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

牛卵母细胞体外成熟（IVM）阶段具有受精后囊胚发育的能力。发育能力即细胞质成熟，是由midkine （MK）促进的。MK是一种视黄酸（RA）诱导的肝素结合生长因子。最近发现，在IVM过程中，牛积云封闭卵母细胞（ceo）的积云细胞发生凋亡，表明ceo积云细胞的凋亡程度可能与其细胞质成熟程度有关。本研究探讨了MK和RA对体外培养期牛ceo细胞质成熟和积云细胞凋亡的影响。结果发现MK和RA具有促进细胞质成熟的活性。结扎介导PCR实验显示，MK-或ra -处理的ceo云细胞核小体间DNA的凋亡程度明显低于未处理的对照组。接下来，利用免疫荧光法在牛ceo的卵母细胞和卵丘细胞中检测到肉毒碱转运体，研究了对多种细胞具有抗凋亡活性的肉毒碱对牛ceo细胞质成熟的影响。研究发现，在IVM过程中，肉碱处理促进了活跃线粒体从皮质区向胞质内的过渡，并促进了细胞浆的成熟。抑制减法杂交分析显示，MK处理可促进云细胞中拓扑异构酶α、凋亡相关基因和卵巢特异性基因OVN6-2的表达。

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.