A cytochemical study of cytoplasmic basic proteins in echinoderm oogenesis

Experimental

Cell Research

42, 429-437

A CYTOCHEMICAL PROTEINS

429

(1966)

STUDY OF CYTOPLASMIC BASIC

IN ECHINODERM

R. DAVENPORT

Zoology Department,

and JANICE

OOGENESIS

C. DAVENPORT

of Illinois, Urbana,

University

Ill.,

U.S.A.

Received May 21, 1965’

CYTOCHEMICAL studies

have revealed

the presence

of basic

proteins

in the

cytoplasm of the oocytes and eggs of several types of animals. Taleporos [13] reported cytoplasmic histones and protamines in the unfertilized eggs of the sea urchin Strongylocentrotus purpuratus. Horn [9] isolated a histone fraction from the alkaline fast green positive cytoplasm of the frog egg. In the sea urchin these cytoplasmic basic proteins are presumed to be bound to the DNA reserves known to occur there. Recently, Davenport and Davenport [5] have demonstrated a basic protein fraction in the young ascidian oocyte which is bound to cytoplasmic RNA. Similar basic proteins have been found in mollusc oocytes [6]. Thus, there appears to be some diversity in the association of cytoplasmic basic proteins with nucleic acids. Since the echinodermata is a large and diverse phylum, it seemed important to ascertain if the occurrence of cytoplasmic basic proteins as described for S. purpuratus was typical of the group as a whole. Consequently, a series of echinoderms of all classes was collected for the purpose of making a comparative study of oogenesis with respect to the occurrence and distribution of basic proteins. Species belonging to the following classes and genera were studied:

ECHINOIDEA: Diadema,

Echinometra,

Echinothrix,

Goniocidaris, Helio-

cidaris, Heterocentrotus, Holopneustes, Paraselenia, Strongylocentrotus, Tripneustes, Moira, Mellita. ASTEROIDEA: Tosia, Nectria, Patiriella, Cocinasterias, Patricia, Culcita, Acanthaster, Linkia, Nardoa, Luidia, Formia. OPHIUROIDEA: Ophionereis, Ophiarachna, maria,

Ophiarthrum, Ophiarthrum, Holothuria,

Sticopus,

Ophidaster,

Ophilolepis, Macriothrix, Ophiomastix, Ophiocoma. HOLOTHUROIDEA: Pentacta, CucuLeptosynapta. CRINOIDEA: Cenolia, Comanthus.

MATERIALS

AND

METHODS

Ovaries were removed from freshly collected animals and small pieces fixed in either acetic-ethanol or 10 per cent neutral buffered formalin. The tissue was then embedded in paraffin and sectioned at 15 cc. 1 Revised version received January 25, 1966. Experimental

Cell Research

42

R. Davenport

430

and Janice

C. Davenport

Nucleic acids were studied by means of the methyl-green pyronin method of Kurnick [IO], the gallocyanin method of Einarson [8], and the Feulgen reaction. Control slides were incubated in crystallin ribonuclease or desoxyribonuclease obtained from Worthington Biochemicals Inc. General proteins were studied by means of bromphenol blue and fast green at low pH. Basic proteins were selectively stained by means of the Alfert and Geschwind [2] alkaline fast green method following removal of both nucleic acids with hot TCA, and following the removal of RNA alone by means of ribonuclease or cold perchloric acid at 4 degrees C for 18 hr. All Fast Green FCF used in this work was obtained from the Allied Chemical Corp. and was certified “93 per cent dye content”. Van Slyke nitrous acid deamination for 30 min at room temperature was used to destroy the e-amino groups of lysine both before and after the removal of nucleic acids [l, 71. RESULTS Holothuroidea Cytoplasmic RNA levels are generally low among the species studied in this class. When sections are stained with basic dyes, the younger oocytes stain to a moderate degree while the older oocytes are only lightly stained (Fig. 1 a). The nucleoli are more heavily stained than the cytoplasm, but not to the degree seen in the other classes. All basic dye binding in the cytoplasm and nucleoli is prevented by ribonuclease treatment. Species belonging to the order Dendrochirota differ from the Aspirochirota and the Apoda by the fact that they have very large eggs with multiple nucleolar systems. The number of nucleoli per oocyte is in some species very high, as many as 37 being counted in serial sections. The cytoplasmic RNA levels seemed to be slightly higher among the species of Dendrochirota than in the other two orders. Staining

for basic proteins

after the removal

of nucleic

acids with hot TCA

results in the staining of the somatic cell nuclei, but only a very light stain in the cytoplasm of the younger oocytes (Fig. 1 d). This plae, almost undetectable, stain in the young oocytes is the only indication of basic proteins outside of the somatic cell nuclei. It is absent from sections which had received

