Rab2 and Rab6 are Implicated in Acrosome Formation during Spermatogenesis in Eriocheir sinensis : Based on Sperm Proteome

Background : Rab proteins are GTP-dependent small proteins that function as regulators of intracellular vesicle transport, fusion, and localization. However, few studies have investigated their function in Decapoda reproduction. The Eriocheir sinensis sperm has no tail and the nuclei are uncondensed. With the acrosome forming the majority of the sperm mass, it provides an ideal model for studying acrosome formation. Methods : We firstly analyzed the sperm proteome using LC-MS/MS. To study the functions of Rab2 and Rab6, related to the Golgi apparatus, in the acrosome formation during spermatogenesis, the genes of Rab2 and Rab6 were cloned based on the testis transcriptome of E.sinensis and poly-clonal antibodies were prepared. The presence of 2 Rab proteins was confirmed in the testis and sperm by western blot. We further observed the characteristics of target 2 Rab proteins using immunofluorescence (IF). Results : A total of 1247 proteins including 7 Rab proteins, Rab1, Rab2, Rab5, Rab6, Rab11, Rab14, and Rab18 were identified in the sperm proteome. The IF results showed that Rab2 co-localizes with GM130, a cis-Golgi matrix protein, in the spermatagonia and spermatocytes. In the early spermatids, Rab2 and Rab6 participate in the formation of pre-acrosomal vesicles. In maturing spermatids, both Rab2 and Rab6 settle on the acrosomal membrane but present different characteristics wrapping the pre-acrosome. In the mature sperm, Rab2 localizes in the perinuclear theca surrounding the nuclei cup, while Rab6 remains on the acrosomal membrane. Conclusions : Our research found 7 Rab proteins based on the analysis of the sperm proteome in E.sinensis , and confirmed the involvement of Rab2 and Rab6 in acrosome formation. These findings provide a foundation for studying the functions of Rab proteins during spermatogenesis in Decapoda animals.


Introduction
Rab proteins are small molecules dependent on guanosine 5'-O-(γ-thio)triphosphate (GTPs) that belong to the Ras superfamily.Over 70 human Rab proteins have been identified [1].The major functions of Rab proteins include managing the cell inner membrane, such as vesicle transport, localization, and fusion [2].In addition, Rab proteins are involved in the maintenance of the morphological function of mitochondria [3], the endoplasmic reticulum [4], lysosomes [5], and other membranous organelles.It has been proposed that around one-third of the Rab proteins are related to the Golgi apparatus [6].The acrosome is a unique membranous organelle that exists in mature sperm and most organelles including the Golgi apparatus are essential for acrosome formation.Until now, 12 Rab proteins, have been found to be involved in male meiosis [7], but only Rab2 [8], Rab3 [9], Rab5 [10], Rab6 [11], Rab7 [12], and Rab27 [13] have been reported to participate in acrosome formation during spermatogenesis.
Rab2 is essential for pre-Golgi trafficking [14], while Rab6, as the most abundant Golgi-associated Rab protein, is involved in Golgi transport [15][16][17].Previous studies have identified Rab2 in the acrosomal matrix of mouse sperm [18].Bae et al. [19] found that Rab2 is mainly located in the midpiece of the mouse sperm and is present during spermatogenesis.Lo et al. [20] found Rab2 plays a significant role in protein delivery to the flagellum in human sperm.Rab6 is involved in all the stages of spermatogenesis and finally settles in the acrosomal inner membrane in the mature mouse sperm [11].However, the roles of Rab2 and Rab6 during spermatogenesis in crustaceans remain unknown.Rab proteins have also been reported to be involved in biological processes such as endocytosis [21], autophagy [22], and organelle interactions [23].Few studies have investigated Rab proteins in crustacea reproduction.Filling these gaps in knowledge will help to better understand the functions and mechanisms of these Rab proteins.
The sperm proteome has been reported in many mammals including humans [24], mice [25], rats [26], and dolphins [27].The proteome of other aquatic animals including the giant grouper [28] and carp [29] has been reported, but information regarding Decapoda species like shrimp and crab is limited.As a Decapoda animal, E.sinensis (Chinese mitten crab) distribute over a wide area globally.The sperm in E.sinensis is unique with a large acrosome surrounded by the uncondensed nuclear cup.Although Eriocheir sinensis sperm have no tail, the apical cap, acrosomal tubule, fibrous layer, lamellar structure, and middle layer are obvious [30].The unique sperm characteristics of E.sinensis make it an ideal experimental model for studying acrosome formation.

