Subunit Structure of Eutherian Sperm Chromatin
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T. E. WAGNER,J. E. SLIWINSKI, and D. B. SHEWMAKER The P-mercaptoethanol induced decondensation of spermatozoon cell nuclei from several Eutherian species has been followed from the intact spermatazoon cell to the solubilized linear unit sperm chromosomal fiber using fluorescence and electron microscopy. Data from nuclease digestion studies in conjunction with electron microscopic evidence indicate that the gross structure of the unit Eutherian sperm chromosomal fiber consists of DNA folded around sperm specific histone multimers spaced regularly along the fiber generating a linear array of sperm nucleosomes connected by short streLches of uncomplexed DNA. The sperm nucleosomes, 80 A in diameter are separated by 20 A filaments of DNA. This structure is remarkably similar to the structure of somatic chromatin although the protein components of the two chromosomes are markedly different. It seems likely that chromosomal fibers, similar to those described herein, may be present in the male pronucleus following fertilization.
Key Words: Sperm chromatin; Subunit structure; Sperm nucleosomes.
INTRODUCTION
The nucleus of the spermatozoon of Eutherian mammals contains the male chromosomal complement in a densely packed, protected state for release to the ovum during fertilization. Although many of the ultrastructural details of chromosomal packaging during spermatogenesis have been observed [ 11 and a recent molecular description of the polymer crystalline organization of the chromatin within mature Eutherian sperm nuclei presented [2,3], little is known about the mechanism of sperm nuclear decondensation following entrance of the sperm headpiece into the ovum, or about the molecular structure of the chromosomal component of the male pronucleus. Because the mature sperm chromosomes are packaged in an inaccessible state [4] within the sperm nucleus and released only after entry into the ovum, there is no period during the natural lifetime of the Eutherian sperm cell when its chromosomal contents can be isolated and studied. Therefore, we have developed a mild procedure to effect the release of sperm chromatin from mature sperm cells. This procedure allows the study of molecular aspects of the decondensation, providing insight into the find events during fertilization. In vitro release also facilitates the purification of highly native chromosomal material which may be structurally very similar to the chromosomal component of the male pronucleus. The induced decondensation of bovine, ovine, equine and human spermatazoon cells have been followed with optical and electron microscopy. The tertiary structure Received August 1977. From the Department of Chemistry, Ohio University, Athens, Ohio. Address reprint requests to Ohio University, Department of Chemistry, Athens, Ohio 4.5701 (T. E. Wagner). @ ARCHIVES OF ANDROLOGY I (January 1978): 3 1 4 1
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of the resulting decondensed chromatin was analyzed using enzymatic and chemical techniques.
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MATERIALS AND METHODS Semen samples were collected from rams, bulls and stallions using an artificial vagina and maintained in extender at liquid N, temperatures. Human semen samples were extended and also maintained at liquid N, temperatures. The viability and functional ability of the animal semen was determined by results of artificial insemination. Normal, live offspring were delivered. Sperm cells were washed into the buffer required for each experiment by sedimentation (1 .SO0 g) and resuspension in the buffer. Incidenr,fuoresc encv microscopy. Sperm cells were incubated at pH 8.5, 0.1M Tris-HC1, in the presence of 10-3M P-mercaptoethanol (equine) or 2 x 10-*M p-mercaptoethanol (bovine, ovine, human) at 4°C. At timed intervals, samples were taken, made 0.1 pgiml in ethidium bromide and observed under incident fluorescence microscopy. Light microscopy was carried out using a No. 100 microscope illuminator (Zeiss) with a 13V, 100 W halogen source. Incident fluorescence microscopy was accomplished using this illuminator in combination with phase contrast optics. Transmission electron microscopy. All samples were spread on grids for electron microscopy. Carbon-coated. freshly glow-discharged, Formvar coated grids were used. To these prepared grids were added 7.5 p1 samples. The samples were placed directly onto the grids, allowed to stand for 2-5 min and then removed by aspiration. The grids were rinsed three times with quartz doubly-distilled H,O by adding and removing 7.5 ~1 aliquots. Positive staining was accomplished with 7.5 ml of uranyl acetate (200 pl of a filtered 5% uranyl acetate solution and 2 ~1 of conc HC1 added to 20 ml of 90% ethanol), followed by rinsing with three volumes of quartz double-distilled H,O after 5 min of staining. The prepared grids were examined in a Hitachi H58 electron microscope at SO KV and photographed at a magnitication of 47,000. Photographic prints were enlarged to a magnification of x 277,000. Pwparation of purified sperm chromatin. Intact, active sperm cells were washed into 0.05M Tris buffer, pH 8.8. The buffer volume used was equal to the volume of the pelleted cells used in each preparation. The resulting suspension was made 5 x 10-*M in P-mercaptoethanol and hand homogenized using a glass-teflon homogenizer prior to incubation at 4°C for 48 hr or until the resulting solution thickened to a viscous gel. The resulting thick suspension, containing dissolved sperm chromatin, some yet uncondensed chromatin, sperm membranes and intact sperm tails, was layered above a solution 1.7M in sucrose and 0.001M in Tris buffer pH 8.0 and centrifuged at 27.000 rpm in an SW-40 rotor for two hr. After centrifugation, most of the sperm membranes and all tails were at the bottom of the centrifuge tube while the soluble sperm chromatin was collected as a transparent thin gel just above the sucrosehuffer boundary. The solubilized chromatin was carefully removed from above the sucrose boundary so as not to contaminate it with the membranous material which also collects within and just below the boundary. The resulting sperm chromatin was dialyzed against 0.001M Tris buffer (pH 8.0) and washed by sedimentation at 15,000 rpm in a Sorval SS-34 head. LVideasi>digestion. Staphylococcal nuclease (micrococcal nuclease) was obtained from Sigma Chemical Company at 6,000 or 10,000 U/mg. A stock solution at 2 mg/ml in H,O was stored at 4" for up to several weeks. Digestions of sperm chromatin were performed in 1 mM Tris-HC1
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(pH 8.0), 0.1 mM CaCI, at 37" C by adding 1/10 to the volume of 1 mM CaCl, and the enzyme to the chromatin solution, which ranged from 0.05 to 3 mg DNA/ml. The nuclease rendered an amount of DNA approximately equal to its own weight acid soluble each minute. The digestion was terminated by the addition of a 10-fold excess of ethylene diamine tetraacetic acid (EDTA) over the Ca++concentration in the digestion mixture and cooling the reaction at 0°C in ice. The amount of digestion resulting from nuclease attack on sperm chromatin was measured as the fraction rendered acid soluble. 100 to 200 p g DNA aliquots of a stopped, freshly suspended nuclease reaction were added to 20 p g bovine plasma albumen (BPA, kept at a 2 mgiml solution) in a centrifuge tube, 0.4 ml of 2M NaCl, 2M perchlonc acid added and the mixture brought to a volume of 1 ml with H,O. After 10 min at O'C, the mixture was centrifuged at 10,OOO x g for 10 min and the of the supernatant measured. (% acid soluble) =
acid soluble A,,, x 0.6 undigested A260 of aliquot
Nucleoprotein polyacrylamide gels. Polyacrylamide gels to separate nucleoprotein components
were run in a slab gel apparatus (Aqueboque Corp.). The gels were run essentially as described by Peacock and Dingman [5] except that the buffer concentration was maintained at 1/10 their concentration to increase the mobility of the weakly charged nucleoprotein material. All separations described were carried out using 8% polyacrylamide gels containing 0.4% bisacrylamide crosslinker. Gels were used after gelling for periods greater than 10 hr. Water cooled gels were pre-electrophoresced and run for 2 hr at 200 V with a running buffer of 1/10 Peacock-Dingman buffer. Samples (-50 pg DNA content in 20 pI) were loaded in 10% glycerol in the 1/10 Peacock-Dingman buffer. After electrophoresis the gels were stained in 0.1 pg/ml of ethidium bromide for 1 hr and photographed under ultraviolet light with an orange filter.
