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Theriogenology 74 (2010) 1149–1160

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Sonic Hedgehog improves in vitro development of porcineparthenotes and handmade cloned embryos

Ngoc Tan Nguyena, David Pei-Cheng Linb, Chawalit Siriboona, Neng-Wen Loc,Jyh-Cherng Jua,*

a Department of Animal Science, National Chung Hsing University, 250 Kuokuang Road, Taichung 402, Taiwan, ROCb School of Medical Laboratory and Biotechnology, Chung Shan Medical University, 110 Chien-Kuo North Road, Taichung 402,

Taiwan, ROCc Department of Animal Science and Biotechnology, Tunghai University, 181 Sec. 3 Taichung Harbor Rd., Taichung 407, Taiwan, ROC

Received 22 December 2009; received in revised form 27 April 2010; accepted 13 May 2010

bstract

This study investigated the expression of Sonic Hedgehog (Shh) signaling pathway and its effect on porcine parthenogeneticPA) embryo development. The Shh receptor Patched (Ptc1) and co-receptor Smoothened (Smo) were expressed at various stagesf PA porcine embryos, at both mRNA and protein levels. Furthermore, the transcriptional activator Gli1 mRNA was first presentn the 2-cell stage embryos, and was readily detected at the 4-cell stage and beyond. Culture medium supplemented with 0.5g/mL Shh optimized blastocyst rates (58.6 vs. 41.1%; P � 0.05) and the total number of cells per blastocyst (56.4 vs. 45.6 cells;� 0.05); however, this response was prevented by simultaneous addition of 1 mM cyclopamine (an Shh inhibitor). Moreover,

lastocysts that developed in medium containing 0.5 �g/mL Shh had lower apoptotic indices and reduced DNA damage (evaluatedy TUNEL and comet assays, respectively). Based on Western-blot analysis, expression of phosphorylated Akt protein inhh-treated blastocysts was higher than that of the control group (1.22- vs. 0.66-fold, P � 0.05), and less total PARP-1/2 proteinas accumulated (0.7-fold, P � 0.05) in treated blastocysts compared to untreated controls. Furthermore, supplementation of Shh

1 �g/mL) also supported development of handmade cloned embryos (50.3 vs. 26.8%; P � 0.05) with reduced apoptotic rates (2.8s. 6.3%; P � 0.05). We inferred that the Shh signaling pathway existed in porcine PA embryos and we concluded that Shhupplementation improved the quality and developmental competence of early PA embryos, at least in part, by increasing cellroliferation and reducing apoptosis of the developing embryos.

2010 Elsevier Inc. All rights reserved.

eywords: Apoptosis; Comet assay; Embryo development; Parthenogenesis; Shh

www.theriojournal.com

eicmamic

. Introduction

In vitro production (IVP) of animal embryos, inombination with other assisted reproductive technolo-ies, has profoundly affected livestock production [1]nd hastened genetic improvement [2]. Although IVP

* Corresponding author. fax: 886-4-2286-0265.

oE-mail address: jcju@dragon.nchu.edu.tw (J.-C. Ju).

093-691X/$ – see front matter © 2010 Elsevier Inc. All rights reserved.oi:10.1016/j.theriogenology.2010.05.016

mbryos have also been intensively used to study pre-mplantation embryo development, the developmentalompetence of IVP porcine embryos remains subopti-al due to poor embryo quality, i.e., low cell number

nd high apoptotic rates [3,4]. It has been proposed thatodification of oocyte maturation and activation/fertil-

zation protocols, as well as the embryo culture system,ould improve IVP efficacy and full-term development

f embryos. For example, embryo culture media sup-

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1150 N.T. Nguyen et al. / Theriogenology 74 (2010) 1149–1160

lemented with growth factors, cytokines, vitamins,nd/or amino acids improved preimplantation embryoevelopment in vitro [5–7].

Hedgehog (Hh) protein is a paracrine factor whichnhanced cell proliferation and differentiation in manyell types [8,9]. Its role in embryo development hasttracted considerable attention over the past decade10,11]. In vertebrates, there are at least three Hh mem-ers, namely, Sonic Hh (Shh), Indian Hh (Ihh), andesert Hh (Dhh) [12–14], among which Shh has been

he most extensively studied. Its signaling pathway isediated through a cell surface receptor system con-

isting of two proteins, the receptor Patched (Ptc) andts co-receptor Smoothened (Smo). In the absence ofh ligand, Ptc suppressed Smo, which in turn inhibitedownstream signaling cascades [15,16]. In contrast, inhe presence of Hh, suppression of Smo was lifted toelay its signaling, and the apparent outcome was thectivation of its downstream transcription factors Gli1,li2, and Gli3 in vertebrates [12,14,17].The roles of Hh signaling during oogenesis have

een examined in murine and porcine ovaries [14,18].n mice, Ihh and Dhh were expressed in granulosa cellsf preantral and antral follicles, and their receptors Ptcnd Smo were detected in theca cells. Consequently,aracrine communication through Hh signaling mayxist between the granulosa cells and the theca cells12,17]. Similarly, Russell et al. [14] reported that Hhigands, including Ihh, Dhh, and Shh, were all ex-ressed in immature and adult mouse ovaries, wheretc (Ptc1, Ptc2) and Smo were also found. In addition,hh had a crucial role in embryo implantation during theeri-implantation period in mice [12,19,20]. Most in-erestingly, we recently reported that Shh and its down-tream molecules Ptc, Smo, and Gli1 were present inorcine ovaries and oocytes [18]. Exogenous Shh sup-lementation in IVM medium promoted nuclear andytoplasmic maturation of oocytes and subsequent em-ryo development. These findings provided the impetuso examine the existence of Shh downstream signalingolecules during early embryogenesis, and the influ-

nce of Shh on embryo development. Aside from theforementioned studies, to our knowledge, there are nourther reports regarding the role of Hh signaling inevelopment of mammalian preimplantation embryos.