Animals
Male E.sinensis were purchased from Panjin, Liaoning province, China.No official approval was required for this study of the crab in China.
Balb/c female mice, used for antibody production, were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd (Beijing, China) and housed in an SPF animal house (constant temperature of 22-25 °C, relative humidity of 40-70%).Male rabbits, used for antibody production, were from Animal Experiment Center of Hebei University, and were provided food and water ad libitum.Experimental protocols were approved by the Institutional Animal Care and Use Committee of Hebei University.The mice and rabbits were managed per the guidelines for Animal Care and Use of Hebei University.

SDS-PAGE of Sperm Protein
Seminal vesicles and accessory glands were obtained from mature male crabs frozen on ice for 15 min before dissection.The spilled spermatophores from seminal vesicles were washed twice using precooled (4 °C) Ca 2+ -free artificial seawater (FASW)-(475 mM NaCl, 12 mM KCl, 30 mM MgCl 2 •6H 2 O, 20 mM Tris, pH 8.2).The liquid was discarded using a straw after spermatophores precipitated on ice.Proteins from the accessory gland were collected after homogenization and centrifugation at 4 °C, 12,000 g, and 5 min to digest the spermatophores.The crude sperm were released within 15 min; all the components were transferred to a centrifuge tube.The supernatant containing released sperm was collected after centrifugation at 4 °C, 200 g, 10 min.After washing 3 times with FASW and centrifugation at 4 °C, 850 g, 10 min, the pure sperm were obtained.
Proteins from crab sperm were extracted on ice using 8 M urea lysate (8 M urea, 3 µM total sialic acid, and 50 mM nicotinamide).Phenylmethylsulfonyl fluoride (PMSF) was added before use (final concentration 1 mM). 1 mL of precooled urea lysate was added to 50 mg sperm and incubated for 5 min on ice, then ultrasonication was conducted at 30% power, 2 s on, 3 s off, for 2 min using an ultrasonic processor (Scientz, Ningbo, China).Then, the proteins were obtained via centrifugation at 4 °C, 12,000 g for 20 min, with the sperm proteins ultimately collected from the supernatant.Proteins were stored at -80 °C after measuring their concentration with a BCA Kit ( P0012S, Beyotime, Beijing, China).
The proteins were mixed with loading buffer (SL1170-1 mL, Coolaber, Beijing, China) and denatured at 100 °C for 5 min.A total of 20 µg of each sample was separated in 15% polyacrylamide gels with 5% stacking gel for SDS-PAGE and the gels were stained for protein with Coomassie Brilliant Blue R-250 (CBB).

