Biomedical subjects

N L First

Publications and source records attributed to N L First.

At least 37 records · Page 2Linked to original sources

PubMed1997

Induction of DNA replication in germinal vesicles and in nuclei formed in maturing mouse oocytes by 6-DMAP treatment.

Immature mouse oocytes (germinal vesicle stage, GV), oocytes at different stages during maturation (prometaphase to anaphase I) and matured oocytes (metaphase II arrested) were cultured in 6-dimethylaminopurine (6-DMAP)-supplemented medium also containing bromodeoxyuridine for the assessment of DNA replication in these cells. Immature oocytes remained arrested at the GV stage and DNA replication was never detected in them. On the other hand, oocytes at the prometaphase to anaphase-telophase I stages responded to 6-DMAP treatment by forming nuclei which synthesised DNA. Mature (metaphase II) oocytes did not respond to 6-DMAP and their chromatin remained condensed. DNA synthesis could even be induced in GV-staged oocytes, but only when they were fused to freshly activated oocytes and incubated in 6-DMAP-supplemented medium.

Adenine↗

PubMed1997

Damaged chromatin does not prevent the exit from metaphase I in fused mouse oocytes.

The presence of checkpoint mechanisms which are able to recognize damaged chromatin and thereafter to prevent exit from metaphase I has been investigated in giant mouse oocytes produced by fusion of a normal metaphase I oocyte with an equivalent oocyte with damaged chromatin. The presence of damaged chromatin did not prevent the onset of anaphase I in both sets of chromatin in the fused cells. Interestingly, fused or unfused cells containing only damaged chromatin failed to enter anaphase and persisted instead in a metaphase-like state. These results demonstrate the fragility of checkpoint controls in mammalian female germ cells.

Animals↗

PubMed1996

Phenotypic variations among paternal centrosomes expressed within the zygote as disparate microtubule lengths and sperm aster organization: correlations between centrosome activity and developmental success.

This study describes a paternal effect on sperm aster size and microtubule organization during bovine fertilization. Immunocytochemistry using tubulin antibodies quantitated with confocal microscopy was used to measure the diameter of the sperm aster and assign a score (0-3) based on the degree of radial organization (0, least organized; 3, most organized). Three bulls (A-C) were chosen based on varying fertility (A, lowest fertility; C, highest fertility) as assessed by nonreturn to estrus after artificial insemination and in vitro embryonic development to the blastocyst stage. The results indicate a statistically significant bull-dependent difference in diameter of the sperm aster and in the organization of the sperm astral microtubules. Insemination from bull A resulted in an average sperm aster diameter of 101.4 microm (76.3% of oocyte diameter). This significantly differs (P < or = 0.0001) from the average sperm aster diameters produced after inseminations from bull B (78.2 microm; 60.8%) or bull C (77.9 microm; 57.8%), which themselves displayed no significant differences. The degree of radial organization of the sperm aster was also bull-dependent. Sperm asters organized by bull A-derived sperm had an average quality score of 1.8, which was higher than that of bull B (1.4; P < or = 0.0005) or bull C (1.2; P < or = 0.0001). Results with bulls B and C were also significantly different (P < or = 0.025). These results indicate that the paternally derived portion of the centrosome varies among males and that this variation affects male fertility, the outcome of early development, and, therefore, reproductive success.

Analysis of Variance↗

PubMed1996

Aspects of follicle and oocyte stage that affect in vitro maturation and development of bovine oocytes.

Success of in vitro maturation (IVM) and production of bovine embryos as related to aspects of follicle source and oocyte size were evaluated. First, it was determined that bovine oocytes continue growing in all follicular sizes studied, including >1- to 15-mm follicles. Populations of oocytes were collected from surface visible (peripheral) and cortical follicles from the same ovaries. When the number of oocytes from both peripheral and cortical follicles was combined, the yield of oocytes was approximately double that collected from 1 ovarian site alone. Oocytes from cortical follicles were smaller than those from the surface population, and the smaller cortical oocytes had a lower potential for both meiotic maturation and embryo development Only cortical oocytes with the largest diameters underwent IVM and subsequently developed to blastocysts at rates comparable to oocytes from peripheral follicles. As the diameter of the oocytes recovered from peripheral follicles increased, so did their developmental potential. When the stage of the estrous cycle was observed, it was found to have no effect on developmental potential. Finally, oocytes which extruded polar bodies at an earlier time during maturation were, on average, larger than those which extruded polar bodies later. The results serve a practical purpose in assisting selection of oocytes capable of developing into blastocysts and they give useful correlates of oocyte competencies based on knowledge of follicle source and oocyte stage.

