PubMed Health⌕ Search

Biomedical subjects

N L First

Publications and source records attributed to N L First.

At least 55 records · Page 3Linked to original sources
PubMed1993

Cell-to-cell coupling in early-stage bovine embryos: A preliminary report.

Gap junction communication has been implicated in providing positional information within an embryo. This positional information is then used to direct the differentiation of the early embryo. To begin to gain an assessment of the cell-to-cell communication observed in the early bovine embryo, fluorescein (5%) was microinjected into single blastomeres of freshly collected embryos. Dye communication was not observed in any of the 8-to 16-cell stage embryos. Very limited dye coupling was observed in compact morula (18%) and expanded blastocysts (25%). Interestingly, none of the expanded blastocysts resulting from in vitro maturation and in vitro fertilization showed any dye coupling. The degree of coupling observed in the bovine embryo was less than that observed in compact morula mouse embryos, where almost all (95%) embryos showed dye coupling. This experimental data is discussed in context with previous electron microscopy data.

Journal Article↗
PubMed1993

Control of germinal vesicle breakdown in bovine x murine hybrid oocytes.

Bovine oocytes cultured in control medium or in medium containing dibutyrylcyclic adenosine monophosphate (dbcAMP) or an inhibitor of cyclic nucleotide phosphodiesterase (3-isobutyl-1-methylxanthine, IBMX) undergo germinal vesicle breakdown (GVBD). On the other hand, mouse oocytes remain arrested at the germinal vesicle (GV) stage when dbcAMP or IBMX is present. When 1 bovine GV stage oocyte is fused to 1 GV stage mouse oocyte, dissolution of both species GV occurred in dbcAMP-supplemented medium. Only when 4 to 5 GV stage mouse oocytes are fused to 1 GV stage bovine oocyte, and these giant cells are cultured in dbcAMP-medium, is maturation arrested with only GVs present in the cytoplasm. The inhibitory effect is more evident in IBMX-supplemented medium. Here nearly 50% of the fused cells exhibit GVs, both mouse and bovine, when 1 cattle GV oocyte is fused to 1 mouse GV oocyte and the fused cells are cultured for 24 h. Moreover, nearly all GVs are well preserved after fusion of 1 bovine oocyte to 2 or more mouse oocytes. When these hybrid cells after 24 h culture in IBMX are then washed and cultured in control medium for a further 24 h, GVBD occurred in all cells. We are of the opinion that this novel approach (ie mixing of sensitive and non-sensitive cytoplasm) may in the future better explain the mechanisms involved in the regulation of mammalian oocyte maturation.

1-Methyl-3-isobutylxanthine↗
PubMed1993

Development of bovine embryos in vitro as affected by energy substrates.

Simple media were developed to study the metabolic requirements of bovine embryos up to Day 7 (Day 0 = day of oocyte aspiration) in vitro. Embryos were derived from oocytes matured and fertilized in vitro. At 45 +/- 2 h post insemination, embryos (> or = 2 cells) were randomly allotted to treatments. Examined in experiments 1 and 3 was the effect of pyruvate concentration in the presence of lactate. In the presence of lactate, pyruvate (0.2-5.0 mM) had no effect (p > 0.05) on the percentage of morulae or blastocysts. However, increasing the concentration of hemicalcium L-lactate from 5 mM to 10 mM decreased (p < 0.001) the percentage of embryos reaching the morula or blastocyst stage (experiment 3). Neither magnesium sulfate (0.5 mM) nor EDTA (10 mM) improved embryo development when added to the medium CR1 (experiment 2). Increasing the calcium level to 5 mM or the lactate level to 10 mM had no effect (p > 0.05) on embryo development (experiment 4). However, the interaction of adding calcium and lactate resulted in a decreased (p < 0.05) percentage of morulae. Determined in experiment 6 were the independent effects of pyruvate, lactate, and glucose on embryo development in vitro. As pyruvate or lactate level was increased from 1 to 10 mM, the percentage of blastocysts was decreased (p < 0.05). These experiments indicate that adding pyruvate to a medium containing lactate is not necessary for development of bovine embryos in vitro.

Animals↗
PubMed1992

In vitro development of bovine one-cell embryos: Influence of glucose, lactate, pyruvate, amino acids and vitamins.