The aims of this study were to investigate the effectsf Shh supplementation in the culture medium on theevelopmental competence of porcine parthenotes andandmade cloned embryos, as well as the embryonic

xpression of Shh signaling molecules in parthenotes. b

. Materials and methods

.1. Chemicals and reagents

All chemicals and reagents used in this study wereurchased from the Sigma-Aldrich (St. Louis, MO,SA), unless otherwise stated.

.2. Oocyte recovery and in vitro maturation (IVM)

Ovaries were obtained from a local abbatoir, main-ained at 35 °C in physiological saline containing pen-cillin (600 IU/mL), and transported to the laboratoryithin 2 h. Cumulus-oocyte-complexes (COCs) were

spirated from medium-sized follicles (3–7 mm in di-meter) with an 18 gauge needle attached to a 10 mLisposable syringe. Only COCs with at least two layersf cumulus cells and homogeneous ooplasm were se-ected; they were rinsed three times in maturation me-ium (North Carolina State University, NCSU-23).wenty to 30 oocytes were randomly allocated to each00 �L droplet of NCSU-23 medium, covered by min-ral oil, and cultured at 39 °C in an incubator contain-ng 5% CO2. For the first 22 h, COCs were cultured inhe NCSU-23 medium supplemented with 10% porcineollicular fluid, cysteine (0.1 mg/mL), eCG (10 IU/mL),nd hCG (10 IU/mL). Thereafter, COCs were placed inormone-free NCSU-23 medium and cultured for andditional 22 h [18].

.3. In vitro embryo culture

.3.1. Parthenogenetically activated embryosAfter 44 h of IVM culture, cumulus cells were re-

oved by gentle pipetting in Dulbecco’s PBS (DPBS)ontaining 0.1% hyaluronidase, and oocytes were thenashed twice in activation medium (0.28 M mannitol,.05 mM Hepes, 0.1 mM CaCl2.2H2O, 0.1 mM MgCl2,nd 0.01% polyvinyl alcohol). For electro-activation,n electrical pulse (2.16 kv/cm, 30 �s) generated by aTX Electro-Cell Manipulator 200 (BTX, San Diego,A, USA) was applied. After being washed twice in.5 mM 6-dimethylaminopurine (6-DMAP), activatedocytes were transferred to porcine zygote mediumPZM-3) containing 6-DMAP and cultured for 4 h,ollowed by intensive washings (four times) withZM-3 medium. Then, activated oocytes were ran-omly allocated to PZM-3 medium containing variousoncentrations of Shh and cultured for 7 d to evaluateheir developmental competence [21].

.3.2. Handmade cloned (HMC) embryos

.3.2.1. Preparation of somatic donor cells. Ear fibro-

last cells were derived from a one-year-old miniature

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1151N.T. Nguyen et al. / Theriogenology 74 (2010) 1149–1160

ilt. After four to six passages in culture, cells wererown to confluence in a petri dish for nucleus trans-lantation [22].

.3.2.2. Chemically assisted enucleation. Enucleationf matured oocytes was performed as described [23],ith minor modifications. After 39 to 40 h of IVM,ocytes were deprived of cumulus cells and only MIIocytes were further cultured for 45 min in maturationedium supplemented with 0.4 �g/mL demecolcine.ubsequently, oocytes were incubated in 3.3 mg/mLronase in TCM-199 medium supplemented with 2%etal bovine serum (FBS) for zona pellucida (ZP) re-oval. Once partial lyses of the ZP and slight defor-ation of the ooplasm was apparent, oocytes wereashed quickly in Hepes-buffered TCM 199 medium

upplemented with 10% FBS (T10). Thereafter, all ma-ipulations were performed on a heated plate (39 °C)nless otherwise indicated. All medium droplets (20L each) for handling oocytes were covered with min-ral oil. Briefly, eight zona-free oocytes with ooplasmicrotrusion were aligned in one drop of Hepes-bufferedCM-199 medium supplemented with 20% FBS (T20)nd 5 �g/mL cytochalasin B (40 �L per drop). Maturedcytes were rotated with a fire-polished glass pipette toocate the ooplasmic protrusion in the petri dish, andriented bisection was performed with an ultra-sharpplitting blade (ESE020, Bioniche Animal Health USA,nc., Georgia, USA) under a stereomicroscope. Lesshan half of the ooplasm on the side with ooplasmicrotrusion was removed from the remaining ooplasm.fter bisection, the larger ooplasts were collected,ashed twice in T10 medium, and then placed into T10rops for fusion with somatic donor cells.

.3.2.3. Cell fusion and activation. Cell fusion waserformed in two steps, as described by Li et al. [23].irst, approximately 100 somatic cells were placed in a10 droplet, and 20 to 30 cytoplasts were placed into

he other droplet. After a short period of equilibration,roups of five ooplasts were transferred to a dropletontaining 1 mg/mL phytohemaglutinin (PHA), andfter approximately 4 to 5 s, each ooplast was quicklyttached to one somatic cell to form an ooplast-somaticell pair, before being transferred to fusion medium0.28 M mannitol and 0.01% polyvinyl alcohol). Fusionas conducted by first applying an AC (0.6 KV/cm) for

lignment, followed by a single DC (2.0 KV/cm, for 9s) for fusion. After fusion, ooplast-somatic cell pairsere carefully transferred to a T10 droplet, followed by

ncubation for 1 h. Fused pairs and the remaining cy-

oplasts were then equilibrated in activation medium. T

or the second step, an identical AC pulse was used tolign the fused pair with an additional bisected ooplast,nd then a DC pulse (0.80 KV/cm, for 90 �s) waspplied for the second fusion. The fused reconstructedocytes were harvested and transferred to PZM-3 me-ium containing 6-DMAP (2.5 mM) for completion ofctivation.

