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Subcellular localization of protein kinase C and affects transcriptional and post-transcriptional processes in four-cell mouse embryos

Department of Biomedical Sciences, University of Guelph, Guelph, Ontario, N1G 2W1 Canada

Correspondence should be addressed to A C Hahnel; Email: ahahnel{at}ovc.uoguelph.ca

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion Acknowledgements References

 
During mouse preimplantation development, two isozymes of protein kinase C (PKC), and , transiently localize to nuclei at the early four-cell stage. In order to study their functions at this stage, we altered the subcellular localization of these isozymes (ratio of nuclear to cytoplasmic concentrations) with peptides that specifically activate or inhibit translocation of each isozyme. The effects of altering nuclear concentration of each isozyme on transcription (5-bromouridine 5'-triphosphate (BrUTP) incorporation), amount and distribution of small nuclear ribonucleoproteins (snRNPs), nucleolar dynamics (immunocytochemistry for Smith antigen (Sm) protein) and the activity of embryonic alkaline phosphatase (EAP; histochemistry) were examined. We found that nuclear concentration of PKC correlated with total mRNA transcription. Higher nuclear concentrations of both PKC and decreased storage of snRNPs in Cajal bodies and decreased the number of nucleoli, but did not affect the nucleoplasmic concentration of snRNPs. Inhibiting translocation of PKC out of the nucleus at the early four-cell stage decreased cytoplasmic EAP activity, whereas inhibiting translocation of PKC increased EAP activity slightly. These results indicate that translocation of PKC and in and out of nuclei at the early four-cell stage in mice can affect transcription or message processing, and that sequestration of these PKC in nuclei can also affect the activity of a cytoplasmic protein (EAP).

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion Acknowledgements References

 
In a signaling cascade, the number or average life time of kinase molecules and kinase–substrate complexes determines the extent of activation of downstream processes. For example, a 100–1000-fold increase in complex formation is an effective switch between the ‘on’ and ‘off’ states of a phosphorylation cascade (Kholodenko et al. 2000). Compartmentation (localization) of signal transduction proteins to cell membranes, cytoskeletal structures, scaffolding proteins or organelles (including the nucleus), can increase the number of collisions and complexes formed, and induce a several-fold activation of the signaling chain. For the protein kinase C (PKC) isozymes, it is hypothesized that localization to a cellular compartment is the major determinant of their specificity and function (Dempsey et al. 2000, Jaken & Parker 2000, Schechtman & Mochly-Rosen 2001).

PKC isozymes concentrate in specific subcellular locations at different stages of preimplantation mouse development (Pauken & Capco 2000, Dehghani & Hahnel 2005). We are particularly interested in PKC and , because they concentrate in early four-cell nuclei and translocate out again by the eight-cell stage (Dehghani & Hahnel 2005). During the two- to eight-cell stages of mouse development nuclear structure changes and there is implementation of transcriptional controls. Transcriptional activation of a large number of genes between the four-and eight-cell stages is known as the ‘mid-preimplantation gene activation’ (MGA), and may be an important event for the formation of different cell lineages at the blastocyst stage (Hamatani et al. 2004). Thus translocation of PKC in and out of nuclei during this stage of development may indicate a function in chromatin remodeling or in regulation of transcription factors. In adult cells, nuclear substrates of activated PKC have been identified; many of them are proteins involved in maintaining chromatin structure and modulating DNA replication or repair (Maraldi et al. 1999). The best-documented nuclear substrates of PKC are histone H1 and lamin B. Indeed, histone H1 has been used in vitro as a substrate to assay PKC activity (Takai et al. 1979). Phosphorylation of lamin B by PKC causes nuclear lamina disassembly at the beginning of mitosis (Hocevar et al. 1993). Moreover, phosphorylation/dephosphorylation reactions mediated by nuclear PKC are capable of affecting both chromatin arrangement and the activity of DNA-regulatory proteins in adult cells (Maraldi et al. 1999), processes which are postulated to be the key events of early development (Wolffe 1996).

