Spotted sea bass (weighing 100.0 ± 5.0 g) were obtained from the Shuangying aquatic breeding factory in Lijin, Shandong. Before the experiment, 100 healthy spotted sea bass were acclimatized in an indoor pool for 2 weeks. The temperature is maintained at 19–21 ℃. The fish were fed twice per day until satiety at 9:00 a.m. and 5:00 p.m., and 1/3 of the water was exchanged every day. Fish sampling was conducted after 0, 1, 6, 12, 24, 48 and 72 h of short-term starvation following a meal, and 0 h was used as the control time point. We sampled three fish randomly after each of the different fasting durations. Each fish was anesthetized with MS-222 (200 mg/L).
The ghrl and its receptors were identified by searching the whole genome sequence database (PRJNA408177) and the transcriptomic database (PRJNA407434) using TBLASTN (1e-5) (Zhang et al. 2017). The available amino acid sequences of ghrl and its receptors in humans and zebrafish retrieved from NCBI (https://www.ncbi.nlm.nih.gov/) were used as queries. The amino acid sequences were predicted using the online tool ORF finder (https://www.ncbi.nlm.nih.gov/orffinder/) and further verified by Smart-BLAST against the NCBI nonredundant (NR) protein sequence database. SignalP 4.1 Server (https://www.cbs.dtu.dk/services/SignalP/) and NeuroPred (http://stagbeetle.animal.uiuc.edu/cgi-bin/neuropred.py) were used to predict the signal peptide and the neuropeptide prohormone cleavage sites, respectively. Potential transmembrane helices in the encoded amino acid sequences were predicted using TMHMM Server v2.0 (https://www.cbs.dtu.dk/services/TMHMM/). To obtain additional information for gene identification and orthology, a syntenic analysis was conducted by comparing the syntenic regions that harbor genes in spotted sea bass with those in other species based on genome information from the Ensembl genome and NCBI databases. The sequences of Ghrl and its receptors in spotted sea bass and several representative vertebrates were used for the phylogenetic analysis. Multiple protein sequence alignments were constructed using DNAMAN 6.0 with the default parameters. The phylogenetic tree was built with MEGA 6 (Tamura et al. 2013).
The gonads, intestine, liver, brain, spleen, skin, gill, kidney, pronephros, stomach, heart, pituitary gland, muscle and fin were collected and stored at - 80 ℃. Total RNA from the samples was extracted using RNAiso Plus reagent (Takara, Otsu, Japan) according to the manufacturer's instructions. The concentration and integrity of the extracted RNAs were examined using the Biodropsis BD-1000 nucleic acid analyzer (OSTC, Beijing) and electrophoresis. Complementary DNA was synthesized using the PrimeScript™ RT Reagent Kit (Takara, Otsu, Japan) with gDNA Eraser (Perfect Real Time) (RR047A Takara) following the manufacturer's instructions. All cDNA products were diluted to 250 ng/μl for quantitative real-time PCR.
The StepOnePlus Real-Time PCR system (Applied Biosystems, USA) was used to detect the mRNA expression of ghrl and its receptors in various tissues of spotted sea bass including stomach tissue at each fasting time point. In addition, we analyzed the mRNA expression of gastrointestinal feeding-related genes (mln, gas, and cck) after static incubation for different times (3, 6, and 12 h) and with different concentrations (10–6, 10–7, and 10–8 mol/L). The specific primers used for quantitative real-time RT-PCR are listed in Table 1. Based on our previous gene expression study of spotted sea bass, 18S ribosomal RNA (18S) was used as the internal positive control for qRT-PCR normalization (Wang et al. 2018). Triplicate RNA samples were used for gene expression profile analysis. Each 20-μl qRT-PCR contained 10 μl of SYBRⓇ FAST qPCR Master Mix (2X), 0.4 μl of ROX, 2 μl of template cDNA, 0.4 μl of each primer and 6.8 μl of nuclease-free water. The entire PCR process consisted of 95 ℃ for 2 min, 40 cycles of 95 ℃ for 15 s and 56 ℃ for 15 s and a final extension at 72 ℃ for 2 min. For the gene qPCR assay, the melting curve analysis showed a single peak, which confirmed the specificity of the PCRs. To analyze the expression of the genes in spotted sea bass tissues, the Ct values of each gene in various tissues were measured and 18S rRNA was used as the reference for the normalization of the relative expression of the genes. The comparative 2-ΔΔCT method was used to analyze the relative mRNA expression levels.
Primer name Primer sequence 5′–3′ ghrl-F ACACCTGTTTGCTGGTCTTTC ghrl-R ATGTGATGTGGTTGGCCTCTG ghs-r1a-F CTACTTGGCGATCTGCTTCC ghs-r1a-R CACTGCGTAATGCGTCATCT ghs-r1a-like-F TTTTGGATGCTGAGATGGGA ghs-r1a-like-R ACTGGATAAGGTGCGGAGGT mln-F TGCTGATGAAGGAGCGAGAA mln-R TCCACCATGTTCCACCTGAG gas-F TGCTAAGAGGGAGAAACTG gas-R TATCTCGCGTTCATCGTC cck-F TGCCAACTACAACCAACCT cck-R GCGTCGTCCAAAGTCCAT 18S-F GGGTCCGAAGCGTTTACT 18S-R TCACCTCTAGCGGCACAA
Table 1. Primers used for quantitative RT-PCR of hormone genes and their receptors
Sea bass stomach samples were fixed overnight at room temperature in buffered 4% paraformaldehyde, dehydrated, embedded in paraffin, and cut in sections of 7 μm thickness. The primers used for probe preparation are listed in Table 2. Specific nonisotopic antisense and sense riboprobes were synthesized using the DIG RNA Labeling Kit (Roche Diagnostics, Mannheim, Germany) according to the manufacturer's instructions. DIG-labeled ISH was performed as described previously (Zhou et al. 2019), and the sections were examined by light microscopy.
Primer name Primer sequence 5′–3′ ghrl probe-F CGCATTTAGGTGACACTATAGAAGCGACCTGTTTGCTGGTCTTT ghrl probe-R CCGTAATACGACTCACTATAGGGAGACATCAAAGTTGTGATGGTCTC
Table 2. Probe primers for in situ hybridization
Spotted sea bass Ghrl was synthesized by GL Biochem, Shanghai, China. The peptide was purified by HPLC to > 97%, dissolved to the required concentration in DMSO (cell-culture grade), and diluted in culture media for in vitro experiments.
The fish were anesthetized with MS-222 (200 mg/L) before sampling. The gastric tissue was washed three times with phosphate-buffered saline (PBS), cut into small pieces (1 mm3) and placed in a 24-well plate containing 1 ml of culture medium 199 (M199), which contained 100 U/ml penicillin, 100 mg/ml streptomycin and 20% FBS. After preincubation at 28 ℃ for 6 h, the medium was replaced with fresh culture medium containing Ghrl (10–6, 10–7, or 10–8 mol/L). After incubation for 3, 6 and 12 h, gastric fragments were collected and stored at - 80 ℃ for subsequent RNA extraction and real-time PCR.
The values are presented as the means ± SEMs (n = 3). The data were analyzed by one-way ANOVA and Duncan's method for multiple comparisons using SPSS 17.0 software. Differences were considered significant at P < 0.05.