LC-MS/MS
LC-MS/MS analyses of sperm proteins were conducted at Jingjie Company (Hangzhou, China).We summarized the steps as follows.The proteins were separated using 10% polyacrylamide gels and SDS-PAGE was finished when the protein migrated 1 cm from the stacking gel.After staining by CBB and then a destaining buffer, the gels were cut into small granules on clean glass.For digestion, the protein solution was reduced with 5 mM dithiothreitol for 30 min at 56 °C and alkylated with 11 mM iodoacetamide for 30 min at room temperature in darkness.The protein sample was then diluted by adding 100 mM triethylammonium bicarbonate (TEAB) to a urea concentration less than 2 M. Finally, trypsin was added at 1:50 trypsin-toprotein mass ratio for the first digestion overnight and 1:100 trypsin-to-protein mass ratio for a second 4 h-digestion.Finally, the peptides were desalted by C18 solid phase extraction (SPE) column.
The desalted and lyophilized peptides were reconstituted with 0.1% formic acid (FA) which contains 2% acetonitrile and loaded onto a home-made reversed-phase analytical column (25-cm length, 75/100 µm i.d.).Peptides were separated with a gradient from 6% to 24% solvent B (0.1% formic acid in acetonitrile) over 70 min, 24% to 35% in 14 min, and climbing to 80% in 3 min then holding at 80% for the last 3 min, all at a constant flow rate of 450 nL/min on a nanoElute Ultra-high-pressure liquid chromatography (UHPLC) system (Bruker Daltonics, Bremen, Germany).
The peptides were subjected to a capillary source followed by the timsTOF Pro (Bruker Daltonics) mass spectrometry.The electrospray voltage applied was 1.60 kV.Precursors and fragments were analyzed at the TOF detector, with an MS/MS scan range from 100 to 1700 m/z.The timsTOF Pro was operated in parallel accumulation serial fragmentation (PASEF) mode.Precursors with charge state 0 to 5 were selected for fragmentation, and 10 PASEF-MS/MS scans were acquired per cycle.

Database Search
The resulting MS/MS data were processed using the MaxQuant search engine (v.1.6.15.0, http://www.maxquant.org/).Tandem mass spectra were searched against the transcriptome of testis and sperm connected with downloaded data that reported data in the SwissProt database on E.sinensis.Reverse decoy databases were used to guarantee the correction rate.Trypsin/P was specified as the cleavage enzyme allowing up to 2 missing cleavages.The mass tolerance for precursor ions was set as 20 ppm in the first search and 5 ppm in the main search, and the mass tolerance for fragment ions was set as 0.02 Da.Carbamidomethyl on Cys was specified as a fixed modification, and acetylation on protein N-terminal and oxidation on Met were specified as variable modifications.The false discovery rate (FDR) was adjusted to <1%.

GO Annotation
Gene Ontology (GO) annotation proteome was derived from the UniProt-GOA database (http://www.ebi.ac.uk/GOA/).Firstly, the identified protein ID was converted to UniProt ID and then mapped to GO IDs by the protein ID.If some identified proteins were not annotated by the UniProt-GOA database, the eggNOG-mapper (v2.0 https://github.com/eggnogdb/eggnog-mapper)was used to annotate the protein's GO function based on the protein sequence alignment method.Then proteins were classified by GO annotation based on three categories: biological process, cellular component, and molecular function.

Subcellular Localization
The cells of eukaryotic organisms are elaborately subdivided into functionally distinct membrane-bound compartments.Some major constituents of eukaryotic cells are the extracellular space, cytoplasm, nucleus, mitochondria, Golgi apparatus, endoplasmic reticulum, peroxisome, vacuoles, cytoskeleton, nucleoplasm, nucleolus, nuclear matrix and ribosomes.There, we used wolfpsort a subcellular localization prediction software to predict subcellular localization.
Rab6 primers were designed with NdeI (underlined) at the forward primer.
Pet-19T-Rab2 and Pet-19T-Rab6 were used as the template using the above primers for PCR.The products were digested with BamH I and XhoI for Rab2, and NdeI and XhoI for Rab6.Rab2 was incorporated in the Pet-28b vector while Rab6 was incorporated in the Pet-21b vector.Recombinant clones for expression are named Pet-28b-Rab2 and Pet-21b-Rab6.All were validated via sequencing by the Sangon Company (Shanghai, China).