Fig. 1.--a: Holothuroidea (Phulloohorus SD.):b: Gnhiuroidea (0Dhoinerei.s schauerik c: Asteroidea (Fyomia elegans); d: Asteroidea (Fromiakl&ans)j e-i: Crinofdea (Cenolia iriciopkra). (a) Gallocganin. Acetic-ethanol fixation. x 250. (b) Gallocyanin. Acetic-ethanol fixation. x 250. (c) Gallocyanin. Acetic-ethanol fixation. x 250. (dj Hot TCA, fast green pH 8.1. Formalin fixation: x 250. (e) Gallocyanin, Acetic-ethanol fixation. x 250. (f) Ribonuclease, gallocyanin. Acetic-ethanol fixation. x 250. (g) Hot TCA, fast green pH 8.1. Formalin fixation. x 400. (h) Nitrous acid deamination, hot TCA, fast green pH 8.1. Acetic-ethanol fixation. x400. (i) Hot TCA, nitrous acid deamination, fast green pH 8.1. Acetic-ethanol fixation. x 400. Experimental

Cell Research 42

Cytoplasmic

29 - 661805

basic proteins

in echinoderm

oogenesis

Experimental

431

Cell Research 42

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R. Davenport

and Janice C. Davenport

no treatment prior to staining, but is present in sections treated with ribonuclease or cold perchloric acid before staining. Ophiuroidea Levels of cytoplasmic RNA staining with basic dyes are moderately high in the young oocytes and quite low in the older ones (Fig. 1 b). All cytoplasmic and nucleolar basic dye binding is prevented by ribonuclease treatment. Staining for basic proteins with alkaline fast green after hot TCA treatment results in the staining of the somatic cell nuclei and a slight green tinge in the cytoplasm of the young oocytes. As in the Holothurians this light cytoplasmic staining is absent in untreated controls, but present after ribonuclease or cold perchloric acid treatment. No nucleolar stain was observed. Asteroidea There is considerable variation in cytoplasmic RNA levels among the members of this class. Many species resemble the Ophiuroidea and the Holothuroidea in that the levels of cytoplasmic RNA are fairly low. In other species, such as Fromia elegans, high concentrations of RNA are observed with basic dye staining both in the cytoplasm and the nucleolus (Fig. 1 c). All basic dye binding in the cytoplasm and nucleoli is prevented by ribonuclease treatment. Staining for basic proteins after hot TCA treatment in all species studied results in only the staining of somatic cell nuclei and the slight staining of the cytoplasm of the younger oocytes noted in the Holothuroids and Ophiuroids (Fig. 1 d). All cytoplasmic staining is absent in untreated controls, but present after the removal of RNA with either ribonuclease or cold perchloric acid. The nucleolus remains unstained with alkaline fast green in all cases. Crinoidea Many species in this class contain oocytes that are heavily pigmented which interferes with staining. In some species, however, the oocytes are Fig. 2..- a-d: Echinoidea, endocyclica (Paraselenia gratiosa); e-h, k; Echinoidea, clypeastroidea (Mellita se.zespanctulofa); i-j: Echinoidea, clypeastroidea (unidentified species). (a) Gallocyanin. Acetic-ethanol fixation. x 250. (b) Ribonuclease, gallocyanin. Acetic-ethanol fixation. x 250. (c) Cold perchloric acid, fast green pH 8.1. Formalin fixation. x 250. (d) Cold perchloric acid, fast green pH 8.1. Formalin fixation. x 400. (e) Gallocyanin. Acetic-ethanol fixation. x 250. (f) Ribonuclease, gallocyanin. Acetic-ethanol fixation. x 250. (P) .” , Cold uerchloric acid. fast green pH 8.1. Formalin-fixatjon. x 400. (h) Cold perchloric acid, fast green pH 8.1. Formaiin fixation. x 400. (i) Gallocyanin. Acetic-ethanol fixation. x 250. (.j) Hot TCA, fast green DH 8.1. Formalin fixation. x 250. (k) Nitrous acid deamination, hot TC& fast green pH 811. A&tic-ethanol fixation. x 400. Experimental

