Developmental ability of vitrified mouse oocytes expressing water channels

Abstracts / Cryobiology 63 (2011) 306–342 stage follicles), seminiferous tubules (permitting more than 8 weeks of tissue viability in vitro post-thawing) and testicular tissue (resulting in resumption of spermatogenesis after xenografting or in vitro culture). These approaches are likely to become reasonable options, especially when explored with other emerging tools, including in vitro follicle and sperm development, as well as germinal vesicle storage Most cryo-tools are considered largely in the context of mammals, but there has been significant progress in other taxa, including birds, amphibians and marine life. For example, our laboratory has recently demonstrated progress in cryopreservation of coral sperm and fish embryos. Finally, it appears that there is the beginning of an appreciation for GRBs beyond the immediate interest of cryobiologists and reproductive biologists. Introduction of biomaterials cryopreservation is finding itself being added as an important strategy to species recovery planning. Cryobiology is being recognized not only as a method for storing and moving important genomes, but as an essential component of being able to monitor gene diversity and diseases in rare populations. In summary, the last 25 years has seen cryobiology gradually emerge as a contributing science to the field of conservation biology, especially of intensively managed, endangered species. Conflicts of interest: None declared. Source of funding: None declared. doi:10.1016/j.cryobiol.2011.09.014

12. Optimization of rhesus macaque ovarian tissue vitrification in a closed system. Alison Y. Ting * 1, Richard R. Yeoman 1, Jacira R Campos 1, Maralee S. Lawson 1, Steve F. Mullen 2, Greg M. Fahy 2, Mary B. Zelinski 1, 1 Division of Reproductive Sciences, Oregon National Primate Research Center, Beaverton, OR, USA2 21st Century Medicine, Inc., Fontana, CA, USA Current clinical practice for ovarian tissue vitrification to preserve fertility in cancer patients utilizes an ‘‘open” system. While allowing samples to be loaded with minimal vitrification solution (VS) to achieve maximum cooling rate, it exposes tissue to direct contact with liquid nitrogen (LN2), a safety risk of cross contamination among specimens. In this study, various conditions were examined for vitrification of macaque ovarian tissue in a closed system using high security tissue straws. The threshold CPA concentration for vitrifying 1 ml VS in a tissue straw was determined by cooling and observing ice formation in VS containing various concentrations of permeating CPAs (glycerol and ethylene glycol [EG]; 1:1; w/v), in the presence of 0.8% or 3% w/v nonpermeating CPAs (supercool X-1000, polyvinylpyrrolidone K12, and supercool Z1000). With the threshold VS determined, macaque (N = 4) ovarian cortical fragments (3  3  0.5 mm3) were exposed to 1/8, 1/4, 1/2, and 1X VS (4-step VS series) for 3, 5 or 10 min/step at 37 °C, RT, or 4 °C (3  3 factorial design), loaded into straws containing 1 ml VS, and cooled in LN2 or LN2 vapor. To warm, straws were submerged into a water bath (40 °C, 1 min) or left at RT (1 min) then submerged into a water bath (20 s). Tissues were transferred to 1, 0.5, 0.25 M sucrose and holding media for CPA dilution (5 min/ step). VS toxicity was examined by omitting cooling and warming procedures. Fresh tissues and tissues subjected to vitrification or toxicity tests were fixed and examined for histology. The threshold concentration for vitrification in the presence of 3% polymers was 53% total CPA; however, 54% total CPA is required with 0.8% polymers. High cooling and warming rates from submerging samples directly into LN2 and water bath led to fracturing of the sample. Cooling rates for 1 ml 53% and 54% total CPA in LN2 vapor were 0.46 ± 0.02 and 0.48 ± 0.02 °C/s, respectively. Histology revealed that stromal and follicular morphology can be preserved by exposing tissue to a 4-step VS protocol for 3 min/step at 37 °C, 5 min/step at RT, or 10 min/step at 4 °C. Overexposure at high temperatures caused cellular toxicity, while insufficient exposure at low temperatures led to tissue devitrification and abnormal stromal and follicular morphology. Tissues exposed to 53% or 54% total CPA in the presence of 3% polymers exhibited degenerating oocytes in primordial and primary follicles, while 0.8% polymers had no apparent toxic effect based on histology. In summary, through careful and systematic experimentation, we identified a vitrification protocol for macaque ovarian tissue in a closed system using 54% total CPA concentration (glycerol, EG) in the presence of 0.8% polymers with gentle cooling and warming. Vitrification of ovarian tissue in a closed system with minimal cellular toxicity can be achieved with effective dehydration and CPA penetration, which are tissue- and CPA-specific and dependent on CPA exposure time and temperature. This is the first demonstration of primate ovarian tissue vitrification in a sealed system and offers a significant technical advancement as a safe and efficient method of fertility preservation. Examination of follicular function after vitrification during follicle culture and heterotopic transplantation is ongoing. Conflict of interest: All authors have no conflict of interest to declare. Funding: Oncofertility Consortium [NIH UL1 RR024926, R01AHD058293, PL1 EB008542], SCCPIR (U54 HD018185), P51RR000163. Acknowledgements: The authors thank Ms. Barbra Mason for tissue processing and sectioning. We are also grateful to the Division of Animal Resources for Surgery and excellent animal care. 21st Century Medicine sells the polymers studied in the present investigations. doi:10.1016/j.cryobiol.2011.09.015

