Introduction

Telocytes (TCs) were discovered by Popescu et al. in 2005 using transmission electron microscopy (TEM) and were named interstitial Cajal-like cells because their structure is similar to that of the interstitial cells of Cajal1. In 2010, researchers renamed them “telocytes” based on their morphology using the Greek suffix “telos”2. The TCs have diverse morphologies, such as spindle, pear, and pike3, and unique cell extensions called telopodes (TPs) that distinguish them from other cells4. The TPs extend from the cell body with fine or expanded segments enriched with mitochondria, endoplasmic reticulum, and secretory vesicles that form mesenchymal networks that facilitate intercellular communication2,5. It is currently believed that TCs play a role in cellular signal transduction6, maintenance of tissue homeostasis7, and inhibition of cellular senescence8, which are involved in the development of different diseases9.

For many years, studies on TCs have been mainly based on the observation of their ultrastructure by TEM; thus, TEM remains the “gold standard” for distinguishing TCs from other interstitial cells10,11. Immunofluorescence (IF) and immunohistochemistry (IHC) are key approaches for TCs identification. The most commonly used markers are stem cell growth factor receptor (C-Kit, also known as CD117), vimentin and platelet derived growth factor receptor α (PDGFRα). The CD117 is a marker of stem cells in the heart12 and is considered an effective marker for TCs13. Thus, the CD117 expression by TCs suggests that it may be associated with the haematopoietic system. Vimentin, a marker of interstitial cells, is an important cytoskeletal protein closely associated with tumour cell growth, invasion, and metastasis14. The PDGFRα is a tyrosine kinase receptor, both of which are widely expressed in mesenchymal cells, often concurrently in TCs15.

In the male reproductive system, Liu et al.16 found that TCs in rat testis form an interactive network connecting peritubular myoid cells and blood vessels, suggesting they have roles similar to those of peritubular myoid cells, such as forming the blood–testis barrier and sperm transport. Pawlick et al.17 found that TCs in mouse testis are always present in the interstitial of the testis and perivascular regions, considered ‘connecting cells’, primarily involved in intercellular signalling. Our previous study identified yak epididymal TCs and speculated that TCs may be a kind of ‘material exchange pump’, which plays an important role in the exchange of material and energy between the epididymal interstitial and trophic blood vessels, and may be related to sperm processing and maturation10.

Tibetan sheep mainly live in the perennially high-cold and low-oxygen regions of the Tibetan Plateau and maintain normal fertility in the harsh natural environment18. The testis and epididymis are important organs in the reproductive system of male animals, and the regulatory roles of different cells are particularly critical. Therefore, the present study was conducted to identify the morphological characteristics of TCs in the testis and epididymis of Tibetan sheep, analyse their ultrastructure and distribution location, explore the functions of TCs in the reproductive system of plateau animals, and provide new clues for analysing the biological roles of TCs in the low-oxygen environment of the plateau.

Results

TEM analysis of TCs

The TEM is a key method for identifying TCs. The TEM revealed that TCs were irregularly shaped, with long shuttled and ellipsoid forms, large nuclei, and clear nucleoli in the testis of the Tibetan sheep. The TCs were distributed outside the basement membrane, the TPs contained alternating dilated and narrow segments, and many secretory vesicles extended outwards (Fig. 1A–C). The cytoplasm of TCs contain secretory vesicles, mitochondria, and endoplasmic reticulum (Fig. 1D). Furthermore, the TCs contact capillaries and mesenchymal cells through TPs, forming special cellular connections (Fig. 1B–C). The TCs found in the epididymis of Tibetan sheep exhibited large nuclei, long TPs, and a significant number of secretory vesicles adjacent to the TPs (Fig. 2A–C). The TCs of the caput epididymis were distributed near the blood vessels; they were pear-shaped and had distinct TPs connected to other mesenchymal cells (Fig. 2A). The TCs of the corpus epididymis were distributed outside the basement membrane; they were spindle-shaped and had particularly elongated TPs that formed tight junctions with the epithelial cells (Fig. 2B). The TCs of the cauda epididymis were densely packed around blood vessels outside the basement membrane, located next to numerous secretory vesicles and had multiple TPs of varying thicknesses. The TCs extend to blood vessels and fibroblasts through TPs, which may form a unique network structure (Fig. 2C).

