Original Article Accepted: July 17, 2014 by M. Schmid Published online: November 25, 2014

Sex Dev 2014;8:364–375 DOI: 10.1159/000369116

Chronology, Magnitude and Duration of Expression of Putative Sex-Determining/ Differentiation Genes in a Turtle with Temperature-Dependent Sex Determination Kayla L. Bieser a, b Thane Wibbels a  

Department of Biology, University of Alabama at Birmingham, Birmingham, Ala., and b Department of Biology, Northland College, Ashland, Wis., USA  

 

Key Words Amh · Dmrt1 · 17β-estradiol · Foxl2 · Letrozole · Lhx9 · Sox9 · Trachemys scripta · TSD

Abstract The red-eared slider turtle (Trachemys scripta) possesses temperature-dependent sex determination (TSD) in which the incubation temperature determines gonadal sex. Although a number of mammalian gene homologues have been identified in reptiles with TSD, the exact sex-determining trigger(s) is not known. To date, the current study represents the most comprehensive simultaneous evaluation of the chronology of mRNA expression profiles of putative sexdetermining/differentiation genes (Dmrt1, Sox9, Amh, Lhx9, and Foxl2) from gonads incubated at male- and female-producing temperatures in T. scripta. Additionally, sex-reversing treatments with 17β-estradiol and letrozole were examined. At a male-producing temperature, Dmrt1 expression was sexually dimorphic by stage 17, Sox9 by 19 and Amh by 21. In contrast, Foxl2 did not significantly increase until after the thermosensitive period at a female-producing temperature. Treatment with 17β-estradiol resulted in reduced gonad size and/or inhibited gonadal development and differentiation. Gene expression was subsequently low in this group. Sex re-

© 2014 S. Karger AG, Basel 1661–5425/14/0086–0364$39.50/0 E-Mail [email protected] www.karger.com/sxd

versal utilizing letrozole failed to produce testes at a femaleproducing temperature and as such, gene expression was comparable to ovary. These results indicate that Dmrt1 and Sox9 are potential triggers for testis differentiation and Amh, Lhx9 and Foxl2 represent a conserved core set of genes in the sex-determining/differentiation pathway of TSD species. © 2014 S. Karger AG, Basel

Great diversity exists among sex-determining mechanisms in amniotic vertebrates, and at first glance they may appear unrelated; but in fact there is a great deal of homology between them. A number of amniotic vertebrates possess chromosomal sex-determining mechanisms, including mammals, birds, snakes, some lizards, and some turtles; however, their sex chromosomes have evolved independently over time and with that, great diversity in the initial sex-determining trigger [Lahn and Page, 1999; Marshall Graves and Shetty, 2001; Ezaz et al., 2006; Graves and Peichel, 2010]. For example, the sex-determining region of the Y chromosome (SRY) is the testis-determining factor in mammals [Sinclair et al., 1990], whereas a double-dose of doublesex and mab-3-related transcript (DMRT1) is reported to trigger testis determination in birds [Smith et al., 2009]. Alternatively, many reptiles, inKayla Bieser Northland College 1411 Ellis Avenue Ashland, WI 54806 (USA) E-Mail kbieser @ northland.edu

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a

 

Ramsey et al., 2007; Barske and Capel, 2010], but they do not demonstrate sexually dimorphic expression, and they continue to function in both ovary and testis beyond determination and differentiation phases. Although Lhx9 shows no sexually dimorphic expression pattern [Bieser et al., 2013], mammalian studies indicate that it is required for early gonadal development and activates the transcription of Sf1 [Birk et al., 2000; Wilhelm and Englert, 2002]. Other genes either associated with testis differentiation, i.e. Dmrt1, Sox9 and Amh, or with ovarian differentiation, i.e. Foxl2, display sexually dimorphic expression patterns during the early phases of sexual differentiation. In chicken, a double-dose of DMRT1 is required for testis differentiation [Smith et al., 2009] as are 2 copies required for mammalian testicular development [Raymond et al., 1999]. Previous studies in turtles with TSD demonstrate that Dmrt1 is expressed early in the TSP at male-producing temperatures (MPT) with expression remaining sexually dimorphic [Murdock and Wibbels, 2003; Rhen et al., 2007; Shoemaker et al., 2007a]. It has been suggested that in birds and reptiles, DMRT1 may be an early regulator of testis differentiation, whereas in mammals SOX9 acts as the early regulator in the sex determination cascade [Murphy et al., 2010]. In mammals, SOX9 is directly activated by SRY, and, in the absence of SRY, SOX9 is sufficient for testis determination [Swain and Lovell-Badge, 1999; Morrish and Sinclair, 2002; Sekido and Lovell-Badge, 2008]. Additionally, SOX9 acts to maintain Sertoli cell differentiation and is responsible for the upregulation of AMH [Swain and Lovell-Badge, 1999]. In species with TSD, the role of Sox9 is unclear. In turtle, Sox9 is shown to be upregulated during the bipotential period, but in alligator it is not upregulated until after testis commitment [da Silva et al., 1996; Moreno-Mendoza et al., 1999; Western et al., 1999; Shoemaker et al., 2007a; Barske and Capel, 2010]. Similarly, in both mammal and turtle, Amh may be regulated by SOX9; however, in chicken and alligator, Amh is expressed prior to Sox9 suggesting that the order of gene expression varies even within species with TSD [Smith et al., 1999c; Shoemaker et al., 2007a]. Therefore, the exact timing of Sox9 in relation to other factors may be a critical turning point in the differentiation of the gonad. While mammals and birds initiate sex determination through the presence or absence of a male-determining gene (i.e. SRY and DMRT1), the expression of aromatase and estrogen production has remained a central focus in many TSD studies. It has been hypothesized that temperature may be regulating aromatase production and in turn stimulating ovarian development through estrogen

