Vol.:(0123456789)1 3
Histochemistry and Cell Biology (2019) 151:115–123
https://doi.org/10.1007/s00418-018-1729-y
ORIGINAL PAPER
Dynamics of WNT signaling components in the human ovary
from development to adulthood
Alisha M. Bothun1
 · Dori C. Woods1
 
Accepted: 12 September 2018 / Published online: 4 October 2018
© Springer-Verlag GmbH Germany, part of Springer Nature 2018
Abstract
WNT signaling has been shown to play a pivotal role in mammalian gonad development and sex differentiation; however,
its role in the developing human ovary has not been investigated. We analyzed a quantitative mass spectrometry dataset to
determine the expression of WNT signaling components between 47 and 137 days of development and in adult ovarian cortex
tissue. WNT signaling was identified within the top ten canonical pathways of proteins detected at every developmental
stage examined. We further examined the specific localization of WNT signaling components glycogen synthase kinase 3
(GSK3B), frizzled 2 (FZD2), and β-catenin (CTNNB1) within ovarian tissue. GSK3B was nearly ubiquitously expressed
during fetal development, while FZD2 was specific to germ cell nests during early development. β-catenin exhibited translocation
from primarily membrane bound during early ovarian development to cytoplasmic and nuclear staining specifically
in early primordial follicles in the fetal ovary. This cytoplasmic and nuclear β-catenin persisted in primordial follicles in
adult ovarian tissue, but returned to membrane-bound localization in secondary follicles. We conclude that WNT signaling
components are expressed in the human ovary from early to mid-gestation and remain in the adult ovary, and observed evidence
for canonical WNT signaling only in the oocytes of primordial follicles. Together, these data are indicative of a role
for canonical WNT signaling via β-catenin nuclear translocation during human follicle formation and follicle maintenance.
Keywords  WNT signaling · Development · Ovary · β-Catenin
Introduction
The processes governing human ovarian development have
been largely described based on histological examination
and comparison to model organisms. In humans, primordial
germ cells (PGCs) are specified and are identifiable
at approximately 3 weeks of development (Witschi 1948)
(Truman et al. 2017). These PGCs proliferate and migrate
between 3 and 4 weeks of development (Witschi 1948)
(Politzer 1933) to colonize the developing gonad at approximately
6 weeks (Makabe and Motta 1989). In mice, germ
cell cysts break down and follicle assembly occurs in the
postnatal period, from 20.5 to 22.5 days post-coitum (dpc),
during which time pregranulosa cells form close associations
with single oocytes and primordial follicles are formed
(Pepling and Spradling 2001). The process of germ cell
nest breakdown and follicle formation is similar in humans;
however, human ovarian follicle formation occurs entirely
during fetal development between 17 and 20 weeks (Kurilo
1981; Konishi et al. 1986; Satoh 1991; Motta et al. 1997).
Because human follicle assembly occurs prenatally, the signaling
mechanisms required for human oocyte meiotic arrest,
granulosa cell specification, and follicle assembly are still
unknown.
Wingless-type mouse mammary tumor virus integration
site (WNT) signaling is a key signaling network implicated
in numerous processes including embryonic development,
tissue regeneration, and cancer (Clevers 2006; Gordon and
Nusse 2006). In canonical WNT signaling, the excreted
WNT ligand binds a frizzled receptor resulting in the accumulation
of non-phosphorylated β-catenin; as β-catenin
accumulates, it translocates to the nucleus where it binds
with lymphoid enhancer factor/T-cell factor (LEF/TCF)
Electronic supplementary material  The online version of this
article (https​://doi.org/10.1007/s0041​8-018-1729-y) contains
supplementary material, which is available to authorized users.
