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Vol. 14, Issue 7, 2768-2780, July 2003
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*Department of Anatomy,
Turku Graduate School of Biomedical Science,
Department of Physiology and Pediatrics,
University of Turku, FIN-20520 Turku, Finland
Submitted October 11, 2002;
Revised February 27, 2003;
Accepted February 27, 2003
Monitoring Editor: Randy Schekman
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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;
Fawcett et al., 1959).
Obviously the function of cytoplasmic bridges is to facilitate the sharing of
cytoplasmic constituents and to allow germ cell differentiation to be directed
by the products of both parental chromosomes
(Erickson, 1973). Despite all
the spermatids (step 1–19 in rats) contain only half of the genome; each
spermatid will finally develop into fully maturing spermatozoa. It is obvious
that the spermatids need an efficient intercellular trafficking system where
the gene products of haploid cells are shared between the neighbor cells.
Braun et al. (1989)
showed with a transgenic mouse strain that chimeric gene products expressed
only by postmeiotic cells are evenly distributed between genotypically haploid
spermatids. However, it has not been previously possible to study either the
mechanisms of this material sharing or the functions of the cytoplasmic
bridges during spermiogenesis. Which gene products are shared between neighbor
male germ cells is not known. Recent findings that there exist many genes that
are expressed only in haploid cells, such as TRA54
(Pereira et al.,
1998), make the role of cytoplasmic bridges in sharing of gene
products even more intriguing.
Another evolutionally conserved feature at haploid phase of germ cell
differentiation is a large perinuclear cytoplasmic organelle, the cheromatoid
body. In Drosophila oocytes, an analogous organelle is called sponge
body (Wilsch-Bräuninger et
al., 1997) or yolk nucleus in human fetal oocytes
(Hertig and Adams, 1967).
Recent investigations have suggested similar functions for these organelles in
both sexes. Antibodies against conserved germline-specific, RNA-binding VASA
proteins demonstrated immunostaining in both yolk nucleus
(Castrillon et al.,
2000) and the chromatoid body
(Toyooka et al.,
2000). However, the functions of these organelles are not clearly
known.
Chromatoid body has been suggested to have a role in transport and storage
of RNA in haploid cells. It has been demonstrated to contain radioactivity
derived from tritiated uridine
(Söderström and Parvinen,
1976a), actin (Walt and
Armbruster, 1984), and RNA
(Figueroa and Burzio, 1998).
It moves rapidly in the cytoplasm of living early spermatids
(Parvinen and Jokelainen,
1974) in both parallel and perpendicular manner related to the
nuclear envelope, suggesting for a transport function of haploid gene products
(Parvinen and Parvinen, 1979).
Obviously these perinuclear organelles of both sexes have an important role
during gametogenesis, because targeted disruption of VASA or its homologue
genes results in sterility and serious defects in germ cell development in
both sexes (Styhler et al.,
1998; Tanaka et al.,
2000).
Here we demonstrate a novel approach to study the transport of small granules between early round spermatids through cytoplasmic bridges ex vivo. We also describe the location of a haploid specific gene product (TRA54) inside the Golgi complex of early spermatids and its transportation in small granules through cytoplasmic bridge into neighbor spermatid. With an inhibition study we demonstrate that the expression of TRA54 is Golgi complex dependent. The subcellular localization of TRA54 in late spermatids was also studied. First we demonstrate a movement of the chromatoid body through the cytoplasmic bridge and propose an intercellular transport function for this organelle. Also new data about the close contacts between the chromatoid body and the nuclear envelope were obtained. Finally, we show that both the movement of cytoplasmic organelles and the integrity of chromatoid body are microtubule dependent.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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) was identified under stereomicroscope, and tubules at stages
I–IV of the cycle (Leblond and
Clermont, 1952) were selected for further analysis. All animal
experiments were approved by the Turku University Committee of Ethics and
Animal Experimentation (permission no. 848/98).
