Biomedical subjects

E S Critser

Publications and source records attributed to E S Critser.

At least 19 recordsLinked to original sources

PubMed2000

Fundamental cryobiology of rat immature and mature oocytes: hydraulic conductivity in the presence of Me(2)SO, Me(2)SO permeability, and their activation energies.

The hydraulic conductivity in the presence of dimethyl sulfoxide Me(2)SO (L(p)(Me(2)SO)), Me(2)SO (P(Me(2)SO)) permeability and reflection coefficient (sigma) of immature (germinal vesicle; GV) and mature (metaphase II; MII) rat oocytes were determined at various temperatures. A temperature controlled micropipette perfusion technique was used to conduct experiments at five different temperatures (30, 20, 10, 4, and -3 degrees C). Kedem and Katchalsky membrane transport theory was used to describe the cell volume kinetics. The cell volumetric changes of oocytes were calculated from the measurement of two oocyte diameters, assuming a spherical shape. The activation energies (E(a)) of L(p)(Me(2)SO) and P(Me(2)SO) were calculated using the Arrhenius equation. Activation energies of L(p)(Me(2)SO) for GV and MII oocytes were 34.30 Kcal/mol and 16.29 Kcal/mol, respectively; while the corresponding E(a)s of P(Me(2)SO) were 19.87 Kcal/mol and 21.85 Kcal/mol, respectively. These permeability parameters were then used to calculate cell water loss in rat oocytes during cooling at subzero temperatures. Based on these values, the predicted optimal cooling rate required to maintain extra- and intracellular water in near equilibrium for rat GV stage oocytes was found to be between 0.05 degrees C/min and 0. 025; while for rat MII oocytes, the corresponding cooling rate was 1 degrees C/min. These data suggest that standard cooling rates used for mouse oocytes (e.g., 0.5-1 degrees C/min) can also be employed to cryopreserve rat MII oocytes. However, the corresponding cooling rate required to avoid damage must be significantly slower for the GV stage rat oocyte. J. Exp. Zool. 286:523-533, 2000.

Animals↗

PubMed2000

Effect of osmotic stress on the developmental competence of germinal vesicle and metaphase II stage bovine cumulus oocyte complexes and its relevance to cryopreservation.

The effects of osmotic stress on germinal vesicle (GV) and metaphase II (MII) stage bovine cumulus oocyte complexes (COCs) were evaluated by first exposing them to various anisotonic NaCl solutions (75, 150, 600, 1200, 2400, and 4800 +/- 5 mOsm/kg) for 10 min and then returning them to isotonic TL-Hepes solution (270 +/- 5 mOsm/kg) at 20 +/- 2 degrees C. Percentages of oocyte maturation, fertilization, polyspermy, cleavage, and blastocyst formation were measured as endpoints. Exposure to anisotonic conditions had a significant (P < 0.05) effect on the developmental competence of both GV and bovine MII COCs. Oocytes at the GV stage were more sensitive to anisotonic stress than MII oocytes (P < 0.05). None of the GV oocytes developed to the blastocyst stage after exposure to hypertonic conditions (2400 or 4800 mOsm solutions), while exposure to hypotonic conditions (75 or 150 mOsm solutions) resulted in significantly lower (P < 0.05) blastocyst formation (9% and 13%, respectively) compared to the isotonic control (25%). A dramatic decrease to 4% development to blastocyst was observed for MII oocytes following exposure to a 4800 mOsm solution. Blastocyst formation of MII oocytes which were exposed to 75, 150, 600, 1200, or 2400 mOsm solutions were similar (15%, 20%, 18%, 14%, and 13%, respectively; P > 0.05), but lower (P < 0.05) than those in the control group (29%). Exposing GV oocytes to anisotonic conditions increased polyspermic fertilization (P < 0.05), although MII oocytes were not similarly affected (P > 0.05). These data support the hypothesis that osmotic stress is detrimental to bovine oocytes and must be considered when developing optimized cryopreservation procedures for these cells. Mol. Reprod. Dev. 55:212-219, 2000.

