The cytology of the gametes and fertilization of Allomyces macrogynus

The gametes and the process of fertilization were examined by light and electron microscopy in the lower eukaryote Allomyces macrogynus. Differences in gamete morphology included the overall larger size and the presence of a larger nuclear apparatus, along with the association of a side-body complex and many more mitochondria in the female gamete. In this species of Allomyces, fertilization was initiated by contact and fusion of specialized regions of the gamete plasma membranes resulting in a binucleate fusion cell surrounded by plasma membrane contributed by both partners. Following plasmogamy, nuclear fusion was initiated by multiple nuclear membrane contacts between adjacent outer membranes. Following inner membrane fusion, small nucleoplasmic bridges were observed which presumably fused with one another and resulted in a single bridge which widened, forming the mature diploid nucleus. After karyogamy, fusion of the nuclear caps did not always occur and zygotes with and without fused caps were observed. Coalescence of the nucleoli completed the events of fertilization, forming a zygote with a single nuclear apparatus (sometimes with two caps) and two flagella. These observations are discussed in relation to fertilization mechanisms and compared to fertilization in other organisms.     

Attraction and transport of male gametes for fertilization

 Two capabilities are critical in attracting and transporting male gametes for fertilization: (1) the pollen tube must locate, enter and discharge its contents at the correct site within the female gametophyte, and (2) once inside the embryo sac, the non-motile male gametes must be transported to the egg and the central cells for double fertilization. This review summarizes current information about evidence for communication between embryo sac and pollen tube and the means by which the non-motile male gametes are transported from the aperture of the pollen tube to the site of gamete fusion. Received: 6 June 1996 / Revision accepted: 9 July 1996     

In vitro fertilization and expression of transgenes in gametes and zygotes

The in vitro fertilization system of maize is the well characterized model system for the fertilization process and early zygotic embryogenesis of higher plants. Application of molecular methods to the in vitro fertilization system led to the isolation of new genes and uncovered specific expression patterns of cell cycle regulators. Recent studies showed that expression of transgenes is possible in gametes and zygotes, thus transgenic approaches might offer an opportunity to unravel the roles of genes during fertilization and early development. The competence of gametes and zygotes to express transgenes will also enable the expression of GFP based reporter genes for the visualization of subcellular components in these cells in vivo. This review focuses on the data concerning the expression of transgenes in gametes and zygotes and describes some examples of recent developments in transgenic technology illustrating the emerging possibilities in experimental design by combining this technology with in vitro fertilization. Received: 20 December 2001 / Revision accepted: 6 June 2001     

Inhibition of in-vitro fertilization of mouse gametes by proteinase inhibitors

Kinetic studies were performed to evaluate the interaction of benzamidine (BD), 4-aminobenzamidine (ABD). 4'-nitrophenyl 4-guanidinobenzoate (NPGB), and 4'-methylumbelliferyl 4-guanidinobenzoate (MUGB) with mouse acrosin. The Michaelis constant of mouse acrosin towards alpha-N-benzoyl-L-arginine ethyl ester and the sensitivity of mouse acrosin to inhibitors differed from those reported for other species. NPGB and MUGB were much more active inhibitors of acrosin than BD and ABD. Plots of percentage fertilization versus acrosin inhibitor concentration were generated for all 4 compounds. Linear dose-response curves were obtained and gave ED50 values (50% inhibition of fertilization) of 230 microM for BD, 27 microM for ABD, 35 nM for MUGB, and 13 nM for NPGB. The relative antifertility activity of the compounds paralleled their inhibitory activity towards mouse acrosin, strongly indicating that the inhibition of fertilization is obtained through the inhibition of acrosin. Since the dose-response curves were linear, the mouse in-vitro fertilization system may be useful to screen acrosin inhibitors for their antifertility potency. MUGB should have low toxicity and may have potential as a contraceptive agent.     

Effects of short-term storage of gametes on fertilization of Pacific herring eggs

