| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Gamete Biology |
ák
Laboratory of Molecular Embryology,3 Mendel University Brno, 613 00 Brno, Czech Republic
Department of Molecular Embryology,4 Institute of Experimental Medicine, Academy of Sciences of the Czech Republic, 142 20 Prague, Czech Republic
Centre for Cell Therapy and Tissue Repair,5 Charles University, 150 18 Prague, Czech Republic
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
early development, gametogenesis, kinases, meiosis, oocyte development
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
In this study, we used the mouse as a model to begin to delineate the potential roles of six functionally interrelated cell cycle regulators (CDK4; CDK6; cyclins D1, D2, and D3; and p27) in developing mammalian oocytes by first determining their quantities and localization in growing and maturing oocytes, as well as in early embryos at several hours after fertilization. The results document that the progression of oocyte development is accompanied by major variations in quantity, structure, and localization of these cell cycle regulators, together suggesting their involvement in some as yet unidentified developmental processes.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
(C57BL/6J X BALB/c)F1 hybrid mice were used throughout the study. They were maintained in the animal facility of the Laboratory of Molecular Embryology, Mendel University Brno (Brno, Czech Republic). Animals were supplied with water and food ad libitum. Ovaries and oviducts were removed from animals after they were killed by cervical dislocation. This method is consistent with the recommendations of the Central Commission for Animal Welfare of the Czech Republic. All cultures/manipulations in this study were carried out in 35 mm tissue culture dishes (Nunclon 153066, Nalge Nunc International, Roskilde, Denmark). To obtain oocytes in about the middle of their growth, ovaries from 13-day-old mice were placed into 1 mg/ml collagenase (Type I, Worthington Biochemical Corporation, Lakewood, NJ) and RQ1 DNase from bovine pancreas (Promega, Madison, WI) in Waymouth media, maintained at 37°C in an atmosphere containing 5% CO2, and gently pipetted in 10-min intervals for a total of 50 min. To obtain fully grown germinal vesicle (GV) stage-arrested oocytes, 22- to 24-day-old female mice were injected by 5 IU equine chorionic gonadotropin (eCG) at 44–48 h before the isolation of oocytes. The oocytes were obtained by puncturing the large antral follicles with 26-gauge needles into Minimum Essential Medium (MEM) with Earles salts supplemented with 3 g/L bovine serum albumin (BSA), 75 mg/L Penicillin-G, and 50 mg/L streptomycin sulphate, followed by collecting oocyte-cumulus cell complexes using glass micropipets. To obtain oocytes at metaphase I (MI), oocyte-cumulus cell complexes were cultured for 7 h in the same media supplemented with 5% fetal calf serum (FCS; Gibco catalog 10106-169, Invitrogen Ltd., Paisley, UK) at 37°C in an atmosphere containing 5% CO2 in air. To obtain in vivo-matured MII oocytes, 22- to 24-day-old female mice were primed for 48 h by 5 IU eCG and then injected by 5 IU human chorionic gonadotropin (hCG) to induce ovulation. At 15 h after hCG injection, ovulated MII oocytes were released from the oviducts into MEM media as above. The cumulus cells were dispersed by brief exposure to 100 µg/ml hyaluronidase at 37°C. For in vitro fertilization, in vivo-matured MII oocytes with their surrounding cumulus cells were combined in drops under oil with sperm that had been capacitated for 3 h. Media (MEM) as above supplemented with 3 g/L BSA instead of FCS was used for both capacitation and fertilization. At 3 h after beginning of insemination, the zygotes were washed free of sperm and released cumulus cells, and they were placed into the same media for further development.
Antibodies and Reagents
Rabbit polyclonal antibody to mouse CDK4 (sc-260); rabbit polyclonal antibody to human CDK6 (sc-177), which cross-reacts with the mouse homolog; mouse monoclonal antibody to mouse cyclin D1 (sc-450); and rabbit polyclonal antibody to mouse cyclin D2 (sc-593) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Mouse monoclonal antibody to mouse p27 (K25020) was purchased from Transduction Laboratories (Lexington, KY). Mouse monoclonal antibody against a C-terminal part of human cyclin D3, which cross-reacts with the mouse homolog (DCS-22), was generously provided by Dr. Jiri Lukas (Danish Cancer Society, Copenhagen, Denmark). Horseradish peroxidase- and FITC-conjugated anti-immunoglobulins were from Sigma Chemical Co. (St. Louis, MO); polyvinylidene difluoride (PVDF) membrane Hybond-P, and chemiluminescence detection reagents (ECL+Plus) were purchased from Amersham (Amersham, Aylesbury, UK). Equine chorionic gonadotropin was from Bioveta (Ivanovice na Hane, Czech Republic), and hCG was purchased from Organon (Oss, Holland). All other chemicals were purchased from Sigma or Fluka (Buchs, Switzerland).
