THE ANATOMICAL RECORD 229:473-481 (1991) Ultrastructure of the Turtle Spermatozoon REX A. HESS, RONALD J. THURSTON, AND DANIEL H. GIST Department of Veterinary Biosciences, University of Illinois, Urbana, Illinois 61801 (R.A.H.);Poultry Science Department, Clemson University, Clemson, South Carolina 29634-0379 (R.J.T.);Department of Biological Sciences, University of Cincinnati, Cincinnati, Ohio 45221 -0006 (D.H.G.) ABSTRACT The turtle spermatozoon is vermiform in shape with a narrow pointed head that is curved. In general, the turtle sperm contains a typical head, midpiece and tail, similar in morphology to that of birds, amphibians and other reptiles. However, several structures are unique. These unusual features include (1)a perforatorial cap over the proximal end of the nucleus, which contains 2-3 rods that are contiguous with intranuclear tubules; (2) a connecting collar of dense material that surrounds the base of the nucleus; (3) a distal centriole containing central microtubules that extend its entire length and having outer triplicate microtubules that open toward the central cavity of the centriole; and (4) unusual spherical mitochondria containing 7-8 outer laminated membranes. Male and female reproductive cycles are not synchro- TEM according to Hess et al. (1986).For SEM, a drop of nized in temperature zone turtles, resulting in the pro- sperm suspended in ethanol was placed on a glass covduction of their respective gametes a t different times of erslip and the sperm were allowed to settle. The samthe year (Licht, 1984). This necessitates a long interval ples were taken through critical point drying in CO, of gamete survival which is accomplished by the stor- and coated with gold a t 10 pA for 440 sec. Photographs age of spermatozoa. This storage is thought to occur in were taken with a JEOL 848 SEM. For TEM, the dethe epididymis (Moll, 1979) but may also occur in the hydrated samples were embedded in low viscosity resin female reproductive tract (Gist and Jones, 1989). Pro- and sectioned. Tannic acid fixation (4% tannic acid in longed survival of sperm in both the male or female 3% glutaraldehyde) was used for negative staining of reproductive systems suggests that turtle sperm may microtubules in the distal centriole (Tilney et al., possess morphological specializations contributing to 1973). Ultrathin sections were stained with uranyl actheir longevity. Past observations of their ultrastruc- etate and lead citrate and photographed with a JEOL ture have not revealed organelles unusually different 1OOCX TEM. from sperm of other species that are capable of longRESULTS term storage (Miranti et al., 1964; Kaplan et al., 1966; Furieri, 1970). The basic structure of the turtle sperSpermatozoa of the turtle Chrysemys picta are long matozoon is well-defined (Furieri, 1970). However, (50-55 pm) and narrow (maximum of 0.9 pm), giving methods for optimum preservation of tissues must be them a vermiform appearance, with a head that is used to attain finer resolution of several structures. In curved and pointed (Figs. la-3). Viewed by SEM, bulgparticular, ultrastructures of the globular mitochon- ing spheroidal mitochondria were conspicuous in the dria, intranuclear tubules, and the distal centriole midpiece (Fig. lb). should be clarified. Therefore, the purpose of the present study was to examine the ultrastructural charHead acteristics of turtle sperm using improved methods of The head is 11-12 pm long by 0.9 pm wide at the fixation and staining. We report the sperm ultrastruc- largest diameter. The tapering anterior one-third of ture of a species (Chrysemys picta) in which oviductal the acrosome extending beyond the tip of the nucleus sperm storage may be a n essential component of the has a dense homogeneous matrix that continues postereproductive cycle (Gist et al., 1990). riorly in a very thin layer that covers the perforatorium and the anterior protrusion of the nucleus (Figs. MATERIALS AND METHODS 2,4,5). The acrosomal membrane is separated from the Turtles (C. picta) were captured in October 1988 from overlying plasmalemma by a space of varying width ponds located in southwestern Ohio. Sperm was col- (Figs. 6-8). At the posterior margin of the acrosome, lected within one week of capture by