Cell Tissue Res DOI 10.1007/s00441-015-2226-4

REGULAR ARTICLE

Enhancement of trypsin-like enzymes by A23187 ionophore is crucial for sperm penetration through the egg vestment of the giant freshwater prawn Atthaboon Watthammawut 1 & Monsicha Somrit 1 & Somluk Asuvapongpatana 1 & Wattana Weerachatyanukul 1

Received: 2 March 2015 / Accepted: 19 May 2015 # Springer-Verlag Berlin Heidelberg 2015

Abstract We report the presence of trypsin-like enzymes preferring Boc-QAR-MCA substrate in sperm collected from different portions of male reproductive tracts of the giant freshwater prawn, Macrobrachium rosenbergii and compare enzyme activities before and after an A23187 calcium ionophore treatment. Fluorogenic enzyme assays revealed that testicular sperm lysates showed high trypsin-like enzyme activity but the activity was relatively low in vas deferens sperm lysates as well as in the live sperm. Upon sperm treatment with A23187, trypsin-like activity was greatly enhanced in distal vas deferens sperm. Substrate- and inhibitor-based localization studies indicated that the sperm trypsin-like enzymes were not of a soluble type but were rather of a membrane-borne type, localized at the anterior spike and upper part of the main body. Notable structural changes were also evident in A23187-induced sperm including extensive ruffling of the sperm membrane structure at the base of the main body thereby supporting the acrosome reaction response in this species. We further proved by substrate inhibition assays that the enhanced trypsin-like enzyme activity participates in sperm penetration through the vitelline envelope, a novel sperm–egg penetration mechanism that is unique in this species.

Keywords Shrimp . Sperm . Calcium . Acrosome reaction . Trypsin

* Wattana Weerachatyanukul [email protected] 1

Department of Anatomy, Faculty of Science, Mahidol University, Rama VI Road, Ratchathewi, Bangkok, Thailand 10400

Introduction Similar to other palaemonid decapods, sperm of the giant freshwater prawn, Macrobrachium rosenbergii, possess an inverted umbrella-shaped structure and a single (unistellate), immotile anterior spike (Lynn and Clark 1983a; Poljaroen et al. 2010). During fertilization, sperm are released from the female open storing sac (thelycum) and allowed to passively contact the egg vestment. Unlike other unistellate sperm, the initial contact of M. rosenbergii sperm to the egg vitelline envelope (VE) occurs at the base of main body instead of the anterior spike. This process is followed by the bending of the anterior spike to penetrate the egg envelope, a process that has been proposed to involve a cytoskeletal-mediated contraction of the spike towards the sperm base (Rios and Barros 1997; Lynn and Clark 1983b). Nevertheless, it is still ambiguous about the subsequent molecular events of how sperm penetrate through the egg envelope following their adherence to the egg vestment. In addition, M. rosenbergii sperm possess unusual morphology and localization of the acrosome, features that are dissimilar to those of other nantantian species such as Sicyonia ingentis (Clark and Griffin 1988), Penaeus monodon (Pongtippatee et al. 2007) and Pandalopsis japonica (Kim et al. 2003). Whether or not M. rosenbergii sperm undergo a virtual acrosome reaction is still a challenging issue to be addressed. Extensive studies in mammals have revealed that sperm binding to egg ligands leads to a clustering of sperm receptors and an activation of downstream cascades of the signaling pathway (Primakoff and Myles 2002; Shur et al. 2004; Litcher et al. 2009). As a consequence of sperm receptor clustering, a calcium-dependent acrosomal exocytosis takes place—a consensus molecular event that is also conserved across marine invertebrates (Collins and Epel 1977; Matsumoto et al. 2008; Li et al. 2010). In most penaeid

Cell Tissue Res

shrimp, the formation of the acrosomal filament (Clark et al. 1981; Clark and Griffin 1993) or its equivalent structure such as the spherical mass (Kruevaisayawan et al. 2008; Pongtippatee et al. 2007) is evident after calcium-induced exocytosis. The formation of the acrosomal filament in Sicyonia ingentis has been proven to be trypsin activitydependent (Griffin and Clark 1990; Lindsay and Clark 1992). Moreover, trypsin-like enzymes among other acrosomal enzymes have been shown to be indispensable for the successful penetration of sperm through the egg VE in many animal species (Clark et al. 1981; Yanagimachi 1994; Yokota and Sawada 2007). The significance of trypsin-like enzymes in other aspects of fertilization has also been reported in invertebrates (Levine and Walsh 1979; Lewis et al. 1982). Here, we connected the gapped information that shrimp sperm trypsin-like enzymes were enhanced and specifically localized to the sperm anterior spike that aided sperm penetration through the egg vitelline envelope.