Western Blot Analysis
A total of 100 oocytes/zygotes were taken for each sample to be loaded onto gel (per one lane). Oocytes/zygotes at indicated developmental stages were freed of surrounding somatic cells by gentle pipetting; washed twice in PBS pH 7.4 containing 1 g/L polyvinylpyrrolidone (PVP; 360 000); lysed in Laemmli sample buffer; boiled for 5 minutes; and stored at -80°C until use. Protein sample made of proliferating primary mouse embryonic fibroblasts (MEFs) was also included into each analysis, both to control authenticity of a particular regulator and to allow for comparison of its amount in oocytes/embryos with its amount in "normal" growing somatic cells. To obtain measurable Western blot signals, 0.2 µg of MEF protein was loaded per lane, which represents approximately 12.5-fold less than the amount of protein in 100 fully grown oocytes. After being separated on 10% SDS/PAGE, the proteins were electrotransferred onto Hybond-P membrane, immunodetected using appropriate primary and secondary antibodies, and visualized by ECL+Plus reagent according to manufacturer's instructions. When required, membranes were stripped in 62.5 mM Tris/HCl pH 6.8, 2% SDS, and 100 mM 2-mercaptoethanol; washed; and reblotted with another antibody from this selection. After immunodetection, each membrane was stained by amido black to confirm protein loading. Resulting films were digitized and the bands were quantified using Intelligent Quantifier (Bio Image, Ann Arbor, MI). For each regulator, the maximal measured density was defined as 1.0, and from this value all other values were calculated.
Indirect Immunofluorescence
Oocytes were freed of somatic cells as for Western blot analysis, and zonae pellucidae were removed by the treatment with 1 mg/ml protease in MEM. After being extensively washed in PBS pH 7.4 containing 1 g/L PVP, oocytes were fixed in 4% paraformaldehyde for 2 h at room temperature (RT) and then permeabilized in 0.2% Triton X-100 in PBS pH 7.4 for 30 minutes at 4°C. Oocytes were then incubated overnight at 4°C with appropriate primary antibody diluted in PBS pH 7.4 containing 3 g/L BSA and 0.01% sodium azide (PBSA), washed three times with PBSA supplemented by 0.5% Tween-20 (PBST), and incubated for 1 h at RT with appropriate secondary antibody diluted in PBSA. After final extensive wash in PBST, oocytes were mounted to Mowiol containing 1,4-diazobicyclo-[2.2.2]-octane (DABCO) to prevent fading. Microscopical analysis was performed using an upright Olympus BX60 microscope equipped with a Fluoview confocal laser scanning unit (Olympus C&S Ltd., Prague, Czech Republic).
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Western blot analysis was employed to determine the quantities of CDK4, CDK6, their partnering D-type cyclins, and the typical regulator of CDK4/cyclin D complexes—p27 CKI. The specificity of all antibodies used here was previously shown in our laboratory on various cell types of mouse origin and/or was recently confirmed by comparing with other reagents, as mentioned below. Oocytes at the following developmental stages were analyzed: 1) growing oocytes, 2) fully grown GV-stage-arrested oocytes, 3) meiotically maturing oocytes at MI, and 4) matured oocytes at MII (Fig. 1). To assess the possible changes associated with the exit of oocyte from MII and/or with the initiation of embryonic development, embryos at 6 h after insemination were also included.
|
As schematized in Figure 1, our analysis covered three well-defined segments of oocyte development: 1) about the second half of growth phase (growing oocytes to GV), 2) meiotic maturation (GV to MII), and 3) exit from MII—completion of meiosis (MII to early embryos). Clearly detectable amounts of the analyzed cell cycle regulators were found in all oocytes/embryos included in this study (Fig. 2). Still, there were major differences in how the quantities of these regulators fluctuated during the developmental periods depicted above.
|
Growth The quantities of CDK4, CDK6, cyclin D1, and cyclin D2 in growing oocytes at Day 13 were lower by about sixfold, threefold, fivefold, and 17-fold, respectively, compared with fully grown GV-stage oocytes (Fig. 2). In contrast, cyclin D3 and p27 did not show appreciable differences between these two stages (Fig. 2). It is of note that the position of cyclin D2 in oocytes is different from that in MEFs. However, this has been confirmed by unrelated monoclonal antibody (DCS-5.2, provided by Dr. Jiri Lukas) not cross-reacting with cyclin D1. Importantly, although the diameter of fully grown oocytes is about 85 µm, the diameter of growing oocytes reaches only 60 µm on average. Therefore, the differences/similarities in the amounts of regulators given above apply only when the same numbers of oocytes and not the amounts of total protein are being compared.
