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Histological aspects and structural characteristics of the testesof Dendropsophus minutus (Anura, Hylidae)

Lia Raquel de Souza Santos a,b, Classius de Oliveira a,b,*a Sao Paulo State University-UNESP, Institute of Biosciences, Humanities and Exact Sciences-IBILCE, Department of Biology,

15054-000 Sao Jose do Rio Preto, Sao Paulo, Brazilb Animal Biology Post-Graduation Program, 15054-000 Sao Jose do Rio Preto, Sao Paulo, Brazil

1. Introduction

The urogenital system of amphibians is formed by primarysexual organs, the gonads, and accessory organs that include ducts.In anuran amphibians, testes are paired, round, compact, andusually yellowish (Goin and Goin, 1962). They are surrounded by athin and fibrous connective tissue capsule, the tunica albuginea,which presents seminiferous structures, namely the seminiferoustubule (Oliveira et al., 2002; Santos and Oliveira, 2007). In general,different cellular types are found in the germ tissue of theseminiferous tubule: spermatogonia at the base of the germepithelium; spermatocytes and spermatids in the cellular differ-entiation sequence; and spermatozoids in the lumen (Oliveira

et al., 2003). In this epithelium, there is also a cystic arrangemi.e. groups of cells associated with Sertoli follicular cells that fospermatocysts or spermatogenetic cysts, where each groupobserved in a specific stage of cellular differentiation (Wake, 19Lofts, 1974; Rastogi et al., 1988; Oliveira et al., 2002, 2003).

Morphofunctional characteristics of Dendropsophus minu

reproduction (Peters, 1872) were presented by Santos and Oliv(2007) who described the annual reproductive cycle basedgonadal anatomical characteristics. In the present studypropose to analyze the testicular morphological arrangemdescribing the histological aspects and structural characteristicgerm cells and their cystic arrangement in D. minutus.

2. Materials and methods

Micron xxx (2008) xxx–xxx

A R T I C L E I N F O

Article history:

Received 11 April 2007

Received in revised form 25 March 2008

Accepted 26 March 2008

Keywords:

Reproduction

Morphology

Spermatogenesis

Germinative cells

Testes

Anura

Dendropsophus minutus

A B S T R A C T

The present study describes morphological aspects of testes and presents a general characterizatio

the seminiferous elements of Dendropsophus minutus (Peters, 1872). Twenty samples of the species w

used; after macroscopic descriptions the testes were submitted to histological routine for microsc

analysis. Anatomically, the testes measured 1.90 � 0.13 mm, and were oval and milky-white. In relatio

microscopic aspects, it was observed in D. minutus, as well as in anuran amphibians, that spermatogen

occurs in the seminiferous tubule where elements of the germ epithelium are organized in spermatogen

cysts. Each cyst contains cells in the same stage of differentiation. Characterization of each cellular

enables the identification and differentiation of germ lineage cells. Spermatogonia I, found at the epith

base, are the largest of the lineage cells and are usually present in association with Sertoli cells present ne

the basal membrane. During the mitotic proliferation phase, cysts containing variable number

spermatocytes II are originated; these spermatocytes are smaller and similar inside the cyst. Spermatocy

are developed after some morphological changes; these spermatocytes are large cells with a spherical nuc

and different degrees of nuclear compaction. Spermatocytes II, highly numerous cells resultant from the

meiotic division, are much smaller than their antecedents. A second meiotic division produces haploid

formation, specifically spermatids I, which through cellular differentiation form spermatids II. Spermati

are elongated and organized in bundles supported by Sertoli cells. Spermatozoids appear during

spermiogenesis process and in their most advanced stage they lose their fascicular organization and

released in the tubular lumen, where they follow a pathway through the duct system.

� 2008 Elsevier Ltd. All rights reser

Contents l is ts ava i lab le at ScienceDirec t

Micron

journal homepage: www.e lsev ier .com/ locate /micron

d inzil;prilion

* Corresponding author at: Department of Biology, Sao Paulo State University-

UNESP, Institute of Biosciences, Humanities and Exact Sciences-IBILCE, Rua

Cristovao Colombo 2265, Jardim Nazareth, 15054-000 Sao Jose do Rio Preto, Sao

Paulo, Brazil. Tel.: +55 17 3221 2387; fax: +55 17 3221 2390.

