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Journal of Clinical Endocrinology & Metabolism , doi:10.1210/jc.2005-2169
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The Journal of Clinical Endocrinology & Metabolism Vol. 91, No. 5 1956-1960
Copyright © 2006 by The Endocrine Society

Isolation and Cultivation of Human Testicular Peritubular Cells: A New Model for the Investigation of Fibrotic Processes in the Human Testis and Male Infertility

Martin Albrecht, Romi Rämsch, Frank M. Köhn, J. Ullrich Schwarzer and Artur Mayerhofer

Institute of Anatomy (M.A., R.R., A.M.), Ludwig-Maximilians-University, and Department of Dermatology and Allergy (F.M.K.), Technical University, 80802 Munich, Germany; and Department of Urology (J.U.S.), Freising Hospital, 85356 Freising, Germany

Address all correspondence and requests for reprints to: Professor Artur Mayerhofer, M.D., Institute of Anatomy, Ludwig-Maximilians-University, Biedersteiner Strasse 29, 80802 Munich, Germany. E-mail: mayerhofer{at}lrz.uni-muenchen.de.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Context: Fibrotic remodeling, especially of the tubule wall, in testes of infertile men is common, but reasons or consequences of these striking changes are not known. Based on cell culture and ex vivo studies, we previously suggested that mast cells via their products tryptase and histamine are involved in the development of fibrosis. However, studies in a relevant human testicular model are required to further test this hypothesis and the mechanisms of testicular fibrosis in general.

Objective: The objective of the study was the isolation, culture, and characterization of adult human testicular peritubular cells.

Patients and Interventions: Peritubular cells were obtained from biopsies of men suffering from obstructive azoospermia (n = 8) and varicocele (n = 2) but displaying normal spermatogenesis.

Results: Explant cultures were obtained from all biopsies. Immunostaining of the cultured cells and corresponding paraffin-embedded tissues with antibodies against markers of fibroblasts (CD90/Thy-1) and smooth muscle cells ({alpha}-smooth muscle actin) clearly proved their origin from the peritubular region. These cells displayed morphological features of myofibroblasts, and gene array analyses as well as immunohistochemistry revealed the predominant expression of extracellular matrix genes and genes coding for basement membrane components. The cultured cells retain receptors for the major mast cell products histamine and tryptase. The addition of histamine (100 µM) and the tryptase agonist peptide SLIGKV (10 µM) resulted in a transient increase in intracellular calcium levels, confirming the functionality of the receptors.

Conclusions: We conclude that human peritubular cells are a novel model for the investigation of paracrine, including mast cell initiated, interactions in the human testis, which will allow the study of fibrotic processes underlying male idiopathic infertility.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
IN THE TESTES of infertile patients, fibrotic thickening of the peritubular region is a common observation and goes in parallel with increased numbers of activated testicular mast cells (MCs) in these regions (1, 2). There is evidence that MCs via their major secretory products tryptase and histamine are crucially and causally involved in these events closely associated with male infertility (2, 3, 4).

To gain deeper insight into how MCs and their secretory products contribute to the fibrotic events leading to male infertility, human studies and thus suitable cell culture models are needed. Although several cell lines and primary cells of rodent peritubular cells have been established (5, 6, 7, 8), studies using adult human peritubular cells have not been performed.

The purpose of this study was therefore to develop a technique that will readily allow isolation and culture of human testicular peritubular cells (HTPCs). Such cells would be of great value for in vitro experiments, e.g. to explore mechanisms involved in MC-peritubular cell interactions and MC involvement in tubular fibrosis and male infertility.


   Materials and Methods
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 Materials and Methods
Results
 Discussion
 References
 
Isolation and culture of primary HTPCs

Testicular tissue was obtained by open biopsy from eight vasectomized men with obstructive azoospermia but displaying normal spermatogenesis and from two men suffering from varicocele accompanied by only a slight reduction of spermatogenesis. Biopsies were obtained from vasectomized patients as a standardized procedure before religation of the spermatic ducts and from the varicocele patients during surgical treatment. Information concerning the state of spermatogenesis was derived by histological analyses of part of the biopsy samples. Ages of the patients were 29, 32, 32, 34, 35, 36, 40, 41, 46 and 47 yr, respectively. All participants granted written informed consent. The study was approved by the local ethics committee.

