Characteristic of PGDS potential regulation role on spermatogenesis in the Chinese mitten crab Eriocheir sinensis

Gene 543 (2014) 244–252

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Characteristic of PGDS potential regulation role on spermatogenesis in the Chinese mitten crab Eriocheir sinensis Di-An Fang a,1, Quan-Zhong Yang b,1, Jin-Rong Duan a,1, Qun Wang c,⁎, Min-Ying Zhang a, Yan-Feng Zhou a, Kai Liu a, Wei-Gang Shi a,⁎ a b c

Scientific Observing and Experimental Station of Fishery Resources and Environment in the Changjiang River, Freshwater Fisheries Research Center, Wuxi, Shanshui Road 9, 214081, China Sanquan College, Xinxiang Medical University, Xinxiang, Henan 453003, China School of Life Science, East China Normal University, Shanghai 200062, China

a r t i c l e

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Article history: Received 19 November 2013 Received in revised form 1 April 2014 Accepted 3 April 2014 Available online 5 April 2014 Keywords: PGDS Spermatogenesis Eriocheir sinensis

a b s t r a c t Prostaglandin D synthase (PGDS) catalyzes the isomerization of PGH2 to produce PGD2 in the presence of sulfhydryl compounds. In this study, a full length PGDS gene comprising 1250 nucleotides from the Chinese mitten crab Eriocheir sinensis (Es-PGDS) was characterized, with a 615 bp open reading frame encoding 204 amino acid residues. Its deduced peptide has high homology with other species' PGDS protein. The Es-PGDS mRNA expression was tissue-related, with the highest expression observed in the hepatopancreas, accessory sex gland, testis and ovaries. We also detected the different stages of tissue expression and the enzyme activity for Es-PGDS in the testis and male crab hepatopancreas. The different expression patterns and its corresponding enzyme activity level indicated that PGDS is involving in the regulation of reproductive action during the period of rapid development in E. sinensis. Furthermore our research could arouse a heat debate on the PGDS reproductive function in invertebrate and further study will be needed to determine the molecular mechanism(s) linking PGDS functions to spermatogenesis and ontogenesis if this gene is to be exploited as a molecular biomarker in further studies of development. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Prostaglandins (PGs) are lipid-derived autacoids generated by sequential metabolism of arachidonic acid by the cyclooxygenase (COX) and prostaglandin synthase enzymes (Wu et al., 2012). These enzymes are named according to the prostanoid that they produce, such that prostaglandin D2 (PGD2) is synthesized by prostaglandin D synthase (PGDS), PGF2a by PGFS, PGI2 by PGIS and PGE2 by PGES (Narumiya et al., 1999). PGDS was discovered 40 years ago, and PGD2 has since proven to be a mediator of sleep (Giacomelli et al., 1996; Urade and Hayaishi, 1999; Urade et al., 1996), platelet aggregation (Brass et al., 1997; Morham et al., 1995), inflammation (Morham et al., 1995), muscle contraction (Cahill et al., 2001), and allergic asthma (Rutella et al.,

Abbreviations: PGDS, prostaglandin D synthase; GSH, glutathione; PGs, prostaglandins; PGD2, prostaglandin D2; COX, cyclooxygenase; L-PGDS, lipocalin-type PGD synthase; H-PGDS, hematopoietic PGD synthase; Es-PGDS, E. sinensis PGDS gene; sqPCR, semi-quantitative RT-PCR; qPCR, real-time quantitative PCR; RACE, rapid amplification of cDNA ends; Gp, gene-specific primer; NJ, neighbor-joining; ISH, in situ hybridization; OCT, optimal cutting temperature; DEPC, diethylpyrocarbonate; PBS, phosphate buffer saline; DIG, digoxigenin; NBT/BCIP, nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate; H&E, hematoxylin–eosin; GST, glutathione S-transferase. ⁎ Corresponding authors. E-mail addresses: [email protected] (Q. Wang), [email protected] (W.-G. Shi). 1 The first three authors contribute equally to the work.

http://dx.doi.org/10.1016/j.gene.2014.04.010 0378-1119/© 2014 Elsevier B.V. All rights reserved.

