Development 103, 129-136 (1988)Printed in Great Britain © The Company of Biologists Limited 1988

129

Two maternally derived X chromosomes contribute to parthenogenetic

inviability

JEFF R. MANN1* and ROBIN H. LOVELL-BADGE2

'Murdoch Institute for Research into Birth Defects, Royal Children's Hospital, Flemington Road, Parkville, Victoria 3052, Australia2MRC Mammalian Development Unit, Wolfson House (University College London), 4 Stephenson Way, London NW1 2HE, UK

^Present address: Department of Cell and Develomental Biology, Roche Institute of Molecular Biology, Nutley, New Jersey, 07110.USA

Summary

In certain extraembryonic tissues of normal femalemouse conceptuses, X-chromosome-dosage compen-sation is achieved by preferential inactivation of thepaternally derived X. Diploid parthenogenones havetwo maternally derived X chromosomes, hence thismechanism cannot operate. To examine whether thiscontributes to the inviability of parthenogenones, XOand XX parthenogenetic eggs were constructed bypronuclear transplantation and their developmentassessed after transfer to pseudopregnant recipients.In one series of experiments, the frequency of post-implantation development of XO parthenogenoneswas much higher than that of their XX counterparts.

This result is consistent with the possibility that twomaternally derived X chromosomes can contribute toparthenogenetic inviability at or very soon after im-plantation. However, both XO and XX partheno-genones showed similar developmental abnormalitiesat the postimplantation stage, demonstrating thatparthenogenetic inviability is ultimately determinedby the possession of two sets of maternally derivedautosomes.

Key words: X chromosome, maternally derived Xchromosome, parthenogenones, mouse embryo, dosagecompensation, preferential inactivation, inviability.

Introduction

Diploid parthenogenetic 1-cell mouse eggs possesstwo maternally derived haploid pronuclei. This is alsothe case in diploid gynogenetic 1-cell eggs (fertilizedeggs in which there is no contribution of paternalchromosomes). Both these types of eggs can undergoa high frequency of development to the blastocyststage and the majority undergoes implantation. How-ever, only a few develop to postimplantation stages,reaching at most about the 25-somite forelimb-budstage (Kaufman & Gardner, 1974; Kaufman et al.1977; Surani & Barton, 1983). Their abnormalitiesresult from the presence of two maternally derivedgenomes (Mann & Lovell-Badge, 1984; Surani et al.1984; McGrath & Solter, 1984), and parthenogenonesand gynogenones are therefore potentially subject toaH deleterious effects involving maternal duplication/paternal deficiency of certain chromosome regions(Searle & Beechey, 1978). In these regions, thereappears to be a differential expression of maternaland paternal gene homologues, determined by differ-

ential gene modifications or 'imprinting' presumablyimparted during gametogenesis (Cattanach & Kirk,1985). These modifications could involve DNA meth-ylation (Swain et al. 1987; Reik et al. 1987; Sapienza etal. 1987).

It is not known at how many loci imprinting isimportant for development, or the time at which eachbecomes critical. The only mechanism involvingimprinting known to occur around the time of implan-tation is the preferential inactivation of the paternallyderived X chromosome, Xp, in the trophectodermand primitive endoderm of XX blastocysts (Takagi etal. 1978; Frels et al. 1979; Frels & Chapman, 1980;Papaioannou & West, 1981; Harper et al. 1982). Thismechanism cannot operate in parthenogenones orgynogenones which have two maternally derived Xchromosomes, XM, and therefore may determine thecharacteristically poor development of trophoblast inpostimplantation-stage parthenogenones and gyno-genones (Endo & Takagi, 1981; Surani et al. 1984,1987). This poor development of trophoblast hasbeen shown to impair significantly the development

