Spermiogenesis in Caecilians Ichthyophis Tricolor and Uraeotyphlus Cf

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

Fig. 1. Early phase of spermiogenesis. TEM. A: Very early phase of spermatids of Ichthyo- phis tricolor. Nucleus (NU) is oval in shape. Cytoplasm contains 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 nucleus (NU) are also shown. Scale bar ϭ 4 ␮m. C: A portion of very early phase spermatid of Uraeo- typhlus cf. narayani. An amorphous 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 spermatids are also shown. Scale bar ϭ 4.4 ␮m. D: More advanced early phase of spermatids of U. cf. narayani showing a Golgi (arrowhead) on top of the nucleus (NU). Mitochondria (MI) and endoplasmic 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 accommodate the acrosomal vesicle (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 spermatid of U. cf. narayani showing fusion of small vesicles, formed in the Golgi area (GA) with the already formed acrosomal vesicle (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 spermiogenesis. 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 apparatus (GA). Scale bar ϭ 1.2 ␮m. B: Uraeotyphlus cf. narayani. Dispersal of the contents of acrosomal vesicle (AV) is depicted. Note the eccentric position of Golgi apparatus (GA) and thickening of nuclear envelope (NE). Scale bar ϭ 1.5 ␮m. C: Ichthyophis tricolor. Centri- oles have shifted in position (arrowhead). Acrosomal vesicle (AV) and Sertoli cell (SC) cytoplasm 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 perforatorium (PM). Endoplasmic reticulum (ER) is also shown. Scale bar ϭ 6 ␮m. E: Uraeotyphlus cf. narayani. Figure shows accumulation of less dense material on top of dense plaque (arrow- heads). Perforatorium (PM), endonuclear canal (EC), acrosome (AC) are also shown. Scale bar ϭ3.5 ␮m. F: Ichthyophis tricolor. Posterior end of nucleus (NU) showing indentation to accommodate 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 76° tioning and transmission electron microscopy (TEM). 57ЈE) in Thiruvananthapuram district and U. cf. narayani from Glutaraldehyde-fixed tissues were postfixed for1hinsimilarly Thodupuzha (Latitude 09° 53ЈN; Longitude 76° 42ЈE) in Idukki buffered 1% osmium tetroxide, rinsed in buffer, dehydrated district of Kerala. Male animals of both species were used during through an ascending series of ethanol, and infiltrated with and the 2-year period of study (2001–2003). Identification of the spe- embedded in thin viscosity epoxy resin (Sigma Chemical, MO). SPERMIOGENESIS IN CAECILIANS 487

Fig. 3. Midphase. TEM. A: Ichthyophis tricolor. Proximal centriole (PC) lies closer to the indentation of the nucleus (arrowhead) and distal centriole (DC) is also seen. Scale bar ϭ 2 ␮m. B: Ichthyophis tricolor. Proximal centriole (PC) is ac- commodated at the indentation of the nucleus (NU). Distal centriole (DC) lies just beneath the proximal centriole. Scale bar ϭ 2.5 ␮m. C: Ichthyophis tricolor. The appearance of chromatoid body (CB) and lamellar body (LB). Nucleus (NU) and a mito- chondrion (MI) are also shown. Scale bar ϭ 3.5 ␮m. D: Uraeo- typhlus cf. narayani. Axial ﬁber (AF) differentiation is shown. The distal centriole (DC) forms the basal body of the axoneme. Mitochondria (MI) have posi- tioned around the developing midpiece. Scale bar ϭ 2.5 ␮m. E: Uraeotyphlus cf. narayani. Axoneme (AX) is clearly evident. Scale bar ϭ 2 ␮m. F: Ichthyophis tricolor. Midpiece differentiation around the axial ﬁber and axoneme (AX). Lamellar body (LB) is also shown. Scale bar ϭ 2 ␮m.

