Molecular Characterization and Expression Profiles of Sp-uchl3 and Sp-uchl5 during Gonad Development of Scylla paramamosain

Ubiquitin C-terminal hydrolases (UCHLs) are a subset of deubiquitinating enzymes, and are involved in numerous physiological processes. However, the role of UCHLs during gonad development has not been studied in crustaceans. In this study, we have first cloned and analyzed expression profiling of Sp-uchl3 and Sp-uchl5 genes from mud crab Scylla paramamosain. The full-length cDNA of Sp-uchl3 is of 1804 bp. Its expression level in the ovary was significantly higher than in other tissues (p < 0.01), and during gonadal development, its expression in both O1 and O5 stages was significantly higher than in the other three stages of ovaries (p < 0.05), while in T3 it was higher than in the former two stages of testes (p < 0.05). Meanwhile, the full-length cDNA of Sp-UCHL5 is 1217 bp. The expression level in the ovary was significantly higher than in other tissues (p < 0.01). Its expression in ovaries was higher than in testes during gonadal development (p < 0.05). The expression level in the O5 stage was the highest, followed by the O3 stage in ovarian development, and with no significant difference in the testis development (p > 0.05). These results provide basic data showing the role of Sp-UCHL3 and Sp-UCHL5 in the gonad development of the crab.


Introduction
The green mud crab Scylla paramamosain is mainly distributed in the southeastern coast of China and is an important economic species in aquaculture. Due to its large size, rapid growth, delicate flavor and abundant nutrient components, the artificial cultivation of the crab has become one of the main aquaculture industries in China [1]. With the development of crab cultivation, the artificial breeding of crabs had been explored by many researchers [2]. However, the aquaculture production currently still relies heavily on wild-caught fry for farming, and the amount of wild-caught fry fluctuates greatly. Insufficient and unstable resources of natural seedlings have become the bottleneck in the health and sustainability of the development of green mud crab aquaculture [3]. Therefore, the fry production techniques related to improving spawning, breeding survival rate and decreasing the cost have become the current main tasks in the aquaculture production of the crab [4]. Thus, it is important to understand the molecular regulatory mechanisms involved in gonad development and gametogenesis in order to provide reliable hatchery technology for production of crab fry. In recent years, many researchers have carried out a lot of work on this aspect. For example, Gong et al. [5] In this study, the full-length cDNAs of uchl3 and uchl5 from S. paramamosain were first identified, and their expressions in different tissues and developing gonad were determined. The cloning and characterization of Sp-uchl3 and Sp-uchl5 transcripts will provide us with useful information to further investigate the molecular mechanism of gonad development in crabs.

Molecular Characterization of Sp-uchl3 and Sp-uchl5
The full-length Sp-uchl3 cDNA is 1804 bp, containing 129 bp in the 5′-untranslated region (UTR), 687 bp in the ORF, and 988 bp in the 3′-UTR with a poly(A) tail. The cDNA sequence and deduced amino acid sequence have been submitted to the NCBI GenBank (accession No. FJ800570). The ORF encodes a polypeptide of 228 amino acids with a predicted molecular mass of 25.35 kDa and a theoretical pI of 4.64. A polyadenylation signal (AATAAA) is located 13 bp upstream of poly(A) tail ( Figure 1). The results of the k-nearest neighbor (k-NN) prediction by PSORTII revealed that Sp-UCHL3 protein may mainly locate in the cytoplasm (65.2%). Sequence analysis revealed that it has a protein kinase C phosphorylation site (S 57 -K 59 ), three casein kinase II phosphorylation sites (T28-D31, S148-E151, T189-D192), three N-myristoylation sites (G 22

