The Japanese Society of Developmental Biologists

Develop. Growth Differ. (2011) 53, 639652

doi: 10.1111/j.1440-169X.2011.01277.x

Original Article

Spatiotemporal expression pattern of gonad-stimulating substance-like peptide of the sea cucumber, Apostichopus japonicus
Hamdy O. Ahmed, Tomoko Katow and Hideki Katow*
Research Center for Marine Biology, Tohoku University, Asamushi, Aomori, Aomori 039-3501, Japan

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 disulde 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 rst in April as two bands of 245 and 190 kDa under reducing conditions. The number of bands increased to ve 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. Key words: breeding season, gonad-stimulating substance-like peptide polypeptide, morula cells, polymorphism, sea cucumber.

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 starsh (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 starshes 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).
*Author to whom all correspondence should be addressed. Email: hkatow@m.tohoku.ac.jp Received 30 November 2010; revised 11 January 2011, 27 January 2011; accepted 27 January 2011. 2011 The Authors Development, Growth & Differentiation 2011 Japanese Society of Developmental Biologists

An in vitro bioassay for GSS that used isolated fragments of starsh 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 transcriptionpolymerase chain reaction (RTPCR) (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 uid 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

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properties of the GSS detected in the coelomic uid, RN, and the gonads remain unknown. In sea cucumbers, MIF has been isolated from the RN of ve 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 modications (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 ltered 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 ne-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 articial 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 oated 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 TrisHCl, 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 nal concentration of 75% (v v) and stored at )20C 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 )80C until use. Each lyophilized sample was dissolved in sodium dodecyl sulfateacrylamide 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 lters (Schleicher and Schuell, Dassel, Germany) at 400 mA for 2 h at 4C. The nitrocellulose blots were blocked with 5% (w v) skim milk in TBST (50 mmol L TrisHCl, 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

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tetrazolium 5-bromo-4-chloro-3-indolyl phosphate (Promega, Madison, WI, USA) in accordance with the manufacturers protocol. To examine the specicity 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 a-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 a-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 twotailed P-value 0.05 was taken to indicate a statistically signicant 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 100C 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, SigmaAldrich Co.). The reaction mixtures were incubated at 37C for 1 h, and the reaction was stopped by adding an equal volume of 2 SDSPAGE sample buffer with 2-mercaptoethanol and heating for 5 min at 100C. The pattern of digestion was examined by immuno-

blotting 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 xed in 4% paraformaldehyde in FSW for 30 min, dehydrated in increasing concentrations of ethanol (30%, 50%, and 70%), and stored at 4C until use. The specimens were dehydrated further in a series of ethanol at increasing concentrations from 80% to twice (20 min each) at 100%, inltrated 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 lm. 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-specic 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 4C. Then, they were washed with PBST three times (10 min each), and incubated with Alexa Fluor 488conjugated 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 immunospecicity 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 lg mL propidium iodide (PI) diluted in PBST for 5 min to locate nuclei. Whole-mount immunohistochemistry Oocytes with follicle cells were xed 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

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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-specic 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-specic bands. Thus, the molecular conformation of the GSSLP in the ovary was different from that in the testis. To examine whether the bands recognized by the antibody were derived from a single GSSLP or from several polymorphic polypeptides, the banding patterns for GSSLP from the RN, testis, and ovary during the empirical breeding season were compared between non-reducing and reducing conditions (Fig. 1B). For the RN extract, the antibody bound to a band of 170 kDa under both non-reducing and reducing conditions (Fig. 1B, lanes 1 and 6, respectively), which suggested that GSSLP is a single peptide in the RN. However, in the testis samples from animals collected during May, two bands were recognized by the anti-GSSL antibody under non-reducing conditions (210 and 78 kDa; Fig. 1B, lane 2), and this increased to three bands (240, 170, and 82 kDa; Fig. 1B, lane 7) under reducing conditions. Furthermore, in the testis

