Oocyte maturation, fertilization, and early embryonic development occur in the absence of gene transcription. Therefore, it is critical to understand at a global level the post-transcriptional events that are driving these transitions. Here we used a systems approach by combining polysome mRNA profiling and bioinformatics to identify RNA-binding motifs in mRNAs that either enter or exit the polysome pool during mouse oocyte maturation. Association of mRNA with the polysomes correlates with active translation. Using this strategy, we identified highly specific patterns of mRNA recruitment to the polysomes that are synchronized with the cell cycle. A large number of the mRNAs recovered with translating ribosomes contain motifs for the RNA-binding proteins DAZL (deleted in azoospermia-like) and CPEB (cytoplasmic polyadenylation element-binding protein). Although a Dazl role in early germ cell development is well established, no function has been described during oocyte-to-embryo transition. We demonstrate that CPEB1 regulates Dazl post-transcriptionally, and that DAZL is essential for meiotic maturation and embryonic cleavage. In the absence of DAZL synthesis, the meiotic spindle fails to form due to disorganization of meiotic microtubules. Therefore, Cpeb1 and Dazl function in a progressive, self-reinforcing pathway to promote oocyte maturation and early embryonic development.

In the preovulatory ovarian follicle, mammalian oocytes are maintained in prophase meiotic arrest until the luteinizing hormone (LH) surge induces reentry into the first meiotic division. Dramatic changes in the somatic cells surrounding the oocytes and in the follicular wall are also induced by LH and are necessary for ovulation. Here, we provide genetic evidence that LH-dependent transactivation of the epidermal growth factor receptor (EGFR) is indispensable for oocyte reentry into the meiotic cell cycle, for the synthesis of the extracellular matrix surrounding the oocyte that causes cumulus expansion, and for follicle rupture in vivo. Mice deficient in either amphiregulin or epiregulin, two EGFR ligands, display delayed or reduced oocyte maturation and cumulus expansion. In compound-mutant mice in which loss of one EGFR ligand is associated with decreased signaling from a hypomorphic allele of the EGFR, LH no longer signals oocyte meiotic resumption. Moreover, induction of genes involved in cumulus expansion and follicle rupture is compromised in these mice, resulting in impaired ovulation. Thus, these studies demonstrate that LH induction of epidermal growth factor-like growth factors and EGFR transactivation are essential for the regulation of a critical physiological process such as ovulation and provide new strategies for manipulation of fertility.The luteinizing hormone (LH) surge plays a central role in promoting a cascade of events in ovarian preovulatory follicles that are essential for the ovulation of a fertilizable oocyte. Acting through LH-chorionic gonadotropin (LH-CG) receptors (LHRs) (LHR is a member of the G protein-coupled receptor superfamily encoded by Lhcgr), LH induces reprogramming of the gene expression profiles of follicular somatic cells (theca and granulosa cells), changes in the secretory properties of the cumulus cells surrounding the oocyte and cumulus expansion, oocyte reentry into the meiotic cell cycle, and follicle rupture (7, 41). LHRs are highly expressed on the granulosa cells lining the antral cavity of preovulatory follicles (mural granulosa cells) and on the external theca cells that are in continuity with the surrounding stroma. However, within preovulatory follicles, oocytes and cumulus cells that are profoundly affected by the LH surge express few or no LHRs and fail to respond when directly exposed to LH in vitro (37).To explain how LH signals are propagated from the periphery toward the cumulus oocyte complex (COC), a model has been proposed whereby factors released by mural granulosa cells function in an autocrine and paracrine manner to transduce the LH effects within the follicle (34). Secretion of bioactive growth factors from the oocyte to affect somatic cells is well established (27,30); conversely, the paracrine signals originating from the somatic cells and affecting oocytes have long been sought but are largely unknown. Recently, we have proposed that intrafollicular release of members of the epidermal growth factor (EGF)-like family (34) may fulfill this ...