To elucidate the effect of nutrient substrates on embryo development, in vitro fertilized bovine one-cell embryos were cultured in a medium similar to synthetic oviduct fluid (SOF) but without glucose and containing 3.3 mM lactate, 0.3 mM pyruvate and 3 mg/ml bovine serum albumin (BSA) at 39 degrees C in 5% CO(2) in air. Results indicated that addition of glucose was not only unnecessary, but it also had a deleterious effect on embryo development to the morula stage. Lactate supported embryo development up to the morula stage as well as pyruvate. Supplementation with 20 amino acids contained in basal medium Eagle's (BME) and minimum essential medium (MEM) improved development to the morula stage dramatically and increased the cell number compared with that of the controls. Addition of the vitamins from MEM to SOF had no beneficial effect. The SOF with amino acids did not increase the frequency of blastocysts 7 days after in-vitro fertilization but did increase the total number of cells compared with that of the controls. Frequency of blastocysts at Day 7 in SOF with amino acids was equivalent to that of co-culture although the total cell number was lower. These results demonstrate that a semi-chemically defined medium can successfully support the development of bovine embryos to the morula stage to a limited extent, but the medium lacks some nutrients or growth factors to fully support development through the blastocyst stage.

Journal Article↗
PubMed1992

Embryonic cytoplasmic extracts rescue murine androgenones to the blastocyst stage.

Androgenones (paternally derived genome) show a significant inability to form a blastocoele cavity. Eighty percent of these embryos die or arrest at earlier stages. Factor(s) from both normal and parthenogenetic late preimplantation embryos injected into each blastomere of androgenetic 4-cell stage can rescue more than twice as many to the blastocyst stage (47.2% versus 19.2% for non-injected androgenones). This factor(s) becomes available beginning at the 4-cell stage and is titratable. Injected total cytoplasmic mRNA will also cause a rescue response. Isolating this specific factor message(s) will permit the eventual cloning of possibly the earliest parentally imprinted gene(s) expressed during development.

Animals↗
PubMed1991

The expression of nuclear lamin A and C epitopes is regulated by the developmental stage of the cytoplasm in mouse oocytes or embryos.

The cytoplasmic regulation of changes of nuclear lamin antigens was examined by transferring 16-cell stage blastomeres into mouse oocytes. Sixteen-cell stage blastomeres were transferred to either pronuclear eggs, enucleated pronuclear eggs or metaphase II oocytes, which were subsequently activated. Pronuclei react with a monoclonal antibody to A/C lamins (J9), whereas nuclei from 16-cell stage blastomeres do not react with J9. However, after transfer of 16-cell stage nuclei to activated metaphase II oocytes, the transferred nuclei acquire the antigen. This is in contrast to 16-cell nuclei that were transferred to intact or enucleated pronuclear eggs; i.e., the nuclei only faintly acquired the A/C epitope. These results suggest that the developmental stage of the cytoplasm regulates the exposure of nuclear lamina epitopes, perhaps by limiting the supply of lamin A/C in the oocyte or because nuclear lamina assembly can only occur at the telophase transition. Furthermore, it appears that there is some exchange of the A/C epitope between (pro)nuclei within the same cell but that the majority of the A/C lamin epitope can be removed from a cell with (pro)nuclear removal.

Animals↗
PubMed1991

Response of porcine oocytes to electrical and chemical activation during maturation in vitro.

These studies were conducted to examine activation of in vitro-matured porcine oocytes in response to an electrical stimulus or to an ionophore. Cumulus-enclosed porcine oocytes were incubated in maturation medium supplemented with either FSH and LH (MM:Exp.1) or pregnant mare serum gonadotropin (PMSG; MM-P: experiments 2-4) at 39 degrees C in 5% CO2:95% air with high humidity. In experiment 1, groups of oocytes were stripped of cumulus and then shampulsed (control) or electrically pulsed with a Zimmerman Cell Fusion unit at 24, 31, 41, 48, and 65 h of incubation. Control oocytes were exposed to the activation medium for 20 sec, whereas oocytes to be pulsed were subjected to a single activation pulse (120 V, 30 microseconds). Oocytes were cultured for an additional 24 h and then fixed and examined. For oocytes pulsed at 24, 31, 41, 48, and 65 h, the proportions which activated were 0, 0, 87, 88, and 83%, respectively. In experiment 2, oocytes were electrically or sham-pulsed with a BTX 200 Embryomanipulation System at 24, 30, and 40 h of incubation and respective proportions of oocytes activating were 27%, 39%, and 72%. In experiment 3, oocytes were subjected to 0, 1, or 2 activation pulses after 41 h of incubation in MM-P. Double-pulsing halved the proportion of activated oocytes (P less than .0001). In experiment 4, oocytes were subjected to 0, 25, 50, or 100 microM ionophore at 48 h of incubation. Proportions of oocytes activated by ionophore were greater than for control (P less than .05), but activation was not increased by increasing dose of ionophore.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

