Glucose and GLP-1 Stimulate cAMP Production via Distinct Adenylyl Cyclases in INS-1E Insulinoma Cells

In β cells, both glucose and hormones, such as GLP-1, stimulate production of the second messenger cAMP, but glucose and GLP-1 elicit distinct cellular responses. We now show in INS-1E insulinoma cells that glucose and GLP-1 produce cAMP with distinct kinetics via different adenylyl cyclases. GLP-1 induces a rapid cAMP signal mediated by G protein–responsive transmembrane adenylyl cyclases (tmAC). In contrast, glucose elicits a delayed cAMP rise mediated by bicarbonate, calcium, and ATP-sensitive soluble adenylyl cyclase (sAC). This glucose-induced, sAC-dependent cAMP rise is dependent upon calcium influx and is responsible for the glucose-induced activation of the mitogen-activated protein kinase (ERK1/2) pathway. These results demonstrate that sAC-generated and tmAC-generated cAMP define distinct signaling cascades

Bicarbonate-responsive “soluble” adenylyl cyclase defines a nuclear cAMP microdomain

Bicarbonate-responsive “soluble” adenylyl cyclase resides, in part, inside the mammalian cell nucleus where it stimulates the activity of nuclear protein kinase A to phosphorylate the cAMP response element binding protein (CREB). The existence of this complete and functional, nuclear-localized cAMP pathway establishes that cAMP signals in intracellular microdomains and identifies an alternate pathway leading to CREB activation

Soluble Adenylyl Cyclase Is Localized to Cilia and Contributes to Ciliary Beat Frequency Regulation via Production of cAMP

Ciliated airway epithelial cells are subject to sustained changes in intracellular CO2/HCO3− during exacerbations of airway diseases, but the role of CO2/HCO3−-sensitive soluble adenylyl cyclase (sAC) in ciliary beat regulation is unknown. We now show not only sAC expression in human airway epithelia (by RT-PCR, Western blotting, and immunofluorescence) but also its specific localization to the axoneme (Western blotting and immunofluorescence). Real time estimations of [cAMP] changes in ciliated cells, using FRET between fluorescently tagged PKA subunits (expressed under the foxj1 promoter solely in ciliated cells), revealed CO2/HCO3−-mediated cAMP production. This cAMP production was specifically blocked by sAC inhibitors but not by transmembrane adenylyl cyclase (tmAC) inhibitors. In addition, this cAMP production stimulated ciliary beat frequency (CBF) independently of intracellular pH because PKA and sAC inhibitors were uniquely able to block CO2/HCO3−-mediated changes in CBF (while tmAC inhibitors had no effect). Thus, sAC is localized to motile airway cilia and it contributes to the regulation of human airway CBF. In addition, CO2/HCO3− increases indeed reversibly stimulate intracellular cAMP production by sAC in intact cells

Capacitation induces changes in metabolic pathways supporting motility of epididymal and ejaculated sperm

Mammalian sperm require sufficient energy to support motility and capacitation for successful fertilization. Previous studies cataloging the changes to metabolism in sperm explored ejaculated human sperm or dormant mouse sperm surgically extracted from the cauda epididymis. Due to the differences in methods of collection, it remains unclear whether any observed differences between mouse and human sperm represent species differences or reflect the distinct maturation states of the sperm under study. Here we compare the metabolic changes during capacitation of epididymal versus ejaculated mouse sperm and relate these changes to ejaculated human sperm. Using extracellular flux analysis and targeted metabolic profiling, we show that capacitation-induced changes lead to increased flux through both glycolysis and oxidative phosphorylation in mouse and human sperm. Ejaculation leads to greater flexibility in the ability to use different carbon sources. While epididymal sperm are dependent upon glucose, ejaculated mouse and human sperm gain the ability to also leverage non-glycolytic energy sources such as pyruvate and citrate

Calcium-sensing soluble adenylyl cyclase mediates TNF signal transduction in human neutrophils

Through chemical screening, we identified a pyrazolone that reversibly blocked the activation of phagocyte oxidase (phox) in human neutrophils in response to tumor necrosis factor (TNF) or formylated peptide. The pyrazolone spared activation of phox by phorbol ester or bacteria, bacterial killing, TNF-induced granule exocytosis and phox assembly, and endothelial transmigration. We traced the pyrazolone's mechanism of action to inhibition of TNF-induced intracellular Ca2+ elevations, and identified a nontransmembrane (“soluble”) adenylyl cyclase (sAC) in neutrophils as a Ca2+-sensing source of cAMP. A sAC inhibitor mimicked the pyrazolone's effect on phox. Both compounds blocked TNF-induced activation of Rap1A, a phox-associated guanosine triphosphatase that is regulated by cAMP. Thus, TNF turns on phox through a Ca2+-triggered, sAC-dependent process that may involve activation of Rap1A. This pathway may offer opportunities to suppress oxidative damage during inflammation without blocking antimicrobial function

Compartmentalization of distinct cAMP signaling pathways in mammalian sperm.

