- Email: [email protected]
ORC4 plays a role in polar body extrusion in the mouse oocyte and zygote
(n¼37): more than 80% of immotile with dead sperm in the culture dish. We compared IVF outcomes between two groups. RESULTS: There were no differences in patients age (group A; 35.122.9 vs. group B; 35.163.1, p¼0.94), number of oocytes (9.25.4 vs. 7.85.5, p¼0.13) and sperm counts (53 x 10^6/mL26.62 vs. 50.13 x 10^6/mL34, p¼0.53). And also fertilization rate did not differ between two groups (72.8% vs. 69.6%, p¼0.32). However, the clinical pregnancy rate in group B was significantly higher than group A (56.3% vs. 75.6%, p¼0.02). CONCLUSION: In this study, dead sperm group showed significantly higher pregnancy rate compared with living sperm group. We suppose that high percentage of dead sperm means good quality sperms burn out the limited amount of nutrients in the culture dish. Therefore, high percentage of sperm death after fertilization on conventional IVF could be a good prediction of IVF outcome. P-192 Tuesday, October 21, 2014 REPRODUCTIVE OUTCOME OF OOCYTE ACTIVATION BY CALCIUM IONOPHORE A23187 FOLLOWING INTRACYTOPLASMIC SPERM INJECTION (ICSI) IN PATIENTS WITH PREVIOUS COMPLETE FERTILIZATION FAILURE. T. Caballero, P. J. Buzzi, M. P. Zappacosta, A. Valcarcel, F. Lorenzo. IFER Instituto de Ginecologıa y Fertilidad, Buenos Aires, Argentina. OBJECTIVE: Fertilization failure after ICSI may be due to the inability of the spermatozoa to trigger oocyte activation which, is characterized by a rise in intracellular calcium concentration. Treatment with calcium ionophore may increase the free intracellular calcium, thereby mimicking physiological cell-signaling mechanisms resulting in oocyte activation.The aim of this study was to evaluate the impact on reproductive outcome of oocyte activation (OA) by calcium ionophore (A23187) in patients with complete fertilization failure following ICSI during previous In Vitro Fertilization (IVF) cycles. DESIGN: Prospective cohort study. MATERIALS AND METHODS: Between January 2011 and December 2013 30 couples who had prior insuccessful ICSI treatments due to complete fertilization failure accepted to enter this study. Following ICSI 217 injected oocytes were activated with calcium ionophore A23187 for twenty minutes. Embryo transfer was performed on day 3. Fertilization and top quality embryos rate, implantation, clinical pregnancy and birth rates were analyzed. RESULTS: Oocyte retrieval in 30 patients resulted in 288 cumulus-oocyte complexes. After denudation, 217 (84.3%) were mature and, consequently, available for ICSI. Fertilization rate was 63.1% (137/217), cleavage rate was 92% (126/137). A mean of 2.9 Day 3 embryos were transferred to 26 patients. Four couples had no embryos to transfer. Clinical pregnancy rate was 38.5% (10/26), miscarriage rate was 20% (2/10). Nine healthy babies were born (one twin pregnancy). CONCLUSION: The clinical use of ionophores in assisted reproduction is still limited by insufficient knowledge about their potential toxic effect on oocytes and embryos. However, healthy children already born seem not to be affected by ionophore treatment in our study. Assisted oocyte activation with calcium ionophore may become a reasonable and efficient treatment in cases of very low or complete fertilization failure. More prospective controlled studies should be performed to confirm these results. EMBRYO BIOLOGY P-193 Tuesday, October 21, 2014 ACCURATE QUANTIFICATION OF SPECIFIC PROTEINS OF INTEREST IN SINGLE HUMAN BLASTOCOELS USING TARGETED MASS SPECTROMETRY. M. Poli,a,b A. Ori,c S. Jaroudi,d T. Child,a,b M. Beck,c D. Wells.a,d aUniversity of Oxford, Oxford, United Kingdom; b Oxford Fertility Unit, Oxford, United Kingdom; cEuropean Molecular Biology Laboratory, Heidelberg, Germany; dReprogenetics UK, Oxford, United Kingdom. OBJECTIVE: In this study we investigate the feasibility of detecting and quantifying specific protein targets secreted within single human blastocoels using Targeted Mass Spectrometry (MS). DESIGN: Prospective Cohort Study. MATERIALS AND METHODS: Blastocoel fluid was removed from 80 Day5/6 fully expanded blastocysts donated to research. The samples were
