8
Reproductive BioMedicine Online (2010) 21, 769-775 www.sciencedirect.com www.rbmonline.com ELSEVIER ARTICLE Developmental competence of parthenogenetic mouse and human embryos after chemical or electrical activation Karen Versieren*, Björn Heindryckx, Sylvie Lierman, Jan Gerris, Petra De Sutter Department of Reproductive Medicine, Ghent University Hospital, De Pintelaan 185, 9000 Ghent, Belgium * Corresponding author. E-mail address: [email protected] (K Versieren). Karen Versieren obtained a MSc degree in biomédical sciences at the Ghent university, Belgium. She joined the research team of the Department of Reproductive Medicine at the Ghent university Hospital in 2007. She is currently working on her PhD. Her major areas of interest include the study of parthenogenetic activation of mouse and human oocytes and somatic cell nuclear transfer for therapeutic stem cell purposes. Abstract Parthenogenetic reconstruction is one major strategy to create patient-specific stem cells. The aim of this study was to find the best artificial activation protocol for parthenogenetic activation of mouse and human oocytes comparing different methods. In a first set of experiments, in-vivo matured mouse oocytes and human failed-fertilized, in-vitro and in-vivo matured oocytes were arti- ficially activated by a chemical (ionomycin) or electrical stimulus. In a second set of experiments, a combination of activating agents (electrical pulses followed by ionomycin or SrClî) was applied in an aim to improve developmental competence. All embryos were evaluated daily until day 6 after activation. Mouse blastocysts were differentially stained to evaluate blastocyst quality. For mouse oocytes and human failed-fertilized oocytes, blastocyst development was significantly higher after electrical activation (P < 0.05). For human in-vitro and in-vivo matured oocytes, blastocyst formation was only obtained after electrical activation of in-vitro matured oocytes. After combining activating agents, no differences in development could be observed. In conclusion, this study revealed that for both mouse and human oocytes development to the blastocyst stage was significantly better after electrical activation compared with chemical activation. Combining activating agents had no further positive effect on developmental potential, w'" "• © 2010, Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved. KEYWORDS: artificial activation, electrical pulses, ionomycin, parthenogenesis, preimplantation development Introduction Somatic cell nuclear transfer (SCNT) and parthenogenesis are two main strategies to create patient-specific cells for future stem cell therapies. In most species, preimplantation development after SCNT has been highly successful (Eggan et al., 2001; Meissner and Jaenisch, 2006; Ogura et al., 2000; Rybouchkin et al., 2002). However, up until now SCNT 1472-6483/$ - see front matter © 2010, Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights resepi/ed. doi:10.1016/j.rbmo.2010.07.001

Parthogenetic Activation

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Reproductive BioMedicine Online (2010) 21, 769-775

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ELSEVIER

ARTICLE

Developmental competence of parthenogeneticmouse and human embryos after chemical orelectrical activation

Karen Versieren*, Björn Heindryckx, Sylvie Lierman, Jan Gerris,Petra De Sutter

Department of Reproductive Medicine, Ghent University Hospital, De Pintelaan 185, 9000 Ghent, Belgium* Corresponding author. E-mail address: [email protected] (K Versieren).

Karen Versieren obtained a MSc degree in biomédical sciences at the Ghent university, Belgium. She joined theresearch team of the Department of Reproductive Medicine at the Ghent university Hospital in 2007. She iscurrently working on her PhD. Her major areas of interest include the study of parthenogenetic activation ofmouse and human oocytes and somatic cell nuclear transfer for therapeutic stem cell purposes.

