EXPERIMENTAL INDUCTION OF CHROMOSOME ABNORMALITIES

Induction experimentale d’anomalies chromosomiques

Experimentelle Induktion von chromosomalen Anomalien

Ondine BOMSEL-HELMREICH *

I ntroduction

As chromosomes are the vectors of genetic inform ation transm itted from one generation to the other, it is to be expected that any alteration in the num ber or organisation of the chromosomes may be of importance.

Chromosome aberrations are specially effective in the perform ance of the reproductive system: reduced fertility or complete sterility, embryonic or fetal m ortality, congenital m alform ations and perinatal death may all result from chromosomal variation.

Many w orkers have contributed in the last 20 years to the exploration of the effects of chrom osom al variation in many fields and some very good reviews are mentioning the different ways of inducing them experimentally (see Fechhei- mer, 1972 for review).

Experiments were m ainly directed in three directions:

— Induction of chromosomal aberrations of any type by exogenous factors,

Induction of physiological situations which are known to increase embryonic m ortality and therefore eventually chromosomal aberrations,

— Induction of program m ed aberrations of one or a complete set of chromosomes.

de ph7 siologie Animale, Centre National de Recherches Zootechni- ques (CNRZ), Institut National de la Recherche Agronomique (INRA), Domaine de Vilvert, 78350 Jouy-en-Josas, France.

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I nduction of chromosomal aberrations of any type by exogenous factors

a) Ionizing radiations

It is well known that ionizing radiations induce chromosome breaks; their effect has been extensively studied on different cell types both in vivo and in culture. The effect of X radiations on m am m alian germ cells is, qualitatively at least, sim ilar to its effects on somatic chromosomes. X rays have been used on testes or samples of semen to produce chrom osom ally abnorm al young (see Russell, 1962 for review).

Griffen and Bunker (1967) applied X rays to the testes of male mice and used them subsequently as sires. A variety of aberrations appeared in the descendants such as translocations, deficiencies or trisomy. But the relative sensitivity of the various spermatogenetic cell types are not the same in all species (Lyon and Smith, 1971) which is also the case for oogenic cells (B aker, 1971). Besides, animals of different genotypes within the same species react also differently to a same X ray dosage (Searle et a l, 1970).

b) Chemicals

Many substances have been used and induce various chromosomal aberrations. Those which excert an effect a t very low concentrations are the ones of prim ary interest. Between many compounds Rohrborn et al. (1971) recently tried mutagens such as triethyleneimminobenzoquinone or triazaquinone (H ansmann and Rohrborn, 1973). They were injected into mice shortly before ovulation. A very high percent of preim plantation embryos had chromosomes w ith structu ral anomalies or were hyper- or hypodiploids.

The interest of these chemicals rem ains in w hat these experim ents show about the structure and functioning of chromosomes and cells. But their action is not necessarily lim ited to DNA or chromosomes bu t many other cellular constituants: perm eability of membranes, enzyme activity, protein synthesis, etc., all of which play a role in embryonic development. Another bu t negative in terest shows the large possibilities of most chemical compounds used in form of food additives, drugs, pesticides or w ater contam inants to induce indesired chromosome aberrations.

I nduction by preferential physiological situations

Another line to induce aberrations is to recreate experimentally physiological situations which are known to increase embryonic m ortality. Aging of gametes is certainly the m ost largely and recently explored situation of these.

The importance of such experiments are confirm ed by the possibility of these situations occuring either in farm animal or hum an reproduction. The use of progestagens to induce ovulation in cows or sheep, the induction of ovulation of eggs for transplant purposes, the im portance of the right timing of mating or insem ination are all involved.

In man, long or irregular cycles appearing in very young or premenopausal women are often related to spontaneaous abortion and chromosomal abnormalities; perhaps through the process of delayed ovulation. Delayed fertilization

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is evidently bound to occur often in the hum an where the possibility of coitus is not lim ited by an oestrus.

a) The aging o f the egg

Aging of the ovum in a preovulatory stage by delaying ovulation causes a threefold increase in chromosomal aberrations of the post im plantation embryos in the ra t (B utcher and F ugo, 1967). Follicular aging of the oocyte of the rabbit (Bomsel-Helmreich, 1972) increases likewise aberrations in the blastocyst: triso- mics, triploids, mosaics, the last being the most frequent, and embryonic m ortality; bu t in a higher proportion as they are observed earlier in development.