cloaca1 electro- the plasmalemma is very closely bound to the outer ejaculation a s described elsewhere (Gist et al., 1990) nuclear membrane, forming a junction that closes off and suspended in physiological saline. Spermatozoa the subplasmalemmal space over the anterior portion were concentrated by centrifugation and resuspended of the head (Fig. 5). In the subacrosomal area, a thin in 3% glutaraldehyde (in 0.1 M cacodylate buffer, pH 7.4) and fixed for 3 hr. After 3 washes in cacodylate buffer, the samples were postfixed in 1% osmium tetroxide for 1 hr. After dehydration through a graded Received November 16, 1989; accepted September 25, 1990 ethanol series, the samples were processed for SEM or 0 1991 WILEY-LISS. INC 474 R. A. HESS ET AL. lntranuclear Tubule Connecting Piece Proximal Centriole Distal Centriole Outer Dense Fibers Fibrous Sheath Fig. 1. a: Illustration of the turtle spermatozoon. b SEM of the head, midpiece and proximal region of the principal piece of the tail. Bar = 1 pm. 475 TURTLE SPERMATOZOON space separates the acrosomal cap from the perforatorium (Figs. 5, 8). The perforatorium consists of two components: (I) a central core of 2-3 electron-dense rods covered by a thin membrane (Figs. 4, 71, and (2) a n outer granular material t h a t covers the thin anterior portion of the nucleus (Figs. 4-5, 8). The electron-dense rods appear continuous with the dense cores of the intranuclear tubules and extend from the nucleus to the subacrosoma1 space at the tip of the perforatorium (Fig. 4). Beginning at the terminus of the acrosomal cap, the nucleus tapers as it penetrates the dense granular material of the perforatorium (Figs. 2,5). The nucleus ends a t the point where the rods of the perforatorium are continuous with the nuclear tubules (Fig. 4). The nucleus contains intensely stained chromatin surrounded by a n adherent membrane (Figs. 2, 5, 10). Paired or occasionally triplicate tubules, 55 pm in diameter and outlined by the nuclear membrane, extend through the center of the nucleus for approximately three-fourths of the length of the nucleus (Figs. 2, 8, 9, 11).I n the anterior portion, these tubules contain a dense core, triangular-shaped in cross section (Fig. 9); but at the terminal end the interior is electron lucent (Figs. 8, 11). Connecting and Middle Pieces A cross-striated connecting piece terminates in a concave implanlation fossa in the caudal end of the nucleus (Figs. 12-15). A connecting collar of finegrained amorphous material is wedged between the first mitochondria and the base of the nucleus (Fig. 12). This material extends laterally and upward while tapering to a point and forming a circumferential collar around the nucleus (Figs. 12, 14-15). When the spermatozoa were sectioned with proper orientation, a proximal centriole perpendicular to the distal centriole could be observed inserted into a vault of the connecting piece (Figs. 12, 13). The midpiece consists of proximal and distal centrioles surrounded by mitochondria (Fig. 12; see Fig. 19). A well-defined annulus separates the midpiece from the tail (see Fig. 19). A majority of spermatozoa taken from the epididymis, and a few from the ejaculate, contained a midpiece cytoplasmic droplet consisting of lipid-like bodies, vacuoles and smooth membranes (described in a separate article; Gist et al., submitted). The centrioles consist of typical nine triplet microtubules arranged in a pinwheel fashion and surrounded by a dense material (Figs. 16, 17). Triplicate microtubules of the distal centriole extend caudally through approximately two-thirds of the midpiece. In the anterior portion of the midpiece, a dense matrix forms a ring around the distal centriole and permeates the space between the microtubules (Fig. 16b). Moving caudally along the centriole, radial links project from this outer dense material, forming spaces between the sets of triplicate microtubules to join dense material surrounding the 2 central microtubules (Fig. 16a,c). Dissolution of the radial links occurs in distal progression (Fig. 17). Finally, only remnants of the dense substance remain attached to each of the outer nine doublets and the central singlet microtubules (Fig. 18). This outer dense fibrous material was lost in the prin- cipal piece of