DNase I nick translation in situ on meiotic chromosomes of the mouse,

Mus musculus*

RAJIVA RAMAN, A. P. SINGH and INDRAJIT NANDA

Cytogenelics Laboratory, Department of Zoology\ Banaras Hindu University, Varanasi-221005, India

•We dedicate this paper to the memory of the late Mcnashc Marcus, who contributed greatly to the development of the in situ DNase Inick-translation method, which enabled us to carry out the present investigation

Summary

DNase-I-sensitive sites have been located on themeiotic chromosomes of the mouse, Mus muscu-lus, by the in situ DNase I nick-translationmethod. We find that: (1) of all the cell typesstudied, pachytene nuclei are the most sensitive toDNase I; (2) in diplotene the nicks occur preferen-tially in the vicinity of chiasmata; (3) the sexchromosomes are also sensitive to the enzymedespite their transcriptional quiescence; and (4)

in the sex bivalent the nicks are primarily ob-served in the putative region of recombination.We conclude that, in addition to discriminatingbetween the transcriptionally active and inactivestates of chromatin, DNase I identifies recombi-nation-specific chromatin changes in meiotic pro-phase.

Key words: DNase-I-sensitive sites, meiotic chromosomes,Mus musculus.

Introduction

It is well established that DNase I preferentiallycleaves transcriptionally active regions of chromatin(Garel & Axel, 1976; Weintraub & Groudine, 1976;Elgin, 1981). Nick translation with this endonucleasethus provides a sensitive method for molecular analysisof functionally distinct fractions of chromatin. In anapparently surprising discovery, Gazit et al. (1982)found that the genes potentially active during inter-phase of the cell cycle retain their DNase I sensitivity atthe following metaphase, the stage at which chromatinis totally inactive but cytologically visible as discretechromosomes. This discovery paved the way for devel-oping methods for cytological mapping of active re-gions of chromosomes by in situ DNase I nick trans-lation (Kercmef al. 1983; Kuo & Plunkctt, 1985). Theefficiency of the method has been demonstrated inseveral studies (Kerem et al. 1984; Sperling et al.1985). While data have been accumulating on thesensitivity of somatic cell chromatin, information onmeiotic cell chromatin is rather sporadic, in spite of thefact that the vital function of recombination of genesand segregation of chromosomes takes place during thisdivision. Of the three detailed studies hitherto pub-Journal of Cell Science 90, 629-634 (1988)

Printed in Great Britain © The Company of Biologists Limited 1988

lished, one on human spermatocytes shows hyper-sensitivity of the XY bivalent at the pairing tips, theputative zone of recombination (Chandley & McBcath,1987). With mouse, Richler et al. (1987) have madesimilar observations to these made with human sper-matocytes, but Separovic & Chandley (1987) havefound the opposite.

In an independent study on the DNase I nick-translated mouse spermatocytes (/;/ situ), we too havefound hypersensitivity at the pairing tips of the XYbivalents. We have also noticed preferential sensitivityto the enzyme of autosomal chiasmata regions, whichsuggests a coincidence between the rccombinogenicactivity of chromatin and its DNase I sensitivity. Theresults are presented in this paper.

Materials and methods

Chromosome preparationThree mice of Park's strain, originally obtained from CentralDrug Research Institute, India, were used for the study.Testis tubules were minced in Eagle's minimal essentialmedium (MEM) to release cells, which were then transferredto 0'56% KC1 for 5min. The cells were then fixed inmethanol:acetic acid (3:1, v/v) fixative for 2-3 min. The

629

hypotonic and fixation treatments were kept to a minimum inorder to retain chromosome organization as close as possibleto their pre-fixed state, and especially to avoid alteration orremoval of chromosomal proteins that result from prolongedfixation. A few drops of the suspension were air-dried onclean, chilled slides. They were immediately used for thenick-translation reaction, but for the intervening period(maximum 30min) they were kept in a refrigerator at 10°C.

DNase I nick translationThe procedure for the nick translation was the same asdeveloped by Kerem et al. (1983). Briefly, each slide wascovered with 20^1 of the chilled nick-translation mixture(50mM-Tris-HCl, pH7-9, 5 mM-MgCl2, 50 / igmr 'BSA(bovine serum albumin), OlmM-DTT (dithiothreitol),10 units ml"1 of DNase-free DNA Pol I; 4jiM each of dATP,dGTP, dCTP; 0 - 3 , U M - [ 3 H ] T T P (sp. act. 53-5Cimmor'))and different concentrations of DNase I ( 0 1 , 0 5 ,l-Ongml"1). No DNase I was added to control slides. Thenick-translation reaction was allowed to proceed for 5 min,following which slides were rinsed in two changes of 2XSSC(SSC is 0-15M-NaCl, 0-015 M-sodium citrate) containing1 mM-EDTA. The dried slides were briefly refixed in metha-nol: acetic acid fixative.

