Phenotype and steroidogenic potential of PDGFRα-positive rat neonatal peritubular cells

Molecular and Cellular Endocrinology 372 (2013) 96–104

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Phenotype and steroidogenic potential of PDGFRa-positive rat neonatal peritubular cells Luise Landreh, Jan-Bernd Stukenborg, Olle Söder, Konstantin Svechnikov ⇑ Department of Women’s and Children’s Health, Pediatric Endocrinology Unit, Q2:08, Karolinska Institutet and University Hospital, SE-17176 Solna, Sweden

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Article history: Received 29 October 2012 Received in revised form 21 March 2013 Accepted 21 March 2013 Available online 30 March 2013 Keywords: Testis Neonatal peritubular cells PDGFRa Stem cells

a b s t r a c t Platelet-derived growth factor receptor a (PDGFRa)-positive peritubular cells (PTCs) are suggested to be putative stem Leydig cells. At present little is known about their phenotype and steroidogenic potential. We isolated highly purified PDGFRa-positive neonatal PTCs by magnetic cell sorting (MACS) from 8dpp rat testes and characterized them in vitro. We have demonstrated that PDGFRa-positive PTCs have a mixed phenotype. They expressed PTC-specific genes (aSma, Myh11), pluripotency markers (Pou5f1, nestin, Lifr) and genes encoding steroidogenic enzymes. Treatment with the cAMP-analog (Bu)2cAMP for 7 days upregulated steroidogenic enzyme gene expression and significantly increased their steroidogenic potential. The main end-point steroid was progesterone due to rapid inactivation of CYP17 and 17bHSD. Long-term culturing of PDGFRa-positive PTCs increased the expression of Myh11, and treatment with (Bu)2cAMP attenuated this process. All together, our findings support the hypothesis that neonatal PDGFRa-positive PTCs are steroidogenic competent progeny of stem Leydig cells (SLCs) which give rise to the adult Leydig cell lineage. Ó 2013 Elsevier Ireland Ltd. All rights reserved.

1. Introduction Earlier morphological studies have shown that peritubular mesenchymal cells located in the outermost line in the peritubular compartment appear to be the precursors for the adult Leydig cell lineage (ALC) (Mendis-Handagama and Ariyaratne, 2001; Hardy et al., 1989; Ariyaratne and Chamindrani Mendis-Handagama, 2000). Moreover, it has been reported that vascular smooth muscle cells and pericytes of testicular blood vessels can also give rise to the ALC lineage (Davidoff et al., 2004), supporting the idea of multifocal origin of Leydig cell precursors in neonatal testis (Haider, 2004, Russell and de Franca; 1995; Habert et al., 2001). It was reported that neonatal peritubular cells abundantly express the PDGFRa (Gnessi et al., 1995) and that PDGFa-deficient mice do not develop the adult Leydig cell lineage (Gnessi et al., 2000), suggesting that PDGFa-mediated signaling plays an important role in the development of these testicular cells. In addition, the PDGFRa has also been demonstrated to be expressed at all stages of Leydig cell development with a peak expression in low differentiated progenitor Leydig cell population (Ge et al., 2005). Taken together all these findings, the PDGFRa seems to be an appropriate marker to isolate and characterize putative SLCs. Indeed, PDGFRa-positive and LHR-negative testicular cells isolated from 7dpp rat testis were shown to express putative Leydig stem cell markers (e.g., c-kit, ⇑ Corresponding author. Tel.: +46 (0)8 517 79528; fax: +46 (0)8 517 75128. E-mail address: [email protected] (K. Svechnikov). 0303-7207/$ - see front matter Ó 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.mce.2013.03.019

LIFR, PDGFRa) and had no steroidogenic competence (Ge et al., 2006). This allows their expansion of numbers during prolonged culture in vitro and expression of Leydig cell-related genes after culturing in differentiation-inducing medium (Ge et al., 2006). It was concluded that PDGFRa-positive spindle-shaped cells are putative SLCs and can give rise to the adult Leydig cell lineage (Ge et al., 2006). However, it was not yet clear whether the PDGFRa positive spindle shaped cells belong to the peritubular cell (PTC) lineage and whether they already possess steroidogenic potential. Thus, the purpose of the present study was to characterize the phenotype and steroidogenic competence of highly purified neonatal PDGFRa-positive testicular cells in vitro.

2. Materials and methods 2.1. Materials Dulbecco’s Modified Eagle’s Medium (DMEM)-Ham’s nutrient mixture F-12 (313330-038), Modified Eagle’s Medium (MEM) (31095-029), Hank’s Balanced Salts Solution (HBSS) without Ca2+ and Mg2+ (14170-088), fetal calf serum (FCS) (26010-066) and antibiotics were obtained from Gibco/BRL (Life Technologies, Paisley, Scotland). Percoll (P1644), HEPES (H0887), hCG (14000 IU/mg), bovine serum albumin (BSA) (A8806) and collagenase type I (C0130) were purchased from Sigma (Sigma Chemical Co., St. Louis, MO).

