THE ANATOMICAL RECORD 223276-282 (1989) Development of Spermatozoa in the Rhea DAVID M. PHILLIPS AND CHERYL S. ASA The Population Council, New York,New York 10021 ABSTRACT We have examined the ultrastructural changes that take place during spermiogenesis in the rhea. Spermatozoa are characterized by a curved head and a midpiece. A thin rod extends from the anterior tip of the spermatozoon through the center of the nucleus. A 3-p-long distal centriole occupies the entire midpiece. The principal piece is characterized by a small fibrous sheath and tiny dense fibers that are only observed in the region of the principal piece, which is immediately behind the annulus. During development a circular manchette surrounds the nucleus of young spermatids. Later the microtubules of the circular manchette become reorganized into a longitudinal manchette. A long distal and short proximal centriole are observed in early round spermatids. The distal centriole becomes associated with the plasma membrane. Later the proximal centriole is observed in association with the nucleus. The area around the centriole pair then accumulates dense material, which is associated with either the centrioles or the circular manchette. The longitudinal manchette forms and then disappears and mitochondria subsequently associate with the distal centriole. The long centriole of the rhea enables this species to develop a midpiece similar to the midpiece of mammalian sperm without the complex intercellular movements that characterize mammalian spermiogenesis. Ultrastructural studies of spermiogenesis and spermatozoa have dealt primarily with insects and mammals. There are very few detailed studies of sperm development in other animal groups. In birds there have been ultrastructural studies of several dozen avian species (Asa and Phillips, 19871, representing a small percentage of the almost 9,000 species. Among avian species examined, even fewer of the investigations have focused on spermiogenesis. The notable exception, for reasons of economic importance, is the domestic chicken (McIntosh and Porter, 1967; Nicander and Hellstrom, 1967; Tingari, 1973; Nagano, 1962). We report here on the ultrastructure of the spermatozoa of the rhea, Rhea americans albisceus, and its development in the testis. The rhea is a large South American, flightless bird, one of the ratites, a group that also includes the ostrich, emu, cassowary, and by some accounts the kiwi (Sibley and Frelin, 1972; Cracraft, 1974, 1981; Sibley and Ahlquist, 1981; Stapel et al., 1984; Jacob and Hoerschelmann, 1985; Houde, 1986). Spermatozoa were prepared from semen collected by massage from a n adult male rhea. Semen was prepared for electron microscopy as described previously (Asa et al., 1986). RESULTS Mature Spermatozoa A curved, tapering head characterizes the spermatozoa of R. americans albisceus. A short midpiece is observed between the head and principal piece (Fig. la). The long principal piece comprises most of the length of the cell. A substantial acrosome fits over the anterior portion of the sperm nucleus. Viewed in both transverse and longitudinal section, the acrosome is seen to be composed of moderately electron-dense homogeneous material (Fig. l b and c). A narrow cylindrical structure extends from the anterior portion of the acrosome to deep within the nucleus (Fig. l b and c). The center of the cylinder is composed of material that is about the same electron density as the acrosome, which is circumscribed by a n electron-lucid region, in turn surrounded MATERIALS AND METHODS by a thin region of moderate electron density (Fig. l b Testicular material was obtained from a zoo specimen and c). The chomatin of the spermatozoa is compact but of R. americans albisceus that had died from accidental not as condensed as is observed in spermatozoa of insects causes. Within minutes of death, testes were removed. or mammals (Fig. la-d). As in many other animals, the neck region of the rhea Small pieces of tissue were fixed overnight in 3%glutaraldehyde buffered in 0.15 M phosphate buffer. Tissues sperm is characterized by a precisely shaped posterior were rinsed in buffer and postfixed in 1%phosphatebuffered OsO4 for 2 hours. After a brief rinse in buffer, Received May 27, 1988; accepted August 22, 1988. testis pieces were dehydrated in ethanol to propylene Cheryl S. Asa’s present address is St. Louis Zoo, Forest Park, St. oxide and embedded in Polybed. Sections cut on a Reich- Louis, MO 63110. ert OmU3 microtome were examined and photographed Address reprint requests to David M. Phillips, The Population Council, 1230 York Avenue, New York, NY 10021. with a Philips 300 microscope. 