Evidences for Red Pigment Concentrating Hormone (RPCH) and Beta-Pigment Dispersing Hormone (β-PDH) Inducing Oocyte Meiotic Maturation in the Chinese Mitten Crab, Eriocheir sinensis

Female individuals (body weight 80-110g) were collected from a local farm in Pudong District, Shanghai. The crabs were kept in a freshwater circulation system supplying sufficient dissolved oxygen and were fed on commercial diet pellets (SC-9011, South Ranch, China) once a day. Various tissues including the muscle, gill, heart, hepatopancreas, thoracic ganglia, brain, eyestalks and ovaries were dissected on ice, quickly frozen in liquid nitrogen, and then stored at -80°C for gene expression analysis. For histological observation or in situ hybridization analysis, the ovarian and brain tissues were fixed in 4% paraformaldehyde solution at 4°C overnight. The ovarian development was divided into three main stages: previtellogenesis (Pvt), vitellogenesis (Vt) (early, Evt; middle, Mvt; late, Lvt), and the final meiotic maturation (30). Germinal vesicle breaks down (GVBD) in the oocyte at the final meiotic maturation was detected in a clearing solution (31).

Total RNA was extracted from tissues using RNAiso Plus reagent (Takara, Kusatsu, Japan) according to the manufacturer’s instructions. The concentration and purity of RNA were quantified by NanoDrop 2000 Spectrophotometers (Thermo Fisher, Massachusetts, USA).

The first strand of cDNA was synthesized with 1 μg of total RNA using the SMARTer RACE cDNA Amplification Kit (Clontech, Kusatsu, Japan). Gene-specific primers (Table S1) were designed by Primer Premier 5.0 software based on the homologous sequence in the brain transcriptome database (32). RACE-PCR amplification was conducted with initial denaturation at 94°C for 30 s, followed by 35 cycles at 94°C for 5 s, 60°C for 30 s, and 72°C for 2 min, then a final elongation at 72°C for 10 min. PCR products were gel-purified (Tiangen, Beijing, China) and ligated to the pGEM^®-T Easy vector (Promega, Madison, WI, USA) at 4°C overnight, then transformed into competent Escherichia coli (DH5α) cells. Eight positive clones were picked for Sanger sequencing.

The open reading frame (ORF) of Es-RPCH and Es-β-PDH were determined using the ORF Finder (https://www.ncbi.nlm.nih.gov/orffinder/↗). The basic physical and chemical properties of the deduced amino acid sequences were analyzed using the ExPASy Molecular Biology server (https://web.expasy.org/protparam/↗). The signal peptide was scanned using the SignalP 5.0 Server program (http://www.cbs.dtu.dk/services/SignalP/↗). Multiple sequence alignment was performed by DNAman software. A phylogenetic tree was generated by MEGAX 64 software using the neighbor-joining methods. Bootstrap analysis of 1000 replicates was carried out to compute the tree branch position confidence (Latin name and protein id are given in Figure S2).

DIG-labeled probes of anti-sense and sense RNA were synthesized by DIG RNA Labeling Kit (Roche Diagnostics, Mannheim, Germany) with the SP6, and T7 RNA polymerases (Takara), respectively. ISH was performed as previously described (33). In brief, the brain tissue sections (~6 µm) were dehydrated and deparaffinized with xylene (three times for 5 min each). After rinsing, each section was treated with 3-5 µg/mL proteinase K at 37°C for 10 min, and then hybridized with DIG-labeled antisense or sense RNA probes two hours at 55°C for 2 hours. After serial washing, the DIG was visualized using colorimetric substrates NBT/BCIP (Roche, Germany) following the manufacturer’s instructions.

The Es-RPCH and Es-β-PDH peptides were chemically synthesized (Jier Biosciences, China). Female individuals were randomly divided into four groups: crabs in group 1 were untreated and served as the blank control group, sacrificed after day 0 post injection. Crabs in group 2, 3 and 4 were respectively injected with 100 μL of PBS (control group), Es-RPCH, and Es-β-PDH every seven days at the base of the fifth walking leg, and then sacrificed after 30 days post injection. The body weight and gonad weight were measured to calculate the gonadosomatic index (GSI) (gonad weight/body weight × 100%). Ovary tissues from each group were sampled for measuring the relative Vg mRNA expression levels and mean oocyte diameter and histological observation.

Ovarian tissues at Lvt stage were selected from the sexually mature crabs (n = 36) and then were dispersed with a pipette to release oocytes. The oocytes were rinsed with DPBS and placed in medium 199 in 24-well culture plates as previously described (34) (containing 100 g/mL of BSA, 100 g/mL of streptomycin, and 100 IU/mL of penicillin G). In the experimental group, the synthesized Es-RPCH or Es-β-PDH was added at the different concentrations of 10^-11 M, 10^-10 M, 10^-9 M, 10^-8 M and 10^-7 M, respectively, while equal volume of PBS was added to the wells in the control group. All the plates were incubated in dark at 24°C. The number of oocytes in GVBD from each well were determined in clearing solution (formaldehyde, ethyl alcohol, acetic acid, 30:60:1) (31).

