Nitric oxide and carbon monoxide production and metabolism in preeclampsia.

OBJECTIVE: To elucidate the regulation of the nitric oxide (NO) and carbon monoxide (CO) pathways in preeclampsia and to evaluate the ratio of asymmetric dimethylarginine (ADMA) to symmetric dimethylarginine (SDMA) as a marker for preeclampsia. METHODS: Maternal plasma and placental samples were obtained from 20 participants with preeclampsia and 23 controls. Enzyme-linked immunosorbent assay was used to measure plasma NO, ADMA, and SDMA as well as placental NO and hemeoxygnase 1 (HO-1). Western blot was used to measure placental dimethylarginine dimethylaminotransferases (DDAH-I and DDAH-II). RESULTS: Placental DDAH-I, placental DDAH-II, placental NO, and placental HO-1 were significantly decreased in participants with preeclampsia. While ADMA and SDMA levels were decreased in preeclampsia, the ADMA-SDMA ratio was not significantly different. CONCLUSIONS: Decreased DDAH and HO with preeclampsia suggest that they are important points in the regulatory pathways of NO and CO production that are altered in preeclampsia. The ADMA-SDMA ratio is not a useful test for preeclampsia.

Endothelial caveolar hub regulation of adenosine triphosphate-induced endothelial nitric oxide synthase subcellular partitioning and domain-specific phosphorylation.

ATP leads to endothelial NO synthase (eNOS)/NO-mediated vasodilation, a process hypothesized to depend on the endothelial caveolar eNOS partitioning and subcellular domain-specific multisite phosphorylation state. We demonstrate herein that, in both the absence and presence of ATP, the uterine artery endothelial caveolae contain specific protein machinery related to subcellular partitioning and act as specific focal "hubs" for NO- and ATP-related proteins. ATP-induced eNOS regulation showed a complex set of multisite posttranslational phosphorylation events that were closely associated with the enzyme's partitioning between caveolar and noncaveolar endothelial subcellular domains. The comprehensive model that we present demonstrates that ATP repartitioned eNOS between the caveolar and noncaveolar subcellular domains; specifically, the stimulatory (PSer635)eNOS was substantially higher in the caveolar pool with subcellular domain-independent increased levels on ATP treatment. The stimulatory (PSer1179)eNOS was not altered by ATP treatment. However, the inhibitory (PThr495)eNOS was regulated predominantly in the caveolar domain with decreased levels on ATP action. In contrast, the agonist-specific (PSer114)eNOS was localized in the noncaveolar pool with increased levels on ATP stimulation. Thus, the endothelial caveolar membrane system plays a pivotal role(s) in ATP-associated subcellular partitioning and possesses the relevant protein machinery for ATP-induced NO regulation. Furthermore, these subcellular domain-specific phosphorylation/dephosphorylation events provide evidence relating to eNOS spatio-temporal dynamics.

Human placental expression of SLIT/ROBO signaling cues: effects of preeclampsia and hypoxia.

Preeclampsia is characterized by dysfunctional endothelium and impaired angiogenesis. Recent studies suggest that the neuronal guidance SLIT/ROBO system regulates tumor angiogenesis. This study investigated if SLIT and ROBO are differentially expressed in healthy term and preeclamptic placentas and if hypoxia regulates SLIT and ROBO expression in placental trophoblast and endothelial cells. Total RNA and protein were extracted from placental tissues of healthy term (n = 5) and preeclamptic (n = 6) pregnancies and used for SLIT/ROBO expression analyses with reverse transcription-polymerase chain reaction (RT-PCR), real-time quantitative-PCR, and immunoblotting. Paraffin-embedded tissues were processed to localize SLIT/ROBO proteins in placental villi by immunohistochemistry. BeWo choriocarcinoma cells and human umbilical vein endothelial cells (HUVEC) were treated with 2% or 10% oxygen or the hypoxia mimetic deferoxamine mesylate (100 µM) to test if hypoxia regulates SLIT/ROBO expression. SLIT2, SLIT3, ROBO1, and ROBO4 mRNA and proteins were detected in the placenta. SLIT2 and ROBO1 proteins localized in the syncytiotrophoblast, and SLIT3, ROBO1, and ROBO4 in capillary endothelium of the placental villi. Levels of ROBO1 and ROBO4 as well as sFLT1 (soluble fms-like tyrosine kinase-1) proteins were significantly greater in preeclamptic placentas compared to normal controls. Hypoxia significantly increased both mRNA and protein levels of SLIT2 in BeWo cells and of SLIT3, ROBO1, and ROBB4 in HUVEC. Thus, trophoblast and endothelial coexpression of SLIT/ROBO suggests an autocrine/paracrine regulatory system for regulating placental function. Differential expression of SLITs and ROBOs in healthy term and preeclamptic placentas and hypoxia regulation of their expressions in placental cells implicate a potential pathophysiological role for this system in preeclampsia.

