MOLECULAR REPRODUCTION AND DEVELOPMENT 72:346–353 (2005)

Mouse Zygotes With One Diploid PronucleusFormed as a Result of ICSI can DevelopNormally Beyond BirthANNA KRUKOWSKA AND ANDRZEJ K. TARKOWSKI*

Department of Embryology, Institute of Zoology, Faculty of Biology, Warsaw University, Warsaw, Poland

ABSTRACT A mouse spermatozoon was in-jected into mouse secondary oocytes (ICSI) in thevicinity of the metaphase spindle. In 22% of oocytesinjected successfully, the maternal chromatin (thehaploid chromatids formed after the second meioticdivision) and paternal chromatin (from the spermnucleus) were surrounded by a common nuclearenvelope to form one diploid bi-parental pronucleus.However, the use of spermatozoa in which BrdU hadbeen incorporated into DNA during spermatogenesisrevealed, that maternal and paternal chromatin occu-pied two separate compartments within the onepronucleus. In the living state, the diploid pronucleuscould be distinguished from a haploid one by itsdistinctly larger size and by a greater number of‘‘nucleolus-like bodies’’—criteria confirmed karylogi-cally at the 1st cleavage division. Such zygotes withone diploid pronucleus were able to develop in vitrointo blastocysts as often as those with two haploidpronuclei [11/29 (38%) vs. 14/35 (40%)]. Seventynine 2-cell embryos developing in vitro from zygoteswith one diploid pronucleus were transplanted to theoviducts of pseudopregnant recipients: two femaleshad six foetuses when killed on the 17th day, and twofemales gave birth to nine young, eight of whichsurvived and developed into normal fertile animals.Mol. Reprod. Dev. 72: 346–353, 2005.� 2005 Wiley-Liss, Inc.

Key Words: mouse; ICSI; zygote; bi-parental diploidpronucleus; BrdU ‘‘labelled’’ sperm; survival to adult-hood

INTRODUCTION

Mammalian oocytes are ovulated and fertilised at themetaphase stage of the second meiotic division. Thepenetrating spermatozoon activates the oocyte, whichis accompanied by a completion of meiosis involvingextrusion of the second polar body (2PB), and formationof a haploid female pronucleus. Coincidentally, thesperm nucleus decondenses and transforms into a malepronucleus. Gametes fusion also results in an immedi-ate extrusion of cortical granules which initiates a blockagainst polyspermy primarily at the level of the zonapellucida. In the mouse, and also in many othermammals (for references see Longo, 1991) the region of

oolemma overlying the metaphase II spindle is devoidof microvilli, and sperm penetration does not occur inthis region (though it can apparently in human eggs—Santella et al., 1992; Van Blerkom et al., 1995).Consequently, as the male and female pronuclei formthey are widely separated, and remain so until theymove into apposition some hours later, following whichthe nuclear envelopes break down allowing the mater-nal and paternal chromosomes to align on the meta-phase plate of the first mitosis.

The success of fertilisation is thus testified by thepresence of two pronuclei and of the second polar body.However, among human oocytes fertilised in vitro(IVF), sometimes one encounters eggs with only onepronucleus. Such eggs have been termed variously asmono-pronuclear, uni-pronuclear, mono-pronucleate,uni-pronucleate, single-nucleated, single-pronucleated,one-pronuclear. Human eggs with one pronucleus arealso often observed following intracytoplasmic sperminjection (ICSI), and according to many authors theirincidence is higher than after natural fertilisation(‘‘classical’’ IVF). In the majority of infertility clinics,human oocytes are inspected for the first time 12–20 hrafter insemination or after ICSI, and eggs with onepronucleus and a 2PB are usually discarded. In a fewcases, however, embryos developing in vitro from sucheggs were later transferred, and late pregnancies or thebirth of healthy babies have been reported. While someof the mono-pronucleate eggs are haploid, its seems thatthe diploid ones are definitely more common (Lim et al.,2000). Correct classification of such zygotes in the livingstate is not easy, however, and the decision as to whetherto transfer such an embryo into the mother’s womb willalways remains as a dilemma.

To our knowledge mouse zygotes with one diploid bi-parental pronucleus have not been described so far. Formany reasons, however, the mouse is a very convenient

� 2005 WILEY-LISS, INC.

