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E. Lee - One of the best experts on this subject based on the ideXlab platform.
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Pig oocytes with a large Perivitelline Space matured in vitro show greater developmental competence after parthenogenesis and somatic cell nuclear transfer.
Molecular reproduction and development, 2013Co-Authors: Joohyeong Lee, Jinyoung You, Geun-shik Lee, Sang-hwan Hyun, E. LeeAbstract:The objective of this study was to examine the developmental competence of pig oocytes in relation to the size of the Perivitelline Space (PVS) of oocytes matured in vitro. Immature oocytes were matured in medium 199 or porcine zygote medium (PZM)-3 containing 108 or 61.6 mM NaCl. In vitro-matured (IVM) oocytes were examined for intracellular glutathione (GSH) level; cyclin-dependent kinase 1 (CDK1), proliferating cell nuclear antigen (PCNA), and extracellular signal-regulated kinase 2 (ERK2) mRNA levels; and developmental competence after parthenogenesis (PA) and somatic cell nuclear transfer (SCNT). IVM oocytes with a larger PVS had higher (P < 0.05) levels of intracellular GSH (1.00 pixels/oocyte vs. 0.57 pixels/oocyte) and blastocyst formation (54.3% vs. 37.3%) after PA than oocytes with a smaller PVS. Culturing oocytes for maturation in PZM-3 with reduced (61.6 mM) NaCl increased (P < 0.05) the size of the PVS (6.4 µm vs. 2.8 µm) compared to control oocytes that were matured in normal PZM-3 containing 108 mM NaCl. Moreover, oocytes with a larger PVS showed higher CDK1, PCNA, and ERK2 mRNA and intracellular GSH levels (1.6 pixels/oocyte vs. 1.2 pixels/oocyte) and increased blastocyst formation after PA (52.1% vs. 40.6%) and SCNT (31.8% vs. 18.2%) than control oocytes. Our results demonstrate that pig oocytes with a large PVS have greater developmental competence after PA and SCNT, which is attributed to improved cytoplasmic maturation based on the enhanced GSH level and transcription factor expression. Further, enlargement of the PVS by culturing in low-NaCl medium improves the developmental competence of pig oocytes.
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67 PIG OOCYTES WITH A LARGE Perivitelline Space MATURED IN VITRO HAVE GREATER DEVELOPMENTAL COMPETENCE AFTER PARTHENOGENESIS AND SOMATIC CELL NUCLEAR TRANSFER
Reproduction Fertility and Development, 2011Co-Authors: Je Sung You, N. Kim, Sang Ook Kang, E. LeeAbstract:The size of Perivitelline Space (PVS) is closely related with the frequency of polyspermic fertilization in pig oocytes. It has been reported that enlargement of PVS is attributed to accumulation of glycoproteins synthesised and secreted from cumulus cells and that culture of immature oocytes in low-salt medium enlarges PVS in pigs. This study examined the developmental competence of pig oocytes after parthenogenetic activation (PA) and somatic cell nuclear transfer (SCNT) in relation to the size of the PVS of oocytes matured in vitro (IVM). Cumulus–oocyte complexes were matured in medium 199 (Experiment 1) or porcine zygote medium (PZM)-3 (Experiment 2) supplemented with pig follicular fluid, cysteine, pyruvate, EGF, insulin, and hormones for the first 22 h and then cultured in hormone-free medium for an additional 22 h. IVM oocytes were activated electrically for PA or used as recipient cytoplasts for SCNT. PA and SCNT embryos were cultured for 7 days in PZM-3 medium supplemented with bovine serum albumin. The intracellular glutathione (GSH) level in IVM oocytes was determined by analysing the fluorescence intensity of oocytes after staining with CellTracker Blue CMF2HC. The expression of CDK1, PCNA, and ERK2 mRNA in IVM oocytes was analysed by RT-PCR. Data were analysed using a general linear model procedure followed by the least significant difference mean separation procedure when the treatments differed at P
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67 pig oocytes with a large Perivitelline Space matured in vitro have greater developmental competence after parthenogenesis and somatic cell nuclear transfer
