Vol. 56, no. 1: 75-83, 2003
CARYOLOGIA
Structural characterization of nuclear phenotypes during Scinax fuscovarius spermatogenesis (Anura, Hylidae) CLASSIUS DE OLIVEIRA1, CARLOS ALBERTO VICENTINI3 and SEBASTIÃO ROBERTO TABOGA1, 2, * 1
Department of Biology and 2 Microscopy and Microanalysis Laboratoty, Institute of Bioscience, Humanities and Exact Sciences, IBILCE/UNESP, Rua Cristóvão Colombo 2265, Jardim Nazareth, 15054 000, São José do Rio Preto, SP, Brazil. 3 Department of Biologic Sciences, Faculty of Sciences, UNESP, Bauru, SP, Brazil.
Abstract - In anuran amphibian Scinax fuscovarius, the spermatogenesis occurs in structures called seminiferous loculi, in which germ epithelium is organized in spermatocysts. Each cyst contains cells in the same stage of cytodifferentiation. Characteristics of each cellular type and their groups made the identification and differentiation of the germ lineage cells possible. In the basis of the epithelium there are the spermatogonia I, the biggest cells and always associated with the Sertoli cell. After the phase of mitotic proliferation, the cysts containing variable number of spermatogonia II are originated, quite smaller and with cellular boundaries a little distinct. After differentiation and growth in volume, the spermatocytes I appear, the nuclei of which are spherical and with different degrees of compaction of the nuclear material. Starting the meiotic process, the spermatocytes II are originated, which by means of the second meiotic division become haploid cells, the spermatids I. These two last spermatocysts are very similar. In this phase, the cells will go through a prominent process of differentiation until they form the spermatids II, which are elongated and begin to be organized in bundles supported by prominent Sertoli cells. With the process of spermiogenesis, spermatozoa appear, usually observed in compact bundles with tails turned to the lumen and their heads fitted in their support cells. In more advanced stages, the spermatozoa can be observed free in the locular lumen, ready to follow the spermatic path. Key words: Anura, histology, Hylidae, nuclear shape, Scinax fuscovarius, spermatogenesis.
INTRODUCTION Among the anamniotes vertebrates, specially the anurans, there are a few papers on gametogenesis, mainly with the species of the neotropical regions. Although there is not a large number of amphibian, it comes true that the taxon presents reproductive strategies very diversified (DUELLMAN and TRUEB 1994). Thus this paper aims at the analysis of some morphological characteristics of the spermatogenetic lineage cells of Scinax fuscovarius. Histologically, testes are constituted by a convoluted seminiferous tubule network, responsible * Corresponding author: fax +55 17 2212390; e-mail:
[email protected]
for the spermatogenesis, surrounded by a fibrous connective tissue layer, the albuginea tunica (LOFTS 1974). In Hyla ranki (TABOGA 1990), Scinax fuscovarius (OLIVEIRA 1996) and also commonly described for another species, testes present this description and the germ tissue is arranged in seminiferous locule delimited by loose connective tissue; however, the formation of intratesticular septa does not occur. As a general characteristic of the histological architecture of the seminiferous elements in amphibian, the germ epithelium is organized in seminiferous locule in the Apoda (WAKE 1969) and Anura (DUELLMAN and TRUEB 1994) or in seminiferous ampoules in the Urodele (HILDEBRAND 1995). However, some authors use indistinctly the terminology “seminiferous tubule”.
