Characterization of a non-human primate model for the study of testicular peritubular cells—comparison with human testicular peritubular cells

Characterization of a non-human primate model for the study of testicular peritubular... Abstract STUDY QUESTION Are monkey testicular peritubular cells (MKTPCs) from the common marmoset monkey (Callithrix jacchus) a suitable translational model for the study of human testicular peritubular cells (HTPCs)? SUMMARY ANSWER MKTPCs can be isolated and propagated in vitro, retain characteristic markers for testicular peritubular cells and their proteome strongly (correlation coefficient of 0.78) overlaps with the proteome of HTPCs. WHAT IS KNOWN ALREADY Smooth-muscle-like peritubular cells form the wall of seminiferous tubules, transport sperm, are immunologically active, secrete a plethora of factors and may contribute to the spermatogonial stem cell niche. Mechanistic studies are hampered by heterogeneity of human samples. STUDY DESIGN, SIZE, DURATION We established a culture method for MKTPCs and characterized these cells from six young adult animals (2–3 years). To examine whether they qualify as a translational model we also examined HTPCs from seven men and compared the proteomes of both groups. PARTICIPANTS/MATERIALS, SETTING, METHODS We used explant cultures to obtain MKTPCs, which express smooth muscle markers (calponin (CNN1), smooth muscle actin (ACTA2)), lack FSH-receptors (FSHR) and LH-receptors (LHCGR), but possess androgen receptors (AR). MKTPCs can be passaged at least up to eight times, without discernable phenotypic changes. Mass-spectrometry-based analyses of the MKTPC and HTPC proteomes were performed. MAIN RESULTS AND THE ROLE OF CHANCE We established a method for isolation and cultivation of MKTPCs, and provide a comprehensive analysis of their protein repertoire. The results let us conclude that MKTPCs are suitable as a non-human primate model to study peritubular cell functions. LARGE SCALE DATA List of identified proteins in MKTPCs by liquid chromatography–tandem mass spectrometry is accessible at the ProteomeXchange (identifier PXD009394). LIMITATIONS, REASON FOR CAUTION This is an in vitro cellular non-human primate model used to provide a window into the role of these cells in the human testis. WIDER IMPLICATIONS OF THE FINDINGS Previous studies with HTPCs from patients revealed a degree of heterogeneity, possibly due to age, lifestyle and medical history of the individual human donors. We anticipate that the new translational model, derived from young healthy non-human primates, may allow us to circumvent these issues and may lead to a better understanding of the role of peritubular cells. STUDY FUNDING AND COMPETION OF INTEREST(S) This work was supported by grants from the Deutsche Forschungsgemeinschaft (MA 1080/27-1; AR 362/9-1; BE 2296/8-1). The authors declare no competing financial interests. testis, cellular model, proteome, marmoset monkey, non-human primate Introduction The human testis in health and disease cannot be readily studied. Cellular models, however, allow insights and human testicular peritubular cells (HTPCs) derived from individual patients represent such a model. These cells form the wall of seminiferous tubules and can be isolated via explant culture from very small fragments obtained during surgical procedures (Albrecht et al., 2006; Schell et al., 2008; Mayerhofer, 2013). They can be passaged and examined by cellular and molecular techniques. The results obtained, in combination with studies on human testicular sections, led to the insight that HTPCs play an important role in the human testis in health and disease. These smooth-muscle-like cells are associated with the transport of immotile sperm, but also possess secretory functions. Glial cell line-derived neurotrophic factor (GDNF) is a factor secreted by these cells, which is required for renewal of spermatogonial stem cells. This indicates a contribution to the spermatogonial stem cell niche (Spinnler et al., 2010), a view supported by recent studies in mice (Chen et al., 2014, 2016). HTPCs also secrete extracellular matrix components, including decorin (DCN) and biglycan (BGN) (Flenkenthaler et al., 2014). The latter is increased in infertile patients and, as we showed, can activate Toll-like receptors (TLRs) of HTPCs. Thereby it induces inflammatory reactions, namely secretion of C–C motif chemokine ligand 2 (CCL2), pentraxin 3 (PTX3) or interleukin 6 (IL6) (Mayer et al., 2016). Taken together, the results support important roles of HTPCs in male fertility and infertility. HTPCs were also shown to be plastic cells and their ability to differentiate towards a steroidogenic, presumably Leydig cell type, became evident (Landreh et al., 2014). The last-mentioned study showed inter-individual differences in the basal and the forskolin-stimulated steroidogenic capacities. Further studies (Welter et al., 2014; Mayer et al., 2016), which examined immunological aspects, revealed that HTPCs derived from individual patients are heterogeneous with respect to the amounts of secreted factors. This may be due to the differences in age, lifestyle and medical history of the patients. While this heterogeneity is instructive and may be of interest in view of an improved understanding of inter-individual differences and personalized treatment option in the future, these differences also make general in-depth mechanistic studies difficult. To circumvent these issues, and to be able to study peritubular cell function and regulation in greater detail, we sought to establish a translational model. In search for suitable model organism, we turned to the common marmoset monkey (Michel and Mahouy, 1990; Mansfield, 2003; Zuhlke and Weinbauer, 2003; Li et al., 2005). Marmosets are non-human primates, which are often used in reproductive research. Here we describe the isolation, cultivation and proteomic characterization of monkey testicular peritubular cells (MKTPCs). We propose that they are an apt model for HTPCs because of their similarity to human and the possibility to control confounding lifestyle issues. Materials and Methods Animals Common marmoset monkeys (Callithrix jacchus) stem from the self-sustaining marmoset monkey colony of the German Primate Center (Deutsches Primatenzentrum; DPZ, Göttingen). All animals used for this study were between 2 and 3 years old, i.e. young adult, sexually mature healthy animals (Li et al., 2005). Marmoset monkey testes were obtained from animals euthanized for scientific purposes unrelated to this study or castrated for colony management purposes. Euthanasia and castration were performed by experienced veterinarians. Parts of the testes were fixed in Bouin’s fixative and embedded in paraffin, for later sectioning and immunohistochemistry, other parts were used for isolation of MKTPCs by explant culture. Ethical approval Organ retrievals from Callithrix jacchus were carried out in accordance with relevant institutional guidelines and legal regulations, namely the German Animal Protection Act. The local ethical committees (Ethikkommission, Technische Universität München, Fakultät für Medizin, München, project number 5158/11) approved the study with human tissues. All experiments were performed in accordance with relevant guidelines and regulations. Isolation and cultivation of MKTPCs Isolation and cultivation of MKTPCs was performed as described in detail previously for human samples (Albrecht et al., 2006). In brief, small pieces of testicular tissue were seeded onto cell culture dishes. The explant cultures were incubated under humidified conditions (37°C, 5% CO2) until the cells started to grow out of the tubular wall. They were cultivated and propagated in Dulbecco’s modified Eagle’s medium (DMEM) high glucose (Gibco, Paisley, UK) containing 10% fetal calf serum (Capricorn Scientific GmbH, Ebsdorfergrund, Germany) and 1% penicillin/streptomycin (Life Technologies, Carlsbad, CA, USA). MKTPCs in passages 2–3 were used for proteomic studies, for RT-PCR cells from early (3) and advanced passages (8) were used. RNA isolation and RT-PCR Total RNA from cultured MKTPC was prepared as described earlier (Welter et al., 2014) using the RNeasy microkit (Qiagen, Hilden, Germany). In brief, a total amount of 200 ng of RNA was subjected to reverse transcription, using random primers (15-mer) and SuperScript II Reverse Transcriptase, 200 U/μl (Invitrogen GmbH, Darmstadt, Germany). Intron-spanning primer pairs amplified specific products for ACTA2, CNN1, AR, GDNF, FSHR, GATA4, LHCGR, INSL3, DCN, BGN, CCL2, PTX3, IL6, CD3e, TPSG1 and CMA1 (Table I). PCR consisted of 35 cycles of denaturing (at 95°C for 60 s) annealing (at 60°C) and extension (at 72°C for 45 s). PCR products were visualized by midori green (NIPPON Genetics EUROPE GmbH, Düren, Germany) staining in agarose (Biozym Scientific GmbH, Oldendorf, Germany) gels. Positive controls consisted of Callithrix jacchus whole testis lysate cDNA (+). Negative controls were performed by excluding reverse transcriptase (−RT), and a non-template reaction (−). The identities of all PCR products were verified by sequencing. Table I Oligonucleotide primers used for PCR studies Gene Gene name Reference ID Nucleotide sequence Amplicon size RPL19 Ribosomal Protein L19 NM_001330200.1 5′-AGG CAC ATG GGC ATA GGT AA-3′ 5′-CCA TGA GAA TCC GCT TGT TT-3′ 199 ACTA2 Actin, Alpha 2, Smooth Muscle, Aorta NM_001613.2 5′-ACC CAG TGT GGA GCA GCC C-3′ 5′-TTG TCA CAC ACC AAG GCA GT-3′ 100 CNN1 Calponin 1 XM_017026289.1 5′-CGA AGA CGA AAG GAA ACA AGG T-3′ 5′-GCT TGG GGT CGT AGA GGT G-3′ 183 AR Androgen Receptor XM_008989422.2 5′-GCC CCT GAT CTG GTT TTC AA-3′ 5′-CCA CTG GAA TAA TGC TGA AGA GT-3′ 163 GDNF Glial Cell Derived Neurotrophic Factor NM_000514.3 5′-GCA GAC CCA TCG CCT TTG AT-3′ 5′-ATC CAC ACC TTT TAG CGG AAT G-3′ 93 FSHR FSH Receptor NM_000145.2 5′-CTG CTC CTG GTC TCT TTG CT-3′ 5′-GGT CCC CAA ATC CTG AAA AT-3′ 208 GATA4 GATA Binding Protein 4 NM_002052 5′-TCC AAA CCA GAA AAC GGA AG-3′ 5′-CTG TGC CCG TAG TGA GAT GA-3′ 187 LHCGR Luteinizing Hormone/Choriogonadotropin Receptor NM_001301843.1 5′-CTG GAT GCC ACG CTG ACT-3′ 5′-ACG CAC TCT GTC CAC TCT-3′ 100 INSL3 Insulin Like 3 XM_017968209.1 5′-ACT TCT CAC CAT CATCGC CA-3′ 5′-GAG GGT CAG CAG GTCTTG TT-3′ 96 CD3e CD3e Molecule XM_009006841.1 5′-ATCTGCATCACTCTGGGCTT-3′ 5′-TGGGCTCATAGTCTGGGTTG-3′ 160 TPSG1 Tryptase Gamma 1 NM_001204310.1 5′-CAT TGT GAG CTG GGG TGA AG-3′ 5′-AGA CCA GCA GAA GGA AGA GG-3′ 182 CMA1 Chymase 1 XM_002753692.4 5′-AAG TTG AAG GAG AAA GCC AGC-3′ 5′-TTC AAC ACA CCT GTT CTT CCC-3′ 125 DCN Decorin XM_002752816.3 5′-ATG AGG CTT CTG GGA TAG GC-3′ 5′-GTC CAG CAA AGT CGT GTC AG-3′ 170 BGN Biglycan XM_008990069.2 5′-GAG ACC CTG AAC GAA CTC CA-3′ 5′-TTG TTG TCC AAG TGC AGC TC-3′ 176 CCL2 C–C Motif Chemokine Ligand 2 NM_002982 5′-CAG CCA GAT GCA ATC AAT GCC-3′ 5′-TGG AAT CCT GAA CCC ACT TCT-3′ 190 PTX3 Pentraxin 3 NM_002852.3 5′-TAG TGT TTG TGG TGG GTG GA-3′ 5′-TGT GAG CCC TTC CTC TGA AT-3′ 110 IL6 Interleukin 6 XM_017975106.1 5′-AAG AGG TAG CTG CCC CAA AT-3′ 5′-AGT GCC TCT TTG CTG CTT TC-3′ 145 Gene Gene name Reference ID Nucleotide sequence Amplicon size RPL19 Ribosomal Protein L19 NM_001330200.1 5′-AGG CAC ATG GGC ATA GGT AA-3′ 5′-CCA TGA GAA TCC GCT TGT TT-3′ 199 ACTA2 Actin, Alpha 2, Smooth Muscle, Aorta NM_001613.2 5′-ACC CAG TGT GGA GCA GCC C-3′ 5′-TTG TCA CAC ACC AAG GCA GT-3′ 100 CNN1 Calponin 1 XM_017026289.1 5′-CGA AGA CGA AAG GAA ACA AGG T-3′ 5′-GCT TGG GGT CGT AGA GGT G-3′ 183 AR Androgen Receptor XM_008989422.2 5′-GCC CCT GAT CTG GTT TTC AA-3′ 5′-CCA CTG GAA TAA TGC TGA AGA GT-3′ 163 GDNF Glial Cell Derived Neurotrophic Factor NM_000514.3 5′-GCA GAC CCA TCG CCT TTG AT-3′ 5′-ATC CAC ACC TTT TAG CGG AAT G-3′ 93 FSHR FSH Receptor NM_000145.2 5′-CTG CTC CTG GTC TCT TTG CT-3′ 5′-GGT CCC CAA ATC CTG AAA AT-3′ 208 GATA4 GATA Binding Protein 4 NM_002052 5′-TCC AAA CCA GAA AAC GGA AG-3′ 5′-CTG TGC CCG TAG TGA GAT GA-3′ 187 LHCGR Luteinizing Hormone/Choriogonadotropin Receptor NM_001301843.1 5′-CTG GAT GCC ACG CTG ACT-3′ 5′-ACG CAC TCT GTC CAC TCT-3′ 100 INSL3 Insulin Like 3 XM_017968209.1 5′-ACT TCT CAC CAT CATCGC CA-3′ 5′-GAG GGT CAG CAG GTCTTG TT-3′ 96 CD3e CD3e Molecule XM_009006841.1 5′-ATCTGCATCACTCTGGGCTT-3′ 5′-TGGGCTCATAGTCTGGGTTG-3′ 160 TPSG1 Tryptase Gamma 1 NM_001204310.1 5′-CAT TGT GAG CTG GGG TGA AG-3′ 5′-AGA CCA GCA GAA GGA AGA GG-3′ 182 CMA1 Chymase 1 XM_002753692.4 5′-AAG TTG AAG GAG AAA GCC AGC-3′ 5′-TTC AAC ACA CCT GTT CTT CCC-3′ 125 DCN Decorin XM_002752816.3 5′-ATG AGG CTT CTG GGA TAG GC-3′ 5′-GTC CAG CAA AGT CGT GTC AG-3′ 170 BGN Biglycan XM_008990069.2 5′-GAG ACC CTG AAC GAA CTC CA-3′ 5′-TTG TTG TCC AAG TGC AGC TC-3′ 176 CCL2 C–C Motif Chemokine Ligand 2 NM_002982 5′-CAG CCA GAT GCA ATC AAT GCC-3′ 5′-TGG AAT CCT GAA CCC ACT TCT-3′ 190 PTX3 Pentraxin 3 NM_002852.3 5′-TAG TGT TTG TGG TGG GTG GA-3′ 5′-TGT GAG CCC TTC CTC TGA AT-3′ 110 IL6 Interleukin 6 XM_017975106.1 5′-AAG AGG TAG CTG CCC CAA AT-3′ 5′-AGT GCC TCT TTG CTG CTT TC-3′ 145 Table I Oligonucleotide primers used for PCR studies Gene Gene name Reference ID Nucleotide sequence Amplicon size RPL19 Ribosomal Protein L19 NM_001330200.1 5′-AGG CAC ATG GGC ATA GGT AA-3′ 5′-CCA TGA GAA TCC GCT TGT TT-3′ 199 ACTA2 Actin, Alpha 2, Smooth Muscle, Aorta NM_001613.2 5′-ACC CAG TGT GGA GCA GCC C-3′ 5′-TTG TCA CAC ACC AAG GCA GT-3′ 100 CNN1 Calponin 1 XM_017026289.1 5′-CGA AGA CGA AAG GAA ACA AGG T-3′ 5′-GCT TGG GGT CGT AGA GGT G-3′ 183 AR Androgen Receptor XM_008989422.2 5′-GCC CCT GAT CTG GTT TTC AA-3′ 5′-CCA CTG GAA TAA TGC TGA AGA GT-3′ 163 GDNF Glial Cell Derived Neurotrophic Factor NM_000514.3 5′-GCA GAC CCA TCG CCT TTG AT-3′ 5′-ATC CAC ACC TTT TAG CGG AAT G-3′ 93 FSHR FSH Receptor NM_000145.2 5′-CTG CTC CTG GTC TCT TTG CT-3′ 5′-GGT CCC CAA ATC CTG AAA AT-3′ 208 GATA4 GATA Binding Protein 4 NM_002052 5′-TCC AAA CCA GAA AAC GGA AG-3′ 5′-CTG TGC CCG TAG TGA GAT GA-3′ 187 LHCGR Luteinizing Hormone/Choriogonadotropin Receptor NM_001301843.1 5′-CTG GAT GCC ACG CTG ACT-3′ 5′-ACG CAC TCT GTC CAC TCT-3′ 100 INSL3 Insulin Like 3 XM_017968209.1 5′-ACT TCT CAC CAT CATCGC CA-3′ 5′-GAG GGT CAG CAG GTCTTG TT-3′ 96 CD3e CD3e Molecule XM_009006841.1 5′-ATCTGCATCACTCTGGGCTT-3′ 5′-TGGGCTCATAGTCTGGGTTG-3′ 160 TPSG1 Tryptase Gamma 1 NM_001204310.1 