Cytophotometric study of nuclear proteins during gametogenesis in Ascaris lumbricoides

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Experimental Cell Research 102 (1976) 191-199

CYTOPHOTOMETRIC DURING

STUDY

GAMETOGENESIS J. PASTERNAK

OF NUCLEAR

PROTEINS

IN ASCARZS LUMBRZCOZDES and R. BARRELL

Department of Biology, University of Waterloo, Waterloo, Ontario N2L 3GI I Canada

SUMMARY Microspectrophotometric analyses of histone and basic nuclear proteins indicated that characteristic changes occur during the development of male and female gametes in Ascaris lumbricoides. The lysine-rich histone/Feulgen-DNA ratio varies slightly during spermatogenesis, whereas the arginine-rich histone/Feulgen-DNA ratio increases prior to meiosis and then dramatically decreases during spermiogenesis. Similarly, both the Sakaguchi-arginine/Feulgen-DNA and fluorodinitrobenzene-lysine plus tyrosine/Feulgen-DNA ratios increase as the spermatocytes develop and than fall after meiosis. Low levels of both histone and basic protein are retained by the nuclear material of the mature sperm. The Feulgen-DNA values of sperm are hypohaploid. Both the arginine-rich and lysine-rich histone/Feulgen-DNA values increase as the primary oocytes mature. Concomitantly, there is a steady accumulation of basic nuclear proteins as revealed by binding of the Sakaguchi reagent and fluorodinitrobenzene. Feulgen stainability is abruptly lost as the primary oocytes reach the terminal quarter of the reproductive tract thereby precluding further quantitation. There is no demonstrable evidence for a nuclear to cytoplasm transfer of basic proteins during either gametogenic process.

Programmed flucutations in protein synthesis occur in many diverse differentiating systems [l-5]. In these cases, differential genie activity accounts for the temporal sequence of many of the biochemical events although the question of how these processes are controlled remains unclear. In addition to somatic cell diversification, macromolecular changes during gametogenesis have been examined in several organisms with special attention to the behaviour of histones during spermiogenesis [6-8]. Similar studies on oogenesis have been rare. Germinal differentiation entails a succession of steps that include the switch from the mitotic cycle to meiosis, the development of chemically and morphologically 13 -761808

distinctive gametes and the preparation of both the germ cell genome and cytoplasm for embryogenesis. Traditionally, the parasitic ascarid nematode has provided material for cytological studies of gametogenesis [9]. Many of the reasons for using such an organism are still applicable. Briefly, the reproductive tracts of the mature sexes are extensive narrow tubes which contain gonocytes in a well-defined linear array. Not only does each gonadal segment carry a cluster of meiocytes that are at the same stage of development; but these worms tend to be prolific gamete producers. In addition, the ascarid nematodes have other intriguing biological properties. For example, the mature oocyte has a paucity of cytoplasmic RNA [lo] with subExp Cell Res 102 (1976)

192

Pasternak and Barrel1

sequent extensive ribosomal RNA (rRNA) synthesis being directed by the male pronucleus after fertilization [ 111. Second, relatively few cleavage divisions are required for the completion of embryogenesis and thereafter cell division tends to be severely restricted in most somatic tissues

[Ql. The disadvantage of these organisms is that they are not readily amenable to either isotope labelling or genetic studies. Notwithstanding, aspects of nucleic acid cytochemistry during gametogenesis in ascarids have been studied [lo, 13, 141. As well, a histone transition during spermiogenesis is suggested by the observation that the non-flagellated sperm of Ascaris contains a “protamine-like” protein (Mackey & Heinen, 1968, as cited in ref. [6]). To date, there has been no complete study of basic protein changes during the various phases of gametogenesis in Ascaris. In this paper we examined cytophotometrically the quantitative changes in DNA, histone and basic nuclear protein content during spermatogenesis and oogenesis in Ascaris lumbricoides. The reactions utilized here include: (1) the Feulgen technique for DNA; (2) the Alfert & Geschwind procedure [ 151, with or without acetylation, for histones; and (3) both the Sakaguchi and l-fluoro-2,4dinitrobenzene (FDNB) techniques for basic nuclear proteins. Discrete patterns of nuclear protein transitions were observed during gametogenesis in both sexes. MATERIALS Preparation

