MOLECULAR REPRODUCTION AND DEVELOPMENT 33:89-98 (1992)
Structural Organization of Surface Domains of Sperm Mitochondria GARY E. OLSON AND VIRGINIA P. WINFREY Department of Cell Biology, Vanderbilt University, Nashville, Tennessee
ABSTRACT Sperm mitochondria are assembled into an organized sheath surrounding the outer dense fibers and axoneme of the flagellar midpiece. Each mitochondrion is arranged so that its surface faces three different cellular organelles including the plasma membrane, neighboring mitochondria and the outer dense fiber-axoneme complex, In this manuscript we present data on structural differentiations of these three different surface domains of the outer mitochondrial membrane. We demonstrate that the apposed surfaces of adjacent mitochondria are joined by a two dimensional network of studs unique to this domain. By contrast, the surface domain facing the outer dense fiber-axoneme complex exhibits a different, but highly ordered structural organization, evident as a parcrystalline network of parallel stripes; this domain is further distinguished by its exclusive association with a midpiece-specific cytoskeletal complex. These differentiations are not seen on the surface domain of mitochondria which faces the plasma membrane, The implications of the mosaic composition of the outer mitochondrial membrane in the assembly and function of the mitochondrial sheath are discussed. o 19% Wiley-Liss, Inc. Key Words: Midpiece mitochondria, Outer mitochondrial membrane, Sperm cytoskeleton
INTRODUCTION Mitochondria of the mammalian spermatozoon are restricted to the midpiece of the flagellum (Fawcett, 1975; Phillips, 1974; Eddy, 1988). The elongate mitochondria wrap i n a helical fashion around the outer dense fiber-axoneme complex to form the cylindershaped mitochondrial sheath (Fawcett, 1975; Woolley, 1970; Phillips, 1977). Within the sheath, adjacent mitochondria associate both end to end and along their lateral surfaces. This positioning of a concentrated array of mitochondria adjacent to the flagellum is believed to represent a n efficient mechanism for provision of energy required for flagellar motility. The mechanisms that generate and maintain this organized arrangement of mitochondria around the midpiece are poorly understood. Mitochondria aggregate around the spermatid midpiece following the migration of the annulus from its original position at the neck to its final destination a t the midpiece-principal piece junction (Phillips, 1974). Two structural specializations have been identified in mature sperm which 0 1992 WILEY-LISS, INC.
could maintain the arrangement of mitochondria within the sheath. First, bridge-like connections between adjacent mitochondria have been noted and second, a midpiece-specific cytoskeletal complex associated with the adaxial surface of the mitochondrial sheath has been identified (Olson and Hamilton, 1976; Olson and Winfrey, 1986; Olson and Winfrey, 1990). Freeze-fracture analysis has demonstrated ordered arrays of particles on the mitochondrial adaxial surface (Friend and Heuser, 1981) but whether these represent specializations for interactions with cytoskeletal components is not known. Germ cell mitochondria exhibit changes in their protein composition and shape during spermiogenesis (Hecht and Bradley, 1981; DeMartino et al., 1979; Clermont et al., 1990), but the relationship of these changes to mitochondrial function in the mature gamete is unclear. Sperm mitochondria undergo further modifications during sperm maturation in the epididymis. One well-established change is that the outer mitochondrial membrane becomes extensively cross linked by disulfide bonds which account for this membranes extraordinary resistance to solubilization by anionic detergents (Calvin and Bedford, 1971). A low-molecular-weight cysteine-rich, selenium-containingprotein, termed mitochondrial capusle protein (MCP), has been identified that is thought to account for outer mitochondrial rnembrane stability (Calvin et al., 1981; Pallini et al., 1979). This protein is synthesized during spermiogenesis (Calvin et al., 1987; Kleene e t al., 1990) but precisely when it associates with mitochondria and how it affects mitochondrial structure andlor function are unclear. The unique arrangement of sperm mitochondria around the sperm midpiece and their structural interactions with each other and with cytoskeletal elements raises the question of whether the outer mitochondrial membrane could be organized into domains of unique structure and, potentially, function. In this paper we define sperm disruption and extraction regimens which facilitate structural examination of the outer mitochondrial membrane. Evidence is presented that the mitochondrial surface is comprised of at least three struc-
Received February 18,1992;accepted April 3,1992. Address reprint requests to Dr. Gary E. Olson, Department of Cell Biology, Vanderbilt University, Nashville, TN 37232.
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turally distinct domains. The potential significance of this mosaic surface organization of the mitochondria is discussed.
MATERIALS AND METHODS Sperm Preparation, Disruption, and Extraction Mature male golden hamsters were maintained on a 14h:lOh 1ight:dark cycle and given free access to food and water. Animals were lethally asphyxiated with CO,, the cauda epididymides were then removed and minced in warm Hank’s solution. Sperm released into the medium were collected and pelleted by centrifugation a t 1,000 g for 10 min. The sperm pellet was resuspended into a n ice-cold Tris-saline-protease inhibitor solution (TNI) composed of 150 mM NaC1,25 mM TrisHCl pH 7.5, 2 mM benzamidine, 1 pgiml leupeptin, 1 pgiml pepstatin, and 0.02% sodium azide. This suspension was centrifuged a t 1,000 g for 10 min to obtain a pellet of washed spermatozoa. All subsequent sperm treatment steps were performed a t 4°C in the presence of the protease inhibitors listed above. Sperm were physically disrupted using either sonication or nitrogen cavitation. For disruption by sonication the sperm pellet was resuspended into TNI and sonicated for three 10-sec intervals at a medium-power setting; phase contrast microscopy demonstrated that although this treatment severed the head-tail junction, the midpiece segments appeared intact. The sperm were then washed three times by centrifugation at 1,000 g, followed by resuspension in TNI. In some experiments, a purified flagellar fraction was prepared by centrifugation of the sonicated sperm suspension for 60 min a t 100,000 g onto discontinuous sucrose gradients comprised of 10 ml sperm suspension, 8 ml55% sucrose, 25 mM Tris-HC1 pH 7.5, 7 ml 70% sucrose, 25 mM Tris-HC1 pH 7.5, and 6 ml 75% sucrose, 25 mM TrisHCl pH 7.5. The enriched flagellar fraction was recovered from the 55%/70%interface; the sperm tail fraction was washed three times by centrifugation at 1,000 g , followed by resuspension with TNI. For disruption by nitrogen cavitation the pellet of intact spermatozoa was resuspended in TNI and subjected to nitrogen cavitation a t a pressure of 500 psi for 15 min as described previously (Olson and Winfrey, 1986). The sperm suspension was then washed three times by centrifugation a t 1,000g followed by resuspension with TNI. Sonicated spermatozoa were further extracted overnight at 4°C in solutions of varying ionic strength and pH which contained dithiothreitol (DTT). The three different solutions used were (1) TNI containing 2-5 mgiml DTT; or (2) 0.5 M NaC1,25 mM Tris-HC1 pH 9.0, 2 mM benzamidine, 1 pgiml leupeptin, 1pgiml pepstatin and 2 mgiml DTT; or (3) 0.5 M NaC1,25 mM CAPS buffer pH 11.0,2 mM benzamidine, 1pg/ml leupeptin, 1 pgiml pepstatin and 2 mgiml DTT. Electron Microscopy Sperm suspensions a t varying stages of fractionation and extraction were prepared for electron microscopy. Sperm fractions were pelleted by centrifugation in a
microcentrifuge for 1 min at 3,000-6,000 g and the pellet fixed in 4% glutaraldehyde in 0.2 M sodium cacodylate pH 7.4. The sample was postfixed with buffered OsO, and dehydrated through a n ethanol series which included a n en bloc staining step of 1%uranyl acetate in 70% ethanol. Following equilibration in propylene oxide, specimens were embedded in Epon resin. Thin sections were stained with uranyl acetate and lead citrate.
