Prostaglandin E2 (PGE2) is an essential intrafollicular regulator of ovulation. In contrast with the one-gene, one-protein concept for synthesis of peptide signaling molecules, production and ...metabolism of bioactive PGE2 requires controlled expression of many proteins, correct subcellular localization of enzymes, coordinated PGE2 synthesis and metabolism, and prostaglandin transport in and out of cells to facilitate PGE2 action and degradation. Elevated intrafollicular PGE2 is required for successful ovulation, so disruption of PGE2 synthesis, metabolism or transport may yield effective contraceptive strategies.
This review summarizes case reports and studies on ovulation inhibition in women and macaques treated with cyclooxygenase inhibitors published from 1987 to 2014. These findings are discussed in the context of studies describing levels of mRNA, protein, and activity of prostaglandin synthesis and metabolic enzymes as well as prostaglandin transporters in ovarian cells.
The ovulatory surge of LH regulates the expression of each component of the PGE2 synthesis-metabolism-transport pathway within the ovulatory follicle. Data from primary ovarian cells and cancer cell lines suggest that enzymes and transporters can cooperate to optimize bioactive PGE2 levels. Elevated intrafollicular PGE2 mediates key ovulatory events including cumulus expansion, follicle rupture and oocyte release. Inhibitors of the prostaglandin-endoperoxide synthase 2 (PTGS2) enzyme (also known as cyclooxygenase-2 or COX2) reduce ovulation rates in women. Studies in macaques show that PTGS2 inhibitors can reduce the rates of cumulus expansion, oocyte release, follicle rupture, oocyte nuclear maturation and fertilization. A PTGS2 inhibitor reduced pregnancy rates in breeding macaques when administered to simulate emergency contraception. However, PTGS2 inhibition did not prevent pregnancy in monkeys when administered to simulate monthly contraceptive use.
PTGS2 inhibitors alone may be suitable for use as emergency contraceptives. However, drugs of this class are unlikely to be effective as monthly contraceptives. Inhibitors of additional PGE2 synthesis enzymes or modulation of PGE2 metabolism or transport also hold potential for reducing follicular PGE2 and preventing ovulation. Approaches which target multiple components of the PGE2 synthesis-metabolism-transport pathway may be required to effectively block ovulation and lead to the development of novel contraceptive options for women. Therapies which target PGE2 may also impact disorders of the uterus and could also have benefits for women's health in addition to contraception.
Abstract
The midcycle surge of LH sets in motion interconnected networks of signaling cascades to bring about rupture of the follicle and release of the oocyte during ovulation. Many mediators of ...these LH-induced signaling cascades are associated with inflammation, leading to the postulate that ovulation is similar to an inflammatory response. First responders to the LH surge are granulosa and theca cells, which produce steroids, prostaglandins, chemokines, and cytokines, which are also mediators of inflammatory processes. These mediators, in turn, activate both nonimmune ovarian cells as well as resident immune cells within the ovary; additional immune cells are also attracted to the ovary. Collectively, these cells regulate proteolytic pathways to reorganize the follicular stroma, disrupt the granulosa cell basal lamina, and facilitate invasion of vascular endothelial cells. LH-induced mediators initiate cumulus expansion and cumulus oocyte complex detachment, whereas the follicular apex undergoes extensive extracellular matrix remodeling and a loss of the surface epithelium. The remainder of the follicle undergoes rapid angiogenesis and functional differentiation of granulosa and theca cells. Ultimately, these functional and structural changes culminate in follicular rupture and oocyte release. Throughout the ovulatory process, the importance of inflammatory responses is highlighted by the commonalities and similarities between many of these events associated with ovulation and inflammation. However, ovulation includes processes that are distinct from inflammation, such as regulation of steroid action, oocyte maturation, and the eventual release of the oocyte. This review focuses on the commonalities between inflammatory responses and the process of ovulation.