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Antibodies against PDGFRa (sc-338), Cyp11 (sc-47652), LIFR (sc-659) and LHR (sc-25828) were purchased from Santa Cruz (Santa Cruz, Biotechnology, Inc., Santa Cruz, CA), the antibody against a smooth muscle actin (a-SMA) (A 2547) and Cyp11 (OBT1756) were obtained from Sigma and an AbD Serotec (AbD Serotec, Martinsried, Germany), respectively. Secondary antibodies were obtained from Jackson Immunoresearch (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) and Santa Cruz, (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). Materials for the magnetic cell separation were bought from Miltenyi biotech (Miltenyi biotech Inc., Bergisch Gladbach, Germany). 2.2. Animals Testes from 8, 20, 40 and 60 dpp Sprague–Dawley rats (Scanbur, Sweden and Charles-River, Germany) were used for the isolation of PDGFRa-positive testicular cells and the adult Leydig cell lineage. The animals were housed under standard conditions. These exper-

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iments were approved by the Northern Stockholm Animal Ethics Committee. 2.3. Magnetic labeling and isolation of PDGFRa-positive testicular cells The magnetic cell separation was performed following the protocol described for fetal Leydig cells (Weisser et al., 2011). In short, the decapsulated testes were incubated in MEM containing 0.1% BSA and 0.25 mg/ml collagenase Type 1 for 20 min by 37 °C. The tubuli were separated mechanically and washed several times in HBSS containing 0.1% BSA. After depletion of the FLC population by using anti-LHR IgGs, the cell suspension was incubated with polyclonal rabbit anti-PDGFRa IgGs (sc-338, Santa Cruz) in HBSS containing 2 mM EDTA and 0.5% BSA (20 ll/107 cells) for 20 min at 4 °C. After washing the cells were labeled with the secondary goat anti-rabbit IgG conjugated with magnetic microbeads (30 ll/107 cells) (Miltenyi Biotec Inc., Bergisch Gladbach, Germany) for 20 min at 4 °C. For the subsequent magnetic separation, the cell

Fig. 1. Fluorescent immunostaining of testicular sections from 8dpp old rats for aSMA, PDGFRa, LIFR and StAR. aSMA-positive neonatal PTCs (B) are immunopositive for PDGFRa (A), double immunostaining (C). Some PTCs express LIFR (D) and StAR (G), double labeling for these proteins with aSMA (F and I, respectively). White arrowheads show co-localization of LIFR and StAR with aSMA. Black arrowhead shows StAR-positive fetal Leydig cells. The respective negative controls are presented in the bottom left corner of the overlay pictures. The scale bar represents 100 lm in figure A–C and 50 lm in figure D–I.

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Fig. 2. Characterization of isolated PDGFRa-positive PTCs for the expression of stem (PDGFRa, LIFR), steroidogenic (Cyp11, StAR) and peritubular cell marker (aSMA). Immunofluorescent staining of freshly isolated PTCs for PDGFRa (A) and aSMA (B), overlay (C). Insert shows negative control (C), size marker: 50 lm. Freshly isolated PDGFRa-positive PTCs are stained for PDGFRa (D) and for Cyp11 (E), double labeling for both proteins (F), the size marker is 50 lm and the negative control is displayed in the upper corner of 2F. In figure G–I the presented PDGFRa-positive cells had been cultured on glass cover slips for 3–5 days before IHC. The cells are positively stained for LIFR (G, size marker: 200 lm), aSMA (H, size marker: 100 lm) and StAR (I, size marker: 100 lm). The corresponding negative controls are shown in the upper right corners. Cell nuclei are stained with 40 ,60 -diamino-2-phenylindole (DAPI).

suspension was poured into a MS separation column and the PDGFRa positive cells were then recovered according to the manufacture’s instruction. 97–98% of the isolated cells were PDGFRa positive after immunohistochemical staining for PDGFRa (Fig. 2A). For culturing, 100 ll of a suspension containing 1  105 cells/ml was plated into each well of a 96-well Falcon plate (Falcon, USA) and treated with hCG (10 ng/ml), (Bu)2cAMP (1 mM), progesterone (1 lM), 17-OH progesterone (1 lM), and androstenedione (1 lM), for different time-points at 37 °C. The steroids were dissolved in DMSO and final concentration of the solvent in the culture media was 0.01%.

by collagenase type 1 treatment and the seminiferous tubules were separated mechanically. In order to obtain purified Leydig cells, this crude suspension was loaded on top of a discontinuous gradient consisting of layers of 20%, 40%, 60% and 90% Percoll solutions dissolved in HBSS and subsequently centrifuged at 800xg for 20 min. The fractions enriched in Leydig cells thus obtained were then placed onto a continuous, self-generating density gradient formed from a 60% solution of Percoll and centrifuged at 20,000g for 30 min at 4 °C. The purity of the resulting Leydig cell preparations was shown to be 90%, as determined by histochemical staining for 3b-HSD.

