in vivo cytogenetics: mammalian germ cells

Mutation Research 455 (2000) 167–189

In vivo cytogenetics: mammalian germ cells

Antonella Russo∗DBSF-Department of Structural and Functional Biology, University of Insubria, Via J.H. Dunant 3, 21100 Varese, Italy

Abstract

This chapter summarizes the most relevant methodologies available for evaluation of cytogenetic damage induced invivo in mammalian germ cells. Protocols are provided for the following endpoints: numerical and structural chromosomeaberrations in secondary oocytes or first-cleavage zygotes, reciprocal translocations in primary spermatocytes, chromosomecounting in secondary spermatocytes, numerical and structural chromosome aberrations, and sister chromatid exchanges(SCE) in spermatogonia, micronuclei in early spermatids, aneuploidy in mature sperm. The significance of each methodologyis discussed. The contribution of novel molecular cytogenetic approaches to the detection of chromosome damage in rodentgerm cells is also considered. © 2000 Elsevier Science B.V. All rights reserved.

Keywords:In vivo; Cytogenetics; Mammalian germ cells

1. Introduction

Cytogenetic analysis of mammalian germ cells con-tributes to the evaluation of genetic risk associatedwith exposure to physical and chemical environmentalagents. Methodologies are available for investigationsin female and male germ cells, by using endpointssuch as structural chromosome aberrations, numericalchromosome aberrations, sister chromatid exchanges(SCE) and micronuclei.

A great effort is required for preparation and ana-lysis of oocytes and zygotes, with respect to the mi-nor difficulties encountered for the analysis of celltypes of spermatogenesis. The specific features char-acterizing the male and female gametogenesis inmammals explain this difference. Spermatogenesis isa continuous process by which an unlimited numberof gametes is produced during the reproductive life ofmale mammals (Fig. 1) [1]. Metaphase spreads canbe obtained easily and in a reproducible way at the

∗ Tel.: +39-0332-421512; fax:+39-0332-421500.E-mail address:[email protected] (A. Russo).

stages of both mitotic (spermatogonia) and meiotic(primary and secondary spermatocytes) divisions. Incontrast, mammalian oogenesis (Fig. 2) is almostcompleted before birth in mammals, resulting in afixed number of oocytes. At birth, oocytes are arrestedat a late and specific stage of the meiotic prophase(dictyate). Resumption of meiosis (oocyte maturation)occurs in adult females under hormonal control [2];oocyte maturation is completed by a second arrest atMII. Mature oocytes resume from this second blockif fertilization occurs [2]. In synthesis, the number ofoocytes available for cytogenetic analysis is stronglylimited and a suitable sample size can be reachedonly with the use of many animals. For the abovereasons, the methodology of preparation of femalegerm cells or early zygotes (namely first-cleavagezygotes) is skill-demanding, time consuming, andhighly expensive. Therefore, the consequences of theexposure of germ cells to environmental mutagenshave been evaluated preferentially on cell types ofmale gametogenesis.

Different hamster species have been demonstratedto be amenable for cytogenetic analysis of female

0027-5107/00/$ – see front matter © 2000 Elsevier Science B.V. All rights reserved.PII: S0027-5107(00)00115-9

168 A. Russo / Mutation Research 455 (2000) 167–189

Fig. 1. Schematic representation of the process of spermatogenesis in rodents.

A. Russo / Mutation Research 455 (2000) 167–189 169

Fig. 2. Schematic representation of the process of oogenesis inrodents.

[3–7] and male germ cells [8–11]. The rat has beenused extensively for the spermatid micronucleus assay[12–15]; with the only exception of this last approach,however, the mouse can be considered the referencespecies for cytogenetic analysis in germ cells andzygotes.

The most relevant methodologies for the evaluationof cytogenetic damage induced in vivo in mammaliangerm cells are presented here. As these assays areonerous from several point of views, literature dataavailable so far are still scarce, and the historicaldatabase is not sufficient for evaluating the optimalprotocol. It must be taken into account also the diffi-culty of identifying a single and exhaustive protocolfor each assay, in view of the complexity and thepeculiarities of gametogenesis processes in male andfemale mammals. This is particularly relevant whenaneuploidy is the effect of interest: it is well knownthat several cells targets can interact with aneugenicagents, and this represents a complicating factor forthe appropriate choice of dose range, time intervals,and other experimental conditions. In fact, in contrastwith the simple dose–effect relationships that can beobtained after exposure to clastogenic agents, those

deriving from treatment with aneugens can result indifferent shape for different chemicals, and in somesituations the effective range of dose can be narrow[16–18]. Consequently, the protocols listed in thispaper, although improved and standardised after theiroriginal formulation, should be considered still in thephase of further validation.

2. Cytogenetic analysis of MII oocytes andfirst-cleavage zygotes

Chromosome analysis of female germ cells orzygotes is complicated by the fact that (a) the clas-sical air-drying protocol cannot be applied on thesmall amount of cells which can be recovered perfemale (less than 40 oocytes per mouse obtained afterhormone-induced superovulation) and (b) the smallnumber of mature oocytes produced per female repre-sents per se a big limitation in terms of time and costs.

The methodology developed by Tarkowski [19] hasbeen considered for many years the reference proto-col for preparation of metaphase spreads from oocytesor early zygotes. Following the method, secondaryoocytes or early zygotes collected from oviducts aremade free from cumulus–granulosa cells by incuba-tion with hyaluronidase, then processed by hypotonictreatment and fixation. The original protocol proposedby Tarkowski required direct fixation of specimenson slides, after seeding cells one-by-one or in smallgroups in a drop of hypotonic solution [19]. In 1987, anew methodology was described by Mailhes and Yuan[20], which consisted in the adaptation of the classi-cal air-drying procedure for processing the very lownumber of oocytes collectable. This approach, basedon the mass harvest procedure originally developed byPayne and Jones [21], implies the isolation of pools ofoocytes from at least 10 superovulated females, theirgradual fixation (which preserves as possible from cellloss), and the use of special microtubes for carryingout the centrifugation steps. The methodology pro-posed by Mailhes and Yuan [20] gives the maximumyield of scorable chromosome spreads with respect tothe initial number of collected oocytes. In particular,at least 40–50% of collected oocytes can be recov-ered on the slide with this method; of these, 30–50%result analyzable [22,23]. This means that about 20females per experimental point are required to guaran-


tee the appropriate sample size (superovulation allowsto isolate 35–40 mature oocytes per female). As al-ready mentioned, the methodologies for chromosomepreparation of female germ cells are either time con-suming and skill-demanding. Although still requiringhigh expertise, the methodology proposed by Mailhesand Yuan [20] has contributed largely in the last 10years to a fast accumulation of data concerning the in-duction of chromosome damage in mammalian germcells.