Preparation of Rab2 and Rab6 Polyclonal Antibodies
Balb/c female mice aged 6 weeks were habituated for 1 week.50 µg Rab2 polypeptide antigen mixed with 50 µg Freund's complete adjuvant (lot number: F5881; Sigma, St. Louis, MO, USA) was injected into the back of the mice for the first immunization.10 days later, a second immunization was performed by emulsifying half of the antigen polypeptide with an equal volume of Freund's incomplete adjuvant (lot number: F5506; Sigma, St. Louis, MO, USA) using intraperitoneal injection.The health status of each animal was observed after each injection.The last two immunizations were prepared as the second injection and injected once weekly.Three days after the last immunization, blood was collected from the eye socket with a glass capillary and the serum was extracted.Anti-Rab2 serum was stored at -80 °C.
Rab6 polyclonal antibodies were prepared similarly, but by using rabbits instead of mice for more anti-serum.Two male New Zealand White rabbits (2 ± 0.3 kg) were housed in 2 pens (1.0 × 0.7 m 2 ) individually at the Animal Experiment Center of Hebei University.A week later, each rabbit was immunized with 0.5 mg of the Rab6 polypeptide mixed with Freund's Complete Adjuvant (lot number: F5881; Sigma, St. Louis, MO, USA) via multi-point subcutaneous injection.The last 3 immunization were performed every 10 days using Freund's Incomplete Adjuvant (lot number: F5506; Sigma, St. Louis, MO, USA), delivered via intraperitoneal injection.Three days after the last immunization, blood was collected from the ear vein and serum was separated.Anti-Rab6 serum was stored at -80 °C.

Enzyme-Linked Immunosorbent Assay (ELISA)
Anti-Rab2 antiserum was collected after centrifugation (12,000 g, 10 min, 4 °C) and quantified by indirect ELISA using the above purified Rab2 as the antigens.The optimal dilution (2 µg/mL) of recombinant protein was used to coat the 96-well plate, and the samples were blocked with 100 µL 10% BSA diluted in washing buffer (0.0015 M KH 2 PO4, 0.008 M Na 2 HPO 4 ꞏ12 H 2 O, 0.14 M NaCl, 0.003 M KCl, 0.05% v/V Tween-20) for 2 h at room temperature.The primary antibody anti-Rab2 serum and preimmune serum were diluted (1:100 to 1:102,400).A total of 100 µL goat to mouse IgG with conjugated HRP (dilution 1:1000, Solarbio, Beijing, China) was added and incubated at 37 °C for 1 h.After washing three times the reaction termination liquid (10% v/V H 2 SO 4 ) was added.Anti-Rab6 antiserum was similarly detected.The difference is that the second polyclonal goat anti-rabbit IgG with conjugated HRP (dilution 1:1000, Solarbio) was used.Finally, the titer of the polyclonal antibody was measured and analyzed based on a 450 nm absorbance value using a multifunctional microplate reader (Molecular Devices, Shanghai, China).

Western Blot Analysis
Western Blot was used to detect the specificity of Rab2 and Rab6 polyclonal antibodies.Proteins from crab testes were extracted on ice using a radioimmunoprecipitation assay (RIPA) lysis buffer (Beyotime, China, cat#P0013B) with 10 µL PMSF to a final concentration of 1 mM.A total of 20 µg of the sample was loaded using 15% sodium dodecyl sulfate-polyacrylamide separation gel electrophoresis (SDS-PAGE) for detection.The gel was cut between 10-25 kD and transferred to a polyvinylidene fluoride membrane (PVDF) membrane.The membranes were incubated with Rab2 polyclonal antibodies overnight at 4 °C after blocking with 5% skim milk in TBST (tris buffered saline and 0.1% Tween-20) for 2 h at room temperature.Then, HRPconjugated goat anti-mouse IgG (dilution 1:1000, Solarbio) after washing 3 times with TBST.Finally, enhanced chemiluminescence (ECL) reagent kit (Solarbio, China, cat#PE0010) was used for detection.Rab6 or GM130 (ab52649) was detected using the same method, the difference is that Rab6 or GM130 antibodies were used instead of Rab2, and the secondary antibodies for HRP-conjugated goat anti-rabbit IgG (dilution 1:1000, Solarbio) were used for detection.β-actin was used as the loading control for the western blot.All results were acquired and analyzed with Image Lab software (v.4.1, Bio-Rad, Hercules, CA, USA).Image J software (v.13,LOCI, University of Wisconsin, Madison, WI, USA) was used for the relative protein quantification analysis.

