699 - 710 - IAS

J. Biosci., Vol. 21, Number 5, September 1996, pp 699–710. © Printed in India.

Age related changes in ovarian follicular kinetics in the Indian skipper frog Rana cyanophlyctis (Schn.) J T KULKARNI and K PANCHARATNA* Department of Zoology, Karnatak University, Dharwad 580003, India MS received 1 February 1996; revised 1 July 1996 Abstract. Changes in ovarian follicular kinetics were studied in relation to aging in the Indian skipper frog Rana cyanophlyctis. Age was determined by skeletochronology, by counting the number of growth rings and lines of arrest of growth from the cross sections of 4th phalange of 4th toe. For follicular kinetics study oocytes were counted under binocular using 10% of Bouin’s fixed ovary and they were classified into first growth phase, medium-sized second growth phase, large-sized second growth phase and atretic follicles. Analysis of phalangeal cross sections indicated that frogs ranging 14–54 g in body weight and 4·9–8·9 cm in body size showed 1–7 year rings. Frogs that weighed 14–16 g showed 1 year ring, and contained immature ovaries; those with 18 g body weight had one to two year rings, in which second growth phase oocytes appeared for the first time in the primiparous ovary. Frogs with 20–54 g body weight showed 2–5 year rings in which ovary contained 5–24% of second growth phase oocytes. Further, body weight, body size, ovarian weight, number and size of second growth phase oocytes and total number of oocytes showed a significant (P < 0·05) positive correlation, while, the number of first growth phase and atretic follicles showed a poor correlation with age. The results suggest that in nature, the age of Rana cyanophlyctis ranges between 1–7 years. Phalangeal growth rings are formed annually. Females attain sexual maturity in 2nd year. Frogs with 2–5 years of age may constitute breeding females. Body weight, body size, ovarian mass, number of second growth phase and total oocytes, and egg size increase with age up to 5 years. Keywords. Aging; ovarian kinetics; frog.

1. Introduction Determination of age of amphibians by skeletochronology method has been reported by many workers (Mina 1974; Hemelaar and Vangelder 1980; Hemelaar 1981, 1983, 1988; Bastien and Leclair 1992; Cherry and Francillon-Vieillot 1992). It is known that analysis of cross sections of phalanges particularly of hind limb show concentric rings called growth rings each corresponding to one year of growth, therefore called as year rings. Each year ring is known to consist a growth ring and a dark line that limits the growth of growth ring the line of arrest of growth (LAG) (Hemelaar 1981). There are skeletochronological studies aimed to determine (i) the age of amphibians (Hemelaar and Vangelder 1980; Hemelaar 1981, 1983; Bastien and Leclair 1992), (ii) relationship between age, body growth/size and attainment of sexual maturity or breeding behaviour (Mina 1974; Smirina 1983; Leclair and Castanet 1987; Ryser 1988; Tejedo 1991; Cherry and Francillon-Vieillot 1992) and (iii) age and longevity (FrancillonVieillot et al 1990). So far no study has been made to investigate the relationship between aging and progression of follicular kinetics in the ovary of amphibians. In most *Corresponding author (Fax, 0836 347884).

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of anurans egg laying is an annual event, it may be of relevance to study age related changes if any, in the ovarian function in terms of production of eggs. Therefore, the present work was initiated to study the effect of aging (age was determined by skeletochronological method) on body size, body mass, ovarian mass, rate of recruitment, growth and degeneration of oocytes in the Indian skipper frog Rana cyanophlyctis. Since, fat bodies are known to be involved in egg production in this species (Saidapur 1989) effect of aging on these organs were also studied. To the best of our knowledge this is the first paper to report determination of age and age related changes in the ovarian follicular kinetics in a tropical anuran. 2. Materials and methods Fifty-four female frogs (R. cyanophlyctis) weighing 14 to 60 g were collected from surrounding areas of Dharwad in August 1994 (breeding season). Soon after their arrival to the laboratory frogs were divided into 6 groups. Group I Group II Group III Group IV Group V Group VI

: : : : : :

Frogs weighing 14–16 g Frogs weighing 18–19 g Frogs weighing 20–29 g Frogs weighing 30–39 g Frogs weighing 40–49 g Frogs weighing 50–60 g

