Putative Role of CFSH in the Eyestalk-AG-Testicular Endocrine Axis of the Swimming Crab Portunus trituberculatus

The experimental animal in this study is Portunus trituberculatus, which belongs to invertebrates and is a kind of crab. In China, ethical approval is not required for experiments on crabs. All of our crabs were purchased from the local aquatic market. Prior to dissection, we anesthetized the crabs on ice. All the experiments comply with the requirements of the governing regulation for the use of experimental animals in Zhejiang Province (Zhejiang provincial government order No. 263, released on 17 August 2009, effective from 1 October 2010) and the Animal Care and Use Committee of Ningbo University. Healthy wild adult swimming crabs with a body weight of 100 to 140 g were purchased from the local aquatic market in Xianxiang, Ningbo. Some individuals in the intermolt period were temporarily reared in a capacious 120 L tank bottom containing 30 cm of fine sand and shelter in our laboratory; the crabs were fed and their water was changed periodically until the 20-day (long-term) and 3-day (short-term) treatment of RNAi and the in vivo recombinant proteins injection was completed. Other crabs were collected for gene cloning and in vitro treatment, respectively. All samples were stored in RNA preservation fluid (Cwbiotech, Taizhou, China) at −80 °C until RNA extraction.

In addition, the testis and AG-related tissues were sampled from the crab and cut into 3–5 mm pieces and fixed in 4% Paraformaldehyde Fix Solution for 24 h at 4 °C. Then, the tissues were dehydrated with serial ethanol, cleared with serial xylene, and finally embedded in paraffin. Tissues were cut into 5–7 μm sections for hematoxylin and eosin (H&E) staining. Next, 30 to 50 mg of testis tissue was placed in a 1.5 mL EP tube containing 300 microliters of PBS and (Protease and phosphatase inhibitor cocktail for general use, 50×) (Beyotime, Shanghai, China), after which the homogenate was centrifuged at 8000 g for 10 min at 4 °C. The supernatant was sucked and stored at −80 °C for western blotting.

Total RNA was isolated from different samples using the RNA-Solv^® reagent (Omega Bio-tek, Norcross, GA, USA) according to the manufacturer’s instructions. RNA concentrations were determined using a NanoDrop 2000 UV Spectrophotometer (Thermo Fisher Scientific, Cheshire, UK). The genomic DNA removed with (10× gDNA Remover Mix) and the first strand of cDNA was synthesized using a HiFiScript gDNA Removal cDNA Synthesis Kit (Cwbiotech, Taizhou, China) according to the manufacturer’s protocol, followed by storage at −80 °C until use.

A keyword-based screening of our RNAseq library (unpublished) yielded a short transcript homologue of the CFSH; this was validated by reverse transcription polymerase chain reaction (RT-PCR) and elongated by the 3′ and 5′ rapid amplification of cDNA ends (RACE) (Clontech, California, USA) using cDNA generated from muscle-extracted RNA. Table 1 shows the primers used in the cDNA clone. The following program was used for each PCR amplification: initial denaturation at 94 °C for 5 min, followed by 35 cycles of 94 °C for 30 s, 55 °C for 30 s and 72 °C for 90 s, with a final elongation at 72 °C for 10 min. All PCR products were analyzed using 1% agarose gel electrophoresis, then ligated with the pMD19-T vector (Takara, Kyoto, Japan) and transformed into competent Escherichia coli cells. After transformation, three positive clones were picked for sequencing (BGI Tech, Shenzhen, China). The amplified sequences from 3′ and 5′ RACE were assembled with the partial cDNA sequence using Vector NTI 10.0 software, and the open reading frame (ORF) was identified using ORF finder (http://www.ncbi.nlm.nih.gov/gorf/↗) (accessed on 22 January 2022). Predictions of the conserved domains were performed using the SMART program (http://smart.emblheidelberg.de/↗) (accessed on 22 January 2022). The 3D structure of the PtCFSH protein was predicted by the Alphafold2 web server (https://alphafold.ebi.ac.uk/↗) (accessed on 15 February 2022) and edited with the PyMOL Software (https://pymol.org/2/↗) (accessed on 27 February 2022). A multiple sequence alignment and phylogenic analysis was performed using ClustalW1.8 and MEGA7.0 (https://www.megasoftware.net/↗) (accessed on 28 February 2022).

