重組與非重組豬胚之體外培養

In vitro Culture of Cloned and Non-cloned Pig Embryos

林子安(Tzy-An Lin) ； 唐品琦(Pin-Chi Tang) ； 朱志成(Jyh-Cherng Ju)

《中華農學會報》 8卷2期 (2007/04) Pp. 113-128

https://doi.org/10.6730/JAAC.200704_8(2).0002

摘要

利用體細胞核轉置技術可成功的產製複製動物，然而其效率仍然往低，除了供核細胞遣傳物質之再程序化或重新整合外，部分原因為不完善之體外培養環境導致胚發育延遲或中止，因此建立－完善之體外培養系統極為重要。影響體外培養系統的因素包括培養液中所含成分、培養液滲透壓與培養環境之氣體濃度等。各種動物胚之體外培養皆有相似之發育阻滯期。在倉鼠胚之研究中發現葡萄糖與磷酸鹽為導致胚體外發育受阻之主要原因，葡萄糖本身對豬胚並沒有明顯之影響，但其與磷酸鹽之交互作用會降低豬胚之體外發育。培養液之成分對不同期別之豬胚發育具有不同程度之影響，通常於1-至4-細胞之早期胚需以丙酗酸與乳酸替代葡萄糖，4-細胞期後則可使用葡萄糖作為能量來源。外源胺基酸依其種類對不同發育階段之豬胚也有不同之影響。早期豬胚使用非極性必需胺基酸會影饗其分裂而導致發育延遲，而非必需胺基酸的添加則可提高早期豬胚分裂與囊胚之擴張。豬胚於體外培養時，於前48h之培養液滲透壓調整為290mOsm，48h後改為256mOsm可顯著提高其囊胚率與囊胚之總細胞數。在與生殖道相同的低氧培養環境(5%O2)中，豬胚細胞質內所產生活性氧化物質(reactive oxygen species, ROS)量較少，可相對的減少氧化性緊迫(oxidative stress)對胚所造成的傷害。在不同培養條件下，包括不同培養液、滲透壓或氧氣濃度對胚所造成之影響程度不同。對於核轉置胚而言亦類似，但由於卵母細胞質對外源性細胞核之重整作用需有不同物質之交換，此可能造成重組胚所需之營養異於一船正常胚者，在此方面之瞭解尚不多，仍需更進一步研究。

關鍵字

重整合 ； 豬受精胚培養液－3 ； 體外培養

並列摘要

Animal cloning can be achieved by somatic cell nuclear transfer (SCNT), although the efficiency remains low. In addition to the requirement for reprogramming the foreign genetic materials, suboptimal in vitro culture system is one of the major causes for developmental retardation. Several factors such as composition of culture medium, sources of energy substrates, exogenous amino acids, osmolarity of medium, and oxygen concentration of culture environment, affect embryonic development during in vitro culture. In vivo development of embryos including transfer to recipient oviducts, co-culture with oviductal epithelium cells, or supplement of follicuar fluid into culture medium could overcome the developmental block of pig embryos in which glucose and phosphate are thought to be attributable. However glucose itself does not hamper development of pig embryos. Instead, the interaction between glucose and phosphate are responsible. Medium components affect embryo development at different stages. Pyruvate and lactate but not glucose are essential for successful development of 1- to 4- celled pig embryos. Supplementation of exogenous amino acids also exerts different influences depending on the types of amino acids and embryonic stages. Non-polar essential amino acids cause retardation non-essential amino acids enhance cleavage and blastocyst expansion. Other factors such as the osmolarity of culture medium and oxygen concentration of culture system could also influence embryo development in vitro due to possible elevation of oxidative stress. In reconstructed pig embryos, reprogramming of foreign nucleus requires complete exchange of nuclear materials in the ooplasm. An incomplete reprogramming of the foreign nuclear materials might result in a shift of metaholi0m of the reconstructed embryos However, the information for nutrition requirement elements to facilitate reprogramming or successful development of reconstructed pig embryos is largely unavailable currently. More studies are required.

並列關鍵字

Reprogramming ； PZM-3 medium ； In vitro culture