the cytosome of differentiating cells in the ovotestes of ... · alkaline phosphatase, and...

453

The Cytosome of differentiating Cells in the Ovotestesof Slugs

By D. PELLUET AND ANNE H. G. WATTS

(From the Zoological Laboratory, Dalhousie University, Halifax, Nova Scotia;

With one plate (fig. i)

SUMMARY

The distribution of ascorbic acid, alkaline phosphatase, and mitochondria has beenfollowed during the differentiation of germinal epithelium cells into spermatogonia,oocytes, and nurse-cells in the ovotestes of slugs. All three substances appear in thecytoplasm of the oocyte and increase during its growth. Occasionally the oocytenucleolus gives a positive test for ascorbic acid. The heads of the mature spermatozoacontain alkaline phosphatase. The cytoplasm of the germinal epithelium, spermato-gonia, and spermatocytes occasionally gives' a positive reaction for ascorbic acid. Thedevelopment of the nurse-cells is accompanied by an increase in the size of the mito-chondria. Attachment of the spermatids results in a decrease in size and number of themitochondria in the nurse-cells. Alkaline phosphatase, ascorbic acid, and mitochondriashow no significant change, either in form or quantity, in the indifferent cells of theovotestis by which one could predict their destiny.

INTRODUCTION

THIS paper contains an account of the distribution of ascorbic acid,alkaline phosphatase, and mitochondria in the developing germ-cells

of certain pulmonate gastropods found in Nova Scotia. The species studiedwere the following almost shell-less slugs, which were collected fromvarious localities: Deroceras laeve (Mueller), D. reticulatum (Miiller), Arionsubfuscus (Draparnaud), A. hortensis (Ferussac), A. circumscriptus (Johnston).All these species have been maintained in the laboratory (Ord and Watts.1948-9).

Among these species, only Deroceras laeve appears to be protogynic (Lams,1909). Eggs appear in the ovotestes about 15 days after hatching. Six monthsafter hatching, the animals are mature and eggs are laid. The Arionidae are notas prolific as the two species of Deroceras, and fewer young animals wereavailable in this family.

OBSERVATIONS

Alkaline phosphatase

The technique used to demonstrate alkaline phosphatase was that otGomori (1939, 1941)- The precautions prescribed by Danielli (1946) werecarefully observed.

The distribution of alkaline phosphatase in the germ-cells appears uniform[Quarterly Journal of Microscopical Science, Vol. 92, part 4, pp. 453-61, Dec. 1951.]

454 Pelhiet and Watts—The Cytosome of differentiating Cells of Slugs

in the five species studied. There was, however, a great deal of variation amongthe individuals. This is in accordance with observations made by Gomori(1941).

The germ-cells of young slugs show a prominent deposit of preformedphosphate, while the phosphatase, when it is present, is represented by a fewgranules of sulphide, restricted to the area of cytoplasm surrounding theoocyte nucleus and to the cytoplasm of the spermatogonia. As the animalsdevelop, the phosphatase activity in the oocyte cytoplasm increases greatly.When the oocytes leave the wall of the acinus, their cytoplasm is full of largeblack dots indicating sites of phosphatase activity (fig. ic). The larger ofthe two nucleoli in the oocyte, sometimes gives a slight diffuse positive reaction(in addition to the reaction given by preformed phosphate), while the nucleusis negative.

The germinal epithelial cells give a weak reaction in the nuclei and cyto-plasm, but more often there is no evidence of phosphatase activity in thesecells.

The spermatocytes occasionally give a slight reaction in the nuclei andcytoplasm. This observation does not agree with that of de Nicola (1949) onthe occurrence of alkaline phosphatase during spermatogenesis in isopods,in which he always found the chromosomes positive. The heads of maturespermatozoa are strongly positive in slugs, whereas in isopods they give onlya slight positive reaction, according to de Nicola.

There is a good deal of difference of opinion on whether alkaline phosphataseis found chiefly in the cytoplasm or chiefly in the nucleus (Gomori, 1941;Danielli, 1946). Schneider (1946) found 50 per cent, of the phosphataseactivity in centrifuged rat liver homogenate in the mitochondrial fraction; andalkaline phosphatase has been found associated with the Golgi structures inepithelial cells (Emmel, 1945; Deane and Dempsey, 1945). Recent papers oninvertebrate material (Day, 1949; Yao, 1950; de Nicola, 1949) all state thatthe reaction is very strong in the nucleus and in the cytoplasm of somatictissues, while Krugelis (1945) finds that the salivary glands of Drosophilashow a strongly positive localization of alkaline phosphatase in the euchro-matic bands.