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RESULTS Incident Fluorescence Microscopy of Sperm Decondensation
In the presence of between and 10-'M P-mercaptoethanol equine, ovine, bovine and human spermatozoa slowly undergo a stepwise nuclear decondensation, swelling and dissolution. Since this decondensation might be similar to events following fertilization within the ovum we have carefully documented the P-mercaptoethanol induced decondensation. Because the swollen sperm headpieces appear transparent and difficult to observe using phase contrast microscopy, samples were stained with ethidium bromide (1 pg/ml) and observed under incident fluorescence microscopy. Of the four species of Eutherian mammals studied, equine sperm cells respond to lower concentrations of P-mercaptoethanol (10-3) resulting in slower and more easily observable decondensation. The ovine, bovine and human sperm cells require concentrations of P-mercaptoethanol as high as 10-'M to induce decondensation. Under these conditions the decondensation process, which is the same as observed with equine sperm, proceeds much more rapidly and does not allow detailed observation of all stages in the decondensation. In Fig. 1 (upper left) is shown the incident fluorescence micrograph of an ethidium stained equine sperm cell prior to P-mercaptoethanol treatment. The micrograph is characteristic of equine sperm cells studied in our laboratory having a head length of -7p and a head width of -4p with a thickness of slightly less than 1p. The internal
T. Wagner, J. Sliwinski, and B. Shewmaker
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FIGURE 1. Fluorescence micrographs of the stages of P-mercaptoethanol induced decondensation of equine spermatazoon nuclei. Upper Left, sperm nucleus prior to P-mercaptoethanol treatment: Upper Right, sperm nucleus at about 6-8 hr after P-mercaptoethanol treatment showing rupture of nuclear membranes; Lower Left, sperm nucleus at about 24 hr after treatment expanded to 6 x the volume of the untreated nucleus; Lower Right, sperm nucleus after 48 hr of exposure to P-mercaptoethanol expanded to greater than 10 X the volume of the untreated nucleus.
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volume of the headpiece of equine sperm cells where the head shape is approximated as an ellipsoidal cylinder with minor axis 2p, major axis 3.5p,and height lp is -45 X lO-lEm3. After approximately 24 hr at pH 8.6 (0.01M Tris buffer) in the presence of 10-3M P-mercaptoethanol, the ellipsoidal cylindrical shape of the equine sperm head swells into an ellipsoid of revolution and bursts its outer membrane structure. Rupture of the headpiece membrane always occurs on an equatorial belt about one-third the length of the headpiece from the point of midpiece attachment. (Fig. 1) Following membrane rupture, the internal contents of the headpiece continue to swell as a unit into a metastable swollen ellipsoidal structure with an internal volume close to 6 times the volume of the headpieces of untreated equine sperm (2.5-3.0 x 10-16m3). Through the light microscope the p-mercaptoethanol induced condensation of sperm nuclei appears as a three-step process: membrane rupture, 6x swelling and slow dissolution. This decondensation is solely caused by p-mercaptoethanol at pH 8.6 and not a result of the prior history of the sperm samples since we have maintained the cells live in liq N, and inseminated mares, ewes and cows with the cells from the same batches used in these experiments and delivered healthy, live offspring. Electron Microscopy of Dissolved Sperm Nuclei
Incubation at 4”C,pH 8.6, in the presence of P-mercaptoethanol for periods in excess of 48 hrs results in apparent dissolution of the sperm head of all species. After this period of time light microscopy is no longer useful as a tool to study the continuing decondensation process. Initial observation of unpurified samples showed primarily a clumped, three-dimensional fibrillar network of high electron density. Although the contents of the sperm headpieces appear completely dissolved under the light microscope they remain extensively crosslinked, probably due to remaining disulfide linkages [6]. In order to prepare samples containing individual fibers disassociated from each other for electron microscopic study, the post 48 hr treated sperm preparations were made 2 X 10-’M in p-mercaptoethanol and hand-homogenized several times over a period of 24 to 48 additional hr. This preparation was then filtered through 0.1 p nucleopore membranes to remove any remaining aggregated material (e.g. , high molecular weight DNA or chromatin fibrils will pass length-wise through nucleopore membranes with pore sizes greater than the diameter of the fiber). Analysis of grids containing samples prepared in this manner showed two distinct types of fibers: long twisted fibers with a diameter of about 100 A and stretches of fibers with repeating knobby stpctures resembling beads on a string wheFe the “string” has a diameter of 15-20 A and the “bead” has a diameter of about 85 A (Fig. 2). Both the 100 h; filament and the repeating knobby fibers are markedly similar to electron micrographs of somatic chromatin [7,8].These structures are also virtually identical to somatic core chromatin from which all chromosomal protein except the arginine rich histones H3 and H4 have been removed [9].Kornberg [ 101 studying calf thymus chromatin suggests that the 100 h; fiber consists of a flexibly jointed chain of 100 A repeating units that are in contact and contain 290 base pairs of DNA, giving a
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packing ratio of about 7: 1. The “beads-on-a-string” structure observed in the electron microscope for purified somatic chromatin is usually described as a linear array of subunits (80 b;) containing 110-120 base pairs of DNA connected by extended, B-form DNA about 240 A (70-80 base pairs) in length, giving a packing ratio of 2: 1 [ l l , 12, 131.
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Micrococcal Nuclease Digestion of Bovine Sperm Chromatin Bovine sperm cells were treated with 2 X 10-2M P-mercaptoethanol at pH 8.6 for -48 hr with intermittent hand homogenization until a thick, viscous mixture was formed. This crude sperm chromatin preparation was then purified on a sucrose gradient. The purified chromatin recovered from the upper shelf of the gradient displayed structures under electron microscopy and appeared to be completely disaggregated (Fig. 2). This material was then dialyzed into 1 mM Tris (pH 8.0), equilibrated at 37”C, made 1 mM in Ca++ and digestion initiated by addition of 1/30th the nucleotide sample mass of staphylococcal nuclease (Sigma). The kinetics of the nuclease digestion of the chromatin sample were studied by removing aliquots of the digestion mixture at timed intervals, quenching the digestion with a 10-fold excess of EDTA over initial Ca++ concentration, and precipitating the undigested DNA with perchloric acid. The perchloric acid soluble digestion products from degraded DNA were then assayed spectrophotometrically to determine the per cent digestion as a function of the time of digestion. Rapid degradation of the sperm chromosomal DNA by micrococcal nuclease took place during the first 15-30 seconds of the reaction followed by a slower digestion of
FIGURE 2. Left, Electron micrograph of positively stained solubilized bovine sperm chromatin showing the 100 A thick fiber structure; Right, Electron micrograph of positively stained solubliized bovine sperm chromatin showing the thin “knobby” fiber structure. ( x 138,500).
