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Programmed Cell Death (Apoptosis) Differs in IVF Versus ICSI Blastocysts from Non-Human Primates
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
Objective: To compare the cell numbers, allocation to the trophectoderm (TE) and inner cell mass (ICM) and proportions of cells undergoing programmed cell death between IVF (in vitro) and ICSI (intracytoplasmic sperm injection) fertilized blastocysts. Design: Mature oocytes from hyperstimulated rhesus monkeys were allocated for fertilization via IVF or ICSI. Materials and Methods: A terminal deoxynucleotidyl transferase-mediated dUPT nick-end labeling (TUNEL) assay kit was used to assay apoptosis. Blastocysts were fixed (2% formaldehyde; pH 7.4; 30 min), rinsed, and permeabilized with 0.1% Triton X-100 — 0.1% Na Citrate (4°C; 2 min). Hoechst 33342 was used to visualize total DNA. Images were obtained by confocal microscopy and 3-dimensional reconstructions of the blastocysts were generated. Reconstruction permitted determination of the total cell number by counting nuclei serially. Cell nuclei were allocated either to TE or ICM depending on their peripheral or interior location in 3-D images, respectively. Data analyzed by Statview Software. Results: ICSI blastocysts have an increased number of both ICM and TE cells over IVF blastocysts (31 6 4 ICM and 200 6 21 TE versus 15 6 0.5 ICM and 120 6 5.1 TE, respectively). However, the ratio of ICM to TE cells in IVF versus ICSI is not significantly different, IVF 5 13% 6 1.3, ICSI 5 15% 6 1.9. The apoptotic ICM dead cell index for IVF blastocysts is higher than for ICSI generated blastocysts (13.2 6 3.3 versus 4.8% 6 2.5, p5.07). In both IVF and ICSI, apoptosis appears to be correlated with cell number. The lower the cell number of the blastocyst, the higher the total dead cell index. Conclusions: Even though morphologically indistinguishable, the viability of blastocysts derived from different fertilization protocols seems to be different. Higher proportions of ICM cells undergoing apoptosis in IVF blastocyst is surprising. The TUNEL assay can be used to reveal differences between embryos and the viability of ICM may depend on the type of manipulation required to generate embryos.
P-313 Gene Expression in Apoptosis Regulated by Nitric Oxide in Human Endometrium. 1M. Vega, 1M. C. Johnson, 1,3A. Villavicencio, 1M. Anido, 2 F. Gabler, 1A. Castro. 1Institute of Maternal and Child Research, 2Department of Anatomy Pathology, San-Borja Arriaran Clinical Hospital, School of Medicine, University of Chile, and 3PLACIRH Fellow. Santiago, Chile. Objectives: Nitric oxide (NO) has been reported to elicit a proapoptotic effect in several tissues. On the other hand, in human endometria DNA fragmentation has been observed predominantly during the secretory phase (SP). The objectives of the present investigation were to study whether NO regulates apoptosis (AP) in the human endometria and its action on the expression of genes involved in cell survival. Design: A prospective study in which endometria samples were obtained from patients undergoing hysterectomy or mini-laparotomy for tubal sterilization throughout the menstrual cycle (proliferative phase endometria, PPE, n56; SP endometria, SPE, n510). Each patient signed a written informed consent and the Institutional Review Board approved this study. Materials and Methods: Tissue slices (10 –20 mg) were frozen, embedded in paraffin or cultured without (basal) or with NOS substrate, L-Arg (1 mM); the same for enzymatic dispersed endometria epithelial cells. AP was assessed by TUNEL (FITC); mRNA expression (bcl-2, bax) by RTPCR and normalized to b-actin mRNA expression (arbitrary units, AU) and bcl-2 also by in situ hybridization; protein expression (bcl-2, Bax) by immunohistochemistry, and Bcl-2 concentration by ELISA. AP and protein expression was semiquantitated according to the degree of positive staining (HScore approach). Results: In tissue slices, AP was 8-fold higher in SPE (HScore52.3) than in PPE (HScore 5 0.3); L-Arg increased AP mainly in epithelial cells of PPE (40%, p,0.05). No changes were observed in SPE. In addition, bcl-2 mRNA and protein were preferentially expressed in glands of PPE and to a lesser extent in SPE. Bax expression was localized predominantly to epithelial cells of PPE (HScore 5 3.1 6 0.4) and in SPE was also strongly detected in stromal cells (HScore 5 3.5 6 0.3). The expression of bcl-2 mRNA was 20% higher in PPE (2.9 6 0.7 AU) than in SPE (2.4 6 0.8 AU), whereas, bax mRNA increased 40% in SPE compared to PPE. In cultures of epithelial cells, L-Arg induced a partial inhibition of bcl-2 mRNA expression, similarly to the effect on Bcl-2 concentration where a 20% decrease of protein concentration was observed with L-Arg. Conclusions: Bcl-2 is mainly expressed in PPE, whereas, Bax increases
FERTILITY & STERILITYt
its expression in SPE, indicating that the relative ratios of these proteins may determine the fate of a cell. In addition, NO induces AP in human endometria, probably by a decrease on bcl-2 mRNA and/or protein expression. Based on the importance of AP as a physiological process for tissue homeostasis, abnormal cell death may be relevant in evaluating embryo implantation failure. FONDECYT 1980899 (Chile).
