Permeability characteristics and osmotic sensitivity of rhesus monkey (Macaca mulatta) oocytes

N. Songsasen1, M.S. Ratterree2, C.A. VandeVoort3, D.E. Pegg4 and S.P. Leibo1,5

1 Department of Biological Sciences, University of New Orleans and Audubon Center for Research of Endangered Species, New Orleans LA 70131, 2 Tulane Regional Primate Research Center, Covington LA 70433, 3 California Regional Primate Research Center, Davis, CA 95617, USA and 4 Department of Biology, University of York, York YO10 5YW, UK


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
BACKGROUND: Permeability characteristics and sensitivity to osmotic shock are principal parameters that are important to derive procedures for the successful cryopreservation of mammalian oocytes. METHODS AND RESULTS: The osmotically inactive volume of rhesus monkey oocytes was determined by measuring their volumes in the presence of hypertonic solutions of sucrose from 0.2 to 1.5 mol/l, compared with their volume in isotonic TALP-HEPES solution. Boyle–van't Hoff plots at infinite osmolality indicated that the non-osmotic volumes of immature and mature oocytes were 20 and 17% respectively. Osmotic responses of oocytes exposed to 1.0 mol/l solutions of glycerol, dimethylsulphoxide (DMSO) and ethylene glycol (EG) were determined. Rhesus monkey oocytes appeared to be less permeable to glycerol than to DMSO or to EG. Sensitivity of oocytes to osmotic shock was determined by exposing them to various solutions of EG (0.1 to 5.0 mol/l) and then abruptly diluting them into isotonic medium. Morphological survival, as measured by membrane integrity, of oocytes diluted out of EG depended significantly on the concentration of EG (P < 0.01). CONCLUSION: Determination of permeability characteristics and sensitivity to osmotic shock of rhesus oocytes will aid in the derivation of procedures for their cryopreservation.

Key words: oocytes/osmotic shock sensitivity/permeability characteristics/rhesus monkey


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Mammalian oocytes have proven to be more difficult to cryopreserve than cleavage-stage embryos (Van Blerkom, 1991Go; Parks and Ruffing, 1992Go; Ludwig et al., 1999Go). Nevertheless, live young have been produced from cryopreserved oocytes of mice (Whittingham, 1977Go; Schroeder et al., 1990Go), humans (Chen, 1986Go; van Uem et al., 1987Go), cattle (Vajta et al., 1998Go) and rabbits (Al-Hasani et al., 1989Go). Despite these successes, the efficiency of methods to cryopreserve oocytes of most species (except for the mouse) is very low (Bernard and Fuller, 1996Go; Ludwig et al., 1999Go; Shaw et al., 2000Go; for reviews). Thus, better methods to cryopreserve mammalian oocytes are still required.

The cryopreservation of oocytes of non-human primates would offer several advantages. First, it would permit the accumulation of oocytes for studies that require large numbers of oocytes to be used simultaneously. Since rhesus monkeys are seasonal breeders, cryopreservation of their oocytes would remove the barrier of reproductive seasonality and allow their use year-round. Cryopreservation of oocytes could also be used to preserve the germplasm of genetically valuable animals, such as a specific animal with unusual immunological characteristics. The same method might be used for endangered species of non-human primates. Furthermore, cryopreservation of non-human primate oocytes could serve as a model for human oocytes.

Despite the potential importance of such a method, only two reports of attempts to cryopreserve oocytes of non-human primates have been made—one with squirrel monkeys (DeMayo et al., 1985Go; Saimiri spp.) and the other with oocytes of an infertile lowland gorilla (Lanzendorf et al., 1992Go; Gorilla gorilla). In both cases, conventional equilibrium freezing with a relatively low concentration of cryoprotectant was used to cryopreserve oocytes. With oocytes of squirrel monkeys, although 50% of the cryopreserved oocytes appeared to be morphologically intact, maturation and fertilization rates were significantly reduced (DeMayo et al., 1985Go). With those of a gorilla, six immature oocytes were cryopreserved in 1.5 mol/l propylene glycol by cooling them at 0.5°C/min to –80°C before plunging them into liquid nitrogen; two oocytes appeared morphologically intact after being thawed, but their developmental competency was compromised (Lanzendorf et al., 1992Go).

Several factors affect survival of oocytes when they are cryopreserved (Van Blerkom, 1991Go; Parks and Ruffing, 1992Go; Critser et al., 1997Go; Parks, 1997Go; Shaw et al., 2000Go). During cryopreservation, in addition to being subjected to subzero temperatures and phase changes of the suspending media, the oocyte must tolerate a sequence of volume excursions. First, it is exposed to an hypertonic solution of a cryoprotective agent (CPA); this causes the oocyte to contract osmotically as intracellular water flows out of the cell because of differences in water activity between the intra- and extracellular compartments. As the CPA diffuses across the plasma membrane, the cell regains its original isotonic volume as water re-enters the cell. The cell undergoes yet another volume change during freezing. As ice crystals form outside the cell, differences in water activity between intra- and extracellular solutions occur, causing the cell to dehydrate (Mazur, 1970Go). Finally, the cell undergoes another volume excursion during warming and removal of the cryoprotectant from the cell. If these volume excursions exceed the limit tolerated by oocytes, cell damage may occur as a consequence of osmotic shock, as has been shown to occur in zygotes, embryos and other types of cells (Leibo, 1986Go, 1992Go; Arnaud and Pegg, 1990Go; Oda et al., 1992Go).