2.4. Isolation of Leydig cells

2.5. Immunohistochemistry

The testes from 20-, 40- and 60dpp rats were used to isolate precursor, immature and adult Leydig cells (PLC, ILC and ALC, respectively) as described previously (Izzo et al., 2010; Svechnikov et al., 2001). Briefly, following decapsulation, testes were disrupted

Testicular tissue was fixed in Bouins Solution (HT 10132, Sigma) at 4 °C overnight and then gradually washed out in 70% ethanol until the solution was clear and no Bouin remaining. The testicular tissue was then embedded in liquid paraffin (Paraplast X-tra;

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3808, Sigma Aldrich) at 61 °C overnight followed by 5–20 lm thick sections were cut and placed on slides (Superfrost plus, J1800AMNZ, Thermo scientific, USA). For IHC the testicular sections were deparaffinized in Xylene (02080, HistoLab, Sweden) and then rehydrated in ethanol (99%, 96% and 70%, respectively). The staining procedure was done following a protocol previously published by van den Driesche et al. (2012). Shortly, after antigen retrieval in citrate buffer (30 min at 96 °C), the sections were blocked in methanol (Merck, Darmstadt, Germany) containing 3% H2O2 (Merck) for 30 min followed by a second blocking with TBS (Santa Cruz) containing 20% chicken serum (Sigma) and 5% BSA (Jackson Immuno Research) (TBS/NChS/ BSA) followed. Staining for PDGFRa, LIFR, aSMA and StAR (kindly provided by D. Stocco, Texas Tech University, USA) (Clark et al., 1994) was conducted at 1:400 at 4 °C overnight, followed by incubation with peroxidase conjugated secondary anti-rabbit antibody in TBS/NChS/BSA for 30 min room temperature (RT) and TyramideFl (Perkin–Elmer–TSA plus Fluorescein System; Perkin Elmer Life Sciences, Boston, MA, USA) according to the manufacturer’s instructions. All sections were incubated with anti-aSMA antibody diluted 1:400 in TBS/NChS/BSA at RT for 1 h followed by incubation with a peroxidase conjugated anti-mouse antibody 1:200 for 30 min at RT and Tyr-Cy5 (Perkin–Elmer–TSA plus Cyanine3 System) used according to the manufacturer’s protocol. Nuclei staining was conducted with Hoechst 33258 (Invitrogen, H1398) at a dilution of 1:1000 in PBS followd by mounting with Dako Faramount aqueos solution (Dako, S3025). Fluorescence pictures were captured using an Eclipse E800 microscope (Nikon, Japan). To stain the freshly isolated cells, the cell suspension was placed directly on polysine slides (Menzel–Gläser, J2800AMNZ, Braunschweig, Germany) and after drying fixed with 4% PFA at 4 °C overnight. After blocking in 10% donkey serum, the cells were incubated with anti-PDGFRa, anti-Cyp11a1 and anti-a-SMA antibodies diluted1:50–1:100 in PBS containing 0.1% BSA at 4 °C overnight followed by incubation with peroxidase-conjugated secondary antibody 1:300–1:500 in 37 °C for 1 h. The negative control was incubated with IgGs (1:100) from the species where the primary antibody was produced in. Some cells were stained for proteins of interest (e.g., LIFR, aSMA, StAR) after culturing on round cover slips for 3–5 days. 2.6. Isolating RNA and producing cDNA Total RNA was extracted from control and (Bu)2cAMP-treated PDGFRa-positive PTCs and from freshly isolated PDGFRa-positive PTCs, PLC, ILC and ALC by RNeasy Mini Kit (74104, Qiagen, Hilden, Germany), according to the protocol provided by the manufacturer. The RNA was pretreated with DNAse (RNase-free DNase Set, Qiagen) according to the manufacturer’s instructions. The amount of total RNA was measured by photometry (BioPhotometer, Hamburg, Germany). The RNA was kept at 80 °C until analysis. Total RNA was further processed using iScript cDNA Synthesis Kit (Bio-Rad Laboratories, Hercules, CA) as proposed in the manufacturer’s protocol. 2.7. Gene expression analysis by qPCR

