Crustacean hyperglycemic hormone (CHH) neuropeptides family: Functions, titer, and binding to target tissues

General and Comparative Endocrinology 166 (2010) 447–454

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Crustacean hyperglycemic hormone (CHH) neuropeptides family: Functions, titer, and binding to target tissues J. Sook Chung a,*, N. Zmora a, H. Katayama b, N. Tsutsui c a

Center of Marine Biotechnology, University of Maryland Biotechnology Institute, Baltimore, MD, USA Department of Applied Biochemistry, Institute of Glycotechnology, Tokai University, 1117 Kitakaname, Hiratsuka, Kanagawa 259-1292, Japan c Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo, Tokyo 113-8657, Japan b

a r t i c l e

i n f o

Article history: Received 1 August 2009 Revised 4 December 2009 Accepted 14 December 2009 Available online 22 December 2009 Keywords: CHH MIH Binding Titer Energy metabolism Molting Osmoregulation Second messengers Vitellogenesis VtG expression

a b s t r a c t The removal of the eyestalk (s) induces molting and reproduction promoted the presence of regulatory substances in the eyestalk (ES), particularly medulla terminalis X-organ and the sinus gland (MTXO– SG). The PCR-based cloning strategies have allowed for isolating a great number of cDNAs sequences of crustacean hyperglycemic hormone (CHH) neuropeptides family from the eyestalk and non-eyestalk tissues, e.g., pericardial organs and fore- and hindguts. However, the translated corresponding neuropeptides in these tissues, their circulating concentrations, the mode of actions, and specific physiological functions have not been well described. The profiles of CHH neuropeptides present in the MTXO–SG may differ among decapod crustacean species, but they can be largely divided into two sub-groups on the basis of structural homology: (1) CHH and (2) molt-inhibiting hormone (MIH)/mandibular organinhibiting hormone (MOIH)/vitellogenesis/gonad-inhibiting hormone (V/GIH). CHH typically elevating the level of circulating glucose from animals under stressful conditions (hyper- and hypothermia, hypoxia, and low salinity) has multiple target tissues and functions such as ecdysteroidogenesis, osmoregulation, and vitellogenesis. Recently, MIH, known for exclusively suppressing ecdysteroidogenesis in Y-organs, is also reported to have an additional role in vitellogenesis of adult female crustacean species, suggesting that some CHH neuropeptides may acquire an extra regulatory role in reproduction at adult stage. This paper reviews the regulatory roles of CHH and MIH at the levels of specific functions, temporal and spatial expression, titers, their binding sites on the target tissues, and second messengers from two crab species: the blue crab, Callinectes sapidus, and the European green crab, Carcinus maenas. It further discusses the diverse regulatory roles of these neuropeptides and the functional plasticity of these neuropeptides in regard to life stage and species-specific physiology. Ó 2010 Elsevier Inc. All rights reserved.

1. Introduction Molting precedes somatic growth during the entire life cycle of arthropods, whereas the pubertal, adult molt is closely associated with reproduction. The presence of regulatory factors within the eyestalk (ES) is suggested by the observations after eyestalk ablation (ESA) (Zeleny, 1905) and the injection of ES extract into ablated animals (Abramowitz et al., 1944). The eyestalk ganglia located within the eyestalk is comprised of a group of neurosecretory perikarya in the medulla terminalis X-organs (MTXO) for synthesis of a battery of crustacean hyperglycemic hormone neuropeptides (CHH) family and the sinus gland (SG) for their storage and release. The induction of molting or reproduction after ESA implies that putative regulatory hormones are most likely inhibitory in nature: molt-inhibiting hormone (MIH) and vitellogenesis/gonad -inhibiting hormone (V/GIH) * Corresponding author. E-mail address: [email protected] (J.S. Chung). 0016-6480/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.ygcen.2009.12.011