There appears to be only one reported study on permeability characteristics of non-human primate oocytes (Younis et al., 1996Go). These authors noted that oocytes of the cynomolgus monkey (Macaca fascicularis) behaved as a perfect osmometer, and that the osmotically inactive cell volumes of germinal vesicle-stage and metaphase II (MII) oocytes were 20 and 10% respectively. Using a cryo-microscope, these authors also determined the ice nucleation parameters and calculated the hydraulic conductivity and its activation energy of cynomolgus oocytes at subzero temperatures.

In the present study, the osmotic behaviour and permeability of oocytes of rhesus monkeys (Macaca mulatta) to three CPAs was determined. Previous studies have shown that rhesus oocytes, like those of the human, are sensitive to chilling injury, in that the meiotic spindle undergoes disassembly when oocytes are exposed to 0°C for >1 min (Zenzes et al., 2001Go; Songsasen et al., 2002Go). Even transient cooling to room temperature has been shown to alter human oocytes (Pickering et al., 1990Go). To avoid possible damage to oocytes used in this study, their osmotic responses were measured at 30°C. The extreme chilling sensitivity of rhesus oocytes suggests that their successful cryopreservation might require that they be vitrified or cooled very rapidly in order to circumvent injury resulting from exposure for several minutes to temperatures near 0°C, as occurs during equilibrium cooling. However, high concentrations of CPAs, such as those commonly used for cryopreservation by vitrification or rapid cooling methods, render oocytes extremely susceptible to osmotic injury. Therefore, the sensitivity of rhesus oocytes to osmotic shock was also determined. Oocytes were collected from female monkeys that had received HCG to induce resumption of meiosis; such oocytes are the most developmentally competent that can be obtained from the rhesus monkey (Schramm and Bavister, 1999Go). This study was predicated on the assumption that determination of permeability characteristics and osmotic shock sensitivity would aid in the derivation of a procedure to cryopreserve rhesus oocytes.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Chemicals used in this study were obtained from Sigma Chemical Company (St Louis, MO, USA), unless otherwise stated.

Animals, superovulation treatment and oocyte retrieval
Adult female rhesus monkeys (Macaca mulatta; ages ranging from 6 to 16 years) used in the present study were housed at the Tulane Regional Primate Research Center and maintained according to recommendations of the Guide for the Care and Use of Laboratory Animals [U.S. Department of Health and Human Service publication no. (NIH) 85–23, revised 1985]. All procedures for handling and treatment of the animals were reviewed and approved in advance by the Animal Care and Use Committees of the Tulane Regional Primate Research Center, the University of New Orleans, and the Audubon Center for Research of Endangered Species. These committees also adhere to guidelines of the U.S. Department of Agriculture.

Monkeys were individually caged in rooms with a 06:00 to 18:00 light cycle at a temperature maintained at 25–27°C, were fed twice daily a diet of Purina monkey chow, and provided with water ad libitum. Animals were monitored daily in the morning, and those that exhibited menses received twice-daily i.m. injections of 37.5 IU of recombinant human FSH (rhFSH; Serono Laboratories, Norwell, MA, USA) for 7–8 days beginning on day 1 or 2 of the menstrual cycle (day 1 = first day of menstruation). Ovaries were examined on day 5 or 6 after 4 days of FSH injections by ultrasonography of sedated monkeys in order to evaluate their follicular response to gonadotrophin. To induce oocyte maturation, a single i.m. injection of 1000 IU of recombinant HCG (rHCG; Serono Laboratories) was given on day 8 or 9 to those monkeys that had responded to rhFSH.

Oocyte aspirations were conducted under strict aseptic conditions. For oocyte collections, females were sedated with acepromazine maleate (0.2 mg/kg body weight) and glycopyrrolate (0.01 mg/kg body weight), anaesthetized with ketamine hydrochloride (10 mg/kg body weight), intubated and maintained on a mixture of isoflurane/oxygen (1.5%) during the procedure. Oocytes were aspirated laparoscopically at 27–32 h after the injection of rHCG from follicles >3 mm in diameter, using a 20-gauge spinal needle. After aspiration, oocytes were placed into warm Tyrode's lactate-pyruvate-HEPES medium (TALP-HEPES; Parrish et al., 1988Go) containing 10 IU/ml heparin. After surgery, the monkeys were given injections of buprenorphine (0.1 mg/kg body weight) as an analgesic for 3 days.

