134. EPIDERMAL GROWTH FACTOR RECEPTOR/MAPK3/1 PATHWAY CROSS-TALK ENABLES GROWTH DIFFERENTIATION FACTOR 9 TO SIGNAL THROUGH SMAD2/3 IN MOUSE GRANULOSA CELLS

2009 ◽  
Vol 21 (9) ◽  
pp. 53
Author(s):  
M. Sasseville ◽  
L. J. Ritter ◽  
T. Nguyen ◽  
D. G. Mottershead ◽  
D. L. Russell ◽  
...  
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Granulosa Cells ◽  
Cross Talk ◽  
Egf Receptor ◽  
Thymidine Incorporation ◽  
Differentiation Factor ◽  
Growth Differentiation Factor 9 ◽  
Epidermal Growth ◽  
Receptor Inhibitor

Oocyte-secreted growth differentiation factor 9 (GDF9) plays a critical role throughout folliculogenesis. It has been shown to control many functions of granulosa cells, including gene expression, steroidogenesis and proliferation. This study investigates the cellular requirements that allow GDF9 to act on granulosa cells. Our results showed that GDF9 (20 ng/ml)-stimulated mouse granulosa cells 3H-thymidine incorporation was inhibited by a type 1 receptor Alk4/5/7 inhibitor (SB431542, 5 μM), by an epidermal growth factor (EGF) receptor inhibitor (AG1478, 5μM) and a MEK1 inhibitor (U0126, 10 μM). Interestingly, activin A- and TGFβ-stimulated 3H-thymidine incorporation shared similar inhibitor sensitivity. Moreover, when denuded oocytes were used as the mitogenic agent, SB431542, AG1478 and U0126 all prevented the increase in 3H-thymidine incorporation. Oocyte-stimulated 3H-thymidine incorporation in secondary follicles and cumulus-oocyte complexes were also sensitive to Alk4/5/7, EGF receptor and MEK1 inhibition. Basal and EGF-stimulated levels of phopho-MAPK3/1 were inhibited by using the EGF receptor inhibitor, but were not affected by inhibition of Alk4/5/7 or by adding GDF9 in granulosa cells. Using granulosa cells transfected with a SMAD3-luciferase reporter construct, GDF9-stimulated SMAD3 response could be inhibited by Alk4/5/7, EGFR and MEK1 inhibitors. Genes involved in cumulus cells expansion (Ptx3 and Has2) were upregulated in granulosa cells by co-culturing with denuded oocytes and that upregulation was inhibited by Alk4/5/7 as well as by EGF receptor inhibition. These results suggest that TGFβ superfamily members signalling through Smad2/3 share a common requirement of EGF receptor-dependant phospho-MAPK3/1 throughout folliculogenesis. These results strongly suggest that, apart from its role in the transmission of the ovulatory LH signal within the ovarian follicle, EGF receptor pathway might serve as modulators of GDF9 action on granulosa cells. Hence the interaction between endocrine and oocyte signalling may be mediated at the level of MAPK and Smad2/3 cross-talk in granulosa cells.

Paracrine effects via the epidermal growth factor receptor in the rodent testis may be mediated by non-Leydig interstitial cells

1993 ◽  
Vol 136 (3) ◽  
pp. 439-NP ◽  
Author(s):  
A. Moore ◽  
I. D. Morris
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Leydig Cell ◽  
Leydig Cells ◽  
Interstitial Cell ◽  
Egf Receptor ◽  
Transforming Growth Factor ◽  
Thymidine Incorporation ◽  
Buoyant Density ◽  
Epidermal Growth

