CONTROL OF COLONY AND UNICELL FORMATION IN A SYNCHRONIZED SCENEDESMUS12

Colony formation in a synchronized Scenedesmus could be controlled by the addition of 0.05% Na₃ citrate or 85 μM EDTA to modified Bristol's medium. No unicells were formed; only S. quadricauda-like or S. longispina-type colonies were observed in young cultures grown in that medium. A colony population could be made completely unicellular in 2 days if grown in soil-Bristol's medium and transferred daily. The pleomorphic Scenedesmus was examined in synchronized culture. When the organism was grown in a defined medium in a 15-hr light /9-hr dark cycle on a roller tube rotator at 2-6 rpm and transferred daily, synchrony of cell division and release of the products were achieved. In a synchronized culture 2 doublings/day were recorded, with most cytoplasmic cleavages and all releasing of daughter cells taking place in the dark period. In many observations with several synchronized strains of Scenedesmus, no fixed pattern of release of daughter products from mother cells or colonies was detected. Colonies or unicells had their full spine complement at the time of release. As a cell or colony aged the spines sometimes increased in thickness. Other Scenedesmus strains were examined to provide supporting data.     

Polymorphism in gloeotaenium (Chlorococcales,Chlorophyceae)

Morphological variations of Gloeotaenium loitlesbergarianum Hansgirg were studied both in cultures and in nature. In cultures, the alga exhibits considerable variation in the number of cells per colony, ranging from unicells to colonies with more than four cells. The characteristic band was also absent in cultures. In nature, colonies resembling the culture material of Gloeotaenium also occur. The morphology of the alga varies depending on the nature and composition of the nutrients available. The study shows that Gloeotaenium may exhibit polymorphism in nature as well.     

Chloroplast ultrastructure,photosynthetic apparatus activities and production of steviol glycosides in <Emphasis Type="Italic">Stevia rebaudiana in vivo</Emphasis> and <Emphasis Type="Italic">in vitro</Emphasis>

The accumulation of steviol glycosides (SGs) in cells of Stevia rebaudiana Bertoni both in vivo and in vitro was related to the extent of the development of the membrane system of chloroplasts and the content of photosynthetic pigments. Chloroplasts of the in vitro plants, unlike those of the intact plants, had poorly developed membrane system. The callus cells grown in the light contained proplastids of almost round shape and their thylakoid system was represented by short thylakoids sometimes forming a little number of grana consisting of 2–3 thylakoids. In cells of the etiolated in vitro regenerants and the callus culture grown in the dark, only proplastids practically lacking the membrane system were observed. All the chloroplasts having developed thylakoids and forming at least a little number of grana were equipped with photochemically active reaction centers of photosystems 1 and 2. Leaves of in vivo plants accumulated greater amount of the pigments than leaves of the in vitro plants. In both the callus culture grown in the light and the etiolated in vitro regenerants, the content of the pigments was one order of magnitude lower than that in leaves of the intact plants. The callus tissue grown in the dark contained merely trace amounts of the pigments. Leaves of the intact and the in vitro plants did not exhibit any significant differences in photosynthetic O₂ evolution rate. However, photosynthetic O₂ evolution rate in the callus cells was much lower than that in the differentiated plant cells. The in vitro cell cultures containing merely proplastids did not practically produce SGs. However, after transferring these cultures in the light, both the formation of chloroplasts and the production of SGs in them were detected.     

The Effect of the Plant Growth Retardants Amo 1618 and CCC on Gibberellin Production in Fusarium monili-forme: Light Stimulated Biosynthesis of Gibberellins

Through the use of a single gene dwarf mutant of Zea mays L., dwarf-1, the interaction of growth retardants with gibberellin biosynthesis was studied in Fusarium monitiforme. It was demonstrated that the growth retardants 2-isopropyl-4-dimcthylamine-5-methyphenyl-1-piperidine-cai'boxylate methyl chloride (Amo 1618) and (2-chloroethyl) trimethylammonium chloride (CCC) are more effective inhibitors of gibberellin biosynthesis in cultures maintained under continuous illumination. Light grown cultures produced significantly more biologically active gibberellin-like materials than dark grown cultures. Stock cultures exposed to light also promoted the subsequent biosynthesis of gibberellins in the dark. Chromatographical analysis of the soluble gibberellins extracted from the culture medium revealed that large amounts of chromatographically detectable A₃ and A₇ were produced in light cultures with only A₇ produced in the dark. Light also induced a greater incorporation of acelate-2-¹⁴C into the gibberellins A₇, A₃ and an unidentified gibberellin. Growth returdants occasionally caused a complete disappearance of chromatographically detectable gibberellins in the dark; however, in the light at no concentration tested was it possible to detect the complete disappearance of gibberellin-like material. A₃ was always detectable. Like higher plants, different strains of F. moniliforme exhibit variation which makes them more or less sensitive to the growth retardants. This variation is interpreted to mean that there may be more than one pathway leading to the synthesis of the gibberellins.     