Animals↗

PubMed2000

Cryobiology of rat embryos II: A theoretical model for the development of interrupted slow freezing procedures.

Current mammalian embryo cryopreservation protocols typically employ an interrupted slow freezing (ISF) procedure. In general, ISF consists of initial slow cooling, which raises the extracellular solute concentration, and results in cell dehydration. Permeating cryoprotective agents (CPAs), such as dimethyl sulfoxide (DMSO), are typically included in the medium to protect the cells against high solute concentrations. As this ISF procedure continues, slow cooling is terminated at an intermediate temperature (T(p)), followed by plunging into liquid nitrogen (LN(2)). If the slow cooling step allowed a critical concentration ([CPA](c)) of CPA to be reached within the cell, the CPA will interact with the remaining intracellular water during rapid cooling, resulting in the majority of the intracellular solution becoming vitrified and preventing damaging intracellular ice formation (IIF). This study presents a theoretical model to develop efficient ISF procedures, on the basis of previously developed data for the rat zygote. The model was used to select values of initial CPA concentrations and slow cooling rates (from initial estimated ranges of 0 to 4 molal DMSO and 0 to 2.5 degrees C/min cooling rates) that would allow the intracellular solute concentration to exceed the critical concentration. The optimal combination was then determined from this range based on minimizing the duration of slow cooling.

Algorithms↗

PubMed1999

Temperature-dependent osmotic behavior of germinal vesicle and metaphase II stage bovine oocytes in the presence of Me2SO in relationship to cryobiology.

Plasma membrane permeability coefficients and their activation energies (Ea) for water (Lp) and dimethyl sulfoxide (PMe2SO) as well as the reflection coefficient (sigma) were determined for germinal vesicle (GV) and metaphase II (MII) bovine oocytes. A micropipette perfusion technique was used with a temperature controlled circulation chamber, which was adapted to a micromanipulator. Experiments were performed at five different temperatures (30, 20, 10, 4 and -3 degrees C). The Kedem and Katchalsky model was assumed and L(p), P(Me2SO) and sigma were estimated. Estimated permeability values from the experimental temperatures were then applied to Arrhenius plots In(Lp) or In(PMe2SO) vs 1/Temperature (K) to estimate the activation energies (Ea) for L(p)Me2SO and P(Me2SO). The estimated E(a) for L(p)Me2SO for GV and MII oocytes were 23.84 Kcal/mol and 8.46 Kcal/mol, respectively. The E(a) for P(Me2SO) were 21.0 Kcal/mol and 23.20 Kcal/mol, respectively. The correlation (r2) for these linear regression plots for GV oocytes were 0.83 and 0.95 for L(p)Me2SO and P(Me2SO), respectively. For MII oocytes, r2 values were 0.95 and 0.99 for L(p)Me2SO and P(Me2SO), respectively. There was a possible discontinuity detected in the Arrhenius plot for L(p)Me2SO for GV oocytes. A significant decrease of the reflection coefficient was observed at 10 degrees C compared to other experimental temperatures. These data provide a fundamental basis that should be taken into account for low temperature preservation of bovine oocytes in the presence of Me2SO.

Animals↗

PubMed1998

Effect of developmental stage on bovine oocyte plasma membrane water and cryoprotectant permeability characteristics.