Journal Article↗

PubMed1996

Nuclear transplantation in mammals: remodelling of transplanted nuclei under the influence of maturation promoting factor.

Whilst the role of Maturation or M-phase Promoting Factor (MPF) as a universal M-phase regulator is well documented, much less attention has been paid to its role in nuclear transplantation experiments and especially to its influence upon remodelling of transplanted nuclei. There is currently wide acceptance that successful nuclear transplantation using differentiated nuclei is possible only in a cytoplasmic environment that is capable of inducing rapid nuclear de-differentiation to a pronuclear-like form. In this review our purpose is firstly, to outline the conditions under which such remodelling can be induced, and secondly, to extend the debate to include a consideration of whether complete nuclear remodelling is an absolute necessity for clonal development.

Animals↗

PubMed1995

Activation of murine oocytes with Ca2+ ionophore and cycloheximide.

A Ca2+ ionophore (A23187, 3 microM) and inhibitor of protein synthesis (cycloheximide, 10 micrograms/ml) were used sequentially as a unique method for activating mouse oocytes in vitro. Brief exposure of oocytes to A23187 followed by 6 hr in cycloheximide resulted in a higher activation rate (93.8%) compared to A23187 or cycloheximide alone (37.7% and 36.5%, respectively) or the two reagents in reverse order (29.8%). The parthenogenones consistently contained a single pronucleus and second polar body, and showed a high degree of developmental potential, as assessed by transfer to recipient females or addition of a male pronucleus followed by transfer to recipients. This method is a useful way of obtaining large numbers of activated haploid mammalian oocytes for further developmental studies.

Animals↗

PubMed1995

Manipulation of bovine sperm metabolism and motility using anoxia and phosphodiesterase inhibitors.

Bovine sperm that were subjected to extended anoxia (2.5 h) in the absence of glycolytic substrates then diluted into oxygenated medium were immotile but metabolically active, producing ATP from lactate via oxidative phosphorylation. In response to anoxia sperm ATP titers dropped from 15-20 mumoles/10(8) cells to 1-2 mumoles/10(8) cells in the first 5 min then remained extremely low until reoxygenation. Cyclic AMP titers declined slowly over the anoxic period, but did not show the same scale of depression as ATP. After dilution and re-oxygenation ATP recovered to pre-anoxia levels within 1 min, and cAMP rose to about the pre-anoxia levels. However, motility, which varied quantitatively and qualitatively between ejaculates prior to anoxic treatment, was substantially depressed after extended anoxia in all cases; progressive motility was almost non-existent in post-anoxic sperm. Addition of isobutylmethylxanthine or Cibacron Blue F3GA, both putative phosphodiesterase inhibitors, stimulated a transient peak of cAMP, which was accompanied by motility stimulation. These techniques provide a protocol to manipulate and dissect the biochemical pathways of motility initiation in mammalian sperm.

1-Methyl-3-isobutylxanthine↗

PubMed1995

Stimulation of bovine sperm motility and respiration by the triazine dye cibacron blue F3GA.

Bovine sperm motility and respiration were stimulated by the triazine dye Cibacron Blue F3GA (CB), which may operate as a nucleotide mimic. CB stimulation of respiration was half-maximal at about 35 microM and respiration reached maximal levels about 1.5 minutes after CB addition. Respiratory stimulation was preceded by a transient increase in cytosolic cAMP. Sperm cAMP titers were elevated from 5 to 10 pmoles/10(8) cells within 30 seconds of CB addition, but rapidly dropped to a stable level of about 7.5 pmoles/10(8) cells. CB was a potent inhibitor of sperm membrane adenylyl cyclase and inhibited respiration in permeabilized cells. Taken together, the data indicated that CB stimulation was not manifested via the cytosol. In addition, a nonpermeant blue dextran preparation synthesized with CB also stimulated sperm respiration and motility. CB inhibited sperm membrane phosphodiesterase activity, suggesting that the transient pulse of cAMP resulted from CB interaction with this enzyme in the sperm membrane.