Abstract Parthenogenetic reconstruction is one major strategy to create patient-specific stem cells. The aim of this study was to findthe best artificial activation protocol for parthenogenetic activation of mouse and human oocytes comparing different methods. In afirst set of experiments, in-vivo matured mouse oocytes and human failed-fertilized, in-vitro and in-vivo matured oocytes were arti-ficially activated by a chemical (ionomycin) or electrical stimulus. In a second set of experiments, a combination of activating agents(electrical pulses followed by ionomycin or SrClî) was applied in an aim to improve developmental competence. All embryos wereevaluated daily until day 6 after activation. Mouse blastocysts were differentially stained to evaluate blastocyst quality. For mouseoocytes and human failed-fertilized oocytes, blastocyst development was significantly higher after electrical activation (P < 0.05).For human in-vitro and in-vivo matured oocytes, blastocyst formation was only obtained after electrical activation of in-vitro maturedoocytes. After combining activating agents, no differences in development could be observed. In conclusion, this study revealed thatfor both mouse and human oocytes development to the blastocyst stage was significantly better after electrical activation comparedwith chemical activation. Combining activating agents had no further positive effect on developmental potential, w'" "•© 2010, Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.KEYWORDS: artificial activation, electrical pulses, ionomycin, parthenogenesis, preimplantation development

Introduction

Somatic cell nuclear transfer (SCNT) and parthenogenesisare two main strategies to create patient-specific cells for

future stem cell therapies. In most species, preimplantationdevelopment after SCNT has been highly successful (Egganet al., 2001; Meissner and Jaenisch, 2006; Ogura et al.,2000; Rybouchkin et al., 2002). However, up until now SCNT

1472-6483/$ - see front matter © 2010, Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights resepi/ed.doi:10.1016/j.rbmo.2010.07.001

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770 K Versieren et al.

in human has resulted in very little success as preimplanta-tion development of reconstructed embryos has been shownto be compromised (French et al., 2008; Hall and Stojkovic,2006; Li et al., 2009).

One of the essential steps determining the success of SCNTis artificial activation of the reconstituted oocytes (De Sousaetal. , 2002; Ma etal., 2009). An optimized activation protocolcan be obtained by studying parthenogenetic activation ofoocytes. For parthenogenesis, oocytes are artificially acti-vated by a wide range of physical and chemical stimuli in anattempt to mimic as closely as possible the Ca^* oscillationsinduced during natural fertilization (Paffoni et al., 2008). Itis now generally assumed that, when entering the oocyte,the spermatozoon introduces a soluble protein factor intothe ooplasm. This sperm factor, identified as phospholipaseC Ç, initiates Ca^* release in the oocyte, leading to Ca *̂ oscil-lations (Heytens et al., 2009; Saunders et al., 2002; Swannet al., 2004). Given most artificial activating agents only causea single and prolonged rise in cytosolic Ca'̂ * concentrations,their efficiency should be further explored (Alberio et al.,2001 ; Kline and Kline, 1992; Whittingham and Siracusa, 1978).

Human oocytes can be successfully activated by differentstimuli but generally, parthenogenetic embryos arrest in theearly stages of preimplantation development (Brevini andGandolfi, 2008; De Sutter et al., 1992, 1994; Rinaudo et al.,1997; Taylor and Braude, 1994; Winston et al., 1991). Mostof these studies were performed using aged failed-fertilizedoocytes after IVF or intracytoplasmic sperm injection (ICSI).In contrast, a few studies have succeeded in parthenogeneticblastocyst formation when in-vivo matured human oocyteswere used (Cibelli et al., 2001; de Fried et al., 2008; Linet al., 2003; Mai et al., 2007; Paffoni et al., 2007; Revazovaet al., 2007). Still, there was a big variance in blastocyst for-mation and not all obtained blastocysts possessed a visibleinner cell mass (ICM).

Due to ethical restrictions, there is a limited availabilityof in-vivo matured human oocytes for research (Müller andLengerke, 2009). Consequently, there is a need to explorealternative oocyte sources, such as failed-fertilized andin-vitro matured human oocytes (De Vos et al., 1999;Mahutte and Arici, 2003; Shu et al., 2007). These oocytesare normally discarded after IVF or ICSI treatment, but theycan be a valuable source of human oocytes for research. Thecurrent study aimed to find the best artificial activation pro-tocol for parthenogenetic activation of mouse and humanoocytes by comparing different methods within one experi-mental setup, excluding all environmental and experimentalvariances. The study first examined the effects of chemicaland electrical artificial activation on the developmentalpotential of in-vivo matured mouse oocytes. Additionally,the effects of the same activation methods on humanoocytes that failed to fertilize after IVF or ICSI treatmentand on in-vitro and in-vivo matured human oocytes wereinvestigated. In a second set of experiments, extra activat-ing agents were added in combined treatments in an aim toimprove developmental competence.

