Ovarian brain-derived neurotrophic factor (BDNF) promotes the development of oocytes into preimplantation embryos -- Data Supplement - HTML Page - 02442SuppText.htslp -- Proceedings of the National Academy of Sciences

Supporting Materials and Methods
Quantitative Real-Time RT-PCR Analyses.
To quantify the transcript levels of brain-derived neurotrophic factor (BDNF), TrkB, and neurotrophin (NT)-4/5 in mouse ovaries during gonadotropin treatment, real-time RT-PCR was performed with a SmartCycler (Cepheid, Sunnyvale, CA) as described in ref. 1. Ovarian mRNA was extracted and reverse-transcribed as described in ref. 2. A total of three mice at each time point was used for mRNA extraction, and the expression levels were determined independently. The primers and hybridization probes for real-time PCR of BDNF, TrkB, NT-4/5, and b -actin were as follows: BDNF: sense 5'-GGTATCCAAAGGCCAACTGA-3', antisense 5'-GCAGCCTTCCTTGGTGTAAC-3', and probe 5'-6-carboxy-fluorescein (FAM)-TACGAGACCAAGTGTAATCCCATGG-6-carboxy-tetramethyl-rhodamine (TAMRA); TrkB: sense 5'-GGTCCTCAGACTGGCCTACA-3', antisense 5'-GCTCCTGGTCCTGTCAACTC-3', and probe, 5'-FAM-CTGACGTCATGACCTTGCTGTCCTT-TAMRA-3'; NT-4/5: sense 5'-GGCTCCATCCTGAACATCAT-3', antisense 5'-GCCATGATCTACCTGCCTGT-3', and probe 5'FAM-CCATGCACGGTGGTACTCATCTGTA-TAMRA-3'; b -actin: sense 5'-TCTGTGTGGATTGGTGGCTCTA-3', antisense 5'-CTGCTTGCTGATCCACATCTG-3', and probe 5'-FAM-CTTGCCCACAGCCTTGGCAGC-TAMRA-3'. In this and the nested RT-PCR test described below, the primers for TrkB were designed to amplify a catalytic kinase domain of the receptor to avoid the amplification of truncated isoforms lacking the catalytic kinase domain (3).

Quantitative PCR was performed in a total reaction volume of 25 m l per tube containing 2´ QuantiTect Probe PCR Master Mix (Qiagen, Valencia, CA), 0.5 m M primer pairs, 0.2 m M TaqMan probe, and suitable dilutions of template cDNA. In all reactions, HotStarTaq DNA polymerase was used under an initial denaturation at 95°C for 15 min, followed by 60 cycles of amplification. Cycling conditions were as follows: denaturation at 94°C for 15 s, and annealing/elongation at 60°C for 60 s. Real-time RT-PCR monitoring was achieved by measuring fluorescent signals at the end of the combined annealing/elongation phase for each cycle. To determine the absolute copy number of target transcripts, cloned plasmid cDNAs were used to generate a calibration curve. A calibration curve was created by plotting the threshold cycle against the known copy number for each plasmid template diluted in log steps from 10⁶ to 10¹ copies. Each run included standard diluted plasmids to generate a calibration curve, a negative control without a template, and samples with unknown mRNA concentrations. Copy numbers for all unknown samples were determined with SMARTCYCLER 2.0 software (Cepheid). To correct for differences in RNA quality and quantity between samples, data were normalized by dividing the copy number for the target cDNA by the copy number for b -actin.

