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Morphological changes in the midpiece of wooly opossum spermatozoa during epididymal transit.

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Morphological Changes in the Midpiece of
Wooly Opossum Spermatozoa during
Epididymal Transit
Department of Anatomy and Laboratory of Human Reproduction and
Reproductive Biology, Harvmd Medical School,
Boston, Massachusetts 021 1 5
Several ultrastructural changes were found to occur in the midpiece region of wooly opossum spermatozoa during epididymal maturation. The
changes include alterations in mitochondrial morphology, development of structural specializations of the plasma membrane, and acquisition OF a prominent
extracellular coating. The lamellar membrane network which is wound about
the periphery of the mitochondria becomes more densely packed during sperm
development and the reticular network of membranes noted in the center of the
mitochondria of immature sperm disappears leaving a homogeneous electron
dense central zone. During epididymal transit the plasma membrane over the
sperm midpiece region shows extensive structural modification. I n cross sections
of paired spermatozoa the plasma membrane of the midpiece regions shows a
very regular, repetitive scalloping. In longitudinal sections the scalloping is observed as continuous parallel ridges which extend slightly obliquely to the flagellar long axis. Each ridge appears to be greater in density than the interridge
areas. In the epididymis a prominent extracellular coating of dense material is
deposited over the midpiece surface; this material is similar in appearance to
dense material seen i n restricted areas of the epididymal lumen. At the proximal
and distal ends of the midpiece the plasma membrane comes into intimate contact with underlying structural specializations and it is suggested that these zones
of fusion may serve to preserve regional differences in membrane composition.
Mammalian spermatozoa undergo morphological and biochemical changes during
their passage through the epididymis; spermatozoa from the initial segments of the
epididymis show poor motility and low
fertilizing capacity whereas mature spermatozoa from the distal regions of the duct
demonstrate both progressive motility and
high fertilizing capacity (see review by
Hamilton, '75; Bedford, '75; Orgebin-Crist
et al.,'75). Ultrastructural studies correlating changes in sperm morphology during
epididymal transit with changes in their
functional capacity have primarily employed eutherian mammals as the model
system (Fawcett and Hollenberg, '63; Fawcett and Phillips, '69), so that while some
studies have described the unique structural features of marsupial spermatozoa
(Phillips, '70; Olson, '75), only little is
known of morphological changes occurring
in them in the epididymis. In a n early
ANAT. REC., 186: 387-404.
study (v. Korff, '02) it was noted that opossum spermatozoa become tightly paired
head-to-head during epididymal passage
and later, at the electron microscope level,
it was shown that the pairing involves a
tight association of only a small zone of
the plasma membranes (Phillips, '70), but
maturational changes in other portions of
the sperm have not been studied. This paper reports new observations on specific
changes that occur during epididymal transit in the ultrastructure of the flagellum of
spermatozoa of the marsupial, Caluromys
Two sexually mature wooly opossums,
Caluromys philander, were anesthetized
with Nernbutal and the testes and epididyReceived Apr. 2, '76. Accepted June 16, '76.
1 Supported by USPHS Grant HD-04290.
2 Population Council Fellow.
piece region. However, at the lateral margins of the nucleus the plasma membrane
does come into relatively close contact
with the membrane covering the cytoplasmic vacuole and appears to insert into the
dense plaque along the nuclear margin
(figs. 3, 4).
The periphery of the midpiece mitochondria of- immature sperm is composed
of prominent laminae of whorled membranes. In contrast, the centers of the mitochondria demonstrate a loosely packed reticular network of paired membranes which
ramify through a (more electron) dense
ground substance (figs. 3 , 5). The mitochondria wrap about the flagellum in a
spiral fashion and the mitochondria of adjacent gyres are interconnected by delicate
strands of filamentous material (fig. 5).
