CHAPTER 5 FEMALE REPRODUCTIVE CYCLE INTRODUCTION The seasonal variation in the ovarian development of lower vertebrates has been reviewed (Lofts, 1974: Rowlands and Weir, 1977; Jones, 1978; Jorgensen et al., 1979). Ovarian cycle of several anurans of temperate zones has been studied (Jorgensen, 1973,1981, 1984 a, 1984 b; 1984 c; Rastogi et al., 1979; Sklavounon et al., 1990). However, studies on the ovarian cycle of tropical species are relatively few (Saidapur and Nadkami, 1974; Promoda and Saidapur, 1984 a, b; Pancharatna and Saidapur, 1985; Jorgensen et al., 1986; Kanamadi and and Jirankali, 1991 a). Though the female reproductive cycle of the tropical toad Bufo melanosrictus has been described by a few workers (Kanamadi and Saidapur, 1982; Kana~nadi and Yamakanmardi, 1988; Kanamadi et al., 1989), no work has been done on the pattern of ovarian activity of the toad in Kerala. Hence, the present work was undertaken to study in detail the female reproductive cycle of B. n~elanostictus inhabiting the Kuttanad region of Kerala.
MATERIALS AND METHODS Adult females were collected monthly from Kuttanad, a natural wetland in Central Kerala from January 1991 to December 1992. Ten animals were used every month for the histological study of seasonal changes in the ovary. They were anaesthetised by using ether and weighed. At autopsy, weight of ovary, oviduct and fat bodies was recorded. Representative pieces of ovaries were fixed in Bouin's fluid, embedded in paraffin-wax, sectioned at 6 Fm thickness and stained with Haematoxylin and Eosin. Gonadosomatic Index was detennined. Diameter of the largest oocyte was determined by measuring the diameter of twenty oocytes per section of each toad. The seasonal changes in ovarian mass were determined by the analysis of variance (Gomez and Gomez, 1976) in order to find out the effect of months and influence of body weight on the ovarian mass. The differences were judged as significant at P<0.05. RESULTS The ovaries of adult toads are paired organs attached to the median surface of kidneys by mesovarium. Each ovary is a hollow sac-like structure, the wall of which is thrown into many folds and consists of a cortical region covered by surface or germinal epithelium. The ovarian stroma is scarce. The oocytes are surrounded by a single layer of flattened granulosa cells. The
follicular epithelium is a single layer throughout the period of maturation unlike the ~nulticellular granulosa tissue found in the higher vertebrates. Body weight The data on the body weight of female toads revealed that there was a seasonal variation in the body weight. The weight of adult toad was higher in October (112.50) and August (103.75) and was lowest in November (26.0; Table 13). Anova table suggests that the seasonal variation in the body weight of females is directly correlated with the ovary weight (Pi0.01). Gonadosomatic Index Gonadosomatic Index (GSI) was found to be fluctuating from January to December. However, it showed an increase from May and the highest value was recorded in August (10.61). In November and December, GSI values exhibited a decrease while they again increased in January and February (Table 13;Fig. 5). They showed reduction in March and April and the lowest value was noted in November (0.32). Ovarian cycle Ovary contains mature and immature ova throughout the year and oogenesis is of continuous type (Plate 11). The collection included individuals
PLATE 2 Photomicrographs of sections of ovary of B. melanostictus (X 100) Sections of ovary of B. melanostictus showing small primary oocytes (Fig. A), FGP oocytes (Figs. B & C) and large yolky oocytes (Figs. D, E and F, x 100).
