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General and Comparative Endocrinology 259 (2018) 176–188 Contents lists available at ScienceDirect General and Comparative Endocrinology journal homepage: www

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General and Comparative Endocrinology 259 (2018) 176–188 Contents lists available at ScienceDirect General and Comparative Endocrinology journal homepage: www.elsevier.com/locate/ygcen Research paper Temperature- vs. estrogen-induced sex determination in Caiman latirostris embryos: Both females, but with different expression patterns of key molecules involved in ovarian development Guillermina Canesini a, Cora Stoker a,b, Germán H. Galoppo a, Milena L. Durando a, María V. Tschopp a, Enrique H. Luque a, Mónica M. Muñoz-de-Toro a, Jorge G. Ramos a,b,⇑ a b Instituto de Salud y Ambiente del Litoral (ISAL), Facultad de Bioquímica y Ciencias Biológicas, Universidad Nacional del Litoral-CONICET, Santa Fe, Argentina Departamento de Bioquímica Clínica y Cuantitativa, Facultad de Bioquímica y Ciencias Biológicas, Universidad Nacional del Litoral, Santa Fe, Argentina a r t i c l e i n f o Article history: Received 17 July 2017 Revised 28 November 2017 Accepted 28 November 2017 Available online 29 November 2017 Keywords: Reptile Sex reversal Steroid hormone receptors Aromatase Apoptosis p63 a b s t r a c t Caiman latirostris is a species with temperature dependent sex determination (TSD), which implies that the incubation temperature of the eggs is the main factor that determines the sex during a thermosensitive period (TSP). However, estrogens play a critical role in this process. The administration of 17b-estradiol (E2) previous to TSP overrides the effects of male incubation temperature, producing phenotypic females. This effect has been defined as sex reversal or estrogen-induced sex determination (E2SD). The aim of the present study is to describe similarities and differences in the effects of TSD and E2SD treatment conditions on ovary development. Our results show that the two treatment conditions studied are able to produce different ovaries. Treatment with E2 modified the expression pattern of estrogen receptor alpha and progesterone receptor, and expression of the enzyme aromatase. Moreover, in E2SD females, the proliferation/apoptosis dynamic was also altered and high expression of TAp63 was observed suggesting the presence of greater DNA damage in germ cells. To the best of our knowledge, this is the first report that describes the morphology of the female gonad of C. latirostris in three stages of embryonic development and shows the expression of TAp63 during the gonad development of a reptile. It is important to emphasize that the changes demonstrated in E2SD female gonads of embryos show that environmental compounds with proven estrogenic activity alter the follicular dynamics of C. latirostris in neonatal as much as in juvenile animals, endangering their reproductive health and possibly bringing consequences to ecology and evolution. Ó 2017 Elsevier Inc. All rights reserved. 1. Introduction Abbreviations: C. latirostris, Caiman latirostris; DAB, diaminobenzidine; E2, 17bestradiol; E2SD, estrogen-induced sex determination; EDCs, endocrine-disrupting compounds; ESRs, nuclear estrogens receptor; ER, estrogen receptor; ERa, estrogen receptor alpha; ERb, estrogen receptor beta; GAM, gonadal-adrenal-mesonephros; IHC, immunohistochemistry.; IOD, integrated optical density; PCNA, proliferating cell nuclear antigen; PR, progesterone receptor; TAp63, isoform TA from p63 tumor suppressor; TSD, temperature-dependent sex determination; TSP, thermo-sensitive period; TUNEL, terminal deoxynucleotidyl transferase-mediated sUTP nick endlabelling; VASA, ATP-dependent RNA helicase. ⇑ Corresponding author at: Instituto de Salud y Ambiente del Litoral (ISAL), Facultad de Bioquímica y Ciencias Biológicas, Universidad Nacional del Litoral, Casilla de Correo 242, Santa Fe 3000, Argentina. E-mail addresses: gcanesini@fbcb.unl.edu.ar (G. Canesini), cstoker@fbcb.unl.edu. ar (C. Stoker), ggaloppo@fbcb.unl.edu.ar (G.H. Galoppo), mdurando@fbcb.unl.edu.ar (M.L. Durando), mvtschopp@fbcb.unl.edu.ar (M.V. Tschopp), eluque@fbcb.unl.edu. ar (E.H. Luque), monicamt@fbcb.unl.edu.ar (M.M. Muñoz-de-Toro), gramos@fbcb. unl.edu.ar (J.G. Ramos). https://doi.org/10.1016/j.ygcen.2017.11.024 0016-6480/Ó 2017 Elsevier Inc. All rights reserved. After oocyte fertilization the sex of the offspring of all crocodilian and many turtle species is determined by the environment. The incubation temperature of the eggs during a critical period of development (thermosensitive period-TSP-) is the main factor that determines the sex of the progeny in the absence of sex chromosomes (Valenzuela et al., 2014). This process is known as temperature dependent sex determination (TSD) (Gilbert, 2000; Lang and Andrews, 1994). TSD initiates a cascade of molecular events that favour the development of one sex or the other altering the control of gene expression and cellular signaling by steroid hormones, hormone receptors and steroidogenic enzymes (Mizoguchi and Valenzuela, 2016). Some molecular events could precede the formation of the histological architecture that characterizes a testis or an ovary (Rhen and Schroeder, 2010). G. Canesini et al. / General and Comparative Endocrinology 259 (2018) 176–188 Estrogens play a critical role in sex determination in crocodilians and turtles. The administration of 17b-estradiol during TSP overrides the effects of male incubation temperature, producing phenotypic females in T. scripta and A. mississippiensis among others (Crain et al., 1997; Crews et al., 1996; Milnes et al., 2002). This effect has been defined as sex reversal or estrogen-induced sex determination (E2SD) (Crews et al., 1991; Holleley et al., 2016; Tousignant and Crews, 1994; Wibbels et al., 1991, 1992). Moreover, the administration of 17b-estradiol during TSP has been proposed as an alternative way to improve the recovery of endangered reptile species, by skewing the population sex ratio to one that favours reproductive females (Crews and Wibbels, 1993). TSD has been demonstrated in Caiman latirostris (Crocodylia: Alligatoridae) following a female-male-female pattern, since eggs incubated at 30–31 °C produced 100% females, 32 °C produced approximately 70% females, and incubation at 33–34 °C produced only males. The transitional range of temperature was >31° to 34 °C to .05). No clutch effects were observed (Q test, p > .05). 3.2. Gross anatomy and histo-morphological changes during gonad development Embryonic gonads were identified in all of the studied stages (22, 24 and 27). The gonads are structures located in the middlelow abdominal cavity symmetrically arranged as a mirror image in the GAM complex. Müllerian ducts were useful for locating them, since they lie dorsolaterally on either side of the GAM complex. The gonads advance in size and differentiation as the embryo’s development progresses (Fig. 3). In stage 22, the GAM complexes consist of paired structures located ventrally and associated with the kidneys, which are in dorsal position (Fig. 3). At this stage the gonadal primordia are facing and very close. In later stages the structures increase in size and begin to separate from each other (Fig. 3). Histologically in stage 22, the gonads under both incubation conditions (30 °C or 33 °C + 1.4 ppm E2) comprise an outer germinal epithelium and underlying medullary cords. In transverse sections, each gonad is composed of a distinct cortex and medulla (Fig. 4A and D). In the cortex, large and weakly stained primordial germ cells (VASA positive cells) are distributed within a pseudostratified to stratified layer of columnar epithelial cells. Germ cells are occasionally observed in the medulla, alone or forming clusters. VASA immunostaining is a useful tool for confirming the identity of germ cells in this early developmental stage. The medulla contains loose cords of unorganized cells separated by dark mesenchymal cells. Empty spaces are seen in the medulla Fig. 2. Characterization of the anti-aromatase antibody by IHC. Representative photomicrographs showing the distribution of aromatase in control C. latirostris