Effect of cumulus cell coculture and oxygen tension on the in vitro developmental competence of bovine zygotes cultured singly

The customary practice in bovine in vitro embryo production (IVP) is to handle oocytes and embryos in groups; although there are several reasons for establishing an IVP system for individual embryos that allows for following a single oocyte from retrieval through development to the blastocyst stage. To date, reports of individual IVP are inconsistent, and in most cases, resulted in unsatisfactory blastocyst rates. The objective of this study was to develop an efficient system for routine in vitro culture of individual bovine embryos. Single culture of zygotes in 2 different culture volumes (20 and 500 μL) yielded less than 3% blastocysts in experiment 1. In an attempt to improve these results, cumulus cells were added to the culture medium in experiment 2, after which blastocyst rates increased from 2.9 to 21.8% (P < 0.05). The third experiment revealed that an atmospheric oxygen tension, which is commonly used with somatic cell coculture, was not beneficial during individual embryo-cumulus cell coculture, because it resulted in lower blastocyst rates (Odds ratio 0.57, P < 0.001) and in lower blastocyst cell numbers (P < 0.05), when compared to culture in 5% oxygen. Grouped vs. single culture and reduced oxygen tension did not have a significant effect on cleavage and hatching rates. In experiment 4, three different cumulus cell coculture conditions during individual culture were tested and compared with the cleavage, blastocyst and hatching rates, and cell number of group culture (73.2%, 36.4%, 66.7% and, 155.1 ± 7.26, respectively). The outcome variables after individual embryo culture on a 5-day-old cumulus cell monolayer (74.1%, 38.2%, 71.9% and 133.4 ± 9.16, respectively), and single culture in the presence of added cumulus cells (69.9%, 31.9%, 66.7% and 137.3 ± 8.01, respectively) were not significantly different from those obtained after group culture (P < 0.05). Though, individual culture in a cumulus cell conditioned medium significantly reduced both the cleavage (59.0%) and blastocyst rates (6.3%). These results demonstrate that single culture of bovine zygotes can be fully sustained by coculture with cumulus cells in a low oxygen environment; implementation of these findings in our IVP system produced blastocysts comparable in quantity and quality to those obtained by group culture. These results were consistently achieved after acquiring experience and expertise in the handling of single zygotes.     

Single in vitro bovine embryo production: coculture with autologous cumulus cells, developmental competence, embryo quality and gene expression profiles

Studies concerning oocyte quality markers, oocyte/embryo metabolism or commercial OPU settings treating donors with low oocyte yields, indicate a need for optimization of IVP protocols to culture single oocytes to the blastocyst stage. However, culture conditions for single oocyte usually impair development, although previous research showed that single oocyte culture on a monolayer of cumulus cells can lead to similar developmental competence than group oocyte culture. Aiming to develop a fully single IVP procedure, Experiment 1 and 2 revealed that individual maturation, fertilization and culture in 20 μL droplets, using a monolayer of heterologous (SSSm, Exp 1) or autologous cumulus cells in coculture (SSSa, Exp 2), resulted in 23.9% and 15.1% of blastocysts 8 days p.i., respectively, which is significantly less compared to regular group IVP (GGGc, 33.5% (Exp 1) and 26.2% (Exp 2), respectively). In a third Experiment, day 7 p.i. blastocyst quality was analyzed in four treatment groups: regular group IVP (GGGc), group IVP with coculture (GGGm), in group produced zygotes, singly cultured on a heterologous cumulus cell monolayer (GGSm) and individually matured and fertilized zygotes, singly cultured on a monolayer (SSSm). Mean cell number and apoptotic cell index, were similar for all treatment groups. Moreover, mRNA abundance relative to H2AFZ was equal for 9 qualitatively linked genes (TP53, BAX, SHC1 SHC, IGF2R, PTGS2, AKR1B1, PLAC8, SLC2A1, and MNSOD). Only GPX1, involved in detoxification and mtDNA protection to oxidative stress, was significantly downregulated (ANOVA, P < 0.05) in singly produced blastocysts (SSSm), compared to the other treatments. In conclusion, a valuable individual IVP system was established and autologous cumulus cells in coculture showed to partly neutralize hampered individual culture conditions. Additionally, to our knowledge this is the first report in which blastocyst quality, in terms of cell number, apoptosis and gene expression, of singly produced embryos was investigated and shown to be similar to in group produced embryos, implicating that the single IVP system can be applied as a tool in oocyte and embryo quality studies.     