Abstract Follicular androgens are important for successful ovulation and fertilization. The classical nuclear androgen receptor (AR) is a transcription factor expressed in the cells of the ovarian ...follicle. Androgen actions can also occur via membrane androgen receptor SLC39A9. Studies in fish ovary demonstrated that androgens bind to SLC39A9 and increase intracellular zinc to regulate ovarian cell function. To determine if SLC39A9 is expressed and functional in the key cell types of the mammalian ovulatory follicle, adult female cynomolgus macaques underwent ovarian stimulation. Ovaries or ovarian follicular aspirates were harvested at 0, 12, 24, and 36 hours after human chorionic gonadotropin (hCG). SLC39A9 and AR mRNA and protein were present in granulosa, theca, and vascular endothelial cells across the entire 40-hour ovulatory window. Testosterone, bovine serum albumin–conjugated testosterone (BSA-T), and androstenedione stimulated zinc influx in granulosa, theca, and vascular endothelial cells. The SLC39A9-selective agonist (−)-epicatechin also stimulated zinc influx in vascular endothelial cells. Taken together, these data support the conclusion that SLC39A9 activation via androgen induces zinc influx in key ovarian cells. Testosterone, BSA-T, and androstenedione each increased proliferation in vascular endothelial cells, indicating the potential involvement of SLC39A9 in ovulatory angiogenesis. Vascular endothelial cell migration also increased after treatment with testosterone, but not after treatment with BSA-T or androstenedione, suggesting that androgens stimulate vascular endothelial cell migration through nuclear AR but not SLC39A9. The presence of SLC39A9 receptors and SLC39A9 activation by follicular androstenedione concentrations suggests that androgen activation of ovarian SLC39A9 may regulate ovulatory changes in the mammalian follicle.
Abstract
Placental growth factor (PGF) is member of the vascular endothelial growth factor (VEGF) family of angiogenesis regulators. VEGFA is an established regulator of ovulation and formation of ...the corpus luteum. To determine whether PGF also mediates aspects of ovulation and luteinization, macaques received gonadotropins to stimulate multiple follicular development. Ovarian biopsies and whole ovaries were collected before (0 hours) and up to 36 hours after human chorionic gonadotropin (hCG) administration to span the ovulatory interval. PGF and VEGFA were expressed by both granulosa cells and theca cells. In follicular fluid, PGF and VEGFA levels were lowest before hCG. PGF levels remained low until 36 hours after hCG administration, when PGF increased sevenfold to reach peak levels. Follicular fluid VEGFA increased threefold to reach peak levels at 12 hours after hCG, then dropped to intermediate levels. To explore the roles of PGF and VEGFA in ovulation, luteinization, and follicular angiogenesis in vivo, antibodies were injected into the follicular fluid of naturally developed monkey follicles; ovariectomy was performed 48 hours after hCG, with ovulation expected about 40 hours after hCG. Intrafollicular injection of control immunoglobulin G resulted in no retained oocytes, follicle rupture, and structural luteinization, including granulosa cell hypertrophy and capillary formation in the granulosa cell layer. PGF antibody injection resulted in oocyte retention, abnormal rupture, and incomplete luteinization, with limited and disorganized angiogenesis. Injection of a VEGFA antibody resulted in oocyte retention and very limited follicle rupture or structural luteinization. These studies demonstrate that PGF, in addition to VEGFA, is required for ovulation, luteinization, and follicular angiogenesis in primates.
Placental growth factor is produced within the primate ovulatory follicle and is necessary for oocyte release, follicle rupture, luteinization, and new vessel formation within the ovulatory follicle.
Ovulation is triggered by the gonadotropin surge that induces the expression of two key genes, progesterone receptor (Pgr) and prostaglandin-endoperoxide synthase 2 (Ptgs2), in the granulosa cells of ...preovulatory follicles. Their gene products PGR and PTGS2 activate two separate pathways that are both essential for successful ovulation. Here, we show that the PGR plays an additional essential role: it attenuates ovulatory inflammation by diminishing the gonadotropin surge-induced Ptgs2 expression. PGR indirectly terminates Ptgs2 expression and PGE2 synthesis in granulosa cells by inhibiting the nuclear factor κB (NF-κB), a transcription factor required for Ptgs2 expression. When the expression of PGR is ablated in granulosa cells, the ovary undergoes a hyperinflammatory condition manifested by excessive PGE2 synthesis, immune cell infiltration, oxidative damage, and neoplastic transformation of ovarian cells. The PGR-driven termination of PTGS2 expression may protect the ovary from ovulatory inflammation.
