- Research article
- Open Access
- Open Peer Review
APC2 is critical for ovarian WNT signalling control, fertility and tumour suppression
- Noha-Ehssan Mohamed1, 2, 3,
- Trevor Hay1,
- Karen R. Reed1,
- Matthew J. Smalley†1Email authorView ORCID ID profile and
- Alan R. Clarke^†1
© The Author(s). 2019
- Received: 1 February 2019
- Accepted: 24 June 2019
- Published: 10 July 2019
Canonical WNT signalling plays a critical role in the regulation of ovarian development; mis-regulation of this key pathway in the adult ovary is associated with subfertility and tumourigenesis. The roles of Adenomatous polyposis coli 2 (APC2), a little-studied WNT signalling pathway regulator, in ovarian homeostasis, fertility and tumourigenesis have not previously been explored. Here, we demonstrate essential roles of APC2 in regulating ovarian WNT signalling and ovarian homeostasis.
A detailed analysis of ovarian histology, gene expression, ovulation and hormone levels was carried out in 10 week old and in aged constitutive APC2-knockout (Apc2−/−) mice (mixed background). Statistical significance for qRT-PCR data was determined from 95% confidence intervals. Significance testing was performed using 2-tailed Student’s t-test, when 2 experimental cohorts were compared. When more were compared, ANOVA test was used, followed by a post-hoc test (LSD or Games-Howell). P-values of < 0.05 were considered statistically significant.
APC2-deficiency resulted in activation of ovarian WNT signalling and sub-fertility driven by intra-ovarian defects. Follicular growth was perturbed, resulting in a reduced rate of ovulation and corpora lutea formation, which could not be rescued by administration of gonadotrophins. Defects in steroidogenesis and follicular vascularity contributed to the subfertility phenotype. Tumour incidence was assessed in aged APC2-deficient mice, which also carried a hypomorphic Apc allele. APC2-deficiency in these mice resulted in predisposition to granulosa cell tumour (GCT) formation, accompanied by acute tumour-associated WNT-signalling activation and a histologic pattern and molecular signature seen in human adult GCTs.
Our work adds APC2 to the growing list of WNT-signalling members that regulate ovarian homeostasis, fertility and suppress GCT formation. Importantly, given that the APC2-deficient mouse develops tumours that recapitulate the molecular signature and histological features of human adult GCTs, this mouse has excellent potential as a pre-clinical model to study ovarian subfertility and transitioning to GCT, tumour biology and for therapeutic testing.
- APC hypomorph
- WNT signalling
- Ovarian fertility
- Ovarian cancer
- Granulosa cell tumour
The canonical WNT signalling pathway is central to numerous biological processes and diseases . Within the ovary, the pathway has been shown to be essential for female sex differentiation during embryogenesis [2–9], however, in the adult ovary its role is less well defined. Conditional deletion of β-catenin within murine granulosa cells of antral follicles did not affect folliculogenesis or ovulation [10, 11], but its removal within oviducts and uteri led to abnormalities therein, with lack of implantation sites rendering mice infertile as a result . Conditional deletion of Wnt4 in ovarian granulosa cells or germline deletion of Fzd4 in mice caused sub-fertility or complete infertility respectively [12, 13], but WNT signalling activity was not measured and it is unclear whether the reported phenotypes were caused by impaired ovarian canonical WNT signalling or by other mechanisms, potentially including non-canonical pathways. In mice with germline deletion of the WNT signalling agonist Fzd1,
17.6% of female mice were infertile and characterized by early follicle depletion, but with no concomitant change in total activated β-catenin levels . Over-activation of canonical WNT signalling also has deleterious effects on ovarian homeostasis. Ovarian amplification of Rspo1 , deletion of Wnt5a (antagonist of canonical WNT signalling)  or expression of dominant stable β-catenin [10, 17] all resulted in up-regulated ovarian WNT signalling and ovarian subfertility caused by disruption of follicle growth [16, 17], ovulation and luteinisation [10, 15]. Taken together, these findings indicate the importance of tight regulation of canonical WNT signalling in growing follicles.
Human ovarian tumours are classified into epithelial ovarian cancers (90%), sex cord-stromal tumours (7%) and germ cell tumours (3%). Granulosa cell tumours (GCTs), which originate from granulosa cells of ovarian follicles, account for more than half of sex cord-stromal tumours . WNT signalling mis-regulation has been implicated in adult GCT formation, as several studies have demonstrated increased β-catenin protein levels therein, with nuclear localisation in some cases [17, 19, 20]. A recent molecular study of GCTs showed epigenetic silencing of DKK3, the gene coding for the WNT-signalling antagonist Dickkopf, implying a need for WNT signalling activation in GCT development (25, 26). Furthermore, GEMMs in which WNT signalling was activated via the introduction of a gain-of-function mutation of R-spondin1 , or a degradation-resistant β-catenin , resulted in 15.8% or 57% of mice developing adult GCTs respectively.
Here, for the first time, we address the importance of APC2 in ovarian folliculogenesis, fertility and GCT formation. The ability of APC2 to regulate the β-catenin/WNT signalling pathway has been demonstrated in Drosophila and in cancer cell lines [21–25]. Structurally, APC2 possesses AXIN1 and β-catenin binding sites, which enable it to destabilize β-catenin, targeting it for degradation and suppressing its transcriptional activity [22, 26], in addition to the APC-basic domain which enables it to regulate cytoskeleton and microtubule association [27–31] and spindle anchoring during mitosis . Importantly, however, in an in vivo setting, APC2-dependent regulation of WNT signalling is tissue-specific, occurring in the liver and intestine but not in the mammary gland [33, 34]. Little is known about how APC2 functions in adult ovaries, but APC2 loss has been reported in epithelial ovarian cancer [28, 35]. Here, we show that Apc2-knockout mice  have a subfertility phenotype associated with an activation of ovarian WNT signalling, and that, on a hypomorphic Apc background [37, 38], loss of APC2 increases the incidence of ovarian GCTs which recapitulate the histologic pattern and molecular signature of human adult GCTs. Not only does this study extend our understanding of the tissue-specific regulation of WNT signalling, but also the APC2-deficient mouse has excellent potential as a pre-clinical model to study ovarian tumour biology and for therapeutic testing.