P-314 Expression of Regulatory Gene Protein p53 in Human Gametes and Preembryos. N. Zaninovic, L. L. Veeck, Z. Rosenwaks. Center for Reproductive Medicine and Infertility, Weill Medical College of Cornell University, New York, NY. Introduction: Human gametes and preembryos may undergo cell arrest and/or apoptosis in vitro. The specific signals that trigger, and proteins that regulate, these events are unknown. The p53 (tumor suppressor gene) protein has the ability to regulate cell arrest and/or apoptosis in response to DNA damage. While normally a nuclear protein, p53 can migrate to the nuclear membrane and cell cytoplasm if activated. The objective of this study was to determine expression of the p53 protein in human gametes and preembryos up to the blastocyst stage. Design: Human gametes and preembryos donated for research were used for this study. Materials and Methods: Expression of the p53 protein was analyzed using immunofluorescence. Specimens were fixed and permeabilizated before incubation with fluorescent conjugated monoclonal antibody which recognizes wild and mutant p53 protein. Results: The p53 protein was expressed in human oocytes either throughout the entire cytoplasm, within the polar body, or peripherally in the oocyte membrane. Some arrested immature oocytes demonstrated poor staining (negative for p53). In some oocytes, cytoplasm did not stain equally exhibiting areas of lower intensity. Bound sperm cells stained negative while cumulus cells expressed protein. In “viable” cleaved preembryos, p53 was localized in the blastomere nucleus and nuclear membrane. In contrast, the entire cytoplasm of arrested preembryos stained positive for p53, indicating activation of the protein. At the blastocyst stage, p53 protein was expressed randomly in the inner cell mass and trophectoderm. Conclusions: (1) P53 is expressed in human oocytes and preembryos up to blastocyst stage. (2) Arrested cleaved preembryos exhibit p53 protein staining throughout the cytoplasm indicating protein activation and cell arrest.
P-315 Expression of Anti-Apoptotic Gene Protein Bcl-2 in Human Gametes and Preembryos. N. Zaninovic, L. L. Veeck, Z. Rosenwaks. Center for Reproductive Medicine and Infertility, Weill Medical College of Cornell University, New York, NY. Objectives: The involvement of programmed cell death (apoptosis) and its regulatory molecules in human preimplantation development has been indicated. The molecular mechanism of apoptosis includes pro-apoptotic and anti-apoptotic bcl-2 gene family members. The bcl-2 protein (associated with mitochondria and cell membranes) functions as one of the most important repressors of cell death in this family. The objective of this study was to determine the expression and localization of the bcl-2 protein in human gametes and preembryos (PEs) up to the blastocyst stage. Design: Research donated human gametes and PEs were studied during oocyte maturation stages, following fertilization, and up to the blastocyst stage of growth. Materials and Methods: Expression of the bcl-2 protein was analyzed using immunofluorescence. Specimens were fixed and permeabilizated before incubation with fluorescent conjugated monoclonal antibody which recognizes bcl-2 protein. Results: The bcl-2 protein was strongly expressed in human oocytes of all maturation stages. The protein was highly expressed throughout the cytoplasm with lower intensity around the metaphase plate. This expression was conserved even after five days in culture when localization shifted to the oocyte membrane. Compared to the surrounding cumulus-corona cells, the expression of the protein was considerably higher within the oocytes. In contrast, bound sperm cells stained negative. At the pronuclear stage, bcl-2
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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
