TISSUE & CELL 1976 8 (3) 471-478 Published by Longman Group Ltd. Printed in Great Britain

THOMAS H. ERMAK

RENEWAL OF THE GONADS IN STYELA CLAVA (UROCHORDATA: ASCIDIACEA) AS REVEALED BY AUTORADIOGRAPHY WITH TRITIATED 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

IN adult invertebrates, germ cells in premeiotic and premitotic DNA synthesis from both the testis and ovary can be labeled with tritiated thymidine and detected with autoradiography. By taking samples of the gonads at increasing time intervals after a brief exposure to tritiated thymidine, the events of gametogenesis can be timed (Durand, 1958 ; Chandley and Bateman, 1962; Holland and Giese, 1965; Tweedell, 1966; Beeman, 1969; Olive, 1972; Hutchings, 1973). In addition to the germ cells, gonadal accessory cells in the ovary can also be labeled.

In ascidians, the accessory cells surround- ing the growing oocytes are the follicle cells and test cells; the test cells are unique to the tunicates. However, the accessory cells have been said to originate from both germ cells in the ovarian wall (Tucker, 1942;

Scripps Institute of Oceanography, University of California at San Diego, La Jolla, California 92037.

Present address: Department of Physiology, University of California, San Francisco, California 94143.

Received 30 June 1975. Revised 10 March 1976.

Kessel and Kemp, 1962) and from blood cells (Mancuso, 1965).

In the present investigation, tritiated thymidine was used to time the events of gametogenesis in the ascidian Styela clava, a

hermaphroditic marine invertebrate. The transformation of germ cells into mature gametes and the differentiation of accessory cells from precursor cells were followed.

Materials and Methods

Specimens of Styefa claw were collected from Mission Bay, San Diego, California, and injected intra-atrially with 1 &i of tritiated thymidine (New England Nuclear Corp.) per gram fresh weight. The aqueous solution of tritiated thymidine (specific activity 6.7 Ci/mmol) was diluted with an equal volume of two times concentrated sea water before use. Three individuals were sacrificed by fixation in Bouin’s fluid at each of the following time intervals: 1 hr, 10 days, 20 days, 30 days, and 60 days. The gonads were dissected out, dehydrated, and em- bedded in paraffin. Seven micron sections were covered with Kodak Nuclear Track Emulsion type NBT-2 by the dipping method and stored at 4°C for periods of

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2 weeks to 2 months. Autoradiograms were developed in Kodak D-l 9 developer (3 min), and sections were stained through the emulsion with hematoxylin.

Results

General morphology

The gonads of Styela have been described most completely by Van Name (1946), Carlisle (1954) and Tucker (1942). The ovaries and testes are separate and are located within the body wall between the atria1 epithelium (an epidermal derivative lining the body cavity) and the muscles of the body wall. Each ovary runs posteriorly from the atria1 siphon (Fig. la) and is flanked on each side by numerous testis

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follicles (Fig. 1 b). Four to nine ovaries occur on the right side of the body while two to five occur on the left side (Abbott and Johnson, 1972).

Each ovary consists of a long tube which is U-shaped in cross section (Fig. lc, e). A short oviduct extends past the ovary and is continuous with the ovarian cavity. The vas efferentia pass from the surface of the testes to the medial surface of the ovary where they form the vas deferens (Fig. 1 b). The vas deferens runs the length of the ovary to the end of the oviduct.

Testis

Each testis follicle of a mature animal consists of a simple or lobulated sac whose 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

spermatogenesis and whose lumen is filled with spermatozoa (Fig. Id). The basal layer of germinal cells consists of spermatogonia. Spermatogonia, spermatocytes, and sperma- tids each form a continuous layer of variable thickness around the circumference of the follicle. The sperm ducts are lined by a ciliated epithelium.

The most advanced germinal cells which incorporate tritiated thymidine are the primary spermatocytes involved in pre- meiotic DNA synthesis. The spermatogonia are presumed to be preparing for mitotic division. At 1 hr after injection, many spermatogonia and primary spermatocytes are labeled (Fig. 2), but no spermatids or spermatozoa are labeled. Usually, most of the spermatogonia and primary spermato- cytes are labeled. However, labeled germinal cells frequently occur clustered into nests. By 10 days after injection, spermatids and spermatozoa are now labeled (Fig. 3). Thus, the testis is a renewing population (as defined by Messier and Leblond, 1960). The time required for a primary spermatocyte to differentiate into a spermatozoan is at most 10 days. It is assumed that the labeled spermatozoa are derived from primary spermatocytes which are labeled at the time of injection. In some testis follicles, labeled spermatozoa are distributed throughout the central cavity. In others, the spermatozoa in the center of the lumen are unlabeled. Some spermatogonia and spermatocytes are also labeled at 10 days, but not as many are labeled as after 1 hr.

