A QUANTITATIVE STUDY OF CHROMATIN DIMINUTION IN EMBRYONIC MITOSES OF CYCLOPS FURCZFER

SIGRID BEERMANN

Max-Planck-lnstitut fur Biologie, Tubingen, Germany

Received March 29, 1966

I T has been reported earlier (S. BEERMANN 1959) that the primordial somatic cells in some copepods become different from the germ line cells by a process

of “chromatin diminution” similar to that which occurs in Ascaris (BOVERI 1899). A good example is Cyclops strenuus diuulsus. As observed by HACKER (1894) and by STICH (1954) the mitotic spindles in the 5th cleavage division of this species contain large amounts of chromatin material, and STICH (1954, 1962) has attributed this to an extra synthesis of DNA. The cytological details indicate quite clearly, however, that the “extra” chromatin simply derives from a dis- integration of heterochromatic chromosome segments: In the diplotene stage of the oocytes the chromosomes of C. strenuus diuulsus all possess long terminal seg- ments of heterochromatin. In developing eggs heterochromatin as such is not visible until the telophase of the 4th cleavage division. During prophase of the 5th division the heterochromatin is seen to become dispersed throughout the nucleus in all somatic cells. This material then fills the spindle and is finally expelled into the cytoplasm. At the same time the chromosomes undergo a drastic reduction in their length and some of them show arm ratios which are different from any of those observed prior to diminution (figures in S. BEERMANN 1959). These changes are irreversible, and no heterochromatin has been found in somatic cells following the 5th cleavage. As expected, the chromosomes in the primordial germ cell and its descendants remain unchanged, and big chromocenters are pres- ent in germ line cells at all stages of development.

In C. strenuus divulsus, as in Ascaris, the observed loss of terminal hetero- chromatin during diminution could simply be due to a fragmentation of the chro- mosome regions involved. However, in Cyclops furcifer, another member of the strenuus species group, the chromosomes have heterochromatic segments not only at their ends but also in the kinetochore regions (Figure l ) , and the latter seem to undergo diminution as well (S. BEERMANN 1959; Figure 2). This would imply a more sophisticated mechanism of diminution. Furthermore, the process of diminution in this species, though similar in its essentials to the occurring in C. strenuus diuulsus, has been found to require two divisions for its completion, SO that the first and major diminution in the 6th cleavage division is followed by a second diminution in the 7th cleavage division (Figure 3 ) . These cytological findings make C . furcifer a particularly interesting species for quantitative and experimental studies on the problem of chromatin diminution. A full account of the cytological events in C . furcifer will be published elsewhere. In this paper Crenetlcs 54: 507-57G August 1966

S. BEERMANN 9

FIGURE i.-The distribution of heterochromatin in the germ line chromosomes of Cyclops furcifer. a, b: Oocyte bivalents at early diakinesis; the bivalent in b is heterozygous for its terminal heterochromatin at one end. c: Interphase nucleus between the 4th and the 5th cleavage division. d: late prophase of the 5th cleavage division. K = heterochromatin adjacent to the kinetochores. T = terminal heterochromatin. Acetic-lactic orcein; magnification 18OOX.

an attempt is made to answer the following three specific questions: (1 ) How much DNA is actually lost and how much synthesis of DNA occurs before or during diminution? (2) What is the quantitative relation, in terms of the elim-

CHROMATIN DIMINUTION IN CYCLOPS 569

FIGURE 2.-A comparison of the cytological appearance of interphase nuclci in C. furcilcr just before and just after diminution. Left: Farly interphase nucleus prior to the 6th clravage. Right: Interphase nucleus prior to the 8th cleavage. Notice the complete lack of chromocentrrs in the terminal (lower) as well as the centric (upper) poles of the nucleus. Acetic-lactic orcein, phase contrast: magnification 1OOOx.

inated heterochromatin, between the two cycles of diminution in C. furcifer? (3) Is it possible to prove that the centromeric heterochromatin in C. furrife. is also subject to diminution?

