MORFOLOGIA

MORPHOLOGICAL CONSIDERATIONS ON THE SEMINIFEROUSSTRUCTURES AND TESTES OF ANURAN AMPHIBIANS:

Bufo crucifer, Physalaemus cuvieri AND Scinaxfuscovarius

Classius de Oliveira I

Aline Cristina Sant' Anna2Paula Munhoz de Omena3

Lia Raquel de Souza Santos4Rodrigo ZierP

ABSTRACT

This study describes the testicular anatomy and the general morphology ofthe seminiferous elementsin three species 01'anurans: Bufo crucifer (Wied, \82\), Physalaemus cuvieri (Fitzinger, \826), ScinaxfÚscovarius (Lutz, 1925). Six samples 01'each species were used and, after a macroscopic examination,their testes were submitted to microscopic analysis. Anatomically, the testes of those three species haddistinct patterns as to shape, size, and color. An unusual characteristic was the presence of pigment-containing cells in the testis 01'P cuvieri, showing a dark brown coloration; pigmented cells were presentonly in the Bidder's organ 01'B. crucifer; however, this pigmentation did not occur in S. jÚscovarius. Wedescribed the histological structure and identified a general organization 01' cellular types 01'spermatogenetic lineage, which were similar among the species. Other morphological aspects 01' themal e gonads were compared with species 01'anurans.

Key words: Anura, Bufo cntc/fer, Physalaemus cuvieri, ScinaxjÚscovarius, testis, spermatogenesis.

RESUMO

Considerações morfológicas sobre os testículos e as estruturas seminíferas de anfíbios anuros: Bufocrucifer, Physalaemus cuvieri e Scinaxfuscovarius

Este trabalho descreve aspectos anatômicos e apresenta uma caracterização morfológica geral doselementos seminíferos, quanto à organização cística do epitélio germinativo, de três espécies de anuros:Bufo crucifer (Wied, 182\), Physalaemus cuvieri (Fitzinger, \826) e Scinax fuscovarius (Lutz, 1925).Foram utilizados seis exemplares de cada espécie que, após as descrições macroscópicas, foram subme-tidos à rotina histológica para análises microscópicas. Anatomicamente, os testículos dessas espéciesapresentam diferentes padrões quanto à forma, ao tamanho e à coloração. Uma característica que sedestaca é a presença de células contendo pigmento no testículo de P cuvieri, conferindo a coloraçãomarrom escura ao órgão, interna e externamente; uma discreta presença de pigmentação ocorre apenasno órgão de Bidder de B. crucifer; entretanto, nenhuma pigmentação ocorre em S. fuscovarius. Emrelação aos aspectos microscópicos também foram analisados, a arquitetura histológica testicular e osdiferentes tipos celulares da linhagem germinativa e conclui-se que, de modo geral, são muito seme-lhantes. A partir da análise do epitélio germinativo cístico desses animais discutimos a formação e odesenvolvimento dos cistos espermatogenéticos em Anura.

Palavras-chave: Anura, Bufo crucifel~ Physalaemus cuvieri, Scinax fuscovarius, testículo, esper-matogênese.

Recebido em: 18.1102; aceito em: 30.04.03.

I Coordenador do Laboratório de Anatomia Comparativa, Departamento de Biologia, Instituto de Biociências, Letras e Ciências Exatas (lBILCE) -

UNESP. CEP 15054-000, São José do Rio Preto, São Paulo, Brasil - E-mail:classius@bio.ibilce.unesp.br2 Discente de Ciências Biológicas, IBILCE. Bolsista PET/SESu-MEC.) Discente de Ciências Biológicas, IBILCE.4 Discente de Ciências Riológicas, UFMS, Três Lagoas.5 Mestrando do Programa de Pós-Graduação em Biologia Animal, IBILCE.

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40 OLIVEIRA, C de, et aI.

