The Differential Effects of the Gonadotropin Receptors on Aromatase Expression in Primary Cultures of Immature Rat Granulosa Cells Are Highly Dependent on the Density of Receptors Expressed and the Activation of the Inositol Phosphate Cascade

Abstract Signaling pathways mediating the divergent effects of FSH and LH on aromatase in immature rat granulosa cells were studied by infecting cells with increasing amounts of adenoviral vectors for the human LH receptor (hLHR) or FSH receptor (hFSHR). Increasing amounts of Ad-hLHR, used at a multiplicity of infection (MOI) of 20 or 200 viable viral particles/cell, increased human chorionic gonadotropin (hCG) binding and hCG-induced cAMP and Akt phosphorylation, but inositol phosphates only increased in response to hCG in cells infected with 200 MOI Ad-hLHR. In contrast, hCG increased aromatase expression in cells infected with 20, but not in cells infected with 200, MOI Ad-hLHR. Cells infected with 20 or 200 MOI Ad-hFSHR showed increased hFSH binding and hFSH-induced Akt phosphorylation, but the hFSH-induced cAMP response was unchanged relative to control cells. However, hFSH was able to stimulate the inositol phosphate cascade in the Ad-hFSHR-infected cells, and the hFSH induction of aromatase was abolished. We also found that activation of C kinase or expression of a constitutively active form of Gαq inhibited the induction of aromatase by hFSH or 8Br-cAMP. We conclude that the differential effects of FSH and LH on aromatase in immature granulosa cells are highly dependent on gonadotropin receptor density and on the signaling pathways activated. We propose that aromatase is induced by common signals generated by activation of the FSHR and LHR (possibly cAMP and Akt) and that the activation of the inositol phosphate cascade in cells expressing a high density of LHR or FSHR antagonizes this induction.

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Phospholipase Cβ3 Mediates LH-Induced Granulosa Cell Differentiation

Endocrinology ◽

10.1210/en.2010-1298 ◽

2011 ◽

Vol 152 (7) ◽

pp. 2857-2869 ◽

Cited By ~ 18

Author(s):

Francesc X. Donadeu ◽

Cristina L. Esteves ◽

Lynsey K. Doyle ◽

Catherine A. Walker ◽

Stephanie N. Schauer ◽

...

Keyword(s):

Cell Differentiation ◽

Granulosa Cells ◽

Inositol Phosphate ◽

Ex Vivo ◽

Receptor Expression ◽

Target Cells ◽

Down Regulation ◽

Protein Levels ◽

Prostaglandin Endoperoxide Synthase ◽

In Cells

Previous studies showed that under certain conditions LH can stimulate not only adenylate cyclase (AC) but also phospholipase Cβ (PLCβ) signaling in target cells; however, the physiological involvement of PLCβ in LH-induced ovarian follicular cell differentiation has not been determined. To address this, ex vivo expression analyses and specific PLCβ targeting were performed in primary bovine granulosa cells. Expression analyses in cells from small (2.0–5.9 mm), medium (6.0–9.9 mm), and ovulatory-size (10.0–13.9 mm) follicles revealed an increase in mRNA and protein levels of heterotrimeric G protein subunits-αs, -αq, -α11, and -αi2 in ovulatory-size follicles, simultaneous with a substantial increase in LH receptor expression. Among the four known PLCβ isoforms, PLCβ3 (PLCB3) was specifically up-regulated in cells from ovulatory-size follicles, in association with a predominantly cytoplasmic location of PLCB3 in these cells and a significant inositol phosphate response to LH stimulation. Furthermore, RNA interference-mediated PLCB3 down-regulation reduced the ability of LH to induce hallmark differentiation responses of granulosa cells, namely transcriptional up-regulation of prostaglandin-endoperoxide synthase 2 and down-regulation of both aromatase expression and estradiol production. Responses to the AC agonist, forskolin, however, were not affected. In addition, PLCB3 down-regulation did not alter cAMP responses to LH in granulosa cells, ruling out a primary involvement of AC in mediating the effects of PLCB3. In summary, we provide evidence of a physiological involvement of PLCβ signaling in ovulatory-size follicles and specifically identify PLCB3 as a mediator of LH-induced differentiation responses of granulosa cells.

