/ . Embryol. exp. Morph. Vol. 70, pp. 29-36, 1982
Printed in Great Britain © Company of Biologists Limited 1982
29
Evidence of male germ cell
redifferentiation into female germ cells in
planarian regeneration
By V. GREMIGNI,1 M. NIGRO AND I. PUCCINELLI
From the Institute of Zoology, University of Pisa, Italy
SUMMARY
The source and fate of blastema cells are important and still unresolved problems in
planarian regeneration. In the present investigation we have attempted to obtain new evidence
of cell dedifferentiation-redifferentiation by using a polyploid biotype of Dugesia lugubris
s.l. This biotype is provided with a natural karyological marker which allows the discrimination of triploid embryonic and somatic cells from diploid male germ cells and from hexaploid
female germ cells. Thanks to this cell mosaic we previously demonstrated that male germ cells
take part in blastema formation and are then capable of redifferentiating into somatic cells.
In the present investigation sexually mature specimens were transected behind the ovaries
and the posterior stumps containing testes were allowed to regenerate the anterior portion
of the body. Along with the usual hexaploid oocytes, a small percentage (3.2%) of tetraploid
oocytes were produced from regenerated specimens provided with new ovaries. By contrast
only hexaploid oocytes were produced from control untransected specimens. The tetraploid
oocytes are interpreted as original diploid male germ cells which following the transection
take part in blastema formation and then during regeneration redifferentiate into female
germ cells thus doubling their chromosome number as usual for undifferentiated cells
entering the female gonad in this biotype.
INTRODUCTION
One of the important and debated problems in planarian regeneration is the
source of blastema cells: the undifferentiated cells from which the removed or
injured tissues are rebuilt. According to some authors (Wolff & Dubois, 1947;
Dubois, 1949; Wolff, 1962; Lender, 1962; Brondsted, 1969; Gabriel, 1970) the
only cells capable of forming the blastema are embryonic reserve cells (the socalled neoblasts) which remain undifferentiated and scattered in the parenchyma
of the adult worm throughout its life. On the contrary, other authors (Hay, 1968;
Coward, 1969) claim that regeneration occurs from differentiated cells which,
following an injury, dedifferentiate and form the blastema. Moreover,
some authors suggest a localized source of blastema cells (Betchaku, 1970;
Bagufia, 1975) while others claim that they can come from any region of the
body (see Brondsted, 1969 for a review).
1
2
Author's address: Istituto di Zoologia, via Volta 4, 56100 Pisa, ITALY.
V. M B 7O
30
V. G R E M I G N I , M. N I G R O AND I. P U C C I N E L L I
Fig. 1. Scheme of the experiments performed to obtain new ovaries regenerated
from a posterior stump containing testes. The spotted area represents the regenerated tissues, p.r.: posterior region.
Previous research (Gremigni & Puccinelli, 1977; Gremigni & Miceli, 1980)
suggests that both types of cell (either embryonic reserve cells or dedifferentiated
cells) can take part in blastema formation and in regeneration as also suggested
by Betchaku (1967) and Pedersen (1972). In the case where cell dedifferentiation
really does occur, a third problem becomes apparent. It concerns the fate of the
dedifferentiated cells which during regeneration might redifferentiate only into
the original type (unipotent cells) or might redifferentiate into any cell type
(totipotent cells), giving rise to a cell transdifferentiation process or metaplasia.
In some of our previous papers (Gremigni & Puccinelli, 1977; Gremigni &
Miceli, 1980; Gremigni, Miceli & Puccinelli, 1980; Gremigni, Miceli &
Picano, 1980) we have tried to obtain direct answers to these questions by
using a biotype of Dugesia lugubris s.l. in which male and female germ cells are
clearly distinguishable from embryonic and somatic cells by means of a stable
karyological marker. The present paper presents evidence for a novel type of
cell 'transdifferentiation' from male to female germ cells.
MATERIAL AND METHODS
The planarians used in this study belong to the triplohexaploid biotype of
Dugesia lugubris s.l. (D. lugubris-polychroa group or subgenus Schmidtea
according to Ball, 1974). They have triploid embryonic and somatic cells
31
Male germ cell ^differentiation in planarians
Table 1. The table represents meiotic oocytes produced from experimental {on
the left) and control {on the right) specimens
Control specimen oocytes
Experimental specimen oocytes
A
A
1
Specimen
Nos
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
Total
Percentage
i
6/i (12") 4/i (8")
35
30
40
34
31
28
33
27
41
33
30
29
35
34
26
40
38
33
32
32
35
39
27
43
31
34
34
31
33
36
1004
96-8
2
1
2
1
1
1
1
0
2
1
1
0
2
1
0
2
1
1
1
1
1
1
1
2
1
2
1
0
1
1
33
3-2
1
Total
Specimen
nos.