By 20 days after injection, labeled spermatozoa fill the lumen of all the testis follicles sectioned and, by both 20 and 30 days, frequently fill the sperm ducts (Fig. 4). At all times after injection, scattered reactions are observed above nuclei in the epithelia of the sperm ducts (Fig. 7), indicat- ing that the sperm ducts belong to an ex- panding population.

Ovary A cross section of an ovary is shown in Fig. le. A single layered germinal epithelium extends along the inner edge of the ovary and is continuous with the ciliated epithelium of the ovary and oviduct. Oocytes become progressively larger as they are displaced further from the germinal epithelium; the smallest oocytes lie within the ovarian wall

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and directly adjacent to it. Cowden (1961) has described the oocytes in Ascidia and Ecteinascidia and, based upon cytochemical criteria, classified the oocytes into three stages; the oocyte stages of Cowden are also applicable to Styeh

Stage I oocytes range from about 15 to 65 p in diameter and possess extremely basophilic cytoplasm. Stage I oocytes have not yet acquired a follicular envelope (defined below) although some oocytes have a few follicle cells surrounding them. Stage II oocytes measure about 75-100 p and are lightly basophilic while stage III oocytes are acidophilic and have a diameter of about 150 p. Stage III oocytes have a completed follicular envelope consisting, from outside to inside, of two layers of follicle cells, the chorion, and a layer of test cells embedded in the cytoplasm of the oocyte (Fig. If). Some test cells also occur in stage II oocytes. The outer follicular epithelium is continuous with the germinal epithelium by a follicle stalk. Both layers of follicle cells completely cover the surface of the oocyte. The test cells, however, never form a complete layer around the oocyte but lie in indentations in the surface of the oocyte (Fig. If).

In the ovary, the germinal cells which incorporate tritiated thymidine into the nuclear DNA include the oogonia and pre- leptotene primary oocytes. The distribution of labeled nuclei is similar at 1 hr, 10 days and 20 days after injection. Several nuclei in the germinal epithelium are labeled (Fig. 5) as well as numerous follicle cells surrounding those stage I oocytes not yet fully covered by a primary follicle. Follicle cells are also labeled in the follicle stalks and around the stage II and stage III oocytes (Figs. 6, 7). The follicle cells around the stage I oocytes, however, are undergoing the greatest cell proliferation. Up until 20 days, no oocytes at any stage and no test cells are labeled (Figs. 5, 6, 7).

At 60 days after injection, a few stage I oocytes are labeled (Fig. 8). Surprisingly, the nuclear region containing the nucleolus is always the most heavily labeled. It seems possible that the chromosomes of stage I oocytes are aggregated around the periphery of the nucleolus, leaving the rest of the nucleus nearly empty of labeled DNA. Such clumping might be an artifact of fixation.

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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, YBS deferens. 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 labeled genial 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 labeled spermatozoa (sp). ae, atrial epithelium; ge, germinal epithelium. x 160.

Fig. 4. Autoradiogram of a sperm duct (vd) 30 days after injection showing labeled spermatozoa (sp). ae, atria1 epitbelium. x 640.

Fig. 5. Autoradiogram of ovarian germinal epithelium (ge) 10 days (same as 1 hr and 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 after injection. In the oocyte, test cells (tc) are not labeled. x 500.

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On the other hand, the nucleolar label might represent extrachromosomal rDNA (Brown and Dawid, 1968; Gall and Pardue, 1969) engaged in the synthesis of rDNA to be utilized during embryogenesis. These possibilities need to be tested by further work (electron microscopy, electron microscopic autoradiography, and enzymatic digestions). A few test cells (Fig. 9) are also labeled at 60 days after injection. The test cells are presumed to originate from follicle cells while the oocytes are presumed to originate from gonial cells in the ovarian wall.

The ciliated epithelium of the ovary has scattered reactions at all times after injection (Fig. 8), indicating that the ciliated epithelium is an expanding population. The ovary, on the other hand, is a renewing population composed of stem cells, follicle cells, test cells and oocytes.

