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Characterization of multilamellar bodies and telocytes within the testicular interstitium of naked mole rat Heterocephalus glabe
Theriogenology 138 (2019) 111e120
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Characterization of multilamellar bodies and telocytes within the testicular interstitium of naked mole rat Heterocephalus glabe Abdul Haseeb a, b, 1, Imran Tarique a, 1, Adeela Iqbal a, Noor samad Gandahi a, Waseem Ali vistro a, Xuebing Bai a, Yu Liang a, Yufei Huang a, Hong Chen a, Qiusheng Chen a, Ping Yang a, * a MOE Joint International Research Laboratory of Animal Health and Food Safety, College of Veterinary Medicine, Nanjing Agricultural University, Jiangsu Province, 210095, China b Faculty of Veterinary and Animal Sciences, University of Poonch Rawalakot, Azad Kashmir, Pakistan
a r t i c l e i n f o
a b s t r a c t
Article history: Received 24 January 2019 Received in revised form 9 July 2019 Accepted 10 July 2019 Available online 10 July 2019
Multilamellar bodies (MLBs) are produced and secreted by many cell types. In this study, we report the existence and ultrastructure of MLBs that are produced by Leydig cells and identification of telocytes in the testicular interstitium of naked mole rat. This study was performed on both breeder and non-breeder male naked mole rats using light microscopy, transmission electron microscopy, and morphometric approaches. In the testicular interstitium, the most prominent cells were Leydig cells, which contained numerous lipid droplets (LDs) in the cytoplasm. We found that MLBs were associated with the LDs of Leydig cells and were secreted into the extracellular or interstitial environment via exocytosis. After their release from Leydig cells, MLBs localized to the space between Leydig cells near blood vessels and attached to telocytes. We also identified telocytes in the testicular interstitium, and their cellular extensions were distributed throughout the interstitium. MLBs were aligned along the cellular extensions of telocytes, and membrane-to-membrane contact was observed between the cellular extensions of telocytes and MLBs, suggesting that telocytes may play a role in the transport of MLBs within the interstitial space. No ultrastructural differences were found in Leydig cells, telocytes, or MLBs between breeder and non-breeder testes. However, morphometric analysis revealed a significant difference in the number of MLBs between the breeder and non-breeder animals. Furthermore, both selective autophagy of LDs and non-selective autophagy were observed in Leydig cells. Typical features of macrolipophagy were also observed, as a few LDs were entirely enclosed by a limiting membrane. Remarkably, autophagy may be a key factor in the biogenesis of MLBs and steroid hormone production. The appearance of MLBs in the testicular interstitium of naked mole rats could thus be related to lipid storage and trafficking. © 2019 Published by Elsevier Inc.
Keywords: Interstitial tissue Leydig cells Telocytes Lipid droplets Autophagy Morphometry
1. Introduction The naked mole rat (Heterocephalus glaber) is the longest living rodent species, with a maximum lifespan of 30 years [1]. Naked mole rats are eusocial, subterranean mammals and they are native to Africa [2]. Both captive and wild colonies of naked mole rats have unique behavioral and reproductive characteristics. Reproductive activities are restricted to one female and 1e3 males in each colony [3,4]. The remaining members of the colony do not breed, but they
* Corresponding author. E-mail address: [email protected] (P. Yang). 1 Authors contributed equally. https://doi.org/10.1016/j.theriogenology.2019.07.010 0093-691X/© 2019 Published by Elsevier Inc.
are not sterile [5]. Those who participate in breeding known as breeder, while rest of them known as non-breeders animals. Spermatogenesis and steroidogenesis occurs both in breeder and non-breeder male naked mole rats [6,7]. Naked mole rats maintain lifelong normal physiological activity without displaying any signs of dysfunction or disease related to age [8]. They also show resistance to spontaneous cancer [1]. The parenchyma of the testis is composed of seminiferous tubules and interstitial tissues [9]. Interstitial tissues consist of highly vascularized, loose connective tissues, including Leydig cells, blood vessels, macrophages, leukocytes, fibroblasts, peritubular cells, and a novel cell type called telocytes [10]. Telocytes are characterized as having a small cellular body and extremely long extracellular
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extensions. They are found in the intestinal space of organs. Telocytes were discovered a decade ago, however their function is still under debate [11]. Leydig cells are the most structurally and functionally prominent cells in the interstitial tissue of the testis [12]. These cells are predominantly enriched within the smooth endoplasmic reticulum, mitochondria, and LDs [13]. Multilamellar bodies (MLBs) are subcellular structures (organelles) that vary in size from 100 to 2400 nm. Morphologically, they consist of concentric membrane layers and often exhibit an electron-dense core. Through electron microscopy studies, many different names have been given to multilamellar membrane structures such as lamellar bodies, lamellar granules, multilamellar bodies, multilamellar lipids, myeloid bodies, membrane-coating granules, Odland bodies, and lysosomal related organelles [14e17]. To avoid confusion during this report, we will use the term MLBs, as all of these terms are still used in the literature depending on the tissue. Under both normal and pathological conditions, MLBs are found in several cell types and are functionally involved in lipid storage and secretion [15]. Excellent work has been done on the MLBs that are secreted by alveolar type 2 cells in the lungs and by the epidermis [15,18,19]. MLBs have also been reported in other tissues, such as the tongue papillae, oral epithelium, digestive tract, and peritoneum [15,20], as well as in lower organisms such as insects and amoebae [21,22]. In the testicular interstitium, structures similar to MLBs (autophagosomes) have been previously reported in Leydig cells as a component of autophagy processes [23,24]. However, no study has described the MLBs as an individual entity in the extracellular spaces of the testicular interstitium. Here, we characterize and report for the first time the presence of MLBs in the testicular interstitium that are produced and secreted by Leydig cells of naked mole rats. Moreover, we identified telocytes in the extended area of the testicular interstitium and describe their interaction with MLBs. Naked mole rats are eusocial animals that show an extreme form of cooperative breeding. Few animals contribute to breeding (known as breeder animals), while other members are either sterile or do not reproduce (known as nonbreeders) [3]. Thus, in the present study, observations were performed on both types of naked mole rats (breeder and nonbreeder) to fully characterize MLBs, Leydig cells, and telocytes. The naked mole rat is known as a super model organism for aging research [1]. Therefore, this research may be useful for future work on studies related to the reproductive physiology of the naked mole rat.