2009). PGDS is synthesized in many organs and has been implicated as a signaling molecule in the mediation or the regulation of various biological processes (Kanaoka et al., 1997). PGDS has been well characterized in mammals and divided into two types including lipocalin-type (L-PGDS) and hematopoietic PGD synthase (H-PGDS) respectively (Irikura et al., 2002; Urade and Hayaishi, 2000). L-PGDS is proved to be dominantly localized in the central nervous system and male genital organs of various mammals (Liu et al., 2007; Saleem et al., 2009; Urade et al., 1985), heart (Eguchi et al., 1997), and male genital organ (Tokugawa et al., 1998). H-PGDS previously termed as the spleen-type PGDS or glutathione (GSH)-requiring enzyme (Urade et al., 1985, 1987), contributes to the production of PGD2 in the peripheral tissues (Inui et al., 1999). The enzyme is localized in antigen-presenting cells and mast cells of a variety of tissues and is involved in the activation and differentiation of various cells including human germ cells (Kanaoka et al., 1997; Reddy et al., 1997; Takeda et al., 2006). Previous studies have shown that the PGDS gene is male specifically expressed in early stages of mouse gonadogenesis (Adams and McLaren, 2002). However, the structure and the regulatory mechanism of PGDS expression in male reproduction remain little knowledge in Decapoda and Arthropoda. The Chinese mitten crab (Eriocheir sinensis) is one of the most important commercially bred species that is native to China (Herborg et al., 2006; Sui et al., 2011; Tepolt et al., 2007). However, with the

D.-A. Fang et al. / Gene 543 (2014) 244–252

development of intensive culture systems, various problems such as sexual precocity have arisen in these populations. Precocious crabs mature and die early at a small size, causing catastrophic losses to farmers and seriously restricting the development of crab aquaculture (Wang et al., 2004, 2012). Sexual precocity results from the early development of sex glands, but the molecular mechanisms causing this phenomenon are still unclear (Jiang et al., 2009b; Kalinina et al., 2008). It is therefore important to gain a greater in-depth knowledge of the gonad developmental processes in crabs in order to formulate strategies to maintain normal growth of this species in aquaculture. In the current study, the E. sinensis PGDS gene (Es-PGDS) was characterized and its mRNA distribution in various tissues and temporal expression pattern in different gonad developmental phases were studied. Furthermore, the enzyme activity of Es-PGDS in the gonads and hepatopancreas was studied. The results showed that PGDS may play a key role in the spermatogenesis, and its further study involving sexual precocity would improve our understanding of the molecular regulation mechanism of reproductive and developmental biology in E. sinensis. 2. Materials and methods 2.1. Animals and tissue preparation Healthy adult crabs (280 ± 46 g for male; 220 ± 32 g for female, in October, 2012, sexually mature individuals in Stage III-2) were collected from Changjiang River, China. Crabs were placed in an ice-bath for 3–5 min until lightly anesthetized. Eight tissues were collected, including the heart, muscle, stomach, intestine, hepatopancreas, accessory sex gland, testis and ovaries, frozen immediately in liquid nitrogen, and stored at −80 °C until nucleic acid extraction. Based on the testis developmental classification of the mitten crab by Du et al. (1984), the mitten crab testis is divided into Stage III-1 (July to August), Stage III-2 (September to November), and Stage IV (December to January) respectively. For the dynamic change detail analysis, testis stages were further sub-divided based on the month of dissection: Stage III-1 (July), Stage III-1 (August); Stage III-2 (September), Stage III-2 (October), Stage III-2 (November); Stage IV-1 (December) and Stage IV-2 (January). Accordingly testes and hepatopancreas at different stages were collected for Es-PGDS relative expression and enzyme activity analysis respectively. 2.2. Isolation of total RNA and cDNA synthesis RNA was extracted using a RNA Extraction Kit (Axygen, USA) according to the manufacturer's protocol. RNA concentration and quality were estimated by spectrophotometry at an absorbance of 260 nm and agarose gel electrophoresis (Bio-Rad, USA), respectively. Total RNA (200 ng) was reverse transcribed using the PrimeScript™ RT-PCR Kit (TaKaRa, Japan) for semi-quantitative RT-PCR (sqPCR) or real-time quantitative PCR (qPCR). 2.3. PGDS gene isolation A partial sequence of Es-PGDS was obtained from the clone that had been isolated from the Chinese mitten crab testis cDNA library, which had been constructed by our laboratory (Jiang et al., 2009a). BLASTx analysis of EST sequences revealed that it has high identity to PGDS in Gallus gallus (NP_990342.1). Sequence of the assembled Es-PGDS contig was confirmed from the 3′ direction using a T7 primer (Table 1). To verify cDNA sequence, a gene-specific primer pair, PG-S (sense) and PG-R (anti-sense) (Table 1), was designed by Primer Premier 5.0 based on the sequence of the assembled contig mentioned above. The PCR reaction was performed in an ABI 2720 Thermal Cycler as the kit instruction (TaKaRa, Japan). Appropriately sized PCR products were gel-purified and ligated into a pGEM-T easy vector (Promega, USA) with T4 DNA