Although rHCG was given to the females to induce resumption of meiosis, the meiotic status of oocytes used in this study ranged from the germinal vesicle stage to MII. In the present study, oocytes were considered to be at one of only two stages: mature oocytes were those that had extruded their first polar body; immature oocytes were those at any stage of meiosis prior to the MII stage. No attempt was made to separate immature oocytes into various categories of maturation, as meiotic status can be determined definitively only after oocytes have been fixed and stained.

A total of 219 oocytes obtained from 11 females undergoing 14 treatment cycles was used in this study. Cumulus–oocyte complexes (COC) were recovered from the aspirated fluids using a stereomicroscope and transported in TALP-HEPES medium at 37°C from Covington LA to the laboratory in New Orleans (approximately a 1.5 h drive) in a MinitübTM portable incubator (Minitüb, Verona, WI, USA). Considerable care was exercised to ensure that the oocytes were maintained at 37°C from the time of collection until their use. In all experiments, oocytes were handled in a room maintained at 30°C. Upon arrival at the laboratory, the COC were placed into TALP-HEPES medium containing 1.5% (w/v) hyaluronidase (Type IV-S from bovine testes) at 30°C for 2 min, and cumulus cells were partially removed by gently pipetting the oocytes a few times. It must be emphasized that cumulus cells were only removed to the point where the oocytes could be visualized in order to avoid the possibility of altering the properties of the oocyte membranes by lengthy exposure to the enzyme. The oocytes were washed three times in TALP-HEPES medium, and then subjected to various treatments as described below.

Determination of the osmotically inactive volume of rhesus oocytes
Three mature and three immature oocytes from two rhesus females were used for these measurements. After cumulus cells had been removed, the oocytes were exposed sequentially to 0.2, 0.5, 0.75, 1.0 and 1.5 mol/l solutions of sucrose (500 to 3000 mOsm) prepared in TALP-HEPES medium for 5 min in each solution at 30°C. The osmolality of each solution was measured using a calibrated vapour pressure osmometer (VAPROTM, Model 5520; Wescor, Inc., Logan, UT, USA). Individual oocytes were transferred into 10 µl droplets of isotonic TALP-HEPES under mineral oil and photographed using a digital camera (Coolpix 950; Nikon Instruments Inc., Lewisville, TX, USA) attached to an inverted microscope (Nikon TE300; Nikon Instruments, Inc.). The oocytes were then rinsed in 2.5 ml of the first hypertonic solution (0.2 mol/l sucrose in TALP-HEPES) and then quickly transferred into a 10 µl droplet of the same solution under oil. After an oocyte had been allowed to equilibrate for 5 min, it was photographed. After being sequentially exposed to the other hypertonic solutions and being photographed, the change in cross-sectional area of each oocyte in each solution was measured. The equilibrium cell volume of oocytes in each hypertonic solution was calculated using the following equation:

where V is the cell volume and A is the cross-sectional area of the oocyte (Leibo, 1980Go). Finally, the mean relative volume of oocytes in each hypertonic solution was plotted as a function of the reciprocal of osmolality to construct a Boyle–van't Hoff plot. The volume of each oocyte in an isotonic solution of TALP-HEPES with an osmolality of 290 mOsm was taken to be 100%. The osmotically inactive volume of the oocytes was determined by extrapolation of the Boyle–van't Hoff plot to an infinitely concentrated solution, i.e. 1x[osmolality]–1 = 0.

Permeability characteristics of rhesus oocytes
A total of 24 oocytes collected on separate occasions from four females was used in this experiment. Permeability characteristics of rhesus oocytes in 1.0 mol/l solutions of dimethylsulphoxide (DMSO), ethylene glycol (EG) and glycerol were assessed at 30°C using a method modified from a previously reported technique (Jackowski et al., 1980Go). On each occasion, 10 µl drops of TALP-HEPES medium and of 1.0 mol/l solutions of DMSO (n = 9 oocytes), EG (n = 10 oocytes) and of glycerol (n = 5 oocytes) overlaid by mineral oil were prepared in Nunclon multiwell dishes (Fisher Scientific, Pittsburgh, PA, USA) and held at 30°C. Individual oocytes were washed in 2.5 ml TALP-HEPES and then quickly placed into a 10 µl drop of the same solution and photographed. Subsequently, the same oocyte was then rinsed briefly in 2.5 ml of a given CPA and immediately placed into a 10 µl drop of the same CPA for 10 min (120 min for glycerol). Oocytes were photographed at 0.5, 1, 2, 3, 4, 5 and 10 min after being exposed to a given CPA. Their cross-sectional areas after exposure to CPAs were measured, and changes in the relative volumes of oocytes were calculated as described above. These volume changes were plotted as a function of the duration of exposure to CPA.