Immunofluorescence
To observe the colocalization of Rab2 and GM130, testes from male crabs were fixed with 4% paraformaldehyde overnight at 4 °C and dehydrated using 30% (w/w) sucrose in 0.1 M PBS, pH 7.4.Frozen sections were permeabilized with 0.5% Triton X-100 at room temperature for 10 min, and blocked with 5% BSA, 10% goat serum, and 0.3 M glycine for 1 h at RT after 3 washes with PBS.Anti-Rab2 antiserum (dilution 1:500) along with GM130 (dilution 1:200, Abcam) were incubated for co-staining overnight at 4 °C.After washing 3 times for 10 min, the secondary antibodies of goat anti-rabbit Alexa Fluor® 488 (1:200, Abcam) and goat anti-mouse Alexa Fluor® 594 secondary antibodies (dilution 1:200, Abcam) were added and incubated at RT in the dark for 1 h at room temperature.After washing 2 times for 5 min with PBS, 6-diamidino-2-phenylindole (DAPI) was used for nuclear staining for 5 min following the last washing.Control sections were incubated with non-immune goat serum (Solarbio, China, cat#SL038) in place of primary antibodies under the same condition described above.
To observe the characteristics of Rab2 during different periods of spermatids, only Rab2 poly-antibodies were used (1:500) and goat anti-mouse Alexa Fluor® 594 secondary antibodies (dilution 1:200, Abcam) were incubated.Rab6 detection was conducted using the method described above.The difference is that anti-Rab6 antiserum was used (1:400) and the secondary antibodies of Alexa Fluor 488conjugated goat anti-rabbit (dilution 1:200) were incubated at RT after washing.Both control sections were incubated without a primary antibody.
All the stained sections were mounted on slides using an anti-fade mounting medium (Southern Biotech, Cat#.0100-01).Images were acquired using an Olympus FV1000-IX81 laser scanning confocal microscope (Olympus, Tokyo, Japan).

SDS-PAGE
The SDS-PAGE result of sperm proteins showed a total of 23 detectable bands (Fig. 1A).Rab proteins represent 21-25 kD and the histones are under 15 kD.

Sperm Proteome
LC-MS/MS was conducted to identify the sperm proteome.Raw data produced by the timsTOF Pro (Bruker Daltonics) mass spectrometry were subsequently searched by using the MaxQuant search engine (v.1.6.15.0).Tandem mass spectra were searched with the transcriptome of testis and sperm in E.sinensis connected with SwissProt database.A total of 26,430 peptides corresponding to 1247 proteins were identified (Supplementary Table 1).As expected, the major proteins fell under either catalytic activity or bind-ing functions.A wide range of protein classes was identified with hydrolase, oxidoreductase, nucleic acid binding, transferase, cytoskeleton protein, and enzyme modulator being the most common.The results could be divided into the biological process (BP), cell component (CC), and molecular function (MF) categories (Fig. 1B).

Detection and Sequencing of Rab2 and Rab6
RNA quality was assessed with 1% agarose gel electrophoresis (Fig. 2A).A band was seen at approximately 360 bp as expected for Rab2 (Fig. 2B) while Rab6 was seen at 450 bp (Fig. 2C).Rab2 was cleaved from Pet-19T-Rab2 with BamH I and XhoI and sequenced after gel-purification; Rab6 was cleaved using NdeI and XhoI from Pet-19T-Rab6 and sequenced.The results of these analyses validated our Rab2 and Rab6 preparation.The structures of Rab2 and Rab6 domains predicted by SWISS-MODEL showed that both two Rab proteins contain β-sheets flanked by α-helices (Fig. 3).

Expression of Rab2 and Rab6
The results of SDS-PAGE showed that Rab2 and Rab6 were purified as expected.A band at 15 kD represents purified Rab2 and a band at 17 kD of Rab6 after Ni column purification (Fig. 4).