E-mail address: classius@ibilce.unesp.br (C. de Oliveira).

0968-4328/$ – see front matter � 2008 Elsevier Ltd. All rights reserved.

doi:10.1016/j.micron.2008.03.006

Please cite this article in press as: de Souza, L.R., de Oliveira, C., HDendropsophus minutus (Anura, Hylidae), Micron (2008), doi:10.10

Twenty adult males of the species D. minutus were collectethe city of Sao Jose do Rio Preto (State of Sao Paulo, Bra20845047.500S and 49819038.800W), between March 2003 and A2004. Specimens were dissected through ventral median incis

istological aspects and structural characteristics of the testes of16/j.micron.2008.03.006

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reproductive organs were exposed for analysis and docu-tation. The testes were removed, fixed in Bouin solution for, dehydrated in increasing alcohol series (Hopwood, 1990), andedded in methacrylate glycol resin (Historesin Leica1).ions of 2 mm were stained with Toluidine blue and borax) (Robinson and Gray, 1990). For morphological analysis, theerials were observed in an Olympus BX 60 microscope andyzed through the Image Pro-plus program at the Center ofoscopy of the Institute. The specimens were fixed in alcoholand deposited in the collection of the Department of ZoologyBotany at Sao Jose do Rio Preto (DZB_SJRP; lot numbers 8904-0 and 8934-8948).

esults

n D. minutus the testes are located in the abdominal cavity,ral to the kidneys, which are closely linked to testes by thedal mesenterium or mesorchium. Testes are milky-white and

, measure 1.90 � 0.13 mm, and weigh 0.002 � 0 g (Fig. 1A). Fattyes are found in their cranial extremity, with several thinnsions and colour varying from whitish to yellowish.estes are externally surrounded by a tunica albugineaposed primarily of collagen fibres, which, as a function ofr minute thickness, permit verification that testes are formedircular or spherical seminiferous units that confer a granularct (Fig. 1B). Germ lineage cells forming the seminiferousle are grouped into cysts composed of cytoplasmaticnsions of Sertoli cells, and appear in different stages oflar differentiation, developing spermatogenetic cysts ormatocysts (Fig. 2). This arrangement is the same for all cysts,pt for the primary spermatogonia or spermatogonia I, which

large and voluminous cells with chromatin granulation and atilobular aspect. These cells have associated follicular cellstoli cells), isolated at the base of the germ epithelium (Fig. 3A).he secondary spermatogonia or spermatogonia II, usuallyd at lobule boundaries, have more pronounced colour and areller than their antecedents (Fig. 3B). After morphologicalges, cells differentiate into two morphologically distinct

spermatocytes: primary spermatocytes or spermatocytes I, whichare large cells though smaller than primary spermatogonia andwith looser chromatin (Fig. 3C); and secondary spermatocytes orspermatocytes II, which are much smaller cells whose populationis very large (Fig. 3D). Spermatocytes are usually observed indifferent stages of the first meiotic division, thus presentingdifferent degrees of nuclear material compaction (Fig. 3E).

Spermatids result from the second meiotic division; theircellular population is highly heterogeneous in appearance, withcells varying from spherical to elongated formats. Two spermatidtypes can be found: round spermatids or spermatids I (Fig. 3F),with a slightly compressed nucleus, which can be differentiatedfrom secondary spermatocytes when some cells are slightlyelongated; and elongated spermatids or spermatids II, whosenucleus presents elongation concomitantly with progressivenuclear compaction (Fig. 3G). At this point, the cystic arrangementis discarded and the cellular population arranges itself into bundlessustained by follicular cells. These Sertoli cells are easily identifiedwhen associated with spermatids II or spermatozoids because theyare always returned to the locular periphery, whereas the germ cellbundles have their tails directed toward the tubular lumen (Fig. 4).

