J. Zool., Lond. (2005) 267, 45–53 C 2005 The Zoological Society of London Printed in the United Kingdom doi:10.1017/S0952836905007235

Seasonal variation in spermatogenic and androgenic activities in a testis ( tricolor)

Mathew Smita1,R.S.Beyo1, Jancy M. George2,M.A.Akbarsha2 and Oommen V.Oommen1* 1 Department of Zoology, University of , Kariavattom – 695 581, Thiruvananthapuram, 2 Department of Science, Bharathidasan University, Thiruchirappalli – 620 024, India (Accepted 20 January 2005)

Abstract are an order of with distribution confined to several of the tropical countries. They are subterranean and practice a form of internal fertilization. Several are viviparous. Published reports on seasonal variation in male reproduction in caecilians are limited to a very few species. A study was undertaken to examine the seasonal variation of testicular activity with respect to spermatogenesis and steroidogenesis in a caecilian, Ichthyophis tricolor, fairly abundant in the of Kerala, India, using light and transmission electron microscopy, terminal deoxynucleotidyl transferase biotin-dUTP nick end labelling (TUNEL) staining and biochemical determination of 3-hydroxy-5-steroid dehydrogenase (5-3β-HSDH) activity. Three phases in spermatogenic activity, active spermatogenesis (July–November), early regression (December–March) and spermatogenic quiescence (April–June) are identified. Apoptosis seems to be the mechanism of germ cell death during the regression phase. Leydig cells are interstitial and not peritubular. Activity of testicular 5-3β-HSDH follows the same pattern as spermatogenic activity, although histometric data on the interstitial tissue reveal higher areas during the regression and quiescent phases. Our findings will be useful in evolving strategies for conservation of caecilians with special reference to Kerala, India.

Key words: caecilian, testis, spermatogenesis, apoptosis, 5-3β-hydroxy-steroid dehydrogenase