exsanguination. The testes were collected through ventral abdominal dissection. The handling of animals and study procedures were in accordance with guidelines of the Animal Research Institute Committee of Nanjing Agriculture University. All the protocols were approved by the Science and Technology Agency of Jiangsu Province (Approval ID: SYXK (SU) 2010-0005). All efforts were made to minimize animal suffering. 2.2. Light microscopy Tissue samples were fixed in 10% neutral buffered formalin overnight. The sample was then dehydrated in an ascending series of ethanol, cleared in xylene, embedded into paraffin wax and sectioned at 5 mm. Tissue sections were stained with hematoxylin and eosin (Harrys hematoxylin for 2 min and 1% eosin for 30 s) and analyzed using an Olympus microscope (BX53, Tokyo, Japan). To measure the interstitial area and the number of Leydig cells, HE photos were imported into ImageJ software. To measure the interstitial tissue area, the total area of the image was first measured, and then the area of seminiferous tubules was determined, delineating the circumference of all the tubules in an image. After that, the area of interstitial tissue was obtained by subtracting the seminiferous tubule area from the total area of the image [26]. The number of Leydig cells was counted using the ImageJ Cell Counter plugin. Morphometric data (interstitial area and number of Leydig cell) was obtained from 40 HE stained photographs (20 per group, cross-section, 1600 1200) taken randomly. 2.3. Ultrastructural analysis Testes samples were cut into 1 mm3 blocks and immersed in 2.5% glutaraldehyde in 0.01 M phosphate-buffered saline (PBS) (4 C, pH 7.4) for 24 h. Samples were rinsed in PBS and post-fixed in an 1% osmium tetroxide solution for 1 h at 37 C. Post fixed samples were dehydrated in ascending concentrations of ethyl alcohol, infiltrated with a propylene oxide-Araldite mixture and embedded in Araldite. The blocks were then sectioned using an ultramicrotome (Reichert Jung, Vienna, Austria). The ultrathin sections of tissue (50 nm) were mounted on copper grids and contrasted with uranyl acetate and lead citrate for 20 min each. Finally, sections were examined and photographed with a transmission electron microscope (TEM) (Hitachi H-7650, Tokyo, Japan). 2.4. Immunohistochemistry (IHC)
2. Materials and methods 2.1. Experimental animals and ethical approval The naked mole rats used in these studies were maintained in the animal house of the College of Veterinary Medicine, Nanjing Agricultural University, China. Animals were housed in artificial Perspex burrow systems, a large plexiglass box (2 1 m2) heated to 24 to 28 C with a constant high relative humidity of 40%e70%. The burrow system consisted of a series of interconnecting clear acrylic plastic tubes with a nest, food and toilet chambers. Food was given ad libitum, included vegetables (sweet potatoes, carrots beet root and cucumber) and fruits (apples, bananas). Weekly, a supplement containing minerals and vitamins was also provided. Routine behavioral observations were carried out to differentiate between the breeder and non-breeder animals. In this research, 2 male breeders and 2 male non-breeders of the same age (15 months) were used. For sample collection, animals were anesthetized according to a standard protocol for naked mole rats with an intramuscular injection of a 6 mg kg1 ketamine and 2.5 mg kg1 xylazine [25], after which the animals were sacrificed by
The tissue was fixed in 10% formalin and embedded in paraffin for sectioning 6 mm. Sections were deparaffinized and then rehydrated. 3% hydrogen peroxide was used to block the further activity of endogenous peroxidase and followed by antigen retrieval. After blocked with 5% bovine serum albumin, the samples were incubated with rabbit anti-CD34 (1:100 dilution; catalog no. BA3414; Boster, Wuhan, China) in a moisture chamber at 4 Cfor 24 h, while PBS (pH 7.2) served as the negative control. After washing, the sections were incubated with biotinylated anti-rabbit/anti-mouse IgG (Boster Bio-Technology, Wuhan, China) for one hour at room temperature. The sections were then incubated with avidinbiotinylated peroxidase complex. Peroxidase activity was revealed using DAB (Boster Bio-Technology, Wuhan, China). The nuclei were stained with hematoxylin. 2.5. Fluorescence microscopy Immunofluorescence (IF) was performed according to a previously described method [27]. Briefly, after completing all of the basic steps (deparaffinization, serum blocking and antigen
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retrieval), the testes tissue slides were incubated with primary antibody (anti-rabbit LC3, 1:75 dilution, 12741, Cell Signaling Technology, Danvers, Massachusetts, USA) at 4 C overnight. Subsequently, the slides were washed in PBS, and the corresponding secondary antibodies (Goat anti-rabbit IgG, 1:75 dilution, ab6721, Abcam, Cambridge, Massachusetts, USA) were added to the sections for 2 h at room temperature. Next, the slides were washed again with PBS and then stained with DAPI to highlight the cell nucleus. For negative control PBS (pH 7.2) was used. Images were obtained using a microscope (BX53, Olympus, Tokyo, Japan) attached to a camera (DP73, Olympus, Tokyo, Japan). 2.6. Morphometric analysis of MLBs To measure the size and number of MLBs, TEM photographs of the same magnifications were imported into ImageJ software. Lines were drawn around the MLBs using the image tool (area of interest (AOI)) to measure the size (mean diameter) of MLBs. The number of MLBs was counted manually. Morphometric data (size and number of MLBs) were obtained from 40 TEM photographs (20 per group, cross-sectioned, 4800 3730) taken randomly. 2.7. Statistical analysis The statistical significance of the results was analyzed by t-test. Differences were considered significant at P < 0.05. Analyses were performed using SAS version 14.0 (SAS Institute). All data are reported as the mean ± S.E.M. 3. Results 3.1. Light microscopy Under a light microscope, a large content of interstitial tissue was observed in the testes of both breeder and non-breeder naked rats (Fig 1ab). Morphometric analysis showed that testes from both breeders and non-breeders contained a substantial content of interstitial area and number of Leydig cells (Fig 1cd, Table 1). Noticeably, the content of interstitial area was significantly higher (p ¼ 0.0005) in non-breeders than in breeders (Fig. 1c, Table 1). The number of Leydig cells within the interstitial tissue was also significantly higher (p ¼ 0.0031) in non-breeder testes than in breeder testes (Fig. 1d, Table 1). 3.2. Localization, biogenesis and association of MLBs The testicular interstitium from both breeder and non-breeder naked rats was observed under a transmission electron microscope. A detailed analysis of the Leydig cells, MLBs and telocytes was performed. In testes from both breeder and non-breeder animals, the Leydig cells contained an oval-shaped nucleus with a prominent nucleolus and cytoplasm with all basic organelles, including endoplasmic reticulum, mitochondria (Figs 2ab and 3 ab), and more prominent LDs (Figs. 2b, 3b and 4 ab). In the cytoplasm of the Leydig cells, MLBs consisted of concentric layers, which were accompanied by LDs (Fig 2ab, 3 ab 4 ab). The association of MLBs with LDs appeared in various forms: MLBs occupying most of the of LD (Fig. 2a), MLBs forming one third of the LDs (Fig 3ab), or MLBs forming half of the LD (Fig. 4a). MLBs are secreted from the cytoplasm of Leydig cells into the extracellular space through exocytosis (Fig. 4a). In addition to being found in the cytoplasm of Leydig cells, MLBs were also found in the space between Leydig cells (Figs. 5a, 6 and 7a) and adjoining the blood vessels (Fig. 5b). Telocytes were identified in the testicular interstitium of both breeder and nonbreeder naked mole rats and were morphologically characterized
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by a cell body and very long cellular extensions (Figs. 5b, 6 and 7abc). The cellular extensions of telocytes consist of podomers (thin segments) and podoms (bead-like portions) (Figs. 5b, 6 and 7bc). Numerous cellular extensions of telocytes were distributed in the interstitial tissue around Leydig cells and blood vessels (Figs. 5b, 6 and 7abc). MLBs were found to be aligned with the cellular extensions of telocytes, and we also observed close membrane-to-membrane contacts between the cellular extensions of telocytes and MLBs (Figs. 6 and 7abc). MLBs were also observed freely in the interstitial space close to cellular extensions of telocytes (Fig. 8ab). Overall, no ultrastructural differences were found in Leydig cells, MLBs or telocytes between breeder and non-breeder naked mole rats. 3.3. Morphology of MLBs We observed morphologically diverse shapes of MLBs. Some MLBs had concentric membrane layers and occasionally had a dense core (Figs. 2e4, 6), and some MLBs showed a mix of concentric layers and amorphous materials (Figs. 5, 7 and 8), while other MLBs that were fused together and surrounded by one membrane or without any outer membrane were also observed (Figs. 2 and 5). 3.4. Morphometric analysis of MLBs Overall, MLBs were observed in both breeder and non-breeder naked mole rats. Morphometry analysis was performed to determine if there were differences in the number or the size of MLBs between breeder and non-breeder animals. The number of intraand extracellular MLBs were greater in the breeder testes compared to non-breeders (Fig. 9ab). The number of intracellular MLBs was significantly higher (p ¼ 0.0005) in breeders than non-breeders (Fig. 9a, Table 1). Similarly, the number of extracellular MLBs was also higher (p ¼ 0.1080) in breeders than in non-breeders (Fig. 9b, Table 1). The size of intra- and extracellular MLBs was not significantly different between breeder and non-breeder animals (Fig 9cd, Table 1). 3.5. Expression of CD34 in the testicular interstitium of naked mole rat testes Under TEM, telocytes were observed in the testicular interstitium of naked mole rat (Figs. 5b, 6, 7abc, and 8 ab). For further confirmation, we performed IHC using CD34 most reliable biomarker for telocytes identification. CD34 was strongly expressed around seminiferous tubules, Leydig cells, blood vessels and with long cellular extensions of telocytes in interstitium spaces (Fig. 10 abc). The distributions of the CD34-positive telocytes in testicular interstitium were agreed with TEM observations. However, unexpected/non-specific staining was also observed within seminiferous tubules of naked mole rat testes. 3.6. Autophagy in Leydig cells Immunofluorescence of LC3 was performed with in order to confirm autophagy activities. Immunofluorescence staining of LC3 showed a strong positive reaction within the interstitial tissues of the testes of naked mole rats (Fig. 11). Under a TEM, MLB associated autophagy activity was also observed in the cytoplasm of Leydig cells. Both selective and nonselective autophagy was found. Some autophagic vacuoles had multiple internal structures or degraded materials, indicating nonselective autophagy (Fig. 2ab, 4ab, 12). More visibly, we observed LDs that were entirely enclosed by a limiting membrane, indicating selective autophagy of LDs (Figs. 1a,
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Fig. 1. Testicular interstitium of naked mole rats. H&E staining of breeder (a) and non-breeder (b) naked mole rat testes. (c) Qunatification of intersititial area (mm2) (n ¼ 20 per group). (d) Qunatification of Leydig cell numbers (n ¼ 20 per group). St ¼ seminiferous tubules, Ic ¼ Interstitial cells, Bv ¼ blood vessel. scale bar ¼ 50 mm.
