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Nondisjunction by failures in the molecular control of oocyte maturation*

I. Hansmann and B. Pabst

Institut fUr Humangenetik, Universitat Gottingen, GoBlerstraBe 12d, D- W -3400 Gottingen, Deutschland

Introduction

The human species is, in comparison with other mammals, characterized by a significantly higher frequency of constitu­tional aneuploidy and it was estimated that at least 10 % of all conceptuses do possess an aneuploid karyotype (Bond and Chandley 1983; Hansmann 1983; for review). In more than 60- 80 % of all families studied so far by using chromosomal heteromorphisms or RFLPs (Restriction Frag­ment Length Polymorphisms), the origin of the trisomic gestation has been assigned to an error at maternal meiosis I (Hansmann et al. 1990). The reasons for the astonishing species specificity in genome mutations originating predo­minantly from maternal meiosis I are still unknown. Most hypotheses, like that of "spindle degeneration" (Mikamo 1968; Alberman et al. 1972), "nucleolus persistence" (Polani et al. 1960; Evans 1967), or the "production line" (Henderson and Edwards 1968), were presented before the ages of molecular biology and considered only morphologi­cal and or mechanical criteria to explain the association of trisomy with maternal age. But also later on, the physiology and molecular control of germ cell maturation and cell division has been neglected completely in the discussion about causes and mechanisms of meiotic aneuploidy.

Despite the hazardous potential of chemical and physical mutagens to increase the frequency of aneuploidy in man (Sankaranarayanan 1982; Bond and Chandley 1983; Hans­mann 1983), it is quite evident that endogenous causes must be responsible for most events of chromosome malsegrega­tion during meiosis. As a result of our studies with oocytes from Djungarian hamsters (Phodopus sungorus) and mice of the random-bred NMRIIHan strain, we proposed a model in which failures within the endocrine and paracrine control of germ cell maturation and meiosis cause an altered develop-

* Main lecture at the 87th meeting of the Anatomische Gesell­schaft, Mainz, March 23 to March 26, 1992.

Ann. Anat. (1992) 174: 485-490

Gustav Fischer Verlag Jena

mental and regulatory program of meiocyte cell division which is afflicted with an increased risk of a default chromo­some/chromatid segregation at either of two meiotic divi­sions. The generation of meiotic aneuploidy by a default germ cell division may be analogous to that causing and/or accompanying tumour cell proliferation (Hansmann et al. 1989). Failures within the control of germ cell division may lead to nondisjunction, chance segregation (both events may occur at meiosis I or II), or pre segregation during meiosis I (Polani and Jagiello 1976; Hansmann and El Nahass 1979; Hansmann et al. 1985; Angell 1991; Angell et al. 1991). The experimental evidence for that proposal will be review­ed shortly in the following sections.

Most experiments have been performed with Djungarian hamster females (Phodopus sungorus, Rodentia, Criceti­nae), an Asiatic hamster inhabitating the plains between Ural mountains, Mongolia, Djungaria and Altai. The diploid chromosome number is 2n = 28 with an advantageous hete­rogeneous chromosome morphology, ranging from large metacentric to small acrocentric chromosomes (Schmid et al. 1986). Nucleolus organizing regions (NORs) are present terminally at the short arm of chromosome 5, 7, 12 and 13. Five chromosome pairs, i.e. number 1, 2, 4, 6 and 7, do possess several extraordinary blocks of interstitial constitu­tive heterochromatin. Thus, the karyotype is not only favou­rable for identifying the origin of the malsegregated bivalent in ovulated oocytes without complicated chromosome band­ing techniques, but also for considering a predisposing influence of constitutive heterochromatin and of NORs on nondisjunction.

Gonadotrophins induce nondisjunction and diploidy in primary oocytes

By studying the spontaneous frequency of nondisjunction during meiosis I in oocytes from several mammalian species

