- Email: [email protected]
Outbred mice conceived by in vitro fertilization (IVF) display increased growth curve and glucose intolerance
O-356 Wednesday, October 19, 2011 05:30 PM KARYOTYPICALLY NORMAL HUMAN EMBRYONIC STEM CELLS (HESC) DERIVED FROM ANEUPLOID PREIMPLANTATION GENETIC DIAGNOSIS (PGD) EMBRYOS. Q. Zhan, N. Zaninovic, Z. Rosenwaks. Center for Reproductive Medicine, Weill Cornell Medical College of Cornell University, New York, NY. OBJECTIVE: Human embryonic stem cells (hESC) derived from chromosomally or genetically abnormal embryos after preimplantation genetic diagnosis (PGD) provide excellent in vitro models to study specific chromosomal and genetic diseases. We evaluated the karyotype of stem cells derived from abnormal embryos after PGD detected by fluorescent in situ hybridization (FISH) on day 3. DESIGN: Comparison of hESC karyotype results with the results of the PGD on the embryos on day 3. MATERIALS AND METHODS: The inner cell mass (ICM) of day 5/6 embryos were isolated by mechanical or non-contact laser methods, cells were cultured on the mouse embryonic fibroblast feeder (MEF) in serumfree hESC medium and condition medium (hESC-CM). hESC were characterized using standard techniques. RESULTS: From 08/2009 to 08/2010, total 57 human embryos from 21 IVF-PGD cycles were donated. Fifty-five embryos, ranging from arrested cleavage stages to 6BA blastocysts, were used for stem cell derivation: 18 at cleavage stage, 37 at blastocyst stage, and 22 with clear ICM. Total 7 hESC lines were derived, with overall efficiency of 12.7% (7/55). All the cell lines had the pluripotent ability to differentiate into derivatives of the three embryonic germ layers. Surprisingly, 5 out of 7 lines were karyotypically normal in all cells analyzed, while 2 remaining lines had predominantly normal cells with low percentage of abnormal cells. None of the aneuploid lines presented the same abnormalities as the original PGD embryos. CONCLUSION: These results indicate that hESC lines can be derived from PGD embryos with limited efficiency. Normal ESC karyotype from apparent aneuploid embryos reveal underlying mosaicism and emerging dominance of the karyotypically normal cells in culture. PGD abnormal embryos can serve as an alternative source for normal euploid lines. Supported by: CRMI
CONCLUSION: Progesterone treatment via a human mitochondrial progesterone receptor (PR-M) decreases cardiac dysfunction in a transgenic male mouse model of heart failure. A lack of effect in ovariectomized females suggests a role for other sex steroids in this process. Supported by: NIH/NICHD R03, Susan Fiery Hughes Foundation, Charles Hammond, MD Research Grant
O-358 Wednesday, October 19, 2011 04:00 PM IMPROVING THE SUCCESS OF IN VITRO MATURATION (IVM) BY CONTROLLING THE RATE OF OOCYTE MATURATION WITH MEIOTIC INHIBITORS USING BOVINE OOCYTE MODEL. T. A. Farghaly, S. A. Mostafa, E. M. Khalifa, J. Liu, J. Goldfarb, A. Ahmady. MacDonald Fertility & IVF Center, UH Case Medical Center, Cleveland, OH; Ob/Gyn.deparment, Womens’Health Center,Assiut University, Assiut, Egypt. OBJECTIVE: To evaluate the effect of delaying the maturation events in vitro to mimic that in vivo by using meiotic inhibitors: maturation promoting factor inhibitor(Roscovitine),phosphodiesterase-3 inhibitor (Cilostamide) and Adenylate cyclase activator (Forskolin) on the meiotic resumption and embryonic development of bovine oocytes. DESIGN: Known meiotic inhibitors were added together to prematuration culture media (PMC) in which bovine oocytes were cultured for 3 or 5 days then transferred to inhibitor free media for 48 hours. MATERIALS AND METHODS: Immature oocytes were cultured in (PMC) consisting of M199 medium containing 10 mM of each maturation inhibitors combined together for either 3 or 5 days and then transferred to maturation media containing 7.5 IU of FSH/LH for 48 hours incubation. In the control group, oocytes were only cultured in the maturation medium supplemented with 7.5 IU of FSH/LH for 48 hours. Mature oocytes were then inseminated with frozen-thawed bull sperm. Fertilization, defined as 2-cell division 48h post-insemination, and blastocyst formation were monitored. Chi- square test was used for statistical analysis RESULTS: A total of 170 oocytes included in this study with there in vitro maturation (IVM) outcome evaluated in the following table:
REPRODUCTIVE BIOLOGY: ANIMAL AND EXPERIMENTAL STUDIES O-357 Wednesday, October 19, 2011 03:45 PM
Groups
n
meiotic arrest (%)
PROGESTERONE VIA A MITOCHONDRIAL PROGESTERONE RECEPTOR (PR-M) PREVENTS HEART FAILURE IN A TRANSGENIC MOUSE MODEL OF AORTIC CONSTRICTION. C. E. Likes, III, K C. Hawkins, L. Mao, Q. Dai, H. A. Rockman, T. M. Price. Obstetrics/Gynecology, Duke University, Durham, NC; Medicine - Cardiology, Duke University, Durham, NC.