Cryopreservation of isolated micropores of spring rapeseed (Brassica napus L.) for in vitro embryo production

Microspore cryopreservation is a potentially powerful method for long-term storage of germplasm for in vitro embryo production in plant species. In this study, several factors influencing embryo production following the ultra-low temperature (–196 °C in liquid nitrogen) storage of isolated microspores of rapeseed (Brassica napus L.) were investigated. Microspores were prepared in cryogenic vials and subjected to various cooling treatments before immersion in liquid nitrogen for varying periods. Efficiency of microspore cryopreservation was reflected by in vitro embryo production from frozen microspores. Of all the cooling treatments, microspores treated with a cooling rate of 0.25% °C/min and a cooling terminal temperature of –35 °C before immersion in liquid nitrogen produced the highest embryo yields (18% and 40% of unfrozen controls in two genotypes, respectively). Fast thawing in a 35 °C water bath was necessary to recover a high number of embryos from microspore samples being frozen at a higher cooling rate, while thawing speed did not affect samples after freezing at a slower cooling rate. The storage density of cryopreserved microspores affected embryo production. Storage at the normal culture density (8×10⁴ microspores/ml) was less efficient for embryo production than at high densities (4×10⁶ microspores/ml and 1.6×10⁷ microspores/ml), although no significant difference was found between the high densities. Evaluation of plant lines derived from frozen microspores indicated no variation in isozyme pattern and no enhanced cold tolerance of these lines. Isolated microspores of B. napus could be stored for extended period for in vitro embryo production.     

Purification, sequence characterization and effect of goat oviduct-specific glycoprotein on in vitro embryo development

Oviduct-specific glycoprotein (oviductin) plays an important role during fertilization and early embryonic development. The oviductin cDNA was successfully cloned and sequenced in goat, which possessed an open reading frame of 1620 nucleotides representing 539 amino acids. Predicted amino acid sequence showed very high identity with sheep (97%) followed by cow (94%), porcine (77%), hamster (69%), human (66%), rabbit (65%), mouse (64%) and baboon (62%). The bioinformatics analysis of the sequences revealed the presence of a signal sequence of 21 amino acids, one potential N-linked glycosylation site at position 402, 21 potential O-linked glycosylation sites and 36 potential phosphorylation sites. The native oviductin was purified from the oviductal tissue, which showed three distinct bands on SDS-PAGE and western blot (MW ∼60-95 kDa). The predicted molecular weight of goat oviductin was 57.5 kDa, calculated from the amino acid sequences. The observed higher molecular weight has been attributed to the presence of large number of potential O-linked glycosylation sites. The lower concentration (10 μg/mL) of oviductin increased the cleavage rate, morula and blastocyst yield significantly (P < 0.05) as compared to higher concentration (100 μg/mL). Goat oviductin retarded the activity of pronase (0.1%) on zona solubility of oocytes significantly (P < 0.01).     