We examined the effect of perturbing intracellular PKC and distributions on mRNA transcription and nuclear structures associated with message processing in order to investigate their role in the four-cell mouse embryo. To alter the intracellular location of the isozymes individually, we used peptides that specifically promote or interfere with activation or translocation of each isozyme. Localization of PKC isozymes to subcellular compartments is mediated by binding to anchoring proteins (Jaken & Parker 2000, Schechtman & Mochly-Rosen 2001). Receptors for activated C kinases (RACKs) anchor active PKCs in the proximity of substrates, and consequently promote substrate phosphorylation. For novel PKCs that are calcium-independent it has been shown that disruption of the intra-molecular interaction between the pseudo-adapter sequence and the adapter-binding site precedes activation, translocation and RACK binding (Ron & Mochly-Rosen 1995, Mochly-Rosen & Gordon 1998, Schechtman et al. 2004). This intra-molecular interaction can be disrupted by so-called translocation-activator peptides that correspond to the pseudo-adapter sequence, and they induce activation of the enzyme and exposure of the RACK-binding site. In this study, we used V1-7 and V1-7, peptides that bind to pseudo-adapter sequences of PKC and PKC respectively (RACK and RACK). Other studies have demonstrated that these peptides induce normal translocation and substrate phosphorylation by the corresponding PKC isozyme (reviewed in Dorn & Mochly-Rosen 2002). The translocation-inhibitor peptides used in this study (V1-1 and V1-2) are derived from the RACK-binding site sequences of PKC isozymes, and work by competition for binding to RACK adapters or other proteins bearing a complementary sequence (Ron et al. 1995). It has been observed in several adult cell types that they block normal physiological translocation of activated PKC in an isozyme-specific manner (reviewed in Dorn & Mochly-Rosen 2002).

The effects of perturbing distribution of PKCs and on mRNA transcription were determined through 5-bromouri-dine 5-’triphosphate (BrUTP) incorporation into nascent RNA, and through nucleolar morphology. The effects on message-processing machinery were assessed through the amount of Smith antigen (Sm) protein (integral components of small nuclear ribonucleoproteins (snRNPs) and of spliceosomes), and through the distribution of Sm proteins in Cajal bodies and speckles. Whereas the emphasis of this research was nuclear effects of these PKC, nuclear sequestration may also have ramifications for cytoplasmic proteins. The effect on a cytoplasmic protein was assessed through embryonic alkaline phosphatase (EAP) activity. Alkaline phosphatases and PKCs are both involved in phosphorylation, although a connection between the two proteins has never been made. However, EAP contains several potential PKC-phosphorylation sites. We have shown previously that EAP is one of the early transcripts of preimplantation mouse embryos (Hahnel et al. 1990), and a lack of EAP affects the development of preimplantation embryos between the four-cell and morula stages (Dehghani et al. 2000).

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion Acknowledgements References

 
Collection and culture of mouse embryos
To collect two-, four- and eight-cell-stage embryos, female CD1 mice (Charles River, Saint Constant, QC, Canada), were super-ovulated and mated, and their oviducts were flushed at 45, 52 and 72 h after human chorionic gonadotropin (hCG) injection, respectively, as described elsewhere (Dehghani & Hahnel 2005). At these time points, most two-cell embryos are in the G₂ phase, and most four-cell embryos are in G₁ or S phases of the cell cycle (Pratt 1987). The cell-cycle phase of eight-cell stage embryos is heterogeneous. Embryos were cultured in 10 µl KSOM/AA medium (Lawitts & Biggers 1993, Ho et al. 1995) and covered with silicone oil in 60-well culture plates (Nunc Brand Products, Roskilde, Denmark) at 37 °C in an atmosphere of 5% CO₂/5% O₂/90% N₂. All treatments were in the same medium and under the same culture conditions.

Induction and suppression of translocation of PKC isozymes
To non-specifically activate PKC and at the two-cell stage, embryos were flushed at 45 h post-hCG and were incubated for 15 min in 100 nM 4ß-phorbol-12-myristate-13-acetate (4ß-PMA; LC Laboratories, Woburn, MA, USA) in KSOM/AA medium. A 30 µM stock solution of 4ß-PMA in ethanol was diluted just prior to use. Control embryos were incubated in 0.33% ethanol. PMA activates all conventional PKCs (, ßI, ßII and ) and novel PKCs (, , and ). The 100 nM concentration of 4ß-PMA has been used to activate PKC in mouse and hamster eggs (Gallicano et al. 1995, 1997). Similar concentrations have been used to induce translocation of PKC isozymes in cardiac myocytes (100 nM; Disatnik et al. 1994, Dorn et al. 1999), mouse oocytes (160 nM; Luria et al. 2000) and rat eggs (80 nM; Raz et al. 1998). In studies of compaction, mouse embryos were treated with 16 nM 4ß-PMA under oil (Winkel et al. 1990). Inactive analogs of PMA were not used, because others have shown non-specific activities (for example, see Doerner et al. 1990, Watanabe et al. 2002).

