Comparison of oocyte development within the follicle in vitro and in vivo has a major impact on research into ovarian physiology and clinical practice. Despite major differences in ovarian physiology between rodents and humans, mice provide a useful model for studies of the endocrine and paracrine mechanisms controlling follicular development. In this study, early preantral follicles were isolated from 12-day-old mice and cultured individually in microdrops under oil during 6, 9 or 12 days. Taking into account previous observations, several oocyte criteria (diameter, chromatin configuration, transcriptional activity, intracytoplasmic calcium signalling and ability to undergo meiosis) were assessed to check that the development pattern of oocytes during follicle growth in vitro was similar to that already observed for oocytes developing in vivo, and that they reached the fertilizable oocyte stage. Results indicate that, during the 12-day-culture period, the oocytes grew until 74.3 ± 4.2 mm, they became transcriptionally quiescent with a surrounded nucleolus (SN) chromatin organization, 50% of them exhibited regular calcium signals and 73.4% of them resumed meiosis. These data demonstrate that the protocol used generates oocytes with characteristics similar to oocytes allowed to mature fully in vivo and that it could be useful to set up the experimental culture of human ovarian follicles.
Amnion and chorion cells from human fetal membranes have been cultured. Chorionic cells secrete renin whereas amnionic cells do not. Renin is secreted by chorionic cells as an inactive form that can be activated by trypsin treatment or acid dialysis. The antigenic and enzymatic properties of activated chorionic renin and kidney renin are similar. Incorporation of [35S]methionine in the culture demonstrates that chorionic renin is secreted as a high molecular weight form, 54K. This 54K inactive renin could represent the proenzyme.
The location of the phospholipase C β1-isoform (PLC-β1) in the mouse oocyte and its role in the resumption of meiosis were examined. We used specific monoclonal antibodies to monitor the in vitro dynamics of the subcellular distribution of the enzyme from the release of the oocyte from the follicle until breakdown of the germinal vesicle (GVBD) by Western blotting, electron microscope immunohistochemistry, and confocal microscope immunofluorescence. PLC-β1 became relocated to the oocyte cortex and the nucleoplasm during the G2/M transition, mainly in the hour preceding GVBD. The enzyme was a 150-kDa protein, corresponding to PLC-β1a. Its synthesis in the cytoplasm increased during this period, and it accumulated in the nucleoplasm. GVBD was dramatically inhibited by the microinjection of anti-PLC-β1 monoclonal antibody into the germinal vesicle (GV) only when this accumulation was at its maximum. In contrast, PLC-γ1 was absent from the GV from the time of release from the follicle until 1 h later, and microinjection of anti-PLC-γ1 into the GV did not affect GVBD. Our results demonstrate a relationship between the relocation of PLC-β1 and its role in the first step of meiosis.
Cell proliferation is controlled by cdk2 which in association with cyclin E and A regulates G1/S transition and S phase progression. cdk2 activation is dependent on its localization in the nucleus where regulatory mediators are found. We report that activation of cdk2 is associated with the formation of cdk2/ MAP Kinase complexes. cdk2 associates with both inactive and activated MAP Kinase. Prevention of MAP Kinase activation by the MEK inhibitor PD98059 inhibits both activation and nuclear localization of cdk2 and S phase entry. These ®ndings indicate that the nuclear translocation of cdk2 is associated with the formation of molecular complexes containing active MAP Kinase and is dependent on MAP Kinase activation. Oncogene (2000) 19, 4184 ± 4189.
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