Publications

Intracellular pH in sperm physiology

Published in Biochemical and Biophysical Research Communications

Intracellular pH (pHi) regulation is essential for cell function. Notably," several unique sperm ion transporters and enzymes whose elimination causes infertility are either pHi dependent or somehow related to pHi regulation. Amongst them are: CatSper"," a Ca 2+ channel; Slo3"," a K + channel; the sperm-specific Na+/H+ exchanger and the soluble adenylyl cyclase. It is thus clear that pHi regulation is of the utmost importance for sperm physiology. This review briefly summarizes the key components involved in pHi regulation"," their characteristics and participation in fundamental sperm functions such as motility"," maturation and the acrosome reaction.

Recommended citation: Nishigaki et al.", 2014 <a href="http://fraroco.github.io/files/NishigakiT_BIC_2014.pdf"">http://fraroco.github.io/files/NishigakiT_BIC_2014.pdf"</a>

Comparative genomic analysis suggests that the sperm-specific sodium/proton exchanger and soluble adenylyl cyclase are key regulators of CatSper among the Metazoa

Published in Zoological Letters

CatSper is a sperm-specific calcium ion (Ca2+) channel," which regulates sperm flagellar beating by tuning cytoplasmic Ca2+ concentrations. Although this Ca2+ channel is essential for mammalian fertilization"," recent bioinformatics analyses have revealed that genes encoding CatSper are heterogeneously distributed throughout the eukaryotes"," including vertebrates. As this channel is activated by cytoplasmic alkalization in mammals and sea urchins"," it has been proposed that the sperm-specific Na+ /H+ exchanger (sNHE"," a product of the SLC9C gene family) positively regulates its activity. In mouse"," sNHE is functionally coupled to soluble adenylyl cyclase (sAC). CatSper"," sNHE"," and sAC have thus

Recommended citation: Romero et al.", 2019 http://fraroco.github.io/files/romero_2020.pdf

FRET-based binding assay between a fluorescent cAMP analogue and a cyclic nucleotide-binding domain tagged with a CFP

Published in FEBS Letters

The cyclic nucleotide-binding domain (CNBD) functions as a regulatory domain of many proteins involved in cyclic nucleotide signalling. We developed a straightforward and reliable binding assay based on intermolecular fluorescence resonance energy transfer (FRET) between an adenosine-3’,5’-cyclic monophosphate analogue labelled with fluorescein and a recombinant CNBD of human EPAC1 tagged with a cyan fluorescence protein (CFP). The high FRET efficiency of this method (~ 80%) allowed us to perform several types of binding experiments with nanomolar range of sample using conventional equipment. In addition," the CFP tag on the CNBD enabled us to perform a specific binding experiment using an unpurified protein. Considering these advantages"," this technique is useful to study poorly characterized CNBDs.

Recommended citation: Romero et al.", 2017 http://fraroco.github.io/files/FRomero_FEBSLETTERS_2017.pdf

How to study a highly toxic protein to bacteria: A case of voltage sensor domain of mouse sperm-specific sodium/proton exchanger

Published in Protein Expression and Purification

Heterologous expression systems have been used as a powerful experimental strategy to study the function of many proteins, particularly ion transporters. For this experiment, it is fundamental to prepare an expression vector encoding a protein of interest. However, we encountered problems in vector preparation of the voltage sensor domain (VSD) of murine sperm-specific Na+/H+ exchanger (sNHE) due to its severe toxicity to bacteria. We overcame the problems by insertion of an amber stop codon or a synthetic intron into the coding sequence of the VSD in the expression vectors. Both methods allowed us to express the protein of interest in HEK293 cells (combined with a stop codon suppression system for amber codon). The VSD of mouse sNHE generates voltage- dependent outward ionic currents, which is a probable cause of toxicity to bacteria. We propose these two strategies as practical solutions to study the function of any protein toxic to bacteria.

Recommended citation: Arcos et al.", 2023. http://fraroco.github.io/files/ArcosC_PEP_2023.pdf

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Robust evaluation of intermolecular FRET using a large Stokes shift fluorophore as a donor

Published in BioTechniques

Fluorescence (or Förster) resonance energy transfer (FRET) is a straightforward and sensitive technique to evaluate molecular interactions. However," most of the popular FRET pairs suffer cross-excitation of the acceptor"," which could lead to false positives. To overcome this problem"," we selected a large Stokes shift (LSS) fluorophore as a FRET donor. As a successful example"," we employed a new FRET pair mAmetrine (an LSS yellow fluorescence protein)/DY-547 (a cyanine derivative) to substitute CFP/fluorescein that we previously employed to study molecular interactions between cyclic nucleotide binding domains and cyclic nucleotides. The new FRET pair is practically free of cross-excitation of the acceptor. Namely"," a change in the fl uorescence spectral shape implies evidence of FRET without other control experiments.

Recommended citation: Santana-Calvo et al.", 2018 http://fraroco.github.io/files/Santana-Calvo_BT_2018.pdf,

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FRET analysis using sperm-activating peptides tagged with fluorescent proteins reveals that ligand-binding sites exist as clusters

Published in Journal of Experimental Biology

Long-range cellular communication between the sperm and egg is critical for external fertilization. Sperm-activating peptides (SAPs) are diffusible components of the outer layer of eggs in echinoderms," and function as chemoattractants for spermatozoa. The decapeptide named speract is the best-characterized sea urchin SAP. Biochemical and physiological actions of speract have been studied with purified or chemically synthesized peptides. In this work"," we prepared recombinant speract fused to a fluorescent protein (FP; FP-speract) using three color variants: a cyan (eCFP)"," a yellow (mVenus) and a large Stokes shift yellow (mAmetrine) FP. Although these fluorescence tags are 20 times larger than speract"," competitive binding experiments using mAmetrine-speract revealed that this FP- speract has binding affinity to the receptor that is comparable (7.6-fold less) to that of non-labeled speract. Indeed"," 10 nmol l −1 eCFP- speract induces physiological sperm responses such as membrane potential changes and increases in intracellular pH and Ca 2+ concentrations similar to those triggered by 10 nmol l −1 speract. Furthermore"," FP-speract maintains its fluorescence upon binding to its receptor. Using this property"," we performed fluorescence resonance energy transfer (FRET) measurements with eCFP-speract and mVenus-speract as probes and obtained a positive FRET signal upon binding to the receptor"," which suggests that the speract receptor exists as an oligomer"," at least as a dimer"," or alternatively that a single speract receptor protein possesses multiple binding sites. This property could partially account for the positive and/or negative cooperative binding of speract to the receptor.

Recommended citation: Arcos-Hernández et al.", 2016 http://fraroco.github.io/files/ArcosC_JEB_2016.pdf