(Skinner, 1985). Additionally, the ventral nerve cords (VNC) may be the source of a stimulatory factor(s), particularly for female reproduction: vitellogenesis/gonad-stimulating hormone (V/GSH) (Charniaux-Cotton and Payen, 1988). The first structural identification of CHH (Kegel et al., 1989) by Edman N-terminal sequencing has expanded into subsequent isolation, purification, and characterization of other neuropeptides from the SG including MIH, CHH, and mandibular organ-inhibiting hormone (MOIH). The functions of not many of these neuropeptides have been confirmed by a bioassay(s), whereas the majority has been named based on the structural similarities (Böcking et al., 2002; Chan et al., 2003; Frajul-Moles, 2006; Lacombe et al., 1999). All these neuropeptides are structurally related and belong to the family of CHH neuropeptides. This CHH family also includes an ortholog of CHH, the insect ion transport peptide (ITP) that has been identified in insects (Dircksen et al., 2008; Drexler et al., 2007; Phillips et al., 1998). Recently, PCR-based cloning strategies have enabled for characterizing a great number of cDNAs encoding CHH neuropeptides

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from the ES and even non-ES tissues, e.g., pericardial organs (PO), subesophageal ganglia, and fore- and hindguts (Chang et al., 1999; Chung et al., 1999; Chung and Zmora, 2008; Dircksen et al., 2001). The sequence homology based on the deduced amino acid sequences of these neuropeptides cDNAs has led into two sub-groups within the family of CHH neuropeptides (Böcking et al., 2002; Chan et al., 2003; Chen et al., 2005; Frajul-Moles, 2006; Lacombe et al., 1999). However, the translated neuropeptides in the tissues and their isoforms that are derived from post-translational modification (Chung and Webster, 1996; Chung et al., 1998; Soyez et al., 1994), their circulating concentrations, the mode of actions, and specific physiological functions have not been well described. This review updates and reports the hormonal status of some of these neuropeptides in light of the traditional definition of a ‘hormone’: titer, the second messengers, and receptor binding on the putative target tissues in terms of physiological relevance. 2. Hormonal roles of CHH neuropeptides and their physiological functions 2.1. Energy metabolism 2.1.1. Expression of CHH neuropeptides and their hemolymph concentrations As alluded to above, the molecular cloning of cDNAs encoding CHH neuropeptides has greatly increased in numbers (GenBank), while relatively less is known about the localization or identification of translated neuropeptides (Azzouna et al., 2003; Böcking et al., 2002; Gu et al., 2001, 2002; Van Herp, 1998). In Carcinus maenas, Cancer pagurus, and Callinectes sapidus, CHHs in MTXO–SG are all assigned to hyperglycemic functions with known-structural sequences of cDNA and neuropeptide, or neuropeptide(s) alone. CHH is present in the SG as two structural isoforms that are derived by the cyclization of N-terminus at a post-translational modification. The cyclized form is the major neuropeptide in SG, along with the highest expression of CHH mRNA in the XO (Chung and Webster, 1996, 2003; Chung et al., 1998; Chung and Zmora, 2008). Like most invertebrates, crustaceans facing with stressful environmental or physiological conditions switch to an alternative anaerobic energy metabolism, i.e., glycolysis, the process of which is modulated by CHH. The life history of a species may be responsible for the difference in the basal, resting levels of CHH, as C. sapidus experiencing a wide range of environmental conditions showed 10 times higher at 10 10M concentration than C. maenas at 10 11M (Chung and Webster, 2005; Chung and Zmora, 2008). Stressful environmental conditions: hypoxia, hypo-/hyperthermia, exercise, hyper-/hyposalinity or infection may signal through enkephalinergic or serotoneric neurons (Charmantier et al., 1997) for the immediate release of CHH from the SG into hemolymph (Chang et al., 1998; Chung and Webster, 2005). Despite CHH has a very short half-life time (T½) of 5–10 min (Chung and Webster, 2008), its release under continuous stressful conditions elevates the levels of glucose and lactate in hemolymphs in 30 min, lasting 2–3 h. When animals return to resting/normal conditions, the elevated glucose and lactate levels slowly reduce to basal levels. It is not yet known in crustaceans, if insulin or insulin/insulin like substance reverse the response of hyperglycemia. In addition, an alternative splice form of CHH, produced by PO also responds to the similar conditions by the simultaneous increases in PO-CHH expression, its release, and tissue specific hyperglycemia (Chung and Zmora, 2008). 2.1.2. Target tissues, binding sites, and second messengers A traditional radiolabeled binding assay revealed that CHH binds to multiple tissues in C. maenas and C. sapidus. The SG-CHH