ABSTRACT The epidermal growth factor (EGF) receptor is expressed in a wide variety of cell types and is known to be present in the testis of many species including man. In the present study, specific 125 I-labelled EGF binding was observed in isolated interstitial cell preparations from both the intact and Leydig cell-depleted rat testis. It was demonstrated that the population of cells to which 125I-labelled EGF binds has a different buoyant density from either of the two adult Leydig cell populations, and remains unchanged in the absence of Leydig cells following in-vivo treatment with ethane dimethane sulphonate (EDS). Cells of this density (1·064 g/ml) identified by electron microscopy were fusiform mesenchymal cells, identical to those suggested by others to be able to differentiate into Leydig cells in vitro, i.e. Leydig cell precursors. In a culture system using two interstitial cell preparations of different buoyant densities from immature rats, both EGF and transforming growth factor-α (TGF-α) caused increased [3H]thymidine incorporation in the less dense cell preparation. TGF-α was more potent than EGF. EGF increased testosterone production in both fractions in amounts which could be related to the amount of 3β-hydroxysteroid dehydrogenase (3β-HSD)-positive cells. This study demonstrated that rat Leydig cells (defined as those cells which bind 125I-labelled human chorionic gonadotrophin, have distinct buoyant densities, are 3β-HSD positive and are sensitive to EDS), do not bind 125I-labelled EGF. Rather, EGF binds to a mesenchymal cell without LH receptors which is resistant to EDS. Growth factors which act via the EGF receptor increased [3H]thymidine incorporation in a Leydig cell-depleted interstitial fraction which may reflect an action upon the progenitor of the mature Leydig cell. Journal of Endocrinology (1993) 136, 439–446

scholarly journals Epidermal Growth Factor (EGF) Receptor Ligands in the Chicken Ovary: I. Evidence for Heparin-Binding EGF-Like Growth Factor (HB-EGF) as a Potential Oocyte-Derived Signal to Control Granulosa Cell Proliferation and HB-EGF and Kit Ligand Expression

Endocrinology ◽  
2007 ◽  
Vol 148 (7) ◽  
pp. 3426-3440 ◽  
Author(s):  
Yajun Wang ◽  
Juan Li ◽  
Crystal Ying Wang ◽  
Amy Ho Yan Kwok ◽  
Frederick C. Leung
Keyword(s):  
Cell Proliferation ◽  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Granulosa Cells ◽  
Egf Receptor ◽  
Follicular Development ◽  
Kit Ligand ◽  
Egfr Ligand ◽  
Epidermal Growth ◽  
Chicken Ovary

There is increasing evidence that epidermal growth factor (EGF) receptor (EGFR) ligand and Kit ligand (KL) play critical roles in controlling follicular development in mammals. Because little is known about their expressions in the ovary of nonmammalian vertebrate, our study aimed to examine the expression, hormonal regulation, and interaction of HB-EGF and KL in the chicken ovary. Using semiquantitative RT-PCR, we demonstrated that ovarian HB-EGF expression increased dramatically with the posthatching ovarian growth. In line with this finding, HB-EGF was shown to be produced primarily by the growing oocytes and capable of stimulating the proliferation of granulosa cells in prehierarchal (3 mm) and preovulatory follicles (F5 and F1). Although HB-EGF expression is mainly restricted to the oocytes, its expression in cultured granulosa cells could be transiently yet strongly induced by HB-EGF and other EGFR ligands including EGF and TGF-α. And the inducing effect of HB-EGF was completely abolished by AG1478 (10 μm) or PD98059 (100 μm), indicating that the action of HB-EGF is mediated by EGFR and intracellular MAPK/ERK signaling pathway. Unlike mammals, only KL-1, not the other three isoforms identified (KL-2, -3, and -4), was detected to be predominantly expressed in the chicken ovary. Interestingly, KL expression in undifferentiated and differentiated granulosa cells could be transiently down-regulated by HB-EGF, implying an intrafollicular communication between growing oocyte and surrounding granulosa cells through the interplay of EGFR ligand and KL. Collectively, our data suggest that HB-EGF is likely a paracrine signal from the oocyte to regulate granulosa cell proliferation and HB-EGF and KL expression during ovarian follicular development.

scholarly journals Paracrine and Autocrine Regulation of Epidermal Growth Factor-Like Factors in Cumulus Oocyte Complexes and Granulosa Cells: Key Roles for Prostaglandin Synthase 2 and Progesterone Receptor

