scholarly journals Stimulatory Effect of Insulin on 5α-Reductase Type 1 (SRD5A1) Expression through an Akt-Dependent Pathway in Ovarian Granulosa Cells

Endocrinology ◽  
2010 ◽  
Vol 151 (10) ◽  
pp. 5030-5037 ◽  
Author(s):  
Pradeep P. Kayampilly ◽  
Brett L. Wanamaker ◽  
James A. Stewart ◽  
Carrie L. Wagner ◽  
K. M. J. Menon
Keyword(s):  
Catalytic Activity ◽  
Mrna Expression ◽  
Granulosa Cell ◽  
Granulosa Cells ◽  
Insulin Treatment ◽  
Reductase Mrna ◽  
Dose Dependent Increase ◽  
Dose Dependent ◽  
Dependent Pathway

Elevated levels of 5α-reduced androgens have been shown to be associated with hyperandrogenism and hyperinsulinemia, the leading causes of ovulatory dysfunction in women. 5α-Dihydrotestosterone reduces ovarian granulosa cell proliferation by inhibiting FSH-mediated mitogenic signaling pathways. The present study examined the effect of insulin on 5α-reductase, the enzyme that catalyses the conversion of androgens to their 5α-derivatives. Granulosa cells isolated from immature rat ovaries were cultured in serum-free, phenol red-free DMEM-F12 media and treated with different doses of insulin (0, 0.1, 1.0, and 10.0 μg/ml) for different time intervals up to 12 h. The expression of 5α-reductase type 1 mRNA, the predominant isoform found in granulosa cells, showed a significant (P < 0.05) increase in response to the insulin treatment up to 12 h compared with control. The catalytic activity of 5α-reductase enzyme was also stimulated in a dose-depended manner (P < 0.05). Inhibiting the Akt-dependent signaling pathway abolished the insulin-mediated increase in 5α-reductase mRNA expression, whereas inhibition of the ERK-dependent pathway had no effect. The dose-dependent increase in 5α-reductase mRNA expression as well as catalytic activity seen in response to insulin treatment was also demonstrated in the human granulosa cell line (KGN). In addition to increased mRNA expression, a dose-dependent increase in 5α-reductase protein expression in response to insulin was also seen in KGN cells, which corroborated well with that of mRNA expression. These results suggest that elevated levels of 5α-reduced androgens seen in hyperinsulinemic conditions might be explained on the basis of a stimulatory effect of insulin on 5α-reductase in granulosa cells. The elevated levels of these metabolites, in turn, might adversely affect growth and proliferation of granulosa cells, thereby impairing follicle growth and ovulation.

scholarly journals Follicle-Stimulating Hormone Inhibits Adenosine 5′-Monophosphate-Activated Protein Kinase Activation and Promotes Cell Proliferation of Primary Granulosa Cells in Culture through an Akt-Dependent Pathway

Endocrinology ◽  
2009 ◽  
Vol 150 (2) ◽  
pp. 929-935 ◽  
Author(s):  
Pradeep P. Kayampilly ◽  
K. M. J. Menon
Keyword(s):  
Cell Cycle ◽  
Cell Proliferation ◽  
Protein Kinase ◽  
Mrna Expression ◽  
Granulosa Cell ◽  
Granulosa Cells ◽  
Ampk Activation ◽  
Cyclin D2 ◽  
Cell Cycle Inhibitor ◽  
Dependent Pathway

FSH, acting through multiple signaling pathways, regulates the proliferation and growth of granulosa cells, which are critical for ovulation. The present study investigated whether AMP-activated protein kinase (AMPK), which controls the energy balance of the cell, plays a role in FSH-mediated increase in granulosa cell proliferation. Cells isolated from immature rat ovaries were grown in serum-free, phenol red free DMEM-F12 and were treated with FSH (50 ng/ml) for 0, 5, and 15 min. Western blot analysis showed a significant reduction in AMPK activation as observed by a reduction of phosphorylation at thr 172 in response to FSH treatment at all time points tested. FSH also reduced AMPK phosphorylation in a dose-dependent manner with maximum inhibition at 100 ng/ml. The chemical activator of AMPK (5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside, 0.5 mm) increased the cell cycle inhibitor p27 kip expression significantly, whereas the AMPK inhibitor (compound C, 20 μm) and FSH reduced p27kip expression significantly compared with control. FSH treatment resulted in an increase in the phosphorylation of AMPK at ser 485/491 and a reduction in thr 172 phosphorylation. Inhibition of Akt phosphorylation using Akt inhibitor VIII reversed the inhibitory effect of FSH on thr 172 phosphorylation of AMPK, whereas ERK inhibitor U0126 had no effect. These results show that FSH, through an Akt-dependent pathway, phosphorylates AMPK at ser 481/495 and inhibits its activation by reducing thr 172 phosphorylation. AMPK activation by 5-amino-imidazole-4-carboxamide-1-β-d-ribofuranoside treatment resulted in a reduction of cell cycle regulatory protein cyclin D2 mRNA expression, whereas FSH increased the expression by 2-fold. These results suggest that FSH promotes granulosa cell proliferation by increasing cyclin D2 mRNA expression and by reducing p27 kip expression by inhibiting AMPK activation through an Akt-dependent pathway. FSH stimulates granulosa cell proliferation by reducing cell cycle inhibitor p27 kip through AMP kinase inhibition.