Grant sponsor: State Committee for Scientific Research; Grantnumber: 3 P04C 075 22.

*Correspondence to: Andrzej K. Tarkowski, Department of Embryol-ogy, Institute of Zoology, Warsaw University, Miecznikowa 1, 02-096Warsaw, Poland. E-mail: akt@biol.uw.edu.pl

Received 30 March 2005; Accepted 1 June 2005Published online 27 July 2005 in Wiley InterScience(www.interscience.wiley.com).DOI 10.1002/mrd.20344

species in which to elucidate the origin and fate of mono-pronucleate zygotes. In this study, we show that whenICSI is used, the deposition of a spermatozoon in thevicinity of the spindle of metaphase II often leads to theextrusion of the second polar body and the formation ofone large pronucleus. We also provide evidence that thispronucleus is indeed diploid and contains both oocyte-and sperm-derived chromatin. Moreover, we show thatthe mono-pronucleate zygotes develop in vitro intoblastocysts, and when transplanted as 2-cell embryosto recipient females, can develop into normal, fertilemice. The reliability of criteria that permitted us todistinguish the mono-pronucleate diploid zygotes frommono-pronucleate haploid parthenotes has been veri-fied with the help of karyological and immunohisto-chemical techniques.

MATERIALS AND METHODS

Animals

Two to six months old F1 (C57Bl/10�CBA/H, or areverse cross) females and 3–6 months old males wereused as donors of oocytes and spermatozoa, respectively.F1 animals are pigmented. Two to six months old albinooutbred females (MIZ and BAMIZ) mated with vasecto-mized MIZ, BAMIZ, and F1 males, or with fertile albinoBAMIZ males, served as recipients.

Media and Chemicals

Oocytes and embryos were manipulated in mediumM2 (Fulton and Whittingham, 1978) supplementedwith bovine serum albumin (fraction V, 4 mg/ml).This medium was also used for culture of oocytes.During microinjection oocytes were kept in BSA-free M2medium supplemented with polyvinyl alcohol (PVA,0,1 mg/ml).

Unless otherwise stated chemicals were from SIGMAALDRICH CHEMIE, Stenheim, Germany.

Ovulated Oocytes

Ovulation was stimulated by intraperitoneal injectionof 10 i.u. PMSG (Folligon, Intervet, Holland) and 10 i.u.of hCG (Chorulon, Intervet, Holland), given 42–50 hrapart. Females were killed 14–15 hr after hCG injec-tion, and cumulus–oocytes complexes were releasedfrom ampullae into a solution of hyaluronidase (150 i.u./ml) in Ca2þ- and Mg2þ-free phosphate-buffered saline(PBS, BIOMED, Poland). Cumulus-free oocytes werethen transferred to M2 medium.

Spermatozoa and Isolation of Sperm Heads

Spermatozoa were obtained by squeezing a caudaepididymidis and the adjacent vas deferens into 1 ml ofBSA-free M2 medium, or of modified NIM medium(nucleus isolation medium, Tateno et al., 2000). The ductcontent was kept for 15 min at 378C to allow dispersionof spermatozoa. Since fertilisation in rodents can beachieved by injection of sperm heads alone, the spermsuspension was sonicated for 2–5 sec (ultrasoundsonicator—VC-50, Sonics & Materials, Inc., Danbury,

CT, USA), during which the sperm vial was kept in ice.Sonicated spermatozoa were then centrifuged and thepellet re-suspended in BSA-free M2 medium or in NIMmedium supplemented with 12% polyvinylpyrrolidone(PVP, MW 360 000). The sperm suspension was used onthe same day, but sometimes the spermatozoa werefrozen at �858C (Ogura et al., 1996), for further ex-periments in which the injected oocytes were studiedonly during the first cell cycle. Only fresh sperm headswere used for ICSI when oocytes were studied for theirdevelopmental potential.

Intracytoplasmic Sperm Injection (ICSI)

ICSI was carried out with the help of piezoelectricmanipulator (PM-10-1, Marthauser Wetzlar, Germany)according to Kimura and Yanagimachi (1995). Theinjection pipette had an inside diameter of 5 mm.