Animal models, fertility and ovulation rate
All experiments were carried out under the authority of UK Home Office personal and project licences and according to ARRIVE guidelines and following local ethical review. Mouse models were maintained on a mixed C57Bl6/J and 129/Ola background in open cages with ad libitum access to food and water. Genotyping for the constitutive knockout allele of Apc2 (Apc2−) and the hypomorphic allele of Apc (Apcfl) [34, 36–38] were performed as previously described [34, 36] (Additional file 1: Table S1). Typically, experiments compared wild type, heterozygous Apc2-deleted and homozygous-Apc2 deleted mice, with a minimum of three animals per groups, unless otherwise specified. The breeding defect of Apc2 knockout animals made it difficult, in some cases, to use large n numbers for analysis; where this is the case it has been clearly indicated in the text. Animals were euthanased for analysis of ovarian tissue by an approved humane method (cervical dislocation) at the times indicated (typically 10 weeks old for functional analysis and 12 or 18 months for tumour studies).
To assess female fertility, retrospective analysis of breeding performance was analysed from cages in which two 7–11 week-old female mice of the experimental genotypes (Apc2+/+, Apc2+/− and Apc2−/−) were housed with a 7–9 week-old male of the same genotype for 3 months (n = 4 cages). Litter sizes were determined at the time of weaning.
To determine ovulation rates, 10 week-old female mice were super-ovulated by a single intraperitoneal injection of 5 IU pregnant mare’s serum gonadotrophin (MSD animal health, UK), followed by 5 IU human chorionic gonadotrophin (MSD animal health, UK), 47 h later . Mice were either euthanased 16–17 (for Cumulus Oophorus Complex retrieval)  or 22–24 h later (for histological analysis) by an approved humane method (cervical dislocation).
Cumulus Oophorus complex (COC) retrieval and characterization
After release from the oviducts, COCs were counted and examined by bright-field microscopy to assess morphology. Oocytes were freed from surrounding cumulus cells by addition of 40 μl of 4 mg/ml collagenase/dispase (Roche, Switzerland), dissolved in DMEM/F12 medium (Mediatech, USA), for 10 min, and examined to determine their integrity  and to measure their diameter .
Histological analysis of ovaries
Follicle counting was performed on ovaries from 10-week-old Apc2+/+ and Apc2−/− mice, either from randomly cycling females staged manually (using the vaginal cytology method) and collected at diestrus stage (n = 4) or 22–24 h post HCG administration (n = 5). Each ovary was serially-sectioned into 100 5 μM sections and each 10th section was stained with H&E. Growing follicles were counted every 10th section, when an oocyte nucleus was visible. Identification and classification of growing follicles and atretic follicles were performed as previously described [43, 44]. The total number of follicles throughout the 10 counted sections was used. Follicle sizes were measured using a minimum of 4 diameters/follicle.
Serum hormonal levels were measured in 10-week-old Apc2+/+ and Apc2−/− mice at diestrus stage using ELISA kits for FSH (Novateinbio, USA) and LH (Enzo Lifesciences, UK).
Tissue sectioning and immunohistochemistry were performed as previously described , using primary antibodies listed in Additional file 1: Table S2. Sections were examined with an Olympus BX43 light microscope and microphotographs taken using a 5 Megapixel HD Microscope Camera (Leica MC170 HD, Germany).
Quantitative RT-PCR analysis
RNA was extracted from whole ovaries or tumour pieces using RNeasy Plus mini extraction kit (Qiagen, Germany) and reverse transcription performed using QuantiTect Reverse transcription kit (Qiagen, Germany). All quantitative real time rtPCR assays were carried out three times using TaqMan® universal master mix II with UNG (Applied Biosystems, USA), Taqman® assays (Additional file 1: Table S3) and QuantStudio™ 7 Flex Real Time PCR system (ThermoFisher, USA), and relative expression levels determined using QuantStudio™ 7 Real Time PCR software.
Statistical significance for qRT-PCR data was determined from 95% confidence intervals . All other statistical analyses were performed using IBM SPSS version 20 (SPSS Inc., Chicago, IL, USA). Significance testing was performed using 2-tailed Student’s t-test, when 2 experimental cohorts were compared. When more were compared, ANOVA test was used, followed by a post-hoc test (LSD or Games-Howell). P-values of < 0.05 were considered statistically significant.
APC2 deficiency results in sub-fertility
Histology of ovaries, oviducts and uteri from 10-week-old virgin Apc2+/+ and Apc2−/− mice revealed no gross morphological differences in the oviducts and uteri (representative images in Additional file 2: Figure S1). No problems were reported during labour in any of the experimental groups; it is therefore unlikely that uterine problems contribute to the observed subfertility phenotype. However, there was a significant decrease in the number of corpora lutea formed in Apc2−/− ovaries (Fig. 1d, e & f), while the total number of growing follicles was increased, but not significantly (Fig. 1g). Morphometric and histochemical analysis of corpora lutea did not reveal any histological differences in these structures between Apc2+/+ and Apc2−/− ovaries (Additional file 3: Figure S2). Collectively, these findings suggest reduced ovulation is the cause of the subfertility observed in APC2-deficient mice.
Subfertility in APC2-deficient female mice is caused by intra-ovarian defects
Given the constitutive nature of the Apc2 gene deletion in our mice, the genotype dose-dependent reduction in fertility, potentially as a result of an ovulation defect, may be due to defects in extra-ovarian regulation of ovarian function, triggered by hypothalamic/pituitary endocrine signals. To address this, follicle stimulating hormone (FSH) and luteinizing hormones (LH) levels in serum from 10-week old virgin Apc2+/+ and Apc2−/− female mice at diestrus stage were analysed by ELISA, but showed no differences (Additional file 4: Figure S3)a, b, suggesting hypothalamic/pituitary signals are not affected by Apc2 deletion.
