We have established transgenic mice expressing the Cre recombinase under the control of the anti-Müllerian hormone (AMH) gene promoter. Cre activity and specificity were evaluated by different means. In AMH-Cre mice, expression of the Cre recombinase mRNA was confined to the testis and ovary. AMH-Cre mice were crossed with reporter transgenic lines and the offspring exhibited Cre-mediated recombination only in the testis and the ovary. In male, histochemical analysis indicated that recombination occurred in every Sertoli cells. In female, Cre-mediated recombination was restricted to granulosa cells, but the protein was not evenly active in every cells. From these results, we conclude that potentially, this transgenic line possessing AMH promoter-driven expression of the Cre recombinase is a powerful tool to delete genes in Sertoli cells only, in order to study Sertoli cell gene function during mammalian spermatogenesis.

Classically, the FSH receptor (FSH-R) mediates its effects through coupling to guanine nucleotide-binding protein alpha S subunit (Galpha(s)) and activation of the cAMP/protein kinase A (PKA) signaling pathway. beta-Arrestins are rapidly recruited to the FSH-activated receptor and play key roles in its desensitization and internalization. Here, we show that the FSH-R expressed in HEK 293 cells activated ERK by two temporally distinct pathways dependent, respectively, on Galpha(s)/PKA and beta-arrestins. Galpha(s)/PKA-dependent ERK activation was rapid, transient, and blocked by H89 (a PKA inhibitor), but it was insensitive to small interfering RNA-mediated depletion of beta-arrestins. beta-Arrestin-dependent ERK activation was slower but more sustained and was insensitive to H89. We identified five Ser/Thr residues in the C terminus of the receptor (638-644) as a major phosphorylation site. Mutation of these residues into Ala (5A FSH-R) significantly reduced the stability of FSH-induced beta-arrestin 1 and 2 interaction when compared with the wild-type receptor. As expected, the 5A FSH-R-mediated cAMP accumulation was enhanced, and its internalization was reduced. In striking contrast, the ability of the 5A FSH-R to activate ERK via the beta-arrestin-dependent pathway was increased. G protein-coupled receptor kinase 5 (GRK5) and GRK6 were required for beta-arrestin-dependent ERK activation by both the wild-type and 5A FSH-R. By contrast, GRK2 depletion enhanced ERK activation by the wild-type FSH-R but not by the 5A FSH-R. In conclusion, we demonstrate the existence of a beta-arrestin-dependent, GRK-regulated mechanism for ERK activation by the FSH-R. A phosphorylation cluster in the C terminus of the FSH-R, identified as a site of beta-arrestin recruitment, positively regulated both desensitization and internalization but negatively regulated beta-arrestin-dependent ERK activation.

Primary cultures of Sertoli cells provide an interesting model to study how signalling pathways induced by a single hormone in a single cell type evolve, depending on the developmental stage. In vivo, follicle-stimulating hormone (FSH) induces proliferation of Sertoli cells in neonate and controls the subsequent di erentiation of the entire population. Molecular mechanisms underlying Sertoli cell pleiotropic responses to FSH have long been investigated. But to date, only cAMP-dependent kinase (PKA) activation has been reported to account for most FSH biological activities in male. Here, we demonstrate that FSH activates the ERK MAP kinase pathway following dual coupling of the FSH-R both to Gs and to Gi heterotrimeric proteins, in a PKA-and also Srcdependent manner. This activation is required for FSHinduced proliferation of Sertoli cells isolated 5 days after birth. Consistently, we show that the ERK-mediated FSH mitogenic e ect triggers upregulation of cyclin D1. In sharp contrast, at 19 days after birth, as cells proceed through their di erentiation program, the ERK pathway is dramatically inhibited by FSH treatment. Taken together, these results show that FSH can exert opposite e ects on the ERK signalling cascade during the maturation process of Sertoli cells. Thus, signalling modules triggered by the FSH-R evolve dynamically throughout development of FSH natural target cells. Oncogene (2001) 20, 4696 ± 4709.

