Original Article

Sex Dev 119

DOI: 10.1159/000XXXXXX

Received: May 15, 2009

Accepted: July 22, 2009
Published online: $$$

Gonadal Morphogenesis and Gene Expression

in Reptiles with Temperature-Dependent
Sex Determination
H. Merchant-Larios V. Daz-Hernndez A. Marmolejo-Valencia
Instituto de Investigaciones Biomdicas, Universidad Nacional Autnoma de Mxico, Ciudad Universitaria,
Mxico City, Mexico

Key Words
Gonadal morphogenesis SF1-CYP19 -sex determination
SOX9-AMH-Sertoli cells Temperature-dependent sex
determination Thermosensitive period

Abstract
In reptiles with temperature-dependent sexual determination, the thermosensitive period (TSP) is the interval in which
the sex is defined during gonadal morphogenesis. One-shift
experiments in a group of eggs define the onset and the end
of the TSP as all and none responses, respectively. Timing for
sex-undetermined (UG) and -determined gonads (DG) differs at male- (MPT) or female-producing temperatures (FPT).
During the TSP a decreasing number of embryos respond to
temperature shifts indicating that in this period embryos
with both UG and DG exist. Although most UG correspond
to undifferentiated gonads, some embryos extend UG after
the onset of histological differentiation. Thus, temperature
affects gonadal cells during the process of morphogenesis,
but timing of commitment depends on individual embryos.
A correlation between gonadal morphogenesis, TSP, and
gene expression suggests that determination of the molecular pathways modulated by temperature in epithelial cells
(surface epithelium and medullary cords) holds the key for a
unifying hypothesis on temperature-dependent sex determination.
Copyright 2009 S. Karger AG, Basel

2009 S. Karger AG, Basel

16615425/09/00000000$26.00/0
Fax +41 61 306 12 34
E-Mail karger@karger.ch
www.karger.com

SXD119.indd 1

Accessible online at:

www.karger.com/sxd

Sexual reproduction of multicellular organisms involves the formation of 2 highly specialized cells known
as germ cells or gametes. In adult animals, females generate oocytes while males produce spermatozoa. In the
19th century, August Weissman coined the concept that
multicellular organisms are essentially formed by 2 kinds
of cells: germ cells and somatic cells. Thus, species survival depends, among other things, on the harmonic interaction between these 2 kinds of cells during the life
cycle of individuals.
In order to produce fertile oocytes or spermatozoa,
gonochoristic animals (individuals who display either
male or female sex, in contrast with hermaphrodites in
which both sexes are simultaneously present in one individual) have evolved great diversity of developmental and
physiological strategies to reach the same end, namely to
differentiate into the male or the female phenotype. At
least 2 aspects must be considered to outline the complex
process of sexual development among gonochoristic species: (a) sex determination and (b) sex differentiation.
Primary sex determination concerns the genetic or environmental factors responsible for the onset of the male
or female pathways that lead to the establishment of the
individuals sex [Bull, 1985]. While genetic sex determination (GSD) is a closed developmental system, environmental sex determination (ESD) is an open system in
which environmental factors (e.g. temperature) modulate
the male or female genetic pathways. It is generally acH. Merchant-Larios
Instituto de Investigaciones Biomdicas, UNAM
Circuito Escolar, Ciudad Universitaria
Apartado Postal 70228, Mxico, D.F. (Mxico)
Tel. +52 55 5616 2668, Fax +52 55 5622 3148, E-Mail merchant @ servidor.unam.mx

29.10.2009 13:19:37

Table 1. Shift experiments of eggs of

L. olivacea from male-producing

temperature (MPT, 26 8 0.5 C) to
female-producing temperature (FPT,
33 8 0.5 C) and the other way round
at different developmental stages

Shift

Days of
Stage
incubation

Eggs
n

Females Males

Response to shifted
temperature, %

MPT to FPT

19
25
27
31
34
38

20/21
21/22
23
23.5
24
25

8
5
12
26
14
9

8
5
11
8
0
0

0
0
1
18
14
9

100
100
91
31
0
0

FPT to MPT

19
22
2527
28

24
24/25
25
26/27

14
22
33
9

0
20
33
9

14
2
0
0

100
9
0
0

cepted that during development, secondary sex determination occurs in the gonadal anlagen. Once secondary
sex determination is established at molecular level in the
morphologically undifferentiated gonad, a complex network of gene expressions leads to differentiation of either
ovaries or testes. As soon as gonadal differentiation occurs, sex differentiation of wolffian and mllerian ducts
and urogenital sinus follows. This process depends on the
endocrine activity of differentiated testes [Jost, 1947].
The goal of the present study is to look for unifying
aspects concerning the role played by temperature in gonadal morphogenesis and gene expression in some species with temperature-dependent sex determination
(TSD). Considering the diversity of temperature regimens and the variety of responses in different species, we
applied a comparative approach using the Olive Ridley
sea turtle (Lepidochelys olivacea) as a reference.

Material and Methods

One-Shift Experiments
In a previous study, 1,000 eggs of L. olivacea were used for oneshift experiments in which 32 C and 27 C were the female-producing temperature (FPT) and the male-producing temperature
(MPT), respectively [Merchant-Larios et al., 1997]. A total of 152
L. olivacea eggs from 2 clutches were used for one-shift experiments; 74 eggs were shifted from MPT (26 C) to FPT (33 C) and
78 eggs were shifted from FPT to MPT at different developmental
stages, as indicated in table 1.
Light and Electron Microscopy
Samples were processed for high-resolution light and electron
microscopy as previously described [Merchant-Larios et al., 1989].
Briefly, samples were fixed in Karnovsky buffer, postfixed in 1%
OsO4 in Zetterqvists buffer, and embedded in Epon 812. Semithin (1 m) and thin (60 nm) sections were stained with toluidine
blue and uranyl acetate/lead citrate, respectively.

