Multiple intersecting pathways are involved in CPEB1 phosphorylation and regulation of translation during mouse oocyte meiosis.

The RNA-binding protein cytoplasmic polyadenylation element binding 1 (CPEB1) plays a fundamental role in regulating mRNA translation in oocytes. However, the specifics of how and which protein kinase cascades modulate CPEB1 activity are still controversial. Using genetic and pharmacological tools, and detailed time courses, we have re-evaluated the relationship between CPEB1 phosphorylation and translation activation during mouse oocyte maturation. We show that both the CDK1/MAPK and AURKA/PLK1 pathways converge on CPEB1 phosphorylation during prometaphase of meiosis I. Only inactivation of the CDK1/MAPK pathway disrupts translation, whereas inactivation of either pathway alone leads to CPEB1 stabilization. However, CPEB1 stabilization induced by inactivation of the AURKA/PLK1 pathway does not affect translation, indicating that destabilization and/or degradation is not linked to translational activation. The accumulation of endogenous CCNB1 protein closely recapitulates the translation data that use an exogenous template. These findings support the overarching hypothesis that the activation of translation during prometaphase in mouse oocytes relies on a CDK1/MAPK-dependent CPEB1 phosphorylation, and that translational activation precedes CPEB1 destabilization.

A genome-wide perspective of the maternal mRNA translation program during oocyte development.

Transcriptional and post-transcriptional regulations control gene expression in most cells. However, critical transitions during the development of the female gamete relies exclusively on regulation of mRNA translation in the absence of de novo mRNA synthesis. Specific temporal patterns of maternal mRNA translation are essential for the oocyte progression through meiosis, for generation of a haploid gamete ready for fertilization and for embryo development. In this review, we will discuss how mRNAs are translated during oocyte growth and maturation using mostly a genome-wide perspective. This broad view on how translation is regulated reveals multiple divergent translational control mechanisms required to coordinate protein synthesis with progression through the meiotic cell cycle and with development of a totipotent zygote.

Alternative cleavage and polyadenylation of the Ccnb1 mRNA defines accumulation of cyclin protein during the meiotic cell cycle.

Progression through the mitotic and meiotic cell cycle is driven by fluctuations in the levels of cyclins, the regulatory subunits controlling the localization and activity of CDK1 kinases. Cyclin levels are regulated through a precise balance of synthesis and degradation. Here we demonstrate that the synthesis of Cyclin B1 during the oocyte meiotic cell cycle is defined by the selective translation of mRNA variants generated through alternative cleavage and polyadenylation (APA). Using gene editing in mice, we introduced mutations into the proximal and distal polyadenylation elements of the 3' untranslated region (UTR) of the Ccnb1 mRNA. Through in vivo loss-of-function experiments, we demonstrate that the translation of mRNA with a short 3' UTR specifies Cyclin B1 protein levels that set the timing of meiotic re-entry. In contrast, translation directed by a long 3' UTR is necessary to direct Cyclin B1 protein accumulation during the MI/MII transition. These findings establish that the progression through the cell cycle is dependent on the selective translation of multiple mRNA variants generated by APA.

The H3.3 chaperone Hira complex orchestrates oocyte developmental competence.

Successful reproduction requires an oocyte competent to sustain early embryo development. By the end of oogenesis, the oocyte has entered a transcriptionally silenced state, the mechanisms and significance of which remain poorly understood. Histone H3.3, a histone H3 variant, has unique cell cycle-independent functions in chromatin structure and gene expression. Here, we have characterised the H3.3 chaperone Hira/Cabin1/Ubn1 complex, showing that loss of function of any of these subunits causes early embryogenesis failure in mouse. Transcriptome and nascent RNA analyses revealed that transcription is aberrantly silenced in mutant oocytes. Histone marks, including H3K4me3 and H3K9me3, are reduced and chromatin accessibility is impaired in Hira/Cabin1 mutants. Misregulated genes in mutant oocytes include Zscan4d, a two-cell specific gene involved in zygote genome activation. Overexpression of Zscan4 in the oocyte partially recapitulates the phenotypes of Hira mutants and Zscan4 knockdown in Cabin1 mutant oocytes partially restored their developmental potential, illustrating that temporal and spatial expression of Zscan4 is fine-tuned at the oocyte-to-embryo transition. Thus, the H3.3 chaperone Hira complex has a maternal effect function in oocyte developmental competence and embryogenesis, through modulating chromatin condensation and transcriptional quiescence.