INTRODUCTION They are a little-studied group among vertebrates. Unlike anurans and urodeles, caecilians practice a form of internal Spermatogenesis is a fairly conserved process throughout fertilization making use of the eversible phallodeum (rear vertebrate series. Reproductive seasonality is the synchro- part of the cloaca) as the phallus. Further, several of the nization of reproductive processes to specific and optimal caecilians are viviparous and some of them totally skip times of the year so as to maximize reproductive success an aquatic phase in the life history (Wake, 1968; Pillai & (Nash, 1999). Most temperate amphibians have an annual Ravichandran, 1999). In the few caecilians so far inves- testicular cycle characterized by a quiescent period (late tigated, the spermatogenic cycle, sperm release, fertili- autumn and winter) and a spermatogenic period (spring zation and hatching are synchronous and seasonal. Sexual and summer) (Paniagua, Fraile & Saez, 1990). Gallien cycle in male caecilians is described in Ichthyophis (1959), while reviewing the evidence for seasonality of re- glutinosus (Ichthyophis beddomei, according to Taylor production in anurans and urodeles, concluded that repro- (1968)) (Sarasin & Sarasin, 1887–90; Seshachar, 1936, duction is invariably correlated with spring and regimes of 1937, 1941, 1943), Gymnopis multiplicata (Wake, 1968, increased or optimum temperature in both temperate and 1977), Dermophis mexicanus (Wake, 1980, 1995) and tropical species. In those cases in which reproduction is Typhlonectes compressicauda (Exbrayat & Sentis, 1982; seasonal, three phases in the testicular activity – sperma- Exbrayat & Delsol, 1985; Exbrayat & Laurent, 1986). togenic, regressive and recrudescent – have been identified Exbrayat & Laurent (1986) suggested a genetic control (Lofts, 1974). Caecilians (Amphibia: ) are of the sexual cycle in T. compressicauda. Seasonality of a unique group of burrowing or secondarily aquatic male reproduction in caecilians is inferred from studies vertebrates confined to several of the tropical countries. on only a few species. Inspired by the rediscovery of a fair abundance of caecilians in the Western Ghats of Kerala, India (Oommen et al., 2000), a study of various *All correspondence to: Oommen V. Oommen, Department of Zoology, University of Kerala, Kariavattom – 695 581, Thiruvananthapuram, Kerala, aspects of the male reproduction of a few species of India. Email: [email protected] caecilians was undertaken; some of the findings have been 46 M. SMITA ET AL. published (Smita, Oommen et al., 2003, 2004; George and computer-assisted histometric analysis of the inter- et al., 2004; Smita, Jancy et al., 2004). In our earlier stitial tissue and biochemical determination of testicular studies, the seasonal pattern of spermatogenesis and the 5-3β-hydroxy-steroid dehydrogenase activity during male Mullerian gland structure and function in caecilians different phases of spermatogenic cycle. The outcome was briefly examined. In this paper, a seasonal variation supports the finding of Anjubault & Exbrayat (1998) in in testicular activity is reported. T. compressicauda. Seasonal regression of the testis, which sets in when the environmental factors become unfavourable (Wake, 1977), should accompany extensive loss of germ cells. MATERIALS AND METHODS In other words, the spermatogenic activity ceases rather abruptly and the cells that do not complete their cellular Animal collection and tissue processing transformation are removed. Cell death in this context could be either through necrosis or apoptosis. Necrosis Ichthyophis tricolor () were collected from involves lysis of cells accompanied by an inflammatory terraced plantations of mixed coconut and rubber from response that would affect the neighbouring cells. Apo- Thekkada (08◦37N, 76◦57E) in the Thiruvananthapuram ptosis, on the other hand, is a physiological process of district in the Western Ghats of Kerala, Southern India, cell death in which individual cells are killed and removed a biodiversity hotspot (Oommen et al., 2000), from June without any impact on the neighbouring cells that need 2000 to June 2002. to be preserved (Wyllie, 1981). Programmed deletion of Monthly samples (3 animals each month) were collected cells by apoptosis is an essential feature of embryonic and dissected to expose the male reproductive system. development, tissue organization and cell turnover in Testis lobes were prepared for routine histological multicellular organisms (Chimini, 2002). There is specu- observation using a light microscope. Tissues representing lation about the stage-specific pattern of apoptosis of different phases in spermatogenic activity were also fixed germ cells from the stage-specific actions of hormones in 2.5% glutaraldehyde and embedded in resin. Semi- or growth factors that are involved in the regulation of thin sections were stained in toluidine blue-O (TBO). spermatogenesis (Leibovitch & Buttyan, 1991; Henriksen, In addition to making observations of the status of Hakovirta & Parvinen, 1995). Given that studies on the spermatogenesis, total area of the cross section and the seasonality of reproduction in caecilians are far too area occupied by lobules in 5 randomly chosen fields limited, it is not known whether the mechanism of germ from the sections of the testis of each animal were cell loss during seasonal regression of the testis involves measured using a Carl Zeiss (Germany) Axioscop-2 apoptosis. Therefore, the occurrence of apoptosis during research microscope, supported with Axiovision software. the seasonal regression of caecilian testis is also investi- The software gives the total area (of the testis section) gated in the present study. photographed and also the areas (of the lobules) It is an established fact that either interstitial or peritu- the boundaries of which were marked, at the given bular Leydig cells are present in the testes of most ver- magnification. By deducting the area of lobules from the tebrates and that they secrete androgens. The hormones are total area of the field, the area of the interstitium was responsible for the development, maintenance and regula- derived. The area of the lobules and that of the interstitium tion of secondary sexual characters, accessory reproduct- was used to calculate the percentage of each, and the ive organs and, to a great extent, spermatogenesis. Several respective means and standard