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Gamete Biology |
in Mouse Oocytes Throughout Meiotic Maturation and Following Egg Activation1
The Jackson Laboratory, Bar Harbor, Maine 04609
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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), a novel isoform of the PKC family, was determined in mouse oocytes undergoing meiotic maturation and following egg activation. The full-length protein was observed as a doublet (76 and 78 kDa) on Western blot analysis. A smaller (47 kDa) carboxyl-terminal fragment, presumably the truncated catalytic domain of PKC-, was also strongly expressed. Both the full-length protein and the catalytic fragment became phosphorylated coincident with the resumption of meiosis and remained phosphorylated throughout metaphase II (MII) arrest. Immunofluorescence staining showed PKC- distributed diffusely throughout the cytoplasm of oocytes during maturation and associated with the spindle apparatus during the first meiotic division. Discrete foci of the protein also localized with the chromosomes in some mature eggs. Following the completion of meiosis, PKC- became dephosphorylated within 2 h of in vitro fertilization or parthenogenetic activation. The protein also accumulated in the nuclei of early embryos and was phosphorylated during M-phase of the initial mitotic cleavage division. By the two-cell stage, expression of the truncated catalytic fragment was minimal. These data demonstrate that the subcellular distribution and posttranslational modification of PKC- is cell cycle dependent, suggesting that its activity and/or function likely vary with the progression of meiosis and egg activation.
gamete biology, in vitro fertilization, kinases, meiosis, oocyte development
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The PKC family consists of 11 different serine/threonine kinases that are subdivided into three groups based on sequence homology as well as on activator and cofactor requirements. These groups include the "conventional" (PKC-
, ßI, ßII, and 
), "novel" (PKC-, 
, 
, µ, and 
) and "atypical" (PKC-
and 
) isoforms [10, for review], which participate in a broad range of somatic cell functions involved in growth, differentiation, and cell cycle progression [11, 12]. Various isoforms are expressed in mouse oocytes, and the modulation of PKC activity reportedly influences the progression of meiosis. Protein expression of PKC-, ß, , , and has been confirmed in both prophase I and MII stage oocytes, while detected mRNAs are limited to fewer isoforms, such as PKC- and [13–16]. PKC function is dependent on the stage of meiotic maturation. Direct activation of PKC in oocytes arrested at prophase I inhibits spontaneous resumption of meiosis, precluding entry into metaphase I (MI) [16–19], while treatment with PKC agonists shortly after germinal vesicle breakdown (GVBD) delays emission of the first polar body [18, 19]. PKC activation at MII, in turn, reportedly promotes entry into interphase by Xenopus, mouse, hamster, and rat eggs [14, 20–24], though complete egg activation and cell cycle resumption might not be induced [25, 26]. The response to PKC is likely dependent on the type and expression pattern of specific isoforms, their substrates, and anchoring proteins known as receptors for activated PKC (RACK) that can target the proteins to particular cellular compartments [27, 28]. Moreover, different PKCs likely mediate unique functions. Hence, an assessment of individual isoforms is needed to accurately define the roles of this kinase family in meiotic maturation and oocyte activation.
