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In vitro growth and maturation of preantral follicles isolated from vitrified mouse ovaries
found in some genes not yet described in the ovary. Among these genes we found an up-regulation at day 15 of GBX-2, Secretagranin III, RHAMM, PAF-3, G- alpha 12, FKBP-65, Neuronatin, Protein Kinase C Delta, NEK-2 and SEK-1. We selected RHAMM and Secretagranin III for follow-up studies using Taqman to confirm the up-regulation of these genes at day 15 of age. By in-situ hybridization, RHAMM gene expression was localized to the granulosa cells of late antral follicles and in the corpus luteum of 60-day old female mouse ovaries. Conclusions: The transcriptional gene data generated by this approach validates the benefit of using 5-day and 15-day old mouse ovaries to identify possible candidate genes such as RHAMM, involved in the transition from secondary to late antral follicles. Supported by: Wyeth.
P-457 In vitro growth and maturation of preantral follicles isolated from vitrified mouse ovaries. Dong Hoon Kim, Hoi Chang Lee, Duck Sung Ko, Ho Joon Lee, Won Il Park, Seung Samuel Kim. Medical Science Institute, Eulji Gen Hosp, Seoul, South Korea; Dept of Physiology, Eulji Univ Sch of Medicine, Seoul, South Korea; Dept of OB/GYN, Eulji Univ Sch of Medicine, Seoul, South Korea. Objective: The aim of this study was to investigate whether oocytes within preantral follicles isolated from vitrified mouse ovaries are viable and can be rescued to undergo growth, maturation, fertilization, and embryo development in vitro. Design: The laboratory animal study was to compare the growth and maturation of oocytes within preantral follicles isolated from vitrified and fresh mouse ovaries. Materials/Methods: The intact ovaries isolated from 12-day-old ICR mice were treated with VS-1 (Leibovitz medium supplemented with 20% FBS, 10% ethylene glycol and 10% DMSO) for 5min. The ovaries were subsequently placed into VS-2 (Leibovitz medium supplemented with 20% FBS, 0.5M sucrose, 20% ethylene glycol and 20% DMSO) and equilibrated either for 5 or 10 min. The treated ovaries were loaded into 0.25ml straws and plunged into LN2. After the rapid thawing, follicles were enzymatically dissociated using collagenase and DNase I. The isolated follicles were cultured on Transwell-COL membrane inserts in six-well cluster dishes containing MEM medium supplemented with 5% FBS, 100 mIU/ml FSH and 10 mIU/ml HMG. The isolated follicles were cultured for 10 days, and the oocytes were matured, fertilized and developed to the blastocyst stage. The maturation to the metaphase II, development to the blastocyst stage and cell numbers were assessed. Results: There was a significant difference (p ⬍0.05) in survival and maturation rates between the 5 min exposure and 10 min exposure groups, but no difference between the 5 min exposure group and fresh control group (Table 1). Oocyte diameter was significantly smaller (p ⬍0.05) in the 5 and 10 min exposure groups (69.4 ⫾ 2.8 and 67.8 ⫾ 3.1) when compared to that of control group (71.7 ⫾ 2.1). Development to the blastocyst after fertilization was lower in the vitrification group (8.6%) than in the fresh control group (12.0%), but there was no statistical significance in the difference of the rates. The mean number of cells per blastocyst was significantly lower (p ⬍0.05) in the vitrification group (41.9 ⫾ 20.2) than in the fresh control group (55.1 ⫾ 22.5). Table 1. Survival and maturation rate of oocytes according to the exposure time in vitrification solution Exposure time
No. of follicles
Survival rate (%)
Maturation rate (%)
232 162 146
144 (62.1)a 113 (69.8)a 61 (41.8)b
88 (37.9)a 63 (38.9)a 38 (26.0)b
Control 5 min 10 min a,b
P ⬍ 0.05 (2 test)
Conclusions: The results show that mouse oocytes within preantral follicles isolated from the vitrified ovary can achieve full maturation and normal fertilization and embryo development.
