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Immunocytochemical study on fibrous sheath formation in mouse spermiogenesis using a monoclonal antibody.

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THE ANATOMICAL RECORD 215: 119-126 (1986)
lmmunocytochemical Study on Fibrous Sheath
Formation in Mouse Spermiogenesis Using a
Monoclonal Antibody
Department of Anatomy, School of Medicine, Kitasato Uniuersity, 1-15-1 Kitasato,
Sagamihara-shi, Kanagawa 228 W.S., 7:N., S. K), Laboratory o f Cell Biology, MitsubishiKasei Institute of Life Sciences, 11 Minamiooya, Machida-shi, Tokyo 194 (YK., H.I?), Division
of Developmental Biology, Imamichi Institute for Animal Reproduction, 1103 Fukaya,
Dejima, Niihari, Ibaraki 300-01 (YK.) Japan
One of the components of the fibrous sheath was localized in the
spermatids by the immunocytochemical method using the monoclonal antibody,
K32, against the fibrous sheath of mouse mature epididymal sperm. The K32
immunoreaction was first detected in the cytoplasm of spermatids at stage 14 and
appeared to increase in intensity a t stage 15. At this stage, the framework structure
of the fibrous sheath was formed completely in the tail, but the positive reaction in
the fibrous sheath was observed only in the proximal portion of the principal piece.
This change in the antigenicity of the fibrous sheath proceeded in a proximal to
distal direction, which was opposite to the mode of formation of the framework
structure in the fibrous sheath. Finally, the entire fibrous sheath strongly reacted
to the K32 antibody at stage 16, while the reaction in the cytoplasm ceased to occur.
These observations indicate that the fibrous sheath matures with immunologically
detectable changes in its components following formation of the framework structure. In consideration of the retrograde progression of the cytoplasmic reaction, the
fibrous sheath components may possibly be transported from the spermatid cytoplasm into the principal piece.
The tail portion of mammalian sperm, consisting of
middle, principal, and end pieces, has a highly complicated internal organization. In the principal piece, axonemal filaments and outer dense fibers, continuous from
the middle piece, are surrounded by the fibrous sheath
(see review by Fawcett, 1975). These complex structures
help propel mammalian sperm through the female reproductive tract. The fibrous sheath in particular is a
structure specific to mammals; its structural components and their functional roles remain to be clarified.
Biochemical analysis of a n isolated rat fibrous sheath
showed the entire structure to be composed predominantly of a single polypeptide with a molecular weight
of 80,000 daltons (Olson et al., 1976). In a similar study,
a fibrous sheath constituent was found in the 74,000dalton component of the mouse sperm tail fraction
Brien and Bellve, 1980a). Electron microscopic studies have shown the fibrous sheath to consist of two
morphologically different components: longitudinal columns and ribs (Sapsford et al., 1970). The longitudinal
columns develop first and are later joined by fine ribs
which subsequently thicken and aggregate to form definitive ribs. These components, which contain filaments
linked by a matrix substance, constitute the framework
of the fibrous sheath. Irons and Clermont (1982a) examined fibrous sheath formation in the rat by electron
microscope autoradiography and found the structurally
0 1986 ALAN R. LISS, INC.
different components to be formed from the distal to
proximal end of the tail by two independent mechanisms. Thus, the fibrous sheath may not be composed of
only a single molecule but may contain minor components formed by different mechanisms.
To analyze the mechanism of fibrous sheath formation, the appearance of each component in the fibrous
sheath during spermiogenesis must be observed. Recently, we obtained a monoclonal antibody which reacted with the tails of mature epididymal sperm (Koyama et al., 1984). As described in the previous paper,
this monoclonal antibody, K32, specifically reacted with
the fibrous sheath of mature sperm. Although complete
characterization of the antigen molecules has yet to be
made, it is certain that this antibody reacts specifically
with one of the fibrous sheath components. Analysis of
the K32 antibody should clarify the mode of the formation of this fibrous sheath. In the present paper, the
formation of the fibrous sheath was examined with a n
electron microscope and immunocytochemical techReceived July 12,1985; accepted December 4,1985.
Y. Koyama’s present address is the Division of Cell Biology, Institute for Comprehensive Medical Science, School of Medicine, FujitaGakuen Health University, Toyoake, Aichi 470-11,Japan.
