Female Sex Hormone May Reduce Production of a Male Growth Regulator in Mud Crabs

The green fluorescence (EGFP) were detected in human embryonic kidney cell line HEK293FT cells after transfection for 24 h, the results showed that EGFP can be detected in cells transfected with pEGFP-N1 (Figure 1A) and pEGFP-pSp-IAG (Figure 1B), but not in cells transfected with pEGFP-1 (Figure 1C). pEGFP-N1 used as a positive control contains cytomegalovirus (CMV) promoter, which can effectively turn on the expression of EGFP. EGFP in cells transfected with pEGFP-pSp-IAG demonstrated promoter activity at 5′-flanking sequence, while pEGFP-1 serving as negative control did not produce any fluorescence. Cells in bright fields are observed and shown in Figure 1D–F, separately.

According to distribution of binding sites for transcription factor in 5′-flanking sequence (Figure 2A), eight plasmids, pGL3-pSp-IAG1 to pGL3-pSp-IAG8, were constructed using pGL3-Basic and various regions of 5′-flanking sequence obtained by fixing the 3′ end and extending the 5′ end (Figure 2B). The sequencing results showed that the regions had no mutation and their lengths were 302, 420, 837, 967, 1235, 1845, 1986, and 2252 bp, respectively. Plasmids were transfected on HEK293FT cells, and luciferase activities of Firefly and Renilla determined to analyze the promoter activity. It was shown that the promoter activities of the eight plasmids were significantly higher than that of negative control (pGL3-basic) (Figure 2C). Compared with the previous plasmid, promoter activities were significantly enhanced in pGL3-pSp-IAG1 (Sox-5-binding site), pGL3-pSp-IAG2 (AP-1-binding site), pGL3-pSp-IAG3 (STAT-binding site), and pGL3-pSp-IAG6 (Sox-5-binding site), while there were no significant differences in pGL3-pSp-IAG4 (GATA-1-binding site, GATA-2-binding site), pGL3-pSp-IAG5 (GATA-1-binding site), and pGL3-pSp-IAG7. The promoter activity of pGL3-pSp-IAG8 (GATA-2-binding site) was significantly reduced than that of pGL3-pSp-IAG7

To analyze the regulation of transcription factor binding sites on promoter activity, SDM were performed on AP-1-binding site (pGL3-pSp-IAG2), STAT-binding site (pGL3-pSp-IAG3), Sox-5-binding site (pGL3-pSp-IAG6) and GATA-2-binding site (pGL3-pSp-IAG8). According to the sequencing results, plasmids with SDM were selected to detect the promoter activity. The results showed that promoter activities were significantly reduced in pGL3-pSp-IAG2-Mut (AP-1-binding site: TGCAGAC) (Figure 3A), pGL3-pSp-IAG3-Mut (STAT-binding site: CGAGATACGAGACGA) (Figure 3B), and pGL3-pSp-IAG6-Mut (Sox-5-binding site: GTCTCGTTAGCCT) (Figure 3C), while there was no significant difference in pGL3-pSp-IAG8-Mut (GATA-2-binding site: GGCTGCATTCTAG) (Figure 3D).

The precipitate was resuspended in binding buffer with 8 M urea, then analyzed by 15% SDS-PAGE (Figure S1A) which showed that rCFSH (~20 kDa) was expressed in E. coli BL21. Figure S1B showed that the purified rCFSH was analyzed by 15% SDS-PAGE, while Figure S1C showed that the renatured protein was analyzed by Western blotting with prepared anti-His-tag rabbit antibody, which confirmed that the antibody could specifically bind to the purified protein. Based on analysis of promoter activity by SDM, AP-1-binding site, STAT-binding site, and Sox-5-binding site may be involved in the regulation of Sp-IAG expression. Therefore, AGs were cultured in vitro and treated with rCFSH to further analyze transcriptional regulation of Sp-IAG expression by rCFSH. qRT-PCR findings showed that expression of Sp-STAT (Figure 4B) and Sp-IAG (Figure 4D) were significantly reduced in AG treated with rCFSH, while there was no difference in the expression of Sp-AP-1 (Figure 4A) and Sp-Sox (Figure 4C).

The role of Sp-STAT was examined by studying the loss of function of this gene by RNAi (dsRNA). Silencing outcomes showed that Sp-STAT-dsRNA administration significantly reduced the expression of Sp-STAT at 4 h, which was approximately 55% of those treated with saline or GFP (green fluorescent protein gene)-dsRNA (Figure 5A). qRT-PCR performed to analyze expression of Sp-IAG showed that the expression of Sp-IAG was also significantly reduced in AG treated with Sp-STAT-dsRNA for 4h (Figure 5B).