FERTILITY & STERILITYÒ
batched in groups of 20, trypsin digested and analyzed by tandem MS. Independent confirmation of the embryonic origin of the identified proteins was achieved via transcriptomic analysis of 18 blastocysts using a combination of RNA amplification and microarray analysis. Initial experiments allowed compilation of a catalogue of detectable blastocoel proteins, after which we selected 26 peptides of interest and designed related Single Reaction Monitoring (SRM) Assays. Subsequently, the SRM Assays and Targeted MS were applied to the analysis of individual blastocoel samples. Homologous synthetic peptides were spiked-in to allow absolute quantification of the targets. Finally, variations in protein profiles across all samples were examined. RESULTS: Analysis of combined tandem MS runs identified 287 proteins that exist within the human blastocoel (false discovery rate of <1%). Comparison of transcriptomic and proteomic data suggests that >80% of proteins found within blastocoel fluid are derived from genes actively transcribed at the blastocyst stage, confirming the high specificity of tandem MS. Using Targeted MS, we succeeded in measuring the concentration of specific proteins in single blastocoels. Interestingly, we observed that the ratio between ‘‘housekeeping’’ and modulated proteins can vary drastically across samples suggesting significant differences between embryos in terms of molecular phenotype. CONCLUSION: This study confirms the feasibility of accurately measuring individual peptides released by the human blastocyst into the blastocoel cavity using Targeted MS. This strategy allows the analysis of proteins of embryonic origin only, avoiding background noise from medium components. Not only has this work provided a catalogue of biologically interesting proteins secreted by embryos nearing the time of implantation, but the data produced is likely to be of relevance to the phenotype of the embryo and may therefore have clinical value. The use of protein concentration ratios as embryo viability/implantation markers is currently under investigation. Supported by: Institutional funding.
P-194 Tuesday, October 21, 2014 ORC4 PLAYS A ROLE IN POLAR BODY EXTRUSION IN THE MOUSE OOCYTE AND ZYGOTE. H. Nguyen, M. Ko, M. A. Ortega, J. Marh, W. S. Ward. Institute for Biogenesis Research John A Burns School of Medicine, Honolulu, HI. OBJECTIVE: Six proteins, ORC1-6, make up the origin recognition complex (ORC) that prepares DNA replication origins for licensing. In somatic cells, the ORC1-6 proteins bind to an unlicensed origin to form a complex, but in mammalian cells four proteins ORC2-5 bind to many origins first and then ORC1 bind to this complex. The complex recruits Cdc6 and Cdt1 and allow licensing to occur by loading Mcm2-7. We tested if ORC1-6 proteins in the mouse oocyte and zygote behave the similarity to the licensing pathways in somatic cells. DESIGN: At different stages of meiosis and the first cell division cycle, embryos were stained for one of five ORC proteins, and MCM7 which associates with chromosomes during cell cycle as a positive control. We used 10 oocytes/group for each experiment. MATERIALS AND METHODS: Immunocytochemistry (ICC) was used to detect ORC1-6 proteins, and cells were analyzed by confocal microscopy. RESULTS: We found that ORC1, 3, 5 and 6 all localized to the area between the separating maternal chromosomes at anaphase II after fertilization, as we had previously reported for ORC2. ORC1, 3 and 5 were not detected in zygotic G1. ORC6 was found in the nucleolar periphery of the pronuclei and polar body nucleus at zygotic G1. Interestingly, in both meiotic divisions, ORC4 surrounds the set of chromosomes that will eventually be discarded in the polar bodies, but not the chromosomes that segregate into the oocyte. ORC4 localizes around the future polar body chromosomes as sphere-like structure in anaphase in both