Regulation of salmonid fish sperm motility by osmotic shock-induced water influx across the plasma membrane.

The motility of salmonid fish sperm is initiated by a decrease in the extracellular K(+) concentration. However, our previous studies revealed that salmonid fish sperm motility could be initiated in the presence of an inhibitory concentration of K(+) by drastic osmotic shock induced by suspension in a hypertonic glycerol solution and subsequent dilution in a hypotonic solution (glycerol-treatment). In the present study, we examined if an osmotic shock-induced water influx is involved in the regulation of salmonid fish sperm motility. HgCl2, a common inhibitor of aquaporins (AQPs), decreased the duration of salmonid fish sperm motility. Dilution of sperm cells in a hypotonic solution increased the cellular volume, whereas HgCl2 inhibited such an increase in cellular volume. Furthermore, the expression of AQP 1a and 10 in rainbow trout testes was confirmed. In contrast, HgCl2 did not affect glycerol-treated sperm motility, indicating that AQPs are not involved in glycerol-treated sperm motility. We also explored the possibility of aquaporin-independent water influx in glycerol-treated sperm by assessing the sperm membrane permeability using propidium iodide. The plasma membrane of glycerol-treated sperm was considerably permeabilized. The cellular volume was decreased in a hypertonic glycerol solution and increased upon subsequent hypoosmotic shock, indicating an AQP-independent water flux across the plasma membrane upon glycerol-treatment. Taken together, these results showed that water influx across the plasma membrane via AQP is crucial for the maintenance of salmonid fish sperm motility under normal conditions, whereas water influx by osmotic shock-induced membrane permeation is critical for the initiation of glycerol-treated sperm motility.

Glycolysis plays an important role in energy transfer from the base to the distal end of the flagellum in mouse sperm.

Many studies have been conducted to elucidate the relationship between energy metabolic pathways (glycolysis and respiration) and flagellar motility in mammalian sperm, but the contribution of glycolysis to sperm motility has not yet been fully elucidated. In the present study, we performed detailed analysis of mouse sperm flagellar motility for further understanding of the contribution of glycolysis to mammalian sperm motility. Mouse sperm maintained vigorous motility in the presence of substrates either for glycolysis or for respiration. By contrast, inhibition of glycolysis by alpha-chlorohydrine caused a significant decrease in the bend angle of the flagellar bending wave, sliding velocity of outer doublet microtubules and ATP content even in the presence of respiratory substrates (pyruvate or ß-hydroxybutyrate). The decrease of flagellar bend angle and sliding velocity are prominent in the distal part of the flagellum, indicating that glycolysis inhibition caused the decrease in ATP concentration threrein. These results suggest that glycolysis potentially acts as a spatial ATP buffering system, transferring energy (ATP) synthesized by respiration at the mitochondria located in the basal part of the flagellum to the distal part. In order to validate that glycolytic enzymes can transfer high energy phosphoryls, we calculated intraflagellar concentration profiles of adenine nucleotides along the flagellum by computer simulation analysis. The result demonstrated the involvement of glycolysis for maintaining the ATP concentration at the tip of the flagellum. It is likely that glycolysis plays a key role in energy homeostasis in mouse sperm not only through ATP production but also through energy transfer.

Incidence and risk factors for falling in patients after total knee arthroplasty compared to healthy elderly individuals.

