Objective: Successful cryopreservation of immature oocytes may be an alternative approach in the ovum bank because cryopreservation will be performed before spindle formation. However, relatively few studies have examined the attempts of freezing germinal vesicle (GV) stage oocytes. There was a report showing that an incidence of chromosomal abnormalities in human GV stage oocytes after freezing and thawing was increased (Park et al., 1997). A recent study showed that MAP kinase plays an important role in controlling chromatin and microtubule behavior. In previous study, we have found that MAP kinase phosphorylation was correlated with cytoskeletal behavior in mouse oocytes after vitrification at the mature stage and that vitrification induced changes in microtubule organization and MAP kinase phosphorylation level in mature oocytes. The main objective of this study was to show a possible correlation between cytoskeletal change and MAP kinase phosphorylation level in mouse oocytes matured in vitro after vitrification at the germinal vesicle stage. Design: Intact immature oocytes from superovulated ICR mouse were collected and divided into three groups: group 1, no treatment (control); group 2, vitrified oocytes, group 3, vitrified oocytes with Taxol™ (cytosketal stabilizer). In vitro-matured oocytes (n=40) in group 1 and those survived and matured (n=40) after vitrification in group 2 and 3 were used for MAP kinase phosphorylation assay. MAP kinase phosphorylation was revealed by western immunoblotting. Materials/Methods: Immature oocytes were pre-equilibrated for 2.5 min in DPBS with 1.5 M EG with and without 1mM Taxol™, and then vitrified with DPBS with 5.5 M or 1.0 M sucrose with and without 1mM Taxol™ for 20 sec. Oocytes were loaded onto the electron microscope copper grid for storing in liquid nitrogen. Stored oocytes were thawed by a five-step method. Control and survived oocytes were cultured in TCM 199 media with 20% FBS for 16 h. In vitro matured oocytes in all groups were used for western immunoblotting using phosphorylated MAP kinase Ab. Results: MAP kinase of oocytes in all groups was presented in a phosphorylated form. There was no change in the expression level of MAP kinases in oocytes matured in vitro after vitrification at the GV stage. However, vitrified oocytes with Taxol™ showed an increased level of MAP kinase phosphorylation. Conclusions: Vitrified oocytes at the immature stage have the potential to maintain the optimal MAP kinase phosphorylation levels, while those vitrified-thawed ones at mature stage decrease them (our previous result). Supported by: Korean Health 21 RND Project, Ministry of Health and Welfare, Korea (01-PJ10-PG6–01GN13–0002).

Objective: The in vitro maturation of immature oocytes retrieved from unstimulated ovaries has been interested in assisted reproductive technology. Oocyte maturation, characterized by germinal vesicle (GV) break-down, formation of first meiotic spindle, extrusion of the first polar body, and arrest in metaphase of the second meiotic division. However, numerous anomalies in the nuclear and cytoplasmic maturation were induced during in vitro maturation. This study was conducted to analyze spindle and chromosome configurations of in vitro maturation failure metaphase I (MI) arrested oocytes collected from PCO patients.Design: Arrested oocytes at the MI stage were collected after 48h culture. The chromosome and spindle configuration were observed by immunostaining.Materials/Methods: Immature oocytes were aspirated using ultrasound guided aspiration method with specially designed injection needle from unstimulated PCO patients. Oocytes with a germinal vesicle were cultured in maturation medium for 48h. Oocytes were removed cumulus cells with hyaluronidase and mechanical pipetting after culture. Oocytes without an intact GV and 1st polar body were defined as metaphase I. Oocytes were used for immunostaining by using monoclonal anti-tubulin antibody and TRITC-conjugated second antibody.Results: Absent (85%) or disorganized (15%) spindle and dispersed chromosomes (100%) were observed in all analyzed oocytes.Conclusions: This result suggested that the MI arrested oocytes was related to the MI spindle, with absence of microtubules and dispersion of the chromosomes. Objective: The in vitro maturation of immature oocytes