Embryonic transcription in in vitro cultured bovine embryos.

The rate of cleavage and the onset of embryonic transcription of bovine embryos cultured in vitro (IVC) has been investigated. Embryos were derived from in vitro matured, in vitro fertilized oocytes (IVM/IVF) to improve developmental synchrony. The rate of cleavage was assessed by morphological evaluation between the one- and eight- to 16-cell stage. The rate of cleavage was found to be equivalent to that reported for in vivo recovered embryos. To assess the onset of embryonic transcription, embryos were cultured to the eight- to 16-cell stage in the presence of alpha-amanitin for various periods of time followed by two-dimensional polyacrylamide gel electrophoresis. Embryos readily cleaved to the eight- to 16-cell stage in the presence of inhibitor. alpha-Amanitin-sensitive protein synthesis was first detected at 36-48 h post-insemination (hpi) and continued up to 84 hpi. We conclude that bovine embryos produced by IVM/IVF/IVC are competent to initiate embryonic transcription at 36-48 h post-insemination and suggest that in vitro-induced cleavage arrest is not due to failure of the embryonic genome to initiate transcription.

Amanitins↗
PubMed1991

Effect of 6-dimethylaminopurine on germinal vesicle breakdown of bovine oocytes.

The effect of 6-dimethylaminopurine (6-DMAP) on germinal vesicle breakdown (GVBD) and maturation in bovine oocytes was investigated in this study. This puromycin analog has been shown to be an inhibitor of phosphorylation. Whereas GVBD occurred in nearly all oocytes (96.8%, 120/124) in control medium, presence of 6-DMAP (2 mM) blocked this process almost completely, irrespective of the presence (98.3% GV, 349/355) or absence (97.1% GV, 165/170) of cumulus cells. When lower concentrations of 6-DMAP were used (100-500 microM), GVBD was observed in 87.9% of oocytes, but their maturation was arrested at late diakinesis-metaphase I stage. The inhibition of GVBD was fully reversible, but most of the metaphase II plates were abnormal (80%). To assess whether the action of 6-DMAP is different from the inhibitors of protein synthesis, metaphase II oocytes were exposed to either cycloheximide or 6-DMAP, respectively. Whereas in cycloheximide-supplemented medium approximately 80% of the oocytes were activated, parthenogenetic activation was much less frequent after incubation in 6-DMAP (14.5%). Fusion studies showed that, even if GVBD occurs in 6-DMAP supplemented medium, the level of the maturation-promoting factor (MPF) is decreased. These experiments may indicate the importance of phosphorylation for GVBD in cattle oocytes.

Adenine↗
PubMed1991

Characterization of developmental arrest in early bovine embryos cultured in vitro.

The susceptibility of early bovine embryos to developmental arrest ("blocking") in vitro was examined. Embryos, obtained from superovulated donors, were cultured in vitro in Ham's F10 culture medium or in vivo in sheep oviducts. Treatments were terminated on Day 7 post-donor estrus (estrus = day 0), and the embryos were evaluated for development. Experiment 1 tested whether the 8- to 16-cell block was reversible. One- to two-cell embryos were cultured in vitro to the 8-cell stage (2 d), then in vivo for 3 d; controls were cultured in vitro or in vivo for 5 d. Forty-two percent (19/45) of in vivo controls developed normally; none (0/55; 0%) of the in vitro controls cleaved past the 9- to 16-cell stage. Only 4% (2/48) of the embryos cultured to eight cells in vitro developed normally after culture in sheep oviducts, indicating that the block was irreversible. Irreversibility was not caused by overt cell death, since 33/33 (100%) of blocked embryos responded positively to fluorescein diacetate vital staining. Experiment 2 tested the effect of in vitro exposure at specific cell stages on subsequent in vivo development. Embryos at the 1- to 2-, 3- to 4-, 5- to 8- and 9- to 16-cell stages were assigned randomly to one of the following treatments: in vivo culture; in vitro culture; or 24 h in vitro culture, followed by in vivo culture. Subsequent in vivo development was affected by 24 h of in vitro culture (P<0.05) only in 3- to 4-cell embryos (11/41, 27% vs 22/41, 54% for in vivo controls). We conclude that 1) the block is a manifestation of in vitro exposure during the four- to eight-cell stage, and 2) the block, while irreversible, is not the result of overt embryonic death.

