/ . Embryol. exp. Morph. Vol. 23, 2, pp. 407-18, 1970
407
Printed in Great Britain
Stimulation of germ cell proliferation in the
planarian Dugesia tigrina (Girard)
By C. V O W I N C K E L 1
From the Department of Zoology, McGill University
Sexual strains of Dugesia tigrina display a seasonal development of sex organs
in late summer or autumn and lay cocoons in the suceeding spring. This is
followed by a period of asexual reproduction in mid-summer. Such a seasonal
alternation of reproductive pathways makes this flatworm an ideal subject of
inquiry into the inductive mechanisms controlling reproduction and its ultimate
coupling to changes in the environment. Asexual strains of this species reproduce
by fission only.
Neurosecretory phenomena appear to be involved in the induction of sexual
development of planarians, as was recently demonstrated by Lender (1964) and
Ude (1964) using aldehyde fuchsin staining techniques. Supporting electronmicrographic evidence came from Morita & Best (1965), Oosaki & Ishii (1965)
and Lentz (1967). Brain regeneration experiments, notably by Grasso (1959)
and Ghirardelli (1965), point in the same direction.
The importance of low temperatures in the control of sexual development was
pointed out by Hyman (1943 a, b) but critical experimentation demonstrating
the involvement of an endocrine factor and its dependence on an environmental
stimulus so far is lacking. In the work reported below an attempt has been made
to pursue a relationship between low water temperature and a subsequent
proliferation of germ cells via the mediation of homogenates.
MATERIALS AND
METHODS
Material. A population of D. tigrina (sexual strain) was collected from the
St Lawrence river in August 1966. Cocoons laid by this population in the
laboratory were isolated and used to determine the presence of germ cells in
newly hatched individuals (Table 1). One parent animal was selected to produce
a clone which provided the sexual strain for the experiments described below.
This clone furnished all experimental animals unless otherwise mentioned.
A population of D. tigrina (asexual strain, not cloned) was obtained from
California Supply House to permit a comparison between the sexual and asexual
strains.
Maintenance. All animals of the sexual strain were maintained in an incubator
1
Author's address: Department of Zoology, McGill University, Montreal, Canada.
408
C. V O W I N C K E L
at 23-24 °C, at a photoperiod of LD 16:8 (light from 0600 to 2200) and fed
regularly three times weekly on beef liver. These conditions are termed standard
maintenance conditions. The asexual population was maintained at room
temperature; light conditions were not controlled.
Methods. An adaptation of Lender's (1956) methods was used for the homogenization experiments. Groups of donor worms were exposed for 1 and 2 days
to a temperature of 14-16 °C and then homogenized. The resulting brei was
diluted with sterilized river water containing 0-04 % Penicillin G, to a concentration of one donor worm to 1 ml of fluid. This homogenate was found to be cell
free when investigated under the light microscope. Donor worms belonged to
the sexual strain in all homogenization experiments.
Recipient worms were decapitated and immersed in the homogenate, one
worm/ml, and kept under standard maintenance conditions for 24 h after which
the homogenate was replaced by a fresh one. Penetration of the worm by substances in the homogenate was encouraged by making a small longitudinal
incision into the anterior end of the worm. All recipient worms were fixed after
four successive exposures to homogenates.
Two controls were run: (1) Donor worms homogenized without preliminary
cold exposure but with otherwise identical treatment of recipient worms and
(2) recipient worms exposed to 0-04% penicillin-water only, without any homogenate, but otherwise receiving identical treatment, including incisions. All
homogenates were prepared in the late afternoon at 23 °C. The experiment was
performed twice.
Donor and recipient worms for each experiment were selected to be of uniform
size (7-8 mm) and in approximately the same stage of the fission cycle. The latter
was determined by the relative length of the regenerating tail.
Histological methods. All material was fixed in Bouin's fluid, embedded in
paraffin wax under vacuum and stained with haematoxylin and eosin. Serial
sections were cut at 16 JLO in order to facilitate counting of germ cells.
Enumeration of germ cells. Planarian germ cells are easily identified histologically. They form characteristic clusters and groups (Figs. 1, 2), as illustrated
by Curtis (1902). These clusters are embedded in the parenchyma dorsolateral
to the nerve cords. In the same general area cells are found which have the same
staining properties, general shape and large size but are not attached to other
germ cells. Since there is a possibility of mistaking neoblasts for germ cells lying
outside clusters, isolated germ cells were not included in the count. It is by their
typical adherence pattern that germ-cell clusters can be definitely identified.
