Open Journal of Cell Biology, 2012, 2, 11-20
doi:10.4236/ojcb.2012.22002 Published Online June 2012 (http://www.SciRP.org/journal/ojcb)
Localization of BRUNOL2 in Rat Spermatogenic Cells as
Revealed by Immunofluorescence and Immunoelectron
Micr oscopic Techniques
Hiroka Yonetamari, Yuko Onohara, Sadaki Yokota
Section of Functional Morphology, Faculty of Pharmaceutical Sciences, Nagasaki International University,
Sasebo, Nagasaki, Japan
Received March 8, 2012; revised April 12, 2012; accepted April 28, 2012
Distribution and localization of a RNA-binding protein, BRUNOL2 in rat spermatogenic cells were studied by dot
blotting of cell fractions, immunofluorescence (IF), and immunoelectron microscopy (IEM). BRUNOL2 distributed in
nuclear (23%), mitochondrial (19%), microsomal (15%), and cytosol fractions (43%). BRUNOL2 was detected in all
spermatogenic cells. In the cytoplasm and nucleoplasm of the spermatogonia, spermatocytes and spermatids, both
diffuse and granular staining patterns were observed. Many cytoplasmic granules were stained also for DDX4 and
DDX25. Large granules in the cytoplasm of elongated spermatids were stained for BRUNOL2 but not for the nuage
proteins. IEM showed that gold signals for BRUNOL2 were concentrated in nuage components including loose
aggregates of small particles, chromatoid body (CB), intermitochondrial cement (IMC), and satellite body (SB). In
addition, many non-nuage structures such as ER-attached small granules, less dense material surrounding connecting
piece of flagellum, reticulated body, mitochondria-associated granules (MAG), granulated body, ribosome aggregate,
and manchette, were stained for BRUNOL2 with different staining intensities. In the nucleus, gold signals were
concentrated in heterochromatin area and nucleolus. The results suggest that BRUNOL2 is one of the nuage proteins
and also associated with the other non-nuage structures, suggesting multiple functions of this protein.
Keywords: BRUNOL2; Immunocytochemistry; Spermatogenesis; Nuage; Nucleus
BRUNOL2 is a RNA-binding protein and belongs to
CELF/BRUNO-like family. Its major function in the
nucleus is a regulatory role in the alternative splicing of
target pre-mRNAs and in the control of translation and
mRNA stability in the cytoplasm [1-3]. CELF/BRUNO-
like protein, CUGBP1, and the related protein Etr-3
regulate the muscle-specific splicing of cardiac troponin
T pre-mRNA [4,5]. The binding of these proteins to
CUG repeats suggests that they play roles in myotonic
dystrophy [4,6]. CELF1/BRUNO-like protein is localized
both in the cytoplasm and the nucleus [6-8]. This dual
localization suggests multiple functions of the protein. In
aged mouse liver, age-specific CUGP1-elF2 complex in-
creases translation of CCAAT/enhancer-binding protein
β . It has been shown that male Cugbp1–/– mice are
infertile and the spermatogenesis is arrested at step 7 .
Thus, it has been suggested that BRUNOL2 plays crucial
role in spermatogenesis but the function of BRUNOL2 is
still unclear. Moreover detailed localization of BRUNOL2
in the spermatogenic cells has not been reported so far. In
this paper, we study the co-localization of BRUNOL2
with known nuage proteins, DDX4 and DDX25, in rat
spermatogenic cells by IF and IEM techniques.
We found that BRUNOL2 was localized to the cytop-
lasm and the nuclei of all spermatogenic cells. In the
cytoplasm, it concentrated in the nuage components, and
in the nuclei, it is associated with heterochromatin and
granulated component of nucleolus attached to XY body.
Such broad localization suggests the multiple functions
of BRUNOL2 in the spermatogenic cells.
2. Materials and Methods
Japanese white rabbits (3 - 4 kg) and male Wistar rats
(180 - 220 g) were obtained from Kyudo Co. Ltd. (Tosu,
Japan) and fed appropriate standard diets and water ad
libitum until use. The animal experiments were performed
in accordance with the guidance for Animal Experiments,
Nagasaki International University Maintaining the Inte-
grity of the Specifications.
Copyright © 2012 SciRes. OJCB
H. YONETAMARI ET AL.
Polypeptide (GMKRLKVQLKRSKNDSKPY) consisting
of 19 amino acids at the C-terminus of mouse CELF-1
(BRUNOL2) was synthetized and to the N-terminus cys-
teine was added to make chemical binding to carrier pro-
tein. The polypeptide (1 mg) was conjugated with egg
albumin (50 mg) by using m-maleimidobenzoyl-N-hy-
drosuccinimide ester. The conjugate was emulsified with
Freund’s complete adjuvant. Rabbits and rats were in-
tracutaneously injected with 200 µg and 30 µg of peptide,
respectively. The injections were carried out 4 times with
2-week interval, and 2 weeks after last injection blood
was collected. The specific antibody was affinity-purified
by peptide-coupled column. Rabbit antibody against mouse
DDX4 and rat antibody against mouse DDX25 were pre-
pared as previously . Rabbit anti-rat IgG was pre-
pared by immunization of rabbits with purified rat IgG
and the specific antibody was purified by affinity column
coupled with rat IgG. Alexa Fluor ®568 or Alexa Fluor
®488 conjugated goat anti-rabbit IgG or goat anti-rat IgG
were obtained from Molecular Probes (Eugene, OR).