Prediction of Sp-UCHL3 and Sp-UCHL5 Three-Dimensional (3D) Structure
To understand the different structures of UCHL3 between S. paramamosain and its closely related species Procambarus clarkii (GenBank: ANI85922.1), we used the template (PDB code: 1uch1A) at the SWISS-MODEL server by a homology modeling method to predict their three-dimensional structures ( Figure 3). Sp-UCHL3 shared 52.21% sequence identity to the template, while Pc-UCHL3 shared 56.19%. The structure of Sp-UCHL3 ( Figure 3A) was based on the 226 amino acid residues (V 2 -T 227 ) of the mature protein, and the Pc-UCHL3 ( Figure 3B) was based on the 226 amino acid residues (V 2 -S 227 ). Both Sp-UCHL3 and Pc-UCHL3 were mainly composed of nine alpha helices, seven beta sheets and several random coils, which contained the protein kinase C (PKC) phosphorylation site and the active site of ubiquitin C-terminal hydrolases family 1. The shape of a random coil located in the seventh alpha helix and the fourth beta sheet was their biggest difference. Meanwhile, the three-dimensional structures of UCHL5 ( Figure 4) in S. paramamosain and its closely related species Hyalella azteca (GenBank: XP_018019441.1), according to the template (PDB code: 3ihrA): Sp-UCHL5 shared 64.15% sequence identity to the template, while Ha-UCHL5 shared 57.14%. The structure of Sp-UCHL5 ( Figure 4A) was based on the 319 amino acid residues (V 3 -K 321 ) of the mature protein, while the Ha-UCHL5 ( Figure 4B) was based on the 312 amino acid residues (A 4 -A 315 ). Both of Sp-UCHL5 and Ha-UCHL5 consisted of thirteen alpha helices, six beta sheets and several random coils, while the conserved sequence of UCHL family 1 (N 8 -Y 213 ) was located between the first beta sheet and the ninth alpha helix. The biggest difference between the Sp-UCHL5 and Ha-UCHL5 was the shape of the random coil located in the sixth alpha helix and the third beta sheet.

Prediction of Sp-UCHL3 and Sp-UCHL5 Three-Dimensional (3D) Structure
To understand the different structures of UCHL3 between S. paramamosain and its closely related species Procambarus clarkii (GenBank: ANI85922.1), we used the template (PDB code: 1uch1A) at the SWISS-MODEL server by a homology modeling method to predict their three-dimensional structures ( Figure 3). Sp-UCHL3 shared 52.21% sequence identity to the template, while Pc-UCHL3 shared 56.19%. The structure of Sp-UCHL3 ( Figure 3A) was based on the 226 amino acid residues (V 2 -T 227 ) of the mature protein, and the Pc-UCHL3 ( Figure 3B) was based on the 226 amino acid residues (V 2 -S 227 ). Both Sp-UCHL3 and Pc-UCHL3 were mainly composed of nine alpha helices, seven beta sheets and several random coils, which contained the protein kinase C (PKC) phosphorylation site and the active site of ubiquitin C-terminal hydrolases family 1. The shape of a random coil located in the seventh alpha helix and the fourth beta sheet was their biggest difference. Meanwhile, the three-dimensional structures of UCHL5 ( Figure 4) in S. paramamosain and its closely related species Hyalella azteca (GenBank: XP_018019441.1), according to the template (PDB code: 3ihrA): Sp-UCHL5 shared 64.15% sequence identity to the template, while Ha-UCHL5 shared 57.14%. The structure of Sp-UCHL5 ( Figure 4A) was based on the 319 amino acid residues (V 3 -K 321 ) of the mature protein, while the Ha-UCHL5 ( Figure 4B) was based on the 312 amino acid residues (A 4 -A 315 ). Both of Sp-UCHL5 and Ha-UCHL5 consisted of thirteen alpha helices, six beta sheets and several random coils, while the conserved sequence of UCHL family 1 (N 8 -Y 213 ) was located between the first beta sheet and the ninth alpha helix. The biggest difference between the Sp-UCHL5 and Ha-UCHL5 was the shape of the random coil located in the sixth alpha helix and the third beta sheet.

