Modulation of the Notch System in Response to Wnt Inhibition
Induces Restoration of the Rat Luteal Function
Paula Accialini1 & Andrés Bechis1 & Griselda Irusta1 & Maria Silvia Bianchi1 & Fernanda Parborell1 & Dalhia Abramovich1 &
Marta Tesone1
Received: 15 December 2018 /Accepted: 13 June 2019
# Society for Reproductive Investigation 2020
Abstract
The aim of this study was to investigate whether the Notch pathway is modulated in response to the downregulation of the Wnt/
Β-catenin system in corpora lutea (CLs) from superovulated rats. To this end, we analyzed the effect of in vitro CLWnt/Β-catenin
inhibition on the expression of Notch members and on luteal function. Mechanically isolated rat CLs were cultured with ICG-
001, a Wnt/B-catenin inhibitor. In this system, Wnt/B-catenin inhibition reduced progesterone production and decreased StAR
protein levels. Besides, Wnt/B-catenin inhibition stimulated the Notch system, evidenced by an increase in Hes1 expression, and
promoted the expression of selected Notch family members. At long incubation times, StAR levels and progesterone concentration reached the control values, effects probably mediated by the Notch pathway. These results provide the first evidence of a
compensatory mechanism between Wnt/B-catenin signaling and the Notch system, which contributes to the homeostasis of luteal
cells.
Keywords WNT/B-catenin . Notch . Corpus luteum . StAR . HES1
Introduction
The corpus luteum (CL) is a temporary endocrine organ essential for the implantation and survival of the embryo.
Although the CL is regulated mainly by the luteinizing hormone (LH), its development and function not only depend on
the activation of the LH receptor but also are acutely regulated
by various signaling mechanisms [1–3]. The main function of
the CL is to secrete progesterone. The limiting step of the
biosynthesis of this hormone is the delivery of cholesterol to
the inner mitochondrial membrane by steroidogenic acute regulatory protein (StAR) [2].
The Notch and Wnt/B-catenin systems are two evolutionarily conserved pathways that regulate cell fate decisions such
as proliferation, differentiation, and cell death [4–6], which are
three processes involved in the development and function of
the CL. In mammals, the Notch system consists of four receptors (Notch1–4) and five ligands [Jagged1–2 and Delta-like
(DLL) 1, 3, and 4], which are expressed on the cell surface and
comprise a juxtacrine signaling system. When Notch signaling is initiated, the receptor undergoes a cleavage mediated by
the presenilin–gamma-secretase complex, which results in the
release of the active intracellular domain of Notch (NICD).
NICD translocates to the nucleus [7] and stimulates the expression of target genes [8, 9] such as the Hes and Hey gene
family. Notch family members have been localized in granulosa, luteal and vascular cells of the rat ovary [10–12].
Particularly, Jagged1, Dll1 and Dll4 ligands, and Notch1–4
receptors are expressed in large and small luteal cells [10, 11,
13–16], while Hes1 is the target gene expressed in CLs [14].
We have previously demonstrated that Notch1, Notch4, and
DLL4 are localized in small and large luteal cells of CLs of
pregnant rats [13]. In addition, we have described that Notch
signaling plays a luteotropic role in promoting both luteal cell
viability and steroidogenesis in the CL. Furthermore, it has
been found that intraovarian Notch system inhibition decreases the circulating progesterone levels, confirming that
Notch has a direct action on luteal function [13]. Moreover,
our group [15] has demonstrated the existence of an interaction between the Notch signaling pathway and progesterone,
Paula Accialini and Andrés Bechis contributed equally to this work.
* Marta Tesone
[email protected]
1 Instituto de Biología y Medicina Experimental (IBYME-CONICET),
Vuelta de Obligado 2490, C1428ADN Buenos Aires, Argentina
Reproductive Sciences
https://doi.org/10.1007/s43032-019-00043-2
which maintain the functionality of the rat CL. The results of
that study provided the first evidence of a cross talk between
the Notch system and progesterone, which upregulate the survival and action of luteal cells.
The Wnt (wingless-type mouse mammary tumor virus
integration site) family comprises an endocrine signaling system which is composed of secreted glycoproteins (WNT ligands) that interact with seven-step transmembrane receptors
called FRIZZLED. When the canonical Wnt pathway is activated, B-catenin translocates to the nucleus, where it interacts
with the TCF/LEF complex and regulates the transcription of
target genes such as Cyclin d1. On the other hand, when the
system is not activated, a destruction complex composed of
adenomatous polyposis coli (APC), AXIN, and glycogen synthase kinase 3β (GSK-3β) is activated and phosphorylates Β-
catenin. As a consequence, this molecule is sent to
proteasomes for its destruction [17].
Wnt family members are expressed in granulosa, theca, and
cumulus cells from developing follicles and in the CLs of rats
and humans [17–19]. In rodents, the abundance of Wnt members in growing follicles increases after human chorionic gonadotropin (hCG) administration [20], and they have been
recently described as intraovarian regulators (reviewed in
Hernandez Gifford 2015). Furthermore, we have previously
demonstrated that the Wnt/Β-catenin system regulates CL
function and may influence the ovulation process in
gonadotropin-treated rats [21]. We have shown that in vivo
ovarian Wnt/Β-catenin inhibition causes a decrease in the percentage of CLs along with the appearance of cystic structures.
Besides, circulating progesterone concentration decreased,
and apoptosis of the CL cells was enhanced, evidencing the
importance of this pathway in ovarian physiology.
