Changes in extracellular osmolality initiate sperm motility in freshwater teleost rosy barb Puntius conchonius

The objective was to investigate the effects of extracellular osmolality and membrane osmotic-sensitive channels on the initiation of sperm motility and to explore mechanisms of sperm initiation in rosy barb (Puntius conchonius). We found that (1) sperm were immotile in seminal plasma and remained quiescent in electrolyte or nonelectrolyte solutions isotonic to seminal plasma; (2) sperm movement was initiated when the sperm were exposed to hypo-osmotic electrolyte or hypo-osmotic nonelectrolyte solutions, and that the responsiveness of sperm to changes in the extracellular osmolalities (100, 200, 250, 270, and 300 mOsm/kg) differed among sperm cells (P < 0.05); (3) sperm movement could be initiated and terminated repeatedly by decreasing and increasing the osmolality (in increments of 100 and 300 mOsm/kg) of a nonelectrolyte mannitol solution, respectively (P < 0.05); (4) gadolinium (20, 40, and 80 μM) inhibited the initiation of sperm motility and abolished the sperm activation caused by the hypo-osmotic media treatment in dose- and time-dependent manners (P < 0.05); and (5) sperm activation in a hypo-osmotic medium and inhibition in an isotonic solution were associated with swelling and shrinkage of the sperm sleeves, respectively. Therefore, we concluded that osmolality was a critical physiologic signal in regulating the initiation and termination of sperm motility in freshwater teleost rosy barb. Furthermore, we inferred that rosy barb sperm were hypo-osmotic–dependent conformers, and the osmotic-sensitive channel could be involved in the mechanism of sperm initiation.     

Survival and Growth of Tetrahymena thermophila in Media that are Conventionally Used for Piscine and Mammalian Cells

The transfer of Tetrahymena thermophila from normosmotic solutions (~20–80 mOsm/kg H₂O) to hyperosmotic solutions (> 290 mOsm/kg H₂O) was investigated. During the first 24 h of transfer from proteose peptone yeast extract (PPYE) to either 10 mM HEPES or PPYE with added NaCl to give ~300 mOsm/kg H₂O, most ciliates died in HEPES but survived in PPYE. Supplementing hyperosmotic HEPES or PPYE with fetal bovine serum (FBS) enhanced survival. When ciliates were transferred from PPYE to a basal medium for vertebrate cells, L‐15 (~320 mOsm/kg H₂O), only a few survived the first 24 h but many survived when the starting cell density at transfer was high (100,000 cells/ml) or FBS was present. These results suggest that nutrients and/or osmolytes in either PPYE or FBS helped ciliates survive the switch to hyperosmotic solutions. FBS also stimulated T. thermophila growth in normosmotic HEPES and PPYE and in hyperosmotic L‐15. In L‐15 with 10% FBS, the ciliates proliferated for several months and could undergo phagocytosis and bacterivory. These cell culture systems and results can be used to explore how some Tetrahymena species function in hyperosmotic hosts and act as opportunistic pathogens of vertebrates.     

A note on the hyperosmotic regulation in the brackish-water clamRangia cuneata

Summary The brackish-water clamRangia cuneata maintains the body fluids hyperosmotic to diluted sea-water of osmolalities less than 200–300 mOsm (Fig. 1). Analyses of pericardial fluid pH, protein concentration and chloride concentration (Table 2, Figs. 3, 2) negate the participation of a Gibbs-Donnan effect in the establishment of the hyperosmolality. The protein concentration of the body fluids decreases with decreasing water osmolality from a mean of 1.3 mg/ml at 416 mOsm to a mean 0.3 mg/ml at 27 mOsm.     

