Neurotensin modulates ovarian vascular permeability via adherens junctions.

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 expression, regulation, and function during the ovulatory period in the mouse ovary†.

The luteinizing hormone (LH) surge induces paracrine mediators within the ovarian follicle that promote ovulation. The present study explores neurotensin (NTS), a neuropeptide, as a potential ovulatory mediator in the mouse ovary. Ovaries and granulosa cells (GCs) were collected from immature 23-day-old pregnant mare serum gonadotropin primed mice before (0 h) and after administration of human chorionic gonadotropin (hCG; an LH analog) across the periovulatory period (4, 8, 12, and 24 h). In response to hCG, Nts expression rapidly increased 250-fold at 4 h, remained elevated until 8 h, and decreased until 24 h. Expression of Nts receptors for Ntsr1 remained unchanged across the periovulatory period, Ntsr2 was undetectable, whereas Sort1 expression (also called Ntsr3) gradually decreased in both the ovary and GCs after hCG administration. To better understand Nts regulation, inhibitors of the LH/CG signaling pathways were utilized. Our data revealed that hCG regulated Nts expression through the protein kinase A (PKA) and p38 mitogen-activated protein kinase (p38MAPK) signaling pathways. Additionally, epidermal-like-growth factor (EGF) receptor signaling also mediated Nts induction in GCs. To elucidate the role of NTS in the ovulatory process, we used a Nts silencing approach (si-Nts) followed by RNA-sequencing (RNA-seq). RNA-seq analysis of GCs collected after hCG with or without si-Nts identified and qPCR confirmed Ell2, Rsad2, Vps37a, and Smtnl2 as genes downstream of Nts. In summary, these findings demonstrate that hCG induces Nts and that Nts expression is mediated by PKA, p38MAPK, and EGF receptor signaling pathways. Additionally, NTS regulates several novel genes that could potentially impact the ovulatory process.

Reduced Endothelin-2 and Hypoxic Signaling Pathways in Granulosa-Lutein Cells of PCOS Women.

Granulosa-lutein cells (GLCs) from PCOS women display reduced HIF-1α and EDN2 levels, suggesting their role in PCOS etiology. Here, we investigated the mechanisms involved in aberrant EDN2 expression in PCOS, and its association with HIF-1α. Various HIF-1α-dependent factors were studied in GLCs from PCOS and compared to normally ovulating women. MicroRNA-210 (miR-210), its target genes (SDHD and GPD1L), and HIF-1α-responsive genes (EDN2 and VEGFA) differed in GLCs from PCOS, compared with those of healthy women. Levels of miR-210-designated hypoxiamiR-and EDN2 were reduced in the PCOS GLCs; concomitantly, GPD1L and SDHD levels were elevated. Cultured GLCs retained low EDN2 expression and had low HIF-1α levels, providing evidence for a disrupted hypoxic response in the PCOS GLCs. However, VEGFA expression was elevated in these cells. Next, miR-210 levels were manipulated. miR-210-mimic stimulated EDN2 twice as much as the miR-NC-transfected cells, whereas miR-210-inhibitor diminished EDN2, emphasizing the importance of hypoxiamiR for EDN2 induction. Intriguingly, VEGFA transcripts were reduced by both miR-210-mimic and -inhibitor, demonstrating that EDN2 and VEGFA are distinctly regulated. Disrupted hypoxic response in the GLCs of periovulatory follicles in PCOS women may play a role in ovulation failure, and in the reduced fertility prevalent in this syndrome.

Neurotensin: a neuropeptide induced by hCG in the human and rat ovary during the periovulatory period†.

Neurotensin (NTS) is a tridecapeptide that was first characterized as a neurotransmitter in neuronal cells. The present study examined ovarian NTS expression across the periovulatory period in the human and the rat. Women were recruited into this study and monitored by transvaginal ultrasound. The dominant follicle was surgically excised prior to the luteinizing hormone (LH) surge (preovulatory phase) or women were given 250 µg human chorionic gonadotropin (hCG) and dominant follicles collected 12-18 h after hCG (early ovulatory), 18-34 h (late ovulatory), and 44-70 h (postovulatory). NTS mRNA was massively induced during the early and late ovulatory stage in granulosa cells (GCs) (15 000 fold) and theca cells (700 fold). In the rat, hCG also induced Nts mRNA expression in intact ovaries and isolated GCs. In cultured granulosa-luteal cells (GLCs) from IVF patients, NTS expression was induced 6 h after hCG treatment, whereas in cultured rat GCs, NTS increased 4 h after hCG treatment. Cells treated with hCG signaling pathway inhibitors revealed that NTS expression is partially regulated in the human and rat GC by the epidermal-like growth factor pathway. Human GLC, and rat GCs also showed that Nts was regulated by the protein kinase A (PKA) pathway along with input from the phosphotidylinositol 3- kinase (PI3K) and mitogen-activated protein kinase (MAPK) pathways. The predominat NTS receptor present in human and rat GCs was SORT1, whereas NTSR1 and NTSR2 expression was very low. Based on NTS actions in other systems, we speculate that NTS may regulate crucial aspects of ovulation such as vascular permeability, inflammation, and cell migration.