RESULTS Surface Domains of Sperm Mitochondria Mitochondria of mammalian sperm are arranged into a cylinder-shaped sheath that surrounds the axial elements of the midpiece. Each mitochondrion is arranged so that its exterior surface faces three different sets of cellular organelles (Figs. 1, 2). These three surface domains include first, the outer-facing (abaxial) surface of the mitochondria that is oriented toward the plasma membrane (termed pms for plasma membrane surface); typically, a thin layer of cytoplasm separates the plasma membrane and mitochondria. The second surface domain consists of the inner-facing (adaxial) surface of the mitochondria that is oriented toward the central outer dense fiber-axoneme complex (termed axs for axoneme-facing surface); this surface is further distinguished by its association with a midpiece-specific cytoskeletal network, the submitochondrial reticulum (Olson and Winfrey, 1986). In cross sections the submitochondrial reticulum appears as a radially arrayed set of electron-dense plaques t h a t adhere to the outer mitochondrial membrane (Fig. 1). The third surface domain includes the lateral surfaces and ends of the mitochondria that are in close apposition with the neighboring mitochondria (termed mms for mitochondria-mitochondria surface); typically the outer membranes of adjacent mitochondria are separated by a space of about 5-6 nm. The mitochondrial interior composed of the inner mitochondrial membrane and the mitochondrial matrix exhibits no apparent regional differences in structure corresponding to the three surface domains. Effects of Physical Disruption on the Mitochondria1 Sheath In intact spermatozoa, sections tangential to the mitochondrial surface can include superimposed views of the plasma membrane, cytoplasm, the outer and inner mitochondrial membranes, and, the mitochondrial matrix. To identify domain-specific specializations of the mitochondrial surface, sperm were physically disrupted and then extracted to strip both cytoplasmic components and inner mitochondrial membrane-matrix elements from the outer mitochondrial membrane. First, either sonication or nitrogen cavitation was employed to partially remove the midpiece plasma membrane and disperse the underlying cytoplasm. These treatments did not disrupt the mitochondrial sheath and the outer and inner mitochondrial membranes as well as the mitochondrial matrix appeared intact. How-
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Fig. 1. Transverse section of intact hamster sperm midpiece and principal piece. The central outer dense fiber (do-axoneme (ax) complex is surrounded by the mitochondrial sheath (m) in the midpiece and by the fibrous sheath (fs)in the principal piece. The three surface domains of the outer mitochondrial membrane include that facing the plasma membrane (pms),the domain facing other mitochondria (mms) and the domain facing the outer dense fiber-axoneme complex (axs). Note that the axs-domain is associated with plates of electron dense
material which comprise the submitochondrial reticulum (smr). The interior of the mitochondria consists of an electron dense matrix and the inner mitochondrial membrane (imm) that exhibits cristae-like infoldings. pm, plasma membrane. ~ 8 1 , 7 5 0 . Fig. 2. Longitudinal section of an intact midpiece again showing the three surface domains (pms, mms, and axs) of individual mitochondria. df = outer dense fibers. X39,750.
ever, the removal of the electron dense cytoplasm facilitated identification of structural interactions between mitochondria. In frontal sections, it is noted that the 5 to 6-nm gap between mitochondria is maintained after physical disruption. Moreover, the mms-domains of adjacent mitochondria are joined by stud-like bridging elements spaced at intervals of about 20 nm center to center (Fig. 3). Transverse sections of mitochondria also reveal the studs between apposed mitochondrial surfaces (Fig. 4).The combination of frontal and transverse views indicates that the network of studs forms a two-dimensional network present throughout the regions of mitochondria-mitochondria contact (Figs. 3,4). These studs are not noted either on the plasma membrane-facing surface or the adaxial-facing surface of the mitochondria which suggests they represent a domain-specific specialization.
The axoneme-facing (axs) domain of the outer mitochondrial membrane maintained its structural association with the submitochondrial reticulum which in longitudinal section appears comprised of longitudinally oriented bands of electron dense material extending the length of the midpiece (Figs. 4, 5 ) . Tangential sections of the mitochondrial surface include contributions from the outer and inner mitochondrial membranes a s well as the mitochondrial matrix, making it difficult to resolve specific membrane specializations.
Effects of Reducing Agents and Elevated pH on Midpiece Structure Sonicated sperm and isolated flagella were incubated in extraction solutions with and without the reducing agent DTT and examined by phase contrast and electron microscopy. By phase contrast microscopy, the
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Fig. 3. Longitudinal section of the midpiece of a sperm subjected to nitrogen cavitation. The mitochondrial sheath remains intact and in frontal views the mms-domain of the outer mitochondrial membrane (om) of adjacent mitochondria are linked by a network of stud-like bridges (5). x63,OOO. Fig. 4. Longitudinal profile of midpiece after nitrogen cavitation. The mitochondria are seen in cross section views and the mms-domain shows the network of studs ( s ) joining successive mitochondria. The
axs-domain remains associated with the longitudinal bands of electron dense material comprising the submitochondrial reticulum (smr). ~71,250. Fig. 5. Longitudinal profile of midpiece subjected to nitrogen cavitation. The continuous electron dense bands of the submitochondrial reticulum (smr) and their association with the axs-domain of the mitochondria are shown. s, network of studs joining the mms-domains. X66,OOO.
midpiece of control samples incubated in TNI buffer appears smooth and individual mitochondria are not resolved; the midpiece segment is distinguished from the thinner principal piece segment by the abrupt reduction in flagellar diameter at the annulus, the diameter of the tail then progressively tapers throughout the principal piece (Fig. 6a). After treatment with solutions
containing DTT the midpiece is no longer smooth but is instead decorated by numerous phase dense vesicles that appear to be derived from the mitochondrial sheath (Fig. 6b-d). In both pH 7.5 and pH 9.0 extraction solutions, some release of single and interconnected aggregates of mitochondrial sheath components from the underlying midpiece were noted (Fig. 6b-d). Extraction
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Fig. 6. Phase contrast photomicrographs showing sperm flagella extracted without (a)and with (b-e) DTT a t pH 7.5 ( a x ) , p.H 9.0 (d) and pH 11.0 (el. cp, connecting piece; an, annulus; pp, principal piece; mp, midpiece. Note that in 6a the midpiece segment appears smooth but that in 6b-e vesicular elements are associated with the mitochondrial sheath. Single vesicles detached from the midpiece are evident
(arrowheads) and in 6d a n interconnected array of mitochondrial sheath elements are partially released from the underlying outer dense fiber-axoneme complex of the midpiece (mp). Note in 6e that after extraction at pH 11.0 many vesicular elements remain interconnected. x 1,560.
