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Research Article |
Animal Development and Biotechnology Group, Division of Animal Physiology, School of Biosciences, The University of Nottingham, Sutton Bonington, Loughborough, Leicestershire LE12 5RD, United Kingdom
ABSTRACT
In general, oocytes arrested at metaphase of the second meiotic division (MII) are used as recipient cytoplasts for nuclear transfer (NT) procedures. MII oocytes contain high levels of maturation-promoting factor (MPF) and mitogen-activated protein kinase (MAPK), which cause nuclear envelope breakdown (NEBD) and premature chromosome condensation (PCC) in the transferred nucleus and have been implicated in nuclear reprogramming. However, the occurrence of NEBD and the extent of PCC are variable between individual oocytes and species and are dependent on donor cell type and cell cycle stage. Enucleation, which removes oocyte cytoplasm, may reduce MPF and MAPK activities and reduce reprogramming; conversely, increasing kinase activities may increase reprogramming. We compared the effects of enucleation of ovine oocytes at anaphase/telophase of the first meiotic division (AI-TI) and at MII. MPF and MAPK activities were maximal at MII; blind enucleation at AI-TI was more efficient than at MII and removed a smaller volume of cytoplasm. Neither protocol significantly affected the activity of either kinase and the fate of the donor nucleus; however, enucleation per se significantly reduced the occurrence of NEBD in NT embryos. Treatment with 10 mM caffeine significantly increased the activities of both kinases and the occurrence of NEBD but did not affect the frequency of development to the blastocyst stage; however, a significant increase in total cell numbers was observed. The results show that caffeine can increase MPF and MAPK activities in ovine oocytes and that this may contribute to an increased reprogramming in NT embryos.
embryo, kinases, meiosis, oocyte development, phosphatases
INTRODUCTION
Nuclear transfer (NT) using somatic cells as donors of genetic material is now a well established but inefficient technique in a range of domestic mammals [1–9]. For development to occur, cell cycle coordination of the donor cell nucleus (karyoplast) and the recipient cell cytoplasm (cytoplast) is essential to prevent DNA damage and maintain ploidy of the reconstructed embryo (for reviews, see Campbell et al. [10] and Campbell and Alberio [11]). The choice of the cell cycle stage of the recipient cytoplast may affect subsequent development; although offspring have been obtained using cytoplasts at telophase of the second meiotic division (TII) and interphase of first cell cycle, MII (metaphase of the second meiotic division) oocytes have typically become the recipient of choice (for reviews, see Campbell et al. [10] and Campbell and Alberio [11]). These contain active maturation-promoting factor (MPF) and have been reported to induce nuclear envelope breakdown (NEBD), premature chromosome condensation (PCC), and dispersion of nucleoli in the transferred nucleus, which may be essential for nuclear reprogramming [12]. In fact, a number of studies have reported increased development and more normal gene expression patterns in embryos reconstructed using MII oocytes as cytoplast recipients when NT occurs before fusion [13, 14], suggesting that NEBD and PCC are beneficial to nuclear reprogramming. However, analysis of studies in a range of species suggests that the occurrence and extent of NEBD and PCC in the donor nucleus are variable between different species, donor cell types [15, 16], reconstruction procedures [17], activation protocols [18], donor animal age [19], and oocyte age [20].
One factor that may affect the resultant cytoplast is the enucleation procedure. In general, this has been achieved by removal of the MII spindle and a small volume of surrounding cytoplasm from matured oocytes (for review, see Li et al. [21]). It has been suggested that enucleation may remove proteins or other factors that are essential for subsequent development, in particular spindle-associated proteins, including nuclear-mitotic apparatus (NuMA) and HSET [22]. Experiments in the mouse have demonstrated that MPF kinase activity may be compartmentalized [23] with the majority of activity remaining with the metaphase plate and not the enucleated oocyte to be used as cytoplast recipient for NT. Such a potential reduction in MPF activity due to enucleation may explain the variable response of donor nuclei to MII cytoplasm and the differences in development reported in previous studies. However, a reduction in MPF activity due to enucleation may be species specific, as studies in porcine oocytes have reported that no decline in oocyte MPF activity was associated with the enucleation procedure [24].