Cell Research 42

Cytoplasmic

basic proteins

in echinoderm

oogenesis

Experimental

433

Cell Research 42

434

R. Davenport

and Janice C. Davenport

unpigmented and high concentrations of RNA can be detected with basic dye staining (Fig. 1 e). This staining is very heavy in the young oocytes, dropping off markedly in the older ones. Ribonuclease treatment abolishes all cytoplasmic and nucleolar staining (Fig. 1 f). Staining for basic proteins after hot TCA treatment results in heavy cytoplasmic staining of the young oocytes, but no stain in the older ones (Fig. lg). The nuclei of the somatic cells are also stained, but the TCA treatment disrupts the tissue sections rather seriously and these were frequently removed from the slide. Sections which had received no treatment before staining were completely unstained. Alkaline fast green staining after the removal of RNA alone with either ribonuclease or cold perchloric acid is identical to that produced by hot TCA treatment. When sections of aceticethanol fixed material are deaminated before the removal of nucleic acids, the cytoplasmic alkaline fast green staining in the young oocytes is diminished somewhat, but still present (Fig. lh). Identical treatment after the removal of nucleic acids completely abolishes all staining (Fig. 1 i). No nucleolar staining with alkaline fast green was observed. Echinoidea A rather diverse staining picture is presented by the Echinoids. In the group known as the endocyclica or true sea urchins, the majority of species show only moderate levels of RNA in both the young and old oocytes similar to the Asteroidea, but some show high concentrations of cytoplasmic RNA in the young oocytes (Fig. 2a). In all the species of echinoids studied, there were extracellular spheres present in the ovary, often in considerable numbers, which stained more intensely than the cytoplasm with basic dyes (Fig. 2 a and 2 e). Basic dye binding in these spheres as well as the cytoplasm is completely prevented by ribonuclease extraction (Figs. 2 b and 2 f). The occurrence of detectable concentrations of basic proteins in the oocytes of this group reflects the diversity observed for cytoplasmic RNA. When sections are stained with alkaline fast green after hot TCA extraction, those echinoids having low levels of cytoplasmic RNA show only traces of basic proteins in the cytoplasm of the young oocytes. In addition, some of the extracellular spheres are also faintly stained. However, in those species having high levels of cytoplasmic RNA, moderate amounts of basic proteins can be detected in both the young oocytes and some of the extracellular spheres after hot TCA extraction, cold perchloric acid extraction, or ribonuclease digestion. These basic proteins are stainable with fast green as high as pH 9.5 (Figs. 2c and 2d). Experimental

Cell Research 42

Cytoplasmic

basic proteins

in echinoderm

oogenesis

435

In the species of clypeastroidea, or sand dollars, cytoplasmic RNA levels are high (Figs. 2 e and 2 i). Extracellular spheres are also evident, some being very large and intensely stained with basic dyes. All basic dye binding is completely prevented by ribonuclease incubation (Fig. 2f). Staining for basic proteins after hot TCA, cold perchloric acid, or ribonuclease treatment reveals the presence of high concentrations of basic proteins in the cytoplasm of the young oocytes (Figs. 2g and 2j) and in some of the extra cellular spheres (Figs. 2 h and 2j). Sections with no pretreatment are completely unstained. Nitrous acid deamination of acetic-ethanol fixed material, following removal of nucleic acids, allows alkaline fast green staining of both the oocytes and the spheres (Fig. 2k). Identical treatment after the removal of nucleic acids completely abolishes all staining. As with the other classes the nucleolus remained unstained with alkaline fast green in all cases.

DISCUSSION

These results indicate that cytoplasmic proteins, stainable with fast green under conditions selective for proteins having an isoelectric point above 8.1, are not a conspicuous feature of oogenesis in the majority of echinoderms. In most groups only a trace of such proteins are detectable. These proteins appear to be bound to cytoplasmic RNA, most likely ribosomal. Differences in staining intensity for RNA are not always reflected in basic proteins staining, however. This may indicate that in some cases only part of the demonstrable basic proteins are bound to RNA. The relationship between the staining of RNA and basic proteins differs markedly from that demonstrated in the ascidian oocyte [5], and in some mollusc oocytes [6]. In the species of Crinoidea examined in this study there are high concentrations of RNA bound basic proteins demonstrable in the young oocytes. In this respect, it resembles more closely the staining picture of ascidian oogenesis than the rest of the echinoderms. The only other echinoderms showing any amount of basic proteins are the clypeasteroids. In these cases they appear to have higher concentrations than the majority of echinoderms, but less than the crinoids. In addition these, and other echinoids have extracellular spheres which give evidence of possessing RNA-bound basic proteins. The results reported here differ from those reported by Taleporos for Strongylocentrotus purpuratus [13]. He found that the oocytes and unfertilized eggs of this species gave strong cytoplasmic alkaline fast green staining and postulated that this staining might be due to cytoplasmic DNA bound histone Experimental

