Journal of Structural Biology 134, 76 81 (2001) doi:10.1006/jsbi.2001.4365, available online at http://www.idealibrary.

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DNA in Human and Stallion Spermatozoa Forms Local Hexagonal Packing with Twist and Many Defects
Nathalie Sartori Blanc,* Alfred Senn, Amelie Leforestier, Francoise Livolant, and Jacques Dubochet*
*Laboratoire dAnalyse ultrastructurale, Batiment Biologie, Universite de Lausanne, CH-1015 Lausanne, Switzerland; Reproductive Medicine Unit, Department of Obstetrics and Gynecology, CHUV, CH-1011 Lausanne, Switzerland; and Laboratoire de Physique des Solides, Batiment 510, Universite Paris Sud, 91405 Orsay Cedex, France Received November 30, 2000, and in revised form April 30, 2001

In human and other mammal sperm nuclei, DNA is packed in a highly condensed state, the structure of which remains unsolved. Cryoelectron microscopy of vitried sections provides a rst direct view of the local arrangement of the nucleoprotamine lament. DNA aligns in parallel in layers and its orientation rotates along a single-twist direction as in a cholesteric liquid crystal. The structure contains numerous defects, which introduce locally double-twist congurations. Destruction of the SS bonds with dithiotrehitol relaxes the twist and favors the extension of the hexagonal close packing of the laments, though keeping constant their interlament distance. 2001 Academic Press Key Words: cholesteric liquid crystal; cryoelectron microscopy; cryo-section; spermatozoid; vitrication.

INTRODUCTION

Human sperm cells have the shape of a tadpole with a attened head. The DNA is packed mainly with protamine, a minor portion of DNA that is associated with histones (Gatewood et al., 1987). As in other mammals, a global orientation of the DNA is revealed by the birefringence properties of the head and the layered structure revealed by freeze fracture electron microscopy (Koehler, 1966, 1972). In stallion sperm, it has been demonstrated that the orientation of the lament is twisting along an axis parallel to the small dimension of the head (Livolant, 1984). In other species, X-ray diffraction showed that the average distance between the laments is about 2.7 to 2.8 nm (Feughelman et al., 1955; Luzzati and Nicolaieff, 1959). Electron microscopy of plastic-embedded samples is of no great help in elucidating the organization of the DNA in spermatozoid heads. In most cases, it shows only a heavily stained substance, sometimes with large cavities. Obviously, the ne structure of the mate1047-8477/01 $35.00 Copyright 2001 by Academic Press All rights of reproduction in any form reserved.

rial does not resist chemical xation and dehydration. Cryoelectron microscopy of vitreous sections gives a better view. The method consists of cooling a small biological specimen in its native state, so rapidly that water is immobilized before it has time to crystallize. The vitreous specimen is then cut at ca. 160C into thin sections of ca. 80 nm thickness, which are observed in a cryoelectron microscope. Though the specimen is unstained and fully hydrated, experience has shown that the lack of contrast is not a limitation; even a single DNA molecule is quite visible when oating in a thin layer of vitreous solution (Dubochet et al., 1994). The limitation of the method comes rst from the technique itself; though it has been nearly 20 years since the rst vitreous sections were obtained, the progress of recent years in high-pressure freezing, cryo-cutting, and observation has been decisive for reproducible high-resolution observation of cells and tissues. The other limitation of the method comes from the complexity of the specimen. In the present case, we are observing laments tightly packed at about 2.7-nm distance in an 80-nm-thick section; this is enough for more than 30 laments to superimpose. Under these conditions, individual laments are visible only in places where they are parallel to the observation direction and straight over a good part of the section thickness. Despite this complication, images can be interpreted by comparing human spermatozoids with stallion spermatozoids, the structure of which is partially known, and with the simpler structure of decondensed spermatozoids. The possibility of determining complex 3D biological structures from observations made on vitreous sections will be greatly improved when combined with powerful tomographic methods (Nicastro et al., 2000).
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electron microscope (Philips CM12, Eindhoven, The Netherlands) at 80 kV, 45 000 magnication, and about 600-nm defocus. Photographs were taken with a minimum dose (about 300 e /nm2) and recorded on Kodak SO 163 lm. Negatives were scanned on a Kodak RFS 3570 professional lm scanner (Rochester, NY) with a 3072 2048 dpi resolution. The contrast of some images was enhanced with Adobe Photoshop F1-4.0. In some cases, ltering was performed with Digital Micrograph 2.5. RESULTS AND DISCUSSION

