ArticlePDF Available

Abstract and Figures

Effects of xenobiotics can be organizational, permanently affecting anatomy during embryonic development, and/or activational, influencing transitory actions during adulthood. The organizational influence of endocrine-disrupting contaminants (EDC's) produces a wide variety of reproductive abnormalities among vertebrates that exhibit temperature-dependent sex determination (TSD). Typically, such influences result in subsequent activational malfunction, some of which are beneficial in aquaculture. For example, 17-αmethyltestosterone (MT), a synthetic androgen, is utilized in tilapia farming to bias sex ratio towards males because they are more profitable. A heavily male-biased hatchling sex ratio is reported from a crocodile population near one such tilapia operation in Guanacaste, Costa Rica. In this study we test the effects of MT on sexual differentiation in American alligators, which we used as a surrogate for all crocodilians. Experimentally, alligators were exposed to MT in ovo at standard ecotoxicological concentrations. Sexual differentiation was determined by examination of primary and secondary sex organs post hatching. We find that MT is capable of producing male embryos at temperatures known to produce females and demonstrate a dose-dependent gradient of masculinization. Embryonic exposure to MT results in hermaphroditic primary sex organs, delayed renal development and masculinization of the clitero-penis (CTP).
Content may be subject to copyright.
Research paper
Methyltestosterone alters sex determination in the American alligator
(Alligator mississippiensis)
Christopher M. Murray
a,
, Michael Easter
b
, Mark Merchant
c
, Justin L. Rheubert
d
, Kelly A. Wilson
d
,
Amos Cooper
e
, Mary Mendonça
a
, Thane Wibbels
f
, Mahmood Sasa Marin
g
, Craig Guyer
a
a
Department of Biological Sciences, Auburn University, 331 Funchess Hall, Auburn, AL 36849, USA
b
Everglades Holiday Park, Fort Lauderdale, FL 33332, USA
c
Department of Chemistry, McNeese State University, Lake Charles, LA, USA
d
Department of Natural Sciences, The University of Findlay, Findlay, OH 45840, USA
e
J. D. Murphree Wildlife Management Area, Texas Parks and Wildlife Department, Port Arthur, TX 77640, USA
f
Department of Biology, University of Alabama at Birmingham, Birmingham, AL 35294, USA
g
Instituto Clodomiro Picado, Facultad de Microbiología, Universidad de Costa Rica, San José, Costa Rica
article info
Article history:
Received 11 February 2016
Revised 3 May 2016
Accepted 7 July 2016
Available online 9 July 2016
Keywords:
Methyltestosterone
Temperature-dependent sex determination
Androgen
Crocodilian
abstract
Effects of xenobiotics can be organizational, permanently affecting anatomy during embryonic develop-
ment, and/or activational, influencing transitory actions during adulthood. The organizational influence
of endocrine-disrupting contaminants (EDC’s) produces a wide variety of reproductive abnormalities
among vertebrates that exhibit temperature-dependent sex determination (TSD). Typically, such influ-
ences result in subsequent activational malfunction, some of which are beneficial in aquaculture. For
example, 17-
a
methyltestosterone (MT), a synthetic androgen, is utilized in tilapia farming to bias sex
ratio towards males because they are more profitable. A heavily male-biased hatchling sex ratio is
reported from a crocodile population near one such tilapia operation in Guanacaste, Costa Rica. In this
study we test the effects of MT on sexual differentiation in American alligators, which we used as a
surrogate for all crocodilians. Experimentally, alligators were exposed to MT in ovo at standard ecotoxi-
cological concentrations. Sexual differentiation was determined by examination of primary and secondary
sex organs post hatching. We find that MT is capable of producing male embryos at temperatures known
to produce females and demonstrate a dose-dependent gradient of masculinization. Embryonic exposure
to MT results in hermaphroditic primary sex organs, delayed renal development and masculinization of
the clitero-penis (CTP).
Ó2016 Published by Elsevier Inc.
1. Introduction
Recent theory maintains that most vertebrates exist somewhere
along a continuum between strict genetic control of sex determi-
nation and strict environmental control (Sarre et al., 2004) and that
placement along this continuum exists as a function of environ-
mental influence on fitness (Charnov and Bull, 1977). Genotypic
sex determination (GSD) is a preset mechanism of primary and
secondary sexual structure differentiation based on the presence
and subsequent expression of a single gene, or lack of that gene,
resulting in production of a default sex (gene absent) or the alter-
nate sex (gene present). In mammals, for example, the SRY (or
SOX9) gene steers development away from a default female pheno-
type via promotion of a male reproductive tract and simultaneous
suppression of a female tract (Kovacs and Ojeda, 2011). While GSD
allows for a heritable sex, it can be influenced by gonadal sex
disorders (Kovacs and Ojeda, 2011).
Environmental sex determination (ESD) is susceptible to many
more external influences (Wibbels et al., 1994). Temperature
dependent sex determination (TSD), a form of ESD, relies on speci-
fic thermal regimes to dictate the expression of aromatase and/or
reductase, thus governing the sex steroid regime of the developing
embryo and subsequent sex characteristics (Wibbels et al., 1994).
Thermal thresholds or ‘critical’ temperatures, that serve as a thermal
pivot between development of one sex or the other, are
http://dx.doi.org/10.1016/j.ygcen.2016.07.007
0016-6480/Ó2016 Published by Elsevier Inc.
Corresponding author at: Department of Biological Sciences, Southeastern
Louisiana University, Hammond, LA 70402, USA.
E-mail addresses: christopher.murray-2@selu.edu (C.M. Murray), M.Easter05@
gmail.com (M. Easter), mmerchant@mcneese.edu (M. Merchant), rheubert@findlay.
edu (J.L. Rheubert), amos.cooper@tpwd.state.tx.us (A. Cooper), mendomt@auburn.
edu (M. Mendonça), twibblels@uab.edu (T. Wibbels), msasamarin@gmail.com
(M.S. Marin), guyercr@auburn.edu (C. Guyer).
General and Comparative Endocrinology 236 (2016) 63–69
Contents lists available at ScienceDirect
General and Comparative Endocrinology
journal homepage: www.elsevier.com/locate/ygcen
species-specific and vary widely among vertebrates with TSD
(Janzen and Paukstis, 1991). TSD mechanisms also vary among
taxa and three main mechanisms exist; male production at low
temperatures and female at high, female production at low tem-
peratures and male at high, and female at low, male at intermedi-
ate, and female at high temperatures (Valenzuela, 2004). A
remarkable series of sex steroid and temperature manipulations
have elucidated some constants among TSD mechanisms; 1) A crit-
ical period exists at which an embryo’s sex determination is sensi-
tive to both temperature and sex steroids; 2) feminization and
masculinization are mediated by steroid-specific receptors; 3) aro-
matase and reductase inhibitors can manipulate sex regardless of
temperature, however with unequal potency; 4) response to estro-
gens can produce female embryos at male-producing tempera-
tures, but androgens can only masculinize embryos at threshold
temperatures; and 5) mixed sex ratio clutches are produced at
threshold temperatures rather than hermaphrodites (Wibbels
et al., 1991, 1994; Wibbels and Crews, 1994; Crews et al., 1994;
Wibbels and Crews, 1995; Crews; 1996).