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Table 1 Sequences of primers. Primers

Sequence

Code

A pair gene-specific primer for verifying PGDS reverse primer 5′-TGCGGCAGCATCTTCCCTTCCA-3′ PGDS forward primer 5′-TCCCGCTGTTGGAACGCCTTGA-3′

PG-F PG-S

Gene-specific primers for cloning the full-length of PGDS cDNA Gene-specific primer pairs for 5′-ATCTTCCCTTCCACCTCCAGCACCG-3′ RACE 5′-GTGCTGGAGGTGGAAGGGAAGATGC-3′

Gp-5 Gp-3

Primers for RT-PCR, probe synthesis and qPCR Es-PGDS 5′ primer 5′-GCAGCATCTTCCCTTCCACCT-3′ Es-PGDS 3′ primer 5′-GCCACCACCACAACATCAACC-3″

Q-R Q-F

β-Actin primers for RT-PCR and qPCR β-Actin R 5′-GGGGTGTTGAAGGTCTCGGA-3′ β-Actin F 5′-CCTCACCCTCAAATACCCCAT-3′

β-R β-F

Primers for sequencing T7 SP6

T7 SP6

5′-TAATACGACTCACTATAGG-3′ 5′-ATTTAGGTGACACTATAGAA-3′

ligase. Positive clones containing inserts of predicted size were sequenced using T7 and SP6 primers (Table 1). 2.4. Rapid amplification of cDNA ends (RACE) The Es-PGDS cDNA sequence was extended using 5′ and 3′ RACE and a pair of gene-specific primer (Gp-5 & Gp-3; Table 1) based upon the verified EST sequence. The SMARTer™ RACE cDNA Amplification Kit (Clontech, USA) was selected according to the protocol for 3′ and 5′ RACE reactions. The PCR program was carried out at 5 cycles at 94 °C for 30 s and 72 °C for 3 min; 5 cycles at 94 °C for 30 s and 68 °C for 30 s, and 72 °C for 3 min; 20 cycles at 94 °C for 30 s, and 68 °C for 30 s, and extend at 72 °C for 3 min. PCR amplicons were separated and visualized on an ethidium bromide stained 1.2% agarose gel and amplicons of the expected size were purified, and cloned as described above. 2.5. Multiple sequence alignment and phylogenetic analysis The full-length multiple alignment of the PGDS sequence was compared with other species' PGDS. Amino acid sequences from various species in crustacean were retrieved from the GenBank and analyzed using the ClustalW 2.0 Multiple Alignment program. A neighbor-joining (NJ) phylogenetic tree was constructed using MEGA software version 4.0 (Tamura et al., 2007). The reliability of the branching was tested using bootstrap re-sampling (with 1000 pseudo replicates). 2.6. PGDS mRNA transcript expression patterns Tissue-dependent mRNA expression and testis developmental cycle quantitative analysis was conducted via quantitative PCR. First-strand cDNA was prepared as described above. qPCR primers (Q-R and Q-F, Table 1) were designed based upon the cloned Es-PGDS cDNA to produce a 208 bp amplicon. The primers β-actin F and β-actin R (β-F, β-R) were designed based on a cloned E. sinensis β-actin cDNA fragment to produce a 276 bp amplicon. PCR reaction was performed according to the PrimeScript Real-time PCR Kit protocol. Samples were run in triplicate, and normalized to the control gene β-actin. All PCR reactions were performed in triplicate using extracted RNA (pooled) of the same concentration. Es-PGDS expression levels were calculated by the 2−ΔΔCt comparative CT method (Livak and Schmittgen, 2001; Peirson et al., 2003). Mean and standard deviations were calculated from triplicate experiments, and presented as the n-fold differences in expression relative to β-actin. Data were analyzed using the CFX Manager™ software version 1.0.