Light Microscopic Evaluation
Studying the Living Male Germ Cells. After identification by
transillumination technique, tubule segments of 0.5–1 mm in length from
stages I-IV were cut under a stereomicroscope and transferred with a pipette
on microscope slides in 15 µl of medium. A coverslip (20 x 20 mm) was
lowered carefully onto the tubule segment, avoiding the formation of air
bubbles. The excess fluid was removed by blotting, which allowed the cells of
the seminiferous epithelium to float out from the tubule. This was monitored
under 40x phase-contrast optics to adjust an optimal slightly flattened
monolayer. The edges of the coverslip were then sealed with paraffin oil to
immobilize the cells. The cells and the cytoplasmic bridges were examined
under a 100x oil immersion phase-contrast optics. The accurate stages of
the cycle of the seminiferous epithelium were identified as described earlier
(Toppari et al.,
1985, Kangasniemi et
al., 1990). Altogether, 16 cytoplasmic bridges were analyzed
for cytoplasmic material exchange.
Image Acquisition and Quantitative Image Analysis. A Kappa CF 8/1 FMC CCD black/white video camera (Kappa, Gleichen, Germany) was attached to a Leica DMRB phase-contrast microscope (Wetzlar, Germany) with a 15-cm extraadapter tube to allow a maximal geometric enlargement. Image sequences were directly digitized and stored into a hard disk for 300 s at a rate of 4–6 pictures per second using a FAST image grabbing system (FAST Multimedia AG, Munich, Germany). The frames from original AVI-files were first converted to bitmap (bmp) format. A custom-made image analysis program developed for Windows95 platform was used in granule and organelle movement analyses by recording the coordinates of the organelles in consecutive frames. The distances were determined in pixels and converted to metric scale (328 pixels = 10 µm). The distances, movement paths, and velocities of the organelles were plotted using Microsoft Excel spreadsheet program (version 97, Microsoft Corporation, Redmond, WA).
Immunofluorescence. After observation of living squash
preparation, it was snap-frozen in liquid nitrogen and fixed with +4°C
ethanol (97%). Then the coverslip was removed and the slides were stored in
cold PBS. Fixed cells were permeabilized with 0.5% Triton X for 10 min
followed by two washes with PBS and PBS/gelatin for 5 min. The cells were then
incubated either with mAb TRA54 1:50
(Pereira et al.
1998), polyclonal anti-Mvh antibody (mouse VASA-homologue,
Toyooka et al. 2000)
1:1000, or anti–heat schock factor (HSF)-2 antibody 1:150
(Alastalo et al. 1998)
for 1–10 h. Control slides were incubated with normal nonimmunized
appropriate animal serum. After two washes with PBS and PBS/gelatin, the
slides were incubated for 1–10 h with fluorescein-conjugated anti-mouse,
-rat, or -rabbit IgG (Jackson ImmunoResearch Laboratories, Inc., West Grove,
PA). From three to six parallel experiments were performed for each group.
Confocal laser scanning microscope Selected stage I-IV seminiferous tubule segments (ca. 2 mm in length) were fixed in + 4°C 4% paraformaldehyde for 4 h. After fixation, the seminiferous tubule segments were stored in PBS at + 4°C. Fixed seminiferous tubules were permeabilized with 0.5% Triton X for 10 min followed by two washes with PBS and PBS/gelatin for 5 min. The seminiferous tubules were then double-labeled with mAb TRA54 (1:50) and HSF-2 (1:150) as described above. Control slides were incubated with normal nonimmunized rat (TRA54) or rabbit (HSF-2) serum. Then fixed and labeled seminiferous tubules were whole-mounted on the microscope slide and observed under a Leica TCS-SP confocal laser scanning microscope equipped with argon-krypton laser (Leica Microsystems Heidelberg GmbH, Heidelberg, Germany).
Electron Microscopy
Tubule segments (ca. 2 mm in length) from stages I–IV of the cycle
were isolated by transillumination
(Parvinen and Vanha-Perttula,
1972) and fixed in 5% glutaraldehyde in s-collidine-HCl buffer
(0.16 M, pH 7.4) at 20°C, postfixed with 1% osmium tetroxide in 1.5%
potassium ferrocyanide, and embedded in Epoxy resin (Glycidether 100, Merck,
Darmstadt, Germany). They were sectioned at 70 nm with a Reichert E
ultramicrotome (Reichert Jung, Vienna, Austria). Uranyl acetate and lead
citrate were used for staining of the sections, before examination with a Jeol
100 SX (Jeol, Tokyo, Japan) electron microscope.
Immunoelectron Microscopy. Seminiferous tubules of stage
I–IV were fixed with 4% paraformaldehyde and 0.1% glutaraldehyde in 0.08
M sodium cacodylate buffer containing 0.05% calcium chloride, pH 7.3, for 4 h.