FIG. 1. Cryo-section of stallion sperm chromatin after partial decondensation with 10 mM DTT. (a,b) Bundles with a hexagonal closed-packed arrangement of DNA laments, checked by the diffractogram in c. (c) Hexagonal packing of the right-side up bundle in a presented in reverse contrast. (d,e) Double-twist bundles of laments seen just along the central axis. (f,g) Similar double-twist bundles seen slightly off-axis. Scale bar of optical diffraction pattern: (2.7 nm) 1.

MATERIAL AND METHODS Frozen stallion sperm was warmed to room temperature, washed twice in EBSSHepes buffer, and centrifuged at 300g for 10 min. The pellet was laid down on a 20% dextran solution (42 kDa, Sigma Chemical Co., St. Louis, MO) prepared in the same buffer or with 4 mM dithiotrehitol (DTT) added. After centrifugation (600g, for 10 min) the supernatant was removed and the compact pellet vitried in a high-pressure freezing apparatus (HPM 010, Balzers, Liechtenstein) (Sartori and Salamin Michel, 1994). For human, fresh ejaculated sperm was laid down in a 20% dextran solution and centrifuged (600g for 20 min). Most of the supernatant was removed and 1 ml of 20% dextran solution in TrisHCl (pH 8.0) was added. For the decondensed samples, 20 mM DTT was added to the buffer. Samples were then incubated at 37C for 15 min and then centrifuged at 600g for 20 min in order to obtain a very compact pellet, which was then vitried as described above. Cryo-sections were prepared with a cryo-ultramicrotome (Reichert Ultracut FCS, Leica, Vienna, Austria) at 160C (Sartori and Salamin Michel, 1994). Humidity (2730%) and temperature (20 22C) were controlled and an antistatic device (Haug, Bienne, Switzerland) was used to prevent the sections from sticking onto the knife. Care was taken to squeeze very strongly between two polished frozen metal surfaces cryo-sections on the 1500-mesh carbon-coated copper grids to avoid most of the drift under the electron beam. Section were then observed in a cryo-

Figure 1 shows bundles of laments obtained by partial DTT decondensation of stallion spermatozoids. For adequately oriented bundles, each single lament is visible. Two different arrangements are observed: hexagonal close packing (Figs. 1a and 1b) and double-twist bundles (Figs. 1d1g). The rst case is simple and sketched in Fig. 2a; at several places, single laments are quite visible in their hexagonal arrangement, and the diffractogram conrms the structure (Fig. 1c). The laments are spaced 2.7 nm apart. By comparison, hexagonal packing of pure DNA observed by the same method shows a distance of 2.3 nm (Richter and Dubochet, 1990). The double-twist bundle is a subtler structure. It forms when elongated molecules tend to associate at an angle rather than in parallel. The orientation of the molecules rotates regularly not only in one direction but in every radial direction from the central axis. The structure is known to exist in blue phase liquid crystals and there is evidence that it can also form with pure DNA (Leforestier and Livolant, 1994). In this case DNA molecules wrap helically around a central core with an angle increasing with the distance to the center (Fig. 2b). For geometrical and energetic reasons, such a local arrangement cannot extend far away (Wright and Mermin, 1989). Figures 1d and 1e show the

FIG. 2. Schematic drawing of a close-packed hexagonal ordering of laments (a) versus double-twist congurations (b) with their corresponding patterns in normal and oblique sections.

