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Ultrastructure and morphometric features of epididymal epithelium in Desmodus rotundus
Micron 102 (2017) 35–43
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Ultrastructure and morphometric features of epididymal epithelium in Desmodus rotundus
MARK
Mariana Moraes de Castroa, Wagner Gonzaga Gonçalvesa, Stéphanie Asséf Millen Valente Teixeiraa, Maria do Carmo Queiroz Fialhob, Felipe Couto Santosa, ⁎ Jerusa Maria Oliveiraa, José Eduardo Serrãoa, Mariana Machado-Nevesa, a b
Departamento de Biologia Geral, Universidade Federal de Viçosa, 36570-900, Viçosa, MG, Brazil Departamento de Morfologia, Universidade Federal do Amazonas, 69077-000, Manaus, AM, Brazil
A R T I C L E I N F O
A B S T R A C T
Keywords: Common vampire bat Morphology Epithelial cells Luminal acidification Reproductive biology
The blood-feeding behavior of Desmodus rotundus made this bat a potential vector of rabies virus and a public health issue. Consequently, the better understanding of its reproductive biology becomes valuable for the development of methods to control its population. In this study, we described morphological aspects of epithelial cells in D. rotundus’ epididymis using light and transmission electron microscopy methods. The duct compartment was the main component of initial segment (83%), caput (90%), corpus (88%) and cauda (80%) regions. The epithelium lining the duct presented a progressive decrease in its height from initial segment to cauda regions. Moreover, the morphology of each cell type was the same along the entire duct. Similarly to rodents, columnar-shaped principal cells were the most abundant cell type throughout the epididymis, followed by basal and clear cells. Differently in rat and mice, the frequency of clear cells did not increase in the epididymis cauda, whereas the proportion of principal and basal cells was greater in this region. Furthermore, D. rotundus presented goblet-shaped clear cells with the nucleus located in the apical portion of the epididymal epithelium. This cellular portion also presented electron-lucid vesicles of different sizes that may correspond to vesicles enriched with proteins related to proton secretion. In addition to the findings regarding clear cells’ structural organization, basal cells presented scarce cytoplasm and no axiopodia. Taken these findings together, we suggest that the mechanism of luminal acidification may have other pathways in D. rotundus than those described in rodents.
1. Introduction Bats from subfamily Desmodontinae are intriguing by their hematophagous feeding habits. Particularly, Desmodus rotundus is one of the three desmodontine species that feeds exclusively on mammal blood, especially cattle (Kotait et al., 2007; Peracchi et al., 2007). The proximity between livestock and D. rotundus has stimulated its geographic range expansion and bat population growth. Consequently, there was a greater incidence of rabies in cattle, since vampire bats may be one of the main transmitters of rabies virus (Delpietro et al., 1992; Schneider et al., 2009; Lee et al., 2012). Little is known about the reproductive biology of D. rotundus. Apparently, there is no influence of either temperature or day length or food availability on reproductive events in D. rotundus when compared to other Neotropical bats (Wilson and Findley, 1970; Krutzsch, 1979; Altrigham, 1998; Neuweiler, 2000). This species may breed year-round due to the abundant feeding resources, and a promiscuous pattern of
⁎
Corresponding author. E-mail address: [email protected] (M. Machado-Neves).
http://dx.doi.org/10.1016/j.micron.2017.08.006 Received 23 April 2017; Received in revised form 25 July 2017; Accepted 22 August 2017 Available online 26 August 2017 0968-4328/ © 2017 Elsevier Ltd. All rights reserved.
mating system (Wilkinson, 1985; Taddei et al., 1991; Alencar et al., 1994; Crichton and Krutzsch, 2000; Freitas et al., 2006). Recently, studies have described the testicular morphometry and spermatogenesis in D. rotundus (Morais et al., 2017) and Neotropical bats (Beguelini et al., 2009; Duarte and Talamoni, 2010; Morais et al., 2012; Morais et al., 2013; Beguelini et al., 2014; Morais et al., 2014). In those animals, however, research focused on the epididymis histophysiology is still scarce (Beguelini et al., 2010; Oliveira et al., 2013; Castro et al., 2017). It is known that spermatozoa produced by testes are immature and unable to fertilize an egg. Thereby, they need to pass through the epididymis to acquire their motility and fertilizing capacity. This male reproductive organ is a long, single, and convoluted duct lined by an epithelium composed of several cell types. Epithelial cells, in turn, are responsible for creating an adequate luminal environment for sperm maturation and storage (Cornwall, 2009; Robaire and Hinton, 2015). Previously, Castro et al. (2017) identified three cell types along the
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used for histology, while the right was used for transmission electron microscopy analysis. In addition, testes were collected for evaluating the sexual maturity of each animal.
entire length of D. rotundus’ epididymis by immunocytochemistry. Aquaporin 9, V-ATPase subunit B1, and cytokeratin 5 were used as cellspecific markers of principal, clear, and basal cells, respectively (Castro et al., 2017). Those authors reported that pencil-shaped V-ATPase-rich cells (narrow cells) were not detected in the initial segment of the bat epididymis, unlike in rodents (Robaire and Hinton, 2015; Castro et al., 2017). While principal cells are responsible for fluid exchange and secretion of different luminal proteins (Cornwall, 2009), clear cells are involved in luminal acidification (Da Silva et al., 2007a). The activities of those cell types might be controlled by basal cells, which regulate electrolyte and water transport in principal cells via paracrine signaling (Leung et al., 2004), and control the V-ATPase-dependent proton secretion by clear cells (Shum et al., 2008). Despite principal, basal and clear cells have been identified in D. rotundus (Castro et al., 2017), the histology and ultrastructure of those cell types cells have not been assessed yet. Therefore, we aimed to describe the morphology, morphometry and ultrastructure of epithelial cells in D. rotundus’ epididymis. We associated light microscopy with transmission electron microscopy in order to create a baseline for future studies in epididymis morphology of vampire bats. This knowledge may generate information about its reproductive biology and, in turn, may support the development of methods to control D. rotundus population.
2.3. Histological procedure for light microscopy Testis (n = 5) and epididymides (n = 5) were immersed in Karnovsky fixative solution (Karnovsky, 1965) for 24 h. Then, they were dehydrated in a crescent ethanol series (70%, 80%, 90%, and 100%), and embedded in 2-hydroxyethyl methacrylate (Historesin®, Leica Microsystems, Nussloch, Germany). Sections with a thickness of 3 μm were stained with toluidine blue-sodium borate (1%), and qualitatively analyzed. Other histological sections were stained with periodic acid Schiff (PAS) reaction for identifying neutral glycoproteins, neutral carbohydrates and glycogen. We performed PAS reaction with amylase digestion and PAS reaction without periodic acid solution for negative controls. All sections were qualitatively analyzed using light microscope (Olympus CX40, Tokyo, Japan). Histological sections of testis were used for identifying sexual maturity of the captured bats. Thus, we evaluated seminiferous tubules regarding the presence of seminiferous epithelium in the Stage VIII of spermatogenic cycle, as well as spermatozoa in the lumen. For morphometric analysis, digital images of the four epididymal regions, initial segment, caput, corpus, and cauda (Fig. 1C), were obtained using a light microscope (Olympus BX53, Tokyo, Japan) equipped with a digital camera (Olympus DP73, Tokyo, Japan) and analyzed with Image-Pro Plus® 4.5 (Media Cybernetics, Silver Spring, MD, USA) software. The mean duct diameter of each epididymal region was obtained by randomly measuring 20 tubular cross sections per animal. These sections were also used to measure the luminal diameter, which is the distance taken from the lamina propria to the lumen. The epithelium height was estimated as the average of four diametrically opposed measurements (Souza et al., 2016). The volumetric proportion of four epididymal regions was obtained by counting 2660 points projected onto 10 images captured from histological slides per animal. Coincident points were registered in duct components (epithelium and lumen) and interduct components (blood vessels and connective tissue). The percentage of points in each component was calculated using the formula: volumetric proportion (%) = (number of points in the component/2660 total points) x 100 (Souza et al., 2016). The relative distribution of epithelial cell types in the four regions was estimated by cell count in different sections per region (Fig. 1C). Cells were counted in 10 sections per region per animal. The result of the crude cell count was corrected by applying Amann’s formula (Amann, 1962), according to Beu et al. (2009).