Importantly, histological analysis post-superovulation demonstrated a significant reduction in the number of corpora lutea in super-ovulated Apc2−/− ovaries compared to Apc2+/+ (Fig. 2 d,e&f). As with unstimulated ovaries, a slight, but non-significant, increase in the number of healthy growing follicles in Apc2−/− ovaries was observed (Fig. 2d,e&g). Taken together, these findings suggest that the subfertility phenotype seen in APC2-deficient female mice is not due to extra-ovarian defects in pituitary gonadotrophin secretion, but rather due to intra-ovarian defects in response to gonadotrophins that result in reduced ovulation. Therefore, expression levels of the ovarian gonadotrophin receptors Fshr and Lhcgr, together with the steroid hormone receptors Pgr, Esr1, Esr2 and Ar, were assessed in ovaries from Apc2+/+ and Apc2−/− mice. Significant over-expression of Lhcgr was evident in Apc2−/− ovaries (Fig. 2h), but the other receptors were unaltered. Importantly, the LH receptor is a target of canonical WNT signalling , and its over-expression has previously been associated with infertility in mice .
APC2 deficiency activates ovarian WNT signalling and upregulates Foxo1 expression
Given the established role of the FOX family of transcription factors as regulators of apoptosis within ovarian granulosa cells [48–50], and their increased expression in granulosa cells of cultured follicles post-WNT signalling activation [51, 52], gene expression levels for Foxo1, Foxo3 and Foxl2 were analysed in Apc2+/+ and Apc2−/− whole ovaries. A significant increase in Foxo1 expression levels was seen in Apc2−/− ovaries (Fig. 4c). Furthermore, the FOXO target genes Bcl6 and Cdkn1b were significantly upregulated in Apc2−/− ovaries compared to controls (Fig. 4d).
The PTEN/PI3K/AKT signalling pathway is an established regulator of FOXO transcriptional activity and post-translational modification . On activation of AKT, FOXO proteins are inactivated by phosphorylation and translocated from nucleus to cytoplasm . In addition, the crosstalk between activated WNT signalling and PTEN, causing the over-expression of the latter, is well established [16, 17, 54]. IHC analysis of PTEN, p-AKT and p-FOXO1,3,4 in Apc2+/+ and Apc2−/− ovaries revealed that PTEN expression was stronger in theca and granulosa cells of Apc2−/− follicles (Fig. 4e). This was accompanied by a reduction in p-AKT immunostaining in Apc2−/− granulosa cells (Fig. 4f) and a consequent reduction in p-FOXO1,3,4 levels (Fig. 4g). Thus, the increased apoptosis seen in Apc2−/− follicles is likely due to upregulation of Foxo1 and its downstream effector genes, secondary to decreased activation of PI3K/p-AKT signalling caused by PTEN upregulation.
APC2-deficient ovaries show impaired vascularisation and steroidogenesis
Negative regulation of follicle steroidogenesis by canonical WNT signalling has also previously been demonstrated . We therefore examined the expression of key enzymes required for steroidogenesis in Apc2 knockout ovaries. We found there was significantly reduced expression of both Cyp17a1 (coding for steroid 17-α-hydroxylase/17,20 lyase) and Cyp19a1 (coding for aromatase) in Apc2−/− ovaries compared to Apc2+/+ ovaries (Fig. 5c).
Therefore, activation of WNT signalling in Apc2 knockout ovaries results in overexpression of PTEN and a reduction in activity of steroidogenesis and angiogenic pathways. These metabolic defects combine to result in a reduced number of follicles maturing to the ovulatory stage.
Long-term activation of WNT signalling by APC/APC2 deficiency, results in ovarian adult granulosa cell tumour formation
Frequency of GCT formation in 12 and 18-month-old Apc2 experimental genotypes on the background of Apcfl/fl. *One animal developed bilateral tumours
Frequency of ovarian GCT
To determine whether active canonical WNT signalling was associated with GCT formation, β-catenin staining was carried out. Tumour cells strongly expressed β-catenin in contrast to the expression seen in non-tumour areas (Fig. 8; compare β-catenin staining in tumour area indicated by black arrow with non-tumour area indicated by red arrow). Due to limitation of available tumour samples, qRT-PCR analysis could only be performed on two WNT signalling target genes (Wif1 and Axin2) using RNA from two Apc2-deficient GCTs, with Apc2-proficient ovaries used as control material. Two independent areas from each tumour were analysed to allow for tumour heterogeneity. Comparison of expression levels demonstrated higher levels of both Wif1 and Axin2 within APC2-deficient tumours (Additional file 6: Figure S5)c,d.
Activation of PI3K/AKT signalling via Pten deletion has been shown to enhance GCT development and progression in mouse models driven by WNT signalling activation [63, 64]. However, phospho-AKT (p-AKT), a marker of active PI3K/AKT signalling, was undetectable within our GCTs (Fig. 8). Furthermore, Apc2+/− and Apc2−/− GCTs showed strong PTEN staining, in contrast to no staining in the Apc2+/+ tumour (Fig. 8). This likely explains the lack of pAKT staining in the APC2-deficient tumours. However, the lack of both pAKT and PTEN staining in the Apc2+/+ tumour may result from the hypomorphic APC allele on its own being an insufficiently strong driver of WNT signalling to activate PI3K/AKT signalling via established cross-talk mechanisms .
Estrogen receptor alpha (ERα) also showed differential staining between APC2-deficient tumours compared to the APC2-proficient tumour analysed. Human ovarian GCTs are also characterized by frequent focal staining for estrogen receptor alpha (ERα) (Fig. 8; Additional file 6: Figure S5)e which suggests that APC2 deficiency not only increases the frequency of GCTs in mice which also carry a hypomorphic Apc allele, but also results in tumours with a greater histological and molecular similarity to human GCTs.