gonads using the polyclonal antibody generated in our laboratory. (A) Specific aromatase immunoreactivity was observed as a dotted cytoplasmic pattern compatible with a mitochondrial located protein in the cytoplasm of oocytes. (B) specific staining was absent when the antibodies were pre-incubated with the corresponding peptide used as immunogen. IHC was developed with DAB and counterstained with Mayers’s hematoxylin. G. Canesini et al. / General and Comparative Endocrinology 259 (2018) 176–188 181 Fig. 3. Anatomical localization of the gonad in each studied stage. Low power photomicrographs representative of GAM (Gonad-Adrenal-Mesonephros) complexes of Caiman latirostris females incubated at 30 °C – female producing temperature in (A) stage 22, (B) stage 24 and (C) stage 27 of embryonic development. The images show the progressive growth in size and differentiation. Gonad (g), adrenal (a), Mullerian duct (md), mesonephros (m) and kidney (k). Transverse tissue sections stained with the trichromic Picrosirius solution and counterstained with Harris hematoxylin. Fig. 4. Structure of gonads at different stages of embryonic development of C. latirostris females produced by temperature and by estrogens. Representative photomicrographs showing the gonads in different stages of development (S22, S24 y S27), and in two conditions of sex determination: by temperature (TSD) and estrogen-induced (E2SD). The arrows show a cluster of germ cells. The inset shows higher magnification of the clusters. Asterisks show irregular cavities that give rise to the lacunaes. Changes are seen in the different incubation conditions (30 °C and 33 °C + E2) from stage 24. The onset of female differentiation implies the clusters delimitation and presence of signs of regression of the medulla which are clear at this stage in both the incubation conditions. E2SD females pose more cellularity in general and less empty spaces in the medulla, difference that is exacerbated in stage 27. Transverse tissue sections stained with the trichromic Picrosirius solution and counterstained with Harris hematoxylin. that could be the first sign of female differentiation of the gonad. In spite of that, there are no consistent signs of gonadal sex differentiation in stage 22 at either 30 °C or 33 °C + E2 (Fig. 4A and D). In stage 24 there is an increase in the size and cellularity of the gonad. Germ cells clusters are more numerous and more organized in both cortex and medulla. The onset of female differentiation implies the clusters delimitation and presence of signs of regression of the medulla which are clear at this stage in both the 30 °C and 33 °C + E2 incubation conditions (Fig. 4B and E). Female gonads in stage 24 obtained by incubation at 30 °C (TSD females) differ from the ones obtained by incubation at 33 °C + 1.4 ppm E2 (E2SD females) in that the latter present more cellularity in general and less empty space in the medulla. Stage 27 in TSD females is characterized by an increase in the number of germ cells grouped in clusters and greater delimitation of the clusters by the stromal cells. The medulla appears in greater regression and the typical lacunaes begin to be observed (Fig. 4C). In E2SD females, stage 27 only differs from the description of TSD females in that the cellularity is exacerbated, and there are less empty spaces because of the increase in extracellular matrix (Fig. 4F). 3.3. Expression of hormonal receptors We assessed by IHC whether the expression of estrogen receptor alpha (ERa) and progesterone receptor (PR) differs between TSD females and E2SD females. ERa was detected as a nuclear protein in embryonic gonads. Its expression was not modified throughout the developmental stages evaluated in TSD females. Whereas in E2SD females an increase was seen at the onset of TSP, that fell towards the end of development, at stages 24 and 27 (p = .0302). The E2SD group showed higher expression of ERa in stage 22 (p = .0286), while in the other stages the expression was lower in this group than in the TSD females (both p < .05) (Fig. 5A–F; Table 2). Similarly, to ERa, PR showed a nuclear