JOuRN&
OF STRUCTURAL
BIOLOGY
103, 13-22 (1990)
Mitochondria-Cytoskeleton GARY Department
Interactions in the Sperm Midpiece’
E. OLSON AND VIRGINIA
of Cell Biology,
Vanderbilt
University,
P. WINFREY Nashville,
Tennessee
37232
Received November 8. 1989
germ cell-specific polypeptides. For example, actin is present in sperm from different species, but it localizes in different domains (Virtanen et al., 1984; Welch and O’Rand, 1985; Flaherty et al., 1986; Camatini et al., 1987). Similarly, antibodies against myosin yield intense immunoreactivity with the neck region but not other domains of human sperm, indicating it is not a major component of cytoskeletal assemblies of the head or midpiece (Virtanen et al., 1984). The intermediate filament protein vimentin has been noted in the equatorial segment of human sperm (Virtanen et al., 1984; Ochs et aZ., 1986) but was not found in bovine spermatozoa (Long0 et al., 1987; Olson et al., 1987). The failure of these somatic cell cytoskeletal polypeptides to display consistent localization between species suggests that they are not major components of the domainspecific cytoskeletal assemblies found in sperm of all mammalian species. The mitochondria of mammalian spermatozoa are restricted to the midpiece segment where they wrap in a helical fashion around the structural elements of the flagellum (Fawcett, 1975; Phillips, 1977; Woolley, 1970). Previously we identified a cytoskeleta1 complex localized within the midpiece which we termed the submitochondrial reticulum (SMR). It is a cylinder-shaped lattice of electron-dense material which is associated with the inner surface of the mitochondrial sheath (Olson and Winfrey, 1986). The SMR terminates distally at the midpieceprincipal piece junction where it fuses with the annulus. In somatic cells the interaction of mitochondria with cytoskeletal elements results in their directed transport and may determine their intracellular distribution (Schilwa, 1986). By analogy, the SMR could participate in mitochondrial migration to the midpiece during spermiogenesis and/or provide a scaffolding which selectively immobilizes mitochondria at this intracellular site. Both biochemical and high resolution structural characterizations are required to define its function. In this study we examine the solubility properties of the SMR and present new data on its substructure and interactions with the mitochondrial membrane.
The mitochondrial sheath of mammalian spermatozoa is adherent to an underlying organized network of electron-dense material termed the submitochondrial reticulum (SMR). In this manuscript we further characterize the substructure of the SMR and the outer mitochondrial membrane and provide new information on their structural interaction. The SMR resists solubilization by detergent and once partially released from the midpiece of extracted spermatozoa, it appears in negatively stained preparations as a network of longitudinally oriented ribbons of fibrillar material which are laterally interconnected. In detergent-extracted specimens the SMR remains attached to the outer mitochondrial membrane thereby suggesting a firm structural interaction. Negatively stained specimens also demonstrate that the outer mitochondrial m.embrane possesses a paracrystalline substructure and it is suggested that ordered arrays of membrane-associated proteins are involved in the structural interaction with the SMR. The potential roles of this cytoskeletal complex during spermiogenesis and in mature sperm are discussed. 6 1999 Academic PRSS, IOC. INTRODUCTION
Domain-specific cytoskeletal complexes have been identified in the acrosomal, postacrosomal, and midpiece segments of the polarized mammalian spermatozoon (for reviews, Olson et al., 1987; Eddy, 1988). These assemblies, which consist of organized arrays of filaments or electron-dense material, associate with specific domains of the plasma membrane and/ or the membrane of specific cytoplasmic organelles. The biochemical composition and function of these cytoskeletal assemblies are poorly understood, but they could contribute to domain-specific membrane functions and/or function in generating the polarized distribution of sperm organelles. Immunohistochemical studies employing antibody probes against somatic cell cytoskeletal polypeptides suggest that the cytoskeletal assemblies of sperm may consist of
‘This
work was supported
by HD20419
and Center Grant
HDO5797. 13
1047-8477190 $3.00 Copyright 0 1990 by Academic Press, Inc. All rights
of reproduction
in any form
reserved.
OLSON AND WINFREY
F ‘IG. 1. Cross section through midpiece region of intact spermatozoa showing mitochondria (m) and plaques of the submitc tchon drial reti culum (r) which adhere to the concave surface of the mitochondria. Note that the plaques of the SMR are positioned eve!r the ! .w bet ween the adjacent outer dense fibers (fJ and the indentation on the abaxial surface of outer dense fibers 1 (f,), 5 (fs), and 6 (fs). x 75 000. F‘IG. 2. Cross section through the midpiece of a sperm briefly extracted in Triton X-100 and DTT. The plaques of the submitc ,chonldrial reti iculum (r) and their radial disposition around the axoneme outer dense fiber complex are clearly shown. X 132 000.
MITOCHONDRIA-CYTOSKELETON
INTERACTIONS
FIG. 3. Longitudinal section of the connecting piece and midpiece of an intact spermatozoon. Note that the submitochondrial reticulum (arrowheads) is composed of continuous bands associated with the outer mitochondrial membrane. Immediately anterior to the proximal-most mitochondrion an aggregate of granular material (g) is associated with a segmented column (s) of the connecting piece. n, nucleus. X 50 000.