at pH 11.0 in the presence of DTT resulted in substantial dissolution of the underlying flagellar fibers, however, mitochondrial sheath components remained intact and appeared as interconnected arrays of phase dense vesicles (Fig. 6e). Electron microscopic analysis revealed specific DTTmediated alterations in mitochondrial sheath structure. Most notably, the inner mitochondrial membrane and its enclosed matrix lost its close association with the outer mitochondrial membrane. These internal mitochondrial components formed rounded vesicles that were displaced toward the pms-domain of the outer mitochondrial membrane and frequently they appeared to bleb from this surface of the mitochondria (Fig. 7; see Figs. 11, 12). Identification of domain-specific specializations of the outer mitochondrial membrane was facilitated by displacement of the inner membranematrix complex as sections tangential to the outer membrane included no additional superimposed structures. The outer mitochondrial membrane of DTT-extracted flagella maintained structural relationships typical of intact mitochondria (Figs. 7-10). Adjacent
mitochondria remained associated via the network of bridges on the mms-domain of the outer mitochondrial membrane (Figs. 9 and 10). Tangential views of the mms-domain reveal that the studs are arrayed as a two dimensional lattice with individual studs spaced about 20 nm center to center (Figs. 8 and 10). Domain-specific structural specializations are also noted on the axoneme-facing surface domain (axs) of the outer mitochondrial membrane of DTT-treated flagella. This surface is decorated by an ordered pattern of parallel stripes extending over the mitochondrial surface (Figs. 7-10). The stripes are spaced 30- to 32-nm center to center and oriented a t an oblique angle relative to the longitudinal axis of the mitochondria. Stripes of adjacent mitochondria exhibit the same orientation (Fig. 7). This striped pattern is noted on the axoneme-facing surface of mitochondria that remain adherent to the midpiece (Figs. 7, 8) as well as those that have been released from the underlying flagellar elements (Figs. 9,lO). Flagella extracted at pH 9.0 (Fig. 11)or pH 11.0 (Fig. 12) also exhibited the striping pattern on the axs-domain.
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Fig. 7. Longitudinal section of flagellum of sperm that was first disrupted by sonication and then extracted a t pH 7.5 with DTT. The outer mitochondrial membranes remain intact and retain the overall organization of the mitochondrial sheath but appear somewhat more irregularly shaped in cross section. The inner mitochondrial membrane and matrix elements (*j are displaced toward the pms-domain of the mitochondrial membrane. Successive mitochondria remain adjoined along their mms-domain and in frontal views it is apparent that the axs-domain of the outer mitochondrial membrane exhibits a repetitive oblique striping pattern (arrowheads). Note the similar orientation of the striping in successive mitochondria of the sheath. The
pms-domain when seen in frontal views (open arrows) does not exhibit this striping pattern. df, dense fibers. X67,500. Fig. 8. Section through the midpiece of a sperm extracted at pH 7.5 with DTT. Note the submitochondrial reticulum (smr) maintains its association with the axs-domain of the mitochondria. Small areas of each mitochondrial surface domain are shown in frontal view. Note that the axs-domain displays a regular striping pattern (arrowheads). Apposed membranes of the the mms-domain (bracketed area) exhibit a two dimensional array of punctate profiles. The pms-domain appears homogenous and no specializations are noted (open arrows). df, outer dense fibers; ax, axoneme. ~66,000.
Thin section analysis of DTT-treated sperm reveals no distinctive differentiations of the plasma membrane-facing (pms) domain of the outer mitochondrial membrane (Figs. 7, 8, 11, 12). The pms-domain appeared to be the most fragile of the three outer mitochondrial membrane domains a s occasional ruptures were noted that appeared to represent the site of exit of
the swollen inner mitochondrial membrane-matrix vesicles (Figs. 11, 12). While the lumen of the outer mitochondrial membrane was approximately 0.2 +m in width, the inner mitochondrial membrane-matrix vesicles measured approximately 0.6 pm in diameter and no cristae-like folds of the inner mitochondrial membrane into the vesicle interior were noted (Fig. 10).
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Fig. 9. High-power view of outer mitochondrial membranes after extraction in TNI containing DTT. Note the studs ( s ) attaching the mms-domains of the adjacent mitochondria and the regular striping pattern (arrowheads) on the axs-domain. ~90,000. Fig. 10.Thin section showing mitochondria that have been released from flagellum of DTT treated flagella. The orientation is equivalent to a cross-sectional view of the flagellum. In areas denoted by brack-
ets, the section includes enface views of the mms-domain and includes the outer membranes of two adjacent mitochondria; in these regions note the two dimensional array ofdots that represent the studs seen in cross sectional views of the membrane. A portion of the axs-domain is also shown and note its regular striped pattern (arrowheads).
DISCUSSION
piece length, mitochondrial size, mitochondrial morphology, and mitochondrial arrangements are evident (Fawcett, 1975; Phillips, 1974). In somatic cells cytoskeletal elements including microtubules and actin filaments have been shown to participate in directed
The spermatozoan midpiece of diverse mammalian species is comprised of the same set of cellular organelles. Although these organelles are arranged upon a common theme, significant species differences in mid-
X90,OOO.
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Fig. 11. Longitudinal section though midpiece segment of sperm extracted at pH 9.0 with D". Note the inner mitochondria membrane-matrix vesicles (imm) which are associated with the pms-domain of the outer mitochondrial membrane. The pms-domain appears homogeneous in frontal views while the axs-domain exhibits the transverse striping (arrowheads). df, outer dense fiber; ax, axoneme. ~54,000. Fig. 12. Slightly oblique cross section through the midpiece of a sonicated sperm extracted at pH 11.0 with DTT. Residual flagellar
material (*) remains within the lumen. Note the outer mitochondrial membranes remain associated with one another but their interior appears mostly empty. The inner mitochondrial membrane-matrix complexes (imm) appear as relatively large, 0.6 Fm diameter, vesicles which bleb from the pms-domain of the outer mitochondrial membrane; arrowheads denote sites of rupture of the pms-domain. Note the regular striping pattern is retained on the axs-domain of the outer mitochondrial membrane. X 54,000.
mitochondrial movements (Schliwa, 19861, but the mechanisms that direct the formation of the spermatid mitochondrial sheath are unresolved.