AND METHODS

of material

Ascaris lumbricoides var. suum was obtained from a local slaughterhouse. In the laboratory, adults were separated, grouped into sexes and thoroughly washed in warm deionized water. Individual live specimens were submerged in 0.14 M NaCI, pinned to a dissecting tray and slit longitudinally. The complete reproductive tract was carefully excised, unravelled and Exp CellRes 102 (1976)

its total length measured. The mature male gonad is about 140 cm long. The female tract consists of two branches, each about 210 cm in length. After removal, each kind of gonoduct was cut into successive 5 cm segments. For the purpose of comparison, the location of gonadal segments from various animals are expressed as a per cent of the total length of the reproductive tract starting from the germinal tip. Gametocytes from some of the isolated gonadal segments were extruded into a drop of distilled water on separate slides. The cells were air-dried overnight at 42°C and fixed in absolute ethanol : glacial acetic acid (3 : 1, v/v), absolute ethanol, 100% methanol or buffered neutral 10% formalin at 5°C. Intact gonadal segments were also fixed and retained for embedding in 93.4% purified glycol methacrylate [16]. Sections were cut to about 5 pm.

Staining procedures Material stained with Feulgen only was hydrolysed for 30-35 min in 5 N HCl at 25°C [17]. The conditions for ootimal hvdrolvsis were checked regularly. After hydrolysis, the preparations were ri&ed once in distilled water, immersed in Schiff s reagent for 2 h, decolourized in fresh sulfite bleach, washed, air-dried and mounted in immersion oil (nn= 1.568: R. P. Cartille Laboratories, Ltd). The alkaline fast green (pH 8.1) procedure of Albert & Geschwind [15] was used to stain basic proteins. Routinely, one set of the material was processed with the pH 8.1 fast green method while a parallel sample was acetvlated in 100% acetic anhvdride in absolute alcohol ai 60°C prior to staining. Both the SakaguchiFeulgen and 1-fluoro-2,4-dinitrobenzene (FDNB)Feulgen double staining procedures were according to Bloch [ 181.The specificities of the various stains were tested by treating preparations with DNase (0.3 mg/ml in 0.004 M MgSO,, pH 7.0) for 12 h at 37°C or with 0.1 N HCI for 3 h at 30°C [ 191. The former treatment yielded Feulgen-negative nuclei. The acid extraction removed alkaline fast green, Sakaauchi and FDNB stainability from most 07 the nuclei examined but not the cytoplasmic regions when present.

Microspectrophotometry Cytophotometric measurements were made with a modified Leitz MPV microscope photometer [20]. Where applicable, either the two-wavelength method [21] or the “plug” method [22] was used. With the Feulgen stain, the criterion of Ea/Eb=0.51-0.53, where Ea and Eb refer to extinctions at the lower and higher wavelengths, was satisfied by 4% and 560 pm. The wavelengths for measuring the alkaline fast green content were 583 and 630 pm. In the doubly stained preparations readings were carried out at 400,410 and 560 pm for FDNB, Sakaguchi and Feulgen stained material, respectively. Routinely, all the material comprising one complete experiment was processed concomitantly. Each analysis was run a minimum of three separate times with congruent qualitative results. Hen red blood cells were used as a standard in all of the experiments. Unless otherwise noted, at least 15 nuclei of each type were measured. Nuclear areas were ascertained photometrically [23].

Nuclear proteins during Ascaris gametogenesis

Table 1. Fast green (pH 8.1) microspectrophotometric

193

analysis of spermatogenic cell

nuclei ofAscaris lumbricoides Gonad region’ (%)

El 22-26

n

Total histone (R.U.fS.E.M.)b

n

20

2.86f0.34

20

E&o.34

20 20

37-41 48-52 59-63 81-85 98-100

5.35f0.45 6.34kO.37 6.33kO.62 1.91f0.88 0.73f0.07

ii 20 19

Acetylated histone (R.U.fS.E.M.)