SXD119.indd 2

Sex Dev 119

Frozen Section in situ Hybridization

A turtle SOX9 fragment of 597 bp and an AMH fragment of
736 bp were generated by PCR amplification of cDNA from MPT
gonads at stage 27. PCR primers were: SOX9-F: 5-AGG AAG TCG
GTG AAG AAC G-3; SOX9-R: 5-CTT GAT GTG TGT CCT
CTG CTG-3; AMH-F: 5-CTG GAG ACG GTC CCT TAT C-3;
AMH-R: 5-GGC CCT CAC AGT TGT TGG-3. The PCR products were cloned into the pGEM-T easy vector system I (Promega).
Fragment identity and insertion orientation were verified by sequencing. Plasmids containing the turtle SOX9 or AMH fragment
were linearized by restriction digestion and used as templates for
either SP6 or T7 RNA polymerase (Promega). In vitro reactions
generated antisense and sense riboprobes, and these were labeled
with digoxigenin-UTP (DIG RNA labeling mix, Boehringer).
Non-incorporated nucleotides were removed by spin column
(DyeEx 2.0 SpinKit, Qiagen). Test or control samples were hybridized with antisense or sense riboprobes, respectively.
Samples for in situ hybridization were fixed overnight in cold
4% paraformaldehyde in PBS, washed in increasing sucrose concentrations (10%, 20%, and 30%) and in 1:1 OCT-30% sucrose at
4 C. Samples were embedded in OCT, frozen in dry ice, and
stored at 70 C. Briefly, 10-m frozen sections were washed and
prehybridized for 2 h at 57 C in hybridization solution (50% formamide, 1.3% SSC pH 7, 5 mM EDTA pH 8, 0.2% Tween 20, 0.5%
CHAPS, 100 g/ml heparin, and 50 g/ml yeast RNA). Riboprobes were added and hybridized overnight. Sections were
blocked in 1% blocking reagent (Roche), incubated with preabsorbed antibody (Anti-digoxigenin-AP Fab fragment, Boehringer) and stained with BM Purple AP Substrate, precipitating
(Roche).
Double Immunofluorescence of SOX9 and AMH
SOX9 and AMH were sequentially detected in 10-m frozen
sections. These were washed in PBS and incubated in 10 mM sodium citrate, pH 6, at 85 C for 45 min. The slides were blocked
with 3% inactivated horse serum and 1% BSA in PBT (1% Triton/
PBS) for 2 h, and subsequently incubated overnight first with rabbit anti-SOX9 primary antibody diluted 1:250 at 4 C. After washing with PBT, sections were incubated in FITC-conjugated donkey anti-rabbit secondary antibody (Chemicon International,
Inc.) for 1 h at room temperature, then sequentially blocked with
10% serum horse and 3% BSA in PBT for 2 h, and incubated over-

Merchant-Larios/Daz-Hernndez/
Marmolejo-Valencia

29.10.2009 13:19:47

night with goat anti-AMH antibody (C-20 Santa Cruz) diluted

1:300 at 4 C. After washing with PBT, sections were incubated in
TRITC-conjugated donkey anti-goat antibody. Negative controls
without the primary antibodies were included. Images were captured and processed with a confocal Pascal LSM5-Zeiss microscope.

The Thermosensitive Period (TSP) and Gonadal

Sexual Determination
Incubation temperature affects the whole developing
embryo; thus, the possibility of an indirect effect on gonadal sex determination should not be completely discarded. Dramatic differences in development rate (time
required to pass from one developmental stage to the
next) have been described in embryos of all species that
show TSD. Figure 1 shows a comparative time-table of the
different developmental rates of L. olivacea embryos. Incubated at female-producing temperature (FPT: 33 C),
embryos develop faster than at male-producing temperature (MPT: 26 C), in which they develop at a slower rate.
At FPT, complete embryonic development lasts only 45
days, while at MPT embryos hatch at around 75 days of
incubation. Thus, at the same chronological age embryos
incubated at different temperatures are at a different developmental stage.
The so-called thermosensitive period (TSP) corresponds to the development stages during which a given
group of embryos responds to temperature [Yntema,
1979; Pieau and Dorizzi, 1981]. However, this definition
fails to clarify the state of each individual embryo during
this period. Individual embryos of a given group (a clutch
in natural conditions or an experimental group) apparently become determined at different times in an all or
none process [Wibbels et al., 1991a]. Developmental stages based on morphological criteria of whole embryos
[Ferguson, 1985; Miller, 1985; Yntema, 1968] are used to
find out the TSP, regardless of differences in the developmental rate caused by FPT or MPT. In L. olivacea, the TSP
is situated between stages 22 and 27. However, the time to
reach these stages at constant temperatures plays a role in
the timing of sex determination.
Temperature shift experiments showed that almost
100% of L. olivacea embryos incubated first at FPT (33 C)
responded to MPT by forming testes when shifted at
stage 24; only around 30% responded at stage 25 and none
at stage 26. On the other hand, all embryos incubated first
at MPT (26 C) responded to FPT at stage 23 by forming
ovaries; around 35% responded at stage 24 and none at
Gene Expression in Embryonary Gonads
of Reptiles with TSD

SXD119.indd 3

70

St: 31

Undetermined period (UP)

65

Thermosensitive period (TSP)

60
55

Sex determined period (DP)

50
45

Days

Results and Discussion

75

St: 31

40
35

St: 25

30

St: 24

25
20
15

St: 26

St: 23

St: 25
St: 24

10
5
0

26C

33C
Temperature

Fig. 1. Correlation between age (days) and developmental stage in

embryos of L. olivacea incubated at 26 C (MPT) or at 33 C (FPT).
According to Millers table, hatching of sea turtles occurs at stage
31. Embryonic development is divided into 3 periods: undetermined (green), thermosensitive (red), and sex determined (blue)
considering percentage of embryos responding to the opposite
temperature in one-shift experiments. All embryos incubated at
FPT respond at stage 24 (undetermined), while some embryos incubated at MPT fail to respond (determined). At MPT all embryos are determined at stage 25, while at FPT this state is reached at
stage 26.

stage 25 (fig. 1). Thus, for these temperatures (26 C and

33 C) all slowly developing embryos incubated first at
MPT are determined as males at stage 25, while their faster developing counterparts incubated first at FPT are all
determined as females one morphological stage ahead, at
stage 26 [Torres-Maldonado et al., 2001]. Figure 1 correlates timing, temperatures, and developmental stages
based on one-shift experiments. Three periods of response to temperature are evident: (a) undetermined
(100% response), (b) TSP (decreasing percentage of response), and (c) determined (zero response). Evidently, all
these parameters are somewhat modified in accordance
with the temperature as has been found using incubation
temperatures of 27 C and 32 C [Merchant-Larios et al.,
1997] instead of 26 C and 33 C used here.
To make sure that most embryos incubated at 26 C
respond to FPT and differentiate as female hatchlings,
embryos must be shifted at stage 23. Thus, older embryos
(chronological dimension) become determined as males
Sex Dev 119

29.10.2009 13:19:48

Fig. 2. Semi-thin section of the genital ridge of L. olivacea at stage

23. This stage corresponds to undetermined embryos incubated

at both temperatures. Ingrowing cells from the cortex are shown
(black arrow). Irregular medullary cords (red arrow) are randomly distributed. Primordial germ cells (yellow asterisks) are surrounded by epithelial cells both at the cortex and the medulla of
the genital ridge. Bar = 20 m.