Aging of the egg in the Fallopian tube by delayed mating, affects the embryos in the same way; bu t the aging process appears already after several hours, whereas it takes more than 40 hours for intrafollicular aging of the oocyte.

The aging of eggs has been mostly observed immediately after fertilization. For this reason digyny and dispermy are the most often described abnormalities, in that they are the easiest to observe. Both have been extensively studied m different species. Their percentage may be quite high: 34 % in the ham ster (Chang and Fernandez-Cano, 1958) and 21 % in the sow for digyny, 10 % for disperm y (Thibault, 1959). The observation of later stages of development shows not only that these eggs are able to develop but also that lesser m itotic anomalies occur in a m uch higher proportion. Austin (1967) obtained 25% triploids and 65% trisom ics and monosomies in the rabbit as did Shaver and Carr (1967) and Vickers (1969) in the mouse. Mixoploids apear as well. In the sow at preim plantation stage, besides a high embryonic m ortality, 25 % of the embryos are morphologically abnorm al and retarded. A certain number are triploid bu t the large m ajority diploid (B omsel-Helmreich, 1967). The relative part played by the environm ental factors which change (age) with delayed mating and the aging of the e§8 per se could be efficiently studied by using in vitro techniques. In the rabbit Thibault (1967) aged eggs in vitro and fertilized them w ith aged sperm. Digyny was observed in m ore than half of the eggs and dispermy in 20 %. This shows a direct effect of aging on the gametes. Fraser and Dandekar (1973) — also in the rabbit — aged the eggs in vitro but fertilized them with fresh sperm and cultivated them for two days. They observed no reduction in the fertilization ra te and no increase in triploid embryos; nevertheless there was a m uch reduced vitality after the first cleavage. The authors supposed that the effects of delayed mating were mainly due to tubal factors. The involvement of tubal environment in impairing development has been recently dem onstrated (Bomsel-Helmreich and S zollosi, 1974).

b) Aging of sperm

The time dependant decrease in the capacity of spermatozoa to fertilize and support norm al embryogenesis is known since a long time. Tesh (1966) dem onstrated both pre-and post im plantation losses; with increasing age of the spermatozoa the losses appeared at an earlier stage. Maurer et al. (1969) reported reduced cleavage rates in cultivated embryos taken from rabbits insem inated 20 hrs. before ovulation. Thibault (1967) described digyny in 40% of freshly ovulated eggs fertilized w ith aged sperm. Martin and Shaver (1972) observed the chromosomal constitution of rabbit blastocysts when sperm was aged in utero for 14-28 hrs.

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Almost ten percent had abnorm al caryotypes: mixoploids (2n/4n), mosaics (2n / 2nd) or chim aeras (XX/XY). I t is interesting tha t aged sperm induces in a young egg digyny which is an anomaly of the egg and may be considered as an incomplete activation. Non-disjunction as late as the first cleavage is also observed. Dispermy appears when the interval between insem ination and ovulation is abnorm ally long in the rabbit (H arper, 1970); it may be related w ith a higher num ber of sperm present being then, a t the site of fertilization. Post-ejaculatory storage of m am m alian spermatozoa before use in artificial insem ination leads to a reduction in fertility and to an increase in embryonic m ortality, in the bull (review by Salisbury and H art, 1970) and in the rabbit (S tranzinger, 1970; Ko- foed-Johnsen et a l, 1969). Storage possibly reduces fertility in such a way, tha t it leads to delayed fertilization, and tha t chromosome aberration occur in the waiting ova.

