P38 participates in spermatogenesis and acrosome reaction prior to fertilization in Chinese mitten crab Eriocheir sinensis

P38 participates in spermatogenesis and acrosome reaction prior to fertilization in Chinese mitten crab Eriocheir sinensis

Gene 559 (2015) 103–111 Contents lists available at ScienceDirect Gene journal homepage: www.elsevier.com/locate/gene P38 participates in spermatog...

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Gene 559 (2015) 103–111

Contents lists available at ScienceDirect

Gene journal homepage: www.elsevier.com/locate/gene

P38 participates in spermatogenesis and acrosome reaction prior to fertilization in Chinese mitten crab Eriocheir sinensis Ming Zhu 1, Wen-Juan Sun 1, Yuan-Li Wang, Qing Li, Hong-Dan Yang, Ze-Lin Duan, Lin He ⁎, Qun Wang ⁎ Laboratory of Immunological Defense & Reproductive Biology, School of Life Science, East China Normal University, No. 500, Dong-Chuan Road, Shanghai, China

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Article history: Received 29 September 2014 Received in revised form 29 October 2014 Accepted 23 November 2014 Available online 26 November 2014 Keywords: Eriocheir sinensis p38 Spermatogenesis Acrosome reaction

a b s t r a c t P38 mitogen-activated protein kinases (MAPKs) comprise a family of serine/threonine protein kinases that play important roles in cellular responses to inflammatory cytokines and environmental stresses. These kinases are involved in controlling cell division, differentiation and death in mammalian testes and therefore are critical to spermatogenesis. To explore their functions in male reproduction of Chinese mitten crabs, Eriocheir sinensis p38 (Es-p38) protein expression was determined in different tissues including testes at different developmental stages by Western blot. Es-p38 was expressed in various tissues, with higher levels in the heart, stomach, gills and testes. Total Es-p38 protein levels increased gradually during spermatogenesis, but phosphorylated Es-p38 was much higher in the spermatid (August–October) than the spermatocyte (July–August) and sperm (October– January) stages. Trypan blue staining and hematoxylin/eosin staining were both used to detect sperm motility and changes in sperm morphology during the acrosome reaction (AR) induced by pre-incubation with A23187 in vitro, activated Es-p38 proteins detected by fluorescent microscopy were translocated gradually to nuclear and apical cap regions, accumulating at the anterior of the acrosomal tubule. The results suggest the involvement of p38 MAPK in spermatogenesis and the AR in E. sinensis. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Mitogen-activated protein kinases play crucial roles in a large number of physiological and pathological processes, such as cell differentiation, proliferation, survival and apoptosis. Their structures and functions have been conserved during evolution from unicellular (e.g., brewers' yeast) to multicellular organisms including humans (Johnson and Lapadat, 2002). Four types of MAPK cascades have been identified in mammals: extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), ERK5 and p38. Each cascade has five components, including three core kinases (MAP3K, MAP2K, and MAPK) and their upstream (MAP4K) and extensive downstream (MAPKAPK) substrates (Almog and Naor, 2008). In response to different stimuli, members of each cascade phosphorylate downstream targets sequentially to activate the final substrate localized either in the cytoplasm or primarily in the nucleus (Roux and Blenis, 2004). However, this chain of events can be counteracted by protein phosphatases, which dephosphorylate MAPKs and render them Abbreviations: MAPKs, mitogen-activated protein kinases; AR, acrosome reaction; ERK, extracellular signal-regulated kinase; JNK, c-Jun N-terminal kinase; MAPK14, p38α; ThrGly-Tyr, threonine–glycine–tyrosine; AV, acrosomal vesicle; AT, acrosomal tubule; AC, apical cap; HE, hematoxylin and eosin; SR, survival rate; ARR, acrosome reaction rate; SARR, spontaneous acrosome reaction rate; N, nucleus; RA, radial arm; ZP, zona pellucida ⁎ Corresponding authors. E-mail addresses: [email protected] (L. He), [email protected] (Q. Wang). 1 Ming Zhu and Wen-Juan Sun are the first two authors and contributed equally to this work.