Caveolin-1 orchestrates fibroblast growth factor 2 signaling control of angiogenesis in placental artery endothelial cell caveolae.

Fibroblast growth factor (FGF) receptor 1 (FGFR1) protein was expressed as the long and short as well as some truncated forms in ovine fetoplacental artery ex vivo and in vitro. Upon FGF2 stimulation, both the long and short FGFR1s were tyrosine phosphorylated and the PI3K/AKT1 and ERK1/2 pathways were activated in a concentration- and time- dependent manner in ovine fetoplacental artery endothelial (oFPAE) cells. Blockade of the PI3K/AKT1 pathway attenuated FGF2-stimulated cell proliferation and migration as well as tube formation; blockade of the ERK1/2 pathway abolished FGF2-stimulated tube formation and partially inhibited cell proliferation and did not alter cell migration. Both AKT1 and ERK1/2 were co-fractionated with caveolin-1 and activated by FGF2 in the caveolae. Disruption of caveolae by methyl-ß-cyclodextrin inhibited FGF2 activation of AKT1 and ERK1/2. FGFR1 was found in the caveolae where it physically binds to caveolin-1. FGF2 stimulated dissociation of FGFR1 from caveolin-1. Downregulation of caveolin-1 significantly attenuated the FGF2-induced activation of AKT1 and ERK1/2 and inhibited FGF2-induced cell proliferation, migration and tube formation in oFPAE cells. Pretreatment with a caveolin-1 scaffolding domain peptide to mimic caveolin-1 overexpression also inhibited these FGF2-induced angiogenic responses. These data demonstrate that caveolae function as a platform for regulating FGF2-induced angiogenesis through spatiotemporally compartmentalizing FGFR1 and the AKT1 and ERK1/2 signaling modules; the major caveolar structural protein caveolin-1 interacts with FGFR1 and paradoxically regulates FGF2-induced activation of PI3K/AKT1 and ERK1/2 pathways that coordinately regulate placental angiogenesis.

Perspectives of SLIT/ROBO signaling in placental angiogenesis.

A novel family of evolutionally conserved neuronal guidance cues, including ligands (i.e., Slit, netrin, epherin, and semaphorin) and their corresponding receptors (i.e., Robo, DCC/Unc5, Eph and plexin/ neuropilin), has been identified to play a crucial role in axon pathfinding and branching as well as neuronal cell migration. The presence of commonalities in both neural and vascular developments has led to some exciting discoveries recently, which have extended the functions of these systems to vascular formation (vasculogenesis) and development (angiogenesis). Some of these ligands and receptors have been found to be expressed in the vasculature and surrounding tissues in physiological and pathological conditions. It is postulated that they regulate the formation and integrity of blood vessels. In particular, it has been shown that the Slit/Robo pair plays a novel role in angiogenesis during tumorigenesis and vascular formation during embryogenesis. Herein we summarize briefly the characteristics of this family of neuronal guidance molecules and discuss the extra-neural expression and function of the Slit/Robo pair in angiogenesis in physiological and pathological settings. We report expression of Robo1 protein in capillary endothelium and co-expression of Slit2 and Robo1 proteins in syncytiotrophoblast in healthy term human placental villi. These cellular expression patterns implicate that the Slit/Robo signaling plays an autocrine and/or paracrine role in angiogenesis and trophoblast functions. We also speculate a possible role of this system in pathophysiological placental angiogenesis.

Estradiol-17beta stimulates specific receptor and endogenous nitric oxide-dependent dynamic endothelial protein S-nitrosylation: analysis of endothelial nitrosyl-proteome.

