Vitellogenin Structure, Processing and Synthetic Control

Aqua-BioScience Monographs Vol. 3, No. 3, pp. 73–110 (2010) www.terrapub.co.jp/onlinemonographs/absm/

Reproductive Mechanisms in Crustacea Focusing on Selected Prawn Species: Vitellogenin Structure, Processing and Synthetic Control

Marcy N. Wilder,1* Takuji Okumura2 and Naoaki Tsutsui1

1Japan International Research Center for Agricultural Sciences 1-1 Ohwashi, Tsukuba, Ibaraki 305-8686, Japan 2National Research Institute of Aquaculture, Fisheries Research Agency Minami-ise, Mie 516-0193, Japan e-mail: [email protected]

Abstract Received on September 9, 2010 Shrimp culture is a significant world-wide industry, with current production levels reach- Accepted on October 25, 2010 ing over 3 million tons per year. The expansion of the industry has given rise to the Online published on December 27, 2010 problems of environmental deterioration due to intensive-scale culture, and the outbreak of disease. While many of these issues are now being sufficiently addressed, the estab- Keywords lishment of sustainable seed production technology is an area that should be given con- • Macrobrachium rosenbergii tinued attention. In this regard, it remains difficult to control reproduction under hatch- • Marsupenaeus japonicus ery conditions for a large number of commercially-important species. At present, an un- • Litopenaeus vannamei derstanding of reproductive mechanisms in Crustacea is not complete, although in recent • Pandalus hypsinotus years, a great deal of knowledge has accumulated on vitellogenin structure, processing, • vitellogenin • vitellin and synthetic site in a number of economically-important species. This monograph will • crustacean reproduction cover the current status of research on vitellogenin in decapod crustaceans, especially • molting prawns and shrimp, and discuss mechanisms of vitellogenin synthetic control, both dem- • vitellogenesis-inhibiting hor- onstrated and postulated. The monograph will also present current knowledge of crusta- mone cean vitellogenin receptors, and cover related facets of reproductive development, such as mechansisms of cortical rod formation and the utilization of vitellin during embryogenesis. Finally, future directions for this research and potential applications to aquaculture will be discussed.

1. Introduction control of disease (especially viral) and artificial seed production, remain areas of importance in relation to In recent years, shrimp culture has become a signifi- the sustainability of the industry. Regarding the latter, cant world-wide industry. Current production levels while the life cycle has been closed (e.g., the ability to reach over 3 million tons per year, corresponding to a obtain larvae from parent spawners, which are in turn market volume of over 10 billion U.S. dollars (FAO reared to maturity and used as parent shrimp) in the 2008). Especially in Southeast Asia, where more than whiteleg shrimp, Litopenaeus vannamei, it remains 75% of the world’s shrimp culture occurs, shrimp farm- difficult to control reproduction under hatchery condi- ing has been considered to be the cause of myriad en- tions for a large number of commercially-important vironmental problems such as the destruction of man- species. Indeed, for Li. vannamei, not more than 30% grove forest, and deterioration of the coastal environ- of all females in a given hatchery will develop mature ment due to efflux from intensive shrimp farms. Thank- ovaries at a given time (personal communication, the fully, much effort has been extended by governments Oceanic Institute, Hawaii, USA); it would be desir- and researchers/technical specialists to address these able to gain a better understanding of reproductive concerns, and there has been a significant ameliora- mechanisms in target species, and use this knowledge tion of the adverse affects of shrimp farming to improve efficiency in hatchery operations. (SEAFDEC 2004). The understanding of reproductive mechanisms in In addition to environmentally-related issues, the Crustacea lags behind that of fish species, with the re-

© 2010 TERRAPUB, Tokyo. All rights reserved. doi:10.5047/absm.2010.00303.0073 74 M. N. Wilder et al. / Aqua-BioSci. Monogr. 3: 73–110, 2010 sult that there are few technologies to control repro- determine the site of vitellogenin gene expression (see duction in the former. Yet, a great deal of knowledge for review, Wilder et al. 2002). has accumulated in the past 15 years, especially re- This monograph will firstly cover the current under- garding the structure and processing of vitellogenin, standing of crustacean vitellogenin structure in decapod and the physiological functioning of eyestalk hor- crustaceans (which include shrimps, prawns, lobsters mones, especially those that control the synchronous and crabs), focusing on prawn species of commercial processes of ovarian development and molting. While importance. This will be followed by a discussion of vitellogenesis commonly occurs in oviparous animal mechanisms of vitellogenin synthesis and its process- groups, it has been shown that crustacean vitellogenins ing to vitellin, including current knowledge of crusta- differ a great deal even from those of insects, which cean vitellogenin receptors. The monograph will also are also arthropods. However, the general process, in cover related facets of reproductive development, such which vitellogenin is produced in specific tissues as a as mechansisms of cortical rod formation and the uti- yolk protein precursor, undergoes processing into lization of vitellin during embryogenesis. Finally, cur- smaller molecules, and is subsequently accumulated rent knowledge concerning the hormonal regulation of in developing oocytes as vitellin to serve as a source vitellogenin synthesis is presented. of nutrients during embryogenesis, is a feature of Crus- tacea as much as it is in insects, amphibians, chickens, 2. Structural and biochemical characterization of and all other groups studied thus far. In addition to their vitellogenin and vitellin nutritive roles in reproduction/embryogenesis, vitellogenin and vitellin are also apparently involved 2-1. Structural aspects: molecular weight, physi- in the importation of minerals, lipids, and other mate- cal properties and amino acid sequence; rials into developing oocytes essential to embryogenic processing mechanisms of vitellogenin to development. vitellin The elucidation of the complete primary structure of vitellogenin in insects preceded that of crustaceans by The earliest studies on vitellin/vitellogenin structure many years (Chen et al. 1997; Sappington and Raikhel utilized immunological and electrophoretic techniques 1998; Tufail et al. 2000), and the induction of to examine molecular weight and subunit composition. vitellogenin gene expression via hormonal action In addition, before molecular biological techniques (ecdysteroids and/or juvenile hormone), has been con- started to be used in this field, various workers char- clusively demonstrated in much earlier studies (Davey acterized amino acid composition, and lipid and car- 1983; Wyatt 1988). The first report of a complete pri- bohydrate content. Many of the species that have been mary sequence in a crustacean species was by the au- studied are commercially important targets of fisher- thors of this monograph, Tsutsui et al. (2000), in the ies and aquaculture activity, while other species serve kuruma prawn, Marsupenaeus japonicus (Fig. 1). This as valuable research models. The first studies examin- seminal study led to the elucidation and comparison ing yolk protein characteristics in Crustacea focused of crustacean vitellogenesis in a great number of sig- on shrimp and prawns and of the genus Penaeus (which nificant prawn/shrimp species, as well as in crab spe- has since been divided into several separate genera), cies, and other crustaceans. However, the exact nature the genera Metapanaeus and Parapenaeus (Tom et al. of vitellogenic control remains somewhat elusive, al- 1987a, b; Chang et al. 1993a; Qiu et al. 1997; Longyant though vitellogenesis-inhibiting hormone (VIH) origi- et al. 2000), and the Macrobrachium sp, especially the nating in the eyestalks has been isolated and tested in giant freshwater prawn Macrobrachium rosenbergii culture (Tsutsui et al. 2007). (Derelle et al. 1986; Chang et al. 1993b; Wilder et al. The earliest studies concerned with crustacean 1994), an economically significant species cultured vitellogenins were based on electrophoretic or immu- widely throughout Southeast Asia. A number of crab nological techniques to examine molecular weight and species, some lobsters, and certain crayfishes have been subunit composition of vitellogenin and vitellin, or studied. examined amino acid content of purified vitellin and Initially, various strategies and methodologies had vitellogenin. In addition, biochemical analysis, to ex- been employed to determine molecular weight and amine the presence of carbohydrates and other moie- subunit composition of vitellogenin/vitellin, with the ties conjugated to vitellogenin, has been conducted to result that although many groups obtained similar re- a certain extent. Initial studies on the elucidation of sults, certain discrepancies were found in regard to the vitellogenin synthetic site employed immunologi- precise molecular weight and composition of the vari- cal techniques, radiolabelling, and light and electron ous subunits. Nevertheless, such information served microscopy. Such studies paved the way for later stud- as the basis for further studies elucidating partial amino ies employing molecular biological techniques that acid sequence (Yang et al. 2000); with the commence- have enabled workers to characterize vitellogenin ment of studies using gene cloning techniques, a wealth cDNA and elucidate primary structure, as well as to of information has thus been obtained on full cDNA

Fig. 1. Deduced amino acid sequence and nucleotide sequences of the 5′ and 3′ UTRs of vitellogenin in the kuruma prawn, Marsupenaeus japonicus. The putative signal sequence is shown in italics (–18~–1). Amino acid sequences elucidated in older studies (fragments obtained by digestion with lysyl-endopeptidase) are underlined with a sold line (LE3, LE19, LE21, and LE46); those obtained in Tsutsui et al. (2000) are underlined with a dotted line (LE101–104; fraction 2). Ser or Thr residues that are possibly phosphorylated by casein kinase II are shaded. Two consensus cleavage sequences targeted by endoproteases of the subtilisin family are shown in white lettering with black background. Positions of nucleotides and amino acids are indicated respectively by numbers of the left and right sides of the figures. The polyadenylation signal is doubly underlined. Accession number AB033719 (GenBankTM/EMBL Data Bank). Reprinted with permission from Zoological Sci- ence, 17, Tsutsui et al., Molecular characterization of a cDNA encoding vitellogenin and its expression in the hepatopancreas and ovary during vitellogenesis in the kuruma prawn, Penaeus japonicus, 651–660, Fig. 3,  2000, Zoological Society of Japan.

Table 1. Amino acid composition of vitellin (molar percentage) in selected crustacean species. ND: not done or undetectable. Reprinted with permission from Adiyodi KG, Adiyodi RG (series eds); Raikhel AS, Sappington TW (eds). Reproductive Biology of Invertebrates. Volume XII—Recent Progress in Vitellogenesis, Wilder MN et al., Yolk proteins of Crustacea, 131– 174, Table 2,  2002, Science Publishers Inc., Enfield, NH, USA.

Amino acid Penaeus Litopenaeus Litopenaeus Metapenaeus Macrobrachium Procambarus Pachygrapsus Artemia monodona semisulcatusb vannameic ensisd rosenbergiie sp.f crassipesg salinah

Asx 7.28 6.67 6.24 9.03 8.26 8.5 9.9 9.1 Threonine 6.08 5.63 6.08 0.14 6.35 5.7 6.3 5.2 Serine 5.96 7.27 7.37 5.07 8.83 8.8 10.6 10.2 Glx 13.06 12.11 11.39 13.77 12.07 8.2 12.1 10.9 Proline 4.83 6.27 5.36 7.98 5.40 4.5 6.3 6.1 Glycine 6.04 8.30 7.05 6.62 8.83 5.3 6.2 11.2 Alanine 10.83 10.62 10.78 13.21 6.99 7.5 8.0 8.7 Valine 8.27 7.64 7.80 9.84 6.92 7.1 8.0 7.6 Methionine 2.59 2.45 2.24 2.00 0.76 2.4 0.0 1.6 Isoleucine 7.06 6.69 6.67 6.82 4.89 6.1 5.7 4.8 Leucine 7.16 7.27 7.35 8.25 8.76 8.0 10.9 6.3 Tyrosine 2.94 3.33 3.16 2.01 2.54 3.8 2.7 2.2 Phenylalanine 3.56 3.70 3.81 4.07 3.37 4.7 4.3 2.7 Histidine 2.52 1.89 2.50 2.08 2.67 3.2 1.4 2.1 Lysine 6.12 5.33 6.26 8.15 7.56 9.2 4.2 7.5 Arginine 4.71 4.80 4.65 0.96 6.35 6.2 3.3 3.9 Cysteine 1.0 ND 1.31 ND ND 0.9 0.0 0.0 Tryptophan ND ND ND ND ND ND ND ND aQuinitio et al. 1990; bTom et al. 1992; cTom et al. 1992; dQiu et al. 1997; eLee et al. 1997; fLui and O’Connor 1976; gFyffe and O’Connor 1974; hDe Chaffoy de Courcelles and Kondo 1980. Asx includes aspartate and asparagines; Glx includes glutamate and glutamine.