scholarly journals High-glucose-induced metallothionein expression in endothelial cells: an endothelin-mediated mechanism

2001 ◽  
Vol 281 (3) ◽  
pp. C899-C907 ◽  
Author(s):  
Margarita D. Apostolova ◽  
Shali Chen ◽  
Subrata Chakrabarti ◽  
M. George Cherian
Keyword(s):  
Oxidative Stress ◽  
Endothelial Cells ◽  
Mrna Expression ◽  
High Glucose ◽  
Umbilical Vein ◽  
Cell Permeability ◽  
Extracellular Glucose ◽  
Dose Dependent Increase ◽  
Dose Dependent ◽  
Dependent Pathway

Vascular endothelial cells are constantly exposed to oxidative stress and must be protected by physiological responses. In diabetes mellitus, endothelial cell permeability is impaired and may be increased by high extracellular glucose concentrations. It has been postulated that metallothionein (MT) can protect endothelial cells from oxidative stress with its increased expression by cytokines, thrombin, and endothelin (ET)-1. In this study, we demonstrate that high glucose concentration can induce MT expression in endothelial cells through a distinct ET-dependent pathway. Exposure of human umbilical vein endothelial cells (HUVEC) to increasing concentrations of glucose resulted in a rapid dose-dependent increase in MT-2 and ET-1 mRNA expression. MT expression may be further augmented with addition of ET-1. Preincubation of the cells with the specific ETB antagonist BQ-788 blocked MT-2 mRNA expression more effectively than the ETA inhibitor TBC-11251. High glucose also increased immunoreactive MT protein expression and induced translocation of MT into the perinuclear area. Perinuclear localization of MT was related to high-glucose-induced reorganization of F-actin filaments. These results demonstrate that an increase in extracellular glucose in HUVEC can lead to a rapid dose-dependent increase in MT-2 mRNA expression and to perinuclear localization of MT protein with changes to the cytoskeleton. These effects are mediated via the ET receptor-dependent pathway.

Effects of recombinant bovine somatotrophin, insulin-like growth factor-I and insulin on bovine granulosa cell steroidogenesis in vitro

1994 ◽  
Vol 143 (1) ◽  
pp. 157-164 ◽  
Author(s):  
J G Gong ◽  
D McBride ◽  
T A Bramley ◽  
R Webb
Keyword(s):  
Granulosa Cell ◽  
Granulosa Cells ◽  
Dependent Manner ◽  
Serum Free ◽  
Factor I ◽  
Igf I ◽  
Serum Free Conditions ◽  
Dose Dependent ◽  
Bovine Somatotrophin ◽  
Cells Cultured

Abstract Our previous studies have demonstrated that physiological concentrations of metabolic hormones, including recombinant bovine somatotrophin (BST), insulin-like growth factor-I (IGF-I) and insulin, can significantly stimulate the proliferation of bovine granulosa cells cultured under serum-free conditions. In this study we investigated the effects of these factors on bovine granulosa cell steroidogenesis using the same culture system. Bovine granulosa cells were obtained from antral follicles classified into three size classes: small, <5 mm; medium-sized, 5–10 mm and large, >10 mm in diameter. Whilst not affecting steroidogenesis by granulosa cells from small and medium-sized follicles, BST (10–1000 ng/ml) stimulated the secretion of both oestradiol and progesterone by granulosa cells from large follicles in a dose-dependent manner. Insulin (1–1000 ng/ml) and IGF-I (10–1000 ng/ml) stimulated the secretion of oestradiol and progesterone by granulosa cells from all three size categories of follicles in a dose-dependent manner. FSH (200 ng/ml) alone increased progesterone secretion by granulosa cells from all three size classes of follicles, but had no effect on oestradiol secretion by granulosa cells. Both IGF-I (200 ng/ml) and insulin (30 ng/ml) acted in synergy with FSH (200 ng/ml) to stimulate steroidogenesis by granulosa cells from all three size categories of follicles, but no such interaction was observed between BST (50 ng/ml) and FSH (200 ng/ml). In conclusion, BST, IGF-I and insulin significantly influence the steroidogenic activity of bovine granulosa cells cultured under serum-free conditions. However, unlike their effects on cell proliferation, the minimal effective concentrations of these factors required to stimulate granulosa cell steroidogenesis were higher than those observed in our previous studies in vivo. Journal of Endocrinology (1994) 143, 157–164

