Telocytes play a key role in prostate tissue organisation during the gland morphogenesis

Abstract Telocytes are CD34‐positive interstitial cells, known to exert several functions, one of which is a role in tissue organisation, previously demonstrated by telocytes in the myocardium. The existence of telocytes in the prostate has recently been reported, however, there is a lack of information regarding the function of these cells in prostate tissue, and information regarding the possible role of these cells in prostatic development. This study used immunofluorescence techniques in prostate tissue and prostatic telocytes in culture to determine the relationship between telocytes and prostate morphogenesis. Furthermore, immunofluorescent labelling of telocytes was performed on prostate tissue at different stages of early postnatal development. Initially, CD34‐positive cells are found at the periphery of the developing alveoli, later in the same region, c‐kit‐positive cells and cells positive for both factors are verified and CD34‐positive cells were predominantly observed in the interalveolar stroma and the region surrounding the periductal smooth muscle. Fluorescence assays also demonstrated that telocytes secrete TGF‐β1 and are ER‐Beta (ERβ) positive. The results suggest that telocytes play a changing role during development, initially supporting the differentiation of periductal and perialveolar smooth muscle, and later, producing dense networks that separate alveoli groups and form a barrier between the interalveolar region and periurethral smooth muscle. We conclude that telocytes play a relevant role in prostate tissue organisation during postnatal development.


Introduction
Prostate development occurs as a result of a complex network of interactions between different molecular signalling pathways. These interactions initially occur between the epithelium of the urogenital sinus (UGE) and mesenchymal urogenital sinus (UGM), leading to the formation of epithelial buds derived from the UGE that invade the UGM. Prostate development begins with the epithelial-mesenchymal interaction of these buds with peripheral condensed mesenchyme, leading to branching, followed by differentiation of the proximal portions of the branches to form the conductive structures of the gland, the prostatic ducts and the distal portions that form the secretory structures, the prostate alveoli [1][2][3][4][5]. The occurrence of such molecular interactions are spatially compartmentalised between different cell types involved in prostate development, such as the progenitor cells of mesenchymal fibroblasts, smooth muscle and basal epithelial cells [6][7][8].
Telocytes differ from other interstitial cells, such as interstitial Cajal cells (ICCs) by their characteristic morphology mentioned above, in addition to the fact that they are CD34-positive cells. ICCs, described in organs of the digestive system, such as the stomach and intestine, possess pacemaker activity via the c-kit receptor [19][20][21], the ligand of which is the stem cell factor (SCF), that acts in a way to facilitate calcium mobilization via a src family kinase and PI3K (Phosphatidylinositol-4,5-bisphosphate 3-kinase) [22]. Telocytes may also exercise pacemaker function by means of the c-Kit, but may also possess a supportive function for the contraction of periductal smooth muscle, possibly by means of c-kit-independent pathways, such as via the SK3 (small conductance calcium-activated potassium Channel 3) pathway [20,23]. The function of telocytes varies between organs [8, 10-12, 17, 18, 24, 25], and their role in the prostate remains elusive, in addition to lack of information regarding the development of these cells in prostate tissue.
This study aimed to evaluate the presence of telocytes during prostate development, and the possible role of telocytes on prostate morphogenesis and tissue organisation in the Mongolian gerbil. This species of rodent is a promising model for studies involving the prostate, as unlike other laboratory rodents, a functional prostate is present in females. Furthermore, this species has greater sensitivity to hormone treatments [4,[26][27][28][29]. For this purpose, immunofluorescence assays were performed on prostatic tissue, in addition to cultured prostatic cells.

