The present studies have been undertaken to investigate the interactions that occur between the nucleus and cytoplasm of ovine oocytes at various stages during meiotic maturation. We report that the nucleus of ovine fully grown dictyate stage oocytes can be efficiently removed by a microsurgical enucleation procedure. It is demonstrated that between the initiation of maturation and germinal vesicle breakdown certain newly syn-thesized polypeptides are selectively sequestered in the oocyte nucleus and the major sequestered polypeptide has a relative molecular mass of 28 000, which represent at least 9 % of the total labelled polypeptides transferred to the oocyte nucleus during the first 4h of maturation. The experiments provide evidence that the removal of the oocyte nucleus at various times before germinal vesicle breakdown (GVBD) does not prevent the major series of changes in protein synthesis that occurs after entry into a metaphase. We conclude therefore that the mixing of the nucleoplasm and cytoplasm is not essential for the initiation or progression of the protein repro-gramming process during maturation. In addition, the experiments show that the development of the ability to condense chromatin during ovine oocyte maturation is independent of the oocyte nucleus. The combined results strongly support the hypothesis that the extensive series of translational changes that occur in oocytes during maturation are controlled by cytoplasmic rather than nuclear factors.

Maturation represents the period during which the oocyte undergoes a series of well-defined intracellular changes including progression from the late diplotene (dictyate) to the metaphase II stage of meiosis (reviewed by Thibault et al. 1987; Wassarman, 1988). This process is initiated in vivo by high levels of circulating luteinizing hormone and in vitro by the release of the fully grown oocyte from its follicle into a suitable culture environment (Pincus and Enzmann, 1935; Chang, 1955; Thibault, 1972). Once initiated, the maturation process is most obviously characterised by the breakdown of the germinal vesicle, condensation of chromatin and extrusion of the first polar body. Extensive reprogramming of protein synthesis also occurs during this period and is an obligatory com-ponent of the differentiation events required for fertilization and early development (reviewed by Moor et al. 1983; Thibault et al. 1987; Wassarman, 1988). The signals that control morphological and biosynthetic remodelling of oocytes during maturation are un-known. However, it is claimed that many of the remodelling changes in mouse oocytes result from the mixing of the nucleoplasm and cytoplasm (Schultz and Wassarman, 1977a,b; Schultz et al. 1978; Wassarman, 1988).

Studies in this report investigate the interactions that occur between the nucleus and cytoplasm of the ovine oocyte at various stages during maturation. The first objective has been to determine the extent of new protein migration to the nucleus in the period before nuclear membrane breakdown. The next series of experiments were carried out to ascertain the role of the nucleus on protein synthesis before GVBD and on protein reprogramming thereafter. It has been of particular interest in this regard to determine whether a causal link exists between intracellular remodelling after GVBD and the intermixing of the karyoplast and cytoplasm. The final objective has been to determine whether extrachromosomal elements in the oocyte nucleus are essential for the condensation of chromatin during maturation.

Preparation of ovine oocytes

Ovaries were obtained from Welsh Mountain sheep pre-viously injected with 1000i.u. of PMSG (Folligon: Intervet Laboratories Ltd, Cambridge, UK) on day 10–13 of oestrous cycle and slaughtered 40–45 h later. Intact, non-atretic follicles (>3 mm in diameter) were dissected from the ovaries and then opened to remove the entire cumulus–oocyte complex. Oocytes were denuded of associated follicle cells by repeated pipetting using a mouth micropipette. All these procedures were performed in PBS at room temperature. In those groups of oocytes designated for immediate enucleation (within 30 min of explantation), follicles were preincubated in PBS containing 7.5 μg ml−1 cytochalasin B (Sigma) for 20 min and then directly opened in the same medium.

Preparation of mouse 8-cell blastomeres

Early 8-cell embryos were obtained from Fj female mice (C57BL×CBA, 4–6 week old) 63–65 h post-HCG injection and the zona pellucida was removed from each embryo by a brief exposure to prewarmed acid tyrode solution (Nicolson et al. 1975). Zona-free embryos were washed thoroughly in PBS dissection medium and then disaggregated by repeated pipetting using fire-polished mouth pipettes. Most blasto-meres were transplanted into ovine oocyte cytoplasts immedi-ately after disaggregation; the remainder were cultured in medium T6 (Quinn et al. 1984) supplemented with 4 mg ml−1 BSA at 37°C in an atmosphere of 5% CO2 in air until required for micromanipulation.