Expression of Putative Sex-Determining Genes in a Turtle with TSD

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cluding most turtles, all crocodilians, tuataras, and some lizards, lack sex chromosomes. These species display temperature-dependent sex determination (TSD) in which the incubation temperature of the egg determines the sex of the organism [Yntema, 1976; Bull and Vogt, 1979; Bull, 1980; Ferguson and Joanen, 1983; Ewert et al., 2004]. Although various evolutionary forces have shaped the variety of sex-determining mechanisms, there is evidence of a conserved core of genes involved in sex determination across amniotic vertebrates. At the morphological and physiological levels, the pattern of gonad development in amniotic vertebrates is highly conserved suggesting similar regulatory mechanisms [Graves and Peichel, 2010; Rhen and Schroeder, 2010]. Regardless of the sex-determining mechanism, the gonads begin as a bipotential primordium, or genital ridge, that develops on the mesonephric portion of the embryonic kidney. During the developmental process, some trigger directs the bipotential primordium to commit to and differentiate into either a testis or ovary [Graves and Peichel, 2010; Rhen and Schroeder, 2010]. It is likely that the initial trigger, be it a single gene or multiple genes, varies among amniotic vertebrates, and that the downstream molecular pathways for testis or ovarian development remain mostly conserved [Barske and Capel, 2008]. In species with TSD, a number of mammalian sex-determining gene homologues have been previously identified and their expression measured [Western et al., 2000; Morrish and Sinclair, 2002; Murdock and Wibbels, 2003; Ramsey et al., 2007; Rhen et al., 2007; Shoemaker et al., 2007a]. It is anticipated that a gene responsible for triggering sex determination in a species with TSD would show sexually dimorphic expression just prior to or early in the temperature-sensitive period (TSP) when the embryo is environmentally sensitive and sex is determined [Bull and Vogt, 1979; Yntema, 1979; Pieau and Dorizzi, 1981; Wibbels et al., 1991a]. Several transcription factors are conserved among amniotic vertebrates and have been implicated in the development of the bipotential gonad prior to sexual differentiation [LIM homeobox 9 (Lhx9), Wilms’ tumor 1 (Wt1) and steroidogenic factor 1 (Sf1)], while others display sexually dimorphic expression at the beginning of the TSP [Dmrt1, sex-determining region Y box 9 (Sox9), forkhead transcription factor 2 (Foxl2), and anti-Müllerian hormone (Amh)]. Potential early regulators of sexual differentiation including Lhx9, Wt1 and Sf1 are expressed prior to and during the early stages of gonadal differentiation [Spotila et al., 1998; Smith et al., 1999b; Birk et al., 2000; Crews et al., 2001; Mazaud et al., 2002; Wilhelm and Englert, 2002;

Methods Egg Procurement and Incubation Red-eared slider turtle eggs were purchased from Kliebert’s Turtle and Alligator Farm (Hammond, La., USA) and brought to the lab within 1–2 days of laying. Eggs were kept in custom-built incubators [Lang et al., 1989] maintained at 26 ° C with 100% humidity until viability was established by candling. Once viability was established, eggs were randomized to minimize clutch effects, equally divided into control and treatment groups, and incubated at 26 ° C or 31 ° C. In this species, incubation of eggs at 26 ° C (MPT) results in the production of only males, whereas incubation at 31 ° C (female-producing temperature, FPT) produces all female hatchlings [Bull et al., 1982; Etchberger et al., 1991; Wibbels et al., 1998]. Temperature inside the incubators was monitored daily using thermistor probes and recorded hourly using HOBO data loggers (Onset Computer Corp., Bourne, Mass., USA). Additionally, egg trays were rotated daily to minimize temperature fluctuations  

 

 

 

 

 

 

 

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Sex Dev 2014;8:364–375 DOI: 10.1159/000369116

