Structure Of Gametes Research Articles

THE DEPENDENCE OF SPERM PROGRESSIVE MOTILITY AND SEMINAL PLASMA BIOCHEMICAL COMPOSITION ON BACTERIAL LOAD IN DUROC BOAR SEMEN

Interactions between spermatozoa and bacterial species in ejaculates comes along with deleterious effects on the structure and function of male gametes. However, the effect of bacterial load on the seminal plasma remains unexplored. Therefore, this study was focused on the identification of bacterial species present in Duroc boar semen (n=18), quantification of the bacterial load (CFU) and alterations to the biochemical parameters of seminal plasma. The computer-assisted semen analysis (CASA) analysed the progressive motility (PRO; motility≥25µm/s) of spermatozoa, their concentration and kinematic parameters. The MALDI Biotyper was used for the bacterial characterization of fresh ejaculates. Seminal plasma was separated and biochemical analysis was carried out using the Randox RX Monza analyzer. Calcium (Ca), magnesium (Mg), total proteins (TP), aspartate transaminase (AST), alanine transaminase (ALT), cholesterol (CHOL), bilirubin (BR), albumins (ALB) and uric acid (UA) were subjected to the Pearson correlation with CFU and PRO. The bacterial identification showed the prevalence of Escherichia coli and Pseudomonas aeruginosa. The correlation analysis showed that the progressive motility of spermatozoa was strongly dependent (-0.799; P<0.01) on the seminal bacterial load. Moreover, a negative significant correlation (-0.810; P<0.001) was detected between CFU and Mg concentration. Also ALT (-0.647; P<0.01) and ALB (-0.484; P<0.05) were negatively affected by the bacterial load. Altogether, the presence of bacterial species may adversely affect the progressive motility as well as the biochemical composition of seminal plasma and thereby create unfavorable conditions for the sperm survival. Further analyses are necessary to reveal interactions between semen and bacteria.

Diversity of Balbiani body formation in internally and externally fertilizing representatives of Osteoglossiformes (Teleostei: Osteoglossomorpha).

During the early stages of oogenesis, the Balbiani body is formed in the primary oocytes. It consists of the Golgi apparatus, endoplasmic reticulum (ER), and numerous mitochondria aggregated with germ plasm, but its form may differ among animals. Hypothetically, during oogenesis oocytes become adapted to future development in two different environments depending on internal or external fertilization. We aimed to investigate, using light and transmission electron microscopy, the development of the Balbiani body during oogenesis in representatives of Osteoglossiformes, one of the most basal Teleostei groups. We analyzed the structure of oogonia and primary oocytes in the internally fertilizing butterflyfish Pantodon buchholzi and the externally fertilizing Osteoglossum bicirrhosum and Arapaima gigas to compare formation of the Balbiani body in relation to modes of fertilization. We demonstrated that the presence of the germ plasm as well as the fusion and fission of mitochondria are the conserved features of the Bb. However, each species exhibited also some peculiar features, including the presence of three types of ooplasm with different electron density and mitochondria-associated membranes in P. buchholzi; annulate lamellae, complexes of the Golgi apparatus, ER network, and lysosome-like bodies in O. bicirrhosum; as well as karmellae and whorls formed by the lamellae of the ER in A. gigas. Moreover, the form of the germ plasm observed in close contact with mitochondria differed between osteoglossiforms, with a "net-like" structure in P. buchholzi, the presence of numerous strings in O. bicirrhosum, and irregular accumulations in A. gigas. These unique features indicate that the extreme diversity of gamete structure observed so far only in the spermatozoa of osteoglossiforms is also characteristic for oocyte development in these basal teleosts. Possible reason of this variability is a period of about 150 million years of independent evolution of the lineages.

Oolemma Receptors in Mammalian Molecular Fertilization: Function and New Methods of Study.

