Endocrine regulation of reproduction in the female blue , Callinectes sapidus by neuropeptides of the XO- Sinus

gland complex

Thesis submitted in partial fulfillment of the requirements for the degree of “DOCTOR OF PHILOSOPHY”

By Nilli Zmora

Submitted to the Senate of Ben-Gurion University of the Negev

January 2010 Beer-Sheva, Israel Endocrine regulation of reproduction in the female blue crab, Callinectes sapidus by neuropeptides of the XO-

Sinus gland complex

Thesis submitted in partial fulfillment of the requirements for the degree of “DOCTOR OF PHILOSOPHY”

By Nilli Zmora

Submitted to the Senate of Ben-Gurion University of the Negev

January 2010 Beer-Sheva, Israel This work was carried out under the supervision of Prof. Amir Sagi Dr. J. Sook Chung Prof. Yonathan Zohar

In the Department of Life Sciences, Faculty of Natural Sciences Ben-Gurion University of the Negev, Israel And In the Center of Marine Biotechnology, University of Maryland Biotechnology Institute, Baltimore, USA

Approved by the advisors: Professor Amir Sagi:______Dr. J. Sook Chung: ______Professor Yonathan Zohar:______

Approved by the Dean of the Kreitman School of Advanced Graduate Studies:______

Table of Contents

1. General background……………………………………………………….…………………… 1 1.1 Ovarian development and vitellogenesis ……….………..……………………….2 1.2 Growth and molting in …………………..…………………….……… 2 1.3 Hormonal regulation of reproduction and molting in crustaceans………….…. 2 1.3.1 Neuropeptides of the hyperglycemic hormone family….…. 4 1.3.1.1 Vitellogeneis/Gonad-inhibiting hormone (V/GIH) and vitellogeneis gonad- stimulatinhormone(V/GSH)…………………………………..… 6 1.3.2 Terpenoids……………………………………………………………………... 6 1.3.3 Steroids…………………………………………………………………….…... 7 1.4 The blue crab, Callinectes sapidus Rathbun……………………….…………... 8 1.4.1 ………………………………………………………….…………..… 8 1.4.2 Life history of C. sapidus 8 1.4.3 Ovarian development in the female…………………………….... 9 1.4.4 Neuropeptide composition in the sinus gland of C. sapidus…………… 9 1.5 The blue crab in the Chesapeake Bay: the problem……………………...……... 10 2. General objectives………………………………………………………………………….… 12 2.1 Specific objectives of the current study………………………………………….. 12 3. Published chapters……………………………………………………………………………. 14 3.1 Characterization of vitellogenesis in the female C. sapidus……………...……. 15 3.2 Regulation of vitellogenesis by neuropeptides of the XOSG complex in the eyestalk……… …………………………………………………………………..……….. 16 3.3 Characterization of the MIH receptor in the hepatopancreas of mature female C. sapidus……………………………………………………………………. 17 4. General discussion and significance………………………………………………….…… 18 5. Literature cited in the general background and general discussion…………………… 22 Acknowledgments

I would like to express my sincere appreciation to the people who have offered me help throughout this dissertation. Their frequent input was extremely useful and important to the success of my studies.

First, I would like to thank my advisors, Professor Amir Sagi, Dr. J. Sook Chung and Professor Yonathan Zohar. Professor Sagi accepted me as a special student, encouraged me throughout the entire process, and professionally, expertly and ‘remotely’ guided me from Beer-Sheva. I am grateful to Dr. Chung, my primary supervisor, who was the first to introduce me to the wonders of crustacean endocrinology and shared with me her tremendous knowledge and experience. At the same time, she gave me the freedom to explore the field in new directions as my research unfolded. Working under Dr. Chung’s supervision was an experience that I will remember and cherish forever. I am also deeply indebted to Professor Zohar, who initiated and led the Blue Crab Advanced Research Consortium project. Prof. Zohar encouraged me to pursue my studies and directed me as I negotiated my way, always being there for help and support. The many years that I worked by his side significantly contributed to improve my abilities as a scientist. Thanks also to Dr. John Trant for providing me the opportunity to initiate my earliest crustacean studies in his laboratory and for his professional help and suggestions with the manuscripts.

I am thankful to John Stubblefield who revised and corrected my manuscripts and dissertation and to Professor Allen Place for his ongoing professional advice. The members of Professor Amir Sagi’s lab group were openly supportive in every sense. They welcomed me as an equal and integral member of the lab, providing all the needed help for someone who resided abroad. I couldn’t have done it without Dr. Rivka Manor, who was of great help in any and all University matters. I am also grateful to Drs. Simy Weil, Eli Aflalo, Assaf Shechter and Shmuel Parnes, who accepted me and treated me as ‘one of their own’.

I would like to thank Ben Gurion University of the Negev for allowing me to pursue my degree as a special student and being accommodating and considerate throughout my graduate process. Thanks to the Center of Marine Biotechnology (COMB), which provided the financial support for the research and allowed me to use it as leverage for my studies.

Finally, and most importantly, I want to extend my love and gratitude to my husband, Oded Zmora, who helped me to achieve my goal. At the professional level, his assistance in the set-up of tank systems and experiments, as well as his work to secure the for my studies, were invaluable. Outside of the lab, his mental support, understanding, and encouragement were more significant than words can express.

Abstract

Reproduction processes are integral to the life cycle of all organisms. Understanding and controlling the reproductive processes has a very advantageous applied value in the management of economically important , including the decapod crustaceans. Ovarian development and vitellogenesis in particular, which are very energy demanding processes, are highly controlled by endocrine factors. To date, the factors involved in the regulation of vitellogenesis in crustaceans and their mechanisms of action are not fully explored. More importantly, despite the large diversity of reproductive strategies and physiology within this group of , only a few general paradigms for hormonal control have been suggested. The blue crab, Callinectes sapidus is an ecologically-important crustacean species that supports commercial fisheries in the US coastal Atlantic states and Gulf of Mexico with a unique female’s reproductive behavior and physiology. However, this species is currently threatened due to local and regional declines in breeding populations. As such, ovarian development and its regulation by neuropeptides of the X-organ sinus gland complex (XOSG) were the subject of the current study. In this study, ovarian development was evaluated by vitellogenesis – the process of production of yolk protein and its absorption in the growing oocytes. Initial work established the foundation for the ensuing hormonal studies by determining the basic profiles of vitellogenesis at both protein and gene expression levels during ovarian development. As a result, the vitellogenin cDNA of C. sapidus (CasVtG) was cloned from the hepatopancreas, correcting previous studies reporting that the ovary is the site of vitellogenesis in this species. Moreover, the results show that, unlike the majority of proteins, vitellogenin (VtG) is accumulated and stored as mRNA in the hepatopancreas and VtG is secreted immediately after translation, suggesting a differential regulation of transcription and translation for this gene. Our next experiments were conducted to identify a vitellogenesis-regulating XOSG-borne neuropeptides in C. sapidus females. An in vitro incubation study identified the molt-inhibiting hormone (MIH) in the regulation of vitellogenesis. The present study revealed that MIH has a positive regulatory role in vitellogenesis at mid/advanced vitellogenesis by stimulating both transcription and translation of VtG. In addition, no inhibitory factor (namely gonadal/vitellogenesis-inhibitory hormone, G/VIH) that was described in other crustacean species was detected in the sinus gland (SG) of the C. sapidus female. These findings contradict the paradigm of G/VIH presence in the SG of all crustaceans, and suggest that VIH or VSH (vitellogenesis-stimulating hormone) activities are species dependent. The presence of G/VSH, and not G/VIH, is probably related to the specific reproductive strategy of the C. sapidus female that features a terminal anecdysis in adulthood. The current study also describes the presence of novel specific binding sites for MIH in the hepatopancreas of vitellogenic females, with increasing abundance that is in correlation with ovarian developmental stage. Comparison of the binding kinetics in the hepatopancreas to the classical target site of MIH, the Y-organ (YO - the molting gland) revealed two different types of receptor: low affinity - high capacity vs. high affinity - low abundance, respectively. Moreover, the hepatopancreas-type receptor utilizes cAMP as a second messenger, while the YO-type activates cGMP. In addition to its novelty, the existence of such a membrane-bound receptor in a tissue other than the YO challenges a second paradigm: that the YO is the exclusive target tissue of MIH. The finding that the MIH receptor in the mature female’s hepatopancreas is different from the YO form in its time of appearance/functionality, its binding kinetics and signal transduction offers a possible model for the widespread, yet unresolved, pleiotropicity mechanism of the CHH neuropeptide family. This model proposes that the multiple functionality of the CHH neuropeptide family may be mediated by multiple receptor forms: each form executing a different function. Altogether, this study presents a comprehensive understanding of vitellogenesis in the female C. sapidus. It also demonstrates that MIH possesses a dual role in adult female, in the most significant processes in the life of crustaceans: molting and reproduction. This work further suggests that in crustaceans the antagonism between somatic growth (which is obtained via molting in all arthropods) and reproduction may require MIH to stimulate vitellogenesis while simultaneously inhibiting molting.

1. General background Arthropods are the most diverse group of taxa, consisting of insects, crustaceans, and arachnids. There are far more species of arthropods than species in all other phyla combined. Members of this phylum have been responsible for the most devastating plagues and famines mankind has known. Yet other species of arthropods are essential for our existence, directly or indirectly (Hickman and Roberts, 1994). A number of important characteristics are shared by most members of this phylum. Arthropods are bilaterally symmetrical protostomes with strongly segmented bodies. The body is covered with an exoskeleton made up primarily of chitin in a protein matrix, lipids and calcium carbonate. Arthropods generally grow by shedding their exoskeletons in a process called ecdysis. Most of the body cavity is an open "hemocoel," or undefined space filled loosely with tissue, sinuses, and hemolymph. The circulatory system is open and consists of a heart, arteries, and the open spaces of the hemocoel. Most arthropods are dioecious and have paired reproductive tissues (ovaries, testes). Fertilization is internal in most but not all groups. Most arthropods lay eggs and development often proceeds with some form of metamorphosis (Brusca and Brusca, 1990; Pearse et al., 1987). The model organism used in the present study is the blue crab, Callinectes sapidus Rathbun (Rathbun, 1930), a decapod crustacean. Its ecological and commercial importance, together with an intriguing reproductive physiology, especially that of females, makes it a species of great interest for reproductive studies. Female C. sapidus ceases molting upon puberty – a rare phenomenon amongst crustaceans. This creates two distinct phases: a molting phase during juvenile stages, and a reproductive phase at adulthood. Since molting and reproduction are both regulated by neuropetides of the XOSG complex, the separation of the reproduction cycles from molt cycles provides an excellent framework for studying the hormonal control of ovarian development in a crustacean species.

1 1.1 Ovarian development and vitellogenesis in crustaceans Oogenesis can be divided into several phases in which the phases that follow the initial phase (characterized by the accumulation of yolk proteins in the growing oocytes and by significant increases in oocyte diameter) are referred to as primary and secondary vitellogenesis (Meusy and Charniaux-Cotton, 1984; Meusy and Payen, 1988). Vitellin (Vn) is the common form of yolk stored in oocytes and a nutrient source for the developing embryos. In many species, vitellogenin (VtG), the precursor molecule of Vn, can be produced in the hepatopancreas and/or the ovary (Charniaux-Cotton, 1985) and transported through the hemolymph in the form of HDL to the developing oocytes, where it is sequestered and modified with the addition of polysaccharides and lipids into Vn (Tsukimura, 2001).

1.2. Growth and molting in crustaceans Molting in crustaceans has been intensively studied and reviewed (Chang, 1991; , 1993; , 1995; Skinner, 1985). The molt cycle is divided into: (1) Premolt, during which ecdysis preparatory events such as the separation of the old cuticle from the underlying hypodermis occur during a process called apolysis. This degradation of the old cuticle continues concurrently up to ecdysis. (2) Ecdysis, the shell-shedding event, carried out through rapid active absorption of water, resulting in an increased body volume, rupture of the partially degraded old cuticle and emergence of the somatically-larger individual enveloped within the new cuticle. (3) Postmolt, during which parts of the new cuticle are being synthesized, calcium is mobilized and deposited in the new cuticle and water is gradually replaced by tissue. (4) Intermolt, during which all processes related to the previous molt have been completed and the cuticle is fully formed and maximally calcified.

1.3. Hormonal control of reproduction and molting in crustaceans

Reproduction In malacostracan crustaceans, neuroendocrine centers are found in the eyestalks, brain and subesophageal ganglion. The neurohemal organs, supplied by several groups

2 of neuroendocrine cells are: the sinus gland that stores and releases neuropeptides produced by the X-organ in the eyestalks; the post-commissural organ and the pericardial organs, which are innervated respectively by neurosecretory perikarya in the brain and thoracic ganglia (Adiyodi and Subramoniam, 1983). Crustaceans regulate gonadal and somatic growth to achieve an optimum balance between the two processes. A general classification divides them into five groups (Raviv et al., 2008): 1) Species that cease to grow after a pubertal molt (terminal anecdysial); mating(s) and oviposition occur with hard exoskeleton. The female C. sapidus belongs to this group. 2) Crustacean species that develop the ovaries between molts; mating and oviposition occur immediately after shedding. 3) Species that mate after shedding when the exoskeleton is soft. 4) In these species, oviposition coincide with mating during intermolt or premolt. 5) Species in which ecdysis, mating and oviposition are uncoupled and independent of each other. Gonadal maturation and sexual receptivity in the female are under neurosecretory/hormonal control (Warner, 1977) by both steroids and peptides (Quackenbush, 1986). While the role of steroids in the control of female reproduction is controversial, the control by neuropeptides is somewhat better understood. Neuroendocrine control of vitellogenesis was first demonstrated by eyestalk ablation, showing a stimulation of secondary vitellogenesis (Panouse, 1943), leading to the classical paradigm of inhibition by sinus gland-borne peptides (Meusy and Charniaux- Cotton, 1984). In addition, the presence of a gonadal-stimulating factor in the thoracic ganglion was also postulated (Eastman-Reks and Fingerman, 1984; Hinsch and Bennet, 1979; Otsu and Hanaoka, 1951).

Molting The molt cycle is a multi-factorially controlled process, in which molting hormones, in the form of ecdysteroids, play a pivotal role. These steroids are synthesized in endocrine steroidogenic glands, the Y-organs (YO) found in the anterior thorax in crustaceans (Lachaise et al., 1993). Ecdysteroid synthesis increases at the onset

3 of premolt, leading to a surge of circulating ecdysteroids. This surge triggers a programmed set of gene expression and protein synthesis in several tissues such as the hypodermis and the hepatopancreas, resulting in the shedding event (Bielfield et al., 1986; Stringfellow and Skinner, 1988; Traub et al., 1987). The complex control of molting is achieved, in major part, by a multi-factorial control of the activity of the YO itself (Lachaise et al., 1993), although the mechanism(s) of control is not completely understood at present. To date, the most established factor is the neuropeptide molt- inhibiting hormone (MIH). A classical paradigm is based on the notion that removal of MIH, the source of inhibition of ecdysteroid production at the YO, allows molting to occur. Endocrine induction of the molting process could be achieved by extirpation of the XOSG complex by eyestalk ablation (Aiken and Waddy, 1987; Sindhukumari et al., 1987). The decreasing neurosecretion of MIH triggers the arrested YO activity, enabling ecdysteroid synthesis and onset of premolt. Recent work in crab and crayfish suggested a refinement of this molt control model. The new approach suggests that during the molt cycle both MIH titers and YO responsiveness (as determined by the signaling pathway in the YO) are changing. These dynamics enable the cyclicity of the molt cycle (Chung and Webster, 2003; Nakatsuji et al., 2000).

1.3.1. Neuropeptides of the crustacean hyperglycemic hormone family The X-organ sinus gland complex (XOSG) produces several neuropeptides belonging to two functionally different families: 1) the chromatins, pigment-dispersing hormone and red pigment-concentrating hormone and 2) members of the crustacean hyperglycemic hormone (CHH) family, which include CHH, molt-inhibiting hormone (MIH), gonad/vitellogenesis-inhibiting hormone (GIH/VIH) and mandibular organ- inhibiting hormone (MOIH). Members of the CHH neuropeptide family share a common basic structure; all mature peptides contain 72-84 amino acid residues with a conserved location of six cysteine residues that form three intra-disulphide bridges (See Fig. 1). It was suggested that the evolution of this group of neuropeptides occurred by gene duplication and mutational events (Chan et al., 2003). It is noteworthy that these neuropeptides are not exclusive to the XOSG complex and are expressed and secreted from other

4 neurosecretory cells in the gut (Chung et al., 1999), pericardial organ (Chung and Zmora, 2008; Dircksen et al., 2001), brain and thoracic ganglion (Chang et al., 1999; Gu et al., 2002).

