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Descriptive morphometry and stereology of the tubular compartment in the wild rodent Hylaeamys megacephalus (Rodentia: Cricetidae) from Central Brazil
Animal Reproduction Science 138 (2013) 110–117
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Descriptive morphometry and stereology of the tubular compartment in the wild rodent Hylaeamys megacephalus (Rodentia: Cricetidae) from Central Brazil Fabiana Cristina Silveira Alves de Melo a,∗ , Tatiane Pires de Sousa a , Kyvia Lugate C. Costa b , Sérgio Luis P. da Matta b , Fabiano Rodrigues de Melo a , Ricardo de Mattos Santa-Rita a a b
Biological Sciences, Federal University of Goiás, Campus Jataí, Jataí, Brazil Department of General Biology, Federal University of Vic¸osa, Vic¸osa, Brazil
a r t i c l e
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Article history: Received 25 June 2012 Received in revised form 7 January 2013 Accepted 28 January 2013 Available online 13 February 2013 Keywords: Seminiferous tubules Spermatogenesis Reproduction Testis
a b s t r a c t Information on reproductive characteristics of wild rodents is scarce in the literature. This study aimed to assess the testis morphometry and stereology of Hylaeamys megacephalus. We used five animals in the study, captured in forest fragments in southwestern Goias State, between April and August 2009. The testes were fixed in Karnovsky solution, dehydrated, and embedded in methacrylate. Two-micrometer-thick sections from each sample were stained with toluidine blue/sodium borate 1%. Images of the testicular parenchyma were obtained from photomicroscope and morphometric and stereological analyses were carried out using the Image Pro-Plus software. The average body weight observed in the specimens of H. megacephalus in the study was 47.84 g, of which, 0.40% is allocated to the gonads (GSI) and 0.36% to the seminiferous tubules (TSI). These parameters suggest promiscuous reproductive behavior, of the polyandrous type, favoring males with higher sperm production and consequently, larger testes. The volume density of the seminiferous tubules was 94.46%, which represented a volume of 0.18 mL. The volume density and volume of the interstitium were 5.54% and 0.011 mL, respectively. The diameter of the seminiferous tubules was 206.5 m and the height of seminiferous epithelium was 71.27 m. H. megacephalus presents 5.06 m of seminiferous tubules and an average of 27.96 m of seminiferous tubules per gram of testis. The mitotic and meiotic indexes showed losses of 85 and 42%, respectively and an overall loss of 90% over the full spermatogenic process. The number of Sertoli cells per testis and per gram of testis was 7.8 × 106 and 95.28 × 106 , respectively. Most of the morphometric parameters evaluated in H. megacephalus in this study are within the range of values described for most mammals. © 2013 Elsevier B.V. All rights reserved.
1. Introduction ∗ Corresponding author at: Federal University of Goiás, BR 364, Km 192 no 3800, zip code 75801-615, Jataí, Goiás, Brazil. Tel.: +55 64 3606 8290; fax: +55 64 3606 8290. E-mail addresses: [email protected] (F.C.S.A. de Melo), [email protected] (T.P. de Sousa), kyvia [email protected] (K.L.C. Costa), [email protected] (S.L.P. da Matta), [email protected] (F.R. de Melo), [email protected] (R.d.M. Santa-Rita). 0378-4320/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.anireprosci.2013.01.013
Nowadays, the Cerrado biome can be described as a hotspot due to the high degree of endemic species and threats mostly as a result of anthropogenic origins mostly as the result of agricultural expansion (Mittermeier et al., 2005). Therefore, studies on mammals, in this environment, have high priority as they could provide information about taxonomic aspects and in addition assist in the
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development of conservation strategies to manage and maintain the current biodiversity (Voss and Emmons, 1996). According to Alves et al. (2009), the distribution of mammals, especially endangered species, has been used to justify the establishment of new protected areas. Recent studies have shown that large diversity and rarity of small mammals indicate that such group could be used as indicators in environmental studies (Bonvicino et al., 2002). According to Buitrón-Jurado and Toba (2007), the ecology of most rodents from South America is not known because of their wide distribution in forested habitats, nocturnal habits, and difficulties in capture and monitoring. The species Hylaeamys megacephalus, Fischer, 1814, belongs to the order Rodentia, Cricetidae Family, and Sigmodontinae Subfamily. The Rodentia order is the most numerous in the Mammalia class, with a long evolutionary history, great diversity, varied habits, and trophic strategies, and is present in all continents, except the Antarctic (Eisenberg and Redford, 1999; Wilson and Reeder, 2005). Rodents influence the dynamics of neotropical forests and are good indicators of both local habitat and landscape changes (Vieira and Monteiro-Filho, 2003). Cricetidae is the richest family in species diversity in Brazil with all members belonging to the Neotropical subfamily Sigmodontinae. H. megacephalus is a wild terrestrial rodent that inhabits forested and open vegetation areas in the Amazon Rainforest, Atlantic Forest, Cerrado, Caatinga, Pantanal, and in Brazil this species is distributed in the eastern Amazonas, Roraima, Pará, Western Amapá, Mato Grosso, Maranhão, Tocantins, Goiás and Distrito Federal, Mato Grosso do Sul, Western Minas Gerais, and Northeastern São Paulo (Carmignotto et al., 2012). The knowledge of the reproductive biology of a species, especially in its basic aspects, can make an important contribution toward the conservation of any species (Guião-Leite et al., 2006). The assessment of the histological parameters of gonads and other reproductive organs becomes an important tool in the analysis of the reproductive conditions of an animal in its natural habitat. These parameters can be related to environmental factors and contribute to the conservation of mammals. Also, the composition of the testicular parenchyma and relative size of the testicles can provide valuable information on the reproductive physiology and mating system of a given species (Kenagy and Trombulak, 1986; Paula et al., 2002). Due to the scarcity of information on the reproductive biology of wild rodents and their associated ecological role, the objective of this study was to describe the testicular morphology of H. megacephalus and expand the knowledge about its reproduction in natural environments. 2. Materials and methods 2.1. Animal capture and histological processing Testes were collected from five adult animals captured (from April to August 2009) in forest fragments in southwestern Goiás (IBAMA License number 11621-1 and Ethics Committee Protocol 233/10 CEP/UFG). The animals were captured through traditional methods using Sherman and
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Tomahawk live traps, spaced at approximately every 10 m along linear ground transects. The testes were removed in the field, rapidly rinsed in saline solution, and fixed in Karnovsky solution (Karnovsky, 1965). Testes were dehydrated and embedded in methacrylate after 24 h of fixation. Two-micrometer sections from each testis were stained with 1% toluidine blue/sodium borate. Images of the testicular parenchyma were obtained using an Olympus AX-70 photomicroscope; the morphometric analyses were carried out using the Image Pro-Plus software. 2.2. Testis morphometry and stereology The volume density of the tubular and interstitial compartments were estimated by projecting 300 points in each digital image (200× magnification) resulting in a total of 2500 points per animal (Fig. 1A). The volume (mL) of each testicular component was estimated considering the percentage occupied by both seminiferous tubules and interstitium multiplied by the testicular parenchyma volume. The parenchyma volume was obtained by subtracting the albuginea weight from the testis weight. Since the testes density is estimated at about 1 in mammals (Johnson and Neaves, 1981; Franc¸a, 1991), the testis weight was considered equal to its volume. The gonadal (GW) and seminiferous tubules (STW) weights were obtained to calculate the gonadosomatic and tubulesomatic indexes, respectively as follows: GW/BW × 100 and STW/BW × 100, where BW = body weight. The average diameter of the seminiferous tubules was obtained from 20 random circular cross-sections from each animal’s sample (100× magnification) (Fig. 1B). The seminiferous epithelium height was measured using the same sections in which the tubule diameter was obtained. The epithelium height was considered as the space between the tunica propria and luminal edge. Two diametrically opposed readings were taken using a digital ruler on each cross section and considering their mean value (Fig. 1B). The total length (TL) of the seminiferous tubules, in meters, was estimated dividing the total volume occupied by the seminiferous tubules (STV) by their mean tubular diameter (R2 , R = tubular diameter/2) (Ortavant et al., 1977; Curtis and Amann, 1981). The TL was divided by the testicular weight to calculate the tubular length per gram of testis. 2.3. Stages of the seminiferous epithelium cycle The stages of the seminiferous epithelium cycle were characterized based on the shape and location of the spermatid nuclei, presence of meiotic divisions, and overall seminiferous epithelium composition (Amann and Almquist, 1962; Courot et al., 1970; Leal and Franc¸a, 2006). This process, known as tubular morphology method, establishes eight stages for the seminiferous epithelium (Berndtson, 1977; Paula et al., 1999). The relative stage frequencies were determined from the characterization and counting of 200 seminiferous tubule cross-sections from each animal randomly chosen (400× magnification).