Several studies have described the existence of an interaction between Notch and Wnt/Β-catenin pathways [22–24] to
regulate development [25], differentiation [26], and angiogenesis [27] in different tissues. In mouse, Wnt modulates Dll1
expression which shows an interaction between these two
pathways to regulate somitogenesis [28]. In addition,
Yamamizu et al. described an interaction among the intracellular domain of Notch, RBPJ, and B-catenin that regulates cell
fate in vascular progenitors [29]. Finally, it was reported that
during T cell development, Notch stimulates the expression of
TCF1, evidencing a synergism between these two pathways
[30, 31]. Regarding the pathological aspect, Notch and Bcatenin are interconnected in different types of cancer. It was
demonstrated that Wnt regulates Notch2 in colorectal cancer
cells [32] and Notch3 in lung cancer cell lines [33] and that
induces Jagged1 expression in ovarian cancer [34]. However,
there is no evidence of their relationship in the rat CL.
Understanding the mechanisms that regulate the functioning
of the CL will contribute to the comprehension of different
pathologies, like the infertility associated to luteal dysfunction. Owing to the lack of a clear etiology, there are a small
number of therapies described that successfully reverse infertility in these patients. For this reason, we consider that studying the interaction between different signaling pathways is
essential for addressing new strategies to improve female reproductive health.
Based on the above, we hypothesize that the Notch pathway is modulated in response to the downregulation of the
Wnt/Β-catenin system. Due to the importance of understanding the molecular network that regulates luteal function, the
objective of this study was to assess the effect of Wnt/Β-
catenin inhibition on the expression of Notch members and
on the luteal function.
Materials and Methods
Animals
Immature female Sprague–Dawley rats (21–23 days old) were
housed at room temperature (21–23 °C) with a 12 h light/12 h
dark photoperiod in an air-conditioned environment with ad
libitum access to food and water. Rats were subcutaneously
injected with equine chorionic gonadotropin (eCG; 25 IU/rat)
and 48 h later with human chorionic gonadotropin (hCG;
10 IU/rat). The animals were euthanized by CO2 aspiration
48 h after hCG administration. The experimental protocols
were approved by the Animal Experimentation Committee
of the Instituto de Biología y Medicina Experimental
(IByME, Buenos Aires, Argentina) (PHS–NIH Approval
Statement of Compliance no. A5072–01) and conducted according to the guide for the care and use of laboratory animals
of the National Institute of Health (USA).
Corpora Lutea Culture
The ovaries were removed, and the CLs were isolated by
ovarian microdissection under a stereoscopic microscope as
previously described [15, 35]. ICG-001 (catalog item
5.04712.0001; Calbiochem-EMD Millipore, Temecula, CA,
USA) dissolved in dimethylsulfoxide (DMSO) was used to
inhibit the Wnt/B-catenin pathway. ICG-001 binds specifically to cyclic AMP response element-binding protein (CBP), a
transcriptional coactivator recruited by B-Catenin, inhibiting
their interaction and thus suppressing the formation of the
TCF/Lef complex. This impairs the expression of Wnt/Bcatenin target genes [36]. In a previous study [21], we have
used XAV939 and ICG-001 to in vivo inhibit WNT/B-catenin
pathway in CL. XAV939 mechanism of action is different
from that of ICG-001; thus, both inhibitors impaired ovulation
and luteal function through a downregulation of the WNT/Bcatenin pathway [21]. Based on these findings and because
ICG-001 mechanism of action is more specific since it directly
suppresses the Wnt/B-catenin-mediated gene transcription,
Reprod. Sci.
we have chosen this inhibitor for our current study. We performed a time-course experiment, where we selected three
time points to analyze the effect of Wnt/B-catenin inhibition
on the expression and protein levels of different genes.
N-(N-(3,5- difluorophenacetyl-L-alanyl) (DAPT, Sigma–
Aldrich) was used to inhibit the Notch pathway, as previously
described [13, 15].
Our experimental conditions consisted of a humidified incubator where the CLs were maintained at 37 °C with 5% CO2
throughout the culture period. Four CLs/well were incubated
for 6, 12, or 18 h in 0.35 mL of DMEM:F12 containing 2.4 g/
L sodium bicarbonate and 0.5% BSA at 37 °C with 0 (control), 20 or 50 μM IGC-001, or 50 μM IGC-001 + 20 μM
DAPT. For each experiment, three ovaries from different rats
were used. Isolated CLs were randomly distributed in the different wells (eight wells/treatment) until reaching four CLs/
well. A replicate (n) was considered as the pooling of two
wells, with n = 4/treatment. After incubations, the eight CLs
from each replicate were pooled for western blot or qPCR, and
the conditioned media from each replicate were frozen for
progesterone determination. The CLs were kept in dry ice
and stored at − 80 °C until processed. The use of CL incubation instead of luteal cell culture has the advantage of preserving the structure of the tissue and thus the interaction between
its molecular components.
Radioimmunoassay (RIA) for Progesterone
Determination
Progesterone concentration was determined as described previously [37], using specific antibodies supplied by Dr. G D
Niswender (Animal Reproduction and Biotechnology
Laboratory, Colorado State University, Fort Collins, CO,
USA). Under these conditions, the intra-assay and interassay coefficients of variations were 8.0 and 14.2%, respectively. The detection limit of the RIA was 25 pg progesterone.
Western Blot Analysis
For protein extracts, the CLs were resuspended in 250 μL
of lysis buffer (20 mM Tris–HCl pH 8, 137 mM NaCl,
1% Nonidet P-40, and 10% glycerol) supplemented with
protease inhibitors (0.5 mM phenylmethylsulfonyl fluoride, 0.025 mM N-CBZ-L-phenylalanine chloromethyl ketone, 0.025 mM N-p-tosyl-lysine chloromethyl ketone,
and 0.025 mM L-1-tosylamide-2-phenylethylchloromethyl ketone), and phosphatase inhibitors
(25 mM sodium fluoride, 0.2 mM sodium orthovanadate,
and 10 mM b-glycerophosphate) and homogenized using
an Ultra-Turrax homogenizer (IKA-Werke GmbH & Co.,
Staufen, Germany). The samples were centrifuged at
10,000 g at 4 °C for 10 min, and the resulting pellets were
discarded. The protein concentration in the supernatant
was measured by the Bradford assay. Then, 20 μg of
protein was boiled for 5 min and then applied to an
SDS–polyacrylamide gel (12–15%). The electrophoresis
was carried out at 25 mA for 1.5 h. The resolved proteins
were transferred onto nitrocellulose membrane for 2 h.
