Cryopreservation of sperm from the coral Acropora humilis

Corals are sensitive to minute changes in their environments, and their continued existence is substantially threatened by the increasing number of destructive anthropogenic activities and unprecedented rates of climate change. Although cryopreservation has been successfully to preserve mammalian gametes for decades, coral cryopreservation was attempted for the first time less than 15 years ago, and freezing protocols exist for only a handful of coral species. The present study developed a cryopreservation protocol for the sperm of the common Indo-Pacific reef-builder Acropora humilis. Colonies of reefs of Sattahip Bay, Chonburi Province, Thailand were collected from 3 m depth with a mesh net during a spawning event. Immediately after collection, the sperm were isolated and subjected to a two-step freezing method featuring DMSO, polyethylene glycol, or methanol as the cryoprotectant. Viability and motility were assessed via a bioluminescence technique and a “computer-assisted semen analysis, and it was found that a 15-min equilibration with 2 M DMSO followed by cooling at 41.7 °C was the optimum cryopreservation protocol for A. humilis sperm. The post-thaw sperm achieved 45% fertilization success, and 35% of the fertilized eggs developed into blastopore larvae. The present optimized protocol can therefore facilitate the preservation of sperm for future propagation efforts of this species and provide an experimental platform for optimizing cryopreservation protocols for gametes of other scleractinian coral species.     

Reliability of wavefront shaping based on coherent optical adaptive technique in deep tissue focusing

Wavefront shaping can compensate the wavefront distortions in deep tissue focusing, leading to an improved penetration depth. However, when using the backscattered signals as the feedback, unexpected compensation bias may be introduced, resulting in focusing position deviations or even no focus in the illumination focal plane. Here we investigated the reliability of wavefront shaping based on coherent optical adaptive technique in deep tissue focusing by measuring the position deviations between the foci in the illumination focal plane and the epi‐detection plane. The experimental results show that when the penetration depth reaches 150 μm in mouse brain tissue (with scattering coefficient ~22.42 mm^?1) using a 488 nm laser and an objective lens with 0.75 numerical aperture, the center of the real focus will deviate out of one radius range of the Airy disk while the optimized focus in the epi‐detection plane maintained basically at the center. With the penetration depth increases, the peak to background ratio of the focus in the illumination focal plane decreases faster than that in the epi‐detection plane. The results indicate that when the penetration depth reaches 150 μm, feedback based on backscattered signals will make wavefront shaping lose its reliability, which may provide a guidance for applications of non‐invasive precise optogenetics or deep tissue optical stimulation using wavefront shaping methods. A, Intensity distribution in the epi‐detection plane and the illumination focal plane before and after correction, corresponding to brain sections with 250 and 300 μm thickness, respectively. Scale bar is 2 μm. B, Averaged focusing deviations in the epi‐detection plane (optimized) and the illumination focal plane (monitored) after compensation. The unit of the ordinate is one Airy disk diameter. Black dashed line represents one Airy disk radius. Bars represent the SE of each measurement set.      

Rat Sertoli and spermatogenic cells express a similar gene, and its product is antigenically related to an outer dense fiber-associated protein.

We have previously reported that a heterodimeric protein secreted by rat Sertoli cells is antigenically related to a protein associated with outer dense fibers of the sperm tail. Therefore, we have explored the possibility that Sertoli and spermatogenic cells express a similar gene encoding a homologous protein. A Sertoli cell heterodimeric protein cDNA probe recognizes specific mRNA in pachytene and round spermatids fractionated by centrifugal elutriation; however, this specific mRNA was less prominent than in cultured Sertoli cells. In agreement with these observations, in situ hybridization experiments show that Sertoli cells are predominantly engaged in active heterodimeric protein mRNA synthesis, while meiotic prophase spermatocytes and spermatids also show significant but less abundant specific mRNA. Immunoblotting experiments demonstrate that, while Sertoli cells synthesize a heterodimeric protein consisting of two disulfide-linked components with molecular masses of 45 and 35 kD, both primary spermatocytes and round spermatids synthesize single 30 kD monomers not associated by disulfide linkage but recognized by antisera to Sertoli cell heterodimeric protein. Immunoblotting and immunogold electron microscopic studies show that antisera to Sertoli cell heterodimeric protein recognize a protein associated with outer dense fibers. This immunoreactivity was abolished by a 5-min pronase treatment, without affecting the integrity of outer dense fibers. Results of this study and previous studies demonstrate that both Sertoli and spermatogenic cells express a similar gene and that an antigenically related product encoded by this gene becomes associated with outer dense fibers during their assembly at spermiogenesis.     