2006 ◽  
Vol 20 (6) ◽  
pp. 1352-1365 ◽  
Author(s):  
Masayuki Shimada ◽  
Inmaculada Hernandez-Gonzalez ◽  
Ignacio Gonzalez-Robayna ◽  
JoAnne S. Richards
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Progesterone Receptor ◽  
Granulosa Cells ◽  
Egf Receptor ◽  
Immune Cell ◽  
Cumulus Cells ◽  
Functional Changes ◽  
Prostaglandin Synthase ◽  
Epidermal Growth

Abstract The molecular bridges that link the LH surge with functional changes in cumulus cells that possess few LH receptors are being unraveled. Herein we document that epidermal growth factor (EGF)-like factors amphiregulin (Areg), epiregulin (Ereg), and betacellulin (Btc) are induced in cumulus oocyte complexes (COCs) by autocrine and paracrine mechanisms that involve the actions of prostaglandins (PGs) and progesterone receptor (PGR). Areg and Ereg mRNA and protein levels were reduced significantly in COCs and ovaries collected from prostaglandin synthase 2 (Ptgs2) null mice and Pgr null (PRKO) mice at 4 h and 8 h after human chorionic gonadotropin, respectively. In cultured COCs, FSH/forskolin induced Areg mRNA within 0.5 h that peaked at 4 h, a process blocked by inhibitors of p38MAPK (SB203580), MAPK kinase (MEK) 1 (PD98059), and PTGS2 (NS398) but not protein kinase A (PKA) (KT5720). Conversely, AREG but not FSH induced Ptsg2 mRNA at 0.5 h with peak expression of Ptgs2 and Areg mRNAs at 4 h, processes blocked by the EGF receptor tyrosine kinase inhibitor AG1478 (AG), PD98059, and NS398. PGE2 reversed the inhibitory effects of AG on AREG-induced expression of Areg but not Ptgs2, placing Ptgs2 downstream of EGF-R signaling. Phorbol 12-myristate 13-acetate (PMA) and adenovirally expressed PGRA synergistically induced Areg mRNA in granulosa cells. In COCs, AREG not only induced genes that impact matrix formation but also genes involved in steroidogenesis (StAR, Cyp11a1) and immune cell-like functions (Pdcd1, Runx1, Cd52). Collectively, FSH-mediated induction of Areg mRNA via p38MAPK precedes AREG induction of Ptgs2 mRNA via ERK1/2. PGs acting via PTGER2 in cumulus cells provide a secondary, autocrine pathway to regulate expression of Areg in COCs showing critical functional links between G protein-coupled receptor and growth factor receptor pathways in ovulating follicles.

Increased soluble EGF after ischemia is accompanied by a decrease in membrane-associated precursors

1993 ◽  
Vol 264 (3) ◽  
pp. F523-F531
Author(s):  
R. P. Schaudies ◽  
J. P. Johnson
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
High Performance ◽  
Egf Receptor ◽  
Thymidine Incorporation ◽  
Biologically Active ◽  
The Third ◽  
Epidermal Growth ◽  
Triton X ◽  
Injured Kidney

We have characterized the distribution of immunoreactive epidermal growth factor (irEGF) in control and ischemia-injured rat kidneys. Kidneys that had undergone ischemic injury contained levels of soluble irEGF that were six times those of uninjured kidneys. The predominant forms of soluble irEGF were native and des-Arg-epidermal growth factor (EGF), both of which are biologically active. Crude membrane fractions from whole kidneys were solubilized in Triton X-100 and tested for irEGF. Amounts of irEGF were slightly decreased in the ischemia-injured kidney membranes. However, when solubilized membrane fractions were digested with trypsin, which generates a single immunoreactive species which appears identical to native EGF, the amount of irEGF in control fractions increased 13-fold and the amount in injured fractions increased only 4-fold as measured by radioimmunoassay. To better characterize the membrane-associated irEGF, Triton X-100-solubilized membrane fractions from control animals were affinity purified and subjected to high-performance liquid molecular sieve chromatography. Three major peaks of material exhibited immunoreactivity to EGF antibodies, bound the EGF receptor, and stimulated [3H]thymidine incorporation in growth-arrested fibroblasts. Trypsin digestion of the two high-molecular-mass peaks enhanced these activities. The third peak eluted with native EGF and showed no change in activity with trypsin addition. We propose that EGF is released from membrane-associated EGF precursors and can then act in an autocrine or paracrine fashion to promote cell growth after ischemia-induced acute renal failure.