MATERIALS AND METHODS Sexually mature male golden hamsters were utilized in the present study. Animals were given free access to food and water and they were maintained on a 14 hr:lO hr 1ight:dark cycle. Animals were either lethally anesthesized with Nembutal or asphyxiated with CO, and the cauda epididymides were removed. The epididymides were placed in either Dulbecco’s or modified Tyrode’s solutions at 36°C and minced with a razor blade. Spermatozoa released into the medium were pelleted by centrifugation at 400g for 5 min and utilized in one of the procedures which follow. In some experiments purified sperm tails were isolated from sonicated sperm suspensions by sucrose density gradient centrifugation (Olson and Sammons, 1980) and extracted as described below. Samples for negative staining were resuspended in a Trissaline solution (TN1 composed of 150 m&f NaCl, 2.5 mM benzamidine, 1 pgiml leupeptin, 1 pg/ml pepstatin, 0.02% sodium azide, and 25 m&f Tris-HCl, pH 7.5) containing 0.5% Triton X100 and 5 mM dithiothreitol (DTT) and extracted at 4°C for 30 min. The sperm were pelleted by centrifugation at 500g for 10 min and then washed three times in TN1 by resuspension and centrifugation. In some experiments the concentration of Triton X-100 was varied between 0.01 and 0.5% and some extractions were performed in the absence of dithiothreitol; variations from the standard extraction regimen are noted in the text. The final pellets were either fixed for thin section analysis as described previously (Olson and Winfrey, 1986) or resuspended in TNI and negatively stained with 1% uranyl acetate. To minimize disruption of extracted sperm by subsequent washing, some sperm samples in the detergent extraction solution were diluted directly into an equal volume of 4% glutaraldehyde in 0.2 M cacodylate buffer, pelleted by centrifugation at 500g for 10 min, and then processed for thin section analysis. RESULTS
Organization
of the submitochondrial
reticulum.
In cross sections of the midpiece the SMR appears as
a set of electron-dense plates, 12 nm in average thickness, which adhere to the inner surface of the mitochondrial sheath (Fig. I). In general these accumulations of electron-dense material are positioned over the gap between the adjacent outer dense fibers and over the indentation which is present on the abaxial surface of the largest outer dense fibers, Nos. 1, 5, and 6 (Figs. 1 and 2). In longitudinal sections these plates appear as continuous ribbons adherent to the outer mitochondrial membrane (Fig. 3). Adjacent ribbons of the SMR are joined by obliquely oriented strands of electrondense material (Olson and Winfrey, 1986) which accounts for the observation that in cross sections the electron-dense plaques appear variable in length (Figs. 1 and 2). The ribbons of the SMR extend beneath the proximal-most mitochondria and terminate (Fig. 3). In some longitudinal sections an aggregation of granular material is present at the anterior end of the mitochondrial sheath; it adheres to both a segmented column of the connecting piece and the adjacent mitochondria (Fig. 3). This granular aggregate may be contiguous with the longitudinally oriented ribbons of the SMR (Fig. 3). Cross sections of the connecting piece reveal that the granular material adheres to the surface of a segmented column and extends laterally until it associates with the mitochondria (Fig. 4). This granular complex appears specifically associated with the segmented column of the connecting piece which gives rise to outer dense fibers 5 and 6.
OLSON AND WINFREY
FIG. 4. Cross section through the connecting piece segment of a spermatozoon extracted briefly with Triton X-100 and DTT. The segmei nted columns (s) of the connecting piece and mitochondria (m) have resisted solubilization. At this level the doublet microtub ules of the iaxoneme are absent but the extended central pair microtubules (cp) are shown in a slightly oblique plane of section. Note that the granul .ar aggregate (g) adheres to both the abaxial surface of a segmented column and to the mitochondria. X 120 000.
Sections tangential to the surface of the mitochondria reveal obliquely oriented, periodically spaced striations associated with the SMR-mitochondrial sheath complex (Fig. 5). These striations are inclined at an angle of about 20” with respect to the mitochondrial long axis and they exhibit a center to center spacing of 29-32 nm. Thin sections also provide direct evidence for structural interactions between the outer mitochondrial membrane and the SMR (Figs. 6 and 7). Appropriately oriented specimens reveal periodic projections, spaced 9-10 nm center to center, which extend from the mitochondrial membrane and insert into the SMR. Negatively strained specimens. The extraction of spermatozoa with Triton X-100 and DTT disrupts the mitochondrial sheath but not the outer dense fiber-axoneme complex (Olson and Linck, 1977). By phase contrast microscopy the midpiece of TritonDTT-extracted spermatozoa is noticeably thinner than in intact spermatozoa and an abrupt thickening is noted at the initiation of the principal piece
segment (Figs. 8 and 9); in some specimens the structural elements of the midpiece fray apart and residual material derived from the mitochondrial sheath is evident. In whole mount specimens of detergent-disrupted spermatozoa the structural interrelationships of the SMR and mitochondrial membrane were clearly visualized. Frequently, the mitochondria of adjacent gyres of the mitochondrial sheath were separated but they remained adherent to the apparently extensible, longitudinally oriented ribbons of the SMR (Fig. 10). The mitochondrial membranes appeared oval-shaped and possessed a paracrystalline substructure (Figs. 11 and 12). Major axes of periodicity extend approximately 20 and 75” with respect to the mitochondrial long axis; mitochondria from successive gyres of the mitochondrial sheath exhibit an identical orientation of these periodic axes. At high magnification it appears that the periodic substructure of the membrane arises from a two-dimensional lattice with a center to center spacing of about 5-6 nm (Figs. 11 and 12). In some specimens a more extensive solubilization