Adenylyl Cyclase Inhibitors↗

PubMed1995

Expression of the cell cycle control protein cdc25 in cleavage stage bovine embryos.

We have examined the synthesis and expression of a homologue of the cell cycle control protein cdc25 by early cleavage stage bovine embryos. cdc25 is the protein phosphatase responsible for activating p34cdc2 by dephosphorylating the threonine 14 (Thr 14) and tyrosine 15 (Tyr 15) residues of p34cdc2. Human cdc25 antibody was utilised in western blots and immunoprecipitations to examine the presence and synthesis of cdc25 in bovine embryos. cdc25 is present as a 52 kDa non-phosphorylated and a 66 kDa presumably phosphorylated form in bovine 1-, 2-, 4- and 8-cell embryos. However, cdc25 is actively synthesised only in 8-cell embryos, indicating that the cdc25 present prior to this stage is inherited from the oocyte. In addition, the synthesis of cdc25 was induced in 2-cell embryos in which cleavage was blocked with the DNA synthesis inhibitor aphidicolin.

Animals↗

PubMed1995

Maintenance of bovine oocytes in prophase of meiosis I by high [cAMP]i.

The effects of high intracellular cAMP concentrations ([cAMP]i) on germinal vesicle maintenance of bovine cumulus-oocyte complexes were investigated, using 8-bromo-3',5'-cAMP (8-Br-cAMP) or an invasive adenylate cyclase from Bordetella pertussis to increase the [cAMP]i. The effects of interactions of these agents with macromolecular supplements in culture medium (fetal calf serum, FCS; polyvinylpyrrolidone, PVP; BSA), and different methods of processing complexes before culture, on subsequent germinal vesicle maintenance by invasive adenylate cyclase were studied. While 8-Br-cAMP was unable to maintain germinal vesicle arrest in the majority of oocytes for 20 h (36% with FCS, 24% with BSA, 18% with PVP), it maintained germinal vesicle arrest in a high proportion of cumulus-enclosed oocytes when BSA or PVP was used (37% with FCS, 52% with BSA, 53% with PVP). The difference in frequency of germinal vesicle maintenance between macromolecular supplements was not related to [cAMP]i when assayed after culture for 2 h with invasive adenylate cyclase. Complexes processed in whole follicular fluid were not maintained in meiotic arrest (26%) when cultured with invasive adenylate cyclase and PVP. Complexes processed in follicular fluid with 3-isobutyl 1-methylxanthine (IBMX) plus invasive adenylate cyclase were arrested at the germinal vesicle stage at high frequencies (65%), while those processed in IBMX or IBMX plus 8-Br-cAMP-supplemented follicular fluid had intermediate (43% and 49%, respectively) frequencies of intact germinal vesicles. Oocyte complexes processed in follicular fluid supplemented with IBMX and invasive adenylate cyclase formed morulae and blastocysts (27.2%), as did oocytes processed in follicular fluid alone (26%). Phosphoprotein profiles showed that control oocytes and 8-Br-cAMP-treated oocytes share a profile that is different from that of oocytes treated with invasive adenylate cyclase. These results show that increased [cAMP]i reversibly maintains bovine oocytes in meiotic arrest for an extended period without the occurrence of the post-translational protein modifications observed during meiotic resumption or transient arrest.

1-Methyl-3-isobutylxanthine↗

PubMed1995

Maintenance of meiotic arrest by increasing [cAMP]i may have physiological relevance in bovine oocytes.

