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Ultrastructure of mammalian spermiogenesis. I. A tubular complex in developing sperm of the cottontop marmoset Sequinus oedipus
© 1970 by Academic Press, Inc.
316
s. ULTRASTRUCTURERESEARCH32, 316-322 (1970)
U l t r a s t r u c t u r e of M a m m a l i a n Spermiogenesis I. A Tubular Complex in Developing Sperm of the Cottontop Marmoset Sequinus oedipus J. B. RATTNER AND B. R. BRINKLEY Section of Cell Biology, Department of Biology, The University of Texas, M. D. Anderson Hospital, and Tumor Institute at Houston, Houston, Texas 77025 Received December 12, 1969 A transitory tubular structure termed a tubular complex appears at the proximal end of the principal piece in mid-spermiogenesis of the cottontop marmoset. The structure is formed by tubules 300-330 A in diameter which are arranged both perpendicular to and helically around the axial filament complex. Disappearance of the structure occurs concomitantly with middle piece formation. The process of spermiogenesis has been reviewed in a variety of mammalian species, but many of the ultrastructural aspects have not been examined in detail (1). In most species, spermiogenesis is characterized by abrupt morphological changes, such as nuclear elongation and condensation, which along with acrosomal cap formation, result in the final shape and form of the sperm head. The surrounding cytoplasm becomes displaced posteriorly, and the distal centriole with its associated segmented neck region elongates to form the tail or axial filament complex (2). An important event associated with tail formation in late spermiogenesis is the development of a middle piece containing mitochondria arranged in a helical sheath around the axial filament complex. In the present study, the events of middle piece formation in a prosimian primate, the cottontop marmoset, are presented in detail. Of particular interest is an elaborate tubular complex composed of hexagonally packed microtubule-like structures. The body, termed a tubular complex (TC), surrounds the proximal end of the principal piece in mid-spermiogenesis and disappears as the middle piece is formed.
FIG. 1. Longitudinal sections showing fate of tubular complex (TC) during middle piece formation. (a) TC prior to mitochondrial sheath formation, x 13,500. (b) TC (arrow) at slightly later stage. x 14,000. (c) TC (arrow) at the end of middle piece formation, x 14,500. (d) TC is no longer present when middle piece is completely formed, x 15,000.
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MATERIALS AND METHODS Testes of the marmoset were removed, and the tunica was dissected to expose the seminiferous tubules. The tubules were carefully diced into small pieces with a sharp scalpel in a petri dish containing 3 % glutaraldehyde buffered at pH 7.4 with phosphate buffer (5). After 1 hour in glutaraldehyde at room temperature, the tubules were rinsed in two changes of phosphate buffer and postfixed for 60 min in 1% osmium tetroxide buffered in a similar manner. The tissues were rapidly dehydrated in a graded series of ethanol and embedded in Epon 812. Thick sections were cut with glass knives and mounted in immersion oil for inspection with the phase microscope. Selected areas were further trimmed and sectioned with a diamond knife using an LKB Ultrotome III. Sections were picked up on 200-mesh, unsupported copper grids or collodion-coated, slotted grids and stained in alcoholic uranyl acetate and then in lead citrate (7). The grids were examined in an Hitachi HU-11C electron miscroscope operated at 75 kV.
RESULTS AND DISCUSSION Spermiogenesis in the cottontop marmoset (Sequinus oedipus oedipus) followed a pattern of differentiation much like that described in other mammalian species (1). Early spermatids consisted of a spherical nucleus containing diffuse chromatin. The surrounding cytoplasm was generally free of rough endoplasmic reticulum and polysomes and contained numerous vacuolated mitochondria. The acrosomal vesicle which formed in association with a large Golgi apparatus developed into the acrosomal cap, which eventually covered the anterior two-thirds of the nucleus. The distal member of a centriole pair, located in a distinct indentation at the posterior margin of the nucleus, formed a basal body which gave rise to the axial filament complex containing typical 9 + 2 microtubules with associated outer fibrils. The tail structure continued to elongate throughout spermiogenesis, and the distal region differentiated into a principal piece with a well-developed fibrous sheath. An amorphous dense body, the ring centriole, formed in association with the plasma membrane just distal to the basal body. The manchette, a microtubular sheath (1, 3) appeared to develop from an amorphous ring at the posterior margin of the acrosomal cap and extended to a region several microns distal to the nucleus. With manchette formation, the cytoplasm became displaced posteriorly, and the nucleus elongated and became greatly condensed. In its final form, the nucleus became extremely condensed, flattened, and oval shaped.
Fio. 2. Higher magnifications of tubular complex during middle piece formation. (a) x 57,000. (b) x 58,550. (c) Ring centriole, x 40,500.