HRP-labeled goat antibodies to rabbit IgG and rat IgG
were purchased from DAKO Japan (Tokyo). Protein
A-gold probe (15 nm gold) was prepared by the method
of de Roe et al. .
2.3. Western Blotting
Rat testes were homogenized in 5 mM MOPS-KOH buffer
(pH 7.4) containing 0.25 M sucrose, 1 mM ethylenedia-
minetetraacetic acid, 1 mM phenylmethylsulfonyl fluo-
ride and a cocktail of protease inhibitors (1 µg/ml) in-
cluding leupeptin, pepstatin, aprotinin, and antipain (me-
dium A) using a Potter-Elvehjem homogenizer. Ten per-
cent homogenate (w/v) was centrifuged at 800 × g for 10
min. The resulting supernatant was centrifuged at 10,000
× g for 20 min and the pellet (mitochondrial fraction)
was suspended in a small volume of medium A. The su-
pernatant was centrifuged at 100,000 × g for 60 min in a
Beckman ultracentrifuge using a SW 41 swing rotor. The
resulting pellets were suspended in medium A and used
as the microsomal fraction, while the supernatant was
used as the cytosol fraction. The cell fractions were
stored at –70˚C. Protein concentrations were determined
by the bicinchoninic acid method (Pierce Chemical,
Rockford, IL) using bovine serum albumin (BSA) as a
standard. The protein concentrations of the fractions were
adjusted to 1 mg/ml, mixed with one volume of sample
buffer for SDS-PAGE, and heated in boiling water for 5
min. Testes of mice and guinea pigs (200 mg of wet
weight each) were homogenized with 1 ml of SDS-PAGE
sample buffer containing 0.1 volume of 0.3 M iodoaceta-
mide and heated in boiling water for 5 min. Ten micro-
grams of each sample were analyzed by western blotting.
Molecular mass of BRUNOL2 was estimated by pre-
stained protein maker (Nippon Genetics Europe GmbH,
2.4. Dot Blot Analysis of BRUNOL2 Content in
Cell Fractions of Rat Testis
The cell fractions including mitochondria, microsomes,
and cytosol were isolated from rat testis homogenate by
differential centrifugation as described above. Nuclei were
isolated by the method of Rickwood and Ford (1983).
Briefly, the crude nuclear fraction (800 × g pellet) was
suspended in medium A containing 1% Triton X-100 and
centrifuged at 800 × g for 10 min. The resulting pellet
was suspended in 2 ml of medium A, mixed with 72.5 %
(w/v) metrizamide (Sigma-Aldrich Japan, Tokyo) in me-
dium A without 0.25 M sucrose and centrifuged at 10,000
× g for 20 min at 5˚C. The nuclei-rich pellicles at the
surface of the metrizamide solution were collected, sus-
pended in a 10-times volume of medium A, and then
centrifuged at 6000 × g for 10 min. The pellet was sus-
pended in 1.6 ml of medium A. Each fraction was diluted
by medium A. The diluted fractions (90 μl) were mixed
with 90 µl of SDS-PAGE sample buffer and 20 μl of 0.3
M iodoacetamide, treated in boiling water for 5 min, and
centrifuged at 10,000 × g for 10 min. The supernatants
were diluted 200-fold and 1 - 5 µl of them was loaded
onto PDVF membranes for immunoblotting. BRUNOL2
protein was visualized using combination of rabbit anti-
BRUNOL, HRP-labeled goat anti-rabbit IgG, and DAB
reaction. Internal standard proteins, histone H2A (nuclei),
ICD1 (mitochondria), PDI (microsomes), and α-tubulin
(cytosol) were visualized using combination of rabbit
antibodies against each protein, HRP-labeled goat anti
rabbit IgG, and DAB reaction. The staining intensity was
measured using a densitometer. The total amount of
BRUNOL2 in each cell fraction was calculated as fol-
lows; the obtained densitometric values were multiplied
by the final volume of each fraction. The data were ob-
tained from three measurements and the average values
and standard deviation were plotted.
2.5. IF Staining of Rat Testis
Rat testes were extracted, embedded in Tissue Tek (Sa-
kura, Japan), and frozen in isopentane cooled by liquid
nitrogen. Frozen sections were cut into 8 µm thick with a
Leitz cryotome and fixed in 4% paraformaldehyde +
0.01% CaCl2 + 0.05M Hepes-KOH (pH 7.4) for 15 min
at RT. After wash in PBS, sections were treated with 0.1
Triton X-100-PBS for 15 min, followed by blocking with
2% fish gelatin-PBS. Sections were then incubated with
anti-BRUNOL2 antibodies for 2 h, followed by 1 h-in-
cubation with Alexa Fluor ®568 conjugated goat
anti-rabbit or rat IgG. For IF control, preimmune serum
Copyright © 2012 SciRes. OJCB
H. YONETAMARI ET AL. 13
was used instead specific antibodies, followed by Alexa
Fluor ®568 conjugated secondary antibodies. Some sec-
tions were stained for DDX4 or DDX25 with combination
of the primary antibodies and Alexa Fluor ®488 conju-
gated secondary antibodies. Nuclei were stained by DAPI.
Sections were mounted by Mowiol-DABCO mixture and
examined with a Nikon Eclipse E600 fluorescence micro-
scope (Nikon, Tokyo, Japan). The images were merged
using Adobe Photoshop®7.0 to visualize cell contours.
Each stage of seminiferous tubules was judged by size and
shape of nuclei stained by DAPI according to stages of the
cycle illustrated by Russell and coworkers .