http://dx.doi.org/10.1016/j.gene.2014.11.050 0378-1119/© 2014 Elsevier B.V. All rights reserved.

inactive (Pearson et al., 2001). Of course, certain phosphorylated substrates must transfer to the nucleus to modulate the activity of transcriptional factors and chromatin remodeling enzymes. Mechanisms of ERK1/ 2 nuclear translocation were elaborated by the discovery of a nuclear translocation sequence that could bind to the shuttling protein importin7 (Plotnikov et al., 2011). Four different isoforms of p38 MAPK are known in mammalian cells, namely p38α (MAPK14), p38β, p38γ and p38δ. P38α and β are ubiquitously expressed, whereas p38γ and δ are differentially expressed in a range of tissues (Cuadrado and Nebreda, 2010). Stress-activated p38 MAPK can be induced by various stimuli, such as ultraviolet light, inflammatory cytokines, osmotic shock and growth factors. Once stimulated, the threonine–glycine–tyrosine (Thr-Gly-Tyr) amino acid motif in the activation loop sequence of p38 MAPK is dually phosphorylated by upstream MAPK kinases (MKK) 3/4/6 (Raingeaud et al., 1996; Ono and Han, 2000). Interestingly, TAB1 appears to be an adaptor protein that can trigger the autophosphorylation and activation of p38α, but it is not a MKK and does not possess catalytic activity itself. The finding of this activation mechanism for p38α, in addition to the prototypic kinase cascade, was unexpected (Ge et al., 2002). The activated p38 proteins may then regulate the activity of downstream protein kinases or transcription factors, such as ATF2, Sap1 and CHOP, to execute different cellular responses (Ono and Han, 2000). Attention has been turning gradually towards p38 MAPK in the reproductive field. Previous studies showed that p38 MAPKs are activated during germ cell apoptosis and mediate the disruption of the blood–

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testis barrier integrity induced by cytokines in mammals (Li et al., 2009). P38 MAPKs have also been detected in the tail of mature human spermatozoa and are implicated in regulating hyperactivated motility and promoting the acrosome reaction (Almog et al., 2008). In a separate study, we were able to successfully clone the full-length p38 cDNA sequence of the Chinese mitten crab Eriocheir sinensis (GenBank accession no. KF582665) and confirmed it to be a homologue of p38α (MAPK14) by sequence alignment (Zhao et al., 2014). E. sinensis, an economically important crab, is a crustacean belonging to the order Decapoda. Its sperm cells have a characteristic acrosome complex and no flagellum. Testis development of E. sinensis can be divided into five stages based on morphology found typically at different times of the year: spermatogonium stage (May–June), spermatocyte stage (July–August), spermatid stage (August–October), sperm stage (October–April in the following year) and dormant stage (April–May) (Du et al., 1988a). The seminiferous ducts contain a number of mature sperm which are encapsulated into spermatophores. Upon entering the female reproductive tract, the spermatophores rupture to release the free sperm, and the male accessory sex gland protein has been shown to aid in this process by digesting the spermatophore wall effectively in the proper environment (Hou et al., 2010). Once sperm cells are released, they must undergo a series of biochemical and physiological processes collectively known as capacitation and AR before fertilizing the egg. The AR of sperm in E. sinensis consists of four main steps: 1) contraction of radial arms; 2) eversion of the acrosomal vesicle (AV); 3) extension of the acrosomal tubule (AT); 4) separation of the lamellar structure from the reacted sperm (Du et al., 1987a). This secretory event is Ca2+-dependent, and artificial treatments that increase [Ca2+] are effective for inducing the AR (Abou-haila and Tulsiani, 2009). In particular, A23187 has been widely used in many studies as an inducer of the AR in vitro. As described above, the p38 cascade has been demonstrated to play vital roles in immunity and reproduction. P38 was confirmed to be involved in Vibrio parahaemolyticus infection and shrimp immunity in Litopenaeus vannamei (He et al., 2013). It also mediated the UV-B radiation-induced oxidative stress in the benthic copepod Tigriopus japonicus (Kim et al., 2014). However, the involvement of the p38 cascade in E. sinensis reproduction remains unclear. Here we established a system through which mature sperm cells could be obtained from spermatophores in vitro, and the AR was successfully, as demonstrated by the morphological variations in sperm during this process, and with a high survival rate. The Es-p38 protein expression pattern was examined in different tissues and developing testes by Western blot using a p38specific antibody. The distribution of Es-p38 protein in the testes and seminal vesicles (spermatocyte and spermatozoa) was also detected separately. Finally, localizations of Es-p38 and phospho-Es-p38 proteins in sperm during different stages of AR were analyzed by immunocytofluorescence. The results suggest that p38 MAPK participates in spermatogenesis and the AR in the Chinese mitten crab. 2. Materials and methods 2.1. Animal and tissue preparation Twenty healthy adult male Chinese mitten crabs were obtained from a local market (Xin'an farmers' market, Shanghai, China) once a month from July to January of the following year. The crabs were raised in wellaerated tanks for several days before the experiment. Crabs were lightly anesthetized in an ice bath for 5 min before dissection. Seven tissues (testes, accessory gland, seminal vesicle, intestines, gills, stomach and heart) were extracted and frozen in liquid nitrogen immediately prior to storage at −80 °C. 2.2. Antibodies The following antibodies used in this study were obtained from commercial sources: anti-p38 (Epitomics, Burlingame, CA, USA), anti-