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Fig. 1. Testicular parenchyma of H. megacephalus. (A) Wide view of testicular parenchyma; left corner, model of the square grid used to calculate the volume density of testicular components (SE, seminiferous epithelium; Int, interstitium). Bar: 87 m. (B) Seminiferous tubule cross section of H. megacephalus showing how the tubular diameter (large arrows) and epithelium height (small arrows) were measured. The average values were considered in each case. (SE, seminiferous epithelium; Int, interstitium; Lu, lumen). Bar: 50 m. (C) Detail of the seminiferous epithelium of H. megacephalus on Stage 1, showing all different germ and somatic cell types (Int, interstitium; Lu, lumen; RSPD, round spermatid; SPT Pl/L, spermatocyte – preleptotene/leptotene; SPT P, spermatocyte – pachytene; SPTGA, type A spermatogonia; S, Sertoli cell nucleus). Bar: 22 m.
2.4. Cell counts The germ cell population that constitutes the seminiferous epithelium in stage 1 (tubular morphology method) was estimated based on the count of cell nuclei within the cross sectioned tubule, including Sertoli cells nucleolus (Swierstra, 1968; Courot et al., 1970; Amann and Schanbacher, 1983). The following celltypes were quantified in 10 seminiferous tubule cross sections: type-A spermatogonia (SPTGA), primary spermatocytes at preleptotene/leptotene (SPT Pl/L) and pachytene (SPT P), round spermatids (RSPD), and Sertoli cells (S) (Fig. 1C). The mean nuclei and nucleoli diameters were
obtained by measuring 30 nuclei from the cells cited above. The counting of different cell types was corrected by the Abercrombie’s formula (1946) further modified by Amann and Almquist (1962) because of size variations. It was possible to quantify the following coefficients based on the corrected values: spermatogonial mitosis (SPT Pl/L/SPTGA), meiotic yield (RSPD/SPT P), spermatogenic yield (RSPD/SPTGA), and Sertoli cell index ((SPTGA + SPTC Pl/L + SPTC P + RSPD)/S). The number of Sertoli cells per testis and per gram of testis was calculated in accordance with Hochereau-de-Reviers and Lincoln (1978). The results are reported as means and standard deviations.
F.C.S.A. de Melo et al. / Animal Reproduction Science 138 (2013) 110–117 Table 1 Biometric, morphometric, and stereological parameters of the testes of H. megacephalus (mean ± standard deviation; n = 5). Parameters
Mean ± SD
Body weight (g) Testicular weight (g) Albuginea weight (g) Parenchyma weight (g) Gonadosomatic index (%) Tubulesomatic index (%) Volume density of seminiferous tubule (%) Volume density of interstitium (%) Volume of seminiferous tubule (mL) Volume of interstitium (mL) Diameter of seminiferous tubule (m) Height of seminiferous epithelium (m) Total length of seminiferous tubule (m) Length of seminiferous tubule per gram of testis (m/g)
47.84 0.19 0.006 0.18 0.40 0.36 94.46
± ± ± ± ± ± ±
8.7 0.09 0.001 0.08 0.15 0.13 1.74
5.54 0.18 0.011 206.5 71.27
± ± ± ± ±
1.74 0.08 0.007 21.40 10.39
5.06 ± 1.62 27.96 ± 6.18
3. Results and discussion The testes of the wild rodent H. megacephalus are paired organs located in the scrotum and surrounded by a thick capsule of connective tissue (testicular albuginea). The testicular parenchyma is divided into two compartments; namely tubular and interstitial. The tubular compartment consists of the seminiferous tubules, which comprises the tunica propria, the seminiferous epithelium, and lumen. The tunica propria has a monolayer of myoid cells; the seminiferous epithelium is composed of spermatogenic cells in various stages of development and Sertoli cells with characteristic irregular nucleus and prominent nucleoli. The interstitial compartment is constituted of Leydig cells, cells and fibers of connective tissue, blood vessels, and lymphatic space. 3.1. Testis stereology The determined values for the biometric, morphometric, and stereological parameters in H. megacephalus are presented in Table 1. The albuginea and mediastinum were deducted from the testicular weight to calculate the testicular parenchyma (Johnson and Neaves, 1981). In H. megacephalus, the albuginea occupied 3.33% of the testicular weight, which is similar to the proportion observed in pacas, Cuniculus paca (3.98%; Carreta Júnior, 2008) and lower than that observed in capybaras, Hydrochoerus hydrochaeris (6.13%; Paula et al., 2002). The gonadosomatic index (GSI) indicates the investment in gonads relative to body weight and is an important reproductive parameter (Franc¸a and Russell, 1998). The GSI of H. megacephalus was 0.40%, which is a significantly higher value than what is reported for H. hydrochaeris (0.12%; Paula et al., 2002) and similar to the value reported in small rodents such as Oligorizomys nigripes (0.50%; Silveira, 2007) and Necromys lasiurus (0.47%; Silva et al., 2010). Because the testicular size is not proportional to body size, the somatic investment in gonadal mass is
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greater in small animals compared to larger body size animals (Kenagy and Trombulak, 1986). The tubulesomatic index (TSI) is a parameter that quantifies the investment of body mass allocated in seminiferous tubules and is an important index to evaluate the influence of reproductive behavior on testicular morphology (Paula et al., 2002). H. megacephalus presented higher TSI values (0.36%) than what is reported for H. hydrochaeris (0.06%; Paula et al., 2002) and C. paca (0.24%; Carreta Júnior, 2008), however, within the range determined in several other species of Rodentia (Romano et al., 2002). The testes weight and size can be used as quantitative indicators of spermatic production because the main testicular component is the seminiferous tubule (Franc¸a and Russell, 1998). The GSI and TSI results in this study suggested that H. megacephalus displays promiscuous reproductive behavior, of the polyandrous type (many males have access to one female), favoring males with higher sperm production and consequently, larger testes (Eisenberg and Redford, 1999). Approximately 95% of the testicular parenchyma in H. megacephalus is constituted by seminiferous tubules, which is a higher percentage than observed in other rodents like C. paca (91%; Carreta Júnior, 2008), Dasyprocta aguti (84.75%; Assis-Neto et al., 2003), and H. hydrochaeris (50%; Paula et al., 2002), and closer to the value reported for small rodents such as N. lasiurus (93%; Silva et al., 2010). The interstitial proportion in H. megacephalus is within the range described for most mammals (Franc¸a and Russell, 1998). The tubular and interstitial volumes in H. megacephalus (0.18 mL and 0.011 mL, respectively) were higher than the values described for O. nigripes (0.052 mL and 0.005 mL; Silveira, 2007) and similar to those described for N. lasiurus (0.16 mL and 0.012 mL; Silva et al., 2010). The tubular measurement is one of the approaches used as an indicator of spermatogenic activity (Navarro et al., 2004; Souza et al., 2005; Mascarenhas et al., 2006; Silva et al., 2006). Although the tubule diameter can reach up to 550 m in some species of marsupials (Woolley, 1975), the values observed in most amniotes varies from 180 to 300 m (Roosen-Runge, 1977). The average tubular diameter in H. megacephalus was 206.6 m, which is closer to the values described in C. paca (202.1 m; Carreta Júnior, 2008), H. hydrochaeris (213 m; Paula et al., 2002), and N. lasiurus (193.47 m; Silva et al., 2010). According to Wing and Christensen (1982), the height of the seminiferous epithelium is more effective than the tubular diameter in the evaluation of sperm production. The height of the seminiferous epithelium in H. megacephalus was 71.27 m, which is within the range of values observed in mammals (60–100 m; Franc¸a and Russell, 1998). The total length of the seminiferous tubules is related to three structural parameters: testes size, tubular diameter, and volume of the seminiferous tubules (Franc¸a and Russell, 1998). The total length of the seminiferous tubules in H. megacephalus was 5.1 m. When considering the differences in testis size among species, the conversion of the total tubular length to total length of the seminiferous tubules per gram of testis, makes the measurement
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Fig. 2. Histological cross-section of seminiferous tubules of H. megacephalus showing stages 1–4 (tubular morphology method). (A) Stage 1; (B) stage 2; (C) stage 3; (D) stage 4. Lu, lumen; S, Sertoli cell; SPT Pl/L, spermatocyte in preleptotene/leptotene; SPT P, spermatocyte in pachytene; Z, spermatocyte in zygotene; D, spermatocyte in diplotene; SPTGA, type A spermatogonia; M, metaphase plate; RSPD, round spermatids; ElSPD, elongating/elongated spermatids.