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2.7. Enzyme assay for PGDS activity Testis tissues (disserted from 5 crabs, mature crabs in October, Stage III-2) were homogenized and pooled (total testis weight about 5 g) in a cuvette containing 0.83 mM glutathione, 5 μg/ml glutathione reductase, 0.50 mM NADPH, 5 mM EDTA, and 100 mM phosphate buffer solution (PBS, pH 7.6). PGDS activity was measured as previous method (Fujimori et al., 2006; Inui et al., 1999; Urade et al., 1985). Samples incubated with the enzyme at 25 °C for 1 min with [1-14C]PGH2 (final concentration of 40 μM, PerkinElmer, Boston, USA) in 50 μl of 100 mM Tris–Cl, pH 8.0, containing 1 mg/ml IgG and 1 mM dithiothreitol (DTT, Roche, China).

with hematoxylin–eosin (H&E). Specimens were photographed with Leica DM4000M optics. 2.10. Statistical analysis A multiple comparison (Duncan's) test was used to compare significant differences in Es-PGDS gene expression between control and tested samples using SPSS 15.0 software. A significant level of P = 0.05 was chosen. 3. Results 3.1. Characterization of Es-PGDS

2.8. Digoxigenin (DIG)-labeled riboprobe synthesis Testis cDNA was obtained as described above and two pairs of primers (Q-F, Q-, Table 1) were designed to amplify a 208 bp Es-PGDS cDNA fragment. PCR was performed at 94 °C for 5 min followed by 30 cycles of 94 °C for 30 s, 58 °C for 30 s, 72 °C for 1 min followed by 72 °C for 10 min. The amplified target fragments were extracted, purified, and cloned as described above. Positive clones were selected using DNA sequencing and extracted using the AxyPrep Plasmid Miniprep Kit (Axygen, Union City, CA, USA), linearized by digestion with SpeI (TaKaRa) and purified. The 20 μl riboprobe transcription mixture included 8 μl, 120 ng/μl linearized plasmid template, 2 μl 10× NTP labeling mixture (10 mM ATP, 10 mM CTP, 10 mM GTP, 6.5 mM UTP, 3.5 mM DIG-11-UTP, pH 7.5), 2 μl 10× transcription buffer, 1 μl 20 U/μl protector RNase inhibitor, 2 μl RNA polymerase SP6 (20 U/μl, Roche), and 5 μl RNase-free water. The riboprobe synthesis reaction was carried out for 2 h at 37 °C in a water bath, then template cDNA was digested using RNase-free DNaseI (10 U/μl) for 20 min at 37 °C. The reaction was stopped by addition of 2 μl 0.2 M EDTA (pH 8.0), riboprobes were precipitated with 75 μl pre-chilled 100% ethanol and 2.5 μl 4 M LiCl at − 20 °C for 2 h, centrifuged at 13,000 g for 20 min, washed with prechilled 70% ethanol, centrifuged at 13,000 g for 5 min, dried at room temperature for 10–15 min and dissolved in 50 μl DEPC-water. Samples of 2 μl were used for agarose electrophoresis and spectrophotometric quantification. 2.9. In situ hybridization (ISH) Testes were dissected from the mature crabs as above described and fixed in 4% paraformaldehyde in phosphate buffer saline (PBS, pH 7.4) at 4 °C overnight. After washing with PBS three times, the samples were dehydrated in 30% saccharose-PBS solutions for 4 h at room temperature then embedded in Tissue-Tek optimal cutting temperature (OCT) compound (Sakura, USA). Standard frozen sections were obtained at 8 μm in thickness with a microtome (Leica, Germany). Sections were washed with diethylpyrocarbonate (DEPC)-treated PBS three times and then treated twice with PBT (PBS containing 0.2% Tween 20) for 5 min each. Subsequently, the sections were permeabilized with proteinase K in DEPC-treated PBT (5–20 ng) at 37 °C for 10–20 min, treated twice with PBS containing 100 mM glycine for 5 min each. The sections were post-fixed with PBT containing 4% paraformaldehyde for 5 min. Pre-hybridization was performed by incubating with hybridization buffer containing 5 × SSC (0.75 M NaCl, 0.05 M sodium citrate), 50% formamide, 0.02% (w/v) sodium dodecyl sulfate and 2% blocking reagent (Boehringer-Mannheim, Germany) at 68 °C for 10 min. The sections were incubated with hybridization buffer containing 50 ng of DIG-labeled Es-PGDS RNA probe at 68 °C for 12–16 h. The hybridized sections were washed three times with 1 × SSC and formamide at 55 °C for 20 min each and then twice with 1× SSC for 15 min each. Detection was performed using anti-DIG-alkaline phosphatase and nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate (NBT/BCIP) as chromogens. For general histology, the frozen testis sections were stained

