Initial Period Of Culture Research Articles

134 Effects of Gas Tension During Culture upon Development of Bovine Embryos from Beef (Nellore) and Dairy (Girolando) Breeds

Culture of bovine embryos is a critical step during in vitro embryo production (IVEP) and, as such, has been the focus of numerous studies on cattle IVEP. Improvements of culture conditions to mimic the in vivo maternal microenvironment involves studying the optimal gas tension for pre-implantation embryonic development. In the commercial conditions, there is great variability in results, in part because of the difference between breeds and donors. The objective of this study was to evaluate the effects of culture in high or low oxygen tension upon the development of embryos from a crossbred dairy breed (Girolando F1; Gir × Holstein) and a beef Bos indicus breed, Nellore. We collected data from an IVEP commercial operation located in a tropical area of southeastern Brazil (Minas Gerais State) from February to May 2017. The study was designed in a 2 × 2 factorial arrangement of treatments: 2 O2 tensions during culture (5%, low O2 v. 20%, high O2) and 2 breeds (Nellore, beef v. Girolando F1, dairy). Thus, the following 4 groups were studied: Nellore-high O2 (n = 86 donors), Nellore-low O2 (n = 107 donors), Girolando F1-high O2 (n = 114 donors), and Girolando F1-low O2 (n = 110 donors). Outcome variables were the number of cleaved embryos 72 h post-insemination (hpi), cleavage rate relative to the total number cumulus–oocyte complexes (COC) put in culture, number and percentage of blastocysts 192 hpi relative to the structures kept in culture. Variables that were not normally distributed were transformed using the formula log(y + 0.05). Data were analysed using the GLM procedure of SAS (SAS Institute Inc., Cary, NC, USA) for the main effects of gas tension (low v. high O2) and breed (Girolando F1 v. Nelore). Results are shown as mean ± SEM. Gas tension affected the number of cleaved embryos (10.52 ± 0.92 v. 8.33 ± 0.72 for high and low O2, respectively; P < 0.01) and cleavage rates (40.58 ± 2.49 v. 44.41 ± 2.88 for high and low O2; P < 0.01 in Nellore), but did not affect these variables in Girolando F1 donors (13.23 ± 1.33 v. 10.76 ± 0.76 cleaved embryos, for high and low O2; P = 0.63; 58.01 ± 2.00 v. 60.19 ± 1.97 cleavage rate, for high and low O2; P = 0.80). Nonetheless, the number and percentage of blastocysts were not affected by gas tension in either breed. Results for Nellore were 4.99 ± 0.56 v. 3.51 ± 0.38 blastocysts in high and low O2, respectively (P = 0.051) and 41.92 ± 3.91% v. 39.81 ± 3.77% blastocysts, in high and low O2 (P = 0.11). For Girolando F1, numbers of blastocysts were 5.84 ± 0.66 v. 4.24 ± 0.39 in high and low O2 (P = 0.19) and percentage of blastocysts 49.14 ± 2.97% v. 49.11 ± 3.40% in high and low O2 (P = 0.46). These results suggest that oxygen tension during culture affects IVEP differently depending on breed. The initial period of culture, recognised as critical in IVEP, seemed more sensitive to high O2 tension, particularly in Nellore.

An insufficient increase of cytosolic free calcium level results postovulatory aging-induced abortive spontaneous egg activation in rat

The present study was aimed to find out whether postovulatory aging-induced abortive spontaneous egg activation (SEA) is due to insufficient increase of cytosolic free Ca(2+) level. Immature female rats (22-24 days old) were subjected to superovulation induction protocol. Eggs were collected 14, 17 and 19 h post-hCG surge to induce in vivo egg aging. The eggs were collected 14 h post-hCG surge and cultured in vitro for 3, 5 and 7 h to induce in vitro egg aging. The morphological changes, rate of abortive SEA, chromosomal status and cytosolic free Ca(2+) levels were analyzed. Postovulatory aging induced morphological features characteristics of abortive SEA in a time-dependent manner in vivo as well as in vitro. The extracellular Ca(2+) increased rate of abortive SEA during initial period of culture, while co-addition of a nifedipine (L-type Ca(2+) channel blocker) protected against postovulatory aging-induced abortive SEA. However, CI induced morphological features characteristics of egg activation (EA) in a dose-dependent manner. As compare to control, an increase of cytosolic free Ca(2+) level (1.42 times) induced abortive SEA, while further increase of cytosolic free Ca(2+) level (2.55 times) induced EA. Our results show that an insufficient cytosolic free Ca(2+) level is associated with postovulatory aging -induced abortive SEA, while furthermore increase is required to induce EA in rat.