INTRODUCTION

In general, anatomically, in anurans the testes arerounded, compact and yellowish, They can presentanatomic variations in form and weight accordingto the reproductive period (OUELLMAN; TRUEB,1994), beside other morphofunctional alterations re-lated to the seasonality of reproduction (LOFTS,1974), as it was described for some species(MONTEIRO; PISANÓ, 1990, 1992; HUANG et aI.,1997; OLIVEIRA; VICENTINI, 1998),

Histologically, the testes are covered with a thincapsule ofconnective tissue, the albuginea tunic, withits traverse for blood vessels, which go to the testicu-lar parenchyma, Th is parenchyma presents a germtissue arranged in seminiferous locules delimited bya loose connective tissue, however an intratesticu-lar septa fonnation does not occur (OLIVEIRA;VICENTINI, 1998),

In vertebrates, the germ tissue is disposed in ahollow structure - e.g. the seminiferous tubules in theamniotes, saclike seminiferous ampoules in the cy-clostomes and urodeles or intermediate structures in

some other groups (HILOEBRANO, 1995), Accordingto Romer and Parsons (1985), the arrangement ofthis tissue, in fish and amphibians, defines some-what spherical structures, the seminiferous ampoule,In amphibians, the terminology used for the des-cription ofthis genninative compartment may change(GRIER, 1992; OLIVEIRA; VICENTINI, 1998),However, as a general characteristic of the histologi-cal architecture of the seminiferous elements in

amphibians, the germ epithelium is organized inseminiferous locules in Apoda (WAKE, 1969) andAnura (OUELLMAN; TRUEB, 1994; OLIVEIRA;VICENTINI, 1998) or in a seminiferous ampoule inUrodela (HILOEBRANO, 1995).

In the testicular parenchyma, the germ tissuepresents spermatogonias located in the base of theepithelium, and, in the sequence of cytodifferentiatioil,spermatocytes, spermatids, and spermatozoa, wh ichare generally found near the lumen. This epitheliumshows a cystic arrangement, i.e., groups of germ cellsjoin with the Sertoli cells forming spermatocysts, acommon characteristic in the amphibians (WAKE,1969; LOFTS, 1974; OLIVEIRA et aI., 2003), as wellas in other anamniotes (GRIER, 1992).

Concerning the morphology of the seminiferouslocules and the cystic arrangement of the germepithelium, we can stress the contributions in thefollowing families: Bufonidae - Nectophrynoides

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occidentalis (ZUBER- VOGELI; XAVIER, 1966);Bufo woodhousei (ATHERTON, 1974); B. arenarum(CAVICCHIA; MOVIGLIA, 1982); B. regularis(ABOELMAGUID; SABRY, 1987); genus Telmatobius(MONTERO; PISANÓ, 1990) and B. melanostictus(HUANG et aI., 1997); Hylidae - Pachyme-

dusa dacnicolor (RASTOGI et aI., 1988); Hylaranki (TABOGA; OOLOER, 1991); H andina(MONTERO; PISANÓ, 1992); H japonica (LEE;KWON, 1992) and Scinax fuscovarius (OLIVEIRA;VICENTINI, 1998; OLIVEIRA et aI., 2003);Leptodactylidae - Physalaemus fuscomaculatus(AOKI et aI., 1969); Caudiverbera caudiverbera(HERMOSILLA et aI., 1983); Odontophrynuscultripes (BÁO et aI., 1991); Bombina bombina(GOLLMANN et aI., 1993); genus Physalaemus(AMARAL et aI., 1999) and Physalaemus cuvieri(OLIVEIRA et aI., 2002).

Thus, we describe some morphological characte-ristics of the seminiferous locules and testes in three

different anuran species of distinct families. Whileanalyzing and comparing these aspects in the referredspecies, we establ ished a generalized description foranurans.

MATERIAL AND METHOD

Five adults of each species were used: Bufonidae- B. crucifer (Wied, ] 82]), Leptodactylidae - Pcuvieri (Fitzinger, 1826), and Hylidae - S fuscovarius(Lutz, 1925). The specimens were collected in Botu-catu (São Paulo State - Brazil), between June andOecember, when the individuais are easily found intheir natural habitat because they were in thereproductive activity period.

