Maternal communication with gametes and embryos

Mechanisms for gametes and embryos to interact with their maternal environment are crucial in achieving reproductive success, both in livestock and the human. Long-range (hormones) and short-range signalling molecules play important roles in mediating cell-cell maternal interactions/communications with gametes and embryos. Slight malfunctions or disturbances of the environment that host this interaction can retard embryonic development. This may lead to creation of a memory for the embryo leading to offspring prone to degenerative diseases in adulthood. Despite an overwhelming amount of research and the literature, not all signalling molecules involved and their relationship with each other are known. Progress in the application of high-throughput genomic and proteomic analytical tools, such as microarrays and quantitative proteomic technologies has had a positive impact on our understanding of various aspects of maternal communication with gametes and embryos. Recent advances point to the presence of a local mechanism in the female reproductive tract capable of recognising the arrival of gametes and embryos and modulating the tract's environment accordingly for the next stage. Further investigations are underway to characterise the details of this system. It is important to consider spatial or temporal components of maternal communication with gametes and embryos that may confer consequences for developmental potential. Finally, it seems that the application of a systems biology approach for creation of an interactome map of maternal communication with gametes and embryos is essential and provides an excellent opportunity for an inter-disciplinary collaboration with engineers and mathematical modellers.     

Refrigeration of rainbow trout gametes and embryos

Prolonged access to early embryos composed of undifferentiated, totipotent blastomeres is desirable in situations when multiple collections of gametes are not possible. The objective of the present study is to examine whether the refrigeration of rainbow trout Oncorhynchus mykiss gametes and early embryos would be a suitable, reliable, and efficient tool for prolonging the availability of early developmental stages up to the advanced blastula stage. The study was conducted continuously during fall, winter, and spring spawning seasons. In all, more than 500 experimental variants were performed involving individual samples from 26 females and 33 males derived from three strains. These strains represented three possible circumstances. In optimal one, gametes from good quality donors were obtained soon after ovulation. In the two non-optimal sources, either donors were of poor genetic quality or gametes were collected from a distant location and transported as unfertilized gametes. A highly significant effect of variability of individual sample quality on efficiency of gamete and embryo refrigeration was revealed. The source of gametes significantly affected viability of refrigerated oocytes and embryos, but not spermatozoa. On average, oocytes from optimal source retained full fertilization viability for seven days of chilled storage, significantly longer than from non-optimal sources. Spermatozoa, regardless of storage method, retained full fertilization ability for the first week of storage. Refrigeration of embryos at 1.4+/-0.4 degrees C significantly slowed the development. Two- week-old embryos were still in blastula stage. Average survival rate of embryos refrigerated for 10 days and then transferred to regular incubation temperatures of 9-14 degrees C was 92% in optimal and 51 and 71% in non-optimal source variants. No effect of gamete and embryo refrigeration on the occurrence of developmental abnormalities was observed. Cumulative refrigeration of oocytes and embryos resulted in an average embryo survival rate of 71% in optimal source variants after 17 days of refrigeration (7 days oocytes+10 days embryos). The study shows that both gamete and embryo refrigeration can be successfully used as an efficient tool for prolonging availability of rainbow trout embryos in early developmental stages.     

Cryopreservation of mammalian embryos

As an innovative method for embryo cryopreservation, vitrification not only reduced the cooling stage duration to a minimum, but also eliminated any injuries cased by extracellular ice, which is a major cause of cell injury. Therefore, if embryos are treated adequately, high survival can be obtained. As a component of a vitrification solution, a permeating cryoprotective agent is essential, and additional inclusion of a macromolecule and a small saccharide makes the solution more effective. The author’s group composed a solution, designated EFS40, with ethylene glycol. Ficoll, and sucrose. This solution proved effective for the cryopreservation of various stages of embryos in many species. In this article, the author describes the detailed procedure for the vitrification of mouse morulae; related information is also described.     

