Ultrastructure and Chromatin Disaggregation of Human Sperm Head With Thioglycolate Treatment

Lung, Ben

doi:10.1083/jcb.52.1.179

Cited by 47 publications

(16 citation statements)

References 22 publications

(15 reference statements)

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“…Recent efforts in this laboratory to define the biochemical composition of sperm chromatin have revealed results which indicate that the DNA in mouse and certain other mammalian sperm must be packaged in a fashion very different from that found in somatic chromatin, a finding that is consistent with the conclusions of circular dichroism (86) and electron microscopy studies (45,57,58) . Determinations of the DNA and protamine content of the mouse sperm nucleus (77), and the volume of the nucleus into which this material must be packed (94), make it clear that the DNA in these sperm can not be packaged in nucleosomes .…”

supporting

confidence: 64%

A model for the structure of chromatin in mammalian sperm.

Balhorn¹

1982

The Journal of Cell Biology

505

269

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DNA in mammalian, and most vertebrate sperm, is packaged by protamines into a highly condensed, biochemically inert form of chromatin. A model is proposed for the structure of this DNA-protamine complex which describes the site and mode of protamine binding to DNA and postulates, for the first time, specific inter- and intraprotamine interactions essential for the organization of this highly specialized chromatin. In this model, the central polyarginine segment of protamine binds in the minor groove of DNA, crosslinking and neutralizing the phosphodiester backbone of DNA while the COOH- and NH2-terminal ends of protamine participate in the formation of inter- and intraprotamine hydrogen, hydrophobic, and disulfide bonds. Each protamine segment is of sufficient length to fill one turn of DNA, and adjacent protamines are locked in place around DNA by multiple disulfide bridges. Such an arrangement generates a neutral, insoluble chromatin complex, uses all protamine sulfhydryl groups for cross linking, conserves volume, and effectively renders the chromatin invulnerable to most external influences.

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supporting

confidence: 64%

A model for the structure of chromatin in mammalian sperm.

Balhorn¹

1982

The Journal of Cell Biology

505

269

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“…The smallest cords about 400-550 A in thickness were uniformly observed. They were of the same thickness as the chromatin bodies and cords measured from the trans mission electron micrographs, and also within similar range to the large fibers described by Lung [15] and Wagner and Yun [25]. These 400-550 A cords probably can merge to form large fascicles (up to 1.400 A) which arc then compacted inside the sperm heads.…”

Section: Discussionsupporting

confidence: 56%

“…Similarly, Lung [15] used thioglycolate at alkaline pH to partially disrupt human sperm heads. The dispersed chromatin exhibited fundamental units of 100 A fibers which could merge to form biggersized fibers of 400-700 A in thickness.…”

Section: Introductionmentioning

confidence: 99%

Transmission and Scanning Electron Microscopic Studies of Human Sperm Heads Extracted with 8 <i>M</i> Urea, 1% Mercaptoethanol and Different Concentrations of Salt

Sobhon¹,

Tanphaichitr²,

Patilantakarnkool³

1984

Cells Tissues Organs

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Transmission (TEM) and scanning (SEM) electron microscopic studies were performed on the human sperm heads extracted with (a) 1% Triton X-100, 1% mercaptoethanol (ME) and (b) 8 M urea, 1% ME together with increasing concentrations of NaCl ranging from 0.2 to 0.6 M. In the TEM study, the extraction of the nuclear material was first observed when the heads were treated with 8 M urea and 1% ME, with the majority of the chromatin remaining as 400–550 Å thick interconnecting cords and oval bodies. At 0.2 M NaCl the cords and bodies were further separated but linked together by extremely thin 20–50 A fibers. Between 0.3 and 0.5 M NaCl the chromatin bodies within the sperm heads began to be extracted, first at the central part and progressively towards the periphery. Finally, at 0.6 M NaCl only the chromatin cords forming the periphery of the heads remained. In the SEM study, the sperm heads remained unbroken up to the treatment with 8 M urea, 1% ME and 0.2 M NaCl. Between 0.3 M and 0.5 M NaCl the majority of heads were disrupted to form interlacing chromatin cords of 400–550Å thick while the unbroken heads exhibited surface with cross-weaving cords. At 0.6 M NaCl all heads were disrupted and the remaining chromatin existed mostly as exoskeleton of former sperm heads. Protein gel electrophoresis showed that histones and nonhistones were removed from the chromatin when the treatment reached 0.2 M NaCl, whereas protamines started to be removed at 0.3 M, and totally removed at 0.6 M NaCl; HP1 was the first protamine fraction to be extracted. It is concluded that the thick cords are nucleoprotamines laced together by nucleohistone fibers; and that the cords forming the peripheral exoskeleton are sturdier than those in the interior of the sperm heads.

