ORIGINAL RESEARCH

published: 22 January 2021

doi: 10.3389/fcell.2020.611503

SHP2 Nuclear/Cytoplasmic
Trafficking in Granulosa Cells Is
Essential for Oocyte Meiotic
Resumption and Maturation
Muhammad Idrees 1 , Vikas Kumar 2 , Myeong-Don Joo 1 , Niaz Ali 3 , Keun-Woo Lee 2 and
Il-Keun Kong 1,4*
1
Division of Applied Life Science (BK21 Four), Institute of Agriculture and Life Science (IALS), Gyeongsang National
University, Jinju, South Korea, 2 Division of Applied Life Science, Department of Bio and Medical Big Data (BK21 Four),
Research Institute of Natural Science (RINS), Gyeongsang National University (GNU), Jinju, South Korea, 3 Institute of Basic
Medical Sciences, Khybar Medical University, Peshawar, Pakistan, 4 The King Kong Corp. Ltd., Gyeongsang National
University, Jinju, South Korea
Edited by:
Karin Lykke-Hartmann,
Aarhus University, Denmark
Src-homology-2-containing phosphotyrosine phosphatase (SHP2), a classic
Reviewed by:
cytoplasmic protein and a major regulator of receptor tyrosine kinases and G
Lucie Němcová, protein-coupled receptors, plays a significant role in preimplantation embryo
Academy of Sciences of the Czech development. In this study, we deciphered the role of SHP2 in the somatic compartment
Republic (ASCR), Czechia
Wang Chao, of oocytes during meiotic maturation. SHP2 showed nuclear/cytoplasmic localization
China Agricultural University, China in bovine cumulus and human granulosa (COV434) cells. Follicle-stimulating hormone
*Correspondence: (FSH) treatment significantly enhanced cytoplasmic SHP2 localization, in contrast to
Il-Keun Kong
[email protected]
the E2 treatment, which augmented nuclear localization. Enhanced cytoplasmic SHP2
was found to negatively regulate the expression of the ERα-transcribed NPPC and
Specialty section: NPR2 mRNAs, which are vital for oocyte meiotic arrest. The co-immunoprecipitation
This article was submitted to
results revealed the presence of the SHP2/ERα complex in the germinal vesicle-stage
Cell Growth and Division,
a section of the journal cumulus–oocyte complexes, and this complex significantly decreased with the
Frontiers in Cell and Developmental progression of meiotic maturation. The complex formation between ERα and SHP2
Biology
was also confirmed by using a series of computational modeling methods. To verify
Received: 30 September 2020
Accepted: 30 November 2020
the correlation between SHP2 and NPPC/NPR2, SHP2 was knocked down via RNA
Published: 22 January 2021 interference, and NPPC and NPR2 mRNAs were analyzed in the control, E2 , and
Citation: FSH-stimulated COV434 cells. Furthermore, phenyl hydrazonopyrazolone sulfonate
Idrees M, Kumar V, Joo M-D, Ali N,
1, a site-directed inhibitor of active SHP2, showed no significant effect on the ERα-
Lee K-W and Kong I-K (2021) SHP2
Nuclear/Cytoplasmic Trafficking in transcribed NPPC and NPR2 mRNAs. Taken together, these findings support a novel
Granulosa Cells Is Essential for Oocyte nuclear/cytoplasmic role of SHP2 in oocyte meiotic resumption and maturation.
Meiotic Resumption and Maturation.
Front. Cell Dev. Biol. 8:611503. Keywords: granulosa cells, SHP2, ER-α, Nppc/Npr2, ERK1/2, COV434 cell line, protein-protein docking, molecular
doi: 10.3389/fcell.2020.611503 dynamics simulations

Frontiers in Cell and Developmental Biology | www.frontiersin.org 1 January 2021 | Volume 8 | Article 611503
Idrees et al. SHP2 Role in Oocyte Maturation

INTRODUCTION The primary function of SHP2 is to positively affect the

intracellular signaling of RTKs and GPCRs in the cytoplasm,
In mammals, dormant oocytes surrounded by somatic cells but unconventional nuclear localization of SHP2 has also been
enter meiotic cell division during embryonic development and identified by several studies (Jakob et al., 2008; Li et al., 2014;
meiotic arrest during the first meiotic prophase (MI) stage for Ran et al., 2017). The nucleus-localized SHP2 is essential for
a prolonged period (Guo et al., 2018). The communication the activity of several nuclear proteins, such as nuclear SHP2,
between the oocyte and its surrounding somatic cells (including which retain telomerase reverse transcriptase in the nucleus
theca, mural granulosa, and cumulus cells) is critical for for telomere lengthening (Jakob et al., 2008). SHP2 is required
maintaining this stage, as meiosis inhibitory signals pass from for signal transducer and activator of transcription 5 nuclear
somatic cells to oocytes via gap junctions. Several molecular translocation and transcriptional activity (Chughtai et al., 2002).
mechanisms in somatic cells work together to preclude the ERα, an essential protein for fertility, also requires nuclear SHP2
activation of meiotic-progression-related machinery in oocytes for DNA binding, as SHP2 enhances the Src kinase-mediated
(Zhang et al., 2010). The estradiol (E2 )–estrogen receptor (ER) tyrosine phosphorylation of ERα in the uterus and facilitates its
system is one of the mechanisms of oocyte meiotic arrest binding to the progesterone promoter region (Ran et al., 2017).
maintained by granulosa cells (Liu et al., 2017). E2 , a steroid Cytoplasmic SHP2 and extra-nuclear ERα also form a complex
hormone, is a critical regulator of oocyte meiotic arrest and exerts and significantly affect mitogen-activated protein (MAP) kinases
its effects by binding to ERα and ERβ (Lubahn et al., 1993). and AKT signaling (Levin, 2009; Li et al., 2014).
Ligand-activated ERα regulates the transcription of natriuretic In this study, we identified a novel role of SHP2 in association
peptide type C (NPPC) and natriuretic peptide receptor 2 with ERα and FSH in mammalian oocyte meiotic resumption.
(NPR2) by directly binding to their promoter regions and Our results provide an experimental proof that SHP2 exists
enhancing their expression in granulosa cells (Liu et al., 2017). in complex with ERα in the nucleus of human granulosa and
The NPPC and NPR2 genes further regulate the production of bovine cumulus cells. Progression in bovine oocyte meiotic
cyclic guanosine monophosphate in granulosa cells, which then maturation significantly reduced the SHP2/ERα complex. The
enters the oocyte via gap junctions, inhibit phosphodiesterase FSH-mediated stimulation of cultured granulosa cells also
3A-induced cyclic adenosine monophosphate degradation, and reduced the SHP2/ERα complex as compared to E2 treatment
maintain oocyte meiotic arrest (Zhang et al., 2010; Kiyosu et al., that markedly enhanced it. Furthermore, FSH increases the
2012; Wigglesworth et al., 2013). cytoplasmic localization of SHP2, and this may be due to FSHR-
Follicle-stimulating hormone (FSH), a negative feedback induced signaling in granulosa cells. Moreover, the E2 activation
regulator of ERα, controls folliculogenesis by stimulating the of ERα not only enhanced the mRNA expression of NPPC and
granulosa cells of early antral follicles (Glidewell-Kenney NPR2 but also increased the nuclear localization of SHP2. The
et al., 2008; El-Hayek et al., 2014). FSH transduces its functional role of SHP2 in oocyte-surrounding somatic cells and
signals via the FSH-specific G protein-coupled receptor its significance in oocyte maturation have never been studied.
(FSHR), depending on intracellular effectors (Casarini and Thus, the elucidation of the SHP2 mechanism could provide a
Crepieux, 2019). By activating mitogen-activated protein potential therapeutic target in the treatment of infertility.
kinase (MAPK)/extracellular signal-regulated kinase (ERK)
and AKT (protein kinase B) signaling pathways, FSH induces
the proliferation and differentiation of granulosa cells to a MATERIALS AND METHODS
pre-ovulatory phenotype (El-Hayek et al., 2014; Donaubauer
et al., 2016). FSH requires several proteins to transduce its The study was conducted in full compliance with the Gyeongsang
signaling in granulosa cells, and SHP2 has been identified to National University Institute of Animal Care Committee
play a key role in FSH signaling (Donaubauer et al., 2016). (GNU-130902-A0059). Most of the chemicals and reagents were
SHP2, a main regulating component of receptor tyrosine obtained from Sigma-Aldrich (St. Louis, MO, USA), unless
kinases (RTKs) and G protein coupled receptors (GPCRs), otherwise noted.
shows ubiquitous expression and has multiple functions
(Qu, 2002; Zhang et al., 2004; Furcht et al., 2014). Evidence
has shown that SHP2 is critical for numerous reproductive Experiment Procedures
tissues (Hu et al., 2015, 2017; Ran et al., 2017; Idrees et al., Experiment 1
2019b, 2020; Kim et al., 2019). By interacting with the To understand the role of SHP2 in the somatic compartment
transmembrane adaptor proteins, SHP2 induces the sustained of oocytes, bovine ovary-derived cumulus–oocyte complexes
activation of the rat sarcoma (RAS) and ERK1/2 signaling (COCs) were used to culture cumulus cells. Primary bovine
pathway, which is essential for oocyte meiotic maturation cumulus cells were separated from pre-ovulatory follicles and
and ovulation (Fan and Sun, 2004; Dance et al., 2008; Fan cultured in α-MEM media. FSH (20 ng/ml) and epidermal
et al., 2009). In addition to folliculogenesis, SHP2 is important growth factor (EGF 25 ng/ml) were used to stimulate the
for embryonic development, as indicated by SHP2-knockout cultured cumulus cells. Phenyl hydrazonopyrazolone sulfonate
embryos which exhibit inner cell mass death and failure to 1 (PHPS1) (5 µM) was used for COCs and cultured cumulus
yield trophoblast stem cells (Saxton et al., 1997; Yang et al., cells according to our previous study (Idrees et al., 2019b).
2006). Samples from various stages of bovine oocytes were used to

