Turkish Journal of Zoology
Turk J Zool
(2016) 40:
© TÜBİTAK
doi:10.3906/zoo-1508-53
http://journals.tubitak.gov.tr/zoology/
Research Article
Reproductive pattern and sex hormones of Calotes emma Gray 1845 and
Calotes versicolor Daudin 1802 (Squamata; Agamidae)
1
2
1
1,
Worawitoo MEESOOK , Taksin ARTCHAWAKOM , Anchalee AOWPHOL , Panas TUMKIRATIWONG *
1
Department of Zoology, Faculty of Science, Kasetsart University, Bangkok, Thailand
2
Sakaerat Environmental Research Station, Thailand Institute of Scientific and Technological Research, Ministry of Science and
Technology, Nakhon Ratchasima, Thailand
Received: 28.08.2015
Accepted/Published Online: 29.03.2016
Final Version: 00.00.2016
Abstract: We monitored testicular and ovarian morphologies, seminiferous tubules, the sexual segments of the kidneys (SSK),
follicular histologies, and male testosterone and female estradiol to define the reproductive pattern of Calotes emma and Calotes
versicolor. Samples were collected monthly at Sakaerat Environmental Research Station in Thailand for 1 year. Testicular hypertrophies
occurred at a time characteristic for each species, with their time course corresponding well to both active spermatogenesis and
the hypertrophied SSK. Gravid females were also found at a time characteristic for each species. In active reproductive females,
oviductal eggs were concomitantly encountered with ovarian vitellogenic follicles. The previtellogenic and vitellogenic follicles
corresponded well to granulosa layer alterations. The distinct large pyriform cells were present in the granulosa layer of previtellogenic
follicles but disappeared from the vitellogenic follicles. Male testosterone levels rose during testicular and SSK hypertrophies,
and female estradiol levels increased during active reproductive stages of late vitellogenic follicles and gestation. We suggest that
the reproductive patterns of C. emma and C. versicolor fall into the same reproductive pattern of annual continual reproduction,
but that the time courses of such events are different in the 2 Calotes, and even in individuals of the same Calotes population.
Key words: Estradiol, sexual segment kidney, spermatogenesis, testosterone, vitellogenesis
1. Introduction
Lizards (including snakes) are the most speciose living
clade of reptiles, with almost 7200 species. Even excluding
snakes, lizards are still the most speciose extant reptiles,
with approximately 4450 species. Lizards, which belong
to the family Agamidae, are classified into 52 genera with
approximately 400 species (Vitt and Caldwell, 2009), 31
species of which are found in Thailand (Laohachinda,
2009). The genus Calotes Cuvier 1817, a genus of
Agamidae, currently contains 23 species (Hartmann et
al., 2013). There are 7 species of Calotes known to occur
on the Southeast Asian mainland: C. chincollium, Vindum
2003; C. htunwini, Zug and Vindum 2006; C. irawadi, Zug
et al. 2006; C. jerdoni, Günther 1870; C. emma, Gray 1845;
C. mystaceus, Duméril and Bibron 1837; and C. versicolor,
Daudin 1802. The latter 3 species are well known to
Indochina, including Thailand (Zug et al., 2006; Hartmann
et al., 2013).
Male reproductive displays are directly correlated
to testicular hypertrophy in the majority of seasonally
breeding vertebrates, and are accompanied by an elevated
*Correspondence: fscipnt@ku.ac.th
level of circulating sex steroids. This is termed an associated
reproductive pattern (Crews, 1984, 1999; Crews et al., 1984;
Licht, 1984; Norris, 2013). In contrast, in a small number
of vertebrate species, such as some turtles, snakes, and bats,
males mate at a time when their gonads are quiescent (QU)
and the levels of circulating sex steroids are low (Garstka
et al., 1982). This pattern of reproduction is referred to as
a dissociated reproductive pattern (Volsøe, 1944; Crews,
1976, 1984, 1999; Lofts, 1977; Licht, 1984; Norris, 2013). In
snakes, especially in the family Colubridae, various terms
have been used to describe the male reproductive patterns
observed in different species. Volsøe (1944) introduced
the term prenuptial spermatogenesis to describe sperm
production that occurs immediately prior to mating, and
postnuptial spermatogenesis to describe sperm production
that occurs after mating. Aldridge et al. (2009) introduced
the term preovulatory spermatogenesis to describe
sperm production immediately prior to ovulation, and
postovulatory spermatogenesis as sperm production that
occurs after ovulation. They assumed that androgenesis
was associated with the development of secondary sex
characters and mating behavior.
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MEESOOK et al. / Turk J Zool
Vitt and Caldwell (2009) added that the currently
known diversity of seasonal patterns of tropical squamate
reproduction suggests that no single explanation
is sufficient. Snakes are included in the same order
(Squamata), but demonstrate a more diverse reproductive
pattern in contrast to lizards. Tumkiratiwong et al. (2012)
studied the reproductive patterns of captive male and
female Naja kaouthia, monocled cobra, Lesson 1831,
suggesting that its reproductive pattern exhibited either
postnuptial spermatogenesis or a dissociated reproductive
pattern.
