Turkish Journal of Zoology Turk J Zool (2016) 40: 691-703 http://journals.tubitak.gov.tr/zoology/ © TÜBİTAK Research Article doi:10.3906/zoo-1508-53

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: 24.10.2016

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 level of circulating sex steroids. This is termed an associated Lizards (including snakes) are the most speciose living reproductive pattern (Crews, 1984, 1999; Crews et al., 1984; clade of reptiles, with almost 7200 species. Even excluding Licht, 1984; Norris, 2013). In contrast, in a small number snakes, lizards are still the most speciose extant reptiles, of vertebrate species, such as some turtles, snakes, and bats, with approximately 4450 species. Lizards, which belong males mate at a time when their gonads are quiescent (QU) to the family Agamidae, are classified into 52 genera with and the levels of circulating sex steroids are low (Garstka approximately 400 species (Vitt and Caldwell, 2009), 31 et al., 1982). This pattern of reproduction is referred to as species of which are found in Thailand (Laohachinda, a dissociated reproductive pattern (Volsøe, 1944; Crews, 2009). The genus Calotes Cuvier 1817, a genus of 1976, 1984, 1999; Lofts, 1977; Licht, 1984; Norris, 2013). In snakes, especially in the family Colubridae, various terms Agamidae, currently contains 23 species (Hartmann et have been used to describe the male reproductive patterns al., 2013). There are 7 species of Calotes known to occur observed in different species. Volsøe (1944) introduced on the Southeast Asian mainland: C. chincollium, Vindum the term prenuptial spermatogenesis to describe sperm 2003; C. htunwini, Zug and Vindum 2006; C. irawadi, Zug production that occurs immediately prior to mating, and et al. 2006; C. jerdoni, Günther 1870; C. emma, Gray 1845; postnuptial spermatogenesis to describe sperm production C. mystaceus, Duméril and Bibron 1837; and C. versicolor, that occurs after mating. Aldridge et al. (2009) introduced Daudin 1802. The latter 3 species are well known to the term preovulatory spermatogenesis to describe Indochina, including Thailand (Zug et al., 2006; Hartmann sperm production immediately prior to ovulation, and et al., 2013). postovulatory spermatogenesis as sperm production that Male reproductive displays are directly correlated occurs after ovulation. They assumed that androgenesis to testicular hypertrophy in the majority of seasonally was associated with the development of secondary sex breeding vertebrates, and are accompanied by an elevated characters and mating behavior. * Correspondence: [email protected] 691 MEESOOK et al. / Turk J Zool

Vitt and Caldwell (2009) added that the currently and histological investigations, and also that annual sex known diversity of seasonal patterns of tropical squamate hormonal levels would reveal the reproductive patterns of reproduction suggests that no single explanation 2 such Calotes species. is sufficient. Snakes are included in the same order (Squamata), but demonstrate a more diverse reproductive 2. Materials and methods pattern in contrast to lizards. Tumkiratiwong et al. (2012) 2.1. Animals studied the reproductive patterns of captive male and We caught the adult lizards monthly over 1 year by hand female Naja kaouthia, monocled cobra, Lesson 1831, or with a noose in the 3 forest types: a dry evergreen suggesting that its reproductive pattern exhibited either forest, a deciduous dipterocarp forest, and an ecotone postnuptial spermatogenesis or a dissociated reproductive forest at Sakaerat Biosphere Reserves of the Sakaerat pattern. Environmental Research Station (14°26′33″–14°32′50″N, There is no detailed information available on testicular 101°50′43″–101°57′21″E; 720–770 m above sea level), and ovarian cycles, plasma sex hormonal profiles during located at Nakhon Ratchasima Province, northeastern gonadal cycles, or male reproduction-associated sexual Thailand. We classified adults of C. emma and C. versicolor segment of the kidney (SSK), especially in Calotes emma. based on external morphological differences (Figure 1). This paper, therefore, monitored annual alterations of The adult stages of the 2 species were diagnosed based on reproductive organs of males and females of 2 Calotes the internal morphology of active male and female gonads species, i.e., C. emma and C. versicolor, and investigated (Figure 1) and expressed in terms of the snout–vent length male and female sex hormones, male testosterone, and (SVL) as: (1) male, C. emma, >5.78 cm; and C. versicolor, female estradiol to define the reproductive patterns of 2 >5.40 cm; (2) female, C. emma, >6.57 cm; and C. versicolor, such Calotes species. We expected that annual male and >5.99 cm. We attempted to collect 10 adult males and female reproductive alterations of the testes, male SSK, females, but in some months the samples were smaller than and the ovaries would require underlying morphological 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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The months that lacked samples in each species are shown the group with the largest-sized follicles were recorded in parentheses as follows: males of C. emma (May, July, from both ovaries. The follicular size was measured with a and August) and C. versicolor (December); females of C. Vernier caliper (0–150 × 0.02 mm). Follicles greater than emma (February to March, May, and December) and C. or equal to 2.5 mm in diameter represented vitellogenic versicolor (March to May, July, October, and December). status (Shanbhag and Prasad, 1993). We anesthetized the samples with diethyl ether and Since ovarian follicular development, ovulation, collected blood samples by puncturing the cardiac and gestation occur asynchronously in a population, chamber; however, it was not possible to acquire samples individuals with differing reproductive statuses are from those lizards with very small cardiac chambers. encountered. Therefore, the data from the reproductive Blood samples were kept at 4 °C in heparinized vials and phase were classified based on the reproductive status centrifuged within 2–3 h following blood collection. The of individuals rather than on a monthly classification as blood was centrifuged at 1600 × g for 10 min. Plasma was follows: (1) the QU stage: previtellogenic follicles sized then aspirated off and frozen at –79 °C for later analysis of <2.5 mm in diameter; (2) the early vitellogenic (EV) plasma levels in male testosterone and in female estradiol. stage: initiation of vitellogenesis with follicles sized 2.5– 2.2. External and internal reproductive morphologies 5.0 mm in diameter and without oviductal eggs; (3) the We measured SVL, which ranges from the snout tip to late vitellogenic (LV) stage: late period of vitellogenesis the anterior margin of the vent. We sacrificed male and with follicles sized >5.0 mm in diameter; (4) the early female lizards to investigate the general reproductive gestation (EG) stage: oviductal eggs with previtellogenic morphologies (Figure 1, bottom middle, male; bottom follicles sized <2.5 mm in diameter; (5) the midgestation right, female) and measured follicular size and testicular (MG) stage: oviductal eggs with vitellogenic follicles sized mass. Each specimen was weighed to the nearest 0.01 g; its 2.5–5.0 mm in diameter; and (6) late gestation (LG) stage: SVL was measured to the nearest 0.05 mm. oviductal eggs with vitellogenic follicles sized >5.0 mm in 2.3. Testicular, male SSK, and ovarian histologies diameter (modified from Radder et al., 2001). Testes, male SSK, and ovaries were excised and fixed in 2.5. Measurement of testosterone a 10% v/v buffered neutral formalin solution, processed The plasma level of testosterone was measured by a125 I by the paraffin technique (Avwioro, 2011). The tissue radioimmunoassay (RIA). We added a 500-µL sample of was cut in a cross-section to 6 µm in thickness using a plasma to 5.0 mL of dichloromethane in a screw-top glass LEICA RM2145 (Nussloch, Germany). Sections were extraction tube. We then capped the mixture and mixed stained with hematoxylin and eosin. The reproductive it for 60 min by gentle inversion with an end-over-end stage of adult females was determined on the basis of rotator, and then centrifuged the sample for 5 min at 1600 the presence or absence of types of pyriform cells in the × g to separate the layers. The upper phase was aspirated granulosa layer. Follicles with the pyriform cells, which without disturbing the interface; 2.0 mL of the lower phase appeared in the granulosa layer, were considered to be was then transferred to a clean 12 × 75 mm glass tube and previtellogenic follicles, and follicles not containing the evaporated to dryness under a gentle