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ORIGINAL ARTICLEEmbryology
Insulin-like growth factor 1 increases
apical fibronectin in blastocysts
to increase blastocyst attachment
to endometrial epithelial cells in vitro
Charmaine J. Green1, Stuart T. Fraser1,2, and Margot L. Day1,*1Disciplineof Physiology, BoschInstitute, SydneyMedicalSchool, Universityof Sydney, K25 Medical FoundationBuilding, Sydney2006, Australia2Discipline of Anatomy and Histology, SydneyMedical School, University of Sydney, K25 Medical Foundation Building, Sydney 2006, Australia
*Correspondence address. Tel: +61-2-9036-3312; Fax: +61-2-9036-3316; E-mail: [email protected]
Submitted on June 23, 2014; resubmitted on October 14, 2014; accepted on October 28, 2014
study question: Does insulin-like growth factor 1 (IGF1) increase adhesion competency of blastocysts to increase attachment to uterineepithelial cellsin vitro?
summaryanswer: IGF1increases apical fibronectin on blastocysts to increaseattachmentand invasion in an in vitro modelof implantation.
what is known already:Fibronectin integrin interactions are important in attachment of blastocysts to uterine epithelial cells atimplantation.
study design, size, duration: Mouse blastocysts (hatched or near completion of hatching) were cultured in serum starved (SS)medium with varying treatments for 24, 48 or 72 h. Treatments included 10 ng/ml IGF1 in the presence or absence of the PI3 kinase inhibitor
LY294002, an IGF1 receptor (IGF1R) neutralizing antibody or fibronectin. Effects of treatments on blastocysts were measured by attachment
of blastocysts to Ishikawa cells, blastocyst outgrowth and fibronectin and focal adhesion kinase (FAK) localization and expression. Blastocysts
were randomly allocated into control and treatment groups and experiments were repeated a minimum of three times with varying numbers
of blastocystsused in each experiment. FAK and integrin protein expression on Ishikawacells wasquantified in the presenceor absence of IGF1.
participants/materials, setting, methods: Fibronectin expression and localization in blastocysts was studied using im-munofluorescence and confocal microscopy. Global surface expression of integrin avb3, b3 and b1 was measured in Ishikawa cells using flow
cytometry. Expression levels of phosphorylated FAK and total FAK were measured in Ishikawa cells and blastocysts by western blot and
image J analysis. Blastocyst outgrowth was quantified using image J analysis.
main results and the role of chance:The presence of IGF1 significantly increased mouse blastocyst attachment to Ishikawacells compared with SS conditions (P, 0.01). IGF1treatment resulted in distinct apical fibronectin staining on blastocysts, which was reduced by
the PI3 kinase inhibitor LY294002. This coincided with a significant increase in blastocyst outgrowth in the presence of IGF1 (P, 0.01) or fibro-
nectin(P, 0.001), which was abolished by LY294002 (P, 0.001). Apical expression of integrinavb3,b3andb1 in Ishikawacellswas unaltered
by IGF1. However, IGF1 increased phosphorylated FAK (P, 0.05) and total FAKexpression in Ishikawa cells. FAK signalling is linked to integrin
activationand can affect the integrins ability to bind and recognizeextracellular matrix proteins suchas fibronectin. Treatment of blastocysts with
IGF1 before co-culture with Ishikawa cells increased their attachment ( P, 0.05). This effect was abolished in the presence of LY294002 (P,
0.001) or an IGF1R neutralizing antibody (P,
0.05).limitations, reasons for caution: This studyuses an invitro modelof attachment thatuses mouseblastocystsand human endo-metrial cells. This involves a species crossover and although this use has been well documented as a model for attachment (as human embryo
numbers are limited) the results should be interpreted carefully.
wider implications of the findings: This study presents mechanisms by which IGF1improves attachment of blastocysts to Ishi-kawa cells and documents forthe first time howIGF1 canincrease adhesion competency in blastocysts. Failure of the blastocyst to implant is the
major cause of human assisted reproductive technology (ART) failure. As growth factors are absent during embryo culture, their addition to
embryo culture medium is a potential avenue to improve IVF success. In particular, IGF1 could prove to be a potential treatment for blastocysts
before transfer to the uterus in an ART setting.
& The Author 2014. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved.
For Permissions, please email: [email protected]
Human Reproduction, Vol.0, No.0 pp. 1 15, 2014
doi:10.1093/humrep/deu309
Hum. Reprod. Advance Access published November 28, 2014
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study funding/competing interest(s): This work was supported by internal funds from the University of Sydney and theBosch Institute. None of the authors has any conflict of interest to declare.
trial registration number: N/A.
Key words: PI3 kinase / fibronectin / implantation / insulin-like growth factor 1 / integrin
Introduction
Unsuccessful embryo implantation is the major cause of human assisted
reproductive technology (ART) failure (Milleret al., 2012). Implantation
is a highly coordinated event in which the receptive endometrium is
primed to receiveadhesion-competentblastocysts.Growthfactorspro-
duced by boththe embryo and the female reproductivetract are thought
to support development of the blastocyst to an adhesion-competent
state, which is synchronizedwith uterine receptivity,to ensure blastocyst
implantation ability (Wang et al., 2000;Armant, 2005). Therefore, an
understanding of how growth factors influence implantation is essential
to improve ART.
Insulin-likegrowthfactor1(IGF1)isoneparticulargrowthfactorthatispresent at the maternalembryo interface in a number of mammalian
species (Murphyet al., 1987;Kapuret al., 1992;Lighten et al., 1998;
Slater and Murphy, 1999). At the time of implantation in the rat IGF1 is
strongly expressed in the basal lamina and the apical surface of uterine
epithelial cells, which are the sites of trophoblast invasion and attach-
ment, respectively (Slater and Murphy, 1999). Additionally IGF1 is
secreted by mouse embryos (Inzunzaet al., 2010). The IGF1 receptor
(IGF1R)is expressedthroughoutmouse preimplantationstagesof devel-
opment (Inzunza et al., 2010) and is expressed apically on trophoblast
cells (Bedzhovet al., 2012), which are the cells that make first contact
with the uterine epithelium. High levels of IGF1, which cause IGF1R
down-regulation, decrease normal blastocyst implantation sites and in-
crease resorption sites (Pintoet al., 2002). Additionally, beads soakedinIGF1elicita decidualresponsein theuterusthat mimicsthatofa blasto-
cyst (Pariaet al., 2001). Despite this knowledge of IGF1/IGF1R expres-
sion in the embryo and uterine tissue, it is not known whether IGF1
affects blastocyst adhesion competency or the mechanisms by which
IGF1 influences early implantation.