After dehydration in ascending series of ethanol, samples were embedded in
LR-White at 50°C for 72 h. Sections of 
100 nm were cut with Reichert
Ultracut E microtome, placed on nickel grids, and incubated in the presence of
monoclonal rat anti-TRA54 antibodies
(Pereira et al.,
1998) or nonimmunized normal rat serum (control slides) in 0.01 M
phosphate buffer, pH 7.4, 0.1% BSA (Sigma), for 60 min at 1:10. This was
followed by goat anti-rat IgG-conjugated 15-nm colloidal gold (Electron
Microscopy Sciences, Fort Washington, PA) labeling (1:30) and counterstaining
with uranyl acetate and lead citrate. Finally, samples were examined with a
Jeol 100 SX (Jeol) electron microscope at 60 kV.
Snap-frozen Method. After observation in living condition
the squash preparations were frozen in liquid nitrogen and processed for
electron microscopy as described earlier
(Parvinen et al.,
1997). Briefly, after dipping in liquid nitrogen, the coverslip
was removed in frozen condition and immediately dipped in 5% glutaraldehyde in
s-collidine-HCl buffer (0.16 M, pH 7.4) at 20°C. The slides were washed
with collidine buffer, dehydrated in graded ethanol series, and treated with
propylene oxide for 2x 5 min and finally infiltrated with propylene
oxide and resin (1:1). The squash preparation was covered with an 8-mm Beem
capsule containing epoxy resin (Glycidether 100; Merck) and polymerized at
60°C for 36 h. The slides with resin blocks were then heated on 70°C
plate for 5 min. The capsule was removed from the slide by dipping into liquid
nitrogen. The preparation was localized to make pyramids including appropriate
areas. Sections were cut close to parallel to the surface, and the subsequent
staining process was continued as described above.
Inhibition Studies with Nocodazole, Vincristine, Cytochalasin D, and
Brefeldin A
The drugs were dissolved in ethanol. Staged tubule segments (I–IV)
were transferred to Petri dishes containing either Dulbecco's MEM alone
supplemented with 25 µl/ml ethanol and 1.0, 10, or 100 µg/ml
concentrations of nocodazole, vincristine, cytochalasin D to disrupt
microtubules and microfilaments or 1.0 and 5.0 µg/ml brefeldin A to destroy
the Golgi complex. The tubules were incubated for 48 h at 34°C in an
atmosphere containing 5% CO2 in air.
Statistical Methods
The velocities inside the cytoplasm and cytoplasmic bridge were compared by
the paired Student's t test. Mann-Whitney U-test was adopted
to compare the distances of the movement path of the analyzed granules and
organelle. p < 0.05 was considered significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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0.5 µm. They moved along defined paths in a
nonrandom manner in varying speeds and had frequent contacts with each other
and with larger organelles, such as Golgi complex and the chromatoid body. We
focused our interest in granules that were close to the intercellular
bridges.
Altogether 20 cytoplasmic bridges were analyzed, 16 of which had exchange
of cytoplasmic material during the observation time. The diameter of the
bridges was 1.9–3.0 µm. Figure
1 shows a characteristic step 2 spermatid, where a small granule
migrates through the bridge and moves back and forth through the cytoplasmic
bridge during 62.05-s observation time.
Figure 1I shows another
granule, which moved through the cytoplasmic bridge and migrated from the
vicinity of chromatoid body of upper spermatid. The average velocity of the
granules transported through the cytoplasmic bridge was 0.44 µm/s inside
the cytoplasm, and the average transit velocity inside the cytoplasmic bridge
was 0.23 µm/s, which is 47.7% lower than that in the cytoplasm (p <
0.001).
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Spermatids Share a Haploid Cell-specific Gene Product TRA54
To study the content of transported granules, germ cell-specific markers
and antibodies against cytoskeleton and haploid cell-specific gene products
were used. Immunostaining of an antigen recognized by a haploid cell-specific
mAb TRA54 was localized in the granules. Translation or addition of a sugar
moiety of the antigenic epitope of TRA54 starts at early spermiogenesis
(Figure 2). Golgi complexes of
step 1–3 spermatids were highly TRA54 positive. Also small granules
inside the cytoplasm of early spermatids were TRA54 positive. Accumulation of
TRA54 into acrosome system is clearly seen in step 3–5 spermatids.
