ISSN 1827-9635 (print) © Firenze University Press ISSN 1827-9643 (online) www.fupress.com/ah Acta Herpetologica 8(1): 1-8, 2013 The oogenic cycle of the Caspian bent-toed gecko, Cyrtopodion caspium (Squamata: Gekkonidae) in Iran Vida Hojati1*, Kazem Parivar2, Eskandar Rastegar-Pouyani3, Abdolhossein Shiravi1 1 PhD in Developmental Biology, Department of Biology, Damghan Branch, Islamic Azad University, Damghan, Iran. *Corresponding author. E-mail: vida.hojati@gmail.com 2 PhD in Developmental Biology, Department of Biology, Science and Research Branch, Islamic Azad University, Tehran, Iran 3 PhD in Molecular Evolution, Department of Biology, Faculty of Science, Hakim Sabzevari University, Sabzevar, Iran Submitted on 2012, 14th June; revised on 2013, 12th January; accepted on 2013, 11th February. Abstract. The Caspian bent-toed gecko, Cyrtopodion caspium, is one of the commonest lizards in northern Iran. It is nocturnal, anthropophile and oviparous. In this study, the reproductive cycle of this species was studied by focusing on oogenesis, from April 5 to October 20, 2011. In total, 70 adult females were obtained from Mazandaran, one of the northern provinces of Iran where the climate is temperate. Ovaries were removed and processed for histological and morphometric studies. The results show that oocyte growth starts in late April and ends in August. Mating starts in spring, especially at the beginning of May, with oviposition occurring from late May to mid August. Females lay 1-2 eggs per clutch with the possibility of producing a secondary clutch later in the season. Maximum reproductive activity occurs in May and peaks in June. There was no significant difference between right and left side of reproductive system. With oogenesis occurring from April through July, C. caspium follows an oogenic cycle typical for temperate species. Keywords. Lizard, gecko, Cyrtopodion caspium, oogenesis, ovary, reproduction. INTRODUCTION Lizards exhibit three general types of reproductive cycles: associated, dissociated and constant (Pough et al., 2001). Associated and dissociated reproductive cycles are characterized by the presence of a discontinuous mat- ing season. In an associated reproductive cycle, gonadal activity increases immediately prior to the mating period in both males and females simultaneously. In this repro- ductive cycle, females have no need to store sperm due to it’s availability during the reproductive season. This cycle is common in species that live in predictable envi- ronments such as in the temperate zones (Diaz et al., 1994), the subtropics (Huang, 1997), and in the seasonal tropics (Censky, 1995a, b). In a dissociated reproductive cycle, gonadal activity is low during the mating period and peaks during the non-mating period. In this type of reproductive cycle, male gonadal activity is shorter than that of females and sperm is stored by the female genital system for some months with fertilization occurring later (Torki, 2006). A dissociated cycle is typically observed in species that live in temperate zones and have a brief mat- ing season (Van Wyk, 1995). A constant reproductive cycle is exhibited by species in which gonadal activity is maintained at nearly maximum level almost year-round. Several species of tropical lizards exhibit a constant reproductive cycle (Jenssen and Nunez, 1994). These relationships between reproductive cycles and climate suggest that reproduction in lizards is affected by envi- ronmental variables such as temperature (Marion, 1982), precipitation (Guillette and Casas-Andrew, 1987), and photoperiod (Licht, 1967). Phylogenetic constraints may also play a major role in shaping the reproductive char- acteristics of lizards (Dunham and Miles, 1985). If so, it may be beneficial to study the reproductive cycles of spe- cies within a single diverse and wide-ranging lineage. 