R

EPRODUCTIONRESEARCH

Sonic hedgehog supplementation of oocyte and embryo culturemedia enhances development of IVF porcine embryos

Ngoc Tan Nguyen1,2, Neng-Wen Lo3, Sing-Ping Chuang1, Ya-Lan Jian1 and Jyh-Cherng Ju1

1Department of Animal Science, National Chung Hsing University, 250 Kuokuang Road, Taichung 402, Taiwan, ROC,2Agricultural Biotechnology Center, Institute of Agricultural Sciences for Southern Vietnam, 121 Nguyen Binh KhiemStreet, Ho Chi Minh City, Vietnam and 3Department of Animal Science and Biotechnology, Tunghai University,181 Section 3, Taichung Harbor Road, Taichung 407, Taiwan, ROC

Correspondence should be addressed to J-C Ju; Email: jcju@dragon.nchu.edu.tw

Abstract

We investigated the expression of sonic hedgehog (SHH) receptor PTCH1 and its co-receptor smoothened (SMO) in fertilized porcine

embryos. Effects of exogenous SHH on embryonic development and expressions of survival- and pluripotency-related genes were also

determined. We found that PTCH1 and SMO are expressed from two-cell to blastocyst embryos. When oocytes or fertilized embryos

were respectively cultured in the maturation or embryo culture medium supplemented with SHH (0.5 mg/ml), their blastocyst rates and

total cell numbers increased (P!0.05) compared with the untreated control. When cultured simultaneously in the in vitro maturation

(IVM) and in vitro culture (IVC) media supplemented with SHH, the oocytes gained increased blastocyst rates and total cell numbers in

an additive manner, with reduced apoptotic indices (P!0.05). Interestingly, SHH treatment did not affect the expression of the BCL2L1

(BCL-XL) gene, yet reduced BAX expression. Blastocysts cultured with various SHH regimes had similar pluripotency-related gene

(POU5F1 (OCT-4) and CDX2) expression levels, but blastocysts derived from SHH treatment during IVM had higher ZPF42 (REX01)

expression (P!0.05). The highest ZPF42 expression was observed in the blastocysts derived from SHH-supplemented IVC and from

dual IVM and IVC treatments. The levels of acetylated histone 3 (AcH3K9/K14) increased in the two-cell and the four-cell embryos

when IVM and/or IVC media were supplemented with SHH (P!0.05). Our findings indicate that SHH conferred a beneficial effect

on preimplantation development of porcine embryos, particularly when both IVM and IVC media were supplemented with SHH, and

the effects may be further carried over from IVM to the subsequent embryonic development.

Reproduction (2011) 142 87–97

Introduction

The porcine animal model has been broadly applied inmany aspects of biochemical research (Prather et al.2003), and porcine embryos hold great promise forembryonic stem cell technologies (Beebe et al. 2009).Despite efforts that have been made to optimize thein vitro production (IVP) system of embryos, the yieldand the quality of IVP embryos are still low whencompared with their in vivo-produced counterparts(Abeydeera et al. 1998, Kim et al. 2006, Ock et al.2007). It is well known that oocyte and embryo qualityhas been closely associated with the expression of aseries of genes during early embryogenesis (Lonerganet al. 2006, Zheng & Dean 2007), exerting a significantinfluence on the pre- and the post-implantationembryonic development to term (Krisher 2004, Sirardet al. 2006, Mtango et al. 2008).

A great majority of improvements in oocyte exploita-tion rely heavily on the accumulated knowledge

q 2011 Society for Reproduction and Fertility

ISSN 1470–1626 (paper) 1741–7899 (online)

concerning the in vitro culture (IVC) system of theoocytes and embryos up to the blastocyst stage (VanLangendonckt et al. 1997, Lonergan et al. 2003, Rizoset al. 2003). By altering the culture conditions of oocytematuration and embryo development, patterns of geneexpression could be manipulated and reprogrammedto closely mimic in vivo conditions, and, as a result,enhance embryo quality. In spite of the common use ofdefined media for in vitro maturation (IVM) of oocytesand IVC of embryos in many species, porcine oocyte-and/or blastocyst-promoting agents to improve theyield and quality of IVP embryos are still under extensiveinvestigation (Thompson 2000). However, little con-sensus has been reached regarding the mechanisms ofaction of growth factors, cytokines, vitamins, or aminoacids added to culture media (Craig et al. 2004, Kimet al. 2006, Gupta et al. 2007, Uhm et al. 2007).

Hedgehog (Hh) signaling molecules exist in the rodent(Wijgerde et al. 2005, Russell et al. 2007, Ren et al.2009) and bovine (Spicer et al. 2009) reproductive

DOI: 10.1530/REP-11-0049

Online version via www.reproduction-online.org

88 N T Nguyen and others

systems and are proposed to function as paracrine factorsto promote cell proliferation and steroid hormoneproduction. On the other hand, Hh signaling, particu-larly of Indian hedgehog (IHH), has been shown topromote the peri-implantation development of embryosin mice (Matsumoto et al. 2002, Takamato et al. 2002,Lee et al. 2006). Recent studies have also reported thatablation of IHH in murine uterus (Franco et al. 2010a) orconsecutive activation of smoothened (SMO; Francoet al. 2010b) led to infertility in female mice. Ourprevious studies in porcine demonstrated that Hhsignaling molecules, including patched1 (PTCH1),SMO, and GLI1, were present in the ovaries, oocytes,and parthenogenetic embryos at different stages.Inclusion of exogenous sonic hedgehog (SHH) in theIVM or IVC medium enhanced oocyte maturation anddevelopment of parthenogenetic embryos (Nguyen et al.2009, 2010). Moreover, embryo culture mediumcontaining SHH also promoted in vitro development ofhandmade cloned embryos (Nguyen et al. 2010). In thisstudy, we investigated the combined effects of SHHtreatments in both IVM and IVC media on thedevelopment of IVF porcine embryos.