role in energy metabolism is mediated via its binding to hepatopancreas (Katayama and Chung, 2009; Kummer and Keller, 1993; Webster, 1993) that is the major metabolic site and the largest internal tissue. Despite different target tissues, the values of binding affinities (KD) are constant at the level of 1.20 13.0  10 10 M, and the maximum number of binding sites (BMAX) ranges between 0.5–9.0  10 10M/mg protein (Table 1). Interestingly, the structural difference between the C-terminal region of CHHs of ES and PO provides tissue specific bindings to hepatopancreas, abdominal muscle, and scaphognathites with similar values of KD and BMAX as shown in Table 1 and Fig. 1 (Katayama and Chung, 2009). When the specific activity of a hormone has not yet been found, a traditional binding study can be useful to locate and identify a putative target tissue where a putative, relevant function may be defined (Katayama and Chung, 2009). The mode of action of CHHs by an in vitro incubation study involves cGMP as a second messenger as its binding causes the large amount of cGMP production in the target tissues, while 8-Br-cGMP mimics CHH induced hyperglycemia (Chung and Webster, 2006). These results suggest that CHH receptor is likely to be a membrane-bound guanylyl cyclase(s) (MGC) particularly in muscles (Goy, 1990). However, the structure of a CHH receptor has not yet been characterized in any crustacean species. 2.2. Molting 2.2.1. Expression of CHH neuropeptides and their hemolymph concentrations Molting is an essential and repeated physiological process for somatic growth in arthropods. Ecdysozoans proceed to the next life stage via molt cycle that consist of intermolt, premolt, ecdysis (shedding of old cuticle), and post molt (Skinner, 1985). Each species experience a species-specific fixed number of juvenile molts during the life cycle to reach adulthood. Some crustaceans, particularly females (C. sapidus, C. bairdi etc.) cease/halt molting at adult stage, as being terminally anecdysial during their reproductive phase, while other species continue to molt during adulthood. As stated, the bilateral eyestalk ablation inducing molting, more precisely the second molt after ablation, implies a low level or absence of MIH or CHH-A (Chang et al., 1990), which in turn stimulates the synthesis and release of ecdysteroids from the Y-organ (YO), molting gland in crustaceans that is an equivalent to the prothoracic gland in insects. Both the expression of MIH (MIH mRNA) in the XO and MIH in the SG are 5–10 times lower than those of CHH (Chung and Webster, 2003). MIH and CHH at >10 9M show the maximal inhibition of 50– 80% in the synthesis and secretion of ecdysteroids by in vitro YO assays. Several in vivo studies have been reported with recombinant (r) MIHs at >10 7M concentrations on the regulation of hemolymph ecdysteroids and molting (Gu et al., 2001, 2002; Nakatsuji and Sonobe, 2004; Okumura et al., 2005). A daily injection of native MIH, native CHH, or both at physiologically relevant concentrations (<10 9M) maintains the low levels of ecdysteroids from the eyestalk ablated C. sapidus where the elevated ecdysteroid level was normally observed (Chung, 2010). Nonetheless, these data from in vivo experiments support the hormonal role of MIH in regulating ecdysteroid in hemolymphs. Considering the very short T½ of MIH in hemolymph, similar to that of CHH (Chung and Webster, 2008), it is likely that the activity of YO is regulated through a brief daily exposure to MIH or CHH. MIH titers in the hemolymph ranges between 10 11–12 M in the following species: C. maenas, P. clarkii, and C. sapidus, where MIH profiles during molt cycle appears to be different and a species specific (Chung and Webster, 2005; Nakatsuji and Sonobe, 2004; Zmora et al., 2009a,b). MIH in hemolymph concentrations in C. maenas and C. sapidus remains constant during molt cycle, whereas,

