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Oocyte-Specific Modulation of Female Pronuclear Development in Mice
DEVELOPMENTAL BIOLOGY 178, 1–12 (1996) ARTICLE NO. 0193
Oocyte-Specific Modulation of Female Pronuclear Development in Mice J. Fulka, Jr.,*,† M. Horska´,* R. M. Moor,† J. Fulka,* and J. Kanka‡ *Institute of Animal Production, CS-104 00 Prague 10, UhrˇıB neˇves, Czech Republic; †Development and Differentiation Laboratory, The Babraham Institute, Babraham, Cambridge CB2 4AT, United Kingdom; and ‡Czech Academy of Sciences, CS-27721 Libeˇchov, Czech Republic
Cell fusion experiments were undertaken to determine both whether oocytes possess, in a stage-specific manner, suppressors of pronuclear development and whether these factors could be used to modulate female pronuclear development. The fusion of telophase II eggs obtained 2 hr after activation (A2 eggs) to germinal vesicle (GV)-staged oocytes (GV 1 A2) suppresses female pronuclear development. The cytoplasm of GV 1 A2 heterokaryons contains, at 10 hr postfusion, a micropronucleus and a GV which is significantly larger (Çx1.6) than normal. Fusion of early pronucleate eggs (3 hr postactivation, A3) to GV-staged oocytes (GV 1 A3) results in the formation of a retarded half-sized pronucleus and a slightly enlarged (Çx1.2) GV at 10 hr postactivation. Normal pronuclear formation occurs when eggs at 4 hr postactivation (A4) are fused to GV oocytes (GV 1 A4). Although pronuclear size is suppressed in heterokaryons formed when GV-staged oocytes are fused to eggs activated 2–3 hr earlier (A2 or A3 stage), entry into S-phase and DNA synthesis is not inhibited even in the micropronuclei. Meiotic progression in the oocyte germinal vesicle is arrested after fusion to activated eggs throughout the period during which the pronucleus is undergoing G1 and S-phase. However, at the end of S-phase in the pronucleate partner, germinal vesicle breakdown occurs, cell cycle progression is accelerated, and both nuclei reach Mphase by 12 hr postactivation. That suppressors of pronuclear development are found only in GV (G2)-staged oocytes, and not in mitotic cells at the same cell cycle stage, was demonstrated by fusing G2-staged blastomeres to A2-staged eggs (G2 mitotic 1 A2). By contrast to the micropronuclei formed in GV 1 A2 heterokaryons, normal pronuclear development occurred in G2 mitotic 1 A2 heterokaryons. Our results show that suppressor activity, present only in GV oocytes, restricts female pronuclear development, but only in the first 3 hr after activation. We propose to use the ability to modulate either female (this study) or male pronuclear formation in studies (i) on imprinting and (ii) on the developmental consequences of early asynchrony between male and female pronuclear development. q 1996 Academic Press, Inc.
INTRODUCTION The first cleavage division is of particular interest from a cell cycle point of view for a variety of reasons. First, it marks the transition from a meiotic to a mitotic form of cell division. Second, the progression of the first cell cycle is not dependent on transcription but relies instead on mRNA and proteins stored during earlier periods of oocyte development. Third, nuclear development during the first cell cycle is unique because of the specialized requirement of forming two haploid pronuclei instead of a single daughter nucleus. This paper focuses on some of the specialized features of pronuclear formation in mouse eggs. A number of papers over the past two decades have reported on male pronuclear development (see Tarkowski, 1982). These studies show that the development of
this pronucleus can be modulated by a variety of experimental procedures including the fertilization of parthenogenetically activated eggs (Komar, 1982; Tarkowski, 1982; Maleszewski, 1992), the fertilization of anucleate oocyte fragments (Tarkowski, 1980; Borsuk, 1991; Szo¨ llo¨ si et al., 1994), or by the induction of polyspermy when a number of sperm compete for a limited series of factors required for chromatin decondensation and male pronuclear development (Witkowska, 1981). These various degrees of sperm decondensation can also be induced outside the egg by using cell-free cytoplasmic extracts (Montag et al., 1992; Banerjee and Hulten, 1994). These extensive studies on male pronuclear development are in sharp contrast to that associated with the formation of the female pronucleus where no comparable information exists. Under normal conditions female pronuclear development is initiated after the completion of telophase II
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and involves both the gradual decondensation of the highly condensed haploid set of female chromosomes and the reassembly of the nuclear membrane. In mouse eggs these processes are completed and full-sized female pronuclei with well-developed nucleoli are formed by 6 hr after activation. The S-phase starts approximately 8 hr postactivation and lasts 4 – 6 hr (Kaufman, 1983). After a relatively long G2-phase the cell enters the first mitotic metaphase (16 – 18 hr postactivation). The first cell cycle has the major advantage that each of its component phases is sufficiently long to allow for detailed analysis to be undertaken. This is not the case in subsequent cycles in which the G1 and G2 phase are extremely short or absent (Pratt, 1987). Moreover, the first cell cycle is extremely important in current human IVF procedures and in nuclear transplantation in animals. The possible asynchrony between the male and female pronuclei is likely to occur especially with protocols involving intracytoplasmic sperm injection (see Van Steirteghem et al., 1993). Surprisingly little is, however, known at present about the effects on embryonic development of retarding or altering the formation of either the male or the female pronucleus. Since techniques for regulating the male pronucleus already exist, our work has been directed toward the study and manipulation of the female pronucleus in mouse eggs. Our results show first that the development of the female pronucleus is influenced by factors present in meiotic but not mitotic cells. We report second that although pronuclei with sizes ranging from micronuclei to normal dimensions can now be produced, these modifications have no effect on entry into S-phase and the completion of DNA synthesis.
MATERIALS AND METHODS Germinal vesicle (GV)-staged oocytes were released from large antral follicles of PMSG (5 – 10 IU)-treated female mice (F1 C57BL/6J 1 CBA) injected 44 hr previously. After cumulus removal, oocytes containing clearly visible GV were placed in T6 Hepes buffered medium (manipulation medium) containing dbcAMP (150 mg/ml) to prevent spontaneous germinal vesicle breakdown (Cho et al., 1974). Ovulated eggs were isolated from oviducts of mice superovulated by intraperitoneal injection of 5 IU of PMSG and 5 IU of hCG given 44 hr apart. Ovulated eggs were recovered 18 – 20 hr post-hCG, denuded of cumulus cells by brief exposure to Hyaluronidase (1 mg/ml in manipulation medium) and thereafter activated by exposure to 7% ethanol for 7 min. The activated eggs were then cultured in TC199 (280 m0smole) containing Na-pyruvate (0.22 mM ), gentamicin (25 mg/ml), and BSA (3 mg/ml) for 2, 3, or 4 hr at 377C in an atmosphere 5% CO2 in air before fused to GV-staged oocytes (Hogan et al., 1986; Fulka and Moor, 1993). Late two-cell stage embryos (G2 stage) were isolated from superovulated animals mated with CBA males. Embryos were flushed from oviducts approximately 48 hr after hCG injection (Hogan et al., 1986). Fusion between cells was induced by polyethyleneglycol (PEG) exactly as described by Fulka et al. (1992). Briefly, before fusion the zonae pellucidae were dissolved by pronase (0.5%). Contact between one immature and one activated oocyte was achieved in PBS
containing phytohemagglutinin (300 mg/ml), using a narrow bore pipette. Cells were thereafter incubated in PEG solution (0.9 g/ ml in TC199) for 45 sec and then washed in manipulation medium and cultured in TC199 supplemented with dbcAMP for 1 hr before further culture in TC199 without dbcAMP for different time intervals (see Results). Heterokaryons consisting of blastomeres fused to activated eggs were produced in the same way. Blastomeres were separated before fusion by vigorous pipetting after previous preincubation in manipulation medium containing cytochalasin D (5 mg/ml), as described by Fulka et al. (1995a). The following combinations were prepared: (i) GV-staged oocyte fused to an activated egg containing a second polar body (2 PB) and obtained 2 hr postactivation (GV 1 A2); (ii) GV oocyte fused to an activated egg (2 PB) at 3 hr postactivation (GV 1 A3); (iii) GV oocyte fused to an activated egg (2 PB) at 4 hr postactivation (GV 1 A4); and (iv) G2-staged blastomere from a two-cell embryo fused to an activated egg (2 PB) obtained 2 hr postactivation (G2 mitotic 1 A2). In preliminary experiments fused cells were periodically observed under a dissecting microscope to determine the time of M-phase onset. Having established that metaphase plates formed at about 12 hr postactivation all subsequent evaluations were made immediately before (10 hr) or after entry into Mphase (12 hr postactivation). In the first series of evaluations heterokaryons were fixed at 10 hr postactivation in aceto-alcohol (1:3), stained with aceto-orcein, and examined by phase contrast microscopy. The diameter of germinal vesicles and pronuclei in control and fused cells was measured using an ocular micrometer. To study in more detail the clear observed differences in pronuclear size and morphology observed at this time a further series of evaluations were carried out using both confocal and electron microscopy. For confocal microscopy, fused cells were fixed and permeabilized for 1 hr in 4% paraformaldehyde and 0.3% Triton X-100 in PBS, washed in PPB buffer [0.1% polyvinylalcohol (Sigma; MW, 30,000 – 70,000) 1% bovine serum albumin (Sigma; Fraction V) in PBS], and incubated in 0.02% propidium iodide in PPB for 20 min to stain nuclei and chromosomes. Following washing in PPB oocytes were transferred in a minimal volume of fluid to microscope slides mounted in antifade mountant (Vectashield) and examined and the images recorded using a Nikon Diaphot equipped with an MRC 500 laser confocal imaging system (Bio-Rad Laboratories, Cambridge, MA). For electron microscopy, cells were fixed for 60 min (2.5% glutaraldehyde and 0.6% paraformaldehyde in 0.06 cacodylate buffer, pH 7.2), postfixed for 60 min in 1% OsO4 , dehydrated in ethanol, and embedded in Epon. Sections were examined in a Jeol JEM CX II 100 electron microscope (Kanka et al., 1991). Between 12 and 14 hr postactivation fused cells were fixed and evaluated after aceto-orcein staining or using confocal microscopy after tubulin labeling. To assess whether DNA synthesis occurs in control pronuclei and in pronuclei in heterokaryons, cells at 6, 7, and 10 hr postactivation were transferred into medium containing bromodeoxyuridine (100 mM; BrDU) for a 1-hr incubation period before being processed by the method of Campbell et al. (1993). Briefly, after incubation in BrDU supplemented medium, cells were fixed in methanol (0207C) for 20 min, washed in PBS, permeabilized in 0.1% Triton X-100 for 15 min, and hydrolyzed in 4 N HCl for 30 min. Cells were then incubated with primary antibody (rat anti-BrDU; Seralabs, Crawley, UK) diluted 1:10 overnight, washed in PBS, and incubated with secondary antibody (FITC rabbit anti-rat) for 2 hr before being mounted on the glass slides with DABCO and evaluated under a fluorescent microscope.