Micromanipulations were carried out in two drops, ina plastic dish under liquid paraffin. The first drop (10 ml)contained oocytes in BSA-free M2 medium with PVA,the second one (5 ml) a suspension of spermatozoa inBSA-free M2 medium or NIM medium with PVP. Thedish was kept on a cooling stage (SEMIC BIOELEK-TRONIKA, Krakow, Poland), and viewed under aNIKON microscope equipped with Hoffman’s modulat-ing contrast. For microinjection, carried out at 128C,oocytes were immobilized with the meiotic spindle in the8–10 o’clock position and the injection pipette wasinserted directly into the area of spindle, which in mouseoocytes with heterogenous granular ooplasm, is clearlyvisible. After injecting the sperm head, excess mediumwas sucked back and the pipette withdrawn. The oocyteswere kept at 128C for 5 min, at room temperature forfurther 5 min, then cultured at 378C in 5% CO2 in air.

Spermatozoa With Incorporated BrdU

Some experiments used spermatozoa whose DNAcontained bromodeoxyuridine (BrdU), the analogof thymidine, incorporated during spermatogenesis(Mayer et al., 2000b). Males received one intraperito-neal injection of BrdU (2 mg/200 ml PBS) and afterwardswere given drinking water with BrdU (0.5 mg/ml) for atleast 35 days before being killed.

Immunocytochemical Detection of SpermWith Incorporated BrdU

Eggs were air-dried at various times after ICSI andstained according to Campbell et al. (1993). BrdUincorporated into the sperm-derived chromatin wasdetected by indirect immunofluorescence using mousemonoclonal anti-BrdU primary antibody and a goatpolyclonal anti-mouse IgG (HþL) fluorescein-conju-gated secondary antibody (both from Caltag Labora-tories, Burlingame, CA, USA). In addition, chromatinwas stained with propidium iodide. The preparationswere mounted in Citifluor (CITIFLUOR, UKC ChemicalLaboratory, London, UK) and examined in fluorescentmicroscope with the help of a CCD camera (VARIOCAM,PCO, Germany). Eggs injected with ‘‘nonlabelled’’

MOUSE ZYGOTES WITH A SINGLE BI-PARENTAL PRONUCLEUS 347

spermatozoa and eggs subjected to the staining proce-dure in which the primary antibody was omitted servedas controls.

Identification of Mono-Pronucleate DiploidZygotes in the Living State

Six hours after ICSI eggs were inspected underinverted microscope for normal fertilisation, as indi-cated by the presence of two pronuclei and two polarbodies (PBs) or just a second PB. In eggs with one or twoPBs and only one pronucleus, the latter’s large size andnumerous ‘‘nucleolus-like bodies’’ identified its pre-sumed diploidy.

Analysis of the Origin of Bi-ParentalSingle Pronuclei

One and a half, 2 or 3 h after ICSI the eggs werestained with Hoechst dye (Bisbenzimid H.33342, Ridel–de-Haen; 2–5 ng/ml of M2þBSA) for 30 min at 378C,placed in tiny drops of the Hoechst solution under liquidparaffin and examined under a fluorescent microscope(Axiovert 135, Zeiss , Germany).

Karyological Studies

Criteria employed for distinguishing diploid mono-pronucleate zygotes were verified by examination ofmetaphase chromosomes of one-cell embryos at thefirst cleavage division, using the air-drying techniqueof Tarkowski (1966). Preparations were stained withGiemsa or were examined immunocytochemically accor-ding to Campbell et al. (1993) (see above).

Permanent Preparations of One-Cell Embryos

After examination in the living state, a representativezygotes were mounted on slides and stained withheamatoxylin (Tarkowski and Wroblewska, 1967).

Culture In Vitro and Transfer of ICSIEmbryos to Recipient Females

Presumed diploid mono-pronucleate zygotes, as wellas normal zygotes with two pronuclei (controls) werecultured in vitro in M2 medium under liquid paraffinat 378C. One group of eggs was cultured for 96 hr toevaluate their potential for development. Another groupwas cultured for 24 hr, then transferred as 2-cellembryos on the day of copulation plug deposition to theoviducts of albino recipients (pseudopregnant or preg-nant) first anaesthetised with an intraperitoneal watersolution of Nembutal (6 mg/g body weight, Abbots). Thepregnant females were either killed on the 17th day orleft till term.