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J.S. Chung et al. / General and Comparative Endocrinology 166 (2010) 447–454 Table 1 Tissue specific binding sites of CHH and MIH: values of BMAX and KM and their titers in decapod crustaceans. Tissues membranes SG-CHH

PO-CHH SG-MIH

10

BMAX (10 a,b

a

b

7.07 2–4 0.86 ± 0.90 2–4 6.50 ± 1.15 2.40 ± 0.42 2.31 ± 0.44 0.78 ± 0.01 0.61 ± 0.70 8.06 1.30 ± 0.20 1.15 ± 0.70 1.3 9.24 4.80

Cam-hepatopancreas Cas-hepatopancreasc Orl-hepatopancreasb Cam-gillsd Cas-gillsc Cam-hindgutd Cas-abdominal musclesc Cas-scaphognathiesc Cam-YOa Cas-abdominal musclesc Cas-scaphognathiesc Cam-YOa Cas-YOe Cas-hepatopancrease

M/mg protein)

KM (10 a

10

M)

b

1.45 4–6 8.10 4–6 5.77 ± 2.05 1.20 ± 0.22 3.54 ± 1.49 13.00 ± 2.00 ND 1.88 11.00 ± 2.00 ND 1.6 4.19 322.00

Titer (M) 10

11

10

11 h

10

12

–10

9

(f,g,h,i)

( ) –10

11 e,i,j

(

)

Cam, C. maenas; Cas, C. sapidus; Orl, O. Limosus. a Webster (1993). b Kummer and Keller (1993). c Katayama and Chung (2009). d Chung and Webster (2006). e Zmora et al. (2009a). f Chang et al. (1998). g Chung and Webster (2005). h Chung and Zmora et al. (2008). i Zmora et al. (2009b). j Nakatsuji and Sonobe (2004).

data do seem fragmentary in explaining the hormonal role of MIH in molt regulation, they may be adequate, when considered a possible differential regulation particularly in life cycle and history of animals between marine and freshwater species.

Scaphognathite Muscle Heart Midgut Hindgut Gills Hepatopancreas

A -4

0

4

8

12

-11

Binding sites (E M/ mg protein)

Scaphognathite Muscle Heart Midgut Hindgut Gills Hepatopancreas

B 0

4

8

12

-11

Binding sites (E M/ mg protein) Fig. 1. Spatial distribution of specific binding sites of [125I] ES-CHH (A) and [125I] PO-CHH (B). (A) The specific binding sites of [125I] ES-CHH were challenged with 10 pmol of cold ES-CHH (solid bar) or cold rPO-CHH (open bar). (B) The specific binding sites of [125I] PO-CHH were competed with 10 pmol of cold rPO-CHH (open bar) or cold ES-CHH. The data are presented as mean ± 1 SE (n = 6) (Katayama and Chung, 2009).

in the freshwater crayfish P. clarkii, its concentration is lower at premolt than intermolt (Chung and Webster, 2005; Nakatsuji and Sonobe, 2004). Adult C. sapidus at premolt shows lower MIH expression in ES than at intermolt (Lee et al., 1998). In contrast, MIH expressions in MTXO of adult C. maenas analyzed using QPCR are constant during molt cycle (Chung and Webster, 2003). While these

