important biological variables that can influence the degree of chemical-induced aneuploidy in...

E L S E V I E R Mutation Research 339 (1995) 155-176 Reviews in Genetic Toxicology

Important biological variables that can influence the degree of chemical-induced aneuploidy in mammalian oocyte and zygotes

J o h n B . M a i l h e s *

Department of Obstetrics and Gynecology, Louisiana State University Medical Center, P. 0. Box 33932, Shreveport, LA 71130, USA

Received 29 April 1995; revised 31 May 1995; accepted 15 June 1995

Abstract

The ability of certain chemicals to increase the frequency of aneuploidy in mammalian oocytes elicits concern about human health and well-being. This concernment exists because aneuploidy is the most prevalent class of human genetic disorders, and very little information exists about the etiology of aneuploidy. Although there are experimental models for studying aneuploidy in female germ cells and zygotes, these models are still being validated because insufficient information exists about the biological variables that can influence the degree of chemical-induced aneuploidy. In this regard, variables such as dose, solvent, use of gonadotrophins, mode and preovulatory time of chemical administration, time of cell harvest relative to the possibility of chemical-induced meiotic delay, criteria for cytogenetic analysis and data reporting, and an introduction to differences between cell types and sexes are presented.

Besides these variables, additional information is needed about the various molecular mechanisms associated with oocyte meiotic maturation and the genesis of aneuploidy. Also, differences between the results from selected chromosome analysis and DNA-hybridization studies are presented. Based upon the various biologic endpoints measured and the differences in cellular physiology and biochemical pathways, agreement among the results from different aneuploidy assays cannot necessarily be expected.

To gain further insight into the etiology of aneuploidy in female germ cells, information is needed about the chemical interactions between endogenous and exogenous compounds and those involved with oocyte meiotic maturation.

Keywords: Aneuploidy; Oocyte; Zygote

I . Introduct ion

A n e u p l o i d y is the most p r eva l en t type o f hu- m a n gene t ic abnormal i ty ; unfo r tuna te ly , we do not know how to r educe its inc idence . Mos t of

* Tel.: 318 675 5382, Fax: 318 675 5442.

the exper imen ta l , aneup lo id l i t e r a tu re compr i ses the effects of causa t ive agents on the induc t ion of aneup lo idy ; however , a d d i t i o n a l i n f o r m a t i o n abou t the var ious mechan i sms and ce l lu lar targets a s soc ia ted with a ne up lo idy p roduc t i on is also ne e de d .

Based on the resul ts f rom ten surveys d e m o n - s t ra t ing tha t 0.31% (204 /64887) of h u m a n new-

0165-1110/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved SSDI 0165-1 110(95)00006-2

156 J.B. Mailhes / Mutation Research 339 (1995) 155-176

borns are aneuploid (Hecht and Hecht, 1987) and that at least 10% of all conceptuses and 40-60% of early spontaneous abortions are aneuploid or polyploid (Bond and Chandley, 1983; Hansmann, 1983; Hook, 1985), information about the etiology of a genetic disorder that is associated with mental and physical retardation, reproductive failure, and certain malignancies is needed. One approach for obtaining information about aneuploidy production is to investigate suspected causative agents and their possible modes of action by using an animal model. We have initially taken this basic approach (Mailhes, 1983; Mailhes and Marchetti, 1994a) and have primarily concen- trated on aneuploidy induction during female meiosis I because approximately 65-85% of human aneuploidies occur during female meiosis I (Hassold and Jacobs, 1984; Hansmann et al., 1990; Antonarakis et al., 1992; Gaulden, 1992; Peterson et al., 1992; Hassold and Sherman, 1993).

Different in vivo and in vitro assays have been employed to study chemical-induced aneuploidy (Parry et al., 1995). A basic premise to any such assay is information about the potential cellular targets and the pharmacokinetics of the chemical being studied. Also, unlike chromosome structural aberration and other mutagenic assays in which DNA damage is evaluated, the various targets for chemical-induced aneuploidy generally involve those organelles and biochemical compounds associated with chromosome segregation and not necessarily DNA per se (Parry and Parry, 1989). Consequently, the results from genotoxic and aneuploid assays may well differ for a partic- ular chemical.

The mammalian oocyte aneuploidy assay is undergoing validation in that variables are still being identified that influence the degree of induced aneuploidy; also, there is a virtual lack of comparative data from different laboratories that employ similar experimental protocols. Identify- ing and describing such variables are deemed essential for discerning the inherent variation associated with aneuploidy production. In essence, the error term in a statistical analysis must be reduced to accurately evaluate treatment effects.

Besides these biological variables, the various physiological and biochemical parameters associ-

ated with oocyte maturation and their influence on chromosome segregation require investigation. Such an approach has already been addressed by Eichenlaub-Ritter and Boll (1989), Hansmann and Pabst (1992), Eichenlaub-Ritter (1993), and Mailhes and Marchetti (1994b). These investigators showed that both intrinsic and extrinsic conditions such as altered endocrine and paracrine control of oocyte maturation, maternal age, and chemical-induced perturbations during the course of oocyte maturation are associated with abnormal chromosome segregation. Although it is important to identify agents that increase the incidence of aneuploidy, it seems equally important to understand the multitude of pathways by which chromosome segregation is controlled. Therefore, the purpose of this review is to discuss several variables that have been shown to alter the level of chemical-induced aneuploidy in oocytes and zygotes and to present areas for future research involving experiments designed to test the general hypothesis that perturbations during the course of oocyte maturation predispose cells to aneuploidy.

In order to appreciate the variables that influence the data obtained from mammalian oocyte aneuploidy assays, a basic understanding of oogenesis is helpful. Unlike males, there is no further postnatal generation of oogonia. Thus, at birth the ovary contains its lifetime supply of oocytes. During fetal development of most mammals, oogonia enter meiotic prophase and progress to diplotene; this stage represents the initial checkpoint of meiosis. During diplotene the chromatin becomes diffuse, and the term dictyate is commonly used to describe this meiotic stage. These primary oocytes, which are housed in primary follicles, remain in dictyate until meiosis resumes in response to gonadotrophins. This transition from dictyate to metaphase II (MII) is commonly termed oocyte meiotic maturation, and it involves nuclear and cytoplasmic maturation of the primary oocyte plus follicular maturation.

Considerable interest has been devoted to the biochemical events and cytologic changes associated with oocyte meiotic maturation. Although these events are summarized in Section 3.2 of this paper, it seems prudent to mention that cyclic

J.B. Mailhes /Mutation Research 339 (1995) 155-176 157

adenosine monophosphate, protein kinases, cy- clins, maturation promoting factor, and other reg- ulatory agents have roles in oocyte meiotic maturation (Schultz et al., 1983a,b; Racowsky, 1993a,b).

At the first meiotic division, the two homolo- gous chromosomes of each tetrad randomly assort to opposite poles during anaphase I. This reductional division normally produces a haploid oocyte and a haploid polar body. Following a brief prophase, the secondary oocyte continues meiotic maturation until it reaches the second checkpoint, namely MII. Upon fertilization, meiosis will continue. During anaphase II the chromatids comprising each chromosome will randomly assort to form an oocyte pronucleus and a second polar body. Subsequently, the haploid male and female pronuclei fuse to form the diploid nucleus of the one-cell zygote. Informa- tion about chromatin and microtubule dynamics during oocyte maturation can be found in Ue- bele-Kallhardt (1978), Tsafriri (1978), Thibault et al. (1987), Messinger and Albertini (1991) and Albertini (1992,1993).

2. Variables that can influence the incidence of induced aneuploidy

2.1. Chemical dose

The shape of aneuploidy dose-response curves varies among different aneugens (Aardema et al., 1992), and their kinetics are not necessarily linear over the entire dose range (Piegorsch et al., 1989). The evaluation of aneuploidy dose-response curves can be strengthened by considering the possibilities that the doses selected for study may not necessarily represent the full spectrum of doses needed for estimating representative func- tions and that aneuploidy may be induced within a narrow dose range in vitro (Rainaldi et al., 1987) and in vivo (Pacchierotti, 1988). For example, when mice were injected with 20, 40, or 60 mg/kg etoposide, the percentages of hyperploidy in MII oocytes were 5.1, 8.5, and 12.0, respectively (Mailhes et al., 1994a). The influence of lower or higher doses on the shape of this dose-

response curve remains unknown. Recently, Parry et al. (1994) presented guidelines for estimating in vitro threshold effects for aneugens that damage the mitotic spindle. Also, Mackay and Elliott (1992) and Fielder et al. (1992) proposed procedures for determining appropriate doses for in vivo genetic toxicology assays based upon a maxi- mum tolerated dose.

Another situation involving oocytes is that the proportion of ovulated metaphase I (MI) oocytes tends to increase with higher doses of spindle poisons. This response results in fewer MII oocytes available for analysis. For chemicals that induce both aneuploidy and ovulated MI oocytes such as colchicine (Mailhes and Yuan, 1987), vinblastine sulfate (Russo and Pacchierotti, 1988; Mailhes and Marchetti, 1993, Mailhes et al., 1993a), griseofulvin (Tiveron et al., 1992; Mailhes et al., 1993b; Marchetti and Mailhes, 1994), and benomyl (Mailhes and Aardema, 1992), the influence of higher doses on aneuploidy production have not been studied due to the relative increase in the proportion of MI oocytes.

Consequently, a discussion of oocyte dose-response curves is limited to the doses chosen by different investigators plus the finding that most of the reported aneugens also induce MI arrest. This latter effect restricts the acquisition of aneuploidy data from relatively high doses. Theoreti- cally, the shape of dose-response curves may also be influenced by the mechanisms (targets af- fected) whereby each compound induces aneuploidy. Unlike the induction of structural chromosome aberrations in which the chromosome is the ultimate target, there exist multiple targets for aneuploidy induction (Liang and Brinkley, 1985). Besides damage to organelles such as microtubules, kinetochores, centrosomes, etc., and alterations to topoisomerase II (Mailhes et al., 1994a), chemical-induced perturbations during oocyte maturation resulting from maternal age (Eichenlaub-Ritter and Boll, 1989) and abnormal hormonal environments (Hansmann and Pabst, 1992) can also predispose oocytes to abnormal chromosome segregation. Furthermore, Pac- chierotti and Mailhes (1991) suggested that the rate of catabolism and the pharmacokinetics of chemicals may influence the shape of aneuploidy


dose-response curves for oocytes in vivo. In essence, the published dose-response curves for chemical-induced aneuploidy in mammalian oocytes display greater variability in shape than those established for the induction of structural chromosomal aberrations following low- and high-LET radiations in somatic cells (Lloyd and Edwards, 1983) and for radiation-induced aneuploidy in mammalian oocytes (Tease, 1988).

2.2. Solvent or suspension

Certainly, the solvent chosen should enable the chemical being studied to form a solution; however, this is not always feasible because the concentration of solvent may be toxic or even aneugenic. For example, a compound may be soluble in either dimethylsulfoxide or ethanol; but, relatively high concentrations of these solvents can be toxic or lethal to mice. Also, ethanol has been reported to elevate aneuploidy levels in mouse zygotes (Kaufman, 1983; Kaufman and Bain, 1984; O'Neill and Kaufman, 1987). At this point, we wish to stress the importance of using concurrent controls that receive the same concentration of solvent and mode of administration as the treated animals.