 

inside the incubators. Embryos were monitored for developmental stage by dissection of 2–3 eggs every few days using standardized staging criteria based upon morphological features of the embryo [Yntema, 1968; Greenbaum, 2002]. According to these guidelines, the TSP in T. scripta is from approximately stages 15 through 21 when the embryo is environmentally sensitive and when sex determination occurs [Wibbels et al., 1991a]. Letrozole and 17β-Estradiol Treatments To induce male-to-female sex reversal in embryos incubated at a MPT, stage 14 embryos were treated with 10 μg 17β-estradiol (Sigma-Aldrich, St. Louis, Mo., USA) in 5 μl 95% ethanol by topical application to the vascular region adjacent to the embryo [Crews et al., 1991]. To induce female-to-male sex reversal in embryos incubated at a FPT, 20 μg letrozole (Sigma-Aldrich) dissolved in 5 μl 95% ethanol was topically applied at stages 14, 15, 17, 19, and 21 [Richard-Mercier et al., 1995]. In all experiments, control groups received 5 μl of 95% ethanol at the same time as drug administration [Crews et al., 1991]. Following drug administration, 12 embryos for each treatment and control group were euthanized and dissected at stages 15, 17, 19, 21, and 23. Adrenal-kidney-gonad complexes were placed in RNAlater (Ambion, Austin, Tex., USA) for storage at –20 ° C. To identify if sex reversal occurred in treatment groups, histological evaluation of gonads from subsets of stage 26 embryos was performed. All eggs and tissue sampling protocols were approved by the University of Alabama at Birmingham Institutional Animal Care and Use Committee (UAB Animal Protocol No. 110107370).  

 

Reverse Transcription Gonads were microdissected from the adrenal-kidney-gonad complexes, and individual pairs of gonads were used for RNA extraction with the RNAqueous-Micro kit (Ambion) following the manufacturer’s protocol. Tissues were homogenized in 100 μl of lysis solution using 2.8-mm ceramic bead tubes (Mobio, Carlsbad, Calif., USA) in an Eppendorf homogenizer at maximum speed for two 45-second bursts. RNA was eluted in 12 μl of elution buffer followed by DNase treatment to remove residual genomic DNA prior to quantification with a Biophotometer Plus with Helma Tray Cell (Eppendorf, Hauppauge, N.Y., USA). Total RNA had 260/280 absorbance ratios of 1.8–2.0 in elution buffer. Immediately following RNA extraction, 100 ng of total RNA from each pair of gonads was reverse transcribed in a 20-μl reaction employing both oligo(dT)15 and random hexamer primers with the 2-step RT-qPCR kit (Promega, Madison, Wis., USA). Quantitative Real-Time PCR Real-time PCR primers were designed for Sox9 and Lhx9 using Integrated DNA Technologies (Coralville, Iowa, USA) PrimerQuestSM software for qPCR primers. Primer sequences for Dmrt1, Amh, Foxl2, and protein phosphatase 1 (PP1) were obtained from previously published sequences [Shoemaker et al., 2007a, b]. Primers were designed across exons when possible to reduce genomic contamination (table 1). All primer pair gene products were sequenced, primer pairs were optimized for annealing temperature, and melting curve analyses were performed to ensure amplification of a single product. Quantitative PCR was standardized by RNA input and normalized by PP1 [Shoemaker et al., 2007a]. Validation of PP1 was conducted between controls and experimental treatments by calculating 2–CT for each experimental condition

Bieser /Wibbels  

 

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[Pieau and Dorizzi, 2004; Lance, 2009]. Ectopic application of estrogens during the bipotential period permanently feminizes the gonad resulting in ovaries regardless of incubation temperature [Wibbels et al., 1991b; Wibbels and Crews, 1992]. A more recent study suggests that ectopic application of estrogen represses Sox9 resulting in feminization and inhibition of the male developmental pathway [Barske and Capel, 2010]. Additional evidence through the use of aromatase inhibitors, which block the conversion of testosterone to estrogen, further supports this hypothesis [Crews and Bergeron, 1994; Rhen and Lang, 1994; Wibbels and Crews, 1994; Belaid et al., 2001; Pieau and Dorizzi, 2004; Warner and Shine, 2008; Barske and Capel, 2010]. Lastly, regulation of aromatase (Cyp19a1) may be through an ovary-specific gene, Foxl2, which is expressed early in the differentiation of the ovary and is thought to positively feedback on aromatase to stimulate ovarian differentiation [Hudson et al., 2005; Rhen et al., 2007]. To date, the majority of studies assessing gene expression in species with TSD have involved different methodologies and assessment at the individual gene level. In the current study, we simultaneously investigated the chronology, magnitude and duration of expression of Dmrt1, Sox9, Amh, Lhx9, and Foxl2 in the red-eared slider turtle, Trachemys scripta. Eggs were incubated at male- and female-producing temperatures with and without the application of sex-reversing drug treatments, 17β-estradiol and letrozole, to further evaluate the chronology of gene expression with particular emphasis on the male-determining pathway in a species with TSD. This study evaluates and extends the results of previous studies on the molecular chronology of TSD in turtles.

Fig. 1. Representative adrenal-kidney-gonad complexes at hatch in T. scripta embryos incubated at a FPT (A), a MPT with application of 17β-estradiol (B), a MPT (C), and a FPT with application of letrozole (D). Tissues shown are still attached to the dorsal wall of the carapace. Gonads (g) are clearly visible in A, C, and D, but barely visible to absent upon application of 17β-estradiol (B). od = Oviduct.

A

B

D

C

Table 1. Quantitative real-time PCR primers and parameters

Gene

Accession No.