5 days 3 days control
54 56 60
93 89 N/A
OBJECTIVE: To determine the effect on cardiac function of a human mitochondrial progesterone receptor (PR-M) using a transgenic mouse model of after-load induced heart failure. DESIGN: Transgenic mouse model MATERIALS AND METHODS: An inducible (TET-On) transgenic mouse model was developed to express human PR-M under the control of the cardiac specific myosin heavy chain 6 promoter. PR-M expression was induced with oral administration of Doxycycline followed in 2 wks by surgical constant thoracic aortic constriction (cTAC) to increase after-load. Intact male and ovariectomized (OVX) female mice were treated with progesterone in oil (2.5 mg/day SQ) starting 1 week prior to cTAC. Echocardiograms were performed prior to and 2 and 4 wks after cTAC with determination of left ventricular dimensions and percent fractional shortening by an investigator blinded to the genotype and treatment. Cardiac parameters were compared with 2-way repeated measures ANOVA. Cardiac PR-M expression was determined by realtime RT-PCR. RESULTS: Male progesterone treated PR-M positive mice (N ¼ 8) showed a significantly smaller left ventricular diameter (LVD) diastolic (mm) at 2 and 4 wks (3.3 vs 3.9 P<0.002, 3.5 vs 4.0 P<0.002), LVD systolic (1.2 vs 1.8 P<0.0001, 1.3 vs 2.0 P<0.0001) and a greater percent fractional shortening (64 vs 55% P<0.002, 62 vs 51% P<0.002) compared to progesterone treated PR-M negative mice (N ¼ 12). Similar results were seen in comparison to other controls; vehicle treated PR-M positive (N ¼ 9) and negative mice (N ¼ 9). There was no difference in cardiac parameters between progesterone treated PR-M positive OVX females (N ¼ 11) and progesterone treated PR-M negative OVX females (N ¼ 22).
FERTILITY & STERILITYÒ
maturation (%)
Fertilization (%)
blastulation (%)
48/50 (96) 45/49 (91) 40/60 (77)
22/48 (46) 18/45 (40) 8/40 (20)
11/22 (50) 9/18 (50) 2/8 (25)
Our preliminary data showed that extended prematuration culture of bovine oocyte in the presence of combination of all the inhibitors significantly improved maturation and fertilization rates in comparison to control (P<0.05).There was no significant differences in these parameters between the 3 day and 5 day groups CONCLUSION: Extended pre-maturation culture of immature oocyte in a combination of maturation inhibitors significantly improves the IVM outcome.If applicable to human oocytes, these findings may improve clinical IVM outcomes.
