Imaging the Zona Pellucida of Canine and Feline Oocytes Using Scanning Electron Microscopy

2009 ◽  
Vol 15 (1) ◽  
pp. 2-14 ◽  
Author(s):  
Matthew O. Lunn ◽  
Shirley J. Wright
Keyword(s):  
Zona Pellucida ◽  
High Vacuum ◽  
Cumulus Cell ◽  
Cumulus Cells ◽  
Future Studies ◽  
Critical Point Drying ◽  
Cacodylate Buffer ◽  
Molecular Aspects ◽  
Potential Use ◽  
Scanning Electron

AbstractUltrastructure of the zona pellucida (ZP) of canine and feline oocytes has not been fully investigated. The objective of the study was to evaluate the potential use of the low vacuum scanning electron microscope (LVSEM) with oocytes. This required development of a method to prepare canine and feline cumulus-oocyte complexes (COCs) for LVSEM to provide ultrastructural information on the ZP. COCs were collected from ovaries, and cumulus cells were either partially or completely removed to reveal the ZP. COCs and zona-intact oocytes were fixed at 4°C for 1 h in 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer, pH 7.4 and subsequently viewed wet or further processed by critical point drying, and viewed uncoated or sputter coated with gold. Although the spongy surface of the ZP was visible at low vacuum in uncoated oocytes, coated oocytes had more details at high vacuum. The ZP surface of canine and feline oocytes contained numerous various-sized, spherical or elliptical pores that narrowed centripetally splitting into several smaller, deep pores. The round to oblong cumulus cells tightly surrounded the ZP. Each corona radiata cumulus cell tapered into a thin projection that entered the ZP. Our detailed techniques will enable future studies connecting ultrastructural and molecular aspects of oocyte maturation and development in mammals.

Taxonomic Identification of Rhabdochona Species Found in Oncorhynchus Clarki Using Scanning Electron Microscopy

1998 ◽  
Vol 4 (S2) ◽  
pp. 1148-1149
Author(s):  
D. Young ◽  
R.A. Heckmann ◽  
J. S. Gardner
Keyword(s):  
Electron Microscopy ◽  
Scanning Electron Microscopy ◽  
Digestive Tract ◽  
Cutthroat Trout ◽  
Parasitic Nematodes ◽  
Buffer System ◽  
Taxonomic Identification ◽  
Critical Point Drying ◽  
Cacodylate Buffer ◽  
Scanning Electron

Adult Rhabdochona nematodes, commonly parasitizing fish, were present in the digestive tracts of cutthroat trout in Little Cottonwood Creek, Utah. Cutthroat trout, Oncorhyncus clarki, are known to serve as both intermediate and definitive hosts for parasitic nematodes. The larval stage parasitizes almost any tissue of its host, but the adult is always found in the digestive tract. Due to the lack of key morphological features, scanning electron microscopy (SEM) was used to identify specific structures leading to the nematode's taxonomic identification.Cutthroat trout were obtained using a rod and reel and were dissected the same day. Nematodes were present in all 12 cutthroat trout residing in all parts of the digestive tract. The nematodes, Rhabdochona sp., were prepared for SEM using the following procedures. First, the parasites were fixed in 2% buffered glutaraldehyde, washed in sodium cacodylate buffer, and post fixed in a 1% solution of osmium tetroxide. The samples were then washed in the same buffer system and dehydrated through a graded alcohol series. Critical-point-drying removed the remaining fluids. Finally, the nematodes were placed on specimen stubs, sputter coated with gold, and each specimen examined with a JOEL-840 high resolution scanning electron microscope with micrographs taken at varying magnifications.

