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The Impact of Cryopreservation on Biophysical and Biochemical Properties and Developmental Rate of Mouse Embryo
Design: The following zygotes were reconstructed: Type 1—two pronuclei (PN) of a zygote from an aged mouse were transferred to enucleated zygote of a young mouse; Type 2—two PN of a young mouse zygote were transferred to the enucleated zygote of an old mouse; Control—two PN were transferred between the zygotes of young mice. Materials and Methods: Young (2 months) and old (19 months) CB6F1 mice were injected with PMSG1hCG and mated with middle aged (10 months) male to obtain zygotes. Following reconstruction, zygotes were cultured in S1 medium to 2 cell embryo stage and then surgically transferred into the oviduct of day 1 pseudopregnant foster mothers. Pregnant mothers delivered normally unless there was only one fetus and then caesarian section was performed at 20 days. Result: Only 26 zygotes were recovered from 8 old mice; in contrast, 8 young mice produced 219 zygotes. Oviduct transfer of 22 Type 1 zygotes produced a live birth rate comparable to that of 23 Type 2 zygotes: 27.3% and 30.4%, respectively. These birth rates were not significantly different than that of 32 control group zygotes.
Type of zygote
Recovered zygotes
Reconstructed zygotes
Fused zygotes
2-Cell embryos
Live offspring
Delivery rate
Type 1 Type 2 Control
26 26 219
23 24 33
23 24 33
22 23 32
6 7 8
27.3% 30.4% 25%
Conclusion: The nucleus and cytoplasm of oocytes from old CB6F1 mice were as competent as those of young mice to support embryonic development to term. Thus, nuclear and cytoplasmic maturation does not appear to be compromised following gonadotropin stimulation of animals of advanced maternal age. Rather, the age-related decline in fertility noted in mice is, at least with this strain, due primarily to the fact that there are fewer oocytes available for ovulation.
P-310 Identification and Localization of Cell Cycle Proteins Implicated in Mammalian Oocyte Maturation. A. Blaszczyk, C. Brockmann, J. A. Grifo, L. C. Krey. Program for In vitro Fertilization, Reproductive Surgery and Infertility, New York University School of Medicine, New York, NY. Objective: Meiosis I, the stage when chromosome non-disjunction most often occurs, closely resembles the G2/M phase transition of the mitotic cell cycle. The rate of aneuploidy increases in oocytes from women of advanced maternal age. Strikingly similar is the increase in aneuploidy in tumors or cancer cells since the risk of developing tumors and cancer also increases with age. The commonality between these two cell types may be a phenomenon that is attributed to common proteins that ensure proper chromosome segregation and cell division. Cell cycle and kinetocore-associated proteins such as MPF, p55, Mad 1-3 and Bub 1-3, initiate the resumption of meiosis and mitosis and assure that, chromosomes are aligned properly on the meiotic/mitotic spindle prior to cytokinesis. The absence or alteration of localization of these proteins may account for the increase in aneuploidy at this specific cell cycle stage. It is not known however if some of these proteins even exist in mammalian oocytes at meiosis. Design: Immunofluorescence microscopy, immunohistochemistry, and immunoblotting of oocyte extracts were used to localize and quantify specific cell cycle proteins known to regulate the G2/M phase transition of mitotic cells. Materials and Methods: CB6F1 female mice were treated with PMSG6 hCG and sacrificed 45– 60 h later to harvest oocytes at GV, MI and MII stages by follicular or ampullar puncture. Oocytes were denuded of cumulus cells; zonae were removed using acid Tyrode’s solution. Oocytes were fixed and stained intact, spun onto microscope slides using a Shandon cytospin, or frozen as extracts for western blot analysis. Ovaries were fixed, embedded and sectioned onto polylysine coated slides. Oocytes were examined by immunohistochemistry or Western blot analyzes with the ECL detection system using the same primary antibodies. Stained specimens were visualized with epifluorescence and digital images were stored for comparison and analysis. Results: The p34 subunit of MPF has been detected in all specimens and interestingly seems to localize to areas which correspond to projections coming from granulosa cells. However, at MI, p34 clearly co-localizes with