PTCH1

2C

4C

8C

M

BL

SMO Negative c

Reproduction (2011) 142 87–97

Results

Experiment 1: expression of Hh signaling molecules atvarious developmental stages of porcine embryos

The expression of Hh receptor PTCH1 and co-receptorSMO at the protein level was detected by immunocyto-chemical staining. PTCH1 (left column) and SMO(middle column) were detected in the IVF-derivedembryos from the two-cell stage to the blastocyststage as shown in Fig. 1 (the positive staining isshown in green). The negative control was derivedfrom embryos stained by secondary antibody, while theprimary antibody was omitted. No detectable signal wasobserved for these embryos (right column).

Experiment 2: the collective effects of SHH on embryodevelopment during IVM and IVC

We have previously reported that SHH enhances nuclearand cytoplasmic maturation of porcine oocytes. Toinvestigate whether there is a combined or accumulativeeffect of SHH on oocyte maturation and embryodevelopment, we tested the correlations of IVM and

ontrol

Figure 1 Detection of the Hh receptor in porcineembryos of different developmental stages byimmunocytochemistry. After fixation in 4% parafor-maldehyde, receptor PTCH1 and co-receptor SMO ofindividual embryos were detected using anti-PTCH1(left column) and anti-SMO antibodies (middle col-umn). Negative control embryos were probed directlywith the secondary antibody instead of the primaryantibody (right column). 2C, two-cell; 4C, four-cell; 8C,eight-cell; M, morula; BL, blastocyst. Green fluor-escence indicates positive staining. Scale bar, 50 mm.

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Table 1 Effects of sonic hedgehog supplementation in maturation and culture media on the development of porcine embryos in vitro.

SHH (0.5 mg/ml)

IVM IVC Total (N)Cleavage rate,

% (n)Blastocyst rate,

% (n)Cell number per

blastocystApoptotic index,

% (n)

K K 226 79.3G3.4a (179) 25.0G1.4a (56) 46.4G3.3a 4.5G0.4a (19)C K 214 84.3G3.1a (181) 37.3G1.2b (79) 57.4G3.7b 3.6G0.3bc (22)K C 220 81.2G2.1a (179) 33.8G2.1b (73) 59.1G2.2ab 3.2G0.3ab (25)C C 214 85.5G1.8a (182) 42.8G1.7c (90) 69.7G3.2c 2.4G0.2c (27)

Numbers in parentheses represent the number of embryos in each category. Data are meanGS.E.M. from six replicates. a–cWithin a column, meanswithout the same superscript letters differed (P!0.05). IVM, in vitro maturation; IVC, in vitro culture.

SHH enhances development of IVF porcine embryos 89

IVC media in the presence or absence of SHH with theend point development of porcine embryos in vitro. Asshown in Table 1, consistent with what we had foundpreviously, presence of SHH in IVM or IVC mediumalone enhanced blastocyst formation (37.3 and 33.8%respectively) compared with the control group (25%,P!0.05). The improvement of blastocyst formationreached the highest level when SHH was included inboth IVM and IVC media (IVMC/IVCC) compared withthe control (42.8 vs 25%, P!0.05) and the other groups,IVMC/IVCK and IVMK/IVCC. In addition, the total cellnumber per blastocyst increased with the presence ofSHH in IVM (54.1G3.7), in IVC (59.1G2.2) and in bothmedia (69.7G3.2) yet remained unaltered in the controlgroup (46.4G3.3, P!0.05). However, no differenceswere observed among the three treatment groups. Theapoptotic indices were significantly reduced when SHHwas present in IVC (3.2G0.3%) or in the IVMC/IVCC

group (2.4G0.2%) compared with the control group(4.6G0.4%). The apoptotic indices were similarbetween the IVMC/IVCK (3.6G0.3%) and the IVMK/IVCC groups, and between the IVMC/IVCK and thecontrol groups (PO0.05).

An additional experiment was designed to evaluatethe fertilization rate after IVM, and the results arepresented in Table 2 and Fig. 2. Sperm penetrationrates were similar among treatment groups (85.5–87.5%). With the presence of SHH in IVM or in bothIVM and IVC media, the monospermic rate (85.0 and

Table 2 Effects of sonic hedgehog supplementation to in vitro maturation a

SHH (0.5 mg/ml)Number of

oocytes examinedPenetration

% (n)aIVM IVC

K K 96 86.4G0.5 (C K 92 85.5G2.2 (K C 88 87.5G1.7 (C C 95 86.5G1.7 (

Numbers in parentheses represent the actual number of oocytes examined.without the same superscript letters differed (P!0.05). IVM, in vitro maturaMonospermic rate (%): number of monospermic oocytes/total penetratedoocytes/total inseminated oocytes!100.

