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Biomedical subjects

R M Moor

Publications and source records attributed to R M Moor.

At least 91 records · Page 5Linked to original sources
PubMed1989

Isolation and preliminary characterization of pig primordial follicles.

An enzymic method for recovering primordial follicles from the pig ovary consists of incubating cortical slices for 2 h with 0.025% collagenase 1A. An average of 185,000 or 419,000 primordial follicles per ovary were recovered from ovaries collected in Cambridge and Kansas, respectively. Following a discontinuous Percoll gradient, primordial follicles can be separated from contaminating somatic cells by mouth pipette or a micromanipulator to collect 100-1500 follicles but for large scale recovery of approximately 30,000 follicles flow cytometry is recommended. Two types of primordial follicles can be distinguished by electron microscopy: peripheral clusters of small oocytes with an incomplete investment of pregranulosa cells and a deeper region of individual oocytes surrounded by a complete layer of pregranulosa cells. The viability of the purified primordial follicles is attested by their ability to synthesize proteins for at least 12 h after incubation with [35S]methionine. Moreover, the primordial follicles showed several polypeptide bands in common with mature oocytes especially with Mr of 60,000-90,000 but with considerable differences from somatic cells.

Animals↗
PubMed1988

Inositol phosphates: proliferation, metabolism and function.

After the initial discovery of receptor-linked generation of inositol(1,4,5)trisphosphate (Ins(1,4,5)P3) it was generally assumed that Ins(1,4,5)P3 and its proposed breakdown products inositol(1,4)bisphosphate (Ins(1,4)P2) and Ins1P, along with cyclic inositol monophosphate, were the only inositol phosphates found in significant amounts in animal cells. Since then, three levels of complexity have been introduced. Firstly, Ins(1,4,5)P3 can be phosphorylated to Ins(1,3,4,5)P4, and the subsequent metabolism of these two compounds has been found to be intricate and probably different between various tissues. The functions of Ins(1,4,5)P3 and Ins(1,3,4,5)P4 are almost certainly to regulate cytosolic Ca2+ concentrations, but the reasons for the labyrinth of the metabolic pathways after their deactivation by a specific 5-phosphatase remain obscure. Secondly, inositol pentakis- and hexakisphosphates have been found in many animal cells other than avian erythrocytes. It has been shown that their synthesis pathway is entirely separate from the inositol phosphates discussed above, both in terms of many of the isomers involved and probably in the subcellular localization; some possible functions of InsP5 and InsP6 are discussed here. Thirdly, cyclic inositol polyphosphates have been reported in stimulated tissues; the evidence for their occurrence in vivo and their possible physiological significance are also discussed.

Animals↗
PubMed1988

In vitro culture of sheep oocytes matured and fertilized in vitro.

Sheep oocytes that matured and fertilized in vitro were cultured to evaluate their cleavage to the 8- to 16- cell stage and further development in five different media as follows: 1) CPMW (TCM199 + 20% ewe serum + 0.4% BSA), 2) Ham's F-10 + 10% ewe serum, 3) Brinster's pyruvate medium + 0.1% glucose (BPM-G), 4) co-culture with sheep oviduct epithelial cells in TCM199 + 10% fetal calf serum, and 5) co-culture with sheep granulosa cells in the same medium as 4. The culture duration was 4 or 7 d for 8- to 16-cell or further development. The proportions of 8- to 16-cell eggs were 1) 16% (8 49 ), 2) 25% (12 49 ), 3) 52% (58 112 ), 4) 63% (105 167 ) and 5) 45% (27 60 ). The co-culture with sheep oviduct cells resulted in a significantly (P < 0.05) higher rate of cleavage than the other media, except BPM-G. The proportion of noncompacted morula (35%, 24 68 ) was also significantly (P < 0.05) higher in the co-culture of sheep oviduct cells than the other media. The 8- to 16-cell eggs produced by BPM-G (n=38) and the co-culture with sheep oviduct cells (n=42) were transferred into the uterus of recipient ewes, but no elongated blastocysts were obtained 13 d later. On the other hand, 8 out of 55 one-cell eggs (15 to 18 h after in vitro insemination) transferred to the oviduct of recipient ewes were elongated blastocysts (24% of 34 recovered eggs). The data show that the co-culture of in vitro fertilized eggs with sheep oviduct epithelial cells could support development of 8- to 16-cell embryos or early morula, but their viability is still questionable.

Journal Article↗
PubMed1988

Regulation of the meiotic cycle in oocytes of domestic mammals.