Our understanding of how hormone sources, storage, and inter-
nal feedback mechanisms affect sexual differentiation mechanisms
has greatly improved as a result of research in temperature-
dependent steroid expression and utilization (Kamel and
Kubajak, 1987; Janzen et al., 1998; Paitz and Bowden, 2011;
Pfannkuche et al., 2011; Paitz et al., 2012). Unfortunately, so has
our understanding of anthropogenic influences on such processes
(Guillette et al., 1995). Numerous industrial and agricultural com-
pounds, when introduced to natural systems, have endocrine-
disrupting affects. Endocrine-disrupting compounds (EDCs) have
negative activational effects on endocrine systems, but more
daunting is the organizational role they play as hormone mimics
during sexual differentiation and embryonic organization
(Guillette et al., 1995). Because sexual differentiation mechanisms
vary among reptiles, these taxa have become model indicators of
EDC exposure. Among reptiles, contaminants that mimic estrogen
are common (e.g. PCBs, Dioxin, Furans, DDE), while environmental
androgens are far more rare, presumably as a result of the aroma-
tizable nature of testosterone and the dominance of the estrogen
pathway associated with TSD (Wibbels and Crews, 1995).
Among reptiles the most notable sentinel taxa for EDCs are
crocodilians (Milnes and Guillette, 2008). Their popularity as
‘model’ organisms emerged because of the Lake Apopka superfund
site, where dicofol, DDT and subsequent metabolites were discov-
ered as contaminants in 1980 (Guillette et al., 1994). Such contam-
inants were deemed potent environmental estrogens after female
alligators displayed unnaturally high 17b-estradiol plasma con-
centrations, polyovular follicles, polynuclear oocytes (Guillette
et al., 1994), and reduced gonadal-adrenal mesonephros (GAM)
aromatase activity (Crain et al., 1997). Males exhibited decreased
plasma testosterone, reduced phallus size (Guillette et al., 1996)
and poorly organized testes (Guillette et al., 1994). This case study
exemplified the utility of crocodilians in understanding the activa-
tional and organizational effects of EDCs.
In Guanacaste, Costa Rica, three large tilapia farms utilize 17-
a
methyltestosterone (MT) to produce all-male offspring that grow
faster and reach larger maximum size than females. Preliminary
data on MT persistence in water and soil was noted during initial
testing of this fish farming practice, however, lipid persistence of
the compound and its effects on vertebrates other than fish are
unknown (Phelps and Popma, 2000; Gupta and Acosta, 2004).
The nearby Tempisque Basin harbors a rapidly expanding popula-
tion of American crocodiles (Crocodylus acutus) that exhibits a
male-biased sex ratio (Bolaños-Montero, 2012; Murray et al.,
2015). Hatchling sex ratios from this population do not match
the ratios predicted by clutch thermal regimes and this sex
ratio bias differs among clutches, with some clutches being
male-biased and others not (Murray et al., 2016). Here, we test
the potential for MT to produce male crocodilian embryos at
female-producing temperatures and histologically analyze
organizational effects of urogenital development from MT exposure
during the experimental assay.
2. Materials and methods
2.1. Experimental assay
For this experiment, 108 American alligator (Alligator mississip-
piensis) eggs were collected from five clutches in June 2013 and 76
eggs from three clutches in June 2014. All eggs were collected at J.
D. Murphree Wildlife Management Area, Port Arthur, TX, within
five days of deposition, as assessed by daily nest monitoring and
the width and length of banding (Masser, 1993). Eggs were trans-
ported to an Auburn University live animal facility and incubated
at 28 °C, a female-producing temperature (Lang and Andrews,
1994). In 2013, eggs were misted with water daily in an incubator
(Fisher Scientific, Isotemp model 655D) to maintain humidity.
However, 50 eggs failed to complete development, likely because
of dehydration. Therefore, eggs in 2014 were maintained at
approximately 100% humidity using a vermiculite substrate and
steam heating. Each year, four eggs were opened periodically to
stage the embryos as described by Ferguson (1985), resulting
in 126 experimental eggs in total. Prior to the temperature-
sensitive period each year (stage 20, when sex determination
occurs; Lang and Andrews, 1994), eggs were randomly assigned
to one of five treatments using a random number generator. Eggs
were randomly dispersed among plastic bins in the incubator with
10–14 eggs per treatment per year. Two treatment groups served
as controls. One control received no treatment while the other
received 5
l
l of 95% ethanol (ETOH) to control for effects of the
vehicle used to deliver MT to all treatment groups. Treatment
groups received 4
l
g/ml, 40
l
g/ml, or 400
l
g/ml of 17
a
-MTin
95% ETOH. These treatments exposed eggs to between 10 and
1000 times the natural amount of testosterone in alligator egg yolk
(Conley et al., 1997), a range of doses standard for ecotoxicological
dose-response assays with sex steroid hormones or related
endocrine-disrupting compounds (Wibbels and Crews, 1995;
Crain et al., 1997). Treatments were applied topically as 5
l
lof
solution deposited on the surface of an egg at stage 21, a technique
that is used to transport compounds inside reptilian eggshells
(Crews et al., 1991,Paitz et al., 2012). Using this method, Crews
et al. (1991) found that at least 90% of applied compound was
incorporated into the embryo. Additional eggs were incubated
separately at male-producing temperatures (32 °C) to serve as
control males for primary and secondary sex organ comparison.
2.2. Methyltestosterone quantification
Upon hatching, yolk samples were collected and frozen to quan-
tify the concentration of 17
a
- MT that reached the embryo. Steroid
hormones were extracted from egg yolk using a 3:2 volume solu-
tion of ethyl acetate and hexane, respectively. Samples were dried
under vacuum at 25 °C and dissolved in 100
l
L assay buffer
supplemented with 10
l
L of DMSO to encourage dissolution.
17
a
-methyltestosterone concentrations were quantified using a
sandwich ELISA kit (MaxSignal
Ò
methyltestosterone kit, Bioo
Scientific, Austin, TX; Rabbit; polyclonal). Cross-reactivity with
testosterone was 0.3% and samples were not analyzed in duplicate
as all other wells were occupied for another study. Optical density
was determined using a Benchmark Plus microtiter plate spec-
trophotometer (Bio-Rad, Hercules, CA) at 450 nm. Hatchlings were
individually marked via caudal scute removal, and snout-to-vent
64 C.M. Murray et al. / General and Comparative Endocrinology 236 (2016) 63–69
length and total length were recorded. The sex of each hatchling
was determined by cloacal examination with an MDS 105 2.7 mm
endoscope. A hatchling was determined to be male if the clitero-
penis (CTP) possessed all of the following character states:
bi-lobed structure, extensive vascularization, and length equal to
or greater than length of the vent (Fig. 2;Allsteadt and Lang, 1995).
2.3. Sexual differentiation analysis
A subset of twenty hatchings was euthanized and preserved for
detailed CTP examination using a Leica M165C stereoscope with
Leica DFC425 camera attachment. These sacrificed individuals
were examined histologically for internal verification of sex and
description of testicular and ovular development among treat-
ments. The remaining surviving hatchlings (n = 16) were housed
at Auburn University and McNeese State University so that sex of
each individual could be verified at five months of growth.