O-359 Wednesday, October 19, 2011 04:15 PM OUTBRED MICE CONCEIVED BY IN VITRO FERTILIZATION (IVF) DISPLAY INCREASED GROWTH CURVE AND GLUCOSE INTOLERANCE. W. Lin, A. Donjacour, L. Xiaowei, K. S. Kolahi, R. Simbulan, P. F. Rinaudo. OB/GYN, CRS, University of California, San Francisco, San Francisco, CA. OBJECTIVE: The Barker hypothesis holds that the developing individual is sensitive to the environment. We have shown that C57BL6/J inbred mice display reduced growth curve and glucose intolerance when cultured in stressful conditions. Here we test long term metabolic alteration of IVF offspring in outbred mice cultured in optimal or suboptimal conditions. DESIGN: Experimental animal model MATERIALS AND METHODS: CF-1 female mice and B6D2F1/J male mice were conceived in vitro and transferred to foster mothers at the
S105
blastocyst stage (IVF group) or conceived in vivo, flushed out at the blastocyst stage and transferred to foster mothers (FB group). Two media were used: Whitten’s medium under 20%O2 (IVFWM group) or KSOM with amino acids, 5% O2 (IVFKAA group). Morphometric parameters were measured at birth and weekly. Adult animals had glucose tolerance test (ipGTT), insulin and body fat contents (DEXA) measured. Parametric tests were used as appropriate. RESULTS: Birth weight of IVF mice was not statistically different from control. However, male IVFWM mice show a higher weight gain starting at 1 week of postnatal life and remained larger than FB and IVFKAA mice up to 28 weeks (P<0.05). Male IVFKAA mice at 8 weeks have lower fat content than IVFWM and FB mice. Importantly, male IVFWM mice have increased glucose levels as tested by ipGTT.
n
Group
Gender
BW(gr)
Fat content (%)
ipGTT AUC (g/dL/min)
Fasting insulin (ng/mL)
20 11 21 19 14 15
FB IVFKAA IVFWM FB IVFKAA IVFWM
Male Male Male Female Female Female
1.71 0.14 1.75 0.19 1.73 0.26 1.73 0.12 1.73 0.20 1.67 0.21
22.99 6.18 18.91 4.20* 22.72 3.92 22.97 6.63 19.12 6.51 22.99 4.78
36.07 15.9 40.68 15.9 52.91 16.4* 17.28 3.3 15.69 4.9 17.35 3.6
5.9 4.3 6.5 3.1 1.4 1.0 1.7 0.8
CONCLUSION: Preimplantation embryo culture is associated with reprogramming of the embryonic genome, resulting in a recognizable adult phenotype. Importantly, we have shown a dose response effect according to the culture conditions used. Supported by: NICHD and ADA to PR
O-360 Wednesday, October 19, 2011 04:30 PM COQ10 TREATMENT CAN IMPROVE FERTILITY AND OOCYTE QUALITY IN OLD MICE. A. Ben-Meir, J. Chong, A. Borrego-Alvarez, K. H. Moley, A. Jurisicova, R. F. Casper. Obstetrics & Gynecology, University of Toronto, Samuel Lunenfeld Research Institute (SLRI), Toronto, ON, Canada; Toronto Centre for Advanced Reproductive Techniques (TCART), Toronto, ON, Canada; Obstetrics and Gynecology, Washington University School of Medicine, St Louis, MO. OBJECTIVE: Oocyte mitochondrial dysfunction with aging may lead to decreased energy production crucial for completion of meiosis and embryo development. We have shown previously that treatment with CoQ10 improved the oocyte mitochondrial performance, ovarian reserve and ovulation rate. The objective of this study was to evaluate whether treatment with CoQ10, a mitochondrial nutrient, can improve chromosomal disjunction and fertility in old mice. DESIGN: Case-control study. MATERIALS AND METHODS: 15 aged mice (9 months old) of ICR strain were treated with 0.042 mg/kg CoQ10 for 12 weeks and were compared to 15 aged matched vehicle-treated mice and 15 young females. Dams were subjected to superovulation and retrieved oocytes were examined for Krebs cycle output (citrate/ATP ratio), chromosomal alignment