Electrical sizing of cells of the halophilic alga Dunaliella parva

Abstract. The cell volume of two strains of the halophilic alga Dunaliella parva was measured by two direct methods and by electrical sizing. The results from the direct methods agreed with each other and were consistently larger than those obtained from electrical sizing. Statistical analysis showed that the cell volume populations obtained by electrical size of D. Parva cells is due to the cell membrane having an unusually low electrical resistance relative to other cell membranes.     

Use of the electronic particle counter to investigate phosphorus on particles in streams

A simple and rapid method is described for the measurement by electronic particle counter of the relative proportions of sediment particles, of diameters 1 to 40 µm, which could enter the stream suspended load. The method is useful to further investigations into nutrients borne upon such material because the amounts of phosphate per unit weight of particles varies through orders of magnitude depending upon the particle size. An example is given to illustrate the application of the method.     

Comparative ultrastructure of spermatozoa from two regular and two irregular New Zealand echinoids

Spermatozoa from four species of echinoids found in New Zealand had morphological characteristics typical of other echinoids, including a conical sperm head with an acrosome‐capped nucleus, a midpiece, and a single long flagellum. The spermatozoa of Fellaster zelandiae, Echinocardium cordatum, Evechinus chloroticus, and Centrostephanus rodgersii also showed statistically significant differences in species‐specific morphological characteristics. Evechinus chloroticus showed the most variable sperm morphology. The irregular urchins (F. zelandiae and E. cordatum) had short, wide sperm heads (head length:width ratios 2.93:1 & 2.97:1, respectively) with a long acrosome complex, while the regular urchins (E. chloroticus and C. rodgersii) had longer, narrower heads with a short acrosome complex (ratios 5.29:1 & 3.37:1). Spermatozoa of E. cordatum from the New Zealand population shared more characteristics with those of conspecifics from the Sea of Japan than those of conspecifics from the Baltic, reflecting the membership of the former two populations in a distinct Pacific clade. Volumetric calculations showed no evidence of phylogenetic grouping. Mitochondria of E. chloroticus spermatozoa were less than half the volume of those of C. rodgersii and E. cordatum, and those of F. zelandiae were intermediate in volume. These volume measurements will be useful in physiological studies of sperm performance and quality.     

Effects of tyrosine kinase inhibitor on the motility and ATP concentrations of fowl spermatozoa

The possible role of tyrosine kinase in the regulation of fowl sperm motility was investigated by using a stable analogue of erbstatin, methyl 2,5-dihydroxycinnamate (2,5-MeC), a specific inhibitor of tyrosine kinase. This inhibited the motility of intact spermatozoa at 30°C in a dose-dependent manner. In contrast, the motility of demembranated spermatozoa was not inhibited by the same concentrations of 2,5-MeC. At 40°C, both intact and demembranated spermatozoa were almost immotile with or without 2,5-MeC. Additionally, intact spermatozoa, stimulated by the addition of Ca²⁺ or calyculin A, a specific inhibitor of protein phosphatases, lost their motility with the subsequent addition of 2,5-MeC at 40°C. However, unlike the motility, the ATP concentrations of spermatozoa were maintained in about 30–35 nmol ATP/10⁹ cells during these incubation periods. The activity of tyrosine kinase of spermatozoa at 30°C, estimated by measuring the phosphorylation of a synthetic peptide substrate, RR-SRC, was 0.17 pmol/min per milligram of protein. This activity was lower than that of fowl testes or chick brain but higher than that of chick liver. These results suggest that tyrosine kinase activity, which is not retained in the axoneme and/or accessory cytoskeletal components, may be involved in the maintenance of flagellar movement of fowl spermatozoa at 30°C. Mol. Reprod. Dev. 49:196–202, 1998. © 1998 Wiley-Liss, Inc.     

Ultimobranchial gland of the domestic fowl

Summary The ultimobranchial gland (UBG) of birds is particularly rich in calcitonin, the hypocalcaemic hypophosphataemic hormone, that is secreted by the C-cells of the mammalian thyroid. The principal cells of the UBG have a striking resemblance with the mammalian C-cells, i.e., they possess small intracytoplasmic dense-core secretory granules, 150–300 nm in diameter. The gland also contains a second, morphologically distinct, endocrine cell type with larger granules, 500–800 nm in diameter. A sensitive immunocytochemical reaction was developed with the use of antibodies against salmon calcitonin. By means of this technique the presence of calcitonin-immunoreactive molecules was demonstrated in both secretory cell types of the UB gland of the chicken. This gland can thus be considered as a homogeneous calcitonin-producing tissue. Whether the secretory products are identical is discussed and differences in the secretory pathways are suggested.     