To detect the expression levels ofin different tissues, primers PtCFSH-RTF/RTR () were designed to generate a 212-bp fragment. Semiquantitative PCR was performed using β-actin as an internal reference to adjust the number of cDNA templates. Amplification was performed with the following program: denaturation at 94 °C for 5 min, followed by 35 cycles of 94 °C for 30 s, 55 °C for 30 s and 72 °C for 90 s, with a final elongation at 72 °C for 10 min. The products were assessed by electrophoresis on 1.5% agarose gel. PtCFSH Table 1

The double-strand RNA for PtCFSH, PtIAG, and green fluorescent protein (GFP) were synthesized as previously described [24]. The plasmid pGEX-4t-2 was digested with restriction endonucleases SmaI and XhoI in order to generate a linearized vector. Primers PtCFSH-GST-F/R with 21-bp (Table 1) extensions were designed and synthesized, which were homologous to the linearized vector 5′ and 3′ ends to generate the amplification products. The purified PCR product was inserted into the corresponding linearized vector by setting up an in-fusion cloning reaction using the ClonExpress II One Step Cloning Kit (Vazyme, Nanjing, China) and then transformed into E. coli cells. The confirmed recombinant plasmids were extracted using the E.Z.N.A. Plasmid Mini Kit I (Omega Bio-Tek) and then transformed into E. coli Rosetta-gami cells to express the rGST-CFSH protein. The vector pGEX-4t-2 as a control was also expressed as the GST tag protein (rGST). The recombinant proteins were purified using the GST-tag Purification Resin (Beyotime, Shanghai, China) and verified three times by SDS-PAGE.

For long-term treatments in vivo, the double-strand RNA of CFSH (dsCFSH) and the recombinant CFSH protein (rCFSH) were injected into crabs at 3 ug/g every three days for 20 days, with dsGFP and rGST serving as controls. The testes and AGs were collected to observe tissue changes. In the short-term RNAi treatment in vivo, dsCFSH, dsIAG, and dsGFP were all injected into different individuals at a dose of 3 ug/g dose, and samples were collected after 48 h. For the in vitro experiments, the desired eyestalks, AGs and testis tissues were all precultured in M199 medium for 1 h. Afterwards, dsCFSH and dsGFP were added at a dose of 3 μg/g dose [20] to Petri dishes (Jet Biofil, Guangzhou, China) containing the eyestalks and AGs, and then co-cultured at 26 °C for 8 h. Additionally, rCFSH and rGST were added to the dishes containing AGs or testes at doses of 10–6 M, respectively. Incubation was continued at 26 °C for 8 h. All samples were collected for qPCR assay and western blot analysis.

We determined the relative gene expression levels using quantitative real-time PCR (qPCR). The template cDNA was prepared as described above and then amplified with qPCR primers (Table 1). PCR was carried out using the SYBR^® Premix Ex Taq™ II Kit (Takara, Kyoto, Japan) according to the manufacturer’s instructions. PCR conditions were as follows: 95 °C for 2 min, followed by 40 cycles of 95 °C for 15 s and 56 °C for 20 s. Relative mRNA expression levels were normalized to β-actin mRNA expression [25] and calculated using the comparative Ct (2^−ΔΔCt) method [26].

For the Western blot assay, the protein concentration of the testis was measured using the BCA Protein Assay Kit (Cwbiotech, Taizhou, China). For SDS-PAGE, 30 mg of protein from each sample was used, followed by electrophoretic transfer to a 0.45 βm PVDF membrane using a Mini Trans-Blot (Bio-Rad, California, USA). After blocking with Y-Tec Rapid Blocking Buffer (Yoche, Shanghai, China) at room temperature for 1 h, the membranes were incubated with Phospho-p44/42 mos/mitogen-activated protein kinase (MAPK) (Erk1/2) or p44/42 MAPK (Erk1/2) (Cell Signaling Technology, Danvers, MA, USA) monoclonal antibodies diluted to 1:1000 in (Y-Tec Stabilizing Antibody Diluent) (Yoche, Shanghai, China) overnight at 4 °C. The membranes were washed three times with TBST (20 mM Tris-HCl, 150 mM NaCl, and 0.05% Tween-20) and then incubated with β-Tubulin Rabbit IgG polyclonal antibody (CST) diluted to 1:2000 in Y-Tec Rapid Blocking Buffer (Yoche, Shanghai, China) at room temperature for 1 h. After washing three times with TBST for 10 min each, the membrane was incubated in a Western Bright ECL substrate (Advansta, California, USA) before exposure to ChemiScope 6000 (CLiNX, Shanghai, China). The protein bands were quantified using the ChemiScope figure software package, and the results were derived from the statistical analysis of three independent experiments.