The progression of meiosis from prophase to metaphase II involves modifications to somatic signals, activation of a master regulator system in the cytoplasm and the manipulation of independent components in the nucleus. Studies on amphibian oocytes suggest that the cytoplasmic component, maturation promoting factor (MPF), is a key component of the master regulator system. A similar and ubiquitous role for MPF in mammalian oocytes is indicated by cell fusion studies. However, the nature of the somatic signals responsible for prophase arrest and the sequence of intracellular events necessary for the synthesis and activation of MPF differ markedly between species. The most fundamental of these differences is in the G2- to M-phase transition where, in rodents, phosphorylation changes alone activate the MPF cascade while in domestic mammals both transcription and new protein synthesis is required for MPF production. One of the proteins (47,000 daltons) involved in the MPF cascade has been tentatively identified in ovine oocytes and has been shown to be dependent upon transcription at the initiation of maturation. The synthesis of additional protein, probably with anti-MPF activity, is thereafter required to drive the cycle from metaphase I to anaphase. The final phase of cell cycle-related protein synthesis occurs immediately before the formation of the metaphase II plate. The Ca2+-sensitive cytostatic proteins synthesized at this stage maintain the MII chromatin in a condensed state and prevent the completion of the meiotic cycle until fertilization when the cytostatic proteins disappear and meiosis is completed.

Animals↗
PubMed1988

Initiation of the cortical reaction in hamster and sheep oocytes in response to inositol trisphosphate.

Microinjection of inositol 1,4,5-triphosphate into sheep and hamster oocytes induces secretion of cortical granules in a dose-dependent manner. In the sheep, this effect is strongly pH-dependent with minimal exocytosis taking place at pH 6.8 but a full cortical reaction occurring at pH8.0. Exocytosis in the hamster is also affected by the pH of the external medium but to a lesser extent. Injection of GTP gamma S also induces exocytosis in both species but is more effective in the hamster. It is suggested that inositol metabolism stimulated by sperm-egg interaction with a GTP-binding protein may be part of the mechanism leading to cortical granule exocytosis and that this may be modulated by the external pH.

Animals↗
PubMed1988

Ram-specific effects on in-vitro fertilization and cleavage of sheep oocytes matured in vitro.

The present experiments were designed to identify possible male-specific effects on early embryonic development in vitro: Sheep oocytes were matured in vitro for 24-26 h and then fertilized in vitro using equal numbers of viable spermatozoa from 1 of 6 Clun Forest rams. At 15-18 h after insemination, oocytes were either fixed and examined for fertilization and polyspermy or further cultured in modified M 199 medium for 3 days in an oviduct epithelial co-culture system. There were significant differences in 5 separate trials between the rams with respect to the rate of fertilization, degree of polyspermy and cleavage rate after monospermic fertilization. The mean rate of fertilization varied from 89% in Ram B to 43% in Ram C while the percentage of polyspermic eggs varied from 5 to 34%. Both the absolute number of embryos cleaving to the 16-cell stage and the calculated percentage of monospermic eggs reaching the 16-cell stage differed markedly between groups of eggs fertilized by different rams. The results indicate that the development of sheep eggs in vitro is differentially affected by the ram from which the spermatozoa are collected.

Animals↗
PubMed1988

Control of protein synthesis during early cleavage of sheep embryos.

Sheep embryos, radiolabelled with [35S]methionine, were studied during the first four cell cycles after fertilization to determine the stage at which the regulation of protein synthesis switches from the direction by maternal mRNA to mRNA transcribed by the embryonic genome. Total protein synthesis was consistently high during the first 2 cleavage divisions, dropped by 95% in the 3rd cell cycle, remained low in the 4th and increased again in the 5th cycle. A consistent pattern of proteins was synthesized during the first 3 cell cycles after fertilization followed by major changes in subsequent cycles. The inhibition of transcription by alpha-amanitin, an inhibitor of polymerase II, did not affect cleavage or protein synthesis during the first 3 cell cycles but blocked cleavage and protein synthesis thereafter. The results indicate that the full activation of transcription in sheep embryos occurs in the 4th cell cycle.

Amanitins↗
PubMed1987

Inositol(1,3,4,5)tetrakisphosphate-induced activation of sea urchin eggs requires the presence of inositol trisphosphate.

