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Theriogenology 75 (2011) 256–267

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Effects of vascular endothelial growth factor on porcinepreimplantation embryos produced by in vitro fertilization and

somatic cell nuclear transferD. Biswasa, E.M. Jungb, E.B. Jeungb, S.H. Hyuna,*

a Laboratory of Veterinary Embryology and Biotechnology, College of Veterinary Medicine, Chungbuk National University,Cheongju-361-763, Chungbuk, South Korea

b Laboratory of Veterinary Biochemistry and Molecular Biology, College of Veterinary Medicine, Chungbuk National University,Cheongju-361-763, Chungbuk, South Korea

Received 27 April 2010; received in revised form 20 August 2010; accepted 20 August 2010

bstract

This study examined the effects of vascular endothelial growth factor (VEGF) on porcine embryos produced by in vitro fertilizationIVF) and somatic cell nuclear transfer (SCNT) at different developmental stages. Four sets of experiments were performed. In the first,upplementation of the in vitro culture medium with 5 ng/mL VEGF was suitable for porcine IVF embryo development, and thelastocyst formation rate was significantly higher than the control and other groups (57.73 � 6.78% (5 ng/mL VEGF) vs. 43.21 �0.22% (control), 42.16 � 10.24% (50 ng/mL VEGF) and 41.91 � 11.74% (500 ng/mL VEGF); P � 0.05). The total cell number afterupplementation with 5 ng/mL VEGF was significantly higher than the control and other groups (151.85 � 39.77 (5 ng/mL VEGF) vs.00.00 � 34.43 (control), 91.2 � 31.51 (50 ng/mL VEGF), and 112.53 � 47.66 (500 ng/mL VEGF); P � 0.05). In the secondxperiment, when VEGF was added at different developmental stages of IVF derived embryos (early stage, days 1–3, late stage, days–7), the blastocyst formation rate and total cell number were significantly higher at the late stage (47.71 � 9.13% and 131.5 � 20.70,

espectively) than in the control (34.32 � 7.44% and 85.50 � 20.41, respectively) and at the early stage (33.60 � 5.78% and 86.75 �5.10, respectively; P � 0.05). There was no significant difference in the blastocyst development rate or total cell number between thehole culture period (days 1–7) and the late stage culture period after supplementation with 5 ng/mL VEGF (P � 0.05). In the third

xperiment, the cleavage rate was significantly higher when SCNT embryos were cultured with VEGF during the whole culture periodhan in the late stage (63.56 � 15.52% vs. 39.72 � 4.94%; P � 0.05), but there was no significant difference between the control andhe early stage culture period (P � 0.05). The blastocyst formation rate was significantly higher at the late stage culture period withEGF than at the early stage culture period (34.40 � 15.06% vs. (16.07 � 5.01%; P � 0.05). There was no significant difference in

he total cell number between the groups (P � 0.05). In experiment 4, using real-time PCR, VEGF mRNA expression was detected inll the developmental stages of IVF and SCNT embryos, but the expression level varied according to the developmental stage. VEGFeceptor, KDR mRNA was detected in all stages IVF and SCNT embryos. However, flt-1 mRNA was not expressed in all embryonictages of IVF and SCNT embryos. These data suggest that VEGF supplementation at the late embryonic developmental stage mightmprove the developmental potential of both IVF and SCNT preimplantation porcine embryos through its receptors.

2011 Elsevier Inc. All rights reserved.

www.theriojournal.com

1

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* Corresponding author. Tel.: �82-43-261-3393; fax: �82-43-267-3150. sE-mail address: shhyun@cbu.ac.kr (S.H. Hyun).

093-691X/$ – see front matter © 2011 Elsevier Inc. All rights reserved.oi:10.1016/j.theriogenology.2010.08.012

. Introduction

Successful implantation requires functionally activembryos [1] and it involves a complex sequence of

ignaling events that are crucial for the establishment of

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257D. Biswas et al. / Theriogenology 75 (2011) 256–267

regnancy. Many molecular mediators participate inhis early feto-maternal interaction, including adhesionolecules, cytokines, growth factors, and lipids [2].rowth factors involved include leukemia inhibitory

actor (LIF), macrophage colony-stimulating factor-1CSF-1), interleukin-1 (IL-1), prostaglandin G (PG),ascular endothelial growth factor (VEGF), glycode-in-A, and insulin-like growth factors (IGFs) and theirinding proteins, and heparin binding-epidermalrowth factor (HB-EGF) [3]. Most of these growthactors are derived from or produced by the endome-rium or embryo. Absence of these factors can lead tomplantation failure or, in rare cases, severe develop-ental abnormalities.The biological effects of VEGF are almost exclu-

ively mediated by two receptor tyrosine kinases thatere isolated from a placental cDNA library in 1990.he fms-like tyrosine kinase (flt-1 or VEGFR-1; 180Da) is a high affinity receptor for VEGF-A, VEGF-B,nd PIGF [4] and flt-1 mRNA is expressed in vascularndothelial cells. The second high affinity receptor isetal liver kinase-1/kinase insert domain-containing re-eptor (flk-1/KDR or VEGFR-2). The expression ofoth receptors is largely restricted to the vascular en-othelium [5].