Fig. 1
figure 1

TEM photograph of telocytes (TCs) in Tibetan sheep testis. (A) The ultrastructure of TCs reveals large nucleus and dendritic TPs lined with numerous secretory vesicles (bar = 5 μm). (B) The ultrastructure of TCs, which are distributed outside the basement membrane, is characterised by large and serrated nucleus. The TPs, which comprise distinct expanded and narrow segments, contain secretory vesicles and mitochondria (bar = 10 μm). (C)The ultrastructure of TCs is characterised by long shuttle-shaped cells with elongated TPs and large nucleus. Secretory vesicles are located adjacent to the TPs (bar = 5 μm). (D) The cytoplasmic ultrastructure of TCs is enriched with secretory vesicles, mitochondria, and endoplasmic reticulum (bar = 2 μm). Nu: nucleus, SV: secretory vesicles, m: mitochondria, ER: endoplasmic reticulum, CAP: capillary, Ley: Leydig cell.

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Fig. 2
figure 2

TEM photograph of TCs in Tibetan sheep epididymis. (A) Ultrastructure of TCs in the caput epididymis is pear-shaped, and is found near blood vessels. Secretory vesicles are present within TPs (bar = 10 μm). (B) The ultrastructure of TCs in the corpus epididymis is distributed in the outer interstitium of the basement membrane. The TCs is long spindle-shaped cell with large nucleus and elongated TPs (bar = 10 μm). (C) The ultrastructure of TCs in the cauda epididymis, which is distributed around blood vessels, comprise multiple TPs and many secretory vesicles around the TPs (bar = 1 μm). Nu: nucleus, SV: secretory vesicles, m: mitochondria, BV: blood vessel, Fb: fibroblast.

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Distribution pattern of TCs

The distribution patterns of the testicular and epididymal TCs in Tibetan sheep were mapped based on TEM observations (Fig. 3).

Fig. 3
figure 3

Distribution pattern of TCs in Tibetan sheep testis and epididymis based on the TCs found in this study. (A) Distribution pattern of testis TCs. The TCs located outside the seminiferous tubules, near the basement membrane and blood vessels, the TCs is long shuttle-shaped with a large nucleus, and the TPs are elongated and surrounded by numerous secretory vesicles. (B) Distribution pattern of epididymis TCs. The TCs is irregularly flattened with multiple TPs of varying thicknesses. There are secretory vesicles excreted next to the TPs, and some TPs extend into blood vessels, possibly participating in substance exchange. TCs: telocytes, SC: Sertoli cells, PMC: peritubular myoid cells, SPG: spermatogonial cell, PSC: primary spermatocyte cell, SSC: secondary spermatocyte cell, SZ: spermatozoa, Mp: macrophages, Ley: Leydig cell, Fb: fibroblast, BV: blood vessel, SV: secretory vesicles, PC: principal cell, m: mitochondria, ER: endoplasmic reticulum. This figure was drawn using MedPeer (www.medpeer.cn).

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Toluidine blue staining analysis of TCs

Microscopic observation revealed interstitial cells with morphological features of TCs in the interstitium of the testis and epididymis of the Tibetan sheep. These cells had thin and elongated cytoplasmic protrusions stained pale blue with toluidine blue (Fig. 4A–D). They are distributed close to the interstitial capillaries and near the basement membrane, and most are spindle-shaped.

Fig. 4
figure 4

Toluidine blue staining of Tibetan sheep testis and epididymis (bar = 50 μm). (A) Testis. (B) Caput epididymis. (C) Corpus epididymis. (D) Caudal epididymis. The red arrow refers to TCs, which are distributed near the interstitial capillary.

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Immunohistochemical analysis of TCs markers

The immunohistochemical results showed that PDGFRα and CD117 were mainly expressed in spermatogonia and Sertoli cells (Fig. 5A1, A2). Vimentin is strongly positively expressed in Sertoli cells in addition to positively expressed in interstitial cells (Fig. 5A3). In the caput epididymis, PDGFR α was expressed positively mainly in the interstitial capillary, and CD117 was expressed positively mainly in epithelial cells. and mesenchymal cells (Fig. 5B1, B2). Vimentin was positively expressed in mesenchymal cells (Fig. 5B3). In the corpus and cauda of epididymis, PDGFRα, CD117, and vimentin were positively expressed in mesenchymal cells, epididymal micro vessels and basement membrane (Fig. 5C1–D3). Each marker identified positive cells shaped similar to TCs around interstitial micro vessels or near the basement membrane. Combined with the location of TCs distribution in TEM, they were identified as TCs. Statistics on the distribution density of TCs markers in different cells of testis and epididymis of Tibetan sheep revealed that PDGFRα was strongly and positively expressed in vascular endothelial cells, CD117 was medium-intensity positively expressed in vascular endothelial cells, and both showed medium-intensity positive expression in Sertoli cells, TCs, and only weak positive expression in Leydig cells and principal cells of the epididymis. Vimentin exhibited strong positive expression in Sertoli cells and vascular endothelial cells, with medium-intensity positive expression in Leydig cells and TCs, and only weak positive expression in principal cells (Table 1).