On a separate occasion, the diameters of four metaphase II oocytes were measured, using an eyepiece micrometer that had been calibrated with a Nikon stage micrometer. The mean and standard error of the mean were calculated. For calculation of the permeability to CPAs, the measured volumes were fitted to a two-parameter model defined by the following equations (Kleinhans, 1998Go):


where J{nu}w = volumetric water flux; Lp = hydraulic conductivity; {pi}e and {pi}i = external and internal osmolality respectively, assuming non-ideal behaviour of the intracellular solutes as previously described (Pegg et al., 1987Go; Arnaud and Pegg, 1990Go); J{nu}s = volumetric flux of CPA; Ps = CPA permeability, Ce and Ci = external and internal concentrations of CPA respectively; and Vs = partial molar volume of the CPA.

Osmotic sensitivity of rhesus oocytes
A total of 189 oocytes from 12 females was used in this experiment. The experiment was divided into two parts. In the first part, oocytes (n = 109, six replicates) were exposed to 0.1 to 2.0 mol/l solutions of EG in TALP-HEPES, whilst in the second part of the experiment, oocytes (n = 80, six replicates) were exposed to 2.0 to 5.0 mol/l solutions of EG. In both experiments, some oocytes were exposed only to TALP-HEPES as dilution controls.

The procedure to determine osmotic sensitivity consisted of the following steps. Oocytes were pipetted into 0.1 ml TALP-HEPES contained in 12x75 mm plastic tubes (Falcon®, Fisher Scientific) that were placed into a 30°C water bath. Then, 0.1 ml volumes of 0.2, 0.8, 1.2, 1.6, 2.0 or 4.0 mol/l EG were added to the plastic tubes, yielding final concentrations of 0.1, 0.4, 0.6, 0.8, 1.0 and 2.0 mol/l EG. After 5 min, all oocytes were rapidly diluted with TALP-HEPES (11-fold dilution) by adding 2.0 ml TALP-HEPES to the plastic tubes. The diluted oocytes were recovered and cultured in CMRL1066 medium (Life Technologies, Grand Island, NY, USA) + 20% fetal bovine serum (Life Technologies) at 37°C for 60 min. The purpose of this brief incubation at 37°C was to allow damaged oocytes to degenerate. A similar procedure was performed in the second part of the experiment, except that the oocytes were exposed to solutions of 2.0, 3.0, 4.0 or 5.0 mol/l EG and allowed to equilibrate for 5 or 10 min at 30°C before dilution. The rationale of using two exposure periods was to determine the effect of equilibration times on the survival of oocytes after dilution.

Survival in both parts of the experiment as judged by membrane integrity of oocytes was assessed using Live/Dead stain® (Molecular Probes, Inc., Eugene, OR, USA) that contains two nucleic acid dyes, SYBR14 and propidium iodide. The staining procedure was modified from that described for bovine spermatozoa (Garner et al., 1994Go). Briefly, an oocyte was placed into a 10 µl droplet of 0.02 mmol/l solution of SYBR14 in TALP-HEPES for 5 min at 37°C. The oocyte was then counterstained with 1 µl of 2.4 mmol/l propidium iodide in water and incubated for an additional 5 min at 37°C, before being examined under a fluorescence microscope.

Statistical analysis
Regression analysis (SigmaStat, version 2.0; SPSS Inc., Chicago, IL, USA) was performed on the volumetric responses of rhesus oocytes exposed to increasing concentrations of sucrose. Comparison of relative volumes of oocytes after exposure to different cryoprotectants for a given time was performed using Student's t-test (SigmaStat). Comparison of the percentage of oocytes that remained membrane-intact after being exposed to various concentrations of EG was conducted using a {chi}2-test (Instat version 3.00 for Windows 95; Graphpad Software, San Diego, CA, USA; www.graphpad.com). The level of significance was set at 5%.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Osmotically inactive volume of rhesus oocytes
Rhesus oocytes contracted isotropically in hypertonic solutions of sucrose. Both mature and immature rhesus oocytes behaved as perfect osmometers over the range of 300 to 3000 mOsm, as their volumes decreased proportionally as the osmolality of sucrose was increased (Figure 1Go). Regression analysis of the data showed a linear relationship between oocyte equilibrium volume and the reciprocal of osmolality. Extrapolation of the data to infinite osmolality gave an osmotically inactive volume of 19.6 ± 1.6% (mean ± SEM, R2 = 0.98) and of 17.0 ± 1.0% (mean ± SEM, R2 = 0.99) of the isotonic volumes of immature and mature oocytes respectively.



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Figure 1. Boyle–van't Hoff plot of immature and mature rhesus monkey oocytes exposed to various concentrations of sucrose. Each point is the mean ± SEM relative volume of three oocytes at each stage considered to be immature or mature. The solid line is the regression fitting of data for immature oocytes; the dashed line is for mature oocytes.

 
Permeability characteristics of rhesus monkey oocytes
The response of oocytes to cryoprotectants was determined by measuring their volume changes as a function of time. Unlike those exposed to hypertonic solutions of sucrose, some oocytes exposed to hypertonic solutions of all three permeating cryoprotectants did not shrink spherically (Figure 2A and BGo). Volume changes during exposure to cryoprotectants were measured only for oocytes that contracted isotropically (compare Figure 2C, D and E, FGo). Rhesus monkey oocytes appeared to be rather impermeable to glycerol, as they had not returned to their isotonic volume even after being exposed to 1.0 mol/l glycerol for as long as 120 min at 30°C. Moreover, all five oocytes exposed to glycerol exhibited non-isotropic shrinkage; thus their volumes could not be measured.



