Cecconi S, Rossi G, De Felici M, Colonna R
Department of Biological Sciences and Technologies, University of L'Aquila, Rome, Italy.
Mol Reprod Dev. 1996 Aug;44(4):540-6. doi: 10.1002/(SICI)1098-2795(199608)44:4<540::AID-MRD14>3.0.CO;2-1.
We have evaluated the possibility that mouse oocyte growth in vitro could be achieved under the influence of soluble compound(s) released by different somatic cell types. For this purpose, zona-free denuded oocytes from 12-day-old mice were cultured on monolayers of NIH-3T3 fibroblasts, which are able to establish gap junctional communications with them, in the presence or absence of media conditioned by preantral granulosa cells or by Sertoli cells, plated at increasing concentrations from 0.3-1 x 10(6) ml-1 cells. After 3 days, no increase in vitellus diameter was recorded from fibroblast-coupled oocytes maintained in culture medium or in the presence of media conditioned by 0.3 x 10(6) ml-1 Sertoli cells. By contrast, increasing proportions of coupled oocytes grew, provided the continuous presence of media conditioned by 0.5 or 1 x 10(5) ml-1 Sertoli cells, or by 0.3, 0.5, and 1 x 10(5) ml-1 preantral granulosa cells. Since the ligand of c-kit, the growth factor KL, promotes the growth in vitro of oocytes cultured in follicles from 8-day-old mice, an antibody against mouse KL was used to evaluate whether in our culture conditions KL might also be responsible for the growth of oocytes from 12-day-old mice. No inhibition of growth was evident in oocytes cultured directly on preantral granulosa or Sertoli-cell monolayers. Furthermore, the growth of fibroblast-coupled oocytes cultured in media conditioned by preantral granulosa cells was not significantly affected by the presence of this antibody during culture. By contrast, a high percentage of oocytes cultured on fibroblasts in the presence of media conditioned by Sertoli cells showed a significant inhibition of growth and no metabolic cooperativity. It was concluded that, besides KL, other bioactive factor(s) released by either preantral granulosa or Sertoli cells can induce a significant stimulation of mouse oocyte growth in vitro.
我们评估了在不同体细胞类型释放的可溶性化合物影响下，小鼠卵母细胞能否在体外生长的可能性。为此，将来自12日龄小鼠的无透明带裸卵培养在能够与它们建立间隙连接通讯的NIH-3T3成纤维细胞单层上，有无由腔前颗粒细胞或支持细胞条件培养基，这些细胞以0.3 - 1×10(6) ml-1细胞的递增浓度接种。3天后，在培养基中培养的或在存在0.3×10(6) ml-1支持细胞条件培养基的情况下，与成纤维细胞偶联的卵母细胞的卵黄直径没有增加。相比之下，只要持续存在由0.5或1×10(5) ml-1支持细胞或由0.3、0.5和1×10(5) ml-1腔前颗粒细胞条件培养基，偶联卵母细胞生长的比例就会增加。由于c-kit的配体生长因子KL可促进8日龄小鼠卵泡中培养的卵母细胞的体外生长，因此使用抗小鼠KL抗体来评估在我们的培养条件下KL是否也可能是12日龄小鼠卵母细胞生长的原因。直接在腔前颗粒细胞或支持细胞单层上培养的卵母细胞中没有明显的生长抑制。此外，在腔前颗粒细胞条件培养基中培养的与成纤维细胞偶联的卵母细胞的生长在培养过程中不受该抗体存在的显著影响。相比之下，在支持细胞条件培养基存在的情况下在成纤维细胞上培养的高比例卵母细胞显示出显著的生长抑制且没有代谢协同作用。得出的结论是，除了KL之外，腔前颗粒细胞或支持细胞释放的其他生物活性因子可以在体外显著刺激小鼠卵母细胞生长。
