ENCICLOPÉDIA BIOSFERA, Centro Científico Conhecer - Goiânia, v.11 n.21; p. 2015 2331 MORPHOMETRY AND STEREOLOGY OF THE INTERSTITIAL COMPARTMENT
IN THE WILD RODENT HYLAEAMYS MEGACEPHALUS (RODENTIA: CRICETIDAE) FROM CENTRAL BRAZIL
Fabiana Cristina Silveira Alves de Melo1, Tatiane Pires de Sousa2; Kyvia Lugate Cardoso Costa3; Sérgio Luis Pinto da Matta3; Fabiano Rodrigues de Melo1
1
Biological Sciences, Federal University of Goiás, Campus Jataí, Jataí, Brazil (facrismelo@yahoo.com.br);
2
Ecology and Conservation Program, State University of Mato Grosso, Campus Nova Xavantina, Nova Xavantina, Brazil;
3
Department of General Biology, Federal University of Viçosa, Viçosa, Brazil.
Recebido em: 31/03/2015 – Aprovado em: 15/05/2015 – Publicado em: 01/06/2015
ABSTRACT
Little is known about the testicular components in wild rodents, particularly the interstitial compartment. The aim of this study was to describe the testis morphometry and stereology of Hylaeamys megacephalus, with emphasis in the interstitial compartment. In this study were used five animals captured in forest fragments in southwestern Goiás. The testes were removed, fixed in Karnovsky, dehydrated, embedded in methacrylate, sectioned with two-micrometer-thick, and stained with 1% toluidine blue/sodium borate. The histological sections of testes were evaluated using light microscopy. The interstitial compartment volume density was 5.5%, consisting predominantly of Leydig cells (3.21%), followed by blood vessels (0.97%), connective tissue (0.83%) and lymphatic space (0.53%). The Leydigossomatic index was 0.015%. The interstitial pattern in H. megacephalus was similar to pattern I of Fawcett’s classification as was found in capybara. The Leydig cell average volume in H. megacephalus was 350.80 µm3, close to the lowest values observed in several domestic, wild and laboratory species. The number of Leydig cells per testis and per gram of testis was higher than observed in other small rodents.
KEYWORDS: Leydig cell, lymphatic space, connective tissue, testes, blood vessel. MORFOMETRIA E ESTEREOLOGIA DO COMPARTIMENTO INTERSTICIAL NO
ROEDOR SILVESTRE HYLAEAMYS MEGACEPHALUS (RODENTIA: CRICETIDAE) DO BRASIL CENTRAL
RESUMO
Pouco se sabe sobre os componentes testiculares em roedores silvestres, particularmente o compartimento intersticial. O objetivo deste estudo foi descrever a morfometria e estereologia testicular de Hylaeamys megacephalus, com ênfase no compartimento intersticial. Neste estudo foram utilizados cinco animais capturados em fragmentos florestais no sudoeste de Goiás. Os testículos foram fixados em Karnovsky, desidratados, embebidos em metacrilato, seccionados com dois micrômetros de espessura e corados com azul de toluidina-borato de sódio 1%. Os cortes histológicos dos testículos foram avaliados ao microscópio de luz. A densidade volumétrica do compartimento intersticial foi de 5,5%, consistindo predominantemente de células de Leydig (3,21%), seguido por vasos sanguíneos
ENCICLOPÉDIA BIOSFERA, Centro Científico Conhecer - Goiânia, v.11 n.21; p. 2015 2332 (0,97%), tecido conjuntivo (0,83%) e espaço linfático (0,53%). O índice Leydigossomático foi de 0,015%. O padrão de organização do tecido intersticial em H. megacephalus foi similar ao padrão I da classificação de FAWCETT et al. (1973), como foi encontrado em capivaras. O volume médio da célula de Leydig em H. megacephalus foi 350,80 µm3, perto dos menores valores observados em diversas espécies domésticas, silvestres e de laboratório. O número de células de Leydig por testículo e por grama de testículo foi maior do que o observado em outros pequenos roedores.
PALAVRAS-CHAVE: célula de Leydig, espaço linfático, tecido conjuntivo, testículos, vaso sanguíneo.