Semithin sections (1 ␮m thickness) were obtained using an ultra- RESULTS microtome (Reichert Jung, Austria) and stained with toluidine blue O (TBO) for light microscopic observation with a Carl Zeiss The development of spermatozoa from round sper- Axio-3 research microscope (Germany). Images were captured by matids is portrayed in stages as early, mid-, and late a computer and processed using Carl Zeiss Axiovision software, phases. Thus, the changes in acrosome, centrioles, with which histometric measurements were also made. Ultrathin sections at 50–80 nm thickness, obtained with a Leica (Germany) nucleus, and mitochondria are depicted concur- ultramicrotome were stained with Reynolds’s 6% aqueous uranyl rently, rather than as each part separately. acetate and lead citrate (Reynolds, 1963) and observed with a Phillips 201 C TEM at 75 kV. Histo/cytometric measurements were also made on TEM images. The quantitative assessment in Early Phase volume change was calculated for round and elongated spermatids. TEM pictures were scanned into the computer and processed In the earliest stage, spermatids are connected by using image analysis software as above. cytoplasmic bridges. The nucleus is spherical or 488 M. SMITA ET AL.

Fig. 4. A–C: Midphase continued. TEM. A: Ichthyophis tricolor. Enlarged picture of Figure 3F showing the structure of lamellar body (LB). Midpiece differentiation starts around the axoneme (AX). Appearance of annulus (AN) is also shown. Scale bar ϭ 1.5 ␮m. B: Ichthyo- phis tricolor. Elongated nucleus (NU), acrosome (AC) lying ahead of it and the establishment of axial ﬁber and axoneme (AX). Scale bar ϭ 5 ␮m. C: Uraeo- typhlus cf. narayani. Elongated nucleus (NU), the acrosome (AC) lying ahead of it, the perforatorium (PM) are shown. The acrosome is embedded in the Sertoli cell (SC). Scale bar ϭ 2 ␮m. D–F: Ichthyophis tricolor. Late phase spermatid. D: Condensa- tion and elongation of spermatids in a cyst, which characterize the late phase. The entire cyst surrounds a Sertoli cell (SC). NU, nucleus; CY, cytoplasm. Scale bar ϭ 20 ␮m. E: A portion of D enlarged. Elongated nucleus (NU) with acrosome (AC) embedded in Sertoli cell (SC) cytoplasm is shown. Scale bar ϭ 15 ␮m. F: Figure shows elongation of the nucleus (NU) as well as condensation of chromatin. Par- allel arrangement of microtubules (MT) and endoplasmic reticulum (ER) are also shown. The acrosome (AC) is embedded in Sertoli cell (SC). Scale bar ϭ 2 ␮m. oval. Cylindrical mitochondria are found peripher- 1D). Several small vesicles, containing a similar ally in the cytoplasm. The endoplasmic reticulum is substance, continue to be produced from the Golgi concentrated at one pole (Fig. 1A). Numerous small apparatus and fuse with the already established vesicles surround the nucleus (Fig. 1B). A typical larger one (Fig. 1E). Consequently, the vesicle in- Golgi complex, consisting of 2–3 stacks of ﬂattened creases in size and forms the acrosomal vesicle (Fig. vesicles and cisternae, is present in the cytoplasm 1F). With an increase in size, the vesicle elongates (Fig. 1C). The earliest of the most prominent and becomes oval-shaped (Fig. 2A) and its contents changes in the transformation of spermatid into become more dispersed (Fig. 2B). The Golgi stacks spermatozoon is marked by the appearance of an shift in position to extend from one side of the apex amorphous material in a vesicle among the Golgi of the acrosomal vesicle. During development and stacks (Fig. 1C). This vesicle shifts away from the attachment of the acrosomal vesicle, the nucleus Golgi apparatus to rest on top of the nucleus (Fig. remains spherical except, for an indentation at the SPERMIOGENESIS IN CAECILIANS 489

