Nuclear transplantation in mammalian eggs and embryos.
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Animals↗
Biomedical subjects
R M Moor
Publications and source records attributed to R M Moor.
Response of avian nuclei to mammalian maturation promoting factor (MPF).
Chicken blastodermal cells (stage X) were fused to mouse enucleated oocytes with either no or high maturation promoting factor (MPF) activity. High MPF levels always induced premature chromosome condensation (PCC) irrespective of the number of nuclei fused to a single oocyte. When a single blastodermal cell was fused to a single oocyte without MPF activity the nucleus remained intact for up to 3 h and thereafter underwent PCC. A quite different situation was observed after multiple fusion of several blastodermal cells to a single oocyte without MPF activity. Here, the transplanted nuclei remained intact even after prolonged culture but underwent extensive swelling. DNA synthesis was detected in almost all unfused blastodermal cells. However, after the fusion of several blastodermal cells to a single oocyte no DNA synthesis could be detected. These results provide further evidence that MPF is the universal cell-cycle regulator in the animal kingdom. Its activity is blocked (or neutralised) after fusion to several S-phase cells. Interestingly, our results further suggest that DNA synthesis is suppressed in meiotic cytoplasm even in the presence of an intact nuclear envelope.
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Association between p34cdc2 levels and meiotic arrest in pig oocytes during early growth.
The molecules involved in determining meiotic competence were determined in porcine oocytes isolated from preantral and antral follicles of different sizes. Oocytes isolated from preantral follicles had a mean diameter of 78 microns, contained diffuse filamentous chromatin in the germinal vesicle and were incapable of progressing from the G2 to the M phase of the cycle even after 72 h in culture. Oocytes from early antral follicles had a mean diameter of 105 microns, showed a filamentous chromatin configuration and about half resumed meiosis but arrested at metaphase I (MI) when cultured. Oocytes from mid-antral (3-4 mm) and large antral follicles (5-6 mm) had mean oocyte diameters of 115 and 119 microns respectively, contained condensed chromatin around the nucleolus and progressed to metaphase II (MII) in 48% and 93% of instances respectively. Analysis of p34cdc2, the catalytic subunit of maturation promoting factor (MPF), by immunoblotting indicates that the inability of small (78 microns) oocytes to resume meiosis is due, at least in part, to inadequate levels of the catalytic subunit of MPF. On the other hand, the inability of intermediate-sized (105 microns) oocytes from antral follicles to complete the first meiotic division by progressing beyond MI appears not to be limited by levels of p34cdc2, which are maximal by this stage. We postulate that an inadequacy of molecules other than p34cdc2 limits progression of MI to MII; the acquisition of these molecules during the final stages of growth may be correlated with the formation of the perinucleolar chromatin rim in the germinal vesicle.
Amino Acid Sequence↗
Chromosome condensation activity (CCA) in bisected C57BL/6JxCBA mouse oocytes.
Chromosome condensation activity (CCA) has been analysed in C57BL/6Jx CBA mouse oocytes bisected (i) shortly after germinal vesicle breakdown (GVBD), (ii) in metaphase I (MI) and (iii) in metaphase II (MII) into two equal halves (nucleated, enucleated) which were thereafter fused to S- or G2-phase 4-cell-stage mouse blastomeres. In nucleated halves, premature chromosome condensation (PCC) in transplanted nuclei was always induced irrespective of the cell cycle stage of the blastomere, whereas in enucleated halves only G2 nuclei underwent PCC after transplantation. Premature chromosome condensation in S-phase nuclei was induced only in enucleated halves produced shortly after GVBD. Although S-phase nuclei transplanted to MI or MII enucleated halves remained intact, their capacity to synthesize DNA was invariably suppressed. When spindles were destroyed by preincubation of the oocytes in colcemid before bisection, both nucleated and enucleated halves produced at MI or MII induced PCC of both G2- or S-phase nuclei. These results demonstrate that chromosome condensation activity in mammalian oocytes is compartmentalized rather than uniformly distributed across the cell, and that the enucleation of mammalian oocytes before nuclear transplantation may, under some conditions, influence the levels of CCA and subsequent response of introduced nuclei to cytoplasmic factors.
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Electrofusion of mammalian oocytes and embryonic cells.
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Animals↗
Nuclear remodelling and early development in cryopreserved, porcine primordial germ cells following nuclear transfer into in vitro-matured oocytes.