Western Blot Analysis
Quantification of Rab2 and Rab6 in the antisera using ELISA confirmed that both poly-antibodies were ready for the following experiments.Proteins extracted from the testes and sperm of E.sinensis were subjected to western blot, and the results showed a band at 22 kd for Rab2 (Fig. 5A) and a band at 26 kd for Rab6 (Fig. 5C), confirming our preparation.A band is shown at 130 kd for GM130 (Fig. 5B), validating this GM130 antibody as suitable for our research (Fig. 5D).Quantification by Image J showed that the expression of Rab2, Rab6 and GM130 in the testes was higher than in sperm (Fig. 5E-G).

Observation of Spermatogenesis
In the crab testis, different stages of germ cells can be recognized according to the unique characteristics of their DAPI-stained nuclei (Fig. 6).Spermatogonia has the largest nuclei with fewer chromatin clumps compared with spermatocytes.At the spermatid stage, the chromatin becomes dense and the clumps disappear.Early spermatids have round donut-like nuclei.Middle spermatids have crescentshaped nuclei that wrap the proacrosome.Late spermatids have cup-like nuclei that hold the acrosome.The spermatozoa in seminal vesicles have a nuclear annulus resembling a ring when observed from above.

Rab2 Co-Localized with GM130 around the Nuclei in Spermatogonia and Spermatocytes
We found that Rab2 (red) and GM130 (green) were colocalized next to the nuclei (blue) as expected in both spermatogonia and spermatocytes (Fig. 7).The nuclei are round in the spermatogonia and spermatocytes.The nuclei in spermatogonia are larger and their chromatin is looser compared with spermatocytes, which show chromatin clumps that are randomly distributed in the nuclei.No signals could be observed in the negative control group.

Rab2 Dynamics during Acrosome Formation
In the early spermatids, Rab2 (red) signals are mainly located at one side of the cell around the nuclei (Fig. 8).The chromatin becomes dense with a central cavern.Rab2 was transferred to the pre-acrosomal vacuoles (PV) in the middle spermatids.At this stage, Rab2 wraps the PV like a long coat with a thickening at the waist compared with the head.The nuclei transform to a crescent shape with a small central aperture.In the late spermatids, the nuclei turn into a ring that wraps the acrosome while Rab2 surrounded the nuclei.In the mature sperm, Rab2 remains surrounding the nuclei.No positive signals were observed in the negative controls.

Rab6 Dynamics during Spermatogenesis and Acrosome Formation
Rab6 settles at one side adjacent to the nuclei in the cytoplasm in spermatogonia and spermatocytes (Fig. 9).In the early spermatids, Rab6 gathers at one side of the cell near the nuclei which indicated that it may participate in the formation of pre-acrosomal vacuoles.These signals transferred to the pre-acrosomal membrane in the middle spermatids, and finally settle on the acrosomal tubule and acrosomal membrane in the mature sperm.