2 Vida Hojati et al. The members of the family Gekkonidae display dif- ferent reproductive patterns, as evidenced by the pres- ence of viviparous and oviparous species (Nogueira et al., 2011). Breeding season is determined by cycles of photo- period, temperature, rainfall and availability of food (Vitt and Pianka, 1994). The Caspian bent-toed gecko, Cyrtopodion caspi- um (Eichwald, 1831), is a nocturnal oviparous lizard of the family Gekkonidae (Anderson, 1999) and com- prises two subspecies in the Caspian Sea region (Szcz- erbak and Golubev, 1996; Anderson, 1999). Cyrtopodion caspium caspium is widely distributed in the eastern part of the Caucasus, Middle Asia including Turkmenistan, Uzbekistan, southern Tajikistan, southwestern Kazakh- stan, northern Afghanistan and Iran (Szczerbak, 2003). The northern border of its range is a line from Komso- molets Bay on the northeastern shore of the Caspian Sea to the northern coast of the Aral Sea and Syr Darya. In Iran it is known to occur in the Mazandaran and Gorgan regions, northern and eastern Khorasan, extending south to Sistan from sea level up to 1700 m (Anderson, 1999). Cyrtopodion caspium insularis (Akhmedov and Szczerbak, 1978) occurs on the island of Vulf in the Caspian Sea and is known only from the type locality. It differs from the nominate subspecies in having the first pair of postmen- tal shields usually separated from each other by gular scales, though there may be a tiny dot-like contact, while in the nominate subspecies they are broadly in contact (Akhmedov and Szczerbak, 1978). Cyrtopodion caspium is one of the most common liz- ards of Iran especially in its northern range. Because no data is available on the reproduction of this species, this project was conducted to characterize its reproductive cycle by focusing on oogenic cycle in northern Iran. MATERIALS AND METHODS Study area The study locality was Sari County (36°32’N, 54°7’E), in the Mazandaran province in northern Iran, located on the southern coast of the Caspian Sea. Sari is situated inland from the Caspian Sea in the semi-tropical coastal plain to the north of the Alborz Mountains. The rainy season lasts about seven months, with an annual precipitation of more than 1,110 mm, giving the countryside a green and lush appearance. The climate of this area is wet and temperate (during this study, the mean temperatures of the coldest and warmest seasons were 1.6 °C and 22.5 °C, respectively), with the most dominant plants being grassy species belonging to the families Asteraceae and Poaceae (Assadi et al., 2005). Sampling Sampling took place periodically every 15 days during the activity period of this species from April 5 to October 20, 2011. All specimens were collected by hand, with the aid of a torch, at night time. Most of the specimens were collected from walls of old buildings and gardens. In total, 70 adult and mature females were captured (five specimens per sampling period) and sexed by the presence of preanal and femoral pores in males and their absence in females. Our observations show that females reached sexual maturity at a body length (SVL) about 42 mm, but we tried to capture specimens which had attained a larger SVL to be sure they were sexually mature adults. Some specimens were kept in terrarium in order to study the frequen- cy of clutch deposition. Methods The specimens were transferred alive to the zoology labo- ratory of Islamic Azad University, Damghan Branch and SVL (south-vent length), TL (tail length), HL (head length) were measured. Then they were anaesthetized by chloroform and anatomized. We observed oviductal eggs and vitellogenic folli- cles simultaneously in most specimens from May to June. ROD (diameter of right ovary), LOD (diameter of left ovary), ROW (weight of right ovary), LOW (weight of left ova- ry), ROV (volume of right ovary), LOV (volume of left ovary), RFN (number of right follicles), LFN (number of left follicles), MaxRF (maximum diameter of right follicles), MaxLF (maxi- mum diameter of left follicles), MinRF (minimum diameter of right follicles), MinLF (minimum diameter of left follicles), MRF (mean diameter of right follicles), MLF (mean diam- eter of left follicles), ROEL (length of right oviductal eggs), LOEL (length of left oviductal eggs), ROEW (width of right oviductal eggs), LOEW (width of left oviductal eggs), ROEWE (weight of right oviductal eggs), LOEWE (weight of left ovi- ductal eggs), ROEV (volume of right oviductal eggs), LOEV (volume of left oviductal eggs), MDFL (mean diameter of fol- licular layer), MDN (mean diameter of nucleus), MNn (mean number of nucleus) and MDn (mean diameter of nucleolus) were measured. Length, width and diameter were measured by dial caliper with an accuracy of 0.02 mm. Weight was measured by a scale with an accuracy of 0.001 g. Gonads, once removed, were inspected for metric and meristic characters. The number, weight, diameter (length and width) and volume of immature, growing and mature follicles and oviductal eggs were inspected in right and left ovaries separately. After fixing the ovaries in 10% formalin, tissues were dehydrated, cleared in Xylene, infil- trated and embedded with paraffin. Sections were made at 5-7 microns, deparaffinized, re-hydrated, stained (Hematoxylin and Eosin) and mounted. The sections were studied by light microscopy at 100× and 400× magnification. Photographs were prepared by digital camera. Data were analyzed by SPSS 17 soft- ware and one-way ANOVA to compare biometric data among monthly samples. 3The oogenic cycle of Cyrtopodion caspium RESULTS In this study, the maximum SVL and TL of females were 68.03 and 91.16 mm, respectively. The maximum HL was 17.56 mm. Body length (SVL) of the smallest mature female was 42.64 mm. The gecko C. caspium in the study area hibernates from late October to early April. Geckos emerge in ear- ly April and begin oogenesis and vitellogenesis by mid April, while mating is observed in early May. Oviposition occurs from from late May to early August. In the ter- rarium, the first egg was observed May 18, and hatching occurred after about 45-50 days post oviposition in July and August, while oogenesis and vitellogenesis stopped by mid August. According to our observations in the field and analyzing the captured animals, oviposition may occur twice a year and clutch size ranges from 1–2 eggs per clutch. The ovaries are paired and vesicular, consisting of 3-8 follicles (Figure 1A). Three types of follicles were observed: immature, growing and mature. In April, ova- ries are white and small with an irregular shape. The growing follicles were observed in late April (Figure 1B). Ovary size increased immediately in May and mature follicles were observed in early May. Ovaries are large in July and August with mature follicles present. Ovary size decreases in September and October until the following April. The follicular layer is multilayered and polymor- phic, composed of three cell types: small, intermediate and the large pyriform cells (Figure 1C). The diameter of follicular layer varies between 30-70 μm in mature and immature follicles, respectively. The mean diameter of follicular layer is 50 μm. The diameter of the nucleus var- ies between 35 to 260 μm in immature and mature fol- licles, respectively. The nucleoli are very large and distin- guishable from April to July and their numbers vary from 2 to 30 in immature and mature follicles, respectively (Figure 1D). The diameter of nucleoli varies between 25 and 70 μm. The activity of nucleoli decreases after July and they disappear in the nucleus. Mature oocytes enter the oviducts, where fertilization occurs. We observed follicullar atresia in some ovaries especially in the post- breeding period. The descriptive statistics of ovarian characters were described in Table 1 and 2. Analysis of variance of shows that weight, SVL and TL of mature females were not significantly different among groups; but ovary weight and volume and mean diameter of