130 J. R. Mann and R. H. Lovell-Badge

of the embryo-proper (Barton et al. 1985).To investigate whether two XM chromosomes con-

tribute to the inviability of gynogenones, we pre-viously studied the developmental potential of XMOgynogenetic eggs (Mann & Lovell-Badge, 1987). Inthese eggs, X-dosage-related functions should beessentially normal, as XMO fertilized eggs are viable.Thus, any developmental abnormalities in XMOgynogenones could be attributed to the lack ofpaternally derived autosomes, Ap. XO gynogeneticeggs showed the same mortality at or very soon afterimplantation as did their XX counterparts, showingthat a lack of AF chromosomes was sufficient to causegynogenetic inviability at this stage. Nevertheless, assuggested, these results could not preclude the possi-bility that the presence of two XM chromosomesmight also have a deleterious effect. Furthermore,postimplantation development of XO or XX gyno-genones was not obtained. Therefore it was notpossible to determine whether normal X dosage couldresult in an improved development of gynogenetictrophoblast.

We have now extended these studies to XO andXX parthenogenetic eggs. Postimplantation develop-ment was obtained from some XO and XX eggs,allowing for a comparison of their developmentalpotential during embryonic development.

Materials and methods

Females more than 8 weeks of age were superovulated byintraperitoneal injection of 5i.u. pregnant mare serumgonadotrophin (Folligon; Intervet) followed about 48 hlater with 5i.u. human chorionic gonadotrophin, hCG(Chorulon; Intervet). Ethanol-induced parthenogenetic ac-tivation of cumulus-denuded eggs was carried out 16-17 hafter hCG injection (Barton et al. 1985) to produce haploidparthenogenetic 1-cell eggs (one pronucleus, 2nd polarbody). The frequency of activation was typically greaterthan 80%. Eggs were cultured in drops of medium M16(Whittingham, 1971) under light paraffin oil (British DrugHouses) at 37°C in 5 % CO2 in air. Pronuclei weretransplanted according to McGrath & Solter (1983) asmodified slightly by Mann & Lovell-Badge (1987). Theprocedure involves fusion of an egg fragment, consisting ofa pronucleus surrounded by a small amount of cytoplasmand plasma membrane, with an egg utilizing Sendai virusplasma membrane fusion. Eggs were transferred at the2-cell stage to the oviducts of recipients on day i ofpseudopregnancy (day of a vaginal plug after mating tovasectomized males of proven sterility). Phosphoglyceratekinase-1, PGK-1, electrophoresis was carried out accordingto Biicher et al. (1980). The enzyme was localized using thevisible tetrazolium-linked staining procedure of Oelshlegel& Brewer (1972).

Source of O eggsFemales [hereafter termed In(X)/X] heterozygous for an X

with a large inversion, In(X)lH (Evans & Phillips, 1975),were used as a source of eggs lacking an X chromosome.Karyotypic analyses have shown that they produce eggs ofwhich about 22 % are O, 72 % carry an X and 6 % carry anX dicentric (Phillips & Kaufman, 1974; P. S. Burgoyne,personal communication; Mann & Lovell-Badge, 1987).Unless otherwise stated, In(X)/X females were producedby mating In(X)/Y males (Murdoch Institute colony;original breeding pairs kindly provided by Dr Paul Bur-goyne, MRC Mammalian Development Unit, UK) toC57BL/6J females.