sequence and deduced primary structure in a very large and were immunologically equivalent to the smaller number of species. two subunits of vitellogenin. At this time, it was pos- To briefly review some of the earliest studies on tulated by Wilder et al. (1994) that vitellogenin is ini- subunit composition/molecular weight, it was first seen tially secreted into the hemolymph from its synthetic that in penaeid prawns and shrimp, vitellogenins gen- site (likely the hepatopancreas) as a large precursor, erally consist of 2–4 subunits while vitellin subunits which is cleaved into the 102 and 90 kDa subunits be- are more numerous, with vitellogenin and vitellin before being taken into the ovary (later confirmed by ing, however, immunologically identical. In Penaeus Okuno et al. 2002, based on elucidation of full cDNA monodon, Chang et al. (1994) found that vitellogenin sequence). has an apparent native molecular weight of 263 kDa, At that time, the native weight of vitellin was esti- and after being taken from the hemolymph into the mated to be 700 kDa by native PAGE (Lee et al. 1997); oocytes, is dissociated into two subunits of 82 and 170 that of vitellogenin could not be established but possi- kDa. Similar results were obtained for this species by bly ranged from 200–800 kDa (Chang et al. 1993b). Longyant et al. (2000). In this regard, Fenneropenaeus More recently, Garcia et al. (2006) found that vitellin chinensis and Penaeus semisulcatus were also examin its native form has a molecular weight of 440 kDa ined, revealing a similar pattern of large vitellogenins in another Macrobrachium species, Macrobrachium and numerous smaller vitellins (Chang and Jeng 1995; borelli, and is composed of 94 kDa and 112 kDa Avarre et al. 2003). Initially, Mar. japonicus was re- subunits. Similarly, in the banana shrimp, Litopenaeus vealed to have large vitellin subunits (Kawazoe et al. merguiensis, vitellin had a native weight of 398 kDa 2000). having subunits of 78 kDa and 87 kDa (Auttarat et al. The giant freshwater prawn, Mac. rosenbergii, was 2006). studied by several groups in terms of vitellin and Prior to the full elucidation of vitellogenin primary vitellogenin subunit composition (Derelle et al. 1986; structure, amino acid composition had been examined Chang et al. 1993b; Wilder et al. 1994; Lee et al. 1997). in a number of species, and as expected, it was seen All of these groups found that vitellogenin was com- that that of vitellogenin and vitellin was very similar posed of three subunits, of molecular weights of around (Chang et al. 1993a, 1994; Chang and Jeng 1995). 200, 100 and 90 kDa (199, 102 and 90 kDa in Wilder Moreover, amino acid composition of vitellin was seen et al. 1994). Further, vitellin consisted of two subunits to be comparable among shrimp and prawns (Table 1). only, both of which had an identical molecular weight Subsequently, several authors examined partial amino

doi:10.5047/absm.2010.00303.0073 © 2010 TERRAPUB, Tokyo. All rights reserved. M. N. Wilder et al. / Aqua-BioSci. Monogr. 3: 73–110, 2010 77 acid sequence of vitellogenin and vitellin. For example, in the giant freshwater prawn, Mac. rosenbergii, (A) Yang et al. (2000) purified the 102 kDa and 90 kDa vitellin subunits of this species using reversed-phase HPLC, and examined their N-terminal amino acid sequences. The 102 kDa band (named as VnB), had the sequence SIDLRQ- - -, while the 90 kDa vitellin band was a mixture of two subunits of APWPSG- - - (named as VnA) and RREEQKV- - - (named as VnC). Vitellogenin was known to have three subunit components of 199 kDa, 102 kDa and 90 kDa, but at that time, it was not possible to examine their N-terminal sequences. However, in a follow-up study also by the authors of this monograph, a method was developed (B) enabling vitellogenin in the hemolymph to be concen- trated in sufficient quantity to carry out sequence analysis (Okuno et al. 2002). It was then seen that the 102 kDa and 199 kDa vitellogenin bands shared the same N-terminal sequence, being identical to that of the 102 kDa vitellin band (Fig. 2B). The sequences of the 90 kDa vitellogenin and vitellin bands were also the same. However, the first breakthrough in terms of elucidation of a full-length cDNA for a crustacean vitellogenin was accomplished by Tsutsui et al. (2000), for the kuruma prawn, Mar. japonicus. In this species, vitellogenin is comprised of subunits of 186 kDa, 128 kDa and 91 kDa (Kawazoe et al. 2000), for which the N-terminal amino acid of the 91 kDa subunit had been deternmined. Tsutsui et al. (2000) used this information and the method of “library walking”, to obtain a full-length cDNA encoding 2,587 amino acids (Fig. 1). This vitellogenin precursor contained 2 consensus Fig. 2. Vitellogenin processing in the giant freshwater prawn cleavage sites R-X-K/R-R targeted by enzymes of the Macrobrachium rosenbergii (daily changes in subunit com- subtilisin family. It was postulated that cleavage oc- position and schematic diagram). (A) Examination of daily curs at the first of these sites, producing the 91 kDa changes in subunit composition of vitellogenin (Vg) in subunit (Tsutsui et al. 2000). This was later confirmed hemolymph and vitellin (Vn) in ovary based on SDS-PAGE by N-terminal amino acid sequence analysis of the 186 and Western blotting in eyestalk ablated female Mac. kDa subunit (Tsutsui et al. 2002). rosenbergii. Lanes 1–12 (top) correspond to 1–12 days after Following the above studies in the kuruma prawn, eyestalk ablation, and show the order of the appearance of the next report elucidating the full cDNA sequence and Vg bands in the hemolymph. Lanes 1, 4, 8 and 12 (bottom) deduced primary sequence of a crustacean species was show corresponding Vn band composition in the ovary dur- for Mac. rosenbergii (Okuno et al. 2002). This cDNA ing ovarian maturation. (B) Schematic representation of synthesis and processing of vitellogenin in Mac. rosenbergii. was 7,800 bp, and encoded a protein corresponding to Vitellogenin is synthesized as a single precursor molecule, 2,537 amino acids containing 3 R-X-K/R-R consensus A–B–C/D, in the hepatopancreas, which is then cleaved into cleavage sites. It was found that the subunits VnA, VnB two subunits A and proB. Subunits A and proB are released and VnC discussed above were connected in a large into the hemolymph, where proB is cleaved to form two vitellogenin precursor in that order (VnA-B-C, postu- subunits B and C/D. The three processed subunits A, B and lated to be 284 kDa; Fig. 2B). Of note, a subunit re- C/D are incorporated into the ovary. Note: as described in ferred to as VnD is also shown in Fig. 2B, but VnC the text, VnC and VnD are identical ovarian subunits that and VnD were determined to be identical ovarian showed slight separation on reversed-phase high perform- subunits that showed slight separation on reversed- ance liquid chromaography due to supposed glycosylation. phase high performance liquid chromatography in Yang Reprinted with permission of John Wiley & Sons, Inc. from et al. 2000 due to supposed glycosylation; hence, sub- Journal of Experimental Zoology Part A, 292, Okuno et al., The deduced primary structure of vitellogenin in the giant sequent discussion refers to VnC only for purposes of freshwater prawn, Macrobrachium rosenbergii, and yolk simplicity. This precursor (VnA-B-C) is initially processing during ovarian maturation, 417–429, Figs. 4 and cleaved at the first of these sites, most likely at the 5,  2002, Wiley-Liss, Inc., a Wiley Company. hepatopancreas prior to secretion into the hemolymph,

Fig. 3. Phylogenetic tree of decapod crustacean VGs. This unrooted tree was generated by PHYLIP software (Felsenstein 1989) using the neighbor-joining method (Saito and Nei 1987). The scale bar corresponds to units of estimated evolutional distance. Bootstrap values were calculated by PHYLIP. Species names and GenBank accession numbers of full-length amino acid sequences used are as follows: Ca. sapidus, DQ314748; Cha. feriatus, AY724676; Che. quadricarinatus, AF306784; Fe. chinensis, DQ354690; Fe. merguiensis, AY499620; Ho. americanus, EF422415; Li. vannamei, AY321153; Mac. rosenbergii, AB056458; Mar. japonicus, AB033719; Me. ensis 1 (MeVg1), AF548364; Me. ensis 2 (MeVg2), AY530205; Pan. hypsinotus, AB117524; Pe. monodon, DQ288843; Pe. semisulcatus, AY051318; Po. trituberculatus, DQ000638; and Up. major, AB365125. HP, hepatopancreas; OV, ovary. Figure drawn for purposes of this monograph.

producing a 90 kDa and a 199 kDa protein, equivalent For example, Mac. rosenbergii vitellogenin harbors a in terms of sequence to VnA and VnB-C, respectively. fairly high degree of identity (52%) to that of Mar. The 199 kDa protein then undergoes a second cleav- japonicus in the VnA region, but is just over 30% in age, yielding a 102 kDa protein equivalent to VnB and the VnB-C regions (Okuno et al. 2002). Mac. an additional 90 kDa protein corresponding to VnC, rosenbergii vitellogenin shows higher identity to that after which all 3 proteins are incorporated into the ova- of the hermaphroditic coonstripe shrimp, Pandalus ries as VnA, B, and C (Okuno et al. 2002). Further- hypsinotus, not surprisingly, as these two species are more, it was seen that the N-terminal sequence of VnA of the same infraorder, i.e., Caridea (Tsutsui et al. in Mac. rosenbergii was 60% identical to that of the 2004). Figure 3 shows a phylogenetic tree based on 91-kDa vitellin in Mar. japonicus, while that of VnB vitellogenin sequence identity of currently known spe- was highly similar to the sequence of the N-terminal cies. Even in decapods other than prawns and shrimp, region of the 186 kDa subunit in Mar. japonicus. such as that of the crayfish Cherax quadricarinatus Thereafter, a full vitellogenin cDNA sequence has (Abdu et al. 2002), a certain level of identity (overall been elucidated in numerous prawn and shrimp spe- 37% with Mar. japonicus) is conserved. Since the re- cies, and other decapod species as well, such as cray- port of Tsutsui et al. (2000), numerous data in other fish and crabs. Thus far, all species have shown high species have been obtained, and have indicated that levels of identity, many of them having the starting shrimp, prawns and other decapod crustacean harbor characteristic sequence APW- - - (see also Auttarat et similar vitellogenin structure. These reports have also al. 2006). Furthermore, the degree of actual identity revealed that almost all species examined thus far un- among species reflects phylogenetic classification, with dergo similar mechanisms of processing during ovar- members of the same family (in the case of Penaeidae) ian maturation. The results of these reports are too having identity levels of around 90% throughout the numerous to detail, and are therefore summarized in entire molecule, for example, the comparison of Pe. Table 2 in an attempt to consolidate knowledge con- semisulcatus and Li. vannamei (Raviv et al. 2006). In cerning vitellogenin structure and processing in known less-related species, the degree of identity is especially prawn and shrimp species. Other decapod species high in the first part of the molecule, but somewhat (crayfish, lobster and crabs) are not shown in the table lower in the middle and latter parts of the molecule. for purposes of simplicity, but full cDNA sequences

2006

. 2007 .

Utarabhand

1995

et al

. 2006 .

. 2003 .

et al

. 2009 .

. 2006b .

et al

References (Authors and year) (Authors and Tiu

Avarre

Chang and Jeng Chang and Xie

Auttarat

Phiriyangkul and Phiriyangkul

information

Mar.

echanisms;

). 203 kDa subunit

Processing m gene structure; other Vitellogenin gene in this species consists of 15 exons and 13 introns. Processing mechanisms not fully confirmed, but likely similar to other species.

Processing mechanisms not fully confirmed, but likely similar to other species. First cleavage occurs between amino acid residues 710 and 711. Second cleavage occurs possibly in the ovary between residues 1795 and 1796.

Processing mechanisms not elucidated, but likely similar to other species (first cleavage after aa 710 if putative 18-amino acid- length signal peptide is excluded).

Vn consists of doublets of A and B on SDS-PAGE. Mature protein (283 kDa) cleaved to 78 kDa and 203 kDa subunits (conserved site as in rosenbergii likely processed to 87 and 104 kDa subunits.

N-terminal amino acid sequence Sequencing ND for subunits; however, APWGADLPRC and SIDSSVISDF equivalent to A and B in other species found from gene cloning.

A: APWGADLP B(1): SIDSSV B(2): SIDSSV B(3): SIDSSV C: VQYTVAEK and MNHPVLPK D: APWGA

Sequencing ND for subunits; however, APWGADLPRC and SIDSSVIADF equivalent to A and B in other species found from gene cloning.

A, B: APWGADVPR C: SIDSSVIADF

Vn. Note: amino acid length represents that of the ORF

length)

AGE unless noted as postulated (*). Polypeptides determined as vitellogenin denoted as

cDNA length (bp);cDNA length (amino acid (2589 aa)

7920 bp (2587 aa)

(2587 aa)

7961 bp (2586 aa)

100,

olypeptides

Summary of data for molecular weight, subunit composition, N-terminal amino acid sequence and processing mecha-

B(2): 207, B(3): B(2): 207, C: 79, D: 72 (Vn)

C: 104 (Vn)

Table 2. Table nisms for selected decapod species. ND: not done; bp: base pairs; aa: amino acid residues; Native: determined by native page; Polypeptides: determined by SDS-P Vg; those determined as vitellin denoted as

Vitellogenin/vitellin (kDa)

ND(Vg)*86 78.6, 106, Approx. 7800 bp

ND A: 74, B(1): 199 (Vg)

ND 85, 91 (Vg) 7761 bp

398 87, B: 78, A:

Native P

shrimp)

Penaeus monodon Penaeus

Penaeus semisulcatus

Fenneropenaeus chinensis

Fenneropenaeus merguiensis

Species (Prawns and

Infraorder Penaeidea

. 2000 .

. 2002 .

. 2000 .

. 1986 .

. 1994 .

. 2002 .

. 2003 .

. 2006 .

. 2004 .

et al

. 1997 .

. 2006a .

et al

Kawazoe Tsutsui Tiu Kung References Raviv Tsang (Authors and year) (Authors and Tsutsui

Derelle Wilder Lee Okuno

information

Mar.

echanisms;

). 179 kDa subunit

). 187 kDa subunit

76 kDa subunit is likely secreted into hemolymph after being Processing m Mature protein (257 kDa) cleaved processed into smaller subunits, gene structure; other to 78 kDa and 179 kDa subunits and is then taken into the ovary. (conserved site as in Multiple genes have been found. rosenbergii Mature protein (287 kDa) cleaved to 91 kDa and 186 kDa (from aa considered to be processed to 113, sequence predicted as 78 kDa and 61 and 42 kDa subunits. 207 kDa, respectively) subunits (conserved site as in rosenbergii likely processed to smaller subunits, harboring B and C sequences at N-terminii.

A-B-C (284 kDa, predicted) produced at hepatopancreas, then cleaved to A (90 kDa), B-C (199 kDa). A, B-C secreted into hemolymph, processed to A, B (102 kDa), C (90 kDa). Then, oocytes take up A, B, C. Note: C and D are identical; see text.

(continued).

A: APYGESTECP B: similar to above N-terminal amino acid A: Postulated to be sequence APWGAD B, C, D, E: Postulated to A: APWGADLPRC be SIDASV B, C: SIDSSVI

A: APWPSGTNLC B: SIDLSQISHL B-C/D: SIDLSQISHL C/D: RREEQKVTGT

Table 2. Table

length)

7677 bp (2559 aa) cDNA length (bp);cDNA length 7970 bp (amino acid (2587 aa) 7970 bp (2587 aa)

7800 bp (2537 aa)

199,

179,

102,

128,

Polypeptides

C: 113, D: 61, E: 42 (Vn) *

other (Vg1) (Vn)C/D: 90

A: 90, B-C/D:

C: 186 (Vn)

B: 102, C/D: 90 (Vg)

(proVg: A-B-C/D)

Vitellogenin/vitellin (kDa)

ND A: 78, B:

ND A: 76, B: 35,

700 A: 90, B:

530 A: 91, B:

Native

ensis

shrimp)

Litopenaeus vannamei

Metapenaeus Macrobrachium rosenbergii

Marsupenaeus japonicus

Species (Prawns and

Infraorder Caridea

. 2004 .

. 2006 .

. 2008 .

et al

Garcia

References (Authors and year) (Authors and

Kang

Tsutsui

Mar.

Pe.

information

Mar. rosenbergii

echanisms;

, or ovary as in

Processing mechanisms not elucidated, but vitellin shows quasi-spherical morphology under electron microscopy.

