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Oogenesis and ovarian development in the freshwater Crab Sodhiana iranica

(Decapoda: Gecarcinuaidae) from the south of Iran

Article  in  Tissue and Cell · December 2014

DOI: 10.1016/j.tice.2014.11.006

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 Tissue and Cell 47 (2015) 213–220

Contents lists available at ScienceDirect

Tissue and Cell

journal homepage: www.elsevier.com/locate/tice

Oogenesis and ovarian development in the freshwater Crab

Sodhiana iranica (Decapoda: Gecarcinuaidae) from the south of Iran
S. Sharifian a,∗ , E. Kamrani a , M. Safaie a , S. Sharifian b
a
Hormozgan University, Faculty of Science, Bandar abbas, Iran
b
Chabhar Maritime University, Faculty of Marine Sciences, Chabhar, Iran

a r t i c l e i n f o a b s t r a c t

Article history: In this study, the reproductive biology of female freshwater crab Sodhiana iranica, oogenesis and ovarian
Received 24 June 2014 development were described. An H-shaped ovary consisting of a pair of long ovarian sacs connected by
Received in revised form a narrow bridge tube was located in the cephalothorax on the dorsal side of the stomach. Females at dif-
18 November 2014
ferent stages of ovarian development were anesthetized and their ovaries were removed, photographed,
Accepted 25 November 2014
fixed, and processed for histological examination. Based on the light microscopic observations of cells’
Available online 18 December 2014
sizes, chromatin patterns, and amount of lipid vesicles, the female germ cells could be classified into seven
different stages: (1) oogonia (Oog), (2) primary oocytes (pOc), (3) early previtellogenic oocyte (Oc1), (4)
Keywords:
Oogenesis late previtellogenic oocyte(Oc2), (5) early vitellogenic oocyte (Oc3), (6) late vitellogenic oocyte (Oc4), and
Ovarian development (7) mature oocyte (mOc). Oog are small oval-shaped cells with irregular-shaped nuclei. Oog undergo first
Sodhiana iranica meiotic division to become primary oocytes. The primary oocytes are small oval-shaped cells with large
The South of Iran nuclei. The secondary oocytes derived from 2nd meiosis and comprise five steps. Four ovarian develop-
ment stages were found for females based on the number and types of oocytes present in each stage:
spent I (Spent), II (Proliferative) and III (Premature) and stage IV (Mature). The ovaries, macroscopically,
varied in size and color during each developmental stage and, microscopically, the ovarian stages dif-
fered in proportion oogonia, and the secondary oocytes. During ovarian stage I, ovary contains primarily
oogonia, primary oocytes and Oc1. In stage II, contains mainly Oc1, Oc2, and Oc3, while in stage III the
predominant cells are Oc4. Mature oocytes appear synchronously in stage IV.
© 2014 Elsevier Ltd. All rights reserved.

1. Introduction and the Mandibulata, have been distinguished based on various

Most of the 6800 species of brachyurans are marine, but a
surprising one-fifth of all crabs on earth are strictly freshwater.
This makes freshwater crabs the largest assemblage within the
Brachyura, which in turn is the most species-rich of all decapods
crustacean groups (Cumberlidge et al., 2009; Ng et al., 2008). Fresh
water crab Sodhiana iranica, is gecarcinid crab which, so far, has
been reported only from Bastak, in southern Iran (Sharifian et al.,
2014). The species inhabits a freshwater spring located in a semi-
mountainous area in Iran (Eellod Area), covered by dense stands
of common reeds and salt cedar trees in the periphery, with mats
of algae on the bottom. S. iranica is fourth species of Sodhiana (Yeo
and Ng, 2012), and is the second one known from Iran.
Two different structural and functional types of ovaries, corre-
sponding to the two major extant arthropod taxa, the Chelicerata
∗ Corresponding author. Tel.:+98 9375982750.
E-mail address: sharifian sana@yahoo.com (S. Sharifian).
descriptions of arthropod ovaries (Makioka, 1988). The gonads of
female crustaceans undergo a sequence of morphological trans-
formation during each reproductive cycle. Such changes exhibit a
number and classes of oocytes that are undergoing various steps
of cellular differentiation. In decapods crustaceans, ovarian devel-
opment is accompanied by changes in color and size during the
reproductive cycle (Arculeo et al., 1995; Cavalli et al., 1997). These
changes are due to the deposition of yolk material in the oocytes,
which results in a rapid increase in oocyte diameter (Sagi et al.,
1995; Tsukimura, 2001)’ and color changes due to the carotenoid
components with specific color changes each being related to a
new maturation stage (Arculeo et al., 1995). These processes play
an important role during embryogenesis (Dall et al., 1995; Linan-
Cabello et al., 2002).
Decapod ovaries are paired organs located in the cephalotho-
rax, which vary in shape, size, and color. Although the macroscopic
characteristics of the ovarian is an important element to char-
acterize the stages of the ovarian development, for an adequate
comprehension of the female reproductive cycle, therefore, it is

