Search PubMed⌕ Search

Biomedical subjects

R M Moor

Publications and source records attributed to R M Moor.

At least 73 records · Page 4Linked to original sources
PubMed1992

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↗
PubMed1992

Effects of protein synthesis on maturation, sperm penetration, and pronuclear development in porcine oocytes.

In vitro matured porcine oocytes were used to test the importance of protein synthesis for sperm penetration, the second meiotic division, and pronuclear development. Experiments were carried out to measure rates of protein synthesis in the presence of protein synthesis inhibitors (35 microM or 350 microM cycloheximide or a combination of inhibitors) (study 1); to test for sperm penetration and pronuclear development when protein synthesis was inhibited during fertilization (study 2); to test for oocyte meiosis, sperm penetration, and female and male pronuclear development when protein synthesis was inhibited during maturation (oocyte maturation in vitro with addition of inhibitor at 0, 24, or 36 hr of culture) (study 3); and to analyze the changes in the pattern of protein synthesis during these phases. Sperm penetration, oocyte meiosis, and female pronuclear development were not affected by the total inhibition of protein synthesis during fertilization. By contrast, inhibiting protein synthesis during maturation severely impaired the completion of meiosis and pronuclear development. Although inhibition of protein synthesis after 36 hr of maturation culture did not totally block male pronuclear development (MPN), the rate of MPN formation was lower than for controls (52% vs. 72%, P less than 0.05). However, protein synthesis was absolutely essential between 24 and 36 hr for the formation of MPN after decondensation. This period of maturation coincided with the dominant phase of protein reprogramming in the oocyte.

Animals↗
PubMed1992

Centrifugal elutriation of porcine oocytes isolated from the ovaries of newborn piglets.

A two step enzymatic procedure for the isolation of ovarian follicles and oocytes at early stages of development from the ovaries of newborn piglets was devised. The isolated oocytes were then separated from the overwhelming majority of the ovarian somatic cells using a centrifugal elutriation system. Twenty to thirty thousand oocytes were routinely collected after elutriation of the cell suspension derived from a pair of ovaries. In the enriched fraction the ratio of oocyte:somatic cells was between 1:1 and 1:3. The validation of the method as an efficient procedure for the isolation of a large, viable, and highly enriched population of oocytes at early stages of development was provided by uptake studies carried out after each step of the isolation and separation technique and by a comparative analysis of the pattern of structural proteins of the enriched fraction and of fully grown porcine oocytes. The results confirmed that the isolated cells were actually oocytes at early stages of development and that they were viable throughout the entire procedure.

Animals↗
PubMed1992

Effects of protein kinase inhibitors on pig oocyte maturation in vitro.

Normal oocyte maturation depends on signal transmission between granulosa cells and the oocyte. We have analysed the effects of inhibiting (I) cyclic AMP-dependent protein kinase (protein kinase A, PK-A), (II) Ca2+/phospholipid-dependent protein kinase (protein kinase C, PK-C) and (III) calmodulin (CaM) on pig oocyte maturation in vitro, protein synthesis and phosphorylation. The inhibition of PK-A using a specific inhibitor H8, decreased the maturation rate (rate of germinal vesicle breakdown, GVBD) of cumulus-enclosed pig oocytes in a dose-dependent manner by approximately 12%, reaching a plateau at 100 microM. The inhibition of PK-C with H7, an inhibitor with some side-effects on PK-A, decreased the maturation rate of cumulus-enclosed oocytes in a dose-dependent manner to a maximum of 20% at a concentration of 100 microM. The calmodulin antagonist W7 up to a concentration of 200 microM had no effects on maturation of cumulus-enclosed pig oocytes. None of the inhibitors (H7, H8 and W7) altered the patterns of protein synthesis of either pig oocytes and cumulus cells after maturation in vitro. Oocyte phosphoprotein patterns were, however, clearly changed by W7. Cumulus cell protein phosphorylation patterns were changed by all 3 agents. Since inhibition of cyclic AMP and Ca2+ phospholipid pathways by PK-A and PK-C blocking chemicals affected only a limited proportion of oocytes (12 and 20%, respectively) and inhibition of Ca2+ binding to CaM was without effect on oocyte maturation, we conclude that these pathways modulate rather than regulate oocyte maturation in the pig.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1992

A comparison of intracellular changes in porcine eggs after fertilization and electroactivation.

