Establishment of an unfed strain of Paramecium bursaria and analysis of associated bacterial communities controlling its proliferation.

The ciliate Paramecium bursaria harbors several hundred symbiotic algae in its cell and is widely used as an experimental model for studying symbiosis between eukaryotic cells. Currently, various types of bacteria and eukaryotic microorganisms are used as food for culturing P. bursaria; thus, the cultivation conditions are not uniform among researchers. To unify cultivation conditions, we established cloned, unfed strains that can be cultured using only sterile medium without exogenous food. The proliferation of these unfed strains was suppressed in the presence of antibiotics, suggesting that bacteria are required for the proliferation of the unfed strains. Indeed, several kinds of bacteria, such as Burkholderiales, Rhizobiales, Rhodospirillales, and Sphingomonadales, which are able to fix atmospheric nitrogen and/or degrade chemical pollutants, were detected in the unfed strains. The genetic background of the individually cloned, unfed strains were the same, but the proliferation curves of the individual P. bursaria strains were very diverse. Therefore, we selected multiple actively and poorly proliferating individual strains and compared the bacterial composition among the individual strains using 16S rDNA sequencing. The results showed that the bacterial composition among actively proliferating P. bursaria strains was highly homologous but different to poorly proliferating strains. Using unfed strains, the cultivation conditions applied in different laboratories can be unified, and symbiosis research on P. bursaria will make great progress.

Spontaneous generation of reactive oxygen species and effect on motility and fertilizability of sea urchin spermatozoa.

We investigated the generation of reactive oxygen species (ROS) by spermatozoa in two species of sea urchin. ROS generation was accompanied by the initiation of motility and respiration and influenced the motility and fertilizability of spermatozoa. The sea urchin performs external fertilization in aerobic seawater. Sperm motility was initiated after spawning through Na+/H+ exchange. ROS generation was dependent on the respiration and sperm concentration and its generation was first observed at initiation of motility, via activation of respiration through ATP/ADP transport. The ROS generation rate increased at higher dilution ratios of spermatozoa, in a manner that was synchronous with the respiratory rate. This phenomenon resembled the previously defined 'sperm dilution effect' on respiration. The loss of motility and fertilizability was induced not only by treatment with hydrogen peroxide but also by sperm dilution. Storage of spermatozoa with a higher dilution ratio also accelerated the decrease in fertilizability. Thus, optimum sea urchin fertilizability is maintained by storage of undiluted spermatozoa on ice, in order to minimize oxidative stress and to maximize longevity.

Sea urchin spermatozoa generate at least two reactive oxygen species; the type of reactive oxygen species changes under different conditions.

Reactive oxygen species (ROS) cause oxidative stress and act as signal transduction molecules in many cells. Spermatozoa from several mammals generate ROS, which are involved in male infertility and signaling during capacitation. In the present study, we investigated ROS generation by sea urchin spermatozoa at the initiation of motility, during dilution with seawater, and following egg jelly treatment. In seawater containing an ROS indicator, 5-(and 6-)chloromethyl-2',7'-dichlorodihydrofluorescein diacetate (CM-H(2)DCFDA), fluorescence increased after the addition of spermatozoa. The ROS generation rate was dependent upon the dilution ratio and respiratory rate of the spermatozoa. Spermatozoa in sodium-free seawater did not increase fluorescence, but fluorescence did increase with the addition of NaCl. Sodium chloride also led to the initiation of sperm motility and respiration. Using the indicator MitoSOX Red, ROS generation was detected from spermatozoa exposed to egg jelly dissolved in seawater, but not in normal seawater. Moreover, the respiratory inhibitor antimycin A prevented CM-H(2)DCFDA-detectable ROS and increased MitoSox-detectable ROS at a higher concentration. These findings revealed that the ROS generated were of different species, possibly hydrogen peroxide (H(2)O(2)) and superoxide anion (O(-)(2)), and their detected levels were altered by egg jelly. We concluded that sea urchin spermatozoa generate at least two species of ROS depending on the physiological conditions to which they are exposed. It is possible that the major ROS from sea urchin spermatozoa changes during the course of fertilization.