A computer-aided measuring system for the characterization of yeast populations combining 2D-image analysis, electronic particle counter, and flow cytometry

An integrated measuring system was developed that directly compares the shape of size distributions of Saccharomyces cerevisiae populations obtained from either microscopic measurements, electronic particle counter, or flow cytometer. Because of its asymmetric mode of growth, a yeast population consists of two different subpopulations, parents and daughters. Although electronic particle counter and flow cytometer represent fast methods to assess the growth state of the population as a whole, the determination of important cell cycle parameters like the fraction of daughters or budded cells requires microscopic observation. We therefore adapted a semiautomatic and interactive 2D-image processing program for rapid and accurate determination of volume distributions of the different sub-populations. The program combines the capacity of image processing and volume calculation by contour-rotation, with the potential of visual evaluation of the cells. High-contrast images from electron micrographs are well suited for image analysis, but the necessary air drying caused the cells to shrink to 35% of their hydrated volume. As an alternative, hydrated cells overstained with the fluorochrome calcofluor and visualized by fluorescence light microscopy were used. Cell volumes calculated from length, and diameter measurements with the assumption of an ellipsoid cell shape were underestimated as compared to volumes derived from 2D-image analysis and contour rotation, because of a deviating cell shape, especially in the older parent cells with more than one bud scar. The bimodal volume distribution obtained from microscopic measurements was identical to the protein distribution measured with the flow cytometer using cells stained with dansylchloride, but differed significantly from the size distribution measured with the electronic particle counter. Compared with the flow cytometer, 2-D image analysis can thus provide accurate distributions with important additional information on, for instance, the distributions of subpopulations like parents, daughters, or budded cells.     

Fate of spermatozoa that do not participate in fertilization in the domestic fowl

Summary The fate of spermatozoa that do not participate in fertilization was investigated by electron microscopy. After artificial insemination, we observed several spermatozoa between the fibers of the outer layer of the vitelline membrane of the ovum. One or more spermatozoa were also found in a phagocytic vesicle of macrophages located in the intercellular space of the mucosal epithelium of the infundibulum or in the outer layer of the vitelline membrane.From these observations, we assume that the superfluous spermatozoa in the lumen of the anterior part of the oviduct might be removed by inclusion into the outer layer of the vitelline membrane and by phagocytosis by macrophages.The authors are greatly indebted to Assoc. Prof. Osamu Koga for his invaluable advice. The authors also wish to thank Mr. Takayuki Mri for his helpful suggestions and technical advice. This investigation was supported by a grant from the Ministry of Education of Japan (156185)     

Spermiogenesis and sperm morphology in the marine gastropod Nucella crassilabrum with an account of morphometric patterns of spermatozoa variation in the family Muricidae

Abstract

Spermatid differentiation and the morphology of mature spermatozoa in Nucella crassilabrum, a muricid snail, was investigated. Five phases of spermiogenesis considering the polarization of organelles, nuclear elongation and chromatin condensation are described. Characteristics observed are compared to those of other muricaceans gastropods. The comparison of the proportional size of the different sperm segments (head, middle- and principal piece) between these species, is emphasized.

Two morphometric patterns of sperm structure are distinguished and their functional significance discussed. The relative size attained by the different sperm segments within each of these patterns, could be the result of the combined effect of at least two types of factors: first, those determining the proportional size (length) of the head and, secondly, those factors conditioning the energy requirements of the gamete thus influencing the relative development of the middle- and principal piece.     

Catecholamine-containing pancreatic islet cells of the domestic fowl

Summary In an attempt to identify pancreatic islet cells emitting formaldehyde-induced fluorescence (FIF), the pancreatic islets of the domestic fowl were studied by combined fluorescence, ultrastructural, silver-impregnation and immunohistochemical methods in the same section or in consecutive semi-thin and ultra-thin sections. The results indicate that islet cells emitting intense FIF exhibit a strongly argyrophil reaction with the Grimelius' silver method and also immunohistochemical reaction with anti-glucagon serum, but not with anti-5-HT serum. Therefore, the fowl islet A cell, a peptide hormone-producing cell, stores simultaneously catecholamine as biogenic amine. The islet B and D cells did not display any FIF, any argyrophil reaction with the Grimelius' silver method, or any immunoreactivity with anti-glucagon or anti-5-HT sera. The fluorescent but non-argyrophil cells dispersed in the exocrine acinus may well be PP cells.     

The passage of spermatozoa through the vitelline membrane in the domestic fowl,Gallus gallus

Summary The developing outer layer of the vitelline membrane of the ovum in the posterior part of the infundibulum of the domestic fowl contains many spermatozoa in nearly parallel orientation with its inner layer. When the acrosomal region of a spermatozoon approaches or contacts the inner layer, promptly undergoes the acrosome reaction. The outer acrosomal membrane and overlying plasma membrane fuse together and the apical region of the acrosome opens, so that the acrosomal contents are released. Meanwhile the spermatozoon remains a time in contact with the surface of the inner layer, and the network of the inner layer just under the tip of the sperm head begins to be dissolved. This dissolution extends downward forming a tunnel, approximately 9 m in diameter. The spermatozoon then passes through the inner layer obliquely via the central region of the tunnel and arrives at the perivitelline space.The authors are greatly indebted to assoc. prof. Dr. Osamu Koga for his valuable advices. The authors also wish to thank Mr. Takayuki Mori for his helpful suggestions and technical advices. This investigation was supported by a grant from the Ministry of Education of Japan (156185)     

Cell surface changes in the proteins of rabbit spermatozoa during epididymal passage

Differences in the exposure of spermatozoa surface components during epididymal passage have been examined using lactoperoxidase-catalyzed ¹²⁵I-iodination or labeling with ¹²⁵I-diazodiiodosulfanilic acid. Labeled surface proteins obtained from caput and cauda epididymides were solubilized in detergent, separated by sodium dodecylsulfate polyacrylamide slab gel electrophoresis, and identified by radiography. Densitometer scans of autoradiograms revealed increased amounts or exposures of surface proteins of ～35,000, ～39,000, ～50,000, and ～78,000 molecular weight on the cauda epididymal spermatozoa.     