Knowledge of bovine oocyte plasma membrane permeability characteristics at different developmental stages in the presence of cryoprotective agents (CPAs) is limited. The objective of this study was to determine the oolema hydraulic conductivity (Lp), cryoprotectant permeability (P[CPA]), and reflection coefficient (sigma) for immature (germinal vesicle stage, GV) and in vitro-matured (metaphase II, MII) bovine oocytes. Two commonly used cryoprotective agents, dimethyl sulfoxide (DMSO) and ethylene glycol (EG), were studied. Osmometric studies were performed using a micromanipulator connected to an inverted microscope at 22 +/- 2 degrees C. Each oocyte was immobilized via a holding pipette, and osmotically induced volume changes over time (dv/dt) were recorded. The Lp values for GV and MII oocytes in DMSO (L(p)DMSO) were 0.70 +/- 0.06 and 1.14 +/- 0.07 microm/min/atm (mean +/- SEM) and in EG (L(p)EG) were 0.50 +/- 0.06 and 0.83 +/- 0.07 microm/min/atm, respectively. Estimates of P(DMSO) for GV and MII oocytes were 0.36 +/- 0.03 and 0.48 +/- 0.03 microm/sec, and PEG values for GV and MII oocytes were 0.22 +/- 0.03, 0.37 +/- 0.03 microm/sec, respectively. The values for GV and MII oocytes in DMSO (sigma[DMSO]) were 0.86 +/- 0.03 and 0.90 +/- 0.04 and in EG (sigma[EG]) were 0.94 +/- 0.03 and 0.76 +/- 0.04, respectively. These data demonstrate that bovine oolema permeability coefficients to water and cryoprotectants change after in vitro maturation. Furthermore, the bovine oocyte P(DMSO) is higher than the P(EG). These results may provide a biophysical basis for developing criteria for choosing optimal CPAs and for minimizing damage during addition and removal of the CPAs. Additionally, these data support the hypothesis that different procedures may be required for optimal cryopreservation of different oocyte developmental stages.

Animals↗

PubMed1998

Membrane permeability characteristics of metaphase II mouse oocytes at various temperatures in the presence of Me2SO.

In this study, the hydraulic conductivity (Lp), Me2SO permeability (PMe2SO), and the reflection coefficients (sigma) and their activation energies were determined for Metaphase II (MII) mouse oocytes by exposing them to 1.5 M Me2SO at temperatures of 30, 20, 10, 3, 0, and -3 degrees C. These data were then used to calculate the intracellular concentration of Me2SO at given temperatures. Individual oocytes were immobilized using a holding pipette in 5 microliters of an isosmotic PBS solution and perfused with precooled or prewarmed 1.5 M Me2SO solutions. Oocyte images were video recorded. The cell volume changes were calculated from the measurement of the diameter of the oocytes, assuming a spherical shape. The initial volume of the oocytes in the isoosmotic solution was considered 100%, and relative changes in the volume of the oocytes after exposure to the Me2SO were plotted against time. Mean (means +/- SEM) Lp values in the presence of Me2SO were (LpMe2SO) at 30, 20, 10, 3, 0 and -3 degrees C were determined to be 1.07 +/- 0.03, 0.40 +/- 0.02, 0.18 +/- 0.01, 7.60 x 10(-2) +/- 0.60 x 10(-2), 5.29 x 10(-2) +/- 0.40 x 10(-2), and 3.69 x 10(-2) +/- 0.30 x 10(-2) microns/min/atm, respectively. The PMe2SO values were 3.69 x 10(-3) +/- 0.3 x 10(-3), 1.07 x 10(-3) +/- 0.1 x 10(-3), 2.75 x 10(-4), +/- 0.15 x 10(-4), 7.83 x 10(-5) +/- 0.50 x 10(-5), 5.24 x 10(-5) +/- 0.50 x 10(-5), and 3.69 x 10(-5) +/- 0.40 x 10(-5) cm/min, respectively. The sigma values were 0.70 +/- 0.03, 0.77 +/- 0.04, 0.81 +/- 0.06, 0.91 +/- 0.05, 0.97 +/- 0.03, and 1 +/- 0.04, respectively. The estimated activation energies (Ea) for LpMe2SO, and PMe2SO, and sigma were 16.39, 23.24, and -1.75 Kcal/mol, respectively. These data may provide the fundamental basis for the development of more optimal cryopreservation protocols for MII mouse oocytes.