http://dx.doi.org/10.1016/j.tice.2014.11.006
0040-8166/© 2014 Elsevier Ltd. All rights reserved.
214 S. Sharifian et al. / Tissue and Cell 47 (2015) 213–220
necessary to describe the maturation of germ cells in the ovaries
(Shinozaki-Mendes et al., 2012).So far, the structure of the female
reproductive of brachyuran crabs have been reported for several
crab species, such as Ranina ranina (Minagawa et al., 1993), Potamon
dehaani (Ando and Makioka, 1999), Maja brachydactyla (Rotllant
et al., 2007), Goniopsis cruentata (Souza and Silva, 2009), Cardis-
oma guanhumi (Souza et al., 2012). The ovarian development has
been investigated based on macroscopic changes in color and size
in decapods crustaceans (López-Greco and Rodríguez, 1999; Santos
and Negreiros-Fransozo, 1999; Swiney and Shirley, 2001; Flores
et al., 2002; Castiglioni and Negreiros-Fransozo, 2006). Ovarian
cycle have also been described microscopically for Brachyura (Joshi
and Khanna, 1982; Castiglioni et al., 2007; Rotllant et al., 2007; Gre-
gati et al., 2010; El-Sherif et al., 2012; Silva et al., 2012). The steps of
oogenesis were also have been studied by groups of investigators
(Meeratana and Sobhon, 2007; Kodama et al., 2009; Shinozaki-
Mendes et al., 2012).
Basic studies on the morphology of reproductive systems are
essential to define the reproductive cycles of animal species. The
development of the ovary is remarkable and the study of this
process is essential to the understanding of reproduction. The
information on ovarian development of a given species is very
important to adequately understand its population dynamics, from
the aspects of reproduction, such as growth characteristics and dif-
ferences among populations. Thus, this information is crucial for
the development and implementation of management strategies.
In the present study, we examined a small freshwater crab, S.
iranica, which produces a small number of large eggs in each spawn-
ing. The aim of this study was to gain a better understanding of the
oogenesis and ovarian development of female S. iranica, through
macroscopic and microscopic descriptions of the reproductive sys-
tem. This crab was chosen since it as a new and threatened species is
requirement of information on all aspects in particular the repro-
duction. In addition, detailed classification of oocyte are still not
clearly defined for gecarcinucid freshwater crab.
2. Materials and methods
2.1. Collection of data
Adult female of S. iranica were handily collected from Eelood
freshwater spring, located in Eelood Area of Hormozgan Province
in the south of Iran (27◦ 13′ N–54◦ 40′ E). The climate of this zone is
tropical with an average yearly temperature of 25 ◦ C. The samp-
ling was done in September 2013. The specimens were brought to
the laboratory alive and anesthetized by cooling for 30 min. Cara-
pace width (CW) was measured using calipers with a precision of
0.01 mm, ranging from 29.57 to 34.86 (mean of 32.42 mm). Termi-
nology is based on Ng (1988). The dorsal portion of the carapace
was removed for the exposure of the reproductive organs, which
were examined and removed from the thoracic cavity. Gonads
were measured in a standard electric balance of 0.0001 g accu-
racy. For the histological description, 20 gonads of mature female
were randomly selected. The stages of gonad development were
investigated using two methods: macroscopic examination of the
consistence, volume and coloration of the reproductive system in
relation to thoracic cavity and microscopic examination of cells
types (Shinozaki-Mendes et al., 2012).
The ovarian sections were prepared according to the method
described by Reddy et al. (2006). The ovaries were fixed in