Feasibility study on the use of hyperosmolar medium for improved antibody production of hybridoma cells in a long-term,repeated-fed batch culture

To test the feasibility of using hyperosmolar medium for improved antibody production in a long-term, repeated fed-batch culture, the influence of various culture conditions (serum concentration and cultivation method) on the hybridoma cells' response to hyperosmotic stress resulting from sodium chloride addition was first investigated in a batch culture. The degree of cell growth depression resulting from hyperosmotic stress was dependent on serum concentrations and cultivation methods (static and agitated cultures). Depression of cell growth was most significant in agitated cultures with low serum concentration. However, regardless of serum concentrations and cultivation methods used, the hyperosmotic stress significantly increased specific antibody productivity (q _MAb). Increasing osmolality from 284 to 396 mOsm kg^–1 enhanced the q_MAb in agitated cultures with 1% serum by approximately 124% while the similar osmotic stress enhanced the q _MAb in static cultures with 10% serum by approximately 153%. Next, to determine whether this enhanced q_MAb resulting from hyperosmotic stress can be maintained after adaptation, long-term, repeated-fed batch cultures with hyperosmolar media were carried out. The cells appeared to adapt to hyperosmotic stress. When a hyperosmolar medium (10% serum, 403 mOsmkg^–1) was used, the specific growth rate improved gradually for the first four batches and thereafter, remained constant at 0.040±0.003 (average ± standard deviation) hr^–1 which is close to the value obtained from a standard medium (10% serum, 284 mOsmkg^–1) in the batch culture. While the cells were adpating to hyperosmotic stress, the q_MAb was gradually decreased from 0.388×10^–6 to 0.265×10^–6 g cell hr^–1 and thereafter, remained almost constant at 0.272±0.014× 10^–6 g cell^–1 hr^–1. However, this reduced q _MAb after adaptation is still approximately 98% higher than the q_MAb obtained from a standard medium in the batch culture.The authors would like to thank Dr.M. Kaminski for providing the hybridoma cell line used in this study. This work was supported by the Korea Science and Engineering Foundation.     

Effect of hypoosmotic stress on hybridoma cell growth and antibody production

To investigate the response of hybridoma cells to hypoosmotic stress, S3H5/gamma2bA2 and DB9G8 hybridomas were cultivated in the hypoosmolar medium [Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% serum] resulting from sodium chloride subtraction. Both hybridomas showed similar responses to hypoosmotic stress in regard to cell growth and antibody production. The cell growth and antibody production at 276 mOsm/kg were comparable to those at 329 mOsm/kg (standard DMEM). Both cells grew well at 219 mOsm/kg, though their growth and antibody production were slightly decreased. When the osmolality was further decreased to 168 mOsm/kg, the cell growth did not occur. When subjected to hyperosmotic stress, both cells displayed significantly enhanced specific antibody productivity (q(Ab)). However, the cells subjected to hypoosmotic stress did not display enhanced q(Ab). Taken together, both hyperosmotic and hypoosmotic stresses depressed the growth of S3H5/gamma2bA2 and DB9G8 hybridomas. However, their response to hypoosmotic stress in regard to q(Ab) was different from that to hyperosmotic stress. (c) 1997 John Wiley & Sons, Inc. Biotechnol Biong 55: 565-570, 1997.     

Effect of sorbitol addition on the physicochemical characteristics of starch–fatty acid systems

Aqueous maize starch dispersions (20%) were heated at 100 °C, in the presence of myristic, palmitic or stearic acid potassium salts as well as of sorbitol added at concentrations up to 60% (dry starch). Flow behaviour measurements at 100 °C indicated that interactions took place between the starch–fatty acid systems and sorbitol resulting in viscosity increase which was more pronounced as the sorbitol content increased. Water solubility measurements showed that a major part of sorbitol was easily extracted by excess water whereas sorption experiments revealed that the moisture uptake rate was proportional to sorbitol content of the starch systems examined. Thermomechanical studies indicated that the starch–fatty acid samples containing sorbitol up to 40% exhibited antiplasticizing behaviour. Scanning electron microscopy studies revealed that at sorbitol concentrations over 30%, free sorbitol crystals were formed on the surface of starch–fatty acid samples, whereas the percentage crystallinity as well as the crystallite size of samples were proportional to sorbitol content.     