c) Polyspermy

Penetration of the ooplasm by more than one spermatozoon has been described in many species including the rat, mouse, ham ster, guineapig, rabbit and pig (for review see Austin and B ishop, 1957; P iko, 1961). The domestic pig seems specially favourable for these studies as polyspermy has been found to reach a striking incidence under a variety of experim ental conditions. Through delayed m ating or insemination (P itkjanen, 1955; H ancock, 1959; Thibault, 1959) an alteration of the egg mem brane seems to occur by which the aged egg is not able any m ore to perform a block to polyspermy. Polyspermy occurs also after injecting progesterone systematically (Day and Polge, 1968) or locally (H unter,1972) shortly before ovulation or after provoking ovulation during the luteal phase of the oestrus cycle (H unter, 1967). The relaxation of the isthm us and uteral- tubal junction occurs after local injection of progesterone. Polyspermy is then thought to originate principally from the increased num ber of sperm atozoa reaching the site of fertilization and an alm ost sim ultaneous penetration of the egg m em brane by two or more spermatozoa before com pletion of the zona reaction. But the direct effect of progesterone on the m em branes of the gametes cannot be excluded.

In the pig surgical resection of the isthm us is followed by the same high percentage of polyspermic eggs (34 %) (H unter et Leglise, 1971) and also bu t to a much lesser extent, in the rabbit. This shows once more a species - specific difference. Logically, the direct deposition of excessive num bers of spermatozoa a t the site of fertilization induces polyspermy as well (H unter, 1972).

But in both dispermic and trisperm ic eggs, a single m ale pronucleus is uniting w ith the female one, the accessory male element (s) remaining in the opposite hem isphere of the egg. This may leave a doubt about the possibility of induction of polyploid embryos, as up to now the descendant of these polyspermic zygotes have not been studied.

I nduction of definite chromosomal aberrations

The obtention of a specifically induced aberration concerning either one chromosome or a whole set should allow genetically b e tte r defined observations.

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a) Aneuploidy

Genetic factors have been used in an extensive line. Recently an experimental system in the mouse using crosses between Mus poschiavinus (tobacco mouse) with the laboratory mouse, perm its the study of the lethal effects of zygotic aneuploidy for certain chromosomes (Tettenborn and Gropp, 1970). Structural heterozygosity of the Robertsonian type may enhance irregular segregation in meiotic anaphase I; as a consequence monosomic and trisom ic gametes occur. Back-crosses of heterozygotes w ith normal laboratory mice are used. It is possible to study fetal m ortality related to the individual specific m etacentrics involved and eventually to the sex of the heterozygous parental carrier dispensing aneu- ploid gametes (Gropp and al, 1972). The efficiency of the system is very high: less than 50 % of preim plantation embryos are eusomic; but aneuploid embryos gradually die off. Hypo- and hypersomic blastocysts are present in equal numbers, which correspond well to the frequency of aneuploids in secondary sperm atocytes of the sires observed a t metaphase (S toll and Gropp, 1974). A small num ber of haploids and triploids occur also. In early post-implantation, monosomies become alm ost completely eliminated while trisom ics and even double trisomics survive distinctively longer, almost to birth. Offspring of female carriers of heterozygosity have a much higher incidence of aneuploidy than that of heterozygotic males.

Trisomic embryos display surprisingly sim ilar pattern of development in all the system of single m etacentric heterozygotes so far studied: retardation of development and general hypotrophy (external and at level of organs) and placenta. Gross m alform ations in contrast occur very rarely (Gropp, 1973).

These experim ents show eventually that in aneuploids one chromosome in excess is less detrim ental to development than the loss of one chromosome and that these losses determ ine in mice a crisis quite early, around implantation. Further, in regard to answer the question: does a spermatozoon transport its genetic load w ith equal efficiency regardless the content, there seems to be no selective process operating against unbalanced genomes between second m etaphase in the male and three- day embryos. A sperm atid with an extra chromosome or w ith one chromosome missing is as likely to m ature into a spermatozoon and take part in fertilization as one with a balanced genome; an egg is not sensitive to an abnorm al genome in the first three days of development (Ford and E vans 1973; Ford and E vans, 1974; Ford, 1972). However, this seems not to apply to all! for instance to sex chromosomes: Morris (1968) has shown that in mice 2n X0 eggs develop norm ally where as 2n Y0 do not develop beyond the first division.

The overwhelming m ajority of trisomic embryos sired by heterozygous males have received the fa ther’s m etacentric chromosomes. Trisomy of another chromosome seems therefore «spontaneous» trisomy and occurs as frequently as in controls (4 % ). This shows tha t the presence of a trivalent does not influence the frequency of o ther abnorm al events; which is different to what seems to happen in other circum stances i.e . in aging eggs or hereditary translocations.