SCENEDESMUS MORPHOGENESIS. TRACE ELEMENTS AND SPINE FORMATION1

When Scenedesmus culture 16, a soil organism with numerous long spines, was grown in axenic culture in a dilute laboratory medium (with approximately 30 mg/liter total salts), spines were not formed. In this medium the organism resembled S. bijugatus, whereas S. longus and S. abundans colonies were typically formed in more concentrated media. Spines were formed on daughter colonies upon transfer to the dilute medium plus additional Fe EDTA or Ca EDTA. No other individual components of the medium did this. Spineless colonies were healthy and green with a well-defined chloroplast, provided the culture was transferred often. In addition to the absence of spines, cultures in the dilute medium had a linear arrangement of cells within a colony, along with terminal cells 20% shorter than median cells. With 4-celled spiny colonies all cells were the same length and were alternately arranged.     

Wall ultrastructure ofScenedesmus culture N 46

Bristles radiating from openings were detected on colonies and unicells ofScenedesmus culture N 46, when examined with transmission and scanning electron microscopes. Although narrower, they correspond in gross appearance and ultrastructure to previously describedScenedesmus bristles. Openings, bordered by a series of props, are unlike those ofScenedesmus culture 614. Additional props are observed scattered independently on the cell wall; ridges are composed of a linear row of props. Sections of cells, or cell walls, reveal an additional prop, situated inside the openings; these props are composed of several tubules. Possible extrusion of bristles through these tubules, as well as the origin of the bristle from the cavity and vesicles immediately under the opening are discussed.     

Selection of a Highly Monensin-Resistant Prevotella bryantii Subpopulation with Altered Outer Membrane Characteristics

Prevotella bryantii cultures treated with monensin grew more slowly than untreated cultures, but only if the monensin concentration was greater than 1 μM. Cultures that were repeatedly transferred (eight transfers or 25 doublings) with monensin always grew rapidly, even at a 10 μM concentration. The amount of monensin needed to facilitate half-maximal potassium depletion (K_d) from monensin-selected cells was 16-fold greater than “unadapted” wild-type cultures (3,200 versus 200 nM). Cells taken from continuous culture had a K_d of 100 nM, and these inocula could not grow in batch culture when the monensin concentration was greater than 300 nM. Continuous cultures treated with monensin nearly washed out, but the surviving cells had a K_d of 1,300 nM. When wild-type cells were transferred in batch culture with 10 μM monensin, the K_d did not reach its maximum value (3,200 nM) until after eight transfers (25 doublings). K_d declined when monensin was removed, and it took eight transfers to reach the control value (200 nM). The most probable number of wild-type cells was 1,000-fold lower than of the monensin-selected cells, but calculations based on relative growth advantage and K_d indicated that the wild-type culture had 1 to 10% highly monensin-resistant cells. Cell pellets of wild-type cultures were more difficult to disperse than were monensin-selected cells, and water-soluble phenol extracts of monensin-selected cells had 1.8-fold more anthrone-reactive material than did the wild type. Wild-type cultures that were washed in Tris buffer (pH 8.0) released little alkaline phosphatase and were agglutinated by lysozyme. Monensin-selected cultures leaked ninefold more alkaline phosphatase and were not agglutinated by lysozyme. Wild-type colonies taken from high-dilution agar roll tubes retained the lysozyme agglutination phenotype even if transferred with monensin, and monensin-selected colonies were never agglutinated. These observations indicated that wild-type P. bryantii cultures had a subpopulation with different outer membrane characteristics and increased monensin resistance.     