deviations (SD) for each authors have reported the occurrence of Leydig cells in month. Ultra-thin sections were subjected to transmission the interstitial tissue of the caecilian testis (Sarasin & electron microscopic (TEM) analysis. Sarasin, 1887–90; Tonutti, 1931; Seshachar, 1936, 1941; Wake, 1968; Anjubault & Exbrayat, 1998). In these stu- dies, Leydig cells were examined from light microscopic, Detection of apoptosis in tissue sections adopting histological, histochemical and histoenzymological per- terminal deoxynucleotidyl transferase biotin-dUTP nick spectives. Seshachar (1941) noticed that interstitial tissue end labelling (TUNEL) assay in Ichthyophis glutinosus was meagre in the testis of animals active in spermatogenesis and more abundant Terminal deoxynucleotidyl transferase biotin-dUTP nick and conspicuous in the testis of spermatogenically quie- end labelling (TUNEL) staining was carried out in paraffin scent animals. Anjubault & Exbrayat (1999), on the other sections of the testis after fixation in neutral buffered hand, adopting histometric and histoenzymological formalin, representing different phases of spermatogenic (5-3β-HSDH) methods, showed that the Leydig cells activity, using the procedure prescribed in the Dead EndTM of Typhlonectes compressicauda exhibit volumetric varia- Colorimetric TUNEL System kit of Promega (Madison, tions characterized by an increase during the breeding WI). Deparaffinized tissue sections were washed by season. Thus, Seshachar (1941) and Anjubault & Exbrayat immersing the slides in 100% ethanol and then rehydrated (1998) differ in their findings. Because two species have by immersing them in a graded series of ethanol. After only been studied for seasonal pattern of the relative washing in 0.85% NaCl followed PBS, the sections were abundance of Leydig cells, more species need to be in- treated with proteinase K solution. The sections were vestigated, adopting more reliable indices. In this study, refixed in 10% buffered formalin in PBS, washed in PBS Ichthyophis tricolor was subjected to a more systematic and then immersed in equilibration buffer. TdT reaction Seasonal variation in testicular activity in a caecilian 47 mix was added over the sections covered with plastic (spermatogonia) to stage Vb (spermateleosis) (Smita, cover slips and the slides were incubated at 37 ◦Cfor Oommen et al., 2004). The lobules were fairly large 60 min inside a humidified chamber. Negative control (225 µm diameter) and round or hexagonal (Fig. 1a, b). slides were incubated with buffer not containing TdT Sertoli cells were extremely tall (90 µm diameter) and enzyme. At the end of the incubation, the cover slips were their cytoplasm contained lesser lysosomes and lipid drop- removed and the reaction was terminated by immersing lets than during the other months of the year. The inter- the slides in 2× SSC. The slides were washed repeatedly stitium, lying outside the lobules contained a few Leydig in PBS. Streptavidin HRP solution at 1:500 dilution, cells around blood capillaries and amidst fibroblasts and was added over the slides and the slides were incubated mast cells. Towards the later part of this phase (i.e. during for 30 min at room temperature. The slides were then September–November), another component, the collect- washed repeatedly in PBS. DAB mix containing DAB ing ductules, appeared in the interstitial areas and es- substrate buffer, DAB chromogen and hydrogen peroxide tablished a connection with the lobules so that the lumen were added over the sections and developed until the of the collecting ductules became continuous with the appearance of a light brown background. The slides were spermiating cysts. There was an active cell proliferation rinsed several times in de-ionized water and mounted in from the epithelium of the collecting ductules at the point 100% glycerol. The sections were observed in Carl Zeiss of contact with the lobules. Dispersed spermatozoa first Axioscop 2 research microscope. An intense brown colour appeared in the collecting ductules during October. of the nuclei indicated apoptosis. Early regression phase Assay of 5-3β-hydroxy-steroid dehydrogenase (5-3β-HSDH) activity From December to March, the lobules gradually regres- sed. They contained fewer cysts representing stages 0 Activity of microsomal NAD-dependent 5-3β-HSDH, to IV, where as cysts in stages Va and Vb were abun- which catalyses the conversion of pregnenolone to pro- dant (Fig. 1c, d). Sertoli cell cytoplasm increased with gesterone, a rate-limiting step in the biosynthesis of abundance of lysosomes and lipid droplets. The interstitial steroid hormones, was assayed in the testis of animals area increased marginally, and the Leydig cells became representing different phases of spermatogenic activity, abundant. The collecting ductules were prominent with according to Shivanandappa & Venkatesh (1997). Tissue sperm in admixture with several irregularly shaped, loose (100 mg) was homogenized in 1 ml of 0.1 M Tris-HCl ◦ cells. The epithelial cell proliferation from the collecting buffer (pH 7.8) and centrifuged at 12 000 × g at 4 C. ductules was highly limited. Enzyme activity was assayed in the supernatant using 0.1 m Tris-HCl buffer (pH 7.8) containing NAD as the hydrogen acceptor and pregnenolone (100 µM) as the Spermatogenically quiescent phase substrate. The reaction was started by adding the enzyme source (100 µl) and incubated at room temperature for During April–June, the lobules were regressed to the mini- 60 min, and stopped by adding 2.0 ml of phthalate buffer mum size (Fig. 1e, f). The intersititial area proportionately (pH 3.0). Turbidity was removed by centrifugation at increased. The lobules consisted essentially of Sertoli 1800 × g for 20 min and the colour of the supernatant was cells, surrounding one or more empty spaces. Germ cell read at 490 nm in a UV-VIS spectrophotometer (Shimadzu, nests representing Stages 0 to II alone were present, con- 1601). Enzyme activity was calculated from the standard fined to the basal and intercalating regions of the Sertoli curve of NADH and expressed as micromoles of NADH cells. In the interstitium, the collecting ductules were formed per h per mg protein. Data were used to calculate less prominent while Leydig cells, fibroblasts and mast the respective means and standard errors (SE), for different cells were more abundant than during the regression phases of reproductive activity. phase.