0 1989 ALAN R. LISS, INC. SPERMATOZOA IN RHEA Fig. 1. The mature spermatozoan of R. americans albisceus. a: Midpiece in longitudinal section. ~48,000.b: Longitudinal section of the acrosome. x 71,000. c: Transverse section of nucleus and acrosome. x 70,000. d: Longitudinal section of the neck region. X52,OOO. c: Trans- 277 verse sections of spermatozoa showing the proximal centriole (right) and distal centriole (left). x 52,000. E Transverse sections through principal pieces. x 105,000.Promixal centriole (p), distal centriole (d), annulus (a), acrosomal rod (r), dense fibers (do, fibrous sheath (D. 278 D.M. PHILLIPS AND C.S. ASA Fig. 2. The circular manchette of young spermatids seen in transverse section (a) and longitudinal section &I). ~80,000.Acrosomal rod (r)and acrosome (ad. portion of the nucleus. Electron-dense material associated with the proximal centriole fits into the contour of the sperm nucleus and associated nuclear membrane (Fig. l a and d). The proximal centriole is short, only about 0.4 pm long, and is inlaid with electron-dense material (Fig. Id and e). The distal centriole is much longer. In fact, because the distal centriole is observed in all cross sections of the midpiece, it apparently extends the entire length of the midpiece. Both centrioles are embedded in electron-dense material. Although the distal centriole displays the characteristically disposed triplet tubules, the central tubules of the flagellum extend into the center of the centriole (Fig. le). The midpiece of the rhea spermatozoa contains about 30 mitochondria. Mitochondria of rhea sperm are typical of sperm mitochondria in that the mitochondria1 matrix is very dense. In the interior of the mitochondrion distal to the centrioles, we observe complex configurations of mitochondrial membranes (Fig. l a , d and el. Transverse sections through the principal piece of rhea spermatozoa reveal a small fibrous sheath. In mammals the fibrous sheath is slightly larger opposite doublet tubules 3 and 8 and is connected t o these two doublets by a thin band of dense material. This is also true of rhea spermatozoa, but the fibrous sheath is only very slightly larger opposite doublets 3 and 8 (Fig. If). Spermatozoa of this bird have very tiny, dense, fibers, and they are present only for a short region at the very anterior portion of the midpiece. The dense fibers are observed as dense structures no larger than microtubules and are associated with the doublet subfibers a and b (Fig. 10. anteriorly (Fig. 2a and b). Anteriorly the circular manchette extends beyond the posterior end of the developing acrosome (Fig. 2a and b). Typically, microtubules are evenly spaced, and bridges are frequently observed between adjacent tubules. At later stages of spermatogenesis manchette microtubules are observed at various angles with respect to the nucleus. In the same section we have observed longitudinal, transverse, and oblique microtubules (Fig. 3a and b). At still later stages of spermiogenesis, the microtubules are seen to be arranged longitudinally (Fig. 4). Usually they are in arrays connected by bridges, similar to the arrangement of manchette tubules such as those observed in mammalian or insect spermatozoa (Phillips, 1974a). Formation of the midpiece Young spermatids contain a pair of centrioles. One is exceptionally long, but the other is very short. The short centriole lies at the base of the long centriole at right angles to it. In young, spherical-shaped spermatids the long centriole becomes associated with the plasma membrane, and the flagellum grows from it (Fig. 5a). Amorphous dense material is associated with the centriole. At a later stage of spermiogenesis the short proximal centriole is observed associated with the nucleus. Golgi and an electron-dense spherical structure are observed adjacent to the short (proximal)centriole (Fig. 5b). Dense material is observed under the plasmalemma, where the flagellum extends from the distal centriole (Fig. 5b). The material is in the analogous position of the annulus or ring centriole, which is observed in developing spermatids of other species except that it is further from the nucleus, as the distal centriole is longer. Sperrniogenesis At a stage