Acquisition of adhesion competence by the blastocyst involves an
accumulation of integrins on the apical surface of trophoblast cells. Im-
plantation depends, in part, on the bridging between integrins on the
endometrium and the blastocyst by extracellular matrix (ECM) proteins
suchas fibronectin (Kaneko etal.,2013). Integrina5b1 subunitsincrease
on theapical surface of adhesion competentmouseblastocysts, andthis
correlates withan increasedability to bindfibronectin (Schultz andArmant,
1995;Schultzet al., 1997;Wang et al., 2002). Most ECM proteins bind to
multiple integrins, for example fibronectin, which binds the a5b1,avb3
and aIIb3 integrins in mouse blastocysts (Sutherlandet al., 1993;Schultz
and Armant 1995;Yelianet al., 1995;Schultzet al., 1997).
The process of integrin activation, which occurs via inside-out signal-
ling, regulates integrin affinity for ECM proteins. This inside-out signalling
is mediated by a number of signalling pathways, such as the PI3 kinase
(PI3K)/Akt pathway (Somanathet al., 2007). Akt is activated by IGF1
in mouse blastocysts (Green and Day, 2013) and treatment of tropho-
blast cells with IGF1 increases fibronectin binding mediated by a5b1
and avb3(Kabir-Salmaniet al., 2002,2003,2004).
The current study investigated the role of IGF1 in attachment, using a
well-characterizedin vitromodel of attachment that involves culture of
blastocysts on Ishikawacells.Ishikawacellsare a welldifferentiated endo-
metrial adenocarcinoma cell line (Nishida et al., 1985;Hannan et al.,
2010) that displays apical adhesiveness (Heneweeret al., 2005) and a
similar integrin expression profile to a receptive endometrium, under
the control of estrogen and progesterone (Lesseyet al., 1996;Castel-
baumet al., 1997). Embryos of different species including mouse, rat
and human (Singhet al., 2010;Kanekoet al., 2011a,2012,2013;Kang
et al., 2014) are known to attach to Ishikawa cells. These characteristics
make Ishikawa cells an invaluable model for the study of implantationin
vitro, especially as the use of human embryos is limited by availability
(Kang et al., 2014). In the present study we demonstrate that IGF1increased apical fibronectin expression on blastocysts and attachment
of blastocysts to Ishikawa cells, as well as blastocyst invasiveness.
These actions of IGF1 were all prevented by inhibition of the PI3K/Akt
pathway. Thesedata suggest an importantrole for IGF1in the acquisition
of blastocyst adhesion competence due to regulation of fibronectin ex-
pression. Furthermore, our results indicate that addition of IGF1 to the
culture system may improve the success of human assisted reproduction
by improving the adhesion competence of blastocysts.
Materials and Methods
Use of animals and ethical approval, ovulationinduction and embryo collection and culture
Procedures involving the use of animals were conducted in accordance with
the Australian Code of Practice for Use of Animals in Research and were
approved by the University of Sydney Animal Care and Ethics Committee.
The Quackenbush Swiss (QS) strain of mice was used (Animal Resource
Centre, Perth, Australia). Mice were housed under a 12-h light: 12-h dark
cycle (light; 06:0018:00), and housed in a conventional holding facility
where the temperature was maintained at 20228C and the humidity
kept at 55%. Mice had free access to water and commercially prepared
pellets. Female mice were caged in groups of 10 whilst male mice were
caged singly. Ovulation induction of female mice (410 weeks old) was
achieved by intraperitoneal injection of 10 I.U. of pregnant mare serum
gonadotrophin (PMSG; Intervet, Sydney, Australia) followed 48 h later by
intraperitoneal injection of 10 I.U. of hCG (Intervet) as previously described
(Nasr-Esfahani et al., 1990). Superovulated female mice were then paired
with a stud male QS mouse (1030 weeks old) overnight. The presence of
a vaginal plug the following day indicated successful mating and this was con-
sidered to be Day 1 of pregnancy.
Female mice were sacrificed on Day 4 of pregnancy, 90 94 h after hCG
administration, in order to recover early blastocysts. The oviducts and
uterine horns were flushed with Hepes-buffered modified synthetic human
tubal fluid medium (Hepes mod-HTF; 300 mosM/l, pH 7.4) containing
0.3 mg/ml bovine serum albumin (BSA; Sigma-Aldrich; St Louis, MO,
USA). Blastocysts were then collected and cultured for 2428 h, to enable
hatching from the zona pellucida (Day 5 Blastocysts), in Potassium simplex
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optimized medium (KSOM; 260 mosM/l, pH 7.4) containing 0.3 mg/ml
BSA. All media was made up from stock solutions prepared from tissue
culture grade reagents (Sigma-Aldrich) as previously described (Green and
Day, 2013). All blastocysts used in this study were collected on Day 4 and
allowed to hatch overnight before treatment (all blastocysts used were
hatched or near completion of hatching).
Recombinant mouse IGF1 (R&D Systems; Minneapolis, MN USA) was
reconstituted at 100mg/ml in sterile phosphate-buffered saline (PBS;
AMRESCO; Solon, OH, USA). Mature mouse IGF1 shares 94% amino acidsequence homology with human IGF1 and exhibits species cross reactivity
(Bell et al., 1986). Specifically, R&D Systems test their mouse IGF1 activity
on human MCF-7 breast cancer cell proliferation. Thus mouse IGF1 was
used in allof theexperiments.IGF1at 10 ng/ml hasbeenusedto seepositive
effects on embryo development (Green and Day, 2013). Higher concentra-
tions, such as 30, 100 and 1000 ng/ml IGF1, are known to have detrimental
effects on blastocysts and cause IGF1R down-regulation (Chi et al., 2000;
Green and Day, 2013), and this range of IGF1 concentrations has been
used in this study to investigate the mechanisms of IGF1 in attachment of
the embryo to uterine epithelial cells.
To determinea directeffect of IGF1 through theIGF1R, an IGF1R neutral-
izing antibody (IGF1RnAb; AF-305-NA; R&DSystems)was reconstituted at
0.2 mg/ml in PBS and used at a final concentration of 2 mg/ml.
LY294002 is a specific inhibitor of PI3K with an IC50 of 1.40 mM (Vlahoset al., 1994). Previously LY294002 has been used at concentrations of
500mM to induce apoptosis in blastocysts and although 250 mM did not in-
crease apoptosis in blastocysts (Riley et al., 2006), this concentration was
shown to decrease blastocyst hatching (Riley et al., 2005). In the present
study we chose to use a 5 mM LY294002, a concentration much closer to
the IC50 for the drug.