Table 1 Distribution densities of TCs markers in different cells of tibetan sheep +: weak positive expression. ++: medium intensity positive expression. +++: strong positive expression.
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Fig. 5
figure 5

Immunohistochemical results of Tibetan sheep testis and epididymis (bar = 50 μm). (A1–D1): The immunohistochemical results for PDGFRα in testis and epididymis. Strong, positive expression for PDGFRα in testicular and epididymal vessels. (A2–D2): The immunohistochemical results for CD117 in testis and epididymis. CD117 were positively expressed in testicular and epididymal interstitial cells and basement membrane. (A3–D3): The immunohistochemical results for vimentin in testis and epididymis. Strong, positive expression of vimentin in testicular Sertoli cells, epididymal mesenchymal cells, basement membranes, and epididymal capillary. Red arrows indicate TCs.

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Double IF analysis of TCs markers

The results of the double IF test observed by fluorescence microscopy showed that CD117 was extensively expressed in spermatogonia and Sertoli cells (Fig. 6A1). In the epididymis, CD117 was strongly expressed in the basement membranes, interstitial cells, and capillaries (Fig. 6A2–A4). Vimentin exhibited strong positive expression in Sertoli cells in the testis (Fig. 7A1) and was widely expressed in the interstitial cells and blood vessels in the epididymis (Fig. 7A2–A4). PDGFRα was mainly expressed in epithelial cells and basement membrane fractions of the testis and epididymis, and especially shows strong positive expression in blood vessels (Figs. 6B1–B4, 7B1–B4). The nuclei of the testis and epididymis were stained blue by 4,6-diamidino-2-phenylindole (DAPI) (Fig. 6C1–C4, 7C1–C4). The co-expression of PDGFRα + CD117 and PDGFRα + vimentin in an orange-yellow colour (Fig. 6D1–D4, 7D1–D4). These cells exhibit distinct cytoplasmic protrusions and vary in morphology. The distribution and morphological features are consistent with the TEM results.

Fig. 6
figure 6

Double IF results of Tibetan sheep testis and epididymis with CD117 and PDGFRα (bar = 50 μm). (A1–A4): The IF results for CD117 in testis and epididymis. (B1–B4): The IF results for PDGFRα in testis and epididymis. (C1–C4): The IF results for DAPI in testis and epididymis. (D1–D4): The double IF co-expression results for CD117 + PDGFRα in testis and epididymis. The TCs were co-expression around interstitial capillary in the testis and epididymis. White arrows indicate TCs.

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Fig. 7
figure 7

Double IF results of Tibetan sheep testis and epididymis with vimentin and PDGFRα (bar = 50 μm). (A1–A4): The IF results for vimentin in testis and epididymis. (B1–B4): The IF results for PDGFRα in testis and epididymis. (C1–C4): The IF results for DAPI in testis and epididymis. (D1–D4): The double IF co-expression results for vimentin + PDGFRα in testis and epididymis. The TCs were co-expressed near the basement membrane of the testis and epididymis. White arrows indicate TCs.

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Discussion

The presence of TCs has been reported in many studies, including the human vaginal mucosa19, mare oviduct20, and human kidney cortex21. We have reported two types of TCs in the yak epididymis; one of which types was closely related to epididymal angiogenesis and microenvironmental substance transport through longer TPs and many secretory vesicles; the other TCs were close to the extra-basal membrane and took the form of long, irregular stripes; shorter TPs were associated with local myxoid cells and participated in the contractile activity of the epididymal ducts, which provided the driving force for spermatozoa10. Therefore, the TCs are important matrix components in the yak epididymis, and their potential impact on tissue homeostasis deserves further investigation.

