Sperm Activating And Attracting Factor Research Articles

The Role of Soluble Adenylyl Cyclase in the Regulation of Flagellar Motility in Ascidian Sperm.

Flagellar motility in sperm is activated and regulated by factors related to the eggs at fertilization. In the ascidian Ciona intestinalis, a sulfated steroid called the SAAF (sperm activating and attracting factor) induces both sperm motility activation and chemotaxis. Cyclic AMP (cAMP) is one of the most important intracellular factors in the sperm signaling pathway. Adenylyl cyclase (AC) is the key enzyme that synthesizes cAMP at the onset of the signaling pathway in all cellular functions. We previously reported that both transmembrane AC (tmAC) and soluble AC (sAC) play important roles in sperm motility in Ciona. The tmAC plays a major role in the SAAF-induced activation of sperm motility. On the other hand, sAC is involved in the regulation of flagellar beat frequency and the Ca2+-dependent chemotactic movement of sperm. In this study, we focused on the role of sAC in the regulation of flagellar motility in Ciona sperm chemotaxis. The immunochemical analysis revealed that several isoforms of sAC protein were expressed in Ciona sperm, as reported in mammals and sea urchins. We demonstrated that sAC inhibition caused strong and transient asymmetrization during the chemotactic turn, and then sperm failed to turn toward the SAAF. In addition, real-time Ca2+ imaging in sperm flagella revealed that sAC inhibition induced an excessive and prolonged Ca2+ influx to flagella. These results indicate that sAC plays a key role in sperm chemotaxis by regulating the clearance of [Ca2+]i and by modulating Ca2+-dependent flagellar waveform conversion.

A novel role for ATP2B in ascidians: Ascidian-specific mutations in ATP2B contribute to sperm chemotaxis.

Sperm chemotaxis, in which sperms are attracted to conspecific eggs via species-specific attractants, plays an important role in fertilization. This phenomenon has been observed in various animals and species-specific sperm attractants have been reported in some species. However, the mechanisms involved in the reception and recognition of the species-specific attractant by the sperms is poorly studied. Previously, we found that the plasma membrane-type Ca2+ /ATPase (PMCA) is the receptor for the sperm-activating and -attracting factor (SAAF) in the ascidian Ciona intestinalis. To determine the role of PMCA in species-specific sperm chemotaxis, we identified the amino acid sequences of PMCAs derived from six Phlebobranchia species. The testis-specific splice variant of PMCA was found to be present in all the species investigated and the ascidian-specific sequence was detected near the 3'-terminus. Moreover, dN/dS analysis revealed that the extracellular loops 1, 2, and 4 in ascidian PMCA underwent a positive selection. These findings suggest that PMCA recognizes the species-specific structure of SAAF at the extracellular loops 1, 2, and 4, and its testis-specific C-terminal region is involved in the activation and chemotaxis of ascidian sperms.

The Roles of Two CNG Channels in the Regulation of Ascidian Sperm Chemotaxis.

Spermatozoa sense and respond to their environmental signals to ensure fertilization success. Reception and transduction of signals are reflected rapidly in sperm flagellar waveforms and swimming behavior. In the ascidian Ciona intestinalis (type A; also called C. robusta), an egg-derived sulfated steroid called SAAF (sperm activating and attracting factor), induces both sperm motility activation and chemotaxis. Two types of CNG (cyclic nucleotide-gated) channels, Ci-tetra KCNG (tetrameric, cyclic nucleotide-gated, K+-selective) and Ci-HCN (hyperpolarization-activated and cyclic nucleotide-gated), are highly expressed in Ciona testis from the comprehensive gene expression analysis. To elucidate the sperm signaling pathway to regulate flagellar motility, we focus on the role of CNG channels. In this study, the immunochemical analysis revealed that both CNG channels are expressed in Ciona sperm and localized to sperm flagella. Sperm motility analysis and Ca2+ imaging during chemotaxis showed that CNG channel inhibition affected the changes in flagellar waveforms and Ca2+ efflux needed for the chemotactic turn. These results suggest that CNG channels in Ciona sperm play a vital role in regulating sperm motility and intracellular Ca2+ regulation during chemotaxis.

Distinct roles of soluble and transmembrane adenylyl cyclases in the regulation of flagellar motility in Ciona sperm.