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FIG. 3. Cryo-section of human sperm chromatin after partial decondensation with 10 mM DTT. (a) Chromatin is homogeneously arranged in the nucleus and some local congurations can be recognized (insets). (b) Optical diffraction pattern of a: the average distance between DNA laments superimpose to the rst maximum of the transfer function. The wide and diffuse ring of the transfer function thus appears reinforced. (c, d, e) Higher magnication of insets in a, the congurations of which may be related to double-twist arrangements. (f,g) Double-twist arrangements from other regions. Scale bar of optical diffraction pattern: (2.7 nm) 1.

characteristic aspect of double-twist bundles seen just along the central axis. Bundles (Fig. 1f) and (Fig. 1g) are seen slightly off-axis. We insist on the fact that the two forms of bundles shown in Fig. 1 do not correspond to native spermatozoids but are the consequence of a rearrangement of nucleoprotamine bers. In human sperm cells treated with DTT, decondensation of chromatin did not reach this extent; chromatin kept a homogeneous density. Nevertheless, similar double-twist congurations were also observed locally (Fig. 3). Only molecules observed in the top view (or close to this orientation) can be recognized individually. Less dened regions are attributed to other orientations of the molecules, from oblique to parallel to the observation plane. As in the stallion sperm, the distance between the laments is about 2.7 nm. This characteristic distance is conrmed in the diffractogram (Fig. 3b).

Typical images of native stallion and human sperm cells are shown in Fig. 4. In human ejaculate, spermatozoids show a variety of shapes and DNA distributions (Chitale and Rathaur, 1995). In this population, we considered only those having a regular shape and in which chromatin is densely and homogeneously condensed. Vitreous sections of native stallion sperm rst show that the nucleus seems homogeneous. No large domains, thick laments, or grains that could be associated with nucleosomes are visible. At higher resolution, the majority of technically good images (thin, good section, correct focus, low electron dose) show no recognizable ordered structure. However, a minority estimated at 20% of the good sections show characteristic thin elongated domains striated perpendicularly to their long axis at 2.7-nm periodicity. The domains are only a few nanometers wide but extend over several tens of nanometers or more. Figure 4a shows such patterns. Since the structure is ne and may be difcult to visualize in print, some typical regions are shown enlarged in (Figs. 4d 4g). The same area is presented with reinforced contrast obtained with a ltered image in Fig. 4c (see legend to Fig. 4). The reality of the structure is conrmed in the diffractogram (Fig. 4b). The striated domains are irregularly spaced though an average distance of ca. 300 nm between the lines is typical (arrows in Fig. 4c). Due to the small size of the recorded surface (about 2 m2), it is generally not possible to determine its orientation in the sperm head. In few cases, when determination of orientation is possible, it is noted that the characteristic striated domains are visible only when the section plan is parallel to the short head axis, namely, when the head is seen as a very elongated structure, ca. 4 m wide. In the vicinity of the nuclear envelope, the long axis of the striated domains is approximately parallel to the envelope (not shown). The present observations are globally in agreement with the cholesteric model of Livolant (1984). Based on the optical activity of the sperm and on freeze-fracture replicas, it was shown that DNA is globally parallel to the large head plane and that its direction rotates along the small head axis with a half-turn period of ca. 33 nm. Vitreous sections complement these images. What we see in Figs. 4a and 4c and in all corroborating sections perpendicular to the major head plane are layers of parallel DNA laments, 2.7 nm apart, aligned approximately along the viewing direction. In between, DNA laments present other orientations, and, as expected, they cannot be resolved. The cholesteric model is thus conrmed. What was not considered before is that the cholesteric structure is remarkably irregular, thus demonstrating the presence of numerous