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13. Cryopreservation of isolated secondary follicles from nonhuman primate using a closed system. Jacira R. Campos *, Alison Y. Ting, Richard R. Yeoman, Maralee S. Lawson, Mary B. Zelinski, Division of Reproductive Sciences, Oregon National Primate Research Center, Oregon Health & Sciences University, Beaverton, OR 97006, USA Cryopreservation of isolated secondary follicles followed by encapsulated 3dimensional (3D) culture to produce a fertilizable oocyte is being investigated as a potential option to restore fertility in young women with cancer. Using a nonhuman-primate model, we established a novel technique for vitrifying isolated secondary follicles using a closed system. Ovaries were obtained from adult rhesus macaques (n = 4) at day 1–4 of the menstrual cycle, the cortex was cut into 1  1  0.5 mm3 pieces, and secondary follicles were isolated mechanically without enzyme digestion. Follicles were randomized (n = 20/group) and vitrified in solutions containing (1) glycerol (GLY) and ethylene–glycol (EG), (2) GLY, EG plus PXZ polymers (P-1000 polyvinyl-pyrrolodine, X-1000 polyvinyl-alcohol and Z-1000 polyglycerol; 21st Century Medicine, Fontana, CA), (3) EG, dimethyl-sulfoxide (Me2SO) and sucrose, (4) EG, Me2SO, sucrose plus PXZ. For groups 1 and 2, isolated follicles were equilibrated for 1 min per step in increasing VS concentrations with (2) or without (1) PXZ. For groups 3 and 4, isolated follicles were equilibrated for 3 min in the first step and 1 min in VS with (4) or without (3) PXZ. Follicles were transferred into 0.25 cc straws which were heat sealed then vitrified in liquid nitrogen (LN2) vapor, and stored in LN2. For warming, straws were left at room temperature for 10 s and plunged into a 40 °C water bath for 5 s. Follicles in Groups 1 and 2 were transferred into holding media containing 1, 0.5, 0.25 M sucrose for 3 min each and 0 M sucrose for 5 min; follicles in groups 3 and 4 were transferred into 1 M sucrose for 1 min, 0.5 M for 3 min and 0 M sucrose for 5 min. Toxicity of the vitrification and warming solutions was tested by omitting follicle exposure to LN2. Fresh and cryopreserved follicles as well as follicles used for toxicity tests were fixed for histological analysis, cultured with bromodeoxyuridine (BrdU) for 48 h to detect DNA synthesis or encapsulated in alginate for 3D culture. Follicular diameter as well as steroid (estradiol, progesterone, androstenedione) and AMH production were measured weekly. Results showed that the vitrification and warming solutions of Groups 1 and 2 had minimal toxicity on follicles, but solutions of Groups 3 and 4 were highly toxic. More (P < 0.05) follicles in Groups 1 (62%) and 2 (65%) survived 5 weeks in 3D culture relative to Groups 3 (32%) and 4 (4%). The proportion of follicles forming an antrum was the greatest (P < 0.05) in Group 2 (50%) compared to Groups 1 (34%) and 3 (6%). No follicles in Group 4 formed an antrum. Isolation of follicles for subsequent cryopreservation is a relatively new approach for fertility preservation. Our results in the nonhuman primate suggest that vitrification with GLY, EG and PXZ polymers in a closed system has potential as a method for cryopreservation of isolated secondary follicles. Whether this technique can yield oocytes capable of fertilization leading to live offspring remains to be determined. Vitrification of secondary follicles would provide an option for fertility preservation in patients where ovarian tissue transplantation poses a risk transmitting of malignant cells. Support: NIH UL1 RR024926, R01AHD058293, PL1 EB008542, P51RR000163, D43TW00068-Fogarty (JRC). Conflicts of interest: None declared. doi:10.1016/j.cryobiol.2011.09.016

