High Osmotic Solution Research Articles

Pretreatment of ovaries with collagenase before vitrification keeps the ovarian reserve by maintaining cell-cell adhesion integrity in ovarian follicles

The mammalian ovarian follicle is comprised of the germ cell or oocyte surrounded by the somatic cells, the granulosa and theca cells. The ovarian stroma, including the collagen-rich matrix that supports the three-dimensional disk-like follicular structure, impacts the integrity of the ovarian follicle and is essential for follicular development. Maintaining follicular integrity during cryopreservation has remained a limiting factor in preserving ovarian tissues for transplantation because a significant proportion of developed follicles in the frozen-thawed ovaries undergo atresia after transplantation. In this study, we show for the first time that during vitrification of the mouse ovary, the attachment of the oocyte to the granulosa cells was impaired by the loss of the cadherin adhesion molecules. Importantly, exposure to a high osmotic solution greatly decreased the ratio of oocyte diameter to the diameter of its follicle but did not alter the collagen-rich matrix surrounding the follicles. By treating ovaries briefly with collagenase before exposure to the hyper-osmotic solution the ratio of oocyte diameter to follicle diameter was maintained, and cadherin adhesion junctions were preserved. When frozen-thawed ovaries were transplanted to the bursa of recipient hosts, pretreatment with collagenase significantly increased serum levels of AMH, the number of intact follicles and the total number of viable offspring compared to frozen-thawed ovaries without collagenase pretreatment, even 6 months after transplantation. Thus, the collagenase pretreatment could provide a beneficial approach for maintaining the functions and viability of cryopreserved ovaries in other species and clinically relevant situations.

Membrane cut‐off does not influence results regarding the measurement of small molecules – a comparative study between 20‐ and 100‐kDa catheters in hepatic microdialysis

Microdialysis is a method used to monitor hepatic tissue metabolism. Membranes with a molecular cut-off of 20 kilodalton (kDa) are currently used to measure the small metabolites glucose, glycerol, lactate and pyruvate. Using membranes with higher cut-off such as 100 kDa allows the possibility of measuring larger molecules but may affect results regarding small molecules. The aim was to compare microdialysis catheters with a cut-off of 20 and 100 kDa in the measurement of small molecules in a pig liver model. Four microdialysis catheters were inserted into the liver of each pig used in the experiment (n = 6). Two catheters with cut-off of 20 kDa were perfused with Ringer acetate, and two catheters with cut-off of 100 kDa: one perfused with Ringer acetate and one with hydroxyethyl starch (Voluven) at a flow rate of 0·3 μl min(-1). Dialysate samples were collected at 40-min intervals and analysed for glucose, glycerol, lactate and pyruvate. Compared to the other catheters, the 100-kDa catheters perfused with Ringer acetate tended to measure higher dialysate concentrations of the small molecules, the difference reaching statistical significance in the case of pyruvate. Concentrations measured by the 100-kDa catheters perfused with Voluven were, however, comparable to the 20-kDa catheters. Microdialysis catheters with membrane cut-off of 20 and 100 kDa can be used equally in hepatic microdialysis for the monitoring of glucose, glycerol, lactate and pyruvate, and lactate/pyruvate ratio if a high osmotic solution (Voluven) is used to perfuse the 100-kDa catheters.

Partial purification of human parathyroid hormone 1-84 as a thioredoxin fusion form in recombinant Escherichia coli by thermoosmotic shock

A modified purification method, thermoosmotic shock (osmotic shock coupled with heat-treatment) for heat-stable proteins, was devised in the purification of Trx-hPTH (1-84) (human parathyroid hormone coupled with thioredoxin as a fusion partner) from E. coli. Thermoosmotic shock can integrate the functions of extraction and crude separation of fusion protein Trx-hPTH (1-84). To improve the purification efficiency, thermoosmotic shock conditions were optimized and achieved as follows: the optimized high osmotic solution containing 20 mM Tris–HCl buffer (pH 8.0), 1 mM EDTA, and 25% sucrose; the low osmotic solution containing 20 mM Tris–HCl buffer (pH 8.0), 1 mM EDTA, and the heat-treatment temperature of 100 °C for 10 min. Using this method, the purity of Trx-hPTH (1-84) was up to 73% and the yield was up to 72%, respectively. In addition, the two separation methods of both thermoosmotic shock and affinity chromatography have been compared, indicating that thermoosmotic shock is an economical and feasible way for the fusion protein separation. Besides, the thermoosmotic shock method may be used for the purification of some proteins of thermal stability without N-terminal His-tag.