and spindle quality. RESULTS: Krebs cycle output was decreased in oocytes of aged dams and treatment with CoQ 10 restored the citrate/ATP ratio to young levels (P¼0.006). DNA misalignment and spindle defects were more common in old oocytes compared to young oocytes (46.7% and 35.7% vs 8.3% and 6.5%; P<0.001) and both were corrected by CoQ10 pre-treatment (11.4% and 6.1%; P<0.001 compared to old oocytes and not different from young oocytes). Comparing breeding performance between old, young and CoQ10 treated old mice revealed a significant (P<0.05) difference between the groups (median of 7 pups, 15 pups and 11.5 pups per litter, respectively). CONCLUSION: Mitochondrial function and spindle alignment were disrupted in oocytes from old mice, and this was corrected by pretreatment with CoQ10, confirming the relationship between energy and spindle quality. CoQ10 treatment translated in improved breeding performance of older dams. We are currently analyzing whether reduction in the expression of the genes involved in CoQ10 synthesis in oocytes could contribute to CoQ10 deficiency in aging females. Supported by: CIHR
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Abstracts
O-361 Wednesday, October 19, 2011 04:45 PM LOSS OF EMBRYONIC POLY(A) BINDING PROTEIN (EPAB) IN MOUSE RESULTS IN FEMALE INFERTILITY DUE IMPAIRED OOCYTE MATURATION AND OVULATION. O. Guzeloglu-Kayisli, M. D. Lalioti, I. Sasson, D. Sakkas, F. Aydiner, E. Seli. Obstetrics, Gynecology, and Reproductive Sciences, Yale University School of Medicine, New Haven, CT. OBJECTIVE: Gene expression during oocyte maturation and early embryogenesis until zygotic genome activation is mainly regulated by translational activation of maternally-derived mRNAs. Embryonic poly(A) binding protein (ePAB) is the predominant poly(A) binding protein during this period in frog, mouse, and human; it stabilizes maternal mRNAs and promotes their translation. We hypothesized that ePAB is required for female fertility. DESIGN: Experimental study. MATERIALS AND METHODS: ePAB knockout (ePAB-/-) mice were generated by targeted deletion of exon 2. Fertility of wild type (WT), heterozygous (ePAB+/-), and ePAB-/- female mice was tested by mating with male mice (male:female; 1:2) with proven fertility (20 weeks total, 12 mice per genotype). Histomorphometric analysis of ovaries was performed to assess folliculogenesis. Oogenesis, oocyte maturation, and fertilization were assessed by superovulation with or without mating, and by in vitro maturation. A polyadenylation (PAT) assay was performed to determine the poly(A)-tail length of mRNAs known to be activated upon oocyte maturation (cyclin B1, c-mos, and tPA) in ePAB-/- and WT mice. RESULTS: ePAB-/- female mice were infertile, while ePAB-/- males and ePAB+/- of both sexes demonstrated normal fertility. Superovulation and mating with WT males showed that ePAB-/- female mice could not generate embryos or mature oocytes in vivo or in vitro. Late antral follicles in the ovaries of ePAB-/- mice exhibited impaired cumulus expansion, and an 8-fold decrease in ovulation (P<0.01) associated with oocyte retention in corpora lutea. Polyadenylation of cyclinB1, c-mos and tPA genes during in vitro maturation was suppressed in ePAB-/- oocytes. CONCLUSION: Our findings indicate that ePAB is necessary for oocyte maturation, effective ovulation and normal reproduction in mammals. Molecular mechanisms of ePAB-mediated mRNA translational activation during development, and their implications for human reproduction remain to be elucidated. Supported by: NIH K08 HD046581 AND R01 HD0446581 (ES).