Primer sequence (5′→3′)

Annealing temperature, ° C

Fragment Sequence obtained from size, bp

Dmrt1

AY316537.1

60

101

Shoemaker et al., 2007a

Sox9

EU914820.1

60

120

current manuscript

Amh

AY235424.1

60

101

Shoemaker et al., 2007a

Lhx9

DQ458799.2

60

127

Bieser et al., 2013

Foxl2

AY155535.1

60

104

Shoemaker et al., 2007b

PP1

DQ848991.1

F: CAACTACTCCCAATACCAGATGGC R: GGCTTCGCAGGCTGTTTTTC F: CAGCATGAGCGAGGTTCACTCT R: CCTTCTCGCTTCAGGTCTTGCT F: CGGCTACTCCTCCCACACG R: CCTGGCTGGAGTATTTGACGG F: CCTTACTTCAATGGCACTGGCACA R: TGTCCAGGTGATCTGCCTCATTCT F: TGGCAGAACAGCATCCGC R: GGGTCCAGCGTCCAGTAGTTG F: CAGCAGACCCTGAGAACTTCTTCCTG R: GCGCCTCTTGCACTCATCAT

60

89

Shoemaker et al., 2007a

[Schmittgen and Livak, 2008]. A 1-way ANOVA was conducted to compare the expression of PP1 between MPT, FPT, MPT treated with 17β-estradiol, and FPT treated with letrozole. Expression of PP1 did not significantly differ across groups, F[3,222] = 1.704, p = 0.17, confirming the validity of its usage as a normalization gene. All gene transcripts were amplified in triplicate from the identical set of 5–12 embryos per sex/stage/treatment (each sample comprising a pair of gonads from the same turtle) using GoTaq qPCR Master Mix (Promega) on an Eppendorf Mastercycler. Each reaction well contained 10 μl GoTaq qPCR Master Mix, 200 nmol of each forward and reverse primer, 4 μl synthesized cDNA isolated from 1 embryo (equivalent to 4 ng of total RNA) and sterile water to bring the total reaction volume to 20 μl. The thermal profile was 95 ° C for 2 min to activate the DNA polymerase, followed by 40 cycles of 2-step PCR (95 ° C for 15 s and 60 ° C for 1 min) and a dissociation cycle to generate a melt curve. Several controls were included on each plate to control for the specific measurement of cDNA synthesized from the reverse transcription reaction. Two negative controls were included, a no-template control in which water was added instead of template, and a

no-reverse-transcriptase control. These controls ensured that the samples were free of genomic and exogenous DNA contamination. Finally, a 2-point standard curve from pooled cDNA was run on each plate as a positive control and interplate calibrator. Efficiencies of real-time PCR primers were calculated from the slope of a standard curve for each gene to validate relative quantification methods.

Expression of Putative Sex-Determining Genes in a Turtle with TSD

Sex Dev 2014;8:364–375 DOI: 10.1159/000369116

 

 

 

 

 

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Statistical Analysis All statistical analyses were done using GraphPad Prism Version 6.04 for Windows 7. Fold change was calculated using the comparative CT method [Livak and Schmittgen, 2001; Schmittgen and Livak, 2008]. Within a single gene, average fold change for the stage 15 male was used as the calibrator for calculating relative expression. For comparisons between genes within a single temperature, the average fold change of stage 15 Dmrt1 at a MPT or FPT was used as the calibrator. All fold changes were log-transformed prior to statistical analyses [Hellemans and Vandesompele, 2011]. A 2-way ANOVA with uncorrected Fisher’s Least Significant Difference multiple comparisons [Motulsky, 2014] was used to exam-

C

A

E B D

Fig. 2. Representative histology at hatch in T. scripta embryos incubated at a MPT with application of 17β-estradiol (A, B), an ovary at a FPT with application of letrozole (C), an ovotestis at a FPT with application of letrozole (D), an ovary at a FPT in the control group (E), and a testis at a MPT in the control group (F). A The

outlined box of the only gonad identified at hatch in 17β-estradioltreated embryos was further magnified and shown in B. od = Oviduct; g = gonad; c = cortex; m = medulla. Scale bar = 100 μm in A, C, D, 50 μm in B, E and 20 μm in F.

ine gene expression differences within a temperature/treatment between stages, within a stage between temperatures/treatments, and within a stage between genes. Significance was assessed at p < 0.05.

FPT (fig. 2C, D). Of the turtles treated with 17β-estradiol, 1 had an identifiable gonad with a developing oviduct (fig. 2A), 4 had a distinct oviduct but no gonad, and 3 had no identifiable gonad or oviduct. In the single turtle with an identifiable gonad, it was greatly reduced in size compared to a non-treated ovary or testis and lacked cortical or medullary organization. In the letrozole-treated group, 5 turtles had an identifiable ovary (fig. 2C), 3 had ovotestes (fig. 2D), and the remaining 5 had a well-developed oviduct, although no gonad was identified. In the letrozole-treated tissues with a well-developed oviduct but no gonad, it was assumed that a gonad developed but was lost during histological processing based upon morphological presence at dissection. Examination of gonads in the female and male control groups receiving ethanol only produced ovary and testis, respectively (fig. 2E, F).

