Mesenchymal stem cells for restoration of ovarian function / 대한생식의학회지

With the progress of regenerative medicine, mesenchymal stem cells (MSCs) have received attention as a way to restore ovarian function. It has been reported that MSCs derived from bone marrow, adipose, umbilical cord blood, menstrual blood, and amniotic fluid improved ovarian function. In light of previous studies and advances in this field, there are increased expectations regarding the utilization of MSCs to restore ovarian function. This review summarizes recent research into potential applications of MSCs in women with infertility or primary ovarian insufficiency, including cases where these conditions are induced by anticancer therapy.

Effect of cell-penetrating peptide-conjugated estrogen-related receptor beta on the development of mouse embryos cultured in vitro / 대한생식의학회지

OBJECTIVE: Estrogen related receptor beta (Esrrb) is a member of the orphan nuclear receptors and may regulate the expression of pluripotency-related genes, such as Oct4 and Nanog. Therefore, in the present study, we have developed a method for delivering exogenous ESRRB recombinant protein into embryos by using cell-penetrating peptide (CPP) conjugation and have analyzed their effect on embryonic development. METHODS: Mouse oocytes and embryos were obtained from superovulated mice. The expression of Oct4 mRNA and the cell number of inner cell mass (ICM) in the in vitro-derived and in vivo-derived blastocysts were first analyzed by real time-reverse transcription-polymerase chain reaction and differential staining. Then 8-cell embryos were cultured in KSOM media with or without 2 microg/mL CPP-ESRRB protein for 24 to 48 hours, followed by checking their integration into embryos during in vitro culture by Western blot and immunocytochemistry. RESULTS: Expression of Oct4 and the cell number of ICM were lower in the in vitro-derived blastocysts than in the in vivo-derived ones (p<0.05). In the blastocysts derived from the CPP-ESRRB-treated group, expression of Oct4 was greater than in the non-treated groups (p<0.05). Although no difference in embryonic development was observed between the treated and non-treated groups, the cell number of ICM was greater in the CPP-ESRRB-treated group. CONCLUSION: Treatment of CPP-ESRRB during cultivation could increase embryos' expression of Oct4 and the formation rate of the ICM in the blastocyst. Additionally, an exogenous delivery system of CPP-conjugated protein would be a useful tool for improving embryo culture systems.

The effect of Ni2+ on the intracellular Ca2+ increase of the mouse early 2-cell embryos / 대한불임학회지

OBJECTIVE: We reported the overcoming effect of Ni2+ on the in vitro 2-cell block of mouse embryos. In this study, we aim to investigate whether Ni2+ should induce intracellular Ca2+ transient in the mouse embryos. MATERIALS AND METHODS: Embryos were collected at post hCG 32hr from the oviduct of the ICR mouse and cultured in M2 medium omitted phenol red. Intracellular Ca2+ was checked by using a confocal laser scanning microscope and fluo-3AM by using various intracellular Ca2+ antagonists. RESULTS: In 1mM Ni2+ treated medium which contained Ca2+(1.71mM), 75.7% of the embryos showed [Ca2+]i transient about 200 sec later. In the Ca2+-free medium, 69.8% of the embryos showed [Ca2+]i transient. In U73122, phospholipaseC(PLC) inhibitor (5uM, 10min) pretreated group, 33.3% of the embryos showed [Ca2+]i transient. Heparine, inositol 1,4,5-triphosphate receptor(IP3R) antagonist preinjected embryos showed no response with 1mM Ni2+. In danthrolene treatment, ryanodine receptor(RyR)-antagonist, 43% embryos showed [Ca2+]i transient but they showed delayed response about 340sec in the presence of Ca2+. CONCLUSIONS: Summing up the above results, Ni2+ seems to induce Ca2+-release from the Ca2+-store even in the Ca2+-free medium. IP3 receptors of the mouse 2-cell embryos might have an essential role for the intracellular Ca2+ increase by Ni2+.

Cryopreservation of Day 3 Mouse Embryos by Vitrification / 대한불임학회지

The use of hormonal stimulation in human in vitro fertilization and embryo transfer (IVF-ET) leads to increased production of embryos for ET. So to avoid high pregnancies and to allow conception in future, unstimulated cycles, cryopreservation of spare embryos is desirable. One of the improvement of cryopreservation methods is vitrification. We cryopreserved mouse day 3 embryos by vitrification using the three different vitrification solution (EFS40, VSll and VS3a). EFS40 solution is consisted of 40% (v/v) ethylene glycol, Ficol170 30% (w/v) and 0.5M sucrose and VSll is 6.0M ethylene glycol and 1.8M glycerol. And VS3a is 6.5M glycerol and 6% (w/v) BSA (bovine serum albumin). First we tested the toxicity of three vitrification solution by exposure to these solution during 3 min. After washing by thawing solution, the survival rates of each groups are 95.5%, 90.9% and 84.4% (EFS40, VS11 and VS3a). High percentages of them developed to expanded blastocyst and hatching embryos in culture 48hrs 94.2%, 97.7%, 100% and 97.4% (no treatment group, EFS40, VS11 and VS3a). So there is no significant differences among the each group. Second, after thawing of vitirfied embryos, the survival rates of each groups are 96.8% (slow freeze), 94.1% (EFS40), 85.5% (VS11) and 80.0% (VS3a, P vs. no freeze or EFS40 is 0.01). Vitrified embryos exhibited a high rate of development in vitro after 48hrs culture. The percentages of each group to blastocyst and hatching embryos are 88.7% (no freeze), 91.8% (slow freeze), 93.4% (EFS40), 87.7% (VS11) and 73.0% (VS3a, P vs. other group is 0.01). The results suggest that there is no significant differences in exposure of various vitrification solution and day 3 mouse embryos can be vitrified in solution EFS40 and VS11 by simple procedure.

Studies on the Participation of Prostaglandin in the Process of Hatchin gin the Mouse / 대한산부인과학회잡지

Most mammalian embryos implant on the uterine endometrium after hatching fromzona pellucida of the expanded blastocyst and pregnancy takes place. The blastocysts produceand control a variety of prostaglandins which activate a few proteolytic enzymes thatdissolve the zona pellucida of the embryos. In the present study, the goal was to investigatethe indomethacin and aspirin(inhibitors of cyclooxygenase pathway which regulates thepathway from arachidonic acid to prostaglandins), which can affect the hatching and implantationof the mouse embryos by the treatment of the different dose level of indomethacinand aspirin in the culture of mouse embryos.The female and male ICR mice, 6~8 weeks and were used for superovulation andmating and M16 was used as a basic culture medium.The above results can be summarized as following:1. Indomethacin seemed to inhibit the development of the mouse embryos and hatchingprocess because of inhibiting or blocking the activation of hatching related enzymes andhigh dose of indomethacin inhibited implantation.2. Aspirin had no effect on the hatching of the embryos at the dose of 0.16 mg/ml.3. FBS seemed to contain a factor which induced outgrowth of the embryo whereasBSA did not and outgrowth did not take place in the BSA contained medium.4. Blastocysts produced enough prostaglandins F2alpha which was needed for thehatching whereas they needed a factor for implantation which might be produced in theendometrium or exuded from the blood.In conclusion the concentration of indomethacin used in the present study inhibithatching of the blastocysts. This seems to be caused by inhibiting the synthesis of theproteins need for hatching. The factor that induce the outgrowth of the blastocysts doesnot seem to be produced in the blastocysts themselves but seem to be present in the FBS.