RESULTS

In order to produce mouse zygotes with a singlebi-parental pronucleus we used ICSI, and we tried todeposit the injected spermatozoon near to the meta-phase spindle in the hope of forming one diploid zygoticpronucleus. To a large extent this aim was realised(Table 1).

TABLE

1.OocytesSubjectedto

Intracyto

plasm

icSperm

Injection(ICSI)

[No.and(%

)]

Ooc

yte

sP

ron

ucl

ear

eggs

Sp

erm

-in

ject

edS

urv

ived

ICS

IF

ragm

ente

dor

inm

etap

hase

IIT

otal

Tw

op

ron

ucl

eior

one

pro

nu

cleu

san

d2P

B2

or3

pro

nu

clei

,2P

Bla

ckin

gIm

med

iate

clea

vage

Tw

op

ron

ucl

eiO

ne

pro

nu

cleu

s(1

n?)

On

ep

ron

ucl

eus

(2n

?)

1,6

34

1,0

98

(67.2

)186

(11.4

)912

(55.8

)(1

00.0

)

627

(38.4

)(6

8.8

)

71

(4.3

)(7

.8)

197

(12.1

)(2

1.6

)

12

(0.7

)(1

.3)

5(0

.3)

(0.5

)

348 A. KRUKOWSKA AND A.K. TARKOWSKI

Six hours after ICSI, eggs were examined under aninverted microscope, and 895 with two pronuclei or onepronucleus, and 2PB extruded, were selected. Thepercentage of mono-pronucleate zygotes varied fromexperiment to experiment (fluctuations from 5% to 40%)but, overall, 30% of the 895 zygotes had only onepronucleus and 2PB. Eggs that had two or threepronuclei but had not extruded the 2PB, and eggs thathad undergone ‘‘immediate cleavage’’ (second meioticdivision that results in the formation of two cells of equalsize) were discarded.

Among 268 sperm-injected mono-pronucleate eggs73.5% had a pronucleus that was definitely larger thanthose in bi-pronucleate zygotes and had always morenucleolus-like bodies (mean: 7.1 as compared to 2.4 perpronucleus in ‘‘normal’’ zygotes) (cf Fig. 2 with Fig. 1). Inthe remaining 26.5% mono-pronucleate eggs the singlepronucleus was similar in size to those in bi-pronucleatezygotes (cf Fig. 3 with Fig. 1) and to the single pronucleiin haploid parthenotes (data not shown). Such apronucleus usually contained one nucleolus-like body(occasionally 2–3 nucleolus-like bodies, mean 1.3). Inthese eggs the ICSI procedure evidently failed (mostprobably the spermatozoon escaped the egg during thewithdrawal of the injecting pipette), but neverthelessthey underwent parthenogenetic activation.

The diploid status of mono-pronucleate eggs with onelarge pronucleus was confirmed by karyological exam-ination of chromosomes at the time of the first cleavagedivision. All embryos examined had one group of 40chromosomes (Fig. 4).

In order to prove that that the large pronuclei were notonly diploid, but were true zygotic nuclei containingboth maternal and paternal genomes, for ICSI we usedspermatozoa with BrdU incorporated into their DNA.The staining of only one pronucleus permitted distinc-tion of the male from the female pronucleus in 28 zygotes

(Fig. 5). In 15 eggs with one large pronucleus thepresence of chromatin reacting with anti-BrdU antibodyand, occupying just a part of the pronucleus, providedunquestionable evidence of its bi-parental character(Fig. 6).

Karyological examination of bi-pronucleate zygotes atthe time of the first cleavage division revealed twohaploid groups of chromosomes, one labelled withantibody against BrdU and one unlabelled and stainedonly with propidium iodide (Fig. 7; seven specimens).The supposedly diploid mono-pronucleate zygotes hadone diploid metaphase plate composed of ‘‘labelled’’ and‘‘unlabelled’’ chromosomes (Fig. 8; six specimens). Thesupposedly haploid mono-pronucleate eggs in whichICSI failed simply had a haploid group of ‘‘unlabelled’’maternal chromosomes stained with propidium iodideonly (Fig. 9; nine specimens).

