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Disposition of the manchette in the normal equine spermatid.

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THE ANATOMICAL RECORD 209:177-183 (1984)
Disposition of the Manchette in the Normal Equine
Spermatid
KAREN L. GOODROWE AND EVERETT HEATH
Department of Veterinary Biosciences, College of Veterinary Medicine,
University of Illinois, Urbana, IL 61801
ABSTRACT
Bielanski and Kaczmarski (1979) reported the presence of
microtubules in the neck region of mature stallion spermatozoa. It was hypothesized that these microtubules are derived from the manchette (a microtubular
organelle present during spermiogenesis). In order to test this hypothesis,
testes from 15 mature stallions were collected, perfused with 2% phosphatebuffered glutaraldehyde, and prepared for transmission electron microscopy.
Spermatozoa from the caudae epididymides of each stallion were prepared in a
similar manner. Spermiogenesis was observed in general, and the presence of
a microtubular manchette was established in this species, juxtapositioned
posterior to the nuclear ring and extending distally into the cytoplasmic collar
which surrounds the prospective midpiece. Interlocking arms between the
microtubules of the manchette were observed in transverse sections at all
levels within the cytoplasmic collar before, during, and after caudal migration
of the nuclear ring. Consequent to caudal migration of the nuclear ring and
the annulus, as well as adluminal movement of the spermatid, the cytoplasmic
collar was transformed into the residual cytoplasm. Within the residual cytoplasm, the manchette remained as a microtubular organelle which undergoes
degeneration. The mature spermatozoa from the caudae epididymides of these
stallions lacked the microtubules reported by Bielanski and Kaczmarski. The
occurrence of such microtubules in the neck region of stallion spermatozoa is
probably a n abnormality.
The ultrastructure of equine spermatozoa
has recently been described by Bielanski and
Kaczmarski (1979). In this report, the authors found that these cells possess many of
the basic characteristics of other mature
mammalian spermatozoa, as well as some
unique features. Of special interest was
the observation of masses of microtubules in
the neck region of equine spermatozoa. This
feature is not common to other species and is
usually considered an abnormality when observed (Heath and Ott, 1982; Pedersen and
Hammen, 1982). Bielanski and Kaczmarski
did not identify the origin of these microtubules. A plausible explanation for their origin would be derivation from the manchette,
which is a microtubular organelle present
during spermiogenesis (Fawcett et al., 1971;
Phillips, 1974). In other mammalian species
this organelle is transient and is often described as “disappearing” during spermio-
0 1984 ALAN R. LISS, INC.
genesis, although it seems unlikely that any
cellular organelle could simply “disappear”
during as detailed and organized a course of
events a s is spermiogenesis.
Since no description concerning the ultrastructure of stallion spermiogenesis was
available, a study of this process was undertaken, placing special emphasis on the presence and disposition of the manchette. By
observing the manchette and its relationships within the spermatid during spermiogenesis, the final location of the organelle
could be determined. This could provide information as to whether or not the manchette is the origin of the microtubules in the
neck region of equine spermatozoa described
by Bielanski and Kaczmarski (1979).
Received March 10,1983; accepted December 8, 1983.
Address reprint requests to Karen L. Goodrowe, National Institutes of Health, Building 14G, Room 102, Bethesda, MD 20205.
178
K.L. GOODROWE AND E. HEATH
MATERIALS AND METHODS
Testes from 15 sexually mature stallions
were collected either upon castration (eight
animals) or at a n abattoir (seven animals).
The ages of these stallions ranged from 3 to
8 years, as determined by examination of the
incisor teeth.
