THE ANATOMICAL RECORD 300:265–276 (2017)

Morphology and Morphometry of the

Ovaries and Uteri of the Amazonian
Freshwater Stingrays
(Potamotrygonidae: Elasmobranchii)
MARIA ISABEL DA SILVA,* MARIA INES ^ BRAGA DE OLIVEIRA,
OSCAR TADEU FERREIRA DA COSTA, AND WALLICE PAXIUBA  DUNCAN
Department of Morphology, Federal University of Amazonas, Manaus, Brazil

ABSTRACT
The reproductive biology of South American freshwater stingrays
(family Potamotrygonidae) is still poorly studied compared to other
marine species. In the present study, we examined the gross anatomy and
histology of six species of potamotrygonids from the Amazon basin and
described the structural asymmetry of the ovaries and their relationship
between ovarian and uterine fecundities. Stereological techniques were
used to quantify the volume of ovarian and epigonal organ tissue associ-
ated with the left and right sides of the Potamotrygon wallacei, a recently
described species, locally known as the cururu ray. This species presented
ovarian asymmetry; the left epigonal organ-ovary complex was 55 times
larger than the right side. The right side was composed of, volumetrically,
7.3% ovarian tissue and 92.7% epigonal organ tissue whereas the left side
was 51.2% of ovarian tissue and 48.8% epigonal organ tissue. In all spe-
cies, six types of follicles were identified in both right and left ovaries.
Uteri were symmetrical and the fecundity ratio between the right and
left sides was 0.9:1.1, respectively. Despite the volumetric difference of
ovarian tissue between the two sides, the uterine fecundity shows that
both ovaries are functional and that ovarian fecundity alone is not an
accurate measure to determine the reproductive potential of freshwater
stingrays. Anat Rec, 300:265–276, 2017. V C 2016 Wiley Periodicals, Inc.

Key words: reproductive biology; potamotrygonid; folliculogen-

esis; epigonal organ-ovary complex; trophonemata

The modern chondrichthyans are primarily marine

and estuarine, with exception of the family Potamotrygo-
nidae, which are obligatorily freshwater batoids adapted
to South American aquatic systems (Carvalho et al.,
2003; Rosa et al., 2010). These stingrays evolved from
marine ancestors during Paleocene-Miocene marine
incursions (Carvalho et al., 2004; Lovejoy et al., 2006;
Adnet et al., 2014).
The family Potamotrygonidae exhibits some unique
physiological and morphological traits as a result of
their conspicuous evolutionary history. For example,
these freshwater stingrays osmoregulate through elec-
trolytes similar to freshwater teleost (Wood et al., 2002;
Duncan et al., 2009). In addition, they have low urea
levels, salt-secreting rectal gland degeneration and
Grant sponsor: Amazonas State Research Support Foundation
(FAPEAM) and National Council of Technological and Scientific
Development (CNPq); Grant number: FAPEAM-PPP No. 389/
2012, FAPEAM-Universal No. 209/2012, and CNPq-Universal
No. 484374/2011-7.
*Correspondence to: Maria Isabel da Silva, Department of
Morphology, Federal University of Amazonas, 6200 Av. General
Rodrigo Octavio – Coroado I, Manaus/AM 69080-900, Brazil. Tel.:
155 92 3305-4231 E-mail: mariaisa21silva@gmail.com
Received 29 February 2016; Revised 15 August 2016;
Accepted 23 August 2016.
DOI 10.1002/ar.23501
Published online 14 October 2016 in Wiley Online Library
(wileyonlinelibrary.com).

C 2016 WILEY PERIODICALS, INC.

V
266
Fig. 1. The collected species of freshwater stingrays from the Potamotrygonidae family: Paratrygon aier-
eba [A], Potamotrygon orbignyi [B], P. scobina [C], P. motoro [D], P. schroederi [E] and P. wallacei [F].
modified electroreceptive organs (Thorson et al., 1983).
However, most of their life history still corresponds to
their marine ancestors: low population growth rate, late
maturation, low fecundity and a long gestation period
(Carrier et al., 2004; Charvet-Almeida et al., 2005).
Despite the general similarities with marine relatives,
the reproductive biology of potamotrygonids is still poorly
characterized (Charvet-Almeida et al., 2005). This family
has approximately 30 described species belonging to four
genera: Heliotrygon, Paratrygon, Plesiotrygon e Potamo-
trygon (Rosa and Lasso, 2014). Because of their diverse
shape, skin patterns and coloration these stingrays are
targeted in fisheries for the international ornamental fish
trade (Rosa et al., 2010). Therefore, knowledge about
their reproductive aspects becomes extremely important
to the conservation and management of these species.
