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Identification of an [alpha]-peptide in Haliotis Rubra with homology to the Lymnaea [alpha]-CDCP spawning peptide.

ABSTRACT It is now understood that a combination of molluscan reproductive peptides are commonly cleaved from a large preprohormone and influence different aspects of spawning behavior. One type of reproductive peptide, known in Lymnaea stagnalis as [alpha]-CDCP, and in Aplysia californica as [alpha]-BCP, acts in egg laying via temperature-dependent autoinhibition or autoexcitation of neuronal cells. In our study, the expression of [alpha]-CDCP-like peptide in the blacklip abalone, Haliotis rubra, was identified by Western blots and immunocytochemistry, using an antiserum developed against [alpha]-CDCP. Western blots of total protein isolated from the central nervous system, cerebral and pleuropedal ganglia, as well as gonad and heptopancreas tissues of sexually mature adults, identified a protein of approximately 100 kDa as well as a range of smaller reactive peptides. This finding suggests that a reproductive [alpha]-peptide is probably synthesized from a single larger precursor protein. The larger peptides were also identified in Western blots of several abalone tissues, lmmunocytochemistry using the same antiserum showed the presence of immunoreactive axons in all the tissues studied, indicating synthesis or transport of products. The function of the abalone [alpha]-CDCP-like peptide is yet to be determined.

KEY WORDS: Haliotis rubra, egg-laying behavior, neuropeptides, [alpha]-CDCP


Gastropods have been commonly used to study the neuronal basis of behavior due to the simplicity of their nervous systems. Examples include Aplysia and Lymnaea, in which reproduction is characterized by a stereotyped behavioral repertoire, controlled by several neuropeptides and culminates in egg laying (Nambu & Scheller 1986, Rothman et al. 1992, Griffond et al. 1992, Van Minnen et al. 1989).

Recombinant DNA technologies have been used to identify and characterize sequences encoding neuropeptides that are associated with the egg laying. These studies have indicated that, like most neuroactive peptides, biologic peptides are initially synthesized and cleaved from a large preprohormone sequence, known as the egg-laying hormone (ELH) preprohormone (Scheller et al. 1982, Scheller et al. 1983, Mahon et al. 1985, Nambu & Scheller 1986). The preprohormone characteristically begins with an initiator methionine residue, a hydrophobic signal sequence, and is followed by a number of cleavage sites (Shyamala et al. 1986). The ELH gene family is defined by the presence of a highly conserved ELH domain, and in Aplysia this corresponds to approximately 36 amino acids, while [beta]-, [alpha]-, [gamma]-, and calfluxin-related bag cell peptides (BCPs) may be present or absent. The A. californica gene family consists of five genes, three of which have been well defined, including a BC precursor isolated from BCs and peptides A and B precursors preferentially expressed in the atrial gland (Scheller et al. 1983). Immunolocalization studies have indicated that ELH expression is not restricted to the BCs and the atrial gland because the abdominal ganglion proper, the pleural, cerebral, and buccal ganglia have also been implicated as immunoreactive cell tissues (Shyamala et al. 1986).

It is now understood that, in Aplysia, a combination of peptides can be produced from a prohormone and each can influence a different aspect of behavior (Dudek & Tobe 1978, Shyamala et al. 1986, Geraerts et al. 1988). One important peptide derived from the precursor, [alpha]-BCP, is produced by the neurosecretory BCs upon excitation, and acts via a temperature-dependent autoinhibition or autoexcitation of the BCs (Rothman et al. 1983, Sigvardt et al. 1986, Redman & Berry 1991). Following release it is rapidly inactivated (Sigvardt et al. 1986). The [alpha]-BCPs are 100% identical in primary sequence across all Aplysiidae species so far examined (Li et al. 1999). The amino acid sequence is Ala-Pro-Arg-Leu-Arg-Phe-Tyr-Ser-Leu.

In Lymnaea, egg laying is also triggered by an array of neuropeptides, encoded by an ovulation hormone gene family, including the caudodorsal cell hormone (CDCH) I and II gene precursors (Vreugdenhil et al. 1988). These precursors produce at least nine caudo-dorsal cell peptides (CDCPs), including a 9 amino acid sequence encoding [alpha]-CDCP. Despite belonging to a different gastropod class to Aplysia, the [alpha]-CDCP retains the residues Arg-Leu-Arg-Phe, as encoded by the [alpha]-BCP gene. The CDCH precursor has a calculated molecular weight of 25 kDa, but due to a high percentage of charged amino acids, it migrates at 35 kDa in pulse-label experiments (Geraerts et al. 1985).