In some cases, an appropriate solvent cannot be found and the compound is simply administered as a suspension. This is the case with the aneugen griseofulvin, which was mixed in olive oil and administered by oral gavage (Tiveron et al., 1992; Marchetti et al., 1992; Mailhes et al., 1993b; Marchetti and Mailhes, 1994). A possible draw- back to using suspensions occurs when they are administered by intraperitoneal (i.p.) injections. We have found that such a treatment modality can result in congelation of the suspension at the injection site. As a result, the desired dosage is questionable. The point of choosing an appropriate solvent and a mode of administration is to increase the likelihood of a uniform distribution of the test chemical throughout the vascular system and ultimately to the target cells.

Besides solvent toxicity and non-uniform distribution of the study compound, the possibility of competitive inhibition between solute and sol-

vent should be considered. An example of competitive inhibition is that between the adenylate cyclase stimulator forskolin (colforsin) and various solvents. Huang et al. (1982) have shown that among the potential solvents (dimethylsulfoxide, ethanol, acetone, n-butanol, t-butanol, dimethyl formamide, dioxane, methanol, and n-propanol) for forskolin, only dimethylsulfoxide at concentrations of 5% or less was noninhibitory to forskolin activity in vivo.

Our interest in forskolin results from several reports (Schultz et al., 1983a; Racowsky, 1984; Sato and Koide, 1984; Chesnel et al., 1994) demonstrating that forskolin induces a transitory delay during oocyte maturation prior to germinal vesicle breakdown. This delay results from the maintenance of relatively high levels of intraoocyte cyclic adenosine monophosphate (cAMP), which requires adenylate cyclase for its synthesis. We discuss later how such a delay may predispose oocytes to aneuploidy.

At this point, we hasten to mention that information about the pharmacokinetics of the compound being studied can contribute to the experimental protocol and also aid when interpreting results. Unfortunately, relevant pharmacokinetic information about a study compound is not always available. Nevertheless, we present an example in which the pharmacokinetics of tamoxifen may influence the experimental results obtained from different cell types. We are conduct- ing experiments designed to test the hypothesis that tamoxifen induces cytogenetic abnormalities in mouse oocytes and bone marrow cells by reducing the levels of intracellular calcium and of calcium-calmodulin complexes, which are needed for orderly chromosome segregation (Hickie et al., 1983; Geiser et al., 1993). Sargent et al. (1994) reported that tamoxifen induces mitotic spindle abnormalities plus numerical and structural chromosome aberrations in rat hepatocytes in vivo. The possibility exists, however, that tamoxifen is active in hepatocytes, and upon subsequent catabolism to inactive metabolites in the liver, is inactive in other cell types. Thus, the cellular pharmacokinetics of a compound requires consideration when evaluating the results obtained from different cell types and species.

ZB. Mailhes / Mutation Research 339 (1995) 155-176 159

2.3. Gonadotrophin dose

The use of pregnant mare's serum (PMS) and human chorionic gonadotrophin (HCG) for increasing the numbers of growing follicles and ovulated oocytes has been routinely employed in female germ cell studies. Although the Aneu- ploidy Methodology and Test Data Review Study Committee advocated the use of 2-5 IU PMS and 2-5 IU HCG 48 h later for superovulating mice (Mailhes et al., 1986), reservations exist about doses outside this range, the ability of gonadotrophins to increase aneuploidy levels over that found in spontaneously ovulating mice, and the synergistic effects of gonadotrophins with certain suspected aneugens on the incidence of aneuploidy. Therefore, relevant results about these issues are presented.

Although the majority of investigators use gonadotrophin doses between 2 and 5 IU, Hans- mann and Jenderny (1983) showed that 10 IU of PMS and of HCG resulted in elevated hyperploidy in N M R I / H A N X C57/B1 F1 mice. This finding contrasted with the results from the other hybrids and the parental strains. Besides the possibility for certain mouse strains and crosses to respond differently to hormones, certain species may be uniquely sensitive to gonadotrophin-induced aneuploidy. Hansmann et al. (1980) reported that Djungarian hamsters ovulate aneuploid oocytes following increasing doses of gonadotrophins. Also, we found that the frequencies of hyperploidy in ICR mouse oocytes were significantly increased (p < 0.05) in control females given 7.5 IU PMS and 5.0 IU HCG relative to those receiving either 5.0 IU PMS and 2.5 IU HCG or 10.0 IU PMS and 7.5 IU HCG (Mailhes et al., 1994b). Thus, the possibility exists that gonadotrophin dosage may influence aneuploidy levels in some strains and species.

Several studies have provided information about the incidences of aneuploidy in superovulated and in spontaneously ovulating mice and hamsters. Hansmann (1978) and Hansmann and E1-Nahass (1979) found similar frequencies of aneuploidy in MII oocytes from three different strains of mice following either spontaneous or hormone-induced ovulation. Hansmann and

Probeck (1979) also reported similar results in Syrian and Chinese hamsters. When aneuploidy was studied in MII oocytes from spontaneously ovulating and in superovulated young and aged mice from different strains, the frequencies of aneuploidy were similar (Golbus, 1981). Another study involving mouse embryos did not find differences in the levels of aneuploidy, triploidy, or mosaicism between spontaneously ovulating and superovulated females (Luckett and Mukherjee, 1986). Finally, Gras et al. (1992) reported that the incidences of aneuploidy did not differ between hormonally induced and naturally ovulated human oocytes.

Limited information exists about the possible synergistic effects between hormone dose and chemical treatment. Hummler and Hansmann (1988) found a positive correlation between the doses of PMS and HCG and carbendazim-induced aneuploidy in Djungarian hamster oocytes. We recently studied the possible synergistic effects of gonadotrophin dosage with the aneugen vinblastine sulfate (VBS) on the frequencies of MI ooCytes, diploid, and aneuploid MII oocytes from ICR mice (Mailhes et al., 1994b). Following 0.2 mg/kg VBS, a significantly (p < 0.05) higher incidence of MI oocytes was detected in each of the groups given 7.5 IU PMS and 5 IU HCG or 10 IU PMS and 7.5 IU HCG as compared to the females receiving 5 IU PMS and 2.5 IU HCG. These results suggest that the gonadotrophin dose may increase the time needed for oocytes to reach MII. Such an alteration in the rate of oocyte maturation has been hypothesized as a predisposition to abnormal chromosome segregation (Eichenlaub-Ritter and Boll, 1989; Hans- mann and Pabst, 1992; Mailhes et al., 1994b). When we compared the frequencies of diploid oocytes among the three different control and VBS groups, only the difference between the 5 IU PMS and 2.5 IU HCG groups was significant (p < 0.01). Diploid oocytes may represent a less pronounced degree of oocyte maturation delay than that represented by MI oocytes. Neverthe- less, gonadotrophin dosage was found to influence the degree of both MI and diploid oocytes. Finally, the females that received 7.5 IU PMS and 5.0 HCG alone or in combination with VBS

160 Z B. Mailhes / Mutation Research 339 (1995) 155-176

had significantly higher (p < 0.05) levels of hyperploidy than did either the lower or higher hormone groups. Since the proportions of ovulated oocytes were similar among all groups and since gonadotrophin dose seems to influence the rate of oocyte maturation in ICR mice, the rec- ommendation was made to use 5 IU PMS and 2.5 IU HCG for superovulation in ICR mice.

Based upon the available information, relatively low doses of gonadotrophins do not increase aneuploidy levels above those detected following spontaneous ovulation. However, the possibility exists that relatively high doses of gonadotrophins may result in elevated spontaneous and chemical-induced aneuploidy in some strains and species. Additional studies involving different mouse strains are needed. Of utmost importance is the need to enhance our understanding about the biochemical and physiological roles that endogenous and exogenous hormones have on oocyte maturation and their possible relationship to abnormal chromosome segregation.

2.4. Mode of treatment

This variable refers to the method of administering the compound of interest by either oral gavage, subcutaneous or intraperitoneal (i.p.) injection. One example in which i.p. t reatment re- suited in higher incidences of MI oocytes and hyperploidy over oral gavage was reported by Mailhes et al. (1990). They found that a 10-fold increase in the oral dose of colchicine was needed to attain MI:MII ratios and hyperploidy levels equivalent to those induced by the i.p. route. Although both modes of colchicine administration resulted in significant (p < 0.05) increases over controls, the differences point out that the quantitative estimates of aneuploidy can be influenced by the mode of treatment.

2.5. Preouulatory time of treatment

The preovulatory time at which a compound is administered in vivo has been shown to influence the degree of induced aneuploidy for triaziquone (RShrborn and Hansmann, 1971), colchicine (Mailhes and Yuan, 1987), carbendazim (Hum-

mler and H a n s m a n n , 1988), griseofulvin (Marchetti and Mailhes, 1994), and etoposide (Mailhes et al., 1994a). Although the study by Mailhes and Yuan (1987) investigated five different preovulatory treatment times and found that the highest incidence of hyperploidy occurred when colchicine was given at the same time as HCG, they cautioned that this treatment time may not be applicable to other doses and compounds. The premise underlying these differences probably involves the exposure of different proportions of susceptible targets plus the ability of treated oocytes to reach MII at the time of harvest.

When MII oocytes are used for studying chemical-induced aneuploidy in vivo, the compound must be administered prior to MI in order for the potential damage to occur during meiosis I and for detection at the following metaphase, namely MII. For those compounds that are considered meiotic spindle poisons, the chemical is commonly given between 3 h pre- and 3 h post-HCG. This time interval considers that the majority of mouse oocytes reach MI between 5 -8 h post- HCG; anaphase I occurs approximately 9 h after HCG in vivo (Edwards and Gates, 1959; Edwards and Searle, 1963; Tiveron et al., 1992; Marchetti and Mailhes, 1994). Subsequently, ovulation occurs during the 11-14 h period following HCG (Donahue, 1972a; Polanski, 1986; Tiveron et al., 1992; Marchetti and Mailhes, 1994). This time frame provides a reference for determining appropriate treatment and oocyte collection times. Actually, the most favorable treatment time is usually unknown due to lack of information about chemical pharmacokinetics relative to accessibil- ity of aneuploidy targets.

The one-cell zygote or first-cleavage (1C1) cytogenetic assay is more comprehensive than the MII oocyte assay. This is because the 1C1 assay can be used for studying both numerical and structural aberrations, the results from treated males and females, and the degree of germ cell damage transmitted to zygotes. For the majority of studies involving 1C1 zygotes, the study compound is also administered prior to MI, but the possibilities of inducing damage during both meiotic divisions and the inability of oocytes with

J.B. Mailhes /Mutation Research 339 (1995) 155-176 161

damaged centromeres to complete anaphase should be considered when the data are compared to MII oocyte data (Mailhes et al., 1990; Marchetti et al., 1992). Recently, Marchetti and Mailhes (1994, 1995) studied the relative sensitivities of meiosis I and II to varying doses of griseofulvin (GF) by analyzing mouse MII oocytes and 1CI zygotes. The design of this study required that GF be administered prior to the formation of the first or second meiotic spindle for analyzing MII oocytes or zygotes, respectively. This protocol increases the probability that the chemical-target interaction will occur prior to the anaphase in which damage would be induced. However, uncertainties about the actual chemical-target interactions still exist because information pertaining to the pharmacokinetics and tis- sue distribution of a potential aneugen are usually unknown.