Column 3column 5

19 19 20 19

1.32kO.29 n.s. 1.76kO.22 3.46fO. 19 2.81?0.26 4.83+0.24

1.54 i.11 1.89 3.52 1.50

;:

0.42+0.04 1.62f0.11

0.21 0.29

o The location of gonadal segments are expressed as per cent of the total length of the reproductive tract starting from the germinal tip. * R.U., rel. units. c n.s., not storable.

tered at the germinal tip (04% of the total length of the reproductive tract starting DNA and nuclear protein contents from the germinal end). Meiotic DNA synduring spermatogenesis in thesis occurs in an adjacent section (from A. lumbricoides 7-11% of the gonad) while primary sperFeulgen dye measurementsof nuclei within matocytes occupy the part of the gonad that sections of the male reproductive tract were extends from about 22-63% of the total made to determine the staging of spermato- length. Secondary spermatocytes are often genesis (fig. 1). Spermatogonia are clus- found in the 75430% region of the gonad. RESULTS

2.0 ,p. ,,: : ,,*’ ‘, ,,*’

1.6 1.2

,’

:

p’ ,:’

.8 .,.: ,c--4

.4 ‘g.....--B ,‘. , 04 0 0

20

40

60

60

100

Fig. I. Abscissa: male gonad length (%); ordinate: Feulgen-DNA content, rel. units. Relative nuclear DNA content during spermatogenesis. The total length of the reproductive tract was converted to 100% and the location of each gonadal segment is designated as a per cent from the germinal tip, i.e., 0%. (See also figs 2-8.) Vertical bars denote ~elDu~t~15 for each sample. The 2C value is 2.18 . .

20

40

\0

.-.60

. 80

100

Fig. 2. Abscissa: male gonad length (%); ordinate: alkaline fast green (pH 8.1)/Feulgen ratio. Changes in alkaline fast green (pH 8.1) histone/ Feulgen-DNA ratio during spermatogenesis. 0, Acetylated alkaline fast green (PH 8.1)/Feulgen ratio; l , total alkaline fast green (pH 8.1) minus acetylated alkaline fast green (pH 8.1) content/Feulgen ratio. Solid curve denotes the regression line (r= -0.24) for data represented by the squares. N~=19 for each sample. Ekp Cell

Res 102 (1976)

194

Pasternak and Barrel1

either location. In fact, the former treatment augmented the intensity of cytophotometric analysis of spermatid nuclei of plasmic stainability. As such, no cytophoto.4scaris lumbricoides metric readings could be taken from this reGonadal gion, whereas the nuclei of cells from all the region” Total histone (R.U.+_S.E.M.) n (%I other zones yielded positive results. The total histone content increases as primary IO-14 20 2.28fO. 16 80-84 20 1.87+0.13 spermatocytes pass along the reproductive 88-92 20 1.82+0.11 tract. After meiosis is completed, there is a 97-100 20 1.55+0.11 reduction in the amount of histone/nucleus. (2 See table 1 for details. To obtain the relative amount of histone/ DNA for various sections of the reproducCells with nuclei containing haploid DNA tive tract, nuclear Feulgen dye contents equivalents or less fill the terminal 20% of were scored in parallel samples. Acetylated the gonoduct. Conventional meiotic stages preparations show an increase in uptake of were not evident, although the changes in the stain in developing primary spermatonuclear Feulgen dye content indicate that cytes followed by a sharp reduction during complete DNA reduction takes place in this the maturation of spermatids, whereas the organism. relative amount of dye-binding attributable The histone content of spermatogenic to lysine-containing basic protein tends to cells was assessed by the extent of fast be fairly constant and low during spermatogreen dye (pH 8.1) bound both prior to and genesis (fig. 2). A slight peak in the lysineafter acetylation of lysine residues (table 1). rich histone to DNA content may occur in Both the nuclei and cytoplasm of spermato- spermatocytes halfway through the reprocytes within the 7-11% segment of the ductive tract. gonad stain with fast green (pH 8.1) and Alkaline fast green staining of postneither acetylation nor extraction with meiotic spermatids was examined in more weak acid reduced the extent of staining in detail (table 2). For these data, an analysis Table 2. Fast green (pH 8.1) microspectro-

