Viability of frozen-thawed mouse embryos is affected by genotype

Embryos from mice of five different genotypes were evaluated for their ability to survive cryopreservation as measured by post-thaw in vitro development. In Study 1, ovulation was induced with a standardized pregnant mares' serum gonadotropin (PMSG)/human chorionic gonadotropin (hCG) regimen, after which females were mated with males of the same genotype to produce incrossed embryos. Four- to 8-cell embryos were frozen in 1.5 M dimethyl sulfoxide (DMSO) at a rate of 0.5 degrees C/min to -80 degrees C and stored in liquid nitrogen. Following thawing at room temperature, embryos were cultured and development was evaluated 24 h later. The mean (+/- SEM) number of 4- to 8-cell embryos/pregnant female by stock/strain were: N:NIH(S), 6.8 +/- 0.8; N:NIH(S)-B, 5.8 +/- 0.5; N:GP(S), 6.5 +/- 0.6; C57BL/6N, 9.7 +/- 1.0; C3H/HeN MTV-, 9.5 +/- 0.9 (P less than 0.05). Post-thaw in vitro development was related to genetic background; the proportion of embryos culturing after thawing was: N:NIH(S), 49%; N:NIH(S)-B, 61%; N:GP(S), 66%; C57BL/6N, 75%; C3H/HeN MTV-, 56% (P less than 0.05). Study 2 was conducted to evaluate the influence of mating various females to males of a genotype known to have a lower post-thaw embryo survival rate. N:NIH(S)-B, N:GP(S), C57BL/6N, and C3H/HeN MTV- female mice were mated with N:NIH(S) males to produce hybrid embryos. Post-thaw embryo survival was reduced (P less than 0.05) in three of the four hybrid groups. Fresh incrossed and hybrid embryos from each study were cultured for 24 h and yielded culture rates ranging from 95% to 99% (P greater than 0.05) among all groups.(ABSTRACT TRUNCATED AT 250 WORDS)     

Factors affecting the survival of frozen-thawed mouse spermatozoa

Mouse epididymal spermatozoa were frozen in solutions containing various compounds with different molecular weights, and the factors affecting the postthawing survival were examined. Monosaccharides (glucose, galactose) had almost no protective effect regardless of the concentration and the temperature of exposure. On the other hand, disaccharides (sucrose, trehalose) and trisaccharides (raffinose, melezitose) resulted in higher survival rates, especially at a concentration of around 0.35 mol/kg H(2)O (0.381-0.412 Osm/kg). Macromolecules, such as PVP10, Ficoll 70, bovine serum albumin, and skim milk had almost no effect, but compounds with a molecular weight of about 800, such as metrizamide and Nycodenz, had some protective effect. When a raffinose solution was supplemented with 10% metrizamide, resulting in an osmolality of approximately 0.400 Osm/kg, a high survival rate was obtained. Solutions at about 0.400 Osm/kg containing trehalose alone, trehalose + metrizamide, raffinose alone, and raffinose + metrizamide, were all effective for sperm freezing; frozen-thawed sperm could fertilize oocytes, and the resultant embryos could develop to live young after transfer. For freezing mouse spermatozoa, aqueous solutions at approximately 0.400 Osm/kg containing a disaccharide or a trisaccharide seem to be effective.     

Effect of cryoprotectant concentration, equilibration time and thawing procedure on survival and development of rapid frozen-thawed mature mouse oocytes