Rapid isolation and susceptibility testing of Leptospira spp. using a new solid medium, LVW agar.

Pathogenic Leptospira spp., the causative agents of leptospirosis, are slow-growing Gram-negative spirochetes. Isolation of Leptospira from clinical samples and testing of antimicrobial susceptibility are difficult and time-consuming. Here, we describe the development of a new solid medium that facilitates more-rapid growth of Leptospira spp. and the use of this medium to evaluate the Etest's performance in determining antimicrobial MICs to drugs in common use for leptospirosis. The medium was developed by evaluating the effects of numerous factors on the growth rate of Leptospira interrogans strain NR-20157. These included the type of base agar, the concentration of rabbit serum (RS), and the concentration and duration of CO(2) incubation during the initial period of culture. The highest growth rate of NR-20157 was achieved using a Noble agar base supplemented with 10% RS (named LVW agar), with an initial incubation at 30°C in 5% CO(2) for 2 days prior to continuous culture in air at 30°C. These conditions were used to develop the Etest for three species, L. interrogans (NR-20161), L. kirschnerii (NR-20327), and L. borgpetersenii (NR-20151). The MICs were read on day 7 for all samples. The Etest was then performed on 109 isolates of pathogenic Leptospira spp. The MIC(90) values for penicillin G, doxycycline, cefotaxime, ceftriaxone, and chloramphenicol were 0.64 units/ml and 0.19, 0.047, 0.5, and 2 μg/ml, respectively. The use of LVW agar, which enables rapid growth, isolation of single colonies, and simple antimicrobial susceptibility testing for Leptospira spp., provides an opportunity for new areas of fundamental and applied research.

Hypercapnia modulates synaptic interaction of cultured brainstem neurons

CO(2) is an important metabolic product whose concentrations are constantly monitored by CO(2) chemoreceptors. However, the high systemic CO(2) sensitivity may not be achieved by the CO(2) chemoreceptors without neuronal network processes. To show modulation of network properties during hypercapnia, we studied brainstem neurons dissociated from embryonic rats (P17-19) in multielectrode arrays (MEA) after initial period (3 weeks) of culture. Spike trains of 33,622 pairs of units were analyzed using peri-event histograms (PEH). The amplitude of peri-central peaks between two CO(2)-stimulated units increased and the peak latency decreased during hypercapnia. Similar enhancement of synaptic strength was observed in those sharing a common input. These phenomena were not seen in CO(2)-unresponsive neurons. The amplitude of peri-central peaks between two CO(2) inhibited units also increased without changing latency. Over 60% CO(2)-stimulated neurons studied received mono-/oligosynaptic inputs from other CO(2)-stimulated cells, whereas only approximately 10% CO(2)-unresponsive neurons had such synaptic inputs. A small number of brainstem neurons showed electrical couplings. The coupling efficiency of CO(2)-stimulated but not CO(2)-unresponsive units was suppressed by approximately 50% with high PCO(2). Inhibitory synaptic projections were also found, which was barely affected by hypercapnia. Consistent with the strengthening of excitatory synaptic connections, CO(2) sensitivity of post-synaptic neurons was significantly higher than presynaptic neurons. The difference was eliminated with blockade of presynaptic input. Based on these indirect assessments of synaptic interaction, our PEH analysis suggests that hypercapnia appears to modulate excitatory synaptic transmissions, especially those between CO(2)-stimulated neurons.

High CO2 chemosensitivity versus wide sensing spectrum: a paradoxical problem and its solutions in cultured brainstem neurons.