Phylogenetic and Homology Analyses of Sp-UCHL3 and Sp-UCHL5
To examine the relationships of UCHL3 and UCHL5 among various animal species, the phylogenetic trees were generated separately by using the MEGA 4.0 neighbor-joining method based on putative amino acid sequences of green mud crab and the other known UCHL3 and UCHL5 retrieved from GenBank. The results showed that the selected UCHL3 sequences were clustered into two groups: chordata and invertebrate. The chordata group consisted of two subgroups: the first subgroup included vertebrate-containing fish (Danio rerio), amphibians (Xenopus laevis), birds (Taeniopygia guttata) and mammals (Orycteropus afer, Bos taurus and Homo sapiens), while the second subgroup included Tunicata (Ciona intestinalis) and Cephalochordata (Branchiostoma belcheri). Furthermore, the invertebrate group consisted of two subgroups: cnidaria and arthropods. Obviously, Sp-UCHL3 belonged to the arthropod group, first being clustered with Procambarus clarkii, and next with Penaeus monodon ( Figure 5). Meanwhile, phylogenetic analysis of the selected UCHL5 amino acid sequences revealed that UCHL5 proteins were formed into two main clusters. Vertebrates, including mammals, birds and amphibians formed a subcluster, and invertebrates including S. paramamosain, Hyalella azteca and Tribolium castaneum formed the other subcluster. Interestingly, Hydra vulgris was grouped to the vertebrates cluster ( Figure 6).

Phylogenetic and Homology Analyses of Sp-UCHL3 and Sp-UCHL5
To examine the relationships of UCHL3 and UCHL5 among various animal species, the phylogenetic trees were generated separately by using the MEGA 4.0 neighbor-joining method based on putative amino acid sequences of green mud crab and the other known UCHL3 and UCHL5 retrieved from GenBank. The results showed that the selected UCHL3 sequences were clustered into two groups: chordata and invertebrate. The chordata group consisted of two subgroups: the first subgroup included vertebrate-containing fish (Danio rerio), amphibians (Xenopus laevis), birds (Taeniopygia guttata) and mammals (Orycteropus afer, Bos taurus and Homo sapiens), while the second subgroup included Tunicata (Ciona intestinalis) and Cephalochordata (Branchiostoma belcheri). Furthermore, the invertebrate group consisted of two subgroups: cnidaria and arthropods. Obviously, Sp-UCHL3 belonged to the arthropod group, first being clustered with Procambarus clarkii, and next with Penaeus monodon ( Figure 5). Meanwhile, phylogenetic analysis of the selected UCHL5 amino acid sequences revealed that UCHL5 proteins were formed into two main clusters. Vertebrates, including mammals, birds and amphibians formed a subcluster, and invertebrates including S. paramamosain, Hyalella azteca and Tribolium castaneum formed the other subcluster. Interestingly, Hydra vulgris was grouped to the vertebrates cluster ( Figure 6).

Phylogenetic and Homology Analyses of Sp-UCHL3 and Sp-UCHL5
To examine the relationships of UCHL3 and UCHL5 among various animal species, the phylogenetic trees were generated separately by using the MEGA 4.0 neighbor-joining method based on putative amino acid sequences of green mud crab and the other known UCHL3 and UCHL5 retrieved from GenBank. The results showed that the selected UCHL3 sequences were clustered into two groups: chordata and invertebrate. The chordata group consisted of two subgroups: the first subgroup included vertebrate-containing fish (Danio rerio), amphibians (Xenopus laevis), birds (Taeniopygia guttata) and mammals (Orycteropus afer, Bos taurus and Homo sapiens), while the second subgroup included Tunicata (Ciona intestinalis) and Cephalochordata (Branchiostoma belcheri). Furthermore, the invertebrate group consisted of two subgroups: cnidaria and arthropods. Obviously, Sp-UCHL3 belonged to the arthropod group, first being clustered with Procambarus clarkii, and next with Penaeus monodon ( Figure 5). Meanwhile, phylogenetic analysis of the selected UCHL5 amino acid sequences revealed that UCHL5 proteins were formed into two main clusters. Vertebrates, including mammals, birds and amphibians formed a subcluster, and invertebrates including S. paramamosain, Hyalella azteca and Tribolium castaneum formed the other subcluster. Interestingly, Hydra vulgris was grouped to the vertebrates cluster ( Figure 6).