O-362 Wednesday, October 19, 2011 05:00 PM EMBRYONIC POLY(A) BINDING PROTEIN (EPAB) PLAYS A KEY ROLE IN CHROMATION REMODELING AND TRANSCRIPTIONAL SILENCING DURING OOCYTE MATURATION. O. Ilbay, O. Guzeloglu-Kayisli, M. D. Lalioti, E. Seli. Obstetrics, Gynecology, and Reproductive Sciences, Yale University School of Medicine, New Haven, CT. OBJECTIVE: Chromatin remodeling occurs in germinal vesicle (GV) stage oocytes in preparation for oocyte maturation. It is characterized by a transition from a non-surrounded nucleolus (NSN) to a surrounded nucleolus (SN) configuration determined by localization and condensation of the chromatin. Transition to SN configuration is also associated with suppression of transcription, characteristic of impending oocyte maturation. ePAB, the predominant poly(A) binding protein in mouse and human oocytes, is a key regulator of oocyte maturation. In this study, we aimed to determine whether ePAB knockout (ePAB-/-) mouse oocytes with defective oocyte maturation have impaired formation of SN configuration and/or transcriptional silencing. DESIGN: Experimental study. MATERIALS AND METHODS: GV stage oocytes were collected from PMSG-primed wild-type (WT) and ePAB-/- mice. To determine chromation configuration, DAPI staining was performed either immediately after oocyte collection or following 18 hours of in vitro maturation. Active transcription was assessed by BrUTP incorporation with or without pre-treatment with aamanitin (RNA polymerase II inhibitor), and nascent transcripts were detected by immunostaining with anti-BrdU antibody. RESULTS: Unlike WT, ePAB-/- mice oocytes failed to proceed to SN chromatin configuration, and exhibited only NSN configuration, both immediately after oocyte collection and following 18 hours of in vitro maturation. BrUTP incorporation revealed that unlike WT oocytes, ePAB-/- oocytes remained transcriptionally active and that this activity was mediated by RNA polymerase II as it was inhibited by a-amanitin.
Vol. 96., No. 3, Supplement, Sepetmber 2011
CONCLUSION: Progesterone treatment via a human mitochondrial progesterone receptor (PR-M) decreases cardiac dysfunction in a transgenic male mouse model of heart failure. A lack of effect in ovariectomized females suggests a role for other sex steroids in this process. Supported by: NIH/NICHD R03, Susan Fiery Hughes Foundation, Charles Hammond, MD Research Grant
O-358 Wednesday, October 19, 2011 04:00 PM IMPROVING THE SUCCESS OF IN VITRO MATURATION (IVM) BY CONTROLLING THE RATE OF OOCYTE MATURATION WITH MEIOTIC INHIBITORS USING BOVINE OOCYTE MODEL. T. A. Farghaly, S. A. Mostafa, E. M. Khalifa, J. Liu, J. Goldfarb, A. Ahmady. MacDonald Fertility & IVF Center, UH Case Medical Center, Cleveland, OH; Ob/Gyn.deparment, Womens’Health Center,Assiut University, Assiut, Egypt. OBJECTIVE: To evaluate the effect of delaying the maturation events in vitro to mimic that in vivo by using meiotic inhibitors: maturation promoting factor inhibitor(Roscovitine),phosphodiesterase-3 inhibitor (Cilostamide) and Adenylate cyclase activator (Forskolin) on the meiotic resumption and embryonic development of bovine oocytes. DESIGN: Known meiotic inhibitors were added together to prematuration culture media (PMC) in which bovine oocytes were cultured for 3 or 5 days then transferred to inhibitor free media for 48 hours. MATERIALS AND METHODS: Immature oocytes were cultured in (PMC) consisting of M199 medium containing 10 mM of each maturation inhibitors combined together for either 3 or 5 days and then transferred to maturation media containing 7.5 IU of FSH/LH for 48 hours incubation. In the control group, oocytes were only cultured in the maturation medium supplemented with 7.5 IU of FSH/LH for 48 hours. Mature oocytes were then inseminated with frozen-thawed bull sperm. Fertilization, defined as 2-cell division 48h post-insemination, and blastocyst formation were monitored. Chi- square test was used for statistical analysis RESULTS: A total of 170 oocytes included in this study with there in vitro maturation (IVM) outcome evaluated in the following table:
REPRODUCTIVE BIOLOGY: ANIMAL AND EXPERIMENTAL STUDIES O-357 Wednesday, October 19, 2011 03:45 PM
Groups
n
meiotic arrest (%)
PROGESTERONE VIA A MITOCHONDRIAL PROGESTERONE RECEPTOR (PR-M) PREVENTS HEART FAILURE IN A TRANSGENIC MOUSE MODEL OF AORTIC CONSTRICTION. C. E. Likes, III, K C. Hawkins, L. Mao, Q. Dai, H. A. Rockman, T. M. Price. Obstetrics/Gynecology, Duke University, Durham, NC; Medicine - Cardiology, Duke University, Durham, NC.
