Reappraising the microscopic anatomy of human testis: identification of telocyte networks in the peritubular and intertubular stromal space

Telocytes are a recently described stromal cell type widely distributed in various organs including the female and male reproductive systems. This study was aimed to investigate for the first time the existence, distribution and characteristics of telocytes in normal human testis by an integrated morphological approach (immunohistochemistry, immunofluorescence and transmission electron microscopy). We found that telocytes displaying typical long and moniliform prolongations and coexpressing CD34 and PDGFRα formed networks in the outer layer of peritubular tissue and around Leydig cells and vessels in the intertubular stroma. Testicular telocytes were immunophenotypically negative for CD31, c-kit/CD117 as well as α-SMA, thus making them clearly distinguishable from myoid cells/myofibroblasts located in the inner layer of peritubular tissue. Transmission electron microscopy confirmed the presence of cells ultrastructurally identifiable as telocytes (i.e. cells with telopodes alternating podomers and podoms) in the aforementioned locations. Intercellular contacts between neighboring telocytes and telopodes were observed throughout the testicular stromal compartment. Telopodes intimately surrounded and often established close contacts with peritubular myoid cells/myofibroblasts, Leydig cells and vessels. Extracellular vesicles were also frequently detected near telopodes. In summary, we demonstrated that telocytes are a previously neglected stromal component of human testis with potential implications in tissue homeostasis deserving further investigation.

been often neglected 1,4,5 . That is not surprising, because during the removal of testicular tissue the seminiferous tubules are shifted against each other so that the loose intertubular stroma frequently disrupts 5 . As a matter of fact, aside from Leydig cells, there is little information concerning the possible existence of different interstitial cell types in the human testis stromal compartment.
Recent works have uncovered the presence of TCs in organs of the female and male reproductive systems from different species of vertebrates 16,29,[42][43][44][45][46][47][48][49] . To date, however, only one report has described the existence of TCs in the testis of the Chinese soft-shelled turtle 50 , while there are no studies on TCs in the mammalian and human testicular stroma. Therefore, in this study we aimed to investigate for the first time the existence, distribution and characteristics of TCs in normal human testis by an integrated morphological approach featuring immunohistochemistry, immunofluorescence and transmission electron microscopy analyses.

