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Studies on the fine structure of spermatids and spermatozoa from the millipede Polydesmus sp.
© 1968 by Academic Press Inc.
60
J. ULTRASTRUCTURERESEARCH23, 60--70 (1968)
Studies on the Fine Structure of Spermotids ond Spermotozoo from the Millipede
Polydesmussp. ~
JAMES F. REGER and DAVID P. COO~R
Department of Anatomy, University of Tennessee Medical Units, Memphis, Tennessee 38103 Received February 7, 1968 This electron microscope study demonstrates changes leading to formation of binary spermatozoa. Spermatids contain Golgi complex, mitochondria, centrioles, microtubules, and ribosomes. Spermiogenesis is characterized by migration of nucleus, Golgi complex, and centrioles to one pole of the cell. The Golgi complex gives rise to an acrosomal vesicle. As spermiogenesis proceeds, there is an increase in phagocytosis and numbers of intracellular multilaminar, multivesicular, and granular bodies leading to cytoplasmic sloughing. Many phagocytic vacuoles fuse and undermine developing, crescent-shaped spermatozoa. During spermiogenesis the acrosome and centrioles fuse with nuclear chromatin to lose their morphological identities. Mature crescent-shaped spermatozoa are found juxtaposed on their concave surfaces. Sperm are characterized by an electron dense nucleus, lack of mitochondria, and an identifiable motile appendage. These observations are compared to earlier studies, and it is concluded that mature spermatozoa of Polydesmus sp. are not binucleate, but adhere at their concave surfaces as binary appearing structures. Light microscope studies of spermatids and spermatozoa from several species of millipedes (2, 4, 5, 8, 9), demonstrated the unusual binary structure of millipede sperm. Neither the mechanisms during spermiogenesis leading to binary structure, nor possible homologies between binary millipede sperm and other spermatozoa are yet completely known (3). Warren (9) suggested that binary sperm in the millipedes Odontopyge sp. and Poratophilus sp. were formed from single spermatids and that mature sperm were composed of a single nucleus and two plasmasomes. Contrariwise, N a t h and Sharma (4) presented evidence that binary sperm in the millipede Thyroglutus malayus were formed following incomplete cytokinesis of secondary maturation divisions. Other morphological changes that occur in the nucleus and cytoplasm of millipede sperm during spermiogenesis are also not yet completely understood (3). The earliest studies by Gilson (2) on Glorneris marginata, Polydesmus complanatus, Iulus sp., 1 This work was supported by U.S.P.H.S. Grants Nos. NB-06285 and ST-1-GM-00202.
MILLIPEDE SPERMATIDS AND SPERMATOZOA
61
Blaniulus guttulatus, a n d Scolopendra dalmatica lacked sufficient r e s o l u t i o n to exa m i n e cytological detail. Oettinger (5) was the first to d e m o n s t r a t e centriolar, m i t o c h o n d r i a l , a n d G o l g i b o d y m o v e m e n t s d u r i n g spermiogenesis in Pachyiulus varius, b u t he failed to describe an acrosome. Likewise Sokoloff (8) in Polyxenus sp. a n d N a t h a n d S h a r m a (4) in Thyroglutus malayus described centriolar, m i t o c h o n d r i a l , a n d G o l g i b o d y m o v e m e n t s b u t failed to describe the d e v e l o p m e n t of an acrosome. The following electron m i c r o s c o p e study was m a d e to show m o r p h o l o g i c a l changes leading to the f o r m a t i o n of millipede b i n a r y s p e r m a t o z o a a n d to describe organelle changes in o r d e r to present evidence for h o m o l o g i e s with other s p e r m a t o z o a . M A T E R I A L S A N D METHODS Both testes and spermatic ducts were isolated from adult male millepedes, Polydesmus sp., directly immersed in cold (0-5°(2) glutaraldehyde (pH 7.6; 0.1 M phosphate buffer), and fixed for an hour. After glutaraldehyde fixation the tissue was washed for an hour in cold (0-5°C) buffer solution (pH 7.6; 0.1 M phosphate) and postfixed for an hour in cold (0-5°C), buffered OsO~ (pH 7.6; 0.1 M phosphate buffer). After fixation the tissue was dehydrated in 5-minute changes of successive 10% grades of methanol (beginning with 50 %), and embedded in Epon 812. Sections, 1-2 ~ thick, were made of the Epon-embedded tissue and stained with Mallory Azure II-Methylene blue for purposes of orientation of spermiogenic tissue prior to thin sectioning. Thin sections were cut with a diamond knife fitted to an LKB Ultrotome II, floated on distilled water, mounted on carbon-coated grids, stained with uranyl acetate and lead citrate and examined with a Hitachi 11 A electron microscope. Micrographs, at initial magnifications of 7500 to 40,000 at exposures of 3-5 seconds, were made on Cronar, Ortholitho, Type A sheet-film and