CO2 central chemoreceptors play an important role in cardiorespiratory control. They are highly sensitive to P(CO2) in a broad range. These two sensing properties seem paradoxical as none of the known pH-sensing molecules can achieve both. Here we show that cultured neuronal networks are likely to solve the sensitivity versus spectrum problem with parallel and serial processes. Studies were performed on dissociated brainstem neurons cultured on microelectrode arrays. Recordings started after a 3 week initial period of culture. A group of neurons were dose-dependently stimulated by elevated CO2 with a linear response ranging from 20 to 70 Torr. The firing rate of some neurons increased by up to 30% in response to a 1 Torr P(CO2) change, indicating that cultured brainstem neuronal networks retain high CO2 sensitivity in a broad range. Inhibition of Kir channels selectively suppressed neuronal responses to hypocapnia and mild hypercapnia. Blockade of TASK channels affected neuronal response to more severe hypercapnia. These were consistent with the pKa values measured for these K+ channels in a heterologous expression system. The CO2 chemosensitivity was reduced but not eliminated by blockade of presynaptic input from serotonin, substance P or glutamate neurons, indicating that both pre and postsynaptic neurons contribute to the CO2 chemosensitivity. These results therefore strongly suggest that the physiological P(CO2) range appears to be covered by multiple sensing molecules, and that the high sensitivity may be achieved by cellular mechanisms via synaptic amplification in cultured brainstem neurons.

Effects of Protease Inhibitors and Antioxidants on In Vitro Survival of Porcine Primordial Germ Cells1

One of the problems associated with in vitro culture of primordial germ cells (PGCs) is the large loss of cells during the initial period of culture. This study characterized the initial loss and determined the effectiveness of two classes of apoptosis inhibitors, protease inhibitors, and antioxidants on the ability of porcine PGCs to survive in culture. Results from electron microscopic analysis and in situ DNA fragmentation assay indicated that porcine PGCs rapidly undergo apoptosis when placed in culture. Additionally, alpha(2)-macroglobulin, a protease inhibitor and cytokine carrier, and N:-acetylcysteine, an antioxidant, increased the survival of PGCs in vitro. While other protease inhibitors tested did not affect survival of PGCs, all antioxidants tested improved survival of PGCs (P: < 0.05). Further results indicated that the beneficial effect of the antioxidants was critical only during the initial period of culture. Finally, it was determined that in short-term culture, in the absence of feeder layers, antioxidants could partially replace the effect(s) of growth factors and reduce apoptosis. Collectively, these results indicate that the addition of alpha(2)-macroglobulin and antioxidants can increase the number of PGCs in vitro by suppressing apoptosis.

Interaction within clusters of dendritic cells and helper T cells during initial Th1 / Th2 commitment

Cytokines are the main agents known to regulate Th1 / Th2 commitment, where they may operate through paracrine activity within clusters of T cells gathered around dendritic cells (DC). An in vitro system is used here to test this possibility, using clusters around DC composed of naive TCR-transgenic ovalbumin peptide 323 – 339-specific CD4+ T cells as targets plus TCR-transgenic pigeon cytochrome C peptide 88 – 104-specific CD4+ polarized Th1 or Th2 cells as inducers. The polarized inducer cells exerted their maximum effect when the two T cell populations were activated within the same cluster, implemented by allowing a single DC to present both their epitopes. This finding thus supports the paracrine hypothesis. The system was then employed to explore the role of individual cytokines by means of inhibition by monoclonal antibodies. Development of Th2 commitment proved strictly dependent on the IL-4 produced by the Th2 inducers. For Th1 commitment, IFN-γ and IL-12 were both needed, but with IFN-γ required only during the initial period of culture. The rapid timing observed under these conditions places constraints on the molecular basis of commitment, and appears accurately to reflect the physiological response in vivo.

Interaction within clusters of dendritic cells and helper T cells during initial Th1/Th2 commitment.

