IMPROVED PROCEDURES FOR THE CLONING AND PURIFICATION OF MICROCYSTIS CULTURES (CYANOPHYTA)1

A cloned axenic culture of Microcystis Kützing was obtained by combining two procedures: a) the disaggregation of multicellular Microcystis colonies by dilution into deionized water, and b) the selective growth of Microcystis in agar media containing Na₂S, which inhibited or killed the associated contaminants. Microcystis growth was stimulated by 0.3–1 mM Na₂SO₃, but not by 0.1–33 mM Na₂SO₄. Although Microcystis cells survived temporary exposure to high Na₂S concentrations, their growth was not stimulated by 1 × 10^?5 to 1.0 M Na₂S. Possible metabolic roles of reduced sulfur compounds are considered. Microcystis colonies disaggregated to unicells at ionic concentrations below 1 mM for univalent cations, 10–100 μM for the divalent cations, and 3–10 μM for Fe³⁺. Higher cation concentrations prompted cell aggregation. With > 100 mM Fe³⁺, the Microcystis capsule appeared rust-colored. Neither nonionic solutes nor anions detectably influenced aggregation. These observations suggest cation interactions with the Microcystis capsule and are discussed with regard to: a) possible siderochrome activity, cation chelation or luxury uptake of cations, b) the questionability of using cell aggregation as a criterion for identifying Microcystis in samples of unknown ionic strength, c) the utility of low ionic strength media in releasing contaminating bacteria from the capsule and in obtaining algal unicells for cloning, and d) a model for cation interactions with the capsule.     

The in vitro Germination of Pollen of Setaria sphacelata

The effects of various substances upon germination and tube growth of pollen of Setaria sphacelata were investigated in hanging-drop culture. Both sucrose (0.6–0.7 M) and boron (1–5 ppm, as borate) are essential for germination. Comparable results were obtained with boric acid, sodium tetraborate and tri-n-butyl borate as boron sources, but sodium tetraphenylboron was inhibitory. Good germination and growth were obtained with raffinose and cellobiose, alone or in combination with sucrose (total 0.5 M); 0.25 M rhamnose, lactose and glycerol were without effect, and xylose, galactose and glucose were slightly inhibitory in the presence of 0.25 M sucrose; 0.25 M arabinose, fructose, mannose, sorbose, maltose, mannitol and sorbitol completely inhibited germination, even in the presence of 0.25 M sucrose. IAA and GA (0.01–10 ppm) could not replace or supplement the effects of borate on germination and growth, indicating the pollen to be self-sufficient in this respect. Riboflavin (0.1–10 ppm) and calcium pantothenate (1–100 ppm) stimulated germination and growth, whereas 0.01–10 ppm of thiamine, pyridoxine, nicotinic acid and ascorbic acid were generally without effect. Although copper sulphate, manganese sulphate, zinc sulphate and ammonium molybdate could not replace boric acid, 1.0 ppm of copper, manganese and zinc stimulated germination and growth in the presence of boron. In no instance were tubes found comparable in length to those required for fertilisation in vivo. It was concluded that the pollen probably requires a complex mixture of substances, including sucrose and borate, before this can be achieved in vitro.     

Inhibitor and Auxin Activity in the Avocado Fruit

The wheat coleoptile elongation bioassay was used for determination of growth-promoting and growth-inhibiting substances in avocado (Persea americana Mill.) fruit tissues, during development and maturation. Growth-promoting activity was found in two zones on paper chromatograms developed with isopropanol: ammonia : water (10:1:1 v/v): Rf 0.30–0.50 and Rf 0.8–0.9. Growth inhibiting activity was found in three different zones: “A” Rf 0.0–0.2, “B” (abscisic acid) Rf 0.6–0.8, and “C” (l-acetoxy-2,4-dihyroxy-n-hepta-deca-16-ene) Rf 0.85–1.0. Higher levels of auxins were found in seed tissues than in the mesocarp. No correlation was found between fruit growth rate and level of extractable auxins in the mesocarp. The amount of abscisic acid (ABA) in the mesocarp was constant during fruit growth. A gradual and consistent increase in 1-acetoxy-2,4-dihydroxy-n-hepta-deca-16-ene was found during fruit growth, reaching a maximum when the fruit attained maturity.     