In order to clarify the origin of diploid zygoticpronuclei, we followed the spatial position of the injectedsperm head in relation to the group of maternaltelophase chromatids, and the fate of both chromatingroups at the time of their transformation into pronu-clei. Examination of more than 200 eggs over thisparticular period (data not shown) revealed that the twogroups of chromatin were often situated very closetogether (Figs. 10 and 11) and later most probablybecame enclosed by the common nuclear envelopeto form one pronucleus (Fig. 12). These observationssuggest that single large pronuclei observed in olderzygotes are constituted at the very beginning of theformation process, and not through the fusion oforiginally independent haploid pronuclei (see ‘‘Discus-sion’’ for various interpretations of this phenomenon inhuman eggs).

Evaluation of the developmental potential of pre-sumptive diploid mono-pronucleate zygotes was carriedout in two steps: first in vitro and, second in vivo. Eleven

Fig. 1. A ‘‘normal’’ zygote with two pronuclei: the female pronucleus is smaller and is situated close to2PB.

Fig. 2. A zygote with a 2PB and a single large pronucleus (presumed diploid) displaying several nucleoli.Fig. 3. A one-cell egg with one pronucleus of the size typical for pronuclei in normal (bi-pronucleate)

zygotes, and a 2PB. Intracytoplasmic sperm injection (ICSI) was unsuccessful but the egg underwentparthenogenetic activation.

MOUSE ZYGOTES WITH A SINGLE BI-PARENTAL PRONUCLEUS 349

of 29 mono-pronucleate zygotes and 14 of 35 bi-pronucleate zygotes developed in vitro into blastocysts,and 5 and 9 respectively hatched from the zonapellucida. In view of this encouraging result, mono-and bi-pronucleate zygotes were cultured in vitro for24 hr only and then transferred as 2-cell embryos topseudopregnant or pregnant recipients. Among the2-cell embryos developing from mono-pronucleatedzygotes 79 were transferred to 14 recipients. Twofemales sacrificed on the 17th day were pregnant andhad one and five foetuses. Two other females gave birth

to nine pups, eight of which grew into fertile adults.Transfer to 18 recipients of 212 embryos developing frombi-pronucleate zygotes resulted in 9 females becomingpregnant and in the birth of 21 young. Clearly, comparedto ‘‘normal’’ zygotes, preimplantation and foetal devel-opment of mono-pronucleate zygotes was not compro-mised.

DISCUSSION

During the first cell cycle in mammalian embryos,each of the parental genomes initially forms a separate

Fig. 4. A diploid metaphase plate from a mono-pronucleate zygote at the first cleavage division.Fig. 5. A zygote with two pronuclei. The larger pronucleus (yellow–green on the red background)

developed from a spermatozoon that had BrdU incorporated into DNA; the smaller female pronucleus,stained only with propidium iodide, is shown in red. The nucleus of the 2PB is visible above the femalepronucleus. The green object close to the 2PB is an artifact.

Fig. 6. A zygote with a single large pronucleus. The sperm-derived chromatin (stained yellow-green)occupies only one half of the pronucleus. The other half is occupied by maternal chromatin (red). Thenucleus of the 2PB (red) is seen above the pronucleus, slightly to the right. The green object close to 2PB isan artifact.

Fig. 7. Two haploid groups of chromosomes from a bi-pronucleate egg similar to that shown in Figure 5,approaching the first cleavage division. The group of paternal chromosomes stained with anti-BrdUantibody and propidium iodide (patches of yellow, green, and red) is shown on the left; the group of maternalchromosomes stained only with propidium iodide (uniformly red) is on the right.

Fig. 8. One diploid metaphase plate showing both ‘labelled’ paternal chromosomes (green and yellowpatches on the red background) and ‘unlabelled’ maternal chromosomes (uniformly red). The metaphaseplate originates from a mono-pronucleate egg similar to that shown in Figure 6, undergoing the firstcleavage division.

Fig. 9. A haploid group of ‘unlabelled’ maternal chromosomes derived from a mono-pronucleate eggsimilar to that shown in Figure 3.