Materials and methods Prawns and sperm collection Blue claw giant freshwater prawns, known to be sexually mature and containing a highly homogeneous sperm population (Poljaroen et al. 2010), were obtained from a farm in Ayutthaya province, Thailand. Prawns were handled according to the guidelines of the Animal Care Committee, Mahidol University, Thailand. They were anaesthetized in ice-cold water until no movement was detected. Sperm were collected from different parts of male reproductive tracts: testis (Tsp), proximal (pVsp) and distal (dVsp) vas deferens. For collecting Tsp, testes were carefully dissected and minced with razor blades. The tissue pieces were gently agitated to release testicular sperm into phosphate-buffered saline (PBS) and filtered through a 45-μm metal sieve (Endercotts, London, UK). Sperm in the vas deferens, which includes both the proximal (coiled–coiled appearance) and distal vas deferens (straight-enlarged portion) including gonopores, were collected and minced into small pieces. The tissues were agitated and filtered as mentioned above. Sperm isolated from all tissues were washed (500g, 4 °C, 10 min) and resuspended in PBS to a final concentration of 1 × 107 cells/ml and used for the following experiments.

sperm were pelleted by centrifugation (500g, 4 °C, 10 min) and both sperm pellets and supernatants were collected. Sperm pellets of Tsp, pVsp and dVsp were either kept live or subjected to vigorous Dounce’s homogenization or extracted with Triton X-114 (Vanichviriyakit et al. 2004). Cellular debris from homogenization was discarded by centrifugation (20,000g, 4 °C, 10 min). All samples including live sperm, their supernatants and sperm homogenates were used for enzyme assays using fluorogenic substrates in black 96-well plates (Thermo Scientific, Rochester, NY, USA). The substrates used were fluorogenic-4-methylcoumarin-7-amide (MCA)-conjugated peptides with trypsin-specific cleavage sites (t-butyloxycarbonyl (Boc)-QAR-MCA, Boc-LSTRMCA and Boc-FSR-MCA) (Peptide Institute, Louisville, KY, USA). The reaction mixture contained 10-μl sperm samples, 80 μl trypsin assay buffer (10 mM Tris-HCl, 5 mM CaCl2, pH 7.5) and 10 μl of 20 μM fluorogenic substrates. Freely-released amidomethyl coumarin (AMC) from cleaved MCA-conjugated substrates were then measured at 5-min intervals for 90 min at an excitation wavelength of 350 nm and an emission wavelength of 460 nm using a Wallac Spectrofluorometer (Perkin Elmer, Waltham, MA, USA). Inhibition of sperm enzyme activities was also performed using trypsin inhibitors including 1 mM soybean trypsin inhibitor (SBTI; Sigma, St. Louis, MO, USA) and 1 mM 4amidinophenyl-methanesulfonylfluoride (APMSF; EMD Merck, Darmstadt, Germany). Inhibitors were pre-incubated with live sperm or their lysates (1 h, room temperature). Pretreated samples were then incubated with the reaction mixtures for measuring the remaining enzyme activities under the same condition mentioned above. In-gel substrate localization For visualizing the localization of enzymes in situ, sperm were allowed to interact with in-gel substrates overlaid on the coverslip as modified from Yi et al. (2001) to mimic the sperm interaction with the eggs. To construct the gel substrate, trypsin QAR substrate at a concentration of 20 μM was mixed with 0.66 % agarose and polymerized to form a thin gel layer on the coverslip. A23187-induced or non-induced sperm were resuspended in the enzyme assay buffer and overlaid on the agarose gel coverslip. After a 90 min incubation, sperm on the coverslips were examined for blue fluorescence under a fluorescence microscope using the excitation/emission filters of 355/460 nm wavelengths.

Fluorogenic enzyme assays Trypsin localization and image reconstruction Measurements of enzyme activities were performed both on live cells, lysate of sperm pellets and the corresponding supernatants as well as membrane extracted with Triton X-114. In either case, live sperm were left untreated or treated with 1– 20 μM A23187 (room temperature, 5 min). Thereafter, the

Sperm were either non-treated or treated with A23187 ionophore following the conditions described above. The method of sperm staining by fluorophore-labeled SBTI was modified from that of Jones and Williams (1990). Briefly, sperm were

Cell Tissue Res

incubated with 15 μg/ml biotinylated-soybean trypsin inhibitor (SBTI) followed by exposure to 1.25 μg/ml avidin conjugated with Alexa 488, emitting green fluorescent signals (Life Technology, Carlsbad, CA, USA). Sperm were centrifuged (500g, 10 min) to wash away excess of avidin, re-suspended and mounted with PBS-glycerol (1:1, v/v). In some cases, they were also pretreated with non-labeled SBTI prior to the incubation with biotinylated SBTI. The samples were counterstained with TOPRO3, a DNA staining dye (Life Technology) and visualized using an Olympus FV1000 confocal microscope with argon and krypton laser lines using a line-by-line and Kalman’s scanning modes. The acquired images were three-dimensionally constructed using Amira™ software (Visage Imaging, Berlin, Germany). Scanning electron microscopy To visualize the structural changes of sperm by scanning electron microscopy (SEM), the A23187-induced or non-induced sperm were fixed with 4 % paraformaldehyde and 2.5 % glutaraldehyde in 50 mM sodium cacodylate buffer pH 7.4. They were dehydrated with increasing concentration of alcohol and subjected to critical point drying and heavy metal coating. The sperm samples were examined under a Hitachi scanning electron microscope at 25 kV. To examine sperm penetration through egg vestment, three conditions of sperm were prepared prior to incubation with freshly spawned eggs (induced by a 14/10 light-dark cycle and maintained in 20 % glycerol in PBS) including (1) noninduced sperm, (2) A23187-induced and (3) sperm pretreated with 1 mM SBTI followed by A23187 induction. The sperm– egg complexes were then fixed with 4 % paraformaldehyde and 2.5 % glutaraldehyde and processed accordingly as mentioned above.

with glutaraldehyde fixative and processed into resin blocks in the same condition mentioned above. The sperm were sectioned, stained with 1 % uranyl acetate and visualized under the electron microscope.