independent of the animal size. H. megacephalus presented 27.9 m of tubule per gram of testis, which is within the pattern value observed in other rodents (Cordeiro-Júnior, 2009; Carreta Júnior, 2008). 3.2. Stages of the seminiferous epithelium cycle and relative stage frequencies The eight stages of the seminiferous epithelium cycle in H. megacephalus were similar to those observed in wild boar, Sus scrofa scrofa (Almeida et al., 2006), white-lipped peccaries, Tayassu pecari (Costa et al., 2007), and brown brocket deer, Mazama gouazoubira (Costa et al., 2011). In H. megacephalus, the nuclei of the Sertoli cells exhibited a nucleolus with approximately 2.9 m in diameter and loose chromatin. Type A spermatogonia were observed from Stage 1 through 8 near the basal lamina (Figs. 2 and 3). Stage 1 was characterized by the presence of round spermatids distributed into three or four cell layers. The preleptotene spermatocytes were observed close to the basal membrane, and the spermatocytes in pachytene were located between the round spermatids and spermatocytes in preleptotene (Fig. 2A). Stage 2 contained spermatids in an initial phase of nuclear elongation and chromatin condensation. Primary spermatocytes under transition from preleptotene to
leptotene, and some spermatocytes in pachytene, were located near the basal lamina (Fig. 2B). Stage 3 was characterized by the presence of elongated spermatids grouped into bunches with their heads oriented toward the basal environment. Two generations of primary spermatocytes were present at this stage: spermatocytes in zygotene and diplotene (Fig. 2C). The occurrence of two meiotic divisions was the most characteristic aspect regarding observations made on metaphase plates in Stage 4. The primary spermatocytes generate the secondary spermatocytes, which divide to produce the round spermatids. In addition, some bunches of elongated spermatids and primary spermatocytes undergoing transition from zygotene to pachytene were observed (Fig. 2D). Two distinct spermatid generations were observed in Stage 5: the round recently formed and elongated spermatids. Some bunches of elongated spermatids were located in the crypts of Sertoli cells. Primary spermatocytes undergoing transition from zygotene to pachytene were observed between the round spermatids and basal environment. The Sertoli cells nuclei with prominent nucleoli generally exhibited their longitudinal axis perpendicularly positioned at the basal lamina (Fig. 3A). All cellular types observed at the stages before Stage 6 were present in this stage except the spermatocytes in
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Fig. 3. Histological cross-section of seminiferous tubules of H. megacephalus showing stages 5–8 (tubular morphology method). (A) Stage 5; (B) stage 6; (C) stage 7; (D) stage 8. Lu, lumen; S, Sertoli cell; SPT Pl/L, spermatocyte in preleptotene/leptotene; SPT P, spermatocyte in pachytene; Z, spermatocyte in zygotene; SPTGA, type A spermatogonia; RSPD, round spermatids; ElSPD, elongating/elongated spermatids; Rb, residual bodies.
zygotene. In general, the spermatid bunches were closer to the tubular lumen than in previous stages, which is a characteristic aspect in this stage. Primary spermatocytes in pachytene were lying in the middle region of the seminiferous epithelium (Fig. 3B). In Stage 7, the bunched groups of elongated spermatids were dissociated from each other and located close to the tubular lumen. Some nuclei in the primary spermatocytes in pachytene were observed in the mid-region of the seminiferous epithelium (Fig. 3C). In Stage 8, the elongated spermatids are at a developmental stage ready to be delivered from the seminiferous epithelium. Residual bodies were lying in the luminal border of the seminiferous epithelium. Spermatocytes in pachytene and round spermatids were also observed. Some spermatocytes in preleptotene were observed near the basal lamina (Fig. 3D). The mean relative frequencies of the eight stages in H. megacephalus are shown in Fig. 4. Stages 1 (20.8%) and 8 (23.3%) showed the highest frequencies, and stages 4 (5.9%) and 7 (5%), the lowest frequencies. Different stages might be grouped into three phases using meiosis as a reference point: the pre-meiotic phase (after spermiation and prior to metaphase in meiosis); the meiotic phase (two meiotic divisions occur and the secondary spermatocytes are present); and the post-meiotic phase (after the completion of meiosis until spermiation) (Franc¸a and Russell, 1998). The pre-meiotic phase (Stages 1–3) represented 38.8%, the
Fig. 4. Mean percentage (±standard deviation) of each stage of the seminiferous epithelium cycle characterized according to tubular morphology system of H. megacephalus. Pre M, pre-meiotic phase; M, meiotic phase; Pos M, post-meiotic phase.
meiotic phase (Stage 4) 5.9%, and the post-meiotic phase (Stages 5–8) 55.3% of the cycle in the studied species (Fig. 4). 3.3. Cell counts The ratios between the corrected number cells used to assess the spermatogenic process and Sertoli cells are presented in Table 2. Three indexes are used to evaluate the different stages of spermatogenesis: (a) the mitotic index, which
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Table 2 Coefficients of the spermatogenic process and Sertoli cells of the testes of H. megacephalus (mean ± standard deviation; n = 5). Parameters
Mean ± SD
Mitotic index (SPT Pl/L/SPTGA) Meiotic index (RSPD/SPT P) Spermatogenic yield (RSPD/SPTGA) Sertoli cell index (SPTGA + SPTC Pl/L + SPTC P + RSPD)/S) Number of Sertoli cells per testis (×106 ) Number of Sertoli cells per gram of the testis (×106 )
9.77 2.29 20.12 22.44
± ± ± ±
3.81 0.28 5.93 8.40
7.82 ± 2.23 95.28 ± 46.59
quantifies the degree of cell loss in the proliferative phase or spermatogonial; (b) the meiotic index, which evaluates the efficiency of two meiotic divisions; and (c) the spermatogenic yield, which evaluates the efficiency of the spermatogenic process (Paula et al., 2002). Most species of mammals have six generations of differentiated spermatogonia, and one type-A spermatogonia will produce sixty-four primary spermatocytes at preleptotene/leptotene if there were no cell loss (Franc¸a and Russell, 1998). In H. megacephalus, each type-A spermatogonia will produce about ten primary spermatocytes at preleptotene/leptotene capable to enter metaphase (mitotic index). If H. megacephalus also presents six spermatogonial generations, the loss in this stage is of approximately 85% in this species, which is within the range of 60–90% observed in most animals (Roosen-Runge, 1973; Costa and Paula, 2003). Theoretically, each primary spermatocyte will produce four round spermatids (1:4) during the meiotic division. The meiotic index in H. megacephalus indicated that each primary spermatocyte at pachytene generated approximately 57% (losses of approximately 43%) of the expected value for round spermatids, which is within the range observed in other rodents (50% in N. lasiurus, Silva and Melo, 2010; 61.5% in C. paca, Carreta Júnior, 2008; and 65% in O. nigripes, Cordeiro-Júnior, 2009). The overall spermatogenesis yield is calculated based on the number of round spermatids because they are not significantly lost during spermiogenesis (Russell and Peterson, 1984). The number of round spermatids and spermatozoa resulting from each type-A spermatogonia is 256 cells, however, in H. megacephalus this number was approximately 20 cells. The overall spermatogenesis yield in H. megacephalus is similar to that of C. paca and D. aguti (20 cells, Carreta Júnior, 2008; 21 cells, Assis-Neto et al., 2003, respectively). This value indicated a cell loss of approximately 90% and suggested that significant losses occurred during the spermatogenic process. The Sertoli cell index in H. megacephalus was 22 germ cells; this value is similar to that described for C. paca (24.36; Carreta Júnior, 2008) and higher than for N. lasiurus (12.20; Silva and Melo, 2010), H. hydrochaeris (12; Paula et al., 2002), and D. aguti (8.0; Assis-Neto et al., 2003). The number of Sertoli cells per testis and per gram of testis in H. megacephalus was 7.82 × 106 and 95.28 × 106 , respectively. These were similar to and higher than values reported in other rodents (Cordeiro-Júnior, 2009).
