Single Cell Restriction Enzyme-Based Analysis of Methylation at Genomic Imprinted Regions in Preimplantation Mouse Embryos.

The methylation of cytosines in DNA is a fundamental epigenetic regulatory mechanism. During preimplantation development, mammalian embryos undergo extensive epigenetic reprogramming, including the global erasure of germ cell-specific DNA methylation marks, to allow for the establishment of the pluripotent state of the epiblast. However, DNA methylation marks at specific regions, such as imprinted gene regions, escape this reprogramming process, as their inheritance from germline to soma is paramount for proper development. To study the dynamics of DNA methylation marks in single blastomeres of mouse preimplantation embryos, we devised a new approach-single cell restriction enzyme analysis of methylation (SCRAM). SCRAM allows for reliable, fast, and high-throughput analysis of DNA methylation states of multiple regions of interest from single cells. In the method described below, SCRAM is specifically used to address loss of DNA methylation at genomic imprints or other highly methylated regions of interest.

Single blastomere removal from murine embryos is associated with activation of matrix metalloproteinases and Janus kinase/signal transducers and activators of transcription pathways of placental inflammation.

Single blastomere removal from cleavage-stage embryos, a common procedure used in conjunction with preimplantation genetic diagnosis (PGD), may affect reproductive outcomes. We hypothesized that negative pregnancy outcomes associated with PGD may be due to impairment of placental signaling pathways. The goal of this study was to determine the molecular mechanisms through which placental signaling is deregulated by blastomere removal. Four-cell stage murine embryos produced by in vitro fertilization were subjected to removal of a single blastomere (biopsied) or to the same manipulations without the blastomere removal (controls). Placental tissues from term (18.5 day) pregnancies obtained after embryo transfer were tested for levels of nitrosative species, interleukin 6, signal transducers and activators of transcription (STAT) 1 and 3, suppressors of cytokine signaling (SOCS) 1, 2 and 3 and matrix metalloproteinases (MMP) 1, 2, 3 and 9. Significant increases in nitrosative stress (P < 0.05), phosphorylative activation of STAT1 (P < 0.05) but not STAT3, lower levels of the inhibitors SOCS2 (P < 0.01) and SOCS3 (P < 0.001) and activation of MMP9 (P < 0.001) were observed in placentas derived from biopsied embryos, compared with controls. Such effects could contribute to greater levels of premature membrane rupture, incorrect parturition, preterm birth and intrauterine growth restriction associated with PGD. This work has determined signaling mechanisms that may be responsible for blastomere removal effects on placental function, with the potential to become targets for improving obstetric and neonatal outcomes in assisted reproduction.

Treating cloned embryos, but not donor cells, with 5-aza-2'-deoxycytidine enhances the developmental competence of porcine cloned embryos.

The efficiency of cloning by somatic cell nuclear transfer (SCNT) has remained low. In most cloned embryos, epigenetic reprogramming is incomplete, and usually the genome is hypermethylated. The DNA methylation inhibitor 5-aza-2'-deoxycytidine (5-aza-dC) could improve the developmental competence of cow, pig, cat and human SCNT embryos in previous studies. However, the parameters of 5-aza-dC treatment among species are different, and whether 5-aza-dC could enhance the developmental competence of porcine cloned embryos has still not been well studied. Therefore, in this study, we treated porcine fetal fibroblasts (PFF) that then were used as donor nuclei for nuclear transfer or fibroblast-derived reconstructed embryos with 5-aza-dC, and the concentration- and time-dependent effects of 5-aza-dC on porcine cloned embryos were investigated by assessing pseudo-pronucleus formation, developmental potential and pluripotent gene expression of these reconstructed embryos. Our results showed that 5-aza-dC significantly reduced the DNA methylation level in PFF (0 nM vs. 10 nM vs. 25 nM vs. 50 nM, 58.70% vs. 37.37% vs. 45.43% vs. 39.53%, P<0.05), but did not improve the blastocyst rate of cloned embryos derived from these cells. Treating cloned embryos with 25 nM 5-aza-dC for 24 h significantly enhanced the blastocyst rate compared with that of the untreated group. Furthermore, treating cloned embryos, but not donor cells, significantly promoted pseudo-pronucleus formation at 4 h post activation (51% for cloned embryos treated, 34% for donor cells treated and 36% for control, respectively, P<0.05) and enhanced the expression levels of pluripotent genes (Oct4, Nanog and Sox2) up to those of in vitro fertilized embryos during embryo development. In conclusion, treating cloned embryos, but not donor cells, with 5-aza-dC enhanced the developmental competence of porcine cloned embryos by promotion of pseudo-pronucleus formation and improvement of pluripotent gene expression.