Materials and methods

All chemicals and reagents were purchased fromSigma-Aldrich (Bornem, Belgium), unless otherwise stated.

Source of mouse oocytes

Mice were purchased from Charles River Laboratories(Brussels, Belgium) and handled according to the guidelinesof the Animal Ethical Committee of the Ghent UniversityHospital. Mice were kept under controlled temperatureand lighting conditions. Food and water were available adlibitum.

Female B6D2F1 hybrid mice aged 7-14 weeks were stimu-lated to superovulate by intraperitoneal injection of 5 lUequine chorionic gonadotrophin (FoUigon; Intervet, Oss,The Netherlands) followed by 5 lU human chorionic gonado-trophin (HCG; Chorulon; Intervet) 48 h later. Oocytes werecollected 14 h post-HCG and freed from cumulus cells by ashort incubation in 200 lU/ml hyaluronidase (type VIII).Oocytes were kept in home-made potassium simplex opti-mized medium (KSOM) supplemented with 0.4% bovine serumalbumin (BSA; Calbiochem, Bierges, Belgium) at 37°C under6% CO2 until artificial activation (Lawitts and Biggers, 1991).

Source of human oocytes

All human oocytes were obtained from stimulated patientsundergoing IVF or ICSI treatment at the study departmentafter written informed consent as approved by the EthicalCommittee of the Ghent University Hospital and the FederalEthical Committee on embryos in vitro.

In-vivo matured oocytes ('in-vivo oocytes') were donatedby one 32-year-old patient whose azoospermic partnershowed absence of spermatozoa in the testicular biopsy atthe day of oocyte retrieval. Failed-fertilized oocytes werecharacterized by the lack of visible pronuclei (PN) and sec-ond polar body (PB) 16-20 h after IVF or ICSI treatment andwere subjected to activation stimuli between 61—64 hpost-HCG.

In-vitro matured oocytes ('IVM oocytes') were obtainedafter immature oocytes were collected from patients whohad at least six mature oocytes available for their infertilitytreatment. Oocytes were denuded with 80 lU/ml hyaluroni-dase (type VIII) and were classified by the presence of agerminal vesicle (GV), absence of both GV and first PB orthe presence of the first PB as GV, Ml or MM, respectively.GV oocytes were matured for 24 or 48 h in TCM-199 mediumsupplemented with 10 ng/ml epidermal growth factor,1 |.ig/ml oestradiol, 10 mlU/ml FSH (Puregon; Organon, Oss,The Netheriands), 0.5 lU/ml HCG (Pregnyl; Organon),1 mmol/l L-glutamine, 0.3 mmol/l sodium pyruvate, 0.8%human serum albumin (Belgian Red Cross, Brussels, Belgium),100 lU/ml penicillin G and 100 ng/ml streptomycin sulphate.Ml oocytes were matured for 24 h in Cook cleavage medium(Cook Ireland Ltd., Limerick, Ireland). In-vitro maturation(IVM) was performed at 37°C under 6% CO2.

