Chromosoma (Berl.) 43, 237--245 (1973) �9 by Springer-Verlag 1973

Histone Synthesis during Meiotic Prophase in L i l i u m *

Yu. F. Bogdanov, A.A. Strokov and S.A. Reznickova

Institute of Molecular Biology, Academy of Sciences, Moscow B-312 and The USSR Research Institute of Essential Oil Plants, Simferopol, U.S.S.t~.

Abstract. Anthers of Lilium candidum L. were cultivated on artificial media containing labelled amino acids. Histones were isolated from meiocytes and frac- tionated by the use of polyaerylamide gel electrophoresis (PAGE). Total histone synthesis was found not to terminate at the end of premeiotic interphase but to continue until at least zygotene. However, the rate of synthesis was reduced during prophase I compared to interphase. Separate fractions were synthesized asynchro- nously during the period from late interphase to zygotene. Tissue specific "histone of meiosis" (FM) was synthesized during late interphase and leptotene.

Introduction

Developmental programming of meiotic cells (meiocytes) is thought to begin several generation before the cells enter meiosis. However, the resulting "critical events" determining typical features of meiocytes take place during the period from premeiotic S-phase to zygotene (Stern and Hotta, 1969). The phenomenology of these events and their sequence during this period were studied using excised anthers or isolated meiocytes in vitro of plants (Tradescantia, Trillium, Lilium) which have synchro- nous meiosis (Taylor, 1950; Stern and Hotta, 1969 ; Reznickova and Bog- danov, 1972).

The best known "critical event" in Lilium meiocytes is the delay in synthesis of a minute satellite DNA fraction (about 0.3% of the genome) until zygotene (Hotta and Stern, 1971). In the mitotic cycle, in contrast, the satellite is replicated during the S-phase. Another biochemical differ- ence between meiosis and mitosis is the delay (comparing gross DNA synthesis) in doubling of the histone portion of the chromosomes. This was determined by cytophotometry of spermatocytes stained with Fast Green at pH 8.2 in three animal species. In all cases histone synthesis was terminated during meiotic prophase I (Bogdanov and Antropova, 1971). On the other hand, in three plant species (Liliaceae) use of the PAGE method indicated that a new histone fraction : "histone of meiosis" (FM), appears in the course of premeiotic interphase and early prophase I, in addition to a standard set of "somat ic" histone fractions (Sheridan and Stern, 1967; Strokov et al., 1973).

* Dedicated to Professor A. A. Prokofieva-Belgovskaia on the occasion of the seventieth anniversary of her birthday.

238 Yu. F. Bogdanov et al.

Thus, two characteristic histone rear rangements are found in meio- cytes: delayed synthesis of to ta l his tone dur ing prophase I, in animals ; and appearance of a specific histone fract ion at the same time, in plants. The reduced q u a n t i t y of to ta l histone in meioyctes a t the beginning of prophase I does mean tha t some par t of DNA has to b e " s t r ipped" at t ha t t ime (Bogdanov and Antropova, 1971). However, the appearance of the new histone fract ion at the same period does mean tha t some new macro- molecular subs t ruc ture is to be bui ld wi th in the chromosomes (Strokov et al., 1973). The evidence for delayed histone synthesis (Bogdanov et al., 1971) is of an indirect na tu re insofar as i t has been derived from estima- t ion of the a m o u n t of dye bound to histone. I t now seems desirable to ob ta in direct evidence for the t ime of premeiotic his tone synthesis. This m a y be achieved by t racing histone synthesis with labelled amino acids, followed by isolation of histones and their f ract ionat ion with P A G E. Here we report some results of such study.

Materials and Methods Excised anthers of Lilium candidum L., which has synchronous meiosis, were

cultivated on artificial media (Reznickova and Bogdanov, 1972) 1. About 100 flower buds (with 6 anthers each) of the same meiotic stage were used in each experiment. Meiotic stages were identified cytologically twice; at the time of explantation of anthers and at the end of each cultivation period. Stages of explantation are indi- cated in the figures. Two experimental designs with labelled amino acids were carried out; 1) cultivation of anthers for 2 hrs on a medium containing 1-1ysine-aH (CEA, France-Italy, 24 Ci/mM, 10 ~zCi/ml) followed by 2 hrs chasing with "cold" media, then freezing with dry ice, and 2) cultivation of anthers for 22 hrs on a radioactive medium containing 1-arginine-laC plus 1-1ysine- 14 C(Prague, Czechoslovakia, 0.080 Ci/ mM, 0.8.fzCi/ml and 0.120 Ci/mM 1.2 ~zCi/ml, respectively), or 1-alanine-14C (Prague, 0.073 Ci/mM, 1.0 ~zCi/ml) then chasing during following 20 hrs with "cold" media, and freezing with dry ice.

