Silvia E. Arranz

Permanent Genetic Resources added to Molecular Ecology Resources Database 1 December 2009-31 January 2010

This article documents the addition of 220 microsatellite marker loci to the Molecular Ecology Resources Database. Loci were developed for the following species: Allanblackia floribunda, Amblyraja radiata, Bactrocera cucurbitae, Brachycaudus helichrysi, Calopogonium mucunoides, Dissodactylus primitivus, Elodea canadensis, Ephydatia fluviatilis, Galapaganus howdenae howdenae, Hoplostethus atlanticus, Ischnura elegans, Larimichthys polyactis, Opheodrys vernalis, Pelteobagrus fulvidraco, Phragmidium violaceum, Pistacia vera, and Thunnus thynnus. These loci were cross‐tested on the following species: Allanblackia gabonensis, Allanblackia stanerana, Neoceratitis cyanescens, Dacus ciliatus, Dacus demmerezi, Bactrocera zonata, Ceratitis capitata, Ceratitis rosa, Ceratits catoirii, Dacus punctatifrons, Ephydatia mülleri, Spongilla lacustris, Geodia cydonium, Axinella sp., Ischnura graellsii, Ischnura ramburii, Ischnura pumilio, Pistacia integerrima and Pistacia terebinthus.

Transmembrane adenylyl cyclase regulates amphibian sperm motility through protein kinase A activation

Sperm motility is essential for achieving fertilization. In animals with external fertilization as amphibians, spermatozoa are stored in a quiescent state in the testis. Spermiation to hypotonic fertilization media triggers activation of sperm motility. Bufo arenarum sperm are immotile in artificial seminal plasma (ASP) but acquire in situ flagellar beating upon dilution. In addition to the effect of low osmolarity on sperm motility activation, we report that diffusible factors of the egg jelly coat (EW) regulate motility patterns, switching from in situ to progressive movement. The signal transduction pathway involved in amphibian sperm motility activation is mostly unknown. In the present study, we show a correlation between motility activation triggered by low osmotic pressure and activation of protein kinase A (PKA). Moreover, this is the first study to present strong evidences that point toward a role of a transmembrane adenyl-cyclase (tmAC) in the regulation of amphibian sperm motility through PKA activation.

Growth hormone receptors in zebrafish (Danio rerio): Adult and embryonic expression patterns

Growth hormone receptor (GHR) is a critical regulator of growth and metabolism. Although two GHRs have been characterized in many fish species, their functional characteristics, mechanisms of regulation and roles in embryonic development remain unclear. The zebrafish (Danio rerio) is an excellent model organism to study both developmental and physiological processes. In the present work, we characterized the complete cDNA sequences of zebrafish GHRs, ghra and ghrb, and their gene structures. We studied the expression of both receptors in adult tissues, and during embryonic development and larval stages by means of RT-PCR and whole-mount in situ hybridization. We determined that both transcripts are maternal ones, with specific expression patterns during development. Both GHR transcripts are mainly expressed in the notochord, myotomes, anterior structures and in the yolk cell. Interestingly, their expression became undetectable at 96h post-fertilization. Unlike other reports in fish, ghrs expression could not be detected in brain when adult tissues were used, and we detected ghrb but not ghra transcripts in muscle. In addition, we determined alternative transcript sequences for ghra with specific domain deletions, and alternative transcripts for ghrb that generate a premature stop codon and codify for truncated isoforms. These isoforms lack intracellular regions necessary for the activation of signal transducers and activators of transcription (STAT) family transcription factors 5.

Diffusible highly glycosylated protein from Bufo arenarum egg-jelly coat: Biological activity

L‐HGP is a highly glycosylated protein from Bufo arenarum egg‐jelly coat that diffuses into the surrounding medium when the strings of oocytes are incubated in saline solutions. L‐HGP was purified from egg water and the estimated percentage of L‐HGP/total protein in egg water was estimated in 30%. In the present study we examine, by indirect immunofluorescence, the effect of L‐HGP on acrosome status of homologous spermatozoa. A high percentage (77%) of sperm lost the acrosome when incubated in 10% Ringer solution buffered with 10 mM Tris‐HCl, pH 7.6, during 60 min, a condition that resembles egg‐jelly osmolarity. The addition of purified L‐HGP to the incubation medium prevents acrosome breakdown. The acrosome integrity is maintained for at least 1 hr. This effect is specific for L‐HGP at concentration ranging from 0.01 to 0.1 mg/ml since neither BSA nor fetuin seems to have similar activity at similar concentrations. The same effect was observed when spermatozoa were incubated in egg water. Preliminary results suggest that L‐HGP binds to B. arenarum spermatozoan membranes. Mol. Reprod. Dev. 56:392–400, 2000.