All the frogs were autopsied on the day of arrival. At autopsy, the body weight (g), body length (snout-vent length in cm), ovary weight (mg) and fat body weight (mg) were recorded. Both the ovaries were excised and fixed in Bouin’s fluid. At the same time the 4th toe (largest) of the right hind limb of each animal was excised and fixed in 10% formalin and preserved in 90% alcohol. The digits were washed in water for 1–2 h, and decalcified in 5% nitric acid. They were then washed overnight with water to remove all the traces of formalin and nitric acid and preserved in 70% alcohol until they were processed further for regular histology (Cherry and FrancillonVieillot 1992). Transverse sections (8 to 10µm thickness) of 4th phalange of the 4th toe of each frog were cut and stained with Harri’s hematoxylin. Growth rings and LAGs were clearly visible in the cross sections. The sections were examined under a compound microscope. Year rings were counted starting from the bone marrow cavity to the outer marginal year ring. Growth rings were light purple in colour and the LAG’s were dark purple in colour in a hematoxylin stained section. For the follicular kinetics study a piece of ovary from middle region weighing 10% of total Bouin’s fixed ovary from each animal was used. The ovarian analysis was done under a dissecting binocular microscope. The oocytes were classified into first growth phase (FGP) (previtellogenic oocytes without any pigmentation), medium sized second growth phase (MSGP) (oocytes showing early signs of pigmentation but without any demarcation between animal and vegetal poles), large sized second growth phase (LSGP) (with a clear demarcation between animal and vegetal poles) and atretic follicles (AF) (previtellogenic AF appear transparent, while yolky AF appear flabby with pigment scars), as described earlier (Pancharatna and Saidapur 1985).

Aging and follicular kinetics in frog

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2.1 Statistical analysis The data were analysed using analysis of variance (ANOVA) followed by studentized range test. The results were judged significant at 5% level of significance (Steel and Torrie 1980). The relationship between age and body mass, age and body length, age and ovarian mass, age and fat body mass age and number of FGP, SGP, AF and largest oocyte diameter were assessed by drawing scatter plots and regression analysis method (Steel and Torrie 1980). The correlation coefficient ‘r’ was calculated in each case by Karl Pearson’s method and significance was tested by ‘t’ test (Steel and Torrie 1980). 3. Results 3.1 Group I Frogs of this group were sexually immature and contained ovaries which were transparent and weighed 116 ± 19 mg (table 1). FGP oocytes formed 87% and remaining 13% of oocytes were atretic (table 2). Oocytes at SGP were completely absent (table 2). The mean largest oocyte diameter was 357 ± 68 ,μm (table 1). Total number of oocytes were 12770 ± 1019 (table 2). Fat bodies weighed 376 ± 110 mg (table 1). Sections of the phalanges of these frogs showed a single growth ring (figure 1A). 3.2 Group II This group consisted frogs at subadult stage with body weight 18–19 g and body length 5·18 ± 0·13 cm (table 1). Ovaries weighed 288 ± 75 mg on an average and SGP oocytes Table 2. Age related changes in the number of different types of oocytes in R. cyanophlyctis.

Values are mean ± SE. * F value significant at 5% level of significance. Significant compared to agroup I; bgroup II; cgroup III; dgroup IV; egroup V. Numbers in parentheses indicate number of frogs in each group.


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(MSGP) appeared for the first time in the ovary that formed 1·3% of total oocytes. Eighty five per cent of oocytes were at FGP and 13·70% of oocytes were atretic (table 2). The mean diameter of largest oocytes was 916 ± 144 μm (table 1), Fat body weights were lower (table 1). Cross sections of the phalanges showed one or two year rings with growth rings and LAGs (figure 1 B). 3.3 Group III Frogs of this group were adults with 20–29 g body weight (table 1). Ovaries contained SGP oocytes that formed 5·52% of total oocytes. FGP formed 71·53% and AF 22·92% (table 2). The mean diameter of largest oocytes was higher (1252 ± 29 μm) compared to group II frogs. Fat bodies formed 323 ± 123 mg on an average (table 1). Two or three growth rings were observed in the cross section of phalanges (figure 1C). 3.4 Group IV Frogs of this group had ovaries that weighed 2172 ± 614 mg with 8% of SGP, 68 % of FGP and 24% of AF (tables l and 2). The mean diameter of largest oocytes was 1310 ± 15 μm. Fat body weights were comparable to group III frogs (table 1).

