Isolation and partial characterization of a basic protein from bovine sperm heads

Coelingh, J.P.; Rozijn, Th.H.; Monfoort, C.H.

doi:10.1016/0005-2795(69)90091-9

Cited by 96 publications

(22 citation statements)

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“…Early studies of mammalian species indicated that there was a shift toward an arginine-rich nucleoprotein during spermiogenesis, and indeed several workers have isolated small basic proteins from spermatozoa. Coelingh et al isolated and sequenced a protein of molecular weight about 6000 from bull spermatozoa which contained large amounts of arginine and cysteine (7,8). Kistler et al have described another nuclear protein (MW about 6200) isolated only from mature rat testes, but which is not present in rat spermatozoa (9, 10).…”

mentioning

confidence: 99%

New histones found in mature mammalian testes.

Shires¹,

Carpenter²,

Chalkley³

1975

Proc. Natl. Acad. Sci. U.S.A.

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Two new histones have been found in sexually mature testes of several mammals. The new histones have been identified as a new F1 (TFI) and F2b (TF2b) on the basis -of their behavior upon chemical fractionation procedures and electrophoresis at several urea concentrations. The new testis-specific histones are absent in very immature animals and in somatic tissues. However, by day 20, in the rat, the histone pattern is that of the adult, displaying nearly a full complement of TF1 and TF2b. The new histone complement evidently is characteristic of prespermatid germ cells. The degree of evolutionary variations in these histones and in other basic sperm proteins, as detected by change in electrophoretic mobility, appears to be much greater than that seen in somatic histones. The process of spermiogenesis involves the production of a haploid genome, packaged in a highly organized and compact fashion. In echinoderms the compact nucleoprotein contains DNA in association with histones which are either identical or similar to those found in somatic cells (1-4). On the other hand, in arthropods and teleosts somatic histones are replaced in mature spermatozoa by a low molecular weight, arginine-rich class of proteins known as protamines (5, 6). Early studies of mammalian species indicated that there was a shift toward an arginine-rich nucleoprotein during spermiogenesis, and indeed several workers have isolated small basic proteins from spermatozoa. Coelingh et al. isolated and sequenced a protein of molecular weight about 6000 from bull spermatozoa which contained large amounts of arginine and cysteine (7,8). Kistler et al. have described another nuclear protein (MW about 6200) isolated only from mature rat testes, but which is not present in rat spermatozoa (9, 10).We initiated a program designed to compare the basic proteins from spermatozoa of a range of mammals. As Protamines were obtained from the headpieces of mature spermatozoa isolated from the testis, intact epididymis, or semen. The headpieces were separated from the tails of the spermatozoa by blending. During the acid extraction of the histones from the nuclei, the sperm heads were precipitated t To whom reprint requests should be made.in the acid-insoluble fraction. The pelleted sperm heads were washed twice with deionized water, suspended in 15-20 ml (1 ml/g of starting tissue) of 5 M guanidine-hydrochloride, 0.5 M 2-mercaptoethanol, and homogenized. The ensuing gel was adjusted to pH 7 with NaOH and sheared in a Virtis model 45 homogenizer at 70 V for 45 sec. The ionic strength was decreased by adding 2.5 volumes of deionized water. Protamines were extracted in 0.2 M H2SO4 at 40 for 2 hr. After centrifugation at 27,000 X g for 20 min, the supernatant was dialyzed against 0.2 M H2SO4 to remove guanidine-hydrochloride.The bull protamine was obtained from spermatozoa isolated from bull semen, utilizing a slightly modified procedure of Marushige and Marushige (13).Electrophoresis of histones was performed on 15% acrylamide gels as described (14). Elec...

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mentioning

confidence: 99%

New histones found in mature mammalian testes.

Shires¹,

Carpenter²,

Chalkley³

1975

Proc. Natl. Acad. Sci. U.S.A.

