CLONINGVolume 1, Number 3, 1999Mary Ann Liebert, Inc.

Comparison of Oocyte-Activating Agents for Mouse Cloning

HIDEFUMI KISHIKAWA, TERUHIKO WAKAYAMA, and RYUZO YANAGIMACHI

ABSTRACT

Since somatic cell components are unable to activate oocytes following injection or fusion,enucleated oocytes receiving adult somatic cells during the cloning process must be activatedartificially for their development. We compared the efficiency of four types of oocyte-acti-vating agents: strontium, ethanol, single electric pulse, and spermatozoa. Although strontiumwas the best in supporting preimplantation development of reconstructed mouse oocytes,there was no significant difference among the four agents with respect to the rate of postim-plantation embryo development and the birth of live offspring.

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INTRODUCTION

M AMMALIAN CLONING using adult somaticcells has been successful in the sheep

(Wilmut et al., 1997), mouse (Wakayama et al.,1998) and cattle (Kato et al., 1998; Wells et al.,1999), but the overall success rate is still ratherlow. Many cloned embryos and fetuses die dur-ing pre- and postimplantation development, andperinatal death of fetuses/young is not uncom-mon. The causes of these problems are yet to bedetermined.

Oocyte activation is a crucial step in the cloningprocedure. Since mammalian somatic cells (in-cluding their nuclei) are unable to activateoocytes (Czolowska et al., 1984; Szollosi et al.,1986, 1988), reconstructed oocytes must be artifi-cially activated for their development. There area variety of agents that activate mammalianoocytes (Graham, 1974; Tarkowski, 1975; Whit-tingham, 1980; Kaufman, 1983; Yanagimachi,1994). The choice of the oocyte-activating methodfor cloning has been largely empirical. Electriccurrent (Wilmut et al., 1997; Baguisi et al, 1999),

electric current plus protein synthesis inhibitor(Kato et al., 1998), calcium ionophore plus pro-tein kinase inhibitor (Cibelli et al., 1998), iono-mycin plus protein kinase inhibitor (Wells et al.,1999), and strontium (Sr2 1 ) (Wakayama et al.,1998) have been used for cloning of the sheep,goat, cattle, and mouse using somatic cells. In thepresent study, we compared four oocyte-activat-ing agents (strontium, ethanol, single electricpulse, and spermatozoa) to see if they are equallyeffective for mouse cloning.

MATERIALS AND METHODS

Animals

Eight- to 12-week old B6D2F1 (C57BL/6 3DBA/2J) females, black eye and black coat, wereused as oocyte, sperm, and cumulus cell donors.All mice were purchased from the National Can-cer Institute and maintained in a temperature-and light-controlled room (14-h light/10-h dark)for at least 2 weeks before use. All animals were

Department of Anatomy and Reproductive Biology, University of Hawaii Medical School, Honolulu, Hawaii.

maintained in accordance with the guidelines ofthe Laboratory Animal Service at the Universityof Hawaii and those prepared by the Committeeon Care and Use of Laboratory Animals of the In-stitute of Laboratory Resources National Re-search Council (DHEW publication no. [NIH] 80-23, revised in 1985). The protocol of our animalhandling and treatment was reviewed and ap-proved by the Animal Care and Use Committeeat the University of Hawaii.

Reagents

Polyvinyl alcohol (PVA; cold-water-soluble,molecular weight ca. 10,000) and polyvinylpyrrolidone (PVP; molecular weight ca. 360,000)were purchased from Sigma Chemical Co. (St.Louis, MO). Bovine testicular hyaluronidase (200USP U/mg) was obtained from ICN Biochemi-cals (Costa Mesa, CA). BSA (fraction V) was pur-chased from Calbiochem (La Jolla, CA) and min-eral oil from Squibb and Sons (Princeton, NJ). Allother reagents were obtained from Sigma unlessotherwise stated.

Media

The medium used for the culture of mouseoocytes after microsurgery was CZB medium(Chatot et al., 1990) supplemented with 5.56mM D-glucose. The medium for collection ofoocytes from oviducts and subsequent oocytetreatments, including micromanipulation, wasa modified CZB [Hepes-CZB, (Kimura andYanagimachi, 1995)] containing 20 mM Hepes-HCl, a reduced amount of NaHCO3 (5 mM), and0.1 mg/mL PVA instead of BSA. CZB was usedunder 5% CO2 in air, and Hepes-CZB was usedunder air.

