- Email: [email protected]
Interleukin-1 (IL-1) Gene Expression in Mouse Embryonic Stem (ES) Cells
The presence of heart beating identified ES cells derived cardiomyocytes, which were isolated by microsurgery to be used as samples for gene expression study. We used FGF5, Gata6 and Brachyury (T) as markers for the presence of ectoderm, endoderm and mesoderm, respectively. RT-PCR and PCR were run with negative controls and 10 l of PCR products were analyzed by 2% gel electrophoresis. RESULTS: None of the 45 undifferentiated ES cells were positive for IL-1; 1 out of 45 was positive for IL-1ra, and this sample was also positive for ectoderm and endoderm primitive markers; 1 out of 45 was positive for IL-1RtI and also for ectoderm and mesoderm primitive markers. Four out of 4 ES cells derived cardiomyocytes were positive for IL-1 and also for IL-1ra. Two out of 4 ES cells derived cardiomyocytes were positive for IL-1RtI. CONCLUSION: The IL-1 family has been correlated to implantation in mammals by many articles, but to our knowledge this is the first time to describe an IL-1 family gene expression study in undifferentiated mouse ES cells. It appeared in samples that have already shown signals of differentiation into mesoderm, ectoderm or endoderm or in ES cells derived cardiomyocytes. Undifferentiated mouse ES cells most likely do not produce major components of IL-1 family. IL-1 may be produced mainly by trophoblast cells in embryo-maternal cross-talk during implantation. Further experiments including others methodologies may provide more information to elucidate the role of IL-1 family during implantation process in mouse. Supported by: Cornell University/EUA and CAPES/Brazil
P-694 Establishment of a Basic System for Single Culture of Preantral Follicles: Relationship Between Retrieval Method and Follicle Development, and in Vitro-Growth of the Follicles and Follicular Oocytes. H. M. Choi, T. S. Lee, Y. S. Moon, Y. J. Han, M. J. Lim. Department of Food and Animal Biotechnology, Seoul, Republic of Korea; Medical Research Center, Seoul National University, Seoul, Republic of Korea.
Supported by: Institute of the Laboratories of Medical Investigation, Clinical Hospital, School of Medicine, University of Sa˜o Paulo. P-693 Interleukin-1 (IL-1) Gene Expression in Mouse Embryonic Stem (ES) Cells. R. L. Tavares, V. Freitas, A. Ferreira, Z. Rosenwaks, K. Xu. Universidade Federal de Minas Gerais, Belo Horizonte, Brazil; Universidade Federal de Sa˜o Paulo, Sa˜o Paulo, Brazil; The Center for Reproductive Medicine and Infertility - Cornell University, New York, NY; The Center for Reproductive Medicine and Infertility - Cornell University, New York, NY. OBJECTIVE: To study the IL-1 family gene expression in undifferentiated mouse embryonic stem (ES) cells and ES cells derived cardiomyocytes. DESIGN: Prospective study MATERIALS AND METHODS: We studied the IL-1 family gene expression in two different groups: undifferentiated mouse ES cells and ES cell derived cardiomyocytes. We used OCT-4 and Nanog as markers of the undifferentiated state of nine new mouse ES cells lines, isolated from the 129 substrain mouse. Zona-free blastocysts were used as a positive control for Oct-4 and Nanog expression and  actin was used as control for the presence of template. Biopsies retrieved at 5, 10, 15, 20 and 25th passages were taken to performe RT-PCR using SuperScript™ III CellsDirect cDNA Synthesis System. PCR amplification was done with Platinum® Taq DNA Polymerase. We used Interleukin-1 (IL-1), IL-1 receptor antagonist (IL-1ra) and IL-1 receptor type I (IL-1RtI) primers to study the IL-1 family.
FERTILITY & STERILITY威
OBJECTIVE: To establish a basic manipulation protocol of preantral follicles for developing novel biotechnology and reproductive medicine. DESIGN: Randomized, prospective study using an animal model. MATERIALS AND METHODS: Two-week old female F1 (C57BL6/ DBA2) mice were sacrificed and the ovaries were removed aseptically. Preantral follicles were retrieved mechanically or enzymatically and subsequently classified into three categories (primary, early secondary and late secondary) by measuring diameter and the typical morphology. All categorized follicles were cultured at 37 °C, 5% CO2 in air atmosphere. Each categorized follicles were cultured individually in 20 l culture droplets overlaid with washed-mineral oil and half of a medium was changed every other day. In vitro-growth of the follicles were monitored daily and maturation of oocytes in cultured follicles was induced by the stimulation with 2.5 IU/ml hCG and 5 ng/ml epidermal growth factor. Oocyte maturation was evaluated by the polar body extrusion and cumulus cell mucification, while capacity of mature oocytes to form pronucleus was monitored after parthenogenetic activation using 10mM SrCl2. A generalized linear model (PROC-GLM) in a Statistical Analysis System (SAS) program was employed and significant differences among groups were determined where the P value was less then 0.05. RESULTS: A mechanical method retrieved more (P⬍0.0001) follicles (339⫾48 vs. 202⫾28) than an enzymatic method. However, the enzymatic method collected more singly-isolated follicles that could be provided for subsequent culture (102⫾26 vs. 202⫾28). The follicles cultured in vitro commonly underwent a step-by-step growth consisting of the follicular, diffuse, pseudoantral and degenerative stages. The retrieval method affected the follicular growth: to reach the maximal incidence of the pseudoantral stage, primary, early secondary and late secondary follicles retrieved by the mechanical method required at least 11, 10 and 7 days, respectively. In the enzymatic method, however, 9 and 6 days for early and late secondary follicles and primary follicles were not possible to reach to the pseudoantral stage. The optimal time of oocyte maturation was 10-11, 7-9 and 5-7 days for primary, early secondary and late secondary follicles, respectively. A general decrease in oocyte diameter was detected in in vitro-derived compared with in vivo-derived oocytes (63.31-65.53 m vs. 75 m) and the enzymatic retrieval typically reduced zona thickness (5.41-5.74 m vs. 7.76 m). Pronuclear formation was detected in 86-94% of oocytes after parthenogenetic activation and no significant difference was detected among groups.