Aggregations of neoblasts never show this typical pattern. For reasons of
simplification the term 'male germ cell' is used here to denote cells which give
rise to spermatogonia and therefore to spermatocytes. This is not meant to
imply that the author has any information available as to the exact stage of
development at which this cell lineage is determined. Thus the cells under
investigation may represent presumptive germ cells in the strict sense.
Germ cell proliferation
409
Counts of germ cells were made from serial sections, summed for the whole
worm and then corrected for the varying length of the specimen, using the
following relationship: C = (G/L) 200, where C = corrected total germ-cell
count of worm, G = the original total germ-cell count of the worm, and L =
the measured length of the specimen. The corrected germ-cell count therefore
expresses the germ-cell count per unit length multiplied by a factor of 200. This
factor returns the count to a value which is representative for a worm of average,
standard length.
RESULTS
The sexual strain during asexual reproduction. The cloned sexual strain was
kept under 'standard maintenance conditions' which correspond to summer
conditions. The fission interval at 24 °C was determined for five groups of
30 animals each. It varied from 7-7 to 9-2 days.
Serial sections of about 80 worms of the clone and over 100 of the mixed
population in the course of almost 2 years showed that male germ cells were
constantly present in three different conditions.
1. Single non-aggregated germ cells were found in a row dorsal or dorsolateral to the nerve cords. Long cytoplasmic extensions of these cells are frequently visible and can usually be traced to other germ cells. This suggests that
germ cells may be motile and seek attachment (Fig. 1 A).
2. Long ribbons or thick cords of germ cells are found in the same general
area, extending parallel to the nerve cords (Figs. 1C and 2 A, B).
3. More or less rounded clusters of germ cells lie more lateral to the cord
(Fig. 1 B). They were termed testicular primordia since they give rise to testis
follicles under proper conditions. The smallest primordia may contain only two
germ cells. The largest primordia are always found farthest laterally and may
display signs of deterioration, i.e. irregular staining qualities and loss of attachment between cells. The maximal size of lateral primordia varies from about 4 to
35 germ cells and apparently depends on the temperature at which the population is maintained. Higher temperatures result in smaller testicular primordia.
At 24 °C the ovaries of sexual strain individuals often contain several large
oocytes. Ovarian development and especially its regression was found to respond
more slowly to environmental variations. For this reason and since ovaries are
usually lost during decapitation their development under experimental conditions was not studied in detail.
Newly hatched worms of the sexual strain. Cocoons and newly hatched individuals were maintained at room temperature and without photoperiod control.
Worms were fixed at daily intervals from the day of hatching. The number of
germ cells associated in the largest testicular primordium of each worm is
given in Table 1. Each entry represents one worm and animals of the same age
in days were usually but not always siblings, i.e. derived from the same cocoon.
Entries where no germ cells could be determined, are to be understood as a lack
410
C. V O W I N C K E L
Fig. 1. (A) Unattached germ cell with long cytoplasmic extensions at arrow, x 2590.
(B) Small testicular cluster of eight cells (arrow) (only four in focus), x 962.
(C) Thick cord of germ cells after 11 days of 10 °C diurnal temperature changes.
(Compare with size of cord in Fig. 2B.) x 814. / = intestine; nc - nerve cord;
v = vitelline gland cells.
Germ cell proliferation
411
of reliable identification due to the great cell density at this age. They do not,
therefore, represent evidence for an absence of germ cells at this stage.
Serial sections show that as early as 2 days after hatching small primordia
were observed in two specimens. Larger clusters are found after 6 days of age.
No trace of ovarian development could be found at this age.
Table 1. Number of germ cells per largest testicular primordium
found in newly hatched worms
Each entry represents one animal
Age ini
days
0*
0
0
1
1
1
2
2
3
3
4
4
4
4
4
4
5
6
Number of
germ cells Unassociated
per largest
germ cells
primordium
observed
-t
—
—
—
—
—
2
2
—
—
—
—
—
2
2
2
—
—
No
Yes
Yes
No
No
Yes
Yes
Yes
No
Yes
No
No
Yes
Yes
Yes
Yes
Yes
Yes
* Age 0 denotes day of hatching.
Age in
days
7
7
7
8
8
8
9
9
9
9
10
11
12
13
14
21
28
35
Number of
germ cells Unassociated
per largest
germ cells
primordium
observed
2
4
4
—
3
5
—
—
—
3
2
2
5
—
—
2
3
2
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
t — denotes no reliable determination.
Asexual strain. Serial sections of ca. 80 mature individuals of the asexual
strain, kept at room temperature and natural photoperiods, were investigated.