Isolation of Ovarian Cells for RT-PCR.
Germinal vesicle-stage oocytes, cumulus cells, and mural granulosa cells were isolated from ovaries of pregnant mare serum gonadotropin (PMSG)-treated immature mice 48 h after treatment as described in ref. 1 before performing RT-PCR. Because of the low number of germinal vesicle-stage oocytes under investigation, hemi-nested PCR (2) was used to amplify transcripts for BDNF, TrkB, and the p75 neurotrophin receptor (NTR). The primers for b -actin were the same as the primers used for real-time RT-PCR, and the primers for BDNF, TrkB, and p75 NTR were as follows. BDNF: sense 5'-CAATCGCTTCATCTTAGGAGTGG-3', antisense 5'-ACCAACATGTAGCTATGATGTAT-3', and hemi-nested sense primer, 5'-CTGTGAAGCCGCTTAACAGACCT-3'; TrkB, sense 5'-TGTCAAGTTCTACGGTGTCTGTG-3', antisense 5'-ATGCACTCTATCACCTCATTGTT-3', and hemi-nested sense primer, 5'-TTCCTATACATGATGCTCTCTGG-3'; p75 NTR, sense 5'-GTGGAGATGGAGATGATATGGAA-3', antisense 5'-GAAGGCAATCTCCAATTAGAAGC-3', and hemi-nested sense primer, 5'-GGAGTCACAGTATGGATCTCAGG-3'. After 30 cycles of PCR, the hemi-nested primer was paired with the antisense primer for another 30 cycles. For positive controls, mouse ovary cDNA was amplified simultaneously. For negative controls, no mRNA was included. BDNF ELISA.
Ovaries for ELISA were obtained from mice 48 h after PMSG treatment and at 3, 5, 7, and 12 h after human chorionic gonadotropin treatment. The ovaries (n = 3 animals) were homogenized in a buffer containing 137 mM NaCl, 20 mM Tris·HCl, 1% Nonidet P-40, 10% glycerol, and a protease inhibitor mixture (Roche Applied Science) before centrifugation at 8,000 ´ g for 5 min at 4°C. The supernatant was removed and stored at –80°C until use. Quantification of BDNF was performed by using the BDNF ImmunoAssay System (Promega) after sample thawing and centrifugation at 8,000 ´ g for 5 min at 4°C. The supernatant was diluted 1:4 in PBS for protein concentration measurement by the DC Protein Assay kit (Bio-Rad). The BDNF ELISA was performed by using 96-well plates coated with a mouse monoclonal anti-BDNF antibody as a capture antibody. A chicken anti-human BDNF polyclonal antibody was used as the primary antibody. The sandwich complex was detected by using an anti-chicken IgY-horseradish peroxidase conjugate, and tetramethylbenzidine was used as the substrate for colorimetric analysis. The reaction was stopped with 1 M HCl before measuring the absorbance at 450 nm. The results were normalized by protein concentrations and expressed as pg of BDNF per mg of ovary. Immunohistochemistry.
To localize BDNF, ovaries were obtained from PMSG-primed mice 7 h after human chorionic gonadotropin treatment and fixed with Bouin’s solution for 1 h at 23°C before transfer to 70% ethanol. Tissues were embedded in paraffin and sectioned at 3-m m intervals. After deparaffinization and dehydration, endogenous peroxidase activities were quenched with 3% hydrogen peroxidase for 10 min at 23°C. After incubation in the TNB blocking buffer (TSA Biotin System; PerkinElmer) for 30 min, slides were incubated with rabbit anti-BDNF antibodies (Chemicon) at a 1:150 dilution for 48 h at 4°C. After three washes in the TNT buffer (0.1 M Tris·HCl, pH 7.5/0.15 M NaCl/0.05% Tween 20), slides were incubated with biotinylated anti-rabbit secondary antibodies (Zymed Laboratories, South San Francisco, CA) for 1 h at 23°C. After three washes, the BDNF signal was amplified by using the TSA biotin system according to the manufacturer’s protocol. Bound antibodies were visualized with a Histostain SP kit (Zymed Laboratories). For negative controls, the primary antibody was replaced by nonimmune rabbit IgG. Immunofluorescence detection of TrkB was performed by using denuded metaphase I-stage oocytes from mice primed with PMSG for 48 h. After removing cumulus cells, denuded oocytes were fixed with 4% paraformaldehyde in PBS for 15 min at 23°C. After three washes in 1% PBS–BSA (BSA, Sigma), the oocytes were subjected to blocking for 30 min at 23°C in Image iT FX Signal Enhancer (Molecular Probes) before incubation with chicken anti-TrkB antibody in 1% PBS–BSA at a 1:500 dilution for 18 h at 4°C. After three washes in Tris-buffered saline, the oocytes were incubated with Cy3-conjugated anti-chicken secondary antibodies (Jackson ImmunoResearch) in 1% PBS–BSA at a 1:800 dilution for 1 h at 23°C. The oocytes were transferred onto a drop of SlowFade Light Antifade (Molecular Probes) on a slide and analyzed under an epifluorescence microscope (Leica Microsystems). For negative controls, the primary antibody was replaced by the nonimmune chicken IgY (Jackson ImmunoResearch). Assay of Glutathione.
Glutathione concentration in oocytes was measured by an enzymatic cycling assay (4, 5). Briefly, oocytes were collected at 16 h after treatment with BDNF and washed three times in PBS. Groups of 30-50 oocytes in 5 m l of PBS were transferred to 0.5-ml microfuge tubes and frozen at –20°C. The frozen samples were thawed in 5 m l of 1.25 M H₃PO₄ (Sigma), and oocytes were ruptured by agitation with a narrow-bore glass pipette. The samples were diluted with 0.49 ml of water and transferred to a glass tube before addition of the colorimetric solutions [0.7 ml of water, 1.2 ml of 0.2 M potassium phosphate buffer containing 10 mM EDTA, pH 7.2, 0.1 ml of 10 m M 5, 5-dithiobis-(2-nitrobenzonic acid), 0.05 ml of 20 units/ml glutathione reductase, and 0.05 ml of 4 m M NADPH (Sigma)]. After addition of NADPH, the absorbance was immediately monitored at 412 nm with a spectrophotometer (Amersham Biosciences) and recorded at 0.5, 2, and 5 min later. The amount of glutathione in each sample was determined based on a standard curve. The glutathione concentration per oocyte was estimated by determining total glutathione content in oocytes and mean cell volume as described in ref. (4). Blastocyst Cell Number Determination.
The number of cells in blastocysts was counted as described in ref. 2. Blastocysts were fixed in 4% of paraformaldehyde at the end of culture, and stained with 5 m g/ml of Hoechst 33342 (Invitrogen) for 30 min at 23°C. After three washes in PBS, individual blastocysts were covered in a drop of SlowFade Light Antifade (Molecular Probes) before counting the total number of nuclei in blastocysts under an epifluorescence microscope. Statistical Analysis.
The Mann–Whitney U test was performed for comparison of embryo development. One-way ANOVA followed by Fisher’s least significant difference test was used to evaluate differences in other experiments. Results are presented as the mean ± SEM of at least three separate experiments.

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