Biol Reprod Keystone Symposia Conference on Frontiers in Reproductive Biology & Regulation of Fertility.
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BOR - Papers in Press, published online ahead of print June 30, 2004.
Biol Reprod 2004, 10.1095/biolreprod.104.031344
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BIOLOGY OF REPRODUCTION 71, 1525–1532 (2004)
DOI: 10.1095/biolreprod.104.031344
© 2004 by the Society for the Study of Reproduction, Inc.

Gamete Biology

Protein Kinase C Activity Regulates the Onset of Anaphase I in Mouse Oocytes1

Maria M. Viveiros2,3, Marilyn O'Brien4, and John J. Eppig4

Center for Animal Transgenesis and Germ Cell Research,3 Department of Animal Biology, School of Veterinary Medicine, University of Pennsylvania, Kennett Square, Pennsylvania 19348 The Jackson Laboratory,4 Bar Harbor, Maine 04609


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The metaphase-to-anaphase I transition is a key step in the completion of meiosis I. In mouse oocytes, competence to exit metaphase I (MI) is developmentally regulated and typically not acquired until the preovulatory stage. The possible role of protein kinase C (PKC) in regulating this critical transition was assessed in both normal oocytes isolated from small antral follicles (18-day-old B6SJLF1 mice), which have not yet developed the capacity to progress to metaphase II (MII), and also oocytes defective in their ability to exit MI despite development to the preovulatory stage (24-day-old CX8 recombinant inbred strains). In both systems, transient suppression of endogenous PKC activity by treatment with a PKC-specific inhibitor, bisindolylmaleimide I (BIM), promoted the onset of anaphase I in a dose-dependent manner, while activation of PKC with the phorbol ester TPA blocked progression to MII. Following a 2-h incubation with BIM, the majority of oocytes progressed to, and arrested at, MII. The resulting MII oocytes were fertilizable in vitro, showing similar cleavage and blastocyst development rates between BIM treated and untreated controls. Transferred embryos resulted in the development of pups to term in both groups. These data demonstrate that PKC plays an important role in regulating the onset of anaphase I in mouse oocytes. Moreover, it is concluded that oocytes isolated from small antral follicles become blocked at MI due to a PKC-mediated signal, suggesting that acquisition of competence to complete meiosis I involves, in part, the control of PKC activity. Similarly, failure to regulate PKC activity at the preovulatory stage likely promotes arrest at MI.

anaphase, fertilization, gamete biology, kinases, meiosis, metaphase I, oocyte development, protein kinase C


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Mammalian oocytes are arrested at prophase of meiosis I during growth, and progressively acquire the capacity to resume and complete meiosis in a stepwise manner that is synchronous with the stage of ovarian follicle development. While small immature oocytes (<60 µm in diameter) recovered from preantral follicles cannot spontaneously resume meiosis when placed in culture, partially grown oocytes from early antral follicles do undergo germinal vesicle breakdown (GVBD). Nevertheless, the majority of these oocytes commonly arrest at metaphase I (MI), failing to undergo the metaphase to anaphase I transition despite extended culture and are therefore considered only partially competent to complete meiosis [1–3]. The molecular mechanisms underlying this block at MI are not known. In general, it is not until the preovulatory stage when full growth is attained (approximately 80 µm) that oocytes become fully competent to complete meiosis I and enter metaphase II (MII).

Entry into anaphase I is dependent on two key processes, 1) the precise regulation of M-phase promoting factor (MPF) activity and 2) appropriate assembly of the first meiotic spindle to which the chromosomes form stable attachments. MPF is a central cell cycle regulator in all eukaryotic cells, comprised of a catalytic p34cdc2 kinase (CDK1) and cyclin B (CYB) regulatory subunit [4]. In mouse oocytes, MPF is activated just before GVBD; its activity continues to increase during meiotic maturation and reaches a plateau toward the end of MI. However, during the MI to MII transition, there is a notable, transient, decrease in MPF activity, as cyclin B is degraded in a ubiquitin-dependent manner [5–8]. Regulation of MPF activity by specific CDK1-interacting proteins is also essential, as recent studies demonstrate that both male and female gametes arrest at the first meiotic metaphase in mice lacking CKS2, a mammalian homologue of the yeast CDK1-binding protein SUC1 [9]. In fully grown oocytes, entry into anaphase I is purportedly initiated by the final alignment of homologous chromosomes [10]. Studies demonstrate that appropriate meiotic spindle assembly and chromosome attachment [11, 12], as well as spindle migration to the oocyte cortex [13], are essential to entry into anaphase I. However, the key molecules and mechanisms that regulate the metaphase to anaphase I transition in mammalian oocytes are not known.