Identification of mRNA-binding proteins during development: Characterization of Bufo arenarum cellular nucleic acid binding protein

Ultraviolet irradiation was used to covalently cross‐link poly(A)+RNA and associated proteins in eggs and embryos of the toad Bufo arenarum. Four major proteins with apparent sizes of 60, 57, 45 and 30–24 kDa were identified. It was observed that the same mRNA‐binding proteins were isolated from eggs to gastrula and neural stages of development. The 30 kDa polypeptide, p30, appeared as the main ultraviolet (UV) cross‐linked protein in the developmental stages analyzed. By means of polyclonal antibodies, it was determined that this polypeptide has a cytoplasmic localization and it was detected in liver, eggs and embryos. The presence of p30 was also analyzed by western blot during oogenesis and development. The 30 kDa polypeptide was present in all stages analyzed but it could not be detected in stages I–II of oogenesis. At the neural stage, the relative amount of p30 began to decrease, reaching its lowest levels after stages 26–30 (tail‐bud in Bufo arenarum). On the basis of purification, immunoprecipitation and western blot assays the 30 kDa protein was identified as the Bufo arenarum cellular nucleic acid binding protein.

Src Kinase Is the Connecting Player between Protein Kinase A (PKA) Activation and Hyperpolarization through SLO3 Potassium Channel Regulation in Mouse Sperm.

Background: Membrane potential (Em) hyperpolarization during sperm capacitation is necessary for acrosome reaction. Results: cSrc is activated downstream of PKA, regulating the SLO3 K+ channel and promoting membrane hyperpolarization. Conclusion: Acrosomal responsiveness of mouse sperm depends on cSrc activation of SLO3. Significance: This represents the first evidence of K+ channel regulation in mouse sperm by a tyrosine kinase, affecting Em. Plasma membrane hyperpolarization is crucial for mammalian sperm to acquire acrosomal responsiveness during capacitation. Among the signaling events leading to mammalian sperm capacitation, the immediate activation of protein kinase A plays a pivotal role, promoting the subsequent stimulation of protein tyrosine phosphorylation that associates with fertilizing capacity. We have shown previously that mice deficient in the tyrosine kinase cSrc are infertile and exhibit improper cauda epididymis development. It is therefore not clear whether lack of sperm functionality is due to problems in epididymal maturation or to the absence of cSrc in sperm. To further address this problem, we investigated the kinetics of cSrc activation using anti-Tyr(P)-416-cSrc antibodies that only recognize active cSrc. Our results provide evidence that cSrc is activated downstream of PKA and that inhibition of its activity blocks the capacitation-induced hyperpolarization of the sperm plasma membrane without blocking the increase in tyrosine phosphorylation that accompanies capacitation. In addition, we show that cSrc inhibition also blocks the agonist-induced acrosome reaction and that this inhibition is overcome by pharmacological hyperpolarization. Considering that capacitation-induced hyperpolarization is mediated by SLO3, we evaluated the action of cSrc inhibitors on the heterologously expressed SLO3 channel. Our results indicate that, similar to SLO1 K+ channels, cSrc blockers significantly decreased SLO3-mediated currents. Together, these results are consistent with findings showing that hyperpolarization of the sperm plasma membrane is necessary and sufficient to prepare the sperm for the acrosome reaction and suggest that changes in sperm membrane potential are mediated by cSrc activation.

Set-up of an infrared fast behavioral assay using zebrafish (Danio rerio) larvae, and its application in compound biotoxicity screening.

Zebrafish (Danio rerio) is increasingly employed for evaluating toxicity and drug discovery assays. Commonly experimental approaches for biotoxicity assessment are based on visual inspection or video recording. However, these techniques are limited for large‐scale assays, as they demand either a time‐consuming detailed inspection of the animals or intensive computing resources in order to analyze a considerable amount of screenshots. Recently, we have developed a simple methodology for tracking the locomotor activity of small animals cultured in microtiter plates. In this work, we implemented this automatic methodology, based on infrared (IR) microbeam scattering, for measuring behavioral activity in zebrafish larvae. We determined the appropriate culture conditions, number of animals and stage of development to get robust results. Furthermore, we validated this methodology as a rapid test for evaluating toxicity. By measuring the effects of reference compounds on larvae activity, we were able to estimate the concentration that could cause a 50% decrease in activity events values (AEC50), showing a strong linear correlation (R2 = 0.91) with the LC50 values obtained with the standard DarT test. The toxicity order of the measured compounds was CuSO4 > 2,4‐dinitrophenol > 3,4‐dichloroaniline > SDS > sodium benzoate > EDTA > K2CrO4; regarding solvents, EtOH ≈ DMSO. In this study, we demonstrate that global swimming behavior could be a simple readout for toxicity, easy to scale‐up in automated experiments. This approach is potentially applicable for fast ecotoxicity assays and whole‐organism high‐throughput compound screening, reducing the time and money required to evaluate unknown samples and to identify leading pharmaceutical compounds. Copyright