2. Material and methods 2.1. Animal capture Adult males (Fig. 1A) were captured using mist nets, positioned near a cave in Itamarati de Minas, Minas Gerais State, Brazil (21°23′S and 42°51′W; 585 m altitude), in October 2014. Permits for field collection were provided by Chico Mendes Institute for Biodiversity Conservation (ICMBio; number 40629-1). This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. All the experimental procedures were reviewed and approved by the Committee on the Ethics and Use of Animal Experiments of UFV (CEUA process number 55/2013). 2.2. Tissue collection Five animals were euthanized using intraperitoneal administration of sodium pentobarbital (40 mg/kg), followed by guillotining (Castro et al., 2017). The entire epididymides were removed, dissected (Fig. 1B), and weighed. The epididymal somatic index (ESI) was obtained by computing the ratio between epididymis weight (EW) and body weight (BW), where ESI = EW/BW x 100. The left epididymis was
Fig. 1. (A) Adult male of Desmodus rotundus. Note the scrotal area (white arrow) out of the abdominal cavity. Dorsal (B) and lateral (C) views of epididymis show four anatomical regions, initial segment (IS), caput, corpus and cauda. VD, vas deferens. Scale bars (A) 1 cm; (B–C) 1 mm. Photos Mariana MachadoNeves (A), and José Lino Neto (B-C).
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Fig. 2. Histological sections of testis (A,B) and epididymis (C-F) of Desmodus rotundus. (A,B) Seminiferous epithelium (SE) composed of Sertoli cells and germ cells especially round spermatid (RS) and elongated spermatid (ES), and lumen (L) with sperm. (B) The intertubular compartment (I) comprised the connective tissue and Leydig cells (white arrow). (C) Initial segment, (D) caput, (E) corpus, and (F) cauda epididymal regions presented a pseudostratified epithelium (E) delimiting lumen (L) with sperm. Note de presence of fibroblast (arrowhead) and blood vessels (asterisk) in the interduct compartment (I), and smooth muscle cells (arrow) surrounding the epididymal duct. Scale bars (A,B) 20 μm, (C-F) 50 μm.
Table 1 Volumetry and morphometry of the epididymal components in Desmodus rotundus. Parameters (n = 5)
Initial segment a
Caput
Corpus a
Cauda a
Epithelium (%) Lumen (%) Blood vessels (%) Connective tissue (%)
75.15 ± 1.64 8.00 ± 0.69a 1.09 ± 0.02a 15.76 ± 2.37ab
74.61 ± 2.56 15.52 ± 1.55b 0.88 ± 0.33a 8.99 ± 0.92a
63.64 ± 1.84 24.79 ± 1.68c 1.00 ± 0.28a 10.57 ± 1.39a
48.17 ± 4.91b 31.84 ± 2.56d 1.24 ± 0.33a 18.75 ± 2.30b
Epithelium height (μm) Tubular diameter (μm) Luminal diameter (μm)
35.39 ± 1.67a 115.45 ± 5.60a 44.66 ± 4.01a
32.66 ± 0.69ab 127.66 ± 3.04a 62.32 ± 2.88ab
28.18 ± 0.97bc 135.60 ± 3.09a 89.31 ± 9.14b
24.11 ± 1.64c 180.80 ± 13.33b 132.58 ± 16.44c
Mean ± SE. Upper case superscript letters (a,b,c,d) in the same row indicate differences among regions (P < 0.05) by Tukey’s HSD test.
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represented approximately 83, 90, 88, and 80% of the total tissue in the initial segment, caput, corpus and cauda regions, respectively. The cauda region presented the lowest proportion of epithelium (F3,16 = 14.607; P = 0), whereas the initial segment showed the lowest proportion of lumen (F3,16 = 37.294; P = 0; Table 1). Furthermore, the epithelium height was greater in the initial segment (F3,16 = 13.007; P = 0) than the corpus and cauda regions (Table 1). Tubular (F3,16 = 15.074; P = 0) and luminal (F3,16 = 5.23; P = 0.01) diameters were higher in the cauda region than the other regions (Table 1; Fig. 2C–F). We identified three epithelial cell types in the epididymis of D. rotundus. Principal cells were the most abundant, followed by basal and clear cells (Fig. 3). The relative distribution of principal cells (F3,16 = 5.23; P = 0.01) and basal cells (F3,16 = 4.246; P = 0.022) was greater in the cauda region compared to the proximal portions of epididymis (Fig. 3). Otherwise, the frequency of clear cells did not differ among regions (F3,16 = 0.819; P = 0.502). With regards to cell histology and ultrastructure, principal cells presented a columnar shape with round nucleus located in their basal portion (Figs. 4 , 5A). Further, the apical portion of this cell type showed a lightly stained area when used the toluidine-blue (Fig. 4). PAS-positive staining was observed in granules presented in the cytoplasm of principal cells, especially in their basal portion, and in the apical membrane in the stereocilia (Fig. 4A'–D'). In the PAS reaction with amylase digestion, we observed slightly PAS-positive staining in the stereocilia, but none in the cytoplasm of principal cells (Fig. 4E). No labeling was detected in the epithelium after PAS staining without periodic acid solution (Fig. 4E'). The supranuclear cytoplasm of principal cells showed several vesicles of different sizes with membranous content (Fig. 6A). Myelin bodies (Fig. 6B) and a highly developed Golgi apparatus (Fig. 6D) were also observed in the apical portion of this cell type, whereas small vesicles were located near to the apical surface (Fig. 6C). Stereocilia were observed in contact with the lumen (Fig. 6C). Further, electron-dense granules were identified in the basal portion of these cells (Fig. 6A). Principal cells’ nucleus showed a non-condensed chromatin (Fig. 6A, B), whereas the perinuclear cytoplasm was rich in electron-dense granules and rough endoplasmic reticulum (Fig. 6E). Mitochondria were mostly found near the cell boundaries (Fig. 6F). Clear cells were observed intercalated among principal cells along the epididymal duct (Fig. 4A-D). This cell type presented a small cytoplasm and nucleus located in the middle portion of the epithelium (Fig. 5B). Its irregular-shaped nucleus showed heterochromatin in its periphery (Fig. 7A). In addition, we found electron-lucid vesicles of different sizes and mitochondria in the apical portion of clear cells (Fig. 7B). Finally, basal cells were located in the basal region of the epithelium (Fig. 4A–D), adjacent to the basement membrane (Fig. 5A). They had scarce cytoplasm (Fig. 5A), and elongated nucleus with clumps of condensed chromatin (Fig. 7C). Further, this cell type did not show body projections going toward the lumen in the duct sections analyzed.
Fig. 3. Relative cell distribution (% of total) in epididymal regions of Desmodus rotundus. Bars represent mean ± SE. Upper case superscript letters (a,b) indicate differences between epididymal regions (P < 0.05) by Tukey’s test.