The blot was preincubated in a blocking buffer (5% nonfat milk, 0.05% Tween-20 in 20 mM TBS pH 8.0) for 1 h
at room temperature and then incubated with the appropriate primary antibody: Cyclin d1 1:50 (Abcam, #
Ab134175, Cambridge, USA), HES1 1:50 (Cell
Signaling, # D6P2U, Danvers, MA, USA), StAR 1:200
(Santa Cruz Biotechnology, # sc-25,806, Santa Cruz,
CA, USA), or B-actin 1:5000 (Cell Signaling, #4967).
The antibodies were diluted in 0.05% Tween-20 in
20 mM TBS pH 8.0 overnight at 4 °C. The blots were
then incubated with anti-rabbit secondary antibody conjugated with HRP (1:1000, Sigma-Aldrich, # A-4914, St.
Louis, MO, USA) and finally detected by chemiluminescence and autoradiography by using X-ray film. Protein
loading was normalized by reprobing the same blots with
antibody against B-actin. Protein content was quantified
by densitometric analysis using Scion Image Software for
Windows (Scion Corporation, Worman’s Mill, CT, USA).
RNA Isolation, Reverse Transcription, and Real-Time
PCR
Total RNA was extracted using TriPure Isolation Reagent
(Roche Diagnostics), and 1 μg of total RNA was converted
into cDNA by using the Transcriptor First Strand cDNA
Synthesis Kit (Lot#23335321, version 06, Roche
Diagnostics GmbH, Mannheim, Germany). The mRNA levels
of Cyclin d1, StAR, Dll4, Jagged1, Notch1, Notch4, Hes1,
Rbpjk, Gapdh, and Cyclophilin were quantitatively measured
by qPCR with the Biorad CFX (Biorad), using the FastStart
Universal SYBR Green (Roche Diagnostics). The program
used was 10 min at 95 °C (1 cycle), followed by 40 cycles
of 15 s at 95 °C, 30 s at 55 °C, and 1 min at 60 °C for all
primers. All the samples were run in duplicate, and negative
controls (no template) were included in all cases. To confirm
the specificity of the signal observed, melting curves were
performed at the end of each run. Glyceraldehyde 3-
phosphate dehydrogenase (Gapdh) and Cyclophilin were used
as endogenous controls. Relative expression was calculated
using the geometrical mean of the two housekeeping genes,
as described by Vandesompele et al. (2002). RT-qPCR assays
were performed using oligonucleotide primers for the genes
mentioned (Table 1). Data were analyzed following the mathematical model of Pfaffl (2001), taking into consideration the
efficiency of each primer set. The efficiencies were calculated
from the slope of a standard amplification curve (E = 10[−1/
slope]) (Table 1).
Reprod. Sci.
Statistical Analysis
Statistical analysis was carried out using GraphPad Prism
Software (version 6.0 for Windows, GraphPad Software, La
Jolla, CA, USA). Data are expressed as the mean ± SEM and
were analyzed using one-way ANOVA following Dunnett’s
as post-test. Pearson’s r coefficient was used to assess the
correlation between HES1 protein content and StAR mRNA
levels.
Values of p < 0.05 were considered significant.
Results
Effect of ICG-001 on Luteal Cyclin d1 Levels
To assess the efficiency of ICG-001, we measured Cyclin d1
mRNA levels by RT-qPCR.
ICG-001 is an inhibitor that binds specifically to the
transcriptional coactivator CBP and thus disrupts the interaction of CBP with B-catenin, suppressing the Wnt/Bcatenin-mediated transcription of target genes, such as
Cyclin d1. Several authors that used ICG-001 to block
Wnt/B-catenin signaling have measured Cyclin d1
mRNA and protein levels to evaluate its efficiency [36,
38–43].
In our experimental model, the expression of Cyclin d1
significantly decreased 6 h after ICG-001 treatment
(Fig. 1a), with no changes at 12 or 18 h. In addition, we
studied the impact of Wnt/B-catenin inhibition on Cyclin d1
protein content after incubation with ICG-001. Cyclin d1
abundance significantly decreased after 12 h of culture with
both inhibitor concentrations used. After 18 h of culture, only
50 μM ICG-001 significantly decreased Cyclin d1 levels
(Fig. 1b).
Effect of Wnt/B-Catenin Pathway Inhibition
on Progesterone Concentration and StAR Levels in CL
Cultures
To study the effect of Wnt/B-catenin pathway inhibition
on luteal function, we measured progesterone concentration in the conditioned media by RIA after 6, 12 or 18 h
of culture with ICG-001. Progesterone concentration significantly decreased after 12 h of culture, while no changes were observed after 6 or 18 h of culture (Fig. 2a). To
elucidate whether the steroidogenic step-limiting regulator
StAR was affected by ICG-001 treatment, we analyzed its
mRNA and protein levels. Surprisingly, StAR mRNA
levels significantly increased after 12 h of culture, while
no changes were observed after 6 or 18 h of culture
(Fig. 2b). Otherwise, as expected StAR protein content
significantly decreased after 12 h of culture with 50 μM
ICG-001, while no changes were observed after 6 or 18 h
of culture (Fig. 2c).