14. Developmental ability of vitrified mouse oocytes expressing water channels. Keisuke Edashige * 1, Yohei Yamaji 1, Shinsuke Seki 1, Kazutsugu Matsukawa 1, Chihiro Koshimoto 2, Magosaburo Kasai 1, 1 College of Agriculture, Kochi University, Kochi 783-8502, India, 2 Frontier Science Research Center, University of Miyazaki, Miyazaki 889-1692, Japan Previously, we showed that the exogenous expression of aquaporin 3 (AQP3), an aquaglyceroporin, improved the tolerance of mouse oocytes to vitrification with a glycerol-based solution. In the present study, we examined conditions suitable for the expression of AQP3 and the ability of vitrified oocytes to develop in vitro and in vivo after fertilization. Cumulus-oocyte complexes were collected from ovaries of ICR mice 48 h after the injecting of eCG. After partial remove of cumulus cells, immature oocytes were injected with 5, 10 or 20 pg of rat AQP3 cRNA or water and cultured for 12 h for maturation. For vitrification, mature oocytes were suspended first in PB1 medium containing 10% v/v glycerol (glycerol/PB1) for 2 min, then in GFS20 for 1 min, and finally in GFS40 in a 250-ll straw for 30 s at 25 °C before being cooled in liquid nitrogen. After warming, oocytes were suspended in PB1 medium containing 2 M sucrose for 30 s and PB1 medium containing 0.5 M sucrose for 5 min before recovery in fresh PB1 medium at 25 °C. Survived oocytes were removed of the zona pellucida, inseminated with ICR sperm, and cultured to blastocysts. For the embryo-transfer experiment, oocytes that had been injected with 5 pg of AQP3 cRNA, matured, and vitrified were removed of the zona pellucida and inseminated with BDF1 sperm. Fertilized oocytes were cultured to expanded blastocysts, and transferred to the uteri of a pseudopregnant ICR mouse. For the membrane-permeability study, immature oocytes injected with 5 pg of AQP3 cRNA were cultured for 6 or 12 h to obtain matur-

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ing and matured oocytes, respectively. Mature oocytes were vitrified, dissected of the zona pellucida partially, inseminated with ICR sperm, and cultured for 8, 24 and 48 h to obtain 1-cell, 2-cell, and 4-cell embryos, respectively. The water-permeability and glycerol-permeability of the oocytes/embryos were measured from their change in volume during exposure to glycerol/PB1 for 20 min at 25 °C, and determined using a two-parameter formalism. After vitrification, 57–61% of oocytes survived regardless of the amount of cRNA injected (5–20 pg). By contrast, no water-injected oocytes (control) survived. When oocytes that survived vitrification were removed of the zona pellucida, fertilized in vitro, and cultured, the proportions that were fertilized and developed into blastocysts were higher when the amount of injected cRNA was 5 pg than 10– 20 pg. When 16 blastocysts were transferred to a pseudopregnant mouse, 5 of them developed to term, demonstrating that oocytes vitrified after injection of AQP3 cRNA retained the ability to develop to term. The water-permeability of cRNA-injected oocytes was higher than that of control oocytes from the maturing stage to the 1-cell zygote stage, whereas glycerol-permeability was higher only at metaphase II. This indicates that AQP3 was expressed for a relatively short period of time. These results demonstrate that the transient expression of water/cryoprotectant channels is effective for cryopreserving cells that have low membrane-permeability, such as mammalian oocytes. This work was supported by JSPS. Conflict of interest: No conflict of interest exists for this work. doi:10.1016/j.cryobiol.2011.09.017