Expression and regulation of high mobility group AT-hook 1 (HMGA1) during ovulation and luteinisation in rat ovary

2019 ◽  
Vol 31 (4) ◽  
pp. 698 ◽  
Author(s):  
Hao-ran Li ◽  
Yan Li ◽  
Yu Liu ◽  
Jiao-jiao Yu ◽  
Fei-xue Li
Keyword(s):  
Mrna Expression ◽  
Granulosa Cell ◽  
Cell Cultures ◽  
Granulosa Cells ◽  
In Situ Hybridisation ◽  
High Mobility ◽  
High Mobility Group ◽  
Female Rats ◽  
Corpora Lutea ◽  
Hmga1 Expression

High mobility group AT-hook 1 (HMGA1) is able to regulate gene expression and function as a tumour suppressor. The spatiotemporal expression pattern of HMGA1 was investigated in this study. Immature female rats (22–23 days old) were treated with 10IU, s.c., pregnant mare’s serum gonadotrophin to stimulate follicular development, followed 48h later by injection with 5IU, s.c., human chorionic gonadotrophin (hCG). Whole ovaries or granulosa cells were collected at various times after hCG administration (n=3 per time point). Real-time polymerase chain reaction and western blot analysis revealed that HMGA1 was highly stimulated in the ovary by 4–12h after hCG treatment. In situ hybridisation analysis demonstrated that Hmga1 mRNA expression was induced in granulosa cells between 8 and 12h after hCG treatment. There was negligible Hmga1 mRNA signal observed in newly forming corpora lutea. In addition, the data indicated that both the protein kinase (PK) A and PKC pathways regulated Hmga1 expression in rat granulosa cells. In rat granulosa cell cultures, upregulation of Hmga1 was dependent on new protein synthesis because Hmga1 was inhibited by cycloheximide. Furthermore, Hmga1 mRNA expression in rat granulosa cell cultures was inhibited by AG1478, whereas NS398 and RU486 had no effect, suggesting that Hmga1 expression was regulated, in part, by the epidermal growth factor pathway. In summary, the findings of this study suggest that induction of Hmga1 may be important for theca and granulosa cell differentiation into luteal cells.

Effect of in vitro copper supplementation on granulosa cell estradiol synthesis and associated genes

2018 ◽  
Author(s):  
Ravi, P.S.P. Gupta, S. Nandi, S. Mondal, Kumar Soni­ ◽  
P.S.P. Gupta ◽  
S. Nandi ◽  
S. Mondal, J.R. Ippala, Avantika Mor, A Mondal ◽  
J.R. Ippala ◽  
...  
Keyword(s):  
Cell Survival ◽  
Mrna Expression ◽  
Granulosa Cell ◽  
Granulosa Cells ◽  
Culture Medium ◽  
Estrus Synchronization ◽  
Ovarian Granulosa Cells ◽  
Effect Of Copper ◽  
Estradiol Synthesis

The study was conducted by supplementing cupric chloride dihydrate to modulate the estradiol synthesis in granulosa cells with a hypothesis of possible use of copper to potentiate or partially replace the hormones for estrus induction / estrus synchronization in future studies. In present study copper at three doses (0.1, 0.5 and 1 mM level in culture medium) were tested to deserve see their effects on in vitro granulosa cell survival, estradiol synthesis and their associated genes of ovarian granulosa cells of goat.There was no effect of copper on the ovarian granulosa cell survival rate. There was a considerable increase in the estradiol level per ml culture medium basis by 6th day of in vitro culture with the second dose of copper i.e. 0.5 mM, but the increase was non-significant (P greator than 0.05). There was no significant effect of copper on estradiol synthesis when expressed on per 30000 cell basis. Effect of copper (0.1 mM and 0.5 mM) on the mRNA expression of genes of aromatase (CYP19A1) and cyclin D2 (CCND2) was estimated. Copper had significantly (P less than 0.05) increased the mRNA expression of CCND2 and CYP19A1in ovarian granulosa cells with only one of the two doses tested i.e. 0.5 mM. Hence, copper can be considered as a potential mineral to supplement along with hormones in estrus induction or estrus synchronization protocols to minimize the use of hormones.