Bouin’s fluid (picric acid/formaldehyde/acetic acid; 75:25:5) for
24 h, immediately after excise and dehydrated with ascending alco-
holic series, cleared in xylene and then embedded in paraffin wax
(m.p. 56–58 ◦ C). The sections were made in 5–7 ␮m thickness,
stained with Hematoxylin followed by counter staining in alcoholic
Eosin. The diameter of 40 oocytes from each ovary was measured
using an ocular micrometer under compound microscope (Olym-
pus).
2.2. Data analysis
The gonadosomatic index was calculated according to the fol-
lowing formula:
GSI = GW(gr)/BW(gr) × 100
where GW is gonad weight and BW is body weight, respectively.
Mean diameter ± SD of 40 oocytes for each ovarian stage and
mean diameter ± SD of oocyte during oogenesis were measured.
Also, GSI (%) for each ovarian stage was measured. Differences of
diameter oocytes among ovarian stages were tested using one-way
ANOVA followed by Tukey’s post-hoc test; differences of diame-
ter oocytes during oogenesis and differences of GSI (%) for each
ovarian stage were tested using Nonparametric test followed by
Kruskal–Wallis test. The significance level was set at ˛ = 0.05 (Zar,
1996). Mean ± standard error (SE) is presented through the text.
3. Results
3.1. Stages of the ovarian development
According to the macroscopic examination of the female gonads
of S. iranica, which could be clearly seen through size and color
gonads, the ovaries could be differentiated into four different
ovarian developmental stages (Fig. 1A–D). These differences were
confirmed by microscopic observations (secondary oocytes diame-
ter significantly differed among stages, (ANOVA, F = 54.73; p < 0.05).
Also, the GSI (%) was significantly different among Stages of the
ovarian development (2 (3) = 41.13; p < 0.05).
The ovarian tissue right after spawning, appears loose, and con-
tains large subcapsular space and germinal cells appear in the
central area of the ovary (Fig. 2A and B). Thickening and folding
of the ovarian capsule is apparent around the ovary. Thereinafter,
the four distinguished stages were described as follows:
3.1.1. Stage I (Spent)
Ovaries are small, smooth, and creamy white in color (Fig. 1A,
left). Collapsed oogenic pouches are found in at the periphery of
ovary, indicating that the spawning has taken place. Ovarian wall
becomes thick and a lot of germinal zone was separately located
throughout the ovary and each consists of numerous oogonia and
primary oocytes (Fig. 1A, right). Previtellogenic oocytes (Oc1–Oc2)
in numbers of much less than the oogonia are predominantly seen
at centeral region than periphery of ovary. There was a few of
residual vitellogenic oocytes (about 10–12 in numbers) also at
peripheral region of ovary, these are the oocytes that are matured
but not released, thereby entering a process of re-absorption by the
ovary. Fc are dispersed at the periphery of ovary in larger num-
bers than germ cells, forming more than one stratum between
the germ cells. There is also a greater space between the sexual
cells, which is filled in by Fc (Fig. 6E). In more flaccid ovaries,
the Fc and Oc1 begin to reorganize to occupy the empty spaces.
Mean diameter of oocytes is 172.85 ± 15.84 ␮m. The GSI (%) was
0.17 ± 0.02.
3.1.2. Stage II (Proliferative)
Ovaries increase in size and are colored yellow. Externally they
appear granular owing to the preponderance of vitellogenic oocytes
in them (Fig. 1B, left). The germinal zone contains a few residual
oogonia and primary oocytes, peripheral zone, with (Oc1, Oc2),
is located more internally, while (Oc3, Oc4) are situated in the
 S. Sharifian et al. / Tissue and Cell 47 (2015) 213–220 215