Journal Article↗
PubMed1991

In vitro fertilization and development of bovine oocytes matured in serum-free medium.

This study was undertaken to investigate the effects of supplementation of serum (fetal calf serum), gonadotropins (LH, FSH, prolactin) and estradiol-17 beta (E2) to culture medium during in vitro maturation of bovine cumulus oocyte complexes on subsequent fertilization and development to the blastocyst stage in vitro. Serum supplementation during bovine oocyte maturation was not required but hormonal supplementation, gonadotropins (LH + FSH) and E2, enhanced the fertilizability and developmental ability of bovine oocytes matured in vitro. The addition of prolactin to maturation medium containing LH, FSH, and E2 did not further enhance frequencies of fertilization and development.

Animals↗
PubMed1991

Genomic potential in mammals.

Embryos of amphibians, fish, sheep, cattle, swine and rabbits have been multiplied by nuclear transfer. Successful nuclear transfer in these species has been accomplished by transfer of a blastomere from a late stage embryo into an enucleated oocyte or egg with large scale multiplication achieved by serial repetition of the procedure using blastomeres from nuclear transfer embryos. This allows the production of clonal lines, which when appropriately selected for performance in a given trait, can be reproduced to capture in the offspring expression of both additive and nonadditive inheritance. The efficiency of producing offspring from nuclear transfer is low in mammals in both frequency of morula or blastocyst produced and maintenance of pregnancy after embryo transfer. In domestic animals the largest number of offspring from one embryo has been eight calves. Embryos as late as the 64-cell stage in cattle and 120-cell blastocyst in sheep have been used successfully as donors of blastomeres. Recloning has also been done in cattle. Potentially, nuclear transfer provides a mechanism for multiplication and production testing of clonal lines, a method for rapid genetic improvement and a means for rapid propagation of a selected genotype.

Animals↗
PubMed1991

Some factors affecting the efficacy of oviduct tissue-conditioned medium for the culture of early bovine embryos.

Oviduct tissue-conditioned medium was evaluated for the culture of IVM-IVF bovine zygotes to the compact morula (cM) and blastocyst (BL) stages. Development was unaffected (P greater than 0.50) by freezing and thawing of conditioned medium: no. cM + BL/no. cleaved ova obtained after culture in nonfrozen, frozen-thawed, and control treatments were 31/148 (21%), 26/124 (21%) and 5/86 (6%), respectively. The greatest proportion of normal development was obtained after a conditioning period of 48 h (P less than 0.05): no. of cM + BL/no. cleaved ova in media conditioned for 5, 24, 48 and 96 h were 23/114 (20%), 38/112 (34%), 43/115 (37%) and 36/125 (29%), respectively. Development declined with increasing dilutions of conditioned media (P less than 0.005): no. cM + BL/no. cleaved ova for 100, 75, 50, 25 and 0% conditioned medium were 32/94 (34%), 29/94 (31%), 17/82 (21%), 10/94 (11%) and 11/73 (15%), respectively. The oestrous cycle stage from which oviducal tissue was obtained did not affect development (P greater than 0.75); no. cM + BL/no. cleaved ova was 21/63 (33%) at oestrus and 21/79 (27%) in the luteal phase.

Animals↗
PubMed1991

Development of one-cell porcine embryos to the blastocyst stage in simple media.