phospho-p38 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), HRPconjugated goat anti-rabbit IgG (Immunology Consultants Laboratory, Portland, OR, USA), FITC-conjugated AffiniPure goat anti-rabbit IgG (Jackson ImmunoResearch Laboratory, West Grove, PA, USA), and rabbit anti-β-actin (Hua'an Biology, Hangzhou, China). 2.3. Western blot analysis A previously described protocol (Ito et al., 2010) with modification was used to extract total proteins from the seven tissue samples, which were collected from crabs and frozen in September, and from testis of different months. Briefly, tissues were homogenized in lysis buffer (Beyotime, Jiangsu, China) containing protease inhibitors on ice for 10 min and centrifuged at 12,000 ×g at 4 °C for 5 min. The supernatant was diluted with a quarter volume of 5 × SDS-PAGE sample loading buffer (Beyotime), heated at 94 °C for 10 min and loaded onto a 12% sodium dodecyl sulfate polyacrylamide gel. Separated proteins were transferred to a polyvinylidene fluoride (PVDF) membrane (Beyotime) after electrophoresis at 120 V for 120 min. Subsequently, the membrane was blocked with 5% bovine serum albumin (BSA) in TBST solution for 60 min at room temperature and then incubated with the primary antibody (diluted at 1:1000) overnight at 4 °C. After washing with TBST three times, the HRP-conjugated secondary antibody (diluted at 1:2000) was added for incubation at room temperature for 2 h. Blots were visualized using the cECL Western Blot Kit (CWBIO, Beijing, China), and photos were acquired by CCD imaging. For subsequent detection of phospho-Es-p38 or β-actin on the same membrane, it was treated with Stripping Buffer (CWBIO) according to the procedures described by the manufacturer and then probed with the corresponding antibody as above. 2.4. Incubation and induction of spermatozoa Bilateral seminal vesicles were resected quickly from five healthy male Chinese mitten crabs and pressed to extrude the spermatophores into Ca2+ free artificial seawater (NaCl 21.63 g/L, KCl 1.12 g/L, H3BO3 0.53 g/L, NaOH 0.19 g/L, MgSO4·7H2O 4.93 g/L; pH = 7.4). The spermatophores in suspension were washed by mixing with a 1 mL pipette and centrifuging at 400 ×g for 5 min at 4 °C. The precipitate was treated with 5–6 volumes of accessory sex gland protein homogenization buffer (15 mg/mL) at 37 °C for approximately 20 min until all the spermatophores were digested. Ruptured spermatophores were centrifuged at the same conditions described above to separate the spermatophore fragments from the spermatozoa, which were adjusted to a final density of 5–6 × 106 spermatozoa/mL for use. A23187 (Sigma, dissolved in DMSO at 2 mg/mL as the stock solution) was added to the prepared spermatozoal suspension to the final concentration of 50 μg/mL at room temperature. A sample of spermatozoa was collected every 30 min (0, 30 min, 60 min, 90 min) and used for further experiments. An identical sample was treated with Ca2+-free artificial seawater instead of A23187 as a control. Sperm viability at different stages was measured by Trypan blue exclusion (Ma et al., 2007). 10 μL of the spermatozoal suspension was mixed with an equal volume of Trypan blue for 5 min and examined under a microscope in random fields of view. At least 200 sperm cells were counted per sample, and all treatments were performed in triplicate. 2.5. Hematoxylin and eosin (HE) staining and immunofluorescence Control and A23187-stimulated sperm samples were smeared on slides, allowed to air dry and fixed with 4% paraformaldehyde for 10 min in preparation for HE staining and immunofluorescence analysis. HE staining was performed according to instructions of the manufacturer of the HE Staining Kit (Solarbio, Beijing, China).