Freshwater stingrays are aplacental viviparous with
histotrophy. Structures called trophonemata work as pla-
cental analogues responsible for the production of the
uterine milk that feeds the embryos (Wourms et al.,
1988). The right ovary is described as rudimentary and
non-functional, because of its smaller size and lack of ova
production (Wourms, 1977; Thorson et al., 1983). In con-
trast, for the Colombian species Potamotrygon magdale-
nae, both ovaries and uteri were described as functional
(Teshima and Takeshita, 1992). Ovarian asymmetry in P.
magdalenae was later reported suggesting that its right
ovary is atrophied similar to other potamotrygonid rays
(Ramos-Socha and Grijalba-Bendeck, 2011). Recently,
detailed descriptions of the morphology of the reproduc-
tive system for this species confirmed that both ovaries
are functional (Pedreros-Sierra et al., 2016). To date, it is
not known if both ovaries of other potamotrygonids are
functional. Data for uterine fecundity in Plesiotrygon iwa-
mae, Paratrygon aiereba, Potamotrygon motoro, P.
DA SILVA ET AL.
orbignyi, P. schoroederi, P. scobina and P. wallacei, recent-
ly described species populary known as the cururu ray,
shows that both uteri store embryos and, consequently,
are functional (Charvet-Almeida et al., 2005).
The association between the epigonal organ and the
ovary is a unique characteristic in elasmobranchs (Lutton
and Callard, 2008). The two tissues are inwardly con-
nected without delimitations, which does not allow their
differentiation (Serra-Pereira et al., 2011). It is common
that all elasmobranchs present asymmetric ovaries
(Wourms et al., 1988). Consequently, the epigonal organ
might also be asymmetric. However, little is known about
this two-organs structure and its tissue distribution on
the left and right sides.
This study presents a detailed description of the gross
anatomy and stereology-based morphometry of ovaries
and uteri of six species of potamotrygonids from the
Amazon basin. The structural asymmetry of ovaries,
ovarian and uterine fecundities are also characterized.
MATERIAL AND METHODS
Area of Capture and Sampling
Twelve adult female stingrays were caught using bottom
longline during the dry season, with permission of Chico
Mendes Institute for Biodiversity Conservation (ICMBio,
the Brazilian environmental agency, license No. 22055-4).
The specimens were measured, weighed and identified
(Fig. 1) according to Rosa and Lasso (2014): Paratrygon
aiereba (N 5 1), Potamotrygon orbignyi (N 5 2) and
Potamotrygon scobina (N 5 1) were collected in the Rio
Jutaı (38280 5100 S/668040 0800 W). Potamotrygon motoro were
collected in both Solim~oes River (N 5 1, 38170 600 S/
608110 900 W) and Rio Negro (N 5 2, 08580 3000 S/628550 2600 W).
Potamotrygon schroederi (N 5 1) and Potamotrygon
 REPRODUCTIVE MORPHOLOGY OF POTAMOTRYGONID RAYS
wallacei “cururu ray” (N 5 4) were also collected in the Rio
Negro. All animals were anaesthetized and euthanized by
spinal contusion in accordance with the institutional
guidelines for protection of animal welfare (Ethical Com-
mittee of Animal Experimentation, CEAA-UFAM protocol
No. 070/2012).
Tissue Collection and Microscopy
Ovaries and uteri were dissected and photographed for
anatomical analyses. The organs were fixed in cold and buff-
ered glutaraldehyde 2.5% solution (pH 7.4) for 24 hours and
then preserved in 70% ethanol. Ovarian and uterine fecun-
dity were estimated by counting the number of visible fol-
licles and intrauterine embryos. In some cases, the uterine
fecundity was counted after natural abortions, a process
caused by stress due to fishing. Ovaries and uteri were
weighed, measured and visible follicles were removed from
the ovaries to measure their diameter. Tissue samples were
dehydrated in alcohol and embedded in historesin (Techno-
vit 7100). Five mm-thick sections were cut using a Leica RM
2125RT microtome and stained with Toluidine Blue (0,1%),
Alcian Blue pH 2.5 and Periodic Acid-Shiff (PAS). Finally,
the images were captured using an Olympus BX51 optic
microscope coupled with a digital camera and the morpho-
metric data was measured using the standard features with-
in ImageJ software (version 2.0) (Schindelin et al., 2015)”.
