Critical tonicity determination of sperm using fluorescent staining and flow cytometry

The use of cryopreserved, rather than fresh, mammalian semen for artificial insemination confers several important medical and/or economic advantages. However, current methods for cryopreservation of both human and bovine spermatozoa result in approximately only a 50% survival rate with thawing, obviously reducing the fertilizing capacity of the semen. A primary consideration during the cooling process is to avoid intracellular ice crystal formation with its lethal consequences to the cell. Current techniques achieve this by controlling the cooling rate. Computation of the time necessary for this dehydration, and hence, the cooling rate, is dependent upon knowledge of the water permeability coefficient (L{sub {rho}}) and its activation energy. The fluorophore, 6-carboxyfluoroscein diacetate (CFDA), which is nonfluorescent, readily crosses the intact plasma membrane. Intracellular esterases hydrolyze CFDA to 6-carboxyfluoroscein, a fluorescent, membrane-impermeable fluorophore. Consequently, spermatozoa with intact plasma membranes fluoresce bright green (Garner et. al., 1986), but those with disrupted membranes do not. Therefore, the purpose of this study was to use loss of CFDA fluorescence to determine the osmolality at which 50% of the spermatozoa will swell and lyse (critical tonicity, CT). These data will then be used to determine the L{sub {rho}} and its activation energy for sperm, thus increasing the knowledge available in cellular cryopreservation. 15 refs., 3 figs

Determination of bull sperm membrane permeability to water and cryoprotectants using a concentration-dependent self-quenching fluorophore

The objective of this study was to determine the membrane permeability characteristics of bovine spermatozoa. These included the hydraulic conductivity (L-p), the permeability coefficients (P-s) of four common cryoprotective agents (CPAs) and the associated reflection coefficients (sigma). Stopped-flow fluorometry was applied in order to capture rapid cell volume changes under anisosmotic conditions in the absence or presence of permeant solutes (CPAs). This technique utilizes a concentration-dependent self-quenching entrapped fluorophore. The resulting cell volume changes were used in three-parameter fitting calculations to compute L-p in the absence of permeant solutes and P-s and L-p in the presence of permeating solutes (CPAs) at 22degreesC. The hydraulic conductivity in the absence of permeating solutes was estimated to be 0.69+/-0.05 mum/min/atm (mean+/-SEM). Hydraulic conductivity (L-p) in the presence of CPAs was 0.91+/-0.27 (mean+/-SEM), 0.29+/-0.04, 0.42+/-0.05, and 0.39+/-0.03 mum/min/atm in the presence of dimethylsulfoxide (Me2SO), glycerol, propylene glycol (PG), and ethylene glycol (EG), respectively. The values for P-s were estimated to be 1.72+/-0.36 1.75+/-0.03, 2.47+/-0.24, and 1.49+/-0.3x10(-3) cm/min for Me2SO, glycerol, PG, and EG, respectively. The data were used to simulate volume excursions during addition and removal of CPA, to predict the different effects of the four CPAs. (C) 2004 Elsevier Inc. All rights reserved

Significant variability among bulls in the sperm membrane permeability for water and glycerol; possible implications for semen freezing protocols for individual males.

The aim of this study was to test the hypothesis that bulls have significant intra-individual differences in the hydraulic conductivity (Lp) and permeability coefficient for glycerol (Ps) of the sperm cell membrane. The permeability parameters were determined at 22, 10, and 0 °C of sperm from 7 Holstein Frisian artificial insemination (AI) bulls, using four ejaculates per bull. A stopped-flow approach was applied to provide temporal resolution sufficient to measure rapid cell volume changes under anisosmotic conditions in the absence or presence of glycerol. This technique utilizes a concentration-dependent self-quenching entrapped fluorophore. The resulting cell volume changes were used in three-parameter fitting calculations to compute Lp in the absence glycerol, and Lp in the presence of glycerol (Lpgly) and Ps. Averaged over all bulls, Lp in the absence of glycerol was 0.28 ± 0.01, 0.15 ± 0.01 and 0.10 ± 0.01 ¿m min-1 atm-1 (mean ± SD) at 22, 10 and 0 °C, respectively, yielding an Arrhenius activation energy (Ea) of 7.39 kcal/mol. The average Lpgly value at 22 °C, was 3.8 times lower than Lp in the absence of glycerol (P <0.05). Lpgly, Ps, and the reflection coefficient (¿) at 22 °C were 0.073 ± 0.015 ¿m min-1 atm-1, 0.80 ± 0.33 × 10-3 cm min-1, and 0.92 ± 0.10 (mean ± SD), respectively. Subsequent experiments were performed at 10 and 0 °C. Activation energies for Lpgly and Ps were 10.08 and 8.77 kcal/mol, respectively. The significant differences between individual bulls in Lp and Ps indicate that individual males may require individual adjustments of the cooling protocol. Application of these data in a theoretical model to simulate the osmotic events during freezing resulted in predicted optimal cooling rates in the range of published empirical value