Meiotic maturation As shown in Figure 2, progression of meiotic maturation introduced changes to the levels of CDK4, CDK6, and cyclin D3, but not to the levels of cyclin D1, cyclin D2, and p27. Specifically, the amount of CDK4 was maintained constantly high in MI oocytes (compared with GV-stage oocytes) to become reduced by about twofold in MII oocytes. In contrast, both CDK6 and cyclin D3 decreased rather gradually from GV to MII, with an intermediate level at MI. Importantly, besides the quantitative changes described above, profound modulations occurred to the electrophoretic mobility of CDK4, CDK6, and p27. Although in GV-stage oocytes CDK4 appeared as a single band corresponding to that detected in MEFs, in MI oocytes its vast majority appeared in a slower position. The slower migrating form of CDK4 also remained predominant in MII oocytes. Two independent lots of the anti-CDK4 polyclonal antibody produced exactly the same results. Similar to CDK4, CDK6 in GV-stage oocytes was represented by only a single band corresponding to that seen in MEFs. Additionally, in the case of CDK6, the faster migrating band occurred in MI and MII oocytes. The change to p27 took place also during the GV to MI transition, with faster migration in GV-stage oocytes and slower migration in MI and MII oocytes. Notably, "genuine" p27, as found in MEFs, corresponded to the slower migrating form of p27 typical for M-phase oocytes and early embryos.
Completion of meiosis: entry to embryogenesis Concerning their quantities, the regulators studied here followed basically two different scenarios. Although the levels of CDK6, cyclin D1, cyclin D2, and cyclin D3 did not undergo any changes, the levels of CDK4 and p27 decreased appreciably (Fig. 2). Notably, an extremely dramatic change—approximately a 40-fold decrease—occurred in the amount of p27. In contrast, down-regulation of CDK4 was only sixfold; thus following the trend entered during meiotic maturation. Besides the profound drop in its quantity, no other changes to p27 were observed, and p27 maintained the electrophoretic mobility typical for MI and MII oocytes. No further changes in the migration of CDK6 in MI and MII oocytes were observed. In contrast, CDK4 re-established its "standard" faster migration, typical for GV-stage oocytes and MEFs (Fig. 2).
Localization of Cell Cycle Regulators
In order to gain more insight into the metabolism of cell cycle regulators, here we also determined their distribution in oocytes/embryos using indirect immunofluorescence followed by confocal microscopy. Oocytes/embryos at the same five stages as above were analyzed, and the representative results are summarized in Figure 3. Generally, localization patterns varied according to both the stage and the regulator.
|
Growth All regulators except for cyclin D1 were clearly concentrated into the nucleus in growing oocytes at Day 13 and in fully grown GV-stage oocytes. Still, there were large differences in whether or not a positive reaction was also observable in the cytoplasm at these two stages. In the case of CDK4, the immunoreaction was solely nuclear in both growing and fully grown GV-stage oocytes. In contrast, cyclin D3 and p27 were invariably observed in the cytoplasm of growing oocytes (but not fully grown oocytes). CDK6 and cyclin D2 showed prominent nuclear reaction that was accompanied by cytoplasmic staining above a background in both growing and fully grown GV-stage oocytes. Rather faint cytoplasmic staining with an obvious submembranous accumulation was typical for cyclin D1 at these two developmental stages.
Meiotic maturation Upon resumption of meiosis, localized (nuclear) staining of all regulators except CDK6 became lost in concert with the disappearance of a formed nucleus. Thus in both MI and MII oocytes these regulators were rather homogenously distributed throughout the cytoplasm. In contrast, an obvious accumulation copying both MI and MII spindles was typical for CDK6. Another notable exception from complete homogeneity was the submembranous accumulation of cyclins D1 and D2 that was most prominent in MII oocytes.
Completion of meiosis: entry to embryogenesis Upon completion of the second meiotic division and with entering embryogenesis (pronuclei formation), nuclear reaction of CDK4 and cyclin D2, but not of CDK6, cyclin D3, and p27, became re-established. Thus at 6 h after insemination, cyclin D3 and p27 were homogenously distributed in cytoplasm. The distribution of cyclin D1 followed the submembranous pattern typical for previous stages.
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Although the mRNAs for CDK4, all D-type cyclins, and also for p27 were found in mouse eggs and early embryos [10], and cyclin D3 and p27 proteins were immunohistochemically confined to oocytes at earlier stages of follicular development [11], this study provides the first unambiguous identification/quantification of these proteins in isolated growing and maturing oocytes. Based on comparison with cycling primary MEFs, the relative amounts of CDK4, CDK6, D-type cyclins, and p27 proteins in oocytes are generally quite significant (see Fig. 4 for the schematic); however, some oocyte-to-oocyte variability may be hidden due to pooling of oocytes for Western blot analysis. It is of note that there is a large difference (two orders of magnitude) in cell volumes of oocytes and fibroblasts. Therefore, when relating the quantities of the regulators to one cell rather then to the total protein, their absolute amounts are from several-fold (for CDK4) to several hundred-fold (for p27) higher in fully grown GV-stage oocytes than in MEFs. Such a different view is meaningful particularly in the case of CDK4, cyclin D3, and p27, which are predominantly nuclear in fully grown GV-stage oocytes.
