International Scholarly Research Network
ISRN Cell Biology
Volume 2012, Article ID 829854, 12 pages
doi:10.5402/2012/829854

Research Article

Morphological Changes of Mammalian Nucleoli during
Spermatogenesis and Their Possible Role in the Chromatoid
Body Assembling

Rita Luiza Peruquetti, Sebastião Roberto Taboga,
and Maria Tercı́lia Vilela de Azeredo-Oliveira

Department of Biology, Sao Paulo State University, UNESP/IBILCE, Rua Cristovao Colombo, 2265,
15054-000 Sao Jose do Rio Preto, SP, Brazil
Department of Biology, Sao Paulo State University, UNESP/IBILCE, Rua Cristovao Colombo, 2265, 15054-000 Sao Jose do Rio Preto,
SP, Brazil

Correspondence should be addressed to Rita Luiza Peruquetti, ritaperuquetti@yahoo.com.br

Received 5 November 2011; Accepted 8 December 2011

Academic Editors: A. Hergovich, P. Lavia, and M. Yamaguchi

Copyright © 2012 Rita Luiza Peruquetti et al. This is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly
cited.

Chromatoid body (CB) is a typical cytoplasmic organelle of germ cells, and it seems to be involved in RNA/protein accumulation
for later germ-cell differentiation. Despite most of the events in mammals spermatogenesis had been widely described in the
past decades and the increase in the studies related to the CB molecular composition and physiology, the origins and functions
of this important structure of male germ cells are still unclear. The aims of this study were to describe the nucleolar cycle
and also to find some relationship between the nucleolar organization and the CB assembling during the spermatogenesis
in mammals. Cytochemical and cytogenetics analysis showed nucleolar fragmentation in post-pachytene spermatocytes and
nucleolar reorganization in post-meiotic spermatids. Significant difference in the number and in the size of nucleoli between
spermatogonia and round spermatids, as well as differences in the nucleolar position within the nucleus were also observed.
Ultrastructural analysis showed the CB assembling in the cytoplasm of primary spermatocytes and the nucleolar fragmentation
occurring at the same time. In conclusion our results suggest that the CB may play important roles during the spermatogenesis
process in mammals and that its origin may be related to the nucleolar cycle during the meiotic cell cycle.

1. Introduction

Spermatogenesis is the biological process of gradual trans-
formation of germ cells into spermatozoon over an extended
period of time within the boundaries of the seminiferous
tubules of the testis. This process involves cellular prolif-
eration by repeated mitotic divisions, duplication of chro-
mosomes, genetic recombination through crossover, reduc-
tion division by meiotic cell division to produce haploid
spermatids, and terminal differentiation of the spermatids
into spermatozoon [1]. This important biological process has
been extensively studied and described, and currently there
are appropriate biological terminologies to describe every
step of the spermatozoon formation [2]. However, there are

some recent important aspects of the spermatogenesis, as, for
example, the assembling and the function of the chromatoid
body (CB), which is not so clearly understood. The CB is
a typical cytoplasmic organelle of haploid germ cells and is
involved in RNA and protein accumulation for later germ-
cell differentiation [3, 4]. Many studies have been carried
out intending to clarify the origins and functions of this
intriguing cytoplasmic structure of male germ cells. Recent
efforts to elucidate the functions of the CB have been
detecting the presence of the Mouse Vasa Homologue protein
(MVH), which is an ATP-dependent DEAD-box RNA heli-
case playing a role in the establishment and differentiation of
germ cells, closely associated with CB in several organisms
[5–8]. Another interesting molecular component of CBs



2 ISRN Cell Biology

is the MAEL protein, an ortholog of Drosophila’s high-
mobility group box protein Maelstrom that is associated not
only with the silenced XY body but also with unsynapsed
autosomes interacting directly with the chromatin remodeler
SNF5/INI1 and chromatin-associated protein SIN3B. The
presence of MAEL in this critical compartment of male
germ cells and its interactions provide a link suggesting the
involvement of the miRNA pathway in meiotic silencing of
unsynapsed chromatin (MSUC) [9]. Despite many efforts
to elucidate the CB functions, its main function is still
unclear. CB origin is also still uncertain although many
theories trying to elucidate it exist. Some authors suggest
that the origin of the CB is nuclear [10, 11]. Others have
proposed that the CB precursor is a dense interstitial material
occurring between mitochondrial clusters [12], or that it is a
result of the mitochondrial products that are released in the
cell cytoplasm [13]. However, still others have suggested that
the CB originates from nuages, a ribonucleoproteic complex
derived from the nucleolus, which migrates to the cytoplasm
during earlier spermatogenesis [14–18].

The nucleolus is a distinct nuclear territory involved in
the compartmentalization of nuclear functions [19]. There-
fore, ribosome biogenesis is the main function attributed
to the nucleolus [20–22], and many studies have shown
that nucleolus also has nonribosomal functions, including
nucleotide modifications of several small RNAs, biosynthesis
of the signal recognition particle and phased sequestration,
and the release of proteins involved in gene silencing,
senescence, and cell division [23]. Moreover, some proteins
that are involved in the control of the cell cycle, such as Net1,
Cdc14, and Sir2, are located in the nucleolar compartments
[24–27], along with the tumor suppressor proteins ARF [28],
p53, and Myc [29]. The nucleolus of germ cells seem to be
involved in meiotic cell-cycle control, because most of the
Pch2 protein, which is required for the meiotic checkpoint
that prevents chromosome segregation when recombination
and chromosome synapses are defective, localizes to the
nucleolus [30].

Since both nucleolar and CB physiology are very impor-
tant to the spermatogenesis process, the aims of the present
study were to describe the nucleolar cycle and also to find
some relationship between nucleolar organization and CB-
assembling process during the spermatogenesis in mammals.
This study was performed by using cytochemical, cytogenet-
ics, and ultrastructural analysis to follow the nucleolar cycle
and the CB assembling in the seminiferous epithelium of
Oryctolagus cuniculus (Mammalia, Lagomorpha).

2. Material and Methods

Investigations were carried out on 5 adult male Oryctolagus
cuniculus (Mammalia, Lagomorpha). The animals were
obtained from Sao Jose do Rio Preto School of Medicine
Vivarium (FAMERP, Sao Jose do Rio Preto, SP, Brazil). All
animals were housed under standard conventional condi-
tions (25◦C, 40–70% relative humidity, 12 h light/12 h dark)
and allowed access to chow and water ad libitum. The

animals were anaesthetized and killed by carbon dioxide gas
inhalation, and after their deaths the animals were dissected,
and their gonads were removed and prepared for each
biological analysis.

2.1. Cytochemical Analysis. Testes of each animal were
removed and immersed in a Karnovsky fixative solution for
36 hours. The material was embedded in glycol-methacrylate
historesin. Sections (1–3 µm thick) were obtained in Leica
RM 2155 microtome. Tissue sections were submitted to
various cytological and cytochemical procedures, including
hematoxylin-eosin (HE) [31], toluidine blue (TB), modi-
fied Critical Electrolyte Concentration for detecting RNA
(CEC) [32], silver-ion impregnation (AgNOR) [33], and
Feulgen reaction [34]. The sections of germ epithelium
were evaluated under an Olympus BX 60 photomicroscope
and documented using Image Pro-Plus; Media Cybernetics
computer software, version 6.1 for Windows for image
analysis.

