Central role of soluble adenylyl cyclase and cAMP in sperm physiology

Cyclic adenosine 3′,5′-monophosphate (cAMP), the first second messenger to be described, plays a central role in cell signaling in a wide variety of cell types. Over the last decades, a wide body of literature addressed the different roles of cAMP in cell physiology, mainly in response to neurotransmitters and hormones. cAMP is synthesized by a wide variety of adenylyl cyclases that can generally be grouped in two types: transmembrane adenylyl cyclase and soluble adenylyl cyclases. In particular, several aspects of sperm physiology are regulated by cAMP produced by a single atypical adenylyl cyclase (Adcy10, aka sAC, SACY). The signature that identifies sAC among other ACs, is their direct stimulation by bicarbonate. The essential nature of cAMP in sperm function has been demonstrated using gain of function as well as loss of function approaches. This review unifies state of the art knowledge of the role of cAMP and those enzymes involved in cAMP signaling pathways required for the acquisition of fertilizing capacity of mammalian sperm. This article is part of a Special Issue entitled: The role of soluble adenylyl cyclase in health and disease.Fil: Buffone, Mariano Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); ArgentinaFil: Wertheimer Hermitte, Eva Victoria. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Estudios Farmacológicos y Botánicos; ArgentinaFil: Visconti, Pablo E.. University Of Massachussets; Estados UnidosFil: Krapf, Dario. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Rosario. Instituto de Biología Molecular y Celular de Rosario; Argentina. Universidad Nacional de Rosario; Argentin

Bloody Women: A critical-creative examination of how female protagonists have transformed contemporary Scottish and Nordic crime fiction

This study will explore the role of female authors and their female protagonists in contemporary Scottish and Nordic crime fiction. Authors including Val McDermid, Denise Mina, Lin Anderson and Liza Marklund are just a few of the women who have challenged the expectation of gender in the crime fiction genre. By setting their novels in contemporary society, they reflect a range of social and political issues through the lens of a female protagonist. By closely examining the female characters, all journalists, in Val McDermid’s Lindsay Gordon series; Denise Mina’s Paddy Meehan series; Anna Smith’s books about Rosie Gilmour; and Liza Marklund’s books about Annika Bengzton, I explore the issue of gender through these writers’ perspectives and also draw parallels between their societies. I document the influence of these writers on my own practice-based research, a novel, The Invisible Chains, set in post-Referendum Scotland. The thesis will examine and define the role of the female protagonist, offer a feminist reading of contemporary crime fiction, and investigate how the rise of human trafficking, the problem of domestic abuse in Scotland and society’s changing attitudes and values are reflected in contemporary crime novels, before discussing the narrative structures and techniques employed in the writing of The Invisible Chains. This novel allows us to consider the role of women in a contemporary and progressive society where women hold many senior positions in public life and examine whether they manage successfully to challenge traditional patriarchal hierarchies. The narrative is split between journalist Megan Ross, The Girl, a victim of human trafficking, and Trudy, who is being domestically abused, thus pulling together the themes of the critical genesis in the creative work. By focusing on the protagonist, the victims and raising awareness of human trafficking and domestic abuse, The Invisible Chains, an original creative work, reflects a contemporary society’s changing attitudes, problems and values

The Non-Trapping Degree of Scattering

We consider classical potential scattering. If no orbit is trapped at energy E, the Hamiltonian dynamics defines an integer-valued topological degree. This can be calculated explicitly and be used for symbolic dynamics of multi-obstacle scattering. If the potential is bounded, then in the non-trapping case the boundary of Hill's Region is empty or homeomorphic to a sphere. We consider classical potential scattering. If at energy E no orbit is trapped, the Hamiltonian dynamics defines an integer-valued topological degree deg(E) < 2. This is calculated explicitly for all potentials, and exactly the integers < 2 are shown to occur for suitable potentials. The non-trapping condition is restrictive in the sense that for a bounded potential it is shown to imply that the boundary of Hill's Region in configuration space is either empty or homeomorphic to a sphere. However, in many situations one can decompose a potential into a sum of non-trapping potentials with non-trivial degree and embed symbolic dynamics of multi-obstacle scattering. This comprises a large number of earlier results, obtained by different authors on multi-obstacle scattering.Comment: 25 pages, 1 figure Revised and enlarged version, containing more detailed proofs and remark

Ca2+ ionophore A23187 can make mouse spermatozoa capable of fertilizing in vitro without activation of cAMP-dependent phosphorylation pathways