bility of genomic DNA contamination that may affect the qPCR results, the isolated RNA samples were pretreated with DNase according to the manufacturer’s instructions. To control effectivity of the process, negative control (RT-) was always added to each qPCR assay. In pilot experiments we found that the expression of b-actin was stable in cultures of PDGFRa-positive PTCs during the course of culturing and comparable between cell types used. All values are normalized to b-actin as a house keeping gene to balance possible irregularities in RNA concentration. For the comparison between different stages of LC development the expression was also normalized to the mean of the expression in PDGFRa-positive PTCs so that the values can be understood as multiplies of the PDGFRa-positive PTCs-gene expression. For an overview of the used primers and the running conditions see Table 1. 2.8. Steroid measurement Culture medium samples were stored at 20 °C prior to analysis of the concentrations of progesterone and testosterone. The levels of the steroids were quantified employing the Coat-a-Count RIA kit (TKPG1, TKTT1, Diagnostic Products Corp., Los Angeles, CA), according to the manufacturer’s instructions. Intraassay and interassay coefficients for testosterone were 6.4% and 4%, respectively. The same parameters for progesterone were 3.2% and 3.9%, respectively. 2.9. Assays of PFLCs proliferation Isolated cells were cultured for 72 h with or without (Bu)2cAMP, PDGFa, LIF and their combinations followed by labeling with 3Hthymidine (Amersham Pharmacia Biotech, UK) at concentration of 1 lCi per well for the last 24 h. The cells were washed and then Table 1 qPCR primer sequences and running conditions, bp-base pair. Oligo

Sequence

Prod. length (bp)

Temp. (°C)

Actb

F: 50 -tgaagatcaagatcattgctcc-30 R: 50 -actcatcgtactcctgcttgc-30 F: 50 -gaacgacctggtgcttcgtaa-30 R: 50 -gattctcgacccatggcatag-30 F: 50 -acctagaggccacaactaacatcc-30 R: 50 -gaggcactgggactagcacct-30 F: 50 -gtcgccatcaggaacctcgagaa-30 R: 50 -caccagcaaagccgccttaca-30 F: 50 -aagacagcagcatcacgggga-30 R: 50 -tgccaaagcgggaggagttgtc-30 F: 50 -tcgacaagacgcagcgtaagcg-30 R: 50 -cgatcagcacgcacagcttcca-30 F: 50 -ggctttctggaccccaagctga-30 R: 50 -taggcaagggggaagggaagga-30 F: 50 -cagcaagaccttttgggtctgt-30 R: 50 -cctgagccctacaatccttcagt-30 F: 50 -gctatggttcccttgggtct-30 R: 50 -ggccaggtaaggatacagca-30 F: 50 -ggctggacacctggcttcaga-30 R: 50 -tggtccgattccaggccca-30 F: 50 -agtgtgtgaggttctcccggtacct-30 R: 50 -tacaacattgagtccatgtctggccag-30 F: 50 -ctgcgccttcaggaatttgcc-30 R: 50 -aatcataatcccagccactgagttcattct-30 F: 50 -agcgtctgtgagggaagcaacg-30 R: 50 -gctttcgccaggacgctcagaa-30 F: 50 -gatagcttcatgagccgacaccca-30 R: 50 -cattggcacgtactgtgtggtgtca-30 F: 50 -gacaggagcaggagggtttgtgg-30 R: 50 -ctccttctaacattgtcaccttggcct-30 F: 50 -ctgctagaccagcccatggac-30 R: 50 -tgatttccttgacatttgggttcc-30

120

60–62

80

60

80

60

111

60

114

60

91

60

112

60

51

62

195

60

142

62

161

62

161

60

150

60

165

60

161

60

90

60

Cyp 11a1 Cyp17a1 Acta2 Myh11 Nr5a1 Nes Insl3 Tspo Pou5f1 Hsd17b3 Lhcgr

The samples for qPCR were prepared using iQ SYBR Green Supermix (170-8882, Bio-Rad Laboratories, Hercules, CA) and the PCR cycles were run at 95 °C for 10 s, 60–62 °C for 45 s, 95 °C for 60 s and 55 °C for 60 s followed by a melting curve from 55 to 95 °C in steps of 0.5 °C and then held at 4 °C (iCycler iQ, Bio-Rad Laboratories, Hercules, CA) after having estimated the best reaction conditions by running a temperature gradient. cDNA samples from 7 or 60 dpp rats were used as positive control. To avoid the possi-

