Studies of meiosis in vitro

DEVELOPMENTAL

BIOLOGY

6, 54-77

Studies

of Meiosis

II. Effect of Inhibiting Prophase MICHIO Department

( 1967 )

DNA

on Chromosome ITO,’

in Vitro

Synthesis Structure

YASUO HOTTA,

during

and Behavior1

AND HERBERT

of Biology, University of California, La Jolla, California 92037 Accepted

December

Meiotic

STERN

San Diego,

8, 1966

INTRODUCTION

The purpose of this paper is to examine the significance of DNA synthesis during meiotic prophase. The evidence that a discrete and distinctive interval of DNA synthesis occurs during the zygotene and pachytene stages of meiosis was presented in an earlier communication (Hotta et al., 1966). Meiosis is thus associated with two intervals of DNA synthesis, one occurring during the conventional Sphase, and the other occurring during the prophase period. The question now being raised is whether the small amount of DNA synthesis detected during meiotic prophase (about O&0.4% of the total DNA) has a functional significance in meiosis. Although a sig@cance may be rationalized by invoking a repair of DNA breaks does not constitute formed during crossing-over, such rationalization proof. Moreover, by limiting the significance of prophase DNA synthesis to crossing-over, we would be prejudging a phenomenon about which we have little information. In attempting to answer this question we have taken advantage of our success in culturing meiotic cells (Ito and Stern, 1967). We have added inhibitors of DNA synthesis to cells at different stages of meiosis and observed the cytological consequences. At the time of explantation, most cells are at the same stage of meiosis, and since the 1 This work was supported by a grant from the National Science Foundation ( GB-3902 ) . 2 Present address: Department of Biology, Nagoya University, Nagoya, Japan. 54

MEIOTIC

DNA

55

SYNTHESIS

cells require several days to progress through the stages of greatest interest to us, zygonema and pachynema, the relationship between DNA inhibition and meiotic development can be readily examined. METHODS

Two varieties of lily have been used in these studies: Lilium longi@rum var. Nellie White and a hybrid, var. Cinnabar. All the quantitative data presented are taken from experiments with Nellie White microsporocytes. The methods used for culturing meiotic cells were described in the preceding paper (Ito and Stern, 1967). Chemical procedures have been outlined elsewhere (Hotta et a!., 1966). RESULTS

Chemical Data on DNA Synthesis and Its Inhibition Prophase

during

Meiotic

The course of DNA synthesis during the meiotic cycle has already been described (Hotta et aZ., 1966). The characteristics of this course which are immediately pertinent to the data presented in this communication may be summarized thus: ( 1) Approximately 99.5% of the DNA in meiotic cells is replicated during the S-phase preceding leptonema. (2) No significant amounts of DNA synthesis have been found to occur between the termination of the S-phase and late leptonema. The duration of this interval varies with different species. In some cases, it may be extremely short. (3) Beginning in zygonema and extending to the termination of pachynema, DNA of distinctive composition is synthesized. (4) No sign&ant amount of DNA synthesis has been observed following the pachytene stage. Selective inhibition of DNA synthesis. Several kinds of inhibitors were tested for their effects on DNA synthesis during meiotic prophase. Of the ones listed here (Table 1) , the most effective on a molar basis was mitomycin C, and the least effective was S-fluorodeoxyuridine. The ineffectiveness of fluorodeoxyuridine at the concentrations tested may reflect the presence of small thymidine pools. This possibility was not investigated. Deoxyadenosine was chosen as the most desirable agent because its action could be reversed by other does inhibit RNA synthesis to a deoxyribosides. Deoxyadenosine greater extent than do the other agents. This effect, however, was not a serious obstacle to the interpretation of its action since com-

56

ITO,

HOTTA,

parisons could be made with selectively. General Cytological Prophuse

AND

STERN

agents which

Eflect of Znhibiting

affected RNA synthesis

DNA Synthesis during

The functional significance of prophase synthesis was first tested by comparing the chemical and cytological effects of the inhibitors listed in Table 1. In the range of concentrations tested, mitomycin C and deoxyadenosine profoundly affected the cytological picture, whereas fluorodeoxyuridine had only slight effects. On the assumption TABLE EFFECT

OF VARIOUS Agent

AGENTS

ON DNA Concentration

Control

-

Deoxyadenosine

bFluorodeoxyuridine Mitomycin

C

4 2 5 1 4 2 1 2 4

x x x x x x x x x

1

SYNTHESIS

10-4 M lo-3M 1O-3 M 10-e M 1O-4 M 1O-3 M 10-b M lo-5M lo-5M

DURING

W-DNA (cpm/lrg)

1920 1716 993 783 172 1955 1712 1685 489 20

MEIOTIC

PROPHASE~

‘% Inhibition (DNA)

yO Inhibition (RNA)

-

-

11 46 61 91 0 11 15 76 99

24 22 11 7 5 6

a A mixture of microsporocytes (Lilium ZongiJEorum var. Nellie White) at eygonema and pachynema was cultured for 3 days in basic medium in the presence of a2P-phosphate and of the various agents indicated in the table. For comparison, some data on the effect of these same agents on RNA synthesis are included. All the procedures used in these experiments for analyzing the DNA have been described elsewhere (Hotta et al., 1966). The inhibitory action of deoxyadenosine on RNA synthesis may be a secondary effect. It will be shown later that deoxyadenosine affects the rate of meiotic development,, and, since RNA synthesis increases during pachynema, the observed decrease in such synthesis could result from a failure of cells to progress through pachynema at a normal rate. Note that a 50-fold increase in concentration of deoxyadenosine does not increase its inhibitory action on RNA.