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FIG. 4. Cryo-sections of native stallion (a g) and human sperm cells (hn). (a,c) Original (a) and ltered (c) images of the same homogeneous chromatin region showing a characteristic layering point out by the series of arrows in c. Thin elongated domains are striated perpendicularly to their long axis with a characteristic 2.7-nm distance. This distance is given by the thin long spots in the corresponding diffraction pattern (b). To obtain the ltered image (c), a mask containing these two spots was applied and the corresponding inverse image calculated and added to the original image (a). (d g) Higher magnications of insets showing the striated domains. (hj) Elongated striated domains can also be recognized (arrows in the ltered image (j)) but they are less regularly distributed. A few regions (insets) are enlarged in kn. Some of them recall the above-mentioned double-twist congurations (n). Scale bar of optical diffraction pattern: (2.7 nm) 1.

defects. Their presence distorts the striated regions and restricts their extension, as schematically drawn in Fig. 5a. The structure of chromatin is the same in the human sperm cell. It has the same

homogeneity and the same periodic distribution of striated regions. These are nevertheless more difcult to recognize and to follow (Figs. 4h 4n). We suspect the density of defects to be even higher.

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FIG. 5. (a) Schematic representation of a cholesteric structure in a perfect planar conguration (on the left) and with numerous defects (on the right). Points and lines represent laments respectively normal and parallel to the observation plane. In between, nails correspond to oblique orientations. The tip of the nail indicates the extremity of the lament pointing to the observer (here, the cholesteric structure is supposed to be left-handed). The period corresponds to half the helical pitch P/2. Note that the core of some defects may remind us of double-twist congurations (enlarged region). (a ) With this geometry, only regions where laments are nearly normal to the cryo-section can be resolved. As drawn here, an obliquity as small as 1 will lead to the formation of striated domains, due to the ratio of the interhelix distance d (2.7 nm) to the section thickness (80 nm). (b) Proposed local 3D structure of nucleolaments in intact sperm cells. With a pitch P/2 of about 30 nm and a nucleolament interdistance d 2.7 nm, the twist angle would be discrete (16.3 on average) between domains extending laterally and corresponding either to monoor multilayers of nucleolaments.

Because the defects are so close, they introduce particular congurations that are related to the doubletwist geometry mentioned above. The question is to understand how the swelling transition occurs and how the bundles shown in Fig. 1 are formed, in which more than 300 DNA laments are closely packed in a quasi-perfect hexagonal structure. As we demonstrated above, they were initially twisted in a small-pitch cholesteric structure. A local hexagonal ordering preexists in the intact sperm head, but it is very local since it does not extend over more than two layers in the direction of the helical axis, as seen for the cryo-sections (see Fig. 4). In dense phases of chiral molecules, the competition between chirality and close hexagonal packing raises theoretical questions (Harris et al., 1999; Kleman, 1985) that may be solved experimentally in multiple ways. In sperm chromatin, this competition is probably very high, owing (i) to the small interhelix distance (2.7 nm) that strongly favors a close-packed hexagonal ordering (the cholesteric to hexagonal transition occurs at 3.15 nm in pure NaDNA solutions (Durand et al., 1992)) and (ii) to the strong chirality of the structure with an average twist angle of ca. 16 between molecules. The coexistence of the two orders as described by Robinson et al. (1958) is therefore prohibited. The twist is not continuous but expelled from the structure along twist walls. The structure must be imagined as a chiral piling of thin hexagonal domains (Fig. 5b). Protamines would be responsible for the stabilization of this structure under tension. The chemical action of DTT apparently relaxes the twist by swelling some regions of the structure, thus allowing the hexagonal order to extend in between. The same process may occur in vivo to allow the rapid decondensation of chromatin after penetration of the sperm nuclei into the oocyte. Nevertheless, such an unwinding of chromatin raises problems that are not understood yet, even if the phenomenon can be restricted to territories of specic chromosomal DNAs as proposed from in situ hybridization data (Haaf and Ward, 1995). The very high density of defect lines present in the structure is also puzzling. At the moment, there is no way to analyze them in detail as was done in other chromatin systems, but we suspect that their 3D distribution may constitute the architecture of the sperm chromatin. Their high density may also be of physiological interest and perhaps help the decondensing agents, such as glutathione (Calvin et al., 1986; Perreault et al., 1988), to diffuse more efciently inside the whole structure.

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