the tail, leaving only well-defined axonema1 units (see Figs. 21, 22). With routine electron microscopic staining, the distal centriole appears to contain doublet microtubules enmeshed by the dense outer ring of matter (Figs. 16a, 17a). With negative staining, the C microtubule of the triplicate is clearly seen (Figs. 16b, 17b). However, within a short distance from its origin, the C microtubule is open to the central axis, forming a longitudinal groove. The mitochondria of turtle sperm form 5 rows of 10 spherical bodies each for a total of 50 mitochondria per spermatozoon (Figs. 12, 16-18). They possess a n unusual structure. The core consists of thick-walled tubular cristae. Surrounding this mitochondria1 core are 7-8 concentric membranes, apparently lipid bilayers (Figs. 16-18). Lucent channels form the interior of the dense branching cristae (Figs. 16-18). The thick outer wall of the core forms a projection to which the concentric membranes are attached (Fig. 18a). The lamination (18 nm in width) preferentially binds lead citrate, creating a peppered appearance with clear spaces between layers (Figs. 17, 18). The plasmalemma of the mid-piece may be directly adjacent to the mitochondria or separated from them by a thin layer of cytoplasm (Figs 12, 16-19). In some areas, this layer is expanded as a n eccentric droplet of residual cytoplasm. Principal and End Pieces of the Tail The nxoncmal complex of the principal piece of the tail is surrounded by several layers of dense granular material of the circumferential fibers (Figs. 19-21). Near the annulus, these fibers are 24 nm in diameter, and several layers thick (Fig. 19), but they are reduced to a single layer near the endpiece (Fig. 20). A finegrained material, covered by the plasmalemma, surrounds the circumferential fibers at the beginning of the principal piece (Fig. 19), but this material stops at the beginning of the endpiece of the flagellum, which consists of the axonemal complex surrounded only by the plasmalemma (Figs. 20, 22). The A microtubule of the axonemal doublets begins to lose its dense core while still in the principal piece. Near the end of the flagellum, the doublet microtubules appear as singlet tubules with lucent cores and eventually their axonema1 organization is also lost (Figs. 22, 23). Out of 15 cross sections of the terminal end piece, 4 contained 20 singlet microtubules (Fig. 23), while others exhibited 6-19 microtubules. DISCUSSION The sauropsid form of the turtle sperm is similar to that of domestic birds, amphibia and other reptiles. However, several structures are unique from what is seen in mammalian and even other reptilian spermatozoa (Afzelius, 1979; Asa e t al., 1986; Boisson and Mattei, 1966; Burgos and Fawcett, 1956; Butler and Gabri, 1984; Carrick and Hughes, 1982; Clark, 1967; Fawcett, 1970; Philips and Asa, 1989; Picheral, 1979; Thurston and Hess, 1987). These unusual features include (1)a perforatorial cap over the proximal end of the nucleus, which contains 2-3 rods that are contiguous with intranuclear tubules; (2) a connecting collar of dense material that surrounds the base of the nucleus; (3) a distal centriole containing central microtu- 476 R. A. HESS ET AL. Figs. 2-10 TURTLE SPERMATOZOON 477 proximal centriole and extends into the implantation fossa at the base of the nucleus. Furieri (1970) did not separate the connecting piece from the surrounding collar and reported transverse microtubules outlining this piece. We did not find these microtubules; therefore, it is possible that the fixation method used in the earlier work (osmium tetroxide as the primary fixative) induced artifacts. In sperm from several avian species, a nonbanded connecting material projects from the triplicate, centriolar microtubules and inserts into the base of the nucleus (Asa et al., 1986; Thurston and Hess, 1987; Phillips and Asa, 1989). The connecting piece of mammalian sperm contains a capitulum located anterior to the centriole (Fawcett, 1970). As in the lizard (Clark, 1967), the toad (Burgos and Fawcett, 19561, and the bird (Thurston and Hess, 1987), turtle sperm contain 2 centrioles, the proximal centriole a t a right angle to the distal. However, the distal centriole in the turtle sperm has 2 central microtubules