The experiments compare intracellular changes in porcine eggs induced by electrical activation with those induced by sperm penetration. Adequate electrostimulation induces changes in both cortical granule exocytosis and protein synthesis similar to those induced by sperm during fertilization. However, ionic changes induced by electrostimulation differ markedly from those initiated at fertilization. Thus, dynamic video imaging using Fura-2 as a Ca2+ probe provides evidence that parthenogenetic activation induced by electrostimulation is initiated by a single sharp rise in the concentration of intracellular free calcium ([Ca2+]i) in the egg. The intracellular Ca2+ transient increase is triggered by an influx of extracellular Ca2+ immediately after electrostimulation. The amplitude of the intracellular Ca2+ transient increase is a function both of the extracellular Ca2+ concentration and of electric field parameters (field strength and pulse duration). Imaging demonstrates further that a single electrical pulse can only induce a single Ca2+ transient which usually lasts three to five minutes; no further Ca2+ transients are observed unless additional electrical stimuli are applied. By contrast, sperm-induced activation is characterised by a series of Ca2+ spikes which continue for at least 3 hours after sperm-egg fusion. The pattern of Ca2+ spiking after fertilization is not consistent during this period but changes both in frequency and amplitude. Overall, the results demonstrate that, although electrostimulation induces both cortical granule exocytosis and protein reprogramming in porcine eggs, it does not reproduce the pattern of [Ca2+]i changes induced by sperm entry at fertilization.

Animals↗
PubMed1992

The fate of DNA injected into mammalian oocytes and zygotes at different stages of the cell cycle.

The stability of exogenous DNA microinjected into the cytoplasm at different stages of the meiotic cycle and after pronuclear formation was examined in ungulate species. Metabolism of the injected 1201 base pair (bp) DNA was examined by Southern blotting. Similar levels of metabolism of the injected DNA were detected in pig, sheep and bovine oocytes before germinal vesicle breakdown, in which about 30-40% of detected DNA was ligated into higher-molecular-weight forms. Porcine metaphase oocytes and pronuclear zygotes showed a reduced ability to ligate the exogenous DNA. In contrast, sheep and bovine metaphase oocytes and zygotes showed increased levels of ligation and, at the pronuclear stage, generated significant amounts of extremely large (greater than 15 kbp) ligation products. These results are discussed in the context of maternal precursors and metabolic activities in the egg.

Animals↗
PubMed1991

Somatic cells and the G2 to M-phase transition in sheep oocytes.

New protein synthesis is essential in sheep oocytes for the progression of the meiotic cycle to metaphase. Inhibition of transcription by alpha-amanitin, a specific inhibitor of RNA polymerase II, arrests meiosis in the G2 phase in cumulus enclosed oocytes but denuded oocytes progress to M-phase. Denuded oocytes injected with alpha-amanitin also undergo maturation. Cumulus-enclosed and denuded oocytes were also cultured on transcriptionally inactive follicle shells to examine the influence of transcription in the granulosa cell compartment on the progression of meiosis. No effect of alpha-amanitin was detected on the maturation rate of both cumulus enclosed and denuded oocytes. We conclude that transcription is required in the cumulus cells to allow translation of stored messages within the oocytes coding for cell-cycle proteins.

Amanitins↗
PubMed1991

Nuclear-cytoplasmic interactions during ovine oocyte maturation.

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

Animals↗
PubMed1991

Secretion of a putative metalloproteinase inhibitor by ovine granulosa cells and luteal tissue.