*	 Dori C. Woods
	d.woods@northeastern.edu
1
	 Laboratory for Aging and Infertility Research, Department
of Biology, Northeastern University, Boston, MA 02115,
USA
116	 Histochemistry and Cell Biology (2019) 151:115–123
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transcription factors (Behrens et al. 1996). Downstream
targets of WNT signaling include genes that promote proliferation
or cell fate determination and differentiation in
stem cells (Reya and Clevers  2005). In addition to the
canonical pathway, WNT signaling via frizzled receptors
has been demonstrated to activate non-canonical signaling,
independent of β-catenin. Non-canonical WNT signaling
encompasses the planar cell polarity pathway, resulting in
regulation of the cytoskeleton, as well as mechanisms mediated
by intracellular calcium release, Rho family GTPases,
heterotrimeric G proteins, and the JNK pathway (Veeman
et al. 2003). Aside from WNT signaling, β-catenin also
forms associations with cadherin complexes to control cellto-cell
contacts and cell migration (Nelson and Nusse 2004).
With respect to gonadogenesis, WNT signaling has been
implicated at several stages of development in the mammalian
gonad. With respect specifically to the establishment
of the germline, Wnt3 is required in mice for appropriate
responsiveness to Bmp4 in the early embryo and subsequent
PGC differentiation (Ohinata et al. 2009; Aramaki et al.
2013). Later in development, during sex determination,
WNT4 is required in both sexes for Mullerian duct formation
and XX gonads lacking either R-spondin 1 (Rspo1) or Wnt4
result in partial sex reversal (Vainio et al. 1999; Tomizuka
et al. 2008; Chassot et al. 2008). Both RSPO1 and WNT4
activate β-catenin signaling, and accordingly, stabilized
β-catenin acts as a pro-ovarian signaling molecule capable
of inducing male to female sex reversal in XY gonads and
rescues the sex reversal observed in Wnt4-null XX gonads
(Maatouk et al. 2008).
It has become clear that the impact of WNT signaling
on the bipotential ovary impacts both germline and somatic
cells. Pregranulosa cells within Wnt4-null and Rspo1-null
ovaries undergo failure to maintain mitotic arrest and exhibit
signs of precocious differentiation and differentiation to Sertoli
cells (Maatouk et al. 2013). In humans, loss of function
mutations in both WNT4 and RSPO1 results in female to
male sex reversal (Mandel et al. 2008; Parma et al. 2006;
Tomaselli et al. 2008). Beyond development, WNT signaling
has also been implicated in cancer and tumor growth;
in human granulosa cell tumors, cells had a higher rate of
nuclear β-catenin than in healthy granulosa cells (Boerboom
et al. 2005). WNT signaling components were also identified
as one of several pathways misregulated in polycystic ovary
syndrome (PCOS) patients (Jansen et al. 2004).
Despite the critical role for WNT signaling in ovarian
development and function, the dynamics of WNT signaling
have not been thoroughly examined during gonadogenesis,
including critical time points associated with
germline differentiation and follicle assembly. Currently, it
is unknown which components of WNT signaling are present
in the human ovary during development, and it is also
not established whether canonical and/or non-canonical
mechanisms of WNT signaling participate in human follicle
assembly. Utilizing a comprehensive quantitative
mass spectrometry data set, we identified WNT signaling
components in the human ovary during stages of PGC
differentiation to primordial follicle formation. We then
characterized β-catenin localization to provide insight
into the role for canonical vs non-canonical WNT signaling
in the formation of the human ovary. We conclude
that β-catenin was primarily membrane bound throughout
development, but that there was evidence for nuclear
β-catenin in oocytes during primordial follicle formation,
indicating canonical WNT signaling may play a role in
follicle assembly and maintenance.
Materials and methods
Ovarian tissue sample collection
All procedures described herein have been reviewed and
approved by the Institutional Review Board (IRB) at
Northeastern University, Saitama Medical University,
and the University of Washington. Human fetal ovarian
tissue was collected after medical termination by the
Laboratory of Developmental Biology at the University of
Washington. Samples were collected between 30 min and
24 h post-procedure. Developmental ages were determined
from prenatal intakes, foot length, Streeter’s stage, and
crown-rump length. Whole ovary tissues were snap frozen
for proteomics analysis or fixed in 4% paraformaldehyde
in PBS and then stored at 4 °C until being processed for
immunohistochemistry. Human adult ovarian cortical tissue
from reproductive aged women was generously provided
to us for our use by Dr. Yasushi Takai at Saitama
Medical University.