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Idrees et al. SHP2 Role in Oocyte Maturation

examine the SHP2/ERα complex via co-immunoprecipitation and co-cultured with mature oocytes for 20 h. On the next day,
(co-IP) and immunofluorescence. the cumulus cells were removed from the presumed zygotes
by vortexing for 3 min and cultured in SOF-BE1 medium
Experiment 2 supplemented with 5 µg/ml insulin, 5 µg/ml transferrin, 5 ng/ml
To analyze the SHP2/ERα complex in granulosa cells, COV434 sodium selenite (cat. 11074547001), 4 mg/ml fatty-acid-free BSA,
human granulosa cell line was used, and co-IP was used and 100 ng/ml EGF. The presumed zygotes were cultured for 8
to detect complex formation. 3-(4,5-Dimethylthiazol-2-yl)-2,5- days, with one-time media replacement after 3 days.
diphenyltetrazolium bromide (MTT) assay showed that the least
effective concentration of PHPS1 in the COV434 cell line was RNA Extraction and Reverse Transcription
3 µM. For FSH and E2 , the same effective concentrations were Quantitative PCR
used as those in a previous study that used the COV434 cell line To generate cDNA, mRNA was extracted from samples using
(Liu et al., 2017). SHP2 was knocked down using small interfering the Dynabeads mRNA Direct Kit (Dynal AS, Oslo, Norway)
RNA (siRNA, 10 nM). The expression of ERα was examined according to the manufacturer’s protocol and as previously
via immunofluorescence, and its transcribed genes (NPPC and described (Idrees et al., 2019b). The concentration of the
NPR2) were analyzed via quantitative real-time polymerase chain purified mRNA was determined spectroscopically (NANO
reaction (RT-qPCR). DROP 2000c Thermo Fisher Scientific) at 260 nm. Superscript
III reverse transcriptase (iScript R cDNA Synthesis Kit from
COC Collection and in vitro Maturation Bio-Rad Laboratories Hercules, CA, USA) was used to reverse-
Local abattoir-derived bovine ovaries were transported to the transcribe the first-strand cDNA. The PCR primers were
laboratory within 2 h after dissection. COCs with follicle diameter designed using the Primer3 (v. 0.4.0) software of the National
ranging from 3 to 6 mm and with a minimum of three layers Center for Biotechnology Information nucleotide database. A
of cumulus cells were collected according to a previously CFX98 instrument (Bio-Rad Laboratories) was used to quantify
described protocol (Idrees et al., 2019b). In brief, ovaries the transcription levels. The reaction was carried out in a final
were washed with Dulbecco’s phosphate-buffered saline (D- volume of 10 µl, comprising 3 µl diluted cDNA, 1X iQ SYBR
PBS), and COCs were aspirated using 18-gauge disposable Green Super mix (iQ SYBR Green Super Mix Kit, Bio-Rad
needles attached to a vacuum pump. The follicular medium Laboratories, cat. 170-8882 Laboratories Hercules, CA, USA),
was diluted using TL-HEPES [10 mM HEPES (H-6147), 2 mM and 0.2 mM each of forward and reverse primers. The samples
calcium chloride (C-7902), 3.2 mM potassium chloride (P-5405), were processed in triplicate according to the manufacturer’s
0.5 mM magnesium chloride (M-2393), 10 mM sodium lactate, guidelines. The relative gene expression was calculated using the
2 mM sodium bicarbonate (S-5761), 114 mM sodium chloride (S- threshold 11 C(t) method. The GAPDH gene was used as an
5886), 0.34 mM sodium bisphosphate (S-5011), 1 µl/ml phenol endogenous control and for the normalization of the expressed
red, 100 IU/ml penicillin, and 0.1 mg/ml streptomycin]. A data. The primers and the PCR conditions for each gene are given
stereomicroscope was used to collect COCs with a minimum in Supplementary Table 4.
of three uniform layers. The retrieved GV-stage oocytes, along
with cumulus cells in groups of 40–50, were cultured in H2 DCFDA Staining for Reactive Oxygen
700 µl of TCM199 media (Invitrogen Corp., Carlsbad, CA, Species
USA) supplemented with 10% (v/v) fetal bovine serum (FBS; To measure the reactive oxygen level (ROS) level,
Gibco BRL, Life Technologies, Grand Island, NY, USA, cat. 2,7-di-chloro-di-hydro-fluorescein di-acetate (H2DCFDA,
16000-044), 10 ng/ml EGF, 10 µg/ml FSH, 1 µg/ml ostradiol-17β, cat. #D6883) staining was performed according to a previously
0.6 mM cysteine, and 0.2 mM sodium pyruvate (Gibco BRL, Life described protocol (Idrees et al., 2019a). In brief, the MII stage
Technologies, Grand Island, NY, USA, cat. 11360-070). Incubator live oocytes were incubated in PBS containing 10 nM H2DCFDA
conditions were set to 38.5◦ C and 5% CO2 for 22–24 h. for 30 min at 38.5◦ C and 5% CO2 . After that, the samples were
washed thrice with PBS and examined under an epifluorescence
In vitro Fertilization and Embryo Culture microscope (Olympus IX71) under 490-nm excitation and
In vitro-matured (MII) COCs were co-cultured with frozen– 525-nm emission filters.
thawed bovine sperm of already proven fertility (Idrees et al.,
2019a). The sperm straw was thawed at 37.0◦ C for 1 min and Cells and Cell Culture
diluted in D-PBS. Subsequently, the sperm suspension was Primary Bovine Cumulus Cell Culture
centrifuged at 750 × g for 5 min at room temperature, and Bovine cumulus cells obtained from 3–6-mm COCs were
the pellet was re-suspended in 500 µl of heparin (20 µg/ml) cultured in previously described media and conditions (Baufeld
diluted in in vitro fertilization (IVF) medium [Tyrode’s lactate and Vanselow, 2013). In brief, cumulus cells were collected
solution supplemented with 6 mg/ml bovine serum albumin immediately after COC isolation and denuded by recurrent
(BSA), 22 µg/ml sodium pyruvate, 100 IU/ml penicillin, and pipetting through a narrow pipette. The collected cumulus cells
0.1 mg/ml streptomycin]. The heparin-diluted sperm suspension were cultured on collagen-coated 24-well-plates with 1.25 ×
was incubated at 38.5◦ C and 5% CO2 for 15 min to facilitate 105 viable cells per well. The serum free α-MEM medium
capacitation. Subsequently, the sperm suspension was diluted was supplemented with HEPES (20 mM), BSA (0.1%), sodium
in IVF medium to a final density of 1.0–2.0 × 106 sperms/ml bicarbonate (0.084%), sodium selenite (4 ng/ml), transferrin

