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Müllerian inhibiting substance inhibits cytochrome P450 aromatase expression in granulosa lutein cells
and the secretory (N⫽12) phases of the menstrual cycle. Total RNA was isolated and subjected to Realtime PCR. The results were analyzed using unpaired Student t-test and Tuckey test (ANOVA) with a probability level of P⬍0.05 considered significant. RESULTS: Using Realtime PCR, we demonstrated the expression of fibromodulin and Abi-2 in leiomyoma and myometrium. The expression of fibromodulin and Abi-2 was significantly elevated in leiomyoma compared to myometrium from proliferative phase (P⬍0.05), but no difference in their expression in these tissues was seen in the secretory phase of the menstrual cycle. The expression of fibromodulin and Abi-2 was significantly reduced in leiomyoma and myometrium from the GnRHa-treated group reaching the level observed in myometrium from proliferative phase (P⫽0.05). CONCLUSION: These results for the first time document the expression of fibromodulin and Abi-2 in leiomyoma and myometrium providing evidence that the expression of these genes is influenced by ovarian steroids and possibly by direct action of GnRHa on myometrial and leiomyoma cells. Since the products of these genes are key regulators of tissue remodeling as well as cell growth and differentiation, we propose that their products may be involved in the regulation mechanisms that influence leiomyoma growth and regression. Supported by: NIH grant HD37243
Wednesday, October 20, 2004 2:30 P.M. O-221
* p⬍0.0003 compared with Control(CTRL) ** p⬍0.02, p⬍0.005, and p⬍0.007 compared with CTRL, CTRL⫹MIS, and FSH, respectively.
* p⬍0.0004 compared with CTRL ** p⬍0.0001 compared with FSH CONCLUSION: MIS inhibited FSH induced E2 production in GLC by suppressing CYP19 gene expression. We propose that MIS may have a role in the pathophysiology of PCOS, since follicular fluid and serum MIS levels in women with PCOS are elevated with correspondingly lower E2 concentrations. Supported by: Kosair Charities and Norton HealthCare Community Trust Fund.
Mu¨ llerian inhibiting substance inhibits cytochrome P450 aromatase expression in granulosa lutein cells. M. P. Grossman, Y. Siow, S. Guo, S. T. Nakajima, M. E. Fallat. University of Louisville, Louisville, KY. OBJECTIVE: To investigate the effects of Mu¨ llerian Inhibiting Substance (MIS) on Cytochrome P450 aromatase (CYP 19) gene expression in granulosa-lutein cells (GLC). DESIGN: In vitro primary cell culture study. MATERIALS AND METHODS: GLC were collected from oocyte aspirates (four women; two male and two tubal factor infertility) undergoing IVF procedures. GLC were cultured on Matrigel-coated cell culture plates (500,000 cells/well) for 7 days at 37°C and 5% CO2. Culture media (Eagle’s medium) supplemented with 10% female fetal calf serum (MIS absent) and 10-7M androstenedione as substrate was used and replaced daily. Spent media were saved and frozen (-80°C) for E2 assay later. Cells were treated with culture media without (CTRL) or with MIS (10ng/mL) (CTRL ⫹ MIS) or with 0.2 IU/mL FSH without or with MIS (FSH ⫹ MIS). On culture day 7, cultured cells were harvested and total RNA extracted using Trizol. Quantitation of CYP19 mRNA was done by reverse transcription (RT) and real-time polymerase chain reaction (PCR). PCR was carried out using customized CYP19 primers and Taqman MGB probes (Applied Biosystem, CA). E2 concentrations in spent media were determined by immunoassay (Diagnostic System Laboratories, TX). Statistical comparison was by ANOVA and post hoc Tukey tests. RESULTS: Data shown are mean ⫾ SD. E2 output represents concentration in spent media and changes in CYP19 gene expression are presented as fold change in CYP19 mRNA levels relative to levels in Control.
FERTILITY & STERILITY威
Wednesday, October 20, 2004 2:45 P.M. O-222 Rescue of zona-free embryos by partial encapsulation with collagen followed by coculture with endometrial cells. H.-C. C. Liu, Z. Y. He, A. Galindo Trias, Z. Rosenwaks. Weill Medical College of Cornell University, New York, NY; Hospital Universitario de la Vall d’Hebron, Barcelona, Spain. OBJECTIVE: Zona-free oocytes, a frequent occurrence when micromanipulation techniques are employed in IVF laboratories, are usually discarded. The purpose of this study was to see whether these embryos can be rescued by encapsulation with artificial zona pellucida. DESIGN: Zona-free mouse embryos encapsulated with sodium alginate (AZP, Group A), partially encapsulated with collagen micro-wells (Group B), zona-free embryos (Group C), and zona-intact embryos as control (Group D), were cocultured with endometrial cells. Embryo development of these studied groups were compared. MATERIALS AND METHODS: Enzymatically isolated mouse endometrial cells were plated in 4-well plates (2 ⫻ 105 cells/well) and cultured with F12/199 medium supplemented with 10% FCS before coculture. Pronuclear-staged embryos were harvested from superovulated female CF1 mice (zona intact, control), treated with pronase to remove the zona pellucida (zona-free). Zona-free embryos were immersed in 1.5% sodium alginate solution, then aspirated with a small amount of sodium alginate and expelled into 1.5% calcium chloride to form microgels (AZP). All zonaintact, zona-free, and AZP embryos were cocultured with endometrial cells plated in 4-well plates directly. For the collagen microwells group, collagen type-I were coated at the surface of the monolayer endometrial cells in the 4-well plates and these micro-wells were formed by introducing small air bubbles in the layer of the collagen gel. The size of the wells is slightly larger then that of the embryos in order to encapsulate the embryos. Zona-free embryos were then transferred to each of the micro-well for coculture (collagen micro-wells group). Embryo development were evaluated every day for 5 days. RESULTS: Zona-free embryos tend to have abnormal early embryo development. They either disassociated into fragments or aggregated to form mosaic embryos. Addition of fully or partially encapsulated zona pellucida (AZP and collagen micro-wells, respectively) did enhance embryo development of these zona-free embryos. Most of the AZP appeared to have abnormal blastomere arrangement and blastocoele collapse and its blasto-
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Wednesday, October 20, 2004 2:30 P.M. O-221
* p⬍0.0003 compared with Control(CTRL) ** p⬍0.02, p⬍0.005, and p⬍0.007 compared with CTRL, CTRL⫹MIS, and FSH, respectively.