In addition to the qualitative analysis of the nucleolar
material distribution, the tissue sections that were subjected
to silver ion impregnation were used for quantitative analysis,
in order to determine the number of nucleoli in the
spermatogonia and round spermatids, and also to measure
the nuclear and nucleolar areas in the same types of cells.

2.1.1. Determination of the Number of Nucleoli in the Sper-
matogonia and Round Spermatids. The number of nucleoli
was determined in all spermatogonia and round spermatids
recorded in this study. Spermatogonia (144.2 ± 9.47) and
round spermatids (169.2±13.81) were considered from each
specimen studied (n = 5). Due to the different amount of
cells analyzed in each cell type, the percentage of the number
of nucleolus in each cell type was calculated.

2.1.2. Measuring the Nuclear and Nucleolar Areas of the
Spermatogonia and Round Spermatids. Spermatogonia and
round spermatids were photodocumented using an Olym-
pus BX 40 photomicroscope and Image Pro-Plus Media
Cybernetics computer software, version 4.5 for Windows.
Next, both nuclear and nucleolar areas of these cells were
measured using Image J-Image Processing and Analysis in
Java software, Version 1.40 (http://rsb.info.nih.gov/ij/) for
image analysis. The cells that had one single nucleolus were
measured immediately, while the total area of those with two
or more nucleoli was calculated by adding up the areas of
each individual nucleolus.

2.2. Data Analysis. Normal distribution of the dataset was
tested using Skewness and Kurtosis analysis [35], and
variance homogenity was tested using the F max test [36].
The number of nucleoli was compared between spermato-
gonia and round spermatids, and also within the same
cell type using the Two-Factor Analysis of Variance, which
was complemented using the LSD multiple comparisons
test [36]. Nuclear and nucleolar areas of spermatogonia
were compared to nuclear and nucleolar areas of round
spermatids using an independent t test [36]. Statistical
significance was considered when P > 0.05.



ISRN Cell Biology 3

go

s

spI

es
stz

gospI

es

(a) (b)

(c)

(d)

(e)

(f)

(g)

(h)

(i)

(j)

(k)

(l)

(m)

(n)

∗

Figure 1: Cytological and cytochemical analysis of the germ epithelium of Oryctolagus cuniculus. (a) Hematoxylin-eosin (HE); (c–e)
toluidine blue (TB); (b, f–h) modified critical electrolyte concentration method (CEC); (i–k) Feulgen reaction; (l–n) silver ion impregnation.
(a and b) General view of the seminiferous tubules, where we can observe the distribution of the somatic and germ cells through the
seminiferous epithelium: s (Sertoli cell); go (spermatogonia); spI (primary spermatocyte); es (round spermatids); stz (elongated spermatids);
∗(residual bodies). (c, f, i, and l) spermatogonia with fully organized nucleoli. (d, g, j, and m) spermatocytes I exhibiting fragmented
nucleolar material disperse through the nuclei. (e, h, k, and n Reassembly of the nucleolar structure could be observed in the nuclei of
round spermatids. Reorganized nucleoli were not observed using the CEC method (h). (f, k, l, and n) arrows (nucleolar corpuscles). Bars: (a
and b) 50 µm; (c–n) 20 µm.



4 ISRN Cell Biology

2.3. Cytogenetic Analysis. Standard meiotic cells were
obtained using the method introduced by [37] and adapted
by [38]. Soon after, the testes were sectioned into small frag-
ments and submitted to hypotonic treatment with 0.075 M
KCl for 20–30 min. The material was then treated with a
fresh fixative solution (methanol : glacial acetic acid, 3 : 1)
for 30 min, and this procedure was performed a minimum
of two times per fragment. After fixation, some fragments
were placed in the well of a depression slide with some
drops of a 50% glacial acetic acid solution and mixed until a
homogeneous cell suspension was obtained. One drop of this
suspension was then deposited onto a clean slide, and heated
to about 38◦C with the aid of a fine-tipped Pasteur pipette.
The drop was then sucked back into the pipette, forming a
cell ring measuring about 1 cm in diameter on the slide, with
the cells preferentially deposited on the border of the ring.
These last two steps were repeated on 2-3 additional fields
of the same slide. Then, the slides were submitted to silver
ion impregnation (AgNOR) [33] to follow the nucleolar
material distribution during the meiotic division in the germ
cells. The preparations were evaluated under an Olympus
BX 60 photomicroscope and documented using Image Pro-
Plus; Media Cybernetics, version 6.1 for Windows computer
software for image analysis.

2.4. Ultrastructural Analysis. Testes fragments of each animal
were removed and sliced into small pieces, and samples
of the germ epithelium were cut and immersed in a 3%
glutaraldehyde plus 0.25% tannic acid solution in Millonig’s
buffer (pH 7.3) containing 0.54% glucose for 24 hours at
room temperature [39]. After being washed with the same
buffer, samples were postfixed in 1% osmium tetroxide for
1 hour at 4◦C, washed in Millonig’s buffer, dehydrated in
a graded acetone series, and embedded in Araldite resin.
Ultrathin silver sections (50–75 nm) were cut using a dia-
mond knife and stained with 2% alcoholic uranyl acetate for
30 min [40], followed by 2% lead citrate in sodium hydroxide
for 10 min [41]. Samples were evaluated using a Leo-Zeiss
906 (Cambridge, UK) transmission electron microscope and
documented using ITEM (Soft Image System—Veleta 2K ×
2K TEM CCD Camera) software for image analysis.

2.5. Ethics Note. This study was approved by the Ethical
Committee for Animal Research (CEEA) of Sao Paulo State
University (UNESP) in Botucatu, Sao Paulo, Brazil, under
protocol number 057/06.