at younger stages (morphological criteria) than the fasterdeveloping females at higher temperatures. In other
words, at the constant temperatures used in the laboratory, care must be taken when comparing levels of gene
expression in gonads from embryos incubated at the same
stage but incubated at different temperatures. That is, to
reliably compare levels of gene expression in undetermined (all responding) and determined (all non-responding) embryos, the first and the last stages of the TSP
at a given temperature range must be chosen since intermediate stages include determined and undetermined
embryos.
In L. olivacea, a reliable approach in embryos incubated at FPT (33 C) is to compare the levels of gene expression of undetermined stage 24 gonads with the gene
expression levels of determined stage 27 gonads. However, to compare levels of gene expression in undetermined and determined embryos incubated at MPT
(26 C), gonads from stages 23 and 26 have to be taken.
Gonads at stages 25 and 24 from embryos incubated at
FPT and MPT, respectively, include a number of sex-determined embryos defined by the primary temperature
prior to the shift to the secondary temperature [Merchant-Larios et al., 1997; our present results].
Thermosensitive Period: Deterministic or Probabilistic
Individual Response?
One-shift experiments indicate that individual eggs of
a given group (from one or several nests) are sex-determined at different times during the TSP. This heterogeneous response may be explained in terms of a determin4

SXD119.indd 4

Sex Dev 119

istic theory, assuming that embryos are functionally different due to the cumulative effects of many small
differences (maternal effects, e.g. yolk steroid levels and
genetic background). In comparison, the probabilistic
theory assumes that there are no relevant individual differences in the response to temperature; rather, temperature-dependent sex determination is a stochastic process,
impossible to predict.
The deterministic theory may be supported by results
in turtles [Yntema, 1979; Etchberger et al., 1991; Mrosovsky and Pieau, 1991] and crocodiles [Lang and Andrews, 1994] demonstrating that clutch of origin is an
important source of variation in the TSP response. Quantitative differences in response include 2 temperature parameters: potency (degrees) and magnitude (duration)
[Wibbels et al., 1991a]. However, since the factor(s) responsible for differences in response to temperature between clutches remains unknown, there is some room for
a probabilistic explanation. If one cell type within the gonad initiates the whole process to organize either testes
or ovaries, the response of such cells to temperature could
be probabilistic. A correlation between timing of gonadal morphogenesis and the so-called temperature-sensitive period may help to clarify this possibility.
The Establishment of the Genital Ridge during
Temperature-Dependent Sex Determination
Results with L. olivacea incubated at 26 C (MPT) or
33 C (FPT) correlate with the well-known fact that the
TSP covers approximately 4 stages (2326) during the
second third of the incubation period. At stage 26, male
and female gonads can barely be distinguished. Then, as
in most amniotic vertebrates, the genital ridge begins to
form as a thickening of the coelomic epithelium on each
side of the hindgut mesentery along the ventromedial
surface of the mesonephric kidneys [Raynaud and Pieau,
1985].
Primordial germ cells (PGCs) migrate to the genital
ridge from the posterior germinal crescent through the
dorsal mesentery [Cuminge et al., 1986; Merchant-Larios
et al., 1989]. Once in the genital ridge, PGCs are incorporated into the thickening coelomic epithelium and thereafter some of them become associated with the ingrowing
medullary cords. As in mammalian gonads, 2 histological arrangements are initially established in the gonadal
primordium of the Olive Ridley sea turtle: epithelial and
stromal tissues (fig. 2). The gradual deposit of a basal
lamina on the condensed epithelial cells segregates them
from loose mesenchymal cells and endothelial blood vessels that constitute the stromal tissue (fig. 3). InvaginaMerchant-Larios/Daz-Hernndez/
Marmolejo-Valencia

29.10.2009 13:19:48

sc

mc
50 m

50 m

50 m

50 m

Fig. 3. Electron micrographs of the sample shown in fig. 2. A De-

posit of a thin basal lamina (arrowheads) on the surface of epithelial cells forming the medullary cords (mc) is barely seen. Part of
a cell located in the stromal tissue (sc) is shown. B High magnification of the area shown in the rectangle marked in (A). The basal lamina on the surface of epithelial cells is shown (arrowheads).
At this stage collagen fibers added to the basal lamina are still
scanty (arrows). Bar in (A) = 1.0 m. Bar in (B) = 200 nm.

tions of the coelomic epithelium and continuity of its basal lamina with the medullary cords suggest that medullary cells in both locations share a common lineage
(fig. 2).
Figures 4AC show the comparable histological features of gonads of L. olivacea at stage 25 at both FPT and
MPT. Although still incipient, the 2 classical topographic regions, medulla and cortex [Witschi, 1967], can already be recognized. The first histological differences between gonads at MPT and FPT are seen at stage 26
(fig. 4D). While medullary cords become conspicuous as
a complex network in gonads at MPT, gonads from embryos incubated at FPT start a fragmentation-like process, and thickening of the surface epithelium forms the
ovarian cortex (fig. 4D). Thereafter, from stage 27 onwards, the medullary cords become seminiferous cords
in testes while they are vestigial in ovaries (not shown).
As stated above, stages 23 and 26 correspond to the onset
and the end of the TSP, respectively. Although the heterogeneous response of gonads at stage 25 incubated at FPT
may be explained by small differences in size and some
histological differences seen in gonads of individual embryos, the heterogeneous response of gonads of individual embryos at stage 24 in MPT cannot be explained in
terms of morphology. Thus, the male pathway becomes
established in early undifferentiated gonads, prior to the
female pathway that remains reversible one more stage.
This timing difference, as revealed in one-shift experiGene Expression in Embryonary Gonads
of Reptiles with TSD

SXD119.indd 5

Fig. 4. Semi-thin sections of gonads of L. olivacea at different stages of development. A and B show gonads from embryos at stage 25

incubated at FPT (33 C). Although both gonads are histologically undifferentiated (medullary cords and surface epithelium
with 1 cell layer), the gonad in (B) is larger than the gonad in (A)
and some medullary cords start fragmentation. At this stage,
around 30% fail to respond when shifted to MPT (26 C). C Gonad
at stage 25 incubated at MPT (26 C). At this stage gonads are determined as testes since all fail to respond when shifted to FPT.
D Gonad at stage 26 incubated at FPT (33 C). At this stage all
ovaries are already determined since they fail to respond when
shifted to MPT. Bar = 50 m.

ments, suggests important differences in setting up the

molecular mechanisms to determine the fate of each sex
in species with TSD.
In contrast to mammalian embryos [Daz-Hernndez
et al., 2008] reptile embryos lack a developmental table
to classify them at equivalent stages. However, figure 5
shows a correlation between the TSP and gonadal morphology using different developmental tables in 3 species
with TSD: Millers table [1985] for L. olivacea, Yntemas
table [1968] for Trachemys scripta, and Fergusons table
[1985] for Alligator mississippiensis. As mentioned above
for L. olivacea, the correlation shown for T. scripta [Wibbels et al., 1991a] and A. mississippiensis [Lang and AnSex Dev 119