BACKGROUND: It is possible that patients who have undergone total knee arthroplasty (TKA) are at a high risk of falling. However, there are insufficient data to confirm the incidence and risk factors for falling in patients after TKA compared with healthy elderly counterparts. The purpose of this study was to elucidate the incidence and risk factors for falling in patients after TKA compared to the age- and gender-matched healthy elderly. METHODS: Subjects who underwent TKA consisted of 252 patients over 60 years of age. Controls were 150 healthy elderly individuals over 60 years of age living independently in the community. A self-administered questionnaire was mailed to patients after TKA and a similar questionnaire was distributed to the controls by investigators during the town-sponsored healthy aging program. The questionnaire included questions for ambulatory ability, functional status in daily living, knee pain, other joint pain and information on falls. RESULTS: Self-administered questionnares were returned by 192 of the 252 patients (76.1%) and 146 of the 150 controls (97.3%). Age and gender matching was performed for respondents between 70 and 80 years of age. There were 81 patients and 80 controls who fulfilled the inclusion criteria, and all of them agreed to participate. In the previous year, 34 of the 81 patients (38.2%) fell. The incidence of falls was significantly higher in patients than controls (23.8%, P = 0.041). In controls, ability to stand up from a chair without using the arms and restriction from joining social activities due to knee pain showed the strongest association with recent falls. In patients, self-reported kyphosis showed the strongest association with recent falls. CONCLUSION: Patients after TKA are more likely to fall than the general Japanese population. Kyphosis showed the strongest association with recent falls in patients after TKA, which was different from the results obtained in the healthy elderly.

Transient Ca2+ mobilization caused by osmotic shock initiates salmonid fish sperm motility.

Salmonid fish sperm motility is known to be suppressed in millimolar concentrations of extracellular K(+), and dilution of K(+) upon spawning triggers cAMP-dependent signaling for motility initiation. In a previous study, however, we demonstrated that suspending sperm in a 10% glycerol solution and subsequent dilution into a low-osmotic solution induced motility independently of extracellular K(+) and cAMP. In the present study, we further investigated the glycerol-induced motility mechanism. We found that treatment with solutions consisting of organic or inorganic ions, as well as glycerol, induced sperm motility in an osmolarity-dependent manner. Elimination of intracellular Ca(2+) by BAPTA-AM significantly inhibited glycerol-treated sperm motility, whereas removal of extracellular Ca(2+) by EGTA did not. Monitoring intracellular Ca(2+), using fluo-4, revealed that intracellular Ca(2+) increased when sperm were suspended in hypertonic solutions, and a subsequent dilution into a hypotonic solution led to a decrease in intracellular Ca(2+) concomitant with motility initiation. In addition, upon dilution of sperm into a hypertonic glycerol solution prior to demembranation, the motility of demembranated sperm was reactivated in the absence of cAMP. The motility recovery suggests that completion of axonemal maturation occurred during exposure to a hypertonic environment. As a result, it is likely that glycerol treatment of sperm undergoing hypertonic shock causes mobilization of intracellular Ca(2+) from the intracellular Ca(2+) store and also causes maturation of axonemal proteins for motility initiation. The subsequent dilution into a hypotonic solution induces a decrease in intracellular Ca(2+) and flagellar movement. This novel mechanism of sperm motility initiation seems to act in a salvaging manner for the well-known K(+)-dependent pathway.

Fall incidence and risk factors in patients after total knee arthroplasty.

PURPOSE: To prospectively investigate the relationship between physical function and falls among elderly patients who underwent total knee arthroplasty (TKA) and to determine the incidence of falls as well as their risk factors. METHODS: A total of 108 patients (17 male, 91 female) over 60 years of age who underwent TKA were enrolled and who were living independently in community. 75 patients fulfilled our inclusion criteria and 74 (8 male, 66 female) of them agreed to participate. Baseline assessment (physical examination, physical performance tests, and self-administered questionnaire) were conducted between 6 and 12 months after the last arthroplasty and the follow-up assessment was performed 6 months after the baseline assessment. Monthly pre-stamped postcards were sent to assess the incidence of falls. RESULTS: Of the 74 patients enrolled, 70 (94.6%) completed a 6-month prospective observation. 23 of 70 patients (32.9%) fell during the observational period. Postoperative range of knee flexion, ranges of knee flexion and extension and ankle plantar flexion were significantly lower in fallers than in non-fallers (P = 0.016, P = 0.037, P = 0.014, respectively). In the multivariate analysis, postoperative range of knee flexion (OR 0.277, 95%CI 0.088-0.869, P = 0.028) and ankle plantar flexion (OR 0.594, 95%CI 0.374-0.945, P = 0.028) were determined to be significant risk factors. CONCLUSION: Elderly people who underwent TKA are considered more likely to fall compared with healthy elderly people. For patients with limited knee flexion and ankle plantar flexion, improvement of ROM by exercise therapy and patient education regarding the prevention of falls and fractures are considered necessary.