Estimation of the proportion of O and Xparthenogenetically activated haploid eggs ofln(X)/X femalesThe following experiment was carried out in order to assesswhether the proportion of O and X eggs produced byIn(X)/X females remained similar following parthenogen-etic activation. In(X)/X females were superovulated andtheir eggs parthenogenetically activated. Fertilized eggswere obtained from (C57BL/6J X CBA/CaH)F, (hereaftertermed B6CBF|) females superovulated 4—5 h later thanIn(X)/X females and mated to males carrying the X-linkedgene Pgk-la (coding for the A electrophoretic form ofPGK-1). In(X)/X females were homozygous Pgk-lh. Dip-loid eggs were then constructed by transplanting the pa-ternal pronucleus of fertilized eggs to haploid parthenogen-etic eggs of In(X)/X mice. Following micromanipulation,the eggs were cultured overnight to cleave to 2-cells, thentransferred to B6CBF, or ARC Swiss recipients (AnimalResources Centre, Murdoch, Western Australia; foun-dation stock of ARC Swiss mice were CD-I mice obtainedfrom Charles River Breeding Labs, USA). These wereautopsied at days 13|-15£ of gestation. Fetuses were sexedby gonad morphology and the PGK-1 allozyme type offemale fetuses determined. Female PGK-1 A fetuses wouldbe derived from O haploid parthenogenetic eggs, whereasall other types of fetuses would be derived from X eggs.

Construction and transfer of diploid XO and XXparthenogenetic eggsTwo series of experiments were carried out.

Series IThis series of experiments were conducted at the MRCMammalian Development Unit, UK (all other experimentsdescribed in this paper were carried out at the MurdochInstitute). In(X)/X females were produced by matingIn(X)/Y males (MRC Mammalian Development Unitcolony) to MF1 (OLAC, UK) females. In(X)/X and(CBA/CaH X C57BL/6J)F, (hereafter termed CBB6F,)females were superovulated, killed 16 h after hCG injec-tion, then cumulus-intact eggs parthenogenetically acti-vated by ethanol treatment (Kaufman, 1982; Cuthbertson,1983). Diploid eggs were then constructed by transplantingthe pronucleus of a haploid egg of an In(X)/X mouse to ahaploid egg of a CBB6F| mouse. The transplantation wasdone in this fashion as eggs of CBB6F, mice were inapparently better condition than those of In(X)/X (MF1hybrid) mice following ethanol activation. If In(X)/Xfemales produce about 22% O, 72% X and 6% X

Two XM chromosomes and parthenogenetic inviability 131

Table 1. Development of haploid parthenogenetic eggs of In(X)/X mice into which a paternal pronucleus wastransplanted

Transferred'

101

Implanted

76 (75 %)

Number of eggs

Developedpostimplantation

36 (36 %)

Male

20

Female

4102t

PGK-1type

AABB

n.d.

Classificationof egg/paternal

pronucleus

O/XX/XX/OX/Y

n.d. = not done.* Into recipients that became pregnant.t These females presumably resulted from the loss or absence in the paternal pronucleus of a sex chromosome and have been

observed in previous experiments (Mann & Lovell-Badge, 1987; Mann, 1987).

dicentric eggs, then these diploids would be in the expectedproportions of 22% XO (viability?), 72% XX (lethalbefore 12i days of gestation) and 6 % X dicentrics [peri-implantation lethal (P. S. Burgoyne, personal communi-cation)]. Eggs were cultured overnight to cleave to 2-cells,then transferred to CBB6F| recipients which were autop-sied at 9 i - l l i days of gestation. Postimplantation-stageparthenogenetic embryos were dissected from the uterusand XOs and XXs identified by chromosome counts.Metaphase spreads were made from the egg cylinder alone,or together with the yolk sac (Burgoyne et al. 1983). XOembryos possess 39 chromosomes. No other monosomy inmice is known to develop after implantation (Epstein,1985).

Series IIDiploid parthenogenetic 1-cell eggs were constructed bytransplanting the pronucleus of a haploid egg of an In(X)/Xmouse to another of the same. The In(X)/X mice used wereC57BL/6J hybrids, from which haploid pronuclear eggs ofgood quality were obtained following parthenogenetic acti-vation. The constructed diploids would be in the expectedproportions of 32 % XO, 52 % XX, 11 % X dicentrics and5 % OO (preimplantation lethal). Eggs were culturedovernight to cleave to 2-cells, then transferred to B6CBF,or ARC Swiss recipients. These were autopsied at day 9i ofgestation. XO and XX parthenogenetic embryos wereidentified as described above. As only these are able todevelop after implantation, the probability of an embryobeing XO or XX, given that XOs and XXs are equallyviable, would be 0-32x1/0-84 = 0-38 and 0-52x1/0-84 =0-62, respectively.