Effect of Wnt/B-Catenin Inhibition on Notch Pathway
Modulation in CL Cultures
Notch is a relevant pathway involved in the regulation of
CL function [13, 15, 44], and it was described as a regulator of StAR expression as well [16, 45]. Based on the
increase observed in StAR mRNA levels and in order to
study the effect of Wnt/B-catenin inhibition on Notch
system modulation, we analyzed the expression of various
Notch pathway members.
Effect of Wnt/B-Catenin Inhibition on Hes1 Expression
Among the different Notch pathway effectors, HES1 was described as the Notch effector expressed in the CL [14].
Therefore, we studied whether ICG-001 treatment modulated
Table 1 Sequences of primers used for RT-qPCR
Gene Forward primer Reverse primer Efficiency
Cyclin d1 5´-TGGGTCGAGAAGAGAAAG 5´-ACGGTCCCTACTTCCAAA 2.04
StARr 5´-ACTGGAAGCAACACTCTAC 5´-CTTTCCTTCTTCCAGCCTTC 1.95
Dll4 5´-ATTACCAGGCAACCTTCTC 5´-CGCTATTCTTGTCCCTGATG 2.17
Jagged1 5´-AGAACCACAGCAACTATCA 5´-GCAACTGCTGACATCAAATC 2.04
Notch1 5´-TGTGGATGAGGAAGACAAG 5´-TTCTGGCAGGGATTAGGT 2.23
Notch4 5´-AATGAGTGTGCCTCTAACC 5´-CATCTGGCACCAGTGAATC 2.18
Hes1 5´-AGGCCACTGCTAATCATAA 5´-GTCTCTCCTAAAGTCCAAGTC 2.25
Rbpjk 5´-GTTTGTCTTTCTGGCTATCT 5´-TGTTCGGAGTGGCATTTAC 1.80
Gapdh 5´-CATCAACGACCCTTCATT 5´-ACTCCACGACATACTCAGCAC 2.02
Cyclophilin 5´-GCGTCTCCTTCGAGCTCTT 5´-AAGTCACCACCCTGGCAC 2.02
The table shows the sequence of primers used for measuring the mRNA levels of different genes
Reprod. Sci.
its expression and protein content. Hes1 mRNA levels significantly increased after 6 h of culture with both inhibitor doses
and after 12 h of culture with 50 μM ICG-001, while no
changes were observed after 18 h of culture (Fig. 3a).
Regarding HES1 protein content, no significant changes were
observed after 6 h of culture, while 50 μM ICG-001 significantly increased HES1 protein abundance after 12 h of culture
(Fig. 3b). In contrast, the higher inhibitor dose caused a decrease in HES1 protein content after 18 h of culture (Fig. 3B).
As changes in both HES1 mRNA and protein levels were
observed after 12 h of culture with ICG-001, this time point
was selected to study the effect of Wnt/B-catenin inhibition on
the expression of the following Notch system members.
Effect of Wnt/B-Catenin Inhibition on Notch Receptor
and Ligand Expression
We next analyzed whether Wnt/B-catenin inhibition modulated the expression of the receptors Notch1 and Notch4.
Notch1 expression significantly increased after treatment
with ICG-001, while no changes were observed in Notch4
expression (Fig. 4a). Then, we studied the expression of
the ligands Jagged1 and Dll4. Jagged1 mRNA levels significantly increased compared to the control, while Dll4
expression was not affected (Fig. 4b).
Effect of Wnt/B-Catenin Inhibition on the Expression
of the Notch Regulator Rbpj-k
Rbpj-k is an important regulator of the Notch pathway, acting
as a co-activator or a co-repressor depending on the presence
or absence of NICD, respectively [7, 46]. Interestingly, Rbpj-k
mRNA levels significantly increased after treatment with
ICG-001 (Fig. 4c).
Analysis of Correlation Between Notch System
Activation and StAR Expression
To analyze whether the increase observed in StAR expression correlates with Notch pathway upregulation, in
first place, we performed an analysis of correlation between HES1 protein content and StAR mRNA levels.
There was a statistically significant positive correlation
between Notch activation and StAR expression with
50 μM ICG-001 (Pearson’s r = 0.86; p < 0.001) (Fig. 5).
Inhibition of Notch System Suppresses
the Compensatory Change in StAR Expression
To confirm whether the increase observed in StAR expression is a consequence of Notch pathway upregulation, we cultured corpora lutea with both ICG-001 and
Fig. 1 Effect of in vitro Wnt/Bcatenin inhibition on luteal Cyclin
d1 levels. (a) Cyclin d1 mRNA
levels after 6, 12, or 18 h incubation with ICG-001, measured by
RT-qPCR. Values were normalized to Gapdh and Cyclophilin.
Data are expressed as the mean ±
SEM. (b) Cyclin d1 protein levels
after 6, 12, or 18 h incubation
with ICG-001 measured by
Western blot. Values were normalized to B-actin and to the
mean of the control group in each
time point analyzed. Data are
expressed as the mean ± SEM.
Representative immunoblots of
protein content are shown in the
lower panel (n = 4.*p < 0.05).
Control (black), 20 μM ICG-001
(dark gray), 50 μM ICG-001
(light gray)
Reprod. Sci.
DAPT, a Notch pathway inhibitor previously used in
our laboratory [13, 15], for 12 h. When examining
Hes1 mRNA levels, we found that co-incubation with
IGC-001+ DAPT efficiently prevented the increase observed in this Notch pathway effector after culturing
with ICG-001 (Fig. 6a). Notably, the co-incubation with
both inhibitors avoided the increase in StAR mRNA
levels observed after 12 h of culture with IGC-
001(Fig. 6b).
Discussion
The involvement of the Notch and Wnt/B-catenin signaling
pathways in the ovarian function has been widely studied. The
Notch pathway promotes the maturation of ovarian follicles
and is critical for luteinization and vascular development [44].