Double activating compounds

In-vivo matured mouse oocytes were randomly distributedto one of the following activation treatments: (i) I + CHX:5 min exposure to 10 |.imol/l ionomycin (I) + 4 h incubationin 10 ng/ml cycloheximide (CHX); (ii) I + DAAAP: 5 minexposure to 10 |.imol/l I + 4 h incubation in 2 mmol/l 6-dime-thylaminopurine (DA^AP); (iii) EP + DMAP: electrical pulses

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Artificial activation of mouse and human oocytes 771

(2x1.6 kV/cm, 30 us each, 100 ms pulse interval; ECM 830;BTX, San Diego, CA, USA)+ 4 h incubation in 2mmol/lDAAAP; and (iv) SrClz: 4 h incubation in 10mmol/l SrCl2 inCa-free KSOM-BSA supplemented with 2 ^ig/ml cytochala-sine D (positive control). Non-treated oocytes were usedas a negative control for spontaneous activation. Humanfailed-fertilized oocytes, IVM oocytes and in-vivo oocyteswere activated by treatments I + CHX and EP + DMAP.

Triple activating compounds

Mouse and human oocytes were randomly distributed to oneof the following treatment groups: (i) EP + DAAAP: electricalpulses + 4 h incubation in 2 mmol/l DMAP; (ii) EP + I + DMAP:electrical pulses + 5 min exposure to 5 nmol/l I + 4 h incuba-tion in 2 mmol/l DMAP; and (iii) EP + SrCl2 + DAAAP: electri-cal pulses + 1 h exposure to 10 mmol/l SrCl2 in Ca-freeKSOM-BSA supplemented with 2 ng/ml cytochalasine D + 4 hincubation in 2 mmol/l DMAP.

Embryo culture

Oocytes were cultured in KSOM-BSA (mouse) or Cook cleav-age medium (human) at 37°C under 6% CO2 and 5% O2. Alloocytes were transferred to Cook blastocyst medium (Cook,Ireland) on day 3.

Blastocyst analysis

To evaluate blastocyst quality, differential staining of ICMand trophectoderm cells (TE) of mouse blastocysts was per-formed (Thouas et al., 2001 ). Briefly, blastocysts were incu-bated in 500 t̂ il HEPES-buffered human tubal fluid medium(Lonza, Verviers, Belgium) with 1% Triton X-100 and100jig/ml propidium iodide for 10 s, transferred to 1 mlchilled 100% ethanol with 25 [.ig/ml bisbenzimide (Hoechst33258) and stored at 4°C overnight. Blastocysts weremounted onto a glass slide in glycerol and covered with acover slip. Numbers of ICM (blue), TE (red) and total cell num-ber (TCN) were counted under a fluorescence microscope.

Statistical analysis

Statistical analysis was performed with InStat from Graph-Pad Software. Activation, cleavage and preimplantationdevelopmental data were analysed by contingency tableanalysis followed by chi-squared test or Fisher's exact testfor independence. The level of significance was set atP < 0.05. Parameters of blastocyst quality (ICM, TE, TCNand ICM/TE rates) were compared using one-way analysisof variance (ANOVA) followed by Tukey post-test when thelevel of significance reached P < 0.05.

Results

Source of human oocytes

A total of 277 GV and 201 Ml human oocytes were collectedfor this study. The maturation rate of GV and Ml oocytesafter 24 h was 44% and 87%, respectively. The global

maturation rate of GV oocytes after 48 h was 72%. Onlyoocytes that reached mature Mil stage were used for artifi-cial activation. Out of the 309 collected failed-fertilizedoocytes, 97 were post-IVF and 212 were post-ICSItreatment.

Double activating compounds

For mouse oocytes, there was no significant difference inactivation and cleavage potential between the differentgroups after chemical or electrical activation (Table 1).However, mórula and blastocyst rate were significantlyhigher in the EP + DAAAP group (95% and 88%) and the SrCl2group (91% and 89%) compared with the I + CHX group (75%and 70%) and the I + DAAAP group (72% and 71%; P < 0.05).

Also for human oocytes, no differences were detected inactivation, cleavage and mórula rate between the twotreatment groups (Table 2). Blastocyst formation offailed-fertilized oocytes was significantly higher in theEP + DMAP group compared with the I + CHX group (10% ver-sus 1% respectively; P<0.05). No differences weredetected in activation efficiency or preimplantation devel-opment between failed-fertilized oocytes originating fromIVF or ICSI treatment. For IVM oocytes, only in theEP + DAAAP group could blastocysts be obtained. Althoughboth of these blastocysts were derived from IVM Ml oocytes,no significant differences in development were foundbetween GV oocytes and Ml oocytes. In-vivo oocytes failedto give rise to blastocyst formation in both the I + CHX groupand the EP + DAAAP group.

