Meiotic chromatin diminution in a vertebrate, the holocephalan fish Hydrolagus collie (Chondrichthyes, Holocephali)

TISSUE & CELL 1984 16 (2) 203-215 @ 1984 Longman Group Ltd.

HUGH P. STANLEY*,

HAROLD E. KASlNSKYt

and NIELS C. BOLSS

MEIOTIC CHROMATIN DIMINUTION IN A VERTEBRATE, THE HOLOCEPHALAN FISH HYDROLAGUS COLLIE (CHONDRICHTHYES, HOLOCEPHALI) Key words: Meiosis.

chromatin

diminution,

vertebrate,

Chondrichthyes.

ABSTRACT. A histochemical, microdensitometric, and electron microscopic study of testes of the ratfish Hydrolagur colliei shows that an instance of the rare phenomenon of germ line chromatin diminution occurs in this vertebrate species. In primary spermatocytes at metaphase I a spherical mass of heterochromatin accumulates at one side of the metaphase plate. At anaphase I the heterochromatic mass is left in the equatorial cytoplasm and is passed into one of the two secondary spermatocytes formed during cytokinesis. As nuclear membranes are being restored, a double membrane envelope is also formed around the heterochromatic mass, which is then termed the ‘chromatin diminution body’ (CDB). At second meiotic division the CDB is included in the cytoplasm of one of the four spermatids and retained there, apparently unchanged, until mid-spermiogenesis. At that time the CDB becomes adherent to the spermatid plasma membrane and is pinched off from the spermatid by a process of apocrine exocytosis, taking a layer of spermatid plasma membrane along with it. Simultaneously this t&membrane CDB is taken into the adjacent Sertoli cell by endocytosis, thereby acquiring a fourth membrane layer, a part of the Sertoli cell plasma membrane. The CDBs are subsequently phagocytized, possibly first fusing with dense, multilaminate bodies in the Sertoli cell cytoplasm. The CDB chromatin mass is strongly positive with the Feulgen method for DNA and the alkaline fast green method for histones. Microdensitometric analysis shows that the discarded chromatin amounts to about 10% of the diploid nuclear content and that it appears to be part of the normal diploid complement rather than DNA amplified during meiosis.

Introduction

or may be redundant chromatin that has been amplified during a previous stage. Most described cases of chromatin diminution occur in somatic cells of invertebrates rather than in germ cells. We here describe a very rare, and in some ways unique, instance of germ line chromatin diminution in a vertebrate, the holocephalan fish, Hydrolagus colliei.

Since the discovery of chromatin diminution in the nematodes by Boveri in 1887, similar phenomena have been described in several taxonomically scattered animal groups (for reviews see Beams and Kessel, 1974; Brown and Chandra, 1977; Swanson et al., 1981). There are numerous variations described in cases of chromatin diminution. Parts of chromosomes are discarded in some species while whole chromosomes or even entire haploid sets are separated from the nucleus in others. Also, the chromatin that is discarded may constitute a normal part of the genome

Materials and Methods Males of the ratfish, Hydrolagus colliei, were collected by trawling operations in waters of the San Juan Islands, Washington, and off the coast of Comox, British Columbia. For electron microscopy, specimens were fixed in 2.5% glutaraldehyde in 0.20M Millonig’s phosphate buffer with 0.17M NaCl for 1.5-2 hr at room temperature (Cloney and Florey, 1968), washed in 0.2 M Millonig’s buffer with 0.17M NaCl three times for 15 min each. Post-fixation was in

*Department of Biology, Utah State University, Logan, Utah, and Friday Harbor Laboratories, Friday Harbor, Washington. tDepartment of Zoology, University of British Columbia, Vancouver, B.C., Canada. *Department of Biology, University of Waterloo, Waterloo, Ontario, Canada. Received 9 August 1983. Revised 10 October 1983. 203

204

STANLEY,

cold 2.0% 0s04 in 0.2 M Millonig’s buffer for 1 hr. Tissues were dehydrated in a cold ethanol series followed by propylene oxide and embedded in Poly-Bed 812 resin A and B (Polysciences, Inc.) in the proportion of 1: 1. Thin sections were stained in half saturated aqueous uranyl acetate for 10 min at 60°C and

