Deficiency in a glutamine-specific methyltransferase for release factor causes mouse embryonic lethality.

Biological methylation is a fundamental enzymatic reaction for a variety of substrates in multiple cellular processes. Mammalian N6amt1 was thought to be a homologue of bacterial N(6)-adenine DNA methyltransferases, but its substrate specificity and physiological importance remain elusive. Here, we demonstrate that N6amt1 functions as a protein methyltransferase for the translation termination factor eRF1 in mammalian cells both in vitro and in vivo. Mass spectrometry analysis indicated that about 70% of the endogenous eRF1 is methylated at the glutamine residue of the conserved GGQ motif. To address the physiological significance of eRF1 methylation, we disrupted the N6amt1 gene in the mouse. Loss of N6amt1 led to early embryonic lethality. The postimplantation development of mutant embryos was impaired, resulting in degeneration around embryonic day 6.5. This is in contrast to what occurs in Escherichia coli and Saccharomyces cerevisiae, which can survive without the N6amt1 homologues. Thus, N6amt1 is the first glutamine-specific protein methyltransferase characterized in vivo in mammals and methylation of eRF1 by N6amt1 might be essential for the viability of early embryos.

Surgical embryo transfer resulted in birth of live offspring in farmed blue fox.

Finland blue fox (Alopex lagopus) has great reputation in pelt industry around the world for its large size and top-ranking fur quality; however, both the herd size and the average survival rate of purebred offspring are rather low in production systems in China. Surgical transfer of blue fox embryos was investigated as a means to increase the population fox and also as a possible means to conserve endangered canine species. The animals were chosen on the basis of synchrony in natural oestrus. During the reproductive season of blue fox, 59 embryos were flushed from 6 farmed donors 9-11 days after the first insemination, and 53 embryos were transferred surgically into the uteri of the 6 paired recipients with natural synchronized oestrous. Two of the recipients littered 46-49 days after embryo transfer; one gave birth to 7 pups and the other 1 pup. This report describes the first successful embryo transfer in the farmed blue fox in China.

Effect of in-straw thawing on in vitro- and in vivo-development of vitrified mouse morulae.

For the purpose of ascertaining parameters to embryo transfer on some domestic animals, mouse morulae were used as a model to investigate the effect of in-straw thawing on in vitro and in vivo-development of vitrified embryos. Embryos were vitrified in 0.25 ml straws preloaded with dilution solution (0.5 M Sucrose) and thawed in the straw by mixing the vitrification solution (Ethylene glycol + Ficoll 70 + Sucrose) and the dilution solution at 25 degrees C. The embryos were randomly divided into six groups and expelled from the straws after they had been suspended in the in-straw mixture for 3 min, 5 min, 8 min, 12 min, 16 min, and 20 min, respectively, and then they were collected under a microscope for in vitro culture or direct transfer. The in vitro developmental rates of the embryos were 92.3% to 98.4% and hatching rates were 64.1% to 75.6% for the groups of 3 min to 16 min, showing no significant differences with those of nonfrozen controls (100%, 76.2%; P > 0.05). While embryos were suspended in the straw for 20 min, the developmental rate (86.6%) and hatching rate (52.4%) were significant lower than those of the control (100%, 76.2%; P < 0.01). When the 168 frozen-thawed embryos (in-straw thawing for 5 min) and 168 fresh embryos were transferred, respectively, the proportion of live fetuses in the pregnant recipients between them (58.7% vs. 54.5%) showed no significant difference (P > 0.05). The data indicate that vitrification with EFS30 and suspension in the in-straw mixture for 3 min to 16 min, when thawing, did not affect the in vitro developmental rate and hatching rate. Moreover, the in vivo developmental rate between vitrified embryos and fresh embryos did not differ significantly. It can be concluded that this method is fit for nonsurgical embryo transfer in some domestic animals with a suggestion that the operation of embryo transfer should be accomplished within 16 min.

Vitrification solution containing DMSO and EG can induce parthenogenetic activation of in vitro matured ovine oocytes and decrease sperm penetration.