Whole animals were fixed in 10% neutral buffered formalin and
preserved in 70% EtOH. The developing urogenital tissues were
excised under a stereomicroscope and placed in 70% EtOH for fur-
ther manipulation. Tissues were washed with deionized water,
dehydrated in a series of increasing concentrations of EtOH, and
placed in paraffin wax overnight to allow the wax to fully infiltrate
the tissues. Tissues were then placed in embedding molds with
paraffin wax and allowed to cure for 24 h. The urogenital tracts
were then serial sectioned sagittally on an American Optical rotary
microtome, and sections were then placed on albuminized slides
and stained with Ehrlich’s Hematoxylin and Eosin.
Slides were viewed at various magnifications using an Olympus
microscope to identify structures of the developing urogenital
system and to determine if gonads were of the male or female cat-
egory for each of the above-mentioned treatments. Representative
slides from each treatment along with any abnormalities were
photographed using an attached digital camera and images were
compiled into composite micrographs using Adobe Photoshop CS5.
2.4. Statistics
G tests for independence were performed on the proportion
of male hatchlings among treatments, the proportion of male
hatchlings among clutches, the proportion of survivors among
treatments, and the proportion of survivors among clutches. A
two-sample T test was used to test for difference in size between
control or treatment individuals. Welch’s two- sample T-tests were
used to determine difference in CTP length and width (Fig. 1)
between treated individuals and control individuals because
sample sizes were low among individual experimental groups.
3. Results
3.1. Sexual differentiation of secondary sex organ
Of 126 experimental or control eggs, only 36 survived long
enough to be categorized as to sex. Survival did not differ among
experimental groups but did differ among clutches (G = 28.3,
df = 7, p = 0.0002). Alligator eggs incubated at a female-producing
temperature and treated with 17
a
- MT produced a significantly
higher proportion of male hatchlings than control groups
(G = 20.2, df = 4, p = 0.0005; Fig. 2). There was no difference in pro-
portion of males among clutches. CTP lengths, but not widths, were
significantly larger in treatment (pooled across treatment groups)
versus the pooled control groups (t = 2.65, df = 10.72, p = 0.02,
Fig. 3). Sixteen surviving treatment eggs were housed for sex con-
firmation five months later. All but one of these was confirmed to
have the same sex as morphologically determined at hatching. The
only hatchling whose sex diagnosis differed was deemed a female
at hatching and a male five months later.
Fig. 1. Schematic illustrating cliteropenis (CTP) width and length measurements
during stereoscopic examination in both A) control females and B) masculinized
CTP at 40
l
g/ml.
Fig. 2. Sex ratios among control, ETOH, 4
l
g/ml, 40
l
g/ml and 400
l
g/ml exper-
imental groups. Percent male based on morphological sexing at hatching was
significantly different among groups (G = 20.2, df = 4, p = 0.0005).
C.M. Murray et al. / General and Comparative Endocrinology 236 (2016) 63–69 65
Stereoscope examination of twenty individuals sacrificed at
hatching revealed novel CTP characters for differentiating males
from females as well as treatment effects on CTP development.
Control females exhibited flatter, non-vascularized structures that
extended from the dorsal cloacal surface in a low-lying ‘‘T” shape
(Fig. 4 A, B). Control male CTPs exhibited a basal bi-lobed structure
with a large projection characterized by a mid-sagittal groove,
presumably for sperm delivery. Four to four hundred
l
g/ml MT
treatment concentrations present a gradient from minimal to
extreme vascularization, bi-lobed shape and projection length
(Fig. 4). Such characters are more pronounced within the 4
l
g/ml
treatment than in control females and nearly mirror control males
for individuals in the 400
l
g/ml treatment group.
3.2. Sexual differentiation of primary sex organ
3.2.1. Control specimens
Alligators sacrificed for histological examination had gonads
that had completely separated from the mesonephros and had
begun differentiation. In controls incubated at female-producing
temperatures, the ovaries extended along the medial border of
the developing mesonephros but were distinctly separated from
the mesonephros by a band of loose connective tissue. Along the
posterior border of the mesonephros separation between the ovary
from the mesonephros was more distinct (Fig. 5A).
Histological analysis of the gonads showed that embryos
incubated at female-producing temperatures had a gonad that is
characterized by a thick band of primordial germ cells (Fig 5A,
Pgc) along the cortical region of the gonad. The medullary region
of the gonad was filled with large open lacunae (Fig 5A, La) and
sparsely arranged cells. Embryos incubated at male producing
temperatures had a gonad that was characterized by a lack of
accumulation of primordial germ cells in the cortical region and
large germ cells organized into seminiferous tubules (Fig 5B, St)
where the germ cells are arranged on the periphery of the tube
to form the lumen (Fig 5C, St).
3.2.2. Treatment specimens
Upon exposure to MT the gonads of embryos incubated at
female producing temperatures developed masculinizing charac-
teristics that seem to follow a continuum with increasing concen-
trations of MT exposure. Individuals treated with 4 mcg/mL of MT
and incubated at female producing temperatures had a gonad that
showed a small band (compared to the control female) of primor-
dial germ cells. The medullary region still contained lacunae
(Fig 5D, La) with sparsely spaced cells but a few accumulations of
cells arranged in tubules (Fig 5D, St) are observed. Individuals trea-
ted with 40 mcg/mL of MT and incubated at female producing tem-
peratures lacked an accumulation of primordial germ cells in the
cortical region (Fig 5E) and a medullary region with large open
lacunae (Fig 5E, La) and instances of germ cell organization similar
to that of seminiferous tubule development (Fig 5E, St). A high dose
of MT of 400 mcg/mL of MT to embryos incubated at female pro-
ducing temperatures exhibited a gonad that was more similar to
the control individuals incubated at male producing temperatures
than the controls individuals incubated at female producing tem-
peratures. The gonad lacked an accumulation of primordial germ
cells in the cortical region, had sparsely located lacunae (Fig 5F,
La) and an accumulation of germ cells organized into seminiferous
tubules.
3.3. Yolk methyltestosterone concentration assay
Sample for yolk analysis were limited due to hatching and cost
logistics. Small samples sizes and prohibited construction of a dose
response curve. However, it is notable that all salvaged yolk sam-
ples (n = 7) contained 17
a
- MT including samples from all three
treatments. The average methyltestosterone (MT) concentration
in yolk samples was 0.05 ± 0.014 ng/g yolk. Yolk MT concentrations
ranged from 0.00004 to 0.09 ng/g yolk. Concentrations did not differ
among treatment groups (F = 1.61, DF = 3, p = 0.35). Yolk was not
obtained from control eggs for comparison. All concentrations
Fig. 3. Clitero-penis (CTP) lengths significantly larger in treatment groups than
control individuals (t = 2.65, df = 10.72, p = 0.02).
Fig. 4. Stereoscope images of experimental alligator clitero-penises at hatching
including control females (A, B), 4 g/ml treatment group (C), 40
l
g/ml treatment
group (D), 400
l
g/ml treatment group (E) and natural male hatchling incubated at
male producing temperatures (F).
66 C.M. Murray et al. / General and Comparative Endocrinology 236 (2016) 63–69
among treatment groups were on the order of ng/mL compared to
the initial
l
g/mL dosage.
4. Discussion
Results presented here indicate that MT has a masculinizing
effect on crocodilian embryos during sexual differentiation and
produces male hatchling alligators at female-producing tempera-
tures. This response is observable in both the gonad and sec-
ondary sex structure (CTP). Additionally, the effect of MT on
developing embryos is dose-dependent. MT does not affect sur-
vivorship of exposed individuals and, regardless of exposure con-
centrations, is barely detectable in yolk late in development.
