Drosophila mushroom body (MB) learning and memory circuitry, but local PKA activity within this well-mapped neuronal network is uncharacterized. Here, we use an in vivo PKA activity sensor (PKA-SPARK) to test spatiotemporal regulatory requirements in the MB axon lobes. We find immature animals have little detectable PKA activity, whereas postcritical period adults show high field-selective activation primarily in just 3/16 defined output regions. In addition to the age-dependent PKA activity in distinct α’/β’ lobe nodes, females show sex-dependent elevation compared with males in these same restricted regions. Loss of neural cell body Fragile X mental retardation protein (FMRP) and Rugose [human Neurobeachin (NBEA)] suppresses localized PKA activity, whereas overexpression (OE) of MB lobe PKA-synergist Meng-Po (human SBK1) promotes PKA activity. Elevated Meng-Po subverts the PKA age-dependence, with elevated activity in immature animals, and spatial-restriction, with striking γ lobe activity. Testing circuit signaling requirements with temperature-sensitive shibire (human Dynamin) blockade, we find broadly expanded PKA activity within the MB lobes. Using transgenic tetanus toxin to block MB synaptic output, we find greatly heightened PKA activity in virtually all MB lobe fields, although the age-dependence is maintained. We conclude spatiotemporally restricted PKA activity signaling within this well-mapped learning/memory circuit is age-dependent and sex-dependent, driven by FMRP-Rugose pathway activation, temporally promoted by Meng-Po kinase function, and restricted by output neurotransmission providing network feedback.Dynamic functional connectivity within brain circuits requires coordination of intercellular signaling and intracellular signal transduction. Critical roles for cAMP-dependent protein kinase A (PKA) signaling are well established in the  Drosophila mushroom body (MB); a network model consolidating ∼2000 Kenyon cells (KCs) into ∼16 output fields per brain hemisphere. We discover heightened PKA activity in just a few circuit regions, with age-dependent and sex-dependent requirements. Learning/memory mutants linked to intellectual and autism spectrum disorders (ASDs) reduce PKA activity. In contrast, PKA-synergist Meng-Po increases PKA activity. Neurotransmission blockade elevates and expands PKA activity, showing network feedback restricts localized signaling.Learning and memory requires the coordination of cell-cell circuit interactions with the appropriate downstream signal transduction. Plasticity regulation via cAMP-dependent protein kinase A (PKA) activation is among the best-characterized signaling pathways, but until recently, it was very challenging to localize PKA signaling within brain circuits. We employ a new PKA biosensor to visualize local signaling in the Drosophila mushroom body (MB) learning/memory circuit model, both PKA and PKA regulators  are highly expressed, and play well-characterized, pivotal roles  is widely recognized to be required for this synaptic regulation . In the al roles . Within al roles . MB lobeal roles . Investial roles . Howeveral roles . New stuDrosophila disease models and human FXS patient cell cultures lacking FMRP (rugose mRNA to positively regulate translation of this A-kinase anchor protein (AKAP) controlling MB PKA activity (Drosophila MB lobes to facilitate memory formation (tsshibire mutant blocking Dynamin-dependent synaptic vesicle (SV) endocytosis can be used to disrupt KC neurotransmission  and Rugose/Neurobeachin (NBEA) are causally linked to intellectual disability and autism spectrum disorder ASD; . FMRP dring FMRP , and FMRing FMRP . FMRP biactivity , and Rugactivity . Downstrormation . Meng-Posmission . The trasmission . Our aimsmission  to visuaDrosophila at 0 d posteclosion (0 dpe) with postcritical period Drosophila adults (7 dpe). Immature animals display little or no detectable PKA-SPARK activity in the MB lobes, whereas mature animals exhibit high levels of spatially-restricted signaling in α’1 and β’1 regions. In both locations, we find females have higher PKA activity. FMRP, Rugose and Meng-Po loss results in strongly reduced PKA activity in both MB lobe regions. In contrast, Meng-Po OE drives PKA activity in immature animals (0 dpe), as strongly as normal mature signaling, and also dramatically increases PKA activity in α’1, β’1, and β’1-adjacent γ3 at maturity (7 dpe). Testing circuit signaling requirements, we targeted KC synaptic output with two tools; temperature-sensitive tsshibire and TNT blockade. The tsshibire animals display both age-dependent and temperature-dependent defects in PKA-SPARK activity, with expanded PKA signaling within the MB lobes at restrictive temperature. Consistently, MB-targeted TNT neurotransmission blockade dramatically expands PKA activity throughout the lobes, although the age-dependence is maintained. These findings indicate that PKA activity in the MB lobes is both age-dependent and sex-dependent in tightly restricted connectivity regions. Moreover, PKA activity is selectively induced during early adulthood in a mechanism requiring FMRP, Rugose, and Meng-Po and can be strongly potentiated by elevated Meng-Po to overcome some spatiotemporal restrictions. Finally, synaptic output suppresses PKA activity to spatially defined MB lobe regions of heightened signaling, suggesting a network feedback mechanism regulates PKA activity.In this study, we compare immature 1118w (BDSC 3605) and RNAi background  were used as controls. Null 50Mdfmr1 was used as the control. The OK107-Gal4 driver line at 2 dpe was used as a negative control for Western blot analyses. UAS responder lines used included: UAS-PKA-SPARK , and insulated UAS-TNT . For acute tsshibire experiments, animals were raised at 20°C until 7 dpe, and then either maintained at 20°C or moved to 33°C for 3 h. For longer tsshibire experiments, animals were raised at 20°C, and then either maintained at this temperature or moved to 25°C or 33°C for 7 d. Recombination and multiallele crossing schemes were performed using standard Drosophila genetic techniques.All animals were reared and maintained on standard food in 12/12 h light/dark cycle cycling incubators at 25°C, unless otherwise noted. A combination of females and males were used for all experiments, except when females and males were compared or mutations were on the X chromosome. For mutant studies, the genetic background dfmr150M and rgFDM. Samples were then incubated 2 h at room temperature (RT) with FITC-conjugated goat anti-GFP , and washed 3× in blocking buffer. Brain preparations were mounted in Fluoromount-G between two #1 coverslips separated by die-cut Oracal 651 spacers. All imaging was done using a Zeiss LSM510 META with a Plan Neofluar 40× oil-immersion (1.3 NA) objective. MB images were collected at 1-μm optical slice thickness for data analyses.g) for 10 min at 4°C. Supernatant was removed and treated with λ Protein Phosphatase following the manufacturer protocol . To equal lysate volumes, 2 × Laemmli  + 2-mercaptoethanol  was added and samples heated to 100°C for 10 min. Precision Plus Protein All Blue Standards  and samples were loaded into 4–15% Mini-PROTEAN TGX Stain-Free Gels , and run at 100V for 10 min, then 180–200 V until completion. Protein was transferred onto Bio-Rad LF PVDF membranes using the Bio-Rad Trans-Blot Turbo system. Membranes were blocked in LI-COR Intercept Blocking Buffer (927-60001) at RT for 1 h, incubated in primary antibodies at RT for 1 h and 10 min, and fluorescent secondary antibodies at RT for 40 min. Labeled membranes were imaged on a LI-COR Odyssey CLx system. Protein bands were standardized to loading control (α-tubulin) and analyzed using ImageStudioLite (LI-COR). Primary antibodies: mouse anti-α-tubulin  and rabbit anti-GFP . Fluorescent secondary antibodies: Alexa Fluor 700 goat anti-rabbit  and DyLight 800 goat anti-mouse , both at 1:10,000.Heads from staged animals  were snap frozen on dry ice and then homogenized in 12-μl 1 × PBS with EDTA-free protease inhibitor . For each lysate, two pooled heads in 24-μl RIPA buffer  were spun  or Brown–Forsythe and Welch ANOVA tests (n > 2 datasets). Datasets failing normality tests were compared with Mann–Whitney tests (n = 2 datasets) or with Kruskal–Wallis tests with Dunn’s correction (n > 2 datasets). Graphs display all individual data points and mean ± SEM. Significance is displayed in the figures as *p < 0.05, **p < 0.005, ***p < 0.001, and not statistically significant . Additional information is presented in the statistical table . Datasets passing the D’Agostino and Pearson normality test were compared with two-tailed Welch’s al table .-separation of phases-based activity reporter of kinase  compared with high levels of signaling at 7 dpe , a very significant increase over the short, one-week critical period  compared with strong signaling at 7 dpe maturity , again a highly significant elevation  compared with the males , a highly significant sex difference  compared with males , again a very significant sex-dependent difference  null mutants also display impaired MB-dependent learning and memory (1118w) to both the 50Mdfmr1  compared with 50Mdfmr1 mutants , a highly significant loss of signaling  compared with 50Mdfmr1 nulls , showing a significant reduction in the localized PKA activity  compared with FDDrg mutants , a very significant reduction  compared with FDDrg nulls , a highly significant loss of signaling  and human FMRP (hFMRP) elevates PKA activity in KC somata  is similar to both hFMRP OE  and ΔRGG-hFMRP OE , albeit with a slight increase following dFMRP OE . In β’1, there is also little early signaling increase at 0 dpe (n = 14), hFMRP OE , ΔRGG-hFMRP OE  and Rg OE , with slight elevation after dFMRP OE  and dFMRP OE , hFMRP OE  and Rg OE , albeit lower in ΔRGG-hFMRP OE  and hFMRP OE , but reduced with dFMRP OE , Rg OE  and especially ΔRGG-hFMRP OE  are highly elevated by Meng-Po OE , a very significant increase  is even more highly increased by Meng-Po OE . This elevated and expanded PKA signaling is more pronounced at maturity (7 dpe), with higher activity in α’1, β’1, and γ3 MBON fields (n = 16), a very significant increase  that is absent in controls . These data show that gain of Meng-Po function strongly expands spatiotemporal PKA activity.Meng-Po (MP) OE causes a profound induction of PKA signaling, strongly subverting normal temporal restrictions compare A and F. N fields F, right.meng-po mutants are lethal before adult eclosion, so MB-specific meng-po RNAi was tested for changes in MB lobe PKA activity . Representative images at all temperatures and quantified data are shown in The spatiotemporally localized PKA activity within a few MBON fields suggests local network regulation. Multiple circuit feedback interactions are proposed to provide modularity to the MB circuit, both within and between signaling compartments . We hyposallele , which isallele . At restild type . At repoMB lobes A, but hatsshibire display greatly elevated PKA activity in both α’1 and β’1, as well as β’2ap (n = 15), more at 25°C , and the most at 33°C [62.2 ± 4.3, n = 17  to 25°C  to 33°C [66.9 ± 3.7, n = 17  are increased at 25°C , but not at 33°C [9.3 ± 2.0, n = 17  and 25°C , but a very significant increase at restrictive 33°C  also increases PKA signaling. The largest elevation in PKA-SPARK puncta number is in α2; a ∼7-fold increase compared with the 20°C control . These findings show that tsshibire impairment of KC neurotransmission