Osmotic Challenge Drives Rapid and Reversible Chromatin Condensation in Chondrocytes

Changes in extracellular osmolality have been shown to alter gene expression patterns and metabolic activity of various cell types, including chondrocytes. However, mechanisms by which physiological or pathological changes in osmolality impact chondrocyte function remain unclear. Here we use quantitative image analysis, electron microscopy, and a DNase I assay to show that hyperosmotic conditions (>400 mOsm/kg) induce chromatin condensation, while hypoosmotic conditions (100 mOsm/kg) cause decondensation. Large density changes (p < 0.001) occur over a very narrow range of physiological osmolalities, which suggests that chondrocytes likely experience chromatin condensation and decondensation during a daily loading cycle. The effect of changes in osmolality on nuclear morphology (p < 0.01) and chromatin condensation (p < 0.001) also differed between chondrocytes in monolayer culture and three-dimensional agarose, suggesting a role for cell adhesion. The relationship between condensation and osmolality was accurately modeled by a polymer gel model which, along with the rapid nature of the chromatin condensation (<20 s), reveals the basic physicochemical nature of the process. Alterations in chromatin structure are expected to influence gene expression and thereby regulate chondrocyte activity in response to osmotic changes.     

Effects of anisosmotic medium on cell volume,transmembrane potential and intracellular K+ activity in mouse hepatocytes

Summary Mouse hepatocytes in primary monolayer culture (4 hr) were exposed for 10 min at 37°C to anisosmotic medium of altered NaCl concentration. Hepatocytes maintained constant relative cell volume (experimental volume/control volume) as a function of external medium relative osmolality (control mOsm/experimental mOsm), ranging from 0.8 to 1.5. In contrast, the relative cell volume fit a predicted Boyle-Van't Hoff plot when the experiment was done at 4°C. Mouse liver slices were used for electrophysiologic studies, in which hepatocyte transmembrane potential (V _m ) and intracellular K⁺ activity (a _K ⁱ ) were recorded continuously by open-tip and liquid ion-exchanger ion-sensitive glass microelectrodes, respectively. Liver slices were superfused with control and then with anisosmotic medium of altered NaCl concentration.V _m increased (hyperpolarized) with hypoosmotic medium and decreased (depolarized) with hyperosmotic medium, and ln [10(experimentalV _m /controlV _m )] was a linear function of relative osmolality (control mOsm/experimental mOsm) in the range 0.8–1.5. Thea _K ⁱ did not change when medium osmolality was decreased 40–70 mOsm from control of 280 mOsm. Similar hypoosmotic stress in the presence of either 60mm K⁺ or 1mm quinine HCl or at 27°C resulted in no change inV _m compared with a 20-mV increase inV _m without the added agents or at 37°C. We conclude that mouse hepatocytes maintain their volume anda _K ⁱ in response to anisosmotic medium; however,V _m behaves as an osmometer under these conditions. Also, increases inV _m by hypoosmotic stress were abolished by conditions or agents that inhibit K⁺ conductance.     

Influence of hyperosmolar basal media on hybridoma cell growth and antibody production

To investigate the influence of hyperosmolar basal media on hybridoma response, S3H5/γ2bA2 and DB9G8 hybridomas were cultivated in a batch mode using hyperosmolar basal media resulting from additional sodium chloride supplementation. The basal media used in this study were IMDM, DMEM, and RPMI 1640, all of which are widely used for hybridoma cell culture. In IMDM, two hybridomas showed different responses to hyperosmotic stress regarding specific MAb productivity (q _MAb), though they showed similar depression of cell growth in hyperosmolar media. Unlike S3H5/γ2bA2 hybridoma, the q _MAb of DB9G8 hybridoma was not enhanced significantly around 390 mOsm kg^?1. The variation of basal media influenced DB9G8 hybridoma response to hyperosmotic stress regarding q _MAb. In IMDM, the q _MAb of DB9G8 hybridoma was increased by more than 200% when the osmolality increased from 281 to 440 mOsm/kg. However, in RPMI 1640 and DMEM, similar amplitude of osmolality increase resulted in less than 100% increase in q _MAb. The variation of basal media also influenced the cell growth in hyperosmolar medium. Both hybridomas were more tolerant against hyperosmotic stress in DMEM than in IMDM, which was found to be due to the high osmolality of standard DMEM. The osmolalities of standard IMDM and DMEM used for inocula preparation were 281 and 316 mOsm kg^?1, respectively. Thus, when the cells were cultivated at 440 mOsm kg^?1, the cells in IMDM experienced higher osmotic shock than in DMEM. By using the inoculum prepared at 317 mOsm kg^?1 in IMDM, S3H5/γ2bA2 cell growth at 440 mOsm kg^?1 in IMDM was comparable to that in DMEM. Taken together, the results obtained from this study show that the selection of basal media is an important factor for MAb production by employing hyperosmotic stress.     