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Abstracts
the meiotic spindle. At MII Bub3 distribution is concentrated as a cloud that encapsulates the chromosomes retained in the egg and extruded in the polar body, a distribution consistent with observations that Bub3 binds to unattached kinetocores of chromosomes. Conclusion: Immunohistochemical characterization of cell cycle proteins in eggs at different developmental stages provides information about the roles of these proteins in the regulation of oocyte maturation and early embryonic development. Some of these proteins may be synthesized in the egg whereas others may be supplied by the surrounding cumulus cells and provide a “second messenger-like” role that mediates gonadotropin stimulation of oocyte maturation. P-311 The Impact of Cryopreservation on Biophysical and Biochemical Properties and Developmental Rate of Mouse Embryo. 1H. J. Ahn, 2I. P. Son, 3 H. C. Kwon, 4H. J. Lee, 3W. I. Park, 1C. K. Min. 1Department of Life Science, College of Natural Science, Ajou University, Suwon, 2Department of Biology, College of Natural Science, Kyonggi University, Suwon, 3Department of Obstetrics and Gynecology, 4Department of Physiology, School of Medicine, Eulji University, Taejon, Korea. Objectives: The aim of this study were to assess biophysical and biochemical changes of the ultrastructure including apoptosis, and to evaluate functional changes such as production of reactive oxygen species (ROS) and embryo development after freezing and thawing of the mouse embryo. Design: Fresh and frozen-thawed 2-cell embryos of ICR mouse were included in control and study group. The cryopreservation was performed using slow-freezing rapid-thawing method. Propanediol with sucrose was used as cryoprotectant, and culture medium for the thawed embryo was mHTF. Materials and Methods: To assess alteration of the plasma membrane fluidity, fluorescent recovery velocity was measured by Argon ion LASER after staining the embryo with fluorecein labeled WGA. Intracellular location and membrane potential of the mitochondria were evaluated by confocal microscope after staining with Rhodamine 123 and JC-1. Relative amount of H2O2 production was measured by H2DCFDA. Cell apoptosis was assessed by Annexin V staining and TUNEL method. Total cell number in the blastocyst was counted by fluorescent microscope after Acrydine Orange staining. Results: Fluorescent recovery velocity was 1.46 6 0.13 sec and 0.28 6 0.04 sec for study and control group respectively (p,0.05), suggesting alteration of the plasma membrane. The distribution pattern of the mitochondria was similar in both groups. However, in arrested embryos, the mitochondria were located mainly near the plasma and nuclear membrane. The membrane potentials (Cm) were represented as color intensity after JC-1 staining. Relative intensity of red color measured at 590 nm (high membrane potential) versus relative intensity of green color measured at 510 nm (low membrane potential) were 17.2 6 3.8 vs. 14.4 6 0.9 and 13.2 6 2.0 vs. 10.8 6 1.2 for control and study group respectively (p, 0.05). The relative amount of produced H2O2 was higher for study group (62.8 6 23.5 vs. 43.2 6 14.5; p,0.05). The expression pattern of Annexin V staining was not significantly different between two groups. However, DNA fragmentation rate was higher for study group (74.7% vs. 32.3%; p,0.05). 74.7% and 33.7% of embryos developed into blastocyst for control and study group (p,0.05). Total cell number in the blastocyst was 95.87619.14 and 42.00 6 11.34 for control and study groups respectively (p,0.05). Conclusions: The process of freezing and/or thawing negatively affected the integrity of cell membrane, function of mitochondria and rate of ROS production. Hampered development of embryo after cryopreservation may be related with these ultrastructural and functional alterations. These results may rationalize the use of antioxidants and anti-apoptotic substances in process of cryopreservation. P-312 Programmed Cell Death (Apoptosis) Differs in IVF Versus ICSI Blastocysts from Non-Human Primates. 1E. Neuber, 1T. Dominko, 1A. W. Chan, 1C. Martinovich, 1,2C. Simerly, 1,2G. Schatten. 1Oregon Regional Primate Research Center, Beaverton, OR 97006; 2Department of Obstetrics and Gynecology and Department of Cell and Developmental Biology, Oregon Health Sciences University, Portland, OR.
