Sayasith Khampoune, Bouchard Nadine, Doré Monique, Sirois Jean
Faculté de Médecine Vétérinaire, Centre de Recherche en Reproduction Animale Département de Biomédecine Vétérinaire, Université de Montréal, Saint-Hyacinthe, Québec, Canada.
Mol Reprod Dev. 2009 Feb;76(2):191-201. doi: 10.1002/mrd.20941.
The objectives of the study were to clone the primary structure of the prostaglandin E2 receptor subtype 2 (PTGER2) cDNA and to characterize its regulation in equine follicles during gonadotropin-induced ovulation. Results from DNA isolation indicated that the equine PTGER2 cDNA encodes a predicted 353-amino acid protein, which is highly similar (76-85%) to known mammalian homologues. The regulation of PTGER2 was studied by semi-quantitative RT-PCR/Southern blot using preparations of theca interna and mural granulosa cells isolated from equine follicles 0-39 hr post-treatment with human chorionic gonadotropin (hCG). Results indicated that a significant increase of PTGER2 mRNA occurred at 24 and 39 hr post-hCG in granulosa cells, and 30 and 33 hr post-hCG in theca cells (P < 0.05). Immunohistochemical staining and immunoblotting performed on equine follicular samples showed a corresponding increase of PTGER2 protein in both cell types after treatment with hCG. Levels of PTGER2 mRNA were also high in uterus, thymus and spleen, but moderate to low in other tested tissues. In the ovary, the expression of PTGER4 mRNA was observed and predominantly occurred in granulosa cells, with highest abundance of transcripts observed at 12 and 39 hr post-hCG. Thus, this study reports for the first time in mares that the ovulatory process is accompanied by the gonadotropin-dependent up-regulation of PTGER2 and PTGER4, which may in turn regulate PGE2-mediated preovulatory effects.
本研究的目的是克隆前列腺素E2受体亚型2（PTGER2）cDNA的一级结构，并表征其在促性腺激素诱导排卵过程中马卵泡中的调控情况。DNA分离结果表明，马PTGER2 cDNA编码一种预测的353个氨基酸的蛋白质，与已知的哺乳动物同源物高度相似（76-85%）。使用从人绒毛膜促性腺激素（hCG）处理后0-39小时的马卵泡中分离的内膜和壁颗粒细胞制备物，通过半定量RT-PCR/ Southern印迹法研究了PTGER2的调控。结果表明，颗粒细胞在hCG处理后24和39小时，内膜细胞在hCG处理后30和33小时，PTGER2 mRNA显著增加（P < 0.05）。对马卵泡样本进行的免疫组织化学染色和免疫印迹显示，hCG处理后两种细胞类型中PTGER2蛋白相应增加。PTGER2 mRNA水平在子宫、胸腺和脾脏中也很高，但在其他测试组织中为中度至低度。在卵巢中，观察到PTGER4 mRNA的表达，主要发生在颗粒细胞中，在hCG处理后12和39小时观察到转录本丰度最高。因此，本研究首次在母马中报道，排卵过程伴随着促性腺激素依赖性的PTGER2和PTGER4上调，这可能反过来调节PGE2介导的排卵前效应。