Fertilization is a key process in biology to the extent that a new individual will be born from the fusion of two cells, one of which leaves the organism in which it was produced to exert its function within a different organism. The structure and function of gametes, and main aspects of fertilization are well known. However, we have limited knowledge about the specific molecules participating in each of the steps of the fertilization process due to the transient nature of gamete interaction. Moreover, if we specifically focus in the fusion of both gametes’ membrane, we might say our molecular knowledge is practically null, despite that molecular mechanisms of cell-to-cell adhesion are well studied in somatic cells. Moreover, between both gametes, the molecular knowledge in the egg is even scarcer than in the spermatozoon for different reasons addressed in this review. Sperm-specific protein IZUMO1 and its oocyte partner, JUNO, are the first cell surface receptor pair essential for sperm–egg plasma membrane binding. Recently, thanks to gene editing tools and the development and validation of in vitro models, new oocyte molecules are being suggested in gamete fusion such as phosphatidylserine recognition receptors. Undoubtedly, we are in a new era for widening our comprehension on molecular fertilization. In this work, we comprehensively address the proposed molecules involved in gamete binding and fusion, from the oocyte perspective, and the new methods that are providing a better understanding of these crucial molecules.

Histological observation of the reproductive system in a viviparous teleost Xenotoca eiseni Rutter 1896 (Cyprinodontiformes: Goodeidae).

Xenotoca eiseni is a viviparous teleost belonging to the family Goodeidae. Here, we report histological observations of the reproductive organs in an adult male, an adult female, a pregnant female with intraovarian embryo and an extracted embryo of X.eiseni. High-resolution images of haematoxylin-eosin-stained sagittal sections revealed the detailed structure of gonads, gametes and reproductive components of the mother-embryo relationship. In the male, mature spermatozoa in the epididymis formed sperm packages. In the female, oogenesis proceeded asynchronously in the ovarian wall, and various stages of oocytes were observed in single ovary. In both sexes, genital openings were located between the anus and anal fin. Developing embryos were observed in an ovary of the pregnant female. Fine structures of components of the mother-to-embryo nutrient supply, ovarian septum and trophotaenia were observed in the pregnant ovary. An immature gonad prior to gamete formation was identified in the extracted embryo. With the aim of supporting the development and extension of studies on this viviparous teleost, we have shared our histological images as raw data in an open online archive, the 'NAGOYA repository (http://hdl.handle.net/2237/00032456)'. Our goal is a comprehensive understanding of the viviparous system in fish using both histological observation and molecular biology methods including genomics and proteomics.

The Potential Role of Seminal Plasma in the Fertilization Outcomes.

For human infertility both male and female factors may be equally important. Searching for molecular biomarkers of male infertility, neglected for decades, and the attempts to explain regulatory mechanisms of fertilization become thus extremely important. Apart from examination of the structure and function of male gametes, also the possible importance of seminal plasma components should be considered. In this article we discuss data that indicate for the substantial significance of active seminal plasma components for conception and achievement of healthy pregnancy. Seminal plasma impact on the storage and cryopreservation of human and animal sperm and regulatory role of glycodelin on human sperm capacitation as well as hypothesized course of female immune response to allogenic sperm and conceptus has been discussed. The possible involvement of carbohydrates in molecular mechanism of fetoembryonic defense has been also mentioned.

Single-cell three-dimensional genome structures of rice gametes and unicellular zygotes.