Lifr Pdgfra Hsd3b1 Star

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lysed with NaOH (5 N) and incorporated radioactivity was measured in a Beckman liquid scintillation counter. 2.10. Statistical analysis The differences between various values were analyzed for statistical significance by Student’s t-test for pairwise comparison, and the one-way analysis of variance (ANOVA) for multi comparison followed by Holm-Sidak analysis or Dunnett’s analysis if the normality test failed using the SigmaStat (v 11.00) package (SPSS, Inc., Chicago, IL). p < 0.05 was considered to be statistically significant. 3. Results 3.1. In situ identification of the PDGFRa-positive neonatal PTCs To confirm that neonatal PTCs belong to peritubular cell lineage and express PDGFRa in situ, we stained sections of neonatal 8dpc rat testes for PDGFRa and aSMA. We found that overwhelming majority of aSMA-positive neonatal PTCs located in the peritubular layer of testicular section (Fig. 1B) were immunoreactive for PDGFRa (Fig. 1A) and co-expressed both aSMA and PDGFRa (Fig. 1C), strongly indicating that these PDGFRa-positive testicular cells belong to the PTC lineage. We also demonstrated that some aSMA-positive PTCs expressed LIFR (Fig. 1D) and co-express LIFR and aSMA (Fig. 1F). Similarly, some of these testicular cells were immunopositive for StAR protein (Fig. 1G) and expressed both StAR and aSMA (Fig. 1I). Therefore, we used magnetic cell separation (MACS) to isolate PDGFRa-positive PTCs for further characterization of their phenotype and steroidogenic potential. 3.2. Phenotype of PDGFRa-positive peritubular cells We observed that more than 98% of isolated cells were immunopositive for PDGFRa (Fig. 2A), abundantly expressed a-SMA (Fig 2B and H) and coexpressed both PDGFRa and a-SMA (Fig. 2C), strongly indicating that these PDGFRa-positive testicular cells belong to the PTC lineage. Some of the PDGFRa-positive PTCs (Fig. 2D) were also immunoreactive for CYP11a1 (Fig. 2E and F) and expressed LIFR and StAR (Fig. 2G and I). In addition, we could also demonstrate that PDGFRa-positive PTCs expressed three pluripotency markers (Lifr, Pou5f1, Nestin) (Fig. 3A and B), suggesting the potential stemness of these cells. This suggestion was further supported by the observation that the expression of Pou5f1, a wellknown pluripotency marker, was higher in PDGFRa-positive PTCs compared to fully differentiated adult Leydig cells, but lower than in neonatal testis that is thought to be rich in spermatogonial stem cells (Fig. 3B). All these findings allow to suggest that PDGFRa-positive neonatal PTCs are weakly differentiated cells with a potential to become steroidogenic. 3.3. Comparative analysis of the expression of Leydig cell-specific genes in PDGFRa-positive PTCs and different developmental stages of adult Leydig cells To investigate the potential of PDGFRa-positive PTCs to differentiate into steroid-producing cells, we further compared the levels of expression of Leydig cell-specific genes in the PDGFRa-positive PTCs and the adult Leydig cell lineage that included populations of PLC, ILC and ALC. We found that PDGFRa-positive PTCs expressed very low but detectable levels of steroidogenic genes and transcription factor Sf-1 compared to the adult Leydig cell lineage. Similarly, those cells expressed negligible transcriptional expression of Insl3 and Lhr that were 200- and 80 times less, respectively, than in the adult population of Leydig cells

Fig. 3. The levels of mRNA expression of representative pluripotency markers in freshly isolated PDGFRa-positive PTCs (A). mRNA levels were determined by qPCR using b-actin as the house keeping gene. Comparative expression levels of Pou5f1 in neonatal testis, PDGFRa-positive PTCs and in fully differentiated adult Leydig cells (B). The mRNA levels are normalized to b-actin and expressed relative to neonatal testis. Mean values ± SEM for three independent PTCs preparations are presented.

(Fig. 4). These findings led to the assumption that PDGFRa-positive PTCs are at an early stage of differentiation into steroidogenic cells and may give rise to the adult Leydig cell lineage. 3.4. The cAMP dependent pathway(s) upregulates steroidogenic gene expression and stimulates steroidogenesis in PDGFRa-positive PTCs Since the cAMP-mediated signaling pathways play an important role in the activation of steroidogenic gene expression and support the sex hormone production in steroid-producing cells (Keeney and Mason, 1992), we further explored the responsiveness of steroidogenic genes in PDGFRa-positive PTCs treated with (Bu)2cAMP. In basal unstimulated conditions primary cultures of the PDGFRa-positive PTCs lost their capacity to express Cyp11, Hsd3b1 and Cyp17 during 7 days of culture, while the Star expression, that also decreased markedly, remained detectable. However, treatment with (Bu)2cAMP increased Star, Cyp11 and Hsd3b1 but not Cyp 17 gene expression after 24 h of incubation and supported this process further during 7 days of culturing the PDGFRa-positive PTCs (Fig. 5). The increased expression of the steroidogenic enzymes induced by (Bu)2cAMP was accompanied by 5.7- and 7.4fold stimulation of progesterone and testosterone production during the first 24 h of incubation, an effect that was significantly attenuated after 7 days of culturing, with no testosterone detected at that time point (Fig. 6A and B). Incubation of the cells with hCG at the same experimental paradigm did not induce steroidogenic activity in those cells (data not shown), indicating that no functionally active LHR positive FLCs were present in primary cultures of the PDGFRa-positive PTCs. In addition, the increased expression of the steroidogenic genes induced by (Bu)2cAMP in PDGFRa-positive PTCs cannot be explained by stimulation of their proliferation since the thymidine assay showed no changes in proliferation due