MITOCHONDRIA-CYTOSKELETON
PIG. 5. Longitudinal section inner surface of the mitochondrial FIG. 6. Cross section showing the submitochondrial reticulum FIG. 7. Longitudinal section ulum (r). X 108 000
INTERACTIONS
of the sperm midpiece in which obliquely oriented striations (arrowheads) are seen associated with the sheath (m). x 60 000. periodically spaced bridges (arrowheads) extending from the outer mitochondrial membrane (omn L)to (r). x 108 000. showing linkages (arrows) between the outer mitochondrial membrane and the submitochondrial n ?tic-
OLSON AND WINFREY
18
of the mitochondrial membrane was noted (Figs. 13 and 14). In these cases organized arrays of parallel striations were noted which retained the overall shape of the mitochondrial membrane and which remained associated with the longitudinally oriented bands of the SMR. These striations were spaced 2932 nm center to center and were oriented at a 20” angle with respect to the flagellar long axis; this corresponded to the pattern noted in thin sections (Fig. 5). Each striation had a highly organized substructure and appeared composed of elongate subunits arranged to create a regular crossbanded pattern (Fig. 14). In the negatively stained specimens the longitudinal ribbons of the SMR appeared composed of aggregated, fibrillar material with no evident periodic substructure (Fig. 14); lateral branches were frequently seen connecting adjacent longitudinal bands of the SMR (Figs. 13 and 14). DISCUSSION
During spermiogenesis mitochondria migrate to the developing spermatid flagellum and ultimately become wrapped in a regular helical pattern around the structural elements of the midpiece (Woolley, 1970; Fawcett, 1975; Phillips, 1977). Two structural mechanisms are apparent in mature sperm which could generate and/or maintain this regular pattern (Olson and Winfrey, 1986): first, adjacent mitochondria are joined to one another by regularly spaced bridging elements which directly link the outer mitochondrial membranes and second, the mitochondria are adherent to the SMR, which is localized exclusively in the midpiece. The SMR is composed of longitudinally oriented, laterally interconnected ribbons of electron-dense material arrayed in a precise spatial distribution around the underlying outer dense fiber-axoneme complex. The SMR is shaped as a cylindrical meshwork which supports the overlying mitochondrial sheath. Structurally unique assemblies are associated with the proximal and distal boundaries of the SMR. At the midpiece-principal piece junction the SMR fuses to the annulus which is composed of a bundle of circumferentially oriented filaments. Proximally the SMR is closely associated with an aggregate of
granular material which is also attached to a segmented column of the connecting piece. These SMRassociated assemblies could function as organizing centers to initiate assembly of the SMR during spermiogenesis. The spatial relationship of the granular complex and annulus in the early spermatid has not yet been examined. However, in published micrographs (Fawcett and Phillips, 1969; Phillips, 19741, the annulus of early spermatids encircles the developing neck region of the flagellum and it appears enlarged and heterogeneous in composition. In contrast, in mature sperm, the annulus appears as a compact, homogeneous band of filaments underlying the plasma membrane. During spermiogenesis a central event in defining the flagellar midpiece and principal piece is the distal migration of the annulus from the proximal end of the flagellum to its final residence at the junction of the midpiece and principal piece. The temporal assembly of the SMR during flagellar development remains to be described but it is possible that the annul.us-associated material contributes to assembly of the granular aggregate associated with the connecting piece and/or to the SMR during its distal migration. In early spermatids most mitochondria occupy a peripheral distribution in the cell, yet after the annulus moves distally, they migrate toward the newly defined midpiece (Phillips, 1974) and it is tempting to speculate that the SMR participates in this process. The polypeptide composition of the SMR, annulus, and connecting piece-associated granular aggregate are presently unknown. In the hamster sperm flagellum, actin has been demonstrated in the neck region but not in the midpiece or principal piece (Flaherty et al., 1988). Therefore actin may be a component of the granular aggregate associated with the connecting piece but it is not a major constituent of the SMR or annulus. Although elements of the complex could be related to other somatic cell cytoskeletal polypeptides, published studies have found no evidence of the intermediate filament polypeptide vimentin in the midpiece of human or bovine spermatozoa (Virtanen et al., 1984; Ochs et al., 1986; Longo et al., 1987) even though the SMR is evident in published micrographs of sperm of these
FIG. 8. Phase contrast photomicrograph of an intact spermatozoon. mp, midpiece. FIG. 9. Phase contrast photomicrograph of Triton-DTT-extracted spermatozoa; compared to intact spermatozoa a thinner midpiece and an abrupt thickening at the midpiece-principal piece junction is noted (arrowheads). pp, principal piece. FIG. 10. Whole mount preparation of a spermatozoon extracted with Triton X-100 and DTT. The insoluble component of the mitochondrial sheath (m) is seen fraying away from the outer dense fibers of the underlying midpiece (mp). Note the ovoid shape of the residual mitochondrial membrane (m). Even though the mitochondria of adjacent gyres are physically separated they maintain a regular alignment due to their attachment to the longitudinally oriented ribbons of the submitochondrial reticulum (r). X 27 000. FIG. 11. High magnification view of specimen shown in Fig. 10. The mitochondrial membrane (m) exhibits a crystalline substructure and adjacent mitochondria are connected by the ribbons of the submitochondrial reticulum (r). x 150 000. FIG. 12. Whole mount preparation showing the crystalline lattice (arrowheads) of the mitochondrial membrane and the longitudinal elements of the submitochondrial reticulum (r). X 150 000.