The application of environmental SEM to marine biology

1992 ◽  
Vol 50 (1) ◽  
pp. 834-835
Author(s):  
Richard Ray
Keyword(s):  
Biological Material ◽  
Marine Biology ◽  
High Vacuum ◽  
Environmental Scanning ◽  
Conductive Film ◽  
Critical Point Drying ◽  
New Development ◽  
High Resolution Images ◽  
Environmental Sem ◽  
Scanning Electron

Preparation of marine biological material for standard scanning electron microscopy (SEM) requires extensive manipulation, including fixation, removal of salts, dehydration, and either air drying or critical-point drying because the SEM operates at high vacuum. Non-conducting samples including biological material must be coated with a conductive film of metal before the specimen can be imaged.The Electroscan Corporation (Wilmington, MA) recently introduced a new development in SEM technology—the environmental scanning electron microscope (ESEM). This instrument uses a unique secondary electron detector capable of forming high resolution images at pressures in the range of 0.1 to 20 torr. If water vapor is used as the specimen environment, wet samples including wet biofilms can be observed directly. This paper illustrates the use of the ESEM to view bacteria, dinoflagellates, and diatoms on surfaces.

Artifacts on the surface of articular cartilage due to drying methods

1978 ◽  
Vol 36 (2) ◽  
pp. 680-681
Author(s):  
I. Hesse ◽  
W. Hesse
Keyword(s):  
Articular Cartilage ◽  
Critical Point ◽  
Femoral Condyle ◽  
Medial Femoral Condyle ◽  
Drying Methods ◽  
Tissue Preparation ◽  
Critical Point Drying ◽  
Cacodylate Buffer ◽  
Drying Techniques ◽  
Scanning Electron

Since the advent of scanning electron microscopy very contrasting studies on the surface topography of normal and pathological cartilage have been published. The existing literature did not respect the influence of tissue preparation, especially the effect of the drying techniques. Thus the results lead to wrong conclusions. The purpose of this paper is to compare the preservation of the surface morphology obtained from three drying techniques, i. e. air-, freeze- and critical-point drying.Our experiments were performed on normal articular cartilage of 50 sheep, on transplanted cartilage of 45 sheep and on articular cartilage taken intraoperatively from 33 patients. Three adequate samples of articular cartilage attached to bone were dissected out of the weightbearing portion of the medial femoral condyle for air-, freeze- and critical-point drying. They were rinsed in 4 changes of cacodylate buffer (pH=7. 2-7. 4).

Human cumulus cell complexes studied in vitro by light microscopy and scanning electron microscopy

Zygote ◽  
1994 ◽  
Vol 2 (2) ◽  
pp. 133-147 ◽  
Author(s):  
Sian Kennedy ◽  
Marjorie A. England ◽  
Carla Mills
Keyword(s):  
Electron Microscopy ◽  
Scanning Electron Microscopy ◽  
Collagen Type ◽  
Cumulus Cell ◽  
Cumulus Cells ◽  
Plastic Substrate ◽  
Thick Sheet ◽  
Type I ◽  
Scanning Electron

SummaryVarious researchers describe the morphology of cumulus cells (CC) in vitro, but few have investigated their behaviour on plastic. Knowledge concerning the behaviour of human CC could be useful in improving the success of in vitro fertilisation procedures. This study aimed to describe the morphology and behaviour of CC in vitro and to investigate movement on a collagen-coated substrate. Following collection some cumulus were mechanically dissected from those surrounding the oocyte. Cumulus aggregates were cultured over 24 h using Earle's medium supplemented with 8% albumin. Substrata were plastic coverslips coated with collagens I, IV, or mixed collagens. Cumulus cultured over corresponding time periods on uncoated coverslips served as controls. Specimens were fixed and prepared for scanning electron microscopy. Over 24 h the controls began exhibiting the morphological features associated with cell movement: cell surface protrusions changed from blebs to microridges, lamellipodia and leading lamellae; cell shape altered from rounded and upright, to flattened. Extracellularmatrix (ECM) transformed from a thick, sheet-like substance to a thin, fibrous material. By 24 h, cells contacting ECM remained rounded showing few features of movement. Collagens enhanced attachment of CC as a monolayer on the substrate. Cell morphology varied according to the collagen type used. On mixed collagens, cells attached rapidly, appearing to be predominantly non-motile. On collagen type I there was less attachment of cells but increased motility. On collagen type IV there was decreased attachment and the cells remained spherical. In conclusion, collagens enhance the settling of cumulus cells on a plastic substrate and the cells exhibit some specificity in attaching to collagens.