Reproduction Fertility and Development, 2011Co-Authors: Je Sung You, N. Kim, Sang Ook Kang, E. LeeAbstract:The size of Perivitelline Space (PVS) is closely related with the frequency of polyspermic fertilization in pig oocytes. It has been reported that enlargement of PVS is attributed to accumulation of glycoproteins synthesised and secreted from cumulus cells and that culture of immature oocytes in low-salt medium enlarges PVS in pigs. This study examined the developmental competence of pig oocytes after parthenogenetic activation (PA) and somatic cell nuclear transfer (SCNT) in relation to the size of the PVS of oocytes matured in vitro (IVM). Cumulus–oocyte complexes were matured in medium 199 (Experiment 1) or porcine zygote medium (PZM)-3 (Experiment 2) supplemented with pig follicular fluid, cysteine, pyruvate, EGF, insulin, and hormones for the first 22 h and then cultured in hormone-free medium for an additional 22 h. IVM oocytes were activated electrically for PA or used as recipient cytoplasts for SCNT. PA and SCNT embryos were cultured for 7 days in PZM-3 medium supplemented with bovine serum albumin. The intracellular glutathione (GSH) level in IVM oocytes was determined by analysing the fluorescence intensity of oocytes after staining with CellTracker Blue CMF2HC. The expression of CDK1, PCNA, and ERK2 mRNA in IVM oocytes was analysed by RT-PCR. Data were analysed using a general linear model procedure followed by the least significant difference mean separation procedure when the treatments differed at P < 0.05. In Experiment 1, oocytes with a larger PVS had higher (P < 0.05) levels of intracellular GSH (1.0 pixels/oocyte v. 0.6 pixels/oocyte) and blastocyst formation (54% v. 37%) after PA than oocytes with smaller PVS. In Experiment 2, maturation culture of oocytes in PZM-3 with reduced (61.6 mM) NaCl concentration significantly increased (P < 0.05) the size of the PVS (5.2 μM v. 3.3 μM) compared with control oocytes that were matured in PZM-3 containing 108 mM NaCl, although the treatment did not alter the nuclear maturation. Moreover, oocytes with increased PVS expressed more CDK1, PCNA, and ERK2 mRNA and had higher (P < 0.05) intracellular GSH levels (1.6 pixels/oocyte v. 1.2 pixels/oocyte) and increased blastocyst formation after PA (52% v. 41%) and SCNT (32% v. 18%) compared with control oocytes. Our results demonstrate that pig oocytes with a large PVS have greater developmental competence after PA and SCNT, which is attributed to improved cytoplasmic maturation resulting from the enhanced GSH level and transcription factor expression and that enlargement of PVS by the culture in low-NaCl medium also improves developmental competence of pig oocytes. This work was supported by grants (#20070301034040 and #20080401034072) from the BioGreen 21 Program (Rural Development Administration, Republic of Korea).
Marcelo Fábio Gouveia Nogueira - One of the best experts on this subject based on the ideXlab platform.
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72 USE OF C57BL/6/EGFP MOUSE TESTICULAR CELLS TO TRAIN Perivitelline Space MICROINJECTION
Reproduction Fertility and Development, 2012Co-Authors: Diego Miranda De Souza, Hugo Fernandes, Patricia V. Silva, Bruno Cazari, P. D. Moco, B. C. S. Campanha, Isabele Picada Emanuelli, Marcelo Fábio Gouveia NogueiraAbstract:The production of embryonic chimeras has been studied as a tool for in vivo pluripotency validation in embryonic stem cells (ESC) as well as to produce transgenic mice. Among the techniques to produce chimeras, one of the most used is microinjection (MI) of ESC into blastocysts or in the Perivitelline Space (PVS) of the embryos with 4 to 8 cells. A well-established training model for this technique could be very useful when ESC are not available, in which injected cells could be easily identified and their subsequent fate could be tracked. Hence, we aimed to test, in mice, a training model for MI in embryos (Swiss Webster, SW) using a pool of EGFP cells derived from testes of the C57BL/6/EGFP strain. Embryos were recovered from prepubertal female SW (n = 20), superstimulated and mated according to a previously described treatment. The MI was performed in the PVS of 4- to 8-cell embryos (collected at 2.5 dpc). When possible, embryos from the same female were randomly allocated to 3 groups: control (C, n = 17), embryos not subjected to MI; perforated (P, n = 15), embryos submitted to perforation by micropipette, without cell injection; and microinjected (MI, n = 32), embryos perforated and submitted to PVS injection with 6 to 8 cells from EGFP testes. After manipulation, embryos from all groups underwent 24 h of in vitro culture (37°C, 5% CO2 and saturated humidity). The viability and quality of the embryos (according to the IETS Manual 1998) and, in group MI, the fluorescence of testicular cells, were evaluated pre- and post-culture. The results were analysed by chi-square test (total frequency observed) and ANOVA (considering the four replicates) with significance being considered when P