The wide variation in the phosphatase reaction of individual germ-cells, incontrast to the apparently uniform results obtained in somatic tissues, suggestsa somewhat capricious distribution of the enzyme, an impression which dis-appears if sufficiently large numbers of animals are sectioned.

FIG. 1. All photographs were taken with a Spencer (American Optical Co.) phase-contrastmicroscope. Each of the scales represents 10 p.

A, oocyte of A. subfuscus, showing the mitochondria. Animal killed in July. Bright contrast.B, part of the oocyte shown in A, photographed under oil-immersion lens with bright field.c, alkaline phosphatase in oocyte of D. laeve, killed in March. Bright field. The apparent

strong positive reaction of the nucleolus is due largely to preformed phosphate.D, ascorbic acid in the cytoplasm of an oocyte of D. reticulatum, 42 days old, killed in Septem-

ber. Bright field. The pigmented epithelium is clearly seen bordering the egg.

Quart. Journ. Micr. Set. New Series, Vol.

FIG. I

D. PELLUET and A. H. G. WATTS

Pelluet and Watts—The Cytosome of differentiating Cells of Slugs 455

Ascorbic acid

The presence of ascorbic acid was determined by the method originated byBourne (1936 a, b).

Few observations have been made on the distribution of ascorbic acid ininvertebrates, in contrast to the many investigations which have been made onvertebrate tissues. Giroud and Ratsimamanga (1936) found that the gonadsof invertebrates were particularly rich in vitamin C, and Day (1949) hasfound the vitamin in the developing eggs of Penplaneta, where it occurred inthe cytoplasm and the nucleolus.

The distribution of ascorbic acid is similar in the germ-cells of the five kindsof slugs, the species-differences being quite slight. In nearly all cases, ascorbicacid seems to be restricted to the cytoplasm, an exception being Deroceraslaeve in which it occurs in the nucleolus and perhaps in the nucleoplasm.

D. reticulatunt and D. laeve differ from the other species in lacking the vita-min in the germinal epithelium and in the male germ-cells. A description of theappearance of ascorbic acid during the development of the cells of the ovotestisin Deroceras laeve and Arion subfuscus will suffice to illustrate the slight speciesdifferences. In D. laeve ascorbic acid appears in the egg about 33 days afterhatching (fig. 2). The granules increase rapidly in number in this species, sothat 2 months after hatching, the ascorbic acid content of the oocytes iscomparable to that found in an adult animal. This is in accordance with thefact that this species reaches maturity early and is protogynic. It will be seenfrom the illustration that an animal 51 days old shows granules of the vitaminin the nucleolus. The lack of vitamin in both male germ-cells and germinalepithelium has been referred to previously.

In Arion subfuscus the black granules representing ascorbic acid appear inthe oocytes 47 days after hatching, and they tend to cluster round the nucleusin this species (fig. 3). The vitamin increases in the oocytes as the animalapproaches maturity, at which time a few granules appear in the male germ-cells and occasionally in the germinal epithelium. (In general the cytoplasmof the germinal epithelium of all species is sparse, making the study of theinclusions difficult.)

The amount of ascorbic acid present in the oocytes of any of the speciesvaries considerably. Individuals of the same age and species, which have beenexposed to exactly the same environmental conditions during their develop-ment, may exhibit all variations from a heavy peppering of granules to com-plete absence of the vitamin in many of their oocytes. Day (1949) found thesame type of variation in the egg of Blatella.

Material in which all the ascorbic acid has been converted to the reducedform shows no increase in the vitamin over that which has been treated bythe ordinary method.

There appears to be a definite relationship between the egg laying seasonand the number and size of the ascorbic acid granules in the oocytes. Arionsubfuscus lays its eggs during late July, August, and early September. Animals

456 Pelluet and Watts—The Cytosome of differentiating Cells of Slugs

J_.O5 MM.Fio. a. Ascorbic acid in oocytes of D. laeve at various ages, A, 33 days, B, 34 days, c, 36

days, D, 51 days (note ascorbic acid in nucleolus). E, 65 days.

killed in February, March, April, or May show very little ascorbic acid in theiroocytes, and the granules are always extremely fine. During June and Julythe granules increase steadily in number and size. Oocytes of animals killedduring the laying season contain many large grains in their cytoplasm. Duringlate September the granules become smaller and fewer. Towards the end ofNovember the ovotestes of animals killed were infested with parasites, and noascorbic acid was observed. No observations could be made in December orJanuary, all the adult animals having died by this time.