29.10.2009 13:19:48

Lepidochelys olivacea (1)

26C
33C
GONAD

ms
22

23

24

25

26

27

28

20

21

Trachemys scripta (2)

26C
31C
GONAD
15

16

17

18

19

ms

Alligator mississippiensis (3)

30C
33C
GONAD
19

20

21

22
23
Stage

24

25

Fig. 5. Correlation between the thermosensitive period (TSP) and

gonadal morphogenesis in 3 species with temperature-dependent
sex determination (TSD). Stage number of each of the 3 species
differs because diverse developmental tables are used according
to different authors: Millers (1985) sea turtle table for L. olivacea;
Yntemas (1968) fresh water turtle for T. scripta, and Fergusons
(1985) for crocodiles for A. mississippiensis. For each species 3 arrows divided in stages are shown. The upper and middle arrows
correspond to percentage of embryos responding to a 2nd incubation temperature (MPT to FPT or the converse) in one-shift experiments. Stages corresponding to the TSP are colored in red,
while yellow and blue indicate stages at which 100% and 0% embryos respond to the 2nd temperature, respectively. The blue lines
drawn through the TSP stages indicate the decreasing number of
individual embryos that respond to the shifted temperature. The
lower arrows designate the morphological state of the gonad at
each developmental stage: white stages correspond to undifferentiated gonads, while grey stages indicate histological distinction
of ovaries and testes. (1) Merchant-Larios et al. [1997]; (2) Wibbels
et al. [1991a]; (3) Smith and Joss [1993]; (4) Lang and Andrews
[1994].

drews, 1994] correspond to one-shift experiments at constant MPT or FPT. Although in the 3 species the onset of
the TSP is labeled when some individual embryos respond to the 2nd temperature and ends when none of
them respond (blue line in fig. 5), each species shows a
different correlation with the morphological state of the
gonads. The most remarkable difference is T. scripta in
which some individuals first incubated at MPT still respond to FPT, forming ovaries 3 stages after histological
differentiation [Wibbels et al., 1991a]. Similarly to L. olivacea [Merchant-Larios et al., 1997], the American alligator shows morphological reversibility only one stage after
6

SXD119.indd 6

Sex Dev 119

Fig. 6. In situ hybridization with a probe of SOX9 on frozen sections of gonads of L. olivacea at stage 27. A and B correspond to
MPT and FPT, respectively. Positive localization of SOX9 delineates the network formed by medullary cords (arrows). Inset is a
high magnification of medullary cords with Sertoli cells expressing SOX9. ms = Mesonephros; g = gonad. Bar in (A) and (B) = 100
m. Bar in inset = 20 m.

the onset of histological differentiation [Smith and Joss,

1993; Lang and Andrews, 1994].
Is the Central Role of Sertoli Cells in Mammalian
Gonadogenesis Conserved in TSD Species?
Regulation of SOX9 expression plays a central role in
sex determination in the gonads of several vertebrates. In
mammals, SOX9 is expressed shortly after SRY (approximately 2 h in mouse) in pre-Sertoli cells and remains expressed in Sertoli cells after birth [Morais da Silva et al.,
1996]. Presumably, signals emitted by Sertoli cells are required for seminiferous cord morphogenesis and Leydig
cell differentiation, i.e., the 2 main features that characterize the functional differentiation of the fetal testis
[Wilhelm et al., 2007].
In L. olivacea embryos incubated at both MPT and
FPT, SOX9 expression occurs in medullary cords at stages 2324. Thereafter, while gonads from embryos incubated at MPT (26 C) maintain SOX9 expression in medullary cords, in gonads at FPT (33 C) few medullary cells
remain SOX9-positive at stage 25 and none at stage 26
and after [Moreno-Mendoza et al., 1999; Torres-Maldonado et al., 2002]. As stated above, if shifted from FPT to
Merchant-Larios/Daz-Hernndez/
Marmolejo-Valencia

29.10.2009 13:19:49

Fig. 7. Testis of L. olivacea at stage 27.

A Semi-thin section showing medullary

cords (mc) and the surface epithelium

formed by a layer of epithelial cells and
some germ cells (arrowheads). B Immunofluorescence with an antibody against
SOX9. Nuclei of Sertoli cells appear green
stained. C Red staining of AMH. D Merging images (B) and (C). E Expression of
AMH detected by ISH in a frozen section.
Positive Sertoli cells are clearly seen. Bar in
(A) = 40 m.

mc

20 m

20 m

20 m

MPT at stage 24, all embryos respond, but at stage 26 or

later almost all fail to respond. Thus, as in mammals, in
L. olivacea there is a correlation between SOX9 downregulation and ovarian determination (fig. 6).
On the other hand, anti-mllerian hormone (AMH) is
a functional marker for Sertoli cell differentiation considering its role in the regression of mllerian ducts in the
male fetus [Josso et al., 2001]. In L. olivacea, AMH detection closely follows that of SOX9 in medullary cords of
embryos incubated at MPT, while it is undetectable in
medullary cords of gonads at FPT prior and after SOX9
extinction (unpublished results). The temporal correlation suggests that both genes in L. olivacea are in the same
pathway where SOX9 up-regulates AMH in pre-Sertoli
cells leading to their differentiation as Sertoli cells (fig. 7).
If this assumption holds true for the Olive Ridley sea turtle, the mechanism(s) mediating sex determination may
be involved in Sertoli cell determination as it occurs in
mammals.
Temperature shift experiments show that testis determination correlates with SOX9 maintenance and both
processes precede ovarian determination. It is tempting
to speculate that FPT promotes ovarian formation in
L. olivacea by a mechanism that down-regulates the constitutive expression of SOX9 in bipotential medullary
cords preventing Sertoli cell differentiation. An ultrastructural study made in developing gonads of the American alligator seems to support this hypothesis [Smith
and Joss, 1993]; however, in contrast with L. olivacea,
SOX9 follows expression of AMH in Sertoli cells [Western
et al., 1999], as seen in chick embryos [Oreal et al., 1998].
If future studies prove that the ultrastructural criteria
used to identify Sertoli cells in A. mississippiensis coincide with the expression of AMH in the same cells, pos-

