TISSUE & CELL 1976 8 (3) 471-478Published by Longman Group Ltd. Printed in Great Britain THOMAS H. ERMAKRENEWAL OF THE GONADS IN STYELACLAVA (UROCHORDATA: ASCIDIACEA) ASREVEALED BY AUTORADIOGRAPHY WITHTRITIATED THYMIDINE ABSTRACT. DNA-synthesizing cells in the gonads of the ascidian Sty& claua were labeled with tritiated thymidine and detected with autoradiography. In the testis, spermatogonia and primary spermatocytes are labeled after 1 hr. Labeled sper- matozoa occur in the lumen of the testis follicles after 10 days and in the suerm ducts after 20 days. In the ovary, only germ cells (oogonia and pre-ieptotene primary oocytes) and follicle cells are labeled after 1 hr. By 60 days, oocytes with basophilic cytoplasm (15-65 p in diameter) are labeled; test cells embedded in larger eosinophilic oocytes (150 p in diameter) are also labeled. Germ cells give rise to both oocytes and follicle cells. Through continued cell division, follicle cells give rise to test cells. Introduction Kessel and Kemp, 1962) and from blood cells (Mancuso, 1965).IN adult invertebrates, germ cells in In the present investigation, tritiatedpremeiotic and premitotic DNA synthesis thymidine was used to time the events offrom both the testis and ovary can be gametogenesis in the ascidian Styela clava, alabeled with tritiated thymidine and detected hermaphroditic marine invertebrate. Thewith autoradiography. By taking samples of transformation of germ cells into maturethe gonads at increasing time intervals after gametes and the differentiation of accessorya brief exposure to tritiated thymidine, the cells from precursor cells were followed.events of gametogenesis can be timed(Durand, 1958 ; Chandley and Bateman,1962; Holland and Giese, 1965; Tweedell, Materials and Methods1966; Beeman, 1969; Olive, 1972; Hutchings, Specimens of Styefa claw were collected1973). In addition to the germ cells, gonadal from Mission Bay, San Diego, California,accessory cells in the ovary can also be and injected intra-atrially with 1 &i oflabeled. tritiated thymidine (New England Nuclear In ascidians, the accessory cells surround- Corp.) per gram fresh weight. The aqueousing the growing oocytes are the follicle cells solution of tritiated thymidine (specificand test cells; the test cells are unique to activity 6.7 Ci/mmol) was diluted with anthe tunicates. However, the accessory cells equal volume of two times concentrated seahave been said to originate from both germ water before use. Three individuals werecells in the ovarian wall (Tucker, 1942; sacrificed by fixation in Bouin’s fluid at each of the following time intervals: 1 hr, 10 days, Scripps Institute of Oceanography, University of 20 days, 30 days, and 60 days. The gonadsCalifornia at San Diego, La Jolla, California 92037. were dissected out, dehydrated, and em- Present address: Department of Physiology,University of California, San Francisco, California bedded in paraffin. Seven micron sections94143. were covered with Kodak Nuclear Track Received 30 June 1975. Emulsion type NBT-2 by the dipping Revised 10 March 1976. method and stored at 4°C for periods of
472 ERMAK2 weeks to 2 months. Autoradiograms were follicles (Fig. 1b). Four to nine ovaries occurdeveloped in Kodak D-l 9 developer (3 min), on the right side of the body while two toand sections were stained through the five occur on the left side (Abbott andemulsion with hematoxylin. Johnson, 1972). Each ovary consists of a long tube which Results is U-shaped in cross section (Fig. lc, e). A short oviduct extends past the ovary and isGeneral morphology continuous with the ovarian cavity. TheThe gonads of Styela have been described vas efferentia pass from the surface of themost completely by Van Name (1946), testes to the medial surface of the ovaryCarlisle (1954) and Tucker (1942). The where they form the vas deferens (Fig. 1b).ovaries and testes are separate and are The vas deferens runs the length of thelocated within the body wall between the ovary to the end of the oviduct.atria1 epithelium (an epidermal derivativelining the body cavity) and the muscles of Testisthe body wall. Each ovary runs posteriorly Each testis follicle of a mature animalfrom the atria1 siphon (Fig. la) and is consists of a simple or lobulated sac whoseflanked on each side by numerous testis wall is composed of cells in various stages of Fig. 1. (a) Gonads on left side of body of Sryela claua. (b) Relationship between ovary (o), testis (t), and sperm ducts. (c) Cross section through part of body wall (x-x’ in (a)) showing tubular ovaries and testis follicles. (d) Cross section through a testis in body wall. (e) Cross section through an ovary in body wall showing oocytes in progressive stages of growth. (f) Cross section through a stage III oocyte showing follicular envelope and follicular stalk (fs). ae, atria1 epithelium; c, chorion; ce, ciliated epithelium; ge, germinal epithelium; if, inner follicle cells; oc, ovarian cavity; of, outer follicle cells; ooc I, stage I oocyte; ooc II, stage II oocyte; ooc III, stage III oocyte; sp, spermatozoa; tc, test cell; vd, vas deferens.