Enhancement of monoclonal antibody production by immobilized hybridoma cell culture with hyperosmolar medium

To determine the effect of hyperosmotic stress on the monoclonal antibody (MAb) production by calcium-alginate-immobilized S3H5/gamma2bA2 hybridoma cells, the osmolalities of medium in the MAb production stage were varied through the addition of NaCI. The specific MAb productivity (q(MAb)) of immobilized cells exposed to abrupt hyperosmotic stress (398 mOsm/kg) was increased by 55% when compared with that of immobilized cells in the control culture (286 mOsm/kg). Furthermore, this enhancement of q(MAb) was not transient. Abrupt increase in osmolality, however, inhibited cell growth, resulting in no increase in volumetric MAb productivity (r(MAb)). On the other hand, gradual increase in osmolality allowed further cell growth while maintaining the enhanced q(MAb) immobilized cells. The q(MAb) immobilized cells at 395 mOsm/kg was 0.661 +/- 0.019 mug/10(6) cells/h, which is almost identical to that of immobilized cells exposed to abrupt osmotic stress. Accordingly, the r(MAb) was increased by ca. 40% when compared with that in the control immobilized cell culture. This enhancement in i(MAb) of immobilized S3H5/gamma2bA2 hybridoma cells by applying gradual osmotic stress suggests the potential of using hyperosmolar medium in other perfusion culture systems for improved MAb production. (c) 1995 John Wiley & Sons, Inc.     

Elver Invasion, Population Structure and Growth of Marbled eels Anguilla marmorata in a Tropical River on Réunion Island in the Indian Ocean

Winter skates, Leucoraja ocellata, exposed to 80% and 50% seawater (SW) exhibited rapid and significant weight gains followed by a slight recovery to new steady state levels within 8 days. Skates were acclimated at each salinity (100% SW [N = 16], 80% SW [N = 8], 50% SW [N = 8]), anesthetized (MS222) and bled from the caudal vein. In 100% SW, skate plasma (930mOsm/kg) was slightly hyperosmotic to the external medium (922mOsm/kg). Plasma osmolality decreased with seawater dilution, but became increasingly hyperosmotic to the bathing media. The environmental dilutions resulted in significant, but disproportionate changes in plasma Cl^–, P^–, Na⁺, Ca⁺, Mg⁺, trimethylamine oxide (TMAO) and urea concentrations. Mean corpuscular [Hb] and milliliter RBC water measurements suggest that skate red cells swelled less at each dilution than predicted for a passive erythrocyte osmometer. Concentrations of the major RBC solutes K⁺, urea, TMAO and Cl^– decreased by 8, 25, 5 and 21%, respectively in 80% SW. In 50% SW, K⁺, urea, TMAO and Cl^– concentrations decreased by 9, 47, 36 and 15%, respectively. Quantitatively, the other measured intracellular electrolytes (Mg⁺, Na⁺, P^– and Ca⁺) also exhibited disproportionate changes in concentration. Our results indicate that L. ocellata is a euryhaline elasmobranch that can tolerate significant reduction in the external salinity through the release of both ions and urea from the extracellular compartments while retaining electrolytes at the expense of urea in the intracellular compartment.     