Cytogenetic analysis of MII oocytes or early zy-gotes can provide evidence for either aneugenic[16,24,25] or clastogenic activity [26–28] of en-vironmental agents. Chromosome analysis of MIIoocytes has been applied mainly for evaluation ofaneuploidy. In fact, the convoluted morphology ofmeiotic chromosomes is not appropriate for detectingstructural aberrations. Metaphases of first-cleavagezygotes can be studied to evaluate the effects ofenvironmental agents upon the second meiotic divi-sion in the female gametogenesis [28,29], but mostimportantly to assess the risk of transmission of un-balanced gametes from both sexes to the progeny[26,27,30,31]. Different studies, including those com-ing from two collaborative projects funded by theEuropean Union, indicate that the evaluation of heri-table chromosomal damage after treatment of malemice can replace the less sensitive dominant lethaltest [32–36].

Hormone-induced ovulation was not used routinelyuntil early 1980s [37], when it was clear from a num-ber of studies that superovulation does not have aninfluence on chromosome segregation and stability[reviewed in [17–38]]. However, it must be taken intoconsideration that appropriate superovulation con-ditions are necessary, as high hormone dosages canlead to synergistic effects with the test compoundand alter the frequencies of ovulated MI oocytes andhyperploid MII oocytes [17–39].

Preliminary knowledge of the kinetics of oocytematuration is necessary for the correct design of theexperiment. As an example, the frequency of segrega-tion errors occurred at the first meiotic division can beevaluated in MII oocytes when maturing oocytes aretreated before the onset of anaphase I. Treatments withaneugenic compounds after anaphase I cannot be de-tected until the metaphase of first cleavage division inthe zygote. It must be considered that rates of oocyte

maturation can differ among mouse strains, being un-der genetic control [40,41]. Therefore, the correct tim-ing of oocyte maturation should be assessed for anystrain used in each laboratory.

The duration of mouse spermatogenesis is knownin details [42,43]. This allows to use the appropri-ate time intervals between treatment and harvestingor treatment and mating to describe the stage-specificresponse of male germ cells from spermatogonia tospermatozoa.

Mouse immature oocytes isolated from untreatedfemales at dioestrus have been also used for cytoge-netic analysis after in vitro treatment [44–46] and afterin vitro fertilization [47]. This approach is particularlysuitable for basic research and it will not be consid-ered here further.

As already discussed, chromosome analysis ofone-cell zygotes can be carried out after treatment ofboth sexes. In principle, the female pronucleus canbe distinguished from the male one because of itslower chromosome condensation [48], thus enablingto evaluate the paternal vs. maternal contributionto transmitted chromosome damage. However, dif-ferent authors reported that this classification canbe inaccurate [49,50] and proposed to calculate thefrequency of chromosome alterations on the basisof the diploid complement of the zygote. Paternaland maternal chromosome complements can be alsodistinguished when C-banding makes evident thehighly heterochromatic Y chromosome. Homozygoustranslocation-carrier mice have been used sometimesto distinguish paternally and maternally derived chro-mosome complements [51–53].

Recently, molecular cytogenetics has been appliedfor a fine evaluation of transmissible chromosomedamage in mouse zygotes [54]; by chromosome paint-ing, stable and unstable chromosome aberrations,induced during the spermatogenesis and transmit-ted to the zygote, can be evaluated simultaneously.Marchetti et al. [35] demonstrated that this approachgives reliable estimates of dominant lethality (bymeans of unstable chromosomal aberrations) and her-itable translocations, which appear in good agreementwith the results coming from the two reference ge-netic assays. The application of chromosome paintingto zygote chromosome preparations can be consideredtherefore a relevant improvement of the sensitivity ofthe assay.


2.1. Protocol for chromosome analysis of MII oocytes

2.1.1. TreatmentsGeneral remarks: animals must be housed under

the following conditions: 12 h light/12 h dark cy-cle, room temperature at 21–23◦C, 45–55% relativehumidity, and standard diet. For treatments, the testchemical solutions should be freshly prepared in theappropriate solvent (e.g. physiological saline, oliveoil). The route of administration should be possibly anintraperitoneal (i.p.) injection; if treatment by gavageis necessary, it is recommended to avoid feeding ani-mals for few hours, because food uptake can interferewith adsorption and metabolism of the compound.Concurrent negative controls must be treated with thesolvent only and maintained at the same experimentalconditions as the treated groups. There is general con-cern to obtain a reduction of the number of animalsrequired for in vivo investigations. In this view, theneed for positive controls in different in vivo assayshas been discussed [55] leading to the conclusionthat concurrentpositive controls are not mandatory(slides coming from treatments with positive controlchemicals can be used instead for verifying the qual-ity of staining and scoring). Anyway, in the case offemale germ cells, it is impractical to use positivecontrols because of the time required for preparationsand analysis, and of the request of high number ofanimals.

Superovulation: 8–12-week-old virgin female miceare injected i.p. with 7.5 IU pregnant mare serum(PMS) to increase the number of mature follicles, and48 h later with 5 IU of human chorionic gonadotrophin(HCG), to induce ovulation.

Chemical exposure: because of the different ratesof oocyte maturation among mouse strains [40], it isrecommended to verify the kinetics of meiotic pro-gression, as well as the effects of dosage on survivaland ovulation, through a preliminary experiment. Fordose–effect relationships, at least three doses shouldbe studied, the highest one showing evidence of toxi-city or cytotoxicity. For aneuploidy studies, multipletreatment time intervals with a single dose can benecessary for a fine and complete characterizationof chemical effects, because of variation of oocyteresponse to the treatment in the course of oocytematuration [17,23]. The most appropriate time fortreatment corresponds to 0–8 h after HCG, since the

onset of anaphase I occurs approximately after thattime [23,56].

2.1.2. Equipments — solutionsDissecting microscope.Dissecting instruments: fine scissors, iris scissors,

straight and curved forceps, microdissecting needle.Extended capillary pipettes (obtained by pulling

pasteur pipettes on the flame).Petri dishes, 30 mm diameter.Multiwell plates (4–6 wells).Microvials obtained from pasteur pipettes (see

Section 2.1.3).Ten milliliter round-bottom centrifuge tubes.Saline solutions: sterile Hank’s balanced salt solu-

tion (HBSS); 0.3% (w/v) trisodium citrate dihydrate(hypotonic solution), prepared freshly or autoclavedfor storing.

Fixative: 3:1 ethanol/glacial acetic acid (methanolcan replace ethanol). Prepare fixative immediatelybefore use.

2.1.3. MethodTen to twenty females can be processed at once

(about 3 h are required to complete the procedure).Seventeen hours after HCG, kill the mice accord-

ing to the local guidelines (cervical dislocation, ormild anaesthesia). The time interval can be slightly in-creased if meiotic delay is expected in response to thetreatment. However, take into consideration that theproportion of degenerated oocytes increases steeplybetween 23–25 h post-HCG [17]. Normal ovulatedoocytes are arrested at MII, thus antimitotic treatmentis not necessary to accumulate metaphases.

Dissect the female and isolate the ovaries and theupper tract of tubes in petri dishes (30 mm diameter)containing HBSS.