scholarly journals Role of FSH and epidermal growth factor (EGF) in the initiation of steroidogenesis in granulosa cells associated with follicular selection in chicken ovaries

Reproduction ◽  
2003 ◽  
pp. 683-691 ◽  
Author(s):  
AG Hernandez ◽  
JM Bahr
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Granulosa Cells ◽  
Egf Receptor ◽  
Mrna Abundance ◽  
Dependent Manner ◽  
Preovulatory Follicle ◽  
Progesterone Secretion ◽  
Chain Cleavage ◽  
Epidermal Growth

In chicken ovaries, one small yellow follicle (SYF) is selected daily from a pool of follicles of similar size and becomes a preovulatory follicle. FSH induces follicular growth and steroidogenesis. Epidermal growth factor (EGF), an intraovarian hormone, suppresses granulosa cell differentiation. This study demonstrates that recruitment of SYFs into the hierarchy of preovulatory follicles is associated with a change in steroidogenic activity in granulosa cells regulated, at least in part, by FSH and EGF. Abundance of P450 side-chain cleavage (P450scc) mRNA was higher in the smallest preovulatory follicle (F6) compared with SYF, whereas FSH and EGF receptor (FSHr and EGFr, respectively) mRNA abundance was similar. FSH increased P450scc mRNA abundance and progesterone secretion and decreased FSHr mRNA in cultured granulosa cells, whereas EGF attenuated or suppressed P450scc mRNA and decreased FSHr mRNA abundance. None of the hormones influenced EGFr mRNA abundance. When used in combination, EGF attenuated or suppressed the stimulatory effect of FSH on the expression of P450scc mRNA and production of progesterone in a dose-dependent manner. The results indicate that (1) selection is associated with an increase in P450scc mRNA; (2) FSH stimulates expression of P450scc mRNA and progesterone secretion in granulosa cells of SYF; and (3) induction of P450scc mRNA and progesterone secretion by FSH is attenuated or blocked by EGF.

scholarly journals Cross talk between epidermal growth factor (EGF) receptor and extra nuclear steroid receptors in cell lines

2010 ◽  
Vol 327 (1-2) ◽  
pp. 19-24 ◽  
Author(s):  
Antimo Migliaccio ◽  
Gabriella Castoria ◽  
Pia Giovannelli ◽  
Ferdinando Auricchio
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Cell Lines ◽  
Cross Talk ◽  
Egf Receptor ◽  
Steroid Receptors ◽  
Epidermal Growth

A novel action of epidermal growth factor in rat granulosa cells: its potentiation of gonadotrophin action

1995 ◽  
Vol 15 (3) ◽  
pp. 283-291 ◽  
Author(s):  
M-A Hattori ◽  
E Yoshino ◽  
Y Shinohara ◽  
R Horiuchi ◽  
I Kojima
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Granulosa Cells ◽  
Egf Receptor ◽  
Egf Receptors ◽  
Down Regulation ◽  
Control Value ◽  
Progesterone Synthesis ◽  
Affinity Labelling ◽  
Epidermal Growth