The first report of neuropeptide involvement in abalone reproduction was by Yahata (1973), who observed induced spawning of abalone after injection of homogenized ppg and visceral ganglia of mature females. Injections of homogenized cerebral ganglia (cg) produced no notable change in the ovaries. More recently, neuro-secretion in the cg, ppg, and visceral ganglia was investigated in H. discus hannai, to determine the role of hormones in the regulation of reproduction (Hahn 1994). He found two types of cells in the cg that appeared to be neurosecretory. In another study involving the cg of H. asinina, two types of neurosecretory cells (NS), N[S.sub.1] and N[S.sub.2], were identified (Upatham et al. 1998). However, there is no experimental proof that the peptides produced in these cells regulate reproduction and growth.

A nucleotide sequence containing an abalone ELH (aELH) has been cloned from H. rubra, and shows high homology with the sequences of the CDCH of Lymnaea and ELH of Aplysia (Wang & Hanna 1998). However, no sequences have been obtained that encode an abalone [alpha]-CDCP-like peptide. Therefore, the current research work aimed to characterize [alpha]-CDCP-like immunoreactive structures within the abalone neural and reproductive systems to determine whether a homologous peptide was present.


Animals and Tissue Preparation

Mature male and female H. rubra (Leach) with ripe gonads were collected from Port Phillip Bay (Victoria) under Fisheries research permits (97/R/049A and RP 626). Tissues were then dissected for analyses of proteins, as well as preparation of tissue sections.

For protein extraction, approximately 200 mg of tissue was macerated, suspended in 2 mL SDS lysis buffer (2% SDS, 50 mM Tris-HCl (pH 7.2), 1 mM [beta]-mercaptoethanol) and boiled for 3 min. The sample was homogenized (Industrial Equipment) and then boiled for a further 5 min. Protein solutions were microcentrifuged (Microcentaur MSE) at 13,000 rpm for l0 min and supernatant collected for storage at -70[degrees]C. Protein concentration was quantified using the Bradford method for protein quantitation (Kruger 1996).

Paraffin sections were prepared for immunocytochemistry by firstly fixing dissected tissues in freshly prepared 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) at 4[degrees]C for 24 h. They were then transferred to phosphate buffer and stored at 4[degrees]C before dehydration in an ascending ethyl alcohol series, xylene, then embedding in paraffin. Serial sections of 8 [micro]m were cut with a microtome (Triangle Biomedical, CUT Series Rotary Microtome) and mounted on gelatin-coated slides. Paraffin sections of H. discus hannai ppg were kindly provided Dr. Kirk Hahn.

Antibodies Against L. Stagnalis [alpha]-CDCP

Antiserum against [alpha]-CDCP peptide was kindly provided by Dr. Gregg Nagle (University of Texas Medical Branch, Galveston, Texas). This polyclonal antibody was made in rabbit and recognizes residues 144-152 (Glu-Pro-Arg-Leu-Arg-Phe-His-Asp-Val) of the L. stagnalis CDCH precursor (Vreugdenhil et al. 1988).

Western Blots

Larger proteins and peptides were separated by 12% SDS-PAGE using the method of Laemmli (1970), then electro-transferred by a Mini-Blot apparatus (Bio-Rad) to 45-[micro]m nitrocellulose sheets (Bio-Rad). After transfer, the membrane was incubated with 5% bovine serum albumin (BSA) in phosphate-buffered saline (PBS; 50 mM Na[H.sub.2]P[O.sub.4] and [Na.sub.2]HP[O.sub.4], 154 mM NaCl, pH 7.4) for 2 h at room temperature (RT), with agitation. The membranes were incubated with the primary antibody, rabbit anti[alpha]-CDCP, at a dilution of 1:1,000 (in Tris-buffered saline [TBS; 20 mM Tris-HCl pH7.5,500 mM NaCl] with 3% BSA) for 2 h. After several washes with PBS, the binding was visualized using HRP-coupled goat antirabbit antibody (Sigma), followed by enhanced chemiluminescence (Amersham) according to the manufacturer's instructions.

Identification of small immunoreactive peptides was performed after 12% SDS PAGE (Gradipore) in Tris-Tricine buffer and subsequent electro-transfer to polyvinylidene fluoride (PVDF) sheets (Bio-Rad). Detection of [alpha]-CDCP-like peptides was performed as for the larger peptides.