A method is described whereby arrays of samples ofClupea pallasi eggs may be stored during their preparation. The high fertilization potential retained by the eggs during short-term storage allows them to be fertilized synchronously when sample preparation is complete. A variation of the dry method of storage retained maximum fertilization potential (80–85%) of the eggs for about 1 hr, and of milt dilution (18 with 17 S sea water), about 7 hr. Following dry storage, eggs fertilized in salinities of 0–45 showed maximum rates of fertilization in salinities of 10–20, and fertilization rates 50% in salinities of 4.5–42. Salinities of fertilization influenced egg diameter, median hatching time, and larval length at hatching in egg samples transferred 21/2 hr after fertilization to an incubation salinity of 17 at 7°C. Fertilization rates were higher (90–95%) for eggs stored in 17 S at 7°C prior to fertilization. Under such wet storage conditions, maximum fertilization pontential was retained for about 2 hr. Highest fertilization rates (95–96%) were obtained for eggs stored and fertilized in salinities of 12–15. For the species and the area of origin considered (British Columbia), wet storage of eggs should result in maximum fertilization when the eggs are stored at 4°C for a period not greater than 2 hr prior to fertilization in the 12–15 S storage medium.Prepared under the auspices of the Canadian-German Scientific and Technical Cooperation Agreement.     

The history of glycobiology in Japan

This mini review surveys the major accomplishments in the field of glycoconjugates research in Japan, which were made after World War II. It describes early movements in the field of glycoconjugate research in Japan, development of the new techniques to investigate structures of the sugar chains of glycoconjugates, studies of the functions of the sugar chain moieties, and the political movement in Japan to support the basic research necessary for the development of glycotechnology. As introduced in this short article, important groundwork for glycobiology was made by Japanese researchers.     

In vitro fertilization of single,isolated gametes of maize mediated by electrofusion

Summary Electrofusion-mediated in vitro fertilization of maize using single sperm and egg cells was performed. Sperm cells were released from pollen grains after rupture of the latter by osmotic shock in the fusion medium (0.55 M mannitol). Egg cells were isolated by enzyme treatment (pectinase, pectolyase, hemicellulase, and cellulase) followed by mechanical isolation. The conditions generally used for the electrical fusion of protoplasts of somatic cells were also applied to the protoplasts of gametic cells of maize. Electrofusion was performed with single pairs of gametes under microscopic observation. The mean fusion frequency was 79%. Isolated egg cells of maize showed protoplasmic streaming during 22 days of culture, but they did not divide. However, after fusion of the sperm with the egg cells, these fused cells did develop, with a mean division frequency of 83%, and grew to multicellular structures. Egg cells and fusion products were cultivated with a maize feeder-cell system.     

Relationship between double fertilization and the cell cycle in male and female gametes of tobacco

Nuclear DNA content of male and female gametes of tobacco was determined using 4,6-diamindino-2-phenylindole and quantitative microfluorimetry. Pollen grains are released with generative cells containing 2C DNA. Mitotic division occurs in the pollen tube 8–12 h after germination. The resulting sperm cells have 1C DNA content during pollen tube elongation in the style. Sperm cells deposited in the degenerated synergid have a DNA content between 1C and 2C, indicating that sperm are in S-phase in the synergid. Concomitant with pollen tube arrival, the egg cell increases in DNA quantity from 1C to between 1C and 2C at 48 h after pollination. In the absence of pollination, S-phase in the egg cell is delayed by up to 36 h. Newly formed zygotes contain nuclear DNA concentrations of 4C at karyogamy and remain at 4C until zygote division. Tobacco displays cell fusion after the completion of S-phase, apparently during G₂. Failure to achieve an optimized system for in vitro fertilization in Nicotiana may reflect the challenges of achieving cell cycle synchrony in gametes isolated from pollen tubes. Receptive gametes are presumably those that pass through the protracted S-phase, reaching G₂ receptivity and cell cycle congruity before fusion.     

Artificial fertilization of gametes from the South African clawed frog,Xenopus laevis

A reproducible and effective method for fertilization eggs of Xenopus laevis was developed based of systematic manipulation of environmental factors. The effects of varying concentrations of individual components of a fertilization medium were tested by measuring jelly swelling, sperm motility, and sperm longevity. Results were used to develop an improved medium for fertilization, consisting of 41.25 mM NaCl, 1.25 mM KCl, 0.25 mM CaCl₂, 0.0625 mM MgCl₂, 0.5 mM Na₂HPO₄, 2.5 mM HEPES, 1.9 mM NaOH, final pH(2°) 7.8.     

Bioinformatics in glycobiology

In comparison with genes and proteins, attention paid to oligosaccharides that modify proteins is still marginal. Accordingly, bioinformatics is so far poorly involved in glycobiology. Some initiatives have been taken, however, to collect in databases all glycobiology-relevant information or to design specific data mining algorithms to infer predictions or identify oligosaccharide structures. In this review, we make a non-exhaustive survey of the available glycobiology-related bioinformatic resources, focussing mainly on those resources that are available through the World Wide Web. Some well-curated databases are identified, but the development of specialised algorithms appears to be limited.