Tissue Distribution of Sp-uchl3 and Sp-uchl5 Transcripts in Female Crab
Tissue distributions of mRNA expression of Sp-uchl3 and Sp-uchl5 in female crab were examined by quantitative real time polymerase chain reaction (RT-qPCR) using 18S rRNA as an internal control gene. The results showed that the Sp-uchl3 ( Figure 7) and Sp-uchl5 ( Figure 8) were differentially expressed in the analyzed tissues including brain, gill, heart, haemocytes, intestine, hepatopancreas, muscle, ovary and stomach. However, their expression levels in the ovary were significantly higher than in other examined tissues (p < 0.01).

Tissue Distribution of Sp-uchl3 and Sp-uchl5 Transcripts in Female Crab
Tissue distributions of mRNA expression of Sp-uchl3 and Sp-uchl5 in female crab were examined by quantitative real time polymerase chain reaction (RT-qPCR) using 18S rRNA as an internal control gene. The results showed that the Sp-uchl3 ( Figure 7) and Sp-uchl5 ( Figure 8) were differentially expressed in the analyzed tissues including brain, gill, heart, haemocytes, intestine, hepatopancreas, muscle, ovary and stomach. However, their expression levels in the ovary were significantly higher than in other examined tissues (p < 0.01).

Expression Profiles of Sp-uchl3 and Sp-uchl5 Transcripts in the Different Stages of Gonad Development
To further understand the developmental expression profiles of Sp-uchl3 and Sp-uchl5 in different gonad developing stages, RT-qPCR was employed to compare their relative expression levels. The results showed that the expression of Sp-uchl3 mRNA was the highest in the O5 stage, followed by O1 and T3 stages, and the lowest in the T2 stage. The expression levels of Sp-uchl3 in both O1 and O5 stages were significantly higher than in the O2, O3 and O4 stages of ovaries (p < 0.05), and its expression in T3 was significantly higher than in the T1 and T2 stages of testes (p < 0.05) ( Figure 9). Meanwhile, the Sp-uchl5 expression level in ovaries was higher than in testes (p < 0.05). The expression level in the O5 stage was the highest, followed by the O3 stage during ovarian development, and there was no significant difference between the O3 and O5 stages, while their expression levels were significantly higher than in testes (p < 0.01). However, the expression level in the O5 stage was much higher than in the O1, O2 and O4 stages (p < 0.05). Moreover, the expression levels in the three stages of testes development were not significantly different from each other (p > 0.05) (Figure 10).

Tissue Distribution of Sp-uchl3 and Sp-uchl5 Transcripts in Female Crab
Tissue distributions of mRNA expression of Sp-uchl3 and Sp-uchl5 in female crab were examined by quantitative real time polymerase chain reaction (RT-qPCR) using 18S rRNA as an internal control gene. The results showed that the Sp-uchl3 (Figure 7) and Sp-uchl5 ( Figure 8) were differentially expressed in the analyzed tissues including brain, gill, heart, haemocytes, intestine, hepatopancreas, muscle, ovary and stomach. However, their expression levels in the ovary were significantly higher than in other examined tissues (p < 0.01).

Expression Profiles of Sp-uchl3 and Sp-uchl5 Transcripts in the Different Stages of Gonad Development
To further understand the developmental expression profiles of Sp-uchl3 and Sp-uchl5 in different gonad developing stages, RT-qPCR was employed to compare their relative expression levels. The results showed that the expression of Sp-uchl3 mRNA was the highest in the O5 stage, followed by O1 and T3 stages, and the lowest in the T2 stage. The expression levels of Sp-uchl3 in both O1 and O5 stages were significantly higher than in the O2, O3 and O4 stages of ovaries (p < 0.05), and its expression in T3 was significantly higher than in the T1 and T2 stages of testes (p < 0.05) (Figure 9). Meanwhile, the Sp-uchl5 expression level in ovaries was higher than in testes (p < 0.05). The expression level in the O5 stage was the highest, followed by the O3 stage during ovarian development, and there was no significant difference between the O3 and O5 stages, while their expression levels were significantly higher than in testes (p < 0.01). However, the expression level in the O5 stage was much higher than in the O1, O2 and O4 stages (p < 0.05). Moreover, the expression levels in the three stages of testes development were not significantly different from each other (p > 0.05) (Figure 10).

