the postimplantation development of spontaneous digynic ... · scribed (piko & bomsel-helmreich,...

Development 101, 383-391 (1987)Printed in Great Britain © The Company of Biologists Limited 1987

383

The postimplantation development of spontaneous digynic triploid

embryos in LT/Sv strain mice

M. H. KAUFMAN and S. SPEIRS

Department of Anatomy, University Medical School, Teviot Place, Edinburgh, EH8 9AG, UK

Summary

When spontaneously ovulating LT/Sv female mice aremated with fertile males, between one third and onehalf of the zygotes analysed at the first cleavage mitosisare found to be triploid. This is due to the fact thatLT/Sv females ovulate both primary and secondaryoocytes, all of which are capable of being fertilized.Fertilization of the former group results in the pro-duction of digynic triploid conceptuses, while theirdiploid littermates result from the fertilization ofnormal secondary oocytes. The present study wastherefore carried out in order to investigate the'spontaneous' level of triploidy in these mice, and toprovide insight into the developmental fate of theLT/Sv triploid embryos, as previous studies had

indicated that in this species triploids invariably fail todevelop beyond the early postimplantation period.This study revealed that when autopsies were carriedout on the 7th and 8th days of gestation, it wasgenerally difficult to distinguish between the kary-ologically normal diploids and the digynic triploidconceptuses when only morphological criteria wereused. However, by the 10th day of gestation, thetriploid conceptuses could usually be readily dis-tinguished from their diploid littermates by theirsmaller size and (occasionally) by their disorganized orabnormal morphological appearance.

Key words: mouse embryo, LT/Sv strain, digynic triploid,postimplantation.

Introduction

Various experimental techniques have been de-scribed which have allowed high rates of triploidy tobe induced in the mouse (for review, see Niemierko& Opas, 1978), though the postimplantation develop-ment of somite-stage mouse, rat and rabbit embryosachieved by these means has only rarely been de-scribed (Piko & Bomsel-Helmreich, 1960; Bomsel-Helmreich & Thibault, 1962; Bomsel-Helmreich,1965; Opas, 1977; Niemerko, 1981). In addition, sincethe spontaneous occurrence of this condition is soinfrequently encountered in these species (Wr6b-lewska, 1971; Baranov, 1976), little information isavailable on the developmental potential of theseconceptuses, though Niebuhr (1974) was able toreport on the developmental fate of several hundredhuman triploids extracted from the world literature.The latter study would appear to indicate that thiscondition is probably considerably more frequentlyencountered in man (see also Boue\ Bou6 & Lazar,

1975) than in the few other mammalian species thathave so far been studied.

While postovulatory ageing of the oocyte withinthe oviduct prior to fertilization is known to beassociated with a decreased efficiency in the variousblocks to polyspermy, this manoeuvre does not ap-pear to be a particularly reliable means of inducingdispermic triploid development either in vivo (Mar-ston & Chang, 1964; Vickers, 1969; Kaufman, 1973;Beatty, 1978; Beatty & Coulter, 1978) or in vitro(Maudlin & Fraser, 1978). While diandric triploidymay also be induced by the fertilization of secondaryoocytes by diploid spermatozoa, the latter appear tohave a reduced level of survivability and impairedmotility compared to haploid spermatozoa within thefemale tract (Mortimer, 1977). In addition to theproduction of dispermic triploids, digynic triploidymay also be induced under these conditions due tothe migration of the second meiotic spindle awayfrom the periphery of the egg, a phenomenon alsoknown to occur in aged oocytes (Szollosi, 1967, 1971;

384 M. H. Kaufman and S. Speirs

Webb, Howlett & Maro, 1986), and consequentinhibition of second polar body extrusion.