2.4. Histological procedure for transmission electron microscopy Fragments from epididymal regions were fixed in 2.5% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.2, at room temperature for 24 h. The samples were then post-fixed in 1% osmium tetroxide in the same buffer, dehydrated in a crescent series of ethanol, and embedded in LR white resin (LR White Resin, London Resin Company, England). The polymerization was performed in gelatin capsules (Electron Microscopy Sciences) at 60 °C for 24 h. Ultrathin sections were counterstained with acetate and lead citrate (Reynolds, 1963). The specimens were mounted on mesh grids, and were analyzed under a Carl Zeiss EM 109 transmission electron microscope (Jena, Thuringia, Germany) at the Center of Microscopy and Microanalysis (NMM-UFV). 2.5. Statistical analysis Results obtained from the quantitative assessments were analyzed by analysis of variance (ANOVA) followed by the post hoc Tukey’s HSD test (Zar, 2010). Differences were considered significant when p < 0.05. Results were expressed as mean ± standard error of the mean (SE). 3. Results The epididymis anatomy showed that it is a highly convoluted duct covered by a dense connective tissue (Fig. 1B). Four anatomical regions were identified, initial segment, caput, corpus, and cauda, with the last one connected to a vas deferens (Fig. 1C). Epididymis weight and epididymal somatic index in D. rotundus were, respectively, 0.057 ± 0.019 g and 0.173 ± 0.055%. Histological analyses showed the D. rotundus testis with a parenchyma composed of tubular and intertubular compartments (Fig. 2A, B). The former presented seminiferous tubules as the main component, and comprised a lumen with sperm and a functional epithelium composed of Sertoli cells and germ cells, especially round and elongated spermatids (Fig. 2A). Further, the intertubular compartment was rich in Leydig cells (Fig. 2B), blood vessels, lymphatic space, and connective tissue. In epididymal sections, each region previously identified presented two compartments, the duct and interduct (Fig. 2C-F). The latter was composed of connective tissue rich in blood vessels, fibroblasts, and smooth muscle cells surrounding each duct section (Fig. 2C–F). While the percentage of blood vessels did not differ among regions (F3,16 = 0.307; P = 0.82), the proportion of connective tissue was greater in the cauda (F3,16 = 8.249; P = 0.002) when compared to the other regions (Table 1). Basically, the duct compartment was mainly composed of the epididymal duct, which was lined by a pseudostratified epithelium delimiting the lumen with sperm (Fig. 2C-F). This compartment
4. Discussion This study describes the histology, morphometry, and ultrastructure of epididymal epithelial cells in adult male D. rotundus. The sexual maturity of each animal used herein was confirmed by the presence of spermatozoa in the epididymal lumen, and the identification of the Stage VIII of spermatogenic cycle in testis. This stage was characterized in D. rotundus by the presence of Sertoli cells, type A spermatogonia, type B spermatogonia in preleptotene, primary spermatocyte in pachytene, round spermatid, and elongated spermatid (Morais et al., 2017). It is well known that spermiation starts when elongated spermatids begin to appear within the lumen in testis and then sperm start to populate the epididymis (Robaire and Hinton, 2015). Herein, three epithelial cell types were identified in D. rotundus’ 38
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Fig. 4. Photomicrographs of epithelium lining the epididymal duct from initial segment (A,A'), caput (B,B'), corpus (C,C’) and cauda (D–E') regions in Desmodus rotundus. (A–D) Toluidine-blue staining shows principal (Pc), basal (Bc) and clear (Cc) cells. Arrowhead, principal cells presented a lightly stained area. (A'–D') Periodic-acid Schiff staining was positive in granules (arrow) located in the cytoplasm of principal cells, and in the glycocalyx present in their stereocilia (asterisk). (E,E') Negative controls of periodic acid Schiff (PAS) staining using amylase digestion (E) and PAS reaction without periodic-acid solution (E'). L, lumen. Scale bars (A–E) 10 μm, (A'–E') 20 μm.
However, findings can be improved when you associate light microscopy with transmission electron microscopy, which is able to image the macromolecular complexes within cells at almost atomic resolution. Both types of microscopy are widely used to visualize epithelial cells
epididymis under light and transmission electron microscopy. Principal, basal and clear cells were observed along the entire duct. In fact, light microscope is an essential tool to study cells even with limited in resolution by the wavelength of visible light (Alberts et al., 2002). 39
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Fig. 5. (A,B) Transmission electron microscopic images from caput region in epididymis of Desmodus rotundus show principal cells (Pc) in abundance and nuclei (n). (A) Bc, basal cell; BM, basement membrane. (B) Cc; clear cells with nuclei (n) located at the apical region of the epithelium. Scale bars 5 μm.
multivesicular bodies or lysosomes in clear cell cytoplasm as reported in gerbil’s epididymis (Domeniconi et al., 2007). In fact, clear cells are well known by their role in the maintenance of the luminal acidification, working together with principal cells (Park et al., 2017) and basal cells (Shum et al., 2009). Especially in the cauda region, acidic pH helps to keep sperm in a dormant state during their storage for long periods (Carr et al., 1985; Pastor-Soler et al., 2005; Da Silva et al., 2007b; Shum et al., 2009; Breton and Brown, 2013). It is for that reason that clear cells usually are present in high frequency in the cauda region of rodents (Shum et al., 2009). Surprisingly, the relative distribution of clear cells in D. rotundus’ epididymis did not increase in this distal region as observed in rats (Shum et al., 2009). In light of this finding, we may suggest that the mechanism of luminal acidification may have other pathways in D. rotundus than those described in rodents (Shum et al., 2008). Another factor that may support this suggestion was the absence of axiopodia in basal cells of D. rotundus’ epididymis. Some studies report that basal cells have high plasticity along the epididymis due to the emission of body projections (called axiopodia) toward the lumen in rats and mice (Shum et al., 2008; Kim et al., 2015) and wild rodents (personal communication). In the present study, however, this cytoplasmic extension was not detected using transmission electron microscopy. Castro et al. (2017) also did not identify axiopodia in the epididymis of vampire bats by immunofluorescence. Roy et al. (2016) have previously detected axiopodia in epididymis from a novel transgenic mouse expressing tdTomato by a stack of ultrathin serial sections under electron microscopy after pre-embedding labeling. The lack of axiopodia in D. rotundus’ epididymis is an intriguing finding, since axiopodia may play a role in the crosstalk between basal and clear cells that culminates in the increasing of luminal acidification by angiotensin II regulation (Shum et al., 2008). Other basal cells’ characteristics observed herein have been previously described in rats (Oke et al., 1989). The abundance of principal cells in the epididymal epithelium of this species was also observed in other bats (Beguelini et al., 2010). Principal cells play an important role in the transepithelial water fluxes, urea, and uncharged solutes due to the presence of aquaporin 9 in their apical membrane (Tsukaguchi et al., 1998; Pastor-Soler et al., 2001; Castro et al., 2017). They also synthesize and release proteins into the lumen and perform an active endocytosis of proteins along the
(Alberts et al., 2002). On the other hand, Castro et al. (2017) used the fluorescence microscopy for identifying the same epithelial cells with cell-specific markers in D. rotundus’ epididymis. This method is most often used to detect specific proteins or other molecules in cells and tissues by coupling fluorescence dyes to antibody molecules (Alberts et al., 2002). In addition, Castro et al. (2017) used primary antibodies for labeling claudin-1. This claudin is highly expressed in cell membranes (Gregory et al., 2001) and permits to define cell limits and consequently their shape. This labeling was essential for characterizing clear cells’ shape in D. rotundus and comparing this data with previous reports using laboratory rodents, which are used as model in epididymis studies (Castro et al., 2017). Based on that, we were clearly able to identify clear cells under light and transmission electron microscopy. In this context, goblet-shaped clear cells were observed with nucleus located in the apical portion of the epithelium in D. rotundus’ epididymis. Similarly to D. rotundus, sigmodontine rodents also showed different clear cells’ shape, which is a columnar cell-shape with the nucleus positioned in the basal portion of the epithelium (Menezes et al., 2017). In rats and mice, in contrast, clear cells shape has been described as a cuboidal-shaped cells in the caput and corpus regions, and large-shaped in the cauda region (Hermo et al., 2005; Piétrement et al., 2006; Shum et al., 2009; Robaire and Hinton, 2015). Thus, we assume that epididymis of vampire bats presents different cells shape in comparison to epididymis from laboratory animals. The latter, indeed, have been raised and selected in lineages that contribute for reducing genetic variation, as well as cellular morphology (Carvalho, 2016), unlike to wild animals and specifically vampire bats. In regard to the fine structure of clear cells, the electron-lucid vesicles observed in the apical portion of those cells may correspond to the vesicles positively labeled for V-ATPase subunit B1 in D. rotundus’ epididymis (Castro et al., 2017). V-ATPase is a ubiquitous protein that acidifies intracellular organelles, and it is also enriched in the plasma membrane of some specialized proton transporting cells (Shum et al., 2008). Specifically, the subunit B1 is responsible for proton secretion across the apical membrane of clear cells, contributing to the luminal acidification (Shum et al., 2009). Although clear cells are also related to endocytic function (Da Silva et al., 2007a, b; Domeniconi et al., 2007; Shum et al., 2008; Breton and Brown, 2013), we were unable to find 40
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Fig. 6. Ultrastructure of principal cells (Pc) in Desmodus rotundus’ epididymis. (A) Columnarshaped cells with electron-dense granules (white arrow) and vesicles (v). (B) black arrow, myelin bodies. (C) Several small vesicles (black arrowhead) near to the apical border, and stereocilia (S) in contact with lumen. (D) GC, Golgi complex. (E) Perinuclear region with endoplasmic reticulum (ER) and electron–dense granules (white arrowhead). (F) M, mitochondria. L, lumen; n, nuclei. Scale bars (A) 5 μm, (B) 2 μm, (C,D,F) 200 nm, (E) 500 nm.