photographically enlarged up to 7 times. OBSERVATIONS W i t h i n each b o d y segment of Polydesmus sp. p a i r e d testes extend v e n t r o l a t e r a l l y f r o m s p e r m a t i c ducts t h a t lie lateral to the ventral nerve cord. E a c h testis contains FIG. 1. Early spermatids (S) of Polydesmussp. are usually interconnected by cytoplasmic bridges (arrows). R, ribosomes, x 22,500. Fro. 2. This micrograph shows spermatids wherein nuclei (N) are now situated to one pole of the cell. Spermatids (S) contain a Golgi complex (G), mitochondria (M), and microtubules (Mt). x 22,500. FIG. 3. A single spermatid (S) to show the location of the Golgi complex (G) and a centriole (C) between nucleus and plasma membrane prior to formation of the acrosomal vesicle (A V, Figs. 4 and 5). Cr, chromatin, x 22,500. Fio. 4. Several spermatids (S) in a later stage than in Fig. 3. Notice the increased electron density and packing of nuclear chromatin (Cr). A V, acrosomal vesicle; N, nucleus, x 22,500. FIo. 5. This spermatid (S) is an example of a cell which failed to undergo any cytokinesis. However, steps in spermiogenesis proceed normally. These include positioning of centrioles (C) and formation of an acrosomal vesicle (A V) at the apex of a triangle-shaped nucleus (N). Electron densities (ED1, ED2) accumulate upon the outer surface of developing spermatids. L, spermatic duct lumen. x 68,000.
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64
JAMES F. R E G E R A N D D A V I D P. C O O P E R
spermatids in different stages of differentiation (Figs. 1-4). Early spermatids are often found interconnected by cytoplasmic bridges (Fig. 1). Early spermatids (S, Figs. 1-5) contain a single Golgi complex (G, Figs. 2 and 3), mitochondria (M, Fig. 2), centrioles (C, Fig. 3), microtubules (Mr, Fig. 2), and single 150-250 A particles (R, Fig. 1) presumed to be ribosomes. Early spermiogenesis is characterized by migration of the nucleus to one pole of the cell (N, Figs. 2 and 4), by changes in overall electron density of the nucleus (N, Fig. 4), and by changes in distribution of nuclear chromatin (compare Cr, Figs. 3 and 4). A Golgi complex (G, Fig. 3) and a centriole (C, Fig. 3) become situated between the nucleus and the plasma membrane in early spermatids. The Golgi complex (G, Fig. 3), in later stages of development, gives rise to an acrosomal vesicle (AV, Figs. 5, 11, and 12). The acrosomal vesicle (AV, Figs. 5, 11 and 12) and centrioles (C, Figs. 5, 10-12 and 14) occupy the same relative position as did the Golgi complex and centrioles in earlier spermatids and are similar in structure to those in other spermatozoa. Two, morphologically different, electron densities (ED1, ED2, Fig. 5) appear on the outer surface of early spermatids at the nuclear level. These densities include a central amorphous electron density (ED1, Fig. 5) at the apex of the triangle-shaped nucleus and peripheral electron densities (ED~, Fig. 5) situated to either side of the convex surface of developing spermatids. When observed at high magnification, the peripheral electron densities (ED2, Fig. 6) are seen to be composed of discrete particles (P, Fig. 6) 70-100 A in diameter space 150-250 A apart. Such particles appear to be a continuation of the outer lamina of the unit membrane. Uninucleate (Figs. 3 and 4) and binuclaete (Fig. 5) spermatids are found in Polydesmus sp. The spermatic duct lumen (L, Fig. 5) contains an electron dense material which appears to become phagocytized (arrow, Fig. 7) by spermatids during the middle stages of spermiogenesis. All the spermatids at this stage of development contain electron dense bodies and surface membrane infoldings (arrow, Fig. 7), which is a prelude to cytoplasmic sloughing, a generally common phenomenon for spermatids. The electron dense bodies observed (Figs. 7-9) are multivesicular (MV, Fig. 7), multilaminar (ML, Fig. 7), and granular (G, Fig. 9). These bodies are all reminiscent of similar structures found in cytoplasm of sloughed spermatids of other animals. During maturation, nuclei of developing spermatids are found directly apposed FiG. 6. Enlarged view of a portion of a developingspermatid to show surfaceelectron densities(EDz) composed of discreteparticles (P) which are 70-100 A in size and which are spaced 150-200 A apart, center to center. × 120,000. FIGS. 7-9. These three electron micrographs demonstrate stages in the dissolution and sloughing of cytoplasm prior to formation of mature sperm. During this process large numbers of intracytoplasmic, electron dense bodies (ML, MV, Fig. 7) accumulate by the probable mechanism of surface phagocytosis (arrow, Fig. 7). The electron dense bodies include multivesicular (MV), granular (G), and multilaminar (ML) structures. × 22,500.