Collection of mature oocytes and cumulus cells

B6D2F1 females were each injected with 5 IUeCG followed by 5 IU hCG 48 h later. Matureoocytes surrounded by cumulus cells were col-lected from oviducts about 14 h after hCG injec-tion. Cumulus cells were dispersed by 5-mintreatment with 0.1% (w/v) bovine testicularhyaluronidase in Hepes-CZB and transferred toHepes-CZB containing about 12% (w/v) PVP,where they were kept at room temperature for upto 4 h before injection. Cumulus-free oocytes wererinsed and kept in CZB medium at 37°C for , 1h under 5% CO2 in air before use.

Enucleation of mature oocytes followed byinjection of cumulus cell nuclei

Cumulus-free oocytes were transferred into adroplet of Hepes-CZB containing 5 m g/mL cy-tochalasin B (CB) in the operation chamber(Kimura and Yanagimachi, 1995) on the micro-scope stage, and kept there for 5–10 min. Enu-cleation was performed by aspirating the meta-phase II chromosome-spindle complex into apipette (8–10 m m in the inner diameter) with aminimal volume of oocyte cytoplasm (Wakayamaet al., 1998). Enucleated oocytes were transferredinto cytochalasin-free CZB and kept there for upto 3 h at 37.5°C before injection of cumulus cellnuclei.

Nucleus injection was carried out according toWakayama et al. (1998). Briefly, cumulus cells ofrelatively small size (9–12 m m) were selected, andtheir nuclei were separated from cytoplasm bygently aspirating in and out of the injectionpipette (inner diameter: 5–7 m m). Isolated nuclei,with or without associated cytoplasm, were indi-vidually injected into oocytes. All operationswere carried out at room temperature in Hepes-CZB. Reconstructed oocytes were kept in CZB for1–3 h at 37°C under 5% CO2 in air before activa-tion. During this period, cumulus cell nucleitransformed into loosely arranged chromosomes(Wakayama et al., 1998).

Oocyte activation

Reconstructed oocytes that had been kept inCZB for 1–3 h were activated in four differentways 18–20 h after hCG administration. Prelimi-nary experiments revealed that the variation inthe interval between nucleus injection and oocyteactivation (1–3 h) did not have any significant ef-fects on the results. In the strontium-activatedgroup, oocytes were treated for 6 h in Ca2 1 -freeCZB containing both 10 mM Sr2 1 and 5 m g/mLCB. Sr2 1 induced oocyte activation, while CB pre-vented extrusion of chromosomes into polar bod-ies so that the complete set of chromosomes fromcumulus cell nucleus was retained (Wakayama etal., 1998). Treatment with Sr2 1 for 30 min wasenough to activate oocytes (Yoshimizu et al.,1998), although prolonged treatment (up to 24 h)did not produce adverse effects (Bos-Mikich et al.,1997). After the Sr2 1 treatment, oocytes werewashed and cultured in CZB. When ethanol wasused as the activating agent, oocytes were treatedfor 5 min at room temperature in Hepes-CZB con-

KISHIKAWA ET AL.154

taining 7% (v/v) ethanol, rinsed with CZB, main-tained in CZB with 5 m g/mL CB for 6 h, then cul-tured in CZB. When an electric pulse was usedto activate, oocytes in Hepes-CZB were trans-ferred to a chamber with electrodes spaced 1 mmapart, and a single electric pulse (direct current,1 kV/cm, 130 m sec) was applied. Immediately af-ter electric pulse application, oocytes were rinsedwith CZB, then maintained in CZB with 5m g/mL CB for 6 h before cultured in CZB. Fi-nally, in sperm-mediated activation, an enucle-ated oocyte was injected with a cumulus cell nu-cleus, left in CZB for 1–3 h, then injected with asingle sperm head. The sperm head was sepa-rated from the tail immediately before injection(Kuretake et al., 1996). Ten min after sperm in-jection, oocytes were transferred to CZB con-taining 5 m g/mL CB. When examined 6 h later,one to three relatively small pseudo-pronuclei ofcumulus cell origin (Wakayama et al., 1998) plusone large pronucleus of sperm origin were seenin each oocyte. The pronucleus was removedwith some accompanied cytoplasm. The oocytewas then cultured in CZB. Differentiation be-tween pseudo-pronuclei (of cumulus cell origin)and sperm pronucleus was sometimes difficult.Such oocytes were discarded and not includedin the data. In a series of experiments, cumulusnucleus–injected oocytes were inseminated withcapacitated, intact spermatozoa (Toyoda et al.,1971). Six hours after insemination, one to threesmall pseudo-pronuclei and one large spermpronucleus were seen commonly within anoocyte. In some instances, the removal of thesperm pronuclei from such oocytes were not suc-cessful because the sperm pronucleus was sotightly associated with a large tail that a fairlylarge amount of cytoplasm had to be removedwith the sperm pronucleus.