INTRODUCTION
The Rodentia order is the most numerous of the Mammalia class and it is present in all continents except the Antarctic. This order has a long evolutionary history, great diversity and varied habits and trophic strategies (BONVICINO et al., 2008). The Cricetidae is the richest family in species diversity in Brazil and all members belong to the Neotropical subfamily Sigmodontinae. The wild rodent Hylaeamys megacephalus has terrestrial habits, inhabiting forested and open vegetation areas in the main Biomes of Brazil, including Amazon Rainforest, Atlantic Forest and Cerrado. In Brazil, this species is founded in Amazonas, Roraima, Pará, Western Amapá, Mato Grosso, Maranhão, Tocantins, Goiás, Distrito Federal, Mato Grosso do Sul, Western Minas Gerais, and Northeastern São Paulo (CARMIGNOTTO et al., 2012).
The mammalian testis can be divided into tubular or spermatogenic compartment and interstitial or intertubular compartment. The tubular compartment contains seminiferous tubules and the interstitial compartment contains blood and lymphatic vessels, nerves, a variable population of cells in the connective tissue, and Leydig cells (MORAIS et al., 2013a). The percentage of the interstitial components is variable in different species (FAWCETT et al., 1973, FRANÇA & RUSSELL, 1998), and the component that has the greater percentage variation is the Leydig cell (COSTA et al., 2010).
FAWCETT et al., (1973) described three patterns of interstitial tissue organization according to the arrangement and the proportion of the elements in this compartment. The first pattern has small volume of Leydig cells, scarce connective tissue, and great part of this compartment is occupied by the lymphatic vessels. In the second pattern the clusters of Leydig cells are widely scattered in a very loose connective tissue stroma drained by conspicuous lymphatic vessels. The third pattern has the Leydig cells occupying almost all the interstitial area, with little connective tissue and few lymphatic vessels. Although little is known about the physiological implications of these variations, they are probably related to the ability of lymphatic vessels in move out of the testes materials vascularly secreted and maintain adequate concentrations of androgens in the testis and blood vessels (FAWCETT et al., 1973).
Little is known about the testicular components in wild rodents, particularly the interstitial compartment. The assessment of 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. The aim of this study was to describe the testicular morphology, morphometry and stereology of the interstitial compartment in H. megacephalus.
ENCICLOPÉDIA BIOSFERA, Centro Científico Conhecer - Goiânia, v.11 n.21; p. 2015 2333 MATERIAL AND METHODS
Animal capture and histological processing
The animals were captured in forest fragments in southwestern Goiás through traditional methods using live animal traps, from April to August 2009 (IBAMA License number 11621-1 and Ethics Committee Protocol 233/10 CEP/UFG).
Five adult animals were anesthetized by intraperitoneal injection of xylazine (1mg/kg) and ketamine (15mg/kg) and killed with an overdose of the same anesthetic. After euthanasia, the testes were removed and fixed in Karnovsky solution, dehydrated and embedded in methacrylate. Two-micrometer sections from each testis were stained with 1% toluidine blue/sodium borate. Images of the testicular parenchyma were obtained using the photomicroscope Olympus AX-70 and the morphometric analyses were carried out using the Image Pro-Plus software. Testis morphometry and stereology
The albuginea from one of the testes was removed and weighed and it was subtracted of the testicular weight to calculate the weight of testicular parenchyma. Since the testicular density is near 1, the weight was considered equal to that testicular volume (COSTA et al., 2010).
The volume density of the tubular and interstitial compartments was estimated by counting 2,500 points projected onto images captured in histological sections per animal. In the interstitial compartment were considered points on nuclei and cytoplasm of the Leydig cells, blood vessels, lymphatic space and connective tissue (Figure 1).
The volume (mL) occupied by the tubular and interstitial compartments was estimated considering the percentage of each compartment multiplied by the testicular parenchyma volume. Choosing only spherical nuclei and evident nucleoli, the average diameter of the Leydig cell nucleus was obtained from the assessment of 50 cells per animal.
FIGURE 1 – Cross section of H. megacephalus testis (ST= seminiferous tubules; IT= interstitial tissue; LC= Leydig cell (cluster); LS= lymphatic space). Bar: 16µm.
ENCICLOPÉDIA BIOSFERA, Centro Científico Conhecer - Goiânia, v.11 n.21; p. 2015 2334 The nuclear volume (µm3) was obtained from the knowledge of the mean nuclear diameter, and the 4/3R3 formula was used (R=nuclear diameter/2). The cytoplasmatic volume (µm3) was estimated multiplying the percentage of cytoplasm by the nuclear volume, divided by the nuclear percentage. The single cell volume (µm3) was estimated by the sum of the nuclear and cytoplasmatic volumes.