5 days 3 days control
54 56 60
93 89 N/A
OBJECTIVE: To determine the effect on cardiac function of a human mitochondrial progesterone receptor (PR-M) using a transgenic mouse model of after-load induced heart failure. DESIGN: Transgenic mouse model MATERIALS AND METHODS: An inducible (TET-On) transgenic mouse model was developed to express human PR-M under the control of the cardiac specific myosin heavy chain 6 promoter. PR-M expression was induced with oral administration of Doxycycline followed in 2 wks by surgical constant thoracic aortic constriction (cTAC) to increase after-load. Intact male and ovariectomized (OVX) female mice were treated with progesterone in oil (2.5 mg/day SQ) starting 1 week prior to cTAC. Echocardiograms were performed prior to and 2 and 4 wks after cTAC with determination of left ventricular dimensions and percent fractional shortening by an investigator blinded to the genotype and treatment. Cardiac parameters were compared with 2-way repeated measures ANOVA. Cardiac PR-M expression was determined by realtime RT-PCR. RESULTS: Male progesterone treated PR-M positive mice (N ¼ 8) showed a significantly smaller left ventricular diameter (LVD) diastolic (mm) at 2 and 4 wks (3.3 vs 3.9 P<0.002, 3.5 vs 4.0 P<0.002), LVD systolic (1.2 vs 1.8 P<0.0001, 1.3 vs 2.0 P<0.0001) and a greater percent fractional shortening (64 vs 55% P<0.002, 62 vs 51% P<0.002) compared to progesterone treated PR-M negative mice (N ¼ 12). Similar results were seen in comparison to other controls; vehicle treated PR-M positive (N ¼ 9) and negative mice (N ¼ 9). There was no difference in cardiac parameters between progesterone treated PR-M positive OVX females (N ¼ 11) and progesterone treated PR-M negative OVX females (N ¼ 22).
FERTILITY & STERILITYÒ
maturation (%)
Fertilization (%)
blastulation (%)
48/50 (96) 45/49 (91) 40/60 (77)
22/48 (46) 18/45 (40) 8/40 (20)
11/22 (50) 9/18 (50) 2/8 (25)
Our preliminary data showed that extended prematuration culture of bovine oocyte in the presence of combination of all the inhibitors significantly improved maturation and fertilization rates in comparison to control (P<0.05).There was no significant differences in these parameters between the 3 day and 5 day groups CONCLUSION: Extended pre-maturation culture of immature oocyte in a combination of maturation inhibitors significantly improves the IVM outcome.If applicable to human oocytes, these findings may improve clinical IVM outcomes.
O-359 Wednesday, October 19, 2011 04:15 PM OUTBRED MICE CONCEIVED BY IN VITRO FERTILIZATION (IVF) DISPLAY INCREASED GROWTH CURVE AND GLUCOSE INTOLERANCE. W. Lin, A. Donjacour, L. Xiaowei, K. S. Kolahi, R. Simbulan, P. F. Rinaudo. OB/GYN, CRS, University of California, San Francisco, San Francisco, CA. OBJECTIVE: The Barker hypothesis holds that the developing individual is sensitive to the environment. We have shown that C57BL6/J inbred mice display reduced growth curve and glucose intolerance when cultured in stressful conditions. Here we test long term metabolic alteration of IVF offspring in outbred mice cultured in optimal or suboptimal conditions. DESIGN: Experimental animal model MATERIALS AND METHODS: CF-1 female mice and B6D2F1/J male mice were conceived in vitro and transferred to foster mothers at the
S105
blastocyst stage (IVF group) or conceived in vivo, flushed out at the blastocyst stage and transferred to foster mothers (FB group). Two media were used: Whitten’s medium under 20%O2 (IVFWM group) or KSOM with amino acids, 5% O2 (IVFKAA group). Morphometric parameters were measured at birth and weekly. Adult animals had glucose tolerance test (ipGTT), insulin and body fat contents (DEXA) measured. Parametric tests were used as appropriate. RESULTS: Birth weight of IVF mice was not statistically different from control. However, male IVFWM mice show a higher weight gain starting at 1 week of postnatal life and remained larger than FB and IVFKAA mice up to 28 weeks (P<0.05). Male IVFKAA mice at 8 weeks have lower fat content than IVFWM and FB mice. Importantly, male IVFWM mice have increased glucose levels as tested by ipGTT.
