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.     

A study on the vitrification of stage III zebrafish (Danio rerio) ovarian follicles

Attempts to cryopreserve fish embryos have been conducted over the past three decades, nevertheless successful cryopreservation protocol for long-term storage still remains elusive. Fish oocytes offer some advantages when compared to embryos, which may help in improving the chances of cryopreservation. In the present study, a series of cryo-solutions were designed and tested for their vitrifying ability using different devices (0.25 ml plastic straw, vitrification block and fibreplug™). Toxicity of vitrification solutions was evaluated by assessing follicle membrane integrity with trypan blue staining. In addition, the effect of vitrification protocol on stage III zebrafish ovarian follicles was investigated by measuring the cytoplasmic ATP content and the mitochondrial distribution and activity using JC-1 probe and confocal microscopy. After vitrification, follicles showed membrane integrity of 59.9 ± 18.4% when fibreplug and V₁₆ (1.5 M methanol + 4.5 M propylene glycol) solution were employed. When vitrified in V₂ (1.5 M methanol + 5.5 M Me₂SO) the membrane integrity decreased to 42.0 ± 21.0%. It was observed that follicles located in the middle of the fragments were more protected from injuries and some of them showed good morphological appearance 2 h post-warming. Mitochondria integrity of granulosa cells layer was clearly damaged by the vitrification protocol and ATP level in the follicles declined significantly after warming. Vitrification of zebrafish follicles in ovarian tissue fragments and its effect at sub-cellular level is reported here for the first time. Information gained from this study will help in guiding development of optimal protocol for cryopreservation of fish oocytes.     

SEX‐SPECIFIC CELL SURFACE STRUCTURE OF ANISOGAMETES: MORPHOLOGICAL CHANGES DURING FERTILIZATION OF BRYOPSIS MAXIMA (ULVOPHYCEAE,CHLOROPHYTA) REVEALED BY ULTRA–HIGH‐RESOLUTION FIELD EMISSION SEM1

Cell surfaces of biflagellate gametes and their morphological changes during fertilization of Bryopsis maxima Okamura were observed using a high‐resolution field emission scanning electron microscope. Male gametes have broad and narrow faces, which are divided into at least five morphologically distinct regions: 1) the apical plate is a plate‐like structure that is approximately 380–530 nm long and approximately 190 nm wide, in the center of the papilla and slightly protruded from the plasma membrane; 2) strips are smooth materials on ridges that originate from the basal part of the papilla and extend downward; 3) the lateral belt is a belt‐shaped structure on the center of the narrower faces; 4) the flagellar surface; and 5) the other region of the cell body has a fine‐grained appearance. In contrast, the entire female gamete surface is rough because of many granular or amorphous cell coats on the plasma membrane. When both gametes were mixed together, the initial fusion proceeded between the broader face of the male gamete and the anterior side of the female one near the basal bodies. Morphology of the male gamete's cell surface changed gradually as fusion proceeded and was covered by the granular materials; that surface closely resembled those of female gametes except for the apical plate. It was present until the planozygote attached itself to the substrate by the papilla. It finally disappeared after settlement. Therefore, these results indicate that gametes of B. maxima have sex‐specific surface structures that change their morphology during fertilization and settlement.     

Eyespot behavior during the fertilization of gametes in Ulva arasakii Chihara (Ulvophyceae, Chlorophyta)

Behavior of the eyespots during the fertilization of Ulva arasakii Chihara was studied using field emission scanning electron microscopy (FE‐SEM). FE‐SEM enabled the visualization of the eyespot of biflagellate male and female gametes. The smaller male gamete has one protruded smaller (1.3 ± 0.15 μm× 1.0 ± 0.29 μm) eyespot and the larger female gamete has a larger (1.6 ± 0.2 μm× 1.1 ± 0.13 μm) one on a posterior position of the cell. The cell membrane over the eyespot region is relatively smooth compared to other parts of the cell body and exhibits hexagonal arranged lipid globules. Because the size of the cell and the morphology of the eyespot are different between male and female gametes, we could follow the fate of the eyespots during the fertilization. The initial cytoplasmic contact and fusion of the gametes takes place at their anterior end, slightly posterior to the flagellar base. The morphology of the fusing gametes followed two clearly distinguishable patterns. About half the gamete pairs lie side‐by‐side with their longitudinal axes nearly parallel, while the rest are oriented anti‐parallel to each other. In all cases, the larger female gamete fused along the same side as the eyespot, while the smaller male gamete fused along the side away from its eyespot. As fusion proceeds, the gamete pair is transformed into the quadriflagellate planozygote, in which the eyespots are positioned side‐by‐side on the region of cell fusion. These observations indicated that the opposite positioning of the eyespot relative to the cell fusion site in male and female gametes is important for the proper arrangement of the eyespots in the planozygote. The significance of this feature in advanced green algae is briefly discussed.     

