Membrane transport properties of mammalian oocytes: a micropipette perfusion technique

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1 Membrane transport properties of mammalian oocytes: a micropipette perfusion technique D. Y. Gao, J. J. McGrath, Jun Tao, C. T. Benson, E. S. Critser and J. K. Critser 1Cryobiology Research Institute, Methodist Hospital of Indiana, Indianapolis, IN 46202, USA; department of Mechanical Engineering, Michigan State University, East Lansing, MI 48829, USA; ^Departments of Physiology/Biophysics and Obstetrics and Gynecology, Indiana University School of Medicine, Indianapolis, IN 46202, USA; and ^Department of Veterinary Clinical Sciences, School of Veterinary Medicine, Purdue University, W. Lafayette, IN 47907, USA 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\pn\10\gm\mdiameter 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 (L p) found to be 1.05, 0.45 and 0.26 \gm\mmin \m\1 atm \m\1at 30\sdeg\C,22\sdeg\Cand 12\sdeg\C,respectively. The corresponding activation energy, Ea, for Lp was calculated to be 13.0 kcal mol\m\1. These values are in agreement with data obtained is that the extracellular by other techniques. One of the major advantages of this technique 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. The second advantage is that the time required for mixing the original cell suspension and perfusion medium is minimized, allowing for accurate control of the extracellular osmolality and ensuring accuracy of the subsequent mathematical formation. This technique also has wide applicability in determining the membrane transport properties of mammalian oocytes, embryos and other cell types. Introduction Cryopreservation offers the possibility of preserving oocytes of patients who are at risk of losing ovarian function through pelvic disease, surgery or clinical treatment involving radio therapy and chemotherapy. Cryopreservation of oocytes cir cumvents many of the ethical and legal objections to human embryo cryopreservation. Because of the relatively short fertile lifespan of mammalian oocytes, successful cryopreservation of oocytes would also provide the potential to supply a large number of viable and developmentally competent oocytes for use in clinical and research work (for example in vitro fertiliz ation, genetic engineering and many other reproductive tech nologies). Although it has been shown that cryopreserved * Correspondence. Received 2 May oocytes from several species have been fertilized in vitro (Trounson, 1986; Friedler et al, 1988), live births have been reported only for mice, rabbits and humans (Whittingham, 1977; Chen, 1986; Van Uem et al, 1987; AlHasani et al, 1989; Vincent et al, 1989; Schroeder et al, 1990; Kono et al, 1991). Cryopreservation procedures for mammalian oocytes have been based primarily on empirical approaches. New and advanced cryopreservation techniques now need to be developed to minimize the potential injury caused by the cryopreservation process and to improve the developmental competence of cryopreserved oocytes. This requires a further understanding of the cryobiology of mammalian oocytes. The addition of cryoprotectant to cells before cooling and its removal after warming may generate deleterious osmotic volume changes of cells if the procedures for addition and dilution are not designed carefully (Penninckx et al, 1984; Critser et al, 1988; Gao et al, 1993). The flux of water

2 and cryoprotectant play critical roles during the freezing process (Muldrew and McGann, 1990, 1994). Therefore, to optimize the procedures for freezing, as well as addition and removal of cryoprotectant, knowledge of various biophysical properties of the cells is needed (Mazur, 1990). These proper ties include permeabilities of the plasma membrane to water (hydraulic conductivity, L ) and to cryoprotectants (for example glycerol, dimethylsulfoxide and 1,2propanediol), their associated reflection coefficients ( ) and activation energies (fa) Several methods have been developed to quantify the plasma membrane permeability to water and solutes. One of these, the microdiffusion chamber method, has proved useful for the determination of the membrane hydraulic conductivity (L ) of a number of cell types when only water flows across the membrane (McGrath and Sherban, 1989; Hunter et al, 1992a). This method has also yielded activation