The highest peak of VEGF expression in the prolif-rative stage of the estrus cycle, and the steady rate ofessel growth during the mid- and late-luteal phases,re important for implantation [6]. The VEGF has beenetected in human oviductal luminal epithelium [7], andEGF mRNA expression was greatest during the peri-vulatory stage, and in the ampullary and infundibularegions [7]. Although VEGF mRNA is expressed more inhe infundibulum and ampulla, VEGF supports fertiliza-ion and early embryo development [7].

Although VEGF is produced primarily during theeri-ovulatory period [8], and human VEGF mRNAan be detected in the unfertilized oocyte to the blas-ocyst stage [9], the effects of VEGF on preimplanta-ion embryos are poorly understood. Additionally, theumbers of blastomeres in the blastocyst increased afterEGF supplementation in the in vitro maturation

IVM) medium, suggesting that, in addition to thenown angiogenic effects, VEGF may affect early em-ryonic viability [10]. Growth factors within tubal fluidave the potential to influence embryonic growth andevelopment [11]. Thus, the present study comparedhe stage-specific embryotropic effects of VEGF onorcine preimplantation embryos derived by IVF and

CNT. 5

. Materials and methods

.1. Chemicals

All chemicals were purchased from Sigma-Aldrichhemical Company (St. Louis, MO, USA), unless oth-rwise stated.

.2. Ovary collection, recovery and in vitroaturation of oocytes

Ovaries of prepubertal gilts were collected from aommercial abattoir and transported to the laboratoryithin 2 h in 0.9% (w/v) NaCl solution supplementedith penicillin-G (100 U/mL) and streptomycin sulfate

100 mg/mL) at 30 to 35 °C. The follicular fluid withocytes was aspirated from 3- to 6-mm antral folliclesith a 10-mL disposable syringe and 20-gauge needle

nd collected in a 15-mL centrifuge tube. Cumulus-ocyte complexes (COC) were recovered under a ste-eoscope microscope; those with at least three layers ofompact cumulus cells and with homogenous cyto-lasm were selected for in vitro maturation. The se-ected COCs were transferred and cultured in 500 �L ofissue culture medium 199 (Life Technologies, Rock-ille, MD, USA) supplemented with 26 mM sodiumicarbonate, 0.91 mM sodium pyruvate, 0.57 mM cys-eine, 10 ng/mL epidermal growth factor, 0.5 IU/mLorcine luteinizing hormone, 0.5 IU/mL porcine folli-le stimulating hormone, 10% (v/v) pFF, 75 �g/mLenicillin-G, and 50 �g/mL streptomycin. The pFF wasspirated from 3–7-mm follicles of prepubertal gilt ova-ies. After centrifugation at 1600� g for 30 min, theupernatants were collected and filtered sequentiallyhrough 1.2- and 0.45-�M syringe filters (Gelman Sci-nces, Ann Arbor, MI, USA). The prepared pFF washen stored at �20 °C until use. For maturation, theelected COCs were washed three times in oocyte mat-ration medium containing hormone supplements, andpproximately 50–60 oocytes were transferred intoach well of a 4-well Nunc dish (Nunc, Roskilde, Den-ark) containing 500 �L of culture medium and equil-

brated at least 2 hrs with 5% CO2 at 39 °C in aumidified atmosphere. After 20–22 h of maturationith hormones, the oocytes were washed two times inmaturation medium without hormone supplements

nd then cultured for 20–22 h without hormone sup-lements at 39 °C under 5% CO2 in air.

