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Fas ligand and tumor suppressor genes in human granulosa cells.
Objective: The presence of hydrosalpinges decreases implantation rates and pregnancy rates in women undergoing in-vitro fertilization and embryo transfer. We studied the effect of hydrosalpinx fluid on the expression of HOXA10, an endometrial transcription factor known to be necessary for implantation. Design: HOXA10 expression in endometrial cell culture was determined by northern blot analysis. Materials/Methods: Hydrosalpinx fluid was aspirated transvaginally from 5 patients at the time of IVF retrieval. The fluid was immediately aliquoted and serial dilutions prepared in Minimum Essential cell culture medium. Ishikawa cells, a well-differentiated endometrial adenocarcinoma cell line were incubated with this fluid at dilutions of 10%, 40% and 50% for 48 hours. Cells incubated in undiluted Minimum Essential cell culture medium were used as controls. Three sets of experiments were performed on each patient sample at each dilution. At 48 hours, the cells were lysed in Trizol, total RNA extracted and analyzed by northern blot using a 32P labeled HOXA10 riboprobe. A 32P labeled G3PDH riboprobe was used as a control for RNA loading. Results: HOXA10 mRNA expression in endometrial cells was found to decrease with increasing concentrations of hydrosalpinx fluid. The decrease was inversely proportional to the concentration of hydrosalpinx fluid. Densitometric analysis of the Northern blot revealed that HOXA10 mRNA expression was significantly different from control at all levels of dilution used, at p ⬍ 0.001 using Student’s t-test. Conclusions: HOXA10 is known to be necessary for implantation. Here we demonstrate that hydrosalpinx fluid decreases the expression of HOXA10 in endometrial cells. This may be a molecular mechanism for diminished implantation rates observed in women with hydrosalpinges. Supported by: NIH HD36887.
Objective: To assess the effect of BSA, glucose and phosphate addition to a chemically defined medium (M16) on the development of mouse one-cell embryos in vitro. Design: Prospective experimental study. Materials/Methods: Harvested mouse (ICR) one-cell embryos were cultured for 48 hours using M16 solution with various concentrations (0-15%) of BSA. First, mouse 1-cell embryos were divided into two groups. One group of them was cultured under the same condition for further 72 hours and the other group was cultured in the media with fixed concentration (0.3%) of BSA, and evaluated the effect of media on the 2-cell block and further embryonic development. Secondly, the effects of removal of glucose and/or phosphate from the BSA conditioned culture media (M16) on the development of mouse embryo were also evaluated. Results: Higher concentration (0.9 –1.5%) of BSA in the culture media during the first 48 hours of 1-cell mouse embryo culture overcome 2-cell block significantly, however, the continued exposure to higher concentrations after 48 hours do not influence further development. Removal of glucose and/or phosphate from the culture media during the early stage (first 48 hours) of culture showed significant overcoming of 2-cell block and increased the blastulation rate, but after the first 48 hours of culture, the absence or presence of glucose and/or phosphate do not influence the further embryonic development. Finally, the positive effects of the removal of glucose and/or phosphate are also augmented with the increased BAS concentration of the culture media. Conclusions: Increased BSA concentration and removal of glucose and/or phosphate during the early stage of culture may have favorable effect on 2-cell block and further development of mouse embryo in vitro.
P-153 P-151 Expression profiles of Fas/Fas ligand and tumor suppressor genes in human granulosa cells. C. Keck, D. Pietrowski. Univ Freiburg, Freiburg, Germany.
Genetic expression of monocarboxylate transporters (MCTs) isoforms and basigin during human oocyte maturation. F. Herubel, Y. Menezo, S. El Mouatassim, E. Servy, R. Frydman. IRH-Laboratoire Marcel Merieux, Bron, France; Augusta Area Reproductive Assoc, Augusta, GA; Hoˆpital Antoine Be´cle`re, Clamart, France.
Objective: Ovarian cell death is an essential process for the homeostasis of ovarian function. Luteolysis by programmed cell death or apoptosis is important for maintenance of ovarian cyclicity. Although there are multiple pathways that can determine cell death or cell survival, crosstalk among protooncogenes, tumor suppressor genes, survival genes and death genes plays an important role in determining the fate of ovarian cells. The objective of this study was to analyse the expression pattern of various tumor suppressor genes (TSG) and the Fas/Fas ligand system with or without stimulation with human chorion gonadotropin (hCG) in human granulosa cells (GC). Design: The expression of the following TSGs involved in different mechanism of apoptosis was studied by RT-PCR: RB-1 (Familial retinoblastoma), VHL (Von-Hippel Lindau), NF-1 (Von Recklinghausen Neurofibromatosis), NF-2 (Neurofibromatosis type 2), Wt-1 (Wilms tumor) p53 (Li Fraumeni), APC (Familial adenomatous polyopsis). Expression of Fas/ Fas ligand was analysed after stimulation with 1 U, 5 U and 20 U hCG. Materials/Methods: GCs were isolated from follicular fluid of patients undergoing IVF therapy as described elsewhere (Keck et al., 1998). RNA was isolated by the method of Chomczynski and Sacchi (1987) and Western Blot analysis was performed as described elsewhere. Results: In GCs derived from IVF patients tumor suppressor genes VHL, NF-1, NF-2, WT-1 p53 and APC are expressed. However, we could not detect any mRNA message for RB-1. In addition we detected mRNA for Fas and Fas ligand in human GC. Stimulation of GC with hCG resulted in an accumulation of Fas ligand protein as shown in Western blot analysis. Conclusions: We conclude that the expression of tumor suppressor genes and the Fas/Fas ligand system is important for regulation of apoptosis. HCG stimulates Fas ligand expression. This suggests that hCG modulates apoptotic mechanisms by interaction with the Fas/Fas ligand system.