energies for membrane hydraulic conductivities that match those determined using alternative methods (Hunter et al, 1992b; Leibo, 1980). How ever, in more complex cases, it is useful to determine the membrane perméabilités of both water and cryoprotectant for the coupled flow of both. Unfortunately, in some cases, the microdiffusion chamber method has limitations associated with mathematical complexity and the unknown physicochemical parameters required for accurate parameter estimations (McGrath et al, 1992a). This is due to difficulties in dis tinguishing between the kinetics of diffusion across the dialysis membrane and the mixing times for flow across cell membranes. Thus, a major motivation for the development of the microperfusion method presented here is to remove the com plexity associated with mathematical modelling of the coupled mass transfer of ternary solutions through the dialysis mem brane in the microdiffusion chamber. Although the results for the coupled flow of water and cryoprotectant determined using the microperfusion method appear reasonable (McGrath et al, 1992b), there is insufficient data for comparison to establish the validity of the microperfusion method. Therefore, as a first step in validating the microperfusion method, a simple experimental case was considered here: that of determining oocyte water permeability when water efflux is induced by exposure of the oocyte to a hypertonic media containing an impermeable solute (NaCl). The basic concept of the microperfusion method involves immobilizing a biological sample (here, single oocytes) in a microvolume of initial solution (12 µ ) followed by rapid perfusion with a much larger volume of new solution (about 1 ml). Before, during and after the perfusion process, the volume of the oocyte and changes in morphology are recorded by a videocamera until osmotic equilibrium is achieved. Videotaped images are processed using a digital image analyser to determine the time dependence of the change in oocyte volume. Statistical parameter estimation is performed to deter mine the passive membrane transport properties of the oocyte membrane. The microperfusion method was applied to determine the L for simple cell shrinkage experiments in which unfertilized mouse oocytes, placed initially in isotonic medium, were exposed to hypertonic medium containing an impermeable solute. The temperature of the sample was controlled by a computeraided control device throughout the experiments at set temperatures of 30 C, 22 C or 12 C The results obtained match previous results. Materials and Methods and activation To study the membrane transport properties energies of an individual cell membrane, the dynamic osmotic response of the cell to changes in extracellular conditions at different temperatures needs to be measured. Four technical requirements must be satisfied if a light microscope is used: (i) the change in extracellular condition must be feasible, rapid and controllable; (ii) the cell must be held at a fixed location under the microscope during the change of the extracellular medium (otherwise, the cell could be either out of focus or disappear entirely from the optical field of the microscope); (iii) the cell response (volume change) to the change of extracellular condition must be measurable and resolvable (in time and space); (iv) the temperature of the cell suspension must be controllable. The microperfusion technique used in this study was designed to meet all these requirements. The methodology is detailed below through a sample study, i.e. determination of the hydraulic permeability coefficient (L ) and associated activation energy ( a) of mouse oocytes. Source of oocytes Female ICR mice (78 weeks old) were obtained from Harlan SpragueDawley (Indianapolis, IN) and used throughout the experiments. The mice were superovulated by injection of 5 iu of pregnant mares' serum gonadotropin (PMSG; Diosynth Inc., Chicago, IL) i.p. followed 48 h later by a second injection of 7.5 iu of hcg (Sigma Co., St Louis, MO) (Critser et al, 1987). Mouse oocytes were collected 1617 h after the hcg injection and used for the experiment within 30 min to 2 h. A modified Tyrode's solution buffered with Hepes (TALPHepes) (Bavister et al, 1983), supplemented with 4 mg BSA ml1 (ph 7.4) and kept at 37 C, was used to wash and maintain the oocytes during