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85.7% respectively) increased significantly comparedwith the control group (67.4%) or the IVMK/IVCC group(68.7%). Similarly, a significant improvement of normalfertilization rate was also found when SHH was added inIVM medium alone (72.6%) or in both media (74.2%;control group, 58.2%).

Experiment 3: effects of SHH treatment in IVM andIVC media on histone acetylation profile

Levels of the histone 3 acetylation or AcH3K9/K14 ofporcine embryos at different stages were determined.As shown in Figs 3 and 4, the level of AcH3K9/K14 wassignificantly increased (P!0.05) at the two-cell stage inthe presence of SHH, either in IVM (130.5G3.7) orin IVC (134.6G3.2) medium alone, and in both media(161.1G4.7) compared with the control group (108.2G4.2). Among the SHH-treated groups, the AcH3K9/K14 levels in the dual SHH-treated embryos (IVMC/IVCC) were significantly higher than those in theembryos with single SHH treatment (IVMC/IVCK orIVMK/IVCC). At the four-cell stage, embryos derivedfrom IVMK/IVCC (214.1G5.8) and IVMC/IVCC treat-ments (223.3G8.0) had a significantly higher level ofacetylation pattern than those of the control (178.9G5.3)or from IVMC/IVCK treatment (182.9G5.0). However,embryos developing up to the blastocyst stage had onlya similar level of acetylation between the control andthe treatment groups.

nd culture media on IVF rates.

rate, Monospermic rate,% (n)

Normal fertilizationrate, %b

73) 67.4G2.7a (57) 58.2G2.5a

80) 85.0G2.1b (68) 72.6G2.3b

77) 68.7G5.5a (54) 60.0G4.2a

83) 83.7G1.4b (71) 74.2G1.2b

Data are meanGS.E.M. from four replicates. a,bWithin a column, meansation; IVC, in vitro culture.oocytes!100. bNormal fertilization rate (%): number of monospermic

Reproduction (2011) 142 87–97

A1 A2

B1 B2

C1 C2

D1 D2

SSP SSP

MII plateMII plate

Hoechst Merged

Figure 2 Representative images of unfertilized (A1 andA2), monospermic (B1 and B2), and polyspermic(C1 and C2 and D1 and D2) oocytes at 10 h post-insemination. Arrowheads indicate pronuclei; SSP,swollen sperm head; yellow arrows indicate first polarbody; red arrows indicate second polar body; MIIplate, chromatin plate of oocytes at the metaphase IIstage. Scale bar, 50 mm.

90 N T Nguyen and others

Experiment 4: effects of SHH supplementation duringIVM and/or IVC on gene expressions of porcineembryos

Expressions of cell survival-related genes (BCL2L1 andBAX) and pluripotency-related genes (POU5F1, CDX2and ZPF42 (REX01)) were analyzed using quantitativereal-time PCR at the blastocyst stage on day 7. As shownin Fig. 5, no significant difference in BCL2L1 geneexpression was found among treatment groups. However,BAX gene expression was significantly reduced in theblastocysts derived from the IVMC/IVCC(0.51-fold) orIVMK/IVCC(0.58-fold) group compared with thosederived from the IVMC/IVCK (0.83-fold) or the controlgroups. There was also a detectable difference in the

Reproduction (2011) 142 87–97

BAX expression between the IVMC/IVCK group andthe control group. Taken together, differential BAXexpression levels in the blastocyst embryos between theSHH-treated groups and the control group were obvious.

In the case of the pluripotency-related genes, thelevels of CDX2 and POU5F1 expressions were similar(PO0.05) between the control and the treatment groups(Fig. 6A and B). However, the levels of ZPF42 expressionwere significantly higher in the IVMC/IVCK (1.13-fold),IVMK/IVCC(1.61-fold), or IVMC/IVCC(1.58-fold) treat-ment groups compared with the untreated control group(P!0.05). However, no difference was found betweenthe SHH-treated embryos in the IVMK/IVCC group andthe IVMC/IVCC group (Fig. 6C, PO0.05).

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IVM–/IVC–

2C

4C

BL

IVM+/IVC– IVM+/IVC+IVM–/IVC+

SHH

Figure 3 Histone 3 acetylation (AcH3K9/K14) levels ofporcine embryos at different developmental stagesunder various regimes of SHH treatment. 2C, two-cellembryo; 4C, four-cell embryo; BL, blastocyst. Scalebar, 50 mm. K/K, absence of SHH in IVM and IVCmedia (control); C/K, presence of 0.5 mg/ml SHHin IVM medium but absence of SHH in IVC medium;K/C, absence of SHH in IVM medium but presenceof 0.5 mg/ml SHH in IVC medium; C/C: presence of0.5 mg/ml SHH in both IVM and IVC media.

SHH enhances development of IVF porcine embryos 91

Similarly, with the presence of SHH in both IVM andIVC media, expression levels of BAX and ZPF42 weresignificantly enhanced. In contrast, the beneficial effectof SHH on BAX and ZPF42 expression levels wasneutralized by the presence of cyclopamine in themedium (Fig. 7).