.3. In vitro fertilization

A 0.25 ml straw of frozen boar semen was thawed at9 °C for 1 min in a water bath and then washed with

mL of phosphate-buffered saline (PBS) by centrifug-

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258 D. Biswas et al. / Theriogenology 75 (2011) 256–267

ng twice (300�g, 2 min). After washing, the spermellet was resuspended in modified Tris-buffered me-ium (mTBM) [12] and incubated at 39 °C under 5%O2 in air until insemination. After 44 h of in vitroaturation, the oocytes were stripped of their cumulus

ells by pipetting with 0.1% hyaluronidase (Sigma-ldrich) and washed three times in mTBM that wasre-equilibrated for 18 h at 39 °C in 5% CO2. Oocytesith evenly dark ooplasm and visible first polar bodiesere selected for all experiments. After washing, 15ocytes were randomly placed in 45-�L droplets ofTBM medium covered with prewarmed mineral oil.fter the appropriate dilution of frozen-thawed sper-atozoa, 5 �L of sperm suspension was added to each

5-�L fertilization droplet to give a final sperm con-entration of 2 � 106 sperm/mL. Gametes were co-ultured for 6 h at 39 °C in a humidified atmosphere of% CO2, 95% air. After 6 h, gametes were removedrom the fertilization drops, washed three or four timesn HEPES-buffered North Carolina State University

edium-23 (NCSU-23) medium and cultured in 25-�Licrodrops (10 gametes/drop) of NCSU-23 coveredith mineral oil and incubated at 39 °C for 168 h under% O2, 5% CO2 and 90% N2. Cleavage and blastocystormation were evaluated under a stereomicroscope at8 and 168 h post-insemination, respectively.

.4. Micromanipulation for somatic cell nuclearransfer, fusion, and activation

Fibroblasts were isolated from fetuses at day 40 ofestation. The head and other soft tissues were removedsing iris scissors and watchmaker’s forceps and dis-arded. After washing twice with dPBS (Invitrogen,arlsbad, CA, USA), the carcass was minced with a

urgical blade on a 100-mm culture dish. The mincedetal tissues were dissociated in DMEM (Invitrogen)upplemented with 0.1% (w/v) trypsin and 1 mMDTA (Invitrogen) for 1–2 hr. Trypsinzed cells wereashed once by centrifugation at 300� g for 10 min

nd subsequently seeded into 100-mm plastic cultureishes. Seeded cells were then cultured for 6–8 days inMEM supplemented with 10% (v/v) fetal bovine se-

um (FBS; Invitrogen), 1 mM sodium pyruvate, 1%v/v) non-essential amino acids (Invitrogen), and 10g/mL penicillin–streptomycin solution at 39 °C in a

umidified atmosphere of 5% CO2 and 95% air. Afteremoving the unattached clumps of cells or explants,he attached cells were further cultured until confluent.ubculturing was done at intervals of 5–7 days by

rypsinization for 2 min using 0.1% trypsin and 0.02%

DTA. The cells were then stored in freezing medium 1

n liquid nitrogen after two passages. The freezingedium consisted of 70% (v/v) DMEM, 10% (v/v)MSO, and 20% (v/v) FBS. Prior to SCNT, the cellsere thawed and subsequently cultured in 10% FBSith DMEM for 3–4 days until 80% confluence. The

ndividual cells were retrieved from the monolayer byrypsinization for �1 min and subsequently used forCNT.

After 44 hr of IVM, Cumulus-cell-free oocytes werencubated for 2 min in a manipulation medium (calciumree TLH-BSA) containing 5�g/ml Hoechst 33343Sigma). Following incubation, the oocytes were trans-erred into a drop of manipulation medium and wereverlaid with warm mineral oil. The zona pellucida wasartially dissected with a fine glass needle to make a slitear the first polar body (PB). The first PB and adjacentytoplasm (�10%), presumably containing the meta-hase-II (M-II) chromosomes, were extruded by squeez-ng the oocytes with the same needle. Enucleation wasonfirmed under an epifluorescence microscope (TE 300,ikon, Tokyo, Japan). Using a fine injecting pipette, a2–15-�m trypsinized fetal fibroblast with a smoothell surface [13] was transferred into the perivitelinepace through the same slit of an enucleated oocyte.he couplets were equilibrated with 0.28 M mannitololution containing 0.5 mM HEPES, 0.1 mM CaCl2,nd MgSO4 for 2–3 min and transferred to a fusionhamber containing two electrodes overlaid with man-itol solution. Membrane fusion was induced by apply-ng an alternating current (AC) field of 2V cycling at 1

Hz for 2 sec, followed by two pulses of 160V/mmirect current (DC) for 50 �sec using a cell fusionenerator (LF101; NepaGene, Chiba, Japan). Activatedocytes were washed 3–4 times with NCSU-23 me-ium supplemented with 4 mg/mL fatty-acid free BSAnd placed in a humidified incubator at 39 °C under 5%O2. After 1 h, the fusion was checked, and fused,roperly shaped oocytes were washed 3–4 times andurther cultured with NCSU-23 medium covered withrewarmed mineral oil and then incubated under 5%

2, 5% CO2, and 90% N2 at 39 °C for 168 h. Cleavagend blastocyst formation were evaluated under a ste-eomicroscope at 48 and 168 h post activation, respec-ively.