Medulla and trans-element of the Golgi complex of early round
spermatids were TRA54 positive in immunoelectron microscopic analysis
(Figure 2E).
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To study whether the transported granules seen ex vivo contain TRA54, the
same cell was studied after fixation and double immunostaining with TRA54 and
Mvh (Mouse VASA-homologue; Toyooka et
al., 2000). The same granule that was seen to move through
the cytoplasmic bridge between living cells was TRA54 positive after
immunostaining (Figure 3).
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The exact localization of the TRA54-positive granule inside the cytoplasmic
bridge was confirmed by immunoelectron microscopy
(Figure 3C). TRA54-positive
granules were also demonstrated inside the cytoplasmic bridge by confocal
laser scanning microscopy of whole mounted seminiferous tubule segments after
double-immunostaining with TRA54 and HSF-2 antibodies
(Figure 3D). HSF-2 was
previously shown to be present in the walls of the cytoplasmic bridges between
spermatids (Alastalo et al.,
1998).
Localizations of TRA54 in late spermatids were studied after immunostaining (Figure 4). The number of TRA54-positive granules inside the cytoplasm decreased after step 5, and at step 10 no TRA54-positive cytoplasmic granules were found. In the elongated spermatids TRA54 was localized at the edge of the acrosome system but the Golgi complexes remained unstained. Immunoelectron microscopy showed the TRA54-positive structure in the middle part of the membranes surrounding the acrosomic system (Figure 4, C and D).
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Chromatoid Body Moves through the Cytoplasmic Bridge between Neighbor
Spermatids
The chromatoid body was demonstrated to move back and forth through the
cytoplasmic bridge between neighbor spermatids with transient contacts with
nuclei of both spermatids (Figure
5). The thresholded image series
(Figure 5, A2–E2) shows
the changing lobulated morphology of the chromatoid body and the close
transient interactions between the chromatoid body and both nuclei. The speed
of the movement of chromatoid body inside the bridge was rather constant, with
a 0.20 µm/s average. Location of chromatoid body inside the cytoplasm and
inside the cytoplasmic bridge was also studied after double-immunostaining
with Mvh (Mouse VASA-homologue) and TRA54 antibodies
(Figure 6). The locations of
the chromatoid body next to the nucleus
(Figure 6A), inside the
cytoplasmic bridge (Figure 6B),
and next to the nucleus of neighbor spermatid
(Figure 6C) were demonstrated.
The transient position of the chromatoid body inside the cytoplasmic bridge
was confirmed by electron microscopy
(Figure 6D).
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The snap-frozen electron microscopy shows a very close relationship between nuclear pore complex with a large contact area between chromatoid body and the nuclear envelope (Figure 6E1). This was observed several (>20) times. Tubule-like structures were found in the space between chromatoid body and nucleus, and they are apparently associated with the outer membrane of the nuclear envelope (Figure 6E2). Figure 6E3 shows how the chromatoid body is connected to nuclear envelope at its sharp outpocketing. A membrane-bound 0.5-µm structure that resembles multive-sicular body is seen in the space between chromatoid body and the nucleus.
Expression of Antigenic Epitope Recognized by TRA54 is Golgi Complex
Dependent In Vitro
To study the role of the Golgi complex in TRA54 expression, a 48-h in vitro
incubation study was performed. Tubule segments (0.5–1 mm) from stage I
of the cycle were selected for incubation. In step 1 spermatids the acrosome
system are not yet developed (Figure
7A), but TRA54 and Mvh immunoreactions showed well-developed Golgi
complex and chromatoid body. In control culture conditions, step 1 spermatids
differentiated to step 3 during 48 h
(Figure 7B); acrosome system
and TRA54-positive granules inside the cytoplasm were seen. However, if stage
I seminiferous tubules were incubated for 48 h in the presence of 1.0 µg/ml
brefeldin A, the Golgi complex was disrupted, and the development of the
acrosome system was prevented (Figure
7C). The spermatids showed no labeling with TRA54, but the
chromatoid body remained intact and showed normal Mvh immunoreaction.