Cytokines are the main agents known to regulate Th1 / Th2 commitment, where they may operate through paracrine activity within clusters of T cells gathered around dendritic cells (DC). An in vitro system is used here to test this possibility, using clusters around DC composed of naive TCR-transgenic ovalbumin peptide 323 - 339-specific CD4(+) T cells as targets plus TCR-transgenic pigeon cytochrome C peptide 88 - 104-specific CD4(+) polarized Th1 or Th2 cells as inducers. The polarized inducer cells exerted their maximum effect when the two T cell populations were activated within the same cluster, implemented by allowing a single DC to present both their epitopes. This finding thus supports the paracrine hypothesis. The system was then employed to explore the role of individual cytokines by means of inhibition by monoclonal antibodies. Development of Th2 commitment proved strictly dependent on the IL-4 produced by the Th2 inducers. For Th1 commitment, IFN-gamma and IL-12 were both needed, but with IFN-gamma required only during the initial period of culture. The rapid timing observed under these conditions places constraints on the molecular basis of commitment, and appears accurately to reflect the physiological response in vivo.

Procedures for the preparation and culture of 'reconstructed' rainbow trout branchial epithelia.

Techniques for the in vitro 'reconstruction' of freshwater rainbow trout branchial epithelia using the primary culture of gill cells on permeable polyethylene terephthalate cell culture filter supports are described. Representing models of the freshwater fish gill, epithelia grown by two separate techniques are composed of branchial pavement cells with or without the inclusion of mitochondria-rich (MR) cells. The generation of epithelia consisting of pavement cells only (via a method called single seeded inserts = SSI) involves an initial period of flask culture during which time MR cells, that appear unable to attach to the culture flask base, are excluded from the general cell populace. Alternately, the generation of a heterogeneous epithelia consisting of both pavement cells and MR cells (via a method called double seeded inserts = DSI) is facilitated by the direct seeding of cells into cell culture filter inserts. Critical to this second procedure is the repeat seeding of filter inserts over a two day period. Repeat seeding appears to allow MR cells to nest amongst the attached cell layer generated by the first day's seeding. The use of cell culture filter supports allows free access to both the apical and basolateral compartment of the epithelium and is ideal for experimental manipulation. Cells are grown under symmetrical conditions (apical media/basolateral media) and epithelium growth is measured as a function of transepithelial resistance (TER). When the epithelia exhibit a plateau in growth they can be subjected to asymmetrical conditions (freshwater apical/media basolateral) in order to assess gill cell function as in vivo.

The origin and efficient derivation of embryonic stem cells in the mouse.

By explanting tissues isolated microsurgically from implanting strain 129 mouse blastocysts individually on STO feeder cells we have established that embryonic stem (ES) cells originate from the epiblast (primitive ectoderm). Isolated early epiblasts yielded ES cell lines at a substantially higher frequency than intact blastocysts regardless of whether they were explanted whole or as strictly single-cell suspensions. When explanted from delayed-implanting 129 blastocysts, epiblasts gave lines consistently in 100% of cases. If primary embryonic fibroblasts rather than STO cells were used as feeders, germline-competent ES cell lines were obtained readily from epiblasts of delayed-implanting blastocysts of several hitherto refractory strains, particularly when recombinant leukemia inhibitory factor was included in the medium during the initial period of culture. Because lines were obtained from the nonpermissive CBA/Ca strain at a rate of up to 56%, this approach to the derivation of germline-competent ES cell lines may not only prove generic for the mouse but also worth pursuing in other species of mammal.

Identification of genes up-regulated in dedifferentiating Nicotania glauca pith tissue, using an improved method for constructing a subtractive cDNA library.

Pith explants of Nicotiana glauca grown in vitro in synthetic medium supplemented with 2,4 dichlorophenoxyacetic acid (2, 4 D), are induced to dedifferentiate. Treatment with actinomycin D within the first 4-8 h of culture (but not later) is lethal and the explants die, implying a requirement for de novo transcription. The genes expressed during the initial period of culture are presumably critical for subsequent cell survival and proliferation, but so far their identity is unknown. We have constructed a subtractive cDNA library, enriched in sequences more abundant in dedifferentiating tissue than in pith. The subtractive library contains approximately seven major species, two of which, NGSUB7 and NGSUB8, are highly abundant. In Northern blots, these two hybridized to mRNA species whose abundance increased significantly but transiently during the first 4 to 8 h of culture. The sequence of NGSUB7 showed no significant homology at a nucleotide or derived amino acid level with any previously reported sequence. NGSUB8 however, showed significant homology over part of the derived amino acid sequence to several yeast and bacterial proteins with DNA binding function. We propose that the two recombinants represent transcripts from two novel genes edeA and edeB, which are expressed early in dedifferentiation.