Stereology
For the species Potamotrygon wallacei, stereological
analyses were performed to estimate the volumetric tis-
sue ratio between ovary and epigonal organ. Full epigonal
organ-ovary complexes were histologically processed, as
described above. The volume was estimated using the
Cavalieri Principle (Howard and Reed, 2010). Briefly, the
length of the immersed tissue in each historesin block
was measured and transected by ten equidistantly spaced
sections (T), beginning at a random starting point within
the first interval. From each section, one five mm-thick
histological section was obtained resulting in ten total
representative sections of the entire organ. The sections
were stained with toluidine blue (0.1%) and photo-
graphed. Over every captured image, a square lattice grid
of points was superimposed by the free software IMOD
(Kremer et al., 1996). The points over epigonal and ovary
tissues were counted separately. The point calculations
for ovaries were the sum of points from the germinal epi-
thelium, corpus luteum, follicles and ovarian stroma, pre-
sented as relative volumes. Absolute volume calculation P
of ovary and epigonal tissue was P obtained as Vovary 5 Pi
ðovaryÞ3 ap 3T and Vepigonaltissue 5 PPiðepigonalÞ3 ap 3T, in
which V is the respective volume, Pi is the total num-
ber of points falling over ovarian or epigonal tissue, ap is
the area represented by each point in the square lattice
grid and T is the distance between sequenced sections
(Howard and Reed, 2010).
Statistical Analysis
Morphometric and stereological results were presented
as mean 6 standard deviation. Left and right organs were
compared in tests for normality and homogeneity and sta-
tistical significance by Mann-Whitney U test with
267
P < 0.05. Calculations of the error coefficient for tissue
volume variance was based on Gundersen et al. (1999):
pffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
Varnoise 1 VarSURS
CE5 P
Pi
Where Varnoise is the inaccuracy of the square lattice
grid calculated by the shape of the average profile, Var-
SURS is the sums of variance areas
P through a systematic
uniform random sampling and Pi is the total number
of points counted over the projected surface area of epi-
gonal organ-ovary complex.
RESULTS
Anatomy and Fecundity of Ovaries and Uteri
Freshwater stingrays presented two ovaries and two
uteri located respectively at the superior and inferior
area of the visceral cavity, parallel to the vertebral col-
umn (Fig. 2A). Ovaries were pale-yellow, elongated, flat
and asymmetrical structures with the left gonad bigger
than the right (Fig. 2B). Macroscopically, the association
of the ovary and epigonal organ was not visible as both
organs appeared as a single mass where bright-yellow
vitellogenic follicles develop during the reproductive
season (Fig. 2C). Ovarian fecundity was also unequal
(Table 1). For all species, independent of their reproduc-
tive stage, the left ovary presented a fecundity of 3-20
follicles with a diameter of 0.93 6 0.47 cm while the
right ovary did not present any visible follicles, except
P. motoro which had only one follicle with a diamter of
2.5 cm (Fig. 2C).
The uteri were white, cylindrical and individual struc-
tures located above the kidneys with no asymmetry
between the right and left sides (Fig. 2D). In adult Pota-
motrygon wallacei, the mass and dimensions of the uteri
were: mass 5 2.26 g, length 5 2.52 6 0.15 cm and width 5
1.37 6 0.17 cm. internally, each organ had a muscular
sphincter controlling the opening for the vaginal canal
and numerous projections called trophonemata arising
from the uterine wall (Fig. 2E). In pregnant specimens,
the embryos were found in contact with the trophone-
mata and immersed in a uterine liquid. The total uterine
fecundity varied from 2 to 3 embryos in the cururu
ray, but reached up to 4 in larger species such as
Potamotrygon motoro and Paratrygon aiereba (Table 1).
Additionally, preliminary data of uterine fecundity of 27
Potamotrygon wallacei showed a proportion of 0.9:1.1
right:left embryos. Therefore, the average fecundity was
1.82 embryos per female (data not shown).
Microscopic Description of Ovaries
The ovarian tissue was found externally to the epigo-
nal organ tissue, however both tissues were continuous
lacking a divisible membrane. The epigonal tissue was
distinguished by a dense aggregation of granulocytes
and lymphocytes while the ovarian tissue had a simple
cuboidal epithelium composed of the vascularized con-
nective tissue of the stroma, which gives support to ovar-
ian follicles during their development (Fig. 3B). The
specimens were found in different reproductive stages.
However, both ovaries presented the following compo-
nents: germinal epithelium, ovarian stroma, oogonia
268 DA SILVA ET AL.
Fig. 2. Anatomy of the female reproductive system in freshwater
stingrays. [A] Anatomical scheme of the reproductive tract. [B,C] Ana-
tomical picture of the epigonal organ-ovary complex of Potamotrygon
wallacei with lack of follicles and Potamotrygon motoro with follicles in
both sides. [D] External and [E] internal anatomy of uteri of Potamotry-
gon motoro, with trophonemata (white arrows). Vertebral column (V);
Right ovary (RO); Left ovary (LO); Right oviductal gland (ROG); Left
oviductal gland (LOG); Right uterus (RU); Left uterus (LU).
cells and follicles in different stages of development.