A TUBULAR COMPLEX IN MAMMALIAN SPERMATIDS
21 --701828 J . Ultrastructure Research
319
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RATTNER AND BRINKLEY
Middle piece formation in the cottontop marmoset occurred in later spermatids after complete condensation of the nucleus had occurred. At the onset, a spindleshaped tubular structure, which we have termed the tubular complex (TC), was observed at the distal third of the presumptive middle piece (Fig. 1 a). In its initial structure, the TC measured 170 m# in length and was composed of tubules arranged in both parallel and helical array around the axial filament complex (Fig. 3 a, b). At this stage, the total length of the presumptive middle piece was 630 m#. Transverse and longitudinal sections of the TC indicated that the tubules were packed in a hexagonal arrangement, which was displaced slightly to one side of the axial filament complex (Figs. 2 a and 3 a, b). The longitudinal tubules extended from the distal edge of the TC to various points along its length. Three-dimensional reconstructions of the structure by means of serial sections indicated that the longitudinal tubules were, in fact, continuous with the tubules which formed a helix around the axial filament complex. The tubules of the TC measured 300-330 A in outside diameter and were larger than the 250-270 A microtubules found in the manchette or the axial filament. By rotational photography (4) each tubule exhibited six subunits, each of which measured 90-100 A in diameter. Small side arms, 50 A in diameter, connected adjacent tubules. The arms were spaced 80-90 A apart along the tubules and established a center-to-center spacing of 600-620 A between adjacent tubules. Precise spacing and consistent side-arm attachments were maintained throughout the appearance of the complex. In a slightly later stage, the TC decreased in length to 150 m# and was composed entirely of helical tubules (Figs. 1b and 2b). It exhibited a triangular appearance in longitudinal section. The base of the triangle contained three tubular helices, with one extending into the anterior region of the collar. Reduction in the length and diameter of the TC signaled the onset of mitochondrial migration into the presumptive middle piece. At this time, the middle piece region measured 660 m/z. Mitochondria (usually four in number) became associated with the ring centriole at its anterior margin distal to the basal body. The ring centriole moved caudally along the middle piece (Fig. 1c), accompanied by the plasma membrane. Subsequently, the mitochondria aggregated around the axial filament complex. As the middle piece formed, the TC was reduced to 70 m# in length, and the total presumptive middle piece region increased to a length of 850 m# (Fig. 2c). As the ring centriole arrived at its final destination to form the annulus, or Jensen's ring, the TC completely disappeared. As shown in Fig. 1 d, the TC is absent from spermatids with mature middle FIG. 3. Serial transverse section of tubular complex showing tubules arranged in both helical and parallel fashionaround the axial filamentcomplex. (a) x 40,000. (b) x 40,000.
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pieces. At completion, the middle piece had a total length of 880 m# and contained approximately 30 mitochondrial profiles. The tubular complex has also been observed in other mammalian species. Nicander (6) and Pedersen (7) have reported a similar structure in developing sperm tails of rabbits. We have observed the structure also in the late spermatid of the North American opossum (Didelphis virginiana) and the nine-banded armadillo (Dasypus novemcinctus) (unpublished data). Since the TC is transitory, it is unlikely that it plays a role in the mature spermatozoan. Its highly ordered "paracrystalline" nature may indicate that it serves as a precurser pool for the assembly of some structure of the mature sperm tail, such as the fibrous sheath, outer fibrils, or microtubules of the axial filament complex. It is generally agreed, however, that these components are well formed before middle piece formation (6). The precise timing of TC dissolution with the migration of the ring centriole and the mitochondria suggests that it may also be involved in middle piece formation, perhaps in defining the distalmost boundary of the middle piece. Although tubular, it is unlikely that these structures are homologous with cytoplasmic microtubules because of their larger size and subunit composition. This study was supported in part by research grants NCI-5T1-CA05047-11 and GM-15887, and Contract No. 69-2139 from the National Institutes of Health. The authors would like to express their appreciation to Miss Patricia Murphy for technical assistance and Mrs. Susan Hampton for her cooperation in obtaining materials for this study. REFERENCES 1. 2. 3. 4. 5. 6.
FAWCE~rT,D. W., Intern. Rev. Cytol. 195, 233 (1958). FAWC~TT,D. W. and PHILLIPS,D. M., Anat. Record 165, 153 (1969). MCINTOSH,J. R. and Porter, K. R., J. CelIBiol. 35, 153 (1967). MARKHAM,R., FRE¥, S. and HILLS, G. J., Virology 20, 88 (1963). MILLON~6,G., J. AppL Phys. 32, 1637 (1961). NICAYOER, L., Proe. 5th Intern. Congr. Electron Microscopy, Philadelphia, 1962. Vol. 2, M-4. Academic Press, New York, 1962. 7. PEOERSEN,H., Z. Zellforsch. Mikros. Anat. 98, 148 (1969).