.5. Embryo evaluation and nuclear staining

The embryos were assessed for cleavage on day 2nd for blastocyst development on day 7. The day ofVF was day 0. Blastocysts considered viable wereashed with 1% PVA for 1 min and then fixed with

00% ethanol containing 10 �g/mL Hoechst 33258 for

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259D. Biswas et al. / Theriogenology 75 (2011) 256–267

t least 5 min. Then, the blastocysts were mounted onlass slides in a drop of 100% glycerol and squashedently with a cover slip. The nuclei were counted usinguorescence microscopy.

.6. Reverse transcription–polymerase chain reactionRT–PCR) and real-time PCR with SYBR Green

Total RNA was extracted using the Trizol reagentInvitrogen, Carlsbad, CA, USA), according to theanufacturer’s protocol, and the total RNA concentra-

ion was determined by measuring the absorbance at60 nm. First-strand complementary DNA (cDNA) wasrepared by subjecting 1 �g of total RNA to reverseranscription using Moloney murine leukemia virusMMLV) reverse transcriptase (Invitrogen). To deter-ine the conditions for logarithmic phase PCR ampli-cation of target mRNA, 1-�g aliquots were amplifiedsing differing numbers of cycles. The housekeepingene, cytochrome oxidase subunit 1 (1A), was PCR-mplified to rule out the possibility of RNA degradationnd to control for the variation in mRNA concentra-ions in the RT reaction. A linear relationship betweenhe PCR product band visibility and the number ofmplification cycles was observed for the targetRNAs. The 1A and target genes were quantified using

2 cycles. The cDNA was amplified in a 20-�L PCReaction, which contained 1 U Taq polymerase (Intronio Technologies, Co., Ltd.), 2 mM dNTP mix, and 10M of each gene-specific primer.

Quantitative real-time PCR was performed with 1L cDNA template added to 10 �L 2� SYBR Premixx Taq (Takara Bio Inc.) containing specific primers atconcentration of 10 pM each. The reactions were

arried out for 32 cycles and the cycling parametersere as follows: denaturation at 95 °C for 30 s, anneal-

ng at 55 °C for 30 s, and extension at 72 °C for 30 s.ll oligonucleotide primer sequences are presented inable 1. The fluorescence intensity was measured at thend of the extension phase of each cycle. The thresholdalue for the fluorescence intensity of all samples waset manually. The reaction cycle at which the PCRroducts exceeded this fluorescence intensity thresholdas deemed the threshold cycle (Ct) in the exponentialhase of the PCR amplification. The expression of eacharget gene was quantified relative to that of the internalontrol gene. The relative quantification was based on aomparison of Cts at constant fluorescence intensity.

The amount of transcript present was inversely re-ated to the observed Ct and, for every two-fold dilutionn the amount of transcript, Ct was expected to increase

y 1. The relative expression (R) was calculated using K

he equation, R � 2-[Ct sample-Ct control]. To determinenormalized arbitrary value for each gene, every data

oint was normalized to the control gene, as well as tots respective control.

.7. Experimental designs for the manipulation ofVF and SCNT embryos

In experiment 1, the effect of different VEGF con-entrations on the in vitro development of IVF embryosas assessed. Following IVF, embryos were randomly

llocated and cultured in NCSU-23 supplemented withifferent concentrations of VEGF (0, 5, 50 and 500g/mL) for the whole culture period (days 1–7) tovaluate preimplantation development.

In experiment 2, the effects of VEGF (5 ng/mL)upplementation at different stages of porcine IVF em-ryo development were evaluated. Four groups wereet up: negative control, with no addition of VEGF tohe culture medium on days 1–7 (1st day of culture isay 1); early stage, with VEGF supplementation onays 1–3; and late stage, with VEGF supplementationn days 4–7. The positive control group was embryosultured with VEGF supplemented on days 1–7.

In experiment 3, the effect of VEGF (5 ng/mL) sup-lementation at different stages of porcine SCNT embryoevelopment were evaluated as in experiment 2.

In experiment 4, IVF and SCNT embryos (mature orused oocytes, 2-, 4-, and 8-cell embryos, morulae andlastocysts) were collected according to their develop-ental stages and VEGF mRNA and its receptors,

able 1equences of oligonucleotide primers used in RT-PCR and real-

ime PCR and predicted sizes of the amplified products.