Covalent adduction of a nitrosyl group to cysteines [S-nitrosylation (S-NO)] is emerging as a key route for nitric oxide (NO) to directly modulate protein functions. Here, we studied the effects of estrogens on endothelial protein S-NO and analyzed the nitrosyl-proteomes by biotin/CyDye switch technique combined with two-dimensional fluorescence difference gel electrophoresis and identified nitrosoproteins by matrix-assisted laser desorption/ionization-time of flight mass spectrometry. Estradiol-17beta (E2) rapidly stimulated protein S-NO in human umbilical vein endothelial cells, maximizing within 10- to 30-min post-E2 (10 nm) exposure. E2-BSA also rapidly stimulated protein S-NO. Both E2 and E2-BSA-induced protein S-NO was blocked by ICI 182,780 and N-nitro-l-arginine-methylester. Human umbilical vein endothelial cells expressed estrogen receptor (ER)alpha and ERbeta; both seemed to be required for E2 stimulation of protein S-NO because: 1) neither ERalpha or ERbeta agonist alone, but their combination, stimulated protein S-NO; and 2) either ERalpha or ERbeta antagonist blocked E2-induced protein S-NO. Numerous nitrosoproteins (spots) were observed on two-dimensional fluorescence difference gel. One hundred spots of interest were picked up; 58 were identified and, of which 15 were novel nitrosoproteins, 28 were up-regulated, 11 were decreased, and the rest were unchanged by E2. Pathway analysis suggested that nitrosoproteins are involved in regulating various endothelial functions, including apoptosis, cell structure and metabolism, redox homeostasis, etc. Thus, estrogens stimulate dynamic endothelial protein S-NO via mechanisms linked to specific ERs possibly on the plasma membrane and endogenous NO. These findings signify a critical next step for the understanding of the biological targets of enhanced NO production by estrogens.

Deciphering mechanisms controlling placental artery endothelial cell migration stimulated by vascular endothelial growth factor.

Vascular endothelial growth factor (VEGF) stimulated fetoplacental artery endothelial (oFPAE) cell migration and activated multiple signaling pathways including ERK2/1, p38MAPK, Jun N-terminal kinase (JNK1/2), v-Akt murine thymoma viral oncogene homolog 1 (Akt1), and c-Src in oFPAE cells. VEGF-induced cell migration was blocked by specific kinase inhibitors of JNK1/2 (SP600125), c-Src (4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d] pyrimidine), and phosphatidylinositol 3-kinase/Akt (wortmannin) but not ERK2/1 (U0126) and p38MAPK (SB203580). VEGF-induced cell migration was associated with dynamic actin reorganization and focal adhesion as evidenced by increased stress fiber formation and phosphorylation of cofilin-1 and focal adhesion kinase (FAK) and paxillin. Inhibition of JNK1/2, c-Src, and phosphatidylinositol 3-kinase/Akt suppressed VEGF-induced stress fiber formation and cofilin-1 phosphorylation. c-Src inhibition suppressed VEGF-induced phosphorylation of focal adhesion kinase, paxillin, and focal adhesion. VEGF-induced cell migration requires endogenous nitric oxide (NO) as: 1) VEGF-stimulated phosphorylation of endothelial NO synthase (eNOS) via activation of Akt, JNK1/2, and Src; 2) a NO donor diethylenetriamine-NO-stimulated cell migration; and 3) NO synthase inhibition blocked VEGF-induced cell migration. Targeted down-regulation and overexpression of caveolin-1 both inhibited VEGF-induced cell migration. Caveolin-1 down-regulation suppressed VEGF-stimulated phosphorylation of Akt, JNK, eNOS, c-Src, and FAK; however, basal activities of c-Src and FAK were elevated in parallel with increased stress fiber formation and focal adhesion. Caveolin-1 overexpression also inhibited VEGF-induced phosphorylation of Akt, JNK, c-Src, FAK, and eNOS. Thus, VEGF-induced placental endothelial cell migration requires activation of complex pathways that are paradoxically regulated by caveolin-1.

Activation of AP-1 transcription factors differentiates FGF2 and vascular endothelial growth factor regulation of endothelial nitric-oxide synthase expression in placental artery endothelial cells.