The cDNA and deduced amino acid sequences were submitted to GenBank under the accession number HM231278.1. The nucleotide sequence of the Es-PGDS gene was comprised of 1250 bp with a 615 bp open reading frame encoding 204 amino acid residues. The initiation ATG codon was indicated at position 137 of the 5′-UTR, and the TAG stop codon at position 751 of the Es-PGDS sequence. The 3′-UTR was shown to contain 499 bp with a 30 bp poly-A signal tail (Fig. 1). The encoded polypeptide was predicted to have a molecular weight of approximately 23.4 kDa and an isoelectric point of 7.75. The predicted polypeptide was submitted to GenBank with the accession number AEF32709. Using InterPro searches, two superfamily specific domains were identified in the Es-PGDS protein sequence (Figs. 1 and 2). It includes GST_N sigma like and GST_C sigma like specific hits which belongs to thioredoxin-like and GST_C family superfamily respectively. Potential phosphorylation sites were identified in Es-PGDS using the Prosite scan provided by PBIL (http://pbil.univ-lyon1.fr/pbil. html) (Gouy and Delmotte, 2008). Es-PGDS contained 5 potential casein kinase II phosphorylation sites and two protein kinase C phosphorylation sites. Blast analysis revealed that Es-PGDS shared high similarity with other species' PGDS, including those from Salmo salar (43%), Meleagris gallopavo (42%), Taeniopygia guttata (42%), Gallus gallus (42%), Rattus norvegicus (41%), Mus musculus (40%), Hymenochirus curtipes (39%) and Otolemur garnettii (39%). Es-PGDS shared N35% identity with PGDS from certain other species and shared N30% identity with glutathione S-transferase (GST) protein. The phylogeny of the GST family is shown in Fig. 3. In the phylogenetic tree, Es-PGDS formed a subcluster with members of the GST required PGDS from the Cyprinidae (Fig. 3). 3.2. Expression patterns of Es-PGDS transcripts The mRNA transcript of Es-PGDS was expressed universally in all the tissues investigated, including the heart, muscle, stomach, intestine, hepatopancreas, accessory sex gland, testis and ovaries (Fig. 4). Expression was greater in the hepatopancreas and testes, with lower levels in the other tissues and the lowest level found in the muscle. Temporal expressions of Es-PGDS in hepatopancreas, testes and ovaries during the crab reproductive cycle were measured using qPCR with β-actin acting as the internal control. For both Es-PGDS and β-actin genes, there was only one peak at the corresponding melting temperature in the dissociation curves, indicating that the PCR was specifically amplified. The temporal expression of the Es-PGDS mRNA transcripts is presented in Fig. 5. During the developmental cycle in the testes, expression of Es-PGDS mRNA in testes was up-regulated during the developmental cycle; high expression was maintained during the spermatogonial multiplication period and peaked at the sperm mature stage (Stage IV). Es-PGDS expression in hepatopancreas increased and was significantly high at Stage III-1 and Stage III-2 (Aug. and Oct.–Nov.), with peak expression being reached in October, which then decreased from Stage III-2 (Nov.) to Stage IV (Dec. and Jan.). Expression of PGDS mRNA in the ovary was low at the early development stage and the rapid growth stage, reached the highest levels in the mature growth

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Fig. 1. The full-length cDNA and deduced amino acid sequence of Es-PGDS. The GST_N sigma like thioredoxin domain (4–86 aa) is shown in underline and GST_C sigma like specific hits (83–190 aa) are shown by dotted-line. The potential casein kinase II phosphorylation sites are shown in bold and two protein kinase C phosphorylation sites are shown in rectangle.

ovary periods (Stage IV-1) and (Stage IV-2), and then gradually decreased to low levels.

Furthermore, the PGDS activity in the testis was relatively higher than in the ovary (Fig. 6).

3.3. The PGDS activity 3.4. Localization of Es-PGDS mRNA The PGDS activity in hepatopancreas and gonads of Chinese mitten crab was differentially regulated along the development stages of testis (Fig. 6). The PGDS activity both in hepatopancreas and gonads increased rapidly and significantly from Stage III-2 to Stage IV. Interestingly in the Stage III the PGDS activity in hepatopancreas was significant higher than that in the gonads (P b 0.05) while in the Stage IV it was lower than that in the testis. The hepatopancreas and gonad PGDS activity was peaked in November (Stage III-2) and in December (Stage IV-1) respectively.