4. Conclusions The present study showed that the wild rodent H. megacephalus has a high investment of body mass allocated in the testis and seminiferous tubules weights. This observation suggests promiscuous reproductive behavior, of the polyandrous type (many males with access to one female), favoring males with higher sperm production and consequently, larger testes. The mitotic and meiotic indexes suggest that the cell loss occurs at approximately 85% during the spermatogonial mitosis, and 43% during the meiotic process in H. megacephalus. The morphometric parameters evaluated in H. megacephalus are within the values described for most mammals. Acknowledgments We thank the Biological Sciences students from the Federal University of Goias – Campus Jatai for the collection of animals, the Structural Biology Laboratory at the Federal University of Vic¸osa for processing the biological material, and the Foundation for Research Support from the State of Goias (FAPEG) for the financial support. References Abercrombie, M., 1946. Estimation of nuclear populations from microtome sections. Anat. Rec. 94, 239–247. Almeida, F.F.L., Leal, M.C., Franc¸a, L.R., 2006. Testis morphometry, duration of spermatogenesis, and spermatogenic efficiency in the Wild boar (Sus scrofa scrofa). Biol. Reprod. 75, 792–799. Alves, A.G., Souza, C.F., Aguiar, C.S., Ferreira, L.R., Aguiar, N.S., Gettinger, D., Martins-Hatano, F., 2009. Levantamento da comunidade de pequenos mamíferos terrestres na área de influência lagoa Mascarenhas, Parauapebas, Pará, Brasil. In: Congresso Brasileiro de Ecologia do Brasil, 9. São Lourenc¸o. Anais Minas Gerais. 4 p. Amann, R.P., Almquist, J.O., 1962. Reproductive capacity of dairy bulls. VIII. Direct and indirect measurement of testicular sperm production. J. Dairy Sci. 45, 774–781. Amann, R.P., Schanbacher, B.D., 1983. Physiology of male reproduction. J. Anim. Sci. 57 (2), 380–403. Assis-Neto, A.C., Melo, M.I.V., Carvalho, M.A.M., Miglino, M.A., Oliveira, M.F., Menezes, D.J.A., Papa, P.C., Kfoury Júnior, J.R., 2003. Análise quantitativa do estabelecimento da espermatogênese em cutias (Dasyprocta aguti) criadas em cativeiros. Braz. J. Vet. Res. Anim. Sci. 40 (3), 175–179. Berndtson, W.E., 1977. Methods for quantifying mammalian spermatogenesis: a review. J. Anim. Sci. 44, 818–883. Bonvicino, C.R., Lindbergh, S.M., Moroja, L.S., 2002. Small non-flying mammals from conserved and altered areas of Atlantic forest and Cerrado: comments on their potential use for monitoring environment. J. Biol. 62 (4B), 765–774. Buitrón-Jurado, G., Toba, M., 2007. Possible asociasión de la ardilla enana Microsciurus flaviventer (Rodentia: Sciuridae) y bandadas mixtas de aves em la Amazonia Ecuatoriana. Mastozoologia Trop. 14 (2), 235–240. Carmignotto, A.P., Vivo, M., Langguth, A., 2012. Mammals of the Cerrado and Caatinga: distribution Patterns of the tropical open biomes of Central South America. In: Patterson, B.D., Costa, L.P. (Eds.), Bones, Clones, and Biomes: The History and Geography of Recent Neotropical Mammals, v. 1. University of Chicago Press, Chicago, pp. 307–350. Carreta Júnior, M., 2008. Avaliac¸ão morfofuncional do processo espermatogênico de pacas (Cunicullus paca, Linnaeus, 1766) adultas. 61 p. Dissertac¸ão (Mestrado) – Programa de Pós-graduac¸ão em Medicina Veterinária, Universidade Federal de Vic¸osa, Vic¸osa-MG. Cordeiro-Júnior, D.A., 2009. Estrutura e func¸ão testiculares em Roedores Silvestres das Famílias Echimyidae (Trinomys moojeni) e Cricetidae (Akodon cursor, Akodon montensis, Necromys lasiurus e Oligoryzomys migripes), da Reserva Particular do Patrimônio Natural do
F.C.S.A. de Melo et al. / Animal Reproduction Science 138 (2013) 110–117 Carac¸a – MG. 109 p. Tese (Doutorado) – Programa de Pós-graduac¸ão em Biologia Celular, Universidade Federal de Minas Gerais, Belo Horizonte – MG. Costa, D.S., Paula, T.A.R., 2003. Espermatogênese em mamíferos. Scientia 4, 53–72. Costa, D.S., Menezes, C.M.C., Paula, T.A.R., 2007. Spermatogenesis in whitelipped peccaries (Tayassu pecari). Anim. Reprod. Sci. 98, 322–334. Costa, K.L.C., Matta, S.L.P., Gomes, M.L.M., Paula, T.A.R., Freitas, K.M., Carvalho, F.A.R., Silveira, J.A., Dolder, H., Mendis-Handagama, S.M.L.C., 2011. Histomorphometric evaluation of the neotropical brown brocket deer Mazama gouazoubira testis, with an emphasis on cell population indexes of spermatogenic yield. Anim. Reprod. Sci. 127, 202–212. Courot, M., Hochereau-de-Reviers, M.T., Ortavant, R., 1970. Spermatogenesis. In: Johnson, A.D., Gomes, W.R., Vandenmark, N.L. (Eds.), The Testis, v. 1. Academic Press, New York, pp. 339–432. Curtis, S.K., Amann, R.P., 1981. Testicular development and establishment of spermatogenesis in Holstein bulls. J. Anim. Sci. 53, 1645–1657. Eisenberg, J.F., Redford, K.H., 1999. Mammals of the neotropics: the central neotropics, v. 3. The University of Chicago, Chicago. Franc¸a, L.R., 1991. Análise morfofuncional da espermatogênese de suínos adultos da rac¸a piau. 185p. Tese (Doutorado) – Programa de Pósgraduac¸ão em Biologia Celular, Universidade Federal de Minas Gerais, Belo Horizonte – MG. Franc¸a, L.R., Russell, L.D., 1998. The testis of domestic animals. In: Regadera, J., Martinez-Garcia (Eds.), Male Reproduction: A Multidisciplinary Overview. Churchill Livingstone, Madrid, pp. 197–219. Guião-Leite, F.L., Paula, T.A.R., Matta, S.L.P., Fonseca, C.C., Neves, M.T.D., Barros, J.B.G., 2006. Cycle and duration of the seminiferous epithelium in puma (Puma concolor). Anim. Reprod. Sci. 90, 307–316. Hochereau-de-Reviers, M.T., Lincoln, G.A., 1978. Seasonal variation in the histology of the testis of the red deer, Cervus elaphus. J. Reprod. Fertil. 54, 209–213. Johnson, L., Neaves, W.B., 1981. Age-related changes in the Leydig cell population, seminiferous tubules, and sperm production in stallions. Biol. Reprod. 24, 703–712. Karnovsky, M.J., 1965. A formaldehyde–glutaraldehyde fixative of high osmolarity for use in electron microscopy. J. Cell Biol. 27, 137A. Kenagy, G.J., Trombulak, S.C., 1986. Size and function of mammalian testes in relation to body size. J. Mammal. 67 (1), 1–22. Leal, M.C., Franc¸a, L.R., 2006. The seminiferous epithelium cycle length in the black tufted-ear marmoset (Callithrix penicillata) is similar to humans. Biol. Reprod. 74, 616–624. Mascarenhas, R.M., Paula, T.A.R., Matta, S.L.P., Lanna, L.L., Fonseca, C.C., Neves, M.T.D., 2006. Morfometria macro e microscópica e índices somáticos dos componentes testiculares de cães sem rac¸a definida, da puberdade à senilidade. Revista Ceres 305 (53), 106–112. Mittermeier, R.A., Gil, P.R., Hoffmann, M., Pilgrim, J., Brooks, T., Mittermeier, C.G., Lamourex, J., Fonseca, G.A.B., 2005. Hotspots Revisited. Earth’s Biologically Richest and Most Endangered Terrestrial Ecorregions. CEMEX e Agrupación Sierra Madre, Washington, USA. Navarro, R.D., Paula, T.A.R., Matta, S.L.P., Fonseca, C.C., Neves, M.T.D., 2004. Efeito da exposic¸ão pré e pós-natal ao ultra-som de baixa intensidade sobre a célula de Leydig e demais componentes do espac¸o intertubular do testículo de camundongos adultos. Revista Ceres 296 (51), 445–455. Ortavant, R., Courot, M., Hochereau-de-Reviers, M.T., 1977. Spermatogenesis in domestic mammals. In: Cole, H.H., Cupps, P.T. (Eds.),
