Meiotic cycle checkpoints in mammalian oocytes.
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Biomedical subjects
J Fulka
Publications and source records attributed to J Fulka.
Cytoplasmic argyrophilic protein(s) in cumulus-oocyte complexes: a preliminary study.
The presence of acidic argyrophilic protein(s) in cumulus cells and oocytes was examined in bovine, ovine, porcine and murine cumulus-oocyte complexes (COCs) using a specific silver-staining method. The COCs were isolated from small (1 mm) or large (3-5 mm) antral follicles; they were fixed immediately after slaughter (first group) or after transfer to the laboratory (ca 60 min, second group) and then silver stained according to Likovskí and Smetana (1981). The argyrophilic proteins were accumulated mainly in foot processes of cumulus cells on oocytes isolated from large bovine follicles. The intensity of the reaction was less evident in small follicles. The intensity of staining gradually disappeared from cumulus cells as the maturation continued. Moreover, we were unable to detect similar strong labelling in mouse, pig and sheep COCs. Such a chronology suggests that argyrophilic protein(s) may play a role in the transition from meiotic arrest to resumption in bovine oocytes. Our results also suggest that preliminary events of maturation may occur just after slaughter, ie a relatively long time before the COCs are isolated and cultured in vitro.
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Influence of cytoplasmic microinjection on meiotic competence in growing pig oocytes.
It is demonstrated that the microinjection of cytoplasm from competent, fully grown oocytes can induce germinal vesicle breakdown in meiotically incompetent growing oocytes. Injection of cytoplasm from oocytes at methaphase I had a similar effect. The maturation in injected growing oocytes was arrested at the premetaphase or metaphase I stage. Induction of germinal vesicle breakdown by cytoplasm microinjection occurs in a dose-dependent manner.
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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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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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Control of germinal vesicle breakdown in bovine x murine hybrid oocytes.
Bovine oocytes cultured in control medium or in medium containing dibutyrylcyclic adenosine monophosphate (dbcAMP) or an inhibitor of cyclic nucleotide phosphodiesterase (3-isobutyl-1-methylxanthine, IBMX) undergo germinal vesicle breakdown (GVBD). On the other hand, mouse oocytes remain arrested at the germinal vesicle (GV) stage when dbcAMP or IBMX is present. When 1 bovine GV stage oocyte is fused to 1 GV stage mouse oocyte, dissolution of both species GV occurred in dbcAMP-supplemented medium. Only when 4 to 5 GV stage mouse oocytes are fused to 1 GV stage bovine oocyte, and these giant cells are cultured in dbcAMP-medium, is maturation arrested with only GVs present in the cytoplasm. The inhibitory effect is more evident in IBMX-supplemented medium. Here nearly 50% of the fused cells exhibit GVs, both mouse and bovine, when 1 cattle GV oocyte is fused to 1 mouse GV oocyte and the fused cells are cultured for 24 h. Moreover, nearly all GVs are well preserved after fusion of 1 bovine oocyte to 2 or more mouse oocytes. When these hybrid cells after 24 h culture in IBMX are then washed and cultured in control medium for a further 24 h, GVBD occurred in all cells. We are of the opinion that this novel approach (ie mixing of sensitive and non-sensitive cytoplasm) may in the future better explain the mechanisms involved in the regulation of mammalian oocyte maturation.
1-Methyl-3-isobutylxanthine↗
Cytochemical and immunocytochemical ultrastructural study of nucleoproteins during nucleologenesis in 8-cell bovine embryos.
The aim of this study was to characterize embryonic nucleologenesis by determining the appearance and localization of acid argyrophilic, basic lysine-rich and histone proteins in 8-cell bovine embryos. Two silver staining techniques, ethanolic phosphotungstic acid (PTA) and immunocytochemical methods using specific antibodies, were applied at the ultrastructural level. The silver-stained proteins were detected at the onset of nucleologenesis on the periphery of the dense nucleolus precursor bodies (NPBs). The amounts of these proteins increased during the transformation of the NPBs into the fibrillogranular nucleolus. At this stage the well-developed dense fibrillar components encircling fibrillar centres showed intense staining. PTA-positive (basic lysine-rich) proteins were present within most nucleolar structures during nucleologenesis as well as in the chromatin. Histones H2B, H3 and H4 were concentrated throughout the chromatin including the nucleolus-associated chromatin. At the onset of nucleologenesis, histones were absent in the NPBs. The first weak histone labelling was detected in the multivacuolated NPBs, both in the fibrous mass as well as inside the vacuoles. Nucleolar histones appeared with the massive penetration of DNA into the NPBs. We suggest that nucleologenesis may serve as a criterion of normal early embryonic development and that the proteins involved in the process of nucleologenesis and transcription could be used as chemical markers of nucleolar function.