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the ideas of Mancuso (1965) and others (see Mancuso, 1965), who believe that the accessory cells are derived from the vascular elements. Mancuso’s scheme seems unlikely since most of the labeled blood cells have been removed from the blood system before any test cells even become labeled (Ermak, 1975). The renewal of the accessory cells is of the order of months and the renewal of the blood cells is of the order of weeks. Although Mansueto (1964) observed the uptake of tritiated thymidine in the test cells of young oocytes in Ciona, 1 have not observed any uptake in the test cells of either stage II or stage 111 oocytes in Styela. In Ciona, as well as in Ascidia, Molgula, and Pyuru, only follicle cells and not test cells are labeled after a 1 hr exposure to tritiated thymidine (Ermak, unpublished results).

According to Tucker (1942), the follicle cells are presumed to first form a single layered primary follicle around the growing oocyte (stage I oocyte) and then, with continued cell division, the inner and outer follicle layer. The test cells apparently originate from the inner follicle epithelium: after the test cells are produced, the chorion is formed between the test cells and the inner follicle layer. At ovulation, the outer follicle layer remains behind in the ovary. The chorion rises from the surface of the egg as the test cells move into the peri- vitelline space. The inner follicle cells become the foam cells of the ovum. The test cells possibly function like the follicle cells in nourishing the growing oocyte (Kessel and Kemp, 1962). They might also play a role in the formation of the larval tunic (Cavey, 1976).

The uptake of tritiated thymidine by the ovary appears similar to that observed in many other invertebrates (Vandenberg, 1963 ; Holland and Giese, 1965; Clark and Olive, 1973) where germinal cells synthesize DNA in the adult. In mammals, by contrast, DNA synthesis in oogonia and primary oocytes only occurs during fetal development (Rud- kin and Griech, 1962; Lima-de-Faria and Borum, 1962) giving the name of a decaying population to the oocytes of the adult mammalian ovary (Lipkin, 1973). The only renewing cell populations in the female reproductive organs of mammals are the linings of the uterus and vagina (Walker, 1960).

Discussion

The time course for the appearance of labeled spermatozoa in the testis of Styela is similar to that observed in other in- vertebrates. The period from DNA synthesis in the primary spermatocyte to the appear- ance of labeled spermatozoa is about a week and a half in fruit flies (Chandley and Bateman, 1962), amphipods (Meusy, 1964), sea urchins (Holland and Giese, 1965), and sea hares (Beeman, 1970). In mammals, the time period for the latter part of spermatogenesis is somewhat longer, about 35 days in the rat (Clermont et al., 1959) and 48 days in man (Heller and Clermont, 1963). The total time of spermatogenesis is longer than the first appearance of labeled sperm since spermatogenesis begins some- where during the production of spermato- gonia. Beeman (1970) in his study of gastro- pod spermatogenesis, points out that cluster- ing of labeled germinal cells indicated local synchronous division and differentiation within each nest of spermatogenic cells; the same pattern occurs in Styela.

The results of the autoradiography in the ovary confirm the belief of Tucker (1942), Kessel and Kemp (1962), and others (see Kessel and Kemp, 1962) who believe that the ovarian germinal cells differentiate into both oocytes and follicle cells, and that the follicle cells further differentiate into test cells. By contrast, my results do not support

RENEWAL OF ASCIDIAN GONADS

Although the events of oogenesis in Styela clava were not followed for more than 60 days, it is assumed that the oocytes fully mature during the same year since its life span is only a year to 18 months and breeding occurs from February to November (Johnson, 1971). Holland and Giese (1965) observed that the label in the sea urchin germ cells did not pass through stages of the oocytes to the mature ova during long term experiments. They suggested that the primary oocytes remain in the dictyotene stage until the following reproductive year when the oocytes grow and mature into ova. In annelids, where oocyte growth occurs in the coelom, very small labeled oocytes have been

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recovered from the coelom after as few as 7 days (Tweedell, 1966) or 18 days (Hutchings, 1973); presumably the remainder of oocyte growth takes at least several more months. In StyeZa clava, germinal cells had differentiated into at least stage I oocytes by 60 days. The total time period for oogenesis is probably on the order of several months.

Acknowledgements

I am indebted to Dr Nicholas D. Holland for his advice, support and criticism. I thank Dr David Epel for his suggestions and Emily Reid for the drawings in this paper.

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