The involvement of PKC in specific meiotic defects that are characteristic of oocytes from strain LTXBO mice has been proposed, and a novel isoform (PKC-) was detected in association with the spindle apparatus during the first meiotic division [29]. Fully grown oocytes from LTXBO mice resume meiosis normally but exhibit an unusually prolonged MI-stage and commonly undergo spontaneous parthenogenetic activation following the first meiotic division [29–32]. PKC- is expressed in a broad range of somatic cell types, and, interestingly, its overexpression can promote cell cycle arrest [33, 34]. PKC- expression has also been confirmed in normal mouse oocytes by Western blot analysis [13, 15, 16], but it is not known whether the protein is posttranslationally modified or whether its association with the meiotic spindle is unique to LTXBO oocytes. The present study was therefore undertaken to fully characterize the protein expression and subcellular distribution profile of PKC- in oocytes that exhibit no overt defects in the progression of meiosis or egg activation.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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All mice were bred and raised in the research colony of the authors at The Jackson Laboratory. Oocytes were recovered from the ovaries of (C57BL/6J x SJL/J) F1 mice, hereafter referred to as B6SJL. Follicle development was stimulated in 21-day-old mice by treatment with 5 IU equine chorionic gonadotropin (eCG), and cumulus-enclosed oocyte complexes (COCs) were isolated 44–48 h later, as previously described [30]. For in vitro oocyte maturation, COCs were cultured in 10- x 35-mm Petri dishes in 3 ml Minimal Essential Medium (MEM) supplemented with 3 mg/ml crystallized bovine serum albumin (BSA; Sigma, St. Louis, MO). All cultures were maintained in MEM/BSA at 37°C in a modular incubation chamber (Billups-Rothenberg, Del Mar, CA) equilibrated with 5% CO2, 5% O2, and 90% N2. Culture duration is specified in different experiments. The stage of meiotic maturation was assessed at the end of culture; the surrounding cumulus cells were removed by repeatedly drawing the complexes in and out of a small-bore pipette, and the denuded oocytes were examined using a stereomicroscope. The oocytes collected following 2, 4, and 6 h of culture had all spontaneously resumed meiosis and undergone germinal vesicle breakdown. Oocytes identified as being at the MI-stage were cultured for 8 h. Progression to MII was indicated by the presence of the first polar body.
Parthenogenetic Activation and Fertilization
Mice were treated with eCG, followed by 5 IU of human chorionic gonadotropin (hCG) approximately 48 h later. MII-stage eggs were recovered from the oviduct 16 h after hCG treatment, and the surrounding cumulus cells were removed by a brief exposure to 0.1% hyaluronidase (Sigma) in MEM/BSA. The denuded eggs were activated by exposure to 8% ethanol in MEM/BSA for 8 min, then washed extensively and incubated at 37°C for an additional 4 and 6 h to evaluate pronucleus (PN) formation. A second group of MII eggs recovered from the oviduct was fertilized in vitro [35]. Development to the two-cell stage was evaluated 24 h following parthenogenetic activation or fertilization. When indicated, two-cell-stage embryos were exposed to 2 µg/ml colcemid (Sigma) in MEM/BSA overnight at 37°C to depolymerize microtubules and arrest blastomeres at M-phase. The embryos remained arrested at the two-cell stage with condensed chromosomes in a metaphase configuration.
Suppression of GVBD
Two methods were used to block the spontaneous resumption of meiosis by fully grown oocytes. On recovery from the ovary, COCs were cultured in MEM/BSA supplemented with 1.0 µM milrinone (Sigma), a specific phosphodiesterase inhibitor, or, alternatively, 150 µM roscovitine (Calbiochem, La Jolla, CA), a selective inhibitor of cyclin-dependent kinases (Cdks) that inhibits CDK1 (also known as p34cdc2) activity. The control group was incubated in medium alone. At the end of an 8-h culture, cumulus cells were removed and the oocytes collected for Western blot analysis. Oocytes cultured in milrinone or roscovitine retained an intact GV, confirming arrest at prophase I.
Phosphatase Treatment
In vivo matured MII-stage eggs were collected from the oviduct and denuded, as previously described. To remove phosphate groups, 1 IU protein phosphatase (Calbiochem) was added to the samples (300 eggs/sample) in phosphatase buffer (50 mM Tris-HCl, 5 mM DTT, 20 mM MgCl2, and 100 µg/ml BSA). The samples were incubated at 30°C for 30 min, and the reaction was terminated by the addition of an equal volume of 2x Laemmli buffer [36].
