an experimental analysis of bivalent interlocking …available (callan & taylor, 1968)...

J. Cell Set. 37, 125-141 (1979)Printed in Great Britain © Company of Biologists Limited 1979

AN EXPERIMENTAL ANALYSIS OF BIVALENT

INTERLOCKING IN SPERMATOCYTES OF

THE NEWT TRITURVS VULGARIS

H. G. CALLAN AND S. M. PEARCEDepartment of Zoology, University of St Andrews,St Andrews, Fife KYib gTS, Scotland

SUMMARY

Interlocked bivalents at ist meiotic metaphase are relatively uncommon in spermatocytesof the newt Triturus vttlgaris, but their frequency of occurrence can be significantly increasedby subjecting newts to a 24-h heat shock. Newt spermatocytes are sensitive to a heat shockat any stage between the end of premeiotic S and mid to late pachytene. The heat shock doesnot cause evident desynapsis, nor does it significantly affect chiasma frequency; therefore theinterlocked condition induced in spermatocytes which were subjected to a heat shock whenthey were in zygotene or pachytene is unlikely to be a consequence of synaptic trapping. Byway of explanation it is suggested that a heat shock may cause telomeres to detach from thenuclear membrane, or from the synaptonemal complex where the latter is attached to themembrane, thus allowing non-homologous chromonemata to become intertwined beforechiasmata have formed. If this explanation is valid, it is then further suggested that the re-combination process which results in chiasma formation probably takes place in chromosomalregions lying outside the synaptonemal complex, rather than inside, between its 2 lateralelements.

INTRODUCTION

When interlocked bivalents, in the simplest case 2 ring bivalents, are observed atist meiotic metaphase, it is generally assumed that this abnormal topological relation-ship came about because 2 pairs of homologous chromosomes trapped one anotherduring synapsis (see Fig. 1). If sufficient chiasmata in the appropriate places areestablished thereafter, the 2 bivalents remain interlocked until they are released atist meiotic anaphase.

There can be no doubt that the trapping of chromosomes during synapsis isresponsible for many of the recorded cases of interlocking; indeed, at the time ofthe telosynapsis/parasynapsis controversy in the early 1900s, reviewed by Wilson(1925), the observation by Gelei (1921, 1922) of interlocked bivalents at zygotenein oocytes of the planarian Dendrocoelum was used as a strong argument in favour ofparasynapsis. There have been several subsequent observations of a similar kind,by Belar (1928) in the snail Viviparus, by Levan (1933) in AUium, by Buss & Hender-son (1971a, b) in heat-shocked locusts, and by John (1976) in the grasshopperChloealtis. All the above are examples of interlocking observed at zygotene orpachytene.

Interlocks have much more frequently been recorded at diplotene or first meiotic9 CEL 37

126 H. G. Callan and S. M. Pearce

metaphase. Darlington (1937) has provided an extensive list of organisms in whichinterlocks have been reported, including Mather's (1933) famous example of complexinterlocking at diakinesis in Lilium regale, which was of crucial import at the time ofthe classical/chiasmatype controversy regarding the relationship between chiasmataand genetical crossing-over. With one exception, all such observations of interlockinghave been attributed to chromosome trapping during synapsis. The exception isprovided by Taylor (1949) who recorded an increase in the frequency of interlocked1st metaphase bivalents of Tradescantia paludosa brought about by excising andculturing anthers in an artificial medium. Taylor observed a marked increase in thefrequency of interlocked 1st metaphase bivalents even in anthers which had been

Fig. 1. Diagrams showing 2 pairs of homologous chromosomes at the beginning ofsynapsis. In urodeles, and many other animals which display a bouquet at zygotene,synapsis starts at both chromosome ends, and these are attached to the nuclearmembrane. The chromosomes are drawn as single lines, and their centromeres areindicated by round granules. In A no interlocking occurs. In B single chromosomesare interlocked. In C both of the smaller homologues are trapped within the largerpair.

excised after synapsis and part of pachytene had been completed on the plant. Taylorwas puzzled as to how this observation could be explained, though he suggested asa possibility that partial desynapsis might follow excision of an anther, and that if itwere followed by chromosome ends 'reuniting' at metaphase, interlocked bivalentscould result. The observations on newt male meiosis to be described in the presentpaper bear out Taylor's contention that bivalent interlocking can be generated inmeiocytes subjected to a heat shock as late as pachytene, i.e. several days aftersynapsis has been completed in normal fashion.