Unpaired spermatozoa
The midpiece of unpaired spermatozoa
from the proximal epididymis is shaped
like an inverted flask, having its greatest
diameter near the proximal end of the
flagellum (figs. 1, 2, 3 ) . The expanded
region of midpiece cytoplasm, which is
anaIogous to the cytoplasmic droplet of
sperm of eutherian mammals, contains a n
array of densely packed, flattened, interconnected, empty vesicles (fig. 3). The nucleus of unpaired spermatozoa is attached
to the tail i n such a way that its plane of
flattening is nearly perpendicular to the
flagellar long axis (fig. 3 ) . A prominent
membrane bound vacuole, located immediately posterior to the nucleus, occupies the
anterior part of the midpiece region and is
limited laterally by a delicate sheath of
cytoplasm (figs. 3, 4 ) . The source of the
vacuole membrane is not clear but the
membrane comes into contact with a dense
plaque, which is probably homologous to
the posterior ring in sperm of eutherian
mammals, on the lateral margin of the nucleus; it is difficult to trace the vacuole
membrane through the dense plaque so i t
is not clear whether it represents the redundant nuclear envelope or not (figs. 3,
4). In contrast to the ground substance of
the epididymal lumen and the sperm cytoplasm, the vacuole generally possesses a
clear matrix interspersed only occasionally
with patches of flocculent material (fig. 3 ) .
No structural specializations are noted
in the plasma membrane covering the mid-
Paired spermatozoa
During epidid ym a1 pass age spermatozoa
become paired head-to-head i n a precise
and regular orientation (figs. 6, 7). As
shown previously (Phillips, '70) the pairing is accompanied by a reorientation of
the head with respect to the tail and the
plasma membranes of the paired spermatozoa are in close apposition only in a narrow zone along the lateral nuclear margin.
The region of membrane association between two spermatozoa does not involve
the electron dense plaque noted in irnmature spermatozoa, since the plaque is present in paired spermatozoa (fig. 7 ) .
Several dramatic alterations in midpiece
morphology are found in the paired spermatozoa when they are compared to immature spermatozoa. The lamellar arrays of
membranes surrounding the mitochondria
are more compacted than in immature
spermatozoa and the core of the mitochondria generally show a homogeneous matrix
devoid of the reticular array of membranes
found in immature sperm (fig. 8 ) .
Paired spermatozoa no longer display
the greatly expanded proximal end of the
midpiece. The array of densely packed,
flattened, interconnected vesicles noted in
immature spermatoma is absent and only
a few single vesicles rcrnain (compare figs.
7, 8 with fig. 3). The post-nuclear vacuole,
which was expansive in immature spermatozoa, is still present although reduced in
size (fig. 7).
The plasma membrane over the mid-
mides removed and fixed by immersion in
4% glutaraldehyde buffered in 0.2 M collidine, pH 7.4, at 4°C for four hours. The
tissue was rinsed in buffer and postfixed
in 1"/o OsO, in collidine at 4°C. After a buffer rinse the tissue was dehydrated through
a n ethanol series and propylene oxide and
embedded in Epon 812. Epididymides were
divided into a proximal (or caput) region
and a distal (or cauda) region, but no attempt was made to further subdivide the
organ. Thin sections cut on a n LKB-Huxley
ultramicrotome were stained with uranyl
acetate and lead citrate and examined in
a Siemens Elmiskop 1A at 80 kv. Photomicrographs of toluidine blue stained thick
sections were taken on a Zeiss photomicroscope.
piece region of paired spermatozoa also
shows structural specializations not seen in
immature spermatozoa. In transverse sections, where the 9
2 axoneme has been
sectioned perpendicular to its long axis,
the plasma membrane is seen to have a
delicately ruffled appearance (fig. 1 0 ) ; but
in transverse sections where the flagellum
is not sectioned exactly perpendicular to
its long axis this ruffling is seen as a repetitive precise scalloping of the plasma
membrane (fig. 11). Areas where the membrane is sectioned slightly obliquely show
hints of increased density at the scallops
(figs. 12, 1 3 ) , and in longitudinal sections
which graze the plasma membrane, parallel electron dense stripes are seen which
correspond in location to each of the
plasma membrane scallops (fig. 1 3 ) .
The external surf ace of the plasma membrane in the midpiece becomes associated
with a discontinuous layer of amorphous
dense material. This material is usually
separated from the plasma membrane by
a n electron lucent space (figs. 8, 10). I n
grazing sections of the midpiece region the
extracellular coating shows heterogeneous
densities with small areas of high density
interspersed in a less dense matrix (fig.
14). The extracellular coating is found
only over the midpiece region and ends
abruptly at the annulus (fig. 8 ) . In favorable sections a further membrane specialization is seen at the end of the midpiece:
the plasma membrane forms a single circumferential infolding that contacts the
underlying dense annulus (fig. 9).
In some areas of the epididymal lumen
deposits of dense material occur which are
strikingly similar in appearance to the material coating the sperm midpiece (fig. 15).