samples from animals obtained during June, four GSSLP bands were recognized under non-reducing conditions, which corresponded to the 210 and 78 kDa bands observed in the May samples together with an additional 75 kDa band and a double band in the region of 210 kDa (210 kDa-a, and 210 kDa-b; Fig. 1B, lane 3). Under reducing conditions, the number of bands recognized in the testis samples from June increased further to ve (245, 240, 190, 90, and 75 kDa; Fig. 1B, lane 8). In the ovary samples, although a similar double-band pattern was observed under both non-reducing and reducing conditions, the overall Mr of the two bands were lower under nonreducing conditions (100 and 85 kDa; Fig. 1B, lanes 4, 5) than under reducing conditions (113 and 100 kDa; Fig. 1B, lanes 9, 10). Thus, overall, the Mr values of the polymorphic bands that corresponded to GSSLP from the ovary and testis were lower under non-reducing conditions than under reducing conditions, which suggested the presence of intra-molecular disulde bonds. Immunoblotting under reducing conditions also suggested that the amino acid sequence AEIDDLAGNIDY, which is the peptide recognized by the anti-GSSL antibody, was present in three to ve polypeptides in the testis and two polypeptides in the ovary. In testis, the GSSLP might comprise multiple polypeptides that contain the amino acid sequence AEIDDLAGNIDY and are linked by disulde bonds (to give the larger bands at around 210 kDa; 210 kDa-a and 210 kDa-b; Fig. 1B, lane 3), whereas the protein bands in the ovary that contained the AEIDDLAGNIDY sequence were two distinctive polypeptides. Pattern of GSSLP expression in the RN and CONR The anti-GSSL antibody bound to a single band of 170 kDa in the RN extract from both males (Fig. 2A, lane 1) and females (Fig. 2A, lane 2). After digestion with PNGase F, no visible changes were seen (Fig. 2A; lane 3 for sample without the enzyme, lane 4 for that with the enzyme), which suggested that there was little or no N-glycosylation of the GSSLP in the RN. The intensity of immunoreaction and the Mr of the 170 kDa polypeptide did not change throughout the period from January (Fig. 2A, lane 5) to August (Fig. 2A, lane 11). Therefore, the expression of GSSLP was not related to the breeding season. In starsh, GSS is known to be transcribed in both the RN and CONR (Mita et al. 2009a,b). To examine whether the expression pattern of GSSLP in the CONR differed from that in the RN in A. japonicus, the CONR was analyzed immunohistochemically. GSSL was detected around the CONR (Fig. 2B, arrow),

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Fig. 1. 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) Immunospecicity 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 identied on 8% sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDSPAGE) gels. Under non-reducing conditions (lanes 15), 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-specic 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 610), 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 ve, 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 1315 kDa larger than those seen under reducing conditions in May and June (lanes 4 and 5). Apparent lower molecular weight bands at <55 kDa region of testis (lanes 2, 3, 7, and 8) are non-specic signal as they were still seen by adsorbed-antibody treatment (A, lane 2).

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Fig. 2. Spatiotemporal expression pattern of gonad-stimulating substance-like peptide (GSSL)-containing polypeptide (GSSLP) in the radial nerve (RN) and the circumoral nerve ring (CONR). (A) Immunoblotting of GSSLP in the RN extract (upper row: lane 1, male; lane 2, female; lane 3, without N-glycosidase F (PNGase F) digestion; lane 4, after PNGase F digestion. Seasonal expression in January (lane 5), February (lane 6), March (lane 7), April (lane 8), May (lane 9), June (lane 10), and August (lane 11, upper row). Immunoreaction of acetylated a-tubulin (aTub) was used as a standard to analyze the GSSLP expression semi-quantitatively (lower row). (B) Confocal microscopic image of a Polywax section shows the immunohistochemical location of GSSL on the hyponeural part (arrow) and the epineural sinus (double arrows) of the CONR (nr). The square (c) indicates the morula cells that are shown at higher magnication in (C). Bar represents 50 lm. (C) Confocal microscopy of the morula cells indicated by the square in (B) and stained with both the anti-GSSL antibody (green) and propidium iodide (magenta indicates the nuclei). Bar represents 20 lm. (D) Immunoblotting of GSSLP in the CONR extract showed a single band of 170 kDa in both sexes (lane 1, male; lane 2, female). 2011 The Authors Development, Growth & Differentiation 2011 Japanese Society of Developmental Biologists

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which was similar to the nding of a previous report on the RN (Katow et al. 2009). As in the RN, GSSL was localized exclusively in the morula cells of the CONR (Fig. 2C). The Mr of GSSLP in the CONR was similar to that in the RN (approximately 170 kDa), and no difference in the molecular conformation of GSSLP was identied between males (Fig. 2D, lane 1) and females (Fig. 2D, lane 2). Seasonal expression pattern in the ovary The two polypeptides of the ovarian GSSLP differed in terms of N-glycosylation. PNGase F digested the 100 kDa band, but the larger 113 kDa band was unchanged visually (Fig. 3A, lanes 1, 2), which indicated that only the smaller peptide was N-glycosylated. 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 a-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 signicantly in July (Fig. 3C). However, the intensity of the 100 kDa polypeptide band changed dynamically over the period from March to July, which
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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 nonspecic. In the animals collected in March, however, the ovarian tubules had acquired previtellogenic oocytes near the peritoneal inner epithelium and were lled 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 lled 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,