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72 use of c57bl 6 egfp mouse testicular cells to train Perivitelline Space microinjection
Reproduction Fertility and Development, 2012Co-Authors: Diego Miranda De Souza, Hugo Fernandes, Patricia V. Silva, Bruno Cazari, P. D. Moco, B. C. S. Campanha, Isabele Picada Emanuelli, Marcelo Fábio Gouveia NogueiraAbstract:The production of embryonic chimeras has been studied as a tool for in vivo pluripotency validation in embryonic stem cells (ESC) as well as to produce transgenic mice. Among the techniques to produce chimeras, one of the most used is microinjection (MI) of ESC into blastocysts or in the Perivitelline Space (PVS) of the embryos with 4 to 8 cells. A well-established training model for this technique could be very useful when ESC are not available, in which injected cells could be easily identified and their subsequent fate could be tracked. Hence, we aimed to test, in mice, a training model for MI in embryos (Swiss Webster, SW) using a pool of EGFP cells derived from testes of the C57BL/6/EGFP strain. Embryos were recovered from prepubertal female SW (n = 20), superstimulated and mated according to a previously described treatment. The MI was performed in the PVS of 4- to 8-cell embryos (collected at 2.5 dpc). When possible, embryos from the same female were randomly allocated to 3 groups: control (C, n = 17), embryos not subjected to MI; perforated (P, n = 15), embryos submitted to perforation by micropipette, without cell injection; and microinjected (MI, n = 32), embryos perforated and submitted to PVS injection with 6 to 8 cells from EGFP testes. After manipulation, embryos from all groups underwent 24 h of in vitro culture (37°C, 5% CO2 and saturated humidity). The viability and quality of the embryos (according to the IETS Manual 1998) and, in group MI, the fluorescence of testicular cells, were evaluated pre- and post-culture. The results were analysed by chi-square test (total frequency observed) and ANOVA (considering the four replicates) with significance being considered when P < 0.05. There was no difference among mortality rates [i.e. % of viable embryos that died after 24 h of culture, of the groups (5.9, 26.7 and 25.0% for C, P and MI, respectively]. The percentage of embryos that maintained or improved quality after 24 h of culture, in comparison with quality evaluation pre-culture, was different (P < 0.01) among groups C, P and MI (94.1, 73.3 and 43.8%, respectively). One chimeric blastocyst was obtained in the MI group (3.1%, 1/32). Considering the proposed conditions, this model for training of MI of EGFP testicular cells in the PVS was feasible and practical to acquire skills, when ESC are not available. Moreover, the method allows easy identification of injected and, eventually, aggregated cellular components. Financial support was received from FAPESP of Brazil.
B. D. Whitaker - One of the best experts on this subject based on the ideXlab platform.
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effects of glucuronic acid and n acetyl d glucosamine supplementation on the Perivitelline Space during the ivm of pig oocytes
Reproduction Fertility and Development, 2020Co-Authors: J. Z. Current, B. D. WhitakerAbstract:The objective of this study was to minimise polyspermic penetration by increasing the Perivitelline Space (PVS) thickness through supplementation of the hyaluronic acid components glucuronic acid and N-acetyl-d-glucosamine (GlcNAc). Oocytes (n=4690) were supplemented during the first 24h and/or the remainder of maturation (final 16-18h) with 0.01mM glucuronic acid and 0.01mM GlcNAc and then evaluated for PVS thickness, hyaluronic acid, glutathione and glutathione peroxidase concentrations. Fertilised oocytes were evaluated for polyspermic penetration and embryo development. The PVS thickness and amount of hyaluronic acid was significantly (P<0.05) greater in oocytes supplemented with 0.01mM glucuronic acid and 0.01mM GlcNAc during the second part or all of maturation compared with the other treatments. In addition, polyspermic penetration was significantly (P<0.05) less in oocytes supplemented with 0.01mM glucuronic acid and 0.01mM GlcNAc during the second part or all of maturation compared with the other treatments. Supplementing 0.01mM glucuronic acid and GlcNAc during maturation significantly (P<0.05) increased the percentage of cleaved embryos by 48h after IVF and blastocysts formed by 144h after IVF compared those not supplemented. These results indicate that supplementing PVS components during maturation decreases polyspermic penetration by increasing PVS thickness.