Cryopreservation of canine embryos

The assisted reproductive techniques (ARTs) such as in vitro fertilization, embryo transfer, and cryopreservation of gametes have contributed considerably to the development of biomedical sciences in addition to improving infertility treatments in humans as well as the breeding of domestic animals. However, ARTs used in canine species have strictly limited utility when compared with other mammalian species, including humans. Although successful somatic cell cloning has been reported, artificial insemination by frozen semen to date is only available for the improved breeding and reproduction for companion and working dogs as well as guide dogs for the blind. We describe here the successful cryopreservation of embryos and subsequent embryo transfer in dogs. Canine embryos were collected from excised reproductive organs after artificial insemination and subsequently cryopreserved by a vitrification method. When the 4-cell to morula stage of cryopreserved embryos were nonsurgically transferred into the uteri of nine recipient bitches using a cystoscope, five recipients became pregnant and four of them delivered a total of seven pups. The cryopreservation of embryos in canine species will facilitate the transportation and storage of genetic materials and will aid in the elimination of vertically transmitted diseases in dogs. In addition, this technique will contribute to the improved breeding of companion and working dogs such as guide dogs, drug-detecting dogs, and quarantine dogs.     

Cryopreservation of mouse half-morulae and chimeric embryos by vitrification.

Mouse half-morulae were cryopreserved less than or equal to 1, 3, 6, and 12 hr after bisection by the vitrification method using 25% glycerol and 25% 1,2-propanediol as cryoprotectant. The developmental rates of the frozen-thawed half-embryos to blastocysts in vitro were 77.8% (63/81), 82.0% (41/50), 92.1% (117/127), and 0% (0/37), respectively. Sixty-one of the half-embryos that had been vitrified 6 hr after the bisection followed by transfer to five recipients resulted in a total of ten (16.4%) normal fetuses. Chimeric mouse embryos constructed by two half-morulae were also vitrified 6 and 16 hr after aggregation. Survivors were obtained from the former case: 40 (80.0%) of 50 frozen-thawed embryos developed in vitro to blastocysts, and, after transfer, six chimeric offspring were obtained from the 34 vitrified chimeric embryos. These results showed that mouse half-morulae and chimeric embryos could be cryopreserved by the vitrification method. It seems possible to manufacture a chimeric mouse embryo of defined genotypic composition that can be analyzed during its frozen state using the identical half-embryos of the components.     

Cryopreservation of murine embryos with trehalose and glycerol

Several concentrations of trehalose (0.0, 0.04, 0.1, 0.25 M) in combination with three concentrations of glycerol (1.0, 1.5, 2.0 M) were evaluated for the cryopreservation of murine embryos. Embryos were transferred through increasing concentrations of glycerol in Dulbecco's phosphate-buffered saline with 10% fetal calf serum (PBS + FCS) to reach the final glycerol concentrations. They were then randomly assigned to one of the concentrations of trehalose. A total of 506 morulae were packaged individually in 0.25-ml plastic straws and cooled from ambient temperature at 1.0 degrees C/min in a programmable methanol freezer. Embryos were seeded at -7 degrees C and then cooled to -25 degrees C at 0.3 degrees C/min before being plunged into liquid nitrogen. After thawing and a one-step dilution of glycerol, embryos were cultured for 48 hr and viability was determined by blastocoel formation. Highest viability (70.0%) after 48 hr in culture was obtained for embryos frozen in 1.5 M glycerol plus 0.10 M trehalose as compared to 31% viability for embryos frozen with glycerol alone. These observations suggest that trehalose can be used in combination with glycerol as a cryoprotectant and that a high rate of viability can be achieved after a one-step dilution of the cryoprotectants.     

Cryopreservation of mammalian ova and embryos by vitrification

Vitrification is a new approach to oocyte and embryo cryoconservation. It consists in the solidification of a solution caused not by crystallization, but by a drastic increase in viscosity during cooling. The application of this approach to cryoconservation of oocytes and embryos of different species depends upon the development of proper procedures and non-toxic media. From the technical point of view, the vitrification method is simple and relatively easily applicable under field conditions. The authors review the current procedures applied to oocytes and embryos of laboratory and farm animals.     

Cryopreservation of somatic embryos and embryonic axes of Camellia japonica L.

Summary Cryopreservation in liquid nitrogen was attempted with both somatic embryos and zygotic embryonic axes of the ornamental Camellia japonica L. Several protective measures were applied to somatic embryos (desiccation, chemical protectors, hardening by culture at low temperatures, encapsulation in alginate beads), but none allowed somatic embryos cultures to survive after 24 h in liquid nitrogen. Embryonic axes, however, were easily cryopreserved by means of the simplest technique: desiccation in a laminar flow hood and direct immersion in liquid nitrogen. Although the causes of the difference in cryopreservability between the two types of material are not known, one might be the difference between their degrees of differentiation and water content.Abbreviations ANOVA analysis of variance - BA N6-benzyladenine - DMSO dimethyl sulfoxide - IBA indole-3-butyric acid - LN liquid nitrogen - MS Murashige and Skoog mineral solution - fwt fresh weight - LSD Least Significant Difference     