Response of wheat to subsoil salinity and temporary water stress at different stages of the reproductive phase

To examine the effects of subsoil NaCl salinity in relation to water stress imposed at different growth stages, wheat was grown in a heavy texture clay soil (vertosol) under glasshouse conditions in polythene lined cylindrical PVC pots (100 cm long with 10.5 cm diameter) with very low salinity level (EC_e 1.0 dS/m; ESP 1.0 and Cl 30 mg/kg soil) in top 10 cm soil (10–20 cm pot zone) and low salinity level (EC_e 2.5 dS/m, ESP 5, and Cl 100 mg/kg soil) in top 10–20 cm soil (20–30 cm pot zone). The plants were exposed to three subsoil salinity levels in the 20–90 cm subsoil (30–100 cm pot zone) namely low salinity (EC_e: 2.5 dS/m, ESP: 5, Cl: 100 mg/kg soil), medium salinity (EC_e: 4.0 dS/m, ESP: 10, Cl: 400 mg/kg) and high salinity (EC_e: 11.5 dS/m, ESP: 20, Cl: 1950 mg/kg) in the subsoil (20–90 cm soil layer: 30–100 cm pot zone). Watering of plants was withheld for 20 days commencing at either early booting or anthesis or mid grain filling, and then resumed until maturity, and these treatments were compared with no water stress. Water stress commencing at anthesis stage had the most depressing effect on grain yield and water use efficiency of wheat followed by water stress at grain filling stage and early booting stage. High subsoil salinity reduced grain yield by 39.1, 24.3%, and 13.4% respectively in plants water-stressed around anthesis, early booting, and mid grain filling compared with 36.6% in well-watered plants. There was a significant reduction in root biomass, rooting depth, water uptake and water use efficiency of wheat with increasing subsoil salinity irrespective of water regimes. Plants at high subsoil salinity had 64% of their root biomass in the top 0–30 cm soil and there was a marked reduction in subsoil water uptake. Roots also penetrated below the non-saline surface into salinised subsoil and led to attain high concentration of Na and Cl and reduced Ca/Na and K/Na ratio of flag leaf at anthesis stage. Results suggest that high subsoil salinity affects root growth and water uptake, grain yield and water use efficiency even in well water plants. Water stress at anthesis stage had the most depressing effect on wheat.     

An energy budget for Simocephalus vetulus (O. F. Muller) (Crustacea : Cladocera)

We discuss the energetics of a cladoceran, Simocephalus vetulus at different temperatures (8.0 ± 1.0, 15.0 ± 1.0, 21.0 ± 1.0 and 28.0 ± 1.0 °C) and food (Chlamydomonas sp.) concentrations (25 × 10³, 50 × 10³, 75 × 10³ and 100 × 10³ cells ml⁻¹). Increase in temperature accelerated ingestion and, to some extent, oxygen consumption. The study revealed a high reproduction efficiency in S. vetulus. Net growth efficiency (ECI) was higher (13.17–41.18%) in pre-adults than in adults (2.71–8.40%). The assimilated energy (A) increased with increasing food concentration at all temperatures. Assimilation efficiency (AD) decreased with increasing food concentrations. The energy used for growth (P) was nearly constant at all food levels because the egested energy increased and assimilation efficiency decreased as food concentration increased.     

