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Regulation of HOX A11 in Trophoblastic Cells
Results: GDF-9 transcript was detected by RT-PCR in the neonatal ovaries that consist of only oocytes and primordial follicles. GDF-9 protein was also detected by Western blotting in the neonatal ovaries but at the lower level compared to 4- and 8-day ones. By Western blot analysis, proand mature forms of GDF-9 protein were observed in all cases. In the cultured ovarian tissues, GDF-9 mRNA was detected in the similar level to that of in vivo tissues. But different patterns of the protein levels in cultured tissues were observed compared to the tissues in vivo at appropriate days. By histological observations, however, the cultured ovarian follicles were healthy and there were growing follicles indicating that the follicular growth has been started in culture. Conclusions: Results from the present study, it is concluded that: 1) To our knowledge, this is the first report of detection of the GDF-9 protein in the neonatal ovaries (primordial follicles) by Western blot analysis. 2) Observed pattern of GDF-9 expression in the cultured mouse ovaries is consistent with a role for GDF-9 in the regulation of folliculogenesis. But the characterization of the difference in the expression pattern of the GDF-9 protein between in vivo and in vitro is under investigation. * Supported by a grant (HMP-98-M-5-0054) of the Good Health R&D Project, Ministry of Health & Welfare, Republic of Korea.
P-319 Reprogramming of Human Embryonic and Somatic Cell Nuclei in Bovine Oocytes. H. Shim, S. H. Jun, Y. H. Kim, J. M. Lim, H. C. Lee, K. Y. Cha. Infertility Medical Center of CHA General Hospital and School of Medicine, Pochon CHA University, Seoul, Korea. Objective: To assess the reprogramming competence of nuclei from human embryonic and somatic cells, the development of reconstituted bovine oocytes after the transfer of human nuclei was assessed. Nucleus donor cells used in this study are known to retain different characteristics. Embryonal carcinoma (EC) cells are embryonic cells, and many of such cells are in S-phase of the cell cycle. Fetal fibroblast (FF) cells are fetal somatic cells, the most of which are in G1-phase. Cumulus (CM) cells are adult somatic cells presumably in G0-phase. Combining with the further isolation of embryonic stem cells from nuclear-transferred (NT) embryos, this study will assist the use of nuclear transfer technology toward stem cell therapy. Design: Development of NT oocytes using various cell types in humans was examined. Materials and Methods: EC cells were purchased from ATCC (American Type Culture Collection; CRL-2073). From consenting patients, FF cells were obtained from the selective abortion, and CM cells from the cumulusoocyte complex of retrieved ova. Various nucleus donor cells were fused to the enucleated MII oocytes using 6V AC pulse for 6 sec followed by 1.8 kV/cm DC pulse for 15 msec. Reconstituted embryos were then activated with 5 mM ionomycin for 4 min and subsequently 1.9 mM 6-dimethylaminopurine for 4 h. NT embryos were cultured in SOF containing 0.3% BSA, and in vitro development of the embryos was monitored. Some of the dividing embryos were analyzed for their chromosomal configuration. Results: The reconstituted oocytes from all three cell types cleaved (71% from EC cells, n587; 61% from FF cells, n551; 64% from CM cells, n550) and developed to the 8-cell stage (31% from EC cells; 4% from FF cells; 22% from CM cells). Chromosomal analysis of NT embryos confirmed that the transferred human nuclei were successfully reprogrammed in bovine oocytes. However, except only 7% of NT embryos derived from EC cells developed to the 16-cell stage, no NT embryos developed beyond the stage of maternal-zygote transition, thought to occur in humans at the 4- to 8-cell stages. Conclusions: Nuclei from human embryonic and somatic cells can be reprogrammed in enucleated bovine oocytes. However, transferred human nuclei supported the development of embryos only until the 8- to 16-cell stages. This might be due to the lack of optimized culture condition, the discrepancy in the timing of embryonic genome activation between two species, or to the incomplete reprogramming of karyoplasts from human cells.
P-320 Regulation of HOX A11 in Trophoblastic Cells. Y. Zhang1, B. Xu2, L. Peterson2, N. Rote1,2, L. Amesse1. Department of Obstetrics and Gynegology, Department of Microbiology, Wright State University, Dayton, OH.