Each testis and epididymis was rinsed with
water. Fluid was then taken from the cauda
epididymidis with a Pasteur pipette through
a small incision, and fixed in 2% phosphatebuffered glutaraldehyde. Each testis was perfused for fixation via the testicular artery
with 150-200 ml of 2% phosphate-buffered
glutaraldehyde. Perfusion of the testis was
completed within 15 minutes of castration
and within 45 minutes postmortem in the
abattoir samples. Samples were obtained immediately by removing a thin slice of testicular parenchyma with a razor blade from the
midventral region of each testis. This tissue
was then diced and held in 2% buffered glutaraldehyde overnight. The samples were
then secondarily fixed with 1% osmium tetroxide, dehydrated in a n alcohol series including 70% ethanol with 0.3% uranyl
acetate, and epoxy-embedded. Epididymal
fluid samples were handled in the same manner after compacting the spermatozoa into a
pellet by the method of Jones (1973).
Thick sections (2-3 pm) of each block were
stained with methylene blue for light microscopy. Seminiferous tubules with open lumens
in stages 4 through 8 of the cycle of the
seminiferous epithelium (Swierstra et al.,
1975) were selected to include the steps of
spermiogenesis in which the manchette is
known to occur and “disappear” in other species. Thin sections of these tubules were collected on 300 mesh grids, stained with uranyl
acetate and lead citrate solutions, and observed in a n RCA-EMU 3G electron microscope operated a t 100 kV.
RESULTS
Microtubules composing the manchette of
equine spermatids were first identified surrounding and paralleling the long axis of the
postacrosomal segment of spermatid nuclei
in stage 4 tubules. The nucleus in these spermatids was elliptical in shape and its chromatin not yet condensed (Fig. 1). In spermatids with more condensed chromatin the
nuclear ring was observed. In these cells the
microtubules of the manchette had begun to
lose their parallel position relative to the
nucleus and become more parallel to each
other (Fig. 2). Once parallel to each other, the
microtubules of the manchette formed a ring
around the postacrosomal segment of the nucleus and around the developing flagellum.
The anterior portion of the manchette appeared to be embedded in the nuclear ring
and its microtubules extended from this position caudally into the cytoplasm. After
chromatin condensation, in stage 6 tubules,
the nuclear ring and the manchette appeared
to have undergone a caudal migration from
the postacrosomal region to a position parallel to the striated columns in the neck and
developing midpiece.
After the presence of the manchette had
been established, transverse sections of the
flagellum through the midpiece and the surrounding cytoplasmic collar (in which the
manchette was located) were used to determine the disposition of the manchette during
spermiogenesis. The cytoplasmic collar can
be defined as that cytoplasm surrounding the
flagellum, bound by plasma membrane both
internally and externally, and therefore separated from the flagellum. This separation is
due to a caudal outfolding of the cytoplasmic
membrane from the region of the annulus, in
which the inner plasma membrane of the
cytoplasmic collar is continuous with the
plasma membrane of the developing principal piece a t the annulus (Fig. 3). In transverse section, the cytoplasmic collar appeared
as a cytoplasmic ring containing a ring of
microtubules composing the manchette (Figs.
4-6).
During early spermiogenesis, the ring of
microtubules in the cytoplasmic collar was
in a compact arrangement with individual
tubules interconnected by dense bridges or
arms (Fig. 4). The microtubules and their
bridges were characteristic of manchette microtubules throughout spermiogenesis. As
spermiogenesis proceeded, the ring of microtubules became less densely arranged and
underwent a change in position relative to
the developing flagellum and inner plasma
membrane of the cytoplasmic collar. The microtubules appeared to spread out peripherally, but the microtubular ring remained
intact and individual microtubules were still
joined to each other by the interconnecting
arms (Fig. 5).
Later, spreading out of the manchette became more pronounced (Fig. 6). The microtubular ring was more peripherally positioned
relative to both the flagellum and the inner
plasma membrane of the cytoplasmic collar.
Most of the microtubules were still joined by
MANCHETTE OF THE EQUINE SPERMATID
Fig. 1. Sagittal section of a spermatid with an elliptical nucleus. The manchette microtubules (arrows) are
parallel to the nucleus and extend caudally into the
cytoplasm. Marker = 1.0 pm.
