Isolation of egg cells and zygotes of Torenia fournieri L. and determination of their surface charge

Zygote ◽  
2008 ◽  
Vol 16 (2) ◽  
pp. 179-186 ◽  
Author(s):  
S.H. Chen ◽  
Y.H. Yang ◽  
J.P. Liao ◽  
A.X. Kuang ◽  
H.Q. Tian
Keyword(s):  
Plasma Membrane ◽  
Molecular Biology ◽  
Surface Charge ◽  
Enzymatic Digestion ◽  
Egg Cell ◽  
Sperm Cells ◽  
Torenia Fournieri ◽  
Successful Isolation

SummaryEgg cells of Torenia fournieri were isolated from embryo sacs 1 day after anthesis using enzymatic digestion or mechanical dissection. About 5% of the egg cells and zygotes (2–3 from 50 ovules) could be mechanically dissected within 2 h. When 0.1% cellulase and 0.1% pectinase were added to the mannitol isolation solution, about 18% of the egg cells (8–10 from 50 ovules) could be isolated within 2 h. The egg cells isolated by mechanical dissection could be used for in vitro fertilization studies without any of the potentially deleterious effects of the enzymes on the plasma membrane of egg cell. The egg cells isolated using enzymatic digestion could be used in the study of the molecular biology of female gamete because more egg cells could be isolated with this technique. Using enzymatic digestion, over 10 zygotes from 50 ovules (over 20%) were isolated from the pollinated ovules. Coupled with our successful isolation of mature sperm cells, the isolation of egg cells of T. fournieri will make in vitro fertilization possible in a dicotyledon plant.

scholarly journals Isolation of living sperm cells and in vitro fusion of Torenia fournieri gametes

2006 ◽  
Vol 12 (4) ◽  
Author(s):  
P. Vági ◽  
K. Imre ◽  
Z. Kristóf
Keyword(s):  
Male Gametophyte ◽  
Embryo Sac ◽  
Pollen Tubes ◽  
Sperm Cells ◽  
Torenia Fournieri ◽  
Isolation Medium ◽  
Vitro Fertilization ◽  
Crucial Part

In contrast to most angiosperms, Torenia contains a naked embryo sac and therefore has been considered since many years as an exciting model plant to study the double fertilization process of flowering seed plants. It is thus not surprising that the isolation of protoplasts from the female gametophyte has been reported already 20 years ago by Mol, the isolation of megaspores and megagametophytes has been published by the authors of this manuscript in 1996 and in 1999. The isolation of the male gametophyte and of sperm cells was published by the authors in 2004. The isolation of viable Torenia sperm cells is a crucial part of the elaboration of an in vitro fertilization system. Torenia sperm cells were isolated from in vivo — in vitro cultured pollen tubes. In this system pollen tubes first grow inside a cut style then follow their elongation in a solid isolation medium. The medium contained agarose in order to detain pollen tube contents. Released sperm cells and enzymatically isolated egg cells were collected and handled using glass micropipettes and transmitted to an electrofusion apparatus or polyethylene glycol containing media for fusion probes.

scholarly journals Visualization and quantification of glycolipid polarity dynamics in the plasma membrane of the mammalian spermatozoon

1994 ◽  
Vol 107 (8) ◽  
pp. 2151-2163 ◽  
Author(s):  
B.M. Gadella ◽  
T.W. Gadella ◽  
B. Colenbrander ◽  
L.M. van Golde ◽  
M. Lopes-Cardozo
Keyword(s):  
Plasma Membrane ◽  
Sperm Head ◽  
Breakdown Product ◽  
Prolonged Storage ◽  
Sperm Cells ◽  
Fluorescence Lifetime Imaging ◽  
Outer Leaflet ◽  
Head Surface ◽  
Sperm Plasma Membrane

