संग्रह और हम्सटर oocytes और माउस भ्रूण के cryopreservation
Summary
इस वीडियो लेख में हम कदम दर कदम कैसे इकट्ठा करने के लिए और उच्च पद पिघलना जीवित रहने की दरों के साथ हम्सटर oocytes cryopreserve पर एक प्रदर्शन प्रस्तुत करते हैं. एक ही प्रक्रिया भी सफलतापूर्वक स्थिर और पिघलना preimplantation विकास के विभिन्न चरणों पर माउस भ्रूण के लिए लागू किया जा सकता है.
Abstract
Embryos and oocytes were first successfully cryopreserved more than 30 years ago, when Whittingham et al. 1 and Wilmut 2 separately described that mouse embryos could be frozen and stored at -196 °C and, a few years later, Parkening et al. 3 reported the birth of live offspring resulting from in vitro fertilization (IVF) of cryopreserved oocytes. Since then, the use of cryopreservation techniques has rapidly spread to become an essential component in the practice of human and animal assisted reproduction and in the conservation of animal genetic resources. Currently, there are two main methods used to cryopreserve oocytes and embryos: slow freezing and vitrification. A wide variety of approaches have been used to try to improve both techniques and millions of animals and thousands of children have been born from cryopreserved embryos. However, important shortcomings associated to cryopreservation still have to be overcome, since ice-crystal formation, solution effects and osmotic shock seem to cause several cryoinjuries in post-thawed oocytes and embryos. Slow freezing with programmable freezers has the advantage of using low concentrations of cryoprotectants, which are usually associated with chemical toxicity and osmotic shock, but their ability to avoid ice-crystal formation at low concentrations is limited. Slow freezing also induces supercooling effects that must be avoided using manual or automatic seeding 4. In the vitrification process, high concentrations of cryoprotectants inhibit the formation of ice-crystals and lead to the formation of a glasslike vitrified state in which water is solidified, but not expanded. However, due to the toxicity of cyroprotectants at the concentrations used, oocytes/embryos can only be exposed to the cryoprotectant solution for a very short period of time and in a minimum volume solution, before submerging the samples directly in liquid nitrogen 5. In the last decade, vitrification has become more popular because it is a very quick method in which no expensive equipment (programmable freezer) is required. However, slow freezing continues to be the most widely used method for oocyte/embryo cryopreservation. In this video-article we show, step-by-step, how to collect and slowly freeze hamster oocytes with high post-thaw survival rates. The same procedure can also be applied to successfully freeze and thaw mouse embryos at different stages of preimplantation development.
Protocol
पशु देखभाल और प्रक्रियाओं बार्सिलोना, स्पेन के विश्वविद्यालय ऑटोनोमा के पशु और मानव अनुसंधान पर आचार समिति द्वारा अनुमोदित दिशा निर्देशों और नियमों के अनुसार आयोजित की गई. I. Oocyte संग्रह …
Discussion
इस प्रोटोकॉल हम्सटर oocytes और माउस भ्रूण सफलतापूर्वक cryopreserved किया जा सकता है. एक बार जम / oocytes और भ्रूण तरल नाइट्रोजन टैंक में अनिश्चित काल के संग्रहीत किया जा सकता है और किसी भी समय या जगह वांछित पर बरामद किया. यह नमूने ?…
Acknowledgements
यह काम स्पेनिश Ministerio डे Educación y (2006-11,792 जैव) Ciencia और Generalitat de Catalunya (2005 SGR00437) द्वारा समर्थित किया गया. लेखक के लिए उनके तकनीकी सहायता के लिए मीडिया की तैयारी में मार्क Puigcerver और Jonatan लुकास धन्यवाद देना चाहूंगा. Servei d'Estabulari से सभी कर्मचारियों को जानवरों के आवास के लिए स्वीकार किया है, और विशेष रूप से जुआन जोस मार्टिन और उनके अतिरिक्त योगदान के लिए इस वीडियो का हिस्सा रिकॉर्डिंग पर जेवियर बेनिटो. एनसीबी पुर्तगाली Fundação पैरा के एक साथी है Ciência ई TECNOLOGIA और एसजी स्पेनिश Ministerio डे Educación y Ciencia के एक साथी है.
