iTRAQ-based quantitative proteomic analysis of embryonic developmental stages in Amur sturgeon, Acipenser schrenckii

The Amur sturgeon, Acipenser schrenckii, is an important aquaculture species in China with annual production of about 150 thousand tons in 2015. In this study, we investigated the regulatory proteins and pathways affecting embryonic development of Amur sturgeon, by analyzing of the differential proteomes among four embryonic developmental stages using isobaric tags for relative and absolute quantitation (iTRAQ), combined with the analysis of effects of microelements and antioxidants on embryonic development. Seventy-four, 77, and 76 proteins were differentially expressed according to iTRAQ analysis between the fertilized egg and blastula, blastula and neurula, and neurula and heart-beat stages, respectively. GO and KEGG enrichment analyses indicated that Gluconeogenesis, Ribosome and Proteasome were the most enriched pathways, which may promote energy formation, immune system protection and protein synthesis process in A. schrenckii. The measurement of microelements indicated that Mn, Cu and Fe were obtained from their parents or water environment in A. schrenckii, while Zn plays vital roles throughout embryonic development. The dramatically high level of malondialdehyde (MDA) across the embryonic development may be the main reason leading to a low hatching rate in A. schrenckii. This study provides the basis for further proteome analysis of embryonic development in A. schrenckii.

The Amur sturgeon (Acipenser schrenckii) is referred to as a 'living fossil' , and is among the most primitive Actinopterygii species. It is distributed in the Amur, Songhua, and Heilong Rivers 1 . Amur sturgeon demonstrate a high growth rate and strong disease resistance, and is the most popular sturgeon aquaculture species in China with annual production of about 150 thousand tons in 2015, accounting for about 15% of the total sturgeon aquaculture production 2 . Several countries have recently shown an interest in Amur sturgeon aquaculture, and this species is now raised widely 3 . However, a low hatching rate represent the main problem affecting the sustainable development of the sturgeon aquaculture industry, especially for that at blastula stage, according to the hatching experience in our laboratory. A full understanding of the regulatory proteins and pathways involved in the embryonic development of A. schrenckii, and the effects of microelements and antioxidants on the embryonic development of A. schrenckii is therefore urgently needed. Several microRNA libraries and mRNA transcriptomes have been constructed for Acipenser species, and sex-related and reproduction-related genes have been investigated [4][5][6][7][8] . However, to the best of our knowledge, no previous studies have analyzed the changes in protein expression in this species.
The embryonic development of A. schrenckii can be divided into nine main stages: fertilized egg, cleavage stage, blastula, gastrula, yolk plug, neurula, formation of the optic bud, heart-beat stage, and hatching stages 9 . Among these, the fertilized egg, blastula, neurula, and heart-beat stages are particularly important stages of embryogenesis; the blastula stage indicates successful fertilization, while the neurula and heart-beat stages represent important steps in organogenesis, especially in relation to the heart and brain 9 .
Isobaric tag for relative and absolute quantization (iTRAQ) profiling is a reliable and accurate method of protein measurement, which is especially useful in annotated-related species. iTRAQ uses a variety of isotope Protein function annotation. The Gene Ontology (GO) and Clusters of Orthologous Groups (COG) tools aim to provide a structured and controlled vocabulary for describing gene products. GO includes three ontologies: cellular component, biological process, and molecular function. GO terms were assigned to 1,005 proteins based on BLAST matches with proteins with known functions. The matched proteins comprised 54 functional groups. The number of proteins in each GO term ranged from 1-841. Biological process mainly included cellular process (785, 14.40%), metabolic process (713, 13.08%), and single-organism process (564, 10.35%) ( Fig. 2A); cellular component included cell part (841, 20.30%), cell (841, 20.30%), organelle (698, 16.85%), and organelle part (495, 11.95%) (Fig. 2B); and molecular function included binding (727, 47.24%) and catalytic activity (496, 32.23%) (Fig. 2C).