2.6. Availability of food during treatment by oral gavage

Whether or not food is available to the experimental animals during the time period from several hours before the initiation of treatment to the time of oocyte harvest has been shown to influence the degree of chemical-induced aneuploidy following oral gavage. Experiments in our laboratory have shown that when mice were given 1500 mg/kg griseofulvin by oral gavage immediately after HCG, the frequencies of ovulated MI oocytes and hyperploid MII oocytes were 29.2% and 4.5%, respectively (Mailhes et al., 1993b). On the other hand, when the food was removed 8-10 h before administering 1500 mg/kg griseofulvin by oral gavage, the frequencies of ovulated MI oocytes and hyperploid MII oocytes were increased to 53.3% and 22.1%, respectively (Marchetti and Mailhes, 1994). Apparently, these differences reflect the ability of food constituents to lower the systemic levels of griseofulvin, thereby reducing the degree of cell progression delay and the target dose to the oocytes.

2. 7. Time of cell harvest

The time interval between administering the chemical treatment and oocyte collection (harvest

time) may range from 12 to 25 h in the mouse (Mailhes and Marchetti, 1994a). We have routinely harvested ovulated oocytes 16-17 h after HCG unless there is chemical-induced meiotic delay as evidenced by ovulated MI oocytes. Fur- thermore, we have found that harvest times of 23 or 25 h post-HCG result in higher proportions of degenerated oocytes, which are not amenable to cytogenetic analysis.

Chemicals that interact with the meiotic apparatus usually result in a dose-dependent increase of ovulated MI oocytes. An association between chemical-induced MI oocytes and aneuploid MII oocytes has been reported for colchicine (Mailhes and Yuan, 1987), vinblastine sulfate (Russo and Pacchierotti, 1988; Mailhes and Marchetti, 1993), griseofulvin (Tiveron et al., 1992; Mailhes et al., 1993b), and benomyl (Mailhes and Aardema, 1992). Such an association may be expected since these spindle toxins disrupt the formation and function of the meiotic spindle. Based upon these findings, the use of elevated frequencies of MI oocytes (Mailhes et al., 1993b) and MI spermatocytes (Adler, 1993) following exposure to spindle toxins has been advocated as an indicator of potential aneuploidy. The available literature has supported this premise for compounds that interact with the meiotic spindle. However, recent results (Mailhes et al., 1994a) have shown that the chemotherapeutic agent etoposide can increase the frequencies of both numerical and structural aberrations in mouse MII oocytes without a concomitant increase in the incidence of ovulated MI oocytes. The distinction between etoposide and spindle toxins probably relates to their modes of action relative to aneuploidy production. Specifically, etoposide inhibits topoisomerase II activity by forming a DNA-topoisomerase II-drug ternary complex (Ross et al., 1984; Liu, 1989) that impairs chromosome segregation during meiosis and increases the probability of structural chromosome damage (Sieber et al., 1978; Murray and Szostak, 1985).

The importance of harvest time upon the frequencies of hyperploid mouse oocytes following chemical-induced meiotic delay was recently demonstrated with griseofulvin (Mailhes et al., 1993b) and with vinblastine sulfate (Mailhes and

162 J.B. Mailhes /Mutation Research 339 (1995) 155-176

Marchetti, 1993). For example, when 2500 mg/kg griseofulvin was given to mice immediately after HCG, the proportions of ovulated MI oocytes were 83.4% and 6.3% when cells were harvested at 17 h and 23 h post-HCG, respectively. The decrease in MI oocytes at the later harvest time was accompanied by a significant (p < 0.01) re- duction in the percentage of hyperploid MII oocytes from 26.7% (17 h) to 2.3% (23h).

2.8. Cell type

Qualitative and quantitative differences exist between male and female germ cells and between somatic and germinal cells in the frequencies of spontaneous and chemical-induced aneuploidy (Adler, 1990,1993; Mailhes, 1987; Mailhes and Marchetti, 1994a). Besides aneuploidy per se, Cliet et al. (1993) showed that the carcinogens dimethylnitrosamine, diethylnitrosamine, 1,1-di- methylhydrazine, and /3-propiolactone induced micronuclei in mouse spermatids, but not in bone marrow cells. Rieder et al. (1993) also stressed the pitfalls of extrapolating conclusions between meiotic and mitotic systems based upon the un- founded assumption of similarities in spindle assembly and function. Therefore, extrapolating results from aneuploid assays to those from micronucleus assays as well as those from different cell types requires caution.

The following examples point out the reported differences in aneuploidy production between oocytes and spermatocytes and between somatic and germ cells. Following in vivo treatment with colchicine, female mice appear to be more sensitive than males to germ cell aneuploidy induction. Miller and Adler (1992) reported that 3 mg/kg colchicine (i.p.) significantly (p < 0.01) increased the frequency of hyperploid MII spermatocytes to 2.5% when cells were collected 14 h after treatment. This response quantitatively differs from that found in females. Mailhes et al. (1990) reported that 0.3 mg/kg colchicine (i.p.) induced 20.8% hyperploid MII oocytes. It is not practical to study aneuploidy induction following 3 mg/kg colchicine (i.p.) and a single harvest time of 17 h post-treatment in females because doses of 2 mg/kg or greater result in only MI ovulated

oocytes (Mailhes and Yuan, 1987). We wish to interject that colchicine-induced aneuploid MII oocytes can be fertilized and transmitted to one- cell zygotes (Albanese, 1988; Mailhes et al., 1990). This finding becomes relevant for estimating genetic risks.

Oocytes are not suitable for detecting the majority of clastogens because an S-phase does not occur between treatment and harvest, and the morphology of oocyte chromosomes (unlike those of 1C1 zygotes) is not conducive for an objective analysis of structural aberrations. In fact, Brewen and Preston (1982) cautioned that analysis of structural aberrations in MII oocytes is highly subjective due to the morphology of oocyte chromosomes and the frequent separation of chromatids of a dyad. Nevertheless, two laboratories have r epo r t ed that t r i az iquone (2,3,5- tris[aziridinyl]-2,5-cyclohexadiene- 1,4-dione) increased the frequency of structural aberrations in mouse MII oocytes (R6hrborn and Hansmann, 1971; Hansmann et al., 1974; Mailhes, 1983). Methotrexate and cyclophosphamide were also found to induce structural aberrations in oocytes (R6hrborn and Hansmann, 1971; Hansmann, 1974). On the other hand, Yuan and Mailhes (1987) failed to confirm that cyclophosphamide increased the frequencies of structural and numerical aberrations in mouse MII oocytes and provided several possible reasons for the conflict- ing results.

Besides these reports involving chromatid acentric fragments and exchanges in oocytes following in vivo chemical treatment, Mailhes et al. (1994a) reported that the chemotherapeutic etoposide (VP-16) preferentially induced centromeric structural damage and, to a lesser ex- tent, chromatid acentric fragments in MII oocytes. Additionally, this topoisomerase II inhibitor increased the frequency of hyperploidy in a manner proposed not to involve the organelles of the spindle apparatus. A subsequent study by Mail- hes et al. (1995) provided information about the relative sensitivities of mouse MII oocytes and 1C1 zygotes to 40 mg/kg etoposide. We found that the incidence of structural aberrations was higher (p < 0.05), and the frequency of aneuploidy was lower in oocytes than in zygotes. The

J.B. Mailhes / Mutation Research 339 (1995) 155-176 163

observed difference in structural aberrations may reflect the possibility that some of the damaged oocytes were not able to successfully complete the various steps required for reaching the one- cell zygote stage of development. For example, structural damage to the centromere may reduce the probabi l i ty of these cells undergoing anaphase. Conversely, the higher level of aneuploidy noted in zygotes than in oocytes may represent the increased probability of malsegrega- tion during both meiotic divisions in zygotes and of only one division in the MII oocyte. This latter possibility recognizes that etoposide was administered before the first meiotic division.

As mentioned earlier, the 1CI assay, as op- posed to the MII assay, can be used for studying the induction of both aneuploidy and structural aberrations following t reatment of either sex or both. An important consideration for both the MII oocyte and the 1C1 assay is the time of chemical treatment. When t reatment occurs prior

to formation of the first meiotic spindle, analysis of oocytes detects aneuploidy induced during meiosis I, whereas analysis of 1C1 zygotes may represent aneuploidy induced during both meiosis I and II if a sufficient dose of the compound persists until the second meiotic division. This consideration plus that involving the possible loss of aneuploid cells prior to the first cleavage has been discussed by Mailhes et al. (1990).

Although aneuploidy can be induced in 1C1 zygotes (Fig. 1) by various compounds such as colchicine (Mailhes et al., 1990), griseofulvin (Marchetti et al., 1992), and etoposide (Mailhes et al., 1995) when administered prior to formation of the first meiotic spindle, we sought to obtain information about the relative sensitivities of mouse meiosis I and II to chemical-induced aneuploidy. In this regard, the reader is referred to the recent studies by Marchett i and Mailhes (1994,1995).

In essence, the 1C1 assay is more versatile than

Fig. 1. Hyperploid first cleavage mouse zygote; n = 21 (female) and n = 20 (male); arrow indicates Y chromosome.


the MII oocyte assay; however, it requires more animals and is technically more difficult to per- form.

2.9. Sex differences

The response of male and female gametes to aneugens and mutagens differs (Mailhes and Marchetti, 1994a; Allen et al., 1995). Such variation may arise from inherent biologic differences in the ontogeny, anatomy, and maturation of male and female germ cells plus the variables discussed in this paper. As mentioned by Eichenlaub-Ritter (1994), anatomic differences exist between the spindles of oocytes and spermatocytes. Unlike spermatocytes, oocytes apparently lose their cen- trioles during embryogenesis and instead have microtubule organizing centers (Eichenlaub- Ritter et al., 1988; Eichenlaub-Ritter and Wink- ing, 1990; Messinger and Albertini, 1991).

The reasons for these reported differences between sexes for chemical-induced aneuploidy in germ ceils have not been resolved. In this context, Tease (1992) has summarized the results from studies involving radiation- and chemical-induced chromosome aberrations in male and female germ cells of mice. He emphasized the problems of using these results for assessing the relative sensitivities between sexes due to the limitations in the quality and quantity of data and the uncertainties associated with comparing results obtained from different assays.

Based upon the available literature, more information about chemical-induced aneuploidy exists for male than for female germ cells. Conse- quently, a larger number of compounds have been reported as aneugenic in males than in females. However, when comparable doses of a compound have been studied in both sexes, the female appears more sensitive to certain chemicals, whereas males seem more sensitive to other compounds. In evaluating the following examples, it is important to consider the differences in experimental protocols, criteria for analysis, and method of data reporting among the various studies.

Both colchicine and vinblastine sulfate (VBS) (one exception) have been shown to elevate aneuploidy levels in spermatocytes and in oocytes with

higher levels found in oocytes. Miller and Adler (1992) reported that 3 m g / k g colchicine (i.p.) significantly increased hyperploidy in MII mouse spermatocytes to 2.5% when cells were collected 14 h after treatment. On the other hand, 0.3 m g /k g colchicine (i.p.) induced 20.8% hyperploidy in mouse MII oocytes (Mailhes et al., 1990). The use of colchicine doses higher than 0.3 m g /k g are impractical in females because over 50% of the ovulated oocytes are blocked in MI. In fact, doses of 2.0 m g /k g colchicine and higher result in 100% ovulated MI oocytes (Mailhes and Yuan, 1987).

Miller and Adler (1992) also showed that 2 m g /k g VBS significantly (p < 0.01) increased hyperploidy in mouse MII spermatocytes to 1.5% when cells were harvested 14 h post-VBS. When comparing these results to those found in females, oocytes again appear more sensitive even at lower doses. For example, Russo and Pac- chierotti (1988) found 17.2% hyperploid MII oocytes following 0.23 m g /k g VBS, and Mailhes et al. (1993a) reported 23.2% and 23.9% hyperploid MII oocytes after 0.2 and 0.25 m g /k g VBS, respectively.