Porcine embryos were flushed from mated donors and examined for cleavage stage. One- and two-cell embryos were randomly allotted to one of the five following in vitro treatments: M199 with Earle's salts, a modified Tyrode's medium (TL), TL supplemented with 10 mM N-2-hydroxyethyl-piperazine-N'-2-ethanesulfonic acid (HEPES) (TLH), TLH supplemented with 5.5 mM glucose (TLHG), or TLH supplemented with 5 mM glutamine (TLHGL). The bicarbonate concentration of TLH, TLHG, and TLHGL was 2 mM, compared with the 25 mM concentration in M199 and TL. Embryos in M199 and TL were incubated in 95% air:5% CO2 at 39 degrees C. Those in the remaining three treatments were incubated in air at 39 degrees C. Embryos incubated in TL and M199 did not develop past the four- to eight-cell stage, whereas the proportions of embryos developing to the compact morula or blastocyst stage by d 7 of culture in the other treatments were as follows: TLHG, 49.1%; TLHGL, 59.4%; TLH, 63.5% (P less than .005). These results indicate that porcine embryos can be cultured from the one-cell stage to blastocyst in a simple HEPES-buffered medium in air. The ability of porcine embryos to develop without supplemental CO2 may be an important finding for use in situations in which embryos must be transported for long periods before embryo transfer.

Animals↗
PubMed1991

Production of embryos by oocyte cytoplast-blastomere fusion in domestic animals.

Embryos of amphibians, sheep, cattle, pigs and rabbits have been multiplied by nuclear transfer. Successful nuclear transfer in these species has been accomplished by transfer of a blastomere from a late-stage embryo into an enucleated oocyte with large scale multiplication by repeating the procedure using blastomeres from the embryos produced from nuclear transfer. This allows the production of clonal lines which, when appropriately selected for performance in a given trait, can be reproduced to capture in the offspring expression of additive and non-additive inheritance. The efficiency of these procedures is high only for amphibian embryos for which as many as 1000 offspring can be made from a blastula- or gastrula-stage embryo and the process repeated 60-100 times with descendant embryos. In domestic animals the largest number of offspring from one embryo has been 8 calves. Embryos as late as the 64-cell stage in cattle and 120-cell blastocyst in sheep have been used successfully as donors of blastomeres. Recloning has also been done in cattle. Nuclear transfer potentially provides a mechanism for multiplication and production testing of clonal lines, a method for rapid genetic improvement and rapid propagation of a selected genotype. Unfortunately the present efficiencies of subsequent embryo development, pregnancy and embryo survival are less than normal. This paper reviews variables contributing to reduced efficiency and research to improve nuclear transfer.

Animals↗
PubMed1990

Nuclear transplantation in the pig embryo: nuclear swelling.

The transfer of nuclei from cleavage stage embryos to enucleated activated meiotic metaphase II oocytes results in a reprogramming of the transferred nucleus such that it behaves as a zygotic nucleus. One estimator of nuclear reprogramming is nuclear swelling after nuclear transfer. The diameter of nuclei after nuclear transfer was not found to be dependent upon the amount of cytoplasm transferred with the donor cell or the amount of cytoplasm in the recipient cell. Nuclei from 4-, 8-, and 16-cell stage embryos swelled to a similar diameter after nuclear transfer (26.9, 27.3, and 27.2 microns, respectively) and this was significantly different from the diameter of contemporary donor embryos (18.3, 14.3, and 13.0 microns, respectively). This is a swelling of 47, 91, and 109%, respectively. Since the degree of nuclear swelling does not appear to be related to cytoplasmic volume it is concluded that the components mediating nuclear swelling are not in a limiting supply.

Animals↗
PubMed1990

Cloning of embryos.

Nuclear transfer for the study of differentiation in amphibians has been used since the 1950s, but not until recently have the same procedures been applied successfully to some mammals. Nuclear transfer, as developed for the amphibian, is successful in sheep, cattle, rabbit, and pig, but not mouse embryos. This fact is discussed in relation to the species-specific timing of the activation of the zygotic genome. Nuclear transfer to an oocyte presumably results in a genomic reprogramming of the transferred nucleus. The limits of differentiation that can be reprogrammed have yet to be determined. Since the cells of early embryos are thought to have identical nuclear genomes, early embryos can be used as a source of donor nuclei; and, when combined with serial nuclear transfer, can theoretically produce an unlimited number of identical offspring. Cloning by splitting does not result in a reprogramming of the genome and is limited in the number of identical offspring that can result. Here we discuss some of the factors to consider concerning micromanipulation and nuclear reprogramming and how they relate to other embryo technologies.

Animals↗