Results
In line with substantial literature 6,7,16,20,51 , the presence and distribution of TCs in the normal adult human testis were firstly explored by CD34 immunohistochemistry and immunofluorescence. As displayed in Fig. 1A-C depicting representative hematoxylin and eosin-stained tissue slides, these analyses were conducted on paraffin-embedded testicular sections which were carefully selected based on the optimal preservation of the peritubular and intertubular connective tissue testified by the presence of typical clusters of Leydig cells in close vicinity to blood capillaries and embedded in an extracellular matrix. Immunoperoxidase-based immunohistochemistry highlighted that CD34-positive cells were arranged in a complex and continuous network distributed throughout the peritubular and intertubular stromal spaces (Fig. 1D,E). These CD34-positive interstitial cells exhibited an elongated/spindle morphology with long and thin varicose cytoplasmic processes surrounding the seminiferous tubules and forming a reticular network around the Leydig cells and blood microvessels in the intertubular stroma ( Fig. 1F-H). Since the CD34 antigen is also expressed by vascular structures that could be misdiagnosed as spindle-shaped interstitial cells especially when appearing as profiles depending on the tissue cutting plane 16 , we further carried out CD34/CD31 double immunofluorescence. As shown in Fig. 2A, the peritubular and intertubular CD34-positive interstitial cells lacked CD31 immunoreactivity, thus making them clearly distinguishable from CD34-positive/CD31-positive vascular endothelial cells. In addition, these CD34-positive stromal cells were immunophenotypically negative for the c-kit/CD117 antigen, whose expression was only detectable in neighboring oval/round-shaped cells, presumably Leydig cells and/or mast cells (Fig. 2B). Keeping into account that spindle-shaped myoid cells/myofibroblasts are well known to be present in the peritubular connective tissue 1,3 , double immunostaining for CD34 and α-smooth muscle actin (α-SMA) was also performed on human testicular specimens. This analysis clearly revealed that two distinct but adjacent cellular entities populated the peritubular interstitium, namely the CD34-negative/α-SMA-positive myoid cells/myofibroblasts located in the inner layer and the CD34-positive/α-SMA-negative cells located in the outer layer of the peritubular stromal compartment (Fig. 3A,B). In particular, the CD34-positive/α-SMA-negative cells formed a continuous network extending from the outer peritubular tissue to the intertubular stroma, where instead the CD34-negative/α-SMA-positive myoid cells/myofibroblasts were absent (Fig. 3A,B). We then characterized more in depth the immunophenotype of these testicular stromal cells by CD34/platelet-derived growth factor receptor α (PDGFRα) double immunofluorescence staining, a cell surface antigenic combination currently considered among the most reliable for the identification of TCs by light microscopy 6,7,16,17,37,46,[52][53][54] . Such double immunolabeling showed coexpression of PDGFRα in all CD34-positive interstitial cells distributed in the outer layer of the peritubular tissue and in the intertubular stroma ( Fig. 4A-F). PDGFRα positivity was also detected in the CD34-negative myoid cells/myofibroblasts bordering the basal side of the seminiferous tubules ( Fig. 4A-F).
Although the aforedescribed immunohistochemical and immunofluorescence findings were rather evocative of the existence of testicular TCs, we next carried out toluidine blue staining of human testis semithin sections followed by transmission electron microscopy analysis of ultrathin sections to definitively confirm the diagnosis of TCs according to the ultrastructural definition by Cretoiu and Popescu 6 (Figs 5A-D and 6A-F). Interstitial cells with very long and thin moniliform cytoplasmic processes forming reticular networks in the same tissue locations identified by CD34/PDGFRα immunostaining were observed in toluidine blue-stained testicular semithin sections ( Fig. 5A-D). Ultrastructurally, testicular TCs exhibited a spindle-shaped, oval or piriform cellular body containing a large euchromatic nucleus surrounded by a small amount of cytoplasm ( Fig. 6A-F). The ultrastructural hallmark of TCs located either in the peritubular or in the intertubular testis stromal space was the presence of telopodes, namely very long, slender and often convoluted cytoplasmic prolongations with a narrow emergence from the cell body and a moniliform silhouette due to the alternation of podomers and podoms ( Fig. 6A-F).
Intercellular contacts between neighboring TCs and telopodes were frequently observed throughout the testicular stromal compartment (Fig. 6B). Moreover, by their telopodes TCs often established close contacts with peritubular myoid cells/myofibroblasts, intertubular Leydig cells and blood-derived mononuclear cells (presumably macrophages), and intimately surrounded the basement membrane of blood microvessels ( Fig. 6C-F). Numerous extracellular vesicles were usually detected nearby telopodes (Fig. 6E, inset).