With the aid of a dissecting microscope, isolateoviducts and transfer them in another petri dish con-taining HBSS. Pierce the wall of the ampulla witha dissecting needle; the cumulus mass of ovulatedoocytes is released in the medium. After isolation ofall cumulus masses, they are transferred with an ex-tended capillary pasteur pipett in a well of a multi-well plate containing hyaluronidase (150 IU/ml HBSS,0.5 ml). After 15–20 min, the oocytes will appearcompletely dissociated from the cumulus–granulosacells.


Isolated oocytes are rinsed three times in HBSS bytransferring the cells in sequence in three differentwells. After then, transfer the oocytes in another wellcontaining the hypotonic solution (0.3% trisodium cit-rate). Incubate at room temperature for 30 min. Beforefixation, evaluate the number of collected oocytes.

Transfer the oocytes in a minimal volume of hypo-tonic solution in a microcentrifuge tube. These tubesare obtained from glass pasteur pipettes, shortened atboth extremities. In particular, the tip of the pasteurpipette is shortened up to few cm, and sealed on theflame to obtain the bottom of the microtube. Be carefulto obtain a uniform surface when sealing, otherwisepelleted cells could be lost or damaged. Microtubesmust be graduated in an approximate cm-scale, in or-der to achieve cell fixation by gradual replacement ofthe small initial volume of hypotonic solution with thefixative.

Add one part of fixative to four parts of hypotonicsolution. Mix gently, by an extended capillary pipette,and fix 5 min at room temperature.

Centrifuge cells at 600 rpm for 2 min (microtubesmust be inserted into standard round-bottom cen-trifuge tubes and positioned in such a way to be firmlymaintained inside during centrifugation).

Remove supernatant but leave 1 cm of volume, thenadd two parts of fixative before resuspending the pel-let. Mix thoroughly by an extended capillary pipetteand fix 5 min at room temperature, then repeat thecentrifugation step.

Remove supernatant up to the 0.5 cm mark. Addfresh fixative in the ratio 7:1; resuspend as above, fixfor 5 min then centrifuge.

Remove supernatant up to the 0.3 cm mark. Addfresh fixative in the ratio 10:1. Mix gently the cellsuspension, and finally prepare slides by conventionalair-drying. Several approaches are in use in differentlaboratories to improve the quality of chromosomespreads. Temperature and humidity can differ in dif-ferent environments and periods of the year, thus theappropriate conditions must be verified in each labo-ratory. The cell suspension can be dropped from avariable distance (10–40 cm) onto microscope slides.These can be maintained at+4◦ or −20◦C for about1 h before use, or kept in distilled water, or used per-fectly dry. What is really critical is to handle perfectlydegreased microscope slides, and this can be obtainedagain in several ways: slides can be soaked for at least

24 h in alcohol–ether solution and wipen with tis-sue immediately before use, or cleaned in detergentor solpho-chromic solution and then rinsed severaltimes in distilled water. Although pre-cleaned slidesare commercially available, they should not be con-sidered as immediately ready for chromosome prepa-rations. It is recommended to handle clean slides withforceps. To obtain well-spread chromosome prepara-tions free of cytoplasm the fixative should evaporatequickly, and this is achieved by gently blowing ontoslides after dropping the cell suspension, or by placingslides on a warm (60◦C) plate. These general sugges-tions apply also to the case of oocyte metaphases.

By contrast-phase analysis, evaluate the number ofoocytes recovered on the slide, recording separatelyMI and MII oocytes. You can verify already at this stepthe number of analyzable vs. not analyzable oocytes(see criteria below), in order to stain only informa-tive slides. Because of the poor morphology of MIIchromosomes, a clear distinction of single chromatidswith respect to whole chromosomes (dyads), must beachieved by C-banding. The procedure is describedin Salamanca and Armendares [57]. Slides must beaged for about a week at room temperature beforestaining.

2.1.4. Scoring criteriaTwo hundred oocytes should be scored per each ex-

perimental condition when possible. However, on thebasis of the effects induced by the treatment, a drasticreduction of the number of collectable MII oocytescan occur; in these cases, a total of 100 MII oocytescan be satisfactory. Metaphases must be scored onlyif chromosome spreading is adequate (avoid eitherscattered metaphases or spreads with overlappingchromosomes). Do not score metaphases with poorC-banding.

Aneuploidy evaluation: the haploid chromosomecontent of the mouse oocyte isn=20. MII oocyteswith 10<n<20 are classified as hypoploid; MIIoocytes with 20<n<30 are classified as hyperploid.Oocytes with a single chromatid in defect or excess(i.e.n=19+1/2,n=20+1/2) are also classified as ane-uploid. Aneuploidy frequency must be calculated asthe ratio between the number of hyperploid oocytes tothe total number of haploid, hypoploid and hyperploidMII oocytes. As it is well known, hypoploid cellsdo not provide a reliable estimate of chromosome


segregation errors as their frequency is influenced bytechnical artefacts.

During the scoring, record also:• the number of non-analyzable MII oocytes;• the number of MI arrested oocytes (note that these

cells are not expected in control animals);• the number of polyploid (2n) oocytes (30–40

chromosomes);• structural abnormalities and premature centromere

separations.The above data can be useful to detect toxic effects

of the treatment and for a full understanding of thecytogenetic effects induced. In several studies the fre-quency of ovulated MI oocytes as well as the propor-tion of diploid MII oocytes have been demonstratedto correlate with the extent of meiotic delay inducedby the treatment [22,56,58]. It has been hypothesizedthat MI oocytes can resume from the meiotic arrestand proceed to MII, eventually contributing to the for-mation of aneuploid or diploid cells [22,56,58]. How-ever, there is still no clear evidence that these cellsare at higher risk of aneuploidy at the delayed mei-otic division, and their fate can be chemical-specific[22,25,31].

Fig. 3. Normal MII oocyte of the mouse (n=20) as appears after C-banding. Courtesy of J.B. Mailhes.

An image of a normal MII oocyte withn=20 isgiven in Fig. 3. Fig. 4 summarizes examples of abnor-mal types of oocytes recovered from treated females.

2.1.5. Statistical analysisChromosome aberration frequencies or aneuploidy

frequencies in treated and control oocytes shouldbe compared by chi-square analysis orG statistics,or Fisher exact test in the case of small samples.A trend test should be applied to verify dose–effectrelationships.

