ABSTRACT
Background: The objective of this study was determination of the changes in the reactive oxygen species [ROS] level, mitochondrial DNA [mtDNA] copy number and enzyme activity and transcription factor A [TFAM] gene expression in oocytes after vitrification
Methods: The oocytes at metaphase II [MII] stage [n=320] were collected from superovulated adult female mice [n=40]. These oocytes were divided into vitrified and non-vitrified groups [n=160 in each group]. After vitrification of oocytes, ROS level, mtDNA copy number; TFAM gene expression and mitochondrial enzymes activity [cytochrome C oxidase and succinate dehydrogenase] were assessed and compared with non-vitrified group. Visualization of the mitochondria was done using Mitotracker green staining under confocal microscope. Data were compared by independent T-test. Values of p<0.05 were considered as statistically significant
Results: The survival rate of oocytes after vitrification and warming was 96.05%. The intensity of cytochrome C oxidase activity, mtDNA copy number and TFAM gene expression in non-vitrified oocytes were significantly lower and the level of ROS was higher in vitrified oocytes in comparison with non-vitrified group [p<0.05]. But the intensity of succinate dehydrogenase activity was not significantly different between the two groups. The pattern of mitochondrial distribution in two groups of study was similar but the intensity of Mitotracker green in non-vitrified oocytes was significantly higher than vitrified oocytes [p<0.05]
Conclusion: This study showed that vitrification of mouse MII oocytes reduced the mtDNA copy number and mitochondrial cytochrome C oxidase activity by increasing ROS level, thus the subsequent embryo development may be affected
ABSTRACT
Objective: This study aimed to evaluate the expression of the genes related to folliculo-genesis after vitrification of mouse ovarian tissues using a two-step in vitro culture
Materials and Methods: In this experimental study, vitrified and non-vitrified ovaries from 7- day old [neonate] female mice were cultured using alpha-Minimum Essential Medium [alpha-MEM] supplemented with 5% fetal bovine serum [FBS] for 7 days. Morphology, surface area of ovaries and percentage of normal follicles were evaluated and compared in both groups. After one-week culture, in non-vitrified group, preantral follicles of cultured ovaries were isolated and cultured in a three-dimensional alginate culture system for 12 days. Then, the collected metaphase [M] II oocytes were inseminated with capacitated spermatozoa derived from 7-8-week old [adult] male NMRI mice. Follicular diameter, oocyte maturation, fertilization, embryo development and the expression of genes related to follicular development [Pcna, Fshr and Cyp17a1,] using real time reverse transcription-polymerase chain reaction [RT-PCR] were assessed at the end of last culture period in both groups
Results: The ovarian area in vitrified group [162468.20 703.78] was less than non-vitrified group [297211.40 6671.71], while the percentage of preantral follicles in vitrified group [18.40%] was significantly lower than those of non-vitrified group [24.50%] on day 7 of culture [P<0.05]. There were no significant differences between the two groups in terms of follicular diameter, expression of genes related to development of follicles, oocyte maturation, fertilization, as well as embryo development [P>0.05]
Conclusion: The results of this study showed that vitrification of ovarian tissue following in vitro culture had negative impact on the survival and development of follicles within the tissue. However, no significant alterations were observed in development, gene expression and hormonal production of in vitro culture of isolated follicles derived from vitrified ovarian tissues as compared to the non-vitrified samples
ABSTRACT
Objective: The present study compared the efficiency of three mouse ovarian tissue culture methods - hanging drop, under mineral oil, and on the insert with regards to improving in vitro ovarian follicular development
Methods: Ovaries from 7-day-old old female NMRI mice sacrificed by cervical dislocation were collected and cultured in alpha-MEM medium supplemented with 5% fetal bovine serum for 7 days in 3 groups [hanging drop, under mineral oil and on the insert]. We evaluated and compared the morphology and surface area of the ovaries and percentage of normal follicles in all groups. After the 7-day culture, the ovaries cultured on the insert showed better growth compared to the other groups. Their preantral follicles were isolated and cultured for 12 days. We assessed the follicular diameter, survival and maturation rate of these oocytes at the end of the last culture period
Results: The percentages of normal follicles in cultured ovaries were 73.85 +/- 2.49% [insert], 51.63 +/- 3.93% [hanging drop], and 40.52 +/- 5.86% [mineral oil] after the 7-day culture. The percentage of preantral follicles significantly increased from 2.1 +/- 0.44 to 24.5 +/- 2.4 in the group cultured on insert [P<0.05], however there was no significant difference in the other groups [P>0.05]. There were significantly increased surface areas of the ovarian tissues after the 7-day culture in all groups [P<0.05]. Ovaries cultured on the insert had a diameter of isolated follicles after 12 days of culture of 410 +/- 7.07 microm and the MII rate was 30.26%
Conclusion: The ovarian growth and morphology were well preserved in tissues cultured on the insert compared to the other culture methods
ABSTRACT
The mitochondria are an important source of adenosine triphosphate [ATP] production in pre-implantation embryo. Therefore, the objective of this study was to investigate the effect of vitrification and in vitro culture of mouse embryos on their mitochondrial distribution and ATP content. The embryos at 2-PN, 4-cell and blastocyst stages were collected from the oviduct of stimulated pregnant mice and uterine horns. Then, the embryos were vitrified with the cryotop method using ethylene glycol and dimethylsulphoxide. After evaluating the survival rates of vitrified embryos, their development to hatching stages were assessed. The ATP content of collected in vivo and in vitro embryos at different stages was measured by luciferin-luciferase bioluminescence assay. The distribution of mitochondria was studied using Mito-tracker green staining under a fluorescent microscope. The survival rates of vitrified embryos at 2-PN, 4-cell and early blastocyst stages were 84.3, 87.87 and 89.89%, respectively. The hatching rates in previous developmental stages in vitrified group were 57.44, 66.73 and 70.89% and in non-vitrified group were 66.32, 73.25 and 75.89%, respectively [P>0.05] The ATP content of in vivo or in vitro collected embryos was not significantly different in both vitrified and non-vitrified groups [P>0.05]. Mitochondrial distribution of vitrified and non-vitrified 2-PN embryos was similar, but some clampings or large aggregation of mitochondria within the vitrified 4-cell embryos was prominent. Vitrification method did not affect the mouse embryo ATP content. Also, the cellular stress was not induced by this procedure and the safety of vitrification was shown