retrieved from unstimulated ovaries has been interested in assisted reproductive technology. Oocyte maturation, characterized by germinal vesicle (GV) break-down, formation of first meiotic spindle, extrusion of the first polar body, and arrest in metaphase of the second meiotic division. However, numerous anomalies in the nuclear and cytoplasmic maturation were induced during in vitro maturation. This study was conducted to analyze spindle and chromosome configurations of in vitro maturation failure metaphase I (MI) arrested oocytes collected from PCO patients. Design: Arrested oocytes at the MI stage were collected after 48h culture. The chromosome and spindle configuration were observed by immunostaining. Materials/Methods: Immature oocytes were aspirated using ultrasound guided aspiration method with specially designed injection needle from unstimulated PCO patients. Oocytes with a germinal vesicle were cultured in maturation medium for 48h. Oocytes were removed cumulus cells with hyaluronidase and mechanical pipetting after culture. Oocytes without an intact GV and 1st polar body were defined as metaphase I. Oocytes were used for immunostaining by using monoclonal anti-tubulin antibody and TRITC-conjugated second antibody. Results: Absent (85%) or disorganized (15%) spindle and dispersed chromosomes (100%) were observed in all analyzed oocytes. Conclusions: This result suggested that the MI arrested oocytes was related to the MI spindle, with absence of microtubules and dispersion of the chromosomes.

Objective: In a previous study, vitrified-thawed oocytes had fewer spindles and more chromosomal abnormalities than fresh oocytes. However, with the addition of a cytoskeleton stabilizer, Taxol™ to vitrification solution, the post-thawed preimplantation development was improved. The result suggests that damage in oocyte cytoskeleton system may be one of the main cryoinjuries so that stabilizing the cytoskeleton system during vitrification is beneficial for the post-thawed survival and the subsequent development of vitrified oocytes (Park et al., 2000). A recent study showed that MAP kinase plays an important role in controlling chromatin and microtubule behavior. However, the relationship of MAP kinase phosphorylation to cytoskeletal behavior during cryopreservation has never been studied. The objective of the present study was to determine whether a relationship exists between cytoskeletal changes and MAP kinase phosphorylation in vitrified-thawed oocytes.Design: Intact mature oocytes from superovulated ICR mouse were collected and divided into four groups: group 1, no treatment (control); group 2, vitrified oocytes, group 3, vitrified oocytes with Taxol™ (cytosketal stabilizer), and group 4, vitrified oocytes with nocodazol (microtubule inhibitor). Oocytes (n=40) in group 1 and those (n=40) survived after vitrification in group 2, 3, and 4 were used for MAP kinase phosphorylation assay. MAP kinase phosphorylation was confirmed by western immunoblotting.Materials/Methods: Oocytes were pre-equilibrated for 2.5 min in DPBS with 1.5 M EG with and without 1mM Taxol™, and then vitrified with DPBS with 5.5 M or 1.0 M sucrose with and without 1mM Taxol™ for 20 sec. Oocytes were loaded onto the electron microscope copper grid for storing in liquid nitrogen. Stored oocytes were thawed by a five-step method. Either fresh or survived oocytes were used for western immunoblotting using phosphorylated MAP kinase antibody.Results: MAP kinase of oocytes in all groups was presented in a phosphorylated from. MAP kinase phosphorylation level was decreased in vitrified-thawed oocytes, but increased in those vitrified with Taxol™.Conclusions: Cryopreservation induces changes in microtubule organization and MAP kinase phosphorylation level in mature oocytes. Stabilizing the cytoskeleton system in mature oocytes with Taxol™ is beneficial for recovery of MAP kinase phosphorylation level after vitrification.Supported by: Korean Health 21 RND Project, Ministry of Health and Welfare, Korea (01-PJ10-PG6-01GN13-0002). Objective: In a previous study, vitrified-thawed oocytes had fewer spindles and more chromosomal abnormalities than fresh oocytes. However, with the addition of a cytoskeleton stabilizer, Taxol™ to vitrification solution, the post-thawed preimplantation development was improved. The