Effects of the Mineral Oil Covered M2 Droplet Culture, M16 Droplet Culture, and DMSO on Release of Mouse Oocytes from Diplotene Arrest / 中国医科大学学报

Objective To study the effects of mineral oil covered M2 culture medium droplet culture, M16 droplet culture, and dimethyl sulfoxide (DMSO) on the morphology and survival rates of mouse oocytes during the release from diplotene arrest. Methods Oocytes were randomly divided into 3 groups and individually cultured for 4 h in M2 covered with mineral oil, M16 covered with mineral oil, and/or M16 only to cause germinal vesicle breakdown (GVBD). The morphological changes and survival rates of oocytes in each group were observed under the microscope. Oocytes were randomly divided into 3 groups and cultured in the medium with 0％, 1％, and 2％ DMSO. The effect of DMSO on oocytes was also observed during the release from diplotene arrest. Results The survival rates of oocytes in M2 covered with mineral oil were higher than those in M16 (P ＜ 0.05). There was no statistical difference with respect to release of mouse oocytes from diplotene arrest between the oocytes in M2 covered with mineral oil and oocytes in M16. The shape of oocytes in M2 with mineral oil was better than that of oocytes in M16. The effect of DMSO on the survival rate of oocytes was similar in the medium with 0％, 1％ and 2％DMSO. But the effect of 2％ DMSO on the release of oocytes was statistically significant (P ＜ 0.01). Conclusion During the release of mouse oocytes from diplotene arrest, oocytes in M2 covered with mineral oil have much better morphology and higher survival rate than those in M16. DMSO (0％, 1％ and 2％) has no effect on the survival rate of oocytes. However, 2％ DMSO is more effective in promoting the release of mouse oocytes from diplotene arrest.

Effect of conditioned medium of mesenchymal stem cells on the in vitro maturation and subsequent development of mouse oocyte

Mesenchymal stem cells (MSCs) secrete a variety of cytokines and growth factors in addition to self-renewal and multiple forms of differentiation. Some of these secreted bioactive factors could improve meiotic maturation in vitro and subsequent embryo developmental potential. The aim of the present study was to determine whether in vitro maturation (IVM) of mouse oocyte with or without cumulus cells could be improved by contact with conditioned medium (CM) of MSCs as well as the efficiency of CM to support follicular growth and oocyte maturation in the ovarian organ of mice cultured on soft agar. The developmental potential of matured oocyte was assessed by blastocyst formation after in vitro fertilization (IVF). Germinal vesicle stage oocytes with or without cumulus cells were subjected to IVM in either CM, Dulbecco's modified Eagle's medium (DMEM), alpha-minimum essential medium (alpha-MEM) or human tubal fluid (HTF). Approximately 120 oocytes were studied for each medium. CM produced a higher maturation rate (91.2%) than DMEM (54.7%), alpha-MEM (63.5%) and HTF (27.1%). Moreover, CM improved embryo development to blastocyst stage significantly more than DMEM and HTF (85 vs 7% and 41.7%, respectively) but there was no significant difference compared with alpha-MEM (85 vs 80.3%). The behavior of cortical granules of IVM oocytes cultured in CM revealed cytoplasmic maturation. Moreover, CM also supported preantral follicles growth well in organotypic culture on soft agar resulting in the maturation of 60% of them to developmentally competent oocytes. The production of estrogen progressively increased approximately 1-fold every other day during organ culture, while a dramatic 10-fold increase in progesterone was observed 17 h after human chorionic gonadotropin stimulus at the end of culture. Thus, CM is an effective medium for preantral follicle growth, oocyte maturation, and sequential embryo development.