Immunofluoresence was performed following dewaxing and rehydration of tissue sections. Blocking was then carried out, first in 1% glycine for 30 min, then in 4% BSA for 30 min. Sections were incubated for 1 h at RT in primary antibody (anti[alpha]-CDCP) diluted 1:500 in PBS, rinsed three times in PBS, then incubated for 1 h at RT with FITC-labeled goat antirabbit secondary antibody (Sigma). Sections were rinsed three more times in PBS, and then mounted in FITC mounting solution (90% glycerol, 4% n-propyl-gallate in 50 mM PBS pH 8.2). The preparations were examined under a fluorescence microscope (Leica) and the images were captured with a Spot cooled CCD camera (Diagnostic Instruments).

Immunoenzyme staining was performed following dewaxing and rehydration of tissue sections. Endogenous peroxidase activity was quenched by incubation of sections in 3% [H.sub.2][O.sub.2] in methanol for 10 min. After washing in PBS, blocking was performed, first in 1% gelatin for 30 min then in 4% BSA for 30 min. Sections were rinsed three times in PBS, incubated for 1 h at RT in primary antibody diluted I:1000 in PBS, then rinsed three times in PBS, and incubated in HRP-labeled goat antirabbit secondary antibody (Sigma) in the dark at RT. After three rinses in PBS, the sections were developed in 3-amino-9-ethylcarbazole (AEC) substrate solution (ICN). When sufficient color occurred, development was stopped by a wash in distilled water, and the sections counterstained in Mayer's haematoxylin. After a further wash the sections were mounted in Faramount aqueous mounting solution (DAKO) and viewed under a Zeiss Axioskop MC 80 microscope. Images were captured on a Spot cooled CCD camera (Diagnostic Instruments).


For each experiment it was important to perform controls to ensure antibody specificity. Immunoreactivity of the respective peptides was not observed following antibody preincubation with crude abalone tissue protein (data mot shown). Also, no immunoreactivity was observed when blots or sections were incubated with no primary antibody (data not shown).



Figure 1A shows the results of Western blots of combined cg and ppg tissues, mature male gonad (mgo), and mature female gonad (fgo) sample separations transferred to nitrocellulose and reacted with anti[alpha]-CDCP as the primary antibody. In the combined cg-ppg sample, a protein was identified of approximately 100 kDa. The mature mgo and fgo also contained the 100 kDa protein, but also contained immunoreactive proteins approximately 48 kDa and 46 kDa in size.


Additional smaller immunoreactive proteins were identified by chemiluminesence detection in Western blots performed using SDS-PAGE in Tris-Tricine buffer and transfer to PVDF membranes (Fig. 1B). The combined cg-ppg sample mow showed the presence of the 48 kDa and 46 kDa proteins, as well as additional smaller ones of approximately 36 kDa and 29 kDa. The mgo contained additional immunoreactive 32 kDa and 22 kDa proteins, whereas the fgo contained additional 36 kDa, 28 kDa, and 23 kDa proteins.

Western blots of fractionated total proteins isolated from the H. rubra heart, foot, rectum, gill, and tentacle also showed a 100 kDa protein that was immunoreactive with anti[alpha]-CDCP (Fig. 1C). As well, the 48 and 46 kDa proteins were also present in all tissues, except the foot tissue.


Immunocytochemistry studies, using an anti[alpha]-CDCP probe on sections of neural and gonadal tissues, showed strong positive results (Figs. 2A to H). The cg and ppg of the mature H. rubra contained numerous immunopositive cells (Figs. 2A to F). Immunoreactive cells of the cg were distributed throughout the cortex, but were mainly located within the outer cells and within the inner cortex (Figs. 2A and B). The outer cells appear to be the neurosecretory N[S.sub.1] and N[S.sub.2] cells identified by Upatham and colleagues (1998). A large number of immunoreactive axons were found to extend into the medulla of the left and right cg, and through the dorsal cerebral commissure.


The ppg also contained abundant immunopositive cells that were distributed within the cortex region (Figs. 2C and D). Many of these cells were likely to be of the NS type because they were located close to the basement membrane. In addition, the immunoreactivity was strong, indicating there was a large amount of reactive protein present. A high density of immunoreactive axons was identified transversing the medial and lateral regions of the medulla in the ganglion mass, and extending into the pedal and cerebro-pleural connectives (Figs. 2E and F). No immunoreactivity was observed within the statocysts. As well, no difference was observed in peptide distribution in the neural tissues between female or male H. rubra.