Utility of gold nanoparticles in luminescence determination of trovafloxacin: comparison of chemiluminescence and fluorescence detection

Two novel sensitive sequential injection chemiluminescence analysis and fluorescence methods for trovafloxacin mesylate detection have been developed. The methods were based on the enhancement effect of gold nanoparticles on luminol–ferricyanide–trovafloxacin and europium(III)–trovafloxacin complex systems. The optimum conditions for both detection methods were investigated. The chemiluminescence signal was emitted due to the enhanced effect of gold nanoparticles on the reaction of luminol–ferricyanide–trovafloxacin in an alkaline medium. The response was linear over a concentration range of 1.0 × 10^–9 to 1.0 × 10^–2 mol/L (%RSD = 1.3), (n = 9, r = 0.9991) with a detection limit of 1.7 × 10^–10 mol/L (S/N = 3). The weak fluorescence intensity signal of the oxidation complex of europium(III)–trovafloxacin was strongly enhanced by gold nanoparticles and detected at λ_ex = 330 and λ_em = 540 nm. Fluorescence detection enabled the determination of trovafloxacin mesylate over a linear range of 1.0 × 10^–8 to 1.0 × 10^–3 mol/L (%RSD = 1.2), (n = 6, r = 0.9993) with a detection limit of 3.3 × 10^–9 mol/L. The proposed methods were successfully applied to the determination of the studied drug in its bulk form and in pharmaceutical preparations. The results were treated statistically and compared with those obtained from other reported methods. Copyright © 2015 John Wiley & Sons, Ltd.     

Dietary arginine requirement of fingerling Indian major carp,Labeo rohita (Hamilton) based on growth,nutrient retention efficiencies,RNA/DNA ratio and body composition

To quantify the optimum dietary arginine requirement of fingerling Indian major carp, Labeo rohita (4.10 ± 0.04 cm; 0.62 ± 0.02 g), an 8‐week growth trial was conducted in eighteen 70‐L indoor circular aqua‐coloured troughs provided with a flow‐through system at 28 ± 1°C. Isonitrogenous (40 g 100 g^?1 crude protein) and isocaloric (4.28 kcal g^?1 gross energy) amino acid test diets containing casein and gelatin as intact protein sources with graded levels of arginine (0.5, 0.75, 1.0, 1.25, 1.50 and 1.75 g 100 g^?1 dry diet) were fed to triplicate groups of fish to apparent satiation at 07:00, 12:00 and 17:30 hours. Growth performance of fish fed the above diets was evaluated on the basis of absolute weight gain (AWG), specific growth rate (SGR), feed conversion ratio (FCR), protein efficiency ratio (PER), protein retention efficiency (PRE) and energy retention efficiency (ERE). Maximum AWG (2.61), SGR (2.80), best FCR (1.35), highest PER (1.85), PRE (37%) and ERE (76%) were recorded at 1.25 g 100 g^?1 dietary arginine. Maximum body protein (18.88 g 100 g^?1) and RNA/DNA ratio (5.20) were also obtained in a 1.25 g 100 g^?1 arginine dry diet. Except for the reduced growth performance in fish fed arginine‐deficient diets, no other deficiency signs were apparent. Based on the broken‐line and second‐degree polynomial regression analysis of the AWG, SGR, FCR, PER, PRE and ERE data, the optimum arginine requirement for fingerling Labeo rohita was found to be in the range of 1.22–1.39 g 100 g^?1 of the dry diet, corresponding to 3.05–3.47 g 100 g^?1 of dietary protein.     

Studies on root nodules of leguminous plants bioproduction of indole acetic acid by a Rhizobium sp. from a twiner Clitoria ternatea L.

The Rhizobium sp. isolated from the root nodules of Clitoria ternatea L., a leguminous twiner, produced a high amount of IAA (16.4 μg/ml) from tryptophan in an unsupplemented basal medium. The production of IAA started simultaneously with the growth and had no different growth and production phase. The growth and production were parallel and increased up to 45–50 h. The IAA production by the Rhizobium sp. was increased by 520% when the medium was supplemented with fructose (1.5%), MnSO₄ (1.0 μg/ml), riboflavin (0.10 μg/ml) and Triton X-100 (0.01%). The possible role of the rhizobial production of IAA on the rhizobia-legume symbiosis is discussed.     