Objective: Pyruvate and lactate are the major energy sources for the preimplantation human embryo, during the first cleavage stages and thus represent important components for embryos culture media. The purpose of this study was to investigate which members of the MCT family were involved in the monocarboxylate uptake by human oocytes. Design: Messenger RNAs of MCT isoforms 1, 2, 4 and basigin, a chaperone protein of MCT 1 and 4, were qualitatively analysed in individual human immature oocytes at the germinal vesicle (GV) stage and in nonfertilized human metaphase II (MII) oocytes. Materials/Methods: Reverse transcription nested polymerase chain reaction ( RT-nested PCR) on GV oocytes were collected from cohorts of oocytes for ICSI, or in MII oocytes in the case of no sperm recovery. Analysis was performed using either poly (dT) probes or 3 specific probes. Results: Transcripts encoding for MCT 1 and MCT 2 were detectable in the majority of the oocytes analyzed. MCT 4 mRNA was not detected in single oocytes and was barely detectable in pools of 5 oocytes. Basigin transcripts were found to be unadenylated at the GV stage, and then fully adenylated at the MII stage of oocytes maturation. Conclusions: MCT 1 and MCT 2 seem to be the two main members for moocarboxylate uptake in human oocytes. These two transcripts are fully polyadenylated at the MII stage. The expression profile of MCT 4 in GV and MII oocytes reflects a very low level of transcription, raising doubts concerning its real biological influence during preimplantation development. Out results concerning basigin confirm that a “last minute” regulatory polyadenylation process occurs during oocyte maturation (EL Moustassim et al, Mol Human Reprod. 1999, Gandolfi et al. 1999). Thus basigin transcripts are translationally inactive until the MII stage and can be considered as markers of cytoplasmic maturation. Supported by: This project was supported by Augusta Area Reproductive Associates and IRH-Laboratoire Marcel Merieux.
P-152
P-154
Effects of BSA, glucose and phosphate on 2-cell block and blastocyst development of 1-cell mouse embryos during in vitro culture. S. H. Kim, Y. H. Lee, J. W. Shin, T. Kim. Korea Univ Hosp, Ansan, Korea.
Expression of Th1 cytokine IL-12 is unaltered in women with hydrosalpinx. S. Y. Brett, J. Thong, T. A. Henderson, D. Niven, A. Williams, H. Critchley. Royal Infirmary of Edinburgh, Edinburgh, UK.
FERTILITY & STERILITY威
S163
Objective: To assess the effect of BSA, glucose and phosphate addition to a chemically defined medium (M16) on the development of mouse one-cell embryos in vitro. Design: Prospective experimental study. Materials/Methods: Harvested mouse (ICR) one-cell embryos were cultured for 48 hours using M16 solution with various concentrations (0-15%) of BSA. First, mouse 1-cell embryos were divided into two groups. One group of them was cultured under the same condition for further 72 hours and the other group was cultured in the media with fixed concentration (0.3%) of BSA, and evaluated the effect of media on the 2-cell block and further embryonic development. Secondly, the effects of removal of glucose and/or phosphate from the BSA conditioned culture media (M16) on the development of mouse embryo were also evaluated. Results: Higher concentration (0.9 –1.5%) of BSA in the culture media during the first 48 hours of 1-cell mouse embryo culture overcome 2-cell block significantly, however, the continued exposure to higher concentrations after 48 hours do not influence further development. Removal of glucose and/or phosphate from the culture media during the early stage (first 48 hours) of culture showed significant overcoming of 2-cell block and increased the blastulation rate, but after the first 48 hours of culture, the absence or presence of glucose and/or phosphate do not influence the further embryonic development. Finally, the positive effects of the removal of glucose and/or phosphate are also augmented with the increased BAS concentration of the culture media. Conclusions: Increased BSA concentration and removal of glucose and/or phosphate during the early stage of culture may have favorable effect on 2-cell block and further development of mouse embryo in vitro.
