Identification of a Putative CFSH Receptor Inhibiting IAG Expression in Crabs

The full length of Sp-SEFIR (GenBank accession number: ON787957↗) cDNA is 2515 base pairs (bp), including a 70 bp 5′ untranslated region (UTR), a 1959 bp ORF and a 486 bp 3′ UTR. The polyadenylation signal (AATAAA) is located at 149 bp upstream of the polyA sequence (Figure A1). The ORF segment was responsible for the synthesis of a polypeptide consisting of 652 amino acids (aa), with a molecular weight of 73801.91 Da and a theoretical isoelectric point (pI) of 5.27. The precursor polypeptide was inferred to possess a 23 aa signal peptide, a 23 aa transmembrane domain, a 123 aa SEFIR domain and two low-compositional complexity regions (11 and 30 aa, respectively) (Figure 1A).

Phylogenetic analysis demonstrated that IL-17R was clustered into five clusters: IL-17RA, IL-17RB, IL-17RC, IL-17RD, as well as IL-17RE. Sp-SEFIR was classified as IL-17RD (Figure 1B).

RT-PCR demonstrated that Sp-SEFIR had a broad distribution across different tissues in males. The Sp-SEFIR mRNA expression level was relatively higher in the Y organ, testis, androgenic gland, stomach, hepatopancreas and muscle (Figure 2A).

The expression profile of Sp-SEFIR in AG throughout AG advancement (stage I–III) was determined using qRT-PCR. The findings revealed that the expression patterns of Sp-SEFIR mRNA were elevated along with the development of AG to reach a peak at stage II before significantly being reduced at stage III (Figure 2B).

Consistent with the previous study, two forms of glandular cells (A and B) were observed in AG [41]. Type A glandular cells have a round, less heterochromatic nucleus, with a lightly stained cytoplasm and indistinct borders (Figure 3A). In type B glandular cells, the cytoplasmic staining is dark and uniform, with hyperchromatic nuclei and well-defined borders (Figure 3A). Sp-SEFIR is mainly located on the membrane of Type B glandular cells (Figure 3B).

Ligand–receptor interaction analysis was conducted using GST pull-down assays. Through prokaryotic expression, we obtained rHisCFSH (20 kDa) (Figure A2), rGSTCFSH (47 kDa) (Figure 4) and rSEFIR (65 kDa) (Figure A3). Results of GST pull-down assay with rGSTCFSH and total protein of AG demonstrated that Sp-SEFIR was a CFSH-binding component of AG (Figure 4). The further GST pull-down assay with rHisCFSH and rSEFIR confirmed that Sp-CFSH could specifically bind to extracellular regions of Sp-SEFIR (Figure 5).

Previous studies have shown that CFSH could suppress IAG and STAT expression in S. paramamosain [27,28]. To study the involvement of Sp-SEFIR in this inhibition, we first carried out in vivo RNA interference experiments. Based on current results, compared to CPS-injected therapy, Sp-SEFIR expression was 42% inhibited (Figure 6A). Meanwhile, the knockdown of Sp-SEFIR significantly induced Sp-IAG and Sp-STAT expression in AG (Figure 6B,C).

To further explore the involvement of Sp-SEFIR in IAG regulation via CFSH, we conducted interference experiments of Sp-SEFIR in vitro. According to the results, the addition of SEFIR-dsRNA inhibited Sp-SEFIR expression by 48% in vitro compared to the PBS therapy (Figure A4A). Furthermore, we confirmed that the addition of rHisCFSH (10⁻⁶ M) could suppress Sp-IAG expression in AG, as previously reported (Figure A4B) [27,28].

Following Sp-SEFIR silencing in the in vitro AG explant culture system, we added rHisCFSH (10⁻⁶ M) and detected the mRNA expression pattern of Sp-IAG and Sp-STAT. The findings demonstrated that the knockdown of Sp-SEFIR could also significantly induce Sp-IAG and Sp-STAT expression in vitro (Figure 7).

According to previous studies, the development stages of AG were classified as follows: Stage I, AG was small and contained fewer secretory cells, which were attached to the spermaduct; Stage II, AG was clearly linear, cells were clustered or cross-linked into cords, and glands expanded into the surrounding connective tissues; Stage III, AG was largest, and tissue hyperplasia occurred in some areas, which contained the largest number of secretory cells; Stage IV, development of AG stopped, AG degenerated rapidly, and its size was smaller than that in stage II and III [41,56].