3. Results

3.1. Cytochemical Analysis. Hematoxylin-eosin (HE) cyto-
logical analysis was used to confirm the spermatogenesis
pattern in the seminiferous tubules (Figure 1(a)). Stadia of
development and type of cells present in the germ epithelium
were ranked according to Leblond and Clermont [42].
Toluidine blue (TB) reaction revealed the cells of the male
germ epithelium with an intense metachromasy in all of the
nuclear domains (euchromatin, heterochromatin, nucleolus,
and chromosomes) (Figures 1(c), 1(d), and 1(e)). The degree

of metachromasy varied according to the compaction of
genetic material, the ploidy of the cell nucleus, and the com-
plexity of the nucleic acids with ribonucleoproteic (RNP)
corpuscles. The TB reaction was performed as a control
for the critical electrocyte concentration (CEC) variant
method for RNA detection. CEC revealed spermatogonia
with metachromatic nucleoli (Figure 1(f)), primary sperma-
tocytes with fragmented nucleoli and nucleolar fragments
surrounding the chromosomes (Figure 1(g)), and round
spermatids with no organized nucleoli (Figure 1(h)). Several
residual bodies were detected in the seminiferous epithelium
lumen (Figure 1(b)). Feulgen reaction is a DNA-specific
method in which all germ cell nuclei are dyed purple and
are categorized according to their degree of ploidy, func-
tional stage, and compaction of chromatin. Heterochromatic
regions were more intensively dyed, and nucleoli were seen
as light spots connected to these heterochromatic regions.
In the spermatogonia (Figure 1(i)), the nucleolar area was
larger than in both primary spermatocytes (Figure 1(j)) and
round spermatids (Figure 1(k)), indicating the occurrence
of fragmentation and reduction of the nucleolar area in the
latter’s cell type. Impregnation using silver ion (AgNOR)
revealed the nucleolar regions in different germ cells.
Spermatogonia were found to possess organized nucleoli
(Figure 1(l)), primary spermatocytes possessed fragmented
nucleoli (Figure 1(m)), and round spermatids possessed
reorganized nucleoli (Figure 1(n)) although they were found
to be smaller than the spermatogonium nucleoli.

3.1.1. Determination of the Number of Nucleolus in the Sper-
matogonia and Round Spermatids. There was a correlation
between the cell type and the number of nucleoli (F =
3.63; P > 0.05). This correlation showed that the number
of spermatogonia with one nucleolus was lower than the
number of round spermatids with the same number of
nucleolus. However, spermatogonia possessed 2, 3, or 4
nucleolus as frequently as round spermatids (Figure 2(a)).

3.1.2. Sizes of the Nuclear and Nucleolar Areas of the Sper-
matogonia and Round Spermatids. There were significant
differences between the nuclear areas of spermatogonia and
round spermatids (t = 9.64; P > 0.05), and also between the
nucleolar area of spermatogonia and round spermatids (t =
10.14; P > 0.05). In both results, the areas of spermatogonia
were larger than the areas of round spermatids (Figure 2(b)).

3.2. Cytogenetic Analysis. Spermatogonia going through
mitotic metaphase were not observed in the cytogenetic
preparations, but the chromosomic number of the species
was established as 2n = 44 according to Korstanjea et al.
[43]. Standard meiotic preparations impregnated using the
silver ion method showed spermatogonia with large central
nucleoli (Figure 3(a)). In primary spermatocytes in the
zygotene stage (Figure 3(b)), fragmented nucleoli and nucle-
olar fragments distributed in the periphery of the nucleus
and also surrounding the chromosomes were observed.
Primary spermatocytes in pachytene stage (Figure 3(c))
possessed nucleolar fragments distributed mainly in the



ISRN Cell Biology 5

100

80

60

40

20

0

1 2 3 4

a;∗
a

b
b

c c
d

d

Spermatogonia
Round spermatids

Number of nucleoli

(a)

7000

500

0
Spermatogonia Earlier spermatids

N
u

cl
ea

r 
ar

ea
 (

n
m

2
)

500

250

0
Spermatogonia Earlier spermatids

N
u

cl
ea

r 
ar

ea
 (

n
m

2
)

∗

∗

(b)

Figure 2: (a) Percentage of germ cells (spermatogonia and round spermatids) of Oryctolagus cuniculus that possessed 1 to 4 nucleoli. The
letters represent a statistically difference in the number of nucleoli in each cell type, and the asterisk represents statistically difference between
both cell types (LSD, P < 0.05). The means (±SD) of the number of spermatogonia and round spermatids possessing 1, 2, 3, or 4 nucleoli
were, spermatogonia: 1 nucleolus 61.33 (±13.72); 2 nucleoli 34.5 (±3.67); 3 nucleoli 17 (±3.42); 4 nucleoli 9.33 (±6.06). Round spermatids:
1 nucleolus 83.16 (±14.51); 2 nucleoli 36.5 (±11.32); 3 nucleoli 16.66 (±3.97); 4 nucleoli 6.33 (±2.94). B: areas (nm2) of the nuclei and
nucleoli of spermatogonia and round spermatids. The asterisk (∗) represents a statistically significant difference between the cell types
(independent t test, P < 0.05).

periphery of the nucleus. In advanced meiotic stages, such
as metaphase I (Figure 3(d)), no nucleolar corpuscles were
observed; however, strong spots of silver impregnation were
observed in some chromosomes probably in the place where
nucleolar organizer regions (NORs) are located. Round
and elongated spermatids were observed possessing 1 to 4
nucleolar corpuscles (Figures 3(e) and 3(f)) preferentially
located in the periphery of the nucleus. Spermatozoon
possessed a strong silver ion impregnation in their middle
piece and tail region (Figure 3(g)).

3.3. Ultrastructural Analysis. Spermatogonia did possess
reticulated nucleoli organized in the nucleus center, but nei-
ther ribonucleoprotein nuages nor initial chromatoid bod-
ies (CB) assembling was observed (Figures 4(a) and 4(b)).
Complete nucleolar disorganization and cytoplasmic accu-
mulation of ribonucleoprotein were observed in the pri-
mary spermatocytes in pachytene stages. Ribonucleoprotein
accumulation in the cytoplasm of primary spermatocytes
was observed both unassociated with other structures
(Figure 4(b)) or in association with mitochondrial aggregates
(Figures 4(c) and 4(d)). Nucleoli fully organized were
observed again in the nucleus of round spermatids (Figures
4(e) and 5(a)), and CBs were observed in association with
mitochondrial aggregates in the anterior nuclear region,
where acrosome formation takes place (Figures 4(e), 4(f),
5(a), and 5(b)). Elongated spermatids were shown also pos-
sessing organized nucleoli, and the migration of the complex

composed by CB and mitochondrial aggregations to the pos-
terior nucleus region, where the mitochondrial sheath and
spermatozoon tail formation take place, was also observed
(Figures 5(c), 5(d), and 5(e)). No dense ri-bonucleoprotein
corpuscles were observed in the residual bodies after the
formation of the mature spermatozoon (Figure 5(f)).

4. Discussion

Most recent studies focused on the molecular composition
and function of the chromatoid body (CB), a structure
present in the cytoplasm of male germ cells, suggest that
this structure acts as a subcellular coordinator of different
RNA-processing pathways and centralizes posttranscrip-
tional mRNA control in the cytoplasm of haploid male
germ cells [7, 44, 45]. The CB seems to be very important
in the spermatogenesis process since mutations in some
proteins located in this structure, such as tudor proteins
(TDR1/MTR-1) and rat histocompatibility antigen (OX3)
cause sterility in rats [46, 47]. Besides of the importance
of this structure, its origins are not fully understudied, and
many different theories were thought aiming to understand
where the CB molecular components come from nucleus
[10, 11], mitochondria [12, 13], and/or nucleolus [14–18].
The nucleolus is a nuclear domain whose main function is
linked to the ribosome synthesis [19]. However, many studies
have shown that the nucleolus also has nonribosomal func-
tions, including mitosis regulation, cell-cycle progression,
cell proliferation, cell stress, biogenesis of ribonucleoprotein



6 ISRN Cell Biology

(a) (b) (c)

(d) (e) (f) (g)

Figure 3: Germ cells of Oryctolagus cuniculus. Silver ion impregnation (AgNOR). (a) Interphasic spermatogonia with organized nucleolus
(arrow). (b) Primary spermatocyte in zygotene stage possessing central and peripheral nucleolar corpuscles (arrows). (c) Primary
spermatocyte in pachytene possessing nucleolar corpucles mainly in the peripheral chromosomal region (arrow). (d) Metaphase I. (e) Round
and elongated spermatids general view. (f) Elongated spermatids possessing peripheral nucleolar corpuscles (arrows). (g) Spermatozoon
with a strong silver ion impregnation in the mitochondrial sheath region. Bars (a, b, c, d, f, and g) 5 µm; (e) 20 µm.

particles, cancer, viral infections, and degenerative diseases
[21]. Nucleolus size also increases and decreases in growing
and resting cells, respectively, and it forms and disperses once
every cell-division cycle [48, 49].

