van Tol H T, Bevers M M
Department of Herd Health and Reproduction, Faculty of Veterinary Medicine, University of Utrecht, The Netherlands.
Mol Reprod Dev. 1998 Nov;51(3):315-21. doi: 10.1002/(SICI)1098-2795(199811)51:3<315::AID-MRD11>3.0.CO;2-1.
The effect of follicular cells and their conditioned media on the FSH-induced oocyte maturation of oocytes surrounded by cumulus cells connected to the membrana granulosa (COCGs) was investigated. COCGs and cumulus oocyte complexes (COCs) were cultured for 22 hr in M199 supplemented with 0.05 IU FSH/ml in either the presence of pieces of theca cell layer or in the presence of pieces of membrana granulosa. COCGs and COCs were also cultured for 22 hr in either theca-cell conditioned medium (CMt) or in granulosa cell conditioned medium (CMg), both supplemented with 0.05 IU FSH/ml. To investigate the importance of cell-cell contacts between granulosa cells and cumulus cells, oocytes were cultured as COCs in CMt, as COCs in CMt supplemented with pieces of membrana granulosa, or as COCGs in CMt. In all groups the medium was supplemented with 0.05 IU FSH/ml. After culture the nuclear status of the oocytes was assessed using orcein staining. Culture of COCGs in the presence of theca cells as well as in CMt resulted in a significantly decreased proportion of oocytes that had undergone germinal vesicle breakdown (GVBD) at the end of the culture period as compared to the control. Of the oocytes that resumed meiosis in the presence of theca cells or in CMt, the proportion of oocytes that progressed up to the MII stage was significantly reduced. This indicates the production of a meiosis-inhibiting factor by theca cells. Culture with COCs instead of COCGs resulted in comparable results although the effect was less pronounced. The significant effect on the progression of meiosis of oocytes cultured as COCGs or as COCs, obtained in the presence of granulosa cells or in CMg, was much weaker than the effect of theca cells or culture in CMt. Culture of COCs in CMt supplemented with layers of membrana granulosa and 0.05 IU FSH/ml, resulted in significantly less oocytes that resumed meiosis as compared to culture of COCs in CMt. Of the oocytes that showed GVBD, the proportion that progressed up to the MII stage was significantly reduced. Attachment of the COCs to the membrana granulosa enhanced this inhibiting action of CMt on the progression of meiosis. It is concluded that theca cells secrete a stable factor that inhibits the progression of FSH-mediated meiosis in oocytes of COCGs.
研究了卵泡细胞及其条件培养基对促卵泡激素（FSH）诱导的、被与颗粒膜相连的卵丘细胞包围的卵母细胞（COCGs）成熟的影响。将COCGs和卵丘卵母细胞复合体（COCs）在补充有0.05 IU FSH/ml的M199中培养22小时，培养环境为存在卵泡膜细胞层碎片或存在颗粒膜碎片。COCGs和COCs也分别在补充有0.05 IU FSH/ml的卵泡膜细胞条件培养基（CMt）或颗粒细胞条件培养基（CMg）中培养22小时。为了研究颗粒细胞与卵丘细胞之间细胞间接触的重要性，将卵母细胞作为COCs在CMt中培养，作为补充有颗粒膜碎片的COCs在CMt中培养，或作为COCGs在CMt中培养。所有组的培养基均补充有0.05 IU FSH/ml。培养后，使用orcein染色评估卵母细胞的核状态。与对照组相比，在卵泡膜细胞存在下以及在CMt中培养COCGs，导致培养期结束时经历生发泡破裂（GVBD）的卵母细胞比例显著降低。在卵泡膜细胞存在下或在CMt中恢复减数分裂的卵母细胞中，进展到MII期的卵母细胞比例显著降低。这表明卵泡膜细胞产生了一种减数分裂抑制因子。用COCs而非COCGs培养得到了类似的结果，尽管效果不那么明显。在颗粒细胞存在下或在CMg中培养作为COCGs或COCs的卵母细胞时，对减数分裂进展的显著影响远弱于卵泡膜细胞或在CMt中培养的效果。在补充有颗粒膜层和0.05 IU FSH/ml的CMt中培养COCs，与在CMt中培养COCs相比，恢复减数分裂的卵母细胞显著减少。在显示GVBD的卵母细胞中，进展到MII期的比例显著降低。COCs与颗粒膜的附着增强了CMt对减数分裂进展的这种抑制作用。结论是，卵泡膜细胞分泌一种稳定因子，抑制COCGs卵母细胞中FSH介导的减数分裂进展。