Address reprint requests to Dr. Yasuhiro Sakai, Department of
Anatomy, School of Medicine, Kitasato University, 1-15-1 Kitasato,
Sagamihara-shi, Kanagawa 228, Japan.
niques to detect changes in its components during tected in the inner region of seminiferous tubules. Some
seminiferous tubules stained strongly but others failed
completely to do so (Fig. 1). The spermatids a t the early
and middle stages showed no positive reaction (Fig. 2).
Morphological technique
Figure 3 shows a tubule containing late spermatids. A
For general electron microscopy, BALB/c strain mice strong reaction with the antibody was evident along the
under nembutal anesthesia were perfused with 5% glu- inner layer of the seminiferous tubule but no reaction
taraldehyde in 0.05 M phosphate buffer (pH 7.2) for 30 products could be detected in the tail. At the last stage
minutes after a short period of perfusion with phos- just before release of the spermatids into the lumen of
phate-buffered saline (PBS; pH 7.2). Small pieces of testes the seminiferous tubules, the tail reacted strongly with
were further fixed with 2% glutaraldehyde in 0.05 M the K32 antibody (Fig. 4).
To demonstrate the precise location of this antigen,
phosphate buffer a t 4°C for 1 hour. After washing, the
samples were postfixed with 1% osmium tetroxide, de- further observations were made with a n electron microhydrated with a graded series of alcohols, and embedded scope, aided by immunoperoxidase staining. In spermain an epoxy resin mixture. Thin sections were observed tids at stage 13, the fibrous sheath could still not be
with a Hitachi H-600 electron microscope after being observed in most regions of the tail though the distal
stained with uranyl acetate and lead citrate. The stages end contained the developing fibrous sheath. At this
of mouse spermiogenesis were determined by the histo- stage, no positive immunoreaction was observed (Fig. 5).
During stage 14, the nucleus of each spermatid conlogical criteria of Oakberg (1956) and fine structural
densed a t its anterior part. In the cytoplasm, mitochoncriteria of Dooher and Bennett (1973).
dria were scattered about, microtubules of the manchette
Preparation of the monoclonal antibody
were prominent, and invading Sertoli cell processes
The isolation of hybridoma clones was described in the could be seen. Numerous cross-section profiles of the
previous paper (Koyama et al. 1984). Briefly, BALB/c cisternae of the endoplasmic reticulum were recognized
female mice were immunized with mature sperm. Spleen in the cytoplasm as vesicles. At this stage, the first
cells were fused with SP2/0-Ag14(SP2) mouse myeloma indication of a n immunoreaction appeared in the cytocells, and hybridoma cells producing antisperm monoclo- plasm of the spermatids but many vesicles and Sertoli
nal antibodies were selected by indirect immunofluores- cell processes failed to do so (Fig. 6). The immunoreactivcence staining of smeared sperm samples. The K32 ity differed from one spermatid to other. Figure 7 shows
hybridoma cells produce IgGl immunoglobulins. Their a spermatid tail at stage 14 and the fibrous sheath to
monoclonality was checked by the two-dimensional gel have formed at the distal portion. Flagellar cytoplasm
electrophoretic analysis (Pearson and Anderson, 1983). containing tubular materials was located just proximal
The hybridoma cells were inoculated into the peritoneal to the developing fibrous sheath. The fibrous sheath and
cavity of pristane-treated BALB/c mice and ascitic fluid flagellar cytoplasm of the spermatid contained no reacwas collected after about 1week. Collected ascitic fluid tion products (Fig. 8).
At stage 15, mitochondria were arrayed along the axwas diluted 1:lOO with PBS and used as the first antiial fibrillar complex to form the mitochondrial sheath of
body for the immunocytochemistry.