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Modulation of Female Pronucleus Size
SCHEME 1. A diagrammatic presentation of the experimental protocol and principal results obtained by fusing G2-staged meiotic oocytes (germinal vesicle, GV) or mitotic-staged blastomeres (G2 mitotic) to eggs obtained 2 (A2), 3 (A3), or 4 (A4) hr postactivation (PA). Analysis at 10 hr postactivation showed that while G2 progression in the oocyte or blastomere partner was invariably inhibited, the development of the pronucleus in the egg partner was affected in a highly stage- and cell-specific manner. Pronuclear development was either prevented or retarded when oocytes were fused to eggs at 2 or 3 hr PA, respectively. Fusions to blastomeres or to oocytes at 4 hr PA were without effect on pronuclear development. However, by 12 hr PA all nuclei had entered M-phase irrespective of previous pronuclear size. For comparison normal rates of development are shown from activation (A) through telophase (B), chromatin decondensation (C), full-size pronuclear development (D), initiation of S-phase (E) to entry into the G2-phase of the first mitotic cycle (F).
All chemicals, unless otherwise stated, were purchased from Sigma. Each experiment has been repeated at least three times.
for the first time that the development of the female pronucleus is amenable to experimental manipulation.
RESULTS
Nuclear and Chromatin Changes at 10 hr Postactivation
Scheme 1 summarizes the experimental protocols and principal findings that will be presented in detail in the following three subsections. The results highlight the unique features of the first cleavage cycle and demonstrate
The effect on pronuclear formation of fusing eggs at the early (A2, 2 hr postactivation), mid (A3, 3 hr postactivation), and late (A4, 4 hr postactivation) G1 stages to GV-staged (meiotic) oocytes is summarized in Table 1.
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TABLE 1 Female Pronuclear Development Following the Fusion of Germinal Vesicle (GV)-Staged Oocytes or G2-Staged Blastomeres (G2 Mitotic) to Eggs Obtained at 2 (A2), 3 (A3), or 4 (A4) hr Postactivation (PA) Female pronuclear development (10 hr PA) Cell–fusion combinations
No. cells used
GV 1 A2 GV 1 A3 GV 1 A4 G2 mitotic 1 A2
120 100 117 30
1 1 1 1
120 100 117 30
No. heterokaryons produced
Micropronuclei
Intermediate pronuclei
Normal pronuclei
100 77 107 25
100 10 0 0
0 60 7 0
0 7 100 25
Note. Micropronuclei: membrane-bound chromatin cluster (diameter not assessed). Intermediate pronuclei: standard morphology but half the normal size (Ç10 mm; n Å 52). Normal pronuclei: indistinguishable from unfused controls (Ç20 mm; n Å 78). Diameter of pronuclei in activated control oocytes at 10 hr postactivation: (Ç21 mm; n Å 50). Germinal vesicle diameter: controls, Ç25 mm, n Å 49; heterokaryons, GV 1 A2, Ç40 mm; n Å 80; GV 1 A3, Ç30 mm, n Å 57; GV 1 A4, Ç26 mm, n Å 91.
From the results in Table 1 it is clear that fusions of eggs at 2, 3, or 4 hr after activation to standardized GV-staged oocytes (G2-phase of meiotic cycle) result in profoundly different postfusion development in the first 10 hr after activation. For comparison the size and morphology of control pronuclear (10 hr) and GV-staged eggs are shown in Figs. 1, 1A, and 1B. Early G1 fusion (GV 1 A2). The results of fusing early G1-phased eggs (2 hr postactivation) to standardized GVstaged partners is shown at the light and ultrastructural levels in Figs. 2 and 3 respectively. In the 8 hr following fusion (10 hr postactivation) four striking events occur. First, the posttelophase II chromosomes form into a tight hypercondensed mass rather than undergoing decondensation as would occur in normal activated eggs at a comparable stage. Second, a clear nuclear membrane forms around the hypercondensed chromatin. However, instead of the normal degree of pronuclear enlargement, the nuclear membrane in the early G1 fusions (GV 1 A2) remains tightly associated around the hypercondensed chromatin and no visible enlargement occurs. The fourth notable morphological change contrasts directly with the third because the GVstaged nucleus increases dramatically in size (Ç11.6, see Table 1) and appears to compensate for the absence of pronuclear enlargement. Mid-G1 fusions (GV 1 A3). Delaying fusion until the mid-G1 phase (3 hr after activation) partially reverses all the nuclear changes observed after early G1 fusion. The G1 chromatin becomes fully decondensed and the resultant pronucleus contains nucleoli of normal appearance (see Fig. 5). From a comparison of Figures 2, 4, and 6 it is, however, evident that the size of the pronucleus in heterokaryons prepared at 3 hr postactivation (GV 1 A3) is still smaller than that of controls (see Table 1). Indeed, light and ultrastructural determinations suggest that pronuclei formed after mid-G1 fusions reach approximately half the size of those in control eggs. It must be, however, noted that a slight enlargement of GV diameter could also be detected (Ç11.2).
Late G1 fusions (GV 1 A4). Both the size and the ultrastructural appearance of pronuclei in heterokaryons formed from eggs obtained 4 hr after activation (GV 1 A4) were indistinguishable at 10 hr from those of controls (see Figs. 1, 1A, 1B, 6, and 7). Fusion of G2 mitotic blastomers with early G1 eggs (G2 mitotic 1 A2). Experiments were undertaken to determine whether the aberrant development of both the pronucleus and the germinal vesicle observed in the GV 1 A2 heterokaryons was specific to meiotic G2 oocytes or whether it was a characteristic of G2 mitotic 1 A2 fusions as well. That the observed aberrant pronuclear development in G1 eggs is induced only by meiotic partners is clear from a comparison of Figs. 2 and 3 (GV 1 A2) with Figs. 8 and 9 (G2-mitotic 1 A2). Neither decondensation nor pronuclear enlargement in A2-staged early (early G1) eggs was prevented by fusion to mitotic cells at the G2-phase of the cycle. By comparison pronuclear formation was completely inhibited by fusion to meiotic G2 cells. We therefore interpret our results as indicating that the GV-staged oocyte has the unique capacity, not present in mitotic cells, of suppressing early pronuclear development.