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β1- and β2-adrenergic receptors (βARs) are highly homologous, yet they play clearly distinct roles in cardiac physiology and pathology. Myocyte contraction, for instance, is readily stimulated by β1AR but not β2AR signaling, and chronic stimulation of the two receptors has opposing effects on myocyte apoptosis and cell survival. Differences in the assembly of macromolecular signaling complexes may explain the distinct biological outcomes. Here, we demonstrate that β1AR forms a signaling complex with a cAMP-specific phosphodiesterase (PDE) in a manner inherently different from a β2AR/β-arrestin/PDE complex reported previously. The β1AR binds a PDE variant, PDE4D8, in a direct manner, and occupancy of the receptor by an agonist causes dissociation of this complex. Conversely, agonist binding to the β2AR is a prerequisite for the recruitment of a complex consisting of β-arrestin and the PDE4D variant, PDE4D5, to the receptor. We propose that the distinct modes of interaction with PDEs result in divergent cAMP signals in the vicinity of the two receptors, thus, providing an additional layer of complexity to enforce the specificity of β1- and β2-adrenoceptor signaling.

In mammalian and amphibian oocytes, the meiotic arrest at the G2/M transition is dependent on cAMP regulation. Because genetic inactivation of a phosphodiesterase expressed in oocytes prevents reentry into the cell cycle, suggesting autonomous cAMP synthesis, we investigated the presence and properties of the G-protein-coupled receptors (GPCRs) in rodent oocytes. The pattern of expression was defined using three independent strategies, including microarray analysis of GV oocyte mRNAs, EST database scanning, and RT-PCR amplification with degenerated primers against transmembrane regions conserved in the GPCR superfamily. Clustering of the GPCR mRNAs from rat and mouse oocytes indicated the expression of the closely related Gpr3, Gpr12, and Edg3, which recognize sphingosine and its metabolites as ligands. Expression of these mRNAs was confirmed by RT-PCR with specific primers as well as by in situ hybridization. That these receptors are involved in the control of cAMP levels in oocytes was indicated by the finding that expression of the mRNA for Gpr3 and Gpr12 is downregulated in Pde3a-deficient oocytes, which have a chronic elevation of cAMP levels. Expression of GPR3 or GPR12 in Xenopus laevis oocytes prevented progesterone-induced meiotic maturation, whereas expression of FSHR had no effect. A block in spontaneous oocyte maturation was also induced when Gpr3 or Gpr12 mRNA was injected into mouse oocytes. Downregulation of GPR3 and GPR12 caused meiotic resumption in mouse and rat oocytes, respectively. However, ablation of the Gpr12 gene in the mouse did not cause a leaky meiotic arrest, suggesting compensation by Gpr3. Incubation of mouse oocytes with the GPR3/12 ligands SPC and S1P delayed spontaneous oocyte maturation. We propose that the cAMP levels required for maintaining meiotic arrest in mouse and rat oocytes are dependent on the expression of Gpr3 and/or Gpr12.