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the substrate to a limit of about 55% of the total substrate DNA (Fig. 3). The results of nuclease digestion are consistent with a structure for bovine sperm chromatin which contains nucleoprotein regions resistant to nuclease digestion and free DNA regions which are markedly susceptible to nuclease digestion. This structural characteristic is consistent with the “beads-on-a-string’’ structure observed under the electron microscope (Fig. 2). Nucleoprotein Gel Electrophoresis of Nuclease Digestion Products of Bovine Sperm Chromatin
Electron microscopy and micrococcal nuclease digestion kinetics (Fig. 3) of purified, solubilized, sperm chromatin suggest a subunit structure for this chromosomal material. From the kinetics of somatic nuclear digestion with staphylococcal nuclease it is apparent that little of the DNA becomes acid soluble when monomer subunit is excised and that most of the starting chromosomal DNA passes through monomer subunit [14,151. The highest concentration of intact subunit monomers in a staphylococcal nuclease digestion of chromatin would be present very early in the digestion. In the case of sperm chromatin, where the initial digestion rate is enhanced, subunit monomer would be most likely present at its highest concentration at about 15-30 sec of digestion. Therefore, a 30-second digestion of bovine sperm chromatin was carried out followed by EDTA quenching and cooling to 0°C. The quenched digestion mixture, containing -0.7 mg/ml of DNA, was maintained at 0°C for several hours until a precipitate appeared. The induced precipitate was redissolved by addition of excess EDTA and warming into a small volume of electrophoresis buffer. Spectrophotometric analysis of this concentrated nucleoprotein solution showed that it contained over 80% of the total initial chromosomal DNA from the digestion mixture. Thus, brief micrococcal nuclease digestion of bovine sperm chromatin cleaves this high molecular weight nucleoprotein polymer to yield insoluble, nuclease-resistant nucleoprotein material. The redissolved nucleoprotein pellet from the 30 sec digestion of
I d
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DIGESTION TIME (MIN.)
FIGURE 3. The kinetics of staphylacoccal nuclease digestion of solubilized bovine sperm chromatin. The amount of digestion was minitored by acid solubility.
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bovine sperm chromatin with staphylococcal nuclease (50 pg) was placed in the sample well of a 7% polyacylamide gel and subjected to a potential of 200 volts for 3 hr using .095MTris-borate buffer at pH 8 . 3 . After electrophoresis the gel was stained with ethidium bromide and photographed under ultraviolet illumination (Fig. 4). This gel electrophoretic separation reveals some high molecular weight nucleoprotein which has not penetrated the gel, a discrete nucleoprotein band about 1.5 cm into the gel and some lower molecular weight digestion products which have smeared along the lower portion of the gel. The presence of a discrete band in the nucleoprotein gel electrophoresis of the digestion products from sperm chromatin treated with micrococcal nuclease strongly supports the existence of a nuclease resistant subunit within sperm chromatin which may be, or be related to. the knobby repeating structures observed in sperm chromatin preparations under the electron microscope (Fig. 2 ) . DISCUSSION
Fluorescence microscopy of several eutherian species of spermatazoa in the presence of P-mercaptoethanol shows the initial gross stages of sperm chromatin decondensation induced by this agent. The nuclear regions of the sperm cells are seen to expand causing rupture of the outer membrane sheath within a discrete equatorial region (-113 the linear distance from the point of midpiece attachment to the opposite end of the headpiece) followed by further expansion of the nuclear contents to about six timcs the initial native volume of the untreated headpiece. The resulting coherent expanded, gel-like, sperm nuclear contents remain in this metastable state until slow disorganization of their structure and dissolution occur. The unusually stepwise nature of the P-mercaptoethanol induced decondensation of Eutherian sperm nuclei suggests that this agent may trigger natural decondensation mechanisms operative within fertilized ova. Within the intact headpieces of Eutherian spermatozoon cells the chromatin contents exist in a close-packed, lamellar structure [ 171. Although no direct molecular measurements have been made on sperm chromatin in this native state, extensive studies of the molecular organization of chromatin fibers within the first stage of P-mercaptoethanol induced decondensation have recently been carried out in our laboratory [ 2 . 3 ] . These studies were carried out on sperm chromatin gels prepared from the 6x volume decondensed sperm headpieces as shown in Fig. 1
FIGURE 4. The polyacrylamide gel electrophoretic separation of the products of a 30 sec staphylacoccal nuclease digestion of solubilized bovine sperm chromatin. The gel has been purposely overloaded to show the absence of any significant products other than the major band near the upper portion of the gel.
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(lower left). A variety of physical probes were used to determine that the chromatin contents of these decondensed nuclei are organized into an ordered cholesteric liquid crystalline array with discrete measured parameters [2,3]. The fluorescence microscopy indicates that this decondensation state, induced under the same conditions used to produce the gels previously studied [2,3], is characterized by nuclear chromosomal contents swollen to 6x the original volume of the native headpiece. This result supports the hypothesis [2] that the liquid crystalline order found for the first decondensation stage of Eutherian sperm is the simple result of molecular rotations within the chromosomal fibers from a pure hexagonal crystalline order [18] existing within the intact headpiece. The theoretical volume change for this hexagonal crystalline to cholesteric liquid Crystalline phase change is 5.7 0.5. The microscopically measured volume change agrees well with this theoretical value. It seems likely that the nucleoprotein fibers within intact sperm cells exist in an ordered hexagonal polymer crystalline arrangement not unlike DNA crystals grown by Giannoni and coworkers [ 181. Apparently this structure is supported by disulfide cross-linkes [6] which are reductively cleaved by P-mercaptoethanol, triggering the phase transition and volume change into the less close packed, liquid crystalline arrangement already described [2,3]. Much of the present weight of evidence supporting the contemporary view of the structure of somatic chromatin has been provided by studies of the regionally selective nuclease digestion of isolated chromatin. This work is based upon the startling observation by Clark and Felsenfeld [19] that digestion of calf thymus chromatin degraded half and only half of the sequences of the DNA component of the chromatin leaving 50% of the DNA undigested. This observation was followed by polyacrylamide gel electrophoretic separation of the undigested nucleoprotein regions and the DNA derived therefrom [ 14,161 which indicated that a discrete nucleoprotein particle remained undigested containing DNA sequences somewhat less