RESUMO

As in most cells, intracellular pH regulation is fundamental for sperm physiology. Key sperm functions like swimming, maturation, and a unique exocytotic process, the acrosome reaction, necessary for gamete fusion, are deeply influenced by pH. Sperm pH regulation, both intracellularly and within organelles such as the acrosome, requires a coordinated interplay of various transporters and channels, ensuring that this cell is primed for fertilization. Consistent with the pivotal importance of pH regulation in mammalian sperm physiology, several of its unique transporters are dependent on cytosolic pH. Examples include the Ca2+ channel CatSper and the K+ channel Slo3. The absence of these channels leads to male infertility. This review outlines the main transport elements involved in pH regulation, including cytosolic and acrosomal pH, that participate in these complex functions. We present a glimpse of how these transporters are regulated and how distinct sets of them are orchestrated to allow sperm to fertilize the egg. Much research is needed to begin to envision the complete set of players and the choreography of how cytosolic and organellar pH are regulated in each sperm function.

Assuntos

RESUMO

Sperm capacitation, crucial for fertilization, occurs in the female reproductive tract and can be replicated in vitro using a medium rich in bicarbonate, calcium, and albumin. These components trigger the cAMP-PKA signaling cascade, proposed to promote hyperpolarization of the mouse sperm plasma membrane through activation of SLO3 K+ channel. Hyperpolarization is a hallmark of capacitation: proper membrane hyperpolarization renders higher in vitro fertilizing ability, while Slo3 KO mice are infertile. However, the precise regulation of SLO3 opening remains elusive. Our study challenges the involvement of PKA in this event and reveals the role of Na+/H+ exchangers. During capacitation, calcium increase through CatSper channels activates NHE1, while cAMP directly stimulates the sperm-specific NHE, collectively promoting the alkalinization threshold needed for SLO3 opening. Hyperpolarization then feeds back Na+/H+ activity. Our work is supported by pharmacology, and a plethora of KO mouse models, and proposes a novel pathway leading to hyperpolarization.

RESUMO

Mitochondria are important organelles in eukaryotic cells and play an essential role in energy production and cell signaling. However, the importance of mammalian sperm mitochondria as an energy source remains to be elucidated because glycolysis is known to be dominant. In this context, one of the functions of mammalian sperm mitochondria is considered as a calcium ion (Ca2+) homeostasis. Previously, the Ca2+ level within the mitochondria of mouse sperm under resting conditions was reported to be high (in the micromolar range) using the fluorescent Ca2+ indicator Calcium Green-5N (CG-5N). To confirm this fact, we performed the semi-quantitative determination of Ca2+ concentration with several Ca2+ indicators. Although we reproduced the previous report of CG-5N, other Ca2+ indicators do not support the result obtained with CG-5N. The results obtained with Rhod-2, Fluo-3, and Fluo-5N indicate that the free Ca2+ concentration in mitochondria is comparable to that of the cytosol at the resting condition and under the condition stimulated by ATP. Although we still do not understand why CG-5N exhibits a distinct result from other indicators, the regulation of Ca2+ concentration in murine sperm mitochondria is analogous to that observed in somatic cells. Namely, the Ca2+ concentrations within sperm mitochondria fluctuate in response to changes in cytosolic Ca2+ levels. Our results contribute to a revised understanding of the role of mitochondria in Ca2+ homeostasis in mammalian sperm.

RESUMO

The membrane potential of a cell (Vm) regulates several physiological processes. The voltage sensor domain (VSD) is a region that confers voltage sensitivity to different types of transmembrane proteins such as the following: voltage-gated ion channels, the voltage-sensing phosphatase (Ci-VSP), and the sperm-specific Na+/H+ exchanger (sNHE). VSDs contain four transmembrane segments (S1-S4) and several positively charged amino acids in S4, which are essential for the voltage sensitivity of the protein. Generally, in response to changes of the Vm, the positive residues of S4 displace along the plasma membrane without generating ionic currents through this domain. However, some native (e.g., Hv1 channel) and mutants of VSDs produce ionic currents. These gating pore currents are usually observed in VSDs that lack one or more of the conserved positively charged amino acids in S4. The gating pore currents can also be induced by the isolation of a VSD from the rest of the protein domains. In this review, we summarize gating pore currents from all families of proteins with VSDs with classification into three cases: (1) pathological, (2) physiological, and (3) artificial currents. We reinforce the model in which the position of S4 that lacks the positively charged amino acid determines the voltage dependency of the gating pore current of all VSDs independent of protein families.