Optimal stocking density for beluga,Huso huso,and ship sturgeon,Acipenser nudiventris during the grow‐out phase

The influence of three different initial stocking densities (SD) in flowthrough systems was evaluated on growth performance and feed utilizaition in beluga, Huso huso, and ship sturgeon, Acipenser nudiventris, juveniles in three different grow‐out phases for 228 days. In each grow‐out phase, fish were randomly distributed in 18 concrete square tanks (2.0 × 1.0 × 1.2 m³; 2 m³ of volume) according to the experimental SD. At the first phase, both fish species were subjected to three SD including 1.5, 3 and 6 kg/m² for 60 days. In the second phase, fish were submitted to three SD including 3, 6 and 9 kg/m², whereas in the third phase, beluga were stocked at 6, 9 and 12 kg/m² and ship sturgeon were stocked at 6, 8 and 10 kg/m² and each phase lasted for 12 weeks. In both species, the growth performance and feed intake (FI) significantly decreased with increasing SD at the end of the first phase (p < 0.05). In the second phase, growth performance and FI in beluga decreased with increasing SD, whereas in ship sturgeon neither growth nor FI were affected by SD. Interestingly, the growth performance and feed utilization in both species were not decreased with increasing SD in the third phase indicating adaptability of these species to high stocking density during grow‐out period. According to the result of this study, the appropriate initial SD for beluga within the range of 100–500, 500–2,000 and 1,500–3,000 g were at 1.5, 6 and 12 kg/m², respectively in an open flow‐throw system. Regarding to ship sturgeon, initial SD at 1.5 and 10 kg/m²recommended for fish within the range of 100–300 and 300–1,200 g, respectively in an open flow‐throw system.     

Kinase requirement for retinal growth cone motility

Since cytoplasmic Ca²⁺ levels are reported to regulate neurite elongation, we tested whether calcium-activated kinases might be necessary for growth cone motility and neurite elongation in explant cultures of goldfish retina. Kinase inhibitors and activators were locally applied by micropipette to retinal growth cones and the responses were observed via phase-contrast videomicroscopy. In some cases, growth rates were also quantifed over several hours after general application in the medium. The selective inhibitors of protein kinase C, calphostin C (0.1–1 μM) and chelerythrin (up to 50 μM), caused no obvious changes in growth cones or neurite elongation, and activators of PKC (phorbols, arachidonic acid, and diacylglycerol) also were generally without effects, although phorbols slowed the growth rate. Inhibitors of protein kinase A and tyrosine kinases also produced no obvious effects. The calmodulin antagonists, calmidazolium (0.1 μM), trifluoperazine (100 μM), and CGS9343B (50 μM), however, caused a reversible growth cone arrest with loss of filopodia and lamellipodia. The growth cone became a club-shaped swelling which sometimes moved a short distance back the shaft, leaving evacuated filaments at points of strong filopodial attachments. A similar reversible growth cone arrest occurred with the general kinase inhibitors: H7 at 200 but not at 100 μM, and staurosporine at 100 but not 10 nM, suggesting possible involvement of a calmodulin-dependent kinase (camK) rather than PKC. The selective inhibitor of camKII, KN-62 (tested up to 50 μM), produced no effects but the specific myosin light-chain kinase (MLCK) inhibitors ML-7 (3–5 μM) and ML-9 (5–10 μM) reversibly reproduced the effect, suggesting that MLCK rather than camKII is necessary for growth cone motility. The MLCK inhibitors' effects both on growth cone morphology and on F-actin filaments (rhodamine-phalloidin staining) were similar to those caused by cytochalasin D (5 μM), and are discussed in light of findings that inhibiting MLCK disrupts actin filaments in astrocytes and fibroblasts. 1994 John Wiley & Sons, Inc.     

