Animals, cell lines and reagents
Mice were maintained on a mixed C57BL/6/129S6/SvEv genetic background and handled in accordance with the NIH Guide for the Care and Use of Laboratory Animals. HEK-293 cells and 293T cells (HEK-293 cells containing the T-antigen from SV40) were obtained from the Tissue Culture Core at Baylor College of Medicine and maintained at 37°C with 5% CO2. Fetal bovine serum (FBS), bovine serum albumin (BSA), anti-FLAG M2 affinity gel, 3× FLAG peptide, FLAG-BAP(bacterial alkaline phosphatase) protein standard, mouse anti-FLAG M2 antibody and chromatography column were purchased from Sigma. DMEM, geneticin (G418), HEPES, penicillin–streptomycin, NuPAGE 4–12% or 12% Bis-Tris gel, Superscript III reverse transcriptase, deoxyribonucleotide triphosphate, oligo (dT)12–18 primers, PureLink PCR purification kit and SilverQuest staining kit were obtained from Invitrogen. Pregnant mare serum gonadotrophin (PMSG) was from Calbiochem. Recombinant human BMP4 (rhBMP4), untagged human BMP15 and soluble BMPR2 ectodomain (BMPR2 ECD) were obtained from R&D. All restriction enzymes were from New England Biolabs. FuGENE6, phosphatase inhibitors and proteinase inhibitors were from Roche. Phospho-SMAD1/5/8 antibody was from Cell Signaling. Phusion Hot Start High-Fidelity DNA polymerase was purchased from Finnzymes. TURBO DNA-free DNase was the product of Ambion. Peroxidase-conjugated goat anti-mouse/rabbit antibody was from Jackson ImmunoResearch. RNeasy Micro Kit containing RLT buffer was obtained from Qiagen. Zymoclean gel DNA recovery kit was purchased from ZYMO Research. SuperSignal West Pico kit and BCA protein assay kit were obtained from Pierce. Taqman Universal PCR master Mix and Taqman gene expression probes were purchased from Applied Biosystems.
Engineering of rhBMP15 cDNA and expression construct
All mutations and restriction sites were introduced by PCR using Phusion Hot Start High-Fidelity DNA polymerase. Overlap extension PCR was performed to obtain the sequence encoding the optimized cleavage site and FLAG-tag followed by the mature domain of hBMP15. Briefly, plasmid containing the native hBMP15 coding sequence was used as a template, and amplified with primers PB1 (5′-gagaaagcttgccgccaccatggtcctcctcag-3′) and PB2 (5′-gagctcaagaccaccactatc-3′). The resultant amplicon, designated as fragment a (Fa), was inserted into the HindIII and BamHI sites of pcDNA3 (designated as construct ABH). The mutation of cleavage site and incorporation of the FLAG-tag were conducted as follows. First, primers PB3 (5′-gcaaaggttctggaataacaag-3′) and PB4 (5′-gccgccgctcaggctccgcttgctccggtgaagag-3′) were used to generate Fb, which was utilized as a template for primers PB3 and PBf5 (5′-cttgtcgtcatcgtccttgtagtcgccgccgctcagg-3′) to produce Fc. Then, Fd, which encodes the FLAG-tag and the mature domain, was derived using primers PBf6 (5′-gactacaaggacgatgacgacaagcaagcagatgg-3′) and PB7 (5′-gagtctcgagtcatctgcatgtac-3′). Lastly, Fd and Fc were combined and subjected to overlap extension PCR using primers PB3 and PB7, and the amplicon (Fe) was cloned into ABH at BamHI and XhoI sites (designated as pQL-rhBMP15). All PCR fragments were purified by using the PureLink PCR purification kit or Zymoclean gel DNA recovery kit. The identities of all cloned sequences were verified by DNA sequencing (Child Health Research Center Molecular Core Laboratory, Baylor College of Medicine).