Sperm mitochondria share common structural features with mitochondria of somatic cells (Fawcett, 19'751, but previous studies have noted some unusual
SURFACE DOMAINS OF SPERM MITOCHONDRIA aspects of their surface structure, For example, filamentous linkages joining adjacent mitochondria have been identified (Olson and Hamilton, 1976; Olson and Winfrey, 1986), but it was not appreciated that these bridging elements form an extensive two-dimensional network between the apposed mitochondrial surfaces as demonstrated in the present study. Also, using the quick-freeze,deep-etch, freeze-fracture technique it has been shown that the adluminal surface of the mitochondria are decorated by an apparently unique, ordered array of particles (Friend and Heuser, 1981);these investigators speculated that these structural specializations could reflect a specific localization of enzymes or transport proteins. The paracrystalline striping pattern identified in the present study on the axonemefacing surface, termed axs-domain, of the outer mitochondrial membrane appears to represent the structural specialization previously noted by freeze fracture; moreover the resistance of this membrane specialization t o the elevated pH and ionic strength extraction regimens employed in the present study raises the possibility that this paracrystalline pattern reflects an ordered arrangement of integral membrane proteins. One additional unique feature of the axs-domain of the outer mitochondrial membrane is its specific adhesion to the submitochondrial reticulum, a midpiece specific cytoskeletal complex (Olson and Winfrey, 1986; Olson and Winfrey, 1990); whether this interaction is mediated by domain-specific polypeptides requires resolution. The specific proteins responsible for generating the domain specific differentiations of the outer mitochondrial membrane remain to be identified. A low-molecular-weight cysteine-rich polypeptide, termed MCP, has been ascribed to the mitochondrial capsule by previous studies (Calvin et al., 1981; Pallini et al., 1979). However the structural differentiations of the mms- and axs-domains persist after extraction with disulfide bond reducing agents, a treatment that efficiently solubilizes MCP (Pallini et al., 1979), so these structural specializations apparently neither represent nor are dependent on MCP for their maintenance. The outer mitochondrial membrane of yeast and somatic cells is comprised of a limited set of polypeptides (Sollner et al., 1989) and some of the major components function as receptors for the import of cytoplasmic polypeptides with appropriate signal sequences (Baker and Schatz, 1991).Although the polypeptide pattern of sperm mitochondria has been analyzed (Hecht and Bradley, 1981) proteins specific to the outer mitochondrial membrane remain to be identified and compared with those identified in other systems. The present study demonstrates that the outer membrane of the midpiece mitochondria possesses three structurally distinct domains. We speculate that these structural specializations reflect differences in protein composition between domains and that these different domains could participate in specific events in the the assembly andlor biochemical function of the mitochondrial sheath. For example, the network of studs located along the paired surfaces of adjacent mitochondria
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(mms-domain) could represent specific adhesion molecules required for the assembly or maintenance of the mitochondrial sheath; alternatively these linkages could function in intermitochondrial communication to coordinate their activities. Similarly, the unique paracrystalline structure of the axoneme-facing surface domain (axs-domain) suggests that it is composed of an assemblage of proteins that may mediate adhesion with the submitochondrial reticulum and may function in targeting and/or maintaining mitochondria a t the developing midpiece. Finally, the mitochondrial surface facing the plasma membrane, the pms-domain, differed from the other domains by the absence of differentiations identifiable by thin section analysis; however its adjacent proximity to the plasma membrane suggests this domain might be specialized for importation of metabolites used for energy generation. If different mitochondrial surface domains possess unique permeability properties, the resultant vectorial flow of specific compounds could concentrate molecules required for motility in the cytoplasm immediately surrounding the axoneme. These various possibilities, although intriguing, will require the identification of domain-specific proteins and the elucidation of their specific protein interactions and enzymatic activities.
ACKNOWLEDGEMENTS This work was supported by NIH grant HD20419 and by Center grant HD05797.
REFERENCES Baker KP, Schatz G (1991): Mitochondria1 proteins essential for viability mediate protein import into yeast mitochondria. Nature 349:205-208. Calvin HI, Cooper GW, Wallace E (1981): Evidence that selenium in rat sperm is associated with a cysteine-rich structural protein of the mitochondrial capsules. Gamete Res 4:139-149. Calvin HI, Grosshans K, Musicant-Shikora SR, Turner SI (1987): A developmental study of rat sperm and testis selenoproteins. J Reprod Fertil81:l-11. Calvin HI, Bedford J M (1971): Formation of disulphide bonds in the nucleus and accessory structures of mammalian spermatozoa during maturation in the epididymis. J Reprod Fertil Suppl. 13:65-75. Clermont Y, Oko R, Hermo L (1990): Immunocytochemical localization of proteins utilized in the formation of outer dense fibers and fibrous sheath in rat spermatids: An electron microscope study. Anat Rec 227:447457. DeMartino C, Floridi A, Marcante ML, Malorni W, Scorza-Barcellona P, Belloci M, Silvestrini B (1979): Morphological, histochemical and biochemical studies on germ cell mitochondria of normal rats. Cell Tissue Res 196:l-22. Eddy EM (1988):The Spermatozoon. In E Knobil, J D Neil1 (eds):“The Physiology of Reproduction.” New York: Raven Press, pp 27-68. Fawcett DW (1975):The mammalian spermatozoon. Dev Biol443394436. Friend DS, Heuser J E (1981):Orderly particle arrays on the mitochondrial outer membrane in rapidly-frozen sperm. Anat Rec 199:159175. Hecht NB, Bradley FM (1981):Changes in mitochondrial protein composition during testicular differentiation in mouse and bull. Gamete Res 4:433-449. Kleene KC, Smith J , Bozorgzadeh A, Harris M, Hahn L, Karimpour I, Gerstel J (1990): Sequence and developmental expression of the mRNA encoding the seleno-protein of the sperm mitochondrial capsule in the mouse. Dev Biol137:395402. Olson GE, Hamilton DW (1976): Morphological changes in the midpiece of wooly opossum spermatozoa during epididymal transit. Anat Rec 186:387404.
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Olson GE, Winfrey VP (1986):Identification of a cytoskeletal network adherent to the mitochondria of mammalian spermatozoa. J Ultrastruct Mol Struct Res 94:131-139. Olson GE, Winfrey VP (1990): Mitochondria-cytoskeleton interactions in the sperm midpiece. J Struct Biol 103:13-22. Pallini V, Baccetti B, Burrini AG (1979): A peculiar cysteine-rich polypeptide related to some unusual properties of mammalian sperm mitochondria. In DW Fawcett, J M Bedford (eds): “The Spermatozoon.” Baltimore: Urban and Schwarzenberg, pp 141-151. Phillips DM (1974):“Spermiogenesis.”New York: Academic Press.
Phillips DM (1977):Mitochondria1 disposition in mammalian spermatozoa. J Ultrastruct Res 58:14&154. Schliwa M (1986): “The Cytoskeleton an Introductory Survey.” Wien New York: Springer-Verlag. Sollner T, Griffiths G, Pfaller R, Pfanner N, Neupert W (1989): MOM19, an import receptor for mitochondria1 precursor proteins. Cell 59:1061-1070. Woolley DM (1970):The midpiece of the mouse spermatozoon: Its form and development as seen by surface replication. J Cell Sci 6:865879.