Production and transfer of diploid XXparthenogenetic eggs produced by suppressing 2ndpolar body extrusionThis was carried out in order to assess the frequency ofpostimplantation development that could be obtained indiploid parthenogenetic eggs produced by suppressing 2ndpolar body extrusion and not involving pronuclear trans-plantation. Immediately following exposure of eggs ofB6CBF| females to ethanol for parthenogenetic activation,

they were cultured for 3h in 5-0^gml ' cytochalasin B tosuppress 2nd polar body formation, washed in 2 ml ofmedium for 5min, then passed through five drops ofmedium under oil. They were cultured for a further 5h,after which diploid eggs (possessing two pronuclei and no2nd polar body) were selected (between 50 % and 70 % ofthe total) and cultured overnight to cleave to 2-cells.Usually, five 2-cell eggs per oviduct were transferred to(ARC Swiss x BALB/c)F, recipients using medium PB1(Whittingham & Wales, 1969). These were autopsied at day9i of gestation.

Results

Estimation of the proportion of O and Xparthenogenetically activated haploid eggs ofIn(X)/X females

In transplanting a paternal pronucleus into haploidparthenogenetic eggs of In(X)/X mice, fusion of theegg fragment occurred in 119 out of 143 (83 %)micromanipulated eggs. All but one cleaved to2-cells. They were transferred to 12 recipients. Onedid not possess implantations at autopsy. The fre-quency of implantation and postimplantation devel-opment of these eggs, and the number of fetuses ofeach type obtained is shown in Table 1. As fourfemale PGK-1 A fetuses (derived from O eggs) wereobtained, at least four OY eggs would also have beenexpected, these being preimplantation lethal. 32fetuses that must have been derived from X eggs werealso obtained. Thus, the ratio of O to X haploidparthenogenetic eggs produced that were able toparticipate in normal development was 8:32 = 1:4.This ratio is similar to that expected from previousestimates of the proportion of O and X eggs producedby In(X)/X females, i.e. 22% O and 72% X eggs;1:3-3. Hence, it is apparent that ethanol activation ofeggs of In(X)/X females does not result in markedly

132 J. R. Mann and R. H. Lovell-Badge

Table 2. Development of diploid XO and XX parthenogenetic eggs

Number

Transferred*

Series I135

(30 XO, 97 XX)t

Series II108

(35 XO, 56 XX)t

of eggs

Implanted

116(86%)

72 (67 %)

Postimplantation-stage

Genotype

XOXX

9

XOXOXOXOXOXO

XX

Developmentalstage

25 somites8 somites

16 somites

egg cylinder-likehead fold14 somites16 somites20 somites25 somites

20 somitesegg cylinder-like

embryos obtained

Numberobtained

(Recipient No.t)

1(01(1)1(2)

5(3 ,3 ,5 ,8 , 14)1(5)1(12)1(11)3 (6, 9. 13)6(5 .7 .8 ,9 . 10. 10)

171(4)4 (3. 4, 4. 6)

* Into recipients that possessed implantations at autopsy.t Expected numbers of XO and XX eggs transferred, calculated from the expected proportions of O and X eggs present (see

Materials and methods section).$ Recipients 1 and 2 were CBBF,, 3-7 ARC Swiss and 8-14 B6CBF,.Series I. Fusion of the egg fragment occurred in 177 out of 208 (85 %) micromanipulated eggs. One did not cleave to 2-cells. These

were transferred to twenty-eight recipients. Seven did not possess implantations of autopsy. XO embryo; chromosomes were countedusing the yolk sac (of twenty-seven spreads, twenty-three had 39, and the remainder had 34-38 chromosomes each). XX embryo;chromosomes were counted using the whole embryo (of twenty-two spreads, eighteen had 40, and the remainder had 39, 38 or 36chromosomes each). The unclassified 16-somite embryo was necrotic; chromosome counts were not attempted.