The deposits are often found between the
branching microvilli at the apices of the
epididymal epithelial cells (fig. 15).
It has been shown in several mammalian
species that spermatozoa acquire both fertilizing capacity and progressive motility
during passage through the epididymis.
Some of the specific morphological and biochemical changes include alteration of the
pattern of energy metabolism, changes in
surface antigenic properties, remodeling
of the acrosorne, and loss of the cytoplasmic droplet (Orgebin-Crist et al., '75; Bed-
ford, '75; Hamilton, '75, for reviews). Most
of the studies have utilized spermatozoa
from eutherian mammals and as a consequence little is known of similar changes
that occur in metatherian mammals. From
a morphological standpoint, it was noted i n
early work (v. Korff, '02) that in some o p s sum species, spermatozoa become tightly
paired during epididymd transit and even
that paired spermatozoa can be recovered
from the uterus of freshly mated females
(Selenka, 1887). Ultrastructural studies
of C a l u r m y s spermatozoa demonstrated
several features of sperm morphology including the expansive cytoplasmic droplet
of immature spermatozoa and the prominent extracellular coat of mature spermatozoa (Phillips, '70). In another opossum
species, Trichosurus vulpecula, a regular
helically wound array of flagellar accessory
fibers is found in close apposition to the
plasma membrane of the spermatozoon
midpiece region (Olson, '75).
The current study shows several new
ultrastructural features of wooly opossum
spermatozoa and demonstrates specific ultrastructural alterations that occur in the
spermatozoan midpiece during epididymal maturation. The mitochondria, which
spiral around the midpiece, show two anatomically distinct regions : in immature
spermatozoa loosely packed lamellar arrays
of membrane compose the periphery of
the mitochondria while their center consists of a sparse reticular network of membranes embedded in a granular matrix. It
is interesting to note that the mitochondria
in adjacent gyres are cross linked by conspicuous filamentous processes. I n paired
spermatozoa the lamellar arrays of membrane about the mitochondrion periphery
tend to be more tightly packed and the
center shows only a granular matrix and
no reticulum of membranes. These ultrastructural changes may provide a basis for
correlating changes in sperm energy metabolism, which have been shown to occur
in sperm of many species during epididyma1 transit (Voglmayr, '75), with morphological changes in a specific part of the
spermatozoon's energy generating apparatus.
Sperm maturation also appears to be associated with general remodeling of the
spermatozoon midpiece region. The midpiece of immature sperm is shaped like an
inverted flask and its anterior end is filled
with a dense network of interconnected
vesicles while mature sperm show a reduction both in the diameter of the proximal
end of the midpiece and in the amount of
intracellular vesicles. The changes in the
plasma membrane along the length of the
midpiece region are quite dramatic. In immature spermatozoa the midpiece plasma
membrane shows no obvious specialization.
However, in flagellar cross sections of
paired spermatozoa the plasma membrane
is fixed into a rigid conformation showing
a very regular repetitive scalloping. In grazing longitudinal section it is seen that each
scallop is continuous and oriented obliquely
along the flagellar long axis, thereby following a spiral path. When viewed in long
section each of the membrane scallops is
associated with material of increased electron density. This material is probably analogous to the membrane associated fibers
which form a very regular helical network
about the midpiece of spermatozoa of another opossum species, Trichosurus vzilpecula (Olson, '75). However, in CaEuromys
spermatozoa no distinct fibers are noted
and the number of membrane scallops exceeds the number of fibers found in the
midpiece of Tric hosurus spermatozoa. Thus
while the function of this midpiece specialization remains obscure it may be a constant feature of spermatozoa of metatherian mammals.
The final midpiece specialization acquired during epididymal maturation is the
prominent extracellular coat. This coating
was first described by Phillips ('70) but in
the present study the coating is shown to
be similar in morphology to dense material
found at the apical margins of some epididymal epithelial cells. Although the composition and origin of this material remains
to be elucidated, it could represent specific
epididymal secretions which become associated with the spermatozoan surface.
Immunological studies have previously
identified antigenic changes occurring in
spermatozoa during epididymal passage
and it has been shown that specific epididyma1 secretions may bind and alter the surface characteristics of the spermatozoon
(Hunter, '69). However, at the ultrastructural level it has been difficult to visualize
alterations in the surface coating of the
spermatozoon. Because of the amount of
material deposited on the midpiece region,
opossum sperm may provide a model system for the biochemical characterization
of epididymal secretions which become related to specific regions of the sperm surface.