Fig. 1. Stages of ovarian development identified macroscopically (left) and microscopically (right). (A) Stage I (spent). (B) Stage II (proliferation). (C) Stage III (premature),
and (D) stage IV (mature). Ov: ovary; sOp: spent oogenesis pouch; Cap: fibromuscular ovarian capsule; Gz: germinal zone.

more peripheral region. Follicular cells increase in number and seen. Mean diameter of oocytes is 297.32 ± 13.95 ␮m. The GSI (%)
become more apparent (Fig. 1B, right). Mean diameter of oocytes was 1.21 ± 0.16.
is 256.42 ± 14.89 ␮m. The GSI (%) was 0.57 ± 0.02.

3.1.4. Stage IV (Mature)

3.1.3. Stage III (Premature)
Ovaries become enlarged, convoluted, and pale orange in color.
Large orange oocytes bulge out on the surface of ovaries (Fig. 1C,
left). Most of the oocytes are in vitellogenesis (Oc3, Oc4) (Fig. 1C,
right). Previtellogenic oocytes (Oc1, Oc2) are rare in the ovaries.
The follicular cells elongate to spindle shape and are less frequently
Ovaries attain a maximum size and deep orange mature
oocytes are visible through the thin ovarian capsule (Fig. 1D,
left). Mature oocytes are present at thorough the ovary (Fig. 1D,
right), and other oocytes (Oc1–Oc4) are absent. Follicular cells are
decreasing in number, while those remaining appear as thin spin-
dle shapes closely surrounding each oocyte. The oogensis pouch
216 S. Sharifian et al. / Tissue and Cell 47 (2015) 213–220

Fig. 2. (A) Ovary right after spawnig with spent ovarian pouch (sOp) and thick and twisted fibromuscular ovarian capsule (Cap). (B) Higher magnification of ovary after
spawning showing highly thick fibromuscular ovarian capsule (Cap) and numerous oogonia (Oog) and follicular cell (Fc) with staining less than oogonia.

boundaries are hardly discernible. Mean diameter of oocytes is are characterized by the nuclei containing dense small cords of
645.01 ± 42.17 ␮m. The GSI (%) was 16.29 ± 5.41. heterochromatin and the nucleolus is not visible. Their very thin
rim of cytoplasm appear blue with H&E stain (Figs. 5A and 4). Mean
3.2. Morphological features of female reproductive system diameter of primary oocytes is 14.83 ± 0.78 ␮m.

The female reproductive organ of S. iranica was an H-shaped 3.4.3. Secondary oocytes (sOc)
ovary consisting of a pair of longitudinal ovarian sacs connected This phase of developing oocytes is undergoing 2nd meiosis and
by a narrow bridge tube which located in the cephalothorax on they are classified into five steps, which include Oc1, Oc2, Oc3, Oc4,
the dorsal side of the stomach. Depending on the female body size, and mOc.
each ovarian sac possesses from approximately 70 to more than
100 oocytes of various steps. Each of these is enclosed in its own
3.4.4. Early previtellogenic oocyte (Oc1)
oogenetic pouch.
In the stage, oocyte increases in volume and acquires a large
amount of basophilic cytoplasm. The nucleus is characterized by
3.3. Internal structure of the ovary
the presence of the intensely bluestained small blocks of hetero-
chromatin, while the rest of the nucleoplasm appears very light.
Histologically, the developing ovary was tightly surrounded by
The nucleolus is small. These cells are found in the periphery of the
ovarian capsule made of fibromuscular tissue. The ovarian wall
germinal zone. They are surrounded by spindle-shaped follicular
consists of a single layer of the ovarian epithelium which invagi-
cells (Figs. 5A and 4). Mean diameter of Oc1 is 78.09 ± 4.64 ␮m.
nated and evaginated to form a number of oogenetic pouches
(Figs. 3 and 4) in various sizes. Each oogenetic pouch contains only
a developing oocyte. The size of the oogenetic pouch depends on
the size of the oocyte it contains. Oogonia and primary oocytes
are localized in the germinal zone, special germ areas of the ovar-
ian epithelium, were located separately at the bases of oogentic
pouches throughout the ovary (Figs. 3 and 4).

3.4. Steps of differentiating oocytes (oogenesis)

The oogonia passes through different maturational stages before

it becomes the mature oocyte. This process involves changes in
the nucleus and cytoplasm. Oocytes diameter significantly differed
during steps of differentiating oocytes (2 (6) = 155.44; p < 0.05):

3.4.1. Oogonia (Oog)

The Oog is an ovoid cell that contains an irregularly shaped
nucleus with deep bluestained heterochromatin. It is notable that
the nucleolus is not noticeable in this stage, while the cytoplasm is
stained faint blue with H&E. The oogonia form clusters in the germi-
nal zone. The oogonia are dividing mitotically, and their daughter
cells increase in size while entering the 1st meiosis (Figs. 5A and
4). Mean diameter of oogonia is 10.96 ± 0.16 ␮m.
Fig. 3. Schematic drawing of cross section of an ovarian sac of S. iranica show-
ing fibromuscular ovarian capsule (Cap); ovarian epithelium (Ep); germinal zone
3.4.2. Primary oocytes (pOc)
(Gz) containing oogonia (Oog) and primary oocytes (pOc); oogenesis pouches (Op)
The oogonia which enter into prophasic activities are termed containing previtellogenic oocytes (Oc1, Oc2), vitellogenic oocytes (Oc3, Oc4) and
as primary oocytes. These cells are undergoing 1st meiosis, and marure oocytes (mOc); central ovarian lumen (Lc).
 S. Sharifian et al. / Tissue and Cell 47 (2015) 213–220 217

