- Email: [email protected]
Bilateral eyestalk ablation of the blue swimmer crab, Portunus pelagicus, produces hypertrophy of the androgenic gland and an increase of cells producing insulin-like androgenic gland hormone
Tissue and Cell 42 (2010) 293–300
Contents lists available at ScienceDirect
Tissue and Cell journal homepage: www.elsevier.com/locate/tice
Bilateral eyestalk ablation of the blue swimmer crab, Portunus pelagicus, produces hypertrophy of the androgenic gland and an increase of cells producing insulin-like androgenic gland hormone Morakot Sroyraya a , Charoonroj Chotwiwatthanakun a,b , Michael J. Stewart c , Nantawan Soonklang d , Napamanee Kornthong a , Ittipon Phoungpetchara a , Peter J. Hanna a,e , Prasert Sobhon a,∗ a
Department of Anatomy, Faculty of Science, Mahidol University, Rama 6 Road, Bangkok 10400, Thailand Mahidol University, Nakhonsawan Campus, Nakhonsawan 60000, Thailand c School of Medicine, Deakin University, Geelong, Victoria 3217, Australia d Department of Preclinical Science, Faculty of Medicine, Thammasat University, Pathumthani 12121, Thailand e Faculty of Science and Technology, Deakin University, Geelong, Victoria 3217, Australia b
a r t i c l e
i n f o
Article history: Received 22 April 2010 Received in revised form 21 June 2010 Accepted 1 July 2010
Keywords: Portunus pelagicus Androgenic gland Insulin-like androgenic gland hormone Eyestalk ablation Hypertrophy
a b s t r a c t The androgenic glands (AG) of male decapod crustaceans produce insulin-like androgenic gland (IAG) hormone that controls male sex differentiation, growth and behavior. Functions of the AG are inhibited by gonad-inhibiting hormone originating from X-organ-sinus gland complex in the eyestalk. The AG, and its interaction with the eyestalk, had not been studied in the blue swimmer crab, Portunus pelagicus, so we investigated the AG structure, and then changes of the AG and IAG-producing cells following eyestalk ablation. The AG of P. pelagicus is a small endrocrine organ ensheathed in a connective tissue and attached to the distal part of spermatic duct and ejaculatory bulb. The gland is composed of several lobules, each containing two major cell types. Type I cells are located near the periphery of each lobule, and distinguished as small globular cells of 5–7 m in diameter, with nuclei containing mostly heterochromatin. Type II cells are 13–15 m in diameter, with nuclei containing mostly euchromatin and prominent nucleoli. Both cell types were immunoreactive with anti-IAG. Following bilateral eyestalk ablation, the AG underwent hypertrophy, and at day 8 had increased approximately 3-fold in size. The percentage of type I cells had increased more than twice compared with controls, while type II cells showed a corresponding decrease. © 2010 Elsevier Ltd. All rights reserved.
1. Introduction Blue swimmer crabs, Portunus pelagicus, have a high economic value, and constitute important marine fisheries in tropical countries (Sumpton et al., 1994; Chande and Mgaya, 2003; Abdel Razek et al., 2006; Chaiyawat et al., 2008). They exhibit sexual dimorphism, with males growing faster and being bigger than females. Therefore, monosex culture of males could increase monetary returns to farmers, and this may be achieved by reversing the sex of females to males (Mires, 1977; Sagi and Aflalo, 2005). The control of male sex differentiation in crustaceans by the androgenic gland (AG) was first described in the amphipod, Orchestia gammarella (Charniaux-Cotton, 1954). The AG plays a major role in the development of the male gonad and secondary sexual characteristics, while inhibiting female secondary charac-
∗ Corresponding author. Tel.: +66 2 2015406; fax: +66 2 3547168. E-mail addresses: [email protected], tokaro [email protected] (P. Sobhon). 0040-8166/$ – see front matter © 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.tice.2010.07.003
teristics. Masculinization of females by implantation of AG has been shown in several isopod species, including O. gammarella (Charniaux-Cotton, 1954), Asellus aquaticus (Balesdent-Marquet, 1958), Armadillidium vulgare (Hasegawa and Katakura, 1985; Suzuki and Yamasaki, 1998), and Porcellio dilatatus and Helleria brevicornis (Juchault and Legrand, 1978). Implantation of AG’s into juvenile female isopods results in the development of male appendages and spermatogenesis, as well as inhibition of female secondary characteristics and vitellogenesis (Charniaux-Cotton, 1954; Katakura, 1960). Conversely, removal of the AG from juvenile or mature males causes degeneration of spermatogenesis and the appearance of vitellogenesis (Charniaux-Cotton, 1954). In decapod crustaceans, removal of the AG from male produces regression of male characteristics, while implantation of AG into juvenile females results in the inhibition of vitellogenesis and development of male sexual characteristics (Nagamine et al., 1980; Nagamine and Knight, 1987; Sagi et al., 1990; Malecha et al., 1992; Lee et al., 1993). Implantation of AG into female mud crabs, Scylla paramamosain, results in ovarian regression, with degeneration of oocytes (Cui et al., 2005).
294
M. Sroyraya et al. / Tissue and Cell 42 (2010) 293–300
The AG is considered to be under inhibitory control of the X-organ-sinus gland complex located in the eyestalk. In Cherax quadricarinatus and Pandalus platyceros, bilateral removal of the eyestalk leads to hypertrophy of the AG (Hoffman, 1968; Khalaila et al., 2002). Moreover, RNA synthesis in the AG increases in eyestalkablated shrimp (Foulks and Hoffman, 1974). The structure of the AG has been studied in many decapods species, including Pachygrapsus crassipes (King, 1964), Ocypoda platytarsis (Thampy and John, 1970), Macrobrachium kistnensis (Mirajkar et al., 1984), Ranina ranina (Minagawa et al., 1994), Penaeus chinensis (Li and Xiang, 1997), Macrobrachium rosenbergii (Okumura and Hara, 2004), Litopenaeus vannamei (Campos-Ramos et al., 2006), S. paramamosain (Liu et al., 2008), and Maja brachydactyla (Simeo et al., 2009). Although the AG gland is generally composed of groups of round or oval cells with round nuclei, the cells in the AG can be classified into 2–3 types according to species. Type I are small cells of approximately 5–12 m in diameter and have a large nucleus/cytoplasm ratio. The cytoplasm stains intensely with hematoxylin. Type II are larger cells of approximately 15–20 m in diameter and have a small nucleus/cytoplasm ratio. The cytoplasm stains lightly with hematoxylin. Type III are the largest cells of approximately 20–25 m in diameter and have prominent cytoplasmic vacuoles. Studies have indicated that the AG secretes a protein hormone, called androgenic gland hormone (AGH). In A. vulgare (Negishi et al., 2001), the ultrastructure of AG cells shows cytoplasm consisting of numerous rough endoplasmic reticulum, Golgi complex, and secretory granules. Similarly, the ultrastructure of the AG cells of P. crassipes (King, 1964), M. rosenbergii (Okumura and Hara, 2004), and Procambarus clarkii (Taketomi, 1986), indicates that the gland secretes a high level of proteins. There have been limited studies of hormones produced by the AG of decapod crustaceans. Using a suppression subtractive hybridization (SSH) technique, a male-specific transcript encoding an insulin-like factor (IAG) was identified in the AG of C. quadricarinatus, and termed Cq-IAG (Manor et al., 2007). The structure of the gene encoding Cq-IAG shares similarity to those of mammals. The Cq-IAG contains a signal peptide, a B chain, a C peptide and an A chain. Using the same SSH technique, a Mr-IAG gene in M. rosenbergii has been recently cloned and characterized (Ventura et al., 2009). Upon silencing of the Mr-IAG gene by RNAi, spermatogenesis was arrested, and growth and development of male secondary sex characteristics were also inhibited. This evidence indicated that IAG plays a role in decapods in a manner similar to AGH in other crustaceans. In decapods, several studies have reported the structure and ultrastructure, as well as the hormone produced by the AG, using molecular cloning. However, there are no reports on changes of the cell populations in the AGs due to eyestalk ablation, or localization of IAG. Therefore, in the present study, we investigated the structure of the AG, particularly under an accelerated hormone production condition caused by bilateral eyestalk ablation, and showed IAG immunolocalization in normal and hypertrophy AGs. Moreover, we also provided the sequence of the Portunus pelagicus IAG (Pp-IAG). We report here that the AG has two main cell types (I and II), and that hypertrophy of the AG occurring from bilateral eyestalk ablation causes increases in the number of type I cells, and the numbers of IAG-producing cells.