than 200 base-pairs in length. These results in conjunction with electron microscopic evidence [7,8] resulted in the “beads-on-a-string” model for chromatin where the DNA folds around histone complexes spaced fairly regularly along the chromosomal fiber. Other nuclease digestion studies of whole nuclei [20] yielded discrete 200 base-pair DNA sequences along with multiples of this 200 base-pair length and led to the flexibly jointed chain model. Both the “beads-on-a-string” model [21] and the 100 A diameter flexibly jointed chain model [20] receive support from electron microscopic observations. Recently, Sollner-Webb and Felsenfeld [22] have shown that both models are real situations at two different stages of disorganization. When examined after positive staining, solubilized sperm chromatin revealed regions of thick (100 A) fibers visually similar to those observed in somatic chromatin and attributed to the flexibly jointed chain model [20] as well as regions of thin “knobby” fibers (Fig. 2). Frequently, views of the regions of thin fibers (Fig. 2) showed ellipsoidal particles about 80 A in diameters connected by thin filaments about 20 A wide, the width of B-form DNA. These views of the thin “knobby” fibers were remarkably similar to observations previously recorded for somatic chromatin [7] and used to support the “beads-on-a-string’’ model. Digestion of solubilized sperm chromatin with micrococcal nuclease degrades only -55% (Fig. 3) of the DNA content of this chromosomal preparation suggesting the presence of nuclease resistant
*
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T. Wagner, J. Sliwinski, and 8. Shewmaker
structures within sperm chromatin like those found in the somatic material [14]. Polyacrylamide gel electrophoretic separation of the undigested, nuclease resistant regions of sperm chromatin clearly shows the presence of a discrete nucleoprotein particle (Fig. 4). This “nucleosome” represents most of the nuclease resistant material in the sperm chromatin limit digest. Electron microscopic evidence combined with the limited extent of nuclease digestion (55%) and the characterization of a discrete nuclease resistant nucleosome particle as the major component of the limit digest together suggest a structure for eutherian sperm chromatin fibers. This structure appears to consist of a DNA molecule folded around sperm histone complexes spaced regularly along the chromosomal fiber in the same manner as has been previously observed in somatic systems [22]. Within the thicker (100 A) fiber also observed under the electron microscope the nucleosome particles in the extended “beads-on-a-string” structure appear to associate directly, “rolling up” the DNA between nucleosomes to form a more condensed flexibly jointed chain. These present studies combined with previous work [2,3] now indicate that decondensation of Eutherian sperm nuclei results from the expansion of an ordered lamellar hexagonal crystalline arrangement of flexibly jointed chains of sperm nucleosomes into a less dense liquid crystalline array [2,3] and finally into separate chains of jointed nucleosomes which may further stretch into fibers of nucleosomes connected by regions of DNA (Fig. 2). The nucleosome protein complement in somatic chromatin is composed of two molecules each of the four histones H2A, H2B, H3 and H4 as an octamer [ 131. Sperm chromatin contains only one basic, histone-like, protein [23] present at a ratio of 16 molecules per 200 base-pairs of DNA. Since the molecular weight of the sperm histone [23] is about half that of the somatic histones the mass of protein per 200 base-pair unit of DNA is about equal in both classes of chromatin. Therefore, the sperm nucleosome may consist of -16 sperm histone proteins associated into a unit structure which is tightly associated with the chromosomal DNA. This stud) was supported by a grant from the National Institute of Child Health and Human Development N o . HD 09042. The authors also wish to thank Dr. James Brazelton for his expert assistance and advice in the use of the transmission electron microscope. REFERENCES
1. Fawcett, D. W. (1958): The structure of
the mammalian spermatozoon. f n t Rct* Cvtol 7, 195-234. 2 . Sipski, M . L. and Wagner, T. E. (1977): The total structure and organization of chromosomal fibers in eutherian sperm nuclei. Biol Rrprod 16, 428-440. 3. Sipski. M . L. and Wagner, T. E. (1977): Probing DNA quaternary ordering with circular dichroism spectroscopy: Studies of equine sperm chromosomal fibers. Biop o l y m c r s 16, 573-582.
4. Daoust, R . and Clermont, Y . (1955): Distribution of nucleic acids in germ cells during the cycle of the seminiferous epithelium in the rat. Arner J Anat %, 225283. 5 . Peacock, A . C. and Dingman, W. C. ( 1967): Resolution of multiple reibonucleic acid species by polyarcylamide gel electrophoresis. Biochern 6, 1818-1827. 6. Calvin, H . I. and Bedford, J . M. (1971): Formation of disulfide bonds in the nucleus and accessory structures of mam-
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malian spermatozoa during maturation in the epididymis. J Reprod Fertil Suppl 13, 65-75. 7. Olins, A. L. and Olins, D. E. (1974): Spheroid Chromatin units ( u bodies). Science 183, 330-332. 8. Kiryanov, G. I., Manamshjan, U. Yu., Polyakov, D. F., and Chentsov, Ju. S. (1976): Levels of granular organization of chromatin fibers. FEBS Let 67, 323-327. 9. Wagner, T. E. (1977): Unpublished data. 10. Kornberg, R. D. (1974): Chromatin structure: a unit of histones and DNA. Science 184, 868-871. 11. Van Holde, K. E., Sahasrabuddhe, C. G. and Shaw, B. R., (1974): A model for particulate structure in chromatin. Nuclei Acids Res 11, 1579- 1585, 12. Axel, R., Melchoir, W., Sollner-Webb, B. and Felsenfeld, G. (1974): Specific sites of interaction between histones and DNA in chromatin. Proc Nut Acad Sci 71, 410141 12. 13. Elgin, S. C. R. and Weinbraub, H. (1975): Chromosomal proteins and chromatin structure. Ann Rev Biochem 44, 725-774. 14. Greil, W., Igo-Kemenes, T. and Zachau, H. G. (1976): Nuclease digestion in between and within nucleosomes. Nucleic Acid Res 3, 2633-2643. 15. Sollner-Webb, B. (1976): The organization of histones in nuclei and chromatin. Ph.D.
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Thesis, Stanford University, USA. 16. Bakayev, V. V., Bakayeva, T. G. and Varshavsky, A. J., (1977): Nucleosomes and subnucleosomes: Hetero-geneity and composition. Cell 11, 619-629. 17. Lung, B. (1968): Whole-mount electron micrscopy of chromatin and membranes in bull and human sperm head. J Ultrastr Res 22, 485-493. 18. Giannoni, G., Padden, F. J., and Keith, H. D. (1969): Crystallization of DNA from Dilute Solution. Proc Nut Acad Aci USA 62, 964-971. 19. Clark, R. J. and Felsenfeld, G. (1971): Structure of chromatin. Nature 229, 107109. 20. Noll, M. (1974): Subunit structure of chromatin. Nature 251, 249-25 1. 21. Senior, M. B., Olins, A. L. and Olins, D. E. (1975): Chromatin fragments resembling u bodies. Science 187, 173-176. 22. Sollner-Webb, B. and Felsenfeld, G. (1975). A comparison of the digestion of nuclei and chromatin by staphyloccal nuclease. Biochem 14, 2915-2920. 23. Coelingh, J. P., Monfoort, C. H., Rozijn, T. H., Gevers Leuven, J. A., Schiphof, R., Steyn-Parve, E. P., Braunitzer, G., Schrank, B., and Ruhfus, A. (1972): The complete amino acid sequence of the basic nuclear protein of bull spermatozoa. Biochem Biophys Acta 285, 1- 14.