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Effects of glucuronic acid and N-acetyl-D-glucosamine supplementation on the Perivitelline Space during the IVM of pig oocytes.
Reproduction fertility and development, 2020Co-Authors: J. Z. Current, B. D. WhitakerAbstract:The objective of this study was to minimise polyspermic penetration by increasing the Perivitelline Space (PVS) thickness through supplementation of the hyaluronic acid components glucuronic acid and N-acetyl-d-glucosamine (GlcNAc). Oocytes (n=4690) were supplemented during the first 24h and/or the remainder of maturation (final 16-18h) with 0.01mM glucuronic acid and 0.01mM GlcNAc and then evaluated for PVS thickness, hyaluronic acid, glutathione and glutathione peroxidase concentrations. Fertilised oocytes were evaluated for polyspermic penetration and embryo development. The PVS thickness and amount of hyaluronic acid was significantly (P
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175 effects of glucuronic acid and n acetyl d glucosamine supplementation on the Perivitelline Space during the in vitro maturation of porcine oocytes
Reproduction Fertility and Development, 2017Co-Authors: J. Z. Current, B. D. WhitakerAbstract:Pig oocytes fertilized in vitro experience high polyspermic penetration rates due to inadequate cortical granule exocytosis into reduced Perivitelline Space (PVS) thickness. The objective of this study was to minimize polyspermic penetration by increasing the PVS thickness through supplementation of its hyaluronic acid components, glucuronic acid (GA), and N-acetyl-d-glucosamine (GlcNAc) during maturation. Oocytes were supplemented during the first 24 h or second 24 h of maturation with 0.01 mM GA and 0.01 mM GlcNAc and then evaluated for nuclear maturation (n = 200), PVS thickness (n = 245), and the amount of hyaluronic acid (n = 245) present. The PVS thickness was determined at the equatorial plane of the oocyte using a micrometer. Hyaluronic acid concentrations were determined using an enzyme-linked immunosorbent assay method. Oocytes (n = 800) were fertilized using frozen-thawed semen and evaluated for fertilization characteristics and subsequent embryonic development at 48 and 144 h for cleavage and blastocyst formation, respectively. The PVS thickness was significantly thicker (P < 0.05) with oocytes supplemented with 0.01 mM GA and 0.01 mM GlcNAc during the first half of maturation (3.20 ± 0.29) and all of maturation (2.78 ± 0.21) compared with no supplementation (2.22 ± 0.13) and supplementation during only the second half of maturation (2.02 ± 0.16). The amount of hyaluronic acid present at 24 h of maturation was significantly greater (P < 0.05) in oocytes supplemented with the PVS components (2.03 ± 0.07 pg/oocyte) compared with no supplementation (0.21 ± 0.02 pg/oocyte). At the end of maturation, oocytes supplemented with 0.01 mM GA and 0.01 mM GlcNAc during the entire maturation had significantly greater (P < 0.05) amounts of hyaluronic acid present (4.16 ± 0.19 pg/oocyte) compared with all other groups. There was no significant difference in penetration rates between the groups. Polyspermic penetration was significantly less (P < 0.05) in oocytes supplemented with 0.01 mM GA and 0.01 mM GlcNAc during the first half of maturation compared with no supplementation or supplementation during only the second half of maturation. Oocytes supplemented with 0.01 mM GA and 0.01 mM GlcNAc during the first half of maturation (87.36 ± 4.01) compared with no supplementation (76.47 ± 5.67) or supplementation during only the second half of maturation (80.23 ± 3.21) had significantly higher percentages (P < 0.05) of male pronuclear formation by 12 h after IVF. Supplementing 0.01 mM GA and 0.01 mM GlcNAc during maturation significantly increased (P < 0.05) the percentage of cleaved embryos by 48 h after IVF and the percentage of those reaching the blastocyst stage by 144 h after IVF compared with those that were not supplemented during maturation (55.00 ± 6.43, 20.00 ± 6.16). There were no significant differences between the supplementation treatment groups at 48 or 144 h after IVF. These results indicate that supplementing GA and GlcNAc to the media during maturation, specifically during the first 24 h, decreases polyspermic penetration by increasing PVS thickness, hyaluronic acid amount, and male pronuclear formation, which improves subsequent embryonic development.