2.2. Protocol for chromosome analysis of one-cellzygotes

2.2.1. Treatments

2.2.1.1. For the evaluation of effects induced duringspermatogenesis.Male mice, 8–12 weeks old, aretreated following the appropriate route and caged 1:1with superovulated virgin females (7.5 IU PMS fol-lowed 48 h later by 5 IU HCG) at multiple time in-tervals, according to the spermatogenesis stage(s) ofinterest [42,43]. In the case of acute treatments, a


Fig. 4. Examples of abnormal oocytes. (A) MII oocyte showing complete centromere separation (n=20, 40 chromatids). (B) MII oocyteshowing partial centromere separation (n=20, 13 dyads and 14 chromatids). (C) MII oocyte showing premature anaphase II (40 chromatids).(D) aneuploid MII oocyte carrying a single chromatid indicated by the arrow (n=21+1/2, 21 dyads and 1 chromatid). (E) MI arrestedoocyte (2n=40, 20 bivalents). (F) diploid MII oocyte (n=40, 40 dyads). Courtesy of J.B. Mailhes.


table of correspondence between stages and time inter-vals can be found in Albanese [26]. Take into accountthat relevant clastogenic effects are mostly induced inlate spermatogenesis stages (spermatid–spermatozoa)[33,36]. General criteria for animal housing, for thechoice of dosage and route of exposure, and for con-sidering negative and positive controls, are discussedin Section 2.1.1.

2.2.1.2. For the evaluation of effects induced duringoocyte development.Eight- to twelve-week-old vir-gin females should be treated in the vicinity of HCG(superovulation of female mice is achieved as de-scribed before). General criteria for animal housing,for the choice of dosage and route of exposure, andfor considering negative and positive controls, are dis-cussed in Section 2.1.1.

Treatment of females with clastogenic agents canbe generally limited to a single time interval corre-sponding to treatment of late maturation stages (in theliterature, time intervals spanning from−24 to +4 hfrom HCG were used). In fact, it has been reportedthat the treatment of early stages of oocyte develop-ment does not lead to the transmission of structuralaberrations to zygotes [27].

For aneuploidy evaluation, treat females within 8h from HCG to detect chromosome segregation er-rors occurring at the first meiotic division (see Section2.1.1). To evaluate aneugenic effects induced at thesecond meiotic division treatment must be done 10 hafter HCG [29].

A pilot experiment can help in the choice of theappropriate time intervals and dosage, and for a pre-liminary evaluation of pre- and post-fertilization toxiceffects of the chemicals.

2.2.2. Equipments — solutionsSee Section 2.1.2.

2.2.3. MethodMating of treated mice with superovulated un-

treated females must be allowed immediately afterHCG injection, for each time interval previously es-tablished. The same is done when treated females aremated with untreated males. 8 h later, check for thepresence of vaginal plug; negative females can bechecked again within 24 h. Twenty-four to twenty-sixhours after HCG, treat mated females with colchicine

(0.2 ml injected i.p. from a 2×10−3 M solution) toarrest zygotes at the metaphase of first-cleavage di-vision. Zygotes are harvested 5 h after colchicineinjection, or later (e.g. 16 h) if the possibility ofchemical-induced delay of the first cleavage divisionexists.

Following the methodology described in Section2.1.3, dissect the female and isolate the ovaries and theupper tract of tubes in petri dishes (30 mm diameter)containing HBSS.

With the aid of a dissecting microscope and a sy-ringe with 30 g blunt-end needle, flush out zygotesfrom the oviducts in HBSS, then transfer them in awell of a multiwell plate, where zygotes are brieflyincubated in hyaluronidase (150 IU/ml HBSS) to re-move the zona pellucida.

After this passage, follow the corresponding stepsdescribed in Section 2.1.3.

Slides prepared for conventional cytogenetic ana-lysis must be C-banded, as discussed in Section 2.1.3,after 1 week of ageing. For chromosome painting ana-lysis, slides should be stored at−20◦ until used. Itis advisable to score slides before storing, to recordthe position of well spread metaphases. Details on theFISH procedure are given in Ref. [54].

2.2.4. Scoring criteriaAt least 200 zygotes must be scored per each

experimental condition tested, under the generalcriteria described in Section 2.1.4. The zygote chro-mosome number is expressed as a diploid comple-ment with 2n=40. For aneuploidy studies, recordzygotes with 30< 2n< 50. Aneuploidy frequenciesare calculated as the proportion of hyperploid zygotes(2n> 40) on the total number of zygotes. For chro-mosome aberration analysis, score diploid (2n= 40)and the eventual hyperploid zygotes (40< 2n< 50);record separately chromosome- and chromatid-typeaberrations, and distinguish breaks from exchangeswithin each class. If chromosome painting is car-ried out, score all the aberrations by analysis ofDAPI stained chromosomes under UV filter; thenuse green and blue light to detect aberrations in-volving the painted chromosomes [54], and clas-sify them according to the PAINT nomenclature[59]. Note that chromosome aberrations detected inone-cell zygotes are mostly of chromosome type[35,54].


Fig. 5. Normal metaphase of first cleavage division of mouse zygote (2n=40). Courtesy of J.B. Mailhes.

During the scoring, record also:• the number of zygotes per female;• the percentage of mated female;• the number of unfertilized oocytes, as a measure of

prefertilization toxicity;• the number of MII arrested zygotes or devel-

opmentally delayed zygotes (i.e. not yet at the

Fig. 6. C-banded chromosomes of male (right, see heterochromaticY chromosome in the center of the metaphase) and female (left)pronuclei in a first-cleavage mouse embryo, after pre-ovulatorytreatment of the parental female with diepoxybutane. Multiplechromatid-type aberrations in the female pronucleus are evident.Courtesy of F. Pacchierotti.

metaphase of first cleavage division), as a measureof post-fertilization toxicity;

• the number of triploid zygotes with 51–60 chromo-somes.Fig. 5 shows a metaphase from a normal diploid zy-

gote at first cleavage division. An example of diploidzygote carrying structural chromosomal aberrations isgiven in Fig. 6. In Fig. 7 an example of chromosomepainting applied on zygote chromosome preparationsis shown.


frequencies in treated and control zygotes shouldbe compared by chi-square analysis orG statistics,or Fisher exact test in the case of small samples.A trend test should be applied to verify dose–effectrelationships.

3. Chromosome analysis of male germ cells

Chromosome spreads from spermatogonia (mitoticdivisions) and from primary or secondary spermato-cytes (meiotic divisions) can be easily obtained fromthe testes of rodents. The methodology for meiotic


Fig. 7. PAINT/DAPI analysis of mouse zygote metaphases. FISH probes were applied to chromosomes 1, 2, 3, and X (bright green) and Y(red). The male chromosomes are clustered to the left and the female chromosomes are on the right. (A) PAINT image showing a dicentricand an acentric fragment. (B) The same metaphase shown with DAPI excitation and emission. Note that one translocated chromosomehas two centromeric regions and the other has none. Courtesy of F. Marchetti and A.J. Wyrobek.

chromosome preparation has been proposed in 1964by Evans et al. [60] and it is still considered the refer-ence protocol. Following Evans’ methodology, germcells are isolated by a simple mechanical disruptionof the tubules.