Series II. Fusion of the egg fragment occurred in 122 out of 137 (89%) micromanipulated eggs. Two did not cleave to 2-cells. 108were transferred into 17 recipients. One did not possess implantations at autopsy and twelve eggs were not transferred due to a lack ofrecipients on one occasion. The nine egg-cylinder-like embryos were very small and disorganized in appearance. Four of these couldnot be identified as the spreads were of insufficient quality. Fifteen embryos were classified as XO according to the presence 39chromosomes in at least ten spreads, with no counts of 40; the mean number of additional spreads counted (all with 36-3Schromosomes) was 1-9. Two more were classified as XO. One was egg-cylinder-like, in which only five spreads could be counted; allhad 39 chromosomes. The other was a 20-somite embryo in which 40 spreads had 39 chromosomes, with one of four non-modalspreads having 40. One embryo was classified as XX; of eight spreads counted, seven had 40 chromosomes and one had 39.

different proportions of O and X parthenogeneticeggs compared to fertilized eggs.

Development of diploid XO and XXparthenogenetic eggs

Series IResults are shown in Table 2. Only three postimplan-tation-stage embryos were obtained. Two of these,the 25-somite and 8-somite embryos, were present inthe same uterine horn of a recipient autopsied at 91days.

Series II22 postimplantation-stage embryos were obtained.Chromosome counts were made in 18 of these; 17were XO and one was XX. Given that the probabilityof an embryo being XO or XX is 0-38 and 0-62,respectively, given that they are equally viable (see

Materials and methods section), then the probabilityof obtaining 17 or more XOs out of 18 embryosis (0-38)17(0-62) x 18!/17!l! + (0-38)'H = 8-3 x 1CT7.Where more than one embryo was obtained in arecipient, there was no obvious tendency for these tobe similar in developmental stage. In all embryos, thedevelopment of the trophoblast was very limited,often represented by only a small piece of necrotictissue. A distinctive Reichert's membrane was oftenpresent, as has been observed previously (Surani etal. 1984), that could be easily separated from thedecidua. This could be attributed to the sparsedevelopment of trophoblast. The extent of develop-ment of the yolk sac appeared consistent with that ofthe embryo proper. Three of the somite-stage par-thenogenetic embryos obtained in the Series II exper-iments are shown in Fig. 1.

Two XM chromosomes and parthenogenetic inviability 133

t,Rm

Fig. I. Somite-stage parthenogenetic embryos obtained in the series II experiments. (A,B) 25-somite XO embryos.(C) 20-somite XX embryo. Trophoblast (t), Reichert's membrane (Rm), yolk sac (ys) and embryo-proper (e). Grossabnormal features were their small size (about half the dimensions of normal embryos) and a poor development oftrophoblast tissue. A curious finding was the large structure observed in the most advanced XO embryos, probably theallantois (indicated by arrows). Bar, 10mm.

Table 3. Development of diploid XXparthenogenetic eggs produced by suppressing 2nd

polar body extrusion

Number of eggs

Transferred* Implanted

Implantation siteswith embryonic

derivates

16t§581-H

12 (75 %)27 (47 %)53 (90 %)

* Into recipients that possessed implantations at autopsy.t Activation of cumulus-intact eggs.t Activation of cumulus-free eggs.§ Unilateral and || bilateral transfer into recipients 15 weeks of

age that had reared one litter.H Bilateral transfer into 8- to 12-week-old nulliparous

recipients.

Development of diploid XX parthenogenetic eggsproduced by suppressing 2nd polar body extrusionThe frequency of implantation and postimplantationdevelopment of these eggs is shown in Table 3. Of 133

eggs transferred to recipients, only three implan-tation sites were obtained in which postimplantationdevelopment was evident. However, only yolk sacvesicles had developed.