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175 EFFECTS OF GLUCURONIC ACID AND N-ACETYL-D-GLUCOSAMINE SUPPLEMENTATION ON THE Perivitelline Space DURING THE IN VITRO MATURATION OF PORCINE OOCYTES
Reproduction Fertility and Development, 2017Co-Authors: J. Z. Current, B. D. WhitakerAbstract:Pig oocytes fertilized in vitro experience high polyspermic penetration rates due to inadequate cortical granule exocytosis into reduced Perivitelline Space (PVS) thickness. The objective of this study was to minimize polyspermic penetration by increasing the PVS thickness through supplementation of its hyaluronic acid components, glucuronic acid (GA), and N-acetyl-d-glucosamine (GlcNAc) during maturation. Oocytes were supplemented during the first 24 h or second 24 h of maturation with 0.01 mM GA and 0.01 mM GlcNAc and then evaluated for nuclear maturation (n = 200), PVS thickness (n = 245), and the amount of hyaluronic acid (n = 245) present. The PVS thickness was determined at the equatorial plane of the oocyte using a micrometer. Hyaluronic acid concentrations were determined using an enzyme-linked immunosorbent assay method. Oocytes (n = 800) were fertilized using frozen-thawed semen and evaluated for fertilization characteristics and subsequent embryonic development at 48 and 144 h for cleavage and blastocyst formation, respectively. The PVS thickness was significantly thicker (P
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The effects of glucuronic acid and N-acetyl-D-glucosamine on in vitro fertilisation of porcine oocytes.
Reproduction fertility and development, 2015Co-Authors: K. Schmidt, A. Clark, A. Mello, C. Durfey, A. Buck, K. Boyd, B. D. WhitakerAbstract:High incidences of polyspermic penetration continue to challenge researchers during porcine in vitro fertilisation (IVF). The aim of this study was to reduce the incidence of polyspermy by increasing the Perivitelline Space thickness with glucuronic acid and N-acetyl-D-glucosamine (GlcNAc) supplementation during oocyte maturation. After maturation, zona pellucida and Perivitelline Space thicknesses, intracellular glutathione concentrations and fertilisation kinetics were measured, in addition to embryonic cleavage and blastocyst formation at 48 h and 144 h after IVF, respectively. There were no significant differences between the treatments for zona pellucida thickness, penetration rates, male pronuclear formation or cortical granule exocytosis. Glucuronic acid supplementation significantly increased (P
Diego Miranda De Souza - One of the best experts on this subject based on the ideXlab platform.
