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P-190 Localization of gap junctional proteins connexin (Cx) 26, Cx32 and Cx43 in the uterus of non-pregnant and early pregnant ewes
of 1.5M dimethyl sulfoxide (DMSO). These data were then used to predict the probability of intracellular ice formation. Design: Individual oocytes were immobilized with a holding pipette using negative pressure on the zona pellucida. Oocytes were first placed into very small amounts (5 #1) of an isosmotic phosphate buffered saline (PBS) solution and perfused with a precooled or prewarmed 1.5M DMSO solution. Oocyte volume changes were recorded throughout the exposure to the CPA. The volumetric changes were calculated from the measurement of the radius diameter of the oocytes, assuming a spherical shape. The initial volume of the oocyte in isosmotic solution was considered 100%, and relative changes in the volume of the oocyte after exposure to CPA were plotted as a function of time. Results: After calculation, mean hydraulic conductivity (Lp) values at 30, 20, 10, 3, 0 and -3°C were found to be 1.07_+0.03 (mean_SEM), 0.40-+0.02, 0.18-+0.006, 0.076-+0.006, 5.29×10-2-+0.4×10 -2 and 3.69×10-2-+0.3 × 10 .2 #m/min/atm. The corresponding DMSO permeability (PDMsO) values were 3.69×10-s-+0.3×10 -3, 1.07×10 -s -+0.1×10 3, 2.75×10-4-+0.15×10-4, 7.83×10-5-+0.5×10-5, 5.24×10-5-+0.5×10 -5 and 3.69×10-5-+0.4×10 -~ cm/min. The corresponding reflection coefficients were 0.70+0.03, 0.77-+0.04, 0.81-+0.06, 0.91-+0.05, 0.97_+0.03 and 1_+0.04. The estimated activation energy (Ea) for Lp was 16.39 Kcal/ mol and 23.25 Kcal/mol for PDMSO. Conclusions: These data provide the basis for the development of optimal protocols for cryopreservation of metaphase II m a m m a l i a n oocytes.
P-189 Evaluation of Follicular Transforming Growth Factor-fl (TGF-fl) Content and TGF-fl Receptor Type II (T~RII) Gene Expression With Relation to Embryonic Potential of H u m a n Oocytes. 1S. G. Kurz, 1'2A. M. Carlson, 1'2'3S.K. Roy, 1C. J. DeJonge, 1j. W. Ramey, 1V. M. Maclin-Collins. 1Olson Center for Women's Health, Depts of OB/GYN and 2Physiology & Biophysics, University of Nebraska Medical Center, Omaha, Nebraska. Objectives: TflRII is essential for TGF-~ action and its expression in granulosa cells is hormonally regulated. The objective was to evaluate if TGFp has an influence on future embryonic potential of the oocytes. Design: The study was done by monitoring follicular TGF-~ concentration, T/~RII mRNA expression in granulosa cells and the microscopic morphology of the embryos. Materials and Methods: Women, <40 years of age, undergoing controlled ovarian hyperstimulation (Leuprolide acetate, FSH and/or FSH/LH) prior to ultrasound-guided transvaginal follicular aspiration of oocytes were included in the study. Granulosa cells and follicular fluid from individual follicular aspirates containing a single oocyte were collected by centrifugation and processed separately. TGFcontent in follicular fluid was detected by a receptorbased ELISA following acid activation. T/~RII mRNA was reversed transcribed and PCR amplified from 300 ng gran-
ulosa cell RNA using a sequence specific primer pair and 21-biotin-dUTP. After transferring to a Zetaprobe membrane the amplicons were identified by chemiluminography and digitized by a Molecular Dynamics laser densitometer. Embryonic morphology was evaluated microscopically. Results: Follicles that produced oocytes with grade 1 embryonic potential had very high TGF-/~ content but lowest T~RII mRNA expression. Follicular TGF-~ content varied considerably among follicles. T~RII mRNA expression was 2 fold higher in follicles, which were smaller or larger than 18-20 mm. Conclusions: High concentration of TGF-/~ may induce overt or premature differentiation of resident granulosa cells, thereby affecting embryonic potential of the oocyte. A decrease in TGF-p receptor gene transcription will result in less receptor synthesis, thus reducing the effect of TGF-f~ on granulosa cells. This regulation may produce a better quality oocyte that will become a grade 1 embryo upon fertilization. Our data also suggest a novel mechanism regulating TGF-/~ influence on follicular development. 3This work was supported by research grants from NIH (HD 18165) and Olson Foundation to S. K. Roy.
