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

Abstract

Using primary cultures of immature rat granulosa cells and adenoviral infections we expressed two mutants of the human lutropin receptor (hLHR) that do not activate the phosphoinositide cascade. One mutant (hLFF) has the extracellular domain of the hLHR and the transmembrane and intracellular domains of the hFSHR. The other (hLHR-L457D) has a leucine to aspartate mutation in residue 457 of transmembrane helix 3.When expressed in immature rat granulosa cells the hLHR stimulates cAMP and inositol phosphate accumulation, transactivates the epidermal growth factor receptor (EGFR), elicits a transient increase in Akt phosphorylation, and a sustained increase in ERK1/2 phosphorylation but aromatase expression is not enhanced. When expressed at comparable densities, hLFF and hLHR-L457D support cAMP accumulation and transient Akt phosphorylation but do not support inositol phosphate accumulation, EGFR transactivation or a sustained phosphorylation of ERK1/2. Cells expressing either of these two mutants respond to hCG with increased aromatase expression.We also show that addition of hCG to cells expressing the hLHR antagonizes the effects of hFSH on aromatase expression whereas addition of hCG to cells expressing the hLHR-L457D mutant does not.These results show that activation of the phosphoinositide cascade is upstream of EGFR transactivation and ERK1/2 phosphorylation and that this pathway is a negative regulator of aromatase expression in granulosa cells.

Lack of Functional GABA_B Receptors Alters <b><i>Kiss1</i></b>, <b><i>Gnrh1 </i></b>and <b><i>Gad1</i></b> mRNA Expression in the Medial Basal Hypothalamus at Postnatal Day 4

“…Whatever the origin of the LH increase in our PND4 mice, it appears to stimulate estrogen synthesis in the gonads, as E 2 content was significantly elevated in testes and ovaries from GABA B1 KO mice, with no alterations in T content. Although the natural stimulus for aromatase is FSH, it has been postulated that LH can induce aromatase synthesis and activity in various tissues, especially when LH receptors are present at low levels [66]. Alternatively, increased E 2 content in gonads of GABA B1 KO mice may be the consequence of the absence of local GABA B Rs, which are normally expressed in ovaries [67, 68] and testes [69], suggesting in this case that GABA B Rs may be negatively modulating aromatase synthesis and/or activity.…”

Transactivation of the Epidermal Growth Factor Receptor Is Involved in the Lutropin Receptor-Mediated Down-Regulation of Ovarian Aromatase Expression in Vivo

“…As summarized above, we reported its involvement in the regulation of Cyp19a1 expression as well as the release of epiregulin in primary cultures of rat granulosa cells (3)(4)(5), whereas others had previously described its role in cumulus expansion (12), oocyte meiotic resumption (12,13), and expression of some granulosa cell-specific genes (14,15). Recently, Richards and co-workers (16) generated a granulosa cell-specific deletion of ERK1/2 and showed that after LHR activation, the granulosa cells in these mice continue to proliferate and to express high levels of Cyp19a1 leading to an inappropriately high level of estradiol throughout the estrous cycle.…”

“…Our previous studies using primary cultures of rat granulosa cells lead us to hypothesize that the EGFR is an important mediator of the LHR-stimulated decrease in ovarian Cyp19a1 expression (2)(3)(4)(5). We proposed that low gonadotropin receptor densities support Cyp19a1 expression because they activate pathways such as cAMP and AKT phosphorylation that enhance Cyp19a1 expression (6 -11).…”

“…Low receptor densities also mediate a transient phosphorylation of ERK1/2 that is weakly inhibitory of Cyp19a1 expression but does not overcome the stimulatory effect of cAMP and phosphorylated AKT (pAKT) (3). Higher gonadotropin receptor densities do not support Cyp19a1 expression because, in addition to the stimulatory pathways (cAMP and pAKT), they also activate the accumulation of inositol phosphates and diacylglycerol, which in turn promote the release of epidermal growth factor (EGF)-like growth factors and the phosphorylation of the EGF receptor (EGFR), thus mediating a sustained phosphorylation of ERK1/2 (3)(4)(5). These pathways of ERK1/2 phosphorylation lead to a stronger and more sustained signal that effectively antagonizes the pathways that stimulate Cyp19a1 expression.…”

Novel signaling mechanisms in the ovary during oocyte maturation and ovulation

“…PIP3 recruits AKT to the membrane, promoting its phosphorylation. An elegant study by Andric and Ascoli using immature rat granulosa cells transfected with mutant LH receptors showed that LH-mediated AKT phosphorylation is independent of cAMP and inositol phopshate production (Andric and Ascoli 2008). PI3K may also be linked to activation of other effectors such as RAS (Ramjaun and Downward 2007).…”