Nagyova E, Scsukova S, Kalous J, Mlynarcikova A
Institute of Animal Physiology and Genetics, Academy of Sciences of the Czech Republic, 27721 Libechov, Czech Republic.
Institute of Experimental Endocrinology, Slovak Academy of Sciences, 83301 Bratislava, Slovakia.
Domest Anim Endocrinol. 2014 Jul;48:7-14. doi: 10.1016/j.domaniend.2014.01.003. Epub 2014 Jan 21.
This study was designed to determine whether inhibition of either cyclooxygenase-2 (COX-2) by indomethacin or progesterone receptor (PR) by PR antagonist, RU486, affects oocyte maturation, progesterone production, and covalent binding between hyaluronan (HA) and heavy chains of inter-α trypsin inhibitor, as well as expression of cumulus expansion-associated proteins (HA-binding protein, tumor necrosis factor α-induced protein 6, pentraxin 3) in oocyte-cumulus complexes (OCCs). The experiments were based on freshly isolated porcine OCC cultures in which the consequences of PR and COX-2 inhibition on the final processes of oocyte maturation were determined. Granulosa cells (GCs) and OCCs were cultured in medium supplemented with FSH/LH (both 100 ng/mL) in the presence/absence of RU486 or indomethacin. Western blot analysis, (3)H-glucosamine hydrochloride assay, immunofluorescence, and radioimmunoassay were performed. Only treatment with RU486 (25 μM) caused a decrease in the number of oocytes that reached germinal vesicle breakdown and metaphase II stage compared with indomethacin (100 μM) or FSH/LH treatment alone after 44 h. All treated OCCs synthesized an almost equal amount of HA. Heavy chains (of inter-α trypsin inhibitor)-HA covalent complexes were formed during in vitro FSH/LH-stimulated expansion in RU486- or indomethacin-treated OCCs. Follicle-stimulating hormone/LH-induced progesterone production by OCCs was increased in the presence of RU486 after 44 h. In contrast, a decrease of FSH/LH-stimulated progesterone production by GCs was detected in the presence of either RU486 or indomethacin after 72 h. We suggest that the PR-dependent pathway may be involved in the regulation of oocyte maturation. Both PR and COX-2 regulate FSH/LH-stimulated progesterone production by OCCs and GCs.
本研究旨在确定吲哚美辛对环氧合酶-2（COX-2）的抑制作用或PR拮抗剂RU486对孕激素受体（PR）的抑制作用是否会影响卵母细胞成熟、孕激素生成、透明质酸（HA）与α-胰蛋白酶抑制剂重链之间的共价结合，以及卵丘-卵母细胞复合体（OCCs）中卵丘扩展相关蛋白（HA结合蛋白、肿瘤坏死因子α诱导蛋白6、五聚体3）的表达。实验基于新鲜分离的猪OCC培养物，其中确定了PR和COX-2抑制对卵母细胞成熟最终过程的影响。颗粒细胞（GCs）和OCCs在添加FSH/LH（均为100 ng/mL）的培养基中培养，同时存在/不存在RU486或吲哚美辛。进行了蛋白质免疫印迹分析、盐酸（3）H-葡萄糖胺测定、免疫荧光和放射免疫测定。与单独使用吲哚美辛（100 μM）或FSH/LH处理相比，仅用RU486（25 μM）处理44小时后，达到生发泡破裂和中期II期的卵母细胞数量减少。所有处理的OCCs合成的HA量几乎相等。在RU486或吲哚美辛处理的OCCs中，体外FSH/LH刺激的扩展过程中形成了重链（α-胰蛋白酶抑制剂）-HA共价复合物。44小时后，在RU486存在的情况下，FSH/LH诱导的OCCs孕激素生成增加。相反，72小时后，在RU486或吲哚美辛存在的情况下，检测到GCs的FSH/LH刺激的孕激素生成减少。我们认为PR依赖性途径可能参与卵母细胞成熟的调节。PR和COX-2均调节FSH/LH刺激的OCCs和GCs的孕激素生成。