Sirtuin-1 inhibits endothelin-2 expression in human granulosa-lutein cells via hypoxia inducible factor 1 alpha and epigenetic modifications†.

Endothelin-2 (EDN2) expression in granulosa cells was previously shown to be highly dependent on the hypoxic mediator, hypoxia inducible factor 1 alpha (HIF1A). Here, we investigated whether sirtuin-1 (SIRT1), by deacetylating HIF1A and class III histones, modulates EDN2 in human granulosa-lutein cells (hGLCs). We found that HIF1A was markedly suppressed in the presence of resveratrol or a specific SIRT1 activator, SRT2104. In turn, hypoxia reduced SIRT1 levels, implying a mutually inhibitory interaction between hypoxia (HIF1A) and SIRT1. Consistent with reduced HIF1A transcriptional activity, SIRT1 activators, resveratrol, SRT2104, and metformin, each acting via different mechanisms, significantly inhibited EDN2. In support, knockdown of SIRT1 with siRNA markedly elevated EDN2, whereas adding SRT2104 to SIRT1-silenced cells abolished the stimulatory effect of siSIRT1 on EDN2 levels further demonstrating that EDN2 is negatively correlated with SIRT1. Next, we investigated whether SIRT1 can also mediate the repression of the EDN2 promoter via histone modification. Chromatin immunoprecipitation (ChIP) analysis revealed that SIRT1 is indeed bound to the EDN2 promoter and that elevated SIRT1 induced a 40% decrease in the acetylation of histone H3, suggesting that SIRT1 inhibits EDN2 promoter activity by inducing a repressive histone configuration. Importantly, SIRT1 activation, using SRT2104 or resveratrol, decreased the viable numbers of hGLC, and silencing SIRT1 enhanced hGLC viability. This effect may be mediated by reducing HIF1A and EDN2 levels, shown to promote cell survival. Taken together, these findings propose novel, physiologically relevant roles for SIRT1 in downregulating EDN2 and survival of hGLCs.

The cAMP pathway promotes sirtuin-1 expression in human granulosa-lutein cells.

Sirtuin-1 (SIRT1), a NAD+-dependent deacetylase, is present in the ovarian granulosa cells (GCs) of various species. This study examined the regulation of SIRT1 expression in human granulosa-lutein cells (hGLCs). Two different, structurally unrelated SIRT1 activators, SRT2104 and resveratrol, dose- and time-dependently enhanced SIRT1 (∼2- and 1.5-fold increase at 50 µmol/L for mRNA and protein levels, respectively), whereas EX-527, an inhibitor of SIRT1 deacetylase activity, significantly suppressed SIRT1 protein induced by these activators. Transfecting cells with SIRT1 siRNA molecules efficiently silenced SIRT1 (∼70 % decrease in 48 h post-transfection). Furthermore, the stimulatory effects of SRT2104 on SIRT1 expression observed in non-transfected or in scrambled siRNA-transfected cells were diminished with SIRT1 silencing. The findings described above imply that SIRT1 autoregulates its own expression. Interestingly, SRT2104 elevated cAMP accumulation (1.4-fold) in the culture media of hGLCs which was further augmented in the presence of hCG (2.2-fold); these effects were evident after 12 h of incubation. This additive effect of hCG and SRT2104 on cAMP accumulation may explain the incremental outcome observed on SIRT1 expression (∼3-fold increase from basal level and ∼1.6-fold stimulation for each compound alone) with these two compounds. SIRT1 knockdown diminished SIRT1 induced by forskolin, providing additional evidence that cAMP promotes SIRT1. These findings imply that by activating adenylyl cyclase (hCG or forskolin) and inhibiting phosphodiesterases (SIRT1 activators), these two signals converge to produce an incremental, positive feedback loop on SIRT1 expression. Such a mechanism highlights the importance of maintaining high SIRT1 levels in human luteinized GCs.

Pentraxin-3 mediates prosurvival actions of interferon tau in bovine luteinized granulosa cells.