The risk of polyspermy in three congeneric sea urchins and its implications for gametic incompatibility and reproductive isolation

Developmental failure caused by excess sperm (polyspermy) is thought to be an important mechanism driving the evolution of gamete-recognition proteins, reproductive isolation, and speciation in marine organisms. However, these theories assume that there is heritable variation in the susceptibility to polyspermy and that this variation is related to the overall affinity between sperm and eggs. These assumptions have not been critically examined. We investigated the relationship between ease of fertilization and susceptibility to polyspermy within and among three congeneric sea urchins. The results from laboratory studies indicate that, both within and among species, individuals and species that produce eggs capable of fertilization at relatively low sperm concentrations are more susceptible to polyspermy, whereas individuals and species producing eggs that require higher concentrations of sperm to be fertilized are more resistant to polyspermy. This relationship sets the stage for selection on gamete traits that depend on sperm availability and for sexual conflict that can influence the evolution of gamete-recognition proteins and eventually lead to reproductive isolation.     

The evolution of conspecific gamete precedence and its effect on reinforcement

Conspecific gamete precedence, the usage of conspecific sperm by a female that mates with both a conspecific and a heterospecific male, has been found in many taxa. We construct a population genetic model to examine the evolution of conspecific gamete precedence and its coevolution with premating isolation in the process of reinforcement. Our findings suggest that conspecific gamete precedence can evolve via a process very similar to reinforcement. We explore the nature of the selection against hybridization necessary to drive this evolution. Moreover, our results confirm the prediction of Marshall et al. (Trends Ecol. Evol. 2002;17:558-563) that conspecific gamete precedence will inhibit the evolution of reinforcement between two species. We further find that reinforcement will inhibit the evolution of conspecific gamete precedence. Both reinforcement and conspecific gamete precedence increase reproductive isolation and contribute to the process of speciation. We discuss factors that may affect which of these phenomena are likely to become predominant between incipient species.     

Role of sialic acid in bovine sperm-zona pellucida binding

Sperm binding activity has been detected in zona pellucida (ZP) glycoproteins and it is generally accepted that this activity resides in the carbohydrate moieties. In the present study we aim to identify some of the specific carbohydrate molecules involved in the bovine sperm-ZP interaction. We performed sperm binding competition assays, in vitro fecundation (IVF) in combination with different lectins, antibodies and neuraminidase digestion, and chemical and cytochemical analysis of the bovine ZP. Both MAA lectin recognising alpha-2,3-linked sialic acid and neuraminidase from Salmonella typhimurium with catalytic activity for alpha-2,3-linked sialic acid, demonstrated a high inhibitory effect on the sperm-ZP binding and oocyte penetration. These results suggest that bovine sperm-ZP binding is mediated by alpha-2,3-linked sialic acid. Experiments with trisaccharides (sialyllactose, 3'-sialyllactosamine and 6'-sialyllactosamine) and glycoproteins (fetuin and asialofetuin) corroborated this and suggest that at least the sequence Neu5Ac(alpha2-3)Gal(beta1-4)GlcNAc is involved in the sperm-ZP interaction. Moreover, these results indicate the presence of a sperm plasma membrane specific protein for the sialic acid. Chemical analysis revealed that bovine ZP glycoproteins contain mainly Neu5Ac (84.5%) and Neu5GC (15.5%). These two types of sialic acid residues are probably linked to Galbeta1,4GlcNAc and GalNAc by alpha-2,3- and alpha-2,6-linkages, respectively, as demonstrated by lectin cytochemical analysis. The use of a neuraminidase inhibitor resulted in an increased number of spermatozoa bound to the ZP and penetrating the oocyte. From this last result we hypothesize that a neuraminidase from cortical granules would probably participate in the block to polyspermy by removing sialic acid from the ZP.