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a Centre de recherche en reproduction animale, Faculté de médecine vétérinaire, Université de Montréal, Saint-Hyacinthe, Canada J2S 7C6
b Department of Obstetrics and Gynecology, McGill University, Montreal, Canada H3A 1A1
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Initial studies in mammals argued for an even more limited developmental potential, with the inner cell mass of the blastocyst and its cultured derivatives being the most advanced stages of development that could be achieved [4,5]. Recent studies have indicated, however, that nuclei derived from cultured cells obtained from mammary tissue of adult ewes [6] and from ovarian granulosa cells of mice [7] are able to support full development to term. These dramatic findings have reopened the question of the irreversibility of nuclear differentiation and of the capability of ooplasm to reprogram somatic nuclei to produce a totipotent state. Nonetheless, the molecular mechanisms underlying the resetting of the developmental program after the transplantation of nuclei into oocytes remain almost entirely unknown.
To better understand the reprogramming of mammalian nuclei, several approaches have been used to characterize the cellular and molecular changes occurring during the early development of nuclear-transplant embryos. One approach has been to analyze the expression of stage-specific proteins and antigens. In mice, differentiated embryonic and endoderm-like somatic nuclei obtained from a teratocarcinoma, when transferred to an enucleated 1-cell embryo, are able to direct synthesis of certain proteins known as the transcription-requiring complex that are markers of embryonic genome activation [8]. Others have observed that ooplasm is able to modify the composition of the nuclear lamina of transplanted mouse 8- and 16-cell blastomere nuclei [9]. Moreover, when pig 16-cell blastomeres are fused to enucleated and activated secondary oocytes, small nuclear ribonuclear proteins disappear, whereas nuclear lamins A/C reappear, reflecting the pattern found at the 1-cell stage [10, 11]. In cattle, ultrastructural studies indicate that complete reprogramming is observed with respect to the blebbing activity of the nuclear envelope and the transcription of heterogeneous nuclear RNA, whereas only partial reprogramming of the nucleolar fine structure occurs [12]. Moreover, effects of the cell cycle stage of host ooplasts on the remodeling and developmental competence of reconstructed embryos has been demonstrated previously using both somatic and blastomere donor nuclei [13, 14].
The most obvious morphological change observed in transplanted nuclei is a rapid swelling reported in amphibians [15] and in mammals, including mice [13], rabbits [14,16], and pigs [17]. Although the precise causes of nuclear swelling are as yet unclear, exchange of both acidic and basic proteins between donor nuclei and cytoplasm has been observed [18, 19]. It may be speculated that ooplasmic proteins imported into the nucleus mediate structural rearrangement of the chromatin, which functionally resets it to a totipotent state. Similar events may occur naturally after fertilization, when ooplasmic nucleoplasmin and core histones mediate remodeling of the sperm chromatin to form the male pronucleus [20, 21].
In addition to the core histones, linker histone H1 appears to be actively involved in the regulation of gene expression during early embryonic development in several species (reviewed by [22]). Oocytes and early embryos of many species lack the somatic form of histone H1, which first becomes detectable on chromatin at about the stage when the embryonic genome becomes transcriptionally active [23–26]. In Xenopus, experimental acceleration or delaying of the timing of the switch from embryonic-type to somatic-type H1 correspondingly altered the time when certain mesoderm-inducing genes could be transcribed [27]. These results strongly support a role for changes in histone H1 in the regulation of early embryonic genome activity.
