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Theriogenology 79 (2013) 709–724
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Theriogenology

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Female germ cell renewal during the annual reproductive cycle in
Ostariophysians fish

Daniel Dantas Wildner a,b, Harry Grier c, Irani Quagio-Grassiotto d,*

aGraduate Program on the Cell and Structural Biology, Institute of Biology, University of Campinas - UNICAMP, Campinas, São Paulo, Brazil
bDepartment of Morphology, Institute of Bioscience, Sao Paulo State University - UNESP, Botucatu, São Paulo, Brazil
c Florida Fish and Wildlife Research Institute, St. Petersburg, Florida, USA
dDepartment of Morphology, Institute of Bioscience, Sao Paulo State University - UNESP, Botucatu, São Paulo, Brazil
a r t i c l e i n f o

Article history:
Received 17 February 2012
Received in revised form 22 November 2012
Accepted 22 November 2012

Keywords:
Reproductive phase
Reproductive cycle
Cell renewal
Oogonia
Early prophase oocyte
* Corresponding author. Tel.: þ55 38800468.
E-mail addresses: iraniqg@ibb.unesp.br, iraniqg@

Grassiotto).

0093-691X
http://dx.doi.org/10.1016/j.theriogenology.2012.11.02

� 2013 Elsevier Inc. Open access under the Else
a b s t r a c t

The objective was to characterize female germ cell renewal during the annual reproductive
cycle in two species of ostariophysian fish with distinct reproductive strategies: a siluri-
form, Pimelodus maculatus, in which oocyte development is group synchronous and the
annual reproductive period is short; and a characiform, Serrasalmus maculatus, with
asynchronous oocyte development and a prolonged reproductive period. These repro-
ductive strategies result in fish determinate and indeterminate fecundity, respectively.
Annual reproductive phases were determined by biometric and histologic analysis of
gonads and interpreted according to new proposals for phase classification and stages of
oocyte development (with special attention to germinal epithelium activity). Histologi-
cally, there were two types of oogonia in the germinal epithelium: single oogonia and
those in mitotic proliferation. Oogonial proliferation and their entry into meiosis resulted
in formation of cell nests (clusters of cells in the ovarian lamellae). Morphometric analysis
was used to estimate germ cell renewal. Based on numbers of single oogonia in the
lamellar epithelium, and nests with proliferating oogonia or early prophase oocytes
throughout the annual reproductive cycle, oogonial proliferation and entrance into meiosis
were more intense during the regenerating phase and developing phase, but decreased
sharply (P < 0.05) during the spawning-capable phase. Oogonial proliferation gradually
recovered during the regressing phase. We concluded that, independent of species or
features of the reproductive cycle, germ cell renewal occurred during the regenerating
phase, ensuring availability of eggs for the spawning event.

� 2013 Elsevier Inc. Open access under the Elsevier OA license.
1. Introduction

In most Teleostei, reproduction is an annual, cyclic
event. During each breeding season, thousands to millions
of eggs are produced during successive spawning events,
depending on species, life history, and body size. In contrast
to most mammals that have a lifetime determinate repro-
duction, in fish, the renewable source of the germ cells
ibb.unesp (I. Quagio-

8
vier OA license.
underlies nondeterminate lifetime reproduction, during
a female fish’s reproductive life history. The renewable
source of oocytes and eggs that allows female teleosts to
produce new follicles during their entire reproductive life
is the germinal epithelium that borders the ovarian lam-
ellae [1,2].

The lamellar germinal epithelium houses oogonia [2–6];
stem cells of the female germinal lineage reside within the
lamellar epithelium [4]. Within this germinal epithelium
oogonia proliferate and form germline cysts. Formation
of these cysts is conserved throughout vertebrate evolution
[7–12]. By definition: “Cysts are groups of cells that form

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D.D. Wildner et al. / Theriogenology 79 (2013) 709–724710
from a single founder cell. The founder cell undergoes
synchronous mitotic divisions that are followed by
incomplete cytokinesis to form a cluster of 2n cells that are
interconnected by intercellular bridges” (see [13] for
review). It is inside cysts that oogonia enter meiosis, giving
rise to oocytes [2,3,6]. Proliferation and differentiation of
oogonia forms nests (cell clusters in the epithelium) [14].
Meiosis progresses and subsequently arrests in diplotene of
the first meiotic division. Diplotene oocytes become
progressively surrounded by prefollicle cells during folli-
culogenesis. At the completion of folliculogenesis, pre-
follicle cells become follicle cells when they, and an oocyte,
are surrounded by a basement membrane, becoming
separated from the cell nest to form a discrete ovarian
follicle [2,3,6]. Near the end of folliculogenesis, the follicle
basement membrane is encompassed by cells from the
ovarian stroma that form a theca. Together, the follicle,
basement membrane and theca form a follicle complex in
which the oocyte develops [2,15–17].

Renewal of gametes, their development, differentiation,
maturation, and release, result in alterations of gonadal
featureswhichdistinguishphasesof theannual reproductive
Fig. 1. Macroscopic features of the Pimelodus maculatus ovary. (A and B) Medial port
the developing phase. Developing oocytes were visible. (E and F) Medial portion of
(G and H) Caudal portion of an ovary in the regressing phase. EF, layer of external
cycle in teleostean fishes [18]. Recognition of reproductive
phases constitutes fundamental knowledge of reproductive
biology in fish and is applied in management of fish stocks.
However, in contrast to males, recognition of female re-
productive phases usually does not consider germinal
epithelium activity. Determination of the reproductive pha-
ses of the females is based on various stages of oocytes
present in the ovarian lamellae, whether they are in primary
or secondary growth, maturation, or undergoing ovulation
[18]. Remarkably, dynamics of female germ cell renewal
throughout the reproductive cycle remain, for themost part,
unknown.