Simplified EM Grid Vitrification Is a Convenient and Efficient Method for Mouse Mature Oocyte Cryopreservation

This study was performed to evaluate the efficiency of simplified EM grid vitrification, skipping the step of removing the cryoprotectant (5.5M EG + 1.0M sucrose) droplet on the grid after loading oocytes, compared to conventional cryopreservation protocols for mouse mature oocytes. Firstly, the recovery, survival, fertilization and hatching rates of simplified EM grid vitrification were compared with those of the slow freezing method using 1.5M DMSO. Then, conventional EM grid vitrification was compared with simplified EM grid vitrification. Simplified EM grid vitrification showed higher survival, fertilization and hatching rates than those of the slow freezing method (85.6% vs. 63.2%; 51.0% vs. 22.3%; 38.7% vs. 12.5%, p < 0.01, respectively). Moreover, simplified EM grid vitrification showed higher recovery, survival and fertilization rates than those of conventional EM grid vitrification (100% vs. 95.0%, p=0.024; 90.0% vs. 78.9%, p=0.033; 56.7% vs. 38.7%, p=0.021, respectively). Hatching rate tended to be higher for simplified EM grid vitrification compared to conventional EM grid vitrification (41.1% vs. 24.1%). In conclusion, simplified EM grid vitrification is a convenient and efficient method for cryopreservation of mouse mature oocytes, compared to conventional EM grid vitrification and slow freezing methods.

Development of Effective Cryopreservation Method for Mouse Oocytes / 대한불임학회지

OBJECTIVE: The purpose of this study was to evaluate the efficacy and effect of various cryopreservation method on the survival and the cytoskeletal stability of metaphase II mouse oocyte. METHODS: Mouse ovulated oocytes were collected and cryopreserved by a modified slow-freezing method with 1.5 M 1,2-propanediol (PrOH)+0.1 M sucrose or by vitrification using cryo loop and EM grid with 40% ethylene glycol+0.6 M sucrose. Four hours after thawing, intact oocytes were fixed and stained with fluorescein isothiocyanate (FITC)-conjugated monoclonal anti-beta-tubulin antibody to visualize spindle and propidium iodide (PI) to visualize chromosome. Spindle morphology was classified as follows: normal (barrel-shaped), slightly and absolute abnormal (multipolar or absent). RESULTS: Survival rate of the frozen-thawed oocytes in vitrification group was significantly higher than that of slow-freezing group (62.7% vs. 24.4%, p<0.01). Vitrification with cryo loop showed significantly higher survival rate than that with EM grid (67.7% vs. 53.5%, p<0.05). On the other hand, proportion of normal spindle and chromosome configurations of the frozen-thawed oocytes between two vitrification group was not significantly different. CONCLUSION: For mouse ovulated oocytes, vitrification with cryo loop may be a preferable procedure compared to slow-freezing method. Further study should be needed to investigate developmental competency of frozen-thawed mouse oocytes.

Effects of Melatonin on the Meiotic Maturation of Mouse Oocytes in vitro / 대한불임학회지

OBJECTIVE: Melatonin, which is secreted by pineal gland play an important role in the regulation of ovarian function via seasonal rhythm and sleep in most mammals. It also has a role in the protection of cells by removing toxic oxygen free radicals brought about by metabolism. In the present study, effects of melatonin on the mouse oocyte maturation were examined using two different culture conditions provided with 5% or 21% oxygen concentration. MATERIAL AND METHOD: Immature mouse oocytes were obtained from the ovarian follicles of 3~4 weeks old ICR strain mice intraperitoneally injected with 5 I.U. PMSG 44 hour before. Under stereomicroscope, morphologically healthy oocytes with distinct germinal vesicle (GV) were liberated from the graafian follicles and collected using mouth-controlled micropipette. They were then cultured for 17 hour at 37degrees C, 5% CO2 and 21% O2 (95% air) or 5% CO2, 5% O2 and 90% N2. New modified Hank's balanced salt solution (New MHBS) was used as a culture medium throughout the experiments. Effects of melatonin were examined at a concentration of 0.0001 micrometer, 0.01 micrometer or 1.0 micrometer. For the prevention of spontaneous maturation of immature oocytes during culture, dibutyryl cyclic AMP (dbcAMP) and/or hypoxanthine were included in the medium. RESULTS: Under 21% oxygen condition, oocytes cultured in the presence of 0.01 micrometer melatonin showed a significantly higher maturation rates, in terms of germinal vesicle breakdown (95.0% vs 89.0%) and polar body formation (88.1% vs 75.4%), compared to those cultured with 0.0001 micrometer or 1.0 micrometer melatonin. However, no difference was observed in oocytes cultured under 5% oxygen whether they were treated with melatonin or not. In the presence of 0.01 micrometer melatonin, oocytes either cultured under 21% or 5% oxygen exhibited no difference in the polar body formation (85.6% vs 86.7%). However, in the absence of melatonin, oocytes cultured under 21% oxygen exhibited lower polar body formation (74.7%). When oocytes were cultured in the presence of dbcAMP alone or with varying concentrations of melatonin, those treated with both compounds always showed better maturation, i.e., germinal vesicle breakdown and polar body formation, compared to those cultured with dbcAMP alone. At the same concentration of melatonin, however, oocytes exposed to 21% oxygen showed poor maturation than those to 5% oxygen. Similar results were obtained from the experiments using hypoxanthine instead of dbcAMP. CONCLUSION: Based upon these results, it is suggested that melatonin could enhance the meiotic maturation of mouse oocytes under 21% oxygen concentration, and release oocytes from the meiotic arrest by dbcAMP or hypoxanthine regardless of the concentration of oxygen, probably via the removal of oxygen free radicals.