Processing m gene structure; other

A-B-C (282 kDa, predicted) produced at hepatopancreas, then cleaved to A (85 kDa), B-C (190 kDa). A, B-C secreted into hemolymph. Taken into occytes as A (85 kDa), B (100 kDa), C (85 kDa), but site of B-C cleavage unclear (in hemolymph as in rosenbergii

A-B-C (289 kDa) produced at hepatopancreas, then cleaved to A (82 kDa), B-C (210 kDa). A, B-C secreted into hemolymph, processed to A, B (115 kDa), C (100 kDa). Oocytes then take up A, B, C (same as

semisulcatus

(VnA

N-terminal amino acid sequence but peptide mass fingerprinting of 94 kDa subunit revealed 42 peptide fragments identical with the N-terminal region of Mar. rosenbergii Vg region)

A: APWPSNLPRC B: SIDFSSLSHL C/D: AQYTRNEQRI

A: VPVWPEAPLC B: SIDFDDLRSL C: VVQYSTGQS

length)

cDNA length (bp);cDNA length (amino acid

7827 bp (2534 aa)

7799 bp (2568 aa)

100,

115,

olypeptides

C/D: 85 (Vn)C/D: 85

A: 85, A: B-C/D: 190 (Vg)B-C/D: 190

(proVg: A-B-C/D)

C: 100 (Vn)

(proVg: A-B-C)

Vitellogenin/vitellin (kDa)

ND A: 85, B:

ND A: 82, B:

440 A: 94, B: 112 (Vn) ND N-terminal sequences ND,

Native P

ajor

Macrobrachium borelli

Pandalus hypsinotus

Upogebia m

Species (Prawns and shrimp) (Prawns and

Infraorder Anomura

doi:10.5047/absm.2010.00303.0073 © 2010 TERRAPUB, Tokyo. All rights reserved. 82 M. N. Wilder et al. / Aqua-BioSci. Monogr. 3: 73–110, 2010 have been obtained for the crayfish, Che. 2-2. Biochemical modifications of vitellogenin quadricarinatus (Abdu et al. 2002), the lobster, and vitellin and secondary structure Homarus americanus (Tiu et al. 2009), the red crab, Charybdis feriatus (Chan et al. 2005; Mak et al. 2005), Crustacean vitellin was first characterized as a lipo- the blue crab, Callinectes sapidus (Zmora et al. 2007), protein by Wallace et al. (1967). It is now known that and the marine crab, Portunus trituberculatus (Yang various modifications occur to vitellogenin and vitel- et al. 2005). All show overall high identity, similar N- lin (especially vitellin), where the molecules may be terminal amino acid sequences in their subunits, and conjugated to lipids, carbohydrates, carotenoids, hor- potentially similar processing mechanisms compared mones, and metals. This area was reviewed extensively with the shrimp and prawn species. by Wilder et al. (2002), and since then, much work in Interestingly, crustacean vitellogenins do not show this field has focused more on the elucidation of gene much similarity to vitellogenins of other animals. In structure and amino acid sequence of vitellogenin than the kuruma prawn, less than 20% identity to sturgeon on the nature of biochemical modifications; hence, the and killifish vitellogenin and 23% identity to tobacco latter will be discussed briefly here, touching upon hornworm apolipophorin were observed in the N- some new results on carbohydrate modification and terminal region (Tsutsui et al. 2000), and similar re- secondary structure. sults were obtained for Pan. hypsinotus (Tsutusi et al. In the earliest studies on lipovitellin as mentioned 2004). Most surprising is that very little identity exists above, Wallace et al. (1967) examined mature ovaries between crustacean and insect vitellogenins and eggs in 6 decapod families, e.g., the crabs Pagurus, (Sappington et al. 2002; Wilder et al. 2002), and of Sasarma, Uca, Libinia and Cancer, and the lobster note, decapod vitellogenins differ yet from those thus Homarus, finding that that yolk proteins contained identified in other crustacean groups such as isopods approximately 30% lipid and were of molecular (Okuno et al. 2000). Crustacean vitellogenins (mean- weights of around 350 kDa. More recently, Tirumalai ing decapod crustaceans) are characterized by the ab- and Subramoniam (1992) analyzed phospholipid con- sence of polyserine domains that feature in many of tent of vitellin in the crab Emerita asiatica, with the known invertebrate and vertebrate vitellogenins. phosphatidyl choline and phosphatidyl serine being the In such species, processing sites targeted by enzymes major species present. Lubzens et al. (1997) analyzed of the subtilisin family are flanked by a series of ser- lipid content in detail in Pe. semisulcatus, finding that ine residues (Barr 1991; Sappington and Raikhel 1998). lipid profiles differed somewhat between vitellogenin This is thought to create a beta-pleat arrangement thus and vitellin. Vitellin contained considerable amounts exposing the processing site in a conformation that can of triacylglycerols and negligible amounts of be more easily cut by the enzyme (Chen et al. 1997). diacylglycerols, differing from vitellogenin in this re- Nevertheless, decapod vitellogenins do contain 2–3 spect. The physiological significance of lipid conju- consensus cleavage sites, e.g., R-X-K/R-R, with ac- gation is still unclear, but this may be a means of pro- tual cleavage occurring at the first of these sites. This viding lipids to the embryo via the ovary. The manner feature is generally conserved throughout decapoda; of how lipids physically associate with vitellogenin and for Mac. rosenbergii, the first cleavage site occurs from vitellin also remains unclarified. Arg707–Arg710, with cleavage occurring between amino Carotenoids are another type of modification found acid residues 710 and 711 (Okuno et al. 2002). This is in crustacean vitellogenin and vitellin. Early studies exactly the same in several other species, for example, showed that they are either directly linked to protein Mar. japonicus (Tsutsui et al. 2002) and Pe. chains through amino groups (carotenoproteins) or es- semisulcatus (Avarre et al. 2003), while other species terified to the fatty acids of the lipovitellin molecules exhibit slight variation in precise location of the cleav- (Cheesman et al. 1967). In terms of function, age site. A conceptual scheme for processing of carotenoids linked to vitellin molecules may protect vitellogenin in Mac. rosenbergii, along with actual developing ovaries and embryos from visible wave- SDS-PAGE data that served as a basis for this elucida- lengths of light or over-radiation (Hairston 1976, 1979; tion, are shown in Figs. 2A, B, reproduced from Okuno Sagi et al. 1995). Late vitellogenic ovaries in matur- et al. (2002). That for other species is not described in ing crayfish Che. quadricaranatus are known to accu- detail in the text, but can be found summarized in Ta- mulate large quantities of beta-carotene (Sagi et al. ble 2. Furthermore, several studies have examined the 1995). The main roles of this type of modification may secondary structure of native vitellogenin, and have be to protect the ovary from radiation, and provide a published data on beta-pleat and alpha-helix composi- source of pigmentation during subsequent embryonic tion using circular dichroism (Garcia et al. 2006). How- development. ever, the focus on such studies related more to how Regarding carbohydrates, glycosylation is considered such structure may be related to uptake at the occytes to be an early step in the post-translational modifica- by the vitellogenin receptor rather than to processing tion of vitellogenin at its synthetic site. One of the first mechanisms. studies on the glycosylation of vitellogenin was con-

doi:10.5047/absm.2010.00303.0073 © 2010 TERRAPUB, Tokyo. All rights reserved. M. N. Wilder et al. / Aqua-BioSci. Monogr. 3: 73–110, 2010 83 ducted by Tirumalai and Subramoniam (1992). These ing ecdysteroids to the ovary in crustaceans; authors found that protein-bound carbohydrates of ecdysteroids are known to be accumulated in develop- Emerita lipovitellin consisted mostly of hexosamines ing ovaries in both crustaceans and insects, and are and hexoses (5 O-linked oligosaccharides and 4 N- thought to be utilized during early embryogenesis be- linked oligosaccharides), but not many similar studies fore the developing embryo is capable of synthesing had been done by other groups. However, Khalaila et its own (Wilder et al. 1990, 1991). Metal binding to al. (2004) performed the first extensive study on the vitellogenin is less-studied, but analysis of the major structure of carbohydrate moieties in vitellogenin us- lipovitellin in Em. asiatica revealed that copper and ing the crayfish, Che. quadricarinatus. calcium are bound to the lipid component of the vitel- Here, the authors had previously elucidated the full lin molecule, whereas iron, sodium, and phosphorus cDNA structure of Che. quadricarinatus (Abdu et al. are bound directly to the protein component of 2002), not surprisingly, finding that there was high lipovitellin (Tirumalai 1996). The physiological roles sequence identity to other decapod species as men- of metal ions bound to vitellogenin remain unclarified, tioned above. Based on this information, it was found but more recently, Abdu et al. (2002) found that that there were 10 putative glycosylation sites. Next, vitellogenin in the crayfish Che. quadricarinatus has the authors used lectin immunoblotting, in-gel calcium-binding properties (in the amino acid residue deglycosylation, and mass spectrometry to identify 2,132–2,584 range), and the authors postulate that this actual sites and determine the structure of glycan moi- property may be related to the transportation of cal- eties conjugated to these sites (Khalaila et al. 2004). cium to the oocyte for purposes of oocyte maturation, Three N-glycosylation sites, at Asn152, Asn160, and fertilization, and embryonic development. Asn2,493, were glycosylated with high-mannose glycans Finally, Garcia et al. (2006) have performed struc- identified as Man5–9GlcNAc2 (Man = mannose; Glc = tural characterization of lipovitellin in Mac. borelli glucose; GlcNac = N-acetylglucosamine) species. using electron microscopy, Matrix Assisted Laser Glycosylation of vitellogenin and vitellin may be im- Desorption Ionization-Time of Flight (MALDI-TOF) portant in the folding, processing, and transport of the mass spectrometry, circular dichroism, and other tech- molecules into the developing oocytes, and the conju- niques. As discussed above, most decapod crustacean gating moieties may also serve as a source of carbohy- vitellins consist of subunits of around 90 kDa and 100 drates during ensuing embryogenesis (Khalaila et al. kDa, and it seems that in the native form, there are two 2004). Roth et al. (2010) performed a similar study of each included in the overall molecule. However, using Mac. rosenbergii. It was found that glycosylation these authors found that vitellin is a hetero-trimer, com- of vitellogenin occurs at 6 N-glycosylation sites: prised of two subunits of 94 kDa, and one subunit of Asn151, Asn159, Asn168, Asn614, Asn660 and Asn2,300. As 112 kDa (however, this does not seem to account for with Che. quadricarinatus, the glycan moieties were the observed native molecular weight of 440 kDa). also hexose type structures rich in mannose; of note, Using circular dichroism, it was estimated that Mac. an unusual mannose N-linked oligosaccharide with a borelli vitellin contains 37.5% alpha-helix, 16.6% beta- glucose cap (Glc1Man9GlcNAc2) was contained. sheets and 20% turns (Garcia et al. 2006). Also inter- Roth et al. (2010) also conducted a bioinformatics esting were the electron microscopy results, showing analysis of putative O- and N-glycosylation sites in 16 that vitellin formed particles of quasi-spherical mor- decapod species in which full vitellogenin amino acid phology. Regarding secondary structure, there have sequence is known. Interestingly, Pleocyemata sub- been very few other studies on crustaceans. In Li. order species, which are egg-carrying, were rich in N- vannamei vitellin, results were somewhat different, glycosylation sites with some O-glycosylation sites, with 25% alpha-helix, 37% beta-sheets and 14% turns while Dendrobranchiata sub-order species, which are (Garcia-Orozco et al. 2002). Structural features such egg-releasing, were lacking in N-glycosylation sites. as these may have significance in conferring proper- These authors postulate that these differences in ties relating to its transport in the hemolymph and/or glycosylation sites and resultant structure of interactions with the vitellogenin receptor, but more vitellogenin may have implications in actual reproduc- information is required to provide insight into these tive strategy. Perhaps differences in structure may have areas. implications in how eggs are physically formed and extruded—for example, in penaied species, cortical rod 3. Vitellogenin synthesis and ovarian development proteins present in mature oocytes form the jelly layer that occurs after egg extrusion (see also below, Sub- 3-1. Determination of vitellogenin synthetic sites section 3-3B). As for other forms of conjugation, in the crab, Em. 3-1A. Earlier studies targeting vitellin or vitellogenin asiatica, ovarian ecdysteroids are found to be conju- polypeptides gated to vitellin (Subramoniam et al. 1999). In this way, There have been many attempts to explore the sites vitellogenin uptake may also be a means of transport- of vitellogenin synthesis in various crustacean species.