2.2.2. Target tissues: binding sites and second messengers The mode of action of MIH appears more intricate than that of CHH. MIH exclusively binds to high-affinity (KD value = 10 10M/ mg protein) receptors in YO where it increases in cGMP levels in C. maenas (Chung and Webster, 1996; Chung and Webster, 2003; Webster, 1993). In addition to cGMP, the transient increase in cAMP is noted prior to significant cGMP accumulation (Saïdi et al., 1994), while cyclic nucleotide analogs and inhibitors of phosphodiestrase(s) mimicked the MIH action in the suppression of YO activities in C. maenas, C. sapidus, and P. clarkii (Nakatsuji et al., 2009, 2006; Okumura, 2006; Saïdi et al., 1994). Furthermore, a nitric oxide (NO)-cGMP pathway is suggested by the findings of the presence of soluble guanylate cyclase (GC) in the YO and the suppression of ecdysteroidogenesis by NO donors and NO synthase stimulator(s) (Covi et al., 2009; Kim et al., 2004; Lee et al., 2007). Given the fact of cGMP involvement in ecdysteroidogenesis in the YO, a MGC is suggested for a candidate for MIH receptor of the YO (Zheng et al., 2006), similar to CHH as described above. Indeed cDNA of MGC(s) recently is isolated from YO which exhibits a molecular weight of 137 kDa on a Western blot analysis, thus implying that it may be a putative receptor for MIH (Zheng et al., 2008). In contrast, the putative receptor of MIH in the YO that was chemically cross-linked with [125I]MIH revealed approximately 51–70 kDa protein (Asazuma et al., 2005; Zmora et al., 2009b). Overall, further studies are required for characterizing receptor(s) and elucidating the specific mode of actions of MIH and CHH on the inhibition of ecdysteroidogenesis in YO. 2.3. Iono–osmoregulation and water uptake 2.3.1. Expression of CHH neuropeptides and their hemolymph concentrations The eyestalk ablated animals are compromised in adapting different salinity, suggesting that the substance(s) involved directly

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or indirectly in iono-osmoregulation is present in the MTXO–SG (Charmantier-Daurers et al., 1994; Mantel and Farmer, 1983), while thoracic ganglia of euryhaline crabs is a neuroendocrine source (Kamemoto, 1991). A direct osmoregulatory role of CHH is suggested by the presence of an ortholog of CHH in arthropods, insect ion transport peptide (ITP) that is involved in regulation of Cl transport on insect ileum, thus assisting water re-absorption (Kamemoto, 1991; Phillips et al., 1998). CHH involvements in gill ion transport are reported in Pchygrapsus marmoratus and in Astacus leptodactylus, that they increase the transepithelial voltage and Na+ influx (Serrano et al., 2003; Spanings-Pierrot et al., 2000). Recently, the cDNA encoding ITP like molecule is found in other tissues including gills of Litopenaeus vannamei (Tiu et al., 2007) where the transcription level was co-related with the salinity. However, the translated neuropeptide has not yet been identified. Therefore, the direct role of CHH in iono–osmoregulation needs to be addressed. Somatic growth in crustaceans is punctuated by molt process as stated above. More specifically, the mechanism of somatic growth of aquatic crustaceans is dependent on a massive water uptake, mostly via drinking, that is equivalent to 60–80% of body volume, resulting in a molt increment, during the process of ecdysis of 2– 3 h. This phenomenon had been described (Baumberger and Olmsted, 1928) as an unknown substance may have driven the process. Earlier attempt to locate/identify the source responsible for the water uptake in the ES failed as the ablated animals proceeded normal ecdysis in C. sapidus (Neufeld and Cameron, 1994). However the structurally identical CHH presence in the brain-gut axis is responsible for this process that is specifically produced in the endocrine cells of fore-and hindgut during premolt of C. maenas (Chung et al., 1999; Webster et al., 2000), implying its expression may be induced by the elevated ecdysteroid in hemolymph. The dramatic and total release of CHH from these cells initiates the ecdysial process with the bursting of the ecdyseal line that is caused by rapid iso-osmotic water uptake. At the mid to completion of ecdysis (animals escape from the old cuticle), the level of CHH in hemolymph reaches the maximal concentration at 10 9M, which is 100 times as high as that at intermolt. This influences the further iso-osmotic water uptake to 1–2 h after ecdysis, completing the increment of 20–50% at molt. It has been suggested that absorption through guts is accountable for 60–70% of total water uptake (Chung et al., 1999), while the gills and the newly laid soft cuticle may be responsible for the rest (Neufeld and Cameron, 1994). The direct effect of CHH has been tested by injecting native CHH into animals at the beginning of ecdysis to determine if the CHH peak induced by injection caused elevated drinking and swelling of the body. Those animals which received an excess amount of CHH, experienced rapid body swelling, failing to retract the appendages from the old cuticle, and did not complete the process successfully. The hormonal status of CHH in the animals’ brain-guts is therefore notable during ecdysis for iso-osmotic water uptake via initial drinking, while the exact mechanism underlying the release and water uptake into other tissues after drinking still needs to be addressed. 2.3.2. Target tissues: binding sites and second messengers The presence of CHH binding sites in gills and guts that is coupled to increase in cGMP levels and a high concentration of CHH in the animals acclimated to low salinity further support its role in iono/osmoregulation as they are the prime iono/osmoregulatory sites (Chung and Webster, 2006). Given the ES-CHH role in energy metabolism, the binding sites of CHH present in gills where exhibits high activities of Na+ + K+-ATPase and carbonic anhydrase (Henry et al., 2003a,b; Lucu and Towle, 2003) may be associated with supplying the energy required for iono/osmoregulation. In light of the structural complexity of gills (Lawson et al., 1994),