Results

Histological Analysis of 17β-Estradiol- and LetrozoleTreated Embryos It was noted upon microdissection that throughout the embryonic developmental period, gonads treated with 17β-estradiol were greatly reduced in size or absent compared to the controls at a MPT or FPT. Therefore, the presumptive gonadal ridge was dissected and used for expression analysis. Histological analysis at embryonic stages 21 and 23 confirmed the presence of a gonadal ridge in some turtles but lack of gonadal differentiation at these stages [Bieser et al., 2013], and the same was confirmed at hatching (fig.  1). Histology was further conducted at hatch in 8 turtles treated with 17β-estradiol at a MPT (fig. 2A, B) and 13 turtles treated with letrozole at a 368

Sex Dev 2014;8:364–375 DOI: 10.1159/000369116

Gene Expression between Treatment Groups during the TSP Expression of Dmrt1 mRNA was influenced by stage (F[4,188] = 57.27, p < 0.0001), temperature/treatment (F[3,188] = 95.04, p < 0.0001), and the interaction beBieser /Wibbels  

 

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F

A

B

C

D

and FPT, a MPT with 17β-estradiol, and a FPT with letrozole. Within each gene, average fold change for the stage 15 male was

used as the calibrator for calculating relative expression. Levels of mRNA are expressed as mean fold change (± SEM). Numbers above columns (A) indicate the number of individual turtles used for the analysis and also apply for B–E.

tween temperature/treatment and stage of development (F[12,188] = 5.665, p < 0.0001) (fig.  3A). Expression of Dmrt1 mRNA at a MPT was significantly greater than all other groups from stage 17 through 23 (p < 0.01). At a MPT, there was a significant 23-fold increase between

stages 15 and 17, and a significant 73-fold increase between stages 19 and 21 (p < 0.01). There was not a significant fold increase between stages 17–19 and 21–23 (p > 0.1) at a MPT. Expression between the FPT and the letrozole-treated group did not differ across all stages

Expression of Putative Sex-Determining Genes in a Turtle with TSD

Sex Dev 2014;8:364–375 DOI: 10.1159/000369116

Fig. 3. Relative expression of Dmrt1 (A), Sox9 (B), Amh (C), Lhx9 (D), and Foxl2 (E) in embryonic gonads during the TSP at MPT

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E

the TSP at a MPT. Relative expression was calculated using the average fold change of stage 15 Dmrt1 at the MPT as the calibrator. Levels of mRNA are expressed as mean fold change (± SEM).

Fig. 5. Chronology of gene expression in embryonic gonads during

the TSP at a FPT. Relative expression was calculated using the average fold change of stage 15 Dmrt1 at the FPT as the calibrator. Levels of mRNA are expressed as mean fold change (± SEM).

(p > 0.05). In embryos treated with 17β-estradiol, expression remained near the baseline for all stages (p ≤ 0.05). Expression of Sox9 mRNA was influenced by stage (F[4,188] = 3.711, p = 0.0062), temperature/treatment (F[3,188] = 132.2, p < 0.0001), and the interaction between temperature/treatment and stage of development (F[12,188] = 27.10, p < 0.0001) (fig. 3B). Expression of Sox9 mRNA at a MPT was significantly greater than all other groups for stages 19, 21 and 23 (p < 0.0001), but did not 370

Sex Dev 2014;8:364–375 DOI: 10.1159/000369116

Chronology of Gene Expression within MPT and FPT Figure 4 shows the chronology of expression for all genes examined at the MPT. Expression of all genes was influenced by stage (F[4,225] = 74.88, p < 0.0001), gene (F[4,225] = 180.2, p < 0.0001), and an interaction between stage and gene (F[16,225] = 15.20, p < 0.0001). At stage 15, Sox9 had a 15-fold higher expression than Dmrt1 (p < 0.0001), but there was no significant difference in expression between Sox9 and Dmrt1 for stages 17 through 23 (p > 0.4). Both Sox9 and Dmrt1 increased across stages. Early in the TSP (stages 15 and 17), Amh, Dmrt1 and Sox9 were expressed much lower than both Foxl2 and Lhx9 (p < 0.0001). By stage 23, Sox9, Dmrt1 and Lhx9 had the Bieser /Wibbels  

 

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Fig. 4. Chronology of gene expression in embryonic gonads during

