Induction of maturation and spermiation in the male European eel: assessment of sperm quality throughout treatment

Weekly injections of human chorionic gonadotropin (hCG; 1·5 IU g⁻¹ W ) induced spermiation in 87–100% farmed male European eels Anguilla anguilla over a 10-week period, producing 3 ml (100 g)⁻¹ milt volume, at 1˙4×10⁹ spermatozoa ml⁻¹ sperm mobility peaked in ninth week of treatment with 97% of males with motile sperm. Gonadotropin-treated males showed earlier but similar morphological changes to control males. Whereas higher sperm density was obtained 6 h after hCG administration, higher percentage of motile cells and longer sperm mobility were observed 24 h after the treatment indicating that this is the optimum time to obtain good sperm after the hormonal induction. Several monounsaturated fatty acids in sperm increased significantly from week 5 to 9 of treatment (when highest volume, density and mobility of milt were recorded), whereas eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA) and total n-3 polyunsaturated fatty acids (PUFAs) were significantly reduced. Fatty acid levels returned to initial values after 13 weeks of treatment. These results are in agreement with the reduction of n-3 PUFAs recently observed in the ovary of female Japanese eel during artificial maturation, and an attempt is made to suggest a physiological explanation of its possible relation with sperm quality.     

Growth, gas exchange and water use efficiency of the facultative hemiparasite Rhinanthus minor associated with hosts differing in foliar nitrogen concentration

The facultative hemiparasitic angiosperm Rhinanthus minor was grown on 11 different host species, and in the absence of a host. The height of R. minor parasitising the legume Trifolium pratense exceeded that of unattached plants by more than an order of magnitude, with performance on grasses and non-legume dicotlyedonous hosts between these two extremes. Light saturated rates of photosynthesis in R. minor on different hosts ranged from 1. 5 to 22. 5 μmol g⁻¹ dry weight min⁻¹, and were positively correlated with growth. Foliar nitrogen concentrations in the parasite exceeded those of the hosts. The former were positively related to light saturated rates of photosynthesis, and inversely related to photosynthetic nitrogen use efficiency. There was no relationship between leaf nitrogen concentration and water use efficiency in R. minor. The data are discussed in relation to studies of nitrogen and water use in mistletoes.     

Intracellular ion levels in erythrocytes and hepatocytes isolated from three teleost species

Intracellular concentrations of Na⁺ and K⁺ were similar (∼75 mmol l⁻¹) in rainbow trout Oncorhynchus mykiss hepatocytes directly following isolation by collagenase digestion, but partial recovery occurred over 6 h with K⁺ levels increasing to 110 mmol l⁻¹ and Na⁺ levels decreasing to 42 mmol l⁻¹. Black bullhead Ameiurus melas hepatocytes exhibited higher intracellular concentrations of K⁺ (90 mmol l⁻¹) than Na⁺ (55 mmol l⁻¹) with no recovery occurring over 6 h following cell isolation. Concentrations of Na⁺, K⁺ and Cl⁻ in eel Anguilla rostrata hepatocytes were similar (∼ 55 mmol l⁻¹) following isolation, with no recovery occurring over time. Erythrocytes from all species apparently did not experience an intracellular ion imbalance following isolation as indicated by high K⁺ levels (<140 mmol l⁻¹) and low Na⁺ levels (<40 mmol l⁻¹) during the entire 24-h monitoring period. Although hepatocytes from all species exhibited an ion imbalance post-isolation, comparison of their in vitro intracellular Na⁺ and K⁺ concentrations with those in plasma demonstrated that directionally correct ion gradients still exist across the cell membrane, albeit differing from those that would be found in the tissue in vivo .     

Inhibitory effect of osmotic concentration, potassium and pH on motility of the sperm of the North American burbot Lota lota maculosa

