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Biomedical subjects

R M Moor

Publications and source records attributed to R M Moor.

At least 19 recordsLinked to original sources
PubMed2003

Enucleolation of porcine oocytes.

Germinal vesicles (GVs) in immature mammalian oocytes contain prominent nucleoli whose role in the process of oocyte maturation is not fully understood. Here we report that the microsurgical removal of nucleoli from immature fully grown porcine oocytes permits germinal vesicle breakdown and chromosome condensation and the enucleolated oocytes mature up to the second metaphase. Interestingly, the enucleolation of growing oocytes which, although unable to mature, resulted in germinal vesicle breakdown and the formation of a cluster of condensed chromatin. These results indicate that the nucleolus in fully grown oocytes is dispensable at least for nuclear maturation. On the other hand, the results obtained in growing oocytes suggest the role of the nucleolus in the cell cycle regulation.

Animals↗
PubMed2001

Nucleus transfer in mammals: how the oocyte cytoplasm modifies the transferred nucleus.

Successful development of clones depends on the reprogramming of transferred nuclei in enucleated oocytes. Thus far, oocytes are the only cells that can convert nuclei, which are already differentiated, into undifferentiated stages resembling pronuclei in freshly fertilized zygotes and that can then complete development of the reconstructed embryos. However, we still don't know exactly how the enucleated oocyte (cytoplast) secures this reprogramming. Oocytes exhibit a number of cytoplasmic activities that may be involved reprogramming. We discuss how these activities may be involved in reprogramming of transferred nuclei.

Animals↗
PubMed2000

Specific regulation of CENP-E and kinetochores during meiosis I/meiosis II transition in pig oocytes.

To understand the mechanisms which regulate meiosis-specific cell cycle and chromosome distribution in mammalian oocytes, the level and the localization of CENP-E and the kinetochore number and direction on a half bivalent were examined during pig oocyte maturation. CENP-E is a kinetochore motor protein whose intracellular level and localization are strictly regulated in the somatic cell cycle. The localizations of CENP-E on meiotic chromosomes from diakinesis stage to anaphase I and at the spindle midzone at telophase I were shown by immunofluorescent confocal microscopy to be similar to those in somatic cells of pig and other species. Further, ultrastructural analysis revealed the presence of CENP-E on fibrous corona and outer plate of kinetochores of the meiotic chromosomes. However, unlike mitosis, CENP-E staining was continuously detected either at the spindle midzone or on the kinetochores of segregated chromosomes during the first polar body emission. Consistent with this, immunoblot analysis revealed that CENP-E level remained high during meiosis I/meiosis II (MI/MII) transition and that some of CENP-E survived through the transition even in cycloheximide-treated oocytes in which cyclin B1 was completely degraded. Furthermore, examinations of CENP-E signals in confocal microscopy and kinetochores in electron microscopy in MI and MII oocytes provide the cytological evidence in mammalian oocytes which suggests that each sister chromatid in a pair has its own kinetochore which localizes side-by-side so that two sister chromatids on a half bivalent are oriented toward and connected to the same pole in MI.

Animals↗
PubMed2000

Localisation of phosphorylated MAP kinase during the transition from meiosis I to meiosis II in pig oocytes.

Mitogen-activated protein kinase (MAPK) has been reported to be involved in oocyte maturation in all animals so far examined. In the present study we investigate the expression and localisation of active phosphorylated MAPKs (p44ERK1/p42ERK2) during maturation of pig oocytes. In immunoblot analysis using anti-p44ERK1 antibody which recognised both active and inactive forms of p44ERK1 and p42ERK2, we confirmed that MAPKs were phosphorylated around the time of germinal vesicle breakdown (GVBD) and the active phosphorylated MAPKs (pMAKs) were maintained until metaphase II, as has been reported. On immunofluorescent confocal microscopy using anti-pMAPK antibody which recognised only phosphorylated forms of MAPKs, pMAPK was localised at the spindle poles in pig mitotic cells. On the other hand, in pig oocytes, no signal was detected during GV stage. After GVBD, the area around condensed chromosomes was preferentially stained at metaphase I although whole cytoplasm was faintly stained. At early anaphase I, the polar regions of the meiotic spindle were prominently stained. However, during the progression of anaphase I and telophase I pMAPK was detected at the mid-zone of the elongated spindle, gradually becoming concentrated at the centre. Finally, at the time of emission of the first polar body, pMAPK was detected as a ring-like structure between the condensed chromosomes and the first polar body, and the staining was maintained even after the metaphase II spindle was formed. The inhibition of MAPK activity with the MAPK kinase inhibitor U0126 during the meiosis I/meiosis II transition suppressed chromosome separation, first polar body emission and formation of the metaphase II spindle. From these results, we propose that the spindle-associated pMAPKs play an important role in the events occurring during the meiosis I/meiosis II transition, such as chromosome separation, spindle elongation and cleavage furrow formation in pig oocytes.