sibly Sertoli cells in non-mammalian vertebrates can be

determined by the same genes but which are expressed in
a different order. Interestingly, AMH up-regulation in
mammals coincides with the onset of seminiferous cord
formation, and this occurs long before its product is required for regression of the Mllerian ducts [Behringer
et al., 1990; Arango et al., 1999; Daz-Hernndez et al.,
2008]. The finding that Amh null mice express Sox9 and
form testis suggests that Amh in mammals is not required
any more for Sertoli cell determination and its precocious
expression in pre-Sertoli cells reflects an evolutionary
remnant. Thus, it is tempting to speculate that non-mammalian species might still require AMH for Sertoli cell
determination and/or differentiation.
In the American alligator, expressed levels of SF1 are
higher in ovaries than in testes [Western et al., 2000], as
found in chick embryos [Smith et al., 1999]. Interestingly,
the fact that higher temperature in crocodiles produces
males [Ferguson and Joanen, 1982; Lang and Andrews,
1994] instead of females, as seen in turtles, indicates that
different temperature-sensitive pathways have evolved in
diverse TSD species. Paleontological and protein sequence studies indicate close phylogenetic affinity between crocodilians and birds [Wang and Conlon, 1993;
Gabriel et al., 2001] and may explain the higher expression of SF1 in testes than in ovaries as seen in turtles and
mammals [Ikeda et al., 1994; Ramsey et al., 2007] (fig. 8).
However, results showing that extremely higher temperatures also produce females in the alligator [Lang and Andrews, 1994] could be due to 2 timing effects on the critical balance of the sex determination pathway. In other
words, in alligators low temperature favors lower levels of
SF1 in pre-Sertoli cells, while in turtles a similar temperature up-regulates its expression in these cells. If, as in

Gene Expression in Embryonary Gonads

of Reptiles with TSD

Sex Dev 119

SXD119.indd 7

29.10.2009 13:19:50

GONAD

22

23

24

25

26

27

28

SOX9
AMH
DAX1
DMRT1

(1)
(2)
(1)
(1)

GONAD

T. scripta
15

16

17

18

19

20

21
(3)
(3)
(3)
(4)
(5)
(6)

SOX9
AMH
DMRT1
WT1
SF1
CYP19
GONAD
SOX9

L. olivacea

A. mississippiensis
19

AMH
DAX1
WT1
SF1
CYP19

20

21

22

23

24

25

(7)
(7)
(8)
(8)
(8)
(9)

Fig. 8. Correlation between gonadal histological state (undifferentiated and differentiated) and gene expression of the Olive Ridley, the red eared snapping turtle, and the American alligator. Excepting DAX1 and WT1, all other genes exhibit sex-dimorphic
expression. Although SOX9 is expressed in undifferentiated stages in turtles, its expression in the alligator is delayed to differentiated testis after AMH expression. DMRT1 expression is higher in
gonads at MPT both before and after histological differentiation
in turtles. CYP19 expression appears higher in differentiated ovaries in T. scripta and A. mississippiensis. Expression of SF1 is higher before and after histological differentiation at MPT in T. scrip-

ta in contrast to A. mississippiensis which shows low levels at both

temperatures in undifferentiated gonads and an increase at FPT
in differentiated ovaries. Arrows are divided into developmental
stages: white and grey stages correspond to undifferentiated and
differentiated gonads, respectively. Lines representing gonads
from embryos at MPT are blue, while gonads at FPT are pink. Line
thickness indicates relative levels of gene expression. (1) TorresMaldonado et al. [2002]; (2) present results; (3) Shoemaker et al.
[2007]; (4) Spotila et al. [1998]; (5) Fleming and Crews [2001]; (6)
Ramsey et al. [2007]; (7) Western et al. [1999]; (8) Western et al.
[2000]; (9) Gabriel et al. [2001].

mammals, SF1 together with other protein partners interacts with putative SOX9 enhancers at different steps of
the sex determination pathway [Sekido and Lovell-Badge,
2008], up- or downregulation of SOX9 in species with diverse regimens of TSD may depend on the presence of
different temperature-sensitive SF1 partners. Thus, low
and high female-producing temperatures in crocodiles
may downregulate SOX9 at 2 different steps.
Recent evidence suggests that Sf1, besides its role in
maintenance of the early genital ridge, is together with
Sry also involved in the regulation of Sox9 expression in
murine gonads. Acting cooperatively, SRY and SF1 first
bind to the enhancer named TESCO (testis-specific enhancer of Sox9 core) to upregulate Sox9. Then, SOX9 and
SF1 also bind the enhancer to maintain Sox9 expression

after SRY has ceased [Sekido and Lovell-Badge, 2008].

Although these authors failed to find testis-specific enhancer (TES) sequences in non-mammalian species, it is
likely that a similar enhancer mechanism exists in TSD
species where SOX9 plays a central role in Sertoli cell differentiation. In contrast to SRY, SF1 is highly conserved
and shows dimorphic expression in gonads of non-mammalian species [Fleming et al., 1999; Smith et al., 1999;
Western et al., 2000]. Thus, a putative temperature-sensitive SF1 partner may bind a TES-like enhancer at MPT to
maintain SOX9 expression in Sertoli cells. Alternatively,
at FPT, interaction and/or binding to a TES-like enhancer might be prevented and consequently follow the ovarian pathway.

SXD119.indd 8

Sex Dev 119

Merchant-Larios/Daz-Hernndez/
Marmolejo-Valencia

29.10.2009 13:19:51

SF1, P450arom (CYP19), and Estrogen Levels in TSD

In species with TSD there is a consistent effect of exogenous estradiol counteracting the effect of MPT to induce ovarian formation instead of testis [Pieau, 1996].
This hormonally induced gonadal differentiation [Gutzke and Chymiy, 1988] also counteracts the male genetic
background in reptiles with GSD [Freedberg et al., 2006].
Moreover, inhibition of aromatase activity, the enzyme
required for estrogen production, in embryos of a parthenogenetic all-female lizard, resulted in differentiation of
testes [Wibbels and Crews, 1994]. These results strongly
point to a highly conserved role of estrogens in sex determination and/or sex differentiation of reptilian species
with diverse mechanisms of sex determination. Extensive
studies in 2 fresh water turtles, Emys orbicularis [review
in Pieau and Dorizzi, 2004] and T. scripta [Wibbels et al.,
1991b], support the hypothesis of a direct or indirect regulation of P450arom (CYP19) (transcriptional, post-transcriptional, and/or enzyme activity) as the putative sensor that mediates temperature-dependent sex determination.
Even though most previous reports failed to show significantly different levels of CYP19 transcripts in mesonephros-adrenal-gonad complexes at MPT or FPT prior
to morphological differentiation [Willingham et al.,
2000; Crews et al., 2001; Gabriel et al., 2001; Murdock and
Wibbels, 2003], recent results show clear differences in
isolated gonads of Chelydra serpentina [Rhen et al., 2007]
and T. scripta [Ramsey and Crews, 2007] (fig. 8). Therefore, the hypothesis that aromatase may play a central
role in gonadal sex differentiation [Pieau et al., 1999] is
still sustained.
The steroidogenic factor (SF1), being a global mediator
of steroidogenesis [Caron et al., 1997], became an obvious
candidate as putative regulator of estradiol levels in gonads of TSD species. Fleming and Crews [2001] found
that temperature and exogenous estradiol modulate the
expression of SF1 in gonads of the red-eared turtle T.
scripta. Its sex-dimorphic expression is detected early in
the undifferentiated genital ridge (stage 15) and remains
higher in testes than in ovaries at all stages. In situ hybridization with radioactive probes revealed that SF1 appears in the medullary cords, probably in pre-Sertoli and
Sertoli cells as in mammals. Even though SRY homologues are absent in non-mammalian species, their mammalian partner, SF1 [Sekido and Lovell-Badge, 2008], is
dimorphically expressed in developing gonads of TSD
reptiles [Western et al., 2000; Fleming and Crews, 2001;
Ramsey et al., 2007]. SF1 acts as transactivator of most
steroidogenic enzymes, including P450arom [Morohashi

