NEUTRAL LIPIDS AS MAJOR STORAGE PRODUCTS IN ZOOSPORES OF THE GIANT KELP MACROCYSTIS PYRIFERA (PHAEOPHYCEAE)1

Neutral lipids, consisting primarily of triacylglycerols, were found to be a major form of carbon reserve in zoospores of the giant kelp Macrocystis pyrifera (L.) C. Ag. The fluorescent stain Nile Red revealed large lipid droplets in the posterior end of the cell, which comprised 20–41% of cellular carbon in newly released spores. Flow cytometric analyses of newly released spores stained with Nile Red revealed considerable variation in the neutral lipid content among spores that was independent of spore size. Lipid droplets were consumed during germination in spores maintained either under constant light or in continual darkness. The availability of light appeared to delay, but did not preclude, lipid use. The rate of lipid use during germination varied considerably among germlings with some cells consuming all of their lipid reserves within 5 h after release. In addition to zoospores, lipid droplets were observed in both male and female gametes. Numerous droplets were observed in eggs, while single lipid droplets were observed in sperm. Neutral lipid droplets were not observed in gametophytes or sporophytes except in developing gametes and spores. Large lipid reserves thus seem to be confined to the microscopic life history stages that presumably have relatively high energy demands. By serving as a supplemental fuel reserve, neutral lipids may be important in extending the effective range of zoospore dispersal.     

ELECTRON MICROSCOPE OBSERVATIONS ON THE FLAGELLAR APPARATUS OF BRYOPSIS MAXIMA (CHLOROPHYCEAE)1

The flagellar apparatus in male gametes of the siphonaceous green alga, Bryopsis maxima Okamura, was studied and compared with that of other green biflagellate cells. The proximal portions of two basal bodies are connected by a single striated proximal band, unique among the biflagellate reproductive cells of green algae studied. Anterior to the flagellar bases is a pair of distal bands different from the single structure in other biflagellate cells. These bands which arise from the distal portion of each basal body, extend upward in the papilla and curve down toward the lower edges of the basal bodies. They seem to have no direct association with each other. Two pairs of distinct flagellar roots, one consisting of 3–5 microtubules and the other of a partially striated fiber of undetermined numbers of microtubules, diverge from the basal body region and extend towards the cell posterior. Their component microtubules are disorganized into single or smaller groups midway over the cell length. The uniqueness of the flagellar apparatus is briefly discussed.     

Plasmodium gallinaceum: transmission-blocking immunity in chickens. I. Comparative immunogenicity of gametocyte- and gamete-containing preparations.

Immunization of chickens by intravenous inoculation of preparations derived from blood infected with Plasmodium galinaceum led to reduced infectivity to mosquitoes (Aedes aegypti) during subsequently induced blood infection but had little effect on the course of asexual parasitemia. Immunization with preparations containing intracellular gametocytes was much less effective than immunization with preparations in which the extracellular gametes had been released during gametogenesis (emergence and exflagellation) prior to inoculation. By far the most effective material were preparations of partially purified gametes of both sexes. Three weekly inoculations of this material resulted in 99.99% suppression of infectivity to mosquitoes during subsequently induced blood infection. Preparations of purified gamete material from which gametes of one or another sex were absent were considerably less effective as transmission-blocking immunogens than the mixed gamete preparation. It is possible that the two sexes of gamete act synergistically to induce transmission-blocking immunity.     

Fertilization mode, sperm characteristics, mate choice and parental care patterns in Artedius spp. (Cottidae)

The reproductive biology of three species in the genus Artedius ( A. harringtoni, A. lateralis and A. fenestralis ) was studied, including parental behaviour, mate choice, fertilization mode, gamete morphology and behaviour, and gonad morphology to help understand the coevolution of reproductive characteristics with potentially different modes of fertilization. In all three species, males guard multiple clutches at oviposition sites. In laboratory mate‐choice experiments, males appearred to prefer to defend sites containing eggs and were better able to attract mates at these sites. There was evidence that internal gomete association, although more pronounced in A. harringtoni , existed in all three species. Sperm behaviour and morphology and histological analysis of gonads suggested that A. fenestralis and A. lateralis were more likely to engage in typical external fertilization. The adoption of eggs and their effect on female mate‐choice patterns appearred to reduce the importance of certainty of paternity in these species, and probably increased the evolutionary stability of male parental care.     