KASINSKY

AND BOLS

then for 5 min in lead citrate (Reynolds, 1963). Sections were examined with a Philips EM 300 electron microscope. For histochemical studies, fragments of testes were fixed in 10% neutral buffered formalin or in absolute ethanol/glacial acetic acid (3: 1) and embedded in Paraplast Plus

Fig. 1. Nine stages of spermiogenesis in the ratfish, Hydroiagus colliei based on the morphology of nucleus and acrosome. Stage I. Nucleus spherical, 4 pm in diameter; chromatin dispersed. Acrosomal vesicle small, not attached to nucleus. Stage 2. Nucleus sphencal but indented by the adherent spherical acrosomal vesicle (A). Nuclear chromatin forms a coarse network attached to the nuclear envelope adjacent to acrosome. Fibrous nuclear sheath (FS) spread slightly outward from acrosomal area. Stage 3. Acrosomal vesicle lenticular in shape. Axial midpiece rod (AR) rudiments attached to nuclear envelope on opposite side from acrosome. Nuclear chromatin attached both at acrosomal and axial midpiece rod attachment sites. Fibrous nuclear sheath covers about one-fourth of nuclear surface. Stage 4. Acrosome broadly conical in shape. Fibrous nuclear sheath covers about one-half the nuclear envelope. Nucleus slightly longer than wide. Stage 5. Nucleus about 7 pm in length with distinct separate chromatin fibers longitudinally orientated. Fibrous nuclear sheath covers two-thirds of nuclear envelope surface. Acrosome a conical cap over pointed antcrlor end of nucleus. Stageb. Nucleus about 10 m in length with distinct chromatin fibers longitudinally orientated but bound side to side into anastomosing sheets. Stage 7. Nucleus about 16 pin length; chromatin fibers Joined m coarser bundles and sheets and showing some spiral arrangement. Fibrous nuclear sheath covers about 90% of nuclear envelope. Nuclear envelope folded posteriorly beyond cxtrnt of chromatin; surrounds anterior part of axial midpiece rod. Nuclear pores on inner fold of nuclear envelope. Stage 8. Nucleus about 18 pm in length with chromatin condensed, formed mto an essentially straight cone tapering anteriorly. Nuclear envelope and fibrous nuclear sheath as m stage 7. Stage 9. Nucleus about 20 m in length. Condensed chromatin head spiral posteriorly. straight anteriorly. Nuclear envelope and fibrous nuclear sheath as in stage 7.

CHROMATIN

DIMINUTION

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205

(Fischer Scientific Co.). DNA was stained by the Feulgen method as outlined by Cullin (1963) and histones by the procedure of Alfert and Geschwind (1953). Microdensitometry studies were performed on cell smears stained by the Feulgen method using either a Vickers M85 scanning microdensitometer or a Zeiss MPMOlK microspectrophotometer. A monochromator was set at 573 nm, the point of greatest absorption. A phase contrast 40X Zeiss objective was used. Measurements were recorded in the transmission mode and subtracted from back-

ground readings taken nearby to give a measure of absorbance. A tentative system for staging the events of spermiogenesis has been constructed based on nuclear and acrosomal morphology. Nine stages are illustrated in Fig. 1 and descriptions in the text are based on these stages. Results At first meiotic metaphase, a spherical mass of heterochromatin about 1.7 pm in average diameter first becomes distinct from the chromosomes (Figs. 2, 3). It usually lies at one edge of the metaphase plate. Microtubules are rarely seen in the immediate vinicity of this chromatin body (CM in Fig. 2) and no kinetochores

or microtubules

have

been observed to be associated as is the case with the nearby metaphase chromosomes. When the nuclear envelopes are established at first meiotic telophase, a double membrane envelope also forms around the chromatin body. We now call this double membrane-bounded heterochromatic mass the chromatin diminution body (CDB). After cytokinesis the CDB is found in the cytoplasm of some secondary spermatocytes (Fig. 4). Its contents remain heterochromatic whereas the secondary spermatocyte nuclei