Seminal plasma factors maintaining North American (NA) burbot Lota lota maculosa sperm quiescent were examined. Sperm were diluted into buffered saline solutions of various compositions and motility assessed. After 1 h in these solutions at 10° C, aliquots of the suspension were diluted with tap water and motility again assessed. Dilution of sperm in an incubation solution containing Ca²⁺ in the absence of K⁺ initiated sperm motility resulting in low motility when sperm were subsequently diluted in tap water. Incubation solutions with osmolalities >200 mOsm kg⁻¹ and containing 12·5 mM K⁺ prevented the onset of sperm motility and were associated with maximal sperm motility upon dilution in tap water. Sperm maintained at lower osmolalities exhibited limited motility upon dilution in tap water indicating interdependence between K⁺ and osmolality in maintaining sperm quiescent in the presence of Ca²⁺. Sperm kept in incubation solution at pH values < c. 7·5 for 1 h demonstrated reduced motility when subsequently diluted in tap water. That motility of sperm was pH sensitive was further indicated by CO₂ inhibition of motility. Therefore, NA burbot sperm are probably maintained in an immotile state, yet with potential for motility, by combination of high K⁺, osmolality and possibly pH. The results from this study differ from published information on sperm quiescence in the temporally and geographically distinct Eurasian burbot Lota lota lota .     

Electroporation of abalone sperm enhances sperm-DNA association

The ability of sperm from the black-footed abalone Haliotis iris to take up foreign DNA in solution has been demonstrated. The efficiency of DNA uptake is related to the conditions of electroporation, including field strength (625 V cm⁻¹, 1000 V cm⁻¹), pulse length (18.6 ms, 27.4ms) and number of pulses (1, 2), and DNA concentration (20, 100 μg ml⁻¹). Sperm motility decreased with increased field strength and pulse number. At a field strength of 625 V cm⁻¹, neither the pulse length nor pulse number enhanced DNA uptake. A 40% enhancement in DNA uptake was observed when the sperm were shocked at 1000 V cm⁻¹ with two long pulses (27.4 ms each). Linear regression analysis revealed that pulse number ( p = 0.013) and field strength ( P =0.039) were the most important factors in sperm–DNA interaction. Higher DNA concentration enhanced sperm DNA uptake irrespective of field strength, pulse length and pulse number. The optimal electroporation conditions for DNA uptake were 1000 V cm', with two pulses of 27.4 ms each, and a DNA concentration of 100 μg ml⁻¹.     

PATTERNS OF INTERSPECIFIC INTERACTIONS IN THE ULVA-DOMINATED INTERTIDAL COMMUNITY IN A SOUTHERN COAST OF KOREA

The reproductive success of marine species with external fertilization depends on environmental conditions during gamete release. There is special interest presently in whether water motion causes sperm limitation under natural conditions. We investigated gamete release of Fucus vesiculosus from an exposed shore to ascertain: 1) when gametes are released during the tidal cycle, 2) when fertilization occurs, and 3) what the natural sperm:egg ratios are. Water samples were collected and concentrated over five minutes every half hour off Pemaquid Point, ME from three replicate sites within each of two locations using a pump-filter device. Immunofluorescence microscopy revealed that gamete release occurred only on the two calmest spring tides. Sperm became present in the water column at the same time as oogonia (30 min⁻¹ h prior to high tide [HT]) and reached peak concentration at exactly HT. The sperm:egg ratio was 76:1 on 8 Oct 1999 and 21:1 on 8 Nov 1999 at exactly 30 min prior to HT and dropped sharply after HT. Gametes continued to be collected for several hours after HT but analysis of pronuclear position in aceto-iron-hematoxylin stained eggs revealed that all fertilization occurred at approximately HT. We modelled the total number of days when reproduction was possible using these results and wind and wave data from the National Data Buoy Center. Our research provides evidence that gamete release by F. vesiculosus occurs at slack HT on calm days and that sperm are not a limiting factor in fertilization for this species.     

Chlorophyll content and the light response curve of shade-adapted conifer needles

Rates of photosynthesis were measured for shade-adapted conifer needles [ Picea abies (L.) Karst., Picea glauca (Moench) Voss, Picea mariana (Mill.) B. S. P., Picea sitchensis (Bong.) Carr., Pinus sylvestris L.] illuminated on i) their top surface, ii) their top and bottom surfaces simultaneously, or iii) uniformly in a light integrating sphere. The rate of bending of the light response curve of photosynthesis was strongly correlated with the amount of chlorophyll per unit of illuminated leaf area, when the amounts of chlorophyll were above 200 mg m⁻². Below 200 mg m⁻² only small differences in the rate of bending occurred and the light response curves took the form of a quasi-Blackman response. At chlorophyll contents below 200 mg m⁻² the rate of bending of the light response curves was insignificantly different from a mean published value for enzymatically isolated cells of angiosperm leaves (I. Terashima and T. Saeki. Ann. Bot. 56: 489–499, 1985). The apparent quantum yield of photosynthesis (based on incident light) also showed a response to chlorophyll content not different from the pattern reported for angiosperm leaves (E. K. Gabrielsen. Physiol. Plantarum 1: 5–37, 1948). Neither CO₂ nor O₂ concentration significantly affected the rate of bending of the light response curves. It is concluded that the light response curve of individual chlorenchyma cells is a quasi-Blackman response and that the rate of bending of the light response curve of intact leaves is a strong function of the development of gradients of light and thus the chlorophyll content of leaves.     