greatly broadens PKA activity in the MB lobes.Mutants reared at low temperature to 0 dpe (20°C), already show some altered MB morphology, including enlarged α/α’ lobe ends A, arrowsas β’2ap D. Quanti, n = 17 E, 20° vs, n = 17 F, 20° vs/γ lobes D. To qua, n = 17 G, 20° vstsshibirestudies suggest that MB synaptic output suppresses PKA activity across MBON fields, but interpretation is complicated by temperature and pleiotropic Dynamin functions. We therefore turned to a stronger, synapse-selective neurotransmission block via targeted transgenic tetanus toxin light chain , but there is little change in the β’1 MBON field , the TNT blockade of KC synaptic transmission has no effect within any MBON field C–I, withn = 31) is replaced with significantly elevated numbers by 7 dpe , but induces many puncta by 7 dpe , and highly active at 7 dpe , as does the adjacent β2 region (0 dpe: 0.2 ± 0.1, n = 32; 7 dpe: 85.1 ± 3.4, n = 58, p < 0.0001; TNT synaptic-silencing of KC output generates strongly elevated PKA signaling in widespread MBON fields that normally lack any detectable PKA activity B. In TNTin vivo (tsshibire (In this study, we explore the spatiotemporal regulation of PKA activity within the MB lobes. PKA signaling initiates in early adulthood, with heightened activity in just 3/16 MB lobe output neuron fields (α’1, β’1, and β’2ap; in vivo . While iin vivo , at the in vivo , PKA actin vivo . There iin vivo , but PKAin vivo , with OEin vivo  and lossin vivo . Networkhibirets  or transhibirets  induces hibirets , 7. ThusmiR-92a in α/β and γ, but not α’/β’, was shown to enhance memory (At a macro level, the α’1 and β’1 regions with the highest localized PKA activity levels  have beee memory , indicate memory , suggeste memory  is consie memory . Importae memory , correlae memory .rugose mRNA to drive KC expression (meng-po RNAi causes the opposite result of reducing localized PKA-SPARK puncta (Two learning and memory proteins, FMRP and Rugose, are needed for full PKA activity in the α’1 and β’1 MB lobe output neuron fields . RNA-binpression . As predpression . In contpression , which ipression . Consistpression . MoreoveK puncta . Based otsshibire and transgenic tetanus toxin tools block KC neurotransmission, but through quite different mechanisms (tsshibire drives PKA activity expansion in the MB γ lobe (tsshibirenor TNT blockade alters early PKA activity (Two different KC synaptic output blocking methods dramatically expand PKA activity signaling in the MB lobes , 7. Bothchanisms . At 33°CB γ lobe . This chB γ lobe . The tetB γ lobe , and theB γ lobe . Neitheractivity , 7, suggactivity . The widdfmr1 mutant circuit defects exacerbated by glutamate exposure (rg mutant social interaction and memory deficits (de facto depression may feedback onto KCs to promote PKA activity signaling. The impact of upstream input onto the MB lobes is an important consideration, including how this circuitry combines with spontaneous MB activity and internal lobe circuitry to determine PKA signaling. Future research directions should attempt to dissect how these different layers of neuromodulation control localized PKA activity signaling within the MB lobe circuit, and between females and males, by manipulating input-specific neuronal activity in targeted transgenic studies.At the MB circuit level, multiple candidate synaptic pathways need to be explored for roles in local PKA activity regulation in different MB lobe output neuron fields. GABAergic inputs to dopaminergic neurons are one likely candidate, since GABA treatment has been shown to correct exposure . Moreoveexposure . Recent deficits . Anotherdeficits . In the Drosophila brain MB learning and memory center is highly induced during early experiential adulthood, with selective upregulation in the α’1, β’1, and β’2ap MB lobe output neuron regions. Age-specific and sex-specific PKA signaling controlled within KCs and downstream of KC output shows that spatiotemporally restricted MB lobe PKA activity is regulated through a combination of both intracellular control and intercellular network-level mechanisms. Importantly, PKA signaling can be precociously promoted and spatial expanded though the activity of the PKA-synergist Meng-Po kinase. Moreover, KC neurotransmission inhibits localized PKA signaling within the MB circuit. Our future studies will be aimed toward generating new genetic responder tools to test KC signaling with both neurotransmission output blockade and activity promotion of upstream and downstream MB circuit components, simultaneously and independently of KCs. We also plan to test PKA activity signaling with the manipulation of specific KC partners, by altering neurotransmission signaling in combination with postsynaptic neurotransmitter receptor mutants to determine network communication cues. Taken together with the current work, these ongoing studies will continue to expand our understanding of circuit-level PKA signaling regulation in normal function and in neurologic disease model contexts.In conclusion, we report here that PKA activity signaling in the


Cellular energy is primarily provided by the oxidative degradation of nutrients coupled with mitochondrial respiration, in which oxygen participates in the mitochondrial electron transport chain to enable electron flow through the chain complex (I–IV), leading to ATP production. Therefore, oxygen supply is an indispensable chapter in intracellular bioenergetics. In mammals, oxygen is delivered by the bloodstream. Accordingly, the decrease in cellular oxygen level (hypoxia) is accompanied by nutrient starvation, thereby integrating hypoxic signaling and nutrient signaling at the cellular level. Importantly, hypoxia profoundly affects cellular metabolism and many relevant physiological reactions induce cellular adaptations of hypoxia-inducible gene expression, metabolism, reactive oxygen species, and autophagy. Here, we introduce the current knowledge of hypoxia signaling with two-well known cellular energy and nutrient sensing pathways, AMP-activated protein kinase (AMPK) and mechanistic target of rapamycin complex 1 (mTORC1). Additionally, the molecular crosstalk between hypoxic signaling and AMPK/mTOR pathways in various hypoxic cellular adaptions is discussed. Therefore, under low oxygen conditions that do not reach the cellular requirement , vertebrates should reprogram the metabolic pathways and the corresponding regulatory circuits in response to the stressful conditions caused by hypoxia, thereby promoting cell survival . Fir. Fir3]. TP ratio ,20. LastTP ratio . AccumulTP ratio . The kinTP ratio . PKB/AktTP ratio . SimilarTP ratio . GlycogeTP ratio , proteinTP ratio , and proTP ratio  are also+-dependent protein deacetylase Sirtuin-1 (SIRT1), which deacetylates and activates PGC-1α ,7647,76]Metabolic regulation by mTORC1. mTORC1 signaling plays a fundamental role in various biosynthetic pathways ,102. mTO2+ and concomitantly activate CaMKKβ independently of any significant change in ATP and AMP level. Indeed, AMPK activation in HeLa and HEK293T cells under hypoxia was blunted by CaMKKβ inhibitor, STO-609 [2O2 was shown to induce oxidation and S-glutathionylation of cysteine residues (Cys299 and Cys304) on AMPKα in HEK293 and lung cells, resulting in the activation of AMPK [2O2 and ischemia induced oxidation of other cysteine residues (Cys130 and Cys174) on α-subunit, which inhibited AMPK through aggregation of AMPK molecules and blockage of Thr172 phosphorylation by upstream kinases [AMPK regulation by hypoxia. Activation of AMPK under hypoxia has been reported in various tissues and cell types via different molecular mechanisms . In gene STO-609 . Additio STO-609 . Alterna STO-609 . ROS are STO-609 ,122. Eve STO-609 , therefo STO-609 . Hypoxia STO-609 ,126. Acc STO-609 ,128,129.2+ and α-ketoglutarate-dependent dioxygenase family, HIF asparaginyl hydroxylase or factor inhibiting HIF1 (FIH1) [2+-CaMKKβ signaling, but not on LKB1 [Hypoxia-inducible transcription factors, HIFs. The life obtaining biological energy from mitochondrial oxidative phosphorylation largely relies on oxygen for proliferation, growth, and survival . Additio1 (FIH1) ,134. In 1 (FIH1) . Notably1 (FIH1) . Additio1 (FIH1) . Interes1 (FIH1) ,139. Thi on LKB1 . However on LKB1 ,141,142  on LKB1 . Conside on LKB1 , Notch-1 on LKB1 , and IκB on LKB1 , FIH1 ma on LKB1  and the  on LKB1 . Notably on LKB1 ,150. Add on LKB1 . However on LKB1 ,153. Sim on LKB1 . This apmTORC1 and REDD1 in hypoxia. Although the activation of AMPK may provide a primary mechanism to inhibit mTORC1 in hypoxia, hypoxia can also directly regulate mTORC1 by a protein regulated in development and DNA damage response-1   155,156156. REDDProtein translation in hypoxia. Along with cellular adaptation at the transcriptional level via HIFs, down-regulation of energy consuming protein translation minimizes unnecessary ATP use and prevents the accumulation of unfolded (or misfolded) proteins by hypoxia, in which protein kinase R (PKR)-like endoplasmic reticulum (ER) kinase (PERK) and mTORC1 play an important role . Accumul+/K+ ATPase) account for 20–70% of the oxygen expenditure of mammalian cells [+/K+ ATPase on the plasma membrane via endocytosis in alveolar epithelial cells. In this condition, hypoxia increases cellular Ca2+ level by calcium release-activated calcium (CRAC) channels. CARC channels are responsible for store-operated calcium (SOC) entry, a major route of Ca2+ influx in nonexcitable cells, and are activated by the depletion of Ca2+ stores in ER [2+ stores in ER, leading to long-lasting calcium signaling [2+ from the ER, and the Ca2+ influx through CRAC channels stimulates CaMKKβ, which is followed by AMPK activation.Mitochondrial respiration and ROS in hypoxia. In hypoxia, mitochondria are first in line to experience the change in oxygen level because the mitochondrial electron transport chain (ETC) is the largest single consumer of intracellular oxygen for the generation of ATP . Althougan cells . This puan cells ,181. Thees in ER . The opeignaling . The ROS+ in cytosol, which is secreted by monocarboxylic transporter (MCT4), sodium-hydrogen (Na+/H+) exchanger (NEH1), and carbonic anhydrase (CAR9) [+ acidifies the tumor microenvironment to inhibit immune responses around cancers (tumor immune evasion) by suppressing the infiltrating T cells [Metabolic adaptation in hypoxia. Hypoxia profoundly influences many key metabolic pathways . Hypoxiae (CAR9) . Extracee (CAR9) ,189. Adde (CAR9) . Notably T cells . Hypoxia T cells ,192. Glu T cells . Additio T cells . Hypoxic T cells . Hypoxia T cells . However T cells . Additio T cells .Autophagy and hypoxia. In hypoxia, cells also activate autophagy for survival. Autophagy  is a catabolic program to remove harmful cellular contents, such as damaged organelles and protein aggregates, from lysosomes. Simultaneously, it provides the energy and new building blocks required to promote cell survival in stressful environments ,195. HypHypoxia is an important pathophysiological condition that can induce massive cellular adaptive responses. It is generally accompanied by nutrient starvation, therefore, hypoxic signaling must be closely linked to nutrient signaling. Indeed, accumulating reports have shed light on the communication of two important signaling pathways, AMPK and mTORC1, with hypoxic signaling. Their crosstalk can be found in a variety of cellular adaptations  to hypoxia. These mechanisms are integral inputs for fine-tuning responses to hypoxic stress.