350 A. KRUKOWSKA AND A.K. TARKOWSKI

nucleus (a pronucleus), and remains physically sepa-rated from its counterpart until the first mitosis. The co-existence of both genomes in one pronucleus, as shownhere, is clearly a deviation from the normal course ofevents, and raises the question of whether their closeapposition has, or has not, any further developmentalconsequences. In the case of human embryos this alsobecomes an issue of fundamental practical importance,because it requires a decision as to whether such zygotesmay be safely used for transfer. In view of the wide-spread development of the programmes of assistedreproduction in man, and their increasing resort toICSI, it is not surprising that mono-pronucleate zygoteswere first observed in man (and so far only in man) andthat this phenomenon has received great attention.Since this paper is the only study of this phenomenon ina nonhuman mammal, we will refer often to clinicalstudies.

The following issues require consideration: first—theorigin and ploidy of mono-pronucleate one-cell embryos,second—criteria for discriminating diploid from haploid

pronuclei and the reliability of this discrimination, andthird—clarification of the consequences of this phenom-enon for further embryonic development.

Origin and Ploidy of Mono-PronucleateOne-Cell Embryos

As mentioned in Introduction, mono-pronucleate one-cell eggs have been described in many studies concernedwith IVF and ICSI in the human, and three routes oftheir origin have been proposed (Plachot and Crozet,1992; Balakier et al., 1993; Munne et al., 1993; Staessenet al., 1993; Dozortsev et al., 1994, 1995; Flaherty et al.,1995; Levron et al., 1995; Sultan et al., 1995; Tesarik andMendoza, 1996; Antinori et al., 1997; Staessen and VanSteirteghem, 1997). (1) Failure of fertilisation andinitiation of haploid development. There are two pos-sible scenarios here. In the first scenario the injectedspermatozoon escapes from the egg during withdrawalof a pipette, but the egg undergoes parthenogeneticactivation. In the second scenario, the spermatozoonfails to transform into a functional male pronucleus and,consequently, to participate in the embryonic genome,but rather activates the egg to haploid gynogeneticdevelopment. (2) Early union of oocyte-derived chroma-tin (telophase chromatids) and sperm nucleus-derivedchromatin into one diploid pronucleus as a result of theirclose apposition and enclosure by a common nuclearenvelope. (3) Fusion of two initially independent haploidpronuclei into a single, diploid pronucleus. Only the twolast routes are pertinent to the subject of this study inthat they are concerned with the origin of the diploidpronucleus composed of both parental genomes. Weleave aside cases (evidently quite frequent in man) ofasynchronous development of two pronuclei, when atthe time of the first inspection only one (larger) pro-nucleus was spotted and the egg was wrongly classifiedas mono-pronucleate. A second inspection after a fewhours sometimes allowed the investigators to distin-guish the second pronucleus and to correct the earlier

Fig. 10. The decondensing sperm nucleus (short arrow) lying closeto the clumped group of telophase chromatids (long arrow).

Fig. 11. Paternal (short arrow) and maternal (long arrow) chromatin closely adhere to each other andbegin to transform into a single, diploid pronucleus.

Fig. 12. An early, putatively diploid, pronucleus.

MOUSE ZYGOTES WITH A SINGLE BI-PARENTAL PRONUCLEUS 351

false classification. However, many human zygotes withasynchronously developing pronuclei may never giverise to normal diploid embryos, because, as shown by ourstudies in the mouse, the lagging pronucleus may not beable ‘‘to catch up’’ and complete DNA replication by thetime of the first mitotic division (Komar, 1982; Borsukand Tarkowski, 1989; Maleszewski, 1992; Maleszewskiet al., 1999).

To our knowledge, fusion of the originally separatepronuclei during the interphase of the first mitotic cellcycle has never been described in nonhuman mammals,and the observations by Tesarik and Mendoza (1996)and Antinori et al. (1997) in human eggs are the onlyclaims of such a possibility. It should be recalled thatin the latter studies spermatids rather than maturespermatozoa were injected, but it is not clear how the useof spermatids could promote fusion of pronuclei. A highincidence of mono-pronucleate eggs was observed alsoby Barak et al. (1998) who also injected spermatidsrather than spermatozoa. Observations described inthe present study strongly suggest that in the mouseat least a single zygotic (bi-parental) pronucleus devel-ops from two closely positioned groups of chromatinthat subsequently become surrounded by a commonnuclear envelope. On several occasions, examinationof sperm-injected eggs shortly after extrusion of the2PB permitted us to view the group of telophasefemale chromatids and the sperm nucleus lying invery close proximity to each other (Figs. 10, 11). ICSIfavours the chance of such positioning (but by no meansguarantees it) when sperm are injected close to the MIIspindle. In this study, among 30% of mono-pronucleatezygotes obtained by ICSI, 22% were diploid and 8%haploid. This incidence of diploid mono-pronucleateeggs was definitely higher than in experiments in whichspermatozoa were injected at random without regardsto the position of the MII spindle (our unpublishedobservations).