Chromatin conformation capture (3C)1 and high-throughput 3C (Hi-C)2 assays allow the study of three-dimensional (3D) genome structures in cell populations or tissues, based on average proximities of folded DNA. However, differences between cells can be observed only by single-cell measurements that avoid ensemble averaging3-5. To study 3D chromatin organization and dynamics before and after fertilization in flowering plants, we analysed the 3D genomes of rice eggs, sperm cells, unicellular zygotes and shoot mesophyll cells. We show that chromatin architectures of rice eggs and sperm cells are comparable to those of mesophyll cells and are reorganized after fertilization. The rice single-cell 3D genomes display specific features of chromosome compartments and telomere/centromere configuration compared to those in mammalian single cells. Active and silent chromatin domains combine to form multiple foci in the nuclear space. Notably, the 3D genomes of the eggs and unicellular zygotes contain a compact silent centre (CSC) that is absent in sperm cells. CSC appears to be reorganized after fertilization, and may be involved in the regulation of zygotic genome activation (ZGA). Our results reveal specific 3D genome features of plant gametes and the unicellular zygote, and provide a spatial chromatin basis for ZGA and epigenetic regulation in plants.

3D Chromatin Structures of Mature Gametes and Structural Reprogramming during Mammalian Embryogenesis

High-order chromatin structure plays important roles in gene expression regulation. Knowledge of the dynamics of 3D chromatin structures during mammalian embryo development remains limited. We report the 3D chromatin architecture of mouse gametes and early embryos using an optimized Hi-C method with low-cell samples. We find that mature oocytes at the metaphase II stage do not have topologically associated domains (TADs). In sperm, extra-long-range interactions (>4 Mb) and interchromosomal interactions occur frequently. The high-order structures of both the paternal and maternal genomes in zygotes and two-cell embryos are obscure but are gradually re-established through development. The establishment of the TAD structure requires DNA replication but not zygotic genome activation. Furthermore, unmethylated CpGs are enriched in A compartment, and methylation levels are decreased to a greater extent in A compartment than in B compartment in embryos. In summary, the global reprogramming of chromatin architecture occurs during early mammalian development.

Probing suitable therapeutic nanoparticles for controlled drug delivery and diagnostic reproductive health biomarker development

Nanomaterial mediated drug delivery represents a highly promising technique while its selectivity for reproductive healthcare application still remains a challenge. Since the delicate structure and functional role of reproductive tissue and gametes require the use of biocompatible nanomedicine/devices that do not affect fertility or the development of resulting offspring, this paper reports an intercomparative study of human spermatozoa interaction with three different nanoparticles (NPs) namely; iron oxide (Fe3O4), multiwalled carbon nanotubes (MWCNT) and graphene platelet nanopowder (GPN) to probe their suitability for drug delivery carrier and biomarker development purposes. ATR–FTIR results revealed that the sperm cell interaction with GPN had maximum amide I absorption for cell proteins and CO stretching of the peptide backbone at the band around 1657cm−1 followed by iron oxide NPs whereas MWCNT had no absorption. These results showed that GPN followed by iron oxide NPs got maximally entrapped by cell membrane protein with maximum disruption but MWCNT exhibited less entrapment but significantly higher internalization which was further validated by morphological analysis of these cell NP interaction by SEM, HRTEM and fluorescence microscopy. The uptake kinetics and penetration mechanism of NPs were examined with isothermal titration calorimetry (ITC). Interestingly, ITC results confirmed ATR–FTIR and morphological observations that the binding of GPN and Fe3O4 NPs with cell was exothermic and their bindings were favored by both negative enthalpy and positive entropy whereas in the case of MWCNT it was endothermic supported by unfavorable positive enthalpy and a favorable entropy change. Hence, it was evident that MWCNT had better internalization efficiency without disrupting the sperm lipid membrane compared to Fe3O4 and GPN NPs. Therefore, this work proposes CNT as promising means.

Comparative analysis of gamete ultrastructure in big-scaled redfin Tribolodon hakonensis (Cyprinidae) from Southern Primorye and Sakhalin

Considerable differences in the structure of gametes of big-scaled redfin Tribolodon hakonensis from Southern Primorye (southern form) and rivers of the Sea of Okhotsk basin (northern form) are found. In spermatozoa of the southern form of T. hakonensis, the mitochondrial apparatus includes up to five mitochondria, and flagellum length is, on average, 36.2 μm. In spermatozoa of the northern form, up to three mitochondria are observed, and flagellum length is, on average, 42.3 μm. In oocytes of the southern form, the zona radiata has well-expressed striation, and radial canals are dense and large. Oocytes of the northern form are characterized by a poor expressed striation, and radial canals are rarely located and small. Observed differences confirm the specificity of gametes in the southern and northern forms of T. hakonensis, and they can be used as additional phylogenetic traits.

