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Prostaglandin E2 and vascular endothelial growth factor A mediate angiogenesis of human ovarian follicular endothelial cells. Trau HA et al. (2016) 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. 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). 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. 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. 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.//////////////////
Angiogenesis in the Primate Ovulatory Follicle Is Stimulated by Luteinizing Hormone via Prostaglandin E2. Trau HA et al. (2015) Rapid angiogenesis occurs as the ovulatory follicle is transformed into the corpus luteum. To determine if luteinizing hormone (LH)-stimulated prostaglandin E2 (PGE2) regulates angiogenesis in the ovulatory follicle, cynomolgus macaques received gonadotropins to stimulate multiple follicular development and chorionic gonadotropin (hCG) substituted for the LH surge to initiate ovulatory events. Before hCG, vascular endothelial cells were present in the perifollicular stroma but not amongst granulosa cells. Endothelial cells entered the granulosa cell layer 24-36 h after hCG, concomitant with the rise in follicular PGE2 and prior to ovulation, which occurs about 40 h after hCG. Intrafollicular administration of the PG synthesis inhibitor indomethacin was coupled with PGE2 replacement to demonstrate that indomethacin blocked and PGE2 restored follicular angiogenesis in a single, naturally developed monkey follicle in vivo. Intrafollicular administration of indomethacin plus an agonist selective for a single PGE2 receptor showed that PTGER1 and PTGER2 agonists most effectively stimulated angiogenesis within the granulosa cell layer. Endothelial cell tracing and three-dimensional reconstruction indicated that these capillary networks form via branching angiogenesis. To further explore how PGE2 mediates follicular angiogenesis, monkey ovarian microvascular endothelial cells (mOMECs) were isolated from ovulatory follicles. The mOMECs expressed all four PGE2 receptors in vitro. PGE2 and all PTGER agonists increased mOMEC migration. PTGER1 and PTGER2 agonists promoted sprout formation while the PTGER3 agonist inhibited sprouting in vitro. While PTGER1 and PTGER2 likely promote the formation of new capillaries, each PGE2 receptor may mediate aspects of PGE2's actions and, therefore, LH's ability to regulate angiogenesis in the primate ovulatory follicle.//////////////////
Identification of new ovulation-related genes in humans by comparing the transcriptome of granulosa cells before and after ovulation triggering in the same controlled ovarian stimulation cycle. Wissing ML 2014 et al.
STUDY QUESTION
Which genes and molecular mechanisms are involved in the human ovulatory cascade and final oocyte maturation?
SUMMARY ANSWER
Up-regulated genes in granulosa cells (GC) represented inflammation, angiogenesis, extracellular matrix, growth factors and genes previously associated with ovarian cancer, while down-regulated genes mainly represented cell cycle and proliferation.
WHAT IS KNOWN ALREADY
Radical changes occur in the follicle during final follicle maturation after the ovulatory trigger: these range from ensuring an optimal milieu for the oocyte in meiotic arrest to the release of a mature oocyte and remodeling into a corpus luteum. A wide range of mediators of final follicle maturation has been identified in rodents, non-human primates and cows.
STUDY DESIGN, SIZE, DURATION
Prospective cohort study including 24 women undergoing ovarian stimulation with the long gonadotrophin-releasing hormone agonist protocol during 2010-2012 at Holbk Fertility Clinic. Nine paired samples of GC and 24 paired samples of follicular fluid (FF) were obtained before and after recombinant human chorionic gonadotrophin (rhCG) administration.
PARTICIPANTS/MATERIALS, SETTING, METHODS
Nine paired (nine arrays before rhCG and nine arrays after rhCG) samples of GC mRNA were amplified and hybridized to Affymetrix Human Gene 1.0 ST GeneChip arrays, compared and bioinformatically analyzed. Eleven selected genes were validated by quantitative reverse transcriptase PCR. FF hormones were analyzed by enzyme-linked immunosorbent assay.