S405
P-694 Establishment of a Basic System for Single Culture of Preantral Follicles: Relationship Between Retrieval Method and Follicle Development, and in Vitro-Growth of the Follicles and Follicular Oocytes. H. M. Choi, T. S. Lee, Y. S. Moon, Y. J. Han, M. J. Lim. Department of Food and Animal Biotechnology, Seoul, Republic of Korea; Medical Research Center, Seoul National University, Seoul, Republic of Korea.
Supported by: Institute of the Laboratories of Medical Investigation, Clinical Hospital, School of Medicine, University of Sa˜o Paulo. P-693 Interleukin-1 (IL-1) Gene Expression in Mouse Embryonic Stem (ES) Cells. R. L. Tavares, V. Freitas, A. Ferreira, Z. Rosenwaks, K. Xu. Universidade Federal de Minas Gerais, Belo Horizonte, Brazil; Universidade Federal de Sa˜o Paulo, Sa˜o Paulo, Brazil; The Center for Reproductive Medicine and Infertility - Cornell University, New York, NY; The Center for Reproductive Medicine and Infertility - Cornell University, New York, NY. OBJECTIVE: To study the IL-1 family gene expression in undifferentiated mouse embryonic stem (ES) cells and ES cells derived cardiomyocytes. DESIGN: Prospective study MATERIALS AND METHODS: We studied the IL-1 family gene expression in two different groups: undifferentiated mouse ES cells and ES cell derived cardiomyocytes. We used OCT-4 and Nanog as markers of the undifferentiated state of nine new mouse ES cells lines, isolated from the 129 substrain mouse. Zona-free blastocysts were used as a positive control for Oct-4 and Nanog expression and  actin was used as control for the presence of template. Biopsies retrieved at 5, 10, 15, 20 and 25th passages were taken to performe RT-PCR using SuperScript™ III CellsDirect cDNA Synthesis System. PCR amplification was done with Platinum® Taq DNA Polymerase. We used Interleukin-1 (IL-1), IL-1 receptor antagonist (IL-1ra) and IL-1 receptor type I (IL-1RtI) primers to study the IL-1 family.
FERTILITY & STERILITY威
OBJECTIVE: To establish a basic manipulation protocol of preantral follicles for developing novel biotechnology and reproductive medicine. DESIGN: Randomized, prospective study using an animal model. MATERIALS AND METHODS: Two-week old female F1 (C57BL6/ DBA2) mice were sacrificed and the ovaries were removed aseptically. Preantral follicles were retrieved mechanically or enzymatically and subsequently classified into three categories (primary, early secondary and late secondary) by measuring diameter and the typical morphology. All categorized follicles were cultured at 37 °C, 5% CO2 in air atmosphere. Each categorized follicles were cultured individually in 20 l culture droplets overlaid with washed-mineral oil and half of a medium was changed every other day. In vitro-growth of the follicles were monitored daily and maturation of oocytes in cultured follicles was induced by the stimulation with 2.5 IU/ml hCG and 5 ng/ml epidermal growth factor. Oocyte maturation was evaluated by the polar body extrusion and cumulus cell mucification, while capacity of mature oocytes to form pronucleus was monitored after parthenogenetic activation using 10mM SrCl2. A generalized linear model (PROC-GLM) in a Statistical Analysis System (SAS) program was employed and significant differences among groups were determined where the P value was less then 0.05. RESULTS: A mechanical method retrieved more (P⬍0.0001) follicles (339⫾48 vs. 202⫾28) than an enzymatic method. However, the enzymatic method collected more singly-isolated follicles that could be provided for subsequent culture (102⫾26 vs. 202⫾28). The follicles cultured in vitro commonly underwent a step-by-step growth consisting of the follicular, diffuse, pseudoantral and degenerative stages. The retrieval method affected the follicular growth: to reach the maximal incidence of the pseudoantral stage, primary, early secondary and late secondary follicles retrieved by the mechanical method required at least 11, 10 and 7 days, respectively. In the enzymatic method, however, 9 and 6 days for early and late secondary follicles and primary follicles were not possible to reach to the pseudoantral stage. The optimal time of oocyte maturation was 10-11, 7-9 and 5-7 days for primary, early secondary and late secondary follicles, respectively. A general decrease in oocyte diameter was detected in in vitro-derived compared with in vivo-derived oocytes (63.31-65.53 m vs. 75 m) and the enzymatic retrieval typically reduced zona thickness (5.41-5.74 m vs. 7.76 m). Pronuclear formation was detected in 86-94% of oocytes after parthenogenetic activation and no significant difference was detected among groups.
S405