Involvement of redox- and phosphorylation-dependent pathways in osmotic adaptation in sperm cells of euryhaline tilapia.

Sperm cells involved in fertilisation must tolerate hypo-osmotic and hyper-osmotic environments. Euryhaline tilapia (Oreochromis mossambicus) can acclimatise to and reproduce in freshwater and seawater because its sperm are able to adapt to these differing osmotic environments. In this study, we found that the dephosphorylation of sperm proteins in O. mossambicus correlated with the activation of flagellar motility when sperm were exposed to hypotonic or hypertonic conditions, and that differences in phosphorylation may reflect adaptations to a given osmotic environment. Of the sperm proteins that were dephosphorylated, the phosphorylation pattern of an 18 kDa protein, identified as the superoxide anion scavenger Cu/Zn superoxide dismutase (Cu/Zn SOD), was different in freshwater- and seawater-acclimatised tilapia sperm. Cu/Zn SOD was distributed from the sperm head to the flagellum. Additionally, differences were observed between freshwater and seawater tilapia in the nitration of tyrosine residues (which might be mediated by SOD) in sperm flagellar proteins in response to osmotic shock. These results demonstrate that reactive-oxygen-species-dependent mechanisms contribute to both osmotic tolerance and the activation of flagellar motility.

The transcriptional coactivators Yap and TAZ are expressed during early Xenopus development.

The Yap and TAZ genes encode highly conserved domains which bind various transcription factors. Yap and TAZ act as transcriptional coactivators to modulate transcriptional activity. The activities of Yap and TAZ are negatively regulated by Hippo signaling via direct phosphorylation. In this study, we describe the expression patterns of Yap and TAZ during the development of Xenopus tropicalis. The Xenopus tropicalis Yap (xtYap) and Xenopus tropicalis TAZ (xtTAZ) genes are expressed maternally. xtYap is widely expressed throughout embryogenesis, particularly in the facial connective tissues, branchial arch, midbrain-hindbrain boundary, otic vesicle, pronephros, notochord, hindgut and tailbud. xtTAZ expression occurs predominantly in the presomitic mesoderm, facial connective tissues, brain, branchial arch, trunk neural crest cells and migrating hypaxial myoblasts. In the muscle lineage, xtTAZ expression is transient and restricted to proliferating cells, the presomitic mesoderm and the edges of the hypaxial myoblasts, with no expression detected in mature muscle cells. These results provide insights into the functions of Yap and TAZ and their regulation by Hippo signaling during early development in Xenopus.

Regulation of sperm flagellar motility activation and chemotaxis caused by egg-derived substance(s) in sea cucumber.

The sea cucumber Holothuria atra is a broadcast spawner. Among broadcast spawners, fertilization occurs by means of an egg-derived substance(s) that induces sperm flagellar motility activation and chemotaxis. Holothuria atra sperm were quiescent in seawater, but exhibited flagellar motility activation near eggs with chorion (intact eggs). In addition, they moved in a helical motion toward intact eggs as well as a capillary filled with the water layer of the egg extracts, suggesting that an egg-derived compound(s) causes motility activation and chemotaxis. Furthermore, demembranated sperm flagella were reactivated in high pH (> 7.8) solution without cAMP, and a phosphorylation assay using (gamma-32P)ATP showed that axonemal protein phosphorylation and dephosphorylation also occurred in a pH-dependent manner. These results suggest that the activation of sperm motility in holothurians is controlled by pH-sensitive changes in axonemal protein phosphorylation. Ca2+ concentration affected the swimming trajectory of demembranated sperm, indicating that Ca2+-binding proteins present at the flagella may be associated with regulation of flagellar waveform. Moreover, the phosphorylation states of several axonemal proteins were Ca2+-sensitive, indicating that Ca2+ impacts both kinase and phosphatase activities. In addition, in vivo sperm protein phosphorylation occurred after treatment with a water-soluble egg extract. Our results suggest that one or more egg-derived compounds activate motility and subsequent chemotactic behavior via Ca2+-sensitive flagellar protein phosphorylation.