Fig. 3. Expression pattern of gonad-stimulating substance-like peptide-containing polypeptide (GSSLP) in the ovary. (A) N-glycosidase F digestion of the two bands of GSSLP from the ovary (lane 1) resulted in a single band with a molecular mass of 113 kDa (lane 2). (B) Seasonal expression pattern of GSSLP in the ovary. GSSLP was not detected in the animals that were collected in February (lane 1), but could be detected weakly in the samples collected in March with the characteristic double-band pattern (lane 2). Both bands were strong in the samples collected from April (lane 3) to May (lane 4), decreased in intensity a little in June (lane 5), and in July the 100 kDa band had disappeared and the 113 kDa band alone could be detected weakly (lane 6). (C) Semi-quantitative analysis of GSSLP expression using immunoreaction of acetylated a-tubulin (aTub) as a standard. Dark columns show the levels of the 113 kDa polypeptide. Gray columns show the levels of the 100 kDa polypeptide. Statistical analysis was conducted to compare samples from March and April, March and June (P > 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. 2011 The Authors Development, Growth & Differentiation 2011 Japanese Society of Developmental Biologists

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Fig. 4. Immunohistochemical location of gonad-stimulating substance-like peptide (GSSL) in the ovary using Polywax sections. (A) February: virtually no GSSL-positive signal was seen in the ovary, except in the morula cells near the peritoneal inner epithelium (pie) and the connective tissue under the epithelium (rectangle and inset). Bar represents 100 lm. (B) March: weak but distinctive GSSL immunopositive signals were detected at the follicular inner epithelium (FIE; arrows) in the ovarian tubules. Bar represents 50 lm. (C) April: arrows show spots of area that stained positive for GSSL. Bar represents 100 lm. (D) May. Bar represents 100 lm. (E) Ovary from animal collected in May double stained with anti-GSSL antibody (green) and propidium iodide (PI; magenta). Rectangles (f) and (i) indicate the regions shown at a higher magnication in (F) and (I). Bar represents 50 lm. (F) High magnication of the morula cells indicated with a rectangle in (E). PI-stained nuclei (magenta) were seen in the GSSL-rich morula cells (green). Bar represents 10 lm. (G) In the ovary from an animal collected in May, immunostaining of the morula cells (rectangle [h]) and around the oocytes was signicantly weaker with adsorbed-antibody-H than with the unadsorbed antibody. Bar represents 50 lm. (H) Higher magnication of the morula cells shown in rectangle (h) in (G) with very weak GSSL immunoreaction. Bar represents 10 lm. (I) Higher magnication of the FIE (green) with a nucleus (magenta) indicated by rectangle (i) in (E). Bar represents 20 lm. (J) Ovary from animal collected in June. Bar represents 100 lm. (K) Double-stained ovary from animal collected in July. No oocytes were seen in the ovarian tubules (ot), which were lled with nuclei and cells that resembled phagocytes (rectangle [l]). Bar represents 100 lm. (L) Higher magnication of the part of the ovarian tubule indicated by rectangle (l) in (K) shows cells with cytoplasm that was immunostained weakly for GSSL and aggregates of PI-positive DNA fragments (arrowheads). Bar represents 30 lm. o, oocytes; ol, ovarian lumen; ot, ovarian tubule; pie, peritoneal inner epithelium; po, previtellogenic oocytes; vo, vitellogenic oocytes.

the fecund ovarian tubules were lled 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 specic. The cytoplasm of the FIE cells was not stained evenly with the antibody, but rather the immunostaining was associated with brillar 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