This study has revealed that APC2-deficiency activates WNT signalling in the ovary during early adulthood, which subsequently disrupts ovarian homeostasis and causes subfertility originating from an ovarian defect. Follicle growth was perturbed in APC2-deficient mice secondary to defective response to gonadotrophins, reduced follicular vascularity, downregulation of genes coding for steroidogenic enzymes and upregulation of Foxo1 expression, which contributed to increased apoptosis of granulosa cells in APC2-deficient follicles. At least 20% of APC2-deficient female mice (on the background of a hypomorphic Apc allele) go on to develop WNT-driven GCT as early as 12 months. These tumours recapitulated human adult GCT histology and molecular features.
Our findings highlight the role of APC2 as an important regulator of WNT signalling in the ovary. Although initial studies performed in Drosophila and on cell lines to functionally-characterize APC2 demonstrated the presence of β-catenin and AXIN1 binding sites in APC2, which enable it to regulate WNT signalling [22, 23, 26, 66–68], in an in vivo mammalian setting, APC2 function is tissue specific. APC2 loss in the mouse small intestine and liver resulted in activation of WNT signalling but not in the mammary glands [33, 34]. Hence, the functions of APC2 cannot be extrapolated from one mammalian system to another without direct experimentation.
The tumour suppressor role of APC2 protein in ovarian granulosa cell tumour formation has been highlighted here for the first time and the current study provides further evidence of the roles of WNT signalling activation in the pathogenesis of ovarian GCT. These findings build on previous work pointing to this role of WNT signalling in clinical data [17, 19, 20, 69], and in GEMMs [15, 17] but as noted above, given the tissue-specific effects of APC2 knockout, could not have been predicted a priori.
The current findings also extend our knowledge of deleterious effects of WNT signalling activation on ovarian homeostasis and fertility [10, 15–17]. We have shown that reduced ovulation observed in APC2-deficient mice is not caused by defects in ovulation and terminal differentiation of granulosa cells (which happen when WNT signalling is activated in antral follicles), but rather caused by restricted follicular growth and failure to reach the pre-ovulatory stage. This phenotype is similar to previous phenotypes published when WNT signalling was activated in pre-antral follicles [16, 17], implying that APC2 activity is required in growing follicles as early as the pre-antral stage.
Given the constitutive nature of the Apc2 null allele, both autonomous and non-autonomous mechanisms are expected to contribute to the phenotypes described. Results of the current study have clearly shown the intra-ovarian origin of the subfertility phenotype described in APC2-deficient mice, and that hypothalamic-pituitary regulation of ovarian function is not contributing to the subfertility phenotype. Although the subfertility is caused by increased apoptosis of granulosa cells, a contribution of endothelial cells to the phenotype was evident. Whether the same phenotype could be reproduced if APC2-deletion was targeted exclusively to granulosa cells (e.g. using Amhr2 or Cyp19a-cre) remains unknown, due to the unavailability of an Apc2 conditional allele. The same applies to GCTs developing in APC2-deficient mice, which – in contrast – displayed enhanced angiogenesis.
It is unlikely that WNT signalling activation is the sole driver of the reported phenotypes and cross talk between WNT signalling and other signalling pathways must also be considered. For example, unlike in early adulthood, FOXO1 expression was absent in APC2-deficient GCT, implying a need to silence FOXO1 and to stop FOXO1-driven granulosa cell apoptosis as a prerequisite for tumourigenesis. It has been previously shown that knocking out Foxo1/Foxo3 leads to the development of GCT in 20% of female mice . However, the cause of the ‘switch’ from FOXO1 being present and granulosa cell apoptosis to absent FOXO1 with granulosa cell proliferation and tumourigenesis was not identified and needs to be further characterized. The high levels of PTEN in granulosa cells of growing follicles might have contributed to increased apoptosis by inhibiting the translocation of FOXO1 outside the nucleus and thus ensuring FOXO1 activates pro-apoptotic target genes. In addition, high PTEN expression levels found in GCT of APC2-deficient ovaries might be responsible for the late development of tumourigenesis, as previously described in other models [60, 63, 64]. It is thus possible to hypothesize that, similar to previously published models, deleting Pten in granulosa cells of APC2-deficient ovaries would lead to rapid tumour development.
This study has caveats. One limitation was that the breeding data available for different genotypes of female Apc2 mice (Apc2+/+, Apc2+/−, Apc2−/−) represented crossings to males of the corresponding genotype, rather than to wild type males. Effects of Apc2-gene dosage on male fertility are not yet characterized, with the caveat that male fertility might be affected in APC2-deficient male mice, and could contribute to the delayed pregnancy and reduced litter size observed in APC2-deficient crosses. However, retrieval and counting of ovulated oocytes post-gonadotrophin administration confirmed that APC2-deficient female mice ovulate less and would be expected to give smaller litter size. Impairment of response to gonadotrophin is mediated by overexpression of Lhcgr, which has been recently reported to cause complete infertility in female mice, with histological analysis revealing that follicles failed to progress beyond the pre-antral stage . Over-expression of Lhcgr in APC2-deficient mice most likely occurs due to canonical WNT signalling activation, as a 3.5-fold increase in Lhcgr expression levels has been reported in granulosa cells transduced with constitutively-active β-catenin, in the presence of FSH . In addition, this early elevation of Lhcgr expression might have contributed to GCT development . Another important caveat to this study was the small numbers of aged Apc2−/− mice available for tumour development studies. This was, unfortunately, an unavoidable consequence of the reduced fertility phenotype in these animals.
