1. 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 Kessel and Kemp, 1962) and from blood
cells (Mancuso, 1965).
IN adult invertebrates, germ cells in In the present investigation, tritiated
premeiotic and premitotic DNA synthesis thymidine was used to time the events of
from both the testis and ovary can be gametogenesis in the ascidian Styela clava, a
labeled with tritiated thymidine and detected hermaphroditic marine invertebrate. The
with autoradiography. By taking samples of transformation of germ cells into mature
the gonads at increasing time intervals after gametes and the differentiation of accessory
a 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 Methods
1966; Beeman, 1969; Olive, 1972; Hutchings, Specimens of Styefa claw were collected
1973). 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 of
labeled. tritiated thymidine (New England Nuclear
In ascidians, the accessory cells surround- Corp.) per gram fresh weight. The aqueous
ing the growing oocytes are the follicle cells solution of tritiated thymidine (specific
and test cells; the test cells are unique to activity 6.7 Ci/mmol) was diluted with an
the tunicates. However, the accessory cells equal volume of two times concentrated sea
have been said to originate from both germ water before use. Three individuals were
cells 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 gonads
California 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 sections
94143. 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
2. 472 ERMAK
2 weeks to 2 months. Autoradiograms were follicles (Fig. 1b). Four to nine ovaries occur
developed in Kodak D-l 9 developer (3 min), on the right side of the body while two to
and sections were stained through the five occur on the left side (Abbott and
emulsion 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 is
General morphology continuous with the ovarian cavity. The
The gonads of Styela have been described vas efferentia pass from the surface of the
most completely by Van Name (1946), testes to the medial surface of the ovary
Carlisle (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 the
located within the body wall between the ovary to the end of the oviduct.
atria1 epithelium (an epidermal derivative
lining the body cavity) and the muscles of Testis
the body wall. Each ovary runs posteriorly Each testis follicle of a mature animal
from the atria1 siphon (Fig. la) and is consists of a simple or lobulated sac whose
flanked 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.
3. RENEWAL OF ASCIDIAN GONADS 413
spermatogenesis 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 and
of germinal cells consists of spermatogonia. Ecteinascidia and, based upon cytochemical
Spermatogonia, spermatocytes, and sperma- criteria, classified the oocytes into three
tids each form a continuous layer of variable stages; the oocyte stages of Cowden are also
thickness around the circumference of the applicable to Styeh
follicle. The sperm ducts are lined by a Stage I oocytes range from about 15 to
ciliated epithelium. 65 p in diameter and possess extremely
The most advanced germinal cells which basophilic cytoplasm. Stage I oocytes have
incorporate tritiated thymidine are the not yet acquired a follicular envelope
primary spermatocytes involved in pre- (defined below) although some oocytes
meiotic 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 p
division. At 1 hr after injection, many and are lightly basophilic while stage III
spermatogonia and primary spermatocytes oocytes are acidophilic and have a diameter
are labeled (Fig. 2), but no spermatids or of about 150 p. Stage III oocytes have a
spermatozoa are labeled. Usually, most of completed follicular envelope consisting,
the spermatogonia and primary spermato- from outside to inside, of two layers of
cytes are labeled. However, labeled germinal follicle cells, the chorion, and a layer of test
cells frequently occur clustered into nests. cells embedded in the cytoplasm of the
By 10 days after injection, spermatids and oocyte (Fig. If). Some test cells also occur
spermatozoa are now labeled (Fig. 3). Thus, in stage II oocytes. The outer follicular
the testis is a renewing population (as epithelium is continuous with the germinal
defined by Messier and Leblond, 1960). The epithelium by a follicle stalk. Both layers
time required for a primary spermatocyte of follicle cells completely cover the surface
to differentiate into a spermatozoan is at of the oocyte. The test cells, however, never
most 10 days. It is assumed that the labeled form a complete layer around the oocyte
spermatozoa are derived from primary but lie in indentations in the surface of the
spermatocytes which are labeled at the time oocyte (Fig. If).
