Rev. Chi!o Anat., 20(3):263-268,2002.
MORPHOLOGICAL OBSERVATIONS ON THE TESTES Physalaemus cuvieri (AMPHIBIA, ANURA) ESTUDIO
MORFOLÓGICO
DE LOS TESTÍCULOS
CIassius de Oliveira;
Cristiani
OF
DE Physalaemus cuvieri (AMPHIBIA, ANURA)
Zanetoni
& Rodrigo Zieri
OLIVEIRA, C.; ZANETONI, C. & ZIERI, R. Morphological observations on the testes of Physalaemus cuvieri (Amphibia, Anura). Rev. Chi!oAnat., 20(3):263-268, 2002. SUMMARY: In general, the testes in the anurans are paired ovoid organs, constituted by a mass of seminiferous structures surrounded by a layer of fibrous connective tissue, which holds a germ epithelium with characteristic cellular types. This study describes the testicular histoJogical architecture and anatomy, as well as the organization and morphology of germ cells. Five male samples, from the Botucatu area (São Paulo State, Brazil), of Physalaemus cuvieri (Leptodactylidae) were used. After macroscopic analyses and obtainment ofthe testicularfragments, the material was submitted to the histological routine to inclusion in paraffin and coloration with haematoxylin/ eosin. Arare peculiarity is the presence of numerous pigment-containing cells randomly distributed in the albuginea tunic and testicular interstitium, giving the testis a dark brown coloration. In the germ tissue the spermatogonia I are biggest spermatogenetic cells. With the cellular differentiation and proliferation, succeded the other cellular types (spermatogonia lI, spermatocytes I and lI, spermatids I and JI, and spermatooza) with a cystic organization, that is, groups of cells associated with Sertoli cells, forming the spermatogenetic cysts or spermatocysts. The spermatogenetic lineage cells were differentiated and identified according to the cellular and cystic morphology. KEY WORDS: 1. Anura; 2. Leptodactylidae;
3. Physalaemus cuvieri; 4. Testis; 5. Spermatogenesis.
INTRODUCTION
In the anurans, the testes are paired ovoid organs constituted by seminiferous tubules convoluted surrounded by the fibrous connective tissue, which canstitutes the albuginea tunic. Cancerning to the histological architecture of the seminiferous elements, to the amphibians and, in a general sense, the germ epithelium may be arranged in seminiferous locules in the Apoda (Wake, 1969) and Anura (Duellman & Trueb, 1994) ar in seminiferous ampoules ar testicular lobules the Urodela. The germ tissue, which constitutes the testicular parenchyma, has different cellular types: spermatagonia in the epithelium boundary; spermatocytes and spermatids in the sequence af the cellular differentiation; and spermatozoa in the lumen or in their adjacencies. In this epithelium, there is a cystic arrangement, that is, groups of sexual cells assaciated with the Sertoli cells forming spermatagenetic cysts or spermatocysts. Therefore, each one of these units c\usters cells
in the same stage of differentiatian and with a synchronism develapment, common characteristic in the amphibians (Wake; Lofts, 1974; Rastogi et ai., 1988; Oliveira et ai., 2002). There are few researches about the organs and structures that constitute the anuran male repraductive system, specifically in animaIs of neatropical areas, like in Brazil. Beside that, even if there is not a large number of amphibians in this taxon, several different reproducti ve strategies will occur (Duellman & Trueb), which seem ta be suggestive afmorphological and functional variations in the reproductive organs. Thus, this study tried to analyze the testes morphological arrangement, describing histological and anatomical aspects, as well as some structural characteristics af the germ cells and their cystic arrangement in the anuran Physalaemus cuvieri (Fitzinger, 1826 - Leptodactylidae).
Department of Biology - Instituto de Biociências, Letras e Ciências Exatas - UNESP, São José do Rio Preto - São Paulo State, Brazil.
263
OLIVEIRA, c.; ZANETONI, C. & ZIE)H, R. Morphological observations on the testes of Physalaemus euvieri (Amphibia, Anura). Rev. Chil. Anaf., 20(3):263-268, 2002.
MATERIAL AND METHOD
Five male samples of Physalaemus cuvieri were used. The specimens were collected in Botucatu (São Paulo State - Brazil), between June and December, when they were in the reproductive activity period. After captured and transported to the laboratory, the individuaIs were anesthetized with ether and submitted to
A
morphological studies. The animaIs were opened through medium incision from the cloaca as far as anterior limbs, exposing the reproductive organs to macroscopic analyses. After the reduction of the testes in small pieces, they were immediately immersed in Bouin fixative solution during 20 hours, washed, and transferred to 70% ethanol solution. Then, the .material was sent to the histological routine to be dehydrated in ethanol, clarified with xylol, and embedded in paraffin. Sections of 6 ~m were stained with haematoxylin/eosin for histological analyses, The individuaIs were appropriately preserved as proof material.