Extracellular vesicle-mediated delivery of molecular compounds into gametes and embryos: learning from nature.

Currently, even the most sophisticated methods of assisted reproductive technology (ART) allow us to achieve live births in only approximately 30% of patients, indicating that our understanding of the fine mechanisms underlying reproduction is far from ideal. One of the main challenges associated with studies of gamete structure and function is that these cells are remarkably resistant towards the uptake of exogenous substances, including 'molecular research tools' such as drugs, biomolecules and intracellular markers. This phenomenon can affect not only the performance of reproductive biology research techniques, but also the outcomes of the in vitro handling of gametes, which forms the cornerstone of ART. Improvement of intra-gamete delivery in a non-aggressive fashion is vital for the investigation of gamete physiology, and the advancement of infertility treatment. In this review, we outline the current state of nanomaterial-mediated delivery into gametes and embryos in vitro, and discuss the potential of a novel exciting drug delivery technology, based upon the use of targeted 'natural' nanoparticles known as extracellular vesicles (EVs), for reproductive science and ART, given the promising emerging data from other fields. A comprehensive electronic search of PubMed and Web of Science databases was performed using the following keywords: 'nanoparticles', 'nanomaterials', 'cell-penetrating peptides', 'sperm', 'oocyte', 'egg', 'embryo', 'exosomes', 'microvesicles', 'extracellular vesicles', 'delivery', 'reproduction', to identify the relevant research and review articles, published in English up to January 2015. The reference lists of identified publication were then scanned to extract additional relevant publications. Biocompatible engineered nanomaterials with high loading capacity, stability and selective affinity represent a potential versatile tool for the minimally invasive internalization of molecular cargo into gametes and embryos. However, it is becoming increasingly clear that the translation of these experimental tools into clinical applications is likely to be limited by their non-biodegradable nature. To allow the subsequent use of these methodologies for clinical ART, studies should utilize biodegradable delivery platforms, which mimic natural mechanisms of molecular cargo trafficking as closely as possible. Currently, EVs represent the most physiological intracellular delivery tools for reproductive science and medicine. These natural mediators of cell communication combine the benefits of engineered nanomaterials, such as the potential for in vitro production, targeting and loading, with the essential feature of biodegradability. We anticipate that future investigations into the possibility of applying EVs for the intentional intracellular delivery of molecular compounds into gametes and embryos will open new horizons for reproductive science and clinical ART, ultimately leading to improvements in patient care.

DIFFERENCES IN THE GENETIC STRUCTURE OF CITRUS TRIPLOID HYBRIDS RECOVERED FROM 2X × 2X AND 4X × 2X SEXUAL HYBRIDIZATIONS

Citrus triploid hybrids can be recovered by 2x X 2x hybridisations as a consequence of the formation of 2n gametes or by interploid hybridisation. Most of the 4x parents used in our program are doubled-diploid (DD), also known as autotetraploid. The genetic structure of diploid gametes and particularly the rate of parental heterozygosity restitution (PHR) depend on the meiotic process by which they were originated. Second-division restitution (SDR) is the 2n gamete formation mechanism involved in 'Fortune' and clementines. With SDR, PHR is positively linked with the distance of each locus to the centromere. With DD parents, PHR depends on the rate of preferential pairing and thus the proportion of disomic versus tetrasomic segregations. We have compared the genetic structure of two populations of diploid gametes of clementine (one population of 2n gametes and one of 2x gametes produced by DD) with SNP and SSR markers covering the 9 citrus chromosomes. The DD displays mostly tetrasomic segregation; however three linkage groups present intermediate segregation and one displays a tendency for disomy. The PHR in the 2n gametes is 2/3 of that obtained in 2x gametes produced by DD. The two methods of triploid production appear complementary in terms of genotypic variability. 4x X 2x hybridizations are more efficient than 2x X 2x for developing new cultivars phenotypically closer to the diploid parent of the DD. Conversely, 2x X 2x hybridisations provides a greater opportunity to select innovative products. (Resume)

Iron and copper in male reproduction: a double-edged sword.