scholarly journals Inhibition of DNA methylation increases follistatin expression and secretion in the human adrenocortical cell line NCI-H295R

2006 ◽  
Vol 188 (2) ◽  
pp. 305-310 ◽  
Author(s):  
Pauliina Utriainen ◽  
Jianqi Liu ◽  
Tiina Kuulasmaa ◽  
Raimo Voutilainen
Keyword(s):  
Dna Methylation ◽  
Mrna Expression ◽  
Aberrant Methylation ◽  
Adrenocortical Cells ◽  
Adrenocortical Tumors ◽  
Specific Pcr ◽  
Methylation Inhibitor ◽  
Peptide Secretion ◽  
Dose Dependent Increase ◽  
Dose Dependent

Activin affects adrenocortical steroidogenesis and increases apoptosis, while follistatin (FS) acts as an activin antagonist by binding to activin, preventing attachment to its receptors. The regulation of FS expression in the adrenal cortex is poorly understood. Adrenocortical tumors often display aberrant methylation. In the present study, we investigated the effect of DNA methylation on FS mRNA expression and peptide secretion in adrenocortical cells. We treated human NCI-H295R adrenocortical cells with the methylation inhibitor 5-Aza-2′deoxycytidine (Azad; 0.1–100 μM for 1, 4 or 7 days) and measured FS mRNA expression by Northern blot and quantitative real time RT-PCR analyses as well as FS secretion by specific ELISA. Methylation-specific PCR showed decreased methylation in the FS promoter region after Azad treatment. A significant (P < 0.05) time- and dose-dependent increase in FS mRNA expression (up to 4.6-fold) and peptide secretion (up to 17.1-fold) was detected after Azad treatment. We conclude that FS gene expression and peptide secretion in NCI-H295R adrenocortical cells are regulated by DNA methylation. Thus, variable methylation in different adrenocortical tumors may influence activin bioactivity and its consequences in steroidogenesis and cell proliferation/apoptosis.

Effect of Tumor Necrosis Factor-α on in vitro Prostaglandin Production in Buffalo Corpus Luteum

2021 ◽  
Author(s):  
M.K. Tripathi ◽  
S. Mondal ◽  
I.J. Reddy ◽  
A. Mor
Keyword(s):  
Mrna Expression ◽  
Corpus Luteum ◽  
Prostaglandin E ◽  
Prostaglandin F ◽  
Important Regulator ◽  
Factor Α ◽  
Dose Dependent Increase ◽  
Dose Dependent ◽  
Necrosis Factor

Background: Corpus luteum plays key role in embryonic survival. Prostaglandins are the important regulator controlling the life span of corpus luteum. The present study investigated the effect of various doses of TNFα on in vitro PGF2α and PGE2 production and expression profiling of PGFS and PGES mRNA in buffalo Corpus Luteum (CL).Methods: Buffalo ovaries with mid-luteal phase CL were collected from the abattoir and CL were enucleated from surrounding tissues. Corpus luteum were finely chopped, rinsed with HBSS (Hanks Balanced Salt Solution) medium; supplemented with gentamycin and 0.1% BSA and incubated at 37°C for 1 hr in HBSS containing 0.1% collagenase. The cell suspension following filtration was washed by HBBS supplemented with gentamycin and 0.1% BSA (bovine serum albumin) and was treated with increasing doses of TNFα (0.1, 0.5 and 1.0 nM) and cultured at 38.5°C, 5% CO2 level for 24 hr. Result: There was dose dependent increase in concentrations of PGF2α and PGE2 with increasing doses of TNFα. The PGFS (prostaglandin F synthase) mRNA expression increased with increasing doses of TNFα. However, there was decrease in PGES (prostaglandin E synthase) mRNA expression at 0.1 nM and 0.5 nM TNFα but PGES mRNA expression increased at 1.0 nM TNFα as compared to control. It can be concluded that TNFα may alter PGES and PGFS mRNA expression and prostaglandin secretion in buffalo CL. 

scholarly journals IL-6 Up-Regulates the Expression of Rat LH Receptors during Granulosa Cell Differentiation