To specifically change the location of PKC and at the two- and four-cell stages, embryos were cultured in isozyme-specific translocation activator peptides and isozyme-specific translocation-inhibitor peptides, which were kindly provided by D Mochly-Rosen (Department of Molecular Pharmacology, Stanford University School of Medicine, Stanford, CA, USA). Translocation of PKC was activated by V1-7, and translocation of PKC was activated by V1-7 (Dorn et al. 1999). Two-cell embryos were cultured for 1 h in 100 nM activator peptide (Dorn et al. 1999) in KSOM/AA medium, and four-cell embryos were cultured in 100 nM activator peptide for 2 h.

The translocation-inhibitor peptides were V1-1 (PKC ) and V1-2 (PKC ; Johnson et al. 1996, Gray et al. 1997, Chen et al. 1999). They were administered at 1 µM in culture medium (Chen et al. 1999) for either 7 h beginning at 45 h post-hCG (mid-two-cell–early four-cell) or 2 h at 52 h post-hCG (early four-cell). The activator and inhibitor peptides are conjugated to Antennapedia carrier peptides. The conjugates have previously been shown to enter cells and to specifically affect PKC or distribution and cell function without compromising cell viability at the concentrations used. It also has been previously shown that scrambled peptides and peptides derived from other parts of the molecules do not affect distribution of the PKC (Dorn et al. 1999). Control embryos were from the same flush as each experimental group and were cultured in a dimer of the carrier Antennapedia peptide.

Immunocytochemistry
For immunocytochemistry, embryos were fixed in 2% paraformaldehyde, permeabilized with 1% Tween 20, and immunostained with antisera as described in Dehghani & Hahnel (2005). Antibodies against PKC were rabbit polyclonal (Research & Diagnostics Antibodies, Berkeley, CA, USA) and used at a final dilution of 1:100. The antibody used to identify Sm protein was a mouse monoclonal antibody against the B/B' and D polypeptides (clone Y12; NeoMarkers, Fremont, CA, USA), and was used at 20 µg/ml. The secondary antibodies were affinity-purified F(ab')₂ fragment of goat anti-rabbit IgG labeled with Rhodamine Red-X (Jackson ImmunoResearch Laboratories, West Grove, PA, USA) for the PKC or goat anti-mouse IgG conjugated with Alexa-488 (Molecular Probes, Eugene, OR, USA) for Sm, and both were used at 20 µg/ml. The procedure for immunostaining for bromodeoxyuridine (BrdU) incorporation is described below. Immunostained embryos were examined with a laser-scanning confocal microscope (BioRad MRC 600) attached to an Optiphot-II Nikon fluorescence microscope (with a 60 x, 1.4 numerical aperture Plan-Apochromat oil-immersion objective). Excitation was with the 488 and 568 nm wavelengths from a krypton/argon laser. Controls for the immunostaining reactions were embryos that had not been incubated in primary antibody. Antibody reaction conditions and confocal settings were kept the same between trials.

Quantification of immunofluorescence due to PKC
Immunofluorescence intensity correlates with PKC concentration, and was quantified in nuclei and cytoplasm of all blastomeres of two-, four- and eight-cell embryos using mid-blastomere optical sections as described elsewhere (Dehghani & Hahnel 2005). There were three trials on different days with each PKC, and five experimental embryos per trial. Nuclear and cytoplasmic regions of each blastomere were identified manually, and the mean fluorescence intensity (mean gray value of all enclosed pixels) for each region measured using Scion Image (Scion Corporation, Frederick, MD, USA). The average background gray value from each section (a region outside of the embryo) was subtracted. For each blastomere, the ratio of nuclear to cytoplasmic fluorescence intensity was calculated (N/C). The data acquired from all treatment groups were checked for normal distribution using the Wilk–Shapiro/Rankit plot procedure. Analysis of variance (ANOVA) did not show any difference (P < 0.05) between trials. Therefore the ratios from all blastomeres in the three trials were pooled, and error bars in the histograms represent S.D. values for the pooled values. Differences between treatment groups were determined using Student’s t-test.

