Mechanisms of nondisjunction induction in drosophila oocytes.

Quantitative and qualitative studies on the induction of no-disjunction and related phenomena can be carried out using the germ cells of Drosophila. X-Irradiation breaks chromosomes and cold-shock disrupts spindles, these two treatments producing different spectra of nondisjunction in oocytes.


Introduction
Several recent articles elaborate on the usefulness of Drosophila as a test system for the detection of environmental mutagens (1)(2)(3). In short "In contast to most other assay systems, where only one class of genetic damage can be studied, special test strains permit the simultaneous assessment of the total spectrum of genetic changes, ranging from recessive lethal or visible mutations, small deletions, translocations, chromosome loss, and dominant lethals to nondisjunction and genetic recombination" (1).
Nondisjunction is one of the first genetic changes which was shown to be induceable by an external agent, x-irradiation (4). A variety of mechanisms have been proposed to account for this phenomenon. These include chromosome stickiness (5), damage to the centromeric region of the chromosome (6), and damage to the spindle (7). The most extensive genetic analysis of the products of induced nondisjunction has been carried out by Parker and coworkers (8,9). Their data are consistent with the model of "heterologous conjunction via chromatid interchanges as the major and perhaps exclusive cause of radiation-induced non-disjunction" (9).
The data supporting the model were all obtained from studies on the sex chromosomes and the fourth chromosomes. Puro (10), working with the major autosomes, has questioned whether induced chromatid rearrangements direct first division segregation. In Drosophila there are many genes which function during the regular meiotic process in the oocyte. Mutations of these genes are interesting for two reasons; firstly, many of them cause nondisjunction and secondly, some of them enhance sensitivity to radiation and mutagenic chemicals (11). There are several ways in which the meiotic process can be disturbed by such mutations. Often this is associated with a reduction in the amount of recombination. While it has been shown that artificially induced conjunctions probably cause nonhomologous chromosomes to segregate, the converse is not true. Homologous chromosomes which are not conjoined by chiasma usually segregate regularly. To explain this phenomenon, Grell (12) has proposed the "distributive pairing" model. According to this model there are two rounds of pairing during meiosis in the oocyte. Firstly the chromosomes pair to permit recombination and thus the formation of regular conjunctions. Then the chromosomes which are nonconjoined form a "distributive pool". Segregation of the chromosomes of this pool is regulated by a specific set of rules. Novitski (13) has argued that the observations can be accounted for by assuming a nonhomologous association of all the chromosomes prior to synapsis. Recent cytological studies have confi.rmed that there is indeed a chromocentral association of all the chromosomes. An example of the complexities which must yet be explained is the behavior ofthe fourth chromosomes and the compound autosomes (14). According to the genetic analysis (15), these are two pairs of chromosomes which segregate independently from the "distributive pool". Cytological analysis showed that the orientation of the fourth chromosomes is disturbed in females carrying compound autosomes.
Compound autosomes segregate regularly in females but at random in males (16), this being an expression of the different mechanisms regulating pairing and segregation in the two sexes. The products of autosomal nondisjunction are generally lethal because of aneuploid unbalance. Studies on induced autosomal nondisjunction in oocytes became feasible when it was shown that males carrying compound autosomes regularly produce 25% nullosomic and 25% disomic gametes. Females with either regular metacentric autosomes or with compound autosomes can be exposed to an agent which is expected to induce aneuploidy. Viable zygotes will form with the corresponding nullosomic or disomic male gamete. Bateman (17), Savontaus (18), and Gibson (19) have studied induced nondisjunction in the oocytes of females with regular second chromosomes. With this technique only exceptional progeny are recovered and frequencies must be estimated. This work has shown that autosomal nondisjunction can be induced by radiation and that the ratio of induced losses to gains is about 6:1, a ratio similar to that which has often been found for rod-X chromosomes.
Clark and Sobels (20) induced nondisjunction by irradiation of females with compound second chromosomes. Theoretically, with this technique there is an uniform viability of regular and exceptional maternal gametes. Therefore, frequencies of induced nondisjunction can be measured directly.

Results and Discussion
Scoring for nondisjunction of two pairs of chromosomes was made possible by adding marked X and Y chromosomes to the stocks with compound second chromosomes. Nondisjunction was induced by irradiating immature oocytes; one-day old females were given an exposure of 1500 R, mated, and then allowed to lay eggs for 4 days. In another set of females nondisjunction was induced by cold treatment of mature oocytes. A summary of the data obtained in these experiments, together with an acomparable set of data on recording nondisjunction (15), is given in Table 1. The first two columns show nondisjunction of only the sex chromosomes, the second two columns show nondisjunction of the second chromosomes, and the next four columns show simultaneous nondisjunction of both pairs of chromosomes.
In the control series (a) almost a third of all the exceptions are nullo-X diplo-2. Secondary nondisjunction (b) is typified by high frequencies of diplo-X nullo-2 and nullo-X diplo-2 progeny. Radiation-induced nondisjunction (c) is characterized by a high ratio of X-loss to X-gain. In mature oocytes cold treatment induces high frequencies of all exceptional classes (d).
The most important point to be made from these data is that nondisjunction can be induced in different ways and significant variations between the distribution of the exceptional progeny classes provides a direct indication that different mechanisms are involved.
There is now general agreement that chromosome breakage is one of the primary events in the process of radiation-induced nondisjunction. The effects of low temperature on mitotic divisions were first demonstrated by Bauer (21), who induced triploidy by giving cold shocks to young embryos. Gloor (22) aged third instar larvae from two stocks for 10 days at approximately 10°C. Polyploidy was induced in the ganglion cells of one of these stocks. Hildreth and Ulrichs (23) cold-aged females for one or two weeks and obtained a significant increase in the frequency of nondisjunction. Tokunaga (24) was able to show that mature oocytes Environmental Health Perspectives are the most sensitive stage for this phenomenon. Clark (25) was able to induce a small increase in the rate of compound-2 nondisjunction by cold aging. The data in Table 1 were obtained with a stock carrying a C(2R)RM, vg chromosome. The distribution of classes is compatible with the interpretation that nonhomologous chromosomes nondisjoin independently. There is no indication that cold aging breaks chromosomes, and Tokunaga found equality of X chromosome losses and gains. The simplest explanation of these observations is that cold aging affects the spindle mechanism (26). Taking into account the theme of this workshop, chemicals which induce only aneuploidies and no other genetic end points, it can be concluded that a simple genetic test for sex chromosome aneuploidy can be used. The studies with complex genetic systems will then be required for an analysis of the mechanism.