Immediately after retrieval in the operating room, the tissue was transferred to Ham’s F12 medium (PAA GmbH, Pasching, Austria) containing 20 mM HEPES (Sigma-Aldrich Chemie GmbH, Schellendorf, Germany), 0.5 g/liter NaHCO3 (Sigma-Aldrich), 15% (vol/vol) fetal calf serum (FCS), 100 U penicillin, and 100 µg/ml streptomycin (all from PAA GmbH). Within 2–3 h, testicular tissue covered with medium was dissected using tweezers under sterile conditions into 1- to 2-mm3 pieces. The tissue was then placed in recalcified human plasma that was positioned in drops of 10–20 µl onto the surface of a plastic cell culture dish. The procedure allowed the specimens to be glued to the bottom of the culture dish. Each biopsy sample yielded sufficient tissue for five to six explants. The explants were incubated for 1–2 h under humidified conditions (37 C, 5% CO2), checked for adherence, and subsequently cultured in Ham’s F12 medium composed as described above. Cells started to grow out of the biopsies after about 1 wk. When the cells covered an area of approximately 1 cm2, which took 2–3 wk, the remnant explant was carefully removed, and cells were allowed to grow for another week before they were trypsinized and subcultured. Subcultured cells were grown in DMEM + 10% FCS (both from PAA GmbH) without antibiotics. For all experiments, cells from passages 3–7 were used. In addition, we found that cells can be grown for at least nine passages and cryopreserved in DMEM containing 10% FCS and 5% dimethylsulfoxide. Thawed and recultured cells show a viability of more than 95%.

Transmission electron microscopy

For ultrastructural studies, HTPCs were cultivated as described, fixed with 4% paraformaldehyde/0.5% glutaraldehyde, and postfixed with 4% OsO4/potassium hexacyanoferrate (II). After embedding in Epon, semithin and ultrathin sections were cut, contrasted with uranylacetate (2%)/lead citrate (2.7%) as described (9, 10), and examined with an EM10 electron microscope (Zeiss, Jena, Germany).

Gene arrays

Gene expression profiles were evaluated, using commercial chemiluminescent human extracellular matrix gene array kits (SuperArray; Biomol GmbH, Hamburg, Germany) as described elsewhere (4). Gene arrays were performed in duplicates.

Immunohistochemistry

After dissecting the testicular material into small pieces, one part was explanted as described above, whereas a remaining part was fixed in Bouin’s solution, embedded in paraffin, and sectioned. In preparation for immunohistochemistry, as previously reported (11), deparaffinized tissue sections of human testes were treated with 3% H2O2 in methanol for 20 min to block endogenous peroxidase activity and then incubated with 5% normal goat serum for 30 min to reduce nonspecific antibody binding. The sections were kept overnight at 4 C with a monoclonal antihuman {alpha}-smooth muscle actin antibody (dilution 1:2000, clone 1A4; Sigma-Aldrich) and a monoclonal antihuman CD90/Thy-1 antibody (dilution 1:50, clone AS02; Dianova, Hamburg, Germany) and probed with a biotin-coupled goat antimouse antibody (1:500). The sites of immunoreaction were visualized by the ABC method (Vectastain elite kit, Vector Laboratories, Burlingame, CA) and addition of 3,3'-diaminobenzidine tetrahydrochloride solution containing H2O2. Controls consisted of nonimmune mouse normal serum (1:5000) or omission of the primary antibody.

Immunofluorescence methods were used as described elsewhere (12) using the monoclonal antibodies mentioned above. The {alpha}-smooth muscle actin antibody was diluted 1:200, whereas the CD90/Thy-1 antibody was used at a 1:50 dilution.

Staining for FSH and LH receptors, indicative of the occurrence of Sertoli cells and Leydig cells in the HTPC cultures, was performed using a polyclonal FSH receptor antibody (Acris GmbH, Hiddenhausen, Germany), diluted 1:500, and a polyclonal LH receptor antibody (Acris GmbH) at the same dilution. Controls consisted of nonimmune mouse/rabbit normal serum (1:5000) or omission of the primary antibody.