As noted earlier, an exception to the aneu- genicity of VBS in spermatocytes stems from a study by Liang et al. (1986). Although these investigators concluded that VBS did not increase hyperploidy in mouse MII spermatocytes, these findings should be evaluated relative to the lengthy interval between treatment and cell harvest. Possibly, the exposed cell population had already completed metaphase II prior to harvest; if so, the spermatocytes actually harvested may have been exposed to a lower effective dose due to chemical catabolism.

When summarizing the results between mouse spermatocytes and oocytes for the two well-known aneugens colchicine and VBS at comparable doses and modes of administration (i.p.), females are more sensitive. However, this finding does not necessarily apply for a selected list of other chemicals. In the following examples, the frequencies of hyperploidy in spermatocytes rarely exceeded 2.5%; this value is considerably lower than those reported for oocytes (Mailhes and Marchetti, 1994a). Nevertheless, the following five examples

J.B. Mailhes /Mutation Research 339 (1995) 155-I 76 165

are intended to demonstrate that differences in aneuploidy induction exist between males and female germ cells. As mentioned earlier, the reasons for these observed differences remain to be elucidated.

Chloral hydrate (CH) is used as an anesthetic. It has been shown to destroy spindle fibers in Pleurodeles waltfii eggs (Sentein and Ates, 1974) and to inhibit spindle formation in Aspergillus nidufans (Mercer and Morris, 1975). When CH was studied in mammalian germ cells, it was found to be aneugenic in spermatocytes (Russo et al., 1984; Miller and Adler, 1992) but not in oocytes (Mailhes et al., 1988, 1993a). Another example is econazole. This fungicide increased the frequency of hyperploidy in mouse spermatocytes (Miller and Adler, 1992), whereas unpub- lished results from our laboratory could not confirm this finding in oocytes even with comparable doses and various treatment times. Similar to these two examples, 6.0 mg/kg cadmium chloride (CD) induced 0.8% hyperploidy in mouse spermatocytes sampled 22 h after CD (Miller and Adler, 1992), but not in oocytes exposed to this same dose (Mailhes et al., 1988). Although Watanabe et al. (1977,, 1979) reported 1.8% (3/164) hyperploid MI1 oocytes following 6.0 mg/kg CD, the dissimilar results from the oocyte studies may have been influenced by the different times of chemical treatment and other experimental variables including scoring criteria. We have recently attempted to restudy CD-induced aneuploidy in oocytes, but found that doses of 8 mg/kg and higher resulted in animal toxicity and death. The fourth example of a compound that has been reported to induce aneuploidy in males but not females is diethylstilbestrol (DES). Al- though DES has been reported to inhibit microtubule assembly and increase the rate of disas- sembly in vitro (Sato et al., 1984; Hartley-Asp et al., 1985; Tucker and Barrett, 1986; Sakakibara et al., 1991), this may not be the sole means of disturbing chromosome segregation especially in females. Estradiol is believed to delay the initiation of oocyte meiotic maturation (Racowsky, 1991,1993a,b); such a delay may predispose oocytes to abnormal chromosome segregation. Also, estrogenic compounds may predispose some

oocytes to missegregation by disturbing the metabolic pathways necessary for normal development of synaptonemal complexes (Masumbuko et al., 1992). Regarding DES-induced aneuploidy in germ cells, Zijno et al. (1986) found that DES increased the frequency of aneuploidy in mouse spermatocytes. A similar response was not found in mouse MI1 oocytes (Mailhes et al., 1988,1993a) or in one-cell zygotes (Becker and Schoneich, 1982). Finally, experiments were conducted in the same laboratory with p-fluorophenylalanine (pFPA) and mouse germ cells (Brook and Chand- ley, 1985,1986). These investigators found that 100 mg/kg pFPA significantly increased aneuploidy levels in MI1 spermatocytes but not in MI1 oocytes. The above five examples suggest that spermatocytes are more prone to chemical-induced aneuploidy than are oocytes. However, these results should be evaluated in light of the reported increased sensitivity of oocytes to aneuploidy following exposure to colchicine and VBS as discussed earlier.

To summarize this brief discussion about differential sensitivities between the sexes for chemical-induced aneuploidy, an overall distinction may not be attainable. For instance, besides inherent biologic variation and that due to experimental protocols, the differences in induced aneuploidy between spermatocytes and oocytes may be chemical-dependent. There exists no biological reason to expect one sex to respond in the same manner across various chemicals when the differences in germ cell anatomy, development and maturation are coupled with the possibilities of sexual differences in chemical pharmacokinet- its and distribution of compounds to the germ cells of both sexes. Also, the diverse molecular mechanisms whereby chemicals ultimately induce aneuploidy, are expected to differ between the sexes, especially when the hypothesis of chemical-induced perturbations during oocyte maturation is added to the previously mentioned possibilities.

2.10. Criteria for cytogenetic analysis and data reporting

All cytogenetic data are not necessarily based upon the same set of criteria for analysis. Subjec-


tivity is introduced into the data because of different scientific backgrounds of the investigators and the quality of cytogenetic preparations. Re- garding the latter, C-banding of chromosomes for aneuploidy analyses seems essential for an objective count of dyad numbers. As pointed out by Mailhes (1987), the distinction between whole chromosomes (dyads) and those separated at the centromeres to form two chromatids is difficult to differentiate in chromosomes that are not C- banded. Without banding or some other means for identifying centromeres, the number of chromosomes cannot be objectively quantified.

When interlaboratory comparisons are attempted between the aneuploidy results from the same cell type, attention should be given to the criteria for analyzing cells and the method of data reporting because these variables can affect the quantitative data. For instance, our recent findings (Mailhes et al., 1993a) qualitatively agree with those of Russo and Pacchierotti (1988) in that VBS increased the frequencies of hyperploid

oocytes, ovulated MI oocytes, and diploid MII oocytes in mice. However, quantitative differences in the data were noted at comparable doses administered in the same manner. The fact that Mailhes et al. (1993a) found higher frequencies of hyperploidy may partially reflect differences in calculating this parameter. We calculated hyperploidy as the number of MII oocytes containing 201/2-291/2 chromosomes divided by the sum of euploid, hypoploid, and hyperploid MII oocytes (excluding diploidy). For example, an oocyte clas- sified as 211/2 contains 21 dyads (chromosomes) plus one unpartnered chromatid (Fig. 2). Con- versely, Russo and Pacchierotti (1988) computed the hyperploid frequency as the number of MII oocytes containing 21 < n < 25 chromosomes divided by the number of euploid plus hyperploid oocytes. However, this difference in calculating hyperploidy still did not account for the observed dispersion of values because when we calculated hyperploidy in the same manner as described by Russo and Pacchierotti (1988), our values were

,-"GI

Fig. 2. Hype rp lo id m e t a p h a s e II m o u s e oocyte; n = 21 i /2 , a r row ind ica tes one u n p a i r e d chromat id .


still higher. They reported 17.2% hyperploidy following 0.23 mg/kg VBS given i.p. 17-18 h prior to harvest. When we calculated the percentage hyperploidy in the same manner (excluding cells containing single chromatids) as these investigators from mice treated 17 h prior to harvest, we obtained 28.4% and 29.6% hyperploidy following i.p. doses of 0.2 and 0.25 mg/kg VBS, respectively. These quantitative differences between the data from the two laboratories could then reflect variation in the sample sizes, scoring criteria, C-banding of chromosomes, harvest times, oocyte processing techniques, and strain of mice. This comparison illustrates the importance of clearly defining the criteria for classifying aneuploid cells and for calculating the frequencies of aneuploidy.

3. Areas for future research

3.1. Cytogenetic analysis and micronuclei

Although techniques for labeling DNA se- quences of specific chromosomes and of pericentric regions with DNA hybridization probes and for identifying kinetochores in micronuclei with antibodies have been employed for various somatic cells (Degrassi and Tanzarella, 1988; Hen- nig et al., 1988; Pinkel et al., 1988; Thompson and Perry, 1988; Eastmond and Tucker, 1989; East- mond and Pinkel, 1990; Brandriff et al., 1991; Weier et al., 1991; Ried et al., 1992; Lynch and Parry, 1993; Miller and Niisse, 1993; Eastmond et al., 1994), the use of such techniques for oocytes have not been reported to our knowledge. How- ever, mouse MII oocyte chromosomes can be labeled with specific molecular probes (J.W. Allen, pers. commun.).

At least three major differences exist among aneuploidy assays employing conventional cytogenetic analysis (chromosome counting), micronucleus analysis (MN), and labeling kinetochores or chromosomal regions with specific probes. First, micronuclei represent chromosome loss events resulting from anaphase lagging, whereas cytogenetic (chromosome analysis) aneuploidy analyses quantitate both hypoploidy (including an unknown contribution from technical artifact) and hyperploidy resulting from nondisjunctional

events. Thus, hyperploidy may not be representative of anaphase lagging. Also, if a micronucleus is formed, it may be randomly incorporated into one of the daughter cells so that one cell becomes trisomic and the other monosomic, whereas the micronuclei will be undetectable if it is incorporated into the cell of origin. In this same light, quantitating the number of chromosomes without distinguishing among the different chromosomes in a cell or counting labeled kinetochores will not detect events in which an equal number of chromosomes is lost and gained. Second, studies involving in situ hybridization (ISH) of DNA with sequence-specific probes followed by quantifica- tion of labeled regions represent aneuploidy for the specific chromosome(s) and not for the entire genome. Such an approach to aneuploidy as- sumes that each chromosome has an equal probability of missegregation. Finally, since micronuclei analyses 'per se' detect whole or partial chromosomes, they may or may not pertain to hypoploidy unless the kinetochores are identified.

Since micronucleus assays and cytogenetic analyses measure different end points, the re- spective data cannot necessarily be expected to yield concordant results. The following examples demonstrate the different conclusions derived from these assays. Adler (1990) reported 'negative' aneuploidy results from the mouse bone marrow micronucleus assay and 'positive' aneuploidy results from the mouse spermatocyte cytogenetic assay for the following chemicals: cadmium chloride, chloral hydrate, diazepam, and econazole. Comparable data from oocytes have not been found. Besides suggesting possible differences in chemical-induced aneuploidy between germ and somatic cells, these results demonstrate that the conclusions derived from micronucleus assays do not necessarily reflect those obtained from cytogenetic aneuploid assays. These diver- gent results are important because the use of antikinetochore antibodies for identifying centromeres in micronuclei from mammalian cells in vitro (Degrassi and Tanzarella, 1988; Hennig et al., 1988; Thompson and Perry, 1988) and in vivo (Gudi et al., 1990,1992; Miller et al., 1991; Witt et al., 1992) have been advocated as a prefatory aneuploid assay.


Efforts to validate aneuploidy assays are essential to provide information for understanding the biological variables that influence the resultant data and to decide upon which assay(s) most accurately reflects the response in human germ and somatic cells. To this end, we wish to point out several variables that can affect the data obtained from micronucleus assays. Miller et al. (1991) reported that the number of kinetochore- labeled micronuclei can be reduced by certain chemical treatments, which result in loss of centromeric antigens. Also, Heddle et al. (1991) found that chromosome bridges, phagocytosis, and apoptosis can reduce the number of micronuclei. Conversely, false positive estimates of hypoploidy can result from the overlap or fusion of DNA hybridization regions and inefficient DNA probe penetration (Eastmond and Pinkel, 1990). The purpose of this brief presentation about selected and different aneuploidy assays is solely to provide a background for understanding that similar results cannot necessarily be expected among the various assays and cell types. In fact, Raimandi et al. (1989) demonstrated that the incidence of hyperploidy in lymphocytes was greater during interphase than in metaphase.