In this study, S. paramamosain at AG development stage I (body weight: 48.4 ± 4.5 g, carapace width: 6.6 ± 0.5 cm), stage II (body weight: 146.7 ± 9.8 g, carapace width: 11.7 ± 1.0 cm) and stage III (body weight: 249.1 ± 13.2 g, carapace width: 14.7 ± 0.5 cm) were selected as experimental materials. Upon arrival at the laboratory, individuals were subjected to a period of acclimatization, during which they were exposed to controlled conditions consisting of 27 ± 1 °C in addition to a salinity level of 26 ± 0.5 ppm.

The transcriptome of S. paramamosain was utilized to obtain the Sp-SEFIR transcript. Moreover, the AG’s total RNA was retrieved by utilizing TRIzol Reagent (Invitrogen, Carlsbad, CA, USA) in accordance with the guidelines provided by the supplier. The 3′ and 5′ untranslated regions (UTR) of Sp-CFSHR were acquired through the utilization of rapid amplification of cDNA ends (RACE) technique, employing the SMART ^TM RACE cDNA Amplification Kit (Clontech, Palo Alto, CA, USA) in accordance with the producer’s guidelines. The validation of open reading frame (ORF) was performed via synthesizing the first-strand cDNA from 1 μg of total RNA utilizing the PrimeScript RT Reagent Kit with gDNA Eraser (TaKaRa, Dalian, China). The ORF of Sp-SEFIR was confirmed by specific primers Sp-SEFIR-OF/OR (Table A1). Utilizing LA Taq polymerase (TaKaRa, Dalian, China), polymerase chain reaction (PCR) was conducted as per these criteria: 94 °C for 5 min; 35 cycles of 94 °C for 30 s, 56 °C for 30 s and 72 °C for 100 s, and then through 72 °C for 10 min final extension. PCR outcomes were observed with 1.5% agarose (Biowest, Kansas City, MO, USA) gel electrophoresis before being connected to the pMD19-T vector (Takara, Dalian, China) for sequencing. Table A1 lists the primer sequences.

The primers utilized for qRT-PCR were obtained from previous investigations [27,28]. The determination of the amplification effect of all primer pairs was conducted prior to their utilization in qRT-PCR tests. The cDNA underwent a dilution of a four-fold magnitude utilizing water that was free of RNases. Components in a 20 μL qRT-PCR reaction system were: 10 μL of 2 × PCR main mixture containing SYBR Green, 2 μL of diluted cDNA, 0.5 μL of every primer and 7 μL of water. The experiment was conducted using a 7500 rapid RT-PCR (Applied Biosystems, CA, USA), and the experimental parameters utilized for the reaction were: 95 °C for 2 min, then 40 cycles of 95 °C for 15 s, 58 °C for 30 s, and 72 °C for 30 s. The 2^−ΔΔCt approach was employed to measure the outcome, with the reference gene being β-actin (GenBank accession number: GU992421↗).

The reverse transcription-PCR (RT-PCR) was utilized to detect the distribution profile of Sp-SEFIR in various tissues (eyestalk ganglion, cerebral ganglion, thoracic ganglion, Y organ, heart, testis, androgenic gland, stomach, hepatopancreas, muscle and epidermis) of S. paramamosain (n = 3). The procedures for extracting total RNA and first-strand cDNAs synthesized in accordance with the guidelines outlined in the relevant Section 4.2. The Sp-SEFIR-F/R was used as a primer, β-actin (GenBank accession no: GU992421↗) was amplified as a positive control, which was achieved utilizing Ex Taq polymerase (TaKaRa, Dalian, China), and the circumstances were: 94 °C for 5 min; 35 cycles of 94 °C for 30 s, 55 °C for 30 s and 72 °C for 30 s before an extension at 72 °C for 5 min. The PCR outcomes underwent analysis through the utilization of 1.5% agarose gel electrophoresis and were subsequently imaged with a UV detector (Geldoc, Thermo Fisher Scientific, Madrid, Spain).

qRT-PCR determined the Sp-SEFIR mRNA expression patterns in AG at stage I–III (n = 5). Total RNA was extracted, first-strand cDNAs were synthesized, and qRT-PCR was conducted as mentioned before (Section 4.2 and Section 4.3).

Immunofluorescence assays for Sp-SEFIR were performed with AG attached to the subterminal portion of ejaculatory ducts. We entrusted Shanghai GL Biochem Co., Ltd. (Shanghai, China) to produce the Sp-SEFIR antibody.