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Idrees et al. SHP2 Role in Oocyte Maturation

(5 µg/ml), insulin (10 ng/ml), androstenedione (2 µM), L- Caspase-3 (Santa Cruz cat. Sc-1225), p-Nf-Kb (Santa Cruz cat.
glutamine (2 mM), non-essential amino acids (1 mM), penicillin Sc-271908), and p-mTOR (Abcam cat. ab84400). The secondary
(100 IU), and streptomycin (0.1 mg/ml). FSH (20 ng/ml) and antibodies used in this study were mouse horseradish peroxidase
EGF (25 ng/ml) were also added to the culture medium to (HRP; Amersham ECL cat. NA931), rabbit HRP (Amersham
stimulate the cells. ECL cat. NA934), mouse-FITC (Santa Cruz cat. sc-516140), and
rabbit-TRITC (Thermo Fisher Scientific, Waltham, MA, USA,
COV434 Cell Culture, Treatment, and cat. A16101).
Transfection
The COV434 human granulosa cell line was kindly gifted by Histological Analysis
Professor Jeehyeon Bae (Chung-Ang University, Seoul, Republic Bovine ovaries collected from the local abattoir were washed
of Korea). Cells were cultured in Dulbecco’s modified Eagle’s with saline and stored at 4◦ C in 20% sucrose diluted in 1×
medium containing 1% penicillin–streptomycin and 10% FBS as PBS solution for 72 h. Thereafter, the ovaries were washed with
previously described (Jin et al., 2016). The incubator conditions saline and stored in 4% paraformaldehyde at 4◦ C for 72 h. The
were set to 37.0◦ C and 5% CO2 air. Cell treatment was performed ovaries were placed in an optimal cutting temperature compound
using the MTT assay in accordance with the manufacturer’s (Sakura Finetek Inc., Torrance, CA, USA) and stored at −80◦ C
protocol (Sigma-Aldrich). In 96-well-plates, COV434 cells were for a minimum of 24 h to make blocks, and 12-µm-thick sections
cultured at a concentration of 0.8 × 104 cells per well, with 200 were taken on probe-on plus charged slides (Fisher, Rock-ford,
µl of media already contained per well. After growing the cells for IL, USA) using a CM 3050C cryostat (Leica, Germany). The slides
24 h, the medium was replaced with a fresh medium containing were stored at −80◦ C until further processing.
no or varying concentrations of PHPS1 and incubated for 24 h.
Subsequently, the cells were incubated with MTT solution for 3 h.
Thereafter, DMSO (100 µl) was added, and the plate was agitated
Immunofluorescence
For immunofluorescence, staining was performed according to
for 20 min. A microplate scanning reader was used to measure
a previously defined protocol (Idrees et al., 2019b). In brief, 4%
absorbance at 550 to 570 nm (L1, value for viable cells) and 620
(v/v) paraformaldehyde in 1 M PBS was used to fix the samples,
to 650 nm (L2, value for debris). The corrected absorbance (A =
which were preserved at 4◦ C for a minimum of 30 min to a
L1–L2) was used to calculate the number of viable/dead cells in
prolonged period. On the staining day, the samples were washed
each well for each group.
twice in 0.3% polyvinyl alcohol in 1× PBS (PBS-PVA) and
LipofectamineTM RNAiMAX Transfection Reagent (Thermo
permeabilized with the proteinase K solution (0.1%) for 5 min.
Fisher Scientific, Waltham, MA USA cat #13778030) was used to
After washing twice with PBS-PVA for 5 min, the samples were
transiently transfect the COV434 cell line. Briefly, lipofectamine
incubated for 90 min in 5% blocking solution (BSA-PBS-PVA).
was diluted in the Opti-MEM medium (Thermo Fisher Scientific,
Primary antibodies were applied to the samples and kept at 4◦ C
Inc.), and the transfection complexes were prepared by diluting
overnight. On the next day, after washing twice with PVA-PBS for
the siRNAs (Santa Cruz products sc-37007 and sc-36488,
10 min, the samples were incubated with secondary antibodies
respectively, Santa Cruz Biotechnology, St. Louis, MO, USA) in
(FITC and TRITC) at room temperature for 90 min. For nuclear
the Opti-MEM medium. The diluted siRNA was added to the
staining, 10 µg/ml 4,6-diamidino-2-phenylindole was applied for
diluted lipofectamine reagent and incubated for 20 min at room
5 min. Thereafter, the samples were washed thrice with PVA-PBS
temperature. The transfection complexes were added to each well
for 5 min, mounted with a fluorescent mounting medium, and
and incubated for 24 h, after which the medium was replaced
covered with a slide cover. A confocal laser scanning microscope
with a fresh one. At 48 h after transfection, the cells were fixed
(Fluoview FV 1000, Olympus, Japan) was used to capture images.
(4% paraformaldehyde) or lysed for the examination of specific
ImageJ analysis software (National Institutes of Health, Bethesda,
protein expression via immunoblotting or immunofluorescence.
MD, USA; https://imagej.nih.gov/ij) was used to measure the
Co-immunoprecipitation and Antibodies relative integrated density of the signals.
To analyze the SHP2/ERα complex, co-immunoprecipitation
was performed according to the manufacturer’s protocol (Pierce Immunoblotting
Co-IP cat. 26149). The SHP2 antibody was used to pull down The samples [MII oocytes (100 per extract) or trypsinized
the protein complex. The SHP2/ERα complex levels in various cells] were washed with D-PBS and dissolved in PRO-PREPTM
samples were analyzed via western blotting. The following (iNtRON Biotechnology, Burlington, NJ, USA, cat. 17081).
primary antibodies were used in this study: β-actin (Santa Subsequently, the samples were sonicated, and the cell lysate was
Cruz cat. sc-47778), SHP2 (Santa Cruz Biotechnology, USA centrifuged at 10,000 × g for 25 min at 4◦ C. The supernatant
cat. sc-271106), ERα (Abcam, Cambridge, Cambs, UK cat. was collected and quantified using the Bradford assay (cat.
ab3575), AKT (Cell Signaling technology cat. 9272), p-AKT (Cell 5000002 Laboratories Hercules, CA, USA), while the settled
Signaling, cat. 9271), p-ERK1/2 (Cell Signaling, cat. CST 9101S), debris was discarded (Idrees et al., 2019a). Equal amounts
ERK1/2 (Cell Signaling, cat. CST 9102S), FOXL2 (LifeSpan of protein (10–20 µg) were fractionated by SDS-PAGE (10
BioScience, Seattle, WA, USA cat. LS-B12865), SF1 (Abcam and 12%) and then transferred to a polyvinylidene fluoride
cat. ab168380), Wnt4 (Santa Cruz cat. sc-376279), octamer- (PVDF) membrane (cat. GE 10600023; Sigma-Aldrich). The
binding transcription factor 4 (OCT4; Santa Cruz cat. Sc-8629), PVDF membrane was blocked with skimmed milk for 1 h and