In the present study, activity of the female germinal
epitheliumwascharacterized in twospeciesofostariophysian
fish with distinct reproductive strategies: a Siluriformes,
Pimelodus maculatus, and a Characiformes, Serrasalmus
maculatus. In P. maculatus, oocyte development is group-
synchronous and the annual reproductive period is short
[19–21], and in S. maculatus, oocyte development is asyn-
chronous, and the reproductive period is prolonged [22–24].
These two types of reproductive strategies result in a fish
of determinate and indeterminate fecundity, respectively
ion of an ovary in regenerating phase. (C and D) Cranial portion of an ovary in
an ovary in spawning-capable phase. Oocytes of various sizes were present.
fat; OC, oocyte; OW, ovarian wall.



D.D. Wildner et al. / Theriogenology 79 (2013) 709–724 711
[25–27]. The Ostariophysi constitutes a basal group in the
Teleostei, comprised of the Orders Cypriniformes, Char-
aciformes, Siluriformes, and Gymnotiformes. Characiformes
and Siluriformes are the predominant fish in the continental
fresh waters of Central and South America. Among them are
large species with great economic importance in Neotropical
regions [28].
Fig. 2. Phases of the reproductive cycle in Pimelodus maculatus. (A) Ovary in the rege
(C) Detail of the epithelium; note the nest with numerous oogonia and a nest with
developing phase. (E) Detail of the lamellae containing primary and secondary grow
a nest with early oocytes. Single oogonia were also present. (G) Ovary in spawnin
secondary and primary growth oocytes. (I) Detail of the epithelium showing a s
regressing phase. (K) Detail of the lamellae containing atretic follicles and primary g
epithelium showing a nest with few oogonia. Nest with oogonia indicated by black a
follicle; BV, blood vessels; FG, full-grown oocyte; LA, ovarian lamellae; MB, muscle
growth oocyte; SG, secondary growth oocyte.
2. Materials and methods

2.1. Fish

Mature female P. maculatus and S. maculatus were
collected monthly, as follows: (1) Jurumirim reservoir, Alto
Paranapanema River, São Paulo State, Brazil, from January
nerating phase. (B) Detail of the lamellae containing primary growth oocytes.
a numerous early oocytes. Single oogonia were also present. (D) Ovary in the
th oocytes. (F) Detail of the epithelium; note the nest with a few oogonia and
g-capable phase. (H) Detail of lamellae containing fully grown oocytes, and
ingle oogonium. A nest with a few oogonia was also present. (J) Ovary in
rowth oocytes. Early secondary growth oocytes were absent. (L) Detail of the
rrowheads and white arrowheads indicate nest with early oocytes. AF, atretic
bundle; OG, oogonium; OL, ovarian lumen; OW, ovarian wall; PG, primary



Fig. 3. Oocyte development in Pimelodus maculatus. (A) Single oogonium scattered in the germinal epithelium. (B) Oogonia proliferation formed cell nests. In the
nest, each oogonium was wrapped by prefollicle cells, giving rise to germline cysts. Oogonium in metaphase of mitosis (black asterisk). (C) Nest with a germline
cyst of oogonia. (D) In the nests, oogonia entering meiosis gave rise to germline cysts with leptotene oocytes. The nucleus of the leptotene oocytes was
basophilic. Note that germline cysts with oocytes (square bracket) and others with a single oogonium coexisted in the same nest. (E) Nest with zigotene oocytes.
(F) Nest with pachytene oocytes. During late pachytene, prefollicle cells extended from the cell layer encompassing the germline cysts, inserted themselves
among oocytes, and progressively surrounded each one of them. (G) Ovarian follicles arrested in diplotene. Early diplotene oocytes became completely sur-
rounded by follicle cells (former prefollicle cells), giving rise to ovarian follicles. Inside the newly formed ovarian follicle, the diplotene oocyte began primary
growth and left the nest. (H) Developing primary growth oocyte. Inset: zona pellucida composed of a single thin layer. (I) Early secondary growth oocyte in
which the first yolk globules and the first vesicles of the cortical alveoli appeared. Inset: zona pellucida composed of two layers. Note the hypertrophic follicular
cells. (J) Developing secondary growth oocyte characterized by progressive deposition of yolk. (K) Full-grown oocyte with cytoplasm completely full of yolk and
cortical alveoli forming a thin peripheral layer. Inset: zona pellucida composed of two layers. (L) Atretic follicles. Note liquefaction of yolk globules (LY). Inset:
high magnification of a nest with oogonia present (black arrowhead). (M) Detail of disintegration and fragmentation of the zona pellucida. Note the phagocytic
follicle cells engulfing yolk (undulated black arrows). Mitosis indicated by black asterisk; black arrowhead indicates nest with oogonia; square brackets indicate
germline cysts; and undulated black arrows indicate fragmentation of the zona pellucida. AF, atretic follicle; BM, basement membrane; BV, blood vessels;
F, follicle cell; FG, full-grown oocyte; LO, leptotene oocytes; LY, liquefaction of the yolk globules; N, nucleus; OF, ovarian follicle; OG, oogonium; OL, ovarian
lumen; PF, prefollicle cells; PG, primary growth oocyte; PO, pachytene oocyte; SG, secondary growth oocyte; Y, yolk globule; ZO, zigotene oocytes; ZP, zona
pellucida.

D.D. Wildner et al. / Theriogenology 79 (2013) 709–724712



D.D. Wildner et al. / Theriogenology 79 (2013) 709–724 713
1996 to December 1997; and (2) Piracicaba River, São
Paulo State, Brazil, from January 2004 to December 2005.
During the first sampling period (from January 1996 to
December 1997), 50 P. maculatus and 55 S. maculatus
were collected. During the second sampling period
(January 2004 to December 2005), 82 P. maculatus and
38 S. maculatus were collected (total of 255 females). All
specimens were anesthetized with 0.1% benzocaine and
euthanized, in accordance with institutional animal care
protocols and approval. For each fish, the total length was
measured to the nearest 0.5 cm and total weight was
determined to the nearest 0.1 g. Soon after death, ovaries
were removed, weighed (nearest 0.01 g), and then fixed
in 2% glutaraldehyde and 4% paraformaldehyde in Sor-
ensen’s phosphate buffer (0.1 mol/L, pH 7.2) for at least
24 hours.