BrUTP incorporation and its quantification by laser-scanning confocal microscopy
Early four-cell embryos (52 h post-hCG) were cultured for 2 h in 1 µM PKC isozyme-specific translocation inhibitor or in 100 nM isozyme-specific translocation activator in KSOM/AA medium and then processed to incorporate BrUTP into nascent mRNA (described by Aoki et al. 1997). Embryos were permeabilized with 0.05% Triton X-100 in physiological buffer (PB; 100 mM potassium acetate, 30 mM KCl, 1 mM MgCl₂, 10 mM Na₂HPO₄, 1 mM ATP, 1 mM dithiotheritol, 0.2 mM PMSF and 50 units RNase inhibitor) and washed briefly three times with PB. Then they were incubated in transcription buffer (100 mM potassium acetate, 30 mM KCl, 2 mM MgCl₂, 10 mM Na₂HPO₄, 2 mM ATP, 0.4 mM each of GTP, CTP and BrUTP) for 10 min at 33 °C. The embryos were washed with PB, treated with 0.2% Triton X-100 in PB for 3 min, washed again in PB and fixed in 3.7% paraformaldehyde in PB overnight. Incorporated BrUTP was detected by immunocytochemistry. Embryos were incubated in 2 µg/ml of a monoclonal antibody to BrdU (Boehringer Mannheim, Mannheim, Germany), washed three times in PBS/BSA (PBS containing 3 mg/ml BSA; 15 min each time), and incubated subsequently in PBS/BSA containing 20 µg/ml goat anti-mouse IgG conjugated with Alexa-488 for 45 min. Embryos were washed with PBS/BSA and mounted on 0.1% poly-lysine-coated slides using Vectashield containing 4'-6-diamidino-2-phenylindole (DAPI; Vector Laboratories, Burlingame, CA, USA). Immunostaining controls were not exposed to primary antibody. To test the specificity of the BrUTP reaction for mRNA, another group of embryos were treated with 1 mg/ml -amanitin in transcription buffer. -Amanatin is a specific inhibitor of RNA polymerases II and III, but not I (Nothias et al. 1995). Any residual staining would have indicated incorporation into rRNA as well as mRNA.

Immunostaining was detected and quantified by laser-scanning confocal microscopy as described above, except that fluorescence from all optical slices of each nucleus was summed and divided by the total number of enclosed pixels. There were three trials with a minimum of 30 embryos per trial. Comparison of the average total nuclear fluorescence between trials by ANOVA did not indicate a significant difference (P > 0.05), and thus all of the values from one treatment were pooled. Differences between treatments were identified by Tukey’s test.

Analysis of the assembly and distribution of snRNP spliceosomes
To identify the effects of the PKC isozymes on the assembly of snRNP spliceosomes, two-cell stage embryos were cultured for 7 h to the four-cell stage in medium containing 1 µM isozyme-specific translocation inhibitor. The snRNP particles contain Sm proteins (seven polypeptides: B/B', D1, D2, D3, E, F and G). The relative concentrations of snRNPs in nuclei were quantified by immunocytochemistry using an antibody that recognizes B/B' and D polypeptides as described above. There were a total of 45 embryos examined, divided between three trials. Since there was no difference between trials by ANOVA (P < 0.05), fluorescence intensity readings from all blastomeres were pooled.

The coefficient of variation of fluorescence intensity for each slice provided information on whether the distribution of snRNP was diffuse within nuclei or collected into discrete granules or speckles. The coefficient of variation of fluorescence intensity was determined by dividing the standard deviation of pixel values in a nucleus by the mean pixel value of the nucleus. This allowed direct comparison of the magnitude of the variation of fluorescence intensity within a subcellular area for embryos with different mean values and different distributions. The mean coefficient of variation was the average of the coefficients of variation from the nuclei of embryos within a treatment group. Lower coefficients of variation correspond to more diffuse staining, whereas higher values correspond to granules of fluorescence or speckles being present. Treatment groups were compared with controls using ANOVA, and significantly different groups were identified by Tukey’s test.

Cajal bodies can also be identified by Sm immunostaining. They vary in size (0.2–1.5 µm) in different cell types and under different growth conditions (Sleeman & Lamond 1999a). The percentage of nuclei with Cajal bodies visible by immunostaining for Sm was determined by examining each 0.5 µm z-step through each nucleus. Different treatments were compared using the ² test. The P value was set for all possible combinations of pairwise tests and determined by Bonferoni’s inequality adjustment (Holm 1979).

Analysis of size and number of nucleoli
The effects of translocation inhibitors of the PKC isozymes on the size and number of nucleoli were determined in the same optical sections that were used for estimating Sm concentrations. Nucleoli were identified as the nuclear areas that did not immunostain with anti-Sm. Blastomeres in different treatment groups were compared using ² test, with the P value determined by Bonferoni’s inequality adjustment as above. The sizes of nucleoli were measured in square pixels on the digitized 0.5 µm z-step confocal images of nuclei using the Optimas program (version 6.0, Media Cybernetics). Results from the experimental groups were compared with the controls by ANOVA, and the significantly different groups identified with Tukey’s test. Phase-contrast microscopy of the same specimens provided qualitative reinforcement of the confocal results.