Components such as the mature follicle, atretic follicle
and corpus luteum were only found during mature
stages and during pregnancy. For each ovarian compo-
nent, the principal morphological characteristics were
described in Table 2 and illustrated in Figures 3 and 4.
Microscopic Description of Uteri
The uterine wall was composed of four concentric
layers: mucosa, submucosa, muscular and serosa (Fig.
5A,B). The mucosa was formed by villi-like extensions
called trophonemata. These structures were composed of
an axis of loose connective tissue rich in fibroblasts,
smooth muscle cells, capillaries and coated by an exter-
nal simple layer of cylindrical epithelium. The
TABLE 1. Ovarian and uterine fecundity (minimum-
maximum) of Paratrygon aiereba, Potamotrygon
orbignyi, P. scobina, P. motoro, P. schoroederi and P.
wallacei
Ovarian
Fecundity
Uterine
Species DiW R L Fecundity
Paratrygon aiereba 84 0 0 4
Potamotrygon orbignyi 31–41 0 3–5 2
Potamotrygon schroederi 28 0 4 np
Potamotrygon scobina 65 0 18 np
Potamotrygon motoro 39–39 0–1 0–20 4
Potamotrygon wallacei 21–23 0 0–6 2–3
DiW 5 disc width (mm); np 5 non-pregnant specimens, R 5 right;
L 5 left.
submucosa was vascularized and composed of loose con-
nective tissue supporting the mucosa layer. The muscu-
lar layer was thick and organized in two layers of
smooth muscles: longitudinal and transversal fibers.
Finally, the serosa layer coated the organ with a loose
and vascularized connective tissue.
We observed four different structures of the trophone-
mata among the specimens: (1) For non-pregnant speci-
mens of P. orbignyi, P. motoro and P. schroederi, the
cylindrical cells from the epithelium were laterally posi-
tioned, presenting nuclei at the basal pole and small
capillaries at the lamina propria (Fig. 5C). (2) For preg-
nant specimens of Paratrygon aiereba and Potamotrygon
wallacei, the epithelium was cuboidal and organized in
numerous short invaginations forming secretory crypts.
Between the crypts, loose connective tissue supported
wide capillaries located at the surface of the trophone-
mata (Fig. 5D). (3) In the non-pregnant specimen P. sco-
bina the secretory crypts were elongated and a thin core
of tissue surrounded the crypts giving support to
capillaries directed to the surface of the trophonemata
(Fig. 5E). (4) In the non-pregnant specimen of Potamo-
trygon wallacei, the crypts were in anastomosis and the
epithelium was breaking down and peeling off (Fig. 5F).
Trophonemata tissue did not have positive staining for
PAS or Alcian blue (data not shown).
Stereological Analysis of Ovary-Epigonal
Complex
The stereological analysis for all specimens of the
Potamotrygon wallacei showed unequal volumetric
proportions for ovarian-epigonal organ association. In
a comparative analysis between the right and left ova-
ries, the two-tissue complex was asymmetrical for the
total and for each individual tissue volume (Fig. 6A).
The right side had a larger proportion of epigonal tis-
sue, 92.7% (0.227 mm3/g 6 0.046) compared to ovarian
tissue, 7.3% (0.018 mm3/g 6 0.013) (Fig. 6B,C). On the
other hand, the left side consisted of 48.8% (0.953
mm3/g 6 0.465) epigonal organ tissue, while 51.2%
(0.983 mm3/g 6 0.468) was ovarian tissue. For ovarian
components (Fig. 6B,C), the right ovary had 90% of
germinal epithelium (P < 0.05). In contrast, the left
ovary presented a majority (62%) of the volume of the
corpus luteum.
REPRODUCTIVE MORPHOLOGY OF POTAMOTRYGONID RAYS 269

Fig. 3.
270 DA SILVA ET AL.
DISCUSSION
Histological Organization of the Female
Reproductive Tract
In all of the studied specimens, both ovaries contained
the same ovarian components described in other elasmo-
branchs: germinal epithelium, ovarian stroma, oogonia
cells and follicles at different stages of development,
atretic follicle and corpus luteum (Hamlett et al, 1999;
Dıaz-Andrade et al., 2009; Dıaz-Andrade et al., 2011;
Serra-Pereira et al., 2011; Galındez et al., 2014; Wehitt
et al., 2015; Pedreros-Sierra et al., 2016).