Localization of Es-PGDS mRNA was studied by ISH. No signal was observed with the negative control sense strand probe (Fig. 7B) when hybridized with the sense PGDS probe. However, the antisense probe gave a positive signal in the sperm cytoplasm of different developmental phases and in the nucleoli located in the periphery of the nucleus (Fig. 7D). In the mature testes the antisense probe gave a strong signal both in the nucleoli and in the cytoplasm (Fig. 7F).

Fig. 2. Comparison of deduced amino acid sequence of PGDS proteins. Identical (*) and similar (. or :) amino acid residues are indicated. Gaps (−) were introduced to maximize the alignment.

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Fig. 3. Phylogenetic analysis of Es-PGDS and other subfamily proteins. A maximum likelihood phylogenetic tree was constructed using MEGA software version 4.0, the reliability of the branching was tested using bootstrap re-sampling (with 1000 pseudo replicates). Es-PGDS clusters in the GSH-requiring subfamily. The putative protein GenBank accession number is shown in parentheses.

4. Discussion PGDS converts PGH2 to PGD2, and the enzyme is also a δ class glutathione transferase (Jowsey et al., 2001; Kanaoka and Urade, 2003; Kanaoka et al., 1997). PGDS was originally found in the rat spleen (Helliwell et al., 2004) and later in other tissues including the testis and ovaries (Helliwell et al., 2004; Wilhelm et al., 2007). PGD2 is known to act via a well-characterized cell membrane G-protein coupled receptor. In zebrafish, the expression pattern suggests that the male

Fig. 4. Expression of Es-PGDS in E. sinensis adult tissues. 1: heart; 2: muscle; 3: stomach; 4: intestine; 5: hepatopancreas; 6: accessory sex gland; 7: testis; 8: ovaries.

specific PGDS/PGD2 mechanism ensures Sertoli cell differentiation and is not an early sex marker (Fujimori et al., 2006; Jowsey et al., 2001). In human, the PGDS gene has response elements for thyroid hormone, estrogen and glucocorticoids (Eguchi et al., 1997). There is evidence that PGDS may function in the control of cell mitogenesis/regulation in human spermatogenesis (Tokugawa et al., 1998). Biochemical and immunohistochemical studies demonstrated that PGDS is abundantly expressed in mast cells in various adult rat tissues including the thymus, stomach, skin, and small intestine (Leone et al., 2002). Mammalian PGDS carries a secretion signal sequence at their amino-terminal portion (Uematsu et al., 2002; Urade and Hayaishi, 2000), and the aminoterminal end of the mature (secreted) protein of mouse PGDS was determined as Asp25 (Chen et al., 2010; Helliwell et al., 2004). Blast analysis revealed the existence of a hydrophobic region in the aminoterminal portion PGDS like that in zebrafish, chicken and mouse PGDS (Fujimori et al., 2006; Jowsey et al., 2001), and the amino-terminal ends for the crab proteins were predicted to be Leu83 and His84, respectively (Figs. 1 & 2). It was speculated that PGDS would act as a carrier of essential molecules for the brain (Koibuchi and Chin, 2000), retina (Heird et al., 1997), fetus (Heird et al., 1997; Sohmer and Freeman, 1996), and sperm development (Sorrentino et al., 1998). In this study, Es-PGDS primary structure (Fig. 1) did not find the essential Cys residue for PGDS activity which is found in mammalian PGDS (Tippin et al., 2012; Urade et al., 1985, 1987). In addition, a partial sequence of the rainbow trout PGDS homologue also does not contain this corresponding Cys residue (Bayne et al., 2001). We suspect that

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Fig. 5. Relative expression of Es-PGDS in hepatopancreas and testes during the testis developmental cycle. The testis developmental cycle can be divided into the following stages: Stage III-1 (July), Stage III-1 (August); Stage III-2 (September), Stage III-2 (October), Stage III-2 (November); Stage IV-1 (December) and Stage IV-2 (January). The bars with different letters are significantly different (P b 0.05, a b b b c; n = 3).