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66
JAMES F. REGER AND DAVID P. COOPER
to the cell surface (Figs. 10-15). They assume a crescentric shape (Figs. 10-15) and are situated proximal to an acrosomal vesicle (AV, Figs. 11 and 12) and centrioles (C, Fig. 10). The nucleus, centrioles, acrosomal vesicle, and scant amounts of spermatid cytoplasm eventually become separated from the main mass of the spermatid cytoplasm by an apparent coalescence of phagocytic vesicles (PV, Figs. 11-14) on the concave surface of the nucleus. During this process the nuclear membrane disappears (NM, Figs. 10 and 13), and centrioles (C, Fig. 14) and acrosomal vesicle (AV, Fig. 13) coalesce with the electron dense chromatin of the maturing nucleus (arrows, Figs. 14 and 15). These areas of coalescence of chromatin, acrosomal vesicle, and centrioles are recognizable in mature sperm (arrows, Figs. 16 and 17) as electron-lucent areas (arrows, Figs. 15-17) within the electron dense chromatin. The mature, crescentric spermatozoa (Figs. 16 and 17) that result are found at lower levels of the spermatic duct juxtaposed on their concave surfaces (Fig. 17). They are characterized by an extremely electron dense nucleus (N, Fig. 17) and by a lack of either mitochondria or an identifiable motile appendage. Such paired sperm, when observed in vitro with the light microscope, appear to be nonmotile. DISCUSSION The fine structure of early spermatids and the mechanisms underlying spermiogenesis in the millipede Polydesmus sp. are generally similar to those of other sperm. However, the structure of mature spermatozoa is completely different. Their binary structure, and the absence of morphologically identifiable acrosome and centrioles is very unusual. Mechanisms underlying adherence of Po!ydesmus sp. spermatozoa on their concave surfaces to produce binary sperm are unknown. Single spermatozoa are rarely observed, and then only in the upper portions of the spermatic duct where spermiogenesis is incomplete. Binary spermatozoa are seen only in the lower portions of the spermatic duct, in the seminal vesicle, and in the seminal receptacle of the female. Similar data from early light microscope studies are available. Gilson (2) shows figures of uninucleated immature spermatids, but only binary spermatozoa. Similarly, the figures of Sokoloff (8), Warren (9), and Nath and Sharma (4) on sperm from other species of millipedes show uninucleate, immature spermatids, but binary mature sperm. One exception is the study of Oettinger (5) on Pachyiulus varius, in which both FIGS. 10-15. These six micrographs demonstrate nuclear changes concomitant with cytoplasmic sloughing prior to formation of mature sperm (see Figs. 16 and 17). Changes include los of nuclear membrane (NM, Figs. 10 and 13); coalescence of acrosomal vesicle (AV, Fig. 13) and centrioles (C, Figs. 12 and 14) with nuclear chromatin (solid arrows, Figs. 14 and 15); and separation of nucleus from cytoplasm by coalescence of pinocytotic vesicles (PV, Figs. 11-14) beneath the nucleus (dashed arrows, Figs. 14 and 15). x 68,000.