Discussion

Two series of experiments were conducted in order todetermine whether a difference exists in the develop-mental potential of diploid XO and XX parthenogen-etic eggs. In the experiments of series I, the majorityof XO and XX parthenogenetic eggs did not developto postimplantation stages. This result was similar tothat obtained with XO and XX gynogenetic eggs(Mann & Lovell-Badge, 1987), and demonstrates thatin these experiments the lack of Ap chromosomes wassufficient to cause the inviability of parthenogenonesat this stage. However, in the experiments of series II,the frequency of postimplantation development ofXO eggs (1 out of an expected 56 transferred) wasmuch greater than in XX eggs (17 out of an expected

134 J. R. Mann and R. H. Lovell-Badge

35 transferred). The probability of obtaining thisresult by chance is extremely low (see Results sec-tion). Therefore, it can be safely concluded that in theexperiments of series II, XO blastocysts were betterable to continue development after implantation thanXX blastocysts. Considering that ethanol activationcan cause aneuploidy in up to 19 % of haploidparthenogenetic eggs (Kaufman, 1982) and that aneu-ploidy often results in death at implantation (Epstein,1985), the frequency of postimplantation develop-ment of euploid XO eggs was likely to be as high asthat obtained following transplantation of pronucleibetween synchronous fertilized eggs, i.e. 67 % (Mann& Lovell-Badge, 1987).

In the experiments of series II then, the increasedfrequency of postimplantation development of XOcompared to XX parthenogenones must havedepended on the absence of a second XM, this beingthe only difference between them. It is evidenttherefore that two XM chromosomes can contributesignificantly to parthenogenetic inviability, the effectbeing manifest at implantation. This result is consist-ent with XX parthenogenones having difficulties inachieving normal X-dosage compensation in thetrophectoderm and primitive endoderm, presumablybecause of a failure of X inactivation in these tissueswhere in normal conceptuses Xp is preferentiallyinactivated. If this explanation is correct, then thesurvival of some XX parthenogenones beyond im-plantation could be due to just enough trophecto-derm and primitive endoderm cells managing toinactivate an X to enable further development. Also,the higher frequency of postimplantation develop-ment of XX parthenogenetic blastocysts subjected toimplantational delay (Kaufman et al. 1977), mayresult from the longer time available to inactivate anX, or from an increased number of cells with aninactive X resulting from an overall increase indelayed blastocyst cell number (Copp, 1982). Theseexplanations are in keeping with observations of ahigh proportion of cells with an inactive X in the yolk-sac endoderm (derived from the primitive endoderm)in postimplantation-stage XX parthenogenones (Ras-tan et al. 1980; Endo & Takagi, 1981).

From the above, it could be predicted that the fewandrogenones that develop to postimplantationstages (Barton et al. 1984) may be of an XPYconstitution, whereas XPXP androgenones may beless viable due to difficulties in achieving normal X-dosage compensation at the time of trophectodermand primitive endoderm formation.

The abnormal development observed in the post-implantation-stage XO parthenogenones was essen-tially equivalent to that reported previously in theirXX counterparts. However, some retardation of XOsrelative to XXs would have been expected, as XO

embryos derived from fertilized eggs are slightlyretarded in comparison to their XX counterparts at 7£days of gestation (Burgoyneef al. 1983). Significantly,the trophoblast was very poorly developed (Fig. 1) ashas been described previously in XMXMAMAM eggs(Surani et al. 1984) (where A refers to autosomes).Thus, it is apparent that the presence of two XM

chromosomes does not contribute significantly to thecharacteristically poor development of trophoblast,or any other developmental abnormality in post-implantation-stage XX parthenogenones. Rather, asX M O A M A P e g g s a r e v j a b l e ? a n d X

M 0A M A M eggs

are not, these abnormalities can be attributed to alack of Ap chromosomes.