Animals↗

PubMed1998

Hydraulic conductivity (Lp) and its activation energy (Ea), cryoprotectant agent permeability (Ps) and its Ea, and reflection coefficients (sigma) for golden hamster individual pancreatic islet cell membranes.

Long-term cryopreservation of islets of Langerhans would be advantageous to a clinical islet transplantation program. Fundamental cryobiology utilizes knowledge of basic biophysical characteristics to increase the understanding of the preservation process and possibly increase survival rate. In this study several of these previously unreported characteristics have been determined for individual islet cells isolated from Golden hamster islets. Using an electronic particle counting device and a temperature control apparatus, dynamic volumetric response of individual islet cells to anisosmotic challenges of 1.5 M dimethyl sulfoxide (DMSO) and 1.5 M ethylene glycol (EG) were recorded at four temperatures (8, 22, 28, and 37 degreesC). The resulting curves were fitted using Kedem and Katchalsky equations which describe water flux and cryoprotectant agent (CPA) flux based on hydraulic conductivity (Lp), CPA permeability (Ps), and reflection coefficient (final sigma) for the membrane. For Golden hamster islet cells, Lp, Ps, and final sigma for DMSO at 22 degreesC were found to be 0.23 +/- 0.06 microm/min/atm, 0.79 +/- 0.32 x 10(-3) cm/min, and 0.55 +/- 0.37 (n = 11) (mean +/- SD), respectively. For EG at 22 degreesC, Lp equaled 0.23 +/- 0.06 microm/min/atm, Ps equaled 0.63 +/- 0.20 x 10(-3) cm/min, and final sigma was 0.75 +/- 0.17 (n = 9). Arrhenius plots (ln Lp or ln Ps versus 1/temperature (K)) were created by adding the data from the other three temperatures and the resulting linear regression yielded correlation coefficients (r) of 0.99 for all four plots (Lp and Ps for both CPAs). Activation energies (Ea) of Lp and Ps were calculated from the slopes of the regressions. The values for DMSO were found to be 12.43 and 18.34 kcal/mol for Lp and Ps (four temperatures, total n = 52), respectively. For EG, Ea of Lp was 11.69 kcal/mol and Ea of Ps was 20.35 kcal/mol (four temperatures, total n = 58).

Animals↗

PubMed1998

Antral follicles develop in xenografted cryopreserved African elephant (Loxodonta africana) ovarian tissue.

The preservation of germ plasm from endangered species could augment captive breeding programs aimed at maintaining genetic diversity. Mammalian female germ plasm (oocytes) is extremely difficult to collect and cryopreserve; however, a promising alternative is the cryopreservation of ovarian tissue. In the present study, athymic nude (nu/nu) Balb/C mice were used to evaluate in vivo viability of cryopreserved ovarian tissue from Institute of Cancer Research genotype (ICR) mice or elephants. Female mice were ovariectomized prior to transplant of cryopreserved-thawed ovarian tissue from ICR mice (n=4) or elephants (n=6). Control mice were sham operated (n=4) or ovariectomized (n=5). Transplants were in the ovarian bursa, enabling in vivo ovulation and pregnancies from allografts. Vaginal cytology was monitored daily, and the intervals between and duration of epithelial cells present in smears were evaluated. Appearance of epithelial cells in sham-operated and allografted mice were at intervals of 4.3+/-0.6 and 3.3+/-0.5 days, lasting for 1.4+/-0.1 and 1.6+/-0.2 days, respectively. Sporadic incidence of epithelial cells in ovariectomized animals occurred at longer intervals (8.6+/-3.8 days). Females with xenografted elephant ovarian tissue demonstrated epithelial cells in vaginal smears at intervals of 4.5+/-1.0 days, for 2.5+/-0.5 days duration, which was significantly longer than the other groups (P < 0.05). Histological evaluation of tissues at the time of epithelial cells in smears demonstrated well-developed antral follicles, although oocytes were of poor morphological appearance or only cumulus-like complexes were seen. The nude mouse model is effective for assessing cryopreserved ovarian tissue xenograft function which can support the development of antral follicles.

