G Model JMIC-1251; No of Pages 5 Micron xxx (2008) xxx–xxx Contents lists available at ScienceDirect Micron journal homepage: www.elsevier.com/locate/micron Histological aspects and structural characteristics of the testes of 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, Brazil b Animal Biology Post-Graduation Program, 15054-000 Sao Jose do Rio Preto, Sao Paulo, Brazil ARTICLE INFO ABSTRACT Article history: The present study describes morphological aspects of testes and presents a general characterization of Received 11 April 2007 the seminiferous elements of Dendropsophus minutus (Peters, 1872). Twenty samples of the species were Received in revised form 25 March 2008 used; after macroscopic descriptions the testes were submitted to histological routine for microscopic Accepted 26 March 2008 analysis. Anatomically, the testes measured 1.90 Æ 0.13 mm, and were oval and milky-white. In relation to microscopic aspects, it was observed in D. minutus, as well as in anuran amphibians, that spermatogenesis Keywords: occurs in the seminiferous tubule where elements of the germ epithelium are organized in spermatogenetic Reproduction cysts. Each cyst contains cells in the same stage of differentiation. Characterization of each cellular type Morphology Spermatogenesis enables the identification and differentiation of germ lineage cells. Spermatogonia I, found at the epithelial Germinative cells base, are the largest of the lineage cells and are usually present in association with Sertoli cells present next to Testes the basal membrane. During the mitotic proliferation phase, cysts containing variable numbers of Anura spermatocytes II are originated; these spermatocytes are smaller and similar inside the cyst. Spermatocytes I Dendropsophus minutus are developed after some morphological changes; these spermatocytes are large cells with a spherical nucleus and different degrees of nuclear compaction. Spermatocytes II, highly numerous cells resultant from the first meiotic division, are much smaller than their antecedents. A second meiotic division produces haploid cell formation, specifically spermatids I, which through cellular differentiation form spermatids II. Spermatids II are elongated and organized in bundles supported by Sertoli cells. Spermatozoids appear during the spermiogenesis process and in their most advanced stage they lose their fascicular organization and are released in the tubular lumen, where they follow a pathway through the duct system. ß 2008 Elsevier Ltd. All rights reserved. 1. Introduction et al., 2003). In this epithelium, there is also a cystic arrangement, i.e. groups of cells associated with Sertoli follicular cells that form The urogenital system of amphibians is formed by primary spermatocysts or spermatogenetic cysts, where each group is sexual organs, the gonads, and accessory organs that include ducts. observed in a specific stage of cellular differentiation (Wake, 1968; In anuran amphibians, testes are paired, round, compact, and Lofts, 1974; Rastogi et al., 1988; Oliveira et al., 2002, 2003). usually yellowish (Goin and Goin, 1962). They are surrounded by a Morphofunctional characteristics of Dendropsophus minutus thin and fibrous connective tissue capsule, the tunica albuginea, reproduction (Peters, 1872) were presented by Santos and Oliveira which presents seminiferous structures, namely the seminiferous (2007) who described the annual reproductive cycle based on tubule (Oliveira et al., 2002; Santos and Oliveira, 2007). In general, gonadal anatomical characteristics. In the present study we different cellular types are found in the germ tissue of the propose to analyze the testicular morphological arrangement, seminiferous tubule: spermatogonia at the base of the germ describing the histological aspects and structural characteristics of epithelium; spermatocytes and spermatids in the cellular differ- germ cells and their cystic arrangement in D. minutus. entiation sequence; and spermatozoids in the lumen (Oliveira 2. Materials and methods * Corresponding author at: Department of Biology, Sao Paulo State University- Twenty adult males of the species D. minutus were collected in UNESP, Institute of Biosciences, Humanities and Exact Sciences-IBILCE, Rua Cristo´ vao Colombo 2265, Jardim Nazareth, 15054-000 Sao Jose do Rio Preto, Sao the city of Sao Jose do Rio Preto (State of Sao Paulo, Brazil; 0 00 0 00 Paulo, Brazil. Tel.: +55 17 3221 2387; fax: +55 17 3221 2390. 20845 47.5 S and 49819 38.8 W), between March 2003 and April E-mail address: [email protected] (C. de Oliveira). 