DNA Synthesis and Cell Cycle Progression Cell cycle events in 10 hr postactivation. Measurement of DNA synthesis in the nuclei of heterokaryons (see Table 2) provided two sets of unambiguous results. First, irrespective of whether the chromatin was in a highly condensed or decondensed form, DNA synthesis invariably occurred in pronuclei derived from activated eggs. The intensity of fluorescence was roughly the same in all types of pronuclei labeled. Second, DNA synthesis was never induced in the GV- or G2-staged mitotic nucleus. Thus, the effect of GV-staged oocytes on pronuclear development was restricted to influencing the chromatin configuration and nuclear morphology of the G1 eggs but did not extend to DNA replication. DNA synthesis could first be detected in pronuclear controls (6/20) and in pronuclei
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Modulation of Female Pronucleus Size
FIG. 1. A pair of unfused (control) cells. Pronuclear egg (top) at 10 hr postinduction of activation; GV-staged oocyte (bottom) is freshly isolated. Phase contrast. Bar, 5 mm. (A) An electron micrograph of a 10-hr-old pronucleus with a typical nucleolus and intact membrane. 15200. (B) An electron micrograph of a GV-staged oocyte. The compact nucleolus and nuclear membrane are evident. 12400.
in fused cells at 6 hr postinduction of activation (GV 1 A2, 5/17; GV 1 A3, 4/16; GV 1 A4, 3/12) and consistently at 7 hr after activation (controls, 8/10; GV 1 A2, 10/12; GV 1 A3, 11/12; GV 1 A4, 12/15). This indicates that the length of S-phase, which lasted at least 4 hr, is unaffected by fusion of activated eggs to GV-staged oocytes. By contrast, the action of the G1:S-staged egg partner was, invariably, to block progression of the meiotic cycle of the oocyte for the first 10–12 hr after fusion. Cell cycle events at 12 hr postactivation. In normally
activated eggs, replication is completed by 12 hr postactivation and after a 6-hr G2-phase, entry into the first mitotic M-phase occurs (Kaufman, 1983). The entry into M-phase in all the fusion combinations was both synchronous and significantly advanced with respect to the controls. In every combination involving a GV-staged oocyte and a pronucleate egg, entry into meiosis occurred at, or soon after, 12 hr postactivation, a time that corresponds closely to the completion of the S-phase in the activated egg partner. That the S-phase was invariably completed by 12 hr after fusion
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FIG. 2. Fusion of germinal vesicle-staged oocytes to eggs 2 hr after activation (GV 1 A2) prevents decondensation of chromatin originating from the early activated egg. While the GV is significantly enlarged, chromatin from the A2-staged egg persists as a condensed cluster within a micropronucleus even after 10 hr in culture. PB, second polar body; GV, germinal vesicle; C, cluster of chromatin within the micropronucleus. Phase contrast. Bar, 4.5 mm.
FIG. 4. Fusion of GV-staged oocytes to eggs 3 hr after activation (GV 1 A3) results at 10 hr after activation in chromatin decondensation, nucleolar formation, and the moderate enlargement of the egg pronucleus (PN) to about half the size of a fully formed pronucleus. The germinal vesicle (GV) from the oocyte partner persists at an almost normal size through the culture period. Phase contrast. Bar, 4 mm.
FIG. 3. An electron micrograph of a micropronucleus in a GV 1 A2 heterokaryon. A cluster of condensed chromatin enclosed within a nuclear membrane is shown. 110,000.
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Modulation of Female Pronucleus Size
FIG. 5. An electron micrograph of an intermediate-sized female pronucleus (PN) and a normal-sized germinal vesicle (GV) in a heterokaryon produced by the fusion of a GV-staged oocyte to an egg 3 hr after activation (GV 1 A3). Apart from the 50% reduction in pronuclear size both the GV and the PN show a normal ultrastructural configuration. 12400.
was evident from an analysis of chromosome structure (Figs. 10 and 11). Neither single-stranded chromosomes nor structures typical of S-phase prematurely condensed chro-
mosomes (Rao et al., 1977) were ever observed. In our fused cells the chromosome morphology invariably resembled that of either mitotic or G2-phase prematurely condensed chromosomes (PCC).
DISCUSSION
FIG. 6. Fusion of GV-staged oocytes to eggs 4 hr after activation results at 10 hr postactivation in the formation of a normal female pronucleus (PN) and the persistence of a standard germinal vesicle (GV). Phase contrast. Bar, 5 mm.
Although it is generally accepted that all the processes associated with fertilization must be perfectly orchestrated in order to ensure normal embryonic development, no definitive information exists on the degree to which development is compromised by asynchronous pronuclear development. Apart from its scientific interest this information is crucially important now that sperm injection procedures are being progressively more widely used in human IVF programs. Indirect evidence suggests that abnormalities in sperm head decondensation result in poor subsequent development (Ogura and Yanagimachi, 1995). However, the correlation between asynchronous pronuclear development and embryonic development has not been subjected to rigorous scientific study even though the capacity to manipulate male pronuclear development has existed for more than a decade (see Tarkowski, 1982). The present paper not only provides the reciprocal capacity, namely, the ability to alter the development of the female pronucleus, but also highlights a number of specialized features of the first cleavage cycle. The ability of mammalian oocytes to transform fertilizing sperm into male pronuclei is acquired in a stage-specific
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FIG. 7. An electron micrograph of a normal female pronucleus (PN) with typical nucleoli and intact membranes and a germinal vesicle (GV) in a heterokaryon formed by the fusion of a GV-staged oocyte with an egg 4 hr after fusion. 12400.
manner. When penetration occurs at the germinal vesicle stage sperm remain intact in the ooplasm until nuclear membrane breakdown (GVBD), when decondensation but
FIG. 8. Fusion of G2-staged blastomeres (G2 mitotic) to eggs 2 hr (A2) after activation (G2 mitotic 1 A2) results at 10 hr postactivation in normal pronuclei which are under phase contrast indistinguishable from their blastomere nuclei partners. Phase contrast. Bar, 4 mm.
no pronuclear formation occurs (Szo¨llo¨si et al., 1990). A further set of stimuli, generated only by egg activation, are required for the induction of male pronuclear formation (Borsuk, 1991). Even after activation, molecules required for full male pronuclear formation are present for a short period only (Borsuk and Tarkowski, 1989). A delay of as little as 30 min between experimental activation and sperm entry results in the formation of an aberrant male pronucleus. When the delay is extended to 3 hr the resultant pronucleus is very small and with longer delays male pronuclear formation is entirely inhibited. Furthermore, Borsuk (1991) has established that the processes of decondensation and male pronuclear formation utilize two entirely different sets of controlling molecules. The first, required for sperm decondensation, is MPF-kinase dependent and present in metaphase II oocytes and is lost at the time of MPF-kinase inactivation. The ability to convert the decondensed sperm into a male pronucleus requires a second stimulus which is independent of MPF-kinase and persists for about 8 hr postactivation. Work on the molecular basis of male pronuclear formation has been paralleled by the development of techniques for the manipulation of male pronuclear size (Tarkowski, 1980). By contrast, no comparable experiments have been carried out on either the formation or the manipulation of the female pronucleus. Results presented in this paper show that the female pronucleus is highly susceptible to manipulation but only during the first 4 hr of development. The size of pronuclei in eggs which did not fuse (fixed 45 min postinduction of fusion) was basically the same as the size of pronuclei in corresponding heterokaryons fixed at 10 hr. This indicates that the swelling of pronuclei is suppressed after fusion of
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Modulation of Female Pronucleus Size
FIG. 9. An electron micrograph showing the characteristic normal ultrastructural appearance of both the pronucleus and the blastomere nucleus in a heterokaryon produced by the fusion of one G2-staged blastomere from a late 2-cell embryo (G2 mitotic) with an egg activated 2 hr previously (A2). 12400.
activated eggs to GV-staged oocytes. In addition, four other features of our findings warrant discussion. The first concerns the type of cellular environment capable of restricting female pronuclear size while the second relates to the effect of cell fusion on DNA synthesis in pronuclei of different sizes. The third point focuses on the effect of the activated egg on meiotic progression in the oocyte partner and the fourth considers the new opportunities arising from the capacity to experimentally manipulate pronuclear development. Having established that a GV-staged meiotic partner blocks female pronuclear formation when fused to an early activated egg we next questioned whether mitotic cells, at
a corresponding G2 cell cycle stage, would impose a similar pronuclear block on newly activated oocytes. The results of fusing G2-staged blastomeres to activated eggs showed clearly that the block to female pronuclear formation is strictly oocyte specific. It is instructive in this regard to contrast the respective nuclear responses of both the meiotic and the mitotic G2-staged cells to fusion with a recently activated (A2) partner. The significant increase in the size of the germinal vesicle following fusion to an A2staged egg partner suggests that in these heterokaryons all the nuclear proteins are preferentially sequestered in the oocyte nucleus to the detriment of the newly forming pronucleus (see Merriam, 1969). This is perhaps not surprising
TABLE 2 DNA Synthesis and Chromosomal Configuration in the Nuclei of Oocyte and Egg Partners Examined at 10 or 12 hr after Egg Activation BrDU staining (10 h PA)
Cell cycle (10 hr PA)
Cell fusion combinations
Number treated heterokaryons
Oocyte GV
Egg PN
Oocyte
GV 1 A2 GV 1 A3 GV 1 A4 G2 mitotic 1 A2
17 18 27 —
0 0 0 —
15 16 25 —
G2 G2 G2 G2
Cell cycle (12 hr PA)
Egg S S S S
(micro pn) (retarded pn) (normal pn) (normal pn)
Oocyte
Egg
M M M M
M M M M
Note. Fusion of either oocyte partners at the germinal vesicle (GV) or G2-staged blastomeres (G2 mitotic) to egg partners obtained 2 (A2), 3 (A3), or 4 (A4) hr postactivation (PA). DNA synthesis, determined by bromodeoxy-uridine uptake (BrDU), and cell cycle progression in both the G2 (GV) and the egg (pronuclei, pn) nuclei are shown.