Transient transfection and selection of stable cell clones
The hBMP15 expression construct (pQL-rhBMP15) was transiently transfected into HEK-293T cells using FuGENE6 transfection reagent according to the manufacturer's instructions. The conditioned medium was assayed for the production of rhBMP15 72 h after transfection. To create the stable cell lines, pQL-rhBMP15 was transfected into HEK-293 cells, and the cells were re-plated at a low density 2 days after transfection in the presence of 800 µg/ml G418. G418-resistant cell colonies were selected after 10–14 days. The colonies were then cultured and analyzed for the expression and secretion of rhBMP15 protein using western blot. The selected stable cell lines were maintained in culture medium containing 200 µg/ml G418.
Protein purification and quantification
Stable cells expressing rhBMP15 were plated in DMEM containing 10% FBS, 100 U/ml penicillin–streptomycin and 200 µg/ml G418. When the cells reached confluency, DMEM containing 2% FBS and 100 U/ml penicillin–streptomycin was used for the production of rhBMP15, and the medium was collected every 2 days from culture dishes for up to 12 days. Purification of rhBMP15 was conducted using anti-FLAG M2 affinity gel according to the manufacturer's protocol. Briefly, the conditioned medium containing rhBMP15 and proteinase inhibitors was incubated overnight at 4°C with the appropriate amount of anti-FLAG M2 affinity gel based on the estimated quantity of recombinant protein in the conditioned medium. After incubation, the resin was collected by centrifugation or filtration through a chromatography column and washed. The rhBMP15 proteins were then eluted with 3× FLAG peptide or 0.1 M glycine HCl (pH 3.5) in TBS (pH 7.5). BSA (1 mg/ml) was added to the protein before being stored at −20°C. The purified proteins were quantified by western blot using FLAG-bacterial alkaline phosphatase (BAP) standards. The control buffer was prepared from the untransfected HEK-293 cell culture using the same protocol. The purity of the purified rhBMP15 was examined by silver staining using a commercially available SilverQuest staining kit.
Generation of human BMP15 polyclonal antibody
A cDNA fragment encoding the mature hBMP15 protein (GenBank accession NM_005448) was subcloned into pET23b containing a His-tag (Novagen), and the His-tagged hBMP15 protein was produced in BL21 cells (Novagen) according to the manufacture's manual. The hBMP15 fusion protein was used to immunize mice null for Bmp15
) (Yan et al., 2001
) to produce the polyclonal antibody according to a protocol consisting of a primary injection and three following boosts. The anti-sera from the mouse were collected and tested by western blot analyses using medium containing rhBMP15 produced from mammalian cells.
The specificity of the anti-hBMP15 antibody was confirmed by a pre-absorption experiment. Briefly, the anti-hBMP15 serum (1:2000) was pre-incubated with rhBMP15 (5 µg/ml) or control buffer overnight at 4°C. Then, the absorbed anti-sera were used to detect 50 ng of rhBMP15 using western blot analysis described below.
Western blots were carried out as previously described (Li et al., 2008b
). In brief, conditioned medium from the transfected/untransfected HEK-293 cells, protein lysates of HEK-293/granulosa cells or purified rhBMP15 were subjected to electrophoresis under reducing or non-reducing conditions on NuPAGE 4–12% or 12% Bis-Tris gel. The separated proteins were then transferred onto nitrocellulose membranes (30 V for 70 min). Membranes were blocked with 3% non-fat milk and incubated with mouse anti-FLAG M2 antibody (1:1000 in 3% milk) or rabbit anti-phospho-SMAD1/5/8 antibody (1:500 in 1% BSA) overnight at 4°C. Membranes were subsequently probed with peroxidase-conjugated goat anti-mouse or rabbit secondary antibody (1:10 000) for 70 min at room temperature. SuperSignal West Pico kit was used to detect the chemiluminescence signal. Quantification of protein signals was performed using NIH Image J software.