450 –/–+/––/++/+

400

350

300

250

Rel

ativ

e flu

ores

cenc

e (p

ixel

s ×

104 )

200

150

100

2-cell

N=

35

N=

30N

=28

N=

30

N=

28N

=31

N=

32

N=

30

N=

25

N=

30

N=

35N

=40

4-cell Blastocyst

50

0

a

a

a

a aa

bb

bb

ca

Figure 4 Histone acetylation (AcH3K9/K14) profile of fertilized porcineembryos after treatment with SHH during IVM and/or IVC. a–cWithinthe embryonic stage, bars with different letters differ (P!0.05). Thesample sizes are shown in each bar. K/K, absence of SHH in IVM andIVC media (control); C/K, presence of 0.5 mg/ml SHH in IVM mediumbut absence of SHH in IVC medium; K/C, absence of SHH in IVMmedium, but presence of 0.5 mg/ml in IVC medium; C/C, presenceof 0.5 mg/ml SHH in both IVM and IVC media.

Discussion

Several lines of evidence indicated the involvement ofHh signaling during oogenesis. It was reported that Hhsignaling molecules were present in mouse (Wijgerdeet al. 2005, Russell et al. 2007, Ren et al. 2009) andbovine ovaries (Spicer et al. 2009), particularly in thetheca and granulosa cells. Ren et al. (2009) reported thata major activation of co-receptor SMO resulted in analteration of theca cell proliferation and prevention ofovulation in mice, although follicles developed normallyto preovulatory stage in response to LH stimulation. Inaddition, we previously reported that SHH and its relatedsignaling molecules (PTCH1, SMO, and GLI1) wereexpressed in porcine ovaries, specifically in the cumulus–oocyte complexes (COCs) and oocytes (Nguyen et al.2009). In contrast, Wijgerde et al. (2005) reported that noSMO was detected in mouse oocytes. Interestingly, wedetected SMO expression in denuded porcine oocytesby western blot (Nguyen et al. 2009), suggesting thatSMO expression could be species-specific. Furthermore,we have recently reported that Hh signaling receptorPTCH1 and SMO are also expressed in porcineparthenotes from the two-cell stage throughout theblastocyst stage at both mRNA and protein levels(Nguyen et al. 2010). In this study, we also found theexpressions of PTCH1 and SMO from two-cell toblastocyst stages of fertilized porcine embryos detectedby immunocytochemistry.

Since the embryonic genome activation (EGA) occursat the four-cell stage of porcine embryos (Jarrell et al.1991), the presence of PTCH1 and SMO at the two-cell

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stage is most likely a maternal legacy. Our previous studydocumented the existence of SHH protein in porcinefollicular fluids from follicles of various sizes (Nguyenet al. 2009). Since Hh signaling was detected in theoocytes and embryos of different developmental stages,we hypothesize that Hh signaling is involved infolliculogenesis as well as in early embryogenesis.In fact, our previous study had demonstratedthat addition of SHH to the IVM medium promotednuclear and cytoplasmic maturation of porcine oocytes,and subsequently enhanced parthenogenetic embryodevelopment (Nguyen et al. 2009). Consistently, wealso found that SHH addition during IVM increased

Reproduction (2011) 142 87–97

2 –/–+/––/++/+

a

b

c c

1.5

Gen

e ex

pres

sion

fold

cha

nge

1

0.5

0BCL2L1 BAX

Figure 5 Expression of cell survival-related genes BCL2L1 and BAX inporcine blastocysts cultured in SHH-supplemented IVM and/or IVCmedia. a–cWithin category, bars with different letters differ (P!0.05).K/K, absence of SHH in IVM and IVC media (control); C/K, presenceof 0.5 mg/ml SHH in IVM medium but absence of SHH in IVC medium;K/C, absence of SHH in IVM medium, but presence of 0.5 mg/ml SHHin IVC medium; C/C, presence of 0.5 mg/ml SHH in both IVM andIVC media.

–/– +/– –/+ +/+

–/– +/– –/+ +/+

POU5F1 (OCT-4)

CDX2

2

A

B

1.5

Gen

e ex

pres

sion

fold

cha

nge

1

0.5

0

2

1.5

Gen

e ex

pres

sion

fold

cha

nge

1

0.5

0

ZPF42

–/– +/– –/+ +/+

a

b

c c2

1.5

Gen

e ex

pres

sion

fold

cha

nge

1

0.5

0

C

Figure 6 Expression of pluripotency-related genes POU5F1 (A), CDX2(B), and ZPF42 (C) in porcine blastocysts cultured under variousregimes of SHH supplementation. a–cWithin each gene, bars withdifferent letters differ (P!0.05). K/K, absence of SHH in IVM and IVCmedia (control); C/K, presence of 0.5 mg/ml SHH in IVM medium butabsence of SHH in IVC medium; K/C, absence of SHH in IVMmedium, but presence of 0.5 mg/ml SHH in IVC medium; C/C,presence of 0.5 mg/ml SHH in both IVM and IVC media.

92 N T Nguyen and others

the blastocyst rate and total cell number per blastocystin this study.