Prevalence of apoptosis RESULTS Haematoxylin and eosin-stained paraffin sections indica- Seasonal testicular histological variations in ted that several germ cells in the testis during the regres- Ichthyophis tricolor sion phase possessed intensely heterochromatic nuclei. Occasionally, the nuclei fragmented into two or more chro- Three different phases of spermatogenic activity were matoid bodies. At a stage in regression, the nuclei even identified from the observation of the testicular lobules indicated marginalization of and/or crescent-shaped chro- of Ichthyophis tricolor over a year, repeated once. matin and these apoptotic morphologies were clearly observed in TBO-stained semi-thin sections and TEM Spermatogenic phase pictures (Fig. 2a–d). TUNEL staining of testicular sections obtained during the early regression phase revealed Between July and November, the lobules had cell nests germ cell nuclei with highly intense reaction, indicating in all stages of the spermatogenic cycle, namely stage 0 apoptosis (Fig. 3). 48 M. SMITA ET AL.

lb ia sp lb

ia sc lc sm lb es ca

lb

sc rs lb

(a) sc (b)

lb

ia

lb va vb cd

vb

lb

(c) vb (d) gc

sc ia

lb

lb lb gc gc (e) (f)

Fig. 1. (a) TBO-stained semi-thin section of testis lobules of Ichthyophis tricolor during the active spermatogenic phase showing the hexagonal lobules (lb) containing germ cells in almost all stages of spermatogenesis including primary spermatocytes (sp), round spermatids (rs), elongating spermatids (es) and spermiated sperm (sm). The highly limited interstitial area (ia), containing Leydig cells, is also shown. Scale bar = 60 µm. (b) TEM of a testis lobe showing three lobules (lb) and the interstitium (ia) lying outside the lobules. The lobules are bounded by myoid cells (arrow). The interstitium contains Leydig cells (lc) and a capillary (ca). Scale bar = 20 µm. Seasonal variation in testicular activity in a caecilian 49

(a) (b)

ch

(c) (d)

Fig. 2. (a) TBO stained semi-thin section of a testis lobule of Ichthyophis tricolor during the regression phase showing germ cells with marginalized chromatin (arrowhead) and cresentic (arrow) chromatin. Scale bar = 20 µm. (b) Low-power transmission electron micrograph of testis lobule in the regression phase showing crescentic chromatin (arrow) and vacuolated cytoplasm of the germ cells. Scale bar = 15 µm. (c) One germ cell as in (b) magnified to show the crescentic chromatin (ch) of the germ cell indicating apoptotic morphology. Scale bar = 3 µm. (d) Same as (e) showing further compaction of the chromatin (arrows). Scale bar = 3 µm.

(c) Haematoxylin & eosin-stained paraffin section of testis lobules (lb) in the early regression phase. The lobules (lb) are decreased in diameter and contain few cysts in the early stage of spermatogenesis. Most of the cysts belong to stages va and vb. Scale bar = 80 µm. (d) Same as (c), but at late regression. The lobules (lb) which are decreased in diameter and contain germ cells (gc) up to stage II and also spermiating cysts (arrowheads). Establishment of contact between a spermiating cyst and the collecting ductule (cd) is indicated (arrow). Note apparent increase in the interstitial area (ia). Scale bar = 40 µm. (e) Haematoxylin & eosin-stained paraffin section of testis lobes during the period of spermatogenic quiescence, showing lobules (lb) containing germ cells (gc) up to stage II. Note the further apparent increase in the interstitial area (ia). Scale bar = 80 µm. f: Same as (e), part magnified showing the lobule (lb) limited in germ cell content (gc) to cells up to stage II associating with the Sertoli cells (sc). A central lumen is shown (arrow). Scale bar = 40 µm. 50 M. SMITA ET AL.