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Fresh testes and seminal vesicles obtained from adult male E. sinensis in September, 2013 were fixed in Bouin's solution, embedded in paraffin and cut into sections of 6-μm thickness. After deparaffinization and dehydration in xylene and graded alcohols, the sections were incubated with 3% H2O2 deionized water for 10 min, washed with distilled water and immersed in PBS for 5 min, followed by blocking in 5% BSA in TBST solution. Two tissues were incubated at 4 °C with anti-p38 (diluted at 1:100) or anti-phospho-p38 (diluted at 1:200) overnight, rinsed three times with PBS and then probed with the FITC-conjugated AffiniPure goat anti-rabbit antibody (diluted at 1:100) at 37 °C for 2 h. Finally, the nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) contained in the mounting medium (ZSGB-BIO, Beijing, China) and observed with a fluorescence microscope (Leica, Germany). The A23187-stimulated sperm smeared on slides also were permeabilized with 0.1% Triton-X-100 in TBST and incubated with antibodies as described above for analysis by immunofluorescence. 2.6. Statistical analysis Statistical analysis was performed using SPSS software (Ver11.0). The data are represented as the mean ± standard error (S.E.). Statistical significance was determined by one-way ANOVA and post hoc Duncan multiple range tests. Significance was set at p b 0.05. 3. Results

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As shown in Fig. 1b, changes in total Es-p38 protein and phosphoEs-p38 protein levels were inconsistent throughout the development of the E. sinensis testes. The expression of total Es-p38 protein did not vary noticeably until November, when it began to rise until the next January. Phospho-Es-p38 was nearly undetectable in July, while it increased significantly in the subsequent three months and decreased to a low level from November to January of the following year. 3.2. Assessment of sperm motility and AR Trypan blue is a cytoactive dye widely used to detect cell survival (Fig. 2I–VIII). Dead sperm cells were stained light blue as observed under an optical microscope (Fig. 2I), while living sperm cells were not colored, except at the acrosomal cap (Fig. 2III, red arrow). Although distinguishing the detailed internal structure of the sperm was difficult by this method, sperm cells at all steps of the AR were observed. Dissociated spermatozoa appeared oval before initiation of the AR, with numerous radial arms stretched out from the cell surface (Fig. 2II, green arrow). The sperm cells shown in Fig. 2VIII were considered to have completed the AR process. After incubation with A23817, the proportion of E. sinensis sperm undergoing the AR increased to nearly 73% at 90 min after induction in a time-dependent manner (Fig. 3). Throughout the in vitro experiment, the sperm maintained a high survival rate (SR) and low spontaneous acrosome reaction rate (SARR), which supported the reliability of our findings.