On the basis of these results, we investigated whether the functional reprogramming of embryonic nuclei that occurs when they are transplanted into ooplasm at different stages after activation is accompanied by changes in their histone H1 complement. We focused our experiments on the bovine system, in which embryonic nuclei transplanted into oocytes support development at a much higher frequency than in the mouse.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Ovaries were collected from a local abattoir, stored in saline at 30–35°C, and brought to the laboratory within 2 h of slaughter. Follicles with diameters between 2 and 8 mm were punctured with a 19-gauge needle, and cumulus-oocyte-complexes (COCs) with several layers of cumulus cells and homogeneous oocyte cytoplasm were washed in Hepes-buffered tissue culture medium (TCM-199; Gibco BRL, Burlington, ON, Canada) supplemented with 10% (v:v) fetal calf serum (FCS; Gibco). Groups of 20 COCs were placed in 100 µl of bicarbonate-buffered TCM-199 supplemented with 10% FCS, 50 µg/ml LH (Ayerst, London, ON, Canada), 0.5 µg/ml FSH (Follitropin-V; Vetrepharm, St-Laurent, PQ, Canada), 1 µg/ml estradiol-17ß (Sigma, St. Louis, MO), 22 µg/ml pyruvate (Sigma), and 50 µg/ml gentamicin (Sigma). After 24 h of maturation in vitro (IVM), oocytes were in vitro fertilized (IVF) using standard protocols [28]. Briefly, COCs were placed in 50-µl drops of Tyrode's medium, supplemented with 0.6% BSA (Fraction V; Sigma), lactate, pyruvate, gentamicin, and 10 µg/ml of heparin. Frozen-thawed spermatozoa were washed and centrifuged through a Percoll gradient and diluted at 106 live spermatozoa/ml. At 20 h postinsemination, COCs were denuded of cumulus cells by brief shaking, and the presumably fertilized zygotes were transferred to 50-µl drops of Menezo B2 medium (MB2; Pharmascience, Paris, France) supplemented with 10% FCS in the presence of bovine oviductal epithelial cells (BOEC). All cultures were performed in drops under equilibrated mineral oil at 39°C in a humidified atmosphere of 5% CO2 in air.
Nuclear Transplantation Protocols
Several nuclear transplantation protocols were compared to assess the ability of oocytes at different cell cycle stages to modify the chromatin of morula-stage nuclei (Fig. 1). In the first protocol (group 1), oocytes were denuded of cumulus cells after 24 h of IVM and placed in PBS containing 7.5 µg/ml cytochalasin B (Sigma), and approximately 30% of the cytoplasm adjacent to the first polar body was removed. After microsurgery, oocytes were placed in medium containing 5 µg/ml Hoechst 33342 for 15 min and exposed briefly to ultraviolet irradiation to verify by the absence of chromatin that enucleation was complete. A single blastomere derived from an in vitro-produced morula at Day 5 after IVF was introduced into the perivitelline space of the enucleated oocyte, and the resulting couplet was placed in a 0.3 M mannitol solution containing 0.1 mM MgSO4 and 0.05 mM CaCl2 and exposed to a 1.5-kV electric pulse lasting 70 µsec. Previous experiments have indicated that exposure to an electric pulse at 26 h causes low rates of activation. After electrical stimulation, oocytes were washed in TCM-199 and 1 h later were exposed to 5 µM ionomycin (Sigma) to induce parthenogenetic activation, washed, and placed in MB2 with BOEC for culture. For group 2, oocytes were matured for 24 h, enucleated, and exposed to ionomycin at 28 h; and a morula nucleus was introduced 1 h later by fusion as described for group 1.
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For groups 3 and 4, oocytes were denuded of cumulus cells at 30 h after IVM, exposed to an activation treatment with ionomycin, and returned to the incubator for 2 h to allow for extrusion of the second polar body. During or immediately after polar body extrusion, telophase II-stage oocytes were exposed for 15 min to medium containing cytochalasin B, and approximately one-tenth of the cytoplasm adjacent to the second polar body was removed [29]. A morula-stage blastomere was introduced either immediately (group 3) or 4 h later (group 4) into the perivitelline space, and the resulting couplet was exposed to an electric pulse to induce fusion, as described above. Although these cells contained no host chromosomes, we refer to them as interphase to indicate that they were used for fusion several hours after oocyte activation.
