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Development of spermatozoa in the rhea.

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THE ANATOMICAL RECORD 223276-282 (1989)
Development of Spermatozoa in the Rhea
The Population Council, New York,New York 10021
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
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).
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
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.
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-
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.
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.
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-
Fig. 3. Transitional stage between the circular and longitudinal
manchettes seen in transverse section (a)and longitudinal section (b).
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
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).
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.
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.
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.
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