JOURNAL OF MORPHOLOGY 231:149–160 (1997)
Oogenesis and the Ovarian Cycle in Salamandra salamandra
infraimmaculata Mertens (Amphibia; Urodela; Salamandridae)
in Fringe Areas of the Taxon’s Distribution
R. SHARON,1 G. DEGANI,2 AND M.R. WARBURG1*
1Department of Biology, Technion, Haifa 32000, Israel
2Golan Research Institute, Haifa University, Kazrin 12900, Israel
ABSTRACT Reproductive cycle and oogenesis were studied in specimens of
Salamandra salamandra infraimmaculata Mertens that inhabit fringe areas
of the taxon’s distribution in the Mediterranean region. Both ovarian mass
and length are correlated significantly with body mass and length. Ovarian
length is also correlated with the number of oocytes. During the oogenetic
cycle six stages in oocyte development were recognized. Three occur during
previtellogenesis: stage 1, in which oogonia divide and form cell nests; stage 2
in which oogonia differentiate into oocytes; and stage 3, in which the oocyte
cytoplasm increases in volume. In the vitellogenic phase two additional
stages, 4 and 5, were recognized: stage 4, in which lipid accumulates in
vacuoles in the periphery followed by the appearance of yolk platelets near the
cytoplasmic margin; and stage 5, in which oocyte volume increases rapidly
due to increased number of yolk platelets until it reaches its maximal size.
During postvitellogenesis one stage was recognized: stage 6, in which the
beginning of maturation is characterized by movement of the nucleus toward
the animal pole. Oogenesis continues year-round. The first four stages were
seen in all ovaries examined. The ovarian cycle is independent of season and
reproductive stage apart from the number of mature, postvitellogenic oocytes
that increases following gestation toward the beginning of spring (March–
April). J Morphol 231:149–160, 1997. r 1997 Wiley-Liss, Inc.
The urodele Salamandra salamandra
(Linnaeus) has a palearctic distribution, with
the center in Central Europe. However, a
few subspecies occur in fringe habitats that
are generally drier and hotter than in the
center. These habitats are in Spain, North
Africa, West Africa, and northern Israel,
which is the southern border. The subspecies S.s. infraimmaculata inhabits the latter
region (Degani, ’86). In these regions the
populations are isolated from each other,
and salamanders are extremely limited by
the availability of water even during their
winter breeding season (Warburg, ’86, ’92).
On the other hand, in Europe, water is available during their entire spring and summer
breeding season. Among the subspecies of S.
salamandra, differences were found in reproductive strategies. Thus, in France, Salamandra salamandra terrestris has a 1 year
reproduction cycle:vitellogenin accumulates
in the oocytes from September, oocytes reach
r 1997 WILEY-LISS, INC.
their maximum size and ovulate in June,
gestation lasts 3–9 months, and parturition
begins from April throughout the summer,
while Salamandra salamandra fastuosa in
the Pyrenees has a biennal cycle:vitellogenesis in the second year after parturition (Joly
et al., ’94). S. salamandra is generally ovoviviparous, but S.s. bernardezi in Spain sometimes produces postmetamorphoic young
(Wake, ’93). Salamandra s. infraimmaculata in Israel delivers 17–200 larvae, mainly
between November and March.
Various aspects of oogenesis in amphibians were described in Chthonerpeton (Berois and De Sa, ’88), Ichthyophis (MasoodParveez and Nadkarni, ’93a,b), Necturus
(Kessel and Panje, ’68), Triturus (Fischer,
’32), Pleurodeles (Bonnafant-Jais and Men-
*Correspondence to: M.R. Warburg, Department of Biology,
Technion–Israel Institute of Technology, Haifa 32000, Israel.
150
R. SHARON ET AL.
tre, ’83), and several anuran species (e.g.,
Rugh, ’51; Del Pino and Sanchez, ’77; Jørgensen, ’73, ’74, ’81, ’84; Xavier et al., ’70),
where oogenesis was described in detail.
Only a few studies have dealt with the
ovarian cycle in Salamandra, some of which
were reviewed recently (Greven and Guex,
’94; Joly et al., ’94). Broek (’33) described the
ovarian epithelium, and Joly and Picheral
(’72) described the follicle before and after
ovulation in S.s. terrestris. However, to date
oogenesis has not been described in any detail in this species, and there has been no
attempt to analyze quantitatively oocyte
number in each stage in order to determine
the ovarian cycle throughout the salamander’s reproductive cycle.