Ricerche Sulla Cultura Sommersa di Cellule Singole di Piante Superiori

Abstract

Research on submerged culture of single cells of higher plants. — The author describes a method which allows to obtain submerged cultures of single cells of Phaseolus vulgaris and Nicotiana tabacum. The medium composition in macroelements in the culture on agar appears to effect to a great extent the ability of tissues to dissociate into single cells in the subsequent liquid culture. In this respect Heller's solution results to be more suitable than Gautheret's and Hildebrandt and Ri-ker's.

Cells are grown at 24 [ddot]C in 300 ml flasks containing 60 ml of broth on a rotary shaker at 220 rpm.

To prevent contaminations some antibacterial agents were added to cultures of Phaseolus vulgaris. Among these Penicillin and Neomycin were not tossic at 20 and 5 ppm concentrations respectively.

The presence of septa, which are observed also in largely vacuolate cells, seems to confirm the ability of single cells to divide.

The optimum 2,4-D concentration for growth decreases from 6 × 10^-8 to 6 × 10^-8 during successive liquid cultures, each of them being inoculated with on amount of the previous one. This fact, showing the adaptation of liquid cultures to decreasing concentrations of the growth hormone, is in agreement with previous observations in solid cultures by several authors.     

Carbon catabolism in continuous cultures and bacteroids of Rhizobium leguminosarum MNF 3841

Bacteroids of R. leguminosarum MNF3841 isolated from pea nodules using Percoll gradients had activities of TCA cycle enzymes up to 6-fold higher than those measured in free-living cells grown on fumarate or sucrose. Activities of sugar catabolic enzymes on the other hand were 2–14-fold lower in isolated bacteroids than in sucrose-grown free-living cells. In continuous culture, cells of strain MNF3841 grown on sucrose under P _i limitation had 2–3-fold higher activities of invertase, glucose-6-phosphate dehydrogenase, the Entner-Doudoroff enzymes and 6-phosphogluconate dehydrogenase, than cells grown on fumarate. With one exception O₂ limited cultures had similar activities of the carbon catabolic enzymes to P _i-limited cultures grown in the same substrate. Glucose-6-phosphate dehydrogenase in O₂-limited cells grown of fumarate was 50% lower than in P _i-limited cells. Co-utilization of fumarate and sucrose occurred with chemostat cultures supplied with both under a variety of conditions.Abbreviations E-D Entner-Doudoroff - EMP Embden-Meyerhof-Parnas - PEPCK phosphoenolpyruvate carboxy kinase - HEPES N-[2-hydroxyethyl]piperazine-N-[2-ethanesulphonic acid]     

STRUCTURE AND FUNCTION OF THE BRISTLES OF PEDIASTRUM BORYANUM (CHLOROPHYTA)1

Tufts of long delicate bristles were detected on mature colonies of Pediastrum boryanum (Turp.) Meneghini. The bristles are not uniform in length and can exceed 100 μm. Bristle number per tuft can exceed eighty. Ultrastructure studies revealed that tufts are an aggregate of hexagonal tubules arranged in staggered rows. Each hexagonal tubule has an inner diameter of 6 nm and is made up of six interconnected subunits 4 nm in diameter that are shared by adjoining tubules. The bristles can be easily removed from mature colonies with a vortex stirrer. Removal of bristles from mature colonies in culture results in the loss of buoyancy and subsequent settling to the bottom of the flask.     

Toxicity of arsenic during high temperature bioleaching of gold-bearing arsenical pyrite

 A moderately thermophilic mixed culture, MT, and the thermophilic Sulfolobus acidocaldarius strain BC were studied for their response to arsenic in a defined medium and also in media containing a pyrite and an arsenical pyrite flotation concentrate. In defined medium, the individual constituents of the MT culture exhibited a high tolerance to arsenite and arsenate compared to S. acidocaldarius strain BC. When grown on increasing concentrations of the pyrite flotation concentrate, both cultures had similar specific leaching rates over the various concentrations of the mineral substrate. In contrast, S. acidocaldarius strain BC exhibited a decreasing specific leaching rate when grown on the arsenical pyrite while the MT culture was not affected. In addition, arsenic added to cultures of S. acidocaldarius strain BC growing with pyrite as a growth substrate inhibited further growth, while added arsenic had no effect on the MT culture growing on the pyrite. These data indicate that the moderately thermophilic, arsenic-resistant MT culture was able to leach arsenical pyrite more efficiently than was the S. acidocaldarius strain BC culture at high concentrations of the mineral. This emphasizes the fact that proper culture selection is an important parameter when developing commercial processes involving arsenic-containing minerals. Received: 21 June 1995/Received revision: 25 August 1995/Accepted: 7 September 1995     

