Portal de Periódicos da CAPES

Spatiotemporal expression pattern of gonad-stimulating substance-like peptide of the sea cucumber, Apostichopus japonicus

2011; Wiley; Volume: 53; Issue: 5 Linguagem: Inglês

10.1111/j.1440-169x.2011.01277.x

1440-169X

Hamdy Omar Ahmed, Tomoko Katow, Hideki Katow,

Protein Hydrolysis and Bioactive Peptides

Development, Growth & DifferentiationVolume 53, Issue 5 p. 639-652 Original ArticleFree Access Spatiotemporal expression pattern of gonad-stimulating substance-like peptide of the sea cucumber, Apostichopus japonicus Hamdy O. Ahmed, Hamdy O. Ahmed Research Center for Marine Biology, Tohoku University, Asamushi, Aomori, Aomori 039-3501, JapanSearch for more papers by this authorTomoko Katow, Tomoko Katow Research Center for Marine Biology, Tohoku University, Asamushi, Aomori, Aomori 039-3501, JapanSearch for more papers by this authorHideki Katow, Corresponding Author Hideki Katow Author to whom all correspondence should be addressed.Email: [email protected]Search for more papers by this author Hamdy O. Ahmed, Hamdy O. Ahmed Research Center for Marine Biology, Tohoku University, Asamushi, Aomori, Aomori 039-3501, JapanSearch for more papers by this authorTomoko Katow, Tomoko Katow Research Center for Marine Biology, Tohoku University, Asamushi, Aomori, Aomori 039-3501, JapanSearch for more papers by this authorHideki Katow, Corresponding Author Hideki Katow Author to whom all correspondence should be addressed.Email: [email protected]Search for more papers by this author First published: 14 June 2011 https://doi.org/10.1111/j.1440-169X.2011.01277.xCitations: 7AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat Abstract The spatiotemporal expression pattern of gonad-stimulating substance-like peptide-containing polypeptide (GSSLP) in the sea cucumber Apostichopus japonicus was examined using immunochemistry. The GSSLP was detected in the gonads from shortly before the empirical breeding season (May and June) to July. On the basis of immunoblotting analysis, GSSLP showed considerable polymorphism among the organs examined in this study, particularly in the gonads, in which the polymorphism was associated with N-glycosylation and the formation of intra-molecular disulfide bonds. In the ovary, GSSLP was expressed from March to June and corresponded to two bands at 113 and 100 kDa under reducing conditions. In July, only the larger band weakly remained. In testis, GSSLP was detected first in April as two bands of 245 and 190 kDa under reducing conditions. The number of bands increased to five in June but decreased to three smeared bands in July. In the radial nerve and circumoral nerve ring, GSSLP corresponded to a single peptide of 170 kDa with little N-glycosylation and its expression level hardly changed throughout a year with no correlation with the breeding season. GSSLP was detected mainly in the morula cells in all the organs examined. In addition, GSSLP was detected in the follicle cells of the ovary and, for a brief period, in the jelly space, but never in the ooplasm. In testis, the morula cells were localized close to the invaginated inner epithelium, but never in the male gametes. In July animals, gonadal morula cells were rarely observed. Introduction In echinoderms, the maturation of oocytes is stimulated by a substance that is found in extracts of the radial nerve (RN): gonad-stimulating substance (GSS) in starfish (Kanatani 1967; Strathmann & Satoh 1969) and sea urchins (Cochran & Engelmann 1972), and maturation-inducing factor (MIF; Maruyama 1985) or gonad-stimulating substance-like peptide (GSSL; Katow et al. 2009) in sea cucumbers. In starfishes and sea urchins, GSS is a heat stable small peptide hormone (Kanatani & Shirai 1969; Cochran & Engelmann 1975) and stimulates spawning (Kanatani & Shirai 1969; Cochran & Engelmann 1972). An in vitro bioassay for GSS that used isolated fragments of starfish ovaries indicated that the concentration of GSS is the same irrespective of the sex of the organism from which it is derived, and also the level does not differ between the RN and the circumoral nerve ring (CONR) (Kanatani & Ohguri 1966). Reverse transcription–polymerase chain reaction (RT–PCR) (Mita et al. 2009a) and in situ hybridization (Mita et al. 2009b) revealed that the mRNA for GSS is transcribed exclusively in the RN/CONR. Despite the year-round presence of GSS in the RN, it is detected in the coelomic fluid exclusively during the natural breeding season (Kanatani & Ohguri 1966). An analysis of the distribution of radioiodinated synthetic GSS peptide using tissue lysates showed that the