In the third protocol (aged metaphase enucleation; group 5), oocytes were matured for 24 h, the chromosomes were removed, and the oocytes were returned to the IVM drops for another 18 h. The extended maturation protocol (aging) is used routinely in nuclear transplantation procedures to improve the developmental competence of reconstructed embryos [30]. After aging (i.e., 42 h of IVM), the ooplasts were cooled to 12°C for 3 h and then fused to a morula-stage blastomere.
Drugs
Stock solutions of drugs were prepared in dimethyl sulfoxide at concentrations of 10 mg/ml, and working solutions of 
-amanitin (100 µg/ml; Boehringer), aphidicolin (1.5 µM; Boehringer), cycloheximide (10 µg/ml, Sigma), and 6-dymethylaminopurine (6-DMAP; 3 mM; Sigma) were prepared by appropriate dilution of stock into MB2 medium supplemented with 10% FCS. Nuclear-transplant embryos were exposed to these drugs as described in the Results.
Synchronization of Blastomeres
Morula-stage embryos at Day 5 after IVF were placed into 0.33 µM nocodazole in MB2 culture medium for 12 h to synchronize cells at metaphase. After nocodazole treatment, embryos were exposed to a 0.1% pronase solution for 3 min to remove the zona pellucida and then disaggregated using a fine bore pipette. Separated blastomeres were placed individually into 10-µl drops and observed hourly to verify the time of cleavage. Cleaved blastomeres were removed (defined as at G1-phase) and fused to interphase ooplasts (group 3). After fusion, the nuclear-transplant embryos were incubated in the presence of 6-DMAP, cycloheximide, or medium alone. In general, blastomeres were fused to oocytes within 1.5–2 h after they had cleaved.
Immunocytochemistry
Groups of nuclear-transplant oocytes and embryos at 1, 3, 6, 9, 12, 16, 24, 48, 96, and 168 h after fusion were fixed in 10% formalin (Sigma) for 20 min, washed, and stored at 4°C in 0.9% saline containing 0.1% Tween-20 (Sigma). To detect somatic histone H1, fixed oocytes and embryos were incubated in a blocking solution (PBS, 3% BSA, 0.5% Triton X-100) for 1 h at room temperature, then transferred to anti-histone H1 antibody (raised in rabbit using histone H1 from rat thymus and affinity-purified [25,31, 32]) diluted 1:50 in blocking solution, and incubated overnight at 4°C. This antibody has previously been shown to recognize somatic H1 subtypes but not H1 subtypes present in mouse and bovine oocytes and early embryos [25,26, 33]. The cells were then washed twice in blocking solution, incubated in fluorescein-conjugated goat anti-rabbit IgG diluted 1:100 in blocking solution for 1 h at room temperature, and washed as above. Specimens were mounted on slides in a mounting medium containing Moviol (Hoechst, Montreal, PQ, Canada), the DNA stain 4',6'-diamindino-2-phenylindole (DAPI; 1 µg/ml), and the anti-fading agent 1,4 diazabicyclo-[2.2.2] octane (DABCO). They were examined using standard epifluorescence optics. Nuclear diameters were measured using an ocular micrometer.
Statistical Analysis
Analysis of nuclear diameters in nuclear-transplant embryos was performed by ANOVA using the Tukey-Kramer HSD test, with the significance level set at 5%. Frequencies of somatic H1 staining among groups of nuclear-transplant embryos were analyzed by chi-square.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The ability of nuclei obtained from blastomeres or differentiated somatic cells, when transplanted into activated oocytes, to direct embryonic development is considered to indicate that these nuclei have been functionally reprogrammed by the oocyte cytoplasm. To investigate whether this presumed reprogramming is accompanied by changes to histone H1 on chromatin, individual blastomeres derived from morula-stage embryos were fused to oocytes whose chromosomes had been removed (ooplasts). Depending on the experiment, the recipient ooplast was parthenogenetically activated at different times before or after introduction of the morula nucleus (Fig. 1). At different times after fusion, the nuclear-transplant embryos were fixed, reacted with an antibody recognizing somatic H1, and examined using immunofluorescence microscopy.