4a and 12).
4. Discussion 4.1. Ultrastructure and possible physiological function of MLBs in the testicular interstitium of naked mole rat In the present study, a population of MLBs were observed inside Leydig cells and were found to be localized to other compartments in the testicular interstitium. Morphologically, almost all MLBs were similar, exhibiting collections of concentric membrane layers [15]. However, ultrastructural morphological differences existed between the MLBs, varying between different tissues as well as within the same tissue. In lung tissues, MLBs appeared as concentric layers both with a dense core or without a dense core, while fusion of MLBs and concentric layers enclosed by an outer limiting layer have also been reported [28]. Three different kinds of MLBs have been reported in amoebae on the basis of morphology. These types are MLBs with concentric layers, with concentric layers with amorphous material and with only amorphous material [22]. Many
Table 1 Morphometric analysis. Data are represented as mean ± S.E.M. (n ¼ 20 per group). Parameters
Breeder
Non-breeder
P value
Interstitial area Leydig cell numbers Number of Intracellular MLBs Number of Extracellular MLBs Size of intracellular MLBs Size of extracellular MLBs
361.3 ± 482.6 9034 ± 8909 10.17 ± 7.542 14.39 ± 2.745 205.6 ± 4.272 670.9 ± 4.709
843.9 ± 47.52 17943 ± 1400 5.5 ± 1.792 9.87 ± 3.452 209.8 ± 27.71 666.2 ± 54.25
0.0005 0.0031 0.0005 0.0108 0.8849 0.935
cells produce and secrete MLBs [15]. Nevertheless, there have not been any studies about MLBs in Leydig cells, particularly extracellular MLBs. In this study, all previously reported ultrastructural characteristics of MLBs were found, including fusion, mix of concentric layers and amorphous material and the presence or absence of a dense core. Therefore, we confirmed these observed structures as MLBs. Although different cell types produce MLBs, the origin of the MLBs differs between cells. Subcellular organelles such as the Golgi apparatus, endoplasmic reticulum, lysosomes, and lipid droplets are all believed to be an origin of MLBs in various tissues [29e32]. In the present study, we observed close association of MLBs with lipid droplets. We also showed another type of association of MLBs with LDs, indicating the step by step biogenesis of MLBs from LDs or may represent partial digestion of LDs by MLBs (Fig. 11). Our findings indicate that MLBs in Leydig cells are derived from LDs. MLBs are metabolically active and are required for the directed transport and conversion of lipids, as well as for interconversion of different lipid species [30]. MLBs contain various cargo, including lipids, hydrolytic enzymes, and several other proteins [33]. MLB biogenesis occurs in the cytoplasm, and they are then secreted into the extracellular or interstitial environment via exocytosis [17,20,32,34]. Following the release by exocytosis, we observed MLBs in different locations of the interstitial environment, including in the space between Leydig cells, around blood vessels and along the cellular extension of telocytes. Given that MLBs are mainly composed of lipids, their main function is lipid storage and direct transport of lipids [15,30]. Therefore, MLBs may be functional in lipid trafficking in the testes of naked mole rats. One important question that arises is why MLBs were not reported previously in testicular interstitium. One possible reason is that Leydig cells are enriched in LDs and cover nearly 60% of the
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Fig. 2. TEM photographs of multilamellar bodies associated with lipid droplets in Leydig cells of a naked mole rat (breeder). (a) Numerous multilamellar bodies (arrowhead) closely associated with lipid droplets (LD) and with membranes around single lipid droplet (arrow). Cytoplasm contains many mitochondria and smooth endoplasmic reticulum (sER). (b) Leydig cell from naked mole rat testes rich in lipid droplets (LD). LD ¼ lipid droplets, N ¼ nucleus, Nu ¼ nucleolus, sER ¼ smooth endoplasmic reticulum, arrowhead ¼ multilamellar bodies. Scale bar ¼ 2 mm.
testicular mass in naked mole rat testes [35e37]. LDs are composed of neutral lipids, including triacylglycerols (TAG) and cholesterol esters (CEs), and are covered by a phospholipid monolayer [38]. Cholesterol accumulation is closely related to the expression of MLBs [39]. Previous studies had suggested that MLBs are related to lipids, lipid metabolism, storage and mobilization. We hypothesized that the expression of MLBs in the Leydig cells of naked mole rats may be involved in regulating the large population of LDs in this species. One other hypothesis is that the expression of MLBs may be associated with a cellular disorder such as cancer or tumor presence, but this is not the case because biological and medical research has shown that naked mole rats display resistance to cancer throughout their life [1]. Naked mole rats have a unique reproductive behavior. Steroidogenesis occurs in the Leydig cells of both breeder and nonbreeder naked mole rats, and in general, the urinary testosterone level is lower in non-breeder then breeder animals [6,7,37]. In this study, MLBs were observed in association with LDs, and the number of MLBs was greater in the testicular interstitium of breeder naked mole rats compared to non-breeders. During steroidogenesis, cholesterol is converted into steroid hormone, and LDs are the primary source of the cholesterol substrate [40]. Thus, in this study, we can infer that the difference in the number of MLBs may be related to the reproductive status of the naked mole rat. Functionally, MLBs are also believed to facilitate cell-to-cell communication [32]. Testicular function is dependent on the communication between different cells within the testis [41].
Communication between cells of a multicellular organism is necessary in order to maintain normal function [42,43]. Cellular communication includes direct cell-to-cell communication and indirect communication through the release of soluble factors that activate nearby or distant target cells [42]. In this study, MLBs were found in the space between Leydig cells, and as such, MLBs may be a novel method of communication among Leydig cells, which could be essential for normal steroidogenesis in naked mole rat testes. 4.2. Telocytes may assist in the transport of MLBs within the interstitial space Interestingly, after their release from Leydig cells, most MLBs are associated with telocytes in the testicular interstitium (Fig 5a and 6abc). Telocytes are described as a novel type of cell consisting of a small cellular body and extremely long extracellular extensions [44,45]. These cells are found in the interstitial space of many organs, including the testis [10,45]. Telocytes were discovered a decade ago, but their function in physiological processes is still a matter of debate [11]. Telocytes form both homocellular contacts, connecting multiple telocytes to one another, and heterocellular contacts, connecting telocytes to other types of cells [46,47]. In the testes, telocytes form heterocellular contact with Leydig cells [10]. Through this contact, telocytes perform several important functions, including cell-to-cell communication [11,47]. Cellular extensions of telocytes are thin, can be up to 1000 mm in length, and are distributed into the interstitial tissues [44,48]. Recent studies had
Fig. 3. TEM photographs of multilamellar bodies and autophagic vacuoles in Leydig cells of a naked mole rat (non-breeder). (a) Leydig cell with mitochondria (M) close to lipid droplets (LD) and multilamellar bodies (arrowhead), two interconnected multilamellar bodies associated with LDs (arrow). (b) Leydig cell containing nucleus with prominent nucleolus and multilamellar body (arrowhead) and autophagic vacuoles (curved arrow). N ¼ nucleus, Nu ¼ nucleolus, M ¼ mitochondria, LD ¼ lipid droplets, scale bar ¼ 2 mm.
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Fig. 4. TEM photograph of lipid droplets (LDs) and associated multilamellar bodies in the cytoplasm of Leydig cells from (a) breeder or (b) non-breeder naked mole rats. (a) Lipid droplets are surrounded by a membrane (arrow). (a). Multilamellar bodies inside lipid droplets and a few autophagic vacuoles (curved arrow). (b). arrowhead ¼ Multilamellar bodies, scale bar ¼ 1 mm.