MATERIALS AND METHODS

C. jurcifer was collected from small ponds in the vicinity of Tubingen. Mature females occur only during springtime. Development within each batch of eggs is synchronous. so that the timing of emliryonic stagrs is easily controllable. The eggs were fixed for 6 to 12 hours in ethanol-acetic acid (3:l). They were squashed in 50% acetic acid and left there for 1 to 2 hours. Cover glasses were removed after freezing on drv ice, and the preparations were then stored for 6 to 8 weeks in isopropyl alcohol at 4°C. The Feulgen reaction was carried out following hydrolysis in 1~ HCI at 59 to 61°C for 8 to 10 minutez. (The Schiff reagent was prepared from Pararoanilin. Merck). The preparations were mounted in Caeclex and mrasurements were made within 14 days.

The DNA measurements were made with n 7 ~ i s s "l'niversnl-Mikrospehtral-Photometer" (UMSP I). In scanning a predefined rectangular microscopic field, the instrument automatically records the absorbancy integral in arbitrary units (see CAsrmssoN 1955). Only those nuclei were measured which had been squashed sntisfactor:ly and which were lying in a homogenrous back- ground. The data obtained with the UMSP I are independent. over a wide range, from geomrtric parameters such as the size of the objects measured. thrir shape, and their distribution. Thus, it has been found repeatedly that difTerent division figures in the same prrparation render the s" absorption integrals regardless of whether the chromsomcs are condensed or swollen, regardless of how they are oriented. and rep;ardlrss of the stage of the mitotic cycle stucliecl, including interphase.

The measurements were made at a wave length of 540 mp with a slit width of 0.5 p. The line distance was 0.5 p. Scanning WAS carried out at a speed of either 20 microns/minute (Tables 1 and 4) or 40 microns/minute (Tables 2 and 3). With the UMSP I the recorded absorbancy integrals are inversely proportional to the scanning velocity. Therefore, the data in Tables 2 and 3 have to he multiplied by 2 in order to ht. comparable to those in Tables 1 and 4. (Further

5 70 S. BEERMANN

FIGURE 3 .Zhromnt in diminution, as s-en at late anaphase of the 6th (a) and the 7th (h) clpavnge division in the s3ma of C. furcifer. The eliminated heterochromatin collects in the equator of the sph:lle. Ma t s i a l eliminated in the 6th cleavage division (“Dl”) is still found as droplets in thp rytoplasm during the 7th division. Acetic-lactic orcein; mamification 18OOx.

comments on the validity of the method, as far as Feulgm spectrophotom2try is concerned, may hc found in KEYL 1Wi).

RESULTS

1) In Cyclops furcifer chromatin diminution begins at the 6th cleavage division. A compirison of 6th division figures with earlier ones (Tables 1 and 4)

TABLE 1

DNA in prediminution stages of C. furcifer (Feulgen absorbancy integrals in arbitrary units)

Menn Stnndnrd Siinilrrr nbuirlmicy ermr of Ploid

1952 iiimwml i i i~rgrnl the nicnn Icrclr

Sperm nuclei 6 124 3.6 1c Single anaphase groups, 5th cleavage i 242 22.05 2c Total nucleus, 6th cleavage 5 470 14.12 4c

CHROMATIN DIMINUTION IN CYCLOPS 571

TABLE 2

DNA in chromosomes and eliminaied chromatin during the 6th and 7th cleavage divisions of C. furcifer (Feulgen absorbancy integrals, arbitrary units)

1965

Mean Standard Number absorbancy error of Ploidy measured integral the mean level

Slide GI0 Single telophase groups. 6th cleavage Elimination chromatin, 6th cleavage Single telophase groups, 7th cleavage Elimination chromatin, 7th cleavage Single telophase groups, late

embryonic divisions Slide GI3

Single telophase groups, 6th cleavage Elimination chromatin, 6th cleavage Single telophase groups, 7th cleavage Elimination chromatin, 7th cleavage Single telophase groups, late

embryonic divisions

16 8

10 6

6

16 8 9 5

5

47 99 33 29

33

69 139 4.9 45

49

0.91 4.65 1.57 2.82

0.01

0.93 5.4 1 .o 1.45

1.23

2c

2C

2 c

TABLE 3

DNA in individual telophases of the 6th cleauage division (Feulgen absorbancy integrals, arbitrary units)

Slide GI0 Chromosomes in the

Eliminated chromatin 88 99 107 92 124 115 104 109

Chromosomes in the

Eliminated chromatin 125 118 147 131 150 126 143 163

twotelophasegroups 51-48 51-47 52-47 4 1 4 1 5 M 3 4 6 4 3 4 . 5 4 48-46

Slide G13

twotelophasegroups 69-67 67-67 74-73 70-70 70-74 63-66 61-65 69-68

TABLE 4

Content of DNA in the kinetochore regions (“upper halves”) as compared to that in the total nucleus in pre- and post-diminution stages of C. furcifer