This study advances our understanding of the role of WNT signalling in ovarian homeostasis and tumourigenesis, and of the role played by APC2 in regulating this pathway. The finding that WNT signalling activation in growing follicles impairs ovulation raises the importance of the assessment of WNT signalling activation in the setting of human female subfertility/infertility. This could provide new insights into the molecular pathogenesis of this condition, and may help in designing new treatment interventions for these patients. Furthermore, our findings extend the list of mutations which cause female subfertility or infertility in early adulthood in mice followed by development of GCT upon aging [15, 17, 47, 60]. It remains to be determined if a similar link exists in humans and, if so, what are the molecular drivers, but APC2 must now be included on the list of candidates which should be investigated in this clinical context. Furthermore, the direct mechanistic link between WNT signalling activation, β-catenin stabilisation and GCT formation warrants further investigation.
We thank Professor Hans Clevers for providing us with Apc2-/- mouse, Professor Owen Sansom for critically reviewing the results, Professor Geraint Williams for training and guidance on histopathological assessment of ovaries, Elaine Taylor for assistance with mouse husbandry, Mark Bishop and Matthew Zverev for technical support and genotyping and Derek Scarborough for histologic preparation of tissues.
This project was funded by Egyptian Ministry of Higher Education (represented by the Egyptian Educational Bureau in London) and Cancer Research UK (ARC Programme grant C1295/A15937).
The research project was designed by NM, TH, MJS and ARC. NM, TH and KRR managed the mouse intercrosses. NM performed all data collection and analysis. The manuscript was drafted by NM and KRR. This manuscript is dedicated to the memory of the late Professor Alan Clarke. All other authors critically reviewed the manuscript and approved the final version submitted.
Ethics approval and consent to participate
Patient studies – not applicable, no human subjects involved. Animals studies – all experiments were carried out under the authority of UK Home Office personal and project licences (30/2737 and 30/3279) and according to ARRIVE guidelines and following local ethical review by the Cardiff University Animal Welfare Ethical Review Panel.
Consent for publication
Not applicable, no human subjects involved.
The authors declare that they have no competing interests. This study was previously made available via a preprint server (http://biorxiv.org/cgi/content/short/516286v1).
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
- Clevers H, Nusse R. Wnt/β-catenin signaling and disease. Cell. 2012;149(6):1192–205.PubMedView ArticleGoogle Scholar
- Biason-Lauber A, Chaboissier MC. Ovarian development and disease: the known and the unexpected. Semin Cell Dev Biol. 2015;45:59–67.PubMedView ArticleGoogle Scholar
- Chassot A-A, Ranc F, Gregoire EP, Roepers-Gajadien HL, Taketo MM, Camerino G, De Rooij DG, Schedl A, Chaboissier M-C. Activation of β-catenin signaling by Rspo1 controls differentiation of the mammalian ovary. Hum Mol Genet. 2008;17(9):1264–77.PubMedView ArticleGoogle Scholar
- Tomizuka K, Horikoshi K, Kitada R, Sugawara Y, Iba Y, Kojima A, Yoshitome A, Yamawaki K, Amagai M, Inoue A, et al. R-spondin1 plays an essential role in ovarian development through positively regulating Wnt-4 signaling. Hum Mol Genet. 2008;17(9):1278–91.PubMedView ArticleGoogle Scholar
- Ottolenghi C, Pelosi E, Tran J, Colombino M, Douglass E, Nedorezov T, Cao A, Forabosco A, Schlessinger D. Loss of Wnt4 and Foxl2 leads to female-to-male sex reversal extending to germ cells. Hum Mol Genet. 2007;16(23):2795–804.PubMedView ArticleGoogle Scholar
- Maatouk DM, DiNapoli L, Alvers A, Parker KL, Taketo MM, Capel B. Stabilization of beta-catenin in XY gonads causes male-to-female sex-reversal. Hum Mol Genet. 2008;17(19):2949–55.PubMedPubMed CentralView ArticleGoogle Scholar
- Jameson SA, Lin YT, Capel B. Testis development requires the repression of Wnt4 by Fgf signaling. Dev Biol. 2012;370(1):24–32.PubMedPubMed CentralView ArticleGoogle Scholar
- Domenice S, Correa RV, Costa EM, Nishi MY, Vilain E, Arnhold IJ, Mendonca BB. Mutations in the SRY, DAX1, SF1 and WNT4 genes in Brazilian sex-reversed patients. Braz J Med Biol Res. 2004;37(1):145–50.PubMedView ArticleGoogle Scholar
- Tomaselli S, Megiorni F, De Bernardo C, Felici A, Marrocco G, Maggiulli G, Grammatico B, Remotti D, Saccucci P, Valentini F, et al. Syndromic true hermaphroditism due to an R-spondin1 (RSPO1) homozygous mutation. Hum Mutat. 2008;29(2):220–6.PubMedView ArticleGoogle Scholar
- Fan H-Y, O'Connor A, Shitanaka M, Shimada M, Liu Z, Richards JS. β-Catenin (CTNNB1) promotes Preovulatory follicular development but represses LH-mediated ovulation and Luteinization. Mol Endocrinol. 2010;24(8):1529–42.PubMedPubMed CentralView ArticleGoogle Scholar
