WNT/β-catenin and p27/FOXL2 differentially regulate supporting cell proliferation in the developing ovary

Abstract

Sexual development is initiated through differentiation of testicular Sertoli cells or ovarian granulosa cells. Although these supporting cells are considered to develop from common bipotential precursors, recent evidence suggests that distinct supporting cell populations are present in the ovary, with one providing granulosa cells of the medullary follicles and the other providing granulosa cells of the cortical follicles, the latter of which support lifelong fertility. Here, we demonstrate that XX fetal gonads contain GATA4 expressing supporting cells that either enter mitotic arrest, or remain proliferative. Blocking WNT signalling reduces XX supporting cell proliferation, while stabilising β-catenin signalling promotes proliferation, indicating that the renewal of pre-granulosa cells is dependent on WNT/β-catenin signalling in the proliferative supporting cell population. In contrast, XX supporting cells express p27 and FOXL2 and are maintained in mitotic arrest. Although FOXL2 is required for maintaining high levels of p27 expression, it is dispensable for entry and maintenance of mitotic arrest in XX supporting cells. Combined our data suggest that both medullary and cortical precursors arise from a common GATA4 expressing cell type. In addition, this work indicates that a balance between supporting cell self-renewal and differentiation is maintained in the developing ovary by relative WNT/β-catenin and p27/FOXL2 activities. This study provides significant new insights into the origin and formation of ovarian follicles and evidence supporting a common fetal origin of medullary and cortical granulosa cells.

FGF9, activin and TGFβ promote testicular characteristics in an XX gonad organ culture model

“…Consistent with the ability of β-catenin to inhibit testis development, GSKi also blocked the formation of laminin-delineated testis cords and the occurrence of SOX9 and AMH-positive cells in E11.5 gonads treated with FAT. However, in a related study, blocking GSK in XX foetal gonads increased supporting cell proliferation in the presence of FGF9 (Gustin et al 2016). Taken together, these observations indicate that although β-catenin blocks FAT induction of testis development, it does so by blocking growth factor induction of testis differentiation genes rather than restricting supporting cell proliferation.…”

“…For analyses of cell proliferation in cultured gonads, EdU was added to the organ culture media at a final concentration of 20 μM for the final 2 h of culture, after which gonads were collected, dissociated into single cells and fixed with 4% PFA in PBS. Flow cytometry was carried out essentially as described , Gustin et al 2016) using cells from 0.5 to 2 gonads/sample, depending on age. For example, in vivo analysis of Sertoli cell proliferation (Fig.…”

“…1) was performed against negative control tissues that did not express that protein or was not treated with EdU: mesonephros or tail (p27 (CDKN1B), STRA8, DPPA4, γH2AX, GATA4 and EdU) or gonad of the opposite sex (SOX9, AMH and FOXL2), depending on the antibody used. Cell cycle analysis was performed using propidium iodide to determine DNA content and EdU incorporation during S-phase to determine proliferation , Gustin et al 2016. Mean individual germ cell staining intensities were acquired using area under the curve in either Pacific Blue 405 nm (405-A) or Alexa-fluor 647 nm (647-A) (Life Technologies) channels for the whole germ cell population isolated using Oct4-GFP fluorescence or MVH staining in the 488 nm channel.…”

“…During early ovarian development, proliferative GATA4-expressing cells activate expression of the cell cycle inhibitor p27KIP1 (p27) and FOXL2 as they form non-proliferative granulosa cells (Mork et al 2012, Rastetter et al 2014, Gustin et al 2016. Although loss of FOXL2 function does not result in female-tomale sex reversal in humans, individuals carrying FOXL2 mutations are susceptible to primary ovarian insufficiency (Crisponi et al 2001).…”

Differential expression of Toll-like receptors in goat dominant and nondominant follicles

“…In addition, previous studies have shown that a somatic missense mutation in the FOXL2 gene, which is expressed in the ovary, have a high correlation to granulosa cell tumors (Schmidt et al, 2004;Gustin et al, 2016). The FOXL2 mutant has also been found to affect the hormone production, apoptosis, and proliferation in vitro (Leung et al, 2016).…”