The Experts below are selected from a list of 360 Experts worldwide ranked by ideXlab platform
John S Davis - One of the best experts on this subject based on the ideXlab platform.
-
trafficking of cholesterol from lipid droplets to mitochondria in bovine luteal cells acute control of Progesterone Synthesis
The FASEB Journal, 2020Co-Authors: John S Davis, Heather Talbott, Andrea S Cupp, Michele R Plewes, Crystal Krause, Emilia Przygrodzka, Jennifer R WoodAbstract:The corpus luteum is a transient endocrine gland that synthesizes and secretes the steroid hormone, Progesterone, which is vital for establishment and maintenance of pregnancy. Luteinizing hormone (LH) via activation of protein kinase A (PKA) acutely stimulates luteal Progesterone Synthesis via a complex process, converting cholesterol via a series of enzymatic reactions, into Progesterone. Lipid droplets in steroidogenic luteal cells store cholesterol in the form of cholesterol esters, which are postulated to provide substrate for steroidogenesis. Early enzymatic studies showed that hormone sensitive lipase (HSL) hydrolyzes luteal cholesterol esters. In this study, we tested whether HSL is a critical mediator of the acute actions of LH on luteal Progesterone production. Using LH-responsive bovine small luteal cells our results reveal that LH, forskolin, and 8-Br cAMP-induced PKA-dependent phosphorylation of HSL at Ser563 and Ser660, events known to promote HSL activity. Small molecule inhibition of HSL activity and siRNA-mediated knock down of HSL abrogated LH-induced Progesterone production. Moreover, western blotting and confocal microscopy revealed that LH stimulates phosphorylation and translocation of HSL to lipid droplets. Furthermore, LH increased trafficking of cholesterol from the lipid droplets to the mitochondria, which was dependent on both PKA and HSL activation. Taken together, these findings identify a PKA/HSL signaling pathway in luteal cells in response to LH and demonstrate the dynamic relationship between PKA, HSL, and lipid droplets in luteal Progesterone Synthesis.
-
effects of il8 and immune cells on the regulation of luteal Progesterone secretion
Reproduction, 2014Co-Authors: Heather Talbott, Abigail Delaney, Pan Zhang, Robert A Cushman, Andrea S Cupp, Xiaoying Hou, John S DavisAbstract:Recent studies suggest that chemokines may mediate the luteolytic action of PGF2α (PGF). Our objective was to identify chemokines induced by PGF in vivo and to determine the effects of IL8 on specific luteal cell types in vitro. Midcycle cows were injected with saline or PGF, ovaries were removed after 0.5 – 4 h and chemokine expression was analyzed by qPCR. In vitro expression of IL8 was analyzed after PGF administration and with cell signaling inhibitors to determine the mechanism of PGF-induced chemokine expression. Purified neutrophils were analyzed for migration and activation in response to IL8 and PGF. Purified luteal cell types (steroidogenic, endothelial and fibroblast cells) were used to identify which cells respond to chemokines. Neutrophils and peripheral blood mononuclear cells (PBMCs) were co-cultured with steroidogenic cells to determine their effect on Progesterone production. IL8, CXCL2, CCL2, and CCL8 transcripts were rapidly increased following PGF treatment in vivo and. The stimulatory action of PGF on IL8 mRNA expression in vitro was prevented by inhibition of p38 and JNK signaling. IL8, but not PGF, TNF, or TGFB1, stimulated neutrophil migration. IL8 had no apparent action in purified luteal steroidogenic, endothelial, or fibroblast cells, but IL8 stimulated ERK phosphorylation in neutrophils. In co-culture experiments neither IL8 nor activated neutrophils altered basal or LH-stimulated luteal cell Progesterone Synthesis. In contrast, activated PBMCs inhibited LH-stimulated Progesterone Synthesis from cultured luteal cells. These data implicate a complex cascade of events during luteolysis involving chemokine signaling, neutrophil recruitment, and immune cell action within the corpus luteum.
-
convergence of 3 5 cyclic adenosine 5 monophosphate protein kinase a and glycogen synthase kinase 3β β catenin signaling in corpus luteum Progesterone Synthesis
Endocrinology, 2009Co-Authors: Lynn Roy, Anthony J Zeleznik, Xiaoying Hou, Claudia A Mcdonald, Chao Jiang, Dulce Maroni, Todd A Wyatt, John S DavisAbstract:Progesterone secretion by the steroidogenic cells of the corpus luteum (CL) is essential for reproduction. Progesterone Synthesis is under the control of LH, but the exact mechanism of this regulation is unknown. It is established that LH stimulates the LH receptor/choriogonadotropin receptor, a G-protein coupled receptor, to increase cAMP and activate cAMP-dependent protein kinase A (PKA). In the present study, we tested the hypothesis that cAMP/PKA-dependent regulation of the Wnt pathway components glycogen synthase kinase (GSK)-3beta and beta-catenin contributes to LH-dependent steroidogenesis in luteal cells. We observed that LH via a cAMP/PKA-dependent mechanism stimulated the phosphorylation of GSK3beta at N-terminal Ser9 causing its inactivation and resulted in the accumulation of beta-catenin. Overexpression of N-terminal truncated beta-catenin (Delta90 beta-catenin), which lacks the phosphorylation sites responsible for its destruction, significantly augmented LH-stimulated Progesterone secretion. In contrast, overexpression of a constitutively active mutant of GSK3beta (GSK-S9A) reduced beta-catenin levels and inhibited LH-stimulated steroidogenesis. Chromatin immunoprecipitation assays demonstrated the association of beta-catenin with the proximal promoter of the StAR gene, a gene that expresses the steroidogenic acute regulatory protein, which is a cholesterol transport protein that controls a rate-limiting step in steroidogenesis. Collectively these data suggest that cAMP/PKA regulation of GSK3beta/beta-catenin signaling may contribute to the acute increase in Progesterone production in response to LH.