Fig. 5. Multilamellar bodies in the space between Leydig cells and near blood vessels in the testicular interstitium of naked mole rat (non-breeder). (a) Multilamellar body releasing from the cell membrane (black arrow) and many MLBs in spaces between Leydig cells (arrowhead). (b) many multilamellar bodies (black arrowhead) adjoining to blood vessel. Two multilamellar bodies enclosed by one membrane (curved arrow). White arrow ¼ cellular extension of telocytes, LD ¼ lipid droplets, sER ¼ smooth endoplasmic reticulum, white arrowhead ¼ Podom, scale bar ¼ 2 mm.
reported presences of telocytes in testicular interstitium of mouse and bank vole (Myodes glareolus). Telocytes in testicular interstitium were possibly involvement in maintaining microenvironment, protein interactions, lipid homeostasis and secretory functions of other interstitial cells [49,50]. In this study, telocytes were identified using the criteria established by Popescu and Faussone-Pellegrini (2010). We found cellular extensions of telocytes between Leydig cells, and MLBs were found closely attached
to the cellular extensions of telocytes. MLBs were also found along the cellular extensions of telocytes. We therefore suggest that the cellular extensions of telocytes may play some role in transporting or guiding MLBs towards their targets in interstitial spaces. Further studies are needed, to analyze the number and functions of telocytes at molecular level in extended interstitial area of naked mole rat testes.
Fig. 6. TEM photograph of telocyte between Leydig cells (non-breeder). A telocyte consisting of a cell body (black arrow) with long cellular extension (curved arrow) having podom (white arrow) and MLBs (arrowhead) along the cellular extension of the telocyte. Scale bar, 4 mm.
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Fig. 7. TEM photograph of the space between testicular Leydig cells showing many multilamellar bodies and their association with telocytes in naked mole rat testes (breeder). Cellular extension of telocytes is clear (black arrow) (a) with multilamellar body attached to the cellular extension (arrowhead) (c). (b,c) higher magnification from (a). LY ¼ Leydig cell, LD ¼ lipid droplets, white arrow ¼ Podom. Scale bar, 2 mm(a) 1 mm (b,c).
Fig. 8. Multilamellar bodies free in testicular interstitial space along with cellular extension of telocytes. (a) low magnification (b) high magnification. Arrow ¼ multilamellar body, arrowhead ¼ Podom. Scale bar, 1 mm.
4.3. MLBs, autophagy and Leydig cells In this study, we observed three main features inside the cytoplasm of Leydig cells: autophagy vacuoles with diverse degraded
material, single LDs enclosed by a limited membrane, and LDassociated MLBs. Previously reported criteria were used in order to differentiate between MLBs and autophagy related structures. Specifically, MLBs were defined as cytoplasmic, membrane-bound
Fig. 9. Number and size of MLBs. Number of intra- and extracellular MLBs (a, b). Size of intra- and extracellular MLBs (c, d). n ¼ 20 per group, P < 0.05.
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Fig. 10. CD34 expression in testicular interstitium of naked mole rat. Localization of CD34 in extended area of testicular interstitium at lower magnification (a). An illustration of Fig b (rectangular area) showing long cellular extension of telocytes stained with CD34 (b). Localizations of CD34 around cluster of Leydig cells (indicated by arrow) and blood vessels (arrowhead) (c). Negative control (d). ST ¼ seminiferous tubules, LY ¼ Leydig cells, arrow ¼ telocytes, arrowhead ¼ blood vessels.
organelles with at least three distinct circumferential concentric membrane lamellae. Autophagic vacuoles were defined by the presence of multiple internal structures surrounded by a limiting membrane, and these could be morphologically distinguished from MLBs [51,52]. We performed immunofluorescence analysis of LC3, the most reliable biomarker for autophagy, in order to confirm autophagy activity [53]. LC3 showed a strong positive reaction with the interstitial tissues of the naked mole rat testes (Fig. 10). The main function of the Leydig cell is to produce the steroid hormone testosterone during a process called steroidogenesis [12]. Several steps are involved in the process of steroidogenesis in order to convert cholesterol into its final steroid hormone products [54]. There are several sources of free cholesterol, but LDs are the preferred source of the cholesterol substrate used in steroidogenesis [40]. The LDs in Leydig cells not only store cholesterol, but they also contain many important enzymes for cholesterol metabolism and the biosynthesis of steroid hormones [55,56]. Autophagy has been reported to be very active in Leydig cells, and it has been hypothesized that autophagy may play a role in testosterone production [23,24,57,58]. A recent finding reported that autophagy
also promotes cholesterol uptake into Leydig cells [59]. Selective autophagy has been implicated in lipid metabolism to provide the cell with cholesterol, a process known as macrolipophagy in hepatocytes [60]. In this study, we observed lipid droplets that were enclosed by a limiting membrane (Figs. 1a, 4a and 9), as in macrolipophagy. We hypothesized that autophagy may play a role in lipid metabolism via macrolipophagy in order to provide cholesterol for steroidogenesis. Following the discovery of MLBs, their biogenesis was under investigation. Older studies reported that autophagy may play a role in the biogenesis of MLBs [28,61,62]. Recent work using Mv1Lu cells lines explored the role of autophagy in the biogenesis of MLBs [51,52]. According to Hariri et al. (2002), the appearance of MLBs was associated with autophagy, and the induction and inhibition of autophagy caused the appearance and disappearance of MLBs, respectively [51]. In this study, we detected autophagy, along with MLBs, in the Leydig cells. Our data support the hypothesis that autophagy plays a role in the biogenesis of MLBs. Autophagy appears to be involved in lipid metabolism via lipophagy and the production of MLBs. Although the role of autophagy in Leydig cells is well established, MLBs may play an
Fig. 11. LC3 localization in testis of naked mole rat. LC3 shows strong expression in extended interstitial tissues. (a) breeder naked mole rat. (b) non-breeder naked mole rat. (c) negative control. LY ¼ Leydig cells/interstitial tissue, ST ¼ seminiferous tubules. Scale bar, 20 mm.
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Fig. 12. Schematic of MLBs, autophagy, and MLB association with telocytes in testicular interstitium of the naked mole rat.
unknown novel role in the regulation of lipid metabolism in Leydig cells.
[7]
5. Conclusion
[8]
This study reported the presence of MLBs and their association with telocytes in the testicular interstitial tissues of the naked mole rat. We proposed that biogenesis of these MLBs occurs inside the Leydig cell, most likely from LDs. Subsequently, MLBs are released by Leydig cells through the cell membrane by the process of exocytosis. After their release from the cell, MLBs attach to the cellular extensions of telocytes. We proposed that telocytes may facilitate the transport of multilamellar bodies to the target cells such as Leydig cells and blood vessels in the interstitial space. We also found that autophagy is active in Leydig cells and may be contributes to the biogenesis of MLBs. Selective breakdown of LDs via macrolipophagy was also found, a process that could provide cholesterol for steroidogenesis. The testes of both breeder and nonbreeder male naked mole rats contained numerous MLBs in testicular interstitium.
[9]
Conflicts of interest The authors have no conflicts of interest to declare.