2. Materials and methods Mature male blue swimmer crabs, P. pelagicus, with a carapace width of 80–90 mm, were reared in rectangular concrete tanks, at the Department of Aquatic Science, Faculty of Science, Burapha University. The rearing conditions were: sea water with salinity at
20 ppm, a temperature of 29 ± 3 ◦ C, constant aeration, a dark:light cycle of 12:12 h, and feeding with boiled fish twice a day. 2.1. Experimental design Mature males were randomly divided into three groups (n = 18 per group): (1) a control (no eyestalk ablation), (2) a unilateral eyestalk-ablated, and (3) a bilateral eyestalk-ablated group. An aseptic technique was used to cut off one eye or two eyes from each experimental male. Each treated crab (including controls) was then placed in a separate basket and reared in a rectangular tank. At days 0, 4, and 8, following the eyestalk ablation, six crabs from each group were sacrificed and the AG from three crabs were collected and prepared for light microscopic observation, and AG from the another three crabs were prepared for immunolocalization of PpIAG. In the control group, an extra two crabs were used for studying gross anatomy of the AG and spermatic duct (SD) and ejaculatory bulb (EB). A 1 ml volume of blue ink was injected into the ejaculatory bulb to assist in locating the AG and studying its gross anatomy within the wall of the EB. 2.2. Light microscopy The carapaces were removed from the crabs, and the distal SD’s and EB’s, with the attached AG’s, were carefully removed. The tissues were fixed in Davidson’s fixative (330 ml 95% ethyl alcohol, 220 ml 100% formalin, 115 ml glacial acetic acid, and 335 distilled H2 O), for 24 h, dehydrated in 70%, 80%, 90%, and 100% ethanol, and transferred to toluene. They were then infiltrated and embedded in paraffin. The embedded tissues were cut at a thickness of 5 m using a rotary microtome (Leica RM2235). The sections were transferred onto gelatin coated slides, deparaffinized with xylene, and then rehydrated in 100%, 95%, 90%, 80%, and 70% ethanol for 5 min each. The sections were then placed in distilled water for 5 min, stained with Harris’s hematoxylin for 1.5 min, and placed in tap water for 15 min. The sections were counterstained with eosin for 2 min, dehydrated in 80%, 90% and 100% ethanol, and then immersed 3 times in xylene. The slides were mounted with permount, and the tissues examined and photographed with a Nikon E600 microscope equipped with a DXM 1200 digital camera, using an ACT-1 software package. 2.3. Cell counting Three AGs from each group were counted by ImageJ software. The numbers of type I and type II cells were counted in cross-sections of an AG, when they possessed complete circular cross-sectional profiles of the associated SD and EB. The quantities of type I and type II were recorded as percentages of the total cells. 2.4. Preparation of an antibody against Cq-IAG The ProtScale Tool from the ExPAsy Server (http://www.expasy.ch/tools/protscale.html) was used to analyzed the position of amino acid to synthesize the peptide for antibody production. A 9-amino acid peptide (RRRNSDTTD), corresponding to the most hydrophilic part of Cq-IAG (aa 128–136, accession number DQ851163.1), was synthesized (GenScript, NJ, USA). Two 8-week old female New Zealand white rabbits were used to produce a polyclonal antibody against the Cq-IAG peptide, as approved by the Animal Care Committee, Faculty of Science, Mahidol University. Before immunization, the synthetic peptide was conjugated to bovine serum albumin (BSA) (Sigma–Aldrich, St. Louise, MO, USA), using 1-ethyl-3-dimethylaminopropyl carbodiimide hydrochloride (EDC) (Sigma–Aldrich) as the linking agent. For the first immunization at day 0, 500 g of the conjugated
M. Sroyraya et al. / Tissue and Cell 42 (2010) 293–300
peptide was mixed with a 1.5 ml volume of the complete Freund’s adjuvant (CFA), and injected subcutaneously into each rabbit using an aseptic technique. For the first to third boosts, at week 2, 4, and 6, 250 g of the conjugated peptide was mixed with a similar volume of the incomplete Freund’s adjuvant (IFA), and then injected using the same protocol. Pre-immune serum was collected from each rabbit before the first immunization. The blood was collected at 2 weeks after the final boost, centrifugated at 5000 g for 15 min, after which the antiserum was collected and stored at −20 ◦ C until used. 2.5. Specificity of the polyclonal antibody against Cq-IAG The rabbit antiserum was pre-absorbed to eliminate anti-BSA by adding 100 mg/ml BSA to the serum, at a dilution of serum to BSA of 1:100 (v/v), and shaking at 4 ◦ C, overnight. To test the antibody specificity, conjugated Cq-IAG-BSA at concentrations of 1, 0.1 and 0.01 mg/ml, as well as pure BSA and the Cq-IAG peptide, were dotted onto nitrocellulose membranes and dried for 45 min at room temperature. The dried membranes were washed 3 times with Tris buffered saline, pH 8.0 (TBS, containing NaCl 8 g, KCl 2 g, Tris 3 g in 1 L of distilled water) for 5 min each. After washing, the membranes were incubated in a blocking solution containing 4% skim
295
milk in TBS with Tween-20 (TBST), for 1 h at room temperature (RT). The membranes were incubated in the primary antibody diluted 1:1500 in blocking solution, for 3 h, at 4 ◦ C. Negative controls were performed by substituting the primary antibody with the rabbit pre-immune serum. After incubation, the membranes were washed 3 times with TBST for 5 min each. HRP-conjugated goat anti-rabbit IgG (SouthernBiotech, USA), diluted 1:10,000 in blocking solution, was added to each membrane, which was then incubated for 1 h at RT. The membranes were then washed 3 times with TBST, for 5 min each. The color signal indicating the location of Pp-IAG was developed with an ECL kit (Amersham Pharmacia Biotech) for 10 s, and followed by exposure to a photographic film. The positive dots on the film were then analyzed.
2.6. Immunoperoxidase staining for Pp-IAG The AG sections from three groups the blue swimmer crabs, i.e., intact, unilateral, and bilateral eyestalk-ablated, were deparaffinized with xylene and then rehydrated in 100%, 95%, 90%, 80%, and 70% ethanol. Endogenous peroxidase in the tissue was reduced by incubating the sections in 3% H2 O2 in 100% methanol for 30 min, and washed 3 times with 0.1 M phosphate buffered saline containing Tween-20 (PBST), pH 7.4, for 5 min each. The sections were
Fig. 1. Location and morphology of the AG in P. pelagicus. (A) The AG of the blue swimming crab is attached to the spermatic duct (SD), which is stained blue, and located between muscles of the fifth walking leg. (B) A stereo-micrograph showing the AG attached to one side of the distal part of spermatic duct (SD) and the ejaculatory bulb (EB). (C) Low power micrograph of the cross-section through the SD, showing the AG attached to one side of the SD, and is covered by a thin layer of connective tissue (CT). (D) A high magnification of the AG displaying two types of cell: type I cells which are located in the periphery of the AG lobule, and type II cells which occupy the central area of the lobule. Both type I and type II are globular cells with round nuclei. Type I cells are small, stained intensely with hematoxylin in cytoplasm, and have dense chromatin (upper inset), but type II cells are large, stained lightly with hematoxylin in cytoplasm, and have loose chromatin (lower inset). These cells are packed together and surrounded by a thin layer of CT. The arrow indicates a binucleated cell located between the zones of type I and type II cells. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
296
M. Sroyraya et al. / Tissue and Cell 42 (2010) 293–300
then incubated in 1% glycine in 0.1 M PBST, pH 7.4, for 15 min and washed 3 times with 0.1 M PBST, pH 7.4, for 5 min each. The sections were incubated for 1 h in the blocking solution consisting of 5% normal goat serum in 0.1 M PBST, pH 7.4, followed by an incubation in 1:100 diluted anti-Cq-IAG overnight, and then 3 washes of 5 min each in PBST. The sections were then incubated with the secondary antibody, HRP-conjugated goat anti-rabbit IgG (SouthernBiotech, USA), diluted at 1:500, for 45 min, at RT. After 3 washes, Nova RED (Zymed) containing H2 O2 was added to the sections to develop the color over 10–30 s, and the reactions stopped by washing in tap water. For negative controls, the primary antibody incubation step was omitted. For a positive control, sections were prepared from C. quadricarinatus AG’s and used for immunoperoxidase staining, following the same protocol. The stained tissues were observed and photographed by a Nikon E600 microscope equipped with a DXM 1200 digital camera, using an ACT-1 software package. 2.7. Cloning and sequencing of Pp-IAG cDNA In order to verify whether the P. pelagisus (Pp)-IAG also contains a peptide that is similar to Cq-IAG peptide that was used to immunize rabbits to generate the anti-IAG antiserum, a cDNA of
Pp-IAG was cloned and compared with Cq-IAG cDNA. Male crabs were dissected to obtain 100 mg of AG and total RNA was prepared from the tissue using Trizol reagent (Invitrogen, USA), following the manufacturer’s protocol. First strand cDNA was then synthesized from 1 g of total RNA, using SuperScriptTM III Reverse Transcriptase (Invitrogen, USA) and SMART primers (SMIV and SMIII+) from a SMART cDNA Library Construction Kit (Clontech, USA). The first strand cDNA was used as a template in RT-PCR. Forward and reverse primers were designed on Cq-IAG (GenBank accession number DQ851163.1), to amplify regions of Pp-IAG. The forward primers used were F1 5 -GTATAACACTCAACGATGCT-3 (nt 541–560) and F2 5 -CTGCCAGTGTCTCCATGTATG-3 (nt 755–775), and the reverse primer were R1 5 -TTCCTGGTAGGTGCGGCA-3 (nt 691–708) and R2 5 -GATTCCGTCCTCCAACTGTTC-3 (nt 1051–1071). The cDNA was used as a template to perform 3 and 5 RACE with a SMARTTM cDNA kit (Clontech, USA), following the manufacturer’s protocol. The primer F2 and primer SMIIIB 5 -GCCGAGGCGGCCGACATGTT3 were used to perform 3 RACE, whereas primer R1 and primer SM2 V 5 -AAGCAGTGGTATCAACGCAGAGT-3 were used to perform 5 RACE. PCR amplifications were performed using one cycle at 94 ◦ C for 2 min, followed by 35 cycles of 30 s at 94 ◦ C, 30 s at 53 ◦ C, and 2 min at 72 ◦ C, and a final step of 72 ◦ C for 10 min. PCR products were
Fig. 2. Histology of the AG in intact (control) and after bilateral eyestalk ablation. (A) A cross-section of the AG in the control group at day 8, showing type I cells at the peripheral region and occupying about 20% of the gland. (B) A cross-section of the AG in a day-4 bilateral eyestalk-ablated male, showing a hypertrophic gland containing an increasing of proportion of type I cells (I). (C) A cross-section of the AG in a day-8 bilateral eyestalk-ablated male, showing a hypertrophic gland containing an obvious larger proportion of type I cells (I) than type II cells (II). (D) A higher magnification of the rectangular area in C, showing bordering zone of type I and type II cells. (E) A hematoxylin-eosin stained section of C. quadricarinatus (Cq) androgenic gland (AG), showing multiple lobules, each surrounded by a thin sheet of connective tissue. (F) A similar section at higher magnification, showing AG cells inside each lobule intensely stained with anti-Cq-IAG.