In vitro zygotic embryo culture of an endangered forest tree Givotia rottleriformis and factors affecting its germination and seedling growth

Summary This study reports a protocol for germination of Givotia rottleriformis (var. Tel. Thella Poniki) using zygotic embryo culture. A 100% germination was obtained by culturing the embryos on Murashige and Skoog medium containing 30 gl^&#x2212;1 sucrose. A sucrose concentration lower or higher than 30 gl^&#x2212;1 resulted in lower germination or promoted callus formation. The seedling growth was promoted by the addition of 100 mgl^&#x2212;1 tyrosine in the medium. Seedlings germinated in the presence of 0.2&#x2013;0.4 mgl^&#x2212;1 &#x03B1;-naphthaleneacetic acid and 0.3&#x2013;0.5 mgl^&#x2212;1 indole-3-butyric acid were abnormal, showing a slender stem with slender roots or forming callus with stout roots. Germination also affected embryo orientation in culture; placing embryos upright on the medium was most beneficial for germination. The in vitro-germinated seedlings were acclimatized in soil under shady conditions with a survival rate of 60&#x2013;70%. These plants were phenotypically normal, healthy, and similar to donor plants. This protocol will be useful for overcoming seed dormancy and for rapid multiplication and conservation of G. rottleriformis using zygotic embryo culture.     

Production of erythropoietin in vitro: A review

Summary The in vitro production of the important regulatory of erythropoiesis, erythropoietin (Epo), is reviewed. It is concluded that it is possible to produce almost routinely small quantities of Epo in tissue culture. Although such procedures offer the potential to provide large quantities of the hormone for clinical use, the optimum culture conditions and mechanisms for triggering Epo production have yet to be resolved. This work was supported by Grants No. 74444 from The John A. Hartford Foundation, and HL 10567 from the National Institutes of Health.     

Proteins produced by barley microspores and their derived androgenic structures promote in vitro zygotic maize embryo formation

Maize zygotes formed in planta were isolated and co-cultured with barley microspores. Evidence suggests that culture with microspores and/or conditioned media in barley promoted zygotic embryo formation. In order to characterise active substances present in the conditioned media, we collected medium at various times after the initiation of culture. We showed that proteins appeared over time and that their quantity increased during the course of the culture. Some proteins were glycosylated as revealed by the ConA peroxidase test. The use of the antigen -glucosyl Yariv reagent has shown that arabinogalactan-proteins (AGPs) were present. In addition, conditioned medium samples were analysed for their oligosaccharide content. New oligosaccharides appear in the course of the culture but they do not seem to affect development. We discuss in details the results in the context of understanding cell-cell interactions between embryo and nurse cells and the possible parallel with that occurs in ovulo between endosperm and embryo.     

The effect of gamma irradiation dose on cucumber (Cucumis sativus L.) haploid embryo production

Pollination with irradiated pollen was the only effective way for the induction of haploid embryo in cucurbits. The possibility of using lower doses of γ rays (Co⁶⁰ source) was studied. The effect of 0.2 and 0.3 kGy of rays was tested on five cucumber lines and three hybrids in the first experiment. It was found that there was hardly any difference between the total number of embryos produced by all studied forms. The highest number of isolated embryos was obtained from hybrids Gy-3 M and BxOg (111 and 188 respectively). The plant regeneration was estimated at 3.3 %. The best two lines and one hybrid were used in the second experiment to find the lowest possible dose of irradiation. The dose 0.05 kGy produced only diploid embryos and was rejected as too low. Other three doses (0.1; 0.2; and 0.3 kGy) effected embryo development in relation to the irradiation applied with the highest number obtained at the lowest dose. However the number of plants regenerated from each combination was similar. The plant regeneration in this experiment was 7.7 %. The effect of 0.1 and 0.3 kGy was tested during the next two years on one highly vigorous variety. It was confirmed that 0.1 kGy stimulated the development of higher number of haploid embryos.     