Nucleolar fragmentation and distribution of its frag-
ments were evaluated by qualitative and quantitative cyto-
chemical and cytogenetics analyses. Ultrastructural analyses
were applied to verify if the nucleolar fragments produced
during the meiotic division play a role in the CB assembling.

Qualitative cytochemical and cytogenetics analysis
showed one to four central nucleolar corpuscles, could
be observed in the nucleus of spermatogonia. In primary
spermatocytes going through pachytene stage no organized
nucleolar corpuscles were observed, and nucleolar fragments
were distributed in the nuclear periphery. One to 4 organized
nucleolar corpuscles could be observed in the nuclear
periphery of the round spermatids after the completion of
the second meiosis (Figures 1 and 3). Two major events could
be noticed regarding the nucleolar cycle during meiotic cell
division progression: the nucleolar fragmentation in the
primary spermatocytes and the peripheral location of
nucleolar corpuscles in the round spermatids.

The phenomenon of nucleolar fragmentation during
prophase I and its subsequent reorganization after meiosis
completion have been described in detail over the past two
decades (e.g., [17, 18, 51–53]). A decrease in the amount of

C23 protein, which is responsible for rRNA transcription,
and a decrease in the amount of B23 protein, which is
responsible for rRNA splicing, were also observed in the
nucleoli of both primary spermatocytes and round sperma-
tids [54]. Those reports suggest that nucleolar fragmentation
occurs during early meiotic prophase I and after the comple-
tion of the meiotic division smaller nucleoli are reorganized
in the nuclei of round spermatids. It also has been shown
that, in most chordates, there is increased nucleolar activity
during prophase I, and that nucleolar activity reaches its peak
during pachytene [49, 55, 56], which helps to explain the
nucleolar fragmentation occurring only after this cell-cycle
stage. Organized nucleoli were not observed in the nucleus
of elongated spermatids or in mature spermatozoon. The
presence of a nucleolus is not necessary for the spermato-
zoon, because, after fecundation, the nucleoli of the male and
female pronuclei in the zygote are both of maternal origin.
Recent studies have suggested that the maternal nucleolus,
associated with other nucleoplasmic products, is essential
for embryonic development [57]. Peripheral nucleoli were
also reported by [58] where the nucleolar position in the
nucleus was linked to the NORs location. Species with
terminal NORs possessed peripheral nucleoli while species
with intercalated NORs possessed central nucleoli. The
authors concluded that the distribution of nucleoli within
the nuclear space of spermatocytes is nonrandom, and it is



ISRN Cell Biology 7

n

nu

(a)

n

∗

(b)

n

m

m

m

m

∗

(c)

n

m

∗

(d)

n

m
m

nu

∗

∗

(e)

n

m

aa

a

∗

(f)

Figure 4: Ultrastructural analysis of Oryctolagus cuniculus germ epithelium. (a) Spermatogonia with central reticulated nucleolus (nu)
organized. (b, c, and d) Primary spermatocytes in the pachytene stage with no organized nucleolus and possessing synaptonemal complex
and ribonucleoprotein accumulation in the cytoplasm (∗). Ribonucleoprotein could join later with mitochondria (m), or it could remain
isolated. (e and f) Round spermatids with reorganized nucleolus (nu) and CB (∗) linked to mitochondria aggregates next to the acrosomal
system region (a). (n: nucleus; nu: nucleolus; m: mitochondria; ∗-chromatoid body; a: acrosomal system). Bars = 10 µm.

consistent with the existence of a species-specific meiotic
nuclear architecture.

Quantitative cytochemical analyses showed that the
number of nucleoli was different between the different
germ cells analyzed because round spermatids had one
single nucleolus more frequently than spermatogonia did
(Figure 2(a)). This result suggests a decrease in the number of
nucleoli during the meiotic division process. Previous studies
reported that the number of nucleoli of germ cells is related

to their number of RONs [49, 59]. Diploid spermatogonia
are therefore expected to have twice as many nucleoli as
the round haploid spermatids, which agree with our result.
There was also a notable difference in the nucleolar area
of the different germ cells. We observed that nuclear and
nucleolar areas of spermatogonia were larger when compared
to nuclear and nucleolar areas of the round spermatids
(Figure 2(b)). The size of a nucleolus is proportional to the
amount of rRNA synthesized [60]; NOR size (the number



8 ISRN Cell Biology

n

a

n

a

(a)

(b)

(c)

(d)

(e) (f)

n

nu

m

a

m

m

∗

n

nu

m

m

ma
a

a

∗∗

n

nu∗

n

a

a

a

a
a∗

n

∗

Figure 5: Ultrastructural analysis of Oryctolagus cuniculus germ epithelium. (a) Round spermatid with reorganized nucleolus (nu) and a
large CB (∗). (b) Round spermatids with reorganized nucleolus (nu) and CB (∗) next to the acrosomal system (a). (c, d, and e) Elongated
spermatids with reorganized nucleolus (nu) and large CB (∗) linked to mitochondria aggregates (m) moving to the nuclear posterior region
(n) where the development of mitochondrial sheath and spermatozoon tail will take place. (f) Spermatozoon in maturation process with no
ribonucleoprotein accumulation. (n: nucleus; nu: nucleolus; ∗-chromatoid body; m: mitochondria; a: acrosomal vesicle). Bars = 10 µm.

of rRNA cistrons) is generally correlated with its expression
level [61]; hypertrophy of the nucleolus is a state in which
rRNA and ribosome synthesis has increased [62]; the size of
the largest nucleoli may correlate with cell-division activity
and also with cellular stages having high-protein demand
[63]. All of these findings may be related to the decrease in
nucleolus size in the round spermatids, but its decrease can
also be explained by the migration of the nucleolar fragments
to the cytoplasm of germ cells in prophase I, where they
may play a role in CB assembling as previously proposed
[14, 16–18]. If nucleolar proteins would play a role in the CB
physiology acting in the RNA processing or if they would be

translocated to the CB because they are targeted for degrada-
tion in late steps of the spermatogenesis process is a question
that still needs to be clarified. However, it has been shown
that nucleolar morphology reestablishment is an important
factor to the reset of lifespan in gametogenesis by eliminating
age-induced cell damage [64], and some nucleolar proteins
could be translocated to the CB during this nucleolar
morphology reestablishment process. We hypothesized here
the nucleolar and other nuclear components role in the
molecular composition and physiology of the CB (Figure 6).

