No abstract

The purpose of the present study was to evaluate the cryogenic effect of antifreeze protein (AFP) on transgenic mouse ovaries which is expressed AFP type III from Ocean pout and the production of live offspring by orthotopic transplantation of cryopreserved mouse ovaries. In this study, whole transgenic and nontransgenic mouse ovaries were vitrified with 20% DMSO and 20% EG in M2 medium supplemented with 0.5 M sucrose. All vitrified and toxicity control and fresh ovaries were transplanted orthotopically into ovariectomized recipients bilaterally. For fresh ovaries transplantation, 5 mice delivered litters of 18 and 19 live pups in first and second matings, respectively. For toxicity control of chemicals, 6 mice delivered litters of 22 and 23 live pups. For nontransgenic mouse ovaries (vitrified) transplantation, 7 mice delivered litters of 22 and 23 live pups. For transgenic mouse ovaries (vitrified) transplantation, 10 mice delivered litters of 35 and 37 live pups. Litter sizes from pups of freshly transplanted ovaries were not significantly different from AFP-transplanted transgenic ovaries but those from nontransgenic-transplanted ovaries were significantly different from the AFP-transplanted transgenic ovaries group (P < 0.05). In this study, for the first time, it was shown that the ovarian tissue of AFP transgenic mice was protected from cryopreservation by vitrification. These results demonstrate that a normal reproductive lifespan can be restored by orthotopic transplantation of AFP transgenic-vitrified ovary.

This study was conducted to determine the additive effects of exogenous growth factors during in vitro oocyte maturation (IVM) and the sequential culture of nuclear transfer (NT) embryos. Oocyte maturation and culture of reconstructed embryos derived from bovine granulosa cells were performed in culture medium supplemented with either epidermal growth factor (EGF) alone or a combination of EGF with insulin-like growth factor-I (IGF-I). The maturation rates of oocytes matured in the presence of EGF or the EGF + IGF-I combination were significantly higher than those of oocytes matured in the presence of only fetal calf serum (FCS) (P 0.05). IGF-I alone or in combination with EGF in sequential embryo culture medium significantly increased the ratio of inner cell mass (ICM) to total blastocyst cells (P < 0.05). Our results showed that the addition of growth factors to IVM and sequential culture media of cloned bovine embryos increased the ICM without changing the total cell number. These unknown and uncontrolled effects of growth factors can alter the allocation of ICM and trophectoderm cells (TE) in NT embryos. A decrease in TE cell numbers could be a reason for developmental abnormalities in embryos in the cloning system.

The cryopreservation of pronuclear‐stage embryos has particular importance in transgenic technology and human assisted reproductive technology (ART). The objective of this study was to improve the efficiency of cryopreservation of pronuclear‐stage mouse embryos. Two vitrification methods (solid surface vitrification (SSV) vs. vitrification in cryotube) have been compared with special emphasis on the effect of the exposure of the embryos to the solutions for various times and the sugar content (trehalose, sucrose, or raffinose) of the vitrification solutions. Pronuclear‐stage embryos were either exposed to 1 M dimethyl sulfoxide (DMSO) + 1 M propylene‐glycol (PG) solution for 2, 5, 10, or 15 min or not exposed to this “equilibration” solution. The vitrification solutions consisted of 2.75 M DMSO and 2.75 M PG in M2 medium supplemented with 1 M trehalose (DPT), 1 M sucrose (DPS), or 1 M raffinose (DPR). In the cryotube method, groups of 15–25 embryos were transferred into a 1.8 ml cryotube containing 30 μl of DPT, DPS, or DPR. After 30 sec, the cryotubes were directly plunged into liquid nitrogen (LN2) and stored for 1 day to 1 month. Vitrified samples were warmed by immersing the cryotubes in a 40°C water bath and then immediately diluted with 300 μl of 0.3 M trehalose, sucrose, or raffinose in M2. In the SSV method, after equilibration 15–20 embryos were exposed to DPT, DPS, or DPR solutions for around 20 sec before being dropped in 2‐μl drops onto a pre‐cooled (−150 to −180°C) metal surface. Vitrified droplets were stored in cryovials in LN2. Warming was performed by transferring the vitrified droplets into 0.3 M solutions of trehalose, sucrose, or raffinose at 37°C, respectively. Results showed that both SSV and cryotube vitrification methods can result in high rates of in vitro blastocyst development (up to 58.3 and 68.5% with DPR, respectively), not statistically different from that of the controls (58.3 and 64.4%). Even without the equilibration step prior to vitrification, relatively high‐survival rates have been achieved, except for the DPS solution. In conclusion, vitrification of pronuclear‐stage mouse embryos can result in high rates of in vitro development to blastocyst, and the use of raffinose in the vitrification solution is advantageous to improve cryosurvival. Mol. Reprod. Dev. 67:186–192, 2004. © 2004 Wiley‐Liss, Inc.