RESULTS
Concerning the gonad macroscopic aspect of Physalaemus cuvieri, as to color, shape, and size, we can conspicuous anatomic variations. However, histologically there is not a difference in the germ epithelium arrangement. Covering the testis we can find a thin capsule of connective tissue and smooth muscle, the albuginea tunic. The blood vessels, which exist in this testicular capsule, are maínly destined to the parenchyma, where a germ epithelium is arranged in seminiferous locules, Delimited by a loose connective tissue, these locules form morphological units, that is, the seminiferous
264
Figure I, A) Testes anatomic aspect of the Physalaemus cuvieri with dark brown pigmentation in the albuginea tunic, Dissected gonad ( ) putting in evidence the internal pigmentation and the units denominated seminiferous [ocules (27x). B) Histology of the seminiferous locule (HlE, 215x) and C) Generalized representation of the cystic germ epithelium: locular wall (I); interlocular tissue (2) with Leydig cells, blood capillary and pigment-containing cells; primary (3) and secondary (4) spermatogonia; primary (5) and secondary (6) spermatocytes; primary (7) and secondary (8) spermatids; spermatozoon bundles (9) and Sertoli cells (10).
Each seminiferous locule has a germ tissue with a lot of spermatogenetic cysts or spermatocysts, that is, c1usters of germ celIs in different stages of ditlerentiation that occur due to a intimate morphofunctional relation with the Sertoli cells. In the cysts, cells are joined in the same differentiation status, therefore, with a development synchrony. Some cysts seIdom present cells with small differences among themselves and, since these asynchronisms are very small, we consider the cysts as a cellular group in which ali the sexual cells are in the same stage of differentiation (Figs. 1 and 2). The different
cellular
types were differentiated and identified according to the morphology and cystic arrangement (Fig. 2), also represented schematicalIy (Fig. 1c).
Spermatogonia af
-
differentiation
three stages were
distinguished. The primordial spermatogonia or primordial germ cells are the most voluminous cells of the spennatogenetic lineage, whose nuclei are irregular and with a multilobular aspect. They present chromatinic granulations, beside a single, eccentric nucleolus. In general, they are very c10se to the locular walI, and are associate to the Sertoli cells which stilI have Figure 2. Detail 01'the histologicaJ arrangement 01'the semini1'erous 10cuJi: I) spermatogonia I; 2) spermatogonia lI; 3) spermatocytes I; 4) spermatocytes 11; 5) spermatids I; 6) spermatids lI; 7) spermatozoon bundles; 8) spermatozoon in the locular lume. (H/E, A and B - 430x; C and D - 850x).
elements of the gonads. In Physalaemus cuvieri, the interlocular tis sue is relatively scarce, presenting a pigmentation in this tissue and also in the albuginea tunic, giving the testis a dark brown coloration. Between the seminiferous units, they have a inter-Iocular tissue composed by Leydig interstitial cells, fibroblasts, pigment-containing cells, blood vessels, and some efferent ductules (Fig. I).
aspect of folIicular cells and the nuc1eus is falciform. It is also common that t~ese spermatogonia be isolated or joined in smalI groups, but this do not
characterize genn cysts. From mitotic divisions, the primary (I) and the secondary (11)spermatogonia are originated. The secondary ones are smaller, more irregular, and they were identified due to the nuclear compactation degree. The spermatogonia I are organized as celIular cysts with a irregular aspect andhard delimitation, they are located near the locular wall. The spennatogonia 11suffer some aIterations, which make thenuc1ei to become more compacted and give 265
OLIVEIRA,
c.; ZANETONI,
C. & ZIERI,
R. Morphological
observatioos
00 the testes of Phvsalael11l1s ellvieri (Amphibia, Ao"ra).
ReI'. Chil. Anat., 20(3):263-268,
2002.
them typical shapes. The Sertoli cells, when associated with the spermatogonia lI, present a nucleus with more elongated shape, still semilunar or falciform.
pigment cells can be found in the different organs, constituting an extracutaneous pigmentary system of unknown function (Zuasti et ai., 1998).