P-190 Localization of Gap J u n c t i o n a l Proteins Connexin (Cx) 26, Cx32 and Cx43 in the Uterus of Non-pregnant and Early Pregnant Ewes. 1A. T. Grazul-Bilska, 1,2j. j. Bilski, ~D. A. Redmer, ~'2L. P. Reynolds. 1Cell Biology Center & 2Department of Animal and Range Sciences, Fargo, ND. Objectives: Gap junctions have been demonstrated to be very important for control of growth of organs and tissues. Gap junctions are formed by proteins named connexins. Evaluation of the presence of connexins is widely used for detection of gap junctions. During each estrous cycle or pregnancy, the uterus grows and regresses. Therefore, gap junctions may play a role in regulation of uterine tissue growth and remodeling. In the present study, we have examined the presence of three connexins in the uterus from several stages of the estrous cycle and early pregnancy in sheep. Design: Presence of Cx26, Cx32 and Cx43 was examined in ovine uterine compartments throughout the estrous cycle and early pregnancy by using immunohistochemistry. Materials and Methods: Uteri were obtained from ewes on days 0, 2, 4, 8, 12 and 15 of the estrous cycle and on days 12, 18, 24, 30 and 40 of pregnancy ( n = 3 - 5 ewes per day); tissue sections were fixed in Carnoy's or formalin solution, embedded in paraffin; and Cx26, Cx32 and Cx43 were immunolocalized by using specific antibodies (Zymed, San Francisco, CA). Results: Cx26, Cx32 and Cx43 were detected in the uterus in all stages examined, and staining appeared to be punctate. Cx26 was detected in the endometrial and myometrial blood vessels and myometrial smooth muscles across all stages. Cx26 staining was weak in endometrial Abstracts
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stroma. In endometrial glands, Cx26 was present in all stages except on days 0, 2 and 4 of the estrous cycle. Cx32 was present in endometrial stroma and blood vessels, and in myometrial smooth muscles across all stages, but in luminal epithelium only during pregnancy. In addition, Cx32 was present in the stroma around endometrial glands across the estrous cycle and on day 12 of pregnancy, and in endometrial glands epithelium at all stages except on days 0, 2 and 4 of the estrous cycle. Cx43 was localized in luminal epithelium, endometrial stroma, endometrial glands and some blood vessels. Distribution of connexins in endometrial stroma and glands, and myometrium was heterogenous within each tissue section. Conclusions: Connexins are expressed in the several compartments of the uterus across the estrous cycle and early pregnancy, which indicates that gap junctions are present in the uterus. Expression of connexins changes in association with growth and development of uterine tissues, suggesting that gap junctions play a role in control of the growth and remodeling of the ovine uterus. Supported in part by N I H grant 1R29 HD30348 to ATGB, and by USDA grant 93-37203-9271 to DAR and LPR. P-191 The Effect o f H u m a n C h o r i o n i c G o n a d o t r o p i n (hCG) o n T r o p h o b l a s t Cell Motility. K. A. Wei, F. D. Yelian. Department of Obstetrics and Gynecology, Wayne State University School of Medicine, Detroit, MI. Objectives: Embryo implantation involves trophoblast cell adhesion to endometrial epithelium and invasion to the interstitium. Both adhesion and motility are important characteristics of trophoblast cells. Our previous studies have demonstrated that a high concentration of hCG inhibits cell adhesion to extracellular matrix protein fibronectin. In this study we are investigating the effect of hCG on trophoblast cell motility. Design: Trophoblast cells were treated with various concentrations of hCG and the cell motility was measured using a modified phagokinetic track motility assay. Materials and Methods: Both HTR-8/SVneo (HTR), an extented-lifespan first trimester trophoblast cell line, and JEG-3, a choriocarcinoma cell line were used in this study. Uniform carpets of gold particles were prepared on glass coverslips in a 6-well