[3H]uridine labellingCell suspensions of two somatic tissues (liver and bonemarrow) and testis in Eagle's MEM were labelled in vitrowith tritiated uridine (sp. act. 13-5 Cimmor 1 ) . While thesomatic cells were labelled for 15 min in lOjuCiml"1, those oftestis were incubated for 2h in 25^Ciml~1. The cells werefixed directly after labelling.

Au toradiographyBoth the nick-translated and [ H]uridine-labelled slides wereexposed for autoradiography. They were treated with chilledtrichloroacetic acid before applying L4-nuclear emulsion(Ilford). All the nick-translated and uridine-labelled slidesfrom liver and bone marrow were exposed for 10-12 days.The uridine-labelled testis slides, however, were kept in thedark for 8 months. The slides were developed in D19bdeveloper for 4 min (12°C) and fixed in acid fixer. They werestained with Giemsa after developing. Silver grains wereremoved after examination of the autoradiograms and thepreparations were restained with Giemsa.

Results

Three experiments were carried out using differentconcentrations of DNase I. Adequate labelling wasobtained even with the lowest concentration(0-lngmP1) and, since there was no remarkabledifference in the labelling pattern among differentconcentrations, 0-1 ng appears to be adequate for nicktranslation in meiocytes. The results presented here aregenerally pooled from experiments with 1-0 and (M ngDNase I. In the control slides (without DNase I),meiocytes were not labelled. Some labelling, 4-10grains, was recorded in a few gonial interphases,presumably due to the utilization of the exogenouslysupplied enzymes and precursors by the already repli-cating S-phase cells. Among the experiments, all but afew spermatids and spermatozoa were labelled.

Pachytene chromosomes are the most DNase-I-sensitive

Grain counts in cells occupying 20 randomly chosenareas in two slides revealed that the extent of labellingin zygotene/pachytene nuclei was considerably higherthan in all the other cell types studied (Table 1).Pachytene grain counts were also higher than inmetaphase I (Table 1) and bone marrow cells (data notshown). This picture contrasted with the actual statesof transcription in the cell types studied. We confirmedit by studying [3H]uridine incorporation in testis andtwo somatic tissues (liver and bone marrow). While a15-day exposure revealed intense labelling of nuclei ofsomatic tissues, it took 8 months to achieve comparablegrain density on spermatogonials in testis. Even aftersuch prolonged exposure, only 30 % of the pachyteneswere sufficiently labelled but their grain count waspoorer than those of the gonials. Post-pachytene cellswere generally unlabelled and the sex bivalent did notincorporate uridine at any stage of meiosis, includingpachytene (Fig. 1). Thus, despite their low transcrip-tion, the pachytene chromosomes were hypersensitiveto DNase I.

Table 1. Pooled grain counts from 20 randomly chosen areas from two slides

Cell typesNo. ofcells

Total graincount

Mean graincount/cell

±S.D.

InterphasesPachytenesSpermatidsSperm heads

Metaphases*

140866275

50

6031

7109

1067676

1493

Ten areas from a slide, each with 0 1 and l-0ngml ' DNase I, were chosen.• Cells counted independently of the 20 areas mentioned above.

43-1 + 6-382-7 ±9-117-2 ± 4 19-0 ±3-0

29-913-0

630 R. Raman et al.

1A ,

Fig. 1. Autoradiograms of (A)DNase-I nick-translated and (B)[3H]uridine-labelled meiotic cells.While the XY bivalent (arrows) islabelled in the nick-translatedpachytene, it is unlabelled in the[3H]uridine-labelled one. Notelabelling on the nick-translatedinterphase nucleus. The fewgrains on the sperm heads havebeen obscured by heavy staining.Bars, 10 /lm.