M. Sroyraya et al. / Tissue and Cell 42 (2010) 293–300
297
analyzed by agarose gel electrophoresis and bands of expected sizes extracted and cloned into pGEM-Teasy vector (Promega, USA). Inserts were sequenced by Macrogen, Korea. 2.8. Statistical analysis of AG cells The percentages of type I and II cells in each group were presented as means and standard deviations. The percentages of type I and II cells in different groups of the same period of sampling were compared and analysed by a one-way analysis of variance (ANOVA). P < 0.05 was considered to be statistically different. 3. Results 3.1. Anatomy of the AG The AG of the blue swimmer crab is attached to the distal part of the spermatic duct (SD) and the ejaculatory bulb (EB), located between the muscles of the fifth pair of walking legs (Fig. 1(A)). It appears as pale yellow patches on one side of the SD and EB (Fig. 1(B)). When viewed in cross-section, the AG is composed of two or three lobules tightly packed together at one side of the SD and EB. Each lobule is surrounded by thin sheets of connective tissue (Fig. 1(C)). On one side the AG abuts the epithelium of the SD and EB, while on the opposite side it is surrounded by a thicker sheet of connective tissue (Fig. 1(C) and (D)). The AG is composed of two main cell types, designated as type I and type II cells (Fig. 1(D)). Type I cells are located on the periphery of each AG lobule under the outer layer of connective tissue, and are small round cells of 5–7 m in diameter. They occupy about 20% of the AG volume. Each cell had a round nucleus containing dense chromatin, and cytoplasm that is stained blue with hematoxylin. Type II are larger cells of 13–15 m in diameter, and occupy about 80% of the AG volume. Each cell possesses a round, vesiculate nucleus (containing mostly euchromatin), with a prominent nucleolus, and the cytoplasm is stained light blue with hematoxylin (Fig. 1(D)). Occasionally, binucleated cells, which could be dividing cells, are found between these two groups (Fig. 1(D)). 3.2. Effects of eyestalk ablation on the AG The structures of the AGs in the unilateral eyestalk-ablated males at day 0, 4, and 8, were not different from the intact (control), and exhibited histological features as described above (Fig. 2(A)). In contrast, the AGs of day-4 and day-8 bilateral eyestalk-ablated crabs were approximately 2-fold and 3-fold larger, respectively, than the controls (Fig. 2(B)–(D)). In particular, the number of type
Fig. 3. Percentages of type I cells in AG: control (), unilateral eyestalk-ablated ( ), and bilateral eyestalk-ablated () male crabs, at days 0, 4, and 8. The columns represent the percentages of type I cells per total number of cells counted under a light microscope, using an Image J program (error bars = SD, n = 6). Asterisk indicates a significant difference when compared among the groups at the same day (P < 0.05).
I cells in the bilateral eyestalk-ablated group had increased significantly in a time-dependent manner. Fig. 3 shows the percentages of type I cell in the gland. At day 0 after eyestalk ablation, the percentage of type I cells among three groups were not different. At day 4 after eyestalk ablation, the number of type I cells in the bilateral eyestalk-ablated group had increased significantly to 35.60 ± 0.03% (P < 0.05), compared with 25.23 ± 0.01% for the control and 22.4 ± 0.03% for the unilateral eyestalk-ablated males. At day 8 after eyestalk ablation, the number of type I cells in the bilateral eyestalk-ablated group had increased significantly to 48.19 ± 0.10% (P < 0.05), compared with 21.91 ± 0.03% for the control and 24.38 ± 0.01% for unilateral eyestalk-ablated males. The percentages of type II were correspondingly opposite to the percentages of type I cells. 3.3. Specificity of the antibody against Cq-IAG The specificity of the pre-absorbed antibody was tested by dot blot analysis. Pre-immune serum showed no positive immunoreactivity on the membrane (Fig. 4(A)), while non-absorbed antibody showed immunoreactivity at the dots with varying amount of BSA and Cq-IAG-BSA conjugate (Fig. 4(B)). In the pre-absorbed antibody with 100 mg/ml BSA, at a dilution of 1:100, the immunoreactivity was detected in the dots containing conjugated peptide, but not BSA (Fig. 4(C)). Therefore, this pre-absorbed antibody was specific to Cq-IAG and used for immunolocalization of Pp-IAG in sections of tissues.
Fig. 4. Dot blot analysis of the specificity of an antibody against Cq-IAG. (A) Negative control using pre-immune serum to detect BSA and Cq-IAG-BSA conjugate. No positive dot is shown on the membrane. (B) Non-absorbed antibody was used as a probe. Both dots containing BSA and Cq-IAG-BSA conjugate at various dilutions show positive signal. (C) Pre-absorbed antibody with 100 mg/ml of BSA at a dilution 1:100 (v/v) was used as a probe. Only conjugated peptide, but not BSA dots were positive to the antibody.
298
M. Sroyraya et al. / Tissue and Cell 42 (2010) 293–300
Fig. 5. Amino acid sequence alignment of IAG in five decapod crustaceans. The IAG factors, species names and GenBank accession numbers are: Pp-IGH, P. pelagicus, HM459854; Cd-IAG, C. destructor, EU718788.1; Cq-IAG, C. quadricarinatus, DQ851163.1; Mr-IAG, M. rosenbergii, FJ409645.1; and Pm-IAG, P. monodon, GU208677.1. Green letters show putative signal peptides, blue letters show putative B and A chains, red letters show putative cleavage sites, black letters show C peptides, gray letters in Pm-IAG is an ambiguous part, shaded gray are conserved C residues. The underlined hydrophilic RRRNSDTTD (aa 128-136) of Cq-IAG corresponds to the sequence of a peptide synthesised by GenScript (USA), and susequently used for antibody production. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
3.4. Cloning and sequencing of Pp-IAG In order to confirm that the antibody produced against the Cq-IAG peptide would also react with a Pp-IAG factor, a deduced sequence of the Pp-IAG factor was identified from AG cDNA. The deduced Pp-IAG sequence is given in Fig. 5, together with GenBank sequences of 4 other decapods crustacean, namely Cd-IAG (C. destructor, EU718788.1), Cq-IAG (C. quadricarinatus, DQ851163.1), Mr-IAG (M. rosenbergii, FJ409645.1), and Pm-IAG (P. monodon, GU208677.1). An alignment of all five sequences showed that PpIAG is 96% similar to Cd-IAG, 86% to Cq-IAG, 23% to Mr-IAG, and 25% to Pm-IAG. The conserved insulin-like regions of signal sequence, A and B chains, C peptides, putative proteolytic cleavage sites, and C residues, are apparent in all but Pm-IAG. Importantly, the CqIAG peptide RRRNSDTTD used to make our antibody was almost identical to the Pp-IAG peptide RRRDSDTTD,which is incidentally identical to a corresponding putative peptide present in Cd-IAG.
3.5. Immunoperoxidase staining of Pp-IAG Immunoperoxidase staining showed that the Cq-IAGimmunoreactivity, which could as well represent Pp immunoreactivity (Pp-IAG-ir), was detected in the AG and not in any parts of the SD or EB of the intact, unilateral and bilateral eyestalk-ablated crabs (Fig. 6(A)–(F)). All negative control sections of all groups showed no Pp-IAG-ir (data not shown), while the positive control showed the immunostaining of cells in the Cq-AG (Fig. 2(F)). In the intact group, a low amount of Pp-IAG-ir was found mostly in peripheral AG cells, with fewer positive cells in the central area of each lobule (Fig. 6(A) and (B)). At higher magnification the positive cells at the periphery of the lobules were identified as type I cell (Fig. 6(B)-inset). A similar pattern of staining was observed in the AG of the unilateral eyestalk-ablated group (data not shown). In the bilateral eyestalk-ablated group, the AG showed
an increased number and more intense Pp-IAG-ir cells at day 4 after the eyestalk ablation. The intense Pp-IAG-ir was detected in the cytoplasm of the type I cells, located at the periphery of each lobule (Fig. 6(C) and (D)) Noticeably, the staining was not even and the high intensity tended to be distributed in the peripheral zone of the cytoplasm (Fig. 6(C), (D) and (D)-inset). Conversely, the type II cells that occupy the central zone of each lobule tended to show a less intense staining. At day 8 after bilateral eyestalk ablation, the Pp-IAG-ir became even more intense than in the 4-day AG (Fig. 6(E) and (F)). In addition, the immunoreactivity was similar in both type I and type II cells, and the staining was even throughout the cytoplasm of both cell types.
4. Discussion One requirement for identifying an insulin-like factor in the androgenic gland of the P. pelagicus (Pp-IAG), and other species, was production of a specific antibody, so we initially produced one against a synthetic 9-amino acid peptide derived from a conserved region of the IAG of C. quadricarinatus and C. destructor. We also produced cDNA from the androgenic gland of the P. pelagicus to obtain a deduced sequence of Pp-IAG (GenBank accession number HM459854). The deduced Pp-IAG sequence (Fig. 5) was 96% similar to Cq-IAG. It varied by only one amino acid in the nonapeptide used to make the antibody, indicating that the antibody against Cq-IAG would react with Pp-IAG in immunohistochemistry. Histologically, the AG of P. pelagicus is composed of two types of cells, and this is similar to M. rosenbergii (Okumura and Hara, 2004), and Penaeus chinensis (Li and Xiang, 1997). In contrast, the presence of three cell types has been reported in S. paramamosain (Liu et al., 2008), and M. kistnensis (Mirajkar et al., 1984). The type III cells, described in the latter two species, appear larger and exhibit more vacuoles inside their cytoplasm. These characteristics imply that
M. Sroyraya et al. / Tissue and Cell 42 (2010) 293–300
299
Fig. 6. Immunoperoxidase staining, with anti-Cq-IAG, of the androgenic gland (AG) in intact (control), unilateral eyestalk-ablated, and bilateral eyestalk-ablated crabs. (A) A low power micrograph of AG cross-section in control group, showing few Pp-IAG-ir positive cells at periphery of the lobules. (B) A high power micrograph of (A) showing mostly Pp-IAG-ir positive type I cells in the periphery of the AG (inset). There are only few positive cells in the central area of the gland. (C) A low power micrograph of an AG cross-section of a bilateral eyestalk-ablated male at day 4, showing intense Pp-IAG-ir in the AG cells, but not in the spermatic duct (SD) and its epithelium (EP). (D) A high power micrograph of (C) showing intense Pp-IAG-ir in the cytoplasm of type I cells. High intensity of Pp-IAG-ir is present at the edge of the cells (inset). (E) A low power micrograph of an AG cross-section of a bilateral eyestalk-ablated crab at day 8, showing very intense IAG-ir in both type I and type II cells. There is no Pp-IAG-ir in the SD and its epithelium (EP). (F) A high power micrograph of (E) showing very intense and evenly distributed Pp-IAG-ir in the cytoplasm of both types of AG cells. Pp-IAG-ir is present throughout the cytoplasm of the cells (inset).
