Four-year-old mature male and female orange-spotted groupers were obtained from the Marine Fisheries Development Center of Guangdong Province (Huizhou, China). Before the experiment, all fish were cannulated through the genital pore to collect gonadal tissue and examined by histology to determine gonadal stage. Fish were individually tagged with a biochip transponder under the dorsal skin for identification. The body weight (BW) and standard length (SL) of each fish were also recorded. All animal handling and experimentation were conducted in accordance with the guidelines and approval of the Animal Research and Ethics Committees of Sun Yat-sen University.

Gonadsomatic index (GSI) was determined for all sacrificed fish according to the following formula: GSI = 100 (gonadal weight/total weight).

Pieces of gonadal tissues were fixed in Bouin's fluid for 24 h at room temperature and then transferred to 70% ethanol prior to dehydration and paraffin embedding. Gonadal tissues were then serially sectioned at a thickness of 7 μm and stained with hematoxylin and eosin for subsequent analysis. Gonadal stage and sex change status were then determined under light microscopy (LEICA DM 1000 LED, Wetzlar, Germany).

Approximately 1 ml of blood was collected from the caudal vein of each fish using a 5 ml non-heparinized syringe and kept in a 1.5 ml microcentrifuge tube until being centrifuged ~3 h later. Serum was separated by centrifugation at 6,000 g for 10 min and then stored at −20°C until further use. The levels of serum cortisol, 11-KT, and E2 were subsequently measured using Enzyme Immunoassay Assay kits (Cayman Chemical Co, Ann Arbor, MI, USA) according to the manufacturer's instructions.

Total RNA was extracted from the tissue samples using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) and reverse transcribed into cDNA by ReverTra Ace qPCR RT Kit (TOYOBO, Osaka, Japan) in accordance with the manufacturer's instructions. Real-time PCR was performed on a Roche Light Cycler 480 real-time PCR System using SYBR Green Real-Time PCR Master Mix (Roche, Germany) according to the manufacturer's protocol. The real-time PCR conditions were as follows: denaturation at 95°C for 5 min, followed by 40 cycles at 95°C for 15 s, 56°C for 20 s, and 72°C for 20 s. The mRNA levels of gr1 (glucocorticoid receptor 1), gr2 (glucocorticoid receptor 2), dmrt1 (doublesex and mab-3-related transcription factor 1), sox9 (sex-determining region y-box 9), amh (anti-Müllerian hormone), hsd11b2 (11β-hydroxysteroid dehydrogenase type 2), cyp11b (11β-hydroxylase), and cyp19a1a (aromatase P450) were then analyzed, with β-actin serving as an internal control. After amplification, fluorescent data were converted to threshold cycle values (Ct). The relative abundance of mRNA transcripts was then evaluated using the formula R = 2^−ΔΔCt, as described previously (19). The sequences encoding for the genes investigated in this study were obtained from transcriptomic data (unpublished data). Table 1 lists the primers used in this study.

Apoptosis during cortisol-induced sex change was detected using a TUNEL Apoptosis Detection Kit (Phygene, Fuzhou, China) in accordance with the manufacturer's instructions. Samples were then analyzed under a light microscope (Nikon IQ50, Tokyo, Japan).

Based on genomic and transcriptomic data (unpublished data) previously obtained for the orange-spotted grouper, we amplified the complete open reading frame (ORF) of gr1 and gr2 using Phanta Max Super-Fidelity DNA Polymerase (Vazyme Biotech, China) and then inserted the ORF into the pcDNA4.0 vector (Invitrogen). Human embryonic kidney (HEK) 293 cells were then cultured in DMEM (Hyclone, USA) supplemented with 10% fetal bovine serum (FBS) (Hyclone, USA) at 37°C in a humidified atmosphere containing 5% CO₂. To confirm the expression of gr1 and gr2 in HEK293 cells, the pcDNA4.0-gr1 and pcDNA4.0-gr2 plasmids were transfected into HEK293 cells using Lipofectamine 3000 reagent (Invitrogen), respectively. At 24 h after transfection, the cells were lysed with RIPA lysis buffer (Beyotime Institute of Biotechnology, China) containing 1% protease inhibitor (Sigma-Aldrich, St. Louis, MO, USA), and total proteins were extracted for Western blotting using an anti-his tag antibody (Proteintech, USA).