Further, methodology used here to determine the sex of hatch-
lings was conservative in the sense that individuals deemed male
were unlikely to be female, but some deemed female could have
been male. This method proved to be accurate when assessing
male versus non-males at hatching (15 out of 16) including the
potential diagnosis of hermaphroditic individuals if some, but
not all, male character criteria are met. However, hatchlings used
to determine the accuracy (male versus non-male) of our
methodology after five months were not histologically analyzed
for hermaphroditic gonads.
Stereoscopic and histological examination reveals a gradient of
characters among treatment individuals from feminine to mascu-
line. As exposure concentrations increase, so does vascularization,
lobature and length of the CTP. Control female CTP morphology
appears to be a non-vascularized laterally folded tissue that is
slightly thickened medially. Control male CTP morphology appears
to be twice as long as control female CTP and is highly vascularized
with a low-lying second lobe posterior to the lengthy primary
structure. The primary structure is characterized by a medial, ante-
rior groove that may serve to assist sperm transport. The gradient
of secondary sex characters noted among MT treatment groups
appears to mirror the relationship between MT exposure and uro-
genital differentiation. Gonads of control hatchlings exhibit differ-
entiated gonads with associated tissues and precursors to gamete
Fig. 5. Histological micrographs of the gonads from control and treatment Alligator mississippiensis embryos. A.) Gonad of a control embryo incubated at female producing
temperature showing an enlarged cortical region of the gonad with a thick band of primordial germ cells (Pgc) and a medullary region with enlarged lacunae. B.) Gonad of
control embryo incubated at male producing temperature showing seminiferous tubule (St) development and a lack of an enlarged cortical region with an accumulation of
primordial germ cells. C.) Enlarged micrograph of the gonad of a male showing the arrangement of cells into seminiferous tubules. D.) Gonad of embryo treated with 4
l
g/ml
of MT showing a medullary region with lacuna (La) that appear less developed than in the control and sparsely located accumulations of cells arranged in tubules (St). E.)
Gonad of embryo treated with 40
l
g/ml of MT showing a reduced cortical region and a diminished accumulation of primordial germ cells (Pgc). The lacunae are enlarged and
are disorganized in appearance with an accumulation of cells arranged in tubules resembling those of seminiferous tubules (St). F.) Gonad of an embryo treated with 400
l
g/
ml of MT showing the lack of a cortical region and primordial germ cells, sparse lacunae (La) and multiple developments of seminiferous tubules (St).
C.M. Murray et al. / General and Comparative Endocrinology 236 (2016) 63–69 67
production. Low MT exposure at female-producing temperatures
results in slight masculinization of the ovary or underdevelopment
of both kidney and gonad. This underdevelopment may be a result
of a both being exposed to low amounts of steroid binding which
could then have an effect on organ development trajectories and
a result in subsequent delay in development. Exposure to higher
concentrations promotes a qualitatively higher ratio of seminifer-
ous tubules to follicles with more gonadal masculinization at higher
MT concentrations. Although the sexing of hatchlings by CTP
morphology was conservative for this study, the gradient of CTP
masculinization appears to be correlated with gonadal masculin-
ization at a fine scale. In future studies, determination of sex based
upon CTP may prove to be more accurate in determining male
versus non-male hatchling than previously thought (See Ziegler
and Olbort, 2007 and Otaño et al., 2010 for review).
The ability of MT to bias structural differentiation towards male
morphology indicates that it is not aromatizable and is, therefore, a
potent androgen. Its effective use in fish farming indicates affinity
as an androgen at the cellular level via steroid ‘swamping,’ or forcing
masculinization by changing relative concentrations of sex steroids
from estrogen rich to androgen rich while the concentrations of
estrogens stays the same (Phelps and Popma, 2000). Results
presented here indicate a potent organizational role for MT as a
sex steroid in vertebrates with temperature-dependent sex
determination.
The minimal detection of MT among experimental yolks
suggests one of two possibilities. First, a limited amount of MT
may have reached the yolk of each treatment egg. Given that
dose-dependence was not observed within yolks, but was observed
in other aspects of this study, limited crossing of MT into the egg
seems unlikely. More likely is a scenario in which we detected only
a portion of MT in yolk samples. Paitz et al. (2012) discovered the
conjugation of maternally-supplied steroid hormones within the
egg of a TSD reptile and further noted accumulation of such
hormones in the albumin. In our analysis, conjugation of experi-
mentally supplied MT may make it invisible to our assay and higher
detected concentrations may be evident in albumin as opposed to
yolk. Because we were unable to sample albumin, this possibility
awaits further testing.
5. Conclusions
Results presented here contradict two observations previously
regarded as constants in the mechanics of TSD. The efficiency of
androgens at producing males has previously been restricted to
threshold temperatures. In addition, our data contradict the
expected result of mixed-sex clutches. Instead, the production of
hermaphrodites was observed in this study. Instead of these pat-
terns, we demonstrate the experimental production of male alliga-
tors at a strong female-producing temperature, as well as
production of dose-dependent hermaphroditic individuals. This
finding is likely a result of the non-aromatizable nature of MT as
opposed to natural androgens used in prior studies. Crews
(1996), in a review of TSD mechanisms, notes the production of
male-biased clutches in turtles as a result of exogenous application
of the non-aromatizable dihydrotestosterone. The male-biased sex
ratio documented in the Tempisque Basin, Costa Rica appears
unrelated to nest thermal regimes (Murray et al., 2016). However,
MT utilized by local tilapia farms is capable of acting as a potent
androgen yielding male-biased sex ratios at hatching. If this repre-
sents the mechanism by which the population of crocodiles of the
Tempisque Basin became male biased, then we predict wild croco-
diles from this site will demonstrate exposure to MT and evidence
of hermaphroditism among females.
References
Allsteadt, J., Lang, J.W., 1995. Sexual dimorphism in the genital morphology of
young American alligators, Alligator mississippiensis. Herpetologica 51,
314–325.
Bolaños-Montero, J.R., 2012. American crocodile (Crocodylus acutus) (Crocodylia:
Crocodylidae) (Cuvier 1807) population status in the Great Tempisque Wetland.
In: Crocodiles. Proceedings of the 21st Working Meeting of the IUCN-SSC
Crocodile Specialist Group. IUCN, Gland, Switzerland, pp. 167–178.
Charnov, E.L., Bull, J., 1977. When is sex environmentally determined? Nature 266,
828–830.
Conley, A.J., Elf, P., Corbin, C.J., Dubowsky, S., Fivizzani, A., Lang, J.W., 1997. Yolk
steroids decline during sexual differentiation in the alligator. Gen. Comp.
Endocrinol. 107, 191–200.
Crain, D.A., Guillette Jr., L.J., Rooney, A.A., Pickford, D.B., 1997. Alterations in
steroidogenesis in alligators (Alligator mississippiensis) exposed naturally and
experimentally to environmental contaminants. Environ. Health Perspect. 105,
528–533.
Crews, D., Bull, J.J., Wibbels, T., 1991. Estrogen and sex reversal in turtles: a dose-
dependent phenomenon. Gen. Comp. Endocrinol. 81, 357–364.
Crews, D., Bergeron, J.M., Bull, J.J., Flores, D., Tousignant, A., Skipper, J.K., Wibbels, T.,
1994. Temperature-dependent sex determination in reptiles: proximate
mechanisms, ultimate outcomes, and practical applications. Dev. Genet. 15,
297–312.
