A soluble adenylyl cyclase from sea urchin spermatozoa

Nomura, Mamoru; Beltrán, Carmen; Darszon, Alberto; Vacquier, Victor D.

doi:10.1016/j.gene.2005.04.034

Cited by 43 publications

(69 citation statements)

References 61 publications

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“…Early work using an antisera raised against the whole 190 kDa sea urchin sperm AC protein, later shown to be susAC [18], indicated it was mainly located in the sperm flagella [21]. However, confocal examination revealed its presence in the head, acrosomal and mitochondrial area as well (Fig.…”

Section: Localization Of Susacmentioning

confidence: 98%

“…All cAMP determinations were done by triplicate. Since susAC is also stimulated by alkaline pH [18], pH was carefully maintained at 7.5 in the bicarbonate experiments.…”

Section: Acrosome Reaction Camp and Adenylyl Cyclase Determinationsmentioning

confidence: 99%

“…The nine isoforms are activated by G αs and all but AC9 are stimulated by forskolin [5]. The S. purpuratus genome predicts that orthologs of mammalian tmACs 1, 2, 3, 5 and 9 are present in this organism (see Materials and methods) besides susAC [18]. To determine if tmACs are functionally present in sea urchin sperm we examined if their AC activity was sensitive to forskolin.…”

Section: Sea Urchin Sperm Membranes Contain Tmac Activitiesmentioning

confidence: 99%

“…All cAMP determinations were done by triplicate. Since susAC is also stimulated by alkaline pH [18], pH was carefully maintained at 7.5 in the bicarbonate experiments.In purified sperm membranes (1-5 µg) or in the crude particulate fraction (5-10 µg) AC activity was determined in 30 µl of the reaction mixture (in mM: 25 Hepes, pH 7.5, 10 MgCl 2 , 100 KCl, 1 IBMX and 0.1% Brij 78). Membranes were incubated at 23 °C in the absence or presence of the tmAC inhibitor.…”

mentioning

confidence: 99%

“…Recently an ortholog of the mammalian sperm sAC was cloned and sequenced from a sea urchin Strongylocentrotus purpuratus testis cDNA library (susAC). Its activity is higher in the presence of Mn 2+ than Mg 2+ , and is stimulated by pH [18]. Furthermore, sea urchin sperm posses G-proteins [15,19,20].…”

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confidence: 99%

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Particulate and soluble adenylyl cyclases participate in the sperm acrosome reaction

Beltrán

Vacquier

Moy

et al. 2007

Biochemical and Biophysical Research Communications

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AbstractcAMP is important in sea urchin sperm signaling, yet the molecular nature of the adenylyl cyclases (ACs) involved remained unknown. These cells were recently shown to contain an ortholog of the mammalian soluble adenylyl cyclase (sAC). Here, we show that sAC is present in the sperm head and as in mammals is stimulated by bicarbonate. The acrosome reaction (AR), a process essential for fertilization, is influenced by the bicarbonate concentration in seawater. By using functional assays and immunofluorescence techniques we document that sea urchin sperm also express orthologs of multiple isoforms of transmembrane ACs (tmACs). Our findings employing selective inhibitors for each class of AC indicate that both sAC and tmACs participate in the sperm acrosome reaction. KeywordsAcrosome reaction; cAMP; Adenylyl cyclases; Sea urchin; Soluble adenylyl cyclase; Sperm; fertilization cAMP, an important second messenger in cell signaling, is fundamental in sperm physiology. Sperm must swim, find the egg and undergo the acrosome reaction (AR) to fertilize it [1]. cAMP levels influence sperm ionic fluxes, motility and the AR [2].Two types of adenylyl cyclases (ACs) are expressed in mammals, transmembrane ACs (tmACs) and soluble AC (sAC) molecularly identified in rat testis [3] and sperm [4]. Restricted to the plasma membrane, tmACs are regulated by heterotrimeric G-proteins and stimulated by forskolin [5]. In contrast, sAC is stimulated by bicarbonate [6] and Ca 2+ [7,8]. In addition to sAC, mouse [9] and human [10] sperm apparently also contain tmACs.Why AC activity, cAMP levels, and cyclic AMP-dependent protein kinase (PKA) activity increase [11] Sea urchin sperm were believed to have only one type of AC and no G proteins [11,13].Recently an ortholog of the mammalian sperm sAC was cloned and sequenced from a sea urchin Strongylocentrotus purpuratus testis cDNA library (susAC). Its activity is higher in the presence of Mn 2+ than Mg 2+ , and is stimulated by pH [18]. Furthermore, sea urchin sperm posses G-proteins [15,19,20]. Here, we show by using specific activators and inhibitors for transmembrane and soluble ACs that both ACs participate in the sperm AR. Moreover, we document the presence of several tmACs and their differential distribution in sperm by immunolocalization and Western blot experiments. PCRs were performed using testis cDNA as template and two oligonucleotide primers. A degenerate sense 5′-TTYGYIGAYATHWSIGG NTT-3′based on rat sAC (Rattus norvegicus), NP067716.1 and an anti-sense 5′-ACGGCTGGCTACATTGACAG-3′; exact match from S. purpuratus coelomocytes AC R61912 EST 030. A 630-bp PCR product was obtained corresponding to one of the two copies of the mammalian adenylyl/guanylyl cyclase catalytic domain. Screening an S. purpuratus testis cDNA Lambda Zap library (Stratagene) with this product yielded two clones containing 26% of the SpAC2 (XP_780688; 479-677) and 25% of the SpAC9 (XP_798394; 509-690) predicted sequences. In addition, we found the predicted sequences for AC1 (SpAC1, XP_787811), AC3 (SpAC3,...