Effect of cytoplasmic lipid content on in vitro developmental efficiency of bovine IVP embryos

The objective was to evaluate the effect of cytoplasmic lipid content on the embryonic developmental efficiency of bovine in vitro embryo production (IVP) embryos. Ovaries from Korean native cows (Bos taurus coreanae) were collected from a local abattoir, and cumulus-oocyte complexes (COCs) were recovered from follicles 2 to 8 mm in diameter. The oocytes were divided into three groups, dependent on their cytoplasm color: pale color (PC), brown color (BC), and dark color (DC). The COCs were fertilized using frozen-thawed semen from a single Hanwoo bull. Based on measurement of the cytoplasmic color intensity of oocytes after 22 h of in vitro maturation (IVM), the DC group had lower (P < 0.05) color intensity than that in the BC and PC groups (56.3 ± 2.7, 93.3 ± 5.1, and 123.9 ± 12.0, respectively). Based on MitoTracker Green FM staining, the number of mitochondria in the DC (170.1 ± 31.2) group was significantly higher than that in the BC (137.5 ± 30.8) and PC (105.5 ± 25.3) groups. The cleavage rate in the DC (81.5%) group was also higher than that in the PC (50.4%) group (P < 0.05), as was the development rate to blastocyst stage (18.9% vs. 9.8%). Finally, cell numbers of blastocysts in the DC (150.8 ± 28.0) group were higher (P < 0.05) than that in the BC (107.6 ± 17.8) and PC (80.5 ± 12.3) groups. In conclusion, cytoplasm color was a useful selection parameter for abattoir-derived oocytes destined for IVP.     

Improved efficiency in apricot breeding: Effects of embryo development and nutrient media on in vitro germination and seedling establishment

Apricot (Prunus armeniaca L.) embryos at three stages of development were cultured on C2d, SBH and WPM media. In vitro culture produced high percentages of germination and seedlings throughout all three developmental stages. Significant media effects were noted for changes in both embryo length and weight during the culture period, as well as number of plants produced. Embryos between 5 and 9 mm (developmental stage I) germinated and developed into plants in a significantly higher percentage than in the other two more mature stages. Therefore, embryo culture can be successfully used as a tool in an apricot breeding program to obtain higher percentages of seedlings from planned hybridization or to overcome a lack of seed germination.     

Effects of arginine,L-alanyl-L-glutamine or taurine on neutrophil (PMN) free amino acid profiles and immune functions <Emphasis Type="Italic">in vitro</Emphasis>

Summary. The objective of this study was to determine the effects of arginine, L-alanyl-L-glutamine (Ala-Gln) or taurine on polymorphonuclear leucocyte (PMN) free amino acid profiles, superoxide anion (O_{2 −}) generation, hydrogen peroxide (H₂O₂) formation and released myeloperoxidase activity (MPO). Arginine led to significant increases in PMN arginine, ornithine, citrulline, aspartate, glutamate and alanine concentrations as well as increased H₂O₂-generation and MPO activity while O_{2 −}-formation was decreased. Ala-Gln caused significant increases in PMN free glutamine, alanine, asparagine, aspartate, glutamate, ornithine, arginine, serine and glycine concentrations and increased PMN immune functions. Taurine significantly increased PMN free taurine profiles, reduced PMN neutral amino acid content and decreased H₂O₂- and O_{2 −}-formation while MPO was increased. Altogether, the pharmacological regimens which enhance the supply of arginine, Ala-Gln or taurine in whole blood significantly affect PMN "susceptible free amino acid pool". This may be one of the determinants in PMN nutrition considerably influencing PMN immune functions. Received October 25, 2000 Accepted March 21, 2001     