Figure 1. Phalangeal cross sections of 4th toe of the right hind limb of female R. cyanophlyctis (× 50 μm). (A) 16 g body weight. Note a single ring. (B) 22 g body mass. Note two growth rings and two LAGs (arrow). (C) 30 g body weight. Note 3 growth rings and 3 LAGs (arrows). (D) 32g body weight. Note 7 LAGs (arrows).

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J T Kulkarni and K Pancharatna Table 3. Age frequencies (%) of the frogs (R. cyanophlyctis) from wild.

Phalanges contained 3 or 4 rings. In one of the frog with body weight 32 g there were 7 rings (figure ID). 3.5 Group V In these frogs ovaries weighed 5305 ± 838 mg and contained 18% of SGP, 57% of FGP and 25% of AF (tables 1 and 2). The mean diameter of largest oocytes was 1343 ± 21 μm (table 1). Fat body weights were higher (table 1) and four or five rings were observed in cross sections of phalanges (table 2). 3.6 Group VI Frogs of this group weighed 52 ± 2 g with body size of 8·15 ± 0·11 cm (table 1). Ovaries weighed 8143 ± 769 mg and contained 24% of SGP, 54% of FGP and 22% of AF (table 2). Oocytes reached their maximum size of 1357 + 14 μm. Fat body weights were slightly reduced compared to group V frogs (table 1). Phalanges contained 4 or 5 rings (table 2). 3.7 Relationship between age vs body mass, body size, ovarian mass, number of different types of oocytes, egg size and fat body mass in R. cyanophlyctis The scatter plot drawn for age vs body mass indicates that there is a positive correlation (r = 0·65, P < 0·05, n = 54) between the two (figure 2). Likewise, there is a significant positive correlation between age and body size (r = 0·57, P < 0·05) and age and ovarian mass (r = 0·64, P < 0·05, n = 36) (figure 2). Figure 3 shows that there is a poor correlation (r = – 0·02) between age and number of FGP oocytes in the ovary, while, the number of SGP oocytes showed a high positive correlation (r = 0.81, P < 0·05) with the age (figure 3). The number of AF seems to be poorly influenced by age (r = 0·40). Scatter plot drawn for age vs egg size indicates a significant positive correlation between the two (r = 0·63, P < 0·05) (figure 4). Total number of oocytes also showed a positive correlation with the age (r = 0·58, P < 0·05). Fat body weights showed a low correlation (r = 0·22) with the age (figure 4). 4. Discussion The production and growth of ovarian follicles in amphibians is known to be influenced by various factors such as hormones, environmental cues, nutrition and


Figure 2. Relationship between age vs body mass, age vs body size and age vs ovarian mass in R. cyanophlyctis.

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Figure 3. Relationship between age vs number of different types of oocytes and age vs atretic follicles in R. cyanophlyctis.


Figure 4. Relationship between age vs largest oocyte diameter, age vs total number of oocytes and age vs fat body mass in R. cyanophlyctis.