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Fig. 4. Comparative mRNA levels of Leydig cell-specific genes in PDGFRa-positive PTCs and the adult Leydig cell lineage. The mRNA levels are normalized to b-actin and expressed relative to PDGFRa-positive PTCs. PLCs are 20dpp progenitor LCs, ILCs are 40dpp immature LCs and ALCs are 60dpp adult LCs. The columns represent the fold changes compared to PDGFRa-positive PTCs. Mean values ± SEM for three independent RNA preparations are denoted. p < 0.05, p < 0.01 and p < 0.001 compared to PTCs.

to treatment with the cAMP analog (data not shown). Taken together, biosynthesis of progesterone but inability to produce testosterone by (Bu)2cAMP-stimulated PDGFRa-positive PTCs after 7 days of culture demonstrated a significant dysfunction of CYP17 despite its detectable expression at that time point. 3.5. Time-dependent inactivation of CYP17 and 17bHSD in primary cultures of PDGFRa-positive PTCs To explore the functionality of CYP17 and 17bHSD in cultured PDGFRa-positive PTCs, the cells were incubated with progesterone, 17OH-progesterone and androstenedione, substrates for the indicated enzymes, for 7 days and testosterone as the end-point steroid was measured at different time points. We found dramatic

attenuation of the capacity of the PDGFRa-positive PTCs to convert progesterone and 17OH-progesterone into testosterone after 48 h (Fig. 7A), an observation that strongly indicates a dysfunction of CYP17 in cultured PDGFRa-positive PTCs. Similarly, androstenedione-supported testosterone production by the PDGFRa-positive PTCs was also significantly (by 5-fold, p < 0.05) decreased after 72 h of culturing (Fig. 7B), indicating a marked attenuation of the activity of 17bHSD in those cells. 3.6. The effect of the cAMP-mediated pathway(s) on the expression of PTC-specific genes in PDGFRa-positive PTCs We further explored whether an activation of the cAMP mediated signaling pathway(s), which triggers the upregulation of

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Fig. 5. Time-dependent changes in steroidogenic gene expression in unstimulated and (Bu)2cAMP treated PDGFRa -positive PTCs. The cells were cultured up to 7 days with or without 1 mM (Bu)2cAMP and the RNA was isolated after 1 day or 7 days of treatment, respectively. The mRNA expression is shown relative to b-actin expression. Each experiment was performed independently three times with similar results. p < 0.05 compared to untreated control, n.d. – non-detectable level of gene expression.

Fig. 6. Effect of (Bu)2cAMP treatment on progesterone (A) and testosterone (B) production by PDGFRa-positive PTCs. The cells were cultured for 7 days with or without 1 mM of (Bu)2cAMP. The steroids were measured by RIA in the cell culture medium at different time points. Mean values ± SEM of three different experiments are shown. p < 0.05, p < 0.01 and p < 0.001 compared to untreated conditions at the same time point. N.d. – non-detectable level of steroid production.

steroidogenic gene expression, may have an effect on the expression of PTC-specific genes, such as myosin heavy chain 11 (Myh 11), a marker for peritubular cells. We observed that long-term culturing of the PDGFRa -positive PTCs in basal unstimulated conditions during 7 days significantly (5-fold, p < 0.05) increased the

Fig. 7. Time-dependent attenuation of Cyp17 and 17bHSD activities in primary cultures of PDGFRa -positive PTCs. The cells were cultured for 1 week with or without progesterone, 17OH-progesterone (A) as well as androstenedione (B) and medium was collected at different time points followed by testosterone production was measured with RIA. Mean values ± SEM from three independent experiments are shown. a, b, c = p < 0.05 compared to testosterone production on day 1, aprogesterone, b-17OH-progesterone, c-androstenedione, respectively. N.d. nondetectable level of steroid production.