Cell Research 42

436

R. Davenport

and Janice C. Davenport

and protamine fractions. He succeeded in extracting “histones” and “protamines” from these unfertilized eggs. S. purparatus was among the species included in the present study. In our hands it gave no evidence of significant quantities of demonstrable cytoplasmic basic proteins in oocytes of any stage, using methods which preclude the staining of non-basic proteins. We have noted, however, that certain brands and batches of Fast Green FCF will give strong staining reactions in the cytoplasm of yolky oocytes and eggs. These dyes can be shown electrophoretically to be impure and therefore cannot be used. That basic proteins exhibiting a strong staining reaction should be bound to a DNA fraction, itself cytochemically undernonstable, does not follow from what is known concerning the relationship of these two compounds. Such cytoplasmic proteins are more likely to be bound to ribosomal RNA, or exist unbound to any nucleic acid. Such basic proteins have now been isolated from a number of sources [3, 4, 11, 121 and appear to be structural components of the ribosome. In nearly all the tissues from which basic ribosomal proteins have been isolated, there is no cytoplasmic alkaline fast green staining. While it has not been established that the strong fast green staining in the young oocytes of some animals is due to ribosomal proteins, it seems likely that this is so. Why these proteins should stain and those of somatic ribosomal population should not remains to be explained. It may be that the ratio of basic proteins to RNA is higher in these oocytes or the proteins are more basic. There is some evidence which indicates that some ribosomal basic proteins may have a lower ratio of basic to acidic amino acids than those of the nucleus [3]. On the other hand, it may simply be that the concentration of ribosomes in the young oocyte reaches a level which permits the visualization of these proteins, while in most somatic cells it does not. In those animals which have no demonstrable cytoplasmic basic proteins in their oocytes, yolk deposition may occur in such a manner that precludes the build up of ribosomal concentration. Experiments are now in progress to decide upon these alternatives.

SUMMARY

A cytochemical study of oogenesis in the echinodermata has revealed the presence of high concentrations of cytoplasmic basic proteins in the young oocytes of the Crinoidea and Clypeastroidea. These basic proteins give evidence of being bound to RNA and become undetectable during the Experimental

Cell Research 42

Cytoplasmic

basic proteins

in echinoderm

oogenesis

437

later stages of oogenesis. Only traces of stainable basic proteins are found in the Holothuroidea, Asteroidea, and Ophiuroidea. This investigation was wholly supported by Public Health Service Research Grant HD 00011-01 from the Institute of Child Health and Human Development.

REFERENCES 1. ALFERT, M., J. Biophys. B&hem. Cytol. 2, 109 (1956). 2. ALFERT, M. and GESCHWIND, I. I., Proc. Nail Acad. Sci. 39, 991 (1953). 3. BUTLER, .I. A. V., COHN, P., and SIMSON, P., Biochim. Biophys. Acta. 38, 386 (1960). 4. CRAMPTON, C. F. and PETERMANN, M. L., J. Biol. Chem. 234,2642 (1959). 5. DAVENPORT, R. and DAVENPORT, J. C., J. Cell Biol. 25, No. 2, P + 1, 319 (1965). 6. Exptl Cell Res. 39, 74 (1965). 7. DEITCH, A., Lab. Invest. 4, 324 (1955). 8. EINARSON, L., Acta. Pathol. Stand. 28, 82 (1951). 9. HORN, E., Proc. Nat1 Acad. Sci. 48, 257 (1962). N. B., Stain Technol. 30, 213 (1955). 10. KURNICK, 11. LESLIE, I., Nafure 189, 260 (1961). 12. SETTERFIELD, G., NEELIN, J. M., NEELIN, E. M. and BAYLEY, S. T., J. Mol. Biol. 2,416 (1960). 13. TALEPOROS, P. P., J. Histochem. Cytochem. 7, 322 (1959).

Experimental

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