.3.2.4. Embryo culture. After incubation with 6-DMAPor 4 h, parthenogenetically activated oocytes werentensively washed with PZM-3 medium and culturedn 100 �L of PZM-3 droplets (20–30 embryos/drop)ontaining 3 mg/mL BSA. In addition, five or six HMCmbryos were cultured in PZM-3 medium containing 5g/mL BSA, based on the WOW system (five or six

mbryos in 20 �L droplets of PZM-3 medium) coveredith mineral oil for 7 d at 39 °C in 5% CO2, 5% O2, and0% N2 [24]. In both systems, various amounts of Shhere added to the PZM-3 medium, in accordance with

he experimental design.

.4. Apoptosis detection by terminal deoxynucleotidylransferase-mediated d-UTP nick end-labelingTUNEL) assay

Embryos were harvested on Day 7 (day of activation �ay 1), washed three times in DPBS supplemented with.1% polyvinyl alcohol (PVA), and then fixed overnightn 4% (v/v) paraformaldehyde at 4 °C, and then incubatedith 0.1% Triton X-100 and 0.1% citrate in DPBS for 1 h

to ensure membrane permeation). Thereafter, fixed em-ryos were incubated in TUNEL reaction medium (In Situell Death Detection Kit, TMR red; Roche, Mannheim,ermany) for another 1 h at 38.5 °C in the dark, and thenashed and stained with Hoechst (10 �g/mL) for 10 min.fter two washes in DPBS/PVA, embryos were mountedn slides with DAKO fluorescent mounting mediumS3023, Dako North America, Inc., California, USA) forbservation (magnification at 20�) under an epifluores-ence microscope (Nikon, Tokyo, Japan). The numbers ofpoptotic nuclei and total numbers of nuclei were deter-ined. The apoptotic index was calculated as follows:poptotic index � (no. TUNEL-positive nuclei/total no.uclei) � 100.

.5. Comet assay

Evaluation of DNA damage was performed byomet assay, as described [25], with minor modifica-ions for whole-embryo analysis. Embryos were col-ected at Day 7 after in vitro culture and incubated in.5% pronase for 20 s to remove the zona pellucida.

hen, they were washed in DPBS supplemented with

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1152 N.T. Nguyen et al. / Theriogenology 74 (2010) 1149–1160

.1% polyvinyl alcohol. A 100 �L droplet of 1% (inPBS) low melting point (LMP) grade agarose waslaced onto the comet slide, covering a layer of 1%w/v in DPBS) normal agarose that had been previouslyoated on the slide. Zona-free embryos were added tohe droplet of LMP agarose and a coverslip was placedn the droplet, spreading the LMP into a thin layer. Thelide was kept on ice for 30 s to allow the LMP agaroseo solidify and then immediately immersed in lysisuffer (2.5 M NaCl, 1 mM EDTA, 10 mM Tris, 10%imethylsulphoxide, and 1% Triton X-100; pH 10) forh at 4 °C. After incubation, the slide was immersed in

lectrophoresis buffer (TAE) for 20 min at 4 °C, fol-owed by electrophoresis at 60 V and 200 mA for 20in. Then, the slide was dipped in 70% ethanol for 5in, dried at room temperature, stained with Hoechst

or 30 min in the dark, and observed under an epiflu-rescence microscope (magnification at 20�) for imagenalysis. The intensity and length of migrated DNAcomet tail) were measured using ImageJ softwareVersion 1.42, National Institutes of Health, Bethesda,

D, USA).

.6. One-step reverse transcription-polymerase chaineaction (RT-PCR)

Embryos at 2-cell, 4- to 8-cell, morula, and blasto-yst stages (n � 160, 120, 80, and 40, respectively)ere snap frozen in liquid nitrogen and stored at �80 °Cending analysis. Total RNAs were isolated using anNA extraction solution (Bio-Mi kit, Bio-Mi, Taiwan)ccording to the manufacturer’s instructions. The ex-racted RNA was analyzed using a semi-quantitativene-step RT-PCR (Qiagen) procedure, as described26], with minor modifications. To detect the transcriptsf Shh signaling molecules including Ptc, Smo, andli1, the total RNA was isolated from each sample andissolved in 20 �L of RNase-free water. For detection

able 1rimer sequences, annealing temperatures, and expected product siz

arget gene Primer Sequences (5= to 3=)

tc1 Forward TAACCATGCTGCGCTGGGReverse TCCATGCTGAGAATTGCA

mo Forward GGAGGAAGAGGAAGAAGReverse AAGTCCGAGTCTGCATCC

li1 Forward CAACACTCAGCTGGACTTReverse AGGCACTAGAGTTGAGGA

apdh Forward GGAGCCAAACGGGTCATCReverse GAGGGGCCATCCACAGTC

f gene expressions, 5 �L (100 ng) of total RNAs was P

sed for each analysis. Total RNAs (100 ng) extractedrom gilt ovarian tissue were used as a positive control.