Seminolipid (sulphogalactosylalkylacylglycerol), the glycolipid that is specific for mammalian germ cells, is located exclusively in the outer leaflet of the sperm plasma membrane. In this study the lateral distribution of seminolipid on sperm heads has been investigated by indirect immunofluorescence labelling and detection with digital imaging fluorescence microscopy. In freshly ejaculated sperm cells this glycolipid was present primarily at the apical ridge subdomain of the plasma membrane of the sperm head. After binding the sperm cells to zona-coated coverslips seminolipid migrated, in 40 minutes, from the apical ridge to the equatorial subdomain of the plasma membrane. A similar redistribution of seminolipid was observed during capacitation of sperm cells in vitro induced by Ca2+ or bovine serum albumin. Comparable migration of seminolipid was also found after prolonged storage of ejaculated sperm cells, albeit at a much slower rate. Addition of arylsulphatase A, an enzyme present in seminal plasma that desulphates seminolipid, significantly enhanced the migration of seminolipid during storage of sperm cells. Its breakdown product desulphoseminolipid (galactosylalkylacylglycerol) appeared highly specifically at the equatorial segment. The measured fluorescence intensity over the sperm head surface correlated linearly with the spatial probe distribution as was checked by fluorescence lifetime imaging microscopy. This paper demonstrates and quantifies for the first time the polarity of seminolipid on the surface of the sperm cell and the dynamic alterations that occur in this polarity during post-ejaculatory events.

scholarly journals Update on mammalian sperm capacitation: how much does the horse differ from other species?

Reproduction ◽  
2019 ◽  
Vol 157 (5) ◽  
pp. R181-R197 ◽  
Author(s):  
Bart Leemans ◽  
Tom A E Stout ◽  
Catharina De Schauwer ◽  
Sonia Heras ◽  
Hilde Nelis ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Zona Pellucida ◽  
Mammalian Species ◽  
Cumulus Cells ◽  
Sperm Cells ◽  
Cholesterol Depletion ◽  
Sperm Capacitation ◽  
Sperm Plasma Membrane ◽  
Species Specific

In contrast to various other mammalian species, conventional in vitro fertilization (IVF) with horse gametes is not reliably successful. In particular, stallion spermatozoa fails to penetrate the zona pellucida, most likely due to incomplete activation of stallion spermatozoa (capacitation) under in vitro conditions. In other mammalian species, specific capacitation triggers have been described; unfortunately, none of these is able to induce full capacitation in stallion spermatozoa. Nevertheless, knowledge of capacitation pathways and their molecular triggers might improve our understanding of capacitation-related events observed in stallion sperm. When sperm cells are exposed to appropriate capacitation triggers, several molecular and biochemical changes should be induced in the sperm plasma membrane and cytoplasm. At the level of the sperm plasma membrane, (1) an increase in membrane fluidity, (2) cholesterol depletion and (3) lipid raft aggregation should occur consecutively; the cytoplasmic changes consist of protein tyrosine phosphorylation and elevated pH, cAMP and Ca2+ concentrations. These capacitation-related events enable the switch from progressive to hyperactivated motility of the sperm cells, and the induction of the acrosome reaction. These final capacitation triggers are indispensable for sperm cells to migrate through the viscous oviductal environment, penetrate the cumulus cells and zona pellucida and, finally, fuse with the oolemma. This review will focus on molecular aspects of sperm capacitation and known triggers in various mammalian species. Similarities and differences with the horse will be highlighted to improve our understanding of equine sperm capacitation/fertilizing events.

Plant gametes as tools for molecular breeding

2002 ◽  
Vol 50 (3) ◽  
pp. 295-301
Author(s):  
B. Barnabás ◽  
Z. Pónya ◽  
F. Bakos ◽  
Keyword(s):  
Molecular Biology ◽  
Essential Role ◽  
Cell Activation ◽  
Molecular Breeding ◽  
Experimental Approach ◽  
Embryo Sac ◽  
Flowering Plants ◽  
Cereal Crops ◽  
Egg Cell

Sexual reproduction plays an essential role in the propagation of Angiosperms. Fertilisation takes place in the embryo sac, which is usually deeply encased in the sporophytic tissues of the ovule. In contrast to animals and primitive plants, the mechanism of egg cell activation in flowering plants has not been discovered fully because of the inaccessibility and complexity of the process of double fertilisation. However, recent advances in plant cell and molecular biology have brought new, powerful technologies to investigate and micromanipulate the reproductive cells of flowering plants including cereal crops. An experimental approach based on various micromanipulation techniques involving in vitro fertilisation (IVF) and microinjection procedures is now available in more and more laboratories. Despite some limitations this offers new possibilities to study cellular and subcellular events preceding or occurring during or after egg cell activation and early embryonic development. Recent achievements in the field of wheat egg cell micromanipulation are presented in this paper.

