- Email: [email protected]
Semiquantitative Analysis of Common Deletion in Mitochondrial DNA of Single Human Oocytes
is expressed around pronuclei where mitochondria are predominantly located. The expression of bcl-2 protein in cleaving PEs was generally observed in the center of PEs, though it varied in some PEs from the whole cytoplasm to the blastomere membrane only in some arrested PEs. Also, no staining was detected in some PEs. At the blastocyst stage, the expression of bcl-2 was concentrated in the area of the inner cell mass (ICM). Conclusions: (1) Bcl-2 protein is highly expressed and conserved in human oocytes. (2) Bcl-2 expression in preembryos varied and showed polarization mostly in the center of the preembryo. (3) Bcl-2 is expressed in the blastocyst primarily in the area of the ICM.
P-316 Regulation of Endometrial Gene Expression by Recombinant Human Chorionic Gonadotropin (rhCG). 1R. S. Hines, 1M. N. Brackin, 1B. D. Cowan, 2C. H. Wyatt, 2S. T. Case. 1Department of Obstetrics and Gynecology, 2Department of Biochemistry, University of Mississippi Medical Center, Jackson, MS. Objective: BeWo choriocarcinoma alters endometrial gene expression via soluble factors in a co-culture model of implantation. We studied the effect of rhCG on gene expression from endometrium grown in culture. Design: Cultured human endometrium was treated with rhCG and analyzed by differential display for alterations in gene expression. Materials and Methods: Endometrial biopsies were obtained from reproductive age women in the luteal phase and cultured until prolactin concentrations increased in the supernatant. rhCG was added to the culture for 4 hours in concentrations of 0, 102, 103, and 104 mIU/mL. Total ribonucleic acid was extracted from control and experimental endometrial cultures. Differential display was performed by creating complimentary deoxyribonucleic acid (cDNA) with reverse transcription using one of three oligo dT 39 anchor primers. Polymerase chain reaction (PCR) of the cDNA was performed with an arbitrary 59 primer, the original 39 anchor primer and [35S] dATP. PCR products were electrophoresed on denaturing polyacrylamide gels prior to autoradiography. cDNAs that were altered by the experimental condition were excised from the gel and blunt-end ligated into a plasmid vector for cloning. The sequence of expressed fragments was determined on a LI-COR 4000 automated DNA sequencer. Results: Differential display revealed a homogenous band pattern throughout most lanes. Three bands were altered in a dose response fashion at 104 mIU/ mL of rhCG. Fragments of 201, 236, and 256 base pairs (bp) were decreased in intensity in the experimental lanes, suggesting downregulation by rhCG. These sequences were subjected to BLAST analysis of the nonredundant and the expressed sequence tag databases. The 201 bp fragment exhibited high probability sequence identity with human chromosome 14 and 17 fragments as well as a cDNA clone from fetal liver. The 236 bp fragment had a significant match to a human clone from chromosome 7. The 256 bp fragment matched cDNA from human ovary. Conclusion: At implantation a communication exists between the trophoblast and the maternal endometrium. Previous differential display and cDNA array experiments with a co-culture of endometrium and BeWo have demonstrated changes in endometrial gene expression as a result of soluble trophoblast factors. hCG can be measured in the supernatant of cultured BeWo. We tested the hypothesis that rhCG could be responsible for some changes demonstrated by BeWo. These experiments support the idea that few of the changes observed in a co-culture model of paracrine perinidatory signaling previously reported by our laboratory are due to hCG alone. A dose dependent response to rhCG was demonstrated and the affected cDNAs may represent factors important in implantation.