There is no detailed information available on testicular
and ovarian cycles, plasma sex hormonal profiles during
gonadal cycles, or male reproduction-associated sexual
segment of the kidney (SSK), especially in Calotes emma.
This paper, therefore, monitored annual alterations of
reproductive organs of males and females of 2 Calotes
species, i.e., C. emma and C. versicolor, and investigated
male and female sex hormones, male testosterone, and
female estradiol to define the reproductive patterns of 2
such Calotes species. We expected that annual male and
female reproductive alterations of the testes, male SSK,
and the ovaries would require underlying morphological
and histological investigations, and also that annual sex
hormonal levels would reveal the reproductive patterns of
2 such Calotes species.
2. Materials and methods
2.1. Animals
We caught the adult lizards monthly over 1 year by hand
or with a noose in the 3 forest types: a dry evergreen
forest, a deciduous dipterocarp forest, and an ecotone
forest at Sakaerat Biosphere Reserves of the Sakaerat
Environmental Research Station (14°26′33″–14°32′50″N,
101°50′43″–101°57′21″E; 720–770 m above sea level),
located at Nakhon Ratchasima Province, northeastern
Thailand. We classified adults of C. emma and C. versicolor
based on external morphological differences (Figure 1).
The adult stages of the 2 species were diagnosed based on
the internal morphology of active male and female gonads
(Figure 1) and expressed in terms of the snout–vent length
(SVL) as: (1) male, C. emma, >5.78 cm; and C. versicolor,
>5.40 cm; (2) female, C. emma, >6.57 cm; and C. versicolor,
>5.99 cm. We attempted to collect 10 adult males and
females, but in some months the samples were smaller than
expected and sometimes we could not collect any samples.
Figure 1. External morphologies of the representatives of 2 Calotes species. Top, C. versicolor: A, no patch of granular scales in front of
forelimb insertion; bottom left, C. emma: B, crescent-shaped patch of small granular scales in front of forelimb insertion, and C, large
postorbital spine present. Bottom middle, dissections of urogenital morphology of male Calotes: T, testis; Vd, vas deferens; K, kidney;
bottom right, female Calotes: OvaF, ovarian follicles; OviE, oviductal eggs. Lines were drawn from a total preparation (in ventral view).
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MEESOOK et al. / Turk J Zool
The months that lacked samples in each species are shown
in parentheses as follows: males of C. emma (May, July,
and August) and C. versicolor (December); females of C.
emma (February to March, May, and December) and C.
versicolor (March to May, July, October, and December).
We anesthetized the samples with diethyl ether and
collected blood samples by puncturing the cardiac
chamber; however, it was not possible to acquire samples
from those lizards with very small cardiac chambers.
Blood samples were kept at 4 °C in heparinized vials and
centrifuged within 2–3 h following blood collection. The
blood was centrifuged at 1600 × g for 10 min. Plasma was
then aspirated off and frozen at –79 °C for later analysis of
plasma levels in male testosterone and in female estradiol.
2.2. External and internal reproductive morphologies
We measured SVL, which ranges from the snout tip to
the anterior margin of the vent. We sacrificed male and
female lizards to investigate the general reproductive
morphologies (Figure 1, bottom middle, male; bottom
right, female) and measured follicular size and testicular
mass. Each specimen was weighed to the nearest 0.01 g; its
SVL was measured to the nearest 0.05 mm.
2.3. Testicular, male SSK, and ovarian histologies
Testes, male SSK, and ovaries were excised and fixed in
a 10% v/v buffered neutral formalin solution, processed
by the paraffin technique (Avwioro, 2011). The tissue
was cut in a cross-section to 6 µm in thickness using a
LEICA RM2145 (Nussloch, Germany). Sections were
stained with hematoxylin and eosin. The reproductive
stage of adult females was determined on the basis of
the presence or absence of types of pyriform cells in the
granulosa layer. Follicles with the pyriform cells, which
appeared in the granulosa layer, were considered to be
previtellogenic follicles, and follicles not containing the
pyriform cells were considered to be vitellogenic follicles
(Tumkiratiwong et al., 2012). Females having vitellogenic
follicles and/or oviductal eggs were considered to be at
the active reproductive stage, while females having only
previtellogenic follicles were considered to be at the QU
reproductive stage. Females with regressed follicles were
considered to be at the postparturient stage. Males with
the appearance of sperm bundles and/or free sperm in
the seminiferous tubules (ST) were assessed as being
at the active reproductive stage, while males without
those attributes were assessed as being at the inactive
reproductive stage (Tumkiratiwong et al., 2012). Males
with SSK that had strongly eosinophilic-stained granules
were regarded as being at the active reproductive stage
(Sever et al., 2002).
