The Journal of Clinical Endocrinology & Metabolism 91(5):1956 –1960 Printed in U.S.A. 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 Ra¨msch, Frank M. Ko¨hn, 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, 85356Freising, Germany Context: Fibrotic remodeling, especially of the tubule wall, in testes
tissues with antibodies against markers of fibroblasts (CD90/Thy-1) of infertile men is common, but reasons or consequences of these and smooth muscle cells (␣-smooth muscle actin) clearly proved their striking changes are not known. Based on cell culture and ex vivo origin from the peritubular region. These cells displayed morpholog- studies, we previously suggested that mast cells via their products ical features of myofibroblasts, and gene array analyses as well as tryptase and histamine are involved in the development of fibrosis.
immunohistochemistry revealed the predominant expression of ex- However, studies in a relevant human testicular model are required tracellular matrix genes and genes coding for basement membrane to further test this hypothesis and the mechanisms of testicular fi- components. The cultured cells retain receptors for the major mast brosis in general.
cell products histamine and tryptase. The addition of histamine (100 ␮M) and the tryptase agonist peptide SLIGKV (10 ␮M) resulted in a Objective: The objective of the study was the isolation, culture, and
transient increase in intracellular calcium levels, confirming the func- characterization of adult human testicular peritubular cells.
tionality of the receptors.
Patients and Interventions: Peritubular cells were obtained from
Conclusions: We conclude that human peritubular cells are a novel
biopsies of men suffering from obstructive azoospermia (n ⫽ 8) and model for the investigation of paracrine, including mast cell initiated, varicocele (n ⫽ 2) but displaying normal spermatogenesis.
interactions in the human testis, which will allow the study of fibrotic
processes underlying male idiopathic infertility. (J Clin Endocrinol
Results: Explant cultures were obtained from all biopsies. Immuno-
Metab 91: 1956 –1960, 2006)
staining of the cultured cells and corresponding paraffin-embedded INTHETESTESofinfertilepatients,fibroticthickeningof mechanisms involved in MC-peritubular cell interactions the peritubular region is a common observation and goes and MC involvement in tubular fibrosis and male infertility.
in parallel with increased numbers of activated testicularmast cells (MCs) in these regions (1, 2). There is evidence that Materials and Methods
MCs via their major secretory products tryptase and hista- Isolation and culture of primary HTPCs mine are crucially and causally involved in these eventsclosely associated with male infertility (2– 4).
Testicular tissue was obtained by open biopsy from eight vasecto- mized men with obstructive azoospermia but displaying normal sper- To gain deeper insight into how MCs and their secretory matogenesis and from two men suffering from varicocele accompanied products contribute to the fibrotic events leading to male by only a slight reduction of spermatogenesis. Biopsies were obtained infertility, human studies and thus suitable cell culture mod- from vasectomized patients as a standardized procedure before religa- els are needed. Although several cell lines and primary cells tion of the spermatic ducts and from the varicocele patients duringsurgical treatment. Information concerning the state of spermatogenesis of rodent peritubular cells have been established (5– 8), stud- was derived by histological analyses of part of the biopsy samples. Ages ies using adult human peritubular cells have not been 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 ap- The purpose of this study was therefore to develop a proved by the local ethics committee.
technique that will readily allow isolation and culture of Immediately after retrieval in the operating room, the tissue was transferred to Ham's F12 medium (PAA GmbH, Pasching, Austria) human testicular peritubular cells (HTPCs). Such cells would containing 20 mm HEPES (Sigma-Aldrich Chemie GmbH, Schellendorf, be of great value for in vitro experiments, e.g. to explore Germany), 0.5 g/liter NaHCO3 (Sigma-Aldrich), 15% (vol/vol) fetal calf serum (FCS), 100 U penicillin, and 100 ␮g/ml streptomycin (all fromPAA GmbH). Within 2–3 h, testicular tissue covered with medium was First Published Online February 14, 2006
dissected using tweezers under sterile conditions into 1- to 2-mm3 Abbreviations: FCS, Fetal calf serum; H1, histamine-1; HTPC, human pieces. The tissue was then placed in recalcified human plasma that was testicular peritubular cell; MC, mast cell; PAR2, protease activated positioned in drops of 10 –20 ␮l onto the surface of a plastic cell culture dish. The procedure allowed the specimens to be glued to the bottom of JCEM is published monthly by The Endocrine Society (http://www.