Cap-MIH ------MMSRTESRYSSQRTWLLSM-VVLAALWSISV------QR CanM-MIH ------MMSRTESRYSSQRTWLLSM-VVLAALWSISV------QR Carm-MIH ------MMSRANSRFSCQRTWLLSV-VVLAALWSFGV------HR CarF-MIH ------MMSRANSRFSCQRTWLLAV-VVLAAIWSSSL------HQ Cas-MIH ------MMSLAHSKFSCQRTRLLAV-VLLAALWSSSL------QQ PorP-MIH ------Cas-CHH MQSIKTVCQITLLVTCMMATLSYTHARSAEGLGRMGRLLASLKSDTVTPLRGFEGETGHP Cap-MOIH1 MTTKCTVMAVVLAACICLQVLPQAYGRSTQGYGRMDKLLATLMGSSEG--GALESASQHS

Cap-MIH ATARVINDDCPNLIGNRDLYKKVEWICEDCS------CanM-MIH ATARVINDDCPNLIGNRDLYKRVEWICEDCS------Carm-MIH AAARVINDECPNLIGNRDLYKKVEWICEDCS------CarF-MIH AAARVFNDDCPNLMGNRDLYKKVEWICDDCA------Cas-MIH AAARVINDDCPNLIGNRDLYKKVEWICDDCA------PorP-MIH ------MGNRDLYKKVEWICDDCA------Cas-CHH LEKRQIYDSSCKGVYDRAIFNELEHVCDDCYNLYRNSRVASGCRENCFDNMMFETCVQEL Cap-MOIH1 LEKRQIFDPSCKGLYDRGLFSDLEHVCKDCY------

Cap-MIH ------NIFRNTGMATLCRKNCFFNEDFLWCVYATERTEEMSQLRQWV CanM-MIH ------NIFRKTGMASLCRRNCFFNEDFVWCVHATERSEELRDLEEWV Carm-MIH ------NIFRKTGMASLCRRNCFFNEDFVWCVHATERSEELRDLEEWV CarF-MIH ------NIFRIPGMASICRKDCFFNEDFLWCVRATERTEEMMQLKQWV Cas-MIH ------NIYRSTGMASLCRKDCFFNEDFLWCVRATERSEDLAQLKQWV PorP-MIH ------NIYRITGMASLCRKDCFFNEDFLWCVRATERSEDMTQLKQWV Cas-CHH FYPEDMLLVRDAIRGDCSNIFRNTGMATLCRKNCFFNEDFLWCVYATERTEEMSQLRQWV Cap-MOIH1 ------NLYRNPQVTSACRVNCYSNRVFRQCMEDLLLMEDFDKYARAI

Cap-MIH GILGAGRE CanM-MIH GILGAGRD Carm-MIH GILGAGRD CarF-MIH RILGAGRM Cas-MIH TILGAGRI PorP-MIH RILGAGRI Cas-CHH GILGAGRE Cap-MOIH1 QTVGKK--

Figure 1: Alignment of CHH family neuropeptides: amino acid sequences of MIH of several including blue crab MIH (blue) and CHH (yellow). The six cysteine residues are highlighted in grey. Cap, Cancer pagurus; CanM, Cancer magister; CarM, Carcinus maenas; CarF, Charybdis feriatus; Cas; Callinectes sapidus; PorP, Portunus pelagicus. Genebank accession numbers: Callinectes sapidus MIH #AAA69029, Callinectes sapidus CHH #ACH85179, Cancer pagurus MIH # CAC39425, Cancer magister MIH #AAC38984, Carcinus maenas MIH #Q27225, Charybdis feriatus MIH # AAC64785, Portunus pelagicus MIH # ABM74397.

5 1.3.1.1 Vitellogenesis/Gonad inhibiting hormone (V/GIH) and vitellogenesis/gonad- stimulating hormone (V/GSH) The sinus gland is the source of V/GIH, as eyestalk removal resulting in precocious gonadal development (first shown by Panouse in Palaemon serratus; (Panouse, 1943). Since then, evidence for vitellogenic inhibitory activity by sinus gland factors has been described in many species (Bomirski and Klek, 1974; Chaves, 2000; Keller, 1992). V/GIH peptide was found in several lobster species (De Klein et al., 1994; Edomi et al., 2002; Ohira et al., 2006; Ollivaux et al., 2006), peneaids (Tsutsui et al., 2007), and the isopod Armadillidum vulgare (Greve et al., 1999). However, the reported effects of V/GIH differed between species. In lobster, V/GIH seems to inhibit the incorporation of VtG into the oocytes (Jugan and Soyez, 1985; Van Herp and Payen, 1991) and in peneaid shrimps V/GIH represses protein and VtG synthesis (Quackenbush, 1989). A second decapod reproductive neurohormone found in the brain and thoracic ganglion, is the gonad-stimulating hormone (V/GSH; (Eastman-Reks and Fingerman, 1984; Otsu, 1963). In the female fiddler crab, Uca pugilator, and in Metapenaeus ensis V/GSH levels correlated with the reproductive state and were highest in advanced vitellogenic females (Eastman-Reks and Fingerman, 1984; Gu et al., 2002). A recent finding identified V/GSH as an isoform of the SG MIH in M. ensis (Gu et al., 2002; Tiu and Chan, 2007). An additional member of the CHH neuropeptide family, found and characterized in Cancer pagurus, is the mandibular organ-inhibiting hormone (MOIH), which inhibits the production and release of methyl farnesoate (MF) from the mandibular organs (Tang et al., 1999; Wainwright et al., 1996). The involvement of MF in the regulation of reproduction is presented in the following section.

1.3.2. Terpenoids The mandibular organ produces a sesquiterpenoid hormone, methyl farnesoate (MF) that has chemical similarity to the insect juvenile hormone. MF was first isolated from Libinia emarginata (Borst et al., 1987). There is some evidence that MF may have a role in larval development by acting as a hormone that retards development, i.e. a

6 ‘juvenilizing’ factor (Borst et al., 1987). In adults, MF may function in a reproductive capacity. However, its function is ambiguous (Borst et al., 1987) with some indications that it may act as ecdysiotropin in Cancer magister (Tamone and Chang, 1992). In M. ensis and Charybdis feriatus, it was found that farnesoic acid (FA), the precursor for MF, stimulates VtG gene expression in vitro (Mak et al., 2005; Tiu and Chan, 2007), and in female Procambarus clarkii, MF injection had a positive effect on oocyte growth (Rodríguez et al., 2002).

1.3.3. Steroids Ecdysteroids are arthropod steroids involved in the regulation of molting (ecdysis). The more active and prominent form is the 20-hydroxyecdysone (20-HE), which is the most common molting hormone. However, in crabs such as C. maenas and C. sapidus the molting hormone is probably a different form, 25-deoxy-20- hydroxyecdysone (ponasterone A; (Chung, 2009; Lachaise et al., 1993; Pis et al., 1995). 20-HE, and the other ecdysteroids are not only involved in the molting process, but are involved in reproduction and vitellogenesis and may have a role in behavior and rhythmic locomotor processes. In addition, it has been suggested that ecdysteroids stimulate vitellogenesis (Eastman-Reks and Fingerman, 1984), ovarian maturation and protein synthesis (Chang, 1995; Oberdörster and Cheek, 2001), although this is still poorly understood. In some species, however, administration of ecdysteroids has inhibited vitellogenesis (Chang and O'Connor, 1988). It appears that the ability of ecdysteroids to promote vitellogenesis in the hepatopancreas is species-dependent (Loeb, 1993), as is the need for ecdysteroids for the completion of vitellogenesis. Correlations between hemolymph ecdysteroid titers and vitellogenesis have been reported (Chang, 1993; Ohira et al., 2006; Young et al., 1993). However, it is still unclear as to whether ecdysteroids directly influence vitellogenesis, or whether their levels during vitellogenesis are simply indicative of the corresponding stage of the molt cycle. Nevertheless, it is widely accepted that circulating ecdysteroids derived from the Y organ do not play a role in ovarian development and are responsible for molting (Ohira et al., 2006; Young et al., 1993). The ecdysteroids that may have a role in regulating ovaria development are most

7 likely those acting in a paracrine manner and are synthesized by ovarian tissues (Gunamalai et al., 2004; Ohira et al., 2006).

1.4. The blue crab, Callinectes sapidus Rathbun 1.4.1 Taxonomy Phylum: Arthropoda Class: Crustacean Subclass: Malcostraca Order: Suborder: Pleocyemata Infraorder: Brachyura Superfamily: Portunoidea Family: Portunidae Subfamily: Portuninae

1.4.2. Life History of the blue crab The life cycle of C. sapidus involves very specific seasonal and geographical breeding and spawning patterns (Hines, 2003; Miller et al., 2005). Most C. sapidus mate in the upper (northern) parts of the Chesapeake Bay in the Eastern Shore of the United States of America during the summer and early fall. During mating, the male transfers spermatophores or “sperm package” to the female, which has just completed her pubertal molt. The female stores the spermatophore in specific sacs, spermathecea, until she is ready to ovulate months later. The inseminated females start to migrate towards the spawning grounds in the lower (southern) higher-salinity regions of the estuary, reaching these waters before the onset of the winter (Aguilar et al., 2005). They hibernate and over-winter until the early spring, during which time the females ovulate, and the eggs are fertilized and deposited on pleopods as an egg mass (“brood”). The embryos develop for 2-3 weeks prior to hatching, which occurs throughout the summer. A typical brood will produce a total of 3-5 million planktonic larvae called zoeae. The hatched zoeae are swept by water currents out of the mouth of the Chesapeake Bay and into the Atlantic waters, and then recruit back to the Bay as megalopae. Megalopae metamorphose into

8 juvenile crabs, which disperse into a wide range of nursery habitats in sub-estuaries of the lower and upper Bay (Lipcius et al., 2005).

1.4.3. Ovarian development in the female Callinectes sapidus Female C. sapidus undergoes a pubertal molt after approximately 18-20 post larval molts (Hard, 1945). It is well accepted that the pubertal molt is terminal in its nature, however this statement is arguable (this issue is discussed in detail in Chapter 3.2.). Mating occurs right after the pubertal molt and at copulation the ovaries are undeveloped, contain only primary oocytes, and appear as thin white strands of tissue connected to the dorsal surface of the spermathecae (Hard, 1945). During the 2-9 month period after the pubertal molt, vitellogenesis takes place and yolk is formed, thus, the ovary increases in size and develops an orange color. Ovarian development in C. sapidus is classified on the basis of weight, size of gametes and gross morphology into several stages described by Lee and Puppione (Lee and Puppione, 1995): Stage 1: Smooth ovaries, weighing less than 1 g, gametes diameter 50 - 60 µM. Stage 2: Ovaries with convoluted exterior, weighing less than 5 g, oocyte diameter 140 - 180 µM. Stage 3: Ovaries are larger, weighing 5 - 10 g, gamete diameter 200 - 250 µM. Stage 4: Large convoluted yellow ovaries of 10 -15 g, oocyte diameter 250 – 330 µM. Stage 5: Ovaries are about 20 g. Stage 6: Ovaries stretched and empty, external brood is bright orange. Stage 7; Ovaries stretched and empty, external brood is dull orange to brown. Stage 8: Ovaries stretched and empty, external brood is grey.

1.4.4. Neuropeptide composition in the sinus gland of the Callinectes sapidus

Neuropeptide composition in the sinus gland of C. sapidus, as obtained by HPLC analysis, revealed two forms of CHH type 1 and type 2, in which type 2 is the major form, and the third peak is MIH (Figure 2). The major CHH (CHH 2) probably possesses

9 a pyroglutamate at the N-terminus via post cyclization of the Glutamate in position 1 of CHH 1 (Chung and Zmora, 2008).

3

Figure 2: CHH neuropeptide profile of single SG extract of C. sapidus on an RP-HPLC C18 column. Peak 1, CHH-I; peak 2, CHHII; Peak 3, MIH. (Chung and Zmora 2008).

1.5. Callinectes sapidus in the Chesapeake Bay: the problem C. sapidus is a common inhabitant of muddy and sandy shores of the East Coast and Gulf Coast of North America, from Massachusetts to Texas (Rathbun, 1930). Since the Chesapeake Bay is the western Atlantic's largest estuary, C. sapidus has become intertwined with the ecology, the economy, and the culture of the region and over time, C. sapidus have become the most recognizable icon of the Chesapeake Bay region. Sustained fishing and environmental deterioration led to almost 70% drop in C. sapidus abundance in the Chesapeake Bay during the last 15 years, from an estimated 900 million crabs down to around 300 million (Bunnell and Miller, 2005; Lipcius et al., 2005; Miller et al., 2005). Consequently, the C. sapidus fishery, which in the early 1990s was a 52,000 ton, $72 million industry, has declined to a 28,000 ton, $61 million harvest in 2004. More alarming than the drop in abundance and harvest of C. sapidus is the concurrent decline in the spawning stock and, in turn, decreased larval abundance and recruitment (Lipcius and Stockhausen, 2002). Detailed analyses conducted over 17 successive years (1988-2004) showed that spawning stock abundance and biomass in the Chesapeake Bay declined by 81% and 84%, respectively, and larval abundance and post- larval recruitment dropped by an order of magnitude (Lipcius and Stockhausen, 2002; Miller et al., 2005). This drastic depletion of C. sapidus “broodstock” resulted in a

10 continuous decline of juvenile abundance in nursery habitats along tributaries of the Chesapeake Bay (Hines, 2003; Lipcius et al., 2005), which are considered to be largely under their carrying capacities (Zohar et al., 2008).

11 2. General Objectives To address the problems raised in chapter 1.5., a multidisciplinary scientific team that consists of aquaculture and hatchery experts, crustacean endocrinologists and pathobiologists, geneticists, benthic and fishery ecologists had gathered (Zohar et al., 2008) and their objectives were to: 1. Understand all elements of the basic biology of the C. sapidus (reproduction, development, nutrition, genetics, immune function) 2. Develop hatchery and nursery technologies for mass production of C. sapidus juveniles. 3. Assess the feasibility of using hatchery-produced juvenile blue crabs to enhance the C. sapidus breeding stocks in the Chesapeake Bay and, in turn, the overall abundance of this species.

2.1 Specific objectives of the current study When scaling up juvenile crab production, it is necessary to be able to fully control and synchronize ovulation, brood production, and hatching. Obtaining such control will necessitate the development of a full understanding of C. sapidus reproduction, at both the gene and hormon levels. Therefore, focusing on female reproduction, the aim of the current study was to study ovarian development and its hormonal control by achieving the following: 1. Study vitellogenesis in the female C. sapidus and develop the tools to track and monitor the process at both gene and protein levels. 2. Study hormonal regulation of vitellogenesis in order to gain the ability to control and manipulate ovarian development by A) detecting hormones and neurohormones along the reproductive axis that are involved in the control of vitellogenesis, B) defining their roles, and determining their mechanism of action. Significance: Accomplishing the above goals will have a broader impact, and will benefit a variety of projects. In fact, the deterioration in water quality in the Chesapeake Bay and accumulation of pollutants has increased the interest in evaluating the impact of endocrine disruptors (Kemp, Boynton et al. 2005). Indeed several groups are focusing on

12 C. sapidus as a model organism for studying endocrine disruption (Levinton et al., 2006; Schlenk and Brouwer, 1993), however, the use of vitellogenesis and ovarian development as biomarkers, as done in other species (Martín-Díaz et al., 2007), was not available in this species up to date.

13 3. Published chapters As mentioned earlier (General objectives, section 1.2.1), one of the objectives of the research was to understand all elements of the basic biology of the C. sapidus with emphasis on reproduction. The study of vitellogenesis and its hormonal control has an important implication on a successful management of C. sapidus population. The current dissertation encompasses three major parts; all three were published in scientific journals: 1. The first article focuses on the understanding of the vitellogenesis process in C. sapidus female, and on obtaining the tools to monitor the process at the gene expression and protein levels. The article “Vitellogenin and its messenger RNA during ovarian development in the female blue crab, Callinectes sapidus: gene expression, synthesis, transport, and cleavage” By Nili Zmora, John Trant, Siu-Ming Chan, and J. Sook Chung was published in “Biology of Reproduction”, 2007, vol: 77, pp:138–146 and is presented in chapter 3.1.

2. The second article deals with hormonal regulation of vitellogenesis by examining different neuropeptides for their possible effect both in vivo and in vitro. The article “Molt-inhibiting hormone stimulates vitellogenesis at advanced ovarian developmental stages in the female blue crab, Callinectes sapidus 1: an ovarian stage dependent involvement “ by Nili Zmora, John Trant, Yonathan Zohar and Chung J Sook was published in “Saline Systems”, 2009, 5:7 and is presented in chapter 3.2.

3. The third article elaborates the findings of the second article by determining the

mechanism and mode of action of the regulatory function with a special attention to the

receptor mediating the hormonal message. An article “Molt-inhibiting hormone

stimulates vitellogenesis at advanced ovarian developmental stages in the female blue

crab, Callinectes sapidus 2: novel specific binding sites in hepatopancreas and cAMP as

a second messenger” by Nili Zmora, Amir sagi, Yonathan Zohar and Chung J Sook was published in “Saline systems”, 2009, 5:6 and is presented in chapter 3.3.

14 Chapter 3.1

Vitellogenin and Its Messenger RNA during Ovarian Development in the Female Blue Crab, Callinectes sapidus: Gene Expression, Synthesis,

Transport, and Cleavage

15 Chapter 3.2

Molt-inhibiting hormone stimulates vitellogenesis at advanced ovarian developmental stages in the female blue crab, Callinectes

sapidus

1: an ovarian stage dependent involvement

16 Chapter 3.3

Molt-inhibiting hormone stimulates vitellogenesis at advanced ovarian developmental stages in the female blue crab, Callinectes sapidus 2: novel specific binding sites in hepatopancreas and cAMP as a second messenger

17 4. General discussion and significance

The popularity and commercial significance of the C. sapidus along the East Coast of the U.S., together with the declining population, make it a species of considerable interest in coastal states such as Maryland, Virginia, North Carolina and Mississippi and federally (Zohar et al., 2008). In addition, the deterioration in water quality in the Chesapeake Bay raised the need for a reliable bioassay for the assessment of the impact on crabs and other inhabitants of the Bay. Indeed, the vitellogenesis process in the C. sapidus female was a topic of interest of several groups prior to the initiation of the current study. In 1995, two independent research groups reported that the ovary is the origin of vitellogenesis in the female C. sapidus (Lee and Watson, 1995; Lee and Walker, 1995). The development of molecular tools, i.e., the cloning of the vitellogenin gene (CasVtG), as well as the biochemical tools in this study (Chapter 3.1) enabled the accurate localization and characterization of the vitellogenesis process. As one of the first crab VtGs to be fully cloned, CasVtG allowed the generation of a more detailed phylogenetic tree. It also supported the previously suggested paradigm that distinguishes pleocyamata from dendrobranchiata with regard to their vitellogenesis site: pleocyamata (crabs amongst them) are characterized by hepatopancreatic vitellogenesis, while vitellogenin originates equally in both the hepatopancreas and ovary in the dendrobranchiata (Raviv et al., 2006). In addition, the general process, including the cleavage pattern, matches the profiles of other decapod crustaceans in both suborders (Avarre et al., 2003; Okuno et al., 2002).