Materials
| Material Name | Tipo | Company | Catalogue Number | Comment |
|---|---|---|---|---|
| French type mini-straw 0.25 ml | Mini-tub | 13407/0010 | ||
| hCG | Farma-Lepori, Spain | 749036.4 | ||
| Hyaluronidase | Sigma | H-3884 | ||
| KSOM-H medium | Ref. [11] | |||
| Liquid Nitrogen | Air Liquide | |||
| Plastic plugs | Mini-tub | 19046 | ||
| PMSG | Intervet, Spain | 1.614/8447 | ||
| Propilenglycol | Fluka | 82280 | ||
| Sucrose | Merck | 107687 | ||
| Surgical Scissors | Aesculap | BC-060R; BG-061R | ||
| Thermo-sealer | SIZ | 220 | ||
| 35 and 90 mm culture dishes | Nunc | 153066; 150350 | ||
| 10 ml tubes | Greiner Bio-one | 163160 | ||
| Nitrogen tank | MVE, Cryogenics | XC 43/28 | ||
| Programmable Freezer | Planner Products Ltd. | Kryo-10 Series III | ||
| Forceps | Aesculap | BD-331R; BD-053R; OC-020R |
Riferimenti
- Whittingham, D. G., Leibo, S. P., Mazur, P. Survival of mouse embryos frozen to -196 and -269 °C. Science. 178, 411-414 (1972).
- Wilmut, I. The effect of cooling rate, warming rate, cryoprotective agent and stage of development on survival of mouse embryos during freezing and thawing. Life Sci. 11, 1071-1079 (1972).
- Parkening, T. A., Tsunoda, Y., Chang, M. C. Effects of various low temperatures, cryoprotective agents and cooling rates on the survival, fertilizability and development of frozen-thawed mouse eggs. J. Exp. Zool. 197, 369-374 (1976).
- Schneider, U. Cryobiological principles of embryo freezing. J. In Vitro Fert. Embryo Transf. 3, 3-9 (1986).
- Vajta, G., Kuwayama, M. Improving cryopreservation systems. Theriogenology. 65, 236-244 (2006).
- Duselis, A. R., Vrana, P. B. Retrieval of mouse oocytes. JoVE. 3, (2007).
- Lassalle, B., Testart, J., Renard, J. P. Human embryo features that influence the success of cryopreservation with the use of 1,2 propanediol. Fertil. Steril. 44, 645-651 (1985).
- Ibáñez, E., Grossmann, M., Vidal, F., Egozcue, J., Santaló, J. Cryopreservation of caprine beta-lactoglobulin transgenic mouse embryos. Cryobiology. 35, 290-298 (1997).
- Dinnyes, A., Wallace, G. A., Rall, W. F. Effect of genotype on the efficiency of mouse embryo cryopreservation by vitrification or slow freezing. Mol. Reprod. Dev. 40, 429-435 (1995).
- Tateno, H., Kamiguchi, Y., Mikamo, K. A freezing and thawing method of hamster oocytes designed for both the penetration test and chromosome assay of human spermatozoa. Mol. Reprod. Dev. 33, 202-209 (1992).
- Biggers, J., McGuinnis, L. K., Raffin, M. Amino acids and preimplantation development of the mouse in the protein-free KSOM. Biol. Reprod. 63, 281-293 (2000).
Citazione di questo articolo
Costa-Borges, N., González, S., Ibáñez, E., Santaló, J. Collection and Cryopreservation of Hamster Oocytes and Mouse Embryos. J. Vis. Exp. (25), e1120, doi:10.3791/1120 (2009).