Animals↗
PubMed2000

Degradation of pig cyclin B1 molecules precedes MAP kinase dephosphorylation during fertilisation of the oocytes.

Pig oocytes at metaphase II were activated by penetration of spermatozoa in cycloheximide-free and cycloheximide-containing fertilisation media. The precise nuclear stage, and the kinetics of degradation of cyclin B1 and dephosphorylation of MAP kinase were assessed after insemination. After maturation culture, 96% of oocytes reached metaphase II. At 6 h after insemination in cycloheximide-free medium, 68% of the oocytes were activated and had progressed to anaphase II or beyond. After 8 h, 89% of the oocytes were activated: a female pronucleus had formed and the heads of penetrating spermatozoa had enlarged and changed to male pronuclei. In the cycloheximide-containing medium, activation of oocytes started earlier than in cycloheximide-free medium. After 4 h, 43% of the oocytes were activated, and the percentage increased to 97% after 6 h. Pig cyclin B1 disappeared in the oocytes at 6 h after insemination in both cycloheximide-containing and cycloheximide-free media. Pig oocytes at metaphase II contained two types of MAP kinase--ERK 1 and ERK 2--in their active phosphorylated forms. At 8 h after insemination ERK 2 changed to the fast-migrating inactive form in the oocytes cultured in both cycloheximide-containing and cycloheximide-free media, although the shift-down was not complete. The change was delayed by 2 h after the degradation of cyclin B1 molecules. These results demonstrate that degradation of pig cyclin B1 molecules corresponds to the transition of the oocytes from metaphase II arrest to anaphase II/telophase II and was followed by MAP kinase dephosphorylation.

Animals↗
PubMed2000

Behaviour of mouse primary spermatocyte nuclei after fusion to enucleated metaphase II oocytes.

Primary spermatocytes originating from prepubertal mouse testes were electrofused to metaphase II (MII)-stage oocytes, enucleated either by the conventional micromanipulation method or by chemical treatment with etoposide and cycloheximide. These experiments were followed by assessment of morphological changes in transferred nuclei using light microscopy, by chromosomal analyses and by screening of hybrids for the presence or absence of DNA synthesis using anti-bromodeoxyuridine antibody and immunofluorescence staining of the hybrids. The results show differences between the two types of ooplasts in susceptibility to activation stimuli. However, when activated, both types of ooplasts gave rise to hybrids of similar morphology. From 35.3% to 63% of activated hybrids originating from chemically or microsurgically enucleated oocytes, respectively, contained one large pronucleus in cytoplasm, 62% or 31.6% hybrids from those two groups, respectively, possessed two smaller pronuclei and a few contained three or four pronuclei. No DNA synthesis was detected in any hybrid containing one or more pronuclei. The chromosome spreads of hybrids with premature chromosome condensation (PCC) morphology (before activation) show that most of the hybrids had a diploid (2n) number of chromosomes. The nature and regularity of the cell division cycle in the hybrids are discussed.

Animals↗
PubMed2000

Activity of maturation promoting factor (MPF) and mitogen-activated protein kinase (MAPK) after parthenogenetic activation of ovine oocytes.