folli- cles were significantly different between groups (P < 0.05, Table 3 and 4). There are no significant differences in the ovarian characters between the left and right side of body (paired t-test, P > 0.05 in all cases). There is no sig- nificant difference between body length (SVL) of mature females and gonadal weight in different months. Oviductal eggs were observed between May 5 and August 5. The largest egg was observed in the left oviduct of a female with SVL = 60.62 mm in early May. Its size, weight and volume were 13.46 × 12.45 mm, 0.819 g and 0.20 mm3, respectively. The mean size and weight of ovi- ductal eggs were 10.27 × 8.46 mm and 0.462 g, respective- ly. There was at least one large oviductal egg in right or left ovary in the adult females, but they usually had two eggs, one in each oviduct (Figure 2A). There were 4 oviductal eggs in a specimen (10.32 × 12.45, 10.70 × 12.48, 5.15 × 5.40 and 5.20 × 5.80 mm) on May 20. Eggs are white and oval shaped and were deposited from late May to early August and they were laid usually in moist and warm holes of the walls of old buildings and gardens as a natu- ral incubator. The egg shells are soft but harden after 8-12 hours after deposition. In this study, the first egg (12.08 × 11.01 mm and 0.755 g) was laid on May 20 and we also observed two additional oviductal eggs (5.83 × 5.42 mm and 12.45 × 12.48 mm) in this female, after dissection. In this study, the number of laid eggs varied from 1-2 per clutch. We observed the first hatchling in 10 July. These results confirm that the incubation period ranges between 45 and 50 days, as we observed the first ovipo- sition on 20 May and the first hatchling appeared on 10 July. One of the embryos (about 35 days old and with total length = 18.10 mm) was studied in early July (Fig- ure 2B). The body length (SVL) and tail length (TL) of the smallest hatchling were 19.11 and 28.15 mm, respec- Fig. 1. Cyrtopodion caspium: A) ovary with growing follicles; B) growing oocyte; C) follicular layer and D) nucleus with nucleoli. IC = interstitial cell; LC = large pyriform cell; SC = small cell; ZP = zona pellucida. Photos by V. Hojati. 4 Vida Hojati et al. Table 1. Descriptive statistics for the examined characters in C. caspium. For abbreviations see Methods. Character N Range Minimum Maximum Mean SE SD Variance W (g) 70 4.915 2.718 7.633 4.681 0.129 1.084 1.175 SVL (mm) 70 25.390 42.640 68.030 58.500 0.474 3.964 15.716 TL (mm) 70 26.670 64.490 91.160 80.298 0.645 5.399 29.149 HL (mm) 70 9.360 8.200 17.560 11.535 0.239 2.000 4.000 ROD (mm) 70 5.990 2.130 8.120 4.32800 0.158 1.321 1.746 LOD (mm) 70 5.440 2.430 7.870 4.16186 0.131 1.098 1.205 ROW (g) 70 7.335 0.005 7.340 0.16587 0.106 0.882 0.780 LOW (g) 70 0.859 0.006 0.865 0.07200 0.020 0.172 0.030 ROV (mm3) 70 0.249 0.011 0.260 0.05516 0.005 0.042 0.002 LOV (mm3) 70 0.210 0.030 0.240 0.05286 0.005 0.040 0.002 RFN 70 5.000 3.000 8.000 5.20000 0.157 1.314 1.728 LFN 70 5.000 3.000 8.000 5.17143 0.132 1.103 1.217 MaxRF (mm) 70 3.800 1.190 4.990 2.407 0.112 0.935 0.875 MaxLF (mm) 70 3.640 1.090 4.730 2.16729 0.097 0.813 0.661 MinRF (mm) 70 1.580 0.340 1.920 0.870 0.0343 0.287 0.083 MinLF (mm) 70 1.220 0.320 1.540 0.890 0.030 0.249 0.062 MRF (mm) 70 2.110 0.830 2.940 1.50857 0.054 0.455 0.207 MLF (mm) 70 1.630 0.720 2.350 1.40586 0.040 0.338 0.114 ROEL (mm) 21 8.170 5.420 13.590 10.674 0.460 2.110 4.448 LOEL (mm) 15 7.630 5.830 13.460 10.4960 0.585 2.267 5.140 ROEW (mm) 21 6.320 5.180 11.500 8.598 0.366 1.677 2.813 LOEW (mm) 15 7.260 5.190 12.450 8.76467 0.559 2.165 4.689 ROEWE (g) 21 0.749 0.069 0.818 0.503 0.054 0.248 0.062 LOEWE (g) 15 0.742 0.077 0.819 0.486 0.067 0.261 0.068 ROEV (mm3) 21 0.130 0.070 0.200 0.133 0.008 0.038 0.001 LOEV (mm3) 15 0.140 0.080 0.220 0.136 0.012 0.0475 0.002 MDFL (μm) 70 40.00 30.00 70.00 49.823 0.7861 6.570 43.169 MDN (μm) 70 94.69 35.81 260.50 121.680 2.791 23.352 545.302 MNn 45 28.00 2.000 30.000 14.6647 0.294 1.972 3.888 MDn ( μm) 45 45.00 25.00 70.00 32.7498 1.407 9.441 89.125 Table 2. Temporal measurements for egg and oviductal characters in C. caspium. Characters April n = 10 May n = 10 June n = 10 July n = 10 August n = 10 September n = 10 October n = 10 Mean diameter of ovary 3.67 4.78 4.42 4.91 3.78 3.84 3.83 Mean diameter of nucleus 152.45 170.46 178.34 118.68 110.52 103.66 101.71 Nucleolus diameter 25-70 25-60 30-60 40-50 – – – Follicles numbers 4-8 4-8 3-7 3-7 4-6 3-6 4-6 Mean diameter of follicles 1.02 1.50 1.74 1.85 1.41 1.59 1.43 Mean size of oviductal eggs – 12.93×10.83 9.08×7.42 9.71×7.64 9.37×7.97 – – Mean weight of oviductal eggs – 0.801 0.331 0.454 0.262 – – 5The oogenic cycle of Cyrtopodion caspium Table 3. ANOVA of ovarian macroscopic characters in C. caspium. For abbreviations see Methods. Character Variance Sum of Squares df Mean Square F P W Between Groups 20.945 13 1.611 1.500 0.147 Within Groups 60.161 56 1.074 Total 81.106 69 SVL Between Groups 149.274 13 11.483 0.688 0.767 Within Groups 935.105 56 16.698 Total 1084.379 69 TL Between Groups 306.479 13 23.575 0.774 0.683 Within Groups 1704.824 56 30.443 Total 2011.303 69 HL Between Groups 12.612 13 0.970 0.930 0.529 Within Groups 58.415 56 1.043 Total 71.028 69 ROD Between Groups 30.161 13 2.320 1.439 0.001 Within Groups 90.296 56 1.612 Total 120.458 69 LOD Between Groups 18.645 13 1.434 1.245 0.004 Within Groups 64.522 56 1.152 Total 83.167 69 ROW Between Groups 14.790 13 1.138 1.634 0.003 Within Groups 38.997 56 0.696 Total 53.787 69 LOW Between Groups 1.092 13 0.084 4.929 0.000* Within Groups 0.954 56 0.017 Total 2.047 69 ROV Between Groups 0.070 13 0.005 5.891 < 0.001 Within Groups 0.051 56 0.001 Total 0.120 69 LOV Between Groups 0.065 13 0.005 6.300 < 0.001 Within Groups 0.045 56 0.001 Total 0.110 69 Fig. 2. Cyrtopodion caspium: A) oviductal eggs and mature follicles and B) 35 days old embryo. Photos by V. Hojati. 6 Vida Hojati et al. tively. The juveniles were commonly observed in July and August. The mean SVL and TL of hatchlings were 22.38 and 30.20 mm, respectively. The maximum activity of oogenesis occurs in May. DISCUSSION Since oogenesis occurred from April through August, the pattern followed an associated reproductive cycle typical of species from temperate areas. The results of our study on the oogenic cycle of C. caspium are reported for the first time from Iran and west southern Asia. The largest female body length in this study was 68.03 mm, whereas in previous studies, it has been reported to be 59.80 mm, in northern Afghanistan (Anderson, 1999). A female collected from northern Afghanistan in mid-April had an oviductal egg about 5 mm long, whereas in the present study we observed oviductal eggs only in early May. This difference is probably due to the different cli- mates of the two regions. All oocytes grow but only some of them enter vitellogenesis and reach the maximum size to later become shelled eggs. The weight, diameter and volume of the ovaries and oocytes increase between May and July and the females are heavier in mid-and- late spring due to the presence of eggs. Clutch size was similar to other gekkonid lizards, such as Cyrtopodion scabrum (Anderson, 1999). The smallest juvenile collect- ed in this present study had a SVL = 19.11 mm in early August, whereas Anderson (1999) reported the smallest juvenile with a SVL = 20.7 mm in late August. Although oocyte growth was accompanied by chang- es in the granulosa layer, zona radiata and thecal layers, the zona pellucida remained unchanged throughout. Based on work on the gecko Hemidactylus mabouia, the small cells of the granulosa layer are differentiated into three distinct cell types: small, intermediate and the large pyriform cells (Moodley and Van Wyk, 2007). Similar to most other squamates, the intermediate and pyriform cells at the onset of vitellogenesis regressed to a single cuboidal epithelium. Following ovulation, the granu- losa layer hypertrophied