Mud crabs (Scylla paramamosain) were obtained from a local market in Xiamen, Fujian Province, China. In this study, crabs at pre-molt stage in autumn were selected. Crabs (carapace width 11.5–14.3 cm, body weight 250–320 g) were anesthetized and dissected, collecting muscles and AGs at stage III of the gland development [16]. Muscles provided genomic DNA while AGs were used for in vitro incubation study. The crabs were reared in tanks (40 × 40 × 60 cm) with seawater (24 L, salinity 26 ± 1 ppm and temperature 27 ± 2 °C), and fed on the white Pacific shrimp (Litopenaeus vannamei) meat. All animal procedures were carried out in strict compliance with the National Institute of Health Guidelines for the Care and Use of Laboratory Animals.

The 5′ region flanking Sp-IAG was amplified by PCR and then cloned in pMD19-T vector (TaKaRa, Dalian, China). Briefly, this region was digested with KpnI and BamHI (TaKaRa, Dalian, China), and ligated in a vector lacking gene promoters (pEGFP-1, YouBio, Changsha, China). The recombinant vector (pEGFP-pSp-IAG) was transfected on HEK293FT cells to determine transcriptional activity. The HEK293FT cells were obtained from the China Center for Type Culture Collection, Wuhan. After three passages, HEK293FT cells were cultured on 24-well plates in high glucose Dulbecco’s modified eagle medium (DMEM, HyClone, Logan, UT, USA) containing 10% FBS (fetal bovine serum) (Gibco, Grand Island, NY, USA) and 1% Penicillin-Streptomycin (Gibco, USA), at 37 °C for 12 h under 5% CO₂. Transient transfection of cells was performed using LipofectamineTM 2000 Reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocols. pEGFP-N1 contains cytomegalovirus (CMV) promoter, which effectively turns on the expression of EGFP. pEGFP-1 and pEGFP-N1 were used as negative and positive controls, respectively. After culture for 24 h, the cells were observed under a fluorescent microscope and green fluorescence of the live cells was observed under a Leica fluorescence microscopy imaging system (Leica, Solms, Germany).

Sequential deletion of fragments flanking 5′ Sp-IAG region were synthesized using primers (Table 1) containing KpnI and XhoI restriction sites (fixing the 3′-terminal and shrinking the 5′-terminal). After digestion with KpnI and XhoI (TaKaRa, Dalian, China), a series of recombinant vectors (pSp-IAG-1 to pSp-IAG-8) were constructed by connecting pGL3-Basic vector (a Luciferase Reporter Vector without promoter, Promega, Madison, WI, USA) with the synthesized fragments described above. The recombinant vectors were verified by sequencing and then transfected on HEK293FT cells using LipofectamineTM 2000 Reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocols [32]. pRL-TK vector was used to normalize the transfection efficiency by co-transfected with the reporter constructs. Cells were collected after a 24 h culture. Firefly and Renilla luciferase activities were measured by Dual-Luciferase Reporter Assay System (Promega, Madison, WI, USA) and chemiluminescence was measured using Varioskan Flash (Thermo Scientific, Waltham, MA, USA). Promoter activity was evaluated by the normalized mean of firefly luciferase action on Renilla luciferase activity.

SDM was performed by overlap extension PCR with primers containing the mutational bases (the transcription factor binding sites are shown in bold, Table 1), and was used to identify the function of transcription elements. pspIAG-2F/Sp-AP-R-mut, pspIAG-3F/Sp-STAT-R-mut, pspIAG-6F/Sp-Sox-R-mut, and pspIAG-8F/Sp-GATA-R-mut were utilized in amplifying pSp-AP-5′, pSp-STAT-5′, pSp-Sox-5′, and pSp-GATA-5′; Sp-AP-F-mut/Sp-STAT-F-mut/Sp-Sox-F-mut/Sp-GATA-F-mut while pspIAG-R was utilized in amplifying pSp-AP-3′, pSp-STAT-3′, pSp-Sox-3′, and pSp-GATA-3′. PCR conditions were 34 cycles, with initial 94 °C for 5 min; and subsequently 94 °C for 30 s, 57 °C for 30 s, 72 °C for 1 min, followed by final extension at 72 °C for 10 min using an ABI 2720 Thermal Cycler (Applied Biosystems). The second reaction was performed in an amplification system containing 2.0 µL 10× Ex Taq Buffer (Mg²⁺ Plus), 0.8 µL dNTP (2.5 mM), 0.1 µL Ex Taq (5U/μL) (TaKaRa, Dalian, China), 2 µL pSp-AP-5′/pSp-STAT-5′/pSp-Sox-5′/pSp-GATA-5, 2 µL pSp-AP-3′/pSp-STAT-3′/pSp-Sox-3′/pSp-GATA-3′, and 12.3 µL water, under initial 94 °C for 5 min; followed by 10 cycles, each at 94 °C for 40 s, 57 °C for 1 min, 72 °C for 1 min, 20 °C for 5 min followed by adding 0.4 µL pspIAG-2F/pspIAG-3F/pspIAG-6F/pspIAG-8F and 0.8 µL pspIAG-R (10μM) to the amplification system. The second reaction conditions were 35 cycles, each at 94 °C for 40 s, 57 °C for 1 min, 72 °C for 1 min, and 72 °C for 10 min. Amplification products were verified by sequencing. Products with SDM were purified and digested with KpnI and XholI (TaKaRa, Dalian, China), then ligated in pGL3-Basic vector. The reporter vectors were transiently transfected on HEK293FT cells and evaluated for relative luciferase activity by Dual-Luciferase Reporter Assay (Promage, Madison, WI, USA). Reporter vector with normal transcription element and pGL3-basic were used as positive and the negative controls, respectively.