There are once m ore species-specific differences: for instance only in mice do XO’s develop normaly; Ford and E vans (1973) obtained ten time m ore mosaics in their born mice than appear in human.

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b) Parthenogenesis

The induction of an alteration of chromosome num ber may concern not only one bu t a complete set of chromosomes. The induction of parthenogenesis, tri- ploidy or tetraploidy by exogenous factors has been largely studied.

Parthenogenic development may appear after exposure to various stimuli, such as hyper- and hypotonic solutions, cooling or heating (for review see Beatty, 1957, 1967). Many experiments were made on the rabbit as suppression of polar body form ation is easily obtained by m oderate cooling; Chalmel (1962) even obtained fertilization (though abnorm al) of the parthenogenetically activated egg.

In the mouse Braden and Austin (1954) employed heat shock as the activating agent but did not continue observation after 70 hrs. Recently Graham (1970) obtained development of mouse eggs up to the blastocyst stage by cultivating cumulus-free eggs in vitro ; Tarkowski et al. (1970) obtained development beyond the stage of im plantation as a result of activation in vivo w ith a electric current. Komar (1973) activated eggs by heat in vitro; fewer eggs developed to morula and blastocyst stage (15%) compared to the two preceding ways of induction (50%).

Whereas Graham (1970), Tarkowski et al. (1970), Witkowska (1973) obtained mostly haploid parthenogenons, Komar (1973) obtained some diploids but her low percentage of success may be explained by the induction of many hypohaploids which seem to be unable to cleave. W hatever the means of obtention, partheno- genetic eggs developped more slowly than controls.

c) Triploids

Triploid embryos have been produced at will by using colchicine or a related compound which suppresses cytokinesis; E dwards (1958; 1961) in the mouse, P iko and Bomsel-Helmreich (1960) in the ra t injected the compound into the m other during ovulation or fertilization. A less damaging way is to use colcemid during a short and precisely defined time i. e. at expulsion of the second polar body, if in vitro fertilization’in the rabbit is used (Bomsel-Helmreich and Thibault, 1962; Bomsel-Helmreich, 1967). The success is alm ost 100 % so tha t embryos which are all of dygynic origin could be studied extensively. They are macroscopically norm al but delayed in development. Most of them die before mid gestation, but some were found in the last quarter of gestation. This shows that under very favourable conditions (genetic and horm onal) a triploid embryo may eventually survive till parturition. This is in accordance w ith the triploid hum an embryos, very num erous in spontaneous abortion; they neither survive till birth , exception made for mosaics 2n/3w.

The genetic pathway to obtain triploids was used by Wroblewska (1971) who crossed inbred females with males of a different strain. They showed all a specific disturbance of embryogenesis, an inhibition of the embryonic part of the egg- cylinder to form; their origin seems to be digynic. But the genetic background seems to be able to modify the morphological expression of the chromosome aberration; since the m ajority of triploids obtained either spontaneously or by the ways mentioned above were retarded in development but otherwise normal.

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Another m anner to induce triploidy in excellent in vivo conditions is suggested by Beatty and Fechheimer (1972); in certain individual rabbits as well as in other species diploid sperm appear quite often in semen and could be well separated from haploid sperm by centrifugation; these could induce triploid zygotes after artificial insemination.

d) Tetraploids

In opposition to triploids or haploids a balanced polyploidy seems to be compatible w ith complete fetal development in mammals; recently Snow (1973) obtained tetraploid mouse embryos by cultivating 2- cell eggs in cytochalasine which prevents cytokinesis w ithout influencing nuclear division. The proportion of treated embryos which develop normally is im portant: they are of norm al dimensions and some develop to term. They go much fu rther than the tetraploids obtained by cell-fusion by Graham (1971). Tarkowski (personal communication) uses also a fusion technique and obtains tetraploid blastocysts. However, most of the tetraploid embryos are abnormal; but as the size of tetraploid cells is larger than diploid ones it is likely tha t the cause of embryonic m alform ations is in the reduced num ber of cells of the blastocysts.