3.1. Expression patterns of Es-p38 protein in various tissues including testes at different developmental stages of E. sinensis

3.3. Morphological changes of E. sinensis sperm during the AR process

The p38 protein was found to be widely expressed in Chinese mitten crabs by Western blot without tissue specificity. The results showed synchronous expression of Es-p38 protein and phospho-Es-p38 protein, with the highest expression levels in the heart and decreasing gradually in the stomach, gills and intestines. Significant expression levels of these total and activated proteins were also observed in the male gonads, including the testes, accessory sex gland and seminal vesicles (Fig. 1a).

HE staining was used to clarify the specific structures and morphological changes of E. sinensis sperm in the AR process (Fig. 4A–F). Two concentric circles were seen in the transverse sections of the mature spermatozoon, the inner ring being the AT and the outer ring a cupshaped nucleus (Fig. 4A). The nuclear cup was seen surrounding the acrosome with an apical cap (AC), AT and AV, consisting of fibrous layer, middle layer and lamellar structures (Fig. 4B) (Du et al., 1987b).

Fig. 1. Characterization of Es-p38 protein in E. sinensis. Tissue proteins extracted with lysis buffer were separated by SDS-PAGE, transferred to a PVDF membrane and probed with anti-p38, anti-phospho-p38 and anti-β-actin (as a positive control) antibodies. (a) Expression of Es-p38 protein in different tissues. (b) Expression of Es-p38 protein in testes during different developmental stages of E. sinensis.

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Fig. 2. Trypan blue staining of sperm induced by A23187 in vivo during different stages of AR. Dead sperm were dyed blue (I). Live sperm were transparent, displaying two concentric circles in transection (II) and were surrounded by multiple radial arms. The four stages of the AR: III, contraction of the radial arms; IV–VI, eversion of the AV; VII, extension of the AT; VIII, separation of the lamellar structure from the reacted sperm. RA, radial arm; AC, apical cap; AT, acrosomal tubule.

Sperm in all four stages of the E. sinensis AR were observed. In the first stage, the radial arms were contracted, and the AC bulged forward to form an aperture (Fig. 4C). In the second stage, the nuclear cup shrank, forcing the AV contents to evert through the aperture and appearing as a dumbbell shape (Fig. 4D). In the third stage, the acrosome tube protruded forward through the AC aperture, causing the bottom of the nucleus to invaginate and converge directly with the AC. The entire nucleus appeared in the shape of a “W” on one side (Fig. 4E). Meanwhile, all of the contents in the AV everted, and parts of the plasmalemma and acrosome membrane began to disintegrate. At the last stage, the lamellar structure started to slough off, ultimately leaving nail-shaped sperm

with a DAPI-stained nucleus, a threadlike AT and an AC between them (Fig. 4F) (Kang and Wang, 2000; Xia et al., 2007). 3.4. Localization of Es-p38 in spermatocytes and mature sperm Based on the results of the Western blot analysis, we explored the potential role of Es-p38 in spermatogenesis by observing its distribution in the testis of E. sinensis. To determine the cellular localization of Es-p38 protein, the testis and seminal vesicle paraffin sections were analyzed by immunofluorescence with antibodies against p38 and phosphop38 as described in the Materials and methods section. We could

Fig. 3. In vitro induction of AR in sperm. Normal E. sinensis spermatozoa were incubated (5–6 × 106/mL) with or without A23187 for the time indicated at room temperature for 90 min and examined every 30 min. Green line represents the percentage (%) of sperm that were still alive at each interval in culture. Red line represents the proportion (%) of sperm completing the AR at each interval in culture. Blue line represents the proportion (%) of sperm that underwent spontaneous AR as a control. Experiments were replicated at least three times, and data are shown as the mean ± SD. Asterisks denote significant differences (p b 0.05).