Ultrastructural analysis did not show evidences of
ribonucleoprotein accumulation, which would originate the



ISRN Cell Biology 9

Transcription

AAA
AAA
AAA
AAA

AAA

RNPs

RNPs

RNPs

RNPs

RNPs

Nucleus

Transcription

AAA
AAA

AAA
AAA

AAA

RNPs

actin

MIWI

MVHDicer                           
Ago                         

miRNA

AcPs

Active 
translation

Translational 
regulation

?

Meiotic 
divisions Spermiogenesis

Mitotic 
divisions

Active transcription Active transcription
Active

A A

P D

MILI

MAEL?

transcription

Nucleolus

TDRD9
TDRD1

Spermatogonium Late first spermatocyte

Nucleus

RNA storage/RNA-binding proteins/translational repression

Round spermatids

DNase/
RNase

Nucleus

Nucleolus

HSP70
KIF17b

Dcp1a

Spermatozoon

Figure 6: Hypothetical schematic view of the nucleolar role on the CB assembly in male germ cells. Spermatogonium is an undifferentiated
male germ cell, originating in a seminal tubule and dividing by mitosis into a new generation of spermatogonia or into two first
spermatocytes. In this type of cell the nucleolus is organized and active. Germ granules and granular localization of PIWI and Tudor
proteins are not well characterized in spermatogonia, but MILI, MAEL, TDRD1, and TDRD9, which are molecular components of CBs,
have been demonstrated in the cytoplasm of spermatogonia and/or early spermatocytes [50]. In late first spermatocytes there is a peak of
nucleolar activity followed by nucleolar disorganization, and some of the nucleolar components become disperses through the cytoplasm.
In this stage there is an intense transcription of mRNA that will be silent for later translation during germ-cell differentiation, and also
many other nuclear products, which will play a role in the CB molecular composition, are in transit nucleus cytoplasm. There is a drastic
inhibition of transcriptional activity during the meiotic divisions, and mRNA storage and translational regulation by RNA-binding proteins
have an important role in the control of the synthesis of many spermatids and spermatozoa proteins [45]. In postmeiotic round spermatids,
nucleolar reorganization is observed, and the transcription is active until the silencing of the haploid genome owing to compaction of the
genome by the replacement of histones with protamines. In this type of cell the CB is condensed to its final shape, and it contains (a)
components that play a role in the RNA processing (Miwi, KIF17b, Dicer, Ago, MVH DCP1a, miRNA–purple shapes); (b) components that
act as proteasome folding and degradation sites (AcPs, HSP70, DNase/RNase–blue shapes). These components possibly play a role also in the
acrosomal system formation; (c) components related to CB movement (actin-red shape). Due to the CB high mobility probably related to
the presence of actin, there is a hypothesis that CB would link to mitochondria aggregates playing a role in the migration of mitochondria to
the nuclear posterior region, where the formation of mitochondrial sheath and spermatozoon tail will take place. (d) The probable function
of nucleolar proteins (light green shape) is still unclear. They could play some physiological role in the RNA metabolism, or even they could
be targeted for degradation. After the spermiogenesis process mature male germ cell does not possess any residue of nucleolar nor even CB
components, indicating that these materials were possibly digested at the end of the spermatogenesis process. RNPs: ribonucleoproteins; P:
pachytene; D: diakinesis; A: acrosome.

CB, in the cytoplasm of spermatogonia. Initial CB assem-
bling was only observed in the cytoplasm of primary
spermatocytes, after the completion of the nucleolar frag-
mentation (Figure 4). It has been described in the liter-
ature that the CB assembling often takes place after the
nucleolar fragmentation [17, 18, 45, 52, 65], which strongly
increase the correlation between both biological process.
Mature CBs were observed isolated or associated with
mitochondria in the cytoplasm of primary spermatocytes
(Figures 4 and 5). This association has been described in
previous studies, which gave rise to the hypothesis that
the CB may originate from intermitochondrial material
[12]. Other authors have suggested that the CB originates
from nuclear genome products and is then supplemented
by mitochondrial genome products [13]. The connection

between the CB and mitochondria may also be explained by
the participation of the CB in the synthesis and transport of
the apocytochrome c, a type of cytochrome c isoform, which
is expressed in the testis tissue [66]. The oxidase cytochrome
c (COXI), an important mitochondrial component, was also
reported in the CB [67]. Detection of the presence of several
mitochondrial components in the CB and related structures
may indicate the attractive possibility that germ cell nuage-
like structures serve as a reconfirmation mechanism for the
symbiotic interaction between mitochondria and the nuclear
genome transmitted to the next generation [6]. Therefore,
in this study, we suggest that the relationship between the
CB and mitochondria can be related to the migration of
these structures to the caudal nuclear region, where the
development of mitochondrial sheath and spermatozoa tail



10 ISRN Cell Biology

take place. This has also been suggested by other authors
[17, 18, 68].

Round spermatids possessed reorganized nucleoli, and
their CBs reach their maximum area. CBs also could be
observed isolated or in association with mitochondria in
the anterior nuclear region, next to the acrosomal system
(Figures 4 and 5). The association between the CB and
the Golgi complex had been previously described by other
authors. Soderstrom and Parvinen [3], Tang et al. [69],
Peruquetti et al. [17], and Peruquetti et al. [18] suggested
that the interaction between these structures could be related
to acrosome formation in round spermatids, because vesicles
are frequently seen moving between the Golgi complex and
the CB. Large CBs linked to mitochondria were observed
in the posterior nuclear region in elongated spermatids at
the same region where the formation of the mitochondrial
sheath and the spermatozoon tail will take place. As the
CBs are seeing in different positions of the germ cells
cytoplasm, it is considered a high-motility organelle, and
this high motility is probably related to the presence of actin
[70, 71] and calcium [72] in the CB molecular composition.
This high mobility was also reported by Parvinen et al.
[11] in living round spermatids of rats, which had CBs
in rapidly changing positions in relation to the nuclear
envelope, Golgi complex, and nuclear pale chromatin areas.
Ribonucleoprotein material from the CB was not observed
in the residual bodies of elongated spermatids, indicating
that this material was possibly digested at the end of the
spermiogenesis process [73, 74].

In conclusion our data suggest that there is a decrease in
the size and in the number of nucleoli during the meiotic
division in the seminiferous tubules of O. cuniculus. Part
of the material originated by nucleolar fragmentation could
migrate to the cytoplasm and be sequestered by the CB. Both
phenomena (nucleolar fragmentation and CB assembly)
occur at the same timing during the meiotic cell cycle, and
nucleolar material originated by nucleolar fragmentation
could be accumulated in the CB for later degradation or
because they would play some role in the CB physiology.
Our results also bring more evidences that the CB may play
important roles during the spermatogenesis process such as
acrosomal system formation and mitochondrial sheath and
spermatozoon tail development.

