Dynamic expression patterns of Irx3 and Irx5 during germline nest breakdown and primordial follicle formation promote follicle survival in mouse ovaries

Abstract

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.

Canonical Wnt/β-catenin activity and differential epigenetic marks direct sexually dimorphic regulation of Irx3 and Irx5 in developing mouse gonads

“…Later in ovarian development, upon germline nest breakdown, Irx3 expression expands to include both somatic cells and oocytes (Fu et al, 2018). β-Catenin is also present in oocytes at this stage as shown by our immunohistochemistry results from ovaries at E14.5 and P7 and supported by previous reports (Figs S1 and S2) (Yan et al, 2019;Bothun and Woods, 2019;Kumar et al, 2016;Usongo et al, 2012;Chassot et al, 2011;Jameson et al, 2012).…”

“…Here, we used in vitro, ex vivo and in vivo approaches to provide a direct link between canonical Wnt/βcatenin signaling and Irx3 and Irx5 expression within the somatic cell population of the developing ovary. In addition, we previously showed that both Iroquois factors emerge in germ cells in late stages of ovarian development (Fu et al, 2018), but here we report that their regulation in this cell type is independent of β-catenin. Based on these data, we developed a model to describe regulation of Irx3 and Irx5 expression within the somatic cell population (Fig.…”

“…Wnt4/Rspo1/β-catenin regulated transcription plays an essential role in ovarian development in somatic cells during sex differentiation. Our lab has previously reported that Irx3 and Irx5 expression increases upon the onset of sex differentiation in the ovary (Fu et al, 2018) and these factors have been linked to canonical Wnt/β-catenin signaling in other tissues (Janssens et al, 2010;Naillat et al, 2010Naillat et al, , 2015. Therefore, we hypothesized that canonical β-catenin regulates Irx3 and Irx5 in the somatic cells of the ovary at this time.…”

“…Developmental regulation of these factors within these systems is context specific, as a number of signaling pathways have been described. Recently, we showed that null mutation of both Irx3 and Irx5 resulted in improper somatic-germ cell connections within follicles, which culminated in oocyte death (Fu et al, 2018). Notably, it has previously been reported that the Wnt4 knockout mouse also exhibited physical gaps between germ and somatic cells within follicles (Vainio et al, 1999), suggesting that Wnt and Iroquois factors may lie in the same pathway.…”

“…Previously, we reported that two Iroquois homeobox transcription factors Irx3 and Irx5 are expressed in the ovary, beginning shortly after sex differentiation. Each exhibits a dynamic profile during the course of germline nest establishment and breakdown through primordial follicle formation, suggesting they play important roles in ovarian development (Kim et al, 2011;Fu et al, 2018). Iroquois factors are highly conserved and are known for their roles in patterning and embryogenesis, along with organization of the spinal cord, limb, bone and heart (Bruneau et al, 2001;Diez del Corral et al, 1999;Gómez-Skarmeta et al, 2001;Gómez-Skarmeta and Modolell, 2002;Lovrics et al, 2014).…”