Ca2+ ionophore A23187 is known to induce the acrosome reaction of mammalian spermatozoa, but it also quickly immobilizes them. Although mouse spermatozoa were immobilized by this ionophore, they initiated vigorous motility (hyperactivation) soon after this reagent was washed away by centrifugation. About half of live spermatozoa were acrosome-reacted at the end of 10 min of ionophore treatment; fertilization of cumulus-intact oocytes began as soon as spermatozoa recovered their motility and before the increase in protein tyrosine phosphorylation, which started 30-45 min after washing out the ionophore. When spermatozoa were treated with A23187, more than 95% of oocytes were fertilized in the constant presence of the protein kinase A inhibitor, H89. Ionophore-treated spermatozoa also fertilized 80% of oocytes, even in the absence of HCO3-, a component essential for cAMP synthesis under normal in vitro conditions. Under these conditions, fertilized oocytes developed into normal offspring. These data indicate that mouse spermatozoa treated with ionophore are able to fertilize without activation of the cAMP/PKA signaling pathway. Furthermore, they suggest that the cAMP/PKA pathway is upstream of an intracellular Ca2+ increase required for the acrosome reaction and hyperactivation of spermatozoa under normal in vitro conditions.Fil: Tateno, Hiroyuki. Asahikawa Medical University. Department of Biological Sciences; JapónFil: Krapf, Dario. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Rosario. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Hino,Toshiaki. Asahikawa Medical University. Department of Biological Sciences; JapónFil: Sánchez Cárdenas, Claudia. Universidad Nacional Autonoma de Mexico. Instituto de Biotecnologia; MéxicoFil: Darszon, Alberto. Universidad Nacional Autonoma de Mexico. Instituto de Biotecnologia; MéxicoFil: Yanagimachi, Ryuzo. University of Hawaii Medical School. Institute for Biogenesis Research; Estados UnidosFil: Visconti, Pablo E.. University Of Massachussets; Estados Unido

The actin cytoskeleton of the mouse sperm flagellum is organized in a helical structure

Conception in mammals is determined by the fusion of a sperm cell with an oocyte during fertilization. Motility is one of the features of sperm that allows them to succeed in fertilization, and their flagellum is essential for this function. Longitudinally, the flagellum can be divided into the midpiece, the principal piece and the end piece. A precise cytoskeletal architecture of the sperm tail is key for the acquisition of fertilization competence. It has been proposed that the actin cytoskeleton plays essential roles in the regulation of sperm motility; however, the actin organization in sperm remains elusive. In the present work, we show that there are different types of actin structures in the sperm tail by using three-dimensional stochastic optical reconstruction microscopy (STORM). In the principal piece, actin is radially distributed between the axoneme and the plasma membrane. The actin-associated proteins spectrin and adducin are also found in these structures. Strikingly, polymerized actin in the midpiece forms a double-helix that accompanies mitochondria. Our findings illustrate a novel specialized structure of actin filaments in a mammalian cell.Fil: Gervasi, Maria Gracia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Estudios Farmacológicos y Botánicos. Universidad de Buenos Aires. Facultad de Medicina. Centro de Estudios Farmacológicos y Botánicos; Argentina. University of Massachussets; Estados UnidosFil: Xu, Xinran. State University of Colorado - Fort Collins; Estados UnidosFil: Carbajal Gonzalez, Blanca. Mount Holyoke College; Estados UnidosFil: 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: Visconti, Pablo E.. University of Massachussets; Estados UnidosFil: Krapf, Diego. State University of Colorado - Fort Collins; Estados Unido

Functional human sperm capacitation requires both bicarbonate dependent-PKA activation and down-regulation of Ser/Thr phosphatases by Src family kinases