Expression Profiles of Sp-uchl3 and Sp-uchl5 Transcripts in the Different Stages of Gonad Development
To further understand the developmental expression profiles of Sp-uchl3 and Sp-uchl5 in different gonad developing stages, RT-qPCR was employed to compare their relative expression levels. The results showed that the expression of Sp-uchl3 mRNA was the highest in the O5 stage, followed by O1 and T3 stages, and the lowest in the T2 stage. The expression levels of Sp-uchl3 in both O1 and O5 stages were significantly higher than in the O2, O3 and O4 stages of ovaries (p < 0.05), and its expression in T3 was significantly higher than in the T1 and T2 stages of testes (p < 0.05) (Figure 9). Meanwhile, the Sp-uchl5 expression level in ovaries was higher than in testes (p < 0.05). The expression level in the O5 stage was the highest, followed by the O3 stage during ovarian development, and there was no significant difference between the O3 and O5 stages, while their expression levels were significantly higher than in testes (p < 0.01). However, the expression level in the O5 stage was much higher than in the O1, O2 and O4 stages (p < 0.05). Moreover, the expression levels in the three stages of testes development were not significantly different from each other (p > 0.05) ( Figure 10).

Discussion
The UPP is the major proteolytic pathway for specific intracellular protein degradation in eukaryotes. Its function is implicated in numerous physiological processes including cell cycle regulation, signal transduction, antigen presenting, apoptosis and so on [18][19][20][37][38][39]. UCHs are important deubiquitinating enzymes which participate in the proteolytic process of polymeric ubiquitin to promote the ubiquitin recirculation process [28,[40][41][42]. They are important for the generation of monomeric ubiquitin, and are the active components of the eukaryotic ubiquitin-dependent proteolytic system [43,44]. This is the first report of cloning and expression profiling of UCHs in crustaceans. The full-length Sp-uchl3 and Sp-uchl5 cDNA sequences from S. paramamosain were cloned successfully, and sequence analysis of these two genes showed that both of them contain UCH family 1 conserved sequences such as cysteine, histidine and aspartate, which are important for the substrate specificity of the enzymes [45][46][47]. The N-terminal amino acid sequence residue domain of Sp-UCHL3 from 87 aa to 103 aa is an active site of UCH family 1. Similarly, Sp-UCHL5 contains a longer domain structure of UCH family 1 from 8 aa to 213 aa. UCHL1 and UCHL3 are the two most predominant isozymes in UCHs, share more than 50% sequence identity, and hydrolyze C-terminal esters and amides of ubiquitin [48,49]; moreover, UCHL5 plays a direct role in recycling ubiquitin from proteasomal degradation [50]. In the 3D-structure analysis, both Sp-UCHL3 and Pc-UCHL3 contained a PKC phosphorylation site and active site of UCH family 1; their similar structures may be responsible for their analogous functions. Meanwhile, although the structure of Sp-UCHL5 and Ha-UCHL5 share a similar conserved sequence of UCHL family 1, there were some differences in this conserved sequence compared with the two closely related species of UCHL3, which may relate to their different evolutionary status. The phylogenetic tree revealed that the both Sp-UCHL3 and Sp-UCHL5 belonged to an independent subgroup with other arthropods individually, which suggested that the evolution of these two proteins is consistent with the evolution of their species. Most UCHs can catalyze the peptide or isopeptide bond at the ubiquitin C-terminus, rather