The accepted knowledge of the hormonal control of vitellogenesis is that both inhibitory and stimulatory factors function in crustaceans. The presence of a gonadal/vitellogenesis-inhibiting hormone has been demonstrated in the eyestalks of a variety of species by eyestalk ablation, and later with the isolation and characterization of a specific CHH family neuropeptide. However, the fact that the C. sapidus female halts molting after the pubertal molt suggest that the control at female maturity may include a component that differs between C. sapidus and other species that continue to molt during maturity. A full discussion as to whether molting is permanently halted or

18 temporarily/reversibly suppressed is included in chapter 3.2. Nevertheless, eyestalk ablation of female C. sapidus did not seem to enhance vitellogenesis (Chung’s laboratory observations in Chapter 3.2., and (Havens and McConaugha, 1990). The present study demonstrated that a G/VIH most likely does not exist in the sinus glands of the mature female C. sapidus and, as such, challenges the generalization of the vitellogenesis- inhibiting activity amongst crustaceans. Moreover, by combining observations of vitellogenesis and neuropeptides profiles in intact wild animals, as well as in vitro studies on the effects of SG-borne neuropeptides on vitellogenesis, it was suggested that the sinus gland of the C. sapidus female contains a vitellogenesis-stimulating hormone (GSH or VSH). This finding is novel, since all the G/VSHs described thus far have been located in the thoracic ganglia and the brain (Eastman-Reks and Fingerman, 1984; Gomez and Nayar, 1965; Gu et al., 2002; Otsu, 1963; Tiu and Chan, 2007). Using an in vitro hepatopancreas incubation system, developed in the Chung laboratory, the G/VSH was identified as the molt-inhibiting hormone (MIH). Attributing to MIH a simultaneous dual role in both the molting and the reproductive axes, i.e., inhibiting molt on one hand and stimulating vitellogenesis on the other, greatly enhances the understanding of the complex crosstalk between molting and ovarian development in crustacean species that continue to molt during maturity. The accepted premise that the two processes are mutually contradicting (Aiken and Waddy, 1980), indicated by the pause in the progress of one process while the other is ongoing, may be executed by one single hormone, the MIH. Thus, MIH may play a pivotal role in managing molting and reproduction, silencing one while allowing the other to proceed, in C. sapidus and potentially other species. The fact that an MIH-like protein with a G/VSH activity has been demonstrated recently in shrimp (Gu et al., 2002; Tiu and Chan, 2007) strongly supports this idea. Considering the energy-demanding migratory behavior of the mature C. sapidus female, ceasing the molting process at maturity seems a prudent solution for efficient energy channeling.

Since the present study demonstrated that MIH acts directly on the hepatopancreas of the mature female C. sapidus to regulate vitellogenesis, its mode of action and the presence of binding sites in this tissue were pursued. As a result, the

19 presence of specific binding sites for MIH with different binding kinetics from the Y- organ receptor was demonstrated. So far, the target tissue for MIH was known to be only the YO, the molting gland, (Asazuma et al., 2005; Webster, 1993), leading to the establishment of the paradigm that MIH binds exclusively to the Y-organ. Thus, the accuracy of two paradigms is now in question: the first regarding the general vitellogenesis-inhibiting activity in the XOSG of crustaceans (Chapter 3.2) and the second that MIH is involved only in molting activities and binds exclusively to the YO (Chapter 3.3). The kinetic studies comparing the binding of MIH to the YO and the hepatopancreas of mature females, revealed not only specific (not displaceable by CHH) binding, but also that the two MIH receptor types are different in terms of affinity and density. To date, the receptors of MIH in the YO and CHH in various tissues were characterized with similar high affinity to their ligands (Asazuma et al., 2005; Chung and Webster, 2006; Webster, 1993). For the first time, a receptor with ~ 80 times lower affinity is described (Chapter 3.3). In addition, although until now all other crustacean neuropeptide receptors have been shown to utilize cGMP as a second messenger (Chung and Webster, 2006; Sedlmeier and Fenrich, 1993; Von Gliscynski and Sedlmeier, 1993; Zheng et al., 2008), we found that the hepatopancreas MIH receptor isoform seems to utilize cAMP.

The structural nature of the CHH neuropeptide family receptor has been a primary focus for several groups (Goy, 1990; Zheng et al., 2006; Zheng et al., 2008), most of which have suggested that the CHH neuropeptide family receptors are guanylate cyclase- type receptors, characterized by the utilization of cGMP and a molecular weight of ~ 120 kDa. Despite significant past effort, this issue remained unresolved. Our data, presented in Chapter 3.3, indicates that the MIH receptor is a GPCR-type receptor rather than a guanylate cyclase-type, based on the findings that its MW is ~ 52 kDa and that the hepatopancreatic-type receptor utilizes cAMP. The novel finding of a dual role for MIH, which is achieved by two different conspecific receptors, sheds light on the mechanism by which part of the well described pleiotropicity of the CHH neuropeptide family is executed. It is expected that the identification and characterization of the first CHH

20 family receptor will be followed by a rapid discovery of multiple receptor isoforms that are distinctive in their localization and signaling.

In conclusion, this research had been an integral contribution toward a greater understanding of the biology of C. sapidus. As part of an enhancement and recovery project for the diminishing C. sapidus breeding populations, a strategy plan for manipulating ovarian development and/or spawning had been proposed. This plan outlines the need to identify a reproductive regulatory factor(s) that may serve as a candidate for the hormonal manipulation of the system. To address this goal, a stepwise study has been conducted. As a first step, vitellogenesis and ovarian development in the female C. sapidus were comprehensively studied. The important regulatory players along the reproductive axis (XOSG-gonads) in the female were identified, positioning MIH in the center of the regulatory process. As a result, the initial hypothesis of regulation of reproduction by a XOSG neuropeptide was confirmed. Our assumption that the reproductive axis will be negatively regulated by this factor was, however, proven incorrect, and was subsequently replaced by an assumption of a stimulatory function. Moreover, MIH arises as a mediator of the antagonism between molting and reproduction, probably not only in the female C. sapidus but also in other species as well. It seems that unlike many crustacean species that continue to molt at adulthood, including the male C. sapidus, the combined reproductive- and molting-specific physiology of the female do not require a specific G/VIH. Reproductive processes such as ovarian development and spawning can be carried on in full capacity without the need to compromise energy demands with molting. The complexity in executing molting and ovarian development cycles in molting adult crustaceans probably requires mediation by both G/VIH and G/VSH. Therefore, it will be interesting to embark on a comparative study on the neuropeptide composition of the SG in adult males and females of species with different strategies and to explore their roles in the regulation of ovarian development and spermatogenesis. The next step of this study could be to test whether MIH is indeed a good candidate for the induction of ovarian development or spawning by MIH injections or, alternatively, by MIH gene silencing.

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29 Saline Systems BioMed Central

Research Open Access Molt-inhibiting hormone stimulates vitellogenesis at advanced ovarian developmental stages in the female blue crab, Callinectes sapidus 2: novel specific binding sites in hepatopancreas and cAMP as a second messenger Nilli Zmora1, Amir Sagi2, Yonathan Zohar1 and J Sook Chung*1

Address: 1Center of Marine Biotechnology, University of Maryland Biotechnology Institute, 701 E Pratt St Columbus Center, Suite 236, Baltimore, MD 21202, USA and 2Department of Life Sciences and the National Institute for Biotechnology in the Negev, Ben Gurion University, Beer Sheva, Israel Email: Nilli Zmora - [email protected]; Amir Sagi - [email protected]; Yonathan Zohar - [email protected]; J Sook Chung* - [email protected] * Corresponding author

Published: 7 July 2009 Received: 9 March 2009 Accepted: 7 July 2009 Saline Systems 2009, 5:6 doi:10.1186/1746-1448-5-6 This article is available from: http://www.salinesystems.org/content/5/1/6 © 2009 Zmora et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract The finding that molt-inhibiting hormone (MIH) regulates vitellogenesis in the hepatopancreas of mature Callinectes sapidus females, raised the need for the characterization of its mode of action. Using classical radioligand binding assays, we located specific, saturable, and non-cooperative binding sites for MIH in the Y-organs of juveniles (J-YO) and in the hepatopancreas of vitellogenic adult females. MIH binding to the hepatopancreas membranes had an affinity 77 times lower than -8 -10 that of juvenile YO membranes (KD values: 3.22 × 10 and 4.19 × 10 M/mg protein, respectively). The number of maximum binding sites (BMAX) was approximately two times higher in the -9 -9 hepatopancreas than in the YO (BMAX values: 9.24 × 10 and 4.8 × 10 M/mg protein, respectively). Furthermore, MIH binding site number in the hepatopancreas was dependent on ovarian stage and was twice as high at stage 3 than at stages 2 and 1. SDS-PAGE separation of [125I] MIH or [125I] crustacean hyperglycemic hormone (CHH) crosslinked to the specific binding sites in the membranes of the J-YO and hepatopancreas suggests a molecular weight of ~51 kDa for a MIH receptor in both tissues and a molecular weight of ~61 kDa for a CHH receptor in the hepatopancreas. The use of an in vitro incubation of hepatopancreas fragments suggests that MIH probably utilizes cAMP as a second messenger in this tissue, as cAMP levels increased in response to MIH. Additionally, 8-Bromo-cAMP mimicked the effects of MIH on vitellogenin (VtG) mRNA and heterogeneous nuclear (hn) VtG RNA levels. The results imply that the functions of MIH in the regulation of molt and vitellogenesis are mediated through tissue specific receptors with different kinetics and signal transduction. MIH ability to regulate vitellogenesis is associated with the appearance of MIH specific membrane binding sites in the hepatopancreas upon pubertal/final molt.

Background ropeptides unique to arthropods. The CHH family mem- The X-organ in the eyestalks of crustaceans produces a bers in crustaceans (CHH, molt-inhibiting hormone family of crustacean hyperglycemic hormone (CHH) neu- (MIH), mandibular organ-inhibiting hormone (MOIH),

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and gonad/vitellogenesis-inhibiting hormone (GIH/ sinus gland factors is described in many species [3,44,45], VIH)), are involved in the regulation of a variety of phys- while gonad stimulatory activity originates in the eyestalk, iological processes [1-6]. It has been established that indi- brain, and thoracic ganglia [13,46,47]. Overall, it appears vidual CHHs are multifunctional, having specific binding that inhibition and stimulation are mediated by neu- sites in multiple target tissues [7,8]. More specifically, in ropeptides of the CHH family [13,44,45,47-53], with one addition to its primary hyperglycemic action [9], CHH exception that attributes the stimulatory action to a small inhibits ecdysteroidogenesis [10]; regulates water uptake peptide of 1000–2000 Da [54]. Further, a study in Marsu- during ecdysis [6]; and inhibits methionine incorporation penaeus japonicus suggests that vitellogenic inhibition is in ovarian fragments, in vitro [11]. It was recently demon- modulated by Ca2+, cAMP, cGMP, and protein kinase C strated that in addition to its traditional molt inhibiting [24]. Much work is still required, however, to define the role, MIH is also involved in the regulation of vitellogen- exact second messengers and the signal transduction path- esis in the mature female Callinectes sapidus [12] and in ways downstream of the neuropeptides that control vitel- Metapenaeus ensis [13]. Since specific hormonal functions logenesis in crustaceans. are accomplished by neuropeptides and their correspond- ing receptors [14], efforts have been made to identify and We have recently reported that MIH levels in the hemol- characterize the CHH neuropeptides family of receptors ymph of female C. sapidus are correlated with vitellogenic by means of binding kinetics [15-18], signal transduction activity, i.e., higher at mid-vitellogenic than at previtello- [15,19-27], and cloning studies [28-31]. However, to genic stage. Furthermore, by the incubation of hepatopan- date, the mechanisms by which the multiple functionality creas fragments in vitro, we demonstrated that MIH acts of these neuropeptides is being executed, are not fully directly in an ovarian stage dependent manner on the understood. hepatopancreas where it stimulates mRNA, heterogene- ous nuclear RNA of vitellogenin (hnVtG RNA = the newly MIH exerts its molt inhibiting activity on the Y-organs transcribed yet unprocessed nuclear VtG mRNA) and VtG (YO) through the suppression of ecdysteroid synthesis translation [12]. Consequently, we proposed that MIH and secretion [32-36] via the down-regulation of protein has a regulatory role in vitellogenesis of the adult female, synthesis [19,22]. It has been reported that MIH binds in addition to its prototypical molt inhibitory function. exclusively to a YO membrane receptor with high affinity This fact imposed a re-examination of the accepted para- in a specific, displaceable, and saturable manner [16,17]. digm that MIH binds exclusively to the membranes of YO Attempts to define the mechanism of MIH signaling [16,17] by verifying the presence of a specific receptor for revealed changes in YO responsiveness throughout a molt MIH in the hepatopancreas of the female C. sapidus. cycle [23,37,38]. While MIH titers in the hemolymph and the number of binding sites of MIH in the YO of Carcinus In the current study, we employed a classical radioligand maenas remained unchanged throughout a molt cycle, the binding assay to 1) locate and characterize specific bind- level of cGMP responding to MIH in this tissue was greater ing sites of MIH in hepatopancreatic membranes of vitel- at intermolt than at premolt stages [38]. This was further logenic females, and 2) determine the molecular weights supported by the finding in Procambarus clarkii that phos- of MIH receptors in the membranes of hepatopancreas phodiesterase activity in the YO at intermolt is markedly and the YO. In addition, we also examined the second low, resulting in an extended cyclic nucleotide life span messenger of MIH in hepatopancreas using an in vitro and in turn, higher levels [23]. incubation assay and radioimmunoassays (RIA). The YO responded to MIH by elevating cGMP levels, while the It has been reported that MIH and CHH act via cGMP as a hepatopancreas responded by increasing cAMP produc- primary second messenger [15,23,26-28,37-39] although tion. Altogether, our results indicate that MIH acts on the the involvement of cAMP, or both cAMP and cGMP [25] YO and the hepatopancreas via a tissue specific receptor. was also suggested. The large increase in cGMP produc- tion in the YO in response to MIH of C. sapidus [23] and Results the stimulation of cGMP in many tissues by CHH in C. MIH binding to the YO membranes maenas [15] have implicated a potential involvement of To determine the maximal number of binding sites of nitric oxide (NO-), soluble guanylate cyclases [20], or MIH in the membranes of the YO, 50 μg of membrane membrane guanylate cyclase type receptors [28,31,40]. proteins were incubated with [125I] MIH from 0.15 to 12.3 However, the structural characterization of a receptor for nM. The non-specific binding was determined using the CHH family of neuropeptides has not yet been eluci- recombinant MIH (rMIH) due to the limited amount of dated in crustaceans nor in insects [41]. native MIH (nMIH). The rMIH was as potent as nMIH in cGMP production in the YO in vitro (unpublished results) Ovarian development in crustaceans is controlled by neu- and produced the same EC50 value in a specific RIA using rohormones [42,43]. The inhibition of vitellogenesis by antibody raised against rMIH [12]. As shown in Fig. 1A,

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from juveniles were also tested, with juvenile YO (J-YO) membranes serving as a reference control. Specific bind- ing of MIH was found only in mature female hepatopan- creas among the tissues tested. MIH binding at ovarian stage 3 was two times higher than in stage 1 and com- prised ~40% of the J-YO control (Fig. 2A). MIH binding to the YO of mature females at stage 3 was equal to that of juveniles (not shown in the figure). The same membranes were tested for [125I] CHH binding by incubating 100 μg with 0.12 pmol [125I] CHH (~182,000 DPM) and unla- beled native CHH at 20 pmol for non-specific binding. No significant difference in CHH binding to the hepato- pancreas was observed between the different ovarian stages, however, CHH binding to juvenile hepatopancreas was two times higher than to J-YO and the other tissues.

A a 100

75

50 c bc 25 b I] MIH binding (% of YO) (%of binding MIH I] 125 [ 0 250 B c 200

125 SaturationtoFigure the membranes 1 curve (A) of and YO displacement of juvenile C. curve sapdius (B) of [ I] MIH 150 Saturation curve (A) and displacement curve (B) of [125I] MIH to the membranes of YO of juvenile C. a a a a a a a 100 sapdius. Bound [125I] MIH was displaced with unlabelled rMIH. The data are presented as mean ± SEM of the tripli- cates. YO) (% of CHH binding I] 50

125 b [ 0 O 1 2 3 P ill le y P Y P P P H sc ar H J- H H H -G u v J- F- F- F- M F M -O F- F the binding sites were saturable with a KD value of 4.19 × -10 -9 10 M/mg protein and a BMAX value of 4.8 × 10 M/mg Tissue protein. Labeled and bound MIH was displaced with cold rMIH [12] at concentrations ranging from 24.4 pM to 10 opancreaspre-vitellogenicFigureMIH specifically 2 with higherbinds to binding memb atranes mid-vitellogenic of mature female stage hepat-than nM (Fig. 1B) with an EC50 value of 0.67 nM. A displace- MIH specifically binds to membranes of mature ment study was carried out on YO membranes using cold female hepatopancreas with higher binding at mid- vitellogenic stage than pre-vitellogenic. A) Specific nMIH, resulting in a similar EC50 value of 0.66 nM. The non-specific binding in these experiments was ~20–30% binding of [125I] MIH to various tissue membranes of vitello- of the total binding. genic females and YO and hepatopancreas of juveniles. B) Specific binding of [125I] CHH to the same membranes. All membranes were prepared from five animals, except for J- MIH binding to the hepatopancreas membranes YO membranes that were prepared from 700 intermolt ani- Membranes (100 μg) of hepatopancreas at ovarian stages mals. F, Females; M, Males; J, juveniles; HP, hepatopancreas; 1 to 3 and gills, abdominal muscle, YO and ovaries that 1, 2, and 3 refer to the ovarian stages; F-ovary- ovarian mem- were pooled from five females at each ovarian stage were brane of females at ovarian stage 3. Results are presented as incubated with 0.4 pmol [125I] MIH (263,000 DPM) with mean ± SEM of the triplicates as % of the J-YO. The alphabet- or without an excess of 20 pmol unlabelled rMIH. Hepat- ical letters show the significant differences at P < 0.05. opancreas membranes prepared from five adult males and

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The ovaries exhibited a binding 20 times lower than J-YO lated KD values were ~77 times greater in the J-YO than (Fig. 2B). female hepatopancreas: 4.19 × 10-10 and 3.22 × 10-8 M/mg -9 protein, respectively. Values of BMAX were 9.24 × 10 M/ A saturation curve was obtained by incubating 0.75 to 60 mg protein for hepatopancreas and 4.80 × 10-9 M/mg pro- nM [125I] MIH with 100 μg hepatopancreas membranes of tein for J-YO. ovarian stage 3 females (Fig. 3A). The calculated KD and -8 - BMAX values were 3.22 × 10 M/mg protein and 9.24 × 10 MIH and CHH effects on cAMP and cGMP levels in the 9 M/mg protein, respectively. Displacement studies were hepatopancreas of vitellogenic females, in vitro conducted using 0.04 nM – 100 nM unlabeled rMIH as The effects of MIH and CHH on levels of cAMP and cGMP well as 12.5 – 100 nM native CHH. rMIH competed with production were tested in vitro in hepatopancreas frag- 125 [ I] MIH on the binding sites with an EC50 value of ments of vitellogenic females at ovarian early stage 2 (E2). 17.35 nM, whereas CHH showed no displacement of As presented in Figs. 5A and 5B, CHH (20 nM) resulted in [125I] MIH (Fig. 3B). The non-specific binding in these a 16 times increase in the levels of cGMP production studies was ~40% for MIH and ~30% for CHH. (from 2 to 32 pmol/mg protein), while MIH at 2 nM had no effect. Cyclic AMP levels did not change with CHH, but The plots obtained for MIH binding to the membranes of increased by 50% in response to 2 nM MIH (from 22 to the J-YO and hepatopancreas were overlaid in order to 33 pmol/mg protein, N = 4), compared to the control demonstrate the difference in the affinities and the which received 1 mM isobutylmethylxanthine (IBMX) number of maximal binding sites (Fig. 4A and 4B). Calcu- alone (Fig. 5B).

membranesstrateFigureKinetic receptor/ligand binding3 of femalestudies C.typical of sapidus [125 I]and MIH at specific ovarian to the binding stagehepatopancreas 3 demon- Kinetic binding studies of [125I] MIH to the hepato- juvenileFigureMIHterized binding by4YO a lowerto female affinity hepatopancreas and higher density membranes compared is charac- to pancreas membranes of female C. sapidus at ovarian MIH binding to female hepatopancreas membranes stage 3 demonstrate receptor/ligand typical and spe- is characterized by a lower affinity and higher density cific binding. Five membrane preparations were pooled and compared to juvenile YO. Overlay of the curves of satu- tested. A) Saturation curve; B) Displacement curve. [125I] ration (A) and displacement (B) of [125I] MIH binding to YO MIH binding sites were displaced with unlabelled rMIH and female hepatopancreas membranes to emphasize the dif- (closed circles) or unlabelled CHH (open circles). The data ferences in values of KD and BMAX between these tissues. YO, are presented as mean ± SEM of the triplicates. closed circles; hepatopancreas, open circles.

