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Comment |
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.//////////////////
A prostaglandin E2 receptor antagonist prevents pregnancies during a preclinical contraceptive trial with female macaques. Peluffo MC 2014 et al.
STUDY QUESTION
Can administration of a prostaglandin (PG) E2 receptor 2 (PTGER2) antagonist prevent pregnancy in adult female monkeys by blocking periovulatory events in the follicle without altering menstrual cyclicity or general health?
SUMMARY ANSWER
This is the first study to demonstrate that a PTGER2 antagonist can serve as an effective non-hormonal contraceptive in primates.
WHAT IS KNOWN ALREADY
The requirement for PGE2 in ovulation and the release of an oocyte surrounded by expanded cumulus cells (cumulus-oocyte expansion; C-OE) was established through the generation of PTGS2 and PTGER2 null-mutant mice. A critical role for PGE2 in primate ovulation is supported by evidence that intrafollicular injection of indomethacin in rhesus monkeys suppressed follicle rupture, whereas co-injection of PGE2 with indomethacin resulted in ovulation.
STUDY DESIGN, SIZE, DURATION
First, controlled ovulation protocols were performed in adult, female rhesus monkeys to analyze the mRNA levels for genes encoding PGE2 synthesis and signaling components in the naturally selected pre-ovulatory follicle at different times after the ovulatory hCG stimulus (0, 12, 24, 36 h pre-ovulation; 36 h post-ovulation, n = 3-4/time point). Second, controlled ovarian stimulation cycles were utilized to obtain multiple cumulus-oocyte complexes (COCs) from rhesus monkeys to evaluate the role of PGE2 in C-OE in vitro (n = 3-4 animals/treatment; =3 COCs/animal/treatment). Third, adult cycling female cynomolgus macaques were randomly assigned (n = 10/group) to vehicle (control) or PTGER2 antagonist (BAY06) groups to perform a contraceptive trial. After the first treatment cycle, a male of proven fertility was introduced into each group and they remained housed together for the duration of the 5-month contraceptive trial that was followed by a post-treatment reversibility trial.
PARTICIPANTS/MATERIALS, SETTING, METHODS
Quantitative real-time PCR, COC culture and expansion, immunofluorescence/confocal microscopy, enzyme immunoassay, contraceptive trial, ultrasonography, complete blood counts, serum biochemistry tests and blood lipid profiles.
MAIN RESULTS AND THE ROLE OF CHANCE
Several mRNAs encoding proteins involved in PGE2 synthesis, metabolism and signaling increase (P < 0.05) in the periovulatory follicle after administration of an ovulatory hCG bolus. PGE2 signaling through PTGER2 induces cumulus cell expansion and production of hyaluronic acid, which are critical events for fertilization. Moreover, chronic administration of a selective PTGER2 antagonist resulted in a significant (P < 0.05 versus vehicle-treated controls) contraceptive effect without altering steroid hormone patterns or menstrual cyclicity during a 5-months contraceptive trial. Fertility recovered as early as 1 month after ending treatment.
LIMITATIONS, REASONS FOR CAUTION
This is a proof-of-concept study in a non-human primate model. Further investigations are warranted to elucidate the mechanism(s) of PTGER2 antagonist action in the primate ovary. Although PTGER2 antagonist treatment did not produce any obvious undesirable effects, improvements in the mode of administration, as well as the efficacy of these compounds, are necessary to consider such a contraceptive for women.
WIDER IMPLICATIONS OF THE FINDINGS
Monitoring as well as improving the efficacy and safety of female contraceptives is an important public health activity. Even though hormonal contraceptives are effective for women, concerns remain regarding their side-effects and long-term use because of the widespread actions of such steroidal products in many tissues. Moreover, some women cannot take hormones for medical reasons. Thus, development of non-hormonal contraceptives for women is warranted.
STUDY FUNDING/COMPETING INTEREST(S)
Supported by Bayer HealthCare Pharmaceuticals, The Eunice Kennedy Shriver NICHD Contraceptive Development and Research Center (U54 HD055744), NIH Office of the Director (Oregon National Primate Research Center P51 OD011092), and a Lalor Foundation Postdoctoral Basic Research Fellowship (MCP). The use of the Leica confocal was supported by grant number S10RR024585. Some of the authors (N.B., A.R., K.-H.F., U.F., B.B. and B.L.) are employees of Bayer Healthcare Pharma.
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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 Holb?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-M?an 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 Sj?and are gratefully acknowledged. None of the authors declared any conflict of interest.
TRIAL REGISTRATION NUMBER
Not applicable.
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Moon et al. (1986) reported that PGE2 increased progesterone accumulation in cultured rat granulosa cells, in part, by decreasing progesterone catabolism to 20 alpha-reduced progestins. In contrast, PGF2 alpha stimulated 20 alpha-hydroxysteroid dehydrogenase activity, thus increasing progesterone catabolism. Combined treatment with PGE2 and PGF2 alpha augmented progesterone accumulation to levels above controls but below those attained with PGE2 alone. Thus, PGE2 and PGF2 alpha exert opposite effects on progesterone production. Reddoch et al. (1982) reported that PGE2 and dibutyryl-cAMP enhance the response of rat granulosa cells to FSH. Eramaa et al. (1996) reported that prostaglandin E2 induces inhibin alpha- and beta A-subunit mRNA and secretion of dimeric inhibin A in cultured human granulosa-luteal cells. Salustri et al. (1985) suggest that the cumulus oophorus expansion-promoting action of granulosa cells is mediated by PGE2. Goverde et al. (1993) reported the enhancement by prostaglandin E2 of cumulus cell outgrowth in vitro.