as in the rhea bird (Phillips and Asa, 19891, rather than a lucent core as in amphibians (Burgos and Fawcett, 19561, lizards (Clark, 19561, nonpasserine birds (Thurston and Hess, 1987), and the more primitive tinamou bird (Asa et al., 1986). Furieri (1970) reported, without illustration, that the turtle sperm contains a distal centriole but also claimed that the anterior portion of this structure was not a centriole. He reported that the opaque material that wraps the anterior portion of the flagellum forms a space alongside the doublet microtubules, giving the appearance of a third microtubule, as in a centriole. In the present study, tannic acid negative staining demonstrated that a third microtubule is present and that this microtubule forms a unique opening along its inner longitudiFig. 2. Acrosomal cap (A) covering the subacrosomal perforatorium nal side. Although it is well established that in cross (P) and twisted intranuclear tubules (T) that pass through the center section the B and C microtubules form partial rings of the nucleus (Nu). X 26,000. (the B ring bound to the A microtubule, and the C ring Fig. 3. The apex of the acrosome (A) is homogenous, consisting of bound to the B microtubule; Dustin, 1984), to our fine-grained, amorphous material anterior to the perforatorium (P). knowledge this is the first report of a microtubule PI, plasmalemma. x 45,000. groove being formed by a space between the protofilaments of the B and C microtubules. Fig. 4. The dense material that surrounds a perforatorial rod (R) may be part of the perforatorium. The rod is continuous with the The mitochondria have unusual configurations and contents of an intranuclear tubule (TI. A, lateral extension of the form poorly defined, but thick, dense cristae cores, suracrosomal cap; S, lucent space between granular material of subacrounded by concentric layers of membranes. The mitorosomal components and the outer acrosomal cap. X 45,000. chondria assume a staggered rows-and-columns arFig. 5. A junction (J) is formed by the adherence of the plasmarangement (10 rows and 5 columns) around the distal lemma (PI)to the nuclear membranes at the base of the acrosomal cap centriole, rather than a helix as in mammalian (Faw(A). The dense granular material of the perforatorium (P)terminates cett, 1975) and avian sperm (Thurston and Hess, 1987). at the junction (*I. T, intranuclear tubule; Nu, nucleus. x 69,000. Mitochondria from invertebrates display peculiar Fig. 6. Cross section of sperm head (level 6, Fig. 3) illustrates hoshapes (ring-shaped, fused, and triangulated; Afzelius, mogeneity of the anterior region of the acrosomal cap (A). The cap is 1979), and Chinese hamster sperm have circumferensurrounded by a swollen plasmalemma (PI). x 45,000. tial cristae (Fawcett, 1970). Opossum spermatozoa have spirals of membranes within the mitochondria1 Fig. 7. Cross section of a sperm head (level 7, Fig.-4) depicting circumferential acrosomal cap (A), perforatorial granular material (P) core (Fawcett, 1970) and membranous configurations and rods (R). X 45,000. are seen in mitochondria of the rhea (Phillips and Asa, 1989). However, no comparable structure such as the Fig. 8. Cross section of nucleus (Nu; level 8, Fig. 5) with two intranuclear tubules (T) containing triangular dense material. A, acroso- lipid bilayers of turtle sperm has been identified in ma1 cap; P, granular material of the perforatorium; Nu, nucleus. sperm from other species. Mitochondria1 modifications x 45,000. are reported in other species such as the bat, in which sperm also overwinter in the female tract (Wimsatt et Fig. 9. High magnification cross section in the apex of the nucleus al., 1966; Uchida and Mori, 1972) but are unlike those (Nu). The intranuclear tubules are outlined by membranes with 910-nm particles dotting the surface (arrows). x 104,000. of the turtle. Under appropriate conditions, mammalian spermatozoa can use phospholipids as a n energy Fig. 10. Cross section in the caudal region of the nucleus (level 10, Fig. 11)showing a single intranuclear tubule (T)surrounded by dense source (Mann, 1966); thus, we speculate that the unusual laminated mitochondria of turtle sperm might chromatin. x 45,000. bules that extend its entire length and having a