meiotic divisions as the two chromosome sets migrate apart. In zygote G1, ORC4 surrounds the nuclei of 1st and 2sd polar bodies, but is absent from the paternal or maternal pronuclei. The absence of ORC4 in the pronuclei was surprising because ORC4 is required for DNA replication in somatic cells. Moreover, we did find ORC2 in zygotic pronuclei, as expected. At mitosis, the ORC4 was absent from the chromosomes at metaphase, but appeared on both sets of separating chromosomes at telophase. At this point, the ORC4 in the polar body also migrated into the nuclei. CONCLUSION: We are currently conducting additional experiments to determine the functional roles of ORC4 in polar body formation. Our results suggest that ORC4 localization can be used to help identify the DNA strands that destined to be expelled in the polar body. They also suggest that ORC4 is
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modified during the first embryonic cell cycle from a protein that is primarily localized to cytoplasmic, perinuclear structure to one that binds to DNA. Supported by: This work was Supported by NIH Grant HD060722 to Dr. W. Steven Ward, and by a grant from the Vietnamese Education Foundation to Hieu Nguyen.
P-195 Tuesday, October 21, 2014 EPIGENETIC ALTERATIONS IN MOUSE BLASTOCYSTS AND ADULT TISSUES FOLLOWING IVF. X. Liu, S. Feuer, A. Donjacour, R. Simbulan, G. Giritharan, P. Rinaudo. Ob-Gyn, UCSF, San Francisco, CA. OBJECTIVE: The Barker hypothesis holds that alterations to homeostasis during critical periods of development predispose individuals to adult-onset chronic diseases1. Previously, we found that mice conceived by IVF display altered growth and altered glucose homeostasis2. Further, it appears that epigenetic alterations might be the molecular mechanism responsible for the observed phenotypic alterations. To explore this possibility, we have assessed epigenetic alterations at selected loci in embryos and adult tissue of IVF and naturally conceived offspring. DESIGN: Experimental study in inbred (C57Bl/6J) and outbred (CF1xB6D2F1) mice. MATERIALS AND METHODS: In vitro-fertilized embryos were cultured in KSOM with amino acids ( 5% O2; 5% CO2) until the blastocyst stage. In vivo-conceived blastocysts flushed out of the uterus were used as control. Some of the blastocysts were used for molecular analysis while others were transferred into pseudopregnant recipient dams; the resulting IVF (n¼26) and control offspring (n¼ 19) were assessed for metabolic alteration and their tissues were collected for molecular and epigenetic analysis. RT PCR, Western blot, DNA promoter methylation using bisulfite sequencing and Chromatin immunoprecipitation of selected histone marks (H4ac; H3K20me3) were performed at selected loci in blastocysts and adult tissues. RESULTS: We identify thioredoxin-interacting protein (TXNIP)—a key molecule involved in integrating cellular nutritional and oxidative states with metabolic response—as a marker for preimplantation stress. Analysis by qPCR confirmed that IVF induces a nearly four-fold increase in Txnip transcription with a comparable increase in TXNIP protein relative to in vivo-generated blastocysts. Examination of the chromatin architecture at the Txnip promoter in IVF blastocysts revealed an enrichment for the active modification acetylated H4 (H4ac). Bisulfite sequencing of the Txnip promoter, detected no changes in CpG methylation between conception conditions. The adipose tissue of IVF mice replicated the molecular and epigenetic findings in the blastocysts, while beta cells, adipose tissue and muscle did not. CONCLUSION: We have identified Txnip as a molecular marker of preimplantation stress in both inbred and outbred mice. Importantly histone modifications as opposed to DNA methylation promoter changes were observed in IVF blastocysts and maintained in adult adipose tissue of inbred mice. This indicates that an epigenetic memory following preimplantation disturbance is maintained in selected non-imprinted loci. Supported by: R01 HD 062803 - 01A1 to PFR.