Objective: Fertility loss due to cancer treatment can be a devastating experience for women and the couple. Undergoing fertility preservation can be a complex decision from both a medical and emotional point of view. The aim of the present study was to evaluate which socio-demographic and psychological factors predict a high motivation to undergo fertility preservation.Methods: Fifty-eight female cancer patients who accessed an Oncofertility Unit completed: a questionnaire to collect socio-demographic characteristics and the level of motivation, the Beck-Depression Inventory-II, the State-Trait Anxiety Inventory-Y, and the Fertility Problem Inventory.Results: Almost half of the sample (44.8%) declared a high motivation. At multiple logistic regression analysis only the “Need for parenthood” subscale of the FPI predicted a high motivation. We alternatively evaluated as possible predictor the construct “Representations about the importance of parenthood”  in place of the two separate subscales. At multiple logistic regression analysis, only this variable predicted a high motivation to undergo fertility preservation.Conclusion: The most important predictor of a high motivation to undergo fertility preservation is the individual desire for parenthood. This implies that, regardless of socio-demographic characteristics, any woman of childbearing age should receive an appropriate counseling about fertility preservation. Advances in cancer diagnosis and treatment have led to the increase of patients’ survival rates, consequently bringing more attention to the long-term side effects of cancer treatments and their impact on quality of life. Among these, gonadotoxicity is considered the principal cause of infertility in young women with cancer .More than 9.2 million women have been diagnosed with cancer in 2020 , with abCurrently, new procedures that allow fertility preservation before cancer treatments are available, such as oocyte or ovarian tissue cryopreservation. In order to better address fertility-related concerns, professional societies such as the American Society of Clinical Oncology have published international guidelines indicating that all young adults and children with cancer should receive timely fertility counseling before treatment to safeguard their reproductive potential .Oncofertility  thus rose as a new interdisciplinary field, which includes fertility preservation discussion and management, the management of sexual dysfunction, hormonal dysfunction, complex contraception, and psychosocial support . It invoDespite best practice recommendations, there are many barriers in providing oncofertility services . A systeHowever, several studies have underlined that receiving specialized counseling about cancer-related infertility and fertility preservation is associated with less regret and greater quality of life in cancer survivors . In factCurrently, the concept of a shared decision-making process, which allows for the provision of all necessary information to the patient in order to guarantee an optimal quality of life, is gaining support. Including patients’ psychological point of view allows them to consider future projects that may occur after the disease and thus endure long-term planning. Since patients are generally referred to oncofertility specialists on an emergency basis, they frequently have limited information, many fears and questions, and varying expectations about the therapeutic process. Consistently, the psychological management of this path is critical, as the psychologist can welcome and support patients before their oncofertility visit.Few studies in the available literature have investigated which variables could intervene in the decision-making process about undergoing fertility preservation techniques in cancer patients. Some of these studies have shown that this decision may be influenced by personal factors , illness factors  and fertility preservation-related factors  . HoweverThe aim of the present study is thus to investigate which sociodemographic and psychological variables predict a high motivation to undergo fertility preservation, first of all, evaluating the sociodemographic and psychological characteristics of female cancer patients that are referred to the Oncofertility Unit of the San Raffaele Hospital before gonadotoxic treatment. Considering that the San Raffaele Hospital is an Italian reference center for fertility preservation, we are not able to intercept female cancer patients who decline such possibility and all the patients who come to the oncofertility examination have usually already been informed by their oncologists about the effects of therapies on fertility and about fertility preservation opportunities. Therefore, they are usually already motivated to undergo fertility preservation techniques. As such, the hypothesis that the present study addressed is that women who decide to undergo fertility preservation may have specific adaptive psychological characteristics that allow them to cope with the disease in the short-term and to preserve the mental space for future planning and thus for possible parenthood.Female patients referred to the San Raffaele Hospital Oncofertility Unit after cancer diagnosis and before gonadotoxic treatment between March 2016 and June 2020 were invited to participate in the study. All patients received oncologic and fertility counseling from a group of specialists including a gynecologic oncologist, a reproductive gynecologist and a psychologist.Eligible women had to meet the following criteria: being at least 18 years old, having been recently diagnosed with cancer, speaking and understanding Italian, having at least an elementary school certificate, and agreeing to voluntarily participate in the research. Following these criteria, 58 women took part in the research.The study was carried out following the guidelines of the San Raffaele Hospital Ethics Committee (protocol N. 149/INT/2019), in accordance with the Declaration of Helsinki. A psychologist illustrated the research objectives during the psychological counseling which precedes the gynecologic examination and obtained written informed consent from every participant.A psychologist collected the sociodemographic characteristics  of the patients. Motivation to undergo fertility preservation was measured on an 10-point Likert scale . Participants were then asked to complete self-report questionnaires measuring psychological symptoms and infertility-related stress.Psychological scales were selected according to the few studies that investigated which factors influence the decision to preserve fertility and identified aspects related to the individual desire for a future pregnancy . MoreoveThe Beck Depression Inventory-II BDI-II– is a selThe State-Trait Anxiety Inventory-Y STAI-Y– is a widThe Fertility Problem Inventory FPI– is a selr = 0.55 and r = 0.64, respectively), between the two subscales “Need for Parenthood” and “Rejection of child-free lifestyle.” In particular, The Italian validation study of the questionnaire  showed tComparisons of the distribution of the psychometric scales with respect to values in the reference populations were performed with the Wilcoxon test. The comparison was made with values of the reference populations for the BDI-II  and the Multiple logistic regression analysis was used to evaluate the possible predictors of a high motivation  to undergo cryopreservation. The covariates considered in the analysis were: sociodemographic variables, state and trait anxiety, the level of depression and either the FPI subscales “Need for parenthood” and “Rejection of a childfree lifestyle” or the FPI construct “Representation about the importance of parenthood.” Each final model was obtained with a backward variable selection.The level of significance was set at 0.05. All statistical analyses were performed using IBM SPSS Statistics version 20 and R version 3.5.0.n = 48) and had no children . In addition, the majority of the sample had never had an abortion in the past  and 53.4% (n = 31) had at least a bachelor’s degree.Descriptive statistics for sociodemographic variables are reported in p = 0.018), with a distribution of the scores  that indicates mainly a minimum level of depression. Trait anxiety was lower than that of the general population (adj. p = 0.007), with a distribution  indicating low anxiety for most of the patients, and state anxiety was lower than that of the population of infertile women (p = 0.003), with a median value  indicating medium anxiety. There was no significant difference in state anxiety levels when compared to the general population (adj. p = 0.124). As regards the FPI, we observed a significant difference between our sample and the population of infertile women only for the subscale “Rejection of childfree lifestyle” (adj. p < 0.001): our sample reported significantly lower scores than that of the population of infertile women.n = 26) declared a high motivation. Multiple logistic regression analysis was performed in order to identify which factors predicted a high motivation to undergo fertility preservation techniques. In the analysis, all collected variables were considered except for the FPI subscales “Social concern,” “Sexual concern,” and “Relational concern.” For the FPI subscales, we only included the “Need for parenthood” and “Rejection of childfree lifestyle” subscales because they reflect the individual investment in parenthood. After backward variable selection, only the “Need for parenthood” subscale of the FPI was retained in the model; the higher the values of the subscale, the higher the probability to have a high motivation . In the literature a moderate correlation was observed between the “Need for parenthood” and the “Rejection of childfree lifestyle” subscales (p < 0.001), suggesting the possibility of a partial overlap between the meanings of the two subscales. Thus, we alternatively evaluated as possible predictor the construct “Representations about the importance of parenthood” .As expected from the design of the study, the patients in the sample declared mostly high levels of motivation to undergo fertility preservation treatments . Thus, in the study, a high motivation was defined as equal to 10. Almost half of the sample  are less likely to be referred to fertility preservation specialists . ConversThe present findings are particularly relevant also in a dyadic perspective, given the literature indicating that the risk of infertility due to cancer treatment can add a further challenge in the early stages of diagnosis and treatment, and can determine important reactions for patients such as depression, anxiety, distress, worry, and a negative impact on sexual well-being, quality of life, and couple relationship . Consideg.perego23@campus.unimib.it.The datasets presented in this article are not readily available because data cannot be shared as participants did not provide written informed consent for it. Requests should be directed to GP, The studies involving human participants were reviewed and approved by the Ethics Committee of the San Raffaele Hospital. The patients/participants provided their written informed consent to participate in this study.VEDM, GP, and PT contributed to the conception and design of the study. PT, PR, MM, and LN organized the database. PR and LN performed the statistical analysis. GP, PT, PR, and MM wrote the first draft of the manuscript. VEDM, GM, AB, RC, EP, and MC revised the manuscript critically. All authors read and approved the final version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Panicum miliaceum L.) is one of the earliest domesticated crops in the world. Weedy broomcorn millet [Panicum ruderale (Kitag.) Chang or Panicum miliaceum subsp. ruderale (Kitag.) Tzvel] is thought to be the descendant of the wild ancestor or the feral type of this cereal. The genealogical relationships and genetic divergence among these taxa have not been clarified. In this study, the genetic diversity and population structure of weedy and cultivated broomcorn millets were investigated by using the high-throughput sequencing technology, i.e., the specific-locus amplified fragment sequencing (SLAF-seq). Our analyses consistently revealed both the wild and the feral genotypes in the weedy broomcorn millets. The single nucleotide polymorphisms (SNPs) at the