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Telocytes: facts, speculations and myths. Folia Med Cracov 2017;57:5e22. [46] Gherghiceanu M, Popescu LM. Heterocellular communication in the heart: electron tomography of telocyte-myocyte junctions. J Cell Mol Med 2011;15: 1005e11. [47] Faussone-Pellegrini MS, Gherghiceanu M. Telocyte's contacts. Semin Cell Dev Biol 2016;55:3e8. [48] Kostin S. Myocardial telocytes: a specific new cellular entity. J Cell Mol Med 2010;14:1917e21. [49] Pawlicki P, Hejmej A, Milon A, Lustofin K, Płachno BJ, Tworzydlo W, et al. Telocytes in the mouse testicular interstitium: implications of G-proteincoupled estrogen receptor (GPER) and estrogen-related receptor (ERR) in the regulation of mouse testicular interstitial cells. Protoplasma 2019;256: 393e408. [50] Milon A, Pawlicki P, Rak A, Mlyczynska E, Płachno BJ, Tworzydlo W, et al. Telocytes are localized to testis of the bank vole (Myodes glareolus) and are affected by lighting conditions and G-coupled membrane estrogen receptor (GPER) signaling. Gen Comp Endocrinol 2019;271:39e48. [51] Hariri M, Millane G, Guimond M-P, Guay G, Dennis JW, Nabi IR. Biogenesis of multilamellar bodies via autophagy. Mol Biol Cell 2000;11:255e68. [52] Lajoie P, Guay G, Dennis JW, Nabi IR. The lipid composition of autophagic vacuoles regulates expression of multilamellar bodies. J Cell Sci 2005;118: 1991e2003. [53] Klionsky DJ, Abdelmohsen K, Abe A, Abedin MJ, Abeliovich H, Acevedo Arozena A, et al. Guidelines for the use and interpretation of assays for monitoring autophagy. Autophagy 2016;12:1e222. [54] Issop L, Rone MB, Papadopoulos V. Organelle plasticity and interactions in cholesterol transport and steroid biosynthesis. Mol Cell Endocrinol 2013;371: 34e46. [55] Wang W, Wei S, Li L, Su X, Du C, Li F, et al. Proteomic analysis of murine testes lipid droplets. Sci Rep 2015;5:12070. [56] Yamaguchi T, Fujikawa N, Nimura S, Tokuoka Y, Tsuda S, Aiuchi T, et al. Characterization of lipid droplets in steroidogenic MLTC-1 Leydig cells: protein profiles and the morphological change induced by hormone stimulation. Biochim Biophys Acta Mol Cell Biol Lipids 2015;1851:1285e95. [57] Tang X. The autophagic activity of Leydig cells in normal rat testes. Shi yan sheng wu xue bao 1988;21:119e29. [58] Tang X, Zhang H, Yi J. Leydig cells–a normal cell model of cellular autophagy. Shi yan sheng wu xue bao 1992;25:39e47. [59] Gao F, Li G, Liu C, Gao H, Wang H, Liu W, et al. Autophagy regulates testosterone synthesis by facilitating cholesterol uptake in Leydig cells. J Cell Biol 2018:2103e19. jcb. 201710078. [60] Singh R, Kaushik S, Wang Y, Xiang Y, Novak I, Komatsu M, et al. Autophagy regulates lipid metabolism. Nature 2009;458:1131e5. [61] SOROKIN SP. A morphologic and cytochemical study on the great alveolar cell. J Histochem Cytochem 1966;14:884e97. [62] Balis J, Conen P. The role of alveolar inclusion bodies in the developing lung. Lab Invest J Tech Methods Pathol 1964;13:1215e29.
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Characterization of multilamellar bodies and telocytes within the testicular interstitium of naked mole rat Heterocephalus glabe Abdul Haseeb a, b, 1, Imran Tarique a, 1, Adeela Iqbal a, Noor samad Gandahi a, Waseem Ali vistro a, Xuebing Bai a, Yu Liang a, Yufei Huang a, Hong Chen a, Qiusheng Chen a, Ping Yang a, * a MOE Joint International Research Laboratory of Animal Health and Food Safety, College of Veterinary Medicine, Nanjing Agricultural University, Jiangsu Province, 210095, China b Faculty of Veterinary and Animal Sciences, University of Poonch Rawalakot, Azad Kashmir, Pakistan
a r t i c l e i n f o
a b s t r a c t
Article history: Received 24 January 2019 Received in revised form 9 July 2019 Accepted 10 July 2019 Available online 10 July 2019
Multilamellar bodies (MLBs) are produced and secreted by many cell types. In this study, we report the existence and ultrastructure of MLBs that are produced by Leydig cells and identification of telocytes in the testicular interstitium of naked mole rat. This study was performed on both breeder and non-breeder male naked mole rats using light microscopy, transmission electron microscopy, and morphometric approaches. In the testicular interstitium, the most prominent cells were Leydig cells, which contained numerous lipid droplets (LDs) in the cytoplasm. We found that MLBs were associated with the LDs of Leydig cells and were secreted into the extracellular or interstitial environment via exocytosis. After their release from Leydig cells, MLBs localized to the space between Leydig cells near blood vessels and attached to telocytes. We also identified telocytes in the testicular interstitium, and their cellular extensions were distributed throughout the interstitium. MLBs were aligned along the cellular extensions of telocytes, and membrane-to-membrane contact was observed between the cellular extensions of telocytes and MLBs, suggesting that telocytes may play a role in the transport of MLBs within the interstitial space. No ultrastructural differences were found in Leydig cells, telocytes, or MLBs between breeder and non-breeder testes. However, morphometric analysis revealed a significant difference in the number of MLBs between the breeder and non-breeder animals. Furthermore, both selective autophagy of LDs and non-selective autophagy were observed in Leydig cells. Typical features of macrolipophagy were also observed, as a few LDs were entirely enclosed by a limiting membrane. Remarkably, autophagy may be a key factor in the biogenesis of MLBs and steroid hormone production. The appearance of MLBs in the testicular interstitium of naked mole rats could thus be related to lipid storage and trafficking. © 2019 Published by Elsevier Inc.
Keywords: Interstitial tissue Leydig cells Telocytes Lipid droplets Autophagy Morphometry
1. Introduction The naked mole rat (Heterocephalus glaber) is the longest living rodent species, with a maximum lifespan of 30 years [1]. Naked mole rats are eusocial, subterranean mammals and they are native to Africa [2]. Both captive and wild colonies of naked mole rats have unique behavioral and reproductive characteristics. Reproductive activities are restricted to one female and 1e3 males in each colony [3,4]. The remaining members of the colony do not breed, but they
* Corresponding author. E-mail address: [email protected] (P. Yang). 1 Authors contributed equally. https://doi.org/10.1016/j.theriogenology.2019.07.010 0093-691X/© 2019 Published by Elsevier Inc.
are not sterile [5]. Those who participate in breeding known as breeder, while rest of them known as non-breeders animals. Spermatogenesis and steroidogenesis occurs both in breeder and non-breeder male naked mole rats [6,7]. Naked mole rats maintain lifelong normal physiological activity without displaying any signs of dysfunction or disease related to age [8]. They also show resistance to spontaneous cancer [1]. The parenchyma of the testis is composed of seminiferous tubules and interstitial tissues [9]. Interstitial tissues consist of highly vascularized, loose connective tissues, including Leydig cells, blood vessels, macrophages, leukocytes, fibroblasts, peritubular cells, and a novel cell type called telocytes [10]. Telocytes are characterized as having a small cellular body and extremely long extracellular
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extensions. They are found in the intestinal space of organs. Telocytes were discovered a decade ago, however their function is still under debate [11]. Leydig cells are the most structurally and functionally prominent cells in the interstitial tissue of the testis [12]. These cells are predominantly enriched within the smooth endoplasmic reticulum, mitochondria, and LDs [13]. Multilamellar bodies (MLBs) are subcellular structures (organelles) that vary in size from 100 to 2400 nm. Morphologically, they consist of concentric membrane layers and often exhibit an electron-dense core. Through electron microscopy studies, many different names have been given to multilamellar membrane structures such as lamellar bodies, lamellar granules, multilamellar bodies, multilamellar lipids, myeloid bodies, membrane-coating granules, Odland bodies, and lysosomal related organelles [14e17]. To avoid confusion during this report, we will use the term MLBs, as all of these terms are still used in the literature depending on the tissue. Under both normal and pathological conditions, MLBs are found in several cell types and are functionally involved in lipid storage and secretion [15]. Excellent work has been done on the MLBs that are secreted by alveolar type 2 cells in the lungs and by the epidermis [15,18,19]. MLBs have also been reported in other tissues, such as the tongue papillae, oral epithelium, digestive tract, and peritoneum [15,20], as well as in lower organisms such as insects and amoebae [21,22]. In the testicular interstitium, structures similar to MLBs (autophagosomes) have been previously reported in Leydig cells as a component of autophagy processes [23,24]. However, no study has described the MLBs as an individual entity in the extracellular spaces of the testicular interstitium. Here, we characterize and report for the first time the presence of MLBs in the testicular interstitium that are produced and secreted by Leydig cells of naked mole rats. Moreover, we identified telocytes in the extended area of the testicular interstitium and describe their interaction with MLBs. Naked mole rats are eusocial animals that show an extreme form of cooperative breeding. Few animals contribute to breeding (known as breeder animals), while other members are either sterile or do not reproduce (known as nonbreeders) [3]. Thus, in the present study, observations were performed on both types of naked mole rats (breeder and nonbreeder) to fully characterize MLBs, Leydig cells, and telocytes. The naked mole rat is known as a super model organism for aging research [1]. Therefore, this research may be useful for future work on studies related to the reproductive physiology of the naked mole rat.