Objective: To compare the cell numbers, allocation to the trophectoderm (TE) and inner cell mass (ICM) and proportions of cells undergoing programmed cell death between IVF (in vitro) and ICSI (intracytoplasmic sperm injection) fertilized blastocysts. Design: Mature oocytes from hyperstimulated rhesus monkeys were allocated for fertilization via IVF or ICSI. Materials and Methods: A terminal deoxynucleotidyl transferase-mediated dUPT nick-end labeling (TUNEL) assay kit was used to assay apoptosis. Blastocysts were fixed (2% formaldehyde; pH 7.4; 30 min), rinsed, and permeabilized with 0.1% Triton X-100 — 0.1% Na Citrate (4°C; 2 min). Hoechst 33342 was used to visualize total DNA. Images were obtained by confocal microscopy and 3-dimensional reconstructions of the blastocysts were generated. Reconstruction permitted determination of the total cell number by counting nuclei serially. Cell nuclei were allocated either to TE or ICM depending on their peripheral or interior location in 3-D images, respectively. Data analyzed by Statview Software. Results: ICSI blastocysts have an increased number of both ICM and TE cells over IVF blastocysts (31 6 4 ICM and 200 6 21 TE versus 15 6 0.5 ICM and 120 6 5.1 TE, respectively). However, the ratio of ICM to TE cells in IVF versus ICSI is not significantly different, IVF 5 13% 6 1.3, ICSI 5 15% 6 1.9. The apoptotic ICM dead cell index for IVF blastocysts is higher than for ICSI generated blastocysts (13.2 6 3.3 versus 4.8% 6 2.5, p5.07). In both IVF and ICSI, apoptosis appears to be correlated with cell number. The lower the cell number of the blastocyst, the higher the total dead cell index. Conclusions: Even though morphologically indistinguishable, the viability of blastocysts derived from different fertilization protocols seems to be different. Higher proportions of ICM cells undergoing apoptosis in IVF blastocyst is surprising. The TUNEL assay can be used to reveal differences between embryos and the viability of ICM may depend on the type of manipulation required to generate embryos.
P-313 Gene Expression in Apoptosis Regulated by Nitric Oxide in Human Endometrium. 1M. Vega, 1M. C. Johnson, 1,3A. Villavicencio, 1M. Anido, 2 F. Gabler, 1A. Castro. 1Institute of Maternal and Child Research, 2Department of Anatomy Pathology, San-Borja Arriaran Clinical Hospital, School of Medicine, University of Chile, and 3PLACIRH Fellow. Santiago, Chile. Objectives: Nitric oxide (NO) has been reported to elicit a proapoptotic effect in several tissues. On the other hand, in human endometria DNA fragmentation has been observed predominantly during the secretory phase (SP). The objectives of the present investigation were to study whether NO regulates apoptosis (AP) in the human endometria and its action on the expression of genes involved in cell survival. Design: A prospective study in which endometria samples were obtained from patients undergoing hysterectomy or mini-laparotomy for tubal sterilization throughout the menstrual cycle (proliferative phase endometria, PPE, n56; SP endometria, SPE, n510). Each patient signed a written informed consent and the Institutional Review Board approved this study. Materials and Methods: Tissue slices (10 –20 mg) were frozen, embedded in paraffin or cultured without (basal) or with NOS substrate, L-Arg (1 mM); the same for enzymatic dispersed endometria epithelial cells. AP was assessed by TUNEL (FITC); mRNA expression (bcl-2, bax) by RTPCR and normalized to b-actin mRNA expression (arbitrary units, AU) and bcl-2 also by in situ hybridization; protein expression (bcl-2, Bax) by immunohistochemistry, and Bcl-2 concentration by ELISA. AP and protein expression was semiquantitated according to the degree of positive staining (HScore approach). Results: In tissue slices, AP was 8-fold higher in SPE (HScore52.3) than in PPE (HScore 5 0.3); L-Arg increased AP mainly in epithelial cells of PPE (40%, p,0.05). No changes were observed in SPE. In addition, bcl-2 mRNA and protein were preferentially expressed in glands of PPE and to a lesser extent in SPE. Bax expression was localized predominantly to epithelial cells of PPE (HScore 5 3.1 6 0.4) and in SPE was also strongly detected in stromal cells (HScore 5 3.5 6 0.3). The expression of bcl-2 mRNA was 20% higher in PPE (2.9 6 0.7 AU) than in SPE (2.4 6 0.8 AU), whereas, bax mRNA increased 40% in SPE compared to PPE. In cultures of epithelial cells, L-Arg induced a partial inhibition of bcl-2 mRNA expression, similarly to the effect on Bcl-2 concentration where a 20% decrease of protein concentration was observed with L-Arg. Conclusions: Bcl-2 is mainly expressed in PPE, whereas, Bax increases
FERTILITY & STERILITYt
its expression in SPE, indicating that the relative ratios of these proteins may determine the fate of a cell. In addition, NO induces AP in human endometria, probably by a decrease on bcl-2 mRNA and/or protein expression. Based on the importance of AP as a physiological process for tissue homeostasis, abnormal cell death may be relevant in evaluating embryo implantation failure. FONDECYT 1980899 (Chile).