Structural Organization of Surface Domains of Sperm Mitochondria GARY E. OLSON AND VIRGINIA P. WINFREY Department of Cell Biology, Vanderbilt University, Nashville, Tennessee
ABSTRACT Sperm mitochondria are assembled into an organized sheath surrounding the outer dense fibers and axoneme of the flagellar midpiece. Each mitochondrion is arranged so that its surface faces three different cellular organelles including the plasma membrane, neighboring mitochondria and the outer dense fiber-axoneme complex, In this manuscript we present data on structural differentiations of these three different surface domains of the outer mitochondrial membrane. We demonstrate that the apposed surfaces of adjacent mitochondria are joined by a two dimensional network of studs unique to this domain. By contrast, the surface domain facing the outer dense fiber-axoneme complex exhibits a different, but highly ordered structural organization, evident as a parcrystalline network of parallel stripes; this domain is further distinguished by its exclusive association with a midpiece-specific cytoskeletal complex. These differentiations are not seen on the surface domain of mitochondria which faces the plasma membrane, The implications of the mosaic composition of the outer mitochondrial membrane in the assembly and function of the mitochondrial sheath are discussed. o 19% Wiley-Liss, Inc. Key Words: Midpiece mitochondria, Outer mitochondrial membrane, Sperm cytoskeleton
INTRODUCTION Mitochondria of the mammalian spermatozoon are restricted to the midpiece of the flagellum (Fawcett, 1975; Phillips, 1974; Eddy, 1988). The elongate mitochondria wrap i n a helical fashion around the outer dense fiber-axoneme complex to form the cylindershaped mitochondrial sheath (Fawcett, 1975; Woolley, 1970; Phillips, 1977). Within the sheath, adjacent mitochondria associate both end to end and along their lateral surfaces. This positioning of a concentrated array of mitochondria adjacent to the flagellum is believed to represent a n efficient mechanism for provision of energy required for flagellar motility. The mechanisms that generate and maintain this organized arrangement of mitochondria around the midpiece are poorly understood. Mitochondria aggregate around the spermatid midpiece following the migration of the annulus from its original position at the neck to its final destination a t the midpiece-principal piece junction (Phillips, 1974). Two structural specializations have been identified in mature sperm which 0 1992 WILEY-LISS, INC.
could maintain the arrangement of mitochondria within the sheath. First, bridge-like connections between adjacent mitochondria have been noted and second, a midpiece-specific cytoskeletal complex associated with the adaxial surface of the mitochondrial sheath has been identified (Olson and Hamilton, 1976; Olson and Winfrey, 1986; Olson and Winfrey, 1990). Freeze-fracture analysis has demonstrated ordered arrays of particles on the mitochondrial adaxial surface (Friend and Heuser, 1981) but whether these represent specializations for interactions with cytoskeletal components is not known. Germ cell mitochondria exhibit changes in their protein composition and shape during spermiogenesis (Hecht and Bradley, 1981; DeMartino et al., 1979; Clermont et al., 1990), but the relationship of these changes to mitochondrial function in the mature gamete is unclear. Sperm mitochondria undergo further modifications during sperm maturation in the epididymis. One well-established change is that the outer mitochondrial membrane becomes extensively cross linked by disulfide bonds which account for this membranes extraordinary resistance to solubilization by anionic detergents (Calvin and Bedford, 1971). A low-molecular-weight cysteine-rich, selenium-containingprotein, termed mitochondrial capusle protein (MCP), has been identified that is thought to account for outer mitochondrial rnembrane stability (Calvin et al., 1981; Pallini et al., 1979). This protein is synthesized during spermiogenesis (Calvin et al., 1987; Kleene e t al., 1990) but precisely when it associates with mitochondria and how it affects mitochondrial structure andlor function are unclear. The unique arrangement of sperm mitochondria around the sperm midpiece and their structural interactions with each other and with cytoskeletal elements raises the question of whether the outer mitochondrial membrane could be organized into domains of unique structure and, potentially, function. In this paper we define sperm disruption and extraction regimens which facilitate structural examination of the outer mitochondrial membrane. Evidence is presented that the mitochondrial surface is comprised of at least three struc-
Received February 18,1992;accepted April 3,1992. Address reprint requests to Dr. Gary E. Olson, Department of Cell Biology, Vanderbilt University, Nashville, TN 37232.
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turally distinct domains. The potential significance of this mosaic surface organization of the mitochondria is discussed.
MATERIALS AND METHODS Sperm Preparation, Disruption, and Extraction Mature male golden hamsters were maintained on a 14h:lOh 1ight:dark cycle and given free access to food and water. Animals were lethally asphyxiated with CO,, the cauda epididymides were then removed and minced in warm Hank’s solution. Sperm released into the medium were collected and pelleted by centrifugation a t 1,000 g for 10 min. The sperm pellet was resuspended into a n ice-cold Tris-saline-protease inhibitor solution (TNI) composed of 150 mM NaC1,25 mM TrisHCl pH 7.5, 2 mM benzamidine, 1 pgiml leupeptin, 1 pgiml pepstatin, and 0.02% sodium azide. This suspension was centrifuged a t 1,000 g for 10 min to obtain a pellet of washed spermatozoa. All subsequent sperm treatment steps were performed a t 4°C in the presence of the protease inhibitors listed above. Sperm were physically disrupted using either sonication or nitrogen cavitation. For disruption by sonication the sperm pellet was resuspended into TNI and sonicated for three 10-sec intervals at a medium-power setting; phase contrast microscopy demonstrated that although this treatment severed the head-tail junction, the midpiece segments appeared intact. The sperm were then washed three times by centrifugation at 1,000 g, followed by resuspension in TNI. In some experiments, a purified flagellar fraction was prepared by centrifugation of the sonicated sperm suspension for 60 min a t 100,000 g onto discontinuous sucrose gradients comprised of 10 ml sperm suspension, 8 ml55% sucrose, 25 mM Tris-HC1 pH 7.5, 7 ml 70% sucrose, 25 mM Tris-HC1 pH 7.5, and 6 ml 75% sucrose, 25 mM TrisHCl pH 7.5. The enriched flagellar fraction was recovered from the 55%/70%interface; the sperm tail fraction was washed three times by centrifugation at 1,000 g , followed by resuspension with TNI. For disruption by nitrogen cavitation the pellet of intact spermatozoa was resuspended in TNI and subjected to nitrogen cavitation a t a pressure of 500 psi for 15 min as described previously (Olson and Winfrey, 1986). The sperm suspension was then washed three times by centrifugation a t 1,000g followed by resuspension with TNI. Sonicated spermatozoa were further extracted overnight at 4°C in solutions of varying ionic strength and pH which contained dithiothreitol (DTT). The three different solutions used were (1) TNI containing 2-5 mgiml DTT; or (2) 0.5 M NaC1,25 mM Tris-HC1 pH 9.0, 2 mM benzamidine, 1 pgiml leupeptin, 1pgiml pepstatin and 2 mgiml DTT; or (3) 0.5 M NaC1,25 mM CAPS buffer pH 11.0,2 mM benzamidine, 1pg/ml leupeptin, 1 pgiml pepstatin and 2 mgiml DTT. Electron Microscopy Sperm suspensions a t varying stages of fractionation and extraction were prepared for electron microscopy. Sperm fractions were pelleted by centrifugation in a
microcentrifuge for 1 min at 3,000-6,000 g and the pellet fixed in 4% glutaraldehyde in 0.2 M sodium cacodylate pH 7.4. The sample was postfixed with buffered OsO, and dehydrated through a n ethanol series which included a n en bloc staining step of 1%uranyl acetate in 70% ethanol. Following equilibration in propylene oxide, specimens were embedded in Epon resin. Thin sections were stained with uranyl acetate and lead citrate.