exsanguination. The testes were collected through ventral abdominal dissection. The handling of animals and study procedures were in accordance with guidelines of the Animal Research Institute Committee of Nanjing Agriculture University. All the protocols were approved by the Science and Technology Agency of Jiangsu Province (Approval ID: SYXK (SU) 2010-0005). All efforts were made to minimize animal suffering. 2.2. Light microscopy Tissue samples were fixed in 10% neutral buffered formalin overnight. The sample was then dehydrated in an ascending series of ethanol, cleared in xylene, embedded into paraffin wax and sectioned at 5 mm. Tissue sections were stained with hematoxylin and eosin (Harrys hematoxylin for 2 min and 1% eosin for 30 s) and analyzed using an Olympus microscope (BX53, Tokyo, Japan). To measure the interstitial area and the number of Leydig cells, HE photos were imported into ImageJ software. To measure the interstitial tissue area, the total area of the image was first measured, and then the area of seminiferous tubules was determined, delineating the circumference of all the tubules in an image. After that, the area of interstitial tissue was obtained by subtracting the seminiferous tubule area from the total area of the image [26]. The number of Leydig cells was counted using the ImageJ Cell Counter plugin. Morphometric data (interstitial area and number of Leydig cell) was obtained from 40 HE stained photographs (20 per group, cross-section, 1600 1200) taken randomly. 2.3. Ultrastructural analysis Testes samples were cut into 1 mm3 blocks and immersed in 2.5% glutaraldehyde in 0.01 M phosphate-buffered saline (PBS) (4 C, pH 7.4) for 24 h. Samples were rinsed in PBS and post-fixed in an 1% osmium tetroxide solution for 1 h at 37 C. Post fixed samples were dehydrated in ascending concentrations of ethyl alcohol, infiltrated with a propylene oxide-Araldite mixture and embedded in Araldite. The blocks were then sectioned using an ultramicrotome (Reichert Jung, Vienna, Austria). The ultrathin sections of tissue (50 nm) were mounted on copper grids and contrasted with uranyl acetate and lead citrate for 20 min each. Finally, sections were examined and photographed with a transmission electron microscope (TEM) (Hitachi H-7650, Tokyo, Japan). 2.4. Immunohistochemistry (IHC)
2. Materials and methods 2.1. Experimental animals and ethical approval The naked mole rats used in these studies were maintained in the animal house of the College of Veterinary Medicine, Nanjing Agricultural University, China. Animals were housed in artificial Perspex burrow systems, a large plexiglass box (2 1 m2) heated to 24 to 28 C with a constant high relative humidity of 40%e70%. The burrow system consisted of a series of interconnecting clear acrylic plastic tubes with a nest, food and toilet chambers. Food was given ad libitum, included vegetables (sweet potatoes, carrots beet root and cucumber) and fruits (apples, bananas). Weekly, a supplement containing minerals and vitamins was also provided. Routine behavioral observations were carried out to differentiate between the breeder and non-breeder animals. In this research, 2 male breeders and 2 male non-breeders of the same age (15 months) were used. For sample collection, animals were anesthetized according to a standard protocol for naked mole rats with an intramuscular injection of a 6 mg kg1 ketamine and 2.5 mg kg1 xylazine [25], after which the animals were sacrificed by
The tissue was fixed in 10% formalin and embedded in paraffin for sectioning 6 mm. Sections were deparaffinized and then rehydrated. 3% hydrogen peroxide was used to block the further activity of endogenous peroxidase and followed by antigen retrieval. After blocked with 5% bovine serum albumin, the samples were incubated with rabbit anti-CD34 (1:100 dilution; catalog no. BA3414; Boster, Wuhan, China) in a moisture chamber at 4 Cfor 24 h, while PBS (pH 7.2) served as the negative control. After washing, the sections were incubated with biotinylated anti-rabbit/anti-mouse IgG (Boster Bio-Technology, Wuhan, China) for one hour at room temperature. The sections were then incubated with avidinbiotinylated peroxidase complex. Peroxidase activity was revealed using DAB (Boster Bio-Technology, Wuhan, China). The nuclei were stained with hematoxylin. 2.5. Fluorescence microscopy Immunofluorescence (IF) was performed according to a previously described method [27]. Briefly, after completing all of the basic steps (deparaffinization, serum blocking and antigen
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retrieval), the testes tissue slides were incubated with primary antibody (anti-rabbit LC3, 1:75 dilution, 12741, Cell Signaling Technology, Danvers, Massachusetts, USA) at 4 C overnight. Subsequently, the slides were washed in PBS, and the corresponding secondary antibodies (Goat anti-rabbit IgG, 1:75 dilution, ab6721, Abcam, Cambridge, Massachusetts, USA) were added to the sections for 2 h at room temperature. Next, the slides were washed again with PBS and then stained with DAPI to highlight the cell nucleus. For negative control PBS (pH 7.2) was used. Images were obtained using a microscope (BX53, Olympus, Tokyo, Japan) attached to a camera (DP73, Olympus, Tokyo, Japan). 2.6. Morphometric analysis of MLBs To measure the size and number of MLBs, TEM photographs of the same magnifications were imported into ImageJ software. Lines were drawn around the MLBs using the image tool (area of interest (AOI)) to measure the size (mean diameter) of MLBs. The number of MLBs was counted manually. Morphometric data (size and number of MLBs) were obtained from 40 TEM photographs (20 per group, cross-sectioned, 4800 3730) taken randomly. 2.7. Statistical analysis The statistical significance of the results was analyzed by t-test. Differences were considered significant at P < 0.05. Analyses were performed using SAS version 14.0 (SAS Institute). All data are reported as the mean ± S.E.M. 3. Results 3.1. Light microscopy Under a light microscope, a large content of interstitial tissue was observed in the testes of both breeder and non-breeder naked rats (Fig 1ab). Morphometric analysis showed that testes from both breeders and non-breeders contained a substantial content of interstitial area and number of Leydig cells (Fig 1cd, Table 1). Noticeably, the content of interstitial area was significantly higher (p ¼ 0.0005) in non-breeders than in breeders (Fig. 1c, Table 1). The number of Leydig cells within the interstitial tissue was also significantly higher (p ¼ 0.0031) in non-breeder testes than in breeder testes (Fig. 1d, Table 1). 3.2. Localization, biogenesis and association of MLBs The testicular interstitium from both breeder and non-breeder naked rats was observed under a transmission electron microscope. A detailed analysis of the Leydig cells, MLBs and telocytes was performed. In testes from both breeder and non-breeder animals, the Leydig cells contained an oval-shaped nucleus with a prominent nucleolus and cytoplasm with all basic organelles, including endoplasmic reticulum, mitochondria (Figs 2ab and 3 ab), and more prominent LDs (Figs. 2b, 3b and 4 ab). In the cytoplasm of the Leydig cells, MLBs consisted of concentric layers, which were accompanied by LDs (Fig 2ab, 3 ab 4 ab). The association of MLBs with LDs appeared in various forms: MLBs occupying most of the of LD (Fig. 2a), MLBs forming one third of the LDs (Fig 3ab), or MLBs forming half of the LD (Fig. 4a). MLBs are secreted from the cytoplasm of Leydig cells into the extracellular space through exocytosis (Fig. 4a). In addition to being found in the cytoplasm of Leydig cells, MLBs were also found in the space between Leydig cells (Figs. 5a, 6 and 7a) and adjoining the blood vessels (Fig. 5b). Telocytes were identified in the testicular interstitium of both breeder and nonbreeder naked mole rats and were morphologically characterized
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by a cell body and very long cellular extensions (Figs. 5b, 6 and 7abc). The cellular extensions of telocytes consist of podomers (thin segments) and podoms (bead-like portions) (Figs. 5b, 6 and 7bc). Numerous cellular extensions of telocytes were distributed in the interstitial tissue around Leydig cells and blood vessels (Figs. 5b, 6 and 7abc). MLBs were found to be aligned with the cellular extensions of telocytes, and we also observed close membrane-to-membrane contacts between the cellular extensions of telocytes and MLBs (Figs. 6 and 7abc). MLBs were also observed freely in the interstitial space close to cellular extensions of telocytes (Fig. 8ab). Overall, no ultrastructural differences were found in Leydig cells, MLBs or telocytes between breeder and non-breeder naked mole rats. 3.3. Morphology of MLBs We observed morphologically diverse shapes of MLBs. Some MLBs had concentric membrane layers and occasionally had a dense core (Figs. 2e4, 6), and some MLBs showed a mix of concentric layers and amorphous materials (Figs. 5, 7 and 8), while other MLBs that were fused together and surrounded by one membrane or without any outer membrane were also observed (Figs. 2 and 5). 3.4. Morphometric analysis of MLBs Overall, MLBs were observed in both breeder and non-breeder naked mole rats. Morphometry analysis was performed to determine if there were differences in the number or the size of MLBs between breeder and non-breeder animals. The number of intraand extracellular MLBs were greater in the breeder testes compared to non-breeders (Fig. 9ab). The number of intracellular MLBs was significantly higher (p ¼ 0.0005) in breeders than non-breeders (Fig. 9a, Table 1). Similarly, the number of extracellular MLBs was also higher (p ¼ 0.1080) in breeders than in non-breeders (Fig. 9b, Table 1). The size of intra- and extracellular MLBs was not significantly different between breeder and non-breeder animals (Fig 9cd, Table 1). 3.5. Expression of CD34 in the testicular interstitium of naked mole rat testes Under TEM, telocytes were observed in the testicular interstitium of naked mole rat (Figs. 5b, 6, 7abc, and 8 ab). For further confirmation, we performed IHC using CD34 most reliable biomarker for telocytes identification. CD34 was strongly expressed around seminiferous tubules, Leydig cells, blood vessels and with long cellular extensions of telocytes in interstitium spaces (Fig. 10 abc). The distributions of the CD34-positive telocytes in testicular interstitium were agreed with TEM observations. However, unexpected/non-specific staining was also observed within seminiferous tubules of naked mole rat testes. 3.6. Autophagy in Leydig cells Immunofluorescence of LC3 was performed with in order to confirm autophagy activities. Immunofluorescence staining of LC3 showed a strong positive reaction within the interstitial tissues of the testes of naked mole rats (Fig. 11). Under a TEM, MLB associated autophagy activity was also observed in the cytoplasm of Leydig cells. Both selective and nonselective autophagy was found. Some autophagic vacuoles had multiple internal structures or degraded materials, indicating nonselective autophagy (Fig. 2ab, 4ab, 12). More visibly, we observed LDs that were entirely enclosed by a limiting membrane, indicating selective autophagy of LDs (Figs. 1a,
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Fig. 1. Testicular interstitium of naked mole rats. H&E staining of breeder (a) and non-breeder (b) naked mole rat testes. (c) Qunatification of intersititial area (mm2) (n ¼ 20 per group). (d) Qunatification of Leydig cell numbers (n ¼ 20 per group). St ¼ seminiferous tubules, Ic ¼ Interstitial cells, Bv ¼ blood vessel. scale bar ¼ 50 mm.