COG annotation analysis classified the proteins identified by iTRAQ analysis into 22 categories (Fig. 3), with 2-164 proteins in each category. Among them, the general function prediction represented the largest functional group, including 164 proteins, followed by Posttranslational modification, protein turnover, chaperones (131), Translation, ribosomal structure and biogenesis (109), and Energy production and conversion (88). Cell motility, RNA processing and modification, and Chromatin structure and dynamics represented the smallest functional categories.

Identification of differentially expressed proteins (DEPs). According to previous studies, DEPs
were defined based on a threshold 1.2-1.5-fold change [20][21][22][23] . In the current study, only proteins expressed in the four embryonic developmental stages with fold changes ≥1.2 or ≤0.83 and with P < 0.05 and FDR < 0.05 (one-way ANOVA) compared with their control groups were considered to be DEPs 12 . Seventy-four proteins were differentially expressed in the fertilized egg relative to the blastula stage, including 53 up-regulated and 21 down-regulated proteins (Supplementary information 1); 77 proteins were differentially expressed in the blastula relative to the neurula stage, including 37 up-regulated and 40 down-regulated proteins (Supplementary information 2); and 76 proteins were differentially expressed in the neurula relative to the heart-beat stage, including . Among these DEPs, 31 were considered as "intersection DEPs", which were differentially expressed in at least two of the comparisons ( Table 1).
The main metabolic pathways of the DEPs are shown in Table 2. The main metabolic pathways enriched among these embryonic developmental stages included Glycolysis/Gluconeogenesis, Ribosome, Metabolic pathways, and Alzheimer's disease, which were the main "intersection metabolic pathways" during embryonic development. Glyceraldehyde-3-phosphate dehydrogenase A (GAPDH) (fertilized egg vs blastula with fold change of 0.41, and neurula vs heart-beat stages with fold change of 1.39) and nicotinamide adenine dinucleotide (NAD)binding (fertilized egg vs blastula with fold change of 0.64, blastula vs neurula with fold change of 0.39) in Gluconeogenesis metabolic pathway, the 60S ribosomal proteins L12 (blastula vs neurula with fold change of 0.76, and neurula vs heart-beat stages with fold change of 0.79), L17 (fertilized egg vs blastula with fold change of 0.64, and neurula vs heart-beat stages with fold change of 0.81), and L22 (fertilized egg vs blastula with fold change of 0.48, and neurula vs heart-beat stages with fold change of 0.69) in Ribosome metabolic pathway, and 26S protease regulatory subunit 6A (fertilized egg vs blastula with fold change of 1.26, and blastula vs neurula with fold change of 0.78) in Proteasome were identified as "intersection DEPs". Microelement contents. The contents of Fe, Mn, and Cu decreased gradually from the fertilized egg to the neurula stage, and then increased again at the heart-beat stage. The Zn content fluctuated during the four stages. The peaks Fe, Mn, Cu, and Zn contents occurred at the fertilized egg, fertilized egg, heart-beat, and blastula stages, respectively (Table 3). Antioxidant contents. Superoxide dismutase (SOD), MDA, catalase (CAT), and glutathione (GSH) showed similar expression patterns during the four embryonic developmental stages. These four antioxidants increased from the fertilized egg to blastula stages, deceased to the neurula stages, and then increased significantly again to the heart-beat stage. The peak GSH, MDA, and SOD contents were observed at the heart-beat stage, while the peak CAT content was observed at the blastula stage ( Table 4). The GSH and SOD levels at heart-beat stage are 10 times and 5 times higher than other stages (P < 0.01). The level of MDA at each developmental stage was hundred folds higher than that of other antioxidants (P < 0.01).