pattern in the embryonic gonads. The expression of PR in TSD females remained low throughout the development stages studied, while in E2SD females its expression was significantly increased towards the end of 182 G. Canesini et al. / General and Comparative Endocrinology 259 (2018) 176–188 Fig. 5. Expression of sex steroid hormone receptors in the embryonic C. latirostris gonads. Histological sections showing representative ERa and PR immunostaining pattern at different embryonic developmental stages and in two conditions of sex determination: by temperature (TSD) and estrogen-induced (E2SD). ERa expression in TSD-females at stages 22, 24 and 27 (A–C). ERa expression in E2SD-females at stages 22, 24 and 27 (D–F). PR expression in TSD-females at stages 22, 24 and 27 (G–I). PR expression in E2SDfemales at stages 22, 24 and 27 (J–L). Lower panel: ERa and PR expressions quantified as percentage of positive area and percentage of positive cells respectively. Values represent mean ± SEM. Asterisks indicate significant differences at p < .05 by Mann-Whitney U test comparing TSD vs. E2SD in each stage of embryonic development. Different letters indicate significant differences at p < .05 by Kruskal-Wallis test followed by Dunn’s post test comparing each treatment condition along the three stages studied (Greek letters for the TSD and Latin letters for E2SD). embryonic development (p = .0082). At stage 27 PR expression was significantly higher in E2SD vs. TSD (p = .0121) (Fig. 5G–L, Table 2). in TSD females (p = .0086) and in E2SD females (p = .0217) (Fig. 6A–F). Aromatase was significantly higher in E2SD animals compared to TSD in the three studied stages (p < .05), Table 2. 3.4. Aromatase expression 3.5. Proliferative activity, DNA damage and apoptosis We detected specific aromatase immunoreactivity as a dotted cytoplasmic pattern compatible with a mitochondrial located protein in embryonic gonads of C. latirostris. The expression of the enzyme showed a decline towards the end of development both Regarding cell proliferation, the expression of PCNA in TSD females remained low throughout development. High expression of PCNA is observed at stage 22 in the E2SD group followed by a 183 G. Canesini et al. / General and Comparative Endocrinology 259 (2018) 176–188 Table 2 Protein expressions by immunohistochemistry. Stage 22 ERa PR Arom PCNA TUNEL p63 Stage 24 Stage 27 TSD E2SD Median Mean ± SEM Median Mean ± SEM p-value Median Mean ± SEM Median Mean ± SEM 8.401 0.359 16.63 0.046 0.292 1.525 15.69 0.8147 41.60 2.326 1.83 13.22 8.35 ± 1.105 0.4 ± 0.38 15.24 ± 1.393 0.043 ± 0.01 0.298 ± 0.12 1.54 ± 0.23 TSD 18.63 ± 4.19 1.13 ± 1.55 40.24 ± 2.88 2.79 ± 0.95 1.83 ± 0.58 13.17 ± 0.78 * 0.02 >0.05 ** 0.002 ** 0.002 * 0.03 * 0.02 5.67 0.309 8.73 0.312 1.32 2.69 E2SD 5.81 ± 0.95 0.51 ± 0.66 9.91 ± 1.24 0.33 ± 0.24 1.06 ± 0.31 2.91 ± 0.21 1.659 0.64 37.50 0.624 5.536 17.53 TSD 2.34 ± 1.73 0.65 ± 0.87 35.93 ± 1.709 0.66 ± 0.41 5.53 ± 131 17.64 ± 0.28 E2SD p-value Median Mean ± SEM Median Mean ± SEM p-value * 0.03 >0.05 ** 0.004 >0.05 * 0.01 * 0.03 6.729 0.925 5.698 0.136 1.506 2.05 6.8 ± 1.36 0.85 ± 0.17 5.740 ± 0.82 0.14 ± 0.17 1.53 ± 0.29 2.15 ± 0.18 0.398 32.23 25.87 0.287 1.49 7.53 1.109 ± 1.36 31.27 ± 7.12 25.09 ± 2.52 0.29 ± 0.25 1.53 ± 0.45 7.42 ± 0.47 * 0.03 0.002 0.004 >0.05 >0.05 * 0.03 ** ** ERa: Estrogen Receptor alfa; PR: Progesterone Receptor; Arom: Aromatase enzyme; PCNA: Proliferating Cellular Nuclear Antigen; TUNEL: Terminal deoxynucleotidyl transferase-mediated sUTP nick end-labelling; p63 (p53 family member). Data shown are presented as the median and mean ± SEM (n = 6/group). * Indicates significant differences at p < .05 and ** indicates significant differences at p < .005 by Mann Whitney U test. Fig. 6. Expression of aromatase in the embryonic C. latirostris gonads. Histological sections showing representative aromatase immunostaining pattern at different embryonic developmental stages in female’s sex determined by temperature (TSD) and estrogen-induced (E2SD). Aromatase expression in TSD-females at stages 22, 24 and 27 (A–C). Aromatase expression in E2SD-females