Of the various experimental approaches that havebeen employed to induce triploid development,exposure of recently fertilized eggs to the cytokinesis-inhibiting effect of cytochalasin would appear to beone of the most efficient means available at thepresent time (see Niemierko, 1975; Niemierko &Opas, 1978). Exposure to phorbol ester (PMA)during fertilization in vitro also produces a highproportion of tripronucleate eggs (Niemierko &Komar, 1985). The incidence of triploidy followingfertilization in vitro is in any case significantly higherthan occurs following fertilization in vivo, probablydue to the higher level of polyspermy (Santalo, Estop& Egozcue, 1986), and may also be related to thedose of PMSG employed (Maudlin & Fraser, 1977).Similarly, the spontaneous level of digynic triploidyencountered in vivo may also be increased followingsuperovulation (Takagi & Sasaki, 1976).

A situation in which a much higher incidenceof spontaneous digynic triploid development occursthan is normally encountered has recently beenreported by O'Neill & Kaufman (1987). Theseauthors mated spontaneously ovulating LT/Sv fe-males with F^hybrid males and analysed thechromosome constitution of fertilized eggs at the firstcleavage mitosis, observing that about one third ofthe resultant zygotes were digynic triploids. Thisfinding was not entirely unexpected, since it hadpreviously been shown by Kaufman & Howlett (1986)that almost one half of the unfertilized eggs isolatedfrom the oviducts of superovulated female LT/Svstrain mice had been ovulated as primary rather thansecondary oocytes. The former group were ovulatedat metaphase of the first meiotic division, had a 4Camount of DNA present and a 2N (diploid) numberof chromosomes. However, it is possible that the useof superovulation may have precluded any mean-ingful estimate being made of the 'spontaneous' levelof triploidy that it was hypothesized might arise in,and consequently be recovered from, LT/Sv strainmice shortly after conception. The present study wastherefore undertaken to establish this information inthe first instance. It was also hoped that it mightprovide insight into the developmental fate of LT/Svtriploid mouse embryos, as previous studies hadindicated that the mouse triploids invariably fail todevelop beyond the early postimplantation period(see Baranov, 1976).

Pregnant LT/Sv strain females were thereforeautopsied between the 7—10th day of gestation, and adetailed karyological and morphological analysis oftheir conceptuses was undertaken.

Materials and methods

8- to 12-week-old randomly cycling female LT/Sv strainmice (MRC, Carshalton) were caged with (C57BL xCBA)F! hybrid males. Early each morning the femaleswere checked for the presence of a vaginal plug and thelatter was taken as evidence of mating. The morning offinding a vaginal plug was considered to be the first day ofpregnancy.

The females were autopsied at about midday on eitherthe 7th, 8th or 10th day of gestation. The decidual swellingswere isolated from the uterine horns and put into phos-phate-buffered saline. The embryos isolated on the 10thday were then dissected free from within their extraembry-onic membranes (i.e. amnion and yolk sac) with finewatchmakers' forceps, while all the conceptuses isolated onthe 7th and 8th day, and those that were developmentallyretarded on the 10th day were generally left intact andtreated as a single unit. The total number of resorptionspresent was noted. All of the morphologically normal andabnormal conceptuses were transferred into '199' tissueculture medium (TC199) with added Colcemid (1 ml of a0-1 % solution of Colcemid in 100 ml of TC199). The totalnumber of embryos that were clearly morphologicallynormal or abnormal or, on the other hand, were consideredto be morphologically normal but retarded in their develop-ment was recorded at this time.

The chromosome constitution of all of the conceptuses,both normal and abnormal, was then determined using amodification of the technique described by Evans, Burten-shaw & Ford (1972). In the case of the developmentallymore advanced embryos that had been isolated from withintheir extraembryonic membranes, only the latter was usedto establish their chromosome constitution (i.e. ploidy).The embryos, on the other hand, were transferred toBouin's fixative for subsequent histological analysis aftertheir crown-rump length and other morphological para-meters had been carefully noted. In the case of the grosslyabnormal conceptuses and the group of developmentallyseverely retarded conceptuses, both the embryo and itsextraembryonic membranes had to be treated as a singleunit in order to maximize the chance of determining theirchromosome constitution. The chromosome preparationswere stained with 5 % buffered Giemsa solution (R.66,G. T. Gurr), then permanently mounted and the mitoticspreads examined under the oil-immersion objective of aLeitz photomicroscope.