Scorable metaphase spreads from primary sperma-tocytes can be obtained in the absence of antimitotictreatment, because this cell stage is relatively fre-quent in the testicular cell population. The specificchromosome configuration in bivalent pairs allowsan immediate detection of reciprocal translocationsamong structural aberrations; reciprocal transloca-tions detected in primary spermatocytes must beinduced in stem cell spermatogonia. The assay ishighly sensitive to the effects of ionizing radiation,while the clastogenic potential of chemical agentscannot be demonstrated by this approach: indeed,a prevalence of breaks of chromatid type and onlyfew chromosome exchanges are generally induced bychemicals. It has been demonstrated in fact a selectiveelimination of aberrant spermatogonia after chemicalexposure [61]. In principle, also chromosome dam-age induced during the different stages of the meioticprophase could be detected by cytogenetic analysisof primary spermatocytes, but again the main mech-anism of induction of chromosome aberrations bychemical agents is a limiting factor. The majority ofchemicals act as S-dependent agents, and premeioticS-phase occurs in the mouse 12 days before mei-

otic divisions [42,43], at preleptotene. Preleptotene isexpected therefore to be the only or the most sensi-tive stage before meiotic divisions, when cytogeneticanalysis is carried out in MI spermatocytes [62]. Inconclusion, reciprocal translocations and other chro-mosome aberrations in MI cannot be considered anuseful endpoint for screening purposes, apart thoseinvestigations in the field of radiobiology.

Metaphase spreads from secondary spermatocytesresult in poor chromosome morphology, and thereforeare not eligible for structural chromosome aberra-tion analysis. Starting from 1980s, the interest forcytogenetic analysis on this cell type has grown be-cause of the possibility to use numerical aberrations,in particular hyperploid secondary spermatocytes, asindicators of nondisjunction events occurred duringthe first meiotic division [16,63]. A number of pub-lished papers and some collaborative efforts have welldemonstrated that an increase of aneuploidy afterexposure of different premeiotic cell stages can be de-tected by chromosomal analysis in MII spermatocytes[16,63,64]. In vivo colchicine treatment is recom-mended to improve either the frequency and the mor-phology of secondary spermatocyte metaphases [65].It has been clearly demonstrated by Liang et al. [65]that the accumulation of a suitable number of meioticmetaphases is achieved at specific conditions (higherconcentrations and longer time intervals with respectto those efficient in the bone marrow compartment),


possibly because of the existence of the blood testisbarrier.

Although spermatogonia mitoses can be found onmicroscopic preparations obtained by Evans’ method-ology, their number is generally very low, as expectedby the fact that these cells form the basal lamina ofseminiferous tubules. Several approaches have beenproposed to improve the yield of spermatogonialmetaphases [66–69]: in our personal experience themost efficient protocol is based on a mild enzymaticdigestion of seminiferous tubules.

Cytogenetic analysis of spermatogonial cells canprovide estimates of structural and numerical chro-mosomal aberrations [62,70–73] and SCE [70,74,75];these endpoints can be useful to compare the sensi-tivity of mitotically dividing, meiotic and postmeioticcell types in the testis, and to demonstrate that thecompound reached the target organ when weak effectsare induced or only some cell stages are sensitive tothe same agent [73,75].

After the micronucleus (MN) assay was introducedfor a fast evaluation of in vivo induced cytogeneticdamage in the mouse, two methodologies were pro-posed for the application of this assay in early sper-matids of rats or mice. MN detected at this cell stageshould have originated during the meiotic divisions.Lähdetie and Parvinen [12] described an approachknown as the dissection method, which is based onthe isolation under the dissecting microscope of shortfragments of tubules, specifically selected amongthose carrying early spermatids. The correct tractsof tubules are identified on the basis of the pecu-liar spatial organization of seminiferous tubules (theso-called seminiferous epithelial wave), then preparedfor the analysis by squash. The dissection method isespecially designed for analysis of rat spermatids, butit can also be applied on mouse cells [76,77]. Themethodology is skill demanding, but provides a highlyhomogeneous population, immediately beyond themeiotic divisions, for MN analysis. The alternativeapproach proposed by Tates et al. [78] is based on thepreparation of a cell suspension from testes tubules,including all germ cell types. To distinguish round(early) spermatids from other cell types, morpholo-gical criteria are coupled with Periodic Acid-Schiff(PAS) staining, which shows the developing acro-some structure. However, the maximum resolution ofpostmeiotic cell stages allowed by PAS differentiation

corresponds to a window of 49 h after the occurrenceof meiotic divisions (the golgi-phase [42]). For thisreason, the sensitivity of the spermatid MN assayis probably lower with the suspension method thanwith the dissection one; on the other hand, the lastmethodology is hardly reproducible without directtraining.

An opportunity to validate the spermatid micronu-cleus assay came from two collaborative projectsfunded by the European Union [34,36]. During thisstage, the original protocol of the suspension methodhas been improved and simplified [34,36]. The resultscoming from the spermatid micronucleus assay inrats and mice were in addition reviewed in the courseof the International Workshop on Genotoxicity TestProcedures held recently in Washington, DC [79].It appears that either clastogenic or aneugenic com-pound can be detected by this assay, and that verygood agreement is reached when the two methods orthe two species are compared. Moreover, molecularcytogenetics can be used to improve the assay bycentromere/telomere localization [76,80,81].

Since the ultimate aim of cytogenetic assays inmammalian germ cells is the evaluation of heritablerisk associated to exposure to environmental fac-tors, the direct inspection of the chromosome contentof mature sperm would be highly informative, andcould take advantage from the huge number of cellsavailable for analysis [82]. Interphase molecular cy-togenetics by multiple probes provided in differentfields the possibility to investigate the chromosomecontent of non-dividing cells; this same approach hasbeen applied on mammalian sperm (including human,mouse and rat sperm) [83–90], and although a ma-jor pitfall is represented by the highly condensationof sperm chromatin, which impairs the penetrationof molecular probes, the methodology has been im-proved and validated leading to reliable results for theevaluation of induced numerical alterations in rodents[85–88,90]. Sperm analysis can contribute to the fastaccumulation of data on the aneugenic effects inducedon both meiotic divisions by environmental agents,giving an accurate risk estimation. In addition, at leastfor human sperm, the possibility to detect structuralchromosome alterations has been demonstrated [84].In conclusion, this novel assay can be considered arelevant improvement in the field of mammalian germcell mutagenesis.


3.1. Protocol for chromosome preparation fromspermatocytes

3.1.1. TreatmentsMice can be treated preferably by i.p injection. Gen-

eral criteria for housing of animals, choice of dosageand route of exposure, use of negative and positivecontrols, are discussed in Section 2.1.1

The choice of appropriate time intervals betweentreatment and sacrifice must be done according to theduration of spermatogenesis and the meiotic or pre-meiotic stage of interest [42,43]. Five animals shouldbe treated per dose and time tested. Before treatments,preferably assign randomly the animals to each exper-imental group.

A pilot experiment can be useful for the choice ofdoses and time intervals between treatment and sacri-fice, especially if the compound is suspected to inducemeiotic delay.