The TEM is the most authoritative method for identifying TCs and confirming the formation of network structures between TCs and mesenchymal cells, which can be observed in testicular mesenchymal tissue22. The TCs form close associations with peritubular myoid cells, fibroblasts, mesenchymal stromal cells, blood vessels, and extracellular vesicles (EVs) through the TPs provided for connectivity16. The TCs reportedly shed EVs that may communicate with neighbouring cells, including Leydig cells and peritubular myoid cells, in humans and softshell turtles23,24. We found that the TCs in the testis of Tibetan sheep were mainly long shuttle-shaped, and were extended with abundant adjacent secretory vesicles, as well as connected to Leydig cells and capillaries through the TPs, which may be involved in androgen transport. In the caput and corpus of the epididymis, the TCs appear pear-shaped, with long TPs having abundant secretory vesicles adjacent to the basement membrane and being intimately connected to the local epithelium, which may be involved in the secretion of epididymal fluid and contractile activity of the epididymal ducts that provide protection for sperm transport. In the cauda epididymis, the TCs have multiple TPs of varying thicknesses that extend to the vascular wall and fibroblasts, which may act as bridges to facilitate the transport of mature spermatozoa. The TCs play multiple roles in organisation, including developmental and postnatal environmental stabilisation, regulation of paracrine intercellular signalling, immunosurveillance, and reparative or regenerative effects by supporting local stem cell maintenance and differentiation. In this study, the TCs network was observed in the peripheral tissues of seminiferous tubules as well as around mesenchymal cells and blood vessels. Further investigation is required to determine its potential impact on intratissue homeostasis.

The TPs can be dyed blue easily using toluidine blue staining, whereas traditional dyeing methods are difficult to implement5,25,26. Consistent with the IHC and IF results, the TCs were observed in the testis and epididymis by toluidine blue staining, they exhibited typical cellular characteristics near the basement membrane and interstitial capillaries.

Although there is currently no single specific marker for TCs, several surface markers have been reported to identify TCs27,28, such as CD117, vimentin, and PDGFRα. We used IHC to identify positive cells for CD117, vimentin, and PDGFRα in the interstitial tissue of testis and epididymis. However, a single immunohistochemical method is insufficient to identify TCs. Therefore, we used double IF to distinguish TCs from other cell types. The vimentin + PDGFRα and CD117 + PDGFRα double-positive cells were found close to the interstitial capillary and basement membrane, characterised by large nuclei and distinct cytoplasmic protrusions. The morphological structure and distribution location were similar to those reported for human29, rabbit30, camel31, and yak10 testicular and epididymal TCs. Combined with the TEM results, they were identified as TCs.

The biological functions of TCs have been reported, such as regulation of intratissue homeostasis, intercellular signalling, and immune surveillance6,7. The CD117 is generally considered a marker protein for TCs because of its stem cell properties. Our results showed that the TCs and Sertoli cells expressed PDGFRα, vimentin, and CD117 with medium-intensity to strong positivity, respectively (Table 1). The PDGFRα is a receptor tyrosine kinase that plays important biological roles in regulating spermatogenesis, angiogenesis, and embryonic development32,33. The PDGFRα has been shown to promote cell proliferation in Sertoli cells and further promote spermatogenesis34. The CD117 is a stem cell factor receptor, and high expression of CD117 is common in hematopoietic stem cells35. The CD117 is important for the migration, proliferation, and survival of spermatogonia36. The PDGFRα and CD117 were medium-intensity positive expression in TCs, and combined TCs were always found near blood vessels, suggesting a possible association with angiogenesis and material transport. Vimentin is a crucial cytoskeletal protein that plays a essentialrole in cell migration and division37. Strong vimentin expression was observed in Sertoli cells, as it is the main structural protein of Sertoli cells, providing its good cytoskeleton, and maintaining the structure of its spermatogenic epithelium38. In this study, vimentin was moderately expressed in TCs, which may facilitate the formation of heterologous cell junctions between TCs, fibroblasts, and peritubular myoid cells via TPs. This junction potentially forms a TCs network that acts as a bridge for interactions with other cells.

The EVs are membrane-bound particles released by different cells and transport bioactive substances, proteins, lipids, and nucleic acids to regulate a wide range of biological functions29. Testicular EVs can pass through the blood–testis barrier and mediate inter-compartment communication between the seminiferous tubules and interstitium23. The TCs in camel efferent ductules have been reported to have more vesicles in spring and fewer vesicles in summer, suggesting that the correlation between TCs and oestrus in animals draws more attention to the link between TCs and animal oestrus5. The EVs characteristics provide ideas for studies exploring the link between TCs and oestrus in animals, granting a research basis for continuing to study TCs in comparison to oestrus or specific functions in the male testis and epididymis pre- and post-sexual maturity.