stream of nitrogen at pyriform cells were considered to be vitellogenic follicles 37 °C. Finally, we reconstituted the extract with 200 µL of (Tumkiratiwong et al., 2012). Females having vitellogenic testosterone buffer. The testosterone extraction procedure follicles and/or oviductal eggs were considered to be at was performed using a Coat A Count Testosterone RIA the active reproductive stage, while females having only Kit (Diagnostic Products, Los Angeles, CA, USA). The previtellogenic follicles were considered to be at the QU intra- and interassay variations expressed as coefficients reproductive stage. Females with regressed follicles were of variation (CVs) were 8.4% and 7.9%, respectively. The considered to be at the postparturient stage. Males with approximate sensitivity of this assay was 40 pg/mL. The the appearance of sperm bundles and/or free sperm in cross-reactivity with androstenedione was 0.5%. The the seminiferous tubules (ST) were assessed as being spiking recovery values averaged 98.3 ± 0.6%. Dilutions at the active reproductive stage, while males without of 50%, 25%, and 12.5% of the undiluted concentration of those attributes were assessed as being at the inactive 7300 pg/mL were 3490 pg/mL, 1700 pg/mL, and 780 pg/ reproductive stage (Tumkiratiwong et al., 2012). Males mL, respectively. with SSK that had strongly eosinophilic-stained granules 2.6. Measurement of estradiol were regarded as being at the active reproductive stage The plasma level of estradiol was measured by a 125I RIA. (Sever et al., 2002). We added 250 µL of plasma to 2.0 mL of diethyl ether 2.4. Categorization of female individuals based on in a screw-top glass extraction tube, and then capped reproductive status and mixed it by gentle inversion with an end-over-end Similar-sized follicles were organized into distinct groups. rotator for 30 min. It was centrifuged for 5 min at 1600 The total number and diameter of follicles belonging to × g to separate the layers. The lower (aqueous) phase was

693 MEESOOK et al. / Turk J Zool frozen using dry ice; the organic phase was then decanted in the ovarian weights, the estradiol levels, and the diameters into another vial and evaporated to dryness under a of the largest follicle between each reproductive stage of the gentle stream of nitrogen at 37 °C. Finally, the extract was various follicular growths of C. emma only because of the reconstituted with 250 µL of estradiol buffer. The estradiol insufficient sample size. There was no statistical analysis of extraction procedure was performed using a Coat A differences in male testosterone levels or testicular masses Count Estradiol (TKE2) RIA kit (Diagnostic Products). based on months due to the small amount of data available. The intra- and interassay variations calculated as CVs were Spearman’s correlation coefficients were used to determine 5.3% and 6.4%, respectively. The approximate sensitivity of relationships between the testicular mass and testosterone, this assay was 10 pg/mL. The specificity of cross-reactivity and the diameter of the largest follicles and estradiol levels. with estradiol was 0.32%. The spiking recovery values The level of significance was set to P < 0.05. averaged 96.8 ± 3.3%. Dilutions of 50%, 25%, 12.5%, and 2.8. Ethical aspects 6.25% of the undiluted concentration of 230.9 pg/mL were This study was approved by the Ethics Committee of 114.4 pg/mL, 60.0 pg/mL, 27.9 pg/mL, and 14.8 pg/mL, the Department of National Parks, Wildlife, and Plant respectively. Conservation, Ministry of Natural Resources and 2.7. Statistical analysis Environment, Thailand (License No. 0909.302/18344). Testicular masses, testosterone levels, ovarian weights, estradiol levels, and diameters of the largest follicle were 3. Results expressed as the mean ± standard error of the mean (SEM). 