Invasive adenylate cyclase (iAC) reversibly inhibits spontaneous maturation of cumulus-enclosed bovine oocytes by increasing the intracellular concentration of cAMP, [cAMP]i. In this study, physiological aspects of maintaining meiotic arrest in bovine oocytes by iAC were investigated. The maintenance of germinal vesicle arrest by iAC in both cumulus-enclosed and denuded bovine oocytes was concentration dependent (r2 = 0.857). Denuded bovine oocytes were more sensitive to maintenance of meiotic arrest by iAC then were cumulus-enclosed oocytes. At the highest concentration, 70% of the cumulus-enclosed and 90% of the denuded bovine oocytes were maintained in meiotic arrest. The iAC increased [cAMP]i in both intact cumulus-oocyte complexes and enclosed oocytes in a concentration-dependent manner (r2 = 0.795). Cumulus-enclosed oocytes maintained in meiotic arrest by iAC retained developmental competence when subsequently cultured in iAC-free medium and then fertilized. The [cAMP]i in bovine complexes decreased precipitously upon release from follicles and remained low for the next 125 min. However, the [cAMP]i of the enclosed oocytes did not change. Bovine oocytes commit to undergo meiosis in a progressive manner. Approximately 10% of the oocytes were already committed when aspirated. This proportion increased to 40% at 2 h and 70% at 5 h. Use of two inhibitors of cAMP-dependent protein kinase A provided further evidence that cAMP functions in mediating meiotic arrest in bovine oocytes. Bovine oocytes, therefore, are sensitive to different cAMP concentrations, and are developmentally competent after iAC-induced arrest, and complexes containing oocytes exhibit a decrease in [cAMP]i before spontaneous maturation. These results suggest that maintenance of meiotic arrest by iAC is accomplished through modulation of cellular machinery, and regulation of oocyte maturation by [cAMP]i may be physiologically relevant.

Adenylyl Cyclases↗

PubMed1994

Production of calves by transfer of nuclei from cultured inner cell mass cells.

We report here the isolation and in vitro culture of bovine inner cell mass (ICM) cells and the use of ICM cells in nuclear transfer to produce totipotent blastocysts that resulted in calves born. Of 15 cell lines represented in this study, 13 were derived from immunosurgically isolated ICM of 3 in vitro produced day 9-10 bovine blastocysts, while 2 lines were derived from single blastocysts. Approximately 70% of attempted cell lines became established cell lines when started from 3 ICMs. The ability to establish cell lines was dependent on the number of ICMs starting the line. Sire differences were noted in the ability of ICMs to establish cell lines and to form blastocysts. The cell lines were cultured as a low cell density suspension in the medium CR1aa plus selenium, insulin, and transferrin (SIT) and 5% fetal calf serum (FCS) for 6-101 days before use in nuclear transfer, at which time some had multiplied to more than 2000 cells. If allowed to aggregate, cells of established cell lines formed embryoid bodies. A total of 659 nuclear transfer clones were made by fusing the ES cells into enucleated oocytes with polyethylene glycol; 460 of these fused, based on cleavage (70%). After culture of the clones for 7 days in vitro in CR1aa/SIT/5% FCS, 109 (24%) of those fused became blastocysts. Thirty-four blastocysts were transferred into uteri of 27 cows, and 13 cows (49%) became pregnant. Four of the 13 cows gave birth to 4 normal calves. DNA typing showed the calves to be derived from the respective sires of the cell lines. The calves were derived from cultures of less than 28 days.

Animals↗

PubMed1994

Microtubule organization in the cow during fertilization, polyspermy, parthenogenesis, and nuclear transfer: the role of the sperm aster.

Microtubule organization in bovine oocytes during fertilization, polyspermy, parthenogenesis, and nuclear transfer is examined with the goal of understanding microtubule activity and the manner in which the centrosome during fertilization and mitotic spindle poles are established. In the unfertilized bovine oocyte, microtubules are detected only in the metaphase-arrested second meiotic spindle; no cytoplasmic asters are observed. After insemination, a small aster of microtubules is seen adjacent to the incorporated sperm head. This aster enlarges and, at the time of pronuclear apposition, fills the cytoplasm. At prophase, the aster splits and forms the poles for the first mitotic spindle, which is anastral, fusiform, and often located eccentrically. During anaphase, asters assemble at each spindle pole. After telophase, these asters develop into the interphase array of microtubules in the daughter blastomeres. During polyspermy, an aster forms from a site between each incorporated sperm head and tail. Multiple mitotic spindles are observed in polyspermic zygotes; multipolar spindles are not seen. Parthenogenetic activation with 5 microns ionomycin followed by a 4-hr incubation in 1.9 mM dimethylaminopurine results in > 80% activation, and antitubulin immunofluorescence microscopy demonstrates that initially disarrayed microtubules are observed and the some microtubules extend from the remnants of the second meiotic spindle. At the time normal for cell division, these parthenogenotes form anastral, barrel-shaped bipolar mitotic spindles. Asters form at the spindle poles at anaphase and the parthenogenotes divide from one to two blastomeres. Nuclear fusion of a morula-derived blastomere to a parthenogenetically activated oocyte results in either a single microtubule aster or at times, two asters, formed in association with the donated nucleus. These results demonstrate that fertilization in the cow is a blending of paternally and maternally derived centrosomal material.