Discussion
The sperm proteome has been reported among mammals, including human [24], mouse [25], rat [26], pig [35], bovine [36], and koala [37].The proteomic analysis of spermatozoa in several species of aquatic animals has been reported, such as the giant grouper [27], bottlenose dolphin [28], and carp [29].However, few have focused on Decapoda animals.In this study, we identified 1247 sperm proteins; as in other species, most were structure proteins, energy metabolism related proteins, cytoskeletal proteins, and enzymes.In the present study, there was a greater quantity of identified sperm proteins compared with other aquatic animals such as the bottlenose dolphin (419) [28] and carp (348) [29], but lower than mammals such as humans (4675) [38] and pigs (1723) [35].The nuclei in E.sinensis is uncondensed and it was thought to be related to the types of histones; we also confirmed that all 5 histones exist in the sperm proteome, which is consistent with our previous findings [31][32][33][34].Similar to the mouse and human sperm proteome [24,25], the sperm proteome in E.sinensis contains many mitochondrial proteins.These results indicate that mitochondria are involved in spermatogensis; more studies are needed to further characterise the mitochondria during acrosome formation.
Rab proteins belong to the Ras superfamily which has GTP-hydrolysis activity.Most are involved in vesicle transport between different membranes including organelles and the cell membrane [1].The acrosome, as a membranous organelle, originates from vesicles related to the Golgi, which highlights the importance of Rab proteins during acrosome formation.In our study, 7 Rab proteins   were identified, including Rab1, Rab2, Rab5, Rab6, Rab11, Rab14, and Rab18.Baker et al. [26] found 858 proteins in the mouse sperm proteome, and the Rab family including Rab2, Rab5, Rab8, Rab11, Rab14 and Rab 22 (but not Rab 6), as a second family of proteins that are highly represented in the sperm.Chauvin further found 2850 proteins using the deep mouse epididymal sperm proteome [25] and increased the number of identified Rab proteins to 23.Published data underline the important role of Rab proteins.It has been reported that Rab2, Rab3, Rab5, Rab6, Rab7, and Rab27 are involved in acrosome formation during spermatogenesis of various species [8][9][10][11][12][13].We identified Rab2 and Rab6 in the sperm proteome in this study; these results are analogous to what is seen in mammals [8,11,18].We did not detect Rab3, but Rab3 is present in the acrosomal region of mouse, rat and human sperm [9,39].Other Rab proteins, related to male fertility, are involved in sperm motility and capacitation [19,40]; more research is needed to fully identify the functions of Rab proteins in germ cells in Decapoda animals.
The involvement of Rab2 and Rab6 in spermatogenesis has been described in mice and cattle [41,42].Rab2 has been ascribed to the formation of pre-acrosomal vesicles in the Golgi and cap phases of spermiogenesis and eventually forms the perinuclear theca in bovine sperm [8].Sim- ilarly, in our study, Rab2 locates on the membrane of preacrosomal membrane in the early and middle spermatids.In the mature sperm, Rab2 transfer to the perinuclear theca around the nuclei, which indicates the importance of Rab2 for sperm maturation.It has been confirmed that Rab2 is important for the maturation of Golgi precursors [43].We found Rab2 co-localized with GM130 in spermatogonia and spermatocytes, confirming that Rab2 is on the Golgi apparatus, which parallels with the somatic cells.It has been found that Rab2, generated from the Golgi apparatus, is essential to autophagy [44,45].Autophagy is essential for spermatogensis, more efforts are necessary for confirming whether Rab2 is related to autophagy during spermatogenesis in E.sinensis.
Rab6, as the most abundant Golgi Rab protein, was found to be essential for shaping the Golgi apparatus in yeast [46].In germ cells, Da Costa [11] found that Rab6 together with Vps13b are located at the Golgi apparatus of all spermatogenic cells, and finally settle down on the acrosomal membrane connected by Vps13b in the spermatozoa.Though the sperm of E.sinensis are unique, we found that Rab6 is involved in pre-acrosome granule formation in the early spermatids, and gradually transfers to the acrosomal membrane in the middle spermatids.In the mature sperm, Rab6 exists on the central tubule alongside the acrosomal In the middle spermatids, the nuclei transformed into a crescent shape, Rab2 wrapped the PV and was thicker at the waist and thinner in the head (D-F).In the mature sperm, Rab2 surrounds the ring nuclei (G-I).In the negative controls, no positive signals were observed (J-L).e-spt, early spermatids; m-spt, middle sper-matids; sp, mature sperm.membrane, indicating the involvement of Rab6 in forming subacrosomal structures.Our results showed that Rab2 and Rab6 are conserved during acrosome formation across species.
Our findings provide a foundation for studying the functions of Rab proteins in acrosome formation during spermatogenesis in Decapoda animals.This work extends our understanding of the sperm proteome in crustaceans.Further research is needed to clarify the functions of other Rab proteins that participatr in spermatogenesis and fertilization in E.sinensis.