3.1. Annual alterations in male and female reproductive The Kolmogorov–Smirnov test and Levene’s test were used morphologies of Calotes to determine if the data were normally distributed and the Annual changes in testicular morphological events homogeneity of variance, respectively. Nonparametric tests between C. emma and C. versicolor are depicted in Figure were used, as all data mentioned above were nonnormal 2. The representative C. emma testes were recrudesced in and heteroscedastic. Therefore, the Kruskal–Wallis H test December, continued to hypertrophy from January to April was used to test for differences in the ovarian weights, the and in June, and completely regressed from September to estradiol levels, and the diameters of the largest follicle November. In the representative C. versicolor, the testes among the reproductive stages of nongestation periods. The became hypertrophied from January to September and Mann–Whitney U test was then used to compare differences 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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Annual changes in ovarian morphological events We found that in the female representative C. emma, (Figure 3) and annual changes in the number of follicular active ovaries contained follicles of vitellogenic stages types and egg types (Table 1) of the representative 2 in January and June, but inactive ovaries contained Calotes species are shown. In the representative C. emma, previtellogenic and atretic follicles in August (Figure 4, we found that in January, April, from June to July, and bottom left and bottom right). In the representative C. from August to November, follicles were in the EV, EG, LV, versicolor, ovaries contained previtellogenic and atretic and QU stages, respectively (Figure 3, top; Table 1). In the follicles in January and both previtellogenic and EV representative C. versicolor, we found that, in January and follicles in June (Figure 5, bottom left and bottom right). February, the ovaries contained only QU follicles; in June Additionally, we found corpus luteum in the ovaries of and August, QU and EV follicles and oviductal eggs; in 1 representative in September (Figure 5, bottom left and September, QU follicles and oviductal eggs; in November, bottom right). We also observed cellular alterations in only QU follicles (Figure 3, bottom; Table 1). the granulosa layer of both C. emma and C. versicolor. It 3.2. Annual histological alterations in ST, SSK, and was demonstrated that, inside the granulosa layer of the ovaries of Calotes previtellogenic follicles, both types of small and pyriform In the male representative C. emma, the ST and SSK cells appeared. However, both such cell types disappeared were hypertrophied from January to April and in June, in the granulosa layer of the vitellogenic follicles (Figures then regressed from September to November, becoming 4 and 5, bottom right). active again in December (Figure 4, top left and top right, 3.3. Annual plasma testosterone levels and testicular respectively). Spermatozoal masses were contained inside masses in Calotes the hypertrophied ST. Spermatogonia initially appeared in Annual variations in plasma testosterone levels and November; additionally, a few types of germ cells occurred, testicular masses between the 2 Calotes species are depicted but there were still no active spermatozoa in December (Figures 6a and 6b). In the representative C. emma, (Figure 4, top left). In the representative C. versicolor, both plasma testosterone levels and testicular masses initially ST and SSK were active from January to September but increased in December, peaked in March, and thereafter were inactive from November to December (Figure 5, top gradually reduced from April to November (Figure 6a). left and top right). 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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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 C. emma <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 ------C. versicolor <2.5 mm 16 28 - - 28 - 19 16 - 10 2.5–5.0 mm - - - - 5 - - - - - >5.0 mm ------Oviductal eggs - - - - 5 - 6 5 - -

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, during their reproductive stages (Table 2). The ovarian peaked in April, and thereafter gradually reduced from weights and the diameters of the largest follicles differed May to November (Figure 6b). The changes in testicular significantly from the QU stage to the LV stage regarding masses tended to correspond well to the changes in plasma the growth of ovarian follicles (P = 0.001) in C. emma. testosterone levels between the 2 species in the genus It was also observed that the growth of ovarian follicles Calotes (r = 0.789, P = 0.001; and r = 0.732, P = 0.001 for C. concomitantly occurred with the growth of oviductal eggs emma and C. versicolor, respectively). during the gestation period of the 2 species.