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Fig. 8. Temporal changes in Myh11 expression in primary cultures of PDGFRapositive PTCs after treatment with and without 1 mM (Bu)2cAMP. The cells were cultured in basal conditions or with 1 mM (Bu)2cAMP for up to 7 days. The amount of mRNA encoding Myh11 was determinate by qPCR and normalized to b-actin expression. Mean values ± SEM of three independent cell cultures are shown.  p < 0.05 compared to the expression in basal conditions at d1.

expression of Myh11, a process that was partly attenuated by (Bu)2cAMP treatment (Fig. 8). 4. Discussion In the present study, we isolated PDGFRa-positive PTCs from 8dpp neonatal rat testis using MACS and explored their phenotype and function in cultures of highly purified cells. We have demonstrated that PDGFRa-positive PTCs have a mixed phenotype, expressing PTC-specific genes (e.g., Myh11, aSma), pluripotency markers (e.g., Pou5f1, Nestin, Lifr) and genes encoding steroidogenic enzymes involved in the biosynthesis of sex steroids. Our study is the first to demonstrate that PDGFRa-positive neonatal PTCs are steroidogenic competent cells that express very low, but detectable levels of steroidogenic enzymes and produce progesterone as the main end-point steroid due to functional inactivity of CYP17 and 17bHSD. Moreover, we found that the cAMP-mediated signaling pathway(s) upregulates the expression of steroidogenic enzymes and increases the steroidogenic potential of PDGFRa-positive neonatal PTCs similar to observations in the adult Leydig cell lineage. The PDGFRa-positive population of neonatal PTCs, which we used in our study, was not contaminated with FLCs since these cells did not produce androgens in response to hCG and did not express functional Lhr and Insl3. Earlier studies on the ontogenesis of ALCs suggested an early recruitment of Leydig cells in the postnatal rat testis from undifferentiated peritubular fibroblast-like cells (Russell and de Franca, 1995; Mancini et al., 1965; Van Straaten and Wensing, 1978). This suggestion was further confirmed by Mendis-Handagama and Ariyaratne (2001) showing that spindle-shaped steroidogenic enzyme-positive cells are exclusively located in the peritubular region of the neonatal testis. It was reported that neonatal peritubular cells abundantly express PDGFRa (Gnessi et al., 1995) and that PDGFRa is restricted to the Leydig cell lineage (Gnessi et al., 2000), which makes PDGFRa an appropriate candidate for the isolation and characterization of this population of neonatal PTCs. Furthermore, Ge and et al. have shown that PDGFRa-positive LHR-negative testicular cells isolated from 7dpp testis express putative Leydig stem cell markers (e.g., LIFR, PDGFRa), expand their numbers during prolonged culture in vitro and start to express Leydig cell-related genes after culturing in differentiationinducing medium, suggesting that those cells may serve as stem Leydig cells (SLCs) (Ge et al., 2006). Micro-array studies have shown that SLCs possess an expression pattern similar to bone marrow stem cells (Stanley et al., 2011). All these findings suggest

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that PDGFRa-positive spindle-shaped cells are putative SLCs and give rise to the adult Leydig cell lineage (Ge et al., 2006; Stanley et al., 2012). Our data have shown that highly purified LHR-negative PDGFRa-positive neonatal testicular cells isolated with MACS do belong to the PTC lineage, express PTC-specific genes and have a low but detectable expression of steroidogenic enzymes and the capacity to synthesize sex steroids. Moreover, this population of PDGFRa-positive PTCs have organized steroidogenesis-related signaling pathway(s), activated expression of steroidogenic enzymes and is thought to be the earlier Leydig cell precursors that are derived from undifferentiated non-steroidogenic PDGFRa-positive PTCs, a population of cells which may serve as SLCs (Ge et al., 2006, Stanley et al.; 2011). However, we cannot exclude that some PDGFRa-positive PTCs are originated from fetal Leydig cells which transformed into silent LHR-negative cells but preserving low but stable transcription of steroidogenic genes. Steroidogenic competence of neonatal PDGFRa-positive PTCs found in our study is thought to be associated with the transcriptional activity of the steroidogenic factor 1 (SF-1) which was expressed in these cells. SF-1 is known to be involved in a program of events that convert non-steroidogenic mesenchymal and embryonic stem cells into differentiated steroidogenic cells (Crawford et al., 1997; Gondo et al., 2004; Tanaka et al., 2007). Its transcriptional activity can be dramatically upregulated by the cAMP-protein kinase A (PKA) signaling pathway (Fan et al., 2004). Thus upregulation of the steroidogenic gene expression observed in the (Bu)2cAMP treated PDGFRa-positive PTCs is thought to be associated with transcriptional activation of SF-1 and its downstream target genes, which control the ultimate regulation of steroidogenesis. In line with this assumption, stable expression of SF-1 in murine embryonic stem cells was shown to alter their morphology, trigger the cAMP-induced expression of the CYP11A1 and consequently promotes the biosynthesis of progesterone as a final end-point steroid (Crawford et al., 1997). Moreover, our finding that the PDGFRa-positive PTCs did not express functional LHR but had operative cAMP-mediated pathway(s) allow to suggest that maturation of this signaling pathway(s) and all its components precede the expression of LHR by the cells and is tightly coupled with SF-1 related downstream target genes. Therefore, one can suggest that hormonal and paracrine factors that have the potential to activate the cAMP-dependent signaling pathway(s) may play an important role in triggering the differentiation of these cells into the adult Leydig cell lineage. One of such factors can be IL-1a, which was shown to increase cAMP levels and to stimulate steroidogenesis in immature rat Leydig cells (Svechnikov et al., 2001). Our findings that native highly purified PDGFRa-positive PTCs isolated after depletion of LHR-positive FLCs have steroidogenic competence are not in agreement with the previous observation that PDGFRa-positive spindle-shaped cells called putative stem Leydig cells are non-steroidogenic and start to express SF-1 and the steroidogenic enzymes only after 1 week of culturing in differentiation media (Ge et al., 2006). While this contradiction may in part be interpreted as the result of differences in cell isolation and culturing approaches, it does reveal that the differentiation pattern of PTCs/PSLCs is more complex than previously thought. Further exploration of the PDGFRa-positive PTCs demonstrated that long-term culturing of the cells upregulates the Myh11 expression, a specific marker for the PTC lineage (Cowan et al., 2010) and that this process is accompanied by a significant attenuation of steroidogenesis in the PDGFRa-positive PTCs, an effect reported earlier for primary cultures of human fetal testicular cells (Cowan et al., 2010). However, upregulation of the expression of steroidogenic enzymes and activation of steroidogenesis in the neonatal PDGFRa -positive PTCs by (Bu)2cAMP attenuated the expression of Myh11, suggesting that the cAMP-mediated signaling pathways may suppress the expression of PTC-specific genes

