**3. Sex steroids and TCs**

96 Sex Steroids

Fig. 9. The schematic drawing of a podom (blue), the dilated portion of a telopode. Note the

Fig. 10. Digitally coloured TEM image shows TC (blue) in human subepicardium, bordering the peripheral cardiomyocytes (CM, highlighted in brown). The TC has three telopodes, illustrating: a) the distinctive dichotomous pattern of branching (arrows); b) Tp are very thin at the emergence from the cell body; c) alternating podoms and podomeres. Note that some portions of podomeres have the same thickness as collagen fibrils, which makes observation

under light microscopy impossible. E – elastin. Scale bar - 2 µm. Reproduced, with

permission, from Popescu et al., 2010b.

podomic endoplasmic reticulum in yellow and the mitochondria in red.

**Branching**, with a dichotomous pattern (Figure 10);

The "sex hormones"— estrogens, progesterone, and androgens—are a special category of steroids. Their actions are mediated by intracellular receptors, generally known as nuclear receptors, acting as ligand regulating transcription factors (slow genomic mechanisms) as well as by membrane-associated receptors and signaling cascades (fast nongenomic mechanism) (Giretti & Simoncini, 2008; Tetel et al., 2009). Sex steroids are involved in the regulation of many functions in human organism, including reproduction and behaviour.

Female genital organs, especially those directly involved in ovum fertilization and embryo implantation - fallopian tubes and uterus - are highly influenced by sex steroids.

Fig. 11. Electron microscopy of non-pregnant human uterus. Note the telocyte covering smooth muscle cells (M). The telopode is digitally coloured in blue, marked with asterisks. Image obtained in 2006. Courtesy of Prof. M. Taggart (Newcastle University, UK) and Dr. Carolyn J.P. Jones (Manchester University, UK).

Connective tissue filling the space between epithelial, muscular and nerve tissue, found in the walls of these organs, abound in different cells. They are generally referred to as stromal

Telocytes in Human Fallopian Tube and Uterus Express Estrogen and Progesterone Receptors 99

Uterine TCs display distinct features which avoid possible confusion with other types of interstitial cells. Methylene blue staining and Golgi impregnation, which was used for the first time by Cajal in 1892 (for ICC identification) are also necessary for identification of TC

Fig. 13. Human myometrium. A. Methylene blue vital staining, before cryofixation (cryosectioning). Note the selective affinity of a telocyte for the blue dye. B. Silver

and intestine, 177-181, Copyright (2006), with permission from Elsevier..

(Figure 16).

impregnation after fixation and paraffin embedding. A pyriform telocyte with a very long, moniliform process. Original magnification: 1000x. Reprinted from European Journal of Pharmacology, 546, L. M. Popescu, C. Vidulescu, A. Curici,L. Caravia, A. A. Simionescu, S. M. Ciontea, S. Simion, Imatinib inhibits spontaneous rhythmic contractions of human uterus

At the myometrial level, TCs establish, through their TPs, vicinity relationships with capillaries and nerve fibers, as well as specialized contacts with other interstitial cells (e.g. macrophages, mast cells, lymphocytes, eosinophils) (Popescu et al., 2005b) (Figure 15). TCs interconnect with each other and with smooth muscle cells (SMC) through cell-to-cell point contacts or gap junctions. Interestingly, we found in uterine myocytes typical 'Ca2+ release units (caveolae, sarcoplasmic reticulum and mitochondria) in the vicinity of gap junctions

presence at tissue level or in cell culture (Figures 13, 14).

cells and are subclassified into fibroblasts, fibrocytes, myofibroblasts, interstitial cells, and mesenchymal cells. In recent years we describe a novel cell type – TCs – with a completely different silhouette. TCs are unequivocally recognized under transmission electron microscope on the basis of their most peculiar feature: TCs have extremely long prolongations, with a very thin and moniliform aspect (Figure 11).