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T. E. WAGNER,J. E. SLIWINSKI, and D. B. SHEWMAKER The P-mercaptoethanol induced decondensation of spermatozoon cell nuclei from several Eutherian species has been followed from the intact spermatazoon cell to the solubilized linear unit sperm chromosomal fiber using fluorescence and electron microscopy. Data from nuclease digestion studies in conjunction with electron microscopic evidence indicate that the gross structure of the unit Eutherian sperm chromosomal fiber consists of DNA folded around sperm specific histone multimers spaced regularly along the fiber generating a linear array of sperm nucleosomes connected by short streLches of uncomplexed DNA. The sperm nucleosomes, 80 A in diameter are separated by 20 A filaments of DNA. This structure is remarkably similar to the structure of somatic chromatin although the protein components of the two chromosomes are markedly different. It seems likely that chromosomal fibers, similar to those described herein, may be present in the male pronucleus following fertilization.
Key Words: Sperm chromatin; Subunit structure; Sperm nucleosomes.
INTRODUCTION
The nucleus of the spermatozoon of Eutherian mammals contains the male chromosomal complement in a densely packed, protected state for release to the ovum during fertilization. Although many of the ultrastructural details of chromosomal packaging during spermatogenesis have been observed [ 11 and a recent molecular description of the polymer crystalline organization of the chromatin within mature Eutherian sperm nuclei presented [2,3], little is known about the mechanism of sperm nuclear decondensation following entrance of the sperm headpiece into the ovum, or about the molecular structure of the chromosomal component of the male pronucleus. Because the mature sperm chromosomes are packaged in an inaccessible state [4] within the sperm nucleus and released only after entry into the ovum, there is no period during the natural lifetime of the Eutherian sperm cell when its chromosomal contents can be isolated and studied. Therefore, we have developed a mild procedure to effect the release of sperm chromatin from mature sperm cells. This procedure allows the study of molecular aspects of the decondensation, providing insight into the find events during fertilization. In vitro release also facilitates the purification of highly native chromosomal material which may be structurally very similar to the chromosomal component of the male pronucleus. The induced decondensation of bovine, ovine, equine and human spermatazoon cells have been followed with optical and electron microscopy. The tertiary structure Received August 1977. From the Department of Chemistry, Ohio University, Athens, Ohio. Address reprint requests to Ohio University, Department of Chemistry, Athens, Ohio 4.5701 (T. E. Wagner). @ ARCHIVES OF ANDROLOGY I (January 1978): 3 1 4 1
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of the resulting decondensed chromatin was analyzed using enzymatic and chemical techniques.
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MATERIALS AND METHODS Semen samples were collected from rams, bulls and stallions using an artificial vagina and maintained in extender at liquid N, temperatures. Human semen samples were extended and also maintained at liquid N, temperatures. The viability and functional ability of the animal semen was determined by results of artificial insemination. Normal, live offspring were delivered. Sperm cells were washed into the buffer required for each experiment by sedimentation (1 .SO0 g) and resuspension in the buffer. Incidenr,fuoresc encv microscopy. Sperm cells were incubated at pH 8.5, 0.1M Tris-HC1, in the presence of 10-3M P-mercaptoethanol (equine) or 2 x 10-*M p-mercaptoethanol (bovine, ovine, human) at 4°C. At timed intervals, samples were taken, made 0.1 pgiml in ethidium bromide and observed under incident fluorescence microscopy. Light microscopy was carried out using a No. 100 microscope illuminator (Zeiss) with a 13V, 100 W halogen source. Incident fluorescence microscopy was accomplished using this illuminator in combination with phase contrast optics. Transmission electron microscopy. All samples were spread on grids for electron microscopy. Carbon-coated. freshly glow-discharged, Formvar coated grids were used. To these prepared grids were added 7.5 p1 samples. The samples were placed directly onto the grids, allowed to stand for 2-5 min and then removed by aspiration. The grids were rinsed three times with quartz doubly-distilled H,O by adding and removing 7.5 ~1 aliquots. Positive staining was accomplished with 7.5 ml of uranyl acetate (200 pl of a filtered 5% uranyl acetate solution and 2 ~1 of conc HC1 added to 20 ml of 90% ethanol), followed by rinsing with three volumes of quartz double-distilled H,O after 5 min of staining. The prepared grids were examined in a Hitachi H58 electron microscope at SO KV and photographed at a magnitication of 47,000. Photographic prints were enlarged to a magnification of x 277,000. Pwparation of purified sperm chromatin. Intact, active sperm cells were washed into 0.05M Tris buffer, pH 8.8. The buffer volume used was equal to the volume of the pelleted cells used in each preparation. The resulting suspension was made 5 x 10-*M in P-mercaptoethanol and hand homogenized using a glass-teflon homogenizer prior to incubation at 4°C for 48 hr or until the resulting solution thickened to a viscous gel. The resulting thick suspension, containing dissolved sperm chromatin, some yet uncondensed chromatin, sperm membranes and intact sperm tails, was layered above a solution 1.7M in sucrose and 0.001M in Tris buffer pH 8.0 and centrifuged at 27.000 rpm in an SW-40 rotor for two hr. After centrifugation, most of the sperm membranes and all tails were at the bottom of the centrifuge tube while the soluble sperm chromatin was collected as a transparent thin gel just above the sucrosehuffer boundary. The solubilized chromatin was carefully removed from above the sucrose boundary so as not to contaminate it with the membranous material which also collects within and just below the boundary. The resulting sperm chromatin was dialyzed against 0.001M Tris buffer (pH 8.0) and washed by sedimentation at 15,000 rpm in a Sorval SS-34 head. LVideasi>digestion. Staphylococcal nuclease (micrococcal nuclease) was obtained from Sigma Chemical Company at 6,000 or 10,000 U/mg. A stock solution at 2 mg/ml in H,O was stored at 4" for up to several weeks. Digestions of sperm chromatin were performed in 1 mM Tris-HC1
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(pH 8.0), 0.1 mM CaCI, at 37" C by adding 1/10 to the volume of 1 mM CaCl, and the enzyme to the chromatin solution, which ranged from 0.05 to 3 mg DNA/ml. The nuclease rendered an amount of DNA approximately equal to its own weight acid soluble each minute. The digestion was terminated by the addition of a 10-fold excess of ethylene diamine tetraacetic acid (EDTA) over the Ca++concentration in the digestion mixture and cooling the reaction at 0°C in ice. The amount of digestion resulting from nuclease attack on sperm chromatin was measured as the fraction rendered acid soluble. 100 to 200 p g DNA aliquots of a stopped, freshly suspended nuclease reaction were added to 20 p g bovine plasma albumen (BPA, kept at a 2 mgiml solution) in a centrifuge tube, 0.4 ml of 2M NaCl, 2M perchlonc acid added and the mixture brought to a volume of 1 ml with H,O. After 10 min at O'C, the mixture was centrifuged at 10,OOO x g for 10 min and the of the supernatant measured. (% acid soluble) =
acid soluble A,,, x 0.6 undigested A260 of aliquot
Nucleoprotein polyacrylamide gels. Polyacrylamide gels to separate nucleoprotein components
were run in a slab gel apparatus (Aqueboque Corp.). The gels were run essentially as described by Peacock and Dingman [5] except that the buffer concentration was maintained at 1/10 their concentration to increase the mobility of the weakly charged nucleoprotein material. All separations described were carried out using 8% polyacrylamide gels containing 0.4% bisacrylamide crosslinker. Gels were used after gelling for periods greater than 10 hr. Water cooled gels were pre-electrophoresced and run for 2 hr at 200 V with a running buffer of 1/10 Peacock-Dingman buffer. Samples (-50 pg DNA content in 20 pI) were loaded in 10% glycerol in the 1/10 Peacock-Dingman buffer. After electrophoresis the gels were stained in 0.1 pg/ml of ethidium bromide for 1 hr and photographed under ultraviolet light with an orange filter.