Micromanipulation

The micromanipulation assembly, preparation of microinstru-ments, set-up of manipulation chamber and microinstruments have already been described by Sun (1989). The manipulation medium was PBS containing cytochalasin B (7.5 μg ml−1), freshly prepared sodium pyruvate (40 μg ml−1) and 10% fetal calf serum (FCS: Sera-Lab Ltd).

(a) Enucleation

Denuded ovine oocytes, previously exposed to manipulation medium for 15 min, were placed singly into microdrops of manipulation medium for enucleation. Each oocyte was held with a holding pipette (overall diameter of 120 μm) and an injection pipette was used to make a small hole in the zona immediately above the germinal vesicle (GV) but without damaging the oolemma. Enucleation was achieved firstly by reorientating the oocyte so that the zona incision was directly apposed to the holding pipette. Thereafter, gentle suction resulted in the intact GV and a small amount of cytoplasm becoming positioned on the outer surface of the zona and attached to the remaining cytoplasm via a small cytoplasmic bridge (Fig. 1). Separation of the karyoplast from the cytoplast was carried out using a mouth pipette (200 μm inner diameter).

Fig. 1.

A fully grown dictyate stage ovine oocyte after micromanipulation. The karyoplast (outside) is still linked to the cytoplast by a cytoplasm bridge through the hole made in the zona pellucida during enucleation.

Fig. 1.

A fully grown dictyate stage ovine oocyte after micromanipulation. The karyoplast (outside) is still linked to the cytoplast by a cytoplasm bridge through the hole made in the zona pellucida during enucleation.

Preliminary experiments were undertaken to determine the effectiveness of the enucleation procedure. A total of 50 oocytes were manipulated and prepared directly thereafter for nuclear examination. The results showed that enucleation was successful in 100 % of oocytes. In an additional series of experiments, oocytes were enucleated and their viability was assessed by incorporation of radioactivity into TCA-precipi-table material. It is found that 65% of oocytes remained viable after enucleation.

A group of 24 karyoplasts obtained by enucleation of oocytes immediately after removal from follicles (n = 18) or after 4h culture (n=6) were fixed for a transmission electron microscopic examination of the nuclear membrane. In no case was the nuclear membrane disrupted by the enucleation procedure (Fig. 2). We conclude therefore that the validity of the enucleation procedure has been established. The pro-cedure is efficient, causes minimal damage to the cytoplasm and removes the nucleus in an intact condition thus eliminating any possibility of leakage of nuclear plasmic components during enucleation.

Fig. 2.

Electron micrograph of an ovine GV karyoplast showing that the nuclear envelope is not damaged by the enucleation process, × 11 100. Inset: high magnification of part of the nucleus, showing that both elements of the nuclear membrane are intact, n, nucleus; c, cytoplasm. × 40 500.

Fig. 2.

Electron micrograph of an ovine GV karyoplast showing that the nuclear envelope is not damaged by the enucleation process, × 11 100. Inset: high magnification of part of the nucleus, showing that both elements of the nuclear membrane are intact, n, nucleus; c, cytoplasm. × 40 500.

In the main experiments, cytoplasts were washed repeat-edly in PBS after manipulation and were then either cultured or radiolabelled directly in accord with experimental require-ments. Oocytes allocated to the experiments on nuclear protein localization were labelled with [35S]methionine for4h before enucleation. In these experiments, the GV was removed with the smallest possible amount of cytoplasm; groups of these GV karyoplasts (>20/group) were washed briefly in Tris-HCl buffer (10 HIM Tris, pH 7.4) before being lyophilized and frozen for electrophoresis.