Animals↗

PubMed1997

Live births after autologous transplant of cryopreserved mouse ovaries.

The fertility of mice after autologous transplantation of ovaries, before or after cryopreservation, was investigated in this study. Female mice were randomly assigned to either sham-operated (n = 14), ovariectomized (n = 11), fresh (n = 12) or cryopreserved (n = 11) ovarian transplant groups. Ovaries were cryopreserved in 1.4 M dimethyl sulphoxide (DMSO) by cooling to -55 degrees C at 0.5 degree C/min (ice nucleation at -7 degrees C), plunged in liquid nitrogen and then thawed at room temperature. Oestrous cyclicity was observed 7 days after sham operation or 15 days after fresh or cryopreserved ovarian transplant. Ovariectomized animals did not demonstrate oestrous cyclicity but were mated, and no pregnancies resulted. Live births were recorded from all sham-operated, all fresh transplant, and 8/11 (73%) cryopreserved transplant animals. Overall mean +/- SEM litter sizes from fresh (4.32 +/- 0.44) and cryopreserved (4.71 +/- 0.57) transplant groups were smaller (P < 0.05) than those of sham-operated animals (12.54 +/- 0.44), although the sizes were not significantly different (P > 0.05) from each other. Animals were mated at least four times, with four litters of live pups from 4/4 sham-operated, 1/10 fresh and 1/9 cryopreserved ovarian transplant animals. Litter sizes from pups of sham-operated and transplant animals were not significantly different from each other. Following autologous transplantation of mouse ovaries, before or after cryopreservation, offspring appeared normal, with high rates of fertility.

Animals↗

PubMed1997

Allogeneic and xenogeneic transplantation of cryopreserved ovarian tissue to athymic mice.

Cryopreserved ovarian tissue has major applications for female oncology patients and for the development of genome resource banks. The objective of the present study was to develop a bioassay of cryopreserved ovarian tissue function after allogeneic and xenogeneic transplantation to ovariectomized athymic nude (nu/nu) Balb/C mice. Transplant function was assessed by examination of vaginal smears, number of live births, and posttransplant histology. Animals were sham operated (group I; n = 4) or ovariectomized (group II; n = 5) or were given transplants of either fresh (group III; n = 3) or cryopreserved (group IV; n = 4) Institute of Cancer Research-strain mouse ovarian tissue or cryopreserved sheep ovarian tissue (group V; n = 7). Vaginal smears were examined 5-7 times per week; the number of days between visualizations of epithelial cells in smears was 4.3 +/- 0.6 for group I, 8.6 +/- 3.8 for group II, 3.4 +/- 0.4 for group III, 3.3 +/- 0.5 for group IV, and 4.6 +/- 0.6 for group V. Epithelial cells were seen for 1.2-1.7 consecutive days; this value was significantly different between groups III and V. Live births were recorded from 3 of 4 animals from group I, 0 of 5 animals from group II, 2 of 3 animals from group III, and 1 of 4 animals from group IV. In vivo function and long-term survival of cryopreserved ovarian tissue after allogeneic or xenogeneic transplant were confirmed by the examination of vaginal cytology, and offspring were derived from allografts.

Animals↗

PubMed1996

Development of a novel microperfusion chamber for determination of cell membrane transport properties.