3.1.2. Equipments — solutionsDissecting instruments: fine scissors, straight and

curved forceps.Petri dishes, 60 mm diameter.Ten milliliter round-bottom centrifuge tubes.Saline solutions: 2.2% (w/v) trisodium citrate dihy-

drate (isotonic solution), 0.9% (w/v) trisodium citratedihydrate (hypotonic solution). Use freshly preparedsolutions; autoclave for storing.

Fixative: 3:1 ethanol/glacial acetic acid; methanolcan be used instead of ethanol. Prepare fixative imme-diately before use.

Colchicine: dissolve 50 mg of colchicine in 10 mlof sterile distilled water to obtain a 0.5% solution.Store in 2 ml vials at−20◦C. After thawing, the so-lution is stable at+4◦C for several weeks and it isready to be injected.

3.1.3. MethodThree hours before killing the animals inject the

mice with 0.3 ml of 0.5% colchicine (for mice of 30 gbody weight the administered dose corresponds to 50mg/kg). This step can be omitted if MI metaphasesmust be analysed; however, colchicine treatmentsteeply improves the MI yield [65]. Note in addi-tion that some authors do not recommend the use ofcolchicine treatment for aneuploidy studies in MIIspermatocytes [87].

Animals must be killed according to the regulatoryguidelines of each country; if possible, cervical dislo-cation is preferred, otherwise light ether anaesthesiacan be used.

Dissect the animal and isolate both testes in a 60 mmpetri dish containing isotonic solution (2.2% sodiumcitrate) at room temperature. Keep testes as free aspossible from fat tissue; this is critical for obtaininghigh quality preparations.

Quickly transfer the testes in another petri dishwith fresh isotonic solution. By using curved for-ceps and fine scissors, make an incision of the tunicaalbuginea. By a gentle pressure of forceps on thesurface of the testis, let seminiferous tubules be re-leased in the medium. After removing the tunica,tease apart the tubules and smash them repeatedlyto allow mechanical disruption of tubules. Dissocia-tion of meiotic germ cells is quickly obtained, as itcan be monitored by the increasing turbidity of themedium.

Transfer the suspension in a 10 ml centrifuge tube,to allow settling of tubular debris, then transfer thesupernatant enriched of meiotic germ cells in a newtube. Centrifuge for 10 min at 600–800 rpm.

Discard the supernatant and resuspend dropwise thepellet in 3 ml of hypotonic saline solution. Incubatefor 12 min at room temperature, then centrifuge againfor 10 min at 600–800 rpm.

Discard the supernatant and resuspend the pellet in5 ml freshly prepared, cold fixative. Incubate for 10min on ice.

Centrifuge for 10 min at 1100 rpm.Repeat the previous two steps.Discard the supernatant and resuspend the pellet in

an appropriate volume of fresh fixative. Spot few dropsof solution on perfectly cleaned slides and air-dry (seeSection 2.1.3). Adjust the cell density if necessary.

Preparations can be stained with Giemsa (8% for10 min) but C-banding is preferable since it cangive a better resolution of meiotic chromosomes (seeSection 2.1.3).

3.1.4. Scoring criteriaAt least 200 MI or MII spermatocytes should be

scored per animal. Metaphase spreads should be se-lected at low magnification (20×) on the basis ofmorphological criteria (well-spread metaphases with-out excessive scattering or isolated chromosomes in


the vicinity). Numerical or structural abnormalities areverified at high magnification (100×).

For structural aberration analysis (MI): score onlyMI with 20 bivalents. Classify reciprocal transloca-tions as chain or ring multivalents. Record the presenceof univalents or chromosome breaks and fragments.

For aneuploidy assay (MII): score MII with18<n<22 chromosomes. The frequency of aneu-ploidy is calculated as the ratio of hyperploid MIIspermatocytes (n>20) to the total MII spermatocytesscored. As already discussed (Section 2.1.4), do notinclude hypoploid MII spermatocytes in the calcula-tion of aneuploidy frequency. An example of haploid(n=20) MII spermatocyte is shown in Fig. 8.

Do not score polyploid MII spermatocytes, themajority of them being technical artifacts due to theexistence of cytoplasmic bridges among synchronousmeiotic cells.

From slides obtained in the absence of antimitotictreatment, the frequency of MI and MII figures can bedetermined with respect to 1000 mid-pachytene nu-clei, to verify the kinetics of the meiotic divisions.Normal MII/MI ratio should be equal to 2 [64,87].Lower values indicate the occurrence of meiotic de-lay [87]. Apart its relevance for preliminary choiceof the appropriate treatment conditions (Section 3.1.1)

Fig. 8. Left: a metaphase spread from a secondary spermatocyte; the arrow indicates the heterochromatic Y chromosome. Right:spermatogonial metaphase showing sister-chromatid differentiation; one SCE is visible.

this information can be useful for a correct interpreta-tion of negative results or weak effects induced by thetreatment, since aneuploid MII oocytes could have notbeen formed before the recovery from possible meioticblock.


frequencies in treated and control animals should becompared by chi-square analysis orG statistics afterpooling individual data. Preliminary chi-square ana-lysis should be done to verify possible inter-individualvariability. A trend test should be applied to verifydose–effect relationships.

Ratios between MII and MI spermatocytes can becompared by applying the Student’st-test on trans-formed mean values (e.g. square root transformation)or a non-parametric test.

3.2. Protocol for chromosome preparation fromspermatogonia

3.2.1. TreatmentsThe cell cycle of differentiating spermatogonia lasts

26–30 h according to a classical paper by Monesi[91]. A single sampling time between 24 and 30 h can


be appropriate to detect clastogenic effects inducedin the spermatogonial differentiating cell population.Five animals should be treated per dose. See alsoSection 2.1.1.

The frequency of SCE can be evaluated in mousespermatogonia as for other in vivo proliferatingcell compartments, if 5-bromodeoxyuridine (BrdU)is administered at a slow rate to lower cytotoxi-city and systemic toxicity, and to counterbalance thefast rate of BrdU metabolism in vivo [92]. How-ever, the very long duration of spermatogonia cellcycle requires further adjustment of the method.Optimal conditions for SCE analysis consist in 56h of exposure to BrdU (two agar-coated 25 mgtablets) implanted subcutaneously [74]. Chemicaltreatments must be done 1 h after tablet implantation[74].

3.2.2. Equipments — solutionsThe following are in addition to the material listed

in Section 3.1.2.Shaking water bath.One hundred-milliliter flasks.TIM (Testis Isolation Medium [93]).Colchicine: to obtain a 10−3 M solution ready to

be injected, make a 1:25 dilution in distilled water ofthe 0.5% solution (prepared as described in Section3.1.2). After thawing, the solution is stable at+4◦Cfor several weeks.

Collagenase: use Type I (Sigma) or collagenase A(Roche Biochemicals).

3.2.3. MethodFour to five hours before sacrifice inject mice

with 0.3 ml of colchicine 10−3 M (for a mouse of30 g body weight, this treatment corresponds to 4mg/kg). This time interval is recommended in viewof the low proliferative rate of spermatogonia. Shorttime intervals require high colchicine concentrations,such as those used for spermatocytes (see Section3.1.3).