Adenylyl cyclase (AC) is a key enzyme that synthesizes cyclic AMP (cAMP) at the onset of the signaling pathway to activate sperm motility. Here, we showed that both transmembrane AC (tmAC) and soluble AC (sAC) are distinctly involved in the regulation of sperm motility in the ascidian Ciona intestinalis. A tmAC inhibitor blocked both cAMP synthesis and the activation of sperm motility induced by the egg factor sperm activating and attracting factor (SAAF), as well as those induced by theophylline, an inhibitor of phoshodiesterase. It also significantly inhibited cAMP-dependent phosphorylation of a set of proteins at motility activation. On the other hand, a sAC inhibitor does not affect on SAAF-induced transient increase of cAMP, motility activation or protein phosphorylation, but it reduced swimming velocity to half in theophylline-induced sperm. A sAC inhibitor KH-7 induced circular swimming trajectory with smaller diameter and significantly suppressed chemotaxis of sperm to SAAF. These results suggest that tmAC is involved in the basic mechanism for motility activation through cAMP-dependent protein phosphorylation, whereas sAC plays distinct roles in increase of flagellar beat frequency and in the Ca2+-dependent chemotactic movement of sperm.

Lipid rafts function in Ca2+ signaling responsible for activation of sperm motility and chemotaxis in the ascidian Ciona intestinalis

Lipid rafts are specialized membrane microdomains that function as signaling platforms across plasma membranes of many animal and plant cells. Although there are several studies implicating the role of lipid rafts in capacitation of mammalian sperm, the function of these structures in sperm motility activation and chemotaxis remains unknown. In the ascidian Ciona intestinalis, egg-derived sperm activating- and attracting-factor (SAAF) induces both activation of sperm motility and sperm chemotaxis to the egg. Here we found that a lipid raft disrupter, methyl-β-cyclodextrin (MCD), inhibited both SAAF-induced sperm motility activation and chemotaxis. MCD inhibited both SAAF-promoted synthesis of intracellular cyclic AMP and sperm motility induced by ionophore-mediated Ca(2+) entry, but not that induced by valinomycin-mediated hyperpolarization. Ca(2+)-imaging revealed that lipid raft disruption inhibited Ca(2+) influx upon activation of sperm motility. The Ca(2+)-activated adenylyl cyclase was clearly inhibited by MCD in isolated lipid rafts. The results suggest that sperm lipid rafts function in signaling upstream of cAMP synthesis, most likely in SAAF-induced Ca(2+) influx, and are required for Ca(2+)-dependent pathways underlying activation and chemotaxis in Ciona sperm.

Second-generation synthesis of endogenous sperm-activating and attracting factor (SAAF) isolated from the ascidian Ciona intestinalis

Stereoselective synthesis of the chemoattractant sperm-activating and attracting factor (SAAF), isolated from the eggs of the ascidian Ciona intestinalis, was achieved via reductive 1,3-transposition of an allylic alcohol and the axial opening of an epoxide as key steps. This second-generation synthesis improved the total yield of SAAF over that of the first-generation synthesis and provided a key intermediate for synthesizing molecular probes of SAAF.

Ca2+ bursts occur around a local minimal concentration of attractant and trigger sperm chemotactic response.

Ca(2+) is known to have important roles in sperm chemotaxis, although the relationship between intracellular Ca(2+) concentration ([Ca(2+)](i)) and modulation of the swimming and chemotactic behavior of spermatozoa has not been elucidated. Using a high-speed Ca(2+) imaging system, we examined the chemotactic behavior and [Ca(2+)](i) in individual ascidian sperm cells exhibiting chemotactic responses toward sperm activating and attracting factor (SAAF), a chemoattractant released by eggs. In this study, we found that transient [Ca(2+)](i) increased in the flagellum (Ca(2+) bursts) concomitantly with a change in the swimming direction in an SAAF gradient field. During the initial phase of the Ca(2+) bursts, the flagellum of the spermatozoon exhibited highly asymmetric waveforms enabling the quick turning of the swimming path. However, the flagellum subsequently changed to symmetric beating, causing the spermatozoon to swim straight. Interestingly, during such responses, [Ca(2+)](i) remained higher than the basal level, indicating that the series of responses was not simply determined by Ca(2+) concentrations. Also, we found that Ca(2+) bursts were consistently evoked at points at which the spermatozoon attained around a temporally minimal value for a given SAAF concentration. We concluded that Ca(2+) bursts induced around a local minimal SAAF concentration trigger a sequence of flagellar responses comprising quick turning followed by straight swimming to direct spermatozoa efficiently toward eggs.