2.6. IEM Staining of Rat Testis
Rat testes were dissected, cut into small tissue block, and
fixed in 4% paraformaldehyde + 0.2% glutaraldehyde in
Hepes-KOH buffer (pH 7.4) for 1 h at 4˚C. The tissue
blocks were dehydrated in graded ethanol series at –20˚C
and embedded in LRWhite, which was polymerized un-
der UV light at –20˚C. Thin sections of LR White-em-
bedded testis tissues were cut with a diamond knife
equipped with a Reichert Ultracut R, mounted on nickel
grids, and incubated with affinity purified antibodies to
BRUNOL2 (1 µg/ml) overnight at 4˚C. Preimmune sera
were used instead of the primary specific antibody for the
control experiments. Sections were contrasted and then
examined with a Hitachi H7650 electron microscope
(Hitachi, Tokyo, Japan) at acceleration voltage of 80 kV.
Stage of seminiferous tubules and step of spermatids
were judged according to stages of the cycle illustrated
by Russell et al. .
3.1. Western Blotting
Our antibodies developed three bands in Western blotting;
molecular mass of a major band was 52 kDa, which was
observed in all testicular cell fractions examined. Minor
two bands had molecular masses of 55 kDa and 73 kDa
(Figure 1(a)). The 55 kDa band was observed in the cy-
tosol fraction, which seemed to be one of BRUNOL2
isoforms, whereas 73 kDa band was detected in the mito-
chondrial fraction. Analysis of cell fractions by dot blot-
ting showed that the cytosolic fraction contained approxi-
mately 43% of total amount of BRUNOL2, followed by
nuclear fraction (23%), mitochondria (19%) and micro-
somes (15%) in sequence (Figure 1(b)). Marker proteins,
histone 2A (H2A), isocitrate dehydrogenase (ICD1),
protein disulfide isomerase (PDI), and α-tubulin (α-Tub)
were detected prominently in nuclear fraction, mitochon-
dria, microsomes, and cytosol, respectively (Figure 1(b),
Figure 1. (a) Western blotti ng analysis of the cytosol fraction
of rat testis homogenate. Lane 1: homogenate; lane 2: nu-
clear fraction; lane 3: mitochondrial fraction; lane 4: micro-
somal fraction, and lane 5: cytosol fraction. Rat anti-RRU-
NOL2 antibody developed three bands. Weakly stained 73
kDa band is detected in homogenate and mitochondrial
fraction; 55 kDa band is seen in homogenate and cytosol
fraction; 52 kDa band showing BRUNOL2 protein is deve-
loped in all cell fractions. (b) Dot blot analysis of cell frac-
tions isolated from rat homogenate. N: nuclear fraction;
Mt: mitochondrial fraction; Mc: microsomal fraction; Cy:
cytosol fraction. Bars in each column are standard deviation.
Dot blot shows marker protein of each cell fraction. H2A:
histone H2A; ICD1: mitochondrial isocitra te dehydrogena se;
PDI: protein disulfide isomerase; α-Tub:α-tubulin.
3.2. IF Staining of Rat Testis for BRUNOL2
Staining for BRUNOL2 was observed in the cytoplasm
and nuclei of all spermatogenic cells with different stain-
ing intensities. Discrete granular staining varied greatly
depending on developing stages. In stage I, small granular
staining was noted in the cytoplasm of step 1 spermatids
and pachytene spermatocytes. A single punctate staining
was seen in the neck region of step 15 spermatids (Figure
2(a)). The nuclei of spermatids at steps 2-5 and pachy-
tene spermatocytes at stages II-V were weakly stained. In
the cytoplasm of spermatids at the same steps, irregu-
larly-shaped large granules were stained, whereas the
cytoplasm of pachytene spermatocytes at these stages
was diffusely stained and contained a few granules (Fig-
ure 2(b)). In pachytene spermatocytes at stages V-IX,
diffuse weak staining was observed in both nucleus and
cytoplasm, but in elongated spermatids at step 9 nume-
rous small granules appeared around nucleus (Figure
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H. YONETAMARI ET AL.
Copyright © 2012 SciRes. OJCB
strong cytoplasmic staining for BRUNOL2 (Figure 4).
When the sections were stained doubly for DDX25 and
BRUNOL2, large cytoplasmic granules with irregular
shapes were stained for both proteins (Figure 5, arrows).
The BRUNOL2-positive nuclear spots were not stained
for DDX25 (Figure 5, circles). These staining reactions
were not observed in the IF control sections incubated
with pre-immune sera (Figures 6(a)-(c)).
2(e)). They disappeared soon; instead, slightly larger gra-
nules appeared in the cytoplasm of steps 10-11 sperma-
tids (Figure 2(f)). In the residual body or cytoplasmic
lobes of step 19 spermatids, round granules were stained
Next, we studied double staining of BRUNOL2 and
DDX4. The results showed that there were three types of
granules; large granules positive for both proteins (Figure
3), small granules positive for only BRUNOL2 located in
the nuclei (Figure 3, arrows), and small granules stained
for only DDX4 (Figure 3, circles). Moreover, fine granu-
lar staining identified as the intermitochondrial cement
(IMC) in previous study  seemed to be buried in the
Figure 3. IF double staining of BRUNOL2 (red) and DDX4
(green) in seminiferous tubule at stage VI. (a) DDX4 stain-
ing; (b) BRUNOL2; (c) Merged (a) and (b). Stained spots in
the nuclei of pachytene spermatocytes are negative for DDX4
(arrows) while small cytoplasmic granules are positive for
only DDX4 (circles). The other large irregularly shaped
granules are stained for both BRUNOL2 and DDX4. Nuclei
are stained with DAPI. Bar = 25 µm.