enlarge and their chromatin becomes highly dispersed (Fig. 5). Secondary spermatocyte nuclei measure about 5.2 m in diameter at this stage and the CDB is about 1.8 pm in diameter. Both the nuclei and the CDBs are strongly positive when stained by the Feulgen method for DNA (Figs. 4,6) and the alkaline fast green method for histones. A smaller number of the early spermatids than of the secondary spermatocytes contain a CDB (Figs. 6, 7). Results of careful counts of nuclei and CDBs made on 10 pm paraffin sections of different cell stages are shown in Table 1. The results support the interpretation that one CDB is formed at each first meiotic division and is included in one of every two secondary spermatocytes thus formed. Subsequently, at second meiotic division, the CDB is retainted in the cytoplasm of one of each four spermatids. The CDBs are retained through approximately half of the spermatid transformation stages as explained below. Secondary spermatocytes are larger than spermatids and are thus more subject to counting errors. This is due to the greater possibility that at the upper and lower surfaces of the sections a nucleus is more likely to be included in the section without its CDB and vice versa. Fig. 8 illustrates a stage 4 spermatid in approximate longitudinal section with a CDB near the posterior pole of the nucleus. The CDB retains its former size and degree of chromatin condensation. By spermatid stage 5 (Figs. 1, 9) the spermatid nuclei have elongated to between 7 and 9 pm and most CDBs have migrated to the anterior pole of the cells. They are often contained in asmall bleb of spermatid cytoplasm and lie directly apposed to the spermatid plasma membrane (Fig. 9). During early stage 6, CDBs first appear isolated in the cytoplasm of the surrounding

Sertoli cells (Fig. 10). The CDBs

Table 1. Distribution of CDBs among stages of spermatogenesis.

For a

description of stages refer to Fig. I

Cell type Secondary spermatocytes Stage 2 spermatids Stage 6 spermatids

Number of nuclei

Number of CDBs

94 223 10s

38 56 24

Ratio nuclei/CDBs 2.4711 3.9911 4.3711

206

STANLEY,

within Sertoli cytoplasm are surrounded by four concentric unit membranes instead of two. The precise mechanism by which the CDBs are transferred from spermatids to Sertoli cells is unknown. Nevertheless it can be inferred that the CDBs are expelled from the mid-stage spermatids by a process of apocrine exocytosis and taken into the Sertoli cells by membrane invagination (endocytosis). The innermost two membranes are the original ‘nuclear’ membranes of the CDBs. Surrounding these membranes is one origi-

KASINSKY

AND BOLS

nating from the spermatid plasma membrane and, lastly. a portion of the invaginated Sertoli cell plasma membrane forms the outermost membrane layer. The latter membrane has a granular layer on the surface facing the spermatids. and that same granular material is retained on the inner surface of the outermost of the four membranes surrounding the discarded chromatin (Fig. 11). It is apparent that most or all CDBs have passed over to the Sertoli cells by a slightly later stage (spermatid nuclei 12- 15 pm in length).

Fig. 2. An equatorial view of a portmn of the metaphase plate ot a diwding pnmary spermatocyte. Metaphase chromosomes are seen at the left and the prospective chromatin diminution mass (CM) at the right. x21 .OOO. Fig. 3. Photomicrograph of a I firn epoxy section through a metaphase viewed from one pole. The chromatin mass to he eliminated is indicated

primary spcrmatocyte by the arrow. x46W.

Fig. 4. Photomicrograph of secondary spermatocyte stained with the Feulgen method for DNA. The nucleus (N) and the chromatin diminution hody (CDB) are both strongly positwc. X4MW). Fig. 5. Electron micrograph of a secondary spermntocytc diminution body (CDB). A double ‘nuclear’ envelope surrounds proper (N). ~20,000.

and accompanying chromatm the CDB as well as the nucleus

Fig. 6. Photomicrograph of an early apermatid stained with the Feulgen method strongly positive nucleus (N) and chromatin diminution body (CDB). x4600 Fig. 7. Electron mlcrograph of an early \permatid (CDB) adjacent to the nucleus (N). X28.000.

(stage 1) with chromntm

\howmg

a

diminution

hod!

