JOURNAL OF MORPHOLOGY 262:484–499 (2004) Spermiogenesis in Caecilians Ichthyophis tricolor and Uraeotyphlus cf. narayani (Amphibia: Gymnophiona): Analysis by Light and Transmission Electron Microscopy Mathew Smita,1 Jancy M. George,2 R. Girija,2 M.A. Akbarsha,2 and Oommen V. Oommen1* 1Department of Zoology, University of Kerala, Kariavattom, Thiruvananthapuram, 695 581, Kerala, India 2Department of Animal Science, Bharathidasan University, Tiruchirappalli, 620 024, Tamil Nadu, India ABSTRACT Spermiogenesis, known as spermateleosis times availability in fair abundance of a few species in lower vertebrates, is the transformation of the round of caecilians has been reported, which has kindled spermatid into a highly specialized spermatozoon with a interest about their ecology, behavior, and reproduc- species-specific structure. Spermateleosis and sperm mor- tive biology (Oommen et al., 2000). phology of two species of caecilians, Ichthyophis tricolor and Uraeotyphlus cf. narayani, from the Western Ghats of The most outstanding contributions on male re- Kerala, India, were studied using light and transmission productive biology of caecilians have come from Se- electron microscopy. Spermateleosis is described in early, shachar (1936, 1937, 1939, 1940, 1941, 1942a,b, mid-, and late phases. During the early phase, the sper- 1943a,b, 1945, 1948; Seshachar and Srinath, 1946), matid nucleus does not elongate, but the acrosome vesicle Wake (1968, 1970a,b, 1972, 1977, 1981, 1994, 1995), is Golgi-derived and its material is produced as a homo- Exbrayat (1986a,b, 2000), and Exbrayat and Dan- geneous substance rather than as discrete granules. In sard (1992, 1993, 1994). Light microscopic observa- development of the acrosome, the centrioles shift in posi- tions on caecilian spermatozoon have been reported tion to the lower half of the cell. The acrosomal vesicles for Ichthyophis glutinosus, Uraeotyphlus narayani, take the full shape of the acrosome with the establishment of the perforatorium in midphase. An endonuclear canal Siphonops annulatus, and Gegeneophis ramaswamii develops and accommodates the perforatorium. The incip- by Seshachar (1939, 1940, 1943, 1945), Chthonerpe- ient flagellum is laid down when the proximal centriole ton indistinctum by de Sa and Berois (1986), and a attaches to the posterior side of the nucleus and the distal few other species by Wake (1994). Ultrastructural centriole connects to the proximal centriole, which forms description of mature spermatozoa of caecilians, to the basal granule of the acrosome. The axial fiber also the best of our knowledge, is confined to appears during midphase. The mitochondria shift in posi- Typhlonectes natans (van der Horst and van der tion to the posterior pole of the cell to commence estab- Merwe, 1991a,b; Jamieson, 1999) and Ichthyophis lishment of the midphase. Late phase is characterized by nuclear condensation and elongation. Consequently, the beddomei, Ichthyophis tricolor, G. ramaswamii, and final organization of the sperm is established with the three species of Uraeotyphlus (Scheltinga et al., head containing the nucleus and the acrosome. The undu- 2003). lating membrane separates the axoneme and axial fiber. Descriptions of spermatogenesis and spermio- Most of the cytoplasm is lost as residual bodies. J. Mor- genesis of caecilians, particularly at the ultra- phol. 262:484–499, 2004. © 2004 Wiley-Liss, Inc. structural level are also rare. Seshachar (1936, 1937, 1939, 1942, 1943a, 1945) for Ichthyophis KEY WORDS: caecilians; testis; spermiogenesis; sperm glutinosus (Ichthyophiidae), Uraeotyphlus narayani (Uraeotyphliidae), Siphonops annulatus (Caeciliidae), Schistometopum gregori Blgr., and The caecilians (Gymnophiona) constitute a unique Gegeneophis carnosus Beddome (Caeciliidae), group of amphibia, which are worm-like limbless animals. Approximately 160 species, 32 genera, and six families of caecilians are currently recognized (Nussbaum and Wilkinson, 1989). Knowledge of Contract grant sponsor: University Grants Commission, New Delhi; Contract grant number: F.3.33/2002 (SR-II) (to O.V.O., M.A.A.); Con- many aspects of caecilian biology lags behind that tract grant sponsor: DST, New Delhi, FIST program. available for frogs and salamanders. The single big- gest obstacle to advances in caecilian biology has *Correspondence to: Dr. Oommen V. Oommen, Professor and Head, been the paucity of material for study, which is Department of Zoology, University of Kerala, Kariavattom, Thiruvan- essentially due to their scattered endemic distribu- anthapuram, 695 581, Kerala, India. E-mail: [email protected] tion confined to certain tropical countries and their Published online in secretive habits resulting from a burrowing mode of Wiley InterScience (www.interscience.wiley.com) life (Scheltinga et al., 2003). However, in recent DOI: 10.1002/jmor.10258 © 2004 WILEY-LISS, INC. SPERMIOGENESIS IN CAECILIANS 485 Fig. 1. Early phase of sper- miogenesis. TEM. A: Very early phase of spermatids of Ichthyo- phis tricolor. Nucleus (NU) is oval in shape. Cytoplasm con- tains cylindrical mitochondria (MI) located peripherally, and also endoplasmic reticulum (ER). Scale bar ϭ 5 ␮m. B: A portion of very early phase of spermatid of I. tricolor. There are small vesicles (VC) in the Golgi area (GA). Mitochondria (MI), centrioles (CT), and nu- cleus (NU) are also shown. Scale bar ϭ 4 ␮m. C: A portion of very early phase spermatid of Uraeo- typhlus cf. narayani. An amor- phous material appearing in vesicles among the centrioles (CT) is present ahead of the Golgi apparatus (GA). Lyso- somes (LY) and a cytoplasmic bridge (CB) connecting sperma- tids are also shown. Scale bar ϭ 4.4 ␮m. D: More advanced early phase of spermatids of U. cf. narayani showing a Golgi (ar- rowhead) on top of the nucleus (NU). Mitochondria (MI) and en- doplasmic reticulum (ER) are also shown. Scale bar ϭ 12 ␮m. E: A portion of more advanced early phase spermatid of U. cf. narayani showing indentation of nucleus (NU) (arrowhead) to ac- commodate the acrosomal vesi- cle (AV). An amorphous material continues to be formed from Golgi apparatus (GA). Scale bar ϭ 5 ␮m. F: A portion of a more advanced early phase sper- matid of U. cf. narayani showing fusion of small vesicles, formed in the Golgi area (GA) with the already formed acrosomal vesi- cle (AV). NU, nucleus. Scale bar ϭ 2.1 ␮m. Wake (1968, 1977, 1995) for Gymnopis multipli- vations by Seshachar (1943a, 1945). Thus, there is cata and Dermophis mexicanus and Exbrayat a paucity of information on ultrastructural descrip- (1986a,b, 2000) for Typhlonectes compressicauda tions of caecilian spermiogenesis/spermateleosis, have made light microscopic descriptions. Re- which can be useful in characterization of species cently, we have described the Sertoli cells (Smita based on sperm morphology (Scheltinga et al., 2003). et al., 2003) and spermatogenesis (Smita et al., We describe the ultrastructural details of spermate- 2004) in Ichthyophis tricolor and Uraeotyphlus cf. leosis and the mature sperm of two species of cae- narayani by combining light and electron micros- cilians. Although the sperm morphology of these two copy and identified stages in spermatogenesis species has been described previously (Scheltinga et comparable to those in the Anura and Urodela. al., 2003), the present article contributes to the ul- However, information on spermateleosis in caecil- trastructural descriptions of spermateleosis, a first ians is available from only light microscopic obser- report of its kind for caecilians. 486 M. SMITA ET AL. Fig. 2. A–C: More advanced stages in early phase spermio- genesis. TEM. A: Uraeotyphlus cf. narayani. Elongation as well as change in shape of acrosomal vesicle (AV). Note modification of the nuclear envelope (NE) and shifting of position of Golgi ap- paratus (GA). Scale bar ϭ 1.2 ␮m. B: Uraeotyphlus cf. narayani. Dispersal of the con- tents of acrosomal vesicle (AV) is depicted. Note the eccentric po- sition of Golgi apparatus (GA) and thickening of nuclear enve- lope (NE). Scale bar ϭ 1.5 ␮m. C: Ichthyophis tricolor. Centri- oles have shifted in position (ar- rowhead). Acrosomal vesicle (AV) and Sertoli cell (SC) cyto- plasm are also shown. Scale bar ϭ 10 ␮m. D–F: Midphase spermatid. TEM. D: Uraeo- typhlus cf. narayani. Figure shows elongation of acrosome (AC) and formation of perforato- rium (PM). Endoplasmic reticu- lum (ER) is also shown. Scale bar ϭ 6 ␮m. E: Uraeotyphlus cf. narayani. Figure shows accumu- lation of less dense material on top of dense plaque (arrow- heads). Perforatorium (PM), en- donuclear canal (EC), acrosome (AC) are also shown. Scale bar ϭ3.5 ␮m. F: Ichthyophis tricolor. Posterior end of nucleus (NU) showing indentation to accom- modate the centriole (CN). Scale bar ϭ 2.5 ␮m. MATERIALS AND METHODS cies was based on matching individuals with reference specimens Animals available at the Natural History Museum, London. Animal collection and tissue processing were accomplished as described previously (Smita et al., 2003). Briefly, Ichthyophis Tissue Processing tricolor (Ichthyophiidae) and Uraeotyphlus cf. narayani (Uraeo- typhliidae) were collected from terraced mixed coconut and rub- The animals were anesthetized using MS-222 and dissected. ber plantations in the Western Ghats of Kerala, southern India, Testes were removed and lobes were fixed in 2.5% glutaraldehyde a biodiversity hotspot (Oommen et al., 2000). Ichthyophis tricolor in sodium cacodylate buffer (pH 7.2) at 4°C for semithin sec- was collected from Thekkada (Latitude 08° 37ЈN; Longitude