the culture dish. Each biopsy sample yielded sufficient tissue for five to endo-society.org), the foremost professional society serving the en-
six explants. The explants were incubated for 1–2 h under humidified conditions (37 C, 5% CO2), checked for adherence, and subsequently Albrecht et al. • Isolation and Culturing of Human Peritubular Cells J Clin Endocrinol Metab, May 2006, 91(5):1956 –1960 cultured in Ham's F12 medium composed as described above. Cells Immunofluorescence methods were used as described elsewhere (12) started to grow out of the biopsies after about 1 wk. When the cells using the monoclonal antibodies mentioned above. The ␣-smooth mus- covered an area of approximately 1 cm2, which took 2–3 wk, the remnant cle actin antibody was diluted 1:200, whereas the CD90/Thy-1 antibody explant was carefully removed, and cells were allowed to grow for was used at a 1:50 dilution.
another week before they were trypsinized and subcultured. Subcul- Staining for FSH and LH receptors, indicative of the occurrence of tured cells were grown in DMEM ⫹ 10% FCS (both from PAA GmbH) Sertoli cells and Leydig cells in the HTPC cultures, was performed using without antibiotics. For all experiments, cells from passages 3–7 were a polyclonal FSH receptor antibody (Acris GmbH, Hiddenhausen, Ger- used. In addition, we found that cells can be grown for at least nine many), diluted 1:500, and a polyclonal LH receptor antibody (Acris passages and cryopreserved in DMEM containing 10% FCS and 5% GmbH) at the same dilution. Controls consisted of nonimmune mouse/ dimethylsulfoxide. Thawed and recultured cells show a viability of more rabbit normal serum (1:5000) or omission of the primary antibody.
Reverse transcription and PCR analysis Transmission electron microscopy RNA extraction was performed using the RNeasy minikit (QIAGEN For ultrastructural studies, HTPCs were cultivated as described, fixed GmbH, Hilden, Germany), followed by reverse transcription using with 4% paraformaldehyde/0.5% glutaraldehyde, and postfixed with 15 or random hexamer primers and PCR amplification (13). The 4/potassium hexacyanoferrate (II). After embedding in Epon, following primers were used: histamine-1 (H1) receptor, 5⬘-CTACAAG- semithin and ultrathin sections were cut, contrasted with uranylacetate GCCGTACGACA-3⬘ and 5⬘-CCTGCTCATCTGTCTTGA-3⬘, yielding a (2%)/lead citrate (2.7%) as described (9, 10), and examined with an EM10 371-bp fragment; histamine-2 receptor, 5⬘-TCTACCGCATGCAA- electron microscope (Zeiss, Jena, Germany).
GATC-3⬘ and 5⬘-CGAGGCTGATCATGAAGA-3⬘ in combination withthe following nested primers: 5⬘-TCATCCTCATCACCGTTG-3⬘ and 5⬘- Gene arrays TGGTAGATGGCAGAGAAG-3⬘, yielding a 155-bp fragment; hista- Gene expression profiles were evaluated, using commercial chemi- mine-3 receptor, 5⬘-ATGTACCCTACGTGCTGA-3⬘ and 5⬘-GTGAT- luminescent human extracellular matrix gene array kits (SuperArray; GAGGAAGTACCAG-3⬘ in combination with the following nested Biomol GmbH, Hamburg, Germany) as described elsewhere (4). Gene primers: 5⬘-CAACATCGTGCTCATCAG-3⬘ and 5⬘-TACTCCCAGCT- arrays were performed in duplicates.
