Trau Heidi A, Brännström Mats, Curry Thomas E, Duffy Diane M
Department of Physiological Sciences, Eastern Virginia Medical School, Norfolk, VA 23501, USA.
Department of Obstetrics and Gynecology, The Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden.
Hum Reprod. 2016 Feb;31(2):436-44. doi: 10.1093/humrep/dev320. Epub 2016 Jan 5.
Which receptors for prostaglandin E2 (PGE2) and vascular endothelial growth factor A (VEGFA) mediate angiogenesis in the human follicle around the time of ovulation?
PGE2 and VEGFA act via multiple PGE2 receptors (PTGERs) and VEGF receptors (VEGFRs) to play complementary roles in follicular angiogenesis.
Production of PGE2 and VEGFA by the follicle are prerequisites for ovulation. PGE2 is an emerging regulator of angiogenesis and has not been examined in the context of the human ovulatory follicle. VEGFA is an established regulator of follicular angiogenesis.
STUDY DESIGN, SIZE, DURATION: Ovarian biopsies containing the ovulatory follicle were obtained from 11 women of reproductive age (30-45 years) undergoing surgery for laparoscopic sterilization. In some cases, women received hCG to substitute for the ovulatory LH surge before ovarian biopsy. In addition, aspirates from four women of reproductive age (18-31 years) undergoing gonadotrophin stimulation for oocyte donation were obtained for isolation of human ovarian microvascular endothelial cells (hOMECs).
PARTICIPANTS/MATERIALS, SETTING, METHODS: Ovarian biopsies were utilized for immunocytochemical detection of von Willebrand factor to identify endothelial cells. hOMECs were cultured with PGE2, PTGER receptor selective agonists, VEGFA, or VEGFR selective agonists. hOMECs were assessed for proliferation by Ki67 immunocytochemistry. hOMEC migration was determined by counting cells which migrated through a porous membrane in vitro. Sprout formation was quantified by determining sprout number and length from photographs take after culture of hOMECs in a 3-dimensional matrix.
Endothelial cells were not observed within the granulosa cell layer of human ovulatory follicles prior to an ovulatory dose of hCG and were first seen amongst granulosa cells 18-34 h after hCG. In vitro, PGE2 enhanced migration and sprout formation but did not alter hOMEC proliferation. Agonists selective for each PTGER increased migration with no change in proliferation. PTGER1 and PTGER2 agonists increased the number of sprouts, while only PTGER1 affected sprout length. VEGFA increased hOMEC proliferation, migration, and formation of structures resembling capillary sprouts. Signaling through VEGFR1 promoted hOMEC migration, proliferation, and the formation of few, long endothelial cell sprouts, while VEGFR2 stimulation promoted hOMEC migration and the formation of many, short sprouts. All effects of treatments in vitro were considered significant at P < 0.05.
LIMITATIONS, REASONS FOR CAUTION: While primary cultures of hOMECs respond to PGE2 and VEGFA differently than other cultured endothelial cells, hOMECs may not respond to PGE2 and VEGFA in vivo as they do in vitro.
Agonists and antagonists selective for PTGER1, PTGER2, VEGFR1, or VEGFR2 may have therapeutic value to promote or prevent ovulation in women.
STUDY FUNDING/COMPETING INTERESTS: This research was supported by grant funding from the Eunice Kennedy Shriver National Institutes of Child Health and Human Development (HD071875 to D.M.D., T.E.C., M.B.). The authors have no conflicts of interest to disclose.
排卵前后人卵泡中，前列腺素E2（PGE2）和血管内皮生长因子A（VEGFA）的哪些受体介导血管生成？
PGE2和VEGFA通过多种PGE2受体（PTGERs）和VEGF受体（VEGFRs）发挥作用，在卵泡血管生成中起互补作用。
卵泡产生PGE2和VEGFA是排卵的先决条件。PGE2是一种新兴的血管生成调节因子，尚未在人排卵卵泡的背景下进行研究。VEGFA是卵泡血管生成的既定调节因子。
研究设计、规模、持续时间：从11名接受腹腔镜绝育手术的育龄妇女（30 - 45岁）中获取含有排卵卵泡的卵巢活检组织。在某些情况下，妇女在卵巢活检前接受hCG以替代排卵时的促黄体生成素高峰。此外，从4名接受促性腺激素刺激以进行卵母细胞捐赠的育龄妇女（18 - 31岁）中获取抽吸物，用于分离人卵巢微血管内皮细胞（hOMECs）。
参与者/材料、环境、方法：利用卵巢活检组织进行免疫细胞化学检测血管性血友病因子，以识别内皮细胞。将hOMECs与PGE2、PTGER受体选择性激动剂、VEGFA或VEGFR选择性激动剂一起培养。通过Ki67免疫细胞化学评估hOMECs的增殖。通过计数体外穿过多孔膜迁移的细胞来确定hOMECs的迁移。通过在三维基质中培养hOMECs后从照片中确定芽的数量和长度来量化芽的形成。
在给予排卵剂量的hCG之前，未在人排卵卵泡的颗粒细胞层内观察到内皮细胞，在hCG后18 - 34小时首次在颗粒细胞中见到。在体外，PGE2增强迁移和芽的形成，但不改变hOMECs的增殖。对每种PTGER具有选择性的激动剂增加迁移，而增殖无变化。PTGER1和PTGER2激动剂增加芽的数量，而只有PTGER1影响芽的长度。VEGFA增加hOMECs的增殖、迁移以及类似毛细血管芽结构的形成。通过VEGFR1的信号传导促进hOMECs的迁移、增殖以及少数长内皮细胞芽的形成，而VEGFR2刺激促进hOMECs的迁移以及许多短芽的形成。体外处理的所有效应在P < 0.05时被认为具有统计学意义。
局限性、谨慎的原因：虽然hOMECs的原代培养对PGE2和VEGFA的反应与其他培养的内皮细胞不同，但hOMECs在体内可能不像在体外那样对PGE2和VEGFA产生反应。
对PTGER1、PTGER2、VEGFR1或VEGFR2具有选择性的激动剂和拮抗剂可能对促进或预防女性排卵具有治疗价值。
研究资金/利益冲突：本研究由尤妮斯·肯尼迪·施赖弗国家儿童健康与人类发展研究所的资助（授予D.M.D.、T.E.C.、M.B.的HD071875）。作者没有利益冲突需要披露。
