Biomedical subjects

C L Treviño

Publications and source records attributed to C L Treviño.

10 recordsLinked to original sources

PubMed2001

Ion transport in sperm signaling.

Ion channels and transporters, key elements in sperm-egg signaling and environmental sensing, are essential for fertilization. External cues and components from the outer envelopes of the egg influence sperm ion permeability and behavior. Combining in vivo measurements of membrane potential, intracellular ions, and second messengers with new molecular approaches and reconstitution strategies are revealing how sperm ion channels participate in motility, sperm maturation, and the acrosome reaction. Sperm are tiny differentiated terminal cells unable to synthesize proteins and difficult to characterize electrophysiologically. Spermatogenic cells, the progenitors of sperm, have become useful tools for probing sperm ion channels since they are larger and molecular biology techniques can be applied. These complementary strategies are opening new avenues to determine how sperm ion channels function in gamete signaling.

Animals↗

PubMed2001

Identification of mouse trp homologs and lipid rafts from spermatogenic cells and sperm.

Intracellular Ca(2+) has an important regulatory role in the control of sperm motility, capacitation, and the acrosome reaction (AR). However, little is known about the molecular identity of the membrane systems that regulate Ca(2+) in sperm. In this report, we provide evidence for the expression of seven Drosophila transient receptor potential homolog genes (trp1-7) and three of their protein products (Trp1, Trp3 and Trp6) in mouse sperm. Allegedly some trps encode capacitative Ca(2+) channels. Immunoconfocal images showed that while Trp6 was present in the postacrosomal region and could be involved in sperm AR, expression of Trp1 and Trp3 was confined to the flagellum, suggesting that they may serve sperm to regulate important Ca(2+)-dependent events in addition to the AR. Likewise, one of these proteins (Trp1) co-immunolocalized with caveolin-1, a major component of caveolae, a subset of lipid rafts potentially important for signaling events and Ca(2+) flux. Furthermore, by using fluorescein-coupled cholera toxin B subunit, which specifically binds to the raft component ganglioside GM1, we identified caveolin- and Trp-independent lipid rafts residing in the plasma membrane of mature sperm. Notably, the distribution of GM1 changes drastically upon completion of the AR.

Acrosome Reaction↗

PubMed1999

Voltage-dependent Ca(2+) channel subunit expression and immunolocalization in mouse spermatogenic cells and sperm.

Though voltage-dependent Ca(2+) channels contribute to the orchestration of sperm differentiation and function, many questions remain concerning their molecular architecture. This study shows that alpha(1A) and alpha(1C) Ca(2+) channel pore-forming subunits are expressed in spermatogenic cells. In addition, it provides what is to our knowledge the first evidence for the presence of the Ca(2+) channel beta auxiliary subunits in spermatogenic cells and sperm. Using RT-PCR we demonstrated the expression of all four known genes encoding the beta subunits in spermatogenic cells. Specific antibodies detected three of these proteins in spermatogenic cells and sperm. In spermatogenic cells both alpha(1) and beta subunits are diffusely distributed throughout the cytoplasm while in sperm they appear to be regionally localized.

Animals↗

PubMed1999

Anion channel blockers differentially affect T-type Ca(2+) currents of mouse spermatogenic cells, alpha1E currents expressed in Xenopus oocytes and the sperm acrosome reaction.

The direct electrophysiological characterization of sperm Ca(2+) channels has been precluded by their small size and flat shape. An alternative to study these channels is to use spermatogenic cells, the progenitors of sperm, which are larger and easier to patch-clamp. In mouse and rat, the only voltage-dependent Ca(2+) currents displayed by these cells are of the T type. Because compounds that block these currents inhibit the zona pellucida-induced Ca(2+) uptake and the sperm acrosome reaction (AR) at similar concentrations, it is likely that they are fundamental for this process. Recent single channel recordings in mouse sperm demonstrated the presence of a Cl(-) channel. This channel and the zona pellucida (ZP)-induced AR were inhibited by niflumic acid (NA), an anion channel blocker [Espinosa et al. (1998): FEBS Lett 426:47-51]. Because NA and other anion channel blockers modulate cationic channels as well, it became important to determine whether they affect the T-type Ca(2+) currents of spermatogenic cells. These currents were blocked in a voltage-dependent manner by NA, 1, 9-dideoxyforskolin (DDF), and 5-nitro-2-(3-phenylpropylamine)benzoic acid (NPPB). The IC(50) values at -20 mV were 43 microM for NA, 28 microM for DDF, and 15 microM for NPPB. Moreover, DDF partially inhibited the ZP-induced AR (40% at 1 microM) and NPPB displayed an IC(50) value of 6 microM for this reaction. These results suggest that NA and DDF do not inhibit the ZP-induced AR by blocking T-type Ca(2+) currents, while NPPB may do so. Interestingly 200 microM NA was basically unable to inhibit alpha1E Ca(2+) channels expressed in Xenopus oocytes, questioning that this alpha subunit codes for the T-type Ca(2+) channels present in spermatogenic cells. Evidence for the presence of alpha1C, alpha1G, and alpha1H in mouse pachytene spematocytes and in round and condensing spermatids is presented.