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FIG. 10. The onset of M-phase occurred approximately 12 hr after activation in all the fusion combinations studied. Typically, one common group of chromosomes was formed containing both the oocyte (arrow) and activated egg chromosomes (arrowheads). Phase contrast. Aceto-orcein staining. 11000.
since it is well established that nuclear proteins, injected into the cytoplasm, are thereafter rapidly translocated to the nucleus (Gurdon, 1970; De Robertis et al., 1978). Included among the proteins which probably locate preferentially in the mammalian cell nucleus are nucleoplasmin-like acidic proteins (Philpott and Leno, 1992), histones, and topoisomerase II, the latter fulfilling both a catalytic and a structural role in chromatin condensation (Adachi et al., 1991). The more difficult current problem is to explain why this postulated preferential sequestering of nuclear proteins does not appear to occur when G2-staged mitotic cells (blastomeres) are fused to early activated (A2) eggs. Our results show that in these heterokaryons the blastomere nuclei do not increase in size and the egg pronucleus develops normally. It will be interesting, at least, to determine whether labeled proteins in newly activated eggs are transported more readily into the nuclei of oocyte fusion partners than into mitotic nuclei after blastomere fusion. Since the content of the GV is known to be required for pronuclear formation it is likely that proteins mentioned above together with some additional GV factors accumulate in newly formed pronuclei. These proteins are necessary for the dispersion of chromatin and its further decondensation. However, the exact nature and role in the process of pronuclear formation are at present unknown. While we show that the size of the female pronucleus can be experimentally manipulated, it is nevertheless notable that DNA synthesis proceeds in an uninterrupted manner even in the smallest sized pronuclei. The onset of S-
phase at 7 hr after activation indicates that this phase lasts at least 4–5 hr in fused cells. The morphology of the two sets of condensed chromosomes at 12–14 hr postactivation provides strong evidence that S-phase is fully completed in our fused cell heterokaryons before progression through G2to M-phase occurs. In no case did we observe the singlestranded structures nor the morphology typical of S-phase prematurely condensed chromosomes (Rao et al., 1977). The morphology of both sets of condensed chromosomes in our fused heterokaryons always resembled the morphology typical of chromosomes in mitosis or prematurely condensed in G2. These findings are in accord with earlier results which demonstrated the DNA synthesis is not suppressed when GV oocytes are fused to S-phase four-cell-stage blastomeres (Fulka et al., 1995b). Thus, while GV oocytes have no DNA suppressive activity, the situation is completely reversed after germinal vesicle breakdown. When S-phase nuclei are fused with such maturing meiotic cytoplasts containing low chromosome condensation activity two events occur. First, DNA synthesis in the S-phase partner is totally suppressed and second, premature chromosome condensation does not occur (Trefil et al., 1995; Fulka et al., 1995c). The effects of the oocyte on female pronuclear size and DNA synthesis in the activated egg are matched by a reciprocal action of the egg on meiotic progression in the oocyte. However, the effects of mitotic partners on meiosis are not uniform and appear to differ depending on the cleavage cycle from which the mitotic cell was obtained. Thus, when a GV-staged oocyte is fused to an S-phase blastomere from a four-cell embryo the onset of GVBD is not delayed (Fulka et al., 1995b). By contrast an entirely different response occurs when GV oocytes are fused to S-phase eggs from the first cleavage cycle. The present experiments demonstrate that in these circumstances cell cycle progression in the oocyte
FIG. 11. The formation of a true spindle during M-phase chromosome organization in heterkaryons at 12 hr postactivation was demonstrated using propidium iodide staining of the chromosomes and confocal microscopy. Original magnification, 11200.
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Modulation of Female Pronucleus Size
is blocked and GVBD occurs only when the activated egg finishes DNA synthesis. This highlights the specificity of the first cell cycle with its protracted G1-phase. Both Adlakha and colleagues (1983) and Balakier and Masui (1986) suggest that this delay is induced by mitotic inhibitors specific for G1-phase cells. The activity of these inhibitors is thought to gradually decrease as the cell cycle progresses toward the G2-phase. Evidently, the block of germinal vesicle breakdown in heterokaryons must be different from that induced by cAMP elevating agents (Eppig, 1993) and its nature is difficult to explain at present. However, when heterokaryons were incubated in medium containing dbcAMP for up to 20 hr no nuclear membrane breakdown and chromosome condensation were detected (not shown). The capacity to selectively manipulate the size of the female pronucleus, while not preventing normal S-phase progression, provides a number of experimental opportunities. The first is to seek explanations for some unexpected phenomena arising from our present findings. For example, why is female pronuclear development limited by G2staged oocytes but not by comparable G2-phased blastomeres? Equally, why do GV nuclei, but not blastomere nuclei at the same G2-phase of the cell cycle, increase significantly in size when fused to a newly activated egg? The second set of new opportunities relates to the current availability of techniques to alter the size of either the male or the female pronucleus. These techniques will provide the means of correlating embryo development with early events associated with pronuclear formation. Pronuclear manipulations will also be useful in certain epigenetic studies (W. Reik and W. Dean, personal discussion). For example, these authors have already shown that the adult phenotype in mice is affected by epigenetic factors (Reik et al., 1993). These authors postulate that some epigenetic effects are likely to be induced by abnormal pronuclear development. We suggest that a combination of the approach presented in this paper together with those associated with male pronuclear manipulation provides a good starting point for future epigenetic studies.
ACKNOWLEDGMENTS J.F. Jr. is supported by GACR 505/95/1371. This publication is also based in part on work sponsored by the U.S.–Czech Science and Technology Joint Fund in cooperation with USDA under Project 95047. We thank Dianne Styles and Linda Notton for editorial assistance and Caroline Lee for carrying out the confocal analysis.
REFERENCES Adachi, Y., Luke, M., and Laemmli, U. K. (1991). Chromosome assembly in vitro. Topoisomerase II is required for condensation. Cell 64, 137–148. Adlakha, R. C., Sahasrabuddhe, C. G., Wright, D. A., and Rao, P. N. (1983). Evidence for the presence of inhibitors of mitotic factors during G1 period in mammalian cells. J. Cell Biol. 97, 1707– 1713.
Balakier, H., and Masui, Y. (1986). Interactions between metaphase and interphase factors in heterokaryons produced by fusion of mouse oocytes and zygotes. Dev. Biol. 117, 102–108. Banerjee, S., and Hulten, M. (1994). Sperm nuclear chromatin transformations in somatic cell-free extracts. Mol. Reprod. Dev. 37, 305–317. Borsuk, E. (1991). Anucleate fragments of parthenogenetic eggs and of maturing oocytes contain complementary factors required for development of a male pronucleus. Mol. Reprod. Dev. 29, 150– 156. Borsuk, E., and Tarkowski, A. K. (1989). Transformation of sperm nuclei into male pronuclei in nucleate and anucleate fragments of parthenogenetic mouse eggs. Gamete Res. 24, 471–481. Campbell, K. H. S., Ritchie, W. A., and Wilmut, I. (1993). Nuclearcytoplasmic interactions during the first cell cycle of nuclear transfer reconstructed bovine embryos: Implications for deoxyribonucleic acid replication and development. Biol. Reprod. 49, 933–942. Cho, W. K., Stern, S., and Biggers, J. D. (1974). Inhibitory effect of dibutyryl cAMP on mouse oocyte maturation in vitro. J. Exp. Zool. 187, 383–386. De Robertis, E. M., Longthorne, R. F., and Gurdon, J. B. (1978). Intracellular migration of nuclear proteins in Xenopus oocytes. Nature 272, 254–256. Eppig, J. J. (1993). Regulation of mammalian oocyte maturation. In ‘‘The Ovary’’ (E. Y. Adashi and P. C. K. Leung, Eds.), pp. 185– 208. Raven Press, New York. Fulka, J., Jr., Jung, T., and Moor, R. M. (1992). The fall of biological maturation promoting factor (MPF) and histone H1 kinase activity during anaphase and telophase in mouse oocytes. Mol. Reprod. Dev. 32, 378–382. Fulka, J., Jr., and Moor, R. M. (1993). Non-invasive chemical enucleation of mouse oocytes. Mol. Reprod. Dev. 34, 427–430. Fulka, J., Jr., Moor, R. M., and Fulka, J. (1995a). Electrofusion of mammalian oocytes and embryonic cells. In ‘‘Methods in Molecular Biology, Animal Cell Electroporation and Electrofusion Protocols’’ (J. A. Nickoloff, Ed.), Vol. 48, pp. 309–316. Humana Press, Totowa, NJ. Fulka, J., Jr., Moor, R. M., and Fulka, J. (1995b). Replicating DNA does not block germinal vesicle breakdown in mouse oocytes. J. Exp. Zool. 272, 245–248. Fulka, J., Jr., Ouhibi, N., Fulka, J., Kanka, J., and Moor, R. M. (1995c). Chromosome condensation activity (CCA) in bisected C57BL/6JxCBA mouse oocytes. Reprod. Fertil. Dev. 7, 1123– 1127. Gurdon, J. B. (1970). Nuclear transplantation and the control of gene activity in animal development. Proc. R. Soc. Lond. Ser. B. 176, 303–314. Hogan, B., Constantini, F., and Lacy, E. (1986). ‘‘Manipulating the Mouse Embryo: A Laboratory Manual,’’ p. 315. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Kanka, J., Fulka, J., Jr., Fulka, J., and Petr, J. (1991). Nuclear transplantation in bovine embryos: Fine structural and autoradiographic studies. Mol. Reprod. Dev. 29, 110–116. Kaufman, M. H. (1983). Early mammalian development: Parthenogenetic studies. In ‘‘Developmental and Cell Biology Series’’ (P. W. Barlow, P. B. Green, and C. C. Wylie, Eds.), Vol. 14. Cambridge Univ. Press, Cambridge, UK. Komar, A. (1982). Fertilization of parthenogenetically activated mouse eggs. I. Behaviour of sperm nuclei in the cytoplasm of parthenogenetically activated eggs. Exp. Cell Res. 139, 361–367. Maleszewski, M. (1992). Behavior of sperm nuclei incorporated into parthenogenetic mouse eggs prior to the first cleavage division. Mol. Reprod. Dev. 33, 215–221.