Cryopreservation of immature embryos of Theobroma cacao

Immature, white zygotic embryos of Theobroma cacao L. (cacao) retained the ability to produce callus and to undergo somatic embryogenesis after slow hydrated freezing and desiccated fast freezing in liquid nitrogen. The highest rate of somatic embryogenesis occurred in embryos which were precultured on a medium containing 3% sucrose, frozen slowly with cryoprotectants before exposure to liquid nitrogen, and recovered on a medium containing 3 mg/liter NAA. Embryos precultured on media containing sucrose increasing to 21% had a higher rate of survival but were less embryogenic after freezing. These results suggest that immature embryos might be used for long-term germplasm storage of T. cacao germplasm.     

Cryopreservation of Bufotes viridis embryos by vitrification

Loss of biodiversity among amphibians is a current concern. Our hypothesis is that the embryos of amphibian species at risk of extinction could be cryopreserved by vitrification, using methods which have proved successful with fish oocyte. To test this hypothesis, samples of four cryoprotectants - methanol (MeOH), dimethyl sulphoxide (Me2SO), propylene glycol (PG) and polyethylene glycol (PEG), some singly, some in combination, were plunged in liquid nitrogen for 5 min to find the best solution for vitrification. To find the least toxic of these solutions, blastulae and stage G17 embryos of Bufotes Viridis, a typical amphibian, were exposed to solutions at different concentrations (0.5–10 M) for different lengths of time (15–30 min), with and without their normal protective jelly coats. In each case the number of survivors, which reached stage G25 was counted. Finally a series of embryos was vitrified in liquid nitrogen using the most efficient and least toxic cryoprotectants.Propylene glycol had the best vitrification characteristics, but MeOH vitrified at higher concentrations. The optimum regime, with the least toxic ctyoprotectants, consisted of 1M Me2SO for 15 min and a combination of 15% PEG_(w/v) + 3M PG + 2M Me2SO for 3 min, with the jelly coat intact, followed by vitrification. This gave a survival percentage of 87.6% immediately after vitrification. Methods designed for cryopreservation of fish embryos make a good starting point for cryopreservation of the embryos of amphibian.     

Cryopreservation of sheep embryos using ethylene glycol

The objective of the study was to evaluate the use of ethylene glycol (EG) for cryopreservation of sheep embryos. A 2 × 2 factorial treatment arrangement examining one-step vs. two-step cryoprotectant addition and removal was used. The one-step cryoprotectant addition involved placement of embryos directly into 1.5 mol EG, whereas the two-step addition utilized an intermediate 10 min exposure to 0.75 mol EG. Similarly, the one-step cryoprotectant removal involved direct placement of thawed embryos into 1.0 mol sucrose, and the two-step procedure included a 10 min exposure to 0.25 mol sucrose before placement in 1.0 mol sucrose. A total of 185 frozen-thawed embryos was placed into in vitro culture for 96 h to determine viability. No differences were observed between cryoprotectant addition or removal techniques, and overall survival was 69%. To validate the results obtained in vitro, a limited number of embryo transfers was performed. Four ewes receiving a total of 11 frozen-thawed embryos produced eight lambs (73% survival) which compared favorably with 74% survival obtained by transferring 19 non-cryopreserved embryos to eight recipients. It is concluded that one-step addition of 1.5 mol ethylene glycol followed by one-step removal in a 1.0 mol sucrose gradient is an appropriate technique for cryopreservation of sheep embryos.     

昆虫卵的超低温冷冻保存

自 Polge等 [1] 首次成功冷冻保存了人精子细胞以来 ,有关细胞冻存的研究取得很大进展 ,与此同时 ,昆虫细胞和组织的冷冻保藏也在脊椎动物细胞冻存技术的基础上 ,逐步建立了一套自己的方法[2 ] 。但这远不能使数量繁多、形式多样的昆虫种质得到有效保存。随着一些哺乳动物如小鼠 [3～ 8]、兔子 [9]、牛 [10 ,11]和人 [12 ,13]卵的冻存成功 ,80年代中期 ,人们开展了对昆虫卵的超低温 (-1 96℃ )冷冻保存研究 [14 ] ,经 1 0多年的努力 ,目前已有果蝇 Drosophilamelanogaster[15,16 ] 和中华蜜蜂 Apis cerana cer-ana[17]的卵经液氮保存后能…