at stages 22, 24 and 27 (D–F). Lower panel: Aromatase expressions quantified as percentage of positive area. Values represent mean ± SEM. Asterisks indicate significant differences at p < .05 by Mann-Whitney U test comparing TSD vs. E2SD in each stage of embryonic development. Different letters indicate significant differences at p < .05 by Kruskal-Wallis test followed by Dunn’s post test comparing each treatment condition along the three stages studied (Greek letters for the TSD and Latin letters for E2SD). significant fall (p < .05) at stage 24, remaining low to stage 27. Cell proliferation at stage 22 is significantly higher in E2SD than TSD (p = .0028) (Fig. 7A–F, Table 2). The TUNEL assay showed in TSD females low apoptosis level at stages 22 and 24 with a significant increase in stage 27 (p < .05). Regarding E2SD females, the percentage of apoptosis at stage 22 was 1.8 ± 0.6. At stage 24 a significant increase occurred (5.5 ± 1.3) with a fall at stage 27 (1.5 ± 0.5, p < .05). At stages 22 and 24 the percentage of apoptosis is higher in E2SD than TSD females (p = .0159; p = .0317), without showing differences towards the end of development (Fig. 7G–L, Table 2). On the other hand, we detected specific p63 immunoreactivity in caiman embryos’ germ cells, staying low throughout the development of TSD females. The co-localization of VASA/p63 confirms the germ cells identity (Fig. 8). Meanwhile, in E2SD females, an increase in stage 24 was observed with respect to the previous stage (p = .0156) followed by a fall towards the end of development 184 G. Canesini et al. / General and Comparative Endocrinology 259 (2018) 176–188 Fig. 7. Proliferation and apoptosis in embryonic C. latirostris gonads of female’s sex determinate by temperature (TSD) and estrogen (E2SD). Histological sections showing representative cell proliferation (PCNA) and apoptosis (by TUNEL assay) immunostaining pattern at different embryonic developmental stages and in two conditions of sex determination: by temperature (TSD) and estrogen-induced (E2SD). Cell proliferation in TSD-females at stages 22, 24 and 27 (A–C). Cell proliferation in E2SD-females at stages 22, 24 and 27 (D–F). Apoptosis in TSD-females at stages 22, 24 and 27 (G–I). Apoptosis in E2SD-females at stages 22, 24 and 27 (J–L). Lower panel: Cell proliferation and apoptosis were quantified as percentage of positive area. Values represent mean ± SEM. Asterisks indicate significant differences at p < .05 by Mann-Whitney U test comparing TSD vs. E2SD in each stage of embryonic development. Different letters indicate significant differences at p < .05 by Kruskal-Wallis test followed by Dunn’s post test comparing each treatment condition along the three stages studied (Greek letters for the TSD and Latin letters for E2SD). (p = .0181). p63 expression was significantly higher in the gonads of E2SD females in all embryonic stages relative to TSD (p < .05) (Fig. 8, Table 2). 4. Discussion In the present study we assessed the similarities and differences of Caiman latirostris gonads in three stages of embryonic development (22, 24 and 27) obtained by TSD and E2SD. The evaluation of histo-morphological characteristics and the expression of different key molecules in embryonic development allowed us to broaden the knowledge about the embryonic ovarian development of this species. Additionally, we demonstrated that E2SD female gonads show alterations in the protein expression of the estrogens and progesterone receptors, as well as in the expression of the enzyme aromatase. We also observed an unbalance between cell proliferation and apoptosis and the possible incidence of DNA damage in germ cells through the expression of p63 immunostaining in these females. All this supports our hypothesis that the changes already observed in ovaries of neonates and juveniles exposed to E2 prior to the thermo-sensitive period (Stoker et al., 2008) could already show changes in early stages of embryonic development. Females are obtained by both TSD and E2SD. It has been demonstrated that the addition of exogenous estrogens in a sensitive period can be used to