68
JAMES F. REGER AND DAVID P. COOPER
immature spermatids and mature spermatozoa are shown as uninucleate cells. However, one figure is shown depicting a binary spermatozoa. It is possible that the electron dense contents of the spermatic duct function some way in the adherence of mature binary sperm. However, their regular adherence only on their concave surface suggests something more specific than mere "stickiness" of luminal contents. The absence of morphologically distinguishable acrosome and centrioles in Polydesmus sp. spermatozoa is in marked contrast to other types of nonmotile, aberrant spermatozoa (1, 6, 7, 10). For although other nonmotile, aberrant sperm (1, 6, 7, 10) lack motile appendages and mitochondria, a typical acrosome and centrioles are present. In lacking morphologically identifiable acrosomes and centrioles, it is possible the spermatozoa of Polydesmus sp. are degenerate, or otherwise nonfunctional. However, sperm taken from the female seminal receptacle, and, therefore, presumed to be functional, are morphologically identical to those found in the spermatic ducts of males. Direct observations on oocyte-spermatozoon fusion will be necessary to prove these nonmotile sperm functional. Sufficient cytological detail is not available in the early light microscope studies of Gilson (2), Sokoloff (8), and Warren (9) to allow comparisons with the present electron microscope study of Polydesmus sp. spermiogenesis. However, comparisons are possible between the results of this study and the early light microscope investigations of Oettinger (5) and Nath and Sharma (4), whose cytological detail is excellent. Oettinger (5), in the millipede PachyiuIus varius described the morphology and move~ ments of centrioles, mitochondria, and Golgi complex but failed to describe the development of an acrosome. However, he shows a structure in several figures in the identical position on the acrosomal vesicle found in sperm of Polydesmus sp. He considers this structure a centrosome and therefore describes a "Doppelcentrosoma" in sperm of Pachyiulus varius (5). These structures were later seen to fuse into the nucleus as shown in his later figures; this is similar to the data on sperm of Polydesmus sp. Nath and Sharma (4) in the millipede Thyroglutus mayalus also described the morphology and movements of centriole, mitochondria, and Golgi complex during spermiogenesis, but failed to describe the formation of an acrosome. They did, however, describe a "ring-like centrosome" which in size, morphology and position is comparable to
Fio. 16. These cells are almost mature spermatozoa and free of sloughed cytoplasmic remnants (Cy). They are found singly and in pairs. Electron lucent areas (arrows). × 15,000. F~o. 17. Low power micrograph of mature sperm found in the seminal receptacle. Mature sperm of PoIydesmus sp. are apposed on their concave surfaces and appear in the light microscope as binary sperm. Notice the electron dense nucleus (N) and central, electron lucent center (arrows). This lies in the same region as did the coalescing acrosome and centrioles of earlier stages (compare with arrows, Figs. 14 and 15). x 12,000.
i
70
JAMES F. REGER AND DAVID P. COOPER
the acrosomal vesicle described in the sperm of Polydesmus sp. Again, as in sperm of Polydesmus sp. and Pachyiulus varius (5), this so-called centrosome was seen to fuse "completely with the nucleus" (4). In fact, several of Nath and Sharma's (4) figures show the centrosome actually within the nucleus. The significance, if any, of the apparent coalescence of acrosome and centrioles within the nucleus in developing sperm of these millipedes is unknown. The authors wish to express their grateful appreciation for the excellent technical assistance given by Mrs. Ann Florendo during the course of this investigation. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10.
FAIN-MAUREL,M. A., Thesis, Facult6 des Sciences, Universit6 de Paris, France (1966). GILSON,G., Cellule 2, 83 (1886). NATH V., Intern Rev. Cytol. 5, 395 (1956). NATH, V. and SHARMA,G. P., Res. Bull. Panjab Univ. 22, 99 (1952). OZTTINGER,R., Arch. Zellforsch. 3, 563 (1909). REGER, J. F., J. Microscopie 3, 559 (1964). - - - - Z. Zellforsch. Microskop. Anat. 75, 579 (1966). SOKOLOEF,J., Zool. Anz. 44, 558 (1914). WARREN, E., Ann Natal. Museum P'mburg. 7, 351 (1934). WERNER, G., Z. Zellforsch. Microskop. Anat. 63, 880 (1964).