We observed that the behavior of the donor nucleus depended on whether it was introduced near the time of oocyte activation or several hours later. When introduced 1 h before or 1 h after activation of the host oocytes, when the oocytes were near metaphase (groups 1 and 2), the morula nucleus remained in interphase in about half of the recipients. In the other half of the recipients, the nucleus underwent chromosome condensation within the oocyte cytoplasm. This condensation was observed regardless of the time postfusion when the cells were fixed. In many cases, the cells contained fragmented or apparently pycnotic chromatin derived from the blastomere nucleus (data not shown). In contrast, when the nucleus was introduced 2.5 h or 6 h after activation, when the oocytes were in interphase (groups 3 and 4), the morula nucleus remained in interphase in every case. It is possible that high metaphase-promoting factor (MPF) activity in the oocytes used for fusion near the time of activation promoted condensation of the donor chromatin, whereas low MPF activity in the oocytes used several hours after activation allowed the donor nuclei to remain at interphase [29].
To examine the fate of the somatic H1 associated with the morula-stage chromatin during residence in the ooplasm, the nuclear-transplant embryos stained using the anti-somatic H1 antibody were examined using epifluorescence. In the cases in which the donor chromatin condensed in the ooplasm, no staining was observed in any case (data not shown). When the donor nucleus remained at interphase, somatic H1 staining was prominent on the donor nuclei during the first several hours after transplantation into oocytes (Fig. 2). In oocytes fixed at later times, however, the intensity of the signal declined, and it ultimately became undetectable. Thus, incubation of morula nuclei in the cytoplasm of activated oocytes led to a loss of immunoreactive somatic H1 from the nuclei.
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The kinetics of loss of immunoreactive somatic H1 from the morula nuclei differed, however, depending on the cell cycle stage of the host cytoplasm (Table 1). When the host oocytes were near metaphase at the time of fusion (activated 1 h before or 1 h after fusion), somatic H1 became undetectable in the donor nucleus after between 3 and 6 h of residence in the ooplasm. However, when the hosts were at early interphase at the time of fusion (activated 2.5 h before fusion), loss of somatic H1 occurred between 6 and 9 h after fusion, and when they were fused later in interphase (activated 6 h before fusion), somatic H1 persisted until 12–16 h postfusion. These results indicate that the loss of immunoreactive somatic H1 from the morula nuclei occurred more rapidly in oocytes that were near metaphase at the time of fusion than in oocytes that were in early or later interphase.
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It may be seen in Figure 2 that the morula nuclei increased in volume during residence in the ooplasm. This raised the possibility that the loss of immunoreactive histone H1 might be linked to nuclear swelling, and, if swelling occurred more slowly in the interphase recipients, this could account for the relatively slow loss of immunoreactive H1 in these ooplasts. To investigate this, we measured the diameter of the nucleus of each embryo at the different times after nuclear transplantation. In some cases, two nuclei were present in a single ooplast, and these were excluded from the calculation. The results are shown in Figure 3, together with the frequency of histone H1 immunoreactivity, which has been obtained from Table 1 and plotted as a histogram. It may be seen that at 6 h after fusion, when somatic H1 was not detectable in the metaphase recipients yet remained in the interphase recipients, there was no significant difference in mean nuclear diameter between these groups. Similarly, at 9 h after fusion, when somatic H1 remained detectable in the 6-h interphase recipients, the mean diameter of these nuclei did not differ from those in the metaphase recipients. By 16 h after fusion, when somatic H1 was not detectable in any group, the mean diameters of the transplanted nuclei were similar in the metaphase and two interphase groups. Thus, the morula nuclei did not enlarge more slowly in the interphase environment than in the metaphase environment, indicating that differential nuclear growth cannot underlie the relatively slow loss of immunoreactive H1 observed in interphase recipients. The results also indicate that, although a general correlation exists between nuclear growth and loss of immunoreactive H1, there appears to be no direct connection between these two processes.