the type III cells may be undergoing degeneration. Alternatively, it has been suggested that the different cell types may just represent different stages of a secretory cycle of a single cell type (Thampy and John, 1970; Veith and Malecha, 1983; Mirajkar et al., 1984). An ultrastructure study in M. rosenbergii lends support to this notion (Okumura and Hara, 2004), as it was shown that both type I and II cells possessed abundant rough endoplasmic reticulum (RER), and that the RER of the type II cells were highly dilated, implying that they were producing proteins for secretion. In addition, secretory granules have been found in the cytoplasm of the A. vulgare AG cells, suggesting that they secrete protein (Negishi et al., 2001). An intense hematoxylin staining of the cytoplasm of type I cells in P. pelagicus, also implies that a high amount of ribosomes and RER is present in the AG cells of this species. This was confirmed by our
data which indicated that Pp-IAG-ir is present in both types of cells, albeit with different staining intensity. This difference may reflect greater synthesis in type I cells. The binucleated cells in the AG of O. platytarsis were attributed to mitotic division (Thampy and John, 1970). It is also interesting to note that in the P. pelagicus AG, most of the binucleated cells are present in the zone between type I and type II cells. It is possible that type I cells are dividing and give rise to type II cells, which then move towards the central area of the AG lobule. The bilateral eyestalk-ablated crabs showed hypertrophy of the AG, whereas the intact and the unilateral eyestalk-ablated crabs did not. This could be because the AG is under the inhibitory control by neuropeptides secreted from the X-organ-sinus gland complex in the eyestalk, as indicated earlier (Hoffman, 1968; Khalaila et
300
M. Sroyraya et al. / Tissue and Cell 42 (2010) 293–300
al., 2002). The AG in unilateral eyestalk-ablated crabs might still receive the inhibitory control from the remaining eyestalk, while in the bilateral eyestalk-ablated crabs this inhibition is completely abrogated, thus resulting in the hypertrophic AG. The bilateral eyestalk ablation significantly increased the percentage of type I cells from an initial 24.14% to 48.19% at day 8 following the treatment. During this time the percentage of type II cells decreased, suggesting that type I cells may be the precursor cells that divide and give rise to type II cells. This idea is supported by a recent study by Liu et al. (2008), in which type I AG cells were found to be a major population in immature mud crabs, S. paramamosain, while type II AG cells were more abundant in mature crabs. This observation indicated that type I cells may differentiate into type II cells during the maturation of the crabs. Our immunoperoxidase data showed that type I cells were immunoreactive in both the intact and eyestalk-ablated males, while type II cells were less immunoreactive. Futhermore, type I cells exhibited stronger staining than type II cells at 4-day post eyestalk ablation, but the staining intensity was the same in both cells types by day 8. This finding suggests that under an absence of inhibitory hormone from the eyestalk, the type I cells in P. pelagicus start to synthesize IAG earlier, and in greater amounts, than type II cells. The type II cells may represent a late stage of the AG cells, and are reaching the final phase of secretion. Further studies, using cell labelling to indicate the type I cell proliferative capacity, simultaneously with immunolabelling (to indicate the cell’s ability to synthesize IAG), are now needed to confirm that there is a single lineage of type I and type II cells in the AG of decapod crustaceans. Acknowledgments This research was supported by a Distinguished Research Professor Grant (co-funded by the Thailand Research Fund, the Commission on Higher Education (CHE), Mahidol University) to P. Sobhon, and a CHE-Ph. D. scholarship to M. Sroyraya. References Abdel Razek, F.A., Taha, S.M., Ameran, A.A., 2006. Ecological observations on the abundance, distribution of holothuroids (Echinodermata: Holothuroidea) in the Red Sea coast, Egypt. Egypt. J. Aquatic Res. 32, 419–430. Balesdent-Marquet, M.L., 1958. Presence of an androgen gland in crustacean isopod, Asellus aquaticus L. Comptes Rendus Hebdomadaires des Seances de l’Academie des Sciences 247, 534–536. Campos-Ramos, R., Garza-Torres, R., Guerrero-Tortolero, D., Maeda-Martnez, M., Obregon-Barboza, H., 2006. Environmental sex determination, external sex differentiation and structure of the androgenic gland in the Pacific white shrimp Litopenaeus vannamei (Boone). Aquac. Res. 37, 1583–1593. Chaiyawat, M., Eungrasamee, I., Raksakulthai, N., 2008. Quality characteristics of blue swimming crab (Portunus pelagicus, Linnaeus 1758) meat fed Gracilaria edulis (Gmelin) Silva. Kasetsart J. (Natural Science) 42, 522–530. Chande, A.I., Mgaya, Y.D., 2003. The fishery of Portunus pelagicus and species diversity of portunid crabs along the coast of Dar es Salaam, Tanzania. Western Indian Ocean. J. Mar. Sci. 2, 75–84. Charniaux-Cotton, H., 1954. Discovery in, an amphipod crustacean (Orchestia gammarella) of an endocrine gland responsible for the differentiation of primary and secondary male sex characteristics. Comptes Rendus Hebdomadaires des Seances de l’Academie des Sciences 239, 780–782. Cui, Z., Liu, H., Lo, S.T., Chu, K.H., 2005. Inhibitory effects of the androgenic gland on ovarian development in the mud crab Scylla paramamosain. Comp. Biochem. Physiol. 104, 343–348. Foulks, N.B., Hoffman, D.L., 1974. The effects of eyestalk ablation and B-ecdysone on RNA synthesis in the androgenic glands of the protandric shrimp, Pandalus platyceros Brandt. Gen. Comp. Endocrinol. 22, 439–447. Hasegawa, Y., Katakura, Y., 1985. Masculinization of female by the newly-formed androgenic glands in the ZW and WW females of the isopod crustacean, Armadillidium vulgare. Zool. Sci. 2, 419–422.
Hoffman, D.L., 1968. Seasonal eyestalk inhibition on the androgenic glands of a protandric shrimp. Nature 218, 170–172. Juchault, P., Legrand, J.J., 1978. Function of the androgen gland in crossed implantations between two species of isopod terrestrial Crustacea, Porcellio dilatatus Brandt and Armadillidium vulgare Latreille: specificity of androgenic hormones and neurohormones implicated in the control of androgenic function. Gen. Comp. Endocrinol. 36, 175–186. Katakura, Y., 1960. Transformation of ovary into testis following implantation of androgenous glands in Armadillidium vulgare, an isopod crustacean. Annot. Zool. 33, 241–244. Khalaila, I., Manor, R., Weil, S., Granot, Y., Keller, R., Sagi, A., 2002. The eyestalkandrogenic gland-testis endocrine axis in the crayfish Cherax quadricarinatus. Gen. Comp. Endocrinol. 127, 147–156. King, D.S., 1964. Fine structure of the androgenic gland of the crab, Pachygrapsus Crassipes. Gen. Comp. Endocrinol. 47, 533–544. Lee, T.H., Shigesawa, R., Yamasaki, F., 1993. Partial masculinization of female Eriocheir japonicus (Brachyura, Grapsidae) by androgenic gland implantation. Suisan Zoshoku 41, 311–319. Li, F., Xiang, J., 1997. Preliminary studies on form, structure and function of androgenic gland in Penaeus chinensis. Chin. Sci. Bull. 42, 499–503. Liu, H., Cheung, K.C., Chu, K.H., 2008. Cell structure and seasonal changes of the androgenic gland of the mud crab Scylla paramamosain (Decapoda: Portunidae). Zool. Stud. 47, 720–732. Malecha, S.R., Nevin, P.A., Ha, P., Barck, L.E., Lamadrid-Rose, Y., Masuno, S., Hedgecock, D., 1992. Sex-ratios and sex-determination in progeny from crosses of surgically sex-reversed freshwater prawns, Macrobrachium rosenbergii. Aquaculture 105, 201–218. Manor, R., Weil, S., Oren, S., Glazer, L., Aflalo, E.D., Ventura, T., Chalifa-Caspi, V., Lapidot, M., Sagi, A., 2007. Insulin and gender: an insulin-like gene expressed exclusively in the androgenic gland of the male crayfish. Gen. Comp. Endocrinol. 150, 326–336. Minagawa, M., Chiu, J.R., Kudo, M., Takashima, F., 1994. Male reproductive biology of the red frog crab, Ranina ranina, off Hachijojima, Izu Islands, Japan. Mar. Biol. 118, 393–401. Mirajkar, M., Sarojini, R., Nagabhushanam, R., 1984. Histophysiology of the androgenic gland in freshwater prawn Macrobrachium kistnensis. J. Anim. Morphol. Physiol. 31, 211–220. Mires, D., 1977. Theoretical and practical aspects of the production of all-male Tilapia hybrids. Bamidgeh 29, 94–101. Nagamine, C., Knight, A.W., 1987. Masculinization of female crayfish, Procambarus clarkii (Girard). Invert. Reprod. 11, 77–87. Nagamine, C., Knight, A.W., Maggenti, A., Paxman, G., 1980. Masculinization of female Macrobrachium rosenbergii (de Man) (Decapoda, Palaemonidae) by androgenic gland implantation. Gen. Comp. Endocrinol. 41, 442–457. Negishi, S., Hasegawa, Y., Nakajima, Y., 2001. Novel structures in secreting the androgenic gland hormone. Zool. Sci. 18, 1237–1243. Okumura, T., Hara, M., 2004. Androgenic gland cell structure and spermatogenesis during the molt cycle and correlation to morphotypic differentiation in the giant freshwater prawn, Macrobrachium rosenbergii. Zool. Sci. 21, 621–628. Sagi, A., Aflalo, E.D., 2005. The androgenic gland and monosex culture of Macrobrachium rosenbergii (De Man): a biotechnological perspective. Aquac. Res. 36, 231–237. Sagi, A., Cohen, D., Milner, Y., 1990. Effect of androgenic gland ablation on morphotypic differentiation and sexual characteristics of male freshwater prawns, Macrobrachium rosenbergii. Gen. Comp. Endocrinol. 77, 15–22. Simeo, C.G., Ribes, E., Rotllant, G., 2009. Internal anatomy and ultrastructure of the male reproductive system of the spider crab Maja brachydactyla (Decapoda: Brachyura). Tissue and Cell 41, 345–361. Sumpton, W.D., Potter, M.A., Smith, G.S., 1994. Reproduction and growth of the commercial sand crab, Portunus pelagicus (L.) in Moreton Bay, Queensland. Asian Fish. Sci. 7, 103–113. Suzuki, S., Yamasaki, K., 1998. Sex reversal by implantations of ethanol-treated androgenic glands of female isopods, Armadillidium vulgare (Malacostraca, crustacea). Gen. Comp. Endocrinol. 111, 367–375. Taketomi, Y., 1986. Ultrastructure of the androgenic gland of the crayfish, Procambarus clarkii. Cell Biol. Int. Rep. 10, 131–136. Thampy, D.M., John, P.A., 1970. On the androgenic gland of the ghost crab Ocypoda platytarsis M. Edwards (Crustacea: Brachyura). Acta Zool. 51, 203–210. Veith, W.J., Malecha, S.R., 1983. Histochemical study of the distribution of lipids, 3aand 3b- hydroxysteroid dehydrogenase, in the androgenic gland of the cultured prawn, Macrobrachium rosenbergii (de Man) (Crustacea: Decapoda). S. Afr. J. Sci. 79, 84–85. Ventura, T., Manor, R., Aflalo, E.D., Weil, S., Raviv, S., Glazer, L., Sagi, A., 2009. Temporal silencing of an androgenic gland-specific insulin-like gene affecting phenotypical gender differences and spermatogenesis. Endocrinology 150, 1278–1286.