n
Group
Gender
BW(gr)
Fat content (%)
ipGTT AUC (g/dL/min)
Fasting insulin (ng/mL)
20 11 21 19 14 15
FB IVFKAA IVFWM FB IVFKAA IVFWM
Male Male Male Female Female Female
1.71 0.14 1.75 0.19 1.73 0.26 1.73 0.12 1.73 0.20 1.67 0.21
22.99 6.18 18.91 4.20* 22.72 3.92 22.97 6.63 19.12 6.51 22.99 4.78
36.07 15.9 40.68 15.9 52.91 16.4* 17.28 3.3 15.69 4.9 17.35 3.6
5.9 4.3 6.5 3.1 1.4 1.0 1.7 0.8
CONCLUSION: Preimplantation embryo culture is associated with reprogramming of the embryonic genome, resulting in a recognizable adult phenotype. Importantly, we have shown a dose response effect according to the culture conditions used. Supported by: NICHD and ADA to PR
O-360 Wednesday, October 19, 2011 04:30 PM COQ10 TREATMENT CAN IMPROVE FERTILITY AND OOCYTE QUALITY IN OLD MICE. A. Ben-Meir, J. Chong, A. Borrego-Alvarez, K. H. Moley, A. Jurisicova, R. F. Casper. Obstetrics & Gynecology, University of Toronto, Samuel Lunenfeld Research Institute (SLRI), Toronto, ON, Canada; Toronto Centre for Advanced Reproductive Techniques (TCART), Toronto, ON, Canada; Obstetrics and Gynecology, Washington University School of Medicine, St Louis, MO. OBJECTIVE: Oocyte mitochondrial dysfunction with aging may lead to decreased energy production crucial for completion of meiosis and embryo development. We have shown previously that treatment with CoQ10 improved the oocyte mitochondrial performance, ovarian reserve and ovulation rate. The objective of this study was to evaluate whether treatment with CoQ10, a mitochondrial nutrient, can improve chromosomal disjunction and fertility in old mice. DESIGN: Case-control study. MATERIALS AND METHODS: 15 aged mice (9 months old) of ICR strain were treated with 0.042 mg/kg CoQ10 for 12 weeks and were compared to 15 aged matched vehicle-treated mice and 15 young females. Dams were subjected to superovulation and retrieved oocytes were examined for Krebs cycle output (citrate/ATP ratio), chromosomal alignment and spindle quality. RESULTS: Krebs cycle output was decreased in oocytes of aged dams and treatment with CoQ 10 restored the citrate/ATP ratio to young levels (P¼0.006). DNA misalignment and spindle defects were more common in old oocytes compared to young oocytes (46.7% and 35.7% vs 8.3% and 6.5%; P<0.001) and both were corrected by CoQ10 pre-treatment (11.4% and 6.1%; P<0.001 compared to old oocytes and not different from young oocytes). Comparing breeding performance between old, young and CoQ10 treated old mice revealed a significant (P<0.05) difference between the groups (median of 7 pups, 15 pups and 11.5 pups per litter, respectively). CONCLUSION: Mitochondrial function and spindle alignment were disrupted in oocytes from old mice, and this was corrected by pretreatment with CoQ10, confirming the relationship between energy and spindle quality. CoQ10 treatment translated in improved breeding performance of older dams. We are currently analyzing whether reduction in the expression of the genes involved in CoQ10 synthesis in oocytes could contribute to CoQ10 deficiency in aging females. Supported by: CIHR
S106
Abstracts
O-361 Wednesday, October 19, 2011 04:45 PM LOSS OF EMBRYONIC POLY(A) BINDING PROTEIN (EPAB) IN MOUSE RESULTS IN FEMALE INFERTILITY DUE IMPAIRED OOCYTE MATURATION AND OVULATION. O. Guzeloglu-Kayisli, M. D. Lalioti, I. Sasson, D. Sakkas, F. Aydiner, E. Seli. Obstetrics, Gynecology, and Reproductive Sciences, Yale University School of Medicine, New Haven, CT. OBJECTIVE: Gene expression during oocyte maturation and early embryogenesis until zygotic genome activation is mainly regulated by translational activation of maternally-derived mRNAs. Embryonic poly(A) binding protein (ePAB) is the predominant poly(A) binding protein during this period in frog, mouse, and human; it