genomic level provided useful evidence to distinguish the wild and the endoferal/exoferal types of weedy broomcorn millets. The genetic divergence revealed between the cultivated broomcorn millet from eastern Eurasia and those from central-western Eurasia was probably derived from either the genetic introgression from weedy broomcorn millets along the spread routes or the founder effect, while the limited gene flow of broomcorn millets from eastern and central-western Eurasia was probably due to the different uses of broomcorn millets and eating habits of the local people.Broomcorn millet ( Panicum miliaceum L.) is an important crop broadly cultivated in the semiarid regions of Eurasia and America due to its short growing season and high tolerance of heat and drought. At present, the broomcorn millet has been used as either staple food, bird feed, or forage and is mainly produced in Russia, Ukraine, China, and India. As its companion weed, the weedy broomcorn millet [Panicum ruderale (Kitag.) Chang or Panicum miliaceum subsp. ruderale (Kitag.) Tzvel] is also commonly found in these areas. Both the weedy and cultivated broomcorn millets contain the same chromosome number (2n = 4x = 36) and can produce fertile offspring by crossing with each other. The morphological characteristics are much similar between weedy and cultivated broomcorn millets with main differences found in pericarp color, plant height, type of panicle, number of branches, seed color, and seed size. One of the most evident morphological variations is the seed color, which is dark yellow in weedy broomcorn millet and yellow or light yellow in cultivated broomcorn millet , which has been widely used as an efficient large-scale genotyping method to identify single nucleotide polymorphisms (SNPs) at low cost due to its reduced representation of deep sequencing without sacrificing the genotyping accuracy . To our –1 for high-throughput sequencing.A total of 106 accessions of (9 weedy and 97 cultivated) broomcorn millets were examined in the present study , providein silico based on the broomcorn millet (Panicum miliaceum L.) reference genome (854.79 Mb)1. Two restriction enzymes (RsaI and HaeIII)  were selected to digest purified genomic DNA. The genome of Oryza sativa2 was selected as the control genome to test the accuracy of the restriction enzyme digestion protocol. DNA fragments of 364–414 bp were selected as SLAFs followed by fragment end reparation, dual-index paired-end adapter ligation, PCR amplification, and target fragment selection for SLAF library construction. The paired-end sequencing was performed on the Illumina HiSeqTM 2,500 sequencing platform  at the Biomarker Technologies Corporation in Beijing, China.The genomic DNA of broomcorn millet was analyzed with specific-locus amplified fragment sequencing (SLAF-seq) . SLAF-se3. The high-quality paired-end reads obtained were mapped onto the reference genome  using the Burrows-Wheeler Aligner (BWA) 0.7.10 with default parameters  containing either adaptor/primer contamination or low-quality bases were filtered using Seqtkrameters . SAMtoolrameters  was usedrameters  was usedp-distance method  and expected heterozygosity (HE) for weedy and cultivated broomcorn millets were calculated using VCFtools software . To estimate the linkage disequilibrium (LD) in weedy and cultivated broomcorn millets, the coefficient of linkage disequilibrium (r2) of seven weedy accessions and seven cultivated accessions randomly selected among a total of 96 cultivated samples was calculated using PopLDdecay 3.40 (FST), across different groups (excluding the “mosaics”) were calculated using pixy  and nucleotide diversity ratios  of weedy and cultivated broomcorn millets 5 and the Swiss-Prot protein database6 using BLASTX program . The distribution of the SLAF-tags on 18 chromosomes of broomcorn millet was shown in K = 5, suggested based on the lowest error rate, was shown in K = 5 was listed in K = 3, 4, 6, and 7 showing lower error rates were provided in A total of 126,822 highly consistent SNPs were identified based on further criteria, i.e., minor allele frequency (MAF) ≥ 0.05 and missing rate ≤ 0.50, to examine the population structure of weedy and cultivated broomcorn millets. The population clustering using admixture proportions from 1 to 10 were performed by the ADMIXTURE program. The result of population clustering based on the optimal clustering number of K = 5 was shown on The geographic distribution of the 106 weedy and cultivated accessions and the results of ADMIXTURE analysis at The genealogical trees were constructed to further explore the genetic relationships among the 106 accessions of weedy and cultivated broomcorn millets attributed to Groups I to V . The genPrincipal component analysis (PCA) was performed on these 106 weedy and cultivated broomcorn millet accessions using EIGENSOFT software . The thrHO), expected heterozygosity (HE), and nucleotide diversity (π) for weedy and cultivated broomcorn millets were listed in HO, HE, and π of weedy broomcorn millets were all significantly higher than those of cultivated broomcorn millets (p < 0.05), indicating that the cultivated broomcorn millet may have experienced significant reduction in genetic diversity during domestication due to the bottleneck effect. The linkage disequilibrium (LD) was estimated and compared between weedy and cultivated broomcorn millets. The coefficient of linkage disequilibrium (r2) decayed to half of its maximum value within 60 and 100 kb in weedy and cultivated broomcorn millets, respectively (The genetic diversity parameters including observed heterozygosity (ectively .FST) across Group I, Groups II + III, and Groups IV + V (FST = 0.330) and between Group I and Groups IV + V (FST = 0.363) than that between Groups II + III and Groups IV + V (FST = 0.035), indicating a relatively closer relationship between cultivated broomcorn millets from eastern and central-western Eurasia than that between weedy and cultivated broomcorn millets.In order to investigate the genetic relationship and divergence between weedy and cultivated broomcorn millets and between the cultivated broomcorn millets from eastern and central-western Eurasia, we calculated the nucleotide diversity (π) and the pairwise fixation index  terms with 20 (47.62%), 11 (26.19%), and 11 (26.19%) revealed in three GO categories, i.e., biological processes, cellular component, and molecular function, respectively . Genes iThe 2,486 genes identified in the selective sweeps were further enriched in the metabolic pathways based on the Kyoto Encyclopedia of Genes and Genomes (KEGG) database . A totalrna17169 (in chromosome CM009694.2) annotated to the DAR1 gene controlling seed and organ size by restricting the period of cell proliferation in Arabidopsis thaliana (Oryza sativa (rna22663 (in CM009695.2) annotated to BLH9 (encoding BEL1-like homeodomain), affecting the development of apical meristem, stem, embryo seat, and inflorescence development in Arabidopsis thaliana  annotated to AP2/ERF gene family, which were involved in diverse types of biological functions, including the increases of seed weight, total seed protein, and total seed oil contents as well as the floral organ development and flowering time in Arabidopsis thaliana  thaliana  and Oryza sativa ; (2) rnathaliana  and Zea Zea mays , and (3)thaliana , diseasethaliana  and respthaliana .HO, HE, and π) of the cultivated broomcorn millets were significantly lower than those of the weedy broomcorn millets across the entire genome (p < 0.05)  . Similar < 0.05) . The dec < 0.05) . TherefoFST across Groups I, II + III, and IV + V identified by ADMIXTURE analysis have consistently shown that there was evident genetic distinction between weedy and cultivated broomcorn millet populations, probably due to domestication bottleneck and artificial selection (indica and 167 kb for japonica)  , soybeanaponica) , and foxaponica) , which aaponica)  and applaponica) . Furtheraponica) .DAR1, BLH9, and AP2/ERF gene family) based on Swiss-Prot and GO annotations and multiple metabolic pathways based on KEGG enrichment analysis. These results indicate that there was reduction in genetic diversity caused by either artificial selection or natural selection/local adaptation in the domestication process of broomcorn millet. Future investigations are necessary to explore the possibility that some of these 2,486 candidate genes may be related to domestication of broomcorn millet. Further studies are needed to investigate and ultimately identify the genes related to domestication in the regions with reduction in genetic diversity of the broomcorn millet genome. To date, studies have identified genes involved in domestication in cereal crops. For example, DAR1 was reported to control seed and organ size by regulating the period of cell proliferation in Arabidopsis thaliana (Oryza sativa (rna17169) identified in the selective sweeps was annotated to the gene of DAR1, suggesting that this gene may be involved in the seed enlargement during domestication of broomcorn millet. Another gene (rna22663) in the selective sweeps was annotated to BLH9 (encoding BEL1-like homeodomain), which was reported to affect the development of apical meristem, stem, and embryo seat in Arabidopsis thaliana. Specifically, the inflorescence of a mutant of this gene in Arabidopsis thaliana showed abnormal internodes, small, deformed, and clustered pods  were also found to be involved in the regulation of inflorescence development in Zea mays  were annotated to the gene family of AP2/ERF, which were associated with a diverse types of biological functions, including the increases of seed weight, total seed protein, and total seed oil contents as well as the floral organ development and flowering time in Arabidopsis thaliana  in Loess Plateau . TherefoK = 5  and became an important food source in the Yellow River Basin. Subsequently, the broomcorn millet spread westward to Hexi Corridor, Xinjiang, and Central Asia at around 5000–4000 BP , 2020 anOur results in the present study provided novel evidence to support the westward spread route, which is an essential path for cultivated broomcorn millets to spread from northern China. As shown in With our above proposal that the cultivated broomcorn millets of Groups IV and V were probably derived from Northeast China, the wide distributions of accessions of cultivated broomcorn millet in Group IV in the vast area of Eurasian steppe from Mongolian Plateau, Kazakhskiy Melkosopochnik, and West Siberian plain, to the Eastern European Plain and the north of Caspian Sea and Black sea suggest that the cultivated broomcorn millets of Group IV might have spread westward along this “Steppe Route.” Furthermore, the cultivated broomcorn millets of Group V showed vast distribution to the south of distribution areas of Group IV, including the areas from Tianshan region to the north of Tarim Basin in Xinjiang, the southern Central Asia, the Western Asia, and the south region of the Caspian Sea and the Black Sea in Europe. The genealogical analysis showed that cultivated broomcorn millets of Group V were closely related to some individuals of Group IV , suggestjaponica rice varieties from 20 countries revealed that the varieties within similar latitudes showed close genetic relationship, while the rice varieties with large difference in latitudes showed increased genetic differentiation . In this first comparative study focusing on the genome-wide variations between weedy and cultivated broomcorn millets, our results showed that both the wild and the feral types were revealed in the weedy broomcorn millets explored in this study. This study has demonstrated that the SNPs at the genomic level provided useful evidence to distinguish the wild and the endoferal/exoferal types of weedy broomcorn millets. We further suggested that the genetic divergence revealed between the cultivated broomcorn millet from eastern Eurasia and those from central-western Eurasia was probably derived from either the genetic introgression from weedy broomcorn millets along the spread routes or the founder effect. The significantly higher genetic variation in weedy broomcorn millet revealed in this study suggests that the wild genetic resources should be preserved as important resources for improvement of crop characteristics in the future crop