60
J. ULTRASTRUCTURERESEARCH23, 60--70 (1968)
Studies on the Fine Structure of Spermotids ond Spermotozoo from the Millipede
Polydesmussp. ~
JAMES F. REGER and DAVID P. COO~R
Department of Anatomy, University of Tennessee Medical Units, Memphis, Tennessee 38103 Received February 7, 1968 This electron microscope study demonstrates changes leading to formation of binary spermatozoa. Spermatids contain Golgi complex, mitochondria, centrioles, microtubules, and ribosomes. Spermiogenesis is characterized by migration of nucleus, Golgi complex, and centrioles to one pole of the cell. The Golgi complex gives rise to an acrosomal vesicle. As spermiogenesis proceeds, there is an increase in phagocytosis and numbers of intracellular multilaminar, multivesicular, and granular bodies leading to cytoplasmic sloughing. Many phagocytic vacuoles fuse and undermine developing, crescent-shaped spermatozoa. During spermiogenesis the acrosome and centrioles fuse with nuclear chromatin to lose their morphological identities. Mature crescent-shaped spermatozoa are found juxtaposed on their concave surfaces. Sperm are characterized by an electron dense nucleus, lack of mitochondria, and an identifiable motile appendage. These observations are compared to earlier studies, and it is concluded that mature spermatozoa of Polydesmus sp. are not binucleate, but adhere at their concave surfaces as binary appearing structures. Light microscope studies of spermatids and spermatozoa from several species of millipedes (2, 4, 5, 8, 9), demonstrated the unusual binary structure of millipede sperm. Neither the mechanisms during spermiogenesis leading to binary structure, nor possible homologies between binary millipede sperm and other spermatozoa are yet completely known (3). Warren (9) suggested that binary sperm in the millipedes Odontopyge sp. and Poratophilus sp. were formed from single spermatids and that mature sperm were composed of a single nucleus and two plasmasomes. Contrariwise, N a t h and Sharma (4) presented evidence that binary sperm in the millipede Thyroglutus malayus were formed following incomplete cytokinesis of secondary maturation divisions. Other morphological changes that occur in the nucleus and cytoplasm of millipede sperm during spermiogenesis are also not yet completely understood (3). The earliest studies by Gilson (2) on Glorneris marginata, Polydesmus complanatus, Iulus sp., 1 This work was supported by U.S.P.H.S. Grants Nos. NB-06285 and ST-1-GM-00202.
MILLIPEDE SPERMATIDS AND SPERMATOZOA
61
Blaniulus guttulatus, a n d Scolopendra dalmatica lacked sufficient r e s o l u t i o n to exa m i n e cytological detail. Oettinger (5) was the first to d e m o n s t r a t e centriolar, m i t o c h o n d r i a l , a n d G o l g i b o d y m o v e m e n t s d u r i n g spermiogenesis in Pachyiulus varius, b u t he failed to describe an acrosome. Likewise Sokoloff (8) in Polyxenus sp. a n d N a t h a n d S h a r m a (4) in Thyroglutus malayus described centriolar, m i t o c h o n d r i a l , a n d G o l g i b o d y m o v e m e n t s b u t failed to describe the d e v e l o p m e n t of an acrosome. The following electron m i c r o s c o p e study was m a d e to show m o r p h o l o g i c a l changes leading to the f o r m a t i o n of millipede b i n a r y s p e r m a t o z o a a n d to describe organelle changes in o r d e r to present evidence for h o m o l o g i e s with other s p e r m a t o z o a . M A T E R I A L S A N D METHODS Both testes and spermatic ducts were isolated from adult male millepedes, Polydesmus sp., directly immersed in cold (0-5°(2) glutaraldehyde (pH 7.6; 0.1 M phosphate buffer), and fixed for an hour. After glutaraldehyde fixation the tissue was washed for an hour in cold (0-5°C) buffer solution (pH 7.6; 0.1 M phosphate) and postfixed for an hour in cold (0-5°C), buffered OsO~ (pH 7.6; 0.1 M phosphate buffer). After fixation the tissue was dehydrated in 5-minute changes of successive 10% grades of methanol (beginning with 50 %), and embedded in Epon 812. Sections, 1-2 ~ thick, were made of the Epon-embedded tissue and stained with Mallory Azure II-Methylene blue for purposes of orientation of spermiogenic tissue prior to thin sectioning. Thin sections were cut with a diamond knife fitted to an LKB Ultrotome II, floated on distilled water, mounted on carbon-coated grids, stained with uranyl acetate and lead citrate and examined with a Hitachi 11 A electron microscope. Micrographs, at initial magnifications of 7500 to 40,000 at exposures of 3-5 seconds, were made on Cronar, Ortholitho, Type A sheet-film and photographically enlarged up to 7 times. OBSERVATIONS W i t h i n each b o d y segment