Discrimination Between Haploid and DiploidPronuclei in the Living Eggs

In the mouse, discrimination between haploid anddiploid mono-pronucleate eggs was relatively easy toachieve on the basis of nuclear size and the number ofnucleolus-like bodies. The validity of this classificationwas confirmed by counting chromosomes of the firstmitotic division, in that eggs with large pronuclei con-taining numerous nucleoli invariably turned out to havea diploid number of chromosomes. In man, discrimina-tion between haploid and diploid pronuclei in mono-nucleate eggs has proven to be much more difficult, anddecisions as regards their transfer back to mothers(Gras and Trounson, 1999) were undertaken withoutcertainty concerning their ploidy. Haploid and diploidpronuclei were distinguished by size and number ofnucleolus-like bodies, but the differences were not aspronounced as in the mouse. In some cases, successfulpreimplantation development in vitro was taken as anadditional evidence in favour of diploidy (Gras andTrounson, 1999).

Are There any Developmental Consequencesof the Co-Existence of Both Parental

Genomes in One Pronucleus?

The birth of healthy children, and several examplesof advanced pregnancies following transfer of mono-pronucleate zygotes (Staessen et al., 1993; Levron et al.,1995; Sultan et al., 1995; Barak et al., 1998; Gras andTrounson, 1999) together provide evidence that theatypical co-existence of maternal and paternal chromo-somes within one pronucleus during the first cell cycledoes not compromise further development up to andbeyond birth. As described in this study, the fact thatadult fertile animals developed from mono-pronucleateeggs, shows that the same is true also in the mouse.Why then are the male and female genomes typicallyseparated in mammals, during the whole of the first cellcycle? In the first cell cycle in the mouse, there is adifference in the timing of activation of the maternaland paternal genomes in regard to: DNA replication(Bouniol-Baly et al., 1997), transcription (Bouniol et al.,1995; Adenot et al., 1997; Aoki et al., 1997), demethyla-tion (Mayer et al., 2000a), and acetylation of histones(Adenot et al., 1997; Arney et al., 2002). It would seem,therefore, that assignment of the two genomes to twoseparated pronuclei could protect this asynchrony(whatever importance it may have for the embryo),which might perhaps be precluded within a singlecommon nucleus (for review see Haaf, 2001). It fact, how-ever, the time course of the activities of maternal andpaternal chromatin seems to be largely, though not com-pletely, autonomous. Our studies on DNA replicationand transcription in diploid mono-pronucleate mouse�hamster one-cell hybrids (Krukowska and Borsuk, un-published results) have shown that species differencesin the timing of these processes are maintained despitethe coexistence of chromosomes in one pronucleus.

Not only do the two genomes coexist in the commondiploid nuclei after the first cleavage division, but theirtopological separation is maintained at least during thenext two cell cycles (2-cell and 4-cell stage), as shown inthe mouse by Mayer et al. (2000b). Although we havenot studied the spatial arrangement of maternal andpaternal chromosomes in 2-cell mouse embryos arisingfrom mono-pronucleate diploid zygotes, it is very likelythat the topological separation continues as in ‘‘normal’’embryos originating from bi-pronucleate zygotes. Inmono-pronucleate zygotes the two parental genomesco-exist within one nucleus right from the beginning ofdevelopment, and although this is a deviation from thenormal course of events it has no harmful effect for thefuture individual.

ACKNOWLEDGMENTS

We wish to thank Professor Marek Maleszewski,Dr. Ewa Borsuk, and Mr. Darek Maluchnik for theirhelp in carrying out experiments and for helpfulcomments on the manuscript. We are most thankful toProfessor Michael Bedford for improving the finalversion of this paper.

352 A. KRUKOWSKA AND A.K. TARKOWSKI

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