Ishikawa cell culture
The Ishikawa cell line was a gift from Professor Chris Murphy, University of
Sydney. Cells were thawed and plated on 10 mm glass coverslips (Menzel
Glaser; Braunschweig, Germany) in 24-well plates (Corning, NY, USA) for
co-culture experiments or directly on to 6 well pates (Corning, NY, USA)
for western blot analysis with Dulbeccos modified Eagles medium
(DMEM; GIBCO, Grand Island, NY, USA) containing 10% (v/v) fetal
bovine serum (FBS; Bovogen Biologicals Pty Ltd, Essendon, VIC, Australia)
and 5000 I.U. of penicillin and 5000 mg of streptomycin per ml (Invitrogen,
Camarillo, CA, USA).
Co-culture of mouse blastocysts with Ishikawa
cells in the presence and absence of IGF1
Attachmentexperiment 1 (Fig. 1) wasperformedwithco-culture ofIshikawa
cells and blastocysts in the presence and absence of IGF1. Ishikawa cells that
werenear confluencewere culturedin eitherserum medium(containing 10%FBS) or in serum starved (SS) medium (0.01% FBS) for 2 h prior to receiving
Figure 1 Timeline of experiments used to study attachment of mouse blastocysts to human Ishikawa cellsin vitro. BL: blastocyst.
IGF1 Increases mouse blastocyst attachmentin vitro 3
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blastocysts.After2 h, SS Ishikawa cells were then cultured in thepresenceor
absence of 10 ng/ml IGF1. Blastocysts were isolated from six mice in each
experiment. Between 4 and 12 Day 5 blastocysts were transferred per well
onto theIshikawa cellsin each ofthe treatmentgroups.Blastocystswereran-
domly allocated to treatment groupsand treatment groupswere performed
in duplicate. Initially, co-cultures were incubated undisturbed for 24 h and
checked for attachment. However as no blastocysts had adhered at 24 h
(up to Day 6), co-cultures were incubated undisturbed for 48 h (up to Day 7).
Blastocyst attachment to Ishikawa cells was determined at 48 h by blowingmedia with a glass mouth pipette at the blastocysts whilst examining their
position under a dissecting microscope (Wild M3 microscope; Leica Micro-
systems, Wetlar, Germany). Embryos that did not float away were consid-
ered to have attached (Singh et al., 2010). Co-culture experiments were
performed in duplicate and repeated three separate times. Data were
pooled from thethree separate experiments and counted as theproportion
of blastocyststhat had attached to the Ishikawacells outof the total number
ofblastocystsadded tothe Ishikawacells. Between 14and 19 mice were used
for each experimental group.
Pretreatment of blastocysts before co-culture
with Ishikawa cells
Attachmentexperiment 2 (Fig. 1) was performed by pre-treating blastocystsbefore their co-culture with Ishikawa cells. Blastocysts were isolated from
between 5 and10 mice in each experiment. Day 5 blastocystswere cultured
in the presence of serum, with or without 2 mg/ml IGF1R nAb or in SS
medium in the presence or absence of 10, 100 or 1000 ng/ml IGF1 or
with 10 ng/ml IGF1 in the presence of 2mg/ml IGF1R nAb or 5mM
LY294002 (PI3 kinaseinhibitor; Selleck, Houston,TX, USA) for24 h. Blasto-
cysts (now day 6 blastocysts) were then washed and cultured on Ishikawa
cells for 24 h in SS medium and assessed for attachment as above. Experi-
ments were repeated three to nine times and the data pooled to calculate
the proportion of attached embryos for each treatment.
Immunofluorescence
Co-cultures, from attachment experiment 1 were examined for total focal
adhesion kinase (FAK) protein expression. Blastocysts were isolated from
between three andfive mice in each experiment. Day5 blastocystswerecul-
tured in the absence of Ishikawa cells at lowembryodensity in SS medium in
the presence or absence of 10 ng/ml IGF1 or 5 mM LY294002 and were
examined for fibronectin expression at 24, 48 or 72 h. Co-cultures or separ-
ately cultured blastocysts were fixed in 4% paraformaldehyde (PFA) for
15 min, washed in PBS + 1 mg/ml Polyvinyl alcohol (PBS + PVA) and then
permeabilised with PBS + PVA+ 0.3% Triton X 100 for 30 min and then
washed with PBS + PVA. Blocking was carried out in PBS + PVA+ 0.1%
Tween-20 + 0.7% BSA (PPTB) for 30 min. Primary and secondary anti-
bodies were diluted in PPTB. Samples were incubated with primary anti-
bodies [rabbit anti-integrin b3; Santa Cruz Biotechnology; #sc-14009,
rabbit anti-FAK; Sigma; #F-2918 or rabbit anti-fibronectin (unattached blas-
tocysts); Novus Biologicals, Littleton, CO, USA; #NBP1-91258, or rabbit
anti-fibronectin antibody (for blastocysts attached to coverslips); Calbio-
chem, EMD Chemicals, Gibbstown, NJ, USA] overnight at 48C and then
washed in PPTB before incubation with the secondary antibody (anti-rabbit
Alexa 488; LifeTechnologies, Carlsbad, CA,USA) for 2 h at room tempera-
ture. Samples were then washed three times in PPTB, with the final wash
lasting 10 min and then mounted in 5 ml Vectashield containing 1.5 mg/ml
4,6-diamidino-2-phenylindole (DAPI; Vector Laboratories, Burlingame,
CA, USA). Isotype controls were performed, in which cells were incubated
with purified rabbit IgG (Sigma) in place of the primary antibody. Fluores-
cence was visualized using the LSM 510 Meta confocal microscope (Carl
Zeiss, Germany) using a 405 nm laser and Argon laser (458, 477, 488 and
514 nm lines) at40 objective. A Z-stack was taken through the co-cultures
or the blastocysts, at 2.5mm intervals. This provides us with sections
throughout the whole blastocyst so that we can visualize the inner cell
mass (ICM) in relation to the rest of the embryo. Images were analysed
using LSM Image Browser software (Carl Zeiss) and the localization of pro-
teins is described in relation to the ICM. An unbiased third party performed
a blinded analysis of the immunofluorescence images.
Western blot analysis
All western blot data on Ishikawa cells are from Ishikawa cells cultured in the
absence ofblastocysts.Near confluentIshikawacells wereallocatedto serum
mediumor SS medium inthe presenceor absence of10 ng/ml IGF1 for48 h.