Ovarian protein, either vitellin or its precursor 3-1B. Confirmation of vitellogenin synthetic site vitellogenin, was considered to be an important mo- based on vitellogenin gene expression studies lecular marker until the late 1990s; hence, As described in Subsection 2-1, full-length cDNAs immunoprecipitation, electrophoresis, Western blot- encoding vitellogenin precursor have been cloned in ting, ELISA, immunohistochemistry, and tissue incu- several crustacean species since 2000, allowing the bation were the main techniques used in this regard. sites of vitellogenin synthesis to be clarified. The con- The combination of tissue incubation with radiolabeled tributions of the hepatopancreas and ovary were shown amino acids and immunoprecipitation using anti- in Mar. japonicus (Tsutsui et al. 2000), Pe. vitellogenin serum was used mostly to define the ori- semisulcatus (Avarre et al. 2003), Li. vannamei (Raviv gin of vitellogenin synthesis. Vitellogenin was detected et al. 2006), Pe. monodon (Tiu et al. 2006b), Fe. in the fat body (adipocytes) of ovariectomized females merguiensis (Phiriyangkul et al. 2007), and of the terrestrial isopod Porcellio dilatatus by immu- Fenneropenaeus chinensis (Xie et al. 2009) by North- nohistochemical analysis, and immunoautoradiography ern hybridization or RT-PCR analysis. The vitellogenin after incubation with [14C]leucine demonstrated that it genes expressed in the hepatopancreas and ovary were was synthesized in that tissue (Picaud 1980). Souty and identical in Mar. japonicus (Tsutsui et al. 2005b) and Picaud (1981) also reported that the fat body of the Pe. semisulcatus (Avarre et al. 2003), but it is likely isopod Idotea balthica basteri was the site of that multiple genes exist in these species. In fact, two vitellogenin synthesis, showing that protein synthe- vitellogenin genes, MeVg1 and MeVg2, were cloned sized de novo by the incubated fat body was in the sand shrimp Metapenaeus ensis; MeVg1 was immunoprecipitated with anti-vitellogenin serum. expressed in both organs, whereas MeVg2 was ex- Electron-microscopic immunocytochemistry of the pressed only in the hepatopancreas (Tsang et al. 2003; amphipod Orchestia gammarella revealed that the Kung et al. 2004); and multiple vitellogenin genes are rough endoplasmic reticulum of the fat body was de- also presumed to exist in Fe. merguiensis (Phiriyangkul veloped in vitellogenic females but not in non- et al. 2007). Detailed localization of vitellogenin vitellogenic females (Meusy et al. 1983a). As supported mRNA was examined by in situ hybridization in Mar. by the results of tissue incubation experiments (Junéra japonicus (Tsutsui et al. 2000); the mRNA was accu- and Croisille 1980), the fat body is considered to be mulated in the parenchymal cells of the hepatopancreas the site of vitellogenin synthesis in this species. Simi- and in the follicle cells of the ovary (Fig. 4). larly, the fat storage cells of thoracopods are responsi- Vitellogenin mRNA signals were detected in the whole ble for lipovitellin synthesis in the anostracan Artemia of the hepatopancreas; cells specific to vitellogenin species (Van Beek et al. 1987). synthesis, such as the “vitellogenocytes” of Paulus and In decapod crustaceans, the sites of vitellogenin syn- Laufer (1987), could not be discerned. In contrast, in thesis are presumed to be the hepatopancreas, ovary, situ hybridization analysis of Mac. rosenbergii sug- and subepidermal adipose tissue (fat body). gested that the R-cells of the hepatopancreas were re- Extraovarian vitellogenin synthesis was shown in the sponsible for vitellogenin synthesis (Jasmani et al. adipose tissue of Palaemon serratus (Meusy et al. 2004). 1983b), Mar. japonicus (Vazquez-Boucard et al. 1986), It is highly likely that the hepatopancreas and ovary Parapenaeus longirostris (Tom et al. 1987b), and are responsible for vitellogenin synthesis in penaeid Scylla serrata (Rani and Subramoniam 1997); and in species, and that the hepatopancreas is the main site of the hepatopancreas of Carcinus maenas and Libinia vitellogenin synthesis in species of the Caridea, emarginata (Paulus and Laufer 1987), Li. vannamei Astacidea, and Portunoidea infraorders. Full-length (Quackenbush 1989), Pe. semisulcatus (Fainzilber et cDNAs encoding vitellogenin were cloned and tissue- al. 1992), Macrobrachium nipponense (Han et al. specific expression of vitellogenin genes was exam- 1994), Sc. serrata (Rani and Subramoniam 1997), and ined by Northern blot or RT-PCR analyses in the giant Mac. rosenbergii (Lee and Chang 1999; Soroka et al. freshwater prawn Mac. rosenbergii (Yang et al. 2000), 2000). Ovarian synthesis of vitellogenin was reported crayfish Che. quadricarinatus (Abdu et al. 2002), in Procambarus sp. (Lui and O’Connor 1976), coonstripe shrimp Pan. hypsinotus (Tsutsui et al. 2004), Pachygrapsus crassipes (Lui and O’Connor 1977), Uca red crab Cha. feriatus (Mak et al. 2005), marine crab pugilator (Eastman-Reks and Fingerman 1985), Mar. Po. trituberculatus (Yang et al. 2005), blue crab japonicus (Yano and Chinzei 1987), Li. vannamei Callinectes sapidus (Zmora et al. 2007), and Ameri- (Quackenbush 1989; Rankin et al. 1989), Pe. can lobster Ho. americanus (Tiu et al. 2009). These semisulcatus (Browdy et al. 1990; Fainzilber et al. gene expression analyses confirm that in decapod crus- 1992), and Ca. sapidus (Lee and Watson 1995; Lee et taceans, vitellogenin is synthesized in both the al. 1996). Whether the main source is intraovarian tis- hepatopancreas and ovary in species of the Penaeidea, sue or extraovarian tissue (or both) has been a contro- and principally in the hepatopancreas in species of the versial subject. Caridea, Astacidea, and Portunoidea infraorders (Fig.

Fig. 4. Localization of vitellogenin mRNA in the hepatopancreas and ovary by in situ hybridization in Marsupeaneus japonicus. Vitellogenin gene expression was observed in (A) follicle cells of the ovary and (C) parenchymal cells of the hepatopancreas. Hybridization with a sense probe resulted in no significant signal in (B) ovary or (D) hepatopancreas. Bars = 20 µm in A and B; 10 µm in C and D. Reprinted with permission from Zoological Science, 17, Tsutsui et al., Molecular characterization of a cDNA encoding vitellogenin and its expression in the hepatopancreas and ovary during vitellogenesis in the kuruma prawn, Penaeus japonicus, 651–660, Fig. 6,  2000, Zoological Society of Japan.

3). The contribution of the subepidermal adipose tis- taneous increases of vitellogenin transcript levels in sue, as examined in the above manner, has not been the hepatopancreas and ovary during vitellogenesis in reported thus far. Mar. japonicus (Tsutsui et al. 2000, 2005b). Levels in the hepatopancreas were low at the previtellogenic 3-2. Dynamics of vitellogenin synthesis stage, increased during the endogenous vitellogenic stage, and remained high during the exogenous Isolation of vitellogenin cDNA has allowed the ex- vitellogenic stage. Those in the ovary were also low at amination of vitellogenin gene expression levels at the previtellogenic stage and increased during the en- different vitellogenic stages, the relationship between dogenous vitellogenic stage and the early exogenous vitellogenin gene expression levels and hemolymph vitellogenic stage, yet they decreased significantly vitellogenin protein levels, and how the presumed during the late exogenous vitellogenic stage. Viewed multiple sites of synthesis contribute to overall in the whole organ, the total amounts of vitellogenin vitellogenin synthesis during vitellogenesis. Northern mRNA were similar between the hepatopancreas and blot and quantitative RT-PCR analyses revealed simul- ovary at each stage, and there was no significant dif-

Fig. 5. Gonadosomatic index (GSI), hemolymph vitellogenin titers, and relative vitellogenin mRNA levels in the hepatopancreas of Macrobrachium rosenbergii females at different stages of the reproductive molt cycle. (A) GSI of intact animals, (B) vitellogenin titers and vitellogenin mRNA levels of intact females, (C) GSI of eyestalk-ablated females, and (D) vitellogenin titers and vitellogenin mRNA levels of eyestalk-ablated females. Values represent the mean ± SEM of five animals. Reprinted with permission of John Wiley & Sons, Inc. from Journal of Experimental Zoology Part A, 293, Jayasankar et al., Dynamics of vitellogenin mRNA expression and changes in hemolymph vitellogenin levels during ovarian maturation in the giant freshwater prawn Macrobrachium rosenbergii, 675–682, Figs. 2 and 3,  2002, Wiley-Liss, Inc., a Wiley Company.

ference in ovarian vitellogenin mRNA levels between index (GSI), and hemolymph vitellogenin levels were the early and late exogenous vitellogenic stages. The examined during the reproductive molt cycle overall gene expression profile showed a good rela- (Jayasanker et al. 2002). In intact animals, vitellogenin tionship with hemolymph vitellogenin levels (Jasmani gene expression levels in the hepatopancreas and et al. 2000). Similarly high vitellogenin gene expres- vitellogenin protein levels in the hemolymph showed sion patterns in both tissues during yolk accumulation a gradual increase during the reproductive molt cycle have been reported in Pe. semisulcatus (Avarre et al. concomitant with increasing GSI (Figs. 5A, B). 2003) and Pe. monodon (Tiu et al. 2006b), although Eyestalk ablation shortened the cycle, accelerating expression was higher in the ovary than in the vitellogenin gene expression and ovarian maturation, hepatopancreas in Li. vannamei (Raviv et al. 2006), although it did not alter the overall pattern of Fe. merguiensis (Phiriyangkul et al. 2007), and Me. vitellogenin mRNA expression (Figs. 5C, D). In addi- ensis (for MeVg1; Tiu et al. 2006a). Vitellogenin gene tion, hemolymph vitellogenin levels and maximum GSI expression patterns in non-penaeid species, in which were higher in eyestalk-ablated animals than in intact the hepatopancreas is the site of vitellogenin synthe- animals, suggesting that eyestalk factors partially regu- sis, were also correlated with vitellogenic stage (Mak late vitellogenin translation, release from the et al. 2005; Zmora et al. 2007). In Mac. rosenbergii, hepatopancreas, and incorporation into the oocyte. changes in vitellogenin mRNA levels, gonadosomatic The relationship between vitellogenin gene expres-

(Tsutsui et al. 2004; Fig. 6). In Che. quadricarinatus, ablation of the androgenic gland induced vitellogenin gene expression in the hepatopancreas of sexually plastic intersex animals that were functional males, suggesting negative regulation by the androgenic gland on vitellogenin transcription (Abdu et al. 2002). More- over, in the mud shrimp Upogebia major, vitellogenin transcripts were detected by RT-PCR in the male hepatopancreas and the ovarian part of the testis, as well as in the female hepatopancreas and ovary (Kang et al. 2008). Further studies are needed to clarify the relationship between regulatory systems for sex determination and those for vitellogenesis.

3-3. Modes of ovarian maturation

3-3A. Microscopic studies of ovarian development in representative prawn species In crustaceans, vitellogenin is accumulated in oocytes as a major yolk protein, vitellin, during vitellogenesis. Yolk accumulation causes a rapid increase in oocyte diameter and size. This process has been histologically investigated in several crustacean species (for reviews, see Charniaux-Cotton and Payen 1988; Meusy and Payen 1988). One of the authors of this monograph has also studied ovarian development in relation to reproductive season and molt cycle in representative prawn species (Okumura et al. 1992, 2004, 2005, 2007; Okumura and Aida 2000). Histological changes in oocytes during ovarian development in Mac. rosenbergii are shown in Fig. 7 (Okumura and Aida 2000). At the center of the ovary, Fig. 6. Northern blot analysis of Pandalus hypsinotus oogonia commence meiotic division I and become vitellogenin mRNA. (A) Hepatopancreas in female prawns. oocytes. While the oocytes remain arrested in prophase (B) Gonads in female prawns. (C) Hepatopancreas and go- I, they accumulate RNA at the previtellogenic stage, nads in male prawns. The number at the top of each lane indicates the GSI of each prawn. PC = positive control (To- oil globules and PAS (periodic acid-Schiff)-positive tal RNA from female hepatopancreas; GSI = 3.1). The ex- vesicles at the endogenous vitellogenic stage, and posure time for B and C was three times longer than that of eosin-positive yolk globules at the exogenous membrane A in order to detect weak signals. Results of vitellogenic stage (Figs. 7A–C, E, F). The endogenous ethidium bromide staining of gels before blotting show equal and exogenous vitellogenic oocytes are enveloped by loading of RNAs. Reprinted with permission of John Wiley follicle cells (Figs. 7A–C). The cytoplasm of the ex- & Sons, Inc. from Journal of Experimental Zoology Part A, ogenous vitellogenic oocytes is stained with anti- 301A, Tsutsui et al., Molecular characterization of a cDNA vitellin antibody (Fig. 7G), indicating that the yolk encoding vitellogenin in the coonstriped shrimp, Pandalus globules contain vitellin. Vitellogenic ovaries are al- hypsinotus and site of vitellogenin mRNA expression, 802– most completely filled with synchronously develop- 814, Fig. 6,  2004, Wiley-Liss, Inc., a Wiley Company. ing oocytes at the exogenous vitellogenic stage. These oocytes are spawned at the next oviposition, and oogonia and oocytes at the previtellogenic and endog- sion and programmed sex change, which is presumed enous vitellogenic stages remain in the ovary (Figs. to be regulated by androgenic gland hormone (AGH), 7A, D). Ovaries similarly develop in Macrobrachium has also been studied. Pa. hypsinotus first matures as nipponense (Okumura et al. 1992) and Pan. hypsinotus a functional male, and then becomes a functional fe- (Okumura et al. 2004). male. Vitellogenin mRNA increased concomitantly In electron microscopic observations of oogenesis with ovarian development in the hepatopancreas of in Pan. hypsinotus (Okumura et al. 2004), the cyto- vitellogenic females, whereas it was not detected ei- plasm of the previtellogenic oocytes contains mito- ther in the hepatopancreas or gonads of immature fe- chondria (Fig. 8A), and the cytoplasm of the endog- males having GSI lower than 1.0 and those of males enous vitellogenic oocytes is filled with rough endo-

Fig. 7. Cross-sections of Macrobrachium rosenbergii ovaries. (A) Periodic acid-Schiff (PAS) staining at the endogenous vitellogenic stage. (B) Hematoxylin and eosin (HE) staining at the early exogenous vitellogenic stage. (C) HE staining at the late exogenous vitellogenic stage. (D) PAS staining just after oviposition. (E) PAS staining at the endogenous vitellogenic stage. (F) OsO4 postfixation at the endogenous vitellogenic stage. (G) Immunohistochemistry at the early exogenous vitellogenic stage using anti-vitellin antibody. AO, regressing atretic oocyte; EF, empty follicle after ovulation; EN, endogenous vitellogenic oocyte; EX, exogenous vitellogenic oocyte; O, oogonium; OG, oil globule; PO, previtellogenic oocyte. Bars: 0.1 mm. Re- printed with permission from Fisheries Science, 66, Okumura and Aida, Hemolymph vitellogenin levels and ovarian development during the reproductive and non-reproductive molt cycles in the giant freshwater prawn Macrobrachium rosenbergii, 678–685, Fig. 2,  2000, The Japanese Society of Fisheries Science.