Reverse transcription and PCR analysis

RNA extraction was performed using the RNeasy minikit (QIAGEN GmbH, Hilden, Germany), followed by reverse transcription using oligo-dT15 or random hexamer primers and PCR amplification (13). The following primers were used: histamine-1 (H1) receptor, 5'-CTACAAGGCCGTACGACA-3' and 5'-CCTGCTCATCTGTCTTGA-3', yielding a 371-bp fragment; histamine-2 receptor, 5'-TCTACCGCATGCAAGATC-3' and 5'-CGAGGCTGATCATGAAGA-3' in combination with the following nested primers: 5'-TCATCCTCATCACCGTTG-3' and 5'-TGGTAGATGGCAGAGAAG-3', yielding a 155-bp fragment; histamine-3 receptor, 5'-ATGTACCCTACGTGCTGA-3' and 5'-GTGATGAGGAAGTACCAG-3' in combination with the following nested primers: 5'-CAACATCGTGCTCATCAG-3' and 5'-TACTCCCAGCTCAGGATG-3', yielding a 158-bp fragment; histamine-4 receptor, 5'-TCTCAGTAGGTGCCAAAG-3' and 5'-AGAATGGCCAGTGACTTG-3' in combination with the following nested primers: 5'-GAGACAGAGGAGAAAGAG-3' and 5'-GGCTCTAAGCAGTTCAAC-3', yielding a 142-bp fragment; and protease activated receptor-2 (PAR2), 5'-CATCCTGCTAGCAGCCTC-3' and 5'-ACCTCTGCACACTGAGGC-3', yielding a 480-bp fragment. Tissue library cDNA (CLONTECH, Palo Alto, CA) was used as positive control in all PCR experiments. Negative controls were performed by omitting the respective input cDNA. The identity of PCR products was verified by commercial sequencing (13).

Calcium measurements

For calcium measurements, HTPCs were grown on glass coverslips in DMEM supplemented with 10% FCS. The cells were loaded with 5 µM fluo-4, AM (Molecular Probes, Eugene, OR) in FCS-free DMEM for 30 min at 37 C and 5% CO2 (for details, see Ref.4). Finally, the cells were transferred into a recording chamber mounted on a TCS SP2 confocal microscope (Leica Microsystems, Wetzlar, Germany). Fluorescence was monitored at 500–540 nm ({lambda}ex = 488 nm) every 2 sec, and the intensity was quantified over single cells. Real-time changes of intracellular calcium levels were recorded during application of 100 µM histamine (Sigma-Aldrich), 100 µM pyrilamine (RBI, Natick, MA), and 10 µM of a PAR2 agonist peptide SLIGKV (SLIGKV-amide; NeoMPS, Strasbourg, France).


   Results
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 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Morphology and characterization of the explant cultures

Explants of human testicular tissue (Fig. 1AGo) were cultured as described in Materials and Methods. Elongated cells became visible extending from the tubules between wk 1 and 2 (Fig. 1Go, B and C). These HTPC cultures were trypsinized and subcultured after 4 wk (Fig. 1DGo) and showed a morphology that was characterized by mainly elongated cells with fibroblast/smooth muscle cell appearance (Fig. 1DGo). This myofibroblastic morphology was retained up to at least nine passages (data not shown).


Figure 1
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  		FIG. 1. Explant cultures of HTPCs. Small pieces of testicular biopsies were seeded onto culture dishes (A). Between wk 1 (B) and 2 (C), cells start growing out of the walls of the seminiferous tubules (depicted with a T). Cells subcultivated after 4 wk display an elongated phenotype (D). Bars, 50 µm.

 
HTPCs were immunonegative for FSH and LH receptors (data not shown), thus excluding contaminations with Sertoli or Leydig cells but stained specifically with antibodies directed against smooth muscle cell and fibroblast markers ({alpha}-smooth muscle actin and CD90/Thy-1; Fig. 2Go, A and B). Immunostaining of the corresponding paraffin-embedded testicular tissue (from which the explant cultures were derived) using the same antibodies resulted in a selective staining of peritubular cells, proving clearly the peritubular origin of HTPCs (Fig. 2Go, E and F).