Deroceras reticulatum begins to lay towards the end of March, though feweggs are laid until late June, when a peak is reached. Laying continues moder-ately through the summer, until a second peak is reached in the latter partof September. After this time the number of eggs laid falls off rapidly. Theascorbic acid content of the oocytes parallels the laying cycle, the granulesseen in sections being larger and more numerous during peak periods, and inmany cases absent entirely from December to March (fig. ID).

D. laeve, a species which seems to lay all the year round, almost always hasa good supply of ascorbic acid in its oocytes.

As we have not yet followed the ascorbic acid during the maturation of theegg, it is not possible to state whether the mature eggs of these slugs will proveto be exceptional like those of Aplysia,in having a high content of ascorbicacid, or will be like the majority ofinvertebrate eggs investigated, whichaccording to Needham (1942) containlittle or none.

It has been stated that ascorbic acidinactivates or inhibits phosphatase andthat therefore these two substances donot occur together in the same cell(Day, 1949; von Euler and Soenson,1934). This is not in accordance withour results, since pieces of the sameovotestis, prepared for either ascorbicacid or alkaline phosphatase, show quitedefinitely that the two substances canexist side by side in the same oocyte; in fact the two show a similar type ofdistribution in the growing eggs. Giri (1939) found that ascorbic acid or Cualone has no effect on the activity of phosphatases, while it is inhibited by theascorbic acid/Cu complex. He suggests that phosphatase activity is regulatedby ascorbic acid as well as by other substances in the cell.

MitochondriaThe methods of Altmann and Benda, as given by McClung (1929), were

used.The vast amount of work carried out on the role of mitochondria in develop-

ing germ-cells might be thought to render yet another account superfluous,even on material not previously studied; but the purpose of this work isnot primarily to follow the changes during sperm-formation, but rather todetermine whether the distribution of ascorbic acid and alkaline phosphatasehas any connexion with the distribution of the mitochondria in the germinalepithelium and germ-cells. One might expect a relationship between alkalinephosphatase and mitochondria, since the latter are now generally believed tobe sites of enzyme activity.

.O5 MM.

FIG. 3. Germ-cells of adult A. subfuscus,showing sites of ascorbic acid. A, oocyte.B, germinal epithelium, c, spermatogonia.

D, spermatocytes.


Owing to a scarcity of young animals of some species, studies of the earlyovotestis were restricted chiefly to Deroceras laeve. In an animal 26 days afterhatching, mitochondria are present in clouds round the nuclei of the young

FIG. 4. Mitochondria in germ-cells of D. laeve. A, oocytes. B, spermatogonia at 26 days.c, spermatogonia at 68 days, D, germinal epithelial cell, E, developing nurse-cell at 26 days,

drawn to right-hand scale.

FIG. 5. Mitochondria in developing oocytes of D. laeve. A, germinal epithelial cell.B-E, growth-stages of oocytes.

oocytes and are scattered through the cytoplasm of the spermatogonia, thenurse-cells, and the germinal epithelium (fig. 4). At 68 days old the area roundthe nucleus has become filled with mitochondria. The ovotestis of Arion sub-fuscus 78 days old presents much the same picture, the only difference beingthat the mitochondria are more numerous in the cytoplasm and do not show a


perinuclear concentration. No differences were observed in the size and num-ber of mitochondria in the germinal epithelial cells in any of the forms studiedby which one could determine whether a cell was to become a spermatogoniumor an oogonium.

The changes accompanying the development of an oocyte from a germinalepithelium cell in an adult specimen of Deroceras laeve are illustrated in fig. 5.There is a steady increase in the number of mitochondria, until they form a

FIG. 6. Mitochondria in A. hortensis. A, germinal epithelium. B, primary spermatocytes.c, first maturation division, D, spermatids. E, second maturation division.

cloud round the nucleus, in which two or more nucleoli appear. The cyto-plasm of the oocyte eventually becomes filled with mitochondria, while theperinuclear concentration persists. The mitochondria, which do not spread tothe periphery of the oocyte until well on in the growth period, often occurunevenly distributed in small groups.

The distribution of mitochondria during the development of a spermato-zoon from an indifferent cell is the same in all five species studied.