In all mammalian species studied so far, sperm capacitation correlates with an increase in protein tyrosine (Tyr) phosphorylation mediated by a bicarbonate-dependent cAMP/PKA pathway. Recent studies in mice revealed however that a Src Family Kinase (SFK) induced inactivation of serine/threonine (Ser/Thr) phosphatases is also involved in the signaling pathways leading to Tyr phosphorylation. In view of these observations and with the aim of getting a better understanding of the signaling pathways involved in human sperm capacitation, in the present work we investigated the involvement of both the cAMP/PKA and SFK/phosphatase pathways in relation to the capacitation state of the cells. For this purpose, different signaling events and sperm functional parameters were analyzed as a function of capacitation time. Results revealed a very early bicarbonate-dependent activation of PKA indicated by the rapid (1 min) increase in both phospho-PKA substrates and cAMP levels (p<0.05). However, a complete pattern of Tyr phosphorylation was detected only after 6 h-incubation at which time sperm exhibited the ability to undergo the acrosome reaction (AR) and to penetrate zona-free hamster eggs. Sperm capacitated in the presence of the SFK inhibitor SKI606 showed a decrease in both PKA substrate and Tyr phosphorylation levels which was overcome by exposure of sperm to the Ser/Thr phosphatase inhibitor okadaic acid (OA). However, OA was unable to induce phosphorylation when sperm were incubated under PKA-inhibitory conditions (i.e. in the absence of bicarbonate or presence of PKA inhibitor). Moreover, the increase in PKA activity by exposure to a cAMP analogue and a phosphodiesterase inhibitor did not overcome the inhibition produced by SKI606. Whereas the presence of SKI606 during capacitation produced a negative effect (p<0.05) on sperm motility, progesterone-induced AR and fertilizing ability, none of these inhibitions were observed when sperm were exposed to SKI606 and OA. Interestingly, different concentrations of inhibitors were required to modulate human and mouse capacitation revealing the species-specificity of the molecular mechanisms underlying this process. In conclusion, our results describe for the first time the involvement of both PKA activation and Ser/Thr phosphatase down-regulation in functional human sperm capacitation and provide convincing evidence that early PKA-dependent phosphorylation is the convergent regulatory point between these two signaling pathways.Fil: Battistone, Maria Agustina. Consejo Nacional de Investigaciones Científicas y Técnicas -conicet. Instituto de Biología y Medicina Experimental (i); ArgentinaFil: Da Ros, Vanina Gabriela. Consejo Nacional de Investigaciones Científicas y Técnicas -conicet. Instituto de Biología y Medicina Experimental (i); ArgentinaFil: Salicioni, A.. University Of Massachussets; Estados UnidosFil: Navarrete, F.. University Of Massachussets; Estados UnidosFil: Krapf, Dario. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico - CONICET - Rosario. Instituto de Biología Molecular y Celular de Rosario; Argentina. Universidad Nacional de Rosario; ArgentinaFil: Visconti, P. E.. University Of Massachussets;Fil: Cuasnicu, Patricia Sara. Consejo Nacional de Investigaciones Científicas y Técnicas -conicet. Instituto de Biología y Medicina Experimental (i); Argentin

The tyrosine kinase FER is responsible for the capacitation-associated increase in tyrosine phosphorylation in murine sperm

Sperm capacitation is required for fertilization. At the molecular level, this process is associated with fast activation of protein kinase A. Downstream of this event, capacitating conditions lead to an increase in tyrosine phosphorylation. The identity of the tyrosine kinase(s) mediating this process has not been conclusively demonstrated. Recent experiments using stallion and human sperm have suggested a role for PYK2 based on the use of small molecule inhibitors directed against this kinase. However, crucially, loss-of-function experiments have not been reported. Here, we used both pharmacological inhibitors and genetically modified mice models to investigate the identity of the tyrosine kinase(s) mediating the increase in tyrosine phosphorylation in mouse sperm. Similar to stallion and human, PF431396 blocks the capacitation-associated increase in tyrosine phosphorylation. Yet, sperm from Pyk2(-/-) mice displayed a normal increase in tyrosine phosphorylation, implying that PYK2 is not responsible for this phosphorylation process. Here, we show that PF431396 can also inhibit FER, a tyrosine kinase known to be present in sperm. Sperm from mice targeted with a kinase-inactivating mutation in Fer failed to undergo capacitation-associated increases in tyrosine phosphorylation. Although these mice are fertile, their sperm displayed a reduced ability to fertilize metaphase II-arrested eggs in vitro.Fil: Alvau, Antonio. University of Massachussets; Estados UnidosFil: Battistone, Maria Agustina. 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: Gervasi, Maria Gracia. University of Massachussets; Estados UnidosFil: Navarrete, Felipe A.. University of Massachussets; Estados UnidosFil: Xu, Xinran. State University of Colorado - Fort Collins; Estados UnidosFil: Sánchez Cárdenas, Claudia. Universidad Nacional Autónoma de México. Instituto de Biotecnología; MéxicoFil: De la Vega Beltran, José Luis. Universidad Nacional Autónoma de México. Instituto de Biotecnología; MéxicoFil: Da Ros, Vanina Gabriela. 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: Greer, Peter. Queens University; CanadáFil: Darszon, Alberto. Universidad Nacional Autónoma de México. Instituto de Biotecnología; MéxicoFil: Krapf, Diego. State University of Colorado - Fort Collins; Estados UnidosFil: Salicioni, Ana María. University of Massachussets; Estados UnidosFil: Cuasnicu, Patricia Sara. 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: Visconti, Pablo E.. University of Massachussets; Estados Unido