2.4. Categorization of female individuals based on
reproductive status
Similar-sized follicles were organized into distinct groups.
The total number and diameter of follicles belonging to
the group with the largest-sized follicles were recorded
from both ovaries. The follicular size was measured with a
Vernier caliper (0–150 × 0.02 mm). Follicles greater than
or equal to 2.5 mm in diameter represented vitellogenic
status (Shanbhag and Prasad, 1993).
Since ovarian follicular development, ovulation,
and gestation occur asynchronously in a population,
individuals with differing reproductive statuses are
encountered. Therefore, the data from the reproductive
phase were classified based on the reproductive status
of individuals rather than on a monthly classification as
follows: (1) the QU stage: previtellogenic follicles sized
<2.5 mm in diameter; (2) the early vitellogenic (EV)
stage: initiation of vitellogenesis with follicles sized 2.5–
5.0 mm in diameter and without oviductal eggs; (3) the
late vitellogenic (LV) stage: late period of vitellogenesis
with follicles sized >5.0 mm in diameter; (4) the early
gestation (EG) stage: oviductal eggs with previtellogenic
follicles sized <2.5 mm in diameter; (5) the midgestation
(MG) stage: oviductal eggs with vitellogenic follicles sized
2.5–5.0 mm in diameter; and (6) late gestation (LG) stage:
oviductal eggs with vitellogenic follicles sized >5.0 mm in
diameter (modified from Radder et al., 2001).
2.5. Measurement of testosterone
The plasma level of testosterone was measured by a 125I
radioimmunoassay (RIA). We added a 500-µL sample of
plasma to 5.0 mL of dichloromethane in a screw-top glass
extraction tube. We then capped the mixture and mixed
it for 60 min by gentle inversion with an end-over-end
rotator, and then centrifuged the sample for 5 min at 1600
× g to separate the layers. The upper phase was aspirated
without disturbing the interface; 2.0 mL of the lower phase
was then transferred to a clean 12 × 75 mm glass tube and
evaporated to dryness under a gentle stream of nitrogen at
37 °C. Finally, we reconstituted the extract with 200 µL of
testosterone buffer. The testosterone extraction procedure
was performed using a Coat A Count Testosterone RIA
Kit (Diagnostic Products, Los Angeles, CA, USA). The
intra- and interassay variations expressed as coefficients
of variation (CVs) were 8.4% and 7.9%, respectively. The
approximate sensitivity of this assay was 40 pg/mL. The
cross-reactivity with androstenedione was 0.5%. The
spiking recovery values averaged 98.3 ± 0.6%. Dilutions
of 50%, 25%, and 12.5% of the undiluted concentration of
7300 pg/mL were 3490 pg/mL, 1700 pg/mL, and 780 pg/
mL, respectively.
2.6. Measurement of estradiol
The plasma level of estradiol was measured by a 125I RIA.
We added 250 µL of plasma to 2.0 mL of diethyl ether
in a screw-top glass extraction tube, and then capped
and mixed it by gentle inversion with an end-over-end
rotator for 30 min. It was centrifuged for 5 min at 1600
× g to separate the layers. The lower (aqueous) phase was
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MEESOOK et al. / Turk J Zool
frozen using dry ice; the organic phase was then decanted
into another vial and evaporated to dryness under a
gentle stream of nitrogen at 37 °C. Finally, the extract was
reconstituted with 250 µL of estradiol buffer. The estradiol
extraction procedure was performed using a Coat A
Count Estradiol (TKE2) RIA kit (Diagnostic Products).
The intra- and interassay variations calculated as CVs were
5.3% and 6.4%, respectively. The approximate sensitivity of
this assay was 10 pg/mL. The specificity of cross-reactivity
with estradiol was 0.32%. The spiking recovery values
averaged 96.8 ± 3.3%. Dilutions of 50%, 25%, 12.5%, and
6.25% of the undiluted concentration of 230.9 pg/mL were
114.4 pg/mL, 60.0 pg/mL, 27.9 pg/mL, and 14.8 pg/mL,
respectively.
2.7. Statistical analysis
Testicular masses, testosterone levels, ovarian weights,
estradiol levels, and diameters of the largest follicle were
expressed as the mean ± standard error of the mean (SEM).
The Kolmogorov–Smirnov test and Levene’s test were used
to determine if the data were normally distributed and the
homogeneity of variance, respectively. Nonparametric tests
were used, as all data mentioned above were nonnormal
and heteroscedastic. Therefore, the Kruskal–Wallis H test
was used to test for differences in the ovarian weights, the
estradiol levels, and the diameters of the largest follicle
among the reproductive stages of nongestation periods. The
Mann–Whitney U test was then used to compare differences
in the ovarian weights, the estradiol levels, and the diameters
of the largest follicle between each reproductive stage of the
various follicular growths of C. emma only because of the
insufficient sample size. There was no statistical analysis of
differences in male testosterone levels or testicular masses
based on months due to the small amount of data available.