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characterized by emptied ovarian tubules that were lled 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-specic 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 to this immunohistochemical observation. To examine the spatial distribution of GSSL around the oocytes, whole-mount immunohistochemistry (WMIHC) was conducted (Fig. 5). The oocytes were found to be surrounded by a layer of FIE (namely, follicle cells) that were connected to the ovarian inner epithelium through the stalk region. However, the

epithelium did not contact the surface of the oocytes directly but was separated from the oocytes by the jelly space (Fig. 5A). In small younger oocytes, which had a wider jelly space, GSSL was detected in the cytoplasm of the FIE cells in the region that was connected to the stalk region (Fig. 5B, double arrows) and in the jelly space on the surface of the oocytes (Fig. 5B,C, green arrow). The intensity of immunostaining of GSSL at the base of the stalk region of the FIE was considerably stronger than that in the distal region (Fig. 5B, white arrow). However, around the larger grown oocytes, GSSL was not detected in the narrower jelly space and thus was not present on the surface of the oocytes (Fig. 5D,E). The intensity of GSSL immunostaining at the FIE was stronger in the grown oocytes than in the younger oocytes (Fig. 5C,E). In both younger and fully-grown oocytes, GSSL was never detected in the ooplasm, which agreed with the results obtained from immunohistochemistry using the Polywax sections. GSSL from both the ovary and isolated follicle cells gave a similar double-band pattern and intensity of immunoreaction upon immunoblotting. These ndings showed that GSSLP had a similar molecular conformation in both the ovary and follicle

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Fig. 5. Double-stained whole-mount immunohistochemistry of oocytes with the follicle cells from animals collected in May. (A) Follicular inner epithelium (FIE) and an oocyte are shown (adapted from Smiley & Cloney 1985). (B) Stacked confocal microscopy image of a complex of a younger oocyte and the FIE. Arrow, germinal vesicle of the oocyte; magenta arrows, nuclei of the FIE cells; green arrows, gonad-stimulating substance-like peptide (GSSL); double-headed white arrow, stalk; white arrow, GSSL-rich base of stalk. Bar represents 50 lm. (C) An optical section showing GSSL on the surface of an oocyte (green arrow) and in the FIE. Magenta arrows, nuclei of FIE cells; white arrowhead, germinal vesicle. (D) Stacked confocal microscopy image of a complex of fully-grown oocytes and FIE. GSSL was seen in association with the FIE. Magenta arrows, nuclei of the epithelium; white arrowhead, germinal vesicle. (E) An optical section of the same oocyte shown in (D) shows a strong GSSL-positive signal in the FIE with GSSL-negative ooplasm. Magenta arrow, nucleus of the epithelium; white arrowhead, germinal vesicle. (F) Immunoblotting shows the double-band pattern of the GSSL polypeptide from the ovary (lane, ovary) and the follicle cells (lane, FC). o, oocyte. Bars in (BD) represent 50 lm. 2011 The Authors Development, Growth & Differentiation 2011 Japanese Society of Developmental Biologists

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(A)

(B)

Fig. 6. Seasonal expression pattern of gonad-stimulating substance-like peptide-containing polypeptide (GSSLP) in the testis. (A) Sample before N-glycosidase F digestion (lane 1) and the appearance of a smaller band of 38 kDa after enzyme digestion (lane 2). (B) Seasonal expression pattern of GSSLP. Before the breeding season, from February to March, GSSLP was not detected (lanes 1, 2). In April, during the pre-breeding season, GSSLP was detected as two bands of 245 240 and 190 kDa (lane 3). However, the intensity of the smaller band was signicantly weaker than that of the larger band. During the breeding season in May, bands of 245 240 and 190 kDa were detected, together with an additional smaller strong band of 90 kDa (lane 4). In animals collected in June, a further smaller band that corresponded to 75 kDa was detected, which gave four bands of GSSLP in total (lane 5). After the breeding season, in July, the two smaller bands were replaced with a broad smeared single band and the two larger bands also showed a broad smeared pattern (lane 6). A band around the 55 kDa region was non-specic as described in Fig. 1B. The lower row shows immunoreaction of acetylated a-tubulin (aTub) for the same samples that were probed with the anti-GSSL antibody (upper row).