The tissues were first preserved in modified Bouin’s fixative (25 mL 37–40% formaldehyde, 75 mL saturation picric acid in addition to 5 mL glacial acetic acid) for one night at 4 °C. Following gradient alcohol drying, tissues were immersed in paraffin and prepared for 5 μm slices. After being dewaxed and rehydrated, parts of tissue sections were subjected to Hematoxylin-Eosin (HE) staining for histological observation. Meanwhile, other tissue sections were repaired in antigen repair solution (EDTA Antigen Retrieval Solution, pH 8.0; Sangon Biotech, Shanghai, China) at 99 °C for 20 min and cooled down to room temperature naturally. After antigen retrieval, slides were blocked with 5% bovine serum antigen (BSA) in 1 × PBS for 30 min at room temperature. After that, the tissue samples were incubated with Sp-SEFIR antibodies (1:500 dilution) or preimmune serum (negative control) at 37 °C for 2 h. Upon using PBS for washing, tissue samples were incubated with Goat Anti-Mouse IgG H&L (Alexa Fluor^® 488) preadsorbed (1:100, Abcam, Cambridge, MA, USA) diluted with 1% BSA in 1 × PBS for 45 min at room temperature. Subsequently, tissue samples were incubated with DAPI (1 μg/mL, Beyotime, Shanghai, China) for nuclear staining. Following washing one time in PBS, the slides were locked up using mounting medium, antifading (Solarbio, Beijing, China) and photographed with the BGIMAGING Cellview 4.11 system.

In this study, we expressed two recombinant Sp-CFSH proteins: Sp-CFSH recombinant protein with only 6 × His tag (rHisCFSH) and Sp-CFSH recombinant protein containing both 6 × His tag and GST (rGSTCFSH). rHisCFSH was expressed and purified according to the previous study [27,28]. rGSTCFSH was also expressed utilizing a prokaryotic expression system. The fragment encoding mature peptide of Sp-CFSH was cloned into the pET-GST vector, utilizing BamH I and Nhe I restriction enzyme sites. The generated constructs (pET-GST-CFSH-His), which included GST and 6 × His tags, were transformed into E. coli TransB (DE3) and induced for 20 h at 16 °C, following the application of isopropyl-beta-D-thiogalactopyranoside (IPTG) at a level of 1 mM. Upon harvest using centrifugation (8000× g, 10 min, 4 °C), the bacteria were disrupted by ultrasonic waves. We selected the supernatant for further purification with glutathione sepharose 4B (Solarbio, Beijing, China) according to the guidelines.

The recombinant protein of Sp-SEFIR extracellular fragment containing GST and 6 × His tag (rSEFIR) was also expressed with the same prokaryotic expression system as rGSTCFSH. The fragment encoding the extracellular segment of Sp-SEFIR was cloned into the pET-GST vector with restriction enzyme sites (EcoR I and Nhe I). The generated constructs (pET-GST-SEFIR-His) were transformed into E. coli TransB (DE3) and induced for 6 h at 25 °C after adding IPTG (0.2 mM final concentration). rSEFIR was purified from the supernatant of crude cell extracts with glutathione sepharose 4B (Solarbio, Beijing, China).

Herein, we conducted two GST pull-down assays to detect the protein–protein interaction between Sp-CFSH and Sp-SEFIR. First, we performed GST pull-down assay with rGSTCFSH and the total protein of AG to detect whether Sp-SEFIR was a CFSH-binding component of AG. After that, a GST pull-down assay with rHisCFSH and rSEFIR was performed, aimed at exploring binding regions of Sp-SEFIR.

The total protein of AG was extracted with PP11-Universal Protein Extraction Reagent (Aidlab, Beijing, China) according to the instructions. rGSTCFSH was obtained as previously described (Section 4.7). Upon being incubated at 4 °C for 30 min, rGSTCFSH was immobilized in the Glutathione Sepharose 4B (Solarbio, Beijing, China). Then, the total protein of AG was added and incubated at 4 °C for 2 h, and the unbound protein was washed away using PBS. A column volume of glutathione elution buffer was added and incubated for 10 min to elute bound proteins, and the supernatant was collected by centrifugation. The proteins that were eluted underwent analysis through the utilization of SDS-PAGE and Western blot techniques, with the aid of the Sp-SEFIR antibody and the anti-His mouse monoclonal antibody. GST protein (with 6 × His tag) was utilized as the negative control.