The animais were dissected and submitted to

morphological studies, after anesthesia with saturationin ether. The animais were opened through mediumincision exposing the reproductive organs to macros-copic analyses. After the reduction of the testes insmall pieces, they were immediately immersed inBouin fixative solution during 20 hours, washed, andtransferred to 70% ethanol solution. Then, the materialwas sent to the histological routine to be dehydratedin increasing concentration of ethanol, submittedto tissular cIarification with xylol, and embeddedin paraffin. Sections of 6 mm were stained withhaematoxylin and eosin for histological analyses. Themorphological testicular arrangement was examinedwith the light microscopy (Zeiss-Jenaval). The indi-viduais were appropriately preserved as proof mate-rial (collectionnumbers: B. crucifer-OZSJRP 6013,

Morphological considerations on the seminiferous . 41

6014,6015,6016,6017,6018; P cuvieri - 6019,6020,6021, 6022, 6023, 6024; S. fuscovarius - 6004 withfifteen samples).

RESULTS AND DISCUSSION

In vertebrates like mammals, the testes are com-prised ofthe albuginea tunic which emits septa, de]i-miting incomplete, strongly marked lobulations whichshelter the seminiferous tubules (HILDEBRAND,1995). ln those animais, the tubules are typicallypermanent structures which produce sperma-tozoa during the reproductive period (ROMER;PARSONS, 1985). To the anurans the structurescontaining the germ epithelium are defined as semi-niferous locules and the testes are not lobulated,common in many urodeles and apodes (DUELLMAN;TRUEB, 1994). In the three anuran species analyzed- B. cruc(fer,P cuvieri, and S.fuscovarius, the testisdo not have septation and the seminiferous unit is alsodefined as seminiferous locules, because histologicallythey do not show a typical tubular aspect similar tothe mammals (OLIVEIRA; VICENTINI, 1998). InH ranki the structure was defined as locular-tubule

(TABOGA; DOLDER, 1991).As established, the species were collected during

the reproductive period, i.e., when the individuaiswere in reproductive activity. The spermatogeneticactivity was afterward confirmed by means of analysisof the histological testicular characteristics. Anato-mically, there were great differences related to thecolor, size, and shape ofthe gonads: B. crucifer had acylindric, milk-white testis with pigmented ovariantissue at the anterior end, Bidder's organ; P cuvieripresented ovoid testis with dark brown pigmentation,including internally; S. fuscovarius had in generalovoid and milk white testis (Fig. 1). A testicularpigmentation is also described for P fuscomaculatus(AOKI et aI., 1969), B. bombina (GOLLMANN et aI.,1993) and P cuvieri (OLIVEIRA et aI., 2002). Thesepigment cells are found in different organs, consti-tuting an extracutaneous pigmentary system ofunknown function (ZUASTI et aI., 1998). Despitethese interspecific anatomic differences, there aremorphologic similarities in the organ, mainly relatedto histological characteristics.

Externally, the gonads show a granular aspect dueto the seminiferous locules, which are observed dueto albuginea tunic transparency. The blood vessels,which exist in this testicular capsule, are mainlydestined to the parenchyma (Figs. 1 and 2). In these

units, the germ epithelium has cellular groups (cystor germ follicles) of ali kinds of cells of sper-matogenetic lineage. This cystic arrangement is acommon characteristic in the amphibians (WAKE,1969; LOFTS, 1974; DUELLMAN; TRUEB, ]994;OLIVEIRA et aI., 2002, 2003). However, it is not anexclusive one because it occurs in other anamniotes(GRIER, 1992). Internally, between the seminiferousunits, they have an interlocular tissue composed byLeydig cells, fibroblasts, blood vessels and someefferent ductules (Fig. 3).