of sperm development when microtubules The manchette of the manchette begin to appear, precisely shaped strucIn young spermatids a circular manchette surrounds tures form in the neck region. The electron-densespherthe developing nucleus. The manchette of microtubules ical structure of the neck region has become more oval can be observed in the anterior midpiece region posteri- in shape. Dense material is also observed associated orly and around the nucleus to the acrosomal region with microtubules of the manchette and the distal cen- SPERMATOZOA IN RHEA Fig. 3. Transitional stage between the circular and longitudinal manchettes seen in transverse section (a)and longitudinal section (b). ~65,000. 279 Fig. 4. The longitudinal manchette seen in transverse section (a) and longitudinal section (b). X 91,000. triole. As the sperm flagellum grows, the cell membrane that is attached to the annulus (ring centriole) is involuted. At a late stage of spermiogenesis the annulus and associated plasma membrane move more distally, until they reach the midpiece (Phillips, 1974a). The spermatid of the rhea accomplishes the same task in what appears to be a much simpler manner by virtue of having a very long distal centriole. The annulus forms a t the distal end of the distal centriole as in mammalian spermatids but it is not near the nucleus because the distal centriole is so long. The annulus never moves, as it does in mammals, and thus the length of the distal centriole becomes the length of the midpiece. Thus the extra-long centriole of bird spermatozoa allows the spermatozoa to form a midpiece without moving the annulus. This type of system would presumably not work in mammals, where in most species a centriole as long as the midpiece would DISCUSSION not fit in a round spermatid. The formation of two manchettes in rhea is unusual. In mammals the midpiece develops differently than in birds. Mammalian spermatids have a short distal cen- In a previous study McIntosh and Porter (1967) carefully triole (Fig. 5c and d). The nuclear membrane is observed to be more precisely shaped at later stages of spermiogenesis, and more dense material is seen associated with the distal centriole. Mitochondria are observed adjacent to the ring centriole. As the nucleus continues to condense, the amount of dense material that can be seen associated with the manchette increases (Fig. 5e and f). Later, when the circular manchette is lost and the longitudinal manchette appears, mitochondria are no longer observed near where the flagellum extends from the distal centriole. The microtubules of the longitudinal manchette extend even beyond what will be the midpiece (Fig. 5g). At a later stage of sperm development, the manchette is no longer present, and numerous mitochondria are observed adjacent to the distal centriole (Fig. 5h). 280 D.M. PHILLIPS AND C.S. ASA Fig. 5. Successive stages showing the development of the midpiece around the distal centriole. The short proximal and long distal centriole of very early spermatids (a) become associated with the nucleus (b). Ordered structures develop in the neck region (c) and mitochondria are observed near the annulus (d). Dense material forms in association with the circular manchette (e,D. Later in spermiogenesis the circular manchette is reorganized into a longitudinal manchette (g). Eventually the longitudinal manchette disappears, and mitochondria are ob. centriole (p), served around the distal centriole (h). ~ 2 5 , 0 0 0Proximal distal centriole (d), annulus (a), and fibrous sheath (0. 281 SPERMATOZOA IN RHEA examined the manchettes of the cock. They found similar circular and longitudinal manchettes and proposed that the circular manchette squeezed the nuclear contents and thus was responsible for nuclear condensation. In the rhea, between the stage of the circular and longitudinal manchettes, microtubules are arranged obliquely. Thus it appears as though the transition from circular to longitudinal manchettes is brought about by a rearrangement of microtubules rather than depolimerization and formation of new microtubules. Work in our laboratory with a number of unusual animal species has shown that spermatid nuclei are quite capable of condensing without a manchette (Asa and Phillips, 1988; Phillips, 1970; Phillips, 197413; Phillips, 1976), and thus the manchette may have other functions in the rhea. There is ongoing debate concerning the relationships of the