Sertoli Cells in the Testis of Caecilians, Ichthyophis Tricolor and Uraeotyphlus Cf. Narayani (Amphibia: Gymnophiona): Light and Electron Microscopic Perspective

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JOURNAL OF MORPHOLOGY 258:317–326 (2003)

Sertoli Cells in the Testis of Caecilians, Ichthyophis tricolor and Uraeotyphlus cf. narayani (Amphibia: Gymnophiona): Light and Electron Microscopic Perspective

Mathew Smita,1 Oommen V. Oommen,1* Jancy M. George,2 and M.A. Akbarsha2

1Department of Zoology, University of Kerala, Kariavattom, Thiruvananthapuram 695 581, Kerala, India 2Departmnt of Animal Sciences, Bharathidasan University, Thiruchirappalli 620 024, Tamilnadu, India

ABSTRACT The caecilians have evolved a unique pat- that surround the cyst/follicle (Fraile et al., 1990; tern of cystic spermatogenesis in which cysts representing Saez et al., 1990; Grier, 1993; Koulish et al., 2002). different stages in spermatogenesis coexist in a testis lob- The follicle cells are known as Sertoli cells when ule. We examined unsettled issues relating to the organi- spermatids form a bundle. Their apical tips become zation of the caecilian testis lobules, including the occur- embedded in the crypts formed by invaginations of rence of a fatty matrix, the possibility of both peripheral and central Sertoli cells, the origin of Sertoli cells from the follicle cell membrane and point towards its follicular cells, and the disengagement of older Sertoli nucleus. This arrangement resembles the Sertoli cells to become loose central Sertoli cells. We subjected the cell / germ cell association in amniotes (Lofts, 1974; testis of Ichthyophis tricolor (Ichthyophiidae) and Uraeo- Bergmann et al., 1982). In anamniotes, with the typhlus cf. narayani (Uraeotyphliidae) from the Western progression of spermatogenesis, isogenic spermato- Ghats of Kerala, India, to light and transmission electron zoa are produced and released by rupture of the cyst microscopic studies. Irrespective of the functional state of into the lumen of the seminiferous lobule. Following the testis, whether active or regressed, Sertoli cells con- spermiation, the Sertoli cell degenerates (Pudney, stitute a permanent feature of the lobules. The tall Sertoli 1993, 1995, 1999). Thus, in the anamniote testis cells adherent to the basal lamina with basally located there is no permanent seminiferous epithelium, or pleomorphic nuclei extend deeper into the lobule to meet at least the Sertoli cells are not permanent, but are at the core. There they provide for association of germ cells at different stages of differentiation, an aspect that has transitory. earlier been misconceived as the fatty matrix. Germ cells On the other hand, in amniotes spermatogenesis up to the 4-cell stage remain in the intercalating region of occurs in the convoluted seminiferous tubules, the Sertoli cells and they are located at the apices of the which are lined by a permanent epithelium in which Sertoli cells from the 8-cell stage onwards. The developing germ cell transformation occurs as a wave along the germ cells are intimately associated with the Sertoli cell length of the tubule. Different discrete stages, each adherent to the basal lamina until spermiation. There are representing a unique cellular association, can be ameboid cells in the core of the lobules that appear to identiﬁed (de Kretser and Kerr, 1994). The germ interact with the germ cells at the face opposite to their cells are translocated during their development to- attachment with the Sertoli cells. Adherence of the Sertoli wards the tubular lumen with the Sertoli cell / germ cells to the basal lamina is a permanent feature of the cell association maintained. A single Sertoli cell is in caecilian testicular lobules. The ameboid cells in the core are neither Sertoli cells nor their degeneration products. contact with several germ cells at different stages of J. Morphol. 258:317–326, 2003. © 2003 Wiley-Liss, Inc. differentiation (Grier, 1993; Bardin et al., 1994). In the amniotic organization, spermatogenesis is non- KEY WORDS: Caecilians; testis; Sertoli cells; spermatogenesis Contract grant sponsor: the University Grant Commission, New Delhi; Contract grant number: f.3.33/2002 (SR-II) dated 5.3.2002 (to OVO, MAA); Contract grant sponsors: DST, New Delhi, FIST pro- During spermatogenesis, whether it be the cystic gramme, and Drs. Mark Wilkinson and David Gower of NHM, Lon- pattern as in the case of ﬁsh and Amphibia, or non- don. cystic as in the case of amniotic vertebrates, germ cells of the testis are intimately associated with *Correspondence to: Dr. Oommen V. Oommen, Professor and Head, somatic Sertoli cells. In cystic spermatogenesis, Department of Zoology, University of Kerala, Kariavattom, 695 581, spermatogenic cysts are produced when follicle cells Thiruvananthapuram, Kerala, India. E-mail: [email protected] associate with the primary spermatogonium. The primary spermatogonium develops synchronously, in close association with the somatic follicle cells DOI: 10.1002/jmor.10155