Crews, D., 1996. Temperature-dependent sex determination: the interplay of
steroid hormones and temperature. Zool. Sci. 13, 1–13.
Ferguson, M.W.J., 1985. Reproductive biology and embryology of the crocodilians.
In: Gans, C., Billet, F., Maderson, P. (Eds.), Biology of the Reptilia. J. Wiley and
Sons, New York, NY, pp. 329–491.
Guillette Jr., L.J., Gross, T.S., Masson, G.R., Matter, J.M., Percival, H.F., Woodward, A.R.,
1994. Developmental abnormalities of the gonad and abnormal sex hormone
concentrations in juvenile alligators from contaminated and control lakes in
Florida. Environ. Health Perspect. 102, 680–688.
Guillette Jr, L.J., Crain, D.A., Rooney, A.A., Pickford, D.B., 1995. Organization versus
activation: the role of endocrine-disrupting contaminants (EDCs) during
embryonic development in wildlife. Environ. Health Perspect. 103, 157–164.
Guillette Jr., L.J., Pickford, D.B., Crain, D.A., Rooney, A.A., Percival, H.F., 1996.
Reduction in penis size and plasma testosterone concentrations in juvenile
alligators living in a contaminated environment. Gen. Comp. Endocrinol. 101,
32–42.
Gupta, M.V., Acosta, B.O., 2004. A review of global tilapia farming practices.
Aquacult. Asia 9, 7–16.
Janzen, F.J., Paukstis, G.L., 1991. Environmental sex determination in reptiles:
ecology, evolution, and experimental design. Q. Rev. Biol. 66, 149–179.
Janzen, F.J., Wilson, M.E., Tucker, J.K., Ford, S.P., 1998. Endogenous yolk steroid
hormones in turtles with different sex-determining mechanisms. Gen. Comp.
Endocrinol. 111, 306–317.
Kamel, F., Kubajak, C.L., 1987. Modulation of gonadotropin secretion by
corticosterone: interaction with gonadal steroids and mechanism of action.
Endocrinology 121, 561–568.
Kovacs, W.J., Ojeda, S.R., 2011. Textbook of Endocrine Physiology, sixth ed. Oxford
University Press, New York, NY.
Lang, J.W., Andrews, H.V., 1994. Temperature-dependent sex determination in
crocodilians. J. Exp. Zool. 270, 28–44.
Masser, M.P., 1993. Alligator Production: Breeding and Incubation. Southern
Regional Aquaculture Center, Publication No. 231.
Milnes, M.R., Guillette, L.J., 2008. Alligator tales: new lessons about environmental
contaminants from a sentinel species. Bioscience 58, 1027–1036.
Murray, C.M., Easter, M., Padilla, S., Garrigós, D.B., Stone, J.A., Bolaños-Montero, J.,
Sasa, M., Guyer, C., 2015. Cohort-Dependent Sex Ratio Biases in the American
Crocodiles (Crocodylus acutus) of the Tempisque Basin. Copeia 103, 541–545.
Murray, C.M., Easter, M., Padilla, S., Sasa Marin, M., Guyer, C., 2016. Regional
warming and the thermal regimes of american crocodile nests in the Tempisque
Basin, Costa Rica. J. Therm. Biol. 60, 49–59.
Otaño, N.B.N., Imhof, A., Bolcatto, P.G., Larriera, A., Noelia, N.O., Piña, C.I., Arambarry,
A., 2010. Sex differences in the genitalia of hatchling Caiman latirostris. Herp.
Rev. 4, 32–35.
Paitz, R.T., Bowden, R.M., 2011. Biological activity of oestradiol sulphate in an
oviparous amniote: implications for maternal steroid effects. Proc. R. Soc. B Biol.
Sci. 278, 2005–2010.
Paitz, R.T., Sawa, A.R., Bowden, R.M., 2012. Characterizing the metabolism and
movement of yolk estradiol during embryonic development in the red-eared
slider (Trachemys scripta). Gen. Comp. Endocrinol. 176, 507–512.
Pfannkuche, K.A., Gahr, M., Weites, I.M., Riedstra, B., Wolf, C., Groothuis, T.G.G.,
2011. Examining a pathway for hormone mediated maternal effects–Yolk
testosterone affects androgen receptor expression and endogenous testosterone
production in young chicks (Gallus gallus domesticus). Gen. Comp. Endocrinol.
172, 487–493.
Phelps, R.P., Popma, T.J., 2000. Sex reversal of tilapia. In: Costa-Pierce, B.A., Rakocy, J.
E. (Eds.), Tilapia Aquaculture in the Americas, second ed. The World
Aquaculture Society, Baton Rouge, Louisiana, United States, pp. 34–59.
Sarre, S.D., Georges, A., Quinn, A., 2004. The ends of a continuum: genetic
and temperature-dependent sex determination in reptiles. BioEssays 26,
639–645.
68 C.M. Murray et al. / General and Comparative Endocrinology 236 (2016) 63–69
Valenzuela, N., 2004. Temperature-dependent sex determination. In: Valenzuela, N.,
Lance, V. (Eds.), Temperature-Dependent Sex Determination in Vertebrates.
Smithsonian Books, Washington, D.C., pp. 1–16.
Wibbels, T., Bull, J.J., Crews, D., 1991. Synergism between temperature and
estradiol: a common pathway in turtle sex determination? J. Exp. Zool. 260,
130–134.
Wibbels, T., Bull, J.J., Crews, D., 1994. Temperature-dependent sex determination: a
mechanistic approach. J. Exp. Zool. 270, 71–78.
Wibbels, T., Crews, D., 1994. Putative aromatase inhibitor induces male sex
determination in a female unisexual lizard and in a turtle with temperature-
dependent sex determination. J. Endocrinol. 141, 295–299.
Wibbels, T., Crews, D., 1995. Steroid-induced sex determination at incubation
temperatures producing mixed sex ratios in a turtle with TSD. Gen. Comp.
Endocrinol. 100, 53–60.
Ziegler, T., Olbort, S., 2007. Genital Structures and Sex Identification in Crocodiles.
Crocodile Specialist Group Newsletter, 26, pp. 16–17.
C.M. Murray et al. / General and Comparative Endocrinology 236 (2016) 63–69 69
... The levels of accumulated MT in all the field collected eggs and in the plasma of wild crocodile hatchlings were similar to those that found in experimentally masculinized hatchlings, even when eggs were exposed to a female-producing temperature [139]. These results strongly suggest that the observed natural bias could result from the masculinizing effects of this synthetic androgen [139][140][141]. ...
... If a female crocodile has accumulated synthetic androgen in its adipose tissues, stored MT could be mobilized during oogenesis and deposit in the yolk, as already suggested [142,143] (Figure 9.6). This would explain the high concentrations of MT found in the eggs of these crocodiles, and these doses have been demonstrated to be able to masculinize an embryo, even under feminizing temperatures [139,141]. ...
... The main hypothesis of these authors relies upon the possible role of biotransporter that wild tilapias could play through a possible MT storage in its adipose tissues (sulfate conjugate). While free MT elimination from aqueous matrices has been well described, little is known about its persistence in a hydrophobic environment, such as the adipose tissue [141]. As tilapia is a common prey for crocodiles, these reptiles will consequently bioaccumulate MT (brought by the biotransporter fish) in their adipose tissues. ...