Spermatocytes - they are the result from the spermatogonia II ditferentiation, the spermatocytes I are cells just smaller than the spermatogonia I. In its nucleus, the chromatin is slightly condensed. The spermatocytes II are haploid cells and quite smaller than the previous, and they are originated in the first meiotic division. Generally, the spermatocytes are observed in different phases of the first meiotic division, presenting different degrees of compactation of the nuclear material. In this stage, the Sertoli cells are more voluminous, the nuclei are less condensed and present the tendency to assume intermediate shape, between elongated and ovoid.
Concerning the general histological architecture observed in Physalaemus cuvieri, it was verified a great similarity to the descriptions of the other species of same family, like Physalaemus fuscomaculatus (Aoki et aI.) and Caudiverbera caudiverbera (Hermosilla et aI., 1983); and also with species of the Hylidae family - Hyla ranki (Taboga & Dolder, 1991), Hyla japonica (Lee & Kwon, 1992); Hyla pulchella andina (Montero & Pisanó, 1992); and Scinax fuscovarius (Oliveira & Vicentini, 1998; Oliveira et al.). In the species that show a continuous spermatogenesis for ali the reproductive period, differerH cellular types may be identified in only one seminiferous locule, that is, ali the cells of the germ lineage occupy the same locule and differentiate themselves simultaneously.
Spermatids
-
when the spermatocytesII pass throughthe
second meiotic division, they form the spermatids. These, in the primary stage or as round spermatids I, are differentiated from the cysts of the spermatocytes II when some cells show themselves slightly elongated. The spermatids II or elongated are cells with compacted and elongated nuclei, whose cystic arrangement is altered to be organized in bundles sustained by the Sertoli cells. In this moment, the nucleus of the Sertoli cells finds itself as oval or rounded, with chromatin loosely distributed, besides the evident and central nucleolus. Spermatozoa - they are characterized by a extraordinary nuclear compactation and a cytoplasmic reduction. The spermatozoon head, represented mainly by the nucleus, is slender; and, in general, it turns to the nucleus of the Sertoli cell, in opposition, the tail has a filamentous appearance and is slightly stain, and, generally, turns to the luminar area of the seminiferous locule. The spermatozoa in development are arranged in bundles very well organized, due to the association with the Sertoli cells. When they reach maturity, they are released from the bundle and reach the locular lumen and the etferent ductule.
DISCUSSION
The unusual dark brown pigmentation of the testes of Physalaemus cuvieri is a peculiarity, which occur due to a presence of numerous pigmented cells (melano-macrophages or typical melanocytes?) randomly distributed in the testicular capsule and interstitium. This unusual characteristic, a testicular pigmentation, rarely is observed in others species, as described in Physalaemus fuscomaculatus (Aoki et aI., 1969) and Bombina bombina (Gollmann et aI., 1993). In the anuran Xenopus laevis, and other lower vertebrates, the
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To Leptodactylids, Telmatobius laticeps and Telmatobius pisanoi (Montero & Pisanó, 1990), the histological arrangement is similar, however a potentially continuous cycle occurs, considering that there is a well-defined phase of the intense spermatogenetic activity and other of rest, as it was also described to the hylidae: Hyla pulchella andina (Montero & Pisanó, 1992). In anurans like Nectophrynoides occidentalis (Zuber- Vogeli & Xavier, 1966), a temperate area bufonidae, the cycle is discontinuous, it influence even more in the gonadal activity and consequently in the dynamic of spermatogenesis. For the neotropical area species, occurs preferentially a continuous reproduction, in which ali the cellular types are present for whole year. With the cystic arrangement, the spermatogenetic lineage cells together with the Sertoli cells form delimited groups by a membranous capsule, which is denominated germ cyst or spermatocyst. We stress the fact that independently of the reproductive cycle type of the species, the cystic arrangement of the germ cells is confirmed as an important characteristic of the anuran amphibians, as well as to other anamniotes vertebrates (Lofts; Grier et aI., 1980; Grier, 1992). In a similar way to what was obtained for Bufo woodhouse (Atherton, 1974), Caudiverbera caudiverbera (Hermosilla et ai.) and Scinaxfuscovarius (Oliveira & Vicentini), we also do not observe any specific area of the testis where predominate certain cellular kinds. It is corroborated by the apparently random distribution of the spermatocysts in the seminiferous structures. Concerning the spermatogenetic lineage cells, the primordial spermatogonia are the biggest cells and rest on the adjacencies of the basallamina of the seminiferous structures, associated with the follicular cells (Lofts). As to the
OLIVEIRA,
C.; ZANETONI,
C. & ZIERI,
R. Morphological
observations
on the testes of Physalaemus
spermatogonial types, similar observations to the Bufo arenarun (Cavicchia & Moviglia, 1983), Hyla ranki (Taboga & Dolder) and to the Scinaxfuscovarius (Oliveira et ai.) were also verified in the species of this research.
cllvieri (Amphibia, Anura).