Effects of Exploitation on Black Bear Populations at White River National Wildlife Refuge

Abstract: We live-trapped American black bears (Ursus americanus) and sampled DNA from hair at White River National Wildlife Refuge, Arkansas, USA, to estimate annual population size (N), growth (γ), and density. We estimated N and γ with open population models, based on live-trapping data collected from 1998 through 2006, and robust design models for genotyped hair samples collected from 2004 through 2007. Population growth was weakly negative (i.e., 95% CI included 1.0) for males (0.901, 95% CI = 0.645–1.156) and strongly negative (i.e., 95% CI excluded 1.0) for females (0.846, 95% CI = 0.711–0.981), based on live-trapping data, with N from 1999 to 2006 ranging from 94.1 (95% CI = 70.3–137.1) to 45.2 (95% CI = 27.1–109.3), respectively, for males and from 151.4 (95% CI = 127.6–185.8) to 47.1 (95% CI = 24.4–140.4), respectively, for females. Likewise, mean annual γ based on hair-sampling data was weakly negative for males (0.742, 95% CI = 0.043–1.441) and strongly negative for females (0.782, 95% CI = 0.661–0.903), with abundance estimates from 2004 to 2007 ranging from 29.1 (95% CI = 21.2–65.8) to 11.9 (95% CI = 11.0–26.9), respectively, for males and from 54.4 (95% CI = 44.3–77.1) to 27.4 (95% CI =24.9–36.6), respectively, for females. We attribute the decline in the number of females in this isolated population to a decrease in survival caused by a past translocation program and by hunting adjacent to the refuge. We suggest that managers restructure the quota-based harvest limits until these growth rates recover.     

Development of sensitive high-performance liquid chromatography with fluorescence detection using 4-(4,5-diphenyl-1H-imidazol-2-yl)benzoyl chloride as a labeling reagent for determination of bisphenol A in plasma samples

A sensitive HPLC method for determination of bisphenol A (BPA) in plasma samples using 4-(4,5-diphenyl-1H-imidazol-2-yl)benzoyl chloride (DIB-Cl) as a fluorescence labeling reagent was developed. The fluorescence labeling reaction was completed within 10 min at room temperature. DIB-Cl reacts with the phenolic hydroxyl group of BPA in the presence of triethylamine (TEA). The DIB-Cl derivative of BPA (DIB-BPA) was separated within 30 min with an ODS column using acetonitrile–water (90:10, v/v) as the isocratic eluent. Calibration graphs were linear over the range of 1.0–100 ng/ml (r=0.999). The detection limit of DIB-BPA was 0.05 ng/ml (2.5 pg) at a signal-to-noise ratio of 3. The relative standard deviations (RSDs) of the method for between-run were 1.0–5.0%. The analytical recoveries of known amounts (1.0 and 100 ng/ml) of BPA-spiked rabbit plasma were around 95%.     

PROBABILITY OF DIVISION OF CELLS IN THE EPIDERMIS OF THE PHLEUM ROOT

Erickson , Ralph O. (U. Pennsylvania, Philadelphia.) Probability of division of cells in the epidermis of the Phleum root. Amer. Jour. Bot. 48(3): 268–274. Illus. 1961.—Photomicrographic records of the growth of a Phleum root, made at R. H. Goodwin's laboratory, in which individual epidermal cells can be identified, have been analyzed to provide estimates of the probability that cells at various distances from the apex will divide. In the apical part of the meristem, from 0μ to about 100μ from the apex, all cells divide (prob. = 1.0). From about 100μ to 275μ, the probability of division falls progressively to 0.0. The relationship of these estimates to rates of cell division and elongation in the same root is discussed.     

The Effect of Methyl Glyoxal on Growth and Cell Division of Chlamydomonas reinhardii

Methyl glyoxal, at concentrations of 1.0–2.0 mM, inhibits growth of the green alga, Chlamydomonas reinhardii. The photosynthetic assimilation of carbon dioxide is also inhibited by the glyoxal. At the lower concentrations (less than 1.0–1.5 mM) protein synthesis Is inhibited, whereas polysaccharide synthesis and assimilation of carbon dioxide into the alcohol–soluble fraction is stimulated; at higher concentrations fl.5–2.5 mM) these latter two processes are also inhibited. Cell division in synchonized cultures of the alga is more sensitive to methyl glyoxal when it is added at the start of the growth cycle than when added late in the growth cycle. However, when added late in the growth cycle, methyl glyoxal delays the onset of cell division by 2 hours. No such delay occurs when cycloheximide is added 4–6 hours before division.     