Chapter
Tilapias are the second largest group of fish produced worldwide, due to their great plasticity and ideal aquaculture traits, particularly the Nile tilapia. On most tilapia farms, sex control is necessary to increase profitability, due to early and continuous reproduction and female mouth‐brooding, and to benefit from the males’ faster growth rate. This review presents the different ways to produce all‐male populations by genetic approaches, such as the use of YY males (or ZZ females in the blue tilapia), and hormonal or high temperature sex reversal treatments, presenting the advantages and drawbacks of each method, as well as protocols for their use. Androgen treatment is still the predominant means to generate monosex male offspring, due to its simplicity, efficiency and price. The hormone amounts currently used worldwide, and its consequences on biodiversity, are discussed, regarding the sustainability of tilapia farming and taking into account growing consumer awareness. Results regarding 17α‐methyltestosterone (MT) accumulation and the possible use of MT‐degrading bacteria are discussed. More sustainable alternative methodologies can be potentiated. Current research status on the sex‐determining loci in Nile, blue, Mozambique and black chin tilapias is presented. Finally, we show the available genetic and phenotypic sex markers that can accelerate progeny testing, rapidly identifying particular genotypes and phenotypes of interest such as YY males or ZZ females, as well as being important for selection programs.
... The levels of accumulated MT in all the field collected eggs and in the plasma of wild crocodile hatchlings were similar to those that found in experimentally masculinized hatchlings, even when eggs were exposed to a female-producing temperature [139]. These results strongly suggest that the observed natural bias could result from the masculinizing effects of this synthetic androgen [139][140][141]. ...
... If a female crocodile has accumulated synthetic androgen in its adipose tissues, stored MT could be mobilized during oogenesis and deposit in the yolk, as already suggested [142,143] (Figure 9.6). This would explain the high concentrations of MT found in the eggs of these crocodiles, and these doses have been demonstrated to be able to masculinize an embryo, even under feminizing temperatures [139,141]. ...
... The main hypothesis of these authors relies upon the possible role of biotransporter that wild tilapias could play through a possible MT storage in its adipose tissues (sulfate conjugate). While free MT elimination from aqueous matrices has been well described, little is known about its persistence in a hydrophobic environment, such as the adipose tissue [141]. As tilapia is a common prey for crocodiles, these reptiles will consequently bioaccumulate MT (brought by the biotransporter fish) in their adipose tissues. ...
... The half-life of MT in water and soil is short, suggesting that MT is unlikely to be an EDC. However, the persistence of this compound in a hydrophobic environment, such as lipids, is poorly known (Phelps and Popma, 2000;Gupta and Acosta, 2004;Murray et al., 2016a) while potential for negative ecological effects have been suggested (Mlalila et al., 2015). Use of this synthetic androgen is a regular practice in tilapia farming, perhaps the most prominent New World fish industry, where it is applied liberally via feed (Gupta and Acosta, 2004). ...
... Specific to the Tempisque drainage, thermal data from nests demonstrate that the male-bias is not a function of temperature effects on hatchling sex determination (Murray et al., 2016b). Additionally, exposure of crocodilian eggs to MT generates male hatchlings when incubated at a temperature that should produce only female offspring (Murray et al., 2016a). The goal of this study is to determine if MT is present in eggs as well as free-ranging hatchling, juvenile and adult crocodiles on the North Pacific versant of Costa Rica. ...
... MT concentration data did not meet the assumptions for the Shapiro-Wilk test of normality. A Wilcoxon signed rank test was performed to assess difference in means between yolk MT concentrations of alligator eggs given doses of MT known to cause sex reversal from female to male (Murray et al., 2016a) and wild yolk concentrations collected for crocodiles sampled during this study. Concentrations of MT in blood plasma and eggs were compared among crocodile cohorts using Kruskal-Wallis H Test. Dunn's post-hoc test was used to elucidate differences among cohorts. ...
Article
Full-text available
Endocrine-disrupting contaminants (EDC's) are well known to alter sexual differentiation among vertebrates via estrogenic effects during development, particularly in organisms characterized by temperature-dependent sex determination. However, substances producing androgenic effects typically lack potency when tested in laboratory settings and are virtually unstudied in field settings. Here, we assay levels of a synthetic androgen, 17α-methyltestosterone (MT), in a heavily male-biased population of American crocodiles in the Tempisque River Basin of Costa Rica based on the recent hypothesis that this chemical is an EDC in developing crocodilian embryos. The presence of MT was documented in all field-collected samples of egg yolk and in plasma of all age classes in among population of crocodiles. Hatchlings exhibited higher plasma MT concentrations (102.1 ± 82.8 ng/mL) than juveniles (33.8 ± 51.5) and adults (25.9 ± 20.8 ng/mL). Among populations, crocodiles captured in the Tempisque River (62.9 ± 73.7 ng/mL) were higher in MT concentration than those from Tarcoles (13.3 ± 11.4 ng/mL) and negative controls (0.001 ± 0.0002 ng/mL). A mechanism for the bio-transport of MT and its subsequent effects is proposed.
... Many studies found that contaminants altered the development of offspring. Common developmental abnormalities included altered Barraza et al. / Environmental Pollution xxx (xxxx) 117470 growth (Beldomenico et al., 2007;Cruze et al., 2015), reduced locomotion (Marco et al., 2004;Wu et al., 2016), altered protein synthesis (Galoppo et al., , 2017, banded eggs (Arukwe et al., 2016), reduced hatchling size (Beldomenico et al., 2007;Garcia-Besne et al., 2015;Neuman-Lee et al., 2015;van de Merwe et al., 2010), altered behavior (Neuman-Lee and Janzen, 2011), and altered morphology and physiology Murray et al., 2016;Nagle et al., 2001). The prevalence of these developmental changes suggests that anthropogenic contaminants have a wide array of sub-lethal effects on offspring development. ...
... Other research found that contaminants can have a masculinizing effect. For example, American alligator eggs exposed to methyltestosterone, a synthetic androgen, were found to produce a dose dependent gradient of males at female-producing temperatures (Murray et al., 2016). These two studies show that increasing doses of exogenous androgen and estrogen can potentially disrupt sex determination across reptile taxa. ...
Article
Threatened or endangered reptiles, such as sea turtles, are generally understudied within the field of wildlife toxicology, with even fewer studies on how contaminants affect threatened species reproduction. This paper aimed to better inform threatened species conservation by systematically and quantitatively reviewing available research on the reproductive toxicology of all reptiles, threatened and non-threatened. This review found 178 studies that matched our search criteria. These papers were categorized into location conducted, taxa studied, species studied, effects found, and chemicals investigated. The most studied taxa were turtles (n = 87 studies, 49%), alligators/crocodiles (n = 54, 30%), and lizards (n = 37, 21%). Maternal transfer, sex steroid alterations, sex reversal, altered sexual development, developmental abnormalities, and egg contamination were the most common effects found across all reptile taxa, providing guidance for avenues of research into threatened species. Maternal transfer of contaminants was found across all taxa, and taking into account the foraging behavior of sea turtles, could help elucidate differences in maternal transfer seen at nesting beaches. Sex steroid alterations were a common effect found with contaminant exposure, indicating the potential to use sex steroids as biomarkers along with traditional biomarkers such as vitellogenin. Sex reversal through chemical exposure was commonly found among species that exhibit temperature dependent sex determination, indicating the potential for both environmental pollution and climate change to disrupt population dynamics of many reptile species, including sea turtles. Few studies used in vitro, DNA, or molecular methodologies, indicating the need for more research using high-throughput, non-invasive, and cost-effective tools for threatened species research. The prevalence of developmental abnormalities and altered sexual development and function indicates the need to further study how anthropogenic pollutants affect reproductive output in threatened reptiles.