For SCE evaluation, implant 2 agar-coated 25 mgBrdU tablets per each mouse subcutaneously; prepara-tion of tablets and implantation procedure is describedin [94]. Treatments must be done 1 h after tablet im-plantation and the animals killed after 56 h of expo-sure to BrdU. This schedule is based on the resultsreported by Russo et al. [74].

Kill the animals according to local regulatory guide-lines. Dissect the animal, and isolate both testes inTIM at 37◦C. Keep testes free from fat tissue to obtainhigh quality preparations.

Isolate seminiferous tubules as described inSection 3.1.3. Transfer the mass of seminifer-ous tubules in a 100-ml flask containing colla-genase (0.5 mg/ml) dissolved immediately beforeuse in TIM. Shake in a water bath for 15 min at37◦C.

At the end of incubation time the germ cells arereleased from the tubules by gently pipetting. Filterthrough a 90-mm nylon membrane, and centrifuge at800 rpm for 10 min.

Resuspend dropwise the pellet in 2.2% sodium cit-rate, then follow the procedure described in Section3.1.3.

Slides can be stained in Giemsa (8%, 10 min).For sister chromatid differentiation, Fluorescence PlusGiemsa (FPG) staining must be done [95].

3.2.4. Scoring criteriaAt least 200 metaphases per mouse should be scored

for chromosome aberration analysis. Record chromo-some and chromatid type of aberrations separately,and for each class distinguish breaks from exchanges.Do not include gaps.

Twenty-five metaphases of 2nd generation sper-matogonia should be scored per animal to calculatethe SCE frequency. Metaphase selection must bedone as described above (Section 3.1.4). In Fig. 8,a spermatogonial metaphase with sister chromatiddifferentiation is shown.

3.2.5. Statistical analysisChromosome aberration frequencies in treated and

control animals should be compared by chi-squareanalysis or G statistics after pooling individualdata. Preliminary chi-square analysis should bedone to verify possible inter-individual variability.A trend test should be applied to verify dose–effectrelationships.

SCE mean values should be calculated per eachexperimental group and used for comparisons by aStudent’st-test on transformed values (e.g. square roottransformation). A dose–effect relationship can beverified by regression analysis on transformed meanvalues.


3.3. Protocol for spermatid micronucleus assay(suspension method)

3.3.1. TreatmentsTreat mice in order to expose cells at the stage

of interest, according to the duration of spermato-genesis [42,43]. Take into account that clastogeniccompounds are expected to give a peak of effect af-ter preleptotene treatment, and aneugenic compoundsshould give the maximum effect when administeredin the vicinity of meiotic divisions. Therefore, a min-imal experimental design requires treatment of thesetwo stages. According to literature, mouse early sper-matids exposed during differentiation divisions ofspermatogonia/preleptotene should be obtained 14–16days from an acute treatment; cells exposed at diaki-nesis/MI/MII can be scored as early spermatids 48 hlater [75,96,97]. For the rat, these intervals must beslightly modified [98–100] according to the diverseduration of meiotic prophase [43]. A repeated dosingregimen at preleptotene has been found to increasethe sensitivity of the assay for weak clastogens (e.g.:4 i.p. injections at 24 h intervals, harvesting 16 dayslater [75,99]). Five animals should be treated perdose. For general remarks see Section 2.1.1.

A pilot experiment aimed to choose the proper doserange and time schedule should be done. The kineticsof progression from meiotic division to spermiogen-esis can be verified by evaluating the ratio betweengolgi and cap phase spermatids (the first two devel-opmental phases of spermiogenesis, identifiable bythe diverse morphology of the acrosomal structure[42]).

3.3.2. Equipments — solutionsDissecting instruments: fine scissors, straight and

curved forceps.Petri dishes (60 mm diameter).Shaking water bath.Cytocentrifuge (optional; Shandon Cytospin is

recommendend).One hundred-milliliter flasks.Fifty-milliliter centrifuge tubes.Ten-milliliter round bottom centrifuge tubes.TIM (Testis Isolation Medium [93]).Phosphate buffered saline (PBS), Ca+2–Mg+2 free.Percoll solution.Helly’s fixative [78].

Collagenase: use Type I (Sigma) or collagenase A(Roche Biochemicals).

3.3.3. MethodKill the animals according to local regulatory guide-

lines. Dissect the animal, and isolate both testes inTIM at 33◦C. Keep testes free from fat tissue to obtainhigh quality preparations.

Isolate seminiferous tubules as described in Sec-tion 3.1.3. Transfer the mass of seminiferous tubulesin a 100-ml flask containing collagenase (0.5 mg/ml)dissolved immediately before use in TIM. Shake in awater bath for 20 min at 33◦C.

At the end of incubation time, transfer the mediumand mild digested tubules in a 50 ml centrifuge tube.Allow sedimentation of tubules, discard the mediumand add fresh TIM (about 10 ml).

Repeat the above step twice, then let the germ cellsbe released from tubules by gently pipetting. Filterthrough a 90-mm nylon membrane, and centrifuge at1000 rpm for 10 min.

Resuspend the pellet in 1 ml TIM, and stratify thisvolume onto 30% Percoll (diluted in TIM). Centrifugeat 2000 rpm for 30 min.

Isolate the layer of sedimented cells and dilute themin 5 ml fresh medium. Centrifuge 10 min at 1000 rpm,then discard the supernatant and resuspend the pelletin 5 ml fresh TIM.

Repeat twice the step above, and finally resuspendin 2 ml fresh medium. Put the cell suspension on ice.

Slides can be prepared by cytocentrifugation. Verifyempirically the appropriate cell densitiy, in order tospin 500ml per slide (a good starting dilution is 1:20from the 2-ml cell suspension). Spin cells on perfectlyclean slides at 800 rpm for 1 min.

Dip immediately the slides in PBS; check the quali-ty of preparations by contrast phase microscopy, butdo not let slides dry at this stage.

Transfer slides in a Coplin jar containing Helly’sfixative within 10 min; incubate for 30 min.

Rinse slides in running tap water for 30 min, thenwash them briefly in distilled water. Transfer prepara-tions in 70% ethanol and maintain at+4◦ C until use.

If a cytocentrifuge is not available, spot few dropsof the cell suspension on perfectly clean slides. Af-ter 15 min (the time necessary for cells to sediment),gradually add Helly’s fixative to slides (first few dropsof fixative are added onto each spot, then the whole


slide is covered with about 2 ml fixative). Incubate for1 h, then rinse the slides as described above.

Helly’s fixative is recommended for the conven-tional cytogenetic analysis requiring PAS reaction tovisualize the developing acrosome. After PAS, slidesmust be counterstained with Mayer’s hemallume, de-hydrated in 70–90–100◦ ethanol, and permanentlymounted.