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•Successful ovulation requires timely expression of PGR in the ovarian granulosa cells•PGR terminates LH-surge-driven PGE2 synthesis by decreasing PTGS2 (COX-2) expression•Impaired PGR expression leads to hyperinflammation and tissue damage in the ovary
A preovulatory LH surge induces the expression of PGR and the production of PGE2, a proinflammatory prostaglandin, in preovulatory granulosa cells. Park et al. show that PGR then suppresses PGE2 synthesis by decreasing the expression of PTGS2. Such PGR-driven termination temporarily confines ovulatory inflammation within a short period.
Neurotensin (NTS) is a 13‐amino acid peptide which is highly expressed in the mammalian ovary in response to the luteinizing hormone surge. Antibody neutralization of NTS in the ovulatory follicle of ...the cynomolgus macaque impairs ovulation and induces follicular vascular dysregulation, with excessive pooling of red blood cells in the follicle antrum. We hypothesize that NTS is an essential intrafollicular regulator of vascular permeability. In the present study, follicle injection of the NTS receptor antagonist SR142948 also resulted in vascular dysregulation. To measure vascular permeability changes in vitro, primary macaque ovarian microvascular endothelial cells (mOMECs) were enriched from follicle aspirates and studied in vitro. When treated with NTS, permeability of mOMECs decreased. RNA sequencing (RNA‐Seq) of mOMECs revealed high mRNA expression of the permeability‐regulating adherens junction proteins N‐cadherin (CDH2) and K‐cadherin (CDH6). Immunofluorescent detection of CDH2 and CDH6 confirmed expression and localized these cadherins to the cell–cell boundaries, consistent with function as components of adherens junctions. mOMECs did not express detectable levels of the typical vascular endothelial cadherin, VE‐cadherin (CDH5) as determined by RNA‐Seq, qPCR, western blot, and immunofluorescence. Knockdown of CDH2 or CDH6 via siRNA abrogated the NTS effect on mOMEC permeability. Collectively, these data suggest that NTS plays an ovulation‐critical role in vascular permeability maintenance, and that CDH2 and CDH6 are involved in the permeability modulating effect of NTS on the ovarian microvasculature. NTS can be added to a growing number of angiogenic regulators which are critical for successful ovulation.
Neurotensin (NTS), a paracrine mediator of ovulation, regulates vascular permeability changes during angiogenesis of the ovulatory follicle. Permeability of follicular microvascular endothelial cells is controlled by adherens junctions, primarily N‐cadherin (CDH2) and K‐cadherin (CDH6) junctions. NTS acts directly at endothelial cells to decrease permeability in a CDH2‐ and CDH6‐dependent manner.
Abstract Wrisley DM, Stephens MJ, Mosley S, Wojnowski A, Duffy J, Burkard R. Learning effects of repetitive administrations of the Sensory Organization Test in healthy young adults. Objectives To ...evaluate the learning effect of multiple administrations of the Sensory Organization Test (SOT) on performance and to begin to establish clinical meaningful change scores for the SOT. Design Descriptive case series. Setting University-affiliated clinic. Participants Healthy young adults (6 men, 7 women; mean age, 24±4y). Intervention All subjects performed the standardized SOT using the SMART EquiTest 5 times over a 2-week period, and 1 month later. Main Outcome Measure Composite and individual SOT test condition standardized equilibrium scores. Results Test-retest reliability (intraclass correlation coefficient model 2,3) of the composite (.67) and equilibrium score (range, .35–.79) were fair to good. Repeated-measures analysis of variance revealed a significant ( P <.05) increase in the composite and equilibrium scores for conditions 4, 5, and 6 over the 5 sessions that plateaued after the third session, and were retained at 1 month. The 95% confidence interval for the composite score change from session 1 to session 4, the plateau of the learning effect, was 3.9 to 8.1. Conclusions Although the findings of this study would indicate that multiple baseline measures are desirable for the more challenging conditions, a composite change of greater than 8 points would indicate change due to rehabilitation.