Fig. 4. Cross sections of stage II ovaries, showing fibromuscular ovarian capsule (Cap); ovarian epithelium (Ep); oogenesis pouches (Op) containing (Oc1, Oc2, Oc3, Oc4, mOc)
and germinal zone (GZ) containing (Oog, pOc).

Fig. 5. Section of steps of differentiating oocytes: (A) ovoid shaped oogonia (Oog) with an irregularly shaped nucleus, primary oocytes (pOc) with large nuclei and unvisible
nucleolus, early previtellogenic oocytes (Oc1) with nucleus containing blocks of heterochromatin and small nucleolus, basophilic cytoplasm surrounded by spindle-shaped
follicular cells. (B) Late previtellogenic oocyte (Oc2) with the decrease of nucleo-cytoplasmic ratio and nucleus with blocks of heterochromatin and prominent nucleolus.
(C) Early vitellogenic oocytes (Oc3) with centrally round nucleus and eosinophilic cytoplasm containing some yolk vesicles distributed randomly at the periphery. (D) Late
vitellogenic oocytes (Oc3) with entirely acidophilic cytoplasm and yolk vesicles occupying most of the cytoplasm. (E) Mature oocytes (mOc) with highly acidophilic cytoplasm
and filled with large yolk vesicles. N:. nuclei; No: nucleolus; Ep: ovarian epithelium; Yv: yolk vesicles; Fc: follicular cell.
218 S. Sharifian et al. / Tissue and Cell 47 (2015) 213–220

Fig. 6. Higher magnification of (A) Late previtellogenic oocyte (Oc2) with ellipsoid shape follicular cell (type I). (B) Early vitellogenic oocyte (Oc3), type I follicular cell is
more flattened and in close apposition to the oocyte surface, while type II is more prominent and more frequently seen, the vitelline envelope (Ve) appears as a very thin
line closes to the oocyte membrane (Om). (C) Dispersed follicular cell at the periphery of spent ovarian pouch (sOp) after spawnig. (D) Fibromuscular ovarian capsule (Cap)
surrounded developing ovary. (E) type II of follicular cell with more ovoid nuclei than type I, oogonia (Oog) with an indistinct nucleus and deeply stained in ovarian stage I.
Fc1: type I follicular cell; Fc2—type II follicular cell; N: nuclei; No: nucleolus.