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72 USE OF C57BL/6/EGFP MOUSE TESTICULAR CELLS TO TRAIN Perivitelline Space MICROINJECTION
Reproduction Fertility and Development, 2012Co-Authors: Diego Miranda De Souza, Hugo Fernandes, Patricia V. Silva, Bruno Cazari, P. D. Moco, B. C. S. Campanha, Isabele Picada Emanuelli, Marcelo Fábio Gouveia NogueiraAbstract:The production of embryonic chimeras has been studied as a tool for in vivo pluripotency validation in embryonic stem cells (ESC) as well as to produce transgenic mice. Among the techniques to produce chimeras, one of the most used is microinjection (MI) of ESC into blastocysts or in the Perivitelline Space (PVS) of the embryos with 4 to 8 cells. A well-established training model for this technique could be very useful when ESC are not available, in which injected cells could be easily identified and their subsequent fate could be tracked. Hence, we aimed to test, in mice, a training model for MI in embryos (Swiss Webster, SW) using a pool of EGFP cells derived from testes of the C57BL/6/EGFP strain. Embryos were recovered from prepubertal female SW (n = 20), superstimulated and mated according to a previously described treatment. The MI was performed in the PVS of 4- to 8-cell embryos (collected at 2.5 dpc). When possible, embryos from the same female were randomly allocated to 3 groups: control (C, n = 17), embryos not subjected to MI; perforated (P, n = 15), embryos submitted to perforation by micropipette, without cell injection; and microinjected (MI, n = 32), embryos perforated and submitted to PVS injection with 6 to 8 cells from EGFP testes. After manipulation, embryos from all groups underwent 24 h of in vitro culture (37°C, 5% CO2 and saturated humidity). The viability and quality of the embryos (according to the IETS Manual 1998) and, in group MI, the fluorescence of testicular cells, were evaluated pre- and post-culture. The results were analysed by chi-square test (total frequency observed) and ANOVA (considering the four replicates) with significance being considered when P
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72 use of c57bl 6 egfp mouse testicular cells to train Perivitelline Space microinjection
Reproduction Fertility and Development, 2012Co-Authors: Diego Miranda De Souza, Hugo Fernandes, Patricia V. Silva, Bruno Cazari, P. D. Moco, B. C. S. Campanha, Isabele Picada Emanuelli, Marcelo Fábio Gouveia NogueiraAbstract:The production of embryonic chimeras has been studied as a tool for in vivo pluripotency validation in embryonic stem cells (ESC) as well as to produce transgenic mice. Among the techniques to produce chimeras, one of the most used is microinjection (MI) of ESC into blastocysts or in the Perivitelline Space (PVS) of the embryos with 4 to 8 cells. A well-established training model for this technique could be very useful when ESC are not available, in which injected cells could be easily identified and their subsequent fate could be tracked. Hence, we aimed to test, in mice, a training model for MI in embryos (Swiss Webster, SW) using a pool of EGFP cells derived from testes of the C57BL/6/EGFP strain. Embryos were recovered from prepubertal female SW (n = 20), superstimulated and mated according to a previously described treatment. The MI was performed in the PVS of 4- to 8-cell embryos (collected at 2.5 dpc). When possible, embryos from the same female were randomly allocated to 3 groups: control (C, n = 17), embryos not subjected to MI; perforated (P, n = 15), embryos submitted to perforation by micropipette, without cell injection; and microinjected (MI, n = 32), embryos perforated and submitted to PVS injection with 6 to 8 cells from EGFP testes. After manipulation, embryos from all groups underwent 24 h of in vitro culture (37°C, 5% CO2 and saturated humidity). The viability and quality of the embryos (according to the IETS Manual 1998) and, in group MI, the fluorescence of testicular cells, were evaluated pre- and post-culture. The results were analysed by chi-square test (total frequency observed) and ANOVA (considering the four replicates) with significance being considered when P < 0.05. There was no difference among mortality rates [i.e. % of viable embryos that died after 24 h of culture, of the groups (5.9, 26.7 and 25.0% for C, P and MI, respectively]. The percentage of embryos that maintained or improved quality after 24 h of culture, in comparison with quality evaluation pre-culture, was different (P < 0.01) among groups C, P and MI (94.1, 73.3 and 43.8%, respectively). One chimeric blastocyst was obtained in the MI group (3.1%, 1/32). Considering the proposed conditions, this model for training of MI of EGFP testicular cells in the PVS was feasible and practical to acquire skills, when ESC are not available. Moreover, the method allows easy identification of injected and, eventually, aggregated cellular components. Financial support was received from FAPESP of Brazil.
Je Sung You - One of the best experts on this subject based on the ideXlab platform.