-
prostaglandin f2α stimulates the expression and secretion of transforming growth factor b1 via induction of the early growth response 1 gene egr1 in the bovine corpus luteum
Molecular Endocrinology, 2008Co-Authors: Edward Arvisais, Chao Jiang, Dongbao Chen, Joy L Pate, Thomas R Hansen, Bo R Rueda, John S DavisAbstract:In most mammals, prostaglandin F2α (PGF2α) is believed to be a trigger that induces the regression of the corpus luteum (CL), whereby Progesterone Synthesis is inhibited, the luteal structure involutes, and the reproductive cycle resumes. Studies have shown that the early growth response 1 (EGR1) protein can induce the expression of proapoptotic proteins, suggesting that EGR1 may play a role in luteal regression. Our hypothesis is that EGR1 mediates the actions of PGF2α by inducing the expression of TGF β1 (TGFB1), a key tissue remodeling protein. The levels of EGR1 mRNA and protein were up-regulated in the bovine CL during PGF2α-induced luteolysis in vivo and in PGF2α-treated luteal cells in vitro. Using chemical and genetic approaches, the RAF/MAPK kinase (MEK) 1/ERK pathway was identified as a proximal signaling event required for the induction of EGR1 in PGF2α-treated cells. Treatment with PGF2α increased the expression of TGFB1 mRNA and protein as well as the binding of EGR1 protein to TGFB1 promoter...
Kiyoshi Okuda - One of the best experts on this subject based on the ideXlab platform.
-
lymphatic involvement in the disappearance of steroidogenic cells from the corpus luteum during luteolysis
PLOS ONE, 2014Co-Authors: Mohamad Omar Alziabi, Tomas J Acosta, Fumio Sekizawa, Dariusz J Skarzynski, Kiyoshi OkudaAbstract:In mammals, the corpus luteum (CL) is an essential endocrine gland for the establishment and maintenance of pregnancy. If pregnancy is not established, the CL regresses and disappears rapidly from the ovary. A possible explanation for the rapid disappearance of the CL is that luteal cells are transported from the ovary via lymphatic vessels. Here, we report the presence of cells positive for 3β-hydroxysteroid dehydrogenase (3β-HSD), an enzyme involved in Progesterone Synthesis, in the lumen of lymphatic vessels at the regressing luteal stage and in the lymphatic fluid collected from the ovarian pedicle ipsilateral to the regressing CL. The 3β-HSD positive cells were alive and contained lipid droplets. The 3β-HSD positive cells in the lymphatic fluid were most abundant at days 22–24 after ovulation. These findings show that live steroidogenic cells are in the lymphatic vessels drained from the CL. The outflow of steroidogenic cells starts at the regressing luteal stage and continues after next ovulation. The overall findings suggest that the complete disappearance of the CL during luteolysis is involved in the outflow of luteal cells from the CL via ovarian lymphatic vessels.
-
hypoxia promotes Progesterone Synthesis during luteinization in bovine granulosa cells
Journal of Reproduction and Development, 2014Co-Authors: Shin Yoshioka, Ryo Nishimura, Kiyoshi OkudaAbstract:In the ovary, follicular vascularization is restricted to the theca cell layer, while the granulosa cell layer and oocyte develop in an avascular environment. As the follicle develops, the blood vessels in the theca cell layer increase in number and size but do not penetrate the granulosa cell layer [1,2,3]. Ovarian blood flow decreases toward ovulation, and gradually increases with luteal development [4]. In addition, the O2 concentration in the follicular fluid in large follicles is less than in small follicles [5]. These conditions seem to represent a physiological hypoxia during follicular growth. Furthermore, immediately after ovulation, the ruptured follicle is also thought to be under a hypoxic condition due to bleeding and immature vascularization [6]. Hypoxia is defined as a reduction in available oxygen whether in a whole organism or in a tissue or cell. Hypoxia response elements of target genes are recognized and regulated by hypoxia-inducible factor 1 (HIF-1), comprising the subunit factors HIF-1α and aryl hydrocarbon receptor nuclear translocator (ARNT; HIF-1β) [7,8,9]. Hypoxia and HIF-1α have been studied on luteal function related to the steroidogenesis at various stages in cows [10,11,12]. Expression of HIF-1α in the corpus luteum (CL) was highest at the early luteal stage in cattle [12], humans [13] and monkeys [14]. In granulosa cells, HIF-1α expression peaks around the time of ovulation [1, 14, 15] and is upregulated by low oxygen conditions (2% O2) in synergy with human chorionic gonadotrophin (hCG), a mimic of luteinizing hormone (LH) [15]. The above findings indicate that the follicle, specifically the granulosa cell layer, is in a hypoxic condition around the time of ovulation. Around the time of ovulation, granulosa cells and theca cells start to be luteinized after an LH surge, and after ovulation, they differentiate into luteal cells and then produce a large amount of Progesterone (P4), which is essential for establishing pregnancy [16]. Luteinization causes important changes in follicular function, as the main product of the luteinized cells is changed from estrogen (E2) to P4. These changes include modifications of the rate-limiting elements of steroid Synthesis. The key protein