- Hernandez Gifford JA, Hunzicker-Dunn ME, Nilson JH. Conditional deletion of Beta-catenin mediated by Amhr2cre in mice causes female infertility. Biol Reprod. 2009;80(6):1282–92.PubMedPubMed CentralView ArticleGoogle Scholar
- Boyer A, Lapointe É, Zheng X, Cowan RG, Li H, Quirk SM, DeMayo FJ, Richards JS, Boerboom D. WNT4 is required for normal ovarian follicle development and female fertility. FASEB J. 2010;24(8):3010–25.PubMedPubMed CentralView ArticleGoogle Scholar
- Hsieh M, Boerboom D, Shimada M, Lo Y, Parlow AF, Luhmann UF, Berger W, Richards JS. Mice null for Frizzled4 (Fzd4−/−) are infertile and exhibit impaired corpora lutea formation and function. Biol Reprod. 2005;73(6):1135–46.PubMedView ArticleGoogle Scholar
- Lapointe E, Boyer A, Rico C, Paquet M, Franco HL, Gossen J, DeMayo FJ, Richards JS, Boerboom D. FZD1 regulates cumulus expansion genes and is required for Normal female fertility in mice. Biol Reprod. 2012;87(5):104.PubMedPubMed CentralView ArticleGoogle Scholar
- De Cian MC, Pauper E, Bandiera R, Vidal VP, Sacco S, Gregoire EP, Chassot AA, Panzolini C, Wilhelm D, Pailhoux E, et al. Amplification of R-spondin1 signaling induces granulosa cell fate defects and cancers in mouse adult ovary. Oncogene. 2016;36(2):208–18.PubMedPubMed CentralView ArticleGoogle Scholar
- Abedini A, Zamberlam G, Lapointe E, Tourigny C, Boyer A, Paquet M, Hayashi K, Honda H, Kikuchi A, Price C, et al. WNT5a is required for normal ovarian follicle development and antagonizes gonadotropin responsiveness in granulosa cells by suppressing canonical WNT signaling. FASEB J. 2016;30(4):1534–47.PubMedView ArticleGoogle Scholar
- Boerboom D, Paquet M, Hsieh M, Liu J, Jamin SP, Behringer RR, Sirois J, Taketo MM, Richards JS. Misregulated Wnt/beta-catenin signaling leads to ovarian granulosa cell tumor development. Cancer Res. 2005;65(20):9206–15.PubMedView ArticleGoogle Scholar
- Colombo N, Peiretti M, Castiglione M. Non-epithelial ovarian cancer: ESMO clinical recommendations for diagnosis, treatment and follow-up. Ann Oncol. 2009;20(Suppl 4):24–6.PubMedGoogle Scholar
- Kilonzo BM, Neff T, Samuelson MI, Goodheart MJ. Wnt signaling in granulosa cell tumors of the ovary. Proc Obstet Gynecol. 2015;4(3):1–1.View ArticleGoogle Scholar
- Stewart CJ, Doherty D, Guppy R, Louwen K, Leung YC. β-Catenin and E-cadherin expression in stage I adult-type granulosa cell tumour of the ovary: correlation with tumour morphology and clinical outcome. Histopathology. 2013;62(2):257–66.PubMedView ArticleGoogle Scholar
- Clevers H. Wnt/beta-catenin signaling in development and disease. Cell. 2006;127(3):469–80.PubMedView ArticleGoogle Scholar
- van Es JH, Kirkpatrick C, van de Wetering M, Molenaar M, Miles A, Kuipers J, Destrée O, Peifer M, Clevers H. Identification of APC2, a homologue of the adenomatous polyposis coli tumour suppressor. Curr Biol. 1999;9(2):105–8.PubMedView ArticleGoogle Scholar
- Roberts DM, Pronobis MI, Poulton JS, Kane EG, Peifer M. Regulation of Wnt signaling by the tumor suppressor adenomatous polyposis coli does not require the ability to enter the nucleus or a particular cytoplasmic localization. Mol Biol Cell. 2012;23(11):2041–56.PubMedPubMed CentralView ArticleGoogle Scholar
- Schneikert J, Vijaya Chandra SH, Ruppert JG, Ray S, Wenzel EM, Behrens J. Functional comparison of human adenomatous polyposis coli (APC) and APC-like in targeting beta-catenin for degradation. PLoS One. 2013;8(7):e68072.PubMedPubMed CentralView ArticleGoogle Scholar
- Sansom OJ, Reed KR, Hayes AJ, Ireland H, Brinkmann H, Newton IP, Batlle E, Simon-Assmann P, Clevers H, Nathke IS, et al. Loss of Apc in vivo immediately perturbs Wnt signaling, differentiation, and migration. Genes Dev. 2004;18(12):1385–90.PubMedPubMed CentralView ArticleGoogle Scholar
- Hamada F, Murata Y, Nishida A, Fujita F, Tomoyasu Y, Nakamura M, Toyoshima K, Tabata T, Ueno N, Akiyama T: Identification and characterization of E-APC, a novel Drosophila homologue of the tumour suppressor APC. Genes to cells : devoted to molecular & cellular mechanisms 1999, 4(8):465-474.Google Scholar
- McCartney BM, Dierick HA, Kirkpatrick C, Moline MM, Baas A, Peifer M, Bejsovec A. Drosophila APC2 is a cytoskeletally-associated protein that regulates wingless signaling in the embryonic epidermis. J Cell Biol. 1999;146(6):1303–18.PubMedPubMed CentralView ArticleGoogle Scholar
- Jarrett CR, Blancato J, Cao T, Bressette DS, Cepeda M, Young PE, King CR, Byers SW. Human APC2 localization and allelic imbalance. Cancer Res. 2001;61(21):7978–84.PubMedGoogle Scholar
- Shintani T, Ihara M, Tani S, Sakuraba J, Sakuta H, Noda M. APC2 plays an essential role in axonal projections through the regulation of microtubule stability. J Neurosci. 2009;29(37):11628–40.PubMedView ArticleGoogle Scholar