Maternal mRNAs with distinct 3' UTRs define the temporal pattern of Ccnb1 synthesis during mouse oocyte meiotic maturation.

The final stages of female gamete maturation occur in the virtual absence of transcription, with gene expression driven by a program of selective unmasking, translation, and degradation of maternal mRNAs. Here we demonstrate that the timing of Ccnb1 mRNA translation in mouse oocytes is dependent on the presence of transcripts with different 3' untranslated regions (UTRs). This 3' UTR heterogeneity directs distinct temporal patterns of translational activation or repression. Inclusion or exclusion of cis-acting elements is responsible for these divergent regulations. Our findings reveal an additional layer of translation control through alternative polyadenylation usage required to fine-tune the timing of meiosis progression.

Atypical ductal hyperplasia on vacuum-assisted breast biopsy: a scoring system to predict the risk of upgrade to malignancy.

RATIONALE AND OBJECTIVES: Our multicentric study analysed clinical, radiologic and pathologic features in patients with atypical ductal hyperplasia (ADH) diagnosed with vacuum-assisted biopsy (VAB), to identify factors associated with the risk of upgrade, to develop a scoring system to support decision making. MATERIALS AND METHODS: Patients with ADH on VAB under stereotactic/tomosynthesis guidance (2012-2022) were eligible. Inclusion criteria were availability of surgical histopathological examination of the entire lesion or radiologic follow-up (FUP) ≥ 24 months. VAB results were compared with surgical pathological results or with imaging FUP evolution to assess upgrade. A backward stepwise linear regression was used to identify predictors of upgrade. The discriminatory power of the model was calculated through the area under the receiver operating curve (ROC-AUC); the Hosmer-Lemeshow test was used to assess model calibration. The points system was developed based on the selected risk factors, and the probability of upgrade associated with each point total was determined. RESULTS: 112 ADH lesions were included: 91 (91/112, 81.3%) underwent surgical excision with 20 diagnosis of malignancy, while 21 (21/112, 18.7%) underwent imaging FUP with one interval change (mean FUP time 48 months). Overall upgrade rate was 18.7% (21/112). Age, menopausal status, concurrent breast cancer, BIRADS classification and number of foci of ADH were identified as risk factors for upgrade. Our model showed an AUC = 0.85 (95% CI 0.76-0.94). The points system showed that the risk of upgrade is < 2% when the total score is ≤ 1. CONCLUSION: Our scoring system seemed a promising easy-to-use decision support tool for management of ADH, decreasing unnecessary surgeries, reducing patients' overtreatment and healthcare costs.

Compensating for Sensing Failures via Delegation in Human-AI Hybrid Systems.

Given the increasing prevalence of intelligent systems capable of autonomous actions or augmenting human activities, it is important to consider scenarios in which the human, autonomous system, or both can exhibit failures as a result of one of several contributing factors (e.g., perception). Failures for either humans or autonomous agents can lead to simply a reduced performance level, or a failure can lead to something as severe as injury or death. For our topic, we consider the hybrid human-AI teaming case where a managing agent is tasked with identifying when to perform a delegated assignment and whether the human or autonomous system should gain control. In this context, the manager will estimate its best action based on the likelihood of either (human, autonomous) agent's failure as a result of their sensing capabilities and possible deficiencies. We model how the environmental context can contribute to, or exacerbate, these sensing deficiencies. These contexts provide cases where the manager must learn to identify agents with capabilities that are suitable for decision-making. As such, we demonstrate how a reinforcement learning manager can correct the context-delegation association and assist the hybrid team of agents in outperforming the behavior of any agent working in isolation.