Maintenance of fertility in cryopreserved Indian gerbil (Tatera indica) spermatozoa

This study is the first attempt at sperm cryopreservation, as well as a further examination of frozen sperm fertility by the hamster test, applied to the maintenance of an Indian gerbil (Tatera indica) colony, which is a newly developing experimental animal.The osmotic tolerance of the spermatozoa was initially investigated by subjection to hypertonicity, up to 620 mOsm/kg, for 5 min at room temperature prior to freezing. Although the percentage of total motile sperm was not affected, that of progressive motile spermatozoa began to drop at 400 mOsm/kg, and a significant decrease was observed at 620 mOsm/kg (p < 0.01). According to these results, the osmolality of the solutions for the freezing experiment, in which 6–22% raffinose was present, was fixed at approximately 400 mOsm/kg. Sperm, suspended in a plastic straw, were frozen in liquid nitrogen vapor for 5 min, followed by immersion in liquid nitrogen. Motile sperm were recovered from all freezing conditions, and high survival was obtained when sperm were frozen in the presence of 14% and 18% raffinose, with a normalized motility higher than 40%. Fertility of cryopreserved Indian gerbil sperm was examined by the zona-free hamster test. Thawed sperm adhered to 88% of the zona-free hamster oocyte surface, and some oocytes were penetrated and exhibited swollen sperm heads or male pronuclei, which we used to define fertilization. Although the fertilization rate of cryopreserved sperm to zona-free hamster eggs was significantly lower than that of fresh sperm (6% vs. 30%, p < 0.01), we demonstrated that thawed Indian gerbil spermatozoa have the ability to maintain their fertility.     

Hyperosmotic stress induces autophagy and apoptosis in recombinant Chinese hamster ovary cell culture

During recombinant Chinese hamster ovary (rCHO) cell culture, various events, such as feeding with concentrated nutrient solutions or the addition of base to maintain an optimal pH, increase the osmolality of the medium. To determine the effect of hyperosmotic stress on two types of programmed cell death (PCD), apoptosis and autophagy, of rCHO cells, two rCHO cell lines, producing antibody and erythropoietin, were subjected to hyperosmotic stress resulting from NaCl addition (310–610 mOsm/kg). For both rCHO cell lines, hyperosmolality up to 610 mOsm/kg increased cleaved forms of PARP, caspase‐3, caspase‐7, and fragmentation of chromosomal DNA, confirming the previous observation that apoptosis was induced by hyperosmotic stress. Concurrently, hyperosmolality increased the level of accumulation of LC3‐II, a widely used autophagic marker, which was determined by Western blot analysis and confocal microscopy. When glucose and glutamine concentrations were measured during the cultures, glucose and glutamine concentrations in the culture medium at various osmolalities (310–610 mOsm/kg) showed no significant differences. This result suggests that induction of PCD by hyperosmotic stress occurred independently of nutrient depletion. Taken together, autophagy as well as apoptosis was observed in rCHO cells subjected to hyperosmolality. Biotechnol. Bioeng. 2010;105: 1187–1192. © 2009 Wiley Periodicals, Inc.     

Effects of elevated pCO2 and osmolality on growth of CHO cells and production of antibody-fusion protein B1: a case study

Partial pressure of CO2 (pCO2) and osmolality as high as 150 mmHg and 440 mOsm/kg, respectively, were observed in large-scale CHO cell culture producing an antibody-fusion protein, B1. pCO2 and osmolality, when elevated to high levels in bioreactors, can adversely affect cell culture and recombinant protein production. To understand the sole impact of pCO2 or osmolality on CHO cell growth, experiments were performed in bench-scale bioreactors allowing one variable to change while controlling the other. Elevating pCO2 from 50 to 150 mmHg under controlled osmolality (about 350 mOsm/kg) resulted in a 9% reduction in specific cell growth rate. In contrast, increasing osmolality resulted in a linear reduction in specific cell growth rate (0.008 h(-1)/100 mOsm/kg) and led to a 60% decrease at 450 mOsm/kg as compared to the control at 316 mOsm/kg. This osmolality shift from 316 to 445 mOsm/kg resulted in an increase in specific production rates of lactate and ammonia by 43% and 48%, respectively. To elucidate the effect of high osmolality and/or pCO2 on the production phase, experiments were conducted in bench-scale bioreactors to more closely reflect the pCO2 and osmolality levels observed at large scale. Increasing osmolality to 400-450 mOsm/kg did not result in an obvious change in viable cell density and product titer. However, a further increase in osmolality to 460-500 mOsm/kg led to a 5% reduction in viable cell density and a 8% decrease in cell viability as compared to the control. Final titer was not affected as a result of an apparent increase in specific production rate under this increased osmolality. Furthermore, the combined effects from high pCO2 (140-160 mmHg) and osmolality (400-450 mOsm/kg) caused a 20% drop in viable cell density, a more prominent decrease as compared to elevated osmolality alone. Results obtained here illustrate the sole effect of high pCO2 (or osmolality) on CHO cell growth and demonstrate a distinct impact of high osmolality and/or pCO2 on production phase as compared to that on growth phase. These results are useful to understand the response of the CHO cells to elevated pCO2 (and/or osmolality) at a different stage of cultivation in bioreactors and thus are valuable in guiding bioreactor optimization toward improving protein production.     