Quantitative proteomics analysis
Ovarian tissue samples were analyzed by LC–MS/MS
as described previously (Bothun et al. 2018). Proteomics
data were retrieved from Mendeley Data (https​://doi.
org/10.17632​/x7zby​j6xh7​.1) and analyzed for WNT signaling
components based on intensity-based absolute quantitation
(iBAQ) values provided by Scaffold (Schwanhausser
et al. 2011). Common proteins were identified as those with
expression at 47, 108, 122, and 137 days of development in
addition to in adult ovarian cortex tissue. PANTHER Gene
Ontology Pathway Analysis was performed to determine
protein pathways present in human ovarian tissue samples
and to identify proteins associated with each pathway (Mi
et al. 2010).
117Histochemistry and Cell Biology (2019) 151:115–123	
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Immunohistochemistry and immunofluorescence
Paraformaldehyde-fixed ovarian tissue was dehydrated,
embedded in paraffin, and 5-micron sections were prepared.
Antigen retrieval was performed in 0.01 M sodium
citrate buffer (pH 6.0) in a pressure cooker, sections were
cooled, washed, and endogenous peroxide activity was
quenched with 3% hydrogen peroxide for 10 min at room
temperature. Sections were blocked for 1 h in TNK Buffer
(0.1 M Tris–HCl, 0.55 M NaCl, 0.1 mM KCl, 0.5% BSA,
0.1% Triton-x100, and 1% normal goat serum). Slides were
stained with primary antibody overnight at 4 °C according
to Table 1. Secondary antibody labeling was performed and
DAB Plus Chromagen was applied prior to nuclear counterstaining
with Weigert’s iron hematoxylin. Secondary
only controls were included to assess background staining
of the DAB substrate (Supplementary Fig. 1a). Slides were
cleared through xylene and mounted with Permount Mounting
Medium prior to imaging.
For immunofluorescence analysis of β-catenin, slides
were processed as described above and blocked in PBSblocking
buffer (PBS, 2% bovine serum albumin, 0.1% Triton
X-100, 1% normal goat serum) for 1 h at room temperature.
Slides were incubated at 4 °C overnight with mouse
anti-B-catenin (1:100, Santa Cruz sc-7963) in PBS-blocking
buffer. Slides were washed with PBS and incubated in antimouse
Alexa Fluor 647 (1:500). Slides were counterstained
with Hoechst 33342, trihydrochloride, trihydrate (Invitrogen)
to visualize cell nuclei. Secondary only controls were
included to assess background signal from the secondary
antibody (Supplementary Fig. 1b). Slides were washed with
PBS and cover-slipped with ProLong Gold Antifade Reagent
(Invitrogen) prior to imaging. All antibodies were validated
by the manufacturer for specificity, utilizing western blotting
for size validation, and, for glycogen synthase kinase-3 beta
(GSK3B), in WT cells and (GSK3B) knockout mice.
Image collection and analysis
Images were taken on an Axiovert Inverted Fluorescence
Microscope (Zeiss) fitted with filters for 640/30-nm excitation
and 690/50-nm emission or 365-nm excitation and
445/50-nm emission.
All reagents were purchased from Thermo Fisher Scientific
unless otherwise noted.
Results and discussion
WNT signaling components were detected
throughout development in human ovarian tissue
Gene Ontology (GO) analysis of quantitative mass spectrometry
identified protein pathways represented in human
ovarian tissue at 47, 108, 122, and 137 days of development
in addition to in adult ovarian cortex tissue (Supplementary
Data). The protein pathways identified in this analysis represent
the most abundant proteins throughout early to midgestation
in the human ovary and were maintained in adult
ovarian tissue. WNT signaling was identified within the top
ten pathways observed with components present in every
developmental stage of ovarian tissue analyzed (Fig. 1a).
24 proteins were identified by GO terms to be related to
WNT signaling (Table 2). In addition to proteins detected at
all stages of development, additional components of WNT
signaling were individually screened for detection in ovarian
tissues from specific stages. Among the family of frizzled
receptors, three were identified throughout the dataset: Frizzled
1 (FZD1), Frizzled 2 (FZD2), and Frizzled 7 (FZD7)
had varying degrees of expression based on iBAQ quantitation
at each stage of development or adult tissue (Fig. 1b).