MITOCHONDRIA-CYTOSKELETON
INTERACTIONS
20
OLSON AND WINFREY
FIG. 13. Whole mount preparation showing an extended view of the outer mitochondrial membrane-submitochondrial reticulum complex. Note bow the adjacent longitudinal ribbons of the SMR are interconnected by lateral branches (r). The mitochondrial membranes display prominent oblique striations (arrows). x 105 000.
species (Escalier, 1984; Olson et al., 1987). Recently a fraction enriched for the SMR has been isolated from bovine spermatozoa and although it contains a
spectrum of polypeptides it is enriched for two polypeptides with a M, between 54,000 and 56,000 (Olson and Winfrey, 1989). It is presently unclear
MITOCHONDRIA-CYTOSKELETON
INTERACTIONS
21
FIG. 14. High magnification view of a specimen showing extensive solubilization of the mitochondrial membrane. The remaining membrane appears as a series of parallel oblique striations spaced at 29- to 32-nm intervals; each striation has a finely cross-banded substructure (arrowheads). The SMR (r) is composed of aggregated fibrillar material. x 140 000.
whether the major polypeptides of the SMR are also common to somatic cells or alternatively represent germ cell-specific polypeptides.
Micrographs of disrupted midpieces with separated mitochondria clearly show that the longitudinal ribbons of the SMR are extensible elements
22
OLSON
AND
which maintain a persistent attachment to the outer mitochondrial membrane. This structural data suggests the likelihood of specific molecular interactions between the SMR and outer mitochondrial membrane. In the present study we demonstrate the highly organized structure of the outer mitochondrial membrane. The outer mitochondrial membrane of sperm has previously been shown to resist solubilization by Triton X-100 (Wooding, 1973) and to become crosslinked by disulfide bonds during sperm maturation in the epididymis (Calvin and Bedford, 1971). The mitochondria also undergo structural remodeling during sperm residence in the epididymis (Woolley, 1970; Olson and Hamilton, 1976). Previously quick-freeze, deep-etch, freezefracture analysis has shown that the outer mitochondrial membrane possesses a high density of integral membrane proteins and that the surface of the mitochondria facing the SMR displays a latticelike arrangement of protein particles (Friend and Heuser, 1931). Our negative stain data indicate that the protein components of the outer mitochondrial membrane are arrayed as a two-dimensional lattice. The prominent oblique striations on the outer mitoehondrial membrane correspond directly in size, spacing, and orientation to the particle arrays seen in freeze-fracture (Friend and Heuser, 1981). ether the ordered array of particles represent integral membrane proteins or proteins adsorbed onto the membrane surface remains unresolved by the freeze-fracture and negative stain analyses. However, since these striations can be seen in tangential thin sections and since thin sections also reveal regularly spaced bridges which connect the outer mitochondrial membrane and SMR, we expect that the striations and their component protein particles reflect a regular distribution of elements which attach the mitochondria to the submitochondrial reticulum. It is evident that mammalian spermatozoa possess a complex cytoskeletal network associated exclusively with the midpiece mitochondria. This complex could play an important role in assembling and maintaining the ordered array of mitochondria. Whether somatic cells which possess abundant or ordered arrays of mitochondria, e.g., photoreceptors of the retina, parietal cells of the stomach, skeletal and cardiac muscle fibers, oxyphil cells of the parathyroid, and ion transporting epithelial cells possess related, but as yet undefined, mitochondria-associated cytoskeletal elements is an intriguing possi-
WINFREY
bility. Identification of the polypeptide components of the SMR, their temporal synthesis, and their assembly into a three-dimensional cytoskeletal complex will provide valuable insights into the structural events of spermiogenesis and on the potential tissue specificity of this cytoskeletal assembly. The expert script.
authors express their appreciation to Vera assistance in the preparation and editing
M. Atwood for of this manu-
REFERENCES C~ATINI,
Biol. 45, CALVIN,
M., CASALE, 274-281.
H. I., AND
A., AND CIFARELLI, BEDFORD,
J. M.
Eur. J. Cell
M. (1987)
(1971) J. Reprod.
Fertil.
Suppl. 13, 65-75. EDDY, E. M. (1988) in KNOBIL, E., AND NEILL, J. D. @%.I, The Physiology of Reproduction, Vol. 1, The Spermatozoon, pp. 2668, Raven Press, New York. ESCALIER, FAWCE~, 184. FAWCE~,
D. (1984) BioZ. Cell D. W., AND PHILLIPS, D. W. (1975)
FLAHERTY, Rec. 216, FLAHERTY, Rec. 221, FRIEND, LONGO,
Biol.
Rec. 165,153-
Dev. Biol. 44, 393-436.
S, P., WINFREY, 504515. S. P., WINFREY, 599-610.
V. P., AND OLSON,
G. E. (1986)
V. P., AND OLSON,
G. E.
D. S., AND HEUSER, J. E. (1981)Anat. F. J., KROHNE, G., AND FRANK& 105, 1105-1120.
GCHS, D., WOLF, 495-504. OLSON, 404.
51, 347-364. D. M. !1969)Anat.
D. P., AND GCHS,
G. E., AND HAMILTON,
(1988)Anat.
Rec. lQQ,15$-175. W. W. (1987) J. Cell Ezp. Cell Res. 167,
R. L. (1986)
D. W. (1976)
Anat.