Target genename

Sequences Predictedproduct

size

EGF 268 bpForward ACC CAT GGC AGA AGG AGA CCA

GAA AReverse TAT GTG CTG GCC TTG GTG AGG

TTT GDR 180 bpForward GAT GCT CGC CTC CCT TTG AReverse AGT TCC TTC TTT CAG TCG CCT

ACAlt-1 180 bpForward CAC CCC GGA AAT CTA TCA GAT CReverse GAG TAC GTG AAG CCG CTG TTG

-A 293 bpForward CAC CGT AGG AGG TCT AAC GReverse GTA TCG TCG AGG TAT TCC G

DR and flt-1mRNA expression was examined. The

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260 D. Biswas et al. / Theriogenology 75 (2011) 256–267

xperiments were repeated three times with equal num-ers of cells (�120 cells) for each developmental stage.

.8. Statistical analyses

Statistical analysis was carried out using the PASWtatistical software. The means of the cleavage rate,evelopmental rate to the blastocyst stage, and numberf nuclei were compared by one-way ANOVA, fol-owed by Duncan’s multiple range test. Data are pre-ented as mean � SEM. P values � 0.05 were deemedo indicate statistical significance.

. Results

.1. Developmental competence of porcine IVFmbryos at different concentrations of VEGF duringn vitro culture (IVC)

We investigated the optimal VEGF concentration inhe medium used to culture the porcine IVF oocytesTable 2). The highest blastocyst rate was obtained with

ng/mL VEGF (57.73 � 6.78%) compared with theontrol (43.21 � 10.22%), 50 ng/mL (42.16 �0.24%), and 500 ng/mL (41.91 � 11.74%) VEGF,ith statistical significance (P � 0.05; Table 2). Thereas no significant difference in the cleavage rate at dayamong the groups (P � 0.05). VEGF supplementation

t 5 ng/mL significantly improved the total cell number151.85 � 39.77) compared with the control (100.00 �4.43) and 50 ng/mL groups (91.2 � 31.51). However,here was no significant difference between the 5

able 2ffect of different VEGF concentrations on in vitro fertilized porcin

Treatment Group Total oocytes � 2 cells (%)

Control 148 117 (79.19 � 5.35)5 ng/mL 158 120 (75.74 � 4.87)50 ng/mL 161 129 (80.86 � 5.52)500 ng/mL 169 129 (76.25 � 10.8)

* Percentage of cleaved embryos.a,b Values with different superscripts in the same column are signifi

able 3he effects of VEGF (5 ng/mL) on preimplantation porcine IVF em

Group Total oocytes � 2 cells (%)

ontrol 123 78 (63.40 � 10.79)ays 1–7 104 66 (62.45 � 12.85)ays 1–3 120 80 (66.73 � 4.54)ays 4–7 126 76 (60.29 � 8.38)

a,b Values with different superscripts in the same column are signifi

* Percentage of cleaved embryos.

g/mL and 500 ng/mL groups in terms of the total cellumber. Thus, 5 ng/mL VEGF was selected as theptimal concentration for porcine in vitro embryo de-elopment.

.2. The effects of VEGF (5 ng/mL) onreimplantation porcine IVF embryos according tohe day

We evaluated the temporal effects of VEGF on theevelopmental ability of porcine IVF embryos (Table). After fertilization, the “good” zygotes were allo-ated randomly into four groups. In the first group, theertilized zygotes were cultured with VEGF for the firstdays (days 1–3) and then cultured without VEGF for

nother 4 days (days 4–7). In the second group, theertilized zygotes were cultured in the absence ofEGF for first 3 days (days 1–3) and then cultured withEGF for another 4 days (days 4–7). The third groupas cultured without VEGF for the entire culture pe-

iod and served as the control group. The fourth groupas cultured with VEGF for the entire culture period

days 1–7). At the end of the culture period, the em-ryos were evaluated for their development rates andell number. As shown in Table 3, there was no sig-ificant difference in the cleavage rate among theseroups (P � 0.05). However, the embryo developmentate was significantly higher when the embryos wereultured with VEGF for days 1–7 or days 4–7, com-ared with the control and the days 1–3 culture (49.69 �.89% or 47.71 � 9.13% vs. 34.32 � 7.44% and 33.60 �

yo development during IVC.

blastocysts (%)* Cell number per blastocyst (mean � SEM)

43.21 � 10.22)a 100 � 34.43a

(57.73 � 6.78)b 151.85 � 39.77b

42.16 � 10.24)a 91.2 � 31.51a

41.91 � 11.74)a 112.53 � 47.66a,b

different (P � 0.05).

ccording to the day.

lastocysts (%)* Cell number per blastocyst (mean � SEM)

4.32 � 7.44)a 85.50 � 20.41a

9.69 � 9.89)b 133.75 � 40.65b

3.60 � 5.78)a 86.75 � 25.10a

7.71 � 9.13)b 131.5 � 20.70b

different (P � 0.05).