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Idrees et al. SHP2 Role in Oocyte Maturation

incubated overnight with a primary antibody at 4◦ C in a two- avoid steric clashes and bad contacts. Subsequently, the energy-
dimensional shaker. Thereafter, it was incubated with HRP- minimized system was subjected to equilibration in two phases.
conjugated secondary antibody at room temperature for 90 min. In the first phase, temperature balancing was performed under
An enhanced chemiluminescence detection reagent (Pierce TM an NVT ensemble for 500 ps at 300 K while using a V-rescale
ECL Western Blotting Substrate, Thermo Fisher Scientific, thermostat (Bussi et al., 2007). In the second phase, pressure
Waltham, MA, USA) was used to detect bound antibodies. equilibration was achieved under the NPT ensemble for 500
Protein ladders (Abcam, USA, cat. ab116029) were used to ps at 1.0 bar using the Parrinello–Rahman barostat (Parrinello
determine the molecular weights of the proteins. ImageJ software and Rahman, 1981). Each system was then subjected to 50 ns
(National Institutes of Health, Bethesda, MD, USA; https:// of production run. The particle mesh Ewald method was used
imagej.nih.gov/ij) was used to detect the optical densities of the to estimate the electrostatics of long-range interactions (Darden
bands on X-ray films (iNtRON, Biotechnology Inc., Burlington, et al., 1993). During simulation, the bond lengths were restrained
NJ, USA). using the Linear Constrain Solver algorithm (Hess et al., 1997).
The MD simulation results were analyzed using the visual
Molecular Docking of ERα and SHP2 molecular dynamics (Humphrey et al., 1996) and DS software
The 3D structural information on the binary complex of programs. Additionally, the binding free energy calculations
human ERα and SHP2 has not been reported yet; therefore, were performed with the molecular mechanics-generalized Born
we performed a molecular docking study to gain an insight surface area (MM-GBSA) algorithm using an online webserver,
into the molecular-level interaction between ERα and SHP2. Hawk Dock (Weng et al., 2019).
The ERα complex structure was not available in Protein
Data Bank (PDB), but a recent study modeled a full-length Statistical Analysis
structure of the ERα comprising the ligand-binding domain At least three separate and independent experiments were
and DNA-binding domain, which is available in the Small- performed to derive data, which were expressed as mean ±
Angle Scattering Biological Data Bank (Valentini et al., 2015; SEM. The western blotting bands and immunofluorescence
Huang et al., 2018). For SHP2, an open conformation structure images were analyzed using Graph Pad Prism 6 (Graph Pad
(PDB ID: 6CRF) was selected from the literature (LaRochelle Software, USA) and Image J software programs. P-values were
et al., 2018) and downloaded from PDB (https://www.rcsb.org/). calculated on the basis of one-way analysis of variance (ANOVA),
The structures of both proteins were prepared using a clean followed by Student’s t-test (significance: ∗ p < 0.05, ∗∗ p < 0.01,
protein protocol and were further minimized using the Discovery ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001).
Studio v2018 software (http://3dsbiovia.com/products/). Water
molecules were removed, and hydrogen atoms were added. For
molecular docking, we utilized HDOCK, the web server for RESULTS
protein–protein and protein–DNA/RNA docking (Yan et al.,
SHP2 Expression in Ovarian Tissue and
2020). During molecular docking, ERα was used as a receptor
and SHP2 as a ligand. Owing to the lack of structural information Dynamic Changes With Growth Factor
for the ERα and SHP2 complex, the docking simulation was Treatment in Cultured Bovine Granulosa
performed with default parameters, without defining the binding Cells
site according to a previously established method (Selent et al., To investigate the protein localization pattern of SHP2 in
2013). The HDOCK server uses a fast Fourier transform- the somatic compartment of a follicle, we first performed the
based approach to sample all possible binding modes globally immunofluorescence staining of bovine ovarian sections with
(Katchalski-Katzir et al., 1992). Additionally, all binding modes an SHP2-specific antibody (Figures 1A,B; Eppig and Handel,
collected were reassessed with iterative-knowledge-based scoring 2012). The results showed the protein expression of SHP2 in
functions (Yan et al., 2017). the granulosa cells of pre-ovulatory follicles and in the ovarian
medulla. The localization pattern analysis revealed that SHP2
Molecular Dynamic Simulation for the ERα was evenly distributed between the nucleus and the cytosol. To
and SHP2 Binary Complex analyze SHP2 localization in cumulus cells, the ovarian immature
The prediction of the correct 3D binding mode for protein– COCs were fixed and immunofluorescent-stained with the SHP2
protein interaction is extremely difficult, as protein-protein antibody (n = 20 COCs per group) (Figure 1C). The results
molecular docking studies have always been challenging. showed that SHP2 had a localization pattern similar to that
Therefore, to refine the docked ERα-SHP2 complex, we identified in granulosa cells. To further confirm the nuclear
subjected the best docked structure to molecular dynamics localization of SHP2, pre-antral-follicle-derived bovine cumulus
(MD) simulations using GROMACS v2018.2 (Pronk et al., cells were cultured in vitro, and SHP2 was evaluated (Figure 1D;
2013). The topology parameters of the protein were prepared Georges et al., 2014). Although SHP2 is a main regulating
by the AMBER99SB-ILDN force field (Lindorff-Larsen et al., component of growth factor receptor intracellular signaling, it
2010). The binary complex was placed into a dodecahedron showed unconventional nuclear localization in granulosa and
box, and the TIP3P water model was used for solvation. The cumulus cells. To examine the function of SHP2, cultured
system was neutralized by 18 Na+ ions and subjected to an cumulus cells were treated with EGF and FSH, and samples were
energy-minimization step with the steepest descent algorithm to prepared for RT-qPCR (Figure 1E). The mRNA expression of

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Idrees et al. SHP2 Role in Oocyte Maturation

FIGURE 1 | Src-homology-2-containing phosphotyrosine phosphatase (SHP2) expression in bovine ovary and upregulation with follicle-stimulating hormone (FSH) or
epidermal growth factor (EGF) treatment. (A) Representative images of SHP2 protein expression (green, fluorescein isothiocyanate) in bovine pre-ovulatory oocyte
granulosa cells. (B) SHP2 protein expression in bovine ovarian medulla. The lower images are zoom-ins of the upper images. (C) SHP2 expression in the cumulus
cells of bovine oocyte (germinal vesicle, GV). (D) SHP2 expression in cultured cumulus cells obtained from GV-stage oocytes. (E) Cultured cumulus cells treated with
FSH or EGF and PTPN11 (SHP2), mitogen-activated protein kinase 1 (MAPK1), and protein kinase B (AKT3) genes analyzed via RT-qPCR (the experiment was
repeated thrice, and data are shown as mean ± SEM. NS, not significant; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

protein tyrosine phosphatase N11 (PTPN11 encoding protein These findings indicate that SHP2 shows nuclear and cytoplasmic
SHP2) and AKT3 genes was found to be statistically significant localization in bovine granulosa and cumulus cells. EGF or FSH
with EGF or FSH stimulation as compared to the control, while treatment can upregulate PTPN11 (SHP2) mRNA expression in
MAP K1 was found to be very significant with FSH treatment cultured cumulus cells (Dance et al., 2008), but the function of
(n = 20 per group; ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗∗ p < 0.0001). the nucleus-localized SHP2 still needs exploration.