No aggregated germ cells in the form of ribbons or as testicular primordia
(condition 2 and 3) were found. Single large cells are frequently observed lying
dorsal to the nerve cord. These resemble the single non-aggregated cells of the
sexual strain. They may or may not represent germ cells.
Effect of low temperatures on germ cells. Under standard maintenance conditions the total number of clustered germ cells per mature worm of the sexual
strain was found to vary from 5 to 329 in 15 individuals. The distribution was
not centered about the mean (114 + 82-2) but scattered about the whole range.
Exposure of sexual strain worms to low water temperatures followed two
different regimes: (A) Large diurnal variations. 23 °C from 6 p.m. to 2 p.m.
followed by a 4 h 10-12 °C fall in temperature, then a swift rise back to 23 °C;
412
C. VOWINCKEL
photoperiod LD 16:8. (B) Constant low temperature. A 10 °C fall in temperature at the beginning of the experiment followed by maintenance at 10-13 °C;
photoperiod LD 16:8.
Under both regimes one worm was fixed every 4th day and the clustered germ
cells counted. The results are represented in Table 2. Under regime (A) worms
showed an immediate increase in numbers of clustered germ cells for 32 days
followed by a decline almost to the level of the control group. Under regime (B)
germ-cell counts increased more slowly, less regularly and to smaller values. The
difference between germ-cell counts under regimes (A) and (B), however, was
not significant while both series had significantly higher counts than the control
group C.
Table 2. Number of clustered germ cells/worm under
different temperature regimes
All control worms were fixed simultaneously at the start. Photoperiod of LD 16:8
throughout experiment. All values corrected for length of worm.
Asexual strain
Sexual strain
A
1
^
Day
Control
S.D.
A
\
SMC*
Regime (A)
Regime (B)
260
148
329
60
29
109
5
78
156
127
68
522
516
475
570
617
602
890
383
394
331
457
475
321
549
4
8
12
16
20
24
28
4
8
12
16
20
23
27
0
0
0
0
0
0
15
1111
258
32 31
0
318
367
413
251
754
549
154
646
36
40
44
48
34
37
—
—
0
0
—
—
—
—
—
—
—
—
—
—
57
110
103
71
Mean
,
114
± 82
554
439
±231
±160
A
C
i
Regime (A)
Mest, sexual strain:
(A) against Control t = 6-6, d.f. = 27, significant at 0001 level.
(B) against Control / = 11-5, d.f. = 27, significant at 0-001 level.
(A) against (B) / = .1 -3, d.f. = 24, difference not significant.
* SMC = standard maintenance conditions.
Asexual strain worms were subjected to a regime similar to (A) : 24 °C from
10 a.m. to 6 p.m. followed by a slow steady drop throughout the night to 13 °C.
The photoperiod was LD 16:8, lights on from 6 a.m. to 10 p.m. One worm was
fixed every 3rd-4th day. The resultant counts of clustered germ cells are also
Germ cell proliferation
413
represented in Table 2. Out of the 10 worms that were investigated only one
developed attached germ cells and in this case there were only 15, a response far
below that of the sexual strain when exposed to low temperatures.
Promotion of germ cell aggregates via homogenates. Worms from the sexual
Table 3. Total number of attached germ cells per recipient worm
after exposure to homogenates
Experiment I
Length
Uncorrected
Worm 1
2
3
4
5
Experiment 2
Corrected
Uncorrected
No donor homogenates—recipienit series 1
238
235
199
217
172
113
268
224
194
291
265
174
152
214
208
Mean, S.E.* 197-9 ±13-8
Corrected
235
114
202
174
147
Homogenate
Worm 1
2
3
4
5
of donor worms NOT cold exposed—recipient series 2
285
244
306
266
228
216
258
240
424
290
309
308
282
253
226
253
313
307
—
-t
Mean,, s.E. 263-2 ±11-5
Homogenate
Worm 1
2
3
4
5
6
of donor worms 1-day-cold exposed—recipient series 3
512
563
543
418
440
441
362
506
524
388
524
435
543
418
845
592
—
—
552
802
551
520
Mean,,S.E.
463-7 ±22-8
Homogenate of donor worms 2-days-cold exposed—recipient series 4
Worm 1
209
261
688
495
2
3
4
5
6
391
000
—
—
313
OOOf
—
—
735
J 81
473
405
315
522
181
383
397
268
Mean, s.E. 352-5 ±12-4
/-test for the difference between the means of groups: difference is significant at the 0001
level for all comparisons except series 3 and 4, where it is 001.