Mechanisms of occurrence of chromosomal aberrations

Despite the num erous ways of induction, not much is known yet about the causal relation of chrom osom al anomalies and abnormal development of m ortality of embryos.

The use of exogenous agents such as X-rays or drugs generally induce chromosome aberrations a t random . When the physiological ways of induction are used in a same situation, alm ost all kinds of abnorm alities occured: digyny as well as dispermy, monomy, trisom y or mosaics. Even the time of occurence of the abnorm ality is not easy to define. Trisomies or monosomies may occur at the first or second meiotic division of the gametes. Triploidy may be induced as well by nonexpulsion of the first or second polar body or dispermy. Mosaics arise at early cleavage but not necessarily at the first. There appears also a notion of connected anomaly, which is the occurence of two different types of aberration occuring subsequently such as 2>z-l/3n-2. This is understandable as well from a genetic point of view than a physiologic one.

Further, the way of induction does not necessarily induce the only chromosomal aberration, but also yet insufficiently explored anomalies of the whole cell. This is easily understood for different exogenous agents but occurs as well in the case of physiological aging. I t explains different aberrations to be determ ined by a some inducer as well as the high embryonic m ortality observed in m ost of the experiments, and which is not always linked to the chromosomal picture.

It is equaly possible tha t a different time of induction as well as a different m anner which produces a same abnorm ality does not allow an equivalent development. This may be the case in dispermy induced in norm al eggs of the pig where the three pronuclei do not fuse, whereas dispermy in an aging egg leads to the syngamy of the three pronuclei and therefore to a triploid embryo.

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The use of identification of the origin of individual chromosomes by the banding technique should be a great help in these problem s. The induction of a specific anomaly by genetic factors or of polyploidy by in vitro experim ents gives a much be tte r control over these variations. Nevertheless they cannot suppress genetic regulations which are still not undestood such as the preferential survival of 3nXXY embryos seem specially morbid.

A m ost puzzling topic remains the alm ost complete lack of inform ation of the causes of embryonic m ortality of chromosomally abnorm al embryos. In some cases the embryo shows a gross developmental m alform ation, but in m ost cases the embryo is only delayed in growth which determ ines death at different periods of embryogenesis. M ortality is also surprising, e. g. 90 % of 2nXO embryos die in utero, 10 % live w ith only m inor anomalies. Very few causal studies have been realized up to now. The m itotic cycle of aneuploids seems to be abnormally long (Cure et al., 1973). Some enzymatic activities of tripoid embryos may be higher than in diploids e. g. glucose-6-phosphodehydrogenase and other X linked enzymes, some may be lower, some of equal activity (B omsel-Helmreich, 1970). But these are only the very first explorations in this direction.

Conclusion

The different ways of inducing experimental anomalies bring all new lights on physiological and genetic research. They give inform ation on behaviour of nuclei and cells with an abnormal genetic constitution, they show the precise circumstances of induction of errors of meiosis or mitosis, which should perm it to avoid these accidents to occur spontaneously; also the critical periods of embryonic m ortality related with abnorm al chromosome num bers. Finally the obtention of planned abnormalities in zygotes which have been little stressed by the induction provides a convenient tool to explore the genetic and metabolic causes of this m ortality, which for the moment is a field still little explored.

SUMMARY

Experiments on the effects of induced chrom osom al variations are directed after three m ajor lines:

1) Exogenous factors such as X-radiations or chemicals induce any type of chromosomal variation, but w ithout possibility of choosing precisely one type.

2) Some physiological situations which may occur spontaneously in farm anim al or hum an reproduction produce a high percentage of embryonic m ortality and chromosomal anomalies in embryos or new-born. The best known situation is the aging of gametes. Aging of sperm before insem ination, aging of intrafollicu- la r oocytes before ovulation, aging of egg or sperm in the fallopian tubes before fertilization induces in all mammals studied the same anomalies: digyny, dispermy and non-disjunction at meiosis or first m itosis after cleavage. The embryos present various anomalies such as monosomies, trisomies, triploidies, mosaics, mixoploids or chimaeras. But on inducing of these preferential physiological situations, once m ore one does not obtain a specific anomaly.