Nevertheless, the future appears promising for aneuploidy assays that utilize ISH and kinetochore-labeling techniques if highly positive corre- lations can be demonstrated with conventional cytogenetic analyses. Overlooking the need for ascertaining the biological relationships between different assays, larger sample sizes, flow cyto- metric methodology, and the simultaneous use of both centromeric and telomeric DNA probes (Miller and Nfisse, 1993), and tandem-labeling with two adjacent DNA probes for distinguishing between chromosome breakage and aneuploidy (Eastmond et al., 1994) are advancing aneuploidy analyses. For additional information about procedures for detecting chemical-induced aneuploidy, the reader is referred to recent reports by Parry and Natarajan (1993) and Parry et al. (1995)

3.2. Physiology and biochemistry of oocyte maturation

Basic to designing experiments for pursuing the hypothesis that disruptions or perturbations

during meiotic oocyte maturation predispose oocytes to chromosome missegregation is an understanding of the current knowledge about the physiology and biochemistry of oocyte maturation. Several relevant references (Schultz et al., 1983a,b; Eppig, 1991; Racowsky, 1991,1993a; Chesnel et al., 1994; Eppig et al., 1994) offer important information, which is not necessarily consistent among species, about the status of this dynamic field of study.

Oocyte meiotic maturation is basically the transition from the dictyate or germinal vesicle stage of meiosis to MII. Donahue (1972b) has described the following events that cytologically comprise oocyte meiotic maturation: chromatin condensation during diakinesis, metaphase I, segregation of homologues and formation of the first polar body at anaphase I, and the transformation to the mature oocyte at MII.

The following overview, although certainly in- complete and restricted to mice and rats, is presented with the intention of pointing out the multitude of possibilities whereby extrinsic and intrinsic perturbations during oocyte maturation may result in an altered (faster or slower) rate of development. Such alterations are hypothesized to disrupt the orderly sequence of interrelated processes essential for normal nuclear and cytoplasmic maturation and predispose oocytes to aneuploidy.

The preovulatory mammalian oocyte is sur- rounded by a dynamic follicular environment containing compounds with independent or interac- tive roles in regulating oocyte meiotic maturation. These interactions between follicular and germinal ceils are mainly regulated by the gonadotrophins FSH and LH. Throughout follicular development, the granulosa cells communicate with the oocyte by means of gap junctions (Anderson and Albertini, 1976; Gilula et al., 1978). Through these channels pass cyclic adenosine monophosphate (cAMP), hormones, and other signals that control oocyte meiotic maturation (Lawrence et al., 1978). Although the role of steroids in modulating oocyte meiotic maturation is under investigation, the possibility exists that steroids regulate the distribution and integrity of gap junctions (Garfield et al., 1988). Progesterone


diminishes gap junction integrity and function (Garfield et al., 1987), whereas estradiol enhances gap junctions (Burghardt et al., 1987).

The arrest of the primary oocyte in dictyate is thought to result from the transmission of cAMP (Eppig, 1989), other purines (Downs et al., 1985; Downs and Eppig, 1987), and estradiol (MacKen- zie and Garfield, 1985; Burghardt et al., 1987) via gap junctions from the granulosa cells to the oocyte.

Since LH receptors have not been found in oocytes (Eppig, 1991), control of oocyte maturation in vivo is thought to arise from the granulosa cells. Overall, three hypotheses have been ad- vanced to account for LH-induced maturation. The first suggests that gonadotrophins induce a maturation-stimulator (growth factors, steroids, prostaglandins) that enhances the activity of a maturation promotion factor (MPF) (Schuetz, 1967). Another suggests that gonadotr0phins reduce the titer of an oocyte maturation inhibitor that reversibly inhibits the maturation of cumulus-enclosed oocytes (Hillensjo et al., 1979). The third proposes that LH physically disrupts the functional integrity of follicular gap junctions; this interrupts the transfer of cAMP from granulosa cells to the oocyte and results in intraoocyte cAMP levels below that required for maintaining meiotic arrest (Dekel and Beers, 1978,1980). Of these hypotheses, the latter has received considerable interest, especially as applied to results from mice and rats. However, the role of cAMP may not be universal in all mammalian species (Racowsky, 1993b). For example, in hamsters (Racowsky and Satterlie, 1985; Racowsky, 1986), pigs (Racowsky and McGaughey, 1982), and sheep (Moore and Heslop, 1981), a decrease in cAMP levels alone does not result in oocyte maturation. Instead, a component in addition to cAMP regu- lates maturation. Such a component may function as an arrester, which decreases in concentration following the LH surge, or a stimulator, which may induce oocyte maturation even in the presence of high cAMP levels.

The available data are consistent with a role for cAMP in maintaining meiotic arrest. How- ever, for the resumption of meiosis, a decrease in the level of intraoocyte cAMP occurs in rats and

mice, but not in several other species studied. Therefore, compounds in addition to cAMP are also involved with regulating meiotic maturation.

Various compounds such as cAMP analogues, cAMP phosphodiesterase inhibitors, hormones, protein synthesis inhibitors, phorbol esters, purines, prostaglandins, growth factors, calcium, calmodulin, forskolin, etc. can alter the normal progression of oocyte meiotic maturation (for review, see Racowsky, 1991,1993a,b). However, a paucity of data are available relating temporal alterations during oocyte maturation with a predisposition to abnormal chromosome segregation. One relevant reference is that by Eichenlaub- Ritter and Boll (1989); they found that oocytes from aged mice progress faster through the first maturation division in vitro and are more prone to aneuploidy than those from younger females. Regarding exogenous chemicals, two reports suggest that caffeine (Prather and Racowsky, 1992) and nicotine (Racowsky et al., 1989) can perturb oocyte maturation and result in abnormal chromosome segregation during meiosis.

Caffeine and its metabolites theophylline and theobromine have been shown to inhibit the activity of cAMP phosphodiesterase (Butcher and Sutherland, 1962). When Prather and Racowsky (1992) studied the effects of different doses of caffeine on hamster oocytes in vitro, they found a dose-dependent decrease in the rate of meiotic progression and an increase in the proportions of diploid and aneuploid MII oocytes. They concluded that caffeine-induced perturbations during oocyte maturation may be responsible for part of the relationship between caffeine intake and reduced fertility in women (Wilcox et al., 1988). Extensive studies have generally shown that caffeine is clastogenic in vitro, but not in vivo (D'Ambrosio, 1994, Nehlig and Debry, 1994, and Mohr et al., 1993 for reviews). Thus, the caffeine-induced effects on oocyte maturation and aneuploidy detected in vitro by Prather and Racowsky (1992) need to be investigated in vivo.

Another common compound to which humans are exposed is nicotine. When hamster oocytes were exposed in vitro to different doses of nicotine, prolongation and complete blockage of the first meiotic division and increased frequencies of


diploid MII oocytes occurred (Racowsky et al., 1989). In light of this report , addit ional studies are needed to investigate the mechanism(s) whereby nicotine causes per turbat ions during female meiosis and the possibility that nicotine induces aneuploidy in vivo. The purpose of pre- senting these two examples involving caffeine and nicotine is twofold: first, both compounds induce per turbat ions during oocyte matura t ion that may be associated with aneuploidy, and second, the relat ionship between caffeine, c A M P phosphodiesterase, and abnormal ch romosome segregat ion alludes to a potential mechanism for aneuploidy induct ion following exposure to a c o m p o u n d that is in widespread usage by humans.

The hypothesis that the etiology of female germ cell aneuploidy rests on the critical timing of different events in oocyte deve lopmen t (Eichenlaub-Ri t te r et al., 1988; Eichenlaub-Ri t te r and Sobek-Klocke, 1993) deserves at tention. Such per turbat ions during oocyte matura t ion can result f rom chemical - induced damage to organelles responsible for cell division, hormonal imbalance, physiologic ageing of the oocyte-fol l ic le complex, interactions between endogenous and exogenous chemicals with those involved in oocyte maturation, and o ther yet unidentif ied mechanisms. In fact, as men t ioned by Eichenlaub-Ri t te r (1994), any condit ion that alters a physiological parameter in the gamete , such as gene expression, protein synthesis and phosphoryla t ion states, or calcium homeostasis , has the potent ial for inducing aneuploidy.

4. Conclusion

The study of aneuploidy is more comprehensive than simply treat ing cells in vitro or adminis- ter ing chemicals to animals and then count ing chromosomes or micronuclei. Instead, information about chemical pharmacokinet ics and distri- but ion among tissues, chemical dose to target cells, cell-cycle kinetics, possible cellular organelles that may be directly or indirectly al tered by t reatment , t r ea tment - induced disequilibrium in biochemical pathways, gonocyte development and maturat ion, and chemical- induced cell-cycle

delay should be ultimately incorpora ted into exper imental protocols. Thus, the biology of the cell system used dictates the experimental protocol. Considering the above information and the physiological and anatomical differences among cell types, extrapolat ing aneuploidy results across different cell types and aneuploid assays requires caution.

Acknowledgements

The constructive comments provided by Dr. Francesco Marchet t i are sincerely appreciated.

References

Aardema, M.J., J.B. Mailhes and R.J. Preston (1992) Status of the mouse metaphase II oocyte assay for detecting chemicals which induce aneuploidy in germ cells, Environ. Mol. Mutagen., 19, Supp. 20, 1.

Adler, I.-D. (1990) Aneuploidy studies in mammals, in: M.L. Mendelson and R.J. Albertini (Eds.), Mutation and the Environment, Part B: Metabolism, Testing Methods and Chromosomes, Wiley-Liss, New York, pp. 285-293.

Adler, I.-D. (1993) Synopsis of the in vivo results obtained with the 10 known or suspected aneugens tested in the CEC collaborative study, Mutation Res., 287, 131-137.

Albanese, R. (1988) Chemically induced aneuploidy in female germ cells, Mutagenesis, 3, 249-255.

Albertini, D.F. (1992) Cytoplasmic microtubular dynamics and chromatin organization during mammalian oogenesis and oocyte maturation, Mutation Res., 296, 57-68.

Albertini, D.F. (1993) The meiotic-to-mitotic cell cycle transition in mammalian eggs, in: F.P. Haseltine and S. Heyner (Eds.), Meiosis II: Contemporary Approaches to the Study of Meiosis, Am. Assoc. Adv. Science Press, Washington D.C., pp. 117-128.

Allen, J.W., U.H. Ehling, M.M. Moore and S.E. Lewis (1995) Germline specific factors in chemical mutagenesis, Muta- tion Res., in press.

Anderson, E. and D.F. Albertini (1976) Gap junctions between the oocyte and companion follicle cells in the mammalian ovary, J. Cell Biol., 71, 680-686.

Antonarakis, S.E., M.B. Peterson, M.G. Mclnnis, P.A. Adels- berger, A.A. Schinzel, F. Binkert, C. Pangalos, O. Raoul, S.A. Salughterhaupt, M. Hafets, M. Cohen, D. Roulsen, S. Schwartz, M. Mikkelsen, L. Tranebjaerg, F. Greenberg, D.I. Hoar, N.L. Rudd, A.C. Warren, C. Metaxotou, C. Bartsocas and A. Chakravarti (1992) The meiotic stage of nondisjunction in trisomy 21: determination by using DNA polymorphisms, Am. J. Hum. Genet., 50, 544-550.