Conclusions
In summary, the findings in the present research showed that a total of 1247 kinds proteins, including 7 Rab proteins, were identified in the sperm proteome in E.sinenesis.Ployantibodies of Rab2 and Rab6 were pre- pared and the the characteristics of the target Rab proteins were illustrated using IF, we found Rab2 and Rab6, though in different patterns, are involved in the acrosome formation during spermatogenesis in E.sinensis.The results showed that Rab proteins are key members of the acrosomal proteins and these findings provide a foundation for further illustrating the functions of Rab proteins during spermatogenesis in Decapoda animals.

Fig. 1 .
Fig. 1.Analysis of sperm proteins.(A) SDS-PAGE of mature sperm protein in E.sinensis.(B) GO annotations of the 1247 identified proteins from the proteome database: biological process, cellular component, and molecular function.(C) Subcelluar localization of the sperm proteome.Pie chart of the subcellular localization of sperm proteins predicted by wolfpsort annotation.

Fig. 2 .
Fig. 2. Detection of total RNA, Rab2, and Rab6.(A) 3 bands of total RNA from the testis in E.sinensis in the gel.(B) A band around 360 bp represents Rab2.(C) A band around 450 bp represents Rab6.M, Marker.

Fig. 5 .
Fig. 5.Western blot analysis using Rab2, Rab6, and GM130 antibodies of E.sinensis testes and sperm proteins.A band at 22 kD represents Rab2 (A), a band at 130 kD represents GM130 (B), and a band at 26 kD represents Rab6 (C).β-actin was used as the loading control for the western blot, represented by a band at 42 kD (D).Expression of Rab2, Rab6, and GM130 in the testes was higher than in sperm (E-G).M, Marker; T, testes proteins; S, sperm proteins.

Fig. 6 .
Fig. 6.Different stages of germ cells of the testis in E.sinens stained by DAPI.The nuclei in spermatogonia are round with fewer chromatin clumps and the chromatin is looser compared with spermatocytes (A).Early spermatids have condensed donutlike chromatin (A-C).Sperm have cup-like nuclei (D).Middle spermatids have crescent nuclei settled at one side of the cell.The nuclei in the late spermatids are a little thicker than in mature sperm, and the radial arms emerge till spermatozoa.SPG (a): spermatogonia; SPC (b): spermatocyte, eSPT (c): early spermatid, mSPT (d): middle spermatid, lSPT (e): late spermatid, SP (f): spermatozoa.The scale bar represents 10 µm.

Fig. 7 .
Fig. 7. Rab2 (red) is co-localized with GM130 (green) in the spermatogonia and spermatocytes during spermatogenesis.The size of the nuclei in spermatogonia is larger (8-10 µm) compared with spermatocytes (5-9 µm), but the chromatin is looser.Rab2 together with GM130 is located in the Golgi apparatus adjacent to the nuclei, both in spermatogonia (A-C) and spermatocytes (D-F).In the negative controls, no positive signals were observed (G-I).spg, spermatogonium; spc, spermatocyte.

Fig. 8 .
Fig. 8. Rab2 (red) was involved in the formation of proacrosomal vacuoles (PV) in spermatids.In the early spermatids, Rab2 localized at one site adjacent to the dense nuclei (A-C).

Fig. 9 .
Fig. 9. Rab6 (green) characteristics during spermatogenesis in E.sinensis.Rab6 was located at one side near the nuclei in the spermatogonia and spermatocytes (A-C).In the early spermatids, the Rab6 signals were more obvious and gathered near the nuclei, indicating that they were involved in the formation of pre-acrosomal vacuoles (D-F).The signals transferred to the preacrosomal membrane in the middle spermatids (G-I) and finally settled on the acrosomal membrane and acrosomal tubule in the mature sperm (J-L).In the negative control, no positive signals were observed (M-O).spg, spermatogonia; spc, spermatocytes, espt, early spermatids, m-spt, middle spermatids, sp, mature sperm.The scale bar represents 10 µm.