CAGGATG-3⬘, yielding a 158-bp fragment; histamine-4 receptor, 5⬘-TCTCAGTAGGTGCCAAAG-3⬘ and 5⬘-AGAATGGCCAGTGACTTG-3⬘in combination with the following nested primers: 5⬘-GAGACAGAG- GAGAAAGAG-3⬘ and 5⬘-GGCTCTAAGCAGTTCAAC-3⬘, yielding a After dissecting the testicular material into small pieces, one part was 142-bp fragment; and protease activated receptor-2 (PAR2), 5⬘-CATC- explanted as described above, whereas a remaining part was fixed in CTGCTAGCAGCCTC-3⬘ and 5⬘-ACCTCTGCACACTGAGGC-3⬘, yield- Bouin's solution, embedded in paraffin, and sectioned. In preparation ing a 480-bp fragment. Tissue library cDNA (CLONTECH, Palo Alto, for immunohistochemistry, as previously reported (11), deparaffinized CA) was used as positive control in all PCR experiments. Negative tissue sections of human testes were treated with 3% H controls were performed by omitting the respective input cDNA. The for 20 min to block endogenous peroxidase activity and then incubated identity of PCR products was verified by commercial sequencing (13).
with 5% normal goat serum for 30 min to reduce nonspecific antibodybinding. The sections were kept overnight at 4 C with a monoclonal antihuman ␣-smooth muscle actin antibody (dilution 1:2000, clone 1A4;Sigma-Aldrich) and a monoclonal antihuman CD90/Thy-1 antibody For calcium measurements, HTPCs were grown on glass coverslips (dilution 1:50, clone AS02; Dianova, Hamburg, Germany) and probed in DMEM supplemented with 10% FCS. The cells were loaded with 5 ␮m with a biotin-coupled goat antimouse antibody (1:500). The sites of fluo-4, AM (Molecular Probes, Eugene, OR) in FCS-free DMEM for 30 immunoreaction were visualized by the ABC method (Vectastain elite min at 37 C and 5% CO2 (for details, see Ref. 4). Finally, the cells were kit, Vector Laboratories, Burlingame, CA) and addition of 3,3⬘-diami- transferred into a recording chamber mounted on a TCS SP2 confocal nobenzidine tetrahydrochloride solution containing H2O2. Controls con- microscope (Leica Microsystems, Wetzlar, Germany). Fluorescence was sisted of nonimmune mouse normal serum (1:5000) or omission of the monitored at 500 –540 nm (␭ ⫽ 488 nm) every 2 sec, and the intensity primary antibody.
was quantified over single cells. Real-time changes of intracellular cal- FIG. 1. Explant cultures of HTPCs. Small pieces of tes-ticular biopsies were seeded onto culture dishes (A). Be-tween wk 1 (B) and 2 (C), cells start growing out of thewalls of the seminiferous tubules (depicted with a T).
Cells subcultivated after 4 wk display an elongated phe-notype (D). Bars, 50 ␮m.
J Clin Endocrinol Metab, May 2006, 91(5):1956 –1960 Albrecht et al. • Isolation and Culturing of Human Peritubular Cells cium levels were recorded during application of 100 ␮m histamine(Sigma-Aldrich), 100 ␮m pyrilamine (RBI, Natick, MA), and 10 ␮m of aPAR2 agonist peptide SLIGKV (SLIGKV-amide; NeoMPS, Strasbourg,France).
Morphology and characterization of the explant cultures Explants of human testicular tissue (Fig. 1A) were cultured as described in Materials and Methods. Elongated cells becamevisible extending from the tubules between wk 1 and 2 (Fig.
1, B and C). These HTPC cultures were trypsinized andsubcultured after 4 wk (Fig. 1D) and showed a morphologythat was characterized by mainly elongated cells with fibro-blast/smooth muscle cell appearance (Fig. 1D). This myofi-broblastic morphology was retained up to at least nine pas-sages (data not shown).
HTPCs were immunonegative for FSH and LH receptors (data not shown), thus excluding contaminations with Sertolior Leydig cells but stained specifically with antibodies di-rected against smooth muscle cell and fibroblast markers(␣-smooth muscle actin and CD90/Thy-1; Fig. 2, A and B).