Interleukin-1β stimulates decidual stromal cell cyclo-oxygenase enzyme and prostaglandin production

The cytokine interleukin-1 (IL-1β) increased prostaglandin production by decidual stromal cells in culture in a time and dose dependent manner. Optimum conditions for stimulation were found to be for 24 hours at a concentration of 100 pg IL-1β/ml. An apparent increase in cyclo-oxygenase enzyme synthesis accompanied the increase in prostaglandin production, and both changes were inhibited by the protein synthesis inhibitor cycloheximide. This implicates protein synthesis in the stimulatory effects of IL-1β, which may be mediated through the increase in cyclo-oxygenase enzyme. A pre-incubation period of 72 hours was found to be necessary to observe the stimulatory effect of IL-1β on prostaglandin production, but this did not seem to be due to any change in the sensitivity of the cells to IL-1β; the increase in the number of cyclo-oxygenase positive cells was the same if IL-1β was added on day 1, day 2 or day 3 of culture, even though prostaglandin production was not stimulated on day 1 or day 2. Cycloheximide increased prostaglandin production on the first two days of culture and had no effect on the third day of culture. This was interpreted as indicating that a factor inhibiting cyclo-oxygenase activity was synthesised during the initial period of culture, which prevented any increase in prostaglandin production following the increase in enzyme synthesis.

Cytophotometric analysis of glycogen, protein and DNA of a glycogen-storing rat hepatoma (N13) cell line

This study examines the behavior of glycogen-storing rat hepatoma (N13) in vitro using cytophotometric techniques. A significant increase in glycogen is observed in these cells after 30 min incubation in a buffered solution containing 0.1 mM glucose, that is 80 times lower than the physiological glucose concentration in rat blood. N13 hepatoma cells grow exponentially in culture using RPMI 1640 tissue culture medium supplemented with 10% fetal bovine serum. During the first day in culture these cells store a large amount of glycogen and this increase is also observed in serum-free cultures. In more prolonged cultures the amount of glycogen per cell gradually becomes lower, although the culturing conditions are maintained. Similar variations of protein are also observed during the initial period of culture. DNA distribution does not show significant changes, although in serum-free cultures an increase in the proportion of cells in S and G2/M phases is observed. The addition of glucagon, epinephrine and cyclic AMP derivatives to serum-free cultures does not impede the storage of glycogen. Nevertheless, addition of either 2 mM N6,O2'-dibutyryl cyclic AMP or 0.1 mM 8-(4-chlorophenylthio)-cyclic AMP blocks the cell cycle at G0/G1 and glycogen content does not decrease after the first day in culture. We believe that this cell line offers an appropriated model to study glycogen metabolism and its involvement in the neoplastic process.

Culture of differentiated and undifferentiated type II cells from fetal rat lung

We have developed a relatively simple and reproducible method for the isolation and culture of both differentiated and undifferentiated type II cells from fetal rat lung. The technique involves an initial period of explant culture in serum and hormone free medium, followed by enzymatic dissociation of the explants, differential adhesion to remove fibroblasts, incubation of the cell pellet to promote aggregation of the type II cells and monolayer culture of the type II cells. The type II cells form clusters which are surrounded by scattered fibroblasts. When the technique was performed with three differential adhesion steps, cultures contained 86.0 ± 1.4% type II cells. To obtain a higher degree of purity and greater yield, two differential adhesions followed by gentle trypsinization of the cultures which selectively removes the isolated fibroblasts was performed. This resulted in cultures with 89.4 ± 1.7% type II cells. The differentiated fetal type II cell cultures were prepared from 19-day fetal rat lungs which were initially maintained in explant culture for 48 h. These differentiated cells demonstrated the characteristic morphologic features of type II cells including lamellar bodies and microvilli. Undifferentiated fetal cells were prepared in a similar manner from 18-day fetal rat lung maintained in explant culture for 24 h. These cells did not contain intracellular osmiophilic granules; the appearance of these granules could, however, be induced by hormones. For this reason they are considered to be pre-type II cells. The viability of the cultured cells was 97%. Both the differentiated and undifferentiated fetal type II cells specifically bound the Maclura pomifera lectin, a type II cell surface marker. The phospholipid profile of the fetal cells was similar to that of adult rat type II cells; the differentiated fetal cells, however, synthesized less phosphatidylcholine than the adult cells did, but more than the undifferentiated fetal cells. The differentiated fetal cells secreted phosphatidylcholine at a basal rate of 0.6% ± 0.1% during a 90-min incubation. There was dose-dependent stimulation of phosphatidylcholine secretion after exposure to terbutaline. Maximum stimulation (76%) was observed at a concentration of 10 μM. This culture system provides a valuable model for studies of the maturation of the undifferentiated fetal type II cell and surfactant metabolism and secretion in the differentiated fetal type II cell.