2.2. Gonadosomatic index

During the first sampling period (from January 1996
to December 1997), the gonadosomatic index (GSI) was
calculated for each fish (50 P. maculatus and 55 S. mac-
ulatus) and was used to establish the gonadal maturation
curve, to estimate the breeding season. The index is
given by GSI ¼ GW/W � 100, where GW ¼ gonad weight,
and W ¼ total weight. These data were grouped and
Fig. 4. Frequency of nests containing proliferating oogonia and early prophase oo
Pimelodus maculatus. (A and B) Regenerating phase, N ¼ 68. (C and D) Developing p
phase, N ¼11.
analyzed (one-way ANOVA, followed by Tukey test)
separately by seasons and phases of the reproductive
cycle.

2.3. Histologic analysis

For light microscopy, the right ovaries from all 225
females (132 P. maculatus and 93 S. maculatus) were
dehydrated in ethanol and embedded in Historesin (Tech-
novit 7100), and the left ovaries were maintained in fixa-
tive. Sections (3 mm) from all right ovaries were stained
with periodic-acid-Schiff/hematoxylin/metanil yellow [29].
The periodic-acid-Schiff method per se or with other dyes
[29] is used to detect neutral polysaccharides. Neutral
polysaccharides are stained magenta. Histologic sections of
15 ovaries of S. maculatus representing all phases of the
reproductive cycle (three per phase) were stained with the
reticulin method, which enhances basement membranes
[6,30,31]. Ovaries were evaluated with a computerized
image analyzer (Leica Qwin 2.5, Leica Microsystems,
Heerbrugg, Switzerland).

Folliculogenesis andoocytedevelopmentweredescribed
in accordance with Grier et al. [2] and Quagio-Grassiotto
et al. [6]. Fecundity was classified as proposed by Hunter
et al. [25], and as used by Murua and Saborido-Rey
[26] and Lowerre-Barbieri et al. [27]. Characterization of
cyte (divided by single oogonia) by each phase of the reproductive cycle in
hase, N ¼ 6. (E and F) Spawning-capable phase, N ¼ 9. (G and H) Regressing



Table 1
Frequency of reproductive phases (1996 plus 1997) and GSI (mean � SEM) by seasons.

Species Season Regenerating Developing Spawning-capable Regressing GSI No.

P. maculatus Summer 40% 0% 40% 20% 2.32 � 0.59A 15
Autumn 100% 0% 0% 0% 0.62 � 0.10B 12
Winter 100% 0% 0% 0% 0.88 � 0.07B 13
Spring 60% 20% 0% 20% 1.26 � 0.20 (A,B) 10

S. maculatus Summer 24% 53% 18% 6% 1.13 � 0.24A 17
Autumn 36% 55% 0% 9% 0.69 � 0.07A 11
Winter 8% 25% 67% 0% 3.19 � 0.56B 12
Spring 20% 13% 60% 7% 3.43 � 0.63B 15

A and B Within a species, means without a common superscript differed (P < 0.05).

D.D. Wildner et al. / Theriogenology 79 (2013) 709–724714
reproductive phases was performed according to Brown-
Peterson et al. [18].

2.4. Morphometric analysis

For morphometric analyses, ovaries from 94 P. mac-
ulatus (50 from the first sampling period and 44 from the
second) and 93 S. maculatus (55 from the first sampling
period and 38 from the second) were used to enumerate
sites of proliferation.

For each fish, three random, histologic sections of the
right ovary were analyzed. In each of these sections, all
single oogonia in the lamellar epithelium were counted.
Also proliferation sites (cell nests) were counted. Numbers
of nests containing proliferating oogonia, and those con-
taining early prophase oocytes, were recorded separately.
The number of nests with oogonia and those containing
Fig. 5. Relationship between ratio of the number of nests containing proliferating o
Pimelodus maculatus throughout the reproductive cycle. The central line of box mark
third quartile, demonstrating the distribution of 50% of the total sample. The upp
minimum and from the third quartile to the maximum value. Different letters indi
early oocytes were each, by their turn, divided by the
number of single oogonia present in the germinal epithe-
lium and the mean for each animal was recorded. The
frequency of these nests (divided by single oogonia) was
graphically represented at each phase of the reproductive
cycle. Analysis of the results was performed by Kruskal–
Wallis nonparametric one-way ANOVA (P < 0.05 was
considered significant).

3. Results

3.1. Reproductive phases in P. maculatus

3.1.1. Regenerating phase
The lowest value of the GSI occurred during the

regenerating phase (0.81 � 0.05, N ¼ 36). Anatomically,
ovaries in this phase were small and oocytes were not
ogonia (A) and early prophase oocytes (B) by the number of single oogonia in
s the median. The bottom of the box marks the first quartile and the tip of the
er and lower rods extend, respectively, from the first quartile value to the
cate differences (P < 0.05).



D.D. Wildner et al. / Theriogenology 79 (2013) 709–724 715
grossly visible (Fig. 1A and B). In histologic sections
(Fig. 2A and B), besides several forming follicles (Fig. 3G),
the ovarian lamellae contained only primary growth
oocytes (Fig. 3H). Scattered among them, there were a few
atretic oocytes. In the regenerating phase, the number of
Fig. 6. Macroscopic featuresof theovary inSerrasalmusmaculatus. (AandB)Medialpor
the early developing subphase. Small oocytes were present. (E and F) Cranial portion
(G andH) Cranial portion of an ovary in the spawning-capable phase. Oocytes (various s
subphase. (K and L) Medial portion of an ovary in regressing phase. EF, external layer o
nests with proliferating oogonia (Figs. 2C and 3B and C),
and nests containing early prophase oocytes (Figs. 2C and
3D–F), reached peak values (Fig. 4A and B) in relation to
each single oogonium (Fig. 3A) present in the germinal
epithelium.
tionofanovary in theregeneratingphase. (CandD)Medialportionofanovary in
of an ovary in the developing phase. Secondary growth oocytes were present.
izes)were present. (I and J)Medial portion of an ovary in the actively-spawning
f fat; OC, oocyte; OL, ovarian lumen; OW, ovarian wall.