Vol. 74, No. 3, Suppl. 1, September 2000
Type of zygote
Recovered zygotes
Reconstructed zygotes
Fused zygotes
2-Cell embryos
Live offspring
Delivery rate
Type 1 Type 2 Control
26 26 219
23 24 33
23 24 33
22 23 32
6 7 8
27.3% 30.4% 25%
Conclusion: The nucleus and cytoplasm of oocytes from old CB6F1 mice were as competent as those of young mice to support embryonic development to term. Thus, nuclear and cytoplasmic maturation does not appear to be compromised following gonadotropin stimulation of animals of advanced maternal age. Rather, the age-related decline in fertility noted in mice is, at least with this strain, due primarily to the fact that there are fewer oocytes available for ovulation.
P-310 Identification and Localization of Cell Cycle Proteins Implicated in Mammalian Oocyte Maturation. A. Blaszczyk, C. Brockmann, J. A. Grifo, L. C. Krey. Program for In vitro Fertilization, Reproductive Surgery and Infertility, New York University School of Medicine, New York, NY. Objective: Meiosis I, the stage when chromosome non-disjunction most often occurs, closely resembles the G2/M phase transition of the mitotic cell cycle. The rate of aneuploidy increases in oocytes from women of advanced maternal age. Strikingly similar is the increase in aneuploidy in tumors or cancer cells since the risk of developing tumors and cancer also increases with age. The commonality between these two cell types may be a phenomenon that is attributed to common proteins that ensure proper chromosome segregation and cell division. Cell cycle and kinetocore-associated proteins such as MPF, p55, Mad 1-3 and Bub 1-3, initiate the resumption of meiosis and mitosis and assure that, chromosomes are aligned properly on the meiotic/mitotic spindle prior to cytokinesis. The absence or alteration of localization of these proteins may account for the increase in aneuploidy at this specific cell cycle stage. It is not known however if some of these proteins even exist in mammalian oocytes at meiosis. Design: Immunofluorescence microscopy, immunohistochemistry, and immunoblotting of oocyte extracts were used to localize and quantify specific cell cycle proteins known to regulate the G2/M phase transition of mitotic cells. Materials and Methods: CB6F1 female mice were treated with PMSG6 hCG and sacrificed 45– 60 h later to harvest oocytes at GV, MI and MII stages by follicular or ampullar puncture. Oocytes were denuded of cumulus cells; zonae were removed using acid Tyrode’s solution. Oocytes were fixed and stained intact, spun onto microscope slides using a Shandon cytospin, or frozen as extracts for western blot analysis. Ovaries were fixed, embedded and sectioned onto polylysine coated slides. Oocytes were examined by immunohistochemistry or Western blot analyzes with the ECL detection system using the same primary antibodies. Stained specimens were visualized with epifluorescence and digital images were stored for comparison and analysis. Results: The p34 subunit of MPF has been detected in all specimens and interestingly seems to localize to areas which correspond to projections coming from granulosa cells. However, at MI, p34 clearly co-localizes with
S192
Abstracts
the meiotic spindle. At MII Bub3 distribution is concentrated as a cloud that encapsulates the chromosomes retained in the egg and extruded in the polar body, a distribution consistent with observations that Bub3 binds to unattached kinetocores of chromosomes. Conclusion: Immunohistochemical characterization of cell cycle proteins in eggs at different developmental stages provides information about the roles of these proteins in the regulation of oocyte maturation and early embryonic development. Some of these proteins may be synthesized in the egg whereas others may be supplied by the surrounding cumulus cells and provide a “second messenger-like” role that mediates gonadotropin stimulation of oocyte maturation. P-311 The Impact of Cryopreservation on Biophysical and Biochemical Properties and Developmental Rate of Mouse Embryo. 