PKC-mediated regulation of cell-cycle transitions has been reported in somatic cells [14, 15]. In mammals, the PKC family is comprised of 10 related isoforms, classified as either conventional (PKC{alpha}, -ßI, -ßII, and -{gamma}), novel (PKC{delta}, -{theta}, -{epsilon}, and -{eta}) or atypical (PKC{lambda} and -{zeta}) based on sequence homology as well as activator and cofactor requirements [16, for review]. Several PKC proteins (PKC{alpha}, -{gamma}, -{delta}, -{lambda}, and -{zeta}) are expressed in normal mouse oocytes during meiotic maturation [17–20], and at least one isoform, PKC{delta}, is phosphorylated upon the resumption of meiosis and associates with the meiotic spindle until the completion of MI [21], suggesting a possible function during the critical transition to MII. Moreover, earlier studies demonstrated that treatment with PKC activators after GVBD can block or delay extrusion of the first polar body in mouse oocytes [22, 23].

A delay or block at MI has been well documented in oocytes from LT/Sv and related mouse strains, such as LTXBO (hereafter referred to as LT oocytes) [24]. Although preovulatory oocytes from LT mice resume meiosis normally, the oocytes remain at MI for an extended period with sustained MPF activity [6], resulting in significant numbers being ovulated as primary oocytes at MI [25, 26]. Disruption in the transition to MII is also associated with parthenogenetic activation in this strain as a high percentage of oocytes, which eventually undergo a delayed MI exit, spontaneously activate following extrusion of the first polar body [24, 27]. The prolonged MI stage in LT oocytes is attributed to a delay in the onset of anaphase I [28, 29], and recent studies indicate a role for PKC in this transition [27]. Preovulatory oocytes from LT mice show an increase in endogenous PKC activity during meiotic maturation, which is highest at the late MI stage. Importantly, suppression of PKC activity promoted entry into anaphase by late MI-stage oocytes, with ensuing progression to and maintenance of MII arrest. In contrast, stimulation of PKC activity further delayed anaphase onset, prolonging the duration of MI, and increased the incidence of parthenogenetic activation [27]. These data suggest that PKC likely plays an important function in regulating MI exit in mouse oocytes.

Based on observations using LT oocytes, we have asked whether the developmental processes whereby normal oocytes develop full meiotic competence involves their ability to regulate PKC. This key question regarding normal oocyte development is addressed using oocytes isolated from early antral follicles, which do not typically mature to MII. In addition, we have used CX8 recombinant inbred mice as a model of abnormal progression of meiosis in preovulatory-stage oocytes to assess the consequences of the failure to acquire competence to regulate PKC activity. CX8 recombinant inbred strains were developed using the parental strains of LT mice (BALB/c and C58/J). Fully grown oocytes from the CX8 strains used in this study arrest at MI but, unlike LT, show a low incidence of parthenogenetic activation, allowing an assessment of PKC function on the metaphase to anaphase I transition without the confounding effects of parthenogenetic activation.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Mouse Strains

Partially competent oocytes were recovered from small antral follicles of 18-day-old (C57BL/6J x SJL/J) F1 mice, in future referred to as B6SJLF1. In addition, fully grown oocytes were recovered from 24-day-old CX8 mice; five different strains were used, CX8-3, -4, -5A, -9, and -15. All CX8 recombinant inbred (RI) strains were derived from BALB/c and C58/J, and originally produced at the Jackson Laboratory. Although otherwise normal, matings between CX8 mice are subfertile, with significantly reduced litter sizes (averaging only 1–4 pups per litter). Breeding of CX8 males to normal B6SJLF1 female mice results in normal litters of 8–10 pups (unpublished data). This suggests that poor CX8 fertility is likely attributed to the MI-arrest phenotype of oocytes from CX8 females [24]. Thus, both partially grown oocytes from normal B6SJLF1 mice [3] and fully grown oocytes from the five specific CX8 RI strains [24] spontaneously resume meiosis when released from the follicular environment; however, the majority fail to progress beyond MI even after prolonged culture. Oocytes recovered from 24-day-old B6SJLF1 mice were used as a control group; by this stage, the oocytes have reached full growth and undergo normal meiotic maturation with the majority progressing to MII within 12 h of culture. All procedures described were reviewed and approved by the Animal Care and Use Committees at the University of Pennsylvania and The Jackson Laboratory and were performed in accordance with the Principles for the Care and Use of Laboratory Animals.