Acknowledgments

The authors would like to thank Dr. Tiago da Silveira
Vasconcelos (Laboratory of Herpetology–UNESP/IB) and
Dr. Thaı́s Billalba Carvalho (Laboratory of Fish Behavior–
UNESP/IBILCE) for their help with statistical analysis. Spe-
cial thanks also go to Mr. Luis Roberto Faleiros, Jr. (Labora-
tory of Microscopy and Microanalysis, UNESP/IBILCE) and
Dr. Rosana Silistino de Souza (Laboratory of Cell Biology,
UNESP/IBILCE) for their help with laboratory techniques.
The authors are indebted to FAPESP (Sao Paulo Research
Foundation–Grants 2005/02919-5 and 2007/04521-4) and
CNPq (Brazilian Research Council, Grant 141375/2006-0)

for financial support and fellowships. They would like to
disclose any conflict of interests in this manuscript.

References

[1] R. A. Hess, “Spermatogenesis, overview,” in Encyclopedia of
Reproduction, vol. 4, Academic Press, New York, NY, USA,
1999.

[2] Y. Clermont, “Kinetics of spermatogenesis in mammals:
seminiferous epithelium cycle and spermatogonial renewal,”
Physiological Reviews, vol. 52, no. 1, pp. 198–236, 1972.

[3] K. O. Soderstrom and M. Parvinen, “Transport of material
between the nucleus, the chromatoid body and the Golgi
complex in the early spermatids of the rat,” Cell and Tissue
Research, vol. 168, no. 3, pp. 335–342, 1976.

[4] P. T. K. Saunders, M. R. Millar, S. M. Maguire, and R. M.
Sharpe, “Stage-specific expression of rat transition protein 2
mRNA and possible localization to the chromatoid body of
step 7 spermatids by in situ hybridization using a nonradioac-
tive riboprobe,” Molecular Reproduction and Development, vol.
33, no. 4, pp. 385–391, 1992.

[5] E. Raz, “The function and regulation of vasa-like genes in
germ-cell development,” Genome Biology, vol. 1, no. 3, pp. 1–6,
2000.

[6] T. Noce, S. Okamoto-Ito, and N. Tsunekawa, “Vasa homolog
genes in mammalian germ cell development,” Cell Structure
and Function, vol. 26, no. 3, pp. 131–136, 2001.

[7] N. Kotaja, S. N. Bhattacharyya, L. Jaskiewicz et al., “The
chromatoid body of male germ cells: similarity with processing
bodies and presence of Dicer and microRNA pathway compo-
nents,” Proceedings of the National Academy of Sciences of the
United States of America, vol. 103, no. 8, pp. 2647–2652, 2006.

[8] N. Kotaja, H. Lin, M. Parvinen, and P. Sassone-Corsi, “Inter-
play of PIWI/Argonaute protein MIWI and kinesin KIF17b in
chromatoid bodies of male germ cells,” Journal of Cell Science,
vol. 119, no. 13, pp. 2819–2825, 2006.

[9] Y. Costa, R. M. Speed, P. Gautier et al., “Mouse MAELSTROM:
the link between meiotic silencing of unsynapsed chromatin
and microRNA pathway?” Human Molecular Genetics, vol. 15,
no. 15, pp. 2324–2334, 2006.

[10] M. Parvinen and L. M. Parvinen, “Active movements of the
chromatoid body. A possible transport mechanism for haploid
gene products,” Journal of Cell Biology, vol. 80, no. 3, pp. 621–
628, 1979.

[11] M. Parvinen, J. Salo, M. Toivonen, O. Nevalainen, E. Soini, and
L. J. Pelliniemi, “Computer analysis of living cells: movements
of the chromatoid body in early spermatids compared with
its ultrastructure in snap-frozen preparations,” Histochemistry
and Cell Biology, vol. 108, no. 1, pp. 77–81, 1997.

[12] D. W. Fawcett, E. M. Eddy, and D. M. Phillips, “Observations
on the fine structure and relationships of the chromatoid body
in mammalian spermatogenesis,” Biology of Reproduction, vol.
2, no. 1, pp. 129–153, 1970.

[13] A. Reunov, V. Isaeva, D. Au, and R. Wu, “Nuage constituents
arising from mitochondria: is it possible?” Development
Growth and Differentiation, vol. 42, no. 2, pp. 139–143, 2000.

[14] D. E. Comings and T. A. Okada, “The chromatoid body in
mouse spermatogenesis: evidence that it may be formed by the
extrusion of nucleolar components,” Journal of Ultrasructure
Research, vol. 39, no. 1-2, pp. 15–23, 1972.



ISRN Cell Biology 11

[15] K. Andersen, “Fine structure of spermatogonia and sperma-
tocytes in the blue fox (Alopex lagopus),” Acta Veterinaria
Scandinavica, vol. 19, pp. 229–242, 1978.

[16] M. Andonov, “Further study of the chromatoid body in rat
spermatocytes and spermadits,” Zeitschrift fur Mikroskopisch-
Anatomische Forschung—Abteilung 2, vol. 104, no. 1, pp. 46–
54, 1990.

[17] R. L. Peruquetti, I. M. Assis, S. R. Taboga, and M. T. V. de
Azeredo-Oliveira, “Meiotic nucleolar cycle and chromatoid
body formation during the rat (Rattus novergicus) and mouse
(Mus musculus) spermiogenesis,” Micron, vol. 39, no. 4, pp.
419–425, 2008.

[18] R. L. Peruquetti, S. R. Taboga, and M. T. V. de Azeredo-
Oliveira, “Characterization of Mongolian gerbil chromatoid
bodies and their correlation with nucleolar cycle during
spermatogenesis,” Reproduction in Domestic Animals, vol. 45,
no. 3, pp. 399–406, 2010.

[19] D. Hernandez-Verdun, “The nucleolus today,” Journal of Cell
Science, vol. 99, no. 3, pp. 465–471, 1991.

[20] S. A. Gerbi, A. V. Borovjagin, and T. S. Lange, “The nucleolus:
a site of ribonucleoprotein maturation,” Current Opinion in
Cell Biology, vol. 15, no. 3, pp. 318–325, 2003.

[21] F. M. Boisvert, S. Van Koningsbruggen, J. Navascués, and A.
I. Lamond, “The multifunctional nucleolus,” Nature Reviews
Molecular Cell Biology, vol. 8, no. 7, pp. 574–585, 2007.

[22] V. Sirri, S. Urcuqui-Inchima, P. Roussel, and D. Hernandez-
Verdun, “Nucleolus: the fascinating nuclear body,” Histochem-
istry and Cell Biology, vol. 129, no. 1, pp. 13–31, 2008.

[23] T. Pederson, “Proteomics of the nucleolus: more proteins,
more functions?” Trends in Biochemical Sciences, vol. 27, no.
3, pp. 111–112, 2002.