1H. J. Ahn, 2I. P. Son, 3 H. C. Kwon, 4H. J. Lee, 3W. I. Park, 1C. K. Min. 1Department of Life Science, College of Natural Science, Ajou University, Suwon, 2Department of Biology, College of Natural Science, Kyonggi University, Suwon, 3Department of Obstetrics and Gynecology, 4Department of Physiology, School of Medicine, Eulji University, Taejon, Korea. Objectives: The aim of this study were to assess biophysical and biochemical changes of the ultrastructure including apoptosis, and to evaluate functional changes such as production of reactive oxygen species (ROS) and embryo development after freezing and thawing of the mouse embryo. Design: Fresh and frozen-thawed 2-cell embryos of ICR mouse were included in control and study group. The cryopreservation was performed using slow-freezing rapid-thawing method. Propanediol with sucrose was used as cryoprotectant, and culture medium for the thawed embryo was mHTF. Materials and Methods: To assess alteration of the plasma membrane fluidity, fluorescent recovery velocity was measured by Argon ion LASER after staining the embryo with fluorecein labeled WGA. Intracellular location and membrane potential of the mitochondria were evaluated by confocal microscope after staining with Rhodamine 123 and JC-1. Relative amount of H2O2 production was measured by H2DCFDA. Cell apoptosis was assessed by Annexin V staining and TUNEL method. Total cell number in the blastocyst was counted by fluorescent microscope after Acrydine Orange staining. Results: Fluorescent recovery velocity was 1.46 6 0.13 sec and 0.28 6 0.04 sec for study and control group respectively (p,0.05), suggesting alteration of the plasma membrane. The distribution pattern of the mitochondria was similar in both groups. However, in arrested embryos, the mitochondria were located mainly near the plasma and nuclear membrane. The membrane potentials (Cm) were represented as color intensity after JC-1 staining. Relative intensity of red color measured at 590 nm (high membrane potential) versus relative intensity of green color measured at 510 nm (low membrane potential) were 17.2 6 3.8 vs. 14.4 6 0.9 and 13.2 6 2.0 vs. 10.8 6 1.2 for control and study group respectively (p, 0.05). The relative amount of produced H2O2 was higher for study group (62.8 6 23.5 vs. 43.2 6 14.5; p,0.05). The expression pattern of Annexin V staining was not significantly different between two groups. However, DNA fragmentation rate was higher for study group (74.7% vs. 32.3%; p,0.05). 74.7% and 33.7% of embryos developed into blastocyst for control and study group (p,0.05). Total cell number in the blastocyst was 95.87619.14 and 42.00 6 11.34 for control and study groups respectively (p,0.05). Conclusions: The process of freezing and/or thawing negatively affected the integrity of cell membrane, function of mitochondria and rate of ROS production. Hampered development of embryo after cryopreservation may be related with these ultrastructural and functional alterations. These results may rationalize the use of antioxidants and anti-apoptotic substances in process of cryopreservation. P-312 Programmed Cell Death (Apoptosis) Differs in IVF Versus ICSI Blastocysts from Non-Human Primates. 1E. Neuber, 1T. Dominko, 1A. W. Chan, 1C. Martinovich, 1,2C. Simerly, 1,2G. Schatten. 1Oregon Regional Primate Research Center, Beaverton, OR 97006; 2Department of Obstetrics and Gynecology and Department of Cell and Developmental Biology, Oregon Health Sciences University, Portland, OR.
Vol. 74, No. 3, Suppl. 1, September 2000