Western Blot Analysis
Samples of 300 denuded oocytes, representing approximately 9 µg of total protein [37], were rinsed and stored frozen (-20°C) in phosphate buffered saline (PBS) supplemented with 1 mg/ml BSA and a protease inhibitor cocktail (1 mM pefabloc together with 10 µg/ml leupeptin, aprotinin, and pepstatin). Prior to analysis, 2x Laemmli buffer was added, and the samples were heated to 100°C for 5 min. Proteins were separated by electrophoresis in 10% polyacrylamide gels containing 0.1% SDS, then transferred onto a hydrophobic polyvinylidene difluoride (PVDF) membrane (Amersham, Piscataway, NJ) for 1 h at 100 V. The membrane was blocked (2% BSA in PBS with 0.1% Tween-20) for 1 h at room temperature or overnight at 4°C. Incubation with an anti-PKC- antibody recognizing either the carboxyl (Santa Cruz Biotechnology, Santa Cruz, CA) or the amino terminal segment (Transduction Laboratories, Lexington, KY) of the protein was carried out for 2 h at room temperature or overnight at 4°C, respectively; this was followed by three (20-min) washes in PBS with 0.1% Tween-20. The membrane was then incubated with a peroxidase-conjugated secondary antibody (Jackson Immuno Research, West Grove, PA) for 1 h, washed, and processed using the ECL-Plus detection system (Amersham).
Immunofluorescence
The subcellular localization of PKC- was evaluated in oocytes during meiotic maturation (GV, MI, and MII) as well as in pronuclear stage zygotes and two-cell embryos. The primary antibody was directed against the carboxyl-terminal end of PKC-. All steps in the fixation and immunostaining procedure were carried out as previously described [29]. FITC conjugated anti-IgG (Jackson Immuno Research) was used as the secondary antibody and the DNA labeled with propidium iodide (Sigma). PKC- expression was assessed using a TCS-NT laser scanning confocal microscope equipped with an air-cooled argon ion laser system (Leica Microsystems).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Is Phosphorylated Coincident with the Resumption of Meiosis
Western blot analysis with an antibody directed against the carboxyl-terminus showed that PKC- was expressed as a doublet (74 and 76 kDa) in GV-stage mouse oocytes as previously reported [13, 16]. In addition, a smaller fragment of PKC-, approximately 47 kDa in size, was also strongly expressed during meiotic maturation (Fig. 1A). Immunostaining with an antibody directed against the N-terminus of the protein failed to detect this smaller fragment (Fig. 1B), indicating that it corresponds to the truncated catalytic fragment of PKC- at the carboxyl-terminal segment of the protein. Both the full-length protein and the catalytic fragment underwent a mobility shift, migrating more slowly, in samples from MI- and MII-stage oocytes (Fig. 1A). The mobility shift occurred within 2–4 h of initial culture (Fig. 2) and correlated with the resumption of meiosis. Blocking GVBD (Fig. 3) with a specific inhibitor of either phosphodiesterase (milrinone) or CDK1 (roscovitine) prevented the appearance of the slower-migrating form of PKC-. Phosphatase treatment of in vivo matured MII eggs prior to SDS-PAGE also eliminated the mobility shift and thus attributed it to the phosphorylation of PKC- (Fig. 3). Protein phosphorylation was not dependent on MAPK activity since oocytes treated with 50 µM of the MEK inhibitor (U0126) during an 8-h culture continued to express phosphorylated PKC- following spontaneous GVBD (data not shown). Phosphorylation of PKC- was observed whether oocytes were matured in vitro (Fig. 1) or in vivo after a superovulation regime (Fig. 3).
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Subcellular Localization of PKC- During Meiotic Maturation
Oocytes from control B6SJL mice express PKC- diffusely throughout the cytoplasm during prophase I arrest (Fig. 4A); this cytoplasmic staining pattern persisted for the duration of meiotic maturation. Additionally, an association of PKC- with the spindle microtubules was initially discerned in oocytes at prometaphase (Fig. 4B) and became more apparent during MI (Fig. 4C) as well as the anaphase I to telophase transition (Fig. 4D). By late telophase, bright PKC- staining was also evident in the midzone of the meiotic spindle (Fig. 4E). In addition, discrete foci of PKC- were observed in association with the chromosomes in approximately 20% (36/182 total) of mature MII eggs (Fig. 4F). However, the protein did not appear to localize with the second meiotic spindle apparatus. This subcellular distribution pattern of PKC- thus appears to be characteristic of typical meiotic maturation and is not exclusive to oocytes from strain LTXBO [29], which exhibit defects in the progression of meiosis.