3

Fig. 3. TUNEL-stained section of testis lobules of Ichthyophis tricolor during the regression phase. TUNEL-positive cells are indicated by arrowheads. Scale bar = 40 µm.

25 120

100 20

80 15

60

10 40 Lobule surface area (%) Interstitial surface area (% )

5 20

0 0 Jul Jul Oct Jan Apr Jun Feb Mar Oct Aug Apr Sep Dec Nov Jan Jun May Feb Mar Sep Aug Nov Dec May

Fig. 4. The relative area of the interstitium of Ichthyophis tricolor Fig. 5. The relative area of the lobules of Ichthyophis tricolor throughout the year. Vertical bars, SD. throughout the year. Vertical bars, SD.

Relative area in sections occupied by lobules and Activity of 5-3β-HSDH interstitium Data on the relative area of interstitium and lobules are Activity of testicular 5-3β-HSDH was highest in presented (Figs 4 & 5). The interstitial area in relation to animals collected in November (active spermatogenesis), the lobular area was lowest in November and highest in at an intermediate level in March (regression, but spermi- June, and that of the lobules was lowest in June and highest ating) and lowest in April (spermatogenically quiescent) in November. (Fig. 6). Seasonal variation in testicular activity in a caecilian 51

0.25 Typhlonectes compressicauda. Data on 5-3β-HSDH activity in Ichthyophis tricolor in this study, however, parallel the observation of Anjubault & Exbrayat (1998) in T. compressicauda, in which histochemical localization 0.2 of 5-3β-HSDH decreased in the spermatogenically quiescent testis. The increased percentage area of the interstitium and decreased 5-3β-HSDH activity during 0.15 spermatogenic quiescence may be explained by extreme regression of the spermatogenic compartment, the magni- tude of regression of the interstitial not matching that of the spermatogenic compartment. The histometric data, as 0.1 presented in Figs 4 & 5, combined with the histological m NAD/mg protein/min