Our laboratory was the first to describe the presence of sex steroid hormone receptors in TCs (D. Cretoiu et al., 2006, S.M. Cretoiu et al., 2009). TCs for cell cultures were obtained from the muscle coat of the fallopian tube and uterus, and analyzed by immunohistochemistry using monoclonal antibodies to determine the presence of estrogen receptor alpha (ERα) and progesterone receptor (PR). TCs were enriched in primary culture by magnetically-activated cell sorting. The magnetic beads conjugated with goat anti-mouse IgG were incubated with monoclonal anti human CD117, considered to be specific for TCs. The cell suspension was then incubated with the magnetic beads and the supernatant was collected as the negative fraction. Culture medium was added to collect the remaining cells and the tube was removed from the magnet. This was considered as the positive fraction. We obtained 1.8 × 105 cells in the positive fraction and 4.8 × 106 cells in the negative fraction. After 9 days, cells grown on coverslips, in primary culture underwent subsequent examination.

## **3.1 TCs as steroid hormone sensors in human myometrium**

TCs have been described in human uterine tissue under different names since 2004 : c-kitpositive cells (Shafik et al., 2004), m-CLIC (Ciontea et al., 2005), Vimentin-positive, c-kitnegative interstitial cells (Duquette et al., 2005), ICLC (Popescu et al., 2007; Hutchings et al., 2009). Our group found that myometrial TCs possessed very long cytoplasmic processes which, by *in vitro* Janus green B staining, were shown to contain numerous mitochondria. (Ciontea et al., 2005). TCs represented approximately 7% of the total cell number on random semi-thin myometrial tissue sections (Figure 12) stained with toluidin blue (Popescu et al., 2006).

Fig. 12. Pregnant human myometrium (39 weeks of gestation). Semi-thin sections (0.5 - 1 µm thick) of uterine muscular layer embedded in Epon resin and stained with toluidine blue. Note the very long process of the TC squeezing between obliquely cut smooth muscle cells. Original magnification 100x. Reproduced, with permission, from Hutchings *et al.*, 2009.

cells and are subclassified into fibroblasts, fibrocytes, myofibroblasts, interstitial cells, and mesenchymal cells. In recent years we describe a novel cell type – TCs – with a completely different silhouette. TCs are unequivocally recognized under transmission electron microscope on the basis of their most peculiar feature: TCs have extremely long

Our laboratory was the first to describe the presence of sex steroid hormone receptors in TCs (D. Cretoiu et al., 2006, S.M. Cretoiu et al., 2009). TCs for cell cultures were obtained from the muscle coat of the fallopian tube and uterus, and analyzed by immunohistochemistry using monoclonal antibodies to determine the presence of estrogen receptor alpha (ERα) and progesterone receptor (PR). TCs were enriched in primary culture by magnetically-activated cell sorting. The magnetic beads conjugated with goat anti-mouse IgG were incubated with monoclonal anti human CD117, considered to be specific for TCs. The cell suspension was then incubated with the magnetic beads and the supernatant was collected as the negative fraction. Culture medium was added to collect the remaining cells and the tube was removed from the magnet. This was considered as the positive fraction. We obtained 1.8 × 105 cells in the positive fraction and 4.8 × 106 cells in the negative fraction. After 9 days, cells grown on

TCs have been described in human uterine tissue under different names since 2004 : c-kitpositive cells (Shafik et al., 2004), m-CLIC (Ciontea et al., 2005), Vimentin-positive, c-kitnegative interstitial cells (Duquette et al., 2005), ICLC (Popescu et al., 2007; Hutchings et al., 2009). Our group found that myometrial TCs possessed very long cytoplasmic processes which, by *in vitro* Janus green B staining, were shown to contain numerous mitochondria. (Ciontea et al., 2005). TCs represented approximately 7% of the total cell number on random semi-thin myometrial tissue sections (Figure 12) stained with toluidin blue (Popescu et al.,

Fig. 12. Pregnant human myometrium (39 weeks of gestation). Semi-thin sections (0.5 - 1 µm thick) of uterine muscular layer embedded in Epon resin and stained with toluidine blue. Note the very long process of the TC squeezing between obliquely cut smooth muscle cells. Original magnification 100x. Reproduced, with permission, from Hutchings *et al.*, 2009.

prolongations, with a very thin and moniliform aspect (Figure 11).

coverslips, in primary culture underwent subsequent examination.