peptide is localized to the fraction of the ovary that is rich in follicle cells and the fraction of the testis that is rich in interstitial cells (Mita et al. 2007). Thus, it appears to be the system that is responsible for the delivery of GSS from the RN to the gonads that is correlated with the breeding season. However, the exact cytological location and molecular properties of the GSS detected in the coelomic fluid, RN, and the gonads remain unknown. In sea cucumbers, MIF has been isolated from the RN of five species, including the present Apostichopus japonicus (Maruyama 1985). Recently, the mRNA of GSSL was isolated from the RN of A. japonicus, and the DNA and protein sequences of GSSL were determined (Katow et al. 2009). Subsequently, an antibody against GSSL was raised and the immunochemical properties of GSSL were characterized. GSSL isolated from the RN is in a 170 kDa polypeptide (GSSLP), and characteristically contains multiple protease-sensitive amino acids. Immunohistochemical analysis shows that GSSL is localized in the morula cells of the hyponeural part and epineural sinus of the RN, and is transported by these cells to the edge of the coelomic epithelium. The cells of the coelomic epithelium are presumed to secrete GSSL into the body cavity (Katow et al. 2009). Around northern Japan, the breeding season of A. japonicus is from approximately late May to early June. However, as for most echinoderms, the neural regulation of the breeding season, particularly the relationship between gametogenesis and neurosecretion, has not been well characterized. Thus, the aims of the study reported herein were as follows: (i) to locate the histological targets of the GSSL that is presumed to be secreted into the body cavity; (ii) to identify the organs and cells in which GSSL can be detected during the breeding season; and (iii) to characterize the molecular conformation and localization of GSSLP in the gonads during the breeding season using immunoblotting and immunohistochemistry with confocal laser scanning microscopy. Materials and methods Adult sea cucumbers of the species A. japonicus were collected monthly throughout a year by scuba divers near the Research Center for Marine Biology. Gonads were detected exclusively in the animals collected from November to the following July, and not detected at all from August to October. The gonads from the animals collected from November to the following February were rudimental. Thus, the animals collected from February to August were subjected to investigation in this study. Immunoblotting The samples for immunoblotting were prepared using a previously described technique with minor modifications (Katow et al. 2009). The RN and CONR (wet weight of approximately 2 g), together with surrounding tissues that included the overlying epithelia of the radial canal and the body wall, were dissected from both males and females. The fragments that corresponded to the ovarian tubules and testicular tubules were dissected under a dissection microscope. They were washed twice with filtered seawater (FSW), and excess seawater was wiped away with paper towel. The tubules were then cut into 5 mm-long pieces on ice using a razor blade. Oocytes with follicle cells were squeezed out gently from fecund tubules of the ovary through ruptures that had been made using fine-tipped forceps, transferred to 15 mL test tubes, and then spun down using a hand centrifuge. The follicular inner epithelium (FIE) was separated from the oocytes by incubating the mixture of cells in artificial seawater that lacked calcium and magnesium for 10 min and gently agitating with a pipette. Then the mixture was stood still in the 15 mL test tubes for 10 min at ambient temperature (AT). During this period, heavy oocytes sunk on the bottom of tubes, while light FIE floated in the supernatant. The FIE in supernatant was collected and transferred to fresh tubes, pelleted in a hand centrifuge and washed once with fresh FSW. The tissue fragments were suspended in 3 mL of lysis buffer (6 mol/L urea, 1% Nonidet P-40, 10 mmol/L Tris–HCl, pH 7.6), and homogenized with a glass homogenizer at 500 rpm for 10 min. The sample was precipitated by adding cold ethanol to a final concentration of 75% (v/v) and stored at −20°C overnight. The precipitates were centrifuged at 1500 g for 10 min, washed twice with 100% cold ethanol, freeze-dried by using a VD-800F Vacuum Freeze Dryer (lyophilized sample; TAITEC, Koshigaya, Japan), and stored at −80°C until use. Each lyophilized sample was dissolved in sodium dodecyl sulfate–acrylamide gel electrophoresis (SDS–PAGE) sample buffer with or without 2-mercaptoethanol at 1 mg/mL for the ovary, isolated FIE, RN, and CONR samples, or 3 mg/mL for the testis samples. The samples were separated on 8% or 10% SDS–PAGE slab gels, and transferred electrophoretically to nitrocellulose filters (Schleicher and Schuell, Dassel, Germany) at 400 mA for 2 h at 4°C. The nitrocellulose blots were blocked with 5% (w/v) skim milk in TBST (50 mmol/L Tris–HCl, pH 7.0, 0.15 mol/L NaCl, and 0.05% Tween-20) for 1 h at room temperature on a rocking deck. The blots were incubated for 2 h with an antibody against GSSL (diluted 1:500 in TBST; Katow et al. 2009). After the blots had been washed three times with TBST (10 min each), they were incubated for 1 h with alkaline phosphatase-conjugated goat anti-mouse IgG antibody (diluted 1:30 000 in TBST; Sigma-Aldrich, St. Louis, MO, USA). After washing with TBST three times (10 min each), binding of the primary antibody was visualized using nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate (Promega, Madison, WI, USA) in accordance with the manufacturer's protocol. To examine the specificity of the GSSL antibody, the antibody (diluted 1:1000 in TBST) was adsorbed with 3 mg/mL antigen peptide (AEIDDLAGNIDY; Katow et al. 2009) to give adsorbed-antibody-B, which was then incubated with the blots of the ovary and testis samples on a 12-channel Mini-Blotter (Sanplatec Co., Osaka, Japan). The purity of the antigen peptide was >50% (immunological grade; Greiner Japan, Tokyo, Japan). To determine the relative intensities of the 113 and 100 kDa bands, a mouse anti-acetylated α-tubulin antibody (Cell Signaling Technology Japan, K.K., Tokyo, Japan) was used to probe the blots that had been made using an aliquot of the same sample as that used to probe the anti-GSSL antibody. The intensities of the 113 and 100 kDa bands were then compared with the intensity of the acetylated α-tubulin band using the image analyzing software ImageJ version 1.37 (public domain imaging software from National Institutes of Health [NIH]). The semi-quantitative analysis was repeated three times using three sets of immunoblots. The data were standardized by setting the amount of immunoreaction for the 113 kDa polypeptide in the samples obtained in March as a relative intensity of "1". The unpaired t-test was applied to compare the samples from (i) March and May; (ii) March and July; and (iii) May and July for the 113 kDa polypeptide, and between (iv) April and May and (v) April and June for the 100 kDa polypeptide, using the public domain GraphPad Software QuickCalcs (http://www.graphpad.com/quickcalcs/ttest1.cfm). A two-tailed P-value ≤0.05 was taken to indicate a statistically significant difference. The numbers of animals examined in each month from January to December were 15, 11, 11, 12, 19, 24, 28, 30, 26, 19, 28, and 20, respectively. N-glycopeptidase F (PNGase F) digestion The lyophilized samples of the ovary, testis, and RN were all dissolved in denaturation buffer (0.5% SDS and 1% 2-mercaptoethanol at 1 mg/mL), heated at 100°C for 10 min, and mixed with 0.1 volumes of 10× reaction buffer (0.5 mol/L sodium phosphate, pH 7.5) and 0.1 volumes of 10% Nonidet P-40 with or without 0.05 volumes of PNGase F (50 units/mg, Sigma-Aldrich Co.). The reaction mixtures were incubated at 37°C for 1 h, and the reaction was stopped by adding an equal volume of 2× SDS–PAGE sample buffer with 2-mercaptoethanol and heating for 5 min at 100°C. The pattern of digestion was examined by immunoblotting using the anti-GSSL antibody as described above. Immunohistochemistry with Polywax-embedded sections Samples of the CONR, ovary, and testis were cut into small pieces (approximately 2 mm long) with razor blades on ice as described above. The pieces were then fixed in 4% paraformaldehyde in FSW for 30 min, dehydrated in increasing concentrations of ethanol (30%, 50%, and 70%), and stored at 4°C until use. The specimens were dehydrated further in a series of ethanol at increasing concentrations from 80% to twice (20 min each) at 100%, infiltrated once with a 1:1 (v/v) mixture of pure ethanol and Polywax for 20 min, and then twice with pure Polywax (20 min each). The samples were embedded in fresh Polywax and sectioned with a microtome at 6 μm. The sections were then dewaxed in 100% ethanol three times (15 min each), air-dried at AT for 10 min, and hydrated in PBST (0.1 mol/L phosphate-buffered saline with 0.1% (v/v) Tween-20) twice (10 min each) in accordance with a previously described method (Katow 1995). The sections were pre-incubated with 1% (w/v) bovine serum albumin in PBST for 30 