FERTILITY & STERILITYt
Objective: Embryonic implantation and placentation is a complex process requiring multiple interactions between embryonic and maternal cells. In our initial studies we showed that HOX A11 is expressed in trophoblasts, and is down regulated as cytotrophoblast differentiated into syncytiotrophoblasts. The dynamics of this down regulation suggest a role for HOX A11 in placental development. To expand on these initial findings, we examined hormonal, cytokine and growth factor regulation of HOX A11 in trophoblastic cell line. Design: To study the effects of exogenous regulatory agents on the expression of HOX A11 in trophoplastic cell lines. Materials and Methods: BeWo and ED27 trophoblastic cell lines were cultured in Ham’s F12 supplemented media. The cultured cells were treated with the following substances: b-Estradiol 5 3 1028M, Progesterone 1 3 1027M, interferon-g (INF-g) 30 ng/ml, and Epidermal Growth Factor (EGF) 50 ng/ml. In some experiments, cyclohexamide was added to culture media before treatment with estradiol. Expression of HOXA11 was determined by reverse transcriptase polymerase chain reaction, Northern, and Western Blotting. Results: Estradiol treatment of both the BeWo and ED27 cell lines showed a dose and time dependent effect on HOX A11. Maximum inhibition of expression of HOA A11 was found at 14 hours exposure to estradiol. HOXA11 expression was not inhibited by the addition of cyclohexamide prior to estradiol treatment. This finding suggests that estradiols effect on HOX A11 gene expression does not require protein synthesis. Treatment of these cell lines with progesterone, INF-g, and EGF had no significant effect on HOX A11 expression. Conclusion: HOX A11 gene expression is down regulated by estradiol in a time and dose specific manner suggesting hormonal control of this gene in trophoblastic cells. Other cytokines and growth factors as well as progesterone had little effect on these trophoblastic derived cells. These results continue to support the role of HOX A11 in trophoblast differentiation.
P-321 The Effect of O2 Tension on the Blastocyst (BL) Development of Mouse Embryos In Vitro. 1T. Mukaida, 1K. Takahashi, 2Y. Yamauchi, 2T. Horiuchi. 1Hiroshima HART Clinic, Hiroshima, 2Department of Bioresources, Hiroshima Pref. University Shobara, Japan. Objectives: The use of sequential media have enabled the extention of human embryo culture to reach blastocyst (BL) efficiently in ART. In order to obtain viable BL, optimal culture conditions are critical. Since we use these sequential media for 5– 6 days, we must consider whether or not the culture condition should be the same as that of conventional 2–3 days culture. Especially, concentration of CO2 and O2 was known to be closely related to pH balance and oxygen radical formations. Design: To investigate the optimal CO2 and O2 tension in sequential media for BL development of mouse embryos. Materials and Methods: Zygotes were collected from ICR mice (Slc, Hamamatsu, Japan), and cultured in groups of 10 in 20 ml drops of medium under mineral oil. All of these embryos were cultured for the first 48 h in G1.2 (IVF Science, Gothenberg, Sweden) to the 8-cell stage, followed by culture for a further 48 h in G2.2 to the BL stage under different O2 (2, 5, 10, 20%), CO2 (5, 7, 10%), and N2. conditions. Level of PCO2 and PO2 in the culture medium was measured by an i-STAT Portable Clinical Analyzer (Abbot Laboratories Inc, USA). In-vitro morphological development was examined every 24 hrs during culture until the hatching stage. Embryo cell number and allocation to the inner cell mass (ICM) and trophectderm (TE) were determined using differential staining with propidium iodide (SIGMA) and bisbenzimide (Hoechst 33258, SIGMA). Differential staining of nuclei was determined under UV light using a fluorescence microscope. Fisher’s