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Merriam, R. W. (1969). Movement of cytoplasmic proteins into nuclei induced to enlarge and initiate DNA or RNA synthesis. J. Cell Sci. 5, 333–349. Montag, M., Tok, V., Liow, S. L., Bongso, A., and Ng, S. C. (1992). In vitro decondensation of mammalian sperm and subsequent formation of pronuclei-like structures for micromanipulation. Mol. Reprod. Dev. 33, 338–346. Ogura, A., and Yanagimachi (1995). Spermatids as male gametes. Reprod. Fertil. Dev. 7, 155–159. Philpott, A., and Leno, G. H. (1992). Nucleoplasmin remodels sperm chromatin in Xenopus egg extracts. Cell 69, 759–767. Pratt, H. P. M. (1987). Isolation, culture and manipulation of preimplantation mouse embryos. In ‘‘Mammalian Development: A Practical Approach’’ (M. Monk, Ed.), pp. 13–42. IRL Press, Oxford/Washington, DC. Rao, P. N., Wilson, B., and Puck, T. T. (1977). Premature chromosome condensation and cell cycle analysis. J. Cell Physiol. 91, 131–142. Reik, W., Ro¨mer, I., Barton, S. C., Surani, M. H., Howlett, S. K., and Klose, J. (1993). Adult phenotype in the mouse can be affected by epigenetic events in the early embryo. Development 119, 933– 942. Szo¨llo¨si, D., Szo¨llo¨si, M. S., Czolowska, R., and Tarkowski, A. K. (1990). Sperm penetration into immature mouse oocytes and nu-
clear changes during maturation: An EM study. Biol. Cell 69, 53–64. Szo¨llo¨si, M. S., Borsuk, E., and Szo¨llo¨si, D. (1994). Relationship between sperm nucleus remodelling and cell cycle progression of fragments of mouse parthenogenotes. Mol. Reprod. Dev. 37, 146–156. Tarkowski, A. K. (1980). Fertilization of nucleate and anucleate egg fragments in the mouse. Exp. Cell Res. 128, 73–77. Tarkowski, A. K. (1982). Nucleo-cytoplasmic interactions in oogenesis and early embryogenesis in the mouse. In ‘‘Progress in Clinical and Biological Research’’ (M. M. Burger and R. Weber, Eds.), Vol. 85 A, pp. 407–416. A. R. Liss, New York. Trefil, P., Horska´, M., Kanka, J., Fulka, J., Jirmanova´, J., Moor, R. M., and Fulka, J., Jr. (1995). Response of avian nuclei to mammalian maturation promoting factor (MPF). Zygote 3, 289–294. Van Steirteghem, A. C., Liu, J., Noris, H., Nagy, Z., Janssenswillen, C., Tournaye, H., Derde, M.-P., Van Assche, E., and Devroey, P. (1993). Higher success rate by intracytoplasmic sperm injection than by subzonal insemination. Report of a second series of 300 consecutive treatment cycles. Hum. Reprod. 8, 1955–1960. Witkowska, A. (1981). Pronuclear development and the first cleavage division in polyspermic mouse eggs. J. Reprod. Fertil. 62, 493–498. Received for publication March 13, 1996 Accepted May 2, 1996
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Oocyte-Specific Modulation of Female Pronuclear Development in Mice J. Fulka, Jr.,*,† M. Horska´,* R. M. Moor,† J. Fulka,* and J. Kanka‡ *Institute of Animal Production, CS-104 00 Prague 10, UhrˇıB neˇves, Czech Republic; †Development and Differentiation Laboratory, The Babraham Institute, Babraham, Cambridge CB2 4AT, United Kingdom; and ‡Czech Academy of Sciences, CS-27721 Libeˇchov, Czech Republic
Cell fusion experiments were undertaken to determine both whether oocytes possess, in a stage-specific manner, suppressors of pronuclear development and whether these factors could be used to modulate female pronuclear development. The fusion of telophase II eggs obtained 2 hr after activation (A2 eggs) to germinal vesicle (GV)-staged oocytes (GV 1 A2) suppresses female pronuclear development. The cytoplasm of GV 1 A2 heterokaryons contains, at 10 hr postfusion, a micropronucleus and a GV which is significantly larger (Çx1.6) than normal. Fusion of early pronucleate eggs (3 hr postactivation, A3) to GV-staged oocytes (GV 1 A3) results in the formation of a retarded half-sized pronucleus and a slightly enlarged (Çx1.2) GV at 10 hr postactivation. Normal pronuclear formation occurs when eggs at 4 hr postactivation (A4) are fused to GV oocytes (GV 1 A4). Although pronuclear size is suppressed in heterokaryons formed when GV-staged oocytes are fused to eggs activated 2–3 hr earlier (A2 or A3 stage), entry into S-phase and DNA synthesis is not inhibited even in the micropronuclei. Meiotic progression in the oocyte germinal vesicle is arrested after fusion to activated eggs throughout the period during which the pronucleus is undergoing G1 and S-phase. However, at the end of S-phase in the pronucleate partner, germinal vesicle breakdown occurs, cell cycle progression is accelerated, and both nuclei reach Mphase by 12 hr postactivation. That suppressors of pronuclear development are found only in GV (G2)-staged oocytes, and not in mitotic cells at the same cell cycle stage, was demonstrated by fusing G2-staged blastomeres to A2-staged eggs (G2 mitotic 1 A2). By contrast to the micropronuclei formed in GV 1 A2 heterokaryons, normal pronuclear development occurred in G2 mitotic 1 A2 heterokaryons. Our results show that suppressor activity, present only in GV oocytes, restricts female pronuclear development, but only in the first 3 hr after activation. We propose to use the ability to modulate either female (this study) or male pronuclear formation in studies (i) on imprinting and (ii) on the developmental consequences of early asynchrony between male and female pronuclear development. q 1996 Academic Press, Inc.
INTRODUCTION The first cleavage division is of particular interest from a cell cycle point of view for a variety of reasons. First, it marks the transition from a meiotic to a mitotic form of cell division. Second, the progression of the first cell cycle is not dependent on transcription but relies instead on mRNA and proteins stored during earlier periods of oocyte development. Third, nuclear development during the first cell cycle is unique because of the specialized requirement of forming two haploid pronuclei instead of a single daughter nucleus. This paper focuses on some of the specialized features of pronuclear formation in mouse eggs. A number of papers over the past two decades have reported on male pronuclear development (see Tarkowski, 1982). These studies show that the development of
this pronucleus can be modulated by a variety of experimental procedures including the fertilization of parthenogenetically activated eggs (Komar, 1982; Tarkowski, 1982; Maleszewski, 1992), the fertilization of anucleate oocyte fragments (Tarkowski, 1980; Borsuk, 1991; Szo¨ llo¨ si et al., 1994), or by the induction of polyspermy when a number of sperm compete for a limited series of factors required for chromatin decondensation and male pronuclear development (Witkowska, 1981). These various degrees of sperm decondensation can also be induced outside the egg by using cell-free cytoplasmic extracts (Montag et al., 1992; Banerjee and Hulten, 1994). These extensive studies on male pronuclear development are in sharp contrast to that associated with the formation of the female pronucleus where no comparable information exists. Under normal conditions female pronuclear development is initiated after the completion of telophase II
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and involves both the gradual decondensation of the highly condensed haploid set of female chromosomes and the reassembly of the nuclear membrane. In mouse eggs these processes are completed and full-sized female pronuclei with well-developed nucleoli are formed by 6 hr after activation. The S-phase starts approximately 8 hr postactivation and lasts 4 – 6 hr (Kaufman, 1983). After a relatively long G2-phase the cell enters the first mitotic metaphase (16 – 18 hr postactivation). The first cell cycle has the major advantage that each of its component phases is sufficiently long to allow for detailed analysis to be undertaken. This is not the case in subsequent cycles in which the G1 and G2 phase are extremely short or absent (Pratt, 1987). Moreover, the first cell cycle is extremely important in current human IVF procedures and in nuclear transplantation in animals. The possible asynchrony between the male and female pronuclei is likely to occur especially with protocols involving intracytoplasmic sperm injection (see Van Steirteghem et al., 1993). Surprisingly little is, however, known at present about the effects on embryonic development of retarding or altering the formation of either the male or the female pronucleus. Since techniques for regulating the male pronucleus already exist, our work has been directed toward the study and manipulation of the female pronucleus in mouse eggs. Our results show first that the development of the female pronucleus is influenced by factors present in meiotic but not mitotic cells. We report second that although pronuclei with sizes ranging from micronuclei to normal dimensions can now be produced, these modifications have no effect on entry into S-phase and the completion of DNA synthesis.