Sperm competition and sperm precedence in the dragonfly Nanophya pygmaea

Abstract. The libellulid dragonfly, Nanophya pygmaea Rambur, has an average ejaculate volume of 0.16 mm³. During successive copulations the volume of sperm stored in the female's sperm storage organs increases in steps equivalent to this volume, suggesting that the sperm competition mechanism in this species is sperm repositioning, i.e. adding an ejaculate to what is already present in the female's sperm storage organ. By using sterile/normal males in double matings with females we have shown that this mechanism results in last male sperm precedence (P²= 0.979).     

Cytoplasmic connection of sperm cells to the pollen vegetative cell nucleus: potential roles of the male germ unit revisited

The male germ cells of angiosperm plants are neither free-living nor flagellated and therefore are dependent on the unique structure of the pollen grain for fertilization. During angiosperm male gametogenesis, an asymmetric mitotic division produces the generative cell, which is completely enclosed within the cytoplasm of the larger pollen grain vegetative cell. Mitotic division of the generative cell generates two sperm cells that remain connected by a common extracellular matrix with potential intercellular connections. In addition, one sperm cell has a cytoplasmic projection in contact with the vegetative cell nucleus. The shared extracellular matrix of the two sperm cells and the physical association of one sperm cell to the vegetative cell nucleus forms a linkage of all the genetic material in the pollen grain, termed the male germ unit. Found in species representing both the monocot and eudicot lineages, the cytoplasmic projection is formed by vesicle formation and microtubule elongation shortly after the formation of the generative cell and tethers the male germ unit until just prior to fertilization. The cytoplasmic projection plays a structural role in linking the male germ unit, but potentially plays other important roles. Recently, it has been speculated that the cytoplasmic projection and the male germ unit may facilitate communication between the somatic vegetative cell nucleus and the germinal sperm cells, via RNA and/or protein transport. This review focuses on the nature of the sperm cell cytoplasmic projection and the potential communicative function of the male germ unit.     

Intracellular pH Changes of Starfish Sperm Upon the Acrosome Reaction

The acrosome reaction is accompanied by ionic changes such as increases in intracellular Ca²⁺ and intracellular pH (pH_i). 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 pH_i-increase using 9-aminoacridine as a probe of pH_i. This paper presents evidence that an oligopeptide(s) is responsible for the pH_i-increase. The pH_i of swimming sperm is 7.4-7.5. Within 20 sec after the addition of jelly, their pH_i 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 pH_i 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 pH_i, 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 pH_i of the sperm. The significance of the pH_i-increase upon the acrosome reaction is discussed.     

高等植物受精过程中雌-雄相互作用的分子调控机制

从广义上讲,被子植物的受精过程是指花粉粒落到柱头上萌发形成花粉管,花粉管穿过柱头沿着引导组织生长进入子房内,最终在胚囊中实现精细胞与卵细胞以及中央细胞分别融合从而起始胚胎和胚乳的发育．被子植物的精细胞由于不具有鞭毛而无法自由移动,因此在受精过程中需要借助于花粉管来将精细胞运送到胚囊中．花粉管通过与雌性的孢子体组织之间的相互作用和识别将精细胞准确地运送到胚珠附近,而最终将精细胞准确地运送到胚囊内的过程则是受到了雌配子体细胞的控制．可以说,受精的成功实现有赖于雌性和雄性细胞之间的持续的识别和相互作用,这种互作具有多样性和阶段特异性．本文将主要综述被子植物受精过程中花粉粒以及花粉管与多种雌性孢子体组织以及雌配子体之间的信号互作研究．     

Are non-reproductive male highveld mole-rats, Cryptomys hottentotus pretoriae physiologically suppressed while in the confines of the natal colony?

The highveld mole-rat, Cryptomys hottentous pretoriae is a social, subterranean, co-operative breeder with a high reproductive skew. The relationship between reproductive status and reproductive physiology in male highveld mole-rats was investigated. Testicular morphometrics, histological parameters, plasma testosterone concentrations and sperm motility parameters were studied in 31 males (14 reproductive and 17 non-reproductive males). Reproductive males were significantly larger than non-reproductive males, with testicular mass and volume corrected for body mass considerably larger for the reproductive males. Circulating plasma testosterone concentrations of reproductive males were not significantly higher than non-reproductive males (reproductive males 10.3±1.8 nmol l⁻¹ vs non-reproductive males 7.3±1.74 nmol l⁻¹). Sperm motility parameters were measured, but no significant differences were found between reproductive and non-reproductive males. All 14 reproductive males had motile sperm, whereas 13 of the 17 (76%) of non-reproductive males possessed motile sperm. A typical ejaculate of a reproductive male contains 48.3% motile sperm characterized by a high percentage of flagellar defects, whereas the non-reproductive male has an ejaculate containing 45.6% motile sperm with a high percentage of head defects. It is apparent that non-reproductive males are not physiologically suppressed from reproducing. Moreover, the non-reproductive males are excluded from incestuous matings as a result of being the offspring of the reproductive female.     

The spermatozoon of the African catfish: fine structure, motility, viability and its behaviour in seminal vesicle secretion

The spermatozoon of the African catfish Clarias gariepinus is a simple organized aquasperm although it reveals very unique characteristics: the cytoplasmic channel is lacking, the mitochondria form a complex structure and the arrangement of the centriolar complex is species specific. Semen has high initial motility rates ( c. 70–90%) and swimming velocities ( c. 120–140 μm s⁻¹), the main swimming type is linear. Motility duration in water is 30 s and is prolonged only to 40 s in NaCl solutions or more complex bu ered motility activating saline solutions. A pH between 7.0 and 9.0 has no e ect on the sperm motility parameters. Motility is completely and reversibly suppressed in electrolyte and non-electrolyte solutions with an osmolality of 200 mosmol kg⁻¹. During immotile storage the sperm viability is influenced by the osmolality and the potassium levels of the storage medium, by the temperature and by the dilution. At optimal conditions (bu ered sperm motility inhibiting saline solution: 150 mmol l⁻¹ NaCl, 2.5 mmol l⁻¹ KCl, 1 mmol l⁻¹ CaCl₂, 1 mmol l⁻¹ MgSO₄, 20 mmol l⁻¹ Tris solution, pH 8.5; dilution rate 1: 5; storage temperature, 4°C) sperm viability persists for >7 days. High viscosity of the pure seminal vesicle secretion completely inhibits the sperm motility. When the seminal vesicle secretion is diluted in water the viscosity decreases and the motility suppressing e ect is neutralized. When semen is mixed with seminal vesicle secretion the sperm viability decreases to zero within 10 min.     

Characterization of sperm motility in sea bass: the effect of heavy metals and physicochemical variables on sperm motility

Computer assisted sperm analysis (CASA) was used to characterize the motility of sea bass Dicentrarchus labrax spermatozoa and to study the effect of several physicochemical variables and heavy metals on sperm swimming performance. Duration of sperm motility in sea bass was very short (<50 s). During the first 20 s all the motility variables measured remained approximately constant, the velocity and linearity of the movement being maximum during this period, while both variables decreased sharply later. While slight variations in pH did not significantly modify sperm swimming performance, changes in osmolality affected all the measured motility variables. Two of the heavy metals tested, Cu²⁺ and Pb²⁺, did not affect sperm motility when the activating media contained up to 100 ppm of the metal salts. In contrast, Hg²⁺ modified the morphology of post-swimming spermatozoa at 0·4–1 ppm (sperm dilution rate 1:39) and completely arrested sperm motility at concentrations as low as 0·1 ppm (sperm dilution rate 1:2500). Assuming a covalent binding to sperm cells, this revealed a finite number of c. 10 million Hg²⁺ binding sites per spermatozoon. Complementary results using demembranated spermatozoa suggested that the main target of HgCl₂ would be located in the plasma membrane and that HgCl₂ would inhibit water channels, hence preventing sperm motility.     