significantly differ between treatment/temperature at stages 15 and 17 (p > 0.1). In the FPT, 17β-estradiol- and letrozole-treated groups, expression of Sox9 was at comparable levels and remained low throughout all stages. Expression of Amh mRNA was influenced by stage (F[4,188] = 4.088, p = 0.0034), temperature/treatment (F[3,188] = 8.822, p < 0.0001), and the interaction between temperature/treatment and stage of development (F[12,188] = 3.010, p = 0.007) (fig. 3C). Expression of Amh was only detectable at a MPT at stages 19, 21 and 23; however, it did not significantly differ from the other groups until stage 21 and 23 (p < 0.01). At the MPT, there was a significant 5-fold increase in expression between stages 17 and 19 (p < 0.01), but increases between stages 19–21 and 21–23 were not significant most likely due to the highly variable expression between turtles. Expression of Lhx9 mRNA was influenced by stage (F[4,187] = 38.72, p < 0.0001), temperature/treatment (F[3,187] = 196.3, p < 0.0001), and the interaction between temperature/treatment and stage of development (F[12,187] = 8.469, p < 0.0001) (fig. 3D). The expression of Lhx9 did not differ between the MPT and the FPT at any embryonic stage or between the MPT and letrozole-treated embryos (p > 0.1). Embryos treated with 17β-estradiol had a significant reduction in Lhx9 mRNA expression compared to the control group at each embryonic stage (p < 0.0001) [Bieser et al., 2013]. Expression of Foxl2 mRNA was influenced by stage (F[4,188] = 5.724, p = 0.0002), temperature/treatment (F[3,188] = 16.69, p < 0.0001), and the interaction between temperature/treatment and stage of development (F[12,188] = 14.91, p < 0.0001 ) (fig. 3E). Expression of Foxl2 did not differ between groups until embryonic stage 21. By stage 23, expression of Foxl2 was 4-fold greater at the FPT than at the MPT and 3-fold higher than the 17β-estradiol and letrozole groups (p < 0.0001).

17β-Estradiol and Letrozole Are Ineffective SexReversal Treatments Several studies in the red-eared slider turtle have utilized 17β-estradiol and aromatase inhibitors to characterize the role of estrogens and aromatase in initiating gonadal differentiation in species with TSD. We initially sought to analyze changes in gene expression patterns to gain an insight on how estrogen may be influencing gene expression and diverting the bipotential gonad to develop as ovary. However, histological analysis at embryonic stages 21, 23 [Bieser et al., 2013] and at hatch produced unexpected results. The 17β-estradiol treatment protocol used involved a single 10-μg dose administered at embryonic stage 14 which has been demonstrated to result in 100% male-to-female sex reversal [Wibbels et al., 1991b]. Other dosing strategies have been employed such as 5 μg delivered at every other stage from embryonic stage 13 through 19 [Barske and Capel, 2010] and 50 μg at embryonic stages 15, 17, 19, or 21 [Dodd and

Wibbels, 2008]. These and previous results in our lab have indicated a reduction in ovary size and Müllerian duct deficiencies, but not the complete absence or nondifferentiation of a gonad [Dodd and Wibbels, 2008; Barske and Capel, 2010]. The current findings demonstrate the complete lack of gonadal development or development of a gonad that is reduced in size and undifferentiated by hatch. Due to difficulties visually identifying a gonad in the 17β-estradiol-treated turtles, the presumptive gonadal ridge region was used for expression analyses [Bieser et al., 2013]. Expression of Dmrt1, Sox9 and Amh were all near baseline in the 17β-estradiol-treated turtles resembling expression similar to or lower than the FPT group as was anticipated if complete sex reversal had occurred and has been previously reported with estrogen treatments [Murdock and Wibbels, 2006; Barske and Capel, 2010]. This does suggest, however, that exogenous estrogenic compounds have the ability to repress male-specific genes required for testis development at a MPT. The most striking loss of gene expression upon 17β-estradiol treatment was for Lhx9 since expression was expected to be similar at both the MPT and FPT. Our results differed from Barske and Capel [2010] who, using different methodology, saw no differences in Lhx9 expression upon 17β-estradiol treatment. They did, however, suggest that exogenous estrogen had a repressive effect on Sox9 resulting in feminization of the gonad, indicating that estrogen signaling represses Sox9 in the developing ovary. This hypothesis is not unreasonable, but under the circumstances of complete lack of gonadal development, it is likely that estrogens have a much greater repressive effect on the genital ridge and bipotential gonad prior to sex determination and differentiation. Interestingly, the expression of Foxl2 in the estrogen-treated turtles did not differ from the expression at a FPT until stage 23. These results are consistent with chicken embryos treated with estrogen and evidence that no estrogen response elements have been found in the chicken FOXL2 promoter [Hudson et al., 2005]. It is also predicted that FOXL2 lies upstream of aromatase; therefore, a response to exogenous estrogenic compounds was not hypothesized [Hudson et al., 2005]. Overall, depending on the specific protocol utilized, the 17β-estradiol treatments may not be resulting in a normal ovarian developmental cascade. It is plausible, then, that exogenous estrogenic compounds could be altering the sex-determining cascade rather than mimicking an endogenous pathway [Murdock and Wibbels, 2006; Ramsey and Crews, 2007, 2009]. Although an uncontrollable variable such as the estrogen

Expression of Putative Sex-Determining Genes in a Turtle with TSD

Sex Dev 2014;8:364–375 DOI: 10.1159/000369116

Discussion

The current study specifically targeted the evaluation of both the chronology and magnitude of expression of a variety of genes relative to one another utilizing the same incubation regimes and tissues. Although a number of these genes have received individual attention in a variety of species, the purpose of this study was to extend the previous findings by providing a comprehensive and simultaneous evaluation of these factors relative to one another within the same individuals. Utilizing quantitative real-time PCR, we examined the expression of Dmrt1, Sox9, Amh, Lhx9, and Foxl2 in gonads of the red-eared slider turtle incubated at MPT and FPT with and without the addition of previously identified sex-reversing chemicals, 17β-estradiol and letrozole.