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intraovarian regulators etc. (Lofts 1974, 1984; Saidapur 1989). Of these, the role of hormones, environmental temperature and nutrition have been well established in the follicular development (Jφrgensen et al 1978; Lofts 1984; Saidapur 1989). Whether the advancement of the age has any effect on the rate of production, growth and degeneration of oocytes in the ovary is not known for any amphibian species due to the paucity of studies. Although, there have been attempts made to understand the relationship between age and onset of sexual maturity, age and breeding behaviour (Mina 1974; Smirina 1983; Leclair and Castanet 1987; Ryser 1988; Tejedo 1991; Cherry and Francillion Vieillot 1992), the influence of aging on ovarian follicular kinetics is not known. The present study is an attempt to understand the age related changes in the ovarian follicular dynamics in a tropical anuran R. cyanophlyctis in which oogenetic activity is continuous in adult females and all the types of oocytes are present throughout the year although quantitative differences occurs in their number, during different months (Pancharatna and Saidapur 1985). For the determination of age, skeletochronology was used. The method used was basically similar to that of Cherry and Francillon–Vieillot (1992) described for Bufo pardalis. However, a slight change was introduced i·e., after the decalcification the toes were processed for regular histology by embedding in paraffin wax and the sections were taken on an ordinary microtome instead of using a cold microtome. The analysis of phalangeal cross sections of R. cyanophlyctis showed growth rings and LAGs (figure 1) as reported earlier for Rana temporaria (Mina 1974; Ryser 1988). Bufo bufo (Hemelaar and Vangelder 1980; Hemelaar 1981, 1983, 1988; Smirina 1983), Triturus cristatus and Triturus marmoratus (Francillon–Vieillot et al 1990), Bufo calamita (Tejedo 1991), B. pardalis (Cherry and Francillon–Vieillot 1992) Rana sylvatica (Bastien and Leclair 1992). Further, in R. cyanophlyctis each year ring was characterized by a relatively broader light purple zone with osteocytes, the growth ring and a limiting line the LAG that stained dark blue with hematoxylin (figure 1). The number of year rings varied from 1–7 in different sized frogs collected from wild (table 1 and figure 1). The frogs at the completion of one year with 14–16g body weight had a ring indicating the year rings may be produced annually as reported earlier for other anurans (Hemelaar 1981, 1983; Ryser 1988; Cherry and Francillon–Vieillot 1992; Tejedo 1991). The frogs at subadult stage (with 18–19 g body weight in which SGP oocytes appeared for the first time in the primiparous ovary) had one completed ring and 2nd was in the process of formation indicating that vitellogenic oocytes are produced in second year of age. This suggests indirectly that in this species, frogs may attain ovarian maturity in the second year. However, whether these frogs are capable of breeding in the same breeding season or not is not known. In B. bufo female toads attain maturity in nature in 3rd year or at an older age, however under experimental conditions in the laboratory, juvenile toads attained maturity in the second year itself (Gibbons and McCarthy 1984; Hemelaar 1988; Jφrgensen 1988). Age frequency study of 54 frogs indicates that frogs with 2–4 rings formed about 78% of population (table 3). These frogs were adults (20–40 g body weight) with 5–18% of SGP oocytes in the ovary (table 2). Frogs with more than 4 rings also had a high percentage (28%) of SGP oocytes. Generally R. cyanophlyctis ranging 25–40 g in body weight, readily respond for artificial induction of ovulation and spawning in the laboratory in breeding season. Therefore, it is possible that frogs with 2–5 years of age may constitute breeding females. Earlier studies on other anurans such as R. cascadae (Briggs and Storm 1970), Bufo pentoni (Francillon–Vieillot et al 1984), R. temporaria


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(Gibbons and McCarthy 1984), B. americanus (Acker et al 1986), Hyla crucifer (Lykens and Forester 1987), Rana iberica (reference in Cherry and Francillon–Vieillot 1992) indicate that generally the age of female anurans at breeding varies from 2–5 years. The percentage of FGP oocytes decreased in ovary with a corresponding increase in SGP number as the age advances. In R. cyanophlyctis body size showed a significant positive correlation with advancement of age as reported in Rana erythraea (Brown and Alcala 1970), B. bufo (Hemelaar 1983), B. pentoni (Francillon–Vieillot et al 1984), H. crucifer (Lykens and Forester 1987), B. calamita (Tejedo 1991). However, body size and age were not correlated in female B. pardalis (Cherry and Francillon–Vieillot 1992). In the present study body weight and ovarian weight also correlated with the age. Among the different types of oocytes, SGP oocyte number and largest oocyte diameter showed a higher positive correlation with age, indicating the SGP oocyte production and growth rate increases with age indicating older frogs may lay larger eggs as reported earlier for female B. calamita in which number of eggs and egg size correlated with body length, body weight and age (Tejedo 1991). AF showed a poor correlation indicating their number may be influenced by factors other than age (figure 3). Atretic follicles were observed in the ovaries of R. cyanophlyctis throughout the year and their number was particularly influenced by hormones (gonadotrophins), nutrition and captivity stress etc. (Saidapur 1989; Pancharatna and Saidapur 1992). The present study indicates the number of AF is not influenced by age. Likewise, number of FGP oocytes and fat body weights not seems to be affected by age (figure 4). In conclusion, the results of the present study clearly suggest in the R. cyanophlyctis growth rings are formed annually or age can be determined by assessing phalangeal cross sections. In nature the frogs range 1–7 years in age. A female frog attains sexual maturity in the 2nd year but majority of breeding females range 2–5 years in their age. Body weight, body size, ovarian weight, number of SGP and egg sizes increase with age from 1–5 years.

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