Anat. Rec. 186, 38%
OLSON, G. E., AND LINCK,
R. W. (1977) J. Ultra&act. Res. 61, 21-43. OLSON, G. E., AND SAMMONS, D. W. (1980) Biol. Reprod. 22,319332. OLSON, G. E., AND WINFREY, V. P. (1986) J. Ultrastract. Mol. Struct. Res. 94, 131-139. OLSON, G. E., WINFREY, V. P., AND FLAHERTY, S. P. (1987) Ann. N.Y.Acad.Sci. 513, 222-246. OLSON,
G. E., AND WINFREY,
PHILLIPS, York.
D. M.
(1974)
PHILLIPS,
D. M. (1977)
V. P. (1989)
Spermiogenesis,
J. Ultrastruct.
Biol.
Repmd. 40,284a.
Academic
Press,
New
Res. 58, 144-154.
SCHLIWA, M. (1986) in ALFERT, M., BEERMAN, W., GOLDSTEIN, L., AND PORTER, K. R. (Ed&, Cell Biology Monographs, Vol. 13, The Cytoskeleton: An Introductory Survey, Springer-VerIag, WienlNew York. VIRTANEN, I., BADLEY, R. A., PAASMJO, T,, AND LEHTO, V.-P. (1984) J. CeZZBioZ. 99, 1083-1091. WELCH, J. E., AND GRAND, M. G. (1985)Dev. BioZ. 109,411417. WOODING, F. B. P. (1973) J. ~~tFa&UCt. Res. 42, 502-516. WOOLLEY,
D. M. (1970)
J. Cell Sci. 6, 665-879.
OF STRUCTURAL
BIOLOGY
103, 13-22 (1990)
Mitochondria-Cytoskeleton GARY Department
Interactions in the Sperm Midpiece’
E. OLSON AND VIRGINIA
of Cell Biology,
Vanderbilt
University,
P. WINFREY Nashville,
Tennessee
37232
Received November 8. 1989
germ cell-specific polypeptides. For example, actin is present in sperm from different species, but it localizes in different domains (Virtanen et al., 1984; Welch and O’Rand, 1985; Flaherty et al., 1986; Camatini et al., 1987). Similarly, antibodies against myosin yield intense immunoreactivity with the neck region but not other domains of human sperm, indicating it is not a major component of cytoskeletal assemblies of the head or midpiece (Virtanen et al., 1984). The intermediate filament protein vimentin has been noted in the equatorial segment of human sperm (Virtanen et al., 1984; Ochs et aZ., 1986) but was not found in bovine spermatozoa (Long0 et al., 1987; Olson et al., 1987). The failure of these somatic cell cytoskeletal polypeptides to display consistent localization between species suggests that they are not major components of the domainspecific cytoskeletal assemblies found in sperm of all mammalian species. The mitochondria of mammalian spermatozoa are restricted to the midpiece segment where they wrap in a helical fashion around the structural elements of the flagellum (Fawcett, 1975; Phillips, 1977; Woolley, 1970). Previously we identified a cytoskeleta1 complex localized within the midpiece which we termed the submitochondrial reticulum (SMR). It is a cylinder-shaped lattice of electron-dense material which is associated with the inner surface of the mitochondrial sheath (Olson and Winfrey, 1986). The SMR terminates distally at the midpieceprincipal piece junction where it fuses with the annulus. In somatic cells the interaction of mitochondria with cytoskeletal elements results in their directed transport and may determine their intracellular distribution (Schilwa, 1986). By analogy, the SMR could participate in mitochondrial migration to the midpiece during spermiogenesis and/or provide a scaffolding which selectively immobilizes mitochondria at this intracellular site. Both biochemical and high resolution structural characterizations are required to define its function. In this study we examine the solubility properties of the SMR and present new data on its substructure and interactions with the mitochondrial membrane.
The mitochondrial sheath of mammalian spermatozoa is adherent to an underlying organized network of electron-dense material termed the submitochondrial reticulum (SMR). In this manuscript we further characterize the substructure of the SMR and the outer mitochondrial membrane and provide new information on their structural interaction. The SMR resists solubilization by detergent and once partially released from the midpiece of extracted spermatozoa, it appears in negatively stained preparations as a network of longitudinally oriented ribbons of fibrillar material which are laterally interconnected. In detergent-extracted specimens the SMR remains attached to the outer mitochondrial membrane thereby suggesting a firm structural interaction. Negatively stained specimens also demonstrate that the outer mitochondrial m.embrane possesses a paracrystalline substructure and it is suggested that ordered arrays of membrane-associated proteins are involved in the structural interaction with the SMR. The potential roles of this cytoskeletal complex during spermiogenesis and in mature sperm are discussed. 6 1999 Academic PRSS, IOC. INTRODUCTION
Domain-specific cytoskeletal complexes have been identified in the acrosomal, postacrosomal, and midpiece segments of the polarized mammalian spermatozoon (for reviews, Olson et al., 1987; Eddy, 1988). These assemblies, which consist of organized arrays of filaments or electron-dense material, associate with specific domains of the plasma membrane and/ or the membrane of specific cytoplasmic organelles. The biochemical composition and function of these cytoskeletal assemblies are poorly understood, but they could contribute to domain-specific membrane functions and/or function in generating the polarized distribution of sperm organelles. Immunohistochemical studies employing antibody probes against somatic cell cytoskeletal polypeptides suggest that the cytoskeletal assemblies of sperm may consist of
‘This
work was supported
by HD20419
and Center Grant
HDO5797. 13
1047-8477190 $3.00 Copyright 0 1990 by Academic Press, Inc. All rights
of reproduction
in any form
reserved.