Animals↗

PubMed1994

Inhibition of protein kinases after an induced calcium transient causes transition of bovine oocytes to embryonic cycles without meiotic completion.

We have examined the response of bovine oocytes matured in vitro for 24 hr to parthenogenic activation using compounds that increase intracellular calcium (ionomycin) or inhibit protein phosphorylation (6-dimethylaminopurine, DMAP). Treatment with ionomycin alone caused resumption of meiosis (57.8 +/- 7.8%) but not pronuclear formation (8.9 +/- 7.3%). DMAP alone did not cause resumption of meiosis or pronuclear formation. Sequential treatment with ionomycin (5 microM for 4 min) immediately followed by DMAP (1.9 mM for 5 hr) resulted in activation that led to pronuclear formation (80.5 +/- 13.1%). Completion of meiosis, however, was bypassed as evidenced by only one polar body and one pronucleus present in activated parthenogenones. It was necessary to incubate the oocytes for at least 3 hr in DMAP to obtain high rates of activation (76.6 +/- 9.8%) and development to blastocysts (21.1 +/- 1.5%). Temporal separation of the two treatments resulted in a decrease in oocytes with one pronucleus and one polar body (uniformly diploid parthenogenones) and an increase in a mixture of diploid and haploid parthenogenones since DMAP was capable of causing transition to interphase of all chromatin configurations after anaphase commenced and prior to metaphase arrest. Parthenotes produced with ionomycin and DMAP that developed to the blastocyst stage had high cell numbers (70 to 88 cells) and were able to cause extended cycles in 33.3% of recipient cattle after nonsurgical transfer to the uterus. Response of the bovine oocyte arrested in metaphase II to different activation stimuli was also found to show age-dependent changes in pattern of activation response and developmental competence.

Adenine↗

PubMed1994

Systems for production of calves from cultured bovine embryonic cells.

The development of totipotent bovine embryonic cell cultures has great value in cattle breeding. They provide: (1) a mechanism for making large numbers of clonal offspring by nuclear transfer; (2) an efficient gene transfer system through the use of selectable markers to select transgenic cells; and (3) a mechanism for site-specific gene transfer or deletion by homologous DNA sequence recombination. Bovine embryonic cell cultures have been established from blastocyst inner cell mass (ICM) cells, morulae and the precompaction 16-20-cell stage. All have exhibited similar morphology to mouse embryonic stem (ES) cells, pluripotency on differentiation and proliferation in culture. Culture systems have consisted of microdrop loose suspension short-term cultures or long-term cultures on bovine or murine fibroblast feeder layers, in either a microdrop or a culture dish. The relative merit of culture systems or media requirements for mitosis and prevention of differentiation have not been determined. At present, totipotency is also unknown for cultured cells of the 16-20-cell stage. For cultured ICM cells, totipotency was demonstrated by the birth of four calves from ICM cells cultured 27 days or less in a loose suspension microdrop. Advanced pluripotency and perhaps totipotency was demonstrated in one fetus in a recently reported study where morulae cells cultured in vitro were chimaerized with non-cultured cells. DNA fingerprinting to associate cell lines with offspring and karyotyping to ascertain chromatin normalcy is important in ES cell research. Data pertaining to the use of each are presented.

Animals↗

PubMed1994

Differences in the role of cyclic adenosine 3',5'-monophosphate during capacitation of bovine sperm by heparin or oviduct fluid.