Contents lists available at ScienceDirect
Tissue and Cell journal homepage: www.elsevier.com/locate/tice
Bilateral eyestalk ablation of the blue swimmer crab, Portunus pelagicus, produces hypertrophy of the androgenic gland and an increase of cells producing insulin-like androgenic gland hormone Morakot Sroyraya a , Charoonroj Chotwiwatthanakun a,b , Michael J. Stewart c , Nantawan Soonklang d , Napamanee Kornthong a , Ittipon Phoungpetchara a , Peter J. Hanna a,e , Prasert Sobhon a,∗ a
Department of Anatomy, Faculty of Science, Mahidol University, Rama 6 Road, Bangkok 10400, Thailand Mahidol University, Nakhonsawan Campus, Nakhonsawan 60000, Thailand c School of Medicine, Deakin University, Geelong, Victoria 3217, Australia d Department of Preclinical Science, Faculty of Medicine, Thammasat University, Pathumthani 12121, Thailand e Faculty of Science and Technology, Deakin University, Geelong, Victoria 3217, Australia b
a r t i c l e
i n f o
Article history: Received 22 April 2010 Received in revised form 21 June 2010 Accepted 1 July 2010
Keywords: Portunus pelagicus Androgenic gland Insulin-like androgenic gland hormone Eyestalk ablation Hypertrophy
a b s t r a c t The androgenic glands (AG) of male decapod crustaceans produce insulin-like androgenic gland (IAG) hormone that controls male sex differentiation, growth and behavior. Functions of the AG are inhibited by gonad-inhibiting hormone originating from X-organ-sinus gland complex in the eyestalk. The AG, and its interaction with the eyestalk, had not been studied in the blue swimmer crab, Portunus pelagicus, so we investigated the AG structure, and then changes of the AG and IAG-producing cells following eyestalk ablation. The AG of P. pelagicus is a small endrocrine organ ensheathed in a connective tissue and attached to the distal part of spermatic duct and ejaculatory bulb. The gland is composed of several lobules, each containing two major cell types. Type I cells are located near the periphery of each lobule, and distinguished as small globular cells of 5–7 m in diameter, with nuclei containing mostly heterochromatin. Type II cells are 13–15 m in diameter, with nuclei containing mostly euchromatin and prominent nucleoli. Both cell types were immunoreactive with anti-IAG. Following bilateral eyestalk ablation, the AG underwent hypertrophy, and at day 8 had increased approximately 3-fold in size. The percentage of type I cells had increased more than twice compared with controls, while type II cells showed a corresponding decrease. © 2010 Elsevier Ltd. All rights reserved.
1. Introduction Blue swimmer crabs, Portunus pelagicus, have a high economic value, and constitute important marine fisheries in tropical countries (Sumpton et al., 1994; Chande and Mgaya, 2003; Abdel Razek et al., 2006; Chaiyawat et al., 2008). They exhibit sexual dimorphism, with males growing faster and being bigger than females. Therefore, monosex culture of males could increase monetary returns to farmers, and this may be achieved by reversing the sex of females to males (Mires, 1977; Sagi and Aflalo, 2005). The control of male sex differentiation in crustaceans by the androgenic gland (AG) was first described in the amphipod, Orchestia gammarella (Charniaux-Cotton, 1954). The AG plays a major role in the development of the male gonad and secondary sexual characteristics, while inhibiting female secondary charac-
∗ Corresponding author. Tel.: +66 2 2015406; fax: +66 2 3547168. E-mail addresses: [email protected], tokaro [email protected] (P. Sobhon). 0040-8166/$ – see front matter © 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.tice.2010.07.003
teristics. Masculinization of females by implantation of AG has been shown in several isopod species, including O. gammarella (Charniaux-Cotton, 1954), Asellus aquaticus (Balesdent-Marquet, 1958), Armadillidium vulgare (Hasegawa and Katakura, 1985; Suzuki and Yamasaki, 1998), and Porcellio dilatatus and Helleria brevicornis (Juchault and Legrand, 1978). Implantation of AG’s into juvenile female isopods results in the development of male appendages and spermatogenesis, as well as inhibition of female secondary characteristics and vitellogenesis (Charniaux-Cotton, 1954; Katakura, 1960). Conversely, removal of the AG from juvenile or mature males causes degeneration of spermatogenesis and the appearance of vitellogenesis (Charniaux-Cotton, 1954). In decapod crustaceans, removal of the AG from male produces regression of male characteristics, while implantation of AG into juvenile females results in the inhibition of vitellogenesis and development of male sexual characteristics (Nagamine et al., 1980; Nagamine and Knight, 1987; Sagi et al., 1990; Malecha et al., 1992; Lee et al., 1993). Implantation of AG into female mud crabs, Scylla paramamosain, results in ovarian regression, with degeneration of oocytes (Cui et al., 2005).
294
M. Sroyraya et al. / Tissue and Cell 42 (2010) 293–300
The AG is considered to be under inhibitory control of the X-organ-sinus gland complex located in the eyestalk. In Cherax quadricarinatus and Pandalus platyceros, bilateral removal of the eyestalk leads to hypertrophy of the AG (Hoffman, 1968; Khalaila et al., 2002). Moreover, RNA synthesis in the AG increases in eyestalkablated shrimp (Foulks and Hoffman, 1974). The structure of the AG has been studied in many decapods species, including Pachygrapsus crassipes (King, 1964), Ocypoda platytarsis (Thampy and John, 1970), Macrobrachium kistnensis (Mirajkar et al., 1984), Ranina ranina (Minagawa et al., 1994), Penaeus chinensis (Li and Xiang, 1997), Macrobrachium rosenbergii (Okumura and Hara, 2004), Litopenaeus vannamei (Campos-Ramos et al., 2006), S. paramamosain (Liu et al., 2008), and Maja brachydactyla (Simeo et al., 2009). Although the AG gland is generally composed of groups of round or oval cells with round nuclei, the cells in the AG can be classified into 2–3 types according to species. Type I are small cells of approximately 5–12 m in diameter and have a large nucleus/cytoplasm ratio. The cytoplasm stains intensely with hematoxylin. Type II are larger cells of approximately 15–20 m in diameter and have a small nucleus/cytoplasm ratio. The cytoplasm stains lightly with hematoxylin. Type III are the largest cells of approximately 20–25 m in diameter and have prominent cytoplasmic vacuoles. Studies have indicated that the AG secretes a protein hormone, called androgenic gland hormone (AGH). In A. vulgare (Negishi et al., 2001), the ultrastructure of AG cells shows cytoplasm consisting of numerous rough endoplasmic reticulum, Golgi complex, and secretory granules. Similarly, the ultrastructure of the AG cells of P. crassipes (King, 1964), M. rosenbergii (Okumura and Hara, 2004), and Procambarus clarkii (Taketomi, 1986), indicates that the gland secretes a high level of proteins. There have been limited studies of hormones produced by the AG of decapod crustaceans. Using a suppression subtractive hybridization (SSH) technique, a male-specific transcript encoding an insulin-like factor (IAG) was identified in the AG of C. quadricarinatus, and termed Cq-IAG (Manor et al., 2007). The structure of the gene encoding Cq-IAG shares similarity to those of mammals. The Cq-IAG contains a signal peptide, a B chain, a C peptide and an A chain. Using the same SSH technique, a Mr-IAG gene in M. rosenbergii has been recently cloned and characterized (Ventura et al., 2009). Upon silencing of the Mr-IAG gene by RNAi, spermatogenesis was arrested, and growth and development of male secondary sex characteristics were also inhibited. This evidence indicated that IAG plays a role in decapods in a manner similar to AGH in other crustaceans. In decapods, several studies have reported the structure and ultrastructure, as well as the hormone produced by the AG, using molecular cloning. However, there are no reports on changes of the cell populations in the AGs due to eyestalk ablation, or localization of IAG. Therefore, in the present study, we investigated the structure of the AG, particularly under an accelerated hormone production condition caused by bilateral eyestalk ablation, and showed IAG immunolocalization in normal and hypertrophy AGs. Moreover, we also provided the sequence of the Portunus pelagicus IAG (Pp-IAG). We report here that the AG has two main cell types (I and II), and that hypertrophy of the AG occurring from bilateral eyestalk ablation causes increases in the number of type I cells, and the numbers of IAG-producing cells.