In contrast to a reduction in kinase activities leading to reduced reprogramming, we hypothesize that increasing kinase activities may increase reprogramming or result in cytoplasts that are less variable in their ability to cause NEBD and PCC. To this end, the objectives of these studies were to measure the MPF and MAPK activities of ovine oocytes matured in vitro and determine the effects of enucleation at different stages during maturation. Oocytes were enucleated at anaphase/telophase of the first meiotic division (AI-TI) and MII, the volume of cytoplasm was removed, and the efficiencies of blind enucleation, the activities of MPF and MAPK, and the fate of the donor nucleus in the resultant cytoplast were determined. The effects of caffeine (a phosphatase inhibitor) on MPF and MAPK activities in nucleated and enucleated oocytes, the fate of the donor nucleus, and the development of embryos reconstructed by nuclear transfer using different cytoplast recipients were then tested.
MATERIALS AND METHODS
All chemicals and reagents were purchased from Sigma-Aldrich unless otherwise stated.
In Vitro Oocyte Maturation
Sheep ovaries were collected at a local slaughterhouse and transported to the laboratory in PBS at 25°C. Oocytes were aspirated from antral follicles 2–3 mm in diameter using a 21-gauge needle, and good-quality oocytes with at least three layers of cumulus cells and a homogeneous cytoplasm were selected for in vitro maturation. Selected oocytes were washed several times in Hepes-buffered TCM 199 (Gibco) containing 10% (v/v) fetal bovine serum (FBS) and then cultured in bicarbonate-buffered TCM 199 (Gibco) supplemented with 10% (v/v) FBS, 5 µg/ml FSH (Vetropharm), 5 µg/ml LH (Vetropharm), 1 µg/ml estradiol, 0.3 mM sodium pyruvate, and 100 µM cysteamine. Groups of 40 oocytes were cultured in 500 µl of maturation medium under mineral oil in four-well dishes (Nunc) at 39°C in a humidified atmosphere of 5% CO2.
Donor Cell Culture
Primary fetal fibroblasts were isolated from a Day 30 ovine fetus as previously described [25] and cultured for two passages in Dulbecco modified Eagle medium (DMEM) supplemented with 1.0% (v/v) ß-mercaptoethanol, 2.0 mM L-glutamine, 1.0% (v/v) penicillin/streptomycin, and 10% FBS. Primary cultures were then stored in liquid N2 until required. For each experiment, cells were thawed and cultured until approximately 80%–90% confluent; quiescence was then induced by reducing the concentration of FBS to 0.1% for a further 3 days. Immediately before use as nuclear donors, a single cell suspension was prepared by trypsinization (0.25% [w/v]). The cells were pelleted and resuspended in DMEM plus 0.1% (v/v) FBS and maintained in this medium at 39°C until used as nuclear donors.
Oocyte Enucleation
Oocytes were enucleated at AI-TI or MII. Prior to enucleation, the cumulus cells were removed, and oocytes were placed into 400 µl of Hepes-buffered synthetic oviduct fluid (H-SOF) medium containing 300 IU/ml hyaluronidase in a 15-ml conical polystyrene tube. The oocytes were incubated at 39°C for 2 min and then vortexed for 4–5 min. For enucleation at AI-TI, cumulus cells were removed at 15 h postonset of maturation (hpm) and for enucleation at MII at 23 hpm. The denuded oocytes were then incubated in H-SOF containing 5 µg/ml Hoechst 33342 for 15 min. Enucleation was carried out at 40x magnification on a Leica DMIRB fitted with Narashige manipulators using a 20-µm (outer diameter) glass micropipette in H-SOF plus 4 mg/ml BSA and 7.5 µg/ml cytochalasin B (CB). For AI-TI enucleation, a portion of cytoplasm was removed from the extrusion cone containing the extruding anaphase/telophase spindle; for MII enucleation, the first polar body and a portion of cytoplasm directly beneath it were aspirated (Fig. 1). Enucleation of oocytes was confirmed by visualization of DNA in the aspirated karyoplast using a short automated exposure to UV light (0.1 sec) and image capture (Simple PCI; Compix Inc.). Control oocytes were sham enucleated by removing an equal volume of cytoplasm.