FGF2 (fibroblast growth factor 2), but not vascular endothelial growth factor (VEGF), stimulates sustained activation of ERK2/1 for endothelial NOS3 (nitric-oxide synthase 3) protein expression in ovine fetoplacental artery endothelial cells (oFPAEC). We deciphered herein the downstream signaling of ERK2/1 responsible for NOS3 expression by FGF2 in oFPAEC. FGF2, but not VEGF, increased NOS3 mRNA levels without altering its degradation. FGF2, but not VEGF, trans-activated sheep NOS3 promoter, and this was dependent on ERK2/1 activation. FGF2 did not trans-activate NOS3 promoters with deletions upstream of the consensus AP-1 site (TGAGTC A, -678 to -685). Trans-activation of wild-type NOS3 promoter by FGF2 was significantly inhibited when either the AP-1 or the cAMP-response element (CRE)-like sequence (TGCGTCA, -752 to -758) was mutated and was completely blocked when both were mutated. EMSA analyses showed that FGF2, but not VEGF, stimulated AP-1 and CRE DNA-protein complexes primarily composed of JunB and Fra1. Chromatin immunoprecipitation assays confirmed JunB/Fra1 binding to NOS3 promoter AP-1 and CRE elements in intact cells. FGF2, but not VEGF, stimulated JunB and Fra1 expressions; all preceded NOS3 up-regulation and were inhibited by PD98059. Down-regulation of JunB or Fra-1, but not c-Jun, blocked FGF2 stimulation of NOS3 expression and NO production. AP-1 inhibition suppressed FGF2 stimulation of NOS3 expression in human umbilical vein EC and uterine artery endothelial cells. Thus, FGF2 induction of NOS3 expression is mainly mediated by AP-1-dependent transcription involving JunB and Fra1 up-regulation via sustained ERK2/1 activation in endothelial cells.

High-throughput caveolar proteomic signature profile for maternal binge alcohol consumption.

Currently, no single marker is sensitive and specific enough to be considered a reliable biomarker for prenatal alcohol exposure. To identify a proteomic signature profile for maternal alcohol consumption, we carried out high-throughput proteomics on maternal endothelial caveolae exposed to moderate binge-like alcohol conditions. In these specialized lipid-ordered microdomains that contain a rich assembly of proteins, we demonstrate that moderate binge-like alcohol resulted in a distinctive maternal caveolar proteomic signature with important proteins being dramatically decreased/knocked out in the alcoholic profile. These proteins span from histones and basic structural proteins like α tubulin to proteins involved in trafficking, deubiquitination, cell signaling, and cell-cell adhesion. The profile also suggests an important role for the mother and the uteroplacental compartment in the pathogenesis of fetal alcohol spectrum disorders (FASD). These data demonstrate that the caveolar proteomic signature created by alcohol shows a promising direction for early detection of FASD.

Compartmentalizing VEGF-induced ERK2/1 signaling in placental artery endothelial cell caveolae: a paradoxical role of caveolin-1 in placental angiogenesis in vitro.

On vascular endothelial growth factor (VEGF) stimulation, both VEGF R1 and R2 receptors were phosphorylated in ovine fetoplacental artery endothelial (oFPAE) cells. Treatment with VEGF stimulated both time- and dose-dependent activation of ERK2/1 in oFPAE cells. VEGF-induced ERK2/1 activation was mediated by VEGFR2, but not VEGFR1, and was linked to intracellular calcium, protein kinase C, and Raf-1. VEGF stimulated oFPAE cell proliferation, migration, and tube formation in vitro. Blockade of ERK2/1 pathway attenuated VEGF-induced cell proliferation and tube formation but failed to inhibit migration in oFPAE cells. Disruption of caveolae by cholesterol depletion with methyl-beta-cyclodextrin or by down-regulation of its structural protein caveolin-1 blunted VEGF-induced ERK2/1 activation, proliferation, and tube formation in oFPAE cells, indicating an essential role of integral caveolae in these VEGF-induced responses. Adenoviral overexpression of caveolin-1 and addition of a caveolin scaffolding domain peptide also inhibited VEGF-stimulated ERK2/1 activation, cell proliferation, and tube formation in oFPAE cells. Furthermore, molecules comprising the ERK2/1 signaling module, including VEGFR2, protein kinase Calpha, Raf-1, MAPK kinase 1/2, and ERK2/1, resided with caveolin-1 in caveolae. VEGF transiently stimulated ERK2/1 activation in the caveolae similarly as in intact cells. Caveolae disruption greatly diminished ERK2/1 activation by VEGF in oFPAE cell caveolae. We conclude that caveolae function as a platform for compartmentalizing the VEGF-induced ERK2/1 signaling module. Caveolin-1 and caveolae play a paradoxical role in regulating VEGF-induced ERK2/1 activation and in vitro angiogenesis as evidenced by the similar inhibitory effects of down-regulation and overexpression of caveolin-1 and disruption of caveolae in oFPAE cells.