Ascidian sperm activating and attracting factor: Importance of sulfate groups for the activities and implication of its putative receptor

The sperm activating and attracting factor (SAAF) from the eggs of the ascidian Cionaintestinalis was identified as the sulfated polyhydroxysterol,3α,4β,7α,26-tetrahydroxy-5α-cholestane-3,26-disulfate. We present a functional analysis of SAAF derivatives that reveals the roles of the various SAAF functional groups. Optical isomerism does not affect SAAF activities. Hydrolysis on one side, i.e. at the sulfate groups of SAAF, decreases the sperm-activating and sperm-attracting activities, while hydrolysis on both sides resulted in the loss of both activities. Biotinylated-SAAF lost its sperm-activating ability, but retained sperm-binding and chemotactic abilities. Thus, the sulfate groups of SAAF are responsible for these activities.

Na+/Ca2+ exchanger modulates the flagellar wave pattern for the regulation of motility activation and chemotaxis in the ascidian spermatozoa

Ion channels and ion exchangers are known to be important participants in various aspects of sperm physiology, e.g. motility activation, chemotaxis, the maintenance of motility and the acrosome reaction in the sperm. We report here on a role of the K+ -independent Na+/Ca2+ exchanger (NCX) on ascidian sperm. Reverse-transcriptase PCR reveals that the NCX is expressed in the testis while immunoblotting and immunolocalization demonstrate that the NCX exists on the sperm in the ascidian Ciona savignyi and C. intestinalis. A potent blocker of the NCX, KB-R7943 was found to block sperm-activating and -attracting factor (SAAF)-induced motility activation, sperm motility and sperm chemotaxis. We further analyzed the effects of this blocker on motility parameters such as the flagellar waveform, curvature, beat frequency, amplitude and wavelength of the sperm flagella. Inhibition of the NCX caused two distinct effects: a low concentration of KB-R7943 induced symmetric bending, whereas a high concentration of KB-R7943 resulted in asymmetric flagellar bending. These findings suggest that the NCX plays important roles in the regulation of SAAF-induced sperm chemotaxis, motility activation and motility maintenance in the ascidian. This study provides new information toward an understanding of Ca2+ transport systems in sperm motility and chemotaxis.

Synthesis and Identification of an Endogenous Sperm Activating and Attracting Factor Isolated from Eggs of the Ascidian Ciona intestinalis: An Example of Nanomolar‐Level Structure Elucidation of Novel Natural Compound.

Abstract A sperm-activating and attracting factor (SAAF) was isolated from the eggs of the ascidian Ciona intestinalis, and its structure was deduced with only approximately 4 μg of the specimen. Based upon the proposed structure, two epimers were synthesized from chenodeoxycholic acid in 16 steps. Comparison between synthetic and natural compounds led to the unambiguous structure determination of SAAF to be (3R,4R,7R,25S)-3,4,7,26-tetrahydroxycholestane-3,26-disulfate. The synthetic pure specimen was also utilized to confirm that both sperm-activation and attraction were elicited by a single compound.

Calmodulin/calmodulin-dependent protein kinase II mediates SAAF-induced motility activation of ascidian sperm.

Ca2+-influx and membrane hyperpolarization by sperm-activating and -attracting factor (SAAF) released from the unfertilized egg of the ascidians Ciona cause a transient increase in cAMP, which triggers activation of sperm motility. We demonstrated here the presence of Ca2+-binding protein, calmodulin (CaM), and CaM-dependent kinase II (CaMKII) in the sperm. CaM antagonist, W-7, and CaMKII inhibitor, KN-93, suppressed SAAF-induced membrane hyperpolarization, increase in cAMP, and activation of sperm motility, but inactive analogues of W-7 and KN-93, namely W-5 and KN-92, respectively, did not. Subsequent addition of K+ ionophore, valinomycin, hyperpolarized the plasma membrane, increased cAMP, and conferred motility to the immotile sperm even in the presence of W-7 and KN-93. Addition of IBMX activated motility of sperm, which has been immobilized by W-7 and KN-93. These suggest that increased [Ca2+]i through influx of Ca2+ by SAAF binds to CaM to activate CaMKII. The activated CaMKII may cause membrane hyperpolarization to increase cAMP, which triggers the activation of sperm motility in Ciona.