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“…The basis of the above method was the extraction procedure of Coelingh et al (1969), who isolated and purified nuclear basic protein on the basis of reductive cleavage of disulphide bonds by f1-mercaptoethanol. This reagent has been used extensively for the lysis of sperm heads (Panyim et al 1970).…”

Section: Discussionmentioning

confidence: 99%

Ethanol Extraction of Basic Proteins From Ejaculated Human Spermatozoa

French¹

1979

Aust. Jnl. Of Bio. Sci.

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A method for the extraction of basic proteins from human ejaculated spermatozoa has been developed It relies on the previously unreported observation that such basic protein is soluble in a solution containing 60% (v/v) ethanol. This unconventional method yields a high percentage of arginine-rich basic protein which is then able to be characterized on the basis of its amino acid composition. This method also allows comparisons to be made between single ejaculates by the banding pattern each displays when subjected to polyacrylamide gel electrophoresis.

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“…In the model proposed by Bril-Petersen and Westenbrink (1963) and extended by Coelingh et al . (1969), the disulfide bonds between protein molecules are distributed throughout the sperm nucleus and maintain a rigid, cross-linked structure which would enclose each DNA molecule and maintain its orientation ; breakage of such bonds would be necessary for loss of birefringence to occur.…”

Section: Mammalian Spermmentioning

confidence: 99%

“…In cephalopod molluscs, such as squid (studied in this paper), and in fish, these basic sperm proteins are called protamines (Miescher, 1897 ;Kossel, 1884Kossel, , 1921 ; they usually contain large amounts of arginine-as much as 70 %Jo 80 % of the amino acid residues in some fish sperm (Felix et al ., 1956) and almost this much in squid sperm (Bloch, 1962) . In mammalian sperm cells the basic proteins have been more resistant to extraction and analysis, but recent work on bull sperm (Coelingh et al ., 1969) shows that the major protein fraction of the nucleus contains a large amount of arginine, as do fish protamines, and differs from them primarily in its large content of half cystine . The components of mammalian sperm nuclei, therefore, may be as simple, and as small in number, as those of cephalopod and fish sperm.…”

Section: Introductionmentioning

confidence: 99%

Birefringence of Spermatozoa

Bearden

Bendet

1972

The Journal of Cell Biology

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In experiments designed to determine the thermal stability and bonding strength of a natural nucleoprotein structure, the loss of birefringence as a function of time and temperature was investigated for both mammalian and nonmammalian sperm nuclei . At a constant temperature, this reaction was found to be first order for both types over a range of temperatures . The methods of chemical kinetics applied to results of these reactions, called birefringence melting reactions, produced values for the enthalpy and entropy of activation in the reactions, which gave some indication of the strength of binding in the nucleoprotein structure ; and these results, plus those on the influence of chemicals on the structure, were consistent with the molecular structures which have been proposed by others for the nucleoprotein complex of sperm nuclei. For both bull and human sperm in ethylene glycol, the ratelimiting step in the melting reactions appeared to be the breakage of disulfide bonds . For squid sperm in ethylene glycol, and bull or squid sperm in ethylene glycol plus /3-mercaptoethanol, the identity of this step was more ambiguous, but a possibility consistent with these and other results would be a cooperative breakage of ionic bonds .head Sato, 1962, 1966 ;Sato and Muller, 1966) ; the interpretation of such measurements has been challenged by others using different experimental methods (Maclnnes and Uretz, 1968 ; Zirwer et al ., 1970), but they still illustrate the potential value of quantitative polarization microscopy as a method for studying the structure of the sperm nucleus.The chromatin of the sperm nucleus of most species consists almost entirely of a complex of negatively charged DNA and positively charged proteins, which are related to the histones of somatic cells but are usually simpler in composition and smaller in molecular weight (Busch,

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Isolation and partial characterization of a basic protein from bovine sperm heads

Cited by 96 publications

References 4 publications

New histones found in mature mammalian testes.

New histones found in mature mammalian testes.

Ethanol Extraction of Basic Proteins From Ejaculated Human Spermatozoa

Birefringence of Spermatozoa

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