It is interesting that the remodeling of
the plasma membrane, described in the
present study, should be restricted to a precise flagellar segment. Previous work has
shown regional variations in the distribution of negative charges and lectin binding sites on the sperm surface (Yanagimachi et al., '72). Furthermore, studies
of membrane fluidity have shown regional variations in the capacity for translational mobility of surf ace antigens over
the sperm surface (A'icolson and Yanagimachi, '74), and freeze fracture studies
have shown regional differences in the
translational mobility of intramembranous
particles (Koehler and Gaddum-Rosse, '75;
Friend and Rudolf, '75). The current study
demonstrates unique properties of the
plasma membrane over the midpiece region of wooly opossum spermatozoa. Since
the plasma membrane comes into contact
with submembranous specializations at
both the anterior and posterior end of the
midpiece, these zones of membrane contact may act as barriers to prevent lateral
diffusion of membrane proteins and preserve regional variations in membrane
Bedford, J. 1975 Maturation, transport and
f a t e of spermatozoa in the epididymis. In:
Handbook of Physiology. Section VII. Endocrinology, Volume V. Male Reproductive System. D. W. Hamilton and R. 0. Greep, eds.
American Physiological Society, Bethesda, pp.
Fawcett, D. W., and R. D. Hollenberg 1963
Changes in t h e acrosome of guinea pig spermatozoa during passage through the epididymis. Z. Zellforsch. Mikroskop Anat., 60: 279292.
Fawcett, D. W., a n d D. M. Phillips 1969 Observations o n the release of spermatozoa and
o n changes in the head during passage through
the epididymis. J. Reprod. Fert. Suppl., 6: 405418.
Friend, D. S . , and I. Rudolf 1975. Acrosomal
disruption in sperm. Freeze-fracture of altered
membranes. J. Cell Biol., 63: 466-479.
Hamilton, D. W. 1975 Structure and function
of the epithelium lining the ductuli efferentes,
ductus epididymis a n d ductus deferens in the
rat. I n : Handbook of Physiology. Section VLI.
Endocrinology. Volume V. Male Reproductive
System. D. W. Hamilton and R. 0. Greep, eds.
American Physiological Society, Bethesda, pp.
Hunter, A. G. 1969 Differentiation of rabbit
sperm antigens from those of seminal plasma.
J. Reprod. Fert., 20: 4 1 3 4 1 8 .
Koehler, J. K., and P. Gaddum-Rosse 1975
Media induced alterations of the membrane associated particles of the guinea pig sperm tail.
J. Ultrastruc. Res., 51; 106-118.
v. Korff, X. 1902 Zur Histogenese der Spermien
von Phalangista vulpina. Archiv f u r Mikroskopische Anatomie, 60: 232-260.
Nicolson, G. L., and R. Yanagimachi 1974 Mobility and restriction of mobility of plasma membrane lectin-binding components. Science, 184:
Olson, G. 1975 Observations on the ultrastructure of a fiber network in the flagellum of
sperm of the bush tailed phalanger, Trichosurus
vulpecula. J. Ultrastruct. Res., 50: 193-198.
Orgebin-Crist, M.-C., B. J. Danzo and J. Davies
1975 Endocrine control of the development
and maintenance of sperm fertilizing ability in
the epididymis. In: Handbook of Physiology.
Section VII. Endocrinology. Volume V. Male Reproductive System. D. W. Hamilton and R. 0.
Greep, eds. American Physiological Society, Bethesda, pp. 319-338.
Phillips, D. M. 1970 Ultrastructure of spermatozoa of the wooly opossum Caluromys philander. J. Ultrastruct. Res., 33: 381-397.
Selenka, E. 1887 Studien uber Entwickelungsgeschichte der Thiere. Viertes Heft Das Opossum (Didelphys virginiana). C. W. Kriedel's
Verlag, Wiesbaden.
Voglmayr, J. K.
Metabolic changes i n
spermatozoa during epididymal transit. In:
Handbook of Physiology. Section VII. Endocrinology. Volume V. Male Reproductive System.
D. W. Hamilton and R. 0. Greep, eds. American
Physiological Society, Bethesda, pp. 437-452.