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Fig. 4. HE staining of sperm induced by A23187 in vivo at different stages of AR. Nuclei were stained blue-violet. Sperm with cup-shape nucleus had transformed to the shape of a nail head (N: nucleus, yellow arrows). AVs were stained red.

distinguish the developmental phase of the sperm by observing the morphology (Hou and Yang, 2013). The cell nucleus was round or oval with dispersed and unequal chromatin at early stages of spermatogenesis, including the spermatogonium, primary spermatocyte and secondary spermatocyte. The shape of the nucleus began to change in the early spermatid phase and invaginated into a cup-like shape in the late spermatid phase (Yu et al., 2009; Sun et al., 2010). Activated Es-p38 proteins were transferred to the nucleus and dispersed in the cytoplasm of spermatocytes (Fig. 5I). Furthermore, high expression levels of phospho-Esp38 were also observed in the nucleus and AC of mature sperm in the seminal vesicles (Fig. 5II). We propose that p38 functions in spermatogenesis of E. sinensis based on its persistent expression in various periods of development. 3.5. Localization of Es-p38 in spermatozoa during AR To track Es-p38 localization in sperm during the AR, DAPI was used to stain the nucleus for comparison. Total Es-p38 proteins were distributed throughout the entire sperm cell, including the nucleus, AT, AC and sperm membrane, but not the AV (Fig. 6I). In sperm during an early AR stage, the localization of activated Es-p38 corresponded with that of total Es-p38, but no obvious signals were observed in the AT region (Fig. 6II.b3, red arrow). As the AR progressed, phospho-Es-p38 proteins gradually gathered at the anterior of the AT (Fig. 6II.c3–d3). Finally in the reacted sperm, the signals were dramatically increased in the AT structures and remained strong in the AC and the W-shaped nucleus (Fig. 6II.e3). The notable accumulation of activated Es-p38 at the anterior of the AT suggests a possible role of this protein in assisting the protruding of the AT during the AR prior to fertilization. Furthermore, its localization at the nucleus and AC implicates functions in promoting the protuberance of the AC and morphological changes of the sperm. Based on these results, we speculate that the activation of p38 is closely related to the AR of E. sinensis. 4. Discussion The male reproductive system of E. sinensis is composed of a pair of testes, a pair of vas deferens, accessory sex gland and ejaculatory duct. Each testis is connected to a vas deferens, and they are fused with

each other forming an “H” shape. The vas deferens is divided into two portions, the far end is the SV which stores spermatophores (Du et al., 1988a; Wang et al., 1996). As mentioned above, spermatogenesis in E. sinensis testes occurs in five major stages with remarkable seasonal patterns. From May to June, the testes begin to develop, and the seminiferous tubules are thin and hollow with newly matured sperm. The seminiferous tubules grow quickly over time (August to October), and spermatids become dominant components from October until the following April, reaching the peak of spermatogenesis and becoming ready for mating. Thereafter, the process ceases gradually, and the reproductive system enters a brief period of rest. In the mature sperm, the cup-shaped nucleus harbors an AV, with many radial arms extending from its body. These polarized unflagellated sperm, which are entirely different morphologically from the flagellated sperm, are commonly seen in crustaceans, including the white shrimp Penaeus vannamei Boone (PerezVelazquez et al., 2001) and mud crab Scylla serrate (Wang et al., 1998; Zhang et al., 2010). In order to fertilize, the E. sinensis sperm must undergo capacitation, which confers the ability to launch the AR and penetrate the zona pellucida (ZP) of oocytes, similar to the process in vertebrates (Du, 1998; Breitbart et al., 2005). For nearly a decade, several MAPKs have been associated with reproductive functions, with many of them being essential or vital for spermatozoa functions. In fact, the observation of sustained and concomitant increase in Tyr phosphorylation and kinase activity of ERK during capacitation of human spermatozoa in vitro prompted us to speculate about the key roles of these molecules in reproduction (Luconi et al., 1998a). ERK was found to be primarily located around the post-acrosomal region and the entire mid-piece of the human sperm tail and to be redistributed to the equatorial segment within the sperm head (Luconi et al., 1998b; Almog et al., 2008). In miniature pig sperm pre-treated with caffeine as an inducer and U0126 as an inhibitor of MEK, a time-dependent phosphorylation of the Raf–MEK–ERK pathway was triggered by the activation of the AR, indicating a positive role of ERK in this process (Kawano et al., 2010). JNK was found to be expressed in the testis and involved in modulating the Sertoli cell tight junction-barrier function and germ cell apoptosis in humans (De Cesaris et al., 1999; Lysiak et al., 2003; Li et al., 2009). P38, an important component of the MAPK signaling transduction pathway, is activated by the dual phosphorylation of Thr and Tyr