Differential staining was performed on obtained mouseblastocysts (Table 3). Numbers of ICM, TE and TCN were sig-nificantly higher in the I + CHX group compared with theother treatment groups. In the I + DMAP, EP + DMAP andSrCl2 groups, ICM and TE were comparable, but TCN was sig-nificantly lower in the SrCl2 group. The ICM/TE ratios werenot different between all activation treatments.

Triple activating compounds

In Tables 4 and 5, the preimplantation development ofmouse and human embryos after the different triple acti-vating compound treatments is presented. There were nodifferences detected in activation, cleavage, mórula andblastocyst rate between the different activation groups.The blastocysts in the group of the human IVM oocytes werederived from one IVM Ml oocyte (EP + DAAAP) and one GVoocyte matured for 24 h (EP + U DMAP).

The mouse blastocysts that were obtained were differen-tially stained. The numbers of ICM, TE and TCN and ICM/TEratios were not significantly different between any treat-ment group (data not shown).

Discussion

Although induced pluripotent stem cells have drawn atten-tion away from SCNT, parthenogenesis and SCNT are stillpromising tools to establish embryonic stem cells for futurepatient-specific cell treatments. One of the crucial steps forboth strategies involves artificial activation. The aim of thisstudy was to find the best activation protocol for mouse and

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772 K Versieren et a l .

Table 1 Preimplantation

Treatment

Non-treatedI + CHXI + DAAAPEP + DAAAP

SrClz

Totaloocytes

7584668483

development of mouse embryos after

Activated oocytes(% of total)

9(12)"83 (99)"65 (98)"83 (99)"79 (95)"

Cleaved oocytes(% of activated)

9 (100)81 (98)61 (94)81 (98)75 (95)

chemical

Mórula

or electrical activation.

Blastocyst(% of activated) (% of activated)

0(0)"62 (75)"47 (72)"79 (95)'72 (91 f

0(0)"58 (70)"46 (71 )"73 (88)'70 (89)'

"•"'Different superscripts within a column indicate significant difference (P< 0.05).CHX = cycloheximide; DAAAP = 6-dimethylaminopurine; EP = electrical pulses; I = ionomycin.

Table 2 Preimplantation development of human

Treatment

1 +CHXff-oocytesIVM oocytesIn-vivo oocytes

EP + DMAPff-oocytesIVM oocytesIn-vivo oocytes

Totaloocytes

85959

8695

7

Activated oocytes(% of total)

76 (89)71 (75)9 (100)

70(81)73 (77)7(88)

embryos after chemical or electrical

Cleaved oocytes(% of activated)

72 (95)62 (87)9 (100)

67 (96)66 (90)7 (100)

Mórula

f?

421

871

a of activated)

(5)(3)(11)

(11)(10)(14)

activation.

Blastocyst

("y

100

720

0 of activated)

{If(Of(0)

(10)"(3)(0)

"•"Different superscripts within a column indicate significant difference (P < 0.05).CHX = cycloheximide; DMAP = 6-dimethylaminopurine; EP = electrical pulses; ff = failed-fertilized; I = ionomycin;IVM = in-vitro matured.

Table 3 Differential staining

Treatment

I + CHX

I + DMAP

EP + DMAP

Totalblastocysts

56467069

of mouse blastocysts after chemical

ICM

26.3019.5917.3615.06

±9.30"±9.20"±6 .52" '± 6.03'

TE

98.09 ± 34.87"75.35 ±20.35"69.27 ±14.51"^'60.55 ±17.35'

or electrical activation.