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RESULTS Incident Fluorescence Microscopy of Sperm Decondensation
In the presence of between and 10-'M P-mercaptoethanol equine, ovine, bovine and human spermatozoa slowly undergo a stepwise nuclear decondensation, swelling and dissolution. Since this decondensation might be similar to events following fertilization within the ovum we have carefully documented the P-mercaptoethanol induced decondensation. Because the swollen sperm headpieces appear transparent and difficult to observe using phase contrast microscopy, samples were stained with ethidium bromide (1 pg/ml) and observed under incident fluorescence microscopy. Of the four species of Eutherian mammals studied, equine sperm cells respond to lower concentrations of P-mercaptoethanol (10-3) resulting in slower and more easily observable decondensation. The ovine, bovine and human sperm cells require concentrations of P-mercaptoethanol as high as 10-'M to induce decondensation. Under these conditions the decondensation process, which is the same as observed with equine sperm, proceeds much more rapidly and does not allow detailed observation of all stages in the decondensation. In Fig. 1 (upper left) is shown the incident fluorescence micrograph of an ethidium stained equine sperm cell prior to P-mercaptoethanol treatment. The micrograph is characteristic of equine sperm cells studied in our laboratory having a head length of -7p and a head width of -4p with a thickness of slightly less than 1p. The internal
T. Wagner, J. Sliwinski, and B. Shewmaker
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FIGURE 1. Fluorescence micrographs of the stages of P-mercaptoethanol induced decondensation of equine spermatazoon nuclei. Upper Left, sperm nucleus prior to P-mercaptoethanol treatment: Upper Right, sperm nucleus at about 6-8 hr after P-mercaptoethanol treatment showing rupture of nuclear membranes; Lower Left, sperm nucleus at about 24 hr after treatment expanded to 6 x the volume of the untreated nucleus; Lower Right, sperm nucleus after 48 hr of exposure to P-mercaptoethanol expanded to greater than 10 X the volume of the untreated nucleus.
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volume of the headpiece of equine sperm cells where the head shape is approximated as an ellipsoidal cylinder with minor axis 2p, major axis 3.5p,and height lp is -45 X lO-lEm3. After approximately 24 hr at pH 8.6 (0.01M Tris buffer) in the presence of 10-3M P-mercaptoethanol, the ellipsoidal cylindrical shape of the equine sperm head swells into an ellipsoid of revolution and bursts its outer membrane structure. Rupture of the headpiece membrane always occurs on an equatorial belt about one-third the length of the headpiece from the point of midpiece attachment. (Fig. 1) Following membrane rupture, the internal contents of the headpiece continue to swell as a unit into a metastable swollen ellipsoidal structure with an internal volume close to 6 times the volume of the headpieces of untreated equine sperm (2.5-3.0 x 10-16m3). Through the light microscope the p-mercaptoethanol induced condensation of sperm nuclei appears as a three-step process: membrane rupture, 6x swelling and slow dissolution. This decondensation is solely caused by p-mercaptoethanol at pH 8.6 and not a result of the prior history of the sperm samples since we have maintained the cells live in liq N, and inseminated mares, ewes and cows with the cells from the same batches used in these experiments and delivered healthy, live offspring. Electron Microscopy of Dissolved Sperm Nuclei
Incubation at 4”C,pH 8.6, in the presence of P-mercaptoethanol for periods in excess of 48 hrs results in apparent dissolution of the sperm head of all species. After this period of time light microscopy is no longer useful as a tool to study the continuing decondensation process. Initial observation of unpurified samples showed primarily a clumped, three-dimensional fibrillar network of high electron density. Although the contents of the sperm headpieces appear completely dissolved under the light microscope they remain extensively crosslinked, probably due to remaining disulfide linkages [6]. In order to prepare samples containing individual fibers disassociated from each other for electron microscopic study, the post 48 hr treated sperm preparations were made 2 X 10-’M in p-mercaptoethanol and hand-homogenized several times over a period of 24 to 48 additional hr. This preparation was then filtered through 0.1 p nucleopore membranes to remove any remaining aggregated material (e.g. , high molecular weight DNA or chromatin fibrils will pass length-wise through nucleopore membranes with pore sizes greater than the diameter of the fiber). Analysis of grids containing samples prepared in this manner showed two distinct types of fibers: long twisted fibers with a diameter of about 100 A and stretches of fibers with repeating knobby stpctures resembling beads on a string wheFe the “string” has a diameter of 15-20 A and the “bead” has a diameter of about 85 A (Fig. 2). Both the 100 h; filament and the repeating knobby fibers are markedly similar to electron micrographs of somatic chromatin [7,8].These structures are also virtually identical to somatic core chromatin from which all chromosomal protein except the arginine rich histones H3 and H4 have been removed [9].Kornberg [ 101 studying calf thymus chromatin suggests that the 100 h; fiber consists of a flexibly jointed chain of 100 A repeating units that are in contact and contain 290 base pairs of DNA, giving a
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T. Wagner, J. Sliwinski, and 8. Shewmaker
packing ratio of about 7: 1. The “beads-on-a-string” structure observed in the electron microscope for purified somatic chromatin is usually described as a linear array of subunits (80 b;) containing 110-120 base pairs of DNA connected by extended, B-form DNA about 240 A (70-80 base pairs) in length, giving a packing ratio of 2: 1 [ l l , 12, 131.
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Micrococcal Nuclease Digestion of Bovine Sperm Chromatin Bovine sperm cells were treated with 2 X 10-2M P-mercaptoethanol at pH 8.6 for -48 hr with intermittent hand homogenization until a thick, viscous mixture was formed. This crude sperm chromatin preparation was then purified on a sucrose gradient. The purified chromatin recovered from the upper shelf of the gradient displayed structures under electron microscopy and appeared to be completely disaggregated (Fig. 2). This material was then dialyzed into 1 mM Tris (pH 8.0), equilibrated at 37”C, made 1 mM in Ca++ and digestion initiated by addition of 1/30th the nucleotide sample mass of staphylococcal nuclease (Sigma). The kinetics of the nuclease digestion of the chromatin sample were studied by removing aliquots of the digestion mixture at timed intervals, quenching the digestion with a 10-fold excess of EDTA over initial Ca++ concentration, and precipitating the undigested DNA with perchloric acid. The perchloric acid soluble digestion products from degraded DNA were then assayed spectrophotometrically to determine the per cent digestion as a function of the time of digestion. Rapid degradation of the sperm chromosomal DNA by micrococcal nuclease took place during the first 15-30 seconds of the reaction followed by a slower digestion of
FIGURE 2. Left, Electron micrograph of positively stained solubilized bovine sperm chromatin showing the 100 A thick fiber structure; Right, Electron micrograph of positively stained solubliized bovine sperm chromatin showing the thin “knobby” fiber structure. ( x 138,500).