To analyze ligand specificity and the downstream signaling pathways of gr1 and gr2, HEK293 cells were seeded into 48-well plates and cultured 24 h prior to transfection. Then, 200 ng/well pcDNA4.0-gr1/pcDNA4.0-gr2, 100 ng/well pGRE-Luc (Beyotime Institute of Biotechnology, China) and 5 ng/well pRL-TK (Promega, WI, USA) were co-transfected. Approximately 12 h after the transfection, the medium was replaced with fresh medium (supplemented with 10% FBS). We then treated the transfected cells with cortisol (Sigma-Aldrich, St. Louis, MO, USA). Luciferase activity was measured after 12 h of hormone treatment using the Dual-Luciferase Reporter Assay System (Promega) in accordance with the manufacturer's instructions.

To confirm whether cortisol was able to control the relative expression of cyp19a1a and amh by binding to GREs within the promoter regions, we amplified a 2,500 bp sequence upstream from the translational start site of cyp19a1a (GenBank Accession Number: JF420889) and amh (GenBank Accession Number: MG017511) and inserted these fragments into the pGL4.1 vector (Invitrogen) using KpnI and XhoI restriction sites. HEK293 cells were then seeded into 48-well plates and cultured for 12 h. Cells were then co-transfected with 200 ng/well of pcDNA4.0/pcDNA4.0-gr1/pcDNA4.0-gr2, 100 ng/well pGL4.1/pGL4.1-cyp19a1a/pGL4.1-amh, and 5 ng/well pRL-TK. Approximately 12 h after transfection, cells were treated with cortisol (1,000 ng/ml). Luciferase activity was measured 12 h later. All experiments were repeated independently three times. Table 1 shows the primers used for cloning.

Data are expressed as the mean ± standard error of the mean (SEM). Differences between groups were evaluated by means of one-way analysis of variance (ANOVA), followed by the Tukey test with statistical significance set at P < 0.05. All statistical tests were performed using SPSS 18.0 (SPSS, Chicago, IL, USA).

Gonadal reprogramming of cortisol-induced female-to-male sex change can be divided into four phases: a female phase, a degenerative phase, an intersex-transitional phase, and a male phase. In brief, the female phase was characterized by the presence of primary oocytes and previtellogenic (cortical alveolar) oocytes in the ovary (Figure 1A). During the degenerative phase, the ovary underwent degeneration and contained numerous atretic oocytes (Figures 1B,C). The intersex-transitional phase, in which female and male germ cells coexisted in the gonad, was characterized by the degeneration of oocytes and a simultaneous proliferation of spermatogonia in spermatogenic cysts (Figure 1D). During the male phase, spermatogenic germ cells were evident in the gonad at various stages of development (Figure 1E). The gonadal stages of fish in the different experimental groups are shown in Table 2.

During the experimental period, the gonads of fish in the control group retained their ovarian status. However, in the groups receiving cortisol injection, the gonads showed structural changes. At 7 dat, all of the cortisol-injection groups were undergoing the degenerative phase and contained many atretic oocytes. At 15 dat, the ovaries had undergone further degeneration, and almost all of the atretic oocytes had been absorbed in the high-dose cortisol and medium-dose cortisol groups. At 30 dat, two fish in the medium-dose cortisol group and all the fish in the high-dose cortisol group had entered the intersex-transitional phase, suggesting that sex change was underway. By 60 dat, two fish in the high-dose cortisol group were found to possess mature testes, indicating that sex change had been completed, and the gonads of the remaining two fish still remained in the intersex-transitional phase. Although atretic oocytes were observed in the low-dose cortisol group, no sex change occurred, and oocytes remained in the primary growth stage throughout the experimental period.

By the end of the experiment, the GSI of the high-dose group (0.16 ± 0.02%) was significantly lower than that of the other groups (control group, 0.48 ± 0.06%; low-dose group, 0.45 ± 0.04%; and medium-dose group, 0.62 ± 0.04%) and was not significantly different from that of the normal males (0.21 ± 0.03%) (Figure 2).