Angiogenesis is essential to both ovulation and the formation of the corpus luteum. The thrombospondin (THBS) family of glycoproteins plays diverse roles in regulation of angiogenesis, but the role ...of these vascular regulators in ovulation and luteinization remain to be elucidated. Using the cynomolgus macaque as a model for human ovulation, we demonstrated that levels of THBS1 mRNA and protein in preovulatory follicle granulosa cells increased after the ovulatory gonadotropin surge, with peak levels just before the expected time of ovulation. THBS1 treatment of monkey ovarian microvascular endothelial cells
in vitro
stimulated migration, proliferation, and capillary sprout formation, consistent with a pro-angiogenic action of THBS1. Injection of an anti-THBS1 antibody into monkey preovulatory follicles reduced rates of follicle rupture and oocyte release in response to an ovulatory gonadotropin stimulus when compared with control IgG-injected follicles. Interestingly, two of three oocytes from anti-THBS1 antibody injected follicles were germinal vesicle intact, indicating that meiosis failed to resume as anticipated. Follicles injected with anti-THBS1 antibody also showed reduced granulosa cell layer expansion, endothelial cell invasion, and capillary formation when compared to control IgG-injected follicles. Overall, these findings support a critical role for THBS1 in follicular angiogenesis, with implications for both successful ovulation and corpus luteum formation.
Context:
The FSH receptor (FSHR) is traditionally thought to play a role in female reproductive physiology solely within the context of ovarian FSHR. However, FSHR is also expressed in endothelial ...cells of the placental vasculature and human umbilical cord vessels, suggesting additional facets of female reproduction regulated by extragonadal FSHR.
Objective:
We sought to determine the functional role of FSHR on human umbilical cord endothelial cells (HUVECs), hypothesizing that activation of the FSHR would stimulate angiogenesis.
Design:
The ability of FSH to stimulate several angiogenic processes in HUVECs was determined.
Setting:
This was a laboratory-based study using commercially prepared HUVECs.
Results:
Tube formation, wound healing, cell migration, cell proliferation, nitric oxide production, and cell survival were stimulated in response to FSH. Quantitative comparisons between HUVECs incubated with maximally stimulatory concentrations of FSH vs vascular endothelial growth factor (VEGF), a well-characterized angiogenic factor, revealed that FSH is as efficacious as VEGF in promoting angiogenic processes. FSH did not provoke increased secretion of VEGF by HUVECs, suggesting the direct stimulation of angiogenic processes by FSH in endothelial cells. In contrast to gonadal cells, the FSHR on HUVECs did not mediate an FSH-stimulated increase in cAMP. However, increased phosphorylation of AKT in response to FSH was observed, suggesting that FSH stimulation of HUVEC FSHR stimulates the PI3K/AKT signaling pathway.
Conclusions:
Our studies reveal a novel role for FSHR in female reproductive physiology. Its ability to promote angiogenesis in placental endothelial cells suggests that the FSHR may have an influential role in pregnancy.
Angiogenesis within the ovarian follicle is an important component of ovulation. New capillary growth is initiated by the ovulatory surge of luteinizing hormone (LH), and angiogenesis is well ...underway at the time of follicle rupture. LH-stimulated follicular production of vascular growth factors has been shown to promote new capillary formation in the ovulatory follicle. The possibility that LH acts directly on ovarian endothelial cells to promote ovulatory angiogenesis has not been addressed. For these studies, ovaries containing ovulatory follicles were obtained from cynomolgus macaques and used for histological examination of ovarian vascular endothelial cells, and monkey ovarian microvascular endothelial cells (mOMECs) were enriched from ovulatory follicles for in vitro studies. mOMECs expressed LHCGR mRNA and protein, and immunostaining confirmed LHCGR protein in endothelial cells of ovulatory follicles in vivo. Human chorionic gonadotropin (hCG), a ligand for LHCGR, increased mOMEC proliferation, migration and capillary-like sprout formation in vitro. Treatment of mOMECs with hCG increased cAMP, a common intracellular signal generated by LHCGR activation. The cAMP analog dibutyryl cAMP increased mOMEC proliferation in the absence of hCG. Both the protein kinase A (PKA) inhibitor H89 and the phospholipase C (PLC) inhibitor U73122 blocked hCG-stimulated mOMEC proliferation, suggesting that multiple G-proteins may mediate LHCGR action. Human ovarian microvascular endothelial cells (hOMECs) enriched from ovarian aspirates obtained from healthy oocyte donors also expressed LHCGR. hOMECs also migrated and proliferated in response to hCG. Overall, these findings indicate that the LH surge may directly activate ovarian endothelial cells to stimulate angiogenesis of the ovulatory follicle. Summary Sentence The ovulatory surge of LH may regulate follicular angiogenesis by acting directly at LHCGRs on ovarian vascular endothelial cells. Graphical Abstract
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