Discussion
To the best of our knowledge, the integrated immunohistochemical, immunofluorescence and transmission electron microscopy approach employed in the present study allows us to clearly demonstrate for the first time that stromal cells with distinctive morphological and immunophenotypical features of TCs are broadly distributed within the human testis interstitium. Indeed, based on current recommendations for the identification of TCs 6 , we found that interstitial cells exhibiting typical telopodes (i.e. very long processes abruptly emerging from the cellular body and featuring the alternation of slenders podomers and small dilated bead-like podoms) and dual immunoreactivity for CD34 and PDGFRα form a continuous reticular network extending from the peritubular area to the intertubular stromal space of normal testes. Specifically, TCs appear to be located in the outer layer of the peritubular connective tissue, surrounding the inner layer of myoid cells/myofibroblasts. Moreover, the intertubular TC network is in close proximity or even directly connected to blood vessels and Leydig cells. Interestingly, an analogous distribution of TCs has been reported in the testes of the soft-shelled turtle 50 , which is in keeping with the suggested remarkable evolutionary conservation of these peculiar stromal cells across different species in the animal kingdom 55 .
As far as human beings are concerned, cells referred to as CD34-positive stromal fibroblastic/fibrocytic cells have previously been noticed in the testicular stroma 56 . However, although there is mounting evidence that CD34-positive stromal cells may indeed correspond to TCs in a variety of organs 51 , it is essential to keep in mind that (i) the CD34 antigen is not specific to TCs, and (ii) at present only transmission electron microscopy permits the definitive identification of TCs 6 . Based on this assumption, we have taken advantage of both dual immunofluorescence staining and ultrastructural analyses to conclusively verify whether the CD34-positive testicular stromal cells could represent TCs. Indeed, we herein demonstrated the presence of TCs in the human testicular stromal space with the aid of that can presently be considered their most specific immunohistochemical marker combination, namely CD34/PDGFRα double immunofluorescence 6,7,16,17,37,46,[52][53][54] . Furthermore, the combination of CD34 and CD31 immunostaining helped us to undoubtedly discriminate between TCs (CD34-positive/CD31-negative) and the endothelium (CD34-positive/CD31-positive) of blood vessels, and also emphasized the frequent  57,58 , the aforementioned evidence prompted us to further carry out CD34/α-SMA double immunofluorescence on human testis samples. Of note, these analyses clearly revealed that the CD34-negative/α-SMA-positive myoid cells/myofibroblasts were selectively located in the inner layer of the peritubular stroma, while the CD34-positive/α-SMA-negative TCs occupied the contiguous outer layer. Collectively, our immunohistochemical data indicated that all human testicular TCs, either in the peritubular or in the intertubular stroma, display the same immunophenotype, that is they are CD34/PDGFRα double-positive and α-SMA-negative. On this basis, we can categorically exclude the existence of a myoid TC subset in the human testis. We also observed that the peritubular CD34-negative/α-SMA-positive myoid cells/myofibroblasts coexpress PDGFRα, which further strengthen the evidence that single immunohistochemistry is not enough and can lead to misleading results when studying TCs by light microscopy. Since the expression of the stemness-related marker c-kit/CD117 has been reported in TCs from some tissues and organs, such as in the heart, skeletal muscles and female reproductive system 6,7,12,14,16,59 , we also double immunolabeled our human testicular sections for CD34/c-kit. Hence, we found that human testicular TCs are c-kit/CD117-negative, as previously documented in other human organs including the gastrointestinal tract and the skin 36,52 . Indeed, we could observe c-kit/CD117 immunoreactivity only in oval/round-shaped stromal cells, presumably Leydig cells and/or mast cells according to literature data 60,61 . At variance with a number of studies 7 , we considered useless to perform vimentin staining, as vimentin can be found almost in every connective tissue cell including fibroblasts, endothelial cells, myoblasts and tissue macrophages among others 62 . Noteworthy, following these in depth immunohistochemical investigations, transmission electron microscopy allowed us to detect cells satisfying the previously established ultrastructural criteria for the identification of TCs 6,20 in the same locations of the testis interstitium disclosed by the CD34/ PDGFRα double labeling. In addition, our electron microscopic observations highlighted that the peritubular and intertubular networks formed by typical telopodes establish intimate relationships with neighboring cells, such as myoid cells/myofibroblasts, endocrine Leydig cells, macrophages and blood capillaries.
Though we are aware of the descriptive/morphological design of the present study, the peculiar spatial TC distribution and their multiple intercellular connections detected in the whole testis stromal space, together with the current knowledge on the presumptive TC roles reported in a variety of organs 6,7,15,16,20,22,26,28,29,41 , allow us to make some interesting speculations on the possible functions of human testicular TCs. For instance, the continuous network of telopodes extending from the peritubular to the intertubular stroma might make a substantial contribution to the morphogenesis and maintenance of the normal three-dimensional architecture of testes. Interestingly, besides their contractile activity, it has been suggested that the peritubular myoid cells/myofibroblasts provide structural support to the seminiferous tubules and are components of the blood-testis barrier 50,63 . Owing to the close spatial association of the telopode network with both the peritubular myoid cells/myofibroblasts and blood vessels, it can be hypothesized that TCs are additional players in the abovementioned functions and likely they participate in the transfer of molecular elements from the interstitial bloodstream to the germinal compartment of seminiferous tubules, thus being potentially involved in the regulation of spermatogenesis. The present study also revealed the presence of numerous TCs that were located in the close vicinity of or even directly connected to Leydig cells by their telopodes, which may suggest that TCs are indirectly involved in the regulation of androgen hormone secretion and release. In this context, it is worth mentioning that the testicular TCs could correspond to the poorly characterized fibroblast-like cells randomly distributed in the intertubular space that have previously been referred to as 'compartmentalizing cells' (or 'Co-cells') by some authors 1,5 . Besides the similar tissue distribution, the fact that such 'Co-cells' were reported to express antigens characteristic for glial cells 1,64 and that TCs have been hypothesized to be microglial-like cells in a recent study 55 might, indeed, further support this assumption. According to several studies on TCs in different organs 6,7,16,[21][22][23][24][25][26][27][28]33 , the testicular TCs might be engaged in justacrine and/or paracrine signaling and deeply influence the behavior of neighboring cells either by direct junctions or indirectly by extracellular vesicle/exosome release, as here suggested by the numerous extracellular vesicles observed nearby telopodes in the testicular stroma. Taken into account that TCs have also been proposed to behave as 'hormonal sensors' in the human female reproductive organs (i.e. uterus and fallopian tube) because they express progesterone and estrogen receptors 42,44,46,65 , we cannot exclude an analogue function in the testes. Finally, other presumptive functions of testicular TCs could include a participation in local tissue immune surveillance, guidance of putative stem/progenitor cells and/or representing themselves a pool of tissue resident mesenchymal progenitors [30][31][32]66 .