MATERIALS AND METHODS Germinal vesicle (GV)-staged oocytes were released from large antral follicles of PMSG (5 – 10 IU)-treated female mice (F1 C57BL/6J 1 CBA) injected 44 hr previously. After cumulus removal, oocytes containing clearly visible GV were placed in T6 Hepes buffered medium (manipulation medium) containing dbcAMP (150 mg/ml) to prevent spontaneous germinal vesicle breakdown (Cho et al., 1974). Ovulated eggs were isolated from oviducts of mice superovulated by intraperitoneal injection of 5 IU of PMSG and 5 IU of hCG given 44 hr apart. Ovulated eggs were recovered 18 – 20 hr post-hCG, denuded of cumulus cells by brief exposure to Hyaluronidase (1 mg/ml in manipulation medium) and thereafter activated by exposure to 7% ethanol for 7 min. The activated eggs were then cultured in TC199 (280 m0smole) containing Na-pyruvate (0.22 mM ), gentamicin (25 mg/ml), and BSA (3 mg/ml) for 2, 3, or 4 hr at 377C in an atmosphere 5% CO2 in air before fused to GV-staged oocytes (Hogan et al., 1986; Fulka and Moor, 1993). Late two-cell stage embryos (G2 stage) were isolated from superovulated animals mated with CBA males. Embryos were flushed from oviducts approximately 48 hr after hCG injection (Hogan et al., 1986). Fusion between cells was induced by polyethyleneglycol (PEG) exactly as described by Fulka et al. (1992). Briefly, before fusion the zonae pellucidae were dissolved by pronase (0.5%). Contact between one immature and one activated oocyte was achieved in PBS
containing phytohemagglutinin (300 mg/ml), using a narrow bore pipette. Cells were thereafter incubated in PEG solution (0.9 g/ ml in TC199) for 45 sec and then washed in manipulation medium and cultured in TC199 supplemented with dbcAMP for 1 hr before further culture in TC199 without dbcAMP for different time intervals (see Results). Heterokaryons consisting of blastomeres fused to activated eggs were produced in the same way. Blastomeres were separated before fusion by vigorous pipetting after previous preincubation in manipulation medium containing cytochalasin D (5 mg/ml), as described by Fulka et al. (1995a). The following combinations were prepared: (i) GV-staged oocyte fused to an activated egg containing a second polar body (2 PB) and obtained 2 hr postactivation (GV 1 A2); (ii) GV oocyte fused to an activated egg (2 PB) at 3 hr postactivation (GV 1 A3); (iii) GV oocyte fused to an activated egg (2 PB) at 4 hr postactivation (GV 1 A4); and (iv) G2-staged blastomere from a two-cell embryo fused to an activated egg (2 PB) obtained 2 hr postactivation (G2 mitotic 1 A2). In preliminary experiments fused cells were periodically observed under a dissecting microscope to determine the time of M-phase onset. Having established that metaphase plates formed at about 12 hr postactivation all subsequent evaluations were made immediately before (10 hr) or after entry into Mphase (12 hr postactivation). In the first series of evaluations heterokaryons were fixed at 10 hr postactivation in aceto-alcohol (1:3), stained with aceto-orcein, and examined by phase contrast microscopy. The diameter of germinal vesicles and pronuclei in control and fused cells was measured using an ocular micrometer. To study in more detail the clear observed differences in pronuclear size and morphology observed at this time a further series of evaluations were carried out using both confocal and electron microscopy. For confocal microscopy, fused cells were fixed and permeabilized for 1 hr in 4% paraformaldehyde and 0.3% Triton X-100 in PBS, washed in PPB buffer [0.1% polyvinylalcohol (Sigma; MW, 30,000 – 70,000) 1% bovine serum albumin (Sigma; Fraction V) in PBS], and incubated in 0.02% propidium iodide in PPB for 20 min to stain nuclei and chromosomes. Following washing in PPB oocytes were transferred in a minimal volume of fluid to microscope slides mounted in antifade mountant (Vectashield) and examined and the images recorded using a Nikon Diaphot equipped with an MRC 500 laser confocal imaging system (Bio-Rad Laboratories, Cambridge, MA). For electron microscopy, cells were fixed for 60 min (2.5% glutaraldehyde and 0.6% paraformaldehyde in 0.06 cacodylate buffer, pH 7.2), postfixed for 60 min in 1% OsO4 , dehydrated in ethanol, and embedded in Epon. Sections were examined in a Jeol JEM CX II 100 electron microscope (Kanka et al., 1991). Between 12 and 14 hr postactivation fused cells were fixed and evaluated after aceto-orcein staining or using confocal microscopy after tubulin labeling. To assess whether DNA synthesis occurs in control pronuclei and in pronuclei in heterokaryons, cells at 6, 7, and 10 hr postactivation were transferred into medium containing bromodeoxyuridine (100 mM; BrDU) for a 1-hr incubation period before being processed by the method of Campbell et al. (1993). Briefly, after incubation in BrDU supplemented medium, cells were fixed in methanol (0207C) for 20 min, washed in PBS, permeabilized in 0.1% Triton X-100 for 15 min, and hydrolyzed in 4 N HCl for 30 min. Cells were then incubated with primary antibody (rat anti-BrDU; Seralabs, Crawley, UK) diluted 1:10 overnight, washed in PBS, and incubated with secondary antibody (FITC rabbit anti-rat) for 2 hr before being mounted on the glass slides with DABCO and evaluated under a fluorescent microscope.
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Modulation of Female Pronucleus Size
SCHEME 1. A diagrammatic presentation of the experimental protocol and principal results obtained by fusing G2-staged meiotic oocytes (germinal vesicle, GV) or mitotic-staged blastomeres (G2 mitotic) to eggs obtained 2 (A2), 3 (A3), or 4 (A4) hr postactivation (PA). Analysis at 10 hr postactivation showed that while G2 progression in the oocyte or blastomere partner was invariably inhibited, the development of the pronucleus in the egg partner was affected in a highly stage- and cell-specific manner. Pronuclear development was either prevented or retarded when oocytes were fused to eggs at 2 or 3 hr PA, respectively. Fusions to blastomeres or to oocytes at 4 hr PA were without effect on pronuclear development. However, by 12 hr PA all nuclei had entered M-phase irrespective of previous pronuclear size. For comparison normal rates of development are shown from activation (A) through telophase (B), chromatin decondensation (C), full-size pronuclear development (D), initiation of S-phase (E) to entry into the G2-phase of the first mitotic cycle (F).
All chemicals, unless otherwise stated, were purchased from Sigma. Each experiment has been repeated at least three times.
for the first time that the development of the female pronucleus is amenable to experimental manipulation.
RESULTS
Nuclear and Chromatin Changes at 10 hr Postactivation
Scheme 1 summarizes the experimental protocols and principal findings that will be presented in detail in the following three subsections. The results highlight the unique features of the first cleavage cycle and demonstrate
The effect on pronuclear formation of fusing eggs at the early (A2, 2 hr postactivation), mid (A3, 3 hr postactivation), and late (A4, 4 hr postactivation) G1 stages to GV-staged (meiotic) oocytes is summarized in Table 1.
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TABLE 1 Female Pronuclear Development Following the Fusion of Germinal Vesicle (GV)-Staged Oocytes or G2-Staged Blastomeres (G2 Mitotic) to Eggs Obtained at 2 (A2), 3 (A3), or 4 (A4) hr Postactivation (PA) Female pronuclear development (10 hr PA) Cell–fusion combinations
No. cells used
GV 1 A2 GV 1 A3 GV 1 A4 G2 mitotic 1 A2
120 100 117 30
1 1 1 1
120 100 117 30
No. heterokaryons produced
Micropronuclei
Intermediate pronuclei
Normal pronuclei
100 77 107 25
100 10 0 0
0 60 7 0
0 7 100 25
Note. Micropronuclei: membrane-bound chromatin cluster (diameter not assessed). Intermediate pronuclei: standard morphology but half the normal size (Ç10 mm; n Å 52). Normal pronuclei: indistinguishable from unfused controls (Ç20 mm; n Å 78). Diameter of pronuclei in activated control oocytes at 10 hr postactivation: (Ç21 mm; n Å 50). Germinal vesicle diameter: controls, Ç25 mm, n Å 49; heterokaryons, GV 1 A2, Ç40 mm; n Å 80; GV 1 A3, Ç30 mm, n Å 57; GV 1 A4, Ç26 mm, n Å 91.