P-314 Expression of Regulatory Gene Protein p53 in Human Gametes and Preembryos. N. Zaninovic, L. L. Veeck, Z. Rosenwaks. Center for Reproductive Medicine and Infertility, Weill Medical College of Cornell University, New York, NY. Introduction: Human gametes and preembryos may undergo cell arrest and/or apoptosis in vitro. The specific signals that trigger, and proteins that regulate, these events are unknown. The p53 (tumor suppressor gene) protein has the ability to regulate cell arrest and/or apoptosis in response to DNA damage. While normally a nuclear protein, p53 can migrate to the nuclear membrane and cell cytoplasm if activated. The objective of this study was to determine expression of the p53 protein in human gametes and preembryos up to the blastocyst stage. Design: Human gametes and preembryos donated for research were used for this study. Materials and Methods: Expression of the p53 protein was analyzed using immunofluorescence. Specimens were fixed and permeabilizated before incubation with fluorescent conjugated monoclonal antibody which recognizes wild and mutant p53 protein. Results: The p53 protein was expressed in human oocytes either throughout the entire cytoplasm, within the polar body, or peripherally in the oocyte membrane. Some arrested immature oocytes demonstrated poor staining (negative for p53). In some oocytes, cytoplasm did not stain equally exhibiting areas of lower intensity. Bound sperm cells stained negative while cumulus cells expressed protein. In “viable” cleaved preembryos, p53 was localized in the blastomere nucleus and nuclear membrane. In contrast, the entire cytoplasm of arrested preembryos stained positive for p53, indicating activation of the protein. At the blastocyst stage, p53 protein was expressed randomly in the inner cell mass and trophectoderm. Conclusions: (1) P53 is expressed in human oocytes and preembryos up to blastocyst stage. (2) Arrested cleaved preembryos exhibit p53 protein staining throughout the cytoplasm indicating protein activation and cell arrest.
P-315 Expression of Anti-Apoptotic Gene Protein Bcl-2 in Human Gametes and Preembryos. N. Zaninovic, L. L. Veeck, Z. Rosenwaks. Center for Reproductive Medicine and Infertility, Weill Medical College of Cornell University, New York, NY. Objectives: The involvement of programmed cell death (apoptosis) and its regulatory molecules in human preimplantation development has been indicated. The molecular mechanism of apoptosis includes pro-apoptotic and anti-apoptotic bcl-2 gene family members. The bcl-2 protein (associated with mitochondria and cell membranes) functions as one of the most important repressors of cell death in this family. The objective of this study was to determine the expression and localization of the bcl-2 protein in human gametes and preembryos (PEs) up to the blastocyst stage. Design: Research donated human gametes and PEs were studied during oocyte maturation stages, following fertilization, and up to the blastocyst stage of growth. Materials and Methods: Expression of the bcl-2 protein was analyzed using immunofluorescence. Specimens were fixed and permeabilizated before incubation with fluorescent conjugated monoclonal antibody which recognizes bcl-2 protein. Results: The bcl-2 protein was strongly expressed in human oocytes of all maturation stages. The protein was highly expressed throughout the cytoplasm with lower intensity around the metaphase plate. This expression was conserved even after five days in culture when localization shifted to the oocyte membrane. Compared to the surrounding cumulus-corona cells, the expression of the protein was considerably higher within the oocytes. In contrast, bound sperm cells stained negative. At the pronuclear stage, bcl-2
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