Conclusions
In summary, our study provides the first comprehensive evidence that TCs are part of the microscopic anatomical structure of the human testis. Since these cells appear to be 'strategically' positioned forming networks that interplay with multiple cell types within the peritubular and intertubular stromal space, we consider that their possible implications in the physiological and pathological processes of the testes should not be further overlooked. Unveiling the roles exerted by testicular TCs, such as through the investigation of diseased tissues, has also the great potential to shed light on their possible therapeutic utility in the setting of different pathological conditions of human testes.

Methods
Human testis specimens. Paraffin-and epoxy resin-embedded normal human testis samples from 12 men aged 18-30 years were selected from the archives of the Section of Anatomy and Histology, Department of Experimental and Clinical Medicine, University of Florence. Archival orchidectomy specimens without evident histopathological lesions were obtained at autopsy (n = 10) or after surgical resection with informed consent (n = 2) as described elsewhere 67 . Immediately after sampling, testicular specimens were divided into small pieces and processed for light and transmission electron microscopy. The study was carried out in accordance with the Declaration of Helsinki and approved by the Hospital Committee for Investigation in Humans (Careggi University Hospital, Florence, Italy).

Histochemistry and immunohistochemistry.
Paraffin-embedded human testis sections (5 µm thick) were deparaffinized and subjected to either routine hematoxylin and eosin staining or immunoperoxidase-based immunohistochemistry for the CD34 antigen using the ready-to-use UltraVision™ Large Volume Detection System Anti-Polyvalent, HRP kit (catalog no. TP-125-HL; Lab Vision, Fremont, CA, USA) according to previously published protocols 17,19,37,39,54 . Briefly, after antigen retrieval in sodium citrate buffer (10 mM, pH 6.0)  sequentially with biotinylated secondary antibodies and streptavidin peroxidase solution (both from Lab Vision) followed by immunoreactivity development using 3-amino-9-ethylcarbazole (catalog no. TA-125-SA; Lab Vision) as chromogen and nuclear counterstaining with hematoxylin. Immunostained tissue sections were examined with a Leica DM4000 B microscope equipped with a Leica DFC310 FX 1.4-megapixel digital color camera and the Leica software application suite LAS V3.8 (Leica Microsystems, Mannheim, Germany).
Transmission electron microscopy. Transmission electron microscopy was carried out as detailed in previously published protocols 17,37 . Epon 812 resin-embedded human testicular specimens were cut with a RMC MT-X ultramicrotome (EMME3, Milan, Italy) and semithin sections (2 μm thick) were stained with a toluidine blue solution in 0.1 M borate buffer and examined under a light microscope. Ultrathin sections (~70 nm thick) of the selected areas were subsequently obtained using a diamond knife and stained with ready-to-use UranyLess solution (Electron Microscopy Sciences, Foster City, CA, USA) followed by an alkaline bismuth subnitrate solution. The stained ultrathin sections were observed and photographed by a high resolution digital camera connected to a JEOL JEM-1010 electron microscope (Jeol, Tokyo, Japan) 17,37 .

Data Availability
All relevant data are within the paper.