The potential role of SHH during oogenesis is evidentin several species including pigs. During in vivomaturation, oocytes are exposed to SHH in the follicularfluid, because SHH was present in porcine follicularfluid. This was supported by our previous studies withsolely parthenogenetic embryos (Nguyen et al. 2009,2010). Interestingly, the yield and quality of IVF-derivedembryos were also further enhanced by the inclusion ofSHH in the IVC medium in addition to the IVM medium,suggesting that an additive or accumulative effect hasbeen exerted by SHH during these courses of develop-ment in porcine embryos.In vitro-produced porcine embryos were particularly

vulnerable to free radicals or subsequent oxidativestresses, due to their high lipid contents (McEvoy et al.2000). This phenomenon may have led to poor embryoviability and, in turn, to progressive embryo losses duringIVP, as evidenced by high incidence of apoptotic ratesin response to suboptimal culture conditions (Nanassyet al. 2008). In this study, we demonstrated that thepresence of SHH in culture medium effectively reducedapoptosis, although the underlying molecularmechanisms remained unclear. Presumably, SHHmight act as a survival factor, perhaps by altering theexpression of some cell survival-related proteins such asAKT (Nguyen et al. 2010). Other reports indicated thatPTCH1, a negative regulator of SHH signaling, maytrigger an apoptotic pathway in the absence of SHH(Thibert et al. 2003, Mille et al. 2009). Similar to thefindings in parthenogenetic embryos, SHH also reducedapoptotic indices, improved blastocyst rates, andincreased cell numbers of fertilized embryos in this

Reproduction (2011) 142 87–97

study. The presence of SHH in IVM and/or IVC media hasno influences on the expression of the anti-apoptoticgene BCL2L1, but the expression of pro-apoptotic geneBAX was reduced with the presence of SHH either in IVCmedium alone or in both IVM and IVC media.Collectively, our data supported the notion that SHHmodulated or prevented apoptosis of blastomeres,which, in turn, improved the blastocyst rate and totalcell numbers of porcine embryos.

It is still unknown whether a certain degree ofpolyspermy is physiologically permissive in porcine,

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–/–

+/+ plus 1µm cyclopamine

+/+

2

1.5

Gen

e ex

pres

sion

fold

cha

nge

1a

aa

a

b

b

BAX ZPF42

0.5

0

Figure 7 Effects of SHH on the expression of BAX and ZPF42 genesin porcine blastocysts in the presence or absence of the inhibitorcyclopamine. a,bWithin each gene, bars with different letters differ(P!0.05). K/K, absence of SHH in IVM and IVC media (control);C/C, presence of 0.5 mg/ml SHH in both IVM and IVC media; C/Cplus cyclopamine, presence of 0.5 mg/ml SHH in both IVM and IVCmedia plus 1 mM/ml cyclopamine.

SHH enhances development of IVF porcine embryos 93

and whether porcine oocytes have any capacity toremove the excessive sperm in the ooplasm (Xia et al.2001). Regardless of the IVC conditions, we found thatthe presence of SHH during oocyte maturation enhancedthe monospermic rate, i.e., normal fertilization rate. It isconceivable that the improved oocyte quality orooplasmic maturation would potentially reduce poly-spermy in the medium containing SHH. Our previousstudy revealed that oocytes matured in mediumcontaining SHH had higher intracellular calcium releaseinduced by IP3 and, in particular, a prolonged duration ofcalcium transient (Nguyen et al. 2009). These physio-logical cues may be of importance for preventing thepolyspermic fertilization in porcine oocytes.

Histone acetylation/deacetylation is a dynamic pro-cess during embryogenesis and differentiation. It isclosely associated with the chromatin structures and,hence, with transcriptional regulation (Kim et al. 2003).We examined whether SHH altered the pattern ofhistone acetylation in various stages of embryos,particularly by the time of EGA of developing embryos.Intriguingly, the results revealed that the levels ofacetylated H3K9/K14 were higher in SHH-treatedembryos in IVMK/IVCC and IVMC/IVCC groups attwo- and four-cell stages compared with the controlgroup. The elevated levels of acetylation of H3k9/K14 inthe SHH-treated embryos during IVM might carry anaccumulative effect of SHH from IVM via an improvedoocyte over to a development-enhanced embryo. It isalso known that oocyte maturation helps to carry out

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a proper epigenetic remodeling of parental chromatin, asreflected by pronuclear methylation and acetylation(Gioia et al. 2005). Ma & Schultz (2008) also noted thatoocyte maturation was accompanied by changes inhistone modification, and the maturation-associatedreduction in acetylation was due to the histonedeacetylase activation and a lack of histone acetyltrans-ferase activity in the oocytes (Kim et al. 2003). Cerveraet al. (2009) reported that high levels of histoneacetylation at the four-cell stage of porcine embryoshelped to render sufficient chromatin accessibility forinitiation of gene transcriptions during subsequent stagesof development. In this study, it appeared that acetyl-ation levels at the four-cell stage were correlated with thereduced expression of pro-apoptotic BAX gene and theenhanced expression of ZPF42 in the blastocyst stage.In addition, we found that expression of POU5F1 andCDX2 transcripts was not changed at the blastocyst stageregardless of the presence of SHH. In contrast toPOU5F1and CDX2, ZPF42, a developmentally regulated genecontaining zinc finger motifs (Hosler et al. 1989), wasexpressed at a higher level in the SHH-treated embryos,although the exact function and mechanisms are notclear. This might have altered the ratio of inner cell massto trophectoderm cells at the blastocyst stage, given thatenhanced histone acetylation led to reallocation of theICM and TE cells in bovine blastocysts (Cervera et al.2009, Ikeda et al. 2009). It is likely that the pattern ofhistone acetylation at the four-cell stage is associatedwith those changes found in this study (Figs 3 and 4). Thisinference is further supported by Ikeda et al. (2009),whose report showed that epigenetic modifications tothe genome during fertilization exerted a carryover effecton cell proliferation and differentiation of preimplanta-tion embryos. Furthermore, the level of POU5F1 proteinexpression would be critical to ZPF42 expression due toa POU/octamer site found in its promoter region(Cervera et al. 2009). However, it is unclear why theZPF42 level was increased without a change in thePOU5F1 transcript level as observed in this study. Morecomplicated mechanisms may be involved in modulat-ing ZPF42 expression of porcine embryos cultured in themedium supplemented with SHH. Previous studies haveshown that SHH promotes the nuclear and cytoplasmicmaturation and the embryonic development through thetrio PTCH1–SMO–GLI1 action (Nguyen et al. 2009,2010). Similarly, in this study, we found that thebeneficial effects of SHH on BAX and ZPF42 geneexpression were prevented by cyclopamine inhibition.However, the correlations of the histone acetylation atthe four-cell stage embryo with the expressions of thesurvival- and pluripotency-related genes (BAX andZPF42) have not been documented.