Pentraxin 3 (PTX3), a multimeric glycoprotein, is implicated in various biological functions. PTX3 was shown to be elevated in the corpus luteum (CL) of early pregnant ewes; however, its role in sheep or other ruminants' CL during this reproductive stage or how it is regulated remain unknown. Here we explored the role of PTX3 and its relationship with interferon-tau (IFNT; the pregnancy recognition signaling molecule during early pregnancy in domestic ruminants) in bovine luteinized granulosa cells (LGCs). IFNT robustly elevated PTX3 expression in bovine LGCs, and significantly stimulated its expression in luteal endothelial cells, along with CL slices; yet, LGCs were the most responsive and sensitive among these luteal models. ALK2/ALK3/ALK6 kinase inhibitor, dorsomorphin, dose-dependently inhibited basal and IFNT-elevated PTX3 expression in LGCs. In contrast, ALK4/5/7 inhibitor, SB431542, did not alter basal and TGFB1-induced PTX3. We found that recombinant human PTX3 itself moderately but significantly increases LGC numbers. Because PTX3 is highly expressed in bovine LGCs, we next examined the impact of lowering endogenous PTX3 levels with siRNA. PTX3 silencing decreased the viable cell numbers and reversed IFNT actions on cell viability, percentage of proliferating cells, and on two key survival/death genes: BIRC5 encoding surviving protein, and FASL - a death-inducing signal. Interestingly, thrombospondin-1, a known luteal proapoptotic factor, was inversely related to PTX3 in LGCs. Together, these findings suggest a novel role for PTX3 during early pregnancy, as mediator of IFNT prosurvival actions supporting CL maintenance during this reproductive stage.

Interferon-Tau Exerts Direct Prosurvival and Antiapoptotic Actions in Luteinized Bovine Granulosa Cells.

Interferon-tau (IFNT), serves as a signal to maintain the corpus luteum (CL) during early pregnancy in domestic ruminants. We investigated here whether IFNT directly affects the function of luteinized bovine granulosa cells (LGCs), a model for large-luteal cells. Recombinant ovine IFNT (roIFNT) induced the IFN-stimulated genes (ISGs; MX2, ISG15, and OAS1Y). IFNT induced a rapid and transient (15-45 min) phosphorylation of STAT1, while total STAT1 protein was higher only after 24 h. IFNT treatment elevated viable LGCs numbers and decreased dead/apoptotic cell counts. Consistent with these effects on cell viability, IFNT upregulated cell survival proteins (MCL1, BCL-xL, and XIAP) and reduced the levels of gamma-H2AX, cleaved caspase-3, and thrombospondin-2 (THBS2) implicated in apoptosis. Notably, IFNT reversed the actions of THBS1 on cell viability, XIAP, and cleaved caspase-3. Furthermore, roIFNT stimulated proangiogenic genes, including FGF2, PDGFB, and PDGFAR. Corroborating the in vitro observations, CL collected from day 18 pregnant cows comprised higher ISGs together with elevated FGF2, PDGFB, and XIAP, compared with CL derived from day 18 cyclic cows. This study reveals that IFNT activates diverse pathways in LGCs, promoting survival and blood vessel stabilization while suppressing cell death signals. These mechanisms might contribute to CL maintenance during early pregnancy.

The cAMP-EPAC Pathway Mediates PGE2-Induced FGF2 in Bovine Granulosa Cells.

During the periovulatory period, the profile of fibroblast growth factor 2 (FGF2) coincides with elevated prostaglandin E2 (PGE2) levels. We investigated whether PGE2 can directly stimulate FGF2 production in bovine granulosa cells and, if so, which prostaglandin E2 receptor (PTGER) type and signaling cascades are involved. PGE2 temporally stimulated FGF2. Accordingly, endoperoxide-synthase2-silenced cells, exhibiting low endogenous PGE2 levels, had reduced FGF2. Furthermore, elevation of viable granulosa cell numbers by PGE2 was abolished with FGF2 receptor 1 inhibitor, suggesting that FGF2 mediates this action of PGE2. Epiregulin (EREG), a known PGE2-inducible gene, was studied alongside FGF2. PTGER2 agonist elevated cAMP as well as FGF2 and EREG levels. However, a marked difference between cAMP-induced downstream signaling was observed for FGF2 and EREG. Whereas FGF2 upregulated by PGE2, PTGER2 agonist, or forskolin was unaffected by the protein kinase A (PKA) inhibitor H89, EREG was significantly inhibited. FGF2 was dose-dependently stimulated by the exchange protein directly activated by cAMP (EPAC) activator; a similar induction was observed for EREG. However, forskolin-stimulated FGF2, but not EREG, was inhibited in EPAC1-silenced cells. These findings ascribe a novel autocrine role for PGE2, namely, elevating FGF2 production in granulosa cells. This study also reveals that cAMP-activated EPAC1, rather than PKA, mediates the effect of PGE2/PTGER2 on the expression of FGF2. Stimulation of EREG by PGE2 is also mediated by PTGER2 but, in contrast to FGF2, EREG was found to be PKA sensitive. PGE2-stimulated FGF2 can act to maintain granulosa cell survival; it can also act on ovarian endothelial cells to promote angiogenesis.