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Activation of the Lutropin/Choriogonadotropin Receptor Inhibits Apoptosis of Immature Leydig Cells in Primary Culture

Endocrinology ◽

10.1210/en.2009-0207 ◽

2009 ◽

Vol 150 (8) ◽

pp. 3766-3773 ◽

Cited By ~ 46

Author(s):

Ping Tai ◽

Koji Shiraishi ◽

Mario Ascoli

Keyword(s):

Leydig Cells ◽

Thymidine Incorporation ◽

Primary Cultures ◽

Drug Induced ◽

Immature Rat ◽

Akt Phosphorylation ◽

Igf I ◽

Epidermal Growth ◽

Induced Apoptosis ◽

Stimulation Of

We used proliferating primary cultures of immature rat Leydig cells expressing the recombinant human LH/choriogonadotropin (CG) receptor (LHR) to test the hypothesis that activation of this receptor inhibits apoptosis. We also compared the effects of LH/CG with epidermal growth factor (EGF) and IGF-I because these have been previously shown to stimulate proliferation and/or inhibit apoptosis in Leydig cells. Human CG (hCG), EGF, and IGF-I stimulated the phosphorylation of ERK1/2 and Akt in primary cultures of immature rat Leydig cells. These three hormones also robustly stimulated thymidine incorporation and inhibited drug-induced apoptosis. Using selective inhibitors of ERK1/2 (UO126) or Akt phosphorylation (LY294002), we show that the ERK1/2 and Akt cascades are both involved in the hCG- and EGF-dependent proliferation of Leydig cells, but only the ERK1/2 cascade is involved in their antiapoptotic actions. The same strategy showed that the proliferative and antiapoptotic actions of IGF-I are mediated entirely by the Akt pathway. These results show that activation of the LHR inhibits apoptosis in Leydig cells and that it does so through stimulation of the ERK1/2 pathway.

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GnRH-induced calcium mobilisation and inositol phosphate production in immature and mature rat ovarian granulosa cells

Journal of Endocrinology ◽

10.1677/joe.0.1490449 ◽

1996 ◽

Vol 149 (3) ◽

pp. 449-456 ◽

Cited By ~ 3

Author(s):

L Anderson ◽

S G Hillier ◽

K A Eidne ◽

F Miro

Keyword(s):

Signal Transduction ◽

Granulosa Cells ◽

Inositol Phosphate ◽

Signal Transduction Pathway ◽

Cyclopiazonic Acid ◽

Calcium Transient ◽

Gnrh Receptor ◽

Immature Rat ◽

Ovarian Granulosa Cells ◽

Stimulation Of

Abstract In rat ovarian granulosa cells the effects of GnRH are determined by the state of granulosa cell development with mainly inhibitory actions in immature cells and stimulatory actions in differentiated mature cells. These developmentally related effects of GnRH may arise from changes in either one or more of the signal transduction pathways activated by GnRH. The present study therefore measured downstream signalling events associated with the activation of the phospholipase C (PLC) signal transduction pathway in both mature and immature rat ovarian granulosa cells. Results showed that GnRH produced similar total inositol phosphate and intracellular calcium ([Ca2+ ]i) responses in both immature and mature granulosa cells. In contrast to the biphasic GnRH-induced [Ca2+]i response in pituitary gonadotropes, stimulation of the endogenously expressed GnRH receptor in both immature and mature granulosa cells produced a prompt monophasic rise in [Ca2+]i. This calcium transient was abolished by pretreating either cell type with a potent GnRH receptor antagonist or the PLC inhibitor U73122, demonstrating a GnRH receptor-specific activation of PLC. Similarly, pretreatment of cells with the [Ca2+]i antagonists thapsigargin or cyclopiazonic acid abolished the GnRH-induced calcium transient, whereas EGTA and nifedipine, a voltage-operated calcium channel (VOCC) antagonist, had no effect. These results suggest that in either immature or mature granulosa cells GnRH mobilises calcium from thapsigargin/cyclopiazonic acid-sensitive [Ca2+]i stores but does not involve the influx of extracellular calcium through VOCCs. We conclude that GnRH-induced stimulation of the PLC signal transduction pathway is independent of the stage of granulosa cell maturity and that alternative mechanisms account for the opposite effects of GnRH on gonadotrophin-induced steroidogenesis in mature and immature rat granulosa cells in vitro. Journal of Endocrinology (1996) 149, 449–456

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Lutropin/Choriogonadotropin Stimulate the Proliferation of Primary Cultures of Rat Leydig Cells through a Pathway that Involves Activation of the Extracellularly Regulated Kinase 1/2 Cascade

Endocrinology ◽

10.1210/en.2007-0160 ◽

2007 ◽

Vol 148 (7) ◽

pp. 3214-3225 ◽

Cited By ~ 62

Author(s):

Koji Shiraishi ◽

Mario Ascoli

Keyword(s):