Immunostaining of the corresponding paraffin-embeddedtesticular tissue (from which the explant cultures were de-rived) using the same antibodies resulted in a selective stain-ing of peritubular cells, proving clearly the peritubular originof HTPCs (Fig. 2, E and F).
Using electron microscopy experiments, electron dense vesicles were detected in the cytoplasm of HTPCs (Fig. 3A).
Several authors (14, 15) previously reported the occurrenceof so-called intracellular collagen fibers in proliferating myo-fibroblasts; the vesicles found in HTPCs may represent or berelated to intracellular collagen fibers, although the finalproof for this is still missing.
HTPCs express several genes coding for proteins of the extracellular matrix and basement membrane, such as col-lagen-I, collagen-IV, collagen-XVIII, fibronectin, secreted FIG. 2. HTPCs show characteristics of smooth muscle cells and fi- protein acidic and rich in cystein (SPARC), and laminin (Fig.
broblasts and originate from the peritubular region. HTPCs are pos- 3B). The synthesis of the proteins collagen-I and fibronectin itive for smooth muscle cell and fibroblast markers [␣-smooth muscle was shown by immunohistochemistry (Fig. 3, C and D).
actin (A) and CD90/Thy-1 (B)]. Sections of the corresponding testic-ular tissue from which the explant cultures were derived contain HTPCs as model to investigate effects of MC products on ␣-smooth muscle actin-positive and CD90/Thy-1-positive cells in the human peritubular cells peritubular region (E and F). In addition to peritubular cells, smoothmuscle cells of small blood vessels are also positive for ␣-smooth To examine the suitability of the established cell culture muscle actin (E, arrowhead). Respective negative controls performed model of human peritubular cells as a system for the inves- by omitting the primary antibody are shown in C, D, G, and H. Bars,40 ␮m.
tigation of paracrine, in particular, MC-mediated effects, thecapacity of HTPCs to react to the two major MC secretory These cells are involved in the development of tubular fi- products histamine and tryptase was investigated. Receptors brosis, a hallmark of infertility in men, which is based on for tryptase (PAR2) and histamine (H1) were shown to be fibrotic remodeling and thickening of the peritubular region expressed in HTPCs by RT-PCR (Fig. 4). Changes in intra- due to increased cell proliferation and extracellular matrix cellular calcium levels were evaluated after stimulation with production (16, 17).
histamine and the PAR2 agonist SLIGKV. Both substances Numbers of testicular MCs (1, 17, 18) and other immune induced a reversible increase in intracellular calcium levels, cells such as macrophages (13) and lymphocytes (17) are proving the response of HTPCs to the two major MC prod- increased in infertile patients, but their involvement in tu- ucts (Fig. 4). The effect of histamine was mediated via H1 bular fibrosis is largely unknown. We and others (2– 4, 17) because the specific H1 blocker pyrilamine was able to inhibit recently provided evidence that activated testicular MCs are histamine-induced calcium fluxes (data not shown).
promoting these fibrotic events and may therefore play acentral role in male idiopathic infertility. This hypothesis is supported by studies showing that MC blockers are able to This paper describes a rapid and economical method for increase fertility in subfertile patients (19 –21).
the isolation and cultivation of human peritubular cells.
Although the proposal of an altered MC-fibroblast inter-

Albrecht et al. • Isolation and Culturing of Human Peritubular Cells J Clin Endocrinol Metab, May 2006, 91(5):1956 –1960 FIG. 4. HTPCs possess functional tryptase and histamine receptorslinked to signal transduction events involving calcium. HTPCs ex-press receptors for tryptase (PAR2) and histamine (H1). IncubatingHTPCs with the PAR2 agonist SLIGKV (10 ␮M) or histamine (100 ␮M)results in an immediate and reversible increase in intracellular cal-cium levels. Note that histamine induces a biphasic signal. One rep-resentative experiment of a single-cell measurement of changes in FIG. 3. HTPCs display a characteristic ultrastructure and express intracellular calcium levels is shown. All experiments were indepen- extracellular matrix and basement membrane genes. HTPCs are dently carried out five times, with at least 10 single-cell determina- characterized by an elongated phenotype containing various electron- tions per experiment. Bold arrowheads denote the addition of buffer.
dense intracytoplasmic vesicles of unknown composition (A, arrows).