Granulosa cell culture and treatment
Isolation and culture of mGCs were performed as described elsewhere (Pangas et al., 2006
; Li et al., 2008b
). Briefly, immature female mice (21–23 days) were primed with 5 IU PMSG (i.p.), and 44–46 h later, large antral follicles were punctured for GC collection. The collection medium was DMEM-F12 containing 0.3% BSA, 100 U/ml penicillin–streptomycin and 10 mM HEPES. To exclude the oocytes, the GC suspension was filtered through a 40 µm nylon mesh and washed twice with collection medium. The mGCs were utilized for the following experiments. (i) SMAD activation analysis: to examine the activation of the SMAD1/5/8 pathway by rhBMP15, mGCs were treated with control buffer, rhBMP15 (100 ng/ml) or untagged hBMP15 (R&D) preincubated with/without the BMPR2 ECD (1 µg/ml), or rhBMP4 (50 ng/ml; positive control for SMAD1/5/8 phosphorylation). Cells were collected after 1 h of treatment. The mGCs were lysed on ice in radio immunoprecipitation assay buffer [50 mM Tris–HCl (pH 7.5), 250 mM NaCl, 0.5% NP-40 and 50 mM NaF] in the presence of proteinase and phosphatase inhibitors. The cell suspensions were then sonicated and cellular proteins collected after centrifugation. Quantification of protein was conducted using BCA protein assay kit. Thirty micrograms of total protein/well were loaded on the gel for western blot analysis. The western blot experiments were repeated twice. (ii) Gene induction assay: the mGCs were treated with rhBMP15 at 0 (control buffer), 30, 50, 100 and 200 ng/ml for a dose–response experiment. For the bioactivity assays, mGCs were treated with rhBMP15 (100 ng/ml), untagged hBMP15 (R&D) or control buffer (control treatment). The control buffer was prepared from the untransfected HEK-293 cell culture medium and contains the same buffer as that in the purified protein. The cells were collected and subjected to RNA isolation after 5 h of treatment.
Reverse-transcription, PCR, and real-time PCR
Total RNAs from HEK-293 cells and primary mGCs (5 h of treatment) were isolated using Qiagen RNeasy Mini Kit and Micro Kit, respectively. On-column DNase digestion was performed to eliminate potential genomic DNA contamination according to the protocol of the manufacturer. Additionally, RNA from HEK-293 cells was further treated with TURBO DNA-free DNase prior to reverse transcription (RT). RT was performed using 200 ng (mGCs) or 1 µg (HEK-293 cells) of total RNA and Superscript III reverse transcriptase in a 20 µl volume. One microlitre of RT product was used for PCR amplification (20 µl reaction volume), which was performed using a DNA Engine PTC-200 Peltier Thermal Cycler. PCR primers were designed using Primer Express (Applied Biosystems, Foster City, CA, USA) and listed in Table . Ten microlitres of the resultant PCR products were separated and visualized on 1% agarose gel containing ethidium bromide. In a parallel experiment, the PCR products (20 µl) were purified and sequenced to confirm their identities (Agencourt Bioscience Corp.). The PCR was repeated twice and DNA sequencing was performed using purified PCR products from one experiment.
Primers for amplification of proprotein convertases
Real-time PCR was conducted using the ABI Prism 7500 Sequence Detection System (Applied Biosystems) (Li et al., 2007
). The reaction was performed in a 20 µl volume using Taqman Universal PCR Master Mix and Taqman gene expression probes (Ptx3
, Mm00477267_g1; Has2
, Mm00515089_m1; Tnfaip6
, Mm00493736_m1; Ptgs2
, Mm00514982_m1; Inha
, Mm03024204_s1; Inhba
, Mm00434339_m1; Inhbb
, Mm01286587_m1; Smad6
, Mm00484738_m1; Smad7
, Mm00484741_m1; Grem1
, Mm03024240_s1; Kitl
, Mm00442972_m1). Gapdh
was used as the internal control and amplified using a Taqman probe (Part no. 4352339E). All real-time PCR analyses were performed in duplicates, and the results were from at least three independent culture experiments. The abundance of mRNA for target genes was normalized relative to that of Gapdh
, and ΔCT was produced by subtracting the mean CT of controls from the CT of each target genes. Fold changes in mRNA expression were calculated using the formula 2−ΔΔCT
(Livak and Schmittgen, 2001
Differences among groups were assessed by ANOVA and the means between individual groups were further compared using Tukey's honest significant difference test. Comparison of means between two groups was made by student's t-test. Data are reported as mean ± standard error of the mean (SEM), and P < 0.05 was considered to be statistically significant.