Cryopreservation of pronuclear-stage embryos would be useful for transgenic technology and genome preservation purposes. We compared a novel vitrification technique (solid surface vitrification, SSV) with another vitrification method in straws for cryosurvival and to generate transgenic progeny from cryopreserved mouse zygotes following microinjection. The SSV solution consisted of 35% ethylene glycol (EG), 5% polyvinyl-pyrrolidone (PVP), and 0.4 M trehalose in M2 supplemented with 4 mg/ml BSA; the in straw vitrification solution was 7 M EG in M2 plus BSA. In experiment I, we compared the effect of the vitrification solutions alone, without cooling. No reduction was detected in survival and cleavage rates. In experiment II, SSV yielded a significantly higher percentage of morphologically normal zygotes (96%) that also cleaved at significantly higher rates (80%) when compared to that following "in straw" vitrification (68 and 66%, respectively). Cleavage rate in the non-vitrified control group (93%) was significantly higher than that of both vitrified groups. Following embryo transfer, there was no difference in the rate of pups obtained from the SSV, "in straw" vitrified, and control groups (97/457, 21%; 15/75, 20% and 56/209, 27%, respectively). In experiment III, SSV vitrified and fresh embryos were used for pronuclear DNA injection. Survival rate of vitrified embryos after microinjection was reduced compared to nonvitrified ones (64 vs. 72%, respectively; P < 0.05); however, development to two-cell stage was not different (76 vs. 72%, respectively). Following embryo transfer of vitrified vs. fresh microinjected embryos, in both cases 10% live pups were generated, including transgenic pups. The results demonstrated that the efficiency of generating transgenic pups from SSV vitrified pronuclear zygotes is comparable to that from fresh embryos.

Vitrification is becoming a preferred method for pre-implantation embryo cryopreservation. The objective of this study was to determine the differentially expressed genes of in vivo- and in vitro-produced bovine embryos after vitrification. In vitro- (IVF) and in vivo-derived (IVV) bovine blastocysts were identified as follows: in vitro-produced fresh (IVF-F), in vitro-produced vitrified (IVF-V), in vivo-derived fresh (IVV-F), in vivo-derived vitrified (IVV-V). The microarray results showed that 53 genes were differentially regulated between IVF and IVV, and 121 genes were differentially regulated between fresh and vitrified blastocysts (P < 0.05). There were 6, 268, 962, and 17 differentially regulated genes between IVF-F × IVV-F, IVF-V × IVV-V, IVF-F × IVF-V, and IVV-F × IVV-V, respectively (P < 0.05). While gene expression was significantly different between fresh and vitrified IVF blastocysts (P < 0.05), it was similar between fresh and vitrified IVV blastocysts. Significantly up-regulated KEGG pathways included ribosome, oxidative phosphorylation, spliceosome, and oocyte meiosis in the fresh IVF blastocyst samples, while sphingolipid and purine metabolisms were up-regulated in the vitrified IVF blastocyst. The results showed that in vitro bovine blastocyst production protocols used in this study caused no major gene expression differences compared to those of in vivo-produced blastocysts. After vitrification, however, in vitro-produced blastocysts showed major gene expression differences compared to in vivo blastocysts. This study suggests that in vitro-produced embryos are of comparable quality to their in vivo counterparts. Vitrification of in vitro blastocysts, on the other hand, causes significant up-regulation of genes that are involved in stress responses.

The aim of this study was to clone native Anatolian Grey cattle by using different donor cell types, such as fibroblast, cartilage and granulosa cells cryopreserved in a gene bank and oocytes aspirated from ovaries of Holstein cows as the recipient cytoplasm source. One male calf from fibroblast, three female calves from granulosa cells and one female calf from cartilage cells were born healthy and at normal birthweights. No calves were lost after birth. The results demonstrated that the cloned calves had the same microsatellite alleles at 11 loci as their nuclear donors. However, the mtDNAs of the five Anatolian Grey cloned calves had different haplotypes from their donor cells and mtDNA heteroplasmy could not be detected in any of the clones. The birth of healthy clones suggests that the haplotype difference between the cell and oocyte donor did not affect the pre- or post-implantation development of the bovine nuclear transfer derived embryos in our study. The results showed that well established nuclear transfer protocols could be useful in conserving endangered species. In conclusion, somatic cell banking can be suggested as a tool in conservation programmes of animal genetic resources.

Embryo transfer is a crucial step in completing the procedures of embryo cryopreservation, in vitro fertilization and transgenic animal production. The success of ET is in uenced by various factors; among which are operational skills, the type and the composition of anaesthetic agents used, and postoperative care. SummaryEmbryo transfer (ET) is among the key factors determining the overall ef ciency of transgenic technology in the mouse. A successful ET depends among other factors on the quality of the transferred embryos, foster mothers and anaesthetic reagents and on the transfer techniques. Anaesthesia-caused deaths and suboptimal ET procedures are factors which reduce the success of transgenic experiments and mouse colony maintenance. Here we compared the effects of two anaesthetic reagents-a ketamine/xylazine combination, and tribromoethanol (Avertin)-on the rates of implantation and development to term of mouse zygotes transferred into the oviducts of CD-1 foster mothers, and evaluated whether hypothermia caused by anaesthetics after the ET operation could be overcome by postoperative incubation of the foster mothers. We established two experimental groups of fosters, one of which was kept at room temperature (RT, 21°C) with the other in an incubator (33°C) overnight after ET. Rates of implantation, resorption and development to normal fetuses were evaluated by sacri cing the foster mothers on the 15th day of their pregnancy. Our results showed that regardless of the anaesthetic reagents used, the rates of implantation and of development to normal fetuses can be signi cantly improved by exposing the foster mothers to warmer temperatures (33°C) immediately after the ET operation. These results may have important implications in increasing the success rate of ET with micromanipulated embryos.