Becker, K. and J. Sch6neich (1982) Expression of genetic


damage induced by alkylating agents in germ cells of female mice, Mutation Res., 92, 447-464.

Bond, D.J. and A.C. Chandley (1983) Aneuploidy, Oxford Monographs on Medical Genetics, No. 11, Oxford Univer- sity Press, Oxford, 198 pp.

Brandriff, B.F., L.A. Gordon and B.J. Trask (1991) DNA sequence mapping by fluorescence in situ hybridisation, Environ. Mol. Mutagen., 18, 259-262.

Brewen, J.G. and R.J. Preston (1982) Cytogenetic analysis of mammalian oocytes in mutagenicity studies, in: T.C. Hsu (Ed.), Cytogenetic Assays of Environmental Mutagens, Allanheld-Osmun, Totowa, N J, pp. 277-287.

Brook, J.D. and A.C. Chandley (1985) Testing of 3 chemical compounds for aneuploidy induction in the female mouse, Mutation Res., 157, 215-220.

Brook, J.D. and A.C. Chandley (1986) Testing for the chemical induction of aneuploidy in the male mouse, Mutation Res., 164, 117-125.

Burghardt, R.C., D. Gaddy-Kurten, R.L. Burghardt, R.C. Kurten and P.A. Mitchell (1987) Gap junction modulation in rat uterus, III. Structure-activity relationships of estro- gen receptor-binding ligands on myometrial and serosal cells, Biol. Reprod., 36, 741-751.

Butcher, R.W. and E.W. Sutherland (1962) Adenosine 3',5'- phosphate in biological materials, 1. Purification and prop- erties of cyclic 3',5'-nucleotide phosphodiesterase and use of this enzyme to characterize adenosine 3',5'-phosphate in human urine, J. Biol. Chem., 237, 1244-1250.

Chesnel, F., K. Wigglesworth and J.J. Eppig (1994) Acquisi- tion of meiotic competence by denuded mouse oocytes: participation of somatic-cell product(s) and cAMP, Dev. Biol., 161, 285-295.

Cliet, I., C. Melcion and A. Cordier (1993) Lack of predictiv- ity of bone marrow micronucleus test versus testis micronucleus test: comparison with four carcinogens, Muta- tion Res., 292, 105-111.

D'Ambrosio, S.M. (1994) Evaluation of the genotoxicity data on caffeine, Regulat. Toxicol. Pharm., 19: 243-281.

Degrassi, F. and C. Tanzarella (1988) Immunofluorescent staining of kinetochores in micronuclei: a new assay for the detection of aneuploidy, Mutation Res., 203, 339-345.

Dekel, N. and W.H. Beers (1978) Rat oocyte maturation in vitro: relief of cyclic AMP inhibition by gonadotrophins, Proc. Natl. Acad. Sci. USA, 75, 4369-4373.

Dekel, N. and W.H. Beers (1980) Development of the rat oocyte in vitro: inhibition of maturation in the presence or absence of the cumulus oophorus, Dev. Biol., 75, 247-254.

Donahue, R.P. (1972a) Fertilization of the mouse oocyte: sequence and timing of nuclear progression to the two-cell stage, J. Exp. Zool., 180, 305-318.

Donahue, R.P. (1972b) The relationship of oocyte maturation to ovulation in mammals, in: J.D. Biggers and A.W. Schuetz (Eds.), Oogenesis, University Park Press, Balti- more, MD, pp. 413-438.

Downs, S.M. and J.J. Eppig (1987) Induction of mouse oocyte maturation in vivo by perturbations of purine metabolism, Biol. Reprod., 36, 431-437.

Downs, S.M., D.L. Coleman, P.F. Ward-Bailey and J.J. Eppig (1985) Hypoxanthine is the principal inhibitor of murine oocyte maturation in a low molecular weight fraction of porcine follicular fluid, Proc. Natl. Acad. Sci. USA, 82, 454-458.

Eastmond, D.A. and D. Pinkel (1990) Detection of aneuploidy-inducing agents in human lymphocytes using fluorescence in situ hybridization with chromosome-specific DNA probes, Mutation Res., 234, 303-318.

Eastmond, D.A. and J.D. Tucker (1989) Identification of aneuploidy-inducing agents using cytokinesis-blocked human lymphocytes and an antikinetochore antibody, Envi- ron. Mol. Mutagen., 13, 34-43.

Eastmond, D.A., D.S. Rupa and L.S. Hasegawa (1994) Detec- tion of hyperploidy and chromosome breakage in interphase human lymphocytes following exposure to the ben- zene metabolite hydroquinone using multicolor fluorescence in situ hybridization with DNA probes, Mutation Res., 322, 9-20.

Edwards, R.G. and A.H. Gates (1959) Timing of the stages of the maturation divisions, ovulation, fertilization and the first cleavage of eggs of adult mice treated with gonadotrophins, J. Endocrin., 18, 292-304.

Edwards, R.G. and A.G. Searle (1963) Genetic radiosensitiv- ity of specific post-dictyate stages in mouse oocytes, Genet. Res., 4, 389-398.

Eichenlaub-Ritter, U. (1993) Studies on maternal age-related aneuploidy in mammalian oocytes and cell cycle control, in: A.T. Sumner and A.C. Chandley (Eds.), Chromosomes Today, Vol. 11, Chapman and Hall, London, pp. 323-336.

Eichenlaub-Ritter, U. (1994) Mechanisms of nondisjunction in mammalian meiosis, in: R.A. Pedersen (Ed.), Current Topics in Developmental Biology, Vol. 29, Academic Press, New York, pp. 281-324.

Eichenlaub-Ritter, U. and I. Boll (1989) Nocadazole sensitivity, age-related aneuploidy and alterations in the cell cycle during maturation of mouse oocytes, Cytogenet. Cell Genet., 52, 170-176.

Eichenlaub-Ritter, U. and I. Sobek-Klocke (1993) Implica- tions of cell cycle disturbances for meiotic aneuploidy: studies on a mouse model system, in: B.K. Vig (Ed.), Chromosome Segregation and Aneuploidy, NATO ASI Series, Vol. H 72, Springer-Verlag, Berlin, pp. 173-182.

Eichenlaub-Ritter, U. and H. Winking (1990) Nondisjunction, disturbances in spindle structure and characteristics of chromosome alignment in maturing oocytes of mice het- erozygous for Robertsonian translocations, Cytogenet. Cell Genet., 54, 47-54.

Eichenlaub-Ritter, U., A.C. Chandley and R.G. Gosden (1988) The CBA mouse as a model for maternal age-related increases in aneuploidy in man: studies on oocyte maturation, spindle formation and chromosome alignment during meiosis, Chromosoma, 96, 220-226.

Eppig, J. (1989) The participation of cyclic adenosine monophosphate (cAMP) in the regulation of meiotic maturation of oocytes in the laboratory mouse, J. Reprod. Fert., Suppl. 38, 3-8.


Eppig, J.J. (1991) Intercommunication between mammalian oocytes and companion somatic cells, BioEssays, 13, 569- 574.

Eppig, J.J., R.M. Schultz, M. O'Brien and F. Chesnel (1994) Relationship between the developmental programs con- trolling nuclear and cytoplasmic maturation of mouse oocytes, Dev. Biol., 164, 1-9.

Fielder, R.J., J.A. Allen, A.R. Boobis, P.A. Botham, J. Doe, D.J. Esdaile, D.G. Gatehouse, G. Hodson-Walker, D.B. Morton, D.J. Kirkland and M. Richold (1992) Dose setting in in vivo mutagenicity assays, Mutagenesis, 7, 313-320.

Garfield, R.E., J.M. Gasc and E.E. Baulieu (1987) Effects of the antiprogesterone RU486 on preterm birth in the rat, Am. J. Obstet. Gynecol., 157, 1281-1285.

Garfield, R.E., M.G. Blennerhassett and S.M. Miller (1988) Control of myometrial contractility: role and regulation of gap junctions, Oxford Rev. Reprod. Biol., 10, 436-490.

Gaulden, M.E. (1992) Maternal age effect: the enigma of Down syndrome and other trisomic conditions, Mutation Res., 296, 69-88.

Geiser, J.R., H.A. Sundberg, B.H. Chang, E.G.D. Muller and T.N. Davis (1993) The essential mitotic target of calmodulin is the ll0-kilodalton component of the spindle pole body in Saccharomyces cerevisiae, Mol. Cell Biol., 13, 7913-7924.

Gilula, N.B., M.L. Epstein and W.H. Beers (1978) Cell-to-cell communication and ovulation. A study of the cumulus- oocyte complex, J. Cell Biol., 78, 58-75.

Golbus, M.S. (1981) The influence of strain, maternal age and method of maturation on mouse oocyte aneuploidy, Cyto- genet. Cell Genet., 31, 84-90.

Gras, L., J. McBain, A. Trounson and I. Kola (1992) The incidence of chromosomal aneuploidy in stimulated and unstimulated (natural) uninseminated human oocytes, Hum. Reprod., 7, 1396-1401.

Gudi, R., S.S. Sandhu and R.S. Athwal (1990) Kinetochore identification in micronuclei in mouse bone-marrow ery- throcytes: an assay for the detection of aneuploidy-inducing agents, Mutation Res., 234, 263-268.

Gudi, R., J. Xu and A. Thilagar (1992) Assessment of the in vivo aneuploidy/micronucleus assay in mouse bone marrow cells with 16 chemicals, Environ. Mol. Mutagen., 20, 106-116.

Hansmann, I. (1974) Chromosome aberrations in metaphase II oocytes, stage sensitivity in the mouse oogenesis to amethopterin and cyclophosphamide, Mutation Res., 22, 175-191.

Hansmann, I. (1978) The. induction of non-disjunction in mammalian oogenesis, in: H.J. Evans and D.C. Lloyd (Eds.), Mutagen-Induced Chromosome Damage in Man, Edinburgh Press, Edinburgh, pp. 316-321.

Hansmann, I. (1983) Factors and mechanisms involved in nondisjunction and X-chromosome loss, in: A.A. Sandberg (Ed.), Cytogenetics of the Mammalian X Chromosome, Part A: Progress and Topics in Cytogenetics, Vol. 3A, Alan R. Liss, New York, pp. 131-170.

Hansmann, I. and E. El-Nahass (1979) Incidence of nondis-

junction in mouse oocytes, Cytogenet. Cell Genet., 24, 115-121.

Hansmann, I. and J. Jenderny (1983) The genetic basis of nondisjunction: increase of hyperploidy in oocytes from F1 hybrid mice, Hum. Genet., 65, 56-60.

Hansmann, I. and B. Pabst (1992) Nondisjunction by failures in the molecular control of oocyte maturation, Ann. Anat., 174, 485-490.

Hansmann, I. and H.D. Probeck (1979) Chromosomal imbalance in ovulated oocytes from Syrian hamsters (Mesocricetus auratus) and Chinese hamsters (Cricetulus griseus), Cytogenet. Cell Genet., 23, 70-76.

Hansmann, I., J. Neher and G. Rfhrborn (1974) Chromosome aberrations in metaphase II oocytes of Chinese hamster (Cricetulus griseus), I. The sensitivity of the preovulatory phase to triaziquone, Mutation Res., 25, 347-359.

Hansmann, I., J. Jenderny and H.D. Probeck (1980) Mecha- nisms of non-disjunction: Gonadotrophin-induced aneuploidy in oocytes from Phodopus sungorus, Eur. J. Cell Biol., 22, 24.