Fig. 8. Electron micrograph ot a stage 4 spermatid with adjacent chromatin dimmution (CDB). The acrosome (A) and flagcllar apparatus (F) are indicated. x24.(X)0.

hod!

Fig. 9. Electron micrograph of a stage 6 spermatid. The CDB is positioned near the acrowmsl end of the nucleus. Chromatin m the nucleus (N) is formed into longitudinal strands. The CDB 15 adherent to the sperm plasma membrane (PM) over part of its surface. Sertoh cell cytoplasm (SC) and Sertoli cell plasma membrane (SPM) closely surround the entire spermatid. A multilaminate body (MB) is positioned nearby. x24.000. Fig. IO. An electron micrograph of a portlon of Sertoli cell cytoplasm m conjunctmn Mlth stage 6 spermatids. A CDB surrounded by four membranes lies free within the Scrtoli cytoplasm. The central two membranes (arrow) are closely adherent to each other. Scrtoli cell mitochrondrin (M). x36.000. Fig. I I. A portion of a CDB at higher magmflcatwn showing the &car&d hetcrochromatm (CH), Sertoli cell cytoplasm (SC) and the four concentric layers of mcmhrane that form the boundary of the chromatin diminution body (numbers l-4). Membrane number 4 show a layer ofgranular material on its inner fact, typical of the surface of Scrtoli cell membrane that faces the spermatids. x91.OW. Fig. 12. Two CDBa in the cytoplasm ot a Sertoli membranes appear to he folding inwards. x32.000.

cell at spermatid

stage 7. The innermost

STANLEY.

Fig.

13. Feulgen-positive

Fig. 14. A presumed have not been observed surrounding

Sertoli

Fig. 15. A higher is at lower between Fig.

The

body

Fig. 17. An clcctron center

of the presumed

Fig.

18. Cross-section (N)

of stage 6 spermatids

chromarin

cytoplasm

cells prior

BOLS

material.

associated

\hwwng

multiple

x43.0(#).

with stage 6apermatids

to this stage in which

CDBs

Such hodick

are transferred

to the

x34.000.

of a portion

magmfication

16. A presumed

cytoplasm

presumed

tn Sertoli

in Sertoli

right surrounded

spcrmatids.

in Scrtoh

with

CDB

cells.

the outermost

chromatin

body

together

AND

BY comparing 1 pm epoxy sections stained by the Feulgen procedure with adjacent thin sections, it was possible to show a positive Feulgen reaction in the structure illustrated in Fig. 13. In this body a chromatin-like material is surrounded by a multilayered membranous cortex. Such structures first appear in the basal cytoplasm of the Sertoli cell during spermatid stages 6 to 7. In the same region of the Sertoli cytoplasm at these stages 6 and 7. multilaminate structures with a central, dense, chromatinlike mass are found (Fig. 14). These structures are similar in size to the CDBs. but rough calculations indicate that they possess at least ten times the membrane surface area found in those CDBs that have just four membrane layers. There are 25-27 membrane layers in the structure (Figs. 14, 15). Multilaminate bodies without the chromatinlike mass in the interior are seen in the Sertoli cytoplasm prior to and during these midspermatid stages. They lie close to the region in which the CDBs are transferred to the Sertoli cells (Fig. 9). It is possible that the CDBs fuse with one or more of these multilaminate bodies shortly after being passed into the Sertoli cell. Such fusion could possibly account for the great increase in membrane layers between stages represented in Figs. 10 and 14. Without definitive tracer

Both the Feulgen reaction for DNA and the alkaline fast green reaction for histones generally become negative in the CDBs at this stage, but continue to be positive in the nuclei. We interpret this observation to mean that the DNA and histones of the CDBs are quickly subjected to biochemical degradation after being phagocytized by the Sertoli cells. No nuclear pores have been observed in the double membrane envelopes surrounding the CDBs. Occasional groups of ribosomes are attached to the outer surface of the double envelope, as is the case with nuclear envelopes. The chromatin does not show any obvious attachment sites to the inner membrane. After entry into Sertoli cells the second and third membranes that surround the CDBs, that is the outer CDB ‘nuclear’ membrane and the membrane putatively derived from a part of the spermatid plasma membrane, are consistently more closely apposed than the innermost and outermost layers are to either of the central two membranes (Figs. 10, 11). This observation may infer a closer bonding of these two membranes. Many of the CDBs that lie within Sertoli cells show greater irregularity in their outlines and show some disruption of the contained chromatin. The inner membranes appear to expand and bleb inwards (Fig. 12).