produce turtle, lizard, and crocodile females. Moreover, it has been proposed as a method for the conservation of endangered turtle species (Crews et al., 1991; Crews and Wibbels, 1993). The gonads in stage 22 show no histological differences, and sex had not been determined yet. VASA expression was a useful tool for identifying germ cells’ location. This tool has also been used in other species for the same purpose (Bachvarova et al., 2009). Female gonads can be distinguished in stage 24, and this is G. Canesini et al. / General and Comparative Endocrinology 259 (2018) 176–188 185 Fig. 8. DNA damage in germ cells of embryonic C. latirostris gonads. Representative photomicrographs of dual immunofluorescence staining for VASA/p63, showing the gonads of an estrogen-induced sex determinate female (E2SD) C. latirostris embryo in stage 24 of development. Histological sections with VASA cytoplasmic immunofluorescence (green), showing the presence of clusters of germ cells and p63 immunofluorescence (red) with nuclear localization, showing the DNA-damage of these germ cells. In the merge, the co-localization of both molecules is shown while the nuclei blue (DAPI +) were negative. Middle panel: p63 expression in female’s sex determined by temperature (TSD) at stages 22, 24 and 27 (C–E). p63 expression in E2SD-females at stages 22, 24 and 27 (F–H). Lower panel: p63 expressions quantified as integrated optical density (IOD) in the germ cell clusters. Values represent mean ± SEM. Asterisks indicate significant differences at p < .05 by Mann-Whitney U test comparing TSD vs. E2SD in each stage of embryonic development. Different letters indicate significant differences at p < .05 by Kruskal-Wallis test followed by Dunn’s post test comparing each treatment condition along the three stages studied (Greek letters for the TSD and Latin letters for E2SD). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) primarily because of the germ cells’ location in well delimited clusters and the regression of the medulla. Smith and Joss (1993) observed a similar result for Alligator mississippiensis. E2SD gonads in stages 24 and 27 differ from TSD gonads in that the former possess more cellularity and fewer ‘‘empty spaces” in the medulla. (Kohno et al., 2015) No differences were found between TSD and E2SD in stage 27 for A. mississippiensis. Contrary to our observations, it was recently published for turtles that exogenous estradiol alters gonadal growth and the timing of temperature sex determination (Diaz-Hernandez et al., 2015). They found diminished cell proliferation and smaller gonads induced by exogenous E2. The differences observed in female gonads between turtles and crocodiles could be because of the temperature (for turtles, male producing temperature is approximately 26 °C while the female producing temperature is approximately 33 °C). Moreover, for alligators and caimans it was demonstrated that the growth is more accelerated at 33 °C (Stoker et al., 2003). Therefore, it is possible that the greater cellularity observed in female gonads obtained by E2SD could be influenced by this accelerated development. As it is known, estrogens play a critical role in determining sex in crocodiles and turtles, as they probably do in most nonmammalian vertebrates. However, it is not clear how estrogens override the influence of temperature during sex determination in these species (Kohno et al., 2015). Most vertebrates studied to date (Katsu et al., 2010), have two forms of nuclear estrogen receptors (ESRs), ERa and ERb, which have different ligand specificity, distribution, and function. In A. mississippiensis the evidence 186 G. Canesini et al. / General and Comparative Endocrinology 259 (2018) 176–188 indicates that ERa is likely the principal ESR involved in sex reversal as well as embryonic Müllerian duct survival (Kohno et al., 2015). ERa expression in C. latirostris gonads was not modified throughout the developmental stages evaluated in TSD. The E2SD group showed higher expression of this receptor at stage 22, when the gonad is still bipotential, whereas towards the end of embryonic