Hansmann, I., F. Beerman, E. Hummler and F. Theuring (1990) Aneuploidy in man and mechanisms of nondisjunction inferred from studying Djungarian hamster oocytes, in: T. Sharma (Ed.), Trends in Chromosome Research, Springer, Berlin, pp. 165-189.

Hartley-Asp, B., J. Deinum and M. Wallin (1985) Diethyl- stilbestrol induces metaphase arrest and inhibits microtubule assembly, Mutation Res., 143, 231-235.

Hassold, T.J and P.A. Jacobs (1984) Trisomy in man, Ann. Rev. Genet., 18, 69-97.

Hassold, T. and S. Sherman (1993) The origin of non-disjunction in humans, in: A. Sumner and A.C. Chandley (Eds.), Chromosomes Today, Vol. 11, Chapman and Hall, Lon- don, pp. 313-322.

Hecht, F. and K. Hecht (1987) Aneuploidy in humans: dimen- sions, demography and dangers of abnormal number of chromosomes, in: B.K. Vig and A.A. Sandberg (Eds.), Aneuploidy, Part A: Incidence and Etiology, Alan R. Liss, New York, pp. 9-49.

Heddle, J.A., M.C. Cimino, M. Hayashi, F. Romagna, M.D. Shelby, J.D. Tucker, Ph. Vanparys and J.T. MacGregor (1991) Micronuclei as an index of cytogenetic damage: Past, present and future, Environ. Mol. Mutagen., 18, 277-291.

Hennig, U.G.G., N.L. Rudd and D.I. Hoar (1988) Kineto- chore immunofluorescence in micronuclei: a rapid method for the in situ detection of aneuploidy and chromosome breakage in human fibroblasts, Mutation Res., 203, 405- 414.

Hickie, R.A., J.W. Wei, L.M. Blyth and D.Y.W. Wong (1983) Cations and calmodulin in normal and neoplastic cell growth regulation, Can. J. Biochem. Cell Biol., 61,934-941.

Hillensjo, T., C.P. Channing, S.H. Pomerantz and A. Schwartz-Kriporer (1979) Intrafollicular control of oocyte maturation in the pig, In Vitro, 15, 32-39.

Hook, E.B. (1985) The impact of aneuploidy upon public health: mortality and morbidity associated with human


chromosome abnormalities, in: V.L. Dellarco, P.E. Voytek and A. Hollaender (Eds.), Plenum Press, New York, pp. 7-33.

Huang, R.D., M.F. Smith and W.L. Zahler (1982) Inhibition of forskolin-activated adenylate cyclase by ethanol and other solvents, J. Cycl. Nucleotide Res., 8, 385-394.

Hummler, E. and I. Hansmann (1988) Pattern and frequency of nondisjunction in oocytes from the Djungarian hamster are determined by the stage of first meiotic spindle inhibition, Chromosoma, 97, 224-230.

Kaufman, M.H. (1983) Ethanol-induced chromosomal abnormalities at conception, Nature (London), 302, 258-260.

Kaufman, M.H. and I.M. Bain (1984) The development potential of ethanol-induced monosomic and trisomic conceptuses in the mouse, J. Exp. Zool., 231, 149-155.

Lawrence, T.S., W.H. Beers and N.B. Gilula (1978) Transmis- sion of hormonal stimulation by cell-to-cell communication, Nature, 272, 501-506.

Liang, J.C. and B.R. Brinkley (1985) Chemical probes and possible targets for the induction of aneuploidy, in: V.L. Dellarco, P.E. Voytek and A. Hollaender (Eds.), Aneu- ploidy: Etiology and Mechanisms, Plenum Press, New York, pp. 491-505.

Liang, J.C., D.A. Sherron and D. Johnson (1986) Lack of correlation between mutagen-induced chromosomal univa- lency and aneuploidy in mouse spermatocytes, Mutation Res., 163, 285-297.

Liu, L.F. (1989) DNA topoisomerase poisons as antitumor drugs, Ann. Rev. Biochem., 58, 351-375.

Lloyd, D.C. and A.A. Edwards (1983) Chromosome aberrations in human lymphocytes: effect of radiation quality, dose and dose rate, in: T. Ishihara and M.S. Sasaki (Eds.), Radiation-Induced Chromosome Damage in Man, Alan R. Liss, New York, pp. 23-49.

Luckett, D.C. and A.B. Mukherjee (1986) Embryonic characteristics in superovulated mouse strains, J. Hered., 77, 39-42.

Lynch, A.M. and J.M. Parry (1993) The cytochalasin-B micronucleus/kinetochore assay in vitro: studies with 10 suspected aneugens, Mutation Res., 287, 71-86.

Mackay, J.M. and B.M. Elliott (1992) Dose-ranging and dose- setting for in vivo genetic toxicology studies, Mutation Res., 271, 97-99.

MacKenzie, L.W. and R.E. Garfield (1985) Hormonal control of gap junctions in the myometrium, Am. J. Physiol., 248, 296-308.

Mailhes, J.B. (1983) Methylmercury effects on Syrian hamster metaphase II oocyte chromosomes, Environ. Mutagen., 5, 679-686.

Mailhes, J.B. (1987) Incidence of aneuploidy in rodents, in: B.K. Vig and A.A. Sandberg (Eds.), Aneuploidy, Part A: Incidence and Etiology, Alan R. Liss, New York, pp. 67-101.

Mailhes, J.B. and M.J. Aardema (1992) Benomyl-induced aneuploidy in mouse oocytes, Mutagenesis, 7, 303-309.

Mailhes, J.B. and F. Marchetti (1993) The relationship be-

tween chemically induced meiotic delay and aneuploidy in mouse oocytes and zygotes, in: B.K. Vig (Ed.), Chromo- some Segregation and Aneuploidy, NATO ASI Series, Springer-Verlag, Berlin, pp. 283-296.

Mailhes, J.B. and F. Marchetti (1994a) Chemically induced aneuploidy in mammalian oocytes, Mutation Res., 320, 87-111.

Mailhes, J.B. and F. Marchetti (1994b) The influence of postovulatory ageing on the retardation of mouse oocyte maturation and chromosome segregation induced by vinblastine, Mutagenesis, 9, 541-545.

Mailhes, J.B. and Z.P. Yuan (1987) Differential sensitivity of mouse oocytes to colchicine-induced aneuploidy, Environ. Mol. Mutagen., 10, 183-188.

Mailhes, J.B., R.J. Preston and K.S. Lavappa (1986) Mam- malian in vivo assays for aneuploidy in female germ cells, Mutation Res., 167, 139-148.

Mailhes, J.B., R.J. Preston, Z.P. Yuan and H.S. Payne (1988) Analysis of mouse metaphase II oocytes as an assay for chemically induced aneuploidy, Mutation Res., 198, 145- 152.

Mailhes, J.B., Z.P. Yuan and M.J. Aardema (1990) Cytoge- netic analysis of mouse oocytes and one-cell zygotes as a potential assay for heritable germ cell aneuploidy, Muta- tion Res., 242, 107-114.

Mailhes, J.B., M.J. Aardema and F. Marchetti (1993a) Investi- gation of aneuploidy induction in mouse oocytes following exposure to vinblastine sulfate, pyrimethamine, diethylstilbestrol diphosphate, or chloral hydrate, Environ. Mol. Mutagen., 22, 107-114.

Mailhes, J.B., F. Marchetti and M.J. Aardema (1993b) Griseo- fulvin-induced aneuploidy and meiotic delay in mouse oocytes: effect of dose and harvest time, Mutation Res., 300, 155-163.

Mailhes, J.B., F. Marchetti, G.L. Phillips, Jr. and D.R. Barn- hill (1994a) Preferential pericentric lesions and aneuploidy induced in mouse oocytes by the topoisomerase II inhibitor etoposide, Teratogen. Carcinogen. Mutagen., 14, 39-51.

Mailhes, J.B., F. Marchetti and D. Young (1994b) Synergism between gonadotrophins and vinblastine sulfate relative to the frequencies of metaphase I, diploid and aneuploid mouse oocytes, Mutagenesis, in press.

Mailhes, J.B., F. Marchetti and D. Young (1995) Numerical and structural chromosome aberrations induced by etoposide (VP16) during oocyte maturation of mice: transmission to one-cell zygotes and persistence during subsequent ovulations, Mutation Res., 25, 33.

Marchetti, F. and J.B. Mailhes (1994) Variation of mouse oocyte sensitivity to griseofulvin-induced aneuploidy and meiotic delay during the first meiotic division, Environ. Mol. Mutagen., 23, 179-185.

Marchetti, F. and J.B. Mailhes (1995) Variation of mouse oocyte sensitivity to griseofulvin-induced aneuploidy during the second meiotic division, Mutagenesis, 10, 113-121.

Marchetti, F., C. Tiveron, B. Bassani and F. Pacchierotti


(1992) Griseofulvin-induced aneuploidy and meiotic delay in female mouse germ cells, II. Cytogenetic analysis of one-cell zygotes, Mutation Res., 266, 151-162.

Masumbuko, M.B., M.M. Freund and R. DeMeyer (1992) Synaptonemal complex alterations in X-irradiated and in oestrogen-treated mice: a comparative study, Mutation Res., 282, 3-12.

Mercer, B. and N.R. Morris (1975) The effect of chloral hydrate upon mitosis in Aspergillus nidulans, J. Gen. Mi- crobiol., 88, 197-199.

Messinger, S.M. and D.F. Albertini (1991) Centrosome and microtubule dynamics during meiotic progression in the mouse oocyte, J. Cell Sci., 100, 289-298.

Miller, B.M. and I.-D. Adler (1992) Aneuploidy induction in mouse spermatocytes, Mutagenesis, 7, 69-76.

Miller, B.M. and M. Niisse (1993) Analysis of micronuclei induced by 2-chlorobenzylidene malonitrile (CS) using fluorescence in situ hybridization with telomeric and centromeric DNA probes and flow cytometry, Mutagenesis, 8, 35-41.

Miller, B.M., H.F. Zitzelsberger, H.UI.G. Weier and I.-D. Adler (1991) Classification of micronuclei in murine ery- throcytes: immunofluorescent staining using CREST antibodies compared to in situ hybridization with biotinylated gamma satellite DNA, Mutagenesis, 6, 297-320.

Mohr, U., M. Emura and M. Riebe-Imre (1993) Experimental studies on carcinogenicity and mutagenicity of caffeine, in: S. Garattini (Ed.), Coffee, Caffeine and Health, Mono- graphs of the Mario Institute for Pharmacological Re- search Milan, Raven Press, Ltd., New York, pp. 359-378.

Moore, R.M. and J.P. Heslop (1981) Cyclic AMP in mammalian follicle cells and oocytes during maturation, J. Exp. Zool., 216, 205-209.

Murray, A.W. and J.W. Szostak (1985) Chromosome segregation in mitosis and meiosis, Ann. Rev. Cell Biol., 1, 289- 315.

Nehlig, A. and G. Debry (1994) Potential genotoxic, mutagenic and antimutagenic effects of coffee: a review, Muta- tion Res., 317, 145-162.

O'Neill, G.T. and M.H. Kaufman (1987) Cytogenetic analysis of first cleavage fertilized mouse eggs following in vivo exposure to ethanol shortly before and at the time of conception, Development, 100, 441-448.

Pacchierotti, F. (1988) Chemically induced aneuploidy in germ cells of mouse, in: B.K. Vig and A.A. Sandberg (Eds.), Aneuploidy, Part B: Induction and Test Systems, Progress and Topics in Cytogenetics, Vol. 7b, Alan R. Liss, New York, pp. 123-140.