rHisCFSH and rSEFIR were obtained as described above (Section 4.7). Upon being incubated at 4 °C for 30 min, rSEFIR was immobilized on the Glutathione Sepharose 4B (Solarbio, Beijing, China). Subsequently, rCFSH was introduced and subjected to incubation at a temperature of 4 °C for 1 h. The protein that did not bind was eliminated through PBS washing. A column volume of glutathione elution buffer was added and incubated for 10 min to elute the bound proteins, and the supernatant was collected by centrifugation (500× g, 5 min, 4 °C). The eluted proteins were analyzed by SDS-PAGE and Western blot using anti-His mouse monoclonal antibody. GST protein (with 6 × His tag) was used as the negative control.

Fragments of Sp-SEFIR and green fluorescent protein gene (GFP) were cloned into pGEMT-Easy Vector to prepare the linearized DNA templates. The dsRNA synthesis was performed using T7 and SP6 polymerase. GFP dsRNA was synthesized as the negative control. Individuals (body weight: 48.4 ± 4.5 g, carapace width: 6.6 ± 0.5 cm, n = 27) in stage I of AG development were equally divided at random into three groups: GFP-dsRNA-injected, SEFIR-dsRNA-injected and crustacean physiological saline (CPS)-injected [57]. Crabs were injected with either 1 μg/g of dsRNA or an equivalent amount of CPS. The injection was repeated 24 h after the first injection. Then, 72 h after first injection, crabs were anesthetized on ice for 5 min and then AGs were dissected. The interference efficiency of Sp-SEFIR and expression levels of Sp-IAG as well as Sp-STAT, were detected using qRT-PCR. Furthermore, the RNAs extraction, cDNA synthesis and qRT-PCR analysis of samples were conducted in accordance with the methods previously outlined.

The in vitro explant culture system was modified and used to investigate the regulatory role of Sp-SEFIR in the inhibitory process of IAG expression mediated by CFSH [27].

Individuals (body weight: 146.7 ± 9.8 g, carapace width: 11.7 ± 1.0 cm, n = 7) at AG stage II were selected for in vitro experiments. The AG explants of the same individual were split into three equal groups and treated with 200 μL L-15 medium containing GFP-dsRNA (1 μg/mL final), SEFIR-dsRNA (1 μg/mL final), or CPS with the identical quantity, respectively. Upon incubating at 26 °C for 6 h, the culture media was changed with 200 μL of L-15 media that contained a concentration of 10⁻⁶ M rCFSH. After a 12 h cultivation period, AG explants were collected to extract total RNA. Expression levels of Sp-IAG, Sp-STAT were detected using qRT-PCR. RNA extraction, cDNA synthesis, as well as qRT-PCR analysis of samples, were performed according to the criteria mentioned above (Section 4.2 and Section 4.3).

ORF of Sp-SEFIR was predicted with the ORF Finder program (https://www.ncbi.nlm.nih.gov/orffinder/↗ (accessed on 7 December 2022)). The amino acid sequences of Sp-SEFIR were subjected to analysis utilizing EXPASY (https://web.expasy.org/protparam/↗ and http://web.expasy.org/compute_pi/↗ (accessed on 7 December 2022)). The signal peptide of Sp-SEFIR was predicted by SignalP 5.0 Server (https://services.healthtech.dtu.dk/service.php?SignalP-5.0↗ (accessed on 8 December 2022)). The transmembrane domain and conserved domain were predicted by SMART (http://smart.embl-heidelberg.de/↗ (accessed on 8 December 2022)) and Phobius (http://www.ebi.ac.uk/Tools/pfa/phobius/↗ (accessed on 8 December 2022)). IL-17R sequences were from published articles and GenBank library (Table A2). In addition, the construction of a phylogenetic tree was carried out utilizing the neighbor-joining approach (NJ) using MEGA 6.0. The process of bootstrap sampling was iterated 1000 times.

Statistical analysis was conducted utilizing SPSS 18.0 program. The provided data exhibited a normal distribution in accordance with the Kolmogorov–Smirnov test. Levene’s test was employed to assess the homogeneity of variance, and significant variations were determined employing one-way analysis of variance (ANOVA), subsequently employing the Duncan test. p < 0.05 was judged significant, while p < 0.01 was judged extremely significant. All findings are expressed as mean ± SEM.