The maturation promoting factor (MPF) and mitogen-activated protein kinase (MAPK) are the key regulators of both meiotic and mitotic cell cycles. Knowledge of the dynamics of these two kinases during the transition from meiosis to mitosis would be of great importance for cloning by nuclear transfer. In this study, experiments were designed to assay the changes of MPF and MAP kinase activity of in vitro matured ovine oocytes after chemical activation and culture in 0 mM or 2 mM 6-dimethylaminopurine (6-DMAP) for 12 h. Moreover, to determine the biological significance of the fluctuations of MPF, activated oocytes were fused with GV-staged partners. The biochemical results showed that the high MPF activity of MII oocytes fell to basal level precipitously within the first hour after activation, started to increase at 6-8 h, rising to 80 +/- 4% of MII after 12 h. MAPK activity decreased to a low level 4 h after activation, increased between 6-12 h, but remained below 30 +/- 3.6% of MII values. The incubation with 6-DMAP had no effect on the kinetics of MPF and MAP kinase activity. Fusion of MII oocytes to GV partners induced rapid breakdown of the GV, whereas no breakdown occurred when GV were fused with eggs in the first hours post activation. Interestingly, the high biochemical levels of MPF activity at 8-12 h after activation were not able to induce GVBD in fusion partners.

Adenine↗
PubMed2000

Manipulating the human embryo: cell cycle checkpoint controls.

Micromanipulation techniques are widely used in assisted human reproduction and it is logical to assume that successes with recent animal cloning will invariably raise the question of human cloning along with its related ethical problems. However, it is often overlooked that even in animals many complications are still associated with this technique. The purpose of our article is to highlight and discuss some of these problems in the context of the eventual use of nuclear and/or cytoplasmic transfer techniques in assisted human reproduction.

Cell Cycle↗
PubMed2000

Spindle formation and dynamics of gamma-tubulin and nuclear mitotic apparatus protein distribution during meiosis in pig and mouse oocytes.

This work focuses on the assembly and transformation of the spindle during the progression through the meiotic cell cycle. For this purpose, immunofluorescent confocal microscopy was used in comparative studies to determine the spatial distribution of alpha- and gamma-tubulin and nuclear mitotic apparatus protein (NuMA) from late G2 to the end of M phase in both meiosis and mitosis. In pig endothelial cells, consistent with previous reports, gamma-tubulin was localized at the centrosomes in both interphase and M phase, and NuMA was localized in the interphase nucleus and at mitotic spindle poles. During meiotic progression in pig oocytes, gamma-tubulin and NuMA were initially detected in a uniform distribution across the nucleus. In early diakinesis and just before germinal vesicle breakdown, microtubules were first detected around the periphery of the germinal vesicle and cell cortex. At late diakinesis, a mass of multi-arrayed microtubules was formed around chromosomes. In parallel, NuMA localization changed from an amorphous to a highly aggregated form in the vicinity of the chromosomes, but gamma-tubulin localization remained in an amorphous form surrounding the chromosomes. Then the NuMA foci moved away from the condensed chromosomes and aligned at both poles of a barrel-shaped metaphase I spindle while gamma-tubulin was localized along the spindle microtubules, suggesting that pig meiotic spindle poles are formed by the bundling of microtubules at the minus ends by NuMA. Interestingly, in mouse oocytes, the meiotic spindle pole was composed of several gamma-tubulin foci rather than NuMA. Further, nocodazole, an inhibitor of microtubule polymerization, induced disappearance of the pole staining of NuMA in pig metaphase II oocytes, whereas the mouse meiotic spindle pole has been reported to be resistant to the treatment. These results suggest that the nature of the meiotic spindle differs between species. The axis of the pig meiotic spindle rotated from a perpendicular to a parallel position relative to the cell surface during telophase I. Further, in contrast to the stable localization of NuMA and gamma-tubulin at the spindle poles in mitosis, NuMA and gamma-tubulin became relocalized to the spindle midzone during anaphase I and telophase I in pig oocytes. We postulate that in the centrosome-free meiotic spindle, NuMA aggregates the spindle microtubules at the midzone during anaphase and telophase and that the polarity of meiotic spindle microtubules might become inverted during spindle elongation.

Animals↗
PubMed1999

Mammalian leukocytes contain all the genetic information necessary for the development of a new individual.