layers of membrane

KASINSKY

by 27 concentric

two

layers

CDB

lying

measures

0.8 m

from a xc(Ion

stained nuclei

a atage Y spermatld

cnlargcd,

14. The chromatm

Granular

of a Scrtoli

in diameter.

and the spcrmntid

through

and a somewhat

shown m Fig.

material

core

1s prcscnt

~154,OtK).

m the cytoplasm

about

micrograph CDB

(arrow).

of the CDB

layers of mcmbranc.

rctamed

cell asaocutcd

with uranyl (not

acetate

shown)

\howmg

chromatin

with

stage Y

x88.000.

the

diminution

stam only.

stain deeply. highly

condcnacd

body (CDB)

Borh the

x55.(X)0. nuclear xZO.OW

212

STANLEY, KASINSKY AND BOLS Table 2. Feulgen microdensitometric

Cell type

analysis of various nuclei and C‘DBs

Number of readings

Red blood cells Pachytene primary spermatocytes Early spermatids CDBs

methods, however, the transformation of a CDB into the multilayered body shown in Fig. 14 must remain speculative. By spermatid stages 8 and Y the Sertoli cytoplasm shows similar bodies much reduced in site (Fig. 16). These bodies are only 0.5-0.9 pm in diameter and their total membrane surface area is only about one-half that of the newly phagocytized CDBs. Uranyl acetate stains the interior core deeply (Fig. 7) just as it stains the condensed nuclei of spermatids. This is circumstantial but not conclusive evidence of their nucleic acid composition. In very rare cases, a CDB is not passed over to a Sertoli cell but is retained within the spermatid. One such example in a stage 9 late spermatid is shown in Fig. 18. This body is about 2.5 pm in average diameter. slightly larger than in previous stages, and the contained chromatin shows a looser and more coarsely fibrous aspect. The data in Table 2 show that chromatin equivalent to about 10% of the diploid genome is sequestered in the CDBs and is expelled from the germ line. Because the amount of discarded chromatin is smaller than the standard deviation of the primary spermatocyte values, a definitive interpretation on the origin of the CDB-DNA is not yet possible. Nevertheless. a doubling of the red blood cell value gives 19.9, only slightly higher than the mean value for primarry spermatocytes. If one quadruples the spermatid mean absorbance value and adds one CDB absorbance value, one gets a total of 20.04, slightly higher than the primary spermatocyte mean, but within the range of standard deviation for these cells. Secondary spermatocytes were rarely encountered in this material, but two cells. each with two verifiable secondary spermatocyte nuclei and one CDB, were measured in toto for light

25 25 2s 2s

Mean absorbance value

9.95 19.32 3.75 143

Standard deviation

Standard error

0.69 1.68 0.64 0.32

0.13 0.34 0. I6 0~06

absorbance. The values were 20.1 and 19.6 respectively. These data are consistent with the interpretation that little or no DNA amplification occurs and that the CDB represents DNA that is a normal part of the diploid genome. Light microscopic observations on testes of Chimaera monstrosa, another member of the family Chimaeridae, also shows the chromatin diminution body typical of Hydrolagus. Furthermore, preliminary work on two species from New Zealand (Stanley. unpublished) indicates that chromatin diminution bodies are present in spermatids of Harriotta raleighana (family Rhinochimaeridae) and Callorhynchus milii (tamily Callorhynchidae). Thus at least one species from each of the three holocephalan families appears to undergo germ-line chromatin diminution. Discussion We define the term chromatin diminution as instances in which deoxyribonucleoprotein is normally and regularly destroyed, either by disintegration within the cytoplasm or, more rarely, by expulsion from the cell. The phenomenon described here in the ratfish is a true case of chromatin diminution because a mass of DNA and histone is separated from the nuclei at first meiotic division and eventually is ejected from the spermatids. The discarded chromatin appears to be left behind as the chromosomes move to the poles at anaphase, apparently because the chromatin to be discarded has no kinetochore sites and no microtubules to mediate its movement. At its first discernible appearance in ratfish. the discarded chromatin is in the form of a single large mass and not as several distinct pieces. We therefore cannot as yet say whether the CDB chromatin is composed