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Reproduction in Domestic Animals. , 3rd ed. Academic Press, New York, pp. 203–227. Paula, T.A.R., Chiarini-Garcia, H., Franc¸a, L.R., 1999. Seminiferous epithelium cycle and its duration in capybaras (Hydrocoerus hydrochaeris). Tissue Cell 31, 327–334. Paula, T.A.R., Costa, D.S., Matta, S.L.P., 2002. Avaliac¸ão histológica do testículo de capivaras (Hydrochoerus hydrochaeris) adultas. Biosci. J. 18 (1), 121–136. Romano, H.S., Pereira, T., Fonseca, C.C., Paula, T.A.R., Neves, M.T.D., Matta, S.L.P., 2002. Avaliac¸ão do testículo e do processo espermatogênico em 5 diferentes espécies de roedores. In: XI Simpósio de Iniciac¸ão Científica. Anais.. v. 1, Vic¸osa, MG: Universidade Federal de Vic¸osa, p. 352–353. Roosen-Runge, E.C., 1973. Germinal-cell loss in normal metazoan spermatogenesis. J. Reprod. Fertil. 35, 339–348. Roosen-Runge, E.C., 1977. The Process of Spermatogenesis in Animals. Academic Press, Cambridge. Russell, L.D., Peterson, R.N., 1984. Determination of the elongate spermatid–Sertoli cell ratio in various mammals. J. Reprod. Fertil. 70, 635–641. Silva, K.C.S., Melo, F.C.S.A., 2010. Morfologia e morfometria testicular de Necromys lasiurus (Cricetidae, Sigmodontinae) em área de Cerrado no Sudoeste goiano. In: Congresso de Pesquisa, Ensino e Extensão da Universidade Federal de Goiás, Goiânia, GO. Anais.. Goiânia: Universidade Federal de Goiás. Silva, K.C.S., Sousa, T.P., Milen, L.C., Melo, F.C.S.A., Matta, S.L.P., Melo, F.R., Santa Rita, R.M., Oliveira, F.C.G., 2010. Testicular morphometry of the wild Necromys lasiurus (Rodentia: Cricetidae). In: Meeting of the Brazilian Society for Cell Biology, São Paulo. Anais. . .São Paulo: Sociedade Brasileira de Biologia Celular, p. 13–14. Silva Jr., V.A., Vieira, C.F.P., Paula, T.A.R., Palma, M.B., Amorim, A.A., Manhães, R.C., 2006. Neonatal treatment with naloxone increases the population of Sertoli cells and sperm production in adult rats. Reprod. Nutr. Dev. 46, 157–166. Silveira, J.A., 2007. Morfologia e morfometria testicular do roedor silvestre Oligoryzomys nigripes (Olfers, 1818) (Rodentia, Muridae). 43 p. Monografia, Universidade Federal de Vic¸osa, Vic¸osa-MG. Souza, P.C., Paula, T.A.R., Natali, A.J., Matta, S.L.P., Costa, D.S., Fonseca, C.C., Sarti, P., 2005. Efeito do exercício crônico voluntário e do sedentarismo, com e sem o uso do esteróide anabólico nandrolona, sobre os componentes do parênquima testicular de ratos adultos. Revista Ceres 300 (53), 305–316. Swierstra, E.E., 1968. Cytology and duration of the cycle of the seminiferous epithelium of the boar: duration of spermatozoa transit through the epididymis. Anat. Rec. 161, 171–186. Vieira, E.M., Monteiro-Filho, E.L.A., 2003. Vertical stratification of small mammals in the Atlantic Rainforest of southeastern Brazil. J. Trop. Ecol. 19, 501–507. Voss, R.S., Emmons, L.H., 1996. Mammalian Diversity in Neotropical Lowland Rainforests: A Preliminary Assessment. Bulletin of the American Museum of Natural History, New York. Wilson, D.E., Reeder, D.M., 2005. Mammal Species of the World: A Taxonomic and Geographic Reference, 3rd ed. The Johns Hopkins University Press, Baltimore. Wing, T.Y., Christensen, A.K., 1982. Morphometric studies on rat seminiferous tubules. Am. J. Anat. 115, 13–25. Woolley, P., 1975. The seminiferous tubules in dasyurid marsupials. J. Reprod. Fertil. 45, 255–261.
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Descriptive morphometry and stereology of the tubular compartment in the wild rodent Hylaeamys megacephalus (Rodentia: Cricetidae) from Central Brazil Fabiana Cristina Silveira Alves de Melo a,∗ , Tatiane Pires de Sousa a , Kyvia Lugate C. Costa b , Sérgio Luis P. da Matta b , Fabiano Rodrigues de Melo a , Ricardo de Mattos Santa-Rita a a b
Biological Sciences, Federal University of Goiás, Campus Jataí, Jataí, Brazil Department of General Biology, Federal University of Vic¸osa, Vic¸osa, Brazil
a r t i c l e
i n f o
Article history: Received 25 June 2012 Received in revised form 7 January 2013 Accepted 28 January 2013 Available online 13 February 2013 Keywords: Seminiferous tubules Spermatogenesis Reproduction Testis
a b s t r a c t Information on reproductive characteristics of wild rodents is scarce in the literature. This study aimed to assess the testis morphometry and stereology of Hylaeamys megacephalus. We used five animals in the study, captured in forest fragments in southwestern Goias State, between April and August 2009. The testes were fixed in Karnovsky solution, dehydrated, and embedded in methacrylate. Two-micrometer-thick sections from each sample were stained with toluidine blue/sodium borate 1%. Images of the testicular parenchyma were obtained from photomicroscope and morphometric and stereological analyses were carried out using the Image Pro-Plus software. The average body weight observed in the specimens of H. megacephalus in the study was 47.84 g, of which, 0.40% is allocated to the gonads (GSI) and 0.36% to the seminiferous tubules (TSI). These parameters suggest promiscuous reproductive behavior, of the polyandrous type, favoring males with higher sperm production and consequently, larger testes. The volume density of the seminiferous tubules was 94.46%, which represented a volume of 0.18 mL. The volume density and volume of the interstitium were 5.54% and 0.011 mL, respectively. The diameter of the seminiferous tubules was 206.5 m and the height of seminiferous epithelium was 71.27 m. H. megacephalus presents 5.06 m of seminiferous tubules and an average of 27.96 m of seminiferous tubules per gram of testis. The mitotic and meiotic indexes showed losses of 85 and 42%, respectively and an overall loss of 90% over the full spermatogenic process. The number of Sertoli cells per testis and per gram of testis was 7.8 × 106 and 95.28 × 106 , respectively. Most of the morphometric parameters evaluated in H. megacephalus in this study are within the range of values described for most mammals. © 2013 Elsevier B.V. All rights reserved.