(R6hrborn and Hansmann 1971; Hansmann and El Nahass 1979; Hansmann and Probeck 1979), we observed that Djungarian hamsters respond to the stimulation with gonado­trophins by ovulating significant numbers of hyperhaploid and diploid oocytes (Hansmann et a1. 1980). This suscepti­bility to gonadotrophins is not a transiently occuring phenomenon, but was observed with females in our breeding colony during the past 15 years. To provoke aneuploidy, females were injected intraperitoneally with PMS (pregnant mares serum) and 48 hrs later with hCG (human chorionic gonadotrophin). Oocytes were recovered from oviducts usu­ally 19hrs later and were prepared cytogenetically for chromosome analysis. The frequency of aneuploidy by meiosis I errors was calculated by counting the number of chromosomes of individually spread oocytes at the meta­phase II-stage. Approximately 70-90% of all females ovu­lated after pretreatment with gonadotrophins; the number of oocytes per female varied between 6-12, as a mean, and depended on the gonadotrophin dose injected. Aneuploid oocytes with a diploid or hyperhaploid karyotype (hypoha­ploidy may be due to preparational artifacts and was not considered) were ovulated by females in all dose groups, ranging from 1.25 IU (international units) up to the highest dose of 20 IU. The frequency of aneuploidy (diploid and hyperhaploid X 2) was strictly related to the dose of gonado­trophins, being lowest at 1.25 IU with 2.0% and highest at 20IU with 17% (Wuttke 1986).

The other species analyzed so far, i.e. mouse, Chinese hamster and Syrian hamster, do not respond to gonado­trophin treatment by the ovulation of aneuploid oocytes (Hansmann and El Nahass 1979; Hansmann and Probeck 1979). One mouse strain (NMRIlHan), however, do respond to gonadotrophins by ovulating significant numbers of diploid oocytes most often arrested at metaphase I (Hans­mann and El Nahass 1979; Hansmann et al. 1983). The expression of this trait, defined as Dipll, is gonadotrophin dose-dependend as well, and is inherited by hybrids of crosses between NMRIIHan and various other strains and by their respective back crosses. An X-chromosomal and an autosomal recessive mode of inheritance of Dipl I was excluded. Furtheron it was shown that the expression of the trait, measured as frequency of diploidy, appeared to be modulated by a maternally transmitted factor either of cyto­plasmic or mitochondrial origin. Strains were also identified exerting an up (C57BU6J, BALB/c) or down modulation (C3H1HeHan) of the Dipl I expression via the maternal mating type (Bartels et a1. 1984; Beermann et a1. 1987). By repeated backcrossing (12 generations), two mouse strains with nearly identical nuclear encoded genes but differing only in their mitochondrial genomes were created (C57BU 6J and NMBI2, the latter obtained from crossing NMRIlHan females with C57BU6J males) and were analyzed for the expression of Dipll. The maternal modulation of the inherit­able meiosis I error Dipll was not only corroborated but was also found to be associated with a mitochondrial inheritance (Beermann et al. 1988). This observation underlines the assumption that also mitochondria do play an important role during oocyte maturation and meiotic divisions (van Bler-

kom and Runner 1984; Beermann and Hansmann 1986). Their physiological contributions to the process of meiotic divisions, however, have not been identified so far.

From the results obtained with Djungarian hamsters and NMRIlHan mice it is presumed that the induceability of aneuploidy by gonadotrophins is a genetically determined species and strain specific phenomenon. The reasons for that specificity and the mechanisms leading to aneuploidy are not known so far. Hence it is still uncertain, whether human oocytes resemble those in susceptible or nonsusceptible species.

Maternal age and nondisjunction

A consistent feature of aneuploidy in man is the strong association of trisomy with maternal age (Penrose 1933; Hassold and Jacobs 1984), which appears to result from an age-dependent increase of meiotic nondisjunction rather than from a decreasing ability to abort trisomic conceptuses in older women (termed relaxed selection by Ame and Lipp­man-Hand 1982) (Hansmann et al. 1985; Hummler et al. 1987, for review). By studying ovulated mouse and Chinese hamster oocytes it was pointed out that nondisjunction dur­ing the 1st meiotic division, though not as drastically as expected, might indeed be more frequent at higher ages in these animal models. From studies with mice it became also evident that strain specificities are active in the generation of age dependent nondisjunction (Hansmann 1983, for review) and that hybrids might show a higher incidence of meiotic aneuploidy than females from their parental strains (Hans­mann and Jenderny 1983). Furtheron, it was suggested that the biological but not the chronological age should influence this age related process of chromosome segregation (Brook et al. 1984). A peculiar relation between nondisjunction and maternal age was furtheron detected in mice from CBA strain. Young and very old females ovulated only normal haploid oocytes (0 % disjunction), whereas oocytes from middle-aged females presented a significantly increased fre­quency (11.8 % nondisjunction) (Martin et al. 1976). Led by this observation of a hump-shaped slope of age-related nondisjunction together with the apparent discrepant mor­phology of mouse and human chromosomes, Hummler et al. (1987) argued about the relevance of the mouse system for studying processes like maternal age effects on nondisjunc­tion in man.