CHH may have multiple functions: glucose mobilization and ionregulation. The specific mechanisms regulating these two processes in these tissues are not known and needs to be studied. 2.4. Ovarian development 2.4.1. Expression CHH neuropeptides and their hemolymph concentrations As stated above, molting and reproduction are two highly energy-dependent processes which appear to be intertwined and well-coordinated by the neuropeptides secreted in the MTXO–SG. Terminally anecdyseal animals reaching adulthood stay as reproductively active, whereas some females continue to molt throughout the reproductively active adult stage. Similar to molting, the ESA induces an increase in ovarian development and precocious spawning (Panouse, 1943), suggesting the presence of VSH in the ES (Van Herp, 1998) and in the brain and ventral nerve cord (Eastman-Reks and Fingerman, 1984; Hinsch and Bennett, 1979; Sarojini et al., 1983). Lack of an adequate bioassay hampered for characterizing and defining the endocrine regulatory role of V/ GIH and V/GSH in crustacean female reproduction, while a co-relationship between the expression of mRNA of GIH and reproductive stages has been reported (de Kleijn et al., 1998). Recently the full length of vitellogenin (VtG) cDNA cloned from a few shrimp and prawn, crayfish, and crabs enabled the study of neuroendocrine regulation of vitellogenesis and ovarian development. Thus, the active role of these neuropeptides has been defined by in vitro and in vivo studies using recombinant or native neuropeptides. A CHH found in MTXO–SG of M. japonicus acts as a GIH by directly inhibiting VtG transcription from the ovarian tissues of immature females (Tsutsui et al., 2005). In contrast, CHH-B found in the MTXO–SG of H. americanus stimulates ovarian maturation, where a GIH, another isoform of CHH suppresses vitellogenesis (Tensen et al., 1989; Van Herp, 1992). In Metapenaeus ensis, an isoform of MIH-B is expressed in the MTXO, VNC, thoracic ganglion, and brain and the injection of rMIH-B increase the levels of VtG expression and VtG in ovary and hepatopancreas (Tiu and Chan, 2007). Nonetheless, the data reported so far present a putative hormonal role of VIH or VSH, as a direct relationship between endogenous circulating levels of these neuropeptides and reproductive stages has not yet been established. Furthermore, the regulatory neuropeptides in the MTXO–SG coordinate the control of molt/ reproduction, yet it has not been clarified how CHH and or MIH share a dual role in molting and reproduction in species that undergo adult molting. Besides the direct action of V/GIH, MOIH and CHH with a dual functions of hyperglycemic and inhibition of methyl farnesoate synthesis are found in the MTXO–SG of C. pagurus and Libinia emarginata, respectively (Liu et al., 1997a,b; Wainwright et al., 1996). Particularly, MOIH is suggested with the ovarian development via regulating the synthesis and release of methyl farnesoate by the mandibular organs (Wainwright et al., 1996). Adult female C. sapidus, C. bairdi and L. emarginata cease molting during reproductive active adult stage and become terminal anecdysis. The regulatory mechanism of sustaining anecdyseal status appears to differ between C. sapidus and the two majidae crabs. Adult C. sapidus female with normal sized YO re-instate molt cycle after ESA while halting reproduction (Havens and McConaugha, 1990), whereas there is no report on ablated C. bairdi female with degenerated YO. The profiles of CHH/MIH in the MTXO–SG of juvenile and adult female of C. sapidus differ only in terms of quantity, suggesting that both are involved in the inhibition of molt process. Surprisingly our recent in vitro study shows that MIH present in the MTXO–SG stimulates heterogeneous nuclear VtG RNA at early vitellogenesis and both VtG transcription and VtG secretion at mid-vitellogenic stage (Zmora et al., 2009a,b). Additionally, MIH