Sperm cell surface characteristics of Plumbago zeylanica L. in relation to transport in the embryo sac

Myosin associated with the male germ cells of angiosperms interacts with actin, promoting transport of the non-motile generative and later sperm cells in the pollen tube. Myosin localizing on the sperm cell plasma membrane seems negligible in Plumbago, as reflected by the absence of: (i) anti-myosin labeling using immunoelectron microscopy, (ii) sperm motility on actin matrices, and (iii) electrophoretic movement changes after addition of antibody. Sperm cells injected directly into actively streaming Nitella internodal cells, however, follow actin bundles and their movement is sensitive to ATP and Mg²⁺. This may be based on simple charge binding since negatively charged latex beads also migrate on actin, whereas neutral or positively-charged latex beads do not. Sperm cells are negatively charged according to capillary microelectrophoresis, whereas killed sperm cells, which are positively charged do not migrate. The sperm cell that normally fertilizes the egg has a higher calculated charge (8.277 × 10³ esu/cm²) compared with the sperm cell that fuses with the central cell (6.120 × 10³ esu/cm²). Received: 15 December 1998 / Accepted: 21 January 1999     

三尖杉属受精作用的研究

三尖杉属的精原细胞有一类似银杏生毛体的星状体结构,它分裂产生的2个精子,在大小与形态上都基本相同,而且在精子细胞质中具有拟核仁颗粒存在。上述结构是三尖杉属的重要特点之一。本属植物的成熟卵细胞特别长,细胞质中有丰富的拟核仁结构,卵核下方具2—3团浓稠的细胞质团,这些结构很像穗花杉的卵细胞。三尖杉属的受精作用,属于有丝分裂后类型,这种类型只在松科和三尖杉科中发现。受精后,卵细胞发生强烈的极性分化,上部细胞质变成高度液泡化;相反,下部细胞质则聚集大量蛋白泡和拟核仁颗粒。     

Electrical recording and ultrastructure of stylet penetration by the greenhouse whitefly

Earlier studies have indicated that interior (physical and/or chemical) properties of a plant may be responsible for feeding-site selection by the greenhouse whitefly, Trialeurodes vaporariorum (Westw.). In order to study the process of feeding-site selection further, stylet-penetration activities and the pathway followed by the stylets in host-plant tissue were investigated using a DC electrical recording method and transmission electron microscopy (TEM). Penetrating whiteflies attached to a gold wire were included in an electrical circuit to record electrical penetration graphs (EPGs). Seven EPG patterns have been distinguished, five of which could be correlated with components of the stylet-penetration process: 1) one with penetration of the leaf surface, 2) one with intercellular penetration and salivary-sheath secretion, 3) one with sieve element penetration and ingestion, 4) one with short penetration of a cell, and 5) one with xylem penetration. The stylet pathway is almost completely intercellular before the phloem is reached and in contrast to aphids, brief symplast punctures are very rare. In general, it takes T. vaporariorum more than half an hour from the start of a penetration to reach a sieve element. Rejection of feeding sites occurs within a few minutes of penetration by adult whiteflies, a time span in which stylets are presumed to penetrate just beyond the epidermis. Properties of the apoplast close to the leaf surface seem therefore to play a major role in feeding-site selection.

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Résumé De précédentes études ont montré que les propriétés internes (physiques et/ou chimiques) d'une plante peuvent induire la sélection du site de nutrition de la mouche blanche de serres, Trialeurodes vaporariorum (Westw.). Afin d'étudier plus avant le processus de sélection du site de nutrition, les activités de piqûre et le chemin suivi par le stylet dans les tissus de la plante-hôte ont été étudiés par une méthode d'enregistrement électrique en courant continu ainsi que par microscopie à transmission d'électrons (TEM). Des aleurodes en activité de piqûre attachées à un fil d'or ont été incluses dans un circuit électrique pour enregistrer des graphes de pénétration électriques (EPG). Sept motifs d'EPG ont été distingués, dont cinq peuvent être corrélés aux composantes du processus de pénétration du stylet: 1) un avec pénétration de la surface foliaire, 2) un avec pénétration interecellulaire et sécrétion d'une gaine de salive, 3) un avec pénétration du phloème, 4) un avec courte pénétration d'une cellule, et 5) un avec pénétration du xylème. Le parcours du stylet est presque entièrement intercellulaire avant que le phloème soit atteint. Contrairement aux pucerons, les ponctions brèves dans le symplasme sont rares. Généralement, T. vaporariorum met plus d'une demi-heure, à partir du début d'une piqûre, pour atteindre un vaisseau. Le rejet des sites de nutrition par les aleurodes adultes se passe quelques minutes après la pénétration du stylet; pendant ce laps de temps, le stylet pénétrerait juste sous l'épiderme. Le rôle des propriétés de l'apoplaste près de la surface foliaire semble donc majeur dans la sélection des sites de nutrition.