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In the constitution of the testicular parenchyma, the germ tissue presents some distinct cellular types: spermatogonia located in the basis of the germ epithelium and, in the sequence of the differentiation, the spermatocytes, spermatids and spermatozoa can be identified; the latter are found in lumen or in adjacent regions. This epithelium has cystic organization, i.e., germ cell groups associate to the Sertoli cells, constituting spermatogenetic cysts or spermatocysts. Therefore, each unity group cells in the same differentiation stage and with a synchrony of development, a common characteristic to the amphibian spermatogenesis (WAKE 1969; LOFTS 1974; DUELLMAN and TRUEB 1994). Being restricted to the hylidae, it can be noticed that there are few studies which have analyzed the spermatogenetic process or the morphological characteristic of the male reproductive system, among these: genus Hyla (FOUQUETTE and DELLAHOUSSAYE 1977), genus Litoria (LEE and JAMIESON 1993), genus Litoria and Cyclorana (M EYER et al. 1997), Hyla japonica (TOYOSHIMA and IWASAWA 1984; LEE and KWON 1992), Hyla pulchella andina (MONTERO and
and TABOGA
PISANÓ 1992), Hyla ranki (TABOGA and DOLDER 1991), Pachymedusa dacnicolor (RASTOGI et al. 1988) and Scinax fuscovarius (=Hyla fuscovaria) (ALMEIDA and CARDOSO 1985; OLIVEIRA 1996; OLIVEIRA and VICENTINI 1998). Thus we have verified that the knowledge about the male reproductive system in the anuran, mainly in species of the family Hylidae, is still precarious. In this study, the histological organization of the seminiferous elements, the main characteristics of different cell types and spermatogenesis are described.
MATERIAL AND METHODS Ten adult males of the specie Scinax fuscovarius (Amphibia; Anura; Hylidae) were used, originating from an area of the Research Sector in Nourishment of Aquatic Organisms in UNESP (Botucatu, São Paulo, Brazil). The animals were obtained through night excursions in the period of June to December, in the age of reproductive activity of the species. As soon as captured, the individuals were anesthetized with ether, sacrificed and sent to the morphological analysis.
Fig. 1 – A – Seminiferous loculus in Scinax fuscovarius. (H/E, 850 X). Indicated: 1 – spermatogonia II – clear type; 2 and 3 – spermatogonia II – dark type; 4 and 5 – spermatocytes I; 6 – prophase in the first meiotic division; 7 and 8 – spermatocytes II in the second meiotic division; 9 – spermatids II. B – Indicated: spermatocytes I (1); spermatocytes II (2); prophase in the second meiotic division (3); spermatids I (4); spermatids II (5); and spermatozoa (6). (H/E, 850X).
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NUCLEAR SHAPE DURING SCINAX FUSCOVARIUS SPERMATOGENESIS
Animals were dissected through median incision and the reproductive system organs were exposed to dropping with the Bouin solution “in situ”. From these animals, small fragments of testes were fixed in Bouin (20 hours), washed and transferred to 70% alcohol solution. Then, the material was sent to the histological routine for dehydration, clarification and embedded in paraffin. From this material slits of 6µm were obtained to subsequent coloration with H/E, Trichromic of Masson and Mallory, analysis of the structures and documentation in light microscopy (Zeiss-Jenaval). The specimen used were fixed in 10% neutral formalin and duly preserved for other descriptions and as proof material.
RESULTS The seminiferous locule are structures bounded by delicate sustaining framework of connective tissue which encloses the germ epithelium
with spermatocysts, structures which group germ cells in distinct stages of differentiation keeping a close morphofunctional relation with the Sertoli cells (Fig. 1A). The analysis of the section of an single seminiferous loculus, in terms of the number and stage of germ epithelium cells differentiation, allow the identification of tens of germ cysts which present different sizes. This arrangement is valid for all the cysts, except for the primary spermatogonia or spermatogonia I which are the trunk cells of this lineage and are located isolatedly in the basis of the epithelium (Fig. 1). Since the beginning of the spermatogenesis, the spermatogonia I are prominent and are associated to the Sertoli cells, still with follicular cells aspects. Later, all the germ cysts will be formed sequentially, so in each loculus there will be spermatocysts in distinct differentiation stages (Fig. 2). Among the spermatogonial cells, three stages of differentiation were distinguished morpho-
Fig. 2 – Interstitial area occupied by several Leydig cells (L). Bordering the seminiferous loculus are some elongated cells, possibly fibroblasts (F). It’s observed also prominent Sertoli cells (S), of spherical nucleus and with an evident central nucleolus. (H/E, 1.850X). Indicated: 1 – spermatogonia I; 2 – spermatogonia II- clear type; 3 – spermatogonia II – dark type; 4 – spermatocytes I; 5 – spermatocytes II; 6 to 8 – spermatids II in progressive stages of differentiation; 9 – spermatozoa.