Fig. 7. Phases of reproductive cycle in the Serrasalmus maculatus. (A) Ovary in the regenerating phase. (B) Detail of lamellae containing primary growth oocytes.
(C) Detail of epithelium containing a nest with numerous oogonia. (D) Ovary in early developing subphase. (E) Detail of the lamellae containing primary and early
secondary growth oocytes. (F) Detail of the epithelium, including nest with numerous oocytes. (G) Ovary in developing phase. (H) Detail of the lamellae con-
taining primary and early to late secondary growth oocytes. (I) Detail of the epithelium showing a nest with numerous early oocytes. (J) Ovary in the spawning-

D.D. Wildner et al. / Theriogenology 79 (2013) 709–724716



D.D. Wildner et al. / Theriogenology 79 (2013) 709–724 717
3.1.2. Developing phase
In the developing phase, the GSI value increased (1.08 �

0.03, N¼ 2). Ovaries were enlarged, and small oocytes were
grossly visible (Fig. 1C and D). With the progression of the
reproductive cycle, some oocytes initiated and others
advanced in vitellogenesis (Fig. 2D and E). During the
developing phase, together with deposition of yolk, cortical
alveoli were progressively appearing and forming a discrete
layer close to the oolemma (Fig. 3I and J). During the
developing phase, additional ovarian follicles were formed
from cell nests. In the germinal epithelium (Fig. 2F), there
were more cell nests that contained proliferating oogonia
than nests with early prophase oocytes or single oogonia in
the epithelium (Fig. 4C and D).

3.1.3. Spawning-capable phase
The maximum value of the GSI was reached in the

spawning-capable phase (4.03 � 1.11, N ¼ 6). In this phase,
ovaries were large (Fig. 1E and F), and oocytes were readily
visible. Full-grown oocytes filled most of the lamellae
(Fig. 2G and H). In the ooplasm of these oocytes, yolk
globules fused to each other, and the cortical alveoli formed
only a single, discrete, and discontinuous layer close to the
oolema (Fig. 3K). Late vitellogenic oocytes and other pre-
vitellogenic oocytes were also present (Fig. 2H). In the
lamellar epithelium (Fig. 2I), the number of nests with
proliferating oogonia was very close to the number of the
single oogonia (Fig. 4E). Nests with early prophase oocytes
were scarce (Fig. 4F).

3.1.4. Regressing phase
Postspawning, during the regressing phase, the value of

GSI decreased (1.42 � 0.33, N ¼ 6). Regressing ovaries were
flaccid, blood vessels were prominent, but oocytes were not
grossly visible (Fig. 1G and H). In the lamellae, atresia of
unovulated oocytes (Fig. 3L and M) coexisted with pre-
vitellogenic oocytes (Fig. 2J and K). In the lamellar epithe-
lium (Fig. 2L), the number of nests containing oogonia
increased and nests with early prophase oocytes were also
observed (Fig. 4G and H).

3.2. Gonadosomatic index versus reproductive phases in
P. maculatus

The GSI of P. maculatus (Table 1) increased progressively
from winter to spring, peaked in summer, and decreased
abruptly in autumn. In addition to reflecting seasonal
changes in oocyte development and production, the strong
decline in GSI from summer to autumn was clear evidence
that the spawning season of P. maculatus was short and
occurred mostly in the summer.

Fish in the spawning-capable phasewere only identified
during summer (Table 1). In addition to reproducing
capable phase. (K) Detail of lamellae containing full-grown oocytes, and secondary a
with only a few oogonia were also present. (M) Ovary in the actively-spawning subp
and primary growth oocytes. Note the postovulatory follicle complex. (O) Detail of
Ovary in regressing phase. (Q) Detail of the lamellae containing atretic follicles, an
with only a few oogonia). Black arrowhead indicates nest with oogonia and white a
LA, ovarian lamellae; MO, maturing oocyte; OG, oogonium; OL, ovarian lumen; OW, o
SG, secondary growth oocyte.
individuals, there were also postspawning individuals with
ovaries in the regression phase and in regenerating phase.
During the autumn and winter, all individuals were in the
regenerating phase, whereas in the spring, individuals in
the developing phase appeared. These data, in combination
with GSI and gonadal histology information, confirmed
that P. maculatus spawned during summer.

3.3. Activity of the germinal epithelium versus reproductive
phases in P. maculatus

It was in the regenerating phase that the highest values
of nests containing proliferating oogonia and nests con-
taining the early prophase oocytes were present (Fig. 5).
Conversely, in fish in the spawning-capable phase, the
number of nests containing proliferating oogonia was
nearest to the number of the single oogonia in the
epithelium (Fig. 5A). Also, the number of nests with oocytes
was very small in relation to all the other phases of the
annual reproductive cycle (Fig. 5B). Postspawning, in the
regressing phase, oogonial proliferation and entrance into
meiosis were higher compared with in the spawning-
capable phase, but with less intensity compared with the
regenerating phase.

