A comparison of the ultrastructure of spray-frozen and freeze-etched or freeze-dried bull and boar spermatozoa with that after chemical fixation

Pfaller, Walter; Rovan, E.; Mairbäurl, Heimo

doi:10.1530/jrf.0.0480285

Cited by 12 publications

(8 citation statements)

References 15 publications

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“…We could observe ultrastructural details of spermatozoa with normal or pathologic morphology, but also we could describe the alterations induced by osmotic processes or to the cold-shock treatment. Several authors have proposed different methods for the treatment of spermatozoa for ultrastructural study (Pfaller et al, 1976;Rozinek, 1977). However, the advantage of using our technique is to avoid centrifugation, and that the ultrastructural details can be defined ''in situ,'' thus preserving the functional relationship of the vesicles and the target cells.…”

Section: Discussionmentioning

confidence: 99%

New method for the treatment of sperm samples for ultrastructural study based on the use of animal tissues as biological containers

Junquera

Colás

Martínez-Ciriano

et al. 2007

Microscopy Res & Technique

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The study of the ultrastructure of spematozoa by means of transmission electron microscopy (TEM) often presents with problems of interpretation according to the method employed, depending on whether samples are either centrifuged previously to the fixation or immersed in viscous gels. The major problems of interpretation are changes in the location of vesicles originated during the maturation process and modifications in the adsorption of seminal plasma proteins to the sperm membrane surface. The aim of our study is to communicate an original new method for the treatment of spermatozoa for ultrastructural study. Our method is based on the use of animal tissues as biological containers, inside which the spermatic suspensions are included. We developed this method using fresh sperm samples taken from mature Rasa Aragonesa rams. As biological container, we used 2.5-cm long segments of the intestine of 1-week-old chickens (Gallus gallus) (diameter around 4 mm). In order to avoid any influence of digestive enzymes of the mucosa on the sperm surface, we put each intestine fragment inside out by means of microdissection forceps under a bifocal optical microscope and cold light. One of the edges was tied with thin suture silk. The sperm suspension was injected in the optimal experimental condition and amount. Finally, the still-open edge of the intestine segment was tied with silk in the same way as the other segment edge. By using this technique, we can perform a suitable morphological study at an ultrastructural level. In addition, the functional relationship of the ultrastructural components of the target cells is correctly preserved.

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Section: Discussionmentioning

confidence: 99%

New method for the treatment of sperm samples for ultrastructural study based on the use of animal tissues as biological containers

Junquera

Colás

Martínez-Ciriano

et al. 2007

Microscopy Res & Technique

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“…It has been employed by many authors in the field of conventional electron microscopy (Sjostrand and Kretzer, 1975;Pfaller et al, 1976;Coulter and Terracio, 1977;Coulter and Elde, 1979;Pfaller, 1979;Schwabe and Terracio, 1980;Terracio and Schwabe, 1981;. For the FD procedure, fresh tissues are quickly frozen and the tissue water is removed by sublimation at high vacuum conditions and low temperatures.…”

Section: Discussionmentioning

confidence: 99%

Some improvement in tissue preparation and colloidal‐gold immunolabeling for electron microscopy

et al. 1986

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The application of freeze-substitution (FS) and freeze-drying (FD) techniques and the protein A-gold-antibody complex immunocytochemical methods are described. The two tissue-preparation techniques produced excellent ultrastructure and topographical fixation of antigens when compared with conventional tissue-preparation techniques. In the FS preparation, however, occasional extragranular immunolabeling was recognized. This may suggest the leakage of antigens from the secretory granules. The FD procedure was considered the best, since such labeling was almost negligible. The protein A-gold-antibody complexes are easily prepared and label the antigens clearly. If the protein A-coated gold particles are saturated with antibodies, there is no interaction between gold particles. Thus, multiple antigens can be determined even in single secretory granules. In fact, we demonstrated intragranular colocalization of immunoreactive oxytocin, labeled with 50-nm gold particles, and immunoreactive methionine-enkephalin, labeled with 15-nm gold particles, in the axonal terminals of the FD-prepared rat neurohypophysis. This study demonstrates the value of the use of gold-antibody complexes for immunocytochemical labeling on FS- or FD-treated tissues.

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“…Bittermann et al (1992) FS (SJF) Paramecium Geanacopoulos and Gear (1988) FF, FE Blood platelets Horowitz et al (1990) FS Nuclei Knoll et al (1991) FF, FS (SJF) Paramecium Lang and Bronk (1978) FF, FE Mitochondria Lang et al (1976) FF, FE Mitochondria Linder and Staehelin (1979) FS Leptomonas Miller and Dahl (1982) FF Liposomes Pfaller and Rovan (1978) FD, FE Spermatozoa Pfaller et al (1976) FD, FE Spermatozoa Plattner (1971) FE Spermatozoa Plattner et al (1972) FF, FE Chlorella, Euglena, spermatozoa Plattner et al (1973) FF, FE Aerobacter, Chlorella, Euglena, Paramecium, Selenastrum, spermatozoa Rand et al (1985) FF Vesicle fusion Williams (1953) FD TMV, red blood cells Fig. 1.…”

Section: Methodsmentioning

confidence: 99%

Spray-freezing freeze substitution (SFFS) of cell suspensions for improved preservation of ultrastructure

Fields

Strout

Russell

1997

Microsc. Res. Tech.

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Some unicellular organisms present challenges to chemical fixations that lead to common, yet obvious, artifacts. These can be avoided in entirety by adapting spray‐freezing technology to ultrarapidly freeze specimens for freeze substitution. To freeze specimens, concentrated suspensions of cells ranging in diameter from 0.5–30 μm were sprayed with an airbrush at 140–200 kPa (1.05–1.5 torr; 20.3–29.0 psi) into a nylon mesh transfer basket submerged in liquid propane. After freezing, the mesh basket containing the frozen sample was lifted out of the chamber, drained and transferred through several anhydrous acetone rinses at 188 K (−85°C). Freeze substitution was conducted in 1% tannic acid/1% anhydrous glutaraldehyde in acetone at 188 K (−85°C), followed by 1% OsO4/acetone at 277 K (4°C). Freeze substitution was facilitated using a shaking table to provide gentle mixing of the substitution medium on dry ice. High quality freezing was observed in 70% of spray‐frozen dinoflagellate cells and in 95% of spray‐frozen cyanobacterial cells. These could be infiltrated and observed directly; however, overall ultrastructural appearance and membrane contrast were improved when the freeze‐substituted cells were rehydrated and post‐fixed in aqueous OsO4, then dehydrated and embedded in either Spurr's or Epon resin. Ultrastructural preservation using this ultrarapid freezing method provided specimens that were consistently superior to those obtainable in even the best comparable chemical fixations. Microsc. Res. Tech. 38:315–328, 1997. © 1997 Wiley‐Liss, Inc.

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A comparison of the ultrastructure of spray-frozen and freeze-etched or freeze-dried bull and boar spermatozoa with that after chemical fixation

Cited by 12 publications

References 15 publications

New method for the treatment of sperm samples for ultrastructural study based on the use of animal tissues as biological containers

New method for the treatment of sperm samples for ultrastructural study based on the use of animal tissues as biological containers

Some improvement in tissue preparation and colloidal‐gold immunolabeling for electron microscopy

Spray-freezing freeze substitution (SFFS) of cell suspensions for improved preservation of ultrastructure

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