S268
Abstracts
P-458 Influences of gonadotropin stimulation on in vitro maturation and embryogenesis of denuded mouse oocytes. Hung Chi Chang, Hui Liu, Jamie Grifo, Lewis C. Krey. Program for IVF, Surg and Infertility, NYU Sch of Medicine, New York, NY. Objective: Denuded germinal vesicle (GV) oocytes are routinely used for GV transfer, a procedure used to study cytoplasmic influences on nuclear function. However, the hormonal background of GV oocytes that most competently mature, fertilize and support embryonic development is unclear. In this study, we examined the influence of gonadotropin priming on follicular development by identifying the GV oocytes with the greatest embryogenic potential. Design: Mice were sacrificed untreated or following treatment with PMSG⫹hCG to generate GV oocytes for in vitro maturation (IVM) and IVF. Progression through IVM, fertilization and early embryonic development was evaluated. Materials/Methods: Female CB6F1 mice (6⫹ weeks old) were used as egg donors and adult CB6F1 males provided the sperm. Group A:no priming; Group B:5 IU PMSG 48h before egg retrieval. Group C:5 IU PMSG ⫹ 5 IU hCG at 48h with retrieval 1h post hCG. Eggs were retrieved by ovarian puncture with a fine needle and cumulus cells were removed by pipetting in HEPES-buffered HTF⫹hyaluronidase (300ug/mL). Denuded GV oocytes were placed in HTF supplemented with 10% fetal calf serum and 50 ug/mL IBMX for 4 – 6h to prevent GV breakdown. A final group (D) of in vivo matured eggs were obtained from mice treated with PMSG⫹hCG and killed after 16h. Mature oocytes (20/drop) were cultured for 4h with ⬃106 sperm in HTF microdrops under oil. At 6 – 8h post-insemination, embryos with 2 pronuclei (PN) and a second polar body were classified as 2PN stage and transferred into G1 microdrops. Four-cell embryos at 48h were transferred into G2 microdrops and monitored to blastocyst stage. Experimental results were analyzed with Chi-square test with significance at p ⬍0.05. Results: The fertilization and blastocyst rates of in vivo matured oocytes were significantly higher than in any IVM group (fertilization: 89 vs 36, 29 and 80% for group D vs A,B and C, respectively; blastocyst: 69 vs 26, 22 and 16%). Gonadotropin treatment did not significantly alter the IVM rate of denuded oocytes (Group A,B,C: 51, 47, 61%) nor the blastocyst rate post-fertilization (above). Curiously, hCG priming 1h before egg retrieval improved the fertilization (Group C vs A,B: 80 vs 36, 29%). GV (n)
PB (n, %)
2PN (n,%)
4-Cell (n,%)
No treatment 296 150 (51) 54 (36) 30 (56) PMSG 48 h 426 202 (47) 58 (29) 31 (53) PMSG 48 h ⫹ hCG 1 h 143 87 (61) 70 (80) 28 (40) In vivo 298 266 (89) 219 (82)
Blastocyst (n,%) 14 (26) 13 (22) 11 (16) 183 (69)
Conclusions: PMSG is routinely used to obtain GV stage oocytes for research on oocyte and embryo function. However, in our denuded mouse oocyte IVM - IVF model system, eggs obtained after PMSG priming do not display improved maturation rates or embryonic developmental competency post-fertilization that are greater than those of eggs from untreated mice. Moreover, embryonic competency in the IVM oocytes was dramatically lower than that of eggs that matured in vivo and were fertilized in vitro. These data agree with our previous report on mouse eggs subjected to IVM and “fertilization” by chemical activation and PN transfer and confirm that IVM impacts adversely on ooplasmic function during the metaphase II anaphase transition at fertilization. The problems with IVM may be due to many factors including an absence of cumulus cell contact during the maturation and IBMX treatment. Supported by: N/A. P-459 Anti-apoptotic actions of IGF-I and TGF-␣ increase cell proliferation and Oct4 gene expression in the mouse blastocyst. Eun Jeong Oh, Il Kyung Jeon, Jong Hyuk Park, Sun Jong Kim, Sung Il Roh, Hyun Soo Yoon. Infertility Research Ctr, MizMedi Hosp, Seoul, South Korea. Objective: Apoptosis during mouse blastocyst formation is a normal feature of development. However, suboptimal culture conditions may in-