(Feulgen absorbancy integrals, arbitrary units)

Number 1964 measured

Total anaphases, 5th cleavage 15 Total interphases between 5th and

6th cleavage 16 “Upper halves” of interphases

between 5th and 6th cleavage 15 Total telophases, late embryonic divisions 15

2 Total metaphase spindles, 6th cleavage

Mean Standard absorbancy error of Ploidy

integral the mean level

428 17.29 4c

386 13.56 4c

131 3.09 (e) 106 2.03 4.c 412 . . . 4c

5 72 S. BEERMANN

reveals that the total DNA content of chromosomes plus eliminated chromatin in 6th division figures is close to, but does not exceed the total DNA content of the preceding division figures. Diminution must therefore be considered as an actual loss of DNA containing material (“chromatin”) from the chromosomes. It does not involve extra replications. The difference in DNA content between post- and pre-diminution, as percent of the total DNA in pre-diminution stages, approaches 75% in the animals studied in 1964 (Table 4). This is probably an extreme value since material from the same population has given lawer values in other years (e.g. 65% in 1965). The possibility that the presence of “extra” chromatin might be a special feature of the female germ line (oocytes and eggs) can be excluded by control measurements on sperm. As shown in Table 1 the DNA content of sperm corresponds to the IC value expected on the basis of DNA constancy in the germ line of both sexes, that is one-fourth of the total DNA of the 5th cleavage division figures.

(2 As mentioned before, the process of chromatin diminution in Cyclops furcifer spreads over two consecutive division cycles. The chromosomes entering the telophase nuclei of the 6th division may still show heterochromatin at their ends as well as in the centromeric regions (cf. Figure 3a). To study the mitotic distribution of this residual portion of the heterochromatin and to test its subse- quent behavior with respect to DNA replication, eggs from the same batch were fixed at the 6th and 7th cleavage divisions, respectively, and processed together on the same slide, along with other embryos in stages either before or after diminution. DNA measurements were made and the results are summarized in Tables 2 and 3. As explained under MATERIAL AND METHODS, the absorbancy integrals in these Tables have to be multiplied by 2 for a direct comparison with the data in the other Tables. The extinction values from slide GI0 consistently fall below those measured in G13, probably because the GI0 material had been exposed to the fixative for a longer time. This does not, however, affect the con- clusions to be drawn from the data. In both series the telophase groups of the 6th division still contain, on the average, 1.4 times the DNA of the telophase groups in the 7th and in later somatic divisions. In terms of the total DNA differ- ence between the 4C values of germ line and soma about 20% of the germ line heterochromatin thus remains unaffected by the first diminution cycle. As shown in Table 3, some individual DNA values for telophases from the 6th division deviate considerably from the mean, and accidental observations sug- gest that the size of the fraction of heterochromatin escaping the first diminution may be influenced by environmental factors. However, in spite of this variation the two daughter chromosome groups always seem to receive equal (or nearly equal) amounts of DNA (Table 3) . Consequently, in the present material each daughter nucleus of the 6th division starts off with a residue of IO%, on the average, of the original 4C amount of heterochromatin in addition to its euchro- matin. This is the amount which would be expected to appear as DNA eliminated in the second diminution cycle if there were no further replication of this material and provided that diminution were complete. The data presented in Table 2 reveal (1 ) that the telophase groups in the 7th division (cf. Figure 3b) are down

CHROMATIN DIMINUTION I N CYCLDPS 5 73

to the final 2C value of the somatic telophases and (2) that the elimination chromatin actually contains twice the DNA expected. Thus in slide G10 the expected value, i.e. the difference between means of single telophase groups from the 6th and from late embryonic divisions, would be 14 arbitrary units, and 20 in slide G13. The actual values found are 29 in G10. and 45 in G13.