6n(12")
37
31
42
35
32
29
34
27
43
34
31
29
37
35
26
42
39
34
33
33
36
40
28
45
32
36
35
31
34
37
1037
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
Total
Percentage
29
35
38
36
42
37
34
31
28
34
32
38
44
40
27
35
34
33
26
30
32
46
28
35
33
33
27
36
35
38
1026
1000
An (8")
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
00
Total
29
35
38
36
42
37
34
31
28
34
32
38
44
40
27
35
34
33
26
30
32
46
28
35
33
33
27
36
35
38
1026
(3/i = 12), diploid male germ cells (2« = 8) and hexaploid female germ cells
(6/i = 24). The haploid set (n = 4) is composed of one large metacentric
chromosome and three acrocentric chromosomes of different lengths (Benazzi,
1957;Gremigni & Puccinelli, 1977, fig. 1, p. 58). The diploid set of chromosomes
in the male line is derived from the elimination of a haploid set occurring in
undifferentiated cells which give rise to spermatogonia within the testes. The
hexaploid set of chromosomes in the female line is derived from a doubling of
the triploid set occurring in undifferentiated cells which give rise to oogonia
within the ovaries. A normal meiosis occurs in both male and female germ
32
V. GREMIGNI, M. NIGRO AND I. PUCCINELLI
(a)
, ,
Fig. 2a. A meiotic oocyte with 12 bivalents produced from a control specimen.
b, A meiotic oocyte with 8 bivalents produced from a regenerated specimen with
new ovaries. The arrows indicate the bivalents derived from synapsis of
metacentric chromosomes.
cells. The oocyte, which is blocked in late prophase I when leaving the ovary,
is usually penetrated by a haploid sperm which causes continuation of the
meiotic division. However, amphimixis does not occur and the sperm nucleus
is extruded within a polar body, or degenerates within the ooplasm (BenazziLentati, 1970). Thus development is pseudogamic and all embryonic cells are
triploid (Benazzi, 1957; Gremigni & Puccinelli, 1977, fig. 2, p. 59). If not penetrated by a sperm, late prophasic oocytes are laid within a capsule (usually
named cocoon) where they remain in pseudometaphase I for a short time (1-2 h)
and then progressively degenerate.The cocoons also contain thousands of vitelline cells.
The specimens used here were originally collected in Lake Iseo (Italy) and then
maintained in our laboratory for some years being fed twice a week on Tubifex.
Newborns from karyologically controlled parents were isolated at birth, numbered and cultured individually so that they reach sexual maturity untouched
and uninjured.
Experimental animals which attained sexual maturity were transected behind
the two ovaries; the anterior fragment was removed while the posterior fragment (containing testes) was allowed the rebuild the anterior area which included
new ovaries (Fig. 1). The regenerated specimens were allowed to reach sexual
maturity once again and the unfertilized eggs deposited inside the cocoon were
karyologically analysed. Each cocoon was pierced and smeared on a slide with
Male germ cell redifferentiation in planarians
33
a drop of acetic carmine and the number of bivalents per oocyte was immediately counted using a 100 x oil immersion objective.
For controls the laid eggs of intact specimens were karyologically analysed
using the same technique. The entire procedure was carried out twice in the
period November-June, 1979-80 and 1980-81. The eggs deposited by both
experimental and control specimens within the last three months of each
season were analysed. On each occasion 15 experimental and 15 control specimens born from five parental couples were used (3 experimental and 3 control
specimens from each couple). In total 30 experimental and 30 control specimens
were studied.
RESULTS
A great majority of experimental specimen oocytes had 12 bivalents typical
of the Iseo population of D. lugubris s.L, but 26 of the 30 regenerated planarians
also produced at least one oocyte with 8 bivalents (Fig. 2b). Among the total
1037 freshly deposited oocytes analysed, 1004 had 12 bivalents (96.8%), while
the other 33 had 8 bivalents (3.2%) (Table 1). In contrast, all control specimen
oocytes (1026) had 12 bivalents (Fig. 2 a).
The two different types of oocytes were usually recognizable at a low magnification on the basis of their different sizes, the oocytes with 12 bivalents being
significantly larger. Mirolli (1956) had previously shown that the oocyte size
in D. lugubris is correlated with the ploidy level.
As mentioned above, the unfertilized oocytes degenerate within a few hours.
During this process the bivalents usually separate into dyads and are progressively extruded from the oocytes within small buds. Thus it was very important
to avoid using aged oocytes deposited from both experimental and control
specimens and also oocytes which did not contain clearly countable and morphologically distinguishable bivalents. These precautions were used to avoid
interpreting as tetraploid those oocytes which, even if they appeared to have
8 bivalents, were really hypohexaploid.
DISCUSSION
Thanks to the natural cell mosaic occurring in the population of D. lugubris
s.l. used, we were previously able to demonstrate (Gremigni & Puccinelli, 1977)
that, following a transection, fragments containing testes formed a blastema
in which a small number of cells with 8 chromosomes were present along with
cells with 12 chromosomes. The cells with 8 chromosomes were interpreted as
young male germ cells while cells with 12 chromosomes could be interpreted as
deriving from either embryonic reserve cells or from dedifferentiated somatic
cells or both. These results were supported by other observations (Banchetti &
Gremigni, 1973) which showed that after transection, testes in the stump
progressively disappear as young male germ cells (most probably spermatogonia)
34
V. GREMIGNI, M. NIGRO AND I. PUCCINELLI
leave the gonads and migrate into parenchymal cell cords directed to the wound.