PLATE - 2
in different stages of ovarian cycle. Higher ovarian weight was recorded in October (14648.75) and August (1 1862.50; Table 13). Table 13. Seasonal variation in the ovarian avctivity of toad B. melanostictus. I hlonth 1 Body wt. I SVL I Fat body wt. / O\ar? nt. / Oviduct nt. 1 CSI 1 Sep 65.0-i-13.39 7.04f 1.20 127515.68 4416.8k33.76 1447.35k63.28 621k 1.33 Oct 1125k12 8.82i1.16 11.385.19 14648.8k100.6 2258.854.85 9.89e.82 Nov 26.0-i-6.77 5.85i0.57 17.0i9.02 56.50k10.2 1 19.0i8.42 0.32S.03 Dec 27.5k1.44 6.05i0.29 28.29i11.54 59.2524.17 l0.5oil.5 0.40i0.09 VR 19.17** 4.58** / 1.74 " 88.7** 34.25** 2.30' (SVL- Snout Vent length) (** Signifies P<0.01) (*Signifies P<0.05) (NS -Not significant) These fluctuations were found to be significant. The ovarian weight was low in November (56.50) and December (59.25). It showed fluctuations from January to May. Then it increased drastically in August and showed a decrease in
September and again increased steeply reaching its maximum in October (Table 13). Oogonia were found in germinal epithelium throughout the year. But they were abundant in ovaries after the spawning. The oogonium divided mitotically and increased in number and later organised into follicular layers. The oocyte then entered previtellogenic primary growth phase. During this phase their size increased and yolk nucleus became visible at the justanuclear position. During the secondary growth of the primary oocyte, the accumulation of yolk was noted. Yolk vesicles were seen at the beginning of yolk deposition. The accumulation of yolk started from periphery and progressed towards the centre. After completion of vitellogenesis the follicles underwent further growth. Some SGP oocytes were found throughout the year but were recruited in large numbers for vitellogenic growth only from March onwards. The diameter of the largest oocyte showed seasonal variation (Fig 6). The diameter of oocytes increased from May and the maximum diameter was noted in October (683.75). The minimum diameter was recorded in the month of November ( I 10.39). The magnitude of seasonal changes in the ovarian mass are directly related to the number of SGP and the pattern of oogenetic activity. It is possible that toads breed only once in the season and almost all the preovulatory follicles
Fig. 5. Seasonal variation in the fat body weight, ovary weight, oviduct and CSI of B. melanostictus +Fatbody +Ovary +Oviduct +GSI wt. irng.) wt. (rngi wt. (mgi 0.1 I Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec Fig.6. Seasonal variation in the diameter of largest oocyte of B. melanostictus
are shed at one time and rarely very few are retained. The ovary weight decreased and the diameter of the largest oocyte declined significantly following the breeding. Based on the seasonal changes in the GSI, diameter of the largest oocytes and gross histological changes in the ovary, the ovarian cycle of B. rnelar~ostictus can be arbitrarily divided into the following phases: 1. Pre-breeding or preparatory phase (January to March). The ovary during this period contained large pre-ovulatory follicles. The number and size of oocytes increased during March-April and this was followed by vitellogenesis which was completed by the end of May. 2. Breeding phase (April to October). During this period the ovaries were gravid and capable of spawning. The weight of ovaries increased drastically and the maximum weight was attained in October. Some ovaries contained the remains of discharged follicles and a large number of atretic follicles in various stages. The onset of monsoon rains in MayIJune acts as a trigger for breeding of toads. Majority of toads breed only once in the season as it was observed that the average ovarian weight and the diameter of the largest oocytes in many individuals declined significantly following the breeding. 3. Post breeding phase (November and December). The period between November and December may be considered as post breeding period. During