In addition to frizzled receptors, glycogen synthase kinase-3
beta (GSK3B), a protein kinase and negative regulator of
WNT signaling, was detected at developmental days 47,
108, 122, and 137, but not in adult tissue (Fig. 1b). Finally,
β-catenin (catenin beta-1, CTNNB1) was detected in all tissues
tested (Fig. 1b).
WNT signaling component localization in early
ovarian development
Based on the proteomics analysis, we performed immunohistochemical
staining to determine localization of several
specific proteins to identify the cell types that expressed each
protein throughout development. GSK3B was detected in
human ovarian tissue at 56 days of development and expression
was detected in germ cell nests as well as in somatic
cells surrounding the germ cell nests (Fig. 2a). At 76, 116,
and 137 days of development, GSK3B localization became
increasingly cytoplasmic, with more intense staining evident
in larger oocytes and the surrounding granulosa cells
Table 1  Antibodies for immunohistochemical analyses
Primary antibody Dilution Detection
Rabbit anti-glycogen synthase kinase-3
beta (CST 12456)
1:500 Rabbit signal stain boost detection reagent
Goat anti-frizzled 2 (Abcam ab109094) 1:100 Anti-goat biotin (1:100), 30 min Streptavidin poly-HRP (1:250) 30 min
118	 Histochemistry and Cell Biology (2019) 151:115–123
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of primordial follicles (Fig. 2a, arrowhead). In adult ovarian
cortex tissue, GSK3B was only faintly detected, with
cytoplasmic expression in oocytes of primordial follicles.
GSK3B plays a role in multiple signaling cascades, and is
responsible for phosphorylation and subsequent destabilization
of β-catenin (Yost et al. 1996). Because of its nearly
ubiquitous expression and localization to all cell types of
the ovary during development, it is likely that GSK3B in
the ovary is participating in several signaling cascades.
However, in adult ovarian tissue, GSK3B was more specifically
detected in oocytes of primordial follicles and was
only faintly detected throughout somatic cells, indicating a
more specialized function in the maintenance of primordial
follicles into adulthood.
While there were multiple frizzled receptors detected
in the proteomics analysis, FZD2 was detected at all time
points of ovarian development assessed. FZD2 was localized
specifically within germ cell nests early in development at
day 56 (Fig. 2b). FZD2 expression at developmental days 76,
122, and 137 maintained specificity to germ cell nests and
was expressed in oocyte cytoplasm in primordial follicles
(Fig. 2b, arrowheads). Adult tissue had only faint cytoplasmic
staining of FZD2 throughout the tissue. Because WNT
signaling depends on both the WNT ligand present in addition
to the specific frizzled receptor it is paired with, we
concluded that likely there are multiple frizzled receptors
participating in WNT signaling in the human ovary. Because
WNT signaling via frizzled receptors is generally understood
Fig. 1  Proteomic analysis of WNT signaling components. Of the core
proteins identified in all tissues by quantitative mass spectrometry, the
top ten pathways by number of components identified are displayed
(a). Individual protein components of WNT signaling were assessed
individually to identify quantitation across tissues. Frizzled 1 (FZD1),
Frizzled 2 (FZD2), Frizzled 7 (FZD7), glycogen synthase kinase 3b
(GSK3B) and β-catenin (CTNNB1) were detected at four stages of
fetal ovarian development (developmental days 47, 108, 122, and
137) and in a representative adult ovarian cortex. Values presented
are intensity-based absolute quantification (iBAQ) values (b)
119Histochemistry and Cell Biology (2019) 151:115–123	
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to lead to either canonical signaling via β-catenin- or to
calcium-mediated signaling, it is likely that WNT signaling
is occurring during these stages through one of these
mechanisms.
