EZZATOLLAH KEYHANI* AND LARRY F. LEMANSKIfLaboratory for Cell Biology and Biochemistry, Institute of Biochemistry andBiophysics, University of Tehran, P.O. Box 314-1700, Tehran, Iran
SUMMARY
Thin-section and freeze-fracture electron microscopy of immature and mature spermatozoaof the Mexican axolotl, Ambystoma mexicanum, revealed numerous small spherical mito-chondria with diameters ranging from 0-15 to 022 fim. Both the spherical form and the smallsize of these mitochondria were confirmed by serial thin-section studies. In mature spermatozoa,the mitochondria are located in the midpiece region, in tight contact with each other, exhibitingan almost crystalline arrangement. They do not surround the midpiece, but form a semi-circular sheet over the sustained filament. The portion of the midpiece on the side of the un-dulating membrane and the flagellum is devoid of mitochondria. The plasma membrane in themidpiece region is tightly apposed to the mitochondria, so that in freeze-fracture or scanningelectron microscopy the mitochondria seem to protrude through the plasma membrane. Wesuggest that the unusual organization of mitochondria in axolotl sperm facilitates the oxidativeprocesses and increases the efficiency of ATP production and/or distribution within the cell.
INTRODUCTION
Among the mitochondria of various cells and tissues, the mitochondria of sperma-tozoa have been the subject of many studies and the unusual development of mito-chondria during spermatogenesis has been reported by many authors. Earlier reports(Meves, 1907; Duesberg, 1910; Bowen, 1920, 1922; Johnson, 1931) describe thevarious steps by which the numerous mitochondria are clustered together and fusedto produce a large spherical mass, termed the nebenkern. The nebenkern is eventuallydisaggregated and, following a series of morphological changes, migrates into themidpiece region closely associated with the flagellum. With the advent of electronmicroscopy the complexity of mitochondrial development during spermatogenesisbecame apparent. The detailed electron-microscopic studies of Kaye (1958), Andre'(1962) and Pratt (1968) demonstrate the progressive fusion of numerous spermatocytemitochondria into 2 interwoven labyrinthine nebenkern and their subsequent re-arrangement and eventual segregation. Other electron-microscopic studies showed anessential similarity between nebenkern from a wide range of species of vertebrates andinvertebrates. The subsequent dissociation of the nebenkern, together with the
• Present address: Johnson Research Foundation G4, University of Pennsylvania, Schoolof Medicine, Philadelphia, Pa 19104, U.S.A., to whom correspondence should be addressed.
f Present address: Anatomy Department, University of Wisconsin-Madison, Bardeen Medi-cal Laboratories, 1215 Linden Drive, Madison, Wise. 53706, U.S.A.
450 E. Keyhani and L. F. Lemanski
elongation of the nucleus and the development of flagella, leads to the formation ofspermatozoa in which mitochondria exhibit various types of organization (Bawa,1964; Reger & Florendo, 1969; Phillips, 1970; Warner, 1971).
In this paper, using thin-section and freeze-fracture transmission electron micro-scopy, and scanning electron microscopy, we describe the structure and unusualorganization of mitochondria in the spermatozoa of the Mexican axolotl, Ambystomamexicanum. Although these mitochondria exhibit a basic structure similar to the mito-chondria from other tissues, e.g. outer and inner membranes and cristae, they arevery small, even smaller than the smallest mitochondria so far described in the litera-ture (Munn, 1974). They do not surround the axial filament (or sustained filament,Burgos & Fawcett, 1956), but rather form a semi-circular sheet over a dense core inthe midpiece region. We suggest the term micromitochondria to designate these verysmall mitochondria.
MATERIALS AND METHODS
Eleven adult male axolotls were killed after anaesthesia in tricaine methanesulphonate (MS222).