ABSTRACT It is well known that epidermal growth factor (EGF) induces down-regulation of LH receptors and desensitization to gonadotrophin stimulation in gonadal cells, including granulosa cells. In a previous study we showed that EGF receptor levels in rat granulosa cells were increased up to fourfold after 96 h of culture with human GH in the presence of FSH, and the present study has evaluated the action of EGF on these cells. The induced EGF receptors were identical in size to the pre-existing receptors as assessed by affinity labelling with 125I-EGF. After 48 h in culture, various amounts of EGF (0·5–10 ng) were added and the cells were cultured for a further 48 h. The addition of EGF caused down-regulation of LH receptors in cells expressing high levels of EGF receptors. However, this down-regulation was less than that in control cells. After the cells were washed, cAMP synthesis in response to human chorionic gonadotrophin (hCG) increased by two to three times the control value and this increase was closely correlated with an increase in EGF receptor content. However, stimulation with cholera toxin or forskolin showed no such augmentation, indicating that it may not be due to quantitative alterations in G proteins and their effector systems. Induction of EGF potentiation required long-term exposure to EGF, for at least more than 24 h. In addition, progesterone synthesis was sensitive to stimulation with lower doses of hCG. These findings indicate that the activation of hGH-induced EGF receptors may potentiate gonadotrophin action in granulosa cells.

Nitric oxide: a modulator for the epidermal growth factor receptor expression in developing ovarian granulosa cells

1996 ◽  
Vol 270 (3) ◽  
pp. C812-C818 ◽  
Author(s):  
M. Hattori ◽  
K. Sakamoto ◽  
N. Fujihara ◽  
I. Kojima
Keyword(s):  
Nitric Oxide ◽  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Granulosa Cells ◽  
Binding Sites ◽  
Egf Receptor ◽  
No Synthase ◽  
Receptor Expression ◽  
Cyclic Monophosphate ◽  
Epidermal Growth

the present study was designed to assess the involvement of nitric oxide (NO) in the regulation of the epidermal growth factor (EGF) receptor during development of rat granulosa cells, which were prepared by puncturing ovaries of diethylstilbestrol-treated rats. The immature cells were cultured for 48 h with follicle-stimulating hormone (FSH) to be transformed into mature cells. A marked accumulation of guanosine 3',5' -cyclic monophosphate (cGMP) was observed during development. The accumulation of cGMP, but not of adenosine 3',5'-cyclic monophosphate (cAMP), was suppressed by specific inhibitors of NO synthase, L-NG-monomethyl-L-arginine (L-NMMA) and L-N(G)-nitro-L-arginine, and a selective inhibitor of the inducible NO synthase, aminoguanidine. The L-NMMA-induced suppression was partially reversed by addition of L-arginine to cultures but not D-arginine, indicating that NO formation is inhibited by competing with analogues of L-arginine for NO synthase. Treatment with 2-(4-carboxyphenyl)-4,4,5,5,-tetramethylimidazoline-1-oxyl-3-oxide , an antagonist of NO, caused suppression in the increase of EGF binding sites, whereas exposure of the cells to sodium nitroprusside (SNP), an NO donor, caused elevation in EGF binding sites, with increasing extra- and intracellular cGMP levels. Analysis of the EGF receptor by affinity labeling with 125I-labeled EGF revealed that the intensity of the cross-linked receptor molecular with a molecular mass of 180 kDa was increased by exposure to SNP. The facilitatory effect of SNP on the EGF receptor was observed when the cAMP-dependent pathway was fully activated by FSH. However, the NO effect may be mediated by a cGMP-independent pathway, as 8-bromo-cGMP did not mimic the action of SNP. These results indicate that the L-arginine-NO system may contribute to the regulation of EGF receptor expression in developing granulosa cells stimulated by FSH.