micromanipulation. The osmolality of the TALPHepes solution was 286 mosmol. The cumulus oophorus cells were separated from the oocytes using hyaluronidase (1 mg ml ). After hyaluronidase treatment, the oocytes were washed using the TALPHepes solution before the microperfusion procedure. Microperfusion Two specific types of micropipettes were made of 1.2 mm glass capillary tubes using needle/pipette a puller (David Kope Instruments, Tujunga, CA) and Microforge MF1 (Technical Products International Inc., St Louis, MO). Type A micropipettes had an 810 µ diameter tip opening and were used to apply negative pressure and held only the zona pellucida of an oocyte during the perfusion process (Fig. 1). (The zona pellucida is a relatively thick, translucent, protein matrix that surrounds the plasma membrane of fully grown mammalian oocytes. It is highly porous and does not serve as a per meability barrier to even very large macromolecules or viruses (Bleil and Wassarman, 1980).) Type B micropipettes had a

3 ^ Micropiperte Zona pellucida Negative pressure to hold zona pellucida Oocyte plasma membrane Fig. 1. Schematic diagram showing a type A micropipette with an 810 µ (internal diameter) tip opening used to apply negative pressure and hold only the zona pellucida of an oocyte. and were used to pm (internal diameter) tip opening collect microvolume amounts of the cell suspension accurately. In each experiment, a 2 µ solution containing one mouse oocyte was collected using the type micropipette filled with mineral oil and transferred onto a 3.5 cm (diameter) cell culture dish. A micromanipulation stage (Frank E. Fryer Co., Scientific Instruments, Chicago, IL) was used and the zona pellucida of an oocyte was held by a type A micropipette under negative pressure. Meanwhile, the oocyte was perfused by adding 1 ml of 900 mosmol NaCl solution into the 2 µ cell suspension (Fig. 2). The negative pressure was controlled using a Hamilton syringe (threaded plunger syringe 170STPLT; Hamilton Co., Reno, NV) linked to the type A micropipette by a plastic tube. The syringe and plastic tube were both filled with mineral oil. The negative pressure was low enough to hold the oocyte during perfusion. (Measurement of the magnitude of negative pressure was not conducted in these experiments.) The same experiment was conducted at different temperatures. The oocytes obtained from five mice were pooled. Twelve or more oocytes were randomly selected for perfusion at each tempera ture. The temperature of the cell suspension during the perfusion process was controlled by a temperature control device (Temperature controller 1221: Frank E. Fryer Co., Scientific Instruments, Chicago, IL). The temperature of the perfusion medium was adjusted using a temperaturecontrolled water bath and waterice mixture. The maximum temperature variation of the cell suspension during each experiment was ± 1 C. The temperature change of the cell suspension was monitored during experiments using a copperconstantan thermocouple (Precision Fine Wire: Omega Engineering, Inc., Stamford, CT). Data collection and analysis The morphology and dimensional change of the oocyte before, during and after the perfusion were observed using an inverted Nikon microscope (Nikon Inc., Garden City, NY) at a magnification of 200 and recorded by a video camera until osmotic equilibrium was achieved. The videotaped images of mouse oocytes were processed using a digital image analyser (The Dynamic Morphology System: Motion Analysis Corp., Santa Rosa, CA) to determine the time dependence of the change in the volume of the oocyte during the perfusion process. Only cells which remained circular in crosssection were analysed. A total of 12 oocytes were analysed at each temperature. The average radius of each oocyte was calculated at experimental times of interest by the image analyser. The average radius was defined as the mean length of the radii from the centroid of the twodimensional oocyte image to its boundary. The cell volumes were computed assuming sphericity. In fact, over 95% of the oocytes maintained spherical shape before, during and after perfusion (Fig. 3). Oocytes that did not maintain spherical shape were excluded from image analysis. Data on cell volume history were entered into the parameter estimation portion of the computer software (McGrath et al, 1992b) as a matrix of time