In a separate series of experiments, cumulus-free oocytes without any treatments (e.g., no enu-cleation and no cumulus cell injection) were acti-vated exactly as described above and cultured inCZB to determine how efficiently Sr2 1 , ethanol,electric pulse, and the spermatozoa are able to ac-tivate intact oocytes.

Examination of oocytes and embryos

About 6 h after the onset of activation treat-ment, cumulus-injected oocytes were examinedusing an inverted microscope with Hoffman interference-contrast optics. Some oocytes were

fixed and stained for cytological details (Yana-gida et al., 1991). Those with distinct pronucleiwere considered activated. Embryos developingin vitro were examined using an inverted micro-scope 74–76 h after activation.

Embryo transfer and examination of fetuses

The embryos reaching the morula or blastocyststage by the third day of culture were transferredinto the uteri of CD-1 (albino) females, which hadbeen mated 3 days previously with vasectomizedmales of the same strain. An average of 5 embryoswere transferred to each uterus. Unless specifi-cally mentioned, all females were euthanized at19.5 day postcoitum (d.p.c.), and the number ofimplantation sites, the number of live fetuses, andthe weights of fetuses and placentas were deter-mined. Live young, all with black eyes as ex-pected, were raised by lactating CD-1 fostermothers.

Statistical analysis

Statistical analysis was done using the x 2 test,Fisher’s exact probability test, and Kruskal-Wal-lis test.

RESULTS

The ability of the four different agents (Sr2 1 ,ethanol, electric pulse, and sperm) to activate in-tact mouse oocytes was assessed. Since theoocytes were activated in the presence of CB, alloocytes developed into diploid parthenogeneticembryos. Regardless of the type of oocyte-acti-vating agents used, the majority (93–99%) of em-bryos reached the morula/blastocyst stage in 3days (Table 1).

Table 2 shows the number of pronuclei in cu-mulus nucleus–injected oocytes 5–6 h after acti-vation in the continuous presence of CB. The ma-jority (78–85%) of activated oocytes had twopronuclei, whereas others had one or threepronuclei. There were no significant differencesin the number of pronuclei per oocyte among fourdifferent groups of oocytes receiving different ac-tivation stimuli.In vitro development of cumulus-injected

oocytes is summarized in Table 3. The proportionof embryos that developed into morulae/blasto-cysts was significantly higher in the Sr2 1 groupthan in others (p , 0.0001). There was no distinct

MOUSE CLONING 155

difference in gross morphology of embryos in allexperimental groups.

When morulae and blastocysts were trans-ferred to foster mothers, six apparently normalterm fetuses were obtained (Table 3). There wereno significant differences in the efficiency of em-bryo development to term among the three ex-perimental groups (Sr2 1 , ethanol, and electricpulse). Placentae of cloned fetuses were larger(210–460 mg) than those of normal fetuses(120–150 mg; Wakayama and Yanagimachi,1999). All offspring developed into normal fertilefemales with the same black eyes and black coatsas nucleus donors. Table 4 summarizes the resultsof experiments in which cumulus-injectedoocytes were activated by spermatozoa. Only onelive fetus at 12.5 d.p.c was obtained in these ex-periments.