© 2003 WILEY-LISS, INC. 318 M. SMITA ET AL. cystic, since the seminiferous epithelium, including sity hotspot (Oommen et al., 2000). Ichthyophis tricolor was col- somatic Sertoli cells, is a permanent entity, in spite lected from Thekkada (Lat. 08° 37Ј N; Long. 76° 57Ј E) in the Thiruvananthapuram district and U. cf. narayani from Thodu- of the fact that in several species the seminiferous puzha (Lat. 09° 53Ј N; Long. 76° 42Ј E) in the Idukki district of epithelium undergoes seasonal changes in relation Kerala. The study was carried out from June 2000 to June 2002. to environmental conditions of temperature, photo- Three animals were sampled per month. Identification of the period, etc. (de Kretser and Kerr, 1994; Bardin et al., species was based on matching with reference specimens avail- 1994). In caecilians, although spermatogenesis is able at the Natural History Museum, London. believed to be cystic, it differs markedly from cystic spermatogenesis in the fish, anurans, and urodeles Tissue Processing in that the different cysts in a lobule represent dif- The specimens studied were sacrificed using MS-222 and were ferent stages of differentiation. dissected. The testes were removed and lobes were fixed in The highly elongated testis in caecilians contains Bouin’s fluid for processing to obtain serial paraffin sections. a number of interconnected lobes, each formed of a Tissues were stained in hematoxylin/eosin or periodic acid- large number of circular to polygonal locules. Each Schiff’s (PAS) / hematoxylin. Lobes were fixed in 2.5% glutaral- lobule, in an actively spermatogenic testis, contains dehyde in sodium cacodylate buffer (pH 7.2) at 4°C for semithin sectioning and transmission electron microscopy (TEM). a large number of cysts/lobules that represent dif- Glutaraldehyde-fixed tissues were postfixed for1hinsimilarly ferent stages of differentiation (Wake, 1968, 1977). buffered 1% osmium tetroxide, rinsed in buffer, dehydrated The lobules are permanent entities and their epithe- through an ascending series of ethanol, and infiltrated with and lium, therefore, is responsible for producing the embedded in epoxy resin (Sigma Chemical Co., St. Louis, MO, USA). Semithin (1 ␮) sections were obtained by use of an ultra- cysts in various stages of spermatogenesis. Efferent tome (Reichert Jung, Austria) and stained with Toluidine blue O ductules connecting the collecting duct and the lu- (TBO) for light microscopic observation in a Carl Zeiss Axio-3 men of the locule have been shown in caecilians microscope (Germany). Images were captured by a computer and (Seshachar, 1936, 1942a; Wake, 1968, 1977, 1995). processed using Carl Zeiss axiovision software. Ultrathin sec- Thus, spermatogenesis in the caecilians is not cystic tions, 50–80 nm thickness, were stained in Reynolds’s lead citrate (Reynolds, 1963) and 6% aqueous uranyl acetate. Electron in the strict sense. micrographs were obtained using a Phillips 201 C transmission Sertoli cells in the caecilian testis, therefore, sug- electron microscope at 75 kV and processed using the image gest an origin and association with the germ cells in analyzer software as above. a different manner than the Sertoli cells in anamniotes where there is cystic spermatogenesis in the RESULTS strict sense. We hypothesized that Sertoli cells in the caecilian testis do not change in relation to the Testicular activity appears to have a cyclical pat- dynamic recruitment and transition of the germ tern from our seasonal study conducted over a span cells and are permanent. However, we found in the of 2 years (unpubl. obs.). Testicular lobules, whether literature (Seshachar, 1936) that 1) Sertoli cells in in active spermatogenesis (Fig. 1A,B) or during re- the caecilian testis are also dynamic and that newer gression of the lobules (Fig. 2A,B), are lined by cells cells arise as parafollicular cells, which later acquire that are comparable in organization to Sertoli cells. a relationship with the wall of the lobule; 2) the Sertoli cells have their basal membrane adherent to parafollicular cells become the Sertoli cells; 3) the the basal lamina of the lobule, developing interdigi- single germ cell surrounded by parafollicular cells tations between them to varying degrees (Fig. 3). develops into a cohort of spermatozoa; and 4) at one The basal lamina is characterized by a thick layer of stage of spermiation the Sertoli cells of the cyst collagen fibers. Intermingled with this layer are become detached from the lobule wall and lie float- fibroblast-like cells, myoid cells, and peritubular ing in the matrix; they are evacuated along with the cells (Fig. 3). spermatozoa (Seshachar, 1942a,b; Bhatta et al., The basally located nucleus of Sertoli cells is typ- 2001). This does not correlate appropriately with the ically pleomorphic, with the nuclear envelope exhib- multiplicity of germ cell cohorts occurring within a iting deeper indentations into which the cytoplasm lobule. Our light and transmission electron micro- extends. The nucleus is fairly darkly stained, with a scopic studies of the testis of Ichthyophis tricolor and thin patch of dense heterochromatin just under- Uraeotyphlus cf. narayani indicate that in the cae- neath the nuclear envelope, followed by dispersed cilian testis Sertoli cells are permanent entities, patches of heterochromatin more internally. It con- maintaining their identity in the lobules of even the tains one or two dense nucleoli (Figs. 3, 4). Depend- fully regressed testis. ing on the status of spermatogenesis (active or regressed) (Figs. 1A,B, 2A,B) apically, the cells protrude deeper into the lobule to surround one or MATERIALS AND METHODS more lumina that contain particulate material and Animals may or may not enclose spermatogenic cysts at very advanced stages of differentiation. Germ cells up to The caecilians used in this study, Ichthyophis tricolor (Ich- thyophiidae) and Uraeotyphlus cf. narayani (Uraeotyphliidae), the 4-cell stage are observed between intercalating were collected from terraced mixed coconut and rubber planta- regions of Sertoli cells (Fig. 5A,B). When the germ tions in the Western Ghats of Kerala, Southern India, a biodiver- cell cohort reaches the 8-cell stage, the germ cells SERTOLI CELLS IN CAECILIANS 319