RESULTS Surface Domains of Sperm Mitochondria Mitochondria of mammalian sperm are arranged into a cylinder-shaped sheath that surrounds the axial elements of the midpiece. Each mitochondrion is arranged so that its exterior surface faces three different sets of cellular organelles (Figs. 1, 2). These three surface domains include first, the outer-facing (abaxial) surface of the mitochondria that is oriented toward the plasma membrane (termed pms for plasma membrane surface); typically, a thin layer of cytoplasm separates the plasma membrane and mitochondria. The second surface domain consists of the inner-facing (adaxial) surface of the mitochondria that is oriented toward the central outer dense fiber-axoneme complex (termed axs for axoneme-facing surface); this surface is further distinguished by its association with a midpiece-specific cytoskeletal network, the submitochondrial reticulum (Olson and Winfrey, 1986). In cross sections the submitochondrial reticulum appears as a radially arrayed set of electron-dense plaques t h a t adhere to the outer mitochondrial membrane (Fig. 1). The third surface domain includes the lateral surfaces and ends of the mitochondria that are in close apposition with the neighboring mitochondria (termed mms for mitochondria-mitochondria surface); typically the outer membranes of adjacent mitochondria are separated by a space of about 5-6 nm. The mitochondrial interior composed of the inner mitochondrial membrane and the mitochondrial matrix exhibits no apparent regional differences in structure corresponding to the three surface domains. Effects of Physical Disruption on the Mitochondria1 Sheath In intact spermatozoa, sections tangential to the mitochondrial surface can include superimposed views of the plasma membrane, cytoplasm, the outer and inner mitochondrial membranes, and, the mitochondrial matrix. To identify domain-specific specializations of the mitochondrial surface, sperm were physically disrupted and then extracted to strip both cytoplasmic components and inner mitochondrial membrane-matrix elements from the outer mitochondrial membrane. First, either sonication or nitrogen cavitation was employed to partially remove the midpiece plasma membrane and disperse the underlying cytoplasm. These treatments did not disrupt the mitochondrial sheath and the outer and inner mitochondrial membranes as well as the mitochondrial matrix appeared intact. How-
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Fig. 1. Transverse section of intact hamster sperm midpiece and principal piece. The central outer dense fiber (do-axoneme (ax) complex is surrounded by the mitochondrial sheath (m) in the midpiece and by the fibrous sheath (fs)in the principal piece. The three surface domains of the outer mitochondrial membrane include that facing the plasma membrane (pms),the domain facing other mitochondria (mms) and the domain facing the outer dense fiber-axoneme complex (axs). Note that the axs-domain is associated with plates of electron dense
material which comprise the submitochondrial reticulum (smr). The interior of the mitochondria consists of an electron dense matrix and the inner mitochondrial membrane (imm) that exhibits cristae-like infoldings. pm, plasma membrane. ~ 8 1 , 7 5 0 . Fig. 2. Longitudinal section of an intact midpiece again showing the three surface domains (pms, mms, and axs) of individual mitochondria. df = outer dense fibers. X39,750.
ever, the removal of the electron dense cytoplasm facilitated identification of structural interactions between mitochondria. In frontal sections, it is noted that the 5 to 6-nm gap between mitochondria is maintained after physical disruption. Moreover, the mms-domains of adjacent mitochondria are joined by stud-like bridging elements spaced at intervals of about 20 nm center to center (Fig. 3). Transverse sections of mitochondria also reveal the studs between apposed mitochondrial surfaces (Fig. 4).The combination of frontal and transverse views indicates that the network of studs forms a two-dimensional network present throughout the regions of mitochondria-mitochondria contact (Figs. 3,4). These studs are not noted either on the plasma membrane-facing surface or the adaxial-facing surface of the mitochondria which suggests they represent a domain-specific specialization.
The axoneme-facing (axs) domain of the outer mitochondrial membrane maintained its structural association with the submitochondrial reticulum which in longitudinal section appears comprised of longitudinally oriented bands of electron dense material extending the length of the midpiece (Figs. 4, 5 ) . Tangential sections of the mitochondrial surface include contributions from the outer and inner mitochondrial membranes a s well as the mitochondrial matrix, making it difficult to resolve specific membrane specializations.
Effects of Reducing Agents and Elevated pH on Midpiece Structure Sonicated sperm and isolated flagella were incubated in extraction solutions with and without the reducing agent DTT and examined by phase contrast and electron microscopy. By phase contrast microscopy, the
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Fig. 3. Longitudinal section of the midpiece of a sperm subjected to nitrogen cavitation. The mitochondrial sheath remains intact and in frontal views the mms-domain of the outer mitochondrial membrane (om) of adjacent mitochondria are linked by a network of stud-like bridges (5). x63,OOO. Fig. 4. Longitudinal profile of midpiece after nitrogen cavitation. The mitochondria are seen in cross section views and the mms-domain shows the network of studs ( s ) joining successive mitochondria. The
axs-domain remains associated with the longitudinal bands of electron dense material comprising the submitochondrial reticulum (smr). ~71,250. Fig. 5. Longitudinal profile of midpiece subjected to nitrogen cavitation. The continuous electron dense bands of the submitochondrial reticulum (smr) and their association with the axs-domain of the mitochondria are shown. s, network of studs joining the mms-domains. X66,OOO.
midpiece of control samples incubated in TNI buffer appears smooth and individual mitochondria are not resolved; the midpiece segment is distinguished from the thinner principal piece segment by the abrupt reduction in flagellar diameter at the annulus, the diameter of the tail then progressively tapers throughout the principal piece (Fig. 6a). After treatment with solutions
containing DTT the midpiece is no longer smooth but is instead decorated by numerous phase dense vesicles that appear to be derived from the mitochondrial sheath (Fig. 6b-d). In both pH 7.5 and pH 9.0 extraction solutions, some release of single and interconnected aggregates of mitochondrial sheath components from the underlying midpiece were noted (Fig. 6b-d). Extraction
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Fig. 6. Phase contrast photomicrographs showing sperm flagella extracted without (a)and with (b-e) DTT a t pH 7.5 ( a x ) , p.H 9.0 (d) and pH 11.0 (el. cp, connecting piece; an, annulus; pp, principal piece; mp, midpiece. Note that in 6a the midpiece segment appears smooth but that in 6b-e vesicular elements are associated with the mitochondrial sheath. Single vesicles detached from the midpiece are evident
(arrowheads) and in 6d a n interconnected array of mitochondrial sheath elements are partially released from the underlying outer dense fiber-axoneme complex of the midpiece (mp). Note in 6e that after extraction at pH 11.0 many vesicular elements remain interconnected. x 1,560.