The thermo-profile for amplification was as follows:everse transcription at 50 °C for 30 min, initial PCRctivation at 95 °C for 15 min, denaturation at 94 °C formin, annealing for 1 min at 55 to 63 °C (depending on

he primer pair used; Table 1), extension at 72 °C for 1in, final extension at 72 °C for 10 min. Overall, 40

ycles were applied. The Hh signaling transcripts (Ptc,mo, Gli1) and GAPDH are shown (Table 1). Since noequence information on Shh signaling molecules ofus scrofa was available in Genbank, primers for Ptc1,mo and Gli1 gene amplification were derived by se-uence alignment between humans and mice (Mus mus-ulus). Consensus sequences were determined ashown (Table 1).

.7. Western blotting

Western-blot analysis was performed as describedreviously [18]. In brief, blastocysts on Day 7 (30lastocysts per treatment for Akt and 15 blastocysts foroly(ADP-ribose) polymerases-1 and �2 [PARP-1/2]etection) were collected and stored at �80 °C inample buffer (100 mM Tris-HCl [pH 6.8], 200 mM-mercaptoethanol, 0.4% sodium dodecylsulfate [SDS],.002% bromophenol blue, 20% glycerol) until use. Toetect receptor Ptc1 and its co-receptor Smo, embryost the 2-cell, 4 to 8-cell, morula, and blastocyst stagesn � 160, 120, 80, and 40, respectively) were used. Forlectrophoresis, samples were boiled for 5 min, cooledn ice, and then loaded on 10% SDS-PAGE gels. Pro-eins were transferred from the gel to nitrocelluloseembranes (catalogue no. HAHY0010, Millipore, Bil-

erica, Ireland) and blocked for 1 h in TBST buffer (20M Tris-HCl, 500 mM NaCl, 0.1% Tween 20) contain-

ng 5% chicken serum. Membranes were then incubatedith primary antibody (at 4 °C for 6 h) for detection of

ne-step RT-PCR analysis of porcine embryos.

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1153N.T. Nguyen et al. / Theriogenology 74 (2010) 1149–1160

echnology Inc., Santa Cruz, California, USA), phospho-kt (1:500; catalogue no. 9271 [Serine 473]; Cell Signal-

ng Technology Inc., Beverly, MA, USA), Patched1:750; sc-13943; Santa Cruz Biotechnology Inc.),moothened (1:750; sc-9016; Santa Cruz) or �-actin (1:000, catalogue no. 4967; Cell Signaling Technologync.). After incubation, membranes were washed fiveimes (10 min each), and then incubated with the second-ry antibody (1:10000; horseradish peroxidase-labelednti-rabbit or anti-mouse immunoglobulin, as appropri-te; Cell Signaling Technology Inc.) for 1 h at roomemperature. After washing three times (5 min each),roteins were detected by the Super®Signal West Picohemiluminescent Subs Kit (Pierce Biotechnology,

nc., Rockford, IL, USA) and visualized on X-ray film.or total Akt protein detection, membranes wereashed in stripping buffer (100 mM �-mercaptoetha-ol, 20% SDS, and 62.5 mM Tris pH 6.7) at 50 °C for5 min to strip off bound antibodies after chemilumi-escent detection. The membrane was reprobed withrimary antibody for total Akt (1:500; catalogue no.272; Cell Signaling Technology Inc.), followed by theforementioned procedures for signal visualization.and intensities, e.g. phosphorylated Akt and PARP-/2, were measured using ImageJ software, and nor-alized to total Akt and �-actin, respectively, before

tatistical analysis.

.8. Specific experiments

.8.1. Experiment 1: Effect of Shh-supplementededium on the development of porcinearthenogenetic embryos

.8.1.1. Dose-response effect of Shh. After 44 h ofVM, matured oocytes were selected for parthenoge-etic activation. Following electrical pulses, activatedocytes were further incubated in PZM-3 medium con-aining 2.5 �M 6-DMAP for 4 h. After four washes inZM-3 medium, activated oocytes were cultured inZM-3 medium supplemented with various concentra-

ions of Shh (0, 0.25, 0.5, 1, and 2 �g/mL; Shh: 461-H, R&D Systems, Inc., Minneapolis, MN, USA) ford to evaluate developmental competence. Cleavage

nd blastocyst rates were recorded on Days 2 and 7,espectively (Day 1 � day of activation). The total cellumber of each Day-7 blastocyst was determined byoechst staining.

.8.1.2. The effects of Shh inhibitor cyclopamine. Toonfirm whether Shh specifically affects embryo devel-pment, activated oocytes were cultured in PZM-3 me-

ium in the presence of Shh, cyclopamine (GR-344, e

iomol, Farmingdale, NY, USA) or both. Activatedocytes were randomly allocated to one of the follow-ng five treatment groups: Control: without Shh; Shh,.5 �g/mL Shh; SCyclo1, 0.5 �g/mL Shh plus 0.5M/mL cyclopamine; SCyclo2, 0.5 �g/mL Shh plus 1M/mL cyclopamine; and Cyclo, 1 �M/mL cyclopam-

ne only. Cleavage and blastocyst rates and total cellumber were determined as described previously.

.8.2. Experiment 2: Detection of Shh signalingranscripts in porcine parthenogenetic embryos

Initially, the expression of Shh signaling transcriptsPtc, Smo, and Gli1) was determined at the mRNAevel in parthenogenetic embryos. Embryos at 2-cell, 4-o 8-cell, morula, and blastocyst stages (n � 160, 120,0, and 40, respectively) were collected for RT-PCRnalysis, and gilt ovarian tissue was used as a positiveontrol. The total RNA extracted from each sample wasissolved in 20 �L of RNase-free water; thereafter, 5L of the resulting solution was used as the template

or each run of the one-step RT-PCR for each gene. Toonfirm the expression of Ptc1 and Smo at the proteinevel, the number of embryos subjected to Western-blotnalysis was equal to that used for RT-PCR.