TCN

124.39 ±36.61"94.93 ± 24.95"86.63 ±15.57"75.61 ± 17.06'

ICM/TE

0.30 ±0.140.27±0.120.26 ±0.120.27±0.13

Values are expressed as mean + SD."•"•'Different superscripts within a column indicate significant difference (P < 0.05).CHX = cycloheximide; DMAP = 6-dimethylaminopurine; EP = electrical pulses; I = ionomycin; ICM = innercell mass; TCN = total cell number; TE = trophectoderm.

Table 4 Preimplantation development of mouse embryos after treatment with triple activating compounds.

Total Activated oocytes Cleaved oocytes Mórula Blastocystoocytes (% of total) (% of activated) (% of activated) (% of activated)

Treatment

Non-treatedEP + DAAAPEP + I + DAAAPEP + SrClz + DAAAP

40454545

4(10)"43 (96)"45 (100)"42 (93)"

4 (100)42 (98)44 (98)42 (100)

0(0)"38 (88)"37 (82)"39 (93)"

"•"Different superscripts within a column indicate significant difference (P < 0.05).DAAAP = 6-dimethylaminopurine; EP = electrical pulses; I = ionomycin.

0 (0)"38 (88)"37 (82)"38 (90)"

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Art i f ic ial act ivat ion of mouse and human oocytes 773

Table 5 Preimplantation development of human

Treatment

EP + DAAAPff-oocytesIVM oocytes

EP + 1 + DAAAPff-oocytesIVM oocytes

EP + SrCl2 + DMAPff-oocytesIVM oocytes

Totaloocytes

4750

4650

4550

embryos after treatment with tr iple activating compounds.

Activated oocytes Cleaved oocytes(% of total)

43 (91)43 (86)

44(96)42(84)

40 (89)40 (80)

(% of activated)

42 (98)42 (98)

42 (95)38 (90)

40 (100)35 (88)

Mórula Btastocyst(% of activated) (% of activated)

9(21)

3m

5(11)6(14)

8(20)2(5)

4 (9 )1 (2)

3(7)1 (2)

5(13)0 (0)

DAAAP = 6-dimethylaminopurine; EP = electrical pulses; ff = failed-fertilized; I = ionomycin; IVM = in-vitro matured.No statistically significant differences were found.

human oocytes comparing di f ferent methods. It was foundthat both mouse and human oocytes can develop into blas-tocysts af ter chemical or electr ical act ivat ion. However,blastocyst format ion was significantly higher af ter electr icalact ivat ion. Combining activat ing agents had no fur therpositive ef fect on preimplantat ion development.

In mice, the most preferred act ivat ion protocol has beenreported to be SrCl2 because i t induces Ca^* oscillations andhas been reported very eff ic ient in both parthenogenesisand SCNT (Bos-Mikich et a l . , 1995; Kline and Kline, 1992;Rybouchkin et a l . , 2002). However, the use of SrCl2 as anactivating agent for human oocytes is st i l l under debate(Kyono et a l . , 2008; Rogers et a l . , 2004; Yanagida et a l . ,2006). Therefore al ternat ive act ivat ing agents are neces-sary. In the current study, SrCl2 was used as a positive con-t ro l for mouse parthenogenesis to compare the eff iciency ofchemical and electr ical act ivat ion. The results suggest thatelectr ical pulses combined wi th DAAAP are as ef fect ive asSrCl2 in terms of mouse oocyte act ivat ion and embryo devel-opment. Blastocyst rate was comparable in both groups(89% for SrCl2 and 88% for EP + DAAAP). On the other hand,act ivat ion w i th ionomycin resulted in similar act ivat ionand cleavage rates but blastocyst format ion was signifi-cantly lower compared w i th the two previous groups (70%for I + CHX and 71% for I + DMAP). The current results con-f i rm that preimplantat ion development can be influencedby the altered calcium patterns caused by the di f ferentactivat ing agents (Ducibella et a l . , 2002; Toth et a l . , 2006).