that deoxyodenosine and mitomycin C were effective because of their inhibitory properties, the conclusion could be drawn that DNA synthesis during zygonema-pachynema had some functional role in meiosis. This conclusion was strengthened by comparing the effectiveness of DNA-inhibitory agents with other agents which affected protein or RNA synthesis. The details of these studies, which are

MEIOTIC

DNA

SYNTHESIS

57

still incomplete, will not be reported here except for one general observation (L. G. Parchman, unpublished). The kinds of cytological effects produced by inhibitors of RNA or protein synthesis were different from those produced by deoxyadenosine or mitomycin C. Exposure of meiotic cells to deoxyadenosine had three principal effects: arrest in the zygotene stage; fragmentation of chromosomes, or chromosome stickiness. The particular type of effect depended to some extent upon the stage of meiosis at which the cells were exposed. No effects were observed in cells which were exposed to deoxyadenosine after pachynema. By contrast, inhibitors of protein or RNA synthesis were effective at all meiotic stages up to and including part of diakinesis. Various abnormalities were produced by these Chromosome segregation was almost always affected; inhibitors. chromosome morphology was usually affected, and frequently, chromosomes would become beaded after 6-7 days in culture. In general, inhibitors of RNA or protein synthesis did not mimic inhibitors of DNA synthesis with respect to the pattern of cytological abnormalities produced. Efectiveness of Deoxyadenosine ities as a Function of Meiotic

in Causing Chromosomal Stage

Abnormal-

The type of abnormality induced by deoxyadenosine is a function of the concentration used and of the meiotic stage at which cells are exposed (Table 2, and Fig. 1). Cells in early zygonema are totally arrested in their development if exposed to 0.01 M deoxyadenosine. At 0.004 M, only 60% of the cells are thus arrested. How far cells can progress through zygonema and still respond in this way is difficult to determine precisely. Neither bud length nor cytological appearance is sufficiently sensitive or exact as an indicator of development to permit accurate correlations with short intervals of a single cytological stage. The probability that only those cells which are in very early zygonema can be arrested by deoxyadenosine is suggested by the data in Table 2. In groups of microsporocytes obtained from several buds of the same length, the responses are much more uniform among cells obtained from the same bud than between cells from different buds. Thus, among the 120 filaments of microsporocytes explanted from five 15-mm buds, 72 showed total arrest, and these were obtained from three of the buds. Microsporocytes from the other two behaved like those from some of the 16-mm buds. We attribute the sharp dif-

TABLE

2

-

-

-

TA

TA TA

TA

6 100

10+ 100 100 100

0

7

3 100

-

0 TA

0

96

T-4* 100

15 (sygonema) l-3 4 5

lo+ 88

lo+ 83

28 100

0 101 100

46 22

30

lo+ 72

96 98

6

88 18

4

16 (sygonema) 3 4

7

32 19

63

2

7

0

100

-

-

-

100

1

IOf 21

63 43

3

99 6

0

5

10+ 40

42 98

95 100 lo+ 74

3

80 11

2

10+ 26

66 49

2

70 3

0

10+ 11

37 54

4

47 7

0

17 (pachynema) 2 3 4

4

51 14

16

1

10+ 8

81 11

0

63 0

0

5

lo+ 26

87 23

2

29 6

0

1

10+ 13

42 20

2

41 5

0

lo+ 7

96 12

---

78 0

0

10+ 5

77 4

36 0

0

18 (pachynema) 3 4 2

lo+ 3

76 3

31 0

0

5

lo+ 4

123 2

2

150 2

0

19 Y~S3llE3) bachy1-5

INTERVALS OF MEIOTIC: PROPHASE’

QMicrosporocytes of Lilium ZoneifEorum(Nellie White) were explanted at the stages shown into basic medium containing 0.004 M deoxyadenosine. Five buds of equal length were selected for each m&tic stage tested. The filaments of microsporocytes obtained from each bud (abont 24) were cultured in separate flasks (designated as “Culture No.“), and samples were removed periodically for cytological analysis. The incidence of abnormalities wss measuredin prophase, metaphase I, anaphase II, and tetrad cells. The higher frequency of abnormalities in anaphase II cells than in tetrads is unexpected, but the difference is apparent rather thsn real becauseof the method of scoring. Tetrads were counted ss normal if 4 nuclei of approximately equal size were present in the aggregate. Thus, if the chromosome fragments did not form micronuclei and if the distribution of chromosomal material among the daughter nuclei w&s approximately equal, the product would appear to be normal and would be counted 8s such. Note that 3 of the 5 cult.uresprepared from X-mm buds showed total arrest. This is discussed in the text. For any particular abnormality, percentage is oaloulsted from the ratio of affected cells over total number of cells at the stage in question. In control cultures, abnormalities occurred with s frequency of the order of 5%. Values in the table which are 5% or less msy be regarded as indicating no effect of treatment. b TA. total arrest.

Abnormal tetrsds (70)

Prophase fragmentation (%) Metaphase I No. observed No. with broken chromatids (‘%) No. of breaks/ cell Anaphase II No. observed No. with broken ohromatids (To) No. of breaks/cell

Bud length Cuk2No . .