Does arthroscopic superior capsule reconstruction using porcine dermal xenograft represent a viable option in case of massive irreparable posterosuperior rotator cuff tear?

INTRODUCTION: The aim of the present study was to evaluate clinical and structural outcomes of patients with a massive irreparable rotator cuff tear treated with arthroscopic superior capsule reconstruction using an acellular porcine dermal xenograft. We hypothesized that this procedure would lead to improvement in clinical and functional results and that structural failure would not influence the final clinical results. MATERIALS AND METHODS: A retrospective analysis on arthroscopic superior capsule reconstruction performed from October 2016 to January 2019 was conducted. The procedure was performed in patients with a massive irreparable posterosuperior rotator cuff tear without a severe glenohumeral arthropathy (Hamada I and II) and complaining a painful pseudoparalysis. Clinical evaluation and MRI study were performed before surgery and after at least 14 months. RESULTS: A comprehensive group of 21 patients with 11 females and 10 males and a mean age of 57 ± 8.5 years underwent arthroscopic superior capsule reconstruction. The graft had a thickness of 1.5 mm in the first 9 cases (43%) since it was used in a single layer. The graft was thereafter doubled for technique evolution in the following 12 cases (57%) achieving a graft thickness of 3 mm. Active ROM significantly improved with a mean increase of active forward flexion from 72.8° ± 7.5° to 120.6° ± 4.5°, active abduction from 68.3° ± 10.2° to 140.2° ± 8.8° and external rotation from 38.2° ± 11.2° to 56.7° ± 6.8° at the last follow-up. The mean Constant score significantly improved from 40.4 ± 6.7 to 73.3 ± 8.2. A graft tear revealed in 52% (11/21) of overall patients was significantly more frequent in single layer graft when compared to double layer (77% vs 33%, p < 0.05). Location and type of graft tear significantly influenced final outcomes. Patients with a healed (graft continuity with bone at medial and lateral insertion) or medial graft tear showed statistically significant better outcomes when compared with patients in which the graft was completely reabsorbed or torn on humeral side (p < 0.05). CONCLUSION: Arthroscopic superior capsule reconstruction using an acellular porcine dermal xenograft may be a viable alternative to treat massive posterosuperior rotator cuff tear in patients with a painful pseudoparalysis without anterosuperior escape. Structural failure may strongly influence final outcomes with significant role played by tear location.

Cyclin B2 is required for progression through meiosis in mouse oocytes.

Cyclins associate with cyclin-dependent serine/threonine kinase 1 (CDK1) to generate the M phase-promoting factor (MPF) activity essential for progression through mitosis and meiosis. Although cyclin B1 (CCNB1) is required for embryo development, previous studies concluded that CCNB2 is dispensable for cell cycle progression. Given previous findings of high Ccnb2 mRNA translation rates in prophase-arrested oocytes, we re-evaluated the role of this cyclin during meiosis. Ccnb2-/- oocytes underwent delayed germinal vesicle breakdown and showed defects during the metaphase-to-anaphase transition. This defective maturation was associated with compromised Ccnb1 and Moloney sarcoma oncogene (Mos) mRNA translation, delayed spindle assembly and increased errors in chromosome segregation. Given these defects, a significant percentage of oocytes failed to complete meiosis I because the spindle assembly checkpoint remained active and anaphase-promoting complex/cyclosome function was inhibited. In vivo, CCNB2 depletion caused ovulation of immature oocytes, premature ovarian failure, and compromised female fecundity. These findings demonstrate that CCNB2 is required to assemble sufficient pre-MPF for timely meiosis re-entry and progression. Although endogenous cyclins cannot compensate, overexpression of CCNB1/2 rescues the meiotic phenotypes, indicating similar molecular properties but divergent modes of regulation of these cyclins.

Genome-wide analysis reveals a switch in the translational program upon oocyte meiotic resumption.