Roles for focal adhesion kinase (FAK) in blastomere abscission and vesicle trafficking during cleavage in the sea urchin embryo.

Is focal adhesion kinase (FAK) needed for embryonic cleavage? We find that FAK is expressed during early cleavage divisions of sea urchin embryos as determined by polyclonal antibodies to the Lytechinus variegatus protein. FAK is absent in eggs and zygotes and then cycles in abundance during the first cleavages after fertilization. It is maximal at anaphase, similar to the destruction and synthesis of cyclin proteins. To investigate whether FAK is needed during early cleavage, we interfered with its function by microinjecting eggs with anti-FAK antibodies or with FAK antisense morpholino oligonucleotides. Both treatments led to regression of the cleavage furrow. FAK knockdown with antibodies or morpholino oligonucleotides also resulted in an over-accumulation of endocytic vesicles. Thus, FAK could be restricting endocytosis or increasing exocytosis in localized areas important for abscission. FAK appears to be necessary for successful cleavage. These results are the first to document a functional role for FAK during embryonic cleavage.

Transcriptome fingerprint of bovine 2-cell stage blastomeres is directly correlated with the individual developmental competence of the corresponding sister blastomere.

To date, gene expression profiles of bovine preimplantation embryos have only been indirectly related to developmental potential due to the invasive nature of such procedures. This study sought to find a direct correlation between transcriptome fingerprint of blastomeres of bovine 2-cell stage embryos with developmental competence of the corresponding sister blastomeres. Isolated blastomeres were classified according to the sister blastomere's development into three groups: two groups displayed developmental incompetency, including those blastomeres whose corresponding sister blastomeres either stopped cleaving after separation (2CB) or were blocked after two additional cleavages before embryonic genome activation (8CB). In the third group were competent blastomeres, which were defined as those whose sister blastomeres developed to the blastocyst stage (BL). As a result, developmental capacity of corresponding sister blastomeres was highly similar. Microarray analysis revealed 77 genes to be commonly differentially regulated among competent and incompetent blastomeres as well as blocked blastomeres. Clustering of differentially expressed genes according to molecular functions and pathways revealed antioxidant activity, NRF2-mediated oxidative stress response, and oxidative phosphorylation to be the main ontologies affected. Expression levels of selected candidate genes were further characterized in an independent model for developmental competence based on the time of first cleavage postfertilization. Moreover, overall results of this study were confirmed by higher developmental rates and more beneficial expression of CAT and PRDX1 when cultured in an antioxidative environment. These results will help us to understand molecular mechanisms defining developmental destination of individual bovine preimplantation embryos.

Multiple functions of FADD in apoptosis, NF-κB-related signaling, and heart development in Xenopus embryos.

FADD is an adaptor protein that transmits apoptotic signals from death receptors. Additionally, FADD has been shown to play a role in various functions including cell proliferation. However, the physiological role of FADD during embryonic development remains to be delineated. Here, we show the novel roles FADD plays in development and the molecular mechanisms of these roles in Xenopus embryos. By whole-mount in situ hybridization and RT-PCR analysis, we observed that fadd is constantly expressed in early embryos. The upregulation or downregulation of FADD proteins by embryonic manipulation resulted in induction of apoptosis or size changes in the heart during development. Expression of a truncated form of FADD, FADDdd, which lacks pro-apoptotic activity, caused growth retardation of embryos associated with dramatic expressional fluctuations of genes that are regulated by NF-κB. Moreover, we isolated a homolog of mammalian cullin-4 (Cul4), a component of the ubiquitin E3 ligase family, as a FADDdd-interacting molecule in Xenopus embryos. Thus, our study shows that FADD has multiple functions in embryos; it plays a part in the regulation of NF-κB activation and heart formation, in addition to apoptosis. Furthermore, our findings provide new insights into how Cul4-based ligase is related to FADD signaling in embryogenesis.