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261D. Biswas et al. / Theriogenology 75 (2011) 256–267

.78%, respectively; P � 0.05). Additionally, the totalell number was significantly increased (P � 0.05)hen VEGF was added during the whole culture periodr the late stage (days 4–7), compared with the controlnd early stage (days 1–3) culture periods.

.3. The effects of VEGF (5 ng/mL) onreimplantation porcine SCNT embryos according tohe day

In experiment 3, the SCNT embryos were culturedith 5 ng/mL VEGF at different developmental stages.here was no significant difference in the cleavage orlastocyst formation rates or the total cell numbermong these groups (Table 4). However, when theeconstructed embryos were cultured with VEGF forhe whole culture period, the cleavage rate was signif-cantly higher than in embryos cultured with VEGFnly on days 1–3 (Table 4; 63.56 � 15.52% vs.6.26 � 9.29%). The blastocyst formation rate wasignificantly higher when the reconstructed embryosere cultured with VEGF during the later stage than the

arly stage (Table 4; 34.40 � 15.06% vs. 16.07 �.01%). There was no significant difference (P � 0.05)n total cell number between the groups.

.4. Expression of VEGF and its receptors KDR andt-1 mRNA in porcine IVF and SCNT embryos

VEGF and its receptors KDR and flt-1 mRNA wasmplified by real-time PCR using SYBR Green. In bothVF and SCNT embryos, VEGF mRNA was detectedn mature or fused oocytes, 2-, 4-, and 8-cell embryos,orulae and blastocysts. VEGF mRNA was quantita-

ively compared by calculating the VEGF/1A mRNAxpression ratio using real-time RT-PCR. In IVF em-ryos, VEGF mRNA expression was detected in alltages of embryonic development, but it increased sig-ificantly at the 4-cell stage and then dramatically de-reased at the 8-cell stage, compared with the 4-celltage. At the morula stage, VEGF mRNA expressionlso increased significantly and remained constant until

able 4he effects of VEGF (5 ng/mL) on preimplantation porcine SCNT e

Group Total fused oocytes � 2 cells (%)

ontrol 79 40 (51.49 � 11.49)a,b

ays 1–7 82 50 (63.56 � 15.52)b

ays 1–3 95 53 (56.26 � 9.29)a,b

ays 4–7 77 31 (39.72 � 4.94)a

* Percentage of cleaved embryos.a,b Values with different superscripts in the same column are signifi

he blastocyst stage (Fig. 1A). In SCNT embryos, basal m

evels of VEGF mRNA were detected in all stages.EGF mRNA expression was statistically significant

P � 0.05) from the 4-cell to the blastocyst stage,ompared with the fused and 2-cell stages. However,he expression was not significant from the 4-cell to theorula stages. In blastocysts, the expression rapidly

ncreased, compared with all other stages (Fig. 1B).The fetal liver kinase-1/ kinase insert domain-con-

aining receptor mRNA (flk-1/KDR) was detected in alltages of preimplantation embryos either derived fromVF or SCNT though real time PCR. The expressionevel was not significantly different. However, the fms-ike tyrosine kinase (flt-1) receptor mRNA was notetected in all stages of embryos derived from eitherVF or SCNT (Fig. 2A,B). The expression level wasot significantly different between the embryonictages.

. Discussion

In vitro production of porcine embryos and liveirths by IVM and IVF are still inefficient comparedith in vivo-produced embryos [14]. Various factors

re related to this inefficiency, including improper cul-ure conditions, underdeveloped embryos, improper ex-ression of several genes that are important for initialmbryonic development [15], and blastocyst qualityrade [16]. The implantation rate increased when ex-anded or hatching blastocysts were transferred, com-ared with small blastocysts [16]. Growth factors, suchs LIF, CSF-1, IL-1, PG, VEGF, glycodelin-A, IGFsnd their binding proteins, and HB-EGF, are involvedn the implantation process [3]. Among these, VEGF isxpressed in the endometrium throughout the menstrualycle, with the highest expression in the glandular ep-thelium at the secretory phase [17,18].

Various growth factors are essential for successfulollicle development, embryo development, and im-lantation. Many previously published reports haveocumented the effects of different growth factors on

according to the day.

blastocysts (%)* Cell number per blastocyst (mean � SEM)

3.97 � 9.94)a,b 100.22 � 48.666.62 � 14.01)a,b 86.92 � 49.986.07 � 5.01)a 99.60 � 48.364.40 � 15.06)b 94.56 � 41.06

different (P � 0.05).

mbryos

Total

9 (213 (29 (1

11 (3

ammalian oocytes [19]. Very few have described

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262 D. Biswas et al. / Theriogenology 75 (2011) 256–267