Vol. 78, No. 3, Suppl. 1, September 2002
P-457 In vitro growth and maturation of preantral follicles isolated from vitrified mouse ovaries. Dong Hoon Kim, Hoi Chang Lee, Duck Sung Ko, Ho Joon Lee, Won Il Park, Seung Samuel Kim. Medical Science Institute, Eulji Gen Hosp, Seoul, South Korea; Dept of Physiology, Eulji Univ Sch of Medicine, Seoul, South Korea; Dept of OB/GYN, Eulji Univ Sch of Medicine, Seoul, South Korea. Objective: The aim of this study was to investigate whether oocytes within preantral follicles isolated from vitrified mouse ovaries are viable and can be rescued to undergo growth, maturation, fertilization, and embryo development in vitro. Design: The laboratory animal study was to compare the growth and maturation of oocytes within preantral follicles isolated from vitrified and fresh mouse ovaries. Materials/Methods: The intact ovaries isolated from 12-day-old ICR mice were treated with VS-1 (Leibovitz medium supplemented with 20% FBS, 10% ethylene glycol and 10% DMSO) for 5min. The ovaries were subsequently placed into VS-2 (Leibovitz medium supplemented with 20% FBS, 0.5M sucrose, 20% ethylene glycol and 20% DMSO) and equilibrated either for 5 or 10 min. The treated ovaries were loaded into 0.25ml straws and plunged into LN2. After the rapid thawing, follicles were enzymatically dissociated using collagenase and DNase I. The isolated follicles were cultured on Transwell-COL membrane inserts in six-well cluster dishes containing MEM medium supplemented with 5% FBS, 100 mIU/ml FSH and 10 mIU/ml HMG. The isolated follicles were cultured for 10 days, and the oocytes were matured, fertilized and developed to the blastocyst stage. The maturation to the metaphase II, development to the blastocyst stage and cell numbers were assessed. Results: There was a significant difference (p ⬍0.05) in survival and maturation rates between the 5 min exposure and 10 min exposure groups, but no difference between the 5 min exposure group and fresh control group (Table 1). Oocyte diameter was significantly smaller (p ⬍0.05) in the 5 and 10 min exposure groups (69.4 ⫾ 2.8 and 67.8 ⫾ 3.1) when compared to that of control group (71.7 ⫾ 2.1). Development to the blastocyst after fertilization was lower in the vitrification group (8.6%) than in the fresh control group (12.0%), but there was no statistical significance in the difference of the rates. The mean number of cells per blastocyst was significantly lower (p ⬍0.05) in the vitrification group (41.9 ⫾ 20.2) than in the fresh control group (55.1 ⫾ 22.5). Table 1. Survival and maturation rate of oocytes according to the exposure time in vitrification solution Exposure time
No. of follicles
Survival rate (%)
Maturation rate (%)
232 162 146
144 (62.1)a 113 (69.8)a 61 (41.8)b
88 (37.9)a 63 (38.9)a 38 (26.0)b
Control 5 min 10 min a,b
P ⬍ 0.05 (2 test)
Conclusions: The results show that mouse oocytes within preantral follicles isolated from the vitrified ovary can achieve full maturation and normal fertilization and embryo development.