et al., 1992; Morohashi, 1999]. Both classical [Pieau, 1974]

and recent [Crews et al., 2001; Pieau and Dorizzi, 2004]
studies in TSD species have shown that estradiol counteracts the effect of MPT and induces ovarian differentiation. Moreover, P450arom inhibitors produce partial or
complete testis differentiation at FPT, which reinforces
the notion that differential levels of estrogenic hormones
are involved in gonadal sex determination and/or sex differentiation [Wibbels et al., 1991b; Lance and Bogart,
1992; Ramsey et al., 2007]. Experimental results in the
red-eared slider turtle (Trachemys scripta elegans) suggest
that estradiol directly or indirectly regulates SF1 expression [Fleming and Crews, 2001].
How do different levels of estradiol induce and/or inhibit growth and differentiation of either medullary cords
or epithelial cortex? Since the action of these 2 concomitant processes implies regulation of cell proliferation and
cell differentiation in both compartments, it is necessary
to know the location of cells expressing genes relevant for
estrogen production. The bipotentiality of epithelial cells
to respond to exogenous estrogen can be explained by the
presence of estradiol receptors (ER) in medullary cords
of undifferentiated gonads of Emys orbicularis incubated
at MPT or FPT [Bergeron et al., 1998]. However, the estrogen experiments in TSD species have raised the question if the sex reversal induced by exogenous estradiol
or aromatase inhibitors actually mimics the endogenous
mechanism that regulates TSD in intact gonads. In fact,
recent results indicate different steroid signaling patterns
of temperature- and estradiol-induced ovarian differentiation [Ramsey and Crews, 2007]. Thus, although the
end result (ovarian formation) is similar, the feminizing
mechanisms of temperature and exogenous estradiol appear to be different.
A morphological foundation for the development of
either sex exists in each individual embryo; thus, sex determination may take place in either direction prior to
irreversible differentiation of ovaries or testes. The actual
molecular mechanism(s) underlying the role of endogenous estradiol on the decision between 2 alternative directions of gonadal development remains to be determined. Considering that gonadal sex determination precedes gonadal sex differentiation, endogenous estradiol
levels within the gonad may affect either of the 2 processes or both. Therefore, clear correlation in timing of endogenous estrogen levels, morphological stage of the
gonad, and final sex outcome assessed in hatchlings
(one-shift experiments) may elucidate the role of temperature-induced sex commitment under physiological levels of estradiol.

Gene Expression in Embryonary Gonads

of Reptiles with TSD

Sex Dev 119

SXD119.indd 9

29.10.2009 13:19:52

Concluding Remarks and Perspectives

Figure 8 summarizes the correlation of the histological state of the gonad with levels of gene expression in 3
species with TSD. Except DAX1 and WT1, all other genes
show dimorphic expression in the 3 species L. olivacea
[Torres-Maldonado et al., 2002; present results], T. scripta [Fleming et al., 1999; Murdock and Wibbels, 2003;
Ramsey et al., 2007; Shoemaker et al., 2007], and A. mississippiensis [Western et al., 1999, 2000; Gabriel et al.,
2001]. However, there is ample diversity in both timing of
expression and levels of temperature-dependent expression between species. In T. scripta [Shoemaker et al.,
2007], AMH is upregulated in undifferentiated gonads
prior to SOX9 upregulation in undifferentiated and differentiated gonads, respectively. While in A. mississippiensis [Western et al., 1999], although AMH upregulation
also precedes SOX9 upregulation, this process occurs in
gonads that have initiated histological differentiation. In
T. scripta [Ramsey et al., 2007] and A. mississippiensis
[Gabriel et al., 2001], dimorphic levels of CYP19 are detected after histological sex differentiation. In contrast,
T. scripta shows higher levels of SF1 in undifferentiated
and differentiated gonads at MPT, while in gonads of
A. mississippiensis levels of SF1 remain constant at FPT
but are lowered at MPT concomitant with the onset of
histological differentiation [Western et al., 2000].
As discussed above, understanding reversibility of
dimorphic gene expression in undifferentiated gonads
prior to histological differentiation is easier than reversibility as soon as ovaries and testes are distinct. Once
medullary cords start regression and surface epithelium
thickens at FPT and seminiferous cords begin to differentiate at MPT, a more complex process of sex reversion
is required. Thus, the mechanism underlying histological
sex reversion in embryos with delayed irreversible sex determination remains to be found.
Molecular mechanisms underlying the process of gonadal morphogenesis in TSD species require decisions
between 2 alternative pathways in epithelial cells accord-

SXD119.indd 10

Acknowledgements
This work was supported by research grants from Consejo Nacional de Ciencia y Tecnologa P46679-Q and PAPIIT-UNAM,
IN212507-3. We are indebted to Isabel Prez-Montfort for English
review and suggestions on the manuscript. We are grateful to
Martha Harfush, Cuauhtemoc Peaflores, and Elpidio Lpez of
the Centro Mexicano de la Tortuga for assistance on collecting
nest and to Direccin General de Vida Silvestre, SEMARNAT, for
capture permits.

Arango NA, Lovell-Badge R, Behringer RR: Targeted mutagenesis of the endogenous mouse
Mis gene promoter: in vivo definition of genetic pathways of vertebrate sexual development. Cell 99:409419 (1999).
Behringer RR, Cate RL, Froelick GJ, Palmiter
RD, Brinster RL: Abnormal sexual development in transgenic mice chronically expressing mullerian inhibiting substance. Nature
345:167170 (1990).