Discussion
This study provides valuable evidence for proteomic changes during embryonic development in A. schrenckii based on iTRAQ profiling. To the best of our knowledge, this is the first proteome-profiling analysis in A. schrenckii, and the results will thus promote further studies to improve our understanding of the regulatory proteins and pathways affecting embryonic development in this species. A total of 1,143 proteins were identified, mostly with biological functions related to Cell part, Cell, Cellular process, Binding, and Metabolic process according to GO assignments, and General function prediction only, Posttranslational modification, protein turnover, chaperones, and Translation, ribosomal structure, and biogenesis in COG analysis. Dramatically more than 1,134 proteins in total were identified by GO assignment and COG analysis, because several sequences were involved in more than one functional category. KEGG analysis can help to detect relationships between different genes. The main KEGG pathways included metabolic pathway, Parkinson's disease, Huntington's disease, and Ribosome.
Gluconeogenesis was the most enriched metabolic pathway across the four developmental stages, resulting in the generation of glucose (C 6 H 12 O 6 ) from non-carbohydrate carbon substrates, and is responsible for maintaining normal blood glucose levels in humans and many other animals 24 , while glycolysis converts glucose into pyruvate (CH 3 COCOO-), with the release of free energy used to form the high-energy molecules ATP (adenosine triphosphate) and NADH (reduced nicotinamide adenine dinucleotide) 25 (Fig. 4). The produced energy is then available for body growth and development. In this study, GAPDHand NAD binding were identified as "intersection DEPs". The product of GAPDH catalyzes an important energy-yielding step in carbohydrate metabolism through the reversible oxidative phosphorylation of glyceraldehyde-3-phosphate in the presence of inorganic phosphate and NAD 26 . The citric acid cycle was the other main enriched pathway involved in the energy formation. The citric acid cycle, also known as the tricarboxylic acid cycle, involves a series of chemical reactions that release stored energy via oxidation of acetyl-CoA derived from carbohydrates, fats, and proteins into carbon dioxide and chemical energy, in the form of ATP 27 . Aconitate hydratase was an important DEP between neurula and heart-beat stages (1.63) which can cleave carbon-oxygen bonds to release ATP 28 .
The ribosome is other main enriched pathway across the four developmental stages, which is a complex molecular machine found within all living cells, serving as the site of biological protein synthesis (translation) 29 (Fig. 5). Ribosomes link amino acids together in the order specified by message RNA (mRNA) molecules. Ribosomes can be divided into small 40S and large 60S ribosomal subunits 29 together composed of four RNA species and approximately 80 structurally distinct proteins. The small ribosomal subunits read the RNA, while the large ribosomal subunits join amino acids to form a polypeptide chain. Each subunit is composed of one or more ribosomal RNA (rRNA) molecules and a variety of ribosomal proteins. In this study, the 60S ribosomal proteins L12, L17, and L22 were identified as "intersection proteins". These ribosomal proteins are encoded by the RPL12, RPL17, and RPL22 genes, respectively, in humans, which each encode a component of the 60S subunit. RPL12 and RPL17 belong to the L11P and L22P families of ribosomal proteins, respectively, located in the cytoplasm, whereas RPL22 belongs to the L22E family of ribosomal proteins. Other main enriched metabolic pathways included the Proteasome. Proteasomes are protein complexes that mainly function to degrade unneeded or damaged proteins by proteolysis 30 . 26S protease regulatory subunit 6A encoded by PSMC3 gene was considered as an "intersection protein". Component of the 26S proteasome plays essential roles in the maintenance of protein homeostasis by removing misfolded proteins, damaged proteins and the unnecessary protein. 26S protease regulatory subunit 6A unfolds ubiquitinated targeted proteins, and translocated them into a proteolytic chamber for degradation. The expression level of 26S protease regulatory subunit 6A was up-regulated at blastula stage, compared to that of fertilized egg and neurula, which is consistent with that the hatching rate at blastula stage was the lowest in Amur sturgeon. Further studies are needed to investigate the roles of these proteins 31 .