Fig. 5. Progression of Late phase. TEM. A: Ichthyophis tricolor. Acrosome with acrosomal vesicle (AV) and a condensed material (CM) at its front end, perforatorium (PM) and Sertoli cell (SC) cytoplasm are shown. Scale bar ϭ 1.5 ␮m. B: Uraeo- typhlus cf. narayani. Elongated nucleus (NU) and the displacement of cytoplasm to the posterior region are shown. Acrosome (AC) is embedded in Sertoli cell (SC). Centriole (CN) and flagellum (FL) are also shown. An ameboid cell (AM) lies outside the cyst. Scale bar ϭ 10 ␮m. C: Ichthyophis tricolor. Elon- gated nucleus with central condensed and peripheral dispersed chromatin is shown. Acrosome (AC) is embedded in Sertoli cell (SC). Parallely arranged micro- tubule (MT) is present in the cytoplasm of spermatid around the nucleus. Scale bar ϭ 2.5 ␮m. D: Ichthyophis tricolor. Fully condensed (CD) and less condensed (LD) regions of the elongating nucleus. Acrosome (AC) is embedded in Sertoli cell (SC), the cytoplasm of which is rich in lysosomes (LY). Microtubules (MT) are also shown. Scale bar ϭ 2.5 ␮m. E: Ichthyophis tricolor. Posterior end of nucleus showing the beginning of redundant nuclear envelope (RN). Fully condensed chromatin (CD), nuclear fibrils (NF), centriole (CN), axial fiber (AF), axoneme (AX) are also shown. Scale bar ϭ 3 ␮m. F: Ichthyophis tricolor. Trans- verse section showing increase in the condensed chromatin (CD). Nuclear fibrils (NF) and mitochondria (MI) are shown. Scale bar ϭ 2 ␮m. attachment site of the acrosome (Fig. 1E). The nu- in acrosomal vesicle size the centrioles shift away clear envelope is modified at the attachment site of towards the lower half of the cell, at which time the the acrosomal vesicle to the nucleus. A layer of fi- axoneme of the flagellum appears (Fig. 1D). Just brous material of medium electron density is sand- before commencement of elongation of the sperma- wiched between the nuclear envelope and the single tid, the centrioles come to lie against the nuclear smooth membrane of the acrosomal vesicle (Fig. 1E). envelope at a lateral position in the cell (Fig. 2C). Concurrently with the activity of the Golgi apparatus, towards the beginning of establishment of the Midphase acrosomal vesicle, the two centrioles lying at right angles to each other move away from their substan- Midphase is characterized by elongation of the tive location on top of the nucleus to a position closer acrosomal vesicle and formation of an electron- to the plasma membrane (Fig. 1B). With an increase dense acrosomal rod, the perforatorium, which ex- 490 M. SMITA ET AL.

Fig. 6. Late phase continued. TEM. A: Uraeotyphlus cf. narayani. Nucleus (NU) is further elongated. Acrosomal vesicle (AV) is embedded in Sertoli cell cytoplasm (SC). An empty space (ES) is formed due to oblit- eration of the less condensed chromatin. Scale bar ϭ 10 ␮m. B: Ichthyophis tricolor. Trans- verse section of nucleus (NU) showing clear space around it. Mitochondria (MI) are present in the cytoplasm. Scale bar ϭ 2.1 ␮m. C: Ichthyophis tricolor. Pos- terior end showing the redundant nuclear envelope (RN) around the nucleus (NU). Cent- riole (CN) and axial fiber (AF) are also shown. Scale bar ϭ 2 ␮m. D: Uraeotyphlus cf. narayani. Caudal portion of nucleus (NU) showing redundant nuclear envelope (RN). Axial fiber (AF) is also shown. Scale bar ϭ 3.5 ␮m. E: Uraeotyphlus cf. narayani. Flagellum consisting of axial fiber (AF), axoneme (AX) and undulating membrane (UM) containing an electron- dense material (ED). Scale bar ϭ 5 ␮m. F: Uraeotyphlus cf. narayani. Flagella lying in the lumen, showing the width of the undulating membrane at the posterior end (arrowhead). Scale bar ϭ 10 ␮m. tends from the posterior end of the acrosome to less dense material is present, and a clear space within the endonuclear canal. The perforatorium is separates the material from the membrane of the formed from a deep indentation of the anterior por- acrosomal vesicle (Fig. 2E). Even before the perfora- tion of the nucleus (Fig. 2D). At the adhesion site of torium is formed, the centrioles migrate to the op- the acrosome with the nucleus, there is an accumu- posite pole of the nucleus, where the acrosome de- lation of dense material, between the two leaflets of velops. The nucleus indents to accommodate the the nuclear envelope. Consequently, the two leaflets proximal centriole (Fig. 2F). The proximal centriole are not distinguishable at this site. Beyond this enlarges and flattens more than the free distal cen- dense material, both around the periphery of the cell triole (Fig. 3A,B) and is oriented perpendicular to and around the endonuclear canal, the two leaflets the long axis of the developing axoneme. This stage are separated by a perinuclear space. Immediately also marks the appearance of a chromatoid body, on top of the dense plaque, at the acrosomal seat, a which is lodged in the indentation formed at the SPERMIOGENESIS IN CAECILIANS 491