4a and 12).
4. Discussion 4.1. Ultrastructure and possible physiological function of MLBs in the testicular interstitium of naked mole rat In the present study, a population of MLBs were observed inside Leydig cells and were found to be localized to other compartments in the testicular interstitium. Morphologically, almost all MLBs were similar, exhibiting collections of concentric membrane layers [15]. However, ultrastructural morphological differences existed between the MLBs, varying between different tissues as well as within the same tissue. In lung tissues, MLBs appeared as concentric layers both with a dense core or without a dense core, while fusion of MLBs and concentric layers enclosed by an outer limiting layer have also been reported [28]. Three different kinds of MLBs have been reported in amoebae on the basis of morphology. These types are MLBs with concentric layers, with concentric layers with amorphous material and with only amorphous material [22]. Many
Table 1 Morphometric analysis. Data are represented as mean ± S.E.M. (n ¼ 20 per group). Parameters
Breeder
Non-breeder
P value
Interstitial area Leydig cell numbers Number of Intracellular MLBs Number of Extracellular MLBs Size of intracellular MLBs Size of extracellular MLBs
361.3 ± 482.6 9034 ± 8909 10.17 ± 7.542 14.39 ± 2.745 205.6 ± 4.272 670.9 ± 4.709
843.9 ± 47.52 17943 ± 1400 5.5 ± 1.792 9.87 ± 3.452 209.8 ± 27.71 666.2 ± 54.25
0.0005 0.0031 0.0005 0.0108 0.8849 0.935
cells produce and secrete MLBs [15]. Nevertheless, there have not been any studies about MLBs in Leydig cells, particularly extracellular MLBs. In this study, all previously reported ultrastructural characteristics of MLBs were found, including fusion, mix of concentric layers and amorphous material and the presence or absence of a dense core. Therefore, we confirmed these observed structures as MLBs. Although different cell types produce MLBs, the origin of the MLBs differs between cells. Subcellular organelles such as the Golgi apparatus, endoplasmic reticulum, lysosomes, and lipid droplets are all believed to be an origin of MLBs in various tissues [29e32]. In the present study, we observed close association of MLBs with lipid droplets. We also showed another type of association of MLBs with LDs, indicating the step by step biogenesis of MLBs from LDs or may represent partial digestion of LDs by MLBs (Fig. 11). Our findings indicate that MLBs in Leydig cells are derived from LDs. MLBs are metabolically active and are required for the directed transport and conversion of lipids, as well as for interconversion of different lipid species [30]. MLBs contain various cargo, including lipids, hydrolytic enzymes, and several other proteins [33]. MLB biogenesis occurs in the cytoplasm, and they are then secreted into the extracellular or interstitial environment via exocytosis [17,20,32,34]. Following the release by exocytosis, we observed MLBs in different locations of the interstitial environment, including in the space between Leydig cells, around blood vessels and along the cellular extension of telocytes. Given that MLBs are mainly composed of lipids, their main function is lipid storage and direct transport of lipids [15,30]. Therefore, MLBs may be functional in lipid trafficking in the testes of naked mole rats. One important question that arises is why MLBs were not reported previously in testicular interstitium. One possible reason is that Leydig cells are enriched in LDs and cover nearly 60% of the
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Fig. 2. TEM photographs of multilamellar bodies associated with lipid droplets in Leydig cells of a naked mole rat (breeder). (a) Numerous multilamellar bodies (arrowhead) closely associated with lipid droplets (LD) and with membranes around single lipid droplet (arrow). Cytoplasm contains many mitochondria and smooth endoplasmic reticulum (sER). (b) Leydig cell from naked mole rat testes rich in lipid droplets (LD). LD ¼ lipid droplets, N ¼ nucleus, Nu ¼ nucleolus, sER ¼ smooth endoplasmic reticulum, arrowhead ¼ multilamellar bodies. Scale bar ¼ 2 mm.
testicular mass in naked mole rat testes [35e37]. LDs are composed of neutral lipids, including triacylglycerols (TAG) and cholesterol esters (CEs), and are covered by a phospholipid monolayer [38]. Cholesterol accumulation is closely related to the expression of MLBs [39]. Previous studies had suggested that MLBs are related to lipids, lipid metabolism, storage and mobilization. We hypothesized that the expression of MLBs in the Leydig cells of naked mole rats may be involved in regulating the large population of LDs in this species. One other hypothesis is that the expression of MLBs may be associated with a cellular disorder such as cancer or tumor presence, but this is not the case because biological and medical research has shown that naked mole rats display resistance to cancer throughout their life [1]. Naked mole rats have a unique reproductive behavior. Steroidogenesis occurs in the Leydig cells of both breeder and nonbreeder naked mole rats, and in general, the urinary testosterone level is lower in non-breeder then breeder animals [6,7,37]. In this study, MLBs were observed in association with LDs, and the number of MLBs was greater in the testicular interstitium of breeder naked mole rats compared to non-breeders. During steroidogenesis, cholesterol is converted into steroid hormone, and LDs are the primary source of the cholesterol substrate [40]. Thus, in this study, we can infer that the difference in the number of MLBs may be related to the reproductive status of the naked mole rat. Functionally, MLBs are also believed to facilitate cell-to-cell communication [32]. Testicular function is dependent on the communication between different cells within the testis [41].
Communication between cells of a multicellular organism is necessary in order to maintain normal function [42,43]. Cellular communication includes direct cell-to-cell communication and indirect communication through the release of soluble factors that activate nearby or distant target cells [42]. In this study, MLBs were found in the space between Leydig cells, and as such, MLBs may be a novel method of communication among Leydig cells, which could be essential for normal steroidogenesis in naked mole rat testes. 4.2. Telocytes may assist in the transport of MLBs within the interstitial space Interestingly, after their release from Leydig cells, most MLBs are associated with telocytes in the testicular interstitium (Fig 5a and 6abc). Telocytes are described as a novel type of cell consisting of a small cellular body and extremely long extracellular extensions [44,45]. These cells are found in the interstitial space of many organs, including the testis [10,45]. Telocytes were discovered a decade ago, but their function in physiological processes is still a matter of debate [11]. Telocytes form both homocellular contacts, connecting multiple telocytes to one another, and heterocellular contacts, connecting telocytes to other types of cells [46,47]. In the testes, telocytes form heterocellular contact with Leydig cells [10]. Through this contact, telocytes perform several important functions, including cell-to-cell communication [11,47]. Cellular extensions of telocytes are thin, can be up to 1000 mm in length, and are distributed into the interstitial tissues [44,48]. Recent studies had
Fig. 3. TEM photographs of multilamellar bodies and autophagic vacuoles in Leydig cells of a naked mole rat (non-breeder). (a) Leydig cell with mitochondria (M) close to lipid droplets (LD) and multilamellar bodies (arrowhead), two interconnected multilamellar bodies associated with LDs (arrow). (b) Leydig cell containing nucleus with prominent nucleolus and multilamellar body (arrowhead) and autophagic vacuoles (curved arrow). N ¼ nucleus, Nu ¼ nucleolus, M ¼ mitochondria, LD ¼ lipid droplets, scale bar ¼ 2 mm.