(3) In addition to the terminal heterochromatin each germ line chromosome of C. furcifer carries a discrete block of heterochromatin either on one or on both sides of the centromere. In interphase nuclei between the 5th and 6th cleavage division the centromeric heterochromatin is situated in one half of the nucleus (the former spindle pole) and the terminal heterochromatin in the other (Figures 1, 2, and 4). Whether or not DNA from the heterochromatin of the kinetochore region will also be removed during diminution can be examined by determining the DNA content of the two nuclear poles separately. This is done in the follow- ing way. In setting the automatic transport for scanning of the preparation each nucleus is divided into an "upper half" containing the kinetochores as well as the major portion of the euchromatin, and a "lower half", containing the termi- nal heterochromatin and the remainder of the euchromatin (Figure 4). The divid- ing line between the two measured areas has been drawn as closely as possible to the terminal heterochromatin. Only those nuclei were selected which showed a clear delimitation of the terminal heterochromatin. This condition is usually met with in primordial germ cells where the heterochromatin tends to fuse into a few large chromocenters. Only fully replicated nuclei were used to provide the data in Table 4. The data are from eight different slides. In addition to eggs in the interphase between the 5th and 6th cleavage division, each slide contained mitotic stages from the 5th division and from late stages of embryonic development. The

19 lines DNA: 120

i

13 lines DNA : 200

-4

FIGURE 4.-Micmpectrophotometric scanning as performed on a Feulgen stained primordial germ cell nucleus at n wave length of 540 mp in order to ohtain separate DNA values for the kinetochore regions ("upper halves") and for the terminal het2rochromat:n. See text and Table 4 for further details. Magnificntion 3 0 0 0 ~ .

5 74 S. B E E R M A N N

data from these eight slides were pooled since the absorbancy integrals of late somatic telophases were found to fall in the same range in all slides. As seen from Table 4, the mean 4C value for nuclei before diminution (5th cleavage) is 428. The mean 4C value for later somatic cells is 106. If the DNA of the hetero- chromatin in the kinetochore regions were not subject to diminution, then the DNA content of the “upper halves” should never exceed, and as a rule fall below the normal 4C amount in late somatic cells. The mean absorbancy integral of “upper halves” is found to be 131. This value is significantly higher (t,,=7.14, P < 0.001) than the mean 4C value (106) of late somatic cells. In fact, it exceeds, by almost 50%, the DNA value expected for “upper halves” if there were no kinetochoric diminution. A systematic measuring error as large as this is excluded with the method and the instrument used (cf. MATERIALS AND METHODS). The result indicates that “upper halves” of pre-diminution nuclei contain a substantial amount of DNA which does not survive the diminution process.

DISCUSSION

In confirmation of previous cytological observations the present data demon- strate that “chromatin diminution” in Cyclops furcifer (and presumably in other copepods as well) involves an actual loss of DNA from the chromosomes. There is no extra synthesis of DNA during this process. This conclusion is in conflict with the views of STICH (1962) whose claim that an “excessive amount of DNA is synthesized and subsequently eliminated” is based on spectrophotometry of Feulgen stained sections using the two wave length method. Disregarding the difference in technique, it seems that STICH, in the interpretation of his data, has overlooked the presence of heterochromatin in all stages of the germ line, and its absence in all somatic cells after diminution. This is true for all members of the Cyclops strenuus group which have been tested (C. strenuus diuulsus, C. strenuus strenuus, C . furcifer, Microcyclops uaricans) and for Canthocamptus staphylinus, a representative of the Harpacticidae.

In C. furcifer, the elimination of the heterochromatin in two consecutive division cycles has been found to involve duplication of the eliminated DNA in each cycle. This result would be expected if the primary events of diminution occurred after the S-phase, or after DNA replication has already been initiated. In fact, the heterochromatin can often be seen to be double or split before it visibly disintegrates in the early prophase of the 6th division. Diminution prior to duplication would imply that DNA is still able to duplicate after it has been detached from the chromosomes. This alternative seems less likely, although it would easily explain the equal mitotic distribution of the heterochromatin which is not eliminated in the 6th division. The equal mitotic distribution of the not eliminated fraction could, in fact, be statistical in nature. Differences which might exist between sister chromatids would tend to become randomized in a population of 44 chromosome ends and 22 kinetochores.

