RESUMEN
Objective: The aim of the present study was to examine whether lysophosphatidic acid [LPA] could decrease cell death and improve in vitro culture [IVC] conditions in cultured vitrified mouse ovarian tissue
Materials and Methods: In this experimental study, we collected and randomly divided 7-day-old mouse ovarian tissues into vitrified and non-vitrified groups. The ovaries were cultured in the presence and absence of LPA for one week. Morphology and follicular development were evaluated by hematoxylin and eosin [H and E] and Masson's trichrome [MTC] staining. The incidence of cell death was assessed by flow cytometry using annexin V/propidium iodide [PI] and a caspase-3/7 assay in all studied groups
Results: The vitrified groups had a significantly decreased follicle developmental rate compared to the non-vitrified groups [P<0.05]. Overall, qualitative and quantitative results showed prominent follicular degeneration in the vitrified groups compared with the respective non-vitrified groups. Both LPA treated groups had a significantly higher proportion of preantral follicles compared to the non-LPA treated groups [P<0.05]. groups had a significantly higher proportion of preantral follicles compared to the non-LPA treated groups [P<0.05]. Flow cytometry analysis results showed significantly greater early and late apoptotic cells in all groups [17.83 +/- 8.80%] compared to necrotic cells [7.97 +/- 0.92%, P<0.05]. The percentage of these cells significantly increased in the vitrified groups compared with non-vitrified groups. LPA treated groups had a lower percentage of these cells compared to non-LPA treated groups [P<0.05]. The lower enzyme activity was observed in non-vitrified [especially in the LPA+ groups] cultured ovaries compared to the vitrified group [P<0.05]
Conclusion: Both vitrification and IVC adversely affected cell survival and caused cell death. We postulated that LPA supplementation of culture medium could improve the developmental rate of follicles and act as an anti-cell death factor in non-vitrified and vitrified ovarian tissues. It could be used for in vitro maturation of ovarian tissue
RESUMEN
Oocyte cryopreservation is one of the most important topics in the field of assisted reproductive technology to preserve women fertility, but relationship between cryopreservation and apoptosis is still a matter of debate. The present study was aimed to investigate the effects of vitrification on apoptosis in mouse oocytes by Cryotop method. A total of 200 germinal vesicle [GV] and 200 metaphase 2 [M2] oocytes were obtained from ovaries and fallopian tubes of NMRI mice, respectively and divided into control and experimental groups. Oocytes in experimental group were vitrified by Cryotop using vitrification medium and were kept in liquid nitrogen for one month. The survival rate of oocytes was evaluated after 2 hour incubation time. Then, the oocyte apoptosis was evaluated by TUNEL technique and compared with those in control group. The data was compared statistically using SPSS software and chi-square test. The survival rates of vitrified GV [93%] and M2 oocytes [88%] showed a significant decrease compared with the control group [P<0.05], but there was no significant difference in survival rate of both vitrified oocyte groups. The incidence of apoptosis in vitrified and control GV oocytes showed no significant difference [13% vs. 7%], but the rate of apoptosis in vitrified M2 oocytes increased significantly not only in comparison with MII control group [25% vs. 5%] but also with vitrified GV oocytes [P<0.05]. The results indicate that vitrification increases apoptosis in mouse M2 oocytes and apoptosis may play a role in M2 oocyte injury after vitrification