Why was the frequency of postimplantation devel-opment of XMOAMAM eggs high in the series IIexperiments, yet low in two previous sets of exper-iments (Mann & Lovell-Badge, 1987 and series I)? Itshould first be noted that XMXMAMAM eggs are alsovariable in terms of the frequency that they willdevelop to postimplantation stages, as well as in thedegree of developmental normality attained. Lowfrequencies of postimplantation development havebeen consistently obtained by the authors (Mann &Lovell-Badge, 1984, 1987 and in the present exper-iments) following transfer of 2-cell eggs to oviducts,and have been also been reported by others followingtransfer of blastocysts to the uterus (Tarkowski et al.1970; Witkowska, 1973; Kaufman & Gardner, 1974).In contrast, a frequency of 25% development tosomite stages has been reported by Kaufman et al.(1977) after inducing delayed implantation in blasto-cysts and up to 22 % developing to various post-implantation stages by Surani & Barton (1983) andSurani et al. (1984) following transfer of 2-cell eggs tooviducts. Concerning the degree of developmentalnormality obtained, at midgestation this can varyfrom disorganized egg-cylinder-like structures toreasonably well-formed embryos that are not re-tarded at least in terms of somite number.

Therefore, the success or failure of parthenogen-etic eggs developing after implantation appears todepend on the penetrance and expressivity of certainloci at which maternal duplication affects develop-ment at the immediate postimplantation stage. Theseloci would appear to be associated with both the Xchromosome and autosomes, as the results of thepresent and previous experiments (Mann & Lovell-Badge, 1987) have demonstrated that the presence of(i) two sets of AM and (ii) two XM chromosomesboth have the potential to affect the viability ofX M X M A M A M e g g s a t t h i s s t a g e T h e g e n e t i c b a c k .

ground may be involved, as in the experiments ofseries II, a different strain combination had been usedcompared to previous experiments (series I; Mann &Lovell-Badge, 1984, 1987). However, we have only

Two XM chromosomes and parthenogenetic inviability 135

ever obtained very low frequencies of postimplan-tation development of XMXMAMAM eggs derivedfrom B6CBF, mice, whereas others have reportedmuch higher frequencies using the same eggs (Surani& Barton, 1983; Surani et al. 1984). Conditions of theexperimental or maternal (recipient) environment,and possibly certain qualities of the egg cytoplasmmay also influence the success of parthenogeneticpostimplantation development. In the experiments ofseries I, two out of the three postimplantation-stageparthenogenones were present in one recipient in thesame uterine horn. Also, Kaufman has described fourvery advanced XX parthenogenones obtained in asingle recipient (Kaufman, 1981). However, in theexperiments of series II, no such pattern was evident.

Given the above, XMOAMAM eggs would have agreater potential to continue development after im-plantation than XMXMAMAM eggs as they have onlyto contend with a lack of Ap chromosomes. Presum-ably therefore, in the experiments of series II, theenvironment and/or genetic background usuallyresulted in a low expressivity of relevant autosomalloci (in XO parthenogenones), but not of X-linkedloci (in XX parthenogenones).

Further definition of the causes for the variabledevelopment of parthenogenones beyond implan-tation awaits future study. Nevertheless, in the exper-iments of series II, there was essentially no variabilityin the experimental and maternal conditions and inthe genetic background of XO and XX parthenogen-etic eggs. Furthermore, the estimated frequency ofpostimplantation development obtained in XOs is atleast double that which has been obtained in XXs inany other study, by ourselves or others, followingtransfer of 2-cell eggs to oviducts. This demonstratesthat, at least under some circumstances, the presenceof two XM chromosomes does have a deleteriouseffect on the viability of parthenogenones at theimmediate postimplantation stage.

We thank Anne McLaren and Paul Burgoyne for helpfuldiscussions.

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{Accepted 11 May 1988)