β‑Catenin localization suggests canonical WNT
signaling is prominent at follicle formation
in oocytes
As canonical WNT signaling is dependent on β-catenin
translocation to the nucleus, we examined β-catenin localization
throughout development. Immunofluorescence
analysis of β-catenin in ovarian tissue from developmental
day 56 revealed localization primarily to cell membranes
within germ cell nests, in addition to expression at the surface
epithelium (Fig. 3a). In tissue at 137 days of development,
β-catenin was detected in the cytoplasm and nucleus
of oocytes within primordial follicles (Fig. 3b, arrow), but it
was not detected in somatic cells (Fig. 3b, arrowhead) or it
was detected only at the membrane in germ cells that were
not yet formed into follicles (Fig. 3b, asterisk). In adult ovarian
cortex, β-catenin was localized specifically to primordial
follicles, where expression was detected at cell membranes
between pregranulosa cells and the oocyte, in the oocyte
cytoplasm, and the oocyte nucleus (Fig. 3c). Expression
was similar in primary follicles (Fig. 3d). Within secondary
follicles, β-catenin was localized at the cell membrane
junctions between granulosa cells and also at the oocyte cell
membrane (Fig. 3e). At this stage, little to no β-catenin was
detected in the oocyte cytoplasm or nucleus (Fig. 3e). This
pattern of β-catenin localization reveals that β-catenin is
nuclear in oocytes primarily during follicle formation and
maintenance of follicles to the primary stage, but becomes
exclusively membrane bound in secondary follicles. Importantly,
we did not observe nuclear β-catenin localization
in somatic cells at any developmental age. There was no
nuclear β-catenin observed in pregranulosa cells, nor of
granulosa cells in primary or secondary follicles. This suggests
that during human ovarian development after sex
determination, β-catenin-dependent canonical WNT signaling
is only active in the oocyte specifically during follicle
formation. The primarily membrane-bound β-catenin and
presence of FZD2 and other WNT signaling proteins prior
to folliculogenesis and in secondary follicles in adulthood
indicate that WNT signaling is occurring through noncanonical
pathways during these developmental stages. The
Table 2  GO term analysis identified proteins involved in WNT signaling present in ovarian tissue from 47 days, 108 days, 122 days, and 137
days of development and in adult tissue
Gene name Protein description
SMARCA5 SWI/SNF-related matrix-associated actin-dependent regulator of chromatin subfamily A member 5; SMARCA5; ortholog
ARID1A AT-rich interactive domain-containing protein 1A; ARID1A; ortholog
GNG12 Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-12; GNG12; ortholog
SMARCC1 SWI/SNF complex subunit SMARCC1; SMARCC1; ortholog
CTNNA1 Catenin alpha-1; CTNNA1; ortholog
GNB1 Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1; GNB1; ortholog
SMARCC2 SWI/SNF complex subunit SMARCC2; SMARCC2; ortholog
FZD7 Frizzled-7; FZD7; ortholog
TBL1XR1 F-box-like/WD repeat-containing protein TBL1XR1; TBL1XR1; ortholog
PPP2R5D Serine/threonine-protein phosphatase 2A 56 kDa regulatory subunit delta isoform; PPP2R5D; ortholog
GNAQ Guanine nucleotide-binding protein G(q) subunit alpha; GNAQ; ortholog
CSNK2B Casein kinase II subunit beta; CSNK2B; ortholog
PLCB3 1-Phosphatidylinositol 4,5-bisphosphate phosphodiesterase beta-3; PLCB3; ortholog
CAD11 Cadherin-11; CDH11; ortholog
ADSS Adenylosuccinate synthetase isozyme 2; ADSS; ortholog
CTNNB1 Catenin beta-1; CTNNB1; ortholog
SMARCB1 SWI/SNF-related matrix-associated actin-dependent regulator of chromatin subfamily B member 1; SMARCB1; ortholog
ACTB Actin, cytoplasmic 1; ACTB; ortholog
CTBP1 C-terminal-binding protein 1; CTBP1; ortholog
CSNK2A2 Casein kinase II subunit alpha’; CSNK2A2; ortholog
PPP2CA Serine/threonine-protein phosphatase 2A catalytic subunit alpha isoform; PPP2CA; ortholog
FZD2 Frizzled-2; FZD2; ortholog
CTBP2 C-terminal-binding protein 2; CTBP2; ortholog
SMARCA2 Probable global transcription activator SNF2L2; SMARCA2; ortholog
120	 Histochemistry and Cell Biology (2019) 151:115–123
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Fig. 2  Immunohistochemical
analysis of WNT signaling
components from developing
and adult human ovarian
tissue. GSK3B and FZD2 were
detected in fetal ovarian tissue
from 56, 76, 116, and 137
developmental days; staining
was primarily localized to
germ cell nests and was mostly
cytoplasmic. FZD2 showed the
highest expression at 56 days of
development, where localization
was specific to germ cell
nests. GSK3B and FZD2 were
detected in primordial follicles
at 137 days of development
(arrowheads). In adult cortex
tissue, GSK3B was detected in
the cytoplasm of oocytes within
primordial follicles (arrows),
while FZD2 was detected
throughout tissue (arrow
indicating a primordial follicle).