.8.3. Experiment 3: Apoptosis of parthenogeneticlastocysts derived from Shh-supplemented cultureedium

.8.3.1. TUNEL assay. Similar to Experiment 1, Day-7lastocysts were subjected to the TUNEL assay, asescribed previously.

.8.3.2. Expression of survival-related proteins. Par-henogenetically activated embryos were cultured in theresence or absence of 0.5 �g/mL Shh, and collected athe blastocyst stage (Day 7). All embryos were stored at

80 °C pending Western blotting analysis, as previ-usly described. Shh was added to the culture medium,nd levels of p-Akt (Serine 473), including total Akt asell as PARP-1/2 protein (full length, downstream of

ctivated caspase-3 protein) were determined.

.8.4. Experiment 4: Evaluation of DNA damage inhe blastomere of parthenogenetic blastocystsupplemented with Shh

This experiment was designed to evaluate the effectf Shh on the reduction of DNA damage. Day-7 blas-ocysts harvested from culture medium, without or with.5 �g/mL Shh, were subjected to a comet assay. Thentensity of each comet tail was measured within aonstant area of a rectangle. The length of the cometail was measured from the putative middle line of the

mbryo to the tip of the tail. These measurements were

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1154 N.T. Nguyen et al. / Theriogenology 74 (2010) 1149–1160

sed as the indicator for the extent of DNA damagencurred by each embryo.

.8.5. Experiment 5: Effect of Shh-supplementededium on development of HMC embryosThis experiment was designed to confirm the effect

f Shh on embryo development using HMC embryos,hich is considered a harsh test for the Shh-supple-ented culture system used. Cloned embryos were re-

onstructed with ear skin fibroblasts and cultured ford in PZM-3 medium supplemented with various con-

entrations of Shh (0, 0.5, 1, or 2 �g/mL). Similar tohat in the parthenogenetically activated embryos, ratesf cleavage and blastocyst formation were determinedn Days 2 and 7, respectively, after activation treat-ent. Blastocyst embryos collected on Day 7 were

valuated with a TUNEL assay to determine total cellumbers and apoptotic indices.

.9. Statistical analysis

All data were subjected to ANOVA, using the Gen-ral Linear Model (GLM) procedure in SAS Version 9SAS Institute, Cary NC, USA), followed by Tukey’s test.ercentile data were arcsine-transformed before ANOVAnalysis, and P � 0.05 was considered significant.

. Results

.1. Experiment 1: Effect of Shh-supplementededium on development of porcine parthenogenetic

mbryos

.1.1. Dose-dependent response of ShhTo optimize the concentration of Shh, activated oo-

ytes were cultured in the IVC medium containing 0,.25, 0.5, 1, or 2 �g/mL Shh. Rates of cleavage andlastocyst formation, and the total cell number perlastocyst after Shh treatments are summarized (Table). Cleavage rate of porcine parthenotes was not sig-ificantly different between the control and the Shh-

able 2ffects of Shh supplementation in the embryo culture medium on po

eplicates).

hh (�g/mL) Total no. Cleavage rate, %

134 93.5 � 1.7 (125).25 134 95.4 � 0.9 (128).5 134 95.7 � 1.3 (128)

134 94.9 � 1.8 (127)134 93.4 � 2.2 (125)

umbers in the parenthesis represent the actual number of embryos

–c Within a column, means without a common superscript differ (P � 0.05

reated groups. However, blastocyst rates were in-reased in the presence of 0.25, 0.5, and 1 �g/mL Shh48.4 � 2.3, 58.6 � 3.4, and 49.6 � 2.8%, respec-ively) with the greatest blastocyst rate and total cellumber (56.4 � 1.7) at 0.5 �g/mL. Embryos culturedn the medium containing the highest concentration (2g/mL) did not have improved development (40.4 �.1 vs. 41.1 � 1.4% in the control group). Similarly, inhe presence of 0.25 or 1 �g/mL Shh, total cell numberser blastocyst (51.5 � 2.1 and 50.7 � 1.8, respectively)ere not significantly different from the control group

45.6 � 2.0). By arbitrarily splitting the data of totalell number into three groups (�40, 40–60, and �60ells), a significantly greater proportion (41.3%) oflastocysts in the 0.5 �g/mL Shh group had total cellounts �60, compared to those in the control (23.8%)nd the 2 �g/mL Shh (18.9%) groups (Fig. 1). Moreover,nly 10.8 and 15.3% of blastocysts in the presence of 0.5nd 1 �g/mL Shh, respectively, had cell counts �40, inontrast to 38.6% in the control group and 51.8% in theroup supplemented with 2 �g/mL Shh.

.1.2. Effect of the Shh inhibitor cyclopamine onarthenogenetic embryo development

To further confirm the favorable effect of Shh onreimplantation embryo development, cyclopamineas used to counteract Shh (0.5 �g/mL) during devel-pment of parthenogenetically activated oocytes (Table). Consistent with the dose-response trial, activatedocytes cultured in the presence of Shh had greaterlastocyst rates and total cell numbers than those inontrol groups (57.5 � 1.2% and 51.5 � 1.4 cells vs.9.9 � 1.8% and 41.5 � 1.7 cells, respectively). Treat-ent of embryos with cyclopamine alone had no sig-

ificant effect on blastocyst rate or total cell number39.2 � 2.1% and 42.0 � 2.0 cells). Although activatedocytes cultured in the presence of 0.5 �g/mL Shh plus.5 �M cyclopamine (SCyclo1 group) appeared to haveo significant effect on these two end points, to some

arthenogenetic embryo development (mean � SEM from five

Blastocyst rate, % (n) Cell no./blastocyst (n)

41.1 � 1.4a (55) 45.6 � 2.0a,b (54)48.4 � 2.3b (65) 51.5 � 2.1b,c (63)58.6 � 3.4c (79) 56.4 � 1.7c (76)49.6 � 2.8b (67) 50.7 � 1.8a,b (65)40.4 � 2.1a (54) 44.3 � 2.5a (49)

tocysts observed.