Pacchierotti, F. and J.B. Mailhes (1991) Aneuploidy dose-re- sponses following radiation or chemical exposures in mammals, in: B.L. Gledhill and F. Mauro (Eds.), New Horizons in Biological Dosimetry, Progress in Clinical and Biologi- cal Research, Vol. 372, Wiley-Liss, New York, pp. 363- 372.

Parry, J.M. and A.T. Natarajan (1993) CEC project-detection of aneugenic chemicals, Mutation Res., 287, 1-139.

Parry, J.M. and E.M. Parry (1989) Induced chromosome aneuploidy: its role in the assessment of the genetic toxicology of environmental chemicals, in: G. Jolles and A. Cordier (Eds.), New Trends in Genetic Risk Assessment, Aca- demic Press, London, pp. 261-296.

Parry, J.M., R.J. Fielder and A. McDonald (1994) Thresholds for aneuploidy-inducing chemicals, Mutagenesis, 9, 503- 504.

Parry, E.M., L. Henderson and J.M. Mckay (1995) Procedures for the detection of chemically induced aneuploidy: rec- ommendations of a UK Environmental Mutagen Society working group, Mutagenesis, 10, 1-14.

Peterson, M.B., M. Frantzen, S.E. Antoniarakis, A.C. War- ren, C. Van-Brockenhoven, A. Chakravarti, T.K. Cox, C. Lund, B. Olsen, H. Poulsen, A. Sand, N. Tommerup and M. Mikkelsen (1992) Comparative study of microsatellite and cytogenetic markers for detecting the origin of the nondisjoined chromosome 21 in Down syndrome, Am. J. Hum. Genet., 51,516-525.

Piegorsch, W.W., K. Zimmermann, S. Fogel, S.G. Whittaker and M.A. Resnick (1989) Quantitative approaches for assessing chromosome loss in Saccharomyces cerevisiae: general methods for analyzing downturns in dose response, Mutation Res., 224, 11-29.

Pinkel, D., J. Landegent, C. Collins, J. Fuscoe, R. Segraves, J. Lucas and J.W. Gray (1988) Fluorescence in situ hybridisation with human chromosome specific libraries: detection of trisomy 21 and translocations of chromosome 4, Proc. Natl. Acad. Sci. USA, 85, 9138-9142.

Polanski, Z. (1986) In vivo and in vitro maturation rate of oocytes from two strains of mice, J. Reprod. Fert., 78, 103-109.

Prather, A.L. and C. Racowsky (1992) Caffeine effects on meiotic maturation in hamster oocytes in vitro, Reprod. Toxicol., 6, 309-318.

Racowsky, C. (1984) Effect of forskolin on the spontaneous maturation and cyclic AMP content of rat oocyte-cumulus complexes, J. Reprod. Fert., 72, 107-116.

Racowsky C. (1986) The releasing action of calcium upon cyclic AMP-dependent meiotic arrest in hamster oocytes, J. Exp. Zool., 239, 263-275.

Racowsky, C. (1991) Gamete resources: origin and production of oocytes, in: R.A. Pederson, A. McLaren and N.L. First (Eds.), Animal Applications of Research in Mammalian Development, Cold Spring Harbor Press, New York, pp. 23-81.

Racowsky, C. (1993a) Follicular control of meiotic maturation in mammalian oocytes, in: B.D. Bavister (Ed.), Preimplan- tation Embryo Development, Springer-Verlag, New York, pp. 22-37.

Racowsky, C. (1993b) Somatic control of meiotic status in mammalian oocytes, in: F.P. Haseltine and S. Heyner (Eds.), Meiosis II, Contemporary Approaches to the Study of Meiosis, Am. Assoc. Adv. Science Press, Washington, D.C., pp. 107-116.

Racowsky, C. and R.W. McGaughey (1982) Further studies of


the effects of follicular fluid and membrana granulosa cells on the spontaneous maturation of pig oocytes, J. Reprod. Fert., 66, 505-512.

Racowsky, C. and R.A. Satterlie (1985) Metabolic, fluorescent dye and electrical coupling between hamster oocytes and cumulus cells during meiotic maturation in vivo and in vitro, Dev. Biol., 108, 191-202.

Racowsky, C., R.C. Hendricks and K.V. Baldwin (1989) Di- rect effects of nicotine on the meiotic maturation of hamster oocytes, Reprod. Toxicol., 3, 13-21.

Raimandi, E., S. Scariolo, A. De Sario and L. De Carli (1989) Aneuploidy assays on interphase nuclei by means of in situ hybridisation with DNA probes, Mutagenesis, 4, 165-169.

Rainaldi, G., L. Flori, C.M. Colella, T. Mariani, A. Piras, S. Simi and M. Simili (1987) Analysis of BrUdR-labelling technique of induced aneuploidy in mammalian cells in culture, Mutation Res., 177, 255-260.

Ried, T., A. Baldini, T.C. Rand and D.C. Ward (1992) Simul- taneous visualization of seven different DNA probes by in situ hybridization using combinatorial fluorescence and digital imaging microscopy, Proc. Natl. Acad. Sci. USA, 89, 1388-1392.

Rieder, C.L., J.G. Ault, U. Eichenlaub-Ritter and G. Sluder (1993) Morphogenesis of the mitotic and meiotic spindle: conclusions obtained from one system are not necessarily applicable to the other, in: B.K. Vig (Ed.), Chromosome Segregation and Aneuploidy, NATO ASI Series, Vol. H 72, Springer-Verlag, Berlin, pp. 183-197.

R6hrborn, G. and I. Hansmann (1971) Induced chromosome aberrations in unfertilized oocytes of mice, Humangenetik, 13, 184-198.

Ross, W.E., T. Rowe, B. Glisson, J. Yalowich and L.F. Lin (1984) Role of topoisomerase II in mediating epipodophyl- lotoxin-induced DNA cleavage, Cancer Res., 44, 5857- 5860.

Russo, A. and F. Pacchierotti (1988) Meiotic arrest and aneuploidy induced by vinblastine in mouse oocytes, Mutation Res., 202, 215-221.

Russo, A., F. Pacchierotti and P. Metalli (1984) Nondisjunc- tion induced in mouse spermatocytes by chloral hydrate, a metabolite of trichloroethylene, Environ. Mutagen., 6, 695-703.

Sakakibara, Y., I. Saito, K. Ichinoseki, T. Oda, M. Kaneko, H. Saito, M. Kodama and Y. Sato (1991) Effects of diethylstilbestrol and its methyl esters on aneuploidy induction and microtubule distribution in Chinese hamster V79 cells, Mutation Res., 263, 269-276.

Sargent, L.M., Y.P. Dragan, N. Bahnub, J.E. Wiley, C.A. Sattler, P. Schroeder, G.L. Sattler, V.C. Jordan and H.C. Pitot (1994) Tamoxifen induces hepatic aneuploidy and mitotic spindle disruption after a single in vivo administration to female Sprague-Dawley rats, Cancer Res., 54, 3357-3360.

Sato, E. and S.S. Koide (1984) Forskolin and mouse oocyte maturation in vitro, J. Exp. Zool., 230, 125-129.

Sato, Y., T. Murai, M. Tsumuray, H. Saito and M. Kadama

(1984) Disruptive effect of diethylstilbestrol on microtubules, Gann, 75, 1046-1048.

Schuetz, A.W. (1967) Action of hormones in germinal vesicle breakdown in frog (Rana pipiens) oocytes, J. Exp. Zool.+ 166, 347-354.

Schultz, R.M., R.R. Montgomery and J.R. Belanoff (1983a) Regulation of mouse oocyte meiotic maturation: implica- tion of a decrease in oocyte cAMP and protein dephos- phorylation in commitment to resume meiosis, Dev. Biol., 97, 264-273.

Schultz, R.M., R.R. Montgomery, P.F. Ward and J.J. Eppig (1983b) Regulation of oocyte maturation in the mouse: possible roles of intercellular communication, cAMP and testosterone, Dev. Biol., 95, 294-304.

Sentein, P. and Y. Ates (1974) Action de l'hydrate de chloral sur les mitoses de segmentation de I'oeuf de Pleurodile, &ude cytologique et ultrastructurale, Chromosoma, 45, 215-244.

Sieber, S.M., J. Whang-Peng, C. Botkin and T. Knutsen (1978) Teratogenic and cytogenetic effects of some plant- derived antitumor agents (vincristine, colchicine, maytan- sine, VP-16-213 and VM-26) in mice, Teratology, 18, 31- 48.

Tease, C. (1988) Radiation-induced aneuploidy in: germ cells of female mammals, in: B.K. Vig and A.A. Sandberg (Eds.), Aneuploidy, Part B: Induction and Test Systems, Alan R. Liss, New York, pp. 141-157.

Tease, C. (1992) Radiation- and chemically-induced chromosome aberrations in mouse oocytes: a comparison with effects in males, Mutation Res., 296, 135-142.

Thibault, C., D. Sz6116si and M. G~rard (1987) Mammalian oocyte maturation, Reprod. Nutr. Dev., 27, 865-896.

Thompson, E.J. and P.E. Perry (1988) The identification of micronucleated chromosomes: a possible assay for aneuploidy, Mutagenesis, 3, 415-418.

Tiveron, C., F. Marchetti, B. Bassani and F. Pacchierotti (1992) Griseofulvin-induced aneuploidy and meiotic delay in female mouse germ cells, I. Cytogenetic analysis of metaphase II oocytes, Mutation Res., 266, 143-150.

Tsafriri, A. (1978) Oocyte maturation in mammals, in: R.E. Jones (Ed.), The Vertebrate Ovary, Plenum Press, New York, pp. 409-442.

Tucker, R.W. and J.C. Barrett (1986) Decreased number of spindle and cytoplasmic microtubules in hamster embryo cells treated with a carcinogen, diethylstilbestrol, Cancer Res., 46, 2088-2095.

Uebele-Kallhardt, B.-M. (1978) Human Oocytes and Their Chromosomes: An Atlas, Springer-Verlag, Berlin, 106 pp.

Watanabe, T., T. Shimada and A. Endo (1977) Mutagenic effects of cadmium on the oocyte chromosomes of mice, Jpn. J. Hyg., 32, 472-481.

Watanabe, T., T. Shimada and A. Endo (1979) Mutagenic effect of cadmium on mammalian oocyte chromosomes, Mutation Res., 67, 349-356.

Weier, H.UI., H.F. Zitzelsberger and J.W. Gray (1991) Non- isotopic labeling of murine heterochromatin in situ by


hybridisation with in vitro synthesized biotinylated mouse satellite DNA, Biotechniques, 10, 498-505.

Wilcox, A., C. Weinberg and D. Baird (1988) Caffeinated beverages and decreased fertility, Lancet, 2: 1453-1456.

Witt, K.L., R. Gudi and J.B. Bishop (1992) Induction of kinetochore positive and negative micronuclei in mouse bone marrow cells by salicylazosulfapyridine and sulfapyri- dine, Mutation Res., 283, 53-57

Yuan, Z.P. and J.B. Mailhes (1987) Aneuploidy determination in C-banded mouse metaphase II oocytes following cyclophosphamide treatment in vivo, Mutation Res., 179, 209-214.

Zijno, A., F. Pacchierotti, S. Quaggia, A. Manca and R. Uccelli (1986) Mitotic and meiotic non-disjunction in the male mouse after treatment with diethylstilbestrol-diphosphate, Atti. Assoc. Genet. Ital., 32, 223-224.

important biological variables that can influence the degree of chemical-induced aneuploidy in...

Documents