1. Introduction ∗ Corresponding author at: Federal University of Goiás, BR 364, Km 192 no 3800, zip code 75801-615, Jataí, Goiás, Brazil. Tel.: +55 64 3606 8290; fax: +55 64 3606 8290. E-mail addresses: [email protected] (F.C.S.A. de Melo), [email protected] (T.P. de Sousa), kyvia [email protected] (K.L.C. Costa), [email protected] (S.L.P. da Matta), [email protected] (F.R. de Melo), [email protected] (R.d.M. Santa-Rita). 0378-4320/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.anireprosci.2013.01.013
Nowadays, the Cerrado biome can be described as a hotspot due to the high degree of endemic species and threats mostly as a result of anthropogenic origins mostly as the result of agricultural expansion (Mittermeier et al., 2005). Therefore, studies on mammals, in this environment, have high priority as they could provide information about taxonomic aspects and in addition assist in the
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development of conservation strategies to manage and maintain the current biodiversity (Voss and Emmons, 1996). According to Alves et al. (2009), the distribution of mammals, especially endangered species, has been used to justify the establishment of new protected areas. Recent studies have shown that large diversity and rarity of small mammals indicate that such group could be used as indicators in environmental studies (Bonvicino et al., 2002). According to Buitrón-Jurado and Toba (2007), the ecology of most rodents from South America is not known because of their wide distribution in forested habitats, nocturnal habits, and difficulties in capture and monitoring. The species Hylaeamys megacephalus, Fischer, 1814, belongs to the order Rodentia, Cricetidae Family, and Sigmodontinae Subfamily. The Rodentia order is the most numerous in the Mammalia class, with a long evolutionary history, great diversity, varied habits, and trophic strategies, and is present in all continents, except the Antarctic (Eisenberg and Redford, 1999; Wilson and Reeder, 2005). Rodents influence the dynamics of neotropical forests and are good indicators of both local habitat and landscape changes (Vieira and Monteiro-Filho, 2003). Cricetidae is the richest family in species diversity in Brazil with all members belonging to the Neotropical subfamily Sigmodontinae. H. megacephalus is a wild terrestrial rodent that inhabits forested and open vegetation areas in the Amazon Rainforest, Atlantic Forest, Cerrado, Caatinga, Pantanal, and in Brazil this species is distributed in the eastern Amazonas, Roraima, Pará, Western Amapá, Mato Grosso, Maranhão, Tocantins, Goiás and Distrito Federal, Mato Grosso do Sul, Western Minas Gerais, and Northeastern São Paulo (Carmignotto et al., 2012). The knowledge of the reproductive biology of a species, especially in its basic aspects, can make an important contribution toward the conservation of any species (Guião-Leite et al., 2006). The assessment of the histological parameters of gonads and other reproductive organs becomes an important tool in the analysis of the reproductive conditions of an animal in its natural habitat. These parameters can be related to environmental factors and contribute to the conservation of mammals. Also, the composition of the testicular parenchyma and relative size of the testicles can provide valuable information on the reproductive physiology and mating system of a given species (Kenagy and Trombulak, 1986; Paula et al., 2002). Due to the scarcity of information on the reproductive biology of wild rodents and their associated ecological role, the objective of this study was to describe the testicular morphology of H. megacephalus and expand the knowledge about its reproduction in natural environments. 2. Materials and methods 2.1. Animal capture and histological processing Testes were collected from five adult animals captured (from April to August 2009) in forest fragments in southwestern Goiás (IBAMA License number 11621-1 and Ethics Committee Protocol 233/10 CEP/UFG). The animals were captured through traditional methods using Sherman and
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Tomahawk live traps, spaced at approximately every 10 m along linear ground transects. The testes were removed in the field, rapidly rinsed in saline solution, and fixed in Karnovsky solution (Karnovsky, 1965). Testes were dehydrated and embedded in methacrylate after 24 h of fixation. Two-micrometer sections from each testis were stained with 1% toluidine blue/sodium borate. Images of the testicular parenchyma were obtained using an Olympus AX-70 photomicroscope; the morphometric analyses were carried out using the Image Pro-Plus software. 2.2. Testis morphometry and stereology The volume density of the tubular and interstitial compartments were estimated by projecting 300 points in each digital image (200× magnification) resulting in a total of 2500 points per animal (Fig. 1A). The volume (mL) of each testicular component was estimated considering the percentage occupied by both seminiferous tubules and interstitium multiplied by the testicular parenchyma volume. The parenchyma volume was obtained by subtracting the albuginea weight from the testis weight. Since the testes density is estimated at about 1 in mammals (Johnson and Neaves, 1981; Franc¸a, 1991), the testis weight was considered equal to its volume. The gonadal (GW) and seminiferous tubules (STW) weights were obtained to calculate the gonadosomatic and tubulesomatic indexes, respectively as follows: GW/BW × 100 and STW/BW × 100, where BW = body weight. The average diameter of the seminiferous tubules was obtained from 20 random circular cross-sections from each animal’s sample (100× magnification) (Fig. 1B). The seminiferous epithelium height was measured using the same sections in which the tubule diameter was obtained. The epithelium height was considered as the space between the tunica propria and luminal edge. Two diametrically opposed readings were taken using a digital ruler on each cross section and considering their mean value (Fig. 1B). The total length (TL) of the seminiferous tubules, in meters, was estimated dividing the total volume occupied by the seminiferous tubules (STV) by their mean tubular diameter (R2 , R = tubular diameter/2) (Ortavant et al., 1977; Curtis and Amann, 1981). The TL was divided by the testicular weight to calculate the tubular length per gram of testis. 2.3. Stages of the seminiferous epithelium cycle The stages of the seminiferous epithelium cycle were characterized based on the shape and location of the spermatid nuclei, presence of meiotic divisions, and overall seminiferous epithelium composition (Amann and Almquist, 1962; Courot et al., 1970; Leal and Franc¸a, 2006). This process, known as tubular morphology method, establishes eight stages for the seminiferous epithelium (Berndtson, 1977; Paula et al., 1999). The relative stage frequencies were determined from the characterization and counting of 200 seminiferous tubule cross-sections from each animal randomly chosen (400× magnification).
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Fig. 1. Testicular parenchyma of H. megacephalus. (A) Wide view of testicular parenchyma; left corner, model of the square grid used to calculate the volume density of testicular components (SE, seminiferous epithelium; Int, interstitium). Bar: 87 m. (B) Seminiferous tubule cross section of H. megacephalus showing how the tubular diameter (large arrows) and epithelium height (small arrows) were measured. The average values were considered in each case. (SE, seminiferous epithelium; Int, interstitium; Lu, lumen). Bar: 50 m. (C) Detail of the seminiferous epithelium of H. megacephalus on Stage 1, showing all different germ and somatic cell types (Int, interstitium; Lu, lumen; RSPD, round spermatid; SPT Pl/L, spermatocyte – preleptotene/leptotene; SPT P, spermatocyte – pachytene; SPTGA, type A spermatogonia; S, Sertoli cell nucleus). Bar: 22 m.
2.4. Cell counts The germ cell population that constitutes the seminiferous epithelium in stage 1 (tubular morphology method) was estimated based on the count of cell nuclei within the cross sectioned tubule, including Sertoli cells nucleolus (Swierstra, 1968; Courot et al., 1970; Amann and Schanbacher, 1983). The following celltypes were quantified in 10 seminiferous tubule cross sections: type-A spermatogonia (SPTGA), primary spermatocytes at preleptotene/leptotene (SPT Pl/L) and pachytene (SPT P), round spermatids (RSPD), and Sertoli cells (S) (Fig. 1C). The mean nuclei and nucleoli diameters were
obtained by measuring 30 nuclei from the cells cited above. The counting of different cell types was corrected by the Abercrombie’s formula (1946) further modified by Amann and Almquist (1962) because of size variations. It was possible to quantify the following coefficients based on the corrected values: spermatogonial mitosis (SPT Pl/L/SPTGA), meiotic yield (RSPD/SPT P), spermatogenic yield (RSPD/SPTGA), and Sertoli cell index ((SPTGA + SPTC Pl/L + SPTC P + RSPD)/S). The number of Sertoli cells per testis and per gram of testis was calculated in accordance with Hochereau-de-Reviers and Lincoln (1978). The results are reported as means and standard deviations.