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1155N.T. Nguyen et al. / Theriogenology 74 (2010) 1149–1160

xtent, the Shh-induced effects on blastocyst formationnd the total cell number were nullified (42.3 � 1.0% and2.4 � 1.4 cells vs. 57.5 � 1.2% and 51.5 � 1.4 cells) athigher concentration of cyclopamine (SCyclo2 group).

.2. Experiment 2: Expression of Shh signalingranscripts in porcine parthenogenetic embryos

The mRNAs of both Shh downstream molecules,tc and Smo, were consistently detected in partheno-enetic embryos, regardless of development stage (Fig.a–c). Initially, Gli1 mRNA was barely detected at the-cell stage, became evident in 4- to 8-cell embryos,nd was prominent at the morula and blastocyst stages.urthermore, GAPDH was amplified to confirm theresence of RNA in each sample (Fig. 2d).

Expressions of Ptc1 and Smo at the protein levelere also confirmed with Western-blots. Both the re-

eptor (Ptc1) and its co-receptor (Smo) were detected atarious stages of embryonic development (Fig. 3;anes 1 to 4 of the upper and the middle panels,

ig. 1. Distribution of the total cell number per parthenogenetically d0.5, 1, and � 2 �g/mL) in PZM-3 medium for 7 d.

–cWithin each cell number category, bars without a common supers

able 3ffects of Shh, and its inhibitor cyclopamine, on development of po

mean � SEM from five replicates).

reatment Total no. Cleavage rate, % (n

ontrol 135 91.9 � 2.0 (124)hh 135 93.4 � 2.6 (126)Cyclo1 135 93.2 � 2.7 (126)Cyclo2 135 91.1 � 1.7 (123)yclo 135 90.2 � 2.5 (122)

he number in the parenthesis represents the actual number of embra,b Within a column, means without a common superscript differ (P

Shh plus 0.5 �M/mL cyclopamine; SCyclo2, 0.5 �g/mL Shh plus 1 �M

espectively; Lane 5 was ovarian tissue, the positiveontrol). However, the expression of Smo was barelyetected at the 2-cell stage. The lower panel was �-ac-in as the loading control.

.3. Experiment 3: Apoptosis of parthenogeneticlastocysts derived from Shh-supplemented cultureedium

Based on a TUNEL assay, embryos derived from thehh-supplemented groups at concentrations 0.25, 0.5, 1.0,nd 2.0 �g/mL yielded apoptotic indices of 5.1, 3.8, 4.7,nd 4.9%, respectively; only embryos derived fromroups exposed to 0.5 and 1 �g/mL Shh had lower indiceshan the control group (7.5%). However, there were noignificant differences among Shh-treated groups.

Expressions of p-Akt and PARP-1/2 protein levelsere determined at various developmental stages (Fig.). The expression of phosphorylated Akt was 0.62-foldf the total protein in the control and 1.22-fold in thehh-treated group (P � 0.05; Fig. 4a–b). The PARP-

porcine blastocyst cultured with various doses of Shh (e 0, 0.25,

iffer (P � 0.05).

arthenogenetic embryos

Blastocyst rate, % (n) Cell no./blastocyst (n)

39.9 � 1.8a (54) 41.5 � 1.7a (48)57.5 � 1.2b (77) 51.5 � 1.4b (65)53.4 � 1.1b (72) 49.3 � 1.2b (56)42.3 � 1.0a (57) 42.4 � 1.4a (48)39.2 � 2.1a (53) 42.0 � 2.0a (44)

stocysts observed.). Control: without Shh; Shh, 0.5 �g/mL Shh; SCyclo1, 0.5 �g/mL

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1156 N.T. Nguyen et al. / Theriogenology 74 (2010) 1149–1160

/2 protein expression in the blastocyst was also de-reased to 0.7-fold in the presence of Shh, as comparedo that in the control group (Fig. 4c–d).

.4. Experiment 4: Shh supplementation reducedNA damage in porcine parthenogenetic blastocysts

The results of comet assay for the whole embryoere expressed as the means of relative intensity and

ength of comet tail from each blastocyst (Table 4). Theelative intensity of the comet tail of migrated DNA frag-ents from blastocysts in the control group (975.35 �

6.69) was higher than that in the Shh-treated group741.41 � 56.0). Similarly, the comet tail in the controlroup (10.35 � 0.47) was longer than that in the Shh-reated group (6.73 � 0.31).