Fig. 7. Late phase continued. TEM. A: Ichthyophis tricolor. Elongating nucleus (NU), axial fiber (AF), axoneme (AX) and undulating membrane (UM) are shown. Scale bar ϭ 8.5 ␮m. B: Uraeotyphlus cf. narayani. Displacement of cytoplasm (CY) to the caudal pole of the nucleus. Mitochondria (MI) are present in the cytoplasm. Microtubules (MT), nucleus (NU), and axial fiber (AF) are also shown. Scale bar ϭ 3.3 ␮m. C: Ichthyophis tricolor. Mitochondria (MI) are confined to the cytoplasm (CY) caudal to the nucleus (NU). Scale bar ϭ 5 ␮m. D: A portion of developing midpiece in C magni- fied. Annulus (AN), undulating membrane (UM), mitochondria (MI), and axial fiber (AF) are also shown. Scale bar ϭ 2.5 ␮m. E: Ichthyophis tricolor. Region of nuclear fossa at the posterior end of nucleus (NU) showing the accommodation of proximal centriole (PC). Distal centriole (DC) forms the attachment site for axial fiber (AF). Spiral arrangement of mitochondria (MI), glycogen (GL) is also shown. Scale bar ϭ 2 ␮m. F: Transverse section of a collecting ductule. EP, epithelium; LU, lumen. The lumen contains mature spermatozoa (SP) and an ameboid cell (AM). Scale bar ϭ 15 ␮m.

posterior end of the nucleus. A lamellar body seen as 4A). The cytoplasm has abundant mitochondria and a curved cap forms between the nuclear envelope endoplasmic reticulum. and the proximal centriole (Fig. 3C). It is composed Thus, before the commencement of condensation of an electron-dense lamella, a more electron-lucent of nuclear chromatin and elongation of nucleus, the striated layer, another electron-dense lamella, a perforatorium of the acrosome and the axoneme of more electron-lucent layer, and yet another electron- the flagellum have begun development (Fig. 4B,C). dense layer. The axial fiber starts differentiation Late Phase much before the nuclear elongation (Fig. 3D,E). The midpiece starts differentiating around the axial fi- Condensation of nuclear chromatin and elonga- ber and the axoneme. Mitochondria and endoplas- tion of the nucleus characterize the late phase (Fig. mic reticulum are prominent in this area (Figs. 3F, 4D,E). The coarse chromatin granules of the nucleus 492 M. SMITA ET AL.