The glycoprotein follicle-stimulating hormone (FSH) acts on gonadal target cells, hence regulating gametogenesis. The transduction of the hormone-induced signal is mediated by the FSH-specific G protein-coupled receptor (FSHR), of which the action relies on the interaction with a number of intracellular effectors. The stimulatory Gαs protein is a long-time known transducer of FSH signaling, mainly leading to intracellular cAMP increase and protein kinase A (PKA) activation, the latter acting as a master regulator of cell metabolism and sex steroid production. While in vivo data clearly demonstrate the relevance of PKA activation in mediating gametogenesis by triggering proliferative signals, some in vitro data suggest that pro-apoptotic pathways may be awakened as a “dark side” of cAMP/PKA-dependent steroidogenesis, in certain conditions. P38 mitogen-activated protein kinases (MAPK) are players of death signals in steroidogenic cells, involving downstream p53 and caspases. Although it could be hypothesized that pro-apoptotic signals, if relevant, may be required for regulating atresia of non -dominant ovarian follicles, they should be transient and counterbalanced by mitogenic signals upon FSHR interaction with opposing transducers, such as Gαi proteins and β-arrestins. These molecules modulate the steroidogenic pathway via extracellular-regulated kinases (ERK1/2), phosphatidylinositol-4,5-bisphosphate 3-kinases (PI3K)/protein kinase B (AKT), calcium signaling and other intracellular signaling effectors, resulting in a complex and dynamic signaling network characterizing sex- and stage-specific gamete maturation. Even if the FSH-mediated signaling network is not yet entirely deciphered, its full comprehension is of high physiological and clinical relevance due to the crucial role covered by the hormone in regulating human development and reproduction.

The IB␣ protein is a key molecular target involved in the control of NF-B/Rel transcription factors during viral infection or inflammatory reactions. This NF-B-inhibitory factor is regulated by posttranslational phosphorylation and ubiquitination of its amino-terminal signal response domain that targets IB␣ for rapid proteolysis by the 26S proteasome. In an attempt to identify regulators of the IB␣ inhibitory activity, we undertook a yeast two-hybrid genetic screen, using the amino-terminal end of IB␣ as bait, and identified 12 independent interacting clones. Sequence analysis identified some of these cDNA clones as Dlc-1, a sequence encoding a small, 9-kDa human homolog of the outer-arm dynein light-chain protein. In the two-hybrid assay, Dlc-1 also interacted with full-length IB␣ protein but not with N-terminal-deletion-containing versions of IB␣. IB␣ interacted in vitro with a glutathione S-transferase-Dlc-1 fusion protein, and RelA(p65) did not displace this association, demonstrating that p65 and Dlc-1 contact different protein motifs of IB␣. Importantly, in HeLa and 293 cells, endogenous and transfected IB␣ coimmunoprecipitated with Myc-tagged or endogenous Dlc-1. Indirect immunofluorescence analyzed by confocal microscopy indicated that Dlc-1 and IB␣ colocalized with both nuclear and cytoplasmic distribution. Furthermore, Dlc-1 and IB␣ were found to associate with the microtubule organizing center, a perinuclear region from which microtubules radiate. Likewise, IB␣ colocalized with ␣-tubulin filaments. Taken together, these results highlight an intriguing interaction between the IB␣ protein and the human homolog of a member of the dynein family of motor proteins and provide a potential link between cytoskeleton dynamics and gene regulation.The cytoskeleton can be considered a complex biological network that integrates signal transduction pathways and responds by altering the pattern of gene expression and by changing cell morphology. Reorganization of structural components within the cytoskeleton can achieve these changes by exposing or masking internal molecular binding sites (32). For example, changes in the arrangement of cytoskeletal filaments may expose sequestered RNA molecules to cytoplasmic enzymes to which they were otherwise resistant (50). Recently, the Rho family of G proteins has become a paradigm of the link between actin cytoskeleton modifications and gene expression in response to growth factors (for a review, see reference 18). For example, phosphatidylinositol 3-kinase-mediated activation of Rac proteins by platelet-derived growth factor or insulin receptors (52) leads to membrane ruffling on the one hand and stimulation of the p21-associated kinases that control JNK/SAPK (18) and the mitogen-activated protein kinases (MAPKs) (25) on the other hand, leading to gene expression.The microtubule cytoskeleton also is involved in modulation of gene expression. Microtubule-disrupting agents such as colchicine have been reported to induce interleukin-1 expression in monocytes (45) or AP1-regulat...