and enzymes in P4 bioSynthesis include steroidogenic acute regulatory protein (StAR; STAR), which transports cholesterol from the outer mitochondrial membrane to the inner mitochondrial membrane, cytochrome P450 side-chain cleavage (P450scc; CYP11A1), which converts cholesterol into pregnenolone and 3β-hydroxysteroid dehydrogenase (3β-HSD; HSD3B), which converts pregnenolone into P4 [17,18,19,20,21]. A common process in luteinization involves rupture and collapse of the follicle at ovulation and also the invasion of some elements, including theca cells and blood vessels [22]. Based on the above findings, luteinization and hypoxia may take place simultaneously. However, it is unclear whether hypoxia contributes to P4 Synthesis during luteinization. In the present study, we hypothesized that hypoxia plays some roles in luteinization by stimulating the P4 generating system. To test this hypothesis, we used a model of bovine luteinizing and non-luteinizing granulosa cells in a culture system. We induced hypoxic conditions (10% and 5% O2) in the culture system and examined P4 production as well as mRNA and protein expression of StAR, P450scc and 3β-HSD. Furthermore, it has been confirmed that the conditions used in the present study are hypoxic by determining the protein expression of HIF-1α, which is known to accumulate in cells and function specifically under hypoxic conditions [23, 24].
-
hypoxia promotes Progesterone Synthesis during luteinization in bovine granulosa cells
Journal of Reproduction and Development, 2014Co-Authors: Shin Yoshioka, Ryo Nishimura, Kiyoshi OkudaAbstract:To determine whether hypoxia has an effect on luteinization, we examined the influence of hypoxia on a model of bovine luteinizing and non-luteinizing granulosa cell culture. The granulosa cells were obtained from small antral follicles (≤ 6 mm in diameter). To induce luteinization, the cells were treated for 24 h with insulin (2 µg/ml), forskolin (10 µM) or insulin in combination with forskolin at 20% O2. After 24 h, Progesterone (P4) production was higher in the treated cells, which we defined as luteinizing granulosa cells, than in non-treated cells, which we defined as non-luteinizing granulosa cells. P4 production by non-luteinizing granulosa cells was not affected by hypoxia (24 h at 10% and 5% O2), while P4 production by granulosa cells treated with insulin in combination with forskolin was significantly increased under hypoxia (24 h at 10% and 5% O2). Because hypoxia affected P4 production by the luteinizing granulosa cells but not by the non-luteinizing granulosa cells, hypoxia seems to promote P4 production during, rather than before, luteinization. In the cells treated with insulin in combination with forskolin, mRNA and protein expression of steroidogenic acute regulatory protein (StAR) and protein expression of 3β-hydroxysteroid dehydrogenase (3β-HSD) increased under 10% O2, while mRNA and protein expressions of key protein and enzymes in P4 bioSynthesis did not increase under 5% O2. The overall results suggest that hypoxia plays a role in progressing and completing the luteinization by enhancing P4 production through StAR as well as 3β-HSD expressions in the early time of establishing the corpus luteum.
-
oxygen concentration is an important factor for modulating Progesterone Synthesis in bovine corpus luteum
Endocrinology, 2006Co-Authors: Ryo Nishimura, Ryosuke Sakumoto, Yuko Tatsukawa, Tomas J Acosta, Kiyoshi OkudaAbstract:Oxygen deficiency caused by a decrease in the blood supply is known to induce various responses of cells. Because luteal blood flow has been shown to decrease during luteolysis, a low-oxygen condition seems to be an integral part of the environment during luteolysis. To determine whether a low-oxygen condition is associated with functional luteolysis, we examined the influence of reduced oxygen tension on the luteal Progesterone (P4) generating system in cultured bovine midluteal cells. Luteal cells obtained from midcycle corpus luteum (d 8-12) were incubated under different O2 concentrations (20, 10, 5, 3% O2) with or without LH for 24 h. P4 production decreased with decreasing O2 concentration but was significantly stimulated by LH regardless of O2 concentration. After 8 h of culture, both basal and LH-stimulated P4 production was significantly lower under 3% O2 than under 20% O2. Low-oxygen condition also inhibited pregnenolone production. Cytochrome P450 side-chain cleavage enzyme (P450scc) mRNA expression, measured by quantitative PCR, decreased under low-oxygen condition in both non-LH-treated and LH-treated cells. Low-oxygen condition did not affect the expressions of steroidogenic acute regulatory protein mRNA or protein, whereas steroidogenic acute regulatory protein mRNA expression was stimulated by LH during 4 h of culture. Low-oxygen condition also did not affect 3 beta-hydroxysteroid dehydrogenase/Delta 5-Delta 4 isomerase mRNA expression or the activity of the enzyme in the cells, regardless of the incubation period. The overall results indicate that a low-oxygen condition decreases P4 Synthesis by attenuating P450scc production and P450scc activity in bovine luteal cells and suggest that oxygen deficiency is an essential condition for the progression of luteolysis in cattle.