In this context, several studies have reported that the blockage
of the Notch system impairs follicular development and induces an inhibition of VEGF-dependent luteal angiogenesis
[12, 47]. Notch inhibition also affects early luteal angiogenesis and subsequent luteal function in marmosets [48].
Regarding Wnt, this family has been recently described as
an intraovarian regulator of follicle growth and CL development (reviewed by Hernandez Gifford [19]). The importance
of Wnt signaling in the CL has been demonstrated in granulosa cell-specific Wnt4-knockout mice, where luteal progesterone synthesis decreases due to a reduction in the StAR expression and overexpression of Wnt4 in mouse granulosa cells
increases the abundance of these transcripts [17]. We have
previously demonstrated the existence of a cross talk between
the Notch system and progesterone, which upregulates the
survival of luteal cells [15]. Moreover, we have demonstrated
that the canonical Wnt pathway is involved in ovulation and
luteal function through the regulation of steroidogenesis, apoptotic proteins, and ovarian vascular development [21].
The interaction between Wnt and Notch signaling was first
described in the development of the wing of Drosophila [49].
In addition, several reports have described that this interaction
regulates development [25] and cell differentiation [26] in
tumor and embryonic fibroblast cells [22–24]. However, the
relationship between the Wnt and Notch systems in luteal
function has not been established yet. In the present study,
we demonstrate for the first time that the Notch pathway is
modulated in response to the downregulation of the Wnt/Β-
catenin system in the rat CL.
To study the effect of Wnt/B-catenin pathway inhibition
over time, mechanically isolated rat CLs were cultured with
the ICG-001 inhibitor. We have previously described that
in vivo Wnt/B-catenin inhibition decreases progesterone production and luteal StAR protein levels [21]. Based on these
results, we measured progesterone concentration in the conditioned media. Progesterone production decreased after Wnt/
B-catenin inhibition, indicating a failure in luteal steroidogenesis. This result correlates with that observed for StAR protein
levels, which consistently decreased after Wnt/B-catenin inhibition. Wnt/B-catenin has been described as a regulator of
StAR in bovine luteal cells, since a reduction in B-catenin
content decreases StAR expression [50]. Surprisingly, StAR
mRNA levels increased after 12 h of culture with ICG-001,
which led us to speculate that another pathway may be
influencing StAR expression. In line with this, Notch was described as a relevant pathway involved in the regulation of CL
function [13, 15, 44]. Besides, Wang et al. [16] showed that
Fig. 2 Effect of in vitro Wnt/B-catenin pathway inhibition on progesterone culture medium concentration and StAR levels. (a) Progesterone
levels in the culture medium after 6, 12, or 18 h incubation with ICG-
001, measured by RIA. Values were normalized to the mean of the control
group in each time point analyzed and data are expressed as the mean ±
SEM (n = 4.*p < 0.05,***p < 0.001). (b) StAR mRNA levels after 6, 12,
or 18 h incubation with ICG-001, measured by RT-qPCR. Values were
normalized to Gapdh and Cyclophilin. Data are expressed as the mean ±
SEM. (c) StAR protein levels after 6, 12, or 18 h incubation with ICG-001
measured by Western blot. Values were normalized to B-actin and to the
mean of the control group in each time point analyzed. Data are expressed
as the mean ± SEM. Representative immunoblots of protein content are
shown in the lower panel (n = 4.*p < 0.05). Control (black), 20 μM ICG-
001 (dark gray), 50 μM ICG-001 (light gray)
Reprod. Sci.
the Notch system stimulates the expression of StAR in mouse
luteal cells. In addition, Prasasya et al. [45] described that the
suppression of progesterone production following Jagged1 or
Rbpj-k knockdown in mouse granulosa cells can be partly
explained by the downregulation of StAR. Based on this information, in first place, we analyzed whether the Notch pathway is modulated as a consequence of Wnt/B-catenin inhibition. To elucidate this, we studied the levels of Hes1, the
Notch target gene described in CL cells [14]. Both Hes1
mRNA and protein levels significantly increased after incubation with ICG-001, suggesting that the Notch pathway is upregulated in response to Wnt/B-catenin inhibition. Notably,
we have also observed an increase in Hes1 protein levels after
in vivo injection of rat ovaries with XAV939, a Wnt/B-catenin
inhibitor with a mechanism of action different from that of
ICG-001 (unpublished results). Hayward et al. [25] proposed
that the Wnt and Notch systems are associated with the activation of alternative fates in vertebrates. In that report, these
authors emphasized an antagonism between these two pathways, as low Wnt signaling tends to be associated with high
Notch signaling and vice versa. Our results suggest a similar
mechanism of action in the rat CL, because blocking Wnt/Bcatenin system impacts on Notch regulation, possibly as a
molecular mechanism to compensate the downregulation of
this essential signaling pathway.
In view of these results and with the purpose of studying
whether Notch system may be regulating StAR expression, in
first place, we performed an analysis of correlation between
HES1 protein content and StAR mRNA levels. The positive
correlation between these two parameters suggests that the
increase observed in StAR expression 12 h after culturing with
ICG-001 may be a consequence of Notch activation, in line
with the observation from Wang et al. [16] who described the
Notch system as a positive regulator of StAR expression in
mouse luteal cells. In light of these results, we performed a
simultaneous inhibition of Wnt/B-catenin and Notch systems.
Remarkably, the incubation with both inhibitors avoided the
increase observed in StAR mRNA levels after 12 h of culture,
thus allowing us to confirm that the Notch pathway is responsible of restoring StAR levels. Accordingly, in our system,
progesterone production reverted to control values after 18 h
of culture, a result likely related to the increase observed in
StAR expression. We propose that Notch system is being upregulated after Wnt/B-catenin inhibition, resulting in an acute
increase in StAR expression in order to balance the reduction
in its protein levels.