culture plate. Five thousand cells were transferred onto each well and incubated with a culture medium containing 10% fetal bovine serum. After the cells attached to the coverslip, the culture medium was replaced with a serum-free medium containing various concentrations of hCG (1, 10, 100, 500, and 1,000 mIU/ ml). Following 24 h incubation at 37°C, the phagokinetic tracks of cell movement were analysed using a computerassisted imaging system. The mean cell motility in each group was determined by measuring 50-100 randomly selected cells and the difference among groups were analyzed using one-way analysis of variance. Results: The mean cell motilities for HTR cells treated with hCG at 0, 1, 10, 100, 500, and 1,000 mIU/ml were 6604-+272, 8626_+413, 8954-+483, 7379-+452, 4724_+260, and 4256_+278 #m S, respectively. The motility for JEG-3 S184
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cells in same treated groups were 2906+220, 2962_+160, 3824_+278, 2460_+201, 1621_+93, and 1672-+115 #m 2, respectively. Statistically significant differences were found among groups (P<0.001). Interestingly, a low concentration ofhCG (1 and 10 mIU/ml) significantly increased HTR cell motility and a high concentration of hCG (500 and 1,000 mIU/ml) significantly decreased cell motility in both HTR and JEG-3 cells (P<0.01). Conclusions: This study demonstrated for the first time that hCG modulates trophoblast cell motility. In conjunction with our previous adhesion study, these data suggest that hCG may play an important role in regulating trophoblast cell function during early embryo implantation. P-192 M i t o c h o n d r i a l DNA (mtDNA) D e l e t i o n s in H u m a n Oocytes ~,2j. A. Barritt, 8B. M. Miller, 3G. C. VanTuyle, 1'2D. W. Matt. 1Department of Obstetrics and Gynecology, 2Department of Anatomy, and 8Department of Biochemistry, Virginia Commonwealth University, Richmond, Virginia. Objectives: Although it is well established that many h u m a n oocytes and pre-implantation embryos fail to fertilize and develop, the intracelhilar mechanisms that contribute to these failures are largely unknown. Currently, we are exploring whether alterations in mitochondrial function of h u m a n oocytes are related to fertilization failure or embryonic developmental arrest. Several degenerative diseases have been associated with mtDNA deletions (dmtDNA), and recent studies have identified a 4977 base pair "common deletion" in unfertilized h u m a n oocytes. The purpose of the current study was to identify the frequency and types of mtDNA deletions in h u m a n oocytes. Materials and Methods: A "nested" two-round PCR approach was developed to amplify the majority of the larger arc region between the two origins of replication ofmtDNA in h u m a n oocytes. The Medical College of Virginia's In Vitro Fertilization Program provided 96 h u m a n oocytes (from 36 patients) that had been discarded because they failed to fertilize 48 hours after insemination or intracytoplasmic sperm injection (ICSI). PCR products amplified from individual oocytes were identified by gel electrophoresis and amplified fragments that were characteristic of dmtDNA were subsequently sequenced. Results: Of the 96 oocytes analyzed, 34 (35%) contained fragments characteristic of deletions. At the time of this report, 15 different deletions from 13 oocytes have been confirmed by sequence analysis. None of these had been previously reported. The sizes of the deleted segments ranged from 5.4 kb to 8.9 kb. Most, but not all, of the deleted segments were flanked by short direct repeats. Conclusion: The current study indicates that a variety of mtDNA deletions other than the "common deletion" exist in h u m a n oocytes that failed to fertilize. The numbers of mitochondria in an oocyte containing dmtDNA, as well as the impact of these dmtDNA on oocyte energy production, fertilization, and embryonic development await future investigations. A portion of this work was supported by DHHS Grant NS 31376.