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titers between early and later vitellogenic stages differ, being significantly higher in mid- vitellogenic stage than in early stage (Zmora et al., 2009a,b). The results suggest that MIH regulates vitellogenesis in a stage-dependent manner: stimulatory at the levels of transcription and translation of VtG at mid-vitellogenesis, but its effect seems different at early stages. This stage dependent effect of MIH appears to be similar to what has been reported in the signaling pathways of VIH in M. japonicus (Okumura, 2006). In light of the regulatory action of MIH in vitellogenesis, MIH may play a dual role in females of crab species especially those exhibit terminal anecdysis upon sexual maturity: sustaining intermolt, while stimulating vitellogenesis (Zmora et al., 2009b). Yet, it is quite clear that some species exhibit a specific form of VIH/VSH in the MTXO and/or VNC, while others seem to utilize CHH or MIH or both for the regulation of molting and reproduction. Given the fact of the regulatory roles of VIH/VSH in reproduction, MIH and CHH may need investigating their temporal expression and release patterns at both juvenile and adult stages of each species.

a 100

A Specific binding (DPM/tube)

25000 75

50

c b

25

bc

0

[125I] CHH binding (% of YO)

B

20000 15000 10000 5000 0

250

0

c

10

20

30

40

50

60

70

[125I] MIH concentration (nM) 200

B 150

100

a

a

a

a

a

a

a

100

50

b 0

3 HP 1 2 le ill ry -HP YO HP HP HP -G usc Ova J M F F F F M F F

J-

Tissue Fig. 2. MIH specifically binds to membranes of mature female hepatopancreas with higher binding at mid-vitellogenic stage than pre-vitellogenic stage. (A) Specific binding of [125I] MIH to various tissue membranes of vitellogenic females and YO and hepatopancreas of juveniles. (B) Specific binding of [125I] CHH to the same membranes. All membranes were prepared from five animals, except for J-YO membranes that were prepared from 700 intermolt animals. F, females; M, males; J, juveniles; HP, hepatopancreas; 1, 2, and 3 refer to the ovarian stages; F-ovary, ovarian membrane of females at ovarian stage 3. Results are presented as mean ± SE of the triplicates as% of the J-YO. The alphabetical letters show the significant differences at P < 0.05 (Zmora et al., 2009a).