Fertilization competence is acquired in the female tract in a process known as capacitation. Capacitation is needed for the activation of motility (e.g. hyperactivation) and to prepare the sperm for an exocytotic process known as acrosome reaction. While the HCO3--dependent soluble adenylyl cyclase Adcy10 plays a role in motility, less is known about the source of cAMP in the sperm head. Transmembrane adenylyl cyclases (tmACs) are another possible source of cAMP. These enzymes are regulated by stimulatory heterotrimeric Gs proteins; however, the presence of Gs or tmACs in mammalian sperm has been controversial. In this manuscript, we used Western blotting and cholera toxin-dependent ADP ribosylation to show Gs presence in the sperm head. Also, we showed that forskolin, a tmAC specific activator, induces cAMP accumulation in sperm from both WT and Adcy10 null mice. This increase is blocked by the tmAC inhibitor SQ-22536 but not by the Adcy10 inhibitor KH7. While Gs immunoreactivity and tmAC activity are detected in the sperm head, PKA is only found in the tail, where Adcy10 was previously shown to reside. Consistent with an acrosomal localization, Gs reactivity is lost in acrosome reacted sperm, and forskolin is able to increase intracellular Ca2+ and induce the acrosome reaction. Altogether, these data suggest that cAMP pathways are compartmentalized in sperm, with Gs and tmAC in the head and Adcy10 and PKA in the flagellum.Fil: Wertheimer Hermitte, Eva Victoria. University Of Massachussets; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Estudios Farmacológicos y Botánicos; ArgentinaFil: Krapf, Dario. Universidad Nacional de Rosario; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Rosario. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: de la Vega Beltran, José L.. Universidad Nacional Autónoma de México; MéxicoFil: Sánchez Cárdenas, Claudia. Universidad Nacional Autónoma de México; MéxicoFil: Navarrete, Felipe. University Of Massachussets; Estados UnidosFil: Haddad, Douglas. University Of Massachussets; Estados UnidosFil: Escoffier, Jessica. University Of Massachussets; Estados UnidosFil: Salicioni, Ana M.. University Of Massachussets; Estados UnidosFil: Levin, Lonny R.. Cornell University; Estados UnidosFil: Buck, Jochen. Cornell University; Estados UnidosFil: Mager, Jesse. University Of Massachussets; Estados UnidosFil: Darszon, Alberto. Universidad Nacional Autónoma de México; MéxicoFil: Visconti, Pablo E.. University Of Massachussets; Estados Unido

Transient exposure to calcium ionophore enables in vitro fertilization in sterile mouse models

Mammalian sperm acquire fertilizing capacity in the female tract in a process called capacitation. At the molecular level, capacitation requires protein kinase A activation, changes in membrane potential and an increase in intracellular calcium. Inhibition of these pathways results in loss of fertilizing ability in vivo and in vitro. We demonstrated that transient incubation of mouse sperm with Ca 2+ ionophore accelerated capacitation and rescued fertilizing capacity in sperm with inactivated PKA function. We now show that a pulse of Ca2+ ionophore induces fertilizing capacity in sperm from infertile CatSper1 (Ca2+ channel), Adcy10 (soluble adenylyl cyclase) and Slo3 (K+ channel) KO mice. In contrast, sperm from infertile mice lacking the Ca 2+ efflux pump PMACA4 were not rescued. These results indicate that a transient increase in intracellular Ca 2+ can overcome genetic infertility in mice and suggest this approach may prove adaptable to rescue sperm function in certain cases of human male infertility.Fil: Navarrete, Felipe A.. University of Massachusetts; Estados UnidosFil: Alvau, Antonio. University of Massachusetts; Estados UnidosFil: Lee, Hoi Chang. University of Massachusetts; Estados UnidosFil: Levin, Lonny R.. Weill Cornell Medical College; Estados UnidosFil: Buck, Jochen. Weill Cornell Medical College; Estados UnidosFil: Leon, Patricia Martin De. University of Delaware; Estados UnidosFil: Santi, Celia M.. University of Washington; Estados UnidosFil: Krapf, Dario. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Mager, Jesse. University of Massachusetts; Estados UnidosFil: Fissore, Rafael A.. University of Massachusetts; Estados UnidosFil: Salicioni, Ana M.. University of Massachusetts; Estados UnidosFil: Darszon, Alberto. Universidad Nacional Autónoma de México. Instituto de Biotecnología; MéxicoFil: Visconti, Pablo E.. University of Massachusetts; Estados Unido

The “Soluble” Adenylyl Cyclase in Sperm Mediates Multiple Signaling Events Required for Fertilization