Fertilization induced changes in sea urchin sperm: mitochondrial deformation and phosphatidylserine exposure.

This study demonstrates that the single mitochondrion of the sea urchin sperm undergoes a shape change at fertilization that is linked to respiration. The mitochondrion swells and shifts to the lateral side of the sperm head on contact with the homologous egg jelly or egg surface; Mg(2+)- or Na(+)-free seawater or respiratory inhibitors also induce this change. During the mitochondrial deformation, the sperm decreases the rate of oxygen consumption and their redox-state of cytochromes is disrupted b-c(1)/c. Simultaneously, the adenine nucleotides content changes precipitously. This suggests that mitochondrial morphology is strongly associated with respiratory activities in the sea urchin sperm. These changes in mitochondrial morphology and function are similar to the mitochondrial changes in apoptotic cells such as swelling, decrease in its membrane potential, and release of cytochrome c. In apoptotic cells, the exposure of phosphatidylserine from the inner to outer leaflet of the plasma membrane is one of prominence phenomena. This change was visualized by staining the sea urchin sperm with Annexin V-Fluorescein. It is possible that mitochondrial deformation is an initial sign of sperm destruction, which like as apoptotic cells.

Ultrastructural Studies on the Behavior of Centrioles during Meiosis of Starfish Oocytes: (oocyte/meiosis/centriole/starfish).

The behavior of centrioles and ultrastructural changes of the nucleus were observed in maturing oocytes of the starfishes, Asterina pectinifera and Asterias amurensis. Observations were focused on the number and behavior of centrioles during two successive meiotic divisions. Examination of serial sections revealed that in meiosis I each division pole has a pair of centrioles, whereas in meiosis II each has only one centriole, confirming the observations by Sluder et al. (1989) on oocytes of Pisaster ocraceus and Asterias forbesi. The first polar body had two centrioles and the second polar body had only one. These results indicate that no duplication of centrioles occurs during the two successive meiotic divisions, and that the egg inherits one centriole from a primary oocyte.

Intracellular pH Changes of Starfish Sperm Upon the Acrosome Reaction: (acrosome reaction/intracellular pH/starfish sperm/egg jelly/9-aminoacridine).

The acrosome reaction is accompanied by ionic changes such as increases in intracellular Ca2+ and intracellular pH (pHi ). Since the two jelly components essential for inducing the acrosome reaction, ARIS and Co-ARIS, were shown to activate Ca-channels (accompanying paper), we examined the jelly components to determine which was responsible for the pHi -increase using 9-aminoacridine as a probe of pHi . This paper presents evidence that an oligopeptide(s) is responsible for the pHi -increase. The pHi of swimming sperm is 7.4-7.5. Within 20 sec after the addition of jelly, their pHi increased rapidly by 0.06 pH unit, then decreased by 0.2-0.3 pH unit, and reached a plateau level within 3 min. Similar changes in pHi were observed on addition of a Pronase digest of ARIS (P-ARIS) and a diffusible fraction of jelly (Fraction M8 ) together. Fraction M8 , but not ARIS or Co-ARIS increased the pHi , and activated sperm respiration in sea water at pH 6.5. The two activities of Fraction M8 depended upon Na+ but not Ca2+ , and were susceptible to Pronase digestion. Fraction M8 is also known to enhance induction of the acrosome reaction by the Ca-ionophore A23187. These results suggest that the egg jelly contains a peptide(s) that is not obligatory for the acrosome reaction but facilitates the reaction by increasing the pHi of the sperm. The significance of the pHi -increase upon the acrosome reaction is discussed.

Acrosome Reaction-Inducing Substance Purified from the Egg Jelly Inhibits the Jelly-Induced Acrosome Reaction in Starfish: An Apparent Contradiction: (acrosome reaction/starfish sperm/egg jelly/ARIS/Co-ARIS).