Capacitation is an important maturational event in the life of a spermatozoan that allows the sperm to undergo a stimulus-induced acrosome reaction. Bovine sperm can be induced to undergo capacitation in vitro by heparin or oviduct fluid, and capacitation can be inhibited by glucose. We found that glucose did not interfere with 3H-heparin binding to sperm. Glucose inhibition of capacitation could be reversed in a dose-dependent manner by 8-bromo-cAMP or by the phosphodiesterase inhibitors isobutylmethylxanthine or caffeine, with ED50S of 25, 32, and 183 microM, respectively. The maximal effect of 8-bromo-cAMP on capacitation was during the first 2 h of a 4-h incubation. Sperm cAMP increased during capacitation with heparin from an initial value of 4.1 +/- 0.1 to 7.3 +/- 1.1 pmol cAMP/20 x 10(6) sperm at 4 h of incubation. Control sperm cAMP at 4 h increased only to 4.9 +/- 0.8 pmol cAMP/20 x 10(6) sperm. There were both similarities and differences in the characteristics of capacitation by heparin or oviduct fluid. Both glucose and protamine sulfate were found to suppress the heparin-dependent cAMP increase and inhibit capacitation. Capacitation by oviduct fluid was inhibited by either glucose or protamine sulfate. A small increase in sperm cAMP was associated with capacitation by oviduct fluid but was not affected by glucose or protamine sulfate.

8-Bromo Cyclic Adenosine Monophosphate↗

PubMed1994

Effect of free amino acids and vitamins on cleavage and developmental rate of bovine zygotes in vitro.

Due to the complicated media used for culturing bovine embryos, most of the nutrient requirements are unknown. Recently, we developed a simple, serum-free medium (CR1) that allows bovine embryos to develop in vitro. Therefore, our objective was to determine whether development of bovine embryos would be improved by the addition of free amino acids and vitamins to CR1. Oocytes were recovered from slaughterhouse ovaries and matured 22 +/- 2 h, following which the oocytes were randomly allotted to treatment. The experiment was a randomized block design with a 2 x 5 factorial treatment structure. The oocytes were fertilized with or without cumulus cells intact. The five fertilization media were 1) Control (CR1 +/- 10 micrograms/mL of phenol red); 2) control + basal medium Eagle (BME) essential amino acids (EAA) + minimum essential medium (MEM) nonessential amino acids (NEA) + MEM vitamins (VIT); 3) control + EAA + NEA; 4) control + EAA + VIT; and 5) control + NEA + VIT. Cleavage rate was greater (P < .001) when cumulus cells remained on the oocytes during fertilization (51.7 vs 73.2% without and with cumulus cells, respectively). The frequency of blastocysts was increased (P < .001) when EAA or NEA were added to CR1; however, adding VIT had no effect or tended (P = .12) to decrease the frequency of embryos attaining the blastocyst stage. This experiment demonstrates that development of bovine embryos in vitro can be improved by the addition of free amino acids to a simple medium. Contrary to work in rodents, the mixture of vitamins in MEM was not beneficial for bovine embryos.

Amino Acids↗

PubMed1993

Gene transfer in bovine blastocysts using replication-defective retroviral vectors packaged with Gibbon ape leukemia virus envelopes.

With this work we demonstrate that murine leukemia virus (MLV)-based replication-defective retroviral vectors encapsidated with Gibbon ape leukemia virus (GaLV) envelopes are significantly more infectious to bovine embryonic trachea (EBTr) cells than vectors encapsidated with murine xenotropic envelope proteins. In a test of internal promoter activity in an MLV retroviral vector, the rat beta-actin promoter was shown to be better than the herpes simplex virus type 1 thymidine kinase (TK) and human cytomegalovirus (CMV) immediate early promoters for the expression of an E. coli beta-galactosidase marker gene in bovine target cells. By co-culture of bovine blastocysts and virus-producing cells, or by culture of embryos in the medium harvested from virus-producing cells, we transferred the E. coli beta-galactosidase gene into trophoblasts and also into inner cell mass (ICM) cells of a bovine embryo through the infection of the MLV-based replication-defective retroviruses encapsidated with GaLV envelope proteins. The infection was confirmed by the expression of the E. coli beta-galactosidase gene under a beta-actin internal promoter. In addition, co-culture of ICM cells with virus-producing cells resulted in differentiation of ICM cells into embryoid bodies expressing the marker genes.

Actins↗