* p⬍0.0004 compared with CTRL ** p⬍0.0001 compared with FSH CONCLUSION: MIS inhibited FSH induced E2 production in GLC by suppressing CYP19 gene expression. We propose that MIS may have a role in the pathophysiology of PCOS, since follicular fluid and serum MIS levels in women with PCOS are elevated with correspondingly lower E2 concentrations. Supported by: Kosair Charities and Norton HealthCare Community Trust Fund.
Mu¨ llerian inhibiting substance inhibits cytochrome P450 aromatase expression in granulosa lutein cells. M. P. Grossman, Y. Siow, S. Guo, S. T. Nakajima, M. E. Fallat. University of Louisville, Louisville, KY. OBJECTIVE: To investigate the effects of Mu¨ llerian Inhibiting Substance (MIS) on Cytochrome P450 aromatase (CYP 19) gene expression in granulosa-lutein cells (GLC). DESIGN: In vitro primary cell culture study. MATERIALS AND METHODS: GLC were collected from oocyte aspirates (four women; two male and two tubal factor infertility) undergoing IVF procedures. GLC were cultured on Matrigel-coated cell culture plates (500,000 cells/well) for 7 days at 37°C and 5% CO2. Culture media (Eagle’s medium) supplemented with 10% female fetal calf serum (MIS absent) and 10-7M androstenedione as substrate was used and replaced daily. Spent media were saved and frozen (-80°C) for E2 assay later. Cells were treated with culture media without (CTRL) or with MIS (10ng/mL) (CTRL ⫹ MIS) or with 0.2 IU/mL FSH without or with MIS (FSH ⫹ MIS). On culture day 7, cultured cells were harvested and total RNA extracted using Trizol. Quantitation of CYP19 mRNA was done by reverse transcription (RT) and real-time polymerase chain reaction (PCR). PCR was carried out using customized CYP19 primers and Taqman MGB probes (Applied Biosystem, CA). E2 concentrations in spent media were determined by immunoassay (Diagnostic System Laboratories, TX). Statistical comparison was by ANOVA and post hoc Tukey tests. RESULTS: Data shown are mean ⫾ SD. E2 output represents concentration in spent media and changes in CYP19 gene expression are presented as fold change in CYP19 mRNA levels relative to levels in Control.
FERTILITY & STERILITY威
Wednesday, October 20, 2004 2:45 P.M. O-222 Rescue of zona-free embryos by partial encapsulation with collagen followed by coculture with endometrial cells. H.-C. C. Liu, Z. Y. He, A. Galindo Trias, Z. Rosenwaks. Weill Medical College of Cornell University, New York, NY; Hospital Universitario de la Vall d’Hebron, Barcelona, Spain. OBJECTIVE: Zona-free oocytes, a frequent occurrence when micromanipulation techniques are employed in IVF laboratories, are usually discarded. The purpose of this study was to see whether these embryos can be rescued by encapsulation with artificial zona pellucida. DESIGN: Zona-free mouse embryos encapsulated with sodium alginate (AZP, Group A), partially encapsulated with collagen micro-wells (Group B), zona-free embryos (Group C), and zona-intact embryos as control (Group D), were cocultured with endometrial cells. Embryo development of these studied groups were compared. MATERIALS AND METHODS: Enzymatically isolated mouse endometrial cells were plated in 4-well plates (2 ⫻ 105 cells/well) and cultured with F12/199 medium supplemented with 10% FCS before coculture. Pronuclear-staged embryos were harvested from superovulated female CF1 mice (zona intact, control), treated with pronase to remove the zona pellucida (zona-free). Zona-free embryos were immersed in 1.5% sodium alginate solution, then aspirated with a small amount of sodium alginate and expelled into 1.5% calcium chloride to form microgels (AZP). All zonaintact, zona-free, and AZP embryos were cocultured with endometrial cells plated in 4-well plates directly. For the collagen microwells group, collagen type-I were coated at the surface of the monolayer endometrial cells in the 4-well plates and these micro-wells were formed by introducing small air bubbles in the layer of the collagen gel. The size of the wells is slightly larger then that of the embryos in order to encapsulate the embryos. Zona-free embryos were then transferred to each of the micro-well for coculture (collagen micro-wells group). Embryo development were evaluated every day for 5 days. RESULTS: Zona-free embryos tend to have abnormal early embryo development. They either disassociated into fragments or aggregated to form mosaic embryos. Addition of fully or partially encapsulated zona pellucida (AZP and collagen micro-wells, respectively) did enhance embryo development of these zona-free embryos. Most of the AZP appeared to have abnormal blastomere arrangement and blastocoele collapse and its blasto-
S89