Lobascio Anna-Maria, Klinger Francesca-Gioia, De Felici Massimo
Department of Public Health and Cell Biology, Section of Histology and Embryology, University of Rome Tor Vergata, Via Montpellier 1, 00173 Rome, Italy.
Int J Dev Biol. 2007;51(2):157-60. doi: 10.1387/ijdb.062203al.
Expression of phosphotidylserine by fetal oocytes in culture renders significant numbers of such cells able to bind AnnexinV-coated microbeads and allows their separation from Annexin V-negative oocytes on a Magnetic Cell Separation (MACS) column in a magnetic field. The majority of oocytes (> or =75%) which bound Annexin V-coated microbeads were viable, as indicated by their propidium iodine (PI) negativity. However, they showed apoptotic morphologies and were found to be TUNEL-positive. On the other hand, AnnexinV-negative oocytes, besides being PI negative, appeared morphologically healthy and TUNEL negative. Moreover, AnnexinV-positive oocytes showed a marked lower ratio of Bcl-xL/Bax transcripts in comparison to AnnexinV-negative oocytes. We conclude that the present method is able to separate fetal oocytes in two distinct populations: AnnexinV-positive oocytes showing features typical of apoptotic cells and AnnexinV-negative oocytes comprising for the most part viable non-apoptotic cells. This procedure should greatly facilitate studies aimed to identify the currently poorly understood molecular pathways governing apoptosis in mammalian fetal oocytes.
培养的胎儿卵母细胞中磷脂酰丝氨酸的表达使大量此类细胞能够结合膜联蛋白V包被的微珠，并使其在磁场中的磁性细胞分离（MACS）柱上与膜联蛋白V阴性的卵母细胞分离。结合膜联蛋白V包被微珠的大多数卵母细胞（≥75%）是活的，这由它们对碘化丙啶（PI）呈阴性表明。然而，它们呈现出凋亡形态，并且被发现TUNEL呈阳性。另一方面，膜联蛋白V阴性的卵母细胞，除了PI呈阴性外，形态上看起来健康且TUNEL呈阴性。此外，与膜联蛋白V阴性的卵母细胞相比，膜联蛋白V阳性的卵母细胞显示出显著更低的Bcl-xL/Bax转录本比率。我们得出结论，目前的方法能够将胎儿卵母细胞分离为两个不同的群体：显示凋亡细胞典型特征的膜联蛋白V阳性卵母细胞和主要由活的非凋亡细胞组成的膜联蛋白V阴性卵母细胞。这个程序应该极大地促进旨在识别目前了解甚少的控制哺乳动物胎儿卵母细胞凋亡分子途径的研究。