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Figure 2. Photomicrographs of rhesus monkey oocytes in isotonic medium (panels A, C, E) and the same oocytes in 1.0 mol/l solutions of ethylene glycol (panels B, D) or dimethylsulphoxide (panel F). Note variation in scale bars (E and F versus A–D).

 
Seven of 10 oocytes exposed to EG shrank isotropically and their volumes were measured. The relationship between the relative volume of rhesus oocytes and time after exposure to EG is shown in Figure 3Go. Among the seven oocytes exposed to EG, three were at the MII stage of maturation, and four had not extruded their first polar body. Immature oocytes contracted to ~60% of their original volume after exposure to EG for 0.5 to 1 min (Figure 3AGo). One of four immature oocytes contracted to only 90% of its isotonic volume and remained at this volume even after 10 min. None of these four oocytes returned to their isotonic volumes after 10 min. MII oocytes responded to exposure to EG by initially shrinking to ~50% of their original volume after 1 min exposure (Figure 3BGo). Although immature oocytes had re-expanded to only 90% of their original volume after 10 min, mature oocytes did appear to reach equilibrium within that time. Nevertheless, there was no significant difference between the two maturational stages (Figure 3CGo).



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Figure 3. Relative volumes of rhesus monkey oocytes as function of time in a 1.0 mol/l solution of ethylene glycol. (A) Immature oocytes; (B) mature oocytes; (C) comparison of immature and mature oocytes. There are missing values that could not be measured at a given time. Mean values in (C) were calculated by the sum of relative volumes divided by the number of oocytes measured at a given time.

 
Five of nine oocytes exposed to DMSO did shrink isotropically, so their volumes could be measured. All oocytes exposed to DMSO returned to their isotonic volume within 10 min, and two of the five oocytes exceeded their original volume somewhat (Figure 4Go), as is to be expected due to the intracellular presence of DMSO. Immature oocytes behaved similarly to those exposed to EG, by decreasing to ~60% of their isotonic volume (Figure 4AGo). One oocyte, which had not extruded a polar body, shrank to 95% of its original volume and remained at that volume for 10 min. This oocyte was obtained from the same female (K226) as the oocyte that exhibited similar behaviour after exposure to EG (Figure 3AGo). Two MII oocytes responded rather differently, as one oocyte shrank substantially after exposure to the CPA, while the other did not (Figure 4BGo). Overall, when results for several oocytes were pooled, there were no differences between MII and immature oocytes in the permeability response to DMSO (Figure 4CGo). The volume of oocytes exposed to DMSO decreased to ~80% after 1 min exposure and then increased to their isotonic volumes within 5 min; the volume of oocytes exposed to EG decreased to ~60% and did not re-expand to their original isotonic volumes until 10 min had elapsed (Figure 5Go). Nevertheless, when pooled responses of several oocytes that had been exposed either to DMSO or to EG were compared, there was no significant difference between them.



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Figure 4. Relative volumes of rhesus monkey oocytes asfunction of time in a 1.0 mol/l solution of dimethylsulphoxide.(A) Immature oocytes; (B) mature oocytes; (C) comparison of immature and mature oocytes. There are missing values that could not be measured at a given time. Mean values in (C) were calculated by the sum of relative volumes divided by the number of oocytes measured at a given time.

 


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Figure 5. Relative volumes of rhesus monkey oocytes as a function of time in 1.0 mol/l solutions of ethylene glycol or dimethylsulphoxide.

 
The mean isotonic volume of four rhesus oocytes in this study calculated from their measured cross-sectional areas was 4.85x105 µm3. The calculated coefficients at 30°C of water permeability (Lp) in the presence of DMSO or EG, as well as of solute permeability (Ps) for both CPAs are shown in Table IGo. There were no significant differences among the water permeability coefficients of immature and mature oocytes, as well as between oocytes exposed to DMSO or to EG.


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Table I. Coefficients at 30°C for hydraulic conductivity (Lp) and solute permeability (Ps) of immature and mature (metaphase II) rhesus monkey oocytes
 
Osmotic sensitivity
Figure 6Go shows the survival, as measured by membrane integrity, of oocytes after being exposed to various concentrations of EG and then being abruptly diluted into isotonic solution. Oocytes tolerated exposure up to 2.0 mol/l EG followed by direct dilution into isotonic medium, since >80% of oocytes were morphologically intact. However, they were damaged when exposed to EG concentrations of >=3.0 mol/l, as indicated by loss of membrane integrity after dilution, especially when oocytes had been exposed to EG for 10 min (P < 0.01). Survival was reduced significantly when the oocytes were exposed to 5.0 mol/l EG for 10 min (P < 0.01). Although exposure of oocytes to solutions of 4.0 or 5.0 mol/l EG for 10 min before dilution resulted in more oocytes with damaged membranes than those exposed for 5 min, the differences were not significant.