The spermatogonia, which develop from the indifferent epithelial cells,have small mitochondria, evenly distributed through their thin layer ofcytoplasm (fig. 43). The spermatogonial cells may go through their growthstages either attached to the acinus wall or free in the lumen. The mitochondriaincrease in number as the cytoplasm increases in volume. When the cellreaches its maximum growth, the nucleus moves to one end of the cell, whichbecomes slightly elongated, and the mitochondria become grouped in a cloudon the side of the nucleus on which the bulk of the cytoplasm lies. At thisstage, several small rods taking the mitochondrial stains appear in this cloud,and the cell may now be considered a primary spermatocyte. The deeplystained rods assume a somewhat diamond-shaped arrangement, enclosing anarea in which the mitochondria are few and very small. The rods, which areeasily visible in live material, are impregnated in Golgi preparations andcorrespond to Parat's lepidosomes (Parat, 1928).


The first maturation division now takes place and fig. 6c illustrates thetypical perinuclear concentration of the mitochondria at diakinesis. Themitochondria sometimes assume the form of short rods during division. Thisagrees with Gatenby's observations on Helix aspersa (Gatenby, 1917). Thesecondary spermatocyte cells are distinguished from the primary spermato-cytes by their much smaller size and lesser concentration of mitochondria.The second maturation divisions were more rarely observed, one of these

FIG. 7. Mitochondria in living preparations. A, B, c, A. subfusats stained by Janus green B1 :io,coo. D, D. reticulalum, unstained, in slug saline. A is a spermatocyte, the rest are sper-

matids.

being shown in fig. 6E. The small spermatids resulting from this divisionincrease rapidly in size, and their cytoplasm becomes pulled out at one end.

The behaviour of the mitochondria during the formation of the maturespermatozoon is essentially the same as that described in Helix by Gatenby

97)In mature spermatozoa, both in live and fixed material, there appears a

double spiral, beginning at the base of the head and running back along the


tail, round the axial filament. The posterior extremity of the spiral has notbeen observed owing to the difficulty in tracing it. The spiral takes up theaniline-fuchsin strongly, and in a cross-section of the tail it appears like one ortwo large mitochondria. It seems possible that this double spiral may beformed from the mitochondria which remain beside the axial filament, by anaggregation of the granules. Cross-sections of the tail which show the two dotsapparently have fewer mitochondria than those in which the dots are absent.

ACKNOWLEDGEMENTS

We are grateful to the Canadian Cancer. Institute for grants for thepurchase of optical and photographic equipment and also to the NationalResearch Council for assistance throughout this work.

REFERENCESANCEL, P., 1903. Arch. Biol., 19, 389.BOURNE, G., 1936a. Anat. Rec, 66, 369.

1936&. Physiol. Rev., 16, 442.DANIELLI, J. F., 1946. Brit. J. exp. Biol., 22, no .DAY, M. F., 1949a. Aus. J. sci. Res. B, 2, 19.

19496. Ibid., 2, 31.DEANE, H. W., and DEMPSEY, E. W., 1945. Anat. Rec., 93, 401.DE NICOLA, M., 1949. Quart. J. micr. Sci., 90, 391.EMMEL, V. M., 1945. Anat. Rec, 91, 39.EULER, H. VON, and SOENSON, T., 1934. Quoted by King, 1936.GATENBY, J. B., 1917. Quart. J. micr. Sci., 248, 556.GIRI, K. V., 1939. Biochem. J., 33, 309.GIROUD, A., and RATSIMAMANGA, A., 1936. Bull. Soc. chim. Biol., 18, 373.GOMORI, G., 1939. Proc. Soc. exp. Biol., 43, 23.

1941. J. cell. comp. Physiol., 17, 71.KING, C. G., 1936. Physiol. Rev., 16, 238.KRUGELIS, E. J., 1945. Bio. Bull., 89, 220.LAMS, H., 1909. Acad. Roy. Belgique, 4.MCCLUNC, C. E., 1929. Handbook of microscopical technique. New York (Hoeber).NEEDHAM, J., 1942. Biochemistry and morphogenesis. Cambridge (Univ. Press).ORD, M. J., and WATTS, A. H. G.( 1948-9. Proc. N. S. Inst. Sci., 22, pt. 3.PARAT, M.( 1928. Arch. d'Anat. micr., 24, 73.SCHNEIDER, W. C, 1946. J. biol. Chem., 165, 583.YAQ, T. 1950. Quart. J. micr. Sci., 91, 89.

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