Synthesis and identification of an endogenous sperm activating and attracting factor isolated from eggs of the ascidian Ciona intestinalis; an example of nanomolar-level structure elucidation of novel natural compound

A sperm-activating and attracting factor (SAAF) was isolated from the eggs of the ascidian Ciona intestinalis, and its structure was deduced with only approximately 4 μg of the specimen. Based upon the proposed structure, two epimers were synthesized from chenodeoxycholic acid in 16 steps. Comparison between synthetic and natural compounds led to the unambiguous structure determination of SAAF to be (3 R,4 R,7 R,25 S)-3,4,7,26-tetrahydroxycholestane-3,26-disulfate. The synthetic pure specimen was also utilized to confirm that both sperm-activation and attraction were elicited by a single compound.

Synthesis of Endogenous Sperm‐Activating and Attracting Factor Isolated from Ascidian Ciona intestinalis.

A chemoattractant candidate named sperm-activating and attracting factor (SAAF) from the eggs of ascidian Ciona intestinalis, was synthesized from chenodeoxycholic acid in 16 steps. The present synthesis led to the unambiguous structure determination of SAAF to be (3R,4R,7R,25S)-3,4,7,26-tetrahydroxycholestane-3,26-disulfate. The synthetic pure specimen was also used to confirm the dual sperm-activating and attracting activity.

Synthesis of endogenous sperm-activating and attracting factor isolated from ascidian Ciona intestinalis

A chemoattractant candidate named sperm-activating and attracting factor (SAAF) from the eggs of ascidian Ciona intestinalis, was synthesized from chenodeoxycholic acid in 16 steps. The present synthesis led to the unambiguous structure determination of SAAF to be (3 R,4 R,7 R,25 S)-3,4,7,26-tetrahydroxycholestane-3,26-disulfate. The synthetic pure specimen was also used to confirm the dual sperm-activating and attracting activity.

A chemoattractant for ascidian spermatozoa is a sulfated steroid.

Sperm chemotaxis toward eggs before fertilization has been demonstrated in many animals and plants, and several peptides and small organic compounds acting as chemoattractants have been identified. We previously showed that sperm of the ascidians Ciona intestinalis and Ciona savignyi are activated and then attracted toward the egg by a common factor released from the egg. In this study, we purified sperm-activating and -attracting factor (SAAF) from the egg-conditioning medium of C. intestinalis by using several steps of column chromatography. Determination of the molecular structure by NMR and MS/MS analysis revealed that SAAF is a previously uncharacterized sulfated steroid: 3,4,7,26-tetrahydroxycholestane-3,26-disulfate. Furthermore, it was shown that the SAAF of C. savignyi was indistinguishable from that of C. intestinalis in terms of the chromatographic behavior and molecular weight, indicating that the same compound might be responsible for sperm activation and chemotaxis in both the species. Furthermore, we established a method for quantitative analysis of sperm chemotaxis and showed that the chemotactic behavior of Ciona sperm is controlled by the "chemotactic turn" associated with decrease in the concentration of SAAF.

Cyclic AMP- and calmodulin-dependent phosphorylation of 21 and 26 kda proteins in axoneme is a prerequisite for SAAF-induced motile activation in ascidian spermatozoa.

Sperm activating and -attracting factor (SAAF), derived from the egg of the ascidian Ciona, activates sperm motility through adenosine 3':5'-cyclic monophosphate (cAMP)-synthesis. A demembranated preparation of intact immotile sperm without SAAF was shown to require cAMP for reactivation. However, a demembranated preparation of intact motile sperm treated with SAAF did not require cAMP for reactivation, suggesting that cAMP is a prerequisite factor for SAAF-dependent activation of sperm motility. Furthermore, a cAMP-dependent protein kinase (PKA) inhibitor, H-89, was found to inhibit sperm motility. During in vivo or in vitro activation of sperm motility by SAAF or cAMP, a 26 kDa axonemal protein and 21 kDa dynein light chain were phosphorylated, respectively, suggesting the involvement of PKA-dependent phosphorylation of these proteins in sperm activation. The calmodulin antagonist, W-7, and an inhibitor of calmodulin-dependent myosin light chain kinase, ML-7, also inhibited the activation of sperm motility. Inhibition was reversed by the addition of phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine. Demembranated preparations of immotile sperm in the presence of W-7 or ML-7 were reactivated by cAMP, suggesting that calmodulin participated in sperm activation and that cAMP synthesis was followed by activation of a calmodulin-dependent mechanism.