2. Materials and methods Mature male blue swimmer crabs, P. pelagicus, with a carapace width of 80–90 mm, were reared in rectangular concrete tanks, at the Department of Aquatic Science, Faculty of Science, Burapha University. The rearing conditions were: sea water with salinity at
20 ppm, a temperature of 29 ± 3 ◦ C, constant aeration, a dark:light cycle of 12:12 h, and feeding with boiled fish twice a day. 2.1. Experimental design Mature males were randomly divided into three groups (n = 18 per group): (1) a control (no eyestalk ablation), (2) a unilateral eyestalk-ablated, and (3) a bilateral eyestalk-ablated group. An aseptic technique was used to cut off one eye or two eyes from each experimental male. Each treated crab (including controls) was then placed in a separate basket and reared in a rectangular tank. At days 0, 4, and 8, following the eyestalk ablation, six crabs from each group were sacrificed and the AG from three crabs were collected and prepared for light microscopic observation, and AG from the another three crabs were prepared for immunolocalization of PpIAG. In the control group, an extra two crabs were used for studying gross anatomy of the AG and spermatic duct (SD) and ejaculatory bulb (EB). A 1 ml volume of blue ink was injected into the ejaculatory bulb to assist in locating the AG and studying its gross anatomy within the wall of the EB. 2.2. Light microscopy The carapaces were removed from the crabs, and the distal SD’s and EB’s, with the attached AG’s, were carefully removed. The tissues were fixed in Davidson’s fixative (330 ml 95% ethyl alcohol, 220 ml 100% formalin, 115 ml glacial acetic acid, and 335 distilled H2 O), for 24 h, dehydrated in 70%, 80%, 90%, and 100% ethanol, and transferred to toluene. They were then infiltrated and embedded in paraffin. The embedded tissues were cut at a thickness of 5 m using a rotary microtome (Leica RM2235). The sections were transferred onto gelatin coated slides, deparaffinized with xylene, and then rehydrated in 100%, 95%, 90%, 80%, and 70% ethanol for 5 min each. The sections were then placed in distilled water for 5 min, stained with Harris’s hematoxylin for 1.5 min, and placed in tap water for 15 min. The sections were counterstained with eosin for 2 min, dehydrated in 80%, 90% and 100% ethanol, and then immersed 3 times in xylene. The slides were mounted with permount, and the tissues examined and photographed with a Nikon E600 microscope equipped with a DXM 1200 digital camera, using an ACT-1 software package. 2.3. Cell counting Three AGs from each group were counted by ImageJ software. The numbers of type I and type II cells were counted in cross-sections of an AG, when they possessed complete circular cross-sectional profiles of the associated SD and EB. The quantities of type I and type II were recorded as percentages of the total cells. 2.4. Preparation of an antibody against Cq-IAG The ProtScale Tool from the ExPAsy Server (http://www.expasy.ch/tools/protscale.html) was used to analyzed the position of amino acid to synthesize the peptide for antibody production. A 9-amino acid peptide (RRRNSDTTD), corresponding to the most hydrophilic part of Cq-IAG (aa 128–136, accession number DQ851163.1), was synthesized (GenScript, NJ, USA). Two 8-week old female New Zealand white rabbits were used to produce a polyclonal antibody against the Cq-IAG peptide, as approved by the Animal Care Committee, Faculty of Science, Mahidol University. Before immunization, the synthetic peptide was conjugated to bovine serum albumin (BSA) (Sigma–Aldrich, St. Louise, MO, USA), using 1-ethyl-3-dimethylaminopropyl carbodiimide hydrochloride (EDC) (Sigma–Aldrich) as the linking agent. For the first immunization at day 0, 500 g of the conjugated
M. Sroyraya et al. / Tissue and Cell 42 (2010) 293–300
peptide was mixed with a 1.5 ml volume of the complete Freund’s adjuvant (CFA), and injected subcutaneously into each rabbit using an aseptic technique. For the first to third boosts, at week 2, 4, and 6, 250 g of the conjugated peptide was mixed with a similar volume of the incomplete Freund’s adjuvant (IFA), and then injected using the same protocol. Pre-immune serum was collected from each rabbit before the first immunization. The blood was collected at 2 weeks after the final boost, centrifugated at 5000 g for 15 min, after which the antiserum was collected and stored at −20 ◦ C until used. 2.5. Specificity of the polyclonal antibody against Cq-IAG The rabbit antiserum was pre-absorbed to eliminate anti-BSA by adding 100 mg/ml BSA to the serum, at a dilution of serum to BSA of 1:100 (v/v), and shaking at 4 ◦ C, overnight. To test the antibody specificity, conjugated Cq-IAG-BSA at concentrations of 1, 0.1 and 0.01 mg/ml, as well as pure BSA and the Cq-IAG peptide, were dotted onto nitrocellulose membranes and dried for 45 min at room temperature. The dried membranes were washed 3 times with Tris buffered saline, pH 8.0 (TBS, containing NaCl 8 g, KCl 2 g, Tris 3 g in 1 L of distilled water) for 5 min each. After washing, the membranes were incubated in a blocking solution containing 4% skim
295
milk in TBS with Tween-20 (TBST), for 1 h at room temperature (RT). The membranes were incubated in the primary antibody diluted 1:1500 in blocking solution, for 3 h, at 4 ◦ C. Negative controls were performed by substituting the primary antibody with the rabbit pre-immune serum. After incubation, the membranes were washed 3 times with TBST for 5 min each. HRP-conjugated goat anti-rabbit IgG (SouthernBiotech, USA), diluted 1:10,000 in blocking solution, was added to each membrane, which was then incubated for 1 h at RT. The membranes were then washed 3 times with TBST, for 5 min each. The color signal indicating the location of Pp-IAG was developed with an ECL kit (Amersham Pharmacia Biotech) for 10 s, and followed by exposure to a photographic film. The positive dots on the film were then analyzed.
2.6. Immunoperoxidase staining for Pp-IAG The AG sections from three groups the blue swimmer crabs, i.e., intact, unilateral, and bilateral eyestalk-ablated, were deparaffinized with xylene and then rehydrated in 100%, 95%, 90%, 80%, and 70% ethanol. Endogenous peroxidase in the tissue was reduced by incubating the sections in 3% H2 O2 in 100% methanol for 30 min, and washed 3 times with 0.1 M phosphate buffered saline containing Tween-20 (PBST), pH 7.4, for 5 min each. The sections were
Fig. 1. Location and morphology of the AG in P. pelagicus. (A) The AG of the blue swimming crab is attached to the spermatic duct (SD), which is stained blue, and located between muscles of the fifth walking leg. (B) A stereo-micrograph showing the AG attached to one side of the distal part of spermatic duct (SD) and the ejaculatory bulb (EB). (C) Low power micrograph of the cross-section through the SD, showing the AG attached to one side of the SD, and is covered by a thin layer of connective tissue (CT). (D) A high magnification of the AG displaying two types of cell: type I cells which are located in the periphery of the AG lobule, and type II cells which occupy the central area of the lobule. Both type I and type II are globular cells with round nuclei. Type I cells are small, stained intensely with hematoxylin in cytoplasm, and have dense chromatin (upper inset), but type II cells are large, stained lightly with hematoxylin in cytoplasm, and have loose chromatin (lower inset). These cells are packed together and surrounded by a thin layer of CT. The arrow indicates a binucleated cell located between the zones of type I and type II cells. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
296
M. Sroyraya et al. / Tissue and Cell 42 (2010) 293–300
then incubated in 1% glycine in 0.1 M PBST, pH 7.4, for 15 min and washed 3 times with 0.1 M PBST, pH 7.4, for 5 min each. The sections were incubated for 1 h in the blocking solution consisting of 5% normal goat serum in 0.1 M PBST, pH 7.4, followed by an incubation in 1:100 diluted anti-Cq-IAG overnight, and then 3 washes of 5 min each in PBST. The sections were then incubated with the secondary antibody, HRP-conjugated goat anti-rabbit IgG (SouthernBiotech, USA), diluted at 1:500, for 45 min, at RT. After 3 washes, Nova RED (Zymed) containing H2 O2 was added to the sections to develop the color over 10–30 s, and the reactions stopped by washing in tap water. For negative controls, the primary antibody incubation step was omitted. For a positive control, sections were prepared from C. quadricarinatus AG’s and used for immunoperoxidase staining, following the same protocol. The stained tissues were observed and photographed by a Nikon E600 microscope equipped with a DXM 1200 digital camera, using an ACT-1 software package. 2.7. Cloning and sequencing of Pp-IAG cDNA In order to verify whether the P. pelagisus (Pp)-IAG also contains a peptide that is similar to Cq-IAG peptide that was used to immunize rabbits to generate the anti-IAG antiserum, a cDNA of
Pp-IAG was cloned and compared with Cq-IAG cDNA. Male crabs were dissected to obtain 100 mg of AG and total RNA was prepared from the tissue using Trizol reagent (Invitrogen, USA), following the manufacturer’s protocol. First strand cDNA was then synthesized from 1 g of total RNA, using SuperScriptTM III Reverse Transcriptase (Invitrogen, USA) and SMART primers (SMIV and SMIII+) from a SMART cDNA Library Construction Kit (Clontech, USA). The first strand cDNA was used as a template in RT-PCR. Forward and reverse primers were designed on Cq-IAG (GenBank accession number DQ851163.1), to amplify regions of Pp-IAG. The forward primers used were F1 5 -GTATAACACTCAACGATGCT-3 (nt 541–560) and F2 5 -CTGCCAGTGTCTCCATGTATG-3 (nt 755–775), and the reverse primer were R1 5 -TTCCTGGTAGGTGCGGCA-3 (nt 691–708) and R2 5 -GATTCCGTCCTCCAACTGTTC-3 (nt 1051–1071). The cDNA was used as a template to perform 3 and 5 RACE with a SMARTTM cDNA kit (Clontech, USA), following the manufacturer’s protocol. The primer F2 and primer SMIIIB 5 -GCCGAGGCGGCCGACATGTT3 were used to perform 3 RACE, whereas primer R1 and primer SM2 V 5 -AAGCAGTGGTATCAACGCAGAGT-3 were used to perform 5 RACE. PCR amplifications were performed using one cycle at 94 ◦ C for 2 min, followed by 35 cycles of 30 s at 94 ◦ C, 30 s at 53 ◦ C, and 2 min at 72 ◦ C, and a final step of 72 ◦ C for 10 min. PCR products were
Fig. 2. Histology of the AG in intact (control) and after bilateral eyestalk ablation. (A) A cross-section of the AG in the control group at day 8, showing type I cells at the peripheral region and occupying about 20% of the gland. (B) A cross-section of the AG in a day-4 bilateral eyestalk-ablated male, showing a hypertrophic gland containing an increasing of proportion of type I cells (I). (C) A cross-section of the AG in a day-8 bilateral eyestalk-ablated male, showing a hypertrophic gland containing an obvious larger proportion of type I cells (I) than type II cells (II). (D) A higher magnification of the rectangular area in C, showing bordering zone of type I and type II cells. (E) A hematoxylin-eosin stained section of C. quadricarinatus (Cq) androgenic gland (AG), showing multiple lobules, each surrounded by a thin sheet of connective tissue. (F) A similar section at higher magnification, showing AG cells inside each lobule intensely stained with anti-Cq-IAG.
M. Sroyraya et al. / Tissue and Cell 42 (2010) 293–300
297
analyzed by agarose gel electrophoresis and bands of expected sizes extracted and cloned into pGEM-Teasy vector (Promega, USA). Inserts were sequenced by Macrogen, Korea. 2.8. Statistical analysis of AG cells The percentages of type I and II cells in each group were presented as means and standard deviations. The percentages of type I and II cells in different groups of the same period of sampling were compared and analysed by a one-way analysis of variance (ANOVA). P < 0.05 was considered to be statistically different. 3. Results 3.1. Anatomy of the AG The AG of the blue swimmer crab is attached to the distal part of the spermatic duct (SD) and the ejaculatory bulb (EB), located between the muscles of the fifth pair of walking legs (Fig. 1(A)). It appears as pale yellow patches on one side of the SD and EB (Fig. 1(B)). When viewed in cross-section, the AG is composed of two or three lobules tightly packed together at one side of the SD and EB. Each lobule is surrounded by thin sheets of connective tissue (Fig. 1(C)). On one side the AG abuts the epithelium of the SD and EB, while on the opposite side it is surrounded by a thicker sheet of connective tissue (Fig. 1(C) and (D)). The AG is composed of two main cell types, designated as type I and type II cells (Fig. 1(D)). Type I cells are located on the periphery of each AG lobule under the outer layer of connective tissue, and are small round cells of 5–7 m in diameter. They occupy about 20% of the AG volume. Each cell had a round nucleus containing dense chromatin, and cytoplasm that is stained blue with hematoxylin. Type II are larger cells of 13–15 m in diameter, and occupy about 80% of the AG volume. Each cell possesses a round, vesiculate nucleus (containing mostly euchromatin), with a prominent nucleolus, and the cytoplasm is stained light blue with hematoxylin (Fig. 1(D)). Occasionally, binucleated cells, which could be dividing cells, are found between these two groups (Fig. 1(D)). 3.2. Effects of eyestalk ablation on the AG The structures of the AGs in the unilateral eyestalk-ablated males at day 0, 4, and 8, were not different from the intact (control), and exhibited histological features as described above (Fig. 2(A)). In contrast, the AGs of day-4 and day-8 bilateral eyestalk-ablated crabs were approximately 2-fold and 3-fold larger, respectively, than the controls (Fig. 2(B)–(D)). In particular, the number of type
Fig. 3. Percentages of type I cells in AG: control (), unilateral eyestalk-ablated ( ), and bilateral eyestalk-ablated () male crabs, at days 0, 4, and 8. The columns represent the percentages of type I cells per total number of cells counted under a light microscope, using an Image J program (error bars = SD, n = 6). Asterisk indicates a significant difference when compared among the groups at the same day (P < 0.05).