Follicle-stimulating hormone (FSH) is a central regulator of male and female reproductive function. Over the last decade, there has been a growing perception of the complexity associated with FSH-induced cellular signaling. It is now clear that the canonical Gs/cAMP/PKA pathway is not the sole mechanism that must be considered in FSH biological actions. In parallel, consistent with the emerging concept of biased agonism, several examples of ligand-mediated selective signaling pathway activation by gonadotropin receptors have been reported. In this context, it is important to gain an integrative view of the signaling pathways induced by FSH and how they interconnect to form a network. In this review, we propose a first attempt at building topological maps of various pathways known to be involved in the FSH-induced signaling network. We discuss the multiple facets of FSH-induced signaling and how they converge to the hormone integrated biological response. Despite of their incompleteness, these maps of the FSH-induced signaling network represent a first step toward gaining a system-level comprehension of this hormone’s actions, which may ultimately facilitate the discovery of novel regulatory processes and therapeutic strategies for infertility and non-steroidal contraception.

The A189 V inactivating mutation of the human FSH receptor (FSHR) leads to subfertility in men and primary ovarian failure in women. This mutation has previously been associated with intracellular retention of the FSHR and impaired cAMP production. Here, we show that the A189 V FSHR stably expressed in HEK293N cells provoked ERK MAP kinases phosphorylation through β-arrestins, independently of the canonical cAMP/PKA pathway. Interesting, both the A189 V and wild-type (Wt) FSHRs selectively activated cAMP-independent ERK phosphorylation when expressed at low plasma membrane densities. These data indicate that the selective intracellular signalling triggered by the A189 V FSHR resulted from reduced membrane expression rather than by switching receptor coupling. Hence, receptor density at the plasma membrane might control the balance between distinct signal transduction mechanisms. Furthermore, our results help to clarify why mutations of FSHβ are more deleterious to human fertility than the FSHR A189 V mutation which preserves parts of receptor signalling repertoire.

FSH is synthesized in the pituitary by gonadotrope cells. By binding to and interacting with its cognate receptor [FSH receptor (FSHR)] in the gonads, this gonadotropin plays a key role in the control of gonadal function and reproduction. Upon activation, the FSHR undergoes conformational changes leading to transduction of intracellular signals, including dissociation of G protein complexes into components and activation of several associated interacting partners, which concertedly regulate downstream effectors. The canonical Gs/cAMP/protein kinase A pathway, considered for a long time as the sole effector of FSHR-mediated signaling, is now viewed as one of several mechanisms employed by this receptor to transduce intracellular signals in response to the FSH stimulus. This complex network of signaling pathways allows for a fine-tuning regulation of the gonadotropic stimulus, where activation/inhibition of its multiple components vary depending on the cell context, cell developmental stage, and concentration of associated receptors and corresponding ligands. Activation of these multiple signaling modules eventually converge to the hormone-integrated biological response, including survival, proliferation and differentiation of target cells, synthesis and secretion of paracrine/autocrine regulators, and, at the molecular level, functional selectivity and differential gene expression. In this mini-review, we discuss the complexity of FSHR-mediated intracellular signals activated in response to ligand stimulation. A better understanding of the signaling pathways involved in FSH action might potentially influence the development of new therapeutic strategies for reproductive disorders.