of injection. In some testis follicles, labeled In the ovary, the germinal cells which
spermatozoa are distributed throughout the incorporate tritiated thymidine into the
central cavity. In others, the spermatozoa nuclear DNA include the oogonia and pre-
in the center of the lumen are unlabeled. leptotene primary oocytes. The distribution
Some spermatogonia and spermatocytes of labeled nuclei is similar at 1 hr, 10 days
are 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 cells
spermatozoa fill the lumen of all the testis surrounding those stage I oocytes not yet
follicles 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 oocytes
reactions are observed above nuclei in the (Figs. 6, 7). The follicle cells around the
epithelia of the sperm ducts (Fig. 7), indicat- stage I oocytes, however, are undergoing the
ing 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 I
A 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 nucleolus
extends along the inner edge of the ovary and is always the most heavily labeled. It seems
is continuous with the ciliated epithelium possible that the chromosomes of stage I
of the ovary and oviduct. Oocytes become oocytes are aggregated around the periphery
progressively larger as they are displaced of the nucleolus, leaving the rest of the
further from the germinal epithelium; the nucleus nearly empty of labeled DNA. Such
smallest oocytes lie within the ovarian wall clumping might be an artifact of fixation.
5. 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 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.
6. 476 ERMAK
On the other hand, the nucleolar label the ideas of Mancuso (1965) and others
might represent extrachromosomal rDNA (see Mancuso, 1965), who believe that the
(Brown and Dawid, 1968; Gall and Pardue, accessory cells are derived from the vascular
1969) engaged in the synthesis of rDNA to elements. Mancuso’s scheme seems unlikely
be utilized during embryogenesis. These since most of the labeled blood cells have
possibilities 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 cells
A few test cells (Fig. 9) are also labeled at is of the order of months and the renewal of
60 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 the
while the oocytes are presumed to originate uptake of tritiated thymidine in the test cells
from 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 of
scattered 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, and
is an expanding population. The ovary, on Pyuru, only follicle cells and not test cells
the other hand, is a renewing population are labeled after a 1 hr exposure to tritiated
composed 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, with
The time course for the appearance of continued cell division, the inner and outer
labeled spermatozoa in the testis of Styela follicle layer. The test cells apparently
is 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 chorion
in the primary spermatocyte to the appear- is formed between the test cells and the
ance of labeled spermatozoa is about a week inner follicle layer. At ovulation, the outer
and 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 the
sea 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 become
the time period for the latter part of the foam cells of the ovum. The test cells
spermatogenesis is somewhat longer, about possibly function like the follicle cells in
35 days in the rat (Clermont et al., 1959) nourishing the growing oocyte (Kessel
and 48 days in man (Heller and Clermont, and Kemp, 1962). They might also play a
1963). The total time of spermatogenesis is role in the formation of the larval tunic
longer than the first appearance of labeled (Cavey, 1976).
sperm since spermatogenesis begins some- The uptake of tritiated thymidine by the
where during the production of spermato- ovary appears similar to that observed in
gonia. 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 DNA
synchronous division and differentiation in the adult. In mammals, by contrast, DNA
within each nest of spermatogenic cells; the synthesis in oogonia and primary oocytes
same pattern occurs in Styela. only occurs during fetal development (Rud-
The results of the autoradiography in the kin and Griech, 1962; Lima-de-Faria and
ovary confirm the belief of Tucker (1942), Borum, 1962) giving the name of a decaying
Kessel and Kemp (1962), and others (see population to the oocytes of the adult
Kessel and Kemp, 1962) who believe that mammalian ovary (Lipkin, 1973). The only
the ovarian germinal cells differentiate into renewing cell populations in the female
both oocytes and follicle cells, and that the reproductive organs of mammals are the
follicle cells further differentiate into test linings of the uterus and vagina (Walker,
cells. By contrast, my results do not support 1960).
7. RENEWAL OF ASCIDIAN GONADS 477
Although the events of oogenesis in recovered from the coelom after as few as
Styela clava were not followed for more 7 days (Tweedell, 1966) or 18 days
than 60 days, it is assumed that the oocytes (Hutchings, 1973); presumably the remainder
fully mature during the same year since its of oocyte growth takes at least several more
life span is only a year to 18 months and months. In StyeZa clava, germinal cells had
breeding 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 for
observed that the label in the sea urchin oogenesis is probably on the order of several
germ cells did not pass through stages of the months.
oocytes to the mature ova during long term
experiments. They suggested that the primary Acknowledgements
oocytes remain in the dictyotene stage until
the following reproductive year when the I am indebted to Dr Nicholas D. Holland for
oocytes grow and mature into ova. In his advice, support and criticism. I thank
annelids, where oocyte growth occurs in the Dr David Epel for his suggestions and Emily
coelom, very small labeled oocytes have been Reid for the drawings in this paper.
31
8. 478 ERMAK
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