To evaluate the validity of the Djungarian hamster system for deciphering age-related processes of aneuploidy, a com­prehensive study about chromosome segregation at meiosis I was performed with more than 2000 ovulated oocytes from three different age groups, i.e. young (10-14 weeks) mid­dle-aged (36.7 weeks) and old (62.4 weeks) (HummIer et a1. 1987). The frequency of nondisjunction (hyperhaploidy x 2) (young: 1. 2 %, middle-aged: 5.6 %; old: 7.2 %) and pre­segregation (default segregation of chromatids at meiosis I) (young: 0%; middle-aged: 1.4%; old: 1.0%) was shown to increase significantly with age. The complete suppression of the 1st meiotic division resulting in the ovulation of diploid oocytes was significantly decreased, however, in middle-

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aged and old females. The latter observation would predict a lower incidence of triploidy in gestations from older females and is in agreement with the inverse maternal age relation of polyploidy in early spontaneous abortions in man (Eiben et al. 1990).

Chromosomal properties determine asynchronous segre­gation and chromosome specific risks for preferential nondisjunction

Due to the favourable and heterogeneous morphology of the Djungarian hamster karyotype it was possible to identify malsegregated bivalents in hyperhaploid ovulated oocytes. Three types of experiments were analyzed in detail to investigate whether each bivalent has the same risk for malsegregation, or whether there exists a bivalent specific­ity. The sample represents hyperhaploid oocytes ovulated after gonadotrophin stimulation (Hansmann et al. 1980 ; Wuttke 1986), after treatment with spindle poisons such as Colchicine (Hummler and Hansmann 1985) and Carben­dazim (MBC, HummIer and Hansmann 1988) during the late meiosis I spindle phase, and hyperhaploid oocytes ovulated from old females (Hummler et al. 1987). Altogether, 93 malsegregated bivalents were identified and were grouped according to size and position of the centro­mere into class A - D (n = 6, chromosome 1 - 5, and X), class E (n = 3, chromosome 6-8), and class F-G (n= 5, chromosome 9-13). Surprisingly, nondisjunction affected most often class A-D bivalents (83.9% of all events), but was rather infrequent in class E (9.7 %) and class F - G bivalents (6.5 %). The data imply that a bivalent in class A - D has a preferential and significantly higher relative risk of 14 % of mal segregation , which is more than 10 times more frequent than the risk of a bivalent class F-G. Class E bivalents have a individual relative risk of only 3.2 %, and a bivalent in class F - G has the lowest risk of 1. 3 %. Nevertheless, it might still be possible that the risks for individual bivalents within each class are considerably heterogenous.

The observed involvement of bivalents from the three classes is significantly different from that expected on the assumption of nondisjunction by chance only. The pattern of preferential nondisjunction is very similar, however, to that theoretically expected when the relative length of each chromosome at mitosis is used for calculating bivalent specific risks (Hansmann et al. 1989). The expected fre­quencies using length at mitosis as a very simplistic deter­minator are 64% for class A-D bivalents, 17.7% for class E, and 11.5% for class F-G. Class A-D, contains very large metacentric and submetacentric chromosomes, some of which do contain interstitial blocks of constitutive heterochromatin. Medium-sized metacentric chromosomes are placed into class E, whereas class F-G is characterized by very small metacentric and acrocentric chromosomes (see e.g. Hummler et al. 1987).