Fig. 2. Sagittal section of a spermatid with an elliptical nucleus and condensing chromatin. The nuclear ring
(N) is present and the manchette microtubules (arrows)
are more parallel to each other than to the nucleus.
Marker = 1.0 pm.
Fig. 3. Sagittal section of a spermatid with condensed
chromatin. The cytoplasmic collar (arrows) is caudal to
the nucleus. The plasma membrane of the tail and the
inner plasma membrane of the cytoplasmic collar are
continuous at the annulus (A). Marker = 1.0 pm.
the characteristic interconnecting arms and
the general integrity of the microtubular ring
appeared intact. However, small breaks in
the ring began to appear (Fig. 6). Finally, the
integrity of the ring was lost, and only random groups of microtubules (still joined by
the characteristic interconnecting arms) were
observed in the cytoplasmic collar (Fig. 7).
Following condensation of the nuclear chromatin but prior to caudal migration of the
annulus, the cytoplasmic collar was eccentrically arranged around the developing fla-
179
180
K.L. GOODROWE AND E. HEATH
MANCHETTE OF THE EQUINE SPERMATID
181
Fig. 8. A lobe of residual cytoplasm containing a longitudinally cut array of microtubules (M). Marker = 1.0
pm.
Fig. 9. A lobe of residual cytoplasm containing a
transversely cut array of microtubules (M) with interlocking arms (arrow) characteristic of the manchette.
Marker = 0.5 pm.
Figs. 4-7. Transverse sections of midpieces and cytoplasmic collars arranged sequentially from earlier (Fig.
4)to later (Fig. 7). The manchette microtubules are identifiable throughout because of their interlocking arms
(small arrows) and are at first densely arranged in a
ringlike structure (Fig. 4) which gradually spreads peripherally (Figs. 5, 6) until breaks in the ring appear
(large arrow, Fig. 6 ) . Finally, only scattered groups of
microtubules are present (Fig. 7). Marker = 0.5 pm.
182
K.L. GOODROWE AND E. HEATH
Fig. 10. Neck region of mature normal equine spermatozoon. The only microtubules present
are those in the proximal centriole (C) and the axoneme (A). Marker = 0.5 pm.
gellum and contained most of the remaining prising the proximal centriole and the axoorganelles (Fig. 3). The cytoplasmic collar neme (Fig. 10).
was eventually transformed into the residual
DISCUSSION
cytoplasm.
The positioning of the microtubules of the
After caudal migration of the annulus, formation of the mitochondria1 helix in the mid- manchette parallel to the nucleus a t the onpiece, and adluminal movement of the set of spermatid elongation has been docuspermatid, the residual cytoplasm of the mented in various species (Fawcett et al.,
spermatids was clearly identifiable due to its 1971; Phillips, 1974). The present study esrelative density compared to nearby Sertoli tablishes that the manchette is formed and
cytoplasm (Fig. 8). Within the residual cyto- arranged in stallion spermiogenesis in a
plasm small masses of microtubules joined manner similar to that of other mammalian
by interconnecting arms characteristic of species.
those from the manchette were present. FigIn contrast to previous reports (Fawcett et
ure 8 depicts a lobe of residual cytoplasm al., 1971; Phillips, 1974), however, it can no
containing a mass of longitudinally cut mi- longer be assumed that the manchette “discrotubules. Figure 9 shows a lobe of residual appears” during spermiogenesis. Rather, this
cytoplasm containing a mass of transversely structure undergoes a n orderly breakdown
cut microtubules which are joined by arms with a definite final location. Alteration of
characteristic of those microtubules originat- the manchette can be attributed to a periphing in the manchette.
eral spreading out of the microtubular ring
Ransmission electron microscopy (TEM) (in relationship to the flagellum and inner
observation of the samples of epididymal cytoplasmic membrane) within the cytospermatozoa of the 15 stallions used in this plasmic collar. This process continues until
study revealed no microtubules in the neck breaks in the integrity of the ring occur.
region, other than those microtubules com- Throughout this process, the majority of
MANCHETTE OF THE EQUINE SPERMATID
these microtubules are joined by interconnecting arms which are characteristic of
manchette microtubules.