Quantitative real-time PCR (qRT-PCR) was performed with gene-specific primers (Table S1) using SYBR^® Premix Ex Taq™ II kit (Takara) in a 20 µL reaction mix containing 4 µL 5× iScript reaction mix (Bio-Rad), 1 μL cDNA template, 0.5 μL forward primer (10 μM), 0.5 μL reverse primer (10 μM) and 14 μL RNase-free ddH₂O. Thermal cycling included an initial denaturation at 95°C for 30 s, followed by 40 cycles at 95°C for 5 s and 55°C for 30 s. To examine product specificity, PCR products were verified by sequencing and melting curve analyses (60°C to 95°C in increments of 0.2°C/s), and a negative control was set without cDNA template. Each reaction was conducted in triplicate. The crab β-actin gene was employed as an internal reference to estimate the relative mRNA expression levels using the 2^−δδCt method (35). The amplification efficiencies for the target gene and the β-Actin gene were approximated 100%.

All data in this study were presented as mean + Standard Deviation (SD), and “n” represents the number of replicates. Kolmogorov-Smirnov and Cochran tests were performed and Student’s t-test or Tukey’s test were utilized to analyze the normally distributed data. One-way analysis of variance (ANOVA) and paired sample T-test were performed to calculate statistically significant values respectively using SPSS statistical software (version 20.0). In all cases, statistical differences among groups were accepted at p <0.05 and indicated with asterisks or double asterisks.

The administrated female crabs with PBS, Es-RPCH and Es-β-PDH were sacrificed at 24 hours post injection for sampling ovarian tissues. Three individuals were sampled in each administrated group for RNA-seq libraries construction. RNA-seq were conducted using an Illumina HiSeq™ 2500/MiSeq (Novogene Biotech,Tianjin, China).

Clean reads were obtained after filtering and removing low quality raw reads. The differentially expressed genes (DEGs) between the Es-RPCH and PBS (Es-β-PDH and PBS) groups were screened by DESeq 2 software using DESeq and BH methods with the negative binomial distribution model methods. The screening criteria used for target DEGs is as follows|log2Fold change|≥0, P ≤ 0.05. The putative target DEGs were annotated by the Gene Ontology (GO) enrichment analysis (http://www.geneontology.org/↗), and the pathway was deduced using the Kyoto Encyclopedia of Gene and Genomes (KEGG) database (https://www.kegg.jp/kegg/pathway.html↗).

Two full-length cDNA sequences of Es-RPCH and Es-β-PDH were cloned by RACE method from the brain of E. sinensis as shown in Figure 1. The 650-base pair (bp) Es-RPCH (GenBank accession number: OK315660↗) contains a 75 bp 5′-untranslated region (UTR), a 245 bp 3′-UTR including a potential polyadenosine (ATTAAA), and a 330 bp ORF encoding a 109 amino acid with a calculated molecular weight of ~11.08 kDa and a predicted isoelectric point of 7.84. Sequence analysis predicted the ORF of Es-RPCH containing a 24-residue signal peptide, a 8-residue mature peptide, and a 73-residue precursor related peptide (RPRP) (Figure 1A). Multiple sequence alignments showed that the Es-RPCH amino acid sequence has high identities with other crustacean RPCHs, sharing the identical mature RPCH peptides, glycine residue preceding the dibasic cleavage site, dibasic cleavage site (KR) and the amidation site (Figure S1A). Phylogenetic analysis showed that Es-RPCH is more closely related to crustacean RPCHs and clustered with insects AKHs, grouped into one GnRH superfamily, which is consistent with traditional classification (Figure S2A).

The Es-β-PDH cDNA (GenBank accession number: OK315661↗) was 535 bp in length, including a 5′-UTR of 36 bp, a 3′-UTR of 253 bp with a potential polyadenosine (ATTAAA), and an ORF of 246 bp encoding a 81 amino acid with a calculated molecular weight of ~9.03 kDa and a predicted isoelectric point of 10.11. The deduced amino acid sequence of Es-β-PDH containing a 25-residue signal peptide, a 33-residue precursor related peptide (PPRP), two proteolytic sites (KR, RR) at positions 59 and 80, and a 18-residue mature peptide (Figure 1B). Sequence alignments showed that the amidation site and two proteolytic sites are identical in different crustacean species, and the PDH mature peptides are conserved in crustaceans (Figure S1B). Phylogenetic analysis showed that Es-β-PDH is more closely related to crustacean β-PDHs than α-PDH, followed by pigment dispersing factor, which is also consistent with traditional classification (Figure S2B).

QRT-PCR detection showed high expression of Es-RPCH and Es-β-PDH mRNA in the eyestalks, and moderate in brain. While weak expression of the Es-RPCH and Es-β-PDH was found in thoracic ganglia and ovary, respectively. No expression was found in other tested tissues (Figure 2).