5′-CAT TGT GAG CTG GGG TGA AG-3′ 5′-AGA CCA GCA GAA GGA AGA GG-3′ 182 CMA1 Chymase 1 XM_002753692.4 5′-AAG TTG AAG GAG AAA GCC AGC-3′ 5′-TTC AAC ACA CCT GTT CTT CCC-3′ 125 DCN Decorin XM_002752816.3 5′-ATG AGG CTT CTG GGA TAG GC-3′ 5′-GTC CAG CAA AGT CGT GTC AG-3′ 170 BGN Biglycan XM_008990069.2 5′-GAG ACC CTG AAC GAA CTC CA-3′ 5′-TTG TTG TCC AAG TGC AGC TC-3′ 176 CCL2 C–C Motif Chemokine Ligand 2 NM_002982 5′-CAG CCA GAT GCA ATC AAT GCC-3′ 5′-TGG AAT CCT GAA CCC ACT TCT-3′ 190 PTX3 Pentraxin 3 NM_002852.3 5′-TAG TGT TTG TGG TGG GTG GA-3′ 5′-TGT GAG CCC TTC CTC TGA AT-3′ 110 IL6 Interleukin 6 XM_017975106.1 5′-AAG AGG TAG CTG CCC CAA AT-3′ 5′-AGT GCC TCT TTG CTG CTT TC-3′ 145 Gene Gene name Reference ID Nucleotide sequence Amplicon size RPL19 Ribosomal Protein L19 NM_001330200.1 5′-AGG CAC ATG GGC ATA GGT AA-3′ 5′-CCA TGA GAA TCC GCT TGT TT-3′ 199 ACTA2 Actin, Alpha 2, Smooth Muscle, Aorta NM_001613.2 5′-ACC CAG TGT GGA GCA GCC C-3′ 5′-TTG TCA CAC ACC AAG GCA GT-3′ 100 CNN1 Calponin 1 XM_017026289.1 5′-CGA AGA CGA AAG GAA ACA AGG T-3′ 5′-GCT TGG GGT CGT AGA GGT G-3′ 183 AR Androgen Receptor XM_008989422.2 5′-GCC CCT GAT CTG GTT TTC AA-3′ 5′-CCA CTG GAA TAA TGC TGA AGA GT-3′ 163 GDNF Glial Cell Derived Neurotrophic Factor NM_000514.3 5′-GCA GAC CCA TCG CCT TTG AT-3′ 5′-ATC CAC ACC TTT TAG CGG AAT G-3′ 93 FSHR FSH Receptor NM_000145.2 5′-CTG CTC CTG GTC TCT TTG CT-3′ 5′-GGT CCC CAA ATC CTG AAA AT-3′ 208 GATA4 GATA Binding Protein 4 NM_002052 5′-TCC AAA CCA GAA AAC GGA AG-3′ 5′-CTG TGC CCG TAG TGA GAT GA-3′ 187 LHCGR Luteinizing Hormone/Choriogonadotropin Receptor NM_001301843.1 5′-CTG GAT GCC ACG CTG ACT-3′ 5′-ACG CAC TCT GTC CAC TCT-3′ 100 INSL3 Insulin Like 3 XM_017968209.1 5′-ACT TCT CAC CAT CATCGC CA-3′ 5′-GAG GGT CAG CAG GTCTTG TT-3′ 96 CD3e CD3e Molecule XM_009006841.1 5′-ATCTGCATCACTCTGGGCTT-3′ 5′-TGGGCTCATAGTCTGGGTTG-3′ 160 TPSG1 Tryptase Gamma 1 NM_001204310.1 5′-CAT TGT GAG CTG GGG TGA AG-3′ 5′-AGA CCA GCA GAA GGA AGA GG-3′ 182 CMA1 Chymase 1 XM_002753692.4 5′-AAG TTG AAG GAG AAA GCC AGC-3′ 5′-TTC AAC ACA CCT GTT CTT CCC-3′ 125 DCN Decorin XM_002752816.3 5′-ATG AGG CTT CTG GGA TAG GC-3′ 5′-GTC CAG CAA AGT CGT GTC AG-3′ 170 BGN Biglycan XM_008990069.2 5′-GAG ACC CTG AAC GAA CTC CA-3′ 5′-TTG TTG TCC AAG TGC AGC TC-3′ 176 CCL2 C–C Motif Chemokine Ligand 2 NM_002982 5′-CAG CCA GAT GCA ATC AAT GCC-3′ 5′-TGG AAT CCT GAA CCC ACT TCT-3′ 190 PTX3 Pentraxin 3 NM_002852.3 5′-TAG TGT TTG TGG TGG GTG GA-3′ 5′-TGT GAG CCC TTC CTC TGA AT-3′ 110 IL6 Interleukin 6 XM_017975106.1 5′-AAG AGG TAG CTG CCC CAA AT-3′ 5′-AGT GCC TCT TTG CTG CTT TC-3′ 145 Immunohistochemistry Immunohistochemistry was performed as described (Schell et al., 2010). Parts of the testicular tissue, which were also used for explant cultures were fixed and embedded in paraffin. Sections (5 μm) of marmoset monkey testicular tissue were incubated with monoclonal mouse antibody against ACTA2 (#A5228, 1:1000; Sigma, St. Louis, MO, USA), monoclonal rabbit antibody against Calponin-1 (C-term) (#1806-1, 1:250; Epitomics, Cambridge, UK) and AR (#5153, 1:400; Cell Signaling, Cambridge, UK). Controls consisted of incubation with non-immune normal goat serum instead of specific antibodies. Hematoxylin counterstained the cell nuclei. Sections were examined with a Zeiss Axiovert microscope, an Insight Camera (18.2 Color Mosaik) and Spot advanced software 4.6 (both from SPOT Imaging Solutions, Sterling Heights, MI, USA). Immunofluorescence MKTPCs were seeded onto cover slips and incubated overnight. They were fixed with 3.7% formaldehyde (Sigma) for 10 min, washed with 0.1% Triton X-100/phosphate buffered saline (PBS) (Sigma) and permeabilized with ice cold 0.2% Triton X-100/PBS for 10 min. Cells were blocked with 0.1% Triton X-100/PBS + 5% goat normal serum (Sigma). The same primary antibodies as for the immunohistochemistry were used and were diluted in 0.1% Triton X-100/PBS + 5% goat normal serum, CNN1 1:100 (Epitomics), ACTA2 1:200 (Sigma), AR 1:100 (Cell Signaling) and incubated for 2 h at room temperature. Secondary antibody for CNN1 and AR, goat anti-rabbit alexa flour 488 1:1000 (Thermo Fisher Scientific, Waltham, MA, USA) for ACTA2, goat anti-mouse alexa fluor 555 1:1000, were incubated for 1.5 h at room temperature. Cells were washed and counterstained with 1.5 μg/ml DAPI for 5 min. For the control, normal goat serum was used instead of the primary antibody. Examination was performed with a fluorescence microscope (Zeiss, Oberkochen, Germany). Human peritubular cell isolation and culture The procedure involving human peritubular cells (HTPCs) isolation and culture from human testicular tissue samples exhibiting normal spermatogenesis was described previously (Albrecht et al., 2006; Schell et al., 2008). The patients (n = 7; aged 39–55 years) had granted written informed consent for scientific purposes. Cells were cultivated in DMEM High Glucose (Gibco) supplemented with 10% fetal bovine serum (Capricorn Scientific) and 1% penicillin/streptomycin (Biochrom, Berlin, Germany) under humidified conditions (37°C, 5% CO2). Cells in passages 3–7 were harvested for proteomic procedures, as described (Flenkenthaler et al., 2014). Nano-liquid chromatography–tandem mass spectrometry Cell pellets were resuspended in 8 M Urea in 50 mM ammonium bicarbonate, sonicated for 5 min at 4°C and homogenized using QIAshredders (Qiagen, Hilden, Germany) at 2500 g for 1 min. Protein concentration was determined using the Pierce 660 nm assay (Thermo Scientific, San Jose, CA). The 10 μg protein were reduced in 4 mM dithiothreitol (DTT) and 2 mM tris(2-carboxyethyl)phosphine for 30 min at 56°C followed by an alkylation step in 8 mM iodoacetamide (IAA) at room temperature in the dark for 30 min. Remaining IAA was quenched at a final concentration of 10 mM DTT. The samples underwent a first digestion step with Lys C (enzyme/substrate: 1/100; Wako, Neuss, Germany) at 37°C for 4 h, were diluted with ammonium bicarbonate to a concentration of 1 M urea and digested overnight with porcine trypsin (enzyme/substrate: 1/50; Promega, Madison, WI, USA) at 37°C. The 2.5 μg of peptides dissolved in 0.1% formic acid (FA) were subjected to liquid chromatography-electrospray ionization–tandem mass spectrometry (LC–MS/MS) analysis. LC was performed on an Ultimate 3000 RS system (Dionex, Sunnyvale, CA, USA). Peptide samples were first trapped on a C18 trap column (μ-Precolumn, C18 PepMap 100, 5 μm, Thermo Scientific, San Jose, CA) at a flow rate of 30 μL/min and separated on a C18 nano-flow column (Acclaim PepMap RSLC, 2 μm, 75 μm × 50 cm) at a flow rate of 0.2 μL/min using the following consecutive gradients: 5–25% B for 285 min and 25–50% B for 30 min (A: 0.1% FA in water, B: 0.1% FA in acetonitrile). Electrospray ionization was done with an uncoated SilicaTip (FS360-20-10-N-20-C15; New Objective, Woburn, MA) and a needle voltage of 2.3 kV. For MS data acquisition, a data dependent top 70 CID method was performed on a 5600+ mass spectrometer (Sciex, Concord, Canada). Data analysis Mass spectrometry (MS) raw data were processed using the MaxQuant software package (version: 1.6.0.1) (Cox and Mann, 2008). The data were searched separately for both species, using the UniProt subset for Callithrix jacchus for MKTPCs and the human Swiss-Prot subset for HTPCs (both: Release 2017/06), each augmented with the MaxQuant common contaminants database. Identification was performed with the ‘match between run’ feature enabled and a target decoy search strategy (resulting in a false discovery rate of 1%). For quantification, the MaxQuant label-free quantification strategy was applied. Data analysis and statistics were performed with Perseus (version: 1.5.8.5). For the scatterplot of MKTPC and HTPC datasets, the protein families contained in both datasets were merged into one matrix using the associated gene name. The ‘circoletto’ graph was done for the 100 most abundant protein identifications of each species with the corresponding online tool hosted by the bioinformatics analysis team (http://tools.bat.infspire.org/circoletto) using the following deviations from the default settings: e-value cutoff 10−15; e-value for coloring; use (score-min)/(max-min) ratio to assign colors. With the blastp online tool (https://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE=Proteins) the top 25 proteins were blasted against the human subset of the Swiss-Prot database. For the spectral counting approach, MS data were searched with Mascot (V 2.4.0) and analyzed with Scaffold (V 4.1.1) using the same databases as for the MaxQuant analysis. For abundance ranks, the averaged spectral counts were used. Functional classification of the proteins was done with the Panther GO analysis online tool (http://pantherdb.org) using gene names and the GO ‘biological process’ and ‘cellular process’ database. Results Isolation and initial characterization Using a similar approach as employed for HTPCs previously (Albrecht et al., 2006), we were able to isolate and culture MKTPCs. Explant cultures of marmoset testicular tissue fragments, small pieces of tissue (1–2 mm), were seeded onto cell culture dishes. The cells started growing out of the walls of the seminiferous tubules within 7–14 days, and small spindle-like cells became visible. They proliferated and cells were propagated for several passages (Fig. 1). Figure 1 View largeDownload slide Testicular tissue-explant culture of MKTPCs. (A) Small pieces of testicular tissue consisting of tubules (depicted with (T)) were placed onto cell culture dishes. The cells grow out from the walls of the seminiferous tubules within 7–14 days, picture was captured on day 10. (B) Cells in passage 1, 8 weeks after tissue extraction. (C) MKTPCs in passage 4, the cells were sub-cultured and propagated for several passages. (Scale bars represent 25 μm). Figure 1 View largeDownload slide Testicular tissue-explant culture of MKTPCs. (A) Small pieces of testicular tissue consisting of tubules (depicted with (T)) were placed onto cell culture dishes. The cells grow out from the walls of the seminiferous tubules within 7–14 days, picture was captured on day 10. (B) Cells in passage 1, 8 weeks after tissue extraction. (C) MKTPCs in passage 4, the cells were sub-cultured and propagated for several passages. (Scale bars represent 25 μm). As shown by immunohistochemistry, sections of the corresponding testicular tissue, from which explant cultures were derived, contain ACTA2-, CNN1- and AR-positive peritubular cells. The corresponding MKTPCs in vitro are likewise positive for CNN1, ACTA2 and AR as shown by immunofluorescence (Fig. 2). Figure 2 View largeDownload slide Expression of peritubular cell markers in situ and in cultured MKTPCs shown by immunohistochemistry. (A–C). Smooth muscle (SM) markers (A), CNN1, (B), ACTA2 and (C), AR were detected in individual testicular tissue sections from MKTPC donor animals (scale bar 50 μm). (D–F) Immunofluorescence of SM-markers, CNN1 (MKTPCs, passage 6) and ACTA2 (MKTPCs, passage 6), and AR (MKTPCs, passage 3) (D1, E1, F1). Higher magnifications, corresponding to D–F, respectively. Insets in A–F show negative controls. Figure 2 View largeDownload slide Expression of peritubular cell markers in situ and in cultured MKTPCs shown by immunohistochemistry. (A–C). Smooth muscle (SM) markers (A), CNN1, (B), ACTA2 and (C), AR were detected in individual testicular tissue sections from MKTPC donor animals (scale bar 50 μm). (D–F) Immunofluorescence of SM-markers, CNN1 (MKTPCs, passage 6) and ACTA2 (MKTPCs, passage 6), and AR (MKTPCs, passage 3) (D1, E1, F1). Higher magnifications, corresponding to D–F, respectively. Insets in A–F show negative controls. Further, as shown by RT-PCR, MKTPCs (all from passages 3 and 8) are positive for smooth muscle (SM) cell markers, including CNN1, ACTA2, and for the peritubular cell marker AR. The cells lack markers for Leydig cells (LHCGR, INSL3) or Sertoli cells (FSHR, GATA4). Mast cell markers like tryptase (TPSG1) and chymase (CMA1) and the T-cell marker CD3e were not detected. Like HTPCs, they express GDNF, extracellular matrix components including DCN and BGN, also the inflammatory molecules CCL2, PTX3 or IL6 (RT-PCR) and are thus similar to HTCPs. The cells can be passaged and the characteristic markers for MKTPCs remain stable for at least up to eight passages (Fig. 3). Figure 3 View largeDownload slide Expression analysis by RT-PCR. (A) Cultured MKTPCs express characteristic markers, SM-markers, e.g. ACTA2, CNN1 and the AR, (B) but lack markers for Leydig cells (LHCGR, INSL3) and Sertoli cells (FSHR, GATA4), which were found in the positive control (+), cDNA from Callithrix jacchus whole testis lysate. (C) Extracellular matrix, DCN and BGN, the inflammatory markers CCL2, PTX3 and IL6 and the neurotrophic factor GDNF were also expressed by MKTPCs. Expression of RPL19 was used as reference gene. Note that immune cell markers (CD3e, TPSG1, CMA1), expressed in the testis, were not found in MKTPCs. Figure 3 View largeDownload slide Expression analysis by RT-PCR. (A) Cultured MKTPCs express characteristic markers, SM-markers, e.g. ACTA2, CNN1 and the AR, (B) but lack markers for Leydig cells (LHCGR, INSL3) and Sertoli cells (FSHR, GATA4), which were found in the positive control (+), cDNA from Callithrix jacchus whole testis lysate. (C) Extracellular matrix, DCN and BGN, the inflammatory markers CCL2, PTX3 and IL6 and the neurotrophic factor GDNF were also expressed by MKTPCs. Expression of RPL19 was used as reference gene. Note that immune cell markers (CD3e, TPSG1, CMA1), expressed in the testis, were not found in MKTPCs. Proteome analysis from MKTPCs and comparison with HTPCs Proteins of MKTPCs from six individual donors were investigated by LC–MS/MS. In total 2437 protein groups were identified (FDR < 1%) in MKTPCs of which 1825 protein groups could be quantified in at least three individual samples. The mass spectrometry proteomics data were deposited in the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD009394. The inter-individual variance was investigated by a multi-scatter plot analysis of all protein label-free quantification (LFQ) intensities between all MKTPC individuals (Fig. 4A). The Pearson correlation coefficient ranged between 0.93 and 0.97. Figure 