P-317 Semiquantitative Analysis of Common Deletion in Mitochondrial DNA of Single Human Oocytes. 1S. H. Lee, 1J. H. Han, 1H. H. Kim, 1S. M. Lee, 2 M. K. Chung, 1K. Y. Cha. 1Department of Obstetrics and Gynecology, Human Genetics Laboratory, 2Embryology Laboratory, of CHA General Hospital of Pochon CHA University, Seoul, Korea. Objectives: Most cells in the body contain between 103 and 104 copies of mitochondrial DNA (mtDNA). This mtDNA plays an essential role in the life cycle via the control of energy production by oxidative phosphorylation. The asymmetric nature of inheritance is exclusively through the female
S194
Abstracts
oocyte. In a mature human oocyte, much higher copy numbers (105) of mtDNA is present to prepare for the energy demands of embryogenesis. There are measurable levels of the so-called “common deletion”, which removes 4977 bp between the ATPase8 and ND5 genes in some oocytes. Not all common deletion becomes pathogenic, but some studies have reported the frequency of deleted mtDNA in oocytes, embryos and have demonstrated that the shift in the mutation decreases with oocyte and embryo development. The purpose of this study was to determine the semi-quantitation of common deletion in oocyte developmental potency. Design: Retrospective cohort study. Materials and Methods: All procedures were reviewed and approved by Pochon CHA University’s Institutional Review Board. Only those oocytes with no evidence of fertilization were used in these experiments. Sixtyseven patients from the CHA IVF-ET program donated a total eight-five unfertilized metaphase II (MII) oocytes derived from mature (Group 1) and immature (Group 2) stage. After removing zona pellucida of oocyte with acidified Tyrode’s solution, a single oocyte was placed in 5 ul of lysis buffer to release DNA. Two rounds of PCR preceded by aliquots of two 2.5 ul were performed for common deletion using primers MT-1 (8239 – 8263) and MT-3 (13551–13575) followed by primers MQ-1 (8421– 8441) and MQ-3 (13500 –13520). For the purpose of semiquantitation, other site within the oocyte mitochondrial genome, 16s rRNA was also amplified to control the DNA in each sample using primers mt16s-F (2515–2537) and mt16s-R (2984 –3003) followed by primres mt16s-1F (2642–2662) and mt16s-1R (2830 –2850). A 10 ul aliquot of the PCR product for common deletion and 15 ul aliquot of 16s rRNA were run on a 2% agarose gel, stained with ethidium bromide and photographed. The relative abundance of the PCR products was determined by the Imaging Densitometry and results were expressed as a ratio of band intensity of common deletion divided by 16s rRNA. Results: A total of 85 unfertilized oocytes from 67 patients were analyzed, in which 42 (49.4%) showed deletion. At developmental stage, the ratio of common deletion divided by 16s rRNA was 24.1553 in Group 2 stage oocytes and 12.5837 in Group 1 stage oocytes. Data were compared with the use of x2 and Anova tests. Statistical significance was defined as P,.05. Conclusions: Although the sample size was too small to conclude definite effects of common deletion, there seemed to be a trend toward decreased amount of common deletion in mature oocytes than in immature oocytes. According to this semi-quantitative analysis, we can hypothesize that the amount of mtDNA deletion may have effect on the oocyte maturity with subsequent fertilization. This finding is supported by reports of Brenner et al., Keefe et al. who described: the potential role of mtDNA together with the effect of altered oxidative phosphorylation and cellular antioxidant systems on the cytoskeleton, fertilization and subsequent embryo development.
P-318 Comparison of the GDF-9 mRNA and Protein Expression Between In Vivo and In Vitro Cultured Mouse Ovaries.* 1S. J. Yoon, 1C. S. Shin, 1,2 K.-H. Baek, 1,2J. J. Ko, 1,2N. Y. Yoon, 1,2K.-A. Lee. 1Infertility Medical Center, CHA General Hospital, 2College of Medicine, Pochon CHA University, Seoul, Korea. Objective: GDF-9 is an oocyte-derived growth factor having a crucial role in the normal follicular development beyond the primary follicle stage in mice. The present study was conducted to evaluate the effect of the in vitro culture of the ovarian tissues on the expression of follicular GDF-9 transcript and proteins. Materials and Methods: ICR mouse ovaries were obtained on the day of birth, postnatal day 4, and day 8 for in vivo control. Neonatal ovaries were cultured in a-MEM medium supplemented with insulin, transferrin, selenium, 10% FBS, and 100 mIU/ml of rhFSH. After 4- and 8-day in vitro culture, ovaries were collected and snap frozen for RT-PCR and Western blot analysis. The GDF-9 cDNA (446 bp) was synthesized by RT-PCR from neonatal mouse ovarian RNA using oligonucleotides 59-GGTTCTATCTGATAGGCGAGG and 59-GGGGCTGAAGGAGGGAGG. The PCR condition was: 94°C for 1 min, 67°C for 1 min, 72°C for 1 min with 30 cycles followed by a final extension of 10 min in 72°C. For Western blot analysis, a commercial affinity-purified goat polyclonal GDF-9 antibody was used. Immunodetection of the protein was achieved using ECL system. Gel bands from films were scanned and quantitatively analyzed by density contrast. To evaluate the culture effect, histological analysis of the ovarian tissues was conducted.