Spectral content of a single non-Brownian trajectory

Time-dependent processes are often analysed using the power spectral density (PSD), calculated by taking an appropriate Fourier transform of individual trajectories and finding the associated ensemble-average. Frequently, the available experimental data sets are too small for such ensemble averages, and hence it is of a great conceptual and practical importance to understand to which extent relevant information can be gained from $S(f,T)$, the PSD of a single trajectory. Here we focus on the behavior of this random, realization-dependent variable, parametrized by frequency $f$ and observation-time $T$, for a broad family of anomalous diffusions---fractional Brownian motion (fBm) with Hurst-index $H$---and derive exactly its probability density function. We show that $S(f,T)$ is proportional---up to a random numerical factor whose universal distribution we determine---to the ensemble-averaged PSD. For subdiffusion ($H<1/2$) we find that $S(f,T)\sim A/f^{2H+1}$ with random-amplitude $A$. In sharp contrast, for superdiffusion $(H>1/2)$ $S(f,T)\sim BT^{2H-1}/f^2$ with random amplitude $B$. Remarkably, for $H>1/2$ the PSD exhibits the same frequency-dependence as Brownian motion, a deceptive property that may lead to false conclusions when interpreting experimental data. Notably, for $H>1/2$ the PSD is ageing and is dependent on $T$. Our predictions for both sub- and superdiffusion are confirmed by experiments in live cells and in agarose hydrogels, and by extensive simulations.Comment: 13 pages, 5 figures, Supplemental Material can be found at https://journals.aps.org/prx/supplemental/10.1103/PhysRevX.9.011019/prx_SM_final.pd

Super-resolution imaging of live sperm reveals dynamic changes of the actin cytoskeleton during acrosomal exocytosis

Filamentous actin (F-actin) is a key factor in exocytosis in many cell types. In mammalian sperm, acrosomal exocytosis (denoted the acrosome reaction or AR), a special type of controlled secretion, is regulated by multiple signaling pathways and the actin cytoskeleton. However, the dynamic changes of the actin cytoskeleton in live sperm are largely not understood. Here, we used the powerful properties of SiR-actin to examine actin dynamics in live mouse sperm at the onset of the AR. By using a combination of super-resolution microscopy techniques to image sperm loaded with SiR-actin or sperm from transgenic mice containing Lifeact-EGFP, six regions containing F-actin within the sperm head were revealed. The proportion of sperm possessing these structures changed upon capacitation. By performing live-cell imaging experiments, we report that dynamic changes of F-actin during the AR occur in specific regions of the sperm head. While certain F-actin regions undergo depolymerization prior to the initiation of the AR, others remain unaltered or are lost after exocytosis occurs. Our work emphasizes the utility of live-cell nanoscopy, which will undoubtedly impact the search for mechanisms that underlie basic sperm functions.Fil: Romarowski, Ana. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Velasco Félix, Ángel G.. Universidad Nacional Autónoma de México; MéxicoFil: Rodriguez, Paulina Torres. Universidad Nacional Autónoma de México; MéxicoFil: Gervasi, Mar?á G.. University Of Massachusetts Amherst;Fil: Xu, Xinran. School Of Biomedical Engineering;Fil: Luque, Guillermina Maria. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Contreras-Jiménez, Gastón. Universidad Nacional Autónoma de México; MéxicoFil: Sánchez-Cárdenas, Claudia. Universidad Nacional Autónoma de México; MéxicoFil: Ramírez-Gómez, Héctor V.. Universidad Nacional Autónoma de México; MéxicoFil: Krapf, Diego. School Of Biomedical Engineering;Fil: Visconti, Pablo E.. University Of Massachusetts Amherst;Fil: 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: Guerrero, Adán. Universidad Nacional Autónoma de México; MéxicoFil: Darszon, Alberto. Universidad Nacional Autónoma de México; MéxicoFil: Buffone, Mariano Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin