Cyclooxygenase 2 (COX-2) encoded by the Cox-2 gene within the periovulatory follicles is a critical mediator of oocyte development. Growth differentiation factor 9 (GDF-9) and bone morphogenetic protein 15 (BMP-15) participate in the modulation of certain target genes in the ovary, possibly influencing the Cox-2 gene expression. However, this relationship has not been characterized in canines. This study aimed to examine the possible relationships among BMP-15, GDF-9, progesterone, and Cox-2 gene expression in granulosa-cumulus cells in dogs. Granulosa cells from antral follicles and their corresponding cumulus-oocyte complexes and follicular fluid (FF) were separately obtained from 56 ovaries collected from adult bitches at estrus (n = 15) and proestrus (n = 13) after ovariohysterectomy. Total RNA extraction was performed in follicular cells, and Cox-2 gene expression was assessed by quantitative PCR analysis. Progesterone, BMP-15, and GDF-9 were determined in the FF samples using ELISA assays. Cumulus-oocyte complexes were subjected to in vitro maturation (IVM) with or without (control) recombinant GDF-9 and BMP-15. After 72 h of culture, Cox-2 transcript analyses were performed in cumulus cells via quantitative PCR. Data were evaluated by ANOVA. An increase (P < 0.05) in Cox-2 messenger RNA levels was observed in follicular cells from follicles at estrus with respect to those at proestrus. However, the levels of BMP-15 and GDF-9 in FF decreased (P < 0.05), whereas progesterone increased (P < 0.05) from the proestrus phase to the estrus phase. The expression of Cox-2 gene in cumulus cells was 4-fold greater (P < 0.01) than that in the control when both growth factors were added to the IVM culture. In conclusion, although BMP-15 together with GDF-9 appears to upregulate the levels of Cox-2 transcripts during IVM, the inverse relationship of these paracrine factors with Cox-2 gene expression and the positive correlation of progesterone with Cox-2 transcripts suggest that the high progesterone levels could be more relevant in the local mechanisms regulating the Cox-2 gene expression.

由排卵期卵泡内的Cox-2基因编码的环加氧酶2 (COX-2)是卵母细胞发育的关键介质。生长分化因子 9 (GDF-9) 和骨形态发生蛋白 15 (BMP-15) 参与调节卵巢中某些靶基因，可能影响Cox-2基因表达。然而，这种关系尚未在犬科动物中得到表征。本研究旨在检查 BMP-15、GDF-9、孕酮和Cox-2之间可能的关系狗颗粒-卵丘细胞中的基因表达。来自窦卵泡的颗粒细胞及其相应的卵丘 - 卵母细胞复合物和卵泡液 (FF) 分别从卵巢子宫切除术后从发情期 (n = 15) 和发情前期 (n = 13) 的成年母犬收集的 56 个卵巢中获得。在滤泡细胞中进行总 RNA 提取，并通过定量 PCR 分析评估Cox-2基因表达。使用 ELISA 测定法测定 FF 样品中的孕酮、BMP-15 和 GDF-9。在有或没有（对照）重组 GDF-9 和 BMP-15 的情况下，对卵丘-卵母细胞复合物进行体外成熟 (IVM)。培养 72 小时后，通过定量 PCR 在卵丘细胞中进行Cox-2转录分析。数据通过方差分析评估。增加（P < 0.05) 在Cox-2信使 RNA 水平中，在来自发情期卵泡的卵泡细胞中观察到相对于发情前期的卵泡细胞。然而，从发情前期到发情期，FF中BMP-15和GDF-9的水平降低（P < 0.05），而孕酮水平增加（P < 0.05）。当两种生长因子都加入到 IVM 培养物中时，Cox-2基因在卵丘细胞中的表达是对照的 4 倍（P < 0.01）。总之，虽然 BMP-15 和 GDF-9 似乎在 IVM 期间上调Cox-2转录物的水平，但这些旁分泌因子与Cox-2的反比关系基因表达和孕酮与Cox-2转录本的正相关表明，高孕酮水平可能与调节Cox-2基因表达的局部机制更相关。