cells (Fig. 5F), which suggested that FIE is the major source of immunopositivity of GSSL in the ovary. Seasonal expression pattern in the testis Given that the 100 kDa ovarian GSSLP was N-glycosylated, we examined whether the three GSSLP found in the testis underwent N-glycosylation. Digestion of the protein with PNGase F resulted in the appearance of a weak band that corresponded to 38 kDa without an apparent decrease in the Mr of any of the three polypeptides (Fig. 6A, lanes 1, 2). Although the origin of the 38 kDa peptide was not identied, the GSSLP of the testis might contain an N-glycosylated component. Similarly to GSSLP in the ovary, the expression of this protein changed dynamically in association with the progress of the breeding season. Although GSSLP was not detected in the testes of animals collected in February or March (Fig. 6B, lanes 1, 2), a clear signal in the region of 245 240 kDa and a weak signal in the 190 kDa region (Fig. 6B, lane 3) were observed in samples collected in April, a month before the empirical breeding season began. In animals collected in May, three bands were detected in the regions that corresponded to 245 240, 190, and 90 kDa (Fig. 6B, lane 4). In samples collected in June, the number of bands increased to four with the addition of a smeared band in the region of 75 kDa (Fig. 6B, lane 5). In samples from July, which was after the end of the breeding season, the four bands could still be detected but all of them were smeared, which suggested that the polypeptides were being degraded (Fig. 6B, lane 6).

Consistent with the immunoblotting results described above, no GSSL could be detected in the testes from the animals collected in March by immunohistochemistry using the Polywax sections, which included the invaginated inner epithelium region (Fig. 7A, arrow). In the testes of animals collected in April, before the breeding season began, punctate strong immunostaining for GSSL was seen along the invaginated inner epithelium (Fig. 7B, arrow). This staining remained strong throughout the breeding season in May (Fig. 7C) and June (Fig. 7D, arrow). In the samples collected during May, the tip regions of the invaginated inner epithelium were stained more strongly than the regions near the junction with the hemal sinus (Fig. 7C, arrow). However, in the testes of animals collected in July, after the breeding season had ended, no GSSL immunoreaction was seen in the invaginated epithelium (Fig. 7E, rectangle and arrow). GSSL was detected sporadically near the hemal sinus region of the invaginated inner epithelium that was associated with morula cells or phagocytes in the samples from July (Fig. 7E, inset). Double staining of samples collected in May during the breeding season revealed large PI-stained nuclei of spermatocytes spermatogonia (Thongkukiatkul et al. 2008), which were aligned closely with the invaginated inner epithelium, and smaller nuclei of spermatids spermatozoa, which were around the area between two epithelia (Fig. 7F), together with GSSL in the morula cells in the invaginated inner epithelium (Fig. 7F, insets [a] and [b]). The GSSL-immunopositive regions did not react with adsorbed-antibody-H (Fig. 7G,H), which indicated that the staining was

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(A)

(B)

(C)

(D)

(F) (E)

(G)

(H)

Fig. 7. Immunohistochemical analysis of the testis using Polywax sections. (A) Gonad-stimulating substance-like peptide (GSSL) was not detected in the testis of animals collected in March. An apparent strong GSSL signal on the surface of the testis was caused by non-specic binding of the antibody. In the animals collected in April (B), May (C), and June (D), GSSL was detected strongly in the invaginated inner epithelium (arrow). (E) In animals collected in July, no immunoreaction was seen in the region of the invaginated inner epithelium (arrow), and a few positive signals were seen near the epithelium. The inset shows a higher magnication image of the double-stained invaginated inner epithelium that is indicated with a rectangle. GSSL (green) was detected in a swollen cell with a single nucleus (magenta) located near the invaginated inner epithelium. (F) Double staining of the testis from an animal collected in May shows GSSL in the morula cells (green) in the invaginated inner epithelium (rectangles [a] and [b]). The larger and smaller PI-positive dots (magenta) in the gonadal tubules are the nuclei of spermatocytes spermatogonia and spermatids, respectively. Insets (a) and (b) are higher magnication images of the regions indicated by rectangles and show morula cells (green). (G) Higher magnication image of double-stained morula cells (green) in the invaginated inner epithelium of the testis of an animal collected in June. (H) The amount of GSSL staining was lower when the cells were incubated with adsorbed-antibody-H (arrow). Arrow in (AE) denotes invaginated inner epithelium; arrow in (GH) denotes morula cells. Bars in (AD), (E), inset of (E), (F), and (GH), and insets (a) and (b) of (F) represent 200, 100, 60, 50, and 10 lm, respectively.

antibody specic and suggested that the morula cells are the major source of GSSL immunopositivity in the testis.

Discussion
Seasonal expression of GSSL in the gonads Active MIF can be isolated from the RN of the sea cucumber A. japonicus throughout the year, including the breeding season (Maruyama 1985), which is similar to ndings for GSS in starsh (Chaet 1966). Consistent with these previous reports, the immunochemical studies reported herein revealed that GSSL was present in the RN of A. japonicus throughout the year. However, if the breeding season is regulated by GSSL in the RN, these observations would suggest that the sea cucumber does not have a discrete breeding season, which is obviously not true.