References

10

ing with their location (cortical or medullary). Proliferation of cells at the surface epithelium originates the medullary cords apparently without losing their epithelial
character. These cells maintain their bipotentiality several days before they become committed, some time after
histological differentiation of the gonad is evident. How
do some degrees of difference in temperature (only 2 C
in the American alligator) [Lance, 2008] lead to the establishment of either the ovarian or the testis pathway? Subtle conformational changes in thermolabile proteins may
cause instability in multimeric molecular complexes required for alternative gene expression in epithelial gonadal cells. This may lead to phenotypic variation due to
environmental conditions. Phenotypic plasticity is defined as the ability of a certain genotype to produce different phenotypes in different environments [Via et al.,
1995]. Thus, it is possible to consider TSD as an example
of phenotypic plasticity. Thermosensitive gene networks
have been investigated in Drosophila where it was found
that temperature interacts with a network of chromatin
regulators involved in chromatin condensation. Temperature modulates the structure of chromatin that can be
compacted and transcriptionally silent or opened and
transcriptionally active [Gibert et al., 2007]. Thus, investigation on epigenetic mechanisms involved in TSD offers new hope to the difficult task of finding a unifying
hypothesis to explain the diverse and somewhat contradictory data presently available.

Sex Dev 119

Bergeron JM, Gahr M, Horan K, Wibbels T,

Crews D: Cloning and in situ hybridization
of estrogen receptor in the developing gonad
of the red-eared slider turtle, a species with
temperature-dependent sex determination.
Dev Growth Differ 40:243254 (1998).
Bull J: Sex determining mechanisms: an evolutionary perspective. Experientia 41: 1285
1296 (1985).

Merchant-Larios/Daz-Hernndez/
Marmolejo-Valencia

29.10.2009 13:19:52

Caron KM, Clark BJ, Ikeda Y, Parker KL: Steroidogenic factor 1 acts at all levels of the reproductive axis. Steroids 62:5356 (1997).
Crews D, Fleming A, Willingham E, Baldwin R,
Skipper J: Role of steroidogenic factor 1 and
aromatase in temperature-dependent sex
determination in the red-eared slider turtle.
J Exp Zool 290:597606 (2001).
Cuminge D, Pieau C, Vasse J, Dubois R: Sur
lorigine des cellules germinales primordiales chez la Cistude dEurope (Emys orbicularis L.) tude exprimentale des stades gastrulens. C R Acad Sci Paris 302: 557560
(1986).
Daz-Hernndez V, Len-del-Ro A, Zamora M,
Merchant-Larios H: Expression profiles of
SRY and SOX9 in rabbit gonads: the classical
model of mammalian sex differentiation.
Sex Dev 2:152166 (2008).
Etchberger RC, Phillips BJ, Ewert AM, Nelson
EC, Prange H: Effects of oxygen concentration and clutch on sex determination and
physiology in red-eared slider turtles
(Trachemys scripta). J Exp Zool 258:394403
(1991).
Ferguson MW: Reproductive biology and embryology of crocodilians; in Gans C, Billett
F, Maderson PFA (eds): Biology of the Reptilian, Vol 14. Development-A, pp 329491
(Wiley, New York 1985).
Ferguson MW, Joanen T: Temperature of egg incubation determines sex in Alligator mississippiensis. Nature 298:850853 (1982).
Fleming A, Crews D: Estradiol and incubation
temperature modulate regulation of steroidogenic factor 1 in the developing gonad
of the red-eared slider turtle. Endocrinology
142:14031411 (2001).
Fleming A, Wibbels T, Skipper JK, Crews D: Developmental expression of steroidogenic factor 1 in a turtle with temperature-dependent
sex determination. Gen Comp Endocrinol
116:336346 (1999).
Freedberg S, Bowden RM, Ewert MA, Sengelaub
DR, Nelson CE: Long-term sex reversal by
oestradiol in amniotes with heteromorphic
sex chromosomes. Bol Lett 2: 378381
(2006).
Gabriel WN, Blumberg B, Sutton S, Place AR,
Lance VA: Alligator aromatase cDNA sequence and its expression in embryos at male
and female incubation temperatures. J Exp
Zool 290:439448 (2001).
Gibert JM, Peronnet F, Schltterer C: Phenotypic plasticity in Drosophila pigmentation
caused by temperature sensitivity of a chromatin regulator network. PLoS Genet 3:226
280 (2007).
Gutzke WH, Chymiy DB: Sensitive periods during embryogeny for hormonally induced sex
determination in turtles. Gen Comp Endocrinol 71:265267 (1988)
Ikeda Y, Shen WH, Ingraham HA, Parker KL:
Developmental expression of mouse steroidogenic factor-1, and essential regulator
of steroid hydroxylases. Mol Endocrinol 8:
654662 (1994).

Gene Expression in Embryonary Gonads

of Reptiles with TSD

SXD119.indd 11

Josso N, di Clemente N, Gouedard L: Anti-mullerian hormone and its receptors. Mol Cell
Endocrinol 179:2532 (2001).
Jost A: Recherches sur la diffrenciation de
lembryon de lapin III. Rle des gonads ftales dans la diffrenciation sexuelle somatique. Arch Anat Microscop Morphol Exp
36:271312 (1947).
Lance VA: Is regulation of aromatase expression
in reptiles the key to understanding temperature-dependent sex determination? J Exp
Zool 309A:17 (2008).
Lance VA, Bogart MH: Disruption of ovarian development in alligator embryos treated with
an aromatase inhibitor. Gen Comp Endocrinol 86:5971 (1992).
Lang JW, Andrews HV: Temperature-dependent
sex determination in crocodilians. J Exp
Zool 270:2844 (1994).
Merchant-Larios H, Villalpando FI, Urruiza CB:
Gonadal morphogenesis under controlled
temperature in the sea turtle Lepidochelys
olivacea. Herpetol Monogr 3:4361 (1989).
Merchant-Larios H, Ruiz-Ramrez S, MorenoMendoza N, Marmolejo-Valencia A: Correlation among thermosensitive period, estradiol response, and gonad differentiation in
the sea turtle Lepiodochelys olivacea. Gen
Com Endocrinol 107:373385 (1997).
Miller JD: Embryology of marine turtles; in
Gans C, Billet F, Maderson PFA (eds): Biology of the Reptilia, Vol 14, pp 270328
(Wiley, New York 1985).
Morais da Silva A, Hacker V, Harley P, Goodfellow A, Swain A, Lovell-Badge R: Sox9 expression during gonadal development implies a
conserved role for the gene in testis differentiation in mammals and birds. Nat Genet 14:
6268 (1996).
Moreno-Mendoza N, Harley V, Merchant-Larios
H: Differential expression of SOX9 in gonads
of the sea turtle Lepidochelys olivacea at
male- or female-promoting temperatures. J
Exp Zool 284:705710 (1999).
Morohashi K: Gonadal and extragonadal function of Ad4BP/SF-1: developmental aspects.
Trends Endocrinol Metab 10: 169173
(1999).
Morohashi K, Honda S, Inomata Y, Handa H,
Omura T: A common trans-acting factor,
Ad4-binding protein, to the promoters of
steroidogenic P-450s. J Biol Chem 267:
1791317919 (1992).
Mrosovsky N, Pieau C: Transitional range of
temperature, pivotal temperatures and thermosensitive stages for sex determination
in reptiles. Amphibia-Reptilia 12: 169179
(1991).
Murdock C, Wibbels T: Cloning and expression
of aromatase in a turtle with temperaturedependent sex determination. Gen Comp
Endocrinol 130:109119 (2003).
Oreal E, Pieau C, Mattei MG, Josso N, Picard JY,
et al: Early expression of AMH in chicken
embryonic gonads precedes testicular SOX9
expression. Dev Dyn 212:522532 (1998).