Fig. 1. A: Testis of Ichthyo- phis tricolor in active spermatogenesis showing lobules containing the germ cell cyst (GC) and the Sertoli cells (SC) with basal nucleus. LM. Toluidine blue- stained semithin section. Scale bar ϭ 200 ␮m. B: A portion of the lobule showing Sertoli cell (SC) associated with germ cell cyst (GC) and also attached to the basal lamina (BL). Peritubu- lar tissue (PT) and myoid cells (MC) are also shown. Scale bar ϭ 20 ␮m. occupy positions around and on top of the Sertoli On completion of differentiation, spermatozoa are cells (Fig. 6A,B) and at one stage, only a few of the disengaged from each other and cease any connec- cells in the cohort have a direct relationship with the tion with the Sertoli cell. They lie free in the heter- Sertoli cell (Fig. 7A). With further germ cell differ- ogeneous matrix of the lumen surrounded by the entiation into elongating spermatids, spermatids re- apical portion of the Sertoli cell (Fig. 8B). As sper- main associated with each other as a compact single- mateleosis proceeds, irregularly shaped cells with layered mass. The more proximal portion of the larger and densely heterochromatic nuclei are seen spermatid that contains the acrosomal vesicle re- in the proximity of the spermatids on the face away mains embedded in the cytoplasm of the Sertoli cell, from the acrosomal vesicle, i.e., in the matrix (Fig. adherent to the basal lamina of the lobule (Figs. 7B, 9A). These cells differ dramatically in organization 8A). compared with the Sertoli cells that remain adher- 320 M. SMITA ET AL. tions comparable to the blood–testis barrier in the Sertoli cells of amniotes could not be identiﬁed. The Sertoli cell / germ cell junctions are invariably gap junctions (Fig. 10A). Processes from germ cells may project deep into the cytoplasm of Sertoli cells (Fig. 5A). The lysosome content of the Sertoli cells appears to vary, depending on the stage of spermatogenesis. The abundance of lysosomes is inversely propor- tional to the stage of spermatogenesis, i.e., few in the testis of an animal in active spermatogenesis and profuse in the regressed testis (Figs. 3, 4). Processes of the Sertoli cells reach deep into the lobule, become part of the lobule, and match descriptions of the stroma/matrix in the caecilian testis (Exbrayat, 1986a,b, 2000; Exbrayat and Dansard, 1994). Even in the most regressed testis, germ cells are present towards the basal portion of the lobule, in close association with Sertoli cells, but only apical portions of Sertoli cells are evident deeper in the lobule (Fig. 11).