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Determination of Karyoplast and Cytoplast Volumes
The diameters of intact oocytes and the aspirated karyoplasts were measured after capturing the images on a Leica DMIRB microscope fitted with a Hammamatsu digital camera and Simple PCI image analysis software (Compix Inc.). Assuming a perfectly spherical oocyte and aspirated karyoplast, volumes were calculated as 4/3
r3.
Caffeine Treatment and Parthenogenetic Activation of In Vitro Matured MII Oocytes
At 23–24 hpm, oocytes were stripped of cumulus cells by vortexing in 400 µl of H-SOF medium containing 300 IU/ml of hyaluronidase for 4–5 min in a 15-ml conical polystyrene tube and then followed by three washes in H-SOF. Good-quality MII oocytes with the first polar body and an evenly pigmented cytoplasm were selected for parthenogenetic activation. Selected oocytes were cultured in maturation medium with different concentrations of caffeine (0, 5, 10, or 20 mM) until 30 hpm, washed in H-SOF, and then immediately activated. Oocytes were activated by a 5-min exposure to 5 µg/ml calcium ionophore (A23187), followed by incubation with 10 µg/ml of cycloheximide (CHXM) and 7.5 µg/ml CB for 5 h at 39°C in a humidified atmosphere of 5% CO2 and 90% N2. Parthenogenetic embryos were cultured and examined as described for reconstructed embryos.
Cell Fusion and Activation
Quiescent primary fetal fibroblasts used as nuclear donors were fused to enucleated cytoplasts with two DC pulses of 1.25 kV/cm for 60 µsec in 0.3 M mannitol without calcium ions using an Eppendorf Multiporator and fusion chamber with a 200 µM electrode gap. Fused couplets were cultured in modified SOFaaci supplemented with 4 mg/ml BSA (mSOFaaciBSA) until activated. Activation was carried out in H-SOF medium containing 5 µg/ml A23187 (Sigma) for 5 min, followed by culture in mSOFaaciBSA medium supplemented with 10 µg/ml of CHXM and 7.5 µg/ml CB for 5 h at 39°C in a humidified atmosphere of 5% CO2, 5% O2, and 90% N2.
Culture of Reconstructed Embryos
Following activation, reconstructed embryos were transferred into mSOFaaciBSA and cultured in a humidified atmosphere of 5% CO2, 5% O2, and 90% N2 at 39°C. On Day 2 of culture, embryo cleavage was assessed and 10% FBS added to the culture medium. On Day 7, development to blastocyst and total cell numbers were assessed.
Analysis of Nuclear and Chromatin Morphology
To evaluate the status of the transferred nuclei, reconstructed embryos were fixed at 2 h following fusion. For fixation, embryos were mounted onto glass slides under coverslips attached with a mixture of vaseline and paraffin wax (9:1) and then placed into methanol:acetic acid (3:1) for 24 h. Fixed embryos were then stained with 1.0% aceto-orcein and examined by phase contrast microscopy (Leica DMRB) at 100x magnification under immersion oil.
MPF and MAPK Assays
The preparation of oocyte lysate and analysis of MPF and MAPK activities were performed as previously described [26] with some modifications. Briefly, groups of 10 cumulus-stripped oocytes were washed several times in Ca2+-free PBS containing 0.1% polyvinyl alcohol and placed into 5 µl of ice-cold lysis buffer. The samples were snap frozen in liquid N2 and stored at –80°C until analyzed. The kinase reaction was started by mixing the oocyte lysate with 5 µl kinase assay buffer containing 2 mg/ml histone H1 (Sigma), 3 mg/ml myelin basic protein (Sigma), 4 µM protein kinase A-inhibiting peptide (Santa Cruz Biotechnology), 4 µM protein kinase C-inhibiting peptide (Promega), and 0.5 µCi (34 µM) [
-32P] ATP (Amersham Pharmacia Biotech). The mixtures were incubated at 37°C for 30 min with gentle shaking. The reaction was stopped by adding 10 µl ice-cold 2x SDS sample buffer. After boiling for 4–5 min, the substrates were separated by standard polyacrylamide gel electrophoresis (SDS-PAGE, 15% gels) using a mini-protean II dual slab cell (Bio-Rad) at 140 V constant voltage for 1.5 h. Gels were dried onto 3-mm filters and exposed to phosphor-screens (Kodak; Amersham Pharmacia Biotech). The phosphor images of gels (screens) were captured, and the kinase activities were quantified using an FX phosphor image analysis system (Bio-Rad).