Figure 2. IF staining of BRUNOL2 protein in rat seminif-
erous tubule. Nuclei are stained with DAPI. (a) Step 15
spermatids. Note that a single punctate staining is present
in the neck region of each spermatid; (b) Stage V tubule.
Large granules in spermatids are stained. The cytoplasm of
spermatogonia, spermatocytes and spermatids is diffusely
stained; (c) Stage VI tubule. Discrete granular staining is
seen in the nuclei (circles) and cytoplasm of step 6 spermat-
ids and diffuse staining is also in the cytoplasm of in sper-
matocytes and spermatids; (d) Stage VII tu bule. Round gra-
nules in the cytoplasm of step 19 spermatids are stained; (e)
Stage IX tubule. Fine granules along periphery of head of
step 9 spermatids are stained; (f) Stage XI tubule. Small
granules in the cytoplasm of step 11 spermatids are stained.
Bar = 25 µm.
Figure 4. Localization of BRUNOL2 (red) and DDX4 (green)
in seminiferous tubule at stage X. (a) Typical IMC contour
is stained for DDX4; (b) BRUNOL2 is not stained in the
IMC contour; (c) Merged (a) and (b). Nuclei are stained
with DAPI. Bar = 25 µm.
Figure 5. Localization of DDX25 (green) and BRUNOL2 (red) in rat seminiferous tubule at stage IV. (a) Granules with ir-
regular shapes are stained for DDX25; (b) Staining for BRUNOL2; (c) Merged (a) and (b). Nuclei are stained with DAPI.
Small spots are stained for BRUNOL2 (circles). A part of DDX25-positive large granules contain an area positive for
RUNOL2 (arrows). Nuclei are st ained with DAPI. Bar = 25 µm. B
H. YONETAMARI ET AL. 15
3.3. IEM Localization of BRUNOL2 in Nuage
and Other Compartments of Spermatogenic
Previously, we have studied the localization of DDX4 in
rat spermatogenic cells and shown the protein is detected
in the four types of nuage components ; 1) small par-
ticles appearing in meiotic cells, 2) intermitochondrial
cement (IMC), 3) loose aggregates of small dense gran-
ules at the perinuclear area, and typical chromatoid bod-
ies (CBs). Nuages of 1)-3) appear in spermatocytes and
CBs occur in spermatids at steps 1 to 11. In the present
study, we focused on the localization of BRUNOL2 in
these structures. Gold particles showing BRUNOL2 sites
were localized to all the nuage components cited above
but labeling intensity was essentially lower than that for
DDX4 . Some of the small particles in meiotic cells
were weakly stained (Figure 7(a)). Loose aggregates were
stained intermediately, compared with the other nuage
components (Figure 7(b)). IMCs were weakly or not
stained for BRUNOL2 (Figure 7(c)). CBs were most
strongly stained among the nuage structures (Figure 8(a)).
They were sometimes connected with unstained materi-
als (Figure 8(b)) and with satellite body (SB) which is
slightly labeled (Figure 8(c)). The CBs migrate to the
cytoplasm at the caudal side of the nucleus and decrease
gradually in their volume. Such CBs were still stained for
BRUNOL2 but the labeling intensity became lower than
that of the CB (Figure 9(a)).
We have found the other BRUNOL2-positive struc-
tures, which were not classified into nuage, in the cyto-
Figure 6. IF control. (a) Section was incubate d with DDX4 preimmune serum, followed by Alexa Fluor 488® conjugated goat
anti- rabbit IgG. No specific staining is observed; (b) Section was incubated with BRUNOL2 preimmune serum, followed by
Alexa Fluor 568® conjugated goat anti-rat IgG. No specific staining is noted; (c) Section was incubated with DDX25 preim-
mune serum, followed by Alexa Fluor 488® conjugated goat anti-rat IgG. No specific staining is seen. Nuclei are stained with
DAPI. Bar = 25 µm.
Figure 7. Localization of BRUNOL2 in various nuage components. (a) Small particles in meiotic cell are weakly labeled with
gold particles (arrows). Some of them attach to mitochondria; (b) Loose aggregates of dense particles are stained; (c) Weak
gold labeling is seen on IMC (arrows). Bar = 0.5 µm for all.
Figure 8. IEM localization of BRUNOL2 in chromatoid bodies (CBs) of spermatids. (a) Typical CB. Gold particles are seen
on the dense material of CB; (b) CB connected with BRUNOL2-negative CB (arrows); (c) CB connected w ith weakly stained
atellite body (circle). Bar = 0.5 µm for all. s
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H. YONETAMARI ET AL.
plasm of spermatids at steps 6-7. They were small granules
without the limiting membrane and closely associated
with ER membrane (Figure 9(b)), and the material sur-
rounding connecting piece between nucleus and flagel-
lum (Figure 9(c)). BRUNOL2-positive structures were
also observed in the cytoplasmic lobe of elongated sper-
matids at step 11-19. They were reticulated body in the
cytoplasm of steps 14-16 spermatids (Figure 9(d)), mi-
tochondria associated granules (MAG, Figure 9(e)), gra-
nulated body (Figure 9(f)), and large aggregates of free
ribosomes (Figure 9(g)). The MAGs were appeared in
the cytoplasmic lobe of the spermatids at steps 11-15 and
relatively heavily labeled (Figure 9(e)). The granulated
bodies were seen in the cytoplasm of step 14-15 sper-
matids and characterized by fine granular matrix and
surrounding less dense small granules. Gold labeling was
noted in the granular matrix (Figure 9(f)). The large ag-
gregates of free ribosomes were one of the constituents
of residual body of the spermatids at step 19. They were
most strongly stained among the non-nuage components
(Figure 9(g)). Furthermore, BRUNOL2 was weakly la-
beled in manchette of step 9 spermatids (Figure 9(h)).