scholarly journals In vitro evaluation of canine spermatozoa cryopreserved in different extenders

2006 ◽  
Vol 58 (6) ◽  
pp. 1116-1122 ◽  
Author(s):  
E.C.S. Oliveira ◽  
G.C. Juliani ◽  
A.P. Marques Jr. ◽  
M. Henry
Keyword(s):  
Plasma Membrane ◽  
Ethylene Glycol ◽  
Egg Yolk ◽  
Dimethyl Formamide ◽  
Sperm Cells ◽  
Fluorescent Staining ◽  
Progressive Motility ◽  
Functional Integrity ◽  
Hypoosmotic Swelling Test

The efficacy of three extenders, tris-egg yolk-5% ethylene glycol (T1), lactose-egg yolk-5% ethylene glycol (T2) and lactose-egg yolk-5% dimethyl formamide (T3) on preserving the viability of post-thawing canine spermatozoa was evaluated. Three ejaculates per dog were obtained of five animals. The semen was packaged in 0.5ml straws and cooled to 4°C for 120min. The straws were frozen 4cm above the nitrogen level for 15min and thawed in water-bath at 37°C for 60sec and at 75°C for 7sec. Progressive motility and vigour were evaluated immediately after thawing (time 0) and at 30, 60, 90 and 120min. Structural and functional integrity of plasma membrane of the spermatozoa were evaluated, respectively, by fluorescent staining probes and hypoosmotic swelling test. Lactose-egg yolk based extenders showed better cryoprotectant capability and dimethyl formamide was an alternative cryoprotectant agent for dog sperm cells.

Characterisation of isolated egg cells,in vitrofusion products and zygotes ofZea maysL. using the technique of image analysis and confocal laser scanning microscopy

Zygote ◽  
1995 ◽  
Vol 3 (1) ◽  
pp. 57-64 ◽  
Author(s):  
Uday K. Tirlapur ◽  
Erhard Kranz ◽  
Mauro Cresti
Keyword(s):  
Image Analysis ◽  
Plasma Membrane ◽  
Sperm Cell ◽  
Laser Scanning Microscopy ◽  
Fusion Product ◽  
Confocal Laser ◽  
Egg Cell ◽  
Nuclear Chromatin ◽  
Fusion Products

SummaryChanges in membrane Ca2+, calcium receptor protein calmodulin, endoplasmic reticulum (ER), mitochondria and cellulose in unfixed, living, isolated egg cells and fusion products of pairs of one egg and one sperm cell ofZea maysL. have been investigated using chlorotetracycline, fluphenazine, immunocytochemical techniques, 3,3'dihexyloxa-carbocyanine iodide (DiOC6(3)) and calcofluor white in conjuction with computer-controlled video image analysis. In addition, confocal laser scannig microscopy has been used in conjuction with ethidium bromide to detect the nature and location of the sperm cell nuclear chromatin before and after karyogamy. Digitised video images of chlorotetra cycline (CTC) fluorescence reveal that egg cells contain high levels of membrane Ca2+in organelles present around the nucleus while the cytosolic signal is relatively low. Intense CTC fluorescence is invariably present just below the plasma membrane of egg cells and a certain degree of regionalised distribution of Ca2+in cytoplasm is also discrnible. Similarly, the fluphenazine (FPZ)-detectable calmodulin (CaM) and that localised immunocytochemically using monoclonal anti-CaM antibodies reveal high levels of Cam in the vicinity of the nucleus in egg cells. Only a few ER profiles and mitochondria could be visualised in the egg cell and no calcofluor fluorescence could be detected. Followingin vitrofertilisation of single isolated eggs substantial changes in the Ca2+levels occur which include an increase in the membrane Ca2+of the fusion product, particularly in the cytosol and around the nucleus. Unlike in the eggs the fine CTC fluorescence signal below the plasma membrane is not detectable in the fusion products. Compared with isolated egg cell protolasts an increase in the CaM level in the cytoplasm was observed in the fusion products. There is a slight increase in the CaM level in the cytoplasm was observed in the fusion products. There is a slight increase in the fluorescene around the fusion product is visible after 16 h in in culture. The sperm cell chromatin in the fusion product is highly condensed, unlike that of the egg cell, and confocally imaged serial optical sections of thein vitrofusion product reveal the occurrence of karyogamy 35 min following gamete fusion. First visual evidence for intermingling of sperm nuclear chromatin in the zygotic nuclei is also provided.