4 View largeDownload slide (A) Multi-scatter plot of log2 Max-Quant LFQ protein intensity values of MKTPCs from six donors. The number in the upper left corner of each individual scatterplot shows the Pearson correlation. (B) Multi-scatter plot of log2 Max-Quant LFQ protein intensity values of HTPCs from seven donors. Figure 4 View largeDownload slide (A) Multi-scatter plot of log2 Max-Quant LFQ protein intensity values of MKTPCs from six donors. The number in the upper left corner of each individual scatterplot shows the Pearson correlation. (B) Multi-scatter plot of log2 Max-Quant LFQ protein intensity values of HTPCs from seven donors. Additionally, a set of HTPCs from seven individual human donors was analyzed using exactly the same parameters and compared to the results of the MKTPCs. From 3374 protein groups, which were identified in HTPCs, 2137 are contained in the MKTPC dataset, demonstrating that 88% (2137 out of 2437) of the proteins in MKTPCs are represented in the HTPC data. The inter-individual variance was again assessed using a multi-scatter plot between all individual donors, which results in a Pearson correlation coefficient between 0.93 and 0.96 (Fig. 4B). The means of the MaxQuant LFQ values were scatter plotted against each other, which results in a correlation coefficient of 0.78 (Fig. 5) between human and marmoset cells. To investigate the variability of this inter-species comparison, each individual of MKTPCs were plotted against all individual HTPC donors. The mean Pearson correlation coefficient was 0.75 with no significant outliers (Grubbs’ test for outliers; 99% confidence) and therefore showing a very homologous correlation pattern (Supplementary Information Fig. S1). Figure 5 View largeDownload slide Scatter plot of averaged log2 LFQ protein intensity values of MKTPCs and HTPCs. Figure 5 View largeDownload slide Scatter plot of averaged log2 LFQ protein intensity values of MKTPCs and HTPCs. From both datasets the 100 most abundant proteins were retrieved and analyzed with the circoletto tool to visualize sequence homology of highly expressed proteins between HTPC and MKTPC (Fig. 6). The resulting network shows sequence homologies (<e-value 10−15) between the most abundant proteins found in both species, of which a vast majority shows high degree of similarity. Furthermore, a BLAST analysis of the 25 most abundant MKTPC using the Human Swiss-Prot database reveals that 22 are showing sequence identities >95% (Table II). Two further proteins could be matched to their human counterparts with >85% identity and represent members of the tropomyosin family, and only one single protein (F7FP14; uncharacterized protein) could not unambiguously be assigned to a human protein. Figure 6 View largeDownload slide Circoletto analysis of the top 100 protein identifications found in HTPC and MKTPC. The left half circle shows HTPC proteins sorted by their abundance from highest at the top to the lowest at the bottom. The right half circle lists the top 100 proteins identified in MKTPC in the same order. Both groups are compared with respect to protein sequence similarity. Proteins are represented by boxes on the circle. The length of the boxes indicates the length of the protein sequences. Sequence similarities between two corresponding proteins (MKTPC vs HTPC) are represented by a ribbon connecting the two boxes (cutoff e-value 10−15). Different ribbon color shades were used for better distinction. Figure 6 View largeDownload slide Circoletto analysis of the top 100 protein identifications found in HTPC and MKTPC. The left half circle shows HTPC proteins sorted by their abundance from highest at the top to the lowest at the bottom. The right half circle lists the top 100 proteins identified in MKTPC in the same order. Both groups are compared with respect to protein sequence similarity. Proteins are represented by boxes on the circle. The length of the boxes indicates the length of the protein sequences. Sequence similarities between two corresponding proteins (MKTPC vs HTPC) are represented by a ribbon connecting the two boxes (cutoff e-value 10−15). Different ribbon color shades were used for better distinction. Table II The top 25 most abundant proteins in MKTPC. Sequence similarities with corresponding human proteins were determined by BLAST analysis and are indicated as ‘% identity’. Callithrix jacchus UniProt accession Callithrix jacchus Gene name Callithrix jacchus protein name Human UniProt accession % Identity F7IH64 ACTA2 Actin, alpha 2, smooth muscle, aorta P62736 100 B0KWW5 FLNA Filamin-A isoform 1,2 P21333 98.79 F7BQY8 VIM Vimentin P08670 99.36 F6ZZ90 MYH9 Myosin heavy chain 9 P35579 99.49 F7I1Q9 TAGLN Transgelin Q01995 99.5 F7I9U1 TPM2 Tropomyosin beta chain isoform 2 P07951 87.32 F7HTQ3 CALD1 Caldesmon 1 Q05682 95.33 F7I0W8 LOC100409006 Uncharacterized protein P63261 100 U3EQM9 ACTN4 Alpha-actinin-4 O43707 99.89 F6YS84 TPM1 Tropomyosin 1 P09493 85.92 U3BZ94 TUBB4B Tubulin beta chain P68371 99.33 U3CYB6 ENO1 Alpha-enolase isoform 1 P06733 99.31 F7I318 LGALS1 Galectin P09382 99.07 F7HZP3 TUBA1B Tubulin alpha chain P68363 100 F6RMA3 ANXA2 Annexin P07355 99.71 F7IHK8 PRDX1 Peroxiredoxin 1 Q06830 97.99 U3EKQ0 ACTN1 Actinin alpha 1 P12814 99.78 F7CXD3 EEF1A1 Elongation factor 1-alpha P68104 100 F6SQW1 PPIA Peptidyl-prolyl cis-trans isomerase P62937 99.78 U3FXT1 HSPA5 78 kDa glucose-regulated protein P11021 99.39 F7HVD2 HSPA8 Heat shock protein family A (Hsp70) member 8 P11142 99.85 F7FP14 Uncharacterized protein Q8N386 62.38 F6YTV2 LMNA Lamin A/C P02545 98.65 F6XL35 PKM Pyruvate kinase P14618 99.06 U3D5Z3 TLN1 Talin-1 Q9Y490 99.21 Callithrix jacchus UniProt accession Callithrix jacchus Gene name Callithrix jacchus protein name Human UniProt accession % Identity F7IH64 ACTA2 Actin, alpha 2, smooth muscle, aorta P62736 100 B0KWW5 FLNA Filamin-A isoform 1,2 P21333 98.79 F7BQY8 VIM Vimentin P08670 99.36 F6ZZ90 MYH9 Myosin heavy chain 9 P35579 99.49 F7I1Q9 TAGLN Transgelin Q01995 99.5 F7I9U1 TPM2 Tropomyosin beta chain isoform 2 P07951 87.32 F7HTQ3 CALD1 Caldesmon 1 Q05682 95.33 F7I0W8 LOC100409006 Uncharacterized protein P63261 100 U3EQM9 ACTN4 Alpha-actinin-4 O43707 99.89 F6YS84 TPM1 Tropomyosin 1 P09493 85.92 U3BZ94 TUBB4B Tubulin beta chain P68371 99.33 U3CYB6 ENO1 Alpha-enolase isoform 1 P06733 99.31 F7I318 LGALS1 Galectin P09382 99.07 F7HZP3 TUBA1B Tubulin alpha chain P68363 100 F6RMA3 ANXA2 Annexin P07355 99.71 F7IHK8 PRDX1 Peroxiredoxin 1 Q06830 97.99 U3EKQ0 ACTN1 Actinin alpha 1 P12814 99.78 F7CXD3 EEF1A1 Elongation factor 1-alpha P68104 100 F6SQW1 PPIA Peptidyl-prolyl cis-trans isomerase P62937 99.78 U3FXT1 HSPA5 78 kDa glucose-regulated protein P11021 99.39 F7HVD2 HSPA8 Heat shock protein family A (Hsp70) member 8 P11142 99.85 F7FP14 Uncharacterized protein Q8N386 62.38 F6YTV2 LMNA Lamin A/C P02545 98.65 F6XL35 PKM Pyruvate kinase P14618 99.06 U3D5Z3 TLN1 Talin-1 Q9Y490 99.21 View Large Table II The top 25 most abundant proteins in MKTPC. Sequence similarities with corresponding human proteins were determined by BLAST analysis and are indicated as ‘% identity’. Callithrix jacchus UniProt accession Callithrix jacchus Gene name Callithrix jacchus protein name Human UniProt accession % Identity F7IH64 ACTA2 Actin, alpha 2, smooth muscle, aorta P62736 100 B0KWW5 FLNA Filamin-A isoform 1,2 P21333 98.79 F7BQY8 VIM Vimentin P08670 99.36 F6ZZ90 MYH9 Myosin heavy chain 9 P35579 99.49 F7I1Q9 TAGLN Transgelin Q01995 99.5 F7I9U1 TPM2 Tropomyosin beta chain isoform 2 P07951 87.32 F7HTQ3 CALD1 Caldesmon 1 Q05682 95.33 F7I0W8 LOC100409006 Uncharacterized protein P63261 100 U3EQM9 ACTN4 Alpha-actinin-4 O43707 99.89 F6YS84 TPM1 Tropomyosin 1 P09493 85.92 U3BZ94 TUBB4B Tubulin beta chain P68371 99.33 U3CYB6 ENO1 Alpha-enolase isoform 1 P06733 99.31 F7I318 LGALS1 Galectin P09382 99.07 F7HZP3 TUBA1B Tubulin alpha chain P68363 100 F6RMA3 ANXA2 Annexin P07355 99.71 F7IHK8 PRDX1 Peroxiredoxin 1 Q06830 97.99 U3EKQ0 ACTN1 Actinin alpha 1 P12814 99.78 F7CXD3 EEF1A1 Elongation factor 1-alpha P68104 100 F6SQW1 PPIA Peptidyl-prolyl cis-trans isomerase P62937 99.78 U3FXT1 HSPA5 78 kDa glucose-regulated protein P11021 99.39 F7HVD2 HSPA8 Heat shock protein family A (Hsp70) member 8 P11142 99.85 F7FP14 Uncharacterized protein Q8N386 62.38 F6YTV2 LMNA Lamin A/C P02545 98.65 F6XL35 PKM Pyruvate kinase P14618 99.06 U3D5Z3 TLN1 Talin-1 Q9Y490 99.21 Callithrix jacchus UniProt accession Callithrix jacchus Gene name Callithrix jacchus protein name Human UniProt accession % Identity F7IH64 ACTA2 Actin, alpha 2, smooth muscle, aorta P62736 100 B0KWW5 FLNA Filamin-A isoform 1,2 P21333 98.79 F7BQY8 VIM Vimentin P08670 99.36 F6ZZ90 MYH9 Myosin heavy chain 9 P35579 99.49 F7I1Q9 TAGLN Transgelin Q01995 99.5 F7I9U1 TPM2 Tropomyosin beta chain isoform 2 P07951 87.32 F7HTQ3 CALD1 Caldesmon 1 Q05682 95.33 F7I0W8 LOC100409006 Uncharacterized protein P63261 100 U3EQM9 ACTN4 Alpha-actinin-4 O43707 99.89 F6YS84 TPM1 Tropomyosin 1 P09493 85.92 U3BZ94 TUBB4B Tubulin beta chain P68371 99.33 U3CYB6 ENO1 Alpha-enolase isoform 1 P06733 99.31 F7I318 LGALS1 Galectin P09382 99.07 F7HZP3 TUBA1B Tubulin alpha chain P68363 100 F6RMA3 ANXA2 Annexin P07355 99.71 F7IHK8 PRDX1 Peroxiredoxin 1 Q06830 97.99 U3EKQ0 ACTN1 Actinin alpha 1 P12814 99.78 F7CXD3 EEF1A1 Elongation factor 1-alpha P68104 100 F6SQW1 PPIA Peptidyl-prolyl cis-trans isomerase P62937 99.78 U3FXT1 HSPA5 78 kDa glucose-regulated protein P11021 99.39 F7HVD2 HSPA8 Heat shock protein family A (Hsp70) member 8 P11142 99.85 F7FP14 Uncharacterized protein Q8N386 62.38 F6YTV2 LMNA Lamin A/C P02545 98.65 F6XL35 PKM Pyruvate kinase P14618 99.06 U3D5Z3 TLN1 Talin-1 Q9Y490 99.21 View Large To review if the most abundant proteins in MKTPCs are also highly abundant in HTPCs, the identified MKTPC and HTPC protein groups were sorted according to their MS/MS spectral count values, a measure for their abundance. Comparison of both datasets revealed that 21 out of the top 25 MKTPC protein groups are contained in the top 50 HTPC protein groups (Table III). Only transgelin (TAGLN), caldesmon (CALD1) and the tropomyosins TPM1 and TPM4 ranked noticeably lower in HTPCs compared to MKTPC. Table III The top 25 most abundant MKTPC protein groups and their number of group members. The abundance ranks of MKTPC protein groups are compared to the ranks of the corresponding protein groups in HTPCs. Rank in MKTPC Rank in HTPC Protein group # Proteins in group 1 2 Actin, cytoplasmic 1 11 2 4 Filamin-A 3 3 7 Myosin 9 5 4 9 Alpha-actinin-4 7 5 1 Vimentin 45 6 3 Tubulin beta 19 7 4 Uncharacterized protein homologue to human neuroblast differentiation-associated protein AHNAK 3 8 63 Transgelin 1 9 93 Tropomyosin alpha-4 chain 5 10 42 Myosin-10 3 11 239 Tropomyosin alpha-1 chain 4 12 23 Filamin-C 2 13 26 Alpha-enolase 6 14 12 Talin-1 1 15 14 Heat shock protein family A (Hsp70) member 5 1 16 113 Caldesmon 3 17 44 Filamin-B 4 18 6 Plectin 2 19 19 Clathrin heavy chain 2 20 11 Tubulin alpha chain 7 21 15 Heat shock protein 90 4 22 21 Cytoplasmic dynein 1 heavy chain 1 1 23 8 Pyruvate kinase 5 24 20 Lamin isoform A 3 25 27 Heat shock protein family A (Hsp70) member 8 3 Rank in MKTPC Rank in HTPC Protein group # Proteins in group 1 2 Actin, cytoplasmic 1 11 2 4 Filamin-A 3 3 7 Myosin 9 5 4 9 Alpha-actinin-4 7 5 1 Vimentin 45 6 3 Tubulin beta 19 7 4 Uncharacterized protein homologue to human neuroblast differentiation-associated protein AHNAK 3 8 63 Transgelin 1 9 93 Tropomyosin alpha-4 chain 5 10 42 Myosin-10 3 11 239 Tropomyosin alpha-1 chain 4 12 23 Filamin-C 2 13 26 Alpha-enolase 6 14 12 Talin-1 1 15 14 Heat shock protein family A (Hsp70) member 5 1 16 113 Caldesmon 3 17 44 Filamin-B 4 18 6 Plectin 2 19 19 Clathrin heavy chain 2 20 11 Tubulin alpha chain 7 21 15 Heat shock protein 90 4 22 21 Cytoplasmic dynein 1 heavy chain 1 1 23 8 Pyruvate kinase 5 24 20 Lamin isoform A 3 25 27 Heat shock protein family A (Hsp70) member 8 3 Table III The top 25 most abundant MKTPC protein groups and their number of group members. The abundance ranks of MKTPC protein groups are compared to the ranks of the corresponding protein groups in HTPCs. Rank in MKTPC Rank in HTPC Protein group # Proteins in group 1 2 Actin, cytoplasmic 1 11 2 4 Filamin-A 3 3 7 Myosin 9 5 4 9 Alpha-actinin-4 7 5 1 Vimentin 45 6 3 Tubulin beta 19 7 4 Uncharacterized protein homologue to human neuroblast differentiation-associated protein AHNAK 3 8 63 Transgelin 1 9 93 Tropomyosin alpha-4 chain 5 10 42 Myosin-10 3 11 239 Tropomyosin alpha-1 chain 4 12 23 Filamin-C 2 13 26 Alpha-enolase 6 14 12 Talin-1 1 15 14 Heat shock protein family A (Hsp70) member 5 1 16 113 Caldesmon 3 17 44 Filamin-B 4 18 6 Plectin 2 19 19 Clathrin heavy chain 2 20 11 Tubulin alpha chain 7 21 15 Heat shock protein 90 4 22 21 Cytoplasmic dynein 1 heavy chain 1 1 23 8 Pyruvate kinase 5 24 20 Lamin isoform A 3 25 27 Heat shock protein family A (Hsp70) member 8 3 Rank in MKTPC Rank in HTPC Protein group # Proteins in group 1 2 Actin, cytoplasmic 1 11 2 4 Filamin-A 3 3 7 Myosin 9 5 4 9 Alpha-actinin-4 7 5 1 Vimentin 45 6 3 Tubulin beta 19 7 4 Uncharacterized protein homologue to human neuroblast differentiation-associated protein AHNAK 3 8 63 Transgelin 1 9 93 Tropomyosin alpha-4 chain 5 10 42 Myosin-10 3 11 239 Tropomyosin alpha-1 chain 4 12 23 Filamin-C 2 13 26 Alpha-enolase 6 14 12 Talin-1 1 15 14 Heat shock protein family A (Hsp70) member 5 1 16 113 Caldesmon 3 17 44 Filamin-B 4 18 6 Plectin 2 19 19 Clathrin heavy chain 2 20 11 Tubulin alpha chain 7 21 15 Heat shock protein 90 4 22 21 Cytoplasmic dynein 1 heavy chain 1 1 23 8 Pyruvate kinase 5 24 20 Lamin isoform A 3 25 27 Heat shock protein family A (Hsp70) member 8 3 To assess similarities between MKTPCs and HTPCs at the functional level, the identified proteins were analyzed with the PANTHER analysis tool using the ‘Biological Process’ and ‘Cellular Process’ Gene Ontology Databases. The proteins of both MKTPC and HTPC show highly similar distributions concerning related biological and cellular processes, suggesting strong biochemical and functional similarities between MKTPC and HTPC (Fig. 7). Figure 7 View largeDownload slide (A) GO analysis using the ‘biological process’ database for MKTPC (left) and HTPC (right) proteomes. (B) GO analysis regarding the ‘cellular process’ subset for MKTPC (left) and HTPC (right). Figure 7 View largeDownload slide (A) GO analysis using the ‘biological process’ database for MKTPC (left) and HTPC (right) proteomes. (B) GO analysis regarding the ‘cellular process’ subset for MKTPC (left) and HTPC (right). Discussion The previous studies in HTPCs provided new insights in to the functions of these testicular cells (Spinnler et al., 2010; Mayerhofer, 2013; Landreh et al., 2014; Welter et al., 2014; Mayer et al., 2016). While a previous proteomic study of HTPCs revealed that they are rather homogeneous with respect to their cellular proteome and their repertoire of secreted factors (Flenkenthaler et al., 2014), further studies showed that their ability to secrete steroids (Landreh et al., 2014) or to respond to stimuli with cytokine secretion (Mayer et al., 2016) occur in a patient-specific fashion. It is likely that the differences are due to age, lifestyle and or medical history, which are beyond control. We hypothesized that cells from a non-human primate species, specifically with a controlled lifestyle, may be an ideal additional model in which to study TPCs. We chose the common marmoset monkey (Callithrix jacchus), which is a well-established model organism for reproductive research, for this model. The testicular structures of marmosets are comparable in several biologically relevant aspects with humans, including characteristics of germ cell development and function (Michel and Mahouy, 1990; Millar et al., 2000; Mansfield, 2003; Zuhlke and Weinbauer, 2003). Importantly, the architecture of the tubular wall shows several layers and therefore comes close to the human situation. Both contrast to rodent testes, in which the wall of the seminiferous tubules consists of a single cell layer of peritubular cells. Finally, the whole genome from Callithrix jacchus was sequenced (Sato et al., 2015), which enables further investigation on the genomic and proteomic level. We successfully isolated and cultured cells from the wall of seminiferous tubules of the testis of young adult, healthy Callithrix jacchus, using the same approach as for HTPCs, namely explant culture. The cells obtained upon initial characterization could be clearly identified as pure peritubular cells. Virtually all MKTPCs were immunoreactive for the smooth muscle markers, CNN1, ACTA2, which were detected only by peritubular cells in situ. In addition, the combination of smooth muscle markers, expression of AR and GDNF and the absence of Sertoli cell markers (FSHR, GATA4) and Leydig cell markers (LHCGR, INSL3) allows this conclusion. Importantly, the expression of the characteristic factors remains stable over at least eight passages. In human testes tryptase-immunoreactive mast cells are found close to the tubular wall (Meineke et al., 2000) and within the layers of peritubular cells and we also detected tryptase-immunoreactive mast cells by immunohistochemistry in Callithrix jacchus testes (data not shown). We therefore explored a possible contamination with immune cells. RT-PCR for mast cell markers tryptase, chymase or the T-cell marker CD3e yielded, however, negative results in MKTPC. We further compared the MKTPC proteome data with a published human macrophage proteome data (Eligini et al., 2015). We found that five proteins coincide, namely Chloride intracellular channel protein 1, Elongation factor 2, Plastin 3, Tubulin alpha 1 chain, Vimentin. These proteins are, however, not specific for macrophages but represent ubiquitously occurring proteins. Apolipoprotein B receptor, which is considered a characteristic macrophage receptor (Hassel et al., 2017), is not detected in our proteome analysis. Thus, isolation of MKTPCs is a practical way to obtain pure testicular peritubular cells of a non-human primate species. We initially tested whether they further resemble HTPCs, and found that they produce DCN and BGN, CCL2, PTX3 and IL6 as well as GDNF. Hence, they resemble HTPCs in this respect (Spinnler et al., 2010; Mayer et al., 2016; Walenta et al., 2018). To further characterize MKTPCs at the protein level, a proteome study of peritubular cells obtained from six individual young adult Callithrix jacchus donors was performed. In order to focus on the most abundant proteins being easily assessable with a single-run LC–MS/MS method, we kept the proteomics workflow as simple as possible and did not use any pre-fractionation at the protein or the peptide level. Nevertheless, the analysis of the acquired mass spectra led to the identification of 2437 MKTPC protein groups (FDR < 1%). For the chosen approach, this represents a fairly high number of protein IDs and reflects the suitability of the Callithrix jacchus database for LC–MS based proteome analysis of MKTPC samples. Additionally, a multi-scatter plot analysis between the donors revealed very reproducible protein expression patterns, demonstrating the robustness of isolation and cultivation methods as well as a low inter-individual variability between the individual donors. For the suitability of the animal model, the similarity between MKTPCs and HTPCs at the proteome level is an important indicator. To assess this, HTPC proteomes from seven human donors were analyzed using exactly the same methodology. Inter-individual correlation analysis shows clear homogeneity and reproducibility similar to MKTPCs, with the latter one being more accessible and generated under monitored conditions. The inter-species scatter plot analysis of protein intensity values between MKTPC and HTPC showed a Pearson correlation coefficient of 0.78 indicating similar abundance patterns of MKTPC and HTPC proteins. A further MKTPC vs HTPC multi-scatter plot analysis at the level of individuals showed in all cases very similar Pearson correlation coefficients with no outliers. Taken together the correlation analyses reveal a clear conformity between MKTPCs and HTPCs on the level of protein expression patterns and a high degree of inter-individual reproducibility of MKTPCs. A circoletto network analysis as well as a BLAST analysis of the 25 most abundant proteins showed high sequence homology between HTPC and MKTPC proteins, indicating a high degree of functional similarity. Using a spectral count quantification approach combined with homology based protein grouping, we could further demonstrate that the broad majority of the 25 most abundant MKTPC protein groups are also highly expressed in HTPCs. Only transgelin (TAGLN), caldesmon (CALD1) and the two tropomyosins (TPM1 and TPM4) showed lower spectral counts in MKTPC. Since all of these four proteins bind to actin this finding may reflect slight differences in the cytoskeleton between MKTPC and HTPCs. Finally, the PANTHER GO analysis of MKTPC and HTPC proteins lead to almost identical results, suggesting a strong resemblance at the level of biological and biochemical processes between MKTPCs and HTPCs. The high similarity at the proteome level elaborated here further approves MKTPC as an excellent non-human primate model to study the biology of HTPCs. In summary, isolation of MKTPCs is a feasible way to obtain primate peritubular cells, which resemble their human counterparts. They are derived from young adults raised under controlled conditions and provide an opportunity to explore functions and regulation of testicular peritubular cells without unknown confounding issues like lifestyle, age, nutrition and the medical history of patients. We anticipate that this may lead to a better understanding of the role of peritubular cells in male (in)fertility, including their role in the spermatogonial stem cell niche and their plasticity. Supplementary data Supplementary data are available at Molecular Human Reproduction online. Acknowledgements The authors thank Astrid Tiefenbacher for skillful technical assistance. Authors’ roles N.S. performed the majority of the cellular experiments and K.G.D. participated in these experiments. J.B.S, F.F., T.F. and G.A performed proteomic studies and evaluated the results. J.U.S, F.-M.K., C.D. and R.B. provided testicular tissues, as well as conceptual input, A.M. conceived of the study, directed the work and supervised the experiments. N.S. and A.M. drafted the article. All authors contributed to and approved the final version. This work was performed in partial fulfillments of the requirements of doctoral degrees (N.S. and J.B.S.) at LMU. Funding Grants from the Deutsche Forschungsgemeinschaft (MA 1080/27-1; AR 362/9-1; BE 2296/8-1). Conflicts of interest The authors declare no competing financial interests. References Albrecht M , Ramsch R , Kohn FM , Schwarzer JU , Mayerhofer A . Isolation and cultivation of human testicular peritubular cells: a new model for the investigation of fibrotic processes in the human testis and male infertility . J Clin Endocrinol Metab 2006 ; 91 : 1956 – 1960 . Google Scholar CrossRef Search ADS PubMed Chen LY , Brown PR , Willis WB , Eddy EM . Peritubular myoid cells participate in male mouse spermatogonial stem cell maintenance . Endocrinology 2014 ; 155 : 4964 – 4974 . Google Scholar CrossRef Search ADS PubMed Chen LY , Willis WD , Eddy EM . Targeting the Gdnf gene in peritubular myoid cells disrupts undifferentiated spermatogonial cell development . Proc Nat Acad Sci USA 2016 ; 113 : 1829 – 1834 . Google Scholar CrossRef Search ADS PubMed Cox J , Mann M . MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification . Nat Biotechnol 2008 ; 26 : 1367 – 1372 . Google Scholar CrossRef Search ADS PubMed Eligini S , Brioschi M , Fiorelli S , Tremoli E , Banfi C , Colli S . Human monocyte-derived macrophages are heterogenous: proteomic profile of different phenotypes . J Proteomics 2015 ; 124 : 112 – 123 . Google Scholar CrossRef Search ADS PubMed Flenkenthaler F , Windschuttl S , Frohlich T , Schwarzer JU , Mayerhofer A , Arnold GJ . Secretome analysis of testicular peritubular cells: a window into the human testicular microenvironment and the spermatogonial stem cell niche in man . J Proteome Res 2014 ; 13 : 1259 – 1269 . Google Scholar CrossRef Search ADS PubMed Hassel B , De Souza GA , Stensland ME , Ivanovic J , Voie O , Dahlberg D . The proteome of pus from human brain abscesses: host-derived neurotoxic proteins and the cell-type diversity of CNS pus . J Neurosurg 2017 ; 20 : 1 – 9 . Google Scholar CrossRef Search ADS Landreh L , Spinnler K , Schubert K , Hakkinen MR , Auriola S , Poutanen M , Soder O , Svechnikov K , Mayerhofer A . Human testicular peritubular cells host putative stem Leydig cells with steroidogenic capacity . J Clin Endocrinol Metab 2014 ; 99 : E1227 – E1235 . Google Scholar CrossRef Search ADS PubMed Li LH , Donald JM , Golub MS . Review on testicular development, structure, function, and regulation in common marmoset . Birth Defects Res B Dev Reprod Toxicol 2005 ; 74 : 450 – 469 . Google Scholar CrossRef Search ADS PubMed Mansfield K . Marmoset models commonly used in biomedical research . Comp Med 2003 ; 53 : 383 – 392 . Google Scholar PubMed Mayer C , Adam M , Glashauser L , Dietrich K , Schwarzer JU , Kohn FM , Strauss L , Welter H , Poutanen M , Mayerhofer A . Sterile inflammation as a factor in human male infertility: involvement of Toll like receptor 2, biglycan and peritubular cells . Sci Rep 2016 ; 6 : 37128 . Google Scholar CrossRef Search ADS PubMed Mayerhofer A . Human testicular peritubular cells: more than meets the eye . Reproduction 2013 ; 145 : R107 – R116 . Google Scholar CrossRef Search ADS PubMed Meineke V , Frungieri MB , Jessberger B , Vogt H , Mayerhofer A . Human testicular mast cells contain tryptase: increased mast cell number and altered distribution in the testes of infertile men . Fertil Steril 2000 ; 74 : 239 – 244 . Google Scholar CrossRef Search ADS PubMed Michel JB , Mahouy G . The marmoset in biomedical research. Value of this primate model for cardiovascular studies . Pathol Biol (Paris) 1990 ; 38 : 197 – 204 . Google Scholar PubMed Millar MR , Sharpe RM , Weinbauer GF , Fraser HM , Saunders PT . Marmoset spermatogenesis: organizational similarities to the human . Int J Androl 2000 ; 23 : 266 – 277 . Google Scholar CrossRef Search ADS PubMed Sato K , Kuroki Y , Kumita W , Fujiyama A , Toyoda A , Kawai J , Iriki A , Sasaki E , Okano H , Sakakibara Y . Resequencing of the common marmoset genome improves genome assemblies and gene-coding sequence analysis . Sci Rep 2015 ; 5 : 16894 . Google Scholar CrossRef Search ADS PubMed Schell C , Albrecht M , Mayer C , Schwarzer JU , Frungieri MB , Mayerhofer A . Exploring human testicular peritubular cells: identification of secretory products and regulation by tumor necrosis factor-alpha . Endocrinology 2008 ; 149 : 1678 – 1686 . Google Scholar CrossRef Search ADS PubMed Schell C , Albrecht M , Spillner S , Mayer C , Kunz L , Kohn FM , Schwarzer U , Mayerhofer A . 15-Deoxy-delta 12-14-prostaglandin-J2 induces hypertrophy and loss of contractility in human testicular peritubular cells: implications for human male fertility . Endocrinology 2010 ; 151 : 1257 – 1268 . Google Scholar CrossRef Search ADS PubMed Spinnler K , Kohn FM , Schwarzer U , Mayerhofer A . Glial cell line-derived neurotrophic factor is constitutively produced by human testicular peritubular cells and may contribute to the spermatogonial stem cell niche in man . Hum Reprod 2010 ; 25 : 2181 – 2187 . Google Scholar CrossRef Search ADS PubMed Walenta L , Fleck D , Frohlich T , von Eysmondt H , Arnold GJ , Spehr J , Schwarzer JU , Kohn FM , Spehr M , Mayerhofer A . ATP-mediated events in peritubular cells contribute to sterile testicular inflammation . Sci Rep 2018 ; 8 : 1431 . Google Scholar CrossRef Search ADS PubMed Welter H , Huber A , Lauf S , Einwang D , Mayer C , Schwarzer JU , Kohn FM , Mayerhofer A . Angiotensin II regulates testicular peritubular cell function via AT1 receptor: a specific situation in male infertility . Mol Cell Endocrinol 2014 ; 393 : 171 – 178 . Google Scholar CrossRef Search ADS PubMed Zuhlke U , Weinbauer G . The common marmoset (Callithrix jacchus) as a model in toxicology . Toxicol Pathol 2003 ; 31 : 123 – 127 . Google Scholar CrossRef Search ADS PubMed © The Author 2018. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Molecular Human Reproduction Oxford University Press