OLSON AND WINFREY
F ‘IG. 1. Cross section through midpiece region of intact spermatozoa showing mitochondria (m) and plaques of the submitc tchon drial reti culum (r) which adhere to the concave surface of the mitochondria. Note that the plaques of the SMR are positioned eve!r the ! .w bet ween the adjacent outer dense fibers (fJ and the indentation on the abaxial surface of outer dense fibers 1 (f,), 5 (fs), and 6 (fs). x 75 000. F‘IG. 2. Cross section through the midpiece of a sperm briefly extracted in Triton X-100 and DTT. The plaques of the submitc ,chonldrial reti iculum (r) and their radial disposition around the axoneme outer dense fiber complex are clearly shown. X 132 000.
MITOCHONDRIA-CYTOSKELETON
INTERACTIONS
FIG. 3. Longitudinal section of the connecting piece and midpiece of an intact spermatozoon. Note that the submitochondrial reticulum (arrowheads) is composed of continuous bands associated with the outer mitochondrial membrane. Immediately anterior to the proximal-most mitochondrion an aggregate of granular material (g) is associated with a segmented column (s) of the connecting piece. n, nucleus. X 50 000.
MATERIALS AND METHODS Sexually mature male golden hamsters were utilized in the present study. Animals were given free access to food and water and they were maintained on a 14 hr:lO hr 1ight:dark cycle. Animals were either lethally anesthesized with Nembutal or asphyxiated with CO, and the cauda epididymides were removed. The epididymides were placed in either Dulbecco’s or modified Tyrode’s solutions at 36°C and minced with a razor blade. Spermatozoa released into the medium were pelleted by centrifugation at 400g for 5 min and utilized in one of the procedures which follow. In some experiments purified sperm tails were isolated from sonicated sperm suspensions by sucrose density gradient centrifugation (Olson and Sammons, 1980) and extracted as described below. Samples for negative staining were resuspended in a Trissaline solution (TN1 composed of 150 m&f NaCl, 2.5 mM benzamidine, 1 pgiml leupeptin, 1 pg/ml pepstatin, 0.02% sodium azide, and 25 m&f Tris-HCl, pH 7.5) containing 0.5% Triton X100 and 5 mM dithiothreitol (DTT) and extracted at 4°C for 30 min. The sperm were pelleted by centrifugation at 500g for 10 min and then washed three times in TN1 by resuspension and centrifugation. In some experiments the concentration of Triton X-100 was varied between 0.01 and 0.5% and some extractions were performed in the absence of dithiothreitol; variations from the standard extraction regimen are noted in the text. The final pellets were either fixed for thin section analysis as described previously (Olson and Winfrey, 1986) or resuspended in TNI and negatively stained with 1% uranyl acetate. To minimize disruption of extracted sperm by subsequent washing, some sperm samples in the detergent extraction solution were diluted directly into an equal volume of 4% glutaraldehyde in 0.2 M cacodylate buffer, pelleted by centrifugation at 500g for 10 min, and then processed for thin section analysis. RESULTS
Organization
of the submitochondrial
reticulum.
In cross sections of the midpiece the SMR appears as
a set of electron-dense plates, 12 nm in average thickness, which adhere to the inner surface of the mitochondrial sheath (Fig. I). In general these accumulations of electron-dense material are positioned over the gap between the adjacent outer dense fibers and over the indentation which is present on the abaxial surface of the largest outer dense fibers, Nos. 1, 5, and 6 (Figs. 1 and 2). In longitudinal sections these plates appear as continuous ribbons adherent to the outer mitochondrial membrane (Fig. 3). Adjacent ribbons of the SMR are joined by obliquely oriented strands of electrondense material (Olson and Winfrey, 1986) which accounts for the observation that in cross sections the electron-dense plaques appear variable in length (Figs. 1 and 2). The ribbons of the SMR extend beneath the proximal-most mitochondria and terminate (Fig. 3). In some longitudinal sections an aggregation of granular material is present at the anterior end of the mitochondrial sheath; it adheres to both a segmented column of the connecting piece and the adjacent mitochondria (Fig. 3). This granular aggregate may be contiguous with the longitudinally oriented ribbons of the SMR (Fig. 3). Cross sections of the connecting piece reveal that the granular material adheres to the surface of a segmented column and extends laterally until it associates with the mitochondria (Fig. 4). This granular complex appears specifically associated with the segmented column of the connecting piece which gives rise to outer dense fibers 5 and 6.
OLSON AND WINFREY
FIG. 4. Cross section through the connecting piece segment of a spermatozoon extracted briefly with Triton X-100 and DTT. The segmei nted columns (s) of the connecting piece and mitochondria (m) have resisted solubilization. At this level the doublet microtub ules of the iaxoneme are absent but the extended central pair microtubules (cp) are shown in a slightly oblique plane of section. Note that the granul .ar aggregate (g) adheres to both the abaxial surface of a segmented column and to the mitochondria. X 120 000.