Eggs regulate sperm flagellar motility initiation, chemotaxis and inhibition in the coral Acropora digitifera, A. gemmifera and A. tenuis.

Corals perform simultaneous mass spawning around the full moon. Most Acropora species release gamete bundles, which are a complex of eggs and sperm, into the seawater. Then, gamete bundles are separated into eggs and sperm. Eggs are fertilized when sperm and eggs come in contact with each other. However, it is still unclear how sperm meet the eggs of the same species in the presence of many eggs of different species and how eggs guard against the fertilization attempts by sperm of different species. In this study, we observed that A. digitifera, A. gemmifera and A. tenuis sperm showed motility initiation/attraction close to eggs. Sperm were completely immotile in seawater, but they began to swim in circular motion when they came in close proximity to eggs, and then approached the eggs in straightforward paths. Sperm flagellar motility was not activated by an egg from different species, suggesting that motility initiation by the egg is species specific. In addition, hybridization among these species did not occur under observed conditions. Furthermore, motility-activated sperm became quiescent when many sperm approached the eggs. This study is the first report to show that the egg secretes immobilization factor(s). Our results suggest that the flagellar motility regulation has evolved to avoid hybridization among different species during the mass spawning.

Microtubule sliding movement in tilapia sperm flagella axoneme is regulated by Ca2+/calmodulin-dependent protein phosphorylation.

Demembranated euryhaline tilapia Oreochromis mossambicus sperm were reactivated in the presence of concentrations in excess of 10(-6) M Ca(2+). Motility features changed when Ca(2+) concentrations were increased from 10(-6) to 10(-5) M. Although the beat frequency did not increase, the shear angle and wave amplitude of flagellar beating increased, suggesting that the sliding velocity of microtubules in the axoneme, which represents dynein activity, rises with an increase in Ca(2+). Thus, it is possible that Ca(2+) binds to flagellar proteins to activate flagellar motility as a result of the enhanced dynein activity. One Ca(2+)-binding protein (18 kDa, pI 4.0), calmodulin (CaM), was detected by (45)Ca overlay assay and immunologically. A CaM antagonist, W-7, suppressed the reactivation ratio and swimming speed, suggesting that the 18 kDa Ca(2+)-binding protein is CaM and that CaM regulates flagellar motility. CaMKIV was detected immunologically as a single 48 kDa band in both the fraction of low ion extract of the axoneme and the remnant of the axoneme, suggesting that CaMKIV binds to distinct positions in the axoneme. It is possible that CaMKIV phosphorylates the axonemal proteins in a Ca(2+)/CaM-dependent manner for regulating the dynein activity. A (32)P-uptake in the axoneme showed that 48, 75, 120, 200, 250, 380, and 400 kDa proteins were phosphorylated in a Ca(2+)/CaM kinase-dependent manner. Proteins (380 kDa) were phosphorylated in the presence of 10(-5) M Ca(2+). It is possible that an increase in Ca(2+) induces Ca(2+)/CaM kinase-dependent regulation, including protein phosphorylation for activation/regulation of dynein activity in flagellar axoneme.

Requirement of the fixed end for spontaneous beating in flagella.