Effects of cadmium and uranium on some in vitro renal targets

Metals are major pollutants not only in occupational settings but also in the general environment. Chronic exposure of workers has been related to severe damage, especially at the renal level. While toxic compounds such as metals are well known to severely impair tubular functions, it is clear that nephrotoxicants can act on various other renal targets, i.e., vascular and glomerular ones.In vitro models are available to assess these toxicities and can be used to better understand the different cell targets. This paper summarizes data obtained in our laboratory after exposure of isolated renal structures such as glomeruli, and cell cultures such as glomerular mesangial and tubular epithelial cells, to cadmium and uranium. Morphometric studies by image analysis of isolated glomeruli and mesangial cultured cells showed that cadmium and uranium induced a dose- and time-dependent glomerular contraction accompanied by disorganization of the cytoskeleton. Classical viability tests demonstrated various factors influencing the metal toxicity. The important roles of pH, extracellular protein concentrations and the nature of the anion accompanying the metal were demonstrated. These data obtained inin vitro models provide better understanding of the cytotoxicity after metal uptake and accumulation in glomerular and tubular cells. Moreover, the glomerular and tubular cytotoxicity they induce may be correlated with severe renal hemodynamic changes in vivo. Finally, we briefly present eventual improvements forin vitro renal models by the use of new cell models such as immortalized human cell lines or by the introduction of porous supports and perifusion devices. This revised version was published online in August 2006 with corrections to the Cover Date.     

Effects of taurine on GABA release from synaptosomes of rat olfactory bulb

Summary Superfusion of synaptosomes prepared from rat olfactory bulb revealed constant basal release of endogenous taurine (Tau), aspartate (Asp), glutamate (Glu) and-aminobutyrate (GABA): their release rates were 110.4 ± 13.0, 30.3 ± 6.7, 93.7 ± 13.1, and 53.3 ± 8.8 pmol/min/mg protein, respectively. The depolarizing-stimulation with 30mM KCl evoked 1.17-, 2.18-, 2.55- and 1.53-fold increases, respectively. Tau release was calcium-independent. However, the perfusion of synaptosomes with Tau (10µM) inhibited the evoked increase in GABA release by 63% without changing basal release, although it did not affect release of Asp and Glu. Phaclofen (10µM, a GABA_B receptor antagonist), but not bicuculline (10µM, a GABA_A receptor antagonist), counteracted the Tau-induced reduction in GABA release. These data suggest that Tau may be abundantly released from nerve endings of rat olfactory bulb and that it may regulate GABA release through the activation of presynaptic GABA_B autoreceptors.     

Effects of carbon sources and auxins on in vitro propagation of banana

The effects of carbon sources (sucrose, glucose, fructose and mannitol) and auxins [indolebutyric acid (IBA) and α-naphthaleneacetic acid (NAA)] on in vitro propagation of banana (Musa spp. AAA) were studied. Over all carbon sources tested, sucrose induced highest frequency of shoot proliferation. Optimal shoot proliferation rates were achieved on the Murashige and Skoog (MS) medium supplemented with sucrose and glucose combination (1:1) at the concentration of 30 g dm⁻³. Similarly, higher frequency of root induction was obtained at IBA and NAA combination (1:1; concentration of 2 mg dm⁻³) than at other concentrations of IBA or NAA alone or their combinations.     

Noninvasive bovine oocyte quality assessment: possibilities of a single oocyte culture

Although bovine embryos are routinely produced in vitro for several decades, there still exists a critical need for techniques to accurately predict the oocyte's developmental competence in a noninvasive way, before the in vitro embryo production procedure. In this review, several noninvasive methods to evaluate oocyte quality are discussed, such as morphological assessment of the cumulus oocyte complex and the use of brilliant cresyl blue. Because an individual oocyte and embryo culture method can possibly generate additional insights into the factors that determine oocyte quality, the second part of this review summarizes the state of the art of bovine single oocyte culture. The optimization of individual in vitro embryo production can obviously accelerate the quest for better noninvasive oocyte quality markers, because more information about the oocyte's requirements and intrinsic quality will be revealed. Although each step of in vitro culture has to be re-examined in light of the hampered production of single embryos, the reward at the end will be substantial. Individual scored oocytes will be traceable along the in vitro embryo production procedure and the final blastocyst outcome can be linked to the original oocyte quality and follicular environment without the bias caused by simultaneously developing embryos.     