(b) Transplantation of mouse blastomeres

For the reconstitution experiments, two or three ovine Mil oocytes or cytoplasts and five to ten mouse blastomeres (from the 8-cell embryos) were placed in each microdrop in the manipulation chamber. After 15 min exposure to the manipu-lation medium a small amount of cytoplasm was removed from each oocyte or cytoplast using the technique of McGrath and Solter (1983). Immediately thereafter a blastomere was injected into the perivitelline space created by the cytoplasmic excision. Nuclear transfers in oocytes were always made to the side opposite to the polar body to facilitate subsequent recognition of oocyte and mouse chromosomes. The partially reconstituted oocytes were thereafter washed carefully in PBS supplemented with 4 mg ml−1 BSA (PBS+BSA) and held in this medium pending cell fusion.

Electrofusion procedures

Fusion of the ovine cytoplasts and murine blastomeres was accomplished using a Zimmerman electrofusion apparatus (GCA, Chicago, USA) and the conditions and media have already been described recently by Sun and Moor (1989). Briefly, the partially reconstituted oocytes were transferred to the fusion chamber in F300 medium, subjected to an alignment current (200 V cm−1) and then fused using a pulse treatment of 1 kVcm−1. After pulse treatment, the fused embryos were washed in PBS+BSA and then cultured in TCM 199 supplemented with 10% FCS until fixation.

Ovine oocyte maturation

Maturation of groups of 10-20 intact oocytes, or cytoplasts were carried out in 35 mm plastic culture dishes (Sterlin, UK) containing 2 ml medium as described by Staigmiller and Moor (1984). The culture medium was TCM 199 supplemented with 10% FCS, and gonadotrophins (NIH-LH-S22, 2.5 μg ml−1; NIH-FSH-S13, 2.5 μg ml−1). In all instances, freshly prepared supplementary granulosa cells (approximately 106 cells ml-1) and sodium pyruvate (40 μg ml−1) were added to the medium in which the oocytes were cultured.

Radiolabelling of oocytes

Groups of ten to fifteen denuded or cumulus-enclosed oocytes were labelled at 37°C for 3–4 h in 50 μl of labelling medium (Moor et al. 1980) containing [35S]methionine (1000 Ci mmol−1, Amersham) at a radioactive concentration of 500 μCi ml−1. After incubation, oocytes were extensively washed in culture medium in order to remove all free label and cumulus-enclosed oocytes were denuded of cumulus cells. Individual oocytes were then briefly washed in 10 mM Tris-HCl, pH 7.4, and transferred to plastic tubes in a minimal volume of the Tris buffer (<2 μl), lyophilized and frozen at −70°C until required for electrophoresis.

Electrophoretic analysis of ovine oocyte proteins

Radiolabelled oocyte polypeptides were analysed on one-dimensional gels as described by Moor et al. (1981). Briefly, after lyophilisation, samples were dissolved in 30 μl of sample buffer (O’Farrell, 1975); an aliquot (10%) was removed for measurement of incorporation of radioactivity into TCA-precipitable material. Equal number of TCA-precipitable counts was applied to an 8–15 % linear gradient SDS– polyacrylamide slab gel and the polypeptides separated for 3 h at a constant current of 20mA per gel. Fluorography was carried out on [35S]methionine-labelled gel according to the methods of Bonner and Laskey (1974) and dried gels were exposed to preflashed Kodak-X-Omat S X-ray film (Laskey and Mills, 1975). Molecular weight determination were made using a 14C-methylated protein mixture (Mr range 14.3×103 to 200×103, Amersham) as standards.

The scanning of one-dimensional gels and quantitative analysis of protein bands were carried out by using a Chromoscan 3 densitometer (Joyce-Loebl, England).

Cytological assessment

After culture, the oocytes and mouse blastomeres were fixed for 24 h in ethanol/acetic acid (3:1), stained with Lacmoid and assessed using phase contrast microscopy.

Rate of meiotic progression during ovine oocyte maturation in vitro

The initiation of meiotic maturation in vitro takes place when the oocyte is released from the follicle into the culture medium. The timing of the nuclear events in the meiotic cycle, while varying by 1 or 2h for individual oocytes, occurs in the following sequence (1) chromatin condensation and the disassembly of the nuclear membrane occur about 8h after explantation (G2 to M-phase transition or GVBD); (2) metaphase I is extended and persists for 8 to 10h (MI phase); (3) a short anaphase and telophase (Al and TI) lead to the formation of the second metaphase plate (Mil) at 22 h after the initiation of maturation.