**3.1 TCs as steroid hormone sensors in human myometrium** 

2006).

Uterine TCs display distinct features which avoid possible confusion with other types of interstitial cells. Methylene blue staining and Golgi impregnation, which was used for the first time by Cajal in 1892 (for ICC identification) are also necessary for identification of TC presence at tissue level or in cell culture (Figures 13, 14).

Fig. 13. Human myometrium. A. Methylene blue vital staining, before cryofixation (cryosectioning). Note the selective affinity of a telocyte for the blue dye. B. Silver impregnation after fixation and paraffin embedding. A pyriform telocyte with a very long, moniliform process. Original magnification: 1000x. Reprinted from European Journal of Pharmacology, 546, L. M. Popescu, C. Vidulescu, A. Curici,L. Caravia, A. A. Simionescu, S. M. Ciontea, S. Simion, Imatinib inhibits spontaneous rhythmic contractions of human uterus and intestine, 177-181, Copyright (2006), with permission from Elsevier..

At the myometrial level, TCs establish, through their TPs, vicinity relationships with capillaries and nerve fibers, as well as specialized contacts with other interstitial cells (e.g. macrophages, mast cells, lymphocytes, eosinophils) (Popescu et al., 2005b) (Figure 15). TCs interconnect with each other and with smooth muscle cells (SMC) through cell-to-cell point contacts or gap junctions. Interestingly, we found in uterine myocytes typical 'Ca2+ release units (caveolae, sarcoplasmic reticulum and mitochondria) in the vicinity of gap junctions (Figure 16).

Telocytes in Human Fallopian Tube and Uterus Express Estrogen and Progesterone Receptors 101

Fig. 15. Rat myometrium: TEM; original magnification 7100x. A multi-contact synapse (MS) between a telocyte and an eosinophil, in the neighbourhood of smooth muscle cells (SMC); m, mitochondria; N, nucleus; db, dense bodies; Note presence of mitochondria (\*) in the synaptic vicinity, typical of chemical synapses. Reproduced, with permission, from Popescu

Fig. 16. Digitally-coloured TEM image of a TC in rat myometrium: TC (blue), smooth muscle cells (Sienna brown). Note the 'Ca2+-release units' (caveolae, sarcoplasmic reticulum and mitochondria) in the cytoplasmic region where smooth muscle sarcolemma comes into close contact with TC plasmalemma. Original magnification: x15000. SMC = smooth muscle cells;

Ht = heterochromatin; Eu = euchromatin; rER = rough endoplasmic reticulum; SR = sarcoplasmic reticulum; m = mitochondria; cav = caveolae (arrowheads). Reproduced from

et al., 2005b.

Ciontea et al., 2005.

Fig. 14. Photographic reconstruction of human TCs in culture establishing contact with smooth muscle cells. From our experience, silver impregnation is one of the choice methods for revealing the typical moniliform aspect of TCs in culture. Inset- the same interlaced distribution of TCs (\*) using methylene blue vital staining. Both methods reveal weaker myocyte staining. Scale bar 10 µm. Reproduced, with permission, from Cretoiu et al., 2006.

Fig. 14. Photographic reconstruction of human TCs in culture establishing contact with smooth muscle cells. From our experience, silver impregnation is one of the choice methods for revealing the typical moniliform aspect of TCs in culture. Inset- the same interlaced distribution of TCs (\*) using methylene blue vital staining. Both methods reveal weaker myocyte staining. Scale bar 10 µm. Reproduced, with permission, from Cretoiu et al., 2006.

Fig. 15. Rat myometrium: TEM; original magnification 7100x. A multi-contact synapse (MS) between a telocyte and an eosinophil, in the neighbourhood of smooth muscle cells (SMC); m, mitochondria; N, nucleus; db, dense bodies; Note presence of mitochondria (\*) in the synaptic vicinity, typical of chemical synapses. Reproduced, with permission, from Popescu et al., 2005b.