Nuclear transfer was conducted in the pig using, as karyoplasts, primordial germ cells which had been cryopreserved. The cytoplasts were presumptive S- or MII-phase, in vitro-matured oocytes, which had been enucleated mechanically. Enucleation was effective in 94.3% of cases. Karyoplasts were introduced into the perivitelline space, in close contact with the cytoplasts, and the complexes fused by electrical stimulation and activation. Activation was successful in 82-88% of nonmanipulated, pulsed oocytes, and in 55% of germ cell-oocyte complexes. The reconstituted embryos were examined for nuclear remodelling and cleavage in vitro. Nuclear swelling was more prominent when MII-phase cytoplasts, rather than S-phase, cytoplasts, were used. After 24 h in culture, the cleavage rate was not significantly different whether blastomeres or primordial germ cells were used as karyoplasts, and whether MII-phase or S-phase cytoplasts were used. However, after 72 h in culture, the developmental rate was higher when MII-phase cytoplasts (75%) were used for the recipients of blastomeres compared with S-phase cytoplasts (38.5%, p < 0.05). Similar tendencies were observed with germ-cell nuclear transfer when inositol was used as medium for electrofusion (60% vs 27.8%, p < 0.05). Furthermore, when MII-phase cytoplasts were used, the nuclear transferred embryos derived from blastomeres developed at a significantly higher rate than from primordial germ cells (37.5%, p < 0.05). We conclude that cryopreserved primordial germ cells are competent to undergo nuclear remodelling and cleavage during 72 h or incubation in vitro to the 4-cell stage, following nuclear transfer to enucleated, activated (S-) or MII-phase oocytes. This experimental system may help to elucidate events in the early development of pig embryo following nuclear transfer using germ cells as karyoplasts.
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MPF components and meiotic competence in growing pig oocytes.
Growing pig oocytes (< or = 90 microns in diameter) are unable to resume meiosis in vitro. The objective of our present experiments has been to identify the reasons for meiotic competence in these cells. By comparing histone H1 kinase activity in growing and fully grown oocytes we demonstrate that incompetence is associated with an inability to activate H1 kinase in growing oocytes. Immunoblotting was used to determine whether this kinase inactivity resulted from a lack of either p34cdc2 protein or B-type cyclin. The results established that each of these cell cycle molecules are present in comparable amounts in both growing and fully grown oocytes. In the third series of studies experiments were carried out in an attempt to induce p34cdc2 activation during growth. Treatment with okadaic acid, an inhibitor of phosphatase 1 and 2A known to stimulate and accelerate the transition into M-phase of the meiotic cycle in a number of different species, was able to induce p34cdc2 kinase activity and facilitated the G2- to M-phase in growing oocytes. We conclude that although growing oocytes in pigs have sufficient key cell cycle components for the G2 to M transition, they remain incapable of converting these components to active maturation-promoting factor (MPF) until growth is virtually completed. Inhibition of phosphatase 1 or 2A induces the formation of active MPF during growth by an as yet unidentified pathway.
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Sister chromatid separation and the metaphase-anaphase transition in mouse oocytes.
The paper reports on the effect of experimentally inhibiting chromatid separation on meiotic progression and maturation-promoting factor (MPF) activity in both metaphase I (Experiment 1) and metaphase II mouse oocytes (Experiment 2) subjected to combinations of inhibitors of (a) protein synthesis, (b) topoisomerase II, and (c) cytokinesis-cytoskeleton integrity. The results from Experiment 1 showed that the inhibition of protein synthesis invariably results in the extrusion of the first polar body and the formation of an interphase nucleus. Furthermore, this inhibition induces a rapid decline in MPF activity. Similarly, in Experiment 2 the exposure of metaphase II oocytes to cycloheximide initiated a rapid fall in MPF activity, progression to anaphase, the extrusion of the second polar body, and the formation of a pronucleus. While the inhibition of protein synthesis hastened progression through the meiotic cycle, the opposite effect was observed when chromatid separation was prevented by etoposide or colcemid treatment. The results in Experiment 1 demonstrated that the inhibition of chromatid separation totally blocked meiotic progression by preventing the metaphase I to anaphase I transition. These oocytes were characterized by the persistence of high MPF activity for extended periods of time (> 20 hr). This activity declined slowly in oocytes exposed both to inhibitors of chromatin separation and protein synthesis. In Experiment 2 the results showed that the prevention of chromatin separation induced changes which paralleled those observed with MI oocytes. The prevention of chromatid separation with either etoposide or colcemid converted the oocytes from being sensitive to activation stimuli to being entirely resistant to standard activation. In addition, MPF activity remained persistently elevated and declined only when protein synthesis was inhibited. The decline in intracellular MPF activity reached basal levels 6 to 10 hr after the addition of cycloheximide and was accompanied by the slow and gradual decondensation of chromatin. Our results are in accord with those from recent experiments in yeast, insects, and amphibia which suggest that chromatid separation provides an essential signal for cell cycle progression beyond M-phase. We postulate first that exist from both metaphase I and metaphase II, and the characteristic reduction in MPF activity at anaphase in mouse oocytes, are initiated by chromosome (chromatid) separation. Second, we suggest that chemically induced chromosome (chromatid) separation block prevents the anaphase to telophase transition by inhibiting MPF degradation. Third, we postulate that the slow escape from metaphase arrest in oocytes treated with both etoposide and cycloheximide reflects a gradual decrease of MPF activity due to normal protein turnover without new synthesis.