Characterization of a non-human primate model for the study of testicular peritubular cells—comparison with human testicular peritubular cells

Loading next page...
 
/lp/ou_press/characterization-of-a-nonhuman-primate-model-for-the-study-of-zLyAPpMLp7
Publisher
Oxford University Press
Copyright
© The Author 2018. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com
ISSN
1360-9947
eISSN
1460-2407
D.O.I.
10.1093/molehr/gay025
Publisher site
See Article on Publisher Site

Abstract

Abstract STUDY QUESTION Are monkey testicular peritubular cells (MKTPCs) from the common marmoset monkey (Callithrix jacchus) a suitable translational model for the study of human testicular peritubular cells (HTPCs)? SUMMARY ANSWER MKTPCs can be isolated and propagated in vitro, retain characteristic markers for testicular peritubular cells and their proteome strongly (correlation coefficient of 0.78) overlaps with the proteome of HTPCs. WHAT IS KNOWN ALREADY Smooth-muscle-like peritubular cells form the wall of seminiferous tubules, transport sperm, are immunologically active, secrete a plethora of factors and may contribute to the spermatogonial stem cell niche. Mechanistic studies are hampered by heterogeneity of human samples. STUDY DESIGN, SIZE, DURATION We established a culture method for MKTPCs and characterized these cells from six young adult animals (2–3 years). To examine whether they qualify as a translational model we also examined HTPCs from seven men and compared the proteomes of both groups. PARTICIPANTS/MATERIALS, SETTING, METHODS We used explant cultures to obtain MKTPCs, which express smooth muscle markers (calponin (CNN1), smooth muscle actin (ACTA2)), lack FSH-receptors (FSHR) and LH-receptors (LHCGR), but possess androgen receptors (AR). MKTPCs can be passaged at least up to eight times, without discernable phenotypic changes. Mass-spectrometry-based analyses of the MKTPC and HTPC proteomes were performed. MAIN RESULTS AND THE ROLE OF CHANCE We established a method for isolation and cultivation of MKTPCs, and provide a comprehensive analysis of their protein repertoire. The results let us conclude that MKTPCs are suitable as a non-human primate model to study peritubular cell functions. LARGE SCALE DATA List of identified proteins in MKTPCs by liquid chromatography–tandem mass spectrometry is accessible at the ProteomeXchange (identifier PXD009394). LIMITATIONS, REASON FOR CAUTION This is an in vitro cellular non-human primate model used to provide a window into the role of these cells in the human testis. WIDER IMPLICATIONS OF THE FINDINGS Previous studies with HTPCs from patients revealed a degree of heterogeneity, possibly due to age, lifestyle and medical history of the individual human donors. We anticipate that the new translational model, derived from young healthy non-human primates, may allow us to circumvent these issues and may lead to a better understanding of the role of peritubular cells. STUDY FUNDING AND COMPETION OF INTEREST(S) This work was supported by grants from the Deutsche Forschungsgemeinschaft (MA 1080/27-1; AR 362/9-1; BE 2296/8-1). The authors declare no competing financial interests. testis, cellular model, proteome, marmoset monkey, non-human primate Introduction The human testis in health and disease cannot be readily studied. Cellular models, however, allow insights and human testicular peritubular cells (HTPCs) derived from individual patients represent such a model. These cells form the wall of seminiferous tubules and can be isolated via explant culture from very small fragments obtained during surgical procedures (Albrecht et al., 2006; Schell et al., 2008; Mayerhofer, 2013). They can be passaged and examined by cellular and molecular techniques. The results obtained, in combination with studies on human testicular sections, led to the insight that HTPCs play an important role in the human testis in health and disease. These smooth-muscle-like cells are associated with the transport of immotile sperm, but also possess secretory functions. Glial cell line-derived neurotrophic factor (GDNF) is a factor secreted by these cells, which is required for renewal of spermatogonial stem cells. This indicates a contribution to the spermatogonial stem cell niche (Spinnler et al., 2010), a view supported by recent studies in mice (Chen et al., 2014, 2016). HTPCs also secrete extracellular matrix components, including decorin (DCN) and biglycan (BGN) (Flenkenthaler et al., 2014). The latter is increased in infertile patients and, as we showed, can activate Toll-like receptors (TLRs) of HTPCs. Thereby it induces inflammatory reactions, namely secretion of C–C motif chemokine ligand 2 (CCL2), pentraxin 3 (PTX3) or interleukin 6 (IL6) (Mayer et al., 2016). Taken together, the results support important roles of HTPCs in male fertility and infertility. HTPCs were also shown to be plastic cells and their ability to differentiate towards a steroidogenic, presumably Leydig cell type, became evident (Landreh et al., 2014). The last-mentioned study showed inter-individual differences in the basal and the forskolin-stimulated steroidogenic capacities. Further studies (Welter et al., 2014; Mayer et al., 2016), which examined immunological aspects, revealed that HTPCs derived from individual patients are heterogeneous with respect to the amounts of secreted factors. This may be due to the differences in age, lifestyle and medical history of the patients. While this heterogeneity is instructive and may be of interest in view of an improved understanding of inter-individual differences and personalized treatment option in the future, these differences also make general in-depth mechanistic studies difficult. To circumvent these issues, and to be able to study peritubular cell function and regulation in greater detail, we sought to establish a translational model. In search for suitable model organism, we turned to the common marmoset monkey (Michel and Mahouy, 1990; Mansfield, 2003; Zuhlke and Weinbauer, 2003; Li et al., 2005). Marmosets are non-human primates, which are often used in reproductive research. Here we describe the isolation, cultivation and proteomic characterization of monkey testicular peritubular cells (MKTPCs). We propose that they are an apt model for HTPCs because of their similarity to human and the possibility to control confounding lifestyle issues. Materials and Methods Animals Common marmoset monkeys (Callithrix jacchus) stem from the self-sustaining marmoset monkey colony of the German Primate Center (Deutsches Primatenzentrum; DPZ, Göttingen). All animals used for this study were between 2 and 3 years old, i.e. young adult, sexually mature healthy animals (Li et al., 2005). Marmoset monkey testes were obtained from animals euthanized for scientific purposes unrelated to this study or castrated for colony management purposes. Euthanasia and castration were performed by experienced veterinarians. Parts of the testes were fixed in Bouin’s fixative and embedded in paraffin, for later sectioning and immunohistochemistry, other parts were used for isolation of MKTPCs by explant culture. Ethical approval Organ retrievals from Callithrix jacchus were carried out in accordance with relevant institutional guidelines and legal regulations, namely the German Animal Protection Act. The local ethical committees (Ethikkommission, Technische Universität München, Fakultät für Medizin, München, project number 5158/11) approved the study with human tissues. All experiments were performed in accordance with relevant guidelines and regulations. Isolation and cultivation of MKTPCs Isolation and cultivation of MKTPCs was performed as described in detail previously for human samples (Albrecht et al., 2006). In brief, small pieces of testicular tissue were seeded onto cell culture dishes. The explant cultures were incubated under humidified conditions (37°C, 5% CO2) until the cells started to grow out of the tubular wall. They were cultivated and propagated in Dulbecco’s modified Eagle’s medium (DMEM) high glucose (Gibco, Paisley, UK) containing 10% fetal calf serum (Capricorn Scientific GmbH, Ebsdorfergrund, Germany) and 1% penicillin/streptomycin (Life Technologies, Carlsbad, CA, USA). MKTPCs in passages 2–3 were used for proteomic studies, for RT-PCR cells from early (3) and advanced passages (8) were used. RNA isolation and RT-PCR Total RNA from cultured MKTPC was prepared as described earlier (Welter et al., 2014) using the RNeasy microkit (Qiagen, Hilden, Germany). In brief, a total amount of 200 ng of RNA was subjected to reverse transcription, using random primers (15-mer) and SuperScript II Reverse Transcriptase, 200 U/μl (Invitrogen GmbH, Darmstadt, Germany). Intron-spanning primer pairs amplified specific products for ACTA2, CNN1, AR, GDNF, FSHR, GATA4, LHCGR, INSL3, DCN, BGN, CCL2, PTX3, IL6, CD3e, TPSG1 and CMA1 (Table I). PCR consisted of 35 cycles of denaturing (at 95°C for 60 s) annealing (at 60°C) and extension (at 72°C for 45 s). PCR products were visualized by midori green (NIPPON Genetics EUROPE GmbH, Düren, Germany) staining in agarose (Biozym Scientific GmbH, Oldendorf, Germany) gels. Positive controls consisted of Callithrix jacchus whole testis lysate cDNA (+). Negative controls were performed by excluding reverse transcriptase (−RT), and a non-template reaction (−). The identities of all PCR products were verified by sequencing. Table I Oligonucleotide primers used for PCR studies Gene Gene name Reference ID Nucleotide sequence Amplicon size RPL19 Ribosomal Protein L19 NM_001330200.1 5′-AGG CAC ATG GGC ATA GGT AA-3′ 5′-CCA TGA GAA TCC GCT TGT TT-3′ 199 ACTA2 Actin, Alpha 2, Smooth Muscle, Aorta NM_001613.2 5′-ACC CAG TGT GGA GCA GCC C-3′ 5′-TTG TCA CAC ACC AAG GCA GT-3′ 100 CNN1 Calponin 1 XM_017026289.1 5′-CGA AGA CGA AAG GAA ACA AGG T-3′ 5′-GCT TGG GGT CGT AGA GGT G-3′ 183 AR Androgen Receptor XM_008989422.2 5′-GCC CCT GAT CTG GTT TTC AA-3′ 5′-CCA CTG GAA TAA TGC TGA AGA GT-3′ 163 GDNF Glial Cell Derived Neurotrophic Factor NM_000514.3 5′-GCA GAC CCA TCG CCT TTG AT-3′ 5′-ATC CAC ACC TTT TAG CGG AAT G-3′ 93 FSHR FSH Receptor NM_000145.2 5′-CTG CTC CTG GTC TCT TTG CT-3′ 5′-GGT CCC CAA ATC CTG AAA AT-3′ 208 GATA4 GATA Binding Protein 4 NM_002052 5′-TCC AAA CCA GAA AAC GGA AG-3′ 5′-CTG TGC CCG TAG TGA GAT GA-3′ 187 LHCGR Luteinizing Hormone/Choriogonadotropin Receptor NM_001301843.1 5′-CTG GAT GCC ACG CTG ACT-3′ 5′-ACG CAC TCT GTC CAC TCT-3′ 100 INSL3 Insulin Like 3 XM_017968209.1 5′-ACT TCT CAC CAT CATCGC CA-3′ 5′-GAG GGT CAG CAG GTCTTG TT-3′ 96 CD3e CD3e Molecule XM_009006841.1 5′-ATCTGCATCACTCTGGGCTT-3′ 5′-TGGGCTCATAGTCTGGGTTG-3′ 160 TPSG1 Tryptase Gamma 1 NM_001204310.1 5′-CAT TGT GAG CTG GGG TGA AG-3′ 5′-AGA CCA GCA GAA GGA AGA GG-3′ 182 CMA1 Chymase 1 XM_002753692.4 5′-AAG TTG AAG GAG AAA GCC AGC-3′ 5′-TTC AAC ACA CCT GTT CTT CCC-3′ 125 DCN Decorin XM_002752816.3 5′-ATG AGG CTT CTG GGA TAG GC-3′ 5′-GTC CAG CAA AGT CGT GTC AG-3′ 170 BGN Biglycan XM_008990069.2 5′-GAG ACC CTG AAC GAA CTC CA-3′ 5′-TTG TTG TCC AAG TGC AGC TC-3′ 176 CCL2 C–C Motif Chemokine Ligand 2 NM_002982 5′-CAG CCA GAT GCA ATC AAT GCC-3′ 5′-TGG AAT CCT GAA CCC ACT TCT-3′ 190 PTX3 Pentraxin 3 NM_002852.3 5′-TAG TGT TTG TGG TGG GTG GA-3′ 5′-TGT GAG CCC TTC CTC TGA AT-3′ 110 IL6 Interleukin 6 XM_017975106.1 5′-AAG AGG TAG CTG CCC CAA AT-3′ 5′-AGT GCC TCT TTG CTG CTT TC-3′ 145 Gene Gene name Reference ID Nucleotide sequence Amplicon size RPL19 Ribosomal Protein L19 NM_001330200.1 5′-AGG CAC ATG GGC ATA GGT AA-3′ 5′-CCA TGA GAA TCC GCT TGT TT-3′ 199 ACTA2 Actin, Alpha 2, Smooth Muscle, Aorta NM_001613.2 5′-ACC CAG TGT GGA GCA GCC C-3′ 5′-TTG TCA CAC ACC AAG GCA GT-3′ 100 CNN1 Calponin 1 XM_017026289.1 5′-CGA AGA CGA AAG GAA ACA AGG T-3′ 5′-GCT TGG GGT CGT AGA GGT G-3′ 183 AR Androgen Receptor XM_008989422.2 5′-GCC CCT GAT CTG GTT TTC AA-3′ 5′-CCA CTG GAA TAA TGC TGA AGA GT-3′ 163 GDNF Glial Cell Derived Neurotrophic Factor NM_000514.3 5′-GCA GAC CCA TCG CCT TTG AT-3′ 5′-ATC CAC ACC TTT TAG CGG AAT G-3′ 93 FSHR FSH Receptor NM_000145.2 5′-CTG CTC CTG GTC TCT TTG CT-3′ 5′-GGT CCC CAA ATC CTG AAA AT-3′ 208 GATA4 GATA Binding Protein 4 NM_002052 5′-TCC AAA CCA GAA AAC GGA AG-3′ 5′-CTG TGC CCG TAG TGA GAT GA-3′ 187 LHCGR Luteinizing Hormone/Choriogonadotropin Receptor NM_001301843.1 5′-CTG GAT GCC ACG CTG ACT-3′ 5′-ACG CAC TCT GTC CAC TCT-3′ 100 INSL3 Insulin Like 3 XM_017968209.1 5′-ACT TCT CAC CAT CATCGC CA-3′ 5′-GAG GGT CAG CAG GTCTTG TT-3′ 96 CD3e CD3e Molecule XM_009006841.1 5′-ATCTGCATCACTCTGGGCTT-3′ 5′-TGGGCTCATAGTCTGGGTTG-3′ 160 TPSG1 Tryptase Gamma 1 NM_001204310.1 5′-CAT TGT GAG CTG GGG TGA AG-3′ 5′-AGA CCA GCA GAA GGA AGA GG-3′ 182 CMA1 Chymase 1 XM_002753692.4 5′-AAG TTG AAG GAG AAA GCC AGC-3′ 5′-TTC AAC ACA CCT GTT CTT CCC-3′ 125 DCN Decorin XM_002752816.3 5′-ATG AGG CTT CTG GGA TAG GC-3′ 5′-GTC CAG CAA AGT CGT GTC AG-3′ 170 BGN Biglycan XM_008990069.2 5′-GAG ACC CTG AAC GAA CTC CA-3′ 5′-TTG TTG TCC AAG TGC AGC TC-3′ 176 CCL2 C–C Motif Chemokine Ligand 2 NM_002982 5′-CAG CCA GAT GCA ATC AAT GCC-3′ 5′-TGG AAT CCT GAA CCC ACT TCT-3′ 190 PTX3 Pentraxin 3 NM_002852.3 5′-TAG TGT TTG TGG TGG GTG GA-3′ 5′-TGT GAG CCC TTC CTC TGA AT-3′ 110 IL6 Interleukin 6 XM_017975106.1 5′-AAG AGG TAG CTG CCC CAA AT-3′ 5′-AGT GCC TCT TTG CTG CTT TC-3′ 145 Table I Oligonucleotide primers used for PCR studies Gene Gene name Reference ID Nucleotide sequence Amplicon size RPL19 Ribosomal Protein L19 NM_001330200.1 5′-AGG CAC ATG GGC ATA GGT AA-3′ 5′-CCA TGA GAA TCC GCT TGT TT-3′ 199 ACTA2 Actin, Alpha 2, Smooth Muscle, Aorta NM_001613.2 5′-ACC CAG TGT GGA GCA GCC C-3′ 5′-TTG TCA CAC ACC AAG GCA GT-3′ 100 CNN1 Calponin 1 XM_017026289.1 5′-CGA AGA CGA AAG GAA ACA AGG T-3′ 5′-GCT TGG GGT CGT AGA GGT G-3′ 183 AR Androgen Receptor XM_008989422.2 5′-GCC CCT GAT CTG GTT TTC AA-3′ 5′-CCA CTG GAA TAA TGC TGA AGA GT-3′ 163 GDNF Glial Cell Derived Neurotrophic Factor NM_000514.3 5′-GCA GAC CCA TCG CCT TTG AT-3′ 5′-ATC CAC ACC TTT TAG CGG AAT G-3′ 93 FSHR FSH Receptor NM_000145.2 5′-CTG CTC CTG GTC TCT TTG CT-3′ 5′-GGT CCC CAA ATC CTG AAA AT-3′ 208 GATA4 GATA Binding Protein 4 NM_002052 5′-TCC AAA CCA GAA AAC GGA AG-3′ 5′-CTG TGC CCG TAG TGA GAT GA-3′ 187 LHCGR Luteinizing Hormone/Choriogonadotropin Receptor NM_001301843.1 5′-CTG GAT GCC ACG CTG ACT-3′ 5′-ACG CAC TCT GTC CAC TCT-3′ 100 INSL3 Insulin Like 3 XM_017968209.1 5′-ACT TCT CAC CAT CATCGC CA-3′ 5′-GAG GGT CAG CAG GTCTTG TT-3′ 96 CD3e CD3e Molecule XM_009006841.1 5′-ATCTGCATCACTCTGGGCTT-3′ 5′-TGGGCTCATAGTCTGGGTTG-3′ 160 TPSG1 Tryptase Gamma 1 NM_001204310.1 5′-CAT TGT GAG CTG GGG TGA AG-3′ 5′-AGA CCA GCA GAA GGA AGA GG-3′ 182 CMA1 Chymase 1 XM_002753692.4 5′-AAG TTG AAG GAG AAA GCC AGC-3′ 5′-TTC AAC