Fig. 16. Digitally-coloured TEM image of a TC in rat myometrium: TC (blue), smooth muscle cells (Sienna brown). Note the 'Ca2+-release units' (caveolae, sarcoplasmic reticulum and mitochondria) in the cytoplasmic region where smooth muscle sarcolemma comes into close contact with TC plasmalemma. Original magnification: x15000. SMC = smooth muscle cells; Ht = heterochromatin; Eu = euchromatin; rER = rough endoplasmic reticulum; SR = sarcoplasmic reticulum; m = mitochondria; cav = caveolae (arrowheads). Reproduced from Ciontea et al., 2005.

Telocytes in Human Fallopian Tube and Uterus Express Estrogen and Progesterone Receptors 103

However, using immunohistochemistry alone, we cannot differentiate between interstitial cells since. For instance, c-kit positive cells could be stem cells, mast cells (Terada, 2009; Cinel et al., 2009), or TCs. TCs have a thin rim of cytoplasm. Their TPs (often up to 100 nm thick) can be undetectable under an immunofluorescence microscope, falling below light microscopy resolution. Electron microscopy is fundamental in identifying TCs with their peculiar appearance, having extremely long and moniliform processes with a dichotomous branching pattern which sometimes gave them a dendritic aspect. Table 1 distinctively

Once TCs were established as cellular components of the hormonally responsive uterine tissue, we addressed whether they might express steroid receptors. Because, *in situ,* it may be difficult to observe morphological differences between the tightly packed myometrial cells and TCs, we chose to dissociate myometrial tissue to determine which cell type(s) expresses ER-α or PR-A. By Immunocytochemistry, we identified two cell types on the basis of their ER and PR immunoreactivity: a. TCs, which showed intense nuclear and weak cytoplasmic immunostaining for both ER and PR, (Figure 18 A, B); b. myocytes and/or fibroblasts which remained relatively unstained (Figure 18 A). Using double immunostaining for CD117/c-kit, we confirmed that cells which stained positive for ER and PR were TCs, (Figure 18 C, D). Double immunofluorescence confirmed the same distribution of PR and ER in c-kit positive

Fig. 18. A-D. Human myometrial cell culture, fourth passage. Immunocytochemical staining for estrogen and progesterone receptors. A. Immunocytochemical detection of estrogen receptor - dark stained nuclei (\*), counterstaining with methyl green for negative nuclei. B. TCs stained positive for progesterone receptor. C. Doublestaining (\*) for CD117/c-kit (red) and estrogen receptor (black). D. Double staining for CD117/c-kit (red) and progesterone receptor (black). Scale bar = 10 μm. Reproduced, with permission, from Cretoiu et al., 2006.

draws a demarcation line between TCs, canonical ICC and fibroblasts.

cells, intense at nuclear level and weak in the cytoplasm (Figure 19 A-H).

Currently, there is no established panel of antibodies for TC immunophenotyping. We previously reported that antibodies against CD117/c-kit result in weak and sometimes inconsistent TC immunostaining. Most of CD117 positive cells co-express CD34 and vimentin (Ciontea et al., 2005, Popescu et al., 2006) (Figure 17).

Fig. 17. Human myometrium cells in culture (the 2nd passage): c-kit (green in A, B), c-kit and CD34 (red and green, respectively, in C) and vimentin (green in D, E). Cells which display the morphologic TC feature (long, moniliform processes) express c-kit and contact adjacent cells (A–C). Some cells suggestive of TCs co-express c-kit and CD34 (C). The characteristic cell processes are immunoreactive for vimentin and establish connections with nearby cells (D and E). Original magnification 60x, nuclear counterstaining with Hoechst 33342 (blue). Reproduced, with permission, from Ciontea et al., 2005.