S268
Abstracts
P-458 Influences of gonadotropin stimulation on in vitro maturation and embryogenesis of denuded mouse oocytes. Hung Chi Chang, Hui Liu, Jamie Grifo, Lewis C. Krey. Program for IVF, Surg and Infertility, NYU Sch of Medicine, New York, NY. Objective: Denuded germinal vesicle (GV) oocytes are routinely used for GV transfer, a procedure used to study cytoplasmic influences on nuclear function. However, the hormonal background of GV oocytes that most competently mature, fertilize and support embryonic development is unclear. In this study, we examined the influence of gonadotropin priming on follicular development by identifying the GV oocytes with the greatest embryogenic potential. Design: Mice were sacrificed untreated or following treatment with PMSG⫹hCG to generate GV oocytes for in vitro maturation (IVM) and IVF. Progression through IVM, fertilization and early embryonic development was evaluated. Materials/Methods: Female CB6F1 mice (6⫹ weeks old) were used as egg donors and adult CB6F1 males provided the sperm. Group A:no priming; Group B:5 IU PMSG 48h before egg retrieval. Group C:5 IU PMSG ⫹ 5 IU hCG at 48h with retrieval 1h post hCG. Eggs were retrieved by ovarian puncture with a fine needle and cumulus cells were removed by pipetting in HEPES-buffered HTF⫹hyaluronidase (300ug/mL). Denuded GV oocytes were placed in HTF supplemented with 10% fetal calf serum and 50 ug/mL IBMX for 4 – 6h to prevent GV breakdown. A final group (D) of in vivo matured eggs were obtained from mice treated with PMSG⫹hCG and killed after 16h. Mature oocytes (20/drop) were cultured for 4h with ⬃106 sperm in HTF microdrops under oil. At 6 – 8h post-insemination, embryos with 2 pronuclei (PN) and a second polar body were classified as 2PN stage and transferred into G1 microdrops. Four-cell embryos at 48h were transferred into G2 microdrops and monitored to blastocyst stage. Experimental results were analyzed with Chi-square test with significance at p ⬍0.05. Results: The fertilization and blastocyst rates of in vivo matured oocytes were significantly higher than in any IVM group (fertilization: 89 vs 36, 29 and 80% for group D vs A,B and C, respectively; blastocyst: 69 vs 26, 22 and 16%). Gonadotropin treatment did not significantly alter the IVM rate of denuded oocytes (Group A,B,C: 51, 47, 61%) nor the blastocyst rate post-fertilization (above). Curiously, hCG priming 1h before egg retrieval improved the fertilization (Group C vs A,B: 80 vs 36, 29%). GV (n)
PB (n, %)
2PN (n,%)
4-Cell (n,%)
No treatment 296 150 (51) 54 (36) 30 (56) PMSG 48 h 426 202 (47) 58 (29) 31 (53) PMSG 48 h ⫹ hCG 1 h 143 87 (61) 70 (80) 28 (40) In vivo 298 266 (89) 219 (82)
Blastocyst (n,%) 14 (26) 13 (22) 11 (16) 183 (69)
Conclusions: PMSG is routinely used to obtain GV stage oocytes for research on oocyte and embryo function. However, in our denuded mouse oocyte IVM - IVF model system, eggs obtained after PMSG priming do not display improved maturation rates or embryonic developmental competency post-fertilization that are greater than those of eggs from untreated mice. Moreover, embryonic competency in the IVM oocytes was dramatically lower than that of eggs that matured in vivo and were fertilized in vitro. These data agree with our previous report on mouse eggs subjected to IVM and “fertilization” by chemical activation and PN transfer and confirm that IVM impacts adversely on ooplasmic function during the metaphase II anaphase transition at fertilization. The problems with IVM may be due to many factors including an absence of cumulus cell contact during the maturation and IBMX treatment. Supported by: N/A. P-459 Anti-apoptotic actions of IGF-I and TGF-␣ increase cell proliferation and Oct4 gene expression in the mouse blastocyst. Eun Jeong Oh, Il Kyung Jeon, Jong Hyuk Park, Sun Jong Kim, Sung Il Roh, Hyun Soo Yoon. Infertility Research Ctr, MizMedi Hosp, Seoul, South Korea. Objective: Apoptosis during mouse blastocyst formation is a normal feature of development. However, suboptimal culture conditions may in-
Vol. 78, No. 3, Suppl. 1, September 2002