ACA CCT GTT CTT CCC-3′ 125 DCN Decorin XM_002752816.3 5′-ATG AGG CTT CTG GGA TAG GC-3′ 5′-GTC CAG CAA AGT CGT GTC AG-3′ 170 BGN Biglycan XM_008990069.2 5′-GAG ACC CTG AAC GAA CTC CA-3′ 5′-TTG TTG TCC AAG TGC AGC TC-3′ 176 CCL2 C–C Motif Chemokine Ligand 2 NM_002982 5′-CAG CCA GAT GCA ATC AAT GCC-3′ 5′-TGG AAT CCT GAA CCC ACT TCT-3′ 190 PTX3 Pentraxin 3 NM_002852.3 5′-TAG TGT TTG TGG TGG GTG GA-3′ 5′-TGT GAG CCC TTC CTC TGA AT-3′ 110 IL6 Interleukin 6 XM_017975106.1 5′-AAG AGG TAG CTG CCC CAA AT-3′ 5′-AGT GCC TCT TTG CTG CTT TC-3′ 145 Gene Gene name Reference ID Nucleotide sequence Amplicon size RPL19 Ribosomal Protein L19 NM_001330200.1 5′-AGG CAC ATG GGC ATA GGT AA-3′ 5′-CCA TGA GAA TCC GCT TGT TT-3′ 199 ACTA2 Actin, Alpha 2, Smooth Muscle, Aorta NM_001613.2 5′-ACC CAG TGT GGA GCA GCC C-3′ 5′-TTG TCA CAC ACC AAG GCA GT-3′ 100 CNN1 Calponin 1 XM_017026289.1 5′-CGA AGA CGA AAG GAA ACA AGG T-3′ 5′-GCT TGG GGT CGT AGA GGT G-3′ 183 AR Androgen Receptor XM_008989422.2 5′-GCC CCT GAT CTG GTT TTC AA-3′ 5′-CCA CTG GAA TAA TGC TGA AGA GT-3′ 163 GDNF Glial Cell Derived Neurotrophic Factor NM_000514.3 5′-GCA GAC CCA TCG CCT TTG AT-3′ 5′-ATC CAC ACC TTT TAG CGG AAT G-3′ 93 FSHR FSH Receptor NM_000145.2 5′-CTG CTC CTG GTC TCT TTG CT-3′ 5′-GGT CCC CAA ATC CTG AAA AT-3′ 208 GATA4 GATA Binding Protein 4 NM_002052 5′-TCC AAA CCA GAA AAC GGA AG-3′ 5′-CTG TGC CCG TAG TGA GAT GA-3′ 187 LHCGR Luteinizing Hormone/Choriogonadotropin Receptor NM_001301843.1 5′-CTG GAT GCC ACG CTG ACT-3′ 5′-ACG CAC TCT GTC CAC TCT-3′ 100 INSL3 Insulin Like 3 XM_017968209.1 5′-ACT TCT CAC CAT CATCGC CA-3′ 5′-GAG GGT CAG CAG GTCTTG TT-3′ 96 CD3e CD3e Molecule XM_009006841.1 5′-ATCTGCATCACTCTGGGCTT-3′ 5′-TGGGCTCATAGTCTGGGTTG-3′ 160 TPSG1 Tryptase Gamma 1 NM_001204310.1 5′-CAT TGT GAG CTG GGG TGA AG-3′ 5′-AGA CCA GCA GAA GGA AGA GG-3′ 182 CMA1 Chymase 1 XM_002753692.4 5′-AAG TTG AAG GAG AAA GCC AGC-3′ 5′-TTC AAC ACA CCT GTT CTT CCC-3′ 125 DCN Decorin XM_002752816.3 5′-ATG AGG CTT CTG GGA TAG GC-3′ 5′-GTC CAG CAA AGT CGT GTC AG-3′ 170 BGN Biglycan XM_008990069.2 5′-GAG ACC CTG AAC GAA CTC CA-3′ 5′-TTG TTG TCC AAG TGC AGC TC-3′ 176 CCL2 C–C Motif Chemokine Ligand 2 NM_002982 5′-CAG CCA GAT GCA ATC AAT GCC-3′ 5′-TGG AAT CCT GAA CCC ACT TCT-3′ 190 PTX3 Pentraxin 3 NM_002852.3 5′-TAG TGT TTG TGG TGG GTG GA-3′ 5′-TGT GAG CCC TTC CTC TGA AT-3′ 110 IL6 Interleukin 6 XM_017975106.1 5′-AAG AGG TAG CTG CCC CAA AT-3′ 5′-AGT GCC TCT TTG CTG CTT TC-3′ 145 Immunohistochemistry Immunohistochemistry was performed as described (Schell et al., 2010). Parts of the testicular tissue, which were also used for explant cultures were fixed and embedded in paraffin. Sections (5 μm) of marmoset monkey testicular tissue were incubated with monoclonal mouse antibody against ACTA2 (#A5228, 1:1000; Sigma, St. Louis, MO, USA), monoclonal rabbit antibody against Calponin-1 (C-term) (#1806-1, 1:250; Epitomics, Cambridge, UK) and AR (#5153, 1:400; Cell Signaling, Cambridge, UK). Controls consisted of incubation with non-immune normal goat serum instead of specific antibodies. Hematoxylin counterstained the cell nuclei. Sections were examined with a Zeiss Axiovert microscope, an Insight Camera (18.2 Color Mosaik) and Spot advanced software 4.6 (both from SPOT Imaging Solutions, Sterling Heights, MI, USA). Immunofluorescence MKTPCs were seeded onto cover slips and incubated overnight. They were fixed with 3.7% formaldehyde (Sigma) for 10 min, washed with 0.1% Triton X-100/phosphate buffered saline (PBS) (Sigma) and permeabilized with ice cold 0.2% Triton X-100/PBS for 10 min. Cells were blocked with 0.1% Triton X-100/PBS + 5% goat normal serum (Sigma). The same primary antibodies as for the immunohistochemistry were used and were diluted in 0.1% Triton X-100/PBS + 5% goat normal serum, CNN1 1:100 (Epitomics), ACTA2 1:200 (Sigma), AR 1:100 (Cell Signaling) and incubated for 2 h at room temperature. Secondary antibody for CNN1 and AR, goat anti-rabbit alexa flour 488 1:1000 (Thermo Fisher Scientific, Waltham, MA, USA) for ACTA2, goat anti-mouse alexa fluor 555 1:1000, were incubated for 1.5 h at room temperature. Cells were washed and counterstained with 1.5 μg/ml DAPI for 5 min. For the control, normal goat serum was used instead of the primary antibody. Examination was performed with a fluorescence microscope (Zeiss, Oberkochen, Germany). Human peritubular cell isolation and culture The procedure involving human peritubular cells (HTPCs) isolation and culture from human testicular tissue samples exhibiting normal spermatogenesis was described previously (Albrecht et al., 2006; Schell et al., 2008). The patients (n = 7; aged 39–55 years) had granted written informed consent for scientific purposes. Cells were cultivated in DMEM High Glucose (Gibco) supplemented with 10% fetal bovine serum (Capricorn Scientific) and 1% penicillin/streptomycin (Biochrom, Berlin, Germany) under humidified conditions (37°C, 5% CO2). Cells in passages 3–7 were harvested for proteomic procedures, as described (Flenkenthaler et al., 2014). Nano-liquid chromatography–tandem mass spectrometry Cell pellets were resuspended in 8 M Urea in 50 mM ammonium bicarbonate, sonicated for 5 min at 4°C and homogenized using QIAshredders (Qiagen, Hilden, Germany) at 2500 g for 1 min. Protein concentration was determined using the Pierce 660 nm assay (Thermo Scientific, San Jose, CA). The 10 μg protein were reduced in 4 mM dithiothreitol (DTT) and 2 mM tris(2-carboxyethyl)phosphine for 30 min at 56°C followed by an alkylation step in 8 mM iodoacetamide (IAA) at room temperature in the dark for 30 min. Remaining IAA was quenched at a final concentration of 10 mM DTT. The samples underwent a first digestion step with Lys C (enzyme/substrate: 1/100; Wako, Neuss, Germany) at 37°C for 4 h, were diluted with ammonium bicarbonate to a concentration of 1 M urea and digested overnight with porcine trypsin (enzyme/substrate: 1/50; Promega, Madison, WI, USA) at 37°C. The 2.5 μg of peptides dissolved in 0.1% formic acid (FA) were subjected to liquid chromatography-electrospray ionization–tandem mass spectrometry (LC–MS/MS) analysis. LC was performed on an Ultimate 3000 RS system (Dionex, Sunnyvale, CA, USA). Peptide samples were first trapped on a C18 trap column (μ-Precolumn, C18 PepMap 100, 5 μm, Thermo Scientific, San Jose, CA) at a flow rate of 30 μL/min and separated on a C18 nano-flow column (Acclaim PepMap RSLC, 2 μm, 75 μm × 50 cm) at a flow rate of 0.2 μL/min using the following consecutive gradients: 5–25% B for 285 min and 25–50% B for 30 min (A: 0.1% FA in water, B: 0.1% FA in acetonitrile). Electrospray ionization was done with an uncoated SilicaTip (FS360-20-10-N-20-C15; New Objective, Woburn, MA) and a needle voltage of 2.3 kV. For MS data acquisition, a data dependent top 70 CID method was performed on a 5600+ mass spectrometer (Sciex, Concord, Canada). Data analysis Mass spectrometry (MS) raw data were processed using the MaxQuant software package (version: 1.6.0.1) (Cox and Mann, 2008). The data were searched separately for both species, using the UniProt subset for Callithrix jacchus for MKTPCs and the human Swiss-Prot subset for HTPCs (both: Release 2017/06), each augmented with the MaxQuant common contaminants database. Identification was performed with the ‘match between run’ feature enabled and a target decoy search strategy (resulting in a false discovery rate of 1%). For quantification, the MaxQuant label-free quantification strategy was applied. Data analysis and statistics were performed with Perseus (version: 1.5.8.5). For the scatterplot of MKTPC and HTPC datasets, the protein families contained in both datasets were merged into one matrix using the associated gene name. The ‘circoletto’ graph was done for the 100 most abundant protein identifications of each species with the corresponding online tool hosted by the bioinformatics analysis team (http://tools.bat.infspire.org/circoletto) using the following deviations from the default settings: e-value cutoff 10−15; e-value for coloring; use (score-min)/(max-min) ratio to assign colors. With the blastp online tool (https://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE=Proteins) the top 25 proteins were blasted against the human subset of the Swiss-Prot database. For the spectral counting approach, MS data were searched with Mascot (V 2.4.0) and analyzed with Scaffold (V 4.1.1) using the same databases as for the MaxQuant analysis. For abundance ranks, the averaged spectral counts were used. Functional classification of the proteins was done with the Panther GO analysis online tool (http://pantherdb.org) using gene names and the GO ‘biological process’ and ‘cellular process’ database. Results Isolation and initial characterization Using a similar approach as employed for HTPCs previously (Albrecht et al., 2006), we were able to isolate and culture MKTPCs. Explant cultures of marmoset testicular tissue fragments, small pieces of tissue (1–2 mm), were seeded onto cell culture dishes. The cells started growing out of the walls of the seminiferous tubules within 7–14 days, and small spindle-like cells became visible. They proliferated and cells were propagated for several passages (Fig. 1). Figure 1 View largeDownload slide Testicular tissue-explant culture of MKTPCs. (A) Small pieces of testicular tissue consisting of tubules (depicted with (T)) were placed onto cell culture dishes. The cells grow out from the walls of the seminiferous tubules within 7–14 days, picture was captured on day 10. (B) Cells in passage 1, 8 weeks after tissue extraction. (C) MKTPCs in passage 4, the cells were sub-cultured and propagated for several passages. (Scale bars represent 25 μm). Figure 1 View largeDownload slide Testicular tissue-explant culture of MKTPCs. (A) Small pieces of testicular tissue consisting of tubules (depicted with (T)) were placed onto cell culture dishes. The cells grow out from the walls of the seminiferous tubules within 7–14 days, picture was captured on day 10. (B) Cells in passage 1, 8 weeks after tissue extraction. (C) MKTPCs in passage 4, the cells were sub-cultured and propagated for several passages. (Scale bars represent 25 μm). As shown by immunohistochemistry, sections of the corresponding testicular tissue, from which explant cultures were derived, contain ACTA2-, CNN1- and AR-positive peritubular cells. The corresponding MKTPCs in vitro are likewise positive for CNN1, ACTA2 and AR as shown by immunofluorescence (Fig. 2). Figure 2 View largeDownload slide Expression of peritubular cell markers in situ and in cultured MKTPCs shown by immunohistochemistry. (A–C). Smooth muscle (SM) markers (A), CNN1, (B), ACTA2 and (C), AR were detected in individual testicular tissue sections from MKTPC donor animals (scale bar 50 μm). (D–F) Immunofluorescence of SM-markers, CNN1 (MKTPCs, passage 6) and ACTA2 (MKTPCs, passage 6), and AR (MKTPCs, passage 3) (D1, E1, F1). Higher magnifications, corresponding to D–F, respectively. Insets in A–F show negative controls. Figure 2 View largeDownload slide Expression of peritubular cell markers in situ and in cultured MKTPCs shown by immunohistochemistry. (A–C). Smooth muscle (SM) markers (A), CNN1, (B), ACTA2 and (C), AR were detected in individual testicular tissue sections from MKTPC donor animals (scale bar 50 μm). (D–F) Immunofluorescence of SM-markers, CNN1 (MKTPCs, passage 6) and ACTA2 (MKTPCs, passage 6), and AR (MKTPCs, passage 3) (D1, E1, F1). Higher magnifications, corresponding to D–F, respectively. Insets in A–F show negative controls. Further, as shown by RT-PCR, MKTPCs (all from passages 3 and 8) are positive for smooth muscle (SM) cell markers, including CNN1, ACTA2, and for the peritubular cell marker AR. The cells lack markers for Leydig cells (LHCGR, INSL3) or Sertoli cells (FSHR, GATA4). Mast cell markers like tryptase (TPSG1) and chymase (CMA1) and the T-cell marker CD3e were not detected. Like HTPCs, they express GDNF, extracellular matrix components including DCN and BGN, also the inflammatory molecules CCL2, PTX3 or IL6 (RT-PCR) and are thus similar to HTCPs. The cells can be passaged and the characteristic markers for MKTPCs remain stable for at least up to eight passages (Fig. 3). Figure 3 View largeDownload slide Expression analysis by RT-PCR. (A) Cultured MKTPCs express characteristic markers, SM-markers, e.g. ACTA2, CNN1 and the AR, (B) but lack markers for Leydig cells (LHCGR, INSL3) and Sertoli cells (FSHR, GATA4), which were found in the positive control (+), cDNA from Callithrix jacchus whole testis lysate. (C) Extracellular matrix, DCN and BGN, the inflammatory markers CCL2, PTX3 and IL6 and the neurotrophic factor GDNF were also expressed by MKTPCs. Expression of RPL19 was used as reference gene. Note that immune cell markers (CD3e, TPSG1, CMA1), expressed in the testis, were not found in MKTPCs. Figure 3 View largeDownload slide Expression analysis by RT-PCR. (A) Cultured MKTPCs express characteristic markers, SM-markers, e.g. ACTA2, CNN1 and the AR, (B) but lack markers for Leydig cells (LHCGR, INSL3) and Sertoli cells (FSHR, GATA4), which were found in the positive control (+), cDNA from Callithrix jacchus whole testis lysate. (C) Extracellular matrix, DCN and BGN, the inflammatory markers CCL2, PTX3 and IL6 and the neurotrophic factor GDNF were also expressed by MKTPCs. Expression of RPL19 was used as reference gene. Note that immune cell markers (CD3e, TPSG1, CMA1), expressed in the testis, were not found in MKTPCs. Proteome analysis from MKTPCs and comparison with HTPCs Proteins of MKTPCs from six individual donors were investigated by LC–MS/MS. In total 2437 protein groups were identified (FDR < 1%) in MKTPCs of which 1825 protein groups could be quantified in at least three individual samples. The mass spectrometry proteomics data were deposited in the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD009394. The inter-individual variance was investigated by a multi-scatter plot analysis of all protein label-free quantification (LFQ) intensities between all MKTPC individuals (Fig. 4A). The Pearson correlation coefficient ranged between 0.93 and 0.97. Figure 4 View largeDownload slide (A) Multi-scatter plot of log2 Max-Quant LFQ protein intensity values of MKTPCs from six donors. The number in the upper left corner of each individual scatterplot shows the Pearson correlation. (B) Multi-scatter plot of log2 Max-Quant LFQ protein intensity values of HTPCs from seven donors. Figure 4 View largeDownload slide (A) Multi-scatter plot of log2 Max-Quant LFQ protein intensity values of MKTPCs from six donors. The number in the upper left corner of each individual scatterplot shows the Pearson