The number of Leydig cells per testis was calculated from the Leydig cell individual volume and the total volume occupied by Leydig cells in the testicular parenchyma. To obtain the number of Leydig cells per gram of testis, the number of Leydig cells was divided by the gonadal weight. The Leydigosomatic index (LSI) was estimated by dividing the volume of Leydig cells in the parenchyma by body weight and multiplying the quotient by 100. This index quantifies the investment in Leydig cells related to the mass of the body.
All data are reported as mean and standard deviation.
RESULTS AND DISCUSSION
The testicular morphometry, volume density and volume of the testicular parenchyma components in H. megacephalus are presented in Table 1.
TABLE 1 - Morphometry and stereology of the testicular parenchyma components in H. megacephalus (mean + standard deviation; n=5). Parameters Mean + SD Body weight (g) 47.84+8.7 Testicular weight (g) 0.195+0.089 Albuginea weight (g) 0.006+0.001 Parenchyma weight (g) 0.189+0.087
Volume density of seminiferous tubule (%) 94.46+1.74 Volume density of interstitial tissue (%) 5.54+1.74 Volume of seminiferous tubule (mL) 0.178+0.081 Volume of interstitial tissue (mL) 0.011+0.007 Volume density in testicular parenchyma (%)
Leydig cells 3.21+2.04
Blood vessel 0.97+0.46
Connective tissue 0.83+0.29
Lymphatic space 0.53+0.96
Volume in testicular parenchyma (mL)
Leydig Cells 0.006+ 0.006
Connective tissue 0.002+ 0.001
Blood vessel 0.002+ 0.001
ENCICLOPÉDIA BIOSFERA, Centro Científico Conhecer - Goiânia, v.11 n.21; p. 2015 2335 The organization of the testis in H. megacephalus was similar to that described for most mammalian studied species (FRANÇA & RUSSELL, 1998). The proportion between the tubular and interstitial compartments is variable, and constitutes one of the main factors responsible for the differenciated efficiency in sperm production observed in several species (BALARINI, 2013).
Approximately 5% of the testicular parenchyma of H. megacephalus is constituted by interstitial tissue, which is a smaller percentage than observed in other rodents like Cuniculus paca 9% (CARRETA JÚNIOR, 2012); Dasyprocta aguti 15.25% (ASSIS NETO et al., 2003); Hydrochoerus hydrochaeris 50% (PAULA et al., 2002), and closer to the value reported for small rodent Necromys lasiurus 7% (SILVA et al., 2010).
The interstitial compartment is composed of Leydig cells, blood and lymphatic vessels, nerves, and a variable population of connective tissue (MORAIS et al., 2013b). The interstitial compartment in H. megacephalus showed Leydig cells grouped in clusters and also found blood vessels, as well as lymphatic space and connective tissue (Figure 1).
The interstitial compartment presents great variation in volume and proportion of its components in different species (FAWCETT et al., 1973, FRANÇA & RUSSELL, 1998, COSTA et al., 2010, MORAIS et al., 2014), but usually the Leydig cell is the most frequent component. In H. megacephalus, the Leydig cell was the most abundant component in the interstitial compartment, followed by blood vessels, connective tissue and lymphatic space (Table 1). This pattern was observed in other wild rodents Akodon cursor, Akodon montensis, Necromys lasiurus and Oligoryzomys nigripes (CORDEIRO JÚNIOR, 2009).
The patterns described by FAWCETT et al., (1973) indicated a great variation related the amount of the Leydig cells and the disposition of the lymphatic vessels. The rodents, in general, present the first pattern described by FAWCETT et al., (1973) where a small amount of Leydig cells and blood vessels are separated from the seminiferous epithelium by a developed lymphatic space. In relation to arrangements of the interstitial tissue components, the distribution in H. megacephalus is similar to that observed in the first pattern described by Fawcett to guinea-pigs and chinchillas, except for the amount of Leydig cells, making possible the inclusion of this species into one more variation of the type I classification like observed in capybara (PAULA et al., 2007).