In the control group, no apoptosis was detected during the experiment (Figures 3A–E). In contrast, gonads in the high-dose cortisol group showed apoptotic signals first at 7 dat in some atretic oocytes (Figures 3F,G,K), and the extent of apoptosis had increased in nearly all the oocytes by 15 dat (Figures 3H,L). However, there was no evidence of apoptosis from 30 to 60 dat (Figures 3I,J), when the gonads entered the intersex-transitional and male phases.

Next, we analyzed short-term changes in serum steroid hormone levels after cortisol injection. A significant increase in serum cortisol levels was observed in the high-dose and medium-dose cortisol groups at 12, 24, 48, and 96 hat and in the low-dose cortisol group at 12 hat (Figure 4A). Compared with the other groups, serum 11-KT levels were significantly higher in the high-dose cortisol group at 12, 24, 48, and 96 hat (Figure 4B). In contrast, there were no significant differences in terms of E2 levels when compared across the different groups after cortisol treatment (Figure 4C).

During long-term cortisol treatment, we observed an elevation of serum cortisol levels in all of the cortisol-treated groups. Furthermore, cortisol levels in the high-dose and medium-dose cortisol groups were significantly higher than those in the control group at all sampling time points (7, 15, 30, and 60 dat), but in the low-dose cortisol group, serum cortisol levels were only significantly higher than those in the control group at 30 dat (Figure 6A). The changes in serum 11-KT levels were consistent with the changes observed in cortisol levels. At each sampling time, serum 11-KT levels in the high-dose cortisol group were significantly higher than those in the other groups and even higher than the levels of males. In the medium-dose cortisol group, serum 11-KT levels were significantly higher than those in the control group at 15 and 60 dat. No significant difference was observed between the low-dose cortisol group and the control group (Figure 6B). In contrast, serum E2 levels showed no significant difference when compared across different groups at most sampling time points; we only observed a significant reduction of E2 in the high-dose cortisol group at 30 dat and 60 dat (Figure 6C).

Next, we analyzed the short-term effects of cortisol injection on the gonadal expression of glucocorticoid receptor genes and sex-related genes. The expression of glucocorticoid receptor genes (gr1, gr2) and male-related genes (dmrt1, sox9, amh and hsd11b2) increased rapidly after cortisol injection (Figures 5A–F). However, cyp11b, the gene encoding the key enzyme responsible for synthesizing the androgen 11-KT, did not change significantly after the injection of cortisol (Figure 5G). The expression of the female-related gene cyp19a1a increased significantly in the low-dose and medium-dose cortisol group at 24 hat but showed no significant difference between the cortisol-treated groups and the control group at 96 hat (Figure 5H).

We also analyzed gene expression profiles during long-term cortisol treatment. Compared with the control group, the expression levels of the glucocorticoid receptor genes (gr1, gr2) increased in all of the cortisol-treated groups (Figures 7A,B). The expression of dmrt1 increased significantly in the high-dose cortisol group from 7 to 60 dat (Figure 7C). Similarly, the expression of sox9 increased significantly in the high-dose cortisol group from 30 to 60 dat and in the medium-dose group at 15 dat (Figure 7D). We also observed a significant increase in amh expression in the high-dose cortisol group from 7 to 60 dat, in the medium-dose group from 7 to 30 dat, and in the low-dose group from 7 to 15 dat (Figure 7E). The expression of hsd11b2 increased significantly in the high-dose cortisol group from 7 to 60 dat and in the medium-dose group from 7 to 15 dat (Figure 7F). In contrast, the expression of cyp11b only increased significantly in the high-dose cortisol group at 60 dat (Figure 7G). It is noteworthy that the expression of the female-related gene cyp19a1a did not change significantly during long-term cortisol treatment (Figure 7H).