.5. Experiment 5: Effects of Shh on development ofMC embryos

The developmental competence of the somatic cellloned embryos (i.e., HMC embryos) under the influ-

ig. 2. The Hedgehog signaling transcripts (Ptc1, Smo, Gli1) were exas amplified to confirm the presence of RNAs in the reaction. Repre36 bp; (c) Gli1: 190 bp; and (d) GAPDH: 233 bp. M: 100 bp markerane 4: blastocyst; and Lane 5: gilt ovarian tissue (positive control)

ig. 3. Representative immunoblots for receptor (Ptc1, upper panevelopment of porcine parthenotes. Three replicates were performed

: morula; Lane 4: blastocyst; and Lane 5: gilt ovarian tissue (positive cont

nce of Shh is shown (Table 5). Similar to Experiment, groups supplemented with Shh at 0.5 and 1 �g/mL43.1 � 2.8 and 50.3 � 1.9%) had higher blastocystates than the control group (26.6 � 1.8%; P � 0.05).mbryos supplemented with 1 �g/mL Shh had thereatest total cell number per blastocyst (57.6 � 2.3),elative to the control group and the 2 �g/mL Shhroup (42.4 � 2.2 and 43.0 � 3.3). In contrast to the 2g/mL Shh-treated group (5.5 � 0.7), blastocysts derived

rom 1 �g/mL Shh supplementation had a significantlyower apoptotic index than control embryos (2.8 � 0.6 vs..3 � 0.7).

. Discussion

In recent years, Hh signaling was detected in murine14,17] and bovine ovaries [27]. Specifically, Hh down-tream molecules Ptc1 and Gli1 were primarily ex-ressed in theca cells, whereas Ihh, Dhh, and ShhRNA were predominately detected in granulosa cells;

in the porcine parthenogenetic embryos. For each sample, GAPDHe images, illustrating the size of amplicons: (a) Ptc: 428 bp; (b) Smo:1: 2-celled embryos; Lane 2: 4 to 8-celled embryos; Lane 3: morula;

co-receptor (Smo, middle panel) expression at various stages ofrker; Lane 1: 2-celled embryos; Lane 2: 4 to 8-celled embryos; Lane

pressedsentativ; Lane

el) and. M: ma

rol).

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1157N.T. Nguyen et al. / Theriogenology 74 (2010) 1149–1160

hey stimulated cell proliferation in vitro [14,17]. Rent al. [28] reported that activation of the Hh signalingathway altered theca cell development and preventedvulation. We previously reported that there were Shh-elated signaling molecules in the porcine ovary, andhat adding Shh to the IVM medium promoted nuclearnd cytoplasmic maturation, and subsequent develop-ent of porcine parthenogenetic embryos [18]. In ad-

ition, Ihh played a crucial role in murine embryomplantation during the peri-implantation period12,19,20]. However, there are apparently no reportsegarding the direct role of Shh in preimplantationmbryo development. We previously reported that Shh

ig. 4. Alteration of phosphorylated Akt and PARP-1 protein levels inre given as fold increases over control, after being normalized to totalA) Representative immunoblots of the phosphorylated Akt (upper paf phospho-Akt levels relative to total Akt protein was performed usength PARP-1 (upper panel) with �-actin (45 kDa) as an internal conystem. Lane 1: control (without Shh); Lane 2: with Shh (0.5 �g/mL,b Bars without a common superscript differ (P � 0.05).

able 4igration of DNA (comet assay) for porcine parthenogenetic

mbryos developing in Shh-supplemented culture mediummean � SEM from five replicates).

roup No.blastocystsexamined*

DNA migration

Intensity(pixel � 103)

Length(pixel � 102)

ontrol 49 975.35 � 76.69a 10.35 � 0.47a

hh (0.5 �g/mL) 46 741.41 � 56.0b 6.73 � 0.38b

* Number of blastocysts positive in the comet assay.a,b Within a column, means without a common superscript differ

h(P � 0.05).

nd its related signaling molecules (e.g. Ptc, Smo, andli1), were expressed in porcine ovaries, specifically in

he cumulus-oocyte complex (COC) and oocytes [18].n the present study, based on RT-PCR analysis, ex-ression of Shh signaling transcripts, including Ptc andmo mRNA, were detected in parthenogenetic embryost various stages of development. Expression of Gli1RNA appeared up-regulated as embryonic develop-ent progressed. The inability to detect Gli1 mRNA

ranscripts in the 2-cell stage embryos was interpreteds degradation of maternal transcripts without replace-ent by embryonic transcripts. Since embryonic ge-

ome activation (EGA) occurred at the 4-cell stage inorcine embryos [29], we inferred that Gli1 transcriptsetected after the 4-cell stage were of embryonic origin.t has been generally accepted that the expression ofli1 is an indicator of Hh signaling activation [10].lthough the physiological significance remains un-

lear, we inferred that Shh signaling may have startedefore EGA in porcine parthenotes.

Although chemically defined embryo culture mediare now available for many species, blastocyst-promot-ng agents are still actively investigated [30]. To im-rove the yield and quality of embryos in vitro, variousrowth factors, cytokines, vitamins, and amino acids

e parthenogenetic blastocysts cultured in media containing Shh. Dataotein or �-actin, and presented as mean � SEM from three replicates.d total Akt protein (60 kDa, lower panel). (B) Densitometry analysisImageJ analysis system. (C) Representative immunoblots of the fullwer panel). (D) ensitometry analysis of PARP-1 levels using ImageJmarker.

porcinAkt prnel) aning thetrol (lo); M:

ave been added to culture media with minimal benefit

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1158 N.T. Nguyen et al. / Theriogenology 74 (2010) 1149–1160