Thin section
The testes and vas deferens were removed separately and immediately diced into i-mmpieces in: (1) 2-5 % or 4% glutaraldehyde buffered with 01 M-phosphate to pH 74 for 4 to24 h; the tissues were rinsed in 01 M-phosphate buffer at pH 7-4 containing 10% sucrose,post-fixed in 2 % osmium tetroxide in o-i M-phosphate buffer, pH 7-4; or (2) 4 % unbufferedosmium tetroxide (Claude, 1961; Keyhani, 1969). The tissues were dehydrated in ethanol andembedded in Epon according to Luft's (1961) procedure. Thick sections were examined undera light microscope to select areas of interest and then thin sections were made on a Porter-BlumMT-2 ultramicrotome with a diamond knife and were collected on 400-mesh grids. In the caseof serial-section studies, each individual section was mounted in a single-hole grid coated withFormvar. The sections were doubly stained with uranyl acetate and lead citrate (Reynolds,1963)-
Freeze-fracture
The vas deferens was carefully removed and flushed with o-i M-phosphate buffer, pH 7-4.The sperm cells were centrifuged and resuspended in a small volume of buffer. The testis wasdiced into i-mm pieces. The diced testis and pelleted sperm were either used unfixed or fixedwith 2-5% glutaraldehyde in o-i M-phosphate buffer, pH 7'4. Fixed or unfixed testes andspermatozoa were incubated in 30 % glycerol for 30 min at 0-4 °C. The suspension of sper-matozoa (approx. 3 fi\) or small pieces of testis were transferred to a side-loading gold-apposedspecimen holder and rapidly quenched in liquid Freon 22 (chlorodifluoromethane), cooled to
Fig. 1. Section through a portion of spermatid (sp) and neighbouring Sertoli cell (sc).The spermatid mitochondria are small and spherical with circular cristae (arrows).Most of them showed an electron-dense bleb protruding outward (arrow). The sizeof spermatid mitochondria should be compared to that of the mitochondria of theSertoli cell on the left, m, mitochondria, x 62000.Fig. 2. Freeze-fracture of the region of immature spermatozoa. The fracture plane wasoutside the nucleus. At this stage of differentiation the formation of flagella wascomplete. The flagellum (/) was attached to the rest of the cytoplasm by the un-dulating membrane (urn). The mitochondria (m) were scattered throughout thecytoplasm, x 29000.
Micromitochondria in axolotl spermatozoa
452 E. Keyhani and L. F. Lemanski
its freezing point by liquid nitrogen. Freeze-fracture was done by standard techniques (Moor& Miihlethaler, 1963) as described by Keyhani & Kriz (i960) and Keyhani (1970), in a DentonDV-50Z freeze-fracture apparatus. Replicas were cleaned in Chlorox (sodium hypochlorite),washed in distilled water and mounted on 4OO-mesh grids.
Scanning electron microscopy
The vas deferens was clamped with surgical threads at approx. 4-mm intervals, and cut inlengths clamped near each end; this precaution prevents the loss of spermatozoa. The clampedpieces of vas deferens were fixed with 2'5 % glutaraldehyde in O'i M-phosphate buffer, pH 7-4.They were dehydrated in ethanol. They were opened at one side so that the inside of the vasdeferens was exposed. The tissue was subsequently dried at the critical point from COj. Thedried tissue was mounted, coated with carbon and gold/palladium, and examined in a CambridgeStereoscan scanning electron microscope.
RESULTS
The mitochondria in the spermatozoa of the Mexican axolotl A. mexicamtm werespherical at the late stage of spermatic! differentiation. Fig. 1 shows the mitochondriain the immature spermatozoa. At this stage of differentiation the elongation of thenucleus and the formation of the tail are complete but the mitochondria have not yetmigrated into the midpiece region. The spherical mitochondria are distributedrandomly throughout the cytoplasm. They are much smaller in size than the mito-chondria of the surrounding Sertoli cells (see Fig. 1). The measured diameter of themitochondria of spermatozoa ranged from 0-15 to 0-22 fim, compared to the 0-2 firnwidth and 1 /an length of the mitochondria of Sertoli cells. However, sperm mito-chondria were similar in fine structure to those of other tissues (Munn, 1974). Theyconsist of an outer and an inner mitochondrial membrane. The cristae are circularand are attached to the inner membrane at one or two points. Many of the mito-chondria exhibit an outwardly protruding electron-dense bleb. The bleb is continuouswith both outer and inner membranes (Fig. i, arrow) and was present regardless ofthe fixation procedure. Freeze-fracture of the sperm at a similar stage of differentia-tion is illustrated in Fig. 2. At this stage the undulating membrane (Burgos & Fawcett,1956) bearing the flagellum is fully differentiated and spans the full length of thespermatozoa. The mitochondria were randomly distributed, and most of them showed
Fig. 3. Longitudinal section through a portion of the midpiece of a spermatozoonnear maturity. At this stage all mitochondria had migrated to the midpiece; some ofthem (arrows) were not yet integrated into the rest of the mitochondrial complex,x 21000.