The germinal epithelium is organized on a single layer
of cuboid cells, similar to other marine and freshwater
viviparous stingrays, but lacking cilia that are present
in marine oviparous rays (Hamlett et al., 1999; Dıaz-
Andrade et al., 2009; Dıaz-Andrade et al., 2011;
Pedreros-Sierra et al., 2016). Oogonia of viviparous rays
are described as small cells, 20 lm in diameter, with
large, evident nuclei, basophilic cytoplasm, located in
nests under the germinal epithelium and found only in
immature and maturing specimens (Dıaz-Andrade et al.,
2009; Dıaz-Andrade et al., 2011; Wehitt et al., 2015).
These same characteristics, but with oogonia occurring
in different reproductive phases, were observed in the
present study. It is possible that this finding corresponds
to a high, lifelong reproductive potential for this group
of potamotrygonids.
In Batoids, folliculogenesis is usually described as
four follicular stages: primordial follicle, primary follicle,
previtellogenic follicle and vitellogenic follicle (Hamlett
et al, 1999; Dıaz-Andrade et al., 2011; Wehitt et al.,
2015; Pedreros-Sierra et al., 2016). In this study, five fol-
licular stages were identified, with two subtypes for
vitellogenic follicles: primordial follicle, primary follicle,
previtellogenic follicle, I and II vitellogenic follicle and
mature follicle. During folliculogenesis, the follicles
increased exponentially in diameter (Table 2). The follic-
ular cells changed from a simple to a pseudostratified
layer composed of cylindrical and globose cells (Serra-
Pereira et al., 2011; Galındez et al., 2014). PAS staining
showed the presence of neutral glycoproteins both inside
globose cells and the surrounding yolk vesicles. Studies
suggest that these glycoproteins are vitelline precursors
for yolk synthesis and the appearance of the theca and
its vascularization also influence yolk storage inside the
ovulum through uptake of hormonal signals (Prisco
et al., 2002; Prisco et al., 2007; Galındez et al., 2014;
Wehitt et al., 2015).
The zona pellucida formation begins during the early
stages of folliculogenesis with varied thickness during
development (Dıaz-Andrade et al., 2009; Galindez et al,
2014). In this study, we observed maximum zona pellu-
cida thickness in previtellogenic follicles, similar to the
Fig. 3. Folliculogenesis and its cellular components. [A] Nest of oogo-
nia (Oo) under the germinal epithelium (blach arrow) stained with tolui-
dine blue. [B] Epigonal organ tissue (EPO) surrounding the follicles and
characterization of follicular epithelium (black arrows), zona pellucida
(asterisk) and theca (t) in formation on primordial follicle (1), primary folli-
cle (2) and previtellogenic follicle (3) stained with toluidine blue. [C] I
vitellogenic follicle surrounded by the theca (t) with complete zona pel-
lucida (asterisk) and follicular epithelium composed by cylindrical (white
arrow) and globose (black arrow) cells stained with toluidine blue. [D] II
pattern found in studies with Mustelus schmitti and
Sympterygia acuta (Dıaz-Andrade et al., 2009; Galındez
et al, 2014).
Records about the presence of corpora lutea in elasmo-
branchs are dated, and there is a lack of information
about the characteristics and developmental aspects of
this structure for both elasmobranchs and particularly for
freshwater stingrays. This study describes the histological
characteristics of the corpora lutea during its development
and regression. Our results correspond to the recent
description of corpora lutea in P. magdalenae by Pedreros-
Sierra et al. (2016). We observed multiple of these struc-
tures in the left ovary in different stages of development
and regression, suggesting that non-simultaneous, multi-
ple ovulations in this group might provide a long lasting
hormone secretion during pregnancy.
The phenomenon of atresia in rays occurs in all stages
of follicular development and consists of the disorganiza-
tion, regression, degradation and cellular death of a folli-
cle during the absorbance of the yolk (Baccari et al.,
1992; Dıaz-Andrade et al., 2011; Galındez et al., 2014;
Wehitt et al., 2015; Pedreros-Sierra et al., 2016). From
primordial to previtellogenic follicles, we observed that
the atresia was characterized by follicular epithelium
disorder, cellular fragmentation and cytoplasm regres-
sion. In vitellogenic follicles, the amount of yolk
decreased in the ovulum cytoplasm, vascularization
increased inside the follicular epithelium and the folded
follicular epithelium tended to collapse. The vasculariza-
tion inside the follicular epithelium becomes important
to yolk evacuation when blood infiltrates bring macro-
phages to accelerate cell degeneration and the formation
of a scar-like structure (Craik, 1978; Galındez et al.,
2014; Wehitt et al., 2015; Pedreros-Sierra et al., 2016).