Oocyte Isolation and Culture Conditions

As indicated, partially competent oocytes were recovered from the ovaries of B6SJLF1 females on Day 18 of postnatal development; the mice received no hormone stimulation to ensure that only oocytes from early antral-stage follicles were collected. For recovery of fully grown oocytes from the CX8 strains, 22-day-old mice were injected with 5 IU equine chorionic gonadotrophin (eCG) to stimulate preovulatory follicle development, and cumulus-enclosed oocyte complexes (COCs) were isolated 44–48 h later. Control 22-day-old B6SJLF1 mice were similarly treated with eCG before recovery of fully grown oocytes.

The COCs were cultured for 16–17 h in minimal essential medium (MEM) supplemented with 3 mg/ml crystallized bovine serum albumin (BSA; Sigma, St. Louis, MO) and 1 mg/ml Fetuin or 5% fetal calf serum (FCS) to prevent hardening of the zona pellucida. All cultures were maintained at 37°C in a modular incubation chamber (Billups-Rothenberg, Del March, CA) equilibrated with 5% CO2, 5% O2, and 90% N2. At the end of culture, the cumulus cells were removed by repeatedly drawing the complexes in and out of a small bore pipette, and denuded oocytes were examined using a stereomicroscope to determine the stage of meiotic maturation. Extrusion of the first polar body indicated progression past the metaphase to anaphase I transition.

Modulation of PKC Activity in MI-Stage Oocytes

The influence of PKC activity on anaphase I onset was tested. After removal of cumulus cells, oocytes that did not extrude a first polar body during the 16- to 17-h maturation period were cultured for an additional 2 h in media alone or media supplemented with increasing concentrations (0.1, 0.5, and 1.0 µM) of bisindolylmaleimide I (BIM) (Calbiochem, La Jolla, CA), a PKC-specific inhibitor; BIM is membrane permeant and acts as a competitive inhibitor of the ATP binding site of PKC proteins with a high selectivity for PKC{alpha}, -ß, -{gamma}, -{delta}, and -{epsilon} [30]. This treatment has been shown to significantly lower endogenous PKC activity in mouse oocytes [27]. In a separate experiment, oocytes were also treated with 0.5 nM of the PKC agonist TPA (Calbiochem), for the initial 30 min only, then washed extensively and cultured in media alone for the remaining 1.5 h. At the end of culture, the oocytes were evaluated to determine the incidence of polar body emission and fixed for subsequent evaluation of chromosome as well as meiotic spindle configurations.

In Vitro Fertilization

The capacity to undergo fertilization and preimplantation development was determined for oocytes that matured to MII in response to BIM treatment and compared with untreated controls. MII eggs were fertilized in vitro with capacitated sperm recovered from sexually mature B6SJLF1 males as previously described [31]. Development to the two-cell and blastocyst stages were evaluated 24 h and 5 days postfertilization, respectively.

As indicated, oocytes from the CX8 strains used in this study normally show a low incidence of parthenogenetic activation; however, because these mice were originally produced using the parental strains of LT mice (BALB/c and C58/J), it was important to confirm that embryos were obtained as a result of successful fertilization and not parthenogenesis. To test this, CX8 oocytes that reached MII in response to BIM treatment were fertilized with sperm from B5/EGFP mice. This transgenic strain carries an enhanced green fluorescent protein (EGFP) tag driven by the ß-actin promoter and expresses the GFP marker in all tissues [32]. Thus, constitutive expression of GFP by blastocysts was used as an indicator of successful fertilization. In addition, two-cell-stage embryos were transferred to recipient females to assess whether embryonic development could continue to term.

Immunocytochemistry

Meiotic spindle and chromosome configurations were evaluated in BIM and TPA-treated, as well as untreated control, oocytes. The samples were fixed with 4% paraformaldehyde in phosphate buffered saline (PBS) supplemented with 1 mg/ml BSA, permeabilized in PBS with 0.1% Triton X-100 for 10 min, then transferred to block solution (PBS with 10% serum). All steps in the immunostaining procedure were carried out in block solution at 37°C. A 1-h incubation with 1 µg/ml anti-ß-tubulin (Sigma) was followed by several washes, then incubation with 3 µg/ml of a FITC-conjugated sheep anti-mouse IgG (Jackson Immuno Research Laboratories, West Grove, PA). The oocytes were subsequently transferred to propidium iodide (1 µg/ml in PBS) for 10 min, washed briefly, and mounted onto poly-L-lysine-coated slides. Negative control samples were evaluated in which the primary antibody was omitted. Fluorescence was assessed using a TCS-NT laser scanning confocal microscope equipped with an air-cooled argon ion laser system (Leica Microsystems).