During oocyte maturation, changes in gene expression depend exclusively on translation and degradation of maternal mRNAs rather than transcription. Execution of this translation program is essential for assembling the molecular machinery required for meiotic progression, fertilization, and embryo development. With the present study, we used a RiboTag/RNA-Seq approach to explore the timing of maternal mRNA translation in quiescent oocytes as well as in oocytes progressing through the first meiotic division. This genome-wide analysis reveals a global switch in maternal mRNA translation coinciding with oocyte re-entry into the meiotic cell cycle. Messenger RNAs whose translation is highly active in quiescent oocytes invariably become repressed during meiotic re-entry, whereas transcripts repressed in quiescent oocytes become activated. Experimentally, we have defined the exact timing of the switch and the repressive function of CPE elements, and identified a novel role for CPEB1 in maintaining constitutive translation of a large group of maternal mRNAs during maturation.

Rotator cuff repair protected with subacromial balloon spacer shows a low rate of non-healing.

PURPOSE: The purpose of this study was to evaluate clinical outcomes and tendon integrity on magnetic resonance imaging (MRI) of chronic posterosuperior rotator cuff tears treated with single-row tensionless repair and subacromial balloon spacer as protection with a minimum follow-up of 2 years. The hypothesis of this study was that this procedure would have acceptable clinical outcomes and tendon-healing rate without increased complications. METHODS: This is a retrospective study of patients with chronic posterosuperior rotator cuff tears repaired with a single-row technique protected with a subacromial balloon device. Patients were followed up for a minimum of 2 years. Clinical outcomes were evaluated with American Shoulder and Elbow Surgeons Standardized Shoulder Assessment Form (ASES) and Numerical Rating Scale (NRS) for pain. MRI study was obtained likewise after at least 2 years to assess tendon-healing rate. Statistical comparison was performed between pre-operative and at least 2-year clinical and imaging follow-up. RESULTS: Thirty-two patients were included in the study with a mean follow-up of 27 ± 7 (range 24-48). The mean age of this cohort was 58 ± 6 (range 41-66) including 15 males and 17 females. The tear size was on average 2.3 cm (range 2-4) and a mean of 2.1 triple-loaded anchors were used (range 2-3). The ASES score significantly increased from a mean of 39 ± 12 points to a mean of 89 ± 12 at the final follow-up (P < 0.001). Similarly, pain significantly reduced from a mean pre-operative NRS of 6.8 ± 1.4 to 0.8 ± 1.5 at the final follow-up (P < 0.001). MRI scans showed that repair occurred in 26 patients (81.3%). Significant higher ASES score was reached at final follow-up in patients with a "healed" (Sugaya I-III) tendon when compared to patients with an evidence of tendon discontinuity on MRI study (Sugaya IV-V), 93 ± 9 and 74 ± 13, respectively (P < 0.001). CONCLUSIONS: Arthroscopic repair of chronic posterosuperior rotator cuff tears using a single-row tensionless repair and subacromial spacer as protection resulted in an 81.3% of tendon integrity at a mean follow-up of 27 months. Clinical outcomes and pain scores significantly improved without severe complications reported after a minimum follow-up of 2 years. LEVEL OF EVIDENCE: III.

Toward a Detailed Evaluation of Wireless Industrial Data Distribution Approaches.

Data distribution is a cornerstone of efficient automation for intelligent machines in Industry 4.0. Although in the recent literature there have been several comparisons of relevant methods, we identify that most of those comparisons are either theoretical or based on abstract simulation tools, unable to uncover the specific, detailed impacts of the methods to the underlying networking infrastructure. In this respect, as a first contribution of this paper, we develop more detailed and fine-tuned solutions for robust data distribution in smart factories on stationary and mobile scenarios of wireless industrial networking. Using the technological enablers of WirelessHART, RPL and the methodological enabler of proxy selection as building blocks, we compose the protocol stacks of four different methods (both centralized and decentralized) for data distribution in wireless industrial networks over the IEEE 802.15.4 physical layer. We implement the presented methods in the highly detailed OMNeT++ simulation environment and we evaluate their performance via an extensive simulation analysis. Interestingly enough, we demonstrate that the careful selection of a limited set of proxies for data caching in the network can lead to an increased data delivery success rate and low data access latency. Next, we describe two test cases demonstrated in an industrial smart factory environment. First, we show the collaboration between robotic elements and wireless data services. Second, we show the integration with an industrial fog node which controls the shop-floor devices. We report selected results in much larger scales, obtained via simulations.