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the substrate to a limit of about 55% of the total substrate DNA (Fig. 3). The results of nuclease digestion are consistent with a structure for bovine sperm chromatin which contains nucleoprotein regions resistant to nuclease digestion and free DNA regions which are markedly susceptible to nuclease digestion. This structural characteristic is consistent with the “beads-on-a-string’’ structure observed under the electron microscope (Fig. 2). Nucleoprotein Gel Electrophoresis of Nuclease Digestion Products of Bovine Sperm Chromatin
Electron microscopy and micrococcal nuclease digestion kinetics (Fig. 3) of purified, solubilized, sperm chromatin suggest a subunit structure for this chromosomal material. From the kinetics of somatic nuclear digestion with staphylococcal nuclease it is apparent that little of the DNA becomes acid soluble when monomer subunit is excised and that most of the starting chromosomal DNA passes through monomer subunit [14,151. The highest concentration of intact subunit monomers in a staphylococcal nuclease digestion of chromatin would be present very early in the digestion. In the case of sperm chromatin, where the initial digestion rate is enhanced, subunit monomer would be most likely present at its highest concentration at about 15-30 sec of digestion. Therefore, a 30-second digestion of bovine sperm chromatin was carried out followed by EDTA quenching and cooling to 0°C. The quenched digestion mixture, containing -0.7 mg/ml of DNA, was maintained at 0°C for several hours until a precipitate appeared. The induced precipitate was redissolved by addition of excess EDTA and warming into a small volume of electrophoresis buffer. Spectrophotometric analysis of this concentrated nucleoprotein solution showed that it contained over 80% of the total initial chromosomal DNA from the digestion mixture. Thus, brief micrococcal nuclease digestion of bovine sperm chromatin cleaves this high molecular weight nucleoprotein polymer to yield insoluble, nuclease-resistant nucleoprotein material. The redissolved nucleoprotein pellet from the 30 sec digestion of
I d
5
10
15
20
25
30
35
40
DIGESTION TIME (MIN.)
FIGURE 3. The kinetics of staphylacoccal nuclease digestion of solubilized bovine sperm chromatin. The amount of digestion was minitored by acid solubility.
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T. Wagner, J. Sliwinski, and
B. Shewmaker
bovine sperm chromatin with staphylococcal nuclease (50 pg) was placed in the sample well of a 7% polyacylamide gel and subjected to a potential of 200 volts for 3 hr using .095MTris-borate buffer at pH 8 . 3 . After electrophoresis the gel was stained with ethidium bromide and photographed under ultraviolet illumination (Fig. 4). This gel electrophoretic separation reveals some high molecular weight nucleoprotein which has not penetrated the gel, a discrete nucleoprotein band about 1.5 cm into the gel and some lower molecular weight digestion products which have smeared along the lower portion of the gel. The presence of a discrete band in the nucleoprotein gel electrophoresis of the digestion products from sperm chromatin treated with micrococcal nuclease strongly supports the existence of a nuclease resistant subunit within sperm chromatin which may be, or be related to. the knobby repeating structures observed in sperm chromatin preparations under the electron microscope (Fig. 2 ) . DISCUSSION
Fluorescence microscopy of several eutherian species of spermatazoa in the presence of P-mercaptoethanol shows the initial gross stages of sperm chromatin decondensation induced by this agent. The nuclear regions of the sperm cells are seen to expand causing rupture of the outer membrane sheath within a discrete equatorial region (-113 the linear distance from the point of midpiece attachment to the opposite end of the headpiece) followed by further expansion of the nuclear contents to about six timcs the initial native volume of the untreated headpiece. The resulting coherent expanded, gel-like, sperm nuclear contents remain in this metastable state until slow disorganization of their structure and dissolution occur. The unusually stepwise nature of the P-mercaptoethanol induced decondensation of Eutherian sperm nuclei suggests that this agent may trigger natural decondensation mechanisms operative within fertilized ova. Within the intact headpieces of Eutherian spermatozoon cells the chromatin contents exist in a close-packed, lamellar structure [ 171. Although no direct molecular measurements have been made on sperm chromatin in this native state, extensive studies of the molecular organization of chromatin fibers within the first stage of P-mercaptoethanol induced decondensation have recently been carried out in our laboratory [ 2 . 3 ] . These studies were carried out on sperm chromatin gels prepared from the 6x volume decondensed sperm headpieces as shown in Fig. 1
FIGURE 4. The polyacrylamide gel electrophoretic separation of the products of a 30 sec staphylacoccal nuclease digestion of solubilized bovine sperm chromatin. The gel has been purposely overloaded to show the absence of any significant products other than the major band near the upper portion of the gel.