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Fig. 5. Localization of p38 in spermatocytes and mature sperm of E. sinensis by immunofluorescent analysis. Es-p38 and phospho-Es-p38 proteins were probed with specific primary antibodies and the appropriate FITC-conjugated secondary antibody (A1, B1, C1, D1), while DAPI was utilized to localize DNA (A2, B2, C2, D2). Tissues were obtained in September, 2013. (I) Activated Es-p38 transferred to the nucleus and were dispersed in the cytoplasm of spermatocytes in the testes (arrows in B3). (II) Activated Es-p38 mainly localized in the nucleus and AC in mature sperm in seminal vesicles (arrows in D3). Diffused chromatin granules were seen in the nucleus (A2, B2).

residues in a Thr-Gly-Tyr motif and transferred from the cytoplasm to the nucleus to phosphorylate other substrates in the course of intracellular signal amplification (Martín-Blanco, 2000). This protein was first noted for its crucial role as a therapeutic target in inflammatory disease (Kumar et al., 2003; Saklatvala, 2004). Its functions in male reproduction, such as the regulation of spermatogenic cell proliferation and androgen secretion in mouse testis, attracted our attention (Jiang et al., 2003). P38 has also been detected at the post-acrosomal region of human sperm and found to be sporadically distributed on the sperm tail, serving as a negative regulator of sperm motility (Almog and Naor, 2010). In the current study, we showed expression patterns of the Es-p38 protein in different tissues (testes, accessory gland, seminal vesicle, intestines, gills, stomach and heart in September, testis from July to January of the following year) and different developmental stages during spermatogenesis of E. sinensis. The results obtained by HE staining and immunofluorescent microscopy provided an association of p38 with spermatogenesis and the AR in mature sperm in E. sinensis. In general, activated Es-p38 proteins appeared to be translocated from the cytoplasm of sperm to the regions of the nucleus and AC, accumulating at

the anterior of the AT gradually as the AR progressed. Given the characteristic expression and dynamic translocation of p38 in E. sinensis, we propose that this protein plays an important role in protraction of the AT during the AR. 4.1. Implications for p38 involvement in spermatogenesis and role in AR of E. sinensis The testes of E. sinensis are composed of numerous winding seminiferous tubules coated by a connective tissue membrane. Epithelial cells line one side of the seminiferous tubule wall, while the germinative zone is on the other side. Maturation of male germ cells within a seminiferous tubule is initiated by the production of spermatocytes near the basement membrane of the germinative zone. They continuously proliferate (through meiosis and mitosis) and enter the lumen to form the germinal zones where germ cells can be found at different developmental stages (Ma et al., 2006). The germinal zones can be seen clearly on transverse sections of seminiferous tubules, which help to divide the development of E. sinensis testes into five stages as we have described above.

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Fig. 6. Localization of p38 in E. sinensis sperm during AR in vivo by immunofluorescent analysis. The Es-p38 (I) and phospho-Es-p38 (II) proteins were probed with specific primary antibodies and the appropriate FITC-conjugated secondary antibody (A2–E2, a2–e2), while DAPI was utilized to localize DNA (A1–E1, a1–e1). Activated Es-p38 protein appeared to translocate to regions of the nucleus and AC, accumulating at the anterior of the AT gradually as the AR progressed (red arrows).