Animals and experimental design
The animals were provided by São Paulo State University (UNESP, São Jos e do Rio Preto). Gerbils were housed in a temperature-controlled (25°C) room on a 12 hrs light/dark cycle. All animals were housed in polyethylene cages, with ad libitum access to filtered water and rodent food. Animal handling and experiments were performed in accordance with the ethical guidelines of UNESP (ethics committee number 115/ 2015 CEUA). Ten adult female and 10 adult male gerbils (Meriones unguiculatus, Muridae: Gerbillinae), aged between 3 and 4 months, were mated. One male and one female were randomly matched to form independent litters. After birth, pups were split into five groups, four of which were used for immunofluorescence and ultrastructural analyses of postnatal prostate development in the gerbil at postnatal day 1 (P1), P7 (postnatal day 7), P14, P30 and P45 (n = 4 males in each group), whereas the sixth group (n = 5 males) formed the group killed at P120 for establishment of the telocyte primary cell culture. All animals were killed by lethal injection of a mixture containing ketamine as an anaesthetic (100 mg/kg body weight; Dopalen, Vetbrands, Jacare ı, Brazil) and xilazine as a muscle relaxant (11 mg/kg body weight; Rompun, Bayer, Brazil).
Isolation and primary culture of telocytes from prostate tissue The isolation and primary culture of TCs from prostate tissue were performed following the protocol described by Bei et al. [30]. After gerbils were killed, the prostate was dissected under sterile conditions and maintained in Hanks' balanced salt solution (HBSS, R21-022-CV; Corning, Corning, NY,USA) supplemented with 100 U/ml penicillin, 100 lg/ml streptomycin (R30-002-CI; Corning) and 0.01 mM HEPES (H3375; Sigma Aldrich, St.Louis, MO,USA). After being transported to the cell culture room and rinsed again with fresh HBSS, the prostates were transferred into a sterile culture dish containing Dubelcco's modified eagle medium nutrient mixture F12 (DMEM/F12; Sigma-Aldrich) supplemented with 0.25 mg/ml collagenase type II (17101-015; Invitrogen, Paisley, Renfrewshire,UK). The prostates were minced into 1 mm 3 pieces, transferred into a 50 ml centrifuge tube, then incubated on an orbital shaker at 37°C for 35 min. with the collagenase solution described above. Following the incubation, 25 ml of ice-cold HBSS was added into the digest to inhibit collagenase activity. The dispersed cells were separated from the non-digested tissue bypassing the mixture through a 40-lm diameter cell strainer, then cells were collected by centrifugation at 1000 r.p.m. for 5 min. at 4°C. The cells were washed once with HBSS, centrifuged, then resuspended in a solution containing 10 ml DMEM/F12 supplemented with 10% foetal bovine serum (FBS, 16000-044,Gibco, Waltham, MA, USA),100 U/ml penicillin and 100 lg/ ml streptomycin. Cells were seeded into sterile culture dishes and cultured in a humidified atmosphere of 5% CO2 at 37°C for 2 hrs to allow fibroblast attachment, in order to separate the fibroblasts from telocytes, as they adhere primarily to culture dish. Later, the unattached cells (containing TCs) were collected and cultured in DMEM/F12 supplemented with 10% FBS, 100 U/ml penicillin and 100 lg/ml streptomycin for 24 hrs, after which the culture medium was changed. Cell cultures were examined using an inverted biological microscope (IX50; Olympus, Tokyo, Japan) and TCs were photographed under 2009 magnification 48 and 96 hrs after seeding.

Ultrastructural analysis
Ultrastructural analysis was performed using the protocol described by Corradi et al. [8]. Fragments of the developing prostate of gerbils at three different ages (P1, P7 and P45) were minced into small pieces and fixed by immersion in 3% glutaraldehyde plus 0.25% tannic acid solution in Millonig's buffer, pH 7.3, containing 0.54% glucose for 24 hrs. After washing with the same buffer, samples were post-fixed with 1% osmium tetroxide for 1 hr, washed in buffer, dehydrated in a graded acetone series and embedded in Araldite resin. Ultrathin sections (50-75 nm) were prepared using a diamond knife and stained with 2% alcoholic uranyl acetate for 30 min. followed by 2% lead citrate in a 1 M sodium hydroxide solution for 10 min. Samples were evaluated by electron microscopy using a LEO -Zeiss 906 TEM at 80 kV.

Immunofluorescence for telocyte cell culture
Immunofluorescence of the telocyte cell cultures was performed using the protocol described by Bei et al. [30]. After three washes with PBS, telocytes were fixed in 4% paraformaldehyde for 30 min., washed with PBS, then permeabilised with 0.5% TritonX-100 for 30 min. Another wash with PBS was performed, after which cells were blocked with 3% BSA for 1 hr. After blocking, cells were incubated overnight at 4°C with ERb (rabbit polyclonal IgG, H-150, sc-8974; Santa Cruz Biotechnology), to evaluate whether this receptor would be expressed in prostatic telocytes, considering the importance of this receptor for late postnatal prostate development [5,32,33] and CD34 (polyclonal mouse, IgG, B-6, sc74499; Santa Cruz Biotechnology), which is the major marker for telocytes. Primary antibodies were diluted 1:100 in 1% BSA, after washing three times with PBS, cells were incubated with goat antimouse FITC-labelled (sc-2011; Santa Cruz Biotechnology) and goat anti-rabbit Texas Red-labelled (sc-2780; Santa Cruz Biotechnology) secondary antibodies diluted 1:200 in 3% BSA for 2 hrs, then stained with DAPI (F36924; Life Technology). Cells were kept in fresh PBS in the dark at 4°C prior to observation. Images were taken under 2009 magnification with a fluorescent inverted microscope (DMI4000 B;Leica). Similar procedures were used for the double immunofluorescence staining for CD34 (mouse polyclonal IgG; Santa Cruz Biotechnology) and TGF-b1 (rabbit polyclonal IgG; Santa Cruz Biotechnology).