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Figure 6. Membrane integrity of rhesus monkey oocytes after being exposed to 0.1 to 5 mol/l ethylene glycol for 5 or 10 min, followed by abrupt dilution into isotonic medium. The respective numbers of oocytes exposed to each solution for 5 min were: 0 (n = 28), 0.1 (n = 19), 0.4 (n = 14), 0.6 (n = 16), 0.8 (n = 15), 1.0 (n = 18), 2.0 (n = 19), 3.0 (n = 8), 4.0 (n = 9), 5.0 (n = 7). Those exposed to each solution for 10 min were: 2.0 (n = 8),3.0 (n = 9), 4.0 (n = 9) and 5.0 (n = 10).

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
In order to develop an effective cryopreservation protocol, it has been argued that the determination of fundamental cryobiological characteristics of each cell type is essential (Critser et al., 1997Go). These characteristics include the hydraulic conductivity (Lp) and the permeability coefficient of the plasma membrane to a cryoprotectant (Ps), as well as the sensitivity of the cell to osmotic shock. Permeability characteristics may vary greatly among cell types and among species for a given cell type (Leibo, 1986Go). For oocytes and embryos of various species, permeability characteristics of the plasma membrane are dependent on the stage of development (Friedler et al., 1988Go; Ruffing et al., 1993Go; Le Gal et al., 1994Go; Critser et al., 1997Go; Agca et al., 1998Go, 2000Go). In the present study, the osmotic responses of two categories of rhesus oocytes obtained from gonadotrophin-stimulated females were determined. It must be reiterated that immature oocytes used in the present study included all meiotic stages prior to MII.

Although NaCl solutions have been commonly used for studies of the osmotic behaviour of oocytes of many species (Leibo, 1980Go; Hunter et al., 1992Go; Ruffing et al., 1993Go; Benson and Critser, 1994Go), solutions of mono- and disaccharides can also be used effectively for the determination of osmotic behaviour of human and mouse oocytes (McWilliams et al., 1995Go). The use of saccharides for this purpose eliminates the deleterious effects observed when oocytes/embryos are exposed to very concentrated electrolyte solutions (Mazur and Schneider, 1986Go). In the present study, the disaccharide sucrose was used to determine the osmotic behaviour of rhesus monkey oocytes. As has been observed with other mammalian species, Boyle–van't Hoff plots showed that rhesus oocytes behaved as `perfect osmometers' over a wide range of osmolalities. Extrapolation of the Boyle–van't Hoff plots to infinite osmolality for immature and mature oocytes yielded osmotically inactive volumes of 20 and 17% respectively. The value for immature oocytes agreed very well with the 20% volume determined for germinal vesicle-stage oocytes of cynomolgus monkeys (Younis et al., 1996Go). It should be noted that the latter authors exposed oocytes to hypertonic solutions of NaCl, whereas the non-electrolyte, sucrose, was used in the present study.

The osmotically inactive volume obtained in the present study was similar for both immature and mature rhesus oocytes. These results differ from those reported for other stages of bovine and cynomolgus monkey oocytes. For example, one group (Ruffing et al., 1993Go) reported that the osmotically inactive volume of immature bovine oocytes was 32%, while that of mature oocytes was 24%. In both of these studies (Ruffing et al., 1993Go; Younis et al., 1996Go), immature oocytes consisted only of germinal vesicle-stage oocytes. However, in the present study, oocytes considered to be immature were all oocytes that had not extruded the first polar body, as they were obtained from females that had been treated with HCG to induce resumption of meiosis.

Permeability to water and its activation energy are among the principal determinants of the response of all types of cells to freezing and thawing (Mazur, 1970Go; Leibo, 1986Go). Coefficients of water permeability have been determined for oocytes of mice (Leibo 1980Go; Hunter et al., 1990Go, 1992Go; Benson and Critser, 1994Go; Paynter et al., 1997Go, 1999aGo), hamsters (Benson and Critser, 1994Go), rats (Agca et al., 2000Go), cattle (Myers et al., 1987Go; Ruffing et al., 1993Go; Agca et al., 1998Go), goats (Le Gal et al., 1994Go, 1995Go), humans (Hunter et al., 1990Go, 1992Go; Newton et al., 1999Go; Paynter et al., 1999bGo, 2001Go) and cynomolgus monkeys (Younis et al., 1996Go). Although a review of permeability properties of oocytes from many species has been published (Critser et al., 1997Go), to our knowledge there have been no reports on rhesus monkey oocytes.