Longevity of the freshwater anostracan Streptocephalus mackini (Crustacean: Anostraca) in relation to food (Chlorella vulgaris) concentration

SUMMARY 1. Temporary ponds are inhabited by a variety of invertebrates, of which anostracans are an important group. We studied the lifetables of male and female anostracan Streptocephalus mackini at 3 algal concentrations (0.5 × 10⁶, 1.0 × 10⁶ and 1.5 × 10⁶ cells mL⁻¹).
2. Regardless of sex, S. mackini showed better survivorship at lower food levels. The longest average lifespan observed was 85 ± 2 days for males fed Chlorella at 0.5 × 10⁶ cells mL⁻¹.
3. Both net reproductive rate and generation time decreased with increasing food level. The highest net reproductive rate was about 120 cysts per female. The longest generation time of about 40 days, observed at 0.5 × 10⁶ cells mL⁻¹, was more than three times that at 1.5 × 10⁶ cells mL⁻¹.
4. The rate of population increase ( r ) was nearly the same (0.31 ± 0.06) at high (1.5 × 10⁶ cells mL⁻¹) and intermediate (1.0 × 10⁶ cells mL⁻¹) food levels. The r -value at low food level (0.5 × 10⁶ cells mL⁻¹ of Chlorella ) was 0.20 ± 0.01 per day.     

β-Adrenergic antagonists and carp (Cyprinus carpio L.) sperm motility

We studied the effects of two β-adrenergic antagonists, atenolol and propranolol, on carp sperm motility. Atenolol (10⁻³−10⁻⁸ m ) has no appreciable effects while propranolol (6 × 10⁻⁵−3 × 10⁻⁶ m ) affects the percentage of sperm motility in a dose-dependent fashion.     

A developmental study of Glycine max cell and protoplast isolation in relation to leaf age and photosynthetic competence

Leaf mesophyll cells were isolated from developing first trifoliate leaves of Glycine max (L.) Merr cv. Fiskeby V using a mechanical isolation procedure combined with low speed centrifugation. Cell yields of 17 ± 1.7% were routinely obtained with 55–75% intactness, as assessed by staining techniques, fluorescence transients and the ability of cells to convert to protoplasts after enzyme treatment. Rates of leaf photosynthesis were maximal in 27-day-old plants [280 μmol O₂ evolved (mg chlorophyll)^-1h^-1], from which isolated cells and protoplasts gave rates of up to 140 μmol O₂ evolved (mg chlorophyll)^-1 h^-1. Results are discussed in relation to leaf development and cell status during the attainment of photosynthetic competence.     

Reproductive characteristics of the male herring in the northern Baltic Sea

The gonad weight and gonadosomatic index of the male Baltic herring Clupea harengus membras L., were highest at the beginning of the reproductive season (April/May), the values decreasing towards the end of it (July/August) during 1988–1991. The decline could not be explained by fish size but may have been due to fish condition. A high individual variation was typical for both gonad weights and gonadosomatic indices in fish of the same size and maturity stage. The mean density of sperm cells was significantly higher in June (34·9 × 10⁹ ml⁻¹) than in July (19·2 × 10⁹ ml⁻¹, Mann-Whitney U= 17; P<0·05), the variation among the males being high in both groups. Electron microscope analysis showed a severe disruption of the mitochondrial elements in males spawning at 22°C.     

Live-cell imaging reveals the dynamics of two sperm cells during double fertilization in Arabidopsis thaliana

Flowering plants have evolved a unique reproductive process called double fertilization, whereby two dimorphic female gametes are fertilized by two immotile sperm cells conveyed by the pollen tube. The two sperm cells are arranged in tandem with a leading pollen tube nucleus to form the male germ unit and are placed under the same genetic controls. Genes controlling double fertilization have been identified, but whether each sperm cell is able to fertilize either female gamete is still unclear. The dynamics of individual sperm cells after their release in the female tissue remain largely unknown. In this study, we photolabeled individual isomorphic sperm cells before their release and analyzed their fate during double fertilization in Arabidopsis thaliana. We found that sperm delivery was composed of three steps. Sperm cells were projected together to the boundary between the two female gametes. After a long period of immobility, each sperm cell fused with either female gamete in no particular order, and no preference was observed for either female gamete. Our results suggest that the two sperm cells at the front and back of the male germ unit are functionally equivalent and suggest unexpected cell-cell communications required for sperm cells to coordinate double fertilization of the two female gametes.