Previous studies indicated that two components of the egg jelly are required for induction of the acrosome reaction in starfish: a sulfated glycoprotein called acrosome reaction-inducing substance (ARIS) and a diffusible organic substance(s) called Co-ARIS. In the present study the sites of action of ARIS and Co-ARIS and their temporal relationships were examined. When sperm had been treated for a few minutes with ARIS, or a crude preparation of Co-ARIS (Fraction M8 ), or inadequate amounts of jelly, or sufficient jelly in low Ca2+ sea water, they did not undergo the acrosome reaction when the deficiencies were corrected. Moreover, they became nonresponsive to the jelly. Pronase digest of ARIS (P-ARIS) but not of Fraction M8 retained this capacity. A steroidal saponin purified as Co-ARIS did not have this capacity. This suggests the presence of a third jelly component, probably an oligopeptide(s), participating in induction of the acrosome reaction. Activation of Ca2+ -uptake seems to be at least one, if not the only, action site of ARIS and Co-ARIS, because ARIS, P-ARIS, and Fraction M8 inhibited jelly-induced Ca2+ -uptake by sperm, and because the calcium ionophore A23187 by-passed the blockage by these components of the jelly-induced acrosome reaction.

Induction of the Acrosome Reaction in Starfish: (acrosome reaction/starfish sperm/egg jelly/Ca-channel/ionophore).

In the starfish, Asterias amurensis, at least two distinct components of the egg jelly are required for inducing the acrosome reaction: a sulfated glycoprotein named acrosome reaction-inducing substance (ARIS) and a diffusible organic substance(s) named Co-ARIS. The following evidence suggested that ARIS and Co-ARIS cooperatively activate CA-channels of the sperm plasma membrane and eventually induce dramatic changes in sperm morphology, the acrosome reaction. 1) Pronase digest of ARIS (P-ARIS) and Co-ARIS, either as a pure or a crude preparation (Fraction M8 ), were fully effective in combination for induction of the acrosome reaction in normal sea water, although they were not effective individually. P- ARIS alone induced the acrosome reaction fully in high Ca2+ sea water and markedly at high pHs, whereas Fraction M8 alone did not induce the reaction even in these conditions. The reaction was not induced by increase in either the Ca2+ concentration or the pH of sea water, but was markedly induced in the absence of jelly components by raising both the pH and Ca2+ concentration together. 2) The ionophore A23187 induced the acrosome reaction appreciably when present alone and fully in the presence of monensin or Fraction M8 . Monesin alone was ineffective. 3) The jelly or a combination of ARIS and Fraction M8 caused abrupt Ca2+ -uptake by the sperm. The Ca-channel blockers verapamil and diltiazem inhibited the jelly-induced acrosome reaction.

Respiration in Eggs of the Echiuroid, Urechis unicinctus, Before and After Fertilization: echiuroid eggs/fertilization/respiration/redox dyes/uncoupler of oxidative phosphorylation.

In eggs of the echiuroid Urechis unicinctus the respiration rate, which is not altered by fertilization, is inhibited by rotenone, antimycin A and cyanide. The respiration in echiuroid eggs is probably mediated by the mitochondrial respiratory chain. In fertilized eggs, the respiration was inhibited by oligomycin and stimulated by the uncouplers of oxidative phosphorylation 2,4-dinitrophenol and carbonylcyanide p-trifluoromethoxyphenylhydrazone, whereas respiration in unfertilized eggs was insensitive to these compounds. Insemination increased the respiratory rate in eggs in the presence of uncouplers and reduced it in the presence of oligomycin. These findings suggest that the capacity of electron transport in mitochondira is elevated by fertilization but becomes latent on fertilization-induced coupling of respiration with oxidative phosphorylation. Strong stimulation of the respiration in unfertilized eggs was induced by dichlorophenol indophenol, phenazine methosulfate and tetramethyl p-phenylenediamine, suggesting possible sites at which electron transport is regulated in unfertilized eggs. The resulting stimulation of respiration in unfertilized eggs was insensitive to uncouplers and oligomycin, but became sensitive to them after fertilization simultaneously with considerable decrease in its rate. Fertilization-induced coupling of the respiration seemed to reduce the respiratory rate enhanced artificially by these redox compounds.