SummaryMammalian fertilization is dependent upon a series of bicarbonate-induced, cAMP-dependent processes sperm undergo as they “capacitate,” i.e., acquire the ability to fertilize eggs. Male mice lacking the bicarbonate- and calcium-responsive soluble adenylyl cyclase (sAC), the predominant source of cAMP in male germ cells, are infertile, as the sperm are immotile. Membrane-permeable cAMP analogs are reported to rescue the motility defect, but we now show that these “rescued” null sperm were not hyperactive, displayed flagellar angulation, and remained unable to fertilize eggs in vitro. These deficits uncover a requirement for sAC during spermatogenesis and/or epididymal maturation and reveal limitations inherent in studying sAC function using knockout mice. To circumvent this restriction, we identified a specific sAC inhibitor that allowed temporal control over sAC activity. This inhibitor revealed that capacitation is defined by separable events: induction of protein tyrosine phosphorylation and motility are sAC dependent while acrosomal exocytosis is not dependent on sAC

Transient Sperm Starvation Improves the Outcome of Assisted Reproductive Technologies

To become fertile, mammalian sperm must undergo a series of biochemical and physiological changes known as capacitation. These changes involve crosstalk between metabolic and signaling pathways and can be recapitulated in vitro. In this work, sperm were incubated in the absence of exogenous nutrients (starved) until they were no longer able to move. Once immotile, energy substrates were added back to the media and sperm motility was rescued. Following rescue, a significantly higher percentage of starved sperm attained hyperactivated motility and displayed increased ability to fertilize in vitro when compared with sperm persistently incubated in standard capacitation media. Remarkably, the effects of this treatment continue beyond fertilization as starved and rescued sperm promoted higher rates of embryo development, and once transferred to pseudo-pregnant females, blastocysts derived from treated sperm produced significantly more pups. In addition, the starvation and rescue protocol increased fertilization and embryo development rates in sperm from a severely subfertile mouse model, and when combined with temporal increase in Ca2+ ion levels, this methodology significantly improved fertilization and embryo development rates in sperm of sterile CatSper1 KO mice model. Intracytoplasmic sperm injection (ICSI) does not work in the agriculturally relevant bovine system. Here, we show that transient nutrient starvation of bovine sperm significantly enhanced ICSI success in this species. These data reveal that the conditions under which sperm are treated impact postfertilization development and suggest that this “starvation and rescue method” can be used to improve assisted reproductive technologies (ARTs) in other mammalian species, including humans.Fil: Navarrete, Felipe A.. University of Massachussets; Estados UnidosFil: Aguila, Luis. University of Massachussets; Estados UnidosFil: Martin Hidalgo, David. University of Massachussets; Estados Unidos. Universidad de Extremadura ; EspañaFil: Tourzani, Darya A.. University of Massachussets; Estados UnidosFil: Luque, Guillermina Maria. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Ardestani, Goli. University of Massachussets; Estados UnidosFil: Garcia Vazquez, Francisco A.. Universidad de Murcia; EspañaFil: Levin, Lonny R.. Cornell University; Estados UnidosFil: Buck, Jochen. Cornell University; Estados UnidosFil: Darszon, Alberto. Universidad Nacional Autónoma de México. Instituto de Biología; MéxicoFil: Buffone, Mariano Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Mager, Jesse. University of Massachussets; Estados UnidosFil: Fissore, Rafael A.. University of Massachussets; Estados UnidosFil: Salicioni, Ana M.. University of Massachussets; Estados UnidosFil: Gervasi, María G.. University of Massachussets; Estados UnidosFil: Visconti, Pablo E.. University of Massachussets; Estados Unido

Mouse sperm energy restriction and recovery (SER) revealed novel metabolic pathways

Mammalian sperm must undergo capacitation to become fertilization-competent. While working on mice, we recently developed a new methodology for treating sperm in vitro, which results in higher rates of fertilization and embryo development after in vitro fertilization. Sperm incubated in media devoid of nutrients lose motility, although they remain viable. Upon re-adding energy substrates, sperm resume motility and become capacitated with improved functionality. Here, we explore how sperm energy restriction and recovery (SER) treatment affects sperm metabolism and capacitation-associated signaling. Using extracellular flux analysis and metabolite profiling and tracing via nuclear magnetic resonance (NMR) and mass spectrometry (MS), we found that the levels of many metabolites were altered during the starvation phase of SER. Of particular interest, two metabolites, AMP and L-carnitine, were significantly increased in energy-restricted sperm. Upon re-addition of glucose and initiation of capacitation, most metabolite levels recovered and closely mimic the levels observed in capacitating sperm that have not undergone starvation. In both control and SER-treated sperm, incubation under capacitating conditions upregulated glycolysis and oxidative phosphorylation. However, ATP levels were diminished, presumably reflecting the increased energy consumption during capacitation. Flux data following the fate of 13C glucose indicate that, similar to other cells with high glucose consumption rates, pyruvate is converted into 13C-lactate and, with lower efficiency, into 13C-acetate, which are then released into the incubation media. Furthermore, our metabolic flux data show that exogenously supplied glucose is converted into citrate, providing evidence that in sperm cells, as in somatic cells, glycolytic products can be converted into Krebs cycle metabolites