Effects of transglutaminase substrates and inhibitors on the motility of demembranated reactivated spermatozoa

The effects of transglutaminase (TGase) substrates putrescine, dansylcadaverine, spermine, etc., and the TGase inhibitor cystamine were tested on the motility of demembranated mammalian spermatozoa. These products blocked within a few seconds the motility of demembranated reactivated spermatozoa at concentrations ranging from 0.25 to 5 mM. These minimal inhibitory concentrations could be decreased 5–150-fold when TGase substrates and inhibitor were incubated with demembranated spermatozoa for 15 min prior to the addition of Mg·ATP. The inhibition was reversed by higher concentrations of Mg·ATP but none of these TGase substrates or inhibitor could inhibit bull sperm dynein ATPase. TGase activities, as measured by the incorporation of ³H-putrescine into TCA-precipitable proteins, were present in both sperm Triton-soluble and -insoluble fractions. On the other hand, amine acceptor protein substrates for the TGase-catalyzed reaction were present only in the insoluble fraction. The Triton-soluble TGase was similar to the known “tissue” TGases; the Triton-insoluble TGase activity was calcium independent. The same TGase substrates and inhibitor that blocked the motility of reactivated spermatozoa also blocked TGase activities. Linear relationships were observed between the concentrations of these substances required to block sperm motility and those to block TGase activities. These data suggest the involvement of a TGase activity in sperm motility.     

The motility of rabbit spermatozoa recovered from the female reproductive tract

The motility of rabbit spermatozoa recovered from the vagina, endocervix, uterus, and four regions of the oviduct was assessed visually by phase-contrast microscopy at intervals from one minute to 16 hours after a single mating. The percentage of motile cells in each sample was dependent on the temperature of recovery, ie, 23° vs 37°C, but was not influenced by the temperature of observation. Spermatozoa in the lower isthmus of the oviduct were the most temperature sensitive population to recovery at 23°C. When all manipulations and observations were performed at 37°C, the percentage of spermatozoa with progressive motility varied according to the region sampled and interval after mating. Populations from the vagina, uterus and upper regions of the oviduct usually had a high proportion of progressively motile cells with vigorous flagellar activity. Fewer spermatozoa showed progressive movement on recovery from the endocervix and lower 2 cm of the tubal isthmus and their flagellar activity was generally depressed. The decrease in flagellar beat frequency noted in the latter regions may be a major factor limiting sperm ascent in the female tract. A unique pattern of “activated” motility was seen exclusively in populations taken from the oviducts at 6 to 16 hours after mating. This motility pattern, consisting of alternating episodes of linear progressive and vigorous nonprogressive movement, may be analogous to the activated motility described for capacitated rodent spermatozoa.     

Characterization of microsatellite loci from the Japanese eel Anguilla japonica

The Japenese eel, Anguilla japonica, is generally assumed to be composed of a single population with wide distribution range, and some genetic studies using allozyme or mitochondrial DNA methods supported this population model. However, one genetic study suggested the existence of multiple populations in this species, and thus, more detailed studies on the population structure is needed. Here we characterized a total of 11 microsatellite markers of the Japanese eel. These will serve as powerful tools for detailed population study for the Japanese eel, though two of them showed the significant departure from the Hardy–Weinberg expectations.     

Initiation,prolongation, and reactivation of the motility of salmonid spermatozoa

The motility of salmonid spermatozoa initiated by dilution of the milt with ovarian fluid or isotonic saline is brief duration; it was believed that it can be activated only once in the life of the spermatozoon. Dilution of the milt with an equal volume of isotonic saline (0.12 M-NaCl) containing 5 mM-3-isobutyl-1-methylxanthine (MIX) prolonged and intensified sperm motiliy. When motility had stopped after initial mobilization with saline or ovarian fluid, it could be reactivated by addition of MIX; reactivated spermatozoa fertilized eggs. Dilution with saline containing K⁺ (24 mEq/liter) did not initiate sperm motility even in the presence of MIX. The spermatozoa were mobilized by subsequent with 0.12 M-NaCl. The concentration of adenosine triphosphate (ATP) in sperm suspensions dropped on dilution with saline and rose as motility ceased, but declined without subsequent recovery following dilution with MIX-saline. The concentration of cyclic adenosine monophosphate (cAMP) rose and fell sharply on initiation of motility and rose again after motility had declined. While salmonid spermatozoa can be mobilized by dilition with saline alone, the effectiveness of MIX in reactivating “spent” spermatozoa supports the assumption that cAMP plays a role in the initiation of sperm motility.     

Ovarian morphology of the Japanese eel in Mikawa Bay

Annual changes in gonadal maturation of female Japanese eel Anguilla japonica in sea water were investigated histologically over 5 years in the Mikawa Bay, Japan, where they occurred throughout the year except in March. Almost all immature Japanese eels (yellow eels) occurred mainly from April to September, and they were rare after November. In contrast, maturing Japanese eels (silver eels) occurred from October to February. The gonado‐somatic index ( I _G) and oocyte diameters of yellow eels were <1·0 and 150 μm, respectively, and oocytes were at the peri‐nucleolus or the oil droplet stages. The I _G and oocyte diameters of silver eels were greater than those of yellow eels and most oocytes developed to the primary yolk globule stage. The numbers of silver eels lacking oocytes at the primary yolk globule stage increased after January in Mikawa Bay, although I _G and oocyte diameters remained unchanged. In contrast, silver eels caught at the mouth of the bay in January possessed oocytes that had advanced to the secondary yolk globule stage. These observations indicate that oocyte development changes seasonally, especially after winter in Mikawa Bay.     

Effects of cyclic AMP on the motility of mature and immature hamster epididymal spermatozoa studied by reactivation of the demembranated cells

Hamster spermatozoa from the caput and cauda epididymides were demembranated with 0.04% Triton X-100 and reactivated with 1 mM ATP. Motility parameters were analysed by video recording and stroboscopic photography. In the absence of added cAMP, reactivated cauda sperm showed percentage motility and forward swimming patterns similar to those of intact cells, but velocities were lower. When 2 or 20 μM cAMP was present, the velocities were increased but there was no effect on beat frequencies or percentage of forward progressing sperm. Cyclic AMP also markedly increased the percentage of cauda sperm which at first displayed nonprogressive “looping” movement. Addition of cAMP to the reactivation medium greatly improved the otherwise feeble and irregular motility of the demembranated caput sperm by increasing the percentage motility and beat frequencies of nonprogressive cells. It also induced forward motility with beat frequencies and velocities similar to cauda sperm reactivated in the absence of cAMP, but looping was never seen, indicating a change in the flagellar apparatus with maturation. The time required for the exhibition of the cAMP effects was reduced when caput sperm were reactivated in extracts of another previously maximally reactivated caput sperm preparation. The results suggest the production of some potent compound(s) by the axonemes for the manifestation of the cAMP effects.     

The onset and maintenance of hyperactivated motility of spermatozoa from the mouse

Estimates were made of the proportion of freely motile mouse spermatozoa displaying hyperactivated motility by an objective photographic method employing stroboscopic illumination under dark-field conditions and examining displacements of the sperm head and bend angles of the sperm tail. In media known to support in vitro fertilisation hyperactivation gradually appeared reaching about 40% by 6 hr incubation, and it was not promoted by 2 mM caffeine or 0.1 mM Bt₂ cAMP or washing the cells free of epididymal fluid. Raising the osmolarity of the medium to 400 mOSM with electrolytes, but not nonelectrolytes, did promote hyperactivation (60% by 2 hr) suggesting that the ionic strength of the medium was important. Hyperactivation in high ionic strength media could be prevented by removing or chelating Ca²⁺, or replacing Ca²⁺ with Ba²⁺ or Mg²⁺, when nonhyperactivated motility was maintained, but Sr²⁺, like Ca²⁺, permitted hyperactivated motility. Hyperactivation in low ionic strength medium could be promoted by the ionophore A23187, suggesting that Ca²⁺ movement into the cells is important. Of a range of glycolytic substrates tested supporting nonhyperactivated motility in the presence of lactate, only glucose supported hyperactivation. Addition to glucose— or Ca²⁺ — free, high ionic strength media after 2 hr increased hyperactivation immediately (glucose) or after a lag of 2 hr (Ca²⁺) suggesting that glucose acts on a Ca²⁺ — primed system. Removal from high ionic strength medium, chelation of Ca²⁺ or inhibition of glucose metabolism did not prevent hyperactivation continuing once it had been initiated, indicating different requirements for initiation and maintenance of this form of motility.     

Temperature effects on the incorporation of strontium in otolith of Japanese eel Anguilla japonica

The effects of temperature on somatic and otolith growth and the incorporation of strontium in otolith of the Japanese eel, were studied in laboratory-reared and field-caught eels. The somatic and otolith growth rates of the eel increased significantly with temperature and were estimated as approximately 0·096 mm t.l, (P<0·01) and 0·36 μm in otolith diameter per degree-day (0·01相似文献   

Recruitment mechanism of the eel, Anguilla japonica, to the Japanese coast

A total of 149 Anguilla japonica elvers collected at 10 locations along the Japanese coast were aged according to otolith daily increments. The interrelationships among age, birth date, body size, pigmentation stage, sampling location and the timing of recruitment were examined in order to determine the recruitment mechanism of elvers to coastal waters.
First catches of eels were earlier in the locations at lower latitudes. Age at recruitment was roughly constant, 218 ± 29 (mean ± s.d.) days old, disregarding the localities, but a weak positive correlation was obtained between age and sampling date or timing of recruitment. Body length at recruitment was also constant, 56.3 ± 2.3 mm s.l., and showed no significant correlation with either locality or recruitment timing. Estimated birth date ranged from April to November, the mean ± s.d. being 22 July 1982 ± 42 days, suggesting a peak season spawning in summer. Birth date was closely related with both the latitude of location and sampling time. Pigmentation developed more at lower latitude. Recruitment mechanism of the Japanese eel was summarized as follows: the earlier-born individual recruits earlier at lower latitude and at younger age, but at a constant body size.     

Computerized analysis of motility, motility patterns and motility parameters of spermatozoa of carp following short-term storage of semen

A computer-aided semen analysis system was used to assess the % motile cells following storage of carp semen in 11 different buffers at 2, 5 or 22° C. BWW and TLP were the most suitable storage buffers because carp semen stored at 5° C in these buffers following activation showed no significant decrease in % motile spermatozoa up to 24 h. But, in most of the other buffers (Fish Ringer, Cytomix, Cortland, FRT, Mannitol, FPS, NAS and TSM) the motility potential was lost by 2 h. Storage was best at pH 6–9 and at 5° C. Carp spermatozoa exhibit three distinct motility patterns, namely 'linear', 'circular' and 'haphazard', the proportion of spermatozoa with a particular motility pattern depending on storage buffer and time. All spermatozoa with a linear trajectory had high VSL, STR and LIN; those moving in circles had low VSL, STR, LIN and BCF and those with a haphazard trajectory were distinct in that they had the highest ALH and their VSL, STR, LIN and BCF were higher than the circular moving spermatozoa and lower than the spermatozoa exhibiting linear trajectory. The study also demonstrates a pronounced time-dependent decrease in VCL, VAP, VSL and ALH of carp spermatozoa following activation with water or low osmolality solutions. This study provides for the first time data related to seven motility parameters of carp spermatozoa and demonstrates how these parameter values could be used to evaluate quality of carp milt following storage in different buffers. It confirms that carp spermatozoa exhibit linear or circular trajectories and provides evidence for a third type of trajectory described as haphazard. All three motility patterns could be discriminated objectively on the seven motility parameters.     

Sperm motility and cryopreservation of spermatozoa in freshwater gobies

Testicular sperm motility and methods for the cryopreservation of spermatozoa in the freshwater goby Rhinogobius sp. CB (Cross Band type) were examined. Spermatozoa were almost immotile upon dilution with 300 mOsm kg⁻¹ of NaCl, KCl and mannitol solutions but began to swim in solutions with concentrations <200 mOsm kg⁻¹. The highest percentage and longest duration of motility was obtained in the 0 and 100–200 mOsm kg⁻¹ solutions, respectively. The highest post-thaw motility, c. 50% of motility before cryopreservation, was obtained when spermatozoa were diluted with an extender of 10% methanol and 90% artificial seminal plasma, cooled at −10·0 ± 1·1° C min⁻¹ (mean ± s . e .) to −50° C and plunged into liquid nitrogen. Spermatozoa were cryopreserved in a 50 μl acrylic haematocrit tube to store the small amount of milt. As the cryopreservation method described above was applicable to the endangered Rhinogobius sp. BI (Bonin Island type), it is probable that this method can be used for other species of freshwater gobies.     

Lipid peroxidation in human spermatozoa as relatd to midpiece abnormalities and motility

The formation of malondialdehyde (MDA), a product of lipid peroxidation (LPO), was measured in human spermatozoa from 27 subjects with normal sperm characteristics. Peroxidation of lipids in washed spermatozoa was induced by catalytic amounts of ferrous ions and ascorbate and malondiaidehyde dctermint-d by thiobarbituric method. MDA formation varied considerably from one sample to another. The studied population showed a strong correlation between lipid peroxidation potential (amount of MDA formed by 10⁸ spermatozoa after 1 hour of incubation) and 1) initial motility r = ?0.623, P = 0.001; and 2) morphologic abnormalities of the midpiece r = 0.405, P = 0.05. These results suggest that poor motility is linked with membrane fragility and that spermatozoa with midpiece abnormalities probably have membrane and/or cytoplasmic antiperoxidant system defects. Because LPO potential is related to the two most important characteristics of fertility, it seems possible that it has the potential to become a good biochemical index of semen quality.     

Determination of the freshwater mark in otoliths of Japanese eel elvers using microstructure and Sr/Ca ratios

The microstructure, in particular checks within the otolith edge, of Anguilla japonica glass-eels and elvers and changes in otolith Sr/Ca ratios were examined to ascertain the environmental history of the eels, especially with regard to the time when glass-eels entered the river, and as a benchmark for count daily increments. The percentage of glass eels and elvers with checks and the mean number of checks within the otoliths of glass-eels caught at four localities, Tosa Bay off Tosa City, the mouth of the Gokase River, the mouth of the Saigo River and the dam of the Tsuri River were 0% (0), 15.0% (0.2), 51.6% (1.0) and 100.0% (4.2), respectively. The Sr/Ca ratios and Sr content peaked in the region where checks were formed and the values decreased rapidly towards the edge of the checks; on the other hand, these decreased gradually in the otolith when checks were not formed. These checks were estimated to be formed by stress when the glass-eels were affected by ambient fresh water within the river. The innermost check was called the freshwater mark in the present study and this mark may be useful as a benchmark in studying the growth history of the eel before and after entering freshwater.     

Opsonic effect of antiserum and complement on phagocytosis by macrophages from the peritoneal cavity of the Japanese eel, Anguilla japonica

Macrophage exudates in the Japanese eel, Anguillu japonica, were induced by intraperitoneal injection with a mixture of Edwnrdsiella bacterin, proteose peptone and liquid paraffin, and the opsonic effect of antiserum and complement on the phagocytic activity of the macrophages was studied.
The macrophages contained a round nucleus with a nucleolus, and an agranular cytoplasm with projecting processes. These cells showed phagocytosis of sheep red blood cells (SRBC). An opsonic effect of antiserum was recognized in terms of a significant increase of macrophage phagocytic activity against SRBC treated with antiserum relative to that against SRBC treated with inactivated normal serum. Complement, however, did not enhance the phagocytic activity of macrophages.     

Sperm motility in the horseshoe crab. IV. Extracellular ions and intracellular pH are not mediators of motility initiation

Upon dilution into sea water, Limulus spermatozoa undergo a brief flurry of motility (duration < 60 sec), after which they are nonmotile until encountering a sperm motility initiating peptide (SMI) that emanates from eggs. Utilizing highly purified SMI extracts and simplified seawater formulations (from which individual ions have been deleted), we found that no specific extracellular ion is required for either dilution-initiated or SMI-initiated motility. Indeed, deletion of one ion (Na⁺) produced dilution-initiated motility of very long duration (several hours). When motility is initiated by SMI (in normal seawater) there is an increase in intracellular pH (pH_i), as indicated by the fluorescent probe, 9-amino acridine; however, this pH, change is not a trigger for motility. As a more general method examining ion movements, the fluorescent probe diS-C₃-(5) was used to qualitatively measure changes in the membrane potential of spermatozoa. Although crude SMI extracts caused membrane depolarization, further purification resulted in an almost complete separation of this activity from SMI, thus showing that SMI activation is apparently an electroneutral event. (The membrane-depolarizing factor has a molecular weight > 30,000 and does not initiate acrosome reactions.) Experiments utilizing the ionophore A23187 and Ca⁺²-blocking agents (verapamil and TMB-8) provided tentative evidence that mobilization of intracellular Ca⁺² may be required for motility initiation. These results show that neither changes in pH_i nor the influx of specific extracellular ions are direct mediators of SMI-initiated motility; however, experiments with pharmacologic agents indicate a possible role for intracellular Ca⁺².     

Inhibitory effect of sodium bicarbonate on the motility of sperm of Japanese eel

Sperm motility of Japanese eel Anguilla japonica was depressed from 59.1 to 1.1% when NaHCO₃ concentration increased from 0 to 150 mM. In 25 mM NaHCO₃, when pH of the medium was 6.2, 7.2, 8.2, 9.2 and 10.2, relative sperm motility was 0, 0, 55.8, 93.7 and 136.6%, respectively to that of the control (0 mM NaHCO₃). The remarkable effect in acid or neutral condition indicates that free-CO₂ (liberated CO₂ and H₂CO₃) is a key factor for the motility inhibiting effect of NaHCO₃.     