µ observations, indicate that during the testicular cycle, the testis was regressed to the minimum size in June when the total area of the lobules in a transverse section of 0.05 the lobes was 78.53 ± 0.65 µm2 and that of the interstitial area was 21.46 ± 0.65 µm2. There was an abrupt transition in the histoarchitecture and the histometric data during 0 July when the area of the lobules increased and that Active Regression Quiescence of the interstitium correspondingly decreased. The trend continued in such a way that maximum area of the lobules Fig. 6. 5-3β-HSDH activity in the testis of Ichthyophis tricolor. (97.56 ± 0.35 µm2) was obtained in November when the Vertical bars, SE. area of the interstitium was minimal (2.43 ± 0.35 µm2). From December onwards, the area of the lobules decrea- sed, to reach the minimum in June with the interstitial area DISCUSSION correspondingly increasing to reach the maximum. The extent of regression of the spermatogenic com- A seasonal pattern in spermatogenesis in Ichthyophis partment does not match that of the androgenic compart- tricolor is reported. The testis is spermatogenically ment. The diameter of the lobules of the fully regressed active between July and November (rainy and post-rainy testis in June was c. 50% of those from a spermatogeni- seasons in Kerala; moisture content of the soil is high; cally fully active testis in November. In the spermato- environmental temperature is modest), when cell nests genically quiescent testis, both the spermatogenic and in all stages of spermatogenesis are present. This is the androgenic compartments are regressed, the former more phase of active spermatogenesis. Though spermiation can dramatically than the latter, giving an illusion that the occur concurrently with active spermatogenesis, fewer cell interstitial area in the spermatogenically quiescent testis nests reach the stage of spermiation. The testis indicates proportionately increased. Data on the activity of 5-3β- clear evidence of spermiation during December–March HSDH seem more reliable than those from histometry. (the period of tropical winter when there is little rainfall, Seasonal testicular regression should necessarily in- yet the moisture content of the soil is high, and the en- volve the death of germ cells and their removal. Seasonal vironmental temperature is low). Concurrently with regression of an organ being physiological, the killing of the changes occurring between December and March, cells connected therewith should not be necrosis, is one the testis starts regression so that between April and June that brings about inflammatory changes in the neighbour- (when there is no or little rainfall, moisture content of the ing cells (Wyllie, 1981). Apoptosis, on the other hand, soil is low, the environmental temperature is the highest) it is a physiological process of programmed cell death is fully regressed in size, and quiescent for spermatogenic involving a cascade of molecular mechanisms so that the activity. This is the phase of spermatogenic quiescence. cells undergo condensation rather than swelling (Steller, An interesting observation in the present study is that 1995). Mitochondria are intact and play a critical role in the testis is regressed and spermatogenically quiescent the progression of events. But in the nucleus, DNA under- in June and it suddenly enters the phase of spermatogenic goes oligonucleosomal fragmentation (Steller, 1995). The activity in July. Thus, a clear phase of recrudescence is not chromatin fragments are attracted towards the nuclear traceable in this animal. As the data in this study are for envelope, which is a marginalization of chromatin. Sub- 2 years, the observation is reliable. Perhaps recrudescence sequently, the fragmented chromatin concentrates towards in I. tricolor is a phenomenon setting in abruptly, and it is a pole of the nucleus to result in crescentic chromatin. probable that we missed animals representing this stage. Cells retract from the surrounding cells and are removed The data for seasonal variation of the interstitium and as apoptotic bodies. Apoptotic bodies are usually phago- Leydig cells are interesting because the percentage area cytosed by macrophages. In the present study, histological, of the lobules and that of the interstitium are inversely transmission electron microscopic and immunocyto- proportional. This aspect matches with the observation chemical evidences are obtained to the effect that during of Seshachar (1941) in Ichthyophis glutinosus and is testicular regression in caecilians, germ cells are killed different from that of Anjubault & Exbrayat (1998) in and removed through an apoptotic mechanism. 52 M. SMITA ET AL.