Sex bivalent is DNase-I-sensitiveThe sex bivalent was the main point of reference forDNase I sensitivity because of its easy discriminationand transcriptional quiescence. Following the nicktranslation, however, it was labelled in all the 200pachytenes and 100 metaphase Is analysed (Figs 1A,2).The average grain count on the sex vesicle in pachy-tenes was 6-3 per cell. Owing to their diffuse nature itwas difficult to identify the labelled region at pachy-tene. At metaphase I, where the overall grain count wasmuch lower (Table 1), the sex bivalent had 1-5grains(Fig. 2A,B). In an attempt to map the distribution ofgrains, the sex bivalent was divided into four segments;namely, the terminally aligned regions of the X and Y,the rest of the Y, the region proximal to the centromereof the X and the interstitial segment of the X. As shownin Fig. 2C, more than half the grains were located onthe region of X-Y terminal alignment, the putative siteof their recombination.

Chiasmata regions are also DNase-I-sensitiveFollowing this indication of preferential labelling of theputative recombination zone of the sex chromosomes,we scrutinized the chiasmata of autosomal bivalents indiplotenes. Although, owing to extremely brief hypo-tonic and fixative treatments, the morphology of thebivalents was not optimal, four diplotene plates werekaryotyped. There were indications of preferentialdistribution of grains on or around chiasmata.

In order to determine whether the preferentialoccurrence of grains on chiasmata was statisticallysignificant, a yf test was done to compare chiasmaticand non-chiasmatic regions and terminal and inter-

stitial chiasmata. For this purpose, photographs of fourGiemsa-stained diplotene plates were given to a personother than the authors, after removing the autoradio-graphic grains, and he was asked to measure lengths inmillimetres or parts thereof of centromeric, interstitialand terminal chiasmata, as well as non-chiasmaticregions. The grains were then mapped on these regionson their autoradiograms. However, there were inherentlimitations in this approach: (1) certain bivalents didnot reveal unambiguous chiasmata through their lengthand had to be omitted from consideration (e.g. bi-valents boxed in Fig. 3); and (2) since telomeric endswere not always distinguishable in bivalents havingterminal or subterminal chiasmata, each such area wastermed a telomeric chiasma (see, for instance, arrow-headed chiasmata in Fig. 3). As a result, particularly insmall bivalents, the total chiasmatic area measured wasmore than the non-chiasmatic area. It was not alwayspossible to distinguish between pericentromeric andterminal regions and, since their grain counts weresimilar, we pooled the two. Thus only three classes,namely terminal chiasmata, interstitial chiasmata andnon-chiasmate regions, were tabulated, and a total of46 bivalents were analysed. Differences in grain fre-quency in different regions were tested statistically.Within the limitations cited above, the difference ingrain frequency between different chiasmata regionswas not significant but that between chiasmate andnon-chiasmate regions was significant in favour of theformer (Table 2). Five pachytene plates were alsokaryotyped to map grains on autosomal bivalents, butthey did not reveal any strikingly sensitive or insensit-ive regions. Owing to excessive condensation, as well as

DNase-I-sensitive sites on meiotic chromosomes 631

'.V*X

2A

B

114

Fig. 2. Nick-translated(A) metaphase I plate and(B) cut-outs of XY bivalents fromdifferent metaphases showingpreferential localization of grainson their attachment site (arrows).In certain autosomal bivalentsalso, their chiasmatic regions arelabelled (broken arrows). Bar,10 jUm. C. A diagrammaticrepresentation of distribution ofgrains on different regions of theXY bivalent computed from 50plates. The numbers beside thehorizontal bars indicate the totalnumber of grains scored.

Table 2. % Test for evaluating randomness of grain distribution in chiasmatic and non-chiasmatic regions ofthe bivalents

Chiasmataregions

Tclomeric/centromericInterstitial

ChiasmataNon-chiasmatic

Length(mm)

281-369-5

350-8364-9

Number

Observed

13631

16761

of grains

Expected

134-033-1

111-86116-36

0168 P>0-95

53-40 P< 0-005

the possibility of tcrininalization of chiasmata, grainswere not mapped on metaphase I cells.

Briefly, the results showed the following: (1) maxi-mal sensitivity of pachytene chromatin; (2) preferentialnicking in the chiasmata regions; and (3) sensitivity toDNase I of the sex bivalent in the putative zone ofrecombination.

Discussion

Mus musculus was chosen for this study because its

632 R. Raman et al.

spermatogenesis has been studied in some detail. It isknown that transcriptional activity declines with theprogression of meiosis and that few transcripts arereleased by post-pachytene cells, that the sex chromo-somes do not transcribe throughout meiosis (Monesi et.al. 1978) and the molecular evidence for X-Y recombi-nation, despite the lack of a chiasma, is unequivocal(Keitges et a/. 1985).