Iron and copper are essential trace nutrients playing important roles in general health and fertility. However, both elements are highly toxic when accumulating in large quantities. Their direct or indirect impact on the structure and function of male gonads and gametes is not completely understood yet. Excess or deficiency of either element may lead to defective spermatogenesis, reduced libido, and oxidative damage to the testicular tissue and spermatozoa, ultimately leading to fertility impairment. This review will detail the complex information currently available on the dual roles iron and copper play in male reproduction.

Genome evolution by matrix algorithms: cellular automata approach to population genetics.

Mammalian genomes are replete with millions of polymorphic sites, among which those genetic variants that are colocated on the same chromosome and exist close to one another form blocks of closely linked mutations known as haplotypes. The linkage within haplotypes is constantly disrupted due to meiotic recombination events. Whole ensembles of such numerous haplotypes are subjected to evolutionary pressure, where mutations influence each other and should be considered as a whole entity—a gigantic matrix, unique for each individual genome. This idea was implemented into a computational approach, named Genome Evolution by Matrix Algorithms (GEMA) to model genomic changes taking into account all mutations in a population. GEMA has been tested for modeling of entire human chromosomes. The program can precisely mimic real biological processes that have influence on genome evolution such as: 1) Authentic arrangements of genes and functional genomic elements, 2) frequencies of various types of mutations in different nucleotide contexts, and 3) nonrandom distribution of meiotic recombination events along chromosomes. Computer modeling with GEMA has demonstrated that the number of meiotic recombination events per gamete is among the most crucial factors influencing population fitness. In humans, these recombinations create a gamete genome consisting on an average of 48 pieces of corresponding parental chromosomes. Such highly mosaic gamete structure allows preserving fitness of population under the intense influx of novel mutations (40 per individual) even when the number of mutations with deleterious effects is up to ten times more abundant than those with beneficial effects.

Genetically based location from triploid populations and gene ontology of a 3.3-mb genome region linked to Alternaria brown spot resistance in citrus reveal clusters of resistance genes.

Genetic analysis of phenotypical traits and marker-trait association in polyploid species is generally considered as a challenge. In the present work, different approaches were combined taking advantage of the particular genetic structures of 2n gametes resulting from second division restitution (SDR) to map a genome region linked to Alternaria brown spot (ABS) resistance in triploid citrus progeny. ABS in citrus is a serious disease caused by the tangerine pathotype of the fungus Alternaria alternata. This pathogen produces ACT-toxin, which induces necrotic lesions on fruit and young leaves, defoliation and fruit drop in susceptible genotypes. It is a strong concern for triploid breeding programs aiming to produce seedless mandarin cultivars. The monolocus dominant inheritance of susceptibility, proposed on the basis of diploid population studies, was corroborated in triploid progeny. Bulk segregant analysis coupled with genome scan using a large set of genetically mapped SNP markers and targeted genetic mapping by half tetrad analysis, using SSR and SNP markers, allowed locating a 3.3 Mb genomic region linked to ABS resistance near the centromere of chromosome III. Clusters of resistance genes were identified by gene ontology analysis of this genomic region. Some of these genes are good candidates to control the dominant susceptibility to the ACT-toxin. SSR and SNP markers were developed for efficient early marker-assisted selection of ABS resistant hybrids.