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When aged oocytes were used as recipients, the loss of immunoreactive H1 also occurred relatively slowly, becoming undetectable between 12 and 16 h postfusion (Table 1). As nuclear swelling also was retarded or inhibited in these oocytes (Fig. 3), it may be that aged cytoplasm differs in several respects from younger cytoplasm, meaning that these results cannot be easily compared with those obtained using metaphase and interphase recipients. The results may suggest, however, that loss of H1 immunoreactivity depends on factors whose activity declines during aging in vitro of inactivated oocytes.
Role of Cellular Synthetic Processes in the Loss of Immunoreactive Histone H1 from Morula Chromatin
To study molecular mechanisms underlying the loss of immunoreactive somatic histone H1 from the chromatin of donor nuclei during residence in activated oocyte cytoplasm, embryos were constructed using telophase-enucleated ooplasts at 2.5 h after activation and then were cultured for 12 h in the presence of an inhibitor of DNA replication (aphidicolin) or transcription (-amanitin), or for 12, 16, or 20 h in the presence of an inhibitor of protein synthesis (cycloheximide) or protein phosphorylation (6-DMAP), before being fixed and processed for immunofluorescence. The 12-h incubation period was chosen because immunoreactive H1 has been lost from morula nuclei in 2.5-h telophase-enucleated recipient ooplasts by this time (Table 1).
Nuclear diameter was significantly reduced in nuclear-transplant embryos exposed to aphidicolin (25.8 vs. 39.4 µm, p 
0.01), suggesting that the extent of nuclear swelling was partially dependent on DNA replication. In contrast, -amanitin had no detectable effect on nuclear diameter (37.9 µm). In nuclear-transplant embryos exposed to either drug, no immunoreactive somatic H1 was detected at 12 h after fusion (aphidicolin: 0/16; -amanitin: 0/14). These results suggest that loss of immunoreactive somatic H1 occurred independently of DNA replication and transcription.
When the nuclear-transplant embryos were incubated in either cycloheximide or 6-DMAP, nuclear swelling was reduced at 12 h postfusion compared to that of controls (Fig. 4). During the next 8 h of incubation, the nuclei of the cycloheximide-treated embryos enlarged to become similar in size to those of controls, whereas the nuclei of 6-DMAP-treated embryos remained relatively small. In the presence of either drug, immunoreactive histone H1 remained detectable in a substantial portion of the donor nuclei after 12 h of residence in ooplasm (Table 2), even though it was undetectable in all control cells processed at the same time. At 16 or 20 h postfusion, however, somatic H1 was no longer detectable in the majority of the cases. These results suggest that the loss of immunoreactive somatic H1 was delayed in the absence of coincident protein synthesis or protein phosphorylation. This effect was transient, however, and variable in that it was manifested only in a proportion of the cases.
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To test whether the variable effects of cycloheximide and 6-DMAP on the loss of immunoreactive somatic H1 were linked to cell cycle asynchrony of the donor morula nuclei, the following experiment was conducted. Embryonic cells were synchronized by using nocodazole to arrest each blastomere when it reached metaphase, then transferred into nocodazole-free medium. One h after cleavage, newborn blastomeres whose nuclei were expected to be at G1 were fused to host oocytes. These nuclear-transplant embryos were cultured in the presence of cycloheximide or 6-DMAP, and then fixed. Non-synchronized cell nuclei were used in parallel, and these results are included in Table 2. It may be seen that, using the G1 nuclei, immunoreactive somatic H1 was no longer detectable by 12 h in most cases despite the presence of cycloheximide or 6-DMAP (Table 2). By contrast, using non-synchronized blastomeres, immunoreactive H1 remained present at 12 h postfusion in between one third and one half of the embryos. These results suggest that loss of immunoreactive somatic histone H1 from G1 nuclei, as compared to that from non-synchronized nuclei, was less dependent on coincident protein synthesis and phosphorylation.