The possibility for a preferential nondisjunction by an

asynchronous segregation of bivalents during anaphase I, with the high risk class of A - D chromosomes acting as late segregators, was analyzed in detail by two experi­ments. In the first study, MBC, known to bind fast and irreversibly to mammalian tubulin (Havercroft et al. 1981), was injected at an early spindle phase (4.5 h post HCG), in contrast to the previously mentioned experiment in which MBC was given during the late phase only (6 h post HCG). The early inhibition of spindle function resulted, as expected, not only in nondisjunction of several bivalents within one oocyte but also in a high frequency of malsegre­gated bivalents from classes E and F-G. The pattern of nondisjunction followed in this study more or less a malsegregation by chance (Humrnler and Hansmann 1988). In a second set of experiments anaphase I configurations in oocytes isolated from preovulatory follicles were analyzed using fluorescence microscopy after fixation of oocytes in situ and staining of the spindle by labelled tubulin anti­bodies and chromosomes by DAPI (Theuring 1986). An asynchronous bivalent separation was observed in several oocytes at anaphase I: Small bivalents were separated and had moved already towards the spindle poles; large biva­lents, however, still rested in a paired configuration at the equatorial plate. In some oocytes medium sized bivalents showed even an intermediate position between large resting bivalents and the small chromosomes, having moved already far away from each other.

The question arises now about the nature of the manyfold chromosomal properties determining the sequence of biva­lent separation. Such properties could be pairing forces along the entire tetravalent, modified e.g. by the number and position of chiasmata, the position and amount of constitutive heterochromatin, or even the composition of DNA sequences. Properties like the presence or absence of rDNA, functioning as Nucleolus Organizing Regions (NORs), the structure and function of centromeric sequences and their associated proteins, or even the struc­ture of telomeric sequences may mediate asynchrony in chromosome segregation as well. Kinetochors possibly may generate also chromosome specific properties, e.g. by nuc­leating for each chromosome characteristically different numbers of pole-kinetochor microtubuli, thereby influenc­ing driving forces. Chromosomal properties may very not only between chromosomes of one species, but also bet­ween karyotypes from different species. And therefore, it is reasonable to predict that the extent of asynchrony in chromosomelchromatid separation at both meiotic divisions is specific for each species and is influenced by an interac­tion of different chromosomal properties. In such a model we would also predict that the mouse with an ordinary karyotype of 40 acrocentric chromosomes would belong to a class of species being characterized by a more synchron­ous type of segregation (see also Hansmann et al. 1983). The Djungarian hamster, in contrast, would belong to species with a more or less extreme asynchrony in bivalent segregation at meiosis I. To evaluate the susceptibility of the human karyotype it would be desirable to investigate the segregation process in human oocytes.

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Rate of meiosis is variable and correlates with aneu­ploidy

Earlier, it was proposed that an altered rate of meiosis in preovulatory oocytes could causally be related with nondis­junction during meiosis I (Crowley et aI. 1979; Hansmann 1983; Hansmann et al. 1988). This would presume that the rate with which oocytes pass through meiosis, starting with the release of germinal vesicle stage and ending a metaph­ase II, does not follow a riged1y timed program, but is variable and characteristically altered in oocytes which fail to segregate their chromosomes properly.

The question of meiotic rate was analyzed in three inde­pendent experiments about the influence of gonadotrophin dose, follicle type and of maternal age. The rate was deter­mined by classifying the meiotic stage after DAPI staining in preovulatory oocytes isolated from Graafian follicles at given time points. In all three studies, significantly varying meiotic rates were observed when compared to the rate in concommitantly analyzed control oocytes (Theuring 1986; Pabst 1987; Willenborg 1991). Accordingly, germinal ves­icle breakdown (GVBD) is the first step within the progres­sion through meiosis showing variation, expressed either as a considerably delayed or enhanced process. Variable furth­eron is the duration with which oocytes remain in chroma­tin mass-metaphase I stage (CMlMI). Both phases, i.e. GVBD and CM/MI, appear to be inversely related, how­ever, with each other. It is deducible from there that in response to the initiating trigger of GVBD in oocytes the duration of the germinal vesicle phase somehow determines the subsequent meiotic phase, at least. It is quite evident that either transcriptional or translational activities during the preceding GVBD-phase is limiting quantitatively and/or qualitatively the subsequent CMlMI-phase. In all three experimental systems in which maternal age, follicle type and gonadotrophin dose were used for investigating aneu­ploidy related processes of oocyte maturation, the fre­quency of aneuploidy was found to be correlated, indeed, with an altered rate of meiosis. The question now arises about the genes and their products governing the cell cycle of oocytes during their transition from GV stage through both meiotic divisions.