F.C.S.A. de Melo et al. / Animal Reproduction Science 138 (2013) 110–117 Table 1 Biometric, morphometric, and stereological parameters of the testes of H. megacephalus (mean ± standard deviation; n = 5). Parameters
Mean ± SD
Body weight (g) Testicular weight (g) Albuginea weight (g) Parenchyma weight (g) Gonadosomatic index (%) Tubulesomatic index (%) Volume density of seminiferous tubule (%) Volume density of interstitium (%) Volume of seminiferous tubule (mL) Volume of interstitium (mL) Diameter of seminiferous tubule (m) Height of seminiferous epithelium (m) Total length of seminiferous tubule (m) Length of seminiferous tubule per gram of testis (m/g)
47.84 0.19 0.006 0.18 0.40 0.36 94.46
± ± ± ± ± ± ±
8.7 0.09 0.001 0.08 0.15 0.13 1.74
5.54 0.18 0.011 206.5 71.27
± ± ± ± ±
1.74 0.08 0.007 21.40 10.39
5.06 ± 1.62 27.96 ± 6.18
3. Results and discussion The testes of the wild rodent H. megacephalus are paired organs located in the scrotum and surrounded by a thick capsule of connective tissue (testicular albuginea). The testicular parenchyma is divided into two compartments; namely tubular and interstitial. The tubular compartment consists of the seminiferous tubules, which comprises the tunica propria, the seminiferous epithelium, and lumen. The tunica propria has a monolayer of myoid cells; the seminiferous epithelium is composed of spermatogenic cells in various stages of development and Sertoli cells with characteristic irregular nucleus and prominent nucleoli. The interstitial compartment is constituted of Leydig cells, cells and fibers of connective tissue, blood vessels, and lymphatic space. 3.1. Testis stereology The determined values for the biometric, morphometric, and stereological parameters in H. megacephalus are presented in Table 1. The albuginea and mediastinum were deducted from the testicular weight to calculate the testicular parenchyma (Johnson and Neaves, 1981). In H. megacephalus, the albuginea occupied 3.33% of the testicular weight, which is similar to the proportion observed in pacas, Cuniculus paca (3.98%; Carreta Júnior, 2008) and lower than that observed in capybaras, Hydrochoerus hydrochaeris (6.13%; Paula et al., 2002). The gonadosomatic index (GSI) indicates the investment in gonads relative to body weight and is an important reproductive parameter (Franc¸a and Russell, 1998). The GSI of H. megacephalus was 0.40%, which is a significantly higher value than what is reported for H. hydrochaeris (0.12%; Paula et al., 2002) and similar to the value reported in small rodents such as Oligorizomys nigripes (0.50%; Silveira, 2007) and Necromys lasiurus (0.47%; Silva et al., 2010). Because the testicular size is not proportional to body size, the somatic investment in gonadal mass is
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greater in small animals compared to larger body size animals (Kenagy and Trombulak, 1986). The tubulesomatic index (TSI) is a parameter that quantifies the investment of body mass allocated in seminiferous tubules and is an important index to evaluate the influence of reproductive behavior on testicular morphology (Paula et al., 2002). H. megacephalus presented higher TSI values (0.36%) than what is reported for H. hydrochaeris (0.06%; Paula et al., 2002) and C. paca (0.24%; Carreta Júnior, 2008), however, within the range determined in several other species of Rodentia (Romano et al., 2002). The testes weight and size can be used as quantitative indicators of spermatic production because the main testicular component is the seminiferous tubule (Franc¸a and Russell, 1998). The GSI and TSI results in this study suggested that H. megacephalus displays promiscuous reproductive behavior, of the polyandrous type (many males have access to one female), favoring males with higher sperm production and consequently, larger testes (Eisenberg and Redford, 1999). Approximately 95% of the testicular parenchyma in H. megacephalus is constituted by seminiferous tubules, which is a higher percentage than observed in other rodents like C. paca (91%; Carreta Júnior, 2008), Dasyprocta aguti (84.75%; Assis-Neto et al., 2003), and H. hydrochaeris (50%; Paula et al., 2002), and closer to the value reported for small rodents such as N. lasiurus (93%; Silva et al., 2010). The interstitial proportion in H. megacephalus is within the range described for most mammals (Franc¸a and Russell, 1998). The tubular and interstitial volumes in H. megacephalus (0.18 mL and 0.011 mL, respectively) were higher than the values described for O. nigripes (0.052 mL and 0.005 mL; Silveira, 2007) and similar to those described for N. lasiurus (0.16 mL and 0.012 mL; Silva et al., 2010). The tubular measurement is one of the approaches used as an indicator of spermatogenic activity (Navarro et al., 2004; Souza et al., 2005; Mascarenhas et al., 2006; Silva et al., 2006). Although the tubule diameter can reach up to 550 m in some species of marsupials (Woolley, 1975), the values observed in most amniotes varies from 180 to 300 m (Roosen-Runge, 1977). The average tubular diameter in H. megacephalus was 206.6 m, which is closer to the values described in C. paca (202.1 m; Carreta Júnior, 2008), H. hydrochaeris (213 m; Paula et al., 2002), and N. lasiurus (193.47 m; Silva et al., 2010). According to Wing and Christensen (1982), the height of the seminiferous epithelium is more effective than the tubular diameter in the evaluation of sperm production. The height of the seminiferous epithelium in H. megacephalus was 71.27 m, which is within the range of values observed in mammals (60–100 m; Franc¸a and Russell, 1998). The total length of the seminiferous tubules is related to three structural parameters: testes size, tubular diameter, and volume of the seminiferous tubules (Franc¸a and Russell, 1998). The total length of the seminiferous tubules in H. megacephalus was 5.1 m. When considering the differences in testis size among species, the conversion of the total tubular length to total length of the seminiferous tubules per gram of testis, makes the measurement
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Fig. 2. Histological cross-section of seminiferous tubules of H. megacephalus showing stages 1–4 (tubular morphology method). (A) Stage 1; (B) stage 2; (C) stage 3; (D) stage 4. Lu, lumen; S, Sertoli cell; SPT Pl/L, spermatocyte in preleptotene/leptotene; SPT P, spermatocyte in pachytene; Z, spermatocyte in zygotene; D, spermatocyte in diplotene; SPTGA, type A spermatogonia; M, metaphase plate; RSPD, round spermatids; ElSPD, elongating/elongated spermatids.
independent of the animal size. H. megacephalus presented 27.9 m of tubule per gram of testis, which is within the pattern value observed in other rodents (Cordeiro-Júnior, 2009; Carreta Júnior, 2008). 3.2. Stages of the seminiferous epithelium cycle and relative stage frequencies The eight stages of the seminiferous epithelium cycle in H. megacephalus were similar to those observed in wild boar, Sus scrofa scrofa (Almeida et al., 2006), white-lipped peccaries, Tayassu pecari (Costa et al., 2007), and brown brocket deer, Mazama gouazoubira (Costa et al., 2011). In H. megacephalus, the nuclei of the Sertoli cells exhibited a nucleolus with approximately 2.9 m in diameter and loose chromatin. Type A spermatogonia were observed from Stage 1 through 8 near the basal lamina (Figs. 2 and 3). Stage 1 was characterized by the presence of round spermatids distributed into three or four cell layers. The preleptotene spermatocytes were observed close to the basal membrane, and the spermatocytes in pachytene were located between the round spermatids and spermatocytes in preleptotene (Fig. 2A). Stage 2 contained spermatids in an initial phase of nuclear elongation and chromatin condensation. Primary spermatocytes under transition from preleptotene to
leptotene, and some spermatocytes in pachytene, were located near the basal lamina (Fig. 2B). Stage 3 was characterized by the presence of elongated spermatids grouped into bunches with their heads oriented toward the basal environment. Two generations of primary spermatocytes were present at this stage: spermatocytes in zygotene and diplotene (Fig. 2C). The occurrence of two meiotic divisions was the most characteristic aspect regarding observations made on metaphase plates in Stage 4. The primary spermatocytes generate the secondary spermatocytes, which divide to produce the round spermatids. In addition, some bunches of elongated spermatids and primary spermatocytes undergoing transition from zygotene to pachytene were observed (Fig. 2D). Two distinct spermatid generations were observed in Stage 5: the round recently formed and elongated spermatids. Some bunches of elongated spermatids were located in the crypts of Sertoli cells. Primary spermatocytes undergoing transition from zygotene to pachytene were observed between the round spermatids and basal environment. The Sertoli cells nuclei with prominent nucleoli generally exhibited their longitudinal axis perpendicularly positioned at the basal lamina (Fig. 3A). All cellular types observed at the stages before Stage 6 were present in this stage except the spermatocytes in
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Fig. 3. Histological cross-section of seminiferous tubules of H. megacephalus showing stages 5–8 (tubular morphology method). (A) Stage 5; (B) stage 6; (C) stage 7; (D) stage 8. Lu, lumen; S, Sertoli cell; SPT Pl/L, spermatocyte in preleptotene/leptotene; SPT P, spermatocyte in pachytene; Z, spermatocyte in zygotene; SPTGA, type A spermatogonia; RSPD, round spermatids; ElSPD, elongating/elongated spermatids; Rb, residual bodies.
