Purpose The goal of the study was to establish the mechanism by which the estrogen concentration difference between the follicular fluid and the serum is maintained. to identify perlecan as the most likely candidate for the major estrogen-binding protein in the follicular fluid. for 10?min to remove cells and cellular debris and frozen shortly after at ?800 C until the time of analysis. Granulosa cells (GCs) were collected from pooled FF as described previously with minor modifications . Briefly the pooled follicular aspirate was centrifuged at 800for 10?min and the resulting cell slurry was diluted twofold Ondansetron HCl with Hanks’ balanced salt solution (HBSS) and then gently layered on top of 3?ml of a Ficoll-paque Plus 50?% (GE Health Care Life Sciences) gradient cushion and then centrifuged at 700for 20?min. GCs were collected from the cell layer at the Ondansetron HCl interface resuspended in 10?ml HBSS and centrifuged. The resulting cell pellets were resuspended in 0.5?ml of culture media and cell number and viability were assessed in a hemocytometer by a Trypan blue exclusion test. The cells (～50 0 cells per well) were plated in a 24-well with Complete Media 199 (Life Technologies) supplemented with 10?% FBS 100 penicillin 100 streptomycin 10 fetal calf serum and 0.25?μg/ml amphotericin B incubated in 37?°C 5.5 CO2. Hormones used were recombinant FSH (Puregon? Merck Canada) in a concentration of 1 1 5 and 10?IU per well insulin (Humulin R? Eli Lilly Indianapolis USA) 0.5 1 5 and 10 units per well hCG (Pregnyl? Merck Canada) 0.5 1 5 and 10 units per well triiodothyronine (T3) (Sigma-Aldrich) 10 Ondansetron HCl and 20?nmol per good hydrocortisone (Sigma-Aldrich) 250 and 500?nmol per well and 17β-estradiol (Sigma-Aldrich) 250 and 500?nmol per well . Following incubation for 48?h the cells were harvested for either RNA or protein extraction and medium was used to measure SHBG by ELISA (MX52001 IBL Hamburg Germany). Reverse transcriptase-PCR for SHBG gene Unless specified all reagents were purchased from Qiagen (Toronto Canada) and were used according to the manufacturer instructions. RNA was extracted from granulosa cells using the RNAeasy mini kit. Commercial human liver and testis RNA (Zyagen San Diego CA) and pooled RNA samples from three individuals were used for reverse transcriptase (RT) reaction using the QuantiTect Reverse Transcription Kit. PCR amplification Bmp8a was carried with the PCR Master Mix Kit using the following primers: SHBG exon 1-forward 5′-TGCTGCTGTTGCTACTACTG; exon 6-reverse 5′-CAAGATGGGTTCTCTGGTGTC; exon 3-forward 5′-AGGATGACTGGTTTATGCTG; exon 6-forward 5′-GACACCAGAGAACCCATCTTG; and exon 8-reverse 5′-ATCTCATGGCTTCTGTTCAGG. PCR reaction products were separated by electrophoresis on a 1?% agarose gel and visualized with SYBR safe DNA gel stain (Life Technologies). Real-time PCR for SHBG Quantitative PCR (qPCR) was performed as described before (Perumalsamy et al. 2010 utilizing the QuantiTect SYBR? Green PCR kit (Qiagen) on the LiteCycler (Roche Mississauga ON Canada). The reaction conditions were as follows: 95?°C for 10?min and then 40?cycles of 95?°C for 30?s 60 for 30?s and 72?°C for 30?s. Comparisons of expression levels were determined by delta CT method normalized to β-actin. Western blot for SHBG Granulosa Ondansetron HCl cells and ovarian protein lysates were prepared in 1?% SDS-radioimmunoprecipitation assay (RIPA) buffer containing a complete protease inhibitor cocktail (Roche). Protein concentration was determined using the bicinchoninic acid (BCA) protein assay and protein samples resolved through 12?% acrylamide gels and transferred to polyvinylidene fluoride (PVDF) membranes. After blocking Ondansetron HCl the blots were probed with rabbit anti-CoQ6 or anti-PDSS2 (1:400 and 1:600 respectively; Proteintech Group Inc). Follicular fluid aspirates obtained from patients undergoing oocyte retrieval were centrifuged and treated with RBC lysis buffer (0.16?M NH4Cl 10 KHCO3 0.1 EDTA) to remove any red blood cells and cell pellet was washed with PBS. GC pellet and OVCAR3 cells were prepared with 1?% SDS-RIPA buffer containing protease inhibitor cocktail (Roche Diagnostics) and protein concentrations were determined using the BCA protein assay. Ondansetron HCl FF was prepared as follows: 9?μl of RIPA lysis buffer with protease inhibitors was added to 3?μl of follicular fluid. The protein lysates were run on a 12?% SDS-PAGE gel and transferred onto PVDF membrane. The membrane was.