The testis was collected at 5, 15 min and 6 h after rCFSH (10⁻⁶ M) and forskolin (10⁻⁵ M) treatment (three replicates per treatment). The cAMP levels and the cGMP levels of the sampled testis at 5 and 15 min were measured using the cAMP ELISA kit (Nanjing Jiancheng Bioengineering Institute of China, Nanjing, China) and cGMP ELISA kit (Nanjing Jiancheng Bioengineering Institute of China, Nanjing, China), respectively. Samples collected after 6 h of treatment were used to detect changes in nitric oxide synthase (NOS) levels using a NOS-typed assay kit (Nanjing Jiancheng Bioengineering Institute of China, Nanjing, China).

At the end of the long-term RNAi and recombinant protein injection experimental period, crabs from four groups with a body width of 12.2–14.6 cm were collected. Testis were dissected from the individuals, cut into 3 mm³ pieces, and fixed in PFS for 24 h at 4 °C. After dehydration in gradient ethanol, they were embedded in paraffin. Paraffin-embedded AGs and testis tissues were sectioned into slices of 5–7 μm. The sections were then stained with H&E and observed under a Nikon Eclipse 80i microscope (Nikon, Tokyo, Japan).

This hypothetical pathway was constructed with reference to the classical insulin pathway and the general neuropeptide signaling pathway [27]. In this paper, we complete the hypothetical pathway by figdraw.

All data are expressed as mean ± SEM and were subjected to a one-way analysis of variance (ANOVA), followed by Student’s t-test or Tukey’s multiple-group comparison test. The data were analyzed using SPSS 24.0 software. Significant differences were accepted at p < 0.05. Asterisk denotes statistically significant differences: * p < 0.05; ** p < 0.01; *** p < 0.001.

We obtained a 1241 bp PtCFSH cDNA (Genbank accession number: ON929327↗) comprising a 19 bp 5′-untranslated region (UTR), 678 bp ORF, and 542 bp 3′-UTR with a poly (A) tail (Figure 1). A sequence analysis of the 678 bp ORF revealed 225 amino acids with a 24 aa signal peptide, 31 aa CFSH-precursor-related peptides, a 2 aa cleavage site and a 166 aa interleukin-17 domain containing eight cysteines, forming four disulfide bonds (Figure 1A,B). The three-dimensional structure of the CFSH was composed of α-helix and connected random coils, as shown by the Alphafold2 results (Figure 1C).

It was shown by multiple sequence alignments of PtCFSH with other crustacean CFSHs that the predicted PtCFSH protein shares significant identities with other CFSHs in the cleavage site “KR”, including “Cysteine” in the interleukin-17 domain (Figure 2A). Furthermore, it was revealed in phylogenetic analysis that the PtCFSH was more closely related to the other CFSH, while it was divided into two branches with subtype CFSH2 (Figure 2B). The transcript levels of PtCFSH were measured in different tissues obtained from the adult male. It was demonstrated by these results that the PtCFSH transcripts were mainly expressed in the eyestalk, with a slight expression in the brain and testis (Supplementary Figure S1).

In order to clarify the inhibitory effect of the CFSH on the AG, dsPtCFSH and rPtCFSH were used to conduct the in vivo and in vitro experiments (,and). Treatments with dsPtCFSH led to a significant down-regulation ofgene expression in the eyestalk, both in vivo and in vitro, showing a high interference efficiency (A andA). The long-term silencing in PtCFSH in vivo caused an increase in the ratio of type I AG cells and a decrease in type II AG cells, while the injection of rPtCFSH showed an opposite effect (E,F and 4D,E). The types of AG cells were classified according to Sroyraya et al. []. Briefly, the type I AG cells have a small globular nucleus containing mainly heterochromatin, while the type II cells are vacuolated with a large cell diameter, containing mostly euchromatin and prominent ribosomes. It was found that the type I cells synthesize IAG in a greater amount than the type II cells []. Consistent with the change in the IAG-producing cells, qPCR analysis showed that theexpression could be reduced by rPtCFSH injection (A), while induced bysilencing (B). Similar results were also observed in in vitro assays (B,C), further confirming the inhibitory role of the CFSH onexpression. Figure 3 Figure 4 Figure 5 Figure 3 Figure 5 Figure 3 ^{28 28} Figure 4 Figure 3 Figure 5 PtCFSH PtIAG PtCFSH IAG