During the cytodifferentiation, the germ cells areintimately associated to the Serto]i cells making cyst:primary (I) spermatogonia, isolatedly and in theepithelium base; secondary (11) spermatogonia, cystswith high degree of variation in the cellular popu-lation, proceeding from mitosis of the previous cellsand, in general, they are observed in the locularperiphery. Primary (I) and secondary (11) sperma-tocytes and primary or round (I) and secondary ore]ongated (11) spermatids, all of them present a greatcellular population, variable localization, and pecu-liar morphologic aspecto Finally, the spermatocystsarranged in spermatozoon bundles near the central areaof the locule, frequently present free spermatozoa inthe lumen of the seminiferous locule (Figs. 3 and 4).

Except for some characteristics of the cells andspecie-specific cystic arrangement, this description, ingeneral, occurs in the three species, and it was reportedin a similar way to some species ofthe same families:Bufonidae - B. arenarum (CAVICCHIA;MOVIGLIA,1982), B. melanostictus (HUANG et aI., 1997),B. regularis (ABDELMAGUID; SABRY, 1987),B. woodhousei (ATHERTON, 1974), N. occidentalis(ZUBER-VOGELI; XAVIER, 1966) and genusTelmatobius (MONTERO; PISANÓ, 1990); Hylidae- H andina (MONTERO; PISANÓ, 1992), Hranki (TABOGA; DOLDER, 1991) P dacnicolor(RASTOGI et aI., 1988) and S. fuscovarius(OLIVEIRA; VICENTINI, 1998; OLIVEIRA etaI., 2003); Leptodactyl idae - C. caudiverbera(HERMOSILLA et aI., 1983), o. cultripes (BÁO etaI., 1991), Pfuscomaculatus (AOKl et aI., 1969) andP cuvieri (OLIVEIRA et aI., 2002). A commoncharacteristic between these species is the fact that thereproductive cyclened as continuous or potentiallycontinuous, according to the established classificationby LOFTS (1974).

ln the germ epithelium of the analyzed species,the cystic distribution apparent]y does not follow adistribution sequence; it is possible to just identify the

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42 OLIVEIRA, C de, et aI.

spermatogonia near to the locule wall and thespermatozoon bundles, in general, near or in thelocular lumen. This description can also be used to B.woodhousei (ATHERTON, 1974) and C. caudiverbera(HERMOSILLA et aI., 1983) for which an aleatorydisposition was also observed.

Concerning formation and development of thecystic structure, it is reported that the interactionbetween the germ cells and Sertoli cells is impor-tant. This was studied before in other species, like:B. regularis (ABDELMAGUlD; SABRY, 1987),C. caudiverbera (HERMOSILLA et aI., 1983),P dacnicolor (RASTOGI et aI., 1988) and Ranacatesbeiana (SPRANDO; RUSSELL, 1988). Accor-ding to these authors, thin extensions of cytoplasm ofthe Sertoli cells comprise completely the germ cells,forming a surrounded wall which also emits cyto-plasmatic process between the cells ofthe cysts.

The Sertoli cells rest on the basal lamina of the

seminiferous tubule (CAVICCHIA; MOVIGLIA,1982; ABDELMAGUlD; SABRY, 1987; RASTOGIet aI., 1988), and continue attached to the wall even inthe more advanced stages of the spermatogenesis(HERMOSILLA et aJ., 1983). With the formation ofthe cysts, a compartmentalization is originated with afundamental role in the cytodifferentiation, providinga structural and functional support to the germ cells(RASTOGI et aI., 1988; BÁO et aI., 1991).

Therefore, the process starts when the sper-matogonia - which are involved by the follicular orSertoli cells, forming the cyst wall- begin in a divisionstage producing their followers, which are also locatedin the interior of the structure. This arrangementwill only be altered in the final stages, during thespermiogenesis, when the cells will pass through anextraordinary elongation until the formation ofthe tailand head of the spermatozoon. The Sertoli cells willsustain the bundles with the heads embedded in their

cytoplasm and the tail will be free and turned to thelumen of the seminiferous locule (Fig. 4). Accordingto Sprando and Russel (1988), at this moment thelumen of the germ cyst joins the Iumen of theseminiferous 10Cllle. Thus, the spermatozoa will bereleased to follow the spermatic path.