P-196 Tuesday, October 21, 2014 ORIGIN RECOGNITION COMPLEX-2 (ORC2) UNIQUELY ASSOCIATES WITH THE MATERNAL AND PATERNAL PRONUCLEI. M. A. Ortega, M. Ko, J. Marh, M. Oshiro, W. S. Ward. Anatomy, Biochemistry and Physiology, University of Hawaii at Manoa, Honolulu, HI. OBJECTIVE: In order to efficiently replicate the genome, potential replication origins are prepared through a series of modifying proteins that begins with ORC2-5 and ends with the binding of inactivated minichromosome maintenance helicases (MCM2-7).1 This process is referred to as ‘‘licensing’’ and marks the DNA site as competent for replication. We have recently shown in the mouse zygote, licensing appears to be completed in ovulated MII maternal DNA, while paternal licensing occurs de novo shortly after pronuclear (PN) formation.2 Since the ORC2 subunit is critical for ORC assembly and nuclear import, we tested whether ORC2 translation in the zygote is required for licensing of the paternal genome.
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ASRM Abstracts
DESIGN: We tested the function of ORC2 translation in the zygote. MATERIALS AND METHODS: MII oocytes were incubated in CHX [40mg/mL] for 30 min followed by 10 mM SrCl2 activation or by injection of sperm to create zygotes, and tested for the presence of ORC2 and MCM7 by immunocytochemistry (ICC) at 2 hrs, 4 hrs, and 8 hrs. Androgenotes were studied under similar conditions. RESULTS: As observed in untreated zygotes, ORC2 localized between the anaphase II maternal DNA on a spindle-like structure, while MCM7 associated more directly to the segregating chromosomes. During G1, 4 hrs after activation, the maternal PN contained both ORC2 and MCM7, which were also observed in untreated embryos. However, unlike normal developing zygotes, by 8 hrs after activation during the time DNA synthesis is typically occurring, the maternal PN becomes depleted of ORC2 but still retains a strong ICC MCM7 signal despite not having undergone replication. We repeated these experiments with zygotes created by ICSI and found similar results with one important exception. In contrast to the maternal PN, under continuous CHX treatment the paternal PN retained ORC2 at 8 hrs after fertilization. When released after 8 hrs, parthenotes stall at one-cell metaphase, however, both ICSI embryos and androgenotes are able to develop considerably further. CONCLUSION: The results, taken together with our previous work, which proposed replication origins were licensed by 2 hrs after fertilization, suggest that continuous translation during zygotic G1 is required to maintain the licensed state of replication origins in the maternal but not paternal pronucleus. To the best of our knowledge, such a surveillance function for ORC2 has not been previously suggested in metazoans. Supported by: NIH grant HD060722.