Under the experimental conditions used in our studies, about 95 % (n=120) of cumulus-enclosed ovine oocytes placed in culture reached Mil and emitted the first polar body. Approximately 90 % (n=45) of oocytes devoid of surrounding follicle cells (denuded oocytes) underwent GVBD, but only about 65% (n=40) of these oocytes emitted a first polar body after 24 h maturation in culture.

Nuclear localisation of proteins synthesized before GVBD

The purpose of this part of the investigation was to determine whether any proteins synthesized between the initiation of maturation and GVBD were specifi-cally associated with the oocyte nucleus. To do that, cumulus-enclosed oocytes were labelled for the first 4h of maturation then separated into cytoplasmic and nuclear fractions for protein analysis. To compensate for the inability to obtain absolutely purified nuclei, comparisons were made in all cases both between intact (control) oocytes and cytoplasts on the one hand and cytoplasts and karyoplasts on the other.

The results of the polypeptide partitioning studies are shown in Fig. 3. Comparison between intact oocytes and cytoplasts showed consistent differences in three low molecular weight polypeptide classes (Mr 9000, 18000 and 28000). Quantitation by densitometry revealed that the three bands (designated a,b,c) accounted respectively for 0.9% (9K band), 2.9% (18K band) and 6.6% (28K band) of total protein synthesis during the first 4h of maturation in control oocytes. In cytoplasts, the two lowest molecular weight bands (a,b) were not detectable whilst band c (28K) was reduced to 3.4 % of total protein synthesis. Comparison between the cytoplast and nuclear fractions showed that bands a, b, c while absent or low in cytoplasts represented 1.3 %, 6.4% and 9.3 % of the total labelled proteins in the nuclear fraction.

Fig. 3.

Identification of the intracellular location of newly synthesized polypeptides in ovine GV oocytes (1–5) and the distribution of these polypeptides in the cytoplasmic (6–9) and nuclear (10) fractions. Ovine cumulus-enclosed oocytes were removed from their follicles and thereafter labelled for 4h with [35S]methionine. They were then denuded and separated into intact (control) oocytes, cytoplasts and karyoplasts as described in the Materials and methods section. The samples were run on 8–15% SDS-gradient gels and the detectable differences among them in the polypeptide composition are indicated by arrows. Each track in the control and cytoplast groups represented a singly oocyte whilst the nuclear track consisted of 12 karyoplasts.

Fig. 3.

Identification of the intracellular location of newly synthesized polypeptides in ovine GV oocytes (1–5) and the distribution of these polypeptides in the cytoplasmic (6–9) and nuclear (10) fractions. Ovine cumulus-enclosed oocytes were removed from their follicles and thereafter labelled for 4h with [35S]methionine. They were then denuded and separated into intact (control) oocytes, cytoplasts and karyoplasts as described in the Materials and methods section. The samples were run on 8–15% SDS-gradient gels and the detectable differences among them in the polypeptide composition are indicated by arrows. Each track in the control and cytoplast groups represented a singly oocyte whilst the nuclear track consisted of 12 karyoplasts.

In addition to the three nuclear-abundant polypep-tide classes (a–c), various other polypeptides appear both in the nuclear and cytoplasmic fractions. The dual appearance of these labelled proteins is at least in part a reflection of our inability to prepare karyoplasts entirely free of cytoplasmic components. However, it can be concluded from these results that certain newly synthesized polypeptides are selectively sequestered in the oocyte nucleus between the initiation of maturation and GVBD.

Role of the oocyte nucleus on protein synthesis before GVBD

Polypeptide profiles in intact oocytes and cytoplasts during the first five hours of maturation were compared to determine whether the nucleus directs protein synthesis before GVBD. The results presented in Fig. 4 showed that enucleated oocytes synthesize the same major pattern of polypeptides as intact controls before the breakdown of the nuclear membrane. It is therefore concluded that the presence of the nucleus is not essential for the synthesis of proteins by ovine oocytes before GVBD.