In addition to the description and quantitative analysis of oogenesis in this species,
we wish to describe the extent to which xeric
environments affect the reproductive cycle
(mainly the ovarian cycle) of Salamandra
salamandra.
MATERIALS AND METHODS
This investigation was based on a rather
limited number of Salamandra s. infraimmaculata females (n 5 18) collected from two
geographicaly close habitats, Tel-Dan and
Mt. Meron. (The reason for this is that this
species is protected. Consequently, we received a special permit from the Board of
Nature Conservation to collect and dissect a
limited number of females during the course
of this study). The study lasted for 18
months, during which salamanders were collected in the vicinity of water or on the way
to their breeding sites during the rainy season (Degani and Warburg, ’78). Average
monthly temperature and monthly precipitation are presented in Table 1. The number of
females collected monthly and their reproductive stage are presented in Figure 1.
Salamanders were weighed, snout-vent
length and body mass were measured, and
the ovaries were excised. Both the length
and weight of the ovaries were taken. Following a longitudinal cut of the ovarial envelope
(which enables us to expose the oocytes that
are connected to the inner side of the envelope), the oocyte number was determined
and their diameters measured. Three groups
of oocytes were distinguished by color: transparent or previtellogenic, yellow or vitellogenic, and orange or atretic.
The ovaries were divided into a number of
sections that were fixed in Bouin, Smith,
Carnoy, Altmann, and Karnowsky fixatives
(Karnowsky, ’65; Humason, ’72). Paraffin
blocks were prepared, sectioned at 5–8 µm,
and stained with the Hematoxylin and Eosin (H1E), Azan, Mason trichrome and Barret’s as well as Periodic Acid Schiff 1 Alcian
Blue (PAS 1 AB), Bromo-Phenol Blue (BPB),
and Para-aldehyde Fuchsin (PF) procedures.
Haematoxylin, Azan, Mason trichrome and
Barret, stain cellular organelles (nucleus,
cytoplasm, yolk, collagen etc). PAS-AB stains
polysaccharides, BPB stains proteins and
PF stains neurosecretory cells (Humason ’72,
Ewen ’62). After staining, the oocyte oogenetic stage was determined. In order to calculate the effect of the treatment, the diameter was measured again, and, based on
both parameters (ovarian mass and oocyte
diameter), an oogenetic scale per female was
determined, thereby obtaining quantitative
data. These data were later analyzed by the
Mann-Whitney test and the Student’s t-test
to describe ovarian cycle and its relationship
to season and reproductive cycle.
TABLE 1. Temperature and precipitation in study habitats
Meron (Galil)
Year
Month
Precipitation
(mm)
1993
IX
X
XI
XII
I
II
III
IV
V
VI
0
11
22
15.4
231.3
118.3
69.8
14
1
0
1994
Tel-Dan
Temperature (°C)
Maximum
28.5
26.1
15.8
14.5
11.1
10.2
14.4
22.7
26.8
28.3
Temperature (°C)
Minimum
Precipitation
(mm)
Maximum
Minimum
17.2
16.9
9.1
8.5
6.5
4.6
6.9
12.7
15.3
16.5
0
17.1
43.4
25.5
153.3
104.9
14.4
8.5
1.3
0
32.1
31.4
22.4
21
17.6
16.6
19.8
27.8
29.9
29.9
17.2
16.7
10.2
9
8.5
7
8.2
12.1
14.1
14.1
OOGENESIS IN SALAMANDER
Fig. 1.
Number of females collected monthly and their reproductive stage.
151
152
R. SHARON ET AL.
RESULTS
Ovary
Ovaries are wrapped in a thin, transparent envelope, and oocytes are attached to the
inner side of the ovaries (Fig. 2). The envelope consists of connective tissue interfolded
Fig. 2. Ovary. A: Ovary enclosed in a thin, transparent envelope (oocyte can be seen through envelope).
Scale bar 5 100 µ. B: Ovary after the transparent
enveloping membrane was slit open. Oocytes can be
with collagen fibers. Both large, rounded,
granular cells and nongranular flat cells
which comprise the germinal epithelium are
seen between the collagenous fibers.
Allometric elements of the ovaries are presented in Table 2. Ovarian length ranges
seen attached to the membrane. Scale bar 5 100 µ. P,
previtellogenic oocyte; Po, postvitellogenic oocyte; V, vitellogenic oocyte.