at pH 11.0 in the presence of DTT resulted in substantial dissolution of the underlying flagellar fibers, however, mitochondrial sheath components remained intact and appeared as interconnected arrays of phase dense vesicles (Fig. 6e). Electron microscopic analysis revealed specific DTTmediated alterations in mitochondrial sheath structure. Most notably, the inner mitochondrial membrane and its enclosed matrix lost its close association with the outer mitochondrial membrane. These internal mitochondrial components formed rounded vesicles that were displaced toward the pms-domain of the outer mitochondrial membrane and frequently they appeared to bleb from this surface of the mitochondria (Fig. 7; see Figs. 11, 12). Identification of domain-specific specializations of the outer mitochondrial membrane was facilitated by displacement of the inner membranematrix complex as sections tangential to the outer membrane included no additional superimposed structures. The outer mitochondrial membrane of DTT-extracted flagella maintained structural relationships typical of intact mitochondria (Figs. 7-10). Adjacent
mitochondria remained associated via the network of bridges on the mms-domain of the outer mitochondrial membrane (Figs. 9 and 10). Tangential views of the mms-domain reveal that the studs are arrayed as a two dimensional lattice with individual studs spaced about 20 nm center to center (Figs. 8 and 10). Domain-specific structural specializations are also noted on the axoneme-facing surface domain (axs) of the outer mitochondrial membrane of DTT-treated flagella. This surface is decorated by an ordered pattern of parallel stripes extending over the mitochondrial surface (Figs. 7-10). The stripes are spaced 30- to 32-nm center to center and oriented a t an oblique angle relative to the longitudinal axis of the mitochondria. Stripes of adjacent mitochondria exhibit the same orientation (Fig. 7). This striped pattern is noted on the axoneme-facing surface of mitochondria that remain adherent to the midpiece (Figs. 7, 8) as well as those that have been released from the underlying flagellar elements (Figs. 9,lO). Flagella extracted at pH 9.0 (Fig. 11)or pH 11.0 (Fig. 12) also exhibited the striping pattern on the axs-domain.
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Fig. 7. Longitudinal section of flagellum of sperm that was first disrupted by sonication and then extracted a t pH 7.5 with DTT. The outer mitochondrial membranes remain intact and retain the overall organization of the mitochondrial sheath but appear somewhat more irregularly shaped in cross section. The inner mitochondrial membrane and matrix elements (*j are displaced toward the pms-domain of the mitochondrial membrane. Successive mitochondria remain adjoined along their mms-domain and in frontal views it is apparent that the axs-domain of the outer mitochondrial membrane exhibits a repetitive oblique striping pattern (arrowheads). Note the similar orientation of the striping in successive mitochondria of the sheath. The
pms-domain when seen in frontal views (open arrows) does not exhibit this striping pattern. df, dense fibers. X67,500. Fig. 8. Section through the midpiece of a sperm extracted at pH 7.5 with DTT. Note the submitochondrial reticulum (smr) maintains its association with the axs-domain of the mitochondria. Small areas of each mitochondrial surface domain are shown in frontal view. Note that the axs-domain displays a regular striping pattern (arrowheads). Apposed membranes of the the mms-domain (bracketed area) exhibit a two dimensional array of punctate profiles. The pms-domain appears homogenous and no specializations are noted (open arrows). df, outer dense fibers; ax, axoneme. ~66,000.
Thin section analysis of DTT-treated sperm reveals no distinctive differentiations of the plasma membrane-facing (pms) domain of the outer mitochondrial membrane (Figs. 7, 8, 11, 12). The pms-domain appeared to be the most fragile of the three outer mitochondrial membrane domains a s occasional ruptures were noted that appeared to represent the site of exit of
the swollen inner mitochondrial membrane-matrix vesicles (Figs. 11, 12). While the lumen of the outer mitochondrial membrane was approximately 0.2 +m in width, the inner mitochondrial membrane-matrix vesicles measured approximately 0.6 pm in diameter and no cristae-like folds of the inner mitochondrial membrane into the vesicle interior were noted (Fig. 10).
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Fig. 9. High-power view of outer mitochondrial membranes after extraction in TNI containing DTT. Note the studs ( s ) attaching the mms-domains of the adjacent mitochondria and the regular striping pattern (arrowheads) on the axs-domain. ~90,000. Fig. 10.Thin section showing mitochondria that have been released from flagellum of DTT treated flagella. The orientation is equivalent to a cross-sectional view of the flagellum. In areas denoted by brack-
ets, the section includes enface views of the mms-domain and includes the outer membranes of two adjacent mitochondria; in these regions note the two dimensional array ofdots that represent the studs seen in cross sectional views of the membrane. A portion of the axs-domain is also shown and note its regular striped pattern (arrowheads).
DISCUSSION
piece length, mitochondrial size, mitochondrial morphology, and mitochondrial arrangements are evident (Fawcett, 1975; Phillips, 1974). In somatic cells cytoskeletal elements including microtubules and actin filaments have been shown to participate in directed
The spermatozoan midpiece of diverse mammalian species is comprised of the same set of cellular organelles. Although these organelles are arranged upon a common theme, significant species differences in mid-
X90,OOO.
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Fig. 11. Longitudinal section though midpiece segment of sperm extracted at pH 9.0 with D". Note the inner mitochondria membrane-matrix vesicles (imm) which are associated with the pms-domain of the outer mitochondrial membrane. The pms-domain appears homogeneous in frontal views while the axs-domain exhibits the transverse striping (arrowheads). df, outer dense fiber; ax, axoneme. ~54,000. Fig. 12. Slightly oblique cross section through the midpiece of a sonicated sperm extracted at pH 11.0 with DTT. Residual flagellar
material (*) remains within the lumen. Note the outer mitochondrial membranes remain associated with one another but their interior appears mostly empty. The inner mitochondrial membrane-matrix complexes (imm) appear as relatively large, 0.6 Fm diameter, vesicles which bleb from the pms-domain of the outer mitochondrial membrane; arrowheads denote sites of rupture of the pms-domain. Note the regular striping pattern is retained on the axs-domain of the outer mitochondrial membrane. X 54,000.
mitochondrial movements (Schliwa, 19861, but the mechanisms that direct the formation of the spermatid mitochondrial sheath are unresolved.