substitution of several amino acid residues including the Cys residue essential for PGDS activity in mammalian PGDS is needed to gain the enzymatic activity. Using InterPro searches and NCBI blast analysis, it is found that EsPGDS belongs to GST superfamily that share both C-terminal domain and N-terminal domain with significant homology to several eukaryotic and prokaryotic PGDS (Helliwell et al., 2004; Irikura et al., 2002). Members of this protein family are cytoplasmic associated and implicated in cellular processes, comprising an N-terminal hydrophobic transmembrane domain and a C-terminal domain (Fujimori et al., 2006; Kanaoka et al., 1997; Liu et al., 2007). In fact, the amino-terminal portion of zebrafish PGDS was not well conserved (23.8–25.6%), whereas the Cterminal segments were better preserved (Chen et al., 2010; Fujimori et al., 2006). The chimeric protein study demonstrated the importance of the amino-terminal portion for acquiring the PGDS activity, which is consistent with a result that the several amino acid residues around the Cys65 are also important for PGDS activity (Kanaoka and Urade, 2003). To understand the evolutional relationship between mammalian and non-mammalian PGDSs in the GSH family, we carried out the phylogenetic analysis shown in Fig. 3. Es-PGDS homologue was separated from other PGDS gene family proteins, and assigned to the same

group with GSH-required PGDSs. The GSTs were previously reported in invertebrates, such as insects, cephalopods, flukes, and nematodes. Thus, Es-PGDS is a novel invertebrate homolog of the GST-required family. Expression profiles of PGDS in chicken, mouse, and human were not preserved except for high-level expression in the heart and testes (Chen et al., 2010; Fujimori et al., 2006). The present results are in good agreement with previous studies demonstrating the high-level expression of mammalian PGDS genes in the brain and testes (Shimamoto et al., 2012; Urade and Hayaishi, 2000). Thus the transcriptional regulatory mechanisms of the PGDS gene in the genital organs are different between non-mammalians and mammalians (Irikura et al., 2002; Leone et al., 2002; Urade and Hayaishi, 2000; Urade et al., 1985). In E. sinensis, EsPGDS transcripts were detected in all the tissues that were examined (Fig. 4). The wide distribution of Es-PGDS among different tissue types may result from its fundamental role in many biological functions (Tippin et al., 2012). Further, the qPCR data showed that the Es-PGDS mRNA transcripts were present in both the testis and ovary throughout the whole reproductive cycle of E. sinensis. A significant increase in the expression of Es-PGDS in testis tissue was increased from Stage III-2 (October) and peaked at Stage IV-2 (January). Under natural conditions, September to October is the period in which the testes of E. sinensis

Fig. 6. The Es-PGDS activity in the hepatopancreas and testis in response to the period of reproductive activity. The testis developmental cycle can be divided into Stage III-1 (July to August), Stage III-2 (September to November), and Stage IV (December to January) respectively. The bars with different letters are significantly different (P b 0.05, a b b b c; n = 5).

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Fig. 7. Hematoxylin–eosin staining and in situ hybridization with DIG-labeled sense and antisense RNA probes in the adult testis. (A) Hematoxylin–eosin staining of the testis. (B) In situ hybridization of the testis showing no Es-PGDS expression in the testis with the control probe. (C) High magnification of spermatocyte staining with H&E. (D) Positive Es-PGDS expression in the cytoplasm of spermatocyte. (E) Spermatid staining with H&E. (F) Es-PGDS expression in the cytoplasm, especially around the nuclear envelope and in the nucleoplasm of spermatid. CT: collecting tubules; St: spermatocyte; Sp: spermatid. Magnification 100× for A and B; 200× for C and 400× for D, E and F. Scale bars: A, B = 20 μm; C = 10 μm; D = 20 μm; E, F = 40 μm. Arrows show hybridization signals of Es-PGDS transcripts.

undergo rapid development (Du et al., 1984), which supports the changing trend in Es-PGDS expression in testes. These observations indicate that E. sinensis must consume a large amount of PGDS during this period to meet the specific physiological requirements of reproduction. Further the mRNA expression level in the ovary was lower than in the testis during the whole reproductive cycle. This result could support that PGDS may play a critical reproduction function in male E. sinensis (Leone et al., 2002). In testes, a higher Es-PGDS transcript synthesis can be detected during spermatogonial developmental and multiplication phases (Fig. 5, Oct. to Nov.), which suggests that Es-PGDS may be a promoter region for spermatogonial multiplication and spermatogenesis (Adams and McLaren, 2002). The highest level of Es-PGDS mRNA observed in the sperm mature period may imply that Es-PGDS is a kind of protein products of spermatogenesis in the crab mitten, which would support its enhancement function of cell proliferation and regulation of sperm metabolism in invertebrates (Tippin et al., 2012). Interestingly, the EsPGDS mRNA transcript expression patterns are different in hepatopancreas. Es-PGDS expression in hepatopancreas was detected with three peaks at Stage III-1 and Stage III-2 (Aug. and Oct.–Nov.) respectively,