Role of the sperm proteasome during fertilization and gamete interaction in the mouse

In this work, we have investigated the role of the sperm proteasome during in vitro fertilization (IVF) and gamete interaction in the mouse. Proteasome activity was measured in extract and intact sperm using a specific substrate. In addition, sperm were treated with specific proteasome inhibitors and evaluated during IVF, binding to the zona pellucida, and progesterone- and zona pellucida-induced acrosome reactions. In other experiments, sperm membrane proteins were obtained resuspending them in Triton X-114, shaking vigorously and let standing by 4 hr. Soluble sperm proteins were partitioned in the aqueous phase and sperm membrane proteins in the detergent phase. In both phases, proteasome activity was measured. Labeling of cell surface sperm proteins was carried out with the cell-impermeable NHS-LC biotin, extracted with Triton X-114, and mixing with avidin-agarose beads. Nonpermeabilized sperm were incubated with an anti-proteasome monoclonal antibody and evaluated by indirect immunofluorescence. The results indicate that sperm extracts as well as intact sperm had proteasome activity; the sperm proteasome was involved in IVF, specifically during sperm-zona pellucida binding and the acrosome reaction; soluble sperm membrane proteins exhibited proteasome activity; biotin experiments indicated the presence of proteasomes on the sperm surface, which was corroborated by indirect immunofluorescence experiments. All these observations indicate that the mouse sperm proteasome participates in the binding to the zona pellucida and the acrosome reaction and that there is a pool of proteasomes located on the sperm head.     

Involvement of the pentose phosphate pathway and redox regulation in fertilization in the mouse

Glucose metabolism is necessary for successful fertilization in the mouse. Both spermatozoa and oocytes metabolize glucose through the pentose phosphate pathway (PPP), and NADPH appears required for gamete fusion. The aims of this study were to further characterize the utilization of glucose by the fertilizing spermatozoon and the fertilized oocyte, to demonstrate the importance of the PPP in different steps of fertilization, and to examine whether the beneficial effect of glucose could be mediated by a NADPH-dependent enzyme involved in redox regulation. By using a fluorescent analog of 2-deoxyglucose, glucose uptake was evidenced in both the head and flagellum of motile spermatozoa. After sperm-oocyte fusion, an increase in glucose uptake by the fertilized oocyte was observed but not before the formation of the male and female pronuclei. By using a microphotometric technique, activity of glucose 6-phosphate dehydrogenase (G6PDH), the key enzyme of the PPP, was localized to the sperm head and midpiece. When epididymal spermatozoa were released into a glucose-containing medium, the NADPH/NADP ratio increased with capacitation. Sperm-oocyte fusion and meiosis reinitiation of the fertilized oocyte was inhibited by the PPP inhibitor 6-aminonicotinamide (6-AN); inhibition of sperm-oocyte fusion was relieved by NADPH. Sperm-oocyte fusion and meiosis reinitiation were also inhibited by diphenylamine iodonium, which is a flavoenzyme inhibitor reported to prevent reactive oxygen species (ROS) generation in mouse spermatozoa and embryos. These findings indicate that the PPP is involved in different steps of fertilization. Subsequent regulation of a NADPH-dependent flavoenzyme responsible of ROS production is envisaged.     

