A Simple Technique for the Purification of Plasma Membranes from Ejaculated Boar Spermatozoa
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Spermatozoa were initially separated from fresh boar ejaculates using a 1.0 M sucrose density gradient. Spermatozoa (1 x 10(8) cells/ml) were subjected to gas cavitation (650 psi, 10 minutes), followed by a 4-step centrifugation technique to yield the final plasma membrane preparation. Purity of the plasma membrane isolate was determined using microscopic techniques (i.e. differential interference contrast and transmission electron microscopy) and marker enzymes for biochemical characterization. Plasma membranes were found to be removed primarily from the periacrosomal region of the sperm. Acrosomes appeared to remain intact on the cavitated spermatozoa. Transmission electron microscopy yielded a homogenous population of 100-200 microns unilamellar vesicles. Enzyme markers specific for plasma, acrosome and mitochondrial membranes substantial the purity observed under visual examination.Keywords:
BOAR
Differential centrifugation
Isopycnic
Differential centrifugation
Sedimentation
Buoyant density
Neutral buoyancy
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Acrosome reaction
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SYNOPSIS The morphological features of boar spermatozoa in freshly ejaculated semen and in semen exposed to various experimental treatments have been studied in stained smears and with the phase‐contrast microscope and the electron‐microscope. The spermatozoon of the boar closely resembles that of other domestic ungulates. The head is approximately 8.5μ long; it is twice as long as it is wide. The mid‐piece is approximately 10μ long and the main tail‐piece is 30μ long. The head is covered anteriorly by a cap‐like structure—the acrosome. Evidence is presented to show that the acrosome has two components: the outer acrosome is the larger, the smaller inner acrosome is a crescentic structure. The segment‐shaped area of overlap between the two parts of the acrosome forms the equatorial segment which is a feature of living boar spermatozoa. The surface of the head behind the acrosome impregnates with silver; this argentophil area is the post‐nuclear cap. Three neck granules can be distinguished at the posterior border of the head and these are apparently connected to three bundles of neck fibres which the head receives from the mid‐piece. The structure of the spermatozoon is altered by post‐mortem changes; the most prominent changes occur in the acrosome. Evidence is presented that the post‐nuclear cap is absent from dead spermatozoa. The development of the boar acrosome has been studied in sections stained by the PAS method and in unfixed tissues with the phase‐contrast microscope. The development of the post‐nuclear cap has been studied in sections impregnated with silver. The morphological features of epididymal spermatozoa have been studied in spermatozoa from different levels of the epididymis. The development of the acrosome is shown to resemble its development in the bull. Two separate components of the acrosome of the spermatid are distinguished. It is suggested that these develop into the two components of the acrosome of the mature spermatozoon. Migration of the cytoplasmic droplet from the neck to the mid‐piece occurs at varying levels in the epididymis, but it is shown that it may occur in the head of the epididymis. The findings are discussed in relation to current ideas about the structure of mammalian spermatozoa.
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Differential centrifugation is used to prepare a "heavy" mitochondrial fraction from liver. These mitochondria are relatively pure, highly coupled, and suitable for respiratory studies. This unit presents protocols for isolation of beef heart mitochondria (also suitable for respiratory studies), skeletal muscle mitochondria, and mitochondria from cultured cells can also be isolated from homogenates by differential centrifugation. Differential centrifugation, which separates cellular organelles based on sedimentation velocity, is a rapid method for preparing mitochondria for metabolic studies.
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Membrane integrity
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A spermhead-defect appearing in 15-20 per cent of the spermatozoa from a Norwegian Landrace boar with a slightly impaired fertility was studied in light and electron microscope. The defect had the shape of a circumferential, somewhat elevated area containing a cystic structure located within the nucleus. In some of the sections observed in electron microscope a communication could be found between the cyst and the acrosome, indicating that a deviating development of the latter could be involved in the formation of the defect. In some spermatozoa the occurrence of the cyst was combined with the presence of an apical defect of the acrosome. From ultrastructural studies of spermatids the development of the cystic defect was found to have reached a rather advanced stage early in the acrosome-phase of the spermateliosis. As a whole the observations correspond very well with those made in connection with the socalled "SME"-defect first found in a Danish Landrace boar.
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Abstract Centrifugation is the use of the centrifugal forces generated in a spinning rotor to separate biological particles, such as cells, viruses, sub‐cellular organelles, macromolecules (principally proteins and nucleic acids) and macromolecular complexes (such as ribonucleoproteins and lipoproteins). The three main methods of separation are differential pelleting, rate‐zonal centrifugation and isopycnic centrifugation. The first two methods separate particles primarily on the basis of size while isopycnic centrifugation separates particles on the basis of their density. The choice of centrifugation method depends on the nature of the particles and often more than one separation technique is required. For example, membrane fractionation often involves first making an enriched fraction from a cell homogenate by differential pelleting followed by isopycnic centrifugation to obtain purified fractions. Key Concepts Modern centrifuges and their rotors can generate centrifugal forces up to a million times that of the Earth's gravity that can separate very small particles. Depending on the centrifugation force used, particles from cells to those as small as macromolecules can be separated. Particles can be separated on the basis of their size using differential pelleting or rate‐zonal centrifugation. Alternatively, particles can be separated on the basis of their density using isopycnic centrifugation. Once cells are broken open by homogenisation, they can be fractionated into a range of defined membrane fractions so that it is possible to analyse the functions of the different components of cells. Many centrifugation purification procedures involve sequential centrifugation steps, often a size separation followed by purification using isopycnic gradient centrifugation.
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Mitochondria are involved in cellular energy metabolism and use oxygen to produce energy in the form of adenosine triphosphate (ATP). Differential centrifugation at low- and high-speed is commonly used to isolate mitochondria from tissues and cultured cells. Crude mitochondrial fractions obtained by differential centrifugation are used for respirometry measurements. The differential centrifugation technique is based on the separation of organelles according to their size and sedimentation velocity. The isolation of mitochondria is performed immediately after tissue harvesting. The tissue is immersed in an ice-cold homogenization medium, minced using scissors and homogenized in a glass homogenizer with a loose-fitting pestle. The differential centrifugation technique is efficient, fast and inexpensive and the mitochondria obtained by differential centrifugation are pure enough for respirometry assays. Some of the limitations and disadvantages of isolated mitochondria, based on differential centrifugation, are that the mitochondria can be damaged during the homogenization and isolation procedure and that large amounts of the tissue biopsy or cultured cells are required for the mitochondrial isolation.
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Mitochondria are involved in cellular energy metabolism and use oxygen to produce energy in the form of adenosine triphosphate (ATP). Differential centrifugation at low- and high-speed is commonly used to isolate mitochondria from tissues and cultured cells. Crude mitochondrial fractions obtained by differential centrifugation are used for respirometry measurements. The differential centrifugation technique is based on the separation of organelles according to their size and sedimentation velocity. The isolation of mitochondria is performed immediately after tissue harvesting. The tissue is immersed in an ice-cold homogenization medium, minced using scissors and homogenized in a glass homogenizer with a loose-fitting pestle. The differential centrifugation technique is efficient, fast and inexpensive and the mitochondria obtained by differential centrifugation are pure enough for respirometry assays. Some of the limitations and disadvantages of isolated mitochondria, based on differential centrifugation, are that the mitochondria can be damaged during the homogenization and isolation procedure and that large amounts of the tissue biopsy or cultured cells are required for the mitochondrial isolation.
Differential centrifugation
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