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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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The fall of biological maturation promoting factor (MPF) and histone H1 kinase activity during anaphase and telophase in mouse oocytes.
Cell fusions have been used to determine the biological activity of the MPF complex in murine oocytes during their progression through anaphase and telophase to metaphase II. Oocytes (1) at metaphase I, (2) during the anaphase-telophase transition, or (3) at metaphase II were fused to germinal vesicle-staged (immature) oocytes. The hybrids were cultured for 1 h in the presence of db cAMP before fixation and nuclear evaluation. Metaphase I oocytes invariably induced germinal vesicle breakdown (GVBD) in the immature partner. By contrast, anaphase/telophase oocytes never induced GVBD in immature oocytes. The capacity to induce GVBD reappears after the formation of the second metaphase plate. In a second study, histone H1 kinase activity was measured during mouse oocyte maturation in single oocytes. H1 kinase activity was low in GV oocytes, increased sharply at MI, declined during anaphase and telophase and increased again at MII. After egg activation, H1 kinase activity was reduced to basal levels. These results provide direct evidence that a drop in activity of MPF in murine oocytes occurs concomitantly with the exit from metaphase I; MPF activity remains low until the cell re-enters metaphase.
Anaphase↗
In vitro fertilization of intact sheep and cattle oocytes with goat spermatozoa.
In an earlier study we reported on the fertilization of in vitro-matured bovine oocytes by ram spermatozoa. The present results extend those observations and demonstrate the penetration of bovine and ovine oocytes matured in culture by goat spermatozoa. Freshly ejaculated and in vitro capacitated goat spermatozoa penetrated 37.1% of bovine and 48.1% of ovine intact oocytes. Monospermic fertilization was detected in about 90% of the oocytes of both species, and the development of pronuclei followed a developmental pattern similar to that of homologous fertilization.
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Pregnancy rate after the transfer of sheep embryos originated from randomly chosen oocytes matured and fertilized in vitro.
Randomly chosen sheep oocytes isolated from 2- to 5-mm follicles of hormonally nonstimulated slaughtered females were matured and fertilized in vitro. Using heparin for the induction of ram sperm capacitation, a fertilization rate close to 80% was recorded. After the transfer of 29 embryos cultured to the 2- to 4-cell stage to 4 recipients, each delivered 1 lamb. In another experiment, 34 2-cell embryos stage were transferred (1 to each oviduct) to 17 synchronized recipients; 8 pregnancies were established and each of 5 recipients delivered a single lamb. The remaining 3 recipients aborted at the third month of gestation. These results show that sheep embryos can be produced in vitro from randomly chosen oocytes and by using relatively simple procedures. However, the viability of the embryos was low, with approximately only 15% developing to term after transfer at the 2-cell stage.
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Development of pronuclei in pig oocytes activated by a single electric pulse.
Pig oocytes were matured in vitro in a modified M-199 medium for 44 h, subjected to electrical stimulation and scored for activation 6 h later. Sham pulsed oocytes, exposed to electroporation medium and an a.c. field, did not develop the female pronucleus any more frequently than occurs spontaneously (8.3% within 50 h of culture). However, a single d.c. pulse proved extremely efficient in activating pig oocytes. Pulses of 0.75-1.65 kV cm-1 lasting 30 or 100 microseconds activated at least 90% of matured oocytes. The developmental pathway taken by the activated oocytes depended on the parameters of the pulse. The lowest effective stimulation (0.45 and 0.60 kV cm-1 for 30 microseconds) frequently produced oocytes that remained in pre-pronuclear stages of activation (29.4 and 42.3%, respectively). Extrusion of the second polar body and creation of one pronucleus was the most frequent type of activation (in up to 88.2% among the activated oocytes). The strongest stimulations used (1.05-1.65 kV cm-1 for 100 microseconds) often yielded oocytes that failed to extrude the second polar body and formed two or more pronuclei (up to 56.3%). Under optimal stimulation (0.75 kV cm-1), the activated oocytes proceed synchronously to interphase of the first mitotic division. Anaphase II is reached within 30 min and telophase Ii at 1 h after application of the pulse. The second polar body is extruded about 2 h after activation. Well-defined swelling pronuclei were found in oocytes 5-6 h after activation. The relationship between the stage of oocyte maturation and susceptibility to activation was investigated. The period of culture in which the oocytes develop the activation competence (32-36 h of culture) overlapped with the period in which the oocytes complete meiosis (28-38 h). This suggests that ageing in meiotic arrest is not essential for pig oocytes to become activated by electric pulses. Activation of pig oocytes was accompanied by release of cortical granules. In sections of control (metaphase II) oocytes, an average of 7.3 intact cortical granules per 10 microns of overlying cytoplasmic membrane was found. This number dropped to 1.5 in 10 microns within 30 min after the pulse.