Fig. 8. Longitudinal sections of anterior portion of sperm of the two species compared. TEM. A: Ichthyophis tricolor. B: Uraeotyphlus cf. narayani. Acrosome (arrow) contains acrosomal vesicle (AV) with three zones, electron-dense homogeneous zone (EZ), moderately homogeneous zone (MZ), and granular zone (GZ). Periperforatorial subacrosomal material (PS) is shown. Perforatorium (PM) is lodged in the endonuclear canal (EC) within the nucleus (NU). Subacrosomal material (SM) and acrosomal base plate (AB) are also shown. Scale bar ϭ 2 ␮m. fuse to form a dense homogeneous mass. The chro- plasm in the posterior half of the cell, but anterior to matin fibers appear to be linked along their length to the flagellum, which has already appeared (Fig. 4B). form anastomosing sheets. Microtubules align par- Condensation of nuclear chromatin and elongation allel to, but outside of the nuclear envelope. Nuclear of the nucleus are concurrent (Fig. 5C). Condensa- condensation and elongation occur during this step tion of chromatin material through formation of (Fig. 4F). The material at the tip of the acrosomal cross-linked chromatin fibers begins at the core of vesicle is condensed (Fig. 5A). There is a clear dis- the nucleus, concomitant with the development of placement of cytoplasm to the posterior region (Fig. the endonuclear canal. The process of condensation 5B,C). Elongation of the caudal cytoplasm is associ- demarcates portions of the nucleus that are fully ated with the development of the middle and prin- condensed and less condensed (Fig. 5D). With in- cipal pieces of the flagellum. Mitochondria shift in creased condensation of the chromatin the portion position and arrange at the periphery of the cyto- with the less-condensed material diminishes (Figs. SPERMIOGENESIS IN CAECILIANS 493

TABLE 1. Features shared by the sperms of Ichthyophis tricolor (Figs. 9,10) and Uraeotyphlus cf. narayani (Figs. 11, 12) Features Descriptions 1. Acrosome complex Composed of an acrosomal vesicle surrounding the perforatorium 2. Acrosomal vesicle Cylindrical and consists of three regions, a moderately homogeneous zone, a granular zone, and basally an electron-dense homogeneous zone. 3. Acrosomal base plate Present in both 4. Anterior barb-like extension Present in both 5. Nucleus Consists of a short cylinder of uniform diameter with condensed chromatin 6. Endonuclear canal Wide in both and the perforatorium is lodged in it. 7. Posterior end of endonuclear canal Rounded 8. Material in endonuclear canal Endonuclear canal is filled with a material that surrounds the perforatorium and is continuous with the acrosomal seat. 9. Midpiece Consists of the centrioles, the anterior part of the flagellum and the mitochondria. 10. Proximal centriole Close to the base of the nucleus and is surrounded by pericentriolar material, which connects it to the nucleolar fossa and the distal centriole 11. Distal centriole Is in the same axis as the proximal cèntriole and forms the basal body of the axoneme. 12. Axial fiber Anterior portion is connected to the distal centriole. 13. Distal centriole A short peripheral fiber is associated with each of the nine triplets. 14. Mitochondria Surrounds the centrioles and the anterior part of the axoneme and the axial fiber. 15. Annulus Thin and distinct; present at the base of the midpiece and marks the beginning of the tail.

4F, 5E). By this time, at the posterior end, the less taining the less-condensed chromatin/nuclear mate- condensed portion begins projecting out beyond the rial (Fig. 6D). nuclear fossa. This is the beginning of the formation The flagellum consists of an axoneme and an axial of a redundant nuclear envelope (Fig. 5E). Contin- fiber lying in the cytoplasm surrounded by the ued condensation of nuclear chromatin results in an plasma membrane (Fig. 6E,F). The axoneme exhib- increase in the area of the nucleus containing the its the normal 9 ϩ 2 microtubular pattern. Proxi- condensed chromatin. The less-condensed chroma- mally, the axoneme and the elongated axial fiber are tin around the fully condensed area is a distinct closely juxtaposed. More caudally, the axoneme is feature and is heterogeneous (Fig. 5F). At one stage separated from the axial fiber by a bulbous undulat- in the condensation of chromatin, the heterogeneous ing membrane (Fig. 7A). An electron-dense material less-condensed portion appears obliterated, result- underlies the plasma membrane. The width of the ing in an almost empty space (Fig. 6A–C). By the undulating membrane decreases as it approaches time when most of the cytoplasm displaces towards the posterior end (Figs. 6F, 7A). The late phase also the caudal pole of the nucleus, the redundant nu- marks the displacement of most of the cytoplasm clear envelope projects further caudally, still con- and the mitochondria arrive at the region caudal to