miR-210 and GPD1L regulate EDN2 in primary and immortalized human granulosa-lutein cells.

Endothelin-2 (EDN2), expressed at a narrow window during the periovulatory period, critically affects ovulation and corpus luteum (CL) formation. LH (acting mainly via cAMP) and hypoxia are implicated in CL formation; therefore, we aimed to elucidate how these signals regulate EDN2 using human primary (hGLCs) and immortalized (SVOG) granulosa-lutein cells. The hypoxiamiR, microRNA-210 (miR-210) was identified as a new essential player in EDN2 expression. Hypoxia (either mimetic compound-CoCl2, or low O2) elevated hypoxia-inducible factor 1A (HIF1A), miR-210 and EDN2 Hypoxia-induced miR-210 was suppressed in HIF1A-silenced SVOG cells, suggesting that miR-210 is HIF1A dependent. Elevated miR-210 levels in hypoxia or by miR-210 overexpression, increased EDN2 Conversely, miR-210 inhibition reduced EDN2 levels, even in the presence of CoCl2, indicating the importance of miR-210 in the hypoxic induction of EDN2 A molecule that destabilizes HIF1A protein, glycerol-3-phosphate dehydrogenase 1-like gene-GPD1L, was established as a miR-210 target in both cell types. It was decreased by miR-210-mimic and was increased by miR-inhibitor. Furthermore, reducing GPD1L by endogenously elevated miR-210 (in hypoxia), miR-210-mimic or by GPD1L siRNA resulted in elevated HIF1A protein and EDN2 levels, implying a vital role for GPD1L in the hypoxic induction of EDN2 Under normoxic conditions, forskolin (adenylyl cyclase activator) triggered changes typical of hypoxia. It elevated HIF1A, EDN2 and miR-210 while inhibiting GPD1L Furthermore, HIF1A silencing greatly reduced forskolin's ability to elevate EDN2 and miR-210. This study highlights the novel regulatory roles of miR-210 and its gene target, GPD1L, in hypoxia and cAMP-induced EDN2 by human granulosa-lutein cells.

Regulation of ovulatory genes in bovine granulosa cells: lessons from siRNA silencing of PTGS2.

Prostaglandin endoperoxide synthase-2 (PTGS2), tumour necrosis factor-alpha-induced protein-6 (TNFAIP6), pentraxin-3 (PTX3), epidermal growth factor-like factors: amphiregulin (AREG) and epiregulin (EREG) are essential for successful ovulation. In this study, we compared the induction of these ovulatory genes in bovine granulosa cells (GCs) in vivo (after LH surge) and in vitro (forskolin (FRS) treatment). These genes were markedly stimulated in GCs isolated from cows 21 h after LH-surge. In isolated GCs, FRS induced a distinct temporal profile for each gene. Generally, there was a good agreement between the in vivo and in vitro inductions of these genes except for PTX3. Lack of PTX3 induction in isolated GCs culture suggests that other follicular compartments may mediate its induction by LH. Next, to study the role of PTGS2 and prostaglandins (PGs) in the cascade of ovulatory genes, PTGS2 was silenced with siRNA. PTGS2 siRNA caused a marked and specific knockdown of PTGS2 mRNA and PGE2 production (70% compared with scrambled siRNA) in bovine GCs. Importantly, PTGS2 silencing also reduced AREG, EREG and TNFAIP6 mRNA levels but not PTX3. Exogenous PGE2 increased AREG, EREG and TNFAIP6 mRNA levels, further confirming that these genes are prostanoid dependent. A successful and specific knockdown of PTGS2 was also achieved in endometrial cells (EndoCs) expressing PTGS2. Then, cholesterol-conjugated PTGS2 (chol-PTGS2) siRNA that facilitates cells' entry was investigated. In EndoCs, but not in GCs, chol-PTGS2 siRNA succeeded to reduce PTGS2 and PGE2 levels even without transfection reagent. PTGS2 knockdown is a promising tool to critically examine the functions of PTGS2 in the reproductive tract.