Discussion
The UPP is the major proteolytic pathway for specific intracellular protein degradation in eukaryotes. Its function is implicated in numerous physiological processes including cell cycle regulation, signal transduction, antigen presenting, apoptosis and so on [18][19][20][37][38][39]. UCHs are important deubiquitinating enzymes which participate in the proteolytic process of polymeric ubiquitin to promote the ubiquitin recirculation process [28,[40][41][42]. They are important for the generation of monomeric ubiquitin, and are the active components of the eukaryotic ubiquitin-dependent proteolytic system [43,44]. This is the first report of cloning and expression profiling of UCHs in crustaceans. The full-length Sp-uchl3 and Sp-uchl5 cDNA sequences from S. paramamosain were cloned successfully, and sequence analysis of these two genes showed that both of them contain UCH family 1 conserved sequences such as cysteine, histidine and aspartate, which are important for the substrate specificity of the enzymes [45][46][47]. The N-terminal amino acid sequence residue domain of Sp-UCHL3 from 87 aa to 103 aa is an active site of UCH family 1. Similarly, Sp-UCHL5 contains a longer domain structure of UCH family 1 from 8 aa to 213 aa. UCHL1 and UCHL3 are the two most predominant isozymes in UCHs, share more than 50% sequence identity, and hydrolyze C-terminal esters and amides of ubiquitin [48,49]; moreover, UCHL5 plays a direct role in recycling ubiquitin from proteasomal degradation [50]. In the 3D-structure analysis, both Sp-UCHL3 and Pc-UCHL3 contained a PKC phosphorylation site and active site of UCH family 1; their similar structures may be responsible for their analogous functions. Meanwhile, although the structure of Sp-UCHL5 and Ha-UCHL5 share a similar conserved sequence of UCHL family 1, there were some differences in this conserved sequence compared with the two closely related species of UCHL3, which may relate to their different evolutionary status. The phylogenetic tree revealed that the both Sp-UCHL3 and Sp-UCHL5 belonged to an independent subgroup with other arthropods individually, which suggested that the evolution of these two proteins is consistent with the evolution of their species. Most UCHs can catalyze the peptide or isopeptide bond at the ubiquitin C-terminus, rather