3.4. Annual plasma estradiol and ovarian cycles in Variations in plasma E2 levels in relation to the Calotes diameters of the largest follicles between the 2 Calotes Variations in ovarian masses and the diameter of the species during reproductive stages are graphically depicted largest follicle were observed between the 2 Calotes species (Figures 7a and 7b). Regarding the groups of follicular sizes

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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 regressed testicular phase. However, the timing of those 2 the QU and the EV stages (U = 9.00, P = 0.090), the QU events appeared asynchronously in the 2 Calotes species, and the LV stages (U = 2.00, P = 0.009), and the EV and the even in individuals of the same population (data not LV stages (U = 2.00, P = 0.083). Additionally, the diameters shown here). The testes were hypertrophied with active of the largest follicles were the difference between the QU spermatozoal production from December to June and and the EV stages (U = 0.00, P = 0.004), the QU and the January to September in the representative C. emma and C. LV stages (U = 0.00, P = 0.004), and the EV and the LV versicolor, respectively. A study by Gouder and Nadkarni stages (U = 0.00, P = 0.021). The correlation coefficient (1979) showed that males of C. versicolor, widely distributed between plasma E2 and the diameter of the largest follicle in India, were spermatogenetically active from April to was 0.82 (P = 0.001) and 0.32 (P = 0.365) of C. emma and September. Active vitellogenic follicles and oviductal C. versicolor, respectively. eggs were concomitantly encountered in C. emma and C. versicolor. Therefore, these 2 Calotes species exhibited 4. Discussion polyautochrony and multiclutches. Radder et al. (2001) Annual variations in the timing of male and female reported that Indian garden lizards, C. versicolor, showed reproductive stages were encountered among populations polyautochrony and multiclutches. The representative of C. emma and C. versicolor, even in individuals of the gravid lizards were found in April in C. emma, and in June same population. Annual changes in testicular sizes and from August to September in C. versicolor. Gravid were categorized into 2 phases in both species: (1) an C. versicolor, whose habitat is in India, was encountered active hypertrophied testicular phase; and (2) an inactive from May to October (Shanbhag and Prasad, 1993). We

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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 0.11 ± 0.09b 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, mid- gestation; 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 hypertrophied stages, respectively, which corresponded in the male and female reproductive morphologies of the 2 well with the timing of the testicular hypertrophied stage Calotes species that the active reproductive events of both mentioned above. In other words, the timing of arrested males and females of the Calotes species lasted nearly 1 spermatogenesis and regressed SSK was in accordance year with only a few months of reproductive arrest, which with that of the regressed testes. Likewise, we confirmed is especially seen in C. versicolor. that the 2 Calotes species have an active reproductive stage According to our investigations on annual histological that is much longer than the inactive reproductive stage. alterations in ST and male SSK timing of the 2 Calotes SSK is present in a variety of male snakes and lizards, species, both ST and SSK were in active spermatogenic and but is absent in both turtles (Regaud and Policard, 1903)

698 MEESOOK et al. / Turk J Zool and crocodilians (Fox, 1952). Bishop (1959) found that small cells. These pyriform cells differentiate from small the testes of the male garter snake, Thamnophis sirtalis, somatic follicular cells early in follicular development were spermatogenically active during the same time as the via the intermediate-cell stage to become nurse cells, in hypertrophied SSK; during the active reproductive period, direct contact with the developing oocytes (Maurizii et the diameter of the SSK tubule was 5 times greater than al., 2004). Differentiation of the small cells into pyriform that of the SSK tubule during the inactive reproductive cells appears to be linked to the progressive appearance period. However, SSK development in female lizards of glycoproteins with