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A 30 **

20

10 cGMP (pmol/mg protein) (pmol/mg cGMP

0 Control CHH MIH Treatment B 40 * 30 cGMPmimicsthetivelyFigure hepatopancreas analog the6 effect mimics of fragmentsMIH the effeon VtGcts of of mRNAstage CHH E2 andand females, hnVtGcAMP respec-RNA analog in cGMP analog mimics the effects of CHH and cAMP 20 analog mimics the effect of MIH on VtG mRNA and hnVtG RNA in the hepatopancreas fragments of stage E2 females, respectively. Hepatopancreas fragments were 10 incubated with 10 μM 8-Bromo- cGMP or 8-Bromo- cAMP

cAMP (pmol/mg protein) followed by QPCR analysis to detect changes in hnVtG 0 (Black) and VtG mRNA (Grey). The results are presented as Control CHH MIH mean ± SEM of % of control (N = 4). *, P ≤ 0.05; **, P ≤ 0.01. Treatment 7A and 7B). Binding of [125I] CHH to hepatopancreas CHHfemaleFigure induces hepatopancreas 5 cGMP and fragments MIH induces cAMP production in membranes revealed a signal at a molecular weight of ~70 CHH induces cGMP and MIH induces cAMP produc- tion in female hepatopancreas fragments. In vitro: A) kDa (Fig. 7B). Assuming a binding ratio of 1 to 1 (ligand: cGMP; B) cAMP. Hepatopancreas fragments were incubated receptor), the estimated size for the MIH receptor in both with 20 nM CHH or 2 nM MIH. The results are presented as the YO and the hepatopancreas is ~51 kDa and ~61 kDa mean ± SEM (N = 4). *, P ≤ 0.05; **, P ≤ 0.01. for the CHH receptor in the hepatopancreas.

Discussion The effects of cyclic nucleotide analogs on VtG Based on our recent finding that MIH acts as a vitellogen- transcriptions: hnVtG RNA and VtG mRNA esis stimulant in addition to its molt inhibitory function The incubation of hepatopancreas fragments at E2 with [12], we demonstrated in the current study the presence of the membrane permeable 8-Bromo-cAMP resulted in a specific binding sites for MIH in the J-YO membranes as 36% increase in the level of hnVtG RNA, while 8-Bromo- well as novel binding sites in hepatopancreatic mem- cGMP had no effect (Fig. 6). VtG mRNA in the hepatopan- branes of vitellogenic female C. sapidus. Kinetic studies creas at E2 decreased to 50% compared to the control with revealed a specific, saturable, and non-cooperative bind- 8-Bromo-cAMP, whereas it remained constant with 8- ing indicative of a receptor-ligand interaction in both tis- Bromo-cGMP (Fig. 6). sues, with differences in the affinity and density of the receptors between the two tissues. In addition, we have Molecular weights of putative MIH receptors in the shown by crosslinking [125I] MIH to its receptor in the YO membranes of the YO and hepatopancreas of vitellogenic and hepatopancreas that the receptors are proteins with female an estimated molecular weight of ~51 kDa. YO and hepatopancreas membranes that were preincu- bated with [125I] MIH or hepatopancreas with [125I] CHH The radioligand receptor assays revealed that the presence and crosslinked with disuccinimidyl suberate (DSS), were of MIH specific binding sites in the hepatopancreas is separated on SDS-PAGE and the signal was detected using found only in adult females and is vitellogenic stage- spe- a phosphorimager. Two bands were observed for each cific. Bound MIH in the hepatopancreas was specifically membrane: a lower one at the expected size of MIH displaced by recombinant MIH (rMIH), but not by CHH (9070.9 Da) or CHH (8478.1 Da) [55]; and, a second (Fig. 3B). Moreover, MIH binding capacity in the hepato- higher band at an estimated size of ~60 kDa for MIH (Figs. pancreas increased as ovarian stage advanced from 1 to 3,

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As shown in Fig. 4, KD values of MIH binding sites in the J-YO and female hepatopancreas membranes were signif- icantly different: 4.19 × 10-10 and 3.22 × 10-8 M/mg pro- tein, respectively. These values indicate that the affinity of the MIH receptor in the hepatopancreas is 77 times lower than that of the J-YO. Similar to what has been reported for C. maenas, CHH binding sites in the YO and hepato- pancreas membranes had calculated KD values of 1.82 × 10-9 and 6 × 10-10 M/mg protein, respectively [17]. These affinities were comparable to those obtained for MIH binding sites in the J-YO (4.19 × 10-10 M/mg protein), all in the 0.1 nM/mg protein range. Thus, it seems that MIH binding sites in the hepatopancreas have the lowest affin- ity to its ligand among those of CHH neuropeptides char- masshepatopancreasinFigureBinding the of hepatopancreas sites7~51 kDa,of [and125 whichI] juvenile MIH is indifferent theYO membraneshave from a similar that of of molecularfemale's [125I] CHH acterized thus far. The low affinity of the hepatopancreas Binding sites of [125I] MIH in the membranes of receptor may require high circulating titers of MIH, with female's hepatopancreas and juvenile YO have a sim- concentrations ranging from 0.01–20 nM for binding ilar molecular mass of ~51 kDa, which is different (Fig. 3A). The level of MIH at ovarian stage 3 was ~0.02 from that of [125I] CHH in the hepatopancreas. A) J- nM [12], which was slightly higher than that of intermolt YO (100 μg); B) female hepatopancreas at ovarian stage 3 (~0.015 nM). Taken together, MIH receptor in the hepat- (200 μg). Membranes were incubated without (total binding, opancreas is featured by low affinity-high binding capac- Tb) or with unlabeled MIH or CHH in an excess of 1 or 10 ity to its ligand (Fig. 4). The presence of different receptors pmol, specified at the top of the lane. YO membranes were for a ligand with different binding kinetics is well known resolved on a 4–15% SDS-PAGE and hepatopancreas mem- for steroid receptors such as glucocorticoid receptor [56], branes on a 10% SDS-PAGE. Labeled ligand and receptor complexes are indicated with arrows. peptides like gonadotropin-releasing hormone receptors [57], and polypeptide receptors such as prolactin receptor [58]. reaching ~40% of that of the J-YO binding, reflecting BMAX To determine whether the MIH signals through cGMP as values (4.8 × 10-9 and 9.24 × 10-9 M/mg protein of J-YO a second messenger in the hepatopancreas as it does in the and hepatopancreas, respectively). The ovarian stage YO [38], we measured in vitro production of cyclic nucle- dependent increase of MIH binding in the hepatopan- otides in hepatopancreas fragments in the presence of 2 creas may be responsible for the ovarian stage dependent nM MIH or 20 nM CHH, which are effective doses in vitel- MIH effect on vitellogenesis[12]. In contrast, CHH bind- logenesis [12]. As expected, intracellular cGMP levels in ing was ubiquitous in the tissues tested except for ovary, the hepatopancreas increased by 16 fold in response to as has been described in C. maenas [15]. Moreover, CHH CHH, but did not change with MIH treatment (Fig. 5A). binding in the hepatopancreas did not differ significantly On the other hand, cAMP levels significantly increased by between ovarian stages and sex (Fig. 2B). The BMAX value 50% with MIH, but did not change with CHH treatment for CHH binding in the hepatopancreas of C. sapidus was (Fig. 5B). The difference in the magnitude of response in 3.67 × 10-10 M/mg protein [18], similar to the value of the increase of cGMP (by CHH) compared to cAMP (by 3.28 × 10-10 M/mg protein obtained for hepatopancreas of MIH) may be due to different basal levels: cAMP, 20 C. maenas [15]. This suggests that at ovarian stage 3, the pmol/mg protein; cGMP, < 1 pmol/mg protein. Hepato- number of MIH receptors in the hepatopancreas is the pancreas fragments incubated with 8- Bromo-cAMP aug- highest ever reported in the binding studies of the family mented hnVtG RNA levels, while 8-Bromo-cGMP had no of CHH neuropeptides, being 30 times higher than that of effect (Fig. 6). These findings are congruent with our pre- the CHH receptor. This high density of MIH receptors in vious results of no effect of CHH and the stimulatory hepatopancreas may cause higher non-specific binding effect of MIH on hnVtG transcription at stage E2 [12]. than in the J-YO, as shown in Fig, 4B and Figs 7A and 7B. Moreover, the membrane permeable 8-Bromo-cAMP and When considering the difference in size of the tissues 8-Bromo-cGMP mimicked the effects of MIH and CHH, between the J-YO (~20 mg) and the hepatopancreas (~7 respectively, on VtG mRNA. VtG mRNA significantly g) in adult females, the total number of MIH binding sites decreased by 60% with MIH and 20% with CHH in hepat- can easily be ~700 times higher in the hepatopancreas opancreas fragments of ovarian stage E2 females[12]. than in the YO. The results obtained from the crosslinking studies showed that the complexes of MIH and its receptor in both the J-

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YO and the hepatopancreas have an estimated size of ~60 It is generally recognized that individual hormones can kDa, resulting in a MW of ~51 kDa for the receptor, which elicit several diverse responses in different organs and tis- is different from that of the CHH receptor in the hepato- sues, as well as in individual cells [14]. Several mecha- pancreas (MW ~61 kDa). Together with the second mes- nisms may be involved in mediating the different signals senger data, this suggests that there is a tissue specific type such as 1) pulses and micropulses (frequency of changes of membrane receptor for MIH, since only one band of hormone circulation or endogenous concentrations); appeared in the autoradiogram in addition to the 2) changes in the activities of hormone-converting unbound neuropeptides (Figs. 7A and 7B). A similar enzymes; 3) selective activation of each receptor type, sub- study estimated the size of the MIH receptor in the YO of type, and isoform; 4) changes in receptors, which may the kuruma prawn as ~70 kDa [16]. Such a difference may activate and suppress different signaling pathways (e.g. lie in the difference in species. phosphorylation, dimerization); and 5) activation and suppression of different nuclear transcription processes The increase in intracellular cAMP in the hepatopancreas [14]. The MIH receptor in the mature female's hepatopan- caused by MIH opposes the possibility that the receptor is creas is clearly different from the one present in the YO in a guanylate cyclase, and instead favors the option of a G terms of its time of appearance/functionality, its binding protein – coupled receptor (GPCR) that acts through a kinetics, and signal transduction. Thus, it fits some of the GTP binding protein to activate a nucleotide cyclase. This mechanisms mentioned above and provides a possible agrees with the calculated MW of the hepatopancreas model for multi-functionality of the CHH neuropeptide putative MIH receptor of ~51 kDa, which is different from family, which is based upon a diverse spatial and tempo- the typical membrane guanylate cyclase having a MW of ral expression, binding kinetics and signal transduction 120–140 kDa [59]. In addition, the similarity in the MWs pathway. Based on this finding, we propose that some of MIH binding sites on the YO and hepatopancreas indi- pleiotropicity of the CHH neuropeptide family might be cates that both MIH receptors may be GPCRs, however, mediated by multiple receptor forms: each form executes each form may activate a different signaling pathway. A a different function. possible scenario is that the hepatopancreas MIH receptor activates the αs subunit of the G protein-adenylyl cyclase, Conclusion while the YO isoform activates soluble guanylyl cyclase In this study, we demonstrated the presence of novel spe- through Ca2+and NO- initiated by an increase in cAMP. cific MIH binding sites in the hepatopancreas of the Support for the latter option is found in a study reporting mature female C. sapidus that are involved in MIH regula- that incubation of the YO of C. maenas and P. clarkii at tion of vitellogenesis. MIH action in the hepatopancreas is premolt stage with MIH resulted in a sustained 60 times probably mediated by cAMP, unlike the YO counterpart increase in cGMP levels, and also a short, transient two that utilizes cGMP as a second messenger. Our result sug- fold increase in cAMP [23,25]. In addition, it was sug- gests that in the female C. sapidus, the antagonism gested that nitric oxide synthase and NO- are possibly between molting and reproduction is mediated by MIH involved in the MIH signaling in the YO of the land crab, that acquires a vitellogenesis stimulating function in addi- Gecarcinus lateralis [20,60]. In this regard, the involvement tion to its traditional molt-inhibitory action. This new of trimeric G proteins in inhibition of protein synthesis in role is obtained through the expression of abundant MIH the YO of C. sapidus, was proposed [29]. Overall, the two specific receptors in the hepatopancreas of the adult suggested signaling pathways may involve a complex net- females. To our knowledge, this is the first description of work of interactions and will require more rigorous inves- an endocrine regulation mechanism of the antagonism tigations. between molting and reproduction in a crustacean spe- cies. As we proposed in Zmora et al. [12], this may occur It was speculated that an extensive gene duplication event in other crustaceans, particularly in those who share a may have occurred to produce multiple CHH isoforms similar life cycle with the female C. sapidus (i.e., a terminal which may show tissue specific expression [61]. Similarly, molt upon puberty). It will be interesting to further exam- receptors including the GPCR family members are ine how the two MIH receptors structurally differ and believed to propagate by gene duplication from a com- determine the specific timing and cues for the hepatopan- mon ancestor [62]. This includes odorant receptors [63], creas MIH receptor appearance or activation. hormone receptors like growth hormone [64], gonado- trophins [65], gonadotrophin releasing hormones [66] Methods and many more. Although the sequences of the MIH Animals receptors in the current study are still unknown, these two Juvenile blue crabs at intermolt stage (carapace width 5– receptors may possibly be products of gene duplication 7.5 cm) were collected by using a seine or a trot line from and share a high degree of structure similarity. the eastern shore of the Chesapeake Bay [67]. Mature females obtained from a local waterman were transferred

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in aerated water and acclimated for 2 to 3 days without binding assay buffer and pelleted by centrifugation at feeding in a 4.5 cubic meter re-circulating tank at ambient 14,000 rpm for 5 min. The pellets were counted in a conditions. Tissue collection was carried out as described gamma-counter (HP counter). Each assay was repeated [68]. The tissues were dissected, rinsed in ice-cold crusta- three or four times in triplicates. cean saline and stored at -80°C until further processing. The effects of MIH and CHH on the levels of cAMP and Purification and quantification of native CHH, native cGMP, in vitro MIH, and recombinant MIH The hepatopancreas of vitellogenic females at stage E2 was Neuropeptides of the sinus glands (SG) and rMIH were excised and washed in 10 volumes of ice-cold Medium purified using RP-HPLC as described [17]. Amino acid 199 (osmolarity 960 mmol/kg, 0.1 mg/ml BSA and 1× analyses were carried out for the quantification of the protease inhibitors cocktail for tissue culture (Sigma), pH purified native MIH, native CHH and rMIH using o- 7.4) for 3 h on ice, with three media changes as described phthalaldehyde pre-column derivatization method as [68]. Quantification of cAMP or cGMP in tissues using outlined [17]. RIAs followed the procedure as described [70]. In brief, fragments of 10 mg each were incubated for 1 h at room Radioligand binding assays temperature in the presence of 2 nM MIH or 20 nM CHH Preparation of membranes in 400 μl Medium199 medium containing 1 mM IBMX, Membranes were prepared from ~700 YO's collected from or only IBMX for the control. The tissues were then dis- juvenile C. sapidus (J-YO, 5–7.5 cm carapace width) at rupted by sonication (Branson) and centrifuged at 14,000 intermolt stage (C4) by following the method as rpm for 10 min at 4°C. Supernatants (100 μl) were trans- described [17]. Hepatopancreas membranes were pre- ferred to tubes containing 900 μl of 0.1 M acetate buffer pared from individual females or males, using ~1 g tissue. (pH 4.75) and immediately acetylated by the addition of Each hepatopancreas or 700 pooled YO were homoge- 20 μl triethylamine (Sigma) and 10 μl acetic anhydride nized in 20 ml ice-cold homogenization buffer (140 mM (Sigma). Fifty or one hundred μl of acetylated samples NaCl, 300 mM sucrose, 10 mM HEPES, and 10 mM ben- were subjected to cAMP and cGMP RIAs. The standards for zamidine (Sigma), pH 7.4) using an Ultra Torax homoge- cAMP and cGMP in the range of 1–1000 fmol/tube were nizer. After an initial centrifugation at 1000 g for 5 min at treated the same as the samples. The final dilution of anti- 4°C, the supernatant was pelleted by centrifugation at bodies was 1: 21,000 for cGMP and 1:4000 for cAMP. 30,000 g for 30 min at 4°C. The pellet was washed in washing buffer (140 mM NaCl and 10 mM HEPES, pH 7.4 2'-O-methyl ester cAMP or cGMP (0.3 nmol) (Sigma) without BSA) by repeating the previous centrifugation were iodinated with Na [125I] (Amersham) using the chlo- step for 15 min and resuspending in the same buffer. Pro- ramine T method as described [70]. The iodinated mate- 125 tein concentration was determined using a DC protein rial was separated from free [ I] on a C18 Sep-Pak quantification kit (BioRad) and the membranes were aliq- cartridge (Waters) by elution with 40% isopropanol [71]. uoted and stored at -80°C until further use. The calculated specific activities were approximately 500– 600 Ci/mmol. MIH and CHH binding studies Binding assays The effects of membrane permeable analogues of cAMP Native [125I] MIH and native [125I] CHH were prepared and cGMP on vitellogenin gene expression, in vitro using the chloramine-T labeling method as described [17] Hepatopancreas tissue was washed as described above or using 1,3,4,6-tetrachloro-3alpha, 6alpha-diphenylglu- and fragments of 10 mg each were incubated in 400 μl coluril (iodogen) coated tubes (Pierce) according to the Medium199 medium containing 10 μM 8-Bromo-cAMP manufacturer's instruction. [125I] MIH or [125I] CHH was or 8-Bromo-cGMP (Alexis) for 1 or 6 h. RNA was extracted separated from free [125I] on a PD 10 column (GE Health- and the levels of hnVtG RNA and VtG mRNA were deter- care) as described [17]. Specific activities were approxi- mined using quantitative PCR analysis (QPCR) analysis as mately 300–500 Ci/mmol. described in Zmora et al. (companion paper 1).