3.4.5. Late previtellogenic oocyte (Oc2) 3.4.8. Mature oocyte (mOc)

Oc2 is characterized by the increase in cell size and the decrease
of nucleo-cytoplasmic ratio. The cytoplasm is enlarged and exhibits
more intensely blue staining with H&E. The nucleus is character-
ized by the presence of dense blocks of heterochromatin scattering
throughout. The nucleolus becomes prominent. The follicular cells
surrounding Oc2 are more elongated as well as increased in
number and size (Figs. 5B and 6A). Mean diameter of Oc2 is
213.41 ± 10.77.
3.4.6. Early vitellogenic oocyte (Oc3)
Oc3 is characterized by the decrease of bluish stain (basophilia)
and the increase of reddish stain (eosinophilia) in the cyto-
plasm. The cytoplasm also contains some yolk vesicles, which
are distributed randomly at the periphery (Figs. 5C and 6B). The
round nucleus is enlarged and centrally located, and exhibits
numerous and variable size heterochromatin blocks scattering
throughout. This step of oocyte has increasing number of type II
follicular cells with oval nuclei. Meanwhile, type I follicular cells
are tightly adhered to the surface of each oocyte and remain
unchanged from their previous stage. Mean diameter of Oc3 is
339.83 ± 4.62 ␮m.
3.4.7. Late vitellogenic oocyte (Oc4)
With further growth of oocyte, cytoplasm becomes entirely
acidophilic. The acidophilia at the periphery is more intense
in comparison to the area surrounding nucleus. Yolk vesi-
cles occupy most of the cytoplasm including the perinuclear
region. The follicular cells type I and II are more apparent and
more elongated in shape (Fig. 5D). Mean diameter of Oc4 is
496.29 ± 17.40 ␮m.
The mOc is characterized by a remarkable increase in cell size,
more than 10 times the early previtellogenic oocytes. The cyto-
plasm becomes highly acidophilic and filled with large yolk vesicles.
The nucleus contains almost entirely euchromatin. The nuclear
membrane is not apparent and frequently disappears. Type I and
type II follicular cells surrounding mOc are fully elongated (Fig. 5E).
Mean diameter of is mature oocyte 841.73 ± 17.08 ␮m.
3.5. Follicular cells
Follicular cells found free, either forming clusters or surround-
ing cells, they have little cytoplasm and the nucleus is strongly
basophilic.
The follicular cells are classified into two types. Type I follicular
cells (Fc1) exhibits ellipsoid shape, and surround Oc2, Oc3, and Oc4.
They become more elongated and flattened around the mOc stage,
and exhibit prominent spindle-shaped nuclei with deeply stained
chromatin. Fc1 adhere closely to oocytes’ surface (Fig. 6A, B and
E). Type II follicular cells (Fc2) first appear during Oc2 step. They
exhibit less change in shape and size from Oc2 to Oc4 steps. Fc2
have lighter stained and more ovoid nuclei, and they form an outer
layer next to Fc1 (Fig. 6B and E). They proliferate and aggregate in
the spaces between oocytes during Oc3 and Oc4 steps. During the
mOc step, the nuclei of both types of follicular cells are quite similar.
However, the Fc1 nucleus is more flattened into spindle shape and
the Fc2 nucleoplasm becomes darker.
4. Discussion
The present investigation of S. iranica showed that the devel-
oping ovary was tightly surrounded by ovarian capsule made of
 S. Sharifian et al. / Tissue and Cell 47 (2015) 213–220
fibromuscular tissue. The ovarian wall consists of a single layer of
the ovarian epithelium which twisted to form a number of ooge-
netic pouches and each oogenetic pouch contains only a developing
oocyte. Ando and Makioka, (1998) recorded the same structure of
ovary for Procambarus clarkii. This simple ovary has some distinct
mandibulate-type features, such as oocytes growing in the ovar-
ian lumen in the oogenetic pouches.Macroscopic and microscopic
analyses of S.iranica showed four ovarian colors during the ovarian
development. These colors were white, yellow, pale orange, and
deep orange. Some authors (El-Sherif et al., 2012; Fyhn and Costlow,
1977) recorded the color changes in the ovary during the matura-
tion of crustaceans and suggested that this phenomenon was due
to the synthesis of carotenoid pigments.
Analysis of the S. iranica ovarian development showed that the
macroscopic classification of ovary is closely related to its devel-
opment and cellular organization. The gradual increase in the size
of the ovarian cells has been attributed to the deposit of lipid in
the ovaries during vitellogenesis (Revathi et al., 2012; Castiglioni
et al., 2007; Gregati et al., 2010). This means that a sheath of follicle
cells which observed around pre-vitellogenic oocytes of S. iran-
ica, could be involved in the process of vitellogenesis. Varadarajan
and Subramoniam (1980) stated that in Clibanarius clibanarius, the
lipoprotein from extraovarian sources when linked to carotenoid
pigments may serve in facilitating the entry of lipoproteins into
oocytes. This observation leads to the idea of extraoocytic produc-
tion of this type of yolk and these cells which are arranged at the
periphery of the oocytes are possibly used for exchanging different
substances that form part of the nutrient stock of the egg (protein,
vitellogenin, and lipids).
In S. iranica, stages of developing oocyte is divided into seven
stages. Shinozaki-Mendes et al. (2012) divided developing oocyte
of C. guanhumi based on shape, nucleus–cytoplasm ratio, and reac-