of Polydesmus sp. p a i r e d testes extend v e n t r o l a t e r a l l y f r o m s p e r m a t i c ducts t h a t lie lateral to the ventral nerve cord. E a c h testis contains FIG. 1. Early spermatids (S) of Polydesmussp. are usually interconnected by cytoplasmic bridges (arrows). R, ribosomes, x 22,500. Fro. 2. This micrograph shows spermatids wherein nuclei (N) are now situated to one pole of the cell. Spermatids (S) contain a Golgi complex (G), mitochondria (M), and microtubules (Mt). x 22,500. FIG. 3. A single spermatid (S) to show the location of the Golgi complex (G) and a centriole (C) between nucleus and plasma membrane prior to formation of the acrosomal vesicle (A V, Figs. 4 and 5). Cr, chromatin, x 22,500. Fio. 4. Several spermatids (S) in a later stage than in Fig. 3. Notice the increased electron density and packing of nuclear chromatin (Cr). A V, acrosomal vesicle; N, nucleus, x 22,500. FIo. 5. This spermatid (S) is an example of a cell which failed to undergo any cytokinesis. However, steps in spermiogenesis proceed normally. These include positioning of centrioles (C) and formation of an acrosomal vesicle (A V) at the apex of a triangle-shaped nucleus (N). Electron densities (ED1, ED2) accumulate upon the outer surface of developing spermatids. L, spermatic duct lumen. x 68,000.
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64
JAMES F. R E G E R A N D D A V I D P. C O O P E R
spermatids in different stages of differentiation (Figs. 1-4). Early spermatids are often found interconnected by cytoplasmic bridges (Fig. 1). Early spermatids (S, Figs. 1-5) contain a single Golgi complex (G, Figs. 2 and 3), mitochondria (M, Fig. 2), centrioles (C, Fig. 3), microtubules (Mr, Fig. 2), and single 150-250 A particles (R, Fig. 1) presumed to be ribosomes. Early spermiogenesis is characterized by migration of the nucleus to one pole of the cell (N, Figs. 2 and 4), by changes in overall electron density of the nucleus (N, Fig. 4), and by changes in distribution of nuclear chromatin (compare Cr, Figs. 3 and 4). A Golgi complex (G, Fig. 3) and a centriole (C, Fig. 3) become situated between the nucleus and the plasma membrane in early spermatids. The Golgi complex (G, Fig. 3), in later stages of development, gives rise to an acrosomal vesicle (AV, Figs. 5, 11, and 12). The acrosomal vesicle (AV, Figs. 5, 11 and 12) and centrioles (C, Figs. 5, 10-12 and 14) occupy the same relative position as did the Golgi complex and centrioles in earlier spermatids and are similar in structure to those in other spermatozoa. Two, morphologically different, electron densities (ED1, ED2, Fig. 5) appear on the outer surface of early spermatids at the nuclear level. These densities include a central amorphous electron density (ED1, Fig. 5) at the apex of the triangle-shaped nucleus and peripheral electron densities (ED~, Fig. 5) situated to either side of the convex surface of developing spermatids. When observed at high magnification, the peripheral electron densities (ED2, Fig. 6) are seen to be composed of discrete particles (P, Fig. 6) 70-100 A in diameter space 150-250 A apart. Such particles appear to be a continuation of the outer lamina of the unit membrane. Uninucleate (Figs. 3 and 4) and binuclaete (Fig. 5) spermatids are found in Polydesmus sp. The spermatic duct lumen (L, Fig. 5) contains an electron dense material which appears to become phagocytized (arrow, Fig. 7) by spermatids during the middle stages of spermiogenesis. All the spermatids at this stage of development contain electron dense bodies and surface membrane infoldings (arrow, Fig. 7), which is a prelude to cytoplasmic sloughing, a generally common phenomenon for spermatids. The electron dense bodies observed (Figs. 7-9) are multivesicular (MV, Fig. 7), multilaminar (ML, Fig. 7), and granular (G, Fig. 9). These bodies are all reminiscent of similar structures found in cytoplasm of sloughed spermatids of other animals. During maturation, nuclei of developing spermatids are found directly apposed FiG. 6. Enlarged view of a portion of a developingspermatid to show surfaceelectron densities(EDz) composed of discreteparticles (P) which are 70-100 A in size and which are spaced 150-200 A apart, center to center. × 120,000. FIGS. 7-9. These three electron micrographs demonstrate stages in the dissolution and sloughing of cytoplasm prior to formation of mature sperm. During this process large numbers of intracytoplasmic, electron dense bodies (ML, MV, Fig. 7) accumulate by the probable mechanism of surface phagocytosis (arrow, Fig. 7). The electron dense bodies include multivesicular (MV), granular (G), and multilaminar (ML) structures. × 22,500.