THE ACCLIMATIVE CHANGES IN PHOTOCHEMISTRY AFTER COLONY FORMATION OF THE CYANOBACTERIA MICROCYSTIS AERUGINOSA1

Microcystis aeruginosa (Kütz.) Kütz. commonly occurs as single cells at early recruitment but forms large colonies in summer. Colony formation will induce many acclimative changes. In this study, we demonstrated the photochemical changes before and after colony formation. In the laboratory, light curves showed that colonies were more responsive to high light than single cells. The values of the maximal slope of electron transport rate (ETR)—light curve (α), relative maximal electron transport rate (rETR_max), and onset of light saturation (I_k) of colonies were significantly higher than those of single cells (P < 0.05), indicating that colonies have higher photosynthetic capability than single cells, especially in high light, where values of rETR_max and I_k of colonies were 2.32 and 2.41 times those of single cells. Moreover, the dark‐light experiments showed that colonial cells can more effectively resist darkness damage. In addition, pigments of colonial cells were higher than those of single cells (P < 0.05). The higher pigment contents probably contribute to higher photosynthetic capability. In the field, the inhibition rate of F_v/F_m in single cells increased significantly faster than that of colonies as light increased (P < 0.05), but nonphotochemical quenching (NPQ) value of colonies was higher (32.4%) than that of single cells at noon, which indicated colonial cells can more effectively resist high‐light inhibition than single cells (P < 0.05). Polysaccharides of colonies were significantly higher compared to those in unicellular cells (P < 0.05) based on their contents and ultrastructural characteristics. This finding implies that colonies could not effectively decrease photoinhibition by negative buoyancy regulation. In fact, NPQ may be an important mechanism for avoiding photodamage. All of these phenomena can help explain the ecological success of colonial M. aeruginosa in eutrophic water.     

Transgenic Murine Dopaminergic Neurons Expressing Human Cu/Zn Superoxide Dismutase Exhibit Increased Density in Culture, but No Resistance to Methylphenylpyridinium-Induced Degeneration

Abstract: Primary dopaminergic neuronal cultures with increased superoxide dismutase (SOD) activity were established for studying the role of superoxide anion (O₂^?) in 1-methyl-4-phenylpyridinium (MPP⁺)-induced degeneration of dopamine (DA) neurons. Mean SOD activity in cultures prepared from transgenic (human) Cu/Zn SOD (hSOD1) mice was 2.46–2.60 times greater than in cultures prepared from nontransgenic control mice. After 1 and 2 weeks in culture, the mean density of DA neurons [number of tyrosine hydroxylase-immunoreactive (TH-ir) cells per visual field] was significantly higher in cultures prepared from transgenic mice compared with those prepared from nontransgenic control mice (4.55–5.63 TH-ir neurons per field in hSOD1 cultures vs. 2.66–2.8 TH-ir neurons per field in control cultures). However, uptake of [³H]DA relative to uptake of [³H]GABA was only slightly greater in hSOD1 cultures than in normal cultures (14.1 nmol of DA/100 nmol of GABA vs. 12.1 nmol of DA/100 nmol of GABA). Resistance to MPP⁺ toxicity was not significantly different from that in normal cultures when based on density of surviving TH-ir cell bodies (EC₅₀ = 0.54 µM in hSOD1 and EC₅₀ = 0.37 µM in normal cultures). A more sensitive measure of DA neuron integrity and function ([³H]DA uptake) also failed to demonstrate increased resistance of hSOD1 cultures to the toxin (EC₅₀ = 73.7 nM in hSOD1 and EC₅₀ = 86.2 nM in controls). These results do not support the hypothesis that neurotoxicity of the active metabolite of MPTP, MPP⁺, is mediated by generation of O₂^? in the cytoplasm. Nevertheless, mesencephalic cultures with increased hSOD1 activity appear to survive better than normal control cultures in the oxidatively stressful environment of cell culture incubators, and such mesencephalic cells may be useful for cell grafting studies in animal models of Parkinson's disease.     