Spermatozoids, in turn, are characterized by extraordinarynuclear compaction and cytoplasmatic reduction. Developingspermatozoids are usually in well-organized bundles due to theirassociation with Sertoli cells where the proximal part or spermheads are anchored in these cells. When in more advanced degreesof maturation, spermatozoids loose their fascicular arrangementand are usually found in the tubular lumen (Fig. 3H).

During the spermatogenic process, it has been verified that thealterations undergone by one cell are experienced by all the othercells found in the same cyst. This trait can be attributed to the closeassociation between germ and follicular cells, which are requiredfor formation of cysts (Fig. 4A) and spermatid bundles (Fig. 4B andC). When the association ends, spermatozoids in the finalmaturation phase become free in the tubular lumen (Fig. 4D).

Fig. 2. General aspect of D. minutus testicular architecture. G1: primary

spermatogonia; G2: secondary spermatogonia; C: spermatocytes in

differentiation; Ez: spermatozoid; tubular wall (arrowhead), intertubular area

(arrow) with Leydig cells and capillaries.

L.R. de Souza Santos, C. de Oliveira / Micron xxx (2008) xxx–xxx

. (A) Dendropsophus minutus testicular localization in the abdominal cavity and

lation to other organs. E: stomach, P: lung, I: intestine, B: spleen, T: testes, R:

y, IP: final portion of the intestine with pigmented melanocytes. (B)

niferous tubular in detail (*).

ase cite this article in press as: de Souza, L.R., de Oliveira, C.,ndropsophus minutus (Anura, Hylidae), Micron (2008), doi:10.10

4. Discussion

Duellman and Trueb (1994) described anuran testes as paired,spherical or oval structures located ventrally to the kidneys; insome species they can be long and extend to the posteriorextremity of the kidney. Testes of D. minutus are located in the

Histological aspects and structural characteristics of the testes of16/j.micron.2008.03.006

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abdominal cavity, in syntopy with the visceral side of kidneys,which are in a position immediately ventral to the dorsal wall ofthe abdominal cavity (Santos and Oliveira, 2007). Similar generalmorphological characterizations of testes have been reported forCaudiverbera caudiverbera (Hermosilla et al., 1983), Scinax fuscov-

arius (Oliveira and Vicentini, 1998), and Eupemphix nattereri

(Oliveira and Zieri, 2005).Males of D. minutus and another species of the Hylidae family, S.

fuscovarius (Oliveira and Vicentini, 1998), have milky-white testesas do most anuran amphibian species. By contrast, among otherspecies, such as Leiuperıdeo E. nattereri, the testes possess darkcolouration, caused by visceral melanocytes, intensely pigmentedstromal cells present in the tunic albuginea and cellular

Leydig cells in mammals are arranged in clusters or isolated inperitubular and perivascular regions (Haider, 2004). These csynthesise a large amount of oxytocin and contribute tocontraction of smooth muscle cells in the lamina propria aroseminiferous tubules (Nicholson and Pickering, 1993 apud Hai2004).

However, the distribution of germ cells in seminiferelements in the specie occurs differently from arrangemedescribed for other vertebrate groups such as mammals, exceptthe primordial germ cells (primary spermatogonia) locatedthe tubular wall and for spermatozoids that predominate inluminal zone (Oliveira et al., 2003). Primary spermatogonia andassociated Sertoli cell develop in agnathan and elasmobra

Fig. 3. Distinct differentiation stages during spermatogenesis in D. minutus. 1: Primary spermatogonia; 2: secondary spermatogonia; 3: primary spermatocytes; 4: prop

of the first meiotic division; 5: secondary spermatocytes; 6: round spermatids; 7: elongated spermatids in initial phase; 8: bundle of elongated spermatids; 9: spermato

arrow: Sertoli cells.

L.R. de Souza Santos, C. de Oliveira / Micron xxx (2008) xxx–xxx

e inred

. Inreyarkart-

interstitium (Oliveira and Zieri, 2005).According to Lofts (1974), and as observed in D. minutus, anuran

testes are surrounded by a fibrous elastic layer consisting ofparenchyma of convoluted seminiferous tubules limited by a germepithelium. The interstitial tissue between tubules is composed ofconnective tissue, blood capillaries, and interstitial Leydig cells.