The Leydig cells represent the endocrine portion of the testes. These cells are responsible for the synthesis of testosterone which is involved in the the spermatogenesis, development and maintenance of the secondary sex characteristics (GILBERT, 2010). Testosterone is still related to the reproductive behavior of the animals, such as libido, demarcation of territory and the status of a male in a colony. The morphometry of the Leydig cell is shown in Table 2.
ENCICLOPÉDIA BIOSFERA, Centro Científico Conhecer - Goiânia, v.11 n.21; p. 2015 2336 TABLE 2 - Morphometry of the Leydig cell in H. megacephalus (mean +
standard deviation; n=5). Parameters Mean + SD Nuclear diameter (µm) 5.94+0.45 Nuclear percentage (%) 37.06+11.64 Cytoplasmatic percentage (%) 62.94+11.64 Nuclear volume (µm3) 111.18+23.66 Cytoplasmatic volume (µm3) 239.61+187.55
Leydig cell volume (µm3) 350.80+206.56
Number of Leydig cells per testis (x106) 9.73+8.65 Number of Leydig cells per gram of testis (x106) 89.58+46.77
Leydigosomatic index (%) 0.015+0.01
The Leydig cell average volume in H. megacephalus was 350.80 µm3, close to the lowest values observed in several domestic, wild and laboratory species (350-2.580 µm3;)(FRANÇA & RUSSELL, 1998, COSTA & PAULA, 2006, AZEVEDO et al., 2010). Variations in the synthesis and secretion of testosterone depend more on the individual capacity of this cell than on their total volume in the testes (EWING et al., 1979). According to ZIRKIN et al. (1980), the productive capacity of the Leydig cell is closely related to the amount of smooth endoplasmic reticulum in this cell.
The Leydig cell number per testis is converted to the Leydig cell number per gram of testis to compare the different species independently of the size of the testes. The numbers of Leydig cells per testis and per gram of testis in H. megacephalus (9.73x106 and 89.58x106, respectively) were higher than observed in other small rodents (5.2 and 19.2x106 A. cursor; 3.1 and 14.6 x106 A. montensis; 4.0 and 17.1x106 N. lasiurus; 1.9 and 33.2x106 O. nigripes;)(CORDEIRO JÚNIOR, 2009). The number of Leydig cells per gram of testis in H. megacephalus has one of the largest values observed in domestic species, which varies from 20x106 to 87x106 cells (FRANÇA & RUSSELL, 1998).
Many factors can influence amount of Leydig cell per animal, such as the number of LH receptors per cell, amount of LH available, amount of testosterone secreted by the Leydig cell, the rate at which the testosterone leaves the testis, and the testosterone metabolism rate (YE et al., 2011). The amount of Leydig cells varies greatly between individuals and between mammalian species, and this variation was not found for other endocrine glands (FAWCETT et al., 1973).
The Leydigosomatic index (ILS) is the percentage of body mass allocated in Leydig cells, allowing comparison of individuals with different body sizes. The ILS in the present study was 0.015%. This value was two times greater than observed in Cuniculus paca (0.007%;)(CARRETA JÚNIOR, 2008) and three times greater than observed in N. lasiurus (0.005%;)(SILVA & MELO, 2010), but lower than the values reported for H. hydrochaeris (0.037%; COSTA et al., 2006), guinea pig, mice and rats
ENCICLOPÉDIA BIOSFERA, Centro Científico Conhecer - Goiânia, v.11 n.21; p. 2015 2337 (0.044, 0.030 and 0.055, respectively;)(RUSSELL, 1996).
According to EWING et al., (1979) the steroid production to regulate tissue physiology and animal behavior in each species is more related to the individual ability of Leydig cell to secrete such hormones than to the proportions, volume, and population differences of these cells in testis.
CONCLUSIONS
This study showed that several morphometric parameters of interstitial tissue evaluated in H. megacephalus are within the values described for most mammals.
H. megacephalus has one of the lowest values of Leydig cell volume than observed for domestic, wild and laboratory species.
The number of Leydig cells per gram of testis in H. megacephalus has one of the largest values observed in domestic species and in other small rodents.
ACKNOWLEDGMENTS
We thank the Biological Sciences students from the Federal University of Goiás – Campus Jataí for the collection of animals, the Structural Biology Laboratory at the Federal University of Viçosa for processing the biological material, and the Foundation for Research Support from the State of Goiás (FAPEG) for the financial support.
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