As shown by the data on “upper halves” of pre-diminution nuclei, there is an actual loss of DNA (presumably in the from of nucleohistone) from the kinet- ochoric chromosome regions during diminution. The amount of DNA involved

CHROMATIN DIMINUTION I N CYCLOPS 5 75

can be estimated to comprise about 10% of the total DNA cast off during the two cycles of diminution, a value which is in agreement with the cytological length ratios between kinetochoric and terminal heterochromatin. Since the chromo- somes remain V-shaped and are not broken during or after the diminution process (cf. Figure 3) , it seems that interstitial DNA can be removed from the chromo- somes without destroying their linear integrity. This situation can be interpreted as pointing to the presence of a continuous axial thread in chromosomes from which chromatin may be detached (cf. the “centipede” model as discussed by TAYLOR 1957). However, a molecular chain model of the chromosome is also compatible with the findings in C. furcifer, Assuming that the chain consists of alternating “axial” and “nonaxial” segments of DNA (BEERMANN 1965), the axial segments might link directly and make the nonaxial segments bulge out to form loops or rings (Figure 5 ) . The latter could be removed without affecting the continuity of the axial chain. The existence of autonomous ring-like chain members in chromosomes has also been postulated by KEYL (1965) in order to explain localized DNA duplications.

FIGURE 5.-A possible molecular model of chromatin diminution, based on an alternating chain-like arrangement of loop-forming (chromomeric) and connecting (interchromomeric) pieces of DNA. Reversible opening-up of the loop, as illustrated by stages 1 to 3, may occur as a regular event in some phases of the chromosome cycle, e.g. in spermiogenesis.

5 76 S. B E E R M A N N

The course of events during the two consecutive cycles of diminution may now be interpreted as follows. In the early prophase (GZ) of the 6th cleavage division a factor is liberated, probably a DNA-ase, which specifically attacks the hetero- chromatic regions of the chromosomes in such a way that only nonaxial segments are removed. The enzyme stops acting in late prophase of the 6th cleavage. The remainder of nonaxial material in the heterochromatin then replicates during the following S-phase before it is again attacked by the same enzyme and com- pletely removed. On this model the fraction of heterochromatin eliminated during the first diminution division may vary widely since a preference of the enzyme for specific regions within the heterochromatin is not postulated. The actual amount of heterochromatin attacked during the first diminution cycle would depend on the enzyme activity available and on the actual duration of this cycle.

The author is indebted to PROFESSOR DR. H. BAUER for making available the UMSP I and to DR. H. G. KEYL for advice and help in carrying out the measurements.

SUMMARY

Quantitative aspects of the process of "chromatin diminution" in early embryo- genesis of the Copepod, Cyclops furcifer (a member of the strenuus group), have been investigated by means of Feulgen microspectrophotometry, using a high resolution automatic scanning and recording system. The data lead to the follow- ing conclusions: (1 ) The chromatin material which is expelled from somatic nuclei during the 6th and 7th cleavage division does not arise by additional, excessive DNA synthesis between the 5th and 6th cleavages. It derives from the disintegration of heterochromatic chromosome regions. (2) Heterochromatin which survives the 6th cleavage replicates regularly along with the euchromatin before it is eliminated in the 7th cleavage division. (3) The centromeric blocks of heterochromatin in C. furcifer are subject to the same elimination as the terminal ones. This removal of interstitial DNA does not result in breakage of the chromosomes.

LITERATURE CITED

BEERMANN, S., 1959 BEERMANN, W., 1965

BOVERI, T., 1899

CASPERSSON, T., G. LQMAKKA, G. SVENSSON, and R. SAFSTROM, 1955

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spectrograph for multiple-line and surface scanning high resolution measurements employ- ing automatized data analysis. Exptl. Cell Res. (Suppl.) 3: 40-51.

Uber generative und embryonale Mitosen, sowie iiber pathologische Kern- teilungsbilder. Arch. mikr. Anat. 43 : 759-787.

Duplikationen von Untereinheiten der chromosomalen DNS wahrend der Evolution von Chironomus thummi. Chromosoma 17 : 139-180.

Stoffe und Stromungen in der Spindel von Cyclops strenuus. Chromosoma 6: 199-236. - 1962 Variations of the desoxyribonucleic acid (DNA) content in em- bryonal cells of Cyclops strenuus. Exptl. Cell Res. 26: 136-I&.

The time and mode of duplication of chromosomes. Am. Naturalist 91: 209-221.

HACKER, V., 1894

KEYL, H. G., 1965

STICH, H., 1954

TAYLOR, J. H., 1957