terminal α-N-acetylgalactosamine has been reported in the genus Cnemidophorus (Del residues on the cell surface, which may be involved in Conte, 1972; Del Conte and Tamayo, 1973) and Scincella fusion between the oocyte and the follicle cell membranes laterale (Sever and Hopkins, 2005). They suggested that as well as maintenance of the differentiated pyriform the females had a low level of natural androgens, which cells. The pyriform cells are connected to the oocyte caused the SSK development (Del Conte and Tamayo, via intercellular bridges containing a cytoskeleton of 1973; Sever and Hopkins, 2005). In the present study, we α-tubulin and cytokeratin microtubules (Maurizii et al., did not monitor the annual seasonal alterations in female 2004). Tumkiratiwong et al. (2012) also demonstrated that SSK. The hypertrophy of the SSK is synchronous with the previtellogenic follicles of the captive monocled cobra, androgen secretion and spermatogenic activity (Sever and Naja kaouthia, had many pyriform cells in the granulosa Hopkins, 2005). Norris (2013) also stated that the SSK of layer, but fewer in the vitellogenic follicles. In this study, sexually active squamates undergoes hypertrophy and is the pyriform cells disappeared when the follicles entered under the influence of androgens. In this study, we did the vitellogenic stage. Andreuccetti (1992) studied the not investigate any alterations in annual SSK with annual differentiation of pyriform cells and their contribution to androgen secretion, but we did demonstrate that the oocyte growth in 3 lizards, namely Tarentola mauritanica, hypertrophy and the regression of SSK changed seasonally Cordylus wittifer, and Platysaurus intermedius, and a and synchronously with the active spermatogenic event colubrid snake, Coluber viridiflavus, and revealed that and the spermatogenic arrest, respectively. In the Iberian pyriform cells differentiate from small follicle cells via rock lizard, Lacerta monticola, SSK secretions form intermediate cells after establishing an intercellular bridge a copulatory plug that adheres to the female’s cloaca with the oocyte. Once pyriform cells are differentiated, following copulation to occlude oviductal openings; they display ultrastructural features indicative of synthetic however, such a plug does not prevent subsequent mating activity, including abundant ribosomes, Golgi membranes, nor does it reduce the female’s attractiveness (Moreira and vacuoles, mitochondria, and lipid droplets. These cellular Birkhead, 2003). components extend to the apex of the cell at the level of With our investigations on annual alterations in female the intercellular bridge, suggesting that constituents of ovarian morphologies between 2 Calotes, we found that pyriform cells may be transferred to the oocyte. Pyriform individuals in the same Calotes species showed different cells and the oocytes may fulfill similar vitellogenic timing of reproductive events throughout a 1-year period functions. The establishment of an intercellular bridge (the data are not shown here). Additionally, there was may represent a crucial event in the development of an quite clear evidence that QU, EV, and LV follicles and integrated system in which pyriform cells and oocytes oviductal eggs overlapped among individuals within cooperate. Norris (2013) reported that the squamate the same populations of both Calotes species. Female granulosa contains the pyriform cells, which are in direct reproductive status is definitely distinguishable between contact with the developing oocyte and are apparently the 2 Calotes species. Gravid lizards were encountered in 1 involved with early steps in oocyte development soon after individual of C. emma in April, and in 3 individuals of C. the onset of vitellogenesis. As ovulation approaches, the versicolor in June, August, and September. Shanbhag et al. granulosa cells, as well as some thecal cells, accumulate (2000) reported that female C. versicolor showed inactive cholesterol-positive lipids, and proliferate and luteinize to reproduction from December to April, and gravidity was form corpora lutea following ovulation. Follicular atresia encountered from May to October. is a common occurrence in reptilian ovaries as in other We found that in the previtellogenic follicles (QU and vertebrates (Norris, 2013), as we found the corpora lutea EV) of females of both Calotes species the granulosa layer in the follicle in accordance