Acrosome Reaction↗

PubMed1998

Localisation of inositol trisphosphate and ryanodine receptors during mouse spermatogenesis: possible functional implications.

During spermatogenesis the activity of intracellular Ca(2+)-release channels is likely to play an important role in different specific cellular functions. Accordingly, messenger RNAs for the three inositol 1,4,5-trisphosphate receptor (IP3R) subtypes were found to be present throughout spermatogenesis. Immunocytochemical analysis revealed distinct distribution patterns of the mature IP3Rs during sperm differentiation. At early stages, IP3Rs are distributed throughout the cytoplasm, and as differentiation proceeds they become selectively localised to the Golgi complex. Consistently, spermatogonia underwent large intracellular Ca2+ release in response to thapsigargin (TG), while smaller responses were detected in late spermatocytes and spermatids. The distribution of IP3Rs and the larger Ca(2+)-release responses found in spermatogonia, suggest that IP3Rs may be involved in cell proliferation at this stage. This notion is supported by our observations in a spermatogenic cell line that depletion of intracellular Ca2+ pools using TG inhibits cell division, and that incubation with an IP3R-I antisense oligonucleotide completely inhibited proliferation. Furthermore, the three genes encoding ryanodine receptor proteins (RyRs) are expressed at all stages of spermatogenesis. However, immunocytochemical studies with specific antibodies against each of the RyR subtypes detected types 1 and 3 in spermatogenic cells and only type 3 in mature sperm. In contrast to IP3Rs, RyRs remain scattered in the cytoplasm throughout differentiation. Functional responses to caffeine and ryanodine were absent in spermatogenic cells and in mature sperm. These findings suggest that IP3Rs have significantly more important roles in spermatogenesis than RyRs, and that one of these roles is crucial for cell proliferation.

Animals↗

PubMed1996

T-type Ca2+ channels and alpha1E expression in spermatogenic cells, and their possible relevance to the sperm acrosome reaction.

There is pharmacological evidence that Ca2+ channels play an essential role in triggering the mammalian sperm acrosome reaction, an exocytotic process required for sperm to fertilize the egg. Spermatozoa are small terminally differentiated cells that are difficult to study by conventional electrophysiological techniques. To identify the members of the voltage-dependent Ca2+ channel family possibly present in sperm, we have looked for the expression of the alpha 1A, alpha 1B, alpha 1C, alpha 1D and alpha 1E genes in mouse testis and in purified spermatogenic cell populations with RT-PCR. Our results indicate that all 5 genes are expressed in mouse testis, and in contrast only alpha 1E, and to a minor extent alpha 1A, are expressed in spermatogenic cells. In agreement with these findings, only T-type Ca2+ channels sensitive to the dihydropyridine nifedipine were observed in patch-clamp recordings of pachytene spermatocytes. These results suggest that low-threshold Ca2+ channels are the dihydropyridine-sensitive channels involved in the sperm acrosome reaction.

Acrosome↗

PubMed1992

Evidence that deprotonation of serine-55 is responsible for the pH-dependence of the parvalbumin Eu3+ 7F0-->5D0 spectrum.

The Eu(III)7F0-->5D0 excitation spectra of the parvalbumins are highly pH-dependent. Below pH 6.0, they exhibit a sharp, partially resolved doublet centered near 5,795 A. However, as the pH is raised, the spectrum becomes increasingly dominated by a much broader signal near 5,784 A. This behavior has been traced to the Eu(III) ion bound at the CD site, but the identity of the moiety undergoing deprotonation remains uncertain. Site-specific mutagenesis studies on the parvalbumin-like protein known as oncomodulin now suggest that the species in question is a liganding serine hydroxyl group. Specifically, replacement of serine-55 by aspartate (the residue present at the corresponding position in the EF site) affords a protein that retains two functional lanthanide binding sites, but fails to undergo the pH-dependent spectral alteration. By contrast, replacement of aspartate-59 by glycine (the corresponding EF site residue) fails to abolish the pH-dependent behavior.