development, the expression fell, suggesting a clear downregulation of ERa in the E2SD females. On the other hand, progesterone receptor (PR) is considered a target molecule of estrogenic action and its regulation differs among species (Boyd-Leinen et al., 1984; Hora et al., 1986); (Giannoukos and Callard, 1996; Schultz et al., 2003). We observed very low expression of PR in the first stages studied in both TSD and E2SD female gonads, but at stage 27, the highest expression of this receptor in E2SD female gonads was observed. Incipient estrogen action could produce both the down-regulation of ERa and the stimulation of PR as in the female reproductive tract of mammals and avian species (Shao et al., 2007; Varayoud et al., 2008; Zhang et al., 2014). The same pattern of results is observed in E2SD females. The enzyme that catalyses estrogen production from an androgen substrate is cytochrome P450 aromatase (Simpson et al., 1994). We observe the same pattern of aromatase expression during development but approximately threefold higher for E2SD than TSD gonads. This pattern was characterized by a high expression of aromatase from the sex determination period with a fall through stage 27. The converse is observed in other crocodilians (Crocodylus porosus and Alligator mississippiensis) where an increase of aromatase is present after TSP in TSD females (Gabriel et al., 2001; Smith and Joss, 1994). For C. porosus the aromatase determination was performed by evaluating of its activity in the whole GAM complex, while for A. mississippiensis, aromatase was assayed by in situ hybridization. It is important to emphasize the generation of the anti-aromatase antibody that allowed us to evaluate the presence of the protein in its actual source. Differential ovarian aromatase activity or gene (cyp19a1) expression occurs in the developing gonad of turtles at female- and male-producing temperatures during the TSP of Emys orbicularis (Desvages and Pieau, 1992), Dermochelys coriacea (Desvages et al., 1993), and Malaclemys terrapin (Jeyasuria and Place, 1998) turtles (Valenzuela and Shikano, 2007). Experiments and assays carried out in female turtles using isolated gonads, provide evidence that the gonads themselves respond to temperature shifts by modifying their sexual differentiation (Pieau and Dorizzi, 2004). Differential aromatase expression in developing gonads late into or after TSP was observed in two crocodilians (TSD), Alligator mississippiensis (Gabriel et al., 2001) and Crocodylus porosus (Smith and Joss, 1994). This lack of endogenous gonadal aromatase expression until late in development led to the conflicting hypothesis that endogenous estrogens are not involved in early ovarian differentiation in TSD species. It was proposed that tissues other than the gonads provide the site for primary thermal sensitivity, as well as the source of estrogen for early ovarian development. In this way aromatase would be a downstream element in TSD’s developmental cascade (Valenzuela and Shikano, 2007). However, it is documented in larvae of pejerrey (Odontesthes bonariensis) at male producing temperature, that E2 treatment caused a marked increase in the expression of cyp19a1 (Fernandino et al., 2008). Moreover, estrogens are synthesized by morphologically undifferentiated female gonads in frogs (Isomura et al., 2011). Gohin et al. (2011) reported that aromatase expression in oocytes of adult Xenopus laevis participates significantly in ovarian estrogen synthesis, suggesting a common feature of vitellogenic vertebrates. We also observed that germ cells express aromatase in C latirostris embryos. To obtain a healthy population of oocytes in adulthood, during the female gonadal differentiation process, germ cells exhibit proliferation, meiosis and apoptosis. We observed high gonadal proliferation in E2SD females at the beginning of TSP followed by a fall in later stages. No modifications were seen in TSD female gonad proliferation throughout development. Our findings are in agreement with those reported by Zhu et al. (2009), who stated that cell division and differentiation often act