Results

(A) Incidence of triploid conceptuses encountered onthe 7th, 8th and 10th days of gestationThe results of the autopsies conducted on the 7th, 8thand 10th days of gestation are presented in detail inTable 1, and representative triploid mitotic spreadsobtained following the disaggregation of the extra-embryonic membranes of two triploid conceptusesare illustrated in Fig. 1A,B. In 18 out of the 21

Triploidy in LT/Sv mice 385

Table 1. Cytogenetic analysis of conceptuses isolated from LT/Sv females on either the 7th, 8th or 10th day ofgestation

Ploidy of conceptuses TotalTotal Total embryos analysed

females Total Total embryos Diploid Triploid Not cytogeneticallyautopsied implantations resorptions recovered (2n) (3n) analysable (% triploid)

Day ofgestation

Group at autopsy

10

I5*

12*

946

119

14

II42

108

6 127 1281 '25

7(14-3)39 (30-8)

106 (23-6)

'One additional female had 12 implants, 11 analysed cytogenetically- all diploid." T w o additional females had 23 implants. 20 analysed cytogenetically - all diploid.

females autopsied, both diploid and triploid con-ceptuses were isolated, while in the remaining 3females all of the embryos isolated were found to bediploid. Out of a total of 190 implantation sitesisolated from females that contained both diploid andtriploid conceptuses, a total of only 16 implantationsites contained resorbing embryos.

The overall incidence of triploidy encounteredbetween the 7-10th days of gestation in this study wastherefore 25% (38/152 conceptuses examined cyto-genetically) which is significantly lower than theincidence of primary oocytes ovulated by LT/Svfemales following exogenous hormonal stimulation(see Kaufman & Howlett, 1986). In the latter study,47-8% out of a total of 295 recently ovulated andparthenogenetically activated eggs analysed werefound to have been ovulated as primary oocytes. In a

more recent study, by O'Neill & Kaufman (1987),33-8% out of a total of 65 fertilized LT/Sv eggsanalysed at the first cleavage mitosis, were assumedto have been ovulated as primary oocytes.

While the figures quoted above are clearly signifi-cantly different, it is unclear whether the reductionobserved is solely due to the reduced incidence oftriploid conceptuses observed at successive stages ofdevelopment. It certainly seems unlikely that thedetrimental effect on their development potential ofpossessing a triploid genome would have manifesteditself as early as the first cleavage division.

A proportion of the resorptions encountered in thisstudy might represent those triploid conceptuses thatfailed to develop beyond the early postimplantationperiod, and this might account for the higher inci-dence of triploid conceptuses encountered at the first

B

Fig. 1. Low (A) and high (B) magnification views of typical mitotic chromosome spreads from the extraembryonicmembranes of two triploid LT/Sv strain embryos. Each mitotic spread illustrated in (A) contains two large metacentricchromosomes. This was the only conceptus in the present study in which such morphologically abnormal chromosomeswere observed.


cleavage compared to the situation observed cyto-genetically in the present study. It is of interest tonote that there was no significant difference observedbetween the total number of conceptuses isolated perfemale in the present study, compared to the numberrecovered in previous studies shortly after ovulationin spontaneously ovulating females (present study,n = 21, 9-90 ± 0-38 (mean ± S.E.M.); previous studies,n = 7, 9-14 ±0-70 (mean ± S.E.M.; G. T. O'Neill,S. Speirs & M. H. Kaufman, unpublished obser-vations)).

(B) Morphological appearance of triploid conceptusesencountered on the 7th, 8th or 10th day of gestation

(1) Isolation of conceptuses on the 7th day ofgestation

Only one female was autopsied on the 7th day ofgestation. All of the conceptuses were at the eggcylinder stage of development and were morphologi-cally indistinguishable from each other. The oneconceptus that was revealed on cytogenetic analysisto have a triploid chromosome constitution appearedin all respects to be morphologically identical to itsdiploid littermates. There was, in addition, no obvi-ous difference between the extraembryonic tissues ofthe triploid conceptus compared to those of its diploidlittermates.