the middle piece, and many small vesicles and Sertoli
lrnmunocytochemical technique
cell processes could be seen in the cytoplasm (Fig. 9). In
Under nembutal anesthesia, BALB/c strain mice were the spermatids at this stage, the fibrous sheath was
perfused with 2% paraformaldehyde for 40 minutes, and completed morphologically along the axial fibrillar comthe fixed testes were washed overnight at 4°C with PBS plex in the principal piece. The proximal end of the
containing 7% sucrose. After washing, cryostat sections fibrous sheath was close to the annulus and no flagellar
about 7 pm thick were prepared. The sections were cytoplasm could be seen. The cytoplasm of spermatids
rinsed with PBS and allowed to react with the monoclo- during this stage showed strong reaction with the K32
nal antibody K32 at room temperature for 0.5-1 hour. antibody (Fig. 10). The outer dense fibers appeared dark
Control samples were treated with ascitic fluid produced but not more so than those of the control. The reaction
by injections of SP2 myeloma cells. After being washed of the mitochondrial membrane seemed to be nonspeagain, the sections were treated with horseradish per- cific, since it occurred only in the region where the
oxidase-labelled antimouse IgG goat immunoglobulin cytoplasmic matrix reacted strongly with the K32 antiprepared by the method of Yamashita et al. (1976) at body. During this stage, some spermatids showed posiroom temperature for 0.5-1 hour. The reaction product tive reaction only in the proximal portion of the fibrous
was colored with a diaminobenzidine-hydrogen perox- sheath of the tail (Fig. 111,but other spermatids did not,
ide solution. Some sections were dehydrated and although formation of the fibrous sheath had already
mounted in Canada balsam. The other sections were been completed.
At stage 16, ultrastructural features of the spermatids
postfixed with 1%osmium tetroxide in 0.1 M phosphate
buffer (pH7.2) at 4°C for 1hour and embedded in epoxy were essentially identical to those of mature sperm. The
resin. For light microscopy, thick sections were stained cytoplasmic lobes of the spermatids became residual
with toluidine blue solution. For electron microscopy, bodies. During this stage, the fibrous sheath reacted
thin sections were made and observed without strongly with the K32 antibody, as shown in Figure 12.
In cross sections of the principal piece of the tail, reaccounterstaining.
tion products were evident both in the longitudinal colRESULTS
umns and ribs (Fig. 12 insert). In the residual cytoplasm,
In the immunocytochemistry of cryostat sections of the however, reaction with the antibody was virtually negtestis, a positive reaction for the K32 antibody was de- ative. In rare cases, small amounts of reaction products
Fig. 1. Cryosections of testis stained with the K32 monoclonal antibody and peroxidase-labelled antimouse y-globulin. Some seminiferous
tubules have strongly reacted with the K32 antibody in the inner cell
layers and other tubules are devoid of any reaction products ( ~ 8 0 ) .
Fig. 2-4. Toluidine blue-counterstained Epon thick sections of cryosections stained by the immunoperoxidase method. Nuclei of germ
cells are darkly stained by toluidine blue.
Fig. 2. Spermatids at early stage of spermiogenesis have not reacted
with the K32 monoclonal antibody ( ~ 2 8 0 ) .
Fig. 3. Spermatids at late spermiogenesis show strong reaction to
the K32 antibody ( x 280).
Fig. 4. Spermatids at just the time of release into the lumen of the
seminiferous tubule show strong reaction with the antibody only in
the tail region ( x 280).
Fig. 5. A stage 13, a spermatid stained with the K32 monoclonal antibody. No positive reaction is
evident (X9,OOO). M, manchette; Nu, nucleus.
Fig. 6. Section of a stage 14 spermatid stained with the K32 monoclonal antibody. A positive reaction is
evident in the cytoplasm but not in many vesicles and Sertoli cell processes (SCP)( X 10,000).An, annulus;
Nu. nucleus.
Fig. 7. Longitudinal sections of the tail region of a stage 14 spermatid. The fibrous sheath (FS) develops in a distal to proximal direction.
The flagellar cytoplasm (FC) containing fibrous material is visible at
the proximal end of the fibrous sheath (X20,OOO).
Fig. 8. Longitudinal section indicating the tail region of a stage 14
spermatid stained with the K32 monoclonal antibody. Fibrous sheath
(FS) and flagellar cytoplasm (FC) are devoid of any reaction products
( x 20,000).
could be seen about aggregated cell organelles.
Control experiments for cytochemical staining were
performed by staining with SP2 instead of K32 ascitic
fluid. No positive reaction deposits could be detected in
any of the stages. In this paper, a spermatid a t stage 15
is shown as representative of stages 13-16 (Fig. 13).
mature sperm. Some change detectable immunologically must take place in the fibrous sheath following
completion of the framework structure. Sapsford et al.
(1970) found the longitudinal columns and ribs to develop by the addition of moderately dense material between converging filaments. Thus, some molecules may
possibly be added to the morphologically completed fibrous sheath. Though we cannot definitely conclude
whether the appearance of the antigen detected by the
K32 antibody is caused by the addition of newly formed
components to the fibrous sheath framework structure
or by modification of preexisting components, it is evident that fibrous sheath formation is completed by a n
additional step so far not detected.