Immunohistochemistry and assays of physiological activity have shown that GSSL (Katow et al. 2009) or MIF (Maruyama 1985) in the sea cucumber and GSS in starsh (Kanatani & Ohguri 1966) are present in various tissues, which include the body wall and the longitudinal muscles. However, MIF (Maruyama 1985) and GSS (Kanatani & Ohguri 1966) derived from these non-RN tissues are much less active than those derived from the RN. GSS mRNA is transcribed almost exclusively in the RN and CONR and not in the gonads (Mita et al. 2009a), but the coelomic uid shows GSS activity only during the breeding season (Kanatani & Ohguri 1966). In fact, the gonads are the targets of GSS from the RN (RN-GSS) and are the only organs in the body cavity that receive GSS during the breeding season. This targeting of GSS has been demonstrated repeatedly by in vitro GSS application studies in starsh (Kanatani 1967), and probably also occurs in sea cucumbers on the basis of similar

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in vitro GSSL application studies (Maruyama 1985; Katow et al. 2009). In the sea cucumber, GSSL could be detected in the ovary from March, i.e. before the beginning of the breeding season. However, the molecular conformation and the expression pattern of GSSLP during the subsequent breeding season differed signicantly from the constant and steady expression of GSSLP in the RN. For example, two distinct polypeptides (113 and 100 kDa) were observed in the ovary as compared with one polypeptide of 170 kDa in the RN and the levels of these polypeptides uctuated during the months from March to July. These observations suggest that the ovary does not simply receive GSSL from the RN (RN-GSSL) through the uid of the body cavity, but rather the ovary might produce its own GSSLP in the form of two polypeptides, one of which is N-glycosylated, and regulate the temporal expression of each peptide. Furthermore, the uctuations in the amount of GSSLP, which were detected by semiquantitative immunoblotting, were correlated with the expression of GSSL in the morula cells at the hemal sinus and FIE, as detected by immunohistochemistry. Therefore, the gonadal GSSLP could be synthesized by these morula cells. The dynamic shift in the distribution of GSSL as the oocytes developed from the stalk region at the base of younger oocytes to the large area of FIE that surrounds fully-grown oocytes might suggest that GSSL is delivered from the morula cells in the hemal sinus to the FIE through the stalk region. However, direct contact between these morula cells and the FIE cannot be involved in the transportation of GSSL. The basal plate of the oocyte (or oolamina) separates the hemal sinus uid from the jelly space that surrounds the oocyte (Smiley & Cloney 1985). Thus, the oolamina might prevent the morula cells from contacting the FIE (Fig. 5A). If RN-GSSL induces the gonads to produce gonad-type GSSLP, then the production of gonadspecic forms of GSSLP can be explained irrespective of how many polymorphic polypeptides are produced in the gonads. Furthermore, with regard to the report that GSS is transcribed only in the RN in starsh, in situ hybridization showed that the transcripts are localized in large cells in the peripheral region of the RN (Mita et al. 2009b). These cells resemble the morula cells of the sea cucumber morphologically (Katow et al. 2009). Thus, RN-GSSL might activate the transcription of a gene for gonad-type GSSLP in the morula cells of the gonads, and the resulting transcripts might undergo alternative splicing to yield the different polymorphic forms of GSSLP (Hypothesis 1; Fig. 8). The RTPCR analysis of starsh GSS was carried out using a single set of primers that were