WIF1, a Wnt pathway inhibitor, regulates SKP2 and c-myc expression leading to G1 arrest and growth inhibition of human invasive urinary bladder cancer cells.

Epigenetic silencing of secreted wingless-type (Wnt) antagonists through hypermethylation is associated with tobacco smoking and with invasive bladder cancer. The secreted Wnt inhibitory factor-1 (WIF1) has shown consistent growth-inhibitory effect on various cancer cell lines. Therefore, we assessed the mechanisms of action of WIF1 by either restoring WIF1 expression in invasive bladder cancer cell lines (T24 and TSU-PR1) or using a recombinant protein containing functional WIF1 domain. Both ectopic expression of WIF1 and treatment with WIF1 domain protein resulted in cell growth inhibition via G(1) arrest. The G(1) arrest induced by WIF1 is associated with down-regulation of SKP2 and c-myc and up-regulation of p21/WAF1 and p27/Kip1. Conversely, reexpression of SKP2 in WIF1-overexpressing TSU-PR1 cells attenuated the WIF1-induced G(1) arrest. Furthermore, inhibition of nuclear Wnt signaling by either dominant-negative LEF1 or short hairpin RNA of TCF4 also reduced SKP2 expression. The human SKP2 gene contains two TCF/LEF1 consensus binding sites within the promoter. Chromatin immunoprecipitation/real-time PCR analysis revealed that both WIF1 and dominant-negative LEF1 expression decreased the in vivo binding of TCF4 and beta-catenin to the SKP2 promoter. Together, our results suggest that mechanisms of WIF1-induced G(1) arrest include (a) SKP2 down-regulation leading to p27/Kip1 accumulation and (b) c-myc down-regulation releasing p21/WAF1 transcription. Additionally, we show that WIF1 inhibits in vivo bladder tumor growth in nude mice. These observations suggest a mechanism for transformation of bladder epithelium on loss of WIF1 function and provide new targets such as SKP2 for intervention in WIF1-deficient bladder cancer.

Differential activation of multiple signalling pathways dictates eNOS upregulation by FGF2 but not VEGF in placental artery endothelial cells.

Fibroblast growth factor (FGF2), but not vascular endothelial growth factor (VEGF), upregulates endothelial nitric oxide synthase (eNOS) protein expression, at least partially, via activation of extracellular signal-regulated kinase 2/1 (ERK2/1) in ovine fetoplacental artery endothelial (oFPAE) cells. Herein we further investigated the temporal effects of FGF2 and VEGF on other signalling pathways including members (Jun N-terminal kinase JNK1/2 and p38MAPK) of mitogen-activated protein kinases (MAPK), phosphatidylinositol-3 kinase/v-akt murine thymoma viral oncogene homologue 1 (PI3K/AKT1), and the tyrosine kinase c-SRC, and examined if either one or more of these pathways play a role in the differential regulation of eNOS by FGF2 and VEGF. We first confirmed that in oFPAE cells, FGF2, but not VEGF, increased eNOS protein. FGF2 stimulated eNOS protein in a time- and concentration-dependent manner, which also depended on cell density. FGF2 provoked sustained (5min to 12h) whereas VEGF only stimulated transient (5min) ERK2/1 phosphorylation. FGF2 was 1.7-fold more potent in stimulating ERK2/1 phosphorylation than VEGF. FGF2 and VEGF only transiently activated JNK1/2 and AKT1 within 5min; however, FGF2 was a stronger stimulus than VEGF. FGF2 and VEGF did not significantly activate p38MAPK at 5min; however, VEGF stimulated p38MAPK phosphorylation at 60min. VEGF but not FGF2 significantly stimulated c-SRC phosphorylation. Inhibitors of MEK-ERK2/1 (PD98059), JNK1/2 (SP600125) and PI3K (wortmannin), but not p38MAPK (SB203580) and SRC (PP2), decreased the FGF2-increased eNOS protein expression. Thus, the FGF2-induced eNOS protein expression requires activation of multiple signalling pathways including ERK2/1, JNK1/2 and PI3K/AKT1. Differences in intensity and temporal patterns of activation of these pathways by FGF2 and VEGF may account for their differential effects on eNOS expression in OFPAE cells.