[Cyto B dependent and ouabain insensitive regulatory volume decrease in bicellular mouse embryo].

Mouse single-cell embryos exhibit robust Regulatory Volume Decrease (RVD). In what manner the very early mammalian embryo following zygote stage is appreciably altered by the anisotonic extracellular solution is, as yet, totally unclear. Little attention was paid to this direction since there was no way to determine the blastomere volume. This work has served to quantitatively investigate the osmotic response of bicellular mouse embryos employing Laser Scanning Microtomography (LSM) followed with three-dimensional reconstruction (3 DR). We have shown that bicellular mouse embryos in hypotonic Dulbecco's experience RVD. Embryonic cells subjected to hyposmolar exhibit rapid osmotic swelling followed by gradual shrinking back toward their original volume. The van't Hoff law defines swelling phase with the effective hydraulic conductivity of 0.3 micron x min(-1) x atm(-1). Water release during RVD in bicellular mouse embryos is abolished by Cytochalasin B (Cyto B) and the volume recovery is insensitive to ouabain treatment.

Life-giving caspases: revealing new roles during mouse embryo preimplantation development.

Caspases, cystein proteases traditionally related to programmed cell death, have recently been found to be involved in vital processes such as cell proliferation, adhesion and differentiation. Although caspases are expressed in mouse embryos before the blastocyst stage, their role is unclear, since apoptosis does not occur significantly before implantation. In this work, we have used mouse preimplantation development as a model to evaluate the existence of non-lethal caspase activities. The use of specific caspase inhibitors during in vitro embryo culture showed that caspase 8 activity, but not caspase 2 or 9, was relevant for development. The inhibition of caspase 8 affected the compaction of morulae and the progression to the blastocyst stage. In agreement with these results, caspase 8 was expressed in mouse embryos, as shown by indirect immunofluorescence and RT-PCR. An in silico approach was used to find putative caspase targets expressed in mouse preimplantation embryos. Large-scale management of sequence data from mouse embryos was used to predict caspase substrates by tools matrix-based on known cleavage sites. A total of 510 potential caspase targets expressed in mouse embryos were identified by this procedure. The functional characterization of these proteins by Gene Onthology associations showed that many of these putative caspase targets were previously related to non-apoptotic functions and only 63 had been previously reported to be actually cleaved by caspases. Interestingly, eleven knockout mice models for caspase substrates identified in our work, i.e. catenin alpha and beta, geminin, pescadillo, calpain-2, have preimplantation lethal phenotypes. This work supports the involvement of caspases in vital functions during mouse preimplantation development and proposes a model in which the regulated cleavage of caspase substrates could account for this role.

Involvement of the PKC family in regulation of early development.

Protein kinase C (PKC) isotypes have been implicated in a number of key steps during gametogenesis, fertilization, and early development. The 11-member family of PKC isotypes, many with different cofactor requirements for activation, can provide for differential activation of the specific kinases. In addition the enrichment of particular PKC isotypes to unique locations within gametes, zygotes, and early embryos likely promotes specific substrate interactions. Evidence exists to indicate involvement of PKC isotypes during sperm capacitation and the acrosome reaction, during resumption of meiosis in the oocytes, regulating the spindle organization in meiosis I and II, at fertilization, in the pronuclei, in the mitotically dividing blastomeres of the embryo, and at the plasma membranes of blastomeres at the time of embryonic compaction. Evidence also exists for crosstalk with other signaling pathways and one or more isotypes of PKC appear to be active at each major developmental transition.

[The influence of potassium 4-toluenethiosulfonate on membrane potential and ATPase activity of plasmatic membranes of loach embryos].