Scanning Electron Microscopy of Blaberus Giganteus (L.) Maxillary Palp Sensilla

1976 ◽  
Vol 34 ◽  
pp. 194-195
Author(s):  
P. Dailey
Keyword(s):  
Osmium Tetroxide ◽  
Ventral Surface ◽  
Maxillary Palp ◽  
Sodium Cacodylate Buffer ◽  
Sodium Cacodylate ◽  
Sensilla Basiconica ◽  
Critical Point Drying ◽  
Cacodylate Buffer ◽  
Maxillary Palps ◽  
Scanning Electron

The maxillary palps of B. giganteus are located on the maxillae which lie between the labrum and labium. They are modified appendages used in feeding. Each appendage bears 5 segments, the most distal of which contains the palp organ or sensory pad (Fig. 1). The maxillary palps were fixed in 2.5% glutaraldehyde in 0.05 M sodium cacodylate buffer (pH 7.1) containing .15 M sucrose and post-fixed in 1% osmium tetroxide in buffer. This was followed by dehydration and critical point drying. Cryofractography was performed on some of the palp organs prior to drying (1).The sensory pad is composed of an exocuticular membrane upon which lie numerous sensilla basiconica in an elliptical pattern on the ventral surface. The membrane enables the sensory pad to expand and, in living specimens it appears dome-like due to the pressure exerted on it by the hemolymph.

scholarly journals A hyaluronidase activity of the sperm plasma membrane protein PH-20 enables sperm to penetrate the cumulus cell layer surrounding the egg.

1994 ◽  
Vol 125 (5) ◽  
pp. 1157-1163 ◽  
Author(s):  
Y Lin ◽  
K Mahan ◽  
W F Lathrop ◽  
D G Myles ◽  
P Primakoff
Keyword(s):  
Plasma Membrane ◽  
Zona Pellucida ◽  
Cell Layer ◽  
Current Model ◽  
Cumulus Cell ◽  
Human Sperm ◽  
Cumulus Cells ◽  
Intact Mouse ◽  
Hyaluronidase Activity ◽  
Pass Through

A typical mammalian egg is surrounded by an outer layer of about 3,000 cumulus cells embedded in an extracellular matrix rich in hyaluronic acid. A current, widely proposed model is that the fertilizing sperm, while it is acrosome intact, passes through the cumulus cell layer and binds to the egg zona pellucida. This current model lacks a well-supported explanation for how sperm penetrate the cumulus layer. We report that the sperm protein PH-20 has a hyaluronidase activity and is present on the plasma membrane of mouse and human sperm. Brief treatment with purified, recombinant PH-20 can release all the cumulus cells surrounding mouse eggs. Acrosome intact mouse sperm incubated with anti-PH-20 antibodies can not pass through the cumulus layer and thus can not reach the zona pellucida. These results, indicating that PH-20 enables acrosome intact sperm to penetrate the cumulus barrier, reveal a mechanism for cumulus penetration, and thus provide the missing element in the current model.

The Critical Point Drying Method Applied to Scanning Electron Microscopy of L-929 Cells

1970 ◽  
Vol 28 ◽  
pp. 278-279
Author(s):  
Charles TurnbiLL ◽  
Delbert E. Philpott
Keyword(s):  
Electron Microscopy ◽  
Carbon Dioxide ◽  
Surface Tension ◽  
Critical Point ◽  
Freeze Drying ◽  
Attractive Alternative ◽  
Crystal Formation ◽  
Critical Point Drying ◽  
Time Required ◽  
Scanning Electron

The advent of the scanning electron microscope (SCEM) has renewed interest in preparing specimens by avoiding the forces of surface tension. The present method of freeze drying by Boyde and Barger (1969) and Small and Marszalek (1969) does prevent surface tension but ice crystal formation and time required for pumping out the specimen to dryness has discouraged us. We believe an attractive alternative to freeze drying is the critical point method originated by Anderson (1951; for electron microscopy. He avoided surface tension effects during drying by first exchanging the specimen water with alcohol, amy L acetate and then with carbon dioxide. He then selected a specific temperature (36.5°C) and pressure (72 Atm.) at which carbon dioxide would pass from the liquid to the gaseous phase without the effect of surface tension This combination of temperature and, pressure is known as the "critical point" of the Liquid.