To investigate the function of PtCFSH on the testicular development of P. trituberculatus, the histological features of the testis and the expression of spermatogenesis-related genes were evaluated after the in vivo PtCFSH silencing and rPtCFSH injection. It was shown that the treatment of rPtCFSH delayed the further conversion of spermatocytes to spermatids, when compared to the controls (Figure 4F,G). In contrast, a gradual transformation of spermatocytes to spermatids in the testis was observed in the PtCFSH silencing group (Figure 3G,H), in parallel with a gradual production of CTFM (Figure 3H). Meanwhile, the mRNA expression of spermatogenesis-related genes in the testis, such as the kinesin-like protein (Kifc-1), ATP-dependent RNA helicase (Vasa), CyclinB and cyclin-dependent kinase-2 (Cdc-2) [29], was reduced by rPtCFSH injection and induced by PtCFSH silencing (Figure 3C and Figure 4B). The combined results suggest that PtCFSH negatively regulates the spermatogenesis of P. trituberculatus.

To find out whether the negative regulatory effect of the CFSH is achieved through IAG, the changes in expression levels of several classical insulin pathway genes, including IAG itself, insulin-like growth factor banding protein-related peptide (IGFBP-rp), insulin-like hormone receptor 1 (IR1), IR2, protein kinase B (Akt), mammalian target of rapamycin (mTOR), and forkhead transcription factors (Foxo) [30,31], were further determined in the testis in the two treatments. In addition, since the insulin pathway activates the phosphorylation of MAPK [32], the change in the phosphorylated MAPK levels were also examined using Western blotting. As expected, the expression of insulin pathway genes and phosphorylated MAPK levels were reduced by rPtCFSH injection and induced by PtCFSH silencing (Figure 3C,D and Figure 4B,C). Therefore, it was speculated that the CFSH can inhibit the production of IAG, which, in turn, affects the testicular development.

Furthermore, in vitro experiments were conducted to explore whether the CFSH had a direct regulatory effect on the testis. It was found that the addition of rPtCFSH into the testis explants also led to a decrease in spermatogenesis-related gene expression and the MAPK phosphorylation level, which means that the CFSH could act directly on the testis (Figure 5D,E). Meanwhile, it was also observed that the expression levels of IAG and insulin pathway genes in the testis were down-regulated (Figure 5D).

A short-term RNAi experiment was performed to verify the existence of the CFSH–IAG–testis endocrine axis. Similar changes in the levels of gene expression and MAPK phosphorylation were observed in short-term injection with dsPtCFSH, when compared to those in long-term treatment (Figure 6A,D). Opposite effects were found in the dsPtIAG injection group (Figure 6B,D), indicating the stimulatory role of IAG in testicular development. To confirm the central role of IAG in this endocrine axis, a mixed injection of dsPtCFSH and dsPtIAG was further performed, and the results were similar to those of the dsPtIAG injection alone (Figure 6C,D). It can be understood that the regulatory effect of dsPtCFSH on the testicular development was blocked after the injection of dsPtIAG, which indicated the existence of the CFSH–IAG–testis endocrine axis.

Most neuropeptides use the G protein-coupled receptor (GPCR) as their membrane receptor, and the signaling system needs the participation of second messengers [27]. To explore which second messengers are involved in the signal transduction of PtCFSH, the levels of cAMP and cGMP, as well as the activity of nitric oxide synthetase (NOS), were determined in in vitro testis experiments. Remarkable down-regulation at 5 min and 15 min after adding rPtCFSH was shown by both cAMP and cGMP levels compared to the control group (Figure 7A,B). In addition, the NOS enzymatic activity was significantly diminished after adding rPtCFSH, suggesting a decrease in the NO level (Figure 7C).