153
OOGENESIS IN SALAMANDER
TABLE 2. Measurements of body and ovaries
N
Snout-vent length (cm)
Body mass (g)
Ovarian length (cm)
Ovarian mass (g)
Oocyte number (per female)
Mean 6 SD
18 animals 11.70 6 1.75
18 animals 52.31 6 20.12
36 ovaries
3.40 6 0.94
36 ovaries
0.48 6 0.24
36 ovaries
218 6 56.5
between 2 and 6.2 cm, and its weight ranges
between 0.17 and 1.13 g. Total number of
oocytes ranges between 108 and 407.
Ovarian mass is significantly correlated
with body mass (r 5 0.697; P , 0.05). Correlations between ovary and snout-vent
length (r 5 0.817; P , 0.05) and between
ovary length and number of oocytes
(r 5 0.741; P , 0.01) are also significant.
However, no significant correlation is found
between ovary mass and oocyte number, although significant correlation is found between ovarian mass and ovarian length
(r 5 0.583; P , 0.05) (see Table 2 for data).
Oogenesis
We distinguished six stages in oogenesis
which we separated into three main phases
in the oogenetic cycle: 1) previtellogenic, 2)
vitellogenic, and 3) postvitellogenic oocytes
(maturation).
During previtellogenesis we found three
stages (Fig. 3): stage 1 in which oogonia
divide and form cell nests (Fig. 3A,B); stage
2 in which oogonia differentiate into oocytes
which possess a large central nucleus containing nucleoli but relatively little ooplasm,
and follicle envelopes are formed which consist of two layers of theca cells and one layer
of granulosa cells differentiated from the
germinal epithelium (Fig. 3C); and stage 3
in which oocytes increase in volume due to
addition of cytoplasm. Two types of cytoplasm are distinguishable: 1) dense cytoplasm at the periphery with a pigmented
ring separating it from 2) the less dense
cytoplasm surrounding the nucleus (Fig. 3D).
In the vitellogenic phase (2) we recognized
two stages (Fig. 4). In stage 1, lipid accumulates in vacuoles forming lipid droplets in
the periphery. Oocyte diameter ranges between 0.2 and 0.6 mm (substage I) (see Fig.
4A). Later, yolk platelets appear at the cytoplasm edges, and the theca fold to form a
space that separates it from the granulosa
(Fig. 4D). Cytoplasm of two densities still
exists. Mean oocyte diameter is 1.12 6 0.07
and ranges between 0.4 and 1.6 mm (substage II). In stage 2, oocyte volume increases
rapidly due to the increased number of yolk
platelets; they are smaller around the edge
and increase in size toward the center. The
cytoplasm becomes homogenous, uniformly
dense, throughout the cell. Nucleoli still surround the nucleus. Mean oocyte diameter is
2.0 6 0.2 mm and ranges between 1.0 and
2.9 mm (substage III) (Fig. 4B). At the end of
this stage the oocyte reaches its maximum
size. A ring of cytoplasm surrounds the
nucleus. Yolk platelets fill the oocyte. Mean
oocyte diameter is 2.66 6 0.32 mm and
ranges between 2.1 and 3.6 mm. The follicle
envelopes attach to the oocyte, and the
granulosa cells flatten (substage IV) (Fig.
4D).
The last stage observed is the beginning of
the third phase, the maturation (postvitellogenic) stage, characterized by movement of
the nucleus toward the animal pole. Mean
oocyte diameter is 3.19 6 0.29 mm, and
ranges between 2.0 and 3.9 mm (Fig. 4C).
Ovarian cycle
The mean number of previtellogenic oocytes in the ovary is 140.47 6 50.49, and the
mean number of vitellogenic oocytes is
75.59 6 19.74. The vitellogenic oocytes comprise 34.86% of the total oocyte number.
There is no correlation between ovarian
mass and number of either previtellogenic
(r 5 0.35), vitellogenic (r 5 0.034), or postvitellogenic (r 5 0.343) oocytes. There is a
significant correlation between ovarian
length and number of previtellogenic oocytes
(r 5 0.697; P , 0.001) as well as the number of vitellogenic oocytes (r 5 0.565;
P , 0.001) but not with the postvitellogenic
(mature) oocytes (r 5 0.16). Significant correlation is found between vitellogenic oocyte
number and total oocyte number (r 5 0.91;
P , 0.001).
Ovarian mass and length are independent
of the season and the reproductive cycle
(Fig. 5). In ovaries of all females examined
we observed the first four stages of oogenesis. The percentage of oocytes reaching the
vitellogenic stage is constant, independent
of season and reproductive stage (Fig. 6).
Over half of the vitellogenic oocytes are at
substage II. Most of the oocytes that reach
substage III continue to substage IV, but
only half of them mature (Table 3). Mature
oocytes increase in number toward March in
gravid and nongravid females (Fig. 7).
DISCUSSION
Ovary
In other amphibians the relative weight of
the ovaries is positively correlated with the
154
R. SHARON ET AL.
Fig. 3. Previtellogenic oocyte development. A: Division of oogonia (arrow). H & E. Scale bar 5 10 µ. B:
Creation of ‘‘cell nest’’ Arrow, cell nest; g, germinal
epithelium. Azan. Scale bar 5 10 µ. C: Differentiation
into oocyte. Arrow, granulosa; g, germinal epithelium.
Azan. Scale bar 5 10 µ. D: Volume of oocyte increases.
Arrows, pigmentation; Cy, cytoplasm; N, nucleus. Azan.
Scale bar 5 50 µ.
Fig. 4. Vitellogenesis (A,B). Postvitellogenesis (maturation) (C). Changes in oocyte envelope (D). A: Substage I.
Lipid accumulates at the periphery of the cytoplasm. Arrows, pigmentation; arrowhead, lipid drops; Cy, cytoplasm;
N, nucleus. Azan. Scale bar 5 10 µ. B: Substage III. Oocyte
is half-filled with yolk. Cy, cytoplasm; N, nucleus. Azan.
Scale bar 5 10 µ. C: Nucleus has shifted to animal pole. N,
nucleus. Azan. Scale bar 5 10 µ. D: Postvitellogenic oocyte
(P). Envelope adheres tightly to the oocyte. It is difficult to
differentiate between the layers. Vitellogenic oocyte (V). The
theca (arrowheads) is folded, and the granulosa cells (arrows) are large and rounded. Azan. Scale bar 5 10 µ.
156
R. SHARON ET AL.
Fig. 5.
Changes in ovarian size (length, mass) by months and during the reproductive cycle.
female’s body weight (Bufo: Reading, ’86;
Rana: Jørgensen, ’81; KyriakopoulouSklavounou and Loumbourdis, ’90). There is
seasonal variation in ovarian mass of Rana
spp. (Jørgensen, ’81; Elmberg, ’91). The increase in the relative weight of Ichthyophis
ovary is related to the increase in oocyte
diameter (Masood-Parveez and Nadkarni,
’93a). The relative ovarian weight in Triturus torosus (5 Taricha torosa) ranges between 0.5 and 4.9% (Miller and Robbins,
’54), and in Triturus (5 Notophthalmus) viridescens between 2.49 and 11.73% (Adams,
’40). Prior to the present study there were no
comparable data available for S. salamandra ovaries.
In the present study the ovarian length
rather than ovarian mass is better correlated with snout-vent length and oocyte number. There is no seasonal variation in ovarian mass and length.
Oogenesis
Oogenesis is the process whereby oogonia
that multiply by mitosis are transformed
into mature oocytes (Rugh, ’51; Lofts, ’74;
Saidapur, ’82). Lofts (’74) recognized in Amphibia primary and secondary phases characterized by the accumulation of yolk and by
growth. Most studies on amphibian oogenesis recognize three main stages: previtellogenesis, vitellogenesis, and postvitellogen-
OOGENESIS IN SALAMANDER
157
Fig. 6. Percentage of vitellogenic oocytes from total number of oocytes by months and
during the reproductive cycle.
esis or maturation. Vitellogenic follicles grow
and bulge out of the ovarian wall (Lofts, ’74).
The vitelline membrane envelopes the egg
after ovulation until fertilization takes place
(Wischnitzer, ’66).
TABLE 3. Vitellogenic and mature oocyte number and
percentage
Development
stage
Vitellogenesis
Stage 1
Substage II
Stage 2
Substage III
Substage IV
Maturation
Percentage from total
number of vitellogenic
Number 6 SD
1 mature oocytes
82.1 6 27.9
55.0
27.9 6 17.7
25.1 6 21.6
14.1 6 17.6
18.7
18.7
9.4
Detailed descriptions of oocyte development were given by Masood-Parveez and
Nadkarni (’93b) for the caeailian Ichthyophis beddomii, by Saidapur (’82) and Berois
and De Sa (’88) for Chthonerpeton indistinctum, and by Xavier et al. (’70) for Rana
nectophrynoides. In Urodela the most detailed description of oogenesis was given by
Bonnafant-Jais and Mentre (’83) for
Pleurodeles waltlii. They distinguished three
stages in oogenesis: previtellogenic with oocytes smaller than 500 µ in diameter, vitellogenic oocytes containing yolk up to 1,000 µ in
diameter, and postvitellogenic stages with oocytes larger than 1,000 µ and more intensely
pigmented due to their denser cytoplasm. Each
stage was divided into substages.