The qual i ty of mouse blastocysts was evaluated by dif fer-ent ial staining and counting the number of cells in ICM andTE. The process of embryonic d i f ferent ia t ion into ICM andTE is essential for implantat ion and normal fur ther develop-ment (Van Soom et a l . , 2001 ). The quali ty of blastocysts wascomparable between all t reatment groups since there wasno dif ference in the ICM/TE rates. In the two groups w i ththe highest blastocyst format ion rate (EP + DAAAP and SrCl2),numbers of ICM and TE were similar, but TCN was signifi-cantly lower in the SrCb group. For this reason, EP + DAAAPcan be considered to be the best act ivat ion technique formouse oocytes in this study. Based on the results in themouse, this study wanted to veri fy if e lectr ical t reatmentwould also be superior than chemical t reatment in human.Because of the scarce avai labi l i ty of human oocytes forresearch, only one chemical t reatment was tested on human

oocytes. Since l i terature has shown a bet ter result forhuman fa i led-fer t i l ized oocytes w i th calcium ionophore fo l -lowed by CHX than by DAAAP, EP + DAAAP was compared w i thI + CHX on human oocytes (McElroy et a l . , 2008).

Human blastocyst format ion af ter art i f ic ia l act ivat ion ofin-vivo matured oocytes has been accomplished mostly byusing a combination of calcium ionophores and DAAAP(Cibelli et a l . , 2001; Paffoni et a l . , 2007). In the currentstudy, no blastocysts were obtained, but the number ofavailable in-vivo matured oocytes was small . In-vivomatured oocytes in this study could only be obtained f romone pat ient whose azoospermic partner showed absenceof spermatozoa in the testicular biopsy on the day of oocyteretr ieval . Since this was the only in fer t i l i ty t reatment cycleof this pat ient , there is no fur ther informat ion on embryoqual i ty af ter ICSI. This might be of importance since thedevelopment of human parthenogenetic embryos may beaf fected by donor peculiarit ies (Revazova et a l . , 2007).Other reasons why blastocysts were not obtained f rom thein-vivo matured oocytes might be the age of the donor(32 years old) and the fact that by the t ime these oocyteswere donated for research (around 6 h af ter oocyteretr ieval) , they were already start ing to age. This mighthave diminished their quali ty since i t has been shown thatthe best donors are l ikely to be younger women and thatoocytes should be used as quickly as possible af ter retr ieval(French et a l . , 2008; Heindryckx et a l . , 2007; Revazovaé t a l . , 2007).

An al ternat ive source of human oocytes for research con-sists of fa i led-fer t i l ized oocytes. Despite the fact that theseoocytes are aged and frequent ly contain a spermatozoon intheir cytoplasm, they form a valuable source of oocytes tostudy act ivat ion. The l i terature shows that parthenogeneticembryos f rom fa i led-fer t i l ized oocytes mostly arrest aroundembryo genome act ivat ion (Brevini and Gandolf i , 2008; DeSutter et a l . , 1992, 1994; Rinaudo et a l . , 1997; Taylor andBraude, 1994; Winston et a l . , 1991). Nevertheless, blasto-cysts f rom fa i led-fer t i l ized oocytes have been producedafter act ivat ion w i th a calcium ionophore combined w i thCHX (McElroy et a l . , 2008). A cleavage rate of 42% wasreported and 14% of cleaved embryos reached the blasto-cyst stage. However, the to ta l number of oocytes used inthe study was very small . In contrast, the current studyobserved a 95% cleavage rate in the I + CHX group but only

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774 K Versieren et al.

1% reached the blastocyst stage. Another study reported ondevelopment of failed-fertilized oocytes after electricalpulses (Zhang et al., 1999). No blastocysts could beobtained, unless repetitive stimulations were used (11%).The current study also reported 10% blastocyst formationof failed-fertiUzed oocytes in the EP + DMAP group.