Similarly all western blots on blastocysts were performed in the absence of
Ishikawa cells. Blastocysts were isolated from at least eight mice in each ex-
periment. Day 5 blastocysts were allocated randomly to serum medium or
SS medium in the presence or absence of 10 ng/ml IGF1 and cultured for
48 h on non-adherent culture dishes (Corning). After 48 h, cells or blasto-
cysts were washed in cold PBS and lysed with lysis buffer (Cell Signalling,
Beverly, MA, USA) + 1 mM PMSF (Sigma). Collections were repeated
until there were 120 embryos to run per lane, per experiment. Protein
assays were carried out on Ishikawa cells according to the manufacturers
instructions (DC protein assay kit, Bio-Rad). Protein samples were diluted
with 6Laemmli buffer (35 mM Tris HCl, pH 6.8, 10.28% (w/v) sodium
dodecyl sulphate (SDS), 36% (v/v) glycerol, 0.05% (w/v) bromophenol
blue; Sigma) and heated for 5 min at 1008C. Proteins were electrophoresed
on an 8% SDS polyacrylamide gel, transferred to a nitrocellulose transfer
membrane (Hy-BLOT, Australia) and then incubated in blocking buffer
(Odyssey; Li-cor Biosciences, Lincoln, NE, USA) overnight at 48C with
gentle shaking. Membranes were probed with primary antibodies (rabbit
anti-E-cadherin; Novus Biologicals; #NB110-56937, rabbit anti-pFAK; Invi-
trogen; #44-624G, or rabbit anti-integrin b3; Santa Cruz Biotechnology;
#sc-14009 or mouse anti-a-tubulin; Sigma; #T9026) overnight at 48C in
blocking buffer+ 0.1% Tween-20. Membranes were washed in Tris-
buffered saline+ Tween 20 (TBST; 10 mM TrisHCl, pH 7.6, 150 mM
NaCl and 0.1% Tween 20) and subsequently incubated for 2 h with either
1:4000 Donkey anti-Rabbit IRDye 800CW (Odyssey) or 1:4000 Donkey
anti-Mouse IRDye 680LT (Odyssey). Proteins were visualized using the
Odyssey infrared imager and Odyssey application software, version 3
(Odyssey). Densitometry was performed using Image-J software (National
Institutes of Health, Bethesda, MD, USA) or Odyssey program.
Flow cytometry
Surface expression of integrin avb3, b3 andb1 on Ishikawa cells,cultured in
theabsence of blastocysts,was analysedby flowcytometry after 48 h culture
inserummedium orSS mediumin thepresenceor absence of10 ng/mlIGF1.
Cells were removed from the culture plate with 0.5% trypsin (Life Technol-
ogies). Cells were resuspended in 600 ml of PBS + 0.3% BSA and incubated
with primary antibodies pre-conjugated (1:200 anti-human integrin avb3;
eBioscience; San Diego, CA, USA; #11-0519-41, anti-human integrinb3;
eBioscience; #11-0619-41 or anti-human integrin b1; eBioscience;
#11-0299-41) or isotype control antibodies (Armenian hamster IgG/RatIgG 2a/2b; Biolegend; San Diego, CA, USA; #78023) for 30 min on ice.
Samples were then washed and resuspended in 200 ml PBS + 0.1% (v/v)
propidium iodide (Sigma). Analyses were performed using the FACS
Calibur (Becton Dickinson, San Jose, CA, USA) and Cell Quest (Becton
Dickinson) and Flow Jo (Treestar, San Carlos, CA, USA) software packages.
A minimum of 10 000 cells were analysed per sample.
Blastocyst outgrowth
Blastocysts were isolated from at least five mice in each experiment. Day 5
blastocysts were cultured on 24-well plates (Corning) that were either un-
coated, or coated with fibronectin. To coat wells, fibronectin was made to
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a concentration of 25mg/ml in PBS, andenough solution wasaddedto cover
eachwellandleftat48C overnight. Blastocysts wereallowed to outgrow,un-
disturbed for 72 h in serum medium or in SS medium in the presence or
absence of 10 ng/ml IGF1 with or without 5 mM LY294002 (Selleck). At
72 h blastocyst outgrowths were micrographed on the Axiovert 35 micro-
scope (Carl Zeiss) using a Axiocam ICc5 camera (Carl Zeiss) and Zen
image software (Carl Zeiss). For those blastocysts that were attached to
the well, the area of outgrowth (arbitrary units), along with blastocyst area
was measured using Image-J software (National Institute of Health) bytracing around the outgrown cells using the freehand area selection tool
andthenthe wand tool toobtainan areavalue in arbitraryunits. Experiments
were repeated three to eight times and the data pooled to calculate the
average area of outgrowth.
Statistical analysis
Embryos collected from multiple mice were randomly allocated between
treatments for all experiments. Chi-squared analysis of the overall propor-
tion of blastocysts attached to Ishikawa cell co-cultures was performed on
pooled proportion data from three separate experiments (Microsoft Office
Excel,Berkshire, UK).Westernblot analysis was performed three times.The
optical density of protein bands was normalized to a-tubulin and expressed
relative to the control, followed by statistical analysis using unpaired t-tests(Microsoft Office Excel). Flow cytometry analysis was performed three
times, followed by statistical analysis of the average fluorescence intensity,
using unpairedt-tests (Microsoft Office Excel). Embryo outgrowth experi-
ments were performed at least three times, followed by statistical analysis
using KruskalWallis test followed by Dunn multiple comparison test for
non-parametric data (GraphPad Prismversion 6.00 for Windows, GraphPad
Software, La Jolla, CA, USA, www.graphpad.com). A P, 0.05 was
considered statistically significant.
Results
Effect of IGF1 on attachment of mouseblastocysts to Ishikawa cells in vitro
Blastocyst attachmentwas assessedin thepresenceor absenceof 10 ng/ml
IGF1 (attachment experiment 1) after 24 and 48 h, before fixation with
PFA (Singhet al., 2010). At 24 h no blastocysts had attached to the Ishi-
kawa cells in any of the treatment groups (data not shown). Therefore
attachment was assessed at 48 h. The proportion of blastocysts that
attached to Ishikawa cells under SS conditions (27 attached/50) was
reduced compared with attachment in the presence of serum (39
attached/47; Fig. 2). The presence of IGF1 increased the proportion
of blastocyst attachment (IGF1: 39 attached/46) compared with attach-
ment in SS conditions (Fig. 2), tothe level seen inthe presenceof serum.
Effect of IGF1 on E-cadherin and integrin b3
protein levels and integrin avb3, b3 and b1
surface expression in Ishikawa cells
As IGF1 increased attachment of blastocysts to Ishikawa cells, the effect
of IGF1 on several adhesion proteins was investigated in Ishikawa cells
by western blot. There was no difference in E-cadherin expression in
Ishikawa cells between the serum, SS or SS plus IGF1 treatment groups
(Fig.3A). Furthermore, IGF1 had no effect on integrin b3 expression
in Ishikawa cells; however, expression of integrin b3 was increased in
the presence of serum (Fig.3B).