TABLE 2. Features different between the sperms of the two species (Figs. 9–12) Ichthyophis tricolor Uraeotyphlus cf. narayani 1. The tip of the acrosome is pointed The tip of the acrosome is rounded 2. The basal homogeneous zone is larger The basal homogeneous zone is smaller 3. The granular and moderately electron-dense homogeneous zones The granular and moderately electron-dense homogeneous are less voluminous. zones are voluminous. 4. The granule size in the granular zone is smaller Granule size is bigger 5. The perforatorium is firmly adherent to the anterior tip of the The perforatorium terminates at the anterior end moderately electron-dense homogeneous zone abruptly, not establishing any contact with the moderately electron-dense homogeneous zone. 6. The subacrosomal material forms a continuous ring around the The subacrosomal material forms a discontinuous ring perforatorium around the perforatorium 7. The anterior end of the acrosomal vesicle is pointed and almost The anterior end of the acrosomal vesicle is larger, triangular and extends down into the subacrosomal area rounded and the granular zone is distinct 8. The periperforatorial subacrosomal material zone is more The periperforatorial subacrosomal material is moderately electron dense and homogeneous homogeneous 9. The layer that fills the endonuclear canal and surrounds the An electron-dense striated layer invests the posterior end perforatorium is continuous with the acrosomal seat of the perforatorium which is homogeneous and electron dense 10. A collar-like structure is not present The outer membrane and the acrosomal base plate fuse and form a collar-like structure. 11. The midpiece consists of well developed peripheral fibers The peripheral fibers are not well developed. 12. Mitochondria are flat and spiral around the tail Mitochondria are spherical, having an extensive array of delicate concentric cristae. 494 M. SMITA ET AL.

Fig. 9. Uraeotyphlus cf. narayani. TEM. A: Longitudinal anterior portion of sperm showing acrosome and nucleus Scale bar ϭ 1.5 ␮m. B–H: Successive transverse sections. Scale bar ϭ 2.5 ␮m (all to the same scale indicated). AV, acrosomal vesicle; EC, endonuclear canal; EZ, electron-dense homogeneous zone; GZ, granular zone; NU nucleus; SM, subacrosomal material; PM, perforatorium. the nucleus (Fig. 7B). The mitochondria are located and the annulus. Mitochondria have concentric or between the nucleus and the annulus, which starts array-like cristae with a central empty space. appearing at the junction between the differentiat- Electron-dense granules are present in the mito- ing middle and principal pieces of the flagellum (Fig. chondria. Glycogen/lipid granules are present 7C,D). At the nuclear fossa, the proximal centriole around the developing midpiece (Fig. 7E). Most of connects to the nucleus, whereas the distal centriole the cytoplasm accumulates as the residual body. As forms the attachment site for the axial fiber (Fig. a rough estimate, the size of the nucleus of the 7E). Mitochondria arrange as a spiral around the mature sperm is reduced to about 4% that of the axoneme and axial fiber, between the distal centriole nucleus of round spermatid. Mature spermatozoa SPERMIOGENESIS IN CAECILIANS 495

Fig. 10. Uraeotyphlus cf. narayani. TEM. A: Longitudinal section (LS) of nucleus and midpiece. Scale bar ϭ 1.5 ␮m. B,C: Transverse sections (TS) at posterior end of nucleus and mid piece. Scale bar ϭ 2.5 ␮m (all to the same scale indicated). D: LS of tail. Scale bar ϭ 5 ␮m. E: TS of tail. Scale bar ϭ 3 ␮m. AF, axial fiber; AX, axoneme; MI, mitochondria; NU, nucleus; UM, undulating membrane; ED, electron-dense material. are released into the lumen that appears in the cyst DISCUSSION (Fig. 7F) and are transferred to the collecting Spermateleosis in caecilians has been described ductule. using the light microscope by Seshachar (1943a, 1945), Exbrayat and Sentis (1982), and Exbrayat (1986a,b). To the best of our knowledge, ours is the Mature Spermatozoon (Fig. 8A,B) first ultrastructural description of spermateleosis in caecilians. Thus, our study reveals new details The general structure of the spermatozoa of Ich- about the formation of the acrosome through Golgi thyophis tricolor and Uraeotyphlus cf. narayani activity. The acrosomal vesicle forms as diffuse ma- have some differences in finer details (Tables 1, 2, terial, as in the anurans (Poirier and Spink, 1971), Figs. 9–12). The spermatozoa are filiform in both. rather than as acrosomic granules, as in amniotes 496 M. SMITA ET AL.