     

Microscopic observations on the interaction of the mycoparasite Verticillium biguttatum with Rhizoctonia solani and other soil-borne fungi

The mycoparasitic interactions of Verticillium biguttatum with Rhizoctonia solani and with a variety of other soil-borne fungi were investigated in dual cultures. V. biguttatum interacted with various soil fungi by appressed growth along the host hyphae and infrequent penetrations. Intracellular growth and subsequent sporulation, however, only occurred with R. solani, a few binucleate Rhizoctonia and Ceratobasidium spp., and Sclerotinia sclerotiorum. Effective mycoparasitism on sclerotia was restricted to those belonging to R. solani.Electron-microscopic observations revealed that V. biguttatum can penetrate the host cell with infection tubes. This process is probably mediated by enzymatic hydrolysis of the cell wall. Subsequently, trophic hyphae develop within the host cytoplasm, ultimately resulting in death of the host cell.     

Membrane potentials as an indication for plant cell penetration by aphid stylets

Electrical penetration graphs (EPGs) of aphids on plants demonstrate distinct periods of lowered potential level: the potential drop (pd). Such pds are produced frequently during the stylet pathway to the phloem. Experimental evidence supports the hypothesis that the pd corresponds with a stylet puncture of a (epidermal or mesophyll) cell membrane. The intracellular potentials of-100 to-180 mV are thought to be measured thereby with the stylets acting as microelectrodes. The short pd of 5–15 s can be described as a sequence of three distinct phases. Besides the short pd, occurring during pattern B and C, long pds are produced during the complete pattern D+E. Pattern D+E may occur on the potential level of the pd (abbr.: D+E (pd)) and on the potential level of the preceding pattern C (abbr.: D+E(c)). Pattern D+E(pd) seems to be related to sieve element penetration, at least in a number of cases. Both pds, short and long, are produced on host and non-host plants, on susceptible and resistant cultivars, using several aphid species.

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Potentiels membraneux comme indication pour des pénétrations intracellulaires des plantes par les stylets des aphides

Résumé L'enrégistrement électrique de la pénétration du stylet des aphides dans les tissus de la plante se caracterise par des périodes distinctes de chute de potentiel (c.p.). Ces c.p. existent fréquemment au cours du trajet du stylet vers le phloème. Une évidence expérimentale soutient l'hypothèse de la correspondance entre la c.p. et la piqûre de la membrane des cellules épidermales et parenchymateuses. Les potentiels intracellulaires de-100 à-180 mV pourraient être mesurés par l'intermédiaire des stylets comme microélectrodes. Les. c.p. brèves, de 5 à 15 s peuvent être décrites comme des séquences composées de 3 phases distinctes. Outre les c.p. brèves pendant les ondes B et C, les pucerons produisent des c.p. durables qui se maintiennent au cours des ondes D+E. Ou bien l'onde D+E peuve apparaite au niveau de potentiel de c.p. (D+E(cp)), ou au niveau de potentiel de l'onde C précédente (D+E(c)). L'onde D+E(cp) semble être reliée à la pénétration des vaisseaux conducteurs, du moins dans certain cas. Les c.p. brêves et durables sont produits sur les plantes hôtes et non-hôtes chez plusiers espèces d'aphids.

     

The spatial association of the sperm cells and vegetative nucleus in the pollen grain ofBrassica

Summary In mature viable pollen ofBrassica oleracea, the pair of sperm cells and the nucleus of the vegetative cell are linked to form a structured unit we term the male germ unit. The sperm cells are held within a common periplasm and have no cell walls. Each sperm cell has a central globular body containing the nucleus surrounded by several evaginations which provide the means for linkage between them. One sperm cell, usually that closest to the nucleus of the vegetative cell contains most of mitochondria profiles (plastids are absent). This sperm cell appears to be linked by its protoplasmic evaginations to the envelope of the vegetative nucleus. The role of this unit in interactions with the female gametic complex is considered.     

Quantitative ultrastructural analysis of barley sperm

Summary Complete serial ultrathin sections of seven sperm pairs, computer-assisted measurements of cell, nuclear and organelle surface areas and volumes, and three-dimensional imagery were used to demonstrate that a process of cytoplasm and organelle elimination occurs during sperm maturation in barley. The number of mitochondria per sperm cell is reduced by 50%; sperm cell surface area and volume are reduced by 30% and 51% respectively. Mean volume and surface area per mitochondrion are significantly less in mature sperms. No examples of mitochondrial fusion or degeneration were observed within sperm cells. These data, along with observations of plasma membrane apposition and vesiculation within cytoplasmic extensions containing mitochondria, support the proposition that cytoplasm and organelle loss results primarily from the formation of cytoplasmic projections that are subsequently discarded from the sperm cell body. Comparisons of the quantitative data, including the number of mitochondria, indicate that differences between sperm cells of a pair are absent to very slight. Spatial organization within the pollen grain is such that the mature sperms, as well as the sperms and vegetative nucleus, are not in close proximity.