Patient samples were obtained with informed consent and approved for screening by the Ethical Approval committees of the respective institutes. The cohort of azoospermic patients from Osaka University, Japan, have been previously described and patients showing obstructive azoospermia or chromosomal abnormalities were excluded : DNA from a control group of healthy Japanese males was obtained from the same source. The thirty azoospermic patient samples from Human Assisted Reproduction Ireland, Rotunda Hospital, Dublin, Ireland showed no Y chromosome microdeletions or chromosome abnormalities: the control group here were thirty proven fertile males . Obstructive azoospermia was excluded for both patient groups.
Tissues were collected from Swiss/To (Harlan, UK) mice, which were maintained in accordance with the Home Office regulations under project licence to CPW.
Analysis of Fkbpl mRNA expression by RT-PCR
Total RNA was extracted using the RNeasy Mini Kit, including the optional DNAse treatment, following the manufacturer's instructions (Qiagen, Crawley, UK) and 1 μg used to make cDNA in a 12.5 μl mixture containing 10 mM Tris HCL (pH 8.3), 0.2 μg Oligo(dT)15 primer (Promega, Southampton, UK), 1.5 mM dNTPs, 1× AMV-RT buffer and 7.5 U AMV reverse transcriptase (Promega, Southampton, UK). Primers specific for Fkbpl (musdirf5ex CTTCCAGGCCTCAACATCAT and musdirR TCCCAGCTCGAAACAGTTCT: chr17 34781824-34782871 NCBI build 37; cDNA 756 nt; DNA 1028 nt) or β-actin (Bact1 GCTGTGCTATGTTGCTCTAGACTTC and Bact2 CTCAGTAACAGTCCGCCTAGAAGC: chr5 143665461-143666180; cDNA 500 nt; DNA 730 nt) were supplied by Invitrogen, Paisley, UK. All primers were checked for uniqueness and location using the BLAT tool available at the UCSC website . PCR was performed in 25 μl containing 1× Taq buffer, 200 μM dNTPs, 0.4 μM primer, 2 U Taq (Invitrogen, Paisley, UK) and 1 μl cDNA. Initial deannealing at 94°C for 3 min was followed by 28 cycles of 45 sec at 94°C, 1 min at 61°C, and 1 min at 72°C and a final elongation for 5 min at 72°C. PCR products were separated on agarose gels and images captured using a Kodak digital camera.
RNA expression analysis using radioactive probes
A Mouse RNA Master Blot array normalised to provide semi-quantitative data on tissue specificity and target mRNA abundance was purchased from BD Biosciences, Cowley, UK. Human and mouse FKBPL coding-region probes were amplified by PCR using: HumanF CTAGGCTCCTGCTGCCGGCTACTG and HumanR TCAGCAGTTGCTTTTTCCAGGTCC; musdirF GAACGAGAAGAACACCGCTC and musdirR TCCCAGCTCGAAACAGTTCT. Northern blotting, radiolabelling of cDNA and detection of mRNA were as previously described .
Nucleotide sequence screening of FKBPL gene
The whole FKBPL gene was directly amplified from patient DNA using primers 5'-GGCTCCAGGGTTAGTTGTCA-3' and 5' CCCAAATCTCACAGCACA GA-3' and purified with the Wizard Gel PCR clean-up kit (Promega, Southampton, UK). Sequencing was done using primers S1 5'-AACCAGTCAGATGCCAGAGG-3', S2 5'-CCTCTGG CATCTGACTGGTT-3', S3 5'-GAACCAGGTTCAGGTCAGC-3', S4 5'-GACTAG CGAGAAGGAAGCC-3' and S5 5'-GGCTTCCTTCTCGCTAGTC-3' on an ABI Prism 3130 at the Centre for Molecular Biosciences Sequencing Facility. Patient sequences were compared with the REFSEQ entry and examined for known variations using the UCSC browser . Zygosity of mutations was confirmed by sequencing TOPO-TA (Invitrogen, Paisley, UK) cloned PCR products.