Reappearance of Immunoreactive Somatic Histone H1 on the Chromatin of Nuclear-Transplant Embryos
During development in vitro of normal bovine embryos, immunoreactive histone H1 first becomes detectable at the 8- to 16-cell stage [26]. We wished to examine whether the nuclear-transplant embryos would exhibit the same developmental regulation (Table 3, Fig. 2). No immunoreactive H1 could be detected in either 2-cell or 4-cell embryos that developed from the nuclear-transplant oocytes. At the 8- to 16-cell stage, however, most blastomere nuclei were immunoreactive. At the blastocyst stage, the nuclei of both the inner cell mass and trophectodermal cells stained positively for somatic H1. No difference in the timing of the reappearance of immunoreactive H1 on the chromatin was apparent among the different experimental groups (Table 3). These results indicate that, in nuclear-transplant embryos, immunoreactive somatic H1 becomes detectable on the chromatin at the same stage as in unmanipulated embryos developing in vitro.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Loss and Reappearance of Immunoreactive Somatic Histone H1 after Nuclear Transplantation
Using an antibody that recognizes the somatic subtypes of histone H1, we observed that immunoreactive H1 disappears from the chromatin of morula-derived blastomere nuclei after transplantation into ooplasts. Thus the ooplasm modifies the morula chromatin in some manner. This modification could include the removal of somatic H1 from the chromatin or alternatively might alter the chromatin such that somatic H1 is masked from the antibody. We favor the first alternative for two reasons. First, in the mouse, the somatic H1 subtypes are not detectable in oocytes by immunoblotting [25]. Unfortunately, thousands of mouse oocytes were required in the latter experiment, an unrealistic option for confirming the loss of somatic H1 from nuclear-transplant embryos. Second, in the amphibian, Xenopus laevis, an extract of egg cytoplasm remodels somatic nuclei by removing the somatic forms of histone H1 [34]. In this case, the somatic H1 is replaced by the oocyte-specific histone H1 variant, B4. Interestingly, somatic H1 is removed from nuclei even in extracts depleted of histone B4, suggesting that this nucleo-protein exchange is not determined by competitive binding of the two proteins [34]. In the experiments reported here, it may be speculated that somatic H1 of the bovine morula nuclei is replaced by an oocyte-specific H1 variant that does not react with the antibody. Although the nature of this putative H1 variant is unknown, mouse oocyte nuclei can be stained using an antibody raised against histone H10 [33].
In addition to the loss of immunoreactive histone H1, the morula nuclei enlarged considerably in the ooplasm. Between 1 and 16 h after fusion, the mean diameter increased approximately 2.5-fold, representing about a 15-fold increase in volume. Early studies in amphibians demonstrated that somatic nuclei transplanted into fertilized egg cytoplasm decondense and increase in volume similarly to pronuclei [35], and this was later shown to be correlated with the import of cytoplasmic proteins into the nuclei [18]. Exchange of nuclear and cytoplasmic non-histone proteins has also been demonstrated after transplantation of somatic nuclei into enucleated oocytes [19, 36]. A similar nuclear swelling has been observed to follow transplantation in many mammalian species [13, 14, 17]. Taken together, these results suggest that numerous modifications to the protein complement of chromatin, as exemplified by the changes in histone H1 reported in this study, may occur when somatic nuclei are exposed to oocyte cytoplasm.