To the best of our knowledge, although the measurements of microelements have been performed in mature tissues in many aquatic species and even in sturgeon species [32][33][34] , the measurements during the embryonic developmental stages have not yet been reported in any aquatic species. Only one previous study has performed the microelements measurement aged from 6 to 12 months in 90 aquatic species, results of which indicated that the levels of microelements were significantly influenced by the culture site and culture conditions 35 . Fe deficiency was associated with decreased enzyme activities in fish, and disturbed growth and immunity 36   Mn, and Cu decreased and then increased again across the four embryonic developmental stages in the current study, while the Zn content gradually increased and then decreased. Mn is an important component of several enzymes in animals, including arginine kinase, proline peptidase, RNA polymerase, superoxide dismutase, and pyruvate carboxylase, which have been shown to promote skeleton development, fertilization, hatchability, and feed conversion 37 . In this study, the peak Mn content occurred at the fertilized egg, and then decreased dramatically with the embryonic development, indicating that Mn was mainly obtained from their parents. Cu is involved in the formation of cytochrome oxidase, lysine hydrochloride, superoxide dismutase, and tyrosinase, which play essential roles in the defensive immune system in fish [37][38][39] . Cu is also involved in regulating the assimilation and utilization of Fe. Cu cannot be synthesized by fish, thus it should be efficiently absorbed from water environment 40 . The peak Cu content occurred during the heart-beat stage in the current study, indicating that the germ cell absorbed Cu from the water environment during embryonic development. Zn is a necessary microelement in fish, with a wide range of physical functions. It is involved in several metabolic processes via alkaline phosphatase, including nucleotide and protein synthesis, energy metabolism, and redox reaction, which can affect substance metabolism, growth, and development in fish 41,42 . In this study, the Zn content was maintained at a high level in all four embryonic stages, indicating that Zn plays vital roles throughout embryonic development in A. schrenckii 43 . Compared to the previous measurement of microelements in skin of farmed A. schrenckii, the contents of Fe and Cu were significantly higher in this study, while Zn was significantly lower 33 . These results further confirmed that Fe and Cu cannot be synthesized by A. schrenckii.
To the best of our knowledge, the measurements of antioxidants during the embryonic developmental stages were not reported in any aquatic species. The measurements of antioxidants were performed under stress condition in sturgeon species, including the changes in food diets and water conditions, in order to analyze the effects of such changes on antioxidant defenses [44][45][46][47][48] . Superoxide dismutase (SOD), Catalase (CAT), Malondialdehyde (MAD), and GSH are important antioxidants in fish, maintaining normal embryonic development through protecting the cytomembrane, DNA, and proteins from damage by reactive oxygen species 49,50 . GSH is involved in maintaining normal immune system functioning, including antioxygenation and antidotal action 51 . SOD is an important antioxidant with essential roles in removing and eliminating the negative effects of free radicals, and SOD content is a vital index indicating senility and death [52][53][54] . The GSH and SOD levels at heart-beat stage are 10 times and 5 times higher than other stages. These results suggested essential roles of GSH and SOD in immune system, which remove negative effects to ensure the normal hatching of A. schrenckii embryos. CAT accounts for about 40% of all expressed peroxidases, and occurs in every tissue, with especially high expression in the liver. CAT can catalyze H 2 O 2 to H 2 O and O 2 , thus preventing H 2 O 2 from binding with O 2 to form hydroxyl radicals, which are very harmful products in biological systems [53][54][55] . MDA is an important product in the peroxidation reaction of membrane lipids. MDA generation dramatically increases membrane damage leading to cell death, and is thus an important index for the analysis of senility and stress-resistance processes 56 . The level of MDA at each developmental stage was hundred folds higher than that of other antioxidants, while the levels of SOD and CAT across the developmental stages were hundred folds lower than that in mature tissues in other sturgeon 44,45 , indicating the significant presence of MDA and the deficiency of SOD, GSH and CAT may be the main reason leading to a low hatching rate in A. schrenckii.