P-197 Tuesday, October 21, 2014 MORPHOKINETICS OF THE CLEAVAGE STAGE MOUSE EMBRYO IS RELATED TO SUBSEQUENT BLASTOCYST METABOLISM AND FETAL VIABILITY. L. Y. S. Lee, G. A. Thouas, D. K. Gardner. Zoology, University of Melbourne, Parkville, VIC, Australia. OBJECTIVE: To investigate the relationship between cleavage stage mouse embryo kinetics and subsequent blastocyst metabolism and viability. DESIGN: Animal experimental study. Morphokinetics of in vitro fertilized mouse zygotes were observed using a time-lapse imaging system and zygotes were identified as ‘fast’ or ‘slow’. Blastocysts were analyzed for carbohydrate and amino acid metabolism followed by assessment of quality and viability. MATERIALS AND METHODS: In vitro fertilized C57BL/6xCBA(F1) mouse zygotes were individually cultured in 2ml drops of G2 medium under Ovoil in a Sanyo time-lapse incubator(5% O2, 6% CO2, 89% N2) with continuous imaging capability. At 72h post fertilization, blastocysts were separated into quartiles derived from cleavage time to 2-cell and cultured for a further 24h. The spent media samples were then frozen and analyzed for amino acid utilization using liquid chromatography-mass spectrometry(LC-MS). Blastocysts were analyzed for carbohydrate utilization and either stained for inner cell mass(ICM) and trophectoderm(TE) cell numbers, cultured further to assess outgrowth potential, or transferred to recipient females for analysis of implantation and fetal development. Data was checked for Gaussian distribution and Student’s t-test or Mann-Whitney test were used accordingly. RESULTS: Embryos observed with a faster time of first cleavage (first quartile, designated ‘fast’) were found on average to be 2.7h ahead of the slower embryos (fourth quartile, designated ‘slow’, 11.50.1h vs. 14.20.1h, p<0.0001). Subsequently on day 5, blastocysts developed from ‘fast’ embryos had more cells in the ICM (17.42.1 vs. 7.42.0, p<0.01), a higher glucose consumption (21.21.2 pmol/h vs. 13.61.0 pmol/h, p<0.0001) and a lower glycolytic rate (54.43.1% vs. 67.35.1%, p<0.05) compared to ‘slow’ embryos. Further LC-MS analysis revealed that ‘fast’ blastocysts consumed more aspartate than ‘slow’ blastocysts (2.20.1 pmol/embryo/h vs. 1.80.1 pmol/embryo/h, p<0.05). Blastocyst outgrowth area was significantly higher in ‘fast’ embryos compared to ‘slow’ embryos (491370.829754 pixels vs. 393385.528972 pixels, p<0.05). There was no significant difference in implantation rate, however ‘fast’ embryos showed higher fetal development per implantation than ‘slow’ embryos (69.6% vs. 40.4%, p<0.01). CONCLUSION: These findings suggest that kinetically different embryos develop into blastocysts with differences in metabolic profiles and potential viability. Ongoing work is now using these viability markers in combination to improve embryo selection efficiency and further improve pregnancy rates.
Vol. 102, No. 3, Supplement, September 2014
FERTILITY & STERILITYÒ
batched in groups of 20, trypsin digested and analyzed by tandem MS. Independent confirmation of the embryonic origin of the identified proteins was achieved via transcriptomic analysis of 18 blastocysts using a combination of RNA amplification and microarray analysis. Initial experiments allowed compilation of a catalogue of detectable blastocoel proteins, after which we selected 26 peptides of interest and designed related Single Reaction Monitoring (SRM) Assays. Subsequently, the SRM Assays and Targeted MS were applied to the analysis of individual blastocoel samples. Homologous synthetic peptides were spiked-in to allow absolute quantification of the targets. Finally, variations in protein profiles across all samples were examined. RESULTS: Analysis of combined tandem MS runs identified 287 proteins that exist within the human blastocoel (false discovery rate of <1%). Comparison of transcriptomic and proteomic data suggests that >80% of proteins found within blastocoel fluid are derived from genes actively transcribed at the blastocyst stage, confirming the high specificity of tandem MS. Using Targeted MS, we succeeded in measuring the concentration of specific proteins in single blastocoels. Interestingly, we observed that the ratio between ‘‘housekeeping’’ and modulated proteins can vary drastically across samples suggesting significant differences between embryos in terms of molecular phenotype. CONCLUSION: This study confirms the feasibility of accurately measuring individual peptides released by the human blastocyst into the blastocoel cavity using Targeted MS. This strategy allows the analysis of proteins of embryonic origin only, avoiding background noise from medium components. Not only has this work provided a catalogue of biologically interesting proteins secreted by embryos nearing the time of implantation, but the data produced is likely to be of relevance to the phenotype of the embryo and may therefore have clinical value. The use of protein concentration ratios as embryo viability/implantation markers is currently under investigation. Supported by: Institutional funding.