outer triplicate microtubules that open toward the central cavity of the centriole; and (4) unusual spherical mitochondria containing 7-8 outer laminated membranes. The head of the turtle sperm is vermiform in shape rather than lance-shaped as described by Furieri (1970). Its acrosome forms a single cap as seen in many avian and mammalian sperm (Fawcett, 1970;Thurston and Hess, 1987). However, the subacrosomal space contains a perforatorium, not a second coaxial cap (Furieri, 1970), that differs considerably from those of other species. The perforatorium consists of rods surrounded by a granular material that forms a cap over the apex of the nucleus. This general structure resembles the perforatorium of lizard sperm (Butler and Gabri, 1984; Clark, 1967; Da Cruz-Landim and Da Cruz-Hofling, 1977; Del Conte, 1976) and those of amphibians (Burgos and Fawcett, 1956; Picheral, 1979) more than the perforatorium of avian sperm (Asa et al., 1986; Thurston and Hess, 1987).Like the rhea (Phillips and Asa 1989) and crested tinamou (Asa et al., 1986), the nucleus of the turtle sperm contains tubules, but they are unique in having a dense core and being lined by nuclear membranes (Sprando and Russell, 1988;Yasuzumi and Yasuda, 1968). The connecting piece of turtle sperm consists of alternating light and dense bands formed anterior to the 478 R. A. HESS E T AL. Fig. 11. Longitudinal section through the nucleus. Tubules (T) extend longitudinally through the center of the nucleus for at least the anterior two-thirds of the nucleus. Nm, nuclear membrane, P1, plasmalemma. x 38,000. Fig. 12. Longitudinal section through the midpiece. The base of the nucleus (Nu) forms a n implantation fossa (14)into which is inserted the banded connecting piece (15)of the neck. A connecting collar (Cc) surrounds the nucleus. Dense material of the collar is continuous with the dense matter of the proximal (Pc) and distal centrioles (16). The spherical, laminated mitochondria (M) are aligned in columns around the centrioles. Outer dense fibers (18)of the axoneme are continuous with the dense regions of the distal centriole. The principal piece of the tail contains a circumferential fibrous sheath (Cf). The numbers function to sustain the sperm during the long storage in the female oviduct. Prominent outer dense fibers are attached to the outer doublet microtubules in the midpiece of turtle indicate levels for approximate cross sections shown in subsequent figures. An, annulus; L, lipid droplet. x 26,000. Fig. 13. High magnification of the neck region. Note the proximal centriole (Pc) in a vault of the connecting piece (Cp). Cc, connecting collar; Dc, distal centriole; M, mitochondria; Nu, nucleus. x 40,000. Fig. 14. Cross section of the nucleus (level 14, Fig. 12). The connecting collar (Cc) extends completely around the nucleus (Nu). 1, lucent area marking the beginning of the implantation fossa. x 45,000. Fig. 15. Cross section of the neck (level 15, Fig. 12). A portion of the connecting piece (Cp), nucleus (Nu), and connecting collar (Cc) is depicted. x 45,000. sperm. These structures are absent or reduced in birds (Thurston and Hess, 1987; Phillips and Asa, 1989) but become pronounced in mammalian sperm (Fawcett, 1975).Thus, it may be argued evolutionarily that these Fig. 16.a: Cross section of midpiece (level 16; Fig. 12) illustrates the electron dense matrix (Dm) surrounding the anterior portion of the distal centriole. Dense material surrounds the central singlet (Cm) and outer triplet (Tm) microtubules and forms radial links between the outer ring and the inner matrix. Five columns of mitochondria (M) surround the distal centriole in the midpiece. x 55,000. b Tannic acid negative staining of a n anterior region of the distal centriole. The triplicate microtubules (a-c) are completely surrounded by the dense matrix (Dm). The b and c microtubules form partial rings that do not open to the matrix. Two central microtubules are also present in the anterior region. x 90,000. c: Tannic acid negative staining of the distal centriole in a region similar to a. The triplicate microtubules are present (a,b,c) but the “c” microtubule opens to the inside and forms a space within the dense matrix (arrow). x 90,000. Fig. 17.a: Cross section of midpiece (level 17, Fig. 12) showing the characteristic dissolution of dense matrix in the caudal portion of the distal centriole. The dense matrix forms incomplete radial spokes near the central microtubules (Cm). Tm, outer triplet microtubules; M, dense matrix of a mitochondrial core. x 55,000. b: Tannic acid negative staining of the caudal region of the distal centriole. The central space that is continuous with the c microtubule (arrow) nearly surrounds the two central microtubules. a-c: Triplicate microtubules. x 90,000. Fig. 18.a: Cross section of midpiece (level 18, Fig. 12). At this level, the dense matrix remains as outer dense fibers (Odf) associated with the outer doublet microtubules (Dm). The dense matrix nearly obscures the a microtubules. The dense wall of the mitochondrial cristae (Mc) protrudes to one side, to which the lamellar membranes are attached (*). The laminated membranes (La), which are layered over the mitochondrial core, are preferentially stained with lead citrate. Cm, central microtubules. x 55,000. b The beginning axonemal complex at level 18, Figure 12. The outer dense fibers (Odf) mask the a microtubules but both a and b microtubules are revealed with tannic acid negative staining. x 90,000. 480 R. A. HESS ET AL. Fig. 19. Longitudinal section of the midpiece-tail junction. A wedge-shaped annulus (An) separates the last mitochondria (M) and the principal piece. Outer dense fibers (Odf) of the axoneme continue for a short distance into the principal piece where a thick layer of circumferential fibers (Cf) form a sheath around the axonemal complex. Cm, central microtubules. X 52,000. Fig. 20. Longitudinal section of the junction between the principal and end pieces of the sperm tail. The circumferential fibers (Cf) are still present in the principal piece but are reduced in thickness. Central microtubules (Cm) are linked by a bridging substance (B). Dm, outer doublet microtubules. X 52,000. fibers developed early in vertebrate species, were lost in modern birds but then retained in the line leading to mammals. The circumferential fibrous sheath of the turtle sperm is similar to that in the rhea bird (Phillips and Asa, 1989) and the Platypus (Carrick and Hughes, 1982). However, the longitudinal thick columns of fibers that are prominent in mammalian sperm are absent in the turtle (Fawcett, 1970). Fig. 21. Cross sections of the principal piece reveal multiple and single layers of the circumferential fibers (Cf) woven around the axoneme. The axonemal complex consists of (A) and (B) microtubules, dynein arms (D) and radial links (Rl). In some doublets, the a microtubule has a lucent core. x 52,000. Fig. 22. Cross section of the endpiece of the tail. Only the plasmalemma surrounds the axoneme a t this level. X 52,000. Fig. 23. Cross section of the endpiece illustrating the dissociation of the axonemal pattern into 20 singlet microtubules. x 52,000. The flagellum exhibits a typical configuration of 9 + 2 microtubules common to avian and mammalian sperm (Fawcett, 1970; Thurston and Hess, 1987). The doublet microtubules became single near the tip of the flagellum, which produces up to 20 singlet microtubules in cross section, as it occurs in some mammalian species (Woolley and Nickels, 1985). However, the dense core of the A microtubule, which is normally lost TURTLE SPERMATOZOON after the formation of single tubules in the endpiece (Woolley and Nickels, 1985),begins to lose its electron density in the principal piece. CONCLUSION The turtle sperm contains several unique structures whose evolutionary origin and functional significance are intriguing. Of particular interest for future study is the function of the perforatorium, the intranuclear tubules, and the laminated mitochondria. ACKNOWLEDGMENTS We are grateful to Lou Ann Miller, of the Veterinary Medicine Electron Microscopy Suite, for outstanding technical assistance, and to the Spring Lawn Association, Cincinnati, Ohio, for the use of their ponds. LITERATURE CITED Afzelius, B.A. 1979 Sperm structure in relation to phylogeny in lower metazoa. In: The Spermatozoon. D.W. Fawcett and J.M. Bedford, eds. Urban and Schwarzenberg, Baltimore, pp. 243-251. Asa, C., D.M. Phillips, and J. 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