Histones were isolated from microsporocytes according to the second method of Stroker et al. (1973), which in turn is a slight modification of the method of Georgiev et al. (1970). Total histene isolated was fractionated by the use of PAGE according to Johns (1967). Densities of the fraction bands in gels stained with Coomassic Bril- liant Blue R (Sigma) were estimated with Chromoscan. Afterwards the gels were cut into pieces containing separate fraction bands, the individual bands were dissolved in a 35 % solution of H~02 placed on filter paper and radioactivity estimated with SL-30 liquid scintillating spectrometer. The radioactivity of non-fractionated total histone was also estimated. The results are expressed in counts per minutes per mg of histone (cpm/mg), unless otherwise specified.

Results

I . Total Histone Synthesis during Prophase I

Line 1 of Fig. 1 presents the incorporat ion of arginine-14C plus lysine- 14C into tota l histone of meiocytes dur ing premeiotic interphase and

1 This type of culture corresponds to" androecia "according to the method described by Rcznickova and Bogdanov (1972).

H/stone Synthesis during Meiotic Prophase in Lilium 239

16

12

m T M

x 8 E o_ o

- 8 O

1 , z ~ 2 -

x - - m

_ ~

i/i ll ~ _- 40 .~ / l i i ~ k - 2o

I I I I LPI EL LL ZP

Fig. 1. Lysine-14C plus arginine-14C incorporation into total h/stone of L. candidum meioeytes during premeiotie interphase to paehytene period. Abscissa: meiotic stages; LPI late premeiotic interphase; EL early leptopene; LL late leptotene; ZP zygotene and pachytene. Left ordinate: counts per minute per mg of total h/stone protein (line 1). Right ordinate: integral of epm/mg of total h/stone in arbi-

trary units (line 2)

early prophase I. I t is easy to see tha t the incorporation, i.e. h/stone syn- thesis, does not terminate in premeiotic interphase, but continues during prophase I. The highest rate of incorporation was observed during the late interphase to early leptotene period. During the period from late leptotene to paehytene, the rate of incorporation decreased markedly

Line 2 of Fig. 1 is a cumulative total based on the data from line 1, and reflects the process of accumulation of total h/stone during the period studied. The sharpest increase in total h/stone was observed during late interphase, and the process terminated in zygotene-pachytene. The period from late interphase to paehytene takes both in situ and in vitro, not less than 3 days (Reznickova and Ostroverkhov, 1970; l~ezniekova and Bog- danov, 1972). We consider Fig. 1 to be direct evidence for delayed total h/stone synthesis during meiotic prophase I in isolated anthers of L. can- didum in vitro.

I I . Synthesis o/Separate H/stone Fractions during Prophase I

Late interphase--leptotene. Anthers 'were explanted onto radioactive medium while in late premeiotic interphase, then transferred to "cold" medium one day later and harvested in leptotene on the second day

240 Yu. s Bogdanov et al.

9000 900 I

7000 700~

5000 500 t-

3000 300 /

2500 25 -

~2000- 200 - ~ /%.x~ 1 l1 ~100

1500 - 150 _ __:. / 80

,'/ Joo 1000 100 ~ l~ ~ 1 / | F ,/ \ / /

/ 500 50 ,,- 11-. .......... j 2 20

0 O ~ O - F2al F2a2 F2B F3 F1 FM

Fig. 2. Lysine-14C plus arginine-14C incorporation into histone fractions of meiocytes during late interphase to leptotene. Abscissa: histone fractions separated by PAGE. Ordinate 1: cpm/mg (line 1). Ordinate 2: molar percent of lysine plus arginine in the histone fractions (line 2). Ordinate 3 : (cpm/mg)/molar percent of arginine plus lysine

in the fractions (line 3)

(design 2). The results of the experiment are presented in Fig. 2. Line 1 reflects incorporation of arginine-14C plus ]ysine-14C into histone fractions isolated from meiocy~es and separated by PAGE. All fractions except F 3 are labelled.