Statistical Analysis
Data were analyzed by one-way analysis of variance. Statistical significance was P < 0.05 unless specified in the text.
RESULTS
Volume of Cytoplasm Aspirated by Enucleation of Oocytes at AI-TI and MII
Ovine oocytes at AI-TI or MII were enucleated 16–18 and 22–24 hpm, respectively (Fig. 1). Immediately after enucleation, the aspirated karyoplast inside the enucleation pipette was checked for the presence of the maternal chromosomes by a short exposure to UV light (0.1 sec). A greater percentage of oocytes at AI-TI (97.8%) were successfully enucleated than at MII (78.0%). A comparison of the volume of the aspirated karyoplasts (Table 1) showed that in our hands enucleation at AI-TI resulted in the production of a significantly smaller karyoplast (15.8 ± 2.4 µm diameter) than enucleation at MII (35.2 ± 3.1 µm diameter) and consequently removed significantly less of the oocyte cytoplasm (0.2% vs. 2.3%, respectively; P < 0.05).
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MPF and MAPK Activities at Different Stages of Maturation
The activities of MPF and MAPK were assayed at three different stages (GV, AI-TI, or MII) during maturation of ovine oocytes in vitro. In immature oocytes (0 hpm; GV), both MPF and MAPK activities were low at basal levels. During maturation, the activities of both kinases increased significantly, reaching maximum activities at MII, 24 h postonset of maturation (Fig. 2).
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Effects of Enucleation on MPF and MAPK Activities
MPF and MAPK activities were measured at AI-TI or MII in intact, enucleated, and sham enucleated oocytes. The activities of MPF and MAPK in intact ovine oocytes were not different from those of enucleated oocytes at the same stages. Additionally, there were no differences in activity of either kinase between enucleated and sham enucleated oocytes (data not shown).
Changes in MPF and MAPK Activities Following Enucleation at AI-TI
Both MPF and MAPK activities increased between AI-TI and MII in unenucleated oocytes. To examine the effects of enucleation at AI-TI on this increase in activity, intact and enucleated oocytes were returned to maturation medium, and culture was continued. The activities of both kinases were measured at 3-h intervals (until 27 hpm). The changes in activities of both MPF and MAPK kinases were identical in intact and enucleated oocytes, reaching maximum levels at 24 hpm (Fig. 5).
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Nuclear Morphology after Fusion to Different Cell Cycle Stage of Oocytes
Groups of embryos were examined at 2 h following fusion and classified into four groups based on nuclear and chromatin morphology as illustrated (Fig. 3). The nucleus remained intact (Fig. 3A). The nuclear DNA was condensed into chromosomes and arranged on a spindle (premature metaphase) (Fig. 3B). The chromatin was condensed into chromosome-like structures but not organized on a spindle (Fig. 3C). The chromatin was condensed into a single mass (Fig. 3D). To test the effect of enucleation of oocytes at different cell cycle phase (AI-TI and MII; 16–18 and 22–24 hpm, respectively) on the fate of the transferred nuclei, primary fetal fibroblast cells (G0/G1 phase of the cell cycle) were fused to enucleated oocytes at 18 or 24 hpm and then assessed 2 h following fusion. As shown in Table 2, the morphology of nuclei transferred into AI-TI and MII stage of oocytes remained intact (70.1% vs. 59.1%); 24.7% of nuclei fused to the MII stage of oocytes formed significantly more condensed chromatin as compared to that to AI-TI (6.9%) (P < 0.05).