[24] W. Shou, J. H. Seol, A. Shevchenko et al., “Exit from mi-
tosis is triggered by Tem1-dependent release of the protein
phosphatase Cdc14 from nucleolar RENT complex,” Cell, vol.
97, no. 2, pp. 233–244, 1999.

[25] A. F. Straight, W. Shou, G. J. Dowd et al., “Net1, a Sir2-
associated nucleolar protein required for rDNA silencing and
nucleolar integrity,” Cell, vol. 97, no. 2, pp. 245–256, 1999.

[26] R. Visintin, E. S. Hwang, and A. Amon, “Cfi 1 prevents
premature exit from mitosis by anchoring Cdc14 phosphatase
in the nucleolus,” Nature, vol. 398, no. 6730, pp. 818–823,
1999.

[27] S. N. Garcia and L. Pillus, “Net results of nucleolar dynamics,”
Cell, vol. 97, no. 7, pp. 825–828, 1999.

[28] M. Carmo-Fonseca, L. Mendes-Soares, and I. Campos, “To be
or not to be in the nucleolus,” Nature Cell Biology, vol. 2, no.
6, pp. E107–E112, 2000.

[29] L. Montanaro, D. Treré, and M. Derenzini, “Nucleolus,
ribosomes, and câncer,” The American Journal of Pathology,
vol. 173, pp. 301–310, 2007.

[30] P. A. San-Segundo and G. Shirleen Roeder, “Pch2 links
chromatin silencing to meiotic checkpoint control,” Cell, vol.
97, no. 3, pp. 313–324, 1999.

[31] M. G. Ribeiro and S. R. Lima, Iniciação às Técnicas de
Preparação de Material Para Estudo e Pesquisa em Morfologia,
SEGRAC Editora e Gráfica Limitada, Belo Horizonte, Brazil,
2000.

[32] M. L. S. Mello, “Cytochemistry of DNA, RNA and nuclear
proteins,” Brazilian Journal of Genetics, vol. 20, no. 2, pp. 257–
264, 1997.

[33] W. M. Howell and D. A. Black, “Controlled silver-staining
of nucleolus organizer regions with a protective colloidal

developer: a 1-step method,” Experientia, vol. 36, no. 8, pp.
1014–1015, 1980.

[34] M. L. S. Mello and B. C. Vidal, Práticas de Biologia Celular,
FUNCAMP Editora Edgard Blücher LTDA, Campinas, Brazil,
1980.

[35] R. R. Ha and J. C. Ha, Integrative Statistics for Behavioral
Science, Pearson Custom Publishing, Boston, Mass, USA,
2007.

[36] J. H. Zar, Biostatiscical Analysis, Prentice Hall, New Jersey, NJ,
USA, 1999.

[37] A. D. Kligerman and S. E. Bloom, “Distribuition of F-bodies,
heterocromatin and nuclear organizers in the genome of the
central mudminnow,” Cytogenetics and Cell Genetics, vol. 18,
pp. 182–196, 1977.

[38] L. A. C. Bertollo and C. A. Mestriner, “The X1X2Y sex
chromosome system in the fish Hoplias malabaricus. II.
Meiotic analyses,” Chromosome Research, vol. 6, no. 2, pp. 141–
147, 1998.

[39] G. Cotta Pereira, F. Guerra Rodrigo, and J. F. David Ferreira,
“The use of tannic acid glutaraldehyde in the study of elastic
and elastic related fibers,” Stain Technology, vol. 51, no. 1, pp.
7–11, 1976.

[40] M. L. Watson, “Staining of tissue sections for electron
microscopy with heavy metals. II. Application of solutions
containing lead and barium,” The Journal of Biophysical and
Biochemical Cytology, vol. 4, no. 6, pp. 727–730, 1958.

[41] J. H. Venable and R. A. Coggeshall, “A simplified lead citrate
stain for use in electron microscopy,” The Journal of Cell
Biology, vol. 25, pp. 407–408, 1965.

[42] C. P. Leblond and Y. Clermont, “Spermiogenesis of rat, mouse,
hamster and guinea pig as revealed by the periodic acid-
fuchsin sulfurous acid technique,” The American Journal of
Anatomy, vol. 90, no. 2, pp. 167–215, 1952.

[43] R. Korstanjea, P. C. M. O’Brienc, F. Yangc et al., “Complete
homology maps of the rabbit (Oryctolagus cuniculus) and
human by reciprocal chromosome painting,” Cytogenetics and
Cell Genetics, vol. 86, pp. 317–322, 1999.

[44] M. Parvinen, “The chromatoid body in spermatogenesis,”
International Journal of Andrology, vol. 28, no. 4, pp. 189–201,
2005.

[45] N. Kotaja and P. Sassone-Corsi, “The chromatoid body: a
germ-cell-specific RNA-processing centre,” Nature Reviews
Molecular Cell Biology, vol. 8, no. 1, pp. 85–90, 2007.

[46] J. R. Head and C. K. Kresge, “Reaction of the chromatoid
body with a monoclonal antibody to a rat histocompatibility
antigen,” Biology of Reproduction, vol. 33, no. 4, pp. 1001–1008,
1985.

[47] S. Chuma, M. Hosokawa, K. Kitamura et al., “Tdrd1/Mtr-
1, a tudor-related gene, is essential for male germ-cell dif-
ferentiation and nuage/germinal granule formation in mice,”
Proceedings of the National Academy of Sciences of the United
States of America, vol. 103, no. 43, pp. 15894–15899, 2006.

[48] C. S. Pikaard, “Transcription and tyranny in the nucleolus:
the organization, activation, dominance and repression of
ribosomal RNA genes,” in The Arabidopsis Book, C. R.
Somerville and E. M. Meyerowits, Eds., American Society of
Plant Biologists, Rockville, Md, USA, 2002.

[49] M. Teruel, J. Cabrero, F. Perfectti, and J. P. M. Camacho,
“Nucleolus size variation during meiosis and NOR activity of
a B chromosome in the grasshopper Eyprepocnemis plorans,”
Chromosome Research, vol. 15, no. 6, pp. 755–765, 2007.

[50] O. Meikar, M. Da Ros, H. Korhonen, and N. Kotaja, “Chroma-
toid bodyand small RNAs in male germ cells,” Reproduction,
vol. 142, no. 2, pp. 195–209, 2011.



12 ISRN Cell Biology

[51] I. K. Takeuchi and Y. K. Takeuchi, “Ethanol-phosphotungstic
acid and bismuth staining of spermatid nucleoli in mouse
spermiogenesis,” Journal of Structural Biology, vol. 103, no. 2,
pp. 104–112, 1990.

[52] R. L. Peruquetti, S. R. Taboga, and M. T. V. De Azeredo-
Oliveira, “Nucleolar cycle and its correlation with chromatoid
bodies in the Tilapia rendalli (Teleostei, Cichlidae) spermatoge-
nesis,” Anatomical Record, vol. 293, no. 5, pp. 900–910, 2010.