* Experiments 1 and 2 were combined for the calculation of the means and standard errors
using corrected values.
t Omitted in the calculation of the mean since this individual obviously did not belong to
the normal experimental distribution (P = 1/106).
X—Denotes an animal which could not be evaluated due to loss or unreliable serial sections.
414
C. V O W I N C K E L
strain, kept under standard maintenance conditions formed the recipient groups.
After decapitation these were immersed in homogenates of whole donor worms
of the same strain (details of experiment described under Methods). As explained
under Methods four groups of five recipients each were exposed to the following
homogenates or culture fluids:
Series 1: No donor homogenate, river water containing 0-04% Penicillin G
only.
Series 2: Homogenate of donor worms which experienced no cold exposure.
Series 3: Homogenate of donor worms which experienced 1 day of cold
exposure (14-16 °C).
Series 4: Homogenate of donor worms which experienced 2 days of cold
exposure (14-16 °C).
Recipients were changed into fresh fluids every day for 4 days and then fixed.
The experiment was performed twice. The resultant germ cell counts and
statistical evaluation are given in Table 3. They show that immersion into homogenates of cold-exposed donors leads to significantly higher counts of clustered
germ cells than immersion into homogenates from donors not exposed to cold
or immersion in river water in the absence of donor homogenate.
The same experiment was repeated with animals from the asexual strain as the
recipients of the homogenate. Four groups of five individuals each were used
again. Donor worms derived from the sexual strain. No clustered germ cells in
any form were found in the asexual recipient groups.
DISCUSSION
In the sexual strain of D. tigrina germ-cell aggregates in the form of small
testicular primordia can be recognized as early as the second day after hatching.
The size of these primordia increases rapidly and in adult worms they are of
normal occurrence. Even at the upper temperature limit for long term healthy
maintenance (ca.21°C for our material, LD 16:8) all adult worms showed
germ-cell clusters (45 specimens investigated).
When sexual worms are exposed to low temperatures with or without diurnal
variations the total number of clustered germ cells rises promptly and remains
at the new level for some weeks. Eventually adaptation to the new conditions
appears to take place and long-term observations indicate that low temperatures
alone will not suffice as stimulus for development of mature testes in incubatorreared animals (C. Vowinckel, in preparation).
The effect of cold exposure on germ cells appears to be transmittable by
homogenates. Inherent in the concept of homogenate activity is the assumption
that a factor from the homogenate enters the recipient worm and causes changes.
Two obvious pathways for entry offer themselves in the present experiment:
diffusion through the epidermis and entry through the daily inflicted wound at
the anterior end.
Germ cell proliferation
415
Bennett, Hebert & Hughes (1967) found that planarians immersed in 0-01 M
solutions of labelled fatty acids, nucleic acid precursors, sucrose and amino acids
efficiently incorporated the label within 2 days. It appears therefore that at least
small molecules can enter the worm's tissue by this route. Decapitation and daily
B
Fig. 2 A, B. Two neighbouring sections showing a total of 8 mitotic figures (arrows)
in a short area of germ-cell cord after exposure to a homogenate of cold-treated
worms, nc = nerve cord. (A) x 925; (B) x 962.
repeated incisions through the developing regeneration blastema would seem to
offer more easy access to macromulecular substances from the homogenate.
Some evidence to support this assumption was found during the cell count. In
416
C. VOWINCKEL
a large number of the worms exposed to homogenates, the majority of germ-cell
clusters were found at the very anterior end of the worm. Frequently these were
in synchronous divisions (Fig. 2A, B). In contrast the 39 intact worms used for
the low temperature experiments (Table 2) all had the greatest cluster density at
the pharyngeal level. All worms under discussion derived from the same clone;
differences between groups therefore should have arisen from environmentalphysiological rather than genetic causes. While there is no proof that a factor
from the homogenate has entered the tissue of the recipients this appears a
plausible explanation.
The three different homogenates all stimulate germ-cell aggregation and/or
proliferation. The most active homogenate is obtained after 24 h cold exposure
of donor worms. Donors not exposed to cold furnish the least active homogenates. The different levels of activity reflect well the effects of cold exposure on
intact worms (Table 2, sexual strain, regime (A) and Control).