to oviductal egg appearances contained 2 types of cells: pyriform and small cells. Uribe et of a representative C. versicolor collected in September. al. (1996) stated that in squamates the follicular epithelium Additionally, as shown in Table 1, several atretic follicles or granulosa initially consists of small cuboidal cells, <2.5 mm in diameter were commonly encountered in C. but differentiates during the previtellogenic phase and emma and C. versicolor. becomes multilayered and polymorphic by the presence of Based on both morphological and histological unique flask-shaped pyriform cells, intermediate cells, and investigations, we found that testes, ST, and SSK were

699 MEESOOK et al. / Turk J Zool concomitantly active and were associated with high decline in P levels in MG with vitellogenic follicles did not levels of plasma testosterone. We also demonstrated that seem to facilitate recruitment or growth of the subsequent there were high correlations between levels of plasma set of vitellogenic follicles in gravid Sceloporus jarrovi testosterone (T) and testicular mass where annual changes (Guillette et al., 1981). occurred in the same direction as the testicular size and In the present study, there were variations in the time of spermatogenetic events among the males of the timing of breeding between the 2 Calotes species and even Calotes species. Radder et al. (2001) reported that in the within populations of the same species. We could not male tropical or oriental garden lizard, C. versicolor, plasma relate the copulation timing of 2 such Calotes species to T is highest during the breeding season, which correlated gonadal activity or sex hormonal surges, as the timing of with testis mass and reproductive behavior. Changes in T natural mating could not be observed during the times we levels are associated with high spermatogenetic activity. collected data. Lizard species that inhabit temperate zones Radder et al. (2001) also stated that the changes in plasma have mostly exhibited seasonal reproduction (Fitch, 1970; T levels during different phases of the male reproductive Licht, 1984; Pianka and Vitt, 2003). The 10 lizard species cycle in C. versicolor follow a reproductive pattern of a that have been studied widely to date exhibit an associated prenuptial type of spermatogenesis that is similar to that reproductive pattern (Lovern, 2011); that is, green anoles, of some other species of lizards: the spiny-tailed lizard, Anolis carolinensis (Crews, 1980; Lovern et al., 2004); Uromastix hardwicki (Arslan et al., 1978a); the viviparous brown anoles, Anolis sagrei (Lee et al., 1989; Tokarz, lizard, Lacerta vivipara (Courty and Dufaure, 1982); the 1998); eastern fence lizards, Sceloporus undulates (Cox western shingleback lizard, Tiliqua (Trachydosaurus) et al., 2005); mountain spiny lizards, Sceloporus jarrovi rugosa (Bourne et al., 1986); the male lizard Podarcis s. (Woodley and Moore, 1999); tree lizards, Urosaurus sicula (Ando et al., 1990); Podarcis s. sicula Raf. (Ando et ornatus (French and Moore, 2008); wall lizards, Podarcis al., 1992); the white-throated savanna monitor, Varanus sicula (Putti et al., 2009); common lizards, Lacerta vivipara albigularis (Phillips and Millar, 1998); and the male brown (Vercken and Clobert, 2008); little striped whiptail lizards, anoles, Anolis sagrei (Tokarz et al., 1998). Cnemidophorus inornatus (Crews, 2005); garden lizards, We found that estradiol (E ) levels increased in 2 Calotes versicolor (Shanbhag, 2003; Lovern, 2011); and vitellogenic females; its high levels were associated with leopard geckos, Eublepharis macularius (Rhen et al., 2005). the presence of the largest vitellogenic follicles in the The temperate Florida populations of the brown anole, 2 Calotes species. Radder et al. (2001) reported that in Anolis sagrei, show a strong seasonality in reproduction female C. versicolor with overlapping reproductive events, (Lee et al., 1989), while the tropical Caribbean (Licht and such as vitellogenesis and gestation, E was at low levels 2 Gorman, 1970; Sexton and Brown, 1977) and Hawaiian when the ovaries were regressed and at high levels at populations of this species (Goldberg et al., 2002) show a vitellogenic follicular recruitment, reaching peak level at the time of preovulatory follicles. The same patterns less-pronounced seasonality, in which reproductively active individuals can be found throughout the year. Although of