designed using the cDNA sequence obtained from mRNA extracted from the RN (Mita et al. 2009a). Therefore, the primer set used might not amplify the alternatively spliced forms of the mRNA. The probes for the in situ hybridization were also synthesized using a single set of primers that included the same forward primer used for the RTPCR and thus might not have detected the alternative forms (Mita et al. 2009b). In contrast, recent studies of polymorphism commonly use multiple sets of primers that have been shown to detect multiple variants (Elzanaty et al. 2006 Heinzen et al. 2008; Prokunina-Olsson et al. 2009). Thus, it is possible that the transcription of GSSLP (possibly GSS) is not restricted to the RN but occurs in other tissues as well, including the gonads. In support of this proposal, peptide hormones such as GSSL bind to their cognate G-protein-coupled receptors, which are embedded in the plasma membrane of their target cells (Koenig 1997), and do not accumulate in the target cells. However, in the present study, GSSL was detected within FIE cells in the ovary and morula cells in the testis. Thus, the present immunohistochemical observations strongly suggest that the positive signal for GSSL in these gonadal cells is derived from protein that is synthesized de novo in these cells, and that GSSLP mRNA is transcribed within these cells. On the other hand, given that the molecular conformation of GSSLP in the FIE cells was identical to that of GSSLP in the ovary and that GSSL was detected in the morula cells, the polypeptide might be transported from the morula cells to the FIE. In FIE cells, GSSLP might be modied chemically to give the 113 and 100 kDa polypeptides of the ovarytype GSSLP (Hypothesis 2). The nding that ovarytype GSSLP were smaller than RN-GSSLP might be explained by proteolytic modication and N-glycosylation of the 100 kDa polypeptide, as will be discussed later in this section in relation to the polymorphic pattern of expression. The immunohistochemical ndings also indicated that GSSL is present on the surface of younger oocytes in the jelly space in addition to within the FIE, which showed a weak immunoreaction. These observations during the early period of oocyte growth and the consistent absence of GSSL in the ooplasm throughout the entire period suggest that GSSL interacts with the surface of young oocytes briey and is then restricted to the follicle cells by a mechanism that has yet to be investigated. The signicance of early direct contact of GSSL with younger oocytes is yet to be studied. In the testis, the results of the immunoblotting analysis described herein showed that the seasonal changes in GSSLP expression were associated with more extensive

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(A)

(B)

(C)

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Fig. 8. Schematic summary of the signaling pathway for gonad-stimulating substance-like peptide containing polypeptide (GSSLP) and Hypothesis 1 for GSSLP signaling. (A) In female and male animals, GSSLP mRNA is transcribed in the morula cells (green) in the radial nerve (RN-GSSLP; Katow et al. 2009), and the resulting polypeptide is secreted into the body cavity and reaches the gonads through the coelomic uid. (B) In the ovary, ovarian GSSLP is synthesized in response to RN-GSSLP in the morula cells in the stalk region of the hemal sinus. The oocyte basal plate at the conjunction of the follicular inner epithelium (FIE) and hemal sinus prevents morula cells to approach the jelly space (sky blue) around the oocyte. Ovarian GSSLP synthesized in the morula cells is transported to the FIE. (C) In the testis, testicular GSSLP is synthesized in the morula cells in the hemal sinus in response to RN-GSSLP and is then transported to the cells of the invaginated inner epithelium. In both the ovary and testis, GSSLP does not contact the gametes directly. (D) Hypothesis 1. RN-GSSLP (blue ovals) encounters the morula cells in the gonads and triggers the production of gonad-type GSSLP (magenta ovals for female GSSLP, yellow ovals for male). The GSSLP then diffuses from the morula cells to either the follicular cells or the invaginated inner epithelial cells and induces these cells to produce maturation-inducing factor (rectangles), which in turn induces oocyte maturation or spermatogenesis (Maruyama 1985).

polymorphism than that observed in the ovary and with the appearance of polypeptides that were larger than RN-GSSLP. To explain these ndings with Hypothesis 2, at least two new properties of GSSLP must be assumed: (i) the presence of multiple repeated AEIDDLAGNIDY peptide sequences in a single RN-GSSLP and (ii) the ability of GSSLP to polymerize to produce larger peptides. Both of these hypothetical properties need to be examined in future studies. The occurrence of extensive polymorphism in the testis was not correlated with the distribution pattern

of GSSL, which was not detected near the male germ cells by immunohistochemistry. This suggested that GSSL does not affect the male germ cells directly, at least not to a degree that can be detected by the immunohistochemical techniques used in the present study. This observation is reminiscent of the presumed role of MIF, which promotes the maturation of oocytes indirectly through biochemically uncharacterized secondary factors that are released from MIF-stimulated follicle cells (Maruyama 1985). Morula cells were frequently detected in the present study, and their