Light microscopy
Phase-contrast microscopy showed the presence of telocytes after 48 hrs in primary culture, with shorter telopodes (Fig. 1A). The formation of telopode networks began after this period (Fig. 1B). Following 96 hrs of primary culture, telocytes showed long telopodes (Fig. 1C), and a telopode network was observed (Fig. 1D) as the monoliform aspect of the telopodes (Fig. 1E).

Ultrastructural analysis
The ultrastructure of the prostate of the neonates on P1 did not attest to the existence of telocytes in the peripheral region of the developing prostate budding, in this region cells with large cytoplasmic processes are verified, in which mitochondria can be seen ( Fig. 2 C-E). Differentiated telocytes with their telopodes of monoliform aspect alternating between thin fibrillar-like segments (arrows) and podoms (dilated, cisternae-like regions) are observed in the interstitium ( Fig. 2A and B). Later in the prostatic development, on P7, an intricate network of thick cytoplasmic processes that is interspersed with the developing smooth muscle cells can be observed, external to this, telocytes are verified, with their extremely thin telopodes that are arranged in a way to involve the developing smooth muscle layer (Fig. 3A, C, E). A cell with a pyramidal cell body with thick cytoplasmic processes surrounding periductal smooth muscle can be observed. In the cytoplasmic processes, mitochondria and dilated rough endoplasmic reticulum are verified, around these cytoplasmic processes it is possible to observe the large deposition of collagen fibrils ( Fig. 3B and D), such cells may constitute differentiating telocytes. Later, differentiated telocytes can be observed at the periphery of the prostatic alveoli, surrounding the differentiated periductal musculature ( Fig. 4E and F), telocytes are also observed forming a network of telopodes in the prostate interstitium ( Fig. 4A and D). In the periphery of some alveoli can also be seen cells similar to the telocytes with triangular cell body and cytoplasmic processes, such processes are thick and carry mitochondria and rough endoplasmic reticulum, in addition to vesicles ( Fig. 4B and C).

Immunofluorescence analysis
The double immunofluorescence assays for CD34/ERb and CD34/ TGF-b1 in telocytes after 96 hrs of primary culture showed the presence of CD34-positive cells and nuclear labelling for ERb ( D). CD34-positive cells in the telocyte culture also showed labelling for TGF-b1. The presence of ERb in the prostatic telocytes indicates that these cells possibly respond to a late pathway activated in prostatic development that leads to a reduction of proliferative activity and stimulates epithelial differentiation. Double immunofluorescence assays were also performed for CD34/CD31 to distinguish telocytes from blood vessels in the interacinar region, as both are CD34 positive. It was verified that blood vessels are positive for CD34 and CD31, and telocytes are exclusively CD34 positive, as well as telocytes show long CD34-positive telopodes demonstrating a morphology different from blood vessels CD34 staining (Fig. 6A-C).
Immunofluorescence assays for a-SMA in the histological sections of Mongolian gerbil prostate on different days of postnatal development were performed to characterise the developmental progression of the periductal and perialveolar muscles. Labelling for a-SMA (green) was observed at the periphery of the prostatic branches on smooth muscle progenitor cells in prostates collected at P3 (Fig. 7A, E and I). Labelling of a-SMA was observed in the developing periductal smooth muscle in the prostate alveoli on P7 (Fig. 7B, F and J). By P30, the periductal smooth muscle had already differentiated (Fig. 7C, G and K), and the lumen of the alveoli had expanded and periductal smooth muscle showed its characteristic conformation (Fig. 7D, H and L).
To evaluate the labelling pattern of the two main markers used for telocyte characterisation, immunofluorescence assays were performed for CD34 and c-kit in histological sections of the prostate of Mongolian gerbil on different days of postnatal development. The immunolabelling for CD34 was found to be dispersed in the early postnatal period, and progressed to concentrate in the periphery of the differentiating alveoli, coinciding with smooth muscle differentiation in the perialveolar region and later it was verified in the region between alveoli and in the region surrounding the periurethral smooth muscle(Figs 8A and E; 9A and E). The immunolabelling for c-kit was found disperse initially, and was arranged adjacent to the prostatic epithelium of the developing alveoli on P14 and P30, involving the periductal smooth muscle, as well as it was verified in the periurethral smooth muscle during this period (Figs 8B and F; 9B and F). Colocalisation of CD34 and c-kit was observed in the periductal region of some cells during early postnatal development of the prostate, and on P30 it was also seen in the perialveolar region. However, these factors do not colocalise on the stromal cells in the adjacent region of the periurethral musculature, as these cells were only positive for CD34. In the periurethral smooth muscle, most immunolabelled cells were c-kit positive, and the minority were CD34 positive or showed colocalisation of c-kit and CD34 (Fig. 9E-H). The possible relationship between telocytes and smooth muscle differentiation was evaluated using immunofluorescence assays for CD34 and TGF-b1 in histological sections of Mongolian gerbil prostate on different days of postnatal development. The telocytes progressively acquired their characteristic phenotype, with the formation of long CD34-positive telopodes, which were observed on the periphery of the developing prostate alveoli on P14. By P30, the telocytes had formed a network that spread to the interalveolar region ( Fig. 10A-C). The immunolabelling of TGF-b1 was concentrated in the periductal/perialveolar region during prostatic postnatal differentiation ( Fig. 10D-F). Colocalisation of CD34 and TGF-b1 labelling was found in the stromal cells of the periductal/perialveolar region throughout postnatal prostate differentiation, whereas a smaller degree of colocalisation was observed in the interalveolar region ( Fig. 10J-L).
The data from the immunofluorescence assays have been represented in a schematic (Fig. 11) that depicts prostate development and the possible role of telocytes.