Human growth hormone augmentation of epidermal growth factor binding sites on rat granulosa cells

1994 ◽  
Vol 142 (1) ◽  
pp. 69-75 ◽  
Author(s):  
M-A Hattori ◽  
Y Shinohara ◽  
E Yoshino ◽  
M Kanzaki ◽  
I Kojima ◽  
...  
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Granulosa Cell ◽  
Granulosa Cells ◽  
Binding Sites ◽  
Egf Receptor ◽  
Scatchard Analysis ◽  
Maximal Effect ◽  
Intact Cells ◽  
Epidermal Growth

Abstract The effect of human GH (hGH) on the regulation of epidermal growth factor (EGF) receptor was investigated during differentiation of FSH-treated rat granulosa cells, which has been reported to be mediated by a cAMP-dependent mechanism. By measuring the binding of [125I]iodo-EGF to the intact cells, FSH was shown to cause increases in the number of EGF binding sites after culture for 72 h. When granulosa cells were cultured with hGH, the number of FSH-induced EGF binding sites was augmented, with a half-maximal effect at about 10 μg hGH/l and a maximal stimulatory concentration of 100 μg/l. The stimulatory effect of hGH was absolutely dependent on insulin which by itself showed stimulatory effects on EGF binding sites. Scatchard analysis of EGF binding sites indicated that treatment with hGH increased the number of EGF binding sites (17 200 sites/cell after treatment with FSH; 31 700 sites/cell after FSH plus hGH), but did not alter the binding affinity. The augmentation was observed after culturing for 48 h and increased progressively with time, reaching 280% of the level after FSH treatment by 120 h. Although progesterone synthesis was increased by hGH, the markers of cell differentiation such as cAMP synthesis and LH binding sites were suppressed, indicating hGH inhibition of the cAMP-mediated signal. The action of hGH on the EGF binding sites was not accompanied by cell proliferation. These findings indicate that hGH has a novel action on the regulation of rat granulosa cell EGF binding sites and that the granulosa cell may possess both cAMP-dependent and -independent mechanisms for expression of EGF binding sites. Journal of Endocrinology (1994) 142, 69–75

scholarly journals Bone morphogenetic protein 15 and growth differentiation factor 9 co-operate to regulate granulosa cell function

Reproduction ◽  
2005 ◽  
Vol 129 (4) ◽  
pp. 473-480 ◽  
Author(s):  
Kenneth P McNatty ◽  
Jennifer L Juengel ◽  
Karen L Reader ◽  
Stan Lun ◽  
Samu Myllymaa ◽  
...  
Keyword(s):  
Growth Factors ◽  
Growth Factor ◽  
Bone Morphogenetic Protein ◽  
Granulosa Cells ◽  
Thymidine Incorporation ◽  
Progesterone Production ◽  
Differentiation Factor ◽  
Morphogenetic Protein ◽  
Growth Differentiation Factor 9 ◽  
Bone Morphogenetic Protein 15

The oocyte-secreted polypeptide growth factors, growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15, also known as GDF9B) have both been shown to be essential for ovarian follicular growth and function. The effects of murine (m) and ovine (o) GDF9 as well as oBMP15, alone or together, on 3H-thymidine uptake and progesterone and inhibin production by granulosa cells from rats were determined. Murine GDF9 stimulated thymidine incorporation by granulosa cells whereas oGDF9 and oBMP15 alone had no effect. However, oBMP15 given together with mGDF9 or oGDF9 was very potent in stimulating 3H-thymidine incorporation by granulosa cells with a greater than 3-fold stimulation compared with any growth factor alone. The synergistic effect of oBMP15 and oGDF9 was almost completely blocked by antibodies generated against these growth factors when administered either alone or in combination. While neither GDF9 (murine or ovine) nor oBMP15 were able to modulate FSH-stimulated progesterone production on their own, FSH-stimulated progesterone production by granulosa cells was potently inhibited when BMP15 and GDF9 were administered together. Immunoreactive α-inhibin levels increased more than 15-fold from granulosa cells when BMP15 and GDF9 were given together whereas consistent stimulatory effects of either growth factor alone were not observed. The effects of GDF9 and BMP15, when added together, were different than those observed for the growth factors alone. Therefore, we hypothesize that within the ovary, these oocyte-secreted growth factors co-operate to regulate proliferation and gonadotropin-induced differentiation of granulosa cells in mammals.

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