rodents (Oke et al., 1989; Domeniconi et al., 2007). While we observed mitochondria in the apical portion of clear cells in vampire bat, Oke et al. (1989) observed this organelle in different portions of principal cells in African giant rats. Differences regarding mitochondria location in the cytoplasm, as well as aspects and organization of secretory and endocytic apparatus, may reflect the functional moment of the cell. Overall, morphometric features observed in each epididymal region in D. rotundus were previously described in the epididymis of other Neotropical bats (Beguelini et al., 2010) and rodents (Serre and Robaire, 1998). The progressive reduction in epithelium height parameter from the proximal to distal regions, as well as the increasing in the tubular and luminal diameters have been reported in gerbil, mice, and Neotropical bats (Domeniconi et al., 2007; Turner et al., 2007; Beguelini et al., 2010). These parameters might be influenced by cellular activity related to absorption, secretion, maintenance of the luminal fluid and sperm concentration during the sperm maturation and storage (Cornwall, 2009; Robaire and Hinton, 2015). Moreover, the higher frequency of principal and basal cells in the cauda region may be related to the epithelium area lining its duct sections, which are
epididymis, which may contribute for a specialized luminal milieu (Turner, 1991; Hermo et al., 1994; Cornwall, 2009; Hermo and Robaire, 2002; Menezes et al., 2017). Furthermore, the presence of PAS-positive granules in principal cells of D. rotundus suggests that this cell type release glycoprotein substances into the epididymal lumen, whereas PAS-positive labeling in stereocilia indicates the presence of glycoproteins associated to plasma cell membrane. The using of PAS reaction with amylase digestion showed that there is also glycogen deposit in the cytoplasm of principal cells as observed in viscacha (Aguilera-Merlo et al., 2009). In the present study, the fine structure of principal cells found in D. rotundus indicates their secretory and absorption functions, with the presence of vesicles with membranous content, electron-dense granules, myelin bodies, well-developed Golgi apparatus, rough endoplasmic reticulum, and stereocilia. All these organelles and cellular structures were previously described in the epididymis of other rodents (Flickinger, 1985; Domeniconi et al., 2007; Lorenzana et al., 2007). Moreover, the location of Golgi apparatus and rough endoplasmic reticulum in the cytoplasm of principal cells is similar in D. rotundus and 41
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Fig. 7. Transmission electron microscopic images of Desmodus rotundus’ epididymis. (A) Nuclei (n) of clear cell (Cc) in the apical region of the epithelium. This cell type was intercalated among principal cells (Pc). (B) Vesicles of different sizes (v) and mitochondria (M) in the apical portion of the clear cell. (C) Basal cell (Bc) with nuclei (n) located in the basal region of the epithelium. L, lumen; arrowhead, sperm; arrow, stereocilia. Scale bars (A) 2 μm, (B) 500 nm, (C)1 μm.
References
characterized by the high diameter. On the contrary, clear cells did not increase in number in order to contribute to the total of epithelial cell population in the cauda region. In conclusion, the microscopy of clear cells and basal cells in D. rotundus’ epididymis showed significant differences when compared to rodent epididymis, except for principal cells. Differently in rats and mice, D. rotundus presented goblet-shaped clear cells with electronlucid vesicles probably related to secretion of proton across membranes. Moreover, basal cells did not present axiopodia. Intriguingly, the frequency of clear cells did not increase in the cauda region as described in rodents. Taken these findings together, we suggest that clear cells may create a low luminal pH using alternative mechanisms of regulation in D. rotundus’ epididymis. The acidic luminal microenvironment is essential to keep sperm quiescent during their maturation and storage in this organ. Furthermore, the knowledge of epididymis histophysiology sets the foundations for comprehensive understanding of reproductive biology in this vampire bat. Finally, the using of multiple microscopy methods is valid for accomplishing a reliable evaluation of cell structural organization, which is a prerequisite for learning how cells function.
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Author contributions Mariana M. Castro and Mariana Machado-Neves conceived and designed the study. Mariana M. Castro, Wagner G. Gonçalves, Stéphanie A. M. V. Teixeira, Felipe C. Santos, and Jerusa M. Oliveira carried out the data acquisition. Mariana M. Castro, Maria C.Q. Fialho, José E. Serrão and Mariana Machado-Neves performed the analyses. Mariana M. Castro and Mariana Machado-Neves drafted the manuscript. Maria C.Q. Fialho and José E. Serrão revised the manuscript.
Acknowledgments The authors thank Luciano C. H. P. Puga, Ana Cláudia F. Souza and Susana P. Ribeiro for their support during the collection of the bats. This work was supported by Brazilian Research Agencies (CAPES, FAPEMIG and CNPq). We thank the Center of Microscopy and Microanalysis (NMM-UFV) for technical assistance. Mariana M. Castro was granted a scholarship from CAPES (visiting graduate student process number − 99999.011031/2013-01). All authors read and approved the final manuscript and declare there is no conflict of interest. 42
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lituratus (Chiroptera: phyllostomidae) in a Brazilian Atlantic forest area. Mammal. Biol. 75, 320–325. Flickinger, C.J., 1985. Autoradiographic analysis of the secretory pathway for glycoprotein in principal cells of the mouse epididymis exposed to H3-fucose. Biol. Reprod. 32, 377–389. Freitas, M.B., Welker, A.F., Pinheiro, E.C., 2006. Seasonal variation and food deprivation in commom vampire bats (Chiroptera: Phyllostomidae). Braz. J. Biol. 66, 1051–1055. Gregory, M., Dufresne, J., Hermo, L., Cyr, D., 2001. Claudin-1 is not restricted to tight junctions in the rat epididymis. Endocrinology 142, 854–863. Hermo, L., Robaire, B., 2002. Epididymal Cell Types and Their Functions. In The Epididymis: from Molecular to Clinical Practice. A Comprehensive Survey of the Efferent Ducts, the Epididymis and the Vas Deferens. pp. 81–102. Hermo, L., Oko, R., Morales, C., 1994. Secretion and endocytosis in the male reproductive tract: a role in sperm maturation. Int. Rev. Cytol. 154, 105–189. Hermo, L., Chong, D.L., Moffatt, P., Sly, W.S., Waheed, A., Smith, C.E., 2005. Region-and cell-specific differences in the distribution of carbonic anhydrases II, III, XII, and XIV in the adult rat epididymis. J. Histochem. Cytochem. 53, 699–713. Karnovsky, M.J., 1965. A formaldehyde-glutaraldehyde fixative of high osmolality for use in electron microscopy. J. Cell. Biol. 15, 127–137. Kim, B., Roy, J., Shum, W.W., Da Silva, N., Breton, S., 2015. Role of testicular luminal factors on basal cell elongation and proliferation in the mouse epididymis. Biol. Reprod. 92, 1–11. Kotait, I., Carrieri, M.L., Carnieli Júnior, P., Castilho, J.G., Oliveira, R.N., Macedo, C.I., Ferreira, K.C.S., Achkar, S.M., 2007. Reservatórios silvestres do vírus da raiva: um desafio para a saúde pública. Bol. Epid. Paul. 40. Krutzsch, P.H., 1979. Male reproductive patterns in nonhibernating bats. J. Reprod. Fert. 56, 333–344. Lee, D.N., Papes, M., van Den Bussche, R.A., 2012. Present and potential future distribution of common vampire bats in the Americas and the associated risk to cattle. PLoS One 7, 1–9. Leung, G.P., Cheung, K.H., Leung, C.T., Tsang, M.W., Wong, P.Y., 2004. Regulation of epididymal principal cell functions by basal cells: role of transient receptor potential (Trp) proteins and cyclooxygenase-1 (COX-1). Mol. Cell. Endocrinol. 216, 5–13. Lorenzana, M.G., López-Wilchis, R., Gómez, C.S., Aranzabal, M.C.U., 2007. A light and scanning eléctron microscopic study of the epididymis active state of the endemic mexican rodent Peromyscus winkelmanni (Carleton) (Rodentia: Muridae). Anat. Histol. Embryol. 