P-194 Tuesday, October 21, 2014 ORC4 PLAYS A ROLE IN POLAR BODY EXTRUSION IN THE MOUSE OOCYTE AND ZYGOTE. H. Nguyen, M. Ko, M. A. Ortega, J. Marh, W. S. Ward. Institute for Biogenesis Research John A Burns School of Medicine, Honolulu, HI. OBJECTIVE: Six proteins, ORC1-6, make up the origin recognition complex (ORC) that prepares DNA replication origins for licensing. In somatic cells, the ORC1-6 proteins bind to an unlicensed origin to form a complex, but in mammalian cells four proteins ORC2-5 bind to many origins first and then ORC1 bind to this complex. The complex recruits Cdc6 and Cdt1 and allow licensing to occur by loading Mcm2-7. We tested if ORC1-6 proteins in the mouse oocyte and zygote behave the similarity to the licensing pathways in somatic cells. DESIGN: At different stages of meiosis and the first cell division cycle, embryos were stained for one of five ORC proteins, and MCM7 which associates with chromosomes during cell cycle as a positive control. We used 10 oocytes/group for each experiment. MATERIALS AND METHODS: Immunocytochemistry (ICC) was used to detect ORC1-6 proteins, and cells were analyzed by confocal microscopy. RESULTS: We found that ORC1, 3, 5 and 6 all localized to the area between the separating maternal chromosomes at anaphase II after fertilization, as we had previously reported for ORC2. ORC1, 3 and 5 were not detected in zygotic G1. ORC6 was found in the nucleolar periphery of the pronuclei and polar body nucleus at zygotic G1. Interestingly, in both meiotic divisions, ORC4 surrounds the set of chromosomes that will eventually be discarded in the polar bodies, but not the chromosomes that segregate into the oocyte. ORC4 localizes around the future polar body chromosomes as sphere-like structure in anaphase in both meiotic divisions as the two chromosome sets migrate apart. In zygote G1, ORC4 surrounds the nuclei of 1st and 2sd polar bodies, but is absent from the paternal or maternal pronuclei. The absence of ORC4 in the pronuclei was surprising because ORC4 is required for DNA replication in somatic cells. Moreover, we did find ORC2 in zygotic pronuclei, as expected. At mitosis, the ORC4 was absent from the chromosomes at metaphase, but appeared on both sets of separating chromosomes at telophase. At this point, the ORC4 in the polar body also migrated into the nuclei. CONCLUSION: We are currently conducting additional experiments to determine the functional roles of ORC4 in polar body formation. Our results suggest that ORC4 localization can be used to help identify the DNA strands that destined to be expelled in the polar body. They also suggest that ORC4 is
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modified during the first embryonic cell cycle from a protein that is primarily localized to cytoplasmic, perinuclear structure to one that binds to DNA. Supported by: This work was Supported by NIH Grant HD060722 to Dr. W. Steven Ward, and by a grant from the Vietnamese Education Foundation to Hieu Nguyen.