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Section: Localization Of Susacmentioning

confidence: 98%

“…All cAMP determinations were done by triplicate. Since susAC is also stimulated by alkaline pH [18], pH was carefully maintained at 7.5 in the bicarbonate experiments.…”

Section: Acrosome Reaction Camp and Adenylyl Cyclase Determinationsmentioning

confidence: 99%

Section: Sea Urchin Sperm Membranes Contain Tmac Activitiesmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Particulate and soluble adenylyl cyclases participate in the sperm acrosome reaction

Beltrán

Vacquier

Moy

et al. 2007

Biochemical and Biophysical Research Communications

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Proteins associated with soluble adenylyl cyclase in sea urchin sperm flagella

Nomura

Vacquier

2006

Cell Motil. Cytoskeleton

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Adenylyl cyclases (ACs) synthesize cAMP and are present in cells as transmembrane AC and soluble AC (sAC). In sperm, the cAMP produced regulates ion channels and it also activates protein kinase-A that in turn phosphorylates specific axonemal proteins to activate flagellar motility. In mammalian sperm, sAC localizes to the midpiece of flagella, whereas in sea urchin sperm sAC is along the entire flagellum. Here we show that in sea urchin sperm, sAC is complexed with proteins of the plasma membrane and axoneme. Immunoprecipitation shows that a minimum of 10 proteins is tightly associated with sAC. Mass spectrometry of peptides derived from these proteins shows them to be: axonemal dynein heavy chains 7 and 9, sperm specific Na+/H+ exchanger, cyclic nucleotide-gated ion channel, sperm specific creatine kinase, membrane bound guanylyl cyclase, cyclic GMP specific phosphodiesterase 5A, the receptor for the egg peptide speract, and alpha- and beta-tubulins. The sAC-associated proteins could be important in linking membrane signal transduction to energy utilisation in the regulation of flagellar motility.

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Roles of Ion Transport in Control of Cell Motility

Stock

Ludwig

Hanley

et al. 2013

Comprehensive Physiology

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Cell motility is an essential feature of life. It is essential for reproduction, propagation, embryonic development, and healing processes such as wound closure and a successful immune defense. If out of control, cell motility can become life-threatening as, for example, in metastasis or autoimmune diseases. Regardless of whether ciliary/flagellar or amoeboid movement, controlled motility always requires a concerted action of ion channels and transporters, cytoskeletal elements, and signaling cascades. Ion transport across the plasma membrane contributes to cell motility by affecting the membrane potential and voltage-sensitive ion channels, by inducing local volume changes with the help of aquaporins and by modulating cytosolic Ca(2+) and H(+) concentrations. Voltage-sensitive ion channels serve as voltage detectors in electric fields thus enabling galvanotaxis; local swelling facilitates the outgrowth of protrusions at the leading edge while local shrinkage accompanies the retraction of the cell rear; the cytosolic Ca(2+) concentration exerts its main effect on cytoskeletal dynamics via motor proteins such as myosin or dynein; and both, the intracellular and the extracellular H(+) concentration modulate cell migration and adhesion by tuning the activity of enzymes and signaling molecules in the cytosol as well as the activation state of adhesion molecules at the cell surface. In addition to the actual process of ion transport, both, channels and transporters contribute to cell migration by being part of focal adhesion complexes and/or physically interacting with components of the cytoskeleton. The present article provides an overview of how the numerous ion-transport mechanisms contribute to the various modes of cell motility.

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A soluble adenylyl cyclase from sea urchin spermatozoa

Cited by 43 publications

References 61 publications

Particulate and soluble adenylyl cyclases participate in the sperm acrosome reaction

Particulate and soluble adenylyl cyclases participate in the sperm acrosome reaction

Proteins associated with soluble adenylyl cyclase in sea urchin sperm flagella

Roles of Ion Transport in Control of Cell Motility

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