Spearman’s correlation coefficients were used to determine
relationships between the testicular mass and testosterone,
and the diameter of the largest follicles and estradiol levels.
The level of significance was set to P < 0.05.
2.8. Ethical aspects
This study was approved by the Ethics Committee of
the Department of National Parks, Wildlife, and Plant
Conservation, Ministry of Natural Resources and
Environment, Thailand (License No. 0909.302/18344).
3. Results
3.1. Annual alterations in male and female reproductive
morphologies of Calotes
Annual changes in testicular morphological events
between C. emma and C. versicolor are depicted in Figure
2. The representative C. emma testes were recrudesced in
December, continued to hypertrophy from January to April
and in June, and completely regressed from September to
November. In the representative C. versicolor, the testes
became hypertrophied from January to September and
regressed from October to November.
Figure 2. Schematics of annual changes in testicular size. Top; C. emma; bottom, C. versicolor. Notes: T, testis; Vd, vas deferens; K,
kidney. Jan–Dec denotes from January to December. All scale bars equal 5 mm.
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MEESOOK et al. / Turk J Zool
Annual changes in ovarian morphological events
(Figure 3) and annual changes in the number of follicular
types and egg types (Table 1) of the representative 2
Calotes species are shown. In the representative C. emma,
we found that in January, April, from June to July, and
from August to November, follicles were in the EV, EG, LV,
and QU stages, respectively (Figure 3, top; Table 1). In the
representative C. versicolor, we found that, in January and
February, the ovaries contained only QU follicles; in June
and August, QU and EV follicles and oviductal eggs; in
September, QU follicles and oviductal eggs; in November,
only QU follicles (Figure 3, bottom; Table 1).
3.2. Annual histological alterations in ST, SSK, and
ovaries of Calotes
In the male representative C. emma, the ST and SSK
were hypertrophied from January to April and in June,
then regressed from September to November, becoming
active again in December (Figure 4, top left and top right,
respectively). Spermatozoal masses were contained inside
the hypertrophied ST. Spermatogonia initially appeared in
November; additionally, a few types of germ cells occurred,
but there were still no active spermatozoa in December
(Figure 4, top left). In the representative C. versicolor, both
ST and SSK were active from January to September but
were inactive from November to December (Figure 5, top
left and top right).
We found that in the female representative C. emma,
active ovaries contained follicles of vitellogenic stages
in January and June, but inactive ovaries contained
previtellogenic and atretic follicles in August (Figure 4,
bottom left and bottom right). In the representative C.
versicolor, ovaries contained previtellogenic and atretic
follicles in January and both previtellogenic and EV
follicles in June (Figure 5, bottom left and bottom right).
Additionally, we found corpus luteum in the ovaries of
1 representative in September (Figure 5, bottom left and
bottom right). We also observed cellular alterations in
the granulosa layer of both C. emma and C. versicolor. It
was demonstrated that, inside the granulosa layer of the
previtellogenic follicles, both types of small and pyriform
cells appeared. However, both such cell types disappeared
in the granulosa layer of the vitellogenic follicles (Figures
4 and 5, bottom right).
3.3. Annual plasma testosterone levels and testicular
masses in Calotes
Annual variations in plasma testosterone levels and
testicular masses between the 2 Calotes species are depicted
(Figures 6a and 6b). In the representative C. emma,
plasma testosterone levels and testicular masses initially
increased in December, peaked in March, and thereafter
gradually reduced from April to November (Figure 6a).
In the representative C. versicolor, plasma testosterone
Figure 3. Schematics of seasonal changes in ovarian size. Top, C. emma; bottom, C. versicolor. Notes: OvaF, ovarian follicles; OviE,
oviductal eggs; Ovi, oviduct. All scale bars equals 5 mm. Jan–Nov denotes from January to November.
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MEESOOK et al. / Turk J Zool
Table 1. Annual changes in numbers of follicular and egg types according to Calotes species.
Follicular size and eggs
Jan
Feb
Apr
May
Jun
Jul
Aug
Sep
Oct
Nov
<2.5 mm
21
-
18
-
18
22
41
33
24
23
2.5–5.0 mm
10
-
-
-
4
1
-
-
-
-
>5.0 mm
-
-
-
-
8
5
-
-
-
-
Oviductal eggs
-
-
4
-
-
-
-
-
-
-
<2.5 mm
16
28
-
-
28
-
19
16
-
10
2.5–5.0 mm
-
-
-
-
5
-
-
-
-
-
>5.0 mm
-
-
-
-
-
-
-
-
-
-
Oviductal eggs
-
-
-
-
5
-
6
5
-
-
C. emma
C. versicolor
Notes: Jan–Nov denotes January to November, respectively.
Figure 4. Photomicrographs of annual changes in C. emma. Top left, testes; top right, male SSK; bottom left, ovaries; bottom
right, granulosa layers (GL). Notes: SZ, spermatozoa; ST, seminiferous tubules; SSK, sexual segments of kidney; AF, atretic
follicle; PF, previtellogenic follicle; VF, vitellogenic follicle; P, pyriform cells; S, small cells.
levels and testicular masses initially increased in January,
peaked in April, and thereafter gradually reduced from
May to November (Figure 6b). The changes in testicular
masses tended to correspond well to the changes in plasma
testosterone levels between the 2 species in the genus
Calotes (r = 0.789, P = 0.001; and r = 0.732, P = 0.001 for C.
emma and C. versicolor, respectively).
3.4. Annual plasma estradiol and ovarian cycles in
Calotes
Variations in ovarian masses and the diameter of the
largest follicle were observed between the 2 Calotes species
6
during their reproductive stages (Table 2). The ovarian
weights and the diameters of the largest follicles differed
significantly from the QU stage to the LV stage regarding
the growth of ovarian follicles (P = 0.001) in C. emma.
It was also observed that the growth of ovarian follicles
concomitantly occurred with the growth of oviductal eggs
during the gestation period of the 2 species.
Variations in plasma E2 levels in relation to the
diameters of the largest follicles between the 2 Calotes
species during reproductive stages are graphically depicted
(Figures 7a and 7b). Regarding the groups of follicular sizes
MEESOOK et al. / Turk J Zool
Figure 5. Photomicrographs of annual changes in C. versicolor. Top left, testes; top right, male SSK; bottom left, ovaries; bottom
right, granulosa layers (GL). Notes: SZ, spermatozoa; ST, seminiferous tubules; SSK, sexual segments of kidney; AF, atretic follicle;
PF, previtellogenic follicle; VF, vitellogenic follicle; P, pyriform cells; S, small cells; CL, corpus luteum.
Figure 6. Annual profiles (mean ± SEM) of testosterone levels and testicular masses (a) C. emma; (b) C. versicolor. Notes: Jan–
Dec denotes January to December. The numbers (in parentheses) represent the number of analyzed samples in each month.
of C. emma, plasma E2 levels were the difference between
the QU and the EV stages (U = 9.00, P = 0.090), the QU
and the LV stages (U = 2.00, P = 0.009), and the EV and the
LV stages (U = 2.00, P = 0.083). Additionally, the diameters
of the largest follicles were the difference between the QU
and the EV stages (U = 0.00, P = 0.004), the QU and the
LV stages (U = 0.00, P = 0.004), and the EV and the LV
stages (U = 0.00, P = 0.021). The correlation coefficient
between plasma E2 and the diameter of the largest follicle
was 0.82 (P = 0.001) and 0.32 (P = 0.365) of C. emma and
C. versicolor, respectively.
4. Discussion
Annual variations in the timing of male and female
reproductive stages were encountered among populations
of C. emma and C. versicolor, even in individuals of the
same population. Annual changes in testicular sizes
were categorized into 2 phases in both species: (1) an
active hypertrophied testicular phase; and (2) an inactive
regressed testicular phase. However, the timing of those 2
events appeared asynchronously in the 2 Calotes species,
even in individuals of the same population (data not
shown here). The testes were hypertrophied with active
spermatozoal production from December to June and
January to September in the representative C. emma and C.
versicolor, respectively. A study by Gouder and Nadkarni
(1979) showed that males of C. versicolor, widely distributed
in India, were spermatogenetically active from April to
September. Active vitellogenic follicles and oviductal
eggs were concomitantly encountered in C. emma and
C. versicolor. Therefore, these 2 Calotes species exhibited
polyautochrony and multiclutches. Radder et al. (2001)
reported that Indian garden lizards, C. versicolor, showed
polyautochrony and multiclutches. The representative
gravid lizards were found in April in C. emma, and in June
and from August to September in C. versicolor. Gravid
C. versicolor, whose habitat is in India, was encountered
from May to October (Shanbhag and Prasad, 1993). We
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MEESOOK et al. / Turk J Zool
Table 2. Changes in ovarian weight and diameter of the largest follicle in Calotes during the
reproduction cycle.
Reproductive stages
N
Ovarian weights
Diameter of the largest follicle
C. emma
(20)
QU
11
0.02 ± 0.00a
1.78 ± 0.06a
EV
4
b
0.11 ± 0.09
3.50 ± 0.33b
LV
4
1.71 ± 0.49c
8.02 ± 0.85c
EG
1
0.02
2.40
MG
-
-
-
LG
-
-
-
C. versicolor
(9)
QU
4
0.02 ± 0.00
1.52 ± 1.99
EV
1
0.2
4.28
LV
-
-
-
EG
3
0.02 ± 0.00
2.22 ± 0.69
MG
2
0.11 ± 0.09
4.36 ± 0.36
LG
-
-
-
Data are presented as mean ± SEM. The differences in superscript alphabets indicate the significant
differences among the reproductive stages of nongestation at P < 0.01 (N, sample sizes).
Notes: QU, quiescent; EV, early vitellogenic; LV, late vitellogenic; EG, early gestation; MG, midgestation; and LG, late gestation.
Figure 7. Changes in the plasma levels of estradiol and the diameter of the largest follicle: (a) C. emma; (b) C. versicolor.
Notes: QU, quiescent; EV, early vitellogenic; LV, late vitellogenic; EG, early gestation; MG, mid-gestation; LG, late gestation.
Data are presented as mean ± SEM. The differences in superscript alphabets (estradiol levels) and in the numbers of asterisks
(diameters of the largest follicles) indicate the significant differences between the various follicular sizes at P < 0.01. The
number (in parentheses) represents the analyzed samples in each month.
are likely to suggest after studying the annual alterations
in the male and female reproductive morphologies of the 2
Calotes species that the active reproductive events of both
males and females of the Calotes species lasted nearly 1
year with only a few months of reproductive arrest, which
is especially seen in C. versicolor.
According to our investigations on annual histological
alterations in ST and male SSK timing of the 2 Calotes
species, both ST and SSK were in active spermatogenic and
8
hypertrophied stages, respectively, which corresponded
well with the timing of the testicular hypertrophied stage
mentioned above. In other words, the timing of arrested
spermatogenesis and regressed SSK was in accordance
with that of the regressed testes. Likewise, we confirmed
that the 2 Calotes species have an active reproductive stage
that is much longer than the inactive reproductive stage.
SSK is present in a variety of male snakes and lizards,
but is absent in both turtles (Regaud and Policard, 1903)
MEESOOK et al. / Turk J Zool
and crocodilians (Fox, 1952). Bishop (1959) found that
the testes of the male garter snake, Thamnophis sirtalis,
were spermatogenically active during the same time as the
hypertrophied SSK; during the active reproductive period,
the diameter of the SSK tubule was 5 times greater than
that of the SSK tubule during the inactive reproductive
period. However, SSK development in female lizards
has been reported in the genus Cnemidophorus (Del
Conte, 1972; Del Conte and Tamayo, 1973) and Scincella
laterale (Sever and Hopkins, 2005). They suggested that
the females had a low level of natural androgens, which
caused the SSK development (Del Conte and Tamayo,
1973; Sever and Hopkins, 2005). In the present study, we
did not monitor the annual seasonal alterations in female
SSK. The hypertrophy of the SSK is synchronous with
androgen secretion and spermatogenic activity (Sever and
Hopkins, 2005). Norris (2013) also stated that the SSK of
sexually active squamates undergoes hypertrophy and is
under the influence of androgens. In this study, we did
not investigate any alterations in annual SSK with annual
androgen secretion, but we did demonstrate that the
hypertrophy and the regression of SSK changed seasonally
and synchronously with the active spermatogenic event
and the spermatogenic arrest, respectively. In the Iberian
rock lizard, Lacerta monticola, SSK secretions form
a copulatory plug that adheres to the female’s cloaca
following copulation to occlude oviductal openings;
however, such a plug does not prevent subsequent mating
nor does it reduce the female’s attractiveness (Moreira and
Birkhead, 2003).
With our investigations on annual alterations in female
ovarian morphologies between 2 Calotes, we found that
individuals in the same Calotes species showed different
timing of reproductive events throughout a 1-year period
(the data are not shown here). Additionally, there was
quite clear evidence that QU, EV, and LV follicles and
oviductal eggs overlapped among individuals within
the same populations of both Calotes species. Female
reproductive status is definitely distinguishable between
the 2 Calotes species. Gravid lizards were encountered in 1
individual of C. emma in April, and in 3 individuals of C.
versicolor in June, August, and September. Shanbhag et al.
(2000) reported that female C. versicolor showed inactive
reproduction from December to April, and gravidity was
encountered from May to October.
We found that in the previtellogenic follicles (QU and
EV) of females of both Calotes species the granulosa layer
contained 2 types of cells: pyriform and small cells. Uribe et
al. (1996) stated that in squamates the follicular epithelium
or granulosa initially consists of small cuboidal cells,
but differentiates during the previtellogenic phase and
becomes multilayered and polymorphic by the presence of
unique flask-shaped pyriform cells, intermediate cells, and
small cells. These pyriform cells differentiate from small
somatic follicular cells early in follicular development
via the intermediate-cell stage to become nurse cells, in
direct contact with the developing oocytes (Maurizii et
al., 2004). Differentiation of the small cells into pyriform
cells appears to be linked to the progressive appearance
of glycoproteins with terminal α-N-acetylgalactosamine
residues on the cell surface, which may be involved in
fusion between the oocyte and the follicle cell membranes
as well as maintenance of the differentiated pyriform
cells. The pyriform cells are connected to the oocyte
via intercellular bridges containing a cytoskeleton of
α-tubulin and cytokeratin microtubules (Maurizii et al.,
2004). Tumkiratiwong et al. (2012) also demonstrated that
the previtellogenic follicles of the captive monocled cobra,
Naja kaouthia, had many pyriform cells in the granulosa
layer, but fewer in the vitellogenic follicles. In this study,
the pyriform cells disappeared when the follicles entered
the vitellogenic stage. Andreuccetti (1992) studied the
differentiation of pyriform cells and their contribution to
oocyte growth in 3 lizards, namely Tarentola mauritanica,
Cordylus wittifer, and Platysaurus intermedius, and a
colubrid snake, Coluber viridiflavus, and revealed that
pyriform cells differentiate from small follicle cells via
intermediate cells after establishing an intercellular bridge
with the oocyte. Once pyriform cells are differentiated,
they display ultrastructural features indicative of synthetic
activity, including abundant ribosomes, Golgi membranes,
vacuoles, mitochondria, and lipid droplets. These cellular
components extend to the apex of the cell at the level of
the intercellular bridge, suggesting that constituents of
pyriform cells may be transferred to the oocyte. Pyriform
cells and the oocytes may fulfill similar vitellogenic
functions. The establishment of an intercellular bridge
may represent a crucial event in the development of an
integrated system in which pyriform cells and oocytes
cooperate. Norris (2013) reported that the squamate
granulosa contains the pyriform cells, which are in direct
contact with the developing oocyte and are apparently
involved with early steps in oocyte development soon after
the onset of vitellogenesis. As ovulation approaches, the
granulosa cells, as well as some thecal cells, accumulate
cholesterol-positive lipids, and proliferate and luteinize to
form corpora lutea following ovulation. Follicular atresia
is a common occurrence in reptilian ovaries as in other
vertebrates (Norris, 2013), as we found the corpora lutea
in the follicle in accordance to oviductal egg appearances
of a representative C. versicolor collected in September.
Additionally, as shown in Table 1, several atretic follicles
<2.5 mm in diameter were commonly encountered in C.
emma and C. versicolor.
Based on both morphological and histological
investigations, we found that testes, ST, and SSK were
9
MEESOOK et al. / Turk J Zool
concomitantly active and were associated with high
levels of plasma testosterone. We also demonstrated that
there were high correlations between levels of plasma
testosterone (T) and testicular mass where annual changes
occurred in the same direction as the testicular size and
time of spermatogenetic events among the males of the
Calotes species. Radder et al. (2001) reported that in the
male tropical or oriental garden lizard, C. versicolor, plasma
T is highest during the breeding season, which correlated
with testis mass and reproductive behavior. Changes in T
levels are associated with high spermatogenetic activity.
Radder et al. (2001) also stated that the changes in plasma
T levels during different phases of the male reproductive
cycle in C. versicolor follow a reproductive pattern of a
prenuptial type of spermatogenesis that is similar to that
of some other species of lizards: the spiny-tailed lizard,
Uromastix hardwicki (Arslan et al., 1978a); the viviparous
lizard, Lacerta vivipara (Courty and Dufaure, 1982); the
western shingleback lizard, Tiliqua (Trachydosaurus)
rugosa (Bourne et al., 1986); the male lizard Podarcis s.
sicula (Ando et al., 1990); Podarcis s. sicula Raf. (Ando et
al., 1992); the white-throated savanna monitor, Varanus
albigularis (Phillips and Millar, 1998); and the male brown
anoles, Anolis sagrei (Tokarz et al., 1998).
We found that estradiol (E2) levels increased in
vitellogenic females; its high levels were associated with
the presence of the largest vitellogenic follicles in the
2 Calotes species. Radder et al. (2001) reported that in
female C. versicolor with overlapping reproductive events,
such as vitellogenesis and gestation, E2 was at low levels
when the ovaries were regressed and at high levels at
vitellogenic follicular recruitment, reaching peak level
at the time of preovulatory follicles. The same patterns
of E2 secretion were found again when the second set of
follicles underwent vitellogenesis (Radder et al., 2001).
Surprisingly, Amey and Whittier (2000) reported that in
female bearded dragons, Pagona barbata, plasma E2 was
low or nondetectable across all reproductive states. In C.
versicolor, E2 levels were low in nonreproductive females
with small previtellogenic follicles and those in the EG
phase (Radder et al., 2001). We do not discuss the level of
the plasma progesterone (P) during the gestation period,
as its level was not detectable in this study. The gravid
lizards in EG exhibited low plasma E2 but high P levels, and
the highest P levels coincided with eggshell production.
P levels declined after eggshell formation as reported in
other gravid individuals in several species of lizards that
do not possess vitellogenic follicles of the subsequent
clutch, including C. versicolor (Radder et al., 2001);
Uromastix hardwicki (Arslan et al., 1978b); Agama atra
(Van Wyk, 1984); Eumeces obsoletus, Scelporus undulatus,
and Crotaphytus collaris (Masson and Guillette, 1987);
and Psammodromus algirus (Diaz et al., 1994). However, a
10
decline in P levels in MG with vitellogenic follicles did not
seem to facilitate recruitment or growth of the subsequent
set of vitellogenic follicles in gravid Sceloporus jarrovi
(Guillette et al., 1981).