breeding.SAMN18616674 to SAMN18616779.The original contributions presented in the study are publicly available. This data can be found here: NCBI with accession numbers of CL and ML: conceptualization and investigation. XZ, MH, and TL: methodology. XZ and MH: software. YX, FS, CL, and ML: validation and formal analysis. ML and PL: resources. CL and YX: data curation. CL, YX, FS, ML, XZ, MH, TL, and PL: writing—original draft preparation. CL, YX, and FS: writing—review and editing. XZ: visualization. YX: supervision, project administration, and funding acquisition. All authors have read and agreed to the published version of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The follicle is the functional unit of the ovary, which is composed of three types of cells: oocytes, granulosa cells, and theca cells. Ovarian follicle development and the subsequent ovulation process are coordinated by highly complex interplay between endocrine, paracrine, and autocrine signals, which coordinate steroidogenesis and gametogenesis. Follicle development is regulated mainly by three organs, the hypothalamus, anterior pituitary, and gonad, which make up the hypothalamic-pituitary-gonadal axis. Steroid hormones and their receptors play pivotal roles in follicle development and participate in a series of classical signaling pathways. In this review, we summarize and compare the role of classical signaling pathways, such as the WNT, insulin, Notch, and Hedgehog pathways, in ovarian follicle development and the underlying regulatory mechanism. We have also found that these four signaling pathways all interact with FOXO3, a transcription factor that is widely known to be under control of the PI3K/AKT signaling pathway and has been implicated as a major signaling pathway in the regulation of dormancy and initial follicular activation in the ovary. Although some of these interactions with FOXO3 have not been verified in ovarian follicle cells, there is a high possibility that FOXO3 plays a core role in follicular development and is regulated by classical signaling pathways. In this review, we present these signaling pathways from a comprehensive perspective to obtain a better understanding of the follicular development process. The ovary is an important organ in the reproductive system of female mammals. The main functions of the ovary are to produce and periodically release oocytes and secrete steroid hormones. Ovarian follicles are composed of different numbers and types of cells, which provide an appropriate environment for oocyte development . The menIn the fourth month of embryonic development, the ovary contains approximately 6–7 million eggs coated with monolayer granulocytes, an area also known as the primitive follicular area . In the Some diseases, such as polycystic ovary syndrome (PCOS) and premature ovarian failure (POF), can also cause ovarian dysfunction. With the abnormal hormonal support associated with ovarian dysfunction, a number of physiological systems, including cardiovascular health and bone density, are affected . The molThe phosphatidylinositol 3-kinases (PI3K) are a family of enzymes that share the function to phosphorylate the 3-hydroxyl group of phosphoinositides , it is aThe forkhead box O (FOXO) transcription factors are negatively regulated by PI3K/AKT signaling pathway . In mamm–/– knockout mice appeared more mature than that of WT littermates.Of all the FOXO family proteins, we are most interested in FOXO3  protein, which was originally known to regulate insulin signaling , yet an The body level of 2,5-Hexanedione  was high in some smokers, drinkers, and those workers who use n-hexane, which will lead to the damage of female reproductive system like early menopause, menstrual disorders, and reduced fertility . In the Previous studies showed that the FOXO3a was closely associated with human longevity. SIRT6, which is a deacetylase, was also shown to be related to human longevity. It was reported that FOXO3a regulated the transcription of SIRT6 by binding and activating nuclear respiratory factor 1 (NRF1) in the mouse. SIRT1, FOXO3a and NRF1 form a complex on the SIRT6 promoter and positively regulates the expression of SIRT6 , which r2+-related mechanisms  on key functions in different pathways . In respIn a recent study, researchers found that the expression level of LNK, a regulator of insulin signaling pathway, was elevated in ovaries of insulin resistant PCOS patients compared with the non-PCOS group, and the overexpression of LNK in ovarian granulosa cell line inhibited insulin induced AKT activation . StudiesNotch signaling is an evolutionarily conserved pathway in a number of cellular processes, including cell proliferation, cell differentiation, migration, adhesion, and apoptosis . Notch sThe vascular system provides nutrition, oxygen and hormone support for ovarian follicles and the CL ; as a reNotch proteins and ligands have been detected in the rodent ovary, and studies have shown that Notch receptors and ligands are expressed in a subset of ovarian vessels. For example, Notch 1 was found to be expressed in the endothelium of the theca layer in the follicular-phase ovary, in endothelial cells from the neovasculature of the corpora lutea, and in mature vessels of the theca layer in the luteal phase . Notch2,in vivo in the primate ovary led to increased luteal angiogenesis and microvascular density , FOXO3 regulates the expression of NOTCH1 and NOTCH3 receptors. Conditional deletion of FOXO3 downregulated Notch signaling, leading to impairment of the self-renewal ability of SCs, while overexpression of the Notch intracellular domain (NICD) could rescue this self-renewal deficit . NF-κB aHedgehog (Hh) signaling is an evolutionarily conserved signaling pathway that regulates embryonic development as well as many essential tissue and cellular properties, such as cell proliferation, differentiation, and survival. Disruption of the Hh pathway results in serious disease . The Hh In the absence of ligand binding, the membrane receptor PTCH1 keeps SMO in an inactive state. Once a ligand binds PTCH1, its inhibition of SMO is unlocked, and signal transduction occurs through the downstream transcription factors GLI1, GLI2, and GLI3 .Ptch1 and Gli1, were found to be expressed in surrounding theca cells, and this expression was inhibited by treatment with the Hh signaling antagonist cyclopamine. The dramatic loss of Hh and induced target gene expression was found to be synchronized in periovulatory follicles, demonstrating the role of Hh signaling in communication between granulosa cells and developing theca cells . FSCs are exquisitely responsive to diet-induced signals such as Hh and insulin signaling. Constitutive Hh signaling drives FSC loss and premature sterility by inducing autophagy in FSCs through a Ptc-dependent, Smo-independent mechanism. During the senescence process, Hh-dependent autophagy increases, which triggers FSC loss and leads to reproductive stagnation .The late endosomal LAMTOR complex is regarded as a convergence point for the RAF/MEK/ERK and PI3K/AKT/mTOR signaling pathways . A recenOvarian follicle development and the subsequent ovulation process are coordinated by highly complex interplay between signaling pathways. In this review, we summarize several classical signaling pathways  and their functions in follicular development. The ovary is composed of three types of cells: oocytes, granulosa cells, and theca cells. Granulosa cells play an essential role in follicular development; they synthesize and secrete mucopolysaccharides, forming a zona pellucida around the oocyte. The cell membrane protuberances of granulosa cells can pass through the zona pellucida, forming a gap connection with the cell membrane of the oocyte. The point of contact between these cell membranes provides a channel for the transmission of information and nutrition to the oocyte.During the preantral follicle phase, follicle-stimulating hormone (FSH), estrogen and androgen receptors appear in granulosa cells, making granulosa cells capable of responding to the corresponding hormones. Five signaling pathways are important for maintaining the function of granulosa cells. WNT2, WNT4, NOTCH2, NOTCH3, Iagged2, IHH, and DHH are expressed in developing granulosa cells . The actWnt2 is increased in cultured granulosa cells (These signaling pathways can work synergistically with hormones. For example, the mRNA level of sa cells ; furthersa cells . In addisa cells . The vassa cells . FinallyIn conclusion, the classical signaling pathway reviewed in this article consists of multiple complex layers, and what we know about the role of FOXO3 and its related signaling pathways in the regulation of follicular development continues to grow. However, many mechanisms remain to be determined to gain a better understanding of these signaling molecules in folliculogenesis.LL, XS, and YS co-wrote the manuscript. LL drew the picture. ZW co-wrote the manuscript and designed the structure of the manuscript. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Endometrial cancer (EC) is currently the most common malignancy of the female genital tract in developed countries. Although it is more common in postmenopausal women, it may affect up to 25% in the premenopausal age and 3–5% under the age of 40 years. Furthermore, in the last decades a significant shift to pregnancy at older maternal ages, particularly in resource-rich countries, has been observed. Therefore, in this scenario fertility-sparing alternatives should be discussed with patients affected by EC. This study summarizes available literature on fertility-sparing management of patients affected by EC, focusing on the oncologic and reproductive outcomes. A systematic computerized search of the literature was performed in two electronic databases (PubMed and MEDLINE) in order to identify relevant articles to be included for the purpose of this systematic review. On the basis of available evidence, fertility-sparing alternatives are oral progestins alone or in combination with other drugs, levonorgestrel intrauterine system and hysteroscopic resection in association with progestin therapies. These strategies seem feasible and safe for young patients with G1 endometrioid EC limited to the endometrium. However, there is a lack of high-quality evidence on the efficacy and safety of fertility-sparing treatments and future well-designed studies are required. Endometrial cancer (EC) is currently the most common malignancy of the female genital tract in developed countries; in Europe, EC has shown a 5-year prevalence of 34.7%  . In 2018Hysterectomy with bilateral salpingo-oophorectomy plus nodal evaluation and with or without peritoneal staging represents the standard therapy of EC ,5,6. AltThus, the standard surgical treatment may not be suitable for patients wishing to maintain their fertility. Therefore, fertility-sparing alternatives should be thoroughly explained to EC women, discussing the oncologic outcomes related to each approach. Fertility-sparing treatments can be proposed to patients with endometrioid intra-epithelial neoplasia (EIN) or grade 1 EC without myometrial invasion . DiffereThe aim of this review is to summarize available evidence on fertility-sparing options for patients affected by EC, focusing on the oncologic and reproductive outcomes.A systematic review of the available evidence, from 1950 until December 2021 (last research 1st May), was conducted consulting two electronic databases (PubMed and MEDLINE) to select relevant articles. All relevant papers were evaluated, and references were evaluated to identify other articles for potential inclusion in the current review. All articles were assessed by two independent reviewers (U.L.R.M. and G.B.) and in the case of discrepancy a third author (F.R.) was asked to participate for consensus. Firstly, eligibility was evaluated considering the titles and abstracts. Full manuscripts were obtained for all selected studies and decision for final inclusion was made after detailed examination of the papers. Randomized controlled trials (RCTs), prospective studies, case–control studies, and retrospective cohort studies were considered for inclusion in the review.Two independent authors  ran a specific literature search. The search included the term endometrial cancer in combination with other keywords and