min at AT to block non-specific binding of the antibody, and then incubated with anti-GSSL antibody (diluted 1:300 in 0.1 mol/L PBST) overnight in a moist chamber at 4°C. Then, they were washed with PBST three times (10 min each), and incubated with Alexa Fluor 488-conjugated goat anti-mouse IgG antibody (diluted 1:500 in PBST; Molecular Probes Inc., Eugene, OR, USA) at AT for 2 h. The sections were washed again three times with PBST (10 min each), mounted in glycerol, and examined under a Micro-Radiance 2000 Confocal Laser Scanning Microscope (Bio-Rad, Hercules, CA, USA). The resulting images were analyzed using ImageJ version 1.37 (NIH). The immunospecificity of the antibody was examined using antibody diluted 1:300 in PBST and adsorbed with 3 mg/mL antigen peptide (adsorbed-antibody-H), and then utilized as described above. In addition to being stained with the anti-GSSL antibody, representative sections were stained with 1 μg/mL propidium iodide (PI) diluted in PBST for 5 min to locate nuclei. Whole-mount immunohistochemistry Oocytes with follicle cells were fixed in 4% paraformaldehyde in FSW, dehydrated, and stored as described above. The samples were then hydrated in a series of decreasing concentrations of ethanol from 50% to 30%, followed by two incubations in PBST (10 min each). Finally, the samples were double-stained with anti-GSSL antibody and PI, and examined under a laser scanning confocal microscope as described above. Results Polymorphic appearance of GSSL-containing polypeptide In this text, GSSL means an AEIDDLAGNIDY (Katow et al. 2009) antigen peptide used for raising the present antibody, and thus was mainly used in describing immunohistochemical localization of the peptide in which one does not know relative molecular mass (Mr) of polypeptides that contain GSSL in the cells or tissues. GSSL-containing polypeptide (GSSLP) means polypeptides that contain GSSL and show various Mr as will be shown in the following text, and thus was used mainly in immunoblotting analysis in which one can easily recognize Mr of a polypeptide. The anti-GSSL antibody only recognized a single band of 170 kDa in the RN extract under reducing conditions, as shown previously (Katow et al. 2009). However, in the testis samples obtained from animals that were collected during May, the antibody bound to three bands, which corresponded to 240, 170, and 82 kDa (Fig. 1A, lane 1). None of these three bands was detected with adsorbed-antibody-B (Fig. 1A, lane 2), which indicated that they were antibody-specific bands. In the ovary samples from animals collected during May, the antibody bound to two bands, which corresponded to 113 and 100 kDa (Fig. 1A, lane 3). The signal for these two bands was considerably weaker with adsorbed-antibody-B (Fig. 1A, lane 4), which indicated that they were also antibody-specific bands. Thus, the molecular conformation of the GSSLP in the ovary was different from that in the testis. Figure 1Open in figure viewerPowerPoint Polymorphic expression pattern of gonad-stimulating substance-like peptide-containing polypeptide (GSSLP) in the radial nerve (RN), testis, and ovary as determined by immunoblotting. (A) Immunospecificity test of the anti-GSSL antibody against GSSLP in the ovary and testis under reducing conditions. The anti-GSSL antibody recognized three bands, which corresponded to 240, 170, and 82 kDa, in the testis (lane 1); these bands were not detected by adsorbed-antibody-B (lane 2). The antibody detected two bands in the ovary, which corresponded to 113 and 100 kDa (lane 3), and were not detected by adsorbed-antibody-B (lane 4). (B) Polymorphism of GSSLP among organs and during the breeding season was identified on 8% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) gels. Under non-reducing conditions (lanes 1–5), the GSSLP from the RN was a single band of 170 kDa (lane 1). In the testis, two bands of 210 and 78 kDa were observed for animals collected in May (lane 2), but, in June, the number of bands was increased to three by the addition of a band that corresponded to 75 kDa (lane 3). A band around 55 kDa region was non-specific immunoreaction, as the bands were seen in adsorbed antibody treatment (A, lane 2). However, in the ovary for animals collected in both May and June, two bands of 100 and 85 kDa were observed (lanes 4 and 5). Under reducing conditions (lanes 6–10), a single band of 170 kDa was detected in the RN extract (lane 6). In the testis, three bands were seen in the samples collected in May and corresponded to 240, 170, and 82 kDa (lane 7). In the samples from June, the number of the bands