2.4

$-q

[j(

0-i

0

oi 0 20

40

60

80

100

Fig. 3. Abscissa: male gonad length (%); ordinate: SakaguchilFeulgen ratio. Changes in Sakaguchi-arghtine/Feulgen-DNA ratio during spermatogenesis. Ns25 for each sample. Exp Cell Res 102 (1976)

. 20

. 40

60

. 80

, 100

Fig. 4. Abscissa: male gonad length (%); ordinare: FDNB/Feulgen ratio. Changes in FDNB-lysine plus tyrosine/FeulgenDNA ratio during spermatogenesis. PI315 for each sample.

Nuclear proteins during Ascaris gametogenesis Table 3. Nuclear and nucleolar sizes during spermatogenesis in Askaris lumbricoides Gonadal region’ cm

Nuclear area (pm*+S.E.M.)

Nucleolar volume (pmWS.E.M.)

04 7-11 22-26 37-4 1 48-52 59-63 81-85 98-100

34.55k1.21 29.48+ 1.45 92.OOrt5.06 111.68k4.22 109.%+3.98 101.17+4.96 5.92kO.24 4.88kO.17

2.41f0.01 2.84kO.02 3.46f0.02 7.55f0.01 6.38+0.01 3.08f0.02 n.a.b na.

’ See table 1 for details. b n.a., Nucleolus absent.

195

sperm from the gonadal region 98-100% contain less bound FDNB than spermatids within the 8 l-85 % segment of the reproductive tract (Student’s t-test, t=89.52, df=14, P
of variance indicated an overall statistical difference among the four means (F3,76= DNA and nuclear protein content 5.75; PcO.01). The Tukey multiple com- during oogenesis in Ascaris parison procedure showed that the mean lumbricoides value for spermatids within the 70-74 % re- The female reproductive tract of Ascaris gion of the gonad differed from that of consists of two extensively coiled tubes, the 97-100% region spermatids (PcO.05). each of which when unravelled extends Identical analyses of the acetylated histone about 210 cm. The ovaries comprise the content of maturing spermatids yielded es- initial 5% of the gonad, the oviducts the sentially the same results. next 85% and the terminal 15% is formed The Sakaguchi-Feulgen reaction was car- by the uteri. The latter structures join to form a short common vagina. Feulgen ried out to determine the arginine-bound protein to DNA ratio during spermato- DNA measurements (fig. 5) indicate that genesis. As shown in fig. 3, the ratio in- the meiotic S phase is completed as the creases as the primary spermatocytes pass along the reproductive tract. A net loss of arginine-containing nuclear protein follows meiosis, with the result that the level of arginine protein/DNA in spermatids is the same as in spermatogonia. Spermatids within the region 8 I-85 % of the gonad contain more arginine-bound protein than found in the sperm of the 98-100 % region of the ger- o-o0 20 40 60 80 100 minal tract (Student’s t-test, t= 102.14, df= Fig. 5. Abscissa: female gonad length (%); ordinate: 14, P~O.001). The nuclear lysine plus (lef) Feulgen dye content, rel. units; (right) nuclear @m*). tyrosine-protein content, as measured by area Feulgen-DNA contents (0) and nuclear area (X) in bound fluorodinitrobenzene, rises as the relation to position within the female reproductive Solid curve represents the regression line (r= primary spermatocytes mature and then tract. -0.04) for Feulgen-DNA values of primary oocytes. falls during spermiogenesis (fig. 4). Mature Na35 for each sample. Exp CeNRes 102 (1976)

196 2.8

Pasternak and Barrel1

1

2.0. 1.6.