This labeling disappeared soon but no longer observed in
the manchette of step 10 spermatids. No staining was
noted in the nuage and the other structures cited above
when the sections were incubated with preimmune serum
instead with the primary antibody against BRUNOL2
3.4. IEM Localization of BRUNOL2 in the
Nuclei of Spermatogenic Cells
As shown by IF staining, BRUNOL2 was localized to the
nuclei of spermatocytes and spermatids so that we ad-
dressed the localization of BRUNOL2 in the nuclei of
differentiating spermatocytes and spermatids. The strong-
Figure 9. IEM localization of BRUNOL2 in various structures. (a) Late CB; (b) ER-attached small granule appearing in
spermatids at steps 6-7; (c) Less dense material surrounding the connecting piece (arrows); (d) Reticulated body of step 15
spermatid; (e) Mitochondria associated granules (MAG); (f) Granulated body; (g) Aggregate of ribosomes; (h) Manchette of
step 9 spermatid (arrows). Bar = 0.25 µm for (b), 0.5 µm for (a), (c), (d), (f), and (h), 1 µm for (e) and (g).
Figure 10. IEM control. Sections were incubated with BRUNOL2 preimmune serum, followe d by incubation w ith rabbit anti-
rat IgG and Protein A-gold probe (15 nm). (a) Loose aggregate; (b) IMC; (c) CB; (d) Late CB; (e) Satellite body; (f) ER at-
tached small granules (arrows); (g) Material surrounding the connecting piece (arrows); (h) Reticulated body; (i) MAG; (j)
ranulated body; (k) Ribosome aggregate ; (l) Manchette (arrows). Bar = 1 µm for all. G
Copyright © 2012 SciRes. OJCB
H. YONETAMARI ET AL. 17
est labeling was noted in the spermatocytes at stages IV -
VI. Heavy gold labeling was observed on the hetero-
chromatin and the granular component of the nucleolus
closely associated with the XY body (Figure 11). The
euchromatin area was weakly or not labeled. The XY
body was intermediately labeled (Figure 11). Gold la-
beling was observed in the nuclei of all spermatogenic
cells including spermatogonia, spermatocytes and sper-
matids with different intensities (Figures 12(a) and (b)).
The BRUNOL2 signals were associated with heterochro-
matin of these cells but not with euchromatin. BRUNOL2
was localized also to the nucleus of Sertoli cells in which
the large nucleolus was strongly stained but the other
area was weakly (Figures 12(c)). The nuclei of Leydig
cells and other cells including fibroblasts, smooth muscle
cells, endothelial cells of blood capillary, myoid cells
were also labeled (data not shown). All these positive
signals were not observed in the IEM control sections
incubated with preimmune sera (data not shown). Sum-
mary of IEM results is shown in Table 1.
4.1. Distribution of BRUNOL2 Molecular
Species in Rat Testicular Cell Fractions
Three molecular species were shown by Western blotting.
The 52 kDa protein is BRUNOL2 and 55 kDa and 73
kDa seem to be isoforms. The 73 kDa molecule seems to
be similar to isoform observed in heart, liver and brain 
and is contained in the mitochondrial fraction by differ-
ential centrifugation. The 55 kDa distributes solely in the
cytosol. Present study showed that in rat testis 43% and
23% of total BRUNOL2 distributed in the cytosol and
nuclear fractions, respectively. These results are largely
consistent with those of IF and IEM staining. The nuclear
fraction examined is confirmed to contain no CBs, many
of which are precipitated into the nuclear fraction by
usual differential centrifugal isolation, so that BRUNOL2
signals in the nuclear fraction are not due to contamina-
tion of CBs. Thus, the major sites of BRUNOL2 in sper-
matogenic cells of rat testis are cytosol and nuclei. BRU-
NOL2 in the mitochondria seems to be derived from co-
precipitated CBs. BRUNOL2 detected in the microsomes
might be due to the ER-attached small granules or mem-
Figure 11. Localization of BRUNOL2 in the nucleus of a pa-
chytene spermatocyte at stage VI. Heavy gold labeling is
observed on dense heterochromatin (arrows) and the gra-
nular component of nucleolus (Large arrows). XY body sur-
rounded by dashed line is intermediately stained. Euchro-
matin is weakly labeled. Inset. Control section incubated
with BRUNOL2 preimmune serum. No gold signal is seen
in XY body (XY), granular component of nucleolus (*), and
euchromatin (EU). Bar = 2 µm and 0.5 µm for inset.
Figure 12. BRUNOL2 localization in the nucleus. (a) Spermatogonia. Heavy gold labeling is seen in heterochromatin (arrows);
(b) Step 6 spermatid. Gold particles are concentrated on heterochromatin (arrows); (c) Sertoli cell. Gold labeling is seen on
nucleolus (arrow s). Bar = 1 µm.
Copyright © 2012 SciRes. OJCB
H. YONETAMARI ET AL.
Table 1. BRUNOL2-positive structures in rat spermatogenic cells and their labeling intensity.