as two mutually exclusive partners that are related but cannot co-exist. An increase in germ cell proliferation could be the defining feature of the determination of females as seen in alligators (Smith and Joss, 1993). It remains unclear whether germ cell apoptosis is involved in gonadal development and sex differentiation. In the gonads of Odontesthes bonariensis larvae during sex differentiation, the TUNEL assay revealed widespread apoptotic signals at male producing temperatures but virtually none at female producing temperatures, suggesting that gonadal apoptosis may have an important role (Yamamoto et al., 2013). Numerous studies show that estrogens are suppressive regulators of ovarian apoptosis that act to ensure the survival of preovulatory follicles in vertebrates (reviewed in Yamamoto et al. (2013)). In our study, the TUNEL assay showed in TSD females low apoptosis levels at stages 22 and 24 with a significant increase in stage 27. In this way, apoptosis would not be related to sex determination in control females. Apoptosis has been related to germ cell cluster remodelling for the beginning of follicle formation (Reviewed in Sun et al. (2017)). We hypothesized that the increase of apoptosis at stage 27 in TSD females is related to this mechanism. In E2SD females the apoptosis showed a peak in stage 24. It could be presumed that this mechanism could be related to sex determination in E2SD. Moreover, we propose that this result could be related to the expression of p63 in germ cells. It is known that the p53 family has been implicated in the pathways by which some cells undergo apoptosis. The TAp63 isoform is the only p53 family member that participates in the oocyte DNA damage response check point in mammals, one of the early events in the apoptotic-mediated selection process (Carroll and Marangos, 2013). We detected specific TAp63 immunoreactivity in caiman embryo germ cells (co-localization VASA/p63), and it stays low throughout development in TSD females. Meanwhile, in E2SD females, an increase in stage 24 was observed, which could be responsible for the greater apoptosis observed in this stage. To our knowledge, this is the first report describing the expression of TAp63 during the gonad development of a reptile. The higher expression of TAp63 would involve increased DNA damage of germ cells in the gonads of E2SD females. In this sense, increased germ cell apoptotic death could be expected. Previous results of our group found an alteration in follicular dynamics in neonatal E2SD females (Stoker et al., 2008). Moreover, juvenile E2SD females of the same species exhibited increased incidence of multi-oocyte follicles (Stoker et al., 2008). Even though the mechanism by which multioocyte follicles develop is unknown, it is postulated to be a consequence of oocyte clusters that did not separate and became enclosed individually in a follicle (Iguchi and Takasugi, 1986; Iguchi et al., 2006). We hypothesized that proliferation/apoptosis imbalance observed in E2SD females could be the cause of these differences observed. In summary, our results show that in the two developmental conditions studied, 30 °C or 33 °C + E2 1.4 ppm applied topically at embryonic stage 20, are able to produce morphologically evaluated ovaries in C. latirostris embryos. Nevertheless, those gonads are different from each other, exhibiting distinct expression patterns of key molecules associated with ovarian development and function. It is important to emphasize that the changes demonstrated in E2SD female embryos reflect on the idea that environmental compounds with proven estrogenic activity could alter the ovarian functionality of adult C. latirostris, endangering G. Canesini et al. / General and Comparative Endocrinology 259 (2018) 176–188 their reproductive health and the balance of wild populations in the ecosystem. Acknowledgments We thank Juan Grant and Juan C. Villarreal for technical assistance and animal care. Field work was done in collaboration with ‘‘Reserva Natural El Cachapé”, Chaco http://www.elcachape.com. ar. 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