Experiments were conducted to develop a simple rapid-freezing protocol for mature mouse oocytes that would yield a high proportion of oocytes with developmental potential. The effects of concentration (3.5, 4.5 and 6.0 M dimethyl sulfoxide (DMSO) all with 0.5 M sucrose) and the duration of exposure (2.5 min vs 45 sec) of oocytes to the cryoprotectant and its extraction after thawing in 2, 3 or 4 steps of descending sucrose concentration were studied. The most effective of the rapid-freezing and thawing protocols (4.5 M DMSO; 45 sec exposure and 3-step thawing) was compared to slow freezing protocols using 1.5 M DMSO and 1.0 M 1,2 propanediol as cryoprotectants. The DMSO concentrations had an effect on survival, fertilization and embryo development using short (45 sec) but not long (2.5 min) exposure. The rate of morphological oocyte survival was significantly higher using 4.5 M DMSO than 3.5 or 6.0 M (92% vs 82 and 73%, respectively). The development of fertilized embryos to blastocysts was also significantly higher at 4.5 M than at 3.5 or 6.0 M (68% vs 42 and 53%, respectively). The extraction of cryoprotectant in 3 or 4 steps of descending sucrose concentration resulted in higher survival (P < 0.01) and fertilization than in 2 steps. The best survival, fertilization and development was achieved with the 3-step procedure. Optimal combinations of conditions were 4.5 M DMSO at 45 sec prefreeze exposure and 3-step extraction of the cryoprotectant. Oocytes frozen by conventional methods had a survival, fertilization and development to blastocyst rate significantly lower than those frozen under the optimal rapid conditions. Thus rapid freezing of mature mouse oocytes with 4.5 M DMSO + 0.5 M sucrose and short prefreeze exposure is effective and has the additional advantage of being less time-consuming than slow freezing methods.     

Involvement of insulin in early development of mouse one-cell stage embryos

Recent studies have suggested that growth factors and hormones play important roles in cell prolif-eration and differentiation during early embryonic development. In the present study, we examined the expression and localization of insulin in the mouse oocytes and one-cell stage embryos by quantitative ELISA, RT-PCR, Western blot and immunofluorescence. In the mouse oocytes and one-cell stage em-bryos, expression of insulin was uniformly distributed in the cytoplasm. We also examined the expres-sion, activity and localization of mTOR (mammalian target of rapamycin) and p70S6K. The expression of mTOR and p70S6K was not significantly different at the cell cycle of mouse one-cell stage embryos. mTOR and S6K were distributed evenly in the cytoplasm at G1, G2 and M phase phase, but at S phase, the distribution of mTOR and S6K was around the pronucleus. At different phases, the activity of mTOR fluctuated. We also used the PI3K specific inhibitor-Wortmannin to investigate the cleavage rate of eggs. The result showed that the rate obviously decreased. When the mTOR specific inhibitor Rapa-mycin was used, the first mitotic division of the mouse one-cell stage embryo was delayed. These re-sults suggested that insulin was expressed both in mouse oocytes and one-cell stage embryos, and may play functional roles in regulation of mouse early embryogenesis by activating the signal pathway of PI3K/PKB/mTOR/S6K.     

Effect of one-step sucrose dilution of glycerol on in-vitro development of frozen-thawed mouse embyros

Several glycerol (GLY) dilution treatments were compared using frozen-thawed early blastocysts from Swiss Webster mice. These treatments consisted of 0.00 (0.00S n = 68), 0.25 (0.25S n = 67), 0.50 (0.50S n = 76), 0.75 (0.75S n = 66), 1.00 (1.00S n = 59), and 1.25 (1.25S n = 60) M of sucrose to remove GLY from embryos in one step. Then the one step procedure was compared with a three-step GLY dilution treatment (C n = 66). Embryos were exposed to 1.5 M of GLY in three-steps, frozen according to a standard freezing technique and stored at -196 degrees C. Embryos were thawed in a 37 degrees C water bath, pooled, and those graded good or excellent were randomly assigned to the experimental groups. The blastocysts were cultured in Whitten's medium microdrops under paraffin oil in a water saturated 5% CO(2) air atmosphere at 37 degrees C. The proportion (%) of embryos developing to expanded blastocysts was lowest (P < 0.05) in treatment 0.00S (63.1 +/- 4.0). The 0.25S (72.0 +/- 4.3) and 0.50S (75.0 +/- 3.1) 0.75S (79.0 +/- 4.4) treatments yielded a similar percentage of expanded blastocysts. The 1.00S treatment (87.0 +/- 4.0) was similar to 0.75S and 1.25S (98.3 +/- 4.0) treatments. The C treatment was superior (P < 0.05) to dilutions done with < 0.75 M sucrose, similar to 0.75S and 1.00S, and inferior (P < 0.05) to 1.25S. This later treatment yielded the highest percentage of expanded blastocysts. The percentage of embryos that hatched in treatments 0.00S, 0.25S, 0.50S, 0.75S and C was lower (P < 0.05) compared to 1.00S and 1.25S. The percentage of embryos and hatched blastocysts increased linearly (P < 0.01) from 0.0 to 1.25 M sucrose. Dilution of GLY with 1 or 1.25 M sucrose yielded better results compared with lower sucrose concentrations or the three-step GLY removal procedure.     