Transcriptional regulation of endothelial nitric oxide synthase expression in uterine artery endothelial cells by c-Jun/AP-1.

Despite extensive studies have shown that increased endothelial nitric oxide synthase (NOS3) expression in the uterine artery endothelial cells (UAEC) plays a key role in uterine vasodilatation, the molecular mechanism controlling NOS3 expression in UAEC is unknown. According to the sheep NOS3 promoter sequence isolated in our laboratory, we hypothesize that the activator protein-1 (AP-1) site in the proximal sheep NOS3 promoter (TGAGTCA, -682 to -676) is important for NOS3 expression. We developed a c-Jun adenoviral expression system to overexpress c-Jun protein into UAEC to investigate the effects of c-Jun/AP-1 on NOS3 expression. Basal levels of c-Jun protein and mRNA were detected in UAEC. c-Jun protein was overexpressed in a concentration and time-dependent fashion in UAEC infected with sense c-Jun (S-c-Jun), but not sham and antisense c-Jun (A-c-Jun) adenoviruses. Infection with S-c-Jun adenovirus (25 MOI, multiplicity of infection) resulted in efficient c-Jun protein overexpression in UAEC up to 3 days. In S-c-Jun, but not sham and A-c-Jun adenovirus infected UAEC, NOS3 mRNA and protein levels were increased (P<0.05) compared to noninfected controls. Increased NOS3 expression was associated with increased total NOS activity. Transient transfections showed that c-Jun overexpression augmented the transactivation of the sheep NOS3 promoter-driven luciferase/reporter constructs with the AP-1 site but not of deletion constructs without the AP-1 site. When the AP-1 site was mutated, c-Jun failed to trans-activate the sheep NOS3 promoter. AP-1 DNA binding activity also increased in c-Jun overexpressed UAEC. Lastly, the pharmacological AP-1 activator phorbol myristate acetate increased AP-1 binding, trans-activated the wild-type but not the AP-1 mutant NOS3 promoter and dose-dependently stimulated UAEC NOS3 and c-Jun protein expression. Hence, our data show that c-Jun/AP-1 regulates NOS3 transcription involving the proximal AP-1 site in the 5'-regulatory region of the sheep NOS3 gene.

Expression of estrogen receptors-alpha and -beta in the pregnant ovine uterine artery endothelial cells in vivo and in vitro.

Estrogen is recognized to be one of the driving forces in increases in uterine blood flow through both rapid and delayed actions via binding to its receptors, ER alpha and ER beta at the uterine artery (UA) wall, and especially in UA endothelium (UAE). However, information regarding estrogen receptor (ER) expression in UAE is limited. This study was designed to test whether ERs are expressed in UAE in vivo, and if they are, whether these receptors are maintained in cultured UA endothelial cells (UAECs) in vitro. By using immunohistochemical and Western blot analyses, we clearly demonstrated ER alpha and ER beta protein expression in pregnant (Days 120-130) sheep UA and UAE in vivo and as well as cultured UAECs in vitro. Reverse transcription-polymerase chain reaction (RT-PCR) amplified both ER alpha and ER beta mRNAs in UA, UAE, and UAECs. Of interest, a truncated ER beta (ER beta2) variant due to a splicing deletion of exon 5 of the ER beta gene was detected in these cells. Quantitative RT-PCR analysis revealed that ER alpha mRNA levels are approximately 8-fold (P < 0.01) higher than that of ER beta in UAECs, indicating that ER alpha may play a more important role than ER beta in the UAEC responses to estrogen. Fluorescence immunolabeling analysis showed that ER alpha is present in both nuclei and plasma membranes in UAECs, and the latter is also colocalized with caveolin-1. The membrane and nuclear ER alpha presumably participate in rapid and delayed responses, respectively, to estrogen on UAE. Taken together, our data demonstrated that UAE is a direct target of estrogen actions and that the UAEC culture model we established is suitable for dissecting estrogen actions on UAE.

Functional and molecular characterization of naturally occurring mutations in the oocyte-secreted factors bone morphogenetic protein-15 and growth and differentiation factor-9.