The influence of novel biologically active substance potassium 4-toluenethiosulfonate in concentration of 4 x 10(-5) M on the changes of electrophysiological parameters of embryonic cells in early development of fish (Misgurnus fossilis L.) was investigated including the changes of membrane potential (TMP) and enzyme activity of plasmatic membranes of loach embryos during the period of synchronous division of blastomers in the early period of development. The evaluation of influence of these matters was studied and aperiodic changes of the level of TMP were shown. The diminishing of amplitude in every period by 7/12 mV and diminishing of growth of maximal values of vibrations of TMP by 39 mV in comparison to control was noticed. It was related to inhibition of some biosynthetic processes and results in the decline of activity of membrane enzyme (Na+, K(+)-ATPase) by 75.5 +/- 4.1% and 78.4 +/- 10.4% both at the action of high (4 x 10(-3) M) and low (4 x 10(-9) M) concentrations, accordingly, at first hours of development with subsequent renewal of its activity to the level of control only for the actions of low concentration.

Chemical enhancement in embryo development and stem cell derivation from single blastomeres.

Several chemicals targeting the mitogen-activated protein (MAP) kinase signaling pathway, which play an important role in regulating cell growth and differentiation, have shown enhancing effects on the development of the inner cell mass (ICM) and the derivation of ES cells. However, investigation of such chemicals on early embryonic development and the establishment of ES cell lines has not been elucidated. This study was aimed to determine if ACTH, MAP2K1 inhibitor [MAP2K1 (I)], and MAPK14 inhibitor [MAPK14 (I)] could enhance the development of the ICM in preimplantation mouse embryos and blastocyst outgrowths, and the establishment of ES cell lines from blastomeres of early embryos. We have demonstrated that both MAP2K1 (I) and MAPK14 (I) delay early embryo development and inhibit the development of embryos from early blastomeres. On the other hand, ACTH had a positive effect on embryos derived from early blastomeres. As a result, 17 ES cell lines were established. Among these ES cell lines, nine and five ES cell lines were established from single blastomeres of two-cell embryos with and without the supplement of ACTH, respectively. In addition to two-cell isolated blastomeres, three ES cell lines were established from blastomeres of four-cell embryos only with the supplement of ACTH. Our results suggest that ACTH can enhance the derivation of ES cells from single blastomere-derived embryos.

Glutamine synthetase is essential in early mouse embryogenesis.

Glutamine synthetase (GS) is expressed in a tissue-specific and developmentally controlled manner, and functions to remove ammonia or glutamate. Furthermore, it is the only enzyme that can synthesize glutamine de novo. Since congenital deficiency of GS has not been reported, we investigated its role in early development. Because GS is expressed in embryonic stem (ES) cells, we generated a null mutant by replacing one GS allele in-frame with a beta-galactosidase-neomycine fusion gene. GS(+/LacZ) mice have no phenotype, but GS(LacZ/LacZ) mice die at ED3.5, demonstrating GS is essential in early embryogenesis. Although cells from ED2.5 GS(LacZ/LacZ) embryos and GS(GFP/LacZ) ES cells survive in vitro in glutamine-containing medium, these GS-deficient cells show a reduced fitness in chimera analysis and fail to survive in tetraploid-complementation assays. The survival of heavily (>90%) chimeric mice up to at least ED16.5 indicates that GS deficiency does not entail cell-autonomous effects and that, after implantation, GS activity is not essential until at least the fetal period. We hypothesize that GS-deficient embryos die when they move from the uterine tube to the harsher uterine environment, where the embryo has to catabolize amino acids to generate energy and, hence, has to detoxify ammonia, which requires GS activity.

p90Rsk is required for G1 phase arrest in unfertilized starfish eggs.