Fig. 4.

Fluorograph of SDS-PAGE gel showing polypeptides synthesized by individual intact oocytes (1–4) and cytoplasts (5–8). Ovine oocytes were denuded immediately after removed from the follicles and divided into a control group of intact oocytes and an enucleated group of oocytes from which nuclei had been removed within 1 h of removal from the follicles. The control oocytes and cytoplasts were labelled with [35S]methionine for 5 h and run on 8–15 % SDS gradient gels.

Fig. 4.

Fluorograph of SDS-PAGE gel showing polypeptides synthesized by individual intact oocytes (1–4) and cytoplasts (5–8). Ovine oocytes were denuded immediately after removed from the follicles and divided into a control group of intact oocytes and an enucleated group of oocytes from which nuclei had been removed within 1 h of removal from the follicles. The control oocytes and cytoplasts were labelled with [35S]methionine for 5 h and run on 8–15 % SDS gradient gels.

Role of the oocyte nucleus in protein reprogramming after GVBD

The extensive programme of protein change that normally occurs after GVBD is blocked both in oocytes that fail to undergo germinal vesicle breakdown in culture and in those that are prevented from doing so by the addition of dibutyryl cyclic AMP (Schultz and Wassarman, 1977a). These authors interpreted their data as showing that nucleoplasmic factors released into the oocyte cytoplasm are essential for the progression of the protein reprogramming process during matu-ration. This interpretation was re-examined in our experiments which were designed as before with a control group of intact oocytes and a group of oocytes enucleated within the first hour after removal from the follicles (see Fig. 5A). The results presented in Fig. 5B show that there are no major detectable differences in the polypeptide patterns between the matured oocytes and cytoplasts. However, the close similarities between the Mil and cytoplast polypeptide patterns contrast sharply with the pattern of polypeptides synthesised by control GV oocytes. It is therefore evident that the extensive programme of protein reprogramming that characterises maturation occurs equally in intact and enucleated oocytes. This conclusion is further streng-thened by the transmission electron microscopic studies which show that the enucleation procedures do not damage the nuclear membrane; uncontrolled leakage of nucleoplasmic contents into the cytoplasm during experimental manipulation can therefore be considered a highly unlikely event.

Fig. 5.

(A) An outline of the procedure used to prepare intact oocytes and cytoplasts for studies on the role of the nucleus in protein reprogramming during maturation. Ovine oocytes were denuded immediately after removal from their follicles and then divided into control and experimental groups. The oocytes in the experimental group were enucleated within 1 h of dissection after which both groups were cultured under optima] conditions for 21 h and then labelled for the final 3h of maturation with [35S]methionine. (B) Fluorographs of polypeptides synthesized by individual GV oocytes (1–3) labelled during the first 5 h of maturation, cytoplasts (4–6) and Mil oocytes (7–9) labelled during the final 3h of ovine oocyte maturation. The preparation of the MII oocytes and cytoplasts is outlined in Fig. 5A. All the samples were run on 8–15% SDS gradient gels.

Fig. 5.

(A) An outline of the procedure used to prepare intact oocytes and cytoplasts for studies on the role of the nucleus in protein reprogramming during maturation. Ovine oocytes were denuded immediately after removal from their follicles and then divided into control and experimental groups. The oocytes in the experimental group were enucleated within 1 h of dissection after which both groups were cultured under optima] conditions for 21 h and then labelled for the final 3h of maturation with [35S]methionine. (B) Fluorographs of polypeptides synthesized by individual GV oocytes (1–3) labelled during the first 5 h of maturation, cytoplasts (4–6) and Mil oocytes (7–9) labelled during the final 3h of ovine oocyte maturation. The preparation of the MII oocytes and cytoplasts is outlined in Fig. 5A. All the samples were run on 8–15% SDS gradient gels.