Note on the Applicability of the F-model in Analysis of Pollen Pool Heterogeneity

Measuring genetic structure of pollen gametes has become the standard approach in studies on plant mating system and pollen flow. However, the popular statistical procedure, TwoGener, provides the point estimate of the pollen pool heterogeneity based on AMOVA, implying any statistical tests regarding pollen pool structure to be based on permutation procedures. In this article, the Bayesian approach was developed based on the Dirichlet-multinomial model (the so-called F-model). Computer simulations revealed the applicability of the Bayesian method in the analysis of pollen pool heterogeneity. Compared with TwoGener, the Bayesian method appeared to have better statistical properties when estimating the inverse of heterogeneity index, mostly due to the relatively high accuracy of the Bayesian estimate when the actual heterogeneity is low. Hence, the Bayesian approach may be preferred, for example, when the effective number of pollen parents is to be assessed. Other applications and potential further developments of the Bayesian method are also briefly discussed.

Occupational, Environmental and Lifestyle Factors Associated With Spontaneous Abortion

Scientific evidence indicates extreme exposure sensitivity of embryos, fetuses, and infants to the persistent environmental/occupational chemicals directly and or indirectly as compared to the same magnitude of exposure in adults. Paternal/maternal exposure to some of these chemicals might have a effect on the gamete structure and function, which might have significant implication for the adverse effect on pregnancy and their outcome. The available data point that some of the organochlorine chemicals such as dichlorodiphenyl trichloroethane (DDT); metals such as lead, mercury; industrial pollutants such as dioxin, organic solvents, radiations; and some of the lifestyle-associated factors such as tobacco smoking (active and passive) and excessive maternal intake of alcohol had adverse effect on pregnancy outcome. The existing data support the hypothesis that, in general, working women have a higher risk of undesirable reproductive outcomes, even though the data are scanty. Studies are needed to find out the effects of those reproductive toxicants on priority basis which have been proved to be toxic in animal studies as well as data on human related to these chemicals are scanty. There is a need to educate the childbearing women to avoid exposure to the known or suspected risk factors and their employers to take measures to reduce the toxicant levels in workplace.

Multilocus half-tetrad analysis and centromere mapping in citrus: evidence of SDR mechanism for 2n megagametophyte production and partial chiasma interference in mandarin cv ‘Fortune’

The genetic structure of 2n gametes and, particularly, the parental heterozygosity restitution at each locus depends on the meiotic process by which they originated, with first-division restitution and second-division restitution (SDR) being the two major mechanisms. The origin of 2n gametes in citrus is still controversial, although sexual polyploidisation is widely used for triploid seedless cultivar development. In this study, we report the analysis of 2n gametes of mandarin cv 'Fortune' by genotyping 171 triploid hybrids with 35 simple sequence repeat markers. The microsatellite DNA allele counting-peak ratios method for allele-dosage evaluation proved highly efficient in segregating triploid progenies and allowed half-tetrad analysis (HTA) by inferring the 2n gamete allelic configuration. All 2n gametes arose from the female genitor. The observed maternal heterozygosity restitution varied between 10 and 82%, depending on the locus, thus SDR appears to be the mechanism underlying 2n gamete production in mandarin cv 'Fortune'. A new method to locate the centromere, based on the best fit between observed heterozygosity restitution within a linkage group and theoretical functions under either partial or no chiasmata interference hypotheses was successfully applied to linkage group II. The maximum value of heterozygosity restitution and the pattern of restitution along this linkage group would suggest there is partial chiasma interference. The implications of such a restitution mechanism for citrus breeding are discussed.