Leydig Cells ◽

Proliferative Response ◽

Recombinant Adenovirus ◽

Inositol Phosphate ◽

Primary Cultures ◽

Steroid Biosynthesis ◽

Immature Rat ◽

Phosphate Accumulation ◽

Inositol Phosphate Accumulation ◽

Immature Cells

Primary cultures of progenitor and immature rat Leydig cells were established from the testes of 21- and 35-d-old rats, respectively. The cell population remained homogeneous after 4–6 d in culture as judged by staining for 3β-hydroxysteroid dehydrogenase, but the cells were unable to bind 125I-human chorionic gonadotropin (hCG) or to respond to hCG with classical LH receptor (LHR)-mediated responses, including cAMP and inositol phosphate accumulation, steroid biosynthesis, or the phosphorylation of ERK1/2. Infection of primary cultures with recombinant adenovirus coding for β-galactosidase showed that approximately 65% of the cells are infected. Infection with adenovirus coding for the human LHR (hLHR) allowed for expression of the hLHR at a density of approximately 25,000 receptors per cell and allowed the cells to respond to hCG with increases in cAMP and inositol phosphate accumulation, steroid biosynthesis, and the phosphorylation of ERK1/2. Although progenitor and immature cells were able to respond to hCG with an increase in progesterone, only the immature cells responded with an increase in testosterone. In addition to these classical LHR-mediated responses, the primary cultures of progenitor or immature rat Leydig cells expressing the recombinant hLHR proliferated robustly when incubated with hCG, and this proliferative response was sensitive to an inhibitor of ERK1/2 phosphorylation. These studies establish a novel experimental paradigm that can be used to study the proliferative response of Leydig cells to LH/CG. We conclude that activation of the LHR-provoked Leydig cell proliferation requires activation of the ERK1/2 cascade.

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Mutations of the lutropin/choriogonadotropin receptor that do not activate the phosphoinositide cascade allow hCG to induce aromatase expression in immature rat granulosa cells

Molecular and Cellular Endocrinology ◽

10.1016/j.mce.2008.01.018 ◽

2008 ◽

Vol 285 (1-2) ◽

pp. 62-72 ◽

Cited By ~ 13

Author(s):

Nebojsa Andric ◽

Mario Ascoli

Keyword(s):

Granulosa Cells ◽

Aromatase Expression ◽

Immature Rat

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Cell-matrix interaction in bone: type I collagen modulates signal transduction in osteoblast-like cells

AJP Cell Physiology ◽

10.1152/ajpcell.1995.268.5.c1090 ◽

1995 ◽

Vol 268 (5) ◽

pp. C1090-C1103 ◽

Cited By ~ 47

Author(s):

J. Green ◽

S. Schotland ◽

D. J. Stauber ◽

C. R. Kleeman ◽

T. L. Clemens

Keyword(s):

Signal Transduction ◽

Signaling Pathways ◽

Type I Collagen ◽

Primary Cultures ◽

Neonatal Rat ◽

Type I ◽

Osteosarcoma Cell ◽

Umr 106 ◽

In Cells

Cell interaction with extracellular matrix (ECM) modulates cell growth and differentiation. By using in vitro culture systems, we tested the effect of type I collagen (Coll-I) on signal transduction mechanisms in the osteosarcoma cell line UMR-106 and in primary cultures from neonatal rat calvariae. Cells were cultured for 72 h on Coll-I gel matrix and compared with control cells plated on plastic surfaces. Agonist-dependent and voltage-dependent rises in cytosolic Ca2+ concentration ([Ca2+]i; measured by fura 2 fluorometry) were significantly blunted in cells cultured on Coll-I compared with cells grown on plastic. In UMR-106 cells, the collagen matrix effect was mimicked by 24-h incubation with soluble Coll-I or short peptides containing the arginine-glycine-aspartate motif. Accumulation of cellular adenosine 3',5'-cyclic monophosphate (cAMP) stimulated by parathyroid hormone, cholera toxin, and forskolin was augmented (50-150%) in cells plated on Coll-I vs. control. The collagen effect on both [Ca2+]i- and adenylate cyclase-signaling pathways in UMR-106 cells was abrogated in the presence of protein kinase C (PKC) depletion or inhibition. Also, Coll-I induced a twofold increase in membrane-bound PKC without changing cytosolic PKC activity. Thus, by altering PKC activity, Coll-I modulates the [Ca2+]i- and cAMP-signaling pathways in osteoblasts. This, in turn, may influence bone remodeling processes.

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