15. Functional survival of mouse oocytes and 8-cell embryos after vitrification in 1, 0.75, 0.5, and 0.33 EAFS vitrification media and warming at an exceedingly high rate of 117,500 °C/min. Shinsuke Seki *, Peter Mazur, Fundamental and Applied Cryobiology Group, Department of Biochemistry and Cellular and Molecular Biology, The University of Tennessee, Knoxville, TN 37996-0840, USA We have reported in several previous Cryobiology meetings and in several recent publications that the ability of mouse oocytes to survive vitrification in a standard EAFS medium depends almost entirely on the warming rate and not on the cooling rate. If the oocytes are warmed at 117,000 °C/min (achieved by using Cryotops), then survivals exceed 70% even when the cooling rate is as low as 95 °C/min. If the warming rate is lowered to 600 °C/min, survival drops to 0% when the cooling rate is 69,000 °C/min. Furthermore, we reported at last year’s meeting, that with this exceedingly high warming rate, one obtains survivals of >90% even if the concentration of EAFS is cut in half. An ability to reduce the solute concentration from the normal 7.4 molal to about half of that could be highly beneficial. All the survival values above are based on morphological normality of the oocyte and its surface membrane and its osmotic integrity after vitrification and warming. Attempts to obtain functional survivals of oocytes have been thwarted by two problems. One is a pronounced difficulty in achieving in vitro fertilization; the other is a ‘‘two-cell block” of development to later stages. The difficulty in achieving IVF and the 2-cell stage can be ameliorated by partial dissection of the zona pellucida (PZD). With this dissection, the percentage of oocytes vitrified in 1, 0.75, and 0.5 EAFS and warmed at 117,500 °C/min that develop to the 2-cell stage after IVF are 81%, 76%, and 66%, respectively. And the percentage of morphological survivors that develop to 2-cell are 88%, 78%, and 70%, respectively. We conducted parallel experiments with precompaction 8-cell embryos. Functional survival here was based on development to expanded blastocysts. No PZD was required. With the very high warming rate of 117,500 °C/min, the percentages of embryos vitrified in 1, 0.75, and 0.5 EAFS that developed to blastocysts were 93%, 92%, and 86%, respectively. And the percentages of morphological survivors that developed to expanded blastocysts were 100%, 92%, and 97%, respectively. Even when the solute concentration of the EAFS was reduced to 33% of normal, we obtained 40% functional survival of these 8-cell embryos. Conflicts of interest: None declared. Source of funding: None declared. doi:10.1016/j.cryobiol.2011.09.018

16. Development of an inexpensive method for long-term storage of mouse embryos at 80 °C. Xu Han *, Hongbin Ma, Yang Liu, Department of Mechanical & Aerospace Engineering, University of Missouri, Columbia, MO 65211, USA, Comparative Medicine Center, University of Missouri, Columbia, MO 65211, USA Current approaches to cryopreservation of embryos from animal models for human health and disease utilize methods that require storage and shipping of samples at very low temperatures, i.e. in liquid nitrogen or its vapor, which are very expensive. To develop an effective and inexpensive method for safe storage and shipping of cryopreserved samples, we investigated the effect of polymer cryoprotectants (Ficoll and polyvinylpyrrolidone) on increasing the devitrification temperatures of Me2SO solutions using differential scanning calorimetry and then developed a cryopreservation media that is thermodynamically stable at 80 °C. Using this media, mouse morula-stage embryos were cooled to 80 °C and stored in a 80 °C freezer

for one week. After rewarming, the percent of embryos developed to the early and hatched blastocyst stage was 927%, which is higher than that of the control group (887%) cryopreserved in liquid nitrogen for the same period of time (P < 0.05). These experimental results provide the proof of concept for using this approach for lowering the costs associated with storage and shipping of cryopreserved animal model embryos. Conflicts of interest: None declared. Source of funding: None declared. doi:10.1016/j.cryobiol.2011.09.019