Currently, there is no established panel of antibodies for TC immunophenotyping. We previously reported that antibodies against CD117/c-kit result in weak and sometimes inconsistent TC immunostaining. Most of CD117 positive cells co-express CD34 and

Fig. 17. Human myometrium cells in culture (the 2nd passage): c-kit (green in A, B), c-kit and CD34 (red and green, respectively, in C) and vimentin (green in D, E). Cells which display the morphologic TC feature (long, moniliform processes) express c-kit and contact adjacent cells (A–C). Some cells suggestive of TCs co-express c-kit and CD34 (C). The

characteristic cell processes are immunoreactive for vimentin and establish connections with nearby cells (D and E). Original magnification 60x, nuclear counterstaining with Hoechst

33342 (blue). Reproduced, with permission, from Ciontea et al., 2005.

vimentin (Ciontea et al., 2005, Popescu et al., 2006) (Figure 17).

However, using immunohistochemistry alone, we cannot differentiate between interstitial cells since. For instance, c-kit positive cells could be stem cells, mast cells (Terada, 2009; Cinel et al., 2009), or TCs. TCs have a thin rim of cytoplasm. Their TPs (often up to 100 nm thick) can be undetectable under an immunofluorescence microscope, falling below light microscopy resolution. Electron microscopy is fundamental in identifying TCs with their peculiar appearance, having extremely long and moniliform processes with a dichotomous branching pattern which sometimes gave them a dendritic aspect. Table 1 distinctively draws a demarcation line between TCs, canonical ICC and fibroblasts.

Once TCs were established as cellular components of the hormonally responsive uterine tissue, we addressed whether they might express steroid receptors. Because, *in situ,* it may be difficult to observe morphological differences between the tightly packed myometrial cells and TCs, we chose to dissociate myometrial tissue to determine which cell type(s) expresses ER-α or PR-A. By Immunocytochemistry, we identified two cell types on the basis of their ER and PR immunoreactivity: a. TCs, which showed intense nuclear and weak cytoplasmic immunostaining for both ER and PR, (Figure 18 A, B); b. myocytes and/or fibroblasts which remained relatively unstained (Figure 18 A). Using double immunostaining for CD117/c-kit, we confirmed that cells which stained positive for ER and PR were TCs, (Figure 18 C, D). Double immunofluorescence confirmed the same distribution of PR and ER in c-kit positive cells, intense at nuclear level and weak in the cytoplasm (Figure 19 A-H).

Fig. 18. A-D. Human myometrial cell culture, fourth passage. Immunocytochemical staining for estrogen and progesterone receptors. A. Immunocytochemical detection of estrogen receptor - dark stained nuclei (\*), counterstaining with methyl green for negative nuclei. B. TCs stained positive for progesterone receptor. C. Doublestaining (\*) for CD117/c-kit (red) and estrogen receptor (black). D. Double staining for CD117/c-kit (red) and progesterone receptor (black). Scale bar = 10 μm. Reproduced, with permission, from Cretoiu et al., 2006.

Telocytes in Human Fallopian Tube and Uterus Express Estrogen and Progesterone Receptors 105

Fig. 19. A-H.Human myometrial cell culture, fourth passage. Immunofluorescent labeling for estrogen (A) and progesterone (E) receptor (red) which appear both inside the nucleus and in cytoplasm. c-kit/CD117 only (green) found in the cytoplasm (B, F) and double labeling for both markers (C, G), where co-expression appears as yellow areas. Hoechst 33342 (blue) for nuclear counterstaining. Phase contrast microscopy focused on the same cells, typical TCs with long, moniliform prolongations (D, H). Scale bar = 2 μm. Reproduced,

with permission, from Cretoiu et al., 2006


Table 1. Morphological aspects, semi-quantitative data concerning the ultrastructural elements (transmission electron microscopy) and specific markers of telocytes compared to archetypal enteric interstitial cells of Cajal (ICC) and fibroblasts. Adapted from Hutchings et al., 2009.