Anaphase↗
Embryonic stem cells in farm animals.
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Animals↗
Changes in intracellular calcium concentration in bovine oocytes following penetration by spermatozoa.
The pattern of changes in intracellular calcium concentration ([Ca2+]i) in bovine oocytes after penetration by spermatozoa was determined. Dynamic video imaging, using Fura-2 as a probe for intracellular free calcium, showed that activation of oocytes by spermatozoa induced multiple transient increases in [Ca2+]i with a spike interval of 24.2 +/- 7.3 min, and that the early transient increases were propagated throughout the oocytes in the form of a wave. Calcium transients at fertilization are involved in the induction of cortical granule exocytosis and the resultant block to polyspermy. The hypothesis that the inhibition of Ca2+ release from inositol trisphosphate-sensitive stores would inhibit exocytosis and increase polyspermy was tested by injecting oocytes before fertilization with heparin, a potent inhibitor of inositol trisphosphate-activated Ca2+ release. There was no significant difference after fertilization in either [Ca2+]i spikes or in polyspermy rates between control and experimental groups injected with low molecular mass heparin up to a final cytoplasmic concentration of 400 mumol l-1. We conclude that inositol trisphosphate-independent Ca2+ stores may be mobilized during the fertilization of bovine oocytes.
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A reversible block at the G1/S border during cell cycle progression of mouse embryos.
Late 2-cell stage mouse embryos were cultured in M-199 plus 100 micrograms/ml Na pyruvate 25 micrograms/ml gentamycin and 0.3% BSA with or without mimosine (200 microM, 150 microM, 100 microM and 50 microM) for a short (4-5 h) or long (18-20 h) culture period; after drug removal subsequent embryo development was evaluated. Late 2-cell stage mouse embryos treated with mimosine were blocked at the 4-cell stage. Autoradiographic studies show that mimosine inhibits cell cycle progression in mouse embryos at the G1/S boundary. The onset of DNA replication occurs within 15 min of releasing the embryos from mimosine block. Embryos pretreated with mimosine at 200 microM and 150 microM for 4-5 h progress after 3-4 days in culture to hatched blastocyst (71% and 79%, respectively) compared with control (90%). However a longer pretreatment (18-20 h) with mimosine at 200 microM was significantly detrimental to the subsequent developmental progression to hatched blastocyst (2% vs 81%, p < or = 0.05); the proportion of degenerated embryos was significantly increased with mimosine at 200 microM and 150 microM compared with control (57% and 28% vs 4%, p < or = 0.05) after 3-4 days in culture. Preliminary studies with mimosine treatment at 100 microM and 50 microM for 18-20 h show that 70% and 37% of the embryos were blocked at 4-cell stage, respectively. These results indicate that mimosine inhibits cell cycle progress in mouse embryos at the G1/S border and thus induces a reversible arrest in a dose- and time-dependent manner.
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Effect of follicle cells and steroidogenesis on maturation and fertilization in vitro of pig oocytes.
Follicle cell numbers, steroidogenesis, and culture conditions were manipulated in a series of experiments designed to identify their effect during in vitro maturation of pig oocytes upon meiotic cycle progression, sperm penetration and male pronuclear formation. In Experiment 1 a static culture system was used to determine the effect of follicle cell numbers on maturation. When the follicle cell number was 3.5 x 10(3) cells per complex or less, cell cycle progression to metaphase II (M-II) and male pronuclear formation were both low (< 30%). Increasing follicle cell numbers to 15 x 10(3) per complex significantly enhanced both meiotic maturation (93%) and male pronuclear formation (83%). A further 10-fold increase in follicle cell numbers (150 x 10(3)), however, reduced subsequent male pronuclear formation (33%). The purpose of Experiment 2 was to determine whether the observed follicle cell effects were influenced by the type of culture system used for oocyte maturation. Comparisons were made between both static and non-static forms of culture. Meiotic progression was significantly depressed when non-static culture systems were used for the maturation of oocyte-cumulus complexes containing low numbers of somatic cells (< 15 x 10(3) cells). However, the addition of somatic cells (follicle shells) to non-static, but not static, culture systems significantly enhanced male pronuclear formation in all groups irrespective of the number of cumulus cells in the oocyte complex. Experiment 3 investigated the effects of the steroid environment on maturation and male pronuclear formation.(ABSTRACT TRUNCATED AT 250 WORDS)
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Noninvasive chemical enucleation of mouse oocytes.