Evaluation of the effects of perturbation of PKC localization on EAP activity
Early four-cell stage embryos (flushed at 52 h post-hCG) were cultured for 2 h in the presence of 100 nM specific activator of PKC and or 1 µM isozyme-specific translocation inhibitor. The embryos were washed, fixed, permeabilized with Triton X-100 as for immunocytochemistry and stained histochemically using an azo-dye coupling reaction (SigmaFast kit; Sigma Chemical Co., St Louis, MO, USA). Since tissue non-specific alkaline phosphatase activity is inhibited by levamisol (0.622 mM) in this kit (Ziomek et al. 1990), only EAP activity is measured in pre-implantation mouse embryos. The effect of activation or inhibition of translocation of PKC and on the amount and distribution of EAP activity was analyzed by imaging the fluorescent product, as for immunocytochemistry, except that fluorescence intensity from all optical sections from each blastomere was summed (sum of all gray values of all the optical slices from each blastomere divided by the total number of enclosed pixels) and the background gray value subtracted. There was no difference by ANOVA (P < 0.05) between the three trials (30 embryos per trial), and all fluorescence intensities for each treatment were pooled. The treatment groups were compared with controls using ANOVA, and differences were identified by Tukey’s test.

Diffuse versus granular cytoplasmic fluorescence due to EAP activity was indicated through the coefficient of variation of fluorescence intensity as described above for nuclear Sm distribution.

	Results

Top Abstract Introduction Materials and Methods Results Discussion Acknowledgements References

 
The subcellular location of PKC and follows a stage-specific pattern which can be modified in vitro
Mouse embryos were flushed from oviducts at 45 h post-hCG (two-cell embryos, presumably G₂ phase), 52 h post-hCG (four-cell embryos, presumably G₁ or S phase) and 72 h post-hCG (early eight-cell embryos, blastomeres heterogeneous for cell-cycle stage; Pratt 1987). The mean ratio of the nuclear to cytoplasmic concentration of PKC and (N/C of fluorescence intensities) increased between late two-cell and early four-cell stage by 23.65% for and 27.36% for (compare Fig. 1A and G, 1D and L), and then decreased between the early four-cell and early eight-cell stage by 9.56% for and 51.23% for (compare Fig. 1G and K, L and P). When late two-cell embryos (flushed at 45 h post-hCG) were activated in vitro with either 4ß-PMA (15 min) or specific peptide activator (1 h), N/C also increased over controls by 11.82–14.73% (P < 0.05; Fig. 1B, C, E, F), and when early four-cell embryos (flushed at 52 h post-hCG) were cultured for 2 h in isozyme-specific activator, N/C decreased relative to controls by 19.13% for and 15.70% for (P < 0.05; Fig. 1H and M). These results suggested that translocation of PKC and in and out of four-cell nuclei is initiated by activation.

View larger version (61K): [in this window] [in a new window]   Figure 1 Immunolocalization of PKC and in two- to eight-cell mouse embryos, and N/C fluorescence intensities after treatment with activators and inhibitors. Embryos were treated with 4ß-PMA or with isozyme-specific activator or inhibitor peptides as described in the Materials and Methods section. Control embryos were similarly cultured in the absence of activator or inhibitor. After each treatment, embryos were immunostained with antibody specific to PKC or . Average ratio of nuclear to cytoplasmic (N/C) fluorescence intensity of all blastomeres in a group (representing relative PKC concentration) was determined. Data are pooled means ±S.D. from these ratios for all embryos in each treatment or control group. There were three trials with five embryos per trial for each treatment. The superscript letters A and a, B and b, and C and c indicate significant differences between a treatment group and its respective control. The 4ß-PMA treatment was analyzed by ANOVA and Tukey’s test; P < 0.05. Isozyme-specific activation and inhibition treatments were analyzed by Student’s t-test; P < 0.05. The percentages represent increased or decreased mean fluorescence intensity compared with the control. For some four-cell stage embryos, only two blastomeres are in the plane of focus.

View larger version (70K): [in this window] [in a new window]   Figure 2 Immunolocalization of PKC during cleavage to the four-cell stage. (A, B) Two-cell embryos with both blastomeres, presumably in G2. (A) is immunostained for PKC , (B) is a two-cell control for the immunostaining reaction (no primary antibody). (C, D) Three-cell embryo: left-hand blastomere in mitosis and right-hand sister blastomeres at early four-cell stage. (C) is immunostained with PKC , (D) is the DAPI-stained image of the embryo depicted in (C), and shows the nuclear morphology. (E, F) Four-cell embryo with two recently cleaved blastomeres with condensed chromatin and two later-stage blastomeres. (E) is immunostained with PKC , (F) is the DAPI-stained image of the embryo in (E). (G, H) Four-cell embryos with decondensed nuclei. (G) is immunostained with PKC , (H) is a four-cell control for the immunostaining reaction (no primary antibody).