EGF in bovine oocytes, and until now, none hasvaluated porcine preimplantation embryo develop-

ig. 1. VEGF mRNA expression pattern in porcine preimplantation embrelative VEGF mRNA expression at different stages was analyzed by reaxpression was normalized to 1A mRNA expression. The letters (a, b, c

O, matured oocyte; BL, blastocyst; FO, fused oocyte.

ent. A complete VEGF system, consisting of the t

igand and two receptors, has been detected in bovineumulus-oocyte-complexes (COC) [10]. However, af-

ived from IVF (A) and SCNT (B) at different developmental stages. TheCR. The data represent the mean � SEM for all samples. VEGF mRNAve the columns indicate statistical significance at P � 0.05.

yos derl-time P, d) abo

er 24 h of IVM, VEGF expression and its receptor

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263D. Biswas et al. / Theriogenology 75 (2011) 256–267

ranscripts in bovine COC were altered in a time-de-endent manner. Our previous study [20] showed that

ig. 2. VEGF receptors, KDR and flt-1 mRNA expression pattern in porevelopmental stages. The relative KDR and flt-1 mRNA expression washe KDR and flt-1 mRNA expression was normalized to 1A mRNA ex

O, matured oocyte; BL, blastocyst; FO, fused oocyte.

ddition of VEGF during IVM, influenced the matura- t

ion rate and also increased the blastocyst rate througharthenogenetic activation. Our present study showed

implantation embryos derived from IVF (A) and SCNT (B) at differentd by real time PCR. The data represent the mean � SEM for all samples.n. Statistical significant were considered as P � 0.05.

cine preanalyzepressio

hat 5 ng/mL VEGF influenced the blastocyst rate

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264 D. Biswas et al. / Theriogenology 75 (2011) 256–267

57.73%) and also increased the total cell number151.85) significantly better than the other groups afterVF. VEGF also improved both nuclear and cytoplas-ic maturation of bovine oocytes through cumulus

ells [21,22] at the same concentration. However, an-ther study demonstrated that 300 ng/mL VEGF influ-nced polar body extraction as well as the cleavage andlastocyst rates [10]. These differences may be theesult of different experimental design, although, inoth cases, VEGF increased the blastocyst rate, consis-ent with our results.

The present study revealed that VEGF influences theotal cell number. Increased total cell number enhancedhe implantation initiation process during early embry-genesis. During implantation, trophoblast cells trans-ort nutrients and other molecules, such as growthactors, to the ovum. Human trophoblasts are extremelynvasive, invading the decidua, the inner third of theyometrium, and the maternal arteries. They are also

he source of VEGF, which promotes angiogenesis andasodilation at the implantation site. The expression ofEGF and its functional receptors by trophoblasts sug-ested that VEGF participates in regulating prolifera-ion, migration, and the metabolic activity of the tro-hoblasts in an autocrine fashion [23]. Trophoblastsroduce proteinases that degrade the intracellular ma-rix to form an implantation cavity. They also controlaternal endocrine functions by producing human cho-

ionic gonadotropin (hCG). hCG “informs” the corpusuteum that fertilization has occurred. Trophoblasts in-erfere with the maternal immune system; the placentavoids the maternal immune system and the tropho-lasts secrete substances that alter the normal maternalmmune responses.

Because porcine pre-implantation embryos weresually cultured for 7 days in the same medium as onhe day of IVF, the day of activation was considereday 0. During its course, the zygote travels through theeproductive tract and secretions from the reproductiveract differ according to the reproductive stage andeproductive region. This indicates that differentrowth hormones and cytokines are essential for propermbryonic development at different embryonic stages.mong these growth factors, VEGF is expressed in theuman endometrium throughout the menstrual cycle,ith a 3-6-fold increase during the secretory phase,

ompared with the proliferative phase [18]. Moreover,EGF mRNA expression in the human oviduct wasighest in the preovulatory stage [7] when oviductalecretion is higher [24]. Furthermore, VEGF and KDR

RNA were significantly higher in the infundibular r

nd ampullar regions than in the isthmus region, sug-esting that they play important roles in the early em-ryotrophic events that occur predomimantly in thempulla during the preovulatory phase [7,25].