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(lower left). A variety of physical probes were used to determine that the chromatin contents of these decondensed nuclei are organized into an ordered cholesteric liquid crystalline array with discrete measured parameters [2,3]. The fluorescence microscopy indicates that this decondensation state, induced under the same conditions used to produce the gels previously studied [2,3], is characterized by nuclear chromosomal contents swollen to 6x the original volume of the native headpiece. This result supports the hypothesis [2] that the liquid crystalline order found for the first decondensation stage of Eutherian sperm is the simple result of molecular rotations within the chromosomal fibers from a pure hexagonal crystalline order [18] existing within the intact headpiece. The theoretical volume change for this hexagonal crystalline to cholesteric liquid Crystalline phase change is 5.7 0.5. The microscopically measured volume change agrees well with this theoretical value. It seems likely that the nucleoprotein fibers within intact sperm cells exist in an ordered hexagonal polymer crystalline arrangement not unlike DNA crystals grown by Giannoni and coworkers [ 181. Apparently this structure is supported by disulfide cross-linkes [6] which are reductively cleaved by P-mercaptoethanol, triggering the phase transition and volume change into the less close packed, liquid crystalline arrangement already described [2,3]. Much of the present weight of evidence supporting the contemporary view of the structure of somatic chromatin has been provided by studies of the regionally selective nuclease digestion of isolated chromatin. This work is based upon the startling observation by Clark and Felsenfeld [19] that digestion of calf thymus chromatin degraded half and only half of the sequences of the DNA component of the chromatin leaving 50% of the DNA undigested. This observation was followed by polyacrylamide gel electrophoretic separation of the undigested nucleoprotein regions and the DNA derived therefrom [ 14,161 which indicated that a discrete nucleoprotein particle remained undigested containing DNA sequences somewhat less than 200 base-pairs in length. These results in conjunction with electron microscopic evidence [7,8] resulted in the “beads-on-a-string” model for chromatin where the DNA folds around histone complexes spaced fairly regularly along the chromosomal fiber. Other nuclease digestion studies of whole nuclei [20] yielded discrete 200 base-pair DNA sequences along with multiples of this 200 base-pair length and led to the flexibly jointed chain model. Both the “beads-on-a-string” model [21] and the 100 A diameter flexibly jointed chain model [20] receive support from electron microscopic observations. Recently, Sollner-Webb and Felsenfeld [22] have shown that both models are real situations at two different stages of disorganization. When examined after positive staining, solubilized sperm chromatin revealed regions of thick (100 A) fibers visually similar to those observed in somatic chromatin and attributed to the flexibly jointed chain model [20] as well as regions of thin “knobby” fibers (Fig. 2). Frequently, views of the regions of thin fibers (Fig. 2) showed ellipsoidal particles about 80 A in diameters connected by thin filaments about 20 A wide, the width of B-form DNA. These views of the thin “knobby” fibers were remarkably similar to observations previously recorded for somatic chromatin [7] and used to support the “beads-on-a-string’’ model. Digestion of solubilized sperm chromatin with micrococcal nuclease degrades only -55% (Fig. 3) of the DNA content of this chromosomal preparation suggesting the presence of nuclease resistant
*
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T. Wagner, J. Sliwinski, and 8. Shewmaker
structures within sperm chromatin like those found in the somatic material [14]. Polyacrylamide gel electrophoretic separation of the undigested, nuclease resistant regions of sperm chromatin clearly shows the presence of a discrete nucleoprotein particle (Fig. 4). This “nucleosome” represents most of the nuclease resistant material in the sperm chromatin limit digest. Electron microscopic evidence combined with the limited extent of nuclease digestion (55%) and the characterization of a discrete nuclease resistant nucleosome particle as the major component of the limit digest together suggest a structure for eutherian sperm chromatin fibers. This structure appears to consist of a DNA molecule folded around sperm histone complexes spaced regularly along the chromosomal fiber in the same manner as has been previously observed in somatic systems [22]. Within the thicker (100 A) fiber also observed under the electron microscope the nucleosome particles in the extended “beads-on-a-string” structure appear to associate directly, “rolling up” the DNA between nucleosomes to form a more condensed flexibly jointed chain. These present studies combined with previous work [2,3] now indicate that decondensation of Eutherian sperm nuclei results from the expansion of an ordered lamellar hexagonal crystalline arrangement of flexibly jointed chains of sperm nucleosomes into a less dense liquid crystalline array [2,3] and finally into separate chains of jointed nucleosomes which may further stretch into fibers of nucleosomes connected by regions of DNA (Fig. 2). The nucleosome protein complement in somatic chromatin is composed of two molecules each of the four histones H2A, H2B, H3 and H4 as an octamer [ 131. Sperm chromatin contains only one basic, histone-like, protein [23] present at a ratio of 16 molecules per 200 base-pairs of DNA. Since the molecular weight of the sperm histone [23] is about half that of the somatic histones the mass of protein per 200 base-pair unit of DNA is about equal in both classes of chromatin. Therefore, the sperm nucleosome may consist of -16 sperm histone proteins associated into a unit structure which is tightly associated with the chromosomal DNA. This stud) was supported by a grant from the National Institute of Child Health and Human Development N o . HD 09042. The authors also wish to thank Dr. James Brazelton for his expert assistance and advice in the use of the transmission electron microscope. REFERENCES
1. Fawcett, D. W. (1958): The structure of
the mammalian spermatozoon. f n t Rct* Cvtol 7, 195-234. 2 . Sipski, M . L. and Wagner, T. E. (1977): The total structure and organization of chromosomal fibers in eutherian sperm nuclei. Biol Rrprod 16, 428-440. 3. Sipski. M . L. and Wagner, T. E. (1977): Probing DNA quaternary ordering with circular dichroism spectroscopy: Studies of equine sperm chromosomal fibers. Biop o l y m c r s 16, 573-582.
4. Daoust, R . and Clermont, Y . (1955): Distribution of nucleic acids in germ cells during the cycle of the seminiferous epithelium in the rat. Arner J Anat %, 225283. 5 . Peacock, A . C. and Dingman, W. C. ( 1967): Resolution of multiple reibonucleic acid species by polyarcylamide gel electrophoresis. Biochern 6, 1818-1827. 6. Calvin, H . I. and Bedford, J . M. (1971): Formation of disulfide bonds in the nucleus and accessory structures of mam-
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malian spermatozoa during maturation in the epididymis. J Reprod Fertil Suppl 13, 65-75. 7. Olins, A. L. and Olins, D. E. (1974): Spheroid Chromatin units ( u bodies). Science 183, 330-332. 8. Kiryanov, G. I., Manamshjan, U. Yu., Polyakov, D. F., and Chentsov, Ju. S. (1976): Levels of granular organization of chromatin fibers. FEBS Let 67, 323-327. 9. Wagner, T. E. (1977): Unpublished data. 10. Kornberg, R. D. (1974): Chromatin structure: a unit of histones and DNA. Science 184, 868-871. 11. Van Holde, K. E., Sahasrabuddhe, C. G. and Shaw, B. R., (1974): A model for particulate structure in chromatin. Nuclei Acids Res 11, 1579- 1585, 12. Axel, R., Melchoir, W., Sollner-Webb, B. and Felsenfeld, G. (1974): Specific sites of interaction between histones and DNA in chromatin. Proc Nut Acad Sci 71, 410141 12. 13. Elgin, S. C. R. and Weinbraub, H. (1975): Chromosomal proteins and chromatin structure. Ann Rev Biochem 44, 725-774. 14. Greil, W., Igo-Kemenes, T. and Zachau, H. G. (1976): Nuclease digestion in between and within nucleosomes. Nucleic Acid Res 3, 2633-2643. 15. Sollner-Webb, B. (1976): The organization of histones in nuclei and chromatin. Ph.D.
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Thesis, Stanford University, USA. 16. Bakayev, V. V., Bakayeva, T. G. and Varshavsky, A. J., (1977): Nucleosomes and subnucleosomes: Hetero-geneity and composition. Cell 11, 619-629. 17. Lung, B. (1968): Whole-mount electron micrscopy of chromatin and membranes in bull and human sperm head. J Ultrastr Res 22, 485-493. 18. Giannoni, G., Padden, F. J., and Keith, H. D. (1969): Crystallization of DNA from Dilute Solution. Proc Nut Acad Aci USA 62, 964-971. 19. Clark, R. J. and Felsenfeld, G. (1971): Structure of chromatin. Nature 229, 107109. 20. Noll, M. (1974): Subunit structure of chromatin. Nature 251, 249-25 1. 21. Senior, M. B., Olins, A. L. and Olins, D. E. (1975): Chromatin fragments resembling u bodies. Science 187, 173-176. 22. Sollner-Webb, B. and Felsenfeld, G. (1975). A comparison of the digestion of nuclei and chromatin by staphyloccal nuclease. Biochem 14, 2915-2920. 23. Coelingh, J. P., Monfoort, C. H., Rozijn, T. H., Gevers Leuven, J. A., Schiphof, R., Steyn-Parve, E. P., Braunitzer, G., Schrank, B., and Ruhfus, A. (1972): The complete amino acid sequence of the basic nuclear protein of bull spermatozoa. Biochem Biophys Acta 285, 1- 14.