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greatest expression. Foxl2 was highly expressed at all stages at a MPT with a 300-fold increase by stage 21 and a subsequent 200-fold decrease by stage 23. Figure 5 shows the chronology of expression for all genes examined at the FPT. Expression of all genes was influenced by stage (F[4,22] = 13.02, p < 0.0001), gene (F[4,224] = 297.4, p < 0.0001), and an interaction between stage and gene (F[16,224] = 3.721, p < 0.0001). Expression of Foxl2 was greater than all genes, except Lhx9, at all stages (p < 0.0001). Amh was not expressed at any stage. Dmrt1 and Sox9 expression were significantly lower than Lhx9 and Foxl2 at all stages (p < 0.0001).

Chronology of Gene Expression at MPT and FPT Of the genes examined, Dmrt1 stands out in this study with its early (embryonic stage 17) and continual sexually dimorphic expression pattern at the MPT. This expression pattern is consistent with a number of other studies in species with TSD [Smith et al., 1999a; Kettlewell et al., 2000; Torres Maldonado et al., 2002; Murdock and 372

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Wibbels, 2003; Rhen et al., 2007; Shoemaker et al., 2007a]. Although Dmrt1 displayed the earliest sexually dimorphic expression, in direct comparison at the MPT, Sox9 was upregulated at embryonic stage 15 prior to Dmrt1. However, expression of Sox9 was not sexually dimorphic until embryonic stage 19. It has been previously hypothesized that DMRT1 may upregulate Sox9 expression [Rhen et al., 2007; Bagheri-Fam et al., 2010]. Sexually dimorphic expression of Dmrt1 prior to Sox9 has been reported in mice, chicken, alligator, and turtle [Morrish and Sinclair, 2002; Smith et al., 2003; Shoemaker-Daly et al., 2010], with the current study supporting this timing of sexual dimorphism. However, the current study also indicates that Sox9 is upregulated prior to Dmrt1 at a MPT with expression of both remaining similar throughout the rest of development. The link between DMRT1 and SOX9 during testis determination is provided by a knockdown study in chicken [Smith et al., 2009]. In chicken, a doubledose of DMRT1 is required for testis differentiation. When DMRT1 is knocked down, there is a significant reduction in SOX9 expression [Smith et al., 2009]. Therefore, it is hypothesized that in species without SRY, DMRT1 may be involved in activating or maintaining SOX9 expression and testis formation [Bagheri-Fam et al., 2010; Murphy et al., 2010]. In the current study, due to the timing of Sox9 expression relative to Dmrt1 at the MPT, it would seem likely that DMRT1 is acting to maintain Sox9 expression rather than initiating its expression. However, since Sox9 is not sexually dimorphic until well into the TSP, it is unlikely the initiator of the bipotential fate but rather a regulator of testis development after testis commitment. Lastly, due to the early sexually dimorphic pattern of Dmrt1, it is a likely candidate for triggering the testicular pathway. While Dmrt1 is sexually dimorphic at embryonic stage 17, Sox9 becomes dimorphic by stage 19 followed by Amh at stage 21. Additionally, at the MPT, expression of Sox9 is significantly greater than Amh from the point Amh is first detected at stage 19 through stage 23. This increase in Amh during the late developmental stages corresponds with the regression of the Müllerian ducts between stages 22 and 23 [Wibbels et al., 1999]. Previous studies have indicated that it is unclear whether Sox9 is expressed prior to Amh or vice versa in the red-eared slider turtle [Shoemaker et al., 2007b; Shoemaker-Daly et al., 2010]. However, studies in chicken, alligator and red-eared slider turtle have shown that sexually dimorphic expression of Amh precedes that of Sox9. This suggests that SOX9 does not upregulate Amh in the red-eared slider turtle [Oreal et al., 1998; Smith et al., 1999c; Western et al., 1999; Bieser /Wibbels  

 