P-189 Evaluation of Follicular Transforming Growth Factor-fl (TGF-fl) Content and TGF-fl Receptor Type II (T~RII) Gene Expression With Relation to Embryonic Potential of H u m a n Oocytes. 1S. G. Kurz, 1'2A. M. Carlson, 1'2'3S.K. Roy, 1C. J. DeJonge, 1j. W. Ramey, 1V. M. Maclin-Collins. 1Olson Center for Women's Health, Depts of OB/GYN and 2Physiology & Biophysics, University of Nebraska Medical Center, Omaha, Nebraska. Objectives: TflRII is essential for TGF-~ action and its expression in granulosa cells is hormonally regulated. The objective was to evaluate if TGFp has an influence on future embryonic potential of the oocytes. Design: The study was done by monitoring follicular TGF-~ concentration, T/~RII mRNA expression in granulosa cells and the microscopic morphology of the embryos. Materials and Methods: Women, <40 years of age, undergoing controlled ovarian hyperstimulation (Leuprolide acetate, FSH and/or FSH/LH) prior to ultrasound-guided transvaginal follicular aspiration of oocytes were included in the study. Granulosa cells and follicular fluid from individual follicular aspirates containing a single oocyte were collected by centrifugation and processed separately. TGFcontent in follicular fluid was detected by a receptorbased ELISA following acid activation. T/~RII mRNA was reversed transcribed and PCR amplified from 300 ng gran-
ulosa cell RNA using a sequence specific primer pair and 21-biotin-dUTP. After transferring to a Zetaprobe membrane the amplicons were identified by chemiluminography and digitized by a Molecular Dynamics laser densitometer. Embryonic morphology was evaluated microscopically. Results: Follicles that produced oocytes with grade 1 embryonic potential had very high TGF-/~ content but lowest T~RII mRNA expression. Follicular TGF-~ content varied considerably among follicles. T~RII mRNA expression was 2 fold higher in follicles, which were smaller or larger than 18-20 mm. Conclusions: High concentration of TGF-/~ may induce overt or premature differentiation of resident granulosa cells, thereby affecting embryonic potential of the oocyte. A decrease in TGF-p receptor gene transcription will result in less receptor synthesis, thus reducing the effect of TGF-f~ on granulosa cells. This regulation may produce a better quality oocyte that will become a grade 1 embryo upon fertilization. Our data also suggest a novel mechanism regulating TGF-/~ influence on follicular development. 3This work was supported by research grants from NIH (HD 18165) and Olson Foundation to S. K. Roy.
P-190 Localization of Gap J u n c t i o n a l Proteins Connexin (Cx) 26, Cx32 and Cx43 in the Uterus of Non-pregnant and Early Pregnant Ewes. 1A. T. Grazul-Bilska, 1,2j. j. Bilski, ~D. A. Redmer, ~'2L. P. Reynolds. 1Cell Biology Center & 2Department of Animal and Range Sciences, Fargo, ND. Objectives: Gap junctions have been demonstrated to be very important for control of growth of organs and tissues. Gap junctions are formed by proteins named connexins. Evaluation of the presence of connexins is widely used for detection of gap junctions. During each estrous cycle or pregnancy, the uterus grows and regresses. Therefore, gap junctions may play a role in regulation of uterine tissue growth and remodeling. In the present study, we have examined the presence of three connexins in the uterus from several stages of the estrous cycle and early pregnancy in sheep. Design: Presence of Cx26, Cx32 and Cx43 was examined in ovine uterine compartments throughout the estrous cycle and early pregnancy by using immunohistochemistry. Materials and Methods: Uteri were obtained from ewes on days 0, 2, 4, 8, 12 and 15 of the estrous cycle and on days 12, 18, 24, 30 and 40 of pregnancy ( n = 3 - 5 ewes per day); tissue sections were fixed in Carnoy's or formalin solution, embedded in paraffin; and Cx26, Cx32 and Cx43 were immunolocalized by using specific antibodies (Zymed, San Francisco, CA). Results: Cx26, Cx32 and Cx43 were detected in the uterus in all stages examined, and staining appeared to be punctate. Cx26 was detected in the endometrial and myometrial blood vessels and myometrial smooth muscles across all stages. Cx26 staining was weak in endometrial Abstracts
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stroma. In endometrial glands, Cx26 was present in all stages except on days 0, 2 and 4 of the estrous cycle. Cx32 was present in endometrial stroma and blood vessels, and in myometrial smooth muscles across all stages, but in luminal epithelium only during pregnancy. In addition, Cx32 was present in the stroma around endometrial glands across the estrous cycle and on day 12 of pregnancy, and in endometrial glands epithelium at all stages except on days 0, 2 and 4 of the estrous cycle. Cx43 was localized in luminal epithelium, endometrial stroma, endometrial glands and some blood vessels. Distribution of connexins in endometrial stroma and glands, and myometrium was heterogenous within each tissue section. Conclusions: Connexins are expressed in the several compartments of the uterus across the estrous cycle and early pregnancy, which indicates that gap junctions are present in the uterus. Expression of connexins changes in association with growth and development of uterine