Figure 2
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  		FIG. 2. HTPCs show characteristics of smooth muscle cells and fibroblasts and originate from the peritubular region. HTPCs are positive for smooth muscle cell and fibroblast markers [{alpha}-smooth muscle actin (A) and CD90/Thy-1 (B)]. Sections of the corresponding testicular tissue from which the explant cultures were derived contain {alpha}-smooth muscle actin-positive and CD90/Thy-1-positive cells in the peritubular region (E and F). In addition to peritubular cells, smooth muscle cells of small blood vessels are also positive for {alpha}-smooth muscle actin (E, arrowhead). Respective negative controls performed by omitting the primary antibody are shown in C, D, G, and H. Bars, 40 µm.

 
Using electron microscopy experiments, electron dense vesicles were detected in the cytoplasm of HTPCs (Fig. 3AGo). Several authors (14, 15) previously reported the occurrence of so-called intracellular collagen fibers in proliferating myofibroblasts; the vesicles found in HTPCs may represent or be related to intracellular collagen fibers, although the final proof for this is still missing.


Figure 3
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  		FIG. 3. HTPCs display a characteristic ultrastructure and express extracellular matrix and basement membrane genes. HTPCs are characterized by an elongated phenotype containing various electron-dense intracytoplasmic vesicles of unknown composition (A, arrows). Gene array analyses reveal the expression of several molecules of the extracellular matrix and basement membrane (B). Collagen-I (C) and fibronectin (D) are two of the proteins typically produced by HTPCs. E and F show respective negative controls performed by omitting the primary antibody. Bars, 5 µm (A) and 35 µm (C–F). Asterisks (B) denote basement membrane proteins. One representative array is shown.

 
HTPCs express several genes coding for proteins of the extracellular matrix and basement membrane, such as collagen-I, collagen-IV, collagen-XVIII, fibronectin, secreted protein acidic and rich in cystein (SPARC), and laminin (Fig. 3BGo). The synthesis of the proteins collagen-I and fibronectin was shown by immunohistochemistry (Fig. 3Go, C and D).

HTPCs as model to investigate effects of MC products on human peritubular cells

To examine the suitability of the established cell culture model of human peritubular cells as a system for the investigation of paracrine, in particular, MC-mediated effects, the capacity of HTPCs to react to the two major MC secretory products histamine and tryptase was investigated. Receptors for tryptase (PAR2) and histamine (H1) were shown to be expressed in HTPCs by RT-PCR (Fig. 4Go). Changes in intracellular calcium levels were evaluated after stimulation with histamine and the PAR2 agonist SLIGKV. Both substances induced a reversible increase in intracellular calcium levels, proving the response of HTPCs to the two major MC products (Fig. 4Go). The effect of histamine was mediated via H1 because the specific H1 blocker pyrilamine was able to inhibit histamine-induced calcium fluxes (data not shown).


Figure 4
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  		FIG. 4. HTPCs possess functional tryptase and histamine receptors linked to signal transduction events involving calcium. HTPCs express receptors for tryptase (PAR2) and histamine (H1). Incubating HTPCs with the PAR2 agonist SLIGKV (10 µM) or histamine (100 µM) results in an immediate and reversible increase in intracellular calcium levels. Note that histamine induces a biphasic signal. One representative experiment of a single-cell measurement of changes in intracellular calcium levels is shown. All experiments were independently carried out five times, with at least 10 single-cell determinations per experiment. Bold arrowheads denote the addition of buffer.

 

   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
This paper describes a rapid and economical method for the isolation and cultivation of human peritubular cells. These cells are involved in the development of tubular fibrosis, a hallmark of infertility in men, which is based on fibrotic remodeling and thickening of the peritubular region due to increased cell proliferation and extracellular matrix production (16, 17).

Numbers of testicular MCs (1, 17, 18) and other immune cells such as macrophages (13) and lymphocytes (17) are increased in infertile patients, but their involvement in tubular fibrosis is largely unknown. We and others (2, 3, 4, 17) recently provided evidence that activated testicular MCs are promoting these fibrotic events and may therefore play a central role in male idiopathic infertility. This hypothesis is supported by studies showing that MC blockers are able to increase fertility in subfertile patients (19, 20, 21).