Vol. 74, No. 3, Suppl. 1, September 2000
P-316 Regulation of Endometrial Gene Expression by Recombinant Human Chorionic Gonadotropin (rhCG). 1R. S. Hines, 1M. N. Brackin, 1B. D. Cowan, 2C. H. Wyatt, 2S. T. Case. 1Department of Obstetrics and Gynecology, 2Department of Biochemistry, University of Mississippi Medical Center, Jackson, MS. Objective: BeWo choriocarcinoma alters endometrial gene expression via soluble factors in a co-culture model of implantation. We studied the effect of rhCG on gene expression from endometrium grown in culture. Design: Cultured human endometrium was treated with rhCG and analyzed by differential display for alterations in gene expression. Materials and Methods: Endometrial biopsies were obtained from reproductive age women in the luteal phase and cultured until prolactin concentrations increased in the supernatant. rhCG was added to the culture for 4 hours in concentrations of 0, 102, 103, and 104 mIU/mL. Total ribonucleic acid was extracted from control and experimental endometrial cultures. Differential display was performed by creating complimentary deoxyribonucleic acid (cDNA) with reverse transcription using one of three oligo dT 39 anchor primers. Polymerase chain reaction (PCR) of the cDNA was performed with an arbitrary 59 primer, the original 39 anchor primer and [35S] dATP. PCR products were electrophoresed on denaturing polyacrylamide gels prior to autoradiography. cDNAs that were altered by the experimental condition were excised from the gel and blunt-end ligated into a plasmid vector for cloning. The sequence of expressed fragments was determined on a LI-COR 4000 automated DNA sequencer. Results: Differential display revealed a homogenous band pattern throughout most lanes. Three bands were altered in a dose response fashion at 104 mIU/ mL of rhCG. Fragments of 201, 236, and 256 base pairs (bp) were decreased in intensity in the experimental lanes, suggesting downregulation by rhCG. These sequences were subjected to BLAST analysis of the nonredundant and the expressed sequence tag databases. The 201 bp fragment exhibited high probability sequence identity with human chromosome 14 and 17 fragments as well as a cDNA clone from fetal liver. The 236 bp fragment had a significant match to a human clone from chromosome 7. The 256 bp fragment matched cDNA from human ovary. Conclusion: At implantation a communication exists between the trophoblast and the maternal endometrium. Previous differential display and cDNA array experiments with a co-culture of endometrium and BeWo have demonstrated changes in endometrial gene expression as a result of soluble trophoblast factors. hCG can be measured in the supernatant of cultured BeWo. We tested the hypothesis that rhCG could be responsible for some changes demonstrated by BeWo. These experiments support the idea that few of the changes observed in a co-culture model of paracrine perinidatory signaling previously reported by our laboratory are due to hCG alone. A dose dependent response to rhCG was demonstrated and the affected cDNAs may represent factors important in implantation.