Ovine granulosa cells respond, through transcriptional mechanisms, to preovulatory concentrations of gonadotrophins by secreting an Mr 30,000 polypeptide. To determine the stages of the oestrous cycle at which this polypeptide is secreted, corpora lutea were collected on Days 3, 7, 10, 13 and 16 (Day 0 = oestrus; n = 4 per group), cut into 1-mm slices, and incubated for 6-7 h with [35S]methionine. Radiolabelled polypeptides of intra- and extra-cellular origin were separated by polyacrylamide gel electrophoresis and quantitated by densitometry. The Mr 30,000 polypeptide was secreted at all stages of the luteal phase tested (Days 3-16), and represented approximately 24% of the total labelled polypeptide present in the medium; polypeptides of approximate Mr 14,000, 25,000 and 46,000 accounted for most of the other secreted proteins. Neither pituitary hormones (LH, FSH, prolactin) nor cholera toxin (chosen to activate adenylate cyclase) affected the rate of production of Mr 30,000 polypeptide, indicating that, once secretion has been initiated in the granulosa cells, it is not readily modulated by hormonal intervention after luteinization. Incubation of luteinized granulosa cells with tunicamycin (inhibits N-linked glycosylation reactions) showed that the secreted polypeptide consists of a heavily glycosylated amino acid backbone of approximately Mr 20,000. Western blot analysis established further that the polypeptide was not an inhibin subunit. However, NH2-terminal amino acid sequencing of the first 25 amino acids revealed a 68% sequence identity between the secreted polypeptide (Mr 30,000) and a human tissue inhibitor of metalloproteinases.

Amino Acid Sequence↗
PubMed1991

Oviduct ampullary epithelium contributes a glycoprotein to the zona pellucida, perivitelline space and blastomeres membrane of sheep embryos.

Previous studies have identified a glycoprotein (sOP 92) that is secreted by sheep oviductal epithelium and subsequently becomes associated with developing embryos. The objective of this study was to more definitively demonstrate the localization of this protein in the oviductal epithelium of cycling ewes and in the embryos (developmental stage ranging between 1 and 8 cells) by using the light and electron microscopic immuno-gold techniques. In the oviduct, sOP 92 was present only in nonciliated cells of the ampulla but not in the isthmus. No major differences in signal intensity, in its organ or intracellular distribution were observed between the postovulatory and the luteal phase of the cycle. At the embryonic level, sOP 92 uniformly covered the surface of the zona pellucida and permeated its width. Moreover, the protein was present in the perivitelline space and on the blastomeres membrane but not in the cytoplasm or in the nucleus. These observations suggest that a marked regional difference in sheep oviduct secretions exists and confirm that there is a close association of these secretions with the developing embryos.

Animals↗
PubMed1991

Derivation of pluripotent, embryonic cell lines from the pig and sheep.

As previously described for the establishment of stable, pluripotent cell lines from pig blastocysts, an analogous cell line was isolated from a sheep blastocyst. There are common features in the morphologies and growth characteristics of the pig and sheep cells in culture; in particular, pig and sheep cells display large nuclei and relatively sparse cytoplasm, as is observed in mouse embryonic stem cells. Furthermore, the morphology of the sheep cells closely resembles that of cells in primary cultures of inner cell masses isolated immunosurgically from sheep blastocysts. This suggests that the sheep cell line represents a primary ectodermal lineage.

Animals↗
PubMed1990

Effect of oviduct cells on the incidence of polyspermy in pig eggs fertilized in vitro.

The consequences of interactions between porcine sperm, eggs, and oviduct cells before and during fertilization in vitro (IVF) has been examined with particular reference to the block to polyspermy. The pattern of polypeptides secreted by porcine oviduct epithelial cells has been determined and its effects on sperm both during pre-fertilization co-culture and during fertilization have been examined. In standard IVF procedures with no oviduct cell involvement, high rates of penetration (91%) were accompanied by equally high rates of multiple sperm penetration (91% of penetrated eggs). Fertilization on oviduct cell monolayers or a combination of 1 h co-culture of sperm and oviduct cells before the addition of in vitro matured oocytes did not reduce polyspermy. However, a sperm-oviduct cell co-culture period of 2.5 h followed by IVF on oviduct cells selectively reduced the rate of polyspermy by 40% and 50% in two separate series of trials (United Kingdom and Japan, respectively): Overall fertilization rates after this treatment were high (95% or 84%, respectively). A 3.5 h period of pre-fertilization co-culture further reduced polyspermy to only 14% of penetrated eggs, but this treatment was accompanied by a sharp drop in the fertilization rate from an overall mean of 88% for all other groups to 19% after 3.5 h co-culture.