To show conservation between abalone species, ppg sections prepared from H. discus hannai were also tested with anti[alpha]-CDCP. Numerous immunopositive fibers were present in the medulla of the ppg of H. discus hannai (Fig. 2G). No reactivity was seen in the cortex, possibly due to Stieve's fixative used to prepare these ganglia, and the antibodies may not have been able to penetrate cortex cells, the antigen destroyed by fixation, or the peptide was not present in these cells. However, as observed in H. rubra, intensely stained axons were observed throughout the medulla, and appeared to form a loop (Fig. 2H).

Immunoreactive material to anti[alpha]-CDCP was localized in the fgo and mgo of H. rubra (Fig. 3A to C). In the fgo, immunopositive protein was demonstrated in special cells and fibers of the trabeculae (Fig. 3A). In addition, a small number of immunoreactive cells and fibers were identified within the gonad capsule, and the hepatopancreas capsule (Fig. 3B), but never within the oocyte nucleus. Control sections corresponding to the same tissue resulted in no positive signals. The level of immunoreactivity in the gonads appeared to differ between individual animals, possibly as a result of oocyte maturation. In the mgo, immunoreactivity was restricted to cells and fibers of the trabeculae (Fig. 3C), as observed in fgo. Also, a small number of immunoreactive cells were located within the gonad capsule and hepatopancreas capsule (data not shown).



The focus of our research involves the cg, ppg, and gonad tissues, due to their importance in gastropod reproduction (Geraerts et al. 1988, Yahata 1973). The difficulty associated with the isolation of the visceral ganglia hindered any experimental analysis on this tissue.

The expression of an [alpha]-CDCP-like peptide in abalone was firstly identified by Western blots. Total protein isolated from abalone tissues confirmed that the anti[alpha]-CDCP probe was reactive to a protein of 100 kDa in the combined cg and ppg tissues, as well as the gonads of H. rubra. Reactive peptides of 46 and 48 kDa were also detected in the gonad tissue. This finding was consistent with peptides being synthesized from a 100-kDa precursor protein, but was larger than the 32 kDa identified in Aplysia (Berry 1981), or 25 kDa as observed in Lymnaea (Geraerts et al. 1985).

Due to the small size of a fully processed [alpha]-CDCP (9 aminoacids), it was expected that a homologous [alpha]-CDCP-like peptide in abalone would not be detected in standard blots. However, a number of possible intermediates were observed and is consistent with the findings in A. californica, that post-translational processing by specific convertases produces peptides of different sizes and these can be post-translationally modified (Fisher et al. 1988). They showed by Western blot analysis that antibodies recognized small final-product peptides and intermediates in the processing pathway, but all contained the sequence encoding the immunogen. Thus, the different sized bands observed in H. rubra Western blots probably represent differential cell processing of the polypeptides. Our results indicate that the abalone precursor of 100 kDa is initially processed to release a 35-kDa protein (aELH immunoreactive, Cummins et al. 2002) and a 48-kDa protein ([alpha]-CDCP immunoreactive, this study), prior to further processing. If this is correct, the size of the precursor is much larger than that shown for the Aplysia ELH egg-laying precursor peptide (32 kDa), and may indicate that the genetic make-up of the H. rubra reproductive precursor is different to Aplysia and Lymnaea homologues. However, the multiple bands may also be the result of expression of a multigene aELH family, as shown in Aplysia species (Scheller et al. 1983), in which different expression occurs in different tissues.

Western blots also showed the [alpha]-CDCP-like peptide distribution in the heart, foot muscle, rectum, gill, and tentacle tissue, suggesting that these peptides may have a broad function in this animal. Similarly, peripheral tissues in L. stagnalis are known to contain immunoreactive material to egg-laying peptides (Van Minnen et al. 1989). However, subsequent immunohistochemical analyses in this laboratory have shown that immunoreactivity is predominantly localized to neural ganglia, therefore the other positive material may only be an indication of transport of protein or peptides.

Previous immunocytochemical studies have successfully used antibodies against Aplysia and Lymnaea egg-laying peptides to demonstrate their presence in the neural systems of other invertebrates. For example, peptides homologous to ELH have been immunolocalized in the neural cells of Busycon and Mytilus (Ram et al. 1998, Croll et al. 1993). Also, [alpha]-CDCP is expressed in the neural network of Helix aspersa, Mytilus edulis, and Busycon canaliculatum (Croll et al. 1993, Ram et al. 1998). In a previous study, peptides immunoreactive to antisera against CDCH, [alpha]-CDCH and [beta]-CDCH, showed positive immunoreactions in tissues of Sarcophaga bullata (Diptera), Leptinotarsa decemlineata (Coleoptera), Locusta migratoria, and Periplaneta americana (Orthoptera) (Theunis et al. 1990). More recently, it has been shown that antisera directed against the same three peptides results have been detected in the central nervous system of the rhyonobdellid leech, Theromyzon tessulatum (Salzet et al. 1997).