DISCUSSION

Under normal conditions, mammalian oocytesare activated by fertilizing spermatozoa. Themechanisms by which a fertilizing spermatozoonactivates an oocyte are still subject to debate.Some investigators maintain that ligand-receptorinteractions of gametes’ plasma membranes trig-

ger G-protein/IP3 signal transduction cascade toliberate Ca2 1 from intracellular Ca2 1 storage sites(Foltz and Schilling, 1993; Schultz and Kopf,1995). Increased intracellular Ca2 1 , which inacti-vates the oocyte’s cytostatic factor (CSF) andmetaphase-promoting factor (MPF), is believed tobe a key event of oocyte activation (Kubiak et al.,1993). Other investigators believe that a specificfactor (or protein) is brought into the oocyte bythe fertilizing spermatozoon and this factor (pro-tein) acts directly on the Ca2 1 -releasing mecha-nism within the oocyte (Swann, 1990; Swann andLai, 1997). The fact that human and mouseoocytes can be activated readily by direct sperminjection into oocytes (Van Steirteghem et al.,1993; Kimura and Yanagimachi, 1995) seems tosuggest that oocyte activation can occur withoutthe interaction of gametes’ plasma membranes.Although the search for the sperm-specific factor(sperm-borne oocyte-activating factor [SOAF]) isin progress (Parrington et al., 1996; Kimura et al.,1998; Sette et al., 1998; Perry et al., 1999), at leastthe mouse SOAF appears or becomes biologicallyactive during spermiogenesis because roundspermatids are unable to activate oocytes, whiletesticular spermatozoa are fully competent to doso (Kimura et al., 1998). Like round spermatids,somatic cells are unable to activate oocytes. For

KISHIKAWA ET AL.156

TABLE 1. PATHOGENETIC DEVELOPMENT OF MOUSE OOCYTES

No. (%) of morulae (M)Oocyte- No. of oocytes No. (%) of and blastocysts (B)activating stimulated oocytes after 3 days ofagent (no. of exp.) activated in vitro culture

M BSr2 1 94 (5) 94 (100) 28 1 65 (98.9)b

EtOH 58 (4) 56 (96.6) 20 1 32 (92.9)b

Electric 61 (4) 60 (98.4) 14 1 42 (93.3)b

Sperma 59 (2) 59 (100) 47 1 8 (93.2)b

aInjected microsurgically.bFisher’s exact probability test: n.s.

TABLE 2. NUMBER OF PRONUCLE I IN CUMULUS-INJECTED OOCYTES ACTIVATED IN THE CONTINUOUS PRESEN CE OF CB

Oocyte- No. of No. (%) ofactivating oocytes oocytesagent examined activated One pronucleus Two pronuclei Three pronuclei

Sr2 1 48 48 (100) 3 41 4EtOH 48 46 (96) 6 39 1Electric 48 46 (96) 9 36 1Sperm heada 52 52 (100) 6 41 5

aInjected microsurgically.bKruskal-Wallis test: n.s.

No. of activated oocytes withb

example, mouse oocytes fused with thymocytesremain unactivated while their nuclei manifest apremature chromosome condensation (Czolow-ska et al., 1984). Thus, the oocytes fused or in-jected with somatic cell nuclei for cloning pur-poses must be activated artificially to allow thereconstructed oocytes to develop further.

Previous studies have focused on the im-provement of activation protocols in nonagedmammalian oocytes by coupling an activationstimulus with the administration of chemical fac-tors known to suppress MPF kinase activity (Szol-losi et al., 1993; Presicce et al., 1994; Loi et al., 1998;Leal and Liu, 1998) because a single calcium in-crease can induce early activation events, whichare characterized by resumption of meiosis andMIII arrest, exocytosis of cortical granules (corti-cal reaction), and the modifications in the glyco-proteins of the zona pellucida, but cannot inducelate events, such as mRNA recruitment, pronu-clear formation, and DNA synthesis and cleavagein young oocytes (Susko-Parrish et al., 1994;Schultz and Kopf, 1995; Soloy et al., 1997). Formouse cloning, we have been using Sr2 1

(Wakayama et al, 1998; Wakayama and Yanagi-machi, 1999). Here, we compared the efficiency

of Sr2 1 -induced activation with ethanol- or elec-tric pulse–induced and sperm-mediated activa-tion. While ethanol and a single electric pulse areknown to induce a single, large Ca2 1 rise (Cuth-bertson et al., 1981; Swann and Ozil, 1994), Sr2 1

(Kline and Kline, 1992) and sperm (Miyazaki etal., 1986) or sperm factors (Swann and Ozil, 1994)induce repetitive Ca2 1 rises which last severalhours. In the present study, we found that Sr2 1 ,ethanol, a single electric pulse, and sperm headsare equally effective in activating mouse oocytes(Tables 1 and 2). The age of oocytes (18–20 h af-ter hCG) could have resulted in the high activa-tion rate.