Compartments Structures Labeling intensityAppearance
IMC +/– SPC
Loose aggregate ++ SPC
CB +++ SPT
late CB + SPT
SB + SPC and early SPT
Cytoplasm ER-attached small granule++ Steps 6-7 SPT
Neck material ++ Steps 14-15 SPT
Reticulated body + Steps 14-16 SPT
MAG ++ Step 17 SPT
Granulated body ++ Steps 14-16 SPT
Ribosome aggregate +++ Step 19 SPT
Manchette + Step 9 SPT
Euchromatin +/– All SPC and round SPT
Nucleus Heterochromatin +++ All SPC and SPT
Nucleolus +++ All SPC and SPT
XY body + Stage VII-IX SPC
SPC: spermatids, SPC: spermatocytes, +/–: weakly stained or not, +: weakly stained, ++: intermedi-
ately stained, +++: strongly stained.
4.2. Localization of BRUNOL2 in the
If staining showed that BRUNOL2 distributed in both the
cytoplasm and the nuclei of spermatogenic cells, includ-
ing spermatogonia, all spermatocytes and spermatids.
The nucleus of Leydig cells was also stained. The double
staining revealed that BRUNOL2-positive irregularly
shaped granules were completely overlapped with DDX4
and DDX25 staining. The results demonstrate that BRU-
NOL2 is localized in the CBs containing DDX4 and
DDX25. In the cytoplasm, small numbers of granules
were solely stained for BRUNOL2. Among these gran-
ules, those appearing in steps 6-7 spermatids seem to
correspond to the ER-attached small granules observed
by IEM. In addition, relatively large granules located in
the cytoplasmic lobe of step 19 spermatids might be con-
sistent with the aggregates of numerous free ribosomes
shown by IEM. Spot staining in the neck region of the
spermatids at steps 13-15 coincides with staining in the
material surrounding connecting piece between nucleus
and flagellum observed by IEM. Many punctate spots in
the nuclei correspond to clustered heterochromatin, nu-
cleoli, and XY body shown by IEM.
4.3. Subcellular Sites for BRUNOL2 Revealed by
The present study showed clearly that BRUNOL2 was
localized to nuage components classified previously by
DDX4 distribution , such as CBs, late CBs, loose
aggregates, IMC, and satellite body (SB). The results
suggest that BRUNOL2 is functioning in these structures.
However, the labeling intensity was consistently lower
than that of DDX4 or DDX25. Especially, the labeling in
the IMC was very weak or null, so that the major func-
tion sites of BRUNOL2 might be the CBs and the loose
aggregates. Several proteins, such as DDX4 [11,14,15],
DDX25 , TDRD1/MTR-1, TDRD6 and TDR7/TRAP
[17,18], RNF17 , MIWI , kinesin KIF17b ,
Maelstrom  have been reported as nuage-constituent
proteins so far. Among these proteins, RNF17 and Mael-
strom are strongly stained in the SB [19,21]. DDX4 
and DDX25  are also stained in this structure but
very weakly. The present study showed that the SB was
weakly labeled for BRUNOL2. The functions of BRUN-
OL2 in the cytoplasm are suggested to be the regulation
of translation and mRNA silencing . The present study
suggests that nuage structures cited above might be the
sites for these functions and BRUNOL2 belongs to one
of nuage proteins.
The present study demonstrated that BRUNOL2 was
localized in several subcellular structures such as ER-
attached small granules, neck material, reticulated body,
MAG, granulated body, and manchette. The most of
these structures have shown to be positively stained for
DDX25 . The ER-attached small granules seem to be
identical to “puffs” described in the cytoplasm of steps 8
-9 spermatids, which are suggested to be assembled to
granulated body . The granulated body is composed
of polypeptides destined for the formation of the outer
Copyright © 2012 SciRes. OJCB
H. YONETAMARI ET AL. 19
dense fiber of flagellum . The reticulated body con-
sists of anastomosed cords and is observed in the cyto-
plasm of steps 14-16 spermatids. The nature and func-
tion of are unclear. The MAG appears in the cytoplasm
of step 17 spermatids and disappears in step 18 spermat-
ids . The function of this structure is unknown. The
ribosome aggregate is composed of numerous free ri-
bosomes and observed in the cytoplasmic lobe of step 19
spermatids and in residual body. The strongest signal for
BRUNOL2 is detected in this structure. It seems that
some population of BRUNOL2 might be tightly bound to
individual free ribosome during spermatogenesis, which
is eventually assembled closely into large aggregates, so
that the labeling intensity is enhanced as observed. The
association of BRUNOL2 with free and membrane-bound
ribosomes is observed in somatic cells such as fibroblasts
and smooth muscle cells (unpublished data). Manchette
is a cytoskeletal complex composed of a sleeve of micro-
tubules and may function in part in alteration of nuclear
shape and transport of materials to spermatid head, cen-
trosome and tail . Several proteins interacting with
manchette microtubules, including Sak57/K5 , Po-
laris  TBP-1 , and motor proteins [29,30] have
been found in spermatids. The present study suggests that
BRUNOL2 may interact directly or indirectly with mi-
The present study showed that the major distribution
sites of BRUNOL2 were the cytoplasm of early to late
spermatocytes and round spermatids and the nuclei of
spermatogonia to round spermatids. Such localization
seems to match to the functions reported, which are the
regulation of transcription . In the nucleus, BRUNOL2
was shown to be closely associated with heterochromatin
but not with euchromatin. Transcription is carried out on
perichromatin fibrils which extend from heterochromatin
[31,32]. BRUNOL2 might be concerned with the activa-
tion of silenced genes at heterochromatin. In addition, the
existence of BRUNOL2 in the XY body and the adjacent
nucleolus suggests some function of BRUNOL2 con-
cerned with the XY body.