Improved in vitro function of islets using small intestinal submucosa

Transplantation of human pancreatic islets has been demonstrated to be a viable alternative to exogenous insulin therapy for diabetes mellitus. However, optimum results require transplantation of islets from two to three pancreas donors after a minimum number of days in culture. This implies that a substantial part of the transplanted islet mass may be nonfunctional. This study investigates the ability of an optimized technique to retain islet function using porcine-derived small intestinal submucosa (SIS) during in vitro culture. Groups of purified human islets were cultured for 3 weeks in modified standard islet culture conditions of CMRL = 1066 tissue culture medium supplemented with 25 mmol/L HEPES, penicillin/streptomycin, and a commercial insulin-transferin-selenium (ITS) supplement. Islets (50 to 200 IE/condition; n = 5 preparations) were cultured in plates containing noncoated Biopore membrane inserts alone, or on inserts that had been covered with SIS. Function was assessed by static incubation with low (4 mmol/L), or high (20 mmol/L) glucose at the end of each week. Glucose-stimulated release of human insulin was measured by radioimmunoassay (Linco, St. Charles, Missouri). Remaining islets were stained and evaluated visually. Neither culture condition resulted in significantly different basal secretion until week 3 ( P = .05). However, by the end of week 2 and for the duration of the experiment thereafter, SIS-treated islets exhibited a higher SI ( P < .05). At the end of the experiment, islets cultured on the SIS exhibited excellent morphology, with greater than 90% staining positive with Dithizone. Islets cultured on the inserts alone lost their initial morphology, becoming “loose” in appearance. The results of this study indicate that SIS enables enhanced function of islets in vitro as compared to non-SIS supported culture conditions

Enhanced recovery of cryopreserved islets using SIS

Although cryopreservation of pancreatic islets would add flexibility to transplantation, the recoveries are only 60% to 90% and function is decreased. Islets are multicellular structures ∼50 to 250 μm in diameter organized into a network of cells and vascular channels. Due to this complexity, islets are more susceptible to damage during cryopreservation than an individual cell. This study investigated porcine small intestinal submucosa (SIS) as a matrix to support islets recovery and function post-thaw. Groups of frozen/thawed human islets (150 IE/condition; n = 4 preparations) were cultured for 5 weeks in plates containing noncoated Biopore membrane inserts alone or inserts covered with SIS. Islets were placed directly on the insert post-thaw (SIS 1), or cultured overnight in standard plates, washed, and then transferred to the SIS (SIS 2). Function was assessed by determining glucose-stimulated release of insulin, which was measured by radioimmunoassay. Analysis of basal insulin secretion showed time and treatment to be significantly different ( P = .0043 and P = .0123, respectively) but without an interaction ( P > .05). The two SIS treatments were not significantly different ( P > .05); however, both SIS 1 and SIS 2 were significantly different from controls ( P = .0108 and P = .0420, respectively). Similar results were obtained for stimulation indices; time and treatment were significantly different ( P = .0161 and P = .0264, respectively) but not an interaction ( P > .05). The two SIS treatments were not significantly different ( P = .05); however, both SIS 1 and SIS 2 differed from controls ( P = .0248 and P = .0407, respectively). The results indicate that SIS enables frozen-thawed islets to exhibit superior post-thaw function compared with a non-SIS–supported condition