Phosphorylation of axonemal 21 kDa and 26 kDa proteins modulates activation of sperm motility in the ascidian,Ciona intestinalis

Protein phosphorylation is highly coupled with sperm motility activation in several animal species. The micro-tubule based flagellar motor protein, dynein, is a candidate for a phosphoprotein related to sperm activation in many animal species (Morisawa & Hayashi, 1985; Hayashiet al., 1987; Dey & Brokaw, 1991; Stephens & Prior, 1992; Inabaet al., 1998, 1999). Sperm motility of the ascidiansCiona intestinalisandC. savignyiis activated by a factor derived from unfertilised eggs named sperm activating and attracting factor (SAAF). SAAF elevates the intracellular cyclic AMP (cAMP) level by a mechanism dependent on membrane hyperpolarisation and extracellular Ca2+(Yoshidaet al., 1994; Izumiet al., 1999). Experiments using demembranatedCionasperm showed that cAMP is required prior to ATP for the activation of axonemal movement (Opreska & Brokaw, 1983; Morisawaet al., 1984; Brokaw, 1985; Dey & Brokaw, 1991; Chaudhryet al., 1995) and that many sperm flagellar proteins including dynein light chain are phosphorylated during incubation of demembranated sperm with ATP and cAMP (Dey & Brokaw, 1991). However, there is no evidence of which proteins are phosphorylated during the SAAF-dependent activation ofCionasperm motility.

Membrane Hyperpolarization by Sperm-Activating and -Attracting Factor Increases cAMP Level and Activates Sperm Motility in the Ascidian Ciona intestinalis

In the ascidian Ciona intestinalis (and C. savignyi), sperm-activating and -attracting factor (SAAF) is released from the egg at fertilization and stimulates both Ca2+ influx and a transient increase in cAMP level of the sperm, leading to the activation of sperm motility (M. Yoshida et al., 1994, Dev. Growth Differ. 36, 589–595). In this paper we show in C. intestinalis that valinomycin, a potassium-selective ionophore, as well as SAAF, activated sperm motility, and this activation was suppressed by extracellular high K+. Membrane potential measurements showed that both SAAF and valinomycin increase K+ permeability of sperm and induce membrane hyperpolarization, the amplitude of which depends on the external K+ concentration. The membrane potential and intracellular K+ concentration of Ciona sperm without SAAF were estimated to be about −50 mV and 560 ± 40 mM, respectively. After treatment with SAAF or valinomycin the membrane potential became almost equal to the equilibrium potential of K+ (−100 mV), and the cAMP level increased in artificial seawater. A potent voltage-dependent K+ channel blocker, MCD peptide, at the concentration of 10 μM blocked SAAF-induced hyperpolarization of the cells, increase in cAMP, and sperm motility. These results suggest that membrane hyperpolarization produced by the opening of K+ channels elevates cAMP synthesis and leads to the activation of sperm motility in Ciona.

Calcium and Cyclic AMP Mediate Sperm Activation, but Ca2+ Alone Contributes Sperm Chemotaxis in the Ascidian, Ciona savignyi: (ascidian/sperm motility/chemotaxis/calcium/cAMP).

Sperm-activating and -attracting factor (SAAF) released from the ascidian, Ciona savignyi, was partially purified from egg seawater with ethanol extraction and separation with the two-phase system of chloroform and water. SAAF did not activate sperm motility and cAMP synthesis in calcium-free seawater (CaFSW), but activated the both in the presence of Ca2+ . Sperm activation by SAAF in Ca2+ -containing medium was inhibited by flunarizine, a T-type Ca2+ channel antagonist, but L-type Ca2+ channel specific antagonists had no effect. Theophylline, a phosphodiesterase inhibitor, induced the increase of cAMP level and sperm activation in CaFSW without SAAF. On the other hand, the theophylline-activated sperm in CaFSW did not exhibit chemotaxis toward the tip of glass capillary containing SAAF, but upon the addition of Ca2+ they were attracted toward SAAF in the same manner as chemotaxis in normal artificial seawater. These results suggest that sperm activation is induced by the increased cAMP level caused by Ca2+ influx through T-type Ca2+ channel, and that Ca2+ alone mediates the sperm chemotaxis in Ciona.