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PKC- Expression Following Egg Activation
Both the expression and the subcellular distribution profiles of PKC- were altered on the completion of meiosis. Notably, PKC- becomes dephosphorylated (Fig. 5A) following fertilization or parthenogenetic activation. The unphosphorylated form of PKC- was initially detected in zygotes within approximately 2 h of activation (Fig. 5B). The protein was also unphosphorylated in two-cell embryos during interphase, at which time the expression of the truncated catalytic domain was markedly reduced. However, when two-cell-stage embryos were treated with colcemid to arrest the blastomeres at M-phase, PKC- again became phosphorylated. Thus PKC- is phosphorylated during both meiotic and mitotic M-phase but unphosphorylated during interphase. Analysis with confocal microscopy revealed that the protein persisted in the cytoplasm but also concentrated in the maternal and paternal pronuclei of early postfertilization zygotes (Fig. 6A). A similar labeling pattern was observed in parthenogenetically activated zygotes with a single female pronucleus (data not shown). Following the first mitotic division, PKC- also concentrated in the nuclei of both blastomeres (Fig. 6B).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, was characterized in fully grown mouse oocytes at various stages of meiotic maturation and following egg activation. Expression of the full-length protein as well as its truncated catalytic domain was evident, and both are phosphorylated coincident with the resumption of meiosis and M-phase entry. PKC- is diffusely distributed in the cytoplasm throughout maturation, and it associates with the spindle apparatus during the first meiotic division. In contrast, during MII, PKC- is observed in a speckled pattern associated with the chromosomes in some but not all mature eggs, while upon egg activation, the protein is dephosphorylated and accumulates in the nuclei of early embryos. This is the first evidence that PKC- is posttranslationally modified and subcellularly distributed in a cell cycle-dependent manner in oocytes and early embryos.
Members of the PKC family are single-chain polypeptides composed of a regulatory and a catalytic domain at the amino- and carboxyl-terminal halves, respectively, each of which contains functionally distinct conserved (C) regions. The regulatory (N-terminal) segment of cPKCs, consisting of a C1 and a C2 region, participates in autoinhibition and transient activation of the enzyme. C1 contains the pseudosubstrate domain and a duplicated cys-rich motif (Cys1 and Cys2) responsive to diacylglycerol (DAG) and to phorbol esters, while the C2 region binds calcium and acidic lipids [10]. Although PKC- shares homology with the cPKCs, it is grouped with the novel PKCs that are calcium independent. It lacks a conventional C2 domain, having instead a C2-like region at the N-terminal end of the molecule, and contains a pseudosubstrate sequence between the C2-like and C1 regions [38]. Interestingly, microinjection of recombinant C1 domain of PKC- stimulates insulin- but not progesterone-induced resumption of meiosis in Xenopus laevis oocytes, suggesting an interaction with the insulin/Ras signaling pathway [39]. In common with other PKCs, the catalytic (carboxyl-terminal) segment of PKC- contains C3 and C4 regions that bind ATP and act as the substrate-binding site, respectively. Occupation of the C4 substrate-binding site by the pseudosubstrate region is proposed to inhibit enzyme activity, while its detachment promotes activation and permits the binding and phosphorylation of target substrates [38].