medical subjects heading terms such as fertility preservation, fertility-sparing, hysteroscopic resection, levonorgestrel, oral progestin. Given the aim of this narrative review, which focuses on different fertility-sparing methods for the treatment of EC, we arbitrarily decided not to use a systematic approach in reporting results; thus, we reported more relevant studies for each kind of treatment to provide the reader a complete and concise overview of the available evidence on the fertility-sparing management for EC.Several types of treatment have been described for the fertility-sparing management of EC; however, no consensus is established on which agent, dose, or duration of treatment is more efficacious. Progestins, metformin, and hysteroscopic resection are the most common investigated modalities to conservatively treat EC .p = 0.001) [Traditionally, the most frequently prescribed drugs for conservative EC treatment are medroxyprogesterone acetate (MPA) or megestrol acetate (MA) and several studies have demonstrated their efficacy and safety ,39,40,41= 0.001) .An alternative to oral progestins is represented by levonorgestrel intrauterine system (LNG-IUS). This device acts through the local release of the second-generation progestin levonorgestrel, thus combining two main advantages: the reduction of systemic adverse effects and the increase of local effectiveness causing endometrial decidualization and atrophy . DiffereMetformin is an insulin sensitizer since it enhances signaling through the insulin receptor, leading to an improvement in insulin resistance, followed by a decrease in circulating insulin levels. Furthermore, evidence indicates that metformin’s key target of action is the inhibition of hepatic gluconeogenesis, causing a secondary decline in insulin levels. Interestingly, Cantrell et al. demonstrated that metformin is a potent inhibitor of cell proliferation in EC cell lines through AMPK activation and subsequent inhibition of the mTOR pathway, paving the way for its potential use for EC prevention and treatment . Subsequn = 120) or G1 EC  were retrospectively enrolled in this study. All women were administered continuous oral progestin associated with hysteroscopic biopsy every 3 months until CR. Overall, 148 women (97.4%) had CR while 3 AEH and 1 EEC patients had a disease progression. The mean time to CR was 6.7 ± 0.3 months . Among 60 women who tried to obtain a pregnancy after achieving CR, 45.0% (27/60) succeeded, 25.0% (15/60) had a live birth, 13.3% (8/60) were still in pregnancy, while 6.7% had a spontaneous miscarriage [An alternative EC fertility-sparing treatment is represented by the combination of hysteroscopic resection followed by progestin therapies. In 2010, a prospective study described for the first time a new technique to maintain fertility in 6 patients with early-stage IA EC with use of hysteroscopic resection combined with 160 mg of MA. This method consisted in a three-step procedure with a pathologic evaluation at every step: the resection of the disease (step 1), the resection of the endometrium next to the disease (step 2), and the resection of the myometrium underlying the disease (step 3). This fertility-sparing surgical technique demonstrated efficacious since both transvaginal ultrasound assessment and diagnostic hysteroscopy at 3, 6, 9, and 12 months after first surgical evaluation were negative for atypia or malignancy. Furthermore, 4/6 women (66%) had live births . In 2011R, 45.0% /60 succecarriage .In 2017, two systematic reviews and meta-analyses on the outcomes of various fertility-sparing treatments for EC were published ,22. Fan Over the last years, there has been a significant shift to pregnancy at older maternal ages, particularly in resource-rich countries . In the The association between epidemiological risk factors and the progression to EC can be justified by the unopposed estrogen hypothesis . In thisPatients wishing to preserve fertility should be referred to tertiary centers. Transvaginal ultrasound performed by an expert sonographer can be used as alternative to MRI . SelectiDespite the effectiveness of different fertility-sparing strategies for EC, some patients do not respond to treatment or they may recur after an initial regression of the disease, demonstrating a risk of progression to invasive cancer. For this reason, great efforts have been spent to identify predictive factors of response to conservative treatment including trials on clinical, pathological, and immunohistochemical characteristics ,75,76, pThe assessment of the response is fundamental, but no universally shared standard protocol has been currently developed. Different follow-up timepoints have been described, the most common being 3 months . EndometFinally, treatment failure and patient recurrence should be considered. In the first case, ESGO/ESTRO/ESP guidelines state that if no response is achieved after 6 months of fertility-sparing therapy, standard surgical treatment is recommended . SimilarOn the basis of available evidence, fertility-sparing strategies seem feasible and safe for young patients with G1 endometrioid EC limited to the endometrium. However, there is a lack of high-quality evidence on the efficacy and safety of fertility-sparing treatments and future well-designed studies are needed to offer stronger evidence on this issue. Furthermore, it is of primary importance that future studies on this topic should also include the molecular classification of endometrial cancer in order to enable early stratification and risk assignment to direct care.Selected and strongly motivated women should be carefully counseled about the non-standard nature of fertility-sparing strategies and only once they have fully understood the potential risks of this management should they start conservative treatment.


Bull fertility is an important economic trait in sustainable cattle production, as infertile or subfertile bulls give rise to large economic losses. Current methods to assess bull fertility are tedious and not totally accurate. The massive collection of functional data analyses, including genomics, proteomics, metabolomics, transcriptomics, and epigenomics, helps researchers generate extensive knowledge to better understand the unraveling physiological mechanisms underlying subpar male fertility. This review focuses on the sperm phenomes of the functional genome and epigenome that are associated with bull fertility. Findings from multiple sources were integrated to generate new knowledge that is transferable to applied andrology. Diverse methods encompassing analyses of molecular and cellular dynamics in the fertility-associated molecules and conventional sperm parameters can be considered an effective approach to determine bull fertility for efficient and sustainable cattle production. In addition to gene expression information, we also provide methodological information, which is important for the rigor and reliability of the studies. Fertility is a complex trait influenced by several factors and has low heritability, although heritability of scrotal circumference is high and that it is a known fertility maker. There is a need for new knowledge on the expression levels and functions of sperm RNA, proteins, and metabolites. The new knowledge can shed light on additional fertility markers that can be used in combination with scrotal circumference to predict the fertility of breeding bulls. This review provides a comprehensive review of sperm functional characteristics or phenotypes associated with bull fertility. Projections indicate that the world population will rise to 9 billion people by 2,050, requiring a 50% increase in food production  to satisBulls are evaluated based on a breeding soundness exam (BSE) that is composed of an inspection of semen characteristics (phenotypes) combined with phenotypic features. Despite great efforts put into evaluating bulls using BSE, bull fertility is deemed suboptimal under field conditions, with a conception rate varying from 20 to 40% . Such diCkb, Kat8, Igf1r, and Tdrd9, which are associated with sperm physiology, such as sperm motility and sperm–egg interaction. Feugang et al.  have been effective in applying dense genetic markers, such as single-nucleotide polymorphism (SNP) markers, to determine genomic regions associated with economically important phenotype such as fertility . There ag et al.  reportedg et al.  showed tertility . BecauseEpigenetics refers to molecular processes that may regulate gene expression (active vs. inactive genes) without alterations in the DNA sequence. Epigenetic modifications, including DNA methylation, histone modifications, and nucleosome positioning, can be transmitted to the daughter cells through cell divisions. Aberrant alterations in the epigenetic profiles may give rise to abnormal gene silencing or activation . TransfoSperm contains diverse proteins present in the sperm membrane, flagellum, cytoplasm, acrosome, and nucleus that play key roles in sperm physiology . Of thesUsing Western blotting and real-time PCR, Velho et al.  postulatCurrently, proteomic approaches are widely used to explore male reproductive physiology . Aslam eThe MDH2 catalyzes the reversible oxidation of malate to oxaloacetate using NAD+/NADH as a cofactor in the citric acid cycle . Aslam eUsing 2D-DIGE analysis of bull sperm, it was shown that ALB, TIMP, spermadhesin-1, and binder of sperm proteins (BSP)-1, 3, and 5, PEBP1, and AKI in sperm and seminal plasma were more abundant in sperm from high fertility bulls, while PSMA6, ELSPbP1, CCT5, CCT8, and CLU were in greater amounts in seminal plasma and in sperm from low-fertility bulls. The expression levels of ZFP34, HSP90, BCL62, IFNRF4, NADHD, histone H1, and TUBB3 were higher in high-fertility bull sperm  Table 2Table 2. Spermadhesin family members interact with carbohydrates, phospholipids, and zona pellucida glycoproteins and participate in sperm–egg binding –55. SperAlbumin has been reported to facilitate cholesterol outflow from sperm membranes and mediates sperm capacitation in the female reproductive tract , 60. MorCholesterol and phospholipids contribute to the regulation of sperm membrane bilayer stability and fluidity. The BSP proteins promote efflux of phospholipids and cholesterol from sperm membranes, thereby, disrupting sperm membrane architecture, resulting in capacitation . MoreoveProteasome subunit alpha type-6 (PSMA6) belongs to proteasome multicatalytic protease degrading polyubiquitinated proteins into small peptides and amino acids . ProteasUsing mass spectrometry coupled with nano HPLC, a total of 1,159 proteins were detected in the bull seminal plasma, of which 29 were abundant in low-fertility bulls, whereas 50 were abundant in high-fertility bulls . While TGalectins  belong to members of ß-galactoside-binding lectins, which can be localized in extracellular spaces and in cellular components such as cell membrane, cytoplasm, and nucleus . They arGomes et al.  examinedSperm delivers, not just the paternal DNA, but other factors, such as cell signaling molecules, RNA, and transcription factors, into the oocyte at the time of fertilization . New cutEfna1, Rbmx, Mlph, Rpl30, and Aqp2, which participate in “extracellular exosome” GO term. Among them, the ephrin A1 (Efna1) protein that is localized on cell surfaces participates in membrane integrity and sperm morphology, and it has been reported that Efna1 is highly present in both seminal plasma and sperm (Rbmx) has been proposed to be a possible splicing factor that modulates spermatogenesis (Aqp2) is expressed in male germ cells, seminiferous epithelium, Leydig cells, and in the male reproductive tract  in bovine sperm were associated with reproductive system functions. They also found that five mRNA transcripts  are related to the immune defense response.Moreover, Wang et al.  also ided et al.  detectedty bulls . Légaré ty bulls  showed tmiR-10a, miR-10b, miR-34c, miR-100, miR-103, miR-196b, miR-365-2, and miR- 2478 consistently exist in bovine spermatozoa , palmitic acid, carbamate, and lactic acid. In addition, four metabolic pathways were found to be associated with differential metabolites, namely, glycolysis or gluconeogenesis, aspartate and glutamate metabolism, pyruvate metabolism, alanine, and β-alanine metabolism. GABA plays an essential role in sperm physiology