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and delay the transition of steroidogenic competent PDGFRa-positive PTCs into non-steroidogenic myoid peritubular cells. Timedependent attenuation of steroidogenic potential of cultured PDGFRa-positive PTCs observed in our experiments is thought to reflect their dedifferentiation, a process that can be triggered by the lack of appropriate hormonal and paracrine milieu that is available in vivo but difficult to reconstruct in in vitro conditions. We suggest that the dedifferentiation process was not accompanied by the elimination of the cells via apoptosis since the expression of Myh11 and steroidogenic genes in PDGFRa-positive PTCs was enhanced after long-term culturing. However, we cannot exclude that loss of steroidogenic competence in these cells was associated with increased proliferation in the course of their culturing. All together, our data indicate that PDGFRa-positive PTCs are present in the rat testis at postnatal day 8 and can give rise to the adult Leydig cell lineage. We suggest that this population of PDGFRa-positive PTCs with steroidogenic potential are progeny of non-steroidogenic SLCs but the mechanisms that trigger the silent SLCs to gradually differentiate are yet unknown, although the SF-1 signaling may play a significant role in this cellular event. Grants/fellowships Financial support was received from Karolinska Institutet, the Frimurare Barnhuset in Stockholm, Stiftelsen Olle Engkvist Byggmästare, Pediatric Research Foundation, Sällskåpet Barnåvard, Vetenskapsradet (VR), VR/FA and Barncancerfonden. J.B.S. is founded by the Deutsche Forschungsgemeinschaft (DFG Grant STU 506/3-1). Disclosure summary None of the authors has received benefits or grants that are relevant for this study from other sources than the research foundations listed above. Acknowledgements The authors would like to thank Dr. Mi Hou, Ahmed Reda, Yvonne Löfgren and Britt Masironi for technical assistance with immunohistochemistry and PCR. References Ariyaratne, H.B., Chamindrani Mendis-Handagama, S., 2000. Changes in the testis interstitium of Sprague Dawley rats from birth to sexual maturity. Biol. Reprod. 62, 680–690. Clark, B.J., Wells, J., King, S.R., Stocco, D.M., 1994. The purification, cloning, and expression of a novel luteinizing hormone-induced mitochondrial protein in MA-10 mouse Leydig tumor cells. Characterization of the steroidogenic acute regulatory protein (StAR). J. Biol. Chem. 269, 28314–28322. Cowan, G., Childs, A.J., Anderson, R.A., Saunders, P.T., 2010. Establishment of longterm monolayer cultures of somatic cells from human fetal testes and expansion of peritubular myoid cells in the presence of androgen. Reproduction 139, 749–757. Crawford, P.A., Sadovsky, Y., Milbrandt, J., 1997. Nuclear receptor steroidogenic factor 1 directs embryonic stem cells toward the steroidogenic lineage. Mol. Cell. Biol. 17, 3997–4006.

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