tion to stains into four stage. Developing oocytes of Macrobrachium
rosenbergii were divided into five stages based on size, yolk accumu-
lation, and follicular cell stage (O’Donovan et al., 1984). Joshi and
Khanna (1982) divided developing oocyte of Potamon koolooense
based on changing in the nucleus and cytoplasm into seven stages.
The diameter of the oocytes appears to be characteristic for each
species, with a broad range of values. What is clear, however, show-
ing an increase in diameter throughout the development process.
For a better understanding of the stages of germ cell development,
it is important to link the somatic content of the ovaries (Souza and
Silva, 2009). According these authors, the fundamental role the FC
play in gonad maturation is the synchronic maturation, thereby
ensuring the amount of MoC necessary for spawning. In addition,
Chang and Shih (1995) reported that the format and size of these
cells are related to the biosynthetic activity. In S.iranica, Oog dif-
ferentiate into primary oocytes is within the germinal zone. The
deposition of yolk in the Oc3 is slow while the oocytes undergo
gradual increase in size. At this stage, yolk protein probably is pro-
duced endogenously (Van Herp, 1992; Tsutsui, 2000). In contrast,
Oc4 and mOc rapidly increases in size and acidophilia due to accu-
mulation of yolk protein which could be derived from extra-ovarian
sources (Chang and Shih, 1995; Sagi et al., 1995). The hepatopan-
creas and adipose tissue were proposed as exogenous sources of
yolk proteins (Tsutsui, 2000; Jasmani et al., 2004). Once the oocytes
reach maturation they become highly enlarged, and they probably
exert strong pressure in the ovary, so much that the thin connec-
tive tissue surrounding oogenic pouches are broken, allowing the
oocytes to be released into the ovarian lumen and pass into the
oviduct.
The ovarian development of S. iranica can be easily determined
externally by direct observation of size and color gonads. This
seems to be similar to that observed in other freshwater crab such
as P. dehaani (Joshi and Khanna, 1982), Sylviocarcinus pictus (Silva
et al., 2012). According to the microscopical examination, El-Sherif
219
et al. (2012) divided developing ovary of Oratosquilla massaven-
sis into six stages. The ovarian development was grouped into five
developmental stages in Uca rapax (Castiglioni et al., 2007). While
Gregati et al. (2010) proposed only four stages for Stenopus hispidus.
Using histological criteria, we also divide the ovarian development
into four stages: stage I (Spent), stage II (Proliferative), stage III (Pre-
mature) and stage IV (Mature). Subsequent studies indicated that
some of these stages could be grouped due to similarities in oocyte
diameter and its chemical composition (Quintero and Gracia, 1998;
Dumont and D’Incao, 2004). The microscopic analyses of four stages
showed a progressive increase in the size of cells over the ovarian
cycle. This seems to be similar to those observed in other crus-
taceans such as P. koolooense (Joshi and Khanna, 1982), S. hispidus
(Gregati et al., 2010), S. pictus (Silva et al., 2012), and O. massavensis
(El-Sherif et al., 2012). The microscopic analyses showed that the
ovarian wall decreased in thickness during the ovarian develop-
ment stages of S. iranica. This was noticed previously by Silva and
Cruz-Landim (2006) who stated that the thickening of the ovarian
wall of Panulirus echinatus and P. laevicauda were reduced, indicat-
ing that they are being stretched to accommodate the growth of
germ cells.
At stage I (Spent stage), ovaries of S. iranica are characterized by
thick ovarian wall and discharged oogensis pouches of ovary. Col-
lapsed ovarian pouches is surrounded with only strands of follicular
cells. Most of the follicular cells return to their original ovoid shape.
This has also been observed for M. rosenbergii (Meeratana and
Sobhon, 2007), Stenopus hispidus (Gregati et al., 2010) and C. guan-
humi (Shinozaki-Mendes et al., 2012). The presence of yolk vesicles
in significant quantities coincides, therefore, with the appearance
of the yellow color in stage II (proliferative) and also the accumula-
tion in significant numbers of previtellogenic oocytes (Oc1, Oc2) is
the distinguishing characteristic of (stage II). The yellow color in S.
hispidus ovaries was also associated to the increase of vesicles yolk
as mentioned by Gregati et al. (2010). Progressive increase in size
of premature (stage III) and mature (stage IV) ovaries is due to the
increased numbers of vitellogenic oocytes (Oc3, Oc4, mOc), whose
sizes are enlarged significantly due to the uptake of yolk protein
from exogenous sources.
In summary, the ovarian development stages of freshwater crab
S. iranica can be determined on the bases of morphological appear-
ance of the ovaries, and the histological structure of the female
ovary. When external morphological characteristics of the ovaries
were compared to histological descriptions, it was possible to
observe modifications that characterize the process in different
developmental stages throughout the ovarian cycle and, conse-
quently, the macroscopic classification of the ovarian stages agrees
with the modifications of the reproductive cells.
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