DISCUSSION Occurrence of Sertoli cells in the lobules of the caecilian testes has been reported for several species, such as Ichthyophis glutinosus, Uraeotyphlus narayani, Gegeneophis carnosus, Siphonops annula- tus, Schistometopum gregori (Seshachar, 1936a, 1942b, 1943, 1945, 1948; Seshachar and Srinath, 1946), Typhlonectes compressicauda (Exbrayat 1986a,b, 1992, 2000; Exbrayat and Dansard, 1992, 1993, 1994), Ichthyophis beddomei (Bhatta et al., 2001), and Typhlonectes natans (van der Horst and van der Merwe, 1991). The general structure of the Sertoli cell (this study) conforms to the description Fig. 2. A: Regressed testis of Ichthyophis tricolor showing germ cell cysts (GC) populating only the peripheral portion of the available for these species of caecilians. There are lobules and the Sertoli cells (SC) with basal nucleus. A lumen two views regarding the disposition of Sertoli cells in (LU) associating with the sperm duct (SD) is also shown. LM. the caecilian testis. According to Seshachar (1942b), H&E. Scale bar ϭ 200 ␮m. B: A portion of a lobule in A magniﬁed, Sertoli cells are found all along the periphery of the showing occurrence of germ cells (GC) and Sertoli cells (SC) even in the fully regressed testis lobule. Peritubular tissue (PT) and lobule just beneath the septum and closely adherent myoid cell (MC) are also shown. Note Sertoli cells (SC) extending to it and in a deeper portion of the lobule. The germ deep into the core of the lobule (arrowheads). Scale bar ϭ 20 ␮m. cell cysts in spermiogenesis remain embedded in a fatty matrix, which has no relationship to or bearing on the Sertoli cells. In the alternative view (Ex- ent to the basement membrane. Sertoli cell cyto- brayat, 1986a,b, 2000; Exbrayat and Dansard, plasm contains elongated rod-shaped mitochondria 1994), what Seshachar (1936, 1942a,b) considered with plicate cristae (Fig. 9B), sparse smooth and as fatty matrix is only hypertrophied cytoplasm of rough endoplasmic reticulum, and spherical vesicles the basal Sertoli cell that connects to the basal as- with homogenous to heterogenous content, which pect of the lobule. Our observation corroborates the probably are lysosomes. Otherwise, the cytoplasm views of Exbrayat (1986a,b, 2000) that portions of generally is less electron-dense than the cytoplasm the Sertoli cells rich in lipid droplets, the so-called of the germ cells. Gap junctions (Fig. 10A), tight “ﬁlamentous matrix,” are only apical portions of the junctions, and desmosomes (Fig. 10B) are seen in- Sertoli cells irrespective of the species. According to frequently between the neighboring Sertoli cells. In- our observation the apical portions are not hypertro- terdigitations between Sertoli cell plasma mem- phied portions of the Sertoli cell, but constitute their branes are rare. However, towards the apical ends, natural organization, i.e., Sertoli cells in caecilian occludens junctions are evident between Sertoli cell testicular lobules are extremely tall and can meet at membranes (Fig. 8B). Lateral membrane specializa- various points in the core of the lobule, providing SERTOLI CELLS IN CAECILIANS 321

Fig. 3. Basal portion of a Ser- toli cell (SC) in the testis lobule of Uraeotyphlus cf. narayani active in spermatogenesis, resting on the basement membrane (BM) and possessing pleomorphic nucleus (NU) containing a nucleolus (NL). A lysosome (LY) is present in the Sertoli cell cytoplasm. Germ cells (GC) are associated with the Sertoli cells. Collagen ﬁbrils (CF) of the basal lamina, peritubular tissue (PT), and myoid cell (MC) are also shown. TEM. Scale bar ϭ 2.5 ␮m.