Rev. Chi!o Allal., 20(3):263-268.
2002.
At the end of the spermatogenesis, the spermatozoa remain united in the bundles, they are still sustained by the Sertoli cells, however with their liberation to the lumen of the seminiferous locule, the arrangement is dissolved and these remain in the spermatic path until the liberation.
The spermatocytes generally are observed in the prophase of the first meiotic division, with different levels of chromatinic and chromosomal condensation. The spermatocytes l are commonly bigger than the spermatogonia II (Lofts; Rastogi et ai. and Oliveira et ai.). With the spermatocytes l division, smaller cells are originated, the spermatocytes lI, which present half the diameter of the origin cells, as we observed in the Physalaemus cuvieri.
Although the architecture of the germ epithelium of ali mentioned species represents a general and typical model for the anurans, the final germ cell- the spermatozoon - shows a great heterogeneity in the species. This can be related to phylogenetic aspects (Kwon & Lee, 1995) and to reproductive strategies of the anurans (Duellman & Trueb).
ln Caudiverbera caudiverbera (Hermosilla et ai.), Odontophrynus cultripes (Báo et ai., 1991), Scinaxfuscovarius (Oliveira et ai.) and in our analysis, the spermatids l, in a first stage, show a spherical nucleus and thin granulation, after the nuclei become oval and the granular chromatin is distributed homogeneously. The following stages are characterized by a cellular and nuclear elongation, which occur simultaneously with the chromatinic condensation, culminating with the spermatids II formation.
ACKNOWLEDGMENTS : We are grateful to the CNPq for the award of Scientific lnitiation Fellowship (PIBlC-CNPq 96 to 98), to Cristiani Zanetoni, to Umberto Jorge Alves de Andrade for schematizing in translucent tracer paper, to Roberta Faria Femandes and Prof. Dr. Álvaro Luiz Hattnher for the translation into English, to Profa. Dra. Rosa Maria da Silva and Dra. Roxana Guadalupe Herrera Álvarez for translation into castellano, and to Prof. Dr. Sebastião Roberto Taboga for the suggestions to the manuscript.
OLIVEIRA, c.; ZANETONI, C. & ZIERI, R. Estudio morfológico de los testículos de Physalaemus cuvieri (Amphibia, Anura). Rev. Chi!oAnat., 20(3):263-268, 2002. RESUMEN: Generalmente, los testículos de anuros constituyen órganos ovoides, pares, formados por masa de estructuras seminíferas, envueltas en una capa de tejido conjuntivo fibroso, que contiene un epitelio germinativo provisto de tipos celulares característicos. En este trabajo se describe Ia anatomía y Ia arquitectura histológica de los testículos, así como también Ia organización y morfología de Ias células germinativas. Se utilizaron cinco ejemplares machos de Ia especie Physalaemus cuvieri (Leptodactylidae), provenientes de Ia zona de Botucatu (São Paulo, Brasil). Tras el análisis macroscópico y Ia obtención de los fragmentos testiculares, el material se sometió a Ias técnicas histológicas rutinarias para su inclusión en parafina y coloración con hematoxilina/eosina. Se constató como rara peculiaridad Ia presencia de numerosas células con pigmento distribuidas de forma aleatoria en Ia túnica albugínea e intersticio testicular, Ias cuales otorgan ai testículo una coloración marrón oscura. En el tejido germinativo Ias espermatogonias I son Ias mayores células espermatogenéticas. En Ia secuencia de Ia diferenciación y proliferación ce]ulares, se siguen ]os demás tipos de células (espermatogonias lI, espermatocitos I y lI, espermátidas I y lI, y espermatozoides) que presentan una organización cística, es decir, grupos de cé]ulas se asocian a ]as célu]as de Sertoli, formando ]os cistos espermatogenéticos o espermatocistos. Se diferenciaron e identificaron Ias células dellinaje espermatogenético, según ]a morfología de ]as células y dei propio cisto. PALABRAS CLAVE: 1. Anura; 2. Leptodactylidae; 3. Physalaemus cuvieri, 4. Testículo; 5. Espermatogénesis.
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OLIVEIRA,
C.; ZANETONI,
C. & ZIERI,
R. Morphological
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Corresponáeru:e to: Prof 'Dr. C[assius áe Ofiveira 'Department of 'Biofogy Instituto áe 'Biociêru:ias, Letras e Ciêru:ias 'hcatas 'llr;{'E5P C'EP: 15040--000 São José áo 'Rio Preto São Paufo State,
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