.5
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66
JAMES F. REGER AND DAVID P. COOPER
to the cell surface (Figs. 10-15). They assume a crescentric shape (Figs. 10-15) and are situated proximal to an acrosomal vesicle (AV, Figs. 11 and 12) and centrioles (C, Fig. 10). The nucleus, centrioles, acrosomal vesicle, and scant amounts of spermatid cytoplasm eventually become separated from the main mass of the spermatid cytoplasm by an apparent coalescence of phagocytic vesicles (PV, Figs. 11-14) on the concave surface of the nucleus. During this process the nuclear membrane disappears (NM, Figs. 10 and 13), and centrioles (C, Fig. 14) and acrosomal vesicle (AV, Fig. 13) coalesce with the electron dense chromatin of the maturing nucleus (arrows, Figs. 14 and 15). These areas of coalescence of chromatin, acrosomal vesicle, and centrioles are recognizable in mature sperm (arrows, Figs. 16 and 17) as electron-lucent areas (arrows, Figs. 15-17) within the electron dense chromatin. The mature, crescentric spermatozoa (Figs. 16 and 17) that result are found at lower levels of the spermatic duct juxtaposed on their concave surfaces (Fig. 17). They are characterized by an extremely electron dense nucleus (N, Fig. 17) and by a lack of either mitochondria or an identifiable motile appendage. Such paired sperm, when observed in vitro with the light microscope, appear to be nonmotile. DISCUSSION The fine structure of early spermatids and the mechanisms underlying spermiogenesis in the millipede Polydesmus sp. are generally similar to those of other sperm. However, the structure of mature spermatozoa is completely different. Their binary structure, and the absence of morphologically identifiable acrosome and centrioles is very unusual. Mechanisms underlying adherence of Po!ydesmus sp. spermatozoa on their concave surfaces to produce binary sperm are unknown. Single spermatozoa are rarely observed, and then only in the upper portions of the spermatic duct where spermiogenesis is incomplete. Binary spermatozoa are seen only in the lower portions of the spermatic duct, in the seminal vesicle, and in the seminal receptacle of the female. Similar data from early light microscope studies are available. Gilson (2) shows figures of uninucleated immature spermatids, but only binary spermatozoa. Similarly, the figures of Sokoloff (8), Warren (9), and Nath and Sharma (4) on sperm from other species of millipedes show uninucleate, immature spermatids, but binary mature sperm. One exception is the study of Oettinger (5) on Pachyiulus varius, in which both FIGS. 10-15. These six micrographs demonstrate nuclear changes concomitant with cytoplasmic sloughing prior to formation of mature sperm (see Figs. 16 and 17). Changes include los of nuclear membrane (NM, Figs. 10 and 13); coalescence of acrosomal vesicle (AV, Fig. 13) and centrioles (C, Figs. 12 and 14) with nuclear chromatin (solid arrows, Figs. 14 and 15); and separation of nucleus from cytoplasm by coalescence of pinocytotic vesicles (PV, Figs. 11-14) beneath the nucleus (dashed arrows, Figs. 14 and 15). x 68,000.