Abstract-Compartmentation of cAMP is thought to generate the specificity of G s -coupled receptor action in cardiac myocytes, with phosphodiesterases (PDEs) playing a major role in this process by preventing cAMP diffusion. We tested this hypothesis in adult rat ventricular myocytes by characterizing PDEs involved in the regulation of cAMP signals and L-type Ca 2ϩ current (I Ca,L ) on stimulation with ␤ 1 -adrenergic receptors (␤ 1 -ARs), ␤ 2 -ARs, glucagon receptors (Glu-Rs) and prostaglandin E 1 receptors (PGE 1 -Rs). All receptors but PGE 1 -R increased total cAMP, and inhibition of PDEs with 3-isobutyl-1-methylxanthine strongly potentiated these responses. When monitored in single cells by high-affinity cyclic nucleotide-gated (CNG) channels, stimulation of ␤ 1 -AR and Glu-R increased cAMP, whereas ␤ 2 -AR and PGE 1 -R had no detectable effect. Selective inhibition of PDE3 by cilostamide and PDE4 by Ro 20-1724 potentiated ␤ 1 -AR cAMP signals, whereas Glu-R cAMP was augmented only by PD4 inhibition. PGE 1 -R and ␤ 2 -AR generated substantial cAMP increases only when PDE3 and PDE4 were blocked. For all receptors except PGE 1 -R, the measurements of I Ca,L closely matched the ones obtained with CNG channels. Indeed, PDE3 and PDE4 controlled ␤ 1 -AR and ␤ 2 -AR regulation of I Ca,L , whereas only PDE4 controlled Glu-R regulation of I Ca,L thus demonstrating that receptor-PDE coupling has functional implications downstream of cAMP. PGE 1 had no effect on I Ca,L even after blockade of PDE3 or PDE4, suggesting that other mechanisms prevent cAMP produced by PGE 1 to diffuse to L-type Ca 2ϩ channels. These results identify specific functional coupling of individual PDE families to G s -coupled receptors as a major mechanism enabling cardiac cells to generate heterogeneous cAMP signals in response to different hormones. Key Words: cAMP Ⅲ heart Ⅲ G-protein-coupled receptor Ⅲ phosphodiesterase C ardiac myocytes express a number of G s -coupled receptors (G s PCRs) that raise intracellular cAMP levels and activate cAMP-dependent protein kinase (PKA) but exert different downstream effects. For instance, ␤ 1 -adrenergic receptor (␤ 1 -AR) stimulation produces a major and sustained increase in force of contraction, accelerates relaxation, and stimulates glycogen phosphorylase. 1 ␤ 2 -AR stimulation also increases contractile force but does not activate glycogen phosphorylase 2 and does not accelerate relaxation 1,3 (see also ref. 2); glucagon receptor (Glu-R) stimulation activates phosphorylase and exerts positive inotropic and lusitropic effects, but the contractile effects fade with time. 4 Finally, prostaglandin E 1 (PGE 1 ) has no effect on contractile activity or glycogen metabolism. 5,6 Such observations led to the proposal that activation of different G s PCRs results in the accumulation of cAMP and phosphorylation of hormone target proteins in distinct compartments. 7 The discovery of A-kinase anchoring proteins, responsible for the subcellular distribution of particulate PKA, 8 and the development of new method...

Since cAMP blocks meiotic maturation of mammalian and amphibian oocytes in vitro and cyclic nucleotide phosphodiesterase 3A (PDE3A) is primarily responsible for oocyte cAMP hydrolysis, we generated PDE3A-deficient mice by homologous recombination. The Pde3a -/-females were viable and ovulated a normal number of oocytes but were completely infertile, because ovulated oocytes were arrested at the germinal vesicle stage and, therefore, could not be fertilized. Pde3a -/-oocytes lacked cAMP-specific PDE activity, contained increased cAMP levels, and failed to undergo spontaneous maturation in vitro (up to 48 hours). Meiotic maturation in Pde3a -/-oocytes was restored by inhibiting protein kinase A (PKA) with adenosine-3′,5′-cyclic monophosphorothioate, Rp-isomer (Rp-cAMPS) or by injection of protein kinase inhibitor peptide (PKI) or mRNA coding for phosphatase CDC25, which confirms that increased cAMP-PKA signaling is responsible for the meiotic blockade. Pde3a -/-oocytes that underwent germinal vesicle breakdown showed activation of MPF and MAPK, completed the first meiotic division extruding a polar body, and became competent for fertilization by spermatozoa. We believe that these findings provide the first genetic evidence indicating that resumption of meiosis in vivo and in vitro requires PDE3A activity. Pde3a -/-mice represent an in vivo model where meiotic maturation and ovulation are dissociated, which underscores inhibition of oocyte maturation as a potential strategy for contraception.

Germ cells divide and differentiate in a unique local microenvironment under the control of somatic cells. Signals released in this niche instruct oocyte reentry into the meiotic cell cycle. Once initiated, the progression through meiosis and the associated program of maternal mRNA translation are thought to be cell-autonomous. Here we show that translation of a subset of maternal mRNAs critical for embryo development is under the control of somatic cell inputs. Translation of specific maternal transcripts increases in oocytes cultured in association with somatic cells and is sensitive to EGF-like growth factors that act only on the somatic compartment. In mice deficient in amphiregulin, decreased fecundity and oocyte developmental competence is associated with defective translation of a subset of maternal mRNAs. These somatic cell signals that affect translation require activation of the PI3K/AKT/mTOR pathway. Thus, mRNA translation depends on somatic cell cues that are essential to reprogram the oocyte for embryo development.