It could be supposed that the factors required for protein reprogramming are synthesized early in matu-ration (see Fig. 3) and are thereafter sequestered in the nucleus where they are inactive. After nuclear mem-brane breakdown (or in cytoplasts), reprogramming may then be facilitated by the interaction of such factors with components in the cytoplasm. The experiments outlined in Fig. 6A were designed to test this possibility by removing the nucleus after protein entry but immediately before membrane disassembly. The results of this experiment (Fig. 6B) demonstrated that the removal of the newly sequestered nuclear proteins just before GVBD did not prevent protein reprogramming.

Fig. 6.

(A) Outline of the procedure for determining the role of nuclear proteins in the reprogramming of protein synthesis during ovine oocyte maturation. Cumulus-enclosed oocytes were cultured for 4h under normal maturation conditions and then denuded and divided into two groups. The oocytes in the experimental group were enucleated between the 4th and 5th hour after explantation, thereafter, the control oocytes and cytoplasts were cultured for another 16 h and then labelled for the final 3 h of maturation with [35S]methionine. (B) Fluorograms of SDS– PAGE gels showing the polypeptides synthesized by individual control oocytes (1–5) and cytoplasts (6–10) labelled during the final 3 h of ovine oocyte maturation. The experiment protocol for this experiment is illustrated in Fig. 6A.

Fig. 6.

(A) Outline of the procedure for determining the role of nuclear proteins in the reprogramming of protein synthesis during ovine oocyte maturation. Cumulus-enclosed oocytes were cultured for 4h under normal maturation conditions and then denuded and divided into two groups. The oocytes in the experimental group were enucleated between the 4th and 5th hour after explantation, thereafter, the control oocytes and cytoplasts were cultured for another 16 h and then labelled for the final 3 h of maturation with [35S]methionine. (B) Fluorograms of SDS– PAGE gels showing the polypeptides synthesized by individual control oocytes (1–5) and cytoplasts (6–10) labelled during the final 3 h of ovine oocyte maturation. The experiment protocol for this experiment is illustrated in Fig. 6A.

In summary, the combined results of the studies described above therefore strongly suggest that the extensive series of translational changes that occur in ovine oocytes during maturation are neither initiated nor sustained by factors from the germinal vesicle.

Role of nuclear components in the acquisition of chromatin condensation capacity in oocytes

To determine whether the capacity for chromatin condensation depends on the mixing of nuclear and cytoplasmic components, interphase nuclei from 8-cell mouse embryos were transplanted into oocytes or cytoplasts cultured previously for 21 h. The morpho-logical changes to the donor nuclei in the reconstituted oocytes were examined 4h after cell fusion.

Tire results showed that fusion between mouse blastomeres and oocytes or cytoplasts occurred within 30 min of pulse treatment. A fusion rate of 80 % (24/30) was observed in the blastomere–oocyte combination and at 4h post-fusion each reconstituted oocyte contained one set of metaphase chromosomes adjacent to the polar body and a set of condensed chromosomes at the opposite pole. This demonstrated that metaphase stage oocytes possess the capacity to induce rapid condensation of interphase chromatin.

In the enucleated experimental group, 77 % (20/26) of cytoplasts fused with mouse blastomeres. Chromo-some analyses 4 h after fusion revealed that three of the fused cytoplasts contained interphase mouse nuclei while, in another three fused cytoplasts, mouse blastomere nuclear membrane breakdown had oc-curred but was accompanied by abnormal chromatin condensation. In the remaining 14 fused cytoplasts (70%), chromatin condensation had occurred in a manner similar to that of the intact controls.

The unfused oocyte-blastomere complexes were also fixed 4 h after pulse treatment for nuclear examination. Each of the unfused oocytes in the control group contained one set of condensed metaphase chromo-somes. By contrast, all (n=12) of the nuclei in the unfused mouse blastomeres contained interphase chromosomes at fixation. It can therefore be concluded that the acquisition of the capacity to condense chromatin occurs in both intact and enucleated oocytes and is not dependent on an interaction between components of the nucleus and cytoplasm.