Ten years ago one of us (H.G. C.) gave 24-h heat shocks to males of Triturusvulgaris (zn = 24) with the hope that the precise stage at which chiasmata areestablished could be determined. Information from autoradiography was alreadyavailable (Callan & Taylor, 1968) concerning the durations of the 5-phase and the4 stages of meiotic prophase, and the experiment was conducted in the anticipationthat a just-sublethal heat shock, if applied during synapsis or at any time up to thestage at which chiasmata are formed, would lead to a significant diminution in chiasmafrequency, as Henderson (1966) had shown to be the case in male meiosis of the locust

Bivalent interlocking in Triturus 127

Schistocerca, whereas a heat shock applied after chiasmata had been establishedwould not be expected to affect chiasma frequency. In the event, the heat shockwas found to have no very dramatic, if indeed any, effect on chiasma frequency inthese newts and the fixed testes from animals which had experienced heat shockswere pooled with other material and used for class teaching. Some years later a fewof the stained preparations were examined again, and in certain of these an unusuallyhigh frequency of interlocked 1st metaphase bivalents was noticed. One such wasphotographed and used as an illustration for a paper by Callan & Perry (1977). As anoutcome of this casual observation it was decided that the heat shock experimentwould be worth repeating, but this time paying attention to what stages of meiosisare sensitive to heat shocks in the sense that they generate interlocked bivalents at1st meiotic metaphase.

First meiotic metaphases in spermatocytes of Triturus vulgaris are particularlyconvenient for scoring the interlocked condition, because the chromosomes arelarge, most of the chiasmata are formed near the ends of the bivalent arm pairs, thecentromeres are median or sub-median, and thus the majority of interlocked ringscan be identified without any ambiguity. It was assumed that in normal circumstancesinterlocking is a rare event; one of us (H.G.C.) had never previously noticed inter-locked bivalents in this species, or in the related Triturus helveticus, which also hasproterminal chiasma localization in male meiosis. It was also assumed that inter-locking would be observed only in those spermatocytes which had been subjectedto heat shocks up to, but not after, zygotene. Both these assumptions proved to bewrong.

MATERIALS AND METHODS

Most of the male specimens of Triturus vulgarit which were used for the heat shock experi-ments in 1968, and all of those used in 1977, were collected from ponds in Tentsmuir forest,northeast Fife, during the spring months, when the newts were aquatic and in full breedingcondition. They were placed in aquaria in a room held at a temperature of about 16 °C(minimum 15 °C, maximum 18 °C) where, as is usual with this species in captivity, thenewts soon lost their breeding condition (the first sign of which is a reduction in height ofthe dorsal crest) and started to climb out of the water. As the newts left the water they weretransferred, 5 at a time, to small plastic tanks (25 x 15 x 10 cm) each containing a wettedpaper tissue and a piece of undulating asbestos/cement roofing material under which thenewts took cover. The tanks were cleaned out once a week, and no attempt was made tofeed the newts once they had become terrestrial.

The capacity of Triturus vulgaris to survive a 24-h heat shock was tested in 1968, using awater-jacketed incubator. Plastic tanks each containing 4 newts were placed inside the incubator,the temperature of which was checked at frequent intervals and found to vary no more than±0-2 °C from the set temperatures. Of 8 newts placed at 33-5 °C, only one was still aliveafter 24 h and this one died a day later. Four newts placed at 30 °C all survived, ao did 4 newtsplaced at 32-5 °C, but of a further 4 placed at 33-0 °C one was dead 24 h later and the remaining3 in poor condition. As it was the intention to use a heat shock from which the newts wouldrecover fully (many were to be kept for a month at or about 16 °C after experiencing theshock) the decision was taken to use a temperature of 31-5 °C for 24 h. All of the experimentalanimals survived this treatment, and were healthy when killed and their testes fixed.

The intention at the outset was to give a 24-h heat shock, then make fixations of 3 newts'testea immediately thereafter ( + 0 days) and similarly of 3 newts' testes at daily intervals

9-2


until +20 days, then at 2-day intervals until +36 days. The 1968 fixations provided somepreparations falling within the period + 12 days to +20 days (see Table 1).

It is impossible to determine from a newt's external features whether its testes will containthe required stage of ist meiotic metaphase; this becomes apparent only after squash pre-parations have been made. Consequently some testes were fixed too early, some too late.Batches of newts were given 24-h heat shocks on 31st May, 2nd June, and 6th, 7th and 15thJuly, 1977, and as far as possible gaps appearing in the earlier fixations, where insufficient orno examples of ist meiotic metaphase were present in the preparations, were made good inlater fixations.

In a study of the time course of male meiosis in Triturus milgaris at 16 °C, carried out in1966 (Callan & Taylor, 1968), it was shown inter alia that spermatocytes take 20 to 21 daysto pass from the end of premeiotic S to ist meiotic metaphase. So as to keep a check on thetime course operating in 1977, when the temperature of the newt room was overall marginallylower owing to a difference in outside temperature and degree of insolation between the2 years, the newts just before being given a heat shock were anaesthetized with MS222 andeach injected with 10 /id of [sH]thymidine, 5 0 Ci/mM.