Internal illuminance and shelter shape affect shelter selection by the Japanese eel Anguilla japonica

The present study evaluated the effects of internal illuminance and shelter shape on shelter selection by Japanese eels to enable the preservation or construction of suitable shelters for the Japanese eel. Japanese eels were able to distinguish a 1.25-fold difference in illumination inside the shelters, and preferred darker shelters. When the internal illumination of two shelters with the same shape was less than one-tenth of the ambient illumination (about 400 lx), shelter selection by Japanese eels was not affected by internal illuminance, even when there was a 10-fold difference in internal illumination between shelters. The width of the shelter was not important, but Japanese eels preferred a deep shelter with a low ceiling and walls that spread to a ‘dead end’. This has important implications on the creation of suitable shelters for Japanese eels.     

Analysis of motility parameters from paddlefish and shovelnose sturgeon spermatozoa

Ninety to 100% of paddlefish Polyodon spathula were motile just after transfer into distilled water, with a velocity of 175 μm s^-1, a flagellar beat frequency of 50 Hz and motility lasting 4–6 min. Similarly, 80–95% of shovelnose sturgeon Scaphirhynchus platorynchus spermatozoa were motile immediately when diluted in distilled water, with a velocity of 200 μm s^-1, a flagellar beat frequency of 48 Hz and a period of motility of 2–3 min. In both species, after sperm dilution in a swimming solution composed of 20 mM Tris–HCl (pH 8·2) and 20 mM NaCl, a majority of the samples showed 100% motility of spermatozoa with flagella beat frequency of 50 Hz within the 5 s following activation and a higher velocity than in distilled water. In such a swimming medium, the time of motility was prolonged up to 9 min for paddlefish and 5 min for sturgeon and a lower proportion of sperm cells had damage such as blebs of the flagellar membrane or curling of the flagellar tip, compared with those in distilled water. The shape of the flagellar waves changed during the motility phase, mostly through a restriction at the part of the flagellum most proximal to the head. A rotational movement of whole cells was observed for spermatozoa of both species. There were significant differences in velocity of spermatozoa between swimming media and distilled water and between paddlefish and shovelnose sturgeon.     

Mouse lipocalin as an enhancer of spermatozoa motility

The 24p3 protein is a 25 kDa glycoprotein that is secreted into the uterine fluid during the proestrous phase of mice. We assessed the effects on spermatozoa motility and on the functions of mouse spermatozoa using the computer-assisted sperm analysis method, cytochemical staining and detection of the protein tyrosine phosphorylation pattern. Compared with the control cells, sperm motility was stimulated by the addition of 24p3 protein into the medium. Introducing 24p3 protein enhanced progressive motility but did not promote the appearance of hyperactivated movement. The presence of 24p3 protein in the medium did not allow the cells to undergo the capacitated protein tyrosine phosphorylation pattern and acrosome reaction. The tyrosine phosphorylation pattern shows phosphoproteins in the range of Mr 50000–106000 correlated with the sperm progressive motility after the addition of 24p3 protein into the medium. Using flow cytometry, we assessed the changes in the intracellular pH and measured the intracellular cAMP concentration with an immunodetection kit. The results indicated that the elevation in intracellular pH from 6.67 to 6.89, increase of intracellular cAMP accumulation, and protein tyrosine phosphorylation might be the factors in enhancement of sperm motility as the 24p3 protein bound to the spermatozoa. The 24p3 protein may have a role in regulating flagellar motility.     

Lipid and fatty acid composition of eggs producing larvae with high survival rate in the Japanese eel

Eggs of Japanese eel Anguilla japonica were obtained by artificial induction of maturation. Fertilization and hatching rates, and survival rates of normal larvae were examined. The levels of total lipids and polar lipids (PL) of high quality (HQ; >80% survival rate at 8 days post-hatch) eggs were significantly lower than low quality (LQ; hatching and survival rates were zero) eggs. The docosahexaenoic acid (DHA) level in PL of HQ eggs, however, was significantly higher than in LQ eggs. In contrast to DHA, arachidonic acid level was negatively correlated with egg quality variables and it was lower in HQ than in LQ eggs. Most of the egg lipid and fatty acid components showed larger variation in LQ than in HQ eggs.