316
s. ULTRASTRUCTURERESEARCH32, 316-322 (1970)
U l t r a s t r u c t u r e of M a m m a l i a n Spermiogenesis I. A Tubular Complex in Developing Sperm of the Cottontop Marmoset Sequinus oedipus J. B. RATTNER AND B. R. BRINKLEY Section of Cell Biology, Department of Biology, The University of Texas, M. D. Anderson Hospital, and Tumor Institute at Houston, Houston, Texas 77025 Received December 12, 1969 A transitory tubular structure termed a tubular complex appears at the proximal end of the principal piece in mid-spermiogenesis of the cottontop marmoset. The structure is formed by tubules 300-330 A in diameter which are arranged both perpendicular to and helically around the axial filament complex. Disappearance of the structure occurs concomitantly with middle piece formation. The process of spermiogenesis has been reviewed in a variety of mammalian species, but many of the ultrastructural aspects have not been examined in detail (1). In most species, spermiogenesis is characterized by abrupt morphological changes, such as nuclear elongation and condensation, which along with acrosomal cap formation, result in the final shape and form of the sperm head. The surrounding cytoplasm becomes displaced posteriorly, and the distal centriole with its associated segmented neck region elongates to form the tail or axial filament complex (2). An important event associated with tail formation in late spermiogenesis is the development of a middle piece containing mitochondria arranged in a helical sheath around the axial filament complex. In the present study, the events of middle piece formation in a prosimian primate, the cottontop marmoset, are presented in detail. Of particular interest is an elaborate tubular complex composed of hexagonally packed microtubule-like structures. The body, termed a tubular complex (TC), surrounds the proximal end of the principal piece in mid-spermiogenesis and disappears as the middle piece is formed.
FIG. 1. Longitudinal sections showing fate of tubular complex (TC) during middle piece formation. (a) TC prior to mitochondrial sheath formation, x 13,500. (b) TC (arrow) at slightly later stage. x 14,000. (c) TC (arrow) at the end of middle piece formation, x 14,500. (d) TC is no longer present when middle piece is completely formed, x 15,000.
l
318
RATTNER AND BRINKLEY
MATERIALS AND METHODS Testes of the marmoset were removed, and the tunica was dissected to expose the seminiferous tubules. The tubules were carefully diced into small pieces with a sharp scalpel in a petri dish containing 3 % glutaraldehyde buffered at pH 7.4 with phosphate buffer (5). After 1 hour in glutaraldehyde at room temperature, the tubules were rinsed in two changes of phosphate buffer and postfixed for 60 min in 1% osmium tetroxide buffered in a similar manner. The tissues were rapidly dehydrated in a graded series of ethanol and embedded in Epon 812. Thick sections were cut with glass knives and mounted in immersion oil for inspection with the phase microscope. Selected areas were further trimmed and sectioned with a diamond knife using an LKB Ultrotome III. Sections were picked up on 200-mesh, unsupported copper grids or collodion-coated, slotted grids and stained in alcoholic uranyl acetate and then in lead citrate (7). The grids were examined in an Hitachi HU-11C electron miscroscope operated at 75 kV.
RESULTS AND DISCUSSION Spermiogenesis in the cottontop marmoset (Sequinus oedipus oedipus) followed a pattern of differentiation much like that described in other mammalian species (1). Early spermatids consisted of a spherical nucleus containing diffuse chromatin. The surrounding cytoplasm was generally free of rough endoplasmic reticulum and polysomes and contained numerous vacuolated mitochondria. The acrosomal vesicle which formed in association with a large Golgi apparatus developed into the acrosomal cap, which eventually covered the anterior two-thirds of the nucleus. The distal member of a centriole pair, located in a distinct indentation at the posterior margin of the nucleus, formed a basal body which gave rise to the axial filament complex containing typical 9 + 2 microtubules with associated outer fibrils. The tail structure continued to elongate throughout spermiogenesis, and the distal region differentiated into a principal piece with a well-developed fibrous sheath. An amorphous dense body, the ring centriole, formed in association with the plasma membrane just distal to the basal body. The manchette, a microtubular sheath (1, 3) appeared to develop from an amorphous ring at the posterior margin of the acrosomal cap and extended to a region several microns distal to the nucleus. With manchette formation, the cytoplasm became displaced posteriorly, and the nucleus elongated and became greatly condensed. In its final form, the nucleus became extremely condensed, flattened, and oval shaped.
Fio. 2. Higher magnifications of tubular complex during middle piece formation. (a) x 57,000. (b) x 58,550. (c) Ring centriole, x 40,500.