36, 230–240. Menezes, T.P., Castro, M.M., Vale, J.A., Moura, A.A.A., Lessa, G., Machado-Neves, M., 2017. Proteomes and morphological features of Calomys tener and Necromys lasiurus (Cricetidae, Sigmodontinae) epididymides. J. Mammal. 98, 579–590. Morais, D.B., Paula, T.A.R., Oliveira, L.C., Freitas, K.M., da Matta, S.L.P., 2012. Cycle of the seminiferous of the bat Molossus molossus, characterized by tubular morphology and acrosomal development. Asian Pac. J. Reprod. 1, 303–307. Morais, D.B., Cupertino, M.C., Goulart, L.S., Freitas, K.M., Freitas, M.B.D., Paula, T.A.R., Matta, S.L.P., 2013. Histomorphometric evaluation of the Molossus molossus (Chiroptera, Molossidae) testis: the tubular compartment and indices of sperm production. Anim. Reprod. Sci. 140, 268–278. Morais, D.B., Barros, M.S., Paula, T.A., Freitas, M.B., Gomes, M.L., Matta, S.L.P., 2014. Evaluation of the cell population of the seminiferous epithelium and spermatic indexes of the bat Sturnira lilium (Chiroptera: Phyllostomidae). PLoS One. 9, 1–9. Morais, D.B., Puga, L.C.H.P., Paula, T.A.R., Freitas, M.B.D., Matta, S.L.P., 2017. The spermatogenic process of the common vampire bat Desmodus rotundus under a histomorphometric view. PLoS One 12, 1–18. Neuweiler, G., 2000. The Biology of Bats. Oxford University Press, New York. Oke, B.O., Aire, T.A., Adeyemo, O., Heath, E., 1989. The ultrastructure of the epididymis of the African giant rat (Cricetomys gambianus, Waterhouse). J. Anat. 165, 75–85.
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Micron journal homepage: www.elsevier.com/locate/micron
Ultrastructure and morphometric features of epididymal epithelium in Desmodus rotundus
MARK
Mariana Moraes de Castroa, Wagner Gonzaga Gonçalvesa, Stéphanie Asséf Millen Valente Teixeiraa, Maria do Carmo Queiroz Fialhob, Felipe Couto Santosa, ⁎ Jerusa Maria Oliveiraa, José Eduardo Serrãoa, Mariana Machado-Nevesa, a b
Departamento de Biologia Geral, Universidade Federal de Viçosa, 36570-900, Viçosa, MG, Brazil Departamento de Morfologia, Universidade Federal do Amazonas, 69077-000, Manaus, AM, Brazil
A R T I C L E I N F O
A B S T R A C T
Keywords: Common vampire bat Morphology Epithelial cells Luminal acidification Reproductive biology
The blood-feeding behavior of Desmodus rotundus made this bat a potential vector of rabies virus and a public health issue. Consequently, the better understanding of its reproductive biology becomes valuable for the development of methods to control its population. In this study, we described morphological aspects of epithelial cells in D. rotundus’ epididymis using light and transmission electron microscopy methods. The duct compartment was the main component of initial segment (83%), caput (90%), corpus (88%) and cauda (80%) regions. The epithelium lining the duct presented a progressive decrease in its height from initial segment to cauda regions. Moreover, the morphology of each cell type was the same along the entire duct. Similarly to rodents, columnar-shaped principal cells were the most abundant cell type throughout the epididymis, followed by basal and clear cells. Differently in rat and mice, the frequency of clear cells did not increase in the epididymis cauda, whereas the proportion of principal and basal cells was greater in this region. Furthermore, D. rotundus presented goblet-shaped clear cells with the nucleus located in the apical portion of the epididymal epithelium. This cellular portion also presented electron-lucid vesicles of different sizes that may correspond to vesicles enriched with proteins related to proton secretion. In addition to the findings regarding clear cells’ structural organization, basal cells presented scarce cytoplasm and no axiopodia. Taken these findings together, we suggest that the mechanism of luminal acidification may have other pathways in D. rotundus than those described in rodents.
1. Introduction Bats from subfamily Desmodontinae are intriguing by their hematophagous feeding habits. Particularly, Desmodus rotundus is one of the three desmodontine species that feeds exclusively on mammal blood, especially cattle (Kotait et al., 2007; Peracchi et al., 2007). The proximity between livestock and D. rotundus has stimulated its geographic range expansion and bat population growth. Consequently, there was a greater incidence of rabies in cattle, since vampire bats may be one of the main transmitters of rabies virus (Delpietro et al., 1992; Schneider et al., 2009; Lee et al., 2012). Little is known about the reproductive biology of D. rotundus. Apparently, there is no influence of either temperature or day length or food availability on reproductive events in D. rotundus when compared to other Neotropical bats (Wilson and Findley, 1970; Krutzsch, 1979; Altrigham, 1998; Neuweiler, 2000). This species may breed year-round due to the abundant feeding resources, and a promiscuous pattern of
⁎
Corresponding author. E-mail address: [email protected] (M. Machado-Neves).
http://dx.doi.org/10.1016/j.micron.2017.08.006 Received 23 April 2017; Received in revised form 25 July 2017; Accepted 22 August 2017 Available online 26 August 2017 0968-4328/ © 2017 Elsevier Ltd. All rights reserved.
mating system (Wilkinson, 1985; Taddei et al., 1991; Alencar et al., 1994; Crichton and Krutzsch, 2000; Freitas et al., 2006). Recently, studies have described the testicular morphometry and spermatogenesis in D. rotundus (Morais et al., 2017) and Neotropical bats (Beguelini et al., 2009; Duarte and Talamoni, 2010; Morais et al., 2012; Morais et al., 2013; Beguelini et al., 2014; Morais et al., 2014). In those animals, however, research focused on the epididymis histophysiology is still scarce (Beguelini et al., 2010; Oliveira et al., 2013; Castro et al., 2017). It is known that spermatozoa produced by testes are immature and unable to fertilize an egg. Thereby, they need to pass through the epididymis to acquire their motility and fertilizing capacity. This male reproductive organ is a long, single, and convoluted duct lined by an epithelium composed of several cell types. Epithelial cells, in turn, are responsible for creating an adequate luminal environment for sperm maturation and storage (Cornwall, 2009; Robaire and Hinton, 2015). Previously, Castro et al. (2017) identified three cell types along the
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used for histology, while the right was used for transmission electron microscopy analysis. In addition, testes were collected for evaluating the sexual maturity of each animal.
entire length of D. rotundus’ epididymis by immunocytochemistry. Aquaporin 9, V-ATPase subunit B1, and cytokeratin 5 were used as cellspecific markers of principal, clear, and basal cells, respectively (Castro et al., 2017). Those authors reported that pencil-shaped V-ATPase-rich cells (narrow cells) were not detected in the initial segment of the bat epididymis, unlike in rodents (Robaire and Hinton, 2015; Castro et al., 2017). While principal cells are responsible for fluid exchange and secretion of different luminal proteins (Cornwall, 2009), clear cells are involved in luminal acidification (Da Silva et al., 2007a). The activities of those cell types might be controlled by basal cells, which regulate electrolyte and water transport in principal cells via paracrine signaling (Leung et al., 2004), and control the V-ATPase-dependent proton secretion by clear cells (Shum et al., 2008). Despite principal, basal and clear cells have been identified in D. rotundus (Castro et al., 2017), the histology and ultrastructure of those cell types cells have not been assessed yet. Therefore, we aimed to describe the morphology, morphometry and ultrastructure of epithelial cells in D. rotundus’ epididymis. We associated light microscopy with transmission electron microscopy in order to create a baseline for future studies in epididymis morphology of vampire bats. This knowledge may generate information about its reproductive biology and, in turn, may support the development of methods to control D. rotundus population.