The MIH binding procedure was followed as stated [69]. Crosslinking [125I] MIH or [125I] CHH to their binding sites Briefly, membranes were incubated in binding assay and visualization buffer (washing buffer containing 1% BSA) with [125I] Juvenile YO membranes (100 μg) were incubated with 0.4 MIH or [125I] CHH for 1 h at room temperature. For dis- pmol [125I] MIH (~263,000 DPM) for total binding or placement and non-specific binding, unlabeled native with additional 1 or 10 pmol/100 μl unlabeled rMIH for CHH or recombinant MIH (rMIH) produced in S2 Dro- nonspecific binding. The membranes were then washed sophila cells (Zmora et al. companion paper 1) was added twice with 1 ml ice-cold washing buffer by centrifugation to the reaction. The membranes were then washed in at 14,000 rpm for 10 min. The pellets were resuspended

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in 100 μl buffer containing DSS (Pierce) at a final concen- Authors' contributions tration of 1 mM for 30 min at room temperature. The reac- NZ carried out the concept, experimental design, and tion was then quenched by the addition of 15 μl of 1 M acquisition, analyses, and interpretation of data, and Tris buffer (pH 7.4) for 15 min at room temperature and drafted and revised the manuscript including tables and centrifuged at 14,000 rpm for 5 min. The pelleted mem- figures. CJS was involved in the acquisition of funding, branes were resuspended in 1× SDS sample loading buffer contributed to concept, experimental design, analyses, (Bio-Rad) and denatured for 5 min at 100°C. Proteins and interpretation of data, and revised the manuscript. AS were separated on a 4–15% SDS-PAGE and briefly stained participated in the discussions and revision of the manu- with Bio-Safe coomassie (Bio-Rad) for visualization. The script. YZ was involved in the acquisition of funding and gel was then dried and exposed to a Phosphor-imager contributed in discussions. screen for 2 h at room temperature and analyzed using a Typhoon 9410 variable mode imager (Molecular Dynam- All authors read and approved the final manuscript. ics). Acknowledgements As for hepatopancreas membranes, the conditions This research was supported by the National Oceanic and Atmospheric described above were applied except that 200 μg of the Administration (NOAA), Chesapeake Bay Program Grant (NA17FU2841) hepatopancreas membranes were incubated with 1 pmol to the Blue Crab Advanced Research Consortium. We would like to thank [125I] MIH (~657,000 DPM) with or without 10 pmol Mr. Mark Saltis for his help and dedication in catching juvenile crabs. We are indebted to Professor J. deVante (Maastricht University) for his gener- 125 cold MIH or 0.12 pmol [ I] CHH (182,500 DPM) and ous gifts of cAMP and cGMP antisera. This is the Center of Marine Biotech- 10 pmol cold CHH for non-specific binding. After nology's contribution number 08-182. crosslinking with 5 mM DSS, membranes were lysed by adding 4% Triton X-100 for 10 min in binding buffer, fol- References lowed by dilution to 1% Triton X-100. The sample was 1. Chan SM, Gu PL, Chu KH, Tobe SS: Crustacean neuropeptide then immunoprecipitated with 4 μl MIH antiserum for 1 genes of the CHH/MIH/GIH family: implications from molec- ular studies. Gen Comp Endocrinol 2003, 134:214-219. h at 4°C, followed by the addition of protein-A magnetic 2. De Klein DPV, Van Herp F: Molecular biology of neurohormone beads (New England Biolabs) to the mixture and incu- precursors in the eyestalk of crustacea. Comp Biochem Physiol B Biochem Mol Biol. 1995, 112(4):573-579. bated for an additional hour. Bound proteins were sepa- 3. Keller R: Crustacean neuropeptides: structure, functions and rated via a magnet apparatus and eluted with 3× SDS comparative aspects. 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Research Open Access Molt-inhibiting hormone stimulates vitellogenesis at advanced ovarian developmental stages in the female blue crab, Callinectes sapidus 1: an ovarian stage dependent involvement Nilli Zmora, John Trant, Yonathan Zohar and J Sook Chung*

Address: Center of Marine Biotechnology, University of Maryland Biotechnology Institute, 701 E. Pratt St. Columbus Center Suite 236, Baltimore, MD 21202, USA Email: Nilli Zmora - [email protected]; John Trant - [email protected]; Yonathan Zohar - [email protected]; J Sook Chung* - [email protected] * Corresponding author

Published: 7 July 2009 Received: 9 March 2009 Accepted: 7 July 2009 Saline Systems 2009, 5:7 doi:10.1186/1746-1448-5-7 This article is available from: http://www.salinesystems.org/content/5/1/7 © 2009 Zmora et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract To understand the hormonal coordination of the antagonism between molting and reproduction in crustaceans, the terminally anecdysial mature female Callinectes sapidus was used as a model. The regulatory roles of crustacean hyperglycemic hormone (CHH) and molt-inhibiting hormone (MIH) in vitellogenesis were examined. A competitive specific RIA was used to measure the levels of MIH and CHH in the hemolymphs of mature females at pre- and mid- vitellogenic stages, and their effects on vitellogenesis at early (early 2, E2) and mid vitellogenesis (3) stages were determined in vitro. A hepatopancreas fragments incubation system was developed and the levels of vitellogenin (VtG), as well as VtG mRNA and heterogeneous nuclear (hn)VtG RNA were determined using RIA or QPCR, respectively. MIH titers were four times higher at mid-vitellogenesis than at pre- vitellogenesis, while CHH levels in the hemolymph were constant. In the in vitro incubation experiments, MIH increased both VtG mRNA levels and secretion at ovarian stage 3. At stage E2, however, MIH resulted in a mixed response: downregulation of VtG mRNA and upregulation of hnVtG RNA. CHH had no effect on any of the parameters. Actinomycin D blocked the stimulatory effects of MIH in stage 3 animals on VtG mRNA and VtG, while cycloheximide attenuated only VtG levels, confirming the MIH stimulatory effect at this stage. MIH is a key endocrine regulator in the coordination of molting and reproduction in the mature female C. sapidus, which simultaneously inhibits molt and stimulates vitellogenesis.

Background only in the juvenile phase and ovarian development and It has been proposed in decapod crustaceans that the high spawning cycles occurring only in adulthood. energy demanding processes of molting and reproduction are mutually antagonistic and do not occur simultane- Ovarian development is a major reproductive process, ously [1]. The antagonism is clearly demonstrated in the during which oocytes grow as a result of vitellogenin pro- female Callinectes sapidus: molting and reproduction occur duction and its accumulation in the form of yolk protein in two distinctive life phases with molting cycles occurring (vitellin) and other cytoplasmic egg proteins in the

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oocytes [2]. In C. sapidus, vitellogenesis occurs primarily tion of ecdysteroids from the YO's by down-regulating in the hepatopancreas after terminal/pubertal molt and protein synthesis [21,22] which in turn leads to low titers requires a relatively short duration of 8–12 weeks for com- of ecdysteroids in the hemolymph [7,23-25]. Reproduc- pletion [3]. tion on the other hand is regulated both negatively and positively by CHH family neuropeptides. The inhibition The phenomenon of reproductive phase accompanied by is exerted by VIH in the eyestalk ganglia [14,26,27], and terminal anecdysis (halt of molting cycles) is found in its presence has been described in a few lobster species only a few decapod crustaceans, including Maja squinado, [28-31], Litopenaeus vannamei [32], and the isopod Arma- Libinia emarginata, and Chionoecetes bairdi [4-7]. Most of dillidium vulgare [33]. Vitellogenesis-stimulating hormone these anecdysial animals are characterized by a degener- (VSH), identified as an isoform of MIH in the sand ated molting gland, the Y-organ (YO), and the resulting shrimp, Metapenaeus ensis [34], positively regulates vitell- low levels of molting hormones (ecdysteroids) in the ogenesis. In addition to the XOSG, it is expressed in the hemolymph [8]. However, the adult C. sapidus female is ventral nerve cord, thoracic ganglia, and brain. This find- distinguished from these animals by having non-degener- ing implies that a MIH like neuropeptide may be involved ated YO's, which retain their normal size and their capa- in the regulation of reproduction. bility to bind and respond to molt-inhibiting hormone (MIH) by elevation of cyclic GMP (cGMP). Furthermore, Considering the roles of CHH and MIH in molting and eyestalk ablation (the removal of the source for MIH) MIH isoform action as a VSH, we aimed to understand induces molting in adult C. sapidus females [9], indicating how the antagonism between molting and reproduction that the YO's also preserve their steroidogenic potential. is hormonally coordinated in the terminally anecdysial Taken together, unlike other anecdysial crustacean spe- female C. sapidus. Our earlier study showed that like other cies, molting cycle in the mature female C. sapidus is tem- crab species, only one form of MIH is identified in the porarily arrested at intermolt stage, in synchrony with the XOSG of C. sapidus [35] and MIH mRNA [36]. In addition, reproductive phase. Since to date the neuroendocrine we found no evidence of a MIH, MIH-like neuropep- mechanism underlying these antagonistic processes has tide(s) or its mRNA in the thoracic ganglia or brain of the not been examined in any crustacean species, the mature adult female (unpublished data). We therefore hypothe- C. sapidus female with its distinctive separate molting and sized that XOSG derived CHH or MIH actively inhibit reproductive phases, serves as an excellent model species molting in the female C. sapidus, while allowing reproduc- to address the issue. tive processes to take place. Specifically, we questioned if MIH or CHH have a role in the regulation of vitellogenesis Molting and reproduction are regulated by the eyestalk of C. sapidus. To address these questions, we first deter- derived crustacean hyperglycemic hormone (CHH) fam- mined the hemolymph circulating concentrations of MIH ily [10,11] including CHH, MIH, mandibular organ- and CHH and their mRNA levels in the XO of mature inhibiting hormone (MOIH) [12], and gonad/vitellogen- female C. sapidus. We also carried out in vitro studies on esis-inhibiting hormone (G/VIH) [13,14]. These neu- hepatopancreas tissue fragments to identify the regulatory ropeptides are synthesized in specific neurosecretory cells roles of MIH and CHH in vitellogenesis, specifically at the of the medullar terminalis X-organ (XO), stored, and levels of VtG mRNA, VtG protein and heterogeneous nuclear released from the sinus gland (SG) in the eyestalk ganglia (hn) VtG RNA. Measuring hnVtG RNA provided an addi- (XOSG complex). The names of these neuropeptides usu- tional tool to examine transcription. We report that MIH ally refer to their primary functions or structural affiliation has a regulatory role in vitellogenesis in the female C. sap- to either CHH or MIH subfamilies. idus, while CHH seems to have no clear involvement. Spe- cifically, MIH upregulated VtG mRNA, hnVtG RNA and CHH and MIH exist in different combinations and VtG secretion in heptopancreas fragments in vitro in the number of isoforms depending on the species. Multiple advanced vitellogenic stage. Interestingly, MIH downreg- isoforms are found in the XOSG of penaeids, crayfish, and ulated VtG mRNA, upregulated hnVtG RNA and had no lobsters [15], whereas in crab species only two isoforms of effect on VtG secretion in the early vitellogenic stage. CHH and one of MIH are detected [16-19]. Moreover, CHH neuropeptides are characterized by pleiotropicity Results and are usually involved in the regulation of several phys- MIH and CHH titers in the hemolymph of reproductive iological processes including molting, reproduction, females osmoregulation, energy metabolism, and stress response Neuropeptides in the hemolymph were determined by [20]. specific radioimmunoassay (RIA) in 10 females at ovarian stages 1 and 3, representing pre-vitellogenic and mid- Molting is controlled by CHH and MIH. High circulating vitellogenic stages, respectively. The results showed a sig- levels of these neuropeptides suppress synthesis and secre- nificant four- fold increase in MIH levels at ovarian stage

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3 (19.6 fmol/ml) compared to stage 1 (5.7 fmol/ml) (Fig. as the optimal incubation time and dose of neuropeptides 1Aa). CHH levels did not differ between the stages and for hepatopancreas fragments in vitro. The stability of RNA ranged between 130 and 150 ± 49 fmol/ml which are ~6 was tested by visualization of the total RNA, determined – 20 times higher than those of MIH (Fig. 1Ab). MIH and by the integrity of 18 S and 28 S ribosomal(r) RNA subu- CHH mRNA levels in the XO of mature females at the nits of the hepatopancreas extracted immediately after same stages did not significantly differ and were 1500– dissection (t = 0 point) and after a 6 h incubation. The 2500 copies/20 ng total RNA for MIH and 15000–40000 results presented in additional file 1 show that 18 S and copies/20 ng total RNA for CHH (Figs. 1Ba and 1Bb). 28 S rRNA bands remained stable and similar to that of t Accordingly, the concentrations of ecdysteroids in the = 0 point after the 6 h incubation. The levels of VtG mRNA hemolymph of these females were low at ovarian stages 1 were further tested by quantitative PCR (QPCR) analysis and 3: 7 to 5 ng/ml, respectively (not shown). on hepatopancreas fragments incubated in plain media. Levels were found to be constant during the incubation In vitro incubation of hepatopancreas fragments period (Fig. 2A). The effects of sinus gland neuropeptides (CHH and MIH) on VtG expression and secretion The effect of different doses of CHH or MIH on VtG To establish an in vitro bioassay, a preliminary experiment mRNA was determined by incubating hepatopancreas was conducted to determine the effect of incubation fragments of stage (early 2) E2 animals for 1 or 6 h. At 1 h period on the stability of total RNA and VtG mRNA as well incubation, MIH at 0.05, 0.2 and 2 nM concentrations

A

B

MIHFigure and 1 CHH levels in the hemolymph and mRNAs in the XO of pre- and mid-vitellogenic females (ovarian stage 1 and 3) MIH and CHH levels in the hemolymph and mRNAs in the XO of pre- and mid-vitellogenic females (ovarian stage 1 and 3). A) hemolymph titers and B) mRNA a) MIH, b) CHH. MIH and CHH neuropeptides in the hemolymph were extracted with 40% isopropanol for the extraction of CHH and 60% isopropanol for the extraction of MIH followed by a spe- cific RIA in 10 females at each stage 1 or 3. MIH and CHH mRNA levels were determined using QPCR in 17 females at stage 1 and 21 females at stage 3 and are presented as copies/20 ng total RNA. Results are presented as mean ± SEM (N = 10). **, = P ≤ 0.01.

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tively (Fig. 2B). CHH at 0.5, 5, and 20 nM showed no dif- A 120 ference from control on VtG mRNA levels at both 1 and 6 h incubation (Fig. 2C). Based on these results, the subse- 80 quent in vitro experiments were carried out at 6 h incuba- tion, unless specified otherwise. 40 % change % The effects of CHH and MIH on vitellogenesis were fur- 0 ther examined on hepatopancreas of females at early and 0420 mid-vitellogenic stages (E2 and 3, respectively). CHH (20 Incubation time (h) nM) significantly reduced VtG mRNA levels by 20% (N = B 21) of control at E2 and had no effect at stage 3 (N = 6) 150 (Fig. 3Ab). MIH however, revealed a stage dependent response. MIH (2 nM) reduced VtG mRNA levels in the hepatopancreas of females at E2 by 60% (N = 21), but 100 caused an increase at stage 3 (N = 6) (Fig. 3Aa). The effect of MIH on VtG secretion from hepatopancreas fragments 50 was stage dependent: at E2, no change was detected (N = 6), while at stage 3, MIH increased the levels of VtG in the mRNA (% (% of control) mRNA media to 204 ± 34% over the control (N = 5) (Fig. 3Ba).