result suggests that damage in oocyte cytoskeleton system may be one of the main cryoinjuries so that stabilizing the cytoskeleton system during vitrification is beneficial for the post-thawed survival and the subsequent development of vitrified oocytes (Park et al., 2000). A recent study showed that MAP kinase plays an important role in controlling chromatin and microtubule behavior. However, the relationship of MAP kinase phosphorylation to cytoskeletal behavior during cryopreservation has never been studied. The objective of the present study was to determine whether a relationship exists between cytoskeletal changes and MAP kinase phosphorylation in vitrified-thawed oocytes. Design: Intact mature oocytes from superovulated ICR mouse were collected and divided into four groups: group 1, no treatment (control); group 2, vitrified oocytes, group 3, vitrified oocytes with Taxol™ (cytosketal stabilizer), and group 4, vitrified oocytes with nocodazol (microtubule inhibitor). Oocytes (n=40) in group 1 and those (n=40) survived after vitrification in group 2, 3, and 4 were used for MAP kinase phosphorylation assay. MAP kinase phosphorylation was confirmed by western immunoblotting. Materials/Methods: Oocytes were pre-equilibrated for 2.5 min in DPBS with 1.5 M EG with and without 1mM Taxol™, and then vitrified with DPBS with 5.5 M or 1.0 M sucrose with and without 1mM Taxol™ for 20 sec. Oocytes were loaded onto the electron microscope copper grid for storing in liquid nitrogen. Stored oocytes were thawed by a five-step method. Either fresh or survived oocytes were used for western immunoblotting using phosphorylated MAP kinase antibody. Results: MAP kinase of oocytes in all groups was presented in a phosphorylated from. MAP kinase phosphorylation level was decreased in vitrified-thawed oocytes, but increased in those vitrified with Taxol™. Conclusions: Cryopreservation induces changes in microtubule organization and MAP kinase phosphorylation level in mature oocytes. Stabilizing the cytoskeleton system in mature oocytes with Taxol™ is beneficial for recovery of MAP kinase phosphorylation level after vitrification. Supported by: Korean Health 21 RND Project, Ministry of Health and Welfare, Korea (01-PJ10-PG6-01GN13-0002).

Objective: Deficiencies in mitochondrial ATP production may be associated with various cellular and chromosomal disorders. In previous study, vitrified-thawed oocytes had lower spindle and chromosome normalities than fresh oocytes. This study was subsequently conducted to examine whether level of mitochondrial ATPase 6 expression changed after vitrification. This gene was chosen because of its central role in ATP production.Design: Intact immature oocytes were collected from superovulated ICR mouse and divided into three groups: group 1, no treatment (control); group 2, vitrified oocytes with grids, group 3, vitrified oocytes with straws. In vitro matured oocytes of group 1 and oocytes that survived and matured after vitrification of group 2, 3 were used for evaluation of ATPase 6 gene expression.Materials/Methods: Immature oocytes were pre-equilibrated in DPBS with 1.5 M EG for 2.5 min. Oocytes were than vitrified with DPBS with 5.5 M EG and 1.0 M sucrose for 30 sec. Oocytes were loaded onto electron microscope copper grids or into conventional 0.25 ml straws for storing in liquid nitrogen. Stored oocytes were thawed by a five-step method. Control and survived oocytes were cultured in the medium containing gonadotropins for 16 h. Control and vitrified-thawed oocytes were compared ATPase 6 gene expression by using reverse transcription polymerase chain reaction. The band intensity of PCR products was measured by using the imaging Densitometer.Results: 1. The intensities of mitochondrial ATPase 6 gene expression in vitrified-thawed oocytes did not change compared with fresh oocytes. 