In conclusion, 74, 77, and 76 proteins were differentially expressed between the fertilized egg and blastula, blastula and neurula, and neurula and heart-beat stages of A. schrenckii development, respectively, according to iTRAQ analysis. On the basis of GO and pathway enrichment analyses of DEPs, Gluconeogenesis and Citrate cycle were considered to provided energy; Ribosome promoted protein synthesis; and Proteasome protected immune system, in order to maintain normal embryonic development of A. schrenckii. Analysis of levels of microelements and antioxidants indicated that Mn, Cu and Fe were obtained from their parents or water environment in A. schrenckii, while the dramatically high level of Zn plays vital roles throughout embryonic development. The dramatically high level of MDA across the embryonic development may be the main reason leading to a low hatching rate in A. schrenckii.

Materials and Methods
Ethics statement. All the fish handling and experimental procedures involved in this study were approved by the Animal Care and Use Committee of the Heilongjiang River Fisheries Research Institute, Chinese Academy of Fishery Sciences, Haerbin, and followed the experimental basic principles. All methods were performed in accordance with relevant guidelines and regulations. All efforts were made under MS222 anesthesia to minimize suffering.
Sample collection. Three healthy male and three female A. schrenckii (body weight 15-25 kg) were obtained from the Amur sturgeon breeding and engineering center, Heilongjiang River Fisheries Research Institute, Chinese Academy of Fishery Sciences. The fish were maintained in aerated freshwater at 14 °C.
A temperature of 16 °C-18 °C was used to hasten spawning in A. schrenckii. Female A. schrenckii received two injections of luteinizing hormone-releasing hormone A2 at 2 μg/kg and 4 μg/kg 12 h after the first injection, respectively. Male A. schrenckii were injected with 1 μg/kg luteinizing hormone-releasing hormone A2 1day prior to the first injection of female A. schrenckii. Mature sperm and eggs were obtained, fertilized for 2 min by a semi-dry technique, and debonded for about 30 min using 30% talcum powder. The fertilized eggs were then transferred to an incubator at 18 °C and oxygen level >6.0 mg/L. The fertilized eggs were observed under a microscope and embryos were collected at the fertilized egg, blastula, neurula, and heart-beat stages in micro tubes, and immediately frozen in liquid nitrogen to prevent total protein degradation, until use for protein extraction for proteome profiling analysis.
Protein preparation and iTRAQ labeling. We analyzed the protein expression profiles of the four embryonic developmental samples of A. schrenckii. At least 100 embryos per group were collected and pooled to eliminate the effects of individual differences. Total proteins were extracted from each developmental sample using a phenol extraction procedure, and the protein concentrations were determined using the Bradford colorimetric method. A total of 100 mg of protein from each sample was lyophilized using a speed vacuum system (Martin Christ, Germany).
iTRAQ labeling was performed using an iTRAQ Reagent 8-Plex kit (Applied Biosystems, Foster City, CA, USA) according to the manufacturer's protocol. A total of 100 mg extracted protein from each sample was dissolved in 0.5 M triethylammonium bicarbonate, incubated with 10 mL 1 mg/ml trypsin (Promega, Madison, WI, USA) at room temperature for 2 h, and labeled using iTRAQ Reagent 8-Plex kit.

LC-MS/MS analysis.