A noninvasive method of enucleating mouse oocytes has been developed and evaluated. Strong chromosome to chromosome binding was induced by culturing early metaphase I oocytes in etoposide supplemented medium. Subsequent expulsion of the entire chromosome complex during polar body extrusion was facilitated by exposing the etoposide treated oocytes to a combination of cycloheximide and etoposide during anaphase and telophase. This simple two-step chemical enucleation procedure yields fully enucleated mouse oocytes in 96% of cases. Chemically enucleated oocytes do not contain maturation promoting factor (MPF) at the end of etoposide-cycloheximide enucleation. MPF levels are, however, restored during subsequent incubation in drug-free medium and, after 15 h of post-enucleation culture, the cytoplasts regain their full capacity for parthenogenetic activation and nuclear remodelling. We believe that this novel enucleation technique will greatly facilitate the research in nuclear transplantation.
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Identification of a 30,000 M(r) polypeptide secreted by cultured ovine granulosa cells and luteal tissue as a tissue inhibitor of metalloproteinases.
Previous studies have revealed that the gonadotropin surge initiates, via transcriptional mechanisms, synthesis and secretion of a 30,000 M(r) polypeptide by the granulosa cells of ovine preovulatory follicles. This polypeptide also appears to be secreted by luteal tissue and has been tentatively identified as a tissue inhibitor of metalloproteinases (TIMP; 68% NH2-terminal amino acid sequence identity to a human TIMP). Therefore, the objective of the present study was to confirm that TIMP is produced by granulosa cells and luteal tissue in the ewe. A series of experiments was conducted to determine whether the 30,000 M(r) polypeptide secreted by granulosa cells and luteal cells is similar to TIMP in biochemical properties (degree of N-linked glycosylation), in biological activity (as ascertained by gelatin zymographic analysis), and in immunoreactivity (as ascertained by Western blot analysis). Incubation of granulosa cells or luteal cells with tunicamycin revealed that the 30,000 M(r) polypeptide is glycosylated. The form lacking N-linked chains had an M(r) (approximately 20,000) similar to that of the unglycosylated form of TIMP in other species. Gelatin zymographic analysis detected significant metalloproteinase inhibitor activity associated with polypeptides of M(r) approximately 21,000 and 30,000 secreted by granulosa cells and luteal cells. Northern hybridization of granulosa cell RNA and Day 10 luteal RNA with an anti-sense TIMP oligonucleotide probe detected an approximately 900-base transcript, which is similar in size to that reported for TIMP mRNA in other species. Finally, Western blot analysis with a rabbit anti-human TIMP antiserum detected immunoreactive polypeptides of M(r) 30,000 secreted by granulosa cells and luteal cells.(ABSTRACT TRUNCATED AT 250 WORDS)
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Effects of the protein phosphorylation inhibitor genistein on maturation of pig oocytes in vitro.
In vitro maturation of cumulus enclosed and denuded pig oocytes was reversibly inhibited by the protein kinase inhibitor genistein. The half-maximal effect on maturation was observed at 40 micrograms ml-1. Genistein inhibited total protein phosphorylation and synthesis with the same dose-response relationship (ED50: 40 micrograms ml-1). Protein phosphorylation and synthesis patterns were changed by effective concentrations of genistein. Pig oocytes were sensitive to genistein during the first 12 h of in vitro maturation. This genistein sensitive period corresponds closely with the period of sensitivity to the protein synthesis inhibitor cycloheximide. Whereas the inhibition of protein synthesis affects only nuclear membrane breakdown and not chromatin condensation, genistein inhibits both events. The results of these experiments suggest that protein phosphorylation and synthesis play major roles during pig oocyte maturation in vitro. It is concluded that genistein inhibited protein phosphorylation is a regulator of chromatin condensation, whereas both new protein synthesis and phosphorylation appear to be required for nuclear membrane disassembly. Caution about this second conclusion is, however, necessary because of the dual action of genistein on both protein phosphorylation and indirectly on protein synthesis.
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Kinetics of MPF and histone H1 kinase activity differ during the G2- to M-phase transition in mouse oocytes.