Line 2 represents the calculated arginine plus lysine content of the fractions. Since there are no published data on the amino acid composi- tion of L i l i u m histone, except for F1 and F l a (F1 4- FM) (Sheridan and Stern, 1967) and since we lacked sufficient L i l i u m histone to perform amino acid analysis, we use here the data on arginine plus lysine content of calf thymus histone fractions F2al , F2a2, F2b and F3 (Hnilica, 1967). This extrapolation is based on the fact tha t the amino acid composition of histone fractions isolated from different animal and plant species vary slightly (Phillips, 1971). The datum for F1 is taken from Sheridan and Stern (1967) and for FM is calculated from the same paper, using the

Histone Synthesis during Meiotic Prophase in Lilium 241

3000 - 3001--

I

ooo-

10oo 100

o I ~ / l _ F2al F2a2 F2B F3 FI FM

Fig. 3. Alanine-140 incorporation into histone fractions of meiocytes during early leptotene to middle leptotene period. Abscissa: ttistone fractions separated by PAGE. Le/t ordinate: cpm/mg of histone (corresponds to broken line). Right ordinate: (cpm/ mg)/molar percent of given amino acid in the fractions (corresponds to solid line)

formula C ~ ~ 2CF1 ~ - -Cm, where CF~r, CF1 a and CF1 are the molar per- centages of FM, F l a and F1, respectively.

Line 3 is an estimate of the number of molecules of each fractions synthesized during the experimental period. I t was obtained by division of cpm/mg of histone (line 1) by molar percents of arginine plus lysine (line 2) for each fraction. During late interphase the highest rateof synthesis was demonstrated by FM histone, F2a2 and F2b are somewhat lower and F2al and F1 lowest. Histone F3 was not synthesized at tha t time.

Early leptotene - - middle leptotene. Anthers with early leptotene meio- cytes were explanted onto medium containing alanine-C 14 and harvested when meiocytes reach not farther than mid leptotene (designe 2). None- theless alanine-14C was used instead of lysine-14C plus arginine-14C, re- calculations of the results obtained as (epm/mg)/molar percent of alanine in the fractions permit us to compare them with the results of the other experiments in which lysine plus arginine were used. Fig. 3 shows tha t F2al histone was the fraction most intensively synthesized during the period studied. F~V[ was less and F2a2, F2b, and F3 were more less inten- sively synthesized fractions. There was no synthesis of F1 histone at all.

Leptotene--zygotene. A similar experiment (design 2) was performed with anthers explanted at late leptotene and harvested at zygotene and

17 Chromosoma (:Berl.), Bd. 43

242 Yu. F. Bogdanov et al.

2500

2000

1500 C:

10oo E

500

- :5ol - -

- o o 1 - ~ , .~

- 5oL-~

- ooF~ i E

- 50 I-

OI

/ \

/ \

1._....// \ / ',.

F2al F2a2 F2B F3 F1 FM

Fig. 4. Lysine-14C plus arginine-14C incorporation into histone fractions of meiocytes during late leptotene to zygotene-pachytene period. Abscissa: ordinates and other

designations are the same as in Fig. 3

2 5001 250

20001- 200

1500 150

1000 100

50O 50

0 0

r

- #

E

E

g I ~/ I I F2al F2a2 F2B F3 F1

/ ~2

I FM

Fig. 5. Lysine-~H incorporation into histone fractions of meioeytes during 2 hrs in middle leptotene. Abscissa, ordinates and other designations the same as in Figs. 3

and 4

paohy%ene. Lysine-14C plus arginine-14C were used as label . The resul ts are shown in Fig . 4. One can see t h a t F1 his tone was the f rac t ion mos t in tens ive ly synthes ized dur ing the per iod s tudied. F M his tone was syn- thes ized somewhat less in tensively , while synthesis of F2a2 and F3 his- tones was very low; F2b and F 2 a l h is tones were no t synthes ized a t all.

Histone Synthesis during Meiotic Prophase in Lilium 243

9000 I

7000

5000 I

3000~-.-

2 500

F 2000 -

1500 -

_~50

!00

150

1000 - 100

500 50

O- 0

/4 I

/ I . J J.

I I

~-. I II - E

- S I I I I I

F2al F2a2 F2B F3 F1

I

FM Fig. 6. Lysine-14C plus arginine-14C incorporation into histone fractions of meiocytes during zygotene to 1st meiotic division period. Abscissa, ordinate and other desig-

nations are the same as in Figs. 3-5

Figure 5 shows the results of the experiment with only two hours cultivation of anthers on radioactive medium (design 1). Anthers were explanted onto radioactive medium while in middle leptotene. The pat tern of fractions labelling was intermediate between these in Figs. 2 and 3. Evidently, F2al and F2b histones were not synthesized during the 2 hrs period studied; FM and F3 histones were most intensively synthesized.