牛精子蛋白质组学技术平台的建立及冻融前后精子差异蛋白初步分析

本研究通过探索不同的精子蛋白制备方法、水化液成分和优化2D电泳程序以建立牛精子蛋白质组学研究技术平台,同时以牛鲜冻精为实验材料通过差异凝胶电泳寻找冻融前后精子蛋白的改变。结果表明：使用改进的热TRIzol法裂解精子细胞制备蛋白,结合优化的2D电泳技术可建立稳定的牛精子蛋白质组学研究技术平台。差异凝胶电泳揭示牛精子在冻融后有质和量的改变：冻融后缺失的蛋白点有20个,表达下调的有2个,表达上调的有10个。作为一项阶段性的实验成果,本研究建立的2D平台和所发现的冻融引起的差异表达蛋白质点为揭示冷冻损伤机理和性控精液的差异蛋白质组学研究奠定了较好的基础。     

Fractionation of bovine spermatozoa for sex selection: A rapid immunomagnetic technique to remove spermatozoa that contain the H-Y antigen

A study was conducted to rapidly fractionate bovine spermatozoa on the basis of cell-surface H-Y antigen (i.e., Y chromosome-bearing spermatozoa). A novel, rapid immunomagnetic method was developed for removal of spermatozoa that bound to anti-H-Y IgG. Fluorescent labeling and flow cytometry were used to measure the efficiency with which spermatozoa binding to anti-H-Y were removed by the immunomagnetic technique. Washed bovine spermatozoa (n=7 bulls) were treated with a mouse monoclonal IgG antibody to H-Y antigen (MoAb 12/49). Fluorescent labeled goat antibody against mouse IgG was added to label those spermatozoa with cell-surface H-Y antigens. Supermagnetized polymer beads coated with an anti-antibody to the MoAb 12/49 were then added to the spermatozoa. After 20 min of incubation, spermatozoa were exposed for 2 min to a magnet, causing the magnetized particles to adhere to the sides of the tube. Nonmagnetized spermatozoa in the supernatent were aspirated and analyzed for fluorescent label by flow cytometry. Approximately 50% of spermatozoa not subjected to immunomagnetic separation were fluorescent labeled, and about one-half of the spermatozoa were observed microscopically to be bound to the magnetized polymer beads prior to magnetic separation (P<0.05). Following magnetic separation, only 1.2% (P<0.05) of the spermatozoa in the magnetic supernatent were fluorescent labeled. Assuming that only Y chromosome-bearing spermatozoa have cell-surface H-Y antigens, the present immunomagnetic fractionation removed almost all of the Y chromosome-bearing spermatozoa, leaving a population that was greater than 98% X chromosome-bearing spermatozoa.     

Phagocytosis of spermatozoa by the ovum of the domestic fowl,Gallus gallus at the time of fertilization

Summary Spermatozoa with intact acrosomes, as well as those coming into contact with the ovum at a smaller angle, and morphologically abnormal spermatozoa reach the plasma membrane of the ovum via an extensively dissolved zone of the inner layer of the vitelline membrane. This zone is assumed to be formed by overlapping of two or more tunnels formed by spermatozoa that had previously come into contact with the ovum.When a spermatozoon comes into contact with the plasma membrane of the ovum, many cytoplasmic processes extend outwards and cover it. Thereafter, the plasma membranes of the processes fuse, thereby phagocytizing the spermatozoon. It is assumed that the phagocytized spermatozoa cannot undergo transformation into male pronuclei and that they degenerate soon after phagocytosis.The authors are greatly indebted to Assoc. Prof. Osamu Koga for his valuable advice. The authors also wish to thank Mr. Takayuki Mori for his helpful suggestions and technical advice. This investigation was supported by a grant from the Ministry of Education of Japan (156185)Previous name: Fukashi Okamura     

Lactate dehydrogenase isozymes in the spermatozoa of the domestic fowl, Gallus domesticus and turkey, Meleagris gallopavo.

1. Electrophoresis of extracts of turkey spermatozoa for lactate dehydrogenase activity revealed the usual five tissue LDHs (LDH-1 to LDH-5). 2. The presence of LDH-X (the spermatozoan-specific isozyme) was not obvious. 3. Only one band was present on electrophoresis of fowl spermatozoan extracts and it coincided with LDH-1 (heart type). 4. Kinetic investigations, the use of inhibitors and the heat-stability test confirmed that the fowl spermatozoan LDH was probably LDH-1 and not LDH-X.