2004. Specimens were dissected through ventral median incision 0968-4328/$ – see front matter ß 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., Histological aspects and structural characteristics of the testes of Dendropsophus minutus (Anura, Hylidae), Micron (2008), doi:10.1016/j.micron.2008.03.006 G Model JMIC-1251; No of Pages 5 2 L.R. de Souza Santos, C. de Oliveira / Micron xxx (2008) xxx–xxx and reproductive organs were exposed for analysis and docu- mentation. The testes were removed, fixed in Bouin solution for 24 h, dehydrated in increasing alcohol series (Hopwood, 1990), and embedded in methacrylate glycol resin (Historesin Leica1). Sections of 2 mm were stained with Toluidine blue and borax (1:1) (Robinson and Gray, 1990). For morphological analysis, the materials were observed in an Olympus BX 60 microscope and analyzed through the Image Pro-plus program at the Center of Microscopy of the Institute. The specimens were fixed in alcohol 70% and deposited in the collection of the Department of Zoology and Botany at Sao Jose do Rio Preto (DZB_SJRP; lot numbers 8904- 8910 and 8934-8948). 3. Results In D. minutus the testes are located in the abdominal cavity, ventral to the kidneys, which are closely linked to testes by the gonadal mesenterium or mesorchium. Testes are milky-white and oval, measure 1.90 0.13 mm, and weigh 0.002 0g(Fig. 1A). Fatty Æ Æ Fig. 2. General aspect of D. minutus testicular architecture. G1: primary bodies are found in their cranial extremity, with several thin spermatogonia; G2: secondary spermatogonia; C: spermatocytes in extensions and colour varying from whitish to yellowish. differentiation; Ez: spermatozoid; tubular wall (arrowhead), intertubular area Testes are externally surrounded by a tunica albuginea (arrow) with Leydig cells and capillaries. composed primarily of collagen fibres, which, as a function of their minute thickness, permit verification that testes are formed spermatocytes: primary spermatocytes or spermatocytes I, which by circular or spherical seminiferous units that confer a granular are large cells though smaller than primary spermatogonia and aspect (Fig. 1B). Germ lineage cells forming the seminiferous with looser chromatin (Fig. 3C); and secondary spermatocytes or tubule are grouped into cysts composed of cytoplasmatic spermatocytes II, which are much smaller cells whose population extensions of Sertoli cells, and appear in different stages of is very large (Fig. 3D). Spermatocytes are usually observed in cellular differentiation, developing spermatogenetic cysts or different stages of the first meiotic division, thus presenting spermatocysts (Fig. 2). This arrangement is the same for all cysts, different degrees of nuclear material compaction (Fig. 3E). except for the primary spermatogonia or spermatogonia I, which Spermatids result from the second meiotic division; their are large and voluminous cells with chromatin granulation and a cellular population is highly heterogeneous in appearance, with multilobular aspect. These cells have associated follicular cells cells varying from spherical to elongated formats. Two spermatid (Sertoli cells), isolated at the base of the germ epithelium (Fig. 3A). types can be found: round spermatids or spermatids I (Fig. 3F), The secondary spermatogonia or spermatogonia II, usually with a slightly compressed nucleus, which can be differentiated found at lobule boundaries, have more pronounced colour and are from secondary spermatocytes when some cells are slightly smaller than their antecedents (Fig. 3B). After morphological elongated; and elongated spermatids or spermatids II, whose changes, cells differentiate into two morphologically distinct nucleus presents elongation concomitantly with progressive nuclear compaction (Fig. 3G). At this point, the cystic arrangement is discarded and the cellular population arranges itself into bundles sustained by follicular cells. These Sertoli cells are easily identified when associated with spermatids II or spermatozoids because they are always returned to the locular periphery, whereas the germ cell bundles have their tails directed toward the tubular lumen (Fig. 4). Spermatozoids, in turn, are characterized by extraordinary nuclear compaction and cytoplasmatic reduction. Developing spermatozoids are usually in well-organized bundles due to their association with Sertoli cells where the proximal part or sperm heads are anchored in these cells. When in more advanced degrees of maturation, spermatozoids loose their fascicular arrangement and are usually found in the tubular lumen (Fig. 3H). During the spermatogenic process, it has been verified that the alterations undergone by one cell