Vitrification of primordial germ cells using whole embryos for gene-banking in loach, Misgurnus anguillicaudatus

In gene-banking, primordial germ cells (PGCs), which are embryonic precursor cells of germ cells, are useful for cryopreservation because PGCs have a potential to differentiate into both eggs and sperm via germ-line chimera. Here, we have established vitrification methods for PGCs cryopreservation using 12- to 17-somite stage embryos in loach, Misgurnus anguillicaudatus, which were dechorionated, removed their yolk and injected with green fluorescent protein (GFP) -nos1 3'UTR mRNA to visualize their PGCs. In order to optimize cryopreservation medium for vitrification, the toxicity of cryoprotectants was analyzed. Different concentrations (2, 3, 4, 5 m) of dimethyl sulfoxide (DMSO), methanol (MeOH), ethylene glycol (EG) and propylene glycol (PG) as cryoprotectants were tested. Then, 5 m DMSO showed significantly-high toxicity. Based on this information, combinations called DMP (2 m (14.2% [v/v]) DMSO, 2 m (8.1% [v/v]) MeOH and 2 m (14.4% [v/v]) PG), DP (2 m (14.2% [v/v]) DMSO and 4 m (28.7% [v/v]) PG) and DE (2.1 m (15% [v/v]) DMSO and 2.7 m (15% [v/v]) EG) were evaluated for their toxicities and efficacy of PGCs cryopreservation using two types of equilibration step: direct immersion of cryopreservation media (one-step) and serial exposure to half and full concentration of cryopreservation media (two-step). Viable PGCs were obtained from post-thaw embryos which were cryopreserved by DP and DE with both 1- and 2-step equilibrations. Despite DP showing the highest toxicity, it gave the highest survival rate of embryonic cells after cryopreservation. When PGCs recovered from vitrified embryos were transplanted into host embryos at the blastula stage, the transplanted PGCs were able to migrate to a host genital ridge similarly as endogenous PGCs. It suggests that our methods could be useful to create a germ-line chimera for the production of gametes from PGCs of cryopreserved embryos.

Effects of different cryopreservation methods on post-thaw culture conditions of in vitro produced bovine embryos

The aim of this work was to evaluate the effect of cryopreservation protocols on subsequent development of in vitro produced bovine embryos under different culture conditions. Expanded in vitro produced blastocysts (n = 600) harvested on days 7-9 were submitted to controlled freezing [slow freezing group: 10% ethylene glycol (EG) for 10 min and 1.2 degrees C/min cryopreservation]; quick-freezing [rapid freezing group: 10% EG for 10 min, 20% EG + 20% glycerol (Gly) for 30 s]; or vitrification [vitrification group: 10% EG for 10 min, 25% EG + 25% Gly for 30 s] protocols. Control group embryos were not exposed to cryoprotectant or cryopreservation protocols and the hatching rate was evaluated on day 12 post-insemination. In order to evaluate development, frozen-thawed embryos were subjected to granulosa cell co-culture in TCM199 or SOFaa for 4 days. Data were analyzed by PROC MIXED model using SAS Systems for Windows (R). Values were significant at p < 0.05. The hatching rate of the control group was 46.09%. In embryos cultured in TCM199, slow freezing and vitrification group hatching rates were 44.65 +/- 5.94% and 9.43 +/- 6.77%, respectively. In embryos cultured in SOFaa, slow freezing and vitrification groups showed hatching rates of 11.65 +/- 3.37 and 8.67 +/- 4.47%, respectively. In contrast, the rapid freezing group embryos did not hatch, regardless of culture medium. The slow freezing group showed higher hatching rates than other cryopreservation groups. Under such conditions, controlled freezing (1.2 degrees C/min) can be an alternative to cryopreservation of in vitro produced bovine embryos.

Post-thaw viability of in vivo-produced canine blastocysts cryopreserved by slow freezing

The objectives were to evaluate the reexpansion blastocoele rate, post-thaw viability, and in vitro development of canine blastocysts cryopreserved by slow freezing in 1.0 m glycerol (GLY) or 1.5 m ethylene glycol (EG). Fifty-one in vivo-produced canine blastocysts were randomly allocated in two groups: GLY (n = 26) and EG (n = 25). After thawing, embryos from MO were immediately stained with the fluorescent probes propidium iodide and Hoechst 33 342 to evaluate cellular viability. Frozen-thawed embryos from M3 and M6 were cultured in SOFaa medium + 10% FCS at 38.5 degrees C under an atmosphere of 5% CO2 with maximum humidity, for 3 and 6 days, respectively, and similarly stained. The blastocoele reexpansion rate (24 h after in vitro culture) did not differ between GLY (76.5%) and EG (68.8%). Post-thaw viable cells rate were not significantly different between GLY and EG (66.5 +/- 4.8 and 57.3 +/- 4.8, respectively, mean +/- SEM), or among MO (62.3 +/- 5.7%), M3 (56.9 +/- 6.0%), and M6 (66.5 +/- 6.0%). In conclusion, canine blastocysts cryopreserved by slow freezing in 1.0 m glycerol or 1.5 m ethylene glycol, had satisfactory blastocoele reexpansion rates, similar post-thawing viability, and remained viable for up to 6 days of in vitro culture. (C) 2012 Elsevier Inc. All rights reserved.

Oocyte meiotic-stage-specific differences in spindle depolymerization in response to temperature changes monitored with polarized field microscopy and immunocytochemistry

Objective: To compare the polymerization status of mouse oocyte spindles exposed to various temperatures at various stages of meiosis. Design: Experimental animal study. Setting: University animal laboratory. Animal(s): CF1 mice. Intervention(s): Immature oocytes matured to metaphase I (MI), telophase I (TI), and metaphase II (MII) were incubated at 37 degrees C (control), room temperature (RT), or 4 degrees C for 0, 10, 30, and 60 minutes. Spindle analysis subsequently was performed using polarized field microscopy and immunocytochemistry. Spindles of TI and MII oocytes that underwent vitrification and warming were analyzed also by immunocytochemistry. Main Outcome Measure(s): Detection of polymerized meiotic spindles. Result(s): At RT, and after 60 minutes at 4 degrees C, a significant time-dependent decrease in the percentage of polymerized meiotic spindles was observed in MI and MII oocytes, but not in TI oocytes. The polymerization of TI spindles at 4 degrees C was similar to that of TI spindles at 4 degrees C that underwent vitrification and warming. Conclusion(s): Significant differences in the microtubule dynamics of MI, TI, and MII oocytes incubated at different temperatures were observed. In particular, meiotic spindles in TI oocytes exhibited less depolymerization than did metaphase spindles. (Fertil Steril (R) 2012; 97: 714-9. (C) 2012 by American Society for Reproductive Medicine.)