We have earlier reported that Inositol (1,3,4,5)tetrakisphosphate microinjection will activate eggs of the sea urchin Lytechinus variegatus provided that it is co-injected with inositol (2,4,5)trisphosphate (Irvine and Moor, Biochem. J. 240, 917-920, 1986). Here we extend these observations to show that inositol (1,3,4,5,6)pentakisphosphate is a partial agonist in this assay and the requirement for the presence of inositol (2,4,5)trisphosphate cannot be bypassed by raised, but sub-threshold, Ca2+ concentrations. A mechanism for the proposed stimulation of Ca2+ entry into the cell requiring both inositol tris- and tetrakisphosphates is presented.

Animals↗
PubMed1987

Cellular origin, hormonal regulation and biochemical characteristics of polypeptides secreted by graafian follicles of sheep.

The pattern, chemical nature and biological role of polypeptides secreted by ovine follicles exposed to gonadotrophins in vivo were studied. Follicles were removed at intervals before ovulation (in-vivo groups), separated into granulosa and cumulus compartments, and incubated for 3 h with radiolabelled amino acids. No differences were detected in the polypeptides (Mr 46,000-60,000) secreted by granulosa and cumulus cells before exposure to the preovulatory LH surge. Each class of polypeptides was characterized by different degrees of phosphorylation, sulphation and glycosylation. Within 15 h of the LH surge the secretion of the Mr 46,000-60,000 polypeptides had ceased and was replaced by a non-sulphated Mr 30,000 secretory product. Significant differences in the secretory pattern of granulosa and cumulus cells were detected after exposure to LH. Intact follicles and granulosa cells were cultured for 24 h (in-vitro groups) and then incubated with radiolabelled amino acids. The profile of polypeptides secreted by intact follicles cultured in the presence or absence of LH corresponded closely with the profile observed in vivo. By contrast, granulosa cells grown as monolayers switched spontaneously to the secretion of Mr 30,000 polypeptides in medium devoid of gonadotrophin. This aberrant secretory switch did not occur in granulosa cells maintained in suspension culture. Inhibition of transcription in follicles exposed to LH prevented both the appearance of the Mr 30,000 polypeptide and the disappearance of the Mr 46,000-60,000 polypeptides. Although the inhibition of steroidogenesis by a variety of steroid enzyme inhibitors was without effect on secretion, evidence was obtained to suggest that one of the secreted polypeptides binds oestradiol-17 beta.

Animals↗
PubMed1987

Stimulation of early embryonic development in the sheep by co-culture with oviduct epithelial cells.

To examine the effects of somatic cell support on the cleavage and viability of fertilized sheep eggs, 434 pronucleate eggs were co-cultured for 3 or 6 days on oviduct cells or fibroblasts and 77 eggs were cultured in medium alone. During the first 3 days in culture 95% of the single-celled eggs cleaved regularly to non-compacted morulae on either of the feeder-layers but only 13% underwent similar regular cleavage in medium alone. Despite the identical cleavage rates in the co-culture groups, only 33% of embryos grown on fibroblasts as compared with 80% of embryos grown on oviduct cells were fully viable as judged by their ability to develop normally after transfer to recipient animals. The viability of embryos in the oviduct group was equal to that obtained after the direct transfer of morulae from donor to recipient sheep. After 6 days in culture 42% of embryos co-cultured with oviduct cells developed into expanded blastocysts as compared with only 4.5% cultured on fibroblasts. In both co-culture groups virtually all the remaining embryos blocked during the 4th cleavage. When transferred, 30% of blastocysts grown from the pronucleate stage on oviduct cells were viable. We conclude that: (1) during the first 3 days after fertilization cleavage will progress at a normal rate on different feeder-layers but oviduct cells appear to be required for the acquisition of full embryonic viability.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1987

Stage-dependent effects of inhibiting ribonucleic acids and protein synthesis on meiotic maturation of bovine oocytes in vitro.

Ribonucleic acid synthesis continues at a low rate within 1 h of germinal vesicle breakdown. To determine if this newly synthesized mRNA is required for the resumption of meiosis in cattle oocytes, cumulus-enclosed oocytes were removed from 2 to 4-mm antral follicles directly into Dulbecco's medium with or without the RNA inhibitor, alpha-amanitin, or the protein synthesis inhibitor, cycloheximide (10 micrograms/ml). They were washed twice and matured for 28 or 48 h in medium 199, with Earle's salts, 2.2 g/L NaHCO3 and L-glutamine supplemented with 20% heated fetal calf serum to which were added gonadotropins (10 micrograms/ml NIH-LH-S18; 10 micrograms/ml NIH-FSH-S8; 20 ng/ml NIH-P-S9), estradiol-17 beta (1.5 micrograms/ml), solcoseryl (30 microliter/ml), and Dibekacin sulfate (100 micrograms/ml) with or without the inhibitors. After 28 h of maturation, 95.8% of control oocytes had undergone germinal vesicle breakdown and formation of a metaphase plate compared with only 1.3% of those continuously exposed to cycloheximide and 38% of those continuously exposed to amanitin. Exposure to cycloheximide or amanitin for only the first 16 h of culture followed by 12 h of inhibitor free culture resulted in 96.6% germinal vesicle breakdown with cycloheximide but only 56.5% germinal vesicle breakdown with amanatin. We conclude newly synthesized mRNA and protein synthesis is required for both full cumulus cell expansion and germinal vesicle breakdown.