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Nuclear Morphology after Fusion to Unactivated Oocytes
In a second series of experiments, primary fetal fibroblasts were fused to groups of AI-TI enucleated and unenucleated MII oocytes at 24 hpm and examined 2 h postfusion. Although the majority of the transferred nuclei did not undergo NEBD, significantly more underwent NEBD in unenucleated than enucleated oocytes (31.8% vs. 11.1%) (P < 0.05); 18.2% of the nuclei in unenucleated oocytes formed unorganized chromosomes as compared to 0.0%, 9.1% formed a premature plate vs. 3.2%, and 4.5% formed a single mass of condensed chromatin vs. 7.9% as compared to the enucleated oocytes (Table 3).
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Effects of Caffeine on MPF and MAPK Activities in Unenucleated MII Oocytes
Matured denuded MII oocytes (24 hpm) were cultured in maturation medium containing 0, 5, 10, or 20 mM caffeine for 6 h and then MPF and MAPK activities determined. The activities of both MPF and MAPK kinases in oocytes treated with 0 and 5 mM caffeine (24–30 hpm) were lower than those in MII oocytes (24 hpm); in contrast, treatment with 10 and 20 mM caffeine increased the activities of both kinases as compared to 24-hpm MII oocytes (Fig. 4).
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Effects of Caffeine Treatment on Development of Ovine Parthenotes
MII oocytes (24 hpm) were treated with 0, 5, 10, and 20 mM caffeine for 2, 4, or 6 h and activated, and development was assessed (Table 4). Treatment with 10 and 20 mM caffeine significantly reduced cleavage at all time periods as compared to 5 mM and control groups; 20 mM caffeine caused a significant reduction in development to the blastocyst stage at all time points. In contrast, although 5 and 10 mM caffeine slightly reduced development to blastocyst, this was not significant, and development after 10 mM caffeine treatment for 6 h was not significantly different to treatment with 5 mM.
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Effects of Caffeine Treatment on MPF and MAPK Activities in Enucleated Oocytes
To determine effects of caffeine treatment on MPF and MAPK kinase activities, enucleated oocytes were cultured in maturation medium supplemented with 10 mM caffeine and groups of oocytes collected of MPF and MAPK every 3 h by 27 hpm. Although MPF and MAPK activities in enucleated 24-hpm oocytes were greater than those of enucleated 15-hpm oocytes, caffeine elevated significantly the activities of both kinases in enucleated oocytes in all groups (P < 0.05) (Fig. 5).
Fate of Donor Cell Nucleus after Fusion to Enucleated Oocytes Treated with Caffeine
To examine the fate of donor cell nucleus transferred to enucleated oocytes treated with caffeine, primary fetal fibroblast cells were fused to oocytes at 24 hpm after incubation with or without 10 mM caffeine from 18 to 24 hpm. AI-TI enucleated oocytes were incubated in the presence or absence of 10 mM caffeine until 24 hpm, fused to donor cells, and examined at 2 h postfusion. In caffeine-treated oocytes, the majority of the transferred nuclei underwent NEBD as compared to the control oocytes (86.2% vs. 11.1%), 36.2% of the nuclei formed a premature metaphase as compared to 3.2% in the control, 1.7% formed unorganized chromosomes vs. 0.0% in the control, and 48.3% formed a single mass of condensed chromatin vs. 7.9% in the control (Table 5).
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Development of NT Embryos Reconstructed Using Caffeine Treated Oocytes as Recipient Cytoplasts
To examine the effects of caffeine treatment on development of NT embryos reconstructed using enucleated ovine oocytes as recipient cytoplasts, three different treated enucleated oocytes were used as cytoplasm recipients; all oocytes were enucleated at 16–18 hpm, and the recipients of A group were fused at 20 hpm, B group recipients were cultured in the maturation medium for 6 h and then fused at 24 hpm, and C group recipients were treated with 10 mM caffeine for 6 h and then fused at 24 hpm. Reconstructed embryos in all groups were activated 1 h following cell fusion. As demonstrated in these studies, each group of the recipient cytoplasts had different MPF and MAPK activities. The fusion rate of reconstructed embryos within the different treatments was significantly different between 20 and 24 hpm (85.3% vs. 73.5% and 70.7%), whereas no significant difference was observed between cleavage (nuclear transfer methods A, B, C: 91.4%, 89.2%, 88.5%) or development to blastocyst (20.2%, 25.2%, 30.0%). Although the development of NT embryos reconstructed using caffeine-treated recipients did not differ from the other groups, caffeine treatment significantly increased the total cell number of blastocyst stage embryos (59.0 ± 8.7%, 66.0 ± 8.5, and 88.8 ± 7.1, respectively) (P < 0.05) (Table 6).