forming laical tissue (Moodley and Van Wyk, 2007). The theca layer differentiated into two layers and septal invasion of the corpus luteum took Table 4. ANOVA of ovarian microscopic characters in C. caspium. For abbreviations see Methods. Character Variance Sum of Squares df Mean Square F P RFN Between Groups 43.200 13 3.323 2.449 0.010 Within Groups 76.000 56 1.357 Total 119.200 69 LFN Between Groups 29.543 13 2.273 2.339 0.014 Within Groups 54.400 56 0.971 Total 83.943 69 MRF Between Groups 4.582 13 0.352 2.033 < 0.001 Within Groups 9.709 56 0.173 Total 14.291 69 MLF Between Groups 4.299 13 0.331 5.164 < 0.001 Within Groups 3.587 56 0.064 Total 7.886 69 MDFL Between Groups 1702.264 13 130.943 5.745 < 0.001 Within Groups 1276.426 56 22.793 Total 2978.690 69 MDN Between Groups 25266.383 13 1943.568 8.806 < 0.001 Within Groups 12359.438 56 220.704 Total 37625.821 69 MNn Between Groups 146.298 8 18.287 26.575 < 0.001 Within Groups 24.773 36 0.688 Total 171.071 44 MDn Between Groups 2984.285 8 373.036 14.329 < 0.001 Within Groups 937.228 36 26.034 Total 3921.513 44 7The oogenic cycle of Cyrtopodion caspium place. At the time of oviposition, corpora lutea regressed to form ovarian scars (corpora albicantia). Follicular atre- sia occurred in previtellogenic follicles (hydration stage) and seldom in vitellogenic follicles. The highest incidence of atresia occurred in the post-breeding period (Moodley and von Wyk, 2007) while atretic follicles were replaced by new growing follicles recruited into the follicular size hierarchy (Jones et al., 1978). The gross morphology, oogenesis and folliculogenesis of the ovaries of C. caspium correspond to the general squa- mate pattern described for oviparous reptiles (Moodley and Van Wyk, 2007). The ovarian stroma was characterized by follicles in different stages of development, and may contain corpora lutea, and corpora atretica. The follicular epitheli- um of the lizard oocytes undergoes structural and morpho- logical modifications throughout oocyte growth. During this process the number of follicle cells increases and the epithelium acquires a multilayered and polymorphic organ- ization which is characterized by the appearance of large follicle cells including intermediate and pyriform cells. The number of large cells also increases during oocyte growth and this increase parallels that of small cells. However, another study indicates that large follicle cells arise from the differentiation of small cells (Filosa et al., 1979). In C. caspium, there was a germinal bed (GB) in each ovary. The germinal bed containing oogonia, oocytes, and primordial follicles, was most active during the vitello- genic period. In this species, the number of laid eggs var- ied from 1-2 per clutch. Another advantage of geckos for comparative analysis of egg shape is that female geckos of all species lay invariant clutches of one or two eggs (Shine and Greer, 1991). Consequently, female geckos have no more than one developing egg in a single ovary at a time. The egg shells harden soon after deposition, thus they undoubtedly offer a better protection against inver- tebrate predators and desiccating environmental condi- tions than a soft shell. In squamate reptiles, the generally flexible and poorly mineralized parchment-like egg shell is clearly plesiomorphic, with hard shells occurring only in the gecko family Gekkonidae (Andrews, 2004). Four gekkotan families (Carphodactylidae, Diplodactylidae, Eublepharidae and Pygopodidae) lay soft-shelled eggs, while their close relatives (Gekkonidae) lay hard-shelled eggs (Doughty, 1997). This highly mineralized egg shell is much more costly to produce, as calcium is a limiting element for most terrestrial organisms. However, within gekkonids, small species lay more elongated eggs than larger species (Kratochvil and Frynta, 2005). In this study, we report more than one egg clutch per year. 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