scholarly journals Glycolipid migration from the apical to the equatorial subdomains of the sperm head plasma membrane precedes the acrosome reaction. Evidence for a primary capacitation event in boar spermatozoa

1995 ◽  
Vol 108 (3) ◽  
pp. 935-946 ◽  
Author(s):  
B.M. Gadella ◽  
M. Lopes-Cardozo ◽  
L.M. van Golde ◽  
B. Colenbrander ◽  
T.W. Gadella
Keyword(s):  
Plasma Membrane ◽  
Acrosome Reaction ◽  
Plasma Membranes ◽  
Phase Segregation ◽  
Sperm Head ◽  
Lipid Phase ◽  
Sperm Cells ◽  
Fluorescence Lifetime Imaging ◽  
Boar Spermatozoa

In order to extend the static information of immunolabelling sulphogalactolipids in fixed boar spermatozoa, a fluorescent sulphogalactolipid analogue, galactose(3-sulphate)-beta 1–1′[(N-lissamine rhodaminyl)-12-aminodode-canoyl]-sphingosine, was incorporated into plasma membranes of living spermatozoa and its lateral distribution over the sperm head was studied. The fluorescent lipid was enriched in the apical ridge subdomain of freshly ejaculated sperm cells. After sperm binding to the zona pellucida the lipid redistributed to the equatorial segment of the sperm surface. A similar shift occurred during capacitation in vitro with 2 mM CaCl2 or with 4% (w/v) bovine serum albumin. The desulphated derivative galactose-beta 1–1′[(N-lissamine rhodaminyl)-12-aminododecanoyl]-sphingosine was also incorporated into the plasma membrane of freshly ejaculated sperm cells and clearly stained the apical ridge subdomain and the (pre)-equatorial subdomains of the sperm heads. The desulphogalactolipid analogue showed a slightly faster migration to the equatorial segment of the sperm plasma membrane than did its sulphated counterpart. The measured fluorescence intensity distributions correlated linearly with the spatial probe distribution, which was checked by fluorescence lifetime imaging microscopy. The observed migration of the incorporated glycolipids precedes the acrosome reaction and is one of the underlying molecular events likely to be important in the process of sperm capacitation. The results of this study suggest that lipid phase segregation is an important driving force for the organization of the sperm head plasma membrane into subdomains.

scholarly journals Isolation of Sperm and Egg Cells from Pepper

2018 ◽  
Vol 143 (4) ◽  
pp. 310-315 ◽  
Author(s):  
Wei Deng ◽  
Yunling Xie ◽  
Yilan Qiu
Keyword(s):  
Generative Cell ◽  
Embryo Sac ◽  
Pollen Tubes ◽  
Enzymatic Digestion ◽  
Sperm Cells ◽  
Free Solution ◽  
In Vitro Method ◽  
Viable Embryo

Pepper (Capsicum annuum) pollen is bicellular and contains a vegetative cell and a generative cell, which divides in pollen tubes to form two sperm cells. Sperm cells of pepper were isolated using an in vivo–in vitro method. Hand-pollinated styles were first grown in vivo for several hours, then cut from their base and cultured in vitro until pollen tubes grew from the cut end. When the pollen tubes were transferred to a breaking solution, sperm cells were released from broken tubes. Viable embryo sac cells of pepper were isolated using enzymatic digestion and mechanical dissection. Isolated ovules were digested using cellulase and pectinase for 40 minutes and then transferred to an enzyme-free solution for mechanical dissection. Three cells of the egg apparatus and a central cell were released from a cut at the chalazal end of each ovule by pressing on the micropylar area of the ovule with a microneedle. Optimal isolation conditions included 11% mannitol, 0.04% CaCl2, 1% bovine serum albumin (BSA), 1% cellulase, 1% pectinase, and 0.3% pectolyase. Using this protocol, populations of pepper egg cells, synergids, and central cells were isolated.

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