Variation of DNA Levels in Euglena Related to pH of Culture Medium1

Variations in DNA levels for log-phase Euglena have been shown to be directly related to the pH of the culture medium. Data are presented here for cultures grown in Cramer and Myer's medium at pH 3.0 and 6.8. DNA levels are ～50% greater for Euglena at the higher pH value, confirming earlier reports.     

A nonhemolytic phospholipase C produced byPseudomonas cepacia

Pseudomonas cepacia Pc224c, a nonhemolytic strain originally isolated from the sputum of a cystic fibrosis patient, produced an extracellular, heat-labile phospholipase C activity, which was measured quantitatively on the synthetic substratep-nitrophenylphophorylcholine. Cell-free supernatants from cultures grown to late log phase in MOPS-minimal salts-Tryptose medium contained specific activity at least 38 times greater than that from cultures grown in Tryptose minimal medium, Tryptic Soy broth, or peptone medium. Production was inhibited by the presence of inorganic phosphate in the medium and enhanced by aeration of the culture. The PLC ofP. cepacia is nonhemolytic and does not exhibit lecithinase activity on egg-yolk agar.     

Effects of Acetazolamide (Diamox), Ethoxzolamide and High Levels of CO2 on Carbonic Anhydrase, Photosystem Activity, and Oxygen Evolution in Euglena gracilis

Investigations using steady-state culture conditions indicate that carbonic anhydrase activity is correlated to the photosynthetic rate in Euglena in some but not all circumstances. When cultures grown with 5% CO₂ were changed to air growth, the photosynthetic rate was independent of the carbonic anhydrase activity. While experiments using the inhibitor acetazolamide indicated a close correlation between photosynthetic capacity and carbonic anhydrase activity, the inhibitor was found to be nonspecific. Acetazolamide altered photosystem activities directly as measured by the photoreduction of DCPIP in chloroplast preparations, whole-cell fluorescence transients of chlorophyll a, and by whole chain photoelectron flow. Ethoxzolamide, another inhibitor of carbonic anhydrase, was also found to inhibit photosystem activities, i.e., the photoreduction of DCPIP, and in vivo photoelectron flow, at high concentrations. Cells grown in 5% CO₂ were less sensitive to the effects of acetazolamide than cells exposed to air. The rate of electron flow in chloroplasts from cells grown with 5% CO₂ and exposed to 10 mM acetazolamide was 2.5-fold faster than that of chloroplasts from air-grown cells exposed to the same concentration of inhibitor. The whole cell chlorophyll a fluorescence transients of cultures grown with high CO₂ were completely different from those of air-grown cells and also showed fewer effects on exposure to acetazolamide. These results suggest a reevaluation of the hypothesis that carbonic anhydrase activity regulates photosynthesis. It is also apparent that results from air-grown and 5% CO₂-grown cultures cannot be directly compared in such studies.     

Culture of blastomeres from in vitro-matured,fertilized, and cultured bovine embryos

A culture system was devised to study the differentiation of bovine blastomeres. Blastomeres (2–13 per well) from embryos produced by in vitro maturation, fertilization, and culture of oocytes obtained from slaughterhouse ovaries were cultured for 10 days in 24-well culture plates on feeder layers in blastomere culture medium (BCM: equal parts tissue culture medium 199 and low-glucose Dulbecco's modified Eagle's medium with 10% fetal bovine serum). Ovine embryonic fibroblasts and STO cells were superior to bovine and mouse embryonic fibroblasts as mitotically inactivated feeder cells. Over five studies in which four blastomeres from an embryo were added to each culture well, an average of one colony per well formed from the blastomeres. The colonies continued to grow throughout the culture period, and most colonies resembled trophectoderm in their cellular characteristics, although some cultures contained a mixture of trophectoderm and endoderm. When the number of blastomeres cultured in each well was varied from 2–8, the number of colonies formed was proportional to the number of blastomeres added. Blastomeres from day 5 and day 6 embryos produced fewer colonies than did those from day 4 embryos, perhaps as a result of differentiation and tighter blastomere adhesion resulting in damage during their separation. The absence of serum did not alter the number of colonies formed. A number of growth factors, including LIF, OM, PDGFα, and FGF4, had no effect on the number of colonies, the size of colonies, or their alkaline phosphatase staining score beyond that provided by the feeder layer or serum when present. Blastomeres did not form colonies in the absence of feeder layers. Mol. Reprod. Dev. 48:238–245, 1997. © 1997 Wiley-Liss, Inc.     