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and TABOGA
Fig. 3 – A – Locular periphery with spermatogonia I (1) associated to Sertoli cells with compacted nuclei (arrowhead). Indicated: spermatogonia II (2); spermatocytes I (3); spermatocytes II (4); spermatids II (5); and a bundle of spermatozoa (E) supported by prominent Sertoli cells. (H/E, 1.700X). B – Indicated: spermatogonia II – clear type (1) and dark (2); spermatocytes I (3) and II (4); spermatids I (5) and II (6). (H/E, 1.700X). C – Limit between two loculi, with some wall elongated cells (arrow) and many Sertoli cells (arrowhead). Indicated: Spermatogonia I (1); spermatogonia II – clear type (2) and dark (3); spermatocytes I (4); beginning of the spermatocytes I meiosis (5); spermatocytes II (6); spermatids II (7); and a bundle of spermatozoa (8). (H/E, 1.850X).
NUCLEAR SHAPE DURING SCINAX FUSCOVARIUS SPERMATOGENESIS
logically. The spermatogonia I are voluminous cells with irregular outline and multilobular aspect. The nucleus often presents chromatin granulation and a single, prominent and eccentric nucleolus. They are also the largest germ lineage cells and usually they are very near the locular wall and scattered (Fig. 2). Around it, the presence of one or more flattened cells is observed, these being follicular cells or Sertoli cells (Fig. 3A). The secundary spermatogonia or spermatogonia II already present similar size and form among the cells of the same cyst, being smaller than the spermatogonia I and the spermatocytes I (Fig. 2). When associated to the spermatogonia II, the Sertoli cell nuclei assume a more elongated form, becoming more conspicuous. As to spermatogonia II, we have identified two types morphologically distinct, a clear type and a dark one. The clear type spermatogonia II appear as a small “stained” cell group and always located near the locular wall. The cells present this aspect because they are originated from the spermatogonial proliferation, which consists of repeated mitotic divisions, and thus they are observed closely associated, forming small groups. The dark type spermatogonia II suffer alterations which make the nuclei very compacted and in “drops” shape, and also cytoplasmatic alterations, which are manifested by the low affinity to the colorings (Figs. 2 and 3). After some mophological alterations, the cells differentiate and give origin to the spermatocytes, which suffer two consecutive meiotic divisions, making possible the identification of the primary (I) and secondary (II) spermatocytes. The spermatocytes I suffer the first meiotic division, culminating with the formation of quite smaller cells, the spermatocytes II, which afterwards undergo the second meiotic division giving origin to the spermatids. Therefore, the spermatocytes present different degrees of nuclear compaction, which first occur in specific regions till it reaches the whole nucleus, a modification that makes it progressively smaller (Fig. 2). Meiotic division figures, like metaphases, are found sometimes; however, they are commonly observed in different stages of the prophase, since this is the longer duration phase of meiosis (Fig. 1). The spermatocytes I are relatively large cells and just smaller than the spermatogonia I. They have in their nuclei a chromatin which is looser