Fig. 8. Transmission electron micrographs of Pandalus hypsinotus ovaries. (A) Previtellogenic oocyte (GSI: 0.60). (B), (C) Endogenous vitellogenic oocyte (GSI: 0.55). (D) Exogenous vitellogenic oocyte and follicle cell (GSI: 1.17). (E), (F) Endo- cytosis in an exogenous vitellogenic oocyte (GSI: 1.12). (G) Exogenous vitellogenic oocyte (GSI: 2.25). (H) Maturing oocyte (GSI: 8.52). Bars: 1 µm. EC, endocytosis; FC, follicle cells; L, lipid droplets; M, mitochondria; MV, microvilli; RER, rough endoplasmic reticulum; VM, vitellin membrane; Y, yolk globules. Reprinted with permission from Zoological Science, 21, Okumura et al., Ovarian development and hemolymph vitellogenin levels in laboratory-maintained protandric shrimp, Pandalus hypsinotus: measurement by a newly developed time-resolved fluoroimmunoassay (TR-FIA), 1037–1047, Fig. 7,  2004, Zoological Society of Japan.

plasmic reticulum, indicating active protein synthesis after the completion of yolk accumulation (Clark et (Figs. 8B, C). The exogenous vitellogenic oocytes con- al. 1990). As in Macrobrachium prawns and shrimp, tain electron-dense yolk globules in addition to mito- oocyte development is classified into the chondria, lipid droplets, and rough endoplasmic reticu- previtellogenic, endogenous vitellogenic, and exog- lum (Figs. 8D, G). Active endocytosis at the oocyte enous vitellogenic stages (Fig. 9). During the oocyte surface suggests that yolk globules are exogenously development, RNA, oil globules, PAS-positive vesi- formed by the uptake of vitellogenin from hemolymph cles, and yolk globules are accumulated in the ooplasm (Figs. 8E, F). The vitellin membrane is formed on the and oocyte diameter and size increase. After the com- surface of the maturing oocytes (Fig. 8H). pletion of yolk accumulation (Fig. 10a), cortical rods In penaeid shrimps, ovarian development is charac- are formed radially around the periphery of the oocyte terized by the formation of cortical rods in the oocytes plasma membrane (Fig. 10b). During their develop-

Fig. 9. Ovarian development of pond-reared Marsupenaeus Fig. 10. Ovarian development of wild mature Marsupenaeus japonicus in (a) December, (b), (c) April, and (d) May. japonicus. Ovarian development is classified into the (a) Oocyte development is classified as previtellogenic oocytes late exogenous vitellogenic stage, (b) early maturation (Pre), endogenous vitellogenic oocytes (Endo), and exog- stage, (c) late maturation stage, and (d) spent stage. CR, enous vitellogenic oocytes (Exo). FC, follicle cell. Bars: 0.1 cortical rod; Endo, endogenous vitellogenic oocyte; Exo, mm. (a), (d) Hematoxylin and eosin staining. (b), (c) exogenous vitellogenic oocyte; Pre, previtellogenic oocyte. Hematoxylin-periodic acid-Schiff staining. Reprinted from Bars: 0.1 mm. Hematoxylin and eosin staining. Reprinted Comparative Biochemistry and Physiology Part A, 147A, from Comparative Biochemistry and Physiology Part A, Okumura et al., Vitellogenin gene expression and 147A, Okumura et al., Vitellogenin gene expression and hemolymph vitellogenin during vitellogenesis, final matu- hemolymph vitellogenin during vitellogenesis, final maturation, and oviposition in female kuruma prawn, ration, and oviposition in female kuruma prawn, Marsupenaeus japonicus, 1028–1037, Copyright (2007), Marsupenaeus japonicus, 1028–1037, Copyright (2007), with permission from Elsevier. with permission from Elsevier.

ment, the germinal vesicle begins to disintegrate at the complete the brooding of the egg mass during the sub- center of the oocyte (germinal vesicle breakdown, sequent intermolt period. While the eggs are being car- GVBD; Fig. 10c) and subsequently migrates toward ried, the ovaries redevelop via yolk accumulation for the oocyte surface. The oocytes that have cortical rods the next oviposition. Gonadosomatic index (GSI) in- are spawned at oviposition, and oogonia and oocytes creases and hemolymph vitellogenin levels are high at the previtellogenic and endogenous vitellogenic during the molt stages C1–D3 (Okumura and Aida 2000; stages remain in the ovary (Fig. 10d). Electron Fig. 11). microscopy reveals that before cortical rod formation, Species that have a defined spawning season show small cortical vesicles appear in the ooplasm and fuse seasonal changes in ovarian development. In Mar. together to form larger vesicles (Hong 1977; Wallis et japonicus, which spawns from April to October in the al. 1990; Carvalho et al. 1999). The fused vesicles then coastal areas of Japan, ovaries develop during March migrate toward the oocyte surface and fuse with the to May (Fig. 12), and hemolymph vitellogenin levels oocyte plasma membrane. The vesicles grow by con- and vitellogenin mRNA levels in the ovary and tinuous fusion and form a rod-like shape. The cortical hepatopancreas increase (Okumura et al. 2007). In the rods thus develop outside the oocyte plasma membrane mid-regions of the Sea of Japan, Pan. hypsinotus in- but under the vitellin membrane. Their content gives habits cold water and requires a long duration (8–14 the appearance of tightly packed feathery elements months) for ovarian development. The hatched larvae under electron microscopic observation (Hong 1977; are released in February–March, yolk accumulation Wallis et al. 1990; Carvalho et al. 1999). starts during April–October, GSI and hemolymph In species that brood an egg mass on the abdomen, vitellogenin levels increase (Fig. 13), and oviposition spawning and molting are coordinated to avoid loss of occurs in June–July of the next year (Okumura et al. the egg mass by untimely ecdysis. Female Mac. 2004). rosenbergii mate and spawn just after ecdysis, and they

10 10 8 /mL) g 6 c (m GSI els Oviposition v 5 Vitellogenin 5 GSI

le 4

nin bc GSI Ecdysis 2 telloge bc bc bc ab ab abc Vi a abab ab 0 0 ABC0 C1 D0 D1 D2 D3 A 0 Molt stage 6

Fig. 11. Hemolymph vitellogenin levels and gonadosomatic index (GSI) in Macrobrachium rosenbergii. Data indicate the mean and SE. Reprinted with permission from Japan 4 Agricultural Research Quaterly (JARQ), 38, Okumura, Per- b spectives on hormonal manipulation of shrimp reproduction, 49–54, Fig. 2,  2004, Japan International Research Center ab for Agricultural Sciences. 2 ab

ab b Vitellogenin (mg/ml) ab a ab ab a ab a 0 Apr Jun Aug Oct Dec Feb Apr Jun Aug Oct 1999 2000 Month

Fig. 13. Seasonal changes in gonadosomatic index (GSI) and hemolymph vitellogenin levels in Pandalus hypsinotus. Points and bars indicate the mean and SD. Points without common letters differ significantly (P < 0.05). Reprinted with permission from Zoological Science, 21, Okumura et al., Ovarian development and hemolymph vitellogenin levels in Fig. 12. Changes in (a) ovarian developmental stage and (b) laboratory-maintained protandric shrimp, Pandalus gonadosomatic index in pond-reared female Marsupenaeus hypsinotus: measurement by a newly developed time- japonicus from December to June. Bars in (b) indicate the resolved fluoroimmunoassay (TR-FIA), 1037–1047, Fig. 10, mean and SD of 14 animals, and bars without common let-  2004, Zoological Society of Japan. ters differ significantly (P < 0.05). Reprinted from Compara- tive Biochemistry and Physiology Part A, 147A, Okumura et al., Vitellogenin gene expression and hemolymph vitellogenin during vitellogenesis, final maturation, and ovi- 150 kDa; Yamano et al. 2003, 2004). Except for their position in female kuruma prawn, Marsupenaeus japonicus, molecular weights, SOP, CRP, and MjTSP show simi- 1028–1037, Copyright (2007), with permission from Elsevier. lar molecular structure in terms of repeated cysteine- rich domains, and proteolytic sites for post- translational modulation and glycosylation, as well as similar deduced amino acid sequences. Yamano et al. 3-3B. Identification of cortical rod proteins in (2004) suggested that they are the products of a single penaeid species, and role in reproduction gene or are encoded by orthologous genes. In penaeid shrimps, after the completion of yolk ac- Messenger RNAs for SOP, CRP, and MjTSP are ex- cumulation, cortical rods are formed radially around pressed in the previtellogenic oocytes, and the respec- the periphery of the oocyte plasma membrane (Clark tive proteins are synthesized and accumulated in the et al. 1990; Wallis et al. 1990). Two major proteins vitellogenic oocytes as yolk materials (Khayat et al. comprising the cortical rods have been purified and 2001; Yamano et al. 2003, 2004; Kim et al. 2004, characterized: shrimp ovarian peritrophin (SOP, 29– 2005). The proteins are first scattered throughout the 35 and 33–36 kDa) in Penaeus semisulcatus (Khayat ooplasm, and then become localized in the cortical rods et al. 2001) or cortical rod protein (CRP, 28.6 and 30.5 during cortical rod formation. kDa) in Mar. japonicus (Kim et al. 2004, 2005); and The endocrine mechanisms regulating the synthesis Mar. japonicus thrombospondin (MjTSP, 130, 140, and of SOP, CRP, and MjTSP have been studied in relation

(A)

(B)

Fig. 14.

doi:10.5047/absm.2010.00303.0073 © 2010 TERRAPUB, Tokyo. All rights reserved. M. N. Wilder et al. / Aqua-BioSci. Monogr. 3: 73–110, 2010 93 to crustacean hyperglycemic hormone-family (CHH- cies that does not form cortical rod structures in the family) peptides. CHH-family peptides are major oocytes (Kim et al. 2007). In addition to the role of neuropeptides produced in the X-organ/sinus gland the jelly layer as a barrier, cortical rod proteins may complex in the eyestalks. CHH, molt-inhibiting hor- have other functions in reproduction. mone (MIH), vitellogenesis-inhibiting hormone (VIH), and mandibular organ-inhibiting hormone (MOIH) are 3-4. Vitellogenin incorporation into oocytes; included in this family due to their similarities in pri- vitellogenin receptors mary structure. CHH-family peptides are divided into two subtypes based on the absence (type-I) or pres- As in other oviparous animals, in Crustacea, ence (type-II) of a glycine residue at position 12 in the vitellogenin is considered to be taken into the devel- mature peptide. In Mar. japonicus, seven CHH-family oping occytes from the hemolymph by the vitellogenin peptides designated as Pej-SGP-I to -VII, were puri- receptor (VgR) via receptor-mediated endocytosis. fied from the sinus glands (Nagasawa et al. 1999; Yang While the mechanisms of the endocytotic internaliza- et al. 1995, 1996, 1997; Fig. 17). Those peptides in- tion of vitellogenin have been well-studied in certain hibited de novo synthesis of SOP, but did not affect oviparous vertebrates (Schneider 1992) and insects SOP mRNA levels in incubated ovarian fragments of (Sappington and Raikhel 1998), such studies in Crus- Pe. semisulcatus (Avarre et al. 2001). One of the au- tacea remain limited. In the earliest work, Jugan and thors of this monograph determined that eyestalk Soyez (1985) demonstrated the uptake of vitellin con- ablation—removal of the source of CHH—increased jugated with colloidal gold by Mac. rosenbergii CRP and MjTSP protein levels but did not affect their oocytes, while Laverdure and Soyez (1988) solubilized mRNA levels in the ovary of Mar. japonicus (Okumura VgR from the oocyte membrane of Ho. americanus. et al. 2007). Taken together with the fact that SOP, CRP, In the latter, using an enzyme linked immunosorbent and MjTSP genes are transcribed and translated at dif- assay, it was shown that binding of vitellogenin with ferent oocyte stages, these results indicate that expres- the solubilized receptors increased at the onset of vitel- sion of these genes is controlled at the translational logenesis, but decreased in older oocytes. Jugan and level by CHH-family peptides. Van Herp (1989) demonstrated that vitellogenin spe- The content of the cortical rods is released around cifically binds to an oocyte membrane protein in the eggs and forms into a jelly layer, which envelopes the crayfish Orconectus limosus. eggs on contact with sea water at spawning (Clark et In more recent work, Warrier and Subramoniam al. 1990). The jelly layer starts dissipating around 45 (2002) purified VgR in the mud crab, Sc. serrata by minutes after spawning, and the hatching envelope HPLC, revealing it to have a molecular weight of 230 forms on the egg surface. The jelly layer is considered kDa. These authors also employed direct binding stud- to function as a barrier against polyspermy and as an ies using [125I]-labelled vitellogenin, finding that the external protective coat from the time of spawning to receptor possesses high affinity for crab vitellogenin × –6 the formation of the hatching envelope (Clark et al. with a dissociation constant of Kd 0.8 10 M. It was 1990). Recently, a CRP homologue (mrCRP, 30 kDa) also seen that the Sc. serrata VgR binds mammalian was found in the ovaries of Mac. rosenbergii, a spe- low density lipoprotein (LDL); this suggested that the

Fig. 14. VgR cloning strategy and sequence for Marsupenaeus japonicus. (A) Cloning strategy of Mar. japonicus ovarian LDLR cDNA. Solid black triangles indicate the primers designed based on the sequence of Penaeus semisulcatus putative ovarian lipoprotein receptor cDNA. Open triangles indicate the primers designed based on the sequence of Mar. japonicus ovarian LDLR. Arrows indicate the universal primers for 5′ and 3′-RACE. Meshwork shows the ligand binding domain (LDL- receptor class A); gray color shows the EGF precursor domain (LDL-receptor class B); The horizontal striped section shows an EGF-like domain; a capital T shows a transmembrane region. (B) Nucleotide sequence of ovarian LDLR cDNA from Mar. japonicus and its deduced amino acid sequence. This nucleotide sequence has been deposited in the DDBJ database (accession number AB304798). Shadowed areas indicate the ligand binding domain (LDL-receptor class A), thinly-outlined rectangles indicate the EGF precursor domain (LDL-receptor class B), the double underline indicates an EGF-like domain and the dotted underline indicates a transmembrane region. Three bold serial circles (SDE) indicate highly conserved residues in the LDL receptor family. Potentially related sequences of SDE are shown in thinly-outlined serial circles. Bold letters indicate YWTD repeats and its potentially related sequence. The single underlines indicate residues homologous to the internalization signal (NPXY). A pair of solid black arrowheads indicates the 5′ upstream probe region, and open arrowheads indicate the 3′ downstream probe region used for library screening. Thickly-outlined rectangles indicate the amino acid residues of the synthetic peptide sequence used for synthesizing ovarian LDLR antibody of Mar. japonicus. Reprinted with permission from Zoological Science, 25, Mekuchi et al., Characterization and expression of the putative ovarian lipoprotein receptor in the kuruma prawn, Marsupenaeus japonicus, 428–437, Figs. 1 and 2,  2008, Zoological Society of Japan.