Fig. 11. Ichthyophis tricolor. Longitu- dinal section (LS) of sperm showing (A)of acrosome TEM. Scale bar ϭ 2 ␮m and successive transverse sections (B–I). Ab- breviations as in Figure 9. Scale bar ϭ 2.5 ␮m (all to the same scale indicated).

(Burgos et al., 1970). Seshachar (1943a, 1945), in a reported their shifting to the caudal region. The light microscopic study, made similar observations. proximal centriole becomes ring-like, while the dis- However, we found that a further increase in the tal one carrying the axial filament moves inwards size of the acrosomal vesicle is through the fusion of and penetrates the nucleus. Consequently, accord- small Golgi-derived vesicles. ing to Seshachar (1943a, 1945), it is the proximal The accumulation of a fibrous material between centriole that lies beneath the distal centriole. In the the outer and inner sheets at the acrosomal seat of present study, we observed that the proximal cent- the nucleus is not previously reported for caecilian riole alone establishes direct contact with the nu- sperm, but occurs during spermiogenesis in many cleus. The distal centriole connects to the proximal other animals, including elasmobranch fish (Stan- centriole. In fact, the axoneme, which connects to ley, 1971). However, in the latter the fibrous nuclear the distal centriole, likely extends towards the prox- sheath extends far beyond the length of the acroso- imal centriole (Scheltinga et al., 2003). Thus, the mal seat, which is not the case in other vertebrates, situation in caecilians appears to conform with that including caecilians. reported in higher vertebrates. Seshachar (1943a, 1945), while describing the The change of organization of the nucleus during changes in the centrioles during spermateleosis in spermateleosis, as observed in the present study, was Ichthyophis glutinosus and Uraeotyphlus narayani, suggested by Seshachar (1943a, 1945) for Ichthyophis SPERMIOGENESIS IN CAECILIANS 497