**Myometrial telocytes**

Oval, heterochromatic under nuclear membrane

++ + + +

n.a. present n.a. n.a.

tion of c-kit, CD34 and connexin 43, lack of prolyl 4-hydroxlase

+++-

+++-

**Fallopian tube** 

Spindle or stellate body Polymorphic

**telocytes Fibroblasts** 

body

Oval, euchromatic with 1-2 visible nucleoli

Prolyl 4 hydroxylase

**Interstitial cells of Cajal** 

**(ICC)**

spindleshaped body

euchromatic

Cytoplasm Smooth ER ++ + + +- Rough ER + + + +++ Golgi apparatus + + +++ Mitochondria +++ ++ + +

Microtubules + + + +

Other structures Caveolae + + ++ -

Gap junctions + + -

Table 1. Morphological aspects, semi-quantitative data concerning the ultrastructural elements (transmission electron microscopy) and specific markers of telocytes compared to archetypal enteric interstitial cells of Cajal (ICC) and fibroblasts. Adapted from Hutchings et

Thin filaments + + + +

Basal lamina 0 +- +- -

c-kit Co-localiza-

Nerve endings +++ + -

Blood vessels n.a. + - Immune cells n.a. +++ -

Cell shape Oval or

Intermediate

Calcium

Immunohistochemical markers

Intercellular contacts

al., 2009.

filaments

releasing units

Smooth muscle

Other interstitial

cells

cells

Nucleus Oval, mostly

Fig. 19. A-H.Human myometrial cell culture, fourth passage. Immunofluorescent labeling for estrogen (A) and progesterone (E) receptor (red) which appear both inside the nucleus and in cytoplasm. c-kit/CD117 only (green) found in the cytoplasm (B, F) and double labeling for both markers (C, G), where co-expression appears as yellow areas. Hoechst 33342 (blue) for nuclear counterstaining. Phase contrast microscopy focused on the same cells, typical TCs with long, moniliform prolongations (D, H). Scale bar = 2 μm. Reproduced, with permission, from Cretoiu et al., 2006

Telocytes in Human Fallopian Tube and Uterus Express Estrogen and Progesterone Receptors 107

TCs with characteristic morphology (one or more very long, thin processes, sometimes with 'beads-on-a-string' appearance, that arise from pyriform, stellate or spindle shaped cell bodies) were found to express c-kit. Some of the TCs co-express CD34, desmin, vimentin and even α-SMA. TCs in the fallopian tube fulfill the ultrastructural identification criteria

Fig. 20. (A) A telocyte compared to (B) a fibroblast from the same TEM ultrathin section of human fallopian tube (digitally coloured images). N = nucleus; Eu = euchromatin; Ht = heterochromatin; rER = rough endoplasmic reticulum; sER = smooth endoplasmic

reticulum; m = mitochondria; v = vacuolae; Ly = lysosomes; arrowheads indicate caveolae.

At least 29 caveolae can be counted in the TC's convoluted process (A, upper part).

Reproduced, with permission, from Popescu et al., 2005a.

and are definitely distinct from fibroblasts (Figure 20).

#### **3.2 TCs of human fallopian tube express ER and PR**

Fallopian tubes are very important in human reproductive medicine playing active roles in such as gamete transport and final maturation, capacitation of sperm, ovum fertilization, early embryo development and delivery of embryo to the uterus. Each anatomic region (infundibulum with fimbria, ampulla, isthmus and intramural segment) seem to perform specific functions. Our discovery of novel interstitial cells in Fallopian tube tissue in 2005 (Popescu, 2005), which we now know to be TCs, has brought more attention to studies of this tissue. TCs are resident (dominantly) in fallopian tube lamina propria and in between smooth muscular fibres. The TCs percentage in the fallopian tube wall discloses the following areas of interest, starting from the basement membrane toward the serosa: area in the lamina propria found in close vicinity of the basement membrane (18±2%); area containing the entire lamina propria thickness (~8%); muscularis per se (7.8±1.2%) and the remaining zone beneath serosa (was not assessed). We concluded that the TC spatial distribution gradient decreases from the sub-epithelial area to the serosa. In lamina propria the percentage of TCs represent on average 11.0±0.6% of all cells. TC cellular bodies can take on various shapes: pyriform (having only one prolongation), (50%); spindle (with two opposite prolongations), (30%), triangular (15%) and other shapes with more than three prolongations (5%).