Recombinant CFSH (rCFSH) was produced as previously described [16]. Briefly, Sp-CFSH was amplified and cloned in pET-His (a prokaryotic expression vector). After sequencing and analysis, the recombinant vector was transfected on E. coli BL21 (DE3), and cultured in Luria-Bertani medium with 0.8 mM isopropyl-D-thiogalactopyranoside for 8 h at 37 °C. After collection and cleaning, the bacteria were suspended in binding buffer and dissociated by ultrasonic wave. Soluble and insoluble fractions were separated by centrifugation at 12,000× g for 10 min at 4 °C. The precipitate containing large amount of rCFSH was resuspended in binding buffer with 8 M urea, then which was analyzed by 15% SDS-PAGE and loaded on a Ni-NTA HisTrapTM FF crude column (GE Healthcare, Danderyd, Sweden) in accordance with the manufacturer’s instructions. Gradient elution was performed with a buffer containing 50 mM imidazole, 100 mM imidazole, 200 mM imidazole and 300 mM imidazole. Eluting solution was collected and examined with 15% SDS-PAGE. Finally, purified rCFSH was denatured with graded urea dialysis and confirmed by Western blotting. The proteins were stored at −80 °C.

CFSH has been shown to inhibit expression of Sp-IAG in S. paramamosain. AGs of one crab were divided into two and cultured according to the established methods [55,56]. First, AGs were rinsed 9 times with saline containing penicillin G (300 IU/mL) and streptomycin (300 µg/mL) (Sigma-Aldrich Co. Louis, MO, USA), then pipetted on a 48-well plate and pre-incubated at 25 °C with 100 μL 2 × L15 medium (Gibco, Grand Island, NY, USA). The medium was changed after one hour for final incubation. Controls were incubated in 2 × L15 medium only while the test samples were incubated in 2 × L15 medium containing 10⁻⁶ M rCFSH (rCFSH group). AGs were collected after 8 h to determine the effects of rCFSH on Sp-AP-1/Sp-STAT/Sp-Sox/Sp-IAG expression. Four biological replicas were prepared in each experiment.

Green fluorescent protein gene (GFP), as an exogenous control, was cloned in pSicoR-EGFP vector [56]. As previously described [57], Sp-STAT and GFP were cloned and purified for synthesis of Sp-STAT-dsRNA and GFP-dsRNA by T7 and SP6 polymerase (Roche, Mannheim, Germany). AGs of one crab were divided into three. AGs were rinsed 9 times with saline containing penicillin G (300 IU/mL) and streptomycin (300 µg/mL) (Sigma-Aldrich Co. Louis, MO, USA), then pipetted on a 48-well plate and pre-incubated at 25 °C with 100 μL 2 × L15 medium (Gibco, Grand Island, NY, USA). The medium were changed after one hour for final incubation. They were incubated in 2 × L15 medium (positive control), 2 × L15 medium containing 1 µg/mL GFP-dsRNA (negative control) or 2 × L15 medium containing 1 µg/mL Sp-STAT-dsRNA, respectively. AGs were collected after either 2 or 4 h to evaluate effects of Sp-STAT mRNA knockdown. Then, samples with obvious interference efficiency were selected to detect the expression of Sp-IAG gene. Four biological replicas were evaluated in the experiments.

Total RNA was extracted from AGs using TRIzol (Invitrogen, USA). Quality and concentration of RNA was measured using a NanoDrop-2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA), and 200 ng of total RNA was reverse-transcribed with random primer using TransScriptII One-step gDNA Removal and cDNA Synthesis SuperMix (TransGen, Beijing, China) according to the manufacturer’s protocol. The cDNAs were diluted four times for quantitative reverse transcription PCR (qRT-PCR) analysis, with β-actin used as a reference gene. Primers in qRT-PCR assay had been previous used as shown in Table 1 [16,54]. Products of the reaction were sequenced to confirm the specificity of qRT-PCR. qRT-PCR was performed with SYBR Select Master Mix (TaKaRa, Dalian, Japan) under the following conditions: an initial 95 °C for 30 s, 40 cycles at 95 °C for 5 s, 58 °C for 30 s, and 72 °C for 30 s. The procedure was carried out in a 7500 Fast Real-time PCR System (Applied Biosystems). Melting curves were analyzed to exclude any non-specific products. qRT-PCR data were calculated by 2^−ΔΔCt method.

The 2^−ΔΔCt method was used to analyze the qRT-PCR data. The statistical analysis was performed using SPSS 22.0, including Duncan’s multiple range tests and one-way ANOVA, and p-value < 0.05 was considered statistically significant.