Figure 8A shows the time schedule of sampling. Table 3 shows the changes in gonadal stage across different groups of fish following the withdrawal of cortisol. In the 30 days cortisol-withdrawal experiment, the gonads of fish in the control group remained in the female phase. In contrast, the gonads of all fish in the cortisol-injection group and in the 30 days cortisol-withdrawal group had reached the intersex-transitional phase prior to cortisol withdrawal (Table 3). At the 60 dat sampling point, the gonads of fish in the control group contained numerous primary oocytes and previtellogenic oocytes (Figure 8B, a). In the cortisol-injection group, the gonad of one fish had almost reached the male phase, containing spermatogenic germ cells at various developmental stages and a few atretic oocytes (Figure 8B, c). The remaining three fish were in the intersex-transitional phase. However, in the cortisol-withdrawal group, only one fish remained in the intersex-transitional phase. In the other three fish, the gonads had been reversed to an ovarian status and featured primary oocytes and previtellogenic oocytes; the male germ cells had disappeared (Figure 8B, e).

In contrast, in the 60 days cortisol-withdrawal experiment, three of the four fish had reached the male phase prior to the withdrawal of cortisol (Table 3). At the 90 dat sampling point, the gonads of fish in the control group were characterized by the presence of primary oocytes (Figure 8B, b). Two of the four fish in the cortisol-injection group had completely changed sex to male (Figure 8B, d). In the cortisol-withdrawal group, one of the three sex-changed males had maintained male characteristics after 30 days of cortisol withdrawal (Figure 8B, f), while the other two had reverted to the intersex-transitional phase.

The levels of serum steroid hormones were further examined after the withdrawal of cortisol treatment. Compared with levels in the negative control fish, the fish receiving cortisol injections had significantly higher levels of serum cortisol and 11-KT (Figures 9A,B) and lower levels of serum E2 (Figure 9C) during the experiments. In the 30 days cortisol-withdrawal experiment, the fish receiving cortisol injection showed high levels of serum cortisol and 11-KT; in contrast, there was a significant decrease in serum cortisol and 11-KT levels in fish after 30 days of cortisol withdrawal. Levels in the cortisol-withdrawal fish were not significantly different from those in the negative control fish (Figures 9A,B). In contrast, serum E2 levels in the cortisol-withdrawal fish were significantly higher than those in the cortisol-injection fish but were not significantly different from those in the negative control fish (Figure 9C). In the 60 days cortisol-withdrawal experiment, we observed a significant decrease in serum cortisol and 11-KT levels in the cortisol-withdrawal fish (Figures 9A,B), but there was no significant change in serum E2 levels across the different treatments (Figure 9C).

Next, we investigated gene expression profiles after cortisol-injection withdrawal. In the 30 days cortisol-withdrawal experiment, the expression levels of the glucocorticoid receptor genes (gr1 and gr2) and sex-related genes (dmrt1, sox9, amh, hsd11b2, cyp11b, and cyp19a1a) were significantly reduced in the cortisol-withdrawal fish and were not significantly different from those in the control fish after cortisol withdrawal (Figures 10A–H). Furthermore, in the 60 days cortisol-withdrawal experiment, the expression levels of gr2, hsd11b2, and cyp19a1a significantly decreased following the withdrawal of cortisol (Figures 10B,F,H). In contrast, although the levels of expression of gr1, dmrt1, sox9, amh, and cyp11b were significantly down-regulated after the withdrawal of cortisol, these levels were still significantly higher than those in the control fish (Figures 10A,C,D,E,G).

After confirming the expression of gr1 and gr2 in HEK293 cells (Figure 11A), the in vitro bioactivity of cortisol was assessed using a homologous receptor binding assay in HEK 293 cells transiently expressing orange-spotted grouper GRs. Using a GRE-driven luciferase assay, we found that luciferase activity exhibited a dose-response curve (Figure 11B). GR1 was activated by high concentrations of cortisol, while, in cells transfected with GR2, high concentrations of cortisol suppressed the expression of downstream GRE-driven luciferase (Figure 11B). We next examined whether the transcription of the female-related gene cyp19a1a and the male-related gene amh was regulated by cortisol/GR signaling in HEK 293 cells. Our data showed that a high concentration of cortisol (1,000 ng/ml) significantly stimulated the transcription of cyp19a1a and amh genes in cells transfected with GR1 or GR2 (Figures 11C,D).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.