P-195 Tuesday, October 21, 2014 EPIGENETIC ALTERATIONS IN MOUSE BLASTOCYSTS AND ADULT TISSUES FOLLOWING IVF. X. Liu, S. Feuer, A. Donjacour, R. Simbulan, G. Giritharan, P. Rinaudo. Ob-Gyn, UCSF, San Francisco, CA. OBJECTIVE: The Barker hypothesis holds that alterations to homeostasis during critical periods of development predispose individuals to adult-onset chronic diseases1. Previously, we found that mice conceived by IVF display altered growth and altered glucose homeostasis2. Further, it appears that epigenetic alterations might be the molecular mechanism responsible for the observed phenotypic alterations. To explore this possibility, we have assessed epigenetic alterations at selected loci in embryos and adult tissue of IVF and naturally conceived offspring. DESIGN: Experimental study in inbred (C57Bl/6J) and outbred (CF1xB6D2F1) mice. MATERIALS AND METHODS: In vitro-fertilized embryos were cultured in KSOM with amino acids ( 5% O2; 5% CO2) until the blastocyst stage. In vivo-conceived blastocysts flushed out of the uterus were used as control. Some of the blastocysts were used for molecular analysis while others were transferred into pseudopregnant recipient dams; the resulting IVF (n¼26) and control offspring (n¼ 19) were assessed for metabolic alteration and their tissues were collected for molecular and epigenetic analysis. RT PCR, Western blot, DNA promoter methylation using bisulfite sequencing and Chromatin immunoprecipitation of selected histone marks (H4ac; H3K20me3) were performed at selected loci in blastocysts and adult tissues. RESULTS: We identify thioredoxin-interacting protein (TXNIP)—a key molecule involved in integrating cellular nutritional and oxidative states with metabolic response—as a marker for preimplantation stress. Analysis by qPCR confirmed that IVF induces a nearly four-fold increase in Txnip transcription with a comparable increase in TXNIP protein relative to in vivo-generated blastocysts. Examination of the chromatin architecture at the Txnip promoter in IVF blastocysts revealed an enrichment for the active modification acetylated H4 (H4ac). Bisulfite sequencing of the Txnip promoter, detected no changes in CpG methylation between conception conditions. The adipose tissue of IVF mice replicated the molecular and epigenetic findings in the blastocysts, while beta cells, adipose tissue and muscle did not. CONCLUSION: We have identified Txnip as a molecular marker of preimplantation stress in both inbred and outbred mice. Importantly histone modifications as opposed to DNA methylation promoter changes were observed in IVF blastocysts and maintained in adult adipose tissue of inbred mice. This indicates that an epigenetic memory following preimplantation disturbance is maintained in selected non-imprinted loci. Supported by: R01 HD 062803 - 01A1 to PFR.
P-196 Tuesday, October 21, 2014 ORIGIN RECOGNITION COMPLEX-2 (ORC2) UNIQUELY ASSOCIATES WITH THE MATERNAL AND PATERNAL PRONUCLEI. M. A. Ortega, M. Ko, J. Marh, M. Oshiro, W. S. Ward. Anatomy, Biochemistry and Physiology, University of Hawaii at Manoa, Honolulu, HI. OBJECTIVE: In order to efficiently replicate the genome, potential replication origins are prepared through a series of modifying proteins that begins with ORC2-5 and ends with the binding of inactivated minichromosome maintenance helicases (MCM2-7).1 This process is referred to as ‘‘licensing’’ and marks the DNA site as competent for replication. We have recently shown in the mouse zygote, licensing appears to be completed in ovulated MII maternal DNA, while paternal licensing occurs de novo shortly after pronuclear (PN) formation.2 Since the ORC2 subunit is critical for ORC assembly and nuclear import, we tested whether ORC2 translation in the zygote is required for licensing of the paternal genome.
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ASRM Abstracts
DESIGN: We tested the function of ORC2 translation in the zygote. MATERIALS AND METHODS: MII oocytes were incubated in CHX [40mg/mL] for 30 min followed by 10 mM SrCl2 activation or by injection of sperm to create zygotes, and tested for the presence of ORC2 and MCM7 by immunocytochemistry (ICC) at 2 hrs, 4 hrs, and 8 hrs. Androgenotes were studied under similar conditions. RESULTS: As observed in untreated zygotes, ORC2 localized between the anaphase II maternal DNA on a spindle-like structure, while MCM7 associated more directly to the segregating chromosomes. During G1, 4 hrs after activation, the maternal PN contained both ORC2 and MCM7, which were also observed in untreated embryos. However, unlike normal developing zygotes, by 8 hrs after activation during the time DNA synthesis is typically occurring, the maternal PN becomes depleted of ORC2 but still retains a strong ICC MCM7 signal despite not having undergone replication. We repeated these experiments with zygotes created by ICSI and found similar results with one important exception. In contrast to the maternal PN, under continuous CHX treatment the paternal PN retained ORC2 at 8 hrs after fertilization. When released after 8 hrs, parthenotes stall at one-cell metaphase, however, both ICSI embryos and androgenotes are able to develop considerably further. CONCLUSION: The results, taken together with our previous work, which proposed replication origins were licensed by 2 hrs after fertilization, suggest that continuous translation during zygotic G1 is required to maintain the licensed state of replication origins in the maternal but not paternal pronucleus. To the best of our knowledge, such a surveillance function for ORC2 has not been previously suggested in metazoans. Supported by: NIH grant HD060722.