Statistical Analysis

All data are presented as mean percentages (±SEM) of a minimum of four independent experimental replicates. For all maturation and fertilization experiments, approximately 50 oocytes were used per group in each replicate. For evaluation of the differences between groups, all percentages were subjected to arcsine transformation. The transformed data were then analyzed by ANOVA and the means compared using a Fisher protected least-significant difference post hoc test using Statview for Macintosh (Abacus Concepts, Inc., Berkeley, CA). Significance was assigned at P < 0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Partially Grown B6SJL and Fully Grown CX8 Oocytes Arrest at MI

Meiotic maturation was assessed at the end of a 16- to 17-h culture period; at this time, approximately 20% of the partially competent oocytes collected from 18-day-old B6SJLF1 females remained arrested at the GV stage and less than 10% progressed to MII (10% of the oocytes isolated from small antral follicles were, therefore, actually fully competent). The majority of partially competent oocytes (approximately 70%) resumed meiosis, but did not extrude the first polar body (Fig. 1A); analysis of meiotic spindle and chromosome configurations (n = 130 total) demonstrated that approximately 21.5% of ooocytes displayed a prometaphase rosette configuration, while the majority (78.5%) typically exhibited condensed chromosomes aligned on a bipolar first meiotic spindle (Fig. 1B), confirming their entry into and arrest at MI. Evaluation of fully grown oocytes from CX8 strains also demonstrated a block at MI after a 16- to 17-h culture. While all CX8 oocytes resumed meiosis, less than 15% progressed to MII (Fig. 1C). The rates of meiotic maturation shown represent the mean for all five strains evaluated (CX8-3, -4, -5A, -9, and -15), as no significant difference was noted among strains. The CX8 oocytes entered MI and assembled a barrel-shaped MI spindle with condensed chromosomes congressed to the metaphase plate (data not shown); this was similar to the partially grown B6SJLF1 group. In contrast, more than 80% of fully grown oocytes recovered from B6SJLF1 mice on Day 24 progressed to MII (Fig. 1C).



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  		FIG. 1. Partially competent oocytes recovered from unprimed 18-day-old B6SJLF1 mice and fully grown CX8 oocytes from 24-day-old eCG-treated mice resume meiosis and assemble the first meiotic spindle, yet arrest at MI. Progression of meiosis (mean ± SEM) was evaluated in oocytes that were cultured cumulus enclosed for 16–17 h. The majority of partially grown B6SJLF1 oocytes entered metaphase I (MI) and successfully assembled the first meiotic spindle, but did not progress to MII (A, B). Condensed chromosomes are shown in red and spindle microtubules in green. Similarly, although fully grown CX8 oocytes resumed meiosis, almost all arrested at MI (C). In contrast, the majority of fully grown Day 24 B6SJLF1 oocytes progressed to MII under the same culture conditions (C). For the CX8 group, the rates of meiotic maturation represent the mean for five strains evaluated, CX8-3, -4, -5A, -9, and -15. An * and different superscripts indicate a statistical difference at P < 0.05. B, original magnification x60

PKC Activity Influences the Onset of Anaphase I

Suppression of endogenous PKC activity promotes the onset of anaphase in MI-stage oocytes from strain LT mice [27]. A similar approach to promote progression to MII was used with genetically and developmentally normal partially competent (Day 18) oocytes from B6SJLF1 mice. Following a 16- to 17-h maturation period, companion granulosa cells were removed and the MI-stage oocytes cultured for 2 h with increasing concentrations of BIM, to suppress PKC activity. By the end of culture, approximately 40% of the untreated (control) cumulus-free oocytes had extruded the first polar body. Interestingly, BIM-treatment further increased (P < 0.05) the incidence of first polar body emission, in a dose-dependent manner (Fig. 2A). In contrast, the incidence of MI arrest was higher (P < 0.05) when oocytes were briefly treated with the PKC agonist TPA (Fig. 2B). Analysis of microtubule and chromosome configurations, after the 2-h culture, demonstrated that 86.5% of all remaining MI-arrested oocytes (n = 110) in the control group maintained condensed chromosomes aligned on an intact meiotic spindle (Fig. 3A). In the TPA-treated group, however, the spindle microtubule configurations were disrupted in 67.8% of all oocytes (n = 137) assessed; ß-tubulin expression was less pronounced and the spindle structure was disorganized, while the chromosomes remained condensed despite poor alignment of the meiotic spindle (Fig. 3B). In contrast, 84.3% of all oocytes (n = 165) treated with the PKC inhibitor BIM underwent the metaphase to anaphase I transition, with most exhibiting a telophase configuration at 2 h (Fig. 3C). With additional time in culture (4 h total), the BIM-treated oocytes had reformed the second meiotic spindle and arrested at MII (Fig. 3D).