Although the proposal of an altered MC-fibroblast interaction in the development of male infertility is very tempting and also supported by one of our previous studies evaluating the effects of MC products on human fetal foreskin fibroblasts (3), the final proof of such an interaction is missing and requires the investigation of human peritubular cell systems.

Regarding the existence of human peritubular cell models, we are aware of only one approach. Cigorraga et al. (22) described a method for the isolation and culture of human peritubular cells from prepubertal patients. However, to the best of our knowledge, there is no in vitro cell culture system of adult human peritubular cells available so far.

In the study presented, we address the lack of human peritubular cell models and describe a method to obtain HTPCs for in vitro studies using testicular biopsy explants. Cells growing out of the explants displayed an elongated phenotype. Immunostaining of the cells and sections of the corresponding testicular tissue for smooth muscle cell- and fibroblast-specific markers revealed the origin of the cells from the peritubular region. In tissue sections, besides the peritubular cells, smooth muscle cells of small blood vessels also stained positive with the anti-{alpha}-smooth muscle actin antibody, confirming the specificity of the used marker. A similar staining pattern was observed, with the fibroblast-specific antibody (CD90/Thy-1) and peritubular cells as well as vascular smooth muscle cells (data not shown). Therefore, we cannot completely exclude the possibility that HTPC cultures contain some vascular smooth muscle cells. Nevertheless, because the explants consisted mainly of seminiferous tubules with very few testicular stroma attached (Fig. 1AGo) and because the cells grew out of the tubule walls (Fig. 1BGo), we expect at most a very small fraction of vascular smooth muscle cells within the HTPC population.

Gene arrays and immunohistochemistry revealed the expression of several genes and proteins coding for basement membrane components (e.g. collagen-IV, fibronectin, laminin, and secreted protein acidic and rich in cystein). Basement membrane proteins have been shown to be expressed by peritubular cells of the rat testis (23, 24), and Pollanen et al. (25) described the presence of laminin and type IV collagen in the myoid cell layers of human seminiferous tubules. Therefore, these results further substantiate the peritubular characteristics of HTPCs.

Tryptase and histamine are typical mediators of activated testicular MCs and are believed to exert various effects on nearby cells in the human testis (1, 2, 16, 17). To examine the suitability of the established cell culture model of human peritubular cells as a system for the investigation of MC- mediated effects, the ability of HTPCs to react to the two major MC secretory products, histamine and tryptase, was investigated. Both the tryptase analog SLIGKV and histamine induced a reversible increase of intracellular calcium levels. Because SLIGKV is specific for the PAR2 receptor (26, 27) and because histamine-induced calcium fluxes in fibroblasts are primarily mediated via the H1 receptor (28), we suggest that these receptors are mainly responsible for the induction of calcium signaling events in HTPCs.

In summary, testicular biopsy material was derived from vasectomized patients displaying normal spermatogenesis. Although we propose that HTPC cultures represent cells of the normal testis, the majority of vasectomized men develop sperm antibodies, which may have an impact on sperm function and fertility (29).

We conclude that isolated HTPCs are a readily available model for adult human peritubular cells because they can be obtained from small amounts of testicular tissue. The established culture system will lead to a better understanding of the pathogenic determinants (30) and significance of fibrotic processes underlying male idiopathic infertility.


   Acknowledgments
 
We thank Dr. Lars Kunz, Dr. Monica Frungieri, and Christoph Schell for discussion and Annette Krieger, Gabriele Terfloth, and Barbara Zschiesche for technical assistance. We especially thank Astrid Tiefenbacher for cell culture work.


   Footnotes
 
This work was supported by the German Research Foundation (Deutsche Forschungsgemeinschaft) Ma 1080/16-1.

M.A., R.R., F.M.K., J.U.S., and A.M. have nothing to declare.

First Published Online February 14, 2006

Abbreviations: FCS, Fetal calf serum; H1, histamine-1; HTPC, human testicular peritubular cell; MC, mast cell; PAR2, protease activated receptor-2.

Received September 30, 2005.

Accepted February 7, 2006.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Meineke V, Frungieri MB, Jessberger B, Vogt H, Mayerhofer A 2000 Human testicular mast cells contain tryptase: increased mast cell number and altered distribution in the testes of infertile men. Fertil Steril 74:239–244[CrossRef][Medline]
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