Figs. 4-6. Longitudinal sections through different levels of the midpiece of a maturespermatozoon showing the arrangement of mitochondria.
Fig. 4. Longitudinal tangential section, x 21000.Fig. 5. Longitudinal section through intermediate region of the midpiece. The
flagellum (/) is seen but the section does not include the sustained filament, x 15 000.Fig. 6. Longitudinal section of midpiece at the sustained filament ($f) region,
x 10000.
Fig. 7. A higher magnification of Fig. 4. Note no blebs are visible in mitochondria ofmature spermatozoa (compare with Fig. 1). x 63 000.
Micromitochondria in axolotl spermatozoa 453
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Figs. 8-11. Serial sections of the midpiece region. The arrows indicate a group ofmitochondria from the top tangential section (Fig. 8), through medial sections (Figs.8-10) and the bottom tangential section (Fig. 11). x 30000.
surface rather than cross-fracture. As maturation progresses the mitochondria migrateinto the midpiece region where they become tightly packed into a highly organized,almost crystalline arrangement. Fig. 3 shows the arrangement of mitochondria in themidpiece region of immature spermatozoa, and should be compared with the maturespermatozoa of Figs. 4-6. In Fig. 3, the mitochondria are uniformly spherical orovoid. The blebs present in the mitochondria in the late stage of spermatid differen-tiation (as seen in Fig. 1) were no longer seen in mature spermatozoa (Figs. 3-6). Thetypical arrangement seen in mature spermatozoa was not yet complete, however,since some mitochondria (arrows in Fig. 3) were not yet integrated into the rest ofthe mitochondrial complex. Figs. 4, 5 and 6 show longitudinal sections through 3distinct midpiece regions exhibiting the arrangement of mitochondria in mature
Figs. 12-16. Freeze-fracture of the midpiece region of mature spermatozoon.Fig. 12. Longitudinal medial fracture of the midpiece region of immature sper-
matozoon, n, nucleus; m, mitochondria, x 22800.Fig. 13. Longitudinal medial fracture of the midpiece region of mature spermato-
zoon. The fracture plane includes surface fracture of the mitochondria and sustainedfilament (rf). x 18000.
Fig. 14. Freeze-fracture of the A(PF)-face of the plasma membrane at the midpieceregion. Note the abundance of IMP when compared to the B(EF)-face (Fig. 15) andthe imprint of the micromitochondria. x 33000.
Fig. 15. Freeze-fracture of the B-face of plasma membrane at the midpiece region.Note a few IMP and the imprint of micromitochondria. x 15000.
Fig. 16. Freeze-fracture of spermatozoon at the midpiece region. The plasmamembrane (pm) has been partially removed, um, undulating membrane; m, mito-chondria, x 24000.