Helly’s fixative does not appear appropriate for thesubsequent application of molecular cytogenetics. Inthis case, slides must be prepared using the cytospinmethod, rinsed in PBS then transferred in absoluteethanol and maintained at−20◦C until use. Localiza-tion of centromeric or telomeric regions can be doneas described in the literature [76,81].

3.3.4. Scoring criteriaSpermatids are small round cells with evident nu-

cleolus and few cytoplasm; PAS positive acrosomeis detected as a pinkish structure. Golgi phase sper-matids have a small globular acrosome (one or twospots), while cap phase spermatids show the acrosomeflattening onto the nucleus membrane [42]. At least2000 golgi phase spermatids must be scored per ani-mal. At the same time, record the number of capphase spermatids. Standard criteria must be followedto score micronuclei: in particular, micronuclei musthave round or oval shape, and staining of same in-tensity of main nucleus. Mayer’s hemallume stainingmakes well evident the nucleus as well as the micronu-cleus boundaries. As usual, micronuclei which do notappear clearly separated from the main nucleus shouldbe discarded; however it must be realized that littlecytoplasm is present in spermatids, therefore micronu-clei with evident membrane can be accepted even ifjuxtaposed to the main nucleus. Record micronucleieither in golgi- and cap-phase spermatids. A cap-phasespermatid carrying a micronucleus is shown in Fig. 9.

The frequencies of micronucleated spermatids mustbe calculated independently for the two developmentalphase scored. A golgi/cap phase ratio can be calculatedas an index of cytotoxicity or meiotic delay.

Acrosomal structure cannot be distinguished ifmolecular cytogenetics is applied, therefore it is ad-visable to carry out both scoring procedures on in-dependent slides. In fact, evaluation of micronucleusfrequencies on early spermatids without distinction ofthe two developmental phases can lead to an under-

Fig. 9. Mouse early spermatid (cap-phase) carrying a micronucleus(indicated by the continuous arrow); the dashed arrow indicatesthe acrosome structure.

estimation of the effect. Molecular cytogenetics canbe applied in a second step for the characterization ofmicronuclei with respect to their possible origin. Anexample of this application is shown in Fig. 10, wherea micronucleated spermatid appears to be originatedfrom a missegregation event since three telomericsequences and one centromeric signal are detected.

3.3.5. Statistical analysisMicronucleus frequencies in treated and control

animals can be compared by chi-square analysis orG statistics after pooling individual data. Preliminarychi-square analysis should be done to verify possi-ble inter-individual variability. A trend test shouldbe applied to verify dose–effect relationships. Ratiosbetween golgi and cap phase spermatids should becompared by applying the Student’st-test on trans-formed mean values (e.g. square root transformation)or a non-parametric test.

3.4. Fluorescent in situ hybridization (FISH)analysis on epididymal sperm

3.4.1. TreatmentsTreat the animals as described in the above sections

(see Section 2.1.1 for general criteria). At least fiveanimals per dose should be considered.


Fig. 10. Mouse early spermatid carrying a micronucleus. (a) DAPI staining, (b) localization of telomeric sequences by green fluorescence(three spots are visible in the MN); (c) localization of centromeric sequences by red fluorescence (one spot visible in the MN. Localizationof sequences obtained by PRINS technique as described in Ref. [81].

3.4.2. Equipments — solutionsDissecting instruments: fine scissors, iris scissors,

straight and curved forceps.Petri dishes, 30 mm diameter.0.5 ml microvials.

Fig. 11. Normal, aneuploid and diploid mouse sperm detected by FISH with probes for chromosome X, Y and 8. Courtesy of I.-D. Adler.

Saline solutions: 2.2% (w/v) trisodium citrate di-hydrate (isotonic solution). 0.1 M Tris–HCl buffer,pH 8.0. Use freshly prepared solutions; autoclave forstoring.

Dithiotreitol.3,5-Diiodosalicylic acid, lithium salt.


3.4.3. MethodKill the mice 22 days after treatment. If rats are

used, modify this time interval according to the dura-tion of postmeiotic stages as reported by Adler [43].Dissect the animals and remove both epididymes.

Isolate the caudae epididymes, place them in apetri dish containing 2.2% sodium citrate and makeseveral incisions with iris scissors. Transfer bothcaudae epididymes in a microvial containing 300ml of 2.2% sodium citrate. Incubate at 32◦C for 30min to allow sperm to be actively released from theepididymes.

Remove the epididymes from the microvial andstore the sperm suspension at−20◦C until slide prepa-ration is required.

To prepare slides, place 5–7ml of the suspensionon perfectly clean slides and smear the drop. Incubateslides at 80◦C for 5 min.

Slides must be aged overnight at room tempera-ture before FISH analysis; slides can be also stored at−20◦C.

Immediately before FISH, sperm decondensationmust be carried out. Selected slides must be main-tained at room temperature for 30 min, then dipped in10 mM dithiotreitol in Tris–HCl buffer for 30 min onice. Transfer slides in 4 mM 3,5-diiodosalicylic acidin Tris–HCl buffer, and incubate for 30 min at roomtemperature.

Air-dry slides completely before FISH, which canbe carried out following a standard protocol.

3.4.4. Scoring criteriaA combination of probes should be used in order to

distinguish aneuploid from diploid sperm. As an ex-ample: by using probes to chromosomes X, Y and 8,normal sperm should be X-8 or Y-8; diploid spermshould be X-Y-8-8 (if deriving from first meiotic di-vision) or X-X-8-8, Y-Y-8-8 (if deriving from secondmeiotic division); disomic sperm should be X-X-8,Y-Y-8, X-Y-8, X-8-8, Y-8-8. Do not score sperm withonly one fluorescent signal (e.g. X-0), since they canbe due to a technical artefact. Fig. 11 gives examplesof the different types of normal and abnormal spermdetected by the FISH approach.

Sperm decondensation can lead to rather diffuseFISH signals, therefore very stringent criteria are nece-ssary to carry out the analysis. Signals belonging tothe same chromosome must appear of same size and

intensity to allow classification of sperm as disomic;in addition, signals must be clearly spatially separated.

At least 10,000 sperm per mouse should bescored.

3.4.5. Statistical analysisFrequencies of aneuploid and diploid sperm can be

compared by chi-square analysis or G statistics afterpooling individual data. Preliminary chi-square ana-lysis should be done to verify possible inter-individualvariability. A trend test should be applied to verifydose–effect relationships.

Acknowledgements

I am particularly grateful to Francesca Pacchierotti(ENEA, Rome, Italy) for helpful suggestions dur-ing the preparation of the manuscript. Many thanksto I.-D. Adler (GSF, Neuherberg, Germany), J.B.Mailhes (Louisiana State University Medical Centre,USA), F. Marchetti–A.J. Wyrobek (Lawrence Liver-more National Laboratories, Livermore CA, USA),and F. Pacchierotti (ENEA, Rome, Italy) for givingme the opportunity to show their beautiful pictures ofgerm cell preparations.

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in vivo cytogenetics: mammalian germ cells

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