2.3. Histological procedure for light microscopy Testis (n = 5) and epididymides (n = 5) were immersed in Karnovsky fixative solution (Karnovsky, 1965) for 24 h. Then, they were dehydrated in a crescent ethanol series (70%, 80%, 90%, and 100%), and embedded in 2-hydroxyethyl methacrylate (Historesin®, Leica Microsystems, Nussloch, Germany). Sections with a thickness of 3 μm were stained with toluidine blue-sodium borate (1%), and qualitatively analyzed. Other histological sections were stained with periodic acid Schiff (PAS) reaction for identifying neutral glycoproteins, neutral carbohydrates and glycogen. We performed PAS reaction with amylase digestion and PAS reaction without periodic acid solution for negative controls. All sections were qualitatively analyzed using light microscope (Olympus CX40, Tokyo, Japan). Histological sections of testis were used for identifying sexual maturity of the captured bats. Thus, we evaluated seminiferous tubules regarding the presence of seminiferous epithelium in the Stage VIII of spermatogenic cycle, as well as spermatozoa in the lumen. For morphometric analysis, digital images of the four epididymal regions, initial segment, caput, corpus, and cauda (Fig. 1C), were obtained using a light microscope (Olympus BX53, Tokyo, Japan) equipped with a digital camera (Olympus DP73, Tokyo, Japan) and analyzed with Image-Pro Plus® 4.5 (Media Cybernetics, Silver Spring, MD, USA) software. The mean duct diameter of each epididymal region was obtained by randomly measuring 20 tubular cross sections per animal. These sections were also used to measure the luminal diameter, which is the distance taken from the lamina propria to the lumen. The epithelium height was estimated as the average of four diametrically opposed measurements (Souza et al., 2016). The volumetric proportion of four epididymal regions was obtained by counting 2660 points projected onto 10 images captured from histological slides per animal. Coincident points were registered in duct components (epithelium and lumen) and interduct components (blood vessels and connective tissue). The percentage of points in each component was calculated using the formula: volumetric proportion (%) = (number of points in the component/2660 total points) x 100 (Souza et al., 2016). The relative distribution of epithelial cell types in the four regions was estimated by cell count in different sections per region (Fig. 1C). Cells were counted in 10 sections per region per animal. The result of the crude cell count was corrected by applying Amann’s formula (Amann, 1962), according to Beu et al. (2009).
2. Material and methods 2.1. Animal capture Adult males (Fig. 1A) were captured using mist nets, positioned near a cave in Itamarati de Minas, Minas Gerais State, Brazil (21°23′S and 42°51′W; 585 m altitude), in October 2014. Permits for field collection were provided by Chico Mendes Institute for Biodiversity Conservation (ICMBio; number 40629-1). This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. All the experimental procedures were reviewed and approved by the Committee on the Ethics and Use of Animal Experiments of UFV (CEUA process number 55/2013). 2.2. Tissue collection Five animals were euthanized using intraperitoneal administration of sodium pentobarbital (40 mg/kg), followed by guillotining (Castro et al., 2017). The entire epididymides were removed, dissected (Fig. 1B), and weighed. The epididymal somatic index (ESI) was obtained by computing the ratio between epididymis weight (EW) and body weight (BW), where ESI = EW/BW x 100. The left epididymis was
Fig. 1. (A) Adult male of Desmodus rotundus. Note the scrotal area (white arrow) out of the abdominal cavity. Dorsal (B) and lateral (C) views of epididymis show four anatomical regions, initial segment (IS), caput, corpus and cauda. VD, vas deferens. Scale bars (A) 1 cm; (B–C) 1 mm. Photos Mariana MachadoNeves (A), and José Lino Neto (B-C).
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Fig. 2. Histological sections of testis (A,B) and epididymis (C-F) of Desmodus rotundus. (A,B) Seminiferous epithelium (SE) composed of Sertoli cells and germ cells especially round spermatid (RS) and elongated spermatid (ES), and lumen (L) with sperm. (B) The intertubular compartment (I) comprised the connective tissue and Leydig cells (white arrow). (C) Initial segment, (D) caput, (E) corpus, and (F) cauda epididymal regions presented a pseudostratified epithelium (E) delimiting lumen (L) with sperm. Note de presence of fibroblast (arrowhead) and blood vessels (asterisk) in the interduct compartment (I), and smooth muscle cells (arrow) surrounding the epididymal duct. Scale bars (A,B) 20 μm, (C-F) 50 μm.
Table 1 Volumetry and morphometry of the epididymal components in Desmodus rotundus. Parameters (n = 5)
Initial segment a
Caput
Corpus a
Cauda a
Epithelium (%) Lumen (%) Blood vessels (%) Connective tissue (%)
75.15 ± 1.64 8.00 ± 0.69a 1.09 ± 0.02a 15.76 ± 2.37ab
74.61 ± 2.56 15.52 ± 1.55b 0.88 ± 0.33a 8.99 ± 0.92a
63.64 ± 1.84 24.79 ± 1.68c 1.00 ± 0.28a 10.57 ± 1.39a
48.17 ± 4.91b 31.84 ± 2.56d 1.24 ± 0.33a 18.75 ± 2.30b
Epithelium height (μm) Tubular diameter (μm) Luminal diameter (μm)
35.39 ± 1.67a 115.45 ± 5.60a 44.66 ± 4.01a
32.66 ± 0.69ab 127.66 ± 3.04a 62.32 ± 2.88ab
28.18 ± 0.97bc 135.60 ± 3.09a 89.31 ± 9.14b
24.11 ± 1.64c 180.80 ± 13.33b 132.58 ± 16.44c
Mean ± SE. Upper case superscript letters (a,b,c,d) in the same row indicate differences among regions (P < 0.05) by Tukey’s HSD test.
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represented approximately 83, 90, 88, and 80% of the total tissue in the initial segment, caput, corpus and cauda regions, respectively. The cauda region presented the lowest proportion of epithelium (F3,16 = 14.607; P = 0), whereas the initial segment showed the lowest proportion of lumen (F3,16 = 37.294; P = 0; Table 1). Furthermore, the epithelium height was greater in the initial segment (F3,16 = 13.007; P = 0) than the corpus and cauda regions (Table 1). Tubular (F3,16 = 15.074; P = 0) and luminal (F3,16 = 5.23; P = 0.01) diameters were higher in the cauda region than the other regions (Table 1; Fig. 2C–F). We identified three epithelial cell types in the epididymis of D. rotundus. Principal cells were the most abundant, followed by basal and clear cells (Fig. 3). The relative distribution of principal cells (F3,16 = 5.23; P = 0.01) and basal cells (F3,16 = 4.246; P = 0.022) was greater in the cauda region compared to the proximal portions of epididymis (Fig. 3). Otherwise, the frequency of clear cells did not differ among regions (F3,16 = 0.819; P = 0.502). With regards to cell histology and ultrastructure, principal cells presented a columnar shape with round nucleus located in their basal portion (Figs. 4 , 5A). Further, the apical portion of this cell type showed a lightly stained area when used the toluidine-blue (Fig. 4). PAS-positive staining was observed in granules presented in the cytoplasm of principal cells, especially in their basal portion, and in the apical membrane in the stereocilia (Fig. 4A'–D'). In the PAS reaction with amylase digestion, we observed slightly PAS-positive staining in the stereocilia, but none in the cytoplasm of principal cells (Fig. 4E). No labeling was detected in the epithelium after PAS staining without periodic acid solution (Fig. 4E'). The supranuclear cytoplasm of principal cells showed several vesicles of different sizes with membranous content (Fig. 6A). Myelin bodies (Fig. 6B) and a highly developed Golgi apparatus (Fig. 6D) were also observed in the apical portion of this cell type, whereas small vesicles were located near to the apical surface (Fig. 6C). Stereocilia were observed in contact with the lumen (Fig. 6C). Further, electron-dense granules were identified in the basal portion of these cells (Fig. 6A). Principal cells’ nucleus showed a non-condensed chromatin (Fig. 6A, B), whereas the perinuclear cytoplasm was rich in electron-dense granules and rough endoplasmic reticulum (Fig. 6E). Mitochondria were mostly found near the cell boundaries (Fig. 6F). Clear cells were observed intercalated among principal cells along the epididymal duct (Fig. 4A-D). This cell type presented a small cytoplasm and nucleus located in the middle portion of the epithelium (Fig. 5B). Its irregular-shaped nucleus showed heterochromatin in its periphery (Fig. 7A). In addition, we found electron-lucid vesicles of different sizes and mitochondria in the apical portion of clear cells (Fig. 7B). Finally, basal cells were located in the basal region of the epithelium (Fig. 4A–D), adjacent to the basement membrane (Fig. 5A). They had scarce cytoplasm (Fig. 5A), and elongated nucleus with clumps of condensed chromatin (Fig. 7C). Further, this cell type did not show body projections going toward the lumen in the duct sections analyzed.