Fig. 4. Ichthyophis tricolor. Basal portion of a Sertoli cell (SC) in the fully regressed testis lobule showing the pleomorphic nucleus (NU) containing a nucleolus (NL). The cytoplasm is rich in lysosomes (LY). TEM. Scale bar ϭ 5 ␮m. 322 M. SMITA ET AL. and Exbrayat and Dansard (1994) proposed two kinds of Sertoli cells, peripheral and central, the latter presumably discharged from the wall of the lobule and lying loose in the lumen. According to Seshachar (1936), the central cells are degenerate germ cells. In our observation, they are cells with irregular shapes and deeply staining densely heterochromatic nuclei. There is no evidence to substanti- ate that these loose cells are produced through disengagement of Sertoli cells from the basal lamina of the lobules. The occurrence of free ameboid cells in the deeper portion of the lobule of caecilian testis is

Fig. 5. A: Ichthyophis tricolor. A 2-cell stage germ cell cyst (GC) associating with the intercalating region of the Sertoli cell (SC). Note portion of a germ cell protruding into the Sertoli cell (arrowheads). TEM. Scale bar ϭ 10 ␮m. B: Uraeotyphlus cf. narayani. A 4-cell stage germ cell cyst (GC) associated with the intercalating region of the Sertoli cell (SC). TEM. Scale bar ϭ 10 ␮m. support for the developing germ cells until spermiation. Exbrayat (1986a,b, Exbrayat (2000), Exbrayat and Dansard (1994), and Bhatta et al. (2001) in their reports, while settling the differences regarding the fatty/ﬁlamentous matrix, have created a new contro- versy by proposing that coinciding with the progression of spermatogenesis, the Sertoli cells leave the periphery of the lobule, when they are pushed to- ward the lumen by a new generation of Sertoli cells newly arising from the follicular cells. These cells, Fig. 6. A: Uraeotyphlus cf. narayani. An 8-cell stage germ cell when pushed further into the lobule, decrease in cyst (GC) conﬁned to the apical aspect of the Sertoli cell (SC). volume, and always surround germ cells that in- TEM. Scale bar ϭ 10 ␮m. B: Uraeotyphlus cf. narayani. An 8-cell stage germ cell cyst (GC) associated with the Sertoli cell (SC) at crease and differentiate. At the end of the cycle, they the intercalating region as well as on top. Spermatids (ST) are degenerate and fall in to the evacuative ductule also seen on the apical aspect of the Sertoli cell (SC). TEM. Scale among the spermatozoa. Exbrayat (1986a,b, 2000) bar ϭ 10 ␮m. SERTOLI CELLS IN CAECILIANS 323 a normal feature. The illustrations of Sertoli cells in Typhlonectes natans reported by van der Horst and van der Merwe (1991) are actually such ameboid cells. Their nature, origin, and role remain to be established, an aspect currently under investigation in our laboratory; however, we consider that they are neither Sertoli cells nor their degeneration products. The view of Pudney (1999) and Pierantoni (1999) that in the anamniotes the Sertoli cells un-

Fig. 8. A: Uraeotyphlus cf. narayani. The acrosomal vesicle of a differentiating spermatid (ST) embedded in the Sertoli cell adherent to the basal lamina. TEM. Scale bar ϭ 5 ␮m. B: Uraeo- typhlus cf. narayani. Spermatozoa (SP), on spermiation, have lost contact with the Sertoli cell (SC). Occludens junctions (OJ) between Sertoli cells, at the apical border, are also shown. TEM. Scale bar ϭ 8 ␮m.

dergo fatty degeneration thus does not appear to hold for caecilians. The most striking observation in the present study is that Sertoli cells are found in the lobules of the testis irrespective of the status of spermatogenesis, viz., active or regressed. In the regressed testis, the lobules are never empty, but contain both Sertoli and germ cells. This substantiates our claim that Fig. 7. A: Ichthyophis tricolor. A cohort of spermatids (ST) in Sertoli cells do not disengage from the basal lamina early spermateleosis associating on top of a Sertoli cell (SC). Note to become loose central cells, but constitute a per- the intimate relationship of the spermatid with the Sertoli cells (arrowheads). TEM. Scale bar ϭ 10 ␮m. B: Same as A, but manent aspect of the lobule. There is also clear spermatids are at an advanced stage of spermateleosis. Scale evidence of Sertoli cells adherent to the basal lam- bar ϭ 10 ␮m. ina, supporting germ cells even in the most ad- 324 M. SMITA ET AL. tion, the germ cells remain adherent to the permanent resident Sertoli cells. Therefore, there is no need for other cells to nutritively support germ cells at advanced stages of spermateleosis. Interestingly, Seshachar (1942b) made the following statement about Sertoli cells in caecilian testis: “They are found not only in the active testis but also in the resting testis and might be considered as permanent cells of the organ,” a view we uphold. However, Seshachar (1942b) contradicted his own statement by stating that follicles are produced from the mouth of the ductules and with division of spermatogonia, migrate from the lobule wall to complete spermatogenesis and the follicle cells are left behind near the