The upregulation observed in the Notch system led us to
study the expression of key members of this pathway. We
studied the expression of the receptors Notch1 and 4 and the
ligands Jagged1 and Dll4. We selected these Notch members
Fig. 3 Effect of in vitro Wnt/Bcatenin inhibition on Hes1 levels.
(a) Hes1 mRNA levels after 6, 12,
or 18 h incubation with ICG-001,
measured by RT-qPCR. Values
were normalized to Gapdh and
Cyclophilin. Data are expressed
as the mean ± SEM. (b) HES1
protein levels after 6, 12, or 18 h
incubation with ICG-001 measured by Western blot. Values
were normalized to B-actin and to
the mean of the control group in
each time point analyzed. Data
are expressed as the mean ± SEM.
Representative immunoblots of
protein content are shown in the
lower panel (n = 4.*p < 0.05,
*p < 0.01,***p < 0.001). Control
(black), 20 μM ICG-001 (dark
gray), 50 μM ICG-001 (light
gray)
Reprod. Sci.
because they are localized in both large and small luteal cells
as well as in endothelial cells [10–14, 16, 47]. In our system,
we observed that Notch1 and Jagged1are the Notch family
members increased after Wnt/B-catenin inhibition.
The receptor-ligand interaction induces a cleavage of the
Notch receptors via the gamma-secretase complex, releasing
NICD. In the nucleus, NICD binds to the transcriptional repressor Rbpj-k, converting it into an activator and inducing
the expression of downstream target genes [7, 46]. In our
system, the expression of the regulator Rbpj-k increased after
treatment with ICG-001, in line with the overall stimulation of
different members of the Notch pathway. Since the expression
of Notch1 increased, the rise observed in Rbpj-k expression
may possibly be required for the NICD1/Rbpj-k interaction,
which is necessary to stimulate Hes1 expression. Taken together, these results strengthen our hypothesis that the Notch
system is upregulated when the Wnt/B-catenin pathway is
inhibited. Experiments are in progress in our laboratory to
further understand the molecular mechanisms by which Wnt
regulates Notch signaling in CL. Nevertheless, it is worth
noting that the involvement of other metabolic pathways cannot be ruled out. Cyclin d1 has been described as a target gene
of the Notch pathway in breast cancer, particularly due to the
interaction between Notch1 and Jagged1 [51]. In this context,
Fig. 4 Effect of in vitro Wnt/B-catenin inhibition on the levels of Notch
family members. mRNA levels of (a) Notch1 and Notch4, (b) Jagged1
and Dll4, and (c) Rbpj-k after 12 h of incubation with ICG-001, measured
by RT-qPCR. Values were normalized to Gapdh and Cyclophilin. Data
are expressed as the mean ± SEM (n = 4.*p < 0.05,**p < 0.01). Control
(black), 20 μM ICG-001 (dark gray), 50 μM ICG-001 (light gray)
Fig. 6 Effect of Wnt/B-catenin and Notch inhibition on Hes1 and StAR
levels. mRNA levels of (a) Hes1 and (b) StAR after 12 h of incubation
with ICG-001 + DAPT, measured by RT-qPCR. Values were normalized
to Gapdh and Cyclophilin. Data are expressed as the mean ± SEM. (n =
4.*p < 0.05). Control (black), 50 μM ICG-001 (light gray), ICG-001 +
DAPT (white)
Fig. 5 Effect of in vitro Wnt/B-catenin inhibition on the correlation between Notch activation and StAR expression. The graph depicts the relationship between HES1 protein levels and StAR expression for the three
time points analyzed. Control (circles), 20 μM ICG-001(triangles) and
50 μM ICG-001 (black squares). The solid line shows the positive correlation with 50 μM ICG-001 (slope = 0.49; r
2 = 0.74; p < 0.001)
Reprod. Sci.
we suggest that the restitution observed in Cyclin d1 mRNA
levels may be partly due to the increase in the expression of
these two members of the Notch pathway.
In summary, in vitro Wnt/B-catenin inhibition affects the
luteal function through a reduction in progesterone production
related to a decrease in StAR protein levels. Besides, Wnt/Bcatenin inhibition stimulates the Notch system, evidenced by
an increase in Hes1 expression, and promotes the expression
of selected Notch family members. Finally, StAR levels and
progesterone concentration restore to control values probably
mediated by the Notch pathway. These results provide the first
evidence of a compensatory mechanism between Wnt/Bcatenin signaling and the Notch system, which contributes to
luteal cells homeostasis.
Funding Information This work was supported by Universidad de
Buenos Aires (Grant number: UBA 01/Q502), Fondo para la
Investigación Científica y Tecnológica (Grant number: PICT 2014-
0429), Consejo Nacional de Investigaciones Científicas y Técnicas
(Grant number: PIP 177), and Rene Baron Foundation, Argentina;
Williams Foundation, Argentina.
Compliance with Ethical Standards
Conflict of Interest The authors declare that they have no conflict of
interest.
References
1. Stouffer RL. The function and regulation of cell populations comprising the corpus luteum during the ovarian cycle.: San Diego:
Elsevier, Academic Press; 2004.
2. Stocco C, Telleria C, Gibori G. The molecular control of corpus
luteum formation, function, and regression. Endocr Rev.
2007;28(1):117–49.
3. Stocco DM, Clark BJ. Regulation of the acute production of steroids in steroidogenic cells. Endocr Rev. 1996;17(3):221–44.
4. Artavanis-Tsakonas S, Rand MD, Lake RJ. Notch signaling: cell
fate control and signal integration in development. Science.
1999;284(5415):770–6.