Discussion
Prostate telocytes are thought to perform a supportive role in the contraction of periductal smooth muscle and in stromal compartmentalisation [8]. We have demonstrated that telocytes in cell culture are ERb positive, suggesting sensitivity to oestrogens, which are associated with differentiation of prostatic epithelium via the ERb signalling pathway, and with reduced stromal proliferation [32][33][34], thus, the telocytes are possibly involved in the developmental stage of morphological differentiation of the prostatic alveoli and ducts. Corroborating this hypothesis, we also found that telocytes produce TGF-b1 in primary culture, which is an antiproliferative paracrine factor involved in differentiation of the periductal and perialveolar smooth muscle [35,36].
In prostate tissue, our ultrastructural data demonstrated that, in the neonates, the telocytes are not yet verified in the periphery of the prostatic budding, they are verified only in the interstitium. At the end of the first week of postnatal life (P7), cells with thick cytoplasmic processes are seen interspersed in the periductal smooth muscle, possibly consisting of undifferentiated telocytes, such cells are surrounded by differentiated telocytes. On the onset of prematuration (P45), telocytes are seen surrounding the prostatic alveoli, cells resembling telocytes with thick cytoplasmic processes are also observed surrounding the prostatic alveoli, such data may indicate a possible supportive role of prostate telocytes on periductal smooth muscle differentiation.
In this sense, the immunofluorescence assays for a-Actin and CD34 corroborate the possible role of telocytes on smooth muscle differentiation at the tissue level, showing that these cells differentiate simultaneously with muscles, in which a-Actin immunolabelling initially disperse around the prostatic buds becomes concentrated on the thin layers of the differentiated perialveolar smooth muscle, at the same time, the immunolabelling for CD34 is verified surrounding the periductal/alveolar region in which smooth muscle differentiates. Moreover, telocytes in the prostatic tissue were also TGF-b1 positive, which validates the data obtained from cell culture, and supports the possible role of prostatic telocytes in the morphogenesis of the gland, particularly in the differentiation of periductal smooth muscle via secretion of TGF-b1. In addition to playing a direct role in prostate morphogenesis via paracrine signalling, prostatic telocytes may also contribute to prostate morphogenesis by stromal compartmentalisation. This would result in the formation of separate stromal microenvironments, as previously observed in the myocardium, in which telocytes are involved in the development of cardiomyocytes, and are spread among cardiomyocyte groups, thereby compartmentalising the myocardium [37].
Our data are suggestive of the formation of telocyte networks between the perinatal period and P30, which form potential barriers between stromal compartments in the prostate and occupy portions of the interalveolar region. Such telocyte networks separate the alveoli groups, as well as separating the interalveolar region from the periurethral smooth muscle, which is an important structure for the establishment of prostate tissue architecture, and consequently, its function. In addition, large deposition of collagen fibrils at the periphery of telopodes may indicate that telocytes, such as fibroblasts, may act directly in the production of extracellular matrix components. In terms of prostatic morphogenesis, our ultrastructural and immunofluorescence data indicate that the telocytes differentiate at different times among prostatic compartments, being present in the interstitium before they are present at the periphery of the prostatic budding and that such cells differentiate together with the smooth muscle cells in the periductal region, at the same time as telopode networks expand through the interalveolar region.
Immunolabelling for CD34 and c-Kit was performed for the detection of prostate telocytes, despite both being telocytes markers, the overlap of these factors in the prostate tissue varies along the developmental time and among the prostate regions. The existence of interstitial cells that were exclusively positive for c-Kit or CD34 labelling may indicate functional differences between such cells. C-kit had been established as a marker for interstitial cells, the ICCs, since before the characterization of telocytes [38]. The primary function assigned to these cells was a pacemaker function in the contraction of smooth muscle [19,20]. C-kit is a receptor that binds SCF, resulting in the mobilisation of calcium via a src family kinase and PI3K [22]. In addition to its functional importance, c-Kit is also essential for the differentiation of ICCs [39,40],as CD34 is a typical marker for a wide variety of blood progenitor cells [41], and more generally, a marker of undifferentiated cells in various tissues [42].