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DISCUSSION
Although the development of embryos reconstructed by NT is dependent on numerous factors, central to development is the quality and developmental competence of the recipient cytoplast. MII oocytes, activated oocytes, zygotes, and two-cell embryos have all been used as recipient cytoplasts with varying degrees of success; however, MII oocytes have become the cytoplast of choice (for review, see Campbell and Alberio [11]). Production of a cytoplast requires removal of the genetic material; it has been suggested that enucleation of oocytes may remove proteins that are essential for development or reduce the levels of cytoplasmic kinases and influence subsequent reprogramming of the donor nucleus (reviewed in Campbell and Alberio [11]). The overall objective of these studies was to produce a more competent cytoplast for NT. To achieve this, we have 1) modified the enucleation procedure to remove a minimum of cytoplasmic volume, 2) examined the effects of enucleation on oocyte kinase activities and the fate of the donor nucleus following fusion, and 3) increased oocyte kinase activities and examined the effects of this on the fate of the donor nucleus and the development of NT embryos.
A comparison of the cytoplasmic volumes removed shows that enucleation at AI-TI removes significantly less of the oocyte cytoplasm than enucleation at MII (0.2% vs. 2.3%). This removal of a smaller volume of oocyte cytoplasm may result in the removal of a smaller percentage of oocyte proteins that are associated with the meiotic spindle. Additionally, proteins associated with the chromatin and the spindle may vary between AI-TI and MII, and therefore a different subset of proteins may be removed. We hypothesize that removal of a smaller volume of cytoplasm may be beneficial to the developmental competence of the recipient cytoplast. Enucleation at AI-TI also had the advantage that a significantly greater percentage of oocytes were enucleated "blind" (without the need for visualization of the DNA by exposure to UV) than when enucleated blind at MII based on the position of the first polar body (PB1) (97.8% vs. 78.0%). The use of PB1 as a marker for the location of the MII plate has been studied in a number of species. Correlation of the MII plate and the position of PB1 varies between species and with oocyte age; in addition, removal of the cumulus cells before oocyte manipulation can further disrupt the relationship between the MII spindle and PB1. These factors can result in a proportion of the oocytes containing residual DNA following enucleation (reviewed in Li et al. [21]). In contrast, at AI-TI, the two groups of chromosomes are much more closely associated with the extrusion cone, and the use of blind enucleation is more reliable. Enucleation of oocytes for use as recipient cytoplasts may remove proteins required for development [22] or reduce the activities of oocyte kinases [27]. We examined the effects of enucleation at AI-TI and MII on the activities of MPF and MAPK. No difference in activity of either kinase was observed at either cell cycle stage when compared to control, enucleated, or sham enucleated oocytes. Furthermore, when oocytes were enucleated at AI-TI, the increase in activities of both kinases observed at 24 hpm in MII oocytes was unchanged. Therefore, oocytes enucleated at AI-TI appeared identical in terms of their kinase activities to oocytes enucleated at MII but retained a greater proportion of the maternal cytoplasm.