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Idrees et al. SHP2 Role in Oocyte Maturation

FIGURE 2 | Src-homology-2-containing phosphotyrosine phosphatase (SHP2)/estrogen receptor alpha (ERα) complex and ERα-dependent gene expression in
cumulus cells. (A) SHP2 and ERα proteins analyzed in germinal vesicle (GV)- and MII-stage cumulus–oocyte complexes (COCs) via immunofluorescence. Both
proteins showed nuclear localization and exited the nucleus with the progression of meiotic maturation. Data are expressed as means ± SEM. GV and MII COCs were
immune-labeled with SHP2 (green) and ERα (red) and counterstained with 10 µg/ml 4′ ,6′ -diamidino-2-phenylindole to visualize DNA (mean ± SEM). (B)
Co-immunoprecipitation of SHP2 and ERα showed that both proteins form a complex at the GV COC stage, and the complex is significantly reduced with oocyte
maturation. (C) Natriuretic peptide C (NPPC) and natriuretic peptide receptor 2 (NPR2) genes analyzed via RT-qPCR in GV, germinal vesicle breakdown (GVBD), and
MII-stage COCs and compared with phenylhydrazonopyrazolone sulfonate 1 (PHPS1) treatment of GVBD- and MII-stage oocytes. (D) CX37 and CX43 genes
analyzed via RT-qPCR in GVBD- and MII-stage COCs and compared with the PHPS1 treatment of GVBD- and MII-stage oocytes. (E) Cultured bovine granulosa cells
were stimulated with follicle-stimulating hormone (FSH) and FSH plus PHPS1, and the p-AKT protein was analyzed along with total AKT. The experiments were
repeated thrice, and data are shown as mean ± SEM. NS, not significant; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

SHP2 Forms a Complex With ERα in GV proteins showed complete cytoplasmic localization. Few studies
Oocyte Cumulus Cells and Is Involved in have identified that SHP2 forms a complex with ERα in the MCF7
ERα Transcriptional Activity and Ishikawa cell lines for activating signaling pathways and ERα
ERα plays a key role in oocyte meiotic resumption and transcription (Li et al., 2014; Ran et al., 2017). To understand
maturation, and several studies have identified the role of nuclear the SHP2 and ERα behavior in cumulus and granulosa cells,
SHP2 in the transcriptional activity of ERα (Li et al., 2014; samples were collected from ovary-retrieved immature COCs,
Ran et al., 2017). To analyze the interaction of SHP2 and 16-h cultured germinal vesicle breakdown (GVBD) COCs, and
ERα in the cumulus cells of GV- and MII-stage oocytes, we 22-h cultured mature (MII) COCs for the Co-IP of SHP2 and
immunostained COCs with specific SHP2 and ERα antibodies ERα proteins (Figure 2B). These data imply that SHP2 exists
(Figure 2A). At the GV stage, SHP2 and ERα were localized in complex with ERα in immature COCs, but the amount of
in the nucleus of cumulus cells, but at the MII stage, both complex becomes significantly reduced with the progression of

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Idrees et al. SHP2 Role in Oocyte Maturation

meiotic maturation. It was previously identified that ERα binds SHP2 would impair the development of subsequent embryos. To
with the promoter regions of NPPC and NPR2 in granulosa do this, we carried out IVF using control and SHP2-inhibited
and cumulus cells and sustains oocyte meiotic arrest (Liu et al., oocytes, and then zygotes were cultured in IVC media for 8
2017). Therefore, we applied PHPS1, a site-specific inhibitor days to check the development. PHPS1 application reduced
of SHP2, to examine ERα transcriptional activity. Contrary to the embryo development by almost 50% [control n = 143
our expectations, the PHPS1-treated COCs showed a higher (39%) vs. PHPS1 n = 108 (21%)]. The SHP2-inhibited oocytes
expression of NPPC/NPR2 as compared to the control COCs, that developed to the blastocyst stage showed significantly
in which the expression of both genes was significantly reduced high apoptosis as identified via nucleus-localized NF-kB and
with meiotic progression (n = 20 per group; ∗ P < 0.05, enhanced caspase-3 expression (Figure 3C). Furthermore, the
∗∗ P < 0.01; Figure 2C). To further confirm, we analyzed the developed blastocysts showed the reduced expression of OCT4
expression of connexin-43 (CX43) and connexin-37 (CX37), as (Supplementary Figure 1B; Simmet et al., 2018). Moreover,
both genes are required for NPPC/NPR2 signaling to mediate SHP2 downstream proteins p-AKT and p-ERK1/2 showed a
oocyte meiotic arrest (Richard and Baltz, 2014). The results significant reduction with PHPS1 treatment as compared to the
indicated a significantly higher expression of both CX37 and control littermates (Supplementary Figure 1C). These findings
CX43 in the control group than in the PHPS1-treated COCs altogether suggest that SHP2-inhibited oocytes, in many cases,
(Figure 2D; n = 20 per group; ∗ P < 0.05, ∗∗ P < 0.01). FSH are unable to develop properly.
activates MAP kinases and AKT signaling in the cumulus cells of
oocytes, which inhibit NPPC/NPR2 (Tsuji et al., 2012; Wang et al.,
2013), while SHP2 inhibition reduced FSH signaling in granulosa FSH Reduces SHP2/ERα Complex in
cells (Donaubauer et al., 2016). We found that FSH stimulation Human Granulosa Cells (COV434)
of cultured cumulus cells enhanced the expression of p-AKT To uncover the SHP2/ERα complex expression, localization,
protein, whereas the combined application of FSH and PHPS1 and involvement in human oocyte meiosis, we cultured an
significantly reduced its expression (∗ P < 0.05, ∗∗ P < 0.01; immortalized human granulosa cell line COV434 that exhibits
Figure 2E). All the above-mentioned results revealed that SHP2 the biological characteristics of normal human granulosa cells
exists in complex with ERα, and this complex shows a significant (Zhang et al., 2000). Granulosa cells were treated with PHPS1
reduction with the progression of oocyte meiotic maturation. at various concentrations, and 3.5 µM was selected as the
Unexpectedly, SHP2 inhibition had no significant effect on ERα least effective concentration via an MTT assay (Figure 4A).
transcriptional activity during COC meiotic maturation, but it Thereafter, SHP2 and fork head box L2 (FOXL2, a surrogate
can significantly reduce FSH intracellular signaling in cultured marker of granulosa cells) antibodies were applied, and the
cumulus cells. results showed that SHP2 had a localization pattern similar
to that previously detected in cultured bovine cumulus cells
(Figure 4B; Georges et al., 2014). PHPS1 application to human
Cumulus Cell Cytoplasmic SHP2 Inhibition granulosa cells had no noticeable effect on nucleus-localized
Deteriorates Oocyte Maturation and SHP2. To analyze the effects of estradiol and FSH on SHP2 and
Embryo Development ERα localization in granulosa cells, we applied E2 (10.0 µM)
SHP2 cytoplasmic localization is essential for FSH-induced and FSH and checked both proteins via immunofluorescence
activation of MAP kinases and AKT signaling, and PHPS1 (Figure 4C). The results indicated that the application of
inhibits active cytoplasmic SHP2 (Donaubauer et al., 2016; E2 highly localized SHP2 in the nucleus and that of FSH
Idrees et al., 2019b). To analyze the effects of FSH signaling translocated it to the cytoplasm. To detect the SHP2/ERα
blockage via SHP2 inhibition on oocyte meiotic maturation, complex in granulosa cells and determine the effects of E2 and
we treated immature COCs with PHPS1 and collected samples FSH on this complex, we performed a co-immunoprecipitation
for immunofluorescence with the progression of meiotic stages assay (Figure 4D). The SHP2/ERα complex was detected in
[oocyte maturation percentage control n = 172 (84%) vs. PHPS1 the control group and was significantly enhanced with E2 ,
n = 185 (58%), n = 50 per group, with four independent but the FSH treatment markedly reduced this complex in
biological replicates] (Figure 3A). The PHPS1-treated COCs granulosa cells. The reduction in the SHP2/ERα complex caused
showed reduced cytoplasmic localization of SHP2 and ERα by the FSH treatment might be due to FSH intracellular
compared to the control, where both proteins were localized signaling in granulosa cells (Li et al., 2014). Therefore, the
in the cytoplasm (n = 30 per group; ∗ P < 0.01). Next, p-ERK1/2 and p-AKT proteins were analyzed via western
we tested p-ERK1/2 and p-AKT signals in the MII oocytes blotting, and the results showed a high expression of these
(Figure 3B), as both proteins are essential for oocyte meiotic proteins in the presence of FSH as compared to the control
maturation and are downstream of SHP2 (Hatch and Capco, and E2 -treated groups (Figure 4E). The above-mentioned results
2001; Andrade et al., 2017). We found a significant reduction in indicated that the SHP2 and ERα complex is present in
the p-ERK1/2 and p-AKT proteins in GVBD and MII oocytes human granulosa cells, and the FSH treatment can reduce
treated with PHPS1 compared to control oocytes. Furthermore, the amount of this complex. Furthermore, E2 acts in an
the MII oocyte also showed a significant enhancement in opposite manner by enhancing the SHP2/ERα complex and
ROS (Supplementary Figure 1A). Given the effects of SHP2 reducing FSH-induced intracellular signaling in the COV434
inhibition on oocyte quality, we speculated whether the loss of cell line.