It has previously been proposed that CD34-positive fibroblast-like ICCs cells were progenitors of c-kit-positive ICCs in the intestine [43,44]. other functions as supporting stromal organisation in the interalveolar region. In the same sense, our ultrastructural data pointed to the existence of cells with thick cytoplasmic processes at the periphery of the periductal smooth muscle on P45, such cells possess triangular cell bodies and may consist of cells similar to ICCs. These cells were described in the prostate prior to the description of prostatic telocytes and to them were assigned the generic name of interstitial cajal-like cells (ICLCs) [45]. The characterization of telocytes was useful to avoid multiple ambiguous terminologies for CD34-positive fibroblastlike cells found in various organs, our data confirm the existence of fibroblast-like CD34 and CD34/c-Kit-positive cells, consisting of prostatic telocytes, however, the data also point to the existence of fibroblast-like cells c-Kit positive and CD34 negative [9][10][11][12][13][14][15][16][17][18], to which the canonical definition of telocytes is not applied. Moreover, in structural terms we obtain some evidence of the existence fibroblast-like cells that possess shorter and thicker cytoplasmic process at the periphery of the developing perialveolar smooth muscle, in which c-Kit-positive/CD34-negative cells are verified.
Moreover, the periurethral smooth muscle that differentiates earlier than periductal/alveolar smooth muscle showed predominantly c-Kit-positive cells, with small interspersed populations of CD34-positive cells and positive cells for both factors. This further supports the possible cell differentiation of telocytes into c-Kit-positive fibroblastlike cells similar to ICCs. These evidence are consistent with the data on the differentiation of ICCs, in which is demonstrated the existence of CD34-positive progenitors, which give rise to CD34 and c-Kit-positive cells and, finally, differentiate into exclusively c-Kit-positive mature ICCs [44,46,47].
The interalveolar region contained predominantly CD34-positive cells on P30, with the formation of networks that separate the clusters of alveoli from each other and separate clusters of alveoli from the periurethral smooth muscle, thus attributing to the exclusively CD34-positive interstitial cells uniquely a role of ICCs progenitor cells is a limited proposition, in view of the evidence that these cells possess many other functions in the tissue organisation and functionality in various organs [8, 10-12, 17, 18, 24, 25]. CD34-positive telocytes that do not express c-Kit may also perform a pacemaker function [48,49] via c-Kit-independent pathways, such as the SK3 pathway, by inhibiting electrical activity. This is unlike the stimulating pathways activated by c-Kit [20,23], and therefore may play a complementary role to the pacemaker activity exerted by c-Kit-positive telocytes or ICCs.
In general terms, our work supports that telocytes play a role in prostate morphogenesis and tissue compartmentalisation, as already demonstrated for myocardium [50], and these cells possibly have a supportive role in the differentiation of periductal/alveolar smooth muscle, as well as contributing by means of a telopode network to tissue compartmentalization of the prostatic stroma in different microenvironments and possibly producing components of the extracellular matrix. In addition, our data point to the existence of fibroblast-like cells in the prostate that are c-Kit positive and CD34 negative, which escape the typical immunolabelling profile of telocytes (CD34 or CD34/c-Kit positive) [9][10][11][12][13][14][15][16][17] And in the face of the immunolabelling profile of these factors during prostate development, in addition to some ultrastructural evidence, it is suggested that some telocytes (CD34 or CD34/c-Kit positive) present at the periphery of the developing smooth muscle of alveoli could differentiate into c-Kit-positive and CD34-negative fibroblast-like cells that resemble ICCs. However, further studies will be needed to test this hypothesis.