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Microtubule Inhibitors Prevented TRA54 Transportation
To study the roles of microfilaments and microtubules in cytoplasmic
organelle movements, seminiferous tubule segments of stage I of the cycle were
incubated for 48 h with microfilament inhibitor, cytochalasin D, and the
microtubule inhibitors, nocodazole and vincristine. Nocodazole and vincristine
at concentrations of 10 µg/ml turned the nonrandom cytoplasmic granules
movement into random Brownian motion, and the chromatoid body disintegrated to
form several small spheres (Figure
8A1). Electron microscopic analysis revealed that these spheres
contained ribosome-like granules covered by a thin layer of chromatoid
material (Figure 8A2). Although
concentration of 100 µg/ml cytochalasin D was toxic to the cells, the
chromatoid body remained intact (unpublished data). The distance between the
initial and the most distant point during the granule movement was measured to
distinguish the Brownian motion from nonrandom granule movement. This value
was significantly smaller (p < 0.01) after incubation of 10 µg/ml
nocodazole when compared with controls. Cytochalasin D at concentrations of 10
µg/ml had also a significant inhibitory effect
(Figure 8A3). After incubation
of stage I tubule segments for 48 h with nocodazole (10 µg/ml), two types
of spermatids developed (Figure
8B): one showed TRA54 immunostaining in Golgi complex, whereas the
other lacked TRA54 labeling. This suggests that microtubule inhibitors block
the transportation of TRA54 between the spermatids. Ca. 90% of the spermatids
were TRA54 positive and 10% negative. The Mvh immunoreaction after nocodazole
or vincristine incubation confirmed the disintegration of the chromatoid body
(Figures 8, B4 and C4), respectively.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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) about sharing of gene
products between spermatids has been recently recognized to be of central
importance. Very likely the products of recently found haploid cell-specific
genes (Penttilä et al.,
1995) are shared between haploid cells. Moreover, certain Y
chromosomal genes are highly transcribed in spermatids
(Hendriksen et al.,
1995) and are demonstrated to have a role in controlling
spermiogenesis (for review, see Delbridge
and Graves, 1999). These data support the hypothesis that also the
products of sex chromosomes are effectively transported between spermatids
ensuring the synchronous development of the germ cells
(Fawcett et al.,
1959). Therefore it is important to understand how this gene
product sharing is regulated and what is the role of certain macromolecules,
such as Golgi complex and chromatoid body, in this process. This study is the
first showing visually in mammals the sharing of cytoplasmic material between
developing spermatids. We report here the sharing of haploid cell-specific
gene product (TRA54; Pereira et
al., 1998) and the transport of the mRNA-rich organelle,
chromatoid body (Figueroa and Burzio,
1998), between the neighbor spermatids. Also the regulation of the
transport and the evaluation on electron microscopic level is demonstrated in
the present study.
Active Role of Cytoplasmic Bridges in Gene Product Sharing
Transient intercellular bridges are seen among a wide variety of cells
before the completion of cytokinesis
(Sanders and Field, 1994).
However, these are distinct from stable intercellular bridges that remain
persistent after incomplete cytokinesis (for review see
Robinson and Cooley, 1996).
Stable intercellular bridges exist both in female and male gametogenesis, in
which the incomplete cytokinesis gives a possibility for sharing of relatively
large cytoplasmic granules. The functions and components of the cytoplasmic
bridges between mammalian gametes are not well understood. Cytoplasmic bridges
show morphological differences in various phases during spermatogenesis
(Weber and Russell, 1987).
Diameter of the bridge increases during spermatogenesis, as spermatogonial
bridges between spermatogonia are 1.0–1.3 µm and 3.0 µm between
step 18 spermatids in rat. At step 1 spermatids the diameter of the bridge is
1.8 µm, which is efficient to allow the passage of the chromatoid body
as demonstrated in the present study. According to passage of cytoplasmic
material and morphological alterations during spermatogenesis in cytoplasmic
bridges, it seems that these channels are active organelles between the cells.
Similarly as in cytoplasmic bridge connecting the ovary and nurse cells in
Drosophila (so-called ring
canals-Bohrmann and Biber,
1994), only few (ca. 30%) of the granules circulating in the
immediate vicinity of the bridge actually were seen to pass through. This
suggests that selection and regulation of material takes place in the
cytoplasmic bridges. This may be due the proteins found inside the cytoplasmic
bridge. In mammals only few proteins have been demonstrated in the walls of
the cytoplasmic bridges: cytoskeletal proteins actin
(Russell et al.,
1987), sak 57 (Tres et
al., 1996), and HSF-2
(Alastalo et al.,
1998). However, molecular structure and the development of
intercellular bridges in Drosophila germ cells are better understood
(Robinson and Cooley, 1996).