Intracellular Delivery of Carbohydrates into Mammalian Cells through Swelling-activated Pathways

Volume changes of human T-lymphocytes (Jurkat line) exposed to hypotonic carbohydrate-substituted solutions of different composition and osmolality were studied by videomicroscopy. In 200 mOsm media the cells first swelled within 1–2 min and then underwent regulatory volume decrease (RVD) to their original isotonic volume within 10–15 min. RVD also occurred in strongly hypotonic 100 mOsm solutions of di- and trisaccharides (trehalose, sucrose, raffinose). In contrast to oligosaccharide media, 100 mOsm solutions of monomeric carbohydrates (glucose, galactose, inositol and sorbitol) inhibited RVD. The complex volumetric data were analyzed with a membrane transport model that allowed the estimation of the hydraulic conductivity and volume-dependent solute permeabilities. We found that under slightly hypotonic stress (200 mOsm) the cell membrane was impermeable to all carbohydrates studied here. Upon osmolality decrease to 100 mOsm, the membrane permeability to monomeric carbohydrates increased dramatically (apparently due to channel activation caused by extensive cell swelling), whereas oligosaccharide permeability remained very poor. The size-selectivity of the swelling-activated sugar permeation was confirmed by direct chromatographic measurements of intracellular sugars. The results of this study are of interest for biotechnology, where sugars and related compounds are increasingly being used as potential cryo- and lyoprotective agents for preservation of rare and valuable mammalian cells and tissues.This revised version was published online in June 2005 with a corrected cover date.     

Effects of different artificial motile activating media on sperms motility of Waigieu seaperch Psammoperca waigiensis throughout a reproductive season

We previously determined changes in sperm quality of Psammoperca waigiensis during its spawning season and the optimal cation concentrations and osmolality for sperm preservation of this species at the peak of the reproductive season. In this study, we went one important step further by assessing the effects of the most adequate medium, considering the dilution ratio, osmolality, and cations (Na⁺, K⁺, Mg²⁺, and Ca²⁺) on the motility of P. wasigiensis sperm collected during the early, peak, and late spawning season. We determined the maximum velocity (VAP), and percentage of sperm motility (MOT), and the duration of sperm motility (DSM). Under optimal dilution, temperature, pH and osmolarity, MOT, VAP, and DSM did not statistically differ during early, peak, and late spawning season. However, under suboptimal external conditions, MOT, VAP, and DSM showed inconsistent trends during different spawning periods. We recommend using one of three different artificial motile activating media: (1) 0.55 M Na⁺, (2) 0.6 M K⁺ or (3) 1200 mOsm/kg for early; or (1) 0.6 M Na⁺, (2) 0.6 M K⁺ or (3) 1100 mOsm/kg for the peak; and (1) 0.65 M Na⁺, (2) 0.55 M K⁺ or (3) 1200 mOsm/kg for late spawning season; all at the dilution of 1:150 (v:v of semen: artificial motile activating medium).     