After analysis of our data, we found a striking coincidence between the expression patterns of total and activated Es-p38 proteins and the timing of testis development. In the spermatid stage, secondary spermatocytes were divided into early haploid spermatids inlaid with a round or oval nucleus through meiosis. Spermatids are known to ultimately differentiate into mature sperm through three indispensable stages: 1) formation of the proacrosomal granule; 2) coalescence of proacrosomal granules to form a large proacrosomal vesicle; and 3) formation of a cup-shape nucleus and the degeneration of the membrane complex (Du et al., 1988b; Sun et al., 2010). In this study, activated Es-p38 proteins were found in the nucleus and detected in the cytoplasm of spermatocytes or spermatids (Fig. 5I.B3, arrows). In addition, Es-p38 appeared to be highly phosphorylated, while the total expression of Es-p38 was maintained at a relatively stable level during this period (Fig. 1b), suggesting that the p38 signaling pathway is involved in the spermatogenesis of E. sinensis. In the sperm stage (October– April of the following year), the seminiferous tubules were full of

sperm waiting to pass into the vas deferens. The activated Es-p38 protein was mainly distributed in the nucleus and AC in mature spermatozoa (Fig. 5I.D3, arrows). After the series of intricate processes of spermatogenesis and spermiogenesis, Es-p38 phosphorylation levels stabilized, while the total amount of Es-p38 protein increased, indicating that non-phosphorylated Es-p38 protein was stored in preparation for subsequent biochemical processes before fertilization. Thus, we reasonably presumed that Es-p38 undertakes certain tasks in the AR of E. sinensis. 4.2. P38 may function in AT protraction during AR In this study, a short period of induction in vivo and immunofluorescence were adopted to further explore the potential role of p38 in mediating the AR. High expression levels of activated Es-p38 were exhibited in the nucleus and AC of mature sperm. This distribution pattern is different from that of p38 in human sperm, which was found to inhibit

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forward and hyperactivated motility as well as take part in the PKCmediated AR of sperm (Almog et al., 2008). The presence and activation of p38 have also been confirmed in bovine spermatozoa to regulate heat-induced sperm damage by post-transcriptional modifications (Rahman et al., 2014). Therefore, we deduced that once activated, p38 moves into the nucleus where it exerts its transcriptional effects by phosphorylating downstream target substrates. This function of p38 is similar to its male-specific role in preventing meiosis in testicular germ cells and inducing mitotic arrest during the germ cell sex differentiation period in mice (Ewen et al., 2010). Gradually enhanced immunostaining was also found at the anterior of the AT on sperm as the AR progressed. This finding is similar to the model in chickens (Lemoine et al., 2009), in which MAPK 14 signals detected in the head region of spermatozoa were demonstrated to be involved to some degree in capacitation and AR processes rather than merely in stimulating the AR. Therefore, we speculate that p38 may also be responsible for the protruding of the AT during the AR in Chinese mitten crabs based on the current observations. However, further mechanistic studies on the p38 MAPK signaling pathway and interactions between multiple pathways in regulating the AR of E. sinensis are still required. In summary, the present study elaborated for the first time the protein expression patterns of p38 in E. sinensis and described the morphological alterations of E. sinensis sperm during the AR. In addition, we tracked the localization of Es-p38 in spermatocytes and mature sperm as well as the migration of Es-p38 after activation during the AR process. These observations suggest that p38 is involved in spermatogenesis of E. sinensis and may play an important role in the protruding of AT during AR before fertilization.

Acknowledgments We thank all members of the Crustacean Laboratory at the East China Normal University for their assistance and helpful suggestions. This work was supported by grants from the National Natural Science Foundation of China (No. 41376157, No. 31172393 and No. 31201974), the Shanghai Natural Science Fund Committee (No. 12ZR1408900) and the Doctoral Fund of Ministry of Education (No. 20120076120011).

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