Least Significant Difference (LSD) test was carried out for statistical analysis. Results: When we changed CO2 concentration under 5% O2 condition, levels of PCO2 and pH were varied from 35.9 6 0.7 mmHg and 7.38 (5.0% CO2), 47.3 6 1.7 mmHg and 7.27 (7.0% CO2), and 63.8 6 2.2 mmHg and 7.15 (10% CO2) respectively. BL development, hatching rate and number of total BL cells, ICM cells, and TE cells did not have any statistical difference in each different CO2 condition. Levels of PO2 were varied from 65.0 6 2.0 mmHg (2.0% O2), 80.3 6 2.8 (5.0% O2), 97.0 6 0.6 (10% O2) and 142 6 0.3 mmHg (20% O2) under 5% CO2 condition. BL development rate did not show a significant difference in each different O2 condition, but hatching rate under 2% O2 condition was significantly lower (1.9%) than under other
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P-319 Reprogramming of Human Embryonic and Somatic Cell Nuclei in Bovine Oocytes. H. Shim, S. H. Jun, Y. H. Kim, J. M. Lim, H. C. Lee, K. Y. Cha. Infertility Medical Center of CHA General Hospital and School of Medicine, Pochon CHA University, Seoul, Korea. Objective: To assess the reprogramming competence of nuclei from human embryonic and somatic cells, the development of reconstituted bovine oocytes after the transfer of human nuclei was assessed. Nucleus donor cells used in this study are known to retain different characteristics. Embryonal carcinoma (EC) cells are embryonic cells, and many of such cells are in S-phase of the cell cycle. Fetal fibroblast (FF) cells are fetal somatic cells, the most of which are in G1-phase. Cumulus (CM) cells are adult somatic cells presumably in G0-phase. Combining with the further isolation of embryonic stem cells from nuclear-transferred (NT) embryos, this study will assist the use of nuclear transfer technology toward stem cell therapy. Design: Development of NT oocytes using various cell types in humans was examined. Materials and Methods: EC cells were purchased from ATCC (American Type Culture Collection; CRL-2073). From consenting patients, FF cells were obtained from the selective abortion, and CM cells from the cumulusoocyte complex of retrieved ova. Various nucleus donor cells were fused to the enucleated MII oocytes using 6V AC pulse for 6 sec followed by 1.8 kV/cm DC pulse for 15 msec. Reconstituted embryos were then activated with 5 mM ionomycin for 4 min and subsequently 1.9 mM 6-dimethylaminopurine for 4 h. NT embryos were cultured in SOF containing 0.3% BSA, and in vitro development of the embryos was monitored. Some of the dividing embryos were analyzed for their chromosomal configuration. Results: The reconstituted oocytes from all three cell types cleaved (71% from EC cells, n587; 61% from FF cells, n551; 64% from CM cells, n550) and developed to the 8-cell stage (31% from EC cells; 4% from FF cells; 22% from CM cells). Chromosomal analysis of NT embryos confirmed that the transferred human nuclei were successfully reprogrammed in bovine oocytes. However, except only 7% of NT embryos derived from EC cells developed to the 16-cell stage, no NT embryos developed beyond the stage of maternal-zygote transition, thought to occur in humans at the 4- to 8-cell stages. Conclusions: Nuclei from human embryonic and somatic cells can be reprogrammed in enucleated bovine oocytes. However, transferred human nuclei supported the development of embryos only until the 8- to 16-cell stages. This might be due to the lack of optimized culture condition, the discrepancy in the timing of embryonic genome activation between two species, or to the incomplete reprogramming of karyoplasts from human cells.
P-320 Regulation of HOX A11 in Trophoblastic Cells. Y. Zhang1, B. Xu2, L. Peterson2, N. Rote1,2, L. Amesse1. Department of Obstetrics and Gynegology, Department of Microbiology, Wright State University, Dayton, OH.