Zygotene First meiotic division. Anthers with zygotene meioeytes were explanted on medium containing lysine-14C plus arginineJ4C accord- ing to design 2, and harvested at one t ime when meiocytes of different anthers reach pachytene, diplotene or first meiotic division (Fig. 6). F2b, F3 and F1 histories synthesized most intensively, while F2al and F2a2 very low. FM was not synthesized during this period.

Discussion

Biochemical evidence indicate tha t 99.7 % of DNA in Li l ium longi- florum meiocytes replicate during the S-period of premeiotic interphase

17"

244 Yu. F. Bogdanov et al.

(Hotta and Stern, 1971). Reznickova and Ostroverkhov (1970) found nearly the same situation in Lilium candidum by the use of cytophotom- etry. Thus, the demonstration of histone synthesis in leptotene and zygotene in L. candidum in the present work means that synthesis of the histone portion of the chromosomes is delayed long after gross DNA syn- thesis. This confirm our previous suggestion that such a delay is typical for classical meiosis in different organisms (Bogdanov et al., 1968). In contrast to DNA which replicates discontinuously during mei- osis (the greater part in S-period and the least in zygotene), total histone synthesis proceeds continuously from S-period to leptotene and then to zygotene. On the other side, when the syntheses of separate histone frac- tions are considered, these appear to be discontinuous. I t is true for example for F1, F2b and F3 histones (Figs. 2-5). F1, and F3 are notably late syn- thesized/ractions; F2al and F2a2 are early synthesized/ractions. This our finding confirms our recent indirect evidence on the "waves" of synthe- sis of separate histone fractions during interphase to middle prophase I in L. candidum meiocytes in situ (Strokov et al., 1973). In agreement with our findings Liapunova and Babadjanian (1973) also found de- layed F1 histone synthesis during meiotic prophase I in a cricket.

Apart from the significance of delayed histone synthesis with respect to the problem of biochemical control of meiosis, we may advocate anthers or meiocytes in vitro as an extremely convenient modell for studying the process of separate histone fraction synthesis. Evidently, the asynchrony of syntheses in various histone fractions in meiocytes provide favorable conditions for a biochemical s tudy of particular fraction synthesis. More- over, a very long duration of the process of histone synthesis which contin- ues from middle premeiotic interphase to zygotene and takes at least 3 days at about 20 ~ C provide further advantage of this model.

Biological significance of delayed and asynchronous histone syn- thesis during premeiotic development of meiocytes may be speculated, but remains still unclear. Further work is in order to settle this question.

Going back to the "critical events" determining typical features of meioeytes mentioned in Introduction, we have to assume that there must be some "critical event(s)" during premeiotic interphase that determine (s) the appearance of FlY[ and the delay of synthesis of other ("somatic") histone fractions until leptotene-pachytene.

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bling after premeiotic DNA synthesis in Triturus vulgaris male meiosis. Chromo- soma (Berl.) 85, 353-373 (1971)

Bogdanov, Yu. F., Liapunova, N.A., Sherudilo, A.I., Antropova, E. N. : Uncoupling of DNA and histone synthesis prior to prophase I of meiosis in the cricket Grillus (Acheta) domesticus. Exp. Cell Res. 52, 59-70 (1968)

Histone Synthesis during Meiotic Prophase in Lilium 245

Georgiev, G. P., Ilyin, Yu. F., Tichoncnko, A. S., Stelmashuk, V. Yu. : The structure of chromosomal deoxyribonueleo-proteins. I. The isolation of soluble DNP by urea treatment and its properties. Molec. Biol. (USSR) 4, 246-255 (1970)

Hnilica, L. S. : Proteins of cell nucleus. In: Proc. Nucleic acid research and molecular biology, voh 7, p. 25-105. London-New York: Academic Press 1967

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Received January 17, 1973 / Accepted by H. Bauer Ready for press April 16, 1973

Dr. Yuri F. Bogdanov, Dr. A.A. Strokov Institute of Molecular Biology Academy of Sciences of USSR Vavilovstr. 32 Moscow B-312, U.S.S.R.

Dr. S.A. Reznickova The USSR Research Institute of Essential Plants Oil Simferopol, U.S.S.R.