Taken together, these findings suggested that SHHplayed a positive role in the development of porcinepre-implantation embryos. Ooplasmic maturation andepigenetic modifications such as histone 3 acetylation

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94 N T Nguyen and others

of the genome pre- and post-fertilization could bepersistent and carried over to the subsequent embryonicdevelopment, in particular, when both IVM and IVCmedia were supplemented with SHH to direct cellsurvival, proliferation, and differentiation during thepreimplantation development of porcine embryos.

Materials and Methods

Chemicals

All chemicals used in this study were purchased from SigmaChemicals, unless otherwise specified.

Oocyte maturation and embryo culture

Procedures for collection of ovaries and oocytes weredescribed previously (Nguyen et al. 2009, 2010). In brief,freshly harvested COCs were rinsed several times and culturedfor 22 h in the NCSU-23 medium supplemented with 10%porcine follicular fluid, cysteine (0.1 mg/ml), eCG (10 IU/ml),and hCG (10 IU/ml). The COCs were further cultured for anadditional 22 h in the same medium without hormones.

Sperm preparation, IVF, and embryo culture

Diluted semen from two boars of proven fertility was suppliedby a porcine artificial insemination center (Taichung, Taiwan),stored at 15 8C, and used for 2 days. Motile sperm wereobtained by centrifugation at 700 g with Percoll (P1644)discontinuous gradient (2 ml of 45% over 2 ml of 90%) for15 min. Spermatozoa were collected from the bottom of the90% fraction and washed in Dulbecco’s PBS (DPBS) supple-mented with 10% fetal bovine serum, followed by centri-fugation at 100 g for 5 min. After centrifugation, the spermpellet was resuspended in modified tris-buffered medium(mTBM) containing 113.1 mM NaCl, 3 mM KCl, 7.5 mMCaCl2.2H2O, 20 mM Tris, 11 mM glucose, 5 mM sodiumpyruvate, and 0.2% (w/v) BSA. After 44 h of maturation,oocytes were freed from cumulus cells, and matured oocyteswith first polar body were washed three times in mTBM thathad been pre-equilibrated for at least 12 h at 39 8C under 5%CO2. After washing, groups of 30 oocytes were randomlyplaced into 45 ml droplets of mTBM covered with pre-warmedmineral oil. After dilution (10!), 5 ml of sperm suspensionwere added to a 45 ml droplet of fertilization medium (mTBM)to give a final sperm concentration of 5!105 sperm/ml. Thegametes were co-cultured for 6 h at 39 8C in an incubatorcontaining 5% CO2 in humidified air. After 6 h, oocytes wereremoved from the fertilization medium and washed three timesin porcine zygote medium-3 (PZM-3) to remove excessivesperm. The presumptive zygotes were then cultured in 100 mldroplets (20–30 oocytes per droplet) of PZM-3 covered withmineral oil for 7 days. Cleavage and blastocyst rates wereevaluated under a stereomicroscope at days 2 and 7 afterinsemination (insemination dayZday 1).

An additional experiment was designed to evaluate thefertilization rate. At 10 h post-insemination, some oocytes wereselected and fixed in the fixative. The fixed oocytes were

Reproduction (2011) 142 87–97

subjected to triple staining and observed under a fluorescencemicroscope for nuclear evaluation, and were classified intothree categories, i.e. unfertilized oocytes, monospermicoocytes (two pronuclei, 2PN), and polyspermic oocytes(more than 2PN or 2PN plus swollen sperm heads). Therepresentative images are presented in Fig. 2.

Detection of histone acetylation and quantification

The effect of SHH on the acetylation of histone 3 at lysine 9 and14 (AcH3K9/K14) during pre-implantation development wasstudied. Embryos at different developmental stages were fixedwith 4% paraformaldehyde (PFA; EMS, Hatfield, Pennsylvania)in DPBS containing 0.1% polyvinyl alcohol (PVA) for 1 h at RT.They were then washed in washing solution (DPBS containing0.1% (w/v) PVA, 1% (w/v) BSA, and 0.1% (v/v) Tween-20) for1 h, and were permeated with 0.5% (v/v) Triton X-100 in DPBSfor 30 min at room temperature and blocked in workingsolution (DPBS–PVA supplemented with 2% BSA) at 4 8Covernight. The embryos were then incubated with anti-acetylhistone 3 (1:300, Cat #9677, Cell Signaling Technology, Inc.Beverly, MA, USA) for 1 h at 37 8C, washed in washing solutionfor 30 min, and then incubated with Alexa Flour 488-conjugated secondary antibody (1:200, Invitrogen) for 1 h atroom temperature (darkness), followed by two washes in thewashing solution. The samples were mounted on slides withmounting medium containing Hoechst. Immunofluorescentlabeling of AcH3K9/K14 was evaluated under an epifluores-cent microscope (Nikon, Tokyo, Japan), and micrographimages were captured. Fluorescence intensity was measuredby analysing the micrographs with ImageJ Software (ImageProcessing and Analysis in Java, Version 1.42 for Windows,NIH, Bethesda, MD, USA) after background subtraction. Dataare presented as a mean of relative fluorescence intensity (pixels).