Please cite this article in press as: de Souza, L.R., de Oliveira, C., HDendropsophus minutus (Anura, Hylidae), Micron (2008), doi:10.10

testes in each germinal compartment. Initially, these cells ardirect contact with interstitial tissue and are not sequestewithin a germinal compartment by the basal membraneprimitive chordates (the hagfish Eptatretus stouti, the lampPetromyzon marinus, the ray Dasyatus americana, and the shCarcharhinus limbatus), separate germinal and interstitial comp

istological aspects and structural characteristics of the testes of16/j.micron.2008.03.006

menarousper(Gri

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ts are established when basal membranes form a groupnd clusters of primary spermatogonia, defining secondarymatogonia that enter meiosis and develop into spermatozoidser, 1992).mphibian cystic spermatogenesis occurs (Lofts, 1974) withproliferation of germ cells, in organized and well-definedps called germ cysts or spermatocysts. Anurans have acular feature that presages amniote testicular evolution. Thel membrane resulted in the development of a permanentration between germinal and interstitial testicular compart-ts in fishes, amphibians, and all amniotes (Grier, 1992). Thee type of spermatogenesis was confirmed in D. minutus (SantosOliveira, 2007) and occurs in all anuran species described to, thus corroborating a fundamental characteristic of sperma-nesis within these follicular structures, where all cells of ap are always in the same differentiation stage (Lofts, 1974;cchia and Moviglia, 1983; Rastogi et al., 1988; Bao et al., 1991).e traits also occur in fishes (Grier et al., 1981; Cruz-Landim., 2003). The general characteristic of a cyst is the formation ofhematotesticular barrier between these cells. It has beenested that this mechanism maintains the compositionalrences between blood or hemolymph and the testicular fluid,lting in the formation of a microenvironment that isopriate for the development of haploid germ cells (CavicchiaMoviglia, 1983; Bergmann et al., 1983).

n cephalochordates and urochordates, Sertoli cell processes doinclude single germ cell clones as found in fishes andhibians. In amphibians, Sertoli cells grow between groupsrconnecting the cells and retaining them in the testicular

Spermatogonia of these species are identified as one of two classes:those clear and pale, usually larger and undifferentiated; and darkones that are smaller and in the process of differentiation. Theprimary spermatogonia of D. minutus are the largest germ lineagecells and rest on the adjacencies of the basal membrane, whereonly one class was identified. Similar observations were docu-mented for Chaunus arenarum (Cavicchia and Moviglia, 1983);Scinax ranki (Taboga and Dolder, 1991); S. fuscovarius (Oliveira andVicentini, 1998), and Odontophrynus cultripes (Bao et al., 1991). Theprimary spermatogonia can go through a mitotic process or splitinto two new and independent primary spermatogonia withfollicular cells on their borders. Or, during the period of intenseactivity, they can divide themselves several times to create a groupof two or more cells that remain together within the same cyst,whose cytoplasm and nuclei, now round, are denominatedsecondary spermatogonia (Lofts, 1974). Among the most notabledifferences that occur during the cytodifferentiation process fromprimary to secondary spermatogonia are size dimunition and greatcytoplasm reduction, with a nucleus that becomes increasinglyless lobulated until it reaches a spherical shape (Rastogi et al.,1988).

According to Oliveira et al. (2003), two types of secondaryspermatogonia are identified in S. fuscovarius. The first ones arecalled secondary spermatogonia of the clear type because theirnuclei are clearer than those of their successors; they are found assmall groups of cells located near the seminiferous tubule wall,usually grouped and with poorly defined contours. The secondaryspermatogonia of the dark type have very compact and intenselycoloured nuclei. Secondary spermatogonia of the dark type were

. Sertoli cell (arrows) with the germinative cells, constituting the arrangement in cyst and bundles of final spermatids in D. minutus. (A) Organized germ cells in cysts; (B

) organized in bundles; (D) some free spermatozoa in the lumen after the release of fascicular formation.