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  		FIG. 2. Modulation of PKC activity influences the onset of anaphase I in partially competent B6SJLF1 oocytes recovered on Day 18 of postnatal development. Cumulus-enclosed oocytes were matured for 16–17 h, then denuded and cultured in media supplemented with increasing concentrations of BIM for an additional 2 h to determine the incidence (mean ± SEM) of first polar body extrusion (A). Treatment with 0.5 nM TPA, for the initial 30 min of culture only, reduced the percent of ooctyes that extruded a polar body after the 2-h culture (B). Different superscripts indicate a statistical difference at P < 0.05



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  		FIG. 3. Analysis of chromosome and microtubule configurations in partially competent B6SJLF1 oocytes following modulation of PKC activity demonstrated that untreated control oocytes (A) remained arrested at MI, while TPA treatment (B) significantly disrupted the meiotic spindle microtubule organization. In sharp contrast, BIM-treated oocytes (C, D) underwent the metaphase to anaphase I transition and segregation of homologous chromosomes, with the majority of oocytes exhibiting telophase configurations at the end of the 2-h culture (C). With further time in culture, the oocytes extruded the first polar body (arrow), assembled a second meiotic spindle, and arrested at MII (D). Chromosomes are shown in red and microtubules in green. Original magnification, (A, B) x60, (C, D) x40

The capacity to enter anaphase I in response to BIM was also tested in fully grown oocytes recovered from mice of CX8 strains. Cumulus-enclosed oocytes were matured for 16–17 h, as previously described, then denuded and cultured for 2 h in media supplemented with 1 µM BIM. Oocytes from five different CX8 strains (CX8-3, -4, -5A, -9, and -15) were evaluated and all responded to PKC suppression with an increase (P < 0.05) in the incidence of polar body emission (Fig. 4). Oocytes from CX8-3, -5A, and -9 mice showed the highest incidence of polar body emission in response to BIM, while those from CX8-15 showed a more limited, yet still significant, response. In summary, approximately 60% of BIM-treated CX8 oocytes progressed to MII, compared with just 10%–15% in the control group. Collectively, these results indicate that normal partially competent oocytes (Day 18, B6SJLF1) as well as fully grown CX8 oocytes, which arrest at MI, are able to enter anaphase I and progress to MII in response to the suppression of PKC activity.



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  		FIG. 4. Suppression of PKC activity promotes the onset of anaphase I in fully grown CX8 oocytes. Cumulus-enclosed oocytes were recovered from eCG primed mice of different CX8 strains (CX8-3, -4, -5A, -9, and -15) on Day 24 of postnatal development and matured for 16–17 h. The oocytes were then denuded and those at the MI-stage cultured in media supplemented with 1 µM BIM for 2 h to determine the incidence (mean ± SEM) of first polar body extrusion. An * indicates a significant (P < 0.05) difference between the control and BIM-treated group

In Vitro Fertilization and Preimplantation Development

The next series of experiments determined whether the oocytes that progressed to MII in response to BIM could be fertilized in vitro and undergo early embryonic development. Although generally very few in numbers (less than 10%), the oocytes recovered from 18-day-old B6SJLF1 mice that reached the MII stage during the initial 16- to 17-h culture were used as a control group showing competence to complete MI. Approximately 45% of the fertilized 17-h MII eggs cleaved to the two-cell stage, of which 45% subsequently developed into blastocysts in vitro (Fig. 5). Thus, only about 20% of all 17-h MII control oocytes were competent to reach the blastocyst stage. In comparison, partially competent MI-stage oocytes at the end of the initial 17-h maturation, which progressed to MII in either medium alone or in response to BIM, exhibited lower (P < 0.05) cleavage and blastocyst development rates (approximately 30%–35%; Fig. 5) relative to the 17-h MII controls. Hence, the developmental potential of partially competent oocytes that fail to progress to MII during the initial 17-h culture was less than that for fully competent oocytes. Nevertheless, the developmental potential of the oocytes originally arrested at MI was similar between the BIM-treated and untreated group, indicating no overtly negative effects of transient culture with BIM. In fact, because treatment with the PKC inhibitor increased the number of oocytes that reached MII, the total number of blastocysts obtained was significantly (P < 0.05) higher in the BIM-treated group (Fig. 5).