zygotene. In general, the spermatid bunches were closer to the tubular lumen than in previous stages, which is a characteristic aspect in this stage. Primary spermatocytes in pachytene were lying in the middle region of the seminiferous epithelium (Fig. 3B). In Stage 7, the bunched groups of elongated spermatids were dissociated from each other and located close to the tubular lumen. Some nuclei in the primary spermatocytes in pachytene were observed in the mid-region of the seminiferous epithelium (Fig. 3C). In Stage 8, the elongated spermatids are at a developmental stage ready to be delivered from the seminiferous epithelium. Residual bodies were lying in the luminal border of the seminiferous epithelium. Spermatocytes in pachytene and round spermatids were also observed. Some spermatocytes in preleptotene were observed near the basal lamina (Fig. 3D). The mean relative frequencies of the eight stages in H. megacephalus are shown in Fig. 4. Stages 1 (20.8%) and 8 (23.3%) showed the highest frequencies, and stages 4 (5.9%) and 7 (5%), the lowest frequencies. Different stages might be grouped into three phases using meiosis as a reference point: the pre-meiotic phase (after spermiation and prior to metaphase in meiosis); the meiotic phase (two meiotic divisions occur and the secondary spermatocytes are present); and the post-meiotic phase (after the completion of meiosis until spermiation) (Franc¸a and Russell, 1998). The pre-meiotic phase (Stages 1–3) represented 38.8%, the
Fig. 4. Mean percentage (±standard deviation) of each stage of the seminiferous epithelium cycle characterized according to tubular morphology system of H. megacephalus. Pre M, pre-meiotic phase; M, meiotic phase; Pos M, post-meiotic phase.
meiotic phase (Stage 4) 5.9%, and the post-meiotic phase (Stages 5–8) 55.3% of the cycle in the studied species (Fig. 4). 3.3. Cell counts The ratios between the corrected number cells used to assess the spermatogenic process and Sertoli cells are presented in Table 2. Three indexes are used to evaluate the different stages of spermatogenesis: (a) the mitotic index, which
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Table 2 Coefficients of the spermatogenic process and Sertoli cells of the testes of H. megacephalus (mean ± standard deviation; n = 5). Parameters
Mean ± SD
Mitotic index (SPT Pl/L/SPTGA) Meiotic index (RSPD/SPT P) Spermatogenic yield (RSPD/SPTGA) Sertoli cell index (SPTGA + SPTC Pl/L + SPTC P + RSPD)/S) Number of Sertoli cells per testis (×106 ) Number of Sertoli cells per gram of the testis (×106 )
9.77 2.29 20.12 22.44
± ± ± ±
3.81 0.28 5.93 8.40
7.82 ± 2.23 95.28 ± 46.59
quantifies the degree of cell loss in the proliferative phase or spermatogonial; (b) the meiotic index, which evaluates the efficiency of two meiotic divisions; and (c) the spermatogenic yield, which evaluates the efficiency of the spermatogenic process (Paula et al., 2002). Most species of mammals have six generations of differentiated spermatogonia, and one type-A spermatogonia will produce sixty-four primary spermatocytes at preleptotene/leptotene if there were no cell loss (Franc¸a and Russell, 1998). In H. megacephalus, each type-A spermatogonia will produce about ten primary spermatocytes at preleptotene/leptotene capable to enter metaphase (mitotic index). If H. megacephalus also presents six spermatogonial generations, the loss in this stage is of approximately 85% in this species, which is within the range of 60–90% observed in most animals (Roosen-Runge, 1973; Costa and Paula, 2003). Theoretically, each primary spermatocyte will produce four round spermatids (1:4) during the meiotic division. The meiotic index in H. megacephalus indicated that each primary spermatocyte at pachytene generated approximately 57% (losses of approximately 43%) of the expected value for round spermatids, which is within the range observed in other rodents (50% in N. lasiurus, Silva and Melo, 2010; 61.5% in C. paca, Carreta Júnior, 2008; and 65% in O. nigripes, Cordeiro-Júnior, 2009). The overall spermatogenesis yield is calculated based on the number of round spermatids because they are not significantly lost during spermiogenesis (Russell and Peterson, 1984). The number of round spermatids and spermatozoa resulting from each type-A spermatogonia is 256 cells, however, in H. megacephalus this number was approximately 20 cells. The overall spermatogenesis yield in H. megacephalus is similar to that of C. paca and D. aguti (20 cells, Carreta Júnior, 2008; 21 cells, Assis-Neto et al., 2003, respectively). This value indicated a cell loss of approximately 90% and suggested that significant losses occurred during the spermatogenic process. The Sertoli cell index in H. megacephalus was 22 germ cells; this value is similar to that described for C. paca (24.36; Carreta Júnior, 2008) and higher than for N. lasiurus (12.20; Silva and Melo, 2010), H. hydrochaeris (12; Paula et al., 2002), and D. aguti (8.0; Assis-Neto et al., 2003). The number of Sertoli cells per testis and per gram of testis in H. megacephalus was 7.82 × 106 and 95.28 × 106 , respectively. These were similar to and higher than values reported in other rodents (Cordeiro-Júnior, 2009).
4. Conclusions The present study showed that the wild rodent H. megacephalus has a high investment of body mass allocated in the testis and seminiferous tubules weights. This observation suggests promiscuous reproductive behavior, of the polyandrous type (many males with access to one female), favoring males with higher sperm production and consequently, larger testes. The mitotic and meiotic indexes suggest that the cell loss occurs at approximately 85% during the spermatogonial mitosis, and 43% during the meiotic process in H. megacephalus. The morphometric parameters evaluated in H. megacephalus are within the values described for most mammals. Acknowledgments We thank the Biological Sciences students from the Federal University of Goias – Campus Jatai for the collection of animals, the Structural Biology Laboratory at the Federal University of Vic¸osa for processing the biological material, and the Foundation for Research Support from the State of Goias (FAPEG) for the financial support. References Abercrombie, M., 1946. Estimation of nuclear populations from microtome sections. Anat. Rec. 94, 239–247. Almeida, F.F.L., Leal, M.C., Franc¸a, L.R., 2006. Testis morphometry, duration of spermatogenesis, and spermatogenic efficiency in the Wild boar (Sus scrofa scrofa). Biol. Reprod. 75, 792–799. Alves, A.G., Souza, C.F., Aguiar, C.S., Ferreira, L.R., Aguiar, N.S., Gettinger, D., Martins-Hatano, F., 2009. Levantamento da comunidade de pequenos mamíferos terrestres na área de influência lagoa Mascarenhas, Parauapebas, Pará, Brasil. In: Congresso Brasileiro de Ecologia do Brasil, 9. São Lourenc¸o. Anais Minas Gerais. 4 p. Amann, R.P., Almquist, J.O., 1962. Reproductive capacity of dairy bulls. VIII. Direct and indirect measurement of testicular sperm production. J. Dairy Sci. 45, 774–781. Amann, R.P., Schanbacher, B.D., 1983. Physiology of male reproduction. J. Anim. Sci. 57 (2), 380–403. Assis-Neto, A.C., Melo, M.I.V., Carvalho, M.A.M., Miglino, M.A., Oliveira, M.F., Menezes, D.J.A., Papa, P.C., Kfoury Júnior, J.R., 2003. Análise quantitativa do estabelecimento da espermatogênese em cutias (Dasyprocta aguti) criadas em cativeiros. Braz. J. Vet. Res. Anim. Sci. 40 (3), 175–179. Berndtson, W.E., 1977. Methods for quantifying mammalian spermatogenesis: a review. J. Anim. Sci. 44, 818–883. Bonvicino, C.R., Lindbergh, S.M., Moroja, L.S., 2002. Small non-flying mammals from conserved and altered areas of Atlantic forest and Cerrado: comments on their potential use for monitoring environment. J. Biol. 62 (4B), 765–774. Buitrón-Jurado, G., Toba, M., 2007. Possible asociasión de la ardilla enana Microsciurus flaviventer (Rodentia: Sciuridae) y bandadas mixtas de aves em la Amazonia Ecuatoriana. Mastozoologia Trop. 14 (2), 235–240. Carmignotto, A.P., Vivo, M., Langguth, A., 2012. Mammals of the Cerrado and Caatinga: distribution Patterns of the tropical open biomes of Central South America. In: Patterson, B.D., Costa, L.P. (Eds.), Bones, Clones, and Biomes: The History and Geography of Recent Neotropical Mammals, v. 1. University of Chicago Press, Chicago, pp. 307–350. Carreta Júnior, M., 2008. Avaliac¸ão morfofuncional do processo espermatogênico de pacas (Cunicullus paca, Linnaeus, 1766) adultas. 61 p. Dissertac¸ão (Mestrado) – Programa de Pós-graduac¸ão em Medicina Veterinária, Universidade Federal de Vic¸osa, Vic¸osa-MG. Cordeiro-Júnior, D.A., 2009. Estrutura e func¸ão testiculares em Roedores Silvestres das Famílias Echimyidae (Trinomys moojeni) e Cricetidae (Akodon cursor, Akodon montensis, Necromys lasiurus e Oligoryzomys migripes), da Reserva Particular do Patrimônio Natural do
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