocytes that determine their reproductive lifespan. During fetal development, individual oocytes are enclosed by a protective layer of granulosa cells to form primordial follicles that will grow, mature, and eventually release the oocyte for potential fertilization. Despite the knowledge that follicles are dysfunctional and will die without granulosa cell-oocyte interactions, the mechanisms by which these cells establish communication is unknown. We previously identified that two members of the Iroquois homeobox transcription factor gene family, Irx3 and Irx5, are expressed within developing ovaries but not testes. Deletion of both factors (Irx3-Irx5EGFP/Irx3-Irx5EGFP) disrupted granulosa cell-oocyte contact during early follicle development leading to oocyte death. Thus, we hypothesized that Irx3 and Irx5 are required to develop cell-cell communication networks to maintain follicle integrity and female fertility. A series of Irx3 and Irx5 mutant mouse models were generated to assess roles for each factor. While both Irx3 and Irx5 single mutant females were subfertile, their breeding outcomes and ovary histology indicated distinct causes. Careful analysis of Irx3- and Irx5-reporter mice linked the cause of this disparity to dynamic spatio-temporal changes in their expression patterns. Both factors marked the progenitor pre-granulosa cell population in fetal ovaries. At the critical phase of germline nest breakdown and primordial follicle formation however, Irx3 and Irx5 transitioned to oocyte- and granulosa cell-specific expression respectively. Further investigation into the cause of follicle death in Irx3-Irx5EGFP/Irx3-Irx5EGFP ovaries uncovered specific defects in both granulosa cells and oocytes. Granulosa cell defects included poor contributions to basement membrane deposition and mis-localization of gap junction proteins. Granulosa cells and oocytes both presented fewer cell projections resulting in compromised cell-cell communication. Altogether, we conclude that Irx3 and Irx5 first work together to define the pregranulosa cell population of germline nests. During primordial follicle formation, they transition to oocyte- and granulosa cell-specific expression patterns where they cooperate in neighboring cells to build the foundation for follicle integrity. This foundation is left as their legacy of the essential oocyte-granulosa cell communication network that ensures and ultimately optimizes the integrity of the ovarian reserve and therefore, the female reproductive lifespan.

Healthy development of ovarian follicles depends on appropriate interactions and function between oocytes and their surrounding granulosa cells. Previously, we showed that double knockout of Irx3 and Irx5 (Irx3/5 DKO) in mice resulted in abnormal follicle morphology and follicle death. Further, female mouse models of individual Irx3 or Irx5 knockouts were both subfertile but with distinct defects. Notably, the expression profile of each gene suggests independent roles for each, first they are colocalized in pre-granulosa cells during development that then progresses to include oocyte expression during germline nest breakdown and primordial follicle formation. Thereafter, their expression patterns diverge between oocytes and granulosa cells coinciding with the formulation and maturation of intimate oocyte-granulosa cell interactions. The objective of this study was to investigate the contributions of Irx5 and somatic cell-specific expression of Irx3 during ovarian development. Our results show that Irx3 and Irx5 contribute to female fertility through different mechanisms and that Irx3 expression in somatic cells is important for oocyte quality and survival. Based on evaluation of a series of genetically modified mouse models, we conclude that IRX3 and IRX5 collaborate in the same cells and then in neighboring cells to foster a healthy and responsive follicle. Long after these two factors have extinguished, their legacy enables these intercellular connections to mature and respond to extracellular signals to promote follicle maturation and ovulation.

Appropriate embryo-uterine interactions are essential for implantation. Besides oocyte abnormalities, implantation failure is a major contributor to early pregnancy loss. Previously, we demonstrated that two members of the Iroquois homeobox transcription factor family, IRX3 and IRX5, exhibited distinct and dynamic expression profiles in the developing ovary to promote oocyte and follicle survival. Elimination of each gene independently caused subfertility, but with different breeding pattern outcomes. Irx3 KO (Irx3LacZ/LacZ) females produced fewer pups throughout their reproductive lifespan which could only be partially explained by poor oocyte quality. Thus, we hypothesized that IRX3 is also expressed in the uterus where it acts to support pregnancy. To test this hypothesis, we harvested pregnant uteri from control and Irx3 KO females to evaluate IRX3 expression profiles and the integrity of embryo implantation sites. Our results indicate that IRX3 is expressed in the endometrial stromal cells at day 4 of pregnancy (D4) with peak expression at D5-6, and then greatly diminishes by D7. Further, histology and immunofluorescence at D7 showed that while embryos were able to attach to the uterus, implantation sites in Irx3 KO pregnant mice exhibited impaired vascularization, abnormal expression of decidualization markers, and disruptions in GJA1 organization in the decidual bed. Finally, we also observed an impaired response of the Irx3 KO uteri to an artificial deciduogenic stimulus, indicating a critical role of this factor in regulating the decidualization program. Together, these data established that IRX3 promotes female fertility via at least two different mechanisms: 1) promoting competent oocytes and 2) facilitating functional embryo-uterine interactions during implantation.

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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 i...