Studies on the promoting effects of cumulus cells on cytoplasmic maturation of bovine immature oocytes during in vitro maturation culture.

牛卵母细胞在体外成熟（IVM）过程中获得了受精后发育为囊胚的能力。中期因子（midkine，MK）促进细胞质成熟，即发育能力。MK是一种视黄酸（RA）诱导的肝素结合生长因子。近年来，研究发现牛卵丘卵母细胞（CEOs）在体外成熟过程中发生了卵丘细胞凋亡，提示CEOs卵丘细胞凋亡的程度可能与其胞质成熟有关。本研究旨在探讨MK和RA对体外成熟牛CEOs细胞质成熟和卵丘细胞凋亡的影响。结果发现MK和RA具有促进细胞质成熟的活性。连接-madiated PCR实验表明，在卵丘细胞的CEO凋亡的核小体间DNA片段化的程度显着降低MK或RA处理的CEO相比，未经处理的对照组。接下来，检查了对各种类型的细胞具有抗凋亡活性的肉毒碱对牛CEOs的细胞质成熟的影响，因为使用免疫荧光方法在CEOs的卵母细胞和卵丘细胞中都检测到肉毒碱转运蛋白。结果表明，在体外成熟过程中，肉毒碱处理促进了活性线粒体从皮层区向胞质内的转移以及CEOs胞质的成熟。抑制性消减杂交结果显示，MK处理后，卵丘细胞中拓扑异构酶α、凋亡相关基因和卵巢特异基因OVN 6 -2的表达增加。

The competence of postfertilization development to blastocysts of bovine oocytes is acquired during in vitro maturation (IVM) period. The developmental competence, referred to as cytoplasmic maturation, is promoted by midkine (MK). MK is a retinoic acid (RA)-inducible and heparin binding growth factor. Recently it was found that apoptosis occurred in cumulus cells of bovine cumulus-enclosed oocytes (CEOs) during IVM, suggesting that the degree of apoptosis in cumulus cells of CEOs may be correlated with their cytoplasmic maturation. The present study was undertaken to examine the effects of MK and RA on cytoplasmic maturation of bovine CEOs and apoptosis of their cumulus cells during IVM period. As a result, it was found that MK and RA had cytoplasmic maturation-promoting activities. Ligation-madiated PCR experiments revealed that the degree of apoptotic internucleosomal DNA fragmentation in cumulus cells of CEOs was significantly lower in MK- or RA-treated CEOs compared to the untreated control group. Next, the effects of carnitine, which has an anti-apoptotic activity to various types of cells, on cytoplasmic maturation of bovine CEOs were examined, because carnitine transporter was detected both in oocytes and cumulus cells of CEOs using immunofluorescence method. It was found that transition of active mitochondria from cortex region into inner cytoplasm as well as cytoplasmic maturation of CEOs were promoted by carnitine treatment during IVM. Finally, suppression subtractive hybridization analysis revealed that expression of some genes such as topoisomerase α, apoptosis related gene and ovary specific gene OVN6-2 in cumulus cells were promoted by MK treatment.