The cell cycle in oocytes generally arrests at a particular meiotic stage to await fertilization. This arrest occurs at metaphase of meiosis II (meta-II) in frog and mouse, and at G1 phase after completion of meiosis II in starfish. Despite this difference in the arrest phase, both arrests depend on the same Mos-MAPK (mitogen-activated protein kinase) pathway, indicating that the difference relies on particular downstream effectors. Immediately downstream of MAPK, Rsk (p90 ribosomal S6 kinase, p90(Rsk)) is required for the frog meta-II arrest. However, the mouse meta-II arrest challenges this requirement, and no downstream effector has been identified in the starfish G1 arrest. To investigate the downstream effector of MAPK in the starfish G1 arrest, we used a neutralizing antibody against Rsk and a constitutively active form of Rsk. Rsk was activated downstream of the Mos-MAPK pathway during meiosis. In G1 eggs, inhibition of Rsk activity released the arrest and initiated DNA replication without fertilization. Conversely, maintenance of Rsk activity prevented DNA replication following fertilization. In early embryos, injection of Mos activated the MAPK-Rsk pathway, resulting in G1 arrest. Moreover, inhibition of Rsk activity during meiosis I led to parthenogenetic activation without meiosis II. We conclude that immediately downstream of MAPK, Rsk is necessary and sufficient for the starfish G1 arrest. Although CSF (cytostatic factor) was originally defined for meta-II arrest in frog eggs, we propose to distinguish ;G1-CSF' for starfish from ;meta-II-CSF' for frog and mouse. The present study thus reveals a novel role of Rsk for G1-CSF.

Reactive oxygen species and Udx1 during early sea urchin development.

Sea urchin fertilization is marked by a massive conversion of molecular oxygen to hydrogen peroxide by a sea urchin dual oxidase, Udx1. This enzyme is essential for completing the physical block to polyspermy. Yet, its expression is maintained during development, as indicated by the presence of both Udx1 mRNA and Udx1 protein enriched at the surface of all non-mesenchymal blastomeres. When hydrogen peroxide synthesis by Udx1 is inhibited, either pharmacologically or by specific antibody injection, cleavage is delayed. Application of exogenous hydrogen peroxide, however, partially rescues a fraction of these defective embryos. We also report an unequal distribution of reactive oxygen species between sister blastomeres during early cleavage stages, suggesting a functional role for Udx1 in intracellular signaling.

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

During mouse preimplantation development, two isozymes of protein kinase C (PKC), delta and epsilon, 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 epsilon correlated with total mRNA transcription. Higher nuclear concentrations of both PKC delta and epsilon 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 delta out of the nucleus at the early four-cell stage decreased cytoplasmic EAP activity, whereas inhibiting translocation of PKC epsilon increased EAP activity slightly. These results indicate that translocation of PKC delta and epsilon 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).

Heparanase improves mouse embryo implantation.

OBJECTIVE: To improve mouse embryonic implantation by recombinant heparanase supplementation. Heparanase, an endoglycosidase-degrading heparan sulfate proteoglycan, may have a role in embryonic implantation because of its enzymatic, angiogenic, and adhesive properties. Increasing endometrial receptivity could improve one of the most difficult pathologies in human fertility. DESIGN: Comparison between mouse blastocysts obtained after 24-hour incubation of morulae with or without heparanase. SETTING: Experimental laboratory in a medical center. ANIMAL(S): Mice. INTERVENTION(S): Morulae were flushed from CB6F1 female mice and incubated for 24 hours at 37 degrees C in M16 medium supplemented with 0.1 mg/mL heparanase (n = 203), with albumin (n = 60), or with medium alone (n = 258). MAIN OUTCOME MEASURE(S): Blastocysts were evaluated by heparanase immunostaining (n = 10), activity assay (n = 283), and transfer to foster mice uterine horns (n = 228). The number of implantation sites was compared. RESULT(S): Immunostaining demonstrated that heparanase is constitutively expressed in mouse morulae and blastocyts. Heparanase supplementation resulted in increased staining and enzymatic activity in blastocyts. Implantation rates for the heparanase, M16 medium, and albumin groups, were 36.9%, 17.8%, and 20%, respectively (P<.01). CONCLUSION(S): Heparanase was found to be constitutively expressed by blastocyst-stage embryos. Moreover, the amount of heparanase was markedly increased by incubation of morulae with recombinant heparanase, evaluated by immunostaining and enzymatic activity. Heparanase supplementation resulted in approximately a twofold increase in embryo implantation rate in vivo. Taken together, these data suggest that heparanase is actively involved in embryo implantation.

[Variance analysis of Cd2+, Zn2+, and Mn2+ effect on membrane Na+,K(+)-ATPase activity of loach embryos].