correlation. (B) Multi-scatter plot of log2 Max-Quant LFQ protein intensity values of HTPCs from seven donors. Additionally, a set of HTPCs from seven individual human donors was analyzed using exactly the same parameters and compared to the results of the MKTPCs. From 3374 protein groups, which were identified in HTPCs, 2137 are contained in the MKTPC dataset, demonstrating that 88% (2137 out of 2437) of the proteins in MKTPCs are represented in the HTPC data. The inter-individual variance was again assessed using a multi-scatter plot between all individual donors, which results in a Pearson correlation coefficient between 0.93 and 0.96 (Fig. 4B). The means of the MaxQuant LFQ values were scatter plotted against each other, which results in a correlation coefficient of 0.78 (Fig. 5) between human and marmoset cells. To investigate the variability of this inter-species comparison, each individual of MKTPCs were plotted against all individual HTPC donors. The mean Pearson correlation coefficient was 0.75 with no significant outliers (Grubbs’ test for outliers; 99% confidence) and therefore showing a very homologous correlation pattern (Supplementary Information Fig. S1). Figure 5 View largeDownload slide Scatter plot of averaged log2 LFQ protein intensity values of MKTPCs and HTPCs. Figure 5 View largeDownload slide Scatter plot of averaged log2 LFQ protein intensity values of MKTPCs and HTPCs. From both datasets the 100 most abundant proteins were retrieved and analyzed with the circoletto tool to visualize sequence homology of highly expressed proteins between HTPC and MKTPC (Fig. 6). The resulting network shows sequence homologies (<e-value 10−15) between the most abundant proteins found in both species, of which a vast majority shows high degree of similarity. Furthermore, a BLAST analysis of the 25 most abundant MKTPC using the Human Swiss-Prot database reveals that 22 are showing sequence identities >95% (Table II). Two further proteins could be matched to their human counterparts with >85% identity and represent members of the tropomyosin family, and only one single protein (F7FP14; uncharacterized protein) could not unambiguously be assigned to a human protein. Figure 6 View largeDownload slide Circoletto analysis of the top 100 protein identifications found in HTPC and MKTPC. The left half circle shows HTPC proteins sorted by their abundance from highest at the top to the lowest at the bottom. The right half circle lists the top 100 proteins identified in MKTPC in the same order. Both groups are compared with respect to protein sequence similarity. Proteins are represented by boxes on the circle. The length of the boxes indicates the length of the protein sequences. Sequence similarities between two corresponding proteins (MKTPC vs HTPC) are represented by a ribbon connecting the two boxes (cutoff e-value 10−15). Different ribbon color shades were used for better distinction. Figure 6 View largeDownload slide Circoletto analysis of the top 100 protein identifications found in HTPC and MKTPC. The left half circle shows HTPC proteins sorted by their abundance from highest at the top to the lowest at the bottom. The right half circle lists the top 100 proteins identified in MKTPC in the same order. Both groups are compared with respect to protein sequence similarity. Proteins are represented by boxes on the circle. The length of the boxes indicates the length of the protein sequences. Sequence similarities between two corresponding proteins (MKTPC vs HTPC) are represented by a ribbon connecting the two boxes (cutoff e-value 10−15). Different ribbon color shades were used for better distinction. Table II The top 25 most abundant proteins in MKTPC. Sequence similarities with corresponding human proteins were determined by BLAST analysis and are indicated as ‘% identity’. Callithrix jacchus UniProt accession Callithrix jacchus Gene name Callithrix jacchus protein name Human UniProt accession % Identity F7IH64 ACTA2 Actin, alpha 2, smooth muscle, aorta P62736 100 B0KWW5 FLNA Filamin-A isoform 1,2 P21333 98.79 F7BQY8 VIM Vimentin P08670 99.36 F6ZZ90 MYH9 Myosin heavy chain 9 P35579 99.49 F7I1Q9 TAGLN Transgelin Q01995 99.5 F7I9U1 TPM2 Tropomyosin beta chain isoform 2 P07951 87.32 F7HTQ3 CALD1 Caldesmon 1 Q05682 95.33 F7I0W8 LOC100409006 Uncharacterized protein P63261 100 U3EQM9 ACTN4 Alpha-actinin-4 O43707 99.89 F6YS84 TPM1 Tropomyosin 1 P09493 85.92 U3BZ94 TUBB4B Tubulin beta chain P68371 99.33 U3CYB6 ENO1 Alpha-enolase isoform 1 P06733 99.31 F7I318 LGALS1 Galectin P09382 99.07 F7HZP3 TUBA1B Tubulin alpha chain P68363 100 F6RMA3 ANXA2 Annexin P07355 99.71 F7IHK8 PRDX1 Peroxiredoxin 1 Q06830 97.99 U3EKQ0 ACTN1 Actinin alpha 1 P12814 99.78 F7CXD3 EEF1A1 Elongation factor 1-alpha P68104 100 F6SQW1 PPIA Peptidyl-prolyl cis-trans isomerase P62937 99.78 U3FXT1 HSPA5 78 kDa glucose-regulated protein P11021 99.39 F7HVD2 HSPA8 Heat shock protein family A (Hsp70) member 8 P11142 99.85 F7FP14 Uncharacterized protein Q8N386 62.38 F6YTV2 LMNA Lamin A/C P02545 98.65 F6XL35 PKM Pyruvate kinase P14618 99.06 U3D5Z3 TLN1 Talin-1 Q9Y490 99.21 Callithrix jacchus UniProt accession Callithrix jacchus Gene name Callithrix jacchus protein name Human UniProt accession % Identity F7IH64 ACTA2 Actin, alpha 2, smooth muscle, aorta P62736 100 B0KWW5 FLNA Filamin-A isoform 1,2 P21333 98.79 F7BQY8 VIM Vimentin P08670 99.36 F6ZZ90 MYH9 Myosin heavy chain 9 P35579 99.49 F7I1Q9 TAGLN Transgelin Q01995 99.5 F7I9U1 TPM2 Tropomyosin beta chain isoform 2 P07951 87.32 F7HTQ3 CALD1 Caldesmon 1 Q05682 95.33 F7I0W8 LOC100409006 Uncharacterized protein P63261 100 U3EQM9 ACTN4 Alpha-actinin-4 O43707 99.89 F6YS84 TPM1 Tropomyosin 1 P09493 85.92 U3BZ94 TUBB4B Tubulin beta chain P68371 99.33 U3CYB6 ENO1 Alpha-enolase isoform 1 P06733 99.31 F7I318 LGALS1 Galectin P09382 99.07 F7HZP3 TUBA1B Tubulin alpha chain P68363 100 F6RMA3 ANXA2 Annexin P07355 99.71 F7IHK8 PRDX1 Peroxiredoxin 1 Q06830 97.99 U3EKQ0 ACTN1 Actinin alpha 1 P12814 99.78 F7CXD3 EEF1A1 Elongation factor 1-alpha P68104 100 F6SQW1 PPIA Peptidyl-prolyl cis-trans isomerase P62937 99.78 U3FXT1 HSPA5 78 kDa glucose-regulated protein P11021 99.39 F7HVD2 HSPA8 Heat shock protein family A (Hsp70) member 8 P11142 99.85 F7FP14 Uncharacterized protein Q8N386 62.38 F6YTV2 LMNA Lamin A/C P02545 98.65 F6XL35 PKM Pyruvate kinase P14618 99.06 U3D5Z3 TLN1 Talin-1 Q9Y490 99.21 View Large Table II The top 25 most abundant proteins in MKTPC. Sequence similarities with corresponding human proteins were determined by BLAST analysis and are indicated as ‘% identity’. Callithrix jacchus UniProt accession Callithrix jacchus Gene name Callithrix jacchus protein name Human UniProt accession % Identity F7IH64 ACTA2 Actin, alpha 2, smooth muscle, aorta P62736 100 B0KWW5 FLNA Filamin-A isoform 1,2 P21333 98.79 F7BQY8 VIM Vimentin P08670 99.36 F6ZZ90 MYH9 Myosin heavy chain 9 P35579 99.49 F7I1Q9 TAGLN Transgelin Q01995 99.5 F7I9U1 TPM2 Tropomyosin beta chain isoform 2 P07951 87.32 F7HTQ3 CALD1 Caldesmon 1 Q05682 95.33 F7I0W8 LOC100409006 Uncharacterized protein P63261 100 U3EQM9 ACTN4 Alpha-actinin-4 O43707 99.89 F6YS84 TPM1 Tropomyosin 1 P09493 85.92 U3BZ94 TUBB4B Tubulin beta chain P68371 99.33 U3CYB6 ENO1 Alpha-enolase isoform 1 P06733 99.31 F7I318 LGALS1 Galectin P09382 99.07 F7HZP3 TUBA1B Tubulin alpha chain P68363 100 F6RMA3 ANXA2 Annexin P07355 99.71 F7IHK8 PRDX1 Peroxiredoxin 1 Q06830 97.99 U3EKQ0 ACTN1 Actinin alpha 1 P12814 99.78 F7CXD3 EEF1A1 Elongation factor 1-alpha P68104 100 F6SQW1 PPIA Peptidyl-prolyl cis-trans isomerase P62937 99.78 U3FXT1 HSPA5 78 kDa glucose-regulated protein P11021 99.39 F7HVD2 HSPA8 Heat shock protein family A (Hsp70) member 8 P11142 99.85 F7FP14 Uncharacterized protein Q8N386 62.38 F6YTV2 LMNA Lamin A/C P02545 98.65 F6XL35 PKM Pyruvate kinase P14618 99.06 U3D5Z3 TLN1 Talin-1 Q9Y490 99.21 Callithrix jacchus UniProt accession Callithrix jacchus Gene name Callithrix jacchus protein name Human UniProt accession % Identity F7IH64 ACTA2 Actin, alpha 2, smooth muscle, aorta P62736 100 B0KWW5 FLNA Filamin-A isoform 1,2 P21333 98.79 F7BQY8 VIM Vimentin P08670 99.36 F6ZZ90 MYH9 Myosin heavy chain 9 P35579 99.49 F7I1Q9 TAGLN Transgelin Q01995 99.5 F7I9U1 TPM2 Tropomyosin beta chain isoform 2 P07951 87.32 F7HTQ3 CALD1 Caldesmon 1 Q05682 95.33 F7I0W8 LOC100409006 Uncharacterized protein P63261 100 U3EQM9 ACTN4 Alpha-actinin-4 O43707 99.89 F6YS84 TPM1 Tropomyosin 1 P09493 85.92 U3BZ94 TUBB4B Tubulin beta chain P68371 99.33 U3CYB6 ENO1 Alpha-enolase isoform 1 P06733 99.31 F7I318 LGALS1 Galectin P09382 99.07 F7HZP3 TUBA1B Tubulin alpha chain P68363 100 F6RMA3 ANXA2 Annexin P07355 99.71 F7IHK8 PRDX1 Peroxiredoxin 1 Q06830 97.99 U3EKQ0 ACTN1 Actinin alpha 1 P12814 99.78 F7CXD3 EEF1A1 Elongation factor 1-alpha P68104 100 F6SQW1 PPIA Peptidyl-prolyl cis-trans isomerase P62937 99.78 U3FXT1 HSPA5 78 kDa glucose-regulated protein P11021 99.39 F7HVD2 HSPA8 Heat shock protein family A (Hsp70) member 8 P11142 99.85 F7FP14 Uncharacterized protein Q8N386 62.38 F6YTV2 LMNA Lamin A/C P02545 98.65 F6XL35 PKM Pyruvate kinase P14618 99.06 U3D5Z3 TLN1 Talin-1 Q9Y490 99.21 View Large To review if the most abundant proteins in MKTPCs are also highly abundant in HTPCs, the identified MKTPC and HTPC protein groups were sorted according to their MS/MS spectral count values, a measure for their abundance. Comparison of both datasets revealed that 21 out of the top 25 MKTPC protein groups are contained in the top 50 HTPC protein groups (Table III). Only transgelin (TAGLN), caldesmon (CALD1) and the tropomyosins TPM1 and TPM4 ranked noticeably lower in HTPCs compared to MKTPC. Table III The top 25 most abundant MKTPC protein groups and their number of group members. The abundance ranks of MKTPC protein groups are compared to the ranks of the corresponding protein groups in HTPCs. Rank in MKTPC Rank in HTPC Protein group # Proteins in group 1 2 Actin, cytoplasmic 1 11 2 4 Filamin-A 3 3 7 Myosin 9 5 4 9 Alpha-actinin-4 7 5 1 Vimentin 45 6 3 Tubulin beta 19 7 4 Uncharacterized protein homologue to human neuroblast differentiation-associated protein AHNAK 3 8 63 Transgelin 1 9 93 Tropomyosin alpha-4 chain 5 10 42 Myosin-10 3 11 239 Tropomyosin alpha-1 chain 4 12 23 Filamin-C 2 13 26 Alpha-enolase 6 14 12 Talin-1 1 15 14 Heat shock protein family A (Hsp70) member 5 1 16 113 Caldesmon 3 17 44 Filamin-B 4 18 6 Plectin 2 19 19 Clathrin heavy chain 2 20 11 Tubulin alpha chain 7 21 15 Heat shock protein 90 4 22 21 Cytoplasmic dynein 1 heavy chain 1 1 23 8 Pyruvate kinase 5 24 20 Lamin isoform A 3 25 27 Heat shock protein family A (Hsp70) member 8 3 Rank in MKTPC Rank in HTPC Protein group # Proteins in group 1 2 Actin, cytoplasmic 1 11 2 4 Filamin-A 3 3 7 Myosin 9 5 4 9 Alpha-actinin-4 7 5 1 Vimentin 45 6 3 Tubulin beta 19 7 4 Uncharacterized protein homologue to human neuroblast differentiation-associated protein AHNAK 3 8 63 Transgelin 1 9 93 Tropomyosin alpha-4 chain 5 10 42 Myosin-10 3 11 239 Tropomyosin alpha-1 chain 4 12 23 Filamin-C 2 13 26 Alpha-enolase 6 14 12 Talin-1 1 15 14 Heat shock protein family A (Hsp70) member 5 1 16 113 Caldesmon 3 17 44 Filamin-B 4 18 6 Plectin 2 19 19 Clathrin heavy chain 2 20 11 Tubulin alpha chain 7 21 15 Heat shock protein 90 4 22 21 Cytoplasmic dynein 1 heavy chain 1 1 23 8 Pyruvate kinase 5 24 20 Lamin isoform A 3 25 27 Heat shock protein family A (Hsp70) member 8 3 Table III The top 25 most abundant MKTPC protein groups and their number of group members. The abundance ranks of MKTPC protein groups are compared to the ranks of the corresponding protein groups in HTPCs. Rank in MKTPC Rank in HTPC Protein group # Proteins in group 1 2 Actin, cytoplasmic 1 11 2 4 Filamin-A 3 3 7 Myosin 9 5 4 9 Alpha-actinin-4 7 5 1 Vimentin 45 6 3 Tubulin beta 19 7 4 Uncharacterized protein homologue to human neuroblast differentiation-associated protein AHNAK 3 8 63 Transgelin 1 9 93 Tropomyosin alpha-4 chain 5 10 42 Myosin-10 3 11 239 Tropomyosin alpha-1 chain 4 12 23 Filamin-C 2 13 26 Alpha-enolase 6 14 12 Talin-1 1 15 14 Heat shock protein family A (Hsp70) member 5 1 16 113 Caldesmon 3 17 44 Filamin-B 4 18 6 Plectin 2 19 19 Clathrin heavy chain 2 20 11 Tubulin alpha chain 7 21 15 Heat shock protein 90 4 22 21 Cytoplasmic dynein 1 heavy chain 1 1 23 8 Pyruvate kinase 5 24 20 Lamin isoform A 3 25 27 Heat shock protein family A (Hsp70) member 8 3 Rank in MKTPC Rank in HTPC Protein group # Proteins in group 1 2 Actin, cytoplasmic 1 11 2 4 Filamin-A 3 3 7 Myosin 9 5 4 9 Alpha-actinin-4 7 5 1 Vimentin 45 6 3 Tubulin beta 19 7 4 Uncharacterized protein homologue to human neuroblast differentiation-associated protein AHNAK 3 8 63 Transgelin 1 9 93 Tropomyosin alpha-4 chain 5 10 42 Myosin-10 3 11 239 Tropomyosin alpha-1 chain 4 12 23 Filamin-C 2 13 26 Alpha-enolase 6 14 12 Talin-1 1 15 14 Heat shock protein family A (Hsp70) member 5 1 16 113 Caldesmon 3 17 44 Filamin-B 4 18 6 Plectin 2 19 19 Clathrin heavy chain 2 20 11 Tubulin alpha chain 7 21 15 Heat shock protein 90 4 22 21 Cytoplasmic dynein 1 heavy chain 1 1 23 8 Pyruvate kinase 5 24 20 Lamin isoform A 3 25 27 Heat shock protein family A (Hsp70) member 8 3 To assess similarities between MKTPCs and HTPCs at the functional level, the identified proteins were analyzed with the PANTHER analysis tool using the ‘Biological Process’ and ‘Cellular Process’ Gene Ontology Databases. The proteins of both MKTPC and HTPC show highly similar distributions concerning related biological and cellular processes, suggesting strong biochemical and functional similarities between MKTPC and HTPC (Fig. 7). Figure 7 View largeDownload slide (A) GO analysis using the ‘biological process’ database for MKTPC (left) and HTPC (right) proteomes. (B) GO analysis regarding the ‘cellular process’ subset for MKTPC (left) and HTPC (right). Figure 7 View largeDownload slide (A) GO analysis using the ‘biological process’ database for MKTPC (left) and HTPC (right) proteomes. (B) GO analysis regarding the ‘cellular process’ subset for MKTPC (left) and HTPC (right). Discussion The previous studies in HTPCs provided new insights in to the functions of these testicular cells (Spinnler et al., 2010; Mayerhofer, 2013; Landreh et al., 2014; Welter et al., 2014; Mayer et al., 2016). While a previous proteomic study of HTPCs revealed that they are rather homogeneous with respect to their cellular proteome and their repertoire of secreted factors (Flenkenthaler et al., 2014), further studies showed that their ability to secrete steroids (Landreh et al., 2014) or to respond to stimuli with cytokine secretion (Mayer et al., 2016) occur in a patient-specific fashion. It is likely that the differences are due to age, lifestyle and or