We demonstrated for the first time that when theorcine IVF and SCNT embryos were cultured withEGF during days 1–7 or days 1–3, the cleavage rates

ncreased slightly and the embryo developmental ratesncreased when both embryo types were cultured withEGF at the late stage (days 4–7) versus the early stage

days 1–3) or the control group. The increased cleavageates might be due to its cytoprotective effects in pre-mplantation embryos [26] when they are cultured withEGF at the early stage. The total cell number in-

reased compared with the control and the early stagesn IVF embryos, but not in SCNT embryos. It wasnclear why the total cell number did not increase inCNT embryos. VEGF is a strong mitogen for vascularndothelial cells derived from arteries, veins, and theymphatics [27], and it also significantly influences theell number for blastocysts from days 7 to 9 in bovinembryos after IVF [10]. The SCNT procedure changeshe total amount of RNA and the mRNA expressionatterns in cloned bovine embryos [28]. The zygoticene activation occurs at the 4- to 8-cell stage in humannd pig preimplantation embryos [29,30]. VEGF maylay an important role in zygotic gene activation fromhe 8-cell stage onwards, because cell cycle and zygoticene activation are crucial for embryo development.lthough embryo development depends on the storagef maternal RNAs and proteins synthesized during oo-enesis, zygotic transcription is an essential event thatccurs at a species-specific time after fertilization. Inhe absence of zygotic transcription, the embryo diesince it no longer supports the requirements for suc-essful development [31]. The present study suggestedhat, VEGF supplementation (5 ng/mL) in IVC mediumt the later stage of embryo development has a benefi-ial effect, because, at this stage, the embryo is moreusceptible to apoptosis.

VEGF mRNA expression has been detected inouse [32] and human blastocysts [33] while VEGF

rotein was detected in human preimplantation em-ryos at different stages by immunofluorescence [9].owever, very few studies have examined VEGFRNA expression in porcine oocytes at different de-

elopmental stages. In the present study, we detected,or the first time, VEGF mRNA in mature and fusedocytes, 2-, 4-, and 8-cell embryos, morulae, and blas-ocysts derived from IVF and SCNT embryos using

eal-time PCR. In cultured human embryos, VEGF se-

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265D. Biswas et al. / Theriogenology 75 (2011) 256–267

retion was increased at the blastocyst stage comparedith embryos at the pronuclear stage [34], althoughEGF mRNA expression did not significantly differ

rom the 8- to the 16-cell stage to the hatched blastocyst35]. However, in our study, VEGF mRNA expressioniffered with the developmental stage in both IVF andCNT embryos. Thus, the detected VEGF mRNA trulyeflects embryonic synthesis and is not a carryover fromhe previously transcribed maternal RNA. Moreover,he switch from maternal to embryonic transcriptionppeared to occur at the 4- to 8-cell stage in porcinembryos [36]. In IVF embryos, VEGF mRNA in-reased significantly at the third cell cycle, significantlyecreased at the 8-cell stage (4th cell cycle), increasedgain at the morula stage and remained constant up tohe blastocyst stage. Unlike IVF embryos, in SCNTmbryos, VEGF mRNA expression significantly in-reased at the 4-cell stage, remained constant up to theorula stage, and then significantly increased again at

he blastocyst stage. Over-expression of VEGF mRNAt the blastocyst stage in SCNT embryos may lead toberrant valvulogenesis as observed in cloned hearts,nd congestion and hemorrhage, as observed in someloned hearts, brains, and spleens. Aberrant VEGF ex-ression may contribute to these defects, because thelone died soon after birth [37]. Moreover, this aberrantEGF expression might explain the abnormal vascular

ormation in the post-implantation period, leading toremature birth or abortion.

In the present study, we detected KDR and flt-1EGF receptors first time in porcine preimplantation

mbryos derived from IVF and SCNT from one cell tolastocyst stages. Although, the receptors proteins,DR, flt-1 and VEGFR-3 were indentified recently inouse in vivo fertilized embryos by indirect immuno-

taining techniques and the detection level was veryow [38]. Biological activities of VEGF in both in vivond in vitro studies appear to be mediated exclusivelyy the flk/KDR receptors [39,40,41]. KDR receptor isritical and essential for vasculogenesis and angiogen-sis during embryo development [42,43] but the activ-ties also mediated by flt-1 receptor [44]. However,mbryonic development with normal angiogenesis wasccurred without flt-1 tyrosine kinase domain [45]. Ourtudy was only dealt with embryo development beforemplantation. VEGF/VEGFR system is involved in theormation of monocytes, retinal progenitor cells, all ofhich are non-vascular endothelial cells [46,47]. In theresent study VEGF significantly influenced the em-ryo developmental potential when VEGF was added

ither in whole culture period (day 1–7) or late culture

eriod (day 4–7) indicated that the activity waschieved by VEGF receptors.

In conclusion, VEGF supplementation at the latertage of embryonic development improved the qualitynd developmental ability of porcine IVF embryos inerms of the blastocyst rate and the blastocyst cellumber. Moreover, it improved the embryo transferechnique and may potentially be applied as a model oftem cell self-renewal.

cknowledgments

This work was supported by a grant (#0070301034040) from the BioGreen 21 Program, Ru-al Development Administration, Republic of Korea.

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