Micromitochondria in axoltl spermatozoa 4S3
456 E. Keyhani and L. F. Lemanski
pm~
Micromitochondria in axolotl spermatozoa 457
spermatozoa. They are longitudinal tangential sections (Fig. 4), and longitudinalmedial sections of 2 different depths in the midpiece region. Fig. 4 also shows theflagellum, while Fig. 6 shows the sustained filament. In all 3 sections, the mitochon-dria were tightly packed. The cytoplasm in this region wa9 no longer abundant andthe plasma membrane was now apposed closely to the mitochondrial layer so thatvisualization of the plasma membrane in this region was difficult in thin sections.Higher magnification (Fig. 7) showed that the spherical mitochondria were closelyapposed to each other. The small size and spherical form of these mitochondria werefurther analysed by serial section as illustrated in Figs. 8-13. In 4 consecutive sections(Figs. 8-11) the entire diameter of the mitochondria was sectioned. Considering thethickness of each section to be 60-80 nm, the diameter of the mitochondria would bec-24-0'32 fim, close to the value obtained by direct measurement of random sections.The longitudinal and medial fracture of the mature spermatozoa were shown inFigs. 12, 13. In both figures the plasma membrane was easily visible. The sphericalmitochondria exhibit an organization essentially similar to that seen in thin section(Figs. 3-7). Only surface fractures of mitochondria were visible. The maximumdiameter of mitochondria in these figures was 0-28 ju,m, close to the values obtainedby random or serial thin section. The tight apposition to the plasma membrane wasmade clearly visible in mature spermatozoa by freeze-fracture (Figs. 14, 15). Figs.14 and 15 are the A(PF) and B(EF)-faces, respectively, of the plasma membrane ofthe midpiece region of mature spermatozoa. The intramembrane particles (IMP)are more abundant in the A(PF) than in the B(EF)-face. In both the A(PF) and B(EF)-faces, it is evident that the plasma membrane is tightly apposed to the mitochondriaso that the mitochondria protrude from the plasma membrane. The plasma membraneand the surface view of the mitochondria can be seen upon partial removal of theplasma membrane during freeze-fracture (Fig. 16). Occasionally mechanical disrup-tion of the plasma membrane loosens the packing of the mitochondria (Fig. 17) andleads to their release from the midpiece (compare with Fig. 18).
While the true surface of the plasma membrane can be visualized by deep-etchingduring the freeze-fracture study, scanning electron microscopy provides easy accessto this surface and furnishes additional information about the relationship betweenthese mitochondria and the plasma membrane. In Figs. 19 (low magnification) and20 (high magnification), the general morphology of mature spermatozoa and the
Fig. 17. Transverse cross-fracture of unfixed spermatozoa at midpiece region. Theplasma membrane (pm) is disrupted. Some mitochondria (m) are liberated, sf, sus-tained filament; um, undulating membrane;/, flagellum. x 32000.Fig. 18. Transverse cross-fracture of spermatozoa at the midpiece region, to becompared with Fig. 17. um, undulating membrane; sf, sustained filament; m, mito-chondria ; / , flagellum. x 31 500.Fig. 19. Low-power scanning electron microscopy of spermatozoa in the vas deferens.Note undulating membrane (um), spanning the entire length of the midpiece and thetail of the spermatozoon, h, head, x 4000.Fig. 20. High magnification of midpiece region as revealed by scanning electronmicroscopy. The imprints of micromitochondria (m) on the plasma membrane arevisible (arrows), um, undulating membrane, x 20000.
458 E. Keyhani and L. F. Lemanski
organization of the mitochondria at the midpiece region, respectively, are revealed byscanning electron microscopy. Fig. 19 shows spermatozoa located in the vas deferens.The epithelium lining the vas deferens contains numerous cilia, which are presumablyinvolved in helping the passage of the spermatozoa.
The undulating membrane starts at the neck portion of the spermatozoon and spansthe entire length of the midpiece and tail. At high magnification (Fig. 20), the imprintsof numerous mitochondria on the plasma membrane of the midpiece region arevisible, giving further indication of the tight contact between mitochondria and plasmamembrane.
DISCUSSION
Both light and electron microscopy have shown that the mitochondria have a varietyof shapes and sizes, including spherical or ovoid, rod-shaped, branched-reticular,cup-shaped and extensive reticular forms (Munn, 1974). The size and shape of mito-chondria are modified by environmental and nutritional factors (Tandler, Erlandson& Wynder, 1968; Keyhani, 1973) as well as in pathological states (Kiesseling &Tobe, 1964; Seljelid & Ericsson, 1965; Keyhani, 1969). The average reported diameterof a mitochondrion varies between 0-5 and 10 fim (Munn, 1974). Random thin-section,serial-section and freeze-fracture studies reported in this paper indicate that A.mexicanum spermatozoa contain the smallest mitochondria ever described. Theiraverage diameter is 0-24 fim. and they are almost equal in size to some pleuropneu-monia agent viruses (Morowitz & Tourtellotte, 1962). Thus the term micromitochon-dria seems appropriate to designate these organelles. These mitochondria arise fromfragmentation of the elongated mitochondria at the stage of spermatid differentiation.