Fig. 12. Ichthyophis tricolor. TEM. Longitudinal section (LS) of nucleus (A) and midpiece (B). Scale bar ϭ 1.2 ␮m (all to the same scale indicated) and successive transverse sections (C–H). Abbre- viations as in Figure 9. Scale bar ϭ 1.5 ␮m (all to the same scale indicated). I: LS of tail. Scale bar ϭ 5 ␮m. J: TS of tail. Scale bar ϭ 2.5 ␮m. glutinosus, Uraeotyphlus narayani, Siphonops annu- dant nuclear envelope for ultimate inclusion into and latus, and Gegeneophis ramaswamii based on light shedding through the residual body. The material ex- microscopic observations. Condensation of chromatin cluded likely is fluid or nuclear proteins. During sper- through formation of nuclear fibrils is the pattern ob- miogenesis nuclear proteins are known to undergo served in other vertebrates. There are two unique as- tremendous changes, from lysine-rich to arginine-rich pects about nuclear condensation of spermatids in the (Courot et al., 1970). Seshachar (1943a) reported a two species of caecilians studied. Unlike in other ver- reduction of about 90% in nuclear volume and sug- tebrates, in which chromatin condensation occurs si- gested that nuclear chromatin of the sperm head ac- multaneously throughout the nucleus, in caecilians it counts for only a small quantity of its original volume. begins at the core of the elongating nucleus, concomi- In our rough estimate, nuclear size of the mature tant with the establishment of the endonuclear canal. sperm is greatly reduced, to about 4% of that of nu- Condensation extends gradually to the periphery, cleus of the round spermatids. Perhaps the pattern of while in the peripheral portion of the nucleus no con- chromatin condensation described earlier has a bear- densation occurs. This, together with the sequential ing on the amount of change in nuclear volume. change in the redundant nuclear envelope, suggests An aspect unique in the present study is the first that nuclear chromatin condensation results in exclu- appearance of the flagellum much before the round sion of a material from the nucleus through the redun- spermatid undergoes any trace of elongation. The 498 M. SMITA ET AL. incipient flagellum, containing at least the axoneme, This zone in I. tricolor is more electron dense than in is short and stumpy, and the axoneme by this time U. cf. narayani. The endonuclear canal is filled with has established contact with the distal centriole. a material that surrounds the perforatorium in both The mitochondria, on their association with the de- the species. This material is continuous with the veloping midpiece, organize into a spiral and the acrosomal seat. It is homogeneous and electron cristae become concentrically lamellate in Ichthyo- dense in I. tricolor but striated in U. cf. narayani. phis tricolor or array-like in Uraeotyphlus cf. As reported in earlier descriptions (van der Horst narayani. During this transformation, perhaps, the and van der Merwe, 1991a,b; Jamieson, 1999; Schel- cristae are displaced into the core due to accumula- tinga et al., 2003), the nucleus in the mature sperm tion of a previously unreported matrix. resembles a relatively short cylinder of constant di- The ultrastructure of mature sperm of Ichthyo- ameter, circular in cross section, with strongly con- phis tricolor and Uraeotyphlus cf. narayani gener- densed chromatin. The anterior tip of the nucleus is ally conforms to descriptions of sperms of these and indented to form the endonuclear canal, which is a few other species of caecilians, Typhlonectes na- longer in Ichthyophis tricolor than in Uraeotyphlus tans (van der Horst and van der Merwe, 1991a,b; cf. narayani.InI. tricolor sperm, the endonuclear Jamieson, 1999), Ichthyophis beddomei, Uraeo- canal is flask-shaped, with a fairly narrow apical typhlus species, and Gegeneophis ramaswamii end, and widens towards a deeper portion to become (Scheltinga et al., 2003). Regarding the acrosome of narrow again and rounded at the posterior end. On caecilian sperm, according to van der Horst and van the other hand, in U. cf. narayani sperm the apical der Merwe (1991a,b; adopted by Jamieson, 1999), half of the endonuclear canal has a uniform diame- the tip of the sperm of T. natans is curved and ter and it narrows to become a pointed base. Also, in hooked, as in the case of urodeles. On the other I. tricolor a distinct layer of cytoplasm invests the hand, in the light microscopic observations of Sesha- sperm nucleus, whereas in U. cf. narayani a cyto- char (1940, 1945), the sperm of Ichthyophis glutino- plasmic investment around the sperm nucleus is sus, U. narayani, Siphonops annulatus, and Gege- hardly discernable. neophis carnosus are spatulate. In our observation, the tip of the sperm of I. tricolor and U. cf. narayani ACKNOWLEDGMENTS is not hooked. In the sperm of I. tricolor the tip of the acrosome is pointed and in U. cf. narayani it is blunt The use of the ultracut and electron microscope and bulbous. Thus, the acrosome in both the species facility of the Wellcome Trust Research Center, is not spatulate at any point, as observed by Schel- Christian Medical College, and Hospital (CMC and tinga et al. (2003). H), Vellore, India, is appreciated. We thank Dr. In describing the sperm of Ichthyophis beddomei, Mark Wilkinson and Dr. David Gower of Natural Scheltinga et al. (2003) found the perforatorium to History Museum, London, and Dr. John G. Measey, end anteriorly in a barb and lacking contact with the Institute of Tropical Ecology, Paris, for support. electron-dense homogeneous zone of the substance of the acrosomal vesicle. In our observation of the LITERATURE CITED sperm of Ichthyophis tricolor, the perforatorium firmly adheres to the anterior tip of the electron- Burgos MH, Vitale-Calpe R, Aoki A. 1970. Fine structure of the testis and its functional significance. In: Johnson AD, Gomes dense homogeneous zone, which would provide for WR, Vandemark NL, editors. The testis. New York London: rigidity of the perforatorium. In fact, the dense ho- Academic Press. p 551–649. mogeneous zone constitutes a barrel-shaped struc- Courot M, Reviers MTH, Ortavant R. 1970. Spermatogenesis. In: ture. 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