Although IGF1 had no effect on total expression of integrinb3 in Ishi-
kawa cells, it is the integrin expression on the apical surface that is
required for interaction with ECM proteins. Therefore, surface expres-
sion ofavb3, b3 and b1 integrins was investigated in Ishikawa cells
using flow cytometry. Integrins avb3, b3 and b1 were all expressed
on the cell surface in Ishikawa cells, as indicated by the greater fluores-
cence seen with the presence of each antibody compared with the
isotype control (Fig.3C). However, there was no difference in surface
expression of integrins avb3,b3 orb1 between any of the treatment
groups (Fig.3C).
Effect of IGF1 on phosphorylation of FAK and
total FAK expression levels in Ishikawa cells
and mouse blastocysts
FAK signalling is linked to integrin activation (Kornberget al., 1992).
Therefore, activationof FAK signallingpathways, indicatedby phosphor-
ylation of FAK (p-FAK) was investigated by western blot analysis in
Ishikawa cells and blastocysts. Phosphorylation of FAK was increased
in Ishikawa cells after 48 h treatment with 10 ng/ml IGF1 (Fig. 4A).Immunofluorescencewas usedto showthat totalFAK expression in Ishi-
kawa cells was increased on the apical surface in co-cultures after treat-
ment with IGF1 for48 h. Similarly, total FAKexpressionwas increasedin
blastocysts that were attached to Ishikawa cells in co-cultures in the
presence of IGF1 in comparison to co-cultures in the absence of IGF1
(Fig. 4B). Therabbit IgG isotype control had no detectableimmunofluor-
escence staining in either the blastocyst or Ishikawa cells (data not
shown). Western blots were performed on unattached blastocysts
and showed no effect on phosphorylation of FAK or total FAK levels in
blastocysts after IGF1 treatment for 48 h (Fig. 4C and D).
Figure 2 Insulin-like growth factor 1 (IGF1) improves attachment of
mouseblastocysts to Ishikawa cells in vitro. Attachmentof blastocysts to
Ishikawa cells at 48 h after culture in serum medium or serum starved
(SS) medium in the presence or absence of 10 ng/ml IGF1. Results
are displayed as the percentage attachment of blastocysts to the Ishi-
kawa cells.Nvalues in parentheses represent the total number of blas-
tocysts added to the Ishikawa cells, pooled from at least threeexperiments. Chi-square analysis was used to compare the treatment
groups. ** indicatesP, 0.01.
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Figure 3 IGF1 has no effect on E-cadherin or integrin b3 protein levels or integrin avb3, b3 and b1 surface expression in Ishikawa cells. Ishikawa cells
wereculturedfor 48 h inserum medium orSS mediumin the presenceor absence of10 ng/ml IGF1. (A) Representativewesternblot showing E-cadherin
expression. Western blots were probedfor anti-E-cadherin (green)and anti-a-tubulin (red).Graph showingaverage E-cadherinband intensities (+SEM,
n 3) normalized to a-tubulin and expressed relative to the SS control. (B) Representative western blot showing integrin b3 expression (green) and
a-tubulin (red).Graph showsaverageintegrinb3 band intensities(+SEM, n 3) normalizedto a-tubulin and expressed relativeto theSS control. Analysis
of variance with Tukeyspost hocanalysis was used to compare the treatment groups. * indicates P, 0.05 (C) Representative flow cytometry histogram
showing surface expression of integrin avb3, b3 and b1 in Ishikawa cells. MW: molecular weight.
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Figure 4 IGF1 increases focaladhesion kinase(FAK) phosphorylationand totalFAK expressionlevels in Ishikawa cells.Ishikawacells or blastocysts were
cultured for 48 h in SS medium in thepresence or absence of 10 ng/ml IGF1. (A) Representative western blot showing phosphorylayed FAK (p-FAK) ex-
pression in Ishikawa cells. Western blots were probed for anti-p-FAK (green) and anti-a-tubulin (red). Graph shows average p-FAK band intensities
(+SEM, n 3) in Ishikawa cells, normalized to a-tubulin and expressed relative to the SS control. (B) Representative confocal image showing total
FAK expression and localization (green) in blastocysts attached to Ishikawa cells. Plain arrows indicate FAK staining on Ishikawa apical cell surface.
Dottedarrow indicatesincreasedFAK staining on IGF1 treated blastocyst. Scalebars represent20 mm. Nine attachment siteswere stained across 3 experi-
mentsin theSS groupand 10 attachment siteswerestained across3 experiments inthe SSplusIGF1 group.(C) Representativewesternblot showing p-FAK
expressionin blastocysts.Westernblotswere probedfor anti-p-FAK (green) andanti-a-tubulin (lowerpanel).One hundredand twentyblastocystswere
run in each lane. Graph shows average p-FAK band intensities ( +SEM,n 3) in blastocysts, normalized to a-tubulin and expressed relative to the SS
control. (D) Representative western blot showing total FAK expression in blastocysts. Western blots were probed for anti-total-FAK (green) and
anti-a-tubulin (lower panel). One hundred and twenty blastocysts were run in each lane. Graph shows average total FAK band intensities (+SEM,
n 3) in blastocysts, normalizedto a-tubulin and expressedrelative to theSS control. Students T-test wasused to compare thecontrol to thetreatment
groups.
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Fibronectin localization in early and late
stage blastocysts
Fibronectin is an important ECM protein that is secreted by blastocysts
butnot by Ishikawacells(Kaneko etal., 2013). Theexpressionand local-
ization of fibronectin in blastocysts was investigated by immunofluores-
cence over 72 h of culture, in the presence or absence of IGF1. Here
we describe the change in fibronectin expression over time as well as
an IGF-induced increase in fibronectin expression over time. Day5 blas-
tocysts wereculturedin SSmedium inthe presenceor absence of 10 ng/
ml IGF1 and collected at 24, 48 and 72 h and then immunostained for
fibronectin. At 24 h (now day 6 blastocyst), fibronectin was located in
the cytoplasm, nucleus, cellcell junctions and on the basal and apical
surface of the cells in the trophectoderm (Fig.5A and B). The ICM also
expressed fibronectin but this was restricted to the cells facing the
blastocoel. IGF1 treatment had no effect on fibronectin localization or
staining intensity at 24 h. IGF1 treatment increased fibronectin staining
intensity at 48 h compared with the SS control (Fig. 5C versus D). At
48 h (Day 7 blastocyst), fibronectin was located in the cytoplasm,
nucleus, cellcell junctions and on the basal and apical surface of the
trophectoderm (Fig.5C and D) and this staining was polarized to one
side of the blastocyst. At 48 h the majority of ICM cells had lost
fibronectin expression. The localization at 48 h was comparable
between the SS control and IGF1 group. At 72 h (Day 8 blastocyst),
there was a distinct polarization of fibronectin in the trophectoderm
on one side of the blastocyst and the localization was cytoplasmic and
punctate. There was no staining in the ICM (Fig. 5E and F). At 72 h in
the IGF1 treated group, fibronectin staining was of a much higher inten-
sity, compared with the SS group (Fig.5E versus F). Additionally, IGF1
treatment resulted in a distinct apical localization of fibronectin. The
polarized fibronectin staining seen at 48 and 72 h in blastocysts partially
coversthe trophoblastcells at theembryonic pole andextends from the
embryonic pole to the abembryonic pole on one side of the blastocyst.