5. Logan CY, Nusse R. The Wnt signaling pathway in development
and disease. Annu Rev Cell Dev Biol. 2004;20:781–810.
6. Bolos V, Grego-Bessa J, de la Pompa JL. Notch signaling in development and cancer. Endocr Rev. 2007;28(3):339–63.
7. Kopan R, Ilagan MX. The canonical Notch signaling pathway:
unfolding the activation mechanism. Cell. 2009;137(2):216–33.
8. Blaumueller CM, Qi H, Zagouras P, Artavanis-Tsakonas S.
Intracellular cleavage of Notch leads to a heterodimeric receptor
on the plasma membrane. Cell. 1997;90(2):281–91.
9. Kopan R, Nye JS, Weintraub H. The intracellular domain of mouse
Notch: a constitutively activated repressor of myogenesis directed
at the basic helix-loop-helix region of MyoD. Development.
1994;120(9):2385–96.
10. Johnson J, Espinoza T, McGaughey RW, Rawls A, Wilson-Rawls J.
Notch pathway genes are expressed in mammalian ovarian follicles.
Mech Dev. 2001;109(2):355–61.
11. Vorontchikhina MA, Zimmermann RC, Shawber CJ, Tang H,
Kitajewski J. Unique patterns of Notch1, Notch4 and Jagged1 expression in ovarian vessels during folliculogenesis and corpus
luteum formation. Gene Expression Patterns : GEP. 2005;5(5):
701–9.
12. Jovanovic VP, Sauer CM, Shawber CJ, et al. Intraovarian regulation
of gonadotropin-dependent folliculogenesis depends on notch receptor signaling pathways not involving Delta-like ligand 4 (Dll4).
Reproductive Biology and Endocrinology : RB&E 2013;11:43.
13. Hernandez F, Peluffo MC, Stouffer RL, Irusta G, Tesone M. Role of
the DLL4-NOTCH system in PGF2alpha-induced luteolysis in the
pregnant rat. Biol Reprod. 2011;84(5):859–65.
14. Murta D, Batista M, Silva E, et al. Differential expression of Notch
component and effector genes during ovarian follicle and corpus
luteum development during the oestrous cycle. Reprod Fertil Dev.
2015;27(7).
15. Accialini P, Hernandez SF, Bas D, et al. A link between Notch and
progesterone maintains the functionality of the rat corpus luteum.
Reproduction. 2015;149(1):1–10.
16. Wang J, Liu S, Peng L, Dong Q, Bao R, Lv Q, et al. Notch signaling
pathway regulates progesterone secretion in murine luteal cells.
Reprod Sci. 2015;22(10):1243–51.
17. Boyer A, Goff AK, Boerboom D. WNT signaling in ovarian follicle
biology and tumorigenesis. Trends Endocrinol Metab. 2010;21(1):
25–32.
18. Hsieh M, Johnson MA, Greenberg NM, Richards JS. Regulated
expression of Wnts and Frizzleds at specific stages of follicular
development in the rodent ovary. Endocrinology. 2002;143(3):
898–908.
19. Hernandez Gifford JA. The role of WNT signaling in adult ovarian
folliculogenesis. Reproduction. 2015;150(4):R137–48.
20. Hsieh M, Mulders SM, Friis RR, Dharmarajan A, Richards JS.
Expression and localization of secreted frizzled-related protein-4
in the rodent ovary: evidence for selective up-regulation in luteinized granulosa cells. Endocrinology. 2003;144(10):4597–606.
21. Accialini P, Irusta G, Bechis A, Bas D, Parborell F, Abramovich D,
et al. Tankyrase inhibition regulates corpus luteum development
and luteal function in gonadotropin-treated rats. Mol Reprod Dev.
2017;84(8):719–30.
22. Foltz DR, Santiago MC, Berechid BE, Nye JS. Glycogen synthase
kinase-3beta modulates notch signaling and stability. Curr Biol.
2002;12(12):1006–11.
23. Gopalakrishnan N, Saravanakumar M, Madankumar P, Thiyagu M,
Devaraj H. Colocalization of beta-catenin with Notch intracellular
domain in colon cancer: a possible role of Notch1 signaling in
activation of CyclinD1-mediated cell proliferation. Mol Cell
Biochem. 2014;396(1–2):281–93.
24. Morris SL, Huang S. Crosstalk of the Wnt/beta-catenin pathway
with other pathways in cancer cells. Genes & Diseases.
2016;3(1):41–7.
25. Hayward P, Kalmar T, Arias AM. Wnt/Notch signalling and information processing during development. Development.
2008;135(3):411–24.
26. Li B, Jia Z, Wang T, Wang W, Zhang C, Chen P, et al.
Interaction of Wnt/beta-catenin and notch signaling in the early stage of cardiac differentiation of P19CL6 cells. J Cell
Biochem. 2012;113(2):629–39.
27. Phng LK, Potente M, Leslie JD, Babbage J, Nyqvist D, Lobov I,
et al. Nrarp coordinates endothelial notch and Wnt signaling to
control vessel density in angiogenesis. Dev Cell. 2009;16(1):70–82.
28. Galceran J, Sustmann C, Hsu SC, Folberth S, Grosschedl R. LEF1-
mediated regulation of Delta-like1 links Wnt and Notch signaling in
somitogenesis. Genes Dev. 2004;18(22):2718–23.
29. Yamamizu K, Matsunaga T, Uosaki H, et al. Convergence of Notch
and beta-catenin signaling induces arterial fate in vascular progenitors. J Cell Biol. 2010;189(2):325–38.
30. Germar K, Dose M, Konstantinou T, Zhang J, Wang H, Lobry C,
et al. T-cell factor 1 is a gatekeeper for T-cell specification in
Reprod. Sci.
response to Notch signaling. Proc Natl Acad Sci U S A.