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“…Various methods have been used to decondense sperm nuclei and to isolate and characterize the sperm chromatin and nuclear proteins (Borenfreund et al, 1961;Henricks & Mayer, 1965;Lung, 1972). Sodium dodecyl sulphate (SDS, an anionic surfactant) and dithiothreitol (DTT, a reagent which specifically cleaves disulphide linkages) have been used to study nuclear stabilization during maturation of mammalian spermatozoa (Calvin & Bedford, 1971;Calvin et al, 1973).…”

Section: (Received 10th December 1974)mentioning

confidence: 99%

“…In spite of this, the nucleus swells and its chromatin begins to decondense very rapidly as the spermatozoon is incorporated into the egg cytoplasm (Austin, 1961;Yanagimachi & Noda, 1970;Bedford, 1972). As yet, the mechanism underlying this phenomenon has not been directly examined.Various methods have been used to decondense sperm nuclei and to isolate and characterize the sperm chromatin and nuclear proteins (Borenfreund et al, 1961;Henricks & Mayer, 1965;Lung, 1972). Sodium dodecyl sulphate (SDS, an anionic surfactant) and dithiothreitol (DTT, a reagent which specifically cleaves disulphide linkages) have been used to study nuclear stabilization during maturation of mammalian spermatozoa (Calvin & Bedford, 1971;Calvin et al, 1973).…”

mentioning

confidence: 99%

Induction of Nuclear Decondensation of Mammalian Spermatozoa in Vitro

Mahi¹,

Yanagimachi²

1975

Reproduction

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The nucleus of the mammalian spermatozoon is an exceptionally stable organelle requiring very rigorous chemical treatments to free its chromatin (Borenfreund et al., 1961). In spite of this, the nucleus swells and its chromatin begins to decondense very rapidly as the spermatozoon is incorporated into the egg cytoplasm (Austin, 1961;Yanagimachi & Noda, 1970;Bedford, 1972). As yet, the mechanism underlying this phenomenon has not been directly examined.Various methods have been used to decondense sperm nuclei and to isolate and characterize the sperm chromatin and nuclear proteins (Borenfreund et al., 1961;Henricks & Mayer, 1965;Lung, 1972). Sodium dodecyl sulphate (SDS, an anionic surfactant) and dithiothreitol (DTT, a reagent which specifically cleaves disulphide linkages) have been used to study nuclear stabilization during maturation of mammalian spermatozoa (Calvin & Bedford, 1971;Calvin et al., 1973). Nuclear stabilization by disulphide (-S-S-) bonding has been studied from an evolutionary standpoint by Bedford & Calvin (1974), who have found that this -S-S-bonding is prominent in the spermatozoa only of eutherian mammals. In the present study, the types of chemicals that can decondense mammalian sperm nuclei were determined, then the common characteristics of these chemicals were evaluated so that possible inferences could be made about the mechanism of nuclear decondensation during fertilization.Epididymal spermatozoa of the golden hamster, rat, guinea-pig and rabbit were obtained by puncturing the excised cauda epididymidis with a needle and squeezing the spermatozoa into a Petri dish containing 0-9% NaCl at 37°C. Pellets of frozen canine semen were prepared according to the techniques of Seager (1969) and were air-shipped from Oregon to Hawaii in liquid nitrogen (-196°C). For these experiments, pellets were thawed rapidly in 0-9% NaCl (37°C), and fresh canine ejaculates were obtained for comparison. Both fresh and frozen-thawed semen were washed by centrifugation two or four times in 0-9% NaCl and resuspended in 0-9% NaCl. The sperm suspension of each species (0-1 ml) was mixed with an equal volume of a test solution in a 5-ml glass test-tube. The final concentration of spermatozoa varied from 105 to 107 spermatozoa/ml. The tubes were sealed with Parafimi (American Can Co., Neenah, Wisconsin) and incubated at 37°C. At the end of incubation, the pH was measured and the degree of nuclear decondensation was determined by 293

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Ultrastructure and Chromatin Disaggregation of Human Sperm Head With Thioglycolate Treatment

Cited by 47 publications

References 22 publications

A model for the structure of chromatin in mammalian sperm.

A model for the structure of chromatin in mammalian sperm.

Transmission and Scanning Electron Microscopic Studies of Human Sperm Heads Extracted with 8 <i>M</i> Urea, 1% Mercaptoethanol and Different Concentrations of Salt

Induction of Nuclear Decondensation of Mammalian Spermatozoa in Vitro

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