When primary fetal fibroblasts were fused to enucleated oocytes, the majority of the donor nuclei remained intact; however, when fused to unenucleated oocytes, fewer nuclei remained intact (88.9% vs. 68.2%). The simplest explanation for this observation is that the fusion stimulus activated the recipient cytoplast and caused a reduction in kinase levels, preventing NEBD. The fusion conditions used in these experiments did not cause activation of unenucleated oocytes as judged by the presence of an intact MII plate at 2 h following fusion. An alternative explanation is that the oocytes may not contain sufficient MPF activity to induce NEBD. Ledda et al. [28] have previously reported that the level of MPF activity at MII is lower in oocytes obtained from prepubertal sheep than mature ewes; furthermore, this lower MPF activity was unable to induce GVBD when fused to GV oocytes, although kinase activity had been sufficient for maturation to MII. Similar lower levels of MPF have also been reported in prepubertal bovine oocytes, which also have a reduced developmental competence [29]. A number of factors are involved in regulating the occurrence of NEBD, including nuclear factors such as cell cycle stage [30, 31] and microtubule-dependent mechanical events [32, 33]. In addition, cytoplasmic factors transferred from the donor cell may alter the regulation of oocyte MPF activity and prevent NEBD [34]. Thus, the volume of donor cytoplasm transferred may be important, and therefore the use of cell types with a lower cytoplasmic volume (i.e., ES cells) or transfer of the donor nucleus by injection with a minimum of cytoplasm may be beneficial for NT.
To examine the effects of the activities of MPF and MAPK on the occurrence of NEBD, oocytes were treated with caffeine, a protein phosphatase inhibitor. Treatment of in vitro matured oocytes for 6 h with 10 mM caffeine did not adversely affect development to the blastocyst stage of parthenogenetically activated oocytes. However, this treatment caused a significant increase in the activities of both MPF and MAPK in enucleated oocytes and resulted in a significant increase in the occurrence of NEBD and PCC in the donor nucleus when treated oocytes were fused to G0/G1 donor cells. The mechanisms responsible for the increase in NEBD and PCC in caffeine-treated oocytes are unknown. The possibility that ovine oocyte MPF levels are simply below that required to induce NEBD is supported by the fact that increasing MPF and MAPK activities increases the occurrence of NEBD. Whether the effects of MPF and/ or MAPK in inducing NEBD occur directly on the donor nucleus or whether one or both of these kinases activates other molecules is unknown.
In NT embryos, the frequency of development to the blastocyst stage did not differ between caffeine-treated or -untreated recipient cytoplasts; however, a significant increase in the total cell number of embryos produced after caffeine treatment was observed. The mechanisms behind this increase in cell number are unknown but may be due to a number of mechanisms, including an increased calcium release, as has been reported in both bovine and porcine oocytes [35, 36], or an increase in the rate of cell division. Previous studies have reported an increase in cell cycle length in bovine NT embryos, resulting in an increased cell number [37] or a decrease in apoptosis. The increase in total cell numbers was restricted to NT embryos reconstructed using caffeine-treated cytoplasts that induced an increased frequency of NEBD and PCC. Previous studies have reported that nuclear reprogramming in bovine somatic nuclei is not affected by the activities of MPF and MAPK [38, 39]; however, these authors used M-phase donor cells in which the chromatin was already condensed. Therefore, MPF and MAPK may be important in the reprogramming of G0/G1 phase cells that contain an intact nucleus by inducing NEBD and PCC.
In conclusion, enucleation at AI-TI provides an efficient method for the blind enucleation of ovine oocytes while removing a minimum of oocyte cytoplasm. Enucleation at AI-TI or MII had no effect on the activities of MPF or MAPK kinases; however, enucleated oocytes were less able to induce NEBD in the donor nucleus after fusion to G0/G1 primary fetal fibroblasts. The treatment of in vitro matured ovine oocytes with 10 mM caffeine for up to 6 h did not affect subsequent development. The same treatment in AI-TI enucleated oocytes increased MPF and MAPK activities, producing a cytoplasmic environment that induced NEBD and PCC in the majority of nuclei transferred, reducing variability between cytoplasts. Furthermore, NT embryos produced using caffeine-treated cytoplasts had increased cell numbers at the blastocyst stage. The mechanisms underlying the increase in cell numbers require further investigation, and the effects on development to term of NT embryos produced in this way needs to be established.
FOOTNOTES
1 Supported by the University of Nottingham. 
2 Correspondence: Keith Campbell, University of Nottingham, School of Biosciences, Sutton Bonington, Loughborough, Leicester LE12 5RD, United Kingdom. FAX: 44 115 951 6302; keith.campbell{at}nottingham.ac.uk
Received: 19 May 2005.
First decision: 7 June 2005.
Accepted: 19 December 2005.
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