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Idrees et al. SHP2 Role in Oocyte Maturation

FIGURE 3 | Cumulus-cell cytoplasmic Src-homology-2-containing phosphotyrosine phosphatase (SHP2) inhibition deteriorates oocyte maturation and embryo
development. (A) SHP2/ERα (GFP-labeled SHP2 and red fluorescent protein-labeled ERα) proteins analyzed via immunofluorescence in cumulus cells during germinal
vesicle breakdown (GVBD) and MII stages of control cumulus–oocyte complexes (COCs) and phenylhydrazonopyrazolone sulfonate 1 (PHPS1)-treated COCs. (B)
Representative images of p-ERK1/2 and p-AKT in GVBD- and MII-stage oocytes in the control and PHPS1-exposed groups. 4′ ,6′ -Diamidino-2-phenylindole was
used to stain the nucleus. (C) Immunofluorescence images of Caspase-3 and phosphorylated NF-Kb in control and PHPS1-treated bovine day 8 blastocysts.
Qualitative analysis showed that PHPS1 treatment enhances p-NF-kB nuclear localization and enhances Caspase-3 expression. All experiments were repeated thrice,
and data are shown as mean ± SEM. NS, not significant; *P < 0.05; **P < 0.01; ***P < 0.001.

Molecular Modeling Studies for Predicting examining the root mean square deviation (RMSD) of the protein
the Binding Mode Between ERα and SHP2 backbone atoms. It was observed that the RMSD values of model
To support our experimental findings, protein–protein docking 1 was significantly lower than those of model 2 and model 3
studies were performed to determine the probable binding mode (Figure 5A), implying that the structure of model 1 was more
between human ERα and SHP2 proteins. For this purpose, stable than the others during the simulation times. Moreover, the
protein–protein/DNA docking was performed using an online binding free energy calculations using the MM-GBSA algorithm
webserver, HDOCK. The 50 models generated were downloaded, was performed to further predict the binding affinity. It was
and the binding modes of all models were then assessed by cluster found that model 1 showed the lowest binding free energy score
analysis in Discovery Studio. The cluster analysis revealed that (−115.2 kcal/mol) among the three models (model 2, −72.47
three different binding modes could be possible for ERα and kcal/mol; model 3, −70.44 kcal/mol)] (Supplementary Table 2).
SHP2. The best model with the lowest docking energy score The MM-GBSA calculations also provide the contribution
from each cluster was selected and subjected to 50 ns of MD of each amino acid in the binding. In model 1, the key
simulation (Supplementary Table 1). The stability of the entire residues involved in complex formation were Trp484, Leu130,
complex structure during the simulation times was analyzed by Thr688, Thr131, and His308 of ERα and Asn336, Trp248,

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Idrees et al. SHP2 Role in Oocyte Maturation

FIGURE 4 | Follicle-stimulating hormone (FSH) reduces the amount of SHP2/ERα complex in human granulosa cells (COV434). (A) To obtain the effective
concentration of phenylhydrazonopyrazolone sulfonate 1 (PHPS1), an MTT assay was performed in COV434 cells, and 3 µM of PHPS1 was identified as the least
effective concentration. (B) Immunofluorescence experiment of FoxL2 (green) and SHP2 (red) identified that SHP2 shows nuclear localization in COV434 cells, and
PHPS1 had no effect on SHP2 nuclear localization. (C) FSH- and E2 -stimulated COV434 cells showed the differential localization of SHP2 and ERα after 24 h.
Estradiol (E2 ) significantly enhanced the nuclear localization of SHP2, while FSH significantly reduced its nuclear localization, compared to that in the control group.
(D) Co-immunoprecipitation identified that SHP2 and ERα form a complex in COV434 cells, and E2 or FSH treatments had a significant effect on this complex. (E)
FSH augmented extracellular signal-regulated kinase (ERK) and AKT signaling in the COV434 cell line compared to that in the control and E2 -treated groups, wherein
signaling was significantly reduced. The experiments were repeated thrice, and data are shown as mean ± SEM. NS, not significant; *P < 0.05; **P < 0.01;
***P < 0.001.

Gln506, Gln256, and Glu379 of SHP2 (Figure 5B). The per- was observed to form 24 hydrogen bonds, five electrostatic
residue contribution for ERα and SHP2 interaction was interactions, and five hydrophobic interactions (Figures 5C,D,
also calculated in model 2 and model 3 and is shown Supplementary Table 3). The residues in chain A and chain B
in Supplementary Figures 2, 3. of ERα interact with the catalytic domain of SHP2 that has been
Furthermore, the binding mode for the ERα and SHP2 reported to have a ligand-binding site ranging from 260 to 510
interaction was analyzed by calculating the average structure amino acids (Hellmuth et al., 2008). Model 1 displayed a higher
from the last 5 ns of the MD simulation trajectory for model number of molecular interactions in this region as compared to
1, model 2, and model 3 (Figure S3). A detailed analysis model 2 and model 3 (Supplementary Table 3). Finally, model 1
revealed that, in model 1, the ERα and SHP2 complex was selected as an ideal model for the ERα and SHP2 interaction

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Idrees et al. SHP2 Role in Oocyte Maturation