Interestingly, mutations in the specific gene, cheerio
(Robinson et al.,
1997), prevent the expansion of cytoplasmic bridges during
oogenesis, resulting obvious malfunction of cytoplasmic material sharing, and
decrease of the size of oocyte and female sterility. On the basis of these
observations and according to the function of the found proteins, it can be
suggested that regulatory mechanisms of the transport and possibly packaging
(chaperone) functions for transported materials occur in the cytoplasmic
bridges. This might explain the decrease of the velocity of the granules,
which we demonstrated to occur inside the cytoplasmic bridges.
An essential question is what gene products are packed in organelles
passing through the cytoplasmic bridges. It is obvious that Golgi complex
plays a main role in vesicle and granule trafficking (for review, see
Lippincott-Schwartz et al.,
2000; Moreno et al.,
2000a). In round spermatids the role of the Golgi complex is
crucial, because of its role in formation of acrosome system, which contains
hydrolytic enzymes involved in fertilization and penetration through zona
pellucida (Hermo et al.
1980; Moreno et al.,
2000b). When the Golgi complex was disturbed by brefeldin A, the
formation of the acrosome system was inhibited
(Ventelä et al.,
2000). This was confirmed here by inhibition of TRA54
immuno-staining. Also no TRA54-positive granules were visible inside the
cytoplasm after disrupting the Golgi complex. This finding emphasizes the role
of the Golgi complex in male germ cell differentiation and in the sharing of
gene products between the spermatids. The lack of TRA54 immunostaining inside
the cytoplasm after addition of brefeldin A supports the idea that the antigen
recognized by the TRA54 antibody is a haploid cell-specific sugar moiety added
in the Golgi complex. Under the immunoelectron microscope the TRA54 labeling
is present only at the trans face of the Golgi complex. It is known that
cisternae of the Golgi complex are highly organized as a series of processing
compartments: the phosphorylation of oligosaccharides exist in
cis-element and completion of glycosylation in
trans-element. Also the sorting of proteins according to their final
destination is performed at the trans-element of the Golgi complex.
On the basis of these data it seems that TRA54 is formed in trans
face of the Golgi complex of early spermatids either after glycosylation or
alteration of configuration of recognized glycoprotein. These findings
demonstrate that the Golgi complex has a novel role during spermiogenesis by
sorting the granules needed not only for formation of one acrosome system but
possibly in several spermatids. The average velocity of the granules moving in
a nonrandom salutatory manner was 0.4 µm/s inside the cytoplasm and 0.2
µm/s inside the cytoplasmic bridge. Similar movement has been demonstrated
in peroxisomes in vivo (Rapp et
al., 1996). As in the present study and also in peroxisomes,
the movements were changed into Brownian movement after
microtubule-depolymerizing agent nocodazole. The effects of nocodazole and
vincristine also demonstrated the importance of the microtubules during
spermatogenesis, probably in part due to the fact that spermatids become
insufficient in their capacity to share cytoplasmic material.
The Function of the Chromatoid Body
It has been proposed that male germ cell-specific organelle chromatoid body
might have a role in storage and transport of haploid cell-specific gene
products (Söderström and
Parvinen, 1976a). However, the ultimate function of the chromatoid
body is still obscure. The concept about storage of haploid gene products in
chromatoid body, originally based on observation about radioactivity
incorporation after incubation with tritiated uridine in this organelle
(Söderström and Parvinen,
1976b) has obtained support from several studies. Figueroa and
Burzio (1998) have showed that
isolated chromatoid bodies contained a complex population of RNAs; mRNA, 5.8
and 5 S rRNA, but no tRNA. Previously it has also been demonstrated that
chromosomal protein histone H4 (Werner and
Werner, 1995), germ cell-specific DNA and RNA binding protein
p48/52 (Oko et al.,
1996), hnRNPs and ribosomal proteins
(Biggiogera et al.,
1990), mouse VASA-homologue
(Tanaka et al., 2000;
Toyooka et al.,
2000), and actin (Walt and
Armbruster, 1984) are localized in the chromatoid body.