Summary and perspectives

The Djungarian hamster system appears to be an appropri­ate animal model for studying mechanisms leading to age related and nonrelated aneuploidy caused by (1) a complete suppression of meiotic division, (2) nondisjunction of indi­vidual bivalents, and (3) presegregation. Due to the com­plexity of cell division such studies have to deal with different levels of meiocyte cell cycle regulation and with several cellular targets:

The endocrine and paracrine control of the somatic gonadal compartment required for the generation of competent germ cells.

The intercellular communication between the surround­ing somatic compartment and the germ cell, e.g. the trigger for the resumption of meiosis. The cascade of intercellular communication within the germ cell aiming to coordinate the cell division machin­ery. In simple terms, the machinery consisting of two cycles one for the nucleus and one for the cytoplasm, both linked physiologically and also mechanically, e.g. by the spindle and microtubuli apparatus.

To approach this complex of cell cycle control experimen­tally we proposed at the beginning of our study a rather simplifying hypothesis (Hansmann 1983; Hansmann et al. 1983) which was based on three assumptions:

1. The oocyte receives an altered trigger for the resumption of meiosis, i.e. for the transition from germinal vesicle phase into meiotic divisions.

2. This altered trigger cause a quantitatively andior qualita­tively asynchronous maturation of the nucleus and cyto­plasm, which would provoke failures in the control of spindle function.

3. The resulting success of chromosome segregation, assumed to follow a bivalent specific order determined e.g. by chromosomal properties, depends on the timing of a transient malfunction/inhibition of spindle pole­kinetochor microtubuli (Hummler et al. 1987).

Meanwhile, we have collected experimental evidence for the validity of the latter two assumptions, namely the exist­ence of an altered meiotic rate (the timing with which oocytes pass through meiosis), associated spindle anomalies, and of an asynchronous and bivalent specific order of chromosome segregation (Theuring 1986; Pabst 1987; Hummler et aI. 1987; Hummler and Hansmann 1988; Hansmann et al. 1989; 1990; Willenborg 1991).

The most critical question which is relevant not only for the understanding of meiotic nondisjunction but also of oocyte maturation per se, has to deal with the molecular nature of the signal triggering GVBD and the resumption of meiosis as well as with the machinery transforming the signal into cell cycle activity. The Maturation Promoting Factor (MPF) represents one significant member of this machinery (Masui and Markert 1971). MPF is considered as a cell cycle regulator, ubiquitously present in mitotic and meiotic cells and functioning as an oscillator responsible e.g. for the transition from G2- to M-phase (Hashimoto and Kishimoto 1988; Choi et aI. 1991; for review). MPF con­sists of two subunits, the p34cdc2 protein and a member of the cyclin protein family A, B, or Gl (Dunphy et aI. 1988; Gautier et al. 1990; Draetta et al. 1990). Its activity is posttranslationally regulated by differential phosphoryla­tion/dephosphorylation of subunits. A degradation of cyc­lins appears to be associated with the transition from M­phase into anaphase (Pines 1991). MPF activity, measured by the capacity to phosphorylate H I-kinase (Arion et al. 1988), is oscillating in oocytes during the resumption of meiosis. A high MPF activity is preceding GVDB and persists up to metaphase I. During the segregation phase

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activity drops drastically below a detectable level and is only measurable again when oocytes reached metaphase II (Hashimoto and Kishimoto 1988). One interesting aspect for understanding the role of MPF for cell cycle control in oocytes, hence also its possible involvement in generating aneuploidy, would be the analysis of meiotic rate and the success of chromosome segregation after a manipulative increase or decrease ofMPF activity within oocytes. At least as important is also the question about the different cellular targets of MPF and about its own regulator. First data point to an important role of the c-mos protooncogene in regulat­ing MPF-associated processes of oocyte maturation (Lorca et al. 1991 ; Weber et al. 1991; Watanabe et al. 1991; Kanki and Donoghue 1991). Nowadays, rnicromethods, such as the Polymerase Chain Reaction of cDNA after reverse transcrip­tion of mRNA (reverse PCR), are potent tools to investigate gene expression in few, or even individual mammalian oocytes during maturation to understand the molecular con­trol of cell cycle regulation. Though, being cumbersome and more time consuming than most studies using somatic cells, the molecular analysis of oocyte maturation in animal mod­els, as those described above, will contribute to understand the mechanisms of genome mutations in the forthcoming years.

Acknowledgements. The experimental work of the authors reviewed here was financiated by grants from the Deutsche For­schungsgemeinschaft.

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