|
Chesnel and Eppig [14] have previously reported that CDK1 and cyclin B accumulated during the growth of mouse oocytes, and that this accumulation seemed to be critical for oocytes to resume meiosis. When we addressed the question of whether or not G1/S regulators accumulate along the progression of oocyte growth, nonuniform patterns were observed. There were several-fold increases in CDK4, CDK6, cyclin D1, and cyclin D2, but essentially no changes in cyclin D3 and p27 when comparing 100 growing oocytes isolated from 13-day-old female mice with 100 fully grown GV-stage oocytes. Still, although fully grown oocytes in our hybrid mice were approximately 85 µm in diameter, growing oocytes recovered from 13-day-old females were only about 60 µm in diameter, which represents about a 2.8-fold difference in terms of their volume. Thus, in fact, the concentrations of only CDK4 and cyclin D2 significantly increase (about twofold and sixfold, respectively) during the investigated period of oocyte growth (schematized in Fig. 4). In Xenopus, cyclin D2 is the only D-type cyclin expressed in oocytes, and it seems to be involved in the reinitiation of meiosis [15]. Whether the massive accumulation of cyclin D2 observed here mirrors such roles in mouse oocytes remains to be investigated. Also, it can only be hypothesized that less pronounced increases in levels of other G1/S regulators indicates their involvement in processes taking place prior to meiotic maturation. Still, recently shown colocalization of CDK4 with replication protein A on the synaptonemal complexes of pairing bivalents in mouse oocytes and spermatogonia [16] may support such a view. Notably, both the finding of Ashley et al. [16] and the data presented in this study contrast with the observations of Zhang et al. [11]. Under their conditions, CDK4 was not seen in oocytes at any stage of follicular development, and the amounts of cyclin D3 and p27 continually decreased during oocyte growth, becoming almost nondetectable in preovulatory oocytes. Since our results are produced by Western blot analysis using isolated oocytes, a failure of the immunohistochemical approach employed by Zhang et al. [11] is a likely reason for such discrepancy.
On resumption of the meiotic cell cycle, oocyte metabolism grossly changes: transcription ceases, stored maternal mRNAs become translated, and various proteins undergo posttranslational modifications. The data presented in this study also distinguish CDK4, CDK6, cyclin D3, and p27 as the proteins that undergo changes during the course of meiotic maturation. It was shown by gene targeting in mice that both CDK4 and p27 are essential for female reproduction [17–21]. However, blastocysts recovered from either CDK4-/- or p27-/- females develop to term when transferred into uteri of nonmutant females [17, 22, 23]. These findings seem to suggest that oocyte development itself does not require either of these proteins. Still, although no obvious defects in oocytes were observed in the absence of either CDK4 or p27, formation of preovulatory follicles and the efficiency of ovulation were affected in both cases [19, 23]. Such abnormalities may reflect some function of p27 in oocytes that is crucial for proper signaling toward companion somatic cells. The well-established role of oocytes in folliculogenesis [24] and a minute level of p27 in granulosa cells [18, 25, 26] support such a hypothesis. Although it is not fully understood, in various cell types the metabolisms of p27 and cyclin D3 are linked together [26–28]. Notably, according to our Western blot and immunofluorescence data, such an interaction might take place also in developing oocytes. Still, the possibility of the existence of thoroughly new interactions of cyclin D3 has been recently suggested by showing that cyclin D3 may serve as a partner for p58PITSLRE during the G2 and M phases [29]. Obviously, successful disruption of cyclin D3 in mice and more detailed biochemical analyses in oocytes are required to validate such hypotheses.
In conclusion, by determining the spatiotemporal expression of the selected G1/S regulators in developing mouse oocytes, this study contributes to the search for proteins that may underlie the functions of female gametes. The levels of these regulators per se, combined with the profound dynamics in their amounts, structure, and localization, strongly suggest their involvement in regulation of oocyte development. Although our results do not provide information on which processes/functions require these proteins, it will be necessary to further exploit mouse oocytes to unravel their molecular interactions, activities, and biochemical nature of the modifications observed in this study. Oocytes from other strains of mice and also from other species should then be studied in order to make the findings more general.
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
2 Correspondence: Ale
Hampl, Laboratory of Molecular Embryology, Mendel University Brno, Zem
dlská 1, 613 00 Brno, Czech Republic. FAX: 420 5 4513 3298; hampl{at}mendelu.cz
Received: 19 March 2003.
First decision: 8 April 2003.
Accepted: 2 September 2003.
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