L.R. de Souza Santos, C. de Oliveira / Micron xxx (2008) xxx–xxx

phery. Mature spermatozoids are released in the testicularen most frequently in direct juxtaposition with spermatoge-c cells (Grier, 1992).n some anurans, such as Pachymedusa dacnicolor (Rastogi et al.,8) and Rana esculenta (Rastogi et al., 1985), the primarymatogonia have a prominent size and a multilobular nucleus.

ase cite this article in press as: de Souza, L.R., de Oliveira, C.,ndropsophus minutus (Anura, Hylidae), Micron (2008), doi:10.10

not evident; however, the same morphological characteristicsfound in secondary spermatogonia of the clear type were observedin D. minutus.

All secondary spermatogonial components of the same cysthave similar shapes and sizes, but do not have distinctivecharacteristics as described in other studies. These cells develop

Histological aspects and structural characteristics of the testes of16/j.micron.2008.03.006

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at a uniform rate and divide themselves synchronically, so thatcysts increase in size and numbers (Lofts, 1974). After differentia-tion, primary spermatocytes appear and are usually observedduring prophase of the first meiotic division, with different degreesof chromatin compaction. With the first meiotic division ofprimary spermatocytes, much smaller cells originate, the second-ary spermatocytes. One distinguishing characteristic of this stageis the gradual appearance of small intercellular spaces thatcoalesce and become more evident in the secondary spermatocytecysts (Hermosilla et al., 1983). According to Lofts (1974), theintercellular vacuoles already seen in primary spermatocytesincrease in size and join in a central vacuole. However, in D.

minutus the central vacuole is not formed even though the cellshave similar morphological characteristics. With the formation ofelongated spermatids, the cystic arrangement is broken for cells toorganize themselves into compact bundles that are still sustainedby the Sertoli cells.

Spermatids start to elongate and arrange themselves in bundlesdue to their association with Sertoli cells (Lofts, 1974). These cellsmaintain a morphofunctional relationship with germ lineage cells,which is still more prominent during spermiogenesis (Cavicchiaand Moviglia, 1983; Rastogi et al., 1988; Bao et al., 1991).Spermatids present a high morphological heterogeneity as they gofrom spherical to elongated cylindrical formats, by means ofnucleo-cytoplasmatic changes inherent to spermiogenesis. In D.

minutus and C. caudiverbera (Hermosilla et al., 1983), in O. cultripes

(Bao et al., 1991), and in S. fuscovarius (Oliveira et al., 2003), the firstspermatids have a spherical nucleus and fine granulation; next, thenucleus becomes oval and the granular chromatin is distributedhomogeneously. The following stages are characterised by cellularand nuclear elongation, which occur at the same time aschromatinic condensation.

For the formation of spermatozoids, cytoplasmic portions areeliminated. This and nucleus compaction are the main reasons fordecrease in cellular volume. In D. minutus, spermatozoids areidentified according to their random arrangement in the semi-niferous lumen, and are free to be conducted to the gametogenicpathways by the efferent ducts.

Testicular morphology and gametogenesis in D. minutus yieldmorphological information similar to that usually described forother anurans. These descriptions are very important for a betterunderstanding of reproductive biology, particularly of neotropicalanurans, for which data remain scarce in the literature.

Acknowledgements

This study is part of the Masters thesis presented by L.R.S.S. forthe Animal Biology Post-graduation Course, IBILCE/UNESP. Theauthors thank Adelina Ferreira for technical assistance. Researchsupport was provided by Fundacao de Amparo a Pesquisa do

Estado de Sao Paulo-FAPESP: grants 02/08016-9 and 05/02919and by Coordenacao de Aperfeicoamento de Pessoal de NSuperior-CAPES (fellowship to L.R.S.S.).

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Please cite this article in press as: de Souza, L.R., de Oliveira, C., Histological aspects and structural characteristics of the testes ofDendropsophus minutus (Anura, Hylidae), Micron (2008), doi:10.1016/j.micron.2008.03.006