Immunocytochemical staining

Patched (PTCH1) and SMO proteins

To detect PTCH1 and SMO, embryos at two-cell, four- to eight-cell, morula, and blastocyst stages were fixed in 4% (w/v) PFAin DPBS supplemented with 0.1% (w/v) PVA for 1 h at roomtemperature. After washing twice in washing solution, embryoswere permeated with 0.5% (v/v) Triton X-100 in DPBS for30 min. The embryos were then blocked in working solutionsupplemented with 5% (v/v) chicken serum at 4 8C overnight.After being washed twice in washing solution, the embryoswere incubated with primary antibody for PTCH1 (1:330;sc-13943; Santa Cruz Biotechnology, Inc., Santa Cruz, CA,USA) or SMO (1:330; sc-9016; Santa Cruz) at 4 8C overnight.They were then washed for 30 min and incubated with AlexaFlour 488-conjugated secondary antibody (1:200, Invitrogen)for 1 h (darkness) and then washed twice with washingsolution. Samples were mounted on slides with mountingmedium containing Hoechst, and images were captured underan epifluorescence microscope (Nikon).

Nuclear and cytoskeleton: triple staining

Triple staining procedure was performed as described by Juet al. (2003). In brief, microtubule stabilization buffer (MTSB,

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SHH enhances development of IVF porcine embryos 95

5!) and working solution (MTSB-XF) containing 0.5 M PIPES,25 mM magnesium chloride, 125 mM EGTA, 0.01% (w/v)aprotinin, 1 mM dithiothreitol, 50% deuterium oxide, 1 mMpaclitaxel, 0.1% (v/v) Triton X-100, and 2% (v/v) formaldehyde.Mounting medium contained 10 mg/ml Hoechst 33342, 50%(v/v) glycerol, and 25 mg/ml sodium azide in DPBS. Oocyteswere transferred into the fixative in an Eppendorf tube and wereincubated in water bath (38 8C) for 1 h, and stored at 4 8C for atleast 1 week before staining. For staining, the fixed oocyteswere incubated with anti-a- and anti-b-tubulin primaryantibodies at 4 8C overnight. The oocytes were then washedthree times with washing solution containing 2% (w/v) BSA,2% goat serum, 0.2% (w/v) milk powder, 0.2% (w/v) sodiumazide, and 0.1% (v/v) Triton X-100 in DPBS. The oocytes weresubsequently incubated with the FITC-conjugated secondaryantibody at 4 8C overnight. After being washed three times,microfilaments of the oocytes were stained with rhodamine–phalloidin (R147; Molecular Probes, Invitrogen) for 1 h forlabeling the filamentous actin and then washed three timesagain. Finally, the oocytes were mounted on the slide withmounting medium containing Hoechst 33342 and then sealedwith fingernail polish, and examined under an epifluorescencemicroscope (Nikon) with magnification at 40!.

RNA purification, reverse transcription and quantitativereal-time PCR

Total RNA was extracted by TRIzol Reagent (Invitrogen) andpurified by RNeasy Mini Kit (Qiagen). Purified RNA wasquantified using a ND-1000 spectrophotometer (NanodropTechnology, Wilmington, DE, USA) and qualitatively analyzedby Bioanalyzer 2100 (Agilent Technology, USA). To prepare acDNA pool from each RNA sample, total RNA (19 ng) wasreverse transcribed using MMLV reverse transcriptase (Pro-mega), and the resulting samples were diluted 2!103 timeswith DNase-free water. Each cDNA pool was stored at K20 8Cuntil further real-time PCR analysis.

Real-time PCR were performed on the Roche LightCyclerInstrument 1.5 using LightCycler FastStart DNA MasterPLUSSYBR Green I kit (Roche Cat. 03 515 885 001). Briefly, the 10 mlreaction mixture contained 2 ml master mix, 2 ml of 0.75 mM or2 ml of 0.50 mM forward primer and reverse primer with 5%

Table 3 Primer sequences, GenBank accession numbers, and expected pro

Target genes Primers Sequences (5 0 to 3 0)

BCL2L1 Forward GCAGGTATTGAACGReverse GCATCTCCTTGTCTA

BAX Forward CTACCAAGAAGTTGReverse ACGGCTGCGATCAT

CDX2 Forward CCTCTCGCCCACAAReverse TCCAACCGCACCTG

ZPF42 Forward GGTCTCAGCCTCAGReverse TGTAGCCAGCGTCC

POU5F1 Forward CGCAACGAGAGGAReverse CGCCAGAGGAAAG

ACTB Forward CCACGCCATCCTGCReverse CCATCTCCTGCTCGA

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DMSO respectively, and 6 ml cDNA sample solutions. Eachsample was performed in triplicate. The RT-PCR wereprogrammed at 95 8C for 10 min, 50 cycles of 95 8C for 10 s,60 8C for 15 s, and 72 8C for 10 s. At the end of the program,a melt curve analysis was performed. At the end of eachRT-PCR run, data were automatically analyzed by the system,and an amplification plot was generated for each cDNAsample. From each of these plots, the LightCycler3 DataAnalysis Software (Roche Diagnostics) automatically calcu-lated CP value (crossing point, the turning point correspondsto the first maximum of the second derivative curve), whichimplied the beginning of an exponential amplification. Thecomparative CT method was applied for quantification of geneexpression levels as described by Wee et al. (2006). Geneexpression fold changes were calculated using the formula2KðDDCT ExpKControlÞ, where

DCT Exp ZCT targetKCT ref of Exp sample

DCT Control ZCT targetKCT ref of control sample

Primer sequences, GenBank accession numbers, andexpected product sizes for the real-time PCR analysis arepresented in Table 3.