Advanced Facial Rejuvenation After Bimaxillary Surgery in Three Different Facial Types.

PURPOSE: Facial aging is the consequence of many mechanisms involving the bones and the "soft tissue" (skin, fat, ligaments, muscles, and periosteum) of the face such as downward migration of the soft tissue, adipose and muscular tissue atrophy, and skeletal resorption. The potential of orthognathic surgery (double jaw surgical advancement) of expanding the skeletal foundation to increase the facial drape support is now recognized and widely popularized by several authors. The aim of this study was to analyze the rejuvenation change of the face after bimaxillary advancement for orthognathic surgery, focusing on the previously mentioned stigmata of the middle and the lower third of the aging face. MATERIALS AND METHODS: A retrospective monocentric chart review was conducted for all patients affected by aging signs of the face who underwent orthognathic surgery between January 2015 and December 2019 at the Face Surgery Center (Parma, Italy). During the postoperative follow-up examination, all patients underwent anthropometric photographs and esthetic assessment to evaluate facial rejuvenation after double jaw surgical advancement. RESULTS: After application of the exclusion criteria, the final study sample included 85 patients (53 females, 32 males). Eighty-three patients (97%) showed a degree of rejuvenation after maxillo-mandibular advancement (MMA); the score of the postoperative face was less than the score of the preoperative face. Two patients reported no significant postoperative change; none reported a more aging face, with a successful "reverse face-lift" occurred in 97% of our cases. CONCLUSION: "Reverse face-lift" by bimaxillary advancement is a surgical procedure which is indicated for a selected group of middle-aged patients with a diagnosis of bimaxillary skeletal retrusion or posterior divergence very motivated to an extreme rejuvenation; this procedure provides support for the facial mask resulting in whole facial rejuvenation.

Elite professional goalkeepers report high rate of sport resumption after shoulder surgery.

PURPOSE: The aim of this study is to evaluate the return to sport after surgical treatment of shoulder injuries in professional goalkeepers in relationship with the mechanism of injury and the pattern of related shoulder lesions. METHODS: Twenty-six shoulders in nineteen elite male professional soccer goalkeepers were retrospectively analyzed considering multiple diseases (instability, rotator cuff, biceps or other tendon injuries). Data was collected for injury modality and context, pathological findings, surgical procedures, time, level of return to sport, and complications. RESULTS: The mechanism of injury was "mild trauma without contact" in 46% of the cases and 54% of injuries happened during training. 11 patients (42%) reported multiple pattern lesions and 9 patients (35%) classic anterior instability lesions. The mean time for return to differentiated training and unrestricted sport activity was 14 and 20.2 weeks, respectively. 15 athletes (62.5%) reported 100% return to sport, 4 (16.7%) to 90%, 1 (4.2%) to 85%, 3 (12.5%) to 80% and 1 athlete to 50%, stopping professional activity. 21% of the cases reported the persistence of some shoulder symptoms. 3 cases experienced a new injury. Patients with classic anterior instability had significantly lower age (30.7 vs 19.8 years, P = 0.001), experienced injury in different context and reported symptoms more frequently compared to multiple lesion patients (4/8 vs 0/10, P = 0.011). CONCLUSION: Professional elite goalkeepers which required shoulder surgery for different causes demonstrated high-rate level of return to play despite the persistence of mild symptoms. The high frequency of multiple lesions, patients' characteristics, injury context and mechanism, increase the concern for injuries in overstressed shoulder for this category of sport. LEVEL OF EVIDENCE: IV.

Give more data, awareness and control to individual citizens, and they will help COVID-19 containment.