Using an antibody specific for the carboxyl-terminal segment of PKC-, we detected the expression of the full-length (74–76 kDa) protein as well as a smaller fragment (47 kDa) in mouse oocytes during meiotic maturation. However, the smaller fragment was not discernible with an antibody specific for the N-terminal segment of the protein, indicating that it likely corresponds to the truncated catalytic fragment of PKC-. Moreover, although the catalytic fragment was strongly expressed in oocytes undergoing meiotic maturation (GV to MII stage), in two-cell-stage embryos the signal was very weak. This expression is unlikely to be a by-product of handling procedures since both oocyte and embryo samples were collected and stored in buffer containing a cocktail of protease inhibitors. A variable region (V) that separates the regulatory and catalytic segments of PKC- can be cleaved by caspase 3 to produce an active catalytic fragment that is no longer subject to regulatory domain control. In somatic cells an active catalytic fragment of PKC- is generated by proteolysis and is often associated with apoptosis [40, 41]. However, the specific activity or significance of the expression of the catalytic fragment in oocytes is currently not known.
In many protein kinase families, such as PKC, enzymatic activity is frequently regulated by protein phosphorylation. The current data demonstrate that PKC- is phosphorylated in oocytes coincident with the resumption of meiosis and remains phosphorylated during both MI and MII. It is not possible, however, to infer a causal relationship between GVBD and phosphorylation of PKC-. Rather, it seems that this posttranslational modification is cell cycle dependent, correlating with high M-phase factor (MPF) activity. Blocking GVBD, by inhibiting CDK1 activity, maintains PKC- in an unphosphorylated state. Likewise, PKC- is dephosphorylated shortly after fertilization, corresponding with the typical decrease in MPF activity. Conversely, entry into mitotic M-phase in early embryos resulted in phosphorylation of this protein, as observed in oocytes upon GVBD. Whether the different regulatory mechanisms that govern meiotic and mitotic M-phase entry influence the manner or rate of PKC- phosphorylation is not known. These precise changes in the phosphorylation status of PKC- suggest possible differences in activity and/or function. Though protein phosphorylation often serves as a molecular switch to regulate kinase activity, it is important to note that this regulation is complex. Whether kinase activity increases or decreases is dependent on the particular amino acid(s) that are phosphorphorylated and is often cell type and substrate specific [38]. PKC-, like other PKC isoforms, also undergoes autophosphorylation, but the functional role of this is still unresolved.
Activation of PKC is often but not exclusively correlated with translocation of the enzyme from the cytosol to the plasma membrane of various cell types [34]. In MII-stage eggs, stimulation with a phorbol ester (TPA), as well as fertilization, results in the translocation of PKC- and -ß to the plasma membrane within minutes [14, 24]. While it is possible that PKC- also accumulates in the plasma membrane following egg activation, it was not investigated since our objective was to characterize this protein during meiosis and after the zygote had reformed a pronucleus and cleaved. During meiotic maturation, PKC- was consistently localized in the cytosol and in addition was distinctly associated with the spindle apparatus during the first meiotic division. Hence, meiotic spindle localization is not unique to oocytes from strain LTXBO or necessarily associated with defects in the progression of meiosis [29] but rather is apparently characteristic of oocytes undergoing normal meiotic maturation. This distinctive distribution profile suggests that potential PKC- target substrates might include microtubules or microtubule-associated proteins. Recent reports indicate that PKC- phosphorylates ß-tubulin from shrimp (Penaeus japonicus) in the presence of the RACK anchoring proteins [42], whereas its overexpression and activation is associated with a decrease in polymerized tubulin and a possible role in oxidant-induced disruption of the microtubule cytoskeleton of intestinal epithelia [43, 44]. Intriguingly, while PKC- associated with the spindle apparatus throughout the first meiotic division, during MII arrest the protein did not localize with the spindle but was instead detected in a speckled pattern associated with the chromosomes in approximately 20% of mature eggs. This might reflect potential differences in the PKC anchoring protein profile or possible target substrates during MI and MII. Moreover, since PKC- was not observed associated with the chromosomes in all eggs, the association may be transitory or may occur only during a specific stage, the synchronous detection of which is precluded as maturing oocytes progress to MII at different times. Following egg activation PKC- undergoes another notable redistribution and accumulates in the pronuclei of zygotes and the nuclei of two-cell embryos. A previous elegant analysis of the PKC isoform profile in preimplantation mouse embryos [15] also reported an enrichment of PKC- in the nuclei of two-cell- as well as some four-cell-stage embryos, yet the protein was excluded from the nucleus at later stages of development. The dynamic distribution of PKC- to various subcelluar compartments suggests that its activity and/or function may vary during meiosis and in early embryos.
