Spanel-Borowski K, Ricken A M, Saxer M, Huber P R
Institute of Anatomy, Basel University, Switzerland.
Mol Cell Endocrinol. 1994 Aug;104(1):11-9. doi: 10.1016/0303-7207(94)90046-9.
The intraovarian axis between granulosa cells and thecal cells is regulated by locally produced autocrine and paracrine factors. Until now, microvascular endothelial cells (MVEC) have not been included in such studies. Bovine granulosa cells from medium-sized antral follicles were plated at low density into the lower compartment of 24-well-culture plates on day 0. MVEC derived from bovine corpus luteum were seeded on appropriate inserts and placed as the upper compartment on day 1. Control granulosa cell cultures and MVEC co-cultures were maintained in serum-containing medium. On day 21, control cultures displayed an epithelioid monolayer and the coculture displayed a multilayer. Histochemical staining for 3 beta-HSD activity and for the lipid droplet stain with the fluorescent dye Nile Red were strong, suggesting augmented steroidogenesis in the multilayer. Yet the progesterone levels of supernants corrected for 10,000 cells were similar in monolayers and in multilayers. Co-cultures contained approximately three times more granulosa cells than control cultures as evaluated with a Coulter counter. Additionally, the occurrence of dead cells was quantified with the fluorescent DNA stain, ethidium homodimer, in 11-day-old control cultures and MVEC co-cultures which were deprived of serum, MVEC, or both for an additional 40 h. Serum and MVEC suppressed the occurrence of granulosa cell death. It is concluded that MVEC produce survival factors for the growth and maintenance of granulosa cells.
颗粒细胞与卵泡膜细胞之间的卵巢内轴受局部产生的自分泌和旁分泌因子调节。到目前为止，微血管内皮细胞（MVEC）尚未纳入此类研究。在第0天，将来自中等大小窦卵泡的牛颗粒细胞以低密度接种到24孔培养板的下室。来自牛黄体的MVEC接种在合适的插入物上，并在第1天作为上室放置。对照颗粒细胞培养物和MVEC共培养物在含血清的培养基中维持。在第21天，对照培养物显示为上皮样单层，而共培养物显示为多层。3β-羟类固醇脱氢酶（3β-HSD）活性的组织化学染色以及用荧光染料尼罗红进行的脂滴染色很强，表明多层中类固醇生成增加。然而，经10000个细胞校正后的上清液孕酮水平在单层和多层中相似。用库尔特计数器评估，共培养物中的颗粒细胞数量约为对照培养物的三倍。此外，在11日龄的对照培养物和MVEC共培养物中，用荧光DNA染料乙锭同二聚体对死亡细胞的发生率进行定量，这些培养物在额外40小时内被剥夺血清、MVEC或两者。血清和MVEC抑制了颗粒细胞死亡的发生。结论是MVEC产生促进颗粒细胞生长和维持的存活因子。