Effects of light, magnesium and sucrose on leaf anatomy, photosynthesis, starch and total sugar accumulation, in kiwifruit culturedin vitro

Shootlets of kiwifruit plants (Actinidia deliciosa) were culturedin vitro. Combinations of light intensity, Mg and sucrose in the cultures showed that an increase of light intensity resulted in a corresponding increase of the relative size of the leaf mesophyll cells and in a decrease of the numbers of chloroplasts and contained starch grains. The addition of sucrose to the substrate media negatively affected the size of the mesophyll cells under normal Mg concentration (35 mg l⁻¹), and positively under high Mg concentration (105 mg l⁻¹ ). Sucrose further resulted in an increase in the numbers of chloroplasts and contained starch grains. The photosynthetic capacity of leaves greatly increased when Mg concentration was enhanced and sucrose was excluded from the nutrient substrate. Total sugar accumulation in all treatments was favoured by normal light intensity and addition of sucrose.     

Effects of in vitro treatments on leaf area growth of potato transplants during acclimatisation

The importance of leaf area of in vitro propagated potato (Solanum tuberosum L.) plantlets for further growth during acclimatisation and the after-effects of in vitro treatments on growth were examined. The in vitro treatments included different levels of alar, nitrogen or mannitol or different temperatures during the last in vitro phase, the rooting phase. Leaf area or ground cover was recorded one day after planting to soil and at the end of the first phase of ex vitro growth, the acclimatisation phase. Regression analysis showed that leaf area of a transplant at the end of acclimatisation phase was positively influenced by leaf area of the same plantlet at the beginning of the phase. The relative increase in leaf area during acclimatisation (increase/early leaf area) was linearly related to the inverse of the early leaf area, indicating almost comparable relative increases for plantlets having larger early leaf areas, but more variable responses for plantlets having smaller early leaf areas. In vitro treatments mainly affected leaf area of transplants through their effects on early leaf area. Adding alar, reducing nitrogen and reducing temperature increased leaf area. Reducing mannitol increased ground cover. A lower nitrogen concentration and higher temperature in some cultivars had slight negative effects on the relative increase in leaf area after acclimatisation. For nitrogen these negative effects were less significant than the positive effects through early leaf area. Results stress the importance of manipulation of leaf area in vitro to enhance plant performance in later stages of growth. This revised version was published online in June 2006 with corrections to the Cover Date.     

In vitro propagation of banana (Musa spp.): initiation,proliferation and development of shoot-tip cultures on defined media

In vitro multiplication of banana (Musa spp.) from shoot-tip explants isolated from lateral suckers is described. Using explants with apical domes, a total of 22 banana cultivars were successfully cultured on a modified Murashige and Skoog's medium containing 6-benzylaminopurine (BA) and indolebutyric acid (IBA). Shoot-tip explants could be induced to produce multiple shoot initials in the presense or absence of apical domes, but the survival rates were higher if apical domes were retained. Cultivars varied widely in their multiplication rates in response to cytokinins, BA being consistently more effective than kinetin (Kn). Although Kn was less effective in this regard, it stimulated vigorous root growth. Rooted plantlets were successfully established in soil.     

Influence of carbon source and concentration on the in vitro development of olive zygotic embryos and explants raised from them

The influence of sucrose or mannitol on in vitro zygotic embryo germination, seedling development and explant propagation of olive tree (Olea europaea L.) was compared. Embryos germinated without sucrose in the medium but for adequate development of the seedlings to yield viable plants, a carbohydrate supply was necessary; both sucrose and mannitol were equally suitable for this purpose. However, when explants obtained from in vitro germinated embryos were cultured with mannitol or sucrose, then the polyalcohol promoted significantly more growth than sucrose by increasing shoot length, pairs of leaves formed, and breaking apical dominance. This improved the in vitro culture of olive plant material, thus allowing new olive clonal lines to be obtained in shorter times. This will assist in future breeding experiments with the species.