Ions and osmolality on post‐thaw sperm motility activation of the endangered Brycon insignis (Characiformes)

The aim of this study was to evaluate the effects of osmolality and the presence of ions on the activation of post‐thaw sperm motility of Brycon insignis. Sperm was frozen under a standardized methodology for this species. In experiment 1, 11 solutions were prepared with reverse osmosis (RO) water (～0 mOsm/kg) and glucose or NaCl adjusted to an osmolality of 50, 100, 150, 200 and 250 mOsm/kg. In experiment 2, six solutions were prepared with RO and adjusted to ~100 mOsm/kg with one of the following chemicals: NaHCO₃, sodium citrate (Na₃C₆H₅O₇), NaCl, KCl, CaCl₂ or glucose (as ion‐free control). Post‐thaw sperm of both experiments was evaluated for motility rate, velocities (curvilinear = VCL, among others) and beat‐cross frequency (BCF). In experiment 1, sperm motility rate and velocities were higher (p < 0.05) when triggered in solutions at osmolalities from 0 to 200 mOsm/kg (62–80% motility; 139–167 µm/s) than that at 250 mOsm/kg (36–44% motility; 94–99 µm/s VCL). BCF was not affected by osmolality and varied from 19 to 24 Hz in all samples. In experiment 2, samples activated in NaHCO₃, citrate, NaCl and KCl solutions yielded higher motility rates (76–85%) and BCF (24–25 Hz) compared to those activated in CaCl₂ (50%; 14 Hz). Samples activated in ion‐free control solution yielded higher motility rate (87%) than those activated in NaHCO₃ and in CaCl₂. Curvilinear velocity was higher in samples activated in NaHCO₃, citrate, KCl and control solutions (144–160 µm/s) than in those activated in CaCl₂ (104 µm/s); samples activated in NaCl yielded intermediate VCL values (127 µm/s). Post‐thaw sperm achieves maximum motility rate and velocities when activated in solutions composed of sodium citrate, NaCl, KCl or glucose. Thus, post‐thaw sperm motility of B. insignis can be triggered in ionic and non‐ionic solutions at osmolality between 0 and 200 mOsm/kg. The use of solutions containing calcium, however, should be avoided.     

Hyperosmotic stress and elevated pCO2 alter monoclonal antibody charge distribution and monosaccharide content

Medium osmolality increases with pCO2 at constant pH. Elevated pCO2 and osmolality inhibit hybridoma growth to similar extents in both serum-containing and serum-free media. The combination of osmolality and elevated pCO2 synergizes to negatively impact cell growth. IgG2a glycosylation by hybridoma cells was evaluated under elevated pCO2 (to 250 mmHg pCO2) and/or osmolality (to 476 mOsm/kg). IgG2a site occupancy did not change significantly under any of the conditions studied, which is consistent with the robust glycosylation of other antibodies produced under various environmental stresses. However, changes were observed in the IgG2a charge distribution. Changes in the isoelectric point (pI) were greater under hyperosmotic stress, increasing by 0.32 and 0.41 pH units at 435 mOsm/kg in serum-containing and serum-free medium, respectively. Hyperosmotic stress also resulted in a concomitant increase in the heterogeneity of the charge distribution. The mean pI in serum-containing medium decreased by 0.16 pH units at 250 mmHg pCO2 when osmolality was controlled at 320 mOsm/kg but increased by 0.20 pH units when the osmolality increased with pCO2 (195 mmHg pCO2-435 mOsm/kg). In serum-free medium, elevated pCO2 did not alter pI, regardless of medium osmolality. In contrast to elevated osmolality at control pCO2, elevated pCO2 did not significantly alter the IgG2a charge heterogeneity under any of the conditions studied. The IgG2a was not sialylated, so sialylation changes were not responsible for changes in the charge distribution. IgG2a galactose content decreased with elevated osmolality, as a result of either elevated NaHCO3 or NaCl. However, when osmolality was controlled at elevated pCO2, the galactose content tended to increase. The mannose content decreased with increasing stress, while the fucose content remained relatively unchanged. It is likely that the observed increases in the pI of murine IgG2a were due to increased organellar pH, which is reflected by increased specific beta-galactosidase activity in the supernatant.