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.     

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。     

Spawning tactics of paired males of the dark chub,Zacco temmincki,reflect potential fitness costs of satellites

Synopsis Paired males of the dark chub, Zacco temmincki, buried released eggs by vibrating their anal fin, but this behavior was prevented and eggs were cannibalized when many satellites (males and females) were present. A number of satellite males also caused a loss to paired males in sperm competition on spawning grounds far from shelters. Paired males followed repeating tactics, which were defined as successive spawning acts at the same redd, in most cases, but occasionally did shifting tactics which refer to spawning acts conducted successively at different redds. The proportion of the shifting tactic was not correlated with the dominance status of paired males. The shifting tactic was not advantageous in performing spawnings frequently. The calculation of the total advantage of both tactics indicated that the shifting tactic itself was not more beneficial than the repeating tactic at any density of satellites. Since a number of satellites stayed around the redd when no spawning pair was present, or pursued a pair or a single dominant male moving between spawning grounds, the occasional shifting tactics of paired males functioned to confuse and disperse satellites. The spawning tactics of paired males apparently reflected potential fitness costs of satellites. Paired males mainly spawned at the redds near shelters, despite the fact that more satellites were present to devour eggs, presumably because they could obtain many females and monopolize fertilizations.     

棉酚对几种动物精子ATP酶抑制作用的比较研究

本文比较了棉酚对几种动物精子ATP酶的抑制作用。实验所测为精子细胞的总ATP酶活力;采用的棉酚浓度为10μM至80μM。20μM时精子酶的相对活力为海胆7604％,大鼠74％,家兔51.6％;40μM时酶的相对活力百分数下降至海胆52.5,大鼠35和家兔29.7;达80μM时酶的相对活力进一步下降至海胆26.7,而大鼠及家兔仅余13.2。结果表明(1)棉酚对精于ATP酶的抑制作用是依赖浓度的变化;(2)各种动物精子ATP酶活力的下降情况显示,大鼠及家兔(哺乳动物)精子ATP酶对棉酚的敏感性比海胆(无脊椎动物)要高些。这种差异性可能对棉酚在临床上的应用具有一定的参考价值。     

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.     

用双相电泳技术分析棉酚对大鼠成熟精子蛋白质组成的影响

本文首次将双相电泳(等电聚焦和SDS电泳)技术应用于对棉酚作用机理的研究。实验结果表明,棉酚能够改变大鼠成熟精子的蛋白质组成;同时也证明,双相电泳这一技术对棉酚的研究是有效的。     

Ultrastructural analysis ofSpinacia oleracea sperm cells isolated from mature pollen grains

Summary In isolated condition, the sperm cells ofSpinacia oleracea are no longer arranged in pairs as in the pollen grain. The vegetative membrane, which surrounds a sperm cell pair in a mature pollen grain, is lost during the isolation procedure. The sperm cells become spherical in shape.The isolated sperm cell is surrounded by an intact plasma membrane. The heterochromatic or euchromatic sperm cell nucleus is located in the cell center. Mitochondria are round to oval and have distinct cristae. Often they are clustered in groups of 5 to 10 mitochondria. Dictyosomes are present in the cytoplasm and consist of 4 to 5 cisterns. Endoplasmatic reticulum is mostly situated at the sperm cell periphery, as single cisterns very near the plasma membrane.From diameters of sectioned sperm cells in electron micrographs, it is possible to calculate the average diameter of the whole sperm cell. This average diameter is 3.66 m with a variation of 3.0 m to 4.2 m, resulting in an average volume of 25.6 m³. The nuclear volume is 12.8 m³ (50.0% of the whole cell) and the mitochondrial volume is 0.7 m³ (2.5% of the whole cell). The frequency distribution of the isolated sperm cells diameters shows only one peak with a normal distribution, indicating that there is no dimorphism in volume.