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lot number could have influenced the results, the most logical explanation is that 17β-estradiol disrupted normal ovarian differentiation. As has been previously noted [Barske and Capel, 2010], the use of aromatase inhibitors, such as letrozole, may not result in complete sex reversal and rather produce ovotestes at the FPT. Although other studies have demonstrated complete sex reversal using 10 μg of letrozole [Crews and Bergeron, 1994; Belaid et al., 2001], in an attempt to produce complete sex reversal, we doubled the dosage from Barske and Capel [2010] and delivered the drug multiple times during embryonic development. Our histological analysis at hatching produced ovaries and ovotestes but no tissues with complete sex reversal to testis. Although we increased the dosage and frequency of letrozole treatments, our results are congruent with previous studies [Barske and Capel, 2010], and incomplete masculinization is not likely due to lot number as has been suggested. Additionally, the expression analyses correspond with the lack of aromatase inhibition and sex reversal. The letrozole treatment group mimicked expression of the FPT for all genes, implying that the gonads did not undergo complete sex reversal to testis and/or that the FPT overrode any reduction in aromatase. If sex reversal had occurred, we anticipated levels of Sox9, Dmrt1 and Amh to resemble the MPT. For all genes, there were only 2 time points, stage 21 and 23, where only in Foxl2 did letrozole treatment alter expression of a gene resulting in a reduction from the FPT. In response to aromatase inhibitors in chicken embryos, FOXL2 reduction was noted but not complete loss of expression [Hudson et al., 2005]. The authors hypothesized that FOXL2 is upstream of aromatase and should not respond to aromatase inhibition but rather may be responding to depleted estrogen or to inhibition from SOX9 and DMRT1 [Hudson et al., 2005]. Therefore, it is possible that letrozole slightly reduced estrogen levels, as possibly evidenced by ovotestes production, but not enough to override the FPT response. Due to the inconsistencies in sex reversal with both 17βestradiol and letrozole, it is imperative that sex reversal be evaluated and verified to ensure that the treatment protocols are effective.

References

Expression of Putative Sex-Determining Genes in a Turtle with TSD

Ramsey et al. [2007] showed sexually dimorphic expression of aromatase in concordance with Foxl2. In summary, using quantitative real-time PCR, we demonstrate the expression patterns of 5 conserved genes in the red-eared slider turtle before, during and after the TSP. Our results indicate that Dmrt1 exhibits the earliest sexual dimorphism by embryonic stage 17 followed by Sox9 and Amh at a MPT representing conserved expression and strong evidence that these genes are required for testis differentiation in a turtle with TSD. This expression pattern is consistent with the hypothesis of DMRT1 maintaining Sox9 expression and SOX9 upregulating Amh. Conversely, Foxl2 was not sexually dimorphic until after the TSP indicating a possible role in both testicular and ovarian pathways. The pattern of expression for Lhx9 suggests that it may be functionally important for the formation of the bipotential gonad but also play a role in both ovarian and testicular development due to its continued expression in both tissues. Although reduced expression was apparent for testis-producing genes upon 17β-estradiol treatments, the changes observed in gonad development warrant further investigations into the exact role of both endogenous estrogen and exogenous estrogenic compounds in the sex-determining/differentiation cascade of TSD vertebrates. The same is true for treatments with aromatase inhibitors due to the lack of complete sex reversal at the MPT. Optimization of sexreversing protocols will be required in order to validate their influence on sex-determining pathways and further enhance our understanding of TSD.

Acknowledgements This research was supported by the Department of Biology University of Alabama at Birmingham and Sigma Xi Grants-InAid of Research. We thank Dr. Karolina Mukhtar in the UAB Department of Biology for her technical assistance; Taylor Roberge and Nickolas Bieser for statistical assistance and manuscript review, and several reviewers for their insightful comments.

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Shoemaker-Daly et al., 2010]. Our data contradict these findings suggesting that Sox9 is sexually dimorphic prior to and expressed higher than Amh. This pattern is consistent with SOX9 upregulating AMH in the mammalian testis. Since mRNA expression levels can only be used as an indicator of molecular regulation, further protein analysis would be necessary to confirm these findings. In mammals, FOXL2 is required for ovarian differentiation and development [Ottolenghi et al., 2005]. FOXL2 has been hypothesized to repress SOX9 expression in all vertebrates because it represses SOX9 in mammals, and it is the first gene that displays female-biased expression in chicken, reptiles and frogs [Ottolenghi et al., 2007; Bagheri-Fam et al., 2010]. Additionally, expression of Foxl2 in the common snapping turtle, mammals and fish has been shown to increase in parallel with aromatase and/or upregulate aromatase suggesting a link between both genes and estrogen [Govoroun et al., 2004; Pannetier et al., 2006; Rhen et al., 2007; Wang et al., 2007]. Although direct comparisons to previous studies cannot be made due to different methodologies [Loffler et al., 2003; Rhen et al., 2007; Shoemaker et al., 2007b; Shoemaker-Daly et al., 2010], the results demonstrate that sexually dimorphic expression of Foxl2 was not observed until embryonic stage 23, and prior to that both MPT and FPT gonads displayed similarly high expression levels. This is slightly later than what has been previously described [ShoemakerDaly et al., 2010], but up to that point, expression levels at both MPT and FPT are similar. If FOXL2 were a global inhibitor of Sox9, one would expect Sox9 repression whenever Foxl2 is highly expressed. However, our evidence suggests that since Foxl2 is expressed more than double that of Sox9 during stages 15–21 at a MPT while Sox9 is increasing, it seems unlikely that FOXL2 is acting as a repressor of Sox9 at least at a MPT. Additionally, due to the high levels of expression at a MPT, future research would benefit from examining a potential role for Foxl2 at a MPT in addition to a FPT. Due to limited quantities of RNA, aromatase was not assessed in this study, but

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differentiation genes in a turtle with temperature-dependent sex determination.

The red-eared slider turtle (Trachemys scripta) possesses temperature-dependent sex determination (TSD) in which the incubation temperature determines...
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