This study was designed to examine the reduced incidence of normal fertilization in vitrified ovine oocytes. After in vitro maturation for 24 h, the oocytes were randomly allocated into three groups: (1) untreated (control), (2) exposed to vitrification solution (VS) without being plunged into liquid nitrogen (toxicity), or (3) vitrified by open-pulled straw method (vitrification). In experiment 1, the treated and control oocytes were matured for another 2 h, and the oocytes were then in vitro fertilized for 12 h to examine sperm penetration. The percentage of monospermy in toxicity group (29.3%) and vitrification group (28.2%) dramatically decreased compared to the control group (45.0%) (P<0.05). To find the mechanism that the VS decreased the monospermy, some treated and control oocytes were used to test the distribution of CG and the resistance of zona pellucida (ZP) to 0.1% pronase E immediately (IVM 24 h), after another 2 h of maturation (IVM 26 h), and after 12 h of in vitro fertilization (IVF 12 h) respectively. Others were used to examine female pronucleus formation after 12 h of culture in fertilization medium with the absence of sperm. The results showed that the percentage of CG completely release in the oocytes (IVM 24 and 26 h) of toxicity group (41.2% and 39.9%) and vitrification group (41.7% and 51.7%) was significantly higher than that of control group (7.1% and 18.4%) (P<0.05). The ZP digestion duration in the oocytes (IVM 26 h) of the toxicity group (435.6 s) and vitrification group (422.3 s) was longer than that of control group (381.6 s) (P<0.05). The percentage of female pronucleus formation in toxicity group (58.7%) and vitrification group (63.9%) was higher than that (8.2%) of control group (P<0.05). The data above demonstrated that the VS containing DMSO and EG could parthenogenetically activate in vitro matured ovine oocytes, resulting in ZP hardening and decreased sperm penetration.

Stepwise in-straw dilution and direct transfer using open pulled straws (OPS) in the mouse: a potential model for field manipulation of vitrified embryos.

In the present study, mouse blastocysts were employed to investigate the feasibility and efficiency of stepwise in-straw dilution and direct transfer using the open pulled straw (OPS) method. In experiment I, the effects of various vitrification solutions (VS) on embryo survival were examined. After thawing, the expanded blastocyst rates (97.59 and 95.05%) and hatching rates (80.48 and 78.95%) achieved in the EDFS30 [15% ethylene glycol (EG), 15% dimethyl sulfoxide (DMSO), Ficoll, and sucrose] and EFS40 [40% EG, Ficoll, and sucrose] groups were no different from those (96.15% and 83.33%) of the control group. However, the rates in the EFS30 [30% EG, Ficoll, and sucrose] (87.80 and 55.43%) and EDFS40 [20% EG, 20% DMSO, Ficoll, and sucrose] (95.69 and 70.97%) groups were significantly lower than those (96.15 and 83.33%) of the control group (P<0.05). In the experiment II, the effects of the volume of VS in the OPS on the survival of embryos after in-straw thawing were investigated. When the length of the VS in the column was less than 1 cm, the in vitro viability of embryos thawed by stepwise in-straw dilution was no different among the experimental and control groups. The embryos could be successfully thawed by immersing the OPS in 0.5 M sucrose for 3 min and then 0.25 M sucrose for 2 min. In experiment III, the effect of immersion time of the OPS in diluent (PBS) on the viability of vitrified embryos was investigated. After in-straw thawing, OPSs were immersed immediately in 1 ml PBS for 0 to 30 min. When the immersion time of the OPSs in PBS was less than 12 min, in vitro development of the in-straw thawed embryos was no different from that of the controls. In experiment IV, in-straw thawed blastocysts were directly transferred to pseudopregnant mice to examine their in vivo developmental viability. The pregnancy (91.67%) and birth rates (42.42%) of embryos in-straw thawed and directly transferred were no different from those of the unvitrified controls (90.90 and 40%) and embryos thawed by the conventional method (84.61 and 46.94%). These results demonstrate that mouse embryos vitrified with OPS could be successfully thawed by stepwise in-straw dilution and transferred directly to a recipient and that this method might be a model for field manipulation of vitrified embryos in farm animals.

[Vitrification of mouse blastocyst by open pulled straw (OPS) method at different temperature].