by inducing the acrosome reaction and sperm hyperactivation. Furthermore, benzoic acid participates in GABA regulation and is highly expressed in high-fertility bull sperm. Because of its participation in lipid metabolism to generate energy, palmitic acid production may be more abundant in high-fertility bull sperm. Higher levels of lactic acid in high-fertility bull sperm might be because anaerobic glycolysis is more efficiently utilized in high-fertility sperm compared with those in low-fertility sperm. Functions of carbamate are considered as potential regulators of intracellular pH in sperm (There were 22 distinct metabolites detectable in bull sperm employing gas chromatography-mass spectrometry (GC-MS) analysis  where main sperm .Velho et al.  studied Citric acid influences the acrosome reaction, sperm transport, and fertilization by being an energy source and regulating semen pH as a chelator for calcium, magnesium, and zinc , 134, suDNA methylation has been the most studied epigenetic mechanism in sperm and is presumed to fulfill a major role in the non-genetic information transfer across generations. Sperm DNA methylation participates in many physiological processes, such as silencing of transposable elements , paternade novo methyltransferases DNMT3A, DNMT3B, and their cofactor DNMT3L. Significant levels of DNA methylation are present at birth and must be sustained by DNMT1 during adulthood across different phases of spermatogenesis  data (486 × coverage) from neighboring CpG sites among 28 distinct bull sperm samples, Liu et al.  identifiz et al.  showed tz et al.  implicatDgat2 and Ldhb) predominantly engaged in lipid storage and metabolism , KIF2A, and KIF2B are involved in spindle bipolarity through induction of depolymerization of microtubules to modulate mitotic dynamics during spermatogenesis  with different fertility by using a custom ChIP on-chip array. For H3K27me3- and H3K4me2-enriched genes, they detected 80 and 84 genes, respectively. Among the H3K4me2-enriched genes, Cct5, Cdc45, Dmc1, Meg3, Mlh1, Prdm14, Pax3, Sox4, Sox14, and Tbx15 have crucial roles in spermatogenesis and embryogenesis. While Cct5, Cdc45, Dmc1, Mlh1, Prdm14, Pax3, Sox4, Sox14, and Tbx15 genes were in greater amounts in sperm from high fertility bulls, Meg3 was enriched in sperm from subfertile bulls.Oliveira et al.  examinedy et al.  determiny et al. , 166. Iny et al. . Verma ey et al.  examinedPax3, Sox4, and Sox14 are genes encoding for transcription factors that participate in supporting embryonic development  gene acts as a long non-coding RNA; therefore, it does not encode a protein. Researchers claimed that Meg3 is involved in p53-mediated transactivation and its suppression of cell proliferation  have enabled high-throughput screening of a wide range of molecular and cellular dynamics in fertility molecules. These approaches also provide means of detecting minute amounts of changes in molecules due to their higher sensitivity. Such attributes of the advanced methods are vitally important for innovative studies to produce new knowledge with transformational and translational values. However, an ejaculate contains many spermatozoa with different phenotypes. Therefore, each spermatozoon should be examined using new high technologies of single-cell analyses such as single-cell metabolomics, proteomics, transcriptomics, and genomics. In addition, these methods should be combined with conventional techniques, such as sperm chromatin structure assay, computer-assisted sperm analyses (CASA), integrity of membranes, flow cytometry, and reactive oxidation stress levels, to determine semen quality using system biology approaches. As an economically important trait, fertility has become more important as there is an urgent need for more efficient, sustainable, and profitable production for food animals to feed the ever-increasing human population in the world. The bull is a unique model for the study of male fertility because of the availability of large amounts of sperm from bulls with reliable fertility phenotypes, and the significant similarities between the bull and other mammals in both sperm biology and genetics.The major advancements in the All authors assisted in the conception of the study, contributed to manuscript revision, read, and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


This observation fueled the widespread hypotheses that these properties extend to living neurons and that reduced stability of microtubules represents a major disease-driving event induced by pathological forms of tau in Alzheimer’s disease and other tauopathies. Accordingly, most research efforts to date have addressed this protein as a substrate, focusing on evaluating how specific mutations, phosphorylation, and other post-translational modifications impact its microtubule-binding and stabilizing properties. In contrast, fewer efforts were made to illuminate potential mechanisms linking physiological and disease-related forms of tau to the normal and pathological regulation of kinases and phosphatases. Here, we discuss published work indicating that, through interactions with various kinases and phosphatases, tau may normally act as a scaffolding protein to regulate phosphorylation-based signaling pathways. Expanding on this concept, we also review experimental evidence linking disease-related tau species to the misregulation of these pathways. Collectively, the available evidence supports the participation of tau in multiple cellular processes sustaining neuronal and glial function through various mechanisms involving the scaffolding and regulation of selected kinases and phosphatases at discrete subcellular compartments. The notion that the repertoire of tau functions includes a role as a signaling hub should widen our interpretation of experimental results and increase our understanding of tau biology in normal and disease conditions.Over four decades ago,  Such dynamics likely impact tau’s availability to interact with kinases and phosphatases on and off the MT surface . Conversely, neuronal survival and homeostasis depend on retrograde FAT (rFAT) of specialized MBOs bearing neurotrophins and degraded materials from peripheral compartments towards the neuronal soma. In the mature brain, aFAT and rFAT are mainly driven by the multi-subunit motor proteins kinesin-1  and cytoplasmic dynein, respectively. Both directions of FAT are regulated, in part, by signaling pathways controlling phosphorylation of these microtubule-based motors  along axons, a cellular process referred to as fast axonal transport (FAT), is critical for appropriate maintenance of neuronal connectivity and survival Black, . The corex vivo preparation uniquely suited for the study of FAT mechanisms , causing aberrant exposure of the N-terminus . These antibodies selectively recognize an epitope within PAD that is exposed in a conformation-dependent manner  or long-term depression (LTD), respectively . Furthermore, PP1 activity is required for the successful execution of pre-mRNA splicing  mRNA translation at the axon-glia interface  to convert it into phosphatidylinositol -triphosphate (PIP3). PIP3 recruits AKT to the cell membrane where it is activated and phosphorylates numerous kinase substrates  that in turn regulate a multitude of cellular processes. Phosphatase and tensin homolog (PTEN) dephosphorylates PIP3, providing a mechanism to terminate insulin-dependent signaling.Signaling pathways activated by insulin play an important role in the maintenance of neuronal function and peripheral metabolism  to regulate cell differentiation, survival, and death (Keshet and Seger, Various reports documented modulatory effects of tau on neurotrophin-based activation of MAPK signaling . For exaThe long-held hypothesis that stabilization of MTs represents tau’s primary biological function is being increasingly challenged by a growing body of independent experimental evidence. Continuing to challenge this dogma will help expand our interpretation of such evidence and advance our understanding of tau biology in health and disease conditions. For example, the ability of tau to interact with MTs may reflect just one of several features enabling a much more diverse set of functions by this protein, such as the regulation of phosphorylation-based signaling pathways in axons and other MT-rich subcellular compartments. On the other hand, MT-independent interactions with additional proteins may facilitate similar functions in modulating pathways in other compartments featuring few or no MTs. This notion appears consistent with tau’s widespread subcellular distribution, its highly dynamic conformational flexibility, and the numerous kinase and phosphatase binding partners identified thus far. Based on these characteristics, we posit that tau functions as a signaling hub, regulating a wide set of cellular processes through the modulation of selected kinases and phosphatases. This function in turn appears to be impacted by specific modification events in tau, including mutations, phosphorylation, truncation, and aggregation, which could have important implications for physiological and disease states. A more dynamic perspective of tau as a multifaceted protein involved in the control of intracellular signaling may also provide a novel framework for the development of therapeutic approaches to treat tauopathies.RM, BC, MA, SB, GM, and NK each contributed to writing and editing this review. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The Ang–Tie signaling pathway is an important vascular signaling pathway regulating vascular growth and stability. Dysregulation in the pathway is associated with vascular dysfunction and numerous diseases that involve abnormal vascular permeability and endothelial cell inflammation. The understanding of the molecular mechanisms of the Ang–Tie pathway has been limited due to the complex reaction network formed by the ligands, receptors, and molecular regulatory mechanisms. In this study, we developed a mechanistic computational model of the Ang–Tie signaling pathway validated against experimental data. The model captures and reproduces the experimentally observed junctional localization and downstream signaling of the Ang–Tie signaling axis, as well as the time-dependent role of receptor Tie1. The model predicts that Tie1 modulates Tie2’s response to the context-dependent agonist Ang2 by junctional interactions. Furthermore, modulation of Tie1’s junctional localization, inhibition of Tie2 extracellular domain cleavage, and inhibition of VE-PTP are identified as potential molecular strategies for potentiating Ang2’s agonistic activity and rescuing Tie2 signaling in inflammatory endothelial cells. Disrupted Tie2 signaling has been linked to many diseases including cancer5, peripheral arterial disease6, ocular diseases, including diabetic retinopathy and diabetic macular edema9, systemic inflammation11, and infectious diseases including COVID-1913.The Angiopoietin (Ang)-Tie signaling pathway is a major endothelial signaling pathway regulating vascular quiescence, permeability, stability, and growth15. Ang2 is primarily considered to be an antagonist of Tie2 and is mostly associated with vascular leakage and inflammation, although it also acts as a context-dependent Tie2 agonist21. Ang1 exists in highly oligomerized forms, whereas Ang2 exists primarily as lower-order multimers , although higher-order oligomeric forms are observed22. Multimeric ligands Ang1 and Ang2 are known to induce the multimerization and subsequent activation of Tie2, and the activation of Tie2 requires tetrameric or higher orders of multimerization22. Ang3 and Ang4 are mouse and human orthologs, respectively, with poorly defined biological functions23.The Ang–Tie signaling pathway consists of multiple ligands, receptors, and molecular regulators, and mechanisms that form a complex reaction network. The Ang family of ligands consists of Ang1, Ang2, Ang3, and Ang4. Ang1 is the natural agonist of Tie2, and it is associated with vascular growth and stability26. A recent study has identified a secreted protein from hepatocytes, leukocyte cell-derived chemotaxin 2, as a ligand of Tie1 in liver fibrogenesis27. Heparan sulfate glycosaminoglycans have also been shown to bind Tie1 and regulate Ang–Tie signaling28. Tie1’s exact mechanisms of action and its effects