Federico Martinez - One of the best experts on this subject based on the ideXlab platform.
-
the heat shock protein 60 promotes Progesterone Synthesis in mitochondria of jeg 3 cells
Reproductive Biology, 2017Co-Authors: Jessica Monrealflores, Maria Teresa Espinosagarcia, Alejandro Garciaregalado, Fabian Arechavaletavelasco, Federico MartinezAbstract:Progesterone Synthesis in human placenta is essential to maintain pregnancy. The limiting step in placental Progesterone Synthesis is cholesterol transport from the cytoplasm to the inner mitochondrial membrane. Multiple proteins located in mitochondrial contact sites seem to play a key role in this process. Previously, our group identified the heat shock protein 60 (HSP60) as part of mitochondrial contact sites in human placenta, suggesting its participation in Progesterone Synthesis. Here, we examined the role of HSP60 in Progesterone Synthesis. Our results show that over-expression of HSP60 in human placental choriocarcinoma cells (JEG-3) and human embryonic kidney 293 cells (HEK293) promotes Progesterone Synthesis. Furthermore, incubation of the HSP60 recombinant protein with intact isolated mitochondria from JEG-3 cells also promotes Progesterone Synthesis in a dose-related fashion. We also show that HSP60 interacts with STARD3 and P450scc proteins from mitochondrial membrane contact sites. Finally, we show that the HSP60 recombinant protein binds cholesterol. Ours results demonstrate that HSP60 participates in mitochondrial Progesterone Synthesis. These findings provide novel insights into Progesterone Synthesis in the human placenta and its role in maintaining pregnancy.
-
membrane potential regulates mitochondrial atp diphosphohydrolase activity but is not involved in Progesterone bioSynthesis in human syncytiotrophoblast cells
Biochimica et Biophysica Acta, 2015Co-Authors: Oscar Floresherrera, Sofia Olverasanchez, Mercedes Esparzaperusquia, Juan Pablo Pardo, Juan L Rendon, Guillermo Mendozahernandez, Federico MartinezAbstract:ATP-diphosphohydrolase is associated with human syncytiotrophoblast mitochondria. The activity of this enzyme is implicated in the stimulation of oxygen uptake and Progesterone Synthesis. We reported previously that: (1) the detergent-solubilized ATP-diphosphohydrolase has low substrate specificity, and (2) purine and pyrimidine nucleosides, tri- or diphosphates, are fully dephosphorylated in the presence of calcium or magnesium (Flores-Herrera 1999, 2002). In this study we show that ATP-diphosphohydrolase hydrolyzes first the nucleoside triphosphate to nucleoside diphosphate, and then to nucleotide monophosphate, in the case of all tested nucleotides. The activation energies (Ea) for ATP, GTP, UTP, and CTP were 6.06, 4.10, 6.25, and 5.26 kcal/mol, respectively; for ADP, GDP, UDP, and CDP, they were 4.67, 5.42, 5.43, and 6.22 kcal/mol, respectively. The corresponding Arrhenius plots indicated a single rate-limiting step for each hydrolyzed nucleoside, either tri- or diphosphate. In intact mitochondria, the ADP produced by ATP-diphosphohydrolase activity depolarized the membrane potential (ΔΨm) and stimulated oxygen uptake. Mitochondrial respiration showed the state-3/state-4 transition when ATP was added, suggesting that ATP-diphosphohydrolase and the F1F0-ATP synthase work in conjunction to avoid a futile cycle. Substrate selectivity of the ATP-diphosphohydrolase was modified by ΔΨm (i.e. ATP was preferred over GTP when the inner mitochondrial membrane was energized). In contrast, dissipation of ΔΨm by CCCP produced a loss of substrate specificity and so the ATP-diphosphohydrolase was able to hydrolyze ATP and GTP at the same rate. In intact mitochondria, ATP hydrolysis increased Progesterone Synthesis as compared with GTP. Although dissipation of ΔΨm by CCCP decreased Progesterone Synthesis, NADPH production restores steroidogenesis. Overall, our results suggest a novel physiological role for ΔΨm in steroidogenesis.