3) There exist some ways to induce definite chrom osom al variations. Embryos

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aneuploid for a known chromosome may be obtained by using genetic factors. Aneuploid sperm for the chromosomes explored up to now is as fertile as euploid sperm. After fertilization, trisom ics and monosomies are present in equal num bers; but monosomies die off before implantation, wheras trisom ics survive after im plantation. The sex of the anomaly-inducing parent is related to the survival of the abnorm al embryo. Nevertheless individual chromosomes (such as sex chromosomes) play also a role in survival.

Parthenogenesis, if relatively easy to induce, allows no development later than m id -gestation. Triploids may be obtained in large numbers, specially by in vitro fertilization techniques, but die before the end of gestation. Tetraploids eventually are able to survive till a fter birth.

Genetic origin such as digyny or dispermy, or cytological factors such as abnorm al cell size, determ ine in polyploids very different survival possibilities. In general, polyploids have no malformation, but seem to die of delay in em- bryogenesis.

The possible m echanisms of occurance of chromosomal aberrations are described, keeping in m ind tha t in general different types of aberration occur simultaneously, but may not necessarily allow an identic development of the embryo. The causes of embryonic m ortality related to chromosomal aberrations are actually alm ost totally unknown. First explorations of this phenomenon are described. But m ost genetic and metabolic causes are still to be discovered.

RESUME

Les experiences destinees a m ettre en evidence les effets d ’une variation induite du nom bre des chromosomes sont menees dans trois directions principales:

1. Les facteurs externes tels que les rayons X ou les substances chimiques sont suceptibles de provoquer tous les types de variations chromosomiques, mais sans possibility de choisir precisem ent un type particulier.

2. Quelques situations physiologiques, qui peuvent se produire spontanem ent au cours de la reproduction des animaux domestiques ou de Thomme provoquent 1’apparition d ’un pourcentage eleve de m ortalite embryonnaire et d ’anomalies chromosomiques chez les embryons ou nouveaux-nes. La mieux connue de ces situations est le vieillissement des gametes. Le vieillissement des spermatozoides avant l’insemination, le vieillisement intrafolliculaire des ovocytes avant l’ovula- tion, ou le vieillissement des spermatozoides ou de 1’oeuf dans les trom pes de Fallope avant la fecondation, provoque chez tous les mammiferes etudies les memes anomalies: digynie, dispermie et non-disjonction lors de la meiose ou de la prem iere m itose de segmentation. Les embryons presentent diverses anomalies telles que des monosomies, des trisomies, des triploidies, des mosaiques, des mixoploidies, des chimeres. Mais, ici encore, l'induction de ces situations physio- logiques preferentielles ne provoque pas 1’apparition d ’une anomalie specifique.

3. Quelques moyens existent cependant pour induire une variation definie du nombre de chromosomes. On peut obtenir des embryons aneuploides pour un chromosome determ ine en m ettan t en oeuvre des facteurs genetiques. Les spermatozoides aneuploides pour les chromosomes etudies jusqu’a ce jou r sont aussi fertiles que les sperm atozoides euploides. Apres la fecondation, les trisomiques

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et les monosomiques sont presents en nom bre egal; mais les monosomiques meu- ren t avant l’implantation, tandis que les trisom iques survivent a celle-ci. Le sexe du parent inducteur de l ’anomalie est en relation avec la survie de l’embryon. II n ’en reste pas moins que des chromosomes determ ines (tels que les chromosomes sexuels) jouent un role aussi dans la survie.

La parthenogenese, si elle est relativam ent facile a induire, ne perm et pas de developpement au dela, de la mi-gestation. Les triploides peuvent etre obtenus en grand nombre, specialement au moyen de techniques de fecondation in vitro, mais m eurent avant la fin de la gestation. Les tetraploides sont parfois capables de survivre ju squ ’a la naissance et au dela.

Les polyploides ont des possibility de survie tres differente suivant leur origine genetique (digynie ou dispermie) ou en fonction de facteurs cytologiques tels une taille cellulaire anormale. En general, les polyploides n ’ont pas de m alform ation mais il semble qu’ils m eurent d ’un retard de l’embryogenese.