158
R. SHARON ET AL.
Fig. 7. Percentage of mature oocytes from total number of vitellogenic oocytes by months
and during the reproductive cycle.
Descriptions of oocytes before and after
ovulation in Salamandra salamandra were
given by Joly and Picheral (’72). The dimensions of yolk platelets increase with oocyte
development (Grodzinski, ’75), and consequently oocyte diameter is 5 mm (Joly and
Picheral, ’72). In our study the maximum
oocyte diameter is 3.9 mm.
Ovarian cycle
The present study gives for the first time
quantitative data about oocyte numbers and
their size and distribution in Salamandra
salamandra. To date most researchers based
their description of the ovarian cycle on ovarian mass (growth curve) or oocyte diameter.
In the present study we have shown great
variation in the size of oocytes belonging to
the same stage of development in different
females. We conclude that in order to give an
accurate description of the ovarian cycle, all
oocytes should be measured and each animal scaled separately for oocyte size at each
stage histologically (see Materials and Methods), and then oocyte number in each stage
of the oogenetic development should be
counted.
The ovarian cycle in amphibians differs
among orders, families, and species. The
factors affecting the ovarian cycle vary. In
Gimnophiona, Masood-Parveez and Nadkarni (’93b) showed a gestation-dependent
ovarian cycle in Ichthyophis beddomei. In
Anura, Jorgensen (’73, ’74, ’81) and Jorgensen et al. (’79) stated that oocyte development is synchronized in Bufo bufo and Rana
temporaria, and that the ovarian cycle is
seasonal, depending on temperature or pre-
159
OOGENESIS IN SALAMANDER
cipitation as well as being gestation-dependent. Redshow (’72) stated that about a third
of the oocytes in the ovary develop into vitellogenic oocytes and that the percentage is
steady irrespective of ovarian mass. Bragg
(’61) stated that oocytes in Scaphiopus toads
are ready all year-round, waiting for heavy
rains and favorable temperature. They are
not season-dependent but depend on favorable conditions. In Urodela, Bonnafant-Jais
and Mentre (’83) showed in Pleurodeles
waltlii that the ovary contains oocytes at all
stages all year-round. In Triturus torosa,
yolk deposition takes place during aestivation and migration to the breeding sites, 5–6
months before ovulation (Miller and Robbins, ’54). Adams (’40) stated that in T. viridescens oocyte development is synchronized;
thus, ovarian mass is low during summer
and high from fall to spring. The ovarian
cycle in other subspecies of Salamandra salamandra depends on season (as in S.s. terrestris) or on gestation (as in S.s. fastuosa), and
oocyte development is synchronized (Joly et
al., ’94). On the other hand, in S.s. infraimmaculata oocytes of previtellogenic and vitellogenic stages are found throughout the year
and throughout the reproductive cycle, and
the percentage of vitellogenic oocytes remains constant. Therefore, ovarian cycle is
not dependent on season or gestation, apart
from oocyte maturation. Oocyte maturation
depends on the season, starting at the onset
of rainfall and increasing toward the end of
winter (March–April). It is an adaptation for
extreme and unpredictable climatic conditions when the female salamander has to be
ready to ovulate practically at any time starting toward the end of the hot and dry summer, when temperatures drop and rain starts
falling. It is a different way to enable survival in a xeric environment for a nonviviparous urodele.
Ovulation in Salamandra salamandra has
been described only once (Joly, ’86) but apparently has not been recorded (5 observed?)
since. This strengthens our impression that
the process of ovulation is extremely rapid
and thus difficult to observe and record. Fertilization takes place when the oocyte passes
through the glandular region of the oviduct
(Siebold, 1858; Schwalbe, 1896; Wahlert, ’54;
Greven, ’80).
In Salamandra s. infraimmaculata, ovulation probably takes place during spring when
temperatures are not too low or too high.
Since water is available only during winter
and parturition occurs mainly between November and March (Sharon et al., ’96), gestation is presumably shorter (less than 1 year)
compared with the European S. salamandra, in which parturition takes place during
the following summer (Joly et al., ’94).
ACKNOWLEDGMENTS
The authors wish to acknowledge the valuable assistance of Ms. Mira Rosenberg during all phases of this study. Partial financial
support came from the J. & A. Taub Biological Research Fund.
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