medical history, which are beyond control. We hypothesized that cells from a non-human primate species, specifically with a controlled lifestyle, may be an ideal additional model in which to study TPCs. We chose the common marmoset monkey (Callithrix jacchus), which is a well-established model organism for reproductive research, for this model. The testicular structures of marmosets are comparable in several biologically relevant aspects with humans, including characteristics of germ cell development and function (Michel and Mahouy, 1990; Millar et al., 2000; Mansfield, 2003; Zuhlke and Weinbauer, 2003). Importantly, the architecture of the tubular wall shows several layers and therefore comes close to the human situation. Both contrast to rodent testes, in which the wall of the seminiferous tubules consists of a single cell layer of peritubular cells. Finally, the whole genome from Callithrix jacchus was sequenced (Sato et al., 2015), which enables further investigation on the genomic and proteomic level. We successfully isolated and cultured cells from the wall of seminiferous tubules of the testis of young adult, healthy Callithrix jacchus, using the same approach as for HTPCs, namely explant culture. The cells obtained upon initial characterization could be clearly identified as pure peritubular cells. Virtually all MKTPCs were immunoreactive for the smooth muscle markers, CNN1, ACTA2, which were detected only by peritubular cells in situ. In addition, the combination of smooth muscle markers, expression of AR and GDNF and the absence of Sertoli cell markers (FSHR, GATA4) and Leydig cell markers (LHCGR, INSL3) allows this conclusion. Importantly, the expression of the characteristic factors remains stable over at least eight passages. In human testes tryptase-immunoreactive mast cells are found close to the tubular wall (Meineke et al., 2000) and within the layers of peritubular cells and we also detected tryptase-immunoreactive mast cells by immunohistochemistry in Callithrix jacchus testes (data not shown). We therefore explored a possible contamination with immune cells. RT-PCR for mast cell markers tryptase, chymase or the T-cell marker CD3e yielded, however, negative results in MKTPC. We further compared the MKTPC proteome data with a published human macrophage proteome data (Eligini et al., 2015). We found that five proteins coincide, namely Chloride intracellular channel protein 1, Elongation factor 2, Plastin 3, Tubulin alpha 1 chain, Vimentin. These proteins are, however, not specific for macrophages but represent ubiquitously occurring proteins. Apolipoprotein B receptor, which is considered a characteristic macrophage receptor (Hassel et al., 2017), is not detected in our proteome analysis. Thus, isolation of MKTPCs is a practical way to obtain pure testicular peritubular cells of a non-human primate species. We initially tested whether they further resemble HTPCs, and found that they produce DCN and BGN, CCL2, PTX3 and IL6 as well as GDNF. Hence, they resemble HTPCs in this respect (Spinnler et al., 2010; Mayer et al., 2016; Walenta et al., 2018). To further characterize MKTPCs at the protein level, a proteome study of peritubular cells obtained from six individual young adult Callithrix jacchus donors was performed. In order to focus on the most abundant proteins being easily assessable with a single-run LC–MS/MS method, we kept the proteomics workflow as simple as possible and did not use any pre-fractionation at the protein or the peptide level. Nevertheless, the analysis of the acquired mass spectra led to the identification of 2437 MKTPC protein groups (FDR < 1%). For the chosen approach, this represents a fairly high number of protein IDs and reflects the suitability of the Callithrix jacchus database for LC–MS based proteome analysis of MKTPC samples. Additionally, a multi-scatter plot analysis between the donors revealed very reproducible protein expression patterns, demonstrating the robustness of isolation and cultivation methods as well as a low inter-individual variability between the individual donors. For the suitability of the animal model, the similarity between MKTPCs and HTPCs at the proteome level is an important indicator. To assess this, HTPC proteomes from seven human donors were analyzed using exactly the same methodology. Inter-individual correlation analysis shows clear homogeneity and reproducibility similar to MKTPCs, with the latter one being more accessible and generated under monitored conditions. The inter-species scatter plot analysis of protein intensity values between MKTPC and HTPC showed a Pearson correlation coefficient of 0.78 indicating similar abundance patterns of MKTPC and HTPC proteins. A further MKTPC vs HTPC multi-scatter plot analysis at the level of individuals showed in all cases very similar Pearson correlation coefficients with no outliers. Taken together the correlation analyses reveal a clear conformity between MKTPCs and HTPCs on the level of protein expression patterns and a high degree of inter-individual reproducibility of MKTPCs. A circoletto network analysis as well as a BLAST analysis of the 25 most abundant proteins showed high sequence homology between HTPC and MKTPC proteins, indicating a high degree of functional similarity. Using a spectral count quantification approach combined with homology based protein grouping, we could further demonstrate that the broad majority of the 25 most abundant MKTPC protein groups are also highly expressed in HTPCs. Only transgelin (TAGLN), caldesmon (CALD1) and the two tropomyosins (TPM1 and TPM4) showed lower spectral counts in MKTPC. Since all of these four proteins bind to actin this finding may reflect slight differences in the cytoskeleton between MKTPC and HTPCs. Finally, the PANTHER GO analysis of MKTPC and HTPC proteins lead to almost identical results, suggesting a strong resemblance at the level of biological and biochemical processes between MKTPCs and HTPCs. The high similarity at the proteome level elaborated here further approves MKTPC as an excellent non-human primate model to study the biology of HTPCs. In summary, isolation of MKTPCs is a feasible way to obtain primate peritubular cells, which resemble their human counterparts. They are derived from young adults raised under controlled conditions and provide an opportunity to explore functions and regulation of testicular peritubular cells without unknown confounding issues like lifestyle, age, nutrition and the medical history of patients. We anticipate that this may lead to a better understanding of the role of peritubular cells in male (in)fertility, including their role in the spermatogonial stem cell niche and their plasticity. Supplementary data Supplementary data are available at Molecular Human Reproduction online. Acknowledgements The authors thank Astrid Tiefenbacher for skillful technical assistance. Authors’ roles N.S. performed the majority of the cellular experiments and K.G.D. participated in these experiments. J.B.S, F.F., T.F. and G.A performed proteomic studies and evaluated the results. J.U.S, F.-M.K., C.D. and R.B. provided testicular tissues, as well as conceptual input, A.M. conceived of the study, directed the work and supervised the experiments. N.S. and A.M. drafted the article. All authors contributed to and approved the final version. This work was performed in partial fulfillments of the requirements of doctoral degrees (N.S. and J.B.S.) at LMU. Funding Grants from the Deutsche Forschungsgemeinschaft (MA 1080/27-1; AR 362/9-1; BE 2296/8-1). Conflicts of interest The authors declare no competing financial interests. References Albrecht M , Ramsch R , Kohn FM , Schwarzer JU , Mayerhofer A . Isolation and cultivation of human testicular peritubular cells: a new model for the investigation of fibrotic processes in the human testis and male infertility . J Clin Endocrinol Metab 2006 ; 91 : 1956 – 1960 . Google Scholar CrossRef Search ADS PubMed Chen LY , Brown PR , Willis WB , Eddy EM . Peritubular myoid cells participate in male mouse spermatogonial stem cell maintenance . Endocrinology 2014 ; 155 : 4964 – 4974 . Google Scholar CrossRef Search ADS PubMed Chen LY , Willis WD , Eddy EM . Targeting the Gdnf gene in peritubular myoid cells disrupts undifferentiated spermatogonial cell development . Proc Nat Acad Sci USA 2016 ; 113 : 1829 – 1834 . Google Scholar CrossRef Search ADS PubMed Cox J , Mann M . MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification . Nat Biotechnol 2008 ; 26 : 1367 – 1372 . Google Scholar CrossRef Search ADS PubMed Eligini S , Brioschi M , Fiorelli S , Tremoli E , Banfi C , Colli S . Human monocyte-derived macrophages are heterogenous: proteomic profile of different phenotypes . J Proteomics 2015 ; 124 : 112 – 123 . Google Scholar CrossRef Search ADS PubMed Flenkenthaler F , Windschuttl S , Frohlich T , Schwarzer JU , Mayerhofer A , Arnold GJ . Secretome analysis of testicular peritubular cells: a window into the human testicular microenvironment and the spermatogonial stem cell niche in man . J Proteome Res 2014 ; 13 : 1259 – 1269 . Google Scholar CrossRef Search ADS PubMed Hassel B , De Souza GA , Stensland ME , Ivanovic J , Voie O , Dahlberg D . The proteome of pus from human brain abscesses: host-derived neurotoxic proteins and the cell-type diversity of CNS pus . J Neurosurg 2017 ; 20 : 1 – 9 . Google Scholar CrossRef Search ADS Landreh L , Spinnler K , Schubert K , Hakkinen MR , Auriola S , Poutanen M , Soder O , Svechnikov K , Mayerhofer A . Human testicular peritubular cells host putative stem Leydig cells with steroidogenic capacity . J Clin Endocrinol Metab 2014 ; 99 : E1227 – E1235 . Google Scholar CrossRef Search ADS PubMed Li LH , Donald JM , Golub MS . Review on testicular development, structure, function, and regulation in common marmoset . Birth Defects Res B Dev Reprod Toxicol 2005 ; 74 : 450 – 469 . Google Scholar CrossRef Search ADS PubMed Mansfield K . Marmoset models commonly used in biomedical research . Comp Med 2003 ; 53 : 383 – 392 . Google Scholar PubMed Mayer C , Adam M , Glashauser L , Dietrich K , Schwarzer JU , Kohn FM , Strauss L , Welter H , Poutanen M , Mayerhofer A . Sterile inflammation as a factor in human male infertility: involvement of Toll like receptor 2, biglycan and peritubular cells . Sci Rep 2016 ; 6 : 37128 . Google Scholar CrossRef Search ADS PubMed Mayerhofer A . Human testicular peritubular cells: more than meets the eye . Reproduction 2013 ; 145 : R107 – R116 . Google Scholar CrossRef Search ADS PubMed Meineke V , Frungieri MB , Jessberger B , Vogt H , Mayerhofer A . Human testicular mast cells contain tryptase: increased mast cell number and altered distribution in the testes of infertile men . Fertil Steril 2000 ; 74 : 239 – 244 . Google Scholar CrossRef Search ADS PubMed Michel JB , Mahouy G . The marmoset in biomedical research. Value of this primate model for cardiovascular studies . Pathol Biol (Paris) 1990 ; 38 : 197 – 204 . Google Scholar PubMed Millar MR , Sharpe RM , Weinbauer GF , Fraser HM , Saunders PT . Marmoset spermatogenesis: organizational similarities to the human . Int J Androl 2000 ; 23 : 266 – 277 . Google Scholar CrossRef Search ADS PubMed Sato K , Kuroki Y , Kumita W , Fujiyama A , Toyoda A , Kawai J , Iriki A , Sasaki E , Okano H , Sakakibara Y . Resequencing of the common marmoset genome improves genome assemblies and gene-coding sequence analysis . Sci Rep 2015 ; 5 : 16894 . Google Scholar CrossRef Search ADS PubMed Schell C , Albrecht M , Mayer C , Schwarzer JU , Frungieri MB , Mayerhofer A . Exploring human testicular peritubular cells: identification of secretory products and regulation by tumor necrosis factor-alpha . Endocrinology 2008 ; 149 : 1678 – 1686 . Google Scholar CrossRef Search ADS PubMed Schell C , Albrecht M , Spillner S , Mayer C , Kunz L , Kohn FM , Schwarzer U , Mayerhofer A . 15-Deoxy-delta 12-14-prostaglandin-J2 induces hypertrophy and loss of contractility in human testicular peritubular cells: implications for human male fertility . Endocrinology 2010 ; 151 : 1257 – 1268 . Google Scholar CrossRef Search ADS PubMed Spinnler K , Kohn FM , Schwarzer U , Mayerhofer A . Glial cell line-derived neurotrophic factor is constitutively produced by human testicular peritubular cells and may contribute to the spermatogonial stem cell niche in man . Hum Reprod 2010 ; 25 : 2181 – 2187 . Google Scholar CrossRef Search ADS PubMed Walenta L , Fleck D , Frohlich T , von Eysmondt H , Arnold GJ , Spehr J , Schwarzer JU , Kohn FM , Spehr M , Mayerhofer A . ATP-mediated events in peritubular cells contribute to sterile testicular inflammation . Sci Rep 2018 ; 8 : 1431 . Google Scholar CrossRef Search ADS PubMed Welter H , Huber A , Lauf S , Einwang D , Mayer C , Schwarzer JU , Kohn FM , Mayerhofer A . Angiotensin II regulates testicular peritubular cell function via AT1 receptor: a specific situation in male infertility . Mol Cell Endocrinol 2014 ; 393 : 171 – 178 . Google Scholar CrossRef Search ADS PubMed Zuhlke U , Weinbauer G . The common marmoset (Callithrix jacchus) as a model in toxicology . Toxicol Pathol 2003 ; 31 : 123 – 127 . Google Scholar CrossRef Search ADS PubMed © The Author 2018. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices)

Journal

Molecular Human ReproductionOxford University Press

Published: Aug 1, 2018

There are no references for this article.

You’re reading a free preview. Subscribe to read the entire article.


DeepDyve is your
personal research library

It’s your single place to instantly
discover and read the research
that matters to you.

Enjoy affordable access to
over 18 million articles from more than
15,000 peer-reviewed journals.

All for just $49/month

Explore the DeepDyve Library

Search

Query the DeepDyve database, plus search all of PubMed and Google Scholar seamlessly

Organize

Save any article or search result from DeepDyve, PubMed, and Google Scholar... all in one place.

Access

Get unlimited, online access to over 18 million full-text articles from more than 15,000 scientific journals.

Your journals are on DeepDyve

Read from thousands of the leading scholarly journals from SpringerNature, Elsevier, Wiley-Blackwell, Oxford University Press and more.

All the latest content is available, no embargo periods.

See the journals in your area

DeepDyve

Freelancer

DeepDyve

Pro

Price

FREE

$49/month
$360/year

Save searches from
Google Scholar,
PubMed

Create lists to
organize your research

Export lists, citations

Read DeepDyve articles

Abstract access only

Unlimited access to over
18 million full-text articles

Print

20 pages / month

PDF Discount

20% off