stabilizes maternal mRNAs and promotes their translation. We hypothesized that ePAB is required for female fertility. DESIGN: Experimental study. MATERIALS AND METHODS: ePAB knockout (ePAB-/-) mice were generated by targeted deletion of exon 2. Fertility of wild type (WT), heterozygous (ePAB+/-), and ePAB-/- female mice was tested by mating with male mice (male:female; 1:2) with proven fertility (20 weeks total, 12 mice per genotype). Histomorphometric analysis of ovaries was performed to assess folliculogenesis. Oogenesis, oocyte maturation, and fertilization were assessed by superovulation with or without mating, and by in vitro maturation. A polyadenylation (PAT) assay was performed to determine the poly(A)-tail length of mRNAs known to be activated upon oocyte maturation (cyclin B1, c-mos, and tPA) in ePAB-/- and WT mice. RESULTS: ePAB-/- female mice were infertile, while ePAB-/- males and ePAB+/- of both sexes demonstrated normal fertility. Superovulation and mating with WT males showed that ePAB-/- female mice could not generate embryos or mature oocytes in vivo or in vitro. Late antral follicles in the ovaries of ePAB-/- mice exhibited impaired cumulus expansion, and an 8-fold decrease in ovulation (P<0.01) associated with oocyte retention in corpora lutea. Polyadenylation of cyclinB1, c-mos and tPA genes during in vitro maturation was suppressed in ePAB-/- oocytes. CONCLUSION: Our findings indicate that ePAB is necessary for oocyte maturation, effective ovulation and normal reproduction in mammals. Molecular mechanisms of ePAB-mediated mRNA translational activation during development, and their implications for human reproduction remain to be elucidated. Supported by: NIH K08 HD046581 AND R01 HD0446581 (ES).
O-362 Wednesday, October 19, 2011 05:00 PM EMBRYONIC POLY(A) BINDING PROTEIN (EPAB) PLAYS A KEY ROLE IN CHROMATION REMODELING AND TRANSCRIPTIONAL SILENCING DURING OOCYTE MATURATION. O. Ilbay, O. Guzeloglu-Kayisli, M. D. Lalioti, E. Seli. Obstetrics, Gynecology, and Reproductive Sciences, Yale University School of Medicine, New Haven, CT. OBJECTIVE: Chromatin remodeling occurs in germinal vesicle (GV) stage oocytes in preparation for oocyte maturation. It is characterized by a transition from a non-surrounded nucleolus (NSN) to a surrounded nucleolus (SN) configuration determined by localization and condensation of the chromatin. Transition to SN configuration is also associated with suppression of transcription, characteristic of impending oocyte maturation. ePAB, the predominant poly(A) binding protein in mouse and human oocytes, is a key regulator of oocyte maturation. In this study, we aimed to determine whether ePAB knockout (ePAB-/-) mouse oocytes with defective oocyte maturation have impaired formation of SN configuration and/or transcriptional silencing. DESIGN: Experimental study. MATERIALS AND METHODS: GV stage oocytes were collected from PMSG-primed wild-type (WT) and ePAB-/- mice. To determine chromation configuration, DAPI staining was performed either immediately after oocyte collection or following 18 hours of in vitro maturation. Active transcription was assessed by BrUTP incorporation with or without pre-treatment with aamanitin (RNA polymerase II inhibitor), and nascent transcripts were detected by immunostaining with anti-BrdU antibody. RESULTS: Unlike WT, ePAB-/- mice oocytes failed to proceed to SN chromatin configuration, and exhibited only NSN configuration, both immediately after oocyte collection and following 18 hours of in vitro maturation. BrUTP incorporation revealed that unlike WT oocytes, ePAB-/- oocytes remained transcriptionally active and that this activity was mediated by RNA polymerase II as it was inhibited by a-amanitin.
Vol. 96., No. 3, Supplement, Sepetmber 2011