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Fig. 4. TEM photograph of lipid droplets (LDs) and associated multilamellar bodies in the cytoplasm of Leydig cells from (a) breeder or (b) non-breeder naked mole rats. (a) Lipid droplets are surrounded by a membrane (arrow). (a). Multilamellar bodies inside lipid droplets and a few autophagic vacuoles (curved arrow). (b). arrowhead ¼ Multilamellar bodies, scale bar ¼ 1 mm.
Fig. 5. Multilamellar bodies in the space between Leydig cells and near blood vessels in the testicular interstitium of naked mole rat (non-breeder). (a) Multilamellar body releasing from the cell membrane (black arrow) and many MLBs in spaces between Leydig cells (arrowhead). (b) many multilamellar bodies (black arrowhead) adjoining to blood vessel. Two multilamellar bodies enclosed by one membrane (curved arrow). White arrow ¼ cellular extension of telocytes, LD ¼ lipid droplets, sER ¼ smooth endoplasmic reticulum, white arrowhead ¼ Podom, scale bar ¼ 2 mm.
reported presences of telocytes in testicular interstitium of mouse and bank vole (Myodes glareolus). Telocytes in testicular interstitium were possibly involvement in maintaining microenvironment, protein interactions, lipid homeostasis and secretory functions of other interstitial cells [49,50]. In this study, telocytes were identified using the criteria established by Popescu and Faussone-Pellegrini (2010). We found cellular extensions of telocytes between Leydig cells, and MLBs were found closely attached
to the cellular extensions of telocytes. MLBs were also found along the cellular extensions of telocytes. We therefore suggest that the cellular extensions of telocytes may play some role in transporting or guiding MLBs towards their targets in interstitial spaces. Further studies are needed, to analyze the number and functions of telocytes at molecular level in extended interstitial area of naked mole rat testes.
Fig. 6. TEM photograph of telocyte between Leydig cells (non-breeder). A telocyte consisting of a cell body (black arrow) with long cellular extension (curved arrow) having podom (white arrow) and MLBs (arrowhead) along the cellular extension of the telocyte. Scale bar, 4 mm.
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Fig. 7. TEM photograph of the space between testicular Leydig cells showing many multilamellar bodies and their association with telocytes in naked mole rat testes (breeder). Cellular extension of telocytes is clear (black arrow) (a) with multilamellar body attached to the cellular extension (arrowhead) (c). (b,c) higher magnification from (a). LY ¼ Leydig cell, LD ¼ lipid droplets, white arrow ¼ Podom. Scale bar, 2 mm(a) 1 mm (b,c).
Fig. 8. Multilamellar bodies free in testicular interstitial space along with cellular extension of telocytes. (a) low magnification (b) high magnification. Arrow ¼ multilamellar body, arrowhead ¼ Podom. Scale bar, 1 mm.
4.3. MLBs, autophagy and Leydig cells In this study, we observed three main features inside the cytoplasm of Leydig cells: autophagy vacuoles with diverse degraded
material, single LDs enclosed by a limited membrane, and LDassociated MLBs. Previously reported criteria were used in order to differentiate between MLBs and autophagy related structures. Specifically, MLBs were defined as cytoplasmic, membrane-bound
Fig. 9. Number and size of MLBs. Number of intra- and extracellular MLBs (a, b). Size of intra- and extracellular MLBs (c, d). n ¼ 20 per group, P < 0.05.
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Fig. 10. CD34 expression in testicular interstitium of naked mole rat. Localization of CD34 in extended area of testicular interstitium at lower magnification (a). An illustration of Fig b (rectangular area) showing long cellular extension of telocytes stained with CD34 (b). Localizations of CD34 around cluster of Leydig cells (indicated by arrow) and blood vessels (arrowhead) (c). Negative control (d). ST ¼ seminiferous tubules, LY ¼ Leydig cells, arrow ¼ telocytes, arrowhead ¼ blood vessels.
organelles with at least three distinct circumferential concentric membrane lamellae. Autophagic vacuoles were defined by the presence of multiple internal structures surrounded by a limiting membrane, and these could be morphologically distinguished from MLBs [51,52]. We performed immunofluorescence analysis of LC3, the most reliable biomarker for autophagy, in order to confirm autophagy activity [53]. LC3 showed a strong positive reaction with the interstitial tissues of the naked mole rat testes (Fig. 10). The main function of the Leydig cell is to produce the steroid hormone testosterone during a process called steroidogenesis [12]. Several steps are involved in the process of steroidogenesis in order to convert cholesterol into its final steroid hormone products [54]. There are several sources of free cholesterol, but LDs are the preferred source of the cholesterol substrate used in steroidogenesis [40]. The LDs in Leydig cells not only store cholesterol, but they also contain many important enzymes for cholesterol metabolism and the biosynthesis of steroid hormones [55,56]. Autophagy has been reported to be very active in Leydig cells, and it has been hypothesized that autophagy may play a role in testosterone production [23,24,57,58]. A recent finding reported that autophagy
also promotes cholesterol uptake into Leydig cells [59]. Selective autophagy has been implicated in lipid metabolism to provide the cell with cholesterol, a process known as macrolipophagy in hepatocytes [60]. In this study, we observed lipid droplets that were enclosed by a limiting membrane (Figs. 1a, 4a and 9), as in macrolipophagy. We hypothesized that autophagy may play a role in lipid metabolism via macrolipophagy in order to provide cholesterol for steroidogenesis. Following the discovery of MLBs, their biogenesis was under investigation. Older studies reported that autophagy may play a role in the biogenesis of MLBs [28,61,62]. Recent work using Mv1Lu cells lines explored the role of autophagy in the biogenesis of MLBs [51,52]. According to Hariri et al. (2002), the appearance of MLBs was associated with autophagy, and the induction and inhibition of autophagy caused the appearance and disappearance of MLBs, respectively [51]. In this study, we detected autophagy, along with MLBs, in the Leydig cells. Our data support the hypothesis that autophagy plays a role in the biogenesis of MLBs. Autophagy appears to be involved in lipid metabolism via lipophagy and the production of MLBs. Although the role of autophagy in Leydig cells is well established, MLBs may play an
Fig. 11. LC3 localization in testis of naked mole rat. LC3 shows strong expression in extended interstitial tissues. (a) breeder naked mole rat. (b) non-breeder naked mole rat. (c) negative control. LY ¼ Leydig cells/interstitial tissue, ST ¼ seminiferous tubules. Scale bar, 20 mm.
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Fig. 12. Schematic of MLBs, autophagy, and MLB association with telocytes in testicular interstitium of the naked mole rat.
unknown novel role in the regulation of lipid metabolism in Leydig cells.
[7]
5. Conclusion
[8]
This study reported the presence of MLBs and their association with telocytes in the testicular interstitial tissues of the naked mole rat. We proposed that biogenesis of these MLBs occurs inside the Leydig cell, most likely from LDs. Subsequently, MLBs are released by Leydig cells through the cell membrane by the process of exocytosis. After their release from the cell, MLBs attach to the cellular extensions of telocytes. We proposed that telocytes may facilitate the transport of multilamellar bodies to the target cells such as Leydig cells and blood vessels in the interstitial space. We also found that autophagy is active in Leydig cells and may be contributes to the biogenesis of MLBs. Selective breakdown of LDs via macrolipophagy was also found, a process that could provide cholesterol for steroidogenesis. The testes of both breeder and nonbreeder male naked mole rats contained numerous MLBs in testicular interstitium.
[9]
Conflicts of interest The authors have no conflicts of interest to declare.
[10]
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