i

1.2

Fig. 6. Abscissa: female gonad length (o/o);ordinate: alkaline fast green (pH 8. l)/Feulgen ratio. Changes in alkaline fast green (PH 8.1) histonel Feulgen-DNA ratio during oogenesis. 0, Acetylated alkaline fast green (pH &l)/Feulgen ratio; B, total alkaline fast green (pH 8.1) minus acetylated alkaline fast green (pH 8.1) content/Feulgen ratio. N~20 for each sample.

oocytes reach the 15% region of the tract. Feulgen positive primary oocytes occupy the region from 15 to 75% of the gonad length; thereafter, the nuclei are insensitive to Feulgen dye but they can be visualized after staining with toluidine blue. With immature Ascaris sperm as a standard, each sample of primary oocytes within various regions of the oviduct has a 4C DNA equivalent (x2 tests; P>O.80 for each test).

A linearly increasing amount of fast green (pH 8.1) binds to oocyte nuclei during maturation (table 4). The nuclei of each oogenic stage stain intensely and evenly with the dye. The cytoplasm of oogonia is clear, whereas small fast green positive particles which are not extracted with 0.1 N HCl accumulate in the cytoplasm of primary oocytes. As oogenesis progresses, an arginine-rich component is amassed in the oocyte nucleus (fig. 6). After the primary oocytes pass the halfway point of the reproductive tract, a lysine-rich component increases within the nuclei while the nuclear arginineprotein content drops slightly (fig. 6). The nuclear SakaguchilFeulgen ratio increases throughout the development of the oocyte (fig. 7). Similarly, the lysine plus Vjrosine protein content/DNA doubles as the primary oocytes mature (fig. 7). Maturing Ascaris oocytes are weakly basophilic after staining with azure B (pH 4.0). The cytoplasm stains faint purple, the nuclei faint blue-green and the single nucleolus purple. The nucleolar volume within oogonia is 3.39f0.01 pm3 while in primary oocytes that are halfway through the reproductive tract it is 11.34kO.02 pm3. Thereafter, the nucleolar mass is reduced and maintained at about 5.51kO.02 pm3.

Table 4. Fast green (pH 8.1) microspectrophotometric Ascaris lumbricoides Gonadal region’ (%I O-3 15-18 30-33 38-40 5&53 61-64 71-74

n 20 iFI z: 20 20

a See table 1 for details. Exp Cell Res 102 (1976)

Total histone (R.U. fS.E.M.) l.%fO. 14 2.29kO.09 7.13kO.91 8.09+0.26 13.08+0.40 16.76kO.86 22.82k1.19

n 20 20 20 ii 20 20

analysis of oogenic cell nuclei of

Acetylated histone (R.U.+S.E.M.)