I cells in the bilateral eyestalk-ablated group had increased significantly in a time-dependent manner. Fig. 3 shows the percentages of type I cell in the gland. At day 0 after eyestalk ablation, the percentage of type I cells among three groups were not different. At day 4 after eyestalk ablation, the number of type I cells in the bilateral eyestalk-ablated group had increased significantly to 35.60 ± 0.03% (P < 0.05), compared with 25.23 ± 0.01% for the control and 22.4 ± 0.03% for the unilateral eyestalk-ablated males. At day 8 after eyestalk ablation, the number of type I cells in the bilateral eyestalk-ablated group had increased significantly to 48.19 ± 0.10% (P < 0.05), compared with 21.91 ± 0.03% for the control and 24.38 ± 0.01% for unilateral eyestalk-ablated males. The percentages of type II were correspondingly opposite to the percentages of type I cells. 3.3. Specificity of the antibody against Cq-IAG The specificity of the pre-absorbed antibody was tested by dot blot analysis. Pre-immune serum showed no positive immunoreactivity on the membrane (Fig. 4(A)), while non-absorbed antibody showed immunoreactivity at the dots with varying amount of BSA and Cq-IAG-BSA conjugate (Fig. 4(B)). In the pre-absorbed antibody with 100 mg/ml BSA, at a dilution of 1:100, the immunoreactivity was detected in the dots containing conjugated peptide, but not BSA (Fig. 4(C)). Therefore, this pre-absorbed antibody was specific to Cq-IAG and used for immunolocalization of Pp-IAG in sections of tissues.
Fig. 4. Dot blot analysis of the specificity of an antibody against Cq-IAG. (A) Negative control using pre-immune serum to detect BSA and Cq-IAG-BSA conjugate. No positive dot is shown on the membrane. (B) Non-absorbed antibody was used as a probe. Both dots containing BSA and Cq-IAG-BSA conjugate at various dilutions show positive signal. (C) Pre-absorbed antibody with 100 mg/ml of BSA at a dilution 1:100 (v/v) was used as a probe. Only conjugated peptide, but not BSA dots were positive to the antibody.
298
M. Sroyraya et al. / Tissue and Cell 42 (2010) 293–300
Fig. 5. Amino acid sequence alignment of IAG in five decapod crustaceans. The IAG factors, species names and GenBank accession numbers are: Pp-IGH, P. pelagicus, HM459854; Cd-IAG, C. destructor, EU718788.1; Cq-IAG, C. quadricarinatus, DQ851163.1; Mr-IAG, M. rosenbergii, FJ409645.1; and Pm-IAG, P. monodon, GU208677.1. Green letters show putative signal peptides, blue letters show putative B and A chains, red letters show putative cleavage sites, black letters show C peptides, gray letters in Pm-IAG is an ambiguous part, shaded gray are conserved C residues. The underlined hydrophilic RRRNSDTTD (aa 128-136) of Cq-IAG corresponds to the sequence of a peptide synthesised by GenScript (USA), and susequently used for antibody production. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
3.4. Cloning and sequencing of Pp-IAG In order to confirm that the antibody produced against the Cq-IAG peptide would also react with a Pp-IAG factor, a deduced sequence of the Pp-IAG factor was identified from AG cDNA. The deduced Pp-IAG sequence is given in Fig. 5, together with GenBank sequences of 4 other decapods crustacean, namely Cd-IAG (C. destructor, EU718788.1), Cq-IAG (C. quadricarinatus, DQ851163.1), Mr-IAG (M. rosenbergii, FJ409645.1), and Pm-IAG (P. monodon, GU208677.1). An alignment of all five sequences showed that PpIAG is 96% similar to Cd-IAG, 86% to Cq-IAG, 23% to Mr-IAG, and 25% to Pm-IAG. The conserved insulin-like regions of signal sequence, A and B chains, C peptides, putative proteolytic cleavage sites, and C residues, are apparent in all but Pm-IAG. Importantly, the CqIAG peptide RRRNSDTTD used to make our antibody was almost identical to the Pp-IAG peptide RRRDSDTTD,which is incidentally identical to a corresponding putative peptide present in Cd-IAG.
3.5. Immunoperoxidase staining of Pp-IAG Immunoperoxidase staining showed that the Cq-IAGimmunoreactivity, which could as well represent Pp immunoreactivity (Pp-IAG-ir), was detected in the AG and not in any parts of the SD or EB of the intact, unilateral and bilateral eyestalk-ablated crabs (Fig. 6(A)–(F)). All negative control sections of all groups showed no Pp-IAG-ir (data not shown), while the positive control showed the immunostaining of cells in the Cq-AG (Fig. 2(F)). In the intact group, a low amount of Pp-IAG-ir was found mostly in peripheral AG cells, with fewer positive cells in the central area of each lobule (Fig. 6(A) and (B)). At higher magnification the positive cells at the periphery of the lobules were identified as type I cell (Fig. 6(B)-inset). A similar pattern of staining was observed in the AG of the unilateral eyestalk-ablated group (data not shown). In the bilateral eyestalk-ablated group, the AG showed
an increased number and more intense Pp-IAG-ir cells at day 4 after the eyestalk ablation. The intense Pp-IAG-ir was detected in the cytoplasm of the type I cells, located at the periphery of each lobule (Fig. 6(C) and (D)) Noticeably, the staining was not even and the high intensity tended to be distributed in the peripheral zone of the cytoplasm (Fig. 6(C), (D) and (D)-inset). Conversely, the type II cells that occupy the central zone of each lobule tended to show a less intense staining. At day 8 after bilateral eyestalk ablation, the Pp-IAG-ir became even more intense than in the 4-day AG (Fig. 6(E) and (F)). In addition, the immunoreactivity was similar in both type I and type II cells, and the staining was even throughout the cytoplasm of both cell types.
4. Discussion One requirement for identifying an insulin-like factor in the androgenic gland of the P. pelagicus (Pp-IAG), and other species, was production of a specific antibody, so we initially produced one against a synthetic 9-amino acid peptide derived from a conserved region of the IAG of C. quadricarinatus and C. destructor. We also produced cDNA from the androgenic gland of the P. pelagicus to obtain a deduced sequence of Pp-IAG (GenBank accession number HM459854). The deduced Pp-IAG sequence (Fig. 5) was 96% similar to Cq-IAG. It varied by only one amino acid in the nonapeptide used to make the antibody, indicating that the antibody against Cq-IAG would react with Pp-IAG in immunohistochemistry. Histologically, the AG of P. pelagicus is composed of two types of cells, and this is similar to M. rosenbergii (Okumura and Hara, 2004), and Penaeus chinensis (Li and Xiang, 1997). In contrast, the presence of three cell types has been reported in S. paramamosain (Liu et al., 2008), and M. kistnensis (Mirajkar et al., 1984). The type III cells, described in the latter two species, appear larger and exhibit more vacuoles inside their cytoplasm. These characteristics imply that
M. Sroyraya et al. / Tissue and Cell 42 (2010) 293–300
299
Fig. 6. Immunoperoxidase staining, with anti-Cq-IAG, of the androgenic gland (AG) in intact (control), unilateral eyestalk-ablated, and bilateral eyestalk-ablated crabs. (A) A low power micrograph of AG cross-section in control group, showing few Pp-IAG-ir positive cells at periphery of the lobules. (B) A high power micrograph of (A) showing mostly Pp-IAG-ir positive type I cells in the periphery of the AG (inset). There are only few positive cells in the central area of the gland. (C) A low power micrograph of an AG cross-section of a bilateral eyestalk-ablated male at day 4, showing intense Pp-IAG-ir in the AG cells, but not in the spermatic duct (SD) and its epithelium (EP). (D) A high power micrograph of (C) showing intense Pp-IAG-ir in the cytoplasm of type I cells. High intensity of Pp-IAG-ir is present at the edge of the cells (inset). (E) A low power micrograph of an AG cross-section of a bilateral eyestalk-ablated crab at day 8, showing very intense IAG-ir in both type I and type II cells. There is no Pp-IAG-ir in the SD and its epithelium (EP). (F) A high power micrograph of (E) showing very intense and evenly distributed Pp-IAG-ir in the cytoplasm of both types of AG cells. Pp-IAG-ir is present throughout the cytoplasm of the cells (inset).