Our use of anti[alpha]-CDCP in immunocytochemistry has demonstrated that an [alpha]-CDCP-like peptide is extensively distributed throughout H. rubra cg and ppg tissues. These results imply that the cg could be important in the regulation of reproduction in gastropods. The same distribution pattern was observed for aELH (Cummins et al. 2002), and enhances the possibility that these peptides are translated as a single precursor. Immunoreactivity appeared to be localized to the N[S.sub.1] or N[S.sub.2]-type cells in the cortex. However, it seemed that the distribution of immunoreactive cells containing aELH and [alpha]-CDCP-like peptide were not concentrated within one specific region or cluster of cells, but they were widely distributed within the cortex cells and axons. These reproductive peptides may be released from much of the surface of the nervous system and not just from well-defined neurohemal organs. This is unlike studies of other molluscs, in which antisera have been shown to react with specific clusters of neuronal populations in the ganglia (i.e., such as the immunoreactivity pattern of antiCDCH in L. stagnalis (Van Minnen et al. 1988). However, we did observe axonal loops within the medulla of ganglia by anti[alpha]-CDCP immunolocalization. It has been suggested that similar loops in L. stagnalis are important sites of synaptic integration (Van Minnen et al. 1988).

Concerning the function of the immunoreactive neurons, it is possible that reproductive peptide colocalization may facilitate their simultaneous release into the surrounding hemolymph, as is the case in other gastropods (Chiu & Strumwasser 1981, Bernheim & Mayeri 1995). Extensive immunoreactive axonal distribution throughout the medulla of the cg and ppg suggests that these may lead to sites of neuropeptide release. In Lymnaea, the CDCs release egg-laying peptides into the hemolymph via neurohemal areas and also from blind ending axons in the medulla of the commissure (Van Minnen et al. 1989). We can also speculate that, similar to Aplysia and Lymnaea, H. rubra reproductive peptides too may control reproduction through autoexcitation or autoinhibition of other neural tissues. However, to fully elucidate their biologic activity, further experiments involving the identification of a precursor sequence, bioassays and electrophysiology would be required. Given that abalone are broadcast spawners (not internal fertilizers), peptide function will invariably be different to Aplysia and Lymnaea. Further investigations are also required to provide a more detailed analysis of the cellular structure of the immunoreactive cells. Although our study using light microscopy predicts that they are of NS type, more definitive ultrastructural studies would provide more precise details.

Despite extensive research being conducted on neuropeptide immunoreactivity within the central nervous system, relatively little research has focused on the reproductive tissue. However, CDCH genes have been immunocytochemically localized to the female part of the reproductive tract and exocrine secretory cells of the male part (Van Minnen & Vreugdenhil 1987). Our study has shown that an [alpha]-CDCP-like peptide is present in the trabeculae of the fgo and in mgo trabeculae of H. rubra. The peptide was only located within certain cell types, which may function as a peptide storage cell for the maturing gonad. It has been suggested that the granulated cells of the gonad trabeculae are the endocrine cells of the gonads (Apisawetakan et al. 2001). Indeed, recent immunocytochemical experiments performed using antiaELH on H asinina gonad tissues, have shown high immunoreactivity in the granulated cells (Chanpoo et al. 2001). The large amount of reproductive-like peptides within the fgo may be a function of the gastropod requirement to release high concentrations of hormone into the hemolymph or gonad to cause maturation of oocytes prior to spawning. It is known in molluscs, that hormone release areas are high in number (Joosse 1988).

The observation of reproductive peptides in the male H. rubra trabeculae is not unusual. Evidence from Drosophila melanogaster shows that when a male mates and releases sperm, he also deposits an ELH that induces egg laying in the female (Park & Wolfner 1995). This hormone is related, by use of cross-reactive antisera, to the ones found in gastropods and other invertebrates. In addition, L. stagnalis CDCH gene products are known to be secreted into the male duct and transferred to the female copulant during copulation, and may function to accelerate the start of egg laying (Van Minnen et al. 1989). Thus, H. rubra trabeculae reproductive peptides may play a similar role, but because they are spawners, this role may be altered.

In summary, these results indicate that [alpha]-CDCP-like peptide could play a significant role in abalone reproduction.


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School of Biological and Chemical Sciences, Deakin University, Geelong, VIC 3217, Australia

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Author:Hanna, Peter J.
Publication:Journal of Shellfish Research
Date:Dec 15, 2004
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