Out of a total of six females autopsied on the 8th dayof gestation, all but one contained a mixture ofdiploid and triploid conceptuses. All of the con-ceptuses that were morphologically abnormal orgrossly retarded in development compared to theirlittermates proved to have a triploid chromosomeconstitution. However, more than one half of thetriploid conceptuses were morphologically indis-tinguishable from their diploid littermates. No obvi-ous differences were observed between the extra-embryonic tissues of the triploids compared to theirdiploid littermates.


By the 10th day of gestation the conceptuses werereadily distinguishable into two principal categories,namely a slightly larger group which containedmorphologically normal-looking viable forelimb-bud-stage embryos with on average about 25 pairs ofsomites present, and a second more heterogeneousgroup of conceptuses. Almost without exception theconceptuses in the latter group were substantiallysmaller in volume and in crown-rump length thanthose that could be assigned to the first group. Theembryos that had successfully 'turned' to adopt the

fetal position occasionally showed evidence of cardio-vascular stasis and morphological abnormalities in-volving the cephalic part of the neural axis. Severalembryos were observed that were still 'unturned'while, in a few instances, partially 'turned' embryoswere observed in which the appearance of thecephalic region was consistent with more advanceddevelopment than the 'unturned' or partially 'turned'state suggested. Only two examples of a completelyempty embryonic sac were observed (the 'triploidysyndrome' described by Wr6blewska, 1971).

Cytogenetic analysis of the extraembryonic mem-branes from the conceptuses isolated on the 10th dayrevealed that those in the first category were in-variably diploid, while the great majority of thosein the second category had a triploid chromosomeconstitution. In fact, only two of the developmentallyretarded embryos had a diploid chromosome consti-tution. In the light of the recent observations on therelationship between the appearance of the extra-embryonic membranes and the genotype of mouseconceptuses (McGrath & Solter, 1984; Surani, Barton& Norris, 1984) it is of interest to note that in the caseof the triploid conceptuses the volume of extraembry-onic membranes present did not appear to be in anyway deficient. This may be related to the fact that thetriploids possessed both paternally and maternallyderived genomes. While a primitive yolk sac circu-lation was occasionally seen in the diploid mem-branes, this was never seen in the triploids. Furtherobservations will obviously be required to establishwhether this difference was significant or not.

Histological examination of ten of the advancedsomite-stage triploid conceptuses all but one of whichhad successfully 'turned' to adopt the fetal positionrevealed that four were morphologically normal,though substantially smaller than their diploid litter-mates. As an indicator of development, three of theseembryos had deeply indented otic pits, while theseven developmentally more advanced embryos hadotocysts present. All six of the morphologically ab-normal embryos examined had either localized orextensive neural tube defects (see Fig. 2). In oneembryo, this consisted of a localized spina-bifida-likelesion in the midabdominal region of the neural axis.Five embryos had neural tube defects involving thecephalic region. These could be divided into threeprincipal groups according to the location of thecephalic lesion present, namely (i) one 'unturned'embryo with a squirrel-type deformity in which thecephalic neural folds had failed to close in the regionsoverlying the fore-, mid- and hindbrain, (ii) twoembryos in which the neural folds had failed to closein the regions overlying the fore- and midbrain and(iii) two embryos in which the neural folds had failedto close in the region overlying the forebrain only. In


one of the embryos in group ii, a localized side-sideduplication of the neural tube was present in themidcaudal region of the neural axis.

In those regions in which a neural tube defect waspresent, the edges of the neural folds were character-istically everted and the neuroepithelial cells roundedup and disorganized. Histological analyses of 12diploid embryos isolated from the same litters wascarried out. None of these embryos had any evidenceof neural tube or cardiovascular abnormalities.