Each dried sample was reconstituted to an average of 0.5 μg/μL using buffer A (5% ACN, 0.1% FA), and followed by centrifuging at 20,000 × g for 10 min to remove impurities. A total of 2.5 μg protein was separated using a LC-20AD HPLC system (Shimadzu, kyoto, Japan). A flow rate of 8 μL/min was used to load the sample onto a Trap column at within 4 min, and then transferred to an analysis column at a flow rate of 300 nL/min with elution buffer B (95% ACN, 0.1% FA) for 5 min, 5%-35% buffer B for 35 min. Buffer B was increased to 60% within 5 min, to 80% within 2 min, maintained for 2 min, finally decreased to 5% within 1 min, and maintained for 10 min. The sample was then transferred to a mass spectrometer (Triple TOF 5600; AB SCIEX) for LC-MS/MS analysis, as described previously 20 . A 2.5 kV ion spray voltage, 15 psi nebulizer gas, 30 psi curtain gas, and interface heater temperature of 150 were used to perform the data analysis. A RP of ≥30,000 FWHM was used to operate the MS for TOF MS scan, and 250 ms was required for survey scan for IDA analysis. Thirty products were collected if the ion scans were more than a threshold of 120 counts/s. The total cycle was conducted at 3.3 s. The Q2 transmission window was set at 100 Da for 100%. 40 GHz multichannel TDC detector containing a four-anode channel was used to detect and sum ions from each scan at a 11 kHz pulser frequency value. iTRAQ adjusted rolling collision energy and sweeping collision energy with 35 ± 5 eV were combined for application to all precursors to ensure collision-induced dissociation. Dynamic exclusion was set for 1/2 of peak width (15 s), and the precursor was then refreshed to the exclusion list. MS/MS analysis was used to select peptides with +2 to +3 charge states. The fragment intensity multiplier was set to 20, with a maximum accumulation time of 2 s. iTRAQ protein identification and quantification. MASCOT 2.3.02 software was used to analyze iTRAQ data. To conduct confident peptide identification, a Mascot probability analysis was used to count the unique peptides with ratios of significance scores (≥20) at the 99% confidence interval. A protein was identified when it contained at least one unique peptide, while at least two unique peptides needed to be matched to a protein for analysis. The median ratio in Mascot was used to perform the weight and normalization of the quantitative protein ratios. The confidence level of the altered expression of proteins was calculated by Protein Pilot as a p-value. We only used ratios with P-values < 0.05 and a false discovery rate (FDR) < 0.05, and only proteins with fold changes of >1.2 were considered as up-regulated proteins or proteins with label ratio of <0.83 were considered as down-regulated proteins.
GO and pathway enrichment analysis. Functional protein annotations according to GO were determined using the Blast2GO program. Hypergeometric tests with a ratio of P < 0.05 were used to identify all and elsewhere differentially expressed proteins in the GO enrichment terms. The proteins were further subjected to functional annotation analysis according to the COG (http://www.ncbi.nlm.nih.gov/COG/) and KEGG databases (http://www.genome.jp/kegg/). Pathways that were significantly enriched compared with differential proteins with P < 0.05 and FDR < 0.05 were used as a threshold to select significant KEGG pathways.
Microelements and antioxidants contents. A total of 1 gA. schrenckii embryos at different stages were dry-ashed and digested by 16 ml nitric acid, 2 ml perchilric acid and 1 ml hydrogen peroxide for overnight. The samples were then incubated at 120 °C for 2 h, and incubated at 150 °C until only 2 ml samples were left. The Fe, Mn, Cu, and Zn contents were then measured by AA6800 atomic absorption spectrophotometry (Shimadzu, Japan) according to the GB/T5009. 12 (13,14,87,90,91,92, 93 and 123)-2003.
Collected different-stage embryos were also diluted in 0.5 ml phosphate-buffered saline, homogenized for 5 min on ice, and centrifuged at 3000 r/min for 10 min. The supernatant was then used for determination of antioxidant contents. The GSH, SOD, MDA, and CAT contents at different embryonic developmental stages were measured using kits from Nanjing Jiancheng Bioengineering Institute, according to the manufacturer's protocols. The measurements of each microelements and antioxidants level were performed in triplicate.

Statistical analysis.
Quantitative data of the measured microelements and antioxidants level were expressed as mean ± standard deviation using Microsoft Excel 2010. The level of significance between different microelements and between different antioxidants was analyzed by one-way analysis of variance (ANOVA) and post hoc Duncan multiple range tests with SPSS software version 17.0. Statistically significant differences were examined by paired t-test. A probability level of 0.05 was considered to be statistically significant (P < 0.05).