Column 3column 5

1.93kO.16 1.54kO.14 6.39&O. 15 7.08fO. 19 12.09+0.37 11.58kO.49 9.83f0.62

0.03 0.65 0.74 1.01 0.99 5.18 12.99

Nuclear proteins during Ascaris gametogenesis

197

Feulgen uptake by the nuclei of primary oocytes occurs abruptly and does not correlate with the build-up of nuclear proteins. If nuclear proteins were masking Feulgenbinding sites, a gradual loss of stainability would be anticipated. Moreover, extraction with acid [ 191failed to restore Feulgen positivity to either mature oocytes or spermatozoa. Finally, a dilution effect due to an increase in nuclear volume alone cannot account for the change in Feulgen staining during oogenesis (see fig. 5). At present Fig. 7. Abscissa: female gonad length (%); ordinate: there is no clear explanation to account for (left) SakaguchilFeulgen ratio: (right) FDNB/Feulgen ratio. variations in the stoichiometry of the FeulChanges in Sakaguchi-arginine/Feulgen-DNA (0) and FDNB-lysine plus tyrosine/Feulgen-DNA (0) dur- gen reaction in various cell types in Ascaris ing oogenesis. Solid curve represents linear regression (see also [30] and references therein). In this line (r=O.95) for closed circles. Dashed curve represents linear regression line (r=O.91) for open context, it should be stressed that if the circles. N320 for each sample. cytophotometric ratios for spermatogenic nuclei were expressed as the amount of chromophoreltheoretical C value, none of Changes in nuclear area during oogenesis the trends described above would be are presented in fig. 5. altered. There is a considerable array of patterns DISCUSSION of basic protein changes during spermatoQuantitative cytophotometry can be used genesis in a variety of animal species [6]. to detect large changes in macromolecular For example, in salmon the somatic histone complement is replaced by protamines [8], expression during various differentiating programs, especially within tissues that are whereas within frog sperm the somatic hisrefractory to direct biochemical analyses. tones are retained [31] and in cancer crabs As shown here, the levels of histone and the sperm nuclei are devoid of all histone [7]. Nor is it unusual for the sperm of some basic nuclear proteins vary quantitatively during gametogenesis in both sexes of forms to maintain somatic histones in addiAscaris. As well, during spermiogenesis tion to a sperm-specific basis protein [32, there is a 30% decrease in the amount of 331.When the data presented here are taken Feulgen dye bound to mature sperm DNA. together, the process in Ascaris has distincThe latter phenomenon is presumably due tive features. (1) Ascarid spermatogonia either to packing effects [25] or other inter- contain equal quantities of arginine- and actions [26] and is probably not the result of lysine-containing histones. (2) An argininea loss of DNA per se. Changes in Feulgen rich histone component accumulates prior stainability have also been observed during to meiosis while the lysine-rich histone to oogenesis in Ascaris and in different nema- DNA ratio remains fairly constant throughtode species [27] as well as during embryo- out spermatogenesis. As well, a build-up of genesis [ 161and spermatogenesis [28, 291in both arginine- and lysine- plus tyrosineother systems. In our case, the loss of bound nuclear proteins precedes meiosis. Exp CeIlRes 102 (1976)

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Pasternak and Barrel1

(3) After meiosis, the relative amount of of basic nuclear proteins during oogenesis is each kind of basic nuclear protein de- sparse, thereby precluding comparisons. creases dramatically until the mature sperm The absence of an appreciable quantity of contains a low level of basic nuclear pro- cytoplasmic basic proteins in the oocytes of teins. This sloughing of nuclear proteins Ascaris resembles the condition found in does not correspond with the acquisition of some echinoderms [39] and in mice [37]. A basic proteins by any sperm-specific cyto- common, although not necessary, feature in plasmic organelles as has been noted in these instances is a low level of cytoplasmic RNA. other organisms [7, 34, 351. In sum, unique temporal sequences of nuIn a number of invertebrates, an argininerich basic protein which is not a protamine clear events accompany both oocyte and becomes prevalent in the sperm during the sperm development in Ascaris. The signifilater stages of spermatogenesis. It is not cance of the changes remains to be deknown whether the arginine-rich fraction in termined. Moreover, since the biological Ascaris sperm is unique to the spermato- reasons for histone shifts during gametogenie process. As noted by Bloch [6], it is genesis in many other organisms are controdifficult to explain the functional basis for versial [40] reiteration of various working the kinds of histone variations encountered hypotheses here would not be germane. during sperm formation within diverse ani- However, a relationship between these mal species. Similarly, the role of each of specific variations and the meiotic process the observed changes during spermato- cannot be overlooked. genesis in Ascaris is enigmatic. The fluctua- This work was supportedby the National Research tions are especially intriguing since ascarid Council of Canada. sperm do not undergo the characteristic REFERENCES morphological streamlining that is typical for many animal species. It is conceivable 1. Gelinas, R E & Kafatos, F C, Proc natl acad sci us 70 (1973) 3764. that the loss of nucleoproteins may be a 2. Newell, P C, Franke, J & Sussman, M, J mol biol prelude to the special transcriptive activity 63 (1972) 373. of the male ascarid pronucleus that occurs 3. Clayton, R M, Curr top dev biol5 (1970) 115. 4. Epstein, C J, Biol reprod 12 (1975) 82. after fertilization and prior to karyogamy 5. Paul. J. Biochemistrv of cell differentiation. Bio-