In contrast to what was described in the placentary
viviparous shark Sphyrna blocliii (Appukuttan, 1978) and
in the aplacentary viviparous shark Mustelus antarcticus
(Storrie et al., 2009), we observed that freshwater sting-
rays do not have uterine compartments. The uterine body
was composed of four concentric tissue layers (Hamlett
et al., 1996; Storrie et al., 2009; Galındez et al., 2010; Col-
onello et al., 2013; Dıaz-Andrade et al., 2013; Wehitt
et al., 2015; Pedreros-Sierra et al., 2016), in which the
two orthogonal muscular layers guarantee uterine multi-
directional expansion (Galındez et al., 2010). The mucosa
morphology changes in accordance with the female repro-
ductive stages (Hamlett et al., 1996; Storrie et al., 2009;
Galındez et al., 2010; Colonello et al., 2013; Dıaz-Andrade
et al., 2013; Pedreros-Sierra et al., 2016). We observed
major changes in the mucosa with the appearance of
secretory cells and an increase of vascularization. Howev-
er, Colonello et al. (2013) described the full uterine
dynamic from the pre-ovulation until post-partum stage
in the ray Myliobatis goodie. Based on their description
vitellogenic follicle with folded follicular epithelium (black arrows). [E]
External (et) and internal (it) theca expanding into the follicular epitheli-
um folds (black arrow) with the zona pellucida skirting along the folds
(asterisk) stained with toluidine blue. [F] Mature follicles of Potamotry-
gon orbignyi visible to the naked eye. [G] Follicular epithelium folds
compressed and united (black arrow) because of the great amount of
stored yolk (y) in the ovum cytoplasm stained with toluidine blue. [H]
Glycoproteic granules surrounding the yolk vesicles (y) and inside the
globose cells (black arrow) stained with Periodic Acid-Schiff.
 REPRODUCTIVE MORPHOLOGY OF POTAMOTRYGONID RAYS 271
TABLE 2. Description and measurements (mean 6 standard deviation) of ovarian components
Component Characteristics Measurements
Germinal Single layer of cuboidal cells from where the oogonias arise (Fig. 3A). –
epithelium
Oogonia Rounded shape cell with a large central nucleus and homogenous cyto- D: 8.21 6 4.16 mm
plasm, found in a cluster under the germinal epithelium (Fig. 3A). ZPt: absent
Primordial Oocytes with a large and conspicuous nucleus, a homogenous cytoplasm and D: 43.34 6 10.05 mm
follicle a discontinuous simple layer of flat follicular cells. This cellular type is ZPt: absent
located between the ovarian epithelium and stroma (Fig. 3B).
Primary Oocytes with a cuboidal follicular layer where large rounded globose cells D: 88.20 6 24.24 mm
follicle appear. The formation of the zona pellucida began at this follicular stage ZPt: 5.54 6 1.29 mm
between the follicular layer and cytoplasm. This cellular type is located in
the ovarian stroma attached by concentric external layers of connective
tissue that compose the follicular theca (Fig. 3B).
Previtellogenic Oocyte with multiple follicular layers composed of globose cells and other D: 196.01 6 30.09 mm
follicle cells varying from cuboidal to cylindrical shape. The cytoplasm starts to ZPt: 7.57 6 1.25 mm
become granular and the follicular theca is evident and attached to the
ovarian stroma (Fig. 3B).
I Vitellogenic Oocyte with a pseudostratified follicular epithelium, cytoplasm presenting D: 295.55 6 67.31 mm
follicle yolk granules and tiny zona pellucida (Fig. 3C). ZPt: 2.56 6 0.52 mm
II Vitellogenic Oocyte visible to the naked eye with follicular layer projecting folds inward- D: 0.36 6 0.11 cm
follicle ly toward the follicle (Fig. 3D). These folds were filled by vascularized ZPt: present, but too
connective tissue expanding from the internal theca (Fig. 3E). The cyto- tin to measure in
plasm was filled by yolk granules organized in vesicles. light microscopy.
Mature follicle Bright yellow oocyte with oval shape and visible to the naked eye (Fig. 3F). D: 0.93 6 0.47 cm
In this follicular final stage, the cytoplasm is fully filled by yolk vesicles ZPt: present, but too
that compressed the expanded folds of the follicular layer (Fig. 3G). Yolk tin to measure in
vesicles were surrounded by the same glycoproteins found in the cyto- light microscopy.
plasm of globose cells, as evidenced PAS coloration (Fig. 3H). This follicle
is ready to be ovulated and impregnated, a principal characteristic for
mature females.