P-197 Tuesday, October 21, 2014 MORPHOKINETICS OF THE CLEAVAGE STAGE MOUSE EMBRYO IS RELATED TO SUBSEQUENT BLASTOCYST METABOLISM AND FETAL VIABILITY. L. Y. S. Lee, G. A. Thouas, D. K. Gardner. Zoology, University of Melbourne, Parkville, VIC, Australia. OBJECTIVE: To investigate the relationship between cleavage stage mouse embryo kinetics and subsequent blastocyst metabolism and viability. DESIGN: Animal experimental study. Morphokinetics of in vitro fertilized mouse zygotes were observed using a time-lapse imaging system and zygotes were identified as ‘fast’ or ‘slow’. Blastocysts were analyzed for carbohydrate and amino acid metabolism followed by assessment of quality and viability. MATERIALS AND METHODS: In vitro fertilized C57BL/6xCBA(F1) mouse zygotes were individually cultured in 2ml drops of G2 medium under Ovoil in a Sanyo time-lapse incubator(5% O2, 6% CO2, 89% N2) with continuous imaging capability. At 72h post fertilization, blastocysts were separated into quartiles derived from cleavage time to 2-cell and cultured for a further 24h. The spent media samples were then frozen and analyzed for amino acid utilization using liquid chromatography-mass spectrometry(LC-MS). Blastocysts were analyzed for carbohydrate utilization and either stained for inner cell mass(ICM) and trophectoderm(TE) cell numbers, cultured further to assess outgrowth potential, or transferred to recipient females for analysis of implantation and fetal development. Data was checked for Gaussian distribution and Student’s t-test or Mann-Whitney test were used accordingly. RESULTS: Embryos observed with a faster time of first cleavage (first quartile, designated ‘fast’) were found on average to be 2.7h ahead of the slower embryos (fourth quartile, designated ‘slow’, 11.50.1h vs. 14.20.1h, p<0.0001). Subsequently on day 5, blastocysts developed from ‘fast’ embryos had more cells in the ICM (17.42.1 vs. 7.42.0, p<0.01), a higher glucose consumption (21.21.2 pmol/h vs. 13.61.0 pmol/h, p<0.0001) and a lower glycolytic rate (54.43.1% vs. 67.35.1%, p<0.05) compared to ‘slow’ embryos. Further LC-MS analysis revealed that ‘fast’ blastocysts consumed more aspartate than ‘slow’ blastocysts (2.20.1 pmol/embryo/h vs. 1.80.1 pmol/embryo/h, p<0.05). Blastocyst outgrowth area was significantly higher in ‘fast’ embryos compared to ‘slow’ embryos (491370.829754 pixels vs. 393385.528972 pixels, p<0.05). There was no significant difference in implantation rate, however ‘fast’ embryos showed higher fetal development per implantation than ‘slow’ embryos (69.6% vs. 40.4%, p<0.01). CONCLUSION: These findings suggest that kinetically different embryos develop into blastocysts with differences in metabolic profiles and potential viability. Ongoing work is now using these viability markers in combination to improve embryo selection efficiency and further improve pregnancy rates.
Vol. 102, No. 3, Supplement, September 2014