SEASONAL CHANGE IN THE DISTRIBUTION OF CELL SIZE OF COCCONEIS SCUTELLUM VAR. ORNATA (BACILLARIOPHYCEAE) IN RELATION TO GROWTH AND SEXUAL REPRODUCTION1

Growth and sexual reproduction of the marine littoral diatom Cocconeis scutellum Ehrenb. var. ornata Grun. were investigated at 30 different combinations of temperature (5, 10, 14, 18, 22° C), irradiance (20, 60, 100 μE·m^?2·s^?1) and daylength (14:10 and 10:14 h LD cycle). Growth occurred at all combinations. The optimal growth was observed at 14–18° C, long daylength and highest-to-moderate irradiance, and at 18° C, short daylength and highest irradiance. Sexual reproduction on the other hand occurred between 5 and 18° C, and the optimal condition was 10–14° C and short daylength. Annual cyclic, and sesonal changes in the distribution of cell size (valve length) were observed in a field population. These changes were characterized by an annual minimum in mean cell size in autumn, an annual maximum in winter, a slight decrease from the mean in spring–middle summer, a rapid decrease from the mean in late summer–early autumn, and appearance of bimodal distribution of cell size in winter. These changes were caused by sexual reproduction in autumn, rapid growth in late summer–early autumn and slow growth in other seasons, and poor viability of small cells near the lower end of the size range.     

The Nutrition of the Freshwater Dinoflagellate Ceratium hirundinella*

A defined medium was devised for a freshwater isolate of the dinoflagellate Ceratium hirundinella. Highest cell yields were produced at 7,700–10,000 lux. The optimum pH range was between 7.0 and 7.5: the optimum temperature 21°C. Ceralium hirundinella tolerated a wide range (per liter) of Ca (0.1–100 mg) and Mg (0.1–50 mg) ion concentrations. The optimum range for growth was 20–30 rns Ca and 10–30 mg Mg. Cells cultured in media lacking Ca often became teratological yet motilp and viable. Variations in the Ca:Mg ratio had little effect on cell yield if the sum of the concentrations of the 2 ions remained the same. Organic as well as inorganic sources of N and P were utilized. NH₄ sources became toxic at elevated levels (7 mg N liter^-1). Methionine was not used as N source. Cells could not be completely depleted of P, but concentrations ≤ 0.01 mg P liter^-1 resulted in poor growth. Vitamin B₁₂, but not thiamine or biotin, was required. Highest cell yields were at a PII-metals concentration of 30 ml liter^-1; a t 100-ml liter^-1 cell yield was very low. Additions (per liter) of Fe (0.5 mg) and Mo (0.1 mg) to the basal medium produced higher cell yields, but Cu (0.1 mg) and V (0.1 mg) inhibited growth.     

Flow‐injection determination of cysteine in pharmaceuticals based on luminol–persulphate chemiluminescence detection

A flow injection (FI) method is reported for the determination of l‐ cysteine, based on its enhancement on chemiluminescence (CL) emission of luminol oxidized by sodium persulphate in alkaline solution. The calibration graph was linear over the range 1.0 × 10^–9–5.0 × 10^–7 mol/L (r² = 0.9992), with relative standard deviations (RSDs) in the range 1.1–2.3% (n = 4). The limit of detection (3σ blank) was 5.0 × 10^–10 mol/L with a sample throughput of 120/h. The method was applied to pharmaceuticals and the results obtained were in reasonable agreement with the amount labelled. The proposed method was also applied to cysteine in synthetic amino acid mixtures. Calibration graphs of N‐acetylcysteine and glutathione over the range 1.0–50 × 10^–8 and 0.5–7.5 × 10^–7 mol/L were also established (r² = 0.998 and 0.9986) with RSDs in the range 1.0–2.0% (n = 4), and the limits of detection (3σ blank) were 5.0 × 10^–9 and 1.0 × 10^–8 mol/L, respectively. Copyright © 2008 John Wiley & Sons, Ltd.     