Before fixation the newts were anaesthetized with MS222, their testes removed and fixedin a freshly prepared mixture of 3 parts absolute ethanol and 1 part glacial acetic acid. Thefixed testes were stored in a refrigerator until preparations were to be made. A small fragmentof testis was placed on a slide cleaned from 95 % ethanol. Time was allowed for residualfixative to evaporate, and then 2 small drops of 0-5 % orcein (G. T. Gurr's synthetic) in 45 %acetic acid were added. The testis fragment was tapped out to dissociate the cells and examined,uncovered, under the low power of a light microscope. If divisions proved to be present, thetestis fragment was tapped out much more extensively, and the slide placed in a moist chamberfor 3 min. The material was now spread out lengthwise over the slide and examined under abinocular dissecting microscope against a white background. Fibrous connective tissue strandswere removed with forceps, and the material covered with a No. 2 22 x 40 mm siliconizedcoverslip cleaned from 95 % ethanol and gas-blasted to remove fluff. The preparation wasnow squashed out firmly between the folds of a folded filter paper circle, placed on solidCO2 in a plastic 'igloo' until well frozen (about 10 min), then the coverslip flipped off witha razor blade and the slide placed at once in 95 % ethanol. After a minute or two the slidewas transferred to absolute ethanol, the preparation then mounted in Euparal under a No. o22 x 40 mm coverslip, and dried on a hot plate.

These stained preparations were later examined under a Zeiss WL microscope fitted witha green interference filter, and sketches made of well-spread ist meiotic metaphases, recordingthe number of chromosome arm pairs associated by chiasmata, the total number of chiasmata,the frequency of pairs of univalent chromosomes, and the number of bivalents interlockedwith one another. Both authors scored 20 different ist meiotic metaphases in this manner;if there was a significant disparity between the 2 sets of observations, which for the mostpart were carried out on different slides, more metaphases were scored. This was deemednecessary only in the cases of 5 out of a total of 81 newts which provided usable material.Photographs of ist meiotic metaphases were taken under a Zeiss planapochromat x 100 oil-immersion objective, N.A. 1.32, using Kodak Pan F film.

Autoradiography was used to check the time course of meiosis in the 1977 fixations, themethodology being just as described by Callan & Taylor (1968).

Testes from a few untreated newts were fixed as controls, one testis in ethanol/acetic acidfor making squash preparations, and the other in Sanfelice's fluid for wax embedding,sectioning, and staining in iron haematoxylin. Likewise one testis from each of the +o-dayanimals, i.e. immediately following heat shock, was fixed in Sanfelice's fluid for sectioning, tocompare with sections of control testes.

OBSERVATIONS

The information collected from the squash preparations is shown in Table 1.One male newt, 77 + 18C, proved to be triploid, and will not concern us here. Thefirst point deserving attention is that there is a low, but appreciable, frequency of


interlocked bivalents in control animals. We thought that the Tentsmuir newtpopulation, which lives close to the sea, might be abnormal in regard to bivalentinterlocking, and so we examined meiosis in preparations from 3 male T. vulgariscollected from a dew pond at Forret Hill, Logie, some 10 km to the southwest ofthe Tentsmuir ponds and 140 m above sea level. However these also showed inter-locking. This makes clear the fact that a cytologist, when studying preparations ofmeiosis for chiasma frequency and distribution, tends to pick out those divisionsin which bivalents are well separated and easily analysable when given the choice!

If we accept from Table 1 that an interlock frequency of up to, say, 10 interlocksin 40 spermatocytes is about the maximum likely to be encountered in normal,control newts (the highest frequency recorded being 7 amongst the 8 controls) thenclearly for the first 3 days following a 24-h heat shock the interlock frequency iswithin the normal range. But from 4 days after the heat shock until 28 days after theheat shock the frequency of interlocks in the majority of the newts (38 out of 56)is much above control level, while from 30 days after the heat shock until + 36 days,which was the latest fixation made, interlock frequency is back to control level.Within the sensitive period, from +4 to +28 days, there is a wide scatter of inter-lock frequencies, ranging up to 55 in 40 spermatocytes of newt 77 + 24C and downto control frequencies (assumed to be 10 and less) in 18 of the heat-shocked animals.This scatter is portrayed in Fig. 2.

The autoradiographic evidence for computing the time course of male meiosisin the newts used in 1977 was less comprehensive than it should have been because,for one reason or another, there was little [3H]thymidine incorporation in many ofthe newts. There was, however, sufficient information to relate the time course in1977 to that obtaining in 1967, from which Callan & Taylor (1968) made theirdetailed analysis. In 1967 the longest interval between [3H]thymidine injection andfixation which showed 'late-label' distribution in pachytene nuclei (i.e. derivingfrom spermatocytes which were labelled at the end of the pre-meiotic 5-phase) was18 days, whereas in 1977 the corresponding longest interval was 20 days plus the24 h spent at 31-5 °C (information from newt 77 + 20A). This indicates that the1977 spermatocytes were taking about 3 days longer to pass from the end of pre-meiotic S to the end of pachytene. This figure is corroborated by information fromnewt 77 + 26A, in which all 1st meiotic metaphases were labelled, and these labelleduniformly, not late-labelled, a condition encountered at its earliest some 23 to 24 daysafter [3H]thymidine injection in the 1967 fixations. In short, the 1977 newts tooksome 15% longer to pass through meiotic prophase, and this factor has been appliedto the data on the durations of the meiotic stages known from the 1967 fixations,giving some 11-12 days for premeiotic S (which laps over into leptotene for about1 day), 7 days for leptotene, 10 days for zygotene, 6 days for pachytene, and 2 daysfor diplotene.