FERTILITY & STERILITYt
Objective: Embryonic implantation and placentation is a complex process requiring multiple interactions between embryonic and maternal cells. In our initial studies we showed that HOX A11 is expressed in trophoblasts, and is down regulated as cytotrophoblast differentiated into syncytiotrophoblasts. The dynamics of this down regulation suggest a role for HOX A11 in placental development. To expand on these initial findings, we examined hormonal, cytokine and growth factor regulation of HOX A11 in trophoblastic cell line. Design: To study the effects of exogenous regulatory agents on the expression of HOX A11 in trophoplastic cell lines. Materials and Methods: BeWo and ED27 trophoblastic cell lines were cultured in Ham’s F12 supplemented media. The cultured cells were treated with the following substances: b-Estradiol 5 3 1028M, Progesterone 1 3 1027M, interferon-g (INF-g) 30 ng/ml, and Epidermal Growth Factor (EGF) 50 ng/ml. In some experiments, cyclohexamide was added to culture media before treatment with estradiol. Expression of HOXA11 was determined by reverse transcriptase polymerase chain reaction, Northern, and Western Blotting. Results: Estradiol treatment of both the BeWo and ED27 cell lines showed a dose and time dependent effect on HOX A11. Maximum inhibition of expression of HOA A11 was found at 14 hours exposure to estradiol. HOXA11 expression was not inhibited by the addition of cyclohexamide prior to estradiol treatment. This finding suggests that estradiols effect on HOX A11 gene expression does not require protein synthesis. Treatment of these cell lines with progesterone, INF-g, and EGF had no significant effect on HOX A11 expression. Conclusion: HOX A11 gene expression is down regulated by estradiol in a time and dose specific manner suggesting hormonal control of this gene in trophoblastic cells. Other cytokines and growth factors as well as progesterone had little effect on these trophoblastic derived cells. These results continue to support the role of HOX A11 in trophoblast differentiation.
P-321 The Effect of O2 Tension on the Blastocyst (BL) Development of Mouse Embryos In Vitro. 1T. Mukaida, 1K. Takahashi, 2Y. Yamauchi, 2T. Horiuchi. 1Hiroshima HART Clinic, Hiroshima, 2Department of Bioresources, Hiroshima Pref. University Shobara, Japan. Objectives: The use of sequential media have enabled the extention of human embryo culture to reach blastocyst (BL) efficiently in ART. In order to obtain viable BL, optimal culture conditions are critical. Since we use these sequential media for 5– 6 days, we must consider whether or not the culture condition should be the same as that of conventional 2–3 days culture. Especially, concentration of CO2 and O2 was known to be closely related to pH balance and oxygen radical formations. Design: To investigate the optimal CO2 and O2 tension in sequential media for BL development of mouse embryos. Materials and Methods: Zygotes were collected from ICR mice (Slc, Hamamatsu, Japan), and cultured in groups of 10 in 20 ml drops of medium under mineral oil. All of these embryos were cultured for the first 48 h in G1.2 (IVF Science, Gothenberg, Sweden) to the 8-cell stage, followed by culture for a further 48 h in G2.2 to the BL stage under different O2 (2, 5, 10, 20%), CO2 (5, 7, 10%), and N2. conditions. Level of PCO2 and PO2 in the culture medium was measured by an i-STAT Portable Clinical Analyzer (Abbot Laboratories Inc, USA). In-vitro morphological development was examined every 24 hrs during culture until the hatching stage. Embryo cell number and allocation to the inner cell mass (ICM) and trophectderm (TE) were determined using differential staining with propidium iodide (SIGMA) and bisbenzimide (Hoechst 33258, SIGMA). Differential staining of nuclei was determined under UV light using a fluorescence microscope. Fisher’s Least Significant Difference (LSD) test was carried out for statistical analysis. Results: When we changed CO2 concentration under 5% O2 condition, levels of PCO2 and pH were varied from 35.9 6 0.7 mmHg and 7.38 (5.0% CO2), 47.3 6 1.7 mmHg and 7.27 (7.0% CO2), and 63.8 6 2.2 mmHg and 7.15 (10% CO2) respectively. BL development, hatching rate and number of total BL cells, ICM cells, and TE cells did not have any statistical difference in each different CO2 condition. Levels of PO2 were varied from 65.0 6 2.0 mmHg (2.0% O2), 80.3 6 2.8 (5.0% O2), 97.0 6 0.6 (10% O2) and 142 6 0.3 mmHg (20% O2) under 5% CO2 condition. BL development rate did not show a significant difference in each different O2 condition, but hatching rate under 2% O2 condition was significantly lower (1.9%) than under other
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