P-153 P-151 Expression profiles of Fas/Fas ligand and tumor suppressor genes in human granulosa cells. C. Keck, D. Pietrowski. Univ Freiburg, Freiburg, Germany.
Genetic expression of monocarboxylate transporters (MCTs) isoforms and basigin during human oocyte maturation. F. Herubel, Y. Menezo, S. El Mouatassim, E. Servy, R. Frydman. IRH-Laboratoire Marcel Merieux, Bron, France; Augusta Area Reproductive Assoc, Augusta, GA; Hoˆpital Antoine Be´cle`re, Clamart, France.
Objective: Ovarian cell death is an essential process for the homeostasis of ovarian function. Luteolysis by programmed cell death or apoptosis is important for maintenance of ovarian cyclicity. Although there are multiple pathways that can determine cell death or cell survival, crosstalk among protooncogenes, tumor suppressor genes, survival genes and death genes plays an important role in determining the fate of ovarian cells. The objective of this study was to analyse the expression pattern of various tumor suppressor genes (TSG) and the Fas/Fas ligand system with or without stimulation with human chorion gonadotropin (hCG) in human granulosa cells (GC). Design: The expression of the following TSGs involved in different mechanism of apoptosis was studied by RT-PCR: RB-1 (Familial retinoblastoma), VHL (Von-Hippel Lindau), NF-1 (Von Recklinghausen Neurofibromatosis), NF-2 (Neurofibromatosis type 2), Wt-1 (Wilms tumor) p53 (Li Fraumeni), APC (Familial adenomatous polyopsis). Expression of Fas/ Fas ligand was analysed after stimulation with 1 U, 5 U and 20 U hCG. Materials/Methods: GCs were isolated from follicular fluid of patients undergoing IVF therapy as described elsewhere (Keck et al., 1998). RNA was isolated by the method of Chomczynski and Sacchi (1987) and Western Blot analysis was performed as described elsewhere. Results: In GCs derived from IVF patients tumor suppressor genes VHL, NF-1, NF-2, WT-1 p53 and APC are expressed. However, we could not detect any mRNA message for RB-1. In addition we detected mRNA for Fas and Fas ligand in human GC. Stimulation of GC with hCG resulted in an accumulation of Fas ligand protein as shown in Western blot analysis. Conclusions: We conclude that the expression of tumor suppressor genes and the Fas/Fas ligand system is important for regulation of apoptosis. HCG stimulates Fas ligand expression. This suggests that hCG modulates apoptotic mechanisms by interaction with the Fas/Fas ligand system.
Objective: Pyruvate and lactate are the major energy sources for the preimplantation human embryo, during the first cleavage stages and thus represent important components for embryos culture media. The purpose of this study was to investigate which members of the MCT family were involved in the monocarboxylate uptake by human oocytes. Design: Messenger RNAs of MCT isoforms 1, 2, 4 and basigin, a chaperone protein of MCT 1 and 4, were qualitatively analysed in individual human immature oocytes at the germinal vesicle (GV) stage and in nonfertilized human metaphase II (MII) oocytes. Materials/Methods: Reverse transcription nested polymerase chain reaction ( RT-nested PCR) on GV oocytes were collected from cohorts of oocytes for ICSI, or in MII oocytes in the case of no sperm recovery. Analysis was performed using either poly (dT) probes or 3 specific probes. Results: Transcripts encoding for MCT 1 and MCT 2 were detectable in the majority of the oocytes analyzed. MCT 4 mRNA was not detected in single oocytes and was barely detectable in pools of 5 oocytes. Basigin transcripts were found to be unadenylated at the GV stage, and then fully adenylated at the MII stage of oocytes maturation. Conclusions: MCT 1 and MCT 2 seem to be the two main members for moocarboxylate uptake in human oocytes. These two transcripts are fully polyadenylated at the MII stage. The expression profile of MCT 4 in GV and MII oocytes reflects a very low level of transcription, raising doubts concerning its real biological influence during preimplantation development. Out results concerning basigin confirm that a “last minute” regulatory polyadenylation process occurs during oocyte maturation (EL Moustassim et al, Mol Human Reprod. 1999, Gandolfi et al. 1999). Thus basigin transcripts are translationally inactive until the MII stage and can be considered as markers of cytoplasmic maturation. Supported by: This project was supported by Augusta Area Reproductive Associates and IRH-Laboratoire Marcel Merieux.
P-152
P-154
Effects of BSA, glucose and phosphate on 2-cell block and blastocyst development of 1-cell mouse embryos during in vitro culture. S. H. Kim, Y. H. Lee, J. W. Shin, T. Kim. Korea Univ Hosp, Ansan, Korea.
Expression of Th1 cytokine IL-12 is unaltered in women with hydrosalpinx. S. Y. Brett, J. Thong, T. A. Henderson, D. Niven, A. Williams, H. Critchley. Royal Infirmary of Edinburgh, Edinburgh, UK.
FERTILITY & STERILITY威
S163