Maturation promoting factor (MPF) is universally recognized as the biological entity responsible for driving the cell cycle from G2- to M-phase. Histone H1 kinase activity is widely accepted as a biochemical indicator of p34cdc2 protein kinase complex activity and therefore MPF activity. In this paper we present results which indicate that during the G2- to M-phase transition in mouse oocytes the dynamic of p34cdc2 related histone H1 kinase activity differs markedly from the biological activity of MPF as measured by classical cell fusion procedures. MPF is activated just before germinal vesicle breakdown (GVBD) whereas histone H1 kinase is activated 5-7 h later coincident with the formation of the definitive first metaphase plate. The biological activity of MPF is merely reduced to about 50% of control levels by a short period of protein synthesis inhibition (1-2 h) and completely suppressed after a prolonged period of inhibition (4-5 h). By contrast, inhibition of protein synthesis in mouse oocytes results in a rapid and complete suppression of histone H1 kinase activity. Therefore, biological MPF and histone H1 kinase activity should not be used in an interchangeable manner during the G2- to M-phase transition in mouse oocytes.
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Early changes in embryonic nuclei fused to chemically enucleated mouse oocytes.
Mouse oocytes were chemically enucleated by subjecting them to etoposide and cycloheximide treatment during the first meiotic division (Fulka, Jr. and Moor, Mol. Reprod. Dev. 34:427-430, 1993) and thereafter electrofused to karyoplasts prepared from: (i) two-cell stage embryos at the G2-phase; (ii) four-cell stage blastomeres (S- or G2-phase); or (iii) embryonic stem (ES) cells. In the first series of experiments we used fusion conditions which do not induce egg activation to define the series of nuclear changes that are initiated immediately following fusion. Although fusion is evident within 5-10 min of induction, nuclei remain visible for up to 20 min prior to chromatin condensation and the formation of metaphase plates (60-90 min post fusion). After activation, the anaphase-telophase transition is completed within 1-2 h, followed thereafter by cleavage of 75% of reconstituted eggs into two equal nucleated blastomeres, irrespective of the origin of the nuclei used for fusion. We conclude from the first study that a protocol involving fusion without activation, followed 90 min later by activation, is likely to be optimal for nuclear transplantation using MII-phase cytoplasts. In the second series of experiments the above optimized protocol was used to study the effects of different cell cycle combinations on chromosome organization in eggs reconstituted by nuclear transplantation. Both G1- and S-phase karyoplasts fused to MII-phase cytoplasts exhibited spindle abnormalities in all eggs studied. Characteristic abnormalities in these cell cycle combinations included chromatin fragmentation and joining or aggregations of chromatin.(ABSTRACT TRUNCATED AT 250 WORDS)
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Protein and nuclear changes in pig eggs at fertilization.
The nuclear restructuring that occurs between insemination and full pronuclear formation in pig eggs is accompanied by posttranslational changes to specific egg proteins. Sperm penetration begins in vitro at 3 hr postinsemination (hpi). By 5 hr, decondensing sperm heads and anaphase II plates are observed in 50% of eggs, and, by 8 hpi, both male and female pronuclei have formed. Three consistent changes to the pattern of newly synthesised proteins are triggered in this period; they affect the 46K, 25K, and 22K polypeptides. Changes are also triggered in the 180-200K polypeptides and in the 14K polypeptides, but these are highly variable. The same changes in the prefertilization pattern were observed when prelabelled eggs were used and new protein synthesis was suppressed. The first and most abrupt change involves the apparent catabolic elimination of a group of 46K unphosphorylated polypeptides (pl 7.3-6.4), whose synthesis was greatest before germinal vesicle breakdown but declined slowly in the final phase of maturation, then declined precipitously after activation. Ageing (beyond maturation) also leads to the disappearance of these polypeptides. The progressive disappearance of a set of 25K polypeptides and the concomitant appearance of a dominant 22K polypeptide is the most characteristic fertilization-induced modification to porcine egg proteins. These modifications begin within 1 hr of sperm penetration or activation, are specific to the pig, and involve heavily phosphorylated polypeptides (25K, pl 6.7-6.0) whose synthesis is begun in the early metaphase I stage. Dual ([35S] and [32P]) labelling, protein blocking experiments, and use of alkaline phosphatase suggest that dephosphorylation selectively affects these 25K polypeptides and is mainly or wholly responsible for converting them (completely within 6 hr) to a single, new (22K, pl 7.6) species that is positively charged. The 25K/22K polypeptide modification has a close temporal relationship with the formation of the male and female pronuclei.
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