Corpus luteum Component present only during pregnancy and described in three different –
developing phases:
Phase 1: the residual follicular layer is conserved with the folds close to
each other and tending to merge tensioned by “bridge” structures (Fig.
4A-B). There are no globose cells and the follicular cells become cylindri-
cal with nuclei positioned at the apical pole giving to this whole structure
a glandular appearance (Fig. 4B).
Phase 2: The follicular folds are completely merged forming a large, oval
and vascularized corpus luteum (Fig. 4C). The nuclei in the cylindrical
cells maintain apical position, but the cytoplasm of these cells shows
white coloration (Fig. 4D).
Phase 3: Represents the devolution of the corpus luteum. This structure
was extremely vascularized, disorganized and with degenerated cells (Fig.
4E-F).
Atretic follicle Oocyte in all described follicular stages identified by irregular conformation –
of the cytoplasm. For follicles in advanced maturation such as vitellogenic
follicle, it was observed with high vascularization inward toward the folds
and presence of blood infiltration. The follicular folds were close to each
other and tended to collapse (Fig. 4G-H).

D 5 diameter; ZPt 5 zona pellucida thickness.

and the recent studies of Pedreros-Sierra et al. (2016) in
P. magdalenae, we were able to describe four types of
trophonemata, each corresponding, respectively, to the
reproductive stages: pre-ovulation, post-ovulation, mid
gestation and post-partum, respectively.
Ovarian asymmetry and its implications for
reproductive potential of freshwater stingrays
Ovarian and epigonal organ asymmetry was observed
in all potamotrygonid species studied. In the cururu ray,
the right ovary was 55 times smaller than the left ovary.
The volumetric distribution of both ovarian and epigonal
organ tissue was also unequal between right and left
sides. On the right side, the epigonal organ predominated
with 90% of the total volume while on the left side ova-
ry and epigonal organ volume was 50% for both tissues.
Interestingly, the right ovary was mostly composed of ger-
minal epithelium and some atretic follicles while the left
ovary was composed primarily of the corpora lutea and
some atretic follicles. These results suggest that in the
analyzed specimens ovulation occurred only in the left
side. However, the presence of germinal epithelium and
atretic follicles in the right side indicates that folliculo-
genesis was occurring as well.
For viviparous elasmobranchs, reproductive fecundity
is quantified by the counting of visible follicles in the
ovary and the number of embryos inside the uteri
(Musick et al., 2000). In a study with seven species of
potamotrygonids, Charvet-Almeida et al. (2005) showed
272 DA SILVA ET AL.

Fig. 4. Evolution of the corpus luteum and follicular atresia stained
with toluidine blue. [A,B] Phase 1: after ovulation the follicular epitheli-
um folds tend to get closer by having bridge-like structures (black
arrow) and cellular nuclei are positioned on the apical pole (asterisk).
[C,D] Phase 2: the folds are connected, the structure has lighter
coloration and the cells still present apical nuclei (asterisk). [E,F]
Phase 3: the vasculature increases (black arrow) and the corpus
luteum cells are fragmented (asterisk). [G,H] Follicular atresia and yolk
absorbance; Vasculature increases inside the folds (black arrow) bring-
ing tissue infiltration (white arrow) and cells are fragmentated.
 REPRODUCTIVE MORPHOLOGY OF POTAMOTRYGONID RAYS 273

Fig. 5. Uterine histology stained with toluidine blue. [A,B] Uterine layers: mucosa (m) with trophonemata
(t), submucosa (sm), transversal (mst) and longitudinal (msl) muscular and serosa (s). [C,F] Structures of
trophonemata with characterization of epithelial cells (asterisk) and capillaries (black arrows) in different
reproductive stages.