This study's data demonstrate that both chemical andelectrical treatment can activate oocytes that failed to fer-tilize after IVF or ICSI and stimulate them to undergo earlyembryonic development. Since blastocyst formation wassignificantly higher in the EP -̂ DAAAP group (10%) comparedwith the I + CHX group (1%), the results suggest thatEP -̂ DAAAP would be the activation treatment of choice forfailed-fertilized oocytes. The data presented here also sup-port the suggestion that artificial activation can be useful incases of failed or low fertilization after ICSI. Since the sper-matozoon is injected directly Into the oocyte, failed fertil-ization after ICSI is mainly the result of an oocyte activationdeficiency (Mahutte and Arici, 2003). Several studies haveshown that normal fertilization and pregnancy could beachieved in patients with previous failed fertilization byusing artificial activation (Heindryckx et al., 2005;Nasr-Esfahani et al., 2008; Rybouchkin et al., 1997;Yanagida et al., 1999). For these assisted oocyte activa-tions, different stimuli were used, but no comparison oftheir efficiency has been reported. The current resultssuggest that electrical pulses may be superior to calciumionophores for assisted oocyte activation techniques.

A last source of interest is the group of human IVM oocytes.Several studies reported decreased rates of fertilizationwhen IVM oocytes are used for ICSI (Balakier et al., 2004;De Vos et al., 1999). Additionally, even 1n the case of normalfertilization, further developmental competence of theembryos is often compromised (Shu et al., 2007). The useof immature human oocytes for parthenogenesis has not beenwell described. One recent study reported formation of oneblastocyst after a combined electrical/chemical activationprotocol (Yu et al., 2009). The current results confirm thesuggestion that IVM oocytes have a decreased developmentalpotential, since only two blastocysts could be obtained afterEP + DAAAP treatment. Activation with I + CHX failed to giverise to blastocyst development from IVM oocytes.

This study showed major differences between the devel-opmental rate of activated mouse and human oocytes. Oneexplanation Is that human oocytes are more prone to usedenvironmental conditions during in-vitro embryo culture.For example, literature has shown that by using a lower oxy-gen concentration and a different culture medium, thepercentage of blastocyst formation after parthenogenesiscan be altered (Revazova et al., 2007). Mouse embryos areless affected by these types of changes and mouse partheno-genetic embryos are known to have a very high developmen-tal potential/nv/tro(Bos-Mikich etal . , 1995; Kline and Kline,1992). Another important difference is the quality of the usedmouse and human oocytes. Mouse oocytes were freshlycollected and activated within 2 h after retrieval. For humanoocytes, only a small number of in-v1vo matured oocyteswas available for this study. This led the current studyto the use of other oocyte sources, such as failed-fertilized oocytes and IVM oocytes, which are of inferior qual-ity. In a second experiment, combined activation protocolswere investigated in an aim to Improve developmental

potential. The results showed no differences in activationrate, cleavage rate or development to the blastocyst stageafter combining the electrical activation with treatment withionomycin or SrCl2. Also the quality of mouse blastocysts wasnot affected.

In conclusion, statistical analysis of blastocyst formationand quality suggests that electrical activation has a highersuccess rate than chemical activation, for mouse oocytes aswell as for human oocytes. Combining the electrical activa-tion in triple activating compound treatments had no furtherpositive influence on developmental competence. Since thebase of this study was to find the best activation protocolfor SCNT, the use of EP -̂ DMAP is proposed as an activationtechnique. However, for the future it might be worthwhileto investigate other activating agents which mimic moreclosely the Ca^* pattern seen during natural fertilization,for instance use of recombinant phospholipase C Ç.

Acknowledgements

The authors would like to thank the colleagues of the IVFlaboratory for being willing to collect human oocytes. K.V.is supported by the Special Research Foundation (BOF, nr.01D32707) of the Ghent University, Belgium. P.D.S. is holderof a fundamental clinical research mandate by the FlemishFoundation of Scientific Research (FWO-Vlaanderen),Belgium.

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Declaration: The authors report no financial or commercialconflicts of interest.

Received 3 March 2010; refereed 24 June 2010; accepted 1 July2010.

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