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  		FIG. 5. Partially grown (Day 18) B6SJLF1 oocytes that mature to MII in response to BIM can be fertilized. Cumulus-enclosed oocytes were matured for 16–17 h, then denuded and cultured in media supplemented with 1 µM BIM for 2 h. Both the untreated control (white bars) and BIM-treated (black bars) oocytes that progressed to MII were then fertilized in vitro with capacitated sperm recovered from sexually mature B6SJLF1 male mice. The rates (mean ± SEM) of cleavage to the two-cell stage and blastocyst development were evaluated 24 h and 5 days postfertilization, respectively. Partially grown oocytes that progressed to MII during the initial 17-h maturation period (hatched bars) were also fertilized. Different superscripts denote a statistical difference at P < 0.05

The developmental potential of fully grown oocytes recovered from 24-day-old mice was also evaluated. As indicated, the majority of oocytes from Day 24 B6SJLF1 mice matured to MII within the initial 16- to 17-h culture. When fertilized, approximately 65% of these in vitro matured eggs cleaved to the two-cell stage and more than 85% of the two-cell embryos successfully developed into blastocysts (Fig. 6). There was no difference in cleavage and blastocyst development rates when these MII oocytes were treated with 1 µM BIM before fertilization (data not shown). Interestingly, fully grown oocytes from CX8 strains that reached MII in response to BIM or medium alone showed similar rates of cleavage (approximately 65%) but lower (P < 0.05) potential to form a blastocyst, compared with oocytes from B6SJLF1 mice (65% versus 90%). Importantly, treatment with BIM did not influence the developmental potential of CX8 oocytes; both cleavage and blastocyst formation rates were similar between BIM-treated and untreated controls (Fig. 6). However, the total number of blastocysts formed was higher (P < 0.05) in the BIM-treated group due to the larger number of oocytes that mature to MII. Cleavage and blastocyst formation rates represent the mean for the three strains, CX8-3, -5A, and -9, evaluated; no significant difference was noted among strains.



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  		FIG. 6. Fully grown oocytes recovered from 24-day-old eCG primed CX8 mice that progress to MII in response to BIM are fertilizable. Cumulus-enclosed oocytes were matured for 16–17 h, then denuded and cultured in media supplemented with 1 µM BIM for 2 h. Control (white bars) and BIM-treated (black bars) oocytes that entered MII were subsequently fertilized in vitro with capacitated sperm recovered from sexually mature B6SJLF1 male mice. The rates (mean ± SEM) of cleavage to the two-cell stage and blastocyst development were evaluated 24 h and 5 days postfertilization, respectively. Fully grown oocytes from eCG primed B6SJLF1 mice, which progressed to MII during the initial 17-h culture (hatched bars) were also fertilized. In the CX8 group, cleavage and blastocyst formation rates represent the mean for three strains evaluated, CX8-3, -5A, and -1;9. Different superscripts denote statistical differences at P < 0.05

To confirm that the blastocysts produced resulted from successful fertilization and not parthenogenesis, CX8 oocytes that progressed to MII in response to BIM were also fertilized in vitro using sperm from B5/EGFP male mice [32]. Our analysis demonstrated that approximately 85% of blastocysts derived from BIM-matured CX8 eggs inseminated with sperm from B5/EGFP mice expressed GFP (Fig. 7, A and B) and were therefore successfully fertilized. The rate of parthenogenetic activation (approximately 10%– 15%) did not differ between the BIM-treated and untreated groups. Moreover, transfer of two-cell embryos to recipient females resulted in live pups that also expressed the GFP marker (Fig. 7C), although it should be noted that the number of mice born from the BIM-treated group was lower (6.25%) than the untreated controls (12.5%). In both groups, the mice developed apparently normally.



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  		FIG. 7. Embryos derived from CX8 oocytes fertilized with sperm from B5/EGFP mice express green fluorescent protein (GFP). To assess possible parthenogenetic activation, oocytes from CX8 mice, which progressed to MII in response to BIM, were fertilized with sperm from mice (B5/EGFP) that ubiquitously express a GFP marker. Analysis of blastocyst-stage embryos under a dissecting microscope (A) and with blue-light excitation (B) confirmed that most blastocysts express GPF and were therefore successfully fertilized. Transfer of the two-cell stage embryos to recipient females resulted in the development of pups to term that also expressed the GFP marker (C). A, B) Original magnification x6.5


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
We demonstrate that PKC plays a significant role in the regulation of anaphase I onset in preovulatory-stage oocytes from CX8 strains that arrest at MI and, importantly, also in developmentally normal oocytes, which have not yet reached full meiotic competence. In both systems, BIM treatment to suppress PKC activity successfully promoted entry into anaphase I. The oocytes subsequently assembled a second meiotic spindle, arrested at MII, and were fertilizable in vitro. PKC-mediated regulation of anaphase I onset is, therefore, not exclusively associated with the meiotic defects that promote MI arrest in oocytes from LT and related CX8 stains; instead, it is a general mechanism functioning in mouse oocytes. The current findings also indicate that, despite typically arresting at MI, partially competent oocytes from normal mice as well as fully grown oocytes from the CX8 strains are functionally able to undergo the metaphase to anaphase I transition and arrest at MII. Thus, these oocytes presumably express the necessary factors required to drive progression to MII, the regulation of which is crucial. Although the mechanisms that underlie arrest at MI remain to be determined, we provide evidence that the PKC pathway serves an important role, likely in negatively regulating the activity of one or more factors required to initiate anaphase I.