VII. Decrease in the Rate of Respiration in the Spermatozoa of the Sea Urchin, Hemicentrotus Pulcherrimus, Caused by Long Chain Fatty Acyl-CoA-induced Inhibition of the Movement: (sperm movement/carnitine/palmitoyl-CoA/sperm-egg interaction/sperm respiration).

Spermatozoa of the sea urchin, Hemicentrotus pulcherrimus, showed marked decrease in respiration, and arrested movement after interaction with the fixed eggs. Immotile spermatozoa that had reacted with fixed eggs contained higher levels of long chain fatty acyl-CoAs than normal motile spermatozoa. On treatment with carnitine, the immotile spermatozoa became motile again and their intracellular concentrations of long chain fatty acyl-CoAs decreased. On incubation with anti-mycin A or CN- for 20 min, the motility of normal spermatozoa decreased gradually but their long chain fatty acyl-CoA content changed only slightly. The decrease in sperm motility in the latter case was probably due to decrease in the level of ATP, resulting from inhibition of respiration by antimycin A or CN- . The motility of spermatozoa extracted with Triton X-100 was restored by ATP and their movement was inhibited by long chain fatty acyl-CoAs, such as myristoly CoA and palmitoyl-CoA, but was not by short chain fatty acyl-CoAs, such as acetyl-CoA, propionyl CoA and butyryl-CoA. Na-palmitate, Na-myristate and CoA did not inhibit the reactivation of extracted spermatozoa by ATP.

Inhibition of Respiration in Sea Urchin Spermatozoa following Interaction with Fixed Unfertilized Eggs VI. Probable Difference between the Species in the Mechanism for the Fixed-Egg-Induced Inhibition of Sperm Respiration: (respiration/sperm/sea urchin/carnitine/sperm-egg interaction).

In spermatozoa of all examined sea urchins, the respiration was inhibited and their motility was lowered by the glutaraldehyde-fixed eggs. The respiration of the fixed-egg-reacted spermatozoa was stimulated by 2, 4 dinitrophenol in Clypeaster japonicus, Hemicentrotus pulcherrimus and pseudocentrotus depressus but was not in Anthocidaris crassispina and Toxopneustes pileolus. Ratio of ADP to ATP was markedly lower in the reacted spermatozoa of the former species than in those of Anthocidaris. The low respiratory rate in the former species probably results from ADP control but does not in the latter species. Tetramethyl-p-phenylenediamine enhanced the respiratory rate in the reacted spermatozoa of the latter species to almost the same rate as in the intact spermatozoa, but elevated slightly in the former species. The inhibition of electron transport in mitochondrial respiratory chain is probably predominant in the latter species. In the former species, the slight inhibition of electron transport does not seem to result in a failure of ADP phosphorylation, and hence the stop of movement probably causes a shortage of ADP. Carnitine, which made the reacted spermatozoa of all species motile, enhanced the respiratory rate only in those of the former species.

Inhibition of Respiration in Sea Urchin Spermatozoa Following Interaction with Fixed Unfertilized Eggs V. Inhibition of Electron Transport in a Span of Mitochondrial Respiratory Chain between Cytochrome b and Cytochrome c in Sea Urchin Spermatozoa, Induced by the Interaction with Glutaraldehyde Fixed Eggs: (sea urchin/sperm/respiration/cytochrome/egg-sperm interaction).

In the sea urchin, Anthocidaris crassispina, α and γ peaks of reduced cytochrome b were distinctly observed but no peaks of cytochrome a and cytochrome c were found in the difference spectra between H2 O2 oxidized and the aerobic suspensions of the immotile spermatozoa, which were obtained by an incubation of the suspension of spermatozoa and the glutaraldehyde-fixed eggs for 15 min at 20°C. A similar profile of difference spectrum was also obtained between the aerobic sperm suspension containing antimycin A and the H2 O2 oxidized one. In Hemicentrorus pulcherrimus, faint peaks of reduced cytochrome a and cytochrome c, as well as evident peaks of cytochrome b, were also found in the difference spectra between aerobic suspension of the fixed-egg-reacted spermatozoa and the H2 O2 oxidized one. In intact swimming spermatozoa of A. crassispina as well as H. pulcherrimus, no peaks of reduced cytochromes were found under aerobic condition. These results suggest that the inhibition of sperm respiration by the fixed eggs is due, at least in part, to the blockage of electron transport in a span between cytochrome b and cytochrome c.