Growth of Salmonella on sprouting alfalfa seeds as affected by the inoculum size, native microbial load and Pseudomonas fluorescens 2-79

Aims: To investigate the growth of salmonellae on sprouting alfalfa seeds as affected by the inoculum size, microbial load and Pseudomonas fluorescens 2–79. Methods and Results: Alfalfa seeds pre‐inoculated with ≤10¹–10³ CFU g^?1 of salmonellae and with or without Ps. fluorescens 2–79 were sprouted in glass jars and the population of salmonellae were determined daily for up to 6 days. The population of salmonellae on germinating seeds reached the maximum 2–3 days after sprouting when total bacterial count reached the maximum (10⁹ CFU g^?1). The population of salmonellae on sprouting seeds not treated with Ps. fluorescens 2–79 showed a net increase of 3–4 log units. However, the population of salmonellae on alfalfa seeds treated with Ps. fluorescens 2–79 showed a net increase of only 1–2 log units. Disinfection of seeds with calcium hypochlorite enhanced the growth of salmonellae. Conclusions: Treatment of seeds with Ps. fluorescens 2–79 reduced the growth of salmonellae by 2–3 log units. Significance and Impact of the Study: The potential of Ps. fluorescens 2–79 as a biological agent for use in control of salmonellae on sprouting seeds was demonstrated and warrants further investigation.     

Biosynthesis of amino acids from hydrocarbons

Optimum conditions for the growth ofPseudomonas arvilla, a hydrocarbon utiliser, have been studied. The microorganism produced economic cell yield at pH 5.7 and 4% kerosene concentration. C₁₀-C₁₆ hydrocarbons were utilised by the strain. The growth was maximum on n-decane. Supplementation of the hydrocarbon medium with 0.5% glucose stimulated the growth. Glutamic acid 16.0 mg; leucine 9.0 mg; valine 10.0 mg; methionine 2.5 mg; arginine 2.5 mg; histidine 1.0 mg were present in 100 ml of the broth. Cell protein contained leucine 13.69%, isoleucine 4.9%, histidine 4.37%, tryptophan 2.33%, methionine 1.8% and arginine 2.70%.     

Effects of recombinant activin A on in vitro culture of mouse preantral follicles

Activins are part of the intragonadal factors that can modulate the actions of gonadotropins and regulate cellular functions during preantral or early antral stages of folliculogenesis in vivo. In a mouse early preantral follicle culture system, activin A production was measured and recombinant bovine activin A (r-ACT A) was added (10 or 50 ng/ml) to recombinant follicle-stimulating hormone (r-FSH)–supplemented (10 or 100 mIU/ml) medium for a 12-day culture period. Specificity of activin A action was ascertained by addition of recombinant human follistatin (r-FS; 20 or 100 ng/ml). Immunoreactive activin A concentrations in mouse follicle–conditioned medium increased by a factor of 20–50, reaching concentrations from 2 to 5 ng/ml at end of culture. In the initial days of culture, additions of r-ACT A to r-FSH-supplemented medium provoked a dramatic volumetric increase and earlier attachment of the follicle. A dose of 100 ng/ml r-FS was able to block the actions of 10 ng/ml but not those caused by 50 ng/ml r-ACT A. In follicle cultures supplemented with 10 mIU/ml r-FSH, additions of r-ACT induced a dose-dependent inhibin (INH) and estradiol (E₂) increase. Basal and human chorionic gonadotropin (HCG)–induced progesterone (P) production were not influenced by r-ACT A or r-FS additions. Addition of r-ACT A decreased (P = 0.017) the intact follicle survival rate and had no influence on final oocyte diameter. In cultures supplemented by 10 mIU/ml r-FSH, additions of r-ACT A did not influence progression and resumption of meiosis I. Use of a higher r-FSH supplementation dose (100 mIU/ml) tended to affect meiosis I adversely (P = 0.052), and r-ACT A addition amplified this effect significantly (P = 0.007). These in vitro experiments demonstrate pronounced effects from r-ACT on r-FSH-mediated follicle survival, growth, and estrogen biosynthesis. Mol. Reprod. Dev. 50:294–304, 1998. © 1998 Wiley-Liss, Inc.