3.4. Reproductive phases and oocyte development in
S. maculatus

3.4.1. Regenerating phase
The lowest value of the GSI occurred during the regen-

erating phase (0.51�0.06, N¼ 12). Anatomically, ovaries in
this phase were small, and oocytes were not grossly visible
(Fig. 6A and B). In histologic sections, they had a thick wall,
numerous blood vessels, and the ovarian lamellae con-
tained only primary growth oocytes (Fig. 7A and B). Scat-
tered among these, therewere a few atretic oocytes. Follicle
formation was intense and occurred in the epithelium
above the basement membrane (Fig. 8G and H). As oocytes
developed, the first vesicles of the cortical alveoli appeared
close to the oolema (Fig. 8I). In the regenerating phase,
oogonial proliferation became evident (Fig. 7C) by
increasing numbers of nests containing oogonia on the
basement membrane in comparison with the number of
single oogonia in the epithelium (Figs. 8A–C, and 9A). Nests
containing the initial prophase oocytes were also present
(Figs. 8D–F and 9B).

3.4.2. Developing phase
During the developing phase, GSI increased (0.95� 0.10,

N ¼ 20). Progressively, ovaries enlarged and small oocytes
were grossly visible (Fig. 6C–F). With the progress of the
reproductive cycle, some oocytes initiated vitellogenesis
(Fig. 7D–I).
nd primary growth oocytes. (L) Detail of epithelium (single oogonium). Nests
hase. (N) Detail of the lamellae containing maturing oocytes, and secondary
the epithelium (single oogonium near a postovulatory follicle complex). (P)
d primary and secondary growth oocytes. (R) Detail of the epithelium (nest
rrowhead, nest with early oocytes. AF, atretic follicle; FG, full-grown oocyte;
varianwall; PG, primary growth oocyte; POC, postovulatory follicle complex;



Fig. 8. Oocyte development in Serrasalmus maculatus. (A) Single oogonium scattered in the germinal epithelium. (B) Oogonia proliferation formed cell nests that
extended into the stroma. Inside the nest, clusters of oogonia were intermingled and individually surrounded by prefollicle cells, forming germline cysts.
(C) Reticulin method showing continuity of the basement membrane under the lamellar epithelium and under the cell nest. (D) In the nests, entry of oogonia into
meiosis gave rise to germline cysts with leptotene oocytes. The nucleus of the leptotene oocytes was basophilic. Note that germline cysts with oocytes and others
with only a single oogonium coexisted in the same nest. (E) Nest with germline cysts containing zigotene oocytes. Cysts with oogonium were also present.

D.D. Wildner et al. / Theriogenology 79 (2013) 709–724718



D.D. Wildner et al. / Theriogenology 79 (2013) 709–724 719
In the early developing subphase (Figs. 6C and D, and 7D
and E), formation of yolk globules began in oocytes, and
vesicles of the cortical alveoli increased in number and size
(Fig. 8J). In the lamellar epithelium (Fig. 7F), in addition to
large nests containing numerous prophase oocytes (Figs.
8D–F and 9D), there were nests with proliferating oogonia
(Fig. 9C) and also with single oogonia.

During the developing phase (Fig. 6E and F), in oocytes,
vesicles of cortical alveoli kept increasing in number and
size, formation of yolk globules progressed (Fig. 8K), and
the layers of the zona pellucida became thicker. Because of
asynchronous vitellogenesis, oocytes in distinct stages and
steps of development were concurrently present in the
lamellae, including previtellogenic ones (Fig. 7G and H). In
the epithelium (Fig. 7I), nests with a few proliferating
oogonia (Fig. 9E) and others with the early prophase
oocytes were present (Figs. 8D and F and 9F). Also, there
were single oogonia, albeit in lower numbers in relation to
proliferating sites.

3.4.3. Spawning-capable phase
The maximum value of GSI occurred during the

spawning-capable phase (4.46 � 0.33, N ¼ 20). In this
phase, ovaries were large (Fig. 6G–J) and oocytes were
readily visible. Full-grown oocytes filled most of the
lamellae (Fig. 7J and K). In the full-grown oocyte, the
ooplasmwas completely full of yolk; yolk globules fused to
each other, and the cortical alveoli formed only a single
layer close to the oolema. The nucleus (or germinal vesicle)
was situated at the center of the oocyte (Fig. 8L). Oocytes in
distinct steps of vitellogenesis and others that were pre-
vitellogenic were present (Fig. 7K). In the lamellar epithe-
lium (Fig. 7L), single oogonia (Fig. 8A) predominated and
some nests with a few proliferating oogonia were present
(Fig. 9G and H).

The actively spawning subphase (Fig. 6I and J) was
marked by oocyte maturation and spawning (Fig. 7M and
N). Nuclear (or germinal vesicle) migration in the direction
of the animal pole signaled the onset of oocyte maturation
(Fig. 8M). In ovarian lamellae, postovulatory follicle
complexes (Fig. 8N and O) appeared concurrent with
maturing and full-grown oocytes (Fig. 7M and N). Post-
spawning, the postovulatory follicle complex remained
(F) Nest with pachytene oocytes. During late pachytene, prefollicle cells extended f
oocytes, and progressively surrounded each one. (G) Ovarian follicles arrested in
completely surrounded by follicle cells (former prefollicle cells), giving risen to o
began primary growth and left the nest. (H) Reticulin method showing that the ov
basement membrane (undulated white arrow). (I) Developing primary growth ooc
composed of two thicker layers. (J) Early secondary growth oocyte with the first yol
pellucida (three layers). (K) Developing secondary growth oocyte, with deposition
plasm completely full of yolk and cortical alveoli forming a thin peripheral layer. No
Maturing oocyte. Note migration of the germinal vesicle or nucleus (NM) towa
postovulatory follicle complex remained attached to the lamellar epithelium. (O)
follicle complex in continuity with the basement membrane of the germinal epith
cell and liquefaction of the yolk globules (LY). (Q) Detail of the disintegration a
phagocytic follicle cells engulfing the yolk (undulated black arrow). Enlarged blood
indicates fragmentation of the zona pellucida; undulated white arrow indicates c
white arrowhead, nest with early oocytes. AF, atretic follicle; BM, basement mem
FG, full-grown oocyte; GE, germinal epithelium; LO, leptotene oocytes; LPO, lat
MO, maturing oocyte; N, nucleus; NM, nuclear migration; OF, ovarian follicle; O
PG, primary growth oocyte; PO, pachytene oocyte; POC, postovulatory follicle com
ZP, zona pellucida.
attached to the lamellar epithelium. It was formed by
follicle cells surrounded by the theca. Also previtellogenic
and vitellogenic oocytes were present (Fig. 7N). In the
actively spawning subphase, the number of single oogonia
in the lamellar epithelium (Figs. 7O and 8A) was high
compared with the amount of nests with proliferating
oogonia and nests with early prophase oocytes (Fig. 9I
and J).