Fig. 3. Relative cell distribution (% of total) in epididymal regions of Desmodus rotundus. Bars represent mean ± SE. Upper case superscript letters (a,b) indicate differences between epididymal regions (P < 0.05) by Tukey’s test.
2.4. Histological procedure for transmission electron microscopy Fragments from epididymal regions were fixed in 2.5% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.2, at room temperature for 24 h. The samples were then post-fixed in 1% osmium tetroxide in the same buffer, dehydrated in a crescent series of ethanol, and embedded in LR white resin (LR White Resin, London Resin Company, England). The polymerization was performed in gelatin capsules (Electron Microscopy Sciences) at 60 °C for 24 h. Ultrathin sections were counterstained with acetate and lead citrate (Reynolds, 1963). The specimens were mounted on mesh grids, and were analyzed under a Carl Zeiss EM 109 transmission electron microscope (Jena, Thuringia, Germany) at the Center of Microscopy and Microanalysis (NMM-UFV). 2.5. Statistical analysis Results obtained from the quantitative assessments were analyzed by analysis of variance (ANOVA) followed by the post hoc Tukey’s HSD test (Zar, 2010). Differences were considered significant when p < 0.05. Results were expressed as mean ± standard error of the mean (SE). 3. Results The epididymis anatomy showed that it is a highly convoluted duct covered by a dense connective tissue (Fig. 1B). Four anatomical regions were identified, initial segment, caput, corpus, and cauda, with the last one connected to a vas deferens (Fig. 1C). Epididymis weight and epididymal somatic index in D. rotundus were, respectively, 0.057 ± 0.019 g and 0.173 ± 0.055%. Histological analyses showed the D. rotundus testis with a parenchyma composed of tubular and intertubular compartments (Fig. 2A, B). The former presented seminiferous tubules as the main component, and comprised a lumen with sperm and a functional epithelium composed of Sertoli cells and germ cells, especially round and elongated spermatids (Fig. 2A). Further, the intertubular compartment was rich in Leydig cells (Fig. 2B), blood vessels, lymphatic space, and connective tissue. In epididymal sections, each region previously identified presented two compartments, the duct and interduct (Fig. 2C-F). The latter was composed of connective tissue rich in blood vessels, fibroblasts, and smooth muscle cells surrounding each duct section (Fig. 2C–F). While the percentage of blood vessels did not differ among regions (F3,16 = 0.307; P = 0.82), the proportion of connective tissue was greater in the cauda (F3,16 = 8.249; P = 0.002) when compared to the other regions (Table 1). Basically, the duct compartment was mainly composed of the epididymal duct, which was lined by a pseudostratified epithelium delimiting the lumen with sperm (Fig. 2C-F). This compartment
4. Discussion This study describes the histology, morphometry, and ultrastructure of epididymal epithelial cells in adult male D. rotundus. The sexual maturity of each animal used herein was confirmed by the presence of spermatozoa in the epididymal lumen, and the identification of the Stage VIII of spermatogenic cycle in testis. This stage was characterized in D. rotundus by the presence of Sertoli cells, type A spermatogonia, type B spermatogonia in preleptotene, primary spermatocyte in pachytene, round spermatid, and elongated spermatid (Morais et al., 2017). It is well known that spermiation starts when elongated spermatids begin to appear within the lumen in testis and then sperm start to populate the epididymis (Robaire and Hinton, 2015). Herein, three epithelial cell types were identified in D. rotundus’ 38
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Fig. 4. Photomicrographs of epithelium lining the epididymal duct from initial segment (A,A'), caput (B,B'), corpus (C,C’) and cauda (D–E') regions in Desmodus rotundus. (A–D) Toluidine-blue staining shows principal (Pc), basal (Bc) and clear (Cc) cells. Arrowhead, principal cells presented a lightly stained area. (A'–D') Periodic-acid Schiff staining was positive in granules (arrow) located in the cytoplasm of principal cells, and in the glycocalyx present in their stereocilia (asterisk). (E,E') Negative controls of periodic acid Schiff (PAS) staining using amylase digestion (E) and PAS reaction without periodic-acid solution (E'). L, lumen. Scale bars (A–E) 10 μm, (A'–E') 20 μm.
However, findings can be improved when you associate light microscopy with transmission electron microscopy, which is able to image the macromolecular complexes within cells at almost atomic resolution. Both types of microscopy are widely used to visualize epithelial cells
epididymis under light and transmission electron microscopy. Principal, basal and clear cells were observed along the entire duct. In fact, light microscope is an essential tool to study cells even with limited in resolution by the wavelength of visible light (Alberts et al., 2002). 39
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Fig. 5. (A,B) Transmission electron microscopic images from caput region in epididymis of Desmodus rotundus show principal cells (Pc) in abundance and nuclei (n). (A) Bc, basal cell; BM, basement membrane. (B) Cc; clear cells with nuclei (n) located at the apical region of the epithelium. Scale bars 5 μm.