We have used leukocytes and oocytes from commercially slaughtered animals to clone a progeny tested Brown Swiss bull. Mononuclear cells were separated from the heparinized blood of the donor male on a Histopaque gradient and cryopreserved. The nuclei of thawed leukocytes were directly microinjected into enucleated Holstein Friesian oocytes that were subsequently activated. Development to morula was 23% and to blastocysts was 17%. Some of the cloned compacting morulae were subjected to a second round of nucleus transfer by fusion of individual blastomeres to enucleated oocytes. Development of these second generation embryos to the blastocyst stage was 19%. Following embryo transfer of 50 blastocysts to 50 recipient heifers (31 from first generation and 19 from second generation), 28 pregnancies were established as evidenced by fetal heartbeat at 35 days. A high proportion of the pregnancies established were lost by day 45. One fetus from a second generation embryo developed to term. The phenotype (Brown Swiss) and DNA analysis (11 microsatellites on 11 different chromosomes) of the resultant normal healthy calf confirmed its identity to the donor sire. The ability to clone animals from hematopoietic cells that can be easily collected and cryopreserved from any donor irrespective of species, age, or sex has important implications for the preservation of genetic resources from a wide variety of animals in the animal breeding and artificial insemination industries and for human medicine.

Animals↗
PubMed1999

Checkpoint control of the G2/M phase transition during the first mitotic cycle in mammalian eggs.

The high incidence of chromosomally abnormal human embryos is frequently assumed to be due to a lack of checkpoint controls operating during early embryogenesis. In our study we have analysed when these mechanisms first become functional. Mouse oocytes treated in late metaphase I with either of two different cyclin-dependent kinase inhibitors [butyrolactone 1 (BL1) or 6-dimethylaminopurine (6-DMAP)] form nuclei in the cytoplasm. BL1-treated eggs enter S-phase at 16-18 h post-treatment and, after completion of DNA synthesis, cleave to 2-cell stage embryos. 6-DMAP treatment results in the rapid initiation of DNA synthesis, its completion by 12 h and then arrest in the G2 phase. Thus, two different cell cycle stages can be obtained at the same time point after the initiation of treatment: G1- after BL1 and G2-staged nuclei after 6-DMAP treatment. That this approach greatly facilitates cell cycle studies has been shown by analysing checkpoint function during the first division. Whilst G2-staged eggs enter M phase within 2-3 h when 6-DMAP is washed out, the onset of M phase is delayed after their fusion to G1 (BL1) cells. Here M phase occurs only after the less advanced nucleus completes DNA replication. Our results indicate that checkpoints in mammalian eggs are functional during the first mitotic cycle.

4-Butyrolactone↗
PubMed1999

UV irradiation of pig metaphase chromosomes: maturation-promoting factor degradation, nuclear cytology and cell cycle progression.

Experiments were designed to test two hypotheses. The first was that irradiation of pig metaphase chromosomes would block the normal sequence of cytological and molecular events associated with activation; the second postulated that damaged DNA would prevent eggs from progressing through the first mitotic cleavage cycle. The experimental protocol involved selectively irradiating the metaphase II plate of pig oocytes with highly focused 254 nm ultraviolet (UV) light, followed by activation using standard electroactivation procedures. The following assessments were made of different groups of eggs: (i) nuclear membrane reassembly; (ii) chromosomal cytology; (iii) changes in maturation-promoting factor kinase (MPF kinase) activity at 1 h intervals after activation; and (iv) mitotic progression of eggs containing damaged chromosomal fragments. UV irradiation neither prevented the reassembly of nuclear membranes required for pronuclear formation nor interfered with the normal pattern of MPF kinase degradation after egg activation. UV irradiation did induce a wide range of chromatin defects, including condensation and dispersal of DNA fragments which, in turn, resulted in the formation of micronuclei in the treated eggs and embryos. The presence of damaged DNA retarded, but did not inhibit, progression through the first mitotic cycle. No evidence was obtained that the subsequent mitotic cycle was adversely affected by the presence of UV-damaged DNA. Overall, these results indicate that early cleavage divisions in pig eggs are not blocked by the presence of damaged, hypercondensed chromatin. In this respect, pig eggs are similar to Xenopus eggs, but are different from bovine eggs. On the basis of these findings it is suggested that focused UV irradiation offers a simple and rapid technique for the non-invasive enucleation of pig oocytes provided that the residual hypercondensed chromatin does not affect later developmental stages.

Analysis of Variance↗
PubMed1998

Cloning by somatic cell nuclear transfer.

The birth of the first cloned mammals, produced by the introduction of somatic cell nuclei into enucleated oocytes, was an impressive and surprising development. Although the ethical debate has been intense, the important scientific questions raised by this work have been inadequately discussed and are still unresolved. In this essay we address three questions about nuclear transplantation in the eggs of mice and domestic animals. First, why were the recent experiments on somatic cell cloning successful, when so many others have failed? Second, were these exceptional cases, or is somatic cloning now open to all? Third, what are the future possibilities for increasing the efficiency and wider applicability of the cloning process?