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67 PIG OOCYTES WITH A LARGE Perivitelline Space MATURED IN VITRO HAVE GREATER DEVELOPMENTAL COMPETENCE AFTER PARTHENOGENESIS AND SOMATIC CELL NUCLEAR TRANSFER
Reproduction Fertility and Development, 2011Co-Authors: Je Sung You, N. Kim, Sang Ook Kang, E. LeeAbstract:The size of Perivitelline Space (PVS) is closely related with the frequency of polyspermic fertilization in pig oocytes. It has been reported that enlargement of PVS is attributed to accumulation of glycoproteins synthesised and secreted from cumulus cells and that culture of immature oocytes in low-salt medium enlarges PVS in pigs. This study examined the developmental competence of pig oocytes after parthenogenetic activation (PA) and somatic cell nuclear transfer (SCNT) in relation to the size of the PVS of oocytes matured in vitro (IVM). Cumulus–oocyte complexes were matured in medium 199 (Experiment 1) or porcine zygote medium (PZM)-3 (Experiment 2) supplemented with pig follicular fluid, cysteine, pyruvate, EGF, insulin, and hormones for the first 22 h and then cultured in hormone-free medium for an additional 22 h. IVM oocytes were activated electrically for PA or used as recipient cytoplasts for SCNT. PA and SCNT embryos were cultured for 7 days in PZM-3 medium supplemented with bovine serum albumin. The intracellular glutathione (GSH) level in IVM oocytes was determined by analysing the fluorescence intensity of oocytes after staining with CellTracker Blue CMF2HC. The expression of CDK1, PCNA, and ERK2 mRNA in IVM oocytes was analysed by RT-PCR. Data were analysed using a general linear model procedure followed by the least significant difference mean separation procedure when the treatments differed at P
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67 pig oocytes with a large Perivitelline Space matured in vitro have greater developmental competence after parthenogenesis and somatic cell nuclear transfer
Reproduction Fertility and Development, 2011Co-Authors: Je Sung You, N. Kim, Sang Ook Kang, E. LeeAbstract:The size of Perivitelline Space (PVS) is closely related with the frequency of polyspermic fertilization in pig oocytes. It has been reported that enlargement of PVS is attributed to accumulation of glycoproteins synthesised and secreted from cumulus cells and that culture of immature oocytes in low-salt medium enlarges PVS in pigs. This study examined the developmental competence of pig oocytes after parthenogenetic activation (PA) and somatic cell nuclear transfer (SCNT) in relation to the size of the PVS of oocytes matured in vitro (IVM). Cumulus–oocyte complexes were matured in medium 199 (Experiment 1) or porcine zygote medium (PZM)-3 (Experiment 2) supplemented with pig follicular fluid, cysteine, pyruvate, EGF, insulin, and hormones for the first 22 h and then cultured in hormone-free medium for an additional 22 h. IVM oocytes were activated electrically for PA or used as recipient cytoplasts for SCNT. PA and SCNT embryos were cultured for 7 days in PZM-3 medium supplemented with bovine serum albumin. The intracellular glutathione (GSH) level in IVM oocytes was determined by analysing the fluorescence intensity of oocytes after staining with CellTracker Blue CMF2HC. The expression of CDK1, PCNA, and ERK2 mRNA in IVM oocytes was analysed by RT-PCR. Data were analysed using a general linear model procedure followed by the least significant difference mean separation procedure when the treatments differed at P < 0.05. In Experiment 1, oocytes with a larger PVS had higher (P < 0.05) levels of intracellular GSH (1.0 pixels/oocyte v. 0.6 pixels/oocyte) and blastocyst formation (54% v. 37%) after PA than oocytes with smaller PVS. In Experiment 2, maturation culture of oocytes in PZM-3 with reduced (61.6 mM) NaCl concentration significantly increased (P < 0.05) the size of the PVS (5.2 μM v. 3.3 μM) compared with control oocytes that were matured in PZM-3 containing 108 mM NaCl, although the treatment did not alter the nuclear maturation. Moreover, oocytes with increased PVS expressed more CDK1, PCNA, and ERK2 mRNA and had higher (P < 0.05) intracellular GSH levels (1.6 pixels/oocyte v. 1.2 pixels/oocyte) and increased blastocyst formation after PA (52% v. 41%) and SCNT (32% v. 18%) compared with control oocytes. Our results demonstrate that pig oocytes with a large PVS have greater developmental competence after PA and SCNT, which is attributed to improved cytoplasmic maturation resulting from the enhanced GSH level and transcription factor expression and that enlargement of PVS by the culture in low-NaCl medium also improves developmental competence of pig oocytes. This work was supported by grants (#20070301034040 and #20080401034072) from the BioGreen 21 Program (Rural Development Administration, Republic of Korea).