Les mecanismes possibles de l’apparition d 'aberrations chromosomiques sont decrits en gardant en memoire que le plus souvant plusieurs types d ’aberrations se produisent simultanement mais qu'ils ne perm ettent pas obligatoirement le meme developpement de l’embryon. Les causes de la m ortalite em bryonnaire liee a des aberrations chromosomiques sont pratiquem ent totalem ent inconnues. Les prem ieres explorations de ce phenomene sont decrites, mais la p lupart des causes d’ordre metaboliques ou genetiques restent a decouvrir.

ZUSAMMENFASSUNG

Die Experimente betreffend die Effekte von induzierten chromosomischen Variationen fiihren in drei hauptsachlichen Richtungen:

1. Exogene Faktoren wie X Strahlen oder chemische Faktoren fiihren zu alien Typen von chromosomischen Variationen, aber ohne die Moglichkeit irgend einen speziellen Typus zu wahlen.

2. Gewisse physiologische Situationen welche spontan bei der Fortplanzung von Haustieren oder vom Menschen auftreten erzeugen einen hohen Prozentsatz embryonaler Sterblichkeit und abnormale chromosomische Effekte in Embryonen und Neugeborenen.

Die am meisten bekannte Situation ist das Altern der Gameten. Das Altern der Spermatozoiden vor der Besamung, das Altern in trafollikularer Ovozyten vor der Ovulation, das Altern von Spermatozoiden und oder Eiern in den Tubae Fallopiae vor der Befruchtung, fiihren bei alien untersuchten Saugetieren zu den gleichen Anomalien: Dygynie, Dispermie, und Non-disjunktion bei Meiose und der ersten Mitose nach der Teilung. Die Embryonen zeigen verschiedene Anomalien wie Monosomien, Trisomien, Triploidien, Mosaiken, Mixoploide oder Chimaeren. Aber bei der Induktion dieser vorziiglichen physiologischen Situationen erhielt man wiederum keine spezifischen Anomalien.

3. Es bestehen verschiedene Moglichkeiten um prezise chromosomische Variationen zu erhalten: aneuploide Embryonen, ein bekanntes Chromosom betreffend konnen erhalten werden indem man genetische Faktoren beniitzt. Aneuploide Spermatozoiden die Chromosomen betreffen die bis je tz t untersucht wurden sind ebenso fruchtbar wie euploide Spermatozoiden. Nach der Befruchtung sind Tri-

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somen und Monosomen in gleicher Zahl vorhanden. Aber die Monosomen gehen vor der Einpflanzung zu Grunde, wahrend die Trisomen bis nach der Einpflan- zung fortbestehen. Das Geschlecht des die Anomalie induzierenden Vorfahrs ist im Zusammenhang m it dem Uberleben des abnormalen Embryos, Nichtsdesto- weniger spielen individuelle Chromosomen (wie z. B. Sex-chromosomen) eine Rolle beim Uberleben.

Parthenogenese ist verhaltnism assig leicht zu erreichen, erlaubt aber keine Entwicklung spater als bis zur Mitte der Tragzeit. Triploide erhalt man in grosser Zahl speziell durch Techniken m it in vitro Befruchtung; sie sterben aber vor dem Ende der Tragzeit. Tetraploide konnen moglisherweise bis nach der Geburt uberleben.

Genetisches Entstehen wie Digynie oder Dispermie oder zytologische Faktoren wie abnoim ale Zellgrosse, bestim m en in den Polyploiden sehr verschiedene Mo- glichkeiten des Uberlebens. Bei Polyploiden bestehen im Allgemeinen keine Missbildungen abr sie scheinen zu Grunde zu gehen durch Verzogerung der Embryogenese.

Der mogliche M echanismus des Erscheinens chromosomischer Abweichungen wird beschrieben und m an legt nahe dass im Allgemeinen verschiedene Typen von Abweichungen gleichzeitig auftreten; aber sie fiihren nicht notwendiger- weise zu identischer Entwicklung. des Embryos. Die Ursachen der Embryonen sterblichkeit die m it chromosomischen Abweichungen in Zusammenhang stehen, sind gegenwattig fast vollig unbekannt. Erste Untersuchungen dieses Phenomens sind beschrieben aber die meisten genetischen und metabolischen Ursachen miissen erst gefunden werden.

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