The evaluation of influences Cd2+, Zn2+, Mn2+ in concentrations 10(-6), 10(-5), 10(-4) M (factor of dose) on the Na+, K(+)-ATP-ase activity in the early period of development (60-330 min.) of loach embryos (time factor) using one- and two-factor analysis of variance has been performed. It has been detected, that the changes of enzyme activity are mainly caused by action of the explored cations and do not depend on time of embryos development. The most influence on activity in the indicated period of embryos development of loach renders Cd2+ in concentration 10(-4) M, relative value of its influence being 95.7% (p = 0.01). Substantial concentration dependence of the Na+, K(+)-ATP-ase activity is exposed to the action of each of cations. The values of the influence of their concentration changes during the studied period of development differ insignificantly for all cations and make 76.2-77.5% (p < 0.01).

[Effect of heavy metal cations on the activity of membrane Na+, K+ -ATPase in embryos of loach (Misgurnus fossilis L.)]. / Aktyvnist' Na+, K+-ATP-azy membran zarodkiv v'iuna (Misgurnus fossilis L.) za diï kationiv vazhkykh metaliv.

The activity of membrane Na+, K+ -ATPase of embryos of loach (Misgurnus fossilis L.) at early stages of development in the normal conditions and under the influence of heavy metal cations has been investigated. It was established, that the influence of such heavy metal cations as Ni2+, Co2+, Sn2+, Zn2+, Mn2+ and Cd2+ in concentrations 10(-6) - 10(-4) M results in reduction of activity of membrane Na+, K+ -ATPase of loach embryos. It was shown, that the inhibition effect is more expressed with the increase of concentrations of heavy metal cations in the incubation medium. The definition of inhibition constants Io.5 has allowed to analyze the sensitivity of Na+, K+ -ATPase to influence of various cations of heavy metals at different stages of blastomer division. Possible mechanisms of influence of heavy metal cations on the activity of membrane Na+, K+ -ATPase of loach embryos have been considered.

[Effect of antibiotics on Na(+),K(+)-ATP-ase activity of the membranes in the Misgurnus fossilis L. embryos]. / Na(+),K(+)-ATFazna aktyvnist' membran zarodkiv v'iuna Misgurnus fossilis L. pry diï antybiotykiv.

The influence of some antibiotics of the fluoroquinolone classes on the Na+, K(+)-ATP-ase activity of loach embryo (Misqurnus fossilis L.) was investigated at different stages of blastomer divisions. It was determined that norofloxacin and borocin induce a dose-dependent decrease in Na+, K(+)-ATP-ase activity. Addition of norofloxacin (5.25 mcg/ml) into the incubation media caused pronounced oscillations in Na+, K(+)-ATP-ase activity, following borocin addition (5,15.25 mcg/ml) this effect was less pronounced. A conclusion was drawn about the influence of the fluoroquinolone on the membrane level.

Effects of PKC activation on the meiotic maturation, fertilization and early embryonic development of mouse oocytes.

Protein kinase C (PKC) is a family of Ser/Thr protein kinase widely distributed in eukaryotes. There is evidence that PKC plays key roles in the meiotic maturation and activation of mammalian oocytes. However, the mechanism of PKC's actions and the PKC isoforms responsible for these actions are poorly understood. In this study, we reveal in mouse eggs and early embryos: (1) the effects of PKC on the meiotic and mitotic cell cycle progression during oocyte maturation, egg activation and embryonic cleavages; (2) the functional importance of classical PKC subclasses in these processes; and (3) the subcellular localization of the PKC alpha isoform during development from GV stage oocytes to the blastocyst stage embryos. The results indicate that the PKC activator phorbol 12-myristate 13-acetate (PMA) inhibits the meiotic resumption of cumulus-free mouse oocytes by a mechanism dependent not only on classical PKC activity but also on other PKC isoforms. PKC activation after germinal vesicle breakdown leads to the inhibition of mitogen-activated protein kinase phosphorylation and the arrest of cell cycle at MI stage. The second polar body emission and the cleavages of early embryos are blocked after prolonged PKC activation. The subcellular localization of PKC alpha isoform in mouse oocytes and embryos is developmental-stage associated. All these results suggest that PKC has multiple functional roles in the cell cycle progression of mouse oocytes and embryos.