Endocrinology ◽  
2014 ◽  
Vol 155 (4) ◽  
pp. 1436-1444 ◽  
Author(s):  
Fumiharu Imai ◽  
Hiroshi Kishi ◽  
Kohshiro Nakao ◽  
Toshio Nishimura ◽  
Takashi Minegishi
Keyword(s):  
Mrna Expression ◽  
Granulosa Cells ◽  
Ovarian Function ◽  
Luciferase Assay ◽  
Dependent Manner ◽  
Signal Transducer ◽  
Female Wistar Rats ◽  
Translational Start ◽  
Transcription 3 ◽  
Dose Dependent

IL-6 is produced in granulosa cells under normal physiological conditions, including during ovulation. However, the roles of IL-6 in ovarian function, including regulation of LH receptor (LHR) expression in granulosa cells, have not been explored in detail. The aim of this study was to identify the mechanism underlying the effect of IL-6 on LHR expression in the granulosa cells of female Wistar rats. Our results indicated that IL-6 clearly enhanced the FSH-induced LHR mRNA expression in a dose-dependent manner and did not stimulate cAMP accumulation by itself. The membrane protein level of LHR, assessed by a binding assay, was increased by FSH and was further enhanced by association with IL-6. Results of the luciferase assay, using promoter constructs of LHR 281 bp upstream of the translational start site, revealed that IL-6 increased the promoter activity induced by FSH, but this effect was not observed with treatment by IL-6 alone. This ability of IL-6 to enhance FSH-induced LHR mRNA expression was blocked by the Janus tyrosine kinase (JAK) pathway inhibitor, but not by the ERK1/2 inhibitor. Thus, we speculated that this IL-6 activity might be mediated by the JAK/signal transducer and activator of transcription pathway. In addition, IL-6 augmented FSH-induced IL-6 receptor α mRNA expression and FSH elevated IL-6 production in granulosa cells, which indicates that IL-6 may positively regulate paracrine and autocrine actions in granulosa cells. These results suggest that IL-6 up-regulates FSH-induced LHR production by increasing mRNA transcription, and JAK/signal transducer and activator of transcription 3 signaling is required for up-regulation by IL-6 in granulosa cells.

scholarly journals IGF1 induces up-regulation of steroidogenic and apoptotic regulatory genes via activation of phosphatidylinositol-dependent kinase/AKT in bovine granulosa cells

Reproduction ◽  
2010 ◽  
Vol 139 (1) ◽  
pp. 139-151 ◽  
Author(s):  
Arul Murugan Mani ◽  
Mark A Fenwick ◽  
Zhangrui Cheng ◽  
Mohan K Sharma ◽  
Dheer Singh ◽  
...  
Keyword(s):  
Mrna Expression ◽  
Granulosa Cell ◽  
Granulosa Cells ◽  
Cellular Proliferation ◽  
Mapk Pathway ◽  
Regulatory Gene ◽  
Follicular Development ◽  
Pi3k Pathway ◽  
Cell Number ◽  
Regulatory Genes

IGF1, a potent stimulator of cellular proliferation, differentiation and development, regulates granulosa cell steroidogenesis and apoptosis during follicular development. Depending upon species and stage of follicular growth, IGF1 acts on granulosa cell steroidogenesis either alone or together with FSH. We examined the mechanism of action of IGF1 in bovine granulosa cells in serum-free culture without insulin to determine its potential role in the regulation of steroidogenic and apoptotic regulatory gene expression and to investigate the interaction of FSH with IGF1 on this mechanism. Bovine granulosa cells treated with IGF1 demonstrated a significant increase in 17β-oestradiol (OE2) production, cell number and in mRNA expression ofCYP11A1,HSD3B1,CYP19A1,BAX, type 1 IGF receptor (IGF1R) andFSHR, while FSH alone had no significant effects. IGF1 or FSH alone or both together had no effect onBCL2expression. IGF1 with FSH resulted in a synergistic increase in granulosa cell number and in mRNA expression ofCYP19A1andIGF1Rwithout altering OE2production. IGF1 stimulated the phosphoinositide 3′-OH kinase (PI3K) but not the MAPK pathway in granulosa cells, as evidenced by increased phosphorylation of AKT but not extracellular-regulated kinase 1/2. Addition of the PI3K pathway inhibitor LY294002 (but not the MAPK pathway inhibitor PD98059) abrogated the increased expression of genes induced by IGF1. IGF1 therefore up-regulates the steroidogenic and apoptotic regulatory genes via activation of PI3K/AKT in bovine granulosa cells. The synergistic action of IGF1 with FSH is of likely key importance for the development of small antral follicles before selection; subsequently, other factors such as LH may also become necessary for continued cell survival.

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