on Tie2 signaling remain incompletely understood. It has been demonstrated to be able to both inhibit and enhance Tie2 activation33. Tie2 activation is also importantly regulated by vascular endothelial protein tyrosine phosphatase (VE-PTP), which dephosphorylates Tie2 to inhibit its vascular stabilizing effects34. The Ang–Tie pathway is also regulated by the extracellular domain cleavage of both receptors. The extracellular domains of both Tie1 and Tie2 are known to be cleaved near the plasma membrane both constitutively and in response to cytokine stimulation, resulting in soluble forms of the receptors (sTie1 and sTie2)37. The endodomains of cleaved Tie1 and Tie2 undergo further proteolytic processing and are subsequently degraded36. sTie2 can act as a ligand trap, further inhibiting the signaling of Tie2, although the physiological role of this process is unclear.The Tie receptor family consists of Tie2, which directly binds the angiopoietins, and Tie1, which does not bind to any of the angiopoietins, but can form heterodimers with Tie2 and modulate its signaling39. Junctional Tie2 activates downstream Akt signaling, promoting endothelial cell stability and survival40. Activated Tie2 also antagonizes pro-permeability signaling by sequestering Src kinase through RhoA and mDia42.In confluent endothelial cells, Ang1 and Ang2 induce Tie2’s subcellular localization towards endothelial cell junctions43; small molecule inhibitors of VE-PTP (AKB-9778 and AKB-9785) promote the activation of Tie2 and vascular stability44; Faricimab, a bispecific antibody designed to simultaneously inhibit Ang2 and VEGF-A, promotes vascular stability and quiescence46; and AXT107, an integrin-binding peptide, promotes Tie2 activation by modulating integrin/Tie interactions20.Therapeutics targeting the Ang–Tie signaling pathway to promote vascular stability and normalization are being explored extensively due to the essential role of the Ang–Tie signaling pathway in a wide variety of diseases. Among many others, an Ang2-binding Tie2 activating antibody (ABTAA) promotes Ang2-stimulated Tie2 activation by forming a complex with Ang2 and Tie2, simultaneously promoting Tie2 activation and inhibiting Ang2’s antagonistic effect49 and the hepatocyte growth factor pathways50. Alawo et al.51 also used a computational model to study interactions of Ang1 and Ang2 with the Tie receptors on the endothelial cell surface. The present study aims to extend the computational model for the Ang–Tie interaction at the endothelial cell surface52 to include junctional localization, homo- and heteromeric receptor interactions, and downstream Ang–Tie signaling to quantitatively investigate the molecular mechanisms for Tie1’s role, the context-dependence of Ang2’s agonist activity, and their implications for downstream signaling.The complexity of the context-dependent interactions and effects of Ang–Tie signaling has created significant challenges to obtaining a quantitative understanding of the molecular control of the Ang–Tie signaling pathway. A systems biology approach to computational modeling of signaling networks allows the complex integration of the molecular mechanisms at a network level. Previously, we have developed computational models of vascular signaling pathways, including the vascular endothelial growth factor (VEGF)In the present study, we developed a mechanistically detailed computational model validated against previously published experimental data to study the molecular control of the Ang–Tie signaling pathway. The model captures and reproduces the ligand–receptor interactions and dynamics, junctional localization, and regulatory mechanisms, including the action of Tie1 and VE-PTP. The model demonstrates that Tie1’s junctional localization and its interaction with Tie2 at the endothelial junctions provide an explanation for its time-dependent effect on activation of Tie2. The model also shows that Tie1’s ability to sustain Tie2 activation is essential for the agonist effects of Ang2. The model provides a mechanistic explanation of how extracellular domain shedding of Tie1 in inflammatory endothelial cells potentiates Ang2’s antagonistic effect on Tie2 activation. The model also shows that inhibiting extracellular domain shedding of Tie2, promoting the junctional localization of Tie1, and inhibiting VE-PTP all have synergistic effects on rescuing Tie2 activation in inflammatory conditions. The model provides a platform to quantitatively study the molecular mechanisms of Ang–Tie signaling and their effects on the reaction network at a systems level and motivates additional experiments to further elucidate the mechanistic aspects of the pathway.53, and detailed reaction rules and molecular reactions of the model are described in the Methods section. All model equations, values of parameters, reaction rules in BioNetGen Language (BNGL) and Systems Biology Markup Language (SBML) computer code are available in the To develop a mechanistically detailed model of the Ang/Tie signaling pathway and its downstream signaling, we included a mass action reaction network of ligand/receptor interactions, regulation by co-receptor Tie1 and phosphatase VE-PTP, receptor trafficking, turnover, subcellular localization to the endothelial cell–cell junctions, as well as downstream signaling. The reaction diagram is shown in Fig. Ang1 molecules are assumed to be in tetrameric form, whereas Ang2 molecules exist in dimeric, trimeric, and tetrameric forms Fig. . Multime42. Phosphorylated Tie2 at the junctions also activates Akt through PI3K/PDK48.The model reactions also include the anti-permeability and pro-stability downstream signaling pathways of Tie2 Fig. . Phospho54 , Ang2 acts mostly as an agonist, contributing to the activation of Tie2. At a high concentration of Ang1, Ang2 antagonizes the activation of Tie2 by Ang1 Fig. . The modect Fig. .Fig. 5Mo56. Global sensitivity analysis demonstrates that the activity of Tie2 at steady-state (60 min post-stimulation) is sensitive to parameters related to extracellular domain cleavage of Tie1 (kcleavetie1), Tie2 trafficking, and turnover , VE-PTP expression , and initial levels of Tie2, Ang1, and Ang2   to calculate the partial rank correlation coefficients (PRCCs) for model outputs activated Tie2, activated Akt, and sequestered Src at 30 min post-stimulation Fig. 56. Globa_0) Fig. . Junctiop-values < 0.05) are shown in Fig. In addition to the sensitive parameters for activated Tie2, activated Akt is also sensitive to initial levels of PTEN, PIP2, PDK1, and the reaction rates of PI3K/Tie2 interaction and PTEN/PIP3 interaction  Fig. . SequestThe model predicts that for endothelial cells in inflammatory states with elevated Tie1 cleavage and high Ang2 concentration, inhibition of Tie2 activity can be rescued by sustaining Tie2 at the cell surface (inhibiting the extracellular domain cleavage of Tie2), promoting Tie1’s junctional localization, and inhibiting VE-PTP. The model also predicts that inhibiting the extracellular domain cleavage of Tie2 and inhibiting VE-PTP synergistically enhance the activation of Tie2 in inflammatory endothelial cells with increased Tie1 cleavage rate and a high level of Ang2. Endothelial cells in the inflammatory state are assumed to have elevated Tie1 cleavage rate and Ang2 concentration. The model predicts that inhibiting Tie2’s cleavage rate and inhibiting VE-PTP by up to 1% alone enhances Tie2 activation by up to threefold. Simultaneous inhibition of Tie2 cleavage and VE-PTP synergistically enhances Tie2 activation by up to 10-fold Fig. . Inhibit57 and an enhancer33 of Tie2’s activation. The exact molecular mechanisms responsible for the dual-action of Tie1 remain incompletely understood. Our model is the first mechanistic computational model with detailed molecular mechanisms of ligand–receptor interactions, junctional localization, and other regulatory mechanisms to quantitatively investigate the seemingly contradictory role of Tie1. The model demonstrates that Tie1 inhibits Tie2 at the cell surface by forming heterodimers with Tie2 but promotes and sustains Tie2’s activation at the endothelial junctions by binding with Tie2 and protecting Tie2 from internalization and dephosphorylation by VE-PTP. The proposed mechanisms capture and reproduce the time-dependent role that Tie1 plays in regulating Tie2’s signaling, consistent with the experimental observation by Savant et al. that Tie1 acutely inhibits but chronically promotes and sustains Tie2’s activation33Tie1 has been reported as both an inhibitor29. We hypothesize that Tie1’s molecular mechanisms proposed by the model, including its heteromeric formation, co-localization, and cross-activation with Tie2 at cell junctions can explain Ang2’s agonistic function in presence of Tie1 and its antagonistic function in absence of Tie1.The model shows that silencing Tie1 chronically inhibits both the Ang1-stimulated and Ang2-stimulated activation of Tie2, and the inhibitory effect of Tie1 silencing is stronger on Ang2-activated Tie2. When simulating Ang2’s activation of Tie2, the model shows that the inhibition of Tie2 activation by Tie1 silencing is stronger compared to Ang1, resulting in a 50% reduction of Tie2 activation, indicating that Tie1’s effect of promoting and sustaining Tie2 activation is essential for Ang2’s activation of Tie2. This observation is consistent with the experimental findings of a recent study by Korhonen et al. demonstrating that Tie1 is required for Ang2’s activation of Tie252. The present model demonstrates Tie1’s role in modulating Ang2’s activity.Previously, our computational model has demonstrated that VE-PTP provides context for Ang2’s agonistic activity by differentially regulating Ang1- and Ang2-stimulated Tie2Ang2 alone is a weak agonist of Tie2 and activates Tie2 and its downstream signaling. Tie1 provides context for Ang2’s agonistic effect by sustaining Ang2-activated Tie2 and protecting it from VE-PTP. The model predicts that silencing Tie1 inhibits sustained Ang2 activation of Tie2, essentially converting it into an antagonist Fig. . Simulat29. The model demonstrates that compared to Ang1, Ang2’s agonistic activity is more sensitive to Tie1’s sustaining of activated Tie2 at the endothelial junctions ; generation of PIP2 (rule 76), its phosphorylation by PI3K (rule 77) and dephosphorylation by PTEN (rule 78); binding of PIP3 to Akt and PDK ; activation of Akt at S473 by mTOR (rule 81); activation of Akt at T308 by PDK (rule 82); dephosphorylation of active Akt . (ix) Activated Tie2 also induces the activation of RhoA (rules 85–86), and its subsequent complex formation of mDia and Src (rules 87–88), preventing Src from phosphorylating VE-Cadherin (rules 89–94)  oli94) Fig. . The comOR rule 8; activat70, Ang2 as a weaker agonist of Tie2 and antagonizes Ang1-activated Tie254, receptor shedding of both Tie1 and Tie236, soluble Tie2 acting as a concentration-dependent inhibitor of Tie271, junctional localization of Tie2 induced by Ang1 and Ang239, Tie1 as a time-dependent regulator of activation of Tie2 and Akt33, VE-PTP as a differential regulator of Ang1- and Ang-activated Tie217, and various downstream signaling of Tie2, including Akt38, RhoA-GTP42, and mDia-Src41.The model parameters were calibrated to experimental data from independent literature sources. Global fitting algorithms, including fmincon and patternsearch in MATLAB’s global optimization toolbox  are used to simultaneously fit all parameters to fit the experimental data on Ang1 and Ang2-induced Tie2 activation, internalization and turnover56. Parameters are sampled from one-tenth to ten times their baseline best-fit values using LHS to obtain the PRCCs. Statistical significance was evaluated by calculating the p-values from the t test with Bonferroni correction. Up to the top ten positively correlated and ten negatively correlated, statistically significant sensitive parameters (Bonferroni-corrected p < 0.05) for model outputs active Tie2, active Akt, and sequestered Src are reported in Fig. Global sensitivity analysis is performed to understand the response of various model outputs to the changes in parameter space based on the methodology described in Marino et alFurther information on research design is available in the Supplementary Data 3Supplementary InformationReporting SummarySupplementary Data 1Supplementary Data 2