68
JAMES F. REGER AND DAVID P. COOPER
immature spermatids and mature spermatozoa are shown as uninucleate cells. However, one figure is shown depicting a binary spermatozoa. It is possible that the electron dense contents of the spermatic duct function some way in the adherence of mature binary sperm. However, their regular adherence only on their concave surface suggests something more specific than mere "stickiness" of luminal contents. The absence of morphologically distinguishable acrosome and centrioles in Polydesmus sp. spermatozoa is in marked contrast to other types of nonmotile, aberrant spermatozoa (1, 6, 7, 10). For although other nonmotile, aberrant sperm (1, 6, 7, 10) lack motile appendages and mitochondria, a typical acrosome and centrioles are present. In lacking morphologically identifiable acrosomes and centrioles, it is possible the spermatozoa of Polydesmus sp. are degenerate, or otherwise nonfunctional. However, sperm taken from the female seminal receptacle, and, therefore, presumed to be functional, are morphologically identical to those found in the spermatic ducts of males. Direct observations on oocyte-spermatozoon fusion will be necessary to prove these nonmotile sperm functional. Sufficient cytological detail is not available in the early light microscope studies of Gilson (2), Sokoloff (8), and Warren (9) to allow comparisons with the present electron microscope study of Polydesmus sp. spermiogenesis. However, comparisons are possible between the results of this study and the early light microscope investigations of Oettinger (5) and Nath and Sharma (4), whose cytological detail is excellent. Oettinger (5), in the millipede PachyiuIus varius described the morphology and move~ ments of centrioles, mitochondria, and Golgi complex but failed to describe the development of an acrosome. However, he shows a structure in several figures in the identical position on the acrosomal vesicle found in sperm of Polydesmus sp. He considers this structure a centrosome and therefore describes a "Doppelcentrosoma" in sperm of Pachyiulus varius (5). These structures were later seen to fuse into the nucleus as shown in his later figures; this is similar to the data on sperm of Polydesmus sp. Nath and Sharma (4) in the millipede Thyroglutus mayalus also described the morphology and movements of centriole, mitochondria, and Golgi complex during spermiogenesis, but failed to describe the formation of an acrosome. They did, however, describe a "ring-like centrosome" which in size, morphology and position is comparable to
Fio. 16. These cells are almost mature spermatozoa and free of sloughed cytoplasmic remnants (Cy). They are found singly and in pairs. Electron lucent areas (arrows). × 15,000. F~o. 17. Low power micrograph of mature sperm found in the seminal receptacle. Mature sperm of PoIydesmus sp. are apposed on their concave surfaces and appear in the light microscope as binary sperm. Notice the electron dense nucleus (N) and central, electron lucent center (arrows). This lies in the same region as did the coalescing acrosome and centrioles of earlier stages (compare with arrows, Figs. 14 and 15). x 12,000.
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JAMES F. REGER AND DAVID P. COOPER
the acrosomal vesicle described in the sperm of Polydesmus sp. Again, as in sperm of Polydesmus sp. and Pachyiulus varius (5), this so-called centrosome was seen to fuse "completely with the nucleus" (4). In fact, several of Nath and Sharma's (4) figures show the centrosome actually within the nucleus. The significance, if any, of the apparent coalescence of acrosome and centrioles within the nucleus in developing sperm of these millipedes is unknown. The authors wish to express their grateful appreciation for the excellent technical assistance given by Mrs. Ann Florendo during the course of this investigation. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10.
FAIN-MAUREL,M. A., Thesis, Facult6 des Sciences, Universit6 de Paris, France (1966). GILSON,G., Cellule 2, 83 (1886). NATH V., Intern Rev. Cytol. 5, 395 (1956). NATH, V. and SHARMA,G. P., Res. Bull. Panjab Univ. 22, 99 (1952). OZTTINGER,R., Arch. Zellforsch. 3, 563 (1909). REGER, J. F., J. Microscopie 3, 559 (1964). - - - - Z. Zellforsch. Microskop. Anat. 75, 579 (1966). SOKOLOEF,J., Zool. Anz. 44, 558 (1914). WARREN, E., Ann Natal. Museum P'mburg. 7, 351 (1934). WERNER, G., Z. Zellforsch. Microskop. Anat. 63, 880 (1964).