Mammalian oocytes undergo protein reprogramming after germinal vesicle breakdown, thus raising import-ant questions about the role of the nucleoplasm in the initiation of the process of reprogramming. In Rana oocytes, Smith and Ecker (1969) and Ecker and Smith (1971) reported that enucleation did not alter the reprogramming of protein synthesis that occurs during progesterone-induced maturation. They concluded that the nucleoplasm was therefore not involved in initiating protein reprogramming during the maturation of amphibian oocytes. By contrast, the results of an extensive series of studies on oocyte maturation in mice have been interpreted in the opposite manner (Schultz and Wassarman, 1977a,b; Schultz et al. 1978; Wassar-man et al. 1981; Wassarman, 1988). These authors draw attention firstly to the temporal relationship between GVBD and protein reprogramming, and second to the fact that oocytes that remain arrested at the dictyate stage, whether naturally or by drugs, do not exhibit maturational changes in protein synthesis. Despite the observation that enucleate fragments of mouse oocytes undergo many, but not all of the protein changes associated with maturation (Schultz et al. 1978), it has nevertheless been firmly concluded that protein repro-gramming during mouse oocyte maturation depends upon the mixing of the nucleoplasm and cytoplasm (see Wassarman, 1988).

The present findings on the role of the nucleus on protein reprogramming after the breakdown of the germinal vesicle are in accord with those obtained in amphibia. It is found that, whether the oocytes are enucleated within the first hour of explantation or during the 4th–5th hour of maturation, no detectable quantitative difference in the patterns of the newly synthesized proteins has been observed between the enucleated and intact oocytes. It is concluded therefore that protein reprogramming during maturation does not depend on the mixing of the nucleoplasm and cyto-plasm in the manner postulated by Wassarman (1988) for the mouse. The results of the experiments are equally unable to support the model of Schultz and colleagues (1978) who suggested that reprogramming of synthesis in murine cytoplasts occurs because an initiator protein, which is normally sequestered in the nucleus until GVBD, accumulates in the cytoplasm after enucleation and at threshold levels triggers reprogramming. By removing the GV immediately before breakdown of the membrane, one should remove the entire store of the putative trigger protein(s) thus blocking or delaying reprogramming. The experiments demonstrate that this pre-GVBD enucleation protocol, like that of enucleation at the initiation of maturation, does not arrest normal reprogramming of protein synthesis. We cannot, however, eliminate the formal possibility that the nucleus is involved in the induction of protein changes that are not detected by one-dimensional polypeptide analysis.

In studies on the partitioning of proteins between the nucleus and cytoplasm before GVBD, we find that a significant proportion (5–10%) of newly synthesized proteins is sequestered in the nucleus. The three most abundant sequestered polypeptides have relative mol-ecular masses of 9000, 18000 and 28000, respectively. Dominant amongst these is the 28K polypeptide which accounts for at least 9 % of the total labelled proteins transferred to the germinal vesicle in the first few hours after the initiation of maturation. It is interesting that a polypeptide of similar relative molecular mass accumu-lates in concentrations of 1000-fold greater than that of the cytoplasm in the nuclei of fully grown mouse oocytes that have been arrested at GV stage by dibutyryl cyclic AMP (Wassarman et al. 1979). The similarities in molecular weight between the mouse and sheep 28K nuclear polypeptide are not matched by the apparent time of synthesis of this polypeptide in the two species. Thus, Wassarman and colleagues (1979) reported that the synthesis of the murine 28K nuclear polypeptide was terminated at the time of GVBD. The observations in the sheep show that the ovine 28K polypeptide is synthesized throughout the maturation period; its presence in GV cytoplasts establishes clearly that its synthesis is independent of the nucleus in this species. However, because it has not been possible at present to obtain entirely pure nuclei from mammalian oocytes with no cytoplasmic contamination, these quantitative studies regarding protein translocation from the cytoplasm to the nucleus should be considered as approximations only. Despite the problem of accuracy, the 28K polypeptide is of particular interest because it is the most abundant newly synthesized nuclear polypeptide both in murine and ovine oocytes. It is important that the nature of this polypeptide and its role on oocyte maturation, fertilization and early embryogenesis should be determined in future exper-iments.