3.4.4. Regressing phase
Postspawning, during the regressing phase, the value of

GSI decreased (0.78� 0.06, N¼ 3). Regressing ovaries were
flaccid. In them, blood vessels were prominent and the
ovary lumen and some oocytes were grossly apparent
(Fig. 6K and L). In atretic follicles (Fig. 8P), follicle cells
become hypertrophic and phagocytic (Fig. 8Q and R), and
zona pellucida fragments (Fig. 8Q) and follicle cells invaded
the oocyte (Fig. 8R). In the ooplasm, yolk globules dis-
integrated, and the yolk became liquefied (Fig. 8P and Q). In
the ovarian lamellae, atretic unovulated oocytes coexisted
with previtellogenic and vitellogenic oocytes (Figs. 7P–R
and 8P). In the lamellar epithelium (Figs. 7R and 8R), the
number of nests containing oogonia increased compared
with the number of single oogonia (Fig. 9K). Nests with the
early prophase oocytes were also present, with a frequency
similar to single oogonia (Fig. 9L).

3.5. Gonadosomatic index versus the reproductive phases in
S. maculatus

Values of GSI of S. maculatus were lowest during
summer and autumn (Table 1). Furthermore, there was
a major incidence of individuals in the developing phase
during summer. In autumn, individuals in the developing
phase were predominant, and there were no spawning-
capable individuals during autumn, consistent with lower
values of the GSI during summer and autumn.

In the winter, spawning-capable fish dominated (Table
1), followed by those in the developing or regenerating
phases. Similarly, in the spring, spawning-capable fish
dominated. These observations were in accordance with
the highest values of the GSI (Table 1), which occurred in
winter and spring, the breeding season of S. maculatus.
rom the cell layer encompassing germline cysts, inserted themselves among
diplotene. Note the presence of chiasmata. Early diplotene oocytes became
varian follicles. Inside the newly formed ovarian follicle, diplotene oocytes
arian follicle remained attached to the germinal epithelium throughout the
yte in which the cortical alveolus began to be formed. Inset: zona pellucida
k globules. In it, vesicles of cortical alveoli increased. Inset: detail of the zona
of yolk and formation of cortical alveoli. (L) Full-grown oocyte with cyto-
te the nucleus (or germinal vesicle) situated at the center of the oocyte. (M)
rd the animal pole. Inset: detail of the micropyle. (N) Postspawning, the
Reticulin method showing the basement membrane of the postovulatory
elium. (P) Unovulated oocyte became atretic. Note the hypertrophic follicle
nd fragmentation of the zona pellucida of an atretic follicle. (R) Detail of
vessels and a nest with oogonia were also present. Undulated black arrow
onnection with the basement membrane of the germinal epithelium; and
brane; BV, blood vessels; CA, cortical alveoli; CH, chiasmata; F, follicle cell;
e pachytene oocyte; LY, liquefaction of the yolk globules; MI, micropyle;
G, oogonium; OL, ovarian lumen; OW, ovarian wall; PF, prefollicle cells;
plex; SG, secondary growth oocyte; Y, yolk globule; ZO, zigotene oocytes;



Fig. 9. Frequency of nests containing proliferating oogonia and early prophase oocyte (divided by single oogonia) during each phase of the reproductive cycle in
Serrasalmus maculatus. (A and B) Regenerating phase, N ¼ 15. (C and D) Early developing subphase, N ¼ 9. (E and F) Developing phase, N ¼ 23. (G and H)
Spawning-capable phase, N ¼ 25. (I and J) Actively-spawning subphase, N ¼ 16. (K and L) Regressing phase, N ¼ 5.

D.D. Wildner et al. / Theriogenology 79 (2013) 709–724720
3.6. Activity of the germinal epithelium versus reproductive
phases in S. maculatus

In S. maculatus, the ratio between the number of nests
with proliferating oogonia and single oocytes was partic-
ularly high in the regenerating phase, signaling that
oogonial proliferation was intense (Fig. 10A). It progres-
sively decreased in the developing phase. In the spawning-
capable phase and actively-spawning subphase, there
were lower values of this ratio (nests with proliferating
oogonia/single oogonia), indicating decreased oogonial
mitotic activity in this phase, because most of the oog-
onia remained quiescent. Postspawning, in the regression
phase, there was an increased number of nests with
oogonia in the epithelium, indicating recovery of oogonial
proliferation.

Nests with early prophase oocytes were always detec-
ted during the regenerating phase, the early-developing
subphase, the developing phase, and the regressing
phase (Fig. 10B). However, these nests were most common
during the developing phase and the early developing
subphase, indicating entrance into meiosis and an intense
production of oocytes. In the spawning-capable phase
and actively-spawning subphase, there were lower values
of this ratio (nests with early prophase oocyte/single
oogonia).



Fig. 10. Relationship between the ratio of the number of nests containing proliferating oogonia (A) and early prophase oocytes (B) by the number of single
oogonia in Serrasalmus maculatus throughout the reproductive cycle. The central line of box marks the median. The bottom of the box marks the first quartile and
the tip of the third quartile, demonstrating the distribution of 50% of the total sample. The upper and lower rods extended, respectively, from the first quartile
value to the minimum and from the third quartile to the maximum value. Different letters indicate differences (P < 0.05).

