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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
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.
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.
2,;


                2
            0
‘el
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.
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).
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
478                                                                                                               ERMAK




                                                         References

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ERMAK, T. H. 1975. An autoradiographic         demonstration     of blood cell renewal in Styela clava (Urochordata:
    Ascidiacea). Experientia, 31, 837-839.
GALL, J. C. and PARDUE, M. L. 1969. Formation and detection of RNA-DNA                      hybrid molecules in cytological
    preparations.    Proc. natn. Acad. Sci. U.S.A., 63, 378-383.
HELLER, C. G. and CLERMONT,Y. 1963. Spermatogenesis                in man: an estimate of its duration. Science, 140,
     184-I 86.
HOLLAND, N. D. and GIESE, A. C. 1965. An autoradiographic                 investigation of the gonads of the purple sea
    urchin (Strongylocentrotus purpuratus). Biol. Bull. mar. biol. Lab., Woods Hole, 128, 241-258.
HUTCHINGS, P. A. 1973. Gametogenesis         in a Northumberland       population of the polychaete Me&ma c’ristotu.
     Mar. Eiol., 18, 199-2 I I.
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    California. Ms. Thesis, University of Nebraska.
KESSEL, R. G. and KEMP, N. E. 1962. An electron microscope study on the oocyte, test-ceils, and follicular
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Ermak styela clava renewal gonads tiss cell 1976

  • 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.
  • 4. 2,; 2 0 ‘el
  • 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 References ABBOTT, D. P. and JOHNSON, J. V. 1972. The ascidians Sty& bnmharti, S. plicata, S. c&a, and S. montrrqr- ensis in California waters. Bull. S. Cal. Acad. Sci., 71,955105. BEEMAN, R. D. 1970. An autoradiographic and phase contrast study of spermatogenesis in the anaspidean apisthobranch Phyllaplysia taylori, Dali, 1900 (Gasrropoda: Opistobranchia). Archs Zoo/. exp. gCn.. 111, S-22. BROWN, D. D. and DAWID, I. 1968. Specific gene amplification in oocytes. Science, 160, 272-280. CARLISLE, D. B. 1954. Styela mommiculata n.sp., a new species of ascidian from the Plymouth area. J. mar. biol. Ass. U.K., 33, 329-334. CAVEY, M. J. 1976. Ornamentation of the larval ascidian tunic by test cells. J. Ultrastruct. Rrs., 55,297-298. CHANDLEY, A. C. and BATEMAN,A. J. 1962. Timing of spermatogenesis in Drosophila melanogas/rr using tritiated thymidine. Nature, Lond., 193, 299-300. CLARK, R. B. and OLIVE, P. J. W. 1973. Recent advances in polychaete endocrinology and reproductive biology. A. Rev. Oceanogr. Mar. Biol., 11,175-222. CLERMONT,Y., LEBLOND, C. P. and MESSIER,B. 1959. Duree de cycle de I’epithelium seminal du rat. Archs Anat. microsc. Morph. exp., 48, 37-55. COWDEN, R. R. 1961. A comparative cytochemical study of oocyte growth and development in two species of ascidians. Acta Emb. Morphol. Exp., 4, 123-141. DURAND, M. 1958. Incorporation de thymidine tritee dans I’ovaire des gryllides. Exp. Cell. Res., 15. 257-259. ERMAK, T. H. 1975. An autoradiographic demonstration of blood cell renewal in Styela clava (Urochordata: Ascidiacea). Experientia, 31, 837-839. GALL, J. C. and PARDUE, M. L. 1969. Formation and detection of RNA-DNA hybrid molecules in cytological preparations. Proc. natn. Acad. Sci. U.S.A., 63, 378-383. HELLER, C. G. and CLERMONT,Y. 1963. Spermatogenesis in man: an estimate of its duration. Science, 140, 184-I 86. HOLLAND, N. D. and GIESE, A. C. 1965. An autoradiographic investigation of the gonads of the purple sea urchin (Strongylocentrotus purpuratus). Biol. Bull. mar. biol. Lab., Woods Hole, 128, 241-258. HUTCHINGS, P. A. 1973. Gametogenesis in a Northumberland population of the polychaete Me&ma c’ristotu. Mar. Eiol., 18, 199-2 I I. JOHNSON, J. V. 1971. The annual growth and reproductive cycle of Styela sp. in the marine del Rey, Venice, California. Ms. Thesis, University of Nebraska. KESSEL, R. G. and KEMP, N. E. 1962. An electron microscope study on the oocyte, test-ceils, and follicular envelope of the tunicate, Molgula manhattensis. J. Ultrastruct. Res., 6, 57-76. LIMA-DE-FARIA, A. and BORUM, K. 1962. The period of DNA synthesis prior to meiosis in the mouse. J. Cell Biol., 14, 381-388. LIPKIN, M. 1973. Proliferation and differentiation of gastrointestinal cells. Physiol. Rev., 53, 891-915. MANCUSO, V. 1965. An electron microscope study of the test cells and follicle cells of Ciona intestinalis during oogenesis. Acta. Embryol. Morphol. Exp., 8, 230-266. MANSUETO,C. 1964. Sulla reproduzione per divisione mitotica delle cellule testali delle Ascidie. Rend. Arc. Naz. Link, 36, 683-689. MESSIER,B. and LEBLOND,C. P. 1960. Cell proliferation and migration as revealed by radioautography after injection of thymidine-H3 into male rats and mice. Am. J. Anat., 106, 247-265. MEUSY, J. 1964. Determination de la durQ de la spermatogbnese d’orchestia gammarella Pallas, crustacea amphipode, par injection de thymidine tritite et autoradiography. Archs. Anat. microsr. Morph. exp., 53,253-260. OLIVE, P. J. W. 1972. Regulation and kinetics of spermatogonial proliferation in Arenicola marine (Annelida, Polychaeta). II. Kinetics. Cell Tissue Kinet., 5, 255-267. RLJDKIN, G. T. and GRIECH, H. P. 1962. On the persistence of oocyte nuclei from fetus to maturity in the laboratory mouse. J. Cell Biol., 12, 169-175. TUCKER, G. H. 1942. The histology of the gonads and development of the egg envelopes of an ascidian (Styelaplicata Lesueur). J. Morph., 70, 81-I 13. TWEEDELL,K. S. 1966. Oocyte development and incorporation of H3-thymidine and HQridine in fectinario (Cistenides) gouldii. Biol. Bull. mar. biol. Lab., Woods Hole, 131, 516-538. VAN NAME, W. G. 1945. The North and South American Ascidians. Bull. Am. Mus. nat. Hist., 84, l-476. VANDENBERG, P. 1963. Synthesis and transfer of DNA, RNA, and protein during vitellogenesis in Rhodnius J. prolixus (Hemiptera). Biol. Bull. mar. biol. Lab., Woods Hole, 125, 556-575. WALKER. B. C. 1960. Renewal of cell populations in the female mouse. Am. J. Anat., 107, 95-105.