-
mitochondrial proteases act on stard3 to activate Progesterone Synthesis in human syncytiotrophoblast
Biochimica et Biophysica Acta, 2015Co-Authors: Mercedes Esparzaperusquia, Oscar Floresherrera, Sofia Olverasanchez, Juan Pablo Pardo, Maria Teresa Espinosagarcia, Hector Floresherrera, Alberto Guevaraflores, Federico MartinezAbstract:Abstract Background STARD1 transports cholesterol into mitochondria of acutely regulated steroidogenic tissue. It has been suggested that STARD3 transports cholesterol in the human placenta, which does not express STARD1. STARD1 is proteolytically activated into a 30-kDa protein. However, the role of proteases in STARD3 modification in the human placenta has not been studied. Methods Progesterone determination and Western blot using anti-STARD3 antibodies showed that mitochondrial proteases cleave STARD3 into a 28-kDa fragment that stimulates Progesterone Synthesis in isolated syncytiotrophoblast mitochondria. Protease inhibitors decrease STARD3 transformation and steroidogenesis. Results STARD3 remained tightly bound to isolated syncytiotrophoblast mitochondria. Simultaneous to the increase in Progesterone Synthesis, STARD3 was proteolytically processed into four proteins, of which a 28-kDa protein was the most abundant. This protein stimulated mitochondrial Progesterone production similarly to truncated-STARD3. Maximum levels of protease activity were observed at pH 7.5 and were sensitive to 1,10-phenanthroline, which inhibited steroidogenesis and STARD3 proteolytic cleavage. Addition of 22( R )-hydroxycholesterol increased Progesterone Synthesis, even in the presence of 1,10-phenanthroline, suggesting that proteolytic products might be involved in mitochondrial cholesterol transport. Conclusion Metalloproteases from human placental mitochondria are involved in steroidogenesis through the proteolytic activation of STARD3. 1,10-Phenanthroline inhibits STARD3 proteolytic cleavage. The 28-kDa protein and the amino terminal truncated-STARD3 stimulate steroidogenesis in a comparable rate, suggesting that both proteins share similar properties, probably the START domain that is involved in cholesterol binding. General significance Mitochondrial proteases are involved in syncytiotrophoblast-cell steroidogenesis regulation. Understanding STARD3 activation and its role in Progesterone Synthesis is crucial to getting insight into its action mechanism in healthy and diseased syncytiotrophoblast cells.
-
Progesterone Synthesis by human placental mitochondria is sensitive to pka inhibition by h89
The International Journal of Biochemistry & Cell Biology, 2011Co-Authors: Cuauhtemoc Gomezconcha, Oscar Floresherrera, Sofia Olverasanchez, Ma Teresa Espinosagarcia, Federico MartinezAbstract:Abstract The transfer of cholesterol to mitochondria, which might involve the phosphorylation of proteins, is the rate-limiting step in human placental steroidogenesis. Protein kinase A (PKA) activity and its role in Progesterone Synthesis by human placental mitochondria were assessed in this study. The results showed that PKA and phosphotyrosine phosphatase D1 are associated with syncytiotrophoblast mitochondrial membrane by an anchoring kinase cAMP protein-121. The 32P-labeled of four major proteins was analyzed. The specific inhibitor of PKA, H89, decreased Progesterone Synthesis in mitochondria while in mitochondrial steroidogenic contact sites protein-phosphorylation was diminished, suggesting that PKA plays a role in placental hormone Synthesis. In isolated mitochondria, PKA activity was unaffected by the addition of cAMP suggesting a constant activity of this kinase in the syncytiotrophoblast. The presence of PKA and phosphotyrosine phosphatase D1 anchored to mitochondria by an anchoring kinase cAMP protein-121 indicated that syncytiotrophoblast mitochondria contain a full phosphorylation/dephosphorylation system.
-
contact sites from human placental mitochondria characterization and role in Progesterone Synthesis
Archives of Biochemistry and Biophysics, 2003Co-Authors: Aida Uribe, Jerome F. Strauss, Federico MartinezAbstract:To understand the functional compartmentalization of human placental mitochondria, we analyzed the composition and steroidogenic activity of contact sites. Several fractions containing contact sites were isolated using osmotic shock treatment and sucrose gradient centrifugation. These fractions contained various proteins and marker enzymes associated with mitochondrial membranes. The fractions containing the cytochrome P450 side chain cleavage system, cholesterol, nicotinamide adenine dinucleotide phosphate-isocitrate dehydrogenase, porin, and adenosine 5(')-triphosphate-diphosphohydrolase activity showed the capacity to synthesize Progesterone. Our observations indicate that all necessary elements and enzymes for steroidogenesis are present and functional in placental mitochondrial contact sites. This organization may facilitate the metabolism of cholesterol delivered to the outer mitochondrial membrane into steroid hormones by the inner mitochondrial membrane cholesterol side chain cleavage system.
Paul E Micevych - One of the best experts on this subject based on the ideXlab platform.
-
estradiol induced Progesterone Synthesis develops post puberty in the rostral hypothalamus and coincides with post pubertal changes in the steroidogenic pathway in female mouse hypothalamic astrocytes
bioRxiv, 2021Co-Authors: Margaret A Mohr, Tina Keshishian, Brennan A Falcy, Blake J Laham, Angela M Wong, Paul E MicevychAbstract:The development of estrogen positive feedback is a hallmark of female puberty. Both estrogen and Progesterone signaling are required for the functioning of this neuroendocrine feedback loop but the physiological changes that underlie the emergence of estrogen positive feedback remain unknown. Only after puberty does estradiol (E2) facilitate Progesterone Synthesis in female hypothalamic astrocytes (neuroP) (Mohr et al. 2018), an event critical for estrogen positive feedback and the LH surge. We hypothesize that prior to puberty, these astrocytes have low levels of membrane estrogen receptor alpha (ER&(alpha)), making them unable to respond to E2 with increased neuroP Synthesis prior to puberty. To test this hypothesis, pure populations of primary astrocyte cultures were derived from female mice at three different stages of development: pre-puberty (postnatal week 3), pubertal onset (week 5), and post-puberty (week 8). Hypothalamic astrocyte responses were measured after treatment with E2. Hypothalamic astrocytes increased Progesterone Synthesis across pubertal development. Prior to puberty, mER&(alpha) expression was low in hypothalamic astrocytes, but expression increased across puberty. The increase in mERα expression in hypothalamic astrocytes also corresponded with an increase in caveolin-1 protein, PKA phosphorylation, and a more rapid [Ca2+]i flux in response to E2. Together, these results indicate that increased mER&(alpha) in hypothalamic astrocytes contributed to the post-pubertal response to E2 that results in neuroP Synthesis, critical for ovulation.