Activation of PKC activity with phorbol esters blocks or delays emission of the first polar body in mouse oocytes that have resumed meiosis [22, 23], suggesting that high PKC activity is associated with the maintenance of M phase. Consistent with this hypothesis, we demonstrated that oocytes recovered from LT mice, known to arrest at MI, express increasing endogenous PKC activity during meiotic maturation, the highest levels of which are detected later in MI. Notably, suppression of PKC activity in LT oocytes promoted MI exit and progression to MII [27], showing a correlation between lower PKC activity and MI exit. However, in addition to a prolonged MI, LT oocytes exhibit a second meiotic abnormality, as a significant number undergo spontaneous activation immediately following delayed extrusion of the first polar body. Thus, parthenogenetic activation and MI arrest are linked in this strain. To assess the potential role of PKC in MI arrest without the activation defect, fully grown oocytes were used from related CX8 stains [24] that are known to arrest at MI, but in which only a small percentage (10%–15%) undergo parthenogenetic activation. Our results demonstrate that, in all five CX8 strains tested, suppression of endogenous PKC activity promoted the onset of anaphase I, with the majority of oocytes subsequently forming a second meiotic spindle and arresting at MII. Thus, PKC activity regulates MI exit in oocytes in which arrest at MI is not linked to an activation defect.

Since the CX8 strains were originally produced using the progenitors of LT mice, it was important to determine whether PKC-mediated regulation of anaphase I onset might be unique to the inherited MI arrest phenotype characteristic of these related strains. Partially competent oocytes recovered from normal stains, which typically arrest at MI, provided a means to address this issue. Our results indicate that BIM treatment to suppress PKC activity in these oocytes also promotes entry into anaphase I, suggesting that a PKC-mediated signal functions generally in mouse oocytes to regulate MI exit. Partially competent oocytes show normal functioning of various parameters similar to oocytes that have reached full size, including the spontaneous resumption of meiosis in culture, followed by a gradual increase in MPF activity [6] and phosphorylation of MAPK [33]. Together with an earlier study [34], the current data also provide evidence of an assembled first meiotic spindle onto which the condensed chromosomes congress. Nonetheless, while entry into and maintenance of MI are apparently normal at this stage of development, the majority of oocytes have not yet acquired the capacity to undergo the metaphase to anaphase I transition and thus complete the first meiotic division. Interestingly, release from MI was previously shown to occur upon oocyte activation [3] or culture with the sterol FF-MAS [35], indicating that arrest at MI by partially competent oocytes is unlikely to be attributed to an insufficiency but rather to the appropriate regulation of factor(s) necessary to drive entry into anaphase. The current results confirm that MI exit is possible and further i) demonstrate successful entry and arrest at MII by partially competent oocytes and ii) provides evidence that PKC plays an important role in regulating one or more factor(s) crucial for anaphase I onset.

Importantly, PKC-mediated regulation of anaphase I in both partially competent oocytes from normal B6SJLF1 mice as well as from fully grown oocytes from CX8 and related LT strains [27] suggest that the mechanism of MI arrest may be similar in the two models. Previous studies have shown that MI arrest in both LT and partially competent oocytes correlates with high MPF activity and elevated cyclin B levels, attributed in part to restricted cyclin B degradation [6]. Suppression of PKC activity in LT oocytes was shown to lower MPF activity and thereby overcome the block at MI [27]. In somatic cells, PKC modulates MPF activity by regulating the expression of specific CDK inhibitor proteins, p21walf1/cip1 and p27kip1 [15, 36]; whether PKC functions similarly in oocytes remains to be determined. Additionally, it is plausible that PKC might interact with the ubiquitin-targeting machinery, which is responsible for eventual cyclin B degradation [7]. The effect of PKC activity on spindle microtubules may also be significant since cyclin B degradation necessitates the presence of an intact spindle [37]. PKC agonists promote the disassembly of spindle microtubules in mouse oocytes at MII [38], and we demonstrate that even a brief treatment with TPA at very low concentrations can significantly disrupt the organization of the first meiotic spindle and induce arrest at MI. Meiotic spindle damage in mouse oocytes promotes the recruitment of the mitotic checkpoint protein MAD2 to the unattached kinetochores and induces arrest at MI with high MPF activity [12]. MAD2 purportedly inhibits the anaphase-promoting complex, which is essential for the ubiquitination of proteins, such as cyclin B, to target it for degradation [39]. Thus, a further assessment of how an increase or decrease in PKC activity influences spindle structure is needed.