D.D. Wildner et al. / Theriogenology 79 (2013) 709–724 721
3.7. Classification of reproductive phases in the Ostariophysi,
taking into account activity of the germinal epithelium

Classification of reproductive phases [18], characteriza-
tion of the stages and steps of oocyte development [2],
descriptions of folliculogenesis [6], and renewal of female
germ cells during the reproductive cycle (current study) are
summarized (Table 2). This table completes previous
studies performed primarily with species of marine Perci-
formes and extends their application to freshwater
Ostariophysi.

4. Discussion

4.1. Germ cell renewal during the reproductive phases

Based on our analysis of female germ cell renewal, in
both P. maculatus and S. maculatus, two types of oogonia
were present in the ovarian lamellar epithelium. The first
was a single oogonium that probably remained quiescent
throughout the reproductive cycle, and the second had
proliferating activity and gave rise to cell nests. According
to Nakamura et al. [4], single oogonia have a prolonged cell
cycle and are stem cell candidates. They also have distinct
morphologic features [6]. In that regard, single oogonia
are dark (ultrastructural studies) and basophilic (his-
tologic studies), whereas proliferating oogonia are clear
and understained, respectively. Dark/basophilic single
oogonia are stem germ cell candidates. This is similar to the
situation observed for spermatogonia in the males [33].
Proliferation of dark/basophilic oogonia gave rise to clear
oogonia that can be committed to oogenesis. In females,
probably during the regenerating phase, dark, single
oogonia divide by mitosis. They produce only clear oogonia
that proliferate and differentiate into oocytes for the next
spawning season.

4.2. Origin of ovarian follicles in fish

Oogenesis in teleost fish has been a continuing focus of
attention. There are considerable newdata onmorphologic,
physiologic, and molecular aspects of the origin of oocytes
and their subsequent development. Although new knowl-
edge has greatly increased comprehension of the repro-
ductive life history of female teleosts [2,17], identification of
the locus in the fish ovary where female germ cell renewal
occurs is very recent. The first information, reported in
Centropomus undecimalis, Perciformes [1], was that the
ovarian lamellar epithelium housed oogonia in adult
females and was a germinal epithelium. Further studies
confirmed the origin of follicles from a germinal epithelium
in fishes [2,6]. Therefore, it is to be expected that the
lamellar epithelium is the source of stem cell candidates of
the germinal lineage in female fish [6]. Elegant studies by
Nakamura et al. [4,5], using transgenic methods and clonal
analysis, clearly demonstrated oogonia stem cells in the
ovarian lamellar epithelium of the Beloniformes, Ory-
ziaslatipes. According to these authors, oogonia stem cell
proliferation formed clusters of germ cells interwoven by
somatic prefollicles cells (sox9b-expressing cells). These



Table 2
Description of reproductive phases in female fish.

Phase Previous terminology Macroscopic and histologic
features

Activity of the germinal epithelium

Regenerating: sexually mature,
reproductively inactive

Resting, regressed, recovering,
inactive

MF: Small ovaries; blood vessels
might be reduced
HF: PG oocytes present advanced to
perinuclear step, with or without
cortical alveoli; thick ovarian wall,
gamma and/or delta atresia,
degenerating POCs might be
present

Proliferation of oogonia is more
intense than in the previous
phases; more nests in the germinal
epithelium and fewer single
oogonia
Indeterminate fecundity: more
nests with oogonia
Determined fecundity: more nests
with oogonia and nests with early
oocytes

Developing: ovaries beginning to
develop, but not ready to spawn

Maturing, early developing, early
maturation, midmaturation,
ripening, previtellogenic

MF: Enlarging ovaries, blood
vessels becoming more distinct
HF: PG, PGca, SGe, and SGl oocytes
present. No evidence of POCs or
SGfg oocytes. Some atresia can be
present
Subphase: early developing: PG
and PGca oocytes only

More nest with oogonia larger than
single oogonia, however fewer
than in regenerating phase
Indeterminate fecundity: more
nests with early prophase oocytes
than nests with oogonia
Determined fecundity: fewer nests
with early prophase oocytes than
nests with oogonia

Spawning-capable: fish are
developmentally and
physiologically able to spawn

Mature, ripe, late developing, late
maturation, late ripening, total
maturation, fully developed,
partially spent, running ripe,
prespawning, gravid, final OM,
vitellogenic, spawning, ovulated

MF: large ovaries, blood vessels
prominent; individual oocytes
visible macroscopically; hydration
HF: SGfg oocytes present or POCs
present in batch spawners; atresia
of vitellogenic and/or hydrated
oocytes might be present; initial
steps of OM can be present; oocytes
with cortical alveoli, SGe, and SGl
can be present in species with
indeterminate fecundity
Subphase: actively-spawning:
oocytes undergoing OMgvm,
OMgvb, hydration (not many
ostariophysian fish), or ovulation

Number of nests with oogonia is
equivalent to number of single
oogonia; nests with early prophase
oocytes are rare
Indeterminate fecundity: might
have more nests with oogonia,
however, fewer than in other
phases
In actively-spawning subphase,
more single oogonia present

Regressing: cessation of spawning Spent, regression, postspawning,
recovering

MF: flaccid ovaries, blood vessels
prominent
HF: atresia (any stage) and POCs
might be present; some PGca and/
or vitellogenic (SGe, SGl) oocytes
might be present

Gradual recovery of oogonia
proliferation and entrance into
meiosis; the number of nests with
oogonia and also with oocytes
increases

Abbreviations: GSI, Gonadosomatic Index; HF, histologic; MF, macroscopic; OM, oocyte maturation; OMgvb, germinal vesicle breakdown; OMgvm, germinal
vesicle migration; PG, primary growth including multiple nucleoli, perinucleolar, and cortical alveolar oocytes; PGca, cortical alveolar; POC, postovulatory
follicle complex; SGl, late secondary growth; SGe, early secondary growth; SGfg, full-grown oocyte.