... La aplicación de esteroides sexuales para alcanzar la reversión sexual se realiza principalmente con las dietas comerciales proporcionadas a los alevines después de la eclosión, antes de la diferenciación sexual, lo cual permite obtener hasta un 100% de machos (Jiménez y Arredondo 2000, Daza et al. 2005. Sin embargo, el uso de grandes volúmenes de esteroides sexuales para obtener poblaciones monosexo ha generado una creciente preocupación por parte de grupos ambientalistas, ya que la acumulación de esteroides en los cuerpos de agua cercanos a las granjas, puede alterar las proporciones sexuales de animales silvestres que habitan dichas zonas (Murray et al. 2016). De igual forma, un creciente número de personas no desean consumir productos que han sido tratados con hormonas o substancias activas (Piferrer 2001, Müller y Hörstgen 2007, Leet et al. 2011. ...
Article
Full-text available
Una alternativa para reducir el uso de hormonas con aplicación inmediata en el cultivo comercial de la tilapia del Nilo, es la evaluación precisa de los tiempos de masculinización en variedades domesticadas. El objetivo del trabajo fue evaluar el porcentaje de masculinización y el crecimiento obtenido en la tilapia del Nilo alimentada con alimento comercial adicionado con 17α-metiltestosterona a diferentes tiempos. Para ello se evaluaron cinco tiempos (0, 10, 15, 20 y 25 días) de administración de hormona a una población mixta de tilapia del Nilo variedad Spring. Para cada tratamiento se utilizaron tres réplicas. Los alevines fueron sembrados a densidad de 0.5 alevines L 1 en un sistema de recirculación cerrado. El cultivo consistió de 30 días en acuarios de acrílico de 85 L y 20 días en tanques exteriores de 1000 L. La determinación del sexo se realizó con la técnica de squash. El crecimiento se determinó por medio del peso húmedo y la longitud total obtenida de las biometrías. Los resultados indican una masculinización del 100% desde los 10 días de aplicación del esteroide, pero los mayores valores de crecimiento se observaron en los grupos que recibieron el esteroide por mayor tiempo. Los resultados muestran que no es necesario administrar el esteroide durante 30 días para lograr una masculinización del 100% en variedades domesticadas y que el esteroide tiene efecto positivo en la tasa de crecimiento de los alevínes, persistiendo por algunas semanas después de terminado el tratamiento.
... Phenotypic toxicity of MT was observed at concentrations down to 4.5 ng/L in zebra fish (Andersen et al. 2006). Moreover, it also impacts the reproductive system in other animals such as the freshwater snails Marisa cornuarietis at 30 ng/L (Janer et al. 2006), the marine calanoid copepod Acartia tonsa at 1.6 μg/L (Watermann et al. 2013), Marmorkrebs marbled crayfish at 100 μg/L (Vogt 2007,;quail Coturnix coturnix japonica at 50 mg/kg feed (Selzsam et al. 2005), and American alligator Alligator mississippiensis at 4 mg/L (Murray et al. 2016). This indicates that MT released from masculinizing ponds potentially induces adverse effect on ecosystems. ...
Article
Full-text available
17α-Methyltestosterone (MT) is widely used synthetic androgenic steroid in the tilapia aquaculture industry for masculinization: a sex reversal process in which hormones are utilized to induce production of male fish. Although MT is beneficial for aquaculture, release of residual MT can cause adverse effects on wild organisms. The aims of this study were to identify MT-degrading bacteria and to characterize their degradation abilities under the conditions experienced in the environment. Nocardioides nitrophenolicus S303, Acinetobacter radioresistens B051, and Ochrobactrum haematophilum B052 were the most efficient MT-degrading bacterial strains, with the shortest degradation half-life of 10–70 h. The MT degradation by Acinetobacter and Ochrobactrum has not been reported before. After comparing their degradation rates and for reason of biosafety, N. nitrophenolicus S303 was selected for further study. Although this strain degraded MT and testosterones, it could not degrade estrogens (estrone, 17β-estradiol, nor 17α-ethinylestradiol). Glucose amendment did not affect the MT degradation rate. No metabolites with androgenic activity were observed after 264-h treatment with this strain under aerobic conditions. Methandrostenolone was found as the major intermediate during 39–90 h. This is the first report indicating the 1,2-dehydrogenase activity in steroid clevage in N. nitrophenolicus. Our study provides important information concerning the application of N. nitrophenolicus S303 to enhance MT degradation in the environment.
... These observations are consistent with a role for disrupted androgen signaling in shaping the functional trajectory of the ovary. Similarly, exposure to 17-α methyltestosterone, another nonaromatizable androgen, can induce formation of testis-like characteristics in alligator embryos incubated at FPT [98], further suggesting a key role for androgens in gonadal differentiation in reptiles and birds. However, the consequences of precocious AR activation have not been well described in either of these systems. ...
Article
Full-text available
Estrogens regulate key aspects of sexual determination and differentiation, and exposure to exogenous estrogens can alter ovarian development. Alligators inhabiting Lake Apopka, FL are historically exposed to estrogenic endocrine disrupting contaminants and are characterized by a suite of reproductive abnormalities, including altered ovarian gene expression and abated transcriptional responses to follicle stimulating hormone. Here, we test the hypothesis that disrupting estrogen signaling during gonadal differentiation results in persistent alterations to ovarian gene expression that mirror alterations observed in alligators from Lake Apopka. Alligator embryos collected from a reference site lacking environmental contamination were exposed to estradiol-17 beta or a non-aromatizable androgen in ovo and raised to the juvenile stage. Changes in basal and gonadotropin-challenged ovarian gene expression were then compared to Apopka juveniles raised under identical conditions. Assessing basal transcription in untreated reference and Apopka animals revealed a consistent pattern of differential expression of key ovarian genes. For each gene where basal expression differed across sites, in ovo estradiol treatment in reference individuals recapitulated patterns observed in Apopka alligators. Among those genes affected by site and estradiol treatment were three aryl hydrocarbon receptor (AHR) isoforms, suggesting that developmental estrogen signaling might program sensitivity to AHR ligands later in life. Treatment with gonadotropins stimulated strong ovarian transcriptional responses, however, the magnitude of responses was not strongly affected by steroid hormone treatment. Collectively, these findings demonstrate that precocious estrogen signaling in the developing ovary likely underlies altered transcriptional profiles observed in a natural population exposed to endocrine disrupting contaminants.