A TUBULAR COMPLEX IN MAMMALIAN SPERMATIDS
21 --701828 J . Ultrastructure Research
319
320
RATTNER AND BRINKLEY
Middle piece formation in the cottontop marmoset occurred in later spermatids after complete condensation of the nucleus had occurred. At the onset, a spindleshaped tubular structure, which we have termed the tubular complex (TC), was observed at the distal third of the presumptive middle piece (Fig. 1 a). In its initial structure, the TC measured 170 m# in length and was composed of tubules arranged in both parallel and helical array around the axial filament complex (Fig. 3 a, b). At this stage, the total length of the presumptive middle piece was 630 m#. Transverse and longitudinal sections of the TC indicated that the tubules were packed in a hexagonal arrangement, which was displaced slightly to one side of the axial filament complex (Figs. 2 a and 3 a, b). The longitudinal tubules extended from the distal edge of the TC to various points along its length. Three-dimensional reconstructions of the structure by means of serial sections indicated that the longitudinal tubules were, in fact, continuous with the tubules which formed a helix around the axial filament complex. The tubules of the TC measured 300-330 A in outside diameter and were larger than the 250-270 A microtubules found in the manchette or the axial filament. By rotational photography (4) each tubule exhibited six subunits, each of which measured 90-100 A in diameter. Small side arms, 50 A in diameter, connected adjacent tubules. The arms were spaced 80-90 A apart along the tubules and established a center-to-center spacing of 600-620 A between adjacent tubules. Precise spacing and consistent side-arm attachments were maintained throughout the appearance of the complex. In a slightly later stage, the TC decreased in length to 150 m# and was composed entirely of helical tubules (Figs. 1b and 2b). It exhibited a triangular appearance in longitudinal section. The base of the triangle contained three tubular helices, with one extending into the anterior region of the collar. Reduction in the length and diameter of the TC signaled the onset of mitochondrial migration into the presumptive middle piece. At this time, the middle piece region measured 660 m/z. Mitochondria (usually four in number) became associated with the ring centriole at its anterior margin distal to the basal body. The ring centriole moved caudally along the middle piece (Fig. 1c), accompanied by the plasma membrane. Subsequently, the mitochondria aggregated around the axial filament complex. As the middle piece formed, the TC was reduced to 70 m# in length, and the total presumptive middle piece region increased to a length of 850 m# (Fig. 2c). As the ring centriole arrived at its final destination to form the annulus, or Jensen's ring, the TC completely disappeared. As shown in Fig. 1 d, the TC is absent from spermatids with mature middle FIG. 3. Serial transverse section of tubular complex showing tubules arranged in both helical and parallel fashionaround the axial filamentcomplex. (a) x 40,000. (b) x 40,000.
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RATTNER AND BRINKLEY
pieces. At completion, the middle piece had a total length of 880 m# and contained approximately 30 mitochondrial profiles. The tubular complex has also been observed in other mammalian species. Nicander (6) and Pedersen (7) have reported a similar structure in developing sperm tails of rabbits. We have observed the structure also in the late spermatid of the North American opossum (Didelphis virginiana) and the nine-banded armadillo (Dasypus novemcinctus) (unpublished data). Since the TC is transitory, it is unlikely that it plays a role in the mature spermatozoan. Its highly ordered "paracrystalline" nature may indicate that it serves as a precurser pool for the assembly of some structure of the mature sperm tail, such as the fibrous sheath, outer fibrils, or microtubules of the axial filament complex. It is generally agreed, however, that these components are well formed before middle piece formation (6). The precise timing of TC dissolution with the migration of the ring centriole and the mitochondria suggests that it may also be involved in middle piece formation, perhaps in defining the distalmost boundary of the middle piece. Although tubular, it is unlikely that these structures are homologous with cytoplasmic microtubules because of their larger size and subunit composition. This study was supported in part by research grants NCI-5T1-CA05047-11 and GM-15887, and Contract No. 69-2139 from the National Institutes of Health. The authors would like to express their appreciation to Miss Patricia Murphy for technical assistance and Mrs. Susan Hampton for her cooperation in obtaining materials for this study. REFERENCES 1. 2. 3. 4. 5. 6.
FAWCE~rT,D. W., Intern. Rev. Cytol. 195, 233 (1958). FAWC~TT,D. W. and PHILLIPS,D. M., Anat. Record 165, 153 (1969). MCINTOSH,J. R. and Porter, K. R., J. CelIBiol. 35, 153 (1967). MARKHAM,R., FRE¥, S. and HILLS, G. J., Virology 20, 88 (1963). MILLON~6,G., J. AppL Phys. 32, 1637 (1961). NICAYOER, L., Proe. 5th Intern. Congr. Electron Microscopy, Philadelphia, 1962. Vol. 2, M-4. Academic Press, New York, 1962. 7. PEOERSEN,H., Z. Zellforsch. Mikros. Anat. 98, 148 (1969).