Specific experiments

Experiment 1: expression of Hh signaling molecules at differentdevelopmental stages of IVF-derived porcine embryos

The expressions of PTCH1 and SMO in IVF-derived two-cell,four- to eight-cell embryos, morulae and blastocysts wereinvestigated by immunocytochemical staining. At least thirtyembryos in each stage were used for detection.

Experiment 2: the collective effects of SHH on embryodevelopment accumulated during IVM and IVC

The optimized dose usage of SHH at 0.5 mg/ml was determinedfrom our previous studies (Nguyen et al. 2009, 2010) and usedthroughout all treatments in this study. Matured oocytes derivedfrom IVM with or without SHH treatment were subjected toIVF. After 6 h co-culture with sperm, the presumptive zygoteswere cultured in PZM-3 and randomly allocated into thefollowing culture groups: no SHH in both IVM and IVC

duct sizes for the qRT-PCR analysis.

GenBankaccession number

Productsize (bp)

AACTCTTCCG AJ_001203 102CGCTCTCCAGCGAGTGTC AJ_606301 85CCTCTGATGTTCAC TC_206866 82TCTTTACCTGTCAGC TC_317123 105TCTTCCTTTTGAG TC_168415 68GATACTGGTCT AK_237086 168AGTCCAG

Reproduction (2011) 142 87–97

96 N T Nguyen and others

(control, IVMK/IVCK), IVM with SHH and IVC withoutSHH (IVMC/IVCK), IVM without SHH and IVC with SHH(IVMK/IVCC), and SHH in both IVM and IVC (IVMC/IVCC).The cleavage and blastocyst rates were recorded at days 2and 7 respectively. The blastocysts were subjected toHoechst staining and TUNEL labeling for the determinationof the total cell number and apoptotic cells respectively(Nguyen et al. 2010). Apoptotic indices were calculated asfollows:

Apoptotic index (%)Z(number of positive nuclei/total cellnumber)!100.

At 10 h post-fertilization, a proportion of presumptivezygotes from each treatment were randomly selected forevaluation of fertilization rate.

Experiment 3: effects of SHH treatment in IVM and IVC mediaon acetylation profile of IVF embryos

Embryos from each treatmentas inExperiment 1werecollectedattwo-cell, four-cell, and blastocyst stages for histone acetylationassay. At least 25 embryos at each stage were used, and therepresentative numbers were given as shown in the Fig. 4.

Experiment 4: effects of SHH treatment in IVM and IVCon gene expressions of IVF embryos

Embryos collected and pooled at day 7 from each treatment(ten embryos per treatment) were incubated with cell lysisbuffer at 42 8C for 1 h, and then stored at K80 8C. Cell survival-related genes (BCL2L1 and BAX), pluripotency marker genePOU5F1, trophectoderm lineage marker CDX2, and inner cellmass lineage marker ZPF42 were analyzed by quantitative real-time PCR.

An additional experiment was designed to confirm thebeneficial effect of SHH on BAX and ZPF42 expression. Afterinsemination, matured oocytes under the presence of 0.5 mg/mlSHH were divided into two groups, in which one group wascultured in the IVC medium supplemented with 0.5 mg/mlSHH, and the other cultured in the medium with 0.5 mg/mlSHH plus 1 mM cyclopamine, an SHH inhibitor. The maturedoocytes cultured without SHH in both IVM and IVC mediawere the control group. The blastocysts harvested at day 7 asmentioned earlier were used to determine the expression levelsof BAX and ZPF42 genes.

Statistical analysis

All data were subjected to ANOVA using the general linearmodel procedure in SAS version 9 (SAS Institute, Cary, NC,USA), followed by Tukey’s test. Percentile data were arcsinetransformed before statistical analysis, and the P!0.05 wasconsidered significantly different between treatment groups.

Declaration of interest

The authors declare that there is no conflict of interest thatcould be perceived as prejudicing the impartiality of theresearch reported.

Reproduction (2011) 142 87–97

Funding

This study was partially supported by grants fromNational Science Council, Executive Yuan, Taiwan, ROC(NSC 98-2628-B005-019-MY3), and was performed in theTissue Engineering and Stem Cells Center, National ChungHsing University, Taichung, Taiwan.

Acknowledgements

The authors would like to thank Dr David Pei-Cheng Lin atChung-Shan Medical University (Taichung-Taiwan) for thecritical reading of this manuscript. We are also grateful toTaichung Meat Market Co. Ltd, Taichung, Taiwan, for kindlyproviding porcine ovaries for years.

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Received 21 February 2011

First decision 12 April 2011

Accepted 12 April 2011

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