INHIBITION OF RESPIRATION IN SEA URCHIN SPERMATOZOA FOLLOWING INTERACTION WITH FIXED UNFERTILIZED EGGS: II. CAPACITY OF THE GLUTARALDEHYDE FIXED UNFERTILIZED EGGS FOR THE INHIBITION OF THE SPERM RESPIRATION.

Glutaradehyde-fixed unfertilized eggs of the sea urchin, Pseudocentrotus depressus, which bound spermatozoa, were capable of causing an inhibition of respiration in 2.3 ± 0.27 × 104 spermatozoa per egg. After stirring a suspension containing fixed eggs (2 × 103 -2 × 104 per ml) and a larger number of spematozoa than 2.5 × 104 per egg for 15 min at 20°c, the fixed eggs isolated from the suspension failed to cause any inhibition of respiration of fresh spermatozoa, and were incapable of binding fresh eggs. Fixed eggs, treated with a smaller number of spermatozoa than 2 × 104 per egg, had a capacity to bind spermatozoa and inhibited sperm respiration. Unless the fixed eggs lost the sperm-binding capacity, they exerted an inhibitory effect on the respiration of spermatozoa.

INHIBITION OF RESPIRATION IN SEA URCHIN SPERMATOZOA FOLLOWING INTERACTION WITH FIXED UNFERTILIZED EGGS: IV. STATE 4 RESPIRATION IN SPERMATOZOA OF HEMICENTROTUS PULCHERRIMUS AFTER THEIR INTERACTION WITH FIXED UNFERTILIZED EGGS.

The respiration of spermatozoa of the sea urchin, Hemicentrotus pulcherrimus, was found to be sensitive to rotenone, antimycin A, and cyanide. This suggests that sperm respiration results from electron transport which spans the whole mitochondrial respiratory chain. The sperm respiration was inhibited by oligomycin and this inhibition was released by 2, 4-dinitrophenol (DNP). DNP did not stimulate the respiration of spermatozoa in a diluted suspension (2 × 108 /ml), where they were swimming vigorously. The ADP level of spermatozoa in the diluted suspension was markedly higher than that in dry sperm. The spermatozoa, which had reacted with unfertilized eggs fixed with glutaraldehyde, were immotile with a quite low respiratory rate. The respiratory rate of the immotile spermatozoa was enhanced by DNP. In the immotile spermatozoa, ADP level was markedly low and the ATP level was as high as that in dry sperm. From these findings, it is concluded that in the swimming spermatozoa respiration coupled with oxidative phosphorylation occurs at the maximum rate. State 3 respiration probably occurs in the swimming spermatozoa. The low respiratory rate of the immotile spermatozoa is assumed to be due to a shortage of ADP and is practically regarded as state 4 respiration.

INHIBITION OF RESPIRATION IN SEA URCHIN SPERMATOZOA FOLLOWIGN INTERACTION WITH FIXED UNFERTILIZED EGGS: III. INHIBITION OF SPERM RESPIRATION BY HEAT STABLE SUBSTANCE REMOVED FROM GLUTARALDEHYDE-FIXED EGGS.

The respiratory rate of spermatozoa of the sea urchin, Pseudocentrotus depressus and Hemicentrotus pulcherrimus, became quite low and spermatozoa was immotile, after sperm suspension containing glutaraldehyde-fixed eggs of homologous species was stirred at 20°C for 15 min. The respiratory rate of fresh spermatozoa, introduced to the suspension of immotile spermatozoa thus obtained, was also reduced markedly. The respiration of fresh spermatozoa was not inhibited by adding them to suspension of intact or acrosome reacted spermatozoa. A heat stable and non-dialyzable substance, which inhibited sperm respiration, was removed from the fixed eggs by vigorously stirring the egg suspension for 10 min, when unfertilized eggs were fixed with insufficient amount of glutaraldehyde (10 ml of 1% glutaraldehyde solution to 1 ml egg pellet).