Effect of long-term selection for early postnatal growth rate on survival and prenatal development of transferred mouse embryos

Reciprocal embryo transfer procedures were performed among mouse selection lines to examine prenatal maternal effects on survival and development of transferred embryos. Mice were from generations 28 and 29 of an experiment to select for (i) increased body weight again from 0 to 10 days (E+); (ii) decreased body weight gain from 0 to 10 days (E-); or (iii) a randomly bred control line (C). A total of 118 embryo transfer procedures performed 12 h after conception resulted in 983 progeny born to 89 litters. There was a 39% overall embryo survival rate and 75% overall pregnancy success rate. Response to superovulation and oestrous synchronization was significantly lower (P < 0.01) in the E+ line. E+ individuals that did superovulate produced an average of 37 oocytes per flush, which was significantly higher than in the control line mice (29 oocytes per flush; P < 0.01). The ability to complete pregnancy successfully was not influenced by uterine environment or embryo-uterine interaction. In contrast, embryo survival in successful pregnancies was significantly affected by uterine environment. There were large maternal effects for body weight and tail length at birth; E+ recipients produced pups that were significantly larger than E- recipient pups (P < 0.01), which in turn were significantly larger than pups gestated by control recipients (P < 0.01).     

Effect of superoxide dismutase on the development of preimplantation mouse embryos

The role of superoxide dismutase (SOD) was tested on preimplantation development of mouse embryos in vitro. The presence of SOD in ovarian antral follicles and in oviductal and uterine secretions was also investigated. Zygotes from superovulated ICR female mice were cultured in modified Whittingham's T6 medium supplemented with SOD (0 to 370 U) or EDTA (100 muM) at 37 degrees C under 5% CO(2) in air. Supplementation of SOD (370 U) significantly promoted the development of zygotes to the blastocyst stage (45%) as compared to that of the controls (1.4%). This favorable effect of SOD was comparable to that of EDTA and completely suppressed by anti-SOD antibody. Blastocysts cultured with SOD consisted of 78.2+/-10.4 blastomeres and possessed as many blastomeres as those (81.6+/-9.3) developing in vivo; blastocysts cultured with EDTA had significantly fewer blastomeres (42.6+/-13.7). These findings suggest that SOD protects embryos against oxidative insults and that it can be an effective substitute for EDTA for supporting mouse embryo development in vitro. The SOD activity was detected in 3 different lumina from mouse reproductive organs, and SOD was identified as a cytosolic Cu,Zn-SOD on photochemically stained polyacrylamide gels. Our results suggest that oxidative injury may be responsible for developmental retardation of preimplantation-stage mouse embryos in vitro and that Cu,Zn-SOD may play a crucial role in protecting embryos against oxygen toxicity in vivo as well as in vitro.     

Whole-embryo culture of E5.5 mouse embryos: development to the gastrulation stage

This study reports establishment of an in vitro culture system for E5.5 mouse embryos that supports development to the gastrulation stage and allows the use of experimental approaches to study gastrulation during mouse embryogenesis. Recent experiments suggest that the extraembryonic tissues may play a critical role for gastrulation from as early as E5.5. To apply whole embryo culture to E5.5 embryos and analyze gastrulation, it is essential to optimize the conditions so that most of the embryos develop to the gastrulation stage in culture. For this purpose, we established a protocol in which embryos were isolated using micromanipulator and cultured with 50-75% rat serum. Although cultured embryos tended to grow a larger extraembryonic portion, more than 80% of them developed the primitive streak and induce mesoderm, which corresponds to the mid-streak stage.     

