Journal:

Article Title: Secretion and Assembly of Zona Pellucida Glycoproteins by Growing Mouse Oocytes Microinjected with Epitope-tagged cDNAs for mZP2 and mZP3 *

doi: 10.1091/mbc.01-09-0440

Figure Lengend Snippet: Gel analysis of epitope-tagged ZP glycoproteins synthesized and secreted by growing mouse oocytes. Two hundred growing oocytes were isolated from 13-d-old mice, microinjected with Myc-mZP2, and cultured in M199-M for ∼5 h. Oocytes were then transferred to Met/Cys-depleted M199-M, supplemented with 4 mCi/ml [35S]Met/Cys. The culture medium was collected after ∼15 h and immunoprecipitated with monoclonal antibodies directed against Myc (9E10). Immunoprecipitates were subjected to 7.5% SDS-PAGE, as described in MATERIALS AND METHODS. Media from uninjected and injected oocytes probed with anti-Myc are shown in A and B, respectively. An arrowhead indicates the position of mZP2. Media were immunoprecipitated once again with an anti-mZP2 polyclonal antibody. As shown in C and D, both injected and noninjected oocytes secreted endogenous mZP2 glycoproteins. A band at ∼70 kDa was observed as a contaminant in all lanes. The positions of molecular mass standards are shown.

Article Snippet: Growing oocytes (50 ± 10 μm in diameter; Sorensen and Wassarman, 1976 ; Eppig and Telfer, 1993 ; Eppig et al. , 1996 ) were released by puncturing ovaries with fine steel needles under a dissecting microscope in a culture dish containing prewarmed Earle's modified medium M199 (Invitrogen) supplemented with bovine serum albumin (BSA; 4 mg/ml) and sodium pyruvate (30 μg/ml; M199-M).

Techniques: Synthesized, Isolation, Cell Culture, Immunoprecipitation, SDS Page, Injection