this period there was a marked decrease in the relative ovarian weight and the diameter of largest oocyte. Also the ovary contained oogonia, previtellogenic follicles and follicles undergoing vitellogenesis. Oviduct cycle The oviduct showed marked seasonal changes in correlation with ovarian weight (Fig 5). The oviduct weight was found to be fluctuating from January to July. It showed marked increase from August to October and the maximum value was recorded in October (2258.8). In November and December it steepily decreased and the lowest value was noted in December (10.50). This variation has been found to be statistically significant (P<O.Ol; Table 13). Fat body cycle Fat body changes were not very significant. The maximum fat body weight was recorded in August (435.75) and lowest weight noticed in October (11.38; Table13). Anova test indicates that the seasonal variation in the fat body weight is not significant but direct correlation between fat body weight and ovarian mass is found in June, July and August (Fig 5). Correlation studies between various factors of ovarian activity in Bufo rnelanostictlrs revealed that body mass vs snout-vent length, ovary weight,
oviduct weight, GSI; snout-vent length vs fat body weight, ovary weight, oviduct weight, GSI; ovary weight vs oviduct weight, GSI and oviduct weight vs GSI are found to be statistically significant. However; body mass vs fat body weight, fatbody weight vs ovary weight, oviduct weight and GSI have not shown significant correlation statistically in the present study (Table 14). Table 14. Correlation coefficient (r) between body weight / ovary weight and various factors of ovarian activity of B. melanostictus Ovary weight vs Oviduct weight Ovary weight vs GSI Oviduct weight vs GSI 0.93 0.79 0.79 Sibmificant correlation
DISCUSSION According to Gallien (1959), the seasonal cycle of reproduction in female amphibians generally shows three phases: 1) breeding phase, 2) ovarian regression and 3) ovarian growth and vitellogenesis. This is generally true of all species inhabiting temperate regions where clear-cut seasonal changes are encountered due to the variations in the environmental temperature. But in the tropical species exposed to environmental fluctuations that are not marked, gametogenesis is the continuous or potentially continuous type. However, they also have an annual reproductive cycle (Lakshrnan, 1965; Sarkar and Rao, 1968; Lofts and Bern, 1972; Saidapur and Nadkami, 1974; Kanamadi and Saidapur. 1982; Promoda and Saidapur, 1984 a, b; Saidapur, 1989). Bzrfo melarzostictus is a tropical species and it is difficult to differentiate the ovarian changes with the season as explained by Gallien (1959) because oogenesis in this toad is a continuous process. In earlier studies it has been concluded that oogenesis occurs continuously year round and the toad may breed twice a year. But studies conducted recently at Bangalore (Jorgensen et al., 1986) and Dhanvad (Kanainadi and Yamakanamaradi, 1988) areas suggest that the ovarian cycles in individual toads are characterised by synchronous growth of the vitellogenetic oocytes and by asynchrony with respect to stage in the ovarian cycle anlong toads. Similar results were obtained when the ovarian cycle of the
laboratory maintained toad was studied (Kanamadi et al., 1989). The results of the present study on the toad inhabiting the Kuttanad region of Kerala provide evidence for the above conclusion. In tropical and subtropical zones such as in Southern India where seasonal changes in ambient temperature are not drastic, the anurans exhibit three different kinds of gametogenetic activity (Saidapur, 1989). They are (1) continuous at the individual level as in Rana cyanoph!~ctis (Pancharatna and Saidapur, 1985), (2) discontinuous (but potentially continuous) as in Rana tigerirza (Promoda and Saidapur, 1984) and (3) continuous at the population level but discontinuous at the individual level as in 5. melanostictus (Jorgensen et al., 1986). In species belonging to the third category, study of annual changes in the ovaries of specimens, caught from wild often lead to erroneous conclusions as the collection include the individuals in different stages of reproductive cycle (Kanamadi et a/., 1989). It is evident from the present study that in 5. melanostictus the oogenesis is of continuous type although breeding seems to be an annual event coinciding with the monsoon rains. Similar observations were made by Kanamadi and Saidapur (1982) and Jorgensen et al. (1986) while studying the pattern of ovarian activity in B. melanostictus from Southern India. Based on the seasonal