CHROMATIN

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same time that nuclear envelopes are constructed around the nuclei of the two secondary spermatocytes. This phenomenon has not been described in any other animal. Second, the chromatin diminution body thus formed is retained in one of the two secondary spermatocytes at cytokinesis and in one of the four spermatids resulting from the meiotic divisions of each primary spermatocyte. Thus it is retained for some time in the germ cells. The enclosure of the segregated chromatin in a double ‘nuclear’ envelope undoubtedly facilitates such retention rather than immediate degradation in the cytoplasm. About midway through the transformation of the spermatids into spermatozoa the CDB is finally expelled by a process of apocrine exocytosis, taking with its original double membrane envelope a layer of tion spermatid plasma membrane. It is imA notable difference between chromatin diminution in Hydrolagus and that in most mediately phagocytized by the closely adheother animals is that diminution occurs in the rent Sertoli cell, adding a fourth membrane germ cell line in Hydrolugus while it is the layer from the plasma membrane of the latter somatic cells in which DNA loss occurs in cell. We know of only one other phenomenon comparable to this, i.e. the extrusion and most other forms. It is only in the dipteran phagocytosis of the erythroblast nucleus in Sciara and the heteropteran Macroceroea most mammals (Bessis, 1964; Weiss, 1965). that we can find examples of true chromatin diminution occurring in the germ cells. In As to the nature of the discarded DNA and the possible function of such loss in the Sciara (Abbott et al., 1981, Metz, 1933,1938) the entire paternally derived haploid set of ratfish, we can only speculate at this time. chromosomes is eliminated at metaphase I in Goldstein and Straus (1978) found that the discarded DNA in Ascaris suum consisted of a polar body-like process. In Macroceroea unique and repetitive sequences in a ratio of (Bannerjee, 1959) fragments of X chromosomes are eliminated at anaphase I. In about 1: 1. Roth and Moritz (1981), however, found that this germ line-limited chromatin in Hydrolagus, only about 10% of the diploid amount of chromatin is discarded, so it the same species consisted entirely of two cannot consist of a haploid set of chromofamilies of tandemly repeated sequences that can be interpreted as satellite DNA. Bantock somes. Among the vertebrates, we can find only (1970) found that germ line-limited chromaone group in which chromatin diminution, tin in the dipteran, Muyetiola, was essential within our prescribed limits of the term, for germ cell production. In Wachtliella occurs: marsupial mammals of the genera (Kunzetal., 1970), another dipteran genus, it Perameles and Isodon (Hayman and Martin, was found to produce RNA during 1965). In these mammals the somatic cells oogenesis. Walker (1971) and Lewin (1982) eliminate one chromosome, the Y in males postulate that satellite DNA may act in some and one X chromosome in females. The germ way to stabilize the processes of synapsis and cell line, however, retains XX and XY crossing over. Such a function is consistent with our observations on Hydrolagus since complements. The discarded chromosomes in these cases, are extruded through the the chromatin is discarded just at the end of nuclear envelope and disintegrate in the meiotic prophase. cytoplasm. We have noted that chromatin diminution Chromatin diminution in Hydrolagus is appears to occur in members of all three unusual in at least two other ways. First, the holocephalan families. It may, therefore, be chromatin that is set aside for elimination is a widespread phenomenon within the group, bounded by a double nuclear envelope at the but no such phenomenon has been recorded

of whole chromosomes or of excised parts of chromosomes. Ohno et al. (1969) described the diploid metaphase karyotype of Hydrolagus colliei as consisting of about 58 chromosomes. He noted a haploid number of four macrochromosomes, several medium-sized chromosomes, and many smaller, dot-like microchromosomes. It is possible that the CDBs in Hydrolagus are composed of an amalgamation of numerous whole microchromosomes. We postulate that this is the case and that therefore the discarded chromatin is part of the normal diploid genome (or tetraploid in the case of late primary spermatocyte) rather than DNA that has been amplified from the genome during meiotic prophase. The microdensitometric studies are consistent with this interpreta-