Discussion
The UPP is the major proteolytic pathway for specific intracellular protein degradation in eukaryotes. Its function is implicated in numerous physiological processes including cell cycle regulation, signal transduction, antigen presenting, apoptosis and so on [18][19][20][37][38][39]. UCHs are important deubiquitinating enzymes which participate in the proteolytic process of polymeric ubiquitin to promote the ubiquitin recirculation process [28,[40][41][42]. They are important for the generation of monomeric ubiquitin, and are the active components of the eukaryotic ubiquitin-dependent proteolytic system [43,44]. This is the first report of cloning and expression profiling of UCHs in crustaceans. The full-length Sp-uchl3 and Sp-uchl5 cDNA sequences from S. paramamosain were cloned successfully, and sequence analysis of these two genes showed that both of them contain UCH family 1 conserved sequences such as cysteine, histidine and aspartate, which are important for the substrate specificity of the enzymes [45][46][47]. The N-terminal amino acid sequence residue domain of Sp-UCHL3 from 87 aa to 103 aa is an active site of UCH family 1. Similarly, Sp-UCHL5 contains a longer domain structure of UCH family 1 from 8 aa to 213 aa. UCHL1 and UCHL3 are the two most predominant isozymes in UCHs, share more than 50% sequence identity, and hydrolyze C-terminal esters and amides of ubiquitin [48,49]; moreover, UCHL5 plays a direct role in recycling ubiquitin from proteasomal degradation [50]. In the 3D-structure analysis, both Sp-UCHL3 and Pc-UCHL3 contained a PKC phosphorylation site and active site of UCH family 1; their similar structures may be responsible for their analogous functions. Meanwhile, although the structure of Sp-UCHL5 and Ha-UCHL5 share a similar conserved sequence of UCHL family 1, there were some differences in this conserved sequence compared with the two closely related species of UCHL3, which may relate to their different evolutionary status. The phylogenetic tree revealed that the both Sp-UCHL3 and Sp-UCHL5 belonged to an independent subgroup with other arthropods individually, which suggested that the evolution Molecules 2018, 23, 213 9 of 15 of these two proteins is consistent with the evolution of their species. Most UCHs can catalyze the peptide or isopeptide bond at the ubiquitin C-terminus, rather than produce monomeric ubiquitin from substrate protein conjugates or disassemble polyubiquitin chains [51]. We found that the Sp-UCHL3 contains the active-site crossover loop of UCHL family 1, which allows binding ubiquitins attached to an unfolded peptide [47], and Sp-UCHL5 may be relevant to its catalytic activity and substrate specificity for cleaving isopeptide ubiquitin chains [52].
UCHL1 and UCHL3 have been reported to play an important regulatory role in oocyte maturation, but UCHL5 is rarely known [53,54]. In this study, we have found that the expression levels of Sp-uchl3 and Sp-uchl5 mRNAs in the ovary were remarkably higher than in other tissues examined. This phenomenon was inconsistent with the report that Sp-ub was ubiquitous in examined tissues [14], but coincided with the tissue expression profiles of Sp-cdc2 and Sp-cycb [13]. Ubiquitous expression of ubiquitin may be because ubiquitin is an important regulatory factor for many critical biological processes [55]. Furthermore, cdc2 and cycb are two important components of the maturation-promoting factor (MPF), which is a key regulator of controlling the G2/M phase transition in the meiotic maturation of oocytes and spermatocytes in animals [56]. Furthermore, the expression patterns in different ovary development stages showed that both Sp-uchl3 and Sp-uchl5 were highest in the O5 stage, and the second-highest expression level of Sp-uchl3 was in the O1 stage and significantly higher than in the O2 to O4 stages. However, there was no significant difference in Sp-uchl5 expression from the O1 to O4 stages. The former research reported that the activity of UCHs could influence cytokinesis on association with the oocyte cortex and meiotic spindle during oocyte maturation [57]. High mRNA expression of UCHs also was detected in the GV and MII stages of pig oocytes [58]. In fish [35] and amphibians [59], the UCHL1 mRNA was highly expressed in the ovary, especially in previtellogenic oocytes, suggesting that it may be important in oocyte growth and maturation. In the toad (Bufo bufo gargarizans), Sun et al. [34] found that tUCH plays an important role in regulating the germinal vesicle breakdown (GVBD) in oocyte maturation. In the mouse, Sekiguchi et al. [33] stated that UCH-L3 was mainly detected in the cytoplasm of mouse ovaries and strongly expressed throughout all stages of oogenesis, which suggested that it participated in the maturation of the oocyte. The rate of cell division in the crab ovary is significantly faster than in other tissues, especially in the process of mud crab ovary maturation. The diameter and number of oocytes increase rapidly [60], and the yolk is quickly accumulated in this stage, which needs a lot of enzymes to regulate intracellular proteins' constant synthesis and degradation in order to meet the requirements of completing cell division [61,62]. UCHs are important deubiquitinating enzymes which are involved in the regulation of numerous biological processes such as cell proliferation and growth, development, and transcriptional regulation [37]. Therefore, the expression patterns of Sp-UCHL3 and Sp-UCHL5 in different tissues and ovarian development suggest that they play important roles in cellular proteins' degradation during oogenesis and ovarian maturation.
Previous studies have demonstrated that the deubiquitinating enzymes play an essential regulatory contribution in spermatogenesis [63]. In humans, UCHL3 plays a pivotal role in the differentiation of spermatocytes into spermatids [64]. In mice, the researchers reported that UCHL3 and UCHL5 mRNAs are mainly expressed in spermatocytes and spermatids during spermatogenesis, and further studies had shown that UCHL3 may play an important role in meiosis that differentiates spermatocytes into spermatozoa, while UCHL1 is required for normal spermatogenesis and sperm quality control [65][66][67]. In the present study, Sp-UCHL3 mRNA expression in T3 was significantly higher than in the T1 and T2 stages (p < 0.05), while Sp-UCHL5 mRNA expressions in the three stages of testes development were not significantly different from each other (p > 0.05), indicating that Sp-UCHL3 and Sp-UCHL5 have different aspects in regulating mechanism of testis development. Furthermore, Sp-UCHL3-rather than Sp-UCHL5-may play a dominant important role in sperm maturation, whose phenomenon coincides with the former reports of mice [48,65].