This is the side that attached to Ishikawa cells in vitro.
The addition of the PI3 kinase inhibitor, 5 mM LY294002, to IGF1
treated blastocysts affected the overall polarization of fibronectin and
compared with the intense apical staining seen in IGF1 treated blasto-
cysts the localization of fibronectin in LY294002 treated blastocysts in
the presence of IGF1, was more cytoplasmic and strong staining in the
cellcell junctions was present (Fig.5F versus G).
To ensure the polarizedfibronectin staining wasfunctionallyrelatedto
attachment, blastocysts treated with IGF1that had adhered to glasscov-
erslips (72 h) were imaged for fibronectin staining. The attached surface
Figure 4 (Continued).
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of the blastocyst expressed fibronectin. The surface of the blastocyst
facing away from the coverslip did not express fibronectin (Fig. 5H).
Effect of IGF1 on blastocyst outgrowth
Fibronectin is known to increase blastocyst outgrowth in serum free
medium to the level seen in the presence of serum ( Armant et al.,
1986). Therefore blastocyst outgrowth was measured after 72 h (now
day 8 blastocysts) of culture on either uncoated wells or wells coated
with fibronectin, in serum medium or SS medium in the presence or
absence of 10 ng/ml IGF1 or 5 mM LY294002. Culture of blastocysts
in serum medium significantly increased the average area of blastocyst
outgrowth compared with SS blastocysts and blastocysts cultured in
thepresenceof IGF1 (Fig. 6H) and this was seen as an increase in troph-
ectoderm spreading across the culture dish (Fig.6A C). Under SS con-
ditions blastocyst outgrowth was increased by the presence of IGF1
when blastocysts were cultured on uncoated wells (Fig. 6B, C and H).
Blastocyst outgrowth in SS media was increased when blastocysts
were cultured on fibronectin coated wells (Fig.6B, D and H). The out-
growthof blastocystsin thepresence of IGF1 wasnot improved by fibro-
nectin (Fig.6C versus E and H). Outgrowth in the presence of IGF1 and
LY294002 on either uncoatedor fibronectin coated wellswas decreased
comparedwith outgrowth in IGF1alone on either uncoatedor fibronec-
tin coated wells, respectively (Fig.6FH).
Effect of pretreatment with IGF1, an IGF1R
nAb or LY294002 on attachment of mouse
blastocysts to Ishikawa cells
In attachment experiment 2, Day 5 blastocysts were cultured in serum
medium containing2 mg/ml IGF1R nAb or in SS medium in thepresence
or absence of 10, 100 or 1000 ng/ml IGF1 or with 10 ng/ml IGF1 in the
presenceof 2mg/mlIGF1RnAbor 5mM LY294002for 24 h.At theend
of thispretreatment periodthe healthof blastocysts cultured in LY294002was assessed and blastocysts (Day 6 blastocysts) were of healthy appear-
ance (Fig.7AD). Blastocysts were washed in SS medium and then cul-
tured on Ishikawa cells for 24 h, under SS conditions and assessed for
attachment (on Day 7). All blastocysts in all treatment groups remained
of normal appearance after the co-culture period (data not shown).
Pretreatment of blastocysts with serum for 24 h did not improve at-
tachmentform the serumstavedgroup (Fig.7E). Pretreatmentof blasto-
cysts with an IGF1R nAb in the presence of serum had no effect on
blastocyst attachment compared with pretreatment with serum
(Fig. 7E). Pretreatment with10 ng/ml IGF1for 24 h increased blastocyst
attachment to Ishikawa cells compared with pretreatment in SS medium
or medium containing serum (Fig. 7E). The positive effect of IGF1 pre-
treatment on attachment was reduced by the IGF1R nAb, to the levelseen in SS conditions (Fig.7E). Additionally, pretreatment of blastocysts
with the PI3K inhibitor LY294002 in the presence or absence of IGF1
reduced blastocyst attachment to Ishikawa cells compared with SS or
IGF1 pretreatment conditions, respectively (Fig. 7E). Pretreatment of
blastocysts with higher concentrations of IGF1 (100 and 1000 ng/ml)
did not increase blastocyst attachment compared with the SS control.
DiscussionA number of growth factors and their receptors are expressed by both
the preimplantation embryo and the reproductive tract and these influ-
ence preimplantation embryo development and implantation (Hardy
and Spanos, 2002;Armant, 2005). In particular, growth factors such as
IGF1, which are produced by the embryo and at the implantation site,
are thought to play a role in the co-ordination of blastocyst implantation
ability with uterine receptivity (Wanget al., 2000;Armant, 2005). In the
present study we used an in vitro model for embryo attachment to inves-
tigate the effect of IGF1 on mouse blastocyst adhesion competency.
Co-culture of blastocysts with Ishikawa cells for 48 h, under SS condi-
tions, decreased blastocyst attachment compared with attachment in
the presence of serum, which is well known to contain a large number
of growth factors. Addition of IGF1 to the co-cultures, in the absence
of serum, increased attachment to a level similar to that observed in
the presence of serum, suggesting that IGF1 alone can support attach-
ment of blastocysts to Ishikawa cellsin vitro. Furthermore, pretreatment
of Day5 blastocysts with IGF1 for 24 h wassufficient to improve blasto-
cyst attachment to Ishikawa cells compared with pretreatment in SS
medium. This suggests that thein vitrocultured embryo can be manipu-
lated by growth factors, normally found in vivo, to increase the blasto-
cysts adhesive ability.
The effects of IGF1 on blastocyst attachment were mediated by the
IGF1R as an IGF1R nAb negated the positive effects of IGF1 pretreat-
ment on blastocyst attachment.In theblastocyst IGF1 is known to phos-
phorylate Akt (Greenand Day, 2013), a signalling molecule downstreamof the PI3K pathway. In the present study the PI3K inhibitor LY294002
prevented the increase in attachment mediated by IGF1. LY294002
also decreased attachment in SS blastocysts, suggesting that even in SS
conditionsthere maybe autocrinegrowth factor stimulation or constitu-
tive activation of the PI3K pathway to influence attachment. In the
present study, pretreatment with serum did not improve blastocyst at-
tachment, in fact attachment in theseconditions was reduced compared
withattachment after IGF1pretreatment. High levels of insulin and other
growth factors that are present in serum may have negative effects on
blastocyst attachment, although serum appears to positively influence
Ishikawa cells to mediate attachment.