The work was supported by the University research fund,
in part by a grant-in-aid (17570158) from the Ministry of
Education, Science, Culture and Sport, and by the Sci-
ence Research Promotion Fund from the Promotion and
Mutual Aid Corporation for Private Schools of Japan.
 A. N. Ladd, N. Charlet-B. and T. A. Cooper, “The CELF
Family of RNA Binding Proteins Is Implicated in
Cell-Specific and Developmentally Regulated Alternative
Splicing,” Molecular and Cellular Biology, Vol. 21, No.
4, 2001, pp. 1285-1296.
 H. Suzuki, Y. Jin, H. Otani, K. Yasuda and K. Inoue,
“Regulation of Alternative Splicing of α-Actinin Tran-
script by Bruno-Like Proteins,” Genes to Cells, Vol. 7,
No. 2, 2002, pp. 133-141.
 C. Barreau, L. Paillard, A. Méreau and H. B. Osborne,
“Mammalian CELF/Bruno-Like RNA-Binding Proteins:
Molecular Characteristics and Biological Functions,” Bio-
chimie, Vol. 88, No. 5, 2006, pp. 515-525.
 L. T. Timchenko, J. W. Miller, N. A. Timchenko, D. R.
DeVore, K. V. Datar, L. Lin, R. Roberts, C. T. Caskey
and M. S. Swanson, “Identification of a (CUG)n Triplet
Repeat RNA-Binding Protein and Its Expression in
Myotonic Dystrophy,” Nucleic Acids Research, Vol. 24,
No. 22, 1996, pp. 4407-4414. doi:10.1093/nar/24.22.4407
 X. H. Lu, N. A. Timchenko and L. T. Timchenko, “Car-
diac Elav-Type RNA-Binding Protein (ETR-3) Binds to
RNA CUG Repeats Expanded in Myotonic Dystrophy,”
Human Molecular Genetics, Vol. 8, No. 1, 1999, pp.
 R. Roberts, N. A. Timchenko, J. W. Miller, S. Reddy, C.
T. Caskey, M. S. Swanson and L. T. Timchenko, “Altered
Phosphorylation and Intracellular Distribution of a
(CUG)n Triplet Repeat RNA-Binding Protein in Patients
with Myotonic Dystrophy and in Myotonin Kinase
Knockout Mice,” Proceedings of the National Academy
of Sciences of the United States of America, Vol. 94, No.
24, 1997, pp. 13221-13226.
 A. N. Ladd and T. A. Cooper, “Multiple Domains Control
the Subcellular Localization and Activity of ETR-3, a
Regulator of Nuclear and Cytoplasmic RNA Processing
Events,” Journal of Cell Science, Vol. 117, No. 16, 2004,
pp. 3519-3529. doi:10.1242/jcs.01194
 J. Wu, C. Li, S. Zhao and B. Mao, “Differential Expres-
sion of the Brunol/CELF Family Genes during Xenopus
Laevis Early Development,” The International Journal of
Experimental Biology, Vol. 54, 2010, pp. 209-214.
 L. T. Timchenko, E. Sailsbury, G.-L. Wang, H. Nguyen, J.
H. Albrecht, J. W. B. Hershey and N. A. Timchencko,
“Age-Specific CUGBP1-eIF2 Complex Increases Transla-
tion of CCAAT/Enhancer-Binding Protein β in Old
Liver,” The Journal of Biological Chemistry, Vol. 281,
No. 43, 2006, pp. 32806-32819.
 C. Kress, C. Gautier-Courteille, H. B. Osborne, C. Babi-
net and L. Pillard, “Inactivation of CUG-BP1/CELF1
Causes Growth, Viability, and Spermatogenesis Defects
in Mice,” Molecular and Cellular Biology, Vol. 27, No. 3,
2007, pp. 1146-1157. doi:10.1128/MCB.01009-06
 Y. Onohara, T. Fujiwara, T. Yasukochi, M. Himeno and
S. Yokota, “Localization of Mouse Vasa Homolog Pro-
tein in Chromatoid Body and Related Nuage Structures of
Mammalian Spermatogenic Cells during Spermatogene-
sis,” Histochemistry and Cell Biology, Vol. 133, No. 6,
2010, pp. 627-639. doi:10.1007/s00418-010-0699-5
Copyright © 2012 SciRes. OJCB
H. YONETAMARI ET AL.
Copyright © 2012 SciRes. OJCB
 C. de Roe, P. J. Courtoy and P. Baudhuin, “A Model of
Protein Colloidal Gold Interactions,” Journal of Histo-
chemistry & Cytochemistry, Vol. 35, No. 11, 1987, pp.
 L. D. Russell, R. A. Ettlin, A. S. P. Hikim and E. D. Clegg,
“Histological and Histopathological Evaluation of Tes-
tis,” Cache River Press, Clearwater, 1990.
 Y. Toyooka, N. Tsunekawa, Y. Matsui, M. Satoh and T.
Noce, “Expression and Intracellular Localization of
Mouse Vasa-Homologue during Germ Cell Develop-
ment,” Mechanisms of Development, Vol. 93, No. 1-2,
2000, pp. 139-149. doi:10.1016/S0925-4773(00)00283-5
 T. Noce, S. Okamoto-Ito and N. Tsunekawa, “Vasa Ho-
molog Genes in Mammalian Germ Cell Development,”
Cell Structure and Function, Vol. 26, No. 3, 2001, pp.