ION FLUXES AND MODIFICATION OF THE EXTRACELLULAR MATRIX DURING GAMETE RELEASE IN FUCOID ALGAE

A rapid phase transition in the extracellular matrix (ECM) of the reproductive tissues (= receptacles) has been proposed to cause gamete release in fucoid algae. We tested this model with cryoanalytical energy dispersive x-ray microanalysis (EDX) scanning electron microscopy (SEM) of rapidly frozen hydrated receptacles of Silvetia compressa Serrão, Cho, Boo et Brawley that were planed to smooth faces using a cryoultramicrotome. Every receptacle typically contained a region(s) of intracellular accumulation of K and Cl and a region(s) of efflux of K and Cl to the ECMs, regardless of treatment (e.g. calm vs. shaken conditions in the light, anthracene-9-carboxylic acid). Although longitudinal variations in [K] and [Cl] between ECM and cells were common, a tissue within any transverse plane of the receptacle was in the same state of efflux or accumulation of K and Cl. The strongest and most extensive effluxes occurred during the time of irreversible commitment to gamete release (i.e. 2–4 min darkness after light potentiation). A receptacle may oscillate rapidly between global states of accumulation and efflux at this time. The polysaccharide collar between the oogonium and stalk cell expanded during Cl and K efflux into the ECM of the conceptacle and condensed when [K] and [Cl] were low in the conceptacle ECM. During periods of strong efflux associated with gamete release, the collar and other components of the exochiton ruptured. Detached gametangia were guided to the pore by the paraphyses. The ECM of the conceptacle is sulfur rich (226 mM) compared with the ECM of the medulla (8 mM). This analysis supports the importance of osmotic modification of the ECM during gamete release and demonstrates that the receptacle is a dynamic signaling organ.     

Differences in ploidy levels of inter-specific hybrids obtained by reciprocal crosses between Primula sieboldii and P. kisoana

Differences in ploidy level were found in inter-specific hybrids obtained by reciprocal crosses between Primula sieboldii and P. kisoana. When P. sieboldii was used as the maternal parent, the inter-specific hybrids were triploids; when P. kisoana was the maternal parent, the inter-specific hybrids were diploids. The possibility of diploid female gamete formation in P. sieboldii is discussed as a causal factor in the production of triploids occasionally found in crosses between diploids of this species. Received: 28 December 1999 / Accepted: 10 January 2000     

Downregulation of egg cell-secreted EC1 is accompanied with delayed gamete fusion and polytubey

One major player known to be essential for successful gamete interactions during double fertilization in Arabidopsis thaliana is the recently identified family of egg cell-secreted EC1 proteins. Both gamete fusion events are affected in EC1-deficient female gametophytes. Here, we show that the number of ovules with unfused sperm cells is considerably higher than the number of undeveloped seeds in the same ec1-RNAi knockdown lines. We found that some sperm cells are able to fuse with the female gametes even 2 to 3 days after pollination, as reflected by delayed embryo and endosperm development, and by polytubey. We propose that the egg cell secretes EC1 proteins upon sperm arrival to promote rapid sperm activation, thereby accelerating gamete fusion and preventing polytubey.     

Seasonal development of five Cephaleuros species (Trentepohliaceae,Chlorophyta) on the leaves of woody plants and the behaviors of their gametes and zoospores

The infection cycle of five Cephaleuros species – C. aucubae, C. biolophus, C. japonicus, C. microcellularis, and C. virescens – was clarified by investigating seasonal development and gamete and zoospore behavior at study sites in Matsue, Shimane Prefecture, Japan. Fresh thalli or lesions appeared on the leaves of various woody plants from August and continued to develop in general from April to September in the following year. Gametangia and zoosporangia matured on 2‐ and 3‐year‐old leaves. Gametes were released in C. microcellularis samples collected from late March to late May and in samples of the other four species collected from late April to late July. Zoospores were released in C. biolophus, C. japonicus, and C. virescens samples collected from mid‐May to early August. In the present study, gametes did not conjugate but germinated like zoospores to produce new plants. After inoculation with zoospores and gametes that behave as asexual spores, fresh thalli or lesions became evident in 2–5 months following inoculation and gametes and zoospores were produced on the developed thalli or lesions the following year; one cycle of the infection was completed per year in the study area.