High concentrations of IGF1 are known to be detrimental to the
blastocyst by decreasing blastocyst hatching (Green and Day, 2013)and increasing apoptosis (Chi et al., 2000). High concentrations of
Figure 5 IGF1 increases apical fibronectin in late stage blastocysts. Representative confocal images of blastocysts stained for fibronectin (Green) and
DAPI (Blue). Day 5 blastocysts were cultured in SS medium for 24 (Aand B), 48 (Cand D) or 72 h (EH) in the absence (A, C and E) or presence
(B, D and F) of 10 ng/ml IGF1 or in the presence of a PI3 Kinase inhibitor (5 mM LY294002 + 10 ng/ml IGF1) (G) or in the presence of 10 ng/ml
IGF1, attached to a coverslip (H). Scale bars represent 20 mm. Red line indicates location of coverslip. Arrow indicates location of the inner cell mass
(ICM). The following total number of blastocysts were analysed, from at least 3 experimental repeats in each treatment; 24 in the SS group (24 h), 21 in
SS plus IGF1 (24 h), 26 in SS (48 h), 30 in SS plus IGF1 (48 h), 40 in SS (72 h), 59 in SS plus IGF1 (72 h), 13 in SS plus IGF1 + LY294002 (48 h) and 15 blas-
tocysts were analysed that were attached to a coverslip (72 h).
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IGF1 have also been shown to decrease normal blastocyst implantation
sitesand increase resorptioncompared withthe levelseen in blastocysts
exposed to physiological IGF1 concentrations (Pinto et al., 2002). In the
presentstudy,pretreatmentof blastocystswithhigh concentrationsof IGF1
did not improve blastocyst development unlike 10 ng/ml IGF1. These
effects could be due to the IGF1R down-regulation over time, in response
to high IGF1 concentrations (Pinto et al., 2002). Taken together these data
highlight our previous finding that the total concentration of IGF1 must be
tightly controlled in order to provide for optimal development.
Both pretreatment of blastocysts with IGF1 as well as co-culture of
blastocysts and Ishikawacells withIGF1 was sufficient to increase attach-
ment. IGF1, therefore, actson the blastocyst to mediate attachment and
may also increase attachment via actions on the Ishikawa cells. Thus the
molecular mechanisms by which IGF1 improved attachment were inves-
tigated in Ishikawa cells and blastocysts. Integrins are present in the api-
cal membraneof endometrial epithelialcells at thetime of implantation in
human,mouseand rat (Sutherland et al., 1993; Aplin et al., 1996; Schultz
etal.,1997; Kaneko etal., 2011a,b). Treatmentof Ishikawa cells withIGF1
for48 h hadno effecton total expression ofeither E-cadherin,or integrin
b3 expression levels. Integrins avb3, b3 and b1 were expressed at the
cell surface in Ishikawa cells; however, IGF1 treatment had no effect onthis surface expression. Although there was no change in integrin local-
izationor expressionin Ishikawacellsby IGF1treatment,total expression
of integrin b3 expression was increased by serum.
Fibronectinisproducedbytheembryo( Wartiovaara etal.,1979; Yoh-
kaichiyaet al., 1988;Thorsteinsdottir, 1992;Kaneko et al., 2013), and is
oneof theimportant bridging ligands, providingthe RGDsitefor therec-
ognition by integrins expressed on the apical surface of both the endo-
metrium and the embryo (Kaneko et al., 2013). IGF1 increased apical
fibronectin expressionin blastocysts after48 and 72 h of treatment, cor-
relating with the time of blastocyst attachment and outgrowth in vitro.
This increase in fibronectin at the apical surface may be responsible for
the increase in attachment of blastocysts to Ishikawa cells as the side of
the blastocyst that expressed fibronectin was the side that attached invitro. The overall polarity of fibronectin appeared to be developmentally
regulated, as fibronectin relocalised from trophectoderm cell cell
contacts (at 24 h culture) to the apical membrane (after 48 h culture)
at the time of attachment in vitro. Together these data suggests that
fibronectin producedby themouseblastocyst is an importantECM pro-
tein for attachment, acting as a possible bridging ligand for the integr-
ins expressed at the maternalembryo interface and that fibronectin
polarization may control the orientation of attachment to an underlying
cell layer.
Furthermore the acquisition of fibronectin on blastocysts from Day 7
onwards may regulate adhesion competency of the blastocyst. This was
demonstrated by the inability of Day 5 blastocysts to attach to Ishikawa
cells after 24 h (Day 6), whereas pretreatment of Day6 blastocystswith
IGF1 before placing them on Ishikawa cells enabled attachment after only
24 h (Day 7).ThisIGF1 inducedincreasein blastocystattachment correlated
with the increase in apical fibronectin on blastocysts by IGF1 and suggests
that attachmentis dependent on theadhesive competencyof theblastocyst,
which is developmentally regulated and can be increased by IGF1.
Fibronectin is also known to increase blastocyst outgrowth and there-
fore, as IGF1 increases fibronectin expression, we hypothesized that
IGF1 would also increase blastocyst outgrowth. We observed an in-
crease in blastocyst outgrowth on wells coated with fibronectin, in the
absence of IGF1, as well as an increase in blastocyst outgrowth in the
Figure 6 IGF1 increases mouse blastocyst outgrowth. Representa-
tivemicrographs showing blastocyst outgrowthafter 72 h. Day 5 blasto-
cysts were cultured on fibronectin (Fn) coated wells (D,E and G), or
uncoated wells (A, B , C and F) in the serum medium (A) or in SS
medium in the absence (B and D) or presence (C, E, F and G) of
10 ng/ml IGF1 and/or in the presence of a PI3 Kinase inhibitor (5 mM
LY294002 + 10 ng/ml IGF1) (F and G) in SS conditions (SS). Scale
bars represent 40 mm. (H) Average outgrowth area of blastocysts +
SEM cultured in serum medium (black bar) or SS medium (white bars)
in the presence or absence of 10 ng/ml IGF1 and/or in the presence
of a PI3 Kinase inhibitor (5 mM LY294002 + 10 ng/ml IGF1), pooled
from at least three experiments. Nvalues are given in parentheses and
represent the total number of blastocysts analysed across three to
eight experiments). Kruskal Wallis test followed by Dunns multiple
comparison test for non-parametric data was used to compare pre-
selected treatment groups. Lines between bars show compared
groups with significance. * indicates P, 0.05, ** indicates P, 0.01
and *** indicatesP, 0.001. AU: arbitrary units.