Animals↗

PubMed1991

Interactions between residues in the oncomodulin CD domain influence Ca2+ ion-binding affinity.

Despite striking sequence homology with rat parvalbumin, oncomodulin exhibits much lower affinity for Ca2+ ion. We are attempting to identify the structural basis for this difference by systematically substituting the parvalbumin residue for the oncomodulin residue at points of nonidentity. In this paper, we examine two mutations in the helical segments flanking the CD ion-binding loop. Replacement of Asp-45 in the C helix by lysine, to produce D45K, reduces the dissociation constant for Ca2+ at the CD site from 0.81 to 0.53 microM. Replacement of Lys-69 in the D helix by glycine, to afford K69G, similarly reduces KCa to 0.59 microM. Both mutations perturb the Eu3+ 7Fo----5Do spectral parameters. We also examine the consequences of simultaneous mutations involving positions 57, 59, 60, and 69. Ca(2+)-binding assays and Eu3+ luminescence measurements indicate that there is a conformational interaction between residues 57 and 69 and that this interaction is modulated by residues 59 and 60. When the mutations at positions 57, 59, 60, and 69 are combined, the resulting variant exhibits a KCa value for the CD site of 0.25 microM, reflecting a 3-fold increase in affinity relative to the wild-type protein. Moreover, the pK alpha governing the interconversion of low and high pH forms of the Eu3+ 7Fo----5Do spectrum is increased to 8.1, very close to the value of 8.25 determined previously for rat parvalbumin. In this paper, we also complete our survey of single mutations in the CD loop by examining L58I. Replacement of Leu-58 by isoleucine reduces the affinity of the CD site for Ca2+, raising KCa to 2.2 microM. Finally, we revise our previous estimate of the KCa value for Y57F downward, from 0.80 to 0.64 microM. The earlier result is believed to have been inflated by heterogeneity in the preparation, a consequence of proteolysis.

Amino Acid Sequence↗

PubMed1990

Site-specific replacement of amino acid residues within the CD binding loop of rat oncomodulin.

Relative to the same site in oncomodulin, the CD ion-binding domain of rat parvalbumin exhibits much greater affinity for Ca2+ and Mg2+. As part of an effort to understand the structural basis for these differences, site-specific variants of oncomodulin have been prepared in which the amino acid residues at positions 52, 54, 57, 59, and 60 have been replaced with the residues present at the corresponding positions in rat parvalbumin. The proteins resulting from the single-site substitutions at residues 52, 54, and 57 are indistinguishable from the wild-type protein on the basis Eu3+ luminescence spectroscopy, and none of the three variants displays increased affinity for Ca2+. By contrast, the substitutions at residues 59 and 60 perturb both the Eu3+ luminescence parameters and the Ca2+ and Mg2+ affinities, and these differences are amplified when both replacements are simultaneously incorporated into the protein. The Eu3+ 7F0----5D0 spectrum of the double variant (D59E/G60E) at pH 5.0, with a maximum at 5796 A and pronounced shoulder at 5791 A, strongly resembles that obtained with pike parvalbumin. Consistent with this increased parvalbumin-like character, KCa is decreased from 0.78 microM (for the wild-type protein) to 0.41 microM, and KMg is decreased from 3.5 to 0.74 mM. Nevertheless, the affinity of the CD ion-binding domain in D59E/G60E for Ca2+ remains almost 2 orders of magnitude lower than the corresponding site in rat parvalbumin, strongly suggesting that residues besides those present in the binding loop are involved in dictating the metal ion-binding properties of the oncomodulin CD site.

Animals↗

PubMed1990

Eu3+ luminescence studies of oncomodulin. The origin of the pH-dependent behavior.

The Eu3+ 7F0----5D0 excitation spectra of parvalbumin and oncomodulin are pH-dependent. Until now, it had been assumed that both the CD and EF ion-binding sites shared this property and that deprotonation of water molecules coordinated to the bound Eu3+ ions might be responsible for the pH dependence. Results obtained with the site-specific variant of oncomodulin known as D59E, in which glutamate replaces the aspartate naturally present at position 59, have necessitated substantial revision of these ideas. It now appears that the pH-dependent behavior is confined to the CD site. Moreover, we observe no corresponding change in the number of O-H oscillators coordinated to the bound Eu3+ ions in the pH range over which we observe the spectroscopic alteration. It is likely that the behavior results from deprotonation of one or more carboxyl groups clustered at the COOH-terminal end of the CD domain.

Amino Acid Sequence↗