Decapitation generally results in the disappearance of sexual structures in
planarians. Grasso (1959) showed that in the absence of the cephalic region
gonads and sexual structures of planarians will regress and Ghirardelli (1965)
noted that they will not redevelop if brain differentiation is prevented after
decapitation. Both Grasso and Ghirardelli conclude that gonadal differentiation
is dependent on a factor issuing from the brain. Their deductions are in accordance with the general theory that neuroendocrine substances control planarian
reproductive physiology. Maintenance of germ cells and their increase in number,
according to this theory, would depend on the constant presence of a factor
issuing from the nervous system. Our observation that homogenates can have
the same effect, both qualitatively and quantitatively as the original stimulus
acting on intact worms, is consistent with this theory.
The asexual strain of D. tigrina does not show germ cell aggregates under
standard maintenance conditions and homogenates capable of stimulating
germ-cell aggregation and/or proliferation in sexual strain worms are inactive
in the asexual strain. Exposure to repeated diurnal temperature variations failed
to stimulate the formation of testicular primordia in 90 % of the asexual strain
individuals. However, it caused the development of small germ-cell clusters in
one individual. The ability to respond is therefore not completely lost in all
members of this strain. In the absence of tests with cloned animals it cannot be
decided whether this is due to genetic or physiological variations within this
population.
Kenk(1940,1941)triedto induce complete development of reproductive organs
in an asexual strain of D. tigrina. Neither temperature nor food variations,
including crushed sexually mature planarians, were capable of induction. However, grafting an anterior-half animal of a sexual strain of D. tigrina on to a
posterior-half of an asexual strain resulted, after a long time interval, in the
development of testes and genital complex in the previously asexual posterior
part. The experiment was repeated by Okugawa (1957) with the same results.
Germ cell proliferation
411
Kenk suggested two possible explanations: (a) that an endocrine substance from
the anterior-half induced the development of sexual organs in the posterior-half;
or (b) that neoblasts migrated from the anterior-half animal into the posteriorhalf and gave rise to the new structures. Of the two possibilities Kenk preferred
the first. Unfortunately no fission products were secured in either experiment;
since these would have developed outside the endocrine influence of the anterior
sexual half, their capacity to develop or not develop sexual structures could
have provided an answer to the problem.
Homogenates of cold-exposed sexual strains in our experiments promoted an
increase in germ cell numbers in the sexual strain but not in the asexual strain.
These cold-exposed homogenates contained the kind of factor which Kenk
assumed to be active in his experiments. However, our asexual strain recipients
were incapable of responding to the factor. According to these results, therefore,
it appears more likely that in Kenk's and Okugwa's experiments the posterior,
asexual portion of the animal was invaded by cellular elements from the anterior
sexual part.
SUMMARY
1. Germ-cell development of mature animals under conditions of asexual
reproduction is described for a sexual clone of Dugesia tigrina. Germ cells are
demonstrated in newly hatched animals of the sexual strain.
2. Large increases of clustered germ cells near the nerve cord can be correlated with low water temperatures.
3. Homogenates of worms exposed to a 10 °C drop in temperature for 24 h
have the capacity to stimulate an increase in the number of aggregated germ
cells.
4. In an asexual strain of this species germ-cell aggregates are missing and
their formation could not be promoted by experimental procedures successful
in the sexual strain. Repeated chilling, however, caused a few germ-cell aggregates in one individual.
RÉSUMÉ
Stimulation de la prolifération des cellules germinales
de la planaire Dugesia tigrina (Girard)
1. On décrit le développement des cellules germinales d'animaux matures
d'un clone sexué de Dugesia tigrina dans des conditions de reproduction asexuée.
On reconnaît des cellules germinales dans des animaux nouvellement éclos de
la souche sexuée.
2. Une importante augmentation des amas de cellules germinales peut être
rapportée à de basses températures de l'eau d'élevage.
3. Des homogénats de vers soumis à une diminution de 10° de la température
ont le pouvoir de déclencher une augmentation du nombre des cellules germinales dans les aggrégats.
27
E M B 23
418
C. V O W I N C K E L
4. Dans des souches asexuées de cette espèce, il n'y a pas d'aggrégats de
cellules germinales et leur formation ne peut être provoquée par les procédés
expérimentaux qui sont efficaces dans la souche sexuée. Des refroidissements
répétés ont cependant provoqué la formation de quelques aggrégats dans un
unique individu.
This work was supported by a McConnell Fellowship of McGill University to the author
and a National Research Council of Canada grant to Professor J. Marsden in whose laboratory the experiments were performed and to whom the author is indebted for valuable advice
and criticism. Dr N. Wolfson's many helpful suggestions and critical reading of the manuscript are gratefully acknowledged. Part of the work reported here was included in a Ph.D.
thesis submitted to McGill University.
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(Manuscript received 16 June 1969, revised 24 October 1969)