FIGURE 5 | Results of molecular modeling studies for predicting the binding mode between human ERα and SHP2. (A) The root mean square deviation values of the
backbone atoms of ERα and SHP2 complexes during the molecular dynamics simulation times. (B) Decomposition of the molecular mechanics-generalized Born
surface area energy for each amino acid of the binding surface from ERα and SHP2. The key interacting residues observed in ERα and SHP2 are highlighted. (C)
Predicted binding mode of ERα and SHP2 complex in surface representation. ERα chain A is shown in blue, chain B in pink, TIF2 in orange, and DNA in yellow. The
SHP2 protein domains are represented in purple (C-SHP2), green (N-SHP2), and cyan (PTP domain). (D) Enlarged view for the contact area between the two proteins.
Interacting amino acids are shown in stick representation. The pink (chain B) and blue (chain A) sticks are from ERα, whereas the cyan (PTP domain) and purple
(C-SHP2) sticks are from the SHP2 protein. The hydrogen bonds are shown as dashed green lines.

because of its stable RMSD throughout the simulation, the lowest via siRNA and verified it at the protein level (∗ P < 0.05;
binding free energy, and the highest number of interactions. Figure 6A). SHP2-knockdown cells were immunofluorescent-
Similar approaches for final model selection have also been used stained with ERα antibody, and the results showed a significant
in previous studies (Selent et al., 2013; Ge et al., 2018; Wu et al., reduction in ERα nuclear localization (Figure 6B). E2 and
2020). FSH have opposing regulatory effects on NPPC/NPR2 genes in
granulosa cells, and all of our above-mentioned results showed
SHP2 Knockdown Reduced ERα that SHP2 plays a role in the transcriptional activity of E2
and in the intracellular signaling of FSH. To directly analyze
Transcription of NPPC/NPR2 and SHP2 interactions with E2 and FSH, the SHP2-knockdown
Aromatase Activity of Human Granulosa granulosa cells were stimulated with E2 and FSH, and the
Cells mRNA expression of NPPC/NPR2 was quantified via RT-qPCR
To validate the direct link between the transcriptional activities (Figure 6C) (Liu et al., 2017). E2 application to granulosa cells
of SHP2 and ERα in granulosa cells, we knocked down SHP2 had a significant effect on ERα transcriptional activity, while

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Idrees et al. SHP2 Role in Oocyte Maturation

FIGURE 6 | Src-homology-2-containing phosphotyrosine phosphatase (SHP2) knockdown reduced estrogen receptor alpha (ERα) transcription of the NPPC and
NPR2 genes and aromatase activity of granulosa cells. (A) SHP2 siRNA was applied to COV434 cell line, and SHP2 protein expression was analyzed via western
blotting. (B) To check the localization of ERα, an immunofluorescence experiment was performed using SHP2-knockdown COV434 cells. The results suggest that
ERα nuclear localization was significantly reduced with SHP2 knockdown. (C) NPPC/NPR2 mRNA expressions were analyzed in follicle-stimulating hormone- and
E2 -stimulated SHP2-knockdown COV434 cell line. (D) To examine the effect of SHP2 knockdown on the functional activity of granulosa cells, the SF1, Wnt4, and
p-mTOR proteins were analyzed via western blotting, and a significant reduction in all proteins was observed with SHP2 knockdown. β-Actin was used as the loading
control for western blotting. Bands were quantified using ImageJ software, and the differences are represented by histograms. The experiments were repeated thrice,
and data are shown as mean ± SEM. NS, not significant; *P < 0.05; **P < 0.01; ***P < 0.001.

SHP2 siRNA abrogated the E2 stimulation of ERα and enhanced cells, taking into account the potential interference with FSH
NPPC/NPR2 mRNA expression (control vs. E2 vs. E2 + SHP2 signaling-mediated transcriptional factor activation. We found
siRNA; ∗ P < 0.05, ∗∗ P < 0.01). Furthermore, SHP2 knockdown that SHP2 knockdown markedly reduced the steroidogenic
interrupted FSH intracellular signaling-induced inhibition of factor-1 (SF1 encoded by the NR5A1 gene) and β-catenin
NPPC and NPR2 genes (control vs. FSH vs. FSH + SHP2 siRNA; pathway protein wingless-type MMTV integration site family
∗ P < 0.05, ∗∗ P < 0.01). Moreover, we examined the effect member 4 (Wnt4) proteins (Figure 6D) (control vs. siRNA vs.
of SHP2 knockdown on the functional activities of granulosa scramble siRNA; ∗ P < 0.05, ∗∗ P < 0.01). SF1 and Wnt4 are the

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Idrees et al. SHP2 Role in Oocyte Maturation

downstream transcriptional factors of FSH signaling that regulate Human immortalized granulosa cells (COV434) also express the
the aromatase activity of granulosa cells (Parakh et al., 2006; SHP2/ERα complex, which shows a different expression with
Pelusi et al., 2008; Boyer et al., 2010). FSH was also found to FSH or E2 supplementation. Furthermore, SHP2 knockdown
activate the mammalian target of rapamycin complex 1 (mTOR), reduced the ERα-transcribed NPPC and NPR2 genes in granulosa
which is necessary for the transcription of several proteins related cells, but PHPS1-mediated SHP2 inhibition had no significant
to follicular development (Alam et al., 2004). Collectively, these effect on the SHP2/ERα complex.
results suggest that SHP2 plays a dual role in the granulosa Intercommunication between the oocyte and its surrounding
and cumulus cells of oocytes. SHP2 interacts with ERα for its granulosa cells is critical for the production of a mature oocyte
transcriptional activity and transduces FSH signaling for meiotic capable of fertilization, as these cells provide 85% of the nutrients,
resumption and maturation. including growth factors, amino acids, and other energy sources,
to the oocyte (Albertini et al., 2001; Eppig, 2001; Sugiura
et al., 2005). Granulosa cells also play a vital role in oocyte
DISCUSSION meiotic arrest and resumption, two critical phenomena that
determine the entire reproductive potential of females (Zhang
In this study, we identified a novel relationship between SHP2 et al., 2010). During the meiotic arrest or pre-ovulatory stage
and ERα in bovine cumulus and human granulosa cells. of ovarian follicles, granulosa cells maintain oocyte meiotic
ERα, a transcriptional regulator of NPPC and NPR2 genes arrest by expressing NPPC and its receptor, NPR2. Several
in granulosa cells, interacts with SHP2 for its transcriptional studies have recognized that the NPPC/NPR2 system is also
activity. The SHP2/ERα complex is highly expressed in the essential for the morphological and genetic health of an oocyte
cumulus cells of immature oocytes (GV stage) but is significantly (Zhang et al., 2015; Celik et al., 2019). A previous study stated
reduced in the cumulus cells of mature oocytes (MII stage). that ERα is the upstream regulator of NPPC/NPR2 governing

FIGURE 7 | Schematic presentation of the Src-homology-2-containing phosphotyrosine phosphatase regulation of oocyte meiotic maturation. Cumulus cells are
shown in light blue, and granulosa cells are shown in reddish green.