Effect of Cytochalasin B on Survivability and in Vitro Development of Mouse Oocyte Frozen by Vitrification / 대한산부인과학회잡지

OBJECTIVE: The purpose of this study was to evaluate the effect of CCB treatment on the survivability and in vitro development of mouse oocyte frozen by vitrification. METHODS: Mouse oocytes retrieved from cycle stimulated by PMSG and hCG were treated by CCB and then exposed to EFS-30. These oocytes were placed onto an EM grid and submerged immediately in liquid nitrogen. Thawing of the oocytes on EM grid was carried out at room temperature for 5 seconds, then the EM grid was placed into 0.75 M sucrose at 37degrees C for 3 minutes. This was followed by 0.5 M and 0.25 M sucrose for 3 minutes, each. We compared the survivability, cleavage and developmental rate of mouse frozen by vitrification between CCB treated and non-treated groups. Chi-square was used to determine statistical significance. statistical significance was defined as p<0.05. RESULTS: Survivability (79.3%) and developmental rate into blastocyst (52.3%) of mouse oocyte were markedly decreased after vitrification. There were no significant differences between CCB treated and non- treated groups regarding survivability of oocyte frozen by vitrification (80.3% vs 78.5%). The developmental rate into 2-cell in CCB treated group was significantly higher than that in non-treated group (69.7% vs 61.9%, p<0.05). The developmental rate into blastocyst in CCB treated group was higher than that in non-treated group (54.9% vs 51.5%), but the difference was not significant. CONCLUSION: Survivability of mouse oocyte could not be affected by CCB treatment and developmental rate into 2-cell was improved in CCB treated group. It is suggested that CCB treatment prior vitrification improve stability of cytoskeleton and then improve fertilization and early stage embryo development.

Studies of Changes of Ca2+-channels Distribution in the Activated Mouse Ova / 대한불임학회지

OBJECTIVE: In muscle and neuronal cells, calcium channels have been classified by electrophysiological and pharmacological properties into (1) voltage-dependent Ca2+-channel(1) P/Q-type Ca2+-channel (2) N-type Ca2+-channel R(3) L-type Ca2+-channel (4) T-type Ca2+-channel (5)R-type Ca2+-channel. The present study was done in order to investigate whether there is any difference in Ca2+-channel distribution between activated and normally fertilized embryos. METHODS: The immunocytochemical method was used to identify the existence of voltage-dependent Ca2+-channels in parthenogenetically activated 2-cell embryos by ethanol and SrCl2 treatment. These 2-cell embryos were obtained by exposure to 6% ethanol for 6 min and to 10 mM SrCl2 for 2h. RESULTS: P/Q-type Ca2+ channels and L-type Ca2+-channels have been identified. Whereas, three type of Ca2+-channel P/Q-type, N-type, L-type have been identified in 2-cell embryos fertilized in vivo. CONCLUSION: Activation by ethanol was faster than those by SrCl2. However, there was difference in DAB staining of the embryos between ethanol and SrCl2 treatment (87.7% and 54.1%). Intensity of staining was also different between ethanol- and SrCl2-treated group. However, it has not been known why there was some difference in DAB staining and staining intensity in the present study.