A novel microperfusion chamber was developed to measure kinetic cell volume changes under various extracellular conditions and to quantitatively determine cell membrane transport properties. This device eliminates modeling ambiguities and limitations inherent in the use of the microdiffusion chamber and the micropipette perfusion technique, both of which have been previously validated and are closely related optical technologies using light microscopy and image analysis. The resultant simplicity should prove to be especially valuable for study of the coupled transport of water and permeating solutes through cell membranes. Using the microperfusion chamber, water and dimethylsulfoxide (DMSO) permeability coefficients of mouse oocytes as well as the water permeability coefficient of golden hamster pancreatic islet cells were determined. In these experiments, the individual cells were held in the chamber and perfused at 22 degrees C with hyperosmotic media, with or without DMSO (1.5 M). The cell volume change was videotaped and quantified by image analysis. Based on the experimental data and irreversible thermodynamics theory for the coupled mass transfer across the cell membrane, the water permeability coefficient of the oocytes was determined to be 0.47 micron. min-1. atm-1 in the absence of DMSO and 0.65 microns. min-1. atm-1 in the presence of DMSO. The DMSO permeability coefficient of the oocyte membrane and associated membrane reflection coefficient to DMSO were determined to be 0.23 and 0.85 micron/s, respectively. These values are consistent with those determined using the micropipette perfusion and microdiffusion chamber techniques. The water permeability coefficient of the golden hamster pancreatic islet cells was determined to be 0.27 microns. min-1. atm-1, which agrees well with a value previously determined using an electronic sizing (Coulter counter) technique. The use of the microperfusion chamber has the following major advantages: 1) This method allows the extracellular condition(s) to be readily changed by perfusing a single cell or group of cells with a prepared medium (cells can be reperfused with a different medium to study the response of the same cell to different osmotic conditions). 2) The short mixing time of cells and perfusion medium allows for accurate control of the extracellular osmolality and ensures accuracy of the corresponding mathematical formulation (modeling). 3) This technique has wide applicability in studying the cell osmotic response and in determining cell membrane transport properties.

Animals↗

PubMed1996

Use of endometrial measurement as an exclusion criterion for in vitro fertilization using clomiphene citrate.

OBJECTIVE: Previous reports have indicated an association between endometrial development and pregnancy outcome for patients treated with clomiphene citrate (CC) in conjunction with intrauterine insemination or intercourse. We expanded the use of CC for ovulation induction in association with in vitro fertilization (IVF). This study was designed to determine if endometrial thickness should be used as an inclusion or exclusion criterion for CC-IVF. STUDY DESIGN: One hundred twenty-eight patients were enrolled in an ovulation-induction regimen using CC for expected IVF-ET between January 1992 and December 1992. A total of 81 patients met inclusion criteria for CC-IVF and had endometrial measurement performed prior to human chorionic gonadotropin administration. Patients were categorized on the basis of endometrial measurement as follows: (A) > 4 - < 7 mm, (B) > or = 7 - < or = 10 mm, and (C) > 10 mm. Standard IVF was performed, and pregnancy rates for each category were evaluated. RESULTS: A total of 23 pregnancies (28% per retrieval) were established. Pregnancy rates were not different by category (P > .10, Fisher's Exact Test): (A) 3/15 (20%), (B) 13/41 (32%), and (C) 7/25 (28%). CONCLUSION: These data suggest that for CC-IVF. endometrial measurement should not be used as an exclusion criterion since pregnancies occurred at comparable frequencies in all the groups.

Adult↗

PubMed1995

Developmental potential of microbiopsied murine blastocysts.

OBJECTIVE: To evaluate the frequency of mouse pups born following blastocyst biopsy and embryo transfer compared to nonbiopsied controls. METHODS: ICR Swiss albino and C57B1/6 mice served as embryo donors. Pregnant mare serum gonadotropin treatment was followed 46-52 hours later by hCG, when donors were paired with fertile males. Mating was confirmed the following day and embryos were collected on the afternoon of day 4. After overnight culture, hatching trophoblast was excised by micromanipulation with a fine-pulled glass pipette. Embryos (206 controls, 206 biopsied) were transferred to 26 pseudopregnant recipients. Alternate mouse strains were used to identify pups born from control or biopsied embryos. RESULTS: The end point was percentage of pups born ((number born/number transferred) x 100), using angular transformation before analysis. There was no significant difference (P > .1) between percent live-born in control (27.7%) or biopsied (34.5%) embryos, nor were there any strain differences. CONCLUSION: These data support the hypothesis that the developmental potential of murine blastocysts is not affected adversely by the biopsy procedure.