Preservation of Plant Cell Surfaces For Scanning Electron Microscopy

1973 ◽  
Vol 31 ◽  
pp. 472-473
Author(s):  
Linda M. Sicko ◽  
Thomas E. Jensen
Keyword(s):  
Electron Microscopy ◽  
Scanning Electron Microscopy ◽  
Critical Point ◽  
Filter Paper ◽  
Freeze Drying ◽  
Cell Surfaces ◽  
Air Drying ◽  
Popular Method ◽  
Critical Point Drying ◽  
Scanning Electron

The use of critical point drying is rapidly becoming a popular method of preparing biological samples for scanning electron microscopy. The procedure is rapid, and produces consistent results with a variety of samples. The preservation of surface details is much greater than that of air drying, and the procedure is less complicated than that of freeze drying. This paper will present results comparing conventional air-drying of plant specimens to critical point drying, both of fixed and unfixed material. The preservation of delicate structures which are easily damaged in processing and the use of filter paper as a vehicle for drying will be discussed.

Remarks on the application of backscattered electron imaging (BEI) to the scanning electron microscopy (SEM) of biological structures

1983 ◽  
Vol 41 ◽  
pp. 484-487
Author(s):  
Etienne de Harven
Keyword(s):  
High Resolution ◽  
Incident Beam ◽  
Spot Size ◽  
Primary Electron ◽  
Critical Point Drying ◽  
Cell Shapes ◽  
High Resolution Images ◽  
Backscattered Electron ◽  
Electron Imaging ◽  
Scanning Electron

Biological ultrastructures have been extensively studied with the scanning electron microscope (SEM) for the past 12 years mainly because this instrument offers accurate and reproducible high resolution images of cell shapes, provided the cells are dried in ways which will spare them the damage which would be caused by air drying. This can be achieved by several techniques among which the critical point drying technique of T. Anderson has been, by far, the most reproducibly successful. Many biologists, however, have been interpreting SEM micrographs in terms of an exclusive secondary electron imaging (SEI) process in which the resolution is primarily limited by the spot size of the primary incident beam. in fact, this is not the case since it appears that high resolution, even on uncoated samples, is probably compromised by the emission of secondary electrons of much more complex origin.When an incident primary electron beam interacts with the surface of most biological samples, a large percentage of the electrons penetrate below the surface of the exposed cells.

High-Vacuum Evaporation and a Cryostage to Observe Fractured Yeast

1988 ◽  
Vol 46 ◽  
pp. 256-257
Author(s):  
William P. Wergin ◽  
Eric F. Erbe ◽  
Eugene L. Vigil
Keyword(s):  
Electron Microscope ◽  
Low Temperature ◽  
Standard Specimen ◽  
High Vacuum ◽  
Specimen Holder ◽  
Sputter Coating ◽  
Fresh Frozen ◽  
Gain Access ◽  
Scanning Electron ◽  
Transfer Device

Investigators have long realized the potential advantages of using a low temperature (LT) stage to examine fresh, frozen specimens in a scanning electron microscope (SEM). However, long working distances (W.D.), thick sputter coatings and surface contamination have prevented LTSEM from achieving results comparable to those from TEM freeze etch. To improve results, we recently modified techniques that involve a Hitachi S570 SEM, an Emscope SP2000 Sputter Cryo System and a Denton freeze etch unit. Because investigators have frequently utilized the fractured E face of the plasmalemma of yeast, this tissue was selected as a standard for comparison in the present study.In place of a standard specimen holder, a modified rivet was used to achieve a shorter W.D. (1 to -2 mm) and to gain access to the upper detector. However, the additional height afforded by the rivet, precluded use of the standard shroud on the Emscope specimen transfer device. Consequently, the sample became heavily contaminated (Fig. 1). A removable shroud was devised and used to reduce contamination (Fig. 2), but the specimen lacked clean fractured edges. This result suggested that low vacuum sputter coating was also limiting resolution.

Sign in / Sign up

Export Citation Format

Share Document