The Effect of Propofol (2,6-diisopropylphenol) and Thiopental Sodium on Fertilization and Early Embryo Development in Mouse IVF Model / 대한마취과학회지

BACKGROUND: Propofol and thiopental sodium are short acting drugs and used as intravenous anesthetics for oocyte retrieval. Anesthetics administered during oocyte retrieval can pass into the follicular fluids and exert a detrimental effect on oocyte fertilizability. The aim of this study was to investigate the exposed concentration and time effect of these drugs on fertilization and early embryo development in a mouse in vitro fertilization (IVF) model. METHODS: Mouse oocytes were exposed in vitro to propofol at 0 (control), 0.09, 0.45, 2.3, 4.5microgram/ml and thiopental sodium at 0 (control), 0.2, 1, 5, 10microgram/ml for 10, 30, 60 minutes, washed, and inseminated. Thereafter, fertilization was assessed. Subsequent in vitro development to the hatched embryo was monitored daily. RESULTS: We found a concentration and time dependent toxic effect of propofol on the fertilizability of oocytes and early embryo development. The fertilization rate of mouse oocytes exposed for 30 minutes to medium containing 0.09microgram/ml of propofol was significantly lower than the control. The fertilization and hatching rate of mouse oocytes exposed for 10 minutes to medium containing 0.45microgram/ml of propofol was not lower than the control. We did not find a toxic effect of thiopental sodium on fertilization, but the hatching rate of fertilized oocytes exposed to medium containing thiopental sodium was significantly lower than the control. CONCLUSIONS: We suggest that the oocyte retrieval procedure should be done as quickly as possible in order to limit the toxic effect of these anesthetics.

The effect of fentanyl and midazolam on in vitro fertilization and early development of mouse embryo / 대한산부인과학회잡지

OBJECTIVE: To assess the effect of fentanyl and midazolam on in vitro fertilization rate and early embryo development in a mouse IVF model. METHODS: Mouse oocytes were exposed in vitro to fentanyl at a concentration of 0(control), 50, 250, 500, 1000, 5000 pg/ml, and midazolam, 0(control), 2.5, 12.5, 25, 50, 250 ng/ml for 30 minutes, washed and inseminated. Thereafter fertilization was assessed. And subsequent in vitro development to the blastocyst stage was monitored daily. RESULTS: Where fertilization occurred, subsequent embryo cleavage and development up to the blastocyst stage was affected significantly by the presence of fentanyl and midazolam solution in the medium(i.e., 14% to 31%, 10% to 35%), in comparison with control group( 60%, 62%). CONCLUSION: It can be concluded from these experiments that even a brief exposure of cumulus enclosed oocytes to a low concentration of fentanyl, midazolam is deleterious to subsequent cleavage.

Effects of Nitric Oxide on the Maturation of Mouse Oocyte in vitro / 대한산부인과학회잡지

OBJECTIVE: Nitric oxide (NO) produced in ovary may contribute to follicle maturation, ovulation, oocyte maturation and luteinization. In this study, the effect of nitric oxide on the spontaneous maturation of mouse oocyte was observed. Method: The index of oocyte maturation was checked by the germinal vesicle breakdown (GVBD) and appearance of polar body (PB) under microscope in the denuded oocytes and oocyte-cumulus complexes (OCCs) from mouse ovarian follicles after 24 hours pregnant-mare serum gonadotropin treatment. RESULTS: The GVBD appeared 50 %, 1 hour and 80 %, 2 hrs after changes of oocytes from dibutyryl cAMP (dbcAMP, 0.5 mM) contained media into dbcAMP-free media. dbcAMP (0.5 mM) completely blocked the GVBD until 24 hrs but dbcGMP (5 mM) delayed the GVBD by 1 hr. Sodium nitroprusside, the NO generator, inhibited the GVBD dose-dependently at 2 hr incubation in denuded and OCCs. The appearance of GVBD was not different between control and dbcGMP or SNP in denuded oocytes and OCCs at 24 hrs incubation. The guanylate cyclase activity in denuded oocyte cytosol was not detected whereas the guanylate cyclase activity in OCCs cytosol was 1.3 nmole/min/mg protein which was increased about 3 times by SNP (100 micrometer). CONCLUSION: These results suggest that the NO in ovary may delay the spontaneous oocyte maturation in early stage by acting on the maturation signaling protein as well as guanylate cyclase.