THE EFFECTS OF TERATOGENIC ANTISERA ON PROTEIN SYNTHESIS AND DEGRADATION BY THE CULTURED RAT CONCEPTUS

Rat embryo culture was initiated on the 10th day of gestation to study embryonic site protein synthesis in normal and pathologic states. Three culture media were employedsrat serum, serum plus teratogenic sheep anti-rat visceral yolk sac (VYS) serum, or rat serum plus normal sheep serum. Two studies were performed: a) 3H-leucine was added to produce incubation periods of 20 min. to 24 hr.; and after an initial period of culture in the presence of 3H-leucine to obtain a constant precursor specific radioactivity, culture was continued in the absence of 3H-leucine for a total of 1-30 hr. Embryos, VYS and culture media were analysed for total radioactivity and 3H-leucine specific radioactivity in the protein-bound and free amino acid fractions. The results suggest that: a) anti-VYS serum inhibited protein synthesis by the embryo and VYS but had no detectable effect on protein degradation; b) anti-VYS serum increased intracellular labeled leucine concentration. The teratogenic mechanism of anti-VYS serum appears to involve both the suppression yolk sac amino acid nutritive support of the embryo and the inhibition of the utilization of amino acids by the embryo for protein synthesis. These techniques provide us with the ability to determine the amino acid requirements which will support normal organogenesis and to quantitate the amino acid deprivation which will result in embryopathology. (Supported by NIH)

Prostaglandin synthesis during the course of limb cartilage differentiation in vitro

In the present study we have used radiometric thin layer chromatography (TLC) and radioimmunoassay (RIA) to examine the synthesis of various prostaglandins (PGs) during the progressive chondrogenic differentiation limb mesenchymal cells undergo in micromass culture. Throughout the 3-day culture period, [3H]arachidonic acid (AA) is metabolized to compounds which comigrate with authentic PGE2, PGF2 alpha, 6-keto-PGF1 alpha, TxB2, and PGD2. In micromass cultures prepared from the cells of whole stage-23/24 wing buds, all 3H-AA metabolites are produced in relatively small amounts during the initial period of culture, i.e. prior to the formation of extensive prechondrogenic cellular aggregates. Concomitant with maximum aggregate formation and the initiation of cartilage differentiation, there is a striking and progressive increase in the production of all the major classes of PGs from 3H-AA. PG production from 3H-AA is also at a maximum during the onset of chondrogenesis in micromass cultures prepared from the distal subridge mesenchymal cells of stage-25 wing buds in which more rapid, extensive, and homogeneous cartilage differentiation occurs. To complement these TLC studies, RIA has been used to examine the amount of various PGs synthesized from endogenous substrates by micromass culture homogenates at various times during in vitro chondrogenesis. These RIA studies also indicate that PG production is highest during periods of culture which coincide with the onset of overt chondrogenesis in both stage-23/24 whole limb and stage-25 subridge mesoderm micromass cultures. RIA indicates that PGE2 is the predominant PG produced from endogenous substrates during 1h incubations at the onset of chondrogenesis, while radiometric TLC indicates compounds which comigrate with PGF2 alpha are the major class of 3H-AA metabolites which accumulate during that time. This qualitative difference very likely reflects metabolism of parent PG compounds during the long (12h) labelling and postlabelling incubations utilized in the TLC analyses. The temporal correlation between PG production and the initiation of chondrogenesis in vitro is consistent with previous studies implicating PGs in the regulation of limb cartilage differentiation.