6,7,31], particularly in porcine embryos. It is notewor-hy that Shh is regarded as a morphogenic paracrineactor which stimulated proliferation and differentia-ion in a wide variety of cell types and tissues [8,14].everal studies have shown that Shh may also act as aurvival factor in neural cells [32,33]. In our previoustudy, Shh supplementation in maturation media im-roved nuclear and cytoplasmic maturation and subse-uent development of porcine embryos [18]. In theresent study, adding Shh to the IVC medium enhancedlasotcyst rates (Table 2) and embryo quality, confirm-ng that Shh functioned as a promoting agent for pre-mplantation embryo development after parthenoge-etic activation. Furthermore, Shh also promoted theevelopment of cloned HMC embryos to the blastocysttage (Table 4). In both cases, the highest dose of Shhsed (2 �g/mL) was not beneficial to embryo develop-ent, consistent with concentration-dependent effects

f paracrine factors or morphogens [8,9,12,17].Given that a greater total cell number in a blastocyst

epresents better embryo quality or viability [34], theotal cell number is also a critical end point for assess-ng in vitro embryo development. In the present study,dding Shh to the culture media improved the total cellumber in both parthenogenetic and HMC porcine em-ryos. This effect could be ascribed to the pro-mito-enic activity of Shh, as reported in mouse granulosaells [14], adult muscle cells [9], and rat gastric muco-al cells [8]. Barnes et al [35] reported that Ptc1 inhib-ted cell division by interacting with phosphorylatedyclin B1, and that this inhibition was overcome byxposure to Shh, which elicited cyclin B1 release fromtc1, leading to MPF translocation to the nucleus andesumption of the cell cycle. Similarly, in the presenttudy, Shh increased blastocyst cell numbers; fur-hermore, the beneficial effect of Shh on embryoevelopment was prevented by 1 �M/mL cyclopam-ne (Table 3).

Apoptosis, in response to suboptimal culture condi-ions and environmental stresses, is a common phenom-

able 5ffects of Shh on the development and apoptosis of porcine handma

hh (�g/mL) Total no. Cleavage rate, % (n) Blast

97 85.7 � 2.9 (87) 26.6.5 103 89.6 � 2.3 (91) 43.1

103 87.4 � 3.9 (91) 50.3102 40.4 � 3.0 (86) 25.8

he number in the parenthesis represents the actual number of embra,b Within a column, means without a common superscript differ (P* Apoptotic indices were calculated as follows: Apoptotic index �

non in IVP embryos [36]. Preimplantation porcine l

mbryos were particularly vulnerable to free radicals orubsequent oxidative stress, due to their high lipid con-ent [37]; this may contribute to poor embryo viabilitynd, in turn, to progressive embryo losses during IVP.n the present study, Shh reduced apoptosis, althoughhe underlying molecular mechanisms remain to beharacterized. We inferred that Shh may act as a sur-ival factor, perhaps by altering the expression of someurvival-related proteins (e.g. Akt and PARPs). That anpoptotic pathway can be triggered by Ptc in the ab-ence of Shh [38,39] was indirectly supported by theeduced apoptotic indices, improved blastocyst rates,nd increased cell numbers of both parthenotes andloned embryos in the present study. Furthermore, Aktromoted cell survival by inhibiting apoptosis by phos-horylating and inactivating several downstream targetolecules, including Bad and caspase-9 [40,41]. That

he Akt phosphorylation level in porcine blastocystsnder the effect of Shh was greater than that in theontrol group provided further support for Shh as aurvival factor during early embryogenesis. Further-ore, when apoptosis of embryonic cells was deter-ined with a comet assay, both the intensity and the

ength of comet tail were consistently reduced in theresence of Shh, suggesting that Shh may reduce DNAtrand breaks.

Another evidence for Shh as an anti-apoptosis factoras that PARP expression was reduced in the present

tudy. The PARPs are nuclear proteins with a centralole in signaling DNA damage and repairs [42,43].ctivation of PARP-1 depends on the occurrence ofNA strand breaks in the cell exposed to DNA-dam-

ging agents [44,45]. For instance, reactive oxygenpecies (ROS)-mediated DNA damage can also triggerARP-1 activation and/or subsequent cell death [46].yun et al. [47] reported that inhibition of PARP-1ttenuated the loss of mitochondrial membrane poten-ial, release of cytochrome c and apoptotic inducingactor (AIF), and cell death caused by ROS-dependentNA damages. When embryos were cultured with Shh,

ed embryos (mean � SEM from eight replicates).

te, % (n) No. cells per blastocyst (n) Apoptotic index*

26) 42.4 � 2.2a (25) 6.3 � 0.7a

45) 50.6 � 2.6ab (39) 4.6 � 0.5a

52) 57.6 � 2.3b (42) 2.8 � 0.6b

26) 43.0 � 3.3a (23) 5.5 � 0.7a

stocysts observed.).f TUNEL-positive nuclei/total No. of nuclei) � 100.

de clon

ocyst ra

� 1.8a (� 2.8b (� 1.9b (� 2.6a (

yos/bla� 0.05

ess PARP-1 protein was accumulated, most likely due

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1159N.T. Nguyen et al. / Theriogenology 74 (2010) 1149–1160

o the reduction of DNA strand breaks in the blas-omeres. However, further studies are required to elu-idate the functional role of PARP-1 in oocytes andmbryos.

In conclusion, we have demonstrated, apparently forhe first time, the existence of Shh signaling moleculesuring preimplantation development of porcine parthe-ogenetic embryos. Adding Shh during early develop-ent improved the quality and developmental compe-

ence of porcine parthenotes and handmade clonedmbryos, at least in part, through its mitogenic and cellurvival activities. These studies have the potential tomprove the quality of IVP porcine embryos, and torovide novel insights into mechanisms underlyingorcine embryo development.

cknowledgements

The first two authors contributed equally to thistudy. This study was partially supported by grantsrom National Science Council, Executive Yuan, Tai-an, ROC (NSC 95-2313-B005-034-MY3 and NSC8-2628-B005-019-MY3), and was performed in theissue Engineering and Stem Cells Center, Nationalhung Hsing University, Taichung, Taiwan.

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