Gene array analyses reveal the expression of several molecules of theextracellular matrix and basement membrane (B). Collagen-I (C) and we cannot completely exclude the possibility that HTPC fibronectin (D) are two of the proteins typically produced by HTPCs.
cultures contain some vascular smooth muscle cells. Never- E and F show respective negative controls performed by omitting theprimary antibody. Bars, 5 ␮m (A) and 35 ␮m (C–F). Asterisks (B) theless, because the explants consisted mainly of seminifer- denote basement membrane proteins. One representative array is ous tubules with very few testicular stroma attached (Fig.
1A) and because the cells grew out of the tubule walls (Fig.
1B), we expect at most a very small fraction of vascular action in the development of male infertility is very tempting smooth muscle cells within the HTPC population.
and also supported by one of our previous studies evaluating Gene arrays and immunohistochemistry revealed the ex- the effects of MC products on human fetal foreskin fibro- pression of several genes and proteins coding for basement blasts (3), the final proof of such an interaction is missing and membrane components (e.g. collagen-IV, fibronectin, lami- requires the investigation of human peritubular cell systems.
nin, and secreted protein acidic and rich in cystein). Base- Regarding the existence of human peritubular cell models, ment membrane proteins have been shown to be expressed we are aware of only one approach. Cigorraga et al. (22) by peritubular cells of the rat testis (23, 24), and Pollanen et described a method for the isolation and culture of human al. (25) described the presence of laminin and type IV col- peritubular cells from prepubertal patients. However, to the lagen in the myoid cell layers of human seminiferous tubules.
best of our knowledge, there is no in vitro cell culture system Therefore, these results further substantiate the peritubular of adult human peritubular cells available so far.
characteristics of HTPCs.
In the study presented, we address the lack of human Tryptase and histamine are typical mediators of activated peritubular cell models and describe a method to obtain testicular MCs and are believed to exert various effects on HTPCs for in vitro studies using testicular biopsy explants.
nearby cells in the human testis (1, 2, 16, 17). To examine the Cells growing out of the explants displayed an elongated suitability of the established cell culture model of human phenotype. Immunostaining of the cells and sections of the peritubular cells as a system for the investigation of MC- corresponding testicular tissue for smooth muscle cell- and mediated effects, the ability of HTPCs to react to the two fibroblast-specific markers revealed the origin of the cells major MC secretory products, histamine and tryptase, was from the peritubular region. In tissue sections, besides the investigated. Both the tryptase analog SLIGKV and hista- peritubular cells, smooth muscle cells of small blood vessels mine induced a reversible increase of intracellular calcium also stained positive with the anti-␣-smooth muscle actin levels. Because SLIGKV is specific for the PAR2 receptor (26, antibody, confirming the specificity of the used marker. A 27) and because histamine-induced calcium fluxes in fibro- similar staining pattern was observed, with the fibroblast- blasts are primarily mediated via the H1 receptor (28), we specific antibody (CD90/Thy-1) and peritubular cells as well suggest that these receptors are mainly responsible for the as vascular smooth muscle cells (data not shown). Therefore, induction of calcium signaling events in HTPCs.
J Clin Endocrinol Metab, May 2006, 91(5):1956 –1960 Albrecht et al. • Isolation and Culturing of Human Peritubular Cells In summary, testicular biopsy material was derived from for human and bovine growth hormones on age-related changes in ovarian vasectomized patients displaying normal spermatogenesis.
morphology in mice. Anat Rec 227:175–186 10. Mayerhofer A, Hohne-Zell B, Gamel-Didelon K, Jung H, Redecker P, Grube
Although we propose that HTPC cultures represent cells of D, Urbanski HF, Gasnier B, Fritschy JM, Gratzl M 2001 ␥-Aminobutyric acid
the normal testis, the majority of vasectomized men develop (GABA): a para- and/or autocrine hormone in the pituitary. FASEB J 15:1089 – sperm antibodies, which may have an impact on sperm func- 11. Fritz S, Fohr KJ, Boddien S, Berg U, Brucker C, Mayerhofer A 1999 Functional
tion and fertility (29).