RENEWAL OF ASCIDIAN GONADS 413spermatogenesis and whose lumen is filled and directly adjacent to it. Cowden (1961)with spermatozoa (Fig. Id). The basal layer has described the oocytes in Ascidia andof germinal cells consists of spermatogonia. Ecteinascidia and, based upon cytochemicalSpermatogonia, spermatocytes, and sperma- criteria, classified the oocytes into threetids each form a continuous layer of variable stages; the oocyte stages of Cowden are alsothickness around the circumference of the applicable to Styehfollicle. The sperm ducts are lined by a Stage I oocytes range from about 15 tociliated epithelium. 65 p in diameter and possess extremely The most advanced germinal cells which basophilic cytoplasm. Stage I oocytes haveincorporate tritiated thymidine are the not yet acquired a follicular envelopeprimary spermatocytes involved in pre- (defined below) although some oocytesmeiotic DNA synthesis. The spermatogonia have a few follicle cells surrounding them.are presumed to be preparing for mitotic Stage II oocytes measure about 75-100 pdivision. At 1 hr after injection, many and are lightly basophilic while stage IIIspermatogonia and primary spermatocytes oocytes are acidophilic and have a diameterare labeled (Fig. 2), but no spermatids or of about 150 p. Stage III oocytes have aspermatozoa are labeled. Usually, most of completed follicular envelope consisting,the spermatogonia and primary spermato- from outside to inside, of two layers ofcytes are labeled. However, labeled germinal follicle cells, the chorion, and a layer of testcells frequently occur clustered into nests. cells embedded in the cytoplasm of theBy 10 days after injection, spermatids and oocyte (Fig. If). Some test cells also occurspermatozoa are now labeled (Fig. 3). Thus, in stage II oocytes. The outer follicularthe testis is a renewing population (as epithelium is continuous with the germinaldefined by Messier and Leblond, 1960). The epithelium by a follicle stalk. Both layerstime required for a primary spermatocyte of follicle cells completely cover the surfaceto differentiate into a spermatozoan is at of the oocyte. The test cells, however, nevermost 10 days. It is assumed that the labeled form a complete layer around the oocytespermatozoa are derived from primary but lie in indentations in the surface of thespermatocytes which are labeled at the time oocyte (Fig. If). of injection. In some testis follicles, labeled In the ovary, the germinal cells whichspermatozoa are distributed throughout the incorporate tritiated thymidine into thecentral cavity. In others, the spermatozoa nuclear DNA include the oogonia and pre- in the center of the lumen are unlabeled. leptotene primary oocytes. The distributionSome spermatogonia and spermatocytes of labeled nuclei is similar at 1 hr, 10 daysare also labeled at 10 days, but not as many and 20 days after injection. Several nuclei are