QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online 14 September 2004
doi: 10.1242/jcs.01375

This Article Summary Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Avazeri, N. Articles by Lefevre, B. Search for Related Content PubMed PubMed Citation Articles by Avazeri, N. Articles by Lefevre, B.

Regulation of spontaneous meiosis resumption in mouse oocytes by various conventional PKC isozymes depends on cellular compartmentalization

Institut National de la Santé et de la Recherche Médicale Unité 566 Commissariat à l'Energie Atomique, 92260 Fontenay-aux-Roses CEDEX, France

View larger version (56K): [in a new window]   Fig. 1. Intracellular redistribution of the various cPKCs isoforms in mouse oocytes during the period preceding germinal vesicle breakdown (GVBD). (A) The photographs show the three different types of immunolabeling observed with the four antibodies, against PKC-, PKC-ßI, PKC-ßII and PKC-, by confocal microscopy of whole mouse oocytes during spontaneous resumption of meiosis. These observations were recorded for a single optical section through the GV. Type I: spots of dense staining in the cytoplasm, intense labeling around the nuclear envelope, no staining in the nucleoplasm; type II: increased labeling in the nucleoplasm; type III: considerable immunoreactivity almost exclusively within the nucleus and around the cortex; isolated spots of fluorescence scattered throughout the cytoplasm. Scale bar, 40 µm. (B) The histograms indicate the distribution of the three different types of immunostaining with respect to time after meiosis reinitiation for each cPKC isoform. White columns: type I; gray columns: type II; black columns: type III. (C) The photographs show the immunolabeling of whole mouse oocytes, observed with the antibodies against PKC-, PKC-ßI and PKC-ßII when the meiotic arrest was maintained by hypoxanthine during 1 to 3 hours.

View larger version (24K): [in a new window]   Fig. 2. Temporal patterns of staining with the various anti-cPKC antibodies in isolated nuclei. For each cPKC studied, the selected photographs are representative of the redistribution of immunostaining observed by confocal microscopy in isolated nuclei. The nuclei were isolated from oocytes cultured for 30 or 90 minutes and then immediately immunostained with an antibody against one of the cPKCs studied. The bottom panel shows control nuclei immunostained with an antibody against lamin A/C. (Left) The nuclear envelope is retained during the nuclear isolation procedure. (Right) Corresponding transmission image of the nucleus. Bar scale, 10 µm.

View larger version (110K): [in a new window]   Fig. 3. Visualization of the subcellular distribution of PKC- and PKC-ßI by electron microscope immunocytochemistry in oocytes 30 and 90 minutes after meiosis reinitiation. Oocytes treated with anti-PKC- antibody 0-30 minutes (A) or 60-90 minutes (B, C) after release from the follicle; Oocytes treated with anti-PKC-ßI antibody 0-30 minutes (D) or 60-90 minutes (E, F) after release from the follicle. For each cPKC isoform, changes in the distribution of gold particles in the cytoplasm (gray columns) and nucleus (black columns) over time are shown in the two histograms: for both principal isoforms, the number of gold particles in the nucleus increased over time; the particles were distributed on the nuclear envelope (NE, white arrow), in the nucleoplasm (Np) and in the interchromatin granules (IG). The number of gold particles remained constant in the cytoplasm (Cy) for PKC-, but decreased with increasing time in culture for PKC-ßI. Black arrows indicate the gold particles on the NE. Magnification, 20,000x; *, statistically significant difference between cytoplasm and nucleus at P<0.05.

View larger version (94K): [in a new window]   Fig. 4. Visualization of the subcellular distribution of PKC-ßII and PKC- by electron microscope immunocytochemistry in oocytes 30 and 90 minutes after meiosis reinitiation. Oocytes treated with anti-PKC-ßII antibody 0-30 minutes (A) or 60-90 minutes (B,C) after release from the follicle. Oocytes treated with anti-PKC- antibody 0-30 minutes (D) or 60-90 minutes (E) after release from the follicle. For each cPKC isoform, changes in the distribution of gold particles in the cytoplasm (gray columns) and in the nucleus (black columns) over time are shown in the two histograms: for PKC-ßII, the number of gold particles increased in the nucleus [in the nucleoplasm (Np) as well as in the interchromatin granules (IG)] and in the cytoplasm (Cy); for PKC-, the nuclear envelope (NE, white arrow) and the nucleus were free of gold particles but the number of gold particles, although smaller than for other isoforms, increased over time. Black arrows indicate the gold particles on the NE. Magnification 20,000x. *, statistically significant difference between cytoplasm and nucleus, P<0.05.

View larger version (35K): [in a new window]   Fig. 5. Western blot analysis of the four cPKCs isoforms. (A) Cellular extracts of GV oocytes cultured for 30 or 90 minutes were subjected to biochemical fractionation (cytoplasmic, nucleoplasmic and nuclear envelope fractions) and analyzed by immunoblotting. For each cPKC isoform, western blots were probed with an antibody against the catalytic domain that reacted equally strongly with dephosphorylated and fully phosphorylated protein. The double asterisk indicates the 80-kDa mature PKC. The single asterisk denotes the presence of the 77-kDa form, which is phosphorylated at the two C-terminal sites. The 74-kDa form, indicated by a hyphen, has either no phosphate groups or a single phosphate on the carboxyl terminus. (B) Control blots demonstrating the purity of the subcellular fractions: ß-tubulin, used as a specific marker of the cytoplasm is absent from the nuclear fraction; lamin A/C, used as a specific marker of the nuclear envelope, is present in the nuclear envelope fraction and absent from the nucleoplasm.

View larger version (46K): [in a new window]   Fig. 6. Effects on spontaneous meiosis resumption or on HX-maintained meiotic arrest of the microinjection into the cytoplasm or into the GV of antibodies specific for the various cPKCs. (A) Control oocytes. Because PKC- is absent from the nucleus, anti-PKC- was used as a control for the microinjection procedure. The microinjection affected neither spontaneous meiosis resumption (-HX) nor maintained meiotic arrest (+HX). (B) Inhibition of the nuclear isoform of each cPKC except PKC- had a negative effect on spontaneous meiotic maturation. By contrast, the inhibition of the cytoplasmic isoforms of these enzymes had no effect on meiosis. (C) Top: oocytes were first cultured for 30 minutes, then microinjected into the cytoplasm and finally maintained in meiotic arrest by adding HX. Inhibition of the cytoplasmic isoforms affected meiotic arrest in different ways: the and ßII isoforms were not involved whereas the ßI and isoforms acted with HX to maintain meiotic arrest, the inhibition of these enzymes resulting in meiosis resumption. Bottom: oocytes were first cultured for 90 min, then microinjected and maintained in meiotic arrest by adding HX. Inhibition of the various cytoplasmic cPKC isoforms had no effect on meiotic arrest. The frequencies±s.e.m. of GVBD were calculated for at least three experiments. *, statistically significant difference versus control, P<0.05. White columns: control oocytes; gray columns: intracytoplasmic microinjection of the antibody; black columns: intranuclear microinjection of the antibody.