While no evidence of cardiovascular stasis wasobserved in the diploid conceptuses, this was ob-served in about one third of the triploids, though in allinstances spontaneous contractions of the heart wereseen. It is possible that the presence of cardiovascularstasis may have been indicative of the impendingdeath of these embryos. While no specific grosscardiac abnormalities were recognized in any of thetriploids, an impression was formed that the heartswere possibly somewhat larger than expected, thoughthis might have been due to the fact that the cephalicregions were slightly smaller than normal in thisgroup.

In only one conceptus out of all of the triploidsstudied, the uniform presence of two metacentricchromosomes was noted. It is unclear whether theserepresented de novo Robertsonian translocations. Inall of the mitotic spreads examined, a minimum offive normal diploid or triploid preparations wasalways present with a total of either 40 or 60 chromo-somes, respectively. However, in most instances,many more spreads were analysed. No examplesof numerically abnormal chromosome preparationswere observed except, that is, from the single con-ceptus with the two metacentric chromosomes indi-cated above (see Fig. 1A).

Discussion

In the present study in which postimplantation con-ceptuses from pregnant LT/Sv strain mice wereexamined both cytogenetically and morphologically,it was apparent that the majority of the digynictriploids induced following the fertilization of ovu-lated primary oocytes developed to the blastocyststage and successfully evoked a decidual response.Furthermore, the majority of the triploids progressedto comparable developmental stages to thoseachieved by normal diploid embryos recovered on the7th or 8th day of gestation. When the conceptuseswere recovered on the 10th day of gestation, thetriploids became increasingly more readily dis-tinguishable from their normal diploid siblings, afinding that is in general agreement with the results ofprevious studies on triploidy in the mouse (recentlyreviewed by Niemierko, 1981).

In two of the experimental studies in each of whichsingle somite-stage triploid mouse conceptuses hadbeen recovered and analysed (Vickers, 1969; Opas,1977) minimal information on their gross morphologyhas been provided. The embryo obtained in theformer study was recovered from a pregnant femalebetween 9|-1H days after delayed fertilization andwas described in the following terms: 'structurallynormal but smaller and paler than its diploid siblings,no heart beat could be detected and it seems likelythat this foetus was dying'. The embryo recovered onthe 12th day of gestation by Opas (1977) was ofapproximately the same developmental age. It had a'beating heart, allantois, yolk sac, but no head'.

More recently, a small number of triploid con-ceptuses has been isolated by Kaufman & Bain (1984)following the exposure of recently mated female miceto an intragastric injection of a dilute solution ofethanol. These embryos were recovered on either the10th or 11th day of gestation and could be dividedinto two easily distinguishable categories, namelythose that were retarded by about 24 h in develop-ment compared to their littermates, but neverthelesswere morphologically normal, and a second groupthat were generally smaller than their normal siblings,but were in addition morphologically grossly abnor-mal. In the present study, an additional class oftriploids has been recognized, namely those thatappeared on gross inspection to be morphologicallynormal in appearance and at a very similar develop-mental stage to their diploid siblings, but had asignificantly reduced crown-rump length and vol-ume.

Despite the relatively limited number of triploidsanalysed in the present study, only two out of thethirty-eight triploid conceptuses recovered demon-strated any of the features of the 'triploidy syndrome'first described in detail by Wr6blewska (1971; see alsoNiemierko, 1981). In addition, it is of interest that inthe few mouse strains in which postimplantationtriploid embryos have been observed, no viableconceptuses have been recovered from females be-yond the 12th day of gestation (Vickers, 1969; Takagi& Oshimura, 1973; Wroblewska, 1971, 1978; Bara-nov, 1976; Opas, 1977; Niemierko, 1981).