Amanitins↗
PubMed1986

Micro-injection of inositol 1,3,4,5-tetrakisphosphate activates sea urchin eggs by a mechanism dependent on external Ca2+.

Micro-injection of submicromolar concentrations of inositol 1,3,4,5-tetrakisphosphate caused a raising of the fertilization envelope in eggs of the sea urchin Lytechinus variegatus. This effect was dependent both on the presence of extracellular Ca2+ and on co-injection with a Ca2+-mobilizing compound, inositol 2,4,5-trisphosphate. Inositol 1,3,4,5-tetrakisphosphate was the most potent compound tested in this assay; removal of the 3- or 5-phosphates or randomization of the phosphates in the inositol ring decreased its potency. These results show that inositol 1,3,4,5-tetrakisphosphate is an intracellular second messenger, and suggest that its function is to control cellular Ca2+ homoeostasis at the plasma membrane.

Animals↗
PubMed1986

Effect of alterations in follicular steroidogenesis on the nuclear and cytoplasmic maturation of ovine oocytes.

The effects of inhibitors of follicular steroidogenesis on biochemical changes occurring in oocytes maturing in vitro were studied using radiolabelling and polyacrylamide gel electrophoresis. These effects were correlated with previously investigated developmental abnormalities induced by the same inhibitors. The most severe effects were generated by inhibition of 17 alpha-hydroxylase with the drug SU10603 which resulted in a greatly increased ratio of progesterone to testosterone and oestrogen. Such treatment halved the rate of meiotic maturation. Treated oocytes were analysed individually on SDS-PAGE gels and quantitative analysis showed that the drug had induced synthetic abnormalities even in those oocytes that resumed meiosis. This conclusion was confirmed by separation of oocyte proteins on two-dimensional gels. The effects of SU were reduced by delaying addition of the drug until 6h after the beginning of maturation but were not alleviated by the addition of exogenous oestrogen to the culture medium. When oocytes from SU-treated follicles were transferred to inseminated, recipient ewes and recovered 24h later, two-dimensional electrophoresis again revealed abnormalities in their protein synthetic patterns. Almost total abolition of steroid secretion by aminoglutethimide (AG) had much less effect on oocyte protein synthesis, although the proportion of oocytes maturing was reduced from 65% to 46%. The aromatase inhibitor, androstatriendione (AST) although eliminating follicular oestrogen secretion, had no effect on the rate of maturation and very little effect on protein synthesis. These results correlate well with the effects of steroid inhibitors on fertilization and early cleavage.

Aminoglutethimide↗
PubMed1986

Protein requirements for germinal vesicle breakdown in ovine oocytes.

The regulation of the cell cycle during the transition from prophase to metaphase I was studied by analysing protein changes and introducing protein blocks during the transition phase. The results show that the progression to metaphase in ovine oocytes is totally dependent on new protein synthesis. By delaying the addition of the inhibitor, cycloheximide, for progressively longer periods after the resumption of meiosis it was established that the required synthesis occurs in the 1-2 h immediately preceding germinal vesicle breakdown (GVBD). The action of cycloheximide was fully reversible: removal of the drug resulted in GVBD between 3 and 4 h later. The synthesis and modification of proteins during these first few hours of maturation were studied by short-term radiolabelling of oocytes with [35S]methionine and [32P]phosphate followed by rapid assessment of their precise nuclear configuration. Changes in phosphorylation of two polypeptides were detected 4-5 h after the beginning of culture, but these changes were not dependent upon protein synthesis. The earliest change in synthesis was the appearance of a new polypeptide 6-8 h after explantation, immediately before GVBD. This polypeptide (Mr 47 X 10(3), pI 5.8) was not significantly phosphorylated and was relatively stable. Oocytes released from cycloheximide treatment began to synthesize this molecule 3-4 h later, again coinciding with GVBD. Synthesis of the polypeptide was suppressed by inhibition of transcription with alpha-amanitin.

Amanitins↗