Bone morphogenetic protein-15 (BMP-15) and growth and differentiation factor-9 (GDF-9) are oocyte-secreted factors that are critical local regulators of ovarian physiology. Recent studies have identified a number of mutations in these genes that cause increased fertility and infertility in heterozygous or homozygous ewes carrying the mutations, respectively. Interestingly, heterozygous ewes with a mutation in both BMP-15 and GDF-9 exhibit higher fertility than those having mutation in only one of the genes. Here, we have produced recombinant human BMP-15 and GDF-9 that carry the mutations identified in those sheep, i.e. I31D and S99I in BMP-15 and S77F in GDF-9. We found that when individually expressed, both BMP-15 mutations had no effect on the processing, secretion, and dimerization of the mature proteins or on the biological activity of the molecules. However, when mutant BMP-15 was co-expressed with wild-type GDF-9, the secretion of BMP-15 and GDF-9 was significantly reduced, suggesting that the mechanisms by which the BMP-15 mutations affect sheep fertility occurs at the level of protein secretion rather than dimerization and biological activity. Moreover, when mutant GDF-9 was co-expressed with mutant BMP-15, the secretion levels of both proteins were significantly lower than those of cells co-expressing wildtype GDF-9 and mutant BMP-15, suggesting a possible mechanism for the extreme fertility observed in the compound heterozygous mutant sheep.

Effect of intracellular interactions on the processing and secretion of bone morphogenetic protein-15 (BMP-15) and growth and differentiation factor-9. Implication of the aberrant ovarian phenotype of BMP-15 mutant sheep.

Bone morphogenetic protein-15 (BMP-15) and growth and differentiation factor-9 (GDF-9) are members of the transforming growth factor-beta superfamily. Both molecules are closely related in their primary structures and share a nearly identical spatiotemporal expression pattern in the oocyte during folliculogenesis in mammals. Here we have established a series of cell lines, which express recombinant BMP-15, GDF-9, or both, and investigated whether they form homodimers and/or heterodimers. We demonstrate the first evidence that both BMP-15 and GDF-9 can form non-covalent homodimers when expressed individually, while when both are co-expressed BMP-15/GDF-9 heterodimers are produced. Interestingly, when GDF-9 and BMP-15 are co-expressed the processing of both proproteins are significantly impaired as compared with that of the singly expressed proproteins, suggesting that the proprotein heterodimer is less susceptible to proteolytic cleavage than the individual homodimers. Since BMP-15 mutant sheep, called Inverdale, exhibit severe defects in ovarian function we have also established stable transformants expressing the mutant BMP-15 (InvBMP-15) alone or together with GDF-9. Although InvBMP-15 was previously predicted to be unable to form homodimers, we show here that it does form non-covalent dimers; however, the processing efficiency of InvBMP-15 proprotein is significantly lower than wild-type BMP-15. Surprisingly, when GDF-9 is co-expressed, the processing and secretion of InvBMP-15 is abolished, and the processing of GDF-9 is also severely impaired, suggesting that the heterodimers of InvBMP-15/GDF-9 proproteins are not susceptible to proteolytic cleavage and thus degrade in the cells. Based on these findings we propose a novel hypothesis that a decrease in GDF-9 secretion may be involved in causing infertility in homozygous Inverdale ewes.

Lack of association between polymorphisms in the testis-specific angiotensin converting enzyme gene and male infertility in an Asian population.

Angiotensin converting enzyme (ACE) is a membrane-bound dipeptidyl carboxy-peptidase that generates vasoconstricting angiotensin II and inactivates vasodilating bradykinin. The ACE gene encodes two isozymes: the somatic isozyme (sACE) is found in many tissues including vascular endothelial cells, whereas the testis-specific isozyme (tACE) is expressed exclusively in developing spermatids and mature sperm. Thus, ACE might have physiological functions in addition to blood pressure regulation. Male mice lacking tACE activity show reduced fertility, indicating its importance in male fertility. In this study, we screened five recently defined tACE gene polymorphisms in 90 Singapore Chinese men with infertility and 84 fertile controls using PCR-based restriction fragment length polymorphism and DNA sequencing. However, only one of these polymorphisms was identified in both patient and control groups, the frequency of which was not significantly different in patients and controls. Thus, these ACE gene polymorphisms are unlikely to contribute to the pathogenesis of male infertility in the Singapore Chinese population.