Fig. 15. Molecular phylogenetic tree between ovarian LDLR of Marsupenaeus japonicus and those of other species. A molecular phylogenetic tree of VgR and/or ovarian LDLR was constructed using the Tree View program (version 1.6.6; Division of Environmental and Evolutional Biology, Institute of Biomedical and Life Science, University of Glasgow). The scale bar shows 1.0 amino acid substitutions per site. Reprinted with permission from Zoological Science, 25, Mekuchi et al., Charac- terization and expression of the putative ovarian lipoprotein receptor in the kuruma prawn, Marsupenaeus japonicus, 428– 437, Fig. 3,  2008, Zoological Society of Japan. (Note: throughout the main text and figure captions, scientific names are abbreviated with the first two letters, or where further specification is necessary, with the first three letters of the genus name. However, in this figure, for purposes of simplicity, all scientific names are indicated with the first letter of the genus name.)

crab receptor belongs to the vertebrate low density li- RNA, leading to decreased VgR protein content in the poprotein receptor (LDLR) family, as do vitellogenin ovary, and higher vitellogenin content in the receptors in nematodes, insects, and vertebrates (Bujo hemolymph, due to the decreased availability of et al. 1994; Sappington et al. 1996; Grant and Hirsh receptor present to serve in the uptake of vitellogenin 1999). However, at this point in time, the VgR gene into the occytes. Regarding sequence information, Pe. had not been cloned; hence sequence information of monodon VgR shared a certain degree of amino acid crustacean VgR could not be compared with that of identity with other known invertebrate (insect) other oviparous animals. receptors, and exhibited two putative internalization More recently, Tiu et al. (2008) carried out the mo- signals (FANPGFG and FENPFF) (Tiu et al. 2008). lecular characterization of VgR in the tiger prawn, The authors of this monograph characterized a puta- Penaeus monodon. This was the first cloning of a cDNA tive ovarian lipoprotein receptor in the kuruma prawn, for this gene in Crustacea. It was found that the VgR Mar. japonicus which was hypothesized to incorporate cDNA was 6.8 kb in length, corresponding to a de- vitellogenin into developing oocytes (Mekuchi et al. duced protein of 1,943 amino acids and molecular 2008). A full-length cDNA of 3,598 bp was obtained weight of 211 kDa. In studies of levels of gene expres- for this molecule, referred to as LDLR, and the de- sion, these authors found that levels are very low in duced amino acid sequence encoded 1,120 amino acid the ovary during early vitellogenesis, but increase to residues. This is a little over half of the size of Pe. maximal levels in females having a GSI of 3–4. Of monodon VgR discussed above; however, these two interest, Tiu et al. (2008) were also able to knock down molecules have not been directly compared in terms VgR expression by injecting VgR double-stranded of sequence identity. The Pe. monodon VgR sequence

sects. In a phenomenon called “patency”, a juvenile hormone (JH)-specific Na/K-ATPase exists in the follicle cells of the ovary, and its enzymatic activity is elevated in the presence of JH. In turn, the follicle cells based on the stoichiometry of the Na/K-ATPase ex- change mechanism, become hypotonic to their surrounding medium, and subsequently “shrink”. This causes spaces between the follicle cells to enlarge, which ables vitellogenin to access the oocyte surface (Davey et al. 1993). This phenomenon, and related knowledge as it concerns Crustacea, is discussed in Fig. 16. Changes in ovarian LDLR mRNA expression of further detail in Subsection 4-1C below. Marsupenaeus japonicus. Lane 1: previtellogenic stage. Lane 2: endogenous vitellogenic stage. Lane 3: early exogenous 4. Regulation of vitellogenin synthesis and uptake; stage. Lane 4: late exogenous stage. Lane 5: maturation stage. utilization of vitellin Values represent the mean ± SEM (n = 5). Variance was sta- tistically evaluated by analysis of variance (ANOVA) with 4-1. Endocrine control of vitellogenin synthesis SPSS software (release 6.1; SPSS Japan Inc., Tokyo, Japan). One-way ANOVA was used to determine significance. Indi- 4-1A. Eyestalk neuropeptides and vitellogenesis in- vidual differences between groups were assessed using hibition Duncan’s multiple range test. Significant differences (P < The existence of a vitellogenesis- or gonad- 0.05) are indicated by differing letters. Reprinted with permission from Zoological Science, 25, Mekuchi et al., Char- inhibiting factor originating in the eyestalks was shown acterization and expression of the putative ovarian lipopro- by means of eyestalk ablation in Pal. serratus (Panouse tein receptor in the kuruma prawn, Marsupenaeus japonicus, 1943), and by sinus gland implantation into eyestalk- 428–437, Fig. 5,  2008, Zoological Society of Japan. ablated Uc. pugilator (Brown and Jones 1948), over 50 years ago. The acceleration of vitellogenesis by eyestalk ablation was observed in a large number of crustaceans (Adiyodi and Adiyodi 1970). Since then, the factor became a target of investigation in many contained characteristic domains of the LDL-receptor crustacean species. As indices of vitellogenesis these family, such as the ligand-binding domain, the epider- studies used GSI (Kl˛ek-Kawinska and Bomirski 1975), mal growth factor (EGF) precursor domain, an EGF- oocyte diameter (Soyez et al. 1987), frequency of re- like domain, and a transmembrane region. These struc- productive molt (Gréve et al. 1999), hemolymph tures are also found in Mar. japonicus and are shown vitellogenin protein levels (Vincent et al. 2001), and in Figs. 14A, B together with a phylogentic tree com- amounts of total protein or vitellogenin synthesized paring LDLR in Mar. japonicus to those of other ovipa- (Quackenbush and Keeley 1988; Quackenbush 1989; rous animals in Fig. 15. In examination of tissue- Aguilar et al. 1992; Lee and Watson 1995; Chaves specific gene expression, it was seen that LDLR was 2000). The complete structure of this factor, termed expressed in the ovary, but not in gill, heart, intestine, vitellogenesis-inhibiting hormone (VIH), was reported muscle, or testis. Furthermore, it was seen that LDLR first in Ho. americanus (Soyez et al. 1991), and sec- mRNA expression was highest in previtellogenic ova- ond in the terrestrial isopod Armadillidium vulgare ries, decreasing in accordance with the progression of (Gréve et al. 1999; Fig. 17). ovarian maturation (Fig. 16). As vitellogenin gene ex- Knowledge of the VIH molecule was originally very pression in the hepatopancreas/ovary generally in- limited because of difficulties in preparing a suitable creases with the progression of ovarian maturation bioassay for vitellogenesis-inhibiting activity. To over- (Jayasankar et al. 2002; Kim et al. 2005), these results come those difficulties, heterologous in vivo or in vitro signified that the receptor is prepared in advance for assays have been used in preference to assays for incorporation of its ligand into the oocytes (Mekuchi hyperglycemic or molt-inhibiting activities. An in vivo et al. 2008). These results were also reflected by in bioassay with Palaemonetes varians to characterize situ hybridization carried out in the same study. Ho. americanus VIH (Soyez et al. 1987) confirmed that It remains that few studies have been carried out on anti-lobster VIH serum cross-reacted with sinus gland shrimp vitellogenin/lipoprotein receptors; future stud- extract of several crustacean species, including Pal. ies using other crustacean species should contribute to varians (Meusy et al. 1987). VIH of the Mexican cray- enhancing knowledge of reproductive function in this fish Procambarus bouvieri was characterized by incu- regard. At the same time, the manner by which bation of ovarian tissue of Li. vannamei (Aguilar et al. vitellogen accesses the oocyte surface to be then taken 1992) and partially sequenced (Huberman et al. 1995). in by pinocytosis has been extensively studied in in- Incubation experiments using Pe. semisulcatus ovary

Fig. 17. Amino acid sequences of CHH-family peptides purified from the sinus gland of Marsupenaeus japonicus. Those of VIH from American lobster (Hoa-VIH) and terrestrial isopod (Arv-VIH) are also shown. Cys residues conserved among CHH-family members are shown in white lettering with black background.

(A) (B)

12000 100

8000 * * 50 ** ** 4000 ** ** ** (% of control) (arbitrary units) VG mRNA levels VG mRNA levels

0 0 Unincubated Incubated 1 0.1 10 0.01 0.001 Control

Unincubated Sinus gland equivalents/ml

Fig. 18. Vitellogenin (VG) mRNA levels in Marsupenaeus japonicus ovarian fragments. (A) Vitellogenin levels in 20-h- incubated fragments and unincubated fragments from the same animals. Vitellogenin mRNA levels in the incubated group were significantly higher than those in the unincubated group (mean ± SEM; n = 9, *P < 0.05). (B) Effects of sinus gland extracts on vitellogenin mRNA levels. Vitellogenin mRNA levels are expressed as percentage changes relative to control values (mean ± SEM; n = 4). Unincubated samples were prepared from ovarian fragments used for incubation at the highest dose. *P < 0.05; **P < 0.01. Reprinted from General and Comparative Endocrinology, 144, Tsutsui et al., The effects of crustacean hyperglycemic hormone family peptides on vitellogenin gene expression in the kuruma prawn, Marsupenaeus japonicus, 232–239, Copyright (2005), with permission from Elsevier.

were used to investigate the vitellogenesis-inhibiting gous sinus gland extracts were more potent than heter- activity of sinus gland peptides in terms of the synthe- ologous ones (Khayat et al. 1998). Similarly, sinus sis of protein or vitellogenin. Six CHHs of Mar. gland extract from Procambarus clarkii reduced pro- japonicus (type-I peptides: Pej-SGP-I, -II, -III, -V, tein synthesis in Pr. clarkii ovaries, but that from -VI, and -VII; Fig. 17) strongly inhibited protein syn- Penaeus species did not (Chaves 2000). thesis including that of vitellogenin. Pej-SGP-IV, a Accumulated information on vitellogenin gene ex- type-II peptide with molt-inhibiting activity also had pression has allowed the effects of vitellogenesis- an inhibitory effect, but its efficacy was weaker than regulating factors to be observed more precisely. In those of type-I peptides (Khayat et al. 1998). Mar. japonicus, the hepatopancreas and ovary are re- Similar results were obtained in an assay of CHH- sponsible for vitellogenin synthesis, and vitellogenin family peptides from the South African spiny lobster mRNA levels in those tissues and hemolymph Jasus lalandii: two type-I peptides (CHH-I and -II) vitellogenin protein levels increase significantly dur- inhibited protein synthesis, but MIH did not. Addition- ing vitellogenesis (Jasmani et al. 2000; Tsutsui et al. ally, C-terminally shortened peptides of CHH-I and 2000). Therefore, ovarian tissue incubation is often -II did not inhibit synthesis (Marco et al. 2002). Dose- used to search for factors affecting vitellogenin gene response analysis of sinus gland extracts of Mar. expression, which could be VIH or vitellogenesis- japonicus and Pe. semisulcatus suggested that homolo- stimulating hormone (VSH). Vitellogenin mRNA lev-

SGP-III (CHH) 100 SGP-IV (MIH) MIH-B 75 *

50 **

(% of control) 25 ** VG mRNA levels ** ** 0 0 10–1310–1210–1110–10 10–9 10–8 10–7 Peptide concentrations (M)

Fig. 19. Effects of Pej-SGP-III, -SGP-IV, and -MIH-B on vitellogenin (VG) mRNA levels in Marsupenaeus japonicus. Levels are expressed as percentage changes relative to control values (mean ± SEM; n = 4–5). *P < 0.05; **P < 0.01. Modified from General and Comparative Endocrinology, 144, Tsutsui et al., The effects of crustacean hyperglycemic hormone family peptides on vitellogenin gene expression in the kuruma prawn, Marsupenaeus japonicus, 232–239, Copyright (2005), with permission from Elsevier.

Liv-SGP-A 100 Liv-SGP-B Liv-SGP-C (Pev20) Liv-SGP-D (Liv-MIH-I) Liv-SGP-E (Pev27) 50 Liv-SGP-F (Pev28) Liv-SGP-G (% of control) VG mRNA levels

0 0 10–13 10–12 10–11 10–10 10–9 10–8 10–7 Peptide concentrations (M)

Fig. 20. Effects of CHH-family peptides from Litopenaeus vannamei on vitellogenin (VG) mRNA levels in Marsupenaeus japonicus ovary. Levels are expressed as averages of percentage changes relative to each control group (n = 4–5). Modified with kind permission from Springer Science + Business Media: Marine Biotechnology, Purification of sinus gland peptides having vitellogenesis-inhibiting activity from the whiteleg shrimp Litopenaeus vannamei, 9, 2007, 360–369, Tsutsui et al., Figs. 4B, C, D, E, F, G and H,  2007, Springer-Verlag.

els in ovarian fragments spontaneously increased dur- family peptides in previtellogenic ovaries and starts to ing the incubation period in this assay system (Tsutsui increase when liberated from inhibition in Mar. et al. 2005a; Fig. 18A), and homologous sinus gland japonicus. extract reduced expression to 20–45% of control lev- The authors of this monograph also used this incu- els at concentrations of 0.001 to 10 sinus gland equiva- bation assay to examine the effects of seven CHH- lents/mL (Fig. 18B). Pej-SGP-III significantly inhib- family peptides purified from sinus glands of Li. ited vitellogenin gene expression in a dose-dependent vannamei on vitellogenin expression (Tsutsui et al. manner, whereas recombinant Pej-SGP-IV, as well as 2007). Type-I peptides (Liv-SGP-A, -B, -C [Pev20 of Pej-MIH-B for which transcript is much more abun- Wang et al. 2000], -E [Pev27], -F [Pev28], and -G) dant in the thoracic and abdominal ganglia than in the inhibited vitellogenin expression, although with dif- eyestalks, reduced vitellogenin expression at 100 nM. ferent efficacies, whereas type-II Pev-SGP-D (recently However, those effects were not significant (Tsutsui et designated as Liv-MIH-I; Chen et al. 2007) did not al. 2005a; Fig. 19). The other five type-I peptides of (Fig. 20). Interestingly, the inhibitory activity of SGP- Mar. japonicus also inhibited vitellogenin expression E, a C-terminally truncated form of SGP-G, was lower with nearly the same efficacy as Pej-SGP-III (Tsutsui, than that of SGP-G by approximately two orders of unpublished data), but no peptide inhibited GAPDH magnitude. These results suggest that the molecular expression. Together, these results show that characteristics required for vitellogenesis-inhibiting vitellogenin expression is suppressed by type-I CHH- activity in penaeid shrimp species are present in type I