In the A/He strain, high rates of triploidy wereencountered following the mating of superovulatedfemales to normal males. Many of these embryosappeared to survive into the early postimplantationperiod (Takagi, 1970; Takagi & Oshimura, 1973).Following this experimental treatment, the averagefrequency of triploidy was found to be in the region of31 % at 2-5-3-5 days after mating, 24 % at 6-5 and 7-5days, and decreased to about 15% at 10-5 and 11-5days. In the majority of cases, the triploid con-ceptuses displayed all the features of the triploidy


B

H


syndrome. One embryo isolated on the 10th day,however, was said to be developing normally, thoughregrettably no further information on this embryowas provided.

In another very extensive study (see Baranov,1976), the spontaneous incidence of triploidy wasestablished in mice from the CBA, C3HA and C57BLinbred strains. The highest incidence of triploidyrecorded was in the C57BL strain in which about 4 %of the conceptuses were found to be triploid. Cyto-genetic evidence was provided which confirmed thehypothesis that the majority of the triploids encoun-tered were digynic in origin. In the same study, themorphology of a considerable number of the triploidsthat survived into the early postimplantation periodwas examined histologically. 10 of the 18 embryosexamined on the 8th and 9th day were at the neurulastage, all appeared to be healthy, though develop-mentally somewhat retarded compared to their dip-loid littermates. The remaining embryos were smallerand morphologically abnormal. When triploid em-bryos were isolated on the 10th day, 25 of 31examined were clearly being resorbed. Of the sixapparently viable embryos 'their head end was much

Fig. 2. Representative intermittent serial transversesections through three LT/Sv triploid embryos isolated atabout midday on the 10th day of gestation.

(A-D) Four sections through the cephalic region of alimb-bud-stage embryo with deeply indented otic pits.Note that the neural folds overlying the entirepresumptive forebrain region (arrows) have failed to fuse,whereas the hindbrain region appears to bemorphologically normal. Bar, 200fim.

(E-F) Two sections through a developmental^ slightlymore advanced limb-bud-stage embryo than thatillustrated in Fig. 2A-D, which possessed a normallylocated pair of otocysts (small arrows). Note that theneural folds overlying the entire presumptive forebrainregion have failed to close. The extent of eversion of themargins of the neural folds (large arrows) and associateddegree of neuroepithelial cellular disorganization isclearly apparent in these sections. Bar (E), 200^m;Bar (F), 400/zm.

(G-H) Two sections through approximately the middleof the embryonic axis of a partially 'turned' embryowhich, in addition, cuts through the proximal part of thetail region. Note in particular the side-side duplication ofthe neural tube (arrow). These neural tubes graduallymerge both cranially and caudally to give, initially, asingle morphologically abnormal neural tube with a singlelumen. However, more distant cranially and caudallyfrom the duplicated segment, a single apparentlymorphologically normal neural tube was present alongthe rest of the 'spinal axis'. In the cephalic region, theneural folds overlying the presumptive fore- and midbrainhad failed to fuse, whereas the neural folds overlying thepresumptive hindbrain region had closed normally. Bar,200 jim.

smoother than normally, the neural crests (sic) wereflattened and asymmetrical, and the anterior andposterior invaginations of the gut were ill defined'(Baranov, 1976). The two triploid embryos isolatedon the 11th day had a sluggishly beating S-shapedheart, several pairs of somites present and wereclearly (from the illustrations published in this paper)at the early headfold stage of develqpment.

The importance of genetic background on thepostimplantation development of triploid mouse em-bryos has clearly been demonstrated by the fact thatboth CBA and A strain conceptuses develop the'triploidy syndrome', whereas 129/Sv and its crossesapparently do not (Wr6blewska, 1978; see alsoBeatty, 1957; Wr6blewska, 1971). Possibly the LT/Svstrain mice used in the present study belong to thissecond category.

The findings reported above are clearly speciesspecific, as triploid (? dispermic) rat embryos havebeen recovered on the 12th day of gestation (Piko &Bomsel-Helmreich, 1960), triploid rabbit embryos onthe 17th day (Bomsel-Helmreich, 1965, 1971), whilehuman triploid fetuses may very rarely survive toterm and beyond (Niebuhr, 1974).