In the present study, there were variations in the
timing of breeding between the 2 Calotes species and even
within populations of the same species. We could not
relate the copulation timing of 2 such Calotes species to
gonadal activity or sex hormonal surges, as the timing of
natural mating could not be observed during the times we
collected data. Lizard species that inhabit temperate zones
have mostly exhibited seasonal reproduction (Fitch, 1970;
Licht, 1984; Pianka and Vitt, 2003). The 10 lizard species
that have been studied widely to date exhibit an associated
reproductive pattern (Lovern, 2011); that is, green anoles,
Anolis carolinensis (Crews, 1980; Lovern et al., 2004);
brown anoles, Anolis sagrei (Lee et al., 1989; Tokarz,
1998); eastern fence lizards, Sceloporus undulates (Cox
et al., 2005); mountain spiny lizards, Sceloporus jarrovi
(Woodley and Moore, 1999); tree lizards, Urosaurus
ornatus (French and Moore, 2008); wall lizards, Podarcis
sicula (Putti et al., 2009); common lizards, Lacerta vivipara
(Vercken and Clobert, 2008); little striped whiptail lizards,
Cnemidophorus inornatus (Crews, 2005); garden lizards,
Calotes versicolor (Shanbhag, 2003; Lovern, 2011); and
leopard geckos, Eublepharis macularius (Rhen et al., 2005).
The temperate Florida populations of the brown anole,
Anolis sagrei, show a strong seasonality in reproduction
(Lee et al., 1989), while the tropical Caribbean (Licht and
Gorman, 1970; Sexton and Brown, 1977) and Hawaiian
populations of this species (Goldberg et al., 2002) show a
less-pronounced seasonality, in which reproductively active
individuals can be found throughout the year. Although
individuals within a population of many tropical lizard
species can breed at any time, no individuals within the
population breed year-round (Lovern, 2011). Additionally,
Vitt and Caldwell (2009) stated that the belief used to be
that tropical squamates had continuous reproduction in
aseasonal tropical environments, or reproduced during
the wet season in a wet–dry seasonal tropical environment.
Many tropical lizard species, namely the anoles Anolis
acutus (Ruibal et al., 1972), Anolis limifrons (Sexton et al.,
1971), and Anolis opalinus (Jenssen and Nunez, 1994), as
well as the gecko Cyrtodactylus malyanus, the flying lizard
Draco melanopogon (Inger and Greenberg, 1966), and the
parthenogenetic, oviparous whiptail lizard Cnemidophorus
nativo (Menezes et al., 2004), showed slightly more
frequent breeding during the wet season than during the
dry season (Jenssen and Nunez, 1994).
Reproductive patterns can be described in a variety of
ways, but not all species fit neatly into such categorizations.
However, 2 general types of reproductive patterns are
MEESOOK et al. / Turk J Zool
recognized in terms of prenuptial and postnuptial
reproductive patterns (Lance, 1998). Prenuptial
reproductive pattern terms, such as gonadal recrudescence,
sex steroid production, and gametogenesis occur in
advance of mating, whereas postnuptial reproductive
patterns occur following mating. In other words, in a highelevation population of Sceloporus grammicus in Parque
Nacional de Zoquiapan in central Mexico, an active
reproductive event occurring in the early fall is described
as dissociated from testicular recrudescence in males but is
associated with the initiation of ovarian recrudescence in
females (Guillette and Casas-Andreu, 1980, 1981; ZunigaVega et al., 2008). This is in contrast to S. grammicus from
Teotihuacan, Mexico, in which testicular recrudescence
and breeding occur in the summer and fall, at the onset
of female ovarian recrudescence (Jimenez-Cruz et al.,
2005). In S. mucronatus from Valle de la Cantimplora,
Mexico, peak testicular recrudescence and mating occur
during the summer, prior to ovarian recrudescence, which
does not occur until several months later (Ortega-Leon
et al., 2009). This is distinct from many fall-breeding
populations elsewhere (Mendez-De La Cruz et al., 1994;
Villagran-Santa Cruz et al., 1994). The examples above
demonstrate that gonadal activity and mating behavior
are clearly variable, but hormone analyses have not been
performed in these species and so endocrine relationships
cannot be assessed at this point.
In conclusion, we suggest that the males and females
of the 2 Calotes species have much more prolonged, active
reproductive phases than inactive reproductive phases.
The reproductive patterns of C. emma and C. versicolor
were classified into the same reproductive pattern of
continual reproduction.
Acknowledgments
We thank the Department of Zoology of Kasetsart
University for financial support. We also thank the staff
of Sakaerat Environmental Research Station, Nakhon
Ratchasima Province, for devoting time for research
collaboration. We also thank Mrs Sureerat Sangkrut for
drawing all illustrations. In addition, we wish to thank the
anonymous referees for many helpful suggestions.
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