These stage durations have been entered in Fig. 2, so that the stage at which thespermatocytes experienced the heat shock, all of them having been scored for inter-locks when they had reached 1st meiotic metaphase, can be appreciated. As regardsthe generation of an abnormally large number of interlocked bivalents, it is apparent

Tab

le I

. In

form

atio

n fr

om s

quas

h pr

epar

atw

m

Y

Mea

n

w

freq

uen

cy

No

. 0

No

. of

M

ean

of c

hro

mos

ome

of p

airs

in

terl

ock

s pe

r in

terl

ock

arm p

airs

of

un

ival

ents

40 s

per

ma-

fr

equ

ency

per

as

soci

ated

per

P

er 40

tocy

tes

sper

mat

ocyt

e V

aria

nce

sper

mat

ocyt

e V

aria

nce

sp

erm

atoc

ytes

Con

trol

new

ts

Mal

e n

o.

Ten

tsm

uir

77A

77B

77c

77D

77E

J4

5e

5515

5517

5519

Hea

t-sh

ock

ed n

ewts

D

ays

afte

r M

ale

no.

he

at s

hock

0.15

0.03

0.08

0.15

0.13

0.18

0.15

0.05

0'00

0'00

O'IO

0.03

0.08

0. I3

O'IO

0.13

0'15

0.79

0.87

0.33

0'33

0'20

0.33

0.60

0.03

Tab

le I

(c

ont.)

Mea

n

freq

uen

cy

No

. N

o.

of

Mea

n

of c

hro

mos

ome

of p

airs

in

terl

ocks

per

in

terl

ock

ar

m p

airs

of

un

ival

ents

D

ays

afte

r 40

spe

rma-

fr

equ

ency

per

as

soci

ated

per

P

er 4

0 M

ale

no.

h

eat

shoc

k to

cyte

s sp

erm

atoc

yte

Var

ianc

e sp

erm

atoc

yte

Var

ianc

e sp

erm

atoc

ytes

Tab

le I

(c

ont.

) u

W

N

Mea

n

freq

uenc

y N

o.

No.

of

Mea

n

of c

hrom

osom

e of

pai

rs

inte

rloc

ks p

er

inte

rlw

k

arm p

airs

of

uni

vale

nts

Day

s af

ter

40 s

perm

a-

freq

uenc

y pe

r as

soci

ated

per

P

er 4

0 M

ale

no.

heat

sho

ck

tocy

tes

sper

mat

ocyt

e V

aria

nce

sper

mat

ocyt

e V

aria

nce

sp

erm

atm

a

68 +

17A

17

I I

0.28

0.2

0

22.8

5 0.

74

I

68 +

17B

17

13

0'

33

0.28

22

.98

1.00

I

68

+ 1

8B

I 8

7 0.

18

0.2

0

zzm

o

0.97

I

77 +

18A

I 8

6 0.1

5

0.13

22

.88

0.54

o

3

68

+ 19A

19

10

0.25

0.

19

22.7

8 0.

54

o

68 +

I~

B

19

10

0.25

0.

29

22.5

0 0.

62

I 9

68

+ 20A

20

9 0.

23

0.23

22

.28

0.82

4

0.60

22

.63

'2 77

+ 20

A

20

25

0.

63

0'55

I

22'2

5 1'

17

0

3

77 +

20B

20

4 I

1.03

0.

64

3

Q

77 + 2

2A

22

30

0.

75

0.73

22

.60

0.60

I

a

R

77 + 2

2B

22

25

0.

63

0.55

2

2.2

0

0.99

I

0.5

I 22

'03

1.13

.t,

0.4

8 4

77 + 2

2C

22

19

77 +

24B

24

23

0.

58

0.67

22

'59

0.42

I

% 77

f 2

4C

24

55

I .3

8 0.

98

22'3

5 0.

62

I 2

2 77

+ 26A

26

10

0.25

0.

29

22.4

0 1-

43

77 + 2

6C

26

I I

0.28

0.

23

21.5

8 7'

44

3 5 cb

0 1

2 3

4 5

6 7

8 9

10

11 1

2 13

14

15

16

17

18 1

9 20

22

24

26

28

3

0

32

E D

ay o

f fi

xati

on f

ollo

win

g 2

4h

hza

t sho

ck

8 I-

PA

CH

YTE

NE

-I-

ZY

GO

TE

NE

-r

LE

~O

TE

NE

+

-I

S-P

HA

SE

Stag

e at

whl

ch t

he s

core

d sp

emat

ccyt

es e

xper

iaxe

d h

eat s

hock

F

ig. 2

. D

iagr

am s

how

ing

the

inte

rloc

k fr

eque

ncie

s ob

serv

ed in

new

ts g

iven

hea

t sh

ocks

at

diff

eren

t st

ages

.