It is well known that any part of a flagellum has the ability to bend. However, it is not clearly understood how flagella generate successive bending waves spontaneously. Some micromanipulation experiments have suggested that the base of the flagellum is required. By contrast, spontaneous bending waves could be generated in computer simulation work if the microtubules were tied together at one end. We hypothesized that the basal structure of flagella can only act as a tied end when the outer doublet microtubules are tightly bound together so as not to slide. We developed a new technique for introducing local inhibition at any position on the demembranated and reactivated flagellum. The flagellum maintained spontaneous beating when the local inhibition was introduced at any position on it. In addition, spontaneous beating occurred without the basal body when an artificial fixed region was introduced to the flagellum. We conclude that the axoneme, a bundle of microtubules, requires the fixed end for spontaneous beating.

Changes in sperm motility in response to osmolality/Ca2+ in three Indonesian fresh water teleosts: goby (Oxyeleotris marmorata), Java carp (Puntius javanicus), and catfish (Clarias batrachus).

Sperm of most fresh water teleosts become motile when released into the hypotonic fresh water environment, but the role of osmolality and Ca2+ on sperm motility is not clear. Osmotic pressure and Ca2+ concentrations increase from fresh water to brackish water. Java carp Puntius javanicus and catfish Clarias batrachus live and reproduce only in fresh water. On the other hand, goby Oxyeleotris marmorata can acclimate and reproduce from fresh water to brackish water. In the present study, sperm motility and trajectory were compared among these three Indonesian endemic species. Sperm of Java carp, goby, and catfish begun to move in the hypotonic condition (< 200 mOsm/kg). However, the response to Ca2+ was different among these teleosts. In the presence of Ca2+, Java carp sperm swam in circular paths and immediately become quiescent, suggesting that Java carp sperm motility is activated in hypotonic aquatic environment without Ca2+. Goby sperm swam straightforward in the presence or absence of Ca2+. Percentages of motile sperm increased in 100-200 mOsm/kg but suppressed by removal of Ca2+. Regarding sperm motility and trajectory, no response was found in catfish sperm. These results suggest that a response to Ca2+ is different among sperm of the three species and suited to their habitat.

Increase in intracellular pH induces phosphorylation of axonemal proteins for activation of flagellar motility in starfish sperm.

Increased intracellular pH ([pH]i) activates dynein in sea urchin and mammalian sperm and induces activation of flagellar motility. It is thought that cAMP-dependent protein phosphorylation is associated with motility activation through increasing [pH]i, but little attention has been given to the cAMP-independent phosphorylation also induced by the [pH]i increase. The present study demonstrates that the increase in [pH]i in starfish sperm induces the phosphorylation of axonemal proteins and activation of flagellar motility independently of cAMP. Flagellar motility of intact sperm was activated when the [pH]i was raised by addition of NH4Cl. Histidine, which is known to activate motility of starfish sperm, also raised the [pH]i during the motility activation. In addition, motility of demembranated sperm flagella was activated in a pH-dependent manner without cAMP. These results indicate that in starfish sperm it is the increase in [pH]i that induces activation of flagellar motility. Moreover, phosphorylation of axonemal proteins (of molecular mass 25, 32 and 45 kDa) was observed during the pH-dependent and cAMP-independent motility activation of demembranated sperm. This suggests that the increase in [pH]i regulates flagellar motility via cAMP-independent phosphorylation of axonemal proteins.

Analysis of flagellar bending in hamster spermatozoa: characterization of an effective stroke.

The mechanism by which flagella generate the propulsive force for movement of hamster spermatozoa was analyzed quantitatively. Tracing points positioned 30, 60, 90, and 120 microm from the head-midpiece junction on the flagellum revealed that they all had zigzag trajectories. These points departed from and returned to the line that crossed the direction of progression. They moved along the concave side (but not the convex side) of the flagellar envelope that was drawn by tracing the trajectory of the entire flagellum. To clarify this asymmetry, the bending rate was analyzed by measuring the curvatures of points 30, 60, 90, and 120 microm from the head-midpiece junction. The bending rate was not constant through the cycle of flagellar bending. The rate was higher when bending was in the direction described by the curve of the hook-shaped head (defined as a principal bend [P-bend]) to the opposite side (R-bend). We measured a lower bending rate in the principal direction (R-bend to P-bend). To identify the point at which the propulsive force is generated efficiently within the cycle of flagellar bending, we calculated the propulsive force generated at each point on the flagellum. The value of the propulsive force was positive whenever the flagellum bent from an R-bend to a P-bend (when the bending rate was lowest). By contrast, the propulsive force value was zero or negative when the flagellum bent in the other direction (when the bending rate was higher). These results indicate that flagellar bending in hamster spermatozoa produces alternate effective and ineffective strokes during propulsion.