CHROMOSOME FRAGMENTATION INDUCED BY DEOXYADENOSINE AT DIFFERENT

2

5

g 1 “i

=i “0

MEIOTIC

DNA

59

SYNTHESIS

ference in response of microsporocytes obtained from buds of the same length to a difference in developmental stage. Since the flower bud elongates from 15 to 17 mm while the microsporocytes progress through zygonema, and since some of the 15 mm but none of the 16 mm buds yield microsporocytes which are arrested by 0.004 M deoxyadenosine, we conclude that such arrest is confined to early zygonema. Since DNA synthesis begins in early zygonema (Hotta et al., 1966), it is probable that the arrest results from a suppression of the initiation of DNA synthesis. Cells which have progressed beyond early zygonema cannot be halted in their meiotic development even in the presence of 0.01 M ARREST

; 2 t a

60 40

PROPHASE FRAGMENT. CHROMATID BREAKS NORMAL

20

FIG. 1. Sensitivity of microsporocytes at different meiotic stages to the presence of deoxyadenosine in the culture medium. Each culture flask contained 15 microsporocyte filaments in a medium which was either 0.002 or 0.004 M with respect to deoxyadenosine. The cells were cultured for 4-7 days prior to examination. The histogram shows the frequency distribution of various meiotic abnormalities in relation to the stage at which the cells were explanted. The left member of each pair of bars indicates the results for the lower concentration of inhibitor. In the actual experiment, microsporocytes were also tested for their response to the following concentrations of deoxyadenosine: 8 x 10-j M; 4 x lo-’ M; and 0.01 M (see Fig. 5). Unlike the procedure used in Table 2, the frequency of abnormalities was calculated in terms of the aggregate properties of each filament of cells (Ito and Stern, 1967). Those in which no meiotic development was evident were classified as “arrested”; those in which more than 50% of the cells showed prophase fragmentation were counted as fragmented; those in which more than 5% of the anaphase II cells showed chromatid breaks were grouped under chromosome breakage, whereas those with less than 5% (the frequency in control preparations) were classified as normal. The percentages in the histogram represent the frequency of a particular type of filament over the total number of filaments. For data on individual cell counts see Table 2. This figure is intended to provide a simplified picture of the general trend of deoxyadenosine action during meiotic development.

60

ITO,

HOTTA,

AND

STERN

deoxyadenosine without manifesting visible chromosomal abnormalities. The characteristic response of cells in mid-zygonema is extensive chromosome fragmentation. In microsporocytes of Nellie White, and less so in Cinnabar, the fragmentation is usually too extreme to permit any meaningful stage identification after several days of culture. Most of the cells appear to be in some stage of prophase, and we therefore refer to this effect as “prophase fragmentation” (Fig. 3). The important conclusion which may be drawn from these observations is that in cells exposed to deoxyadenosine during the interval following the initiation of DNA synthesis and ending prior to completion of zygonema, chromosome structure becomes so altered as to make progress beyond prophase impossible. Cells exposed to deoxyadenosine in late zygonema or early pachynema, stages which we have found difficult to distinguish cytologically, show relatively few shattered prophase chromosomes. Most cells progress through metaphase I and later stages although the chromosomes show various abnormalities. The absence of any prominent effect by deoxyadenosine on chromosome segregation is noteworthy. As discussed earlier, inhibitors of RNA and protein synthesis usually have marked effects on chromosome segregation ( Parchman, unpublished). Cells exposed to deoxyadenosine from mid-pachynema on show a decreasing susceptibility to the action of the inhibitor. By late pachynema, the cells are almost entirely unaffected. Since prophase DNA synthesis terminates at the end of pachynema (Hotta et al., 1966), the ineffectiveness of deoxyadenosine at this stage may be attributed to the completion of DNA synthesis. An alternative interpretation is that the cells become impermeable to deoxyadenosine, but we consider this alternative to be most unlikely. A variety of amino acid analogs and inhibitors of protein or RNA synthesis produce abnormalities if administered in late pachynema or diplonema ( Hotta and Stem, 1963, and unpublished observations of Parchman ) . Monosaccharides remain toxic to cells at these stages (Ito and Stern, 1967). Labeled precursors of RNA or protein can be shown to. penetrate the cells. We consider these observations to be sufficient proof that low molecular weight solutes can penetrate the cells at the conclusion of the pachytene stage. The set of experiments described in this section serves to demonstrate that the effects of deoxyadenosine on meiotic development

MEIOTIC

DNA

61

SYNTHESIS

depend upon the stage at which the cells are first exposed. Cells at early zygonema which are explanted into a deoxyadenosine-containing medium, do not develop further. Although the nuclei of affected cells show no gross cytological abnormalities, the cells die after 4-5 days in culture. In cells exposed to deoxyadenosine during midzygonema, the chromosomes eventually fragment and do not progress beyond prophase. Cells treated in late zygonema or early pachynema progress through anaphase I with relatively few chromosome breaks but with frequent cases of chromosome stickiness. A very prominent effect is the appearance of breaks at the anaphase II stage. In general, the variety of chromosomal abnormalities is greatest in cells exposed during these two adjacent stages. As cells progress beyond early pachynema, they show a decreasing susceptibility to the action of deoxyadenosine. Once they reach the end of pachynema, they are no longer affected in their development by the presence of deoxyadenosine. The results thus clearly show that deoxyadenosine is effective in producing abnormalities during that interval of meiosis when DNA synthesis is occurring. They also show that cells become less susceptible to the action of deoxyadenosine during the later stages of pachynema. This decrease in susceptibility could be explained by the fact that the cells have already synthesized most of the DNA, thereby reducing the possibilities of damage. This explanation, however, does not take into account the equally significant fact that the chromosomes are progressively undergoing structural changes during zygonema-pachynema. What needs to be explored is whether the structural changes, themselves render the chromosomes more or less sensitive to deoxyadenosine action. We have no direct evidence for such a relationship, but a more detailed examination of the cytological abnormalities does provide strong circumstantial evidence in its favor. The Nature adenosine

of the Cytological

Abnormalities

Induced

by Deoxy-

Zygonema arrest. The cytological features of such arrest have already been described. The one point to be emphasized is that this type of response to deoxyadenosine is unique to cells in early zygonema. Why cells do not recover from this effect is unclear. Deoxyadenosine has no general toxic effect on the cells, for they continue to elaborate cell wall as long as they survive. The only explanation