VtG 0 CHH had no significant effect on VtG secretion at E2 and 0 0.05 0.2 2 5 3 stages (N = 5)(Fig. 3Bb). C MIH (nM) To clarify the results obtained for stage E2, the effect of 150 CHH and MIH on VtG transcription was also tested on de novo synthesis of VtG mRNA by measuring heterogeneous nuclear RNA of VtG (hnVtG RNA). The levels of hnVtG 100 RNA increased to 270% over the control with MIH, whereas CHH had no effect after a 1 h incubation. This 50 effect disappeared at the 2 h incubation period (N = 3) (Fig. 4A). Since a difference in hnVtG RNA was only meas- mRNA (% (% of control) mRNA urable at the 1 h but not 2 h incubation, experiments test-

VtG 0 ing hnVtG RNA were set for 1 h. In a larger scale set of 0 0.5 5 20 experiments with a 1 h incubation, MIH caused a ~2.5 CHH (nM) fold increase (256 ± 86%) in hnVtG RNA levels in the hepatopancreas of females at E2, while CHH had no effect EvaluationexperimentFigure 2 and optimization of the hepatopancreas incubation (N = 11) (Fig. 4B). Evaluation and optimization of the hepatopancreas incubation experiment. A) Incubation period had no The effects of co-incubation of actinomycin D and cycloheximide with effect on the stability of VtG mRNA in hepatopancreas frag- MIH on VtG expression and VtG production ments of females at early ovarian stage 2 (E2). Bars, normal- To test whether the changes in VtG secretion by MIH is ized VtG mRNA; dashed line, total RNA. MIH had a more associated with its impact on VtG mRNA, hepatopancreas pronounced decreasing effect on VtG mRNA levels than fragments of females at ovarian stage 3 were co-incubated CHH (0 – 20 nM) in hepatopancreas fragments of females at E2 incubated for 1 and 6 h, B) Incubation with 0 to 5 nM MIH with 2 nM MIH and a transcription inhibitor (actinomy- and C) Incubation with 0 to 20 nM CHH. circles, 1 h incuba- cin D) or a translation inhibitor (cycloheximide). These tion; squares, 6 h incubation. Data are presented as mean ± inhibitors were tested at two different concentrations: 0.5 SEM (N = 3). and 10 μM. MIH increased VtG levels by 51% compared to the control and MIH + 0.1% v/v EtOH increased by 45% (N = 5). Actinomycin D at 0.5 and 10 μM, in the had no effect, however 5 nM resulted in a non-statistically presence of MIH, reduced VtG levels to 60% and 49% of significant decrease of 25% of control in VtG mRNA. Dur- MIH treatment, respectively (Fig. 5A), which were similar ing the 6 h incubation, the effect was more pronounced, to the media control. MIH + 10 μM AD decreased VtG although still statistically insignificant due to the low N mRNA to 51% of MIH treatment (Fig. 5B). number (N = 3). MIH at 0.2, 2, and 5 nM showed a trend of dose dependent effect, where VtG mRNA levels Cycloheximide at 0.5 and 10 μM co-incubated with MIH dropped to 62%, 62%, and 51% of the control, respec- (+ EtOH), reduced VtG levels to ~35% of MIH (+ EtOH)

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MIHstageFigure stimulates early 3 2 (E2) VtG while expression CHH shows and secretion no significant in hepatopancreas effect of females at ovarian stage 3 and downregulates VtG mRNA at MIH stimulates VtG expression and secretion in hepatopancreas of females at ovarian stage 3 and downregu- lates VtG mRNA at stage early 2 (E2) while CHH shows no significant effect. A) VtG mRNA and B) VtG in the medium. a) 2 nM MIH treatment and b) 20 nM CHH treatment. The effects on VtG mRNA and protein secretion were tested in females at E2 (N = 21 and N = 6, respectively) and stage 3 (N = 6 and N = 5, respectively). Results are presented as mean ± SEM of % of control. * = P ≤ 0.05; *** = P ≤ 0.001. and ~54% of the media control, but had no effect on VtG three major neuropeptides in the SG. CPRP fraction at 5% mRNA levels (Fig. 6A and 6B). HnVtG RNA was not meas- and 25% SG equivalents and purified native CPRP at 25 ured in this particular set of experiments as a different nM showed no significant effect on VtG expression experimental setup is required (i.e., 1 h incubation time, (Fig. 7). see Fig. 4A). Discussion The effect of other sinus gland factors on VtG mRNA levels The present study aimed to gain a better understanding of The pooled HPLC fractions of sinus-glands (SG) from how XOSG derived neuropeptides regulate the antago- stage 3 vitellogenic females without CHH, MIH, and crus- nism between vitellogenesis and molting in the terminally tacean hyperglycemic hormone precursor related- peptide anecdysial mature female C. sapidus. We determined the (CPRP) were tested using an in vitro hepatopancreas incu- titers of CHH and MIH in the hemolymph of mature bation to determine whether other sinus gland-derived females at different vitellogenic stages and tested their substances had an effect on VtG expression. No effect was effect on vitellogenesis, in vitro. Hemolymph titers of found with 5% and 25% of SG equivalent (minus CHH, CHH in these females were comparable to those meas- MIH, and CPRP) on VtG expression in the hepatopancreas ured in juvenile animals [35] and remained unchanged at fragments (Fig. 7). CPRP was also tested, as it is one of the ovarian stages 1 and 3. However, the detection of MIH in

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MIHovarianFigure upregulates stage4 E2 afterhnVtG 1 RNA h incubation in hepatopancreas of females at MIH upregulates hnVtG RNA in hepatopancreas of females at ovarian stage E2 after 1 h incubation. A) Changes in hnVtG RNA were measured after 1 h or 2 h incu- Co-incubationincreasecreasFigure fragments 5 of VtG of proteinof MIH ovarian and (A) actinomycinstage and VtG3 females mRNA D (AD) (B) inblocks hepatopan- the bation with either 20 nM CHH (black bars) or 2 nM MIH Co-incubation of MIH and actinomycin D (AD) (grey bars) and are measurable only at 1 h incubation period blocks the increase of VtG protein (A) and VtG (N = 3). B) hnVtG RNA change in response to 20 nM CHH or mRNA (B) in hepatopancreas fragments of ovarian 2 nM MIH (N = 11). Results are presented as mean ± SEM of stage 3 females. The concentration of MIH was 2 nM and % of control. * = P ≤ 0.05; ** = P ≤ 0.01. those of actinomycin D were 0.5 and 10 μM. Results are pre- sented as mean ± SEM of % of control (N = 5). The alphabet- ical letters show the significant differences at P < 0.05. the hemolymph of mature females was initially surprising considering they ceased molt. Hemolymph MIH levels at mid-vitellogenic ovarian stage 3 were four times higher VtG mRNA levels, and VtG translation, determined by than those of stage 1 (Fig. 1A), implying a putative role for changes in VtG secretion as this protein does not accumu- MIH in vitellogenesis. MIH and CHH mRNA levels were late in the hepatopancreas but is being secreted immedi- statistically indifferent in both ovarian stages, with MIH ately following translation [3]. CHH had no consistent exhibiting high variation within each group (Fig. 1B). effects on vitellogenesis: it decreased VtG mRNA by 20% These results indicate that the expression level of these only at stage E2 and had no effect on VtG secretion at both neuropeptides is not correlated with their secretion from E2 and 3 stages (Figs. 3Ab and 3Bb). However, with MIH the SG, as shown in other studies [22,36]. treatment, both VtG mRNA and VtG protein levels exhib- ited different responses at the two ovarian stages tested. The in vitro hepatopancreas fragments incubation system While VtG mRNA decreased at stage E2, it slightly was used to further determine whether circulating CHH increased at stage 3 in response to MIH (Fig. 3Aa). VtG and MIH play a regulatory role in vitellogenesis in C. sap- secretion did not differ from control treatment at stage E2 idus [5,27,28,32,34,37,38]. Their effect was tested at the but increased at stage 3 (Fig. 3Ba). These results imply that levels of VtG gene transcription by measuring changes in MIH may have an inhibitory role at early ovarian stages,

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inFigureSinuscaused vitro gland no 7 change extract in (SGE) VtG mRNA without in thehepatopancreas known neuropeptides of stage 3 Sinus gland extract (SGE) without the known neu- ropeptides caused no change in VtG mRNA in hepat- opancreas of stage 3 in vitro. Hepatopancreas fragments were incubated with sinus gland extract from which CPRP, CHH, and MIH were removed (SGE(-)) at 25% and 5% sinus gland equivalent; CPRP HPLC fraction (CPRP F) at 25% and 5% sinus gland equivalent; and at 25 nM CPRP. Control level is indicated by a dashed line and data are presented as mean ± SEM (N = 5).

(B)FigureCo-incubationof MIH on6 VtG of protein MIH and (A) cycloh and hadeximide no effect reversed on VtG the mRNA effect Stimulation of vitellogenesis by SG-MIH has not been Co-incubation of MIH and cycloheximide reversed reported in decapod crustaceans, with the exception of the effect of MIH on VtG protein (A) and had no MIH-B in M. ensis [34]. To verify the stimulatory effect of effect on VtG mRNA (B). The concentration of MIH was 2 MIH on vitellogenesis at ovarian stage 3, actinomycin D nM and those of cycloheximide were 0.5 and 10 μM. Results (a general inhibitor of eukaryotic transcription) and are presented as mean ± SEM of % of control (N = 5). The cycloheximide (a general inhibitor of eukaryotic transla- alphabetical letters show the significant differences at P < tion), were separately co-incubated with MIH. The addi- 0.05. tion of actinomycin D and cycloheximide to MIH treatment resulted in a decrease to 60% and 35% of VtG and a stimulatory role at mid ovarian stages. To determine levels, respectively, compared to MIH alone (equivalent to whether MIH indeed downregulates VtG gene transcrip- 95% and 54% of control levels) (Figs. 5 and 6). Actinomy- tion at stage E2, we further tested the activation of VtG cin D co-incubated with MIH, decreased VtG mRNA levels gene by measuring the short-lived hnVtG (= pre VtG by 49%, indicating that the respective decrease in VtG lev- mRNA). The increase of hnVtG RNA levels in response to els is likely to be a result of the reduced VtG transcription MIH at this stage indicated that the VtG gene is activated (Fig. 5A). The decrease of VtG mRNA with MIH/actinomy- and thus its transcription is being enhanced (Fig. 4). These cin D to control levels suggests pre-existing VtG mRNA in results were corroborated by a different set of experiments the hepatopancreas fragments. Furthermore, cyclohex- in which incubation of hepatopancreas fragments with imide probably blocked the translation of the newly MIH membrane permeable cyclic nucleotides at stage E2 induced VtG mRNA as well as the pre-existing VtG mRNA, resulted in the same response [39]. The reason for the con- resulting in lower levels of VtG protein compared to the tradicting response of VtG mRNA and hnVtG RNA is control. In addition, the unchanged levels of VtG mRNA unclear and will require further study. with MIH/cycloheximide imply that the transcription

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ropeptide in non-XOSG tissues of the female C. sapidus, suggests that the vitellogenesis-stimulatory role is carried out by the SG-MIH. To our knowledge, the current study is the second description of the effect of MIH neuropep- tide on VtG mRNA in the hepatopancreas of a mature vitellogenic female crustacean.

In light of the mutual antagonism of molt and reproduc- tion, the coordinated control of these two processes in crustacean species that continue to molt after puberty may require a complex regulatory system to allow both events [1]. The control may be exerted by multiple CHH and MIH isoforms, which indeed are found in non- crab spe- cies such as penaeids, crayfish, and lobsters [15]. It also can be controlled by pleiotropic neuropeptides in species C.cityFigure sapidus to MIH 8 MIH and antibody rMIH and demonstrates does not cross high react and equalwith CHH specifi- carrying only a few neuropeptide forms, such as C. maenas C. sapidus MIH antibody demonstrates high and equal [17,18] and Necora (unpublished observation) that do specificity to MIH and rMIH and does not cross react molt during their adulthood. Low ecdysteroid circulating with CHH. Standard curves of MIH, rMIH and CHH with levels were also observed in another anecdysial crab, the MIH antibody. Neuropeptides at concentrations ranging from C. opillio [42], suggesting that terminal anecdysis is associ- 0.25 pM to 1 nM were applied to RIA using anti-MIH raised ated with remarkably low ecdysteroids levels (lower than against rMIH produced in Drosophila S2 cells at 1:20,000 final intermolt juveniles levels) in the hemolymph. Neverthe- dilution. Close circles- nMIH, open circles- rMIH, triangles- less, the two groups (terminally anecdysial or those that CHH. Inset- Parallelism of nMIH and diluted hemolymph molt during their adulthood) may differ only in the dura- tested by MIH RIA and shown in linear regressions. Close tion of the intermolt stage, i.e., extended intermolt period circles- nMIH, open circles- hemolymph. in species like the female C. sapidus. Altogether, based on the low ecdysteroid level in the hemolymph compared to high MIH and CHH levels, and the potentially functional machinery was not affected. Overall, these results demon- YO, we conclude that the activity of the YO is suppressed strate that MIH stimulation occurs at transcription and by MIH in the mature female C. sapidus. Furthermore, our translation of VtG at ovarian stage 3. results suggest that the SG of adult female C. sapidus does not contain another vitellogenic regulatory factor(s) Support evidence for MIH regulation in vitellogenesis is (Fig. 7). provided by the appearance of specific binding sites for MIH in the hepatopancreas of mature female C. sapidus. Conclusion The abundance of the binding site increased gradually The current study tested the possible regulatory roles of with ovarian development and was higher at stage 3 than SG-MIH, CHH and CPRP in vitellogenesis, in vitro. The at stages 1 and 2 [39]. results show that MIH regulates vitellogenesis in a vitello- genic stage dependent manner: MIH is stimulatory at the It is tacitly accepted that V/GIH inhibits vitellogenesis, levels of both transcription and translation of VtG at mid- while vitellogenin/gonad-stimulating hormone (V/GSH) vitellogenesis but its effect seems different at early stages. enhances vitellogenesis processes. However, CHHs were In light of the role of MIH in vitellogenesis, we infer that shown to inhibit general mRNA synthesis in the ovaries of MIH may play a dual role in females of crab species that vitellogenic female Penaeus semisulcatus [40]. The few exhibit a terminal anecdysis upon sexual maturity in their cases reporting the decrease of VtG mRNA levels by V/GIH life cycle: maintaining intermolt, while regulating vitello- and CHH have been shown in ovaries of immature genesis. penaeid females [28,32,41]. Although these studies were conducted on ovarian tissue and not hepatopancreas, they Methods support our finding of decreased VtG mRNA levels and Animals indicate that this effect may be more common at early Female C. sapidus were captured in the Rhode River (a trib- stages of ovarian development. utary of the Chesapeake Bay, USA) transported in aerated water, and held in recirculating 4.5 cubic meter tanks at The observation that MIH positively regulates vitellogene- environmental conditions matching the Bay for 2–5 days sis at stage 3 is concordant with a previous report in which before dissection. Ovarian development was staged based the non SG-MIH isoform stimulated vitellogenesis in the on ovarian weight and gamete size according to criteria shrimp M. ensis [34]. The lack of MIH or MIH-like neu- established previously [43]. Stage early 2 (E2) represents

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the beginning of vitellogenesis where oocyte size is small Quantitative PCR (QPCR) analysis of VtG in the and similar to those at stage 1, but vitellin (yellow in hepatopancreas and VtG ELISA color) starts to accumulate within the oocyte. This stage is Total RNA was extracted using the RNeasy mini kit (Qiagen). the earliest point of detecting both VtG mRNA and VtG cDNA preparation and quantitative PCR (QPCR) analysis protein [3]. were conducted using the procedure described for the meas- urement of hepatopancreas VtG mRNA and with normaliza- Batch purification and quantification of native CHH, tion against arginine kinase [3]. HnVtG RNA was measured native MIH, and recombinant MIH by QPCR analysis using a pair of primers located in intron #1 Neuropeptides of sinus glands (SG) were purified using of the vitellogenin gene based on a 1539 bp partial sequence RP-HPLC as described [44]. Amino acid analyses were car- amplified from genomic DNA (GenBank accession number ried out for the quantification of the purified native MIH, EU293808): Forward primer – native CHH, and recombinant MIH (rMIH) using the o- 5'GTTCCCTGCCTGGCTTCA3'; Reverse primer – phthalaldehyde pre-column derivation method as 5'CGGCTGTCGAGGTGATTATGA3'. This partial VtG gene described [16]. served as a template for the cRNA used in the standard curve. To avoid genomic DNA amplification, total RNA was treated CHH and MIH concentrations in the hemolymph with 1 unit/1 μg RQ1 DNase (Promega) prior to reverse tran- Hemolymph samples were collected and frozen at -20°C scription, and the results obtained from cDNA amplification until processed. Neuropeptides were extracted from 1 ml were corrected by subtracting the levels of non-reverse tran- hemolymph by adding isopropanol to 40% (v/v) for scribed RNA amplification (no-RT control). CHH extraction followed by centrifugation at 2000 rpm for 30 min at 4°C. The resulting pellet was re-extracted Vitellogenin levels in the incubation medium were deter- with 60% isopropanol (for MIH extraction) and pelleted mined by a competitive VtG ELISA. The media proteins as before. Supernatants were pooled and dried under vac- were precipitated with 60% ammonium sulfate prior to uum (SpeedVac, Jouan) before being analyzed by RIAs as the ELISA procedure as described [3]. When VtG levels described [45]. The antiserum used in MIH RIA was gen- were measured in the tissues and media, both were erated in rabbits (Proteintech Group Inc.) against recom- homogenized together in a "tissuelyser" (Qiagen) before binant MIH with a His Tag at its C terminus, produced in ammonium sulphate precipitation. an S2 Drosophila cell expression system. Both native [125I] MIH and [125I] CHH were prepared using a chloramine-T Incubation in vitro of hepatopancreas fragments with labeling method. [125I] MIH or [125I] CHH was separated neuropeptides, actinomycin D or cycloheximide from free [125I] on a PD 10 column (GE Healthcare) as Hepatopancreas tissue was removed from vitellogenic described [16]. Specific activities were approximately females and washed in 10 volumes of ice-cold Medium- 300–500 Ci/mmol. A standard curve of MIH RIA ranging 199 medium (Sigma) (adjusted with NaCl to osmolarity from 0.1–1000 fmol/tube was prepared with native MIH of 960 mmol/kg), with gentle agitation on ice for 2–3 h, and a final dilution of MIH antiserum at 1:20,000 (Fig. 8). and 3 medium exchanges. Hepatopancreas fragments The sensitivity of typical MIH RIAs was between 1.2 10-12 (~10 mg each) were directly weighed into 400 μl medium -11 - and 2.5 × 10 M, with an ED50 value of 5.73 ± 0.02 × 10 containing 100 μg/ml BSA, 1× protease inhibitors cocktail 10 M (N = 3) and the detection limit of 1.2 × 10-12 M. A for tissue culture (Sigma), and with or without the tested serial dilution of hemolymph was run in parallel to the neuropeptide in 24 well plates (Corning). The plates were native MIH (Fig. 8 insert). The detailed descriptions for incubated at 23–24°C with gentle agitation for 6 h. To test antiserum production and RIA of CHH were previously if other factors in the SG have roles in vitellogenesis, all provided [35]. The ED50 value of CHH was 4.00 ± 0.02 × HPLC fractions of SG extracts were pooled excluding crus- 10-10 M (N = 3). The RIA data were analyzed and plotted tacean hyperglycemic hormone precursor related peptide using the SOFTMax PRO v1.2.0 software (Molecular (CPRP), CHH, and MIH, and were also examined. Devices). To further define the specific action of MIH, hepatopancreas Ecdysteroid Radioimmunoassay fragments were exposed to actinomycin D (Sigma) or Hemolymph samples (10 μl) were analyzed for the total cycloheximide (Research Products international Corp.) at a concentration of ecdysteroid using an Ecd-RIA with ecdys- final concentration of 0.5 and 10 μM in the presence of MIH. one specific antiserum and [3H] PoA (Perkin Elmer) and The actinomycin D results were compared to those of MIH Ecd antiserum, as described [18]. Ecdysone served as a treatment. The cycloheximide results were compared to standard at concentrations ranging from 2.5 ng to 30 pg/ those of MIH + 0.1% v/v EtOH treatment, since EtOH was tube. The results were analyzed using the AssayZap pro- used as a vehicle for this reagent. In these experiments, VtG gram (Biosoft) and the EC50 value of Ecd-RIA was 100 ± levels were measured in the medium and tissue and the 10 pg/tube (n = 10). results were obtained as μg/mg total protein of both tissue

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and medium combined, before conversion into % of con- Acknowledgements trol. For the measurement of hnVtG RNA in the hepatopan- This research was supported by the National Oceanic and Atmospheric creas, which indicates a de novo synthesis of newly Administration (NOAA), Chesapeake Bay Program Grant (NA17FU2841) transcribed and unprocessed mRNA, and reflects the actual to the Blue Crab Advanced Research Consortium. We would like to thank transcription rates [46,47], incubation periods of 1 and 2 h Mr. J. Stubblefield for reading the manuscript and Professor E. Chang (Bod- ega Bay Marine Laboratory, University of California, Davis) for Ecdysone were initially compared. Consequently, a 1 h incubation antiserum. This article is Center of Marine Biotechnology contribution # period was set for all experiments measuring hnVtG RNA. 08-181.