INHIBITION OF RESPIRATION IN SEA URCHIN SPERMATOZOA FOLLOWING INTERACTION WITH FIXED UNFERTILIZED EGGS.: I. CHANGE IN THE RESPIRATORY RATE OF SPERMATOZOA IN THE PRESENCE OF FIXED EGGS.

The respiratory rate of spermatozoa of the sea urchins, Anthocidaris crassispina, Clypeaster japonicus and Pseudocentrotus depressus, decreases markedly in the presence of homologous unfertilized eggs fixed with glutaraldehyde. No decrease in the rate of respiration occurs in the presence of fixed fertilized eggs. Fixed unfertilized eggs of different sea urchin species do not cause any change in the rate of sperm respiration. Spermatozoa adhere only to the fixed unfertilized eggs of the same species and are removed by a stirring for 5 min on a magnetic stirrer. The spermatozoa thus removed, are immotile and their respiratory rate is quite lower than that of motile spermatozoa in a control suspension stirred for 5 min. Intact spermatozoa adhere to the fixed eggs, from which the attached spermatozoa have been removed, and the respiratory rate of the spermatozoa also becomes quite low.

CHANGE IN THE GLYCOGEN CONTENT OF SEA URCHIN EGGS DURING EARLY DEVELOPMENT.

During development of eggs of the sea urchins, Pseudocenrotus depressus and Anthocidaris crassispina, the glycogen level is maintained from the time of fertilization to the swimming blastula stage and then decreases rapidly in the early gastrula stage. During development of eggs of Clypeaster japonicus. Hemicentrotus pulcherrimus and Mespilia globulus the glycogen content decreases slowly from the time of fertilization to the mesenchyme blastula stage, and then more rapidly during gastrulation. The amounts of glycogen mobilized in the embryos from the time of fertilization to the morula stage correspond to 67% of the amount of O2 consumed in Mespilia eggs, 62% in Clypeaster eggs, 30% in Hemicentrotus eggs and 0-4% in Anthocidaris and Pseudocentrotus eggs. The main energy source in early development seems to differ in different species. When eggs and embryos were incubated with [14 C]glucose for 10min, considarable 14 C-radioactivity accumulated in the glycogen fraction. The rate of [14 C]glucose incorporation into glycogen increased gradually during the first 6 hr after fertilization (up to the morula stage), decreases during the next 4 hr (up to the early blastula stage), and then increased again.

PRODUCTION AND UTILIZATION OF GLUCOSE 1-PHOSPHATE IN THE EGGS OF THE SEA URCHIN ANTHOCIDARIS CRASSISPINA.

Concentrations of G1P, G6P, UDPG, UTP and PPi were measured in the eggs of the sea urchin, Anthocidaris crassispina. Activities of phosphorylase a (EC 2.4.1.1), phosphoglucomutase (EC 2.7.5.1), UDPG pyrophosphorylase (EC 2.7.7.9) and pyrophosphatase (EC 3.6.1.1) were also estimated. Levels of G1P and G6P increase following fertilization, but concentrations of UDPG and UTP in unfertilized eggs are very similar to those in fertilized eggs. PPi is undetectable. In unfertilized and fertilized eggs, the G1P level is very low as compared with the G6P level and is far less than that expected from the equilibrium constant in a reaction catalyzed by phosphoglucomutase. Since the phosphoglucomutase activity is higher by about 20 times than the phosphorylase a activity, G1P is probably produced in the reverse reaction, catalyzed by phosphoglucomutase, rather than in the reaction catalyzed by phosphorylase. The G1P thus produced seems to be utilized thoroughly in the reaction catalyzed by UDPG pyrophosphorylase. The reaction seems to be irreversible and tends to go to UDPG production in sea urchin eggs, since the PPi level is negligible due to high pyrophosphatase activity. The utilization of G1P in the reaction catalyzed by UDPG pyrophosphorylase seems to keep the G1P level low.