Animals and Tissue Collection
S. paramamosain at different stages of the ovary and testis development was purchased from a local market of Jimei, Xiamen, China. Various tissues including ovary, muscle, heart, gill, brain, stomach, hepatopancreas, intestine and haemocytes from the mature female crab were immediately frozen in liquid nitrogen, stored at −80 • C for total RNA extraction. According to external morphology, color, gonadosomatic index (GSI) and histological feature [15,61], ovarian development was classified into five stages: proliferation (stage I), pre-vitellogenesis (stage II), primary vitellogenesis (stage III), secondary vitellogenesis (stage IV), and tertiary vitellogenesis (stage V). The male crabs were grouped into three stages: spermatocytes (stage I), spermatids (stage II) and mature sperms (stage III). Five crabs at each developmental stage were used for the experiments. All of the study design and animal experiments were conducted in accordance with guidelines of Jimei University's Animal Care and Use Committee (2011-59).

RNA Isolation and cDNA Synthesis
Total RNA was isolated from these tissues obtained above as described in the previous study [68], and then treated with RNase-free DNase I at 37 • C for 30 min to eliminate residual DNA contamination. For the first-strand cDNA, 3 µg of total RNA from each sample was reverse transcribed using oligo-dT-adaptor primer and M-MLV reverse transcriptase (Promega, Madison, WI, USA) at 42 • C for 90 min.

cDNA Cloning for Sp-uchl3 and Sp-uchl5
Based on the transcriptomics database from our laboratory, the partial cDNA sequences of Sp-uchl3 and Sp-uchl5 were obtained. The missing 5 and 3 sequences of Sp-uchl3 and Sp-uchl5 were obtained using a SMART TM -RACE cDNA amplification kit according to the protocol recommended by the manufacturer (BD Biosciences Clontech, Palo Alto, CA, USA). The amplified fragments were resolved by electrophoresis on the agarose gel, and were purified with a gel extraction kit (Generay, Shanghai, China), then inserted into pMD-19T vector (Takara, Beijing, China), propagated in E. coli (JM109) competent cells and sequenced. The open reading frames (ORFs) of Sp-uchl3 and Sp-uchl5 were confirmed by head-to-toe PCR with three different cDNA templates. We have designed the RACE primers of target genes according to the principle of SMART TM -RACE cDNA amplification. All the primers used in this experiment are listed in Table 1.

RT-qPCR
The mRNA distributions of both Sp-uchl3 and Sp-uchl5 in various tissues and in different stages of gonad development were analyzed by RT-qPCR, and these reactions were performed in the Applied Biosystems 7500 real-time system (ABI 7500) using SYBR green PCR Master Mix as recommended by the manufacturer (ABI). Primers used for RT-qPCR are listed in Table 1. Three separate individuals at least, in each gonad development stage and tissues distribution, were tested; all samples were repeated in triplicate for RT-qPCR analysis. The data of the expression levels of Sp-uchl3 and Sp-uchl5 were calculated using 2 −∆∆Ct that was normalized with 18S rRNA as described previously [13], and expressed as mean and standard error of the mean (SEM) of RQ value stated. Statistical analysis of the normalized CT values was performed with one-way analysis of variance and Student's t-test conducted using Statistical Package for the Social Sciences 13.0 (SPSS 13.0) software (IBM Corporation, New York, NY, USA). A significant difference was accepted at p < 0.05, or most significant at p < 0.01

Conclusions
In this paper, we have successfully identified the full-length cDNAs of Sp-uchl3 and Sp-uchl5, and demonstrated that the expression levels of Sp-uchl3 and Sp-uchl5 in the ovary were remarkably higher than in other tissues. Further analysis of the expression of these two genes in the gonad development showed that the expression of Sp-uchl3 was the highest in the O5 and O1 stages, whereas the expression of Sp-uchl5 in the O5 stage was the highest, and lower in the three stages of testis. These results suggested that the two genes of UCHs may play different roles in gonad development of the crab.