Effects of static load on the weight and protein content in the leg muscles of the mouse: a simulation of prolonged standing in the workplace.

To simulate the effects of prolonged standing in the workplace on the leg muscles, we subjected mice to centrifugation for 6 wk. The absolute wet weight of leg muscles and internal organs of mice were measured after exposure to 3G by centrifugation for 6 wk and at 2 wk after removal of centrifugation. The weight of the soleus muscle (antigravity muscle) significantly increased after 6-wk exposure to centrifugation, but it decreased to its control weight 2 wk after removal of centrifugation. In contrast, the wet weights of the anterior tibial muscle, liver, and kidneys of mice centrifuged for 6 wk were significantly lower than those of the control mice; they had returned to control levels 2 wk after removal of centrifugation. It was therefore suggested that prolonged standing enlarged the leg muscles but its effect did not last for a long period of time after stopping prolonged standing. Western blot analysis of proteins extracted from the soleus muscle showed that vinculin and alpha-actinin in the centrifuged mice increased slightly, but there were no differences in the heat shock protein 70 (HSP70) and desmin levels between the centrifuged mice and control mice. No difference in HSP 70 suggested that muscle damage did not exist after 6 wk centrifugation.

Identification of 36 kDa phosphoprotein in fibrous sheath of hamster spermatozoa.

In our previous studies (Fujinoki et al., 2001, 2003), we reported that two types of 36 kDa proteins, designated 36K-A protein and 36K-B protein, obtained from hamster sperm flagella, are associated with motility activation and phosphorylated in a cAMP-dependent manner at serine residues. In the present experiments, we focused on the hamster (Mesocricetus auratus) 36K-A protein, which was analyzed by peptide mass finger printing and amino acid sequencing. The results suggest that 36K-A protein is a pyruvate dehydrogenase E1 component beta subunit lacking the N-terminal 30 amino acids. Moreover, our results suggest that 36 K-A protein is localized in the fibrous sheath of the principal piece of hamster spermatazoa.

Glycolysis plays a major role for adenosine triphosphate supplementation in mouse sperm flagellar movement.

The mammalian sperm must be highly motile for a long time to fertilize a egg. It has been supposed that ATP required for sperm flagellar movement depends predominantly on mitochondrial respiration. We assessed the contribution of mitochondrial respiration to mouse sperm motility. Mouse sperm maintained vigorous motility with high beat frequency in an appropriate solution including a substrate such as glucose. The active sperm contained a large amount of ATP. When carbonyl cyanide m-chlorophenylhydrazone (CCCP) was applied to suppress the oxidative phosphorylation in mitochondria, the vigorous motility was maintained and the amount of ATP was kept at the equivalent level to that without CCCP. When pyruvate or lactate was provided instead of glucose, both sperm motility and the amount of ATP were high. However, they were drastically decreased when oxidative phosphorylation was suppressed by addition of CCCP. We also found that sperm motility could not be maintained in the presence of respiratory substrates when glycolysis was suppressed. 2-Deoxy-d-glucose (DOG) had no effect on mitochondrial respiration assessed by a fluorescent probe, 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolylcarbocyanine iodide (JC-1), but, it inhibited motility and decreased ATP content when pyruvate or lactate were provided as substrates. The present results suggest that glycolysis has an unexpectedly important role in providing the ATP required for sperm motility throughout the length of the sperm flagellum.