Fig. 3. Two ring bivalents interlocked (arrow) in a ist meiotic metaphase from newt77 + 9CFig. 4. Two ring bivalents interlocked (arrow) in a ist meiotic metaphase from newt77 + 20A.


that newt spermatocytes are sensitive to a heat shock from some time towards theend of premeiotic S until the last day or two of pachytene, i.e. some 4 days aftersynapsis has been completed.

As interlocked bivalents will be recognized at ist meiotic metaphase only if theappropriate chiasmata are formed after interlocking has occurred, such chiasmatamust have formed right at the end of pachytene. This finding is entirely in keepingwith classical cytological doctrine, but the finding that interlocks can be generatedbetween bivalents which had already completed synapsis is not.

A 'ring' bivalent is one in which both arm pairs are associated by one or morechiasmata, whereas a 'rod' bivalent is associated in one arm pair only. Some typicalphotographs of ist metaphases including interlocked bivalents are shown in Figs. 3to 6. Fig. 3 shows a simple ring through ring interlock, Fig. 4 likewise, Fig. 5 showsa rod interlocked with a ring, and Fig. 6 shows 3 rings interlocked with one another.

At the outset we expected to be confronted with the problem of 'losing' potentiallyinterlocked bivalents because insufficient chiasmata might be formed in some of thenewts, and that to make sense of the data we might need to make some allowance forthis. Thus 2 rod bivalents, each of which has formed only a single chiasma, areunlikely to remain interlocked at ist meiotic metaphase unless the centromeres ofboth assume non-disjunctional orientations, and strain against one another, in themanner depicted by Buss & Henderson (1971a, b) for locusts; in the newts weencountered no examples of such ' orientational' interlocking. However it will beapparent from Table 1 that there is no great variation in mean frequency of associatedchromosome arm pairs per spermatocyte throughout the fixations, the overwhelmingmajority falling between 21-0 and 22-9, with low variance values.

Moreover, and rather to our surprise, plenty of rod bivalents remain recognizablythreaded through rings (we scored 94 rods through rings, 8-8%, compared with979 rings through rings, 91-2%) at ist meiotic metaphase. The mean of the meanfrequencies of chromosome arm pairs associated per spermatocyte is 22-08, andtherefore if pairs of univalents are neglected (their numbers are, except for themaverick newt 68+13B, trivial, as can be seen from the right-hand column ofTable 1), the average frequency of ring bivalents per spermatocyte is 10-08, and ofrod bivalents 1-92. If we were dealing with a large 'population' of ring and rodbivalents mixed in the aforesaid proportions, we would expect to find rings associatedin pairs at a frequency of io-o82, as compared with single rods associated with singlerings at a frequency of 2 x I-92X 10-08; however only one half of the latter wouldlead to interlocks because each rod bivalent has, by definition, only one of its 2 armpairs available for interlocking with a ring. On a percent basis these expectationsare therefore 84 and 16% respectively.

Professor R. M. Cormack has pointed out to us that these probability calculationsare not strictly applicable to our data, because each spermatocyte contains a limitednumber of bivalents, 12, ring and rod bivalents occur in different numbers perspermatocyte (12:0, 11 : i , 10:2, 9:3, 8:4, 7:5 and 6:6), and that therefore theexpected ring/ring and continuing ring/rod associations must be separately assessedfor each; these expectations are respectively 11:0, 10:1, 9:2, 8:3, 7:4, 6:5 and 5:6.

H. G. Callan and S. M. Pearce

Fig. 5. A rod bivalent, which has formed 2 chiasmata in one arm pair, interlockedwith a ring bivalent (arrow) in a ist meiotic metaphase from newt 77 + 14B.Fig. 6. Three ring bivalents interlocked with one another (arrow) in a ist meioticmetaphase from newt 77 + 8B.


The known percentage frequency of occurrence of 12 rings: 0 rods is 4-06, of11 rings: 1 rod is 39-47, of 10 rings: 2 rods is 32-54, of 9 rings: 3 rods is 15-24, of8 rings: 4 rods is 6-o6, of 7 rings: 5 rods is 2-22, and of 6 rings: 6 rods is 0-42. Whenthese percentages are applied as weighting factors to the relevant expectations, theoverall expected frequency of ring/ring associations is 82-9% and of ring/rodassociations 17-1%. The interlock frequencies actually found, 91-2% and 8-8%respectively, allow us to infer that about one half of potential ring/rod interlocksare retained up to 1st meiotic metaphase, the other half presumably dissociating asthe bivalents move on to the division spindle.

