Blots were revealed with enhanced chemiluminescent reagents

n, zonae pellucidae were removed with acidic Tyrode’s solution. Oocytes were fixed in a prewarmed solution, containing 100 mM HEPES, 50 mM EGTA, 10 19088077 mM MgSO4, 0.2% Triton X-100, 3.7% formaldehyde, for 30 minutes at room temperature. Fixed oocytes were then permeabilized for 2 hours in PBS with 0.2% Triton X-100, and then incubated in PBS with 10% FBS for at least 30 minutes. Interval washes were done in PBS with 0.1% polyvinylpyrrolidone. Incubation with primary antibody was overnight at 4uC, followed by one hour incubation with secondary antibody at room temperature. The rabbit SPIN1 antibody and the mouse tubulin antibody were used at 1:100 dilutions as primary antibodies. Hoechst 33342 dye was used to stain DNA. Mammalian Cell Culture and Transfection HEK293T cells were cultured in DMEM supplemented with 10% FBS. Plasmids prepared using QIAGEN maxiprep kit were transfected into HEK293T cells with Lipofectamine 2000. Histology and Immunohistochemistry For immunodetection, dissected ovaries were first fixed using PBS with 4% formaldehyde at 4uC for 12 hours, then processed into tissue blocks and cryo-sectioned. The tissue sections were incubated in PBS with 10% FBS for at least 30 minutes, and then with primary antibodies recognizing SPIN1 and SERBP1 for 4 hours at room temperature. Secondary antibodies conjugated with Alexa Fluor-488 or 594 were added after three washes with PBS containing 0.1% Tween 20. DNA was stained using Hoechst 33342. To reveal the histological structure of the gonad grafted under the kidney capsule, the tissue was processed into wax blocks, and then sectioned and stained with hematoxylin and eosin. Molecular Cloning To fuse mouse Spin1, Serbp1, and Habp4 with Myc or HA tags, the cDNAs were amplified and cloned into pCMV6-AN. Primers used are listed in Confocal Microscopy Fixed oocytes were imaged on glass bottom dishes with a Zeiss LSM510 confocal 10037488 microscope equipped with a 40X EC Plan-NeoFluor 1.3 NA oil immersion objective lens. Images were acquired using LSM510 software and analyzed using ImageJ. Reverse Transcription-PCR and Real-time Quantitative PCR Total RNAs of fully grown oocytes were purified using a PicoPure RNA Isolation Kit, and reverse transcribed to cDNAs using the SuperScript III reverse transcriptase and oligo-dT. The synthesized cDNA was amplified in a Scopoletin site sequence-specific manner, using the TaqMan PreAmp Master mix kit, prior to analysis with the Universal Probe Library for gene Luciferase Reporter Assay Roles of SPIN1 in Mouse Oocytes 3 Roles of SPIN1 in Mouse Oocytes 4 Roles of SPIN1 in Mouse Oocytes 5 Roles of SPIN1 in Mouse Oocytes 6 Roles of SPIN1 in Mouse Oocytes transfected into HEK293T cells with pCMV6-AN plasmids containing Myc, Myc-Spin1, Myc-Spin1Y155F, or HA-Serbp1, respectively. The luciferase activity of empty vectors pmirGLO and pCMV6-AN was used to normalize the expression level. Since Spin1 mutants exhibit early post-natal lethality, we grafted ovaries of E18.5 Spin1 mutant and wild type littermates under the kidney capsules of C57BL6/J adult mice, to study SPIN1 function during oocyte growth and maturation. The procedure of grafting ovaries under adult kidney capsules has previously been shown to support ovarian growth and development. Grafted ovaries were removed and sectioned after 21 days following stimulation by PMSG. Staining the ovarian sections showed that follicles at different stages including pre-antral follicles, early antral follicles, and antral follicles were presen