In general, control treatments were tested in sextuplicate and References all other treatments in quadruplicates. At the end of the 1. Aiken DE, Waddy SL: Reproductive biology. In The Biology and experiment, the tissue fragments were frozen at -80°C until Management of Lobsters: Physiology and Behavior Volume 1. Edited by: Cobb JS, Philips BF. New York: Academic Press; 1980:215-276. further analyses for VtG mRNA and hnVtG RNA QPCR, as 2. Adiyodi RG, Subramoniam T: Oogenesis, oviposition and described above. The results of several independent experi- oosorption. In Reproductive Biology of Invertebrates Volume 1. Edited by: Adiyodi KG, Adiyodi RG. New York: John Wiley & Sons Ltd; ments were pooled, compared, and converted to % of con- 1983:443-495. trol due to high individual variations in VtG levels. 3. Zmora N, Trant MJ, Chan SM, Chung JS: Vitellogenin and its mes- senger RNA during ovarian development in the female blue crab, Callinectes sapidus: Gene expression, synthesis, trans- Statistical analysis port, and cleavage. Biol Reprod 2007, 77:138-146. The data obtained from QPCR, RIA, and ELISA is pre- 4. Passano LM: Molting and its control. In The Physiology of Crustacea sented as the mean ± SEM. The results were subjected to Volume 1. Edited by: Waterman TH. New York: Academic Press, Inc; 1960:473-536. GraphPad Instat 3 program (GraphPad Software). The 5. Hinsch GW: Some factors controlling reproduction in the spi- data were analyzed using one-way ANOVA followed by der crab, Libinia emarginata. Biol Bull 1972, 143:358-366. 6. Donaldson WE, Cooney RJ, Hilsinger JR: Growth, age and size at Tukey-Kramer multiple comparison test. In all cases, a sta- maturity of Tanner crab, Chionectes bairdi M.J. Rathbun, in tistical difference was accepted at P ≤ 0.05. the northern gulf of Alaska (Decapoda, Brachyura). Crustacea 1981, 40:286-301. 7. Skinner DM: Molting and regeneration. In The Biology of Crustacea Abbreviations Edited by: Bliss DE, Mantel LH. New York: Academic Press; MIH: molt-inhibiting hormone; CHH: crustacean hyperg- 1985:43-146. 8. Carlisle DB, Knowels FG: Endocrine Control in Crustaceans London and lycemic hormone; QPCR: quantitative PCR; VtG: vitello- New York: University Press; 1959. genin mRNA; ELISA: enzyme linked immunosorbent 9. Havens KJ, McConaugha JR: Molting in the mature female blue assay; RIA: radioimmunoassay; VtG: vitellogenin protein. crab, Callinectes sapidus Rathbun. Bull Mar Sci 1990, 46(1):37-47. 10. Van Herp F, Soyez D: Arthropoda – crustacea. In Reproductive Competing interests Biology of Invertebrates: Progress in Reproductive Endocrinology Volume 8. The authors declare that they have no competing interests. Edited by: Adiyodi KG, Adiyodi RG, Adams TS. John Wiley & sons; 1977:247-275. 11. Fingerman M: Crustacean endocrinology: a retrospective, pro- Authors' contributions spective, and introspective analysis. Physiol Zool 1997, 70(3):257-269. NZ carried out the concept, experimental design, and 12. Wainwright G, Webster SG, Wilkinson MC, Chung JS, Rees HH: acquisition, analyses, and interpretation of data, and Structure and significance of mandibular organ-inhibiting drafted and revised the manuscript including tables and hormone in the crab, Cancer pagurus. Involvement in multi- hormonal regulation of growth and reproduction. J Biol Chem figures. JMT participated in the discussions and revision of 1996, 271(22):12749-12754. the manuscript. YZ involved in the acquisition of funding 13. De Kleijn DPV, Van Herp F: Molecular biology of neurohormone and contributed to discussions. JSC was involved in the precursors in the eyestalk of crustacea. Comp Biochem Physiol B, Biochem Mol Biol 1995, 112:573-579. acquisition of funding, contributed to the concept, exper- 14. Keller R: Crustacean neuropeptides: structure, functions and imental design, analyses, and interpretation of data, and comparative aspects. Experientia 1992, 48:439-448. 15. Chan SM, Gu PL, Chu KH, Tobe SS: Crustacean neuropeptide revised the manuscript. All authors read and approved the genes of the CHH/MIH/GIH family: implications from molec- final manuscript. ular studies. Gen Comp Endocrinol 2003, 134:214-219. 16. Webster SG: High-affinity binding of putative moult-inhibiting hormone (MIH) and crustacean hyperglycemic hormone Additional material (CHH) to membrane bound receptors on the Y- organ of the shore crab Carcinus maenas. Proc R Soc Lond, B, Biol Sci 1993, B 251:53-59. Additional file 1 17. Webster SG: Amino acid sequence of putative moult-inhibit- ing hormone from the crab Carcinus maenas. Proc Biol Sci. Ribosomal RNA stability during 6 h incubation of hepatopancreas 1991, 244(1311):247-252. fragments. Gel electrophoresis of total RNA, extracted from hepatopan- 18. Chung JS, Webster SG: Does N-terminal pyroglutamate resi- creas fragments before and after 6 h incubation, demonstrates that ribos- due have any physiological significance for crab hyperglyc- omal RNA remains intact after 6 h incubation. emic neuropeptides? Eur J Biochem 1996, 240:358-364. Click here for file 19. Webster S, Keller R: Physiology and biochemistry of crusta- cean neurohormonal peptides. In Peptides and amines in inverte- [http://www.biomedcentral.com/content/supplementary/1746- brates Edited by: Thorndyke M, Goldsworthy G. Cambridge 1448-5-7-S1.ppt] Cambridge University Press; 1987. 20. Böcking D, Dircksen H, Keller R: The crustacean neuropeptides of the CHH/MIH/GIH family: structures and biological activ-

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ities. In The Crustacean Nervous System Edited by: Wiese K. Berlin: 40. Khayat M, Yang WJ, Aida K, Nagasawa H, Tietz A, Funkenstein B, Springer-Verlag; 2002:84-97. Lubzens E: Hyperglycaemic hormones inhibit protein and 21. Mattson MP, Spaziani E: Regulation of Y-organ ecdysteroidogen- mRNA synthesis in in vitro – incubated ovarian fragments of esis by molt-inhibiting hormone in crabs: involvement of the marine shrimp Penaeus semisulcatus. Gen Comp Endocrinol cyclic AMP-mediated protein synthesis. Gen Comp Endocrinol 1998, 110:307-318. 1986, 63:414-423. 41. Tsutsui N, Katayama H, Ohira T, Nagasawa H, Wilder MN, Aida K: 22. Imayavaramban L, Dhayaparan D, Devaraj H: Molecular mecha- The effects of crustacean hyperglycemic hormone-family nism of molt-inhibiting hormone (MIH) induced suppression peptides on vitellogenin gene expression in the kuruma of ecdysteroidogenesis in the Y-organ of mud crab: Scylla ser- prawn, Marsupenaeus japonicus. Gen Comp Endocrinol 2005, rata. 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In Recent Advances in Arthropod Endocrinology cal activity of moult-inhibiting hormone (MIH) and crusta- Edited by: Coast GM, Webster SG. Cambridge: Cambridge University cean hyperglycaemic hormone (CHH) in the crab Carcinus Press; 1998:33-52. maenas. Eur J Biochem 2003, 270:3280-3288. 26. Chaves AR: Effect of X-organ sinus gland extract on [(35)S] 45. Chung JS, Webster SG: Dynamics of in vitro release of molt- methionine incorporation to the ovary of the red swamp inhibiting hormone and crustacean hyperglycemic hormone crawfish Procambarus clarkii. Comp Biochem Physiol, Part A Mol in the shore crab, Carcinus maenas. Endocrinology 2005, Integr Physiol 2000, 126:407-413. 146:5545-5551. 27. Bomirski A, Klek-Kawi'nska E: Ovary-inhibiting hormone activ- 46. Sugimoto K, Mori K, Uchida K, Kobayashi D, Itoi K: Quantitative ity in shrimp (Crangon crangon) eyestalks during the annual analysis of thyroid-stimulating hormone messenger RNA reproductive cycle. 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Panouse JB: Influence de l'ablation du pedoncule oculaire sur Your research papers will be: la croissance de l'ovaire chez la crevette leander serratus. C R Acad Sci III, Sci Vie 1943, 217:553-555. available free of charge to the entire biomedical community 39. Zmora N, Sagi A, Zohar Y, Chung J: Molt-inhibiting hormone peer reviewed and published immediately upon acceptance stimulates vitellogenesis at advanced ovarian developmental stages in the female blue crab, Callinectes sapidus 2: novel cited in PubMed and archived on PubMed Central specific binding sites in hepatopancreas and cAMP as a sec- yours — you keep the copyright ond messenger. Saline Systems. 2009, 5(1):6. Submit your manuscript here: BioMedcentral http://www.biomedcentral.com/info/publishing_adv.asp

Page 11 of 11 (page number not for citation purposes) BIOLOGY OF REPRODUCTION 77, 138–146 (2007) Published online before print 4 April 2007. DOI 10.1095/biolreprod.106.055483

Vitellogenin and Its Messenger RNA During Ovarian Development in the Female Blue Crab, Callinectes sapidus: Gene Expression, Synthesis, Transport, and Cleavage1

Nili Zmora,3 John Trant,3 Siu-Ming Chan,4 and J. Sook Chung2,3

Center of Marine Biotechnology,3 University of Maryland Biotechnology Institute, Baltimore, Maryland 21202 Department of Zoology,4 University of Hong Kong, Hong Kong SAR, People’s Republic of China

ABSTRACT major yolk protein, vitellin (VT), which is a lipo-glyco- carotenoprotein. VTG in non-mammalian vertebrates and Blue crab vitellogenin (VTG) cDNA encodes a precursor that, several invertebrates is produced in an extraovarian tissue together with two other Brachyuran VTGs, forms a distinctive and then transported as a high-density lipoprotein (HDL) into cluster within a phylogenetic tree of crustacean VTGs. Using the ovary [4, 5]. VTG is serologically identical to VT and is quantitative RT-PCR, we found that VTG was primarily found in the hemolymph of most crustacean species during expressed in the hepatopancreas of a vitellogenic female, with ovarian development [6–12], including the female blue crab minor expression in the ovary. VTG expression in the hepato- [13]. Thereafter, it is internalized into the oocytes through pancreas correlated with ovarian growth, with a remarkable receptor-mediated endocytosis [14] and undergoes several 8000-fold increase in expression from stage 3 to 4 of ovarian development. In contrast, the VTG levels in the hepatopancreas modifications, such as specific proteolytic cleavage, to become and hemolymph decreased in stage 4. Western blot analysis and VT [15]. N-terminal sequencing revealed that vitellin is composed of It is well established that the livers of several vertebrate three subunits of ;78.5 kDa, 119.42 kDa, and 87.9 kDa. The species [16, 17] and the fat bodies of insects [18] are the sites processing pathway for VTG includes an initial hepatopancreatic of VTG synthesis. However, the source of yolk proteins in cleavage of the primary precursor into ;78.5-kDa and 207.3- crustaceans remains controversial, with evidence for hepato- kDa subunits, both of which are found in the hemolymph. A pancreatic [19–21], as well as intraovarian synthesis [3, 22– second cleavage in the ovary splits the ;207.3-kDa subunit into 25]. ;119.4-kDa and ;87.9-kDa subunits. The hemolymph VTG Ovarian development, as well as reproductive physiology profiles of mated and unmated females during ovarian develop- and behavior, have been the subjects of several studies in the ment indicate that early vitellogenesis and ovarian development female blue crab [26–28]. It has been established that the do not require mating, which may be essential for later stages, as female blue crab undergoes a terminal molt at puberty and VTG decreased to the basal level at stage 4 in the unmated group immediately mates thereafter, usually from May to October in but remained high in the mated females. Our results encompass the estuaries of Chesapeake Bay. After mating, the females comprehensive overall temporal and spatial aspects of vitello- migrate to high salinity waters, where egg maturation, genesis, which may reflect the reproductive physiology of the fertilization, and spawning occur. Spawning usually takes female blue crab, e.g., single mating and anecdysis in adulthood. place 1–2 mo after mating and can occur multiple times [26, behavior, ovary, oocyte development 28]. A female-specific lipoprotein associated with vitellogen- esis, which is composed of 48% lipid (mainly phosphatidyl- INTRODUCTION choline), 50% protein, and 2% carbohydrate, has been detected in the hemolymph of blue crabs that are undergoing ovarian Ovarian development and egg maturation are crucial development [29]. Subsequently, two lipoproteins, which are processes for the success of reproduction. In crustaceans, as cleavage products of ovarian VT, have been detected in in other oviparous animals, ovarian development includes a developing blue crab embryos [30]. Furthermore, using in vitro growth process that consists of two consecutive phases: the incubation followed by Western blot or immunohistochemical primary phase is characterized by primary oocyte recruitment analyses, it has been suggested that the ovary is the exclusive from oogonia [1], and the secondary phase features growth of site of vitellogenesis in the female blue crab [31, 32]. oocytes as a result of the accumulation of yolk proteins and During the last decade, molecular methods have been used other cytoplasmic egg proteins [2]. Yolk proteins are the most to investigate vitellogenesis in crustaceans. The rapidly important source of nutrients for developing embryos in increasing list of characterized crustacean VTG genes includes oviparous animals and may constitute 60–90% of the total those from penaeids [15, 23, 33–35], crayfish [36], prawns [19, egg proteins [3]. Vitellogenin (VTG) is the precursor for the 37], and two brachyuran species [38, 39]. From these reports, data have emerged indicating that VTG expression takes place 1Supported by a Program Grant (NA17FU2841) from the NOAA in the hepatopancreas of Pleocyamata (including brachyurans), Chesapeake Bay Office to the Blue Crab Advanced Research while in the Dendrobranchiata, both the hepatopancreas and the Consortium. ovary express VTG [15, 33–35]. However, the relative 2Correspondence: J. Sook Chung, Center of Marine Biotechnology, contribution of each tissue during vitellogenesis, either at the University of Maryland Biotechnology Institute, 701 E. Pratt Street, mRNA or protein level, remains unclear. In addition, although Baltimore MD 21202. FAX: 410 234 8896; e-mail: [email protected] VTG expression has been observed in the hepatopancreases of all the species mentioned above, the presence of the VTG protein in this tissue has yet to be demonstrated [15, 37]. Received: 10 July 2006. The conclusion that the ovary is the sole site of First decision: 28 August 2006. Accepted: 14 March 2007. vitellogenesis in the blue crab [31, 32] is not consistent with Ó 2007 by the Society for the Study of Reproduction, Inc. the latest suggestion of hepatopancreatic expression in ISSN: 0006-3363. http://www.biolreprod.org Pleocyamata [37]. Therefore, we aimed to confirm and 138 VITELLOGENESIS IN THE BLUE CRAB 139 complement the original data, derived from protein analyses, For the phylogenetic analysis, a Neighbor Joining Tree was constructed with data obtained using a combined biochemical and using the MEGA phylogeny package [44] with the pair-wise deletion option molecular approach. In addition, because the blue crab and 250 bootstrap repeats. To choose the closest outgroup, a PSI Blast search was iterated three times [45] using the blue crab VTG sequence as a query, and spawning stocks in Chesapeake Bay (and along the US the alignment was optimized manually according to the PSI Blast alignment Atlantic coastline) have sustained a severe and persistent results. decline beginning in 1992 [40, 41], it is of interest to better understand vitellogenesis, ovarian development, and the Purification of VT and Generation of Polyclonal Antibodies control of these processes, so as to develop strategies for the recovery of this ecologically and commercially important Ovarian tissue (2 g) from a vitellogenic female blue crab (stage 4) was species. homogenized in 10 ml of extraction buffer (20 mM Tris [pH 7.5], 1 mM PMSF), cleared by centrifugation at 8000 3 g for 10 min at 48C, and In the present study, we isolated the full-length cDNA of the precipitated in 50% ammonium sulfate. The pellet was resuspended in blue crab vitellogenin from the female hepatopancreas and extraction buffer and the precipitation step was repeated twice. generated specific VT antibodies (anti-VT serum), to monitor The resulting pellet was resuspended in 1 ml of elution buffer (PBS [pH the temporal and spatial profiles of vitellogenesis. More 7.5], 1 mM EDTA) and the proteins were separated on a 56 3 1.6 cm Bio-gel P- specifically, we measured VTG expression in the hepatopan- 200 size exclusion column (Bio-Rad, Hercules, CA) at a flow rate of 0.1 ml/ creas and ovary, as well as the VTG levels in the min. The protein elution profile was monitored at 280 nm and 3-ml fractions were collected. Protein fractions that were yellow in color were stored for hepatopancreas, ovary, and hemolymph during ovarian devel- further analysis. The purity of VT was verified on 4–15% PAGE under native opment. We also followed the cleavage pattern and transport of and denaturing conditions and further confirmed by N-terminal sequencing of VTG from the hepatopancreas through the hemolymph to the its subunits (see Supplemental Fig. 1 online at www.biolreprod.org and at ovary. In addition, a comparison of hemolymph VTG levels in http://combshare.umbi.umd.edu/zmora/zmora.html). This purified protein was mated and unmated females suggests the involvement of semen then used to generate antiserum in rabbits (Sigma-Genosys Laboratories). deposition in the regulation of vitellogenesis. Northern Blot Analysis MATERIALS AND METHODS Total RNA (20 lg) from the ovaries and hepatopancreases of females at different stages of ovarian development were electrophoresed in a 1.2% agarose Animals gel that contained formaldehyde, and the samples were transferred to a nylon Final-stage juvenile female blue crabs (prepubertal) at the premolt stage membrane (Immobilon; Millipore Inc., Bedford, MA). For prehybridization, the were obtained from local fishermen in Chesapeake Bay or from the membrane was incubated with hybridization buffer (50% formamide, 53 SSC, Aquaculture Research Center at the Center of Marine Biotechnology 7% SDS, 2 mM EDTA, 1% blocking reagent) for 3 h at 608C, and then (Baltimore, MD). The crabs were held in 20-ppt artificial seawater at 228C hybridized for 16 h with a digoxigenin (DIG)-labeled antisense riboprobe. The (16L:8D) and fed daily with a combination diet of frozen squid and pelleted sea riboprobes, which were generated from the VTG cDNA clone using SP6 or T7 bream (EWOS, Surrey, BC, Canada). Wild crabs were acclimated for 10–14 RNA polymerase, corresponded to nucleotides 206-1508 of VTG cDNA and days before experimentation in a 4-m3 tank. For the monitoring of VTG were used at a final concentration of 20 ng/ml. After hybridization, the hemolymph levels, females were kept individually in a 30 3 30 cm membrane was subjected to a 30-min wash in 23 SSC, 0.1% SDS at 688C, a compartment in a 0.5-m3 tank. Animal collections and experiments were 30-min wash in 0.53 SSC, 0.1% SDS at 688C, and a final wash in 0.13 SSC, performed from late March to early October, 2005. Prepubertal (ripe 0.1% SDS at 688C for 30 min. Hybridized probes were visualized with an AP- prepubertal, approaching final molt) and pubertal females were distinguished conjugated anti-DIG antibody (1:20 000) and the CDP-Star chemiluminescent based on rounded abdomen shape and other signs of the premolt stage [42, 43]. detection system (Roche Diagnostics GmBH, Mannheim, Germany). The Ovarian developmental stage was determined based on ovarian weight and hybridized membrane was exposed to BioMax MS Kodak film for 5–30 min. gamete size, according to criteria established by Lee and Puppione [29], in Ribosomal RNA was visualized with ethidium bromide. which stage 1 is previtellogenic, stages 2 to 5 are vitellogenic, and 6 to 8 are postspawning stages. Histology Hemolymph samples were diluted 1:1 with anticoagulant buffer (0.3 M NaCl, 30 mM sodium citrate, 26 mM citric acid, 2 mM EDTA [pH 7.4]) and Sample preparation. Hepatopancreatic and ovarian tissues were fixed in stored at 208C. Tissues for RNA extraction were dissected from ice-cold Bouin solution for 24 h, and then dehydrated gradually through a series of anesthetized animals, snap-frozen on dry ice, and stored at 808C. increasing alcohol concentrations. Tissues were cleared and embedded in Paraplast according to conventional procedures. Sections (6-lm thickness) Amplification, Cloning, and Sequencing of VTG were prepared on 3-aminopropyl triethoxysilane (APTES)-coated slides. Immunohistochemistry. Sections were deparaffinized, rehydrated, and Total RNA was extracted from the hepatopancreas and ovary of a rinsed in distilled water before incubation with 0.3% hydrogen peroxide in PBS vitellogenic female using Trizol reagent (Invitrogen, Carlsbad, CA) according (pH 7.4) for 30 min at room temperature. After washing in PBST (PBS, 0.5% to the manufacturer’s instructions. Total RNA was quantified in a NanoDrop Tween-20), the slides were blocked in 10% normal goat serum and incubated UV/visible spectrophotometer (NanoDrop Technologies, Wilmington, DE). overnight with anti-VT serum diluted 1:3000 (or preimmune serum for the First-strand cDNA was generated from 1 lg total RNA using 50- and 30- RACE negative control), followed by incubation with HRP-conjugated goat anti-rabbit (SMART RACE cDNA Amplification Kit; BD Biosciences, Mountain View, IgG (Vector Laboratories, Burlingame, CA) at 1:5000 for 1 h. Sections were CA). The initial degenerate primers were designed according to the conserved then washed and incubated with 3,30diaminobenzidine (DAB; Sigma Chemical amino acid domains MYKYVEA and GNMGVMTP of the VTG proteins of Co., St. Louis, MO). Photographs were taken using an Olympus micro/DP70 penaeids and C. feriatus (GenBank accession nos. AB191486, DQ288843, camera mounted on a Zeiss light microscope (model Axioplan2), and converted AY321153, AB176641, and AY724676). An amplicon of 718 bp was initially into computer images using the Olympus DPController program. generated from hepatopancreas cDNA by PCR using the degenerate primers In situ hybridization. Deparaffinized and rehydrated sections were VitpF1 (50-ATGTAYAARTAYGTNGARGC-30) and VitpR1 (50- incubated with 0.2 M HCl for 20 min, washed in PBS, treated with proteinase GGNGTCATNACNCCCATRTTNCC-30) and the Advantage cDNA polymer- K (10 lg/ml in 50 mM Tris-HCl [pH 7.5], 50 mM EDTA) for 15 min, and ase mix (BD Biosciences), and this fragment was cloned in the TOPO TA acetylated in 0.1 M triethanolamine-HCl/0.25% (v/v) acetic anhydride. The cloning vector (Invitrogen). A 50-RACE fragment was amplified using the sections were covered with 500 ll of hybridization buffer (50% formamide, 53 reverse primer Vitfin1 (50-CGCAGGCTTCTGGGCTCCAGCTC-30) and the SSC, 50 lg/ml denatured salmon sperm DNA) and incubated for 2 h at 588C. adapter primer (from the kit) from a 50-RACE hepatopancreas cDNA library, After prehybridization, the slides were incubated overnight at 588C in fresh and analyzed similarly. A 6990-bp 30-RACE fragment was amplified using hybridization buffer that contained 40 ng/ml denatured antisense or sense DIG- primer Vitfin2 (50-GAGCTGGAGCCCAGAAGCCTGCG-30) and the adapter labeled riboprobes, which were prepared as described above. After hybridiza- primer with the GeneAmp XL PCR kit (Applied Biosystems, Branchburg, NJ). tion, the sections were washed for 30 min in 23 SSC at 258C and for 1 h each in The amplicon was ligated into a vector using the TOPO TA XL cloning kit 23 SSC, 0.43 SSC, and 0.13 SSC at 658C. The slides were then incubated in (Invitrogen). The full-length cDNA sequence of VTG was constructed from the 1% blocking reagent [Roche] and 2% normal sheep serum in buffer I (100 mM overlapping cDNA clones. Tris-Cl [pH 7.5], 150 mM NaCl) with AP-coupled anti-DIG antibody (Roche) 140 ZMORA ET AL.