-
membrane estrogen receptors stimulate intracellular calcium release and Progesterone Synthesis in hypothalamic astrocytes
The Journal of Neuroscience, 2010Co-Authors: Naheed Hamid, Galyna Bondar, Eric R Prossnitz, Paul E MicevychAbstract:In hypothalamic astrocytes obtained from adult female rats, estradiol rapidly increased free cytoplasmic calcium concentrations ([Ca 2+ ] i ) that facilitate Progesterone Synthesis. The present study demonstrated that estradiol (1 nm) significantly and maximally stimulated Progesterone Synthesis within 5 min, supporting a rapid, nongenomic mechanism. The group I metabotropic glutamate receptor (mGluR1a) antagonist LY 367385 [( S )-(+)-a-amino-4-carboxy-2-methylbenzeneacetic acid] attenuated both the estradiol-induced [Ca 2+ ] i release and Progesterone Synthesis. To investigate membrane-associated estrogen receptors (mERs), agonists for ERα, ERβ, STX-activated protein, and GPR30 were compared. The selective ERα agonist propylpyrazole triole (PPT) and STX most closely mimicked the estradiol-induced [Ca 2+ ] i responses, where PPT was more potent but less efficacious than STX. Only high doses (100 nm) of selective ERβ agonist diarylpropionitrile (DPN) and GPR30 agonist G-1 induced estradiol-like [Ca 2+ ] i responses. With the exception of DPN (even at 100 nm), all agonists stimulated Progesterone Synthesis. The PPT- and STX-induced [Ca 2+ ] i release and Progesterone Synthesis were blocked by LY 367385. While the G-1-stimulated [Ca 2+ ] i release was blocked by LY 367385, Progesterone Synthesis was not. Since GPR30 was detected intracellularly but not in the membrane, we interpreted these results to suggest that G-1 could activate mGluR1a on the membrane and GPR30 on the smooth endoplasmic reticulum to release intracellular calcium. Although STX and G-1 maximally stimulated [Ca 2+ ] i release in astrocytes from estrogen receptor-α knock-out (ERKO) mice, estradiol in vivo did not stimulate Progesterone Synthesis in the ERKO mice. Together, these results indicate that mERα is mainly responsible for the rapid, membrane-initiated estradiol-signaling that leads to Progesterone Synthesis in hypothalamic astrocytes.
-
membrane estrogen receptor α interacts with metabotropic glutamate receptor type 1a to mobilize intracellular calcium in hypothalamic astrocytes
Endocrinology, 2009Co-Authors: Omid R Hariri, Galyna Bondar, Paul E MicevychAbstract:Estradiol, acting on a membrane-associated estrogen receptor-α (mERα), induces an increase in free cytoplasmic calcium concentration ([Ca2+]i) needed for Progesterone Synthesis in hypothalamic astrocytes. To determine whether rapid estradiol signaling involves an interaction of mERα with metabotropic glutamate receptor type 1a (mGluR1a), changes in [Ca2+]i were monitored with the calcium indicator, Fluo-4 AM, in primary cultures of female postpubertal hypothalamic astrocytes. 17β-Estradiol over a range of 1 nm to 100 nm induced a maximal increase in [Ca2+]i flux measured as a change in relative fluorescence [ΔF Ca2+ = 615 ± 36 to 641 ± 47 relative fluorescent units (RFU)], whereas 0.1 nm of estradiol stimulated a moderate [Ca2+]i increase (275 ± 16 RFU). The rapid estradiol-induced [Ca2+]i flux was blocked with 1 μm of the estrogen receptor antagonist ICI 182,780 (635 ± 24 vs. 102 ± 11 RFU, P < 0.001) and 20 nmof the mGluR1a antagonist LY 367385 (617 ± 35 vs. 133 ± 20 RFU, P < 0.001). Whereas the mGluR1a ...