150 (a) previtellogenic ovary 150 (b) vitellogenic ovary

100 100

50 50

0 0 0 X M 0 X M 0.1 10 10 M m 0.1 10 10 1000 IB 1 1000 + 0. +IBM0.1 m

150 (c) previtellogenic ovary 150 (d) vitellogenic ovary Fig. 21. Effects of (A) rHoa-VIH-OH and (B) rHoa-VIH- amide on vitellogenin (VG) mRNA levels in Marsupenaeus 100 100 japonicus ovary. Relative levels (vitellogenin/GAPDH mRNA) are expressed as percentage changes relative to each 50 50 control group (mean ± SEM; n = 5). *P < 0.05 relative to 0 nM group. Reprinted from Peptides, 27, Ohira et al., Pro- 0 0 0 X M 0 X M 0.1 10 10 0.1 10 10 duction and characterization of recombinant vitellogenesis- 1000 1000 IBM +IBM0.1 m + 0.1 m inhibiting hormone from the American lobster Homarus americanus, 1251–1258, Copyright (2006), with permission 150 (e)150 (f) from Elsevier. previtellogenic previtellogenic vitellogenic vitellogenic 100 100

Relative expression level of VTG (% control VTG/GAPDH) 50 50 peptides having a carboxyamide C-terminal moiety. Ho. americanus VIH (Hoa-VIH; Fig. 17) has an 0 0 amidated C-terminus (Soyez 1997). The biological sig- 0 1 0 1 0 1 0 1 100 100 0.1 0.1 nificance of this amide moiety was examined by the IBMX (mM) assay system employed above (Ohira et al. 2006). A recombinant Hoa-VIH with a free C-terminus (rHoa- Fig. 22. Dose-response effects of pharmacological agents— VIH-OH), showed no effect on vitellogenin gene ex- (a), (b) dibutyl-cAMP (dbcAMP), (c), (d) dibutyl-cGMP (dbcGMP), (e) forskolin, and (f) IBMX—on vitellogenin pression at 4 to 400 nM, whereas a carboxy-terminal- (VTG) mRNA levels in previtellogenic and vitellogenic ovar- amidated rHoa-VIH (rHoa-VIH-amide) significantly ian fragments of Marsupenaeus japonicus incubated for 24 inhibited vitellogenin mRNA levels at 400 nM (50.9%), hours. Bars indicate mean and SD of 4 samples. Asterisks compared with the control (Fig. 21), suggesting the indicate significant difference from control (P < 0.05). Re- importance of C-terminal amidation in Hoa-VIH. Its printed from General and Comparative Endocrinology, 148, efficacy was definite, but much lower than those of Okumura, Effects of cyclic nucleotides, calcium ionophore, sinus gland extracts and Pej-SGP-III from Mar. and phorbol ester on vitellogenin mRNA levels in incubated japonicus (Figs. 18B, 19, respectively). This result was ovarian fragments of the kuruma prawn Marsupenaeus probably due to the utilization of a heterologous in vivo japonicus, 245–251, Copyright (2006), with permission from assay. Elsevier. Recently, the gene silencing effect of double-stranded RNA (dsRNA) has been utilized to clarify the biological functions of various genes, including those encod- 4-1B. Mode of action of VIH on the ovary: experi- ing crustacean neuropeptides. In Me. ensis, this tech- ments using secondary messengers in ovarian incu- nique showed the gonad stimulatory effects of MeMIH- bation to examine vitellogenin gene expression B. MeMIH-B is expressed in nervous tissues, and its Vitellogenin synthesis in the ovary is directly inhib- expression pattern in the eyestalk was correlated with ited by vitellogenesis-inhibiting hormone (VIH) the reproductive cycle (Gu et al. 2002). Injection of (Tsutsui et al. 2005a). Because actions of peptide hor- recombinant MeMIH-B increased vitellogenin gene ex- mones are mediated by intracellular signaling path- pression in the hepatopancreas and ovary, and ways, the actions of VIH are expected to be mediated vitellogenin protein in hemolymph and ovary; injec- via secondary messengers in the ovary. Thus, Okumura tion of MeMIH-B-dsRNA decreased them (Tiu and (2006) determined whether cyclic nucleotides, Ca2+, Chan 2007). In Pe. monodon, a cDNA encoding Pem- and protein kinase C are involved in the regulation of GIH was cloned, and the administration of GIH-dsRNA vitellogenin mRNA levels in the ovary of Mar. increased vitellogenin gene transcript levels japonicus. (Treerattrakool et al. 2008). Ovarian fragments of Mar. japonicus were incubated

Cell Adenylyl membrane Ca2+ cyclase Phospho- PIP2 lipase C IP3 Gs Cell G Guanylyl membrane DAG Gi cyclase ER cAMP cGMP GTP ATP 2+ PDE Ca 2+ PKC Ca /Calmodulin AMP GMP Protein phospholylation PKA PKG cAMP Vitellogenin expression Protein phospholylation

Vitellogenin expression Fig. 24. Schematic diagram of signaling pathways for vitellogenin gene expression via calcium ion and protein kinase C (PKC) in ovary. DAG, diacylglycerol; ER, endo- Fig. 23. Schematic diagram of signaling pathways for plasmic reticulum; G, G protein; IP3, inositol 1,4,5-triphos- vitellogenin gene expression via cyclic nucleotides in ovary. phate; PIP2, phosphatidylinositol bisphosphate; VIH, vitel- Gi, inhibitory G protein; Gs, stimulatory G protein; PDE, logenesis-inhibiting hormone. “+” and “–” indicate stimu- phosphodiesterase; PKA, protein kinase A; PKG, protein latory and inhibitory effects, respectively. Reprinted with kinase G, VIH, vitellogenesis-inhibiting hormone. “+” and permission from Bulletin of Fisheries Resesarch Agency, 26, “–” indicate stimulatory and inhibitory effects, respectively. Okumura, Intracellular signaling pathways for vitellogenin Reprinted with permission from Bulletin of Fisheries synthesis in the ovary of the kuruma prawn, Penaeus Resesarch Agency, 26, Okumura, Intracellular signaling path- (Marsupenaeus) japonicus, 135–141, Fig. 5,  2008, Fish- ways for vitellogenin synthesis in the ovary of the kuruma eries Research Agency, Japan. prawn, Penaeus (Marsupenaeus) japonicus, 135–141, Fig. 4,  2008, Fisheries Research Agency, Japan.

kinase A or C inhibitors, phosphatidylinositol 3-kinase with pharmacological agents, and vitellogenin mRNA inhibitors) will clarify the specific pathways. levels in the fragments were determined after 24 hours by quantitative RT-PCR. A23187 (calcium ionophore), 4-1C. Role of vitellogenesis-promoting hormones: dibutyl-cAMP (cAMP analogue), dibutyl-cGMP putative vitellogenesis-stimulating hormone, bio- (cGMP analogue), forskolin (adenylate cyclase acti- genic amines, and juvenoids vator), 3-isobutyl-1-methylxanthine (IBMX, phos- While the existence of inhibitory hormones in Crus- phodiesterase inhibitor), and phorbol 12-myristate 13- tacea has been conclusively demonstrated and their acetate (PMA, protein kinase C activator) significantly characteristics elucidated, an understanding of posi- decreased vitellogenin mRNA levels in a dose- tive mechanisms controlling crustacean reproduction dependent manner (Fig. 22). Addition of IBMX in com- remains elusive. There is a history of several decades bination with cyclic nucleotides dramatically reduced of work in this regard, but in contrast to the above dis- vitellogenin mRNA levels. These results suggest that cussed for eyestalk hormones, a definitive factor that cyclic nucleotides, Ca2+, and protein kinase C are in- induces vitellogenesis, analogous to the role of juve- volved in the signaling pathways that regulate nile hormone/ecdysteroids in insects, has not been fully vitellogenin mRNA levels in the ovaries (Figs. 23, 24). identified. The signaling pathways of peptide hormones in- The earliest work in this area suggests that a puta- volved in ecdysteroid synthesis in Y-organs have been tive vitellogenesis-stimulating factor originates in the intensively investigated (for reviews, see Mattson brain and thoracic ganglion. Hinsch and Bennett (1979) 1986; Spaziani et al. 2001). By analogy with the cel- found that thoracic ganglion implants stimulated vitel- lular signaling in Y-organ cells, cAMP and cGMP in logenesis in immature spider crabs Libinia emarginata. the ovarian follicle cells probably mediate the action In the shrimp, Paratya compressa, brain and thoracic of VIH, and Ca2+ and protein kinase C probably in- ganglia extracts stimulated ovarian development both hibit vitellogenin synthesis independently of VIH. Fur- in vitro and in vivo (Takayanagi et al. 1986). Similar ther tests on signal transduction in vitellogenin syn- results were obtained in implantation experiments in thesis using pharmacological inhibitors (e.g., protein the whiteleg shrimp, Li. vannamei (Yano 1988; Yano

doi:10.5047/absm.2010.00303.0073 © 2010 TERRAPUB, Tokyo. All rights reserved. 100 M. N. Wilder et al. / Aqua-BioSci. Monogr. 3: 73–110, 2010 and Wyban 1992), and crab, Potamon koolooense may exist in Crustacea, but has not been confirmed. (Joshi 1989). At this time, the putative VSH could not The actual existence of a juvenoid substance in Crus- be fully chemically characterized, but appeared to be tacea was first confirmed by Laufer et al. (1987). MF a 10 kDa peptide that is inactivated by trypsin (Yano was detected in the hemolymph of Li. emarginata us- 1998). ing gas chromatography-mass spectrometry (GC-MS) There is also evidence that the neurotransmitter se- and the mandibular organs were confirmed as its site rotonin, also referred to as 5-hydroxytryptamine (5- of production. Suggestive of its role as a reproductive HT), is responsible for triggering the relevant neuroen- hormone, MF secretion was highest in individuals ac- docrine organs to release VSH. In the red swamp cray- tively undergoing vitellogenesis. Thereafter, MF was fish, Procambarus clarkii, the effects of brain or tho- found in the giant freshwater prawn, Mac. rosenbergii racic ganglia on ovarian maturation in vitro were en- (Sagi et al. 1991; Wilder et al. 1994), mud crab, Sc. hanced by the administration of 5-HT (Sarojini et al. serrata (Tobe et al. 1989), crayfish, Pr. clarkii (Landau 1995). Similar results were obtained in the fiddler crab, et al. 1989), and lobster, Ho. americanus (Borst et al. Uc. pugilator (Fingerman 1997) and the whiteleg 1987), among other species. However, to date, there shrimp, Li. vannamei (Vaca and Alfaro 2000). are no reports of JH in a crustacean species. In some of the most recent work in this area, A number of studies have suggested that juvenoids Meeratana et al. (2006) injected 5-HT intramuscularly have positive effects on ovarian development in Crus- into adult female Mac. rosenbergii, or alternatively, 5- tacea, for example JH in the paddy fieldcrab, HT-primed culture medium of different tissues includ- Paratelphusa hydrodromous (Sasikala and ing brain, thoracic ganglion, muscle and eyestalk. At a Subramoniam 1991), and MF in the whiteleg shrimp, dose of 1 µg 5-HT/g body weight, ovarian GSI was Li. vannamei (Tsukimura and Kamemoto 1991). In increased to 5.79% compared to 1.59% for the control Mac. rosenbergii, vitellogenin production could not be group. However, doses of 5-HT above 1 µg/g up to 50 stimulated by the injection of MF into eyestalk-ablated µg/g did not cause any further increases in GSI. At the juvenile males and females (Wilder et al. 1994), but same time, regarding the injection of tissue medium, MF application did increase DNA and protein synthe- it was seen that culture medium of 5-HT-primed tho- sis in in vitro-incubated pre-vitellogenic ovaries racic ganglion caused significant advancement of ovar- (Soroka et al. 1993). In the red swamp crayfish Pr. ian stage, both in terms of GSI and histological exami- clarkii, MF strongly stimulated oocyte maturation nation (with brain culture medium having some effect (Laufer et al. 1998). as well) (Meeratana et al. 2006). Such research strongly In several recent studies, MF, its precursor, farnesoic indicates the presence of a vitellogenesis-stimulating acid (FA) which is also found in Crustacea, and sev- factor in the thoracic ganglion, and perhaps the brain, eral other compounds, have been tested in vitro to ex- the release of which is brought about by 5-HT. Of note, amine their effects on vitellogenin gene expression in Sosa et al. (2004) have cloned a crustacean serotonin the ovary and/or hepatopancreas. In Mak et al. (2005), receptor for three species, e.g., the giant freshwater low concentrations of FA stimulated gene expression prawn, Mac. rosenbergii, the crayfish, Pr. clarkii, and in the hepatopancreas of the red crab Cha. feriatus, the Pacific spiny lobster, Panulirus interrruptus. while MF and JH III were effective only at high con- Rodriguez-Sosa et al. (1997) have demonstrated the centrations. Interestingly, in examination of the dose- presence of 5-HT in the eyestalk ganglia of Pr. clarkii, dependency of FA concentration on gene expression, suggesting its role as a neurotransmitter. while 0.001 µM and 0.01 µM concentrations stimu- On the other hand, there is a body of thought that lated expression above the control, expression de- methyl farnesoate (MF), the precursor of juvenile hor- creased at concentrations over 0.1 µM (Mak et al. mone III in insects, may stimulate reproduction in Crus- 2005). The same group has investigated the effects of tacea. Methoprene, a JH mimic, seemed to promote these substances in the shrimp Me. ensis (Tiu et al. vitellogenesis in Li. emarginata (Payen and Costlow 2006a), and the lobster, Ho. americanus (Tiu et al. 1977; Hinsch 1981). In insects, one mode of action of 2009, 2010). While results differ according to species JH in reproduction is to induce “patency”, the devel- and reproductive size, a similar trend is seen, espe- opment of spaces between follicle cells that allows cially with regard to the effects of FA. Of note, in Tiu access of hemolymph, and therefore vitellogenin, to et al. (2010), the molting hormone, 20- the oocyte surface (Ilenchuk and Davey 1982, 1987; hydroxyecdysone, when administered together with Davey et al. 1993). More specifically, a JH-specific FA, synergistically increased vitellogenenin gene ex- Na/K-ATPase is found in insect follicles; exposure to pression. More research of this nature is needed to char- JH elevates its activity, with the result that the follicle acterize the effects of juvenoids in other crustacean cells to shrink and intercellular gaps form. In brine species; moreover, to date, it appears that no research shrimp, Artemia salina larvae, MF and methoprene has demonstrated any conclusive effect of juvenoids elevate Na/K-ATPase activity in larval homogenates on vitellogenin gene expression in vivo. (Ahl and Brown 1991); therefore, such a mechanism