Tubal TC immunophenotyping was performed by correlating morphology with immunohistochemistry using a panel of 15 antibodies (Table 2).


Table 2. Summary of immunohistochemical results for telocytes from human fallopian tube. The intensity of telocyte reactivity was assessed semi-quantitatively using an adaptation of the Quick score method. Intensity: Negative (no staining of any cellular part at high magnification):–; Occasionally weak positive: +; Low (only visible at high magnification):+; Medium (readily visible at low magnification):+ +; High (strikingly positive at high magnification):+ + +; Strong (strikingly positive even at low magnification):+ + + +.

Fallopian tubes are very important in human reproductive medicine playing active roles in such as gamete transport and final maturation, capacitation of sperm, ovum fertilization, early embryo development and delivery of embryo to the uterus. Each anatomic region (infundibulum with fimbria, ampulla, isthmus and intramural segment) seem to perform specific functions. Our discovery of novel interstitial cells in Fallopian tube tissue in 2005 (Popescu, 2005), which we now know to be TCs, has brought more attention to studies of this tissue. TCs are resident (dominantly) in fallopian tube lamina propria and in between smooth muscular fibres. The TCs percentage in the fallopian tube wall discloses the following areas of interest, starting from the basement membrane toward the serosa: area in the lamina propria found in close vicinity of the basement membrane (18±2%); area containing the entire lamina propria thickness (~8%); muscularis per se (7.8±1.2%) and the remaining zone beneath serosa (was not assessed). We concluded that the TC spatial distribution gradient decreases from the sub-epithelial area to the serosa. In lamina propria the percentage of TCs represent on average 11.0±0.6% of all cells. TC cellular bodies can take on various shapes: pyriform (having only one prolongation), (50%); spindle (with two opposite prolongations), (30%), triangular (15%) and

Tubal TC immunophenotyping was performed by correlating morphology with

**Antibody Clone Dilution Source IHC positivity**  CD117/c-kit polyclonal 1:100 DAKO + + + + CD34 QBEnd10 1:100 Biogenex + + + S-100 polyclonal 1:500 DAKO + + α-SMA 1A4 1:1500 Sigma + CD57 NK1 1:50 DAKO + nestin 5326 1:100 Santa Cruz + desmin D33 1:50 DAKO – vimentin V9 1:50 DAKO + NSE BBS/NC/VI-H14 1:50 DAKO +

**3.2 TCs of human fallopian tube express ER and PR** 

other shapes with more than three prolongations (5%).

immunohistochemistry using a panel of 15 antibodies (Table 2).

GFAP 6F2 1:50 DAKO–

CD68 PG-M1 1:50 DAKO + CD62P 1E3 1:25 DAKO – CD1a CD1a-235 1:30 Novocastra – Chromo A LK2H10 1:50 Novocastra – PGP9.5 10A1 1:40 Novocastra –

Table 2. Summary of immunohistochemical results for telocytes from human fallopian tube. The intensity of telocyte reactivity was assessed semi-quantitatively using an adaptation of the Quick score method. Intensity: Negative (no staining of any cellular part at high magnification):–; Occasionally weak positive: +; Low (only visible at high magnification):+;

Medium (readily visible at low magnification):+ +; High (strikingly positive at high magnification):+ + +; Strong (strikingly positive even at low magnification):+ + + +. TCs with characteristic morphology (one or more very long, thin processes, sometimes with 'beads-on-a-string' appearance, that arise from pyriform, stellate or spindle shaped cell bodies) were found to express c-kit. Some of the TCs co-express CD34, desmin, vimentin and even α-SMA. TCs in the fallopian tube fulfill the ultrastructural identification criteria and are definitely distinct from fibroblasts (Figure 20).

Fig. 20. (A) A telocyte compared to (B) a fibroblast from the same TEM ultrathin section of human fallopian tube (digitally coloured images). N = nucleus; Eu = euchromatin; Ht = heterochromatin; rER = rough endoplasmic reticulum; sER = smooth endoplasmic reticulum; m = mitochondria; v = vacuolae; Ly = lysosomes; arrowheads indicate caveolae. At least 29 caveolae can be counted in the TC's convoluted process (A, upper part). Reproduced, with permission, from Popescu et al., 2005a.