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Nuclear transplantation in bovine embryo: fine structural and autoradiographic studies.
Nucleolar fine structure, "blebbing" activity of nuclear envelope, and activation of heterogeneous nuclear RNA (hnRNA) synthesis were studied in bovine reconstructed embryos obtained by electrofusion of a single eight-cell blastomere with an enucleated oocyte. Developmental progress of nucleolar fine structure and hnRNA synthesis are arrested during three cell cycles following fusion. The activation of both appears during the eight-cell stage of the reconstructed embryo, after the same number of cell cycles after fusion as in nonmanipulated bovine embryos after fertilization. "Blebbing" activity of nuclear envelope, which is already absent in original blastomeres, reappears after fusion and continues for the next two cell cycles. From the present results, it can be concluded that the donor nuclei are arrested after fusion in morphology and function. Their reactivation corresponds to the developmental pattern typical for normal bovine embryos.
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Effect of 6-dimethylaminopurine on germinal vesicle breakdown of bovine oocytes.
The effect of 6-dimethylaminopurine (6-DMAP) on germinal vesicle breakdown (GVBD) and maturation in bovine oocytes was investigated in this study. This puromycin analog has been shown to be an inhibitor of phosphorylation. Whereas GVBD occurred in nearly all oocytes (96.8%, 120/124) in control medium, presence of 6-DMAP (2 mM) blocked this process almost completely, irrespective of the presence (98.3% GV, 349/355) or absence (97.1% GV, 165/170) of cumulus cells. When lower concentrations of 6-DMAP were used (100-500 microM), GVBD was observed in 87.9% of oocytes, but their maturation was arrested at late diakinesis-metaphase I stage. The inhibition of GVBD was fully reversible, but most of the metaphase II plates were abnormal (80%). To assess whether the action of 6-DMAP is different from the inhibitors of protein synthesis, metaphase II oocytes were exposed to either cycloheximide or 6-DMAP, respectively. Whereas in cycloheximide-supplemented medium approximately 80% of the oocytes were activated, parthenogenetic activation was much less frequent after incubation in 6-DMAP (14.5%). Fusion studies showed that, even if GVBD occurs in 6-DMAP supplemented medium, the level of the maturation-promoting factor (MPF) is decreased. These experiments may indicate the importance of phosphorylation for GVBD in cattle oocytes.
Adenine↗
Influence of dbcAMP on the inhibitory effect of cumulus cell factor(s).
The factor(s) produced by porcine cumulus cells (cumulus cell factor (s): CCF) was described as quantitatively inhibiting the maturation of oocytes in vitro (Petr et al, 1989). When 1, 10, 20 or 40 cumulus oocyte complexes (COCs) were cultured in a droplet of medium (vol 10 microliters), germinal vesicle breakdown (GVBD) was observed in 85, 78, 57 or 19% of the oocytes, respectively. GVBD was observed in 82, 84, 80 or 90% of cumulus-free oocytes, respectively, when they were cultured at the same numbers per 10-microliters droplet. When 1, 10, 20 or 40 cumulus-free oocytes were cultured under the same conditions in a medium containing 140 dbcAMP per ml, 61, 63, 60 or 58% of them were observed at GVBD. However, when COCs were cultured in a 10 microliter droplet of medium with 140 micrograms of dbcAMP per ml, GVBD occurred in 64, 42, 9 or 0% respectively. Based on these results, we can conclude that dbcAMP exerted a further inhibitory effect on GVBD in pig oocytes cultured under the influence of inhibitory factor(s) from cumulus cells. On the other hand, dbcAMP was shown to partly overcome the effect of CCF on GVBD in porcine oocytes. This suggestion was based on the finding that a 6-h pre-culture of COCs in a medium with 1,000 micrograms of dbcAMP significantly decreased the subsequent effect of CCF (GVBD: 44%) compared with those pre-cultured in a medium with 140 micrograms of dbcAMP/ml (GVBD:5%) or without dbcAMP (GVBD: 15%).(ABSTRACT TRUNCATED AT 250 WORDS)
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Regulation of processes involved in mammalian oocyte maturation.
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