When the nuclear-transplant embryos were incubated to permit further development, immunoreactive H1 reappeared at the 8- to 16-cell stage, which is the stage when it first becomes detectable in unmanipulated bovine embryos [26]. This time corresponds to the major activation of embryonic gene expression. Indeed, a close temporal relationship exists in several species between the transition to the somatic form of histone H1 and the initiation of major transcriptional activity. In the mouse, in which the major transcriptional activation occurs at the 2-cell stage [37], somatic H1 becomes detectable at the 2- or 4-cell stage [25]. In Xenopus embryos, somatic H1 begins to accumulate and B4 declines near the mid-blastula transition, when there is a major increase in transcriptional activity [38]. This developmentally regulated change in the Xenopus histone H1 subtype plays an important role in regulating the pattern of activity of specific genes [27]. The fact that the reappearance of immunoreactive H1 in nuclear-transplant embryos precisely matches that of unmanipulated embryos implies that this chromatin modification serves a specific function during early mammalian embryogenesis as well.
Effects of the Cell Cycle Stage on Loss of Immunoreactive Histone H1
We found that the immunoreactive histone H1 was lost from the morula chromatin more rapidly when the host ooplasts were near metaphase than when they were near telophase or further in the first embryonic cell cycle. This suggests that the cytoplasmic activities that remove immunoreactive histone H1 from the morula chromatin may decline in efficiency as the cell progresses into the first embryonic cell cycle. Several previous studies have suggested that reprogramming of somatic nuclei in oocytes may occur more efficiently in metaphase cytoplasm than in interphase cytoplasm. When thymocyte nuclei were transplanted to murine oocytes, they developed into pronuclear-like structures when the hosts were obtained at metaphase but retained their somatic-like nuclear morphology when the hosts were at interphase [13, 39]. In cattle, metaphase ooplasm extinguished transcriptional activity of morula nuclei more efficiently than interphase cytoplasm [40] and, in the rabbit, host ooplasts at the metaphase stage allowed for more extensive nuclear swelling and better development of nuclear-transplant embryos [14]. Most recently, Wakayama et al. [7], using cumulus granulosa cell nuclei to clone mice, found that development was improved by prolonged exposure of donor nuclei to metaphase-stage ooplasm before activation, although this was not compared with telophase or interphase cytoplasm. Considering these results together with our observation that immunoreactive histone H1 is lost more rapidly in metaphase cytoplasm than in interphase cytoplasm, it may be proposed that metaphase cytoplasm more rapidly molecularly remodels foreign nuclei and this enhances their developmental competence.
Molecular Mechanisms Controlling Loss of Immunoreactive Histone H1
Neither aphidicolin nor -amanitin detectably influenced the timing of somatic histone H1 disappearance, implying that the controlling mechanisms do not depend on DNA replication or transcription. This contrasts with the reappearance of somatic histone H1 on chromatin during early embryogenesis, which requires both DNA synthesis and transcription [25, 26]. Our results suggest that, if the immunoreactive histone H1 is replaced by an oocyte-specific H1 variant, this variant must be present in the ooplasm before the first round of DNA replication, or it is synthesized through a replication-independent mechanism. In this connection, Wiekowski et al. [41] observed that in activated eggs certain histone H1 subtypes are synthesized only at the late 1-cell stage, implying that if histone H1 is assembled into nascent chromatin during the first embryonic S-phase, it is present in the oocyte cytoplasm before fertilization.
Inhibitors of protein synthesis or kinase activities delayed the loss of immunoreactive histone H1 from the morula chromatin in a large proportion of the reconstructed oocytes. Although the identity of protein kinases that may participate in this process is entirely speculative, metaphase-arrested oocytes, which effect a rapid loss of immunoreactive H1, have high MPF activity. It has previously been shown that thymocyte nuclear membranes break down under the influence of residual MPF in newly activated oocytes, although a new nuclear membrane subsequently forms as the cell cycle progresses [39]. Furthermore, in amphibian egg extracts, permeabilization of the nuclear envelope of interphase nuclei by addition of chemical reagents or MPF allowed access to the chromatin of cytoplasmic factors capable of modifying chromatin activity [42, 43]. Thus, the faster removal of somatic histone H1 from morula nuclei exposed to ooplasts at metaphase or immediately after activation might be due to MPF activity on the nuclear membrane, allowing rapid accessibility of ooplasmic factors to the donor chromatin or a faster exit of immunoreactive histone H1 through the nuclear membrane.