The rapid dynamics of COVID-19 calls for quick and effective tracking of virus transmission chains and early detection of outbreaks, especially in the "phase 2" of the pandemic, when lockdown and other restriction measures are progressively withdrawn, in order to avoid or minimize contagion resurgence. For this purpose, contact-tracing apps are being proposed for large scale adoption by many countries. A centralized approach, where data sensed by the app are all sent to a nation-wide server, raises concerns about citizens' privacy and needlessly strong digital surveillance, thus alerting us to the need to minimize personal data collection and avoiding location tracking. We advocate the conceptual advantage of a decentralized approach, where both contact and location data are collected exclusively in individual citizens' "personal data stores", to be shared separately and selectively (e.g., with a backend system, but possibly also with other citizens), voluntarily, only when the citizen has tested positive for COVID-19, and with a privacy preserving level of granularity. This approach better protects the personal sphere of citizens and affords multiple benefits: it allows for detailed information gathering for infected people in a privacy-preserving fashion; and, in turn this enables both contact tracing, and, the early detection of outbreak hotspots on more finely-granulated geographic scale. The decentralized approach is also scalable to large populations, in that only the data of positive patients need be handled at a central level. Our recommendation is two-fold. First to extend existing decentralized architectures with a light touch, in order to manage the collection of location data locally on the device, and allow the user to share spatio-temporal aggregates-if and when they want and for specific aims-with health authorities, for instance. Second, we favour a longer-term pursuit of realizing a Personal Data Store vision, giving users the opportunity to contribute to collective good in the measure they want, enhancing self-awareness, and cultivating collective efforts for rebuilding society.

How to Prevent Mandibular Lower Border Notching After Bilateral Sagittal Split Osteotomies for Major Advancements: Analysis of 168 Osteotomies.

PURPOSE: Mandibular ramus bilateral sagittal split osteotomy (BSSO) has been the most commonly used technique in orthognathic surgery for mandibular advancement. However, a common complication of BSSO has been the occurrence of visible and palpable osseous defects at the inferior border of the mandible. The aim of the present study was to determine whether bone grafting of the osseous defect at surgery would reduce the defect at 1 year postoperatively compared with no bone grafting. MATERIALS AND METHODS: The present retrospective cohort study evaluated patients who had undergone mandibular ramus BSSO for 10 mm or more of advancement. The primary predictor variable was BSSO surgery with bone grafting of the defect (graft group [GG]) versus no bone graft (no graft group [NGG]). The size of the mandibular ramus inferior border defect was the outcome variable considered within the framework of a 1-year postoperative cone beam computed tomography (CBCT) analysis. Gender, age, and the amount of advancement were also considered in the multilevel regression analyses. RESULTS: From January 2012 to November 2016, 84 patients (168 osteotomies) had undergone BSSO surgery with 10 mm or more of mandibular advancement at the Facesurgery Center (Parma, Italy). Their mean age was 27.4 years (range, 17 to 44 years). Of the 84 patients, 40 had undergone BSSO with bilateral bone grafts (GG). The monocortical block of the iliac crest bone was used as the bone homograft. The final residual defect was measured at 1 year postoperatively on CBCT scans. The GG and NGG had presented with a mean final defect of 0.7 mm (range, 0 to 4.5 mm) and 3.0 mm (range, 0 to 5.5 mm), respectively. Complete absence of the defect was achieved in 72% of the osteotomies in the GG and 9% of the osteotomies in the NGG. CONCLUSIONS: The use of an iliac crest bone allograft block in the gap between 2 segments during mandibular advancement of 10 mm or more substantially reduced the size and incidence of inferior border defects.

International Union of Basic and Clinical Pharmacology. CI. Structures and Small Molecule Modulators of Mammalian Adenylyl Cyclases.