and molecular characterization of a muscarinic receptor type and evidence for We conclude that isolated HTPCs are a readily available expression of choline-acetyltransferase and vesicular acetylcholine transporterin human granulosa-luteal cells. J Clin Endocrinol Metab 84:1744 –1750 model for adult human peritubular cells because they can be 12. Mayerhofer A, Hemmings Jr HC, Snyder GL, Greengard P, Boddien S, Berg
obtained from small amounts of testicular tissue. The estab- U, Brucker C 1999 Functional dopamine-1 receptors and DARPP-32 are ex-
lished culture system will lead to a better understanding of pressed in human ovary and granulosa luteal cells in vitro. J Clin EndocrinolMetab 84:257–264 the pathogenic determinants (30) and significance of fibrotic 13. Frungieri MB, Calandra RS, Lustig L, Meineke V, Kohn FM, Vogt HJ,
processes underlying male idiopathic infertility.
Mayerhofer A 2002 Number, distribution pattern, and identification of mac-
rophages in the testes of infertile men. Fertil Steril 78:298 –306
14. Dominguez-Malagon H 2004 Intracellular collagen and fibronexus in fibro-
matosis and other fibroblastic tumors. Ultrastruct Pathol 28:67–73 15. Welsh RA, Meyer AT 1967 Intracellular collagen fibers. In human mesenchy-
We thank Dr. Lars Kunz, Dr. Monica Frungieri, and Christoph Schell mal tumors and inflammatory states. Arch Pathol 84:354 –362 for discussion and Annette Krieger, Gabriele Terfloth, and Barbara 16. Apa DD, Cayan S, Polat A, Akbay E 2002 Mast cells and fibrosis on testicular
Zschiesche for technical assistance. We especially thank Astrid Tiefen- biopsies in male infertility. Arch Androl 48:337–344 bacher for cell culture work.
17. Hussein MR, Abou-Deif ES, Bedaiwy MA, Said TM, Mustafa MG, Nada E,
Ezat A, Agarwal A 2005 Phenotypic characterization of the immune and mast
Received September 30, 2005. Accepted February 7, 2006.
cell infiltrates in the human testis shows normal and abnormal spermatogen- Address all correspondence and requests for reprints to: Professor esis. Fertil Steril 83:1447–1453 18. Yamanaka K, Fujisawa M, Tanaka H, Okada H, Arakawa S, Kamidono S 2000
Artur Mayerhofer, M.D., Institute of Anatomy, Ludwig-Maximilians- Significance of human testicular mast cells and their subtypes in male infer- University, Biedersteiner Strasse 29, 80802 Munich, Germany. E-mail: tility. Hum Reprod 15:1543–1547 19. Cayan S, Apa DD, Akbay E 2002 Effect of fexofenadine, a mast cell blocker,
This work was supported by the German Research Foundation (Deut- in infertile men with significantly increased testicular mast cells. Asian J sche Forschungsgemeinschaft) Ma 1080/16-1.
Androl 4:291–294 M.A., R.R., F.M.K., J.U.S., and A.M. have nothing to declare.
20. Hibi H, Kato K, Mitsui K, Taki T, Yamada Y, Honda N, Fukatsu H, Yamamoto
M 2001 The treatment with tranilast, a mast cell blocker, for idiopathic oli-
gozoospermia. Arch Androl 47:107–111 21. Matsuki S, Sasagawa I, Suzuki Y, Yazawa H, Tateno T, Hashimoto T, Nakada
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2. Albrecht M, Frungieri MB, Gonzalez-Calvar S, Meineke V, Kohn FM, May-
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23. Konrad L, Albrecht M, Renneberg H, Ulrix W, Hoeben E, Verhoeven G,
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Microsoft word - pharmacy warfarin standing order signed.doc

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