labeled as after 1 hr. in the germinal epithelium are labeled By 20 days after injection, labeled (Fig. 5) as well as numerous follicle cellsspermatozoa fill the lumen of all the testis surrounding those stage I oocytes not yetfollicles sectioned and, by both 20 and fully covered by a primary follicle. Follicle 30 days, frequently fill the sperm ducts cells are also labeled in the follicle stalks and(Fig. 4). At all times after injection, scattered around the stage II and stage III oocytesreactions are observed above nuclei in the (Figs. 6, 7). The follicle cells around theepithelia of the sperm ducts (Fig. 7), indicat- stage I oocytes, however, are undergoing theing that the sperm ducts belong to an ex- greatest cell proliferation. Up until 20 days,panding population. no oocytes at any stage and no test cells are labeled (Figs. 5, 6, 7).Ovary At 60 days after injection, a few stage IA cross section of an ovary is shown in oocytes are labeled (Fig. 8). Surprisingly,Fig. le. A single layered germinal epithelium the nuclear region containing the nucleolusextends along the inner edge of the ovary and is always the most heavily labeled. It seemsis continuous with the ciliated epithelium possible that the chromosomes of stage Iof the ovary and oviduct. Oocytes become oocytes are aggregated around the peripheryprogressively larger as they are displaced of the nucleolus, leaving the rest of thefurther from the germinal epithelium; the nucleus nearly empty of labeled DNA. Suchsmallest oocytes lie within the ovarian wall clumping might be an artifact of fixation.
kg. 7. Autoradiogram of a stage III oocyte (ooc III) 10 days after injection showing several labeled follicle cells (fc) but no labeled test cells (tc). vd, YBSdeferens. x 500. Fig. 8. Autoradiogram of an ovary 60 days after injection showing a labeled stage I oocyte (ooc 1). labeled follicle cells (fc), and labeled cells in the ciliated epitbelium (ce). oc, ovarian cavity. x 640. Fig. 9. Autoradiogram of a stage III oocyte (ooc III) 60 days after injection showing labeled test cells (tc). x 500. Fig. 2. Autoradiogram of two testis follicles I hr after injection showing labeledgenial cells. ae, atrial epithelkn; ge, germinal epithelium; In, lymph nodule; m.muscle; sp, spermatozoa. x 160. Fig. 3. Autoradiogram of testis follicles 10 days after injection showing labeledspermatozoa (sp). ae, atrial epithelium; ge, germinal epithelium. x 160. Fig. 4. Autoradiogram of a sperm duct (vd) 30 days after injection showing labeledspermatozoa (sp). ae, atria1 epitbelium. x 640. Fig. 5. Autoradiogram of ovarian germinal epithelium (ge) 10 days (same as 1 hrand 20 days) after injection showing labeled genial cells. ooc I, stage I oocyte; ooc II,stage II oocyte. x 500. Fig. 6. Autoradiogram of a stage II oocyte (ooc II) and follicles cells (fc) IO days afterinjection. In the oocyte, test cells (tc) are not labeled. x 500.