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localization and morphology are reminiscent of similar cells found in the testis of starsh. In the latter, secondary factor (1-methyladenine) is not produced by male gametes but rather by a group of free large interstitial cells that measure 710 lm in diameter and are associated closely with the germinal epithelium (namely, the invaginated inner epithelium of the sea cucumber testis) (Kubota et al. 1977). The signicance of the polymorphism of the GSSLP remains to be investigated in future studies. However, it has been suggested that a polymorphic hydra gene of a subgroup of the species Drosophila melanogaster is involved in male fertility through affecting late-stage spermatogenesis (Chen et al. 2007), and there have been other reports on the polymorphism of the Deleted in Azoospermia gene (Kim et al. 2009) and the H2BFWT (H2B histone family, member W, testisspecic) gene (Lee et al. 2009) in human testis and transferrin in the seminal plasma of carp (Cyprinus carpio) (Wojtczak et al. 2007). Therefore, the polymorphism examined in this study could also be involved in spermatogenesis to some extent. Expression pattern in the RN and CONR The similar molecular properties of the GSSLP isolated from the RN and CONR in the present study were simply due to the fact that these two regions are united anatomically and developmentally (Nakano et al. 2006; Mashanov et al. 2007). The continuous expression of RN CONR-GSSLP as a single polypeptide with similar relative intensity before, during, and after the breeding season strongly suggests that this protein is not involved in the regulation of gametogenesis during the breeding season, at least not to a degree that can be measured by current molecular approaches. Instead, gametogenesis could be regulated by the response of the target organs to RN-GSSL to produce gonad-type GSSLP, with the onset of the breeding season being determined by the time at which a particular accumulated seawater temperature is reached, a stimulus that has been used empirically by numerous marine hatcheries. Polymorphic expression pattern The unexpected expression pattern of GSSLP that was identied in this study arose from numerous polymorphic forms of GSSLP that differed among the organs we examined. Our previous report of the protein structure of GSSLP implicated proteolysis in the production of the polypeptide (Katow et al. 2009). In starsh, the cDNA for GSS encodes 116 amino acids, whereas its product is comprised of only 44 amino

acids owing to proteolytic cleavage (Mita et al. 2009b). Thus, both GSSLP and GSS are proteolytic products of larger precursor GSSLP and GSS proteins, respectively. This suggests that GSS could also have polymorphic forms in the gonads. There has been an intriguing observation with respect to the potential posttranslational processing of the protein: the ADAM (a disintegrin and metalloproteinase) proteins can produce several distinctive smaller proteins from a precursor protein through a multiple proteolysis process that is known as ectodermal shedding (Edwards et al. 2008). However, ADAM proteins only produce smaller proteins, whereas the Mr of the GSSLP did not simply decrease, but also increased during the breeding season in the testis. Thus, posttranslational proteolysis alone, as postulated in Hypothesis 2 above, might not be able to explain the observed polymorphism. As a consequence, Hypothesis 2 can be excluded as a likely explanation for the expression pattern of GSSLP that was observed in this study. The fact that the polypeptides with different Mr values were all recognized by the anti-GSSL antibody, and therefore presumably contained the same amino acid epitope, might suggest alternative splicing of the GSSLP mRNA, as proposed in Hypothesis 1. Such alternative splicing has been reported for proteins such as APOBEC3H, a member of the APOBEC3 family of cytidine deaminases, for which alternative splicing is common in humans and results in variants with distinct C-terminal regions (Harari et al. 2009). Although it remains controversial whether different polymorphic products of a gene have different functional roles (Keyt et al. 1996; Huhtaniemi & Themmen 2005), polymorphic proteins in the gonads have been examined extensively with respect to human pathology. For example, the close relationship of variants of the 5a-reductase gene with sperm concentration and motility has been investigated (Elzanaty et al. 2006), as well as the consequences of gonadotropin polymorphism on the functional activities of the protein (Huhtaniemi & Themmen 2005). The polymorphic forms of GSSLP that were evaluated in the present study also showed characteristic tissue-specic patterns, as have been seen for the transcription factor 7-like 2 (TCF7L2) gene in humans. One splice form of TCF7L2 is unique to pancreatic islets, the pancreas, and colon, and is not found in other tissues. It has been shown that alternative TCF7L2 exons are incorporated into the transcript in a tissue-specic manner (Prokunina-Olsson et al. 2009). The characteristic expression pattern of polymorphic forms of GSSLP in the testis described herein was correlated closely with the progression of the breeding season. To date, this observation of polymorphism associated with the maturation of oocytes in

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echinoderms is unique to the GSSLP of the sea cucumber. Future analysis of the function and developmental signicance of polymorphic forms of GSSLP in the ovary and the testis might shed new light on, and further understanding of, the neural regulation of the breeding season in the sea cucumber.

Acknowledgments
This research was funded by the Egyptian Government Scholarship for PhD Program to HOA. The present project was supported in part by Aomori Research, Art and Culture Foundation to HK. We thank Mr M. Washio, Research Center for Marine Biology, Tohoku University, for collecting sea cucumbers throughout this study.

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