Yanagimachi, R., Y. D. Noda, M. Fujimoto and
G. L. Nicolson 1972 The distribution of negative surface charges on mammalian spermatozoa. Am. J. Anat., 135: 497-520.
1 Photomicrograph of seminiferous tubule showing released spermatozoa
in the tubule lumen. Note the anterior expansion of the midpiece
(arrows) and the delicate strands of cytoplasm that extend around the
peripheral margins of the vacuole to attach to the lateral margins of
the flattened nucleus.
Photomicrograph of spermatozoa from proximal segment of the epididymis. The spermatozoa are still unpaired and appear similar jn
morphology to spermatozoa just released from the seminiferous tubule.
Gary E. Olson and David W. Hamilton
Thin section of head and proximal midpiece region of unpaired spermatozoon. Note the expanded area of cytoplasm filled with interconnected, flattened vesicles (Ve) and the thin strands of cytoplasm
( C ) which surround the vacuole (Va). While the epididymal luminal
contents show a finely granular appearance the vacuole is clear and
devoid of contents. The mitochondria ( M ) show a lamellar arrangement of membranes around their periphery while their cores contain
a reticular network of paired membranes. N, nucleus; A, acrosome.
x 17,000.
High magnification view of nucleus of immature spermatozoon. Note
how the plasma membrane ( P M ) and membrane limiting the intracellular vacuole ( V ) converge a t the lateral margin of the nucleus
and are embedded in an electron dense plaque (P). x 72,000.
High magnification electron micrograph of midpiece mitochondria of
immature spermatozoa as seen i n longitudinal section. Fine filamentous linkages extend between adjacent mitochondria and fuse to
the outer mitochondria1 membranes (arrows). x 50,000.
Gary E. Olson and David W. Hamilton
Photomicrograph of distal segment of the epididymis where the spermatozoa show characteristic head-to-head pairing (arrows). The midpiece region of the sperm n o longer possess the large anterior expansion noted in immature spermatozoa.
Electron micrograph showing nuclei of paired spermatozoa. The
plasma membranes of the paired spermatozoa come into close contact
in a single narrow zone (arrows). The dense plaque is still retained
a t the lateral margin of the nucleus where the plasma membrane
comes into close association with the membrane lining the vacuole
( P ) . Note also the apparent pinocytotic vesicles ( V ) being formed
by the plasma membrane. x 27,000.
Gary E . Olson and David W. Hamilton
Longitudinal section showing both the midpiece and principal piece
regions of a single spermatozoon from a region of the epididymis
where the sperm are paired. The midpiece has acquired a prominent
extracellular coating ( C ) . The mitochondria (MT) show a homogeneous granular core and the periphery is formed by a tightly
packed lamellar array of membranes. The outer dense fibers ( O D F )
show a very regular periodicity set at a n angle of 70"-80" to their
long axis. At the annulus (An) the plasma membrane has a n underlying layer of dense material. x 43,000.
9 High magnification micrograph showing the annulus which extends as
a plate-like layer of dense material under the last mitochondria1 layer
as well as a dense lainellae underlying the plasma membrane. Note
that at one point the plasma membrane apparently fuses to the underlying material of the annulus (arrow). x 63,000.
Gary E. Olson and David W. Hamilton
10 Transverse section of midpiece region of spermatozoon from tubule
of epididyniis where sperm were paired. In certain areas the plasma
membrane is seen to be regularly scalloped (arrows). C, extracellular
coat. x 49,000.
Slightly oblique section of midpiece region showing the very regular
repetitive scalloping of the plasma membrane ( P M ) . x 50,000.
Oblique section of midpiece region so that the scallops of the plasma
membrane appear as periodically spaced densities (arrows). x 62.000.
Gary E. Olson and David W. Hamilton
40 I
Grazing longitudinal section of sperm midpiece region so that the
scallops viewed i n cross section now appear as equally spaced electron
dense stripes (arrows). x 61,000.
Grazing longitudinal section of the extracellular coating of the midpiece region. The coating material shows electron dense aggregates
(arrows) embedded i n a less dense matrix. Note how the plasma
membrane ( P M ) shows periodically spaced ruffling. x 49,000.
15 Thin section of apex of epididymal epithelial cells and epididymal
lumen from region of the epididymis showing paired spermatozoa. The
surface microvilli ( M V ) show extensive branching patterns. Large
deposits of electron dense material (arrows) are found i n the epididymal lumen. X 26,000.
Gary E. 'Olson and David W. Hamilton
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woolf, transit, change, opossum, epididymal, morphological, spermatozoa, midpiece
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