MAIN RESULTS AND THE ROLE OF CHANCE
Eleven hundred and eighty-six genes were differentially expressed (>2-fold, P<0.0001, false discovery rate <0.0012) when comparing GC isolated before and 36 h after hCG, among those were genes known to be expressed at ovulation, i.e. ADAMTS1 and HAS2. Many new ovulation-related genes were revealed, such as CD24, ANKRD22, CLDN11 and FBXO32. FF estrogen, androstenedione and anti-Mllerian hormone decreased significantly while progesterone increased, accompanied by radical changes in the expression of steroidogenic genes (CYP17A, CYP19A, HSD11B1 and HSD11B2, StAR). Genes related to inflammation, angiogenesis, extracellular matrix formation, growth factors and cancer were up-regulated while cell cycle genes were massively down-regulated. Seventy-two genes previously described in connection with ovarian cancer were among the highly regulated genes. In silico analysis for top upstream regulators of the ovulatory trigger suggested-besides LH-TNF, IGF1, PGR, AR, EGR1 (early growth response 1), ERK1/2 (extracellular signal regulated kinase 1/2) and CDKN1A (cyclin-dependent kinase inhibitor 1A) as potential mediators of the LH/hCG response.
LIMITATIONS, REASONS FOR CAUTION
The present dataset was generated from women under hormonal stimulation. However, comparison with a macaque natural cycle whole follicle ovulation dataset revealed major overlap, supporting the idea that the ovulation-related genes found in this study are relevant in the human natural cycle.
WIDER IMPLICATIONS OF THE FINDINGS
These data will serve as a research resource for genes involved in human ovulation and final oocyte maturation. Ovulation-related genes might be good candidate biomarkers of follicle and oocyte health. Further, some of the ovulation-related genes may serve as future ovarian cancer biomarkers.
STUDY FUNDING/COMPETING INTEREST(S)
Grants from the Research Fund of Region Sjlland are gratefully acknowledged. None of the authors declared any conflict of interest.
TRIAL REGISTRATION NUMBER
Not applicable.
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PTGER1 and PTGER2 receptors mediate regulation of progesterone synthesis and type 1 11{beta}-hydroxysteroid dehydrogenase activity by prostaglandin E2 in human granulosa lutein cells. Chandras C et al. In luteinizing granulosa cells, prostaglandin E(2) (PGE(2)) can exert luteotrophic actions, apparently via the cAMP signalling pathway. In addition to stimulating progesterone synthesis, PGE(2) can also stimulate oxidation of the physiological glucocorticoid, cortisol, to its inactive metabolite, cortisone, by the type 1 11beta-hydroxysteroid dehydrogenase (11betaHSD1) enzyme in human granulosa-lutein cells. Having previously shown these human ovarian cells to express functional G-protein coupled, E-series prostaglandin (PTGER)1, PTGER2 and PTGER4 receptors, the aim of this study was to delineate the roles of PTGER1 and PTGER2 receptors in mediating the effects of PGE(2) on steroidogenesis and cortisol metabolism in human granulosa-lutein cells. PGE(2)-stimulated concentration-dependent increases in both progesterone production and cAMP accumulation (by 1.9 +/- 0.1- and 18.7 +/- 6.8-fold respectively at 3000 nM PGE(2)). While a selective PTGER1 antagonist, SC19220, could partially inhibit the steroidogenic response to PGE(2) (by 55.9 +/- 4.1% at 1000 nM PGE(2)), co-treatment with AH6809, a mixed PTGER1/PTGER2 receptor antagonist, completely abolished the stimulation of progesterone synthesis at all tested concentrations of PGE(2) and suppressed the stimulation of cAMP accumulation. Both PGE(2) and butaprost (a preferential PTGER2 receptor agonist) stimulated concentration-dependent increases in cortisol oxidation by 11betaHSD1 (by 42.5 +/- 3.1 and 40.0 +/- 3.0% respectively, at PGE(2) and butaprost concentrations of 1000 nM). Co-treatment with SC19220 enhanced the ability of both PGE(2) and butaprost to stimulate 11betaHSD1 activity (by 30.2 +/- 0.2 and 30.5 +/- 0.6% respectively), whereas co-treatment with AH6809 completely abolished the 11betaHSD1 responses to PGE(2) and butaprost. These findings implicate the PTGER2 receptor-cAMP signalling pathway in the stimulation of progesterone production and 11betaHSD1 activity by PGE(2) in human granulosa-lutein cells.
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