[53] R. L. Peruquetti, S. R. Taboga, L. R. D. S. Santos, C. de Oliveira,
and M. T. V. de Azeredo-Oliveira, “Nucleolar cycle and
chromatoid body formation: is there a relationship between
these two processes during spermatogenesis of Dendropsophus
minutus (Amphibia, Anura)?” Micron, vol. 42, no. 1, pp. 87–
96, 2011.

[54] M. Biggiogera, S. H. Kaufmann, J. H. Shaper, N. Gas, F.
Amalric, and S. Fakan, “Distribution of nucleolar proteins B23
and nucleolin during mouse spematogenesis,” Chromosoma,
vol. 100, no. 3, pp. 162–172, 1991.

[55] M. Schmid, C. Loser, J. Schmidtke, and W. Engel, “Evo-
lutionary conservation of a common pattern of activity of
nucleolus organizers during spermatogenesis in vertebrates,”
Chromosoma, vol. 86, no. 2, pp. 149–179, 1982.

[56] F. Wachtler and A. Stahl, “The nucleolus: a structural and
functional interpretation,” Micron, vol. 24, no. 5, pp. 473–505,
1993.

[57] B. Lefèvre, “The nucleolus of the maternal gamete is essential
for life,” BioEssays, vol. 30, pp. 613–616, 2008.

[58] S. Berrı́os, R. Fernández-Donoso, J. Pincheira, J. Page, M.
Manterola, and M. Cristina Cerda, “Number and nuclear
localisation of nucleoli in mammalian spermatocytes,” Genet-
ica, vol. 121, no. 3, pp. 219–228, 2004.

[59] M. Guo, D. Davis, and J. A. Birchler, “Dosage effects on gene
expression in a maize ploidy series,” Genetics, vol. 142, no. 4,
pp. 1349–1355, 1996.

[60] T. Caspersson, Cell Growth and Cell Function, A Cytochemical
Study, WW Norton, New York, NY, USA, 1950.

[61] I. Schubert and G. Kunzel, “Position-dependent NOR activity
in barley,” Chromosoma, vol. 99, no. 5, pp. 352–359, 1990.

[62] K. Nakamoto, A. Ito, K. Watabe et al., “Increased expression of
a nucleolar Nop5/Sik family member in metastatic melanoma
cells: evidence for its role in nucleolar sizing and function,”
American Journal of Pathology, vol. 159, no. 4, pp. 1363–1374,
2001.

[63] W. Mosgoeller, “Nucleolar ultrastructure in vertebrates,” in
The Nucleolus, M. O. J. Olson, Ed., Kluwer, New York, NY,
USA, 2004.

[64] E. Ünal, B. Kinde, and A. Amon, “Gametogenesis eliminates
age-induced cellular damage and resets life span in yeast,”
Science, vol. 332, pp. 1554–1557, 2011.

[65] R. Paniaguea, M. Nistal, P. Amat, and M. C. Rodriguez, “Ultra-
structural observations on nucleoli and related structures
during human spermatogenesis,” Anatomy and Embryology,
vol. 174, no. 3, pp. 301–306, 1986.

[66] R. A. Hess, L. A. Miller, J. D. Kirby, E. Margoliash, and
E. Goldberg, “Immunoelectron microscopic localization of
testicular and somatic cytochromes c in the seminiferous
epithelium of the rat,” Biology of Reproduction, vol. 48, no. 6,
pp. 1299–1308, 1993.

[67] C. M. Haraguchi, T. Mabuchi, S. Hirata et al., “Chromatoid
bodies: aggresome-like characteristics and degradation sites
for organelles of spermiogenic cells,” Journal of Histochemistry
and Cytochemistry, vol. 53, no. 4, pp. 455–465, 2005.

[68] J. T. Soley, “Centriole development and formation of the
flagellum during spermiogenesis in the ostrich (Struthio
camelus),” Journal of Anatomy, vol. 185, no. 2, pp. 301–313,
1994.

[69] X. M. Tang, M. F. Lalli, and Y. Clermont, “A cytochemical
study of the Golgi apparatus of the spermatid during spermio-
genesis in the rat,” American Journal of Anatomy, vol. 163, no.
4, pp. 283–294, 1982.

[70] H. Walt and B. L. Armbruster, “Actin and RNA are compo-
nents of the chromatoid bodies in spermatids of the rat,” Cell
and Tissue Research, vol. 236, no. 2, pp. 487–490, 1984.

[71] G. Aumuller and J. Seitz, “Immunocytolchemical localization
of actin and tubulin in rat testis and spermatozoa,” Histochem-
istry, vol. 89, no. 3, pp. 261–267, 1988.

[72] M. D. Andonov and G. N. Chaldakov, “Morphological
evidence for calcium storage in the chromatoid body of rat
spermatids,” Experientia, vol. 45, no. 4, pp. 377–378, 1989.

[73] B. Sud, “Morphological and histochemical studies of the
chromatoid body and related elements in the spermatogenesis
of rat,” Quarterly Journal of Microscopical Science, vol. 102, pp.
273–292, 1961.

[74] S. Yokota, “Historical survey on chromatoid body research,”
Acta Histochemica et Cytochemica, vol. 41, no. 4, pp. 65–82,
2008.



Submit your manuscripts at
http://www.hindawi.com

Hindawi Publishing Corporation
http://www.hindawi.com Volume 2014

 Anatomy 
Research International

Peptides
International Journal of

Hindawi Publishing Corporation
http://www.hindawi.com Volume 2014

Hindawi Publishing Corporation 
http://www.hindawi.com

 International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporation
http://www.hindawi.com Volume 2014

Molecular Biology 
International 

Hindawi Publishing Corporation
http://www.hindawi.com

Genomics
International Journal of

Volume 2014

The Scientific 
World Journal
Hindawi Publishing Corporation 
http://www.hindawi.com Volume 2014

Hindawi Publishing Corporation
http://www.hindawi.com Volume 2014

Bioinformatics
Advances in

Marine Biology
Journal of

Hindawi Publishing Corporation
http://www.hindawi.com Volume 2014

Hindawi Publishing Corporation
http://www.hindawi.com Volume 2014

Signal Transduction
Journal of

Hindawi Publishing Corporation
http://www.hindawi.com Volume 2014

BioMed 
Research International

Evolutionary Biology
International Journal of

Hindawi Publishing Corporation
http://www.hindawi.com Volume 2014

Hindawi Publishing Corporation
http://www.hindawi.com Volume 2014

Biochemistry 
Research International

Archaea
Hindawi Publishing Corporation
http://www.hindawi.com Volume 2014

Hindawi Publishing Corporation
http://www.hindawi.com Volume 2014

Genetics 
Research International

Hindawi Publishing Corporation
http://www.hindawi.com Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporation
http://www.hindawi.com

Nucleic Acids
Journal of

Volume 2014

Stem Cells
International

Hindawi Publishing Corporation
http://www.hindawi.com Volume 2014

Hindawi Publishing Corporation
http://www.hindawi.com Volume 2014

Enzyme 
Research

Hindawi Publishing Corporation
http://www.hindawi.com Volume 2014

International Journal of

Microbiology