274
Fig. 6. Stereology of ovary and epigonal organ. [A] Volumetric proportion of ovaries and epigonal
organs for the right and left sides. On the right side, the volume of ovarian tissue is significantly smaller
than the volume of epigonal tissue: P 5 0.029 (Mann-Whitney, asterisk). [B,C] Volumetric percentage of
ovary-epigonal organ association and ovarian components in the right and left side, respectively.
that the ovarian fecundity varied from 1-13 mature fol-
licles while uterine fecundity varied from 1-16 embryos
in total. However, these data were not separately exam-
ined from right and left sides of the ovaries or uteri. In
our study, the number of mature follicles varied from 1-
20. Follicles were present in the ovaries of specimens
before, during and after pregnancy with a right:left
ovarian fecundity proportion of 0:6. In Potamotrygon
wallacei, the uterine fecundity varied from 2-4. Although
the right:left uterine fecundity is difficult to estimate
due to spontaneous abortion after capture, we were able
to estimate the right:left uterine fecundity as 0.9:1.1 for
this species. It was observed that the uterine fecundity
in freshwater stingrays was smaller than ovarian fecun-
dity, as reported by Charvet-Almeida et al. (2005). Inter-
estingly, the fertility of the ovaries and uteri (right and
left) did not match; ovarian fecundity was unequal
between right and left ovaries, but uterine fecundity
seemed slightly equilibrated between both uteri.
In this study, the contradiction between ovarian and
uterine fecundity plays an important role in the under-
standing of ovarian functionality. According to Hamlett
and Koob (1999), Carrier et al (2004) and Lutton et al
(2005), the ovarian fecundity in elasmobranchs varies
according to the reproductive status; both oviparous
elasmobranchs ovaries are functional while viviparous
species have only one functional ovary with the other
being rudimentary. Being viviparous, potamotrygonids
are described with a non-functional right ovary
(Wourms, 1977; Thorson et al., 1983). However, studies
with Potamotrygon magdalenae have shown that both
ovaries are functional, with the left producing more ova
(Teshima and Takeshita, 1992; Pedreros-Sierra et al.,
2016). In our studied species, we observed that the right
ovary has a reduced size and partial or no ovarian activ-
ity because of its reduced or absent follicular production.
However, observing that the uteri are non-connected
DA SILVA ET AL.
chambers with equal uterine functionality and fecundity
(Charvet-Almeida et al., 2005), there is no longer strong
evidence to classify the right ovary as non-functional.
Because its ova often develop into embryos in the right
uterus, we determined the right ovary to be functional in
potamotrygonids. Similarly, in P. magdalenae, although
the right ovary is smaller and produces smaller follicles
compared with the left ovary, corpora lutea were observed
in both ovaries indicating that ovulation occurs eventually
in both functional gonads (Pedreros-Sierra et al., 2016).
We believe that the size-reduction of right ovaries might
be due to anatomical pressures from the spiral valve posi-
tioned over the gonad, limiting space for its development.
This hypothesis, however, still requires investigation.
Additionally, we investigated which fecundity (ovarian
or uterine) actually represents the real reproductive
potential in this group. For the ovarian fecundity, the
excessive follicular production does not seem to promote
any trade-off to the animal but instead wastes energy.
However, it is possible to hypothesize reasonable trade-
offs in this situation if: (1) the large number of ova bal-
ances the chances of failure on fecundation and/or fetus
development; (2) these species have a biennial reproduc-
tive cycle. Neither of these hypotheses were confirmed
for this group, but studies with Dasyatis centroura and
Dasyatis imbricate found gestation periods equal to or
shorter than 6 months, suggesting bi-annual cycles
(Struhsaker, 1969; Devadoss, 1978). Potamotygon
motoro, P. orbignyi and Potamotrygon wallacei also pre-
sent short gestation periods (Charvet-Almeida et al.,
2005) and these species were found with both mature
ova and embryos at the same time. Thus, it might be
possible that the extra number of ovarian follicles during
pregnancy will be directed to ovulation after the birth of
embryos to the beginning of a new gestation. Fahy et al.
(2007) made similar observations in Urobatis jamaicen-
sis and argued that the maximization of ova production
 REPRODUCTIVE MORPHOLOGY OF POTAMOTRYGONID RAYS
might be a strategy to increase brood survivorship. Stud-
ies with captive specimens of P. motoro and P. circularis
(synonymous of P. motoro, Potamotrygon constellata or P.
orbignyi) have also observed more than one reproductive
cycle in a year (Thorson et al., 1983). However, this is a
topic that requires additional data and more analysis
before its confirmation.
In summary, the ovarian and uteri components of
potamotrygonid rays are similar to other elasmobranchs.
In all potamotrygonid species, we identified five follicu-
lar phases in both ovaries. In the left ovary, we observed
multiple corpora lutea in different stages. In fact, all
Potamotrygonid rays presented ovarian asymmetry. In
Potamotrygon wallacei, the left epigonal organ-ovary
complex was 55 times larger than the right side, but the
right ovary was mostly composed by germinal epitheli-
um. At present, our collected data support the function-
ality of the right ovary and we strongly recommend
future analysis of the left/right quantification for ovarian
and uterine fecundity in order to obtain a better under-
standing of organ asymmetry and fecundity distribution.
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