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shown that attachment of rat blastocysts to Ishikawa cells in vitro is
reduced by pre-incubation of blastocysts and or Ishikawa cells with the
RGD-blocking peptide (Kanekoet al., 2013). In outgrowth studies, the
FN-120 fragment in fibronectin, which contains the RGD sequence,
promotes blastocyst outgrowth, whereas the FN-50 fragment, which is
lacking the RGD sequence, impairs blastocyst outgrowth (Yelianet al.,
1995). Furthermore, function blocking antibodies to integrin b1 and
b3 also reduced fibronectin binding on trophoblast cells and inhibited
blastocyst outgrowth in mice (Schultz and Armant, 1995;Yelianet al.,
1995). In human extravillous trophoblast cells IGF1 treatment increasesfibronectin binding and this is also blocked by an RGD-blocking peptide
(Kabir-Salmaniet al., 2002).
Integrin signallingis a complex process thatinvolvesboth alterations in
integrin affinity for ECM through pathwaysdownstream of growth factor
signalling (inside-out signalling), and signalling downstream of integrin-
ECM interactions (outside-insignalling) (Sastry and Horwitz, 1993). FAK
is a major componentof theoutside-in integrin signal transduction path-
way to regulate a variety of cellular events(reviewed in (Schaller, 2001)).
FAK is notonlyactivated by integrin-ECMligation, butalso is phosphory-
latedby hormonesand growth factors,including insulin and IGF-1 (Baron
et al., 1998,Casamassima and Rozengurt, 1998). In the present study
treatment of Ishikawacells withIGF1 for 48 h increasedphosphorylation
of FAK at Tyr397. Total FAK was also increased by IGF1 and its ex-
pression was apical. In the endometrium FAK is up-regulated in the
early proliferative to mid secretory stage, suggesting a role of FAK in im-
plantation (Orazizadeh et al., 2009). As FAKcan regulateintegrinactiva-
tion to promote integrin binding (Michael et al., 2009), the up-regulation
of FAK phosphorylation at Tyr397 in Ishikawa cells by IGF1 may play
a role in integrin recognition of ECMproteins secretedby theblastocyst,
and can explain the increase in attachment seen on Ishikawa cells,
although IGF1 did not increase surface expression or total expression
of integrins.
IGF1 treatment for 2 h has been shown to phosphorylate FAK in
trophoblast cells obtained from human 610 week placental tissue
(Kabir-Salmani et al., 2002). In the present study total FAK staining
after IGF1 treatment in blastocysts attached to Ishikawa cells appeared
to be stronger,although this wasnot quantified. However in unattached
blastocysts no change in phosphorylated or total FAK after IGF1 treat-
ment was observed, suggesting that FAK phosphorylation is dependent
on attachment status (Baronet al., 1998).
Akt is another signalling mediator that is necessary for integrin acti-
vation and has been shown to regulate fibronectin assembly through ac-tivation of integrin a5b1(Somanathet al., 2007). Akt is known to be
activated by IGF1 in mouse blastocysts (Green and Day, 2013). In the
present study, inhibition of PI3K prevented the polarized distribution
of fibronectin in blastocysts treated with IGF1. PI3K inhibition also
decreasedblastocystoutgrowthin thepresenceof IGF1as wellas attach-
ment of blastocysts to Ishikawa cells, possibly due to the decrease in
apical fibronectin. However, the overall intensity of fibronectin staining
remained high in thepresence of thePI3Kinhibitor, suggesting that fibro-
nectin synthesis in the blastocyst is via a signalling pathway other than
PI3K.
In summary, the present study demonstrated that IGF1 improved the
adhesion competency of the mouse blastocyst. IGF1 up-regulated fibro-
nectin expression on the apical surface of the trophoblasts, via activation
of the PI3K/Akt pathway and we propose that this increases blastocyst
adhesion competency (Fig.8). IGF1 also activated FAK expression and
signalling in Ishikawa cells, potentially regulating integrin activation in
orderto primethe cells forinteraction with thefibronectinon theblasto-
cyst (Fig.8). These findings provide evidence for the important role that
IGF1 plays in vivo, in the co-ordination of blastocyst adhesion compe-
tency and uterine receptivity. This study used a heterologous yet well
documented model to study adhesion of mouse blastocysts to the
human Ishikawa cell line in response to IGF1. This system would be
Figure 8 Schematic diagram proposing mechanisms by which IGF1 increases blastocyst adhesion competency and mediates attachment to Ishikawa
cells.IGF1 increases attachmentof mouseblastocysts to Ishikawa cells in vitro andhas actions on both theIshikawacellsand theblastocysts. IGF1 increases
apicalfibronectin on blastocystsand thismay bridgeintegrinspresent on theapical surface of theblastocyst and Ishikawacells. ThePI3 kinase(PI3K) inhibitor
LY294002 reduces apical fibronectin and pretreatment of blastocysts with LY294002 reduces attachment. IGF1 increases focal adhesion kinase (FAK)expression and phosphorylation and this may increase the integrins ability to bind and recognize extracellular matrix proteins such as fibronectin.
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useful to studyIGF1 adhesion regulationof human blastocysts or a spher-
oidal human trophoblast cell line such as JArcells. At thetime of implant-
ation in the human there is a sufficient amount of IGF1 present in the
uterineand luminal secretions (Lighten etal.,1998); however, thismater-
nal IGF1 is absent during thein vitro culture of embryos. As failure of the
embryo to implant successfullyis a majorcauseof IVFfailure(Milleret al.,
2012),ourresults,alongwithothers(Lighten etal.,1998; GreenandDay,
2013) indicate that pretreatment of human blastocystswith IGF1 before
their introduction back into the uterus may be a possible avenue to
improve implantation rates in assisted reproduction.
Acknowledgements
We would like to thank Professor Chris Murphy (University of Sydney,
Australia) for the Ishikawa cell line and integrin b3 and total FAK anti-
bodies; Dr Steve Assinder (University of Sydney, Australia) for the
E-cadherin antibody; Dr Matthew Naylor (University of Sydney, Austra-
lia)for the p-FAK antibody;Professor Frank Lovicu (Universityof Sydney,
Australia) for the fibronectin antibody. We also thank Dr Louise Cole
and Dr Yingying Su (Advanced Microscopy Facility, Bosch Institute, Uni-
versity of Sydney) for help with confocal microscopy and Dr AngelesSanchez-Perez (Live Cell Analysis facility, Bosch Institute, University of
Sydney) for help with flow cytometry.
Authors roles
C.J.G designed the study, carried out experimental work, analysed data
and prepared manuscript. S.T.F gave technical assistance in running and
analysingflow cytometry experiments as wellas experimental design and
editing of manuscript. M.L.D designed the study, gave technical advice
and critical editing of manuscript.
FundingThis work was supportedby internal funds from theUniversity of Sydney
and the Bosch Institute.
Conflict of interest
The authors declare that there is no conflict of interest that could be
perceived as prejudicing the impartiality of the research reported.
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IGF1 Increases mouse blastocyst attachmentin vitro 15