The change of HSP47, collagen specific molecular chaperone, expression in rat skeletal muscle may regulate collagen production with gravitational conditions.

It is well known that unloading of skeletal muscle with spaceflight leads skeletal muscle atrophy. However, it remains unclear how the extracellular matrix within the muscle and the connective tissues such as tendon and ligament respond to reduced mechanical load including microgravity, although they have been thought to play important roles in both the transmission of force and the signal transduction between cells and tissues. Type-I collagen and type-IV collagen, both of the major components of extracellular matrix and connective tissues. We focused on change of these collagen synthesis with mechanical load. To obtain an insight into the effects of gravitational changing on the protein metabolism of collagen in skeletal muscle during mechanical unloading, reloading after unloading, we investigated changes in the amount of Heat shock protein 47 (HSP47), has been postulated to be a collagen-specific molecular chaperone localized in the ER (Nagata et al, 1992). Western blot analysis revealed that HSP47 in rat soleus muscle decreases at 5 days after hindlimb suspension (HS). On the other hand, HSP47 in rat soleus muscle increases at 5 days after hypergravity (HG) induced by the centrifugation. RT-PCR analysis showed HSP47 mRNA decreased with HS earlier, as compared with collagen type-I and type-IV mRNA. From these results, the amount of HSP47 changing by gravitational condition may effect on signal transfers in the primary stage of adaptation and the change of HSP47 expression in skeletal muscle may regulate collagen production with gravitational conditions.

Relationship between the stress of hyper-gravity and food intake and growth rate in mouse.

A new method was introduced to assess the effects of hyper-gravity on secretion of corticosterone, one of the major stress hormone, in mouse. The hormone was extracted from feces of the animal and measured by means of ELISA. The amount of corticosterone was high at the beginning of breeding under the hyper-gravity, 3 G. It decreased down to the level of the ground control within 2 weeks. Increases both in the growth rate of the body weight and the food intake were closely related to the decrease in the amount of corticosterone. It is likely that hyper-gravity affects the growth rate via internal secretion.

Acclimation of sperm motility apparatus in seawater-acclimated euryhaline tilapia Oreochromis mossambicus.

Euryhaline tilapia Oreochromis mossambicus can reproduce in freshwater and in seawater. Regulation of sperm motility appears to be modulated during acclimation of the fish from freshwater to seawater, being independent of extracellular Ca2+ in freshwater and dependent on extracellular Ca2+ in seawater. In the presence of extracellular Ca2+, sperm of seawater-acclimated tilapia (SWT) showed motility even in a hypertonic environment, whereas sperm of freshwater-acclimated tilapia (FWT) were not motile. The Ca2+ indicator, fluo-3, revealed that intracellular Ca2+ concentration, [Ca2+]i, of SWT sperm increased only in the presence of extracellular Ca(2+) in hypotonic or hypertonic conditions. Since the increased [Ca2+]i in FWT sperm occurred under hypotonic conditions via intracellular Ca2+ stores, it is likely that tilapia modulate their source of increasing [Ca2+]i from intracellular stores (in FWT sperm) to extracellular stores (in SWT sperm). Experiments using demembranated sperm revealed that Ca2+ is necessary for activation of motility, suggesting that Ca2+ plays a key role in motility regulation in SWT sperm. We detected three phosphoproteins associated with the activation of sperm motility. Serine and threonine residues of two proteins of 15 kDa and 18 kDa became dephosphorylated in hypotonic conditions but remained phosphorylated in hypertonic conditions, suggesting that these protein phosphorylations were not only related to motility activation under hypertonic conditions but also resistant to osmotic pressure. The threonine residue(s) of a 41 kDa protein was also phosphorylated in dry sperm, even in FWT sperm in motility-feasible hypotonic conditions. It is likely that acclimation of the motility apparatus is associated with modulation of the flow of Ca2+ to increase [Ca2+]i and protein phosphorylation.