and corresponding cell radii. Thermodynamic modelling and mathematical formulation for water across the oocyte membrane The classical formulation of coupled, passive membrane transport was developed by Kedem and Katchalsky (1958) using the theory of linear irreversible thermodynamics. The formulation includes two coupled firstorder nonlinear ordinary equations which describe the total transmembrane volume flux and the transmembrane permeable solute flux, respectively. To test the validity of the microperfusion technique in the present study, experiments were designed to determine the total transmembrane volume flux caused by water transpor tation only (the oocyte was perfused by hyperosmotic NaCl solution without permeating solutes). The corresponding mathematical equations describing the kinetics of water transport through the cell membrane are as follows: 1 dv(f). ; L Jv (CehC,JRT (1) v ' * jr salt salt' salt sale, / V(0) vh\ \y(f) J _ V(0) Cl (3) Csah(0) 286 mosmol (4) C^u 900 mosmol (5)

4 '), (a) Micropipette to hold the zona pellucida Perfusion solution Cell culture plate Objective lens of light microscope (b) Micropipette to hold the zona pellucida 2 µ of cell suspension medium with one mouse oocyte Cell culture plate Objective lens of light microscope Fig. 2. Schematic diagram showing the zona pellucida of an oocyte held by a type A micropipette under negative pressure (a), and the oocyte perfused by addition of 1 ml of 900 mosmol NaCl solution into the 12 µ cell suspension (b). where Jv the total cell volume flux, R gas constant ( atm mole ' K J), T " temperature (K), t time (s), V cell volume (pm3), Ac cell surface area (pm2), L hydraulic conductivity or water permeability coefficient x (pm min atm Csait concentration (osmolality) of salts, Vh osmoticallyinactive cell volume, 0 initial con intracellular, dition (f 0), ' e extracellular, and Cl were measured for each individual oocyte. It was assumed that (i) extracellular concentration of nonpermeating NaCl was con stant, and (ii) the mixture of solutions during perfusion was instantaneous. The initial volume, V(0), and membrane surface area, Ac, of each oocyte were calculated from the experimental data. It was assumed that Ac was a variable (as a function of spheric cell volume) during volume shrinkage of the oocyte. The osmotically inactive volume, Vh, was taken to be 20% of the initial oocyte volume. This value is based on results (Leibo, 1980; Hunter et al, 1992b) that are reproducible with quite small standard deviations about the mean values. Parameter estimation methods In all cases to be discussed here, the BoxKanemasu method of parameter estimation was applied (Beck and Arnold, 1977). The method of estimating hydraulic conductivity (L ) is based on Gauss linearization with ordinary least squares (McGrath et al, 1992b). Results Mouse oocytes were initially equilibrated to an isotonic saline solution (286 mosmol). After perfusion at 12 ± 1 C, 22 ± 1 C and 30 ± 1 C, respectively, the oocytes were subjected to a onestep change in extracellular osmolality to 900 mosmol. The morphology and volume of the oocyte in its osmotic equilibrium states before and after perfusion are demonstrated in Fig. 3. From microscopic observation and image analysis, it was found that (i) oocytes shrank immediately in response to

5 ( f) UT) Uro)xexp (6) where L ( ) is the water permeability coefficient of the oocyte plasma membrane at any temperature T(K), L_(T0) is any one of the known L values at the corresponding temperature, T0, determined in the present study (for example, when : T0 22 C 295K, Lp (22 C) 0.45 pm min atm J). " " Discussion microperfusion technique was used to determine the hydraulic permeability, L of the ICR mouse oocyte, as well as The, the associated activation energy. The values obtained conform to data obtained by other techniques, including the photomicroscopic method (Leibo, 1980) and the microdiffusion technique (Hunter et al, 1992b); these are the only two optical techniques of which we are aware for the measurement of the membrane permeability of individual cells. In the investigation by Leibo (1980), a mean I of 1 x atm 0.44 ± 0.03 (mean ± sem) pm min (20 C) was deter mined for the unfertilized oocyte. This correlates well with that determined by the microperfusion technique and associated software of (mean ± sd) pm min1 