Fine structural observations of spermatids showed the
formation of the fibrous sheath to start at the distal end
of the tail and extend in a proximal direction (Sapsford
et al., 1970; Irons and Clermont, 1982a). The flagellar
cytoplasm containing filamentous material appears
temporarily at the proximal end of the developing fi-
In the present study, formation of the fibrous sheath
of mouse sperm was observed during spermiogenesis
immunocytochemically with the aid of the antifibrous
sheath monoclonal antibody K32. A positive immunoreaction with this antibody was detected in the fibrous
sheath and cytoplasm of late-stage spermatids.
Antigens in the fibrous sheath of the tail first appeared at stage 15, following completion of the fibrous
sheath framework structure. Thus, the K32 antibody
does not react with the framework structure of the fibrous sheath. But as indicated in the previous paper,
this antibody certainly reacts with the fibrous sheath of
Fig. 12. Section of stage 16 spermatids stained with the K32 monoclonal antibody. A positive reaction is evident in the fibrous sheath of
the tail but the residual cytoplasm (RC) is virtually devoid of reaction
products (x8,OOO). Longitudinal column and rib structures of the fibrous sheath in the insert are equally stained (~30,000).
Fig. 13. Control section of a stage 15 spermatid stained with SP2 FS, fibrous sheath; Nu, nucleus; ODF, outer dense fiber.
instead of K32 ascitic fluid. No positive reaction is evident ( ~ 8 , 0 0 0 ) .
brous sheath. A direct transformation of the filamentous
material to dense elements of the fibrous sheath is suggested by De Kretser (lg6’). The flage11ar
proceeds in a proximal direction during formation of the
fibrous sheath (Rattner and Brinkley, 1970;Wartenberg
Fig. 10. Section of a stage 15 spermatid stained with the K32 mono- and Holstein, 1975; Irons and Clermont, 1982a) and
clonal antibody. A strong reaction is apparent in the cytoplasm. Negative reaction regions in the cytoplasm are Sertoli cell processes (SCP). disappears with the formation Of the framework strutThe outer dense fibers (ODF) appear electron dense but not more so ture and €%&ddkhment of the principal piece. The data
than those of the control ( X 11,000).
of the present study indicate the appearance of the antigen detected by the K32 antibody proceeds in a proxiFig. 11. Immuno-electron micrograph of the proximal tail region of a
direction*This direction is Opposite to that
stage 15 spermatid. The fibrous sheath at the proximal side is stained
by the K32 antibody, but no reaction deposits are evident in the distal Of the mode of fibrous Sheath formation previously
side ( X 15,000).An, annulus.
Fig. 9.Section of a stage 15 spermatid. The nucleus has uniformly
condensed and mitochondria surround the axial fibrillar complex in
the middle piece of the sperm tail. Developed framework structure of
the fibrous sheath is seen in the principal piece. A Sertoli cell process
(SCP)is enclosed in the spermatid cytoplasm (x5,OOO). An, Annulus.
Besides the reaction in the tail, K32 immunoreactive
sites were detected in the cytoplasm of spermatids before
appearing in the fibrous sheath. An autoradiographic
study of Irons and Clermont (1982a) demonstrates that
only a small number of silver grains can be recognized
in the fibrous sheath 1 hour after a n intratesticular
injection of 3H-proline, but this region becomes heavily
labelled 2 or 4 days later. Such attempts to determine
the sites of the synthesis of the fibrous sheath and outer
dense fiber proteins appear to be unsuccessful because
of the nonspecific nature of labelled precursors (Irons
and Clermont, 1982a,b). But Irons and Clermont’s
(1982a) study does indicate that components of the fibrous sheath may possibly be transported from other
areas in the cell to the tail. According to O’Brien and
Bellve (1980b), tail structural proteins are mainly synthesized at the middle or late stages of spermiogenesis.
The present study shows that reaction deposits disappear from the cytoplasm at the last stage of spermiogenesis when the positive reaction in the fibrous sheath
becomes strong. Thus, some fibrous sheath components
may be transported from the cytoplasm of the spermatids into the tail portion. Biochemical identification of
the antigens which react with the K32 antibody will be
necessary for confirmation of this possibility.
The authors wish to thank the members of the Electron Microscope Laboratory a t Kitasato University for
their very capable technical assistance.
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