E2 secretion were found again when the second set of follicles underwent vitellogenesis (Radder et al., 2001). individuals within a population of many tropical lizard Surprisingly, Amey and Whittier (2000) reported that in species can breed at any time, no individuals within the population breed year-round (Lovern, 2011). Additionally, female bearded dragons, Pagona barbata, plasma E2 was low or nondetectable across all reproductive states. In C. Vitt and Caldwell (2009) stated that the belief used to be that tropical squamates had continuous reproduction in versicolor, E2 levels were low in nonreproductive females with small previtellogenic follicles and those in the EG aseasonal tropical environments, or reproduced during phase (Radder et al., 2001). We do not discuss the level of the wet season in a wet–dry seasonal tropical environment. the plasma progesterone (P) during the gestation period, Many tropical lizard species, namely the anoles Anolis as its level was not detectable in this study. The gravid acutus (Ruibal et al., 1972), Anolis limifrons (Sexton et al., 1971), and Anolis opalinus (Jenssen and Nunez, 1994), as lizards in EG exhibited low plasma E2 but high P levels, and the highest P levels coincided with eggshell production. well as the gecko Cyrtodactylus malyanus, the flying lizard P levels declined after eggshell formation as reported in Draco melanopogon (Inger and Greenberg, 1966), and the other gravid individuals in several species of lizards that parthenogenetic, oviparous whiptail lizard Cnemidophorus do not possess vitellogenic follicles of the subsequent nativo (Menezes et al., 2004), showed slightly more clutch, including C. versicolor (Radder et al., 2001); frequent breeding during the wet season than during the Uromastix hardwicki (Arslan et al., 1978b); Agama atra dry season (Jenssen and Nunez, 1994). (Van Wyk, 1984); Eumeces obsoletus, Scelporus undulatus, Reproductive patterns can be described in a variety of and Crotaphytus collaris (Masson and Guillette, 1987); ways, but not all species fit neatly into such categorizations. and Psammodromus algirus (Diaz et al., 1994). However, a However, 2 general types of reproductive patterns are

700 MEESOOK et al. / Turk J Zool recognized in terms of prenuptial and postnuptial populations elsewhere (Mendez-De La Cruz et al., 1994; reproductive patterns (Lance, 1998). Prenuptial Villagran-Santa Cruz et al., 1994). The examples above reproductive pattern terms, such as gonadal recrudescence, demonstrate that gonadal activity and mating behavior sex steroid production, and gametogenesis occur in are clearly variable, but hormone analyses have not been advance of mating, whereas postnuptial reproductive performed in these species and so endocrine relationships patterns occur following mating. In other words, in a high- cannot be assessed at this point. elevation population of Sceloporus grammicus in Parque In conclusion, we suggest that the males and females Nacional de Zoquiapan in central Mexico, an active of the 2 Calotes species have much more prolonged, active reproductive event occurring in the early fall is described reproductive phases than inactive reproductive phases. as dissociated from testicular recrudescence in males but is The reproductive patterns ofC . emma and C. versicolor associated with the initiation of ovarian recrudescence in were classified into the same reproductive pattern of females (Guillette and Casas-Andreu, 1980, 1981; Zuniga- continual reproduction. Vega et al., 2008). This is in contrast to S. grammicus from Teotihuacan, Mexico, in which testicular recrudescence Acknowledgments and breeding occur in the summer and fall, at the onset We thank the Department of Zoology of Kasetsart of female ovarian recrudescence (Jimenez-Cruz et al., University for financial support. We also thank the staff 2005). In S. mucronatus from Valle de la Cantimplora, of Sakaerat Environmental Research Station, Nakhon Mexico, peak testicular recrudescence and mating occur Ratchasima Province, for devoting time for research during the summer, prior to ovarian recrudescence, which collaboration. We also thank Mrs Sureerat Sangkrut for does not occur until several months later (Ortega-Leon drawing all illustrations. In addition, we wish to thank the et al., 2009). This is distinct from many fall-breeding anonymous referees for many helpful suggestions.

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