Assuntos

RESUMO

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.

Assuntos

RESUMO

Amino acid-derived isoindolines are synthetic compounds that were created with the idea of investigating their biological actions. The amino acid moiety was included on the grounds that it may help to avoid toxic effects. Recently, the isoindoline MDIMP was shown to inhibit both cardiac excitation-contraction coupling and voltage-dependent calcium channels. Here, we revealed that MDIMP binds preferentially to low-voltage-activated (LVA) channels. Using a holding potential of -90 mV, the following IC50 values were found (in micromolars): >1000 (CaV2.3), 957 (CaV1.3), 656 (CaV1.2), 219 (CaV3.2), and 132 (CaV3.1). Moreover, the isoindoline also promoted both accelerated inactivation kinetics of high-voltage-activated Ca2+ channels and a modest upregulation of CaV1.3 and CaV2.3. Additional data indicate that although MDIMP binds to the closed state of the channels, it has more preference for the inactivated one. Concerning CaV3.1, the compound did not alter the shape of the instantaneous current-voltage curve, and substituting one or two residues in the selectivity filter drastically increased the IC50 value, suggesting that MDIMP binds to the extracellular side of the pore. However, an outward current failed in removing the inhibition, which implies an alternative mechanism may be involved. The enantiomer (R)-MDIMP [methyl (R)-2-(1,3-dihydroisoindol-2-yl)-4-methylpentanoate], on the other hand, was synthesized and evaluated, but it did not improve the affinity to LVA channels. Implications of these findings are discussed in terms of the possible underlying mechanisms and pharmacological relevance. SIGNIFICANCE STATEMENT: We have studied the regulation of voltage-gated calcium channels by MDIMP, which disrupts excitation-contraction coupling in cardiac myocytes. The latter effect is more potent in atrial than ventricular myocytes, and this could be explained by our results showing that MDIMP preferentially blocks low-voltage-activated channels. Our data also provide mechanistic insights about the blockade and suggest that MDIMP is a promising member of the family of Ca2+ channel blockers, with possible application to the inhibition of subthreshold membrane depolarizations.

Assuntos

RESUMO

Intracellular pH (pH i ) plays a crucial role in mammalian sperm physiology. However, it is a challenging task to acquire quantitative single sperm pH i images due to their small size and beating flagella. In this study, we established a robust pH i imaging system using the dual-emission ratiometric pH indicator, SNARF-5F. Simultaneous good signal/noise ratio fluorescence signals were obtained exciting with a green high-power LED (532 nm) and acquiring with an EM-CCD camera through an image splitter with two band-pass filters (550-600 nm, channel 1; 630-650 nm, channel 2). After in vivo calibration, we established an imaging system that allows determination of absolute pH i values in spermatozoa, minimizing cell movement artifacts. Using this system, we determined that bicarbonate increases non-capacitated human pH i with slower kinetics than in mouse spermatozoa. This difference suggests that distinct ionic transporters might be involved in the bicarbonate influx into human and mouse spermatozoa. Alternatively, pH i regulation downstream bicarbonate influx into spermatozoa could be different between the two species.

RESUMO

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.

Assuntos

RESUMO

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.

Assuntos

RESUMO

Progesterone is a physiological agonist for mammalian sperm, modulating its flagellar movement and facilitating the acrosome reaction. To study the initial action of progesterone, we developed a caged analog with a photosensitive group: nitrophenylethanediol, at position 20. Using this compound combined with stroboscopic illumination, we performed Ca(2)(+) imaging of human spermatozoa and analyzed the effects of progesterone on the intracellular Ca(2)(+) concentration ([Ca(2)(+)](i)) of beating flagella for the first time. We observed a transient [Ca(2)(+)](i) increase in the head and the flagellum upon photolysis of the caged progesterone and an increase in flagellar curvature. Detailed kinetic analysis revealed that progesterone elicits an increase in the [Ca(2)(+)](i) immediately in the flagellum (mid-piece and principal piece), thereafter in the head with a short time lag. This observation is different from the progesterone-induced Ca(2)(+) mobilization in mouse spermatozoa, where the Ca(2)(+) rise initiates at the base of the sperm head. Our finding is mostly consistent with the recent discovery that progesterone activates CatSper channels in human spermatozoa, but not in mouse spermatozoa.

Assuntos