PKC- is reported to influence a broad range of functions, depending on the cell type [38, for review]. Though the present study provides a comprehensive analysis of PKC- expression and subcellular distribution during oocyte maturation and egg activation, its specific function(s) cannot be inferred. Attempts to suppress PKC- activity with an antagonist (rottlerin) noted to preferentially target novel PKCs such as PKC- were inconclusive, as exposure to even extremely low doses of this compound were toxic to oocytes (data not shown) and precluded its use. Recently, however, mice with a targeted null mutation for Prkcd, the gene encoding PKC- have been generated [45–47]. These mice show problems with B-cell autoreactivity and hyperproliferation and develop arteriosclerotic lesions but are reportedly fertile. Nevertheless, a detailed study of these mice may provide important clues to the role of PKC- in gametes. The current findings provide the first evidence that this kinase is posttranslationally modified and exhibits distinct cell cycle-dependent subcellular distribution profiles during meiotic maturation and in early stage embryos. Identifying potential target substrates for PKC- will be key in helping to define its function(s) in mammalian oocytes.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence. FAX: 610 925-8121; viveiros{at}vet.upenn.edu
3 Current address: University of Pennsylvania, School of Veterinary Medicine, Department of Animal Biology, Center for Animal Transgenesis and Germ Cell Research, 382 West Street Road, Kennett Square, PA 19348-1692
Received: 2 May 2003.
First decision: 30 May 2003.
Accepted: 17 June 2003.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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subspecies. Natl Acad Sci U S A 1992 89:10159-10163) isoform of PKC (PKC-) causes iNOS and NO upregulation: key mechanism for oxidant-induced carbonylation, nitration and disassembly of the microtubule cytoskeleton and hyperpermeability of barrier of intestinal epithelia. J Pharmacol Exp Ther 2003 305:482-494This article has been cited by other articles:
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  H. Dehghani and A. C Hahnel Expression profile of protein kinase C isozymes in preimplantation mouse development Reproduction, October 1, 2005; 130(4): 441 - 451. [Abstract] [Full Text] [PDF]
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H. Dehghani, C. Reith, and A. C Hahnel Subcellular localization of protein kinase C {delta} and {varepsilon} affects transcriptional and post-transcriptional processes in four-cell mouse embryos Reproduction, October 1, 2005; 130(4): 453 - 465. [Abstract] [Full Text] [PDF]
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Z.-Y. Zheng, Q.-Z. Li, D.-Y. Chen, H. Schatten, and Q.-Y. Sun Translocation of phospho-protein kinase Cs implies their roles in meiotic-spindle organization, polar-body emission and nuclear activity in mouse eggs Reproduction, February 1, 2005; 129(2): 229 - 234. [Abstract] [Full Text] [PDF]
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 M. M. Viveiros, M. O'Brien, and J. J. Eppig Protein Kinase C Activity Regulates the Onset of Anaphase I in Mouse Oocytes Biol Reprod, November 1, 2004; 71(5): 1525 - 1532. [Abstract] [Full Text] [PDF]
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 G. Halet, R. Tunwell, S. J. Parkinson, and J. Carroll Conventional PKCs regulate the temporal pattern of Ca2+ oscillations at fertilization in mouse eggs J. Cell Biol., March 29, 2004; 164(7): 1033 - 1044. [Abstract] [Full Text] [PDF]
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H.-Y. Fan and Q.-Y. Sun Involvement of Mitogen-Activated Protein Kinase Cascade During Oocyte Maturation and Fertilization in Mammals Biol Reprod, March 1, 2004; 70(3): 535 - 547. [Abstract] [Full Text] [PDF]
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