Histological section showed that the mitten crab brain can be classified into protocerebrum, deutocerebrum, and tritocerebrum (36). Middle horizontal section of the brain in Figure 3A showed the locations of neuronal clusters (numbers) and neuropils. A strong positive signal of Es-RPCH was visualized in the cell clusters 6 in the protocerebrum, middle signal in cell clusters 8 in the protocerebrum, 9 and 10 in the deutocerebrum, but a weaker signal in cell clusters 17 in the tritocerebrum (36) (Figures 3B, F). In addition, a strong positive signal of Es-β-PDH was visualized in the cell clusters 8, 9, and 10, but weaker in cell clusters 6 and 17 (Figures 3D, G–I). No positive signal was detected in negative control sections with the sense-strand RNA probes (Figures 3C, E).

As shown in Figure 4, qRT-PCR indicated that the abundance of Es-RPCH and Es-β-PDH mRNA were the highest in the eyestalks, and moderate in the brain at each ovarian developmental stage (Figure 4A). The relative expression level of Es-RPCH and Es-β-PDH mRNA were all dramatically increased in the eyestalks and brain and reach the highest at Vt stage, and then decrease at GVBD stage (P <0.01) (Figure 4B).

To investigate the exact role of the Es-RPCH and Es-β-PDH in the ovarian maturation in E. sinensis, the female individuals at Pvt stage were selected for injection of the synthesized Es-RPCH and Es-β-PDH peptides. At the beginning of experiment, the nucleus in the Pvt oocytes were transparent and were termed germinal vesicle (GV), while the ooplasm and nucleolus were notably stained in dark blue color by hematoxylin (Figure 5A). After administrated with injection of 30 days, the oocytes were visualized at Mvt stage in PBS, Es-RPCH, and Es-β-PDH groups. Due to the appearance of yolk, the ooplasm was stained in red by eosin (Figures 5B–D). Notably, in the Es-RPCH injection group, the GV visually migrated toward peripheral cytoplasmic membrane as compared with that of the Es-β-PDH and PBS groups (Figures 5C, D), in which GV situated in the center of oocyte. The GSI index and Vg expression in the ovary were all significantly decreased in the Es-RPCH and Es-β-PDH injection group when compared with the PBS control group (Figures 5E, G). The mean oocytes diameter was also smaller in the Es-RPCH group, but no significant differences between Es-β-PDH and the control groups (Figure 5F).

To determine whether the Es-RPCH and Es-β-PDH can induce GVBD, oocytes at Lvt stage were selected to culture with the synthesized Es-RPCH and Es-β-PDH peptides. Results showed that the GVBD index in the Es-RPCH and Es-β-PDH group were all dramatically increased at the concentration of 10^-11 M, 10^-10 M, 10^-9 M, 10^-8 M, and 10^-7 M as compared with control groups (p < 0.05). There was a significant dosage effect with the increasing concentration of Es-RPCH or Es-β-PDH. In addition, the GVBD index in Es-RPCH group was higher than in the Es-β-PDH in each concentration gradient (Figure 6).

To probe the molecular basis of induction of GVBD by Es-RPCH and Es-β-PDH, nine ovarian cDNA libraries from the crabs administrated with PBS, Es-RPCH and Es-β-PDH were constructed and sequenced by Illumina HiSeq 4000 platform. More than 42 million raw reads were generated, and then about 33 million clean reads of every group were obtained after removing adapters as well as filtering the low-quality sequences. The Q20 percentage of each sample was over 97%, the Q30 percentage was over 92%, and the CG content was below 53.96% (Table S2), indicating that the sequencing data are high quality and credibility.

In comparison with PBS group, a total of 1976 differentially expressed genes (DEGs) including 1002 up- and 974 down-regulated genes were identified in the Es-RPCH group, as well as1714 DEGs including 885 up- and 829 down-regulated genes in the Es-β-PDH group (Figure 7). GO analysis indicated DEGs from the Es-RPCH and Es-β-PDH groups were all classified into three ontologies: molecular function, biological process, and cellular component. Respectively, DEGs in the Es-RPCH group were mainly assigned into DNA binding, DNA integration and biosynthetic processes (Figure S3A), while in the Es-β-PDH group DEGs were mainly assigned into DNA integration, small molecule metabolism process, and monosaccharide metabolism process (Figure S3C). In addition, KEGG pathway analysis showed that DEGs in the Es-RPCH group were statistically enriched some pathways, including TGF‐β signaling pathway, oocyte meiosis, and Wnt signaling pathway, and so on. (Figure S3B), while in the Es-β-PDH group the DEGs were mainly enriched into TGF‐β signaling pathway, pyruvate metabolism and insect hormone biosynthesis, and so on (Figure S3D). Interestingly, some DEGs related to meiosis such as cell division cycle 2 kinase (Cdc2) and cyclin proteins as well as 5-HT-R were identified to be highly expressed in the Es-RPCH and Es-β-PDH groups as compared with the PBS control (Figure 8A). Additionally, the DEGs related to gonadal maturation such as foxl2, retinoid-X receptor (RXR), neuropeptide F-R, and GnRHR were also detected to be highly expressed in the Es-RPCH and Es-β-PDH groups (Figure 8B).