Seasonal changes in the testis can be correlated with that during the regression of the testis, the germ cells die environmental factors. Caecilians, similar to other am- through apoptosis. phibians, are ectothermic vertebrates and most have a bur- rowing mode of life. The moisture content of the soil is a limiting factor in this mode. When the moisture con- Acknowledgements tent of the soil goes down, the animals go deep into the soil, which will restrict their foraging. Further, egg laying/ We are grateful for permission to use the ultracut and live birth would require not only moisture, but also stag- the electron microscope facilities of the Wellcome Trust nant/running water for the eggs to hatch and or the young Research Center, Christian Medical College and Hospital to survive. Therefore, the animal is spermatogenically (CMC & H), Vellore, and the help of Dr M. Radhakrishna and androgenically active during the months when the Pillai (Regional Cancer Centre, Thiruvananthapuram), climatic conditions are modest, and there is no shortage in TUNEL staining. We thank the University Grants of water in the environment. Regression coincides with Commission (UGC), New Delhi, (F.3.33/2002 (SR-II), low temperature, but it is during this phase that spermia- and the Kerala State Council for Science and Technology tion occurs. Spermatogenic quiescence coincides with (KSCSTE), Thiruvananthapuram, for financial support, rigorous climatic conditions of high temperature, little and the Department of Science and Technology (DST), stagnant/running water and poor moisture content of the New Delhi, for the instrumentation facility under the FIST soil. These inferences clearly match those of Exbrayat programme. We also thank Dr Mark Wilkinson and Dr (2000), Exbrayat & Sentis (1982), Exbrayat & Flatin David Gower of the Natural History Museum, London, (1985) and Wake (1995) who found rainfall and environ- and Dr John G. Measey, Institute of Tropical Ecology, mental temperature to be the decisive factors influencing Paris, for other support. the seasonal reproductive cycle in caecilians. Comparing of the patterns of seasonal spermatogenic cycles in Ichthyophis glutinosus (Seshachar, 1936), Typh- REFERENCES lonectes compressicauda (Exbrayat & Sentis, 1982), Gym- nopis multiplicata (Wake, 1995), Dermophis mexicanus Anjubault, E. & Exbrayat, J. M. (1999). Yearly cycle of Leydig- (Wake, 1995) and Ichthyophis tricolor, the present study like cells in testes of Typhlonectes compressicaudus (Amphibia: is pertinent because the different species have different Gymnophiona). In Current Studies in Herptetology: 53–58. geographic distributions. Even among the two species Miaud, C. & Guyetant, R. (Eds). France: Le Bourget Du Lac. of Ichthyophis, I. glutinosus is found more along the Chimini, G. (2002). Apoptosis: repulsive encounters. Nature (Lond.) 418: 11. northern parts of the Western Ghats of India whereas Exbrayat, J. M. (2000). Les gymnophiones, ces curieux Amphibiens. I. tricolor is distributed along the southern parts. The testis Paris: Boubee. of G. multiplicata is active in spermatogenesis for 10– Exbrayat, J. M. & Delsol, M. (1985). Reproduction and growth 11 months of the year and exhibits a brief regression fol- of Typhlonectes compressicauda, a viviparous Gymnophione. lowed by recrudescence in January–February (Wake, Copeia 4: 95. 1995). The pattern in D. mexicanus consists of recrude- Exbrayat, J. M. & Flatin, J. (1985). Les cycles de reproduction chez les Amphibiens Apodes. Influence des variations saisonnieres.` scence with extensive interstitial tissue present in each Bull. Soc. Herpetol. Fr. 110: 301–305. lobe. In January, the animals illustrate more advanced Exbrayat, J. M. & Laurent, M. T. (1986). Quelques observations spermatogenesis, when the interstitial tissue is markedly sur la reproduction elevage´ de Typhlonectes compressicauda, reduced. In March, the collecting ductules begin to form Amphibien Apode vivipare. Possibilite´ de rhythmes endogens. and cell nests that include spermatids are present. In April, Bull. Soc. Herpetol. Fr. 40: 52–62. more nests with spermatids are present. In May, there is a Exbrayat, J. M. & Sentis, P. (1982). Homogen´ eit´ e´ du testicule et large increase in interstitial tissue. Spermatids and mature cycle annual chez Typhlonectes compressicaudus (Dumeril´ et Bibron, 1841) Amphibien Apode vivipare. C. R. Acad. Sci. Paris sperm migrate into the ducts. June to July is characterized 292: 417–420. by extensive interstitial tissue. Cell nests contain primary Gallien, L. (1959). Endocrine basis for reproductive adaptations in spermatocytes in June and either primary or secondary amphibia. In Comparative endocrinology: 479–487. Gorbman, spermatocytes in July. The testis is regressed in August A. (Ed.). New York: Wiley. and the interstitial tissue is limited. The lobules largely George, J. M., Smita, M., Oommen, O. V. & Akbarsha, M. A. evacuate, with little stroma, but numbers of peripheral (2004). Histology and ultrastructure of male mullerian gland of primary spermatocytes are present. Thus, most caecilians narayani (Amphibia: Gymnophiona). J. Morphol. 260: 33–56. breed seasonally in correlation with the rainy season. Both Henriksen, H., Hakovirta, H. & Parvinen, M. (1995). Testosterone oviparous and viviparous caecilians have a reproductive inhibits and induces apoptosis in the rat seminiferous tubules in biology that is environmentally cued, but determined by a stage-specific manner: in situ quantification in squash pre- the pattern of seasonal changes in the environmental paration after administration of ethane dimethane sulfonate. factors prevalent in their respective geographic areas Endocrinology 136: 3285–3291. (Wake, 1995). Leibovitch, I. & Buttyan, R. (1991). Sulfated glycoprotein-2 induced endogenous resistance to ischaemia and reperfusion Thus, our study shows that the spermatogenic and injury in the seminiferous tubules. Am. J. Reprod. Immunol 26: androgenic activities in the testis of Ichthyophis tricolor 114–117. follow a seasonal pattern, coinciding with the factors in the Lofts, B. (1974). Reproduction. In Physiology of the Amphibia: environment, particularly rainfall. The study also shows 107–218. Lofts B, (Ed.). New York: Academic Press. Seasonal variation in testicular activity in a caecilian 53