tinamou, ratites, Galliformes (chickens, quail), and Anseriformes (ducks) (Sibley and Frelin, 1972; Cracraft, 1974, 1981; Sibley and Ahlquist, 1981; Stapel et al., 1984; Jacob and Hoerschelmann, 1985; Houde, 1986). Most agree that the tinamou and ratites are the most primitive forms, the tinamou perhaps representing the most ancient lineage. The most numerous, the passerine or song birds, are the most recent type, with the others falling somewhere in between. It is interesting, therefore, to consider spermatozoan features as taxonomic characters (Asa and Phillips, 1988). The spermatozoa of the tinamou do not have dense fibers (Asa et al., 1986). Rhea spermatozoa have very small dense fibers present only in the most anterior portion of the midpiece. Dense fibers of the spermatozoa of the domestic chicken (Bakst and Howarth, 1975) and mallard duck (Humphreys, 1972) are not prominent but are larger than those of the rhea. In further contrast, dense fibers of chicken and duck spermatozoa are present in the distal, not proximal, portion of the midpiece, beyond the termination of the distal centriole, which does not extend the entire length of the midpiece (Lake et al., 1968; Bakst and Howarth, 1975). Spermatozoa of the jacana, a Charadriform, apparently not closely related to ratites, chickens, or ducks (Stapel et al., 1984) also have small dense fibers in the midpiece. Our investigation of spermatozoa of the whitenaped crane (Phillips et al., 1987)revealed a n expecially small midpiece devoid of dense fibers. The crane is a member of the Gruiformes, a n order whose taxonomic position relative to the avian groups already mentioned is unestablished, but is probably not as primitive as the tinamou, ratites, Galliformes, or Anseriformes. An interesting observation reported by Mattei et al. (1972) is the disappearance of dense fibers from spermatozoa of the dove a t maturation. Because so few studies have addressed development of spermatozoa, it is not known if this phenomenon may be widespread taxonomically. Passerine birds are divided into two groups, the oscines, which are the more highly evolved, and the suboscines, which include birds such as the tyrant flycatchers and wood peewee (Asa and Phillips, 1987). The development of dense fibers is most pronounced in spermatozoa of the oscines, in which they are large and extend through almost the entire length of the cell (Furieri, 1961, 1962, 1963; Sugioka and Yasazumi, 1966; Humphreys, 1972; Henley et al., 1978). These dense fibers are very uniform in shape, in contrast to those of mammalian spermatozoa (Phillips, 1974b). In rhea spermatozoa, a n electron-dense structure lies directly behind the acrosome. We have called this structure a n acrosomal rod, however, in other species it has been referred to as the perforatorium, although its function is unknown. An acrosomal rod (perforatorium) has been described in most non-passerine birds (Nagano, 1962; Humphreys, 1972,1975; Bakst and Howarth, 1975; Thurston et al., 1982; Asa et al., 1986; Asa and Phillips, 1987). However, sperm of a few species apparently lack this structure (Saita et al., 1982, 1983). Overall, spermatozoa of the tinamou and rhea are very similar. However, tinamou spermatozoa contain large numbers of glycogen particles in the principal piece (Asa et al., 1986), and these are not present in rhea spermatozoa. A possible role of glycogen might be to nourish spermatozoa during storage. Sperm storage has been described in a wide range of passerine and non-passerine birds (Hatch, 1983; Shugart, 19881, however, it is not known if sperm storage occurs in the reproductive tracts of the male or female tinamou and rhea. ACKNOWLEDGMENTS We would like to thank Vanaja Zacharopoulos and Susan Warren for their excellent technical help. We are also grateful to Drs. Janet Stover and Emil Dolensek of the New York Zoological Society. The work was supported in part by a grant from the Andrew W. Mellon Foundation to Dr. David M. Phillips and a J.S. Noyes Foundation Fellowship to Dr. C.S. Asa. LITERATURE CITED Asa, C.S. and D.M. Phillips 1987 Ultrastructure of avian sperm: A short review. In: New Horizons in Sperm Cell Research. H. Mohri ed. Gordon and Breach Science Publishers, New York, pp. 365-373. Asa, C.S. and D.M. Phillips 1988 Nuclear shaping in spermatids of the Thai leaf frog Megophrys montana. Anat. Rec., 220:276-290. Asa, C.S., D.M. Phillips, and J. 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