In general, the responses of rhesus monkey oocytes to cryoprotectants were similar to those of oocytes of other species. The oocytes initially shrank when they were exposed to an hypertonic solution and reached a minimum volume within 1 min. Thereafter, the oocytes re-expanded as the cryoprotectant entered the cell, accompanied by water uptake. None of the oocytes that was exposed to glycerol shrank isotropically; thus, their volumetric changes could not be measured. However, it is clear that glycerol moved across the plasma membrane of rhesus oocytes very slowly. The oocytes partially regained their isotonic volume, but did not reach equilibrium even after exposure to glycerol for 120 min. This result was similar to that reported for mouse oocytes (Jackowski et al., 1980Go). These differences suggest that the permeability characteristics of a given cell type, e.g. oocytes, may vary among species.

Seven of 10 oocytes exposed to EG contracted isotropically, and five of nine oocytes exposed to DMSO did so. Irregular shrinkage of oocytes after exposure to cryoprotectants has also been observed in other species (Hunter et al., 1990Go; Ruffing et al., 1993Go; Le Gal et al., 1995Go). Exposure of mouse oocytes to 1.5 mol/l DMSO caused depolymerization of microfilaments, which was associated with disruption of the actin network and alteration of the cell surface (Vincent et al., 1990aGo). However, mouse oocytes have been found to tolerate exposure to 4.0 to 8.0 mol/l EG for 5 min without any visible alteration of microfilaments (Hotamisligil et al., 1996Go). Unlike mouse oocytes, rabbit oocytes and zygotes seem to tolerate exposure to DMSO, since exposure to this cryoprotectant did not cause depolymerization of actin (Vincent et al., 1989Go, 1990bGo). The effects of cryoprotectants on the organization of the cytoskeleton of macaque oocytes have previously been studied using glycerol, but not DMSO or EG (Younis et al., 1996Go). The latter authors showed that exposure of cynomolgus oocytes to glycerol resulted in depolymerization of F-actin around the cortex of oocytes. It is speculated therefore that DMSO and EG might also affect the organization of microfilaments of rhesus oocytes, which in turn may cause the plasma membrane of some oocytes to collapse upon exposure to these cryoprotectants.

Oocytes exposed to 1.0 mol/l DMSO reached equilibrium after 5 min, whereas those exposed to EG required 10 min to return to their initial isotonic volume. This result was similar to that described for bovine oocytes at 22°C (Agca et al., 1998Go), although these authors reported differences between germinal vesicle stage and MII oocytes. In that study, osmotic responses of germinal vesicle-stage oocytes suggested that they were more permeable to DMSO than MII oocytes, whereas the opposite was true for oocytes in EG. Substantial differences in permeability characteristics between mature and immature oocytes have also been observed in the goat (Le Gal et al., 1994Go) and rat (Agca et al., 2000Go). In the present study, no significant differences were found in permeability responses between mature and immature oocytes. This was most likely due to the small number of oocytes that contracted spherically and could be assessed. There was no clear evidence that cumulus cells surrounding oocytes were responsible for these differences. As shown in Figure 2Go, oocytes in Figure 2AGo were surrounded by fewer cumulus cells than those in Figures 2C and 2EGo; all three oocytes had not extruded the polar body. However, the oocytes in Figures 2C and 2EGo had contracted spherically, whereas that in Figure 2AGo had not. Moreover, even among oocytes of the same meiotic stage there was a large variation in their responses to exposure to DMSO and EG (Figures 3 and 4GoGo). Thus far, it has not been possible to identify factors that may have contributed to differences in permeability responses among individual oocytes, though this may well be due to the genetic heterogeneity of rhesus monkeys used. As shown in a previous report for bovine oocytes (Ruffing et al., 1993Go), sizeable differences exist among the osmotic responses of individual oocytes, suggesting that there may be considerable variation in permeability characteristics among oocytes of all species other than inbred strains of mice. Among outbred ICR mice, it has been reported that, although there was no significant variability within animals, the water permeability of oocytes collected from different females differed significantly (Benson and Critser, 1994Go). In contrast, the water permeability of oocytes from different females of the genetically homogeneous hybrid strain, B6D2F1, did not differ significantly (Leibo, 1980Go).

In the present study, the osmotic responses of rhesus oocytes were measured only at 30°C. Previous studies had shown rhesus oocytes to be very sensitive to chilling injury (Songsasen et al., 2002Go); hence it was concluded provisionally that the successful cryopreservation of these oocytes would require them to be cooled at high rates in order to circumvent any injury that might result from exposure to temperatures near 0°C for several minutes, as occurs during standard methods of equilibrium cooling. This suggests that mathematical modelling of the response of rhesus oocytes when cooled to subzero temperatures by equilibrium cooling is unlikely to yield a practicable procedure, as such a method would inevitably expose oocytes to damaging temperatures near 0°C for more than 1 min.

After having determined the hydraulic conductivity of several rhesus oocytes, these values were compared with those values reported previously. The mean value of Lp for immature and mature rhesus oocytes was ~1 µm/min/atm at 30°C (see Table IGo). In order to make a comparison, values for human oocytes of 0.43 µm/min/atm at 3°C (McGrath et al., 1995Go) and 0.78 µm/min/atm at 22°C (Newton et al., 1999Go) were used, together with a value for cynomolgus monkey oocytes of 0.23 µm/min/atm at 0°C (Younis et al., 1996Go). (In the latter report, Lp was given as 3.8x10–14 m3/N/s; although not specified, the reference temperature was 0°C; M.Toner and A.I.Younis, personal communication.)