214

STANLEY,

in the other subclass of chondrichthyan fish, the Elasmobranchii, although spermatogenesis is quite similar in the two groups (Boisson et al., 1968; Stanley 1971a, b, and personal observation). Beams and Kessel(l974) offer a plausible explanation for the fact that chromatin diminution occurs in some species and not in others that are closely related. Chromatin diminution would appear to be a rarely used alternative form of genome silencing, the more usual method being heterochromatization. This is especially clear in the coccids (see reviews by Brown and Nur, 1964; Brown and Chandra, 1977; Swanson et a/., 1981). Chromatin diminution early in embryogenesis would seem to be advantageous in that replication of that chromatin would not be necessary cell generation after cell generation. If it is advantageous over heterochromatization, however, why is the practice not more prevalent? Chromatin diminution has been reported in a variety of animal forms including nematodes (Boveri, 1887; Tobler et a/., 1973), copepods (Beerman, 1959), mites (Cooper, 1939; Nelson-Rees et al, 1980), coccids (Hughes-Schrader, 1948), hemipterans (Bannerjee, 1959) and various species of dipterans (Beermann. 1956; Fux, 1974; Geyer-Duzinska, 19.59; Metz, 1933, 1938; Nicklas, 1959). Certain marsupial mammals (Hayman and Martin, 1965) also exhibit diminution of parts of the normal genome. Redundant DNA in amphibian oocytes (Brown and Dawid, 1968) and in the macronuclei of several species of ciliate

KASINSKY

AND BOLS

protozoa (Cleffmann, 1980; Dysart, 1960; Meyer and Lipps, 1980) also is subject to degradation. From the widely dispersed occurrence of chromatin diminution and the quite varied amounts and nature of the DNA discarded, as well as the diversity of types of cells and parts of the life cycle in which it occurs, chromatin diminution has apparently arisen independently a number of times during evolution. Loss of chromatin from germ cells is extremely rare in the animal kingdom. One may speculate that in the present case the lost chromatin might serve a limited structural function rather than an essential genetic function. It will be of interest to determine whether ratfish oocytes also show diminution or instead supply to the zygote the type of chromatin lost from the male germ line. Alternatively, the retained chromatin may preserve a few copies of the diminished chromatin as has been reported in Ascaris suutn (Roth and Moritz, 1981). Cytogenetic analysis is currently being pursued in our laboratories.

Acknowledgements The authors wish to thank Dr Charles Laird and MS Linda Wilkinson. University of Washington, Dr William Campbell and Dr Charles Ferris, Utah State University, for assistance and use of equipment for microdensitometry. We also thank Dr Lloyd Bennett, Utah State University, for assistance in statistical analysis.

References Abbott, A. G., Hess, J. E. and Gerbi. S. A. lY81. Spermatogenesis in Sciaro copruphilu. I. Chromosome orientation on the monopolar spindle of meiosis. Chromosomu. 83, l- 18. Alfert, M. and Geschwind. I. 1953. A selective method for the basic proteinsof cell nuclei. Proc mtn. Acud. Ser. U.S.A., 39,991-999. Bannerjee. M. R. 1959. Chromosome elimination during meiosis in the males of Macrocerveu (Lohrra) grandis (Gray) (Pyrrhocoridae, Heteroptera). Proc. mol. Sot. Lond.. 12, 1-8. Bantock, C. R. 1970. Experiments on chromosome elimination in the gall midge May&da destructor. J. Embryol. exp. Morph., 24, 257-286. Beams, H. W. and Kessel, R. G. 1974. The problem of cell determinants. fnr. Rev. Cytol.. 39, 413-479. Beermann. S. 1959. Chromatin-Diminution bei Copepoden. Chromosoma, 10, 504-514. Beerman, W. 1956. Nuclear differentiation and functional morphology of chromosomes. Cold Spring Hub. Symp. qunnt. Biol., 21, 217-232.

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