Sex Dev 119

Pieau C: Diffrenciation du sexe en function de

la temprature chez les embryons dEmys orbicularis L. (Chlonien); effets des hormones
sexuelles. Annls Embryol Morph 7: 365394
(1974).
Pieau C: Temperature variation and sex determination in reptiles. BioEssays 18: 1926
(1996).
Pieau C, Dorizzi M: Determination of temperature sensitive stages for sexual differentiation of the gonads in embryos of the turtle,
Emys orbicularis. J Morphol 170: 373382
(1981).
Pieau C, Dorizzi M: Oestrogens and temperature
dependent sex determination in reptiles: all
is in the gonads. J Endocrinol 181: 367377
(2004).
Pieau C, Dorizzi M, Richard-Mercier N: Temperature-dependent sex determination and
gonadal differentiation in reptiles. Cell Mol
Life Sci 55:887900 (1999).
Ramsey M, Crews D: Adrenal-kidney-gonad
complex measurements may not predict gonad-specific changes in gene expression patterns during temperature-dependent sex determination in the red-eared slider turtle
(Trachemys scripta elegans). J Exp Zool
307A:463470 (2007).
Ramsey M, Shoemaker C, Crews D: Gonadal expression of Sf1 and aromatase during sex determination in the red-eared slider turtle
(Trachemys scripta), a reptile with temperature-dependent sex determination. Differentiation 75:978991 (2007).
Raynaud A, Pieau C: Embryonic development of
the genital system; in Gans C, Billett F (eds):
Biology of the Reptilian, Vol 15, Development-B, pp 149300 (Wiley, New York
1985).
Rhen T, Metzger K, Sachroeder A, Woodward R:
Expression of putative sex-determining
genes during the thermosensitive period of
gonadal development in the snapping turtle,
Chelydra serpentina. Sex Dev 1: 255270
(2007).
Sekido R, Lovell-Badge R: Sex determination involves synergistic action of SRY and SF1 on a
specific Sox9 enhancer. Nature 453:930934
(2008).
Shoemaker C, Ramsey M, Queen J, Crews D: Expression of Sox9, Mis and Dmrt1 in the gonad
of a species with temperature-dependent sex
determination. Dev Dyn 236: 10551063
(2007).
Smith CA, Joss MP: Gonadal sex differentiation
in Alligator mississippiensis, a species with
temperature-dependent sex determination.
Cell Tissue Res 273:149162 (1993).
Smith CA, Smith MJ, Sinclair AH: Expression of
chicken steroidogenic factor-1 during gonadal sex differentiation. Gen Comp Endocrinol 113:187196 (1999).
Spotila LD, Spotila JR, Hall SE: Sequence and expression analysis of WT1 and Sox9 in the
red-eared slider turtle, Trachemys scripta. J
Exp Zool 281:417427 (1998).

11

29.10.2009 13:19:52

Torres-Maldonado L, Moreno-Mendoza N, Landa A, Merchant-Larios H: Timing of SOX9

downregulation and female sex determination in gonads of the sea turtle Lepidochelys
olivacea. J Exp Zool 290:498503 (2001).
Torres-Maldonado L, Piedra LA, Moreno-Mendoza N, Valencia MA, Martinez AM, Merchant-Larios H: Expression profiles of Dax1,
Dmrt1, and Sox9 during temperature sex determination in gonads of the sea turtle Lepidochelys olivacea. Gen Comp Endocrinol
129:2026 (2002).
Via S, Gomulkievicz R, de Jong G, Scheiner SM,
Schlichting CD, et al: Adaptive phenotypic
plasticity: consensus and controversy. Trend
Ecol Evol 10: 212217 (1995).
Wang Y, Conlon JM: Neuroendocrine peptides
(NPY, GRP, VIP, somatostatin) from the
brain and stomach of the alligator. Peptides
14:573579 (1993).

12

SXD119.indd 12

Sex Dev 119

Western PS, Harry JL, Graves JA, Sinclair AH:

Temperature-dependent sex determination
in the American Alligator: Amh precedes
Sox9 expression. Dev Dyn 216: 411419
(1999).
Western PS, Harry JL, Marshall Graves JA, Sinclair AH: Temperature-dependent sex determination in the American Alligator: expression of SF1, WT1 and DAX1 during gonadogenesis. Gene 241:223232 (2000).
Wibbels T, Crews D: Putative aromatase inhibitor induces male sex determination in a female unisexual lizard and in a turtle with
temperature-dependent sex determination. J
Endocrinol 141:295299 (1994).
Wibbels T, Bull JJ, Crews D: Chronology and
morphology of temperature-dependent sex
determination. J Exp Zool 260: 371381
(1991a).
Wibbels T, Bull JJ, Crews D: Synergism between
temperature and estradiol: a common pathway in turtle sex determination? J Exp Zool
260:130134 (1991b).

Wilhelm D, Palmer S, Koopman P: Sex determination and gonadal development in mammals. Physiol Rev 87: 128 (2007).
Willingham E, Baldwin R, Skipper JK, Crews D:
Aromatase activity during embryogenesis in
the brain and adrenal-kidney-gonad of the
red-eared slider turtle, a species with temperature-dependent sex determination. Gen
Comp Endocrinol 119:202207 (2000).
Witschi E: Biochemistry of sex differentiation in
vertebrate embryos; in Weber R (ed): The
Biochemistry of Animal Development, pp
193225 (Academic Press, New York 1967).
Yntema CL: A series of stages in the embryonic
development of Chelydra serpentina. J Morphol 125: 219252 (1968).
Yntema CL: Temperature levels and periods of
sex determination during incubation of eggs
of Chelydra serpentina. J Morphol 159:1727
(1979).

Merchant-Larios/Daz-Hernndez/
Marmolejo-Valencia

29.10.2009 13:19:52