Animals↗

PubMed1995

Growth of mouse pups derived from biopsied blastocysts.

OBJECTIVE: To test the hypothesis that removal of cells from the pre-embryo affects intrauterine growth, as reflected in birth weight or growth to weaning. METHODS: ICR Swiss albino and C57Bl/6 mice served as embryo donors to provide two strains of distinct coat colors. Alternate strains were assigned to control treatment or blastocyst biopsy of trophectoderm. Embryos from both treatments were transferred to a different uterine horn within each pseudopregnant recipient. A total of 412 embryos (206 controls and 206 biopsied blastocysts) were transferred to 26 recipients, which resulted in the birth of 122 pups. RESULTS: There was no significant difference (P > .1) in birth weight or growth to weaning, defined as [[(weaning weight-birth weight)/birth weight)] x 100], between male and female pups. There was a significant difference (P < .01) between pups derived from ICR (birth weight 1.9 +/- 0.02 g; growth to weaning 544 +/- 14%; n = 72) versus C57Bl/6 (birth weight 1.43 +/- 0.03 g; growth to weaning 460 +/- 14%; n = 50) embryos. However, there was no significant difference (P > .1) in birth weight or growth to weaning for pups derived from biopsied embryos (birth weight 1.70 +/- 0.03 g; growth to weaning 502 +/- 14%; mean +/- standard error of the mean; n = 67) or control embryos (birth weight 1.71 +/- 0.05 g; growth to weaning 520 +/- 17%; n = 55). CONCLUSIONS: These data support the null hypothesis that embryo biopsy does not result in reduced birth weight or altered subsequent growth to weaning.

Animals↗

PubMed1995

Prevention of osmotic injury to human spermatozoa during addition and removal of glycerol.

Use of a cryoprotective agent is indispensable to prevent injury to human spermatozoa during the cryopreservation process. However, addition of cryoprotective agents to spermatozoa before cooling and their removal after warming may create severe osmotic stress for the cells, resulting in injury. The objective of this study was to test the hypothesis that the degree (or magnitude) of human sperm volume excursion can be used as an independent indicator to evaluate and predict possible osmotic injury to spermatozoa during the addition and removal of cryoprotective agents. Glycerol was used as a model cryoprotective agent in the present study. To test this hypothesis, first the tolerance limits of spermatozoa to swelling in hypo-osmotic solutions (iso-osmotic medium diluted with water) and to shrinkage in hyperosmotic solutions (iso-osmotic medium with sucrose) were determined. Sperm plasma membrane integrity was measured by fluorescent staining, and sperm motility was assessed by computer-assisted semen analysis before, during and after the anisosomotic exposure. The result indicate firstly that motility was much more sensitive to anisosmotic conditions than membrane integrity, and secondly that motility was substantially more sensitive to hypotonic than to hypertonic conditions. Based on the experimental data, osmotic injury as a function of sperm volume excursion (swelling or shrinking) was determined. The second step, using these sperm volume excursion limits and previously measured glycerol and water permeability coefficients of human spermatozoa, was to predict, by computer simulation, the cell osmotic injury caused by different procedures for the addition and removal of glycerol. The predicted sperm injury was confirmed by experiment. Based on this study, an analytical methodology has been developed for predicting optimal protocols to reduce osmotic injury associated with the addition and removal of hypertonic concentrations of glycerol in human spermatozoa.

Buffers↗

PubMed1995

High water permeability of human spermatozoa is mercury-resistant and not mediated by CHIP28.