The fact that the triploids in the present studyresulted from the fertilization of spontaneously ovu-lated primary oocytes which apparently were capableof developing during the preimplantation periodin synchrony with their normal diploid littermateswould undoubtedly have favoured their chance ofsurvival into the postimplantation period. Whethertheir digynic genotype conferred any additional de-velopmental advantage has yet to be established, butthis would seem to be unlikely.

The only other mice that are apparently capable ofregularly ovulating a significant number of primaryoocytes are from the NMRI/Han strain (Jenderny elat. 1980; Hansmann, Jenderny & Probeck, 1983) inwhich up to 12 % of their eggs are ovulated as primaryoocytes. However, even this relatively high level,though considerably lower than observed in theLT/Sv strain females, is only observed after theexposure of the NMRI/Han females to exogenousgonadotrophins (Bartels, Jenderny & Hansmann,1984). However, for reasons that are not entirelyclear, when Ta/O females of C3H x 101 backgroundwere mated to males of the NMRI/Han strain and theadult Fj hybrids treated with exogenous gonado-trophins, analysis of their ovulated oocytes revealedthat a low level of diploidy (10%) occurred in theoocytes from XX mice, but a high level of diploidywas observed (24-6 %) in oocytes from females withthe XO karyotype (Beermann, Franke & Hansmann,1986). In the LT/Sv mice, our findings would appearto indicate that significant numbers of primaryoocytes are ovulated in both spontaneously ovulating


females and following exogenous hormonal stimu-lation.

The most advanced triploid mouse embryosencountered on the 10th day of gestation in thepresent study appeared to be developmentally sub-stantially more advanced than those previouslydescribed by Baranov (1976). In the present study,the majority of the triploid embryos recovered on the10th day of gestation was found to be at a similardevelopmental stage to their normal diploid litter-mates. A proportion of the morphologically abnor-mal embryos, however, while still viable and with abeating heart at the time of their isolation, was clearlyquite unhealthy, often having areas of vascular stasisand would almost certainly have died 24-36 h later,had they been left to develop in utero. Preliminaryhistological analysis of the triploid conceptuses hasrevealed that they had several common features, inaddition to their small size. More than half of thetriploid conceptuses displayed anomalies of closure ofthe neural tube, principally involving the cephalicneural folds, the latter ranging from isolated closuredefects overlying the presumptive forebrain to exten-sive lesions involving the entire cephalic region.While no obvious gross cardiac abnormalities wereidentified, in a proportion of these embryos, therewas evidence of vascular stasis. It appears likelytherefore that the present observations are consistentwith previous observations on triploidy in the mouse(see Beatty & Fischberg, 1949, 1951; Edwards, 1954),where disturbances of organogenesis were assumedto have resulted from a reduced total embryonic cellnumber associated with an increase in the volume oftheir individual component cells. Takagi & Sasaki(1976) estimated that from their experimentalfindings the cell-cycle time during the preimplan-tation period was about 10 % longer in triploid mouseembryos than in their diploid siblings. They predictedfrom these values that the total cell number of triploidembryos would become half that of their diploidsiblings by about the time of the 10th cleavage (about5-5 days p.c.) and a quarter at the time of the 20thcleavage (about 10-5 days p.c). In fact, similarfindings were reported by Snow (1975, 1976) duringhis investigations into the postimplantation develop-ment of tetraploid mouse embryos. We hope toquantify this difference in cell number and cellvolume between control (diploid) and triploid con-ceptuses, and describe their histological features inmore detail when sufficient numbers of viable LT/Svembryos become available to us.

The work described here was supported by grants fromthe National Fund for Research into Crippling Diseases(Action Research for the Crippled Child), the CancerResearch Campaign, the Melville Trust, and an equipmentgrant from the Scottish Home and Health Department (ref.

no. K/MRS/50/C866). We thank Miss Corinne Arnott forassistance with the wax histology.

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(Accepted 22 June 1987)

the postimplantation development of spontaneous digynic ... · scribed (piko & bomsel-helmreich,...

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