-
estradiol regulation of Progesterone Synthesis in the brain
Molecular and Cellular Endocrinology, 2008Co-Authors: Paul E Micevych, Kevin SinchakAbstract:Abstract Steroidogenesis is now recognized as a global phenomenon in the brain, but how it is regulated and its relationship to circulating steroids of peripheral origin have remained more elusive issues. Neurosteroids, steroids synthesized de novo in nervous tissue, have a large range of actions in the brain, but it is only recently that the role of neuroProgesterone in the regulation of arguably the quintessential steroid-dependent neural activity, regulation of the reproduction has been appreciated. Circuits involved in controlling the LH surge and sexual behaviors were thought to be influenced by estradiol and Progesterone synthesized in the ovary and perhaps the adrenal. It is now apparent that estradiol of ovarian origin regulates the Synthesis of neuroProgesterone, and it is the locally produced neuroProgesterone that is involved in the initiation of the LH surge and subsequent ovulation. In this model, estradiol induces the transcription of Progesterone receptors while stimulating Synthesis of neuroProgesterone. Although the complete signaling cascade has not been elucidated, many of the features have been characterized. The Synthesis of neuroProgesterone occurs primarily in astrocytes and requires the interaction of membrane-associated estrogen receptor-α with metabotropic glutamate receptor-1a. This G protein-coupled receptor activates a phospholipase C that in turn increases inositol trisphosphate (IP3) levels mediating the release of intracellular stores of Ca2+ via an IP3 receptor gated Ca2+ channel. The large increase in free cytoplasmic Ca2+ ([Ca2+]i) stimulates the Synthesis of Progesterone, which can then diffuse out of the astrocyte and activate estradiol-induced Progesterone receptors in local neurons to trigger the neural cascade to produce the LH surge. Thus, it is a cooperative action of astrocytes and neurons that is needed for estrogen positive feedback and stimulation of the LH surge.
-
estradiol stimulates Progesterone Synthesis in hypothalamic astrocyte cultures
Endocrinology, 2007Co-Authors: Paul E Micevych, Victor V Chaban, Julie Ogi, Phoebe Dewing, Kevin SinchakAbstract:The brain synthesizes steroids de novo, especially Progesterone. Recently estradiol has been shown to stimulate Progesterone Synthesis in the hypothalamus and enriched astrocyte cultures derived from neonatal cortex. Estradiol-induced hypothalamic Progesterone has been implicated in the control of the LH surge. The present studies were undertaken to determine whether hypothalamic astrocytes derived from female neonatal or female postpubertal rats increased production of Progesterone in response to an estradiol challenge. Estradiol induced Progesterone Synthesis in postpubertal astrocytes but not neonatal astrocytes. This estradiol action was blocked by the estrogen receptor antagonist ICI 182,780. Previously we had demonstrated that estradiol stimulates a rapid increase in free cytosolic Ca(2+) ([Ca(2+)](i)) spikes in neonatal cortical astrocytes acting through a membrane estrogen receptor. We now report that estradiol also rapidly increased [Ca(2+)](i) spikes in hypothalamic astrocytes. The membrane-impermeable estradiol-BSA construct also induced [Ca(2+)](i) spikes. Both estradiol-BSA and estradiol were blocked by ICI 182,780. Depleting intracellular Ca(2+) stores prevented the estradiol-induced increased [Ca(2+)](i) spikes, whereas removing extracellular Ca(2+) did not prevent estradiol-induced [Ca(2+)](i) spikes. Together these results indicate that estradiol acts through a membrane-associated receptor to release intracellular stores of Ca(2+). Thapsigargin, used to mimicked the intracellular release of Ca(2+) by estradiol, increased Progesterone Synthesis, suggesting that estradiol-induced Progesterone Synthesis involves increases in [Ca(2+)](i). Estradiol treatment did not change levels of steroid acute regulatory protein, P450 side chain cleavage, 3beta-hydroxysteroid dehydrogenase, and sterol carrier protein-2 mRNAs as measured by quantitative RT-PCR, suggesting that in vitro, estradiol regulation of Progesterone Synthesis in astrocytes does not depend on transcription of new steroidogenic proteins. The present results are consistent with our hypothesis that estrogen-positive feedback regulating the LH surge involves stimulating local Progesterone Synthesis by hypothalamic astrocytes.
Robert C. Tuckey - One of the best experts on this subject based on the ideXlab platform.
-
Progesterone Synthesis by the human placenta
Placenta, 2005Co-Authors: Robert C. TuckeyAbstract:One of the essential roles of the human placenta is to produce the steroid hormone Progesterone, which is required for the maintenance of pregnancy. The rate-determining step of placental Progesterone Synthesis is the conversion of cholesterol to pregnenolone by cytochrome P450scc (CYP11A1) in placental mitochondria in a reaction requiring electrons delivered via adrenodoxin reductase and adrenodoxin. Pregnenolone is converted to Progesterone by type 1 3beta-hydroxysteroid dehydrogenase located in the mitochondrion. Progesterone Synthesis by the human placenta displays notable differences from steroid Synthesis in the classical steroid producing tissues such as the adrenal cortex and corpus luteum. One important difference is that the placenta lacks short term modulation of steroid Synthesis and does not express the steroidogenic acute regulatory (StAR) protein. The most notable difference between the placenta and other steroidogenic tissues is that electron supply to P450scc limits the rate at which cholesterol is converted to pregnenolone in the placenta. The limiting component for electron delivery to P450scc is the concentration of adrenodoxin reductase in the mitochondrial matrix which is insufficient to maintain the adrenodoxin pool in a fully reduced state. Evidence suggests that placental mitochondria have a near-saturating cholesterol concentration for P450scc, likely provided by the StAR-like protein MLN64, and cholesterol translocation to the P450scc is not a major site of regulation of Progesterone Synthesis. Cyclic AMP stimulates Progesterone Synthesis by the human placenta but uncertainty remains regarding the key hormones that control cyclic AMP levels. The mechanism of regulation of adrenodoxin reductase levels in the human placenta remains to be studied.