D.D. Wildner et al. / Theriogenology 79 (2013) 709–724722
clusters were designated as a germinal cradle, which were
present between epithelial cells of the germinal epithelium
and the basement membrane bordering the stromal
compartment [4]. In reality, the germinal cradles defined by
Nakamura et al. [4,5] topologically corresponded to cell
nests described by Selman and Wallace [32]. Therefore,
germinal cradles or nests are niches of stem cells of the
female germinal lineage [4], and these constitute epithelial
niches [34]. By definition, a niche “consists of a local tissue
microenvironment capable of housing andmaintaining one
or more stem cells” [34]. In these niches, stem cells lie
dormant; they can be activated at particular stages during
the reproductive life cycle [34]. This is relevant for oogonial
stem cells throughout the reproductive cycle of teleostean
fish.

To generate new knowledge in this area, we have used,
as a model, two species of Ostariophysi with distinctly
different reproductive strategies to describe the early female
germ cell behavior throughout the annual reproductive
cycle. In seasonal breeders, P. maculatus and S. maculatus,
external environmental factors trigger internal physiologic
mechanisms activating oogonial proliferation and differen-
tiation that give rise to ovarian follicles. Production and
development of these follicles result in a series of gonadal
transformations (reproductive phases).

4.3. Reproductive phases

As proposed by Brown-Peterson et al. [18], reproductive
phases in adult female fish include: regenerating phase,
developing phase, spawning-capable phase, and regression
phase. The description of annual reproductive phases was
described primarily for marine, perciform fishes that
produce pelagic eggs. However, the classification by Brown-
Peterson et al. [18] was flexible enough to contemplate
different reproductive strategies and also some subphases,
e.g., early developing subphase and actively-spawning
subphase. However this classification lacked adaptation



D.D. Wildner et al. / Theriogenology 79 (2013) 709–724 723
to freshwater ostariophysian fish that produce demersal
eggs.Ourproposition, basedonanalyses reproductive cycles
in the Siluriformes, P. maculatus, and the Characiformes,
S. maculatus, was intended to eliminate this gap by helping
to promote the universality of the characterization of the
reproductive phases of female fish, as proposed by Brown-
Peterson et al. [18].

Until now, apparently no classification of the phases of
the female reproductive cycle in fish, including Brown-
Peterson et al. [18], had considered the activity of the
ovarian lamellar germinal epithelium. In that regard, our
data characterizing female germcell renewal, inP.maculatus
and S. maculatus, are original and promote a new under-
standing of the female reproductive cycle.

4.4. Conclusions

In the Siluriformes, namely P. maculatus (determinate
annual fecundity and a short spawning season), the regen-
erating phase comprised the longest period during the
annual reproductive cycle. During this phase, oogonial
proliferation was more intense, as was the entrance of
oogonia intomeiosis. Most oocytes for the spawning season
were produced during the regenerating phase. In the
developing phase, as oocytes were recruited into vitello-
genesis, oogonial proliferation and entrance of oocytes
into meiosis decreased relative to other phases. Oogonial
activity was sharply decreased during the spawning-
capable phase, but their proliferation was recovered
during the regressing phase. Conversely, in the Characi-
formes, as studied in S. maculatus (prolonged spawning
season and indeterminate annual fecundity), oogonial
proliferation was more intense during the regenerating
phase, whereas entrance intomeiosis was more frequent in
the developing phase. In this species, given the prolonged
spawning season, the spawning-capable females, whose
oocyte stock were not depleted, continued to have discreet
oogonial proliferating activity, but their entrance into
meiosis became rare. At the time of oocyte release (actively-
spawning subphase), most oogonia remained quiescent.
Similarly, in P. maculatus and S. maculatus, mitotic activity
of the oogonia decreased sharply during the spawning-
capable phase. Gradually, oogonial proliferation was
recovered during the regressing phase. In summary, inde-
pendent of the species or features of the reproductive cycle,
in P. maculatus and S. maculatus (and perhaps generally in
fish), germ cell renewal occurred during the regenerating
phase, ensuring availability of eggs for the next spawning
event.

Acknowledgments

The authors thank Mirtes Costa (Sao Paulo State
University) for statistical advice and Rafael H. Nóbregafor
valuable suggestions. This study was supported by the
following Brazilian Agencies: FAPESP (Fundação de Apoio à
Pesquisa do Estado de São Paulo); CNPq (Conselho Nacional
de Desenvolvimento Científico e Tecnológico); and CAPES
(Coordenação de Aperfeiçoamento de Pessoal de Nível
Superior).
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	Female germ cell renewal during the annual reproductive cycle in Ostariophysians fish
	1. Introduction
	2. Materials and methods
	2.1. Fish
	2.2. Gonadosomatic index
	2.3. Histologic analysis
	2.4. Morphometric analysis

	3. Results
	3.1. Reproductive phases in P. maculatus
	3.1.1. Regenerating phase
	3.1.2. Developing phase
	3.1.3. Spawning-capable phase
	3.1.4. Regressing phase

	3.2. Gonadosomatic index versus reproductive phases in P. maculatus
	3.3. Activity of the germinal epithelium versus reproductive phases in P. maculatus
	3.4. Reproductive phases and oocyte development in S. maculatus
	3.4.1. Regenerating phase
	3.4.2. Developing phase
	3.4.3. Spawning-capable phase
	3.4.4. Regressing phase

	3.5. Gonadosomatic index versus the reproductive phases in S. maculatus
	3.6. Activity of the germinal epithelium versus reproductive phases in S. maculatus
	3.7. Classification of reproductive phases in the Ostariophysi, taking into account activity of the germinal epithelium

	4. Discussion
	4.1. Germ cell renewal during the reproductive phases
	4.2. Origin of ovarian follicles in fish
	4.3. Reproductive phases
	4.4. Conclusions

	Acknowledgments
	References