the type III cells may be undergoing degeneration. Alternatively, it has been suggested that the different cell types may just represent different stages of a secretory cycle of a single cell type (Thampy and John, 1970; Veith and Malecha, 1983; Mirajkar et al., 1984). An ultrastructure study in M. rosenbergii lends support to this notion (Okumura and Hara, 2004), as it was shown that both type I and II cells possessed abundant rough endoplasmic reticulum (RER), and that the RER of the type II cells were highly dilated, implying that they were producing proteins for secretion. In addition, secretory granules have been found in the cytoplasm of the A. vulgare AG cells, suggesting that they secrete protein (Negishi et al., 2001). An intense hematoxylin staining of the cytoplasm of type I cells in P. pelagicus, also implies that a high amount of ribosomes and RER is present in the AG cells of this species. This was confirmed by our
data which indicated that Pp-IAG-ir is present in both types of cells, albeit with different staining intensity. This difference may reflect greater synthesis in type I cells. The binucleated cells in the AG of O. platytarsis were attributed to mitotic division (Thampy and John, 1970). It is also interesting to note that in the P. pelagicus AG, most of the binucleated cells are present in the zone between type I and type II cells. It is possible that type I cells are dividing and give rise to type II cells, which then move towards the central area of the AG lobule. The bilateral eyestalk-ablated crabs showed hypertrophy of the AG, whereas the intact and the unilateral eyestalk-ablated crabs did not. This could be because the AG is under the inhibitory control by neuropeptides secreted from the X-organ-sinus gland complex in the eyestalk, as indicated earlier (Hoffman, 1968; Khalaila et
300
M. Sroyraya et al. / Tissue and Cell 42 (2010) 293–300
al., 2002). The AG in unilateral eyestalk-ablated crabs might still receive the inhibitory control from the remaining eyestalk, while in the bilateral eyestalk-ablated crabs this inhibition is completely abrogated, thus resulting in the hypertrophic AG. The bilateral eyestalk ablation significantly increased the percentage of type I cells from an initial 24.14% to 48.19% at day 8 following the treatment. During this time the percentage of type II cells decreased, suggesting that type I cells may be the precursor cells that divide and give rise to type II cells. This idea is supported by a recent study by Liu et al. (2008), in which type I AG cells were found to be a major population in immature mud crabs, S. paramamosain, while type II AG cells were more abundant in mature crabs. This observation indicated that type I cells may differentiate into type II cells during the maturation of the crabs. Our immunoperoxidase data showed that type I cells were immunoreactive in both the intact and eyestalk-ablated males, while type II cells were less immunoreactive. Futhermore, type I cells exhibited stronger staining than type II cells at 4-day post eyestalk ablation, but the staining intensity was the same in both cells types by day 8. This finding suggests that under an absence of inhibitory hormone from the eyestalk, the type I cells in P. pelagicus start to synthesize IAG earlier, and in greater amounts, than type II cells. The type II cells may represent a late stage of the AG cells, and are reaching the final phase of secretion. Further studies, using cell labelling to indicate the type I cell proliferative capacity, simultaneously with immunolabelling (to indicate the cell’s ability to synthesize IAG), are now needed to confirm that there is a single lineage of type I and type II cells in the AG of decapod crustaceans. Acknowledgments This research was supported by a Distinguished Research Professor Grant (co-funded by the Thailand Research Fund, the Commission on Higher Education (CHE), Mahidol University) to P. Sobhon, and a CHE-Ph. D. scholarship to M. Sroyraya. References Abdel Razek, F.A., Taha, S.M., Ameran, A.A., 2006. Ecological observations on the abundance, distribution of holothuroids (Echinodermata: Holothuroidea) in the Red Sea coast, Egypt. Egypt. J. Aquatic Res. 32, 419–430. Balesdent-Marquet, M.L., 1958. Presence of an androgen gland in crustacean isopod, Asellus aquaticus L. Comptes Rendus Hebdomadaires des Seances de l’Academie des Sciences 247, 534–536. Campos-Ramos, R., Garza-Torres, R., Guerrero-Tortolero, D., Maeda-Martnez, M., Obregon-Barboza, H., 2006. Environmental sex determination, external sex differentiation and structure of the androgenic gland in the Pacific white shrimp Litopenaeus vannamei (Boone). Aquac. Res. 37, 1583–1593. Chaiyawat, M., Eungrasamee, I., Raksakulthai, N., 2008. Quality characteristics of blue swimming crab (Portunus pelagicus, Linnaeus 1758) meat fed Gracilaria edulis (Gmelin) Silva. Kasetsart J. (Natural Science) 42, 522–530. Chande, A.I., Mgaya, Y.D., 2003. The fishery of Portunus pelagicus and species diversity of portunid crabs along the coast of Dar es Salaam, Tanzania. Western Indian Ocean. J. Mar. Sci. 2, 75–84. Charniaux-Cotton, H., 1954. Discovery in, an amphipod crustacean (Orchestia gammarella) of an endocrine gland responsible for the differentiation of primary and secondary male sex characteristics. Comptes Rendus Hebdomadaires des Seances de l’Academie des Sciences 239, 780–782. Cui, Z., Liu, H., Lo, S.T., Chu, K.H., 2005. Inhibitory effects of the androgenic gland on ovarian development in the mud crab Scylla paramamosain. Comp. Biochem. Physiol. 104, 343–348. Foulks, N.B., Hoffman, D.L., 1974. The effects of eyestalk ablation and B-ecdysone on RNA synthesis in the androgenic glands of the protandric shrimp, Pandalus platyceros Brandt. Gen. Comp. Endocrinol. 22, 439–447. Hasegawa, Y., Katakura, Y., 1985. Masculinization of female by the newly-formed androgenic glands in the ZW and WW females of the isopod crustacean, Armadillidium vulgare. Zool. Sci. 2, 419–422.
Hoffman, D.L., 1968. Seasonal eyestalk inhibition on the androgenic glands of a protandric shrimp. Nature 218, 170–172. Juchault, P., Legrand, J.J., 1978. Function of the androgen gland in crossed implantations between two species of isopod terrestrial Crustacea, Porcellio dilatatus Brandt and Armadillidium vulgare Latreille: specificity of androgenic hormones and neurohormones implicated in the control of androgenic function. Gen. Comp. Endocrinol. 36, 175–186. Katakura, Y., 1960. Transformation of ovary into testis following implantation of androgenous glands in Armadillidium vulgare, an isopod crustacean. Annot. Zool. 33, 241–244. Khalaila, I., Manor, R., Weil, S., Granot, Y., Keller, R., Sagi, A., 2002. The eyestalkandrogenic gland-testis endocrine axis in the crayfish Cherax quadricarinatus. Gen. Comp. Endocrinol. 127, 147–156. King, D.S., 1964. Fine structure of the androgenic gland of the crab, Pachygrapsus Crassipes. Gen. Comp. Endocrinol. 47, 533–544. Lee, T.H., Shigesawa, R., Yamasaki, F., 1993. Partial masculinization of female Eriocheir japonicus (Brachyura, Grapsidae) by androgenic gland implantation. Suisan Zoshoku 41, 311–319. Li, F., Xiang, J., 1997. Preliminary studies on form, structure and function of androgenic gland in Penaeus chinensis. Chin. Sci. Bull. 42, 499–503. Liu, H., Cheung, K.C., Chu, K.H., 2008. Cell structure and seasonal changes of the androgenic gland of the mud crab Scylla paramamosain (Decapoda: Portunidae). Zool. Stud. 47, 720–732. Malecha, S.R., Nevin, P.A., Ha, P., Barck, L.E., Lamadrid-Rose, Y., Masuno, S., Hedgecock, D., 1992. Sex-ratios and sex-determination in progeny from crosses of surgically sex-reversed freshwater prawns, Macrobrachium rosenbergii. Aquaculture 105, 201–218. Manor, R., Weil, S., Oren, S., Glazer, L., Aflalo, E.D., Ventura, T., Chalifa-Caspi, V., Lapidot, M., Sagi, A., 2007. Insulin and gender: an insulin-like gene expressed exclusively in the androgenic gland of the male crayfish. Gen. Comp. Endocrinol. 150, 326–336. Minagawa, M., Chiu, J.R., Kudo, M., Takashima, F., 1994. Male reproductive biology of the red frog crab, Ranina ranina, off Hachijojima, Izu Islands, Japan. Mar. Biol. 118, 393–401. Mirajkar, M., Sarojini, R., Nagabhushanam, R., 1984. Histophysiology of the androgenic gland in freshwater prawn Macrobrachium kistnensis. J. Anim. Morphol. Physiol. 31, 211–220. Mires, D., 1977. Theoretical and practical aspects of the production of all-male Tilapia hybrids. Bamidgeh 29, 94–101. Nagamine, C., Knight, A.W., 1987. Masculinization of female crayfish, Procambarus clarkii (Girard). Invert. Reprod. 11, 77–87. Nagamine, C., Knight, A.W., Maggenti, A., Paxman, G., 1980. Masculinization of female Macrobrachium rosenbergii (de Man) (Decapoda, Palaemonidae) by androgenic gland implantation. Gen. Comp. Endocrinol. 41, 442–457. Negishi, S., Hasegawa, Y., Nakajima, Y., 2001. Novel structures in secreting the androgenic gland hormone. Zool. Sci. 18, 1237–1243. Okumura, T., Hara, M., 2004. Androgenic gland cell structure and spermatogenesis during the molt cycle and correlation to morphotypic differentiation in the giant freshwater prawn, Macrobrachium rosenbergii. Zool. Sci. 21, 621–628. Sagi, A., Aflalo, E.D., 2005. The androgenic gland and monosex culture of Macrobrachium rosenbergii (De Man): a biotechnological perspective. Aquac. Res. 36, 231–237. Sagi, A., Cohen, D., Milner, Y., 1990. Effect of androgenic gland ablation on morphotypic differentiation and sexual characteristics of male freshwater prawns, Macrobrachium rosenbergii. Gen. Comp. Endocrinol. 77, 15–22. Simeo, C.G., Ribes, E., Rotllant, G., 2009. Internal anatomy and ultrastructure of the male reproductive system of the spider crab Maja brachydactyla (Decapoda: Brachyura). Tissue and Cell 41, 345–361. Sumpton, W.D., Potter, M.A., Smith, G.S., 1994. Reproduction and growth of the commercial sand crab, Portunus pelagicus (L.) in Moreton Bay, Queensland. Asian Fish. Sci. 7, 103–113. Suzuki, S., Yamasaki, K., 1998. Sex reversal by implantations of ethanol-treated androgenic glands of female isopods, Armadillidium vulgare (Malacostraca, crustacea). Gen. Comp. Endocrinol. 111, 367–375. Taketomi, Y., 1986. Ultrastructure of the androgenic gland of the crayfish, Procambarus clarkii. Cell Biol. Int. Rep. 10, 131–136. Thampy, D.M., John, P.A., 1970. On the androgenic gland of the ghost crab Ocypoda platytarsis M. Edwards (Crustacea: Brachyura). Acta Zool. 51, 203–210. Veith, W.J., Malecha, S.R., 1983. Histochemical study of the distribution of lipids, 3aand 3b- hydroxysteroid dehydrogenase, in the androgenic gland of the cultured prawn, Macrobrachium rosenbergii (de Man) (Crustacea: Decapoda). S. Afr. J. Sci. 79, 84–85. Ventura, T., Manor, R., Aflalo, E.D., Weil, S., Raviv, S., Glazer, L., Sagi, A., 2009. Temporal silencing of an androgenic gland-specific insulin-like gene affecting phenotypical gender differences and spermatogenesis. Endocrinology 150, 1278–1286.