In vivo imaging of C. elegans ASH neurons: cellular response and adaptation to chemical repellents.

ASH sensory neurons are required in Caenorhabditis elegans for a wide range of avoidance behaviors in response to chemical repellents, high osmotic solutions and nose touch. The ASH neurons are therefore hypothesized to be polymodal nociceptive neurons. To understand the nature of polymodal sensory response and adaptation at the cellular level, we expressed the calcium indicator protein cameleon in ASH and analyzed intracellular Ca(2+) responses following stimulation with chemical repellents, osmotic shock and nose touch. We found that a variety of noxious stimuli evoked strong responses in ASH including quinine, denatonium, detergents, heavy metals, both hyper- and hypo-osmotic shock and nose touch. We observed that repeated chemical stimulation led to a reversible reduction in the magnitude of the sensory response, indicating that adaptation occurs within the ASH sensory neuron. A key component of ASH adaptation is GPC-1, a G-protein gamma-subunit expressed specifically in chemosensory neurons. We hypothesize that G-protein gamma-subunit heterogeneity provides a mechanism for repellent-specific adaptation, which could facilitate discrimination of a variety of repellents by these polymodal sensory neurons.

Heat shock treatment protects osmotic stress–induced dysfunction of the blood-brain barrier through preservation of tight junction proteins

The blood-brain barrier (BBB) is a specialized structure in the central nervous system (CNS), which participates in maintenance of a state of cerebrospinal fluid homeostasis. The endothelial cells of the cerebral capillaries and the tight junctions between them form the basis of the BBB. Research has shown that destruction of the BBB is associated with diseases of the CNS. However, there is little research on how the BBB might be protected. In this study, we used a high osmotic solution (1.6 M D-mannitol) to open the BBB of rats and Evans blue dye as a macromolecular marker. The effect of heat shock treatment was evaluated. The results show that increased synthesis of heat shock protein 72 (Hsp72) was induced in the heated group only. BBB permeability was significantly less in the heat shock-treated group after hyperosmotic shock. The major tight junction proteins, occludin and zonula occludens (ZO)-1, were significantly decreased after D-mannitol treatment in the nonheated group, whereas they were preserved in the heated group. The coimmunoprecipitation studies demonstrated that Hsp72 could be detected in the precipitates of brain extract interacting with anti-ZO-1 antibodies as well as those interacting with anti-occludin antibodies in the heated group. We conclude that the integrity of tight junctions could be maintained by previous heat shock treatment, which might be associated with the increased production of Hsp72.

Electrochemical estimation of protoplast population

The protoplast concentration of Bacillus licheniformis FDO150 was estimated with a combined electrode composed of a platinum anode, a platinum cathode and a saturated calomel electrode (S.C.E.). The anode was adjusted at +0.4 V vs. S.C.E. When the electrode was inserted in a protoplast suspension, the anodic current increased slightly. Addition of dichlorophenol-indophenol (DCIP) caused considerable increase in the output current. When protoplasts (0.5 mg ml −1 wet cells) were suspended in a high osmotic solution, the rate of current increase was 0.8 μA min −1. On the other hand, no anodic current was obtained when protoplasts were suspended in a low osmotic pressure solution. However, in the case of whole cells of B. licheniformis, an increase of anodic current was observed in a low as well as a high osmotic solution. The difference of the rates of current increase obtained both in a high and low osmotic solutions was correlated with protoplast concentration in the range from 10 −2 to 10 −5 unit, where 1 unit is defined as the frequency of osmotically sensitive cells.