Primary antibodies were
labeled and detected with DAB
substrate (brown). Nuclei were
counterstained with hematoxylin
(blue). Scale bar = 50 µm
121Histochemistry and Cell Biology (2019) 151:115–123	
1 3
identification of β-catenin-dependent WNT signaling coincident
with follicle formation in humans is consistent with
its role in mouse development, where stabilized β-catenin
disrupts male XY development and induces follicle formation
(Maatouk et al. 2008).
Conclusion
Proteins involved in WNT signaling were detected in the
human ovary from early to mid-gestation and remained
detectable in follicles of adult ovaries. The detection of frizzled
receptors indicated that either canonical WNT signaling
via β-catenin or non-canonical calcium signaling may be
occurring; and as we detected multiple frizzled receptors
(FZD1, FZD2, and FZD7) the ligand-receptor pairs likely
change throughout development or are cell-type specific.
We observed evidence for canonical WNT signaling during
follicle formation based on the nuclear translocation of
β-catenin, the signal transducer of WNT signaling, shifting
from membrane localization to cytoplasmic and nuclear
localization in the oocytes of newly formed primordial follicles.
There was no indication of canonical WNT signaling
occurring in somatic cells of the ovary; however, the adult
tissue studied did not contain follicles of more advanced
stage than secondary. Additionally, it is possible that WNT
signaling is occurring through non-canonical pathways
throughout the ovary, based on the abundance of frizzled
receptor expression throughout development.
Female germ cell development in humans is a process
that, due to its timing during early development, is particularly
difficult to study. Establishing the signaling mechanisms
that are occurring during germ cell differentiation and
follicle development is key to understanding disorders of the
Fig. 3  β-catenin expression and localization throughout ovarian
development. Immunofluorescence analysis reveals β-catenin localization
is cell type and development stage specific. β-catenin was
exclusively membrane bound throughout all cell types at 56 days
of development. Scale bar 20 µm (a). At 137 days of development,
β-catenin was undetected in some somatic cells (arrowhead), membrane
bound in PGCs and somatic cells (asterisk) and was cytoplasmic
and nuclear in the oocytes of newly assembled primordial follicles
(arrow). Scale bar 20  µm (b). In adult tissue, β-catenin was
detected in the cytoplasm and nucleus within oocytes of primordial
follicles and at cell membranes between granulosa cells. Scale bars
50 µm and 20 µm respectively (c, d). In secondary follicles in adult
tissue, β-catenin was only detected at the membranes between granulosa
cells, at the oocyte membrane, and showed some punctate staining
in the cytoplasm of the oocyte. Scale bar 50 µm (e)
122	 Histochemistry and Cell Biology (2019) 151:115–123
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ovary that have far reaching consequences for hormone production
and fertility. Determining the physiological role of
WNT signaling in human folliculogenesis may uncover new
mechanisms for determining pathologies caused by dysregulated
WNT signaling (Wood et al. 2004; Aydos et al. 2016).
Additionally, as the field of stem cell biology progresses,
establishing in vitro models of human ovarian cellular lineages
relies on translation from basic biological studies to
characterize protein and gene expression cascades needed
for these processes (Truman et al. 2017).
Acknowledgements  The authors would also like to thank Monika
Izdebski, Joseph Maglio, and Kena Patel for invaluable technical help
in preparing samples for histological analysis. This material is based
upon work supported by the National Science Foundation under Grant
Number 1750996 to D.C.W. The ‘Laboratory of Developmental Biology’
was supported by NIH Award Number 5R24HD000836 from the
Eunice Kennedy Shriver National Institute of Child Health and Human
Development.
Compliance with ethical standards 
Conflict of interest All authors declare that they have no conflict of
interest.
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