Evidence for non-disomic inheritance in a Citrus interspecific tetraploid somatic hybrid between C. reticulata and C. limon using SSR markers and cytogenetic analysis

Artificial tetraploid somatic hybrids have been developed for sterile triploid citrus breeding by sexual hybridization between diploid and tetraploid somatic hybrids. The genetic structure of diploid gametes produced by tetraploid genotypes depends on the mode of chromosome association at meiosis. In order to evaluate tetraploid inheritance in a tetraploid interspecific somatic hybrid between mandarin and lemon, we performed segregation studies using cytogenetic and single sequence repeat molecular markers. Cytogenetic analysis of meiosis in the somatic hybrid revealed 11% tetravalents and 76% bivalents. Inheritance of the tetraploid hybrid was analyzed by genotyping the triploid progeny derived from a cross between a diploid pummelo and the tetraploid somatic hybrid, in order to derive genotypes of the meiospores produced by the tetraploid. A likelihood-based approach was used to distinguish between disomic, tetrasomic, and intermediate inheritance models and to estimate the double reduction rate. In agreement with expectations based the cytogenetic data, marker segregation was largely compatible with tetrasomic and inheritance intermediate between disomic and tetrasomic, with some evidence for preferential pairing of homoeologous chromosomes. This has important implications for the design of breeding programs that involve tetraploid hybrids, and underscores the need to consider inheritance models that are intermediate between disomic and tetrasomic.

Sperm–egg adhesion and fusion in mammals

Fertilisation is an orchestrated, stepwise process during which the participating male and female gametes undergo irreversible changes, losing some of their structural components while contributing others to the resultant zygote. Following sperm penetration through the egg coat, the sperm plasma membrane fuses with its oocyte counterpart, the oolemma. At least two plasma membrane proteins essential for sperm-oolemma fusion--IZUMO and CD9 on the male and female gametes, respectively--have been identified recently by classical cell biology approaches and confirmed by gene deletion. Oolemma-associated tetraspanin CD81, closely related to CD9, also appears to have an essential role in fusion. Additional proteins that may have nonessential yet still facilitating roles in sperm-oolemma adhesion and fusion include oolemma-anchored integrins and oocyte-expressed retroviral envelope proteins, sperm disintegrins, and sperm-borne proteins of epididymal origin such as CRISP1 and CRISP2. This review discusses these components of the gamete fusion mechanism within the framework of gamete structure, membrane biology, cell signalling and cytoskeletal dynamics, and revisits the topic of antipolyspermy defence at the oolemma level. Harnessing the mechanisms of sperm-egg fusion is of importance to animal biotechnology and to human assisted fertilisation, wherein male patients with reduced sperm fusibility have been identified.

ASYMMETRY OF EYESPOT AND MATING STRUCTURE POSITIONS IN ULVA COMPRESSA (ULVALES, CHLOROPHYTA) REVEALED BY A NEW FIELD EMISSION SCANNING ELECTRON MICROSCOPY METHOD(1).

Gametes of the marine green alga Ulva compressa L. are biflagellate and pear shaped, with one eyespot at the posterior end of the cell. The species is at an early evolutionary stage between isogamy and anisogamy. In the past, zygote formation of green algae was categorized solely by the relative sizes of gametes produced by two mating types (+ and -). Recently, however, locations of cell fusion sites and/or mating structures of gametes have been observed to differ between mating types in several green algae (asymmetry of cell fusion site and/or mating structure positions). To use this asymmetry for determining gamete mating type, we explored a new method, field emission scanning electron microscopy (FE-SEM), for visualizing the mating structure of U.compressa. When gametes were subjected to drying stress in the process of a conventional critical-point-drying method, a round structure was observed on the cell surfaces. In the mating type MGEC-1 (mt(+) ), this structure was located on the same side of the cell as the eyespot, whereas it was on the side opposite the eyespot in the mating type MGEC-2 (mt(-) ). The gametes fuse at the round structures. TEM showed an alignment of vesicles inside the cytoplasm directly below the round structures, which are indeed the mating structures. Serial sectioning and three-dimensional construction of TEM micrographs confirmed the association of the mating structure with flagellar roots. The mating structure was associated with 1d root in the MGEC-1 gamete but with 2d root in the MGEC-2 gamete.