Partitioning of CO2 Fixation in the Colonial Cyanobacterium Microcystis aeruginosa: Mechanism Promoting Formation of Surface Scums

Constraints on inorganic carbon (C_i) availability stimulated buoyancy in natural, photosynthetically active populations of the colonial blue-green alga (cyanobacterium) Microcystis aeruginosa. In nonmixed eutrophic river water and cultures, O₂ evolution determinations indicated C_i limitation of photosynthesis, which was overcome either by CO₂ additions to the aqueous phase or by exposure of buoyant colonies to atmospheric CO₂. Microautoradiographs of M. aeruginosa colonies revealed partitioning of ¹⁴CO₂ fixation and photosynthate accumulation between peripheral and internal cells, particularly in large colonies. When illuminated colonies were suspended in the aqueous phase, peripheral cells accounted for at least 90% of the ¹⁴CO₂ assimilation, whereas internal cells remained unlabeled. However, when ¹⁴CO₂ was allowed to diffuse into colonies 15 min before illumination, a more uniform distribution of labeling was observed. Resultant differences in labeling patterns were most likely due to peripheral cells more exclusively utilizing CO₂ when ambient C_i concentrations were low. Among colonies located at the air-water interface, internal cells showed an increased share of photosynthate production when atmospheric ¹⁴CO₂ was supplied. This indicated that C_i transport was restricted in large colonies below the water surface, forcing internal cells to maintain a high degree of buoyancy, thus promoting the formation of surface scums. At the surface, C_i restrictions were alleviated. Accordingly, scum formation appears to have an ecological function, allowing cyanobacteria access to atmospheric CO₂ when the C_i concentration is growth limiting in the water column.     

Inhibitory effect of ethanol on growth and solute accumulation by Saccharomyces cerevisiae as affected by plasma-membrane lipid composition

Incorporation of ethanol (1.0 or 1.25 M) into exponential-phase cultures of Saccharomyces cerevisiae NCYC 366 growing anaerobically in a medium supplemented with ergosterol and an unsaturated fatty acid caused a retardation in growth rate, which was greater when the medium contained oleic rather than linoleic acid. Ethanol incorporation led to an immediate drop in growth rate, and ethanol-containing cultures grew at the slower rate for at least 10 h. Incorporation of ethanol (0.5 M) into buffered (pH 4.5) cell suspensions containing d-[6-³H] glucose, d-[1-¹⁴C] glucosamine, l-[U-¹⁴C] lysine or arginine, or KH₂ ³²PO₄ lowered the rate of solute accumulation by cells. Rates of accumulation of glucose, lysine and arginine were retarded to a greater extent when cells had been grown in the presence of oleic rather than linoleic acid. This difference was not observed with accumulation of phosphate. Ethanol was extracted from exponential-phase cells by four different methods. Cells grown in the presence of linoleic acid contained a slightly, but consistently, lower concentration of ethanol than cells grown in oleic acid-containing medium. The ethanol concentration in cells was 5–7 times greater than that in the cell-free medium.     

The lack of an inhibitory effect of hyaluronate on chondrogenesis in chick limb-bud mesoderm cells grown in culture

The effect of hyaluronate on chondrogenesis in cultures of chick limb-bud mesoderm cells, derived from stage 20–21, 23–24 and 26 embryos grown at different cell densities and in 3 different culture media, was studied. The results show that hyaluronate at a concentration of 500 μg/ml, does not consistently produce an inhibition of chondrogenesis in cultures of stage 20–21, 23–24 or 26 limb-bud mesoderm cells in contrast to what has been reported by Toole et al. (1972). It was demonstrated that under optimal conditions, stage 26 cells grown in the absence of hyaluronate do not form as many cartilage colonies in culture as do cells from stage 20–21 or 23–24 embryos. It was determined that culture medium composed of Eagle's MEM supplemented with 7% horse serum, 3% fetal calf serum and 5% 10-day chick embryo extract supported chondrogenesis significantly better than Ham's F-12 supplemented with 10% fetal calf serum. Our results suggest that the inhibition of chondrogenesis by hyaluronate reported earlier is most likely due to the sub-optimal conditions of growth medium, cell density and embryonic stage than to the hyaluronate treatment.