Hermosilla et aI. (1983) reported that the germcells remain directly or indirectly attached to the loculewall. For this reason, we suppose that after thespermatozoa liberation, the Sertoli cell regresses till itacquires the characteristics of a follicular cell, whichmay restart the process as soon as it joins the primor-dial genn cell. Although this might be a possibility

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concerning these cells, these questions can not beelucidated by the present study.

Despite the architecture ofthe germ epithelium ofali mentioned species represents general and typicalmodel for the anurans, the final germ cell ~ thespermatozoon - shows a great heterogeneity in thespecies. This can be related to phylogenetic aspects(KWON; LEE, 1995) and to reproductive strategiesofthe anurans (DUELLMAN; TRUEB, 1994).

Regarding the reproduction, the presence ofdeveloped spermatozoa during ali the reproductiveperiod suggests a capacity of reproduction occurringa few times in a single season. Thus, the gametogenicactivity occurs simultaneously in ali the locules;however, while some seminiferous locules sheltermainly free spermatozoa, ready to be spread, othershave bigger cellular populations in the previous stagesofthe cytodifferentiation. This allows for each indivi-dual and to the population, as a reproductive strategy,a great chance of success in the appropriate momentsduring that only season. Our observations are corro-borated by Rossa-Feres and Jim (1994), who putin evidence preferential periods of reproduction,based on the occurrence of tadpole and male invocal ization.

ACKNOWLEDGMENTS

We are grateful to Benedito Rinaldo Cardana (Departamentode Zoologia - UNESP - Botucatu) for identificatíon ofthe species.to Umberto Jorge Alves de Andrade for schematizing intranslucent tracer paper, to Roberta Faria Fernandes and Prol'. Df.Álvaro Luiz Hattnher (Departamento de Letras Modernas,UNESP, São José do Rio Preto) for the translation into English.This work was supported by FAPESP (grant n° 02/08016-9).

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CAVICCHIA,.T. c.; MOVIGLIA, G. A. Fine structure ofthe testisin the toad (Bufo arenarum Hensel): a freeze-fracture study.Anatomical Record, New York, v. 203, n. 4, p. 463-474,1982.

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44 OLIVEIRA, C. de, et a!.

Fig. 1. A. Cylindric testes (1) af B. crucifer with Bidder's argan (arrow) in the cranial extremity(3,2x). B. Ovaid testes af P. cuvieri with dark brown pigmentatian (6,4x). C. Ovaid testes afS. fuscovarius unprovide af the pÍgmentatian (3,2x).

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Morphological considerations on the seminiferous ... 45

t

A

t

B

Fig. 2. Testes anatomic aspect of the B. crucifer (A - 7x) and P cuvieri (B - 17x):testes (t), fat body (I) and Bidder's organ (arrowhead). The pigmentation in P cuvieriemphasize the units denominated seminiferous locules.

Fig. 3. Histological testicular arrangement in S.fuscovarius. A. interstitial tissue (i) with various Leydig cells, blood capillary (arrowhead)and efferent ductules (arrow). (H/E, 850x). B. Seminiferous locuIes with the cystic germ epithelium: primary (1) and secondary (2 and 3)spermatogonia; primary (4), in meiotic division (5) and secondary (6) spermatocytes; secondary spermatids (7); spermatozoa (8). Indicatedalso the Leydig (L) and Sertoli (S) cells. (H/E, 680x).

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46 OLIVEIRA, C. de, et a!.

Fig. 4. The schematic shows the generalized representation of the cystic germ epithelium for the three species:1) spermatogonia I; 2) spermatogonia II; 3) spermatogonia II; 4) spermatocytes I; 5) spermatocytes I in divisionmeiotic; 6) spermatocytes II; 7) spermatocytes II in division meiotic; 8) spermatids I; 9) spermatids II;10) spermatozoon bundles. The cysts are always connected to the locular wall by the Sertoli cells.

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