Mice carrying a null mutation for either of the two cyclooxygenase (COX) isoenzymes, necessary for prostanoid production, exhibit several isotype-specific reproductive abnormalities. Mice deficient in COX-1 are fertile but have decreased pup viability, whereas mice deficient in COX-2 fail to ovulate and have abnormal implantation and decidualization responses. Davis et al. (1999) reported that wild-type and COX-1(-/-) mice ovulated in response to PMSG/hCG; very few COX-2(-/-) animals responded to this regimen. The defect in ovulation in COX-2 mutants was attributed to both an abnormal cumulus oophorum expansion and subsequent stigmata formation. Gonadotropin stimulation and concurrent treatment with PGE2 or interleukin-1beta resulted in ovulation of COX-2(-/-) mice comparable to that in COX-2(+/+).
Cyclooxygenase-2 derived prostaglandin E2 directs oocyte maturation by differentially influencing multiple signaling pathways. Takahashi T et al. The process of oocyte maturation which impacts ovulation and fertilization is complex and requires an integration of the endocrine, paracrine, juxtacrine and autocrine signaling pathways. This process involves an intimate interaction between the oocyte and encircling cumulus cells within a follicle, a unique venue for somatic and germ cell communication. Cumulus cell expansion and resumption of meiosis with germinal vesicle breakdown (GVBD) are major events in oocyte maturation. Cyclooxygenase-2 (COX-2) derived prostaglandin E2 (PGE2) is a known critical mediator of oocyte maturation, but the diverse function of this lipid mediator in oocyte maturation, ovulation and fertilization has not been fully appreciated. We show here that gonadotropins in coordination with PGE2 signaling via its cell surface G-protein coupled EP2 and EP4 receptor subtypes directs cumulus cell expansion and survival, and oocyte meiotic maturation by differentially impacting PKA, MAPK, NF-B and PI3K/Akt pathways. This study is unique in the sense that it provides evidence for new site- and event-specific involvement of these signaling pathways under the influence of COX-2 derived PGE2 during the critical stages of this somatic-germ cell interaction, an absolute requirement for oocyte maturation.
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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Mutations |
2 mutations
Species: mouse
Mutation name: None
type: null mutation
fertility: infertile - ovarian defect
Comment: Reproductive failure and reduced blood pressure in mice lacking the EP2 prostaglandin E2 receptor. Tilley SL 1999 et al.
Prostaglandins (PGs) are bioactive lipids that modulate a broad spectrum of biologic processes including reproduction and circulatory homeostasis. Although reproductive functions of mammals are influenced by PGs at numerous levels, including ovulation, fertilization, implantation, and decidualization, it is not clear which PGs are involved and whether a single mechanism affects all reproductive functions. Using mice deficient in 1 of 4 prostaglandin E2 (PGE2) receptors -- specifically, the EP2 receptor -- we show that Ep2(-/-) females are infertile secondary to failure of the released ovum to become fertilized in vivo. Ep2(-/-) ova could be fertilized in vitro, suggesting that in addition to previously defined roles, PGs may contribute to the microenvironment in which fertilization takes place. In addition to its effects on reproduction, PGE2 regulates regional blood flow in various vascular beds. However, its role in systemic blood pressure homeostasis is not clear. Mice deficient in the EP2 PGE2 receptor displayed resting systolic blood pressure that was significantly lower than in wild-type controls. Blood pressure increased in these animals when they were placed on a high-salt diet, suggesting that the EP2 receptor may be involved in sodium handling by the kidney. These studies demonstrate that PGE2, acting through the EP2 receptor, exerts potent regulatory effects on two major physiologic processes: blood pressure homeostasis and in vivo fertilization of the ovum.
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Species: mouse
Mutation name: None
type: null mutation
fertility: subfertile
Comment: Abortive expansion of the cumulus and impaired fertility in mice lacking the prostaglandin E receptor subtype EP(2). Hizaki H 1999 et al.
Female mice lacking the gene encoding the prostaglandin (PG) E(2) receptor subtype EP(2) (EP(2)(-/-)) become pregnant and deliver their pups at term, but with a much reduced litter size. A decrease in ovulation number and a much reduced fertilization rate were observed in EP(2)(-/-) females without difference of the uterus to support implantation of wild-type embryos. Treatment with gonadotropins induced EP(2) mRNA expression in the cumulus cells of ovarian follicles of wild-type mice. The immature cumuli oophori from wild-type mice expanded in vitro in response to both follicle-stimulating hormone and PGE(2), but the response to PGE(2) was absent in those from EP(2)(-/-) mice. Cumulus expansion proceeded normally in preovulatory follicles but became abortive in a number of ovulated complexes in EP(2)(-/-) mice, indicating that EP(2) is involved in cumulus expansion in the oviduct in vivo. No difference in the fertilization rate between wild-type and EP(2)(-/-) mice was found in in vitro studies using cumulus-free oocytes. These results indicate that PGE(2) cooperates with gonadotropin to complete cumulus expansion for successful fertilization.
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