... Specifically, six immune factors serving as indicators capable of reflecting H. erectus immune capacity (Lin et al. 2016b), including monocytes/leucocytes (M/L), leucocytes phagocytic rate (LPR), immunoglobulin M (Ig M), interleukin-2 (IL-2), interferon-α (IFN-α), and lysozyme (LZM), were analyzed. With regard to sex steroids, four important steroids' involvement in seahorse reproduction, i.e., testosterone (T), 11-ketotestosterone (11-KT), 17βe s t r a d i o l ( E 2 ) , a n d 1 7 α -h y d r o x y -2 0 βdihydroprogesterone (17α-20β-P) (Oconer et al. 2003;Poortenaar et al. 2004;Scobell and MacKenzie 2011), together with 11β-hydroxytestosterone (11β-OHT), another androgen for reproduction regulation being detected in the pipefishes (Syngnathidae, a close relative of the seahorse) (Mayer et al. 1993) and many other fishes (Rosenblum et al. 1985), and 17α-methyltestosterone (17α-MT), an androgen association with sex reversal in many fishes (Murray et al. 2016), were analyzed. Moreover, given the significant morphological changes of the brood pouch during pregnancy, in the present study, the variations of immune metabolic activity of the brood pouch epithelium at different pregnant stages were also examined through ultrastructural observations of the abundance of immune metabolism-related cellular components, such as cytoplasmic granules, mitochondria, endoplasmic reticulum, lysosomes, and exocytosis, an important process that involves the transportation of the secreted cytoplasmic granules out of the cell (Gandasi and Barg 2014). ...
Article
Full-text available
To better understand the endocrine- and immune-response pattern during reproduction in a fish species having parental care behaviors and also to accumulate the endocrine- and immune-related data for future explanations of the low reproductive efficiency in seahorse species, the variations of immune factors and sex steroids in the plasma of the male lined seahorse Hippocampus erectus at different breeding stages, i.e., pre-pregnancy, pregnancy (early, middle, and late periods), and post-pregnancy, were investigated in the present study. The immune factors included monocytes/leucocytes (M/L), leucocyte phagocytic rate (LPR), immunoglobulin M (Ig M), interleukin-2 (IL-2), interferon-α (IFN-α), and lysozyme (LZM). The sex steroids included testosterone (T), 11-ketotestosterone (11-KT), 11β-hydroxytestosterone (11β-OHT), 17α-methyltestosterone (17α-MT), 17β-estradiol (E2), and 17α-hydroxy-20β-dihydroprogesterone (17α-20β-P). Moreover, the immune metabolic activity of epithelium cells in the brood pouch at different breeding stages was also analyzed through ultrastructural observations of the abundance of cytoplasmic granules, mitochondria, endoplasmic reticulum, lysosomes, and exocytosis. The results show that a higher immune level was observed during pregnancy, particularly in the early and middle periods, and a lower immune level was noted during pre-pregnancy. Correspondingly, the epithelium cells in the brood pouch also showed a stronger immune metabolic activity during pregnancy and weaker activity during pre-pregnancy. Four sex steroids of T, 11β-OHT, 17α-MT, and E2 were higher during pre-pregnancy and lower during post-pregnancy, whereas 11-KT and 17α-20β-P, which were positively correlated with part immune factors, were higher during pregnancy. No negative correlations between sex steroids and immune factors were observed. In conclusion, the higher immune competence during pregnancy may indicate that parental care could improve immunity, which may be the major factor for no immunosuppressive effect of sex steroids during reproduction in the seahorse H. erectus, unlike noncaregiving fishes in which inhibitions of sex steroids on immunity are frequently observed. Moreover, higher 11-KT and 17α-20β-P during pregnancy than during pre-pregnancy and post-pregnancy may suggest that these two steroids are also involved in parental care regulation.
Article
Full-text available
Sexual identification of crocodilians is important in population studies and provides useful information for conservation and management plans and monitoring populations over time. It is possible to distinguish between male and female Caiman latirostris by cloacal palpation or eversion of the penis in individuals larger than 75 cm total length, but smaller animals possess a barely differentiable cliteropenis. In hatchlings, sex determination methods involve surgical examination, necropsy, or analysis of cranial dimorphism, which cannot be applied in the field. We classified hatchlings of C. latirostris by observing the color and shape of their genitals. The penis is a milky white organ with a rounded shape at the tip and a purple hue at the end, whereas the clitoris is shorter, whitish, and has a pointed end. The procedure was tested on hatchlings from three nests at the Proyecto Yacar study area (Santa Fe province); half of the eggs of each nest were incubated at a constant temperature of 31C (producing females) and the other half at 33C (producing males). To observe the sexual organs by cloacal inspection, we used a modified instrument whose function during palpation is like that of a finger applied in large animals to evert the penis or clitoris. In the first days after hatching we correctly scored the sex of 80% of the individuals. The number of correct identifications was slightly lower for males than for females. This technique might be a useful tool for field studies, as it allows the sex of small caimans to be estimated in situ.
Article
A number of strategies have emerged that appear to relate to the evolution of mechanisms for sexual determination in vertebrates, among which are genetic sex determination caused by sex chromosomes and environmental sex determination, where environmental factors influence the phenotype of the sex of an individual. Within the reptile group, some orders such as: Chelonia, Crocodylia, Squamata and Rhynchocephalia, manifest one of the most intriguing and exciting environmental sexual determination mechanisms that exists, comprising temperature-dependent sex determination (TSD), where the temperature of incubation that the embryo experiences during its development is fundamental to establishing the sex of the individual. This makes them an excellent model for the study of sexual determination at the molecular, cellular and physiological level, as well as in terms of their implications at an evolutionary and ecological level. There are different hypotheses concerning how this process is triggered and this review aims to describe any new contributions to particular TSD hypotheses, analyzing them from the "eco-evo-devo" perspective.
Article
Full-text available
A male-biased sex ratio of 3:1 has been reported for a population of American crocodiles (Crocodylus acutus) in the Tempisque River Basin, Guanacaste, Costa Rica. If confirmed, this would constitute one of the largest male-biased sex ratios reported for any population of a member of the genus Crocodylus. Here, we examine the aforementioned population of C. acutus and report on sex ratios of hatchling, juvenile, and adult age classes within a sample of 474 crocodiles captured in the Tempisque Basin between May 2012 and June 2014. Hatchling sex ratio is exceptionally male biased (3.5:1), an imbalance that is maintained in juveniles but is reduced in adults (1.5:1). Mark–recapture data document that juvenile males disperse from the study site, potentially to avoid competition, a process that reduces male bias in the adult age class. An increased role of males in human–crocodile conflict may be a result of juvenile males dispersing to human-inhabited areas.
Article
Full-text available
Tilapias are an important component of subsistence fisheries for thousand of years but have gained prominence in recent years in areas where they are not endemic. Introductions of better performing tilapia species/strains and development of techniques to manage unwanted reproduction have spurred significant developments that led to success in tilapia farming. Of the 70 species of tilapias, 9 are used in farming and of these, Nile tilapia (Oreochromis niloticus) is the main cultured species and responsible for the significant increase in global tilapia aquaculture production. Tilapia contributed 1.27 million metric tons in 2000 or 3.57% of global aquaculture production. The major producing countries are China, Egypt, Thailand, Philippines and Indonesia.
Article
Full-text available
Wildlife species have been recognized as sentinels of environmental health for decades. In fact, ecological data on various wildlife populations provided the impetus for banning some organochlorine pesticides over the last few decades. Alligators are important sentinels of ecosystem health in the wetlands of the southeastern United States. Over the last 15 years, a series of studies have demonstrated that environmental exposure to a complex mixture of contaminants from agricultural and municipal activities alters the development and functioning of alligators' reproductive and endocrine systems. Further studies of basic developmental and reproductive endocrinology in alligators and exposure studies performed under controlled laboratory conditions support the role of contaminants as causal agents of abnormalities in gonadal steroidogenesis and in reproductive tract development. These studies offer potential insight into environmentally induced defects reported in other wildlife and human populations exposed to a wide array of endocrine-disruptive contaminants.