Permeability of the rhesus monkey oocyte membrane to water and common cryoprotectants

Abstract

Successful cryopreservation of oocytes of the rhesus monkey (Macaca mulatta) would facilitate the use of this valuable animal model in research on reproduction and development, while providing a stepping stone towards human oocyte cryopreservation and the conservation of endangered primate species. To enable rational design of cryopreservation techniques for rhesus monkey oocytes, we have determined their osmotic and permeability characteristics in the presence of dimethylsulfoxide (DMSO), ethylene glycol (EG), and propylene glycol (PROH), three widely used cryoprotectants. Using nonlinear regression to fit a membrane transport model to measurements of dynamic cell volume changes, we estimated the hydraulic conductivity (L(p)) and cryoprotectant permeability (P(s)) of mature and immature oocytes at 23.5 degrees C. Mature oocyte membranes were most permeable to PROH (P(s) = 0.56 +/- 0.05 microm/sec) and least permeable to DMSO (P(s) = 0.24 +/- 0.02 microm/sec); the permeability to EG was 0.34 +/- 0.07 microm/sec. In the absence of penetrating cryoprotectants, mature oocytes had L(p) = 0.55 +/- 0.05 microm/min/atm, whereas the hydraulic conductivity increased to 1.01 +/- 0.10, 0.61 +/- 0.07, or 0.86 +/- 0.06 microm/min/atm when mature oocytes were exposed to DMSO, EG, or PROH, respectively. The osmotically inactive volume (V(b)) in mature oocytes was 19.7 +/- 2.4% of the isotonic cell volume. The only statistically significant difference between mature and immature oocytes was a larger hydraulic conductivity in immature oocytes that were exposed to DMSO. The biophysical parameters measured in this study were used to demonstrate the design of cryoprotectant loading and dilution protocols by computer-aided optimization.

Figure 1

Typical shrink-and-swell response of a mature rhesus monkey oocyte during exposure to a penetrating cryoprotectant additive. A: Oocyte held in isotonic medium within the tip of an outer pipette, just prior to cryoprotectant exposure. Subsequent images show the same oocyte after exposure to a 1.1 M solution of ethylene glycol for 5 sec (B), 1 min (C), and 16 min (D). Scale bar represents 20 µm. [See color version online at www.interscience.wiley.com.]

Figure 2

Volume of mature (closed symbols; solid line) and immature (open symbols; dashed line) rhesus monkey oocytes, normalized to the isotonic cell volume, as a function of time exposed to hypertonic PBS. Symbols and error bars represent the average and standard error, respectively, of the normalized volumes measured at each timepoint. Whereas the data are from experiments with four mature and two immature oocytes, measurements could not always be made at each timepoint, resulting in missing values; the number of measurements averaged at each time-point is indicated by the size of the corresponding circular symbol (the smallest symbols represent n=1, and the largest represent n=4). The solid and dashed curves represent model predictions for mature and immature oocytes, respectively, using averaged values of the hydraulic conductivity (see Table 1) and osmotically inactive volume fraction (see text).

Figure 3

Volume of mature (closed symbols; solid line) and immature (open symbols; dashed line) rhesus monkey oocytes, normalized to the isotonic cell volume, as a function of time exposed to DMSO (A), EG (B), or PROH (C). Symbols and error bars represent the average and standard error, respectively, of the normalized volumes measured at each timepoint. Whereas the data are from experiments with four to seven mature oocytes and two to four immature oocytes, measurements could not always be made at each timepoint, resulting in missing values; the number of measurements averaged at each timepoint is indicated by the size of the corresponding circular symbol (the smallest symbols represent n=2, and the largest represent n=7). The solid and dashed curves represent model predictions for mature and immature oocytes, respectively, using averaged values of the membrane permeability parameters (see Table 1) and osmotically inactive volume fraction (see text).

Figure 4

Correlation of rhesus oocyte viability with the predicted maximum volume excursion during exposure to 0.1–5.0 M EG for 5 (circles) or 10 min (squares), followed by abrupt dilution in isotonic media. Viability data and oocyte permeability characteristics are adapted from Songsasen et al. (2002a). Closed symbols represent the predicted maximum volume reduction during EG addition, and open symbols indicate the predicted maximum volume increase during the subsequent dilution step.

Figure 5

Predicted response of rhesus monkey oocytes during addition of PROH to achieve a final intracellular concentration of 1.5 M. A: Intracellular concentration of PROH. B: Oocyte volume, normalized to the isotonic cell volume; the horizontal dotted line indicates the lower bound of the assumed range of tolerable volume excursions (75–125% of the isotonic volume). Dashed curves represent the response to a nonoptimized single-step CPA addition process, in which oocytes are transferred directly to a solution containing 1.5 M PROH. Solid curves represent the response to an optimized two-step addition process, in which oocytes are first exposed to a solution containing 1M PROH for 1.9 min before transfer to the 1.5 M PROH solution.

Figure 6

Predicted response of rhesus monkey oocytes during dilution of PROH, which is initially present at an intracellular concentration of 1.5 M. A: Intracellular concentration of PROH. B: Oocyte volume, normalized to the isotonic cell volume; the horizontal dotted lines indicate the upper and lower bounds of the assumed range of tolerable volume excursions (75–125% of the isotonic volume). Dashed curves represent the response to a nonoptimized single-step dilution process, in which oocytes are transferred directly to isotonic medium. Solid curves represent the response to an optimized two-step dilution process, in which oocytes are first exposed to a solution containing 0.6 M PROH for 6.5 min before transfer to isotonic medium. Dash-dotted curves represent the response to an optimized two-step dilution method that uses sucrose as an osmotic buffer: oocytes are first exposed to a solution containing 1Msucrose for 32 sec before transfer to isotonic medium.