Nash, J. P. (1999). Seasonal reproduction, fish. In Encyclopedia of analysis by light and transmission electron microscopy. J. reproduction 4: 329–340. Knobil, E. & Neill, J. D. (Eds). New Morphol. 262: 484–499. York: Academic Press. Smita, M., Oommen, O. V., Jancy, M. G. & Akbarsha, M. A. (2003). Oommen, O. V., Measey, G. J., Gower, D. J. & Wilkinson, M. Sertoli cells in the testis of caecilians, Ichthyophis tricolor and (2000). Distribution and abundance of the caecilian Gegeneophis Uraeotyphlus cf . narayani (Amphibia: Gymnophiona): light and ramaswamii (Amphibia: Gymnophiona) in Southern Kerala. electron microscopic perspective. J. Morphol. 258: 317–326. Curr. Sci 79: 1386–1389. Smita, M., Oommen, O. V., Jancy, M. G. & Akbarsha M. A. Paniagua, R., Fraile, B. & Saez, F. J. (1990). Effects of photoperiod (2004). Stages in spermatogenesis of two species of caecilians and temperature on testicular functions in amphibians. Histol. Ichthyophis tricolor and Uraeotyphlus cf. narayani (Amphibia: Histopathol. 5: 365–378. Gymnophiona): light and electron microscopic study. J. Morphol. Pillai, R. S & Ravichandran, M. S. (1999). Gymnophiona of India, 261: 92–104. a taxonomic study. Rec. Zool. Surv. India 172: 1–117. Steller, H. (1995). Mechanisms and genes of cellular suicide. Sarasin, R. & Sarasin, P. (1887–90). Zur Entwicklungsgeschi- Science 267: 1445–1449. chte und Anatomie der ceylonesischen Blindwuhle¨ Ichthyophis Taylor, E. H. (1968). The caecilians of the world. A taxonomic glutinosus L. In Ergebnisse naturwissenschaftlicher Forschun- review. Lawrence, KS: University of Kansas Press. gen auf Ceylon: 1–263. Wiesbaden: C. W. Kreidel’s Verlag. Tonutti, E. (1931). Beitrag zur Kenntnis der Gymnophionen. XV. Seshachar, B. R. (1936). The spermatogenesis of Ichthyophis Das Genital System. Morphol. Jahrb. 70: 101–130. glutinosus L. The spermatogonia and their division. Z. Zellforsch. Wake, M. H. (1968). Evolutionary morphology of the caecilian Mikr. Anat 24: 662–706. urogenital system. Part I. The gonads and fat bodies. J. Morphol. Seshachar, B. R. (1937). The spermatogenesis of Ichthyophis 126: 291–332. glutinosus (Linn.). II. The meiotic divisions. Z. Zellforsch. Mikr. Wake, M. H. (1977). The reproductive biology of caecilians: Anat. 27: 133–158. an evolutionary perspective. In The reproductive biology of Seshachar, B. R. (1941). The interstitial cells in the testis of Ichthyo- amphibians: 73–101. Taylor, D. H. & Guttman, S. I. (Eds). phis glutinosus Linn. Proc. Indian. Acad. Sci 13B: 244–254. NewYork: Plenum Press. Seshachar, B. R. (1943). The spermatogenesis of Ichthyophis Wake, M. H. (1980). Reproduction, growth and population structure glutinosus (Linn.), III. Spermateleosis. Proc. Nat. Inst. Sci. India of the Central American caecilian Dermophis mexicanus. 9: 271–285. Herpetology 36(3): 244–256. Shivanandappa, T. and Venkatesh, S. (1997). A colorimetric Wake, M. H. (1995). The spermatogenic cycle of Dermophis assay method for 3β-hydroxy 5-steroid dehydrogenase. Anal. mexicanus (Amphibia: Gymnophiona). J. Herpetol. 29: 119–122. Biochem 254: 57–61. Wyllie, A. H. (1981). Cell death: a new classification separating Smita, M., Jancy, M. G., Girija, R., Akbarsha, M. A. & Oommen, apoptosis from necrosis. In Cell death in biology and pathology: O. V. (2004). Spermiogenesis in caecilians Ichthyophis tricolor 9–34. Bowen, I. D. & Lockshin, R. A. (Eds). London: Chapman & and Uraeotyphlus cf. narayani (Amphibia: Gymnophiona): Hall.