Results Presence of trypsin- like enzymes in shrimp sperm Enzyme assays using fluorescent substrates showed that the lysates of testicular sperm (Tsp) possessed a significantly higher trypsin-like activity (158.02 ± 48.03 μmole/μg protein/min) than those of sperm collected from the proximal vas deferens (pVsp) and the distal vas deferens (dVsp including spermatophoric sperm) (Fig. 1a). Among the three trypsin substrates, Tsp lysates exerted the highest affinity towards the QAR substrate, which was ~4- to 5-fold more than the other trypsin substrates, FSR and LSTR. We further investigated whether the enzymes were localized to the sperm plasma membrane as they are crucial for digesting a path for the sperm entry into the egg proper. Fluorometric enzyme assays using fluorescent QAR substrates were carried out with live Tsp, pVsp and dVsp samples. Among the three sperm samples, live dVsp hydrolyzed QAR

Inhibitor-based ultrastructural localization of trypsin-like enzymes For transmission electron microscopy (TEM), sperm were fixed with the same fixative as SEM and post-fixed with 1 % tannic acid. The samples were then dehydrated and embedded in Spurr’s Resin. Ultrathin sections (~70 nm) were counterstained with uranyl acetate and Reynold’s lead citrate prior to examining under a FEI Tecnai20 transmission electron microscope operated at 80 kV. The images were acquired by a Gatan CCD camera at a resolution of 1024 × 1024 pixels. In order to examine the localization of trypsin-like enzymes on the sperm surface, live A23187-induced sperm were incubated with 15 μg/ml biotinylated SBTI (room temperature, 2 h). They were then exposed to 1.25 μg/ml streptavidin conjugated to HRP and diaminobenzidine (DAB) in 0.1 % H2O2 followed by incubating with 0.1 % NiCl2 (room temperature, 2 h). After extensive washing with PBS, sperm were fixed

Fig. 1 Fluorogenic enzyme assay of trypsin-like activities in three different sperm samples. The activities were measured either in sperm lysates using three different fluorescent trypsin substrates: 200 nM of QAR, FSR and LSTR (a) or in live sperm (b). Note that trypsin-like activities are substantially detected in Tsp lysates but are relatively low in the corresponding live sperm, while the surface trypsin-like activity is found to be highest in sperm collected from distal vas deferens (dVsp)

Cell Tissue Res

trypsin substrate, giving an enzyme activity of 11.13 ± 1.65 μmole/μg protein/min (calculated from triplicate experiments), which was >6-fold higher than those obtained from pVsp and Tsp (1.74 ± 0.47 and 0.47 ± 0.03 μmole/μg protein/ min, respectively) (Fig. 1b). Calcium ionophore A23187 enhanced trypsin-like activity in M. rosenbergii sperm We searched for an optimal concentration of A23187 in enhancing enzyme activities in dVsp sperm (which are located distal-most in the tract and readily to be released for fertilization). It was found that A23187 at a concentration of 20 μM generated maximal sperm trypsin-like enzyme activity (as measured in the sperm pellet) of 73.90 ± 2.23 μmole/μg protein/ min (Fig. 2a). Trypsin-like activities at 1 μM and 5 μM A23187 in the sperm pellets were 17.77 ± 2.82 and 36.60 ± 1.32 μmole/ μg protein/min, respectively. Membrane fractions extracted by Triton X-114 from the dVsp induced by A23187 showed a considerably high trypsin-like activity of 87.48 ± 4.94 μmole/ μg protein/min. In contrast, the trypsin-like enzyme activity of the membrane fraction extracted from the non-induced sperm was 5.66 ± 1.14 μmole/μg protein/min (Fig. 2b). It is noteworthy that the supernatants of live dVsp either non-induced or induced by A23187 showed minimal trypsin-like enzyme activity of 1.83 ± 0.55 μmole/μg protein/min (Fig. 2b), thus indicating minimal cell damage that might cause the leakage of enzymes into the medium during sperm preparation. Two inhibitory assays for calcium and enzyme activities were conducted. For calcium flux inhibition, sperm were treated with 1 mM EDTA + 20 μM A23187. The result revealed a decrease in sperm trypsin-like activity by ~65 % as compared to that of A23187 treated sperm. Furthermore, the addition of 2 mM extracellular Ca2+ (without A23187) did not enhance the activity of the enzymes (Fig. 2b). Interestingly, the activities of trypsin-like enzymes in the supernatants were as low as