Although we scored total chiasma frequencies as well as frequencies of arm pairassociations, for the sake of simplicity mean chiasma frequencies and their variancesare omitted from Table 1. The biggest difference between mean chiasma frequencyand mean frequency of arm pair associations was 4-9 (Logie control 55 /$) but innone of the other newts was the mean excess more than 2, and the variances of meanchiasma frequencies were much the same as the variances of mean arm pair associations.Evidently a 24-h heat shock at 31-5 °C, whenever delivered, has little or no effecton the frequency with which chromosome arm pairs are associated by chiasmata, noron total chiasma frequency, in T. vulgaris.

When scoring interlocked bivalents it is hard to avoid the preconception thatbecause some spermatocytes contain several interlocks and others, the great majority,none, some spermatocytes may be particularly susceptible to the influence of a heatshock, but others less so or not at all. This preconception is manifestly false, for it isapparent from Table 1 that the variances of mean interlock frequency per sperma-tocyte follow the means remarkably closely, in other words each interlock is a chanceevent, which may or may not be accompanied by other interlocks in the samespermatocyte on a purely random basis.

We encountered 17 whole bivalent interlocks, i.e. where one entire ring wasthreaded through another, out of the total of 1073 interlocks scored. We found nointerlocks between ring bivalents where both arm pairs of both bivalents wereassociated by 2 chiasmata, and where an interlock might have been expected proxi-mally, i.e. within the rings including the centromeres. We found only 2 interlockswhere a simple ring was threaded through the proximal ring, including the centro-meres, of another bivalent which had formed 2 chiasmata in each of its arm pairs.Finally, we found no interlocks involving the region between 2 neighbouring chiasmata inonearmpairinanybivalentswhere2chiasmatahadformedwithin one or both arm pairs.

DISCUSSION

White (1973) has suggested that interlocking ' . . .must be regarded as an extremelyrare accident of meiosis whose frequency of occurrence is two or three orders ofmagnitude lower than would be the case on the hypothesis of random leptoteneorientation'. John (1976) on the contrary, has demonstrated that interlocking isfrequent in male meiosis of the grasshopper Chloealtis conspersa (this has beenconfirmed by H.G.C.) and that it occurs, although less frequently, in 5 other species


of grasshopper. We now find interlocking to be not infrequent in Triturus vulgarismale meiosis. Nevertheless White is substantially correct in his claim. It is astonishingthat interlocking brought about by synopsis is not of common occurrence in all thoseorganisms, probably the great majority, in which synapsis begins at both arm endsof each pair of homologues, and proceeds from these ends towards the centralregions. All text-book illustrations of the origin of interlocked bivalents (see forexample, White, 1973, p. 154; Rees & Jones, 1977, p. 45) merely show 2 pairs ofsynapsed chromosomes trapped together, without illustrating the relationships ofthe ends of these synapsed chromosomes to the nuclear membrane, as is shown inFig. 1. There are organisms whose chromosomes usually synapse from one endonly (for example Bombyx mori females studied by Rasmussen, 1977) and inwhich one can visualize how an intruding chromosome or bivalent might be pro-gressively excluded as synapsis proceeds, but even in such organisms the exchangeof pairing partner in triploids, and the successful completion of synapsis aroundloops in inversion heterozygotes, clearly demonstrates the inadequacy of the zip-fastener explanation that is sometimes offered for synapsis, and which thereforecannot be held to account for the rarity of interlocking.

Several cytologists have claimed that interlocking is rare because homologouschromosomes already become pre-aligned with one another during the premeioticmitoses, and retain their alignment during interphase because homologous telomeresare attached, alongside one another, to the nuclear membrane. Evidence for theattachment of telomeres to the nuclear membrane, or rather of the ends of the lateralelements (axial cores) of the synaptonemal complex to the membrane, is entirelyconvincing, whereas evidence of pre-alignment of homologues is, to say the least,conflicting. Indeed there cannot be any pre-alignment of homologues in haploidorganisms, such as the Ascomycetes, where the homologues do not meet for synapsisuntil gamete nuclei fuse.

To account for the rarity of interlocking Rees & Jones (1977) propose that align-ment of homologues occurs during premeiotic interphase when ' . . .the chromosomesare in vigorous movement'; homologues become associated together at or near onepair of telomeres, which then move through the nucleus pulling the homologouspair of chromosomes towards one another and disentangling them from others, afterwhich the telomeres attach to the nuclear membrane and consolidate the disentangledstate. We find such a mechanism difficult to conceive in view of the lengths of chromo-somes at premeiotic interphase, lengths which are admittedly not known with precisionbut which are assuredly greater than chromosome lengths at, for example, leptoteneor pachytene. Moreover if one examines mid-zygotene in particularly favourablematerial, such as the spermatocytes of the plethodont salamander Batrachosepsattenuatus, introduced to us by Dr J. Kezer of the University of Oregon, but alreadywell known to Janssens (1909), homologous chromosomes are manifestly not lyingclose together except where they are synapsed. At any Y-fork marking the limit ofthe synapsed region, the as yet unsynapsed regions generally form an obtuse anglewith respect to one another, i.e. they are approaching one another from widelyseparate nuclear domains.