Altering N/C of PKC and affects transcription
The N/C of PKC and in early four-cell embryos was increased or decreased in relation to controls by 2 h incubation in isozyme-specific translocation inhibitor or translocation activator, respectively. At the end of the treatment, mRNA transcription was measured through BrUTP incorporation into nascent transcripts (Fig. 3). Higher N/C for PKC resulted in more nascent mRNA after 2 h in culture (P < 0.05), and lower N/C resulted in less mRNA synthesis (P < 0.05). Higher nuclear PKC after treatment with peptide inhibitor increased mRNA production (P < 0.05), but lower N/C after treatment with peptide activator did not result in a significant decrease in mRNA synthesis. These results show a direct correlation between nuclear PKC and RNA polymerase II-derived transcription at the four-cell stage. Incorporation was unlikely due to DNA synthesis, since cellular dNTPs are lost following permeabilization. The conditions used are not permissive for rRNA synthesis (Aoki et al. 1997), and incubation of four-cell embryos in BrUTP in the presence of the transcriptional inhibitor, -amanitin, resulted in no detectable signal (Fig. 3A).

View larger version (27K): [in this window] [in a new window]   Figure 3 Effects of specific inhibition and activation of translocation of PKC and on BrUTP incorporation in four-cell stage embryos. (A) Four-cell stage embryos after incorporation of BrUTP. Incorporation was identified by immunocytochemistry with an antibody against BrdU. The embryo on the right was part of the group that was treated with -amanitin, showing specificity of BrUTP incorporation. Scale bar, 20 µm. (B) Average fluorescence intensities of all four-cell stage nuclei in treatment and control groups. The control group was cultured alongside the experimentals but without activator or inhibitor. There were three independent trials per treatment with at least 30 embryos per trial. Error bars, S.D. * Indicate groups that are significantly different from control (analyzed by ANOVA and Tukey’s test; P < 0.05). Captions under each bar indicate inhibitor or activator peptide used and effect of each treatment on N/C concentrations of the respective PKC. V1-1, PKC translocation inhibitor; V1-7, PKC translocation activator, V1-2, PKC translocation inhibitor; V1-7, PKC translocation activator.

Higher N/C of PKC and did not affect concentration or distribution of Sm proteins, but decreased the number of Sm-containing Cajal bodies and nucleoli

View larger version (33K): [in this window] [in a new window]   Figure 4 Effects of inhibition of translocation of PKC and on the number of Sm-containing Cajal bodies in four-cell stage embryos. (A) Embryo with (left-hand panel) and without (right-hand panel) detectable Cajal bodies (shown by arrows). Cajal bodies were identified by immunocytochemistry with anti-Sm. Scale bar, 20 µm. (B) Percentage of nuclei with detectable Cajal bodies. There were three independent trials with at least 45 embryos per trial. * Indicate groups that are sig-nificantly different from control (analyzed by 2 test; P < 0.008). This P value has been set for six possible combinations of pairwise tests (Bonferoni’s inequality adjustment; Holm 1979). Captions under each bar indicate inhibitor peptide used and effect of each treatment on N/C concentrations of the respective PKC. Details of inhibitors are given in the Fig. 3 legend.

View larger version (42K): [in this window] [in a new window]   Figure 5 Inhibition of translocation of PKC and decreases the number of nucleoli in four-cell stage embryos. (A) Nucleoli are identified by nuclear regions devoid of Sm protein by immunocytochemistry. A control embryo is shown in the left-hand panel, and an embryo treated with PKC translocation inhibitor is shown in the right-hand panel. Scale bar, 20 µm. (B) Percentages of nuclei with one, and two or more nucleoli in the different treatment groups. These were the same embryos as used for Fig. 4. Controls were treated as experimentals except for absence of inhibitor. * Indicate the groups that are significantly different from control (analyzed by 2 test; P < 0.008). This P value has been set for six possible combinations of pairwise tests (Bonferoni’s inequality adjustment; Holm 1979). Captions under each bar indicate inhibitor peptide used and effect of each treatment on N/C concentrations of the respective PKC. Details of inhibitors are given in the Fig. 3 legend.