Animals↗
PubMed1998

Nuclear and cytoplasmic determinants involved in the regulation of mammalian oocyte maturation.

The requisite endpoint of mammalian oocyte maturation, whether in vivo or in vitro, is a metaphase II oocyte which is able to be fertilized and which can eventually support normal embryonic development. Oocytes which have been matured in vivo basically fulfill these criteria. On the other hand, a completely different situation exists when these cells are isolated from the ovaries and cultured in vitro. If they are too small (growing oocytes), they do not undergo maturation, or, if more advanced, will mature only to the metaphase I stage. Even in fully grown oocytes which are able to mature to metaphase II, the developmental potential after fertilization is disappointingly low, for reasons which remain unknown. The complexity of certain factors (nuclear, cytoplasmic or arising from our current culture systems) undoubtedly influences both the ability of oocytes to mature fully, as well as their developmental potential after fertilization.

Animals↗
PubMed1998

Development of parthenogenetic and cloned ovine embryos: effect of activation protocols.

Preliminary experiments carried out on ovine oocytes were designed to establish correlations between activation protocols and subsequent rates of embryonic development. The best activation protocols were thereafter used in studies on ovine parthenogenesis and cloning. The first study established that chemical activators induce pronuclear development at a slightly higher rate than physical activation (ionomycin, 96%; ethanol, 95%; electro activation, 80%). Inhibition of second polar body extrusion and one single pronucleus were observed in the majority of the oocytes (approximately 90%) treated for 3 h with 6-dimethylaminopurine (6-DMAP) following either ionomycin or ethanol activation. While over 80% of these oocytes cleaved after transfer to the oviducts of recipients, progression to the blastocyst stage was higher after ionomycin as compared with ethanol activation (58% vs. 19%). The ionomycin plus 6-DMAP activation protocol was used to produce parthenogenetic blastocysts whose subsequent development was monitored both by ultrasonography and by direct fetal examination. Over 70% of parthenogenotes were viable on Day 21 of pregnancy but dead by Day 25. The effects of 6-DMAP on nuclear remodeling and fetal development of cloned embryos was then investigated. Control cloned embryos underwent nuclear envelope breakdown (NEBD), premature chromatin condensation (PCC), and inhibition of DNA synthesis. By contrast, reconstructed embryos treated with 6-DMAP exhibited intact nuclear membranes, interphase chromatin, and no interference on DNA synthesis. Moreover, cloned embryos developed to blastocyst stage in higher percentage after 6-DMAP treatment (83% vs. 25%). We conclude that ionomycin followed by 6-DMAP incubation yields high percentages of diploid parthenogenetic embryos that develop to Day 25 before dying. Cloned embryos activated by the ionomycin-6-DMAP protocol develop readily to term.

Adenine↗
PubMed1997

Role of secreted proteins and gonadotrophins in promoting full maturation of porcine oocytes in vitro.

Experiments were designed to identify the extent to which follicle cells and hormones contribute to the developmental competence of porcine oocytes matured in vitro. Oocyte-cumulus complexes were collected from ovaries by dissection and cultured in 2 ml of TCM199-based medium in 5% CO2 in humidified air at 38.5 degrees C. This basic maturation system was supplemented, for either the first 24 hr only or for the 48-hr culture period, with 1) everted follicle shell alone, 2) gonadotrophic hormones alone, or 3) both follicle shells and hormones. The effect of these treatments was evaluated on 1) meiotic maturation rates, 2) the capacity of matured eggs to undergo activation and early cleavage, and 3) changes to the profile of proteins secreted into the culture medium. The results showed that 1) supplementation with either follicle shell or hormones alone increased the rates of meiotic maturation over the nonsupplemented control group, and 2) combined follicle shell and hormonal supplementation yielded the highest rates for maturation, activation, and cleavage but only when hormonal supplementation was removed after the first 24 hr of culture. Proteins of 30, 37, 45, and 46 kD, but of unknown function, were secreted during the first 24 hr into the culture medium in groups supplemented with follicle shells. The addition of hormones did not affect this pattern of secreted proteins. It is possible that some secreted proteins may act to facilitate full maturation of pig oocytes.

Animals↗