476 ERMAKOn the other hand, the nucleolar label the ideas of Mancuso (1965) and othersmight represent extrachromosomal rDNA (see Mancuso, 1965), who believe that the(Brown and Dawid, 1968; Gall and Pardue, accessory cells are derived from the vascular1969) engaged in the synthesis of rDNA to elements. Mancuso’s scheme seems unlikelybe utilized during embryogenesis. These since most of the labeled blood cells havepossibilities need to be tested by further work been removed from the blood system before(electron microscopy, electron microscopic any test cells even become labeled (Ermak,autoradiography, and enzymatic digestions). 1975). The renewal of the accessory cellsA few test cells (Fig. 9) are also labeled at is of the order of months and the renewal of60 days after injection. The test cells are the blood cells is of the order of weeks.presumed to originate from follicle cells Although Mansueto (1964) observed thewhile the oocytes are presumed to originate uptake of tritiated thymidine in the test cellsfrom gonial cells in the ovarian wall. of young oocytes in Ciona, 1 have not The ciliated epithelium of the ovary has observed any uptake in the test cells ofscattered reactions at all times after injection either stage II or stage 111oocytes in Styela.(Fig. 8), indicating that the ciliated epithelium In Ciona, as well as in Ascidia, Molgula, andis an expanding population. The ovary, on Pyuru, only follicle cells and not test cellsthe other hand, is a renewing population are labeled after a 1 hr exposure to tritiatedcomposed of stem cells, follicle cells, test thymidine (Ermak, unpublished results).cells and oocytes. According to Tucker (1942), the follicle cells are presumed to first form a single Discussion layered primary follicle around the growing oocyte (stage I oocyte) and then, withThe time course for the appearance of continued cell division, the inner and outerlabeled spermatozoa in the testis of Styela follicle layer. The test cells apparentlyis similar to that observed in other in- originate from the inner follicle epithelium:vertebrates. The period from DNA synthesis after the test cells are produced, the chorionin the primary spermatocyte to the appear- is formed between the test cells and theance of labeled spermatozoa is about a week inner follicle layer. At ovulation, the outerand a half in fruit flies (Chandley and follicle layer remains behind in the ovary.Bateman, 1962), amphipods (Meusy, 1964), The chorion rises from the surface of thesea urchins (Holland and Giese, 1965), and egg as the test cells move into the peri-sea hares (Beeman, 1970). In mammals, vitelline space. The inner follicle cells becomethe time period for the latter part of the foam cells of the ovum. The test cellsspermatogenesis is somewhat longer, about possibly function like the follicle cells in35 days in the rat (Clermont et al., 1959) nourishing the growing oocyte (Kesseland 48 days in man (Heller and Clermont, and Kemp, 1962). They might also play a1963). The total time of spermatogenesis is role in the formation of the larval tuniclonger than the first appearance of labeled (Cavey, 1976).sperm since spermatogenesis begins some- The uptake of tritiated thymidine by thewhere during the production of spermato- ovary appears similar to that observed ingonia. Beeman (1970) in his study of gastro- many other invertebrates (Vandenberg, 1963 ;pod spermatogenesis, points out that cluster- Holland and Giese, 1965; Clark and Olive,ing of labeled germinal cells indicated local 1973) where germinal cells synthesize DNAsynchronous division and differentiation in the adult. In mammals, by contrast, DNAwithin each nest of spermatogenic cells; the synthesis in oogonia and primary oocytessame pattern occurs in Styela. only occurs during fetal development (Rud- The results of the autoradiography in the kin and Griech, 1962; Lima-de-Faria andovary confirm the belief of Tucker (1942), Borum, 1962) giving the name of a decayingKessel and Kemp (1962), and others (see population to the oocytes of the adultKessel and Kemp, 1962) who believe that mammalian ovary (Lipkin, 1973). The onlythe ovarian germinal cells differentiate into renewing cell populations in the femaleboth oocytes and follicle cells, and that the reproductive organs of mammals are thefollicle cells further differentiate into test linings of the uterus and vagina (Walker,cells. By contrast, my results do not support 1960).
RENEWAL OF ASCIDIAN GONADS 477 Although the events of oogenesis in recovered from the coelom after as few asStyela clava were not followed for more 7 days (Tweedell, 1966) or 18 daysthan 60 days, it is assumed that the oocytes (Hutchings, 1973); presumably the remainderfully mature during the same year since its of oocyte growth takes at least several morelife span is only a year to 18 months and months. In StyeZa clava, germinal cells hadbreeding occurs from February to November differentiated into at least stage I oocytes(Johnson, 1971). Holland and Giese (1965) by 60 days. The total time period forobserved that the label in the sea urchin oogenesis is probably on the order of severalgerm cells did not pass through stages of the months.oocytes to the mature ova during long termexperiments. They suggested that the primary Acknowledgementsoocytes remain in the dictyotene stage untilthe following reproductive year when the I am indebted to Dr Nicholas D. Holland foroocytes grow and mature into ova. In his advice, support and criticism. I thankannelids, where oocyte growth occurs in the Dr David Epel for his suggestions and Emilycoelom, very small labeled oocytes have been Reid for the drawings in this paper. 31
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