Previous time-lapse cinemicrographic observations have shown two main
components of the rapid movement of the chromatoid body in early spermatids,
directed either parallel or perpendicular in relation to the nuclear envelope
(Parvinen and Parvinen, 1979;
Parvinen et al.,
1997). The significance of these movements is not clear but it is
possible that the parallel movements over the haploid nucleus are needed for
collection of gene products from various parts of the haploid nucleus from
different chromosome territories (Cremer
et al. 1993). The significance of the perpendicular
component of the movement of chromatoid body has been more difficult to
understand. However, the demonstration that chromatoid body moves through the
intercellular bridge suggests for the significance of this type of movement.
Finally, our electron microscopic study demonstrated for the first time the
location of chromatoid body inside the cytoplasmic bridge between two
spermatids. Previously, a special RNA-rich structure called sponge body has
been found from Drosophila oocytes
(Wilsch-Bräuninger et al.,
1997). It located inside the nurse cells, inside the oocytes and
inside the cytoplasmic bridge between nurse cells and oocytes, suggesting that
this subcellular structure is transported between these cells. Therefore the
authors propose that sponge body is needed in assembly and transport of mRNA
during Drosophila oogenesis. These observations suggest for a number
of similarities between chromatoid body and sponge body.
The snap-freeze-fixed preparations revealed a multitude of details in the
interrelationship between chromatoid body and early haploid nucleus that have
not observed earlier. Parvinen et al.
(1997) showed that in living
condition, chromatoid body has pinocytosis-like transient engulfments toward
nuclear pale chromatin areas. Snap-frozen preparations revealed a large
contact area between the chromatoid body and nuclear envelope, and several
intermediate organelles that obviously are sensitive for conventional fixation
processes. In the present study we found that the chromatoid body has close
contacts with several multivesicular body- or lysosome-like organelles that
may be mediators of both nucleus-chromatoid body and spermatid-spermatid
material transport. All these phenomena were observed in multiple (>20)
specimens in several different cells. This study further demonstrated that the
space between the chromatoid body and nuclear envelope is rich of vesicles and
granules. The existence of these structures was anticipated by observations on
early spermatids ex vivo.
It has been reported previously that the movement of chromatoid body is
inhibited with vincristine in a dose-dependent manner
(Mali et al., 1990).
In both light and electron microscopic studies we were able to demonstrate
that both microtubule inhibitors vincristine and nocodazole disintegrates the
chromatoid body and induces a clear separation of chromatoid material and
ribosome-like granules. Surprisingly, after 
-tubulin
immunocytochemistry, no microtubules were found inside the chromatoid body
(unpublished data). However, after FITC-phalloidin staining chromatoid bodies
were positive (unpublished data), supporting the previous data that actin is a
component of chromatoid body (Walt and
Armbruster, 1984). It might be that actin inside the chromatoid
body is involved in mRNA binding to the cytoskeletal framework, which has been
demonstrated to exist in many type of cells (for review, see
Jansen, 1999;
Ornelles et al.,
1986). Actin is not involved in the normal movement of chromatoid
body, because in our experiments cytochalasin D was ineffective. These results
suggest that microtubules are involved in the normal integrity of the
chromatoid body, in its movements, and in the normal Golgi
complex–derived granule traffic in the cytoplasm of spermatids and
between neighbor cells. Recently Morales et al.
(1998) presented
immunocytochemical evidence that testis-brain RNA-binding protein (TB-RBP)
moves from the nucleus to the cytoplasm and through intercellular bridges in
rat spermatids, supporting the hypothesis that small granules may be involved
in transport of gene products at mRNA level. TB-RBP has also been demonstrated
to suppress in vitro the translation of mRNA and to bind specific mRNAs to
microtubules (Han et al.,
1995), which further supports the importance of microtubules in
inter- and intracellular material transport.
The present study demonstrates that novel digital techniques for quantification of organelle movements in living spermatogenic cells are useful for clarification of the possible function of certain organelles, such as the chromatoid body and Golgi complex in living cells. Particularly the demonstration that whole chromatoid body is able to pass the cytoplasmic bridge opens new insights about the function of this organelle.
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ACKNOWLEDGMENTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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Footnotes |
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Online version of this article contains video material. Online version is
available at
www.molbiolcell.org. 
Corresponding author. E-mail address:
satuve{at}utu.fi.
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REFERENCES |
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