透明质酸与猪卵母细胞体外培养成活率及减数分裂的关系

Relationship between Hyaluronic Acid and Viability of Porcine Oocyte Cumulus Complexes Cultured in vitro, Meiosis

从猪卵巢表面 ２～６ ｍ ｍ 直径卵泡中抽取的卵丘卵母细胞复合体（ Ｃ Ｏ Ｃｓ），依据其外观形态，分为Ⅰ、Ⅱ、Ⅲ、Ⅳ类 ４ 种。Ⅰ类 Ｃ Ｏ Ｃｓ 在体外培养 ０～２０ ｈ，受培养液中 Ｅ Ｃ Ｇ 和 ｈ Ｃ Ｇ 的刺激，合成透明质酸并分泌到培养液中，培养液中透明质酸的含量由（９８５±４５０） μｇ／ Ｌ急剧上升到（２ ００００±１４６２） μｇ／ Ｌ。培养 ２０～４４ ｈ 期间，培养液中不加 Ｅ Ｃ Ｇ 和 ｈ Ｃ Ｇ，透明质酸分泌速度明显降低；培养 ４４ ｈ，培养液中透明质酸含量为（６０４５±９４０） μｇ／ Ｌ。卵丘扩展程度，在 ２０～４４ ｈ 最为强烈。各类卵母细胞在体外培养成活率显著不同，Ⅰ类为最高，其次为Ⅱ类和Ⅲ类，Ⅳ类 Ｃ Ｏ Ｃｓ 为退化卵母细胞。卵泡液能够维持体外培养卵母细胞的成活。去除卵泡液成分，卵母细胞在培养 ２０ ｈ 死亡率达（６０２±１４５）％ ，培养 ４４ ｈ 后上升至（９０５±８６）％ 。添加外源透明质酸，可以显著地降低卵母细胞死亡率。本研究结果表明，猪卵母细胞体外培养中，添加激素 Ｅ Ｃ Ｇ 和ｈ Ｃ Ｇ，能够显著刺激透明质酸的分泌。同时，分泌的透明质酸不仅存在于扩展的卵丘中间，还能够分泌到培养液中。卵丘细胞?

Cumulus oocyte complexes were aspirated from pig ovaries surface follicles with a diameter of 2 6 mm. According to morphology, the oocytes could be divided into four categories: ClassⅠ,Ⅱ,Ⅲ and Ⅳ. COCs from class Ⅰ synthesized hyaluronic acid(HYA) and secreted to the medium when stimulated by PMSG and hCG during 0 20 h of maturation. Hyaluronic acid content in maturation medium increased from (98.5±45.0) μg/L to (2 000.0±146.2) μg/L. During 20 44 h of maturation, the speed of synthesis of hyaluronic acid slowed down and the content of HYA decreased to (604.5±94.0) μg/L at 44 h of maturation. Cumulus expansion occurred during 44 h of maturation, but showed a stronger expansion during 20 44 h of maturation. Viability of class Ⅰ oocytes was the highest followed by class Ⅱ and Ⅲ, class Ⅳ oocytes were degenerated. Follicular fluid sustained the viability of oocytes . Removal of follicular fluid made mortality of oocytes increase to (60.2±14.5)% at 20 h of maturation, and to (90.5±8.6)% at 44 h of maturation. Addition of hyaluronic acid could significantly decrease the mortality of oocytes. The results showed that during maturation in vitroX pig cumulus oocyte complexes secreted HYA by the stimulation of PMSG and hCG. Meanwhile secreted hyaluronic acid not noly existed among the expanded cumulus oorphorus, but also secreted into the medium. Cumulus expansion seemed to be related to the concentration of hyaluronic acid aggregated around the oocytes, not proportionate to that in the medium. Addition of HYA could prevent death of oocytes in the follicle follocle fluid free medium.