with the highest expression at Stage III-2 in October. Surprisingly, the expression patterns in testes and in hepatopancreas were completely opposite (Fig. 5). This interesting result could indicate that the PGDS protein was transported from hepatopancreas to testes so as to meet the reproductive need during the stages of gonad fast maturation, leaving a deficit in the hepatopancreas. Once the reproductive stages have passed, the crab would again store PGDS in the hepatopancreas to meet the following physiological demands of reproduction. Furthermore, the expression level is lower in the ovary indicating the more important relevance between PGDS and spermatogenesis biology. Taken together, dramatic transformations and sperm cellular differentiation take place in all these development periods, so spermatogenesis is associated with dynamic expression levels of Es-PGDS. Expression results of mRNA should be complemented by the enzyme activities to provide a better understanding of the enzymatic and transcriptional regulations (Wu et al., 2012). It was well-characterized that PGDS is involved in normal metabolism and function (Chen et al., 2010; Tippin et al., 2012). Such complexity in GST-dependent metabolism requires specific mechanisms for the control of the expression of these proteins (Saleem et al., 2009; Tippin et al., 2012). The results of the PGDS

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activity were not coincident with the change trend of the PGDS expression level in the testis, but the PGDS activity change trend in the hepatopancreas after Stage III-2 (November) was consistent with the PGDS expression pattern. As we all know, expression of a gene can be controlled at many levels, including transcription, mRNA splicing, translation and post-translational events (Shimamoto et al., 2012; Tippin et al., 2012). No matter how different, the dynamic expression of Es-PGDS transcript and protein level in both the testicular and hepatopancreas tissues strongly indicated that a strong relationship exists between it and the gonad function. In both mice and chicken embryos the expression of PGDS is initiated in the male gonad whereas PGDS is absent in the female gonads for several days before expression also can be detected in the female gonads (Liu et al., 2007; Saleem et al., 2009; Urade et al., 1985). Expression of PGDS RNA was detected in the crab by ISH in the gonads. The results revealed that PGDS transcripts mainly localized to the periphery of the nucleus membrane in the spermatocyte and spermatid (Fig. 7), which shows that the transcripts are abundant in male germ cells from the beginning of the meiotic phase until the end of the post-meiotic phase (Rutella et al., 2009). These findings are in agreement with data from other species and suggest that the PGDS is primarily needed at the initial steps of spermatogenesis (differentiation of spermatogonia and spermatocytes), during which most of the cell divisions occur (Liu et al., 2007; Saleem et al., 2009; Urade et al., 1985). The presence of PGDS in the stages associated with high mitotic activity (i.e. spermatogonia and spermatocytes) suggests that there exists very active cytoplasmic protein assembly machinery that generates further proteins needed for cell division (Fujimori et al., 2006). It has been suggested that PGDS regulates microfilament organization in a manner dependent on its phosphorylation and oligomeric status (Uematsu et al., 2002; Urade and Hayaishi, 2000). Therefore, it is reasonable to speculate that Es-PGDS could regulate mitosis and meiosis in crustacean gonad development. In conclusion, our study provided quantitative evidence supporting that Es-PGDS may play a key role in the regulation of spermatogenesis during the period of rapid gonadal development in E. sinensis, and indicates that the testis and hepatopancreas tissues are involved in the absorption and utilization processes of PGDS. Our present study also provides an opportunity to elucidate the comparable transcriptional regulatory mechanism involved in the tissue or stage-specific expression, especially in the hepatopancreas and testes during spermatogenesis. In the future, the molecular mechanism(s) linking PGDS functions involving sex differentiation and ontogenesis such as sexual precocity need to be determined in further studies of reproductive biology in crustacean. Conflict of interest None. Acknowledgments This study was supported by grants from the Public Welfare Agricultural Scientific Research (No. 201203065), the Basic Research Funds from Freshwater Fisheries Research Center (No. 2013JBFR02), and the National Infrastructure of Fishery Germplasm Resources (No. 2014DKA3047003). References Adams, I.R., McLaren, A., 2002. Sexually dimorphic development of mouse primordial germ cells: switching from oogenesis to spermatogenesis. Development 129, 1155–1164. Bayne, C.J., Gerwick, L., Fujiki, K., Nakao, M., Yano, T., 2001. Immune-relevant (including acute phase) genes identified in the livers of rainbow trout, Oncorhynchus mykiss, by means of suppression subtractive hybridization. Developmental and Comparative Immunology 25, 205–217.

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