As had been performed for rhesus oocytes in the present study, both groups (McGrath et al., 1995Go; Newton et al., 1999Go) had determined Lp values for human oocytes exposed to DMSO. In all these cases, observations had been made at a single temperature; hence, in order to make this comparison an Arrhenius plot was constructed (Figure 7Go) in which the solid line is the calculated regression of the four values indicated by the solid points. The open symbols are the values for human oocytes determined by others (Paynter et al., 1999bGo, 2001Go), and these were not used for the regression calculation. The fact that the four independent measurements yielded a straight line suggests that they were consistent with each other. The Arrhenius plot yields an activation energy of 6.85 kcal/mol for Lp of primate oocytes. Recent determinations of Lp values for human oocytes in the presence of DMSO and propylene glycol yielded respective activation energies of 15 kcal/mol and 11 kcal/mol (Paynter et al., 1999bGo,2001Go). Although there were discrepancies in the activation energy for Lp between primate oocytes (human and rhesus monkey) plotted in the present study and that reported previously for human oocytes (Paynter et al., 1999bGo, 2001Go), these values were lower than those of 23.52 and 22.48 kcal/mol reported for mouse oocytes in the presence of DMSO and propylene glycol respectively (Paynter et al., 1999aGo). In the present study, the Lp of rhesus oocytes in the presence of DMSO was 0.96 µm/min/atm, which was higher than 0.64 µm/min/atm for mouse oocytes measured at the same temperature. When comparing the Lp and activation energy values for the two species, it was concluded that, in the presence of DMSO, rhesus oocytes appeared to be more permeable to water than did mouse oocytes.



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Figure 7. Arrhenius plot of hydraulic conductivity (Lp) of primate oocytes. {blacklozenge}, values for rhesus monkey oocytes (data derivedfrom the present observations); {blacktriangledown}, data for human oocytes at 22°C {equiv} 3.39x10–3 °K (Newton et al., 1999Go); •, data for human oocytes at 3°C {equiv} 3.62x10–3 °K (McGrath et al., 1995Go); {blacksquare}, data for cynomolgus monkey oocytes at 0°C {equiv} 3.66x10–3 °K (Younis et al., 1996Go). Open symbols are data for human oocytes (Paynter et al., 1999, 2001Go); {triangleup}, oocytes in propylene glycol; {circ}, oocytes in dimethylsulphoxide.

 
Most vitrification solutions contain high concentrations of cryoprotectants (Rall, 1987Go); these may create osmotic stresses and cause osmotic injury upon dilution. In the present study, it was shown that rhesus oocytes tolerate exposure up to 2.0 mol/l EG followed by direct dilution into isotonic medium, but that they are damaged when diluted directly from 4 or 5 mol/l EG. This finding agrees with that reported by others (Oda et al., 1992Go) for mouse zygotes. With bovine oocytes, exposure to a freezing medium containing a high concentration of cryoprotectants compromised their fertilizing ability and developmental competency (Arav et al., 1993Go; Martino et al., 1996Go). It was also shown (Oda et al., 1992Go) that osmotic injury of zygotes could be reduced by dilution from an hypertonic solution into isotonic medium at an elevated temperature, and could be prevented by the use of sucrose as an osmotic buffer at 37°C. These authors suggested that osmotic shock is more critical to the survival of ova than inherent toxic consequences of exposure to concentrated solutes. Therefore, it is suggested that the step-wise dilution or use of mono- or disaccharides as an `osmotic buffer' may be preferable for the successful recovery of rhesus oocytes after vitrification. Moreover, the choice of cryoprotectant will also be important for the successful cryopreservation of rhesus oocytes.

In the present study, the permeability responses and sensitivity to osmotic shock of rhesus oocytes were determined. Rhesus oocytes were similar to oocytes of other species in that they behaved as a perfect osmometer; moreover, they appeared rather impermeable to glycerol but permeable to DMSO and EG. The results obtained in the present study should aid in the development of a method by which may rhesus oocytes be cryopreserved.


    Acknowledgements
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
The authors thank Drs Il-Jeoung Yu and Richard Harrison for their contributions to these research investigations, and Melissa Johnston and SueAnn Schneider for technical assistance. The staff of the Tulane Regional Primate Research Center are also thanked for their care of the animals. These studies were supported by Grant NIH-5RO1 RR13439 awarded to C.A.V. The monkeys were provided through the support of Grant NIH-5P51 RR00164-39 awarded to the Tulane Regional Primate Research Center.


    Notes
 
5 To whom correspondence should be addressed. E-mail: sleibo{at}acres.org Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
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Submitted on January 3, 2002; accepted on March 22, 2002.