P-317 Semiquantitative Analysis of Common Deletion in Mitochondrial DNA of Single Human Oocytes. 1S. H. Lee, 1J. H. Han, 1H. H. Kim, 1S. M. Lee, 2 M. K. Chung, 1K. Y. Cha. 1Department of Obstetrics and Gynecology, Human Genetics Laboratory, 2Embryology Laboratory, of CHA General Hospital of Pochon CHA University, Seoul, Korea. Objectives: Most cells in the body contain between 103 and 104 copies of mitochondrial DNA (mtDNA). This mtDNA plays an essential role in the life cycle via the control of energy production by oxidative phosphorylation. The asymmetric nature of inheritance is exclusively through the female
S194
Abstracts
oocyte. In a mature human oocyte, much higher copy numbers (105) of mtDNA is present to prepare for the energy demands of embryogenesis. There are measurable levels of the so-called “common deletion”, which removes 4977 bp between the ATPase8 and ND5 genes in some oocytes. Not all common deletion becomes pathogenic, but some studies have reported the frequency of deleted mtDNA in oocytes, embryos and have demonstrated that the shift in the mutation decreases with oocyte and embryo development. The purpose of this study was to determine the semi-quantitation of common deletion in oocyte developmental potency. Design: Retrospective cohort study. Materials and Methods: All procedures were reviewed and approved by Pochon CHA University’s Institutional Review Board. Only those oocytes with no evidence of fertilization were used in these experiments. Sixtyseven patients from the CHA IVF-ET program donated a total eight-five unfertilized metaphase II (MII) oocytes derived from mature (Group 1) and immature (Group 2) stage. After removing zona pellucida of oocyte with acidified Tyrode’s solution, a single oocyte was placed in 5 ul of lysis buffer to release DNA. Two rounds of PCR preceded by aliquots of two 2.5 ul were performed for common deletion using primers MT-1 (8239 – 8263) and MT-3 (13551–13575) followed by primers MQ-1 (8421– 8441) and MQ-3 (13500 –13520). For the purpose of semiquantitation, other site within the oocyte mitochondrial genome, 16s rRNA was also amplified to control the DNA in each sample using primers mt16s-F (2515–2537) and mt16s-R (2984 –3003) followed by primres mt16s-1F (2642–2662) and mt16s-1R (2830 –2850). A 10 ul aliquot of the PCR product for common deletion and 15 ul aliquot of 16s rRNA were run on a 2% agarose gel, stained with ethidium bromide and photographed. The relative abundance of the PCR products was determined by the Imaging Densitometry and results were expressed as a ratio of band intensity of common deletion divided by 16s rRNA. Results: A total of 85 unfertilized oocytes from 67 patients were analyzed, in which 42 (49.4%) showed deletion. At developmental stage, the ratio of common deletion divided by 16s rRNA was 24.1553 in Group 2 stage oocytes and 12.5837 in Group 1 stage oocytes. Data were compared with the use of x2 and Anova tests. Statistical significance was defined as P,.05. Conclusions: Although the sample size was too small to conclude definite effects of common deletion, there seemed to be a trend toward decreased amount of common deletion in mature oocytes than in immature oocytes. According to this semi-quantitative analysis, we can hypothesize that the amount of mtDNA deletion may have effect on the oocyte maturity with subsequent fertilization. This finding is supported by reports of Brenner et al., Keefe et al. who described: the potential role of mtDNA together with the effect of altered oxidative phosphorylation and cellular antioxidant systems on the cytoskeleton, fertilization and subsequent embryo development.
P-318 Comparison of the GDF-9 mRNA and Protein Expression Between In Vivo and In Vitro Cultured Mouse Ovaries.* 1S. J. Yoon, 1C. S. Shin, 1,2 K.-H. Baek, 1,2J. J. Ko, 1,2N. Y. Yoon, 1,2K.-A. Lee. 1Infertility Medical Center, CHA General Hospital, 2College of Medicine, Pochon CHA University, Seoul, Korea. Objective: GDF-9 is an oocyte-derived growth factor having a crucial role in the normal follicular development beyond the primary follicle stage in mice. The present study was conducted to evaluate the effect of the in vitro culture of the ovarian tissues on the expression of follicular GDF-9 transcript and proteins. Materials and Methods: ICR mouse ovaries were obtained on the day of birth, postnatal day 4, and day 8 for in vivo control. Neonatal ovaries were cultured in a-MEM medium supplemented with insulin, transferrin, selenium, 10% FBS, and 100 mIU/ml of rhFSH. After 4- and 8-day in vitro culture, ovaries were collected and snap frozen for RT-PCR and Western blot analysis. The GDF-9 cDNA (446 bp) was synthesized by RT-PCR from neonatal mouse ovarian RNA using oligonucleotides 59-GGTTCTATCTGATAGGCGAGG and 59-GGGGCTGAAGGAGGGAGG. The PCR condition was: 94°C for 1 min, 67°C for 1 min, 72°C for 1 min with 30 cycles followed by a final extension of 10 min in 72°C. For Western blot analysis, a commercial affinity-purified goat polyclonal GDF-9 antibody was used. Immunodetection of the protein was achieved using ECL system. Gel bands from films were scanned and quantitatively analyzed by density contrast. To evaluate the culture effect, histological analysis of the ovarian tissues was conducted.
Vol. 74, No. 3, Suppl. 1, September 2000