A novel integral membrane protein with an apparent molecular mass of 28 kDa (CHIP28) was first isolated from human erythrocytes and is now recognized as a water channel protein. The expression of this protein has been found in several other cell types that all require high water permeability for their functions. Recent studies have shown that the water permeability (Lp) of human spermatozoa is among the highest reported for mammalian cells. Together with the low activation energy of human spermatozoa for Lp, this suggests that CHIP28 water channel may be present in the plasma membrane of human spermatozoa. However, our current studies do not support this hypothesis. Results from Western blot analysis on human sperm plasma membrane proteins, performed through use of an antibody against human erythrocyte CHIP28 protein, indicated that human spermatozoa do not express CHIP28 protein on their cell surface (n = 10). Consistent with the Western blot finding, mercuric chloride (HgCl2), a known water channel blocker, failed to reduce the osmotic water permeability of human spermatozoa. The calculated Lp values were 1.30 +/- 0.29 micron/min/atm (n = 16; mean +/- SEM) for the control group and 1.31 +/- 0.29 (n = 9; mean +/- SEM), 1.04 +/- 0.27 (n = 11; mean +/- SEM), and 1.34 +/- 0.19 (n = 6; mean +/- SEM), respectively, for the 10 microM, 30 microM, and 50 microM HgCl2-treated groups. These Lp values are not different (p > 0.05). In contrast, the same concentration of HgCl2 significantly blocked the osmotic water transport across the membrane of human erythrocytes.(ABSTRACT TRUNCATED AT 250 WORDS)

Aquaporin 1↗

PubMed1995

The effect of collection temperature, cooling rate and warming rate on chilling injury and cryopreservation of mouse spermatozoa.

The experiments presented here identify several factors that affect survival (motility) of cryopreserved mouse spermatozoa after freezing and thawing. Among these factors are: (i) the temperature at which spermatozoa are collected, (ii) the cooling rate to 0 degrees C and (iii) the warming rate from -196 degrees C to ambient. When excised epididymides were cooled to near 0 degrees (1-4 degrees C) and spermatozoa collected and mixed with cryoprotectant at that temperature, motilities after subsequent freezing and thawing were 8-10 times higher than when the spermatozoa were collected from the epididymides at 22 degrees C. In addition, the survival rates of spermatozoa warmed at rates ranging from 150 to 2000 degrees C min-1 were about five times higher than those in suspensions warmed at about 7500 degrees C min-1. The combination of a low collection temperature and the lower warming rates resulted in approximately 50% motility relative to unfrozen controls. Motility was reduced to 6-8% when the collection temperature was 22 degrees C, and to approximately 10% when frozen suspensions of spermatozoa collected in the cold were rapidly warmed from -196 degrees C. When spermatozoa collected at 22 degrees C were abruptly cooled to 0 degrees C, 40-80% of the cells suffered an irreversible loss of motility after warming. In contrast, when spermatozoa were cooled to 0 degrees C at 1 degree C min-1 and warmed (either rapidly or slowly), motilities were similar to those of uncooled controls (75-90%).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1994

Membrane transport properties of mammalian oocytes: a micropipette perfusion technique.

A perfusion technique using micropipette methodology was developed to determine quantitatively the membrane transport properties of mammalian oocytes. This method eliminates modelling ambiguities inherent in microdiffusion, a closely related technology, and should prove to be especially valuable for study of the coupled transport of water and cryoprotectant through mammalian oocytes and embryos. The method is described and evidence given for validity of the method for the simple case of uncoupled flow of water through the mouse oocyte membrane. The zona pellucida of a mouse oocyte was held by a micropipette with an 8-10 microns diameter tip opening and perfused by hyperosmotic media. The kinetic volume change of the cell was videotaped and quantified by image analysis. Experimental data and mathematical modelling were used to determine the hydraulic conductivity of the oocyte membrane (Lp) found to be 1.05, 0.45 and 0.26 microns min-1 atm-1 at 30 degrees C, 22 degrees C and 12 degrees C, respectively. The corresponding activation energy, Ea, for Lp was calculated to be 13.0 kcal mol-1. These values are in agreement with data obtained by other techniques. One of the major advantages of this technique is that the extracellular osmotic condition can be changed readily by perfusing a single cell with a prepared medium. To study the response of the same cell to different osmotic conditions, the old perfusion medium can be removed easily and the cell reperfused with a different medium.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