Conclusions

We demonstrated for the first time that the TCs present in the testis and epididymis of Tibetan sheep, their distribution and morphology exhibited typical TEM and biomarker characteristics of TCs, and a considerable number of secretory vesicles were present in the vicinity of TPs. The TCs may be involved in the composition of the blood–testis and blood–epididymis barriers and play a role in the stability of the microenvironment and transport of spermatozoa. The findings of this study provide new insights into the potential role of TCs in the reproductive system of plateau animals and establish a novel foundation for further investigation of the biological properties of TCs in low-oxygen environments.

Methods

Animals

The testis and epididymis of 2-year-old male Tibetan sheep (n = 8) were collected from the designated slaughterhouses in Xining City, Qinghai Province, China. The samples were removed by testicular castration according to ethical principles, and the epididymis was divided into the caput, corpus, and cauda based on anatomical and structural characteristics. All experimental animals were approved by the Animal Care and Use Committee of the Veterinary College of Gansu Agricultural University (ratification number: GSAU-Eth-VMC-2022-017), and all experiments were performed in accordance with the relevant guidelines and regulations.

Main drugs and reagents

All the experimental antibodies were purchased from commercial suppliers. Rabbit polyclonal antibodies against CD117(bs-10005R), vimentin (bs-0756R), and an IHC test kit (PV-0023) were purchased from Beijing BIOSS Antibodies, Ltd. (Beijing, China). Mouse monoclonal antibody PDGFRα (14-1401-82) from Thermo Fisher Scientific Inc. (Waltham, USA). Fluorescent secondary antibodies (ab150113, Alexa Fluor®488; ab150079, Alexa Fluor®647) were purchased from Abcam (Cambridge, UK). The DAB colour reagent kit (PA110) was provided by Beijing TIANGEN Biotechnology Ltd. (Beijing, China).

Transmission electron microscopy

The testis and epididymis tissues samples of Tibetan sheep fixed in 2.5% glutaraldehyde were divided into 0.5 × 0.5 × 0.5 cm pieces, rinsed in phosphate buffer solution (PBS) and fixed in 1% osmium acid for 3 h. The tissues were washed six times with PBS for 10 min each. The pieces were dehydrated using graded acetone, followed by overnight immersion in a mixture of Spurr epoxy resin and acetone. The tissues were embedded in epoxy resin and sectioned into ultrathin slices of 50 nm using an ultrathin sectioning machine. The slices were attached to a copper mesh and stained with 1% uranyl acetate and lead citrate for 20 min. The processed specimens were examined under a transmission electron microscope.

Toluidine blue stain

Paraffin sections were routinely deparaffinised and dehydrated, then stained with a drop of 50 µL of toluidine blue staining solution for 20 min, rinsed slightly in running water. Subsequently, the sections were stained with 95% ethanol under the microscope with controlled colour separation, gradient alcohol dehydration, xylene transparency, and sealed with neutral resin, and the results were observed under the microscope and images were captured.

Immunohistochemistry

The Tibetan sheep testis and epididymis tissue samples were fixed in 4% paraformaldehyde, embedded in paraffin, and cut into 4-µm thin sections. The tissue sections were deparaffinised and dehydrated using decreasing concentrations of ethyl alcohol (100, 100, 95, 95, 85, and 75%). The antigen was then repaired with citrate at 80℃ for 15 min. Then, the sections were blocked by 3% hydrogen peroxide for 15 min at 37 °C. The specimens were then blocked with 5% goat serum albumin at 37 °C for 30 min, followed by incubation with rabbit antibody CD117, vimentin (1:400), and mouse antibody PDGFRα (1:400) antibodies at 4 °C for 12 h. After washing three times with PBS, the sections were incubated with biotinylated goat anti-rabbit or goat anti-mouse IgG for 15 min at 37 ℃. Horseradish enzyme-labelled streptavidin solution was added, and the cells were washed with PBS three times. The DAB colour-developing solution was added for 5 min. Nuclei were stained with haematoxylin. The sections were observed under a microscope.

Double immunofluorescence

IHC was repeated until the primary antibody was added. Add proportionally mixed antibody PDGFRα + CD117 and PDGFRα + vimentin, then incubate overnight at 4 °C. Subsequently, after washing with PBS, samples were incubated with fluorescent secondary antibodies AF488 and AF647 (1:800) at 37 °C for 1 h. Nuclei were stained with DAPI (1:1000). The sections were then rinsed with PBS and sealed using an anti-fluorescence-quenching sealer. Finally, images were captured using a fluorescence microscope.

Statistical analysis

Six different fields of view (1000×) were randomly selected for each IHC section, analysing the distribution density of CD117, PDGFR α, and vimentin by semi-quantitative analysis.