The investigation of UPR (Unfolding protein response) signaling cascade in oocyte and embryos before and after cryopreservation and its impact on preimplantation embryo development

PURPOSE In this thesis, we aimed to investigate the comparative impact of endoplasmic reticulum (ER) stress in normal mature oocyte and preimplantation embryo development and in differently cryopreserved oocytes and preimplantation embryos using Immunofluorescence staining and Western blot techniques at the protein level and RT-PCR technique at the RNA level.MATERIAL-METHOD We used C57BL/6 female mice for our experimental animal model. Following standard superovulation protocol and in vivo fertilization in C57BL/6 mice embryos at two cell stage were collected. Embryos were then divided into 3 groups; I. Control (n=6), II. Tunicamycin (0.5 µg/ml; n=6) and III. Cycloheximide (10 µg/ml; n=6) and incubated in vitro conditions during the preimplantation period ( from day 2 to day 4) to achieve the comparative development analysis among the groups in a day to day basis. In the second set of the experiments embryos in two cell stage were divided into three groups; I. control (n=12), II. short time exposure (4 h) of tunicamycin and III. long time exposure (72 h) of tunicamycin to investigate the role of short- and long-term effects of UPR stressors on preimplantation embryo development. Similarly, in the third set of our experiments embryos and mature oocytes (MII) from obtained in vivo conditions as described above were analyzed in three groups as I. Control (no freezing-thawing procedure), II. Vitrification and III. Slow freezing-thawing groups. Following morphological evaluation embryos and oocytes in all groups were analyzed for ER stress molecules BiP at the protein and mRNA level and XBP-1 and sXBP-1 at the mRNA level.RESULTS Embryos treated with cycloheximide were arrested at two cells stage during the preimplantation development while control and tunicamycin groups were first divided to 8-cell stage and then followed with compaction stage without significant morphological differences between these two groups. However, there was a significant difference in the number of blastocyst formed between two groups on the day of blastocyst formation (on day 4). None of tunicamycin-treated embryos could achieve blastocyst formation with a clear inner cell mass and outer cell mass while almost all embryos in the control group could reach to the blastocyst stage. Moreover, blastocyst formation rate in control, short-term and long-term tunicamycin treated groups was 100%, 33% and 0%, respectively. In RT-PCR analysis, sXBP-1 mRNA level was significantly increased with long-term tunicamycin treatment compared to short-term and control groups. Embryos and oocytes cryopreserved with both slow freezing and vitrification techniques showed higher BiP immunofluorescence staining compared to that of control embryos and oocytes. Moreover, BiP expression was increased in both protein and RNA level in slow frozen and vitrified oocytes and embryos compared to that in control oocytes and embryos as determined by Western blot and RT-PCR. Compared to XBP-1 form, sXBP1 was predominant with significantly higher level in frozen-thawed oocytes and embryos compared to that in control oocytes and embryos. Moreover, embryos from slow freezing group showed higher BiP expression compared to embryos from vitrification group.CONCLUSION In conclusion, these results suggest that activation of ER mediated UPR signaling in developing preimplantation embryos is extremely important to complete preimplantation development. Either long-term or short-term activation of UPR mediated signaling molecules prevents the embryos to progress from compacting stage to blastocyst stages suggesting that even a shot time activation of ER stress molecules could inhibit embryonic cell differentiations. Our freeze-thaw experiments also suggest that these processes could activate ER-mediated UPR signaling and in turn could have a negative impact on later stage of preimplantation embryo development.