- Shintani T, Takeuchi Y, Fujikawa A, Noda M. Directional neuronal migration is impaired in mice lacking adenomatous polyposis coli 2. J Neurosci. 2012;32(19):6468–84.PubMedView ArticleGoogle Scholar
- Nakagawa H, Koyama K, Murata Y, Morito M, Akiyama T, Nakamura Y. EB3, a novel member of the EB1 family preferentially expressed in the central nervous system, binds to a CNS-specific APC homologue. Oncogene. 2000;19(2):210–6.PubMedView ArticleGoogle Scholar
- McCartney BM, McEwen DG, Grevengoed E, Maddox P, Bejsovec A, Peifer M. Drosophila APC2 and Armadillo participate in tethering mitotic spindles to cortical actin. Nat Cell Biol. 2001;3(10):933–8.PubMedView ArticleGoogle Scholar
- Daly C. The roles of the Apc proteins in homeostasis and tumourigenesis. Cardiff: Cardiff University; 2013.Google Scholar
- Daly CS, Shaw P, Ordonez LD, Williams GT, Quist J, Grigoriadis A, Van Es JH, Clevers H, Clarke AR, Reed KR. Functional redundancy between Apc and Apc2 regulates tissue homeostasis and prevents tumorigenesis in murine mammary epithelium. Oncogene. 2016;36(13):1793–803.PubMedPubMed CentralView ArticleGoogle Scholar
- Perets R, Wyant GA, Muto KW, Bijron JG, Poole BB, Chin KT, Chen JYH, Ohman AW, Stepule CD, Kwak S. Transformation of the fallopian tube secretory epithelium leads to high-grade serous ovarian Cancer in Brca; Tp53; Pten Models. Cancer Cell. 2013;24(6):751–65.PubMedPubMed CentralView ArticleGoogle Scholar
- van der Meer M, Baumans V, Hofhuis FM, Olivier B, van Zutphen BF. Consequences of gene targeting procedures for behavioural responses and morphological development of newborn mice. Transgenic Res. 2001;10(5):399–408.PubMedView ArticleGoogle Scholar
- Buchert M, Athineos D, Abud HE, Burke ZD, Faux MC, Samuel MS, Jarnicki AG, Winbanks CE, Newton IP, Meniel VS, et al. Genetic dissection of differential signaling threshold requirements for the Wnt/beta-catenin pathway in vivo. PLoS Genet. 2010;6(1):e1000816.PubMedPubMed CentralView ArticleGoogle Scholar
- Shibata H, Toyama K, Shioya H, Ito M, Hirota M, Hasegawa S, Matsumoto H, Takano H, Akiyama T, Toyoshima K, et al. Rapid colorectal adenoma formation initiated by conditional targeting of the Apc gene. Science. 1997;278(5335):120–3.PubMedView ArticleGoogle Scholar
- Luo C, Zuñiga J, Edison E, Palla S, Dong W, Parker-Thornburg J. Superovulation strategies for 6 commonly used mouse strains. J Am Assoc Lab Anim Sci. 2011;50(4):471–8.PubMedPubMed CentralGoogle Scholar
- Zudova D, Wyrobek AJ, Bishop J, Marchetti F. Impaired fertility in T-stock female mice after superovulation. Reproduction. 2004;128(5):573–81.PubMedView ArticleGoogle Scholar
- Kiyosu C, Tsuji T, Yamada K, Kajita S, Kunieda T. NPPC/NPR2 signaling is essential for oocyte meiotic arrest and cumulus oophorus formation during follicular development in the mouse ovary. Reproduction. 2012;144(2):187–93.PubMedView ArticleGoogle Scholar
- Brown C, LaRocca J, Pietruska J, Ota M, Anderson L, Smith SD, Weston P, Rasoulpour T, Hixon ML. Subfertility caused by altered follicular development and oocyte growth in female mice lacking PKB alpha/Akt1. Biol Reprod. 2010;82(2):246–56.PubMedPubMed CentralView ArticleGoogle Scholar
- Balla A, Danilovich N, Yang Y, Sairam MR. Dynamics of ovarian development in the FORKO immature mouse: structural and functional implications for ovarian reserve. Biol Reprod. 2003;69(4):1281–93.PubMedView ArticleGoogle Scholar
- Visser JA, Durlinger AL, Peters IJ, van den Heuvel ER, Rose UM, Kramer P, de Jong FH, Themmen AP. Increased oocyte degeneration and follicular atresia during the estrous cycle in anti-Mullerian hormone null mice. Endocrinology. 2007;148(5):2301–8.PubMedView ArticleGoogle Scholar
- Cumming G, Fidler F, Vaux DL. Error bars in experimental biology. J Cell Biol. 2007;177(1):7–11.PubMedPubMed CentralView ArticleGoogle Scholar
- Law NC, Weck J, Kyriss B, Nilson JH, Hunzicker-Dunn M. Lhcgr expression in granulosa cells: roles for PKA-phosphorylated β-catenin, TCF3, and FOXO1. Mol Endocrinol. 2013;27(8):1295–310.PubMedPubMed CentralView ArticleGoogle Scholar
- Hai L, McGee SR, Rabideau AC, Paquet M, Narayan P. Infertility in female mice with a gain-of-function mutation in the luteinizing hormone receptor is due to irregular estrous Cyclicity, anovulation, hormonal alterations, and polycystic ovaries. Biol Reprod. 2015;93(1):16.PubMedPubMed CentralView ArticleGoogle Scholar
- Shen M, Lin F, Zhang J, Tang Y, Chen WK, Liu H. Involvement of the up-regulated FoxO1 expression in follicular granulosa cell apoptosis induced by oxidative stress. J Biol Chem. 2012;287(31):25727–40.PubMedPubMed CentralView ArticleGoogle Scholar
- Kim JH, Yoon S, Park M, Park HO, Ko JJ, Lee K, Bae J. Differential apoptotic activities of wild-type FOXL2 and the adult-type granulosa cell tumor-associated mutant FOXL2 (C134W). Oncogene. 2011;30(14):1653–63.PubMedView ArticleGoogle Scholar
- Matsuda F, Inoue N, Maeda A, Cheng Y, Sai T, Gonda H, Goto Y, Sakamaki K, Manabe N. Expression and function of apoptosis initiator FOXO3 in granulosa cells during follicular atresia in pig ovaries. J Reprod Dev. 2011;57(1):151–8.PubMedView ArticleGoogle Scholar