tissues, suggesting that gap junctions play a role in control of the growth and remodeling of the ovine uterus. Supported in part by N I H grant 1R29 HD30348 to ATGB, and by USDA grant 93-37203-9271 to DAR and LPR. P-191 The Effect o f H u m a n C h o r i o n i c G o n a d o t r o p i n (hCG) o n T r o p h o b l a s t Cell Motility. K. A. Wei, F. D. Yelian. Department of Obstetrics and Gynecology, Wayne State University School of Medicine, Detroit, MI. Objectives: Embryo implantation involves trophoblast cell adhesion to endometrial epithelium and invasion to the interstitium. Both adhesion and motility are important characteristics of trophoblast cells. Our previous studies have demonstrated that a high concentration of hCG inhibits cell adhesion to extracellular matrix protein fibronectin. In this study we are investigating the effect of hCG on trophoblast cell motility. Design: Trophoblast cells were treated with various concentrations of hCG and the cell motility was measured using a modified phagokinetic track motility assay. Materials and Methods: Both HTR-8/SVneo (HTR), an extented-lifespan first trimester trophoblast cell line, and JEG-3, a choriocarcinoma cell line were used in this study. Uniform carpets of gold particles were prepared on glass coverslips in a 6-well culture plate. Five thousand cells were transferred onto each well and incubated with a culture medium containing 10% fetal bovine serum. After the cells attached to the coverslip, the culture medium was replaced with a serum-free medium containing various concentrations of hCG (1, 10, 100, 500, and 1,000 mIU/ ml). Following 24 h incubation at 37°C, the phagokinetic tracks of cell movement were analysed using a computerassisted imaging system. The mean cell motility in each group was determined by measuring 50-100 randomly selected cells and the difference among groups were analyzed using one-way analysis of variance. Results: The mean cell motilities for HTR cells treated with hCG at 0, 1, 10, 100, 500, and 1,000 mIU/ml were 6604-+272, 8626_+413, 8954-+483, 7379-+452, 4724_+260, and 4256_+278 #m S, respectively. The motility for JEG-3 S184
Abstracts
cells in same treated groups were 2906+220, 2962_+160, 3824_+278, 2460_+201, 1621_+93, and 1672-+115 #m 2, respectively. Statistically significant differences were found among groups (P<0.001). Interestingly, a low concentration ofhCG (1 and 10 mIU/ml) significantly increased HTR cell motility and a high concentration of hCG (500 and 1,000 mIU/ml) significantly decreased cell motility in both HTR and JEG-3 cells (P<0.01). Conclusions: This study demonstrated for the first time that hCG modulates trophoblast cell motility. In conjunction with our previous adhesion study, these data suggest that hCG may play an important role in regulating trophoblast cell function during early embryo implantation. P-192 M i t o c h o n d r i a l DNA (mtDNA) D e l e t i o n s in H u m a n Oocytes ~,2j. A. Barritt, 8B. M. Miller, 3G. C. VanTuyle, 1'2D. W. Matt. 1Department of Obstetrics and Gynecology, 2Department of Anatomy, and 8Department of Biochemistry, Virginia Commonwealth University, Richmond, Virginia. Objectives: Although it is well established that many h u m a n oocytes and pre-implantation embryos fail to fertilize and develop, the intracelhilar mechanisms that contribute to these failures are largely unknown. Currently, we are exploring whether alterations in mitochondrial function of h u m a n oocytes are related to fertilization failure or embryonic developmental arrest. Several degenerative diseases have been associated with mtDNA deletions (dmtDNA), and recent studies have identified a 4977 base pair "common deletion" in unfertilized h u m a n oocytes. The purpose of the current study was to identify the frequency and types of mtDNA deletions in h u m a n oocytes. Materials and Methods: A "nested" two-round PCR approach was developed to amplify the majority of the larger arc region between the two origins of replication ofmtDNA in h u m a n oocytes. The Medical College of Virginia's In Vitro Fertilization Program provided 96 h u m a n oocytes (from 36 patients) that had been discarded because they failed to fertilize 48 hours after insemination or intracytoplasmic sperm injection (ICSI). PCR products amplified from individual oocytes were identified by gel electrophoresis and amplified fragments that were characteristic of dmtDNA were subsequently sequenced. Results: Of the 96 oocytes analyzed, 34 (35%) contained fragments characteristic of deletions. At the time of this report, 15 different deletions from 13 oocytes have been confirmed by sequence analysis. None of these had been previously reported. The sizes of the deleted segments ranged from 5.4 kb to 8.9 kb. Most, but not all, of the deleted segments were flanked by short direct repeats. Conclusion: The current study indicates that a variety of mtDNA deletions other than the "common deletion" exist in h u m a n oocytes that failed to fertilize. The numbers of mitochondria in an oocyte containing dmtDNA, as well as the impact of these dmtDNA on oocyte energy production, fertilization, and embryonic development await future investigations. A portion of this work was supported by DHHS Grant NS 31376.