% Binding

[125I] MIH binding (% of YO)

A

2.4.2. Target tissues: binding sites and putative second messengers The roles of VIH/VSH in vitellogenesis and ovarian development suggest ovary and/or hepatopancreas for their putative target tissues. The binding characteristics and the mode of action of these neuropeptides have been little studied, while multiple second messengers for V/GIH are suggested: cyclic nucleotides, Ca2+, and protein kinase C in vitellogenesis of M. japonicus (Okumura, 2006). In C. sapidus, the regulatory action of MIH in vitellogenesis is supported by the presence of its ovarian stage-dependent specific binding sites in the hepatopancreas as shown in Figs. 2 and 3, which possibly modulate a cAMP pathway (Zmora et al., 2009b). Table 1 summarizes the different binding kinetics of MIH noted in terms of the values of BMAX and KD in these tissues, compared to the multiple binding sites of CHH in various tissues of a few decapod crustacean species. The suggested molecular weight of MIH receptor in the YO of C. sapidus and M. japonicus is 51–70 kDa, respectively (Asazuma et al., 2005; Zmora et al., 2009b). MIH receptor located in hepatopancreas of adult vitellogenic female C. sapidus is 51 kDa protein, (Fig. 4), is similar to that in YO. However, it seems that these receptors utilize different second messengers for their actions as describe above. The size of CHH receptor in hepatopancreas is

75

50

25

0 0.01

0.1

1

10

100

MIH concentration (nM) Fig. 3. Binding characteristics of MIH in membranes of (A) YO of C. maenas and C. sapidus (B) hepatopancreas of female C. sapidus at ovarian stage 3. Five membrane preparations were pooled and tested. (A) Saturation curve. (B) Displacement curve. [125I] MIH binding sites were displaced with unlabelled rMIH (closed circles) or unlabelled CHH (open circles). The data are presented as mean ± SE of the triplicates (Zmora et al., 2009a).

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A Marker (kDa) 150

B MIH Tb

1

10

Marker (kDa)

Tb

SG-CHH 10

Tb 10

200 150

100

100

75

75

50

MIH

50

37 25 20 15

37

25

20

Fig. 4. Determination of the molecular size of receptors of (A) MIH and CHH on membranes of juvenile YO (100 lg) and adult female hepatopancreas (200 lg) and (B) CHH on juvenile hepatopancreas. Binding sites of [125I] MIH in the membranes of female hepatopancreas and juvenile YO have a similar molecular mass of 51 kDa, which is different from that of [125I] CHH in the hepatopancreas. Tb = total binding; 1 or 10 pmol of unlabeled MIH or CHH, specified at the top of the lane. YO membranes were resolved on a 4– 15% SDS–PAGE and hepatopancreas membranes on a 10% SDS–PAGE. Labeled ligand and receptor complexes are indicated with arrows (Zmora et al., 2009a).

slightly larger 61 KDa (Fig. 4). Further characterization of these receptors remains to be determined. 3. Conclusion In this review, existing physiological and endocrinological data of the CHH neuropeptides family are piercing together in order to better understand their regulatory roles in various crustacean physiologies. It is likely that many of these neuropeptides can be multifunctional like CHH and MIH. A GSH activity shown by MIH in adult female C. sapidus, is life-stage dependent and portrays the importance of life stage in studying functionality of neuropeptides. In general, however, the functionality of these neuropeptides seem to be incomplete, fragmentary and sometimes conflicting in their involvement in crustacean physiology, as the data have been gleaned from using many different species with different life histories. A multi-disciplinary approach to redefine the hormonal roles of the CHH neuropeptides family in terms of expression, hemolymph titer and their mode of actions including receptor characterization are imperative and essential in understanding regulatory mechanisms underlying the diverse physiology of crustaceans. Furthermore, using functional genomics like RNA interference (RNAi) and manipulating an ortholog shared in ecdysozoan species that genome is known may provide an insight into their precise actions during a particular stage. Acknowledgments This work was supported by the National Oceanic and Atmospheric Administration (NOAA), Chesapeake Bay Program Grant (NA17FU2841) to the Blue Crab Advanced Research Consortium and Binational Agricultural Research Development (MB-8714-08). Dr. H. Katayama was supported by a Research Fellowship of the Japanese Society for the Promotion of Science for Young Scientists. This article is contribution no. 09-208 of the Center of Marine Biotechnology, University of Maryland Biotechnology Institute, MD, USA.

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