Effect of culture media on in vitro development of cloned mouse embryos

The effect of simple and sequential embryo culture media on the preimplantation development of mouse nuclear transfer (NT) embryos reconstructed with cumulus cell nuclei using a mechanical NT technique was studied. Blastocyst formation rate was evaluated using CZB medium and the sequential media G1/G2 and KSOM/G2. Arrested two- and three-cell NT embryos were Hoechst-stained to check for nuclear abnormalities. Nonmanipulated and sham-manipulated parthenogenetic embryos served as controls for, respectively, the medium and the handling technique. Rates of blastocyst formation for medium and handling control embryos were similar in CZB (58% and 61%), in G1/G2 (94% and 85%), and in KSOM/G2 (88% and 84%). Development of NT embryos was significantly impaired from the two-cell stage onwards, reaching the blastocyst stage at a rate of 5% in CZB, 14% in G1/G2, and 28% in KSOM/G2. Arrested two- and three-cell stage NT embryos showed a high rate of binucleation. These data demonstrate not only that NT embryos are more sensitive to in vitro culture conditions than parthenogenetic control embryos but also that selection of culture media can influence the preimplantation development of NT embryos.     

Effects of equilibration time, precooling and developmental stage on the survival of mouse embryos cryopreserved by vitrification

Factorial experiments were carried out to examine the effects of equilibration time, precooling and developmental stage on the postthaw in vitro survival of vitrified mouse embryos. Eight-cell embryos, compacted morulae, or blastocysts were cryopreserved using vitrification Solution 1 (VS1; 10% glycerol + 20% propylene glycol), and vitrification Solution 2 (VS2; 25% glycerol + 25% propylene glycol) in phosphate buffered saline + 10% calf serum. Each embryo stage group was first equilibrated in VS1 for 5, 10 or 20 min and then exposed to either a precooled ( approximately 4 degrees C) or nonprecooled ( approximately 20 degrees C) VS2 in a 0.25-ml straw before they were plunged directly into liquid nitrogen. Results of this study showed an interaction between precooling, equilibration time and developmental stage which affect significantly post-thaw embryo survival (P< 0.05). High survival rates were obtained after 10 min equilibration in VS1 irrespective of the embryo developmental stage. Precooling of the VS2 significantly improved the survival mainly of blastocysts. However, eight-cell and morula-stage embryos also showed high survival rates when they were exposed to precooled VS2 after 5 min equilibration in VS1. It was further observed that morulae usually exhibit high survival rates, and vitrification conditions are more critical for early and advanced stage embryo development.     

Influence of cell cycle stage at nuclear transplantation on the development in vitro of mouse embryos

Nuclei were transplanted from embryos of mice at different stages of the 1st and 2nd cell cycle to oocytes enucleated at various times after fertilization. After transfer of pronuclei, a greater proportion of embryos developed to blastocysts if donor and recipient embryos were at the same stage of the cell cycle (synchronous transfer = 94%, asynchronous transfer = 76%). By contrast, when 2-cell blastomere nuclei were fused to the cytoplasm of enucleated zygotes, there was a significant effect of both cytoplast and karyoplast cell cycle stage on the development of the reconstituted embryos. Karyoplasts and cytoplasts derived from embryos at later stages of the cell cycle had greater potential to support development to blastocysts in vitro. It is suggested that the secretion of stage-specific messengers and the timing of nuclear membrane breakdown are the main factors causing the karyoplast and cytoplast effects, respectively.     

Osmotic consequences of cryoprotectant permeability and its relation to the survival of frozen-thawed embryos

The freezing of a living cell involves a complex physicochemical process of heat and water transport between the cell and its surrounding medium. Embryos survive cryopreservation only in the presence of a cryoprotectant in concentrations between 1 and 2M. During the addition and dilution of a permeating cryoprotectant, the cell undergoes osmotic changes in cell size. As a consequence, if the addition or particularly the dilution are carried out inappropriately, the viability of cells can be affected. Equations which model the influx and efflux of cryoprotectants in cells can be used to calculate the optimum and most practical addition and removal method. However, the equations require the permeability coefficient of the cryoprotectant, a quantity that has only experimentally determined for a few of the developmental stages of two species.