62

ITO,

HOYTA,

AND

STERN

which can be offered is that early zygonema represents a critical stage in meiotic development and that if essential events are suppressed the cells ultimately lose the capacity to coordinate their metabolism. Whatever these events might be, they are not evident through the light microscope. If structural changes are involved, these would have to be sought at a higher level of resolution. Studies with the electron microscope do indicate that inhibition of DNA synthesis in early zygonema prevents the formation of the synaptinemal complex (Roth and Ito, 1966). If so, the essential event might be chromosome synapsis, but this alone cannot explain the eventual loss of viability. Asynaptic mutants are known in several organisms, but the gametes remain viable even though chromosome segregation is abnormal. We can only conclude that early zygonema is a physiologically distinctive stage of the meiotic cycle. Most probably, this distinctiveness is related to the initiation of synapsis, but the experiments thus far performed reveal little about the nature of the relationship. Chromosome fragmentation. An adequate description of the meiotic abnormalities induced by deoxyadenosine would be that they fall into one of two classes, zygonema arrest or chromosome damage. For in the total picture of deoxyadenosine effects on cells beyond early zygonema, the outstanding feature is the damage suffered by chromosomes. There can be no question that the chromosomes in cells from mid-zygonema to mid-pachynema are extremely susceptible to damage. How significant each particular type of damage is to deoxyadenosine action will now be considered, but we may anticipate our conclusion by stating that the particular cytological effects are secondary consequences of this action. The main reason for considering the two types of chromosome damage, breakage and stickiness, separately is the convenience of analyzing the factors which appear to be responsible for their occurrence. Broken chromosomes are evident at virtually all meiotic stages, but it is probably significant that, except for the few cells which reach metaphase I after explantation at mid-zygonema, the highest frequencies of breakage are seen either in prophase or anaphase II chromosomes (Table 2, Figs. 2 and 3). The two characteristics which appear to be important in interpreting the mechanisms of breakage are the time interval required for the manifestation of breakage and the cytological appearance of the breaks.

MEIOTIC

DNA

FIG. 2.

SYNTHESIS

63

64

11-0,

HOTTA,

AND

STEHN

FIG. 3. Types of chromosome damage induced by 0.004 M deoxyadenosine in microsporocytes of Nellie White. (A) Prophase fragmentation. (B) Sticky anaphase I. (C) Sticky anaphase II. Note also the chromosome fragmentation. (D ) Breaks in diakinesis chromosomes. As discussed in the text, such breaks are relatively rare. (E) Breaks in metaphase I chromosomes. Note the chromatid breaks. (F) Fragmentation of anaphase I chromosomes. (G) Fragmentation of anaphase II chromosomes. As discussed in the text, such fragmentation is the common event in cells which have been exposed to deoxyadenosine from late zygonema or early pachynema. (H) L at e anaphase II chromosomes showing some fragmentation. (I) Telophase with some chromosome fragments. Magnification: Approximately X 640. have originated during pachynema, for breaks which were exposed to deoxyadenosine during Less obvious but equally definite is the fact phase chromosomes is also a delayed effect of

are rare even in cells late pachynema. that breakage of prodeoxyadenosine. Very

MEIOTIC

DNA

SYNTHESIS

65

Breakage is a delayed and not an immediate effect of deoxyadenosine action. Several lines of evidence point to this conclusion. Breakage of anaphase II chromosomes is the most obvious example of a delayed effect. In order to damage anaphase II chromosomes, cells must be exposed to deoxyadenosine no later than pachynema. When cells treated in mid-pachynema reach metaphase I, occasional chromatid or chromosome breaks may be seen (Table 2)) but when the cells reach anaphase II, the percentage of those with breaks is at least ten times greater than the percentage of metaphase I cells which show broken chromosomes or chromatids. It is evident from the photographs in Fig. 3 that the breaks do not result from a tearing of the chromosomes at anaphase separation. The appearance of breaks is delayed either because of the time required by other processes to effect them, or because of some change in the internal organization of the chromosomes during second division which renders them particularly susceptible to breakage. The cytological events which occur between metaphase I and anaphase II do not point to any particular change in the chromosomes which might account for this higher susceptibility. Except for the centromeric region the chromatids are already separate at anaphase I, and it is difficult to see how centromere division would directly affect internal organization elsewhere in the chromosome. However, the important point is that the susceptibility of the chromatids to breakage during second division must

FIG. 2. Effects of deoxyadenosine on cultured meiotic cells. (A) and (B) Microsporocytes of Nellie White explanted at early zygonema into a basic medium containing 0.004 M deoxyadenosine. The cells were fixed after 5 days of culture. Various degrees of prophase fragmentation are evident. Two of the cells (B) also show some chromosome stickiness. Approximate magnification: x 500. (C) Microsporocytes of Cinnabar explanted into a deoxyadenosine medium at early mid-zygonema. Prophase fragmentation and sticky anaphase chromosomes are

evident. Magnification: X300. (D) Nellie White microsporocytes showing anaphase II fragmentation after being exposed to deoxyadenosine from early pachynema. Magnification: x 300. (E) Fragmentation of Nellie White chromosomes during second division. An anaphase bridge may be seen. The frequency of such bridges may be increased considerably by exposing late zygonema or early pachynema cells to deoxyadenosine for 3 days and then transferring them to a thymidine medium. This phenomenon is discussed in the text. Magnification: x300. (F) Nellie White microsporocytes (second division) cultured in the presence of a mixture of deoxyadenosine and adenosine. The failure of adenosine to antagonize the action of deoxyadenosine is discussed in the text. Magnification: X 500.