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or presents a slight degree of condensation. At this stage, the Sertoli cells are slightly elongated. The spermatocytes II are haploid cells and quite smaller than the previous ones. Because they enter quickly in the second meiotic division, these cells are rarely observed just after their formation; thus, usually, they are already in the meiotic prophase, forming cysts with a large cellular density (Fig. 3). The spermatid cysts present cell populations of very heterogeneous aspects, in which we find cells that vary from a spherical shape to an elongated one, making possible to distinguish two basic morphological types: round spermatids (I) and elongated spermatids (II). The spermatids I cysts are distinguished only from some spermatocytes II cysts, when they present some slight elongated cells (Figs. 1B and 3B). The spermatids II are cells whose nuclei suffer a big elongation, concomitantly to a progressive nuclear compaction. At this moment, the cystic organization begins to break in order to be arranged in bundles. The spermatids II are associated to very prominent Sertoli cells, which have a round nucleus, loosely distributed chromatin and with an evident nucleolus, usually in a central position. The Sertoli cells are easily identified when they are associated to the spermatids II and to the spermatozoa, since they are always turned to the locular periphery, while the compact bundle of germ cells are intensely dyed and are directed to the locular lumen (Figs. 2 and 3). The spermatozoa are arranged in bundles by the cytoplasmatic extensions of the Sertoli cells, so the proximal part or the spermatozoa head is stuck in these cells, a characteristic which is only ultra-structurally evident (unpublished data). The tail has a filamentous aspect and is dyed intensely by the Eosin; in the bundles the tail is free and turns to the locular center. These bundles are concentrated predominantly in the central region of the seminiferous loculus (Figs. 2, 3A and 3C). A progressive sequence of the cellular differentiation which occurs in the spermatogenetic process (Fig. 4), demonstrates that the alterations of a cell also occur in all the other cells of the same cyst. It is also possible to watch the alteration of the Sertoli cells which, first, have a follicular cell aspect (Fig. 4.1), afterwards assuming an oval shape (Fig. 4.10) and finally the nucleus becomes spherical and with a single central nucleolus (Figs. 4.11, 4.13 and 4.18).
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DISCUSSION In the amphibian, the spermatogenesis is cystic, i.e., the proliferation of the germ cells occurs
and TABOGA
in organized and well defined groups, which receive the denomination of germ cysts. In several other species, the cryptic spermatogenesis has also been described. It is fundamental character-
Fig. 4 – Distinct cytodifferentiation stages during the spermatogenesis process. The cellular types were separated based in the form, dimension and tinctorial affinity of the nuclei. The arrows (10, 11, 13 and 18) indicate Sertoli cells. (H/E, 850X). 1 – primary spermatogonia (arrowhead); 2 – secondary spermatogonia –clear type (arrowhead); 3 and 4 – spermatogonial differentiation; 5 – secondary spermatogonia – dark type; 6 – transition to spermatocytes; 7 – primary spermatocytes; 8 and 9 – prophase of the first meiotic division; 10 – secondary spermatocytes; 11 to 14 – prophase of the second meiotic division; 15 – round spermatids; 16 and 17 – spermatids in cellular elongating; 18 – elongated spermatids; 19 – immature spermatozoa; 20 – mature spermatozoa.