atm"1. Simi larly, Hunter et al (1992b) predicted the mean L value for the mouse oocyte at 20 C to be (mean ± sd) pm min atm1. The a value for mouse oocyte of 13.0 kcal mol1, determined using the microperfusion technique over the tem perature range of 1230 C, was similar to those determined by Leibo (1980) for fertilized (13.3 kcal mol1) and unfertilized (14.5 kcal mol ) oocytes. The study at lower temperatures by Toner el al (1990) calculated a similar a for unfertilized mouse oocytes of 13.3 kcal mol further corroborating the present data. A single oocyte that had been suspended in a small amount (2 µ ) of original solution (286 mosmol) was perfused by a much larger amount (1000 µ ) of hyperosmotic solution (900 mosmol). This ensured a rapid mixing of two solutions and a very quick exposure of the oocyte to the perfusion medium (900 mosmol). The mathematical modelling of the cell kinetic osmotic response was therefore simpler in comparison with the microdiffusion chamber technique (McGrath et al, 1992a) because the mathematical formulation and computation of water and solute diffusion across a dialysis membrane were avoided. As shown in Eqn 5, the extracellular osmolality was assumed to be a constant (900 mosmol), i.e. it was assumed that extracellular osmolality was instantaneously changed from the original 286 mosmol to 900 mosmol during the perfusion process. The accuracy of this assumption was supported by the video image observation and by McGann et al (1982), who estimated that the mixing time of two small amounts of solution (< 10 ml) during perfusion was less than 0.3 s. This assumption was also supported by agreement between the L and a values determined by the current method and by alternative techniques. The current study has shown that the microperfusion technique is a very useful and powerful method for studying the membrane transport properties of mouse oocytes. Pre sumably this technique can also be used for studying oocytes 3. Morphology and volume changes of an oocyte in osmotic equilibrium before and after perfusion with 900 mosmol NaCl solution Fig. at (2) 22 C. (a) In isotonic medium (286 mosmol); (1) type A micropipette, zona pellucida of a mouse oocyte and (3) mouse oocyte. (b) In 900 mosmol medium., the extracellular osmotic change, indicating a fast mix between the perfusion medium (1 ml) and the original cell suspension (2 µ ); (ii) because the zona pellucida was held by a micro pipette, the oocyte was well focused in the microscope view field during and after perfusion, ensuring precision in the measurement of changes in cell volume, especially during early initial cell shrinkage; and (iii) during shrinkage, most oocytes maintained the spherical shape satisfying the basic assumption of the image analysis. Figure 4 shows the measured volume change of four oocytes as a function of time at 22 C. A comparison of measured and predicted volume histories of one of the four oocytes (best fit between predicted and measured volume history) is shown in Fig. 5. The predicted history or curve was obtained from Eqns 1 and 2, when the L was adjusted to provide the best fit of the theoretical curve to the experimental data points for that particular oocyte. The deter mined average L (mean ± sd, 12) is 0.26 ± 0.03, 0.45 ±0.11, pm min"1 atm' at 12, 22 and 30 C, respectively. Figure 6 shows an Arrhenius plot of the L data. There is a between ln(l ) and 1000/T over the tem perature range studied (the correlation coefficient of the linear regression was determined to be 0.98). The a was calculated from these data to be 13.0 kcal mol" From the a one can compute or estimate the L value at any temperature, T, by the linear relationship. following equation (Arrhenius relation):