15 30 Estradiol-17 Jun Control Nov Estradiol-17 Ablated 10 20 5 10 0 15 Estriol 0 10 15 Estriol 5 10 0 100 Progesterone 5

50 0 40 Progesterone

0 Hemolymph levels (pg/mL) 10 Hemolymph levels (pg/mL) Testosterone 20

0 0 0231 4 10 11-Ketotestosterone GSI

5 Fig. 26. Correlation between gonadosomatic index (GSI) and hemolymph levels of estradiol-17β, estriol, and progesterone in control and eyestalk-ablated female Marsupenaeus 0 japonicus. Dashed lines indicate the limit of detection. Re- 0482 6 10 GSI printed with permission from Fisheries Science, 70, Okumura and Sakiyama, Hemolymph levels of vertebrate-type steroid hormones in female kuruma prawn Marsupenaeus Fig. 25. Correlation between gonadosomatic index (GSI) and japonicus (Crustacea: Decapoda: Penaeidae) during the natu- hemolymph levels of estradiol-17β, estriol, progesterone, tes- ral reproductive cycle and induced ovarian development by tosterone, and 11-ketotestosterone in female Marsupenaeus eyestalk ablation, 372–380, Fig. 7,  2004, The Japanese japonicus under the natural reproductive cycle. Undetectable Society of Fisheries Science. levels are shown as 0 pg/mL. Dashed lines indicate the limit of detection. Reprinted with permission from Fisheries Sci- ence, 70, Okumura and Sakiyama, Hemolymph levels of vertebrate-type steroid hormones in female kuruma prawn To determine the functions of vertebrate-type ster- Marsupenaeus japonicus (Crustacea: Decapoda: Penaeidae) oid hormones in female shrimp reproduction, one of during natural reproductive cycle and induced ovarian de- the authors of this monograph examined the correla- velopment by eyestalk ablation, 372–380, Fig. 1,  2004, tion between hemolymph hormone levels and ovarian The Japanese Society of Fisheries Science. development in female Mar. japonicus (Okumura and Sakiyama 2004). Hemolymph samples were taken at different ovarian developmental stages, and steroid 4-1D. Possible roles of vertebrate-type steroid hor- levels were measured. Levels of estradiol-17β, estriol, mones in vitellogenesis progesterone, testosterone, and 11-ketotestosterone In oviparous vertebrates, steroid hormones play im- were not significantly related to ovarian development portant roles in the regulation of vitellogenesis. By (GSI, 0.80–9.39; Fig. 25). Furthermore, levels of analogy with the function of steroid hormones in ver- estradiol-17β, estriol, and progesterone did not differ tebrates, the occurrence of vertebrate-type steroid hor- significantly between control non-vitellogenic female mones in crustaceans have been examined, and prawns and female prawns under vitellogenesis induced estradiol-17β, progesterone, 17α- by bilateral eyestalk ablation (removal of the source hydroxyprogesterone, and testosterone have been found of VIH; Fig. 26). These results suggest that these (for reviews, see Fingerman et al. 1993; Huberman vertebrate-type steroid hormones do not play an im- 2000; Subramoniam 2000). portant role in ovarian development in Mar. japonicus.

The above results are supported by recent advances lipovitellin. in genome-wide surveys. Genes encoding estrogen In the mole crab, Em. asiatica, lipovitellins I and II receptors are lacking in the fruit fly Drosophila are progressively cleaved proteolytically into their con- melanogaster and the ascidian Ciona intestinalis. This stituent polypeptides during embryonic development absence suggests that innovations in steroid hormone until they are finally utilized as a source of amino ac- receptors occurred in the vertebrate lineage (Yagi et ids (Subramoniam 1991). This is also a time of intense al. 2003). On the other hand, there are several reports esterase activity in this species (Subramoniam 1991). that the administration of vertebrate-type steroid hor- Of note, the proteolytic products of the vitellins gradu- mones stimulates vitellogenesis in crustaceans (for re- ally lose their PAS staining properties; this suggests views, see Fingerman et al. 1993; Huberman 2000; that carbohydrate prosthetic groups have become dis- Subramoniam 2000). To be able to elucidate the roles associated from vitellin (Tirumalai 1996). Furthermore, of vertebrate-type steroid hormones in shrimp ovarian the activity of two glycosidases, glucosidase and ga- development, further research is necessary. lactosidase, increases in embryos as PAS staining sub- sides in vitellin fractions (Gunamalai 1993). These 4-2. Role and utilization of vitellin during embryo- glycosidases may be required to release bound glucose genesis and galactose from the glycolipid and oligosaccharide components of the major yolk proteins during embryo- Penaeid shrimp and prawns are an exception among genesis in Em. asiatica. decapod Crustacea. These species broadcast their eggs More recently, the utilization of lipovitellin in the into the surrounding medium following spawning, and eggs and embryos of Mac. borelli has been extensively eggs generally hatch within about 1 day. However, most studied by Garcia et al. (2008). This species broods an female decapods brood their eggs externally, with the egg mass for approximately 39 days, and this time egg mass being attached to reproductive setae for a frame is divided into seven embryonic stages. The au- length of time that varies according to species. For thors found that the lipovitellin discussed above exists example, the shrimp Pal. serratus exhibits a 110-day in its native form as a 440 kDa protein in ovaries, and embryonic period (Spindler et al. 1987), while in Mac. is also found in developing eggs. During early embryo- rosenbergii, this period lasts for only 18 days (Wilder genesis, this protein decreases in quantity, but is con- et al. 1990). In the crabs, Cancer magister and Cancer sumed slowly, after which it is consumed rapidly dur- anthonyi, eggs are brooded for 90 and 40 days, respec- ing stages 6 and 7 (Garcia et al. 2008). Furthermore, tively (Okazaki and Chang 1991). during the early embryonic stages, lipovitellin consists Therefore, it is necessary that the newly-spawned egg of the 112 kDa and 94 kDa subunits also identified in contains a reserve of nutrient sources which serve the the ovary, but during the late stages, is broken down to developing embryo until it hatches and is able to feed. smaller subunits of 70 kDa, and finally 43 kDa. A simi- Vitellin is the main provider of nutrition during this lar study was also conducted for the blue crab, Ca. time, and is often referred to as lipovitellin when dis- sapidus (Walker et al. 2006), revealing that lipovitellin cussed in this context. The egg should also possess is used in much the same way, although the embryonic hydrolytic enzymes and proteases to break down the period is shorter (10–13 days). Such studies at present lipovitellin and make lipids, proteins, and amino acids have not been conducted for other shrimp and prawn available for usage. In the brine shrimp, Ar. salina species, but it is likely that other Macrobrachium ge- (which is in the Class Branchiopoda, not Malacostraca nus species exhibit similar patterns of vitellin usage to which decapods belong), the utilization of during embryonic development. lipovitellin in eggs has been studied extensively; egg lipovitellin contains primarily two apoprotein subunits 5. Conclusions and future perspectives having molecular weights of 68 kDa and 190 kDa. During embryonic development, these subunits un- In decapod Crustacea, the process of vitellogenesis, dergo proteolytic cleavage to give rise to smaller where yolk proteins are synthesized, transformed into peptides (De Chaffoy de Courcelles and Kondo 1980). smaller molecules, and taken into maturing oocytes, is Under unfavorable conditions, Ar. salina produces dor- a central feature of reproduction. As discussed above, mant gastrulae capable of resuming development when the vitellogenic site is principally the hepatopancreas, incubated under appropriate conditions (Vallejo et al. or both the hepatopancreas and ovary. While 1981). A vitellin-bound trypsin-like protease (Ezquieta vitellogenin molecules in some animal groups exhibit and Vallejo 1985) is activated concomitantly with the a certain degree of homology, such as those proteins yolk granule dissolution that takes place during hy- found in animal groups such as nematodes, insects and dration of the dry cyst. In the case of Artemia yolk amphibians (Chen et al. 1997), it is now known that utilization, protease activity appears to be a pro- crustacean vitellogenins differ somewhat from those grammed developmental event with possible control of other animals. Most notably, crustacean vitellogenins mechanisms conferred by its association with are lacking in polyserine domains. However, in the

doi:10.5047/absm.2010.00303.0073 © 2010 TERRAPUB, Tokyo. All rights reserved. M. N. Wilder et al. / Aqua-BioSci. Monogr. 3: 73–110, 2010 103 numerous decapod species for which full cDNA se- japonicus) is under 2,000 tons, while about 25,000 tons quence is known, all vitellogenins show a characterstic is provided by conventional fisheries activity, mainly length of around 7,800 bp and an ORF with a typical of cold-water species (MAFF 2008). size of about 2,534–2,589 amino acid residues (see The use of re-circulating aquaculture systems as a Table 2). means of promoting sustainable shrimp culture with- Vitellogenesis is under the negative control of out impacting the environment has received a great deal eyestalk hormones of the CHH family. Vitellogenesis- of attention in recent years. In Japan, the authors of inhibiting hormone (VIH) has been isolated and char- this monograph have been involved in the experimen- acterized, and conclusively demonstrated to inhibit tal phase of setting up re-circulating culture systems vitellogenin mRNA expression in vitro (Tsutsui et al. for Li. vannamei; the system now operates on a 2007). On the other hand, while a great deal of research commerical basis (owned by Myoko Yuki-Guni Suisan, has demostrated evidence for a putative vitellogenesis- Co. Ltd.) in Myoko City, Niigata Prefecture, an inland stimulating hormone (VSH), perhaps a peptide or pro- mountainous area. The system has proved to be stable tein hormone originating from the brain and/or tho- for the low-salinity culture of Li. vannamei starting racic ganglion, a factor having these properties remains from PL15 (Jayasankar et al. 2009), and the resultant to be isolated and fully identified. The work of Sarojini product, Myoko Yuki Ebi® (“Myoko Snow Shrimp”), et al. (1995) using the crayfish Pr. clarkii, strongly has been shown to be of high quality in terms of the indicates that serotonin has a role in promoting vitel- content of flavor-producing amino acids (Okutsu et al. logenesis via its effects as a neurotransmitter causing 2010). However, it remains necessary to import seed the release of a putative VSH. However, this type of from Thailand or Hawaii, and the authors are now aim- work has not been extensively conducted by other re- ing to develop improved seed production methodol- search groups or in other species. Such experiments ogy making use of knowledge of the reproductive should be attempted by others in order to fully reveal mechanisms of this species. the nature of the positive mechanisms affecting repro- Recently, Treerattrakool et al. (2008, 2010) have duction in decapod Crustacea. The same may be put conducted studies using double-stranded RNA in Pe. forth for the other categories of work on positive fac- monodon to investigate VIH (named gonad-inhibiting tors described in this monograph—e.g., on juvenoids, hormone: GIH in these reports) function, and as a second messenger substances, even vertebrate-type means of inducing ovarian maturation and spawning steroids. on-site in Thailand, where this species is an important Nevertheless, a great deal of progress has been made target of culture. The results of Treerattrakool et al. in understanding vitellogenesis in Crustacea. In addi- (2010) show that the use of GIH-dsRNA to create GIH- tion to the knowledge concerning vitellogenin struc- knockdown shrimp, is as effective as unilateral eyestalk ture and processing, much has been learned about its ablation in promoting ovarian maturation and spawn- biochemical features—secondary structure, conjugat- ing. The results are very promising, and appear to be ing moieties, and so forth, as discussed in Subsection almost at the level of being able to be commercially 2-2. There is also a great deal of new information avail- utilized in the field. However, there is still little infor- able on the crustacean vitellogenin receptor, and how mation on how VIH itself fluctuates in the hemolymph, vitellin is utilized during embryogenesis after the egg although the work of de Klejin et al. (1998) has dem- mass is spawned up until hatching. Of particular note, onstrated the dynamics of CHH and GIH storage in vitellogenin is a high-density lipoprotein and has the the sinus glands and their release into the hemolymph additional role of lipid transport; obviously, lipid ac- in the lobster Ho. americanus in relation to reproduc- cumulation is a primary feature of crustacean eggs, tion. More information of this nature in other commer- where it serves as the source of energy during embryo- cially important species may lead to even more effec- genesis (Pandian 1970; Subramoniam 1991). tive means of exploiting, for example, the use of In terms of basic science, the full elucidation of crus- dsRNA methodology to artificially control maturation. tacean reproductive mechanisms is of course, highly Of course, if a stimulatory factor influencing vitello- interesting and necessitated. It is certain that many genesis can be conclusively demonstrated, hormonal similarities, yet many more differences, will be found treatment could also be used in combination with the in comparison to what is known in other arthropods, environmental control of light, salinity and water tem- for example, insects. There also exists much potential perature in the hatchery. for this knowledge to be applied to further aquacultural In conclusion, further research on the hormonal con- development—to control reproduction and stabilize trol of vitellogenesis in Cruatacea is expected to pro- seed production operations in the hatchery, as put forth vide greater insight into reproductive mechanisms in in the Introduction. Japan as a country consumes general, and have many new, exciting applications in 300,000 tons per year of shrimp; yet its production aquaculture, helping to thus provide greater based on aquaculture (of the kuruma prawn, Mar. sustainability to this important industry.

Acknowledgments Chicken oocyte growth is mediated by an eight ligand We thank Prof. Katsumi Aida, Professor Emeritus, The binding repeat member of the LDL receptor family. EMBO University of Tokyo, Japan, for extensive support and en- J. 1994; 13: 5165–5175. couragement. A part of the work conducted by the authors Carvalho F, Sousa M, Oliveira E, Carvalheiro J, Baldaia L. of this monograph was supported by the Research and De- Ultrastructure of oogenesis in Penaeus kerathurus (Crus- velopment Program for New Bio-industry Initiatives of the tacea, Decapoda). III. Cortical vesicle formation. J. Bio-oriented Technology Research Advancement Institution Submicrosc. Cytol. Pathol. 1999; 31: 57–63. (BRAIN) of Japan. The authors also acknowledge the part- Chan S-M, Mak AS, Choi C-L, Ma THT, Hui JH, Tiu SHK. nership of International Mariculture Technology Co., Ltd. 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