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Idrees et al. SHP2 Role in Oocyte Maturation

oocyte meiotic arrest in granulosa cells (Liu et al., 2017). ERα state. During the inactive or auto-inhibition state, the N-terminal
is a ligand-activated transcription factor, and we found that SH2 domain blocks the PTP domain, while in the active state,
it requires nucleus-localized SHP2 for the regulation of NPPC the SH2 domain binds to specific phosphotyrosine sites on the
and NPR2 genes in cumulus and granulosa cells. A previous adaptor proteins of receptors (Hof et al., 1998; Neel et al., 2003).
study detected the ERα and SHP2 complex in breast tissues Active cytoplasmic SHP2 interacts with the transmembrane
and confirmed its cytoplasmic localization and involvement in adaptor proteins of RTKs and GPCRs and activates MAP kinases,
triggering MAP kinases and AKT signaling (Li et al., 2014). cell cycle controller p34 (CDC2), and PI3K/AKT signaling, which
In our study, the co-immunoprecipitation analysis revealed are involved in oocyte GVBD and meiotic progression (Wehrend
that ERα forms a physiological complex with SHP2 in the and Meinecke, 2001; Lin et al., 2009). SHP2 plays a critical role
cumulus cells of GV-stage oocytes, and this complex becomes in FSH receptor-induced ERK1/2 and PI3K/AKT signaling in
significantly reduced with the progression of oocyte meiotic granulosa cells (Donaubauer et al., 2016). In our study, we found
maturation in vitro. Furthermore, to determine the localization significantly enhanced SHP2 cytoplasmic localization during in
of ERα/SHP2 complex, we found that the cumulus cells of vitro oocyte maturation or with the cells (cumulus or granulosa)
immature (GV) oocytes showed nuclear localization of the exposed to FSH. It may be possible that, during LH surge, SHP2
ERα/SHP2 complex, but those of mature (MII) oocytes showed move toward the cytoplasm and transduce FSH signaling in
cytoplasmic localization of both proteins. The nucleus-localized granulosa cells (Figure 7), but this mechanism of SHP2 needs
SHP2’s role in ERα transcriptional activity has been previously further verification in in vivo animal models. Furthermore,
identified in uterine tissues, but it dephosphorylates ERα and SHP2 knockdown significantly affected FSHR signaling, targeting
recruits it to the target gene without making a complex (Ran downstream transcriptional factors such as SF1, Wnt4, and p-
et al., 2017). We found a physiological complex of SHP2/ERα in mTOR, which regulate the aromatase activity of granulosa cells
granulosa cells, and the knockdown of SHP2 highly affected ERα- and play a key role in follicular development (Alam et al., 2004;
targeted NPPC and NPR2 genes. Additionally, cultured granulosa Parakh et al., 2006; Pelusi et al., 2008; Boyer et al., 2010).
cells (COV434) express the SHP2/ERα complex, and the FSH
and E2 stimulation of granulosa cells has significant effects on
this complex. CONCLUSION
To analyze the probable structure of the SHP2/ERα complex,
Taken together, our findings clearly demonstrate that nuclear
we applied a series of computer modeling methods, including
SHP2 is involved in ERα transcriptional activity for the
protein–protein molecular docking, molecular dynamic
promotion of NPPC and NPR2 genes related to oocyte meiotic
simulation, and MM-GBSA calculations, to identify the binding
arrest. Furthermore, FSH or growth factors restrict SHP2
modes of ERα and SHP2. Similar approaches for protein–protein
interaction with ERα by promoting its export from the nucleus to
interaction or complex generation have been used in previous
transduce RTKs or GPCR-dependent signaling in oocyte somatic
studies (Selent et al., 2013; Ge et al., 2018; Wu et al., 2020).
cells during meiotic resumption. These findings could contribute
Initially, 50 models of the SHP2/ERα complex were generated
to opening new avenues of research to understand the process of
via the online webserver HDOCK. Subsequently, the cluster
oocyte meiosis in the mammalian ovary.
analysis in Discovery Studio revealed that three different binding
modes could be possible for ERα and SHP2. We further applied
MD simulations to select the best model for the interaction DATA AVAILABILITY STATEMENT
between SHP2 and ERα. Based on the RMSD values, binding
free energies, and key residue interaction numbers, model 1 The original contributions generated for the study are included
was selected as the most probable candidate for the SHP2/ERα in the article/supplementary material, further inquiries can be
complex with DNA. In our wet lab data, we found that PHPS1, directed to the corresponding author/s.
a site-directed inhibitor of SHP2, has no substantial effects
on SHP2 nuclear localization and its involvement with ERα
transcriptional activity (Hellmuth et al., 2008). After observing ETHICS STATEMENT
the key residues that are involved in ERα (Leu130, Thr131,
The animal study was reviewed and approved by All
His308, Trp484, and Thr688) and SHP2 (Trp248, Gln256,
experiments, including surgical procedures, were approved
Asn336, Glu379, and Gln506) complex formation, we found that
by the Gyeongsang National University Institute of Animal Care
PHPS1 binds to SHP2 in a similar range (Hellmuth et al., 2008).
Committee (GNU-130902-A0059).
Therefore, PHPS1 is unable to bind with SHP2 and inhibit its
nuclear localization in cumulus and granulosa cells.
SHP2 not only interacts with ERα to regulate meiotic arrest, AUTHOR CONTRIBUTIONS
but it also transduces FSH receptor signaling in granulosa cells.
SHP2 is a classic cytoplasmic protein, is a core component of MI designed the research and analyzed the data. MI and VK
growth factor and cytokine signal transduction, and is essential performed the research, M-DJ provided reagents and helped in
for oocyte maturation and embryo development (Saxton et al., the experiments. MI, VK, and NA wrote the paper. K-WL and
1997; Idrees et al., 2019b; Kim et al., 2019). SHP2 is mostly found I-KK reviewed the paper and supervised the study. All authors
in two states, an inactive or auto-inhibition state and an active contributed to the article and approved the submitted version.

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Idrees et al. SHP2 Role in Oocyte Maturation

FUNDING ACKNOWLEDGMENTS
This work was partly supported by the National Research We thank Prof. Jeehyeon Bae (Chung-Ang University, Seoul,
Foundation of Korea (NRF), a grant funded by the Korean Republic of Korea) for giving us the COV434 cell line.
government (MSIT; Grant No. 2020R1A2C2006614), Korea
Institute of Planning, a scholarship from the BK21 Four SUPPLEMENTARY MATERIAL
Program, the Bio & Medical Technology Development
Program of the National Research Foundation (NRF), and The Supplementary Material for this article can be found
fund granted by the Korean government (MSIT) (Grant online at: https://www.frontiersin.org/articles/10.3389/fcell.2020.
No. NRF-2018M3A9A70-57263). 611503/full#supplementary-material

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cell line (COV434). Mol. Hum. Reprod. 6, 146–153. doi: 10.1093/molehr/6. Conflict of Interest: I-KK was employed by company The King Kong Corp. Ltd.
2.146
Zhang, J., Wei, Q., Cai, J., Zhao, X., and Ma, B. (2015). Effect of C-type natriuretic The remaining authors declare that the research was conducted in the absence of
peptide on maturation and developmental competence of goat oocytes any commercial or financial relationships that could be construed as a potential
matured in vitro. PLoS ONE 10:e0132318. doi: 10.1371/journal.pone.013 conflict of interest.
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Zhang, M., Su, Y. Q., Sugiura, K., Xia, G., and Eppig, J. J. (2010). Granulosa cell Copyright © 2021 Idrees, Kumar, Joo, Ali, Lee and Kong. This is an open-access
ligand NPPC and its receptor NPR2 maintain meiotic arrest in mouse oocytes. article distributed under the terms of the Creative Commons Attribution License (CC
Science 330, 366–369. doi: 10.1126/science.1193573 BY). The use, distribution or reproduction in other forums is permitted, provided
Zhang, S. Q., Yang, W., Kontaridis, M. I., Bivona, T. G., Wen, G., the original author(s) and the copyright owner(s) are credited and that the original
Araki, T., et al. (2004). Shp2 regulates SRC family kinase activity and publication in this journal is cited, in accordance with accepted academic practice.
Ras/Erk activation by controlling Csk recruitment. Mol. Cell 13, 341–355. No use, distribution or reproduction is permitted which does not comply with these
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