changes in the GSI and diameter of the largest oocyte and gross histological changes in the ovary, the ovarian cycle in the toad inhabiting Kerala can be broadly divided into pre-breeding period, breeding period and post breeding period. The ovarian cycle in amphibians is regulated by both intrinsic and extrinsic factors. The intrinsic factors include hypophyseal gonadotrophins and ovarian oestrogen. The extrinsic factors are temperature, light, rainfall and relative humidity which serve as the environmental synchronisers of gametogenetic and breeding activities (Saidapur, 1989). Studies on environmental control on amphibian reproduction have revealed that temperature plays a primary role in stimulating gametogenesis (Pancharatna and Patil, 1997) and also high temperature was found to stimulate vitellogenic growth of oocytes. Environmental temperature plays a key role in regulating gonadal cycle in temperate amphibians by affecting the sensitivity of germinal epithelium as well as the gonadotropin content of the pituitary glands (Lofts, 1974, 1984). The role of temperature has been studied in three tropical species such as Rana tigerina (Pancharatna and Saidapur, 1990), Pol~pedatus maculatus (Kanamadi and Jirankali, 1993) and R. cyanoplzlyctis (Pancharatna and Kulkami, 1993, Kulkami and Pancharatna, 1994). In some species rainfall regulates the timing of breeding and reproductive behaviour (Lofts, 1984; Whinier and Crews, 1987;
Saidapur, 1989). The role of photoperiod in the control of amphibians is inconclusive due to limited number of studies available (Whittier and Crews. 1987). In the present study, toads in different stages of ovarian cycle are found for most part of the year. It suggests that individual toads attain sexual maturity at different times, leading to an extended breeding phase within the population of toads as noted by all the previous investigators. Even though rainfall has no effect on the gametogenesis, it seems to trigger breeding activity in Bufo melanostictus. The breeding activity includes migration to breeding grounds, chorusing, mating and oviposition by the breeders. Toads are reported to breed during monsoon months i.e.. June-August in Dhanvad (Kanamadi and Saidapur, 1982 b) and August-October in Bangalore (Jorgensen et al., 1986). In B. melanostictus maintained in the laboratory, completion of ovarian cycle requires nine to ten months and the individual toads breed only once a year (Kanamadi et al., 1989). The rainfall seems to trigger breeding activity in B. rnelanostictus as rainfall extends from MayJJune to December in the study area. However. rainfall may not have any effect on gametogenesis. Among Indian anurans, annual changes in the oviduct have been studied in R. tigerina (Promoda and Saidapur, 1984 b), R. cyanophlyctis (Pancharatna
and Saidapur, 1985) and Rana hexadactyla (George and Andrews, 1993). In these species the oviduct undergoes marked seasonal changes that are correlated with the ovarian cycle. This suggests that the seasonal variation in oviduct weight is related to the number of SGP oocytes, the primary determinant of ovarian weight (Saidapur, 1989). Further the annual variation in the oviduct weight is maximum in R. tigerina due to the distinct changes in ovarian mass, whereas in R. cyanophlyctis and R. hexadactyla these changes are comparatively less drastic. The present study revealed that the oviduct cycle in B. melanostictus is directly related to the ovarian cycle. The weight of the oviduct increased 12 fold in R. cyanophlyctis (Pancharatna and Saidapur, 1985), 200 fold in R. tigerina and 500 fold in P. maculatus (Kanamadi and Jirankali, 1991 a). In the laboratory maintained B. melanostictus, the weight of oviduct increased 24 fold (Kanamadi et al., 1989). But in the present study weight of the oviduct of the toad increased by 225 fold. Fat bodies in anurans are primarily known to serve as nutritional reserve, especially during hibernation, Involvement of fat bodies in gametogenesis was suggested in females of Notophthalmus viridescens (Adams and Rae, 1929), Amphiunia means (Rose, 1967), Rana esculenta (Pierantoni et al., 1984a), Rana tigerina (Promoda and Saidapur, 1984 a,c) and Rana cyanophlyctis (Prasadmurthy and Saidapur, 1987). In R. cyanophly;ctis (Pancharatna and
Saidapur, 1985), R. tigerina (Promoda and Saidapur, 1984) and P. maculatus (Kanamadi and Jirankali, 1991 a), fat bodies showed seasonal variation which are inversely related to ovarian mass. There exists confusion with regard to actual role of abdominal fat bodies in the reproduction of anurans (Saidapur and Hoque, 1996). In the present study, seasonal variation in the abdominal fat body was not significant. However, during the breeding season it showed a direct correlation with the ovarian weight. This suggests that it merely serves as an energy reservoir in female reproduction. This is in agreement with the findings of Jorgensen et al. (1979) and George and Andrews (1993). In male toad also, statistical analysis shows no significant correlation between fat body cycle and testicular cycle(chapter 4).