increased to five, which corresponded to 245, 240, 190, 90, and 75 kDa (lane 8). In the ovary, although the double-band pattern remained under non-reducing conditions, the relative molecular masses of the bands were 113 and 100 kDa (lanes 9 and 10), namely 13–15 kDa larger than those seen under reducing conditions in May and June (lanes 4 and 5). Apparent lower molecular weight bands at 0.05), and March and July (P < 0.05) for the 113 kDa peptide, and April and May (P = 0.067) and June and July (P < 0.05) for the 100 kDa peptide. Vertical bars are standard deviations (SD). Data are means ± SD. In contrast to the expression of GSSLP in the RN, detection of the protein in the ovary was associated closely with the breeding season. In February, no GSSLP was detected in the ovary (Fig. 3B, lane 1 of upper row). On the basis of semi-quantitative analysis of GSSLP using acetylated α-tubulin as a standard (Fig. 3B, lower row), a band of approximately 113 kDa (Fig. 3C, darker columns) appeared from March and remained at a similar intensity through June, but then decreased significantly in July (Fig. 3C). However, the intensity of the 100 kDa polypeptide band changed dynamically over the period from March to July, which encompassed the breeding season. The intensity of the 100 kDa band was considerably stronger in May, in effect, during the breeding season, than in March and April (Fig. 3B, lane 4, C), and decreased thereafter in June (Fig. 3B, lane 5). In July, no immunoreaction of the 100 kDa polypeptide was detected (Fig. 3B, lane 6, C, gray columns). In the ovaries from animals collected in February, the ovarian tubules were empty and no GSSL was detected immunohistochemically, except in a few morula cells near the peritoneal inner epithelium and the connective tissue under the epithelium (Fig. 4A, rectangle and inset). Regarding the above negative immunoblotting results from animals collected in February; an apparent extracellular immunopositive signal at the connective tissue was nonspecific. In the animals collected in March, however, the ovarian tubules had acquired previtellogenic oocytes near the peritoneal inner epithelium and were filled with numerous vitellogenic oocytes. The FIE around the vitellogenic oocytes showed a weak immunopositive signal for GSSL (Fig. 4B). In the animals collected in April, the positive signal for GSSL at the FIE had increased considerably around the fully-grown oocytes that filled the ovarian tubules (Fig. 4C). GSSL-immunopositive spots that resembled morula cells were also found in the peritoneal inner epithelium in the ovaries of animals collected in May (Fig. 4C, arrows). In these animals, the fecund ovarian tubules were filled with fully-grown oocytes that were lined with FIE with a strong immunopositive signal for GSSL (Fig. 4D). On the basis of double staining with the anti-GSSL antibody and PI, the morula cells were also major GSSL-positive components in the ovary. These cells were located on the side of the ovarian lumen of the peritoneal inner epithelium (Fig. 4E, rectangle [f], F). Adsorbed-antibody-H did not bind to these morula cells (Fig. 4G, rectangle [h], H), which indicated that the immunopositive signal in these cells was antibody specific. The cytoplasm of the FIE cells was not stained evenly with the antibody, but rather the immunostaining was associated with fibrillar features (Fig. 4E, rectangle (i), I). In the animals collected in June, overall, the GSSL-immunopositive features in the ovary remained similar to those observed in the samples collected in May, except for a slight visible decrease in the intensity of the signal (Fig. 4J). In the animals collected in July, the ovary was reduced in size as compared with previous months and was characterized by emptied ovarian tubules that were filled with numerous small cells with scant cytoplasm, and by the absence of GSSL-immunopositive FIE and morula cells (Fig. 4K). An apparent extracellular immunopositive signal in the connective tissue was caused by non-specific binding of the antibody, as described above for the ovaries from the animals collected in February. However, large cells that showed weak staining for GSSL in the cytoplasm and fragmented PI-positive grains, which were presumably aggregates of DNA fragments, were seen in the ovarian tubules (Fig. 4K rectangle [l], L, arrowheads). The morphology of these cells resembled that of phagocytes, and the decreased intensity of the 113 kDa polypeptide and the disappearance of the 100 kDa polypeptide (Fig. 3B, lane 6) in the ovarian samples from July, as shown by immunoblotting, could be related