Telocytes in Human Fallopian Tube and Uterus Express Estrogen and Progesterone Receptors 109

Fig. 22. A-F. Human Fallopian tube cell culture, sixth passage. Immunouorescent labeling for c-kit/FITC (A) and ER-α/Alexa Fluor 546 (B), and superimposed images to show colocalization (C). c-kit uorescence of TC (D) and PR-A uorescence (E). Superimposed labeling for both markers (F), where c-kit (green) is localized only in the cytoplasm and PR-A is expressed in the TC nuclei (red). Scale bar = 5 μm. With kind permission from Springer Science+Business Media: Journal of Molecular Histology, Interstitial Cajal-like cells of human Fallopian tube express estrogen and progesterone receptors, 40, 2009, 387-394,

Recently, some of the TCs located on the extracellular matrix of blood vessels were described as having a primary cilium (Cantarero et al., 2011). The presumed functions of such a nonmotile cilium could be:- organizer of the mitotic spindle (Alieva et al., 2004), sensory organelle involved in signal transduction - hedgehog pathway (Singla et al., 2006), mechanical sensing and mechano-chemical conversion in endothelial cells (Egorova et al., 2011; Nauli et al., 2008). By analogy, we can presume that TCs could be involved in the signaling process if located near the stromal colony-forming cells/units in human endometrium or might act as stretch

sensors if located near smooth muscle structures in both Fallopian tubes and uterus.

Cretoiu, S.M.;Cretoiu, D.;Suciu, L.&Popescu, L.M., figure 5.

**4. Possible TC roles** 

**4.1 TCs and signaling processes** 

TCs were enriched in primary culture by magnetically-activated cell sorting, after being identified as c-kit positive cells with characteristic morphology. Indeed TCs were found in a higher percentage after magnetic cell sorting: approximately 30 ± 0.8% (n = 516) compared to 9.9 ± 0.9% (n = 324) (Popescu, 2005). The sorted populations underwent subsequent passages, because according to our previous experience, the number of TCs increases with each passage.

*In vitro* double staining on Fallopian tube samples showed that desmin-positive cells (SMC) tested negative for ER-α or PR-A and c-kit-positive cells (telocytes) tested positive for ER-α or PR-A (Fig. 21 A,B). Moreover, double staining for c-kit (green fluorescence) and PR-A or ER-α (red fluorescence) revealed that only cells positive for c-kit were also positive for ER-α and PR-A at nuclear level (Fig. 22 A-F). PR-A expression at nuclear level was more intense than for ER-α. SMC were weakly positive or completely negative.

Fig. 21. A,B. Human Fallopian tube cell culture, fourth passage. The expression of ER-α (A) and PR-A (B) demonstrated by immunocytochemical staining. TCs (arrows) stained positive for ER and PR (brown nuclei). Scale bar = 5 μm. With kind permission from Springer Science+Business Media: Journal of Molecular Histology, Interstitial Cajal-like cells of human Fallopian tube express estrogen and progesterone receptors, 40, 2009, 387-394, Cretoiu, S.M.;Cretoiu, D.;Suciu, L.&Popescu, L.M., figure 4.

Fig. 22. A-F. Human Fallopian tube cell culture, sixth passage. Immunouorescent labeling for c-kit/FITC (A) and ER-α/Alexa Fluor 546 (B), and superimposed images to show colocalization (C). c-kit uorescence of TC (D) and PR-A uorescence (E). Superimposed labeling for both markers (F), where c-kit (green) is localized only in the cytoplasm and PR-A is expressed in the TC nuclei (red). Scale bar = 5 μm. With kind permission from Springer Science+Business Media: Journal of Molecular Histology, Interstitial Cajal-like cells of human Fallopian tube express estrogen and progesterone receptors, 40, 2009, 387-394, Cretoiu, S.M.;Cretoiu, D.;Suciu, L.&Popescu, L.M., figure 5.