-
molten globule structure and steroidogenic activity of n 218 mln64 in human placental mitochondria
Endocrinology, 2004Co-Authors: Robert C. Tuckey, Himangshu S Bose, Inez Czerwionka, Walter L MillerAbstract:Progesterone Synthesis by the human placenta requires the conversion of mitochondrial cholesterol to pregnenolone by cytochrome P450scc. Most steroidogenic tissues use the steroidogenic acute regulatory protein (StAR) to deliver cholesterol to the inner mitochondrial membrane where P450scc is located, but StAR is not expressed in the human placenta. However, the human placenta does express MLN64, which has a C-terminal domain homologous to StAR that can also transport cholesterol. We investigated the ability of bacterially expressed N-218 MLN64 and N-62 StAR to transport cholesterol between artificial membranes and to its inner membrane site of use in placental mitochondria. Urea denaturation experiments show that N-218 MLN64 undergoes a pH-dependent and denaturant-dependent structural transition to a molten globule state, as reported previously for N-62 StAR. N-218 MLN64 stimulated cholesterol transfer between artificial phospholipid vesicles with an initial rate of 6.5 mol/min·mol N-218 MLN64. Both N-21...
-
Placental cytochrome P450scc (CYP11A1): comparison of catalytic properties between conditions of limiting and saturating adrenodoxin reductase.
The Journal of steroid biochemistry and molecular biology, 2002Co-Authors: Robert C. Tuckey, Madeleine J. HeadlamAbstract:Abstract The mitochondrial side-chain cleavage of cholesterol, catalysed by cytochrome P450scc, is rate-limiting in the Synthesis of Progesterone by the human placenta. Cytochrome P450scc activity is in turn limited by the concentration of adrenodoxin reductase (AR) in placental mitochondria. In order to better understand which components of the cholesterol side-chain cleavage system are important in the regulation of placental Progesterone Synthesis, we have examined their effects on P450scc activity with both saturating and limiting concentrations of AR. The present study reveals that decreasing the AR concentration causes a decrease in the K m of cytochrome P450scc for cholesterol, facilitating saturation of the enzyme with its substrate. Decreasing AR resulted in P450scc activity becoming less sensitive to changes in P450scc concentration. The adrenodoxin (Adx) concentration in mitochondria from term placentae is near-saturating for P450scc and under these conditions, we found that decreasing AR reduces the K m of P450scc for adrenodoxin. Increasing either the cholesterol or P450scc concentration increased the amount of AR required for P450scc to work at half its maximum velocity. A relatively small increase in AR can support considerably higher rates of side-chain cleavage activity when there is a coordinate increase in AR and P450scc concentrations. We conclude from this study that cholesterol is near-saturating for cytochrome P450scc activity in placental mitochondria due to the P450scc displaying a low K m for cholesterol resulting from the low and rate-limiting concentration of AR present. This study reveals that it is unlikely that cholesterol or adrenodoxin concentrations are important regulators of placental Progesterone Synthesis but AR or coordinate changes in AR and P450scc concentrations are likely to be important in its regulation.
-
The concentration of adrenodoxin reductase limits cytochrome P450scc activity in the human placenta
European journal of biochemistry, 1999Co-Authors: Robert C. Tuckey, Jade SadleirAbstract:We have previously reported that cytochrome P450scc activity in the human placenta is limited by the supply of electrons to the P450scc [Tuckey, R. C., Woods, S. T. & Tajbakhsh, M. (1997) Eur. J. Biochem. 244, 835‐839]. The aim of the present study was to determine whether it is adrenodoxin reductase, adrenodoxin or both which limits cytochrome P450scc activity and hence Progesterone Synthesis in the placenta. We found that the concentrations of adrenodoxin reductase and adrenodoxin in placental mitochondria were both considerably lower than the concentrations of these proteins in the bovine adrenal cortex. When P450scc activity assays were carried out at high mitochondrial protein concentrations, we found that the addition of exogenous adrenodoxin reductase to sonicated mitochondria rescued pregnenolone Synthesis to a level above that for intact mitochondria, showing that adrenodoxin is near-saturating in vivo. In contrast, pregnenolone Synthesis by sonicated mitochondria was almost zero even after the addition of human adrenodoxin. This shows that the concentration of endogenous adrenodoxin reductase was insufficient to support appreciable rates of pregnenolone Synthesis, even when concentrated mitochondrial samples were used. Comparative studies with human and bovine adrenodoxin reductase have revealed that a twofold higher concentration of human adrenodoxin reductase is required for maximal P450scc activity in the presence of saturating human adrenodoxin. Thus, not only is the adrenodoxin concentration low in placental mitochondria, but the amount required for maximal P450scc activity is higher than that for the bovine reductase. Overall, the data indicate that the adrenodoxin reductase concentration limits the activity of P450scc in placental mitochondria and hence determines the rate of Progesterone Synthesis.