[ill. It is difficult to relate the changes in the histone and basic nuclear protein contents during oocyte maturation in Ascaris with either specific nuclear events during oogenesis or some attribute of embryonic development. The rises in basic nuclear protein levels coincide with increases in cytoplasmic components of the maturing ascarid oocyte [36]. Moreover, a progressive accretion of nuclear-bound basic proteins has also been found in oocytes of some other invertebrates [37, 381. But, in general the existing data on the behaviour Exp Cell Res 102 (1976)

chemistry ser. 1, vof. 9 (ed J Paul). Butterworth, London (1974). 6. Bloch, D P, Genetics, suppl. 61 (1%9) 93. 7. Vaughn, J C & Thomson, L A, J cell bio152 (1972) 322. 8. Marushige, K & Dixon, G H, Dev biol 19 (1%9) 397. 9. Hughes, A, A history of cytology, p. 55. AbelardSchuman, London (1959). 10. Pasteels, J, Arch bio159 (1948) 405. 11. Kaulenas, M S & Fairbairn, D, Exp cell res 52 (1968) 233. 12. Nigon, V, Trait6 de zoologie (ed P P Grass?) vol. 4, p. 218. Masson, Paris (1%5). 13. Swartz, F J, Henry, M & Floyd, A, J exp zoo1 164 (1%7) 297. 14. Mortiz, K B, Introduction to quantitative cytochemistry (ed G L Wied & G FBahr) vol. 2, p. 57. Academic Press, New York (1970). 15. Alfert, M & Geschwind, I I, Proc natl acad sci US 39 (1953) 991.

Nuclear proteins during Ascaris gametogenesis 16. Sin, W C & Pasternak, J, Chromosoma 32 (1970) 191. 17. Fand, S B, Introduction to quantitative cytochemistry(edGLWied&GFBahr)vol.2,p.209. Academic Press, New York (1970). 18. Bloch, D P, Protoplasmatol5 (1966) (3d) 1. 19. Noeske, K, J histochem cytochem 19 (1971) 169. 20. Pasternak, J & Haight, M, Chromosoma 49 (1975) 279. 21. Patau, K, Chromosoma 5 (1952) 341. 22. Swift, H, Physiol zoo1 23 (1950) 169. 23. Garcia, A M & Iorio, R, Introduction to quantitative cytochemistry (ed G L Wied) p. 215. Academic Press, New York (1966). 24. Flax. M H & Himes. M H. Phvsiol zoo1 25 (1952) 297. ’ 25. Gledhill, B L, Gledhill, M P, Rigler, R & Ringertz, N R, Exp cell res 41 (1966) 652. 26. Rasch, R W & Rasch, E M, J histochem cytochem 21 (1973) 1053. Mont& L, Parasitology 53 (1963) 273. ii: Bloch, D P & Hew, H Y C, J biophys biochem cytol7 (1960) 69. 29. Gledhill, B L, Introduction to quantitative cyto__

.

30. 31. 32. 33. 34. 35. 36. 37. 38.

,

39. 40.

199

chemistry (ed G L Wied & G F Bahr) vol. 2. D. 125. Academic Press, New York (1970). Andersson, G K A & Kiellstrand. P T T. Histo’ chemistry 43 (1975) 123. ” Zirkin, B R, Chromosoma 3 l(l970) 231. Subirana, J A, Exp cell res 63 (1970) 253. Tessier, A & Pallotta, D, Exp cell res 82 (1973) IfI? ___. Das, N K, Micou-Eastwood, J & Alfert, M, J cell bio135 (1%7) 455. Vaughn, J C, J cell bio13 1 (1966) 257. Foor, W E, J parasit 58 (1972) 524. Cowden, R R, Histochemistry 6 (1966) 226. Rosenbaum, R M & Rolon, C I, J histochem cytochem 7 (1959) 322. Davenport, R & Davenport, J C, Exp cell res 42 (1966) 429. Hnilica, L S, The structure and biological function of histones. Chemical Rubber Co. Press, Cleveland (1972).

Keceived March 3, 1976 Accepted May 3, 1976

Exp CellRes 102 (1976)