2011;108(50):20060–5.
31. Wang H, Zou J, Zhao B, Johannsen E, Ashworth T, Wong H, et al.
Genome-wide analysis reveals conserved and divergent features of
Notch1/RBPJ binding in human and murine T-lymphoblastic leukemia cells. Proc Natl Acad Sci U S A. 2011;108(36):14908–13.
32. Ungerback J, Elander N, Grunberg J, Sigvardsson M, Soderkvist P.
The Notch-2 gene is regulated by Wnt signaling in cultured colorectal cancer cells. PLoS One. 2011;6(3):e17957.
33. Li C, Zhang Y, Lu Y, Cui Z, Yu M, Zhang S, et al. Evidence of the
cross talk between Wnt and notch signaling pathways in non-smallcell lung cancer (NSCLC): Notch3-siRNA weakens the effect of
LiCl on the cell cycle of NSCLC cell lines. J Cancer Res Clin
Oncol. 2011;137(5):771–8.
34. Chen X, Stoeck A, Lee SJ, Shih Ie M, Wang MM, Wang TL.
Jagged1 expression regulated by Notch3 and Wnt/beta-catenin signaling pathways in ovarian cancer. Oncotarget. 2010;1(3):210–8.
35. Hernandez F, Peluffo MC, Bas D, Stouffer RL, Tesone M. Local
effects of the sphingosine 1-phosphate on prostaglandin F2alphainduced luteolysis in the pregnant rat. Mol Reprod Dev.
2009;76(12):1153–64.
36. Grigson ER, Ozerova M, Pisklakova A, Liu H, Sullivan DM,
Nefedova Y. Canonical Wnt pathway inhibitor ICG-001 induces
cytotoxicity of multiple myeloma cells in Wnt-independent manner.
PLoS One. 2015;10(1):e0117693.
37. Irusta G, Parborell F, Peluffo M, Manna PR, Gonzalez-Calvar SI,
Calandra R, et al. Steroidogenic acute regulatory protein in ovarian
follicles of gonadotropin-stimulated rats is regulated by a
gonadotropin-releasing hormone agonist. Biol Reprod.
2003;68(5):1577–83.
38. Emami KH, Nguyen C, Ma H, Kim DH, Jeong KW, Eguchi M,
et al. A small molecule inhibitor of beta-catenin/CREB-binding
protein transcription [corrected]. Proc Natl Acad Sci U S A.
2004;101(34):12682–7.
39. Chung R, Wong D, Macsai C, Piergentili A, del Bello F, Quaglia W,
et al. Roles of Wnt/beta-catenin signalling pathway in the bony
repair of injured growth plate cartilage in young rats. Bone.
2013;52(2):651–8.
40. Maftouh M, Belo AI, Avan A, Funel N, Peters GJ, Giovannetti E,
et al. Galectin-4 expression is associated with reduced lymph node
metastasis and modulation of Wnt/beta-catenin signalling in pancreatic adenocarcinoma. Oncotarget. 2014;5(14):5335–49.
41. Rong M, Chen S, Zambrano R, Duncan MR, Grotendorst G, Wu S.
Inhibition of beta-catenin signaling protects against CTGF-induced
alveolar and vascular pathology in neonatal mouse lung. Pediatr
Res. 2016;80(1):136–44.
42. Feng Y, Liang Y, Ren J, Dai C. Canonical Wnt signaling promotes
macrophage proliferation during kidney fibrosis. Kidney Diseases.
2018;4(2):95–103.
43. Li F, Liu Y, Cai Y, Li X, Bai M, Sun T, et al. Ultrasound irradiation
combined with hepatocyte growth factor accelerate the hepatic differentiation of human bone marrow mesenchymal stem cells.
Ultrasound Med Biol. 2018;44(5):1044–52.
44. Vanorny DA, Mayo KE. The role of notch signaling in the mammalian ovary. Reproduction. 2017;153(6):R187–204.
45. Prasasya RD, Mayo KE. Notch signaling regulates differentiation
and steroidogenesis in female mouse ovarian granulosa cells.
Endocrinology. 2018;159(1):184–98.
46. Lai EC. Keeping a good pathway down: transcriptional repression
of Notch pathway target genes by CSL proteins. EMBO Rep.
2002;3(9):840–5.
47. Garcia-Pascual CM, Zimmermann RC, Ferrero H, et al. Delta-like
ligand 4 regulates vascular endothelial growth factor receptor 2-
driven luteal angiogenesis through induction of a tip/stalk phenotype in proliferating endothelial cells. Fertil Steril. 2013;100(6):
1768–76 e1761.
48. Fraser HM, Hastings JM, Allan D, Morris KD, Rudge JS, Wiegand
SJ. Inhibition of delta-like ligand 4 induces luteal
hypervascularization followed by functional and structural
luteolysis in the primate ovary. Endocrinology. 2012;153(4):
1972–83.
49. Hing HK, Sun X, Artavanis-Tsakonas S. Modulation of wingless
signaling by Notch in Drosophila. Mech Dev. 1994;47(3):261–8.
50. Roy L, McDonald CA, Jiang C, Maroni D, Zeleznik AJ, Wyatt TA,
et al. Convergence of 3′,5′-cyclic adenosine 5′-monophosphate/protein kinase A and glycogen synthase kinase-3beta/beta-catenin signaling in corpus luteum progesterone synthesis. Endocrinology.
2009;150(11):5036–45.
51. Cohen B, Shimizu M, Izrailit J, Ng NF, Buchman Y, Pan JG, et al.
Cyclin d1 is a direct target of JAG1-mediated Notch signaling in
breast cancer. Breast Cancer Res Treat. 2010;123(1):113–24.
Reprod. Sci.