NUCLEAR SHAPE DURING SCINAX FUSCOVARIUS SPERMATOGENESIS
istic that is common to Amphibian testis. It is the event of spermatogenesis inside the follicular structures where each group of cells are the same stage of differentiation (LOFTS 1974; RASTOGI et al. 1988; BÁO et al. 1991). Also in Scinax fuscovarius the germ cyst consists of a agglomeration of germ cells which suffer a synchronic maturation, along with the supporting follicular cells or Sertoli cells. This structure is also described in other anamniotes vertebrates (GRIER 1992). In anuran like Hyla ranki (TABOGA 1990) and Scinax fuscovarius, which have a continuous spermatogenesis along all the breeding season, it becomes possible the identification of different cellular types in a single seminiferous loculus, i.e., all the germ lineage cells occupy the same loculus and differentiate simultaneously. According to LOFTS (1974), the primary spermatogonia are the largest germ lineage cells and rest on the basal lamina adjacencies of the seminiferous elements, associated with follicular cells. Similar observations were documented in Scinax fuscovarius and in another species: Bufo arenarum (CAVICCHIA and MOVIGLIA 1983); Caudiverbera caudiverbera (H ERMOSILLA et al. 1983); Pachymedusa dacnicolor (RASTOGI et al. 1988); Hyla ranki (TABOGA and DOLDER 1991) and Odontophrynus cultripes (BÁO et al. 1991). In Hyla ranki, the volume of the primary spermatogonia can reach the triple of the other cells in differentiation and its big nucleus, very irregular and multilobed, results in a reduction of the cytoplasmatic area (TABOGA 1990). The primary spermatogonia of Pachymedusa dacnicolor (RASTOGI et al. 1988) and Rana esculenta (RASTOGI et al. 1985) have a prominent size with the pleomorphic multilobuled nucleus. These two last species present the spermatogonia divided in two classes, in terms of morphological and tinctorial characteristics, thus being denominated: pale, usually larger and indifferentiated; and dark, which are smaller and are in differentiation. Our observations in Scinax fuscovarius allow us to assert that the spermatogonia characteristic are similar to those described for most of the anuran; however, based on morphological and tinctorials characteristics, we identified only one type of primary spermatogonia, which are large cells of multilobuled nucleus and located near the basal membrane of the seminiferous loculus. The spermatogonia I, through a mitotic process, are divided some times to form a cell group which stays united inside the cyst, whose cells
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have cytoplasm and basophilous nucleus and become the spermatogonia II. We believe that in the vertebrates which have a cystic spermatogenesis, the establishment of the germ cyst must occur in the beginning of the process and immediately after the first mitotic divisions. According to LOFTS (1974), the development of the secondary spermatogonia cysts define a “multiplication season”, in which a primary spermatogonia may have originated a cyst containing about two hundred cells. In Pachymedusa dacnicolor, all the divisions inside a spermatogonia cyst occur in synchronic way and the larger cysts contain more than two hundred cells, whose nuclei present a lobular aspect, which tend to a spherical format with the formation of the primary spermatocytes (RASTOGI et al. 1988). In the course of multiplication season, we identified two spermatogonia II groups containing tens of cells in Scinax fuscovarius. The clear type-spermatogonia II have clearer nuclei than their successors; however, they are observed as a “stained” aspect cell group and always located near the seminiferous loculus wall. The dark typespermatogonia II have cytoplasm with low affinity to the colorings and much more compacted and intensely dyed nucleus, which often has “drop” shape. This last cellular type forms germ cysts larger which can extend till the seminiferous loculus lumen. Among the more remarkable differences which occur during the formation of the spermatogonia I in II, the size diminution and the large cytoplasm reduction stand out, with a nucleus which becomes more and more lobuled until it reaches a spherical form during the transition of the secondary speramatogonia into the primary spermatocyte. According to L OFTS (1974), this last transformation marks the beginning of the cyst “maturation season”. The spermatocytes are usually observed in the prophase in the first meiotic division, with different degrees of chromosomal condensation. With the division of the spermatocytes I, much smaller cells are originated, the spermatocytes II, which have a diameter around half of their origin cells, as we notice in Scinax fuscovarius and as also described for Hyla ranki (TABOGA 1990) and Pachymedusa dacnicolor (RASTOGI et al. 1988). As to Caudiverbera caudiverbera (HERMOSILLA et al. 1983) and other species (LOFTS 1974), intercellular vacuoles are observed in the primary spermatocytes, which grow in size and adhere in