Although a single Ca2 1 rise induced by eitheran electric pulse (Sun et al., 1992) or calciumionophore (Colonna et al., 1989) is sufficient forthe oocytes to undergo cortical granule exocyto-sis and the resumption of the meiotic division, re-peated Ca2 1 rises may be beneficial for later de-velopment of embryos (Ozil, 1990; Vitullo andOzil, 1992). A recent study by Bos-Mikich et al.(1997) showed that mouse blastocysts parth-enogenetically developed from Sr2 1 -activatedoocytes have significantly larger number of innercell mass (ICM) cells than those developed from

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TABLE 3. DEVELOPMENT OF ENUCLEATED OOCYTES INJECTED WITH CUMULUS CELL NUCLEI

Total no. of No. (%) of No. (%) ofcumulus- morulae (M) morulae (M)injected and blastocysts (B) and blastocysts (B)

Oocyte- oocytes after 3 days of transferred No. of Total no. of No. of Fetal andactivating activated in vitro culture (no. of foster mothers) pregnant implantation live (placenta)agent (no. of exp.) (M 1 B) (M 1 B) recipients sites fetuses weights, mg

Sr2 1 416 (17) 172 1 70 (58.2)a 150 1 69 (21) 2 23 2b 1,490 (210)1,580 (300)

EtOH 596 (17) 134 1 57 (32.1)a 131 1 57 (16) 1 25 2b 1,540 (250)1,660 (360)

Electric 556 (17) 145 1 50 (35.1)a 126 1 47 (17) 2 8 2b 1,510 (310)1,600 (460)

ap , 0.0001, using x 2 test.bFisher’s exact probability test: n.s.

At 19.5 d.p.c.

TABLE 4. DEVELOPMENT OF ENUCLEATED OOCYTES INJECTED WITH

CUMULUS CELL NUCLEI AND ACTIVATED BY SPERMATOZOA

No. (%) of oocytes No. of (%) morulae No. of morulaeOocyte- No. of reconstructed with a distinct and blastocysts and blastocysts No. of No. ofactivating oocytes activated pronucleus derived 3 days after transferred pregnant livemethod by spermatozoa from sperm in vitro culture (no. of foster mothers) recipients fetuses

Sperm injection 56 51 (91.1) 7 (15.9) 7 (1) 0 0Insemination 87 58 (66.7) 8 (14.5) 8 (1) 1 1

At 12.5 d.p.c.

ethanol-activated oocytes. The larger ICM cellnumber is believed to be beneficial for the postim-plantation embryos (Bos-Mikich et al., 1997). Al-though Sr2 1 and sperm head factor were the bestin supporting preimplantation development invitro of reconstructed oocytes (Table 3), we didnot find any dramatic differences among fouroocyte-activating agents as far as the postim-plantation development of transferred embryosis concerned (Tables 3 and 4). All live cloned an-imals we have today have developed from re-constructed oocytes activated by artificial stimuli.Thus far, one of the most commonly used stim-uli is a single electric pulse (Wilmut et al., 1997;Baguisi et al., 1999; Kato et al., 1998), which in-duces a single intracellular Ca2 1 rise. Althoughrepetitive Ca2 1 rises during oocyte activationmay be better for later embryonic development,they are not essential for embryonic developmentto term. For mouse cloning, we have been usingSr2 1 (Wakayama et al, 1998; Wakayama andYanagimachi, 1999), but other chemical or phys-ical agents, including ethanol and electric pulse,also can be used. However, Sr2 1 activation is themethod of choice for egg activation in mousecloning studies because of the clear benefits topreimplantation development.

ACKNOWLEDGMENT

This study was supported by a research fundfrom ProBio America, Inc.

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Address reprint requests to:H. Kishikawa, M.D.

Department of UrologyOsaka University Medical School

2-2 Yamadaoka, Suita-CityOsaka, 565-0871

Japan

E-mail: kishi@uro.med.osaka-u.ac.jp

Received for publication June 28, 1999; acceptedas revised September 1, 1999.

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