17. Determination of the compositions of intracellular lipid droplets in porcine oocytes and investigation of the mechanisms of their thermal mechanical damages during cryopreservation. Xu Han *, Yang Liu, Department of Mechanical & Aerospace Engineering, University of Missouri, Columbia, MO 65211, USA, Comparative Medicine Center, University of Missouri, Columbia, MO 65211, USA Intracellular lipids play a major role in the cryosensitivity of lipid-rich cell types, such as porcine oocytes. The aim of this investigation was to characterize the difference between the thermal expansion behaviors of intracellular lipids and cryoprotectant agent (CPA) solutions. Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) was applied to identify the intracellular lipid components of single pig embryos. The MALDI-MS results were then used to determine the molar percentages of the major triglyceride components in the intracellular lipid droplets. A phantom, i.e. an artificial lipid mixture, was produced with the same composition of the intracellular lipid droplets in pig embryos. A cryomicroscopic method was developed to measure their thermal expansion coefficients by optically determining their relative volume change during cooling. Using this information, we subsequently investigated whether this difference might cause mechanical damage to the lipid-rich cell types during cryopreservation processes. The results showed that the thermal expansion coefficients of intracellular lipids and CPA (e.g. ethylene glycol and Me2SO) solutions are quite different. Based on a simple thermodynamic model, it is predicted that during cooling processes, the different thermal expansion of intracellular lipid and intracellular solutions generates significant thermal stress (>10 MPa) on the surface of lipid vesicles in porcine oocytes and may cause the destruction of their lipid-reticulum connections. This model also provides a physical explanation for the morphological change of lipid vesicles of rewarmed porcine oocytes. Conflicts of interest: None declared. Source of funding: None declared. doi:10.1016/j.cryobiol.2011.09.020

18. Osmotic properties and membrane phase behavior during cooling of stallion sperm. Harriëtte Oldenhof * 1, Marina Gojowsky 1,2, Willem F Wolkers 3, Harald Sieme 1, 1 Clinic for Horses – Unit for Reproductive Medicine, University of Veterinary, Medicine Hannover, Hannover, Germany, 2 National Stud Lower Saxony, Celle, Germany, 3 Institute of Multiphase Processes, Leibniz Universität Hannover, Hannover, Germany Stallion sperm is reputed to display a large male to male variability in its ability to survive freezing and thawing. The underlying mechanisms associated with this large individual variation in cryosensitivity are not known. Knowledge on cellular properties, such as osmotic tolerance and membrane permeability to water, that correlate with freezability may help to rationally design customized freezing protocols for individuals. In order to determine the osmotic tolerance limits and osmotic behavior of stallion sperm a flow cytometer was used that allowed for simultaneous assessment of both the cell volume based on the Coulter principle and the permeability of the plasma membrane for the fluorescent dye propidium iodide. A Boyle van ’t Hoff plot of sperm volume data in anisotonic media showed that stallion sperm behave as linear osmometers between 150 and 600 mOsm kg 1. Viability, however, drops below 50% at 150 mOsm kg 1. Inclusion or exclusion of different (non-viable) subpopulations, greatly affects Boyle van ’t Hoff behavior and thereby determination of the osmotic inactive volume. Fourier transform infrared spectroscopy was used to determine membrane properties of stallion sperm in a wide supra- and sub-zero temperature regime. Membranes undergo a weakly cooperative phase transition from 30 to 10 °C during cooling. Upon extracellular ice formation membranes undergo a sharp phase transition to a highly ordered gel phase. The extent and rate of this freezinginduced membrane phase change is dependent on the ice nucleation temperature and the presence of cryoprotective agents. The membrane hydraulic permeability, Lp, and corresponding activation energy for water transport during freezing, ELp, were derived from freezing-induced membrane phase behavior determined by FTIR. Permeating cryoprotective agents such as glycerol decrease the activation energy for water transport. Non permeating cryoprotective agents have lesser effects on ELp, but also modulate water transport processes during freezing by controlling cellular and membrane dehydration. This work is supported by funding from the Deutsche