From the results in Table 1 it is clear that fusions of eggs at 2, 3, or 4 hr after activation to standardized GV-staged oocytes (G2-phase of meiotic cycle) result in profoundly different postfusion development in the first 10 hr after activation. For comparison the size and morphology of control pronuclear (10 hr) and GV-staged eggs are shown in Figs. 1, 1A, and 1B. Early G1 fusion (GV 1 A2). The results of fusing early G1-phased eggs (2 hr postactivation) to standardized GVstaged partners is shown at the light and ultrastructural levels in Figs. 2 and 3 respectively. In the 8 hr following fusion (10 hr postactivation) four striking events occur. First, the posttelophase II chromosomes form into a tight hypercondensed mass rather than undergoing decondensation as would occur in normal activated eggs at a comparable stage. Second, a clear nuclear membrane forms around the hypercondensed chromatin. However, instead of the normal degree of pronuclear enlargement, the nuclear membrane in the early G1 fusions (GV 1 A2) remains tightly associated around the hypercondensed chromatin and no visible enlargement occurs. The fourth notable morphological change contrasts directly with the third because the GVstaged nucleus increases dramatically in size (Ç11.6, see Table 1) and appears to compensate for the absence of pronuclear enlargement. Mid-G1 fusions (GV 1 A3). Delaying fusion until the mid-G1 phase (3 hr after activation) partially reverses all the nuclear changes observed after early G1 fusion. The G1 chromatin becomes fully decondensed and the resultant pronucleus contains nucleoli of normal appearance (see Fig. 5). From a comparison of Figures 2, 4, and 6 it is, however, evident that the size of the pronucleus in heterokaryons prepared at 3 hr postactivation (GV 1 A3) is still smaller than that of controls (see Table 1). Indeed, light and ultrastructural determinations suggest that pronuclei formed after mid-G1 fusions reach approximately half the size of those in control eggs. It must be, however, noted that a slight enlargement of GV diameter could also be detected (Ç11.2).
Late G1 fusions (GV 1 A4). Both the size and the ultrastructural appearance of pronuclei in heterokaryons formed from eggs obtained 4 hr after activation (GV 1 A4) were indistinguishable at 10 hr from those of controls (see Figs. 1, 1A, 1B, 6, and 7). Fusion of G2 mitotic blastomers with early G1 eggs (G2 mitotic 1 A2). Experiments were undertaken to determine whether the aberrant development of both the pronucleus and the germinal vesicle observed in the GV 1 A2 heterokaryons was specific to meiotic G2 oocytes or whether it was a characteristic of G2 mitotic 1 A2 fusions as well. That the observed aberrant pronuclear development in G1 eggs is induced only by meiotic partners is clear from a comparison of Figs. 2 and 3 (GV 1 A2) with Figs. 8 and 9 (G2-mitotic 1 A2). Neither decondensation nor pronuclear enlargement in A2-staged early (early G1) eggs was prevented by fusion to mitotic cells at the G2-phase of the cycle. By comparison pronuclear formation was completely inhibited by fusion to meiotic G2 cells. We therefore interpret our results as indicating that the GV-staged oocyte has the unique capacity, not present in mitotic cells, of suppressing early pronuclear development.
DNA Synthesis and Cell Cycle Progression Cell cycle events in 10 hr postactivation. Measurement of DNA synthesis in the nuclei of heterokaryons (see Table 2) provided two sets of unambiguous results. First, irrespective of whether the chromatin was in a highly condensed or decondensed form, DNA synthesis invariably occurred in pronuclei derived from activated eggs. The intensity of fluorescence was roughly the same in all types of pronuclei labeled. Second, DNA synthesis was never induced in the GV- or G2-staged mitotic nucleus. Thus, the effect of GV-staged oocytes on pronuclear development was restricted to influencing the chromatin configuration and nuclear morphology of the G1 eggs but did not extend to DNA replication. DNA synthesis could first be detected in pronuclear controls (6/20) and in pronuclei
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Modulation of Female Pronucleus Size
FIG. 1. A pair of unfused (control) cells. Pronuclear egg (top) at 10 hr postinduction of activation; GV-staged oocyte (bottom) is freshly isolated. Phase contrast. Bar, 5 mm. (A) An electron micrograph of a 10-hr-old pronucleus with a typical nucleolus and intact membrane. 15200. (B) An electron micrograph of a GV-staged oocyte. The compact nucleolus and nuclear membrane are evident. 12400.
in fused cells at 6 hr postinduction of activation (GV 1 A2, 5/17; GV 1 A3, 4/16; GV 1 A4, 3/12) and consistently at 7 hr after activation (controls, 8/10; GV 1 A2, 10/12; GV 1 A3, 11/12; GV 1 A4, 12/15). This indicates that the length of S-phase, which lasted at least 4 hr, is unaffected by fusion of activated eggs to GV-staged oocytes. By contrast, the action of the G1:S-staged egg partner was, invariably, to block progression of the meiotic cycle of the oocyte for the first 10–12 hr after fusion. Cell cycle events at 12 hr postactivation. In normally
activated eggs, replication is completed by 12 hr postactivation and after a 6-hr G2-phase, entry into the first mitotic M-phase occurs (Kaufman, 1983). The entry into M-phase in all the fusion combinations was both synchronous and significantly advanced with respect to the controls. In every combination involving a GV-staged oocyte and a pronucleate egg, entry into meiosis occurred at, or soon after, 12 hr postactivation, a time that corresponds closely to the completion of the S-phase in the activated egg partner. That the S-phase was invariably completed by 12 hr after fusion
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FIG. 2. Fusion of germinal vesicle-staged oocytes to eggs 2 hr after activation (GV 1 A2) prevents decondensation of chromatin originating from the early activated egg. While the GV is significantly enlarged, chromatin from the A2-staged egg persists as a condensed cluster within a micropronucleus even after 10 hr in culture. PB, second polar body; GV, germinal vesicle; C, cluster of chromatin within the micropronucleus. Phase contrast. Bar, 4.5 mm.
FIG. 4. Fusion of GV-staged oocytes to eggs 3 hr after activation (GV 1 A3) results at 10 hr after activation in chromatin decondensation, nucleolar formation, and the moderate enlargement of the egg pronucleus (PN) to about half the size of a fully formed pronucleus. The germinal vesicle (GV) from the oocyte partner persists at an almost normal size through the culture period. Phase contrast. Bar, 4 mm.
FIG. 3. An electron micrograph of a micropronucleus in a GV 1 A2 heterokaryon. A cluster of condensed chromatin enclosed within a nuclear membrane is shown. 110,000.
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FIG. 5. An electron micrograph of an intermediate-sized female pronucleus (PN) and a normal-sized germinal vesicle (GV) in a heterokaryon produced by the fusion of a GV-staged oocyte to an egg 3 hr after activation (GV 1 A3). Apart from the 50% reduction in pronuclear size both the GV and the PN show a normal ultrastructural configuration. 12400.
was evident from an analysis of chromosome structure (Figs. 10 and 11). Neither single-stranded chromosomes nor structures typical of S-phase prematurely condensed chro-
mosomes (Rao et al., 1977) were ever observed. In our fused cells the chromosome morphology invariably resembled that of either mitotic or G2-phase prematurely condensed chromosomes (PCC).
DISCUSSION
FIG. 6. Fusion of GV-staged oocytes to eggs 4 hr after activation results at 10 hr postactivation in the formation of a normal female pronucleus (PN) and the persistence of a standard germinal vesicle (GV). Phase contrast. Bar, 5 mm.
Although it is generally accepted that all the processes associated with fertilization must be perfectly orchestrated in order to ensure normal embryonic development, no definitive information exists on the degree to which development is compromised by asynchronous pronuclear development. Apart from its scientific interest this information is crucially important now that sperm injection procedures are being progressively more widely used in human IVF programs. Indirect evidence suggests that abnormalities in sperm head decondensation result in poor subsequent development (Ogura and Yanagimachi, 1995). However, the correlation between asynchronous pronuclear development and embryonic development has not been subjected to rigorous scientific study even though the capacity to manipulate male pronuclear development has existed for more than a decade (see Tarkowski, 1982). The present paper not only provides the reciprocal capacity, namely, the ability to alter the development of the female pronucleus, but also highlights a number of specialized features of the first cleavage cycle. The ability of mammalian oocytes to transform fertilizing sperm into male pronuclei is acquired in a stage-specific
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FIG. 7. An electron micrograph of a normal female pronucleus (PN) with typical nucleoli and intact membranes and a germinal vesicle (GV) in a heterokaryon formed by the fusion of a GV-staged oocyte with an egg 4 hr after fusion. 12400.
manner. When penetration occurs at the germinal vesicle stage sperm remain intact in the ooplasm until nuclear membrane breakdown (GVBD), when decondensation but
FIG. 8. Fusion of G2-staged blastomeres (G2 mitotic) to eggs 2 hr (A2) after activation (G2 mitotic 1 A2) results at 10 hr postactivation in normal pronuclei which are under phase contrast indistinguishable from their blastomere nuclei partners. Phase contrast. Bar, 4 mm.