To examine the efficiency of EDFS and EFS for blastocyst cryopreservation and to provide information for preparation of vitrification medium at different temperature, the present investigation was conducted to establish a model for vitrification of domestic animal's blastocyst and human blastocyst using mouse blastocyst as a model and OPS (open pulled straw) as the container. When ambient temperature was 25 degrees C and heating plate temperature was 37 degrees C, mouse blastocysts were vitrified using 1-step OPS method, after thawing, the expanded blastocyst (EB) rates of EDFS40 and EFS40 group (92.31%, 92.30%) were not different from that of the control (97.26%) (P > 0.05), however, the hatched blastocyst (HB) rate of EDFS40 group (59.62%) was significantly lower than that of the control (83.56%); When 2-step method was adopted at the same temperature, mouse blastocyst was successfully cryopreserved with EDFS30 and EFS40, the post-thawing EB rate (95.69%, 95.05%) and HB rate(80.48%, 78.95%) of both EDFS30 and EFS40 groups did not differ from those of the control (P > 0.05). When ambient temperature was 25 degrees C and heating plate was free, the EB rate (85.96%) and HB (75.44%) of embryos vitrified with EDFS40 using 1-step OPS method were not statistically different from those of the control (96.05% and 82.89%, respectively) (P > 0.05); the EB and HB rates (92.03%-95.31%, 67.19%-76.76%) of embryos vitrified with EDFS30, EDFS40 and EFS40 using 2-step OPS method were similar to those of the control (P > 0.05). According to in vitro development of vitrified embryos, the promising group was chosen for embryo transfer to examine in vivo development of vitrified embryos. When vitrified blastocysts were transferred into uterus of pseudopregnant mouse, the pregnancy rate of receipts (90.90%) and birth rate of foster mother (37.33%) were similar to those of fresh embryos (91.67% and 42.33%) (P > 0.05). All data demonstrated that mouse blastocyst was successfully cryopreserved with EDFS30, EDFS40 and EFS40 at different temperature and using different procedures.

Vitrification of mouse embryos at various stages by open-pulled straw (OPS) method.

This study was performed to pursue the optimal condition for the cryopreservation of mouse morulae by a two-step OPS method and to investigate the feasibility of the optimal condition for vitrification of embryos at other developmental stages. First, the mouse morulae were vitrified in OPS using one-step procedure-that is, embryos were vitrified after direct exposure to EDFS30 (15% ethylene glycol (EG), 15% dimethyl sulfoxide (DMSO), Ficoll and sucrose), or two-step method-that is, embryos were first pretreated in 10%E + 10%D (10% EG and 10% DMSO in mPBS) for 30 sec, then exposed to EDFS30 for 15 to 60 sec, respectively. After vitrification and warming, the embryos were morphologically evaluated and assessed by their development to blastocysts, expanded/hatched blastocysts, or to term after transfer. The result showed that all the vitrified-warmed morulae had similar blastocyst rate compared to that of control (91.7% vs. 100%), and the highest developmental rate to expanded blastocysts (100%) or hatched blastocysts (62.3%) was observed when the morulae were pretreated with 10%E + 10%D for 0.5 min, exposed to EDFS30for 25 sec before vitrification and warming in 0.5 M sucrose for 5 min. After transfer, the survival rate (33.1%) in vivo of the vitrified morulae was higher (P > 0.05) than that of the fresh embryos (24.6%). Secondly, embryos at different stages were cryopreserved and thawed following the above program. Most (93.4 to 100%) of the embryos recovered after vitrification were morphologically normal at all the developmental stages. The blastocyst rates of the vitrified one-cell (52.5 to 66.7%) and the two-cell (63.3 to 68.9%) embryos were lower (P < 0.05) than those of the vitrified four-cell embryos (81.7 to 86.4%), the eight-cell embryos (90.0 to 93.3%), morulae (96.7 to 100%), and the expanded blastocysts rate (98.3 to 100.0%) of the vitrified early blastocysts. The highest survival rate in vivo of vitrified embryos were from the early blastocysts (40.4%), which was similar to that of fresh embryos (48.6%). The data demonstrate that the optimal protocol for the cryopreservation of morulae was suitable for the four-cell embryos to early blastocyst stages and that the early blastocyst stage is the most feasible stage for mouse embryo cryopreservation under our experimental conditions.