Sperm mitochondria share common structural features with mitochondria of somatic cells (Fawcett, 19'751, but previous studies have noted some unusual
SURFACE DOMAINS OF SPERM MITOCHONDRIA aspects of their surface structure, For example, filamentous linkages joining adjacent mitochondria have been identified (Olson and Hamilton, 1976; Olson and Winfrey, 1986), but it was not appreciated that these bridging elements form an extensive two-dimensional network between the apposed mitochondrial surfaces as demonstrated in the present study. Also, using the quick-freeze,deep-etch, freeze-fracture technique it has been shown that the adluminal surface of the mitochondria are decorated by an apparently unique, ordered array of particles (Friend and Heuser, 1981);these investigators speculated that these structural specializations could reflect a specific localization of enzymes or transport proteins. The paracrystalline striping pattern identified in the present study on the axonemefacing surface, termed axs-domain, of the outer mitochondrial membrane appears to represent the structural specialization previously noted by freeze fracture; moreover the resistance of this membrane specialization t o the elevated pH and ionic strength extraction regimens employed in the present study raises the possibility that this paracrystalline pattern reflects an ordered arrangement of integral membrane proteins. One additional unique feature of the axs-domain of the outer mitochondrial membrane is its specific adhesion to the submitochondrial reticulum, a midpiece specific cytoskeletal complex (Olson and Winfrey, 1986; Olson and Winfrey, 1990); whether this interaction is mediated by domain-specific polypeptides requires resolution. The specific proteins responsible for generating the domain specific differentiations of the outer mitochondrial membrane remain to be identified. A low-molecular-weight cysteine-rich polypeptide, termed MCP, has been ascribed to the mitochondrial capsule by previous studies (Calvin et al., 1981; Pallini et al., 1979). However the structural differentiations of the mms- and axs-domains persist after extraction with disulfide bond reducing agents, a treatment that efficiently solubilizes MCP (Pallini et al., 1979), so these structural specializations apparently neither represent nor are dependent on MCP for their maintenance. The outer mitochondrial membrane of yeast and somatic cells is comprised of a limited set of polypeptides (Sollner et al., 1989) and some of the major components function as receptors for the import of cytoplasmic polypeptides with appropriate signal sequences (Baker and Schatz, 1991).Although the polypeptide pattern of sperm mitochondria has been analyzed (Hecht and Bradley, 1981) proteins specific to the outer mitochondrial membrane remain to be identified and compared with those identified in other systems. The present study demonstrates that the outer membrane of the midpiece mitochondria possesses three structurally distinct domains. We speculate that these structural specializations reflect differences in protein composition between domains and that these different domains could participate in specific events in the the assembly andlor biochemical function of the mitochondrial sheath. For example, the network of studs located along the paired surfaces of adjacent mitochondria
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(mms-domain) could represent specific adhesion molecules required for the assembly or maintenance of the mitochondrial sheath; alternatively these linkages could function in intermitochondrial communication to coordinate their activities. Similarly, the unique paracrystalline structure of the axoneme-facing surface domain (axs-domain) suggests that it is composed of an assemblage of proteins that may mediate adhesion with the submitochondrial reticulum and may function in targeting and/or maintaining mitochondria a t the developing midpiece. Finally, the mitochondrial surface facing the plasma membrane, the pms-domain, differed from the other domains by the absence of differentiations identifiable by thin section analysis; however its adjacent proximity to the plasma membrane suggests this domain might be specialized for importation of metabolites used for energy generation. If different mitochondrial surface domains possess unique permeability properties, the resultant vectorial flow of specific compounds could concentrate molecules required for motility in the cytoplasm immediately surrounding the axoneme. These various possibilities, although intriguing, will require the identification of domain-specific proteins and the elucidation of their specific protein interactions and enzymatic activities.
ACKNOWLEDGEMENTS This work was supported by NIH grant HD20419 and by Center grant HD05797.
REFERENCES Baker KP, Schatz G (1991): Mitochondria1 proteins essential for viability mediate protein import into yeast mitochondria. Nature 349:205-208. Calvin HI, Cooper GW, Wallace E (1981): Evidence that selenium in rat sperm is associated with a cysteine-rich structural protein of the mitochondrial capsules. Gamete Res 4:139-149. Calvin HI, Grosshans K, Musicant-Shikora SR, Turner SI (1987): A developmental study of rat sperm and testis selenoproteins. J Reprod Fertil81:l-11. Calvin HI, Bedford J M (1971): Formation of disulphide bonds in the nucleus and accessory structures of mammalian spermatozoa during maturation in the epididymis. J Reprod Fertil Suppl. 13:65-75. Clermont Y, Oko R, Hermo L (1990): Immunocytochemical localization of proteins utilized in the formation of outer dense fibers and fibrous sheath in rat spermatids: An electron microscope study. Anat Rec 227:447457. DeMartino C, Floridi A, Marcante ML, Malorni W, Scorza-Barcellona P, Belloci M, Silvestrini B (1979): Morphological, histochemical and biochemical studies on germ cell mitochondria of normal rats. Cell Tissue Res 196:l-22. Eddy EM (1988):The Spermatozoon. In E Knobil, J D Neil1 (eds):“The Physiology of Reproduction.” New York: Raven Press, pp 27-68. Fawcett DW (1975):The mammalian spermatozoon. Dev Biol443394436. Friend DS, Heuser J E (1981):Orderly particle arrays on the mitochondrial outer membrane in rapidly-frozen sperm. Anat Rec 199:159175. Hecht NB, Bradley FM (1981):Changes in mitochondrial protein composition during testicular differentiation in mouse and bull. Gamete Res 4:433-449. Kleene KC, Smith J , Bozorgzadeh A, Harris M, Hahn L, Karimpour I, Gerstel J (1990): Sequence and developmental expression of the mRNA encoding the seleno-protein of the sperm mitochondrial capsule in the mouse. Dev Biol137:395402. Olson GE, Hamilton DW (1976): Morphological changes in the midpiece of wooly opossum spermatozoa during epididymal transit. Anat Rec 186:387404.
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Olson GE, Winfrey VP (1986):Identification of a cytoskeletal network adherent to the mitochondria of mammalian spermatozoa. J Ultrastruct Mol Struct Res 94:131-139. Olson GE, Winfrey VP (1990): Mitochondria-cytoskeleton interactions in the sperm midpiece. J Struct Biol 103:13-22. Pallini V, Baccetti B, Burrini AG (1979): A peculiar cysteine-rich polypeptide related to some unusual properties of mammalian sperm mitochondria. In DW Fawcett, J M Bedford (eds): “The Spermatozoon.” Baltimore: Urban and Schwarzenberg, pp 141-151. Phillips DM (1974):“Spermiogenesis.”New York: Academic Press.
Phillips DM (1977):Mitochondria1 disposition in mammalian spermatozoa. J Ultrastruct Res 58:14&154. Schliwa M (1986): “The Cytoskeleton an Introductory Survey.” Wien New York: Springer-Verlag. Sollner T, Griffiths G, Pfaller R, Pfanner N, Neupert W (1989): MOM19, an import receptor for mitochondria1 precursor proteins. Cell 59:1061-1070. Woolley DM (1970):The midpiece of the mouse spermatozoon: Its form and development as seen by surface replication. J Cell Sci 6:865879.