Work on amphibian oocytes has not only demon-strated marked differences between the protein compo-sition of the nucleus and cytoplasm but has been extended to a detailed characterisation of many of the GV proteins (Hill et al. 1973; Feldherr, 1975; Merriam and Hill, 1976; Clark and Merriam, 1977; De Robertis et al. 1978; Franke et al. 1979; Mills et al. 1980; Krohne and Franke, 1980). For example, nucleoplasmin, the most abundant nuclear protein, binds histones and transfers them to DNA during nucleosome assembly (Laskey et al. 1978; Mills et al. 1980; Krohne and Franke, 1980; Kleinschmidt et al. 1985; Dilworth et al. 1987). Not only have the nuclear proteins in amphibian oocytes been characterised but their mode of entry into the nucleus has also been analysed. Results have shown that uptake is highly selective (Gurdon, 1970; Bonner, 1975a,b ; De Robertis et al. 1978) and is dependent upon nuclear signal sequences first identified in nucleoplas-min and now known for a variety of nuclear proteins (Krohne and Franke, 1980; Dingwall et al. 1982; Kalderon et al. 1984; Lanford and Butel, 1984; Hall et al. 1984; Davey et al. 1985; Moreland et al. 1985; Lanford et al. 1986; Richardson et al. 1986; Burglin and De Robertis, 1987). Although still imprecisely under-stood, protein entry into the nucleus requires ATP (Newmeyer et al. 1986a,b) and involves two distinct steps; the protein first binds to the nuclear pore and is then translocated into the nucleus (Dingwall and Laskey, 1986; Newmeyer and Forbes, 1988; Richardson et al. 1988). Our objective is to carry out similar studies on nuclear proteins in mammalian oocytes as the required techniques become available.

Studies on ovine oocytes show that the synthesis of new proteins immediately before GVBD is obligatory for the G2 to M transition (Moor and Crosby, 1986). Moreover, the synthesis of proteins before GVBD requires transcriptional activity in the first 2h after the induction of maturation (Osborn and Moor, 1983). These authors were, however, unable with certainty to determine whether transcription was required within the oocyte or only in the associated follicle cells. The present results show that the synthesis of protein before GVBD occurs in enucleated oocytes and is therefore independent of nuclear control.

In additional experiments, it is shown that cytoplasts, in common with their intact controls, develop the ability during maturation to condense chromatin. The conclusion that condensation ability does not depend on components from the GV is in accord with the findings of Ziegler and Masui (1976) on amphibian oocytes and Balakier and Masui (1986) on mouse oocytes. It is well established that chromatin conden-sation ability is detected at the time of nuclear membrane breakdown and is mediated by the ubiqui-tous protein complex called Maturation Promoting Factor (MPF: reviewed by Masui and Clarke, 1979; Tarkowski, 1982; Gerhart et al. 1984). In the mouse the activation of MPF occurs in the absence of both transcription and translation (Wassarman, 1988). How-ever, as indicated previously new protein synthesis is essential for full condensation of chromatin in other mammalian oocytes including those of the sheep (Moor and Crosby, 1986). The enucleation studies in this investigation indicate that transcription cannot be required after the first 30 min following the initiation of maturation. However, we are not able to exclude the possibility that an extremely early and short burst of transcription is required in the oocyte before this time for chromatin condensation. Indeed, Osborn and Moor (1983) showed that early transcription is an obligatory event both in intact follicles and in oocyte–cumulus complexes for the occurrence of chromatin conden-sation and for the disassembly of the nuclear mem-brane. Our present, interpretation, based on the enucleation data and on new evidence from exper-iments on transcriptional inhibition (R.M. Moor, unpublished observations), strongly suggest that the early transcription occurs in the follicle cells rather than in the oocyte. It appears that this somatic trigger initiates translational change within the oocyte which results in the formation of active MPF. The results show further that condensation factors in ovine oocytes are capable of acting in a non-species specific manner on chromatin from mouse blastomeres. Although the capacity to condense chromatin develops in cytoplasts, our experiments were.not designed to test whether the condensed chromatin would thereafter retain stability and the correct spatial arrangement within the develop-ing nucleus. The possible role of GV components on post-condensation organisation within the nucleus offers interesting possibilities for future study.

We thank Xiuying Huang for the electron microscope studies, Stuart Laurie and Neville Clarke for advice and Linda Notton for typing the manuscript.

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