no pronuclear formation occurs (Szo¨llo¨si et al., 1990). A further set of stimuli, generated only by egg activation, are required for the induction of male pronuclear formation (Borsuk, 1991). Even after activation, molecules required for full male pronuclear formation are present for a short period only (Borsuk and Tarkowski, 1989). A delay of as little as 30 min between experimental activation and sperm entry results in the formation of an aberrant male pronucleus. When the delay is extended to 3 hr the resultant pronucleus is very small and with longer delays male pronuclear formation is entirely inhibited. Furthermore, Borsuk (1991) has established that the processes of decondensation and male pronuclear formation utilize two entirely different sets of controlling molecules. The first, required for sperm decondensation, is MPF-kinase dependent and present in metaphase II oocytes and is lost at the time of MPF-kinase inactivation. The ability to convert the decondensed sperm into a male pronucleus requires a second stimulus which is independent of MPF-kinase and persists for about 8 hr postactivation. Work on the molecular basis of male pronuclear formation has been paralleled by the development of techniques for the manipulation of male pronuclear size (Tarkowski, 1980). By contrast, no comparable experiments have been carried out on either the formation or the manipulation of the female pronucleus. Results presented in this paper show that the female pronucleus is highly susceptible to manipulation but only during the first 4 hr of development. The size of pronuclei in eggs which did not fuse (fixed 45 min postinduction of fusion) was basically the same as the size of pronuclei in corresponding heterokaryons fixed at 10 hr. This indicates that the swelling of pronuclei is suppressed after fusion of
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Modulation of Female Pronucleus Size
FIG. 9. An electron micrograph showing the characteristic normal ultrastructural appearance of both the pronucleus and the blastomere nucleus in a heterokaryon produced by the fusion of one G2-staged blastomere from a late 2-cell embryo (G2 mitotic) with an egg activated 2 hr previously (A2). 12400.
activated eggs to GV-staged oocytes. In addition, four other features of our findings warrant discussion. The first concerns the type of cellular environment capable of restricting female pronuclear size while the second relates to the effect of cell fusion on DNA synthesis in pronuclei of different sizes. The third point focuses on the effect of the activated egg on meiotic progression in the oocyte partner and the fourth considers the new opportunities arising from the capacity to experimentally manipulate pronuclear development. Having established that a GV-staged meiotic partner blocks female pronuclear formation when fused to an early activated egg we next questioned whether mitotic cells, at
a corresponding G2 cell cycle stage, would impose a similar pronuclear block on newly activated oocytes. The results of fusing G2-staged blastomeres to activated eggs showed clearly that the block to female pronuclear formation is strictly oocyte specific. It is instructive in this regard to contrast the respective nuclear responses of both the meiotic and the mitotic G2-staged cells to fusion with a recently activated (A2) partner. The significant increase in the size of the germinal vesicle following fusion to an A2staged egg partner suggests that in these heterokaryons all the nuclear proteins are preferentially sequestered in the oocyte nucleus to the detriment of the newly forming pronucleus (see Merriam, 1969). This is perhaps not surprising
TABLE 2 DNA Synthesis and Chromosomal Configuration in the Nuclei of Oocyte and Egg Partners Examined at 10 or 12 hr after Egg Activation BrDU staining (10 h PA)
Cell cycle (10 hr PA)
Cell fusion combinations
Number treated heterokaryons
Oocyte GV
Egg PN
Oocyte
GV 1 A2 GV 1 A3 GV 1 A4 G2 mitotic 1 A2
17 18 27 —
0 0 0 —
15 16 25 —
G2 G2 G2 G2
Cell cycle (12 hr PA)
Egg S S S S
(micro pn) (retarded pn) (normal pn) (normal pn)
Oocyte
Egg
M M M M
M M M M
Note. Fusion of either oocyte partners at the germinal vesicle (GV) or G2-staged blastomeres (G2 mitotic) to egg partners obtained 2 (A2), 3 (A3), or 4 (A4) hr postactivation (PA). DNA synthesis, determined by bromodeoxy-uridine uptake (BrDU), and cell cycle progression in both the G2 (GV) and the egg (pronuclei, pn) nuclei are shown.
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FIG. 10. The onset of M-phase occurred approximately 12 hr after activation in all the fusion combinations studied. Typically, one common group of chromosomes was formed containing both the oocyte (arrow) and activated egg chromosomes (arrowheads). Phase contrast. Aceto-orcein staining. 11000.
since it is well established that nuclear proteins, injected into the cytoplasm, are thereafter rapidly translocated to the nucleus (Gurdon, 1970; De Robertis et al., 1978). Included among the proteins which probably locate preferentially in the mammalian cell nucleus are nucleoplasmin-like acidic proteins (Philpott and Leno, 1992), histones, and topoisomerase II, the latter fulfilling both a catalytic and a structural role in chromatin condensation (Adachi et al., 1991). The more difficult current problem is to explain why this postulated preferential sequestering of nuclear proteins does not appear to occur when G2-staged mitotic cells (blastomeres) are fused to early activated (A2) eggs. Our results show that in these heterokaryons the blastomere nuclei do not increase in size and the egg pronucleus develops normally. It will be interesting, at least, to determine whether labeled proteins in newly activated eggs are transported more readily into the nuclei of oocyte fusion partners than into mitotic nuclei after blastomere fusion. Since the content of the GV is known to be required for pronuclear formation it is likely that proteins mentioned above together with some additional GV factors accumulate in newly formed pronuclei. These proteins are necessary for the dispersion of chromatin and its further decondensation. However, the exact nature and role in the process of pronuclear formation are at present unknown. While we show that the size of the female pronucleus can be experimentally manipulated, it is nevertheless notable that DNA synthesis proceeds in an uninterrupted manner even in the smallest sized pronuclei. The onset of S-
phase at 7 hr after activation indicates that this phase lasts at least 4–5 hr in fused cells. The morphology of the two sets of condensed chromosomes at 12–14 hr postactivation provides strong evidence that S-phase is fully completed in our fused cell heterokaryons before progression through G2to M-phase occurs. In no case did we observe the singlestranded structures nor the morphology typical of S-phase prematurely condensed chromosomes (Rao et al., 1977). The morphology of both sets of condensed chromosomes in our fused heterokaryons always resembled the morphology typical of chromosomes in mitosis or prematurely condensed in G2. These findings are in accord with earlier results which demonstrated the DNA synthesis is not suppressed when GV oocytes are fused to S-phase four-cell-stage blastomeres (Fulka et al., 1995b). Thus, while GV oocytes have no DNA suppressive activity, the situation is completely reversed after germinal vesicle breakdown. When S-phase nuclei are fused with such maturing meiotic cytoplasts containing low chromosome condensation activity two events occur. First, DNA synthesis in the S-phase partner is totally suppressed and second, premature chromosome condensation does not occur (Trefil et al., 1995; Fulka et al., 1995c). The effects of the oocyte on female pronuclear size and DNA synthesis in the activated egg are matched by a reciprocal action of the egg on meiotic progression in the oocyte. However, the effects of mitotic partners on meiosis are not uniform and appear to differ depending on the cleavage cycle from which the mitotic cell was obtained. Thus, when a GV-staged oocyte is fused to an S-phase blastomere from a four-cell embryo the onset of GVBD is not delayed (Fulka et al., 1995b). By contrast an entirely different response occurs when GV oocytes are fused to S-phase eggs from the first cleavage cycle. The present experiments demonstrate that in these circumstances cell cycle progression in the oocyte
FIG. 11. The formation of a true spindle during M-phase chromosome organization in heterkaryons at 12 hr postactivation was demonstrated using propidium iodide staining of the chromosomes and confocal microscopy. Original magnification, 11200.
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Modulation of Female Pronucleus Size
is blocked and GVBD occurs only when the activated egg finishes DNA synthesis. This highlights the specificity of the first cell cycle with its protracted G1-phase. Both Adlakha and colleagues (1983) and Balakier and Masui (1986) suggest that this delay is induced by mitotic inhibitors specific for G1-phase cells. The activity of these inhibitors is thought to gradually decrease as the cell cycle progresses toward the G2-phase. Evidently, the block of germinal vesicle breakdown in heterokaryons must be different from that induced by cAMP elevating agents (Eppig, 1993) and its nature is difficult to explain at present. However, when heterokaryons were incubated in medium containing dbcAMP for up to 20 hr no nuclear membrane breakdown and chromosome condensation were detected (not shown). The capacity to selectively manipulate the size of the female pronucleus, while not preventing normal S-phase progression, provides a number of experimental opportunities. The first is to seek explanations for some unexpected phenomena arising from our present findings. For example, why is female pronuclear development limited by G2staged oocytes but not by comparable G2-phased blastomeres? Equally, why do GV nuclei, but not blastomere nuclei at the same G2-phase of the cell cycle, increase significantly in size when fused to a newly activated egg? The second set of new opportunities relates to the current availability of techniques to alter the size of either the male or the female pronucleus. These techniques will provide the means of correlating embryo development with early events associated with pronuclear formation. Pronuclear manipulations will also be useful in certain epigenetic studies (W. Reik and W. Dean, personal discussion). For example, these authors have already shown that the adult phenotype in mice is affected by epigenetic factors (Reik et al., 1993). These authors postulate that some epigenetic effects are likely to be induced by abnormal pronuclear development. We suggest that a combination of the approach presented in this paper together with those associated with male pronuclear manipulation provides a good starting point for future epigenetic studies.
ACKNOWLEDGMENTS J.F. Jr. is supported by GACR 505/95/1371. This publication is also based in part on work sponsored by the U.S.–Czech Science and Technology Joint Fund in cooperation with USDA under Project 95047. We thank Dianne Styles and Linda Notton for editorial assistance and Caroline Lee for carrying out the confocal analysis.
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