Additionally, metaphase-related kinases could affect the phosphorylation pattern of proteins that mediate the loss of immunoreactive histone H1. Hyperphosphorylated nucleoplasmin removes basic proteins from Xenopus sperm more rapidly than the hypophosphorylated form [21, 34, 44]. Remodeling of the sperm nucleus into the paternal pronucleus is also associated with phosphorylation of core histones [45]. Additionally, phosphorylation of histone H1 can affect its stability within the chromatin [46], suggesting that kinases active in the ooplasm close to the time of activation may affect histone H1 interaction with chromatin by changing its phosphorylation state. These studies suggest that the effect of ooplast cell cycle on the kinetics of somatic H1 disassembly may in part reflect differential activities of protein kinases.
Although both protein synthesis and kinase inhibitors delayed the timing of H1 disassembly in a proportion of the nuclei when non-synchronized cells were used, they had little apparent effect when nuclei were obtained from cells shortly after release from a metaphase block. We infer that these synchronized nuclei were at G1 and that, in the non-synchronized cells, the nuclei from which loss of immunoreactive histone H1 was not delayed by cycloheximide or 6-DMAP were also at G1. This suggests that on-going protein synthesis and phosphorylation were required for the timely removal of immunoreactive histone H1 from S-phase or G2 nuclei, but not from G1 nuclei.
As the cell fusion technique that we used introduced the morula cell cytoplasm as well as the nucleus, it is possible that donor cells in G1 contain remnants of metaphasic kinase activities that, as discussed above, could promote the removal of histone H1 from the chromatin. Alternatively, the chromatin structure of G1 nuclei may differ from S-phase and G2 nuclei. Phosphorylation of H1 subtypes is minimal at G1 and increases thereafter until mitosis, when it is maximal [47]. These cell cycle-related changes in H1 histone modification levels imply that H1 modulates chromatin condensation and decondensation, possibly affecting their stability in chromatin. If these modifications have not been fully reversed in the G1 nuclei, as compared to the S or G2 nuclei, this could explain their different responses to the cycloheximide and 6-DMAP. An additional possibility is that histone H1 is more tightly associated with replicated chromatin than with unreplicated chromatin. Although the difference between G1 and later-stage nuclei that influences their response to cycloheximide and 6-DMAP remains speculative, it is interesting to note that a similar comparison of donor nuclei at different stages of the cell cycle revealed that nuclei obtained at G1 were better able to support embryonic development to the blastocyst stage than were nuclei obtained at later stages [48]. These results are consistent with the view that more rapid molecular alteration of foreign chromatin in ooplasm is correlated with enhanced developmental potential.
Together, these findings identify a specific modification of morula-stage chromatin that accompanies its functional reprogramming after transplantation to oocyte cytoplasm. Further characterization of the molecular mechanisms underlying chromatin reprogramming will enhance our understanding of the role that oocyte cytoplasm plays in the restructuring of gametic chromatin during early embryogenesis. Moreover, knowledge of the factors involved could enhance our ability to completely reverse nuclei from terminally differentiated and/or neoplastic cells to totipotency.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Lawrence C. Smith, Centre de recherche en reproduction animale (CRRA), Faculté de médecine vétérinaire, Université de Montréal, C.P. 5000, Saint-Hyacinthe, Canada J2S 7C6. FAX: 450 778 8103; smithl{at}medvet.umontreal.ca
Accepted: February 23, 1999.
Received: November 13, 1998.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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D. H. Betts, V. Bordignon, J. R. Hill, Q. Winger, M. E. Westhusin, L. C. Smith, and W. A. King Reprogramming of telomerase activity and rebuilding of telomere length in cloned cattle PNAS, January 30, 2001; 98(3): 1077 - 1082. [Abstract] [Full Text] [PDF]
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