multivesicular bodies or lysosomes in clear cell cytoplasm as reported in gerbil’s epididymis (Domeniconi et al., 2007). In fact, clear cells are well known by their role in the maintenance of the luminal acidification, working together with principal cells (Park et al., 2017) and basal cells (Shum et al., 2009). Especially in the cauda region, acidic pH helps to keep sperm in a dormant state during their storage for long periods (Carr et al., 1985; Pastor-Soler et al., 2005; Da Silva et al., 2007b; Shum et al., 2009; Breton and Brown, 2013). It is for that reason that clear cells usually are present in high frequency in the cauda region of rodents (Shum et al., 2009). Surprisingly, the relative distribution of clear cells in D. rotundus’ epididymis did not increase in this distal region as observed in rats (Shum et al., 2009). In light of this finding, we may suggest that the mechanism of luminal acidification may have other pathways in D. rotundus than those described in rodents (Shum et al., 2008). Another factor that may support this suggestion was the absence of axiopodia in basal cells of D. rotundus’ epididymis. Some studies report that basal cells have high plasticity along the epididymis due to the emission of body projections (called axiopodia) toward the lumen in rats and mice (Shum et al., 2008; Kim et al., 2015) and wild rodents (personal communication). In the present study, however, this cytoplasmic extension was not detected using transmission electron microscopy. Castro et al. (2017) also did not identify axiopodia in the epididymis of vampire bats by immunofluorescence. Roy et al. (2016) have previously detected axiopodia in epididymis from a novel transgenic mouse expressing tdTomato by a stack of ultrathin serial sections under electron microscopy after pre-embedding labeling. The lack of axiopodia in D. rotundus’ epididymis is an intriguing finding, since axiopodia may play a role in the crosstalk between basal and clear cells that culminates in the increasing of luminal acidification by angiotensin II regulation (Shum et al., 2008). Other basal cells’ characteristics observed herein have been previously described in rats (Oke et al., 1989). The abundance of principal cells in the epididymal epithelium of this species was also observed in other bats (Beguelini et al., 2010). Principal cells play an important role in the transepithelial water fluxes, urea, and uncharged solutes due to the presence of aquaporin 9 in their apical membrane (Tsukaguchi et al., 1998; Pastor-Soler et al., 2001; Castro et al., 2017). They also synthesize and release proteins into the lumen and perform an active endocytosis of proteins along the
(Alberts et al., 2002). On the other hand, Castro et al. (2017) used the fluorescence microscopy for identifying the same epithelial cells with cell-specific markers in D. rotundus’ epididymis. This method is most often used to detect specific proteins or other molecules in cells and tissues by coupling fluorescence dyes to antibody molecules (Alberts et al., 2002). In addition, Castro et al. (2017) used primary antibodies for labeling claudin-1. This claudin is highly expressed in cell membranes (Gregory et al., 2001) and permits to define cell limits and consequently their shape. This labeling was essential for characterizing clear cells’ shape in D. rotundus and comparing this data with previous reports using laboratory rodents, which are used as model in epididymis studies (Castro et al., 2017). Based on that, we were clearly able to identify clear cells under light and transmission electron microscopy. In this context, goblet-shaped clear cells were observed with nucleus located in the apical portion of the epithelium in D. rotundus’ epididymis. Similarly to D. rotundus, sigmodontine rodents also showed different clear cells’ shape, which is a columnar cell-shape with the nucleus positioned in the basal portion of the epithelium (Menezes et al., 2017). In rats and mice, in contrast, clear cells shape has been described as a cuboidal-shaped cells in the caput and corpus regions, and large-shaped in the cauda region (Hermo et al., 2005; Piétrement et al., 2006; Shum et al., 2009; Robaire and Hinton, 2015). Thus, we assume that epididymis of vampire bats presents different cells shape in comparison to epididymis from laboratory animals. The latter, indeed, have been raised and selected in lineages that contribute for reducing genetic variation, as well as cellular morphology (Carvalho, 2016), unlike to wild animals and specifically vampire bats. In regard to the fine structure of clear cells, the electron-lucid vesicles observed in the apical portion of those cells may correspond to the vesicles positively labeled for V-ATPase subunit B1 in D. rotundus’ epididymis (Castro et al., 2017). V-ATPase is a ubiquitous protein that acidifies intracellular organelles, and it is also enriched in the plasma membrane of some specialized proton transporting cells (Shum et al., 2008). Specifically, the subunit B1 is responsible for proton secretion across the apical membrane of clear cells, contributing to the luminal acidification (Shum et al., 2009). Although clear cells are also related to endocytic function (Da Silva et al., 2007a, b; Domeniconi et al., 2007; Shum et al., 2008; Breton and Brown, 2013), we were unable to find 40
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Fig. 6. Ultrastructure of principal cells (Pc) in Desmodus rotundus’ epididymis. (A) Columnarshaped cells with electron-dense granules (white arrow) and vesicles (v). (B) black arrow, myelin bodies. (C) Several small vesicles (black arrowhead) near to the apical border, and stereocilia (S) in contact with lumen. (D) GC, Golgi complex. (E) Perinuclear region with endoplasmic reticulum (ER) and electron–dense granules (white arrowhead). (F) M, mitochondria. L, lumen; n, nuclei. Scale bars (A) 5 μm, (B) 2 μm, (C,D,F) 200 nm, (E) 500 nm.
rodents (Oke et al., 1989; Domeniconi et al., 2007). While we observed mitochondria in the apical portion of clear cells in vampire bat, Oke et al. (1989) observed this organelle in different portions of principal cells in African giant rats. Differences regarding mitochondria location in the cytoplasm, as well as aspects and organization of secretory and endocytic apparatus, may reflect the functional moment of the cell. Overall, morphometric features observed in each epididymal region in D. rotundus were previously described in the epididymis of other Neotropical bats (Beguelini et al., 2010) and rodents (Serre and Robaire, 1998). The progressive reduction in epithelium height parameter from the proximal to distal regions, as well as the increasing in the tubular and luminal diameters have been reported in gerbil, mice, and Neotropical bats (Domeniconi et al., 2007; Turner et al., 2007; Beguelini et al., 2010). These parameters might be influenced by cellular activity related to absorption, secretion, maintenance of the luminal fluid and sperm concentration during the sperm maturation and storage (Cornwall, 2009; Robaire and Hinton, 2015). Moreover, the higher frequency of principal and basal cells in the cauda region may be related to the epithelium area lining its duct sections, which are
epididymis, which may contribute for a specialized luminal milieu (Turner, 1991; Hermo et al., 1994; Cornwall, 2009; Hermo and Robaire, 2002; Menezes et al., 2017). Furthermore, the presence of PAS-positive granules in principal cells of D. rotundus suggests that this cell type release glycoprotein substances into the epididymal lumen, whereas PAS-positive labeling in stereocilia indicates the presence of glycoproteins associated to plasma cell membrane. The using of PAS reaction with amylase digestion showed that there is also glycogen deposit in the cytoplasm of principal cells as observed in viscacha (Aguilera-Merlo et al., 2009). In the present study, the fine structure of principal cells found in D. rotundus indicates their secretory and absorption functions, with the presence of vesicles with membranous content, electron-dense granules, myelin bodies, well-developed Golgi apparatus, rough endoplasmic reticulum, and stereocilia. All these organelles and cellular structures were previously described in the epididymis of other rodents (Flickinger, 1985; Domeniconi et al., 2007; Lorenzana et al., 2007). Moreover, the location of Golgi apparatus and rough endoplasmic reticulum in the cytoplasm of principal cells is similar in D. rotundus and 41
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Fig. 7. Transmission electron microscopic images of Desmodus rotundus’ epididymis. (A) Nuclei (n) of clear cell (Cc) in the apical region of the epithelium. This cell type was intercalated among principal cells (Pc). (B) Vesicles of different sizes (v) and mitochondria (M) in the apical portion of the clear cell. (C) Basal cell (Bc) with nuclei (n) located in the basal region of the epithelium. L, lumen; arrowhead, sperm; arrow, stereocilia. Scale bars (A) 2 μm, (B) 500 nm, (C)1 μm.
References
characterized by the high diameter. On the contrary, clear cells did not increase in number in order to contribute to the total of epithelial cell population in the cauda region. In conclusion, the microscopy of clear cells and basal cells in D. rotundus’ epididymis showed significant differences when compared to rodent epididymis, except for principal cells. Differently in rats and mice, D. rotundus presented goblet-shaped clear cells with electronlucid vesicles probably related to secretion of proton across membranes. Moreover, basal cells did not present axiopodia. Intriguingly, the frequency of clear cells did not increase in the cauda region as described in rodents. Taken these findings together, we suggest that clear cells may create a low luminal pH using alternative mechanisms of regulation in D. rotundus’ epididymis. The acidic luminal microenvironment is essential to keep sperm quiescent during their maturation and storage in this organ. Furthermore, the knowledge of epididymis histophysiology sets the foundations for comprehensive understanding of reproductive biology in this vampire bat. Finally, the using of multiple microscopy methods is valid for accomplishing a reliable evaluation of cell structural organization, which is a prerequisite for learning how cells function.
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Author contributions Mariana M. Castro and Mariana Machado-Neves conceived and designed the study. Mariana M. Castro, Wagner G. Gonçalves, Stéphanie A. M. V. Teixeira, Felipe C. Santos, and Jerusa M. Oliveira carried out the data acquisition. Mariana M. Castro, Maria C.Q. Fialho, José E. Serrão and Mariana Machado-Neves performed the analyses. Mariana M. Castro and Mariana Machado-Neves drafted the manuscript. Maria C.Q. Fialho and José E. Serrão revised the manuscript.
Acknowledgments The authors thank Luciano C. H. P. Puga, Ana Cláudia F. Souza and Susana P. Ribeiro for their support during the collection of the bats. This work was supported by Brazilian Research Agencies (CAPES, FAPEMIG and CNPq). We thank the Center of Microscopy and Microanalysis (NMM-UFV) for technical assistance. Mariana M. Castro was granted a scholarship from CAPES (visiting graduate student process number − 99999.011031/2013-01). All authors read and approved the final manuscript and declare there is no conflict of interest. 42
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