Animals↗
PubMed1990

Maintenance and differentiation in culture of pluripotential embryonic cell lines from pig blastocysts.

Cell lines were established from explanted blastocysts of domestic pigs; the cells could be maintained indefinitely when grown on mouse fibroblast feeder cell layers. They differentiate spontaneously at high densities, or when allowed to form aggregates when cultured on a non-adhesive substratum. Their appearance and differentiative behaviour resembles that of mouse embryonic stem cell lines. We are currently attempting to establish whether these cultures represent primary ectodermal lineages which would be of particular relevance to developmental and transgenic studies.

Animals↗
PubMed1989

Characterization of proteins secreted by sheep oviduct epithelial cells and their function in embryonic development.

The role in early development of proteins secreted by oviduct epithelial cells has been investigated. Secreted proteins devoid of serum contamination have been produced by the surgical removal and immediate incubation of oviduct cells in [35S]methionine-containing medium. After electrophoretic separation, secreted polypeptides could be divided into those that were secreted uniformly throughout the oestrous cycle and a second class that showed a cyclical pattern of secretion. The first class of proteins represented a small proportion of total output whilst the predominant second class was composed mainly of polypeptides of Mr 92 and 46 x 10(3), respectively. Both of these polypeptide species, referred to as sheep oviduct proteins 92 and 46 (SOP 92, SOP 46), are detected only during the first 4 to 5 days after oestrus when the embryos are located in the oviduct. Oviduct cells collected at oestrus and maintained thereafter in culture secrete the same pattern of proteins and follow the same time course as their counterparts in vivo. The interaction between the oviduct proteins and the developing embryo was studied firstly by determining whether any of the secreted proteins bound to the zona pellucida. The results of iodination studies showed that two polypeptides of Mr 92 and 46 x 10(3), respectively, were bound to the zona pellucida of eggs removed from the oviduct but were absent from eggs that had not had contact with the oviduct epithelium. That these newly acquired proteins represent SOP 92 and 46 is suggested by their electrophoretic mobility and their ability to bind to the zona of follicular eggs when added in vitro and by the fact that they both disappear from the zonae of embryos after exit from the oviduct. The collection of unlabelled secreted proteins enabled us to produce a monoclonal antibody, which was used in the second series of experiments on oviduct-embryo interactions. The results confirmed that SOP 92 binds to the zona pellucida and moreover showed that this protein crosses the zona and becomes associated with the individual blastomeres of the developing embryo. These findings provide evidence that the mammalian oviduct probably plays a direct role in supporting embryonic development through specific polypeptides produced by its epithelium.

Animals↗
PubMed1989

Translational capacity of sheep oocytes microinjected with messenger RNA.

Sheep oocytes were microinjected with tobacco mosaic virus RNA (TMV-RNA) and isotopically labelled with L-[35S]methionine. Total incorporation of labelled methionine was similar in TMV-RNA-injected and in carrier-injected control oocytes, whether injections were performed during the period of high protein synthesis at maturation or during the period of reduced synthesis at a time equivalent to the mid-cleavage transition (48 h after germinal vesicle breakdown). Varying the amount of TMV-RNA injected from 2.5 to 10 pg had little effect on the overall level of amino acid incorporation. Furthermore TMV-RNA appeared to be very stable in oocytes and eggs; the proportion of total polypeptide synthesis directed by TMV-RNA did not diminish during the first 48 h after injection. Synthesis of most endogenous proteins was uniformly reduced to compensate for the synthesis of TMV-polypeptides. Our results suggest, therefore, that the translational capacity of sheep oocytes is fully saturated during maturation.

Animals↗