 C.-H. Tsai-Morris, Y. Sheng, E. Lee, K.-J. Lei and M. L.
Dufau, “Gonadotropin-Regulated Testicular RNA Heli-
case (GRTH/Ddx25) is Essential for Spermatid Devel-
opment and Completion of Spermatogenesis,” Proceed-
ings of the National Academy of Sciences of the United
States of America, Vol. 101, No. 17, 2004, pp. 6373-6378.
 S. Chuma, M. Hiyoshi, A. Yamamoto, M. Hosokawa, K.
Takamune and N. Nakatsuji, “Mouse Tudor Repeat-
1(MTR-1) is a Novel Component of Chromatoid Bod-
ies/Nuages in Male Germ Cells and Forms a Complex
with snRNPs,” Mechanisms of Development, Vol. 120,
No. 9, 2003, pp. 970-990.
 M. Hosokawa, M. Shoji, K. Kitamura, T. Tanaka, T.
Noce, S. Chuma and N. Nakatsuji, “Tudor-Related Pro-
tein TDRD 1/MTR-1, TDRD6 and TDR7/TRAP: Domain
Composition, Intracellular Localization, and Function in
Male Germ Cells in Mice,” Developmental Biology, Vol.
301, No. 1, 2007, pp. 38-52.
 J. Pan, M. Goodheart, S. Chuma, N. Nakatsuji, D. C.
Page and P. J. Wang, “RNF17, a Component of the
Mammalian Germ Cell Nuage, is Essential for Spermio-
genesis,” Development, Vol. 132, 2005, pp. 4029-4039.
 N. Kotaja, H. Lin, M. Parvienen and P. Sassone-Corsi,
“Interplay of PIWI/Argonaute Protein MIWI and Kinesin
KIF17b in Chromatoid Bodies of Male Germ Cells,”
Journal of Cell Science, Vol. 119, No. 119, 2006, pp.
 S. F. C. Soper, G. W. van der Heijden, T. C. Hardiman,
M. Goodheart, S. L. Marten, P. de Boer and A. Bortvin,
“Mouse Maelstrom, a Component of Nuage, is Essential
for Spermatogenesis and Transposon Repression in
Meiosis,” Developmental Cell, Vol. 15, No. 2, 2008, pp.
 Y. Onohara and S. Yokota, “Expression of DDX25 in
Nuage Components of Mammalian Spermatogenic Cells:
Immunofluorescence and Immunoelectron Microscopic
Study,” Histochemistry and Cell Biology, Vol. 137, No. 1,
2011, pp. 37-51.
 Y. Clermont, R. Oko and L. Hermo, “Cell Biology of
Mammalian Spermatogenesis,” In: C. Desjardins and L. L.
Ewing, Eds., Cell and Molecular Biology of the Testis,
Oxford University Press, New York, 1993, pp. 332-376.
 Y. Clermont, R. Oko and L. Hermo, “Immunocytoche
mical Localization of Proteins Utilized in the Formation
of Outer Dense Fibers and Fibrous Sheath in Rat Sper-
matids: An Electron Microscopic Study,” The Anatomical
Record, Vol. 227, No. 4, 1990, pp. 447-457.
 A. L. Kierszenbaum, “Intramanchette Transport (IMT):
Managing the Making of the Spermatid Head, Centro-
some, and Tail,” Molecular Reproduction and Develop-
ment, Vol. 63, No. 1, 2002, pp. 1-4.
 L. L. Tres and A. L. Kierszenbaum, “Sak57, an Acidic
Keratin Initially Present in the Spermatid Manchette be-
fore Becoming a Compound of Paraaxonemal Structures
of the Developing Tail,” Molecular Reproduction and
Development, Vol. 44, No. 3, 1996, pp. 395-407.
 P. D. Taulman, C. J. Hycraft, D. F. Balkovetz and B. K.
Yoder; “Polaris, a Protein Involved in Left-Right Axis
Patterning, Localizes to Basal Bodies and Cilia,” Molecu-
lar Biology of the Cell, Vol. 12, No. 3, 2001, pp. 589-599.
 E. Rivkin, E. B. Cullinan, L. L. Tres and A. L. Kier-
szenbaum, “A Protein Associated with the Manchette
during Rat Spermiogenesis is Encoded by a Gene of the
TBP-1-Like Subfamily with Highly Conserved ATPase
and Protease Domain,” Molecular Reproduction and De-
velopment, Vol. 48, No. 1, 1997, pp. 77-89.
 A. Junco, B. Bhullar, H. A. Tarnasky and F. A. van der
Hoorn, “Kinesin Light Chain KLC3 Expression in Testis
is Restricted to Spermatids,” Biology of Reproduction,
Vol. 64, No. 5, 2001, pp. 1320-1330.
 M. G. Miller, D. J. Mulholand and W. A. Vogt, “Rat Tes-
tis Motor Proteins Associated with Spermatid Transloca-
tion (Dynein) and Spermatid Flagella (Kinesin-II),” Biol-
ogy of Reproduction, Vol. 60, No. 4, 1999, pp. 1047-1056.
 D. L. Spector, “Nuclear Organization and Gene Expres-
sion,” Experimental Cell Research, Vol. 229, No. 2, 1996,
pp. 189-197. doi:10.1006/excr.1996.0358
 M. Labrador and V. G. Corces, “Setting the Boundaries
of Chromatin Domains and Nuclear Organization,” Cell,
Vol. 111, No. 2, 2002, pp. 151-154.