Sections tangential to the surface of the mitochondria reveal obliquely oriented, periodically spaced striations associated with the SMR-mitochondrial sheath complex (Fig. 5). These striations are inclined at an angle of about 20” with respect to the mitochondrial long axis and they exhibit a center to center spacing of 29-32 nm. Thin sections also provide direct evidence for structural interactions between the outer mitochondrial membrane and the SMR (Figs. 6 and 7). Appropriately oriented specimens reveal periodic projections, spaced 9-10 nm center to center, which extend from the mitochondrial membrane and insert into the SMR. Negatively strained specimens. The extraction of spermatozoa with Triton X-100 and DTT disrupts the mitochondrial sheath but not the outer dense fiber-axoneme complex (Olson and Linck, 1977). By phase contrast microscopy the midpiece of TritonDTT-extracted spermatozoa is noticeably thinner than in intact spermatozoa and an abrupt thickening is noted at the initiation of the principal piece
segment (Figs. 8 and 9); in some specimens the structural elements of the midpiece fray apart and residual material derived from the mitochondrial sheath is evident. In whole mount specimens of detergent-disrupted spermatozoa the structural interrelationships of the SMR and mitochondrial membrane were clearly visualized. Frequently, the mitochondria of adjacent gyres of the mitochondrial sheath were separated but they remained adherent to the apparently extensible, longitudinally oriented ribbons of the SMR (Fig. 10). The mitochondrial membranes appeared oval-shaped and possessed a paracrystalline substructure (Figs. 11 and 12). Major axes of periodicity extend approximately 20 and 75” with respect to the mitochondrial long axis; mitochondria from successive gyres of the mitochondrial sheath exhibit an identical orientation of these periodic axes. At high magnification it appears that the periodic substructure of the membrane arises from a two-dimensional lattice with a center to center spacing of about 5-6 nm (Figs. 11 and 12). In some specimens a more extensive solubilization
MITOCHONDRIA-CYTOSKELETON
PIG. 5. Longitudinal section inner surface of the mitochondrial FIG. 6. Cross section showing the submitochondrial reticulum FIG. 7. Longitudinal section ulum (r). X 108 000
INTERACTIONS
of the sperm midpiece in which obliquely oriented striations (arrowheads) are seen associated with the sheath (m). x 60 000. periodically spaced bridges (arrowheads) extending from the outer mitochondrial membrane (omn L)to (r). x 108 000. showing linkages (arrows) between the outer mitochondrial membrane and the submitochondrial n ?tic-
OLSON AND WINFREY
18
of the mitochondrial membrane was noted (Figs. 13 and 14). In these cases organized arrays of parallel striations were noted which retained the overall shape of the mitochondrial membrane and which remained associated with the longitudinally oriented bands of the SMR. These striations were spaced 2932 nm center to center and were oriented at a 20” angle with respect to the flagellar long axis; this corresponded to the pattern noted in thin sections (Fig. 5). Each striation had a highly organized substructure and appeared composed of elongate subunits arranged to create a regular crossbanded pattern (Fig. 14). In the negatively stained specimens the longitudinal ribbons of the SMR appeared composed of aggregated, fibrillar material with no evident periodic substructure (Fig. 14); lateral branches were frequently seen connecting adjacent longitudinal bands of the SMR (Figs. 13 and 14). DISCUSSION
During spermiogenesis mitochondria migrate to the developing spermatid flagellum and ultimately become wrapped in a regular helical pattern around the structural elements of the midpiece (Woolley, 1970; Fawcett, 1975; Phillips, 1977). Two structural mechanisms are apparent in mature sperm which could generate and/or maintain this regular pattern (Olson and Winfrey, 1986): first, adjacent mitochondria are joined to one another by regularly spaced bridging elements which directly link the outer mitochondrial membranes and second, the mitochondria are adherent to the SMR, which is localized exclusively in the midpiece. The SMR is composed of longitudinally oriented, laterally interconnected ribbons of electron-dense material arrayed in a precise spatial distribution around the underlying outer dense fiber-axoneme complex. The SMR is shaped as a cylindrical meshwork which supports the overlying mitochondrial sheath. Structurally unique assemblies are associated with the proximal and distal boundaries of the SMR. At the midpiece-principal piece junction the SMR fuses to the annulus which is composed of a bundle of circumferentially oriented filaments. Proximally the SMR is closely associated with an aggregate of
granular material which is also attached to a segmented column of the connecting piece. These SMRassociated assemblies could function as organizing centers to initiate assembly of the SMR during spermiogenesis. The spatial relationship of the granular complex and annulus in the early spermatid has not yet been examined. However, in published micrographs (Fawcett and Phillips, 1969; Phillips, 19741, the annulus of early spermatids encircles the developing neck region of the flagellum and it appears enlarged and heterogeneous in composition. In contrast, in mature sperm, the annulus appears as a compact, homogeneous band of filaments underlying the plasma membrane. During spermiogenesis a central event in defining the flagellar midpiece and principal piece is the distal migration of the annulus from the proximal end of the flagellum to its final residence at the junction of the midpiece and principal piece. The temporal assembly of the SMR during flagellar development remains to be described but it is possible that the annul.us-associated material contributes to assembly of the granular aggregate associated with the connecting piece and/or to the SMR during its distal migration. In early spermatids most mitochondria occupy a peripheral distribution in the cell, yet after the annulus moves distally, they migrate toward the newly defined midpiece (Phillips, 1974) and it is tempting to speculate that the SMR participates in this process. The polypeptide composition of the SMR, annulus, and connecting piece-associated granular aggregate are presently unknown. In the hamster sperm flagellum, actin has been demonstrated in the neck region but not in the midpiece or principal piece (Flaherty et al., 1988). Therefore actin may be a component of the granular aggregate associated with the connecting piece but it is not a major constituent of the SMR or annulus. Although elements of the complex could be related to other somatic cell cytoskeletal polypeptides, published studies have found no evidence of the intermediate filament polypeptide vimentin in the midpiece of human or bovine spermatozoa (Virtanen et al., 1984; Ochs et al., 1986; Longo et al., 1987) even though the SMR is evident in published micrographs of sperm of these
FIG. 8. Phase contrast photomicrograph of an intact spermatozoon. mp, midpiece. FIG. 9. Phase contrast photomicrograph of Triton-DTT-extracted spermatozoa; compared to intact spermatozoa a thinner midpiece and an abrupt thickening at the midpiece-principal piece junction is noted (arrowheads). pp, principal piece. FIG. 10. Whole mount preparation of a spermatozoon extracted with Triton X-100 and DTT. The insoluble component of the mitochondrial sheath (m) is seen fraying away from the outer dense fibers of the underlying midpiece (mp). Note the ovoid shape of the residual mitochondrial membrane (m). Even though the mitochondria of adjacent gyres are physically separated they maintain a regular alignment due to their attachment to the longitudinally oriented ribbons of the submitochondrial reticulum (r). X 27 000. FIG. 11. High magnification view of specimen shown in Fig. 10. The mitochondrial membrane (m) exhibits a crystalline substructure and adjacent mitochondria are connected by the ribbons of the submitochondrial reticulum (r). x 150 000. FIG. 12. Whole mount preparation showing the crystalline lattice (arrowheads) of the mitochondrial membrane and the longitudinal elements of the submitochondrial reticulum (r). X 150 000.