Our observation on the preferential sensitivity of therecombination zone of the XY bivalent is in agreementwith that of Richler et al. (1987) but contrasts with

I)00

Iff * * 60

00

O<544

44n n

$!n ««

Fig. 3. Karyotypical arrangement of two nick-translated diplotenes. Bivalents with and without grains have been arrangedside-by-side. These two plates were among the four diplotenes that provided data for Table 2. Boxed bivalents wereomitted from the calculations for grain distribution. Arrowheads indicate chiasmata whose location cannot be distinguishedbetween terminal and subtermina! category. For details see the text and Table 2. Bars, 10 ftm.

another study on the mouse (Separovic & Chandley,1987), which identifies instead the early replicating X-inactivation centre (Xce) as the most sensitive site.Though the interstitial region of the X, which includedthe Xcc, was also occasionally labelled in our prep-arations it was not possible for us to identify anyspecific region, perhaps due to low grain count. Wecannot explain the difference between our observationsand those of Separovic & Chandley (1987). Inciden-tally, in two other mammals, man (Chandley &McBeath, 1987) and the musk shrew, Suncus murinus(Raman & Nanda, 1985), studied by this method

hypersensitivity of the X-Y recombination zone toDNase I has also been indicated.

The observation that pachytene may have possiblythe most sensitive chromatin of all the somatic andgerm cell types is consistent with the biochemicallyanalysed DNase I sensitivity in the rat (Rao et al. 1985)and in microsporocytes of the lily plant (Hotta et al.1985). It is believed that DNase I identifies active orpotentially active domains of transcription in chroma-tin, but the hypersensitivity of pachytene chromatindoes not seem to imply this. Compared with the prc-meiotic germ cells and proliferating somatic cells,

DNase-I-sensitive sites on meiotic chromosomes 633

much less transcription occurs in pachytene and itcontinues to decline in post-pachytene cell types rightup to zygote formation. This implies, therefore, thatthe pachytene sensitivity is quite disproportionate to itstranscriptional potential. It should therefore be reason-able to assume that factor(s) besides transcriptionalpotential contribute to the hypersensitivity of thepachytene chromatin. Considering that recombination,which takes place during pachytene, requires intensenicking and repairing activity, it is likely that an alteredchromatin organization is required to facilitate nickformation. It is most plausible that the same alterationsmust also make the chromatin DNase-I-sensitive. Thatthis could indeed be so has been demonstrated in thelily, where the zyg and psn DNA, the DNAs syn-thesized during zygotene and pachytene, respectively,are the most sensitive to DNase I during the stage oftheir synthesis (Hotta et al. 1985). The unique featureof thzpsn DNA, which flanks the P-DNA domains inmeiocytes, is that it provides sites for the endogenousmeiotic endonuclease for recombinational nicks. Thisproperty is achieved by a qualitative change in chroma-tin organization of psn DNA brought about by theremoval of histones and binding with/>sn RNA and psnprotein (Stern & Hotta, 1983). It is most likely that thesame region is hypersensitive to DNase I in lily.Although equally exhaustive studies on mouse meiosisare lacking, since most molecular events of meiosisseem to be evolutionarily conserved, we suggest thatthe hypersensitivity of pachytene in the mouse iscaused by the chromatin changes brought about forrecombination as well as transcription. We furthersuggest that the altered conformation of recombino-genic regions of chromatin is retained till later in celldivision and thus leads to preferential sensitivity ofperichiasmate regions in diplotene, the stage closest topachytene. The sensitivity of what appears to be therecombination zone of the XY bivalent even at meta-phase I goes to strengthen the suggestion that DNase Iidentifies recombinogenic chromatin for creating nicks.

We are extremely grateful to Professor Karl Sperling andDr R. D. Wegner, both from the Institute of HumanGenetics, Berlin (West), for demonstrating the method ofDNase I nick translation and donating most of the chemicals.Thanks are also due to Dr Mercy J. Raman for criticism andsuggestions.

Funds for the work were provided by a DST-sponsoredgrant and a DAE-sponsored block grant to R.R.; A.P.S. is aJunior Research Fellow in the Special Assistance Programmeto the Department of Zoology, BHU.

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(Received 11 January 1988 - Accepted 19 April 1988)

634 R. Raman et al.