Photographs in the report of Dym and Cavicchia (1978: Figs. 11, 13) demonstrate a decrease in the number of microtubules
surrounding the flagellum in progressively
later steps of spermiogenesis in macaque
monkeys. These photographs also suggest a
breakdown of the integrity of the manchette
similar to what has been described here for
the stallion. Rattner and Brinkley (1972)
have reported a decrease in the number of
microtubules surrounding the tail in rodent
spermiogenesis, again similar to some of the
findings here. However, because of the scattering of microtubules consequent to breakdown of the integrity of the microtubular
ring of the manchette, it is difficult to determine if there is a n actual decrease in the
absolute number of these microtubules in the
stallion.
Once breaks in the microtubular ring of
the manchette occur, the microtubules scatter in groups throughout the cytoplasmic collar. Within these groups, the microtubules
are still identifiable as of manchette origin
due to being joined by the interconnecting
arms. Once the relationship between the flagellum and the cytoplasm changes due to
annulus migration, the manchette microtubules are still identifiable in the residual
cytoplasm due to the presence of the interconnecting arms between tubules. deKretser
(1969) hypothesized that in human spermiogenesis the final location of the manchette
microtubules is the residual cytoplasm. He
did not, however, show microtubules in the
residual cytoplasm joined by the characteristic interconnecting arms.
Since the sequence of events and structures
present in stallion spermiogenesis is quite
183
similar to those of other species studied (Phillips, 19741, it can be assumed that this disposition of the manchette of the stallion is
typical for other mammalian species a s well.
It should also be reemphasized that no microtubules were observed in the neck region
of spermatozoa from the cauda epididymidis
of the 15 stallions in this study. Therefore,
we conclude that microtubules, other than
those of the proximal centriole and axoneme,
are not normal structures of the neck region
of equine spermatozoa. Rather, we propose
that mature spermatozoa showing microtubules in the neck region should be considered
abnormal, as they are in other species (Heath
and Ott, 1982; Pedersen and Hammen, 1982).
LITERATURE CITED
Bielanski, W., and F. Kaczmarski (1979) Morphology of
spermatozoa in semen from stallions of normal fertility. J. Reprod. Fertil. [Suppl.], 27t39-45.
deKretser, D.M. (1969) Ultrastructural features of human spermiogenesis. Z. Zellforsch., 98t477-505.
Dym, M., and J.C. Cavicchia (1978) Functional morphology of the testis. Biol. Reprod., 18:l-15.
Fawcett, D.W., W.A. Anderson, and D.W. Phillips (1971)
Morphogenetic factors influencing the shape of the
sperm head. Dev. Biol., 26t220-251.
Heath, E., and R.S. Ott (1982) Diadedcrater defect in
spermatozoa of a bull. Vet. Rec., 11Ot5-6.
Jones, R.C. (1973) Preparation of spermatozoa for electron and light microscopy. J. Reprod. Fertil., 33t145149.
Pedersen, H., and R. Hammen (1982) Ultrastructure of
human spermatozoa with complete subcellular derangement. Arch. Androl., 9t251-259.
Phillips, D.M. (1974) Ed. Spermiogenesis. Academic
Press, New York.
Rattner, J.B., and B.R. Brinkley (1972)Ultrastructure of
mammalian spermiogenesis. 111. The organization and
morphogenesis of the manchette during spermiogenesis. J. Ultrastruct. Res., 41t209-218.
Swierstra, E.E., B.W. Pickett, and M.R. Gebauer (1975)
Spermatogenesis and duration of transit of spermatozoa through the excurrent ducts of stallions. J. Reprod.
Fertil. [Suppl.l, 23t53-57.
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