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; spermato- genesis 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 ci- trate (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 anam- niotes 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