MITOCHONDRIA-CYTOSKELETON
INTERACTIONS
20
OLSON AND WINFREY
FIG. 13. Whole mount preparation showing an extended view of the outer mitochondrial membrane-submitochondrial reticulum complex. Note bow the adjacent longitudinal ribbons of the SMR are interconnected by lateral branches (r). The mitochondrial membranes display prominent oblique striations (arrows). x 105 000.
species (Escalier, 1984; Olson et al., 1987). Recently a fraction enriched for the SMR has been isolated from bovine spermatozoa and although it contains a
spectrum of polypeptides it is enriched for two polypeptides with a M, between 54,000 and 56,000 (Olson and Winfrey, 1989). It is presently unclear
MITOCHONDRIA-CYTOSKELETON
INTERACTIONS
21
FIG. 14. High magnification view of a specimen showing extensive solubilization of the mitochondrial membrane. The remaining membrane appears as a series of parallel oblique striations spaced at 29- to 32-nm intervals; each striation has a finely cross-banded substructure (arrowheads). The SMR (r) is composed of aggregated fibrillar material. x 140 000.
whether the major polypeptides of the SMR are also common to somatic cells or alternatively represent germ cell-specific polypeptides.
Micrographs of disrupted midpieces with separated mitochondria clearly show that the longitudinal ribbons of the SMR are extensible elements
22
OLSON
AND
which maintain a persistent attachment to the outer mitochondrial membrane. This structural data suggests the likelihood of specific molecular interactions between the SMR and outer mitochondrial membrane. In the present study we demonstrate the highly organized structure of the outer mitochondrial membrane. The outer mitochondrial membrane of sperm has previously been shown to resist solubilization by Triton X-100 (Wooding, 1973) and to become crosslinked by disulfide bonds during sperm maturation in the epididymis (Calvin and Bedford, 1971). The mitochondria also undergo structural remodeling during sperm residence in the epididymis (Woolley, 1970; Olson and Hamilton, 1976). Previously quick-freeze, deep-etch, freezefracture analysis has shown that the outer mitochondrial membrane possesses a high density of integral membrane proteins and that the surface of the mitochondria facing the SMR displays a latticelike arrangement of protein particles (Friend and Heuser, 1931). Our negative stain data indicate that the protein components of the outer mitochondrial membrane are arrayed as a two-dimensional lattice. The prominent oblique striations on the outer mitoehondrial membrane correspond directly in size, spacing, and orientation to the particle arrays seen in freeze-fracture (Friend and Heuser, 1981). ether the ordered array of particles represent integral membrane proteins or proteins adsorbed onto the membrane surface remains unresolved by the freeze-fracture and negative stain analyses. However, since these striations can be seen in tangential thin sections and since thin sections also reveal regularly spaced bridges which connect the outer mitochondrial membrane and SMR, we expect that the striations and their component protein particles reflect a regular distribution of elements which attach the mitochondria to the submitochondrial reticulum. It is evident that mammalian spermatozoa possess a complex cytoskeletal network associated exclusively with the midpiece mitochondria. This complex could play an important role in assembling and maintaining the ordered array of mitochondria. Whether somatic cells which possess abundant or ordered arrays of mitochondria, e.g., photoreceptors of the retina, parietal cells of the stomach, skeletal and cardiac muscle fibers, oxyphil cells of the parathyroid, and ion transporting epithelial cells possess related, but as yet undefined, mitochondria-associated cytoskeletal elements is an intriguing possi-
WINFREY
bility. Identification of the polypeptide components of the SMR, their temporal synthesis, and their assembly into a three-dimensional cytoskeletal complex will provide valuable insights into the structural events of spermiogenesis and on the potential tissue specificity of this cytoskeletal assembly. The expert script.
authors express their appreciation to Vera assistance in the preparation and editing
M. Atwood for of this manu-
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