2. There was no difference in the level of ATP ase 6 gene expression between oocytes vitrified with grids and straws.Conclusions: The aspects of ATPase 6 gene expression in vitrified-thawed oocytes suggest that vitrification of oocytes by using grids or straws at GV stage may have comparatively good strategy.Supported by: Korean Health 21 RND Project, Ministry of Health and Welfare, Korea (01-PJ10-PG6-01GN13-0002). Objective: Deficiencies in mitochondrial ATP production may be associated with various cellular and chromosomal disorders. In previous study, vitrified-thawed oocytes had lower spindle and chromosome normalities than fresh oocytes. This study was subsequently conducted to examine whether level of mitochondrial ATPase 6 expression changed after vitrification. This gene was chosen because of its central role in ATP production. Design: Intact immature oocytes were collected from superovulated ICR mouse and divided into three groups: group 1, no treatment (control); group 2, vitrified oocytes with grids, group 3, vitrified oocytes with straws. In vitro matured oocytes of group 1 and oocytes that survived and matured after vitrification of group 2, 3 were used for evaluation of ATPase 6 gene expression. Materials/Methods: Immature oocytes were pre-equilibrated in DPBS with 1.5 M EG for 2.5 min. Oocytes were than vitrified with DPBS with 5.5 M EG and 1.0 M sucrose for 30 sec. Oocytes were loaded onto electron microscope copper grids or into conventional 0.25 ml straws for storing in liquid nitrogen. Stored oocytes were thawed by a five-step method. Control and survived oocytes were cultured in the medium containing gonadotropins for 16 h. Control and vitrified-thawed oocytes were compared ATPase 6 gene expression by using reverse transcription polymerase chain reaction. The band intensity of PCR products was measured by using the imaging Densitometer. Results: 1. The intensities of mitochondrial ATPase 6 gene expression in vitrified-thawed oocytes did not change compared with fresh oocytes. 2. There was no difference in the level of ATP ase 6 gene expression between oocytes vitrified with grids and straws. Conclusions: The aspects of ATPase 6 gene expression in vitrified-thawed oocytes suggest that vitrification of oocytes by using grids or straws at GV stage may have comparatively good strategy. Supported by: Korean Health 21 RND Project, Ministry of Health and Welfare, Korea (01-PJ10-PG6-01GN13-0002).

Objective: Selection of an oocyte cryopreservation method is a prerequisite factor for developing an effective bank system. In previous study, we had examined the effect of vitrification with ethylene glycol (EG)-based cryoprotectants on mouse oocytes in grid, and found higher post-thawed survival and subsequent embryo development than conventional slow freezing methods. The disadvantage of vitrification by using grids is that oocytes with the vitrification solution directly contact with liquid nitrogen during cooling or storage. To protect the risk of LN2 contamination, this study was conducted to develop effective vitrification method by using straws. Design: Intact immature oocytes were collected from superovulated ICR mouse and divided into three groups : group 1, no treatment (control) ; group 2, vitrified oocytes with grids, group 3, vitrified oocytes with straws. The morphological survival and spindle configuration were compared in mouse oocytes matured in vitro after vitrification with grids and straws at the germinal vesicle stage. Materials/Methods: Immature oocytes were pre-equilibrated in DPBS with 1.5 M EG for 2.5 min. Oocytes were then vitrified with DPBS with 5.5 M EG and 1.0 M sucrose for 30 sec. Oocytes were loaded onto electron microscope copper grids and plunged into LN2. The straws were filled with 50 ml of vitrification medium (VM), air, 50 ml of VM containing oocytes, air, and 100 ml of VM. The straws were sealed and immersed directly into LN2. Stored oocytes were thawed by a five-step method. Control and survived oocytes were cultured in TCM 199 media with 20% FBS for 16 h. In vitro matured oocytes in group 1 and oocytes that survived and matured after vitrification in group 2, 3 were used for immunostaining by using monoclonal anti-tubulin antibody and TRITC-conjugated second antibody. Results: Rates of survival were 87.3% in group 2 and 86.5% in group 3. Incidence of spindle abnormalities were 19.5%, 27.5%, and 26.0% in group1, group 2, and group 3, respectively. There was no difference in the survival and spindle abnormalities between group 2 and group 3. Conclusions: Vitrification method of oocytes using straws provides a simple, rapid and effective strategy for preventing the risk of LN2 contamination during storage. Supported by: Korean Health 21 RND Project, Ministry of Health and Welfare, Korea (01-PJ10-PG6-01GN13-0002).