To determine the mechanism(s) by which PKC activity influences anaphase onset will necessitate an assessment of the specific PKC isoforms and potential target substrates that are expressed in mouse oocytes. Ten related PKC isoforms comprise the mammalian PKC family, several of which (PKC{alpha}, -{gamma}, -{delta}, -{lambda}, and -{zeta}) are expressed in mouse oocytes [17–20]. Detailed analysis of the expression of PKC{delta} during meiotic maturation demonstrates that the protein becomes phosphorylated upon the resumption of meiosis and, interestingly, associates with the spindle during the first meiotic division in oocytes from normal mice [21] as well as the LT strain [27]. PKC{delta} also associates with the second meiotic spindle specifically during anaphase II following egg activation [40]. Therefore, the subcellular localization of this kinase suggests a possible role in anaphase onset. Studies are needed to determine whether the expression and/or activity of PKC{delta} or possibly other PKC isoforms function to regulate MI exit. Moreover, it is important to note that, while BIM treatment promoted the onset of anaphase I in the majority of partially competent oocytes, those from the CX8-15 strain demonstrated a more limited response. Thus, in addition to a PKC-mediated signal(s), other possible factors such as calcium/calmodulin kinase II also likely play a role in the metaphase to anaphase I transition [41, 42].

The final stages of oocyte growth in antral follicles are essential not only for the acquisition of full meiotic competence, but also for oocytes to acquire the ability to cleave and form an embryo upon fertilization, commonly referred to as developmental competence [3, 43, 44]. Although not necessarily interdependent, meiotic and developmental competence seemingly develop together, as fully grown oocytes able to complete meiotic maturation exhibit the highest developmental potential. The sterol FF-MAS increased the frequency of partially competent oocytes maturing to MII and also improved their preimplantation developmental competence [35]. Nonetheless, meiotic and developmental capacities are separable. In the current study, while BIM treatment to suppress PKC activity promoted progression to MII in partially grown oocytes, no difference in developmental potential was noted and the resultant eggs showed lower developmental potential when fertilized compared with fully grown oocytes. In addition, similar rates of blastocyst formation between BIM-treated and control oocytes suggested that lowering PKC activity had no overtly negative effects on subsequent fertilization. These findings indicate that PKC specifically targets the mechanisms involved in the regulation of MI exit and not the processes affecting subsequent preimplantation development.

Suppression of PKC activity also promoted CX8 oocyte maturation to MII, without affecting developmental potential. Our analyses indicate that the subfertility of CX8 strains is primarily attributable to the MI arrest phenotype. CX8 oocytes that progressed to MII in either the presence or absence of BIM demonstrated comparable rates of cleavage to the two-cell stage relative to in vitro-matured fully grown control oocytes. Blastocyst development rates were also significant, but lower than the B6SJLF1 controls. Moreover, in vitro fertilization of CX8 oocytes that matured to MII in response to BIM treatment with sperm carrying a GFP marker confirmed that the majority of blastocyst-stage embryos were attributable to successful fertilization. Importantly, transfer of these embryos to recipient females resulted in development of healthy pups to term. The low incidence (approximately 15%) of spontaneous parthenogenetic activation in these strains is consistent with previous studies [24].

In summary, partially competent oocytes recovered from early antral follicles can express the necessary factors for completion of meiosis I. While the molecular mechanisms that restrain these oocytes at MI are not fully understood, evidence is provided that PKC plays an important role since suppression of PKC activity promotes entry into anaphase I. These findings suggest that a PKC-mediated signal regulates one or more factors that are necessary for the onset of anaphase I. Therefore, control of PKC activity may be an important component of the developmental program required for the acquisition of full meiotic competence during the final stages of oocyte growth. Moreover, these mechanisms are likely to be disrupted in oocytes from the CX8 strains that typically arrest at MI despite reaching full growth at the preovulatory stage.


   ACKNOWLEDGMENTS
 
The authors thank Drs. R. De La Fuente, M.A. Handel, and R. Taft for their helpful comments and critical reading of this manuscript.


   FOOTNOTES
 
1 Support was provided by grants CA 62392 from the NCI and HD21970 from the NICHD to J.J.E. Scientific Resources of The Jackson Laboratory are supported in part by a Cancer Center Core Grant (CA 34196) from the NCI. Back

2 Correspondence. FAX: 610 925 8121; viveiros{at}vet.upenn.edu Back

Received: 26 April 2004.

First decision: 20 May 2004.

Accepted: 22 June 2004.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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