In discussion Dr E. M. De Robertis has suggested to us that a possible explanationfor the rarity of interlocking is that the telomeres of single chromosomes may betemporarily joined to one another at the end of the premeiotic mitosis, so thateukaryotic chromosomes are for a time circular, like the chromonemes of prokaryotes.The joined telomeres become anchored to the nuclear membrane and associatedwith the joined telomeres of the homologous chromosome which is anchored nearby.Thereafter the chromosomes decondense, but so long as their telomeres remainunited and homologous ends remain in association, they will occupy nuclear domainswhich exclude the possibility of trapping other chromosomes during the latersynapsis. Such an explanation for the rarity of interlocking is by no means ruledout by the knowledge that, once synapsis has begun, bivalent arm ends are indis-putably separate from one another (see, in particular, the electron-microscopic studyof synapsis in Locusta by Moens, 1973) because a careful examination of leptotenein Batrachoseps neither shows nor fails to show the ends of individual chromosomes.When based on a light-microscopic study of sections or squash preparations of eventhe most favourable materials, observational difficulties preclude any firm statementon this question. It would be a subject worthy of study by more refined techniques.

Buss & Henderson (1971a, b) found that exposure of male Locusta migratoria to40 °C for several days induced bivalent interlocking that was detectable both atpachytene and at 1st meiotic metaphase. Their analysis was complicated by thefact that exposure to 40 °C also reduces chiasma frequency in Locusta, therebydiminishing the frequency of interlocks detectable at 1st metaphase; and to complicatematters still further, after an initial and severe drop in chiasma frequency lastingfor several days, when interlocks would be expected to be most often 'lost', chiasmafrequencies rise back to near normality and it is only when they do that interlocksbecome detectable. Despite this complication Buss & Henderson interpret theirobservations by claiming that heat treatment applied to spermatocytes in leptotene,zygotene or pachytene does not induce interlocking, but that the sensitive stageoccurs earlier, ' . . . at some period between the telophase of the last spermatogonialmitosis and the main premeiotic S phase'. They go on to argue that chromosomepairing must therefore be a 2-step process (see also Maguire, 1977), the first of thesebeing an initial alignment of homologues at premeiotic interphase or the final mitotictelophase, and that it is this initial alignment which is sensitive to heat and which, ifit goes awry, will inevitably lead to interlocking at the later stage of zygotene synapsis.Buss & Henderson, like Rees & Jones, consider that the attachment of telomeres tothe nuclear membrane stabilizes the mutual alignment of homologues that is saidto be achieved prior to synapsis and during the first step of the pairing process, butthey discount an alternative possible explanation for the generation of interlocks,that the heat treatment may cause telomeres to detach from the nuclear membrane,allowing non-homologous chromonemata to intertwine and thus become interlocked.

We on the contrary contend that our observations on Triturus support the oppositeconclusion. We have shown that interlocks can be generated by a 24-h heat shockapplied to spermatocytes at any stage from late premeiotic S until mid-pachytene.Our information regarding spermatocytes exposed to heat shock earlier in the

140 H. G. CaUan and S. M. Pearce

premeiotic S phase is too scanty to warrant a statement that these are insensitive,though what information we have suggests that this may be so. However our signi-ficant finding is that interlocks can be generated in spermatocytes which hadalready completed synapsis, under normal conditions, before they experienced a heatshock.

At first sight this finding seems hard to explain. Like Taylor (1949) we thoughtof the possibility that the heat shock might cause desynapsis, followed by recoveryand a second synapsis, preceding which non-homologous chromosomes might havebecome intertwined, but the appearance of pachytene stages in sectioned controltestes, and in testes fixed immediately after the removal of newts from a heatedincubator, proved to be identical. Heat shock does not appear to induce desynapsisin Tritiums.

The likeliest explanation for our findings is that a heat shock causes telomeres todetach from the nuclear membrane, or from the lateral elements of the synaptonemalcomplex where these are attached to the nuclear membrane, allowing non-homologouschromonemata to become intertwined. Thereafter the interlocked condition will bemaintained provided recombination between homologues occurs distal to the region ofintertwining, in the ' cloud' of chromonemata outside the synaptonemal complex, notinside the complex between its 2 lateral elements. Despite the fact that unambiguousevidence for the presence of DNA between the lateral elements of the synaptonemalcomplex is lacking, some students of meiosis assert that recombination occurs withinthe complex (Moens, 1968; von Wettstein, 1971; Stern, Westergaard & von Wett-stein, 1975) whereas others, and notably the original discoverer of the synaptonemalcomplex, M. J. Moses, are less dogmatic. Moses (1977) writes: 'There is presentlyno direct evidence from which to conclude whether crossing over takes place inchromatin immediately associated with the synaptonemal complex or in that moreperipheral to it'. We think we may have provided indirect evidence in favour of theperiphery.