- Stapp AD, Gomez BI, Gifford CA, Hallford DM, Hernandez Gifford JA. Canonical WNT signaling inhibits follicle stimulating hormone mediated steroidogenesis in primary cultures of rat granulosa cells. PLoS One. 2014;9(1):e86432.PubMedPubMed CentralView ArticleGoogle Scholar
- Li L, Ji SY, Yang JL, Li XX, Zhang J, Zhang Y, Hu ZY, Liu YX. Wnt/beta-catenin signaling regulates follicular development by modulating the expression of Foxo3a signaling components. Mol Cell Endocrinol. 2014;382(2):915–25.PubMedView ArticleGoogle Scholar
- Tzivion G, Dobson M, Ramakrishnan G. FoxO transcription factors; regulation by AKT and 14-3-3 proteins. Biochim Biophys Acta (BBA) – Mol Cell Res. 2011;1813(11):1938–45.View ArticleGoogle Scholar
- Tanwar PS, Kaneko-Tarui T, Lee H-J, Zhang L, Teixeira JM. PTEN loss and HOXA10 expression are associated with ovarian endometrioid adenocarcinoma differentiation and progression. Carcinogenesis. 2013;34(4):893–901.PubMedView ArticleGoogle Scholar
- Taddei A, Giampietro C, Conti A, Orsenigo F, Breviario F, Pirazzoli V, Potente M, Daly C, Dimmeler S, Dejana E. Endothelial adherens junctions control tight junctions by VE-cadherin-mediated upregulation of claudin-5. Nat Cell Biol. 2008;10(8):923–34.PubMedView ArticleGoogle Scholar
- Robinson RS, Woad KJ, Hammond AJ, Laird M, Hunter MG, Mann GE. Angiogenesis and vascular function in the ovary. Reproduction. 2009;138(6):869–81.PubMedView ArticleGoogle Scholar
- Tanwar PS, Zhang L, Kaneko-Tarui T, Curley MD, Taketo MM, Rani P, Roberts DJ, Teixeira JM. Mammalian target of rapamycin is a therapeutic target for murine ovarian endometrioid adenocarcinomas with dysregulated Wnt/beta-catenin and PTEN. PLoS One. 2011;6(6):e20715.PubMedPubMed CentralView ArticleGoogle Scholar
- van der Horst PH, van der Zee M, Heijmans-Antonissen C, Jia Y, DeMayo FJ, Lydon JP, van Deurzen CH, Ewing PC, Burger CW, Blok LJ. A mouse model for endometrioid ovarian cancer arising from the distal oviduct. Int J Cancer. 2014;135(5):1028–37.PubMedView ArticleGoogle Scholar
- Gao Y, Vincent DF, Davis AJ, Sansom OJ, Bartholin L, Li Q. Constitutively active transforming growth factor beta receptor 1 in the mouse ovary promotes tumorigenesis. Oncotarget. 2016;7(27):40904–18.PubMedPubMed CentralView ArticleGoogle Scholar
- Liu Z, Ren YA, Pangas SA, Adams J, Zhou W, Castrillon DH, Wilhelm D, Richards JS. FOXO1/3 and PTEN depletion in granulosa cells promotes ovarian granulosa cell tumor development. Mol Endocrinol. 2015;29(7):1006–24.PubMedPubMed CentralView ArticleGoogle Scholar
- D'Angelo E, Mozos A, Nakayama D, Espinosa I, Catasus L, Munoz J, Prat J. Prognostic significance of FOXL2 mutation and mRNA expression in adult and juvenile granulosa cell tumors of the ovary. Mod Pathol. 2011;24(10):1360–7.PubMedView ArticleGoogle Scholar
- Kaspar HG, Crum CP. The utility of immunohistochemistry in the differential diagnosis of gynecologic disorders. Arch Pathol Lab Med. 2015;139(1):39–54.PubMedView ArticleGoogle Scholar
- Lague MN, Paquet M, Fan HY, Kaartinen MJ, Chu S, Jamin SP, Behringer RR, Fuller PJ, Mitchell A, Dore M, et al. Synergistic effects of Pten loss and WNT/CTNNB1 signaling pathway activation in ovarian granulosa cell tumor development and progression. Carcinogenesis. 2008;29(11):2062–72.PubMedPubMed CentralView ArticleGoogle Scholar
- Richards JS, Fan HY, Liu Z, Tsoi M, Lague MN, Boyer A, Boerboom D. Either Kras activation or Pten loss similarly enhance the dominant-stable CTNNB1-induced genetic program to promote granulosa cell tumor development in the ovary and testis. Oncogene. 2012;31(12):1504–20.PubMedView ArticleGoogle Scholar
- Jefferies MT, Cox AC, Shorning BY, Meniel V, Griffiths D, Kynaston HG, Smalley MJ, Clarke AR. PTEN loss and activation of K-RAS and beta-catenin cooperate to accelerate prostate tumourigenesis. J Pathol. 2017;243(4):442–56.PubMedPubMed CentralView ArticleGoogle Scholar
- Ahmed Y, Nouri A, Wieschaus E. Drosophila Apc1 and Apc2 regulate wingless transduction throughout development. Development. 2002;129(7):1751–62.PubMedGoogle Scholar
- Akong K, Grevengoed EE, Price MH, McCartney BM, Hayden MA, DeNofrio JC, Peifer M. Drosophila APC2 and APC1 play overlapping roles in wingless signaling in the embryo and imaginal discs. Dev Biol. 2002;250(1):91–100.PubMedView ArticleGoogle Scholar
- McCartney BM, Price MH, Webb RL, Hayden MA, Holot LM, Zhou M, Bejsovec A, Peifer M. Testing hypotheses for the functions of APC family proteins using null and truncation alleles in Drosophila. Development. 2006;133(12):2407–18.PubMedView ArticleGoogle Scholar
- Xu Y, Li X, Wang H, Xie P, Yan X, Bai Y, Zhang T. Hypermethylation of CDH13, DKK3 and FOXL2 promoters and the expression of EZH2 in ovary granulosa cell tumors. Mol Med Rep. 2016;14(3):2739–45.PubMedView ArticleGoogle Scholar