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a central vacuole, transforming the cyst in a hollow bladder with the spermatids, which initially are spherical and relatively small, arranged alongside the internal wall. In Scinax fuscovarius the formation of this central vacuole does not occur; however the cells present characteristics morphologically similar. After the meiosis, with the formation of the spermatids I, this spherical and relatively small cells start to elongate and, with the growing of the tail filaments, the cyst wall breaks, imprisoning them in the Sertoli cells. This organization in buncdles is related in several species and it is observed that the cells keep a fundamental morphofunctional relationship with the germ lineage cells, which is still more evident during the spermiogenic process (CAVICCHIA and MOVIGLIA 1983; RASTOGI et al. 1988; TABOGA 1990; BÁO et al. 1991). In Caudiverbera caudiverbera (HERMOSILLA et al. 1983), Odontophrynus cultripes (BÁO et al. 1991) and also Scinax fuscovarius, the spermatids I, in a first stage present a spherical nucleus and slender granulation, then the nuclei become oval and the granular chromatin is homogeneously distributed. The following stages are characterized by cellular elongation and compaction of the nuclear material, which occur simultaneously to the chromatinic condensation. The morphological aspects of the spermiogenesis have been described and discussed, in details, by many authors and in a broad variety of species, including the anuran (FURIERI 1975; RASTOGI et al. 1988; BÁO et al. 1991). Although these organisms present spermatozoa with large form varieties, they are usually constituted of four main structures: one terminal achrosome, better designated as achrossomal cover; a head, where the extremely compacted nucleus is located; medium or intermediary part (neck), which contains many mitochondria; and the tail or tail filament, with the flagellum arranged linearly with several accessory parts, mainly the axial rod (NICANDER 1970; POIRIER and SPINK 1971; FOUQUETTE and DELAHOUSSAYE 1977; DUELLMAN and TRUEB 1994). In regards to the spermatozoa head, some anuran present uncommon types: Bombina variegata (FURIERI 1975) is pear-shaped, with part of the flagellum on the head; Megophrys montana (ASA and PHILLIPS 1988) and Xenopus laevis (REED and STANLEY 1972) with helicoidal head; Racophorus arboreus and Racophorus schlegelii (MIZUHIRA et
OLIVEIRA, VICENTINI
and TABOGA
al. 1986) snail-shaped or spiral-shaped. For Chiromantis xerampelina (MAINOYA 1981), the snailshaped head may present ten to twelve spiral turns. In Scinax fuscovarius, the head has a high degree of chromatin condensation and intense cytoplasmatic reduction, its format is elongated and pointed, as described before in this species by ALMEIDA and CARDOSO (1985). Similar observations were made for Caudiverbera caudiverbera, Odontophrynus cultripes, Pachymedusa dacnicolor, Rana clamitans and Rana pipiens. Concerning the position and distribution of the germ cells along their differentiation inside the seminiferous elements, it’s not observed a sequence of distribution like the one described for the mammalians, except the primordial germ cells located near the locular wall and the spermatozoa in the luminar zone. This situation, which also occurs in Scinax fuscovarius and in many other species, is probably due to the cystic spermatogenesis which occurs in the anamniotes, which implies in a compartmentalization of the germ epithelium, although it occurs in a different way in the amniotes. According to GRIER (1992)’s observations about the chordates cystic spermatogenesis, the anuran have a structural factor which predicts an amniota testis evolution, which present one testis with post-spermatocystic tubular arrangement. According to ROSSA-FERES and JIM (1994), the breeding season of Scinax fuscovarius (=Hyla fuscovaria) occurs in the hot and wet season, based in the period in which the adult males vocalize and, mainly because of the frequency of the tadpole in development and egg-laying initial stage. Our observations collate these results from the specimen assessment period to the histologic analysis, since the gonad has always presented mature or in final phase of maturing spermatozoa. This fact suggests that the spermatozoa are being produced through a relatively long period and so contribute to the species. These ecological information are important in the morphofunctional analysis, since behavioral reproductive strategies can be varied in terms of the structures and the testicular behavior. Acknowledgments – This work was part of a Doctoral’s Thesis by C.O. who was granted a Fellowship from RUNESP (Brazil). The authors thank Angélica Farias Marini and Prof. Dr. Álvaro Luiz Hattnher (Dept. de Letras Modernas) for the English version.
NUCLEAR SHAPE DURING SCINAX FUSCOVARIUS SPERMATOGENESIS
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