Fig. 9. A: Uraeotyphlus cf. narayani. A cohort of differentiating spermatids (ST), associating with the Sertoli cell (SC) at the acrosomal phase (arrowheads); loose ameboid cells (AC) are present on the face of the spermatid cohort opposite the attachment with the Sertoli cell. Spermatozoa (SP) lie loose in testis lumen. TEM. Scale bar ϭ 10 ␮m. B: Ichthyophis tricolor. Mito- chondria (MI) in the apical cytoplasm of the Sertoli cell (SC). Note association of an early differentiating spermatid (ST) with the Sertoli cell and a desmosome (DS) between two Sertoli cell membranes in the testis. TEM. Scale bar ϭ 4 ␮m. vanced stages of spermateleosis in testes active in spermatogenesis. It is also logical to assume that loose and degenerating cells cannot provide support to developing spermatozoa. We speculate that if the Sertoli cells lose connection with the basal lamina, they will lose access to blood vessels and peritubular tissue and hence can no longer support the germ Fig. 10. A: Ichthyophis tricolor. Gap junctions between germ cells. Therefore, extraordinary structural intricacy cell (GC) and Sertoli cell (SC) (arrowheads) and between Sertoli cells (arrows). TEM. Scale bar ϭ 8 ␮m. B: Ichthyophis tricolor. A of the lobular epithelial components is the most ob- desmosome (DS) between two Sertoli cell (SC) membranes. The vious evidence of interdependence of germ cells and Sertoli cell cytoplasm abounds with lysosomes (LY) and endoplas- Sertoli cells in the caecilian testis. Until spermia- mic reticulum (arrowheads). TEM. Scale bar ϭ 1.5 ␮m. SERTOLI CELLS IN CAECILIANS 325

Fig. 11. Uraeotyphlus cf. narayani. Germ cells associated with Sertoli cells (SC) in the fully regressed testis. Note the extensive lobulation of the pleomorphic Sertoli cell nucleus. TEM. Scale bar ϭ 10 ␮m. periphery in their original position and become the permanent epithelium. This also explains how a lob- Sertoli cells. We believe that the Sertoli cells consti- ule can have at the same time cysts representing tute a permanent feature of the caecilian testis and cohorts each at different stages of differentiation, a the need for generation and establishment of newer feature intermediate between the characteristic cys- Sertoli cells does not arise. We are examining the tic and noncystic patterns of spermatogenesis. possibility that the ameboid cells in the lobules are This report refutes the view that the caecilian produced at the mouth of the collecting ductules to testis contains a fatty matrix in the lobules; what fulfill a yet undetermined role. was presumed to be the matrix is only the apical The adult Sertoli cell number is established dur- portions of the Sertoli cells. As the Sertoli cells are ing the process of larval development and before adherent to the basal lamina in the caecilian testis sexual maturation. Once established, they usually and constitute a permanent feature, this study ques- do not divide. Thus, there is no recruitment of fresh tions the occurrence of central Sertoli cells. In this Sertoli cells in the adult testis (Russell, 1993, 1999; respect the caecilian testis may be considered simi- Bardin et al., 1994; Griswold and Russell, 1999). lar to that of amniotes. Our observation in the present study suggests that the Sertoli cells in the caecilian testis match those of ACKNOWLEDGMENTS the amniotes with respect to permanency and support of the germ cells until spermiation. We thank the ultracut and electron microscope Our observations prompt us to propose that with facility of Wellcome Trust Research Center, Chris- respect to seasonality of reproduction and cystic pat- tian Medical College and Hospital (CMC&H), Vel- tern of spermatogenesis, the caecilian testis has a lore, India. 326 M. SMITA ET AL. LITERATURE CITED Pierantoni R. 1999. Male reproductive system, amphibians. In: Knobil E, Neill JD, editors. Encyclopedia of reproduction, vol. 3. Bardin CW, Cheng CY, Mustow NA, Gunsalus GL. 1994. The New York: Academic Press. p 10–15. Sertoli cell. In: Knobil E, Neill JD, editors. The physiology of Pudney J. 1993. Comparative cytology of the nonmammalian reproduction, 2nd ed. New York: Raven Press. p 1291–1333. Sertoli cell. 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