In contrast, the more mature male germ cells (certainly spermatids and
spermatozoa) degenerate. Later on, it was demonstrated that the blastema cells
with 8 chromosomes were found in areas of mature regenerated specimens where
gonads are not present and are not to be formed (Gremigni, Miceli & Puccinelli,
1980; Gremigni, Miceli & Picano, 1980). These results strongly supported the
hypothesis that original male germ cells can transform into somatic cells during
regeneration and the validity of this hypothesis has been recently confirmed
(Gremigni & Miceli, 1980) by using the cytophotometric technique which
allows the discrimination between differentiated somatic cells with either
triploid or diploid interphase nuclei.
In the present research we asked whether male germ cells are also able to
redifferentiate into female germ cells during regeneration. The results obtained
show that all oocytes produced from control, untransected specimens had 12
bivalents as constant in the population used, while about 3 % of the oocytes
produced from regenerated specimens had 8 bivalents. These tetraploid oocytes
are an absolutely unusual phenomenon not only in the intact specimens of the
population used, but also in all other natural biotypes of the species studied
up to now. In the D. lugubris-polychroa group oocytes with 8 bivalents have
only been obtained from experimental interracial hybrids (Benazzi-Lentati,
1970).
The following interpretation is suggested to explain the results. After transection, a number of young male germ cells with 8 chromosomes leave the
testes and migrate into the blastema as previously demonstrated (Gremigni &
Puccinelli, 1977). Then during regeneration some of these diploid cells migrate
into the new female gonad primordium. Here they differentiate into oogonia
thus doubling their chromosome number and giving rise to tetraploid oocytes
with 8 bivalents.
The precise nomenclature of the ^differentiation process of male germ cells is
controversial. Although spermatogonia are by nature poorly differentiated cells,
in the population used the undifferentiated cells undergo a chromosomal elimination when they become spermatogonia. This process could be interpreted either
as the first step in cellular differentiation or as a determination process as recently
suggested by Baguna (1981). In any case male germ cells which enter the blastema, even if not specialized, have already undergone an important and stable
nuclear differentiation and nevertheless they are able to change their fate during
regeneration giving rise to a peculiar transdifferentiation process. More generally
on the basis of the previously quoted and the present data, we think it is legitimate to suggest that in planarians, where embryonic reserve cells most likely
participate in blastema formation, those differentiated cells which are not
irreversibly specialized can also migrate to the blastema, where they become
totipotent again, and then redifferentiate into any cell type depending on the
morphogenetic influences they encounter (Wolff, Lender & Ziller-Sengel, 1964).
Male germ cell redijferentiation in planarians
35
To conclude and to attempt to answer the key questions on planarian regeneration as clearly pointed out by Slack (1980), we think that: (1) the blastema is
formed both from cells drawn from all parts of the body and from cells local
to the wound; (2) the regenerate is formed by both de novo differentiation of
reserve cells and by dedifferentiation and ^differentiation of functional cells;
and that (3) all blastema cells are similarly pluripotent cells, whatever their
source might be, each therefore being able to become any cell type in the regenerate thus giving rise to transdifferentiation.
ITALIAN SUMMARY
L'origine e il destino delle cellule del blastema costituiscono problemi importanti e tuttora
non risolti nella rigenerazione delle planarie. Con questa indagine abbiamo tentato di ottenere
nuovi dati in favore dello sdifferenziamento-ridifferenziamento cellulare mediante l'uso di un
biotipo poliploide di Dugesia lubugris s.l.. Questo biotipo e provvisto di un marcatore cariologico naturale che permette di riconoscere le cellule embrionali e somatiche triploidi da
quelle germinali maschili diploidi e femminili esaploidi. Grazie a questo mosaico cellulare
abbiamo precedentemente dimostrato che cellule germinali maschili partecipano alia formazione del blastema e sono successivamente capaci di ridifferenziarsi in cellule somatiche. In
questa ricerca individui sessualmente maturi sono stati tagliati dietro gli ovari e i monconi
posteriori contenenti i testicoli hanno rigenerato il frammento anteriore con nuovi ovari.
Accanto ai normali ovociti esaploidi, una piccola percentuale (3.2%) di ovociti tetraploidi e
stata prodotta dagli individui rigenerati, mentre gli individui di controllo non sezionati hanno
prodotto solo ovociti esaploidi. Gli ovociti tetraploidi sono interpretati come originali
cellule maschili che in seguito al taglio hanno contribuito alia formazione del blastema e
successivamente durante la rigenerazione si sono ridifferenziate in cellule germinali femminili
duplicando il loro corredo cromosomico come tipico delle cellule indifferenziate che entrano
nella gonade femminile in questo biotipo.
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{Received 25 January 1982)