Each mitochondrion at the late stage of spermatid differentiation shows a blebprotruding outward. One explanation for the presence of blebs would be that theyrepresent involutions of portions of the mitochondria during fragmentation leading tothe formation of small spherical organelles.
An important question is whether, despite such extensive modifications, the mito-chondria of axolotl spermatozoa preserve their functional capacity, e.g. respirationand oxidative phosphorylation. An attempt to determine the spectral properties andmeasure substrate oxidation has not been successful owing to the difficulty of obtainingadequate amounts of material for biochemical studies. Light-microscopic cytochemicalstudies using 3,3'-diaminobenzidine to stain cytochrome c oxidase, however (Selig-man, Karnovsky, Wasserkrug & Hanker, 1968; Keyhani, 1972), showed a positivereaction only at the level of the midpiece, the region containing mitochondria. Bythis method the length of the midpiece containing the mitochondria was estimated tobe 80-100 /tm. Cross-fracture of the midpiece indicated that a row of about 10 mito-chondria partially covers the midpiece region. Considering that each mitochondrionon average has a diameter of 0-24/tm, the number of mitochondria in the midpiececan be estimated to be between 3200 and 4000.
According to Bishop (1962), spermatozoa of animals reproducing by internal ferti-lization, e.g. mammals and birds, utilize glycolytic substances under both anaerobic
Micromitochondria in axolotl spermatozoa 459
and aerobic conditions, and respiratory substrates in the presence of oxygen. In-vertebrate sperm, as well as those of frogs, which reproduce by external fertilization,are oxygen-dependent and depend principally on aerobic metabolism. Since the axolotl,like the frog, reproduces by external fertilization, its spermatozoa may be oxygen-dependent and contain respiratory pigments to generate ATP; thus the mitochondriamay function in a manner similar to those from other tissues despite their small size.The small size of the mitochondria, however, may be an indication of the low meta-bolic activity of these organelles, since in some tissues, e.g. insect flight muscle (Lennie& Birt, 1967; Smith, 1968; Afzelius & Gonnert, 1972), the high metabolic activity isparallel to the large size of the mitochondria. Another possibility is that the small size,high number and unusual organization of mitochondria in axolotl sperm facilitate theoxidative process, presumably, by increasing substrate accessibility, efficiency of ATPproduction and/or ATP distribution within the cell. Thus the wide range of diff-erences in the fine structure of spermatozoa among various species, particularly withrespect to the organization of their mitochondria, suggests extreme evolutionaryadaptation to different environmental conditions. Indeed the spermatozoa of variousspecies exhibit a wide range of tolerance toward fluctuations in the concentration ofsalt, pH, cations, etc. The sperm of the marine teleost Gillichthys, for example, with-stand a range of osmotic pressure equivalent to 17-200% sea-water (Weisel, 1948).Cytochrome c from mitochondria of various species leaks out after the mitochondriaare incubated in hypotonic medium (Jacobs & Sanadi, i960; Keyhani & Keyhani,1975). Cytochrome c from rabbit epididymal spermatozoa, however, despite thecomplete removal of the plasma membrane, remains firmly bound to the mitochondriaupon exposure of spermatozoa to hypotonic medium (Keyhani & Storey, 1973).Therefore, it is reasonable to assume that the unusual organization of mitochondria inthe spermatozoa in particular, and the structure of the sperm cell in general, are areflection of the adaptations necessarily evolved in order to cope with the exigencies ofthe variable conditions with which the spermatozoa must contend.
We thank Mr Massumi for taking care of the colony of axolotls. This investigation wassupported in part by the University of Tehran, Iran.
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