Whether all interlocked bivalents in Triturus vulgaris originate in similar fashionis debatable. We imagine that whole bivalent interlocks, of which we found only17 in a total of 1073, must have arisen by trapping during synapsis, much as depictedby Rasmussen (1977) in Bombyx mori oocytes. However telomere detachment andintertwining of chromonemata could account for all the rest, whether detachmentoccurs prior to synapsis, in which case synaptic trapping, i.e. foreign chromonematalying within synaptonemal complexes, might result, or after synapsis, and involvingonly the chromonemata peripheral to the complexes.

REFERENCES

BELAR, K. (1928). Die cytologischen Grundlagen der Vererbung. Berlin: Springer.Buss, M. E. & HENDERSON, S. A. (1971a). Induced bivalent interlocking and the course of

meiotic chromosome synapsis. Nature, New Biol. 234, 243-246.Buss, M. E. & HENDERSON, S. A. (1971&). The induction of orientational instability and

bivalent interlocking at meiosis. Chromosoma 35, 153-183.CALLAN, H. G. & PERRY, P. E. (1977). Recombination in male and female meiocytes contrasted.

Phil. Trans. R. Soc. Ser. B 277, 227-233.


CALLAN, H. G. & TAYLOR, J. H. (1968). A radioautographic study of the time course of malemeiosis in the newt Triturus vulgaris. J. Cell Sci. 3, 615-626.

DARLINGTON, C. D. (1937). Recent Advances in Cytology. 2nd edn. London: Churchill.GELEI, J. (1921). Weitere Studien iiber die Oogenese des Dendrocoelum lacteum. II. Die

Langskonjugation der Chromosomen. Arch. Zellforsch. 16, 88-169.GELEI, J. (1922). Weitere Studien iiber die Oogenese des Dendrocoelum, lacteum. III. Die

Konjugations-frage der Chromosomen in der Literatur und meine Befunde. Arch. Zell-forsch. 16, 299-370.

HENDERSON, S. A. (1966). Time of chiasma formation in relation to the time of deoxyribo-nucleic acid synthesis. Nature, Lond. 211, 1043-1047.

JANSSENS, F. A. (1909). Spermatog6nese dans les batraciens. V. La thiorie de la chiasmatypie,nouvelle interpretation des cineses de maturation. Cellule 25, 387-411.

JOHN, B. (1976). Myths and mechanisms of meiosis. Chromosoma 54, 295-325.LEVAN, A. (1933). Cytological studies in Allium. IV. Allium fistulosum. Sv. bot. Tidskr. 27,

211-232.MACUIRE, M. P. (1977). Homologous chromosome pairing. Phil. Trans. R. Soc. Ser. B277,

245-258.MATHER, K. (1933). Interlocking as a demonstration of the occurrence of genetical crossing-

over during chiasma formation. Am. Nat. 67, 476-479.MOENS, P. B. (1968). The structure and function of the synaptinemal complex in Lilium

longiflorum sporocytes. Chromosoma 23, 418-451.MOENS, P. B. (1973). Quantitative electron microscopy of chromosome organization at meiotic

prophase. Cold Spring Harb. Symp. quant. Biol. 38, 99-107.MOSES, M. J. (1977). The synaptonemal complex and meiosis. In Molecular Human Cyto-

genetics, ICN-UCLA Symposia on Molecular and Cellular Biology, vol. 7 (ed. R. S. Sparkes,D. Comings & C. F. Fox), pp. 101-125. New York and London: Academic Press.

RASMUSSEN, S. W. (1977). Meiosis in Bombyx mori females. Phil. Trans. R. Soc. Ser.BzT],343-35°-

REES, H. & JONES, R. N. (1977). Chromosome Genetics. London: Arnold.STERN, H., WESTERCAARD, M. & VON WETTSTEIN, D. (1975). Presynaptic events in meiocytes

of Lilium longiflorum and their relation to crossing-over: a preselection hypothesis. Proc.natn. Acad. Sci. U.S.A. 72, 961-965.

TAYLOR, J. H. (1949). Increase in bivalent interlocking and its bearing on the chiasma hypo-thesis of metaphase pairing. J. Hered. 40, 65—69.

VON WETTSTEIN, D. (1971). The synaptinemal complex and four-strand crossing over. Proc.natn. Acad. Sci. U.S.A. 68, 851-855.

WHITE, M. J. D. (1973). Animal Cytology and Evolution, 3rd edn., p. 154. Cambridge:Cambridge University Press.

WILSON, E. B. (1925). The Cell in Development and Heredity, 3rd edn. London & New York:Macmillan.

(Received 4 September 1978)

CBL 37

an experimental analysis of bivalent interlocking …available (callan & taylor, 1968)...

Documents