Adenylyl cyclases (ACs) generate the second messenger cAMP from ATP. Mammalian cells express nine transmembrane AC (mAC) isoforms (AC1-9) and a soluble AC (sAC, also referred to as AC10). This review will largely focus on mACs. mACs are activated by the G-protein Gαs and regulated by multiple mechanisms. mACs are differentially expressed in tissues and regulate numerous and diverse cell functions. mACs localize in distinct membrane compartments and form signaling complexes. sAC is activated by bicarbonate with physiologic roles first described in testis. Crystal structures of the catalytic core of a hybrid mAC and sAC are available. These structures provide detailed insights into the catalytic mechanism and constitute the basis for the development of isoform-selective activators and inhibitors. Although potent competitive and noncompetitive mAC inhibitors are available, it is challenging to obtain compounds with high isoform selectivity due to the conservation of the catalytic core. Accordingly, caution must be exerted with the interpretation of intact-cell studies. The development of isoform-selective activators, the plant diterpene forskolin being the starting compound, has been equally challenging. There is no known endogenous ligand for the forskolin binding site. Recently, development of selective sAC inhibitors was reported. An emerging field is the association of AC gene polymorphisms with human diseases. For example, mutations in the AC5 gene (ADCY5) cause hyperkinetic extrapyramidal motor disorders. Overall, in contrast to the guanylyl cyclase field, our understanding of the (patho)physiology of AC isoforms and the development of clinically useful drugs targeting ACs is still in its infancy.

Functional selectivity of GPCR-directed drug action through location bias.

G-protein-coupled receptors (GPCRs) are increasingly recognized to operate from intracellular membranes as well as the plasma membrane. The ß2-adrenergic GPCR can activate Gs-linked cyclic AMP (Gs-cAMP) signaling from endosomes. We show here that the homologous human ß1-adrenergic receptor initiates an internal Gs-cAMP signal from the Golgi apparatus. By developing a chemical method to acutely squelch G-protein coupling at defined membrane locations, we demonstrate that Golgi activation contributes significantly to the overall cellular cAMP response. Golgi signaling utilizes a preexisting receptor pool rather than receptors delivered from the cell surface, requiring separate access of extracellular ligands. Epinephrine, a hydrophilic endogenous ligand, accesses the Golgi-localized receptor pool by facilitated transport requiring the organic cation transporter 3 (OCT3), whereas drugs can access the Golgi pool by passive diffusion according to hydrophobicity. We demonstrate marked differences, among both agonist and antagonist drugs, in Golgi-localized receptor access and show that ß-blocker drugs currently used in the clinic differ markedly in ability to antagonize the Golgi signal. We propose 'location bias' as a new principle for achieving functional selectivity of GPCR-directed drug action.

Dynamic secretion during meiotic reentry integrates the function of the oocyte and cumulus cells.

The differentiation of the female gamete into a developmentally competent oocyte relies on the protected environment of the ovarian follicle. The oocyte plays a key role in establishing this microenvironment by releasing paracrine factors that control the functions of surrounding somatic cells. Growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15) are secreted during follicle growth and play pivotal roles in this local regulation. The current view is that the function of these secreted factors declines in the periovulatory period when the oocyte reenters the meiotic cell cycle. Here, we provide evidence that oocyte reentry into meiosis is instead associated with a shift in the pattern of secretion with a new set of bioactive molecules synthesized before ovulation. Using interleukin 7 (IL7) as a prototypic secreted factor, we show that its secretion is dependent on activation of mRNA translation in synchrony with the cell cycle and that its translation is under the control of somatic cells. IL7 is part of a local feedback loop with the soma because it regulates cumulus cell replication. Similar conclusions are reached when IL7 secretion is measured in human follicular fluid during in vitro fertilization cycles. IL7 concentration in the follicular fluid correlates with the oocyte ability to reach the MII stage of maturation. These findings are consistent with the hypothesis that a new set of local factors is secreted by the oocyte during ovulation. These dynamic secretions are likely critical for promoting the final stages of maturation and oocyte developmental competence.

Genome-wide analysis of translation reveals a critical role for deleted in azoospermia-like (Dazl) at the oocyte-to-zygote transition.

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.