Generation of expression constructs and reporter assays
N-terminal GFP-tagged constructs were generated by cloning into pcDNA3.1/NT-TOPO-GFP (Invitrogen, Paisley, UK) using the primers fkbplF0: GAGACCCCACCA CTCAATAC and fkbplR0: TCAGCCAAACATCTTGCCC. LNCaP cells (4 × 104 per well) were seeded in 24-well plates coated with fibronectin (Invitrogen, Paisley, UK) and maintained in phenol red-free RPMI1640 with 10% charcoal-dextran stripped FBS and 10 mM HEPES at 5% CO2 for 24 h. Cells were then co-transfected for 6 h using lipofectamine (Invitrogen, Paisley, UK) with pPA6.1Luc reporter construct, GFP-FKBPL-WTl or a pcDNA3.1 empty vector control together with the pBIND Renilla transfection efficiency control. The transfection mix was then replaced by the RPMI media with or without 10 nM R1881 and luciferase activity assessed 24 h post-transfection using the Dual-Glo assay system (Promega, Southampton, UK).
Western blot analysis
Whole cell protein extracts were obtained by lysis with extraction buffer (50 mM Tris-HCl, 150 mM NaCl, 5 mM EDTA,10% glycerol, 1% Igepal,1% protease inhibitor cocktail for mammalian cells (Sigma)), followed by centrifugation to remove debris. A 30 μg aliquot was fractionated on a 7.5% SDS-polyacrylamide gel (Biorad, Hemel Hempstead, UK) and transferred to a nitrocellulose membrane (GE Healthcare, Amersham, UK). Membranes were blocked for 1 h at RT with 5% preimmune serum in Tris-buffered saline with 0.2% Tween-20 (TBST) prior to incubation with a 1:800 dilution of anti-FKBPL rabbit polyclonal IgG (cat. no. 10060-1-AP, Proteintech Group, Chicago, USA) in TBST. After washing with TBST, membranes were exposed to a horseradish peroxidise (HRP) conjugated secondary antibody (sc-3837, Santa Cruz Biotechnology, Santa Cruz, USA) and signal detected using ECL reagents (GE Healthcare, Amersham, UK).
Paraffin-embedded sections of a fertile human testis were obtained from ProSci Inc, Poway, California, USA. For FKBPL and FKBP52, tissue was dewaxed and rehydrated through an alcohol series prior to antigen retrieval by heating in 10 mM citrate buffer (pH 6.0) using a 600 W microwave oven (2 × 3 min). Blocking was carried out with 10% serum in PBS-Tween20 (PBST) for 1 h at RT followed by 15 min with 1% hydrogen peroxide to remove endogenous peroxidase activity. Sections were washed twice with PBST then incubated overnight with 1:100 rabbit anti-human FKBPL (ProteinTech Group Inc, Chicago) or 1:250 dilution of anti-FKBP52 (cat#10011442, Cayman Chemical Company, Ann Arbor, Michigan, USA) at 4°C, followed by 1 h incubation with 1:2000 goat anti-rabbit IgG-HRP. Immunostaining was carried out with DAB Substrate Plus kit (Zymed, South San Francisco, USA), before counterstaining with hematoxylin (Sigma, Welwyn Garden City UK), dehydrating and mounting in Mowiol (Calbiochem, Nottingham, UK). For AR, immunostaining was essentially as described previously for this antibody (PG21, Millipore, Billerica, MA) , with a Vectastain ABC kit (Vector, Peterborough, UK) used for signal amplification and the Impact DAB kit (Vector) used for staining.
To examine differences in mutation frequencies between patient and control groups, Chi-squared values were calculated from a contingency table (Yate's method) and used to calculate significance values (p) by Fisher's exact method in the PRISM application (Graphpad, San Diego, USA).