6 _ O X o E î ' r * co E o Z 0.6 s w fi. * * m < Time (s) Fig. 4. Experimental data showing four different mouse oocyte volume changes in 900 mosmol NaCl solution as a function of time at 22 C The normalized cell volume is defined as the cell volume divided by the original cell volume in the isotonic condition. 1.1 t E o Time (s) mouse oocyte exposed to 900 mosmol NaCl solution at 22 C. A are based on the best fit Fig. 5. Shrinking osmotic response of a comparison of measured ( ) and predicted () volume histories. The predictions L determined here for individual oocytes. The normalized cell volume is defined as the cell volume divided by the original cell volume in the isotonic condition. that have a zona and embryos of other mammalian species pellucida. In addition, the technique should be applicable to any cell type or cell system that has an outer 'shell', or nonliving cell wall, to which the pipette can be affixed, which includes many nonmammalian oocytes and embryos (for example, fish, insects, amphibians, echinoderms, algae and higher plant cells). It may also be applicable to smaller cells like yeast if powerful enough microscope objectives are used. A further application may be the measurement of osmotic volume changes in small, multicellular mammalian systems such as Islets of Langerhans, fetal pancreata, or corneas. (Even if the cells in contact with the pipette are damaged, the cells

7 /T(K) Fig. 6. Arrhenius plot of the L data (mean ± sd; 12), i.e. ln(lp) versus 1000/ (T: temperature) (r2 098). apart from the pipette should still be alive and function well in osmotic response.) The following major advantages of the microperfusion technique are confirmed or expected. First, the extracellular osmotic condition can readily be changed 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 easily be removed and the cell reperfused with a different perfusion medium. Second, the time required for mixing the original cell suspension and the perfusion medium is minimized by the quick perfusion process, ensuring the ac curate control of the extracellular osmolality and the accuracy of the subsequent mathematical formulation and modelling. Finally, the technique has wide applicability in the determi nation of membrane transport properties not only of mam malian oocytes and embryos but also of other cell types. This work was supported by Methodist Hospital of Indiana, Inc. The authors thank K. Vernon for assistance with manuscript preparation. References AlHasani S, Kirsch J, Diedrich K, Blanke S, van der Ven H and Krebs D (1989) Successful embryo transfer of cryopreserved and in vitro fertilized rabbit oocytes Human Reproduction Bavister BD, Leibfried ML and Lieberman G (1983) Development of preimplan tation embryos of the golden hamster in a defined culture medium Biology of Reproduction Beck JV and Arnold KJ (1977) Parameter Estimation in Engineering and Science. John Wiley & Sons, New York Blei! JD and Wassarman PM (1980) Structure and function of the zona pellucida: identification and characterization of the proteins of the mouse oocyte's zona pellucida Developmental Biology Chen C (1986) Pregnancy after human oocyte cryopreservation Lancet i Critser JK, HuseBenda AR, Aaker DV, Arneson BW and Ball GD (1987) Cryopreservation of human spermatozoa. I. Effects of holding procedure and seeding on motility, fertilizability and acrosome reaction Fertility and Sterility Critser JK, HuseBenda AR, Aaker DV, Arneson BW and Ball GD (1988) Cryopreservation of human spermatozoa. III. The effect of cryoprotectants on motility Fertility and Sterility (1988) Cryopreservation of embryos and Friedler S, Giudice LC and Lamb EJ ova Fertility and Sterility Gao DY, Ashworth E, Watson PF, Kleinhans KW, Mazur and Critser JK (1993) Hyperosmotic tolerance of human spermatozoa: separate effects of glycerol, sodium chloride, and sucrose on spermolysis Biology of Reproduction Hunter JE, Bernard A, Fuller BJ, McGrath JJ and Shaw RW (1992a) Plasma membrane water permeability of human oocytes: the temperature depen dence of water transport in individual cells Journal of Cellular Physiology Hunter JE, Bernard A, Fuller BJ, McGrath JJ and Shaw RW (1992b) Measurements of the membrane water permeability (L ) and its temperature dependence (activation energy) in human fresh and failedto fertilize oocytes and mouse oocytes Cryobiology Kedem O and Katchalsky A (1958) Thermodynamic analysis of the permeability of biological membranes to nonelectrolytes Biochimica et Biophysica Acta Kono, Kwon and Nakahara (1991) Development of vitrified mouse oocytes after in vitro fertilization Cryobiology Leibo SP of fertilized and (1980) Water permeability and its activation energy unfertilized mouse ova Journal of Membrane Biology McGann LE, Turner AR and Turc JM (1982) Microcomputer interface for rapid measurement of average volume using an electronic particle counter Medical and Biological Engineering and Computing

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