Role of anther on pollen embryogenesis in anther culture of Nicotiana tabacum L.

In order to define the role of anther on the pollen embryogenesis in anther culture of tobacco, the presence or absence of starch granules, amount of soluble sugars and content of free amino acids in cultured anthers were investigated through comparing with those in in vivo anthers. Starch granules in the tissues of cultured anthers disappeared rapidly, and the amount of soluble sugars also decreased soon after the onset of culture. Only a very small amount of soluble sugars was detected during the first 6 days of culture although sucrose was supplemented to the medium. On the 8th day of anther culture, the amount of soluble sugars, especially sucrose increased. It is known that pollen embryogenesis of tobacco can be induced under condition of sugar starvation in the culture medium. These results indicate that in the initial period of culture, the conchtions of the anther loculus are favorable for the induction of pollen embryogenesis. From the results of free amino acid analysis of cultured anthers, it was revealed that large amounts of glutamine and asparagine were present in cultured anthers on the 10 th day of culture when cell division leading to embryo formation began in cultured anther, although no amino acid was supplemented to the medium. It has been reported that certain amino acids such as glutamine and asparagine were essential to undergo cell dlvision leading to embryo formation from pollen grains of tobacco. Therefore, the results indicate that the amino acids required for embryo formation are supplied to pollen grains by cultured anthers. The present results revealed that the factors required for the process of pollen embryogenesis were successively provided to pollen grains by cultured anthers. It is concluded that satisfactory conditions of cultured anthers are the key points of the efficient formation of pollen embryos in anther culture of tobacco.

Establishment and characterization of a new clonal strain derived from human benign gastric teratoma

Various kinds of cell-types such as squamous epithelial cells, columnar epithelial cells, sarcomatous cells and fibroblast-like cells proliferated in vitro during the initial period of the primary culture from the human benign gastric tridermal teratoma. The fibroblast-like cells designated HOGT clonal strain were established from a single cell of primary cultures. The HOGT clonal strain grew well and 70 serial passages were successively carried out within 17 months. Although the HOGT cells are karyologically normal, when transplanted into the subcutis of nude mice they produced a benign tridermal teratoma. The results obtained from cell culture and heterotransplantation of teratoma strain enabled us to hypothesize that the graft (tridermal teratoma) in mice may arise from a pluripotent stem cell.

Formation of Adventitious Buds in Stem Internodes of Populus spp. Cultured in vitro on Basal Medium: Influence of Endogenous Properties of Explants

Stem-internodes of the following poplar species formed adventitious buds when cultured in vivo on basal medium in the absence of exogenous growth regulators: Populus nigra, P. deltoides, P. tacamahaca, P. trichocarpa, P. alba, P. wilsonii and Populus hybrid TT32 (P. trichocarpa × P. tacamahaca). The number of buds formed per internodal explant varied with each genotype. Adventitious buds differentiated in tissue produced by cambium and phloem cells. Capacity for bud formation was highest at the physiological apical end of explants. Morphogenesis was reduced in explants cultured with an axillary bud. Formation of adventitious buds was greatest during the initial period of explant culture; bud-formation was reduced or absent in re-cultured explants. Bud-forming capacity was related to length of explant; explants 2mm in length were unresponsive whereas a mean of eight buds were obtained per 8mm explant. Internodes close to the shoot apex failed to produce buds and morphogenic competence increased with increasing distance from the apex.

The effects of inhibitors of cell wall synthesis on tobacco protoplast development

The effect of 2,6‐dichlorobenzonitrile (DB) and coumarin on tobacco protoplast development has been studied using a combination of flow microfluorimetry and quantitative fluorescence microscopy. The results indicated that, over the initial period of culture, DB largely inhibited cellulose production at the cell surface, but did not inhibit DNA synthesis or protein accumulation by the protoplasts. Continuous culture of protoplasts in DB resulted in the accumulation of an increased capacity for cellulose synthesis as expressed following the removal of inhibitor. The possible sites of the specific inhibitory action of DB are discussed.