Characterization and biological properties of L-HGP, a glycoprotein from the amphibian oviduct with acrosome-stabilizing effects

Background information. The role of the jelly coat that surrounds the amphibian oocytes has been widely discussed, but is poorly understood. The presence of the jelly coat is essential for fertilization. However, the structure and function of the molecules that comprise the jelly coat have not been thoroughly documented. L‐HGP (low‐molecular‐mass highly glycosylated protein) is a highly glycosylated protein that is present in the jelly coat of the toad, Bufo arenarum, oocytes and diffuses to the surrounding media. L‐HGP, when purified from egg water, protects the sperm acrosome from breakdown induced by hypotonic solutions.

Egg Water from the Amphibian Bufo arenarum Modulates the Ability of Homologous Sperm to Undergo the Acrosome Reaction in the Presence of the Vitelline Envelope

Abstract Sperm from the toad Bufo arenarum must penetrate the egg jelly before reaching the vitelline envelope (VE), where the acrosome reaction is triggered. When the jelly coat is removed, sperm still bind to the VE, but acrosomal exocytosis is not promoted. Our previous work demonstrated that diffusible substances of the jelly coat, termed “egg water” (EW), triggered capacitation-like changes in B. arenarum sperm, promoting the acquisition of a transient fertilizing capacity. In the present work, we correlated this fertilizing capacity with the ability of the sperm to undergo the acrosome reaction, further substantiating the role of the jelly coat in fertilization. When sperm were exposed to the VE, only those preincubated in EW for 5 or 8 min underwent an increase in the intracellular Ca2+ concentration ([Ca2+]i), which led to acrosomal exocytosis. Responsiveness to the VE was not acquired on preincubation in EW for 2 or 15 min or in Ringer solution regardless of the preincubation time. In contrast, depletion of intracellular Ca2+ stores (induced by thapsigargin) promoted [Ca2+]i rise and the acrosome reaction even in sperm that were not exposed to EW. Acrosomal exocytosis was blocked by the presence of Ca2+ chelators independent of whether a physiological or pharmacological stimulus was used. However, Ni2+ and mibefradil prevented [Ca2+]i rise and the acrosome reaction of sperm exposed to the VE but not of sperm exposed to thapsigargin. These data suggest that the acrosomal responsiveness of B. arenarum sperm, present during a narrow period, is acquired during EW incubation and involves the modulation of a voltage-dependent Ca2+ channel.

Calcineurin regulates progressive motility activation of Rhinella (Bufo) arenarum sperm through dephosphorylation of PKC substrates.

Animals with external fertilization, as amphibians, store their sperm in a quiescent state in the testis. When spermatozoa are released into natural fertilization media, the hypotonic shock triggers activation of sperm motility. Rhinella (Bufo) arenarum sperm are immotile in artificial seminal plasma (ASP, resembling testicular plasma tonicity) but acquire in situ flagellar beating upon dilution. However, if components from the egg shelly coat are added to this medium, motility shifts to a progressive pattern. Recently, we have shown that the signal transduction pathway required for in situ motility activation involves a rise in intracellular cAMP through a transmembrane adenylyl cyclase and activation of PKA, mostly in the midpiece and in the sperm head. In this report, we demonstrate that activation of calcineurin (aka PP2B and PPP3) is required for the shift from in situ to progressive sperm motility. The effect of calcineurin is manifested by dephosphorylation of PKC substrates, and can be promoted by intracellular calcium rise by Ca2+ ionophore. Both phosphorylated PKC substrates and calcineurin localized to the flagella, indicating a clear differentiation between compartmentalization of PKA and calcineurin pathways. Moreover, no crosstalk is observed between these signaling events, even though both pathways are required for progressive motility acquisition as discussed. J. Cell. Physiol. 229: 1378–1386, 2014.