66

ITO,

HOlTA,

AND

STERN

few breaks are seen in cells after 1 day or even 2 days of exposure to the agent. The number of breaks increases with time after the initial 2-day period. This pattern of behavior is implicit in the curves shown in Fig. 7, which summarize studies, to be discussed later, on antagonists to deoxyadenosine action. The curves of immediate interest are those which show that cells exposed to deoxyadenosine for 2 days and then returned to a deoxyadenosine-free medium develop no significant amount of chromosome breaks. Thus, even if lesions are produced soon after exposure, they are subject to repair. Whether or not repairs do occur, the important fact is that the breakages observed cytologically are delayed products of ADR action. A second characteristic of breakage which is important to an identification of mechanisms is the cytological appearance of the chromosomes in the regions where breaks are apparent. Frequently, thin strands may be seen to connect apparently detached pieces. This, of course, is not always so but the experiments provide no basis for separately classifying real and apparent breaks according to origin. Most probably, actual breaks result from the apparent ones. The feature common to both types of breaks is the presence of morphologically abnormal regions at various points along the length of the chromosome. The major conclusion which can be drawn from observing chromosome fragmentation is that deoxyadenosine induces lesions in the chromosome which eventually develop as structurally deficient regions. Chromosome stickiness. This abnormality is defined as an adhesion between chromosomes or chromatids. Occasionally, the adhesion may be evident in a clumping of chromosomes at metaphase; usually, it is evident in anaphase bridges (Figs. 2 and 3). As pointed out earlier, the relative frequencies with which breakage and stickiness occur are different in Nellie White and Cinnabar microsporocytes. Stickiness obviously results from some type of abnormal cross-linking between otherwise separate chromosomes or chromatids, so that the processes which act on the lesions produced by deoxyadenosine must be different from those which lead to breaks. We have no evidence for two distinct types of primary lesions which would lead to correspondingly distinctive types of abnormalities. Conceivably, the stage of meiosis at which deoxyadenosine acts might be a factor in determining the eventual response, but if a relationship does exist, it is unlikely to be a simple one because the two varieties of lily microsporocytes

MEIOTIC

DNA

SYNTHESIS

67

have different response patterns. The evidence which we do have clearly indicates that breakage and stickiness are not mutually exclusive events even within a single cell. In many cells in which sticky chromosomes are prominent, fragments may be seen and, conversely, cells with fragmented chromosomes also show some stickiness (Figs. 24). The conclusion we therefore draw is that whatever the combination of factors leading to a particular cytological abnormality, the primary consequence of inhibiting DNA synthesis by deoxyadenosine is in a disruption of the normal organization of the chromosome. Abnormalities in relation to deoxyadenosine concentration. The difficulty in assigning distinctive primary lesions to each type of abnormality is made even more apparent on comparing the effects of different concentrations of deoxyadenosine (Fig. 5). As would be expected, the total number of abnormalities appearing at the end of the culture period rises as the concentration of deoxyadenosine is increased (Fig. 5A). Also, the percentage of cells showing a particular type of abnormality increases with increase in deoxyadenosine concentration (Fig. 5B). If, however, a particular abnormality were produced by deoxyadenosine at only one particular meiotic stage, one would expect the concentration of deoxyadenosine to affect the frequency of that particular abnormality, but not to affect the kinds of abnormalities. This is not the case. Early pachynema cells treated with different concentrations of deoxyadenosine (Fig. 5C ) show differences in the relative frequencies of abnormalities. Prophase fragmentation, for example, is not detected in cells treated with 0.002 M deoxyadenosine, but occurs in 50% of the prophase cells which had been treated with 0.008 M deoxyadenosine. Results such as these suggest that the kind of abnormality produced depends at least in part upon the degree to which DNA synthesis has been inhibited. Apparently, DNA synthesis must be inhibited to a high degree in late zygonema-early pachynema cells in order to produce prophase fragmentation. The observed relationship between deoxyadenosine concentration and abnormality indicates that the primary lesions produced are cumulative in their effect. Additional evidence in favor of this conclusion is to be found in the frequencies of anaphase II breaks produced by different concentrations of deoxyadenosine at different meiotic stages (Fig. 5B). For any particular concentration of deoxy-

68

ITO,

HOI-I-A,

AND

STERN

FIG. 4. Reversal of deoxyadenosine action by thymidine. All cells were exposed to deoxyadenosine from mid-zygonema. (A) Cells cultured in an equimolar mixture of deoxyadenosine and thymidine for 5 days. Comparatively few chromosome breaks can be seen. Some aberrant segregation is however evident. It is attributed to an interference with centromere function, but the phenomenon is not yet well understood. ( B ), ( C), and ( D) : Cells were cultured for 3 days in the presence of 0.004 M deoxyadenosine and then transferred to a medium containing 0.004M thymidine for 2 days. The chromosomes show very few breaks. Magnifications: (A) and (B) x300; (C) and (D) x640.

MEIOTIC

DNA

69

SYNTHESIS

adenosine, the earlier the stage of treatment, the higher the frequency of anaphase II breaks. Such a comparison is meaningful only for low concentrations of inhibitor, since the higher the concentration the fewer the cells treated at the younger stages which reach anaphase II. Nevertheless, the fact that those cells treated in mid-zygonema with 0.001 M deoxyadenosine which reach anaphase II have about 3 times the frequency of breaks as those treated in early pachynema with the same concentration, rules out the probability that anaphase A Total

B An&II

C BI leaks

Types

of Abnormalities

60 5Y

60 40

DEOXYADENOSINE

CONCENTRATION

(mM1

FIG. 5. Relationship of type and frequency of abnormalities to concentration of deoxyadenosine. Microsporocytes of Nellie White were cultured in basic medium supplemented with the indicated concentrations of deoxyadenosine. Numbers adjacent to curves indicate bud lengths at which the microsporocytes were explanted. (A) Total abnormalities include all abnormalities irrespective of origin. Cells explanted in early zygonema, unless grown in media supplemented with amino acids, show a high proportion of cells with erratic chromosome segregation (Ito and Stern, 1967). Thus, cells from the 15-mm bud show 60% of the meiotic products to be abnormal even in the absence of deoxyadenosine. (B ) The frequency of breaks in anapase II chromosomes relative to the total number of anaphase II figures. (C) The proportions of different abnormalities (anaphase II breaks, metaphase I breaks, prophase fragmentation, and abnormal tetrads) in microsporocytes explanted from a 17-mm bud. Each abnormality is expressed as a percentage of the total number of cells at the stage in question. At least 100 cells were counted for any particular stage in each preparation.

II fragmentation is effected only during pachynema. The main difference between cells treated in mid-zygonema and those treated in early pachynema is in the failure of most of the younger cells to proceed to anaphase II. A simple relationship has thus not been found between stage of treatment and abnormality produced. The frequency of anaphase II

70

ITO,

HOT-l-A,

AND

STERN

breaks would appear to depend upon the number of primary lesions produced in the course of exposure to deoxyadenosine. Prophase fragmentation, on the other hand, occurs as though it were dependent upon a threshold number of lesions. A doubling of deoxyadenosine concentration (from 4 to 8 mM) results in a tenfold increase in the number of prophase cells affected (from 5 to 50%). However, the frequency of metaphase I breaks is only slightly affected. For reasons which must lie either in the internal organization of chromosomes or in some metabolic parameter of the cells, chromosomes break either during meiotic prophase or during second division. Physiological

Aspects of Deoxyadenosine

Action

In view of the fact that the cytological abnormalities are a delayed response to deoxyadenosine treatment, the question arises whether the primary lesions produced by deoxyadenosine are irreversible or whether they are subject to repair. Bearing on this question is the observation that deoxyadenosine invariably prolongs the prophase stage (Fig. 6). The rate of meiosis is unaffected in cells exposed to deoxyadenosine at stages later than mid-pachynema. Cells which have nearly or entirely completed DNA synthesis are neither susceptible to

Di Path.

w--o

f 116, , 012345676

,

,

,

control +ADR ,

,

,

J

9 DAYS

The effect of 0.004 M deoxyadenosineon the meiotic rate of cultured White). The vertical bars indicating the spread of stages have been omitted from the controls explanted at 16 and 17 mm to facilitate reading of the curves. The microsporocytes were explanted on day 0 at the stages indicated. The main result of the experiments is the demonstration that deoxyadenosine prolongs prophase but not later stages. FIG. 6.

microsporocytes (Nellie

hlEIOTIC

DKA

SYNTHESIS

71

chromosome damage nor slowed down in meiotic development. Those which are undergoing DNA synthesis are clearly slowed down in their progress through prophase, but not through subsequent stages. Cells exposed to deoxyadenosine at mid-zygonema reach diakinesis approximately 1% days later than the controls, but both treated and untreated cultures progress from diakinesis to the tetrad stage in about 3X days. These results again point to a unique relationship between DNA synthesis and prophase development. To examine the possibility of reversing the presumed primary lesions produced by deoxyadenosine, various nucleosidic compounds were tested for their antagonistic effects on deoxyadenosine action (Table 3). Several of these have been reported to reverse the inhibition by deoxyadenosine of DNA synthesis (Munch-Petersen, 1960; Nichols et al., 1964). At the concentrations tested, none of the ribosidic compounds tested had a sufficiently pronounced effect on deoxyadenosine action (Table 3). Guanosine deoxyriboside had an appreciable antagonistic effect, but thymidine was the most effective antagonist (Fig. 4). Deoxycytidine was unique inasmuch as it completely reversed the effects of deoxyadenosine on prophase fragmentation but also produced a distinctive type abnormality, a clumping of chromosomes at the metaphase stage. This effect occurred regardless of whether the cells were exposed to deoxycytidine alone or to a combination of deoxycytidine and deoxyadenosine. We have not made any further studies of the deoxycytidine phenomenon. For the purpose of examining the possibility of repair, the use of thymidine appeared to be sufficient. To determine whether the primary lesions induced by deoxyadenosine could be repaired, meiotic cells were first exposed to deoxyadenosine and then, after different intervals of time, were transferred either to basic medium or to basic medium supplemented with thymidine (Fig. 7). The tests were made on cells which were explanted either in mid-zygonema or in late zygonema-early pachynema. The first group, which characteristically showed a high degree of prophase fragmentation was cultured for 6 days. The second group which characteristically showed a high frequency of anaphase II breakages was cultured for a total of 5 days. Three principal observations were made in these experiments. 1. Mid-zygonema cells (16-mm buds) cultured for up to 3 days in the presence of deoxyadenosine and then transferred to basic medium

72

ITO,

HO’ITA,

AND

STERN

with or without thymidine showed little or no prophase fragmentation. This result might suggest that deoxyadenosine does not act on the chromosomes during the first 3 days and that the transfer removes the deoxyadenosine before it effects lesions. The explanation, howTABLE REVERSAL Series

1

2

3

4

OF DEOXYADENOSINE

components ADR + + ADR + + ADR + + CDR GDR TDR

EFFECTS COW.

3 BY VARIOUS

NUCLEOSIDIC

COMPOUNDS~

(m.W

Pro.

MI

AI1

Tet.

4 4 4 4 4 4 4 4 8 4 4 4

12 6 8 13 0 1 5 0.5 0 0 0.3 0

9 5 6 7 -

84 68 67 66

1 5 0.3 0.3

34 56 21 7

0.3 0

2 3

62 50 53 56 100 41 42 32 16 100 2 4

Adenosine Cytidine CDR GDR TDR TDR

-

a The purpose of this experiment was to determine the usefulness of the compounds listed in reversing the effects of deoxyadenosine (ADR). All tests were performed with microsporocyte filaments extruded from 17-mm buds of Lilium ZongijZorum (Nellie White). The components listed were added to the basic medium in the concentrations shown. Each component or combination of components was tested in 4 culture flasks each containing 8 microsporocyte filaments. No less than 100 cells were counted from any single flask for each of the meiotic stages listed. All abnormalities-prophase fragmentation (Pro.), metaphase I breaks (MI), anaphase II breaks (AII), abnormal tetrads (!Z’et)-are expressed as a percentage of the total number of cells at the particular stage in question. CDR, GDR, and TDR represent deoxycytidine, deoxyguanosine, and thymidine, respectively. Numbers are not shown for the effects of deoxycytidine on metaphase and anaphase because this compound causes a clumping of the chromosomes at about metaphase I. The viability of cells thus affected is very low. Although each of the compounds showed some effect in reversing deoxyabenosine action, thymidine appeared to be the most suitable for the experiments described in the text. Higher concentrations of the other compounds were not tested.

ever, would be inconsistent with inferences which can be drawn from the cytological progress of the cells in deoxyadenosine media. Mid-zygonema cells cultured in the presence of deoxyadenosine reach early pachynema in 2% days and mid-pachynema in 3 days (Fig. 6). If deoxyadenosine were to begin acting when chromosomes reach

MEIOTIC

DNA

73

SYNTHESIS

mid-pachynema, the relative insensitivity of cells exposed at midpachynema would have to be explained by supposing that a 3-day lag in penetration of deoxyadenosine exists at all stages of meiosis. This is inconsistent with various observations. The rapid penetration of tritium-labeled solutes has already been discussed; cells may be labeled with thymidine, various ribosides or amino acids after 1 day of exposure. Even more pertinent, perhaps, is the evidence that mid-

2

0

DAYS IN ADR PRIOR TO TRANSFER FIG. 7. Reversibility of the effects of deoxyadenosine (ADR) on chromosome breakage. Microsporocyte filaments were extruded from Nellie White buds of the same length and ten filaments were placed in each flask which contained basic medium plus 0.004 M deoxyadenosine. After one or more days of culture, the medium was replaced with either basic medium or medium supplemented with 0.004 M thymidine ( TDR). Each point on the curves represents the analysis of the microsporocytes from a single flask. Solid circles refer to 16mm buds ( mid-zygonema) and open circles to 17-mm buds (early pachynema). The analysis of anaphase bridge frequencies was done on 17-mm buds. All frequencies are expressed as percentages of abnormal cells over the total number of cells in the stage analyzed. “0” days refer to cultures maintained throughout in basic medium. All cultures were maintained for at least 6 days, and filaments were removed periodically for cytological analysis.

zygonema cells are much retarded in their progress through prophase after being cultured for 3 days in the presence of deoxyadenosme (Fig. 6). We therefore interpret the low incidence of prophase fragmentation in cells which have been removed from deoxyadenosine after 3 days to repair mechanisms within the cells. This interpre-

74

ITO,

HOTTA,

AND

STERN

tation is consistent with the observation that transfer to a thymidine medium results in fewer abnormalities than transfer to a thymidinefree medium. 2. Very few anaphase II breaks are observed in cells which have been exposed to deoxyadenosine for one or two days. An appreciable reduction in the number of cells with anaphase II breaks occurs if transfer is made after 3 days of exposure to deoxyadenosine. It is significant, however, that the reduction in anaphase breaks is accompanied by an increase in anaphase bridges. Thymidine, which is more effective in reducing the frequency of breaks is correspondingly more effective in increasing the number of bridges. After 4 days of exposure to deoxyadenosine, only a slight reduction in the frequency of anaphase breaks occurs and very few anaphase bridges are formed. Since early-pachynema cells reach late pachynema in 3 days, it would appear that repair during late pachynema is abnormal and anaphase bridges result. 3. Mid-zygonema cells show a negligible reduction in abnormalities if transferred after 5 days of exposure to deoxyadenosine, and early pachynema cells behave similarly after 4 days of exposure. Both groups of cells have progressed through pachynema at these times. The conclusion may therefore be drawn that once the cells have passed pachynema, repair is no longer possible. This behavior correlates with the chemical evidence that prophase DNA synthesis terminated at the end of pachynema (Hotta et al., 1966). DISCUSSION

The cytological studies reported here together with the chemical studies reported elsewhere (Hotta et al., 1966) not only establish the actuality of DNA synthesis during meiotic prophase, but also demonstrate the importance of such synthesis to meiotic development. The principal question arising from these studies is the specific nature of the role which DNA synthesis plays in meiosis. The effects of deoxyadenosine on meiotic cells are clearly circumscribed. Chromosome contraction, anaphase separation, and cell wall synthesis are only indirectly affected. On the other hand, synapsis and something we may loosely call “internal chromosome organization” are profoundly affected. The assumption is made that these two effects result from a partial or total inhibition of DNA synthesis. The

MEIOTIC

DNA

SYNTHESIS

75

circumstantial evidence in support of this assumption is considerable. Inhibition of prophase DNA synthesis by deoxyadenosine has been demonstrated chemically; abnormalities are induced only during the interval of DNA synthesis; other deoxyribosides can reverse the effects of deoxyadenosine if applied during the interval of DNA synthesis; inhibitors of RNA or protein synthesis do not mimic the action of deoxyadenosine. Virtually all the chromosomal abnormalities observed may be attributed to the presence of gaps along the DNA chain. Where gaps persist, abnormalities in structure would be expected to be present and these would ultimately lead to breaks. Where the free ends of DNA undergo abnormal repair, a cross-linking of sister or homologous strands would be expected to occur, and these would lead to various degrees of chromosome stickiness. One might also postulate an abnormal deposition of proteins in the region of DNA gaps which would lead to similar forms of stickiness. The critical question is the origin of the gaps rather than the processes that modify them. By itself, inhibition of DNA synthesis does not produce gaps. The gaps could arise either from special enzymes which induce breaks in association with crossing-over, or from regions in the chromosome which do not replicate until meiotic prophase. Inhibition of DNA synthesis would prevent repair of breaks or would leave the regions of delayed synthesis in the chromosome incompletely replicated (see also Nichols et al., 1964). Cytogenetic evidence would lead to the conclusion that gaps must arise in association with crossing-over. If so, the question reduces itself to whether all the abnormalities produced by deoxyadenosine are due to such gaps. This possibility is rejected for the following reasons: (1) Inhibition of synapsis in early zygonema by deoxyadenosine cannot be rationalized in terms of preexisting gaps. (2) Crossingover would be expected to begin after the chromosomes have paired can be induced ( i.e., in pachynema), but prophase fragmentation only in early to mid-zygonema. (3) The chemical evidence that zygonema is associated with the synthesis of a high SC DNA and pachynema with a synthesis of DNA similar in composition to the total DNA points to two types of replication ( Hotta et al., 1966). In view of these considerations, we favor the possibility that DNA synthesis during zygonema represents a delayed replication of certain chromosome regions, and that DNA synthesis during pachynema rep-

76

ITO,

HOITA,

AND

STERN

resents a repair of breaks associated with crossing-over. Initiation of DNA synthesis is a precondition for pairing; completion of DNA synthesis is necessary for the maintenance of chromosome integrity. TO give these conclusions a concrete meaning, some model of chromosome organization must be invoked. The model of choice can be described briefly. Its essential feature is the presence of an “axial element,” a feature which has been proposed by a number of people (Taylor, 1963). We wish to attach two specific properties to this hypothetical axis. First, the principal substance of this axis is DNA. Second, the axis is so situated in the structure of the chromosome that its duplication is essential to the ultimate formation of chromatids. A variety of detailed models could be proposed for axial organization, and most of them could probably be used to explain the phenomena observed. The axis could be no more than a set of “linkers” between genetically active segments of a DNA filament (Taylor, 1963), or it could be an uninterrupted axis subtending discrete DNA units. The only requirement we wish to impose is a functional one, namely, that axial replication is a distinctive condition for chromatid formation. Given this model we interpret our data to indicate that in meiotic cells, replication of the axial element is delayed until zygonema. This delay is considered to be essential for those events to occur which make possible the formation of the synaptinemal complex as pairing proceeds. We attribute prophase fragmentation to an incomplete replication of the axis during zygonema which makes certain regions of the chromosome susceptible to degradation or to abnormal repair. We attribute the abnormalities which arise in cells treated toward the end of zygonema or later, to a disturbance of normal repair mechanisms associated with crossing-over. The model and the scheme have no direct experimental proof. They are proposed because of the plausibility and because they focus on one important characteristic of the meiotic cell. This characteristic is the delayed synthesis of a small fraction of the total nuclear DNA which appears to be intimately related to the critical events occurring during the protracted prophase of meiosis. The delay is a distinguishing developmental feature between meiotic and mitotic cells. In mitosis, axial replication may occur in either late S-phase or in G-2. In meiosis, a regulatory mechanism exists which inhibits the replication from occurring until after prophase has begun. The essentials

MEIOTIC

DNA

SYNTHESIS

of this scheme are very much like those proposed by Darlington his “Precocity Theory of Meiosis” some thirty years ago ( 1937).

77 in

SUMMARY

Inhibitors of DNA synthesis interfere with the meiotic cycle if administered to cells during zygonema and/or pachynema. The cytological effects correlate with the chemical evidence for a synthesis of DNA during that same interval. The main effects of inhibiting DNL4 synthesis are either zygonema arrest or fragmentation of chromosomes. The type of abnormality depends upon the time at which inhibition is effected. Zygonema arrest occurs only if DNA synthesis is inhibited at the time of its initiation which is coincident with the initiation of pairing. Fragmentation of prophase chromosomes occurs only if DNA synthesis is inhibited during mid-zygonema. Inhibition of DNA synthesis during late zygonema or early pachynema does not interfere with the first meiotic division but results in chromosome fragmentation during the second division. The relevance of these observations to chromosome structure is discussed. REFERENCES DAHLINGTON, C. D. ( 1937). “Recent Advances in Cytology,” (2nd ed.). Blakiston, Philadelphia, Pennsylvania. HOTTA, Y., and STERN, H. (1963). Inhibition of protein syntheses during meiosis and its bearing on intracellular regulation. J. Cell Biol. 16, 259-279. HOTTA, Y., ITO, M., and STERN, H. (1966). Synthesis of DNA during meiosis. Proc. Natl. Acad. Sci. U.S. 56, 1184-1191. ITO, M., and STERN, H. (1967). Studies of meiosis in vitro. I. In vitro culture of meiotic cells. Develop. Bid. 15, 36-53. MUNCH-PETERSEN, A. ( 1960). Formation in vitro of deoxyadenosine triphosphate from deoxyadenosine in Ehrlich ascites cells. Biochem. Biophys. Res. Commun. 3, 392-396. NICHOLS, W. W., LEVAN, A., and KIHLMAN, B. A. ( 1964). Chromosome breakages associated with viruses and DNA inhibitors. In “Cytogenetics of Cells in Culture” (R. J. C. Harris, ed. ), pp. 255-271. Academic Press, New York. ROTH, T. F., and ITO, M. ( 1966). DNA synthesis during meiotic prophase in relation to pairing and disjunction. J. Cell Bid. 31, 96A. TAYLOR, J. H. ( 1963). The replication and organization of DNA in chromosomes. In “Molecular Genetics” (J. H. Taylor, ed.), Part I, pp. 65-112. Academic Press, New York.