View larger version (47K): [in this window] [in a new window]   Figure 6 Effects of inhibition of translocation of PKC and on the size of nucleoli in four-cell stage embryos. (A) Phase-contrast images of control (a) and embryos treated with PKC translocation-inhibitor peptide (b and c). (B) Nucleoli of varying sizes are shown by arrows. Scale bar, 20 µm. (C) Average size of nucleoli (square pixels) in different groups. The embryos used were the same as in Figs 4 and 5, and nucleoli were identified by the absence of Sm protein by immunocytochemistry. * Indicate the groups that are significantly different from control (analyzed by ANOVA and Tukey’s test; P < 0.05). Captions under each bar indicate inhibitor peptide used and effect of each treatment on N/C concentrations of the respective PKC. Details of inhibitors are given in the Fig. 3 legend.

Higher N/C of PKC and affected the level and pattern of EAP activity

View larger version (44K): [in this window] [in a new window]   Figure 7 Effects of inhibition of translocation of PKC and on the expression level and distribution of EAP activity in four-cell stage embryos. (A) Four-cell stage embryos histochemically stained for alkaline phosphatase activity. Scale bar, 20 µm. (B) Average fluorescence intensity from all blastomeres in a treatment group. (C) Average coefficient of variation of fluorescence intensity of all blastomeres in each treatment group. There were three independent trials with at least 30 embryos per trial. Controls were treated as experimentals except for absence of activator or inhibitor peptide. * Indicate the groups that are significantly different from control (analyzed by ANOVA and Tukey’s test; P < 0.05). Captions under each bar indicate inhibitor peptide used and effect of each treatment on N/C concentrations of the respective PKC. Details of inhibitors are given in the Fig. 3 legend.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion Acknowledgements References

In this study, isozyme-specific activation at the late two-cell or early four-cell stage resulted in translocation of the respective isozyme into (two-cell) or out of (four-cell) nuclei. Since the movements were reminiscent of translocation of these enzymes between two- and four-cell and four- and eight-cell embryos, respectively, we assumed that the PKC isoforms were activated prior to translocation in each case. This is what has been described in the literature in many studies with adult cells (Teruel & Meyer 2000, Mackay & Mochly-Rosen 2001). However, the peptides that specifically inhibit translocation of activated PKC or did not prevent translocation of either of these PKC into nuclei between the two- and four-cell stages; 7 h in inhibitor still resulted in increased N/C ratios. The peptides did inhibit subsequent translocation of these PKC out of four-cell nuclei; early four-cell embryos were cultured in inhibitor for 2 h and N/C ratios remained elevated. Assuming that the inhibitor peptides were active for the full 7-h incubation, then transport of these PKC out of four-cell nuclei seems to be activation- and RACK-binding-site-dependent, whereas transport into the nuclei is not. These results were unexpected. Therefore we collected embryos undergoing division from the two- to four-cell stage. PKC does not seem to enter nuclei until they reformed at the end of mitosis. At this point (early four-cell stage), activation induces movement out of nuclei. These two pieces of evidence suggest that it is catalytically inactive PKC and that translocate into four-cell nuclei at the end of mitosis, and that PKC and associate with other types of anchoring proteins or substrates such as receptors for inactivated C kinases, or RICKs. Therefore, PKC or may not be affecting four-cell nuclei through a kinase activity. There is evidence for non-kinase activities of PKC. Translocation to the nucleus of a kinase-negative mutant of PKC was sufficient to induce apoptosis in smooth muscle cells (Goerke et al. 2002). The possibility that PKC associates with nucleoli is interesting in light of its effect on nucleolar number at the four-cell stage, although it still needs to be proven that the ‘bright spots’ are nucleoli.

Effects on transcription
The direct correlation between nuclear concentration of PKC and total mRNA transcription (BrUTP incorporation into nascent transcripts) suggests that this isozyme is involved in transcription at the early four-cell stage. Culture in the peptide inhibitor of PKC translocation increased N/C by 18.18% over controls, and mRNA synthesis increased by 24.78% over controls in embryos similarly cultured in inhibitor. The peptide activator of PKC decreased N/C by 15.7%, and mRNA synthesis decreased by 20.23% over controls in embryos similarly cultured in activator. The fact that only the inhibitor of PKC affected mRNA synthesis suggests a less-direct or less-specific interaction. It has been shown that a small change in the local concentration of a signal-transduction protein can lead to a large response through its associated signaling chain (Bray 1998). Thus, the amount of change induced by altered PKC localization could be sufficient to modify transcriptional activity. Modification could be of DNA-regulatory proteins (Balsalobre et al. 2000) or chromatin structure (Thomson et al. 1999), or result from alteration to the cell cycle (Livneh & Fishman 1997, Viveiros et al. 2001, Quan et al. 2003). Indeed, many nuclear substrates of PKC are involved in chromatin structure and in modulating DNA replication or repair (Maraldi et al. 1999). The BrUTP results are supported by decrease in number of nucleoli with higher N/C of PKC and (Table 1). The decrease may be due to joining of multiple small nucleoli into a single nucleolus (Figs 5 and 6), a hallmark of active transcription in preimplantation embryos (King et al. 1988, Komeili & O’Shea 2000).

View this table: [in this window] [in a new window]   Table 1 Summary of findings in four-cell-stage mouse embryos treated with PKC and translocation inhibitors.

Effects on EAP expression
The results of this study indicate that concentration of PKC and into nuclei affects the cytoplasmic expression of EAP at the four-cell stage (Table 1), an interesting observation since this is when absence of EAP begins to produce a phenotype (Dehghani et al. 2000). Inhibition of PKC translocation into the cytoplasm at the early four-cell stage decreased EAP activity at sites of intercellular contact. In contrast, inhibition of translocation of PKC into the cytoplasm at the early four-cell stage increased EAP activity. There were no antibodies to mouse EAP, so we could not determine whether activity or protein levels were affected. A three-dimensional model of EAP structure based on the known structure of bacterial alkaline phosphatase suggests that the EAP active site is surrounded by three PKC-specific phosphorylation sequences. Therefore it is not unreasonable to hypothesise that the PKC are involved in the activation or degradation of EAP. The effects of these PKC on EAP activity clearly demonstrate specificity in the actions of the two pairs of activating and inhibiting peptides, and suggest that sequestration of PKC and into nuclei may also be important to cytoplasmic as well as nuclear function.

Summary
Various members of the PKC family are expressed in pre-implantation mouse embryos, but little is known of specific isozyme functions. It has become clear that sub-cellular localization of a PKC isozyme is critical to its normal function. In this study, we increased the ratio of nuclear to cytoplasmic concentrations of PKC and in early four-cell mouse embryos with peptides that specifically inhibit translocation of these PKC. Previous research has demonstrated the specificity of the peptides at the concentrations used, and their effect on translocation and physiological processes in other cells. The relative concentration of each of these PKC was determined by measuring relative fluorescence intensity due to antibody binding. Reaction conditions and confocal settings were carefully maintained, and there were multiple trials over several days. Using relative fluorescence intensity to follow translocation of PKC has previously been used in adult cells (for example, see Almholt et al. 1999, Ron et al. 1999, Echevarria et al. 2003, Schechtman et al. 2004), but not in preimplantation embryos. Yet it is particularly suitable for studying preimplantation embryos where paucity of material makes subcellular fractionation combined with Western blotting impractical.

Using this approach, processes and molecules affected by PKC and in early four-cell mouse embryos were identified. More studies are required to elucidate the pathways through which these PKC exert their effects. Particular attention will have to be paid to whether the action of each PKC is directly on a nuclear pathway. PKC targets in nuclei include transcription factors, chromatin-associated proteins and structural proteins. Thus PKC effects on transcription and PKC and effects on Cajal bodies and nucleoli likely are through nuclear pathways. However an indirect effect through the cytoplasm has not been ruled out. The results show that sequestration of PKC and in four-cell nuclei affects EAP activity in the cytoplasm. It also will be important to determine the activation state of the PKC over time. These results suggest that the PKC and that accumulates in early four-cell nuclei may not be activated. If so, an explanation of the effects described in this study will need to invoke a pathway that does not require activation. However, we may have missed activation with the long culture periods, perhaps controlled by the cell cycle, as described in oocytes by Viveiros et al.(2003).

	Acknowledgements

Top Abstract Introduction Materials and Methods Results Discussion Acknowledgements References

 
We thank Dr Mochly-Rosen for providing us with the isozyme-specific translocation inhibitors and activators of PKC. This work was supported by an NSERC Canada grant to A C H. H D was supported by a scholarship from MCHE Iran. The authors declare that there is no conflict of interest that would prejudice the impartiality of this scientific work.

	Footnotes

 
Received 15 November 2004
First decision 6 January 2005
Revised manuscript received 22 March 2005
Accepted 21 June 2005

(H Dehghani is now at Programme in Cell Biology, The Research Institute, The Hospital for Sick Children, Toronto, Ontario, M5G 1X8 Canada)

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