on 6% SDS-PAGE, the proteins were transferred to a PVDF membrane (Pierce) and stained with Coomassie blue. Positive bands with the expected molecular masses were excised and N-terminally sequenced on a Perkin Elmer/Applied Biosystems Edman sequencer at the synthesis and sequencing facilities of Johns Hopkins University (Baltimore, MD).

ELISA Purified VT was used to develop a competitive ELISA and to serve as a standard. The VT ELISA was based on the procedure of Lee and Watson [47], with a linear standard curve range of 50–1600 ng/ml and a detection limit of 20 ng/ml. Specifically, microplates (Costar Corp., Cambridge, MA) were coated overnight at 48C with 100 ll of 200 ng/ml VT in 50 mM bicarbonate buffer (pH 9.6). After washing with PBST, the microplates were blocked for 1 h at 378C with 200 ll of blocking buffer (PBST plus 1% BSA). Unknown and standard samples were incubated overnight at 48C with anti-VT serum in blocking buffer at a final dilution of 1:20 000. Thereafter, the samples were dispensed into the wells (100 ll/well) and incubated for an additional 2 h at 378C. After washing as described above, the plates were incubated with 100 ll of 1:5000 HRP- conjugated goat anti-rabbit IgG (Vector Laboratories, Burlingame, CA) in blocking buffer for 1 h at 378C. Color was developed using 200 ll of 3 mg/ml FIG. 1. Phylogenetic analysis (Neighbor Joining) of blue crab and other 0 deduced crustacean VTG amino acid sequences. Rainbow trout (O. 2,2 -azino-bis(3-ethylbenzo-acid-6-sulfonic acid) diammonium salt (ABTS; mykiss) VTG served as an outgroup. Genetic distance was tested with 250 Sigma) in 0.1 M citrate buffer (pH 4.1) for 15–20 min at room temperature. bootstrap repeats. All bootstrap values were 100%. The following Absorbance was measured at 405 nm using an automated microplate reader sequences (GenBank accession no.) were included: Fenneropenaeus (Thermo-Max; Molecular Devices, Menlo Park, CA). merguiensis (AAR88442); Penaeus semisulcatus (AAL12620); Litopenaeus vannamei (AAP76571); Marsupenaeus japonicus (BAD97832); Metape- Quantitative RT-PCR naeus ensis (AAT01139); Cherax quadricarinatus (AAG17936); Macro- brachium rosenbergii (BAB69831); Pandalus hypsinotus (BAD1958); First-strand cDNA was generated from hepatopancreatic or ovarian total Charybdis feriatus (AAU93694); Portunus trituberculatus (AAX94762); RNA as described above, using random hexamers as the primers and MMLV- Callinectes sapidus (DQ314748); and Oncorhyncus mykiss (CAA63421). RT (Promega, Madison, WI). Total RNA (3 lg) for each sample was subjected to DNase treatment (2 U of RQ1; Promega) to eliminate gDNA contamination. Standards were prepared from sense VTG cRNA generated from the VTG (150 mU/ml in buffer I). Color was developed using NBT/BCIP plus cDNA clone using T7 RNA polymerase (Roche Diagnostics) followed by suppressor (Pierce Biotechnology, Rockford, IL). The sections were examined purification in a ChromaSpin-100 column (BD Biosciences). The resulting and photographed as described above. sense cRNA was quantified using the RiboGreen RNA quantification kit (Molecular Probes, Eugene, OR) and converted into copy numbers based on the molecular mass of the RNA fragment. Standards that ranged from 480 to 4.8 3 SDS-PAGE and Western Blot Analyses 106 copies were reverse-transcribed as described above. Proteins from ovaries, hemolymph, and hepatopancreases of females at Duplicate cDNA aliquots (20 ng of total RNA) from each sample served as different stages of ovarian development were prepared as follows. Ovaries were templates in PCR with SYBR Green PCR core reagent (Applied Biosystems, homogenized in an extraction buffer (0.3 M sucrose, 140 mM Tris-HCl [pH Foster City, CA) that contained 200 nM of the gene-specific primers TaqvitF 0 0 0 7.4]), precipitated in 50% ammonium sulfate, and resuspended in PBS. HDL (5 -TGTACAGCTGAAAGGCGTGG-3 ) and TaqvitR (5 -CATGGGCCGA- 0 was isolated from the hemolymph, to avoid interference of hemocyanin, as GAACAGTCA-3 ). Amplification reactions were carried out with the ABI described by Shechter et al. [46]. Hepatopancreatic proteins were prepared as Prism 7700 Sequence Detection System at 508C for 4 min, 958C for 10 min, described for the ovarian preparation, layered onto 1.35 M sucrose, and followed by 40 cycles of 958C for 15 sec and 608C for 60 sec. The copy number centrifuged at 160 000 3 g for 6 h at 48C. The upper fraction was collected for in each sample was determined by comparing CT (threshold cycle) values [48] analysis. Protein concentrations were determined using the RC Dc protein assay to the standard (run on every plate) and normalized against the abundance of 0 kit (Bio-Rad). Each protein sample (0.5 lg) was subjected to 7% SDS-PAGE arginine kinase mRNA using the TaqAKF (5 -ACCACAAGGGTTTCAAG- 0 0 0 and the gel was stained with Coomassie blue (GelCode, Bio-Rad) or transferred CAG-3 ) and TaqAKR (5 -GGTGGAGGAAACCTTGGACT-3 ) primers [49]. to a nitrocellulose membrane at 100 V for 1 h in transfer buffer (25 mM Tris [pH 8.3], 192 mM glycine, 20% methanol). After blocking in PBST that Statistical Analyses contained 5% skimmed milk, the membrane was incubated with anti-VT serum (1:20 000 in blocking buffer) for 1 h at room temperature. After three washes in The data obtained from QRT-PCR and ELISA are presented as mean 6 PBST for 15 min, the membrane was incubated with HRP-goat anti-rabbit IgG SEM. The results were examined using one-way ANOVA followed by the (1:40 000). The signal was developed using the Super-Signal West-Pico Tukey multiple range test. In all cases, statistical difference was accepted at P chemiluminescence detection kit (Pierce) and detected by exposure to BioMax , 0.05. MS Kodak film for 30 sec to 2 min. RESULTS Preparation of VTG/VT Subunits for N-terminal Sequencing Characterization of VTG cDNA Ovarian VT subunits were separated on 7% SDS-PAGE, as described above. In order to obtain 10 lg of the pure hemolymph and hepatopancreas The full-length cDNA sequence of VTG was determined by VTG subunits, previously extracted HDLs were immunoprecipitated using anti- overlapping the 50-RACE and 30-RACE cDNA fragments. The VT serum and Protein A magnetic beads (New England Biolabs, Ipswich, MA), complete VTG cDNA, which is composed of a 28-nt 50-UTR, a according to the manufacturer’s protocol. In brief, 30 lg hemolymph HDL 0 (equivalent to ; 100 ll of hemolymph) and 1 mg hepatopancreatic HDL 113-nt 3 -UTR, and a 7689-nt ORF, encodes a precursor of (equivalent to 25 mg of tissue) diluted four times with PBS that contained 13 2563 amino acids (aa), which includes an 18-aa signal peptide protease inhibitor cocktail (Sigma) were preincubated with 25 ll of Protein A (GenBank accession no. DQ314748) with a predicted size of magnetic beads for 1 h at 48C, in order to remove any proteins that were 282 kDa. The deduced amino acid sequence contains four nonspecifically bound to Protein A. Once the beads were removed using a putative subtilisin-like protein endopeptidase motifs (RXXR), magnet, the sample was incubated with 20 ll anti-VT serum, followed by the nine putative N-glycosylation sites at amino acids 159, 658, addition of 20 ll Protein A magnetic beads, each for 1 h at 48C. After incubation, the beads were retained on a magnetic rack and washed four times 885, 979, 994, 1451, 1631, 1863, and 1938 (analyzed at http:// with 1 ml PBST. Proteins bound to Protein A were finally eluted by 3 min of www.cbs.dtu.dk/cgi-bin) but no O-glycosylation sites. Serine heating at 708Cin30llof33 Laemmli sample loading buffer. After separation residues constitute 5% of the total amino acid composition of VITELLOGENESIS IN THE BLUE CRAB 141

FIG. 2. Northern blot analysis of tissue- specific expression of VTG at different stages of ovarian development. A)RNA from the hepatopancreas. B) RNA from the ovary. Lane 1, stage 1; lane E2, early stage 2; lane 3, midstage 2; lane 4, midstage 3. Lower panel, ribosomal RNA (rRNA) visu- alized by ethidium bromide staining. Tran- script size is indicated on the left.

VTG, including one hepta-serine, three tri-serines, and twelve hepatopancreas tubule. No signal was observed in stage 1 di-serines. The VTG cDNA shares 87% and 80% identity with female or male hepatopancreases (Fig. 3, C and D). the nucleotide and deduced amino acid sequence of the Using immunohistochemistry (ICC), VTG was detected in Japanese blue crab (P. trituberculatus) vitellogenin [38, 39], the epithelial cells of hepatopancreatic tubules of a vitellogenic respectively. We also performed a phylogenetic analysis of the female at stage 3, as well as in the adjacent hemocytes (Fig. VTG proteins, to construct a Neighbor Joining Tree using the 3E), but not in male hepatopancreases (Fig. 3F). No positive rainbow trout VTG as an outgroup, as described in Materials signal was detected in previtellogenic hepatopancreas nor when preimmune serum was used (data not shown). and Methods. The Brachyurans VTG formed a distinctive separate cluster from the other crustacean VTG proteins (Fig. 1). Proteolysis of Blue Crab VTG Northern blot analysis (Fig. 2A) revealed that a VTG The results of Western blot analysis of HDLs extracted from transcript of ;7.8 kb in length was present only in the the hepatopancreas, hemolymph, and total protein extracts of hepatopancreases of vitellogenic females and only at stages 2 ovaries at stages 1 and 3 were stage-dependent, and differential and 3, not stage 1. No signal was detected in the ovary at any band patterns were noted in these tissues (Fig. 4, A and B). In developmental stage (Fig. 2B), in female muscle, or in the male the hepatopancreas, three bands with molecular masses of ;95 hepatopancreas (data not shown). kDa, ;80 kDa, and ;78 kDa were detected by the anti-VT serum, while in the hemolymph, two bands were detected The spatial and cellular distributions of VTG expression (Figs. 4, A and B, lanes 3 and 4). In addition, a faint ; 250- (Fig. 3, A and B), as determined by RNA in situ hybridization, kDa band cross-reacted with the anti-VT serum (Fig. 4, A and were congruent with the results of the Northern blot analysis, B, lane 4). In the ovary, bands of ;95 kDa and ;78 kDa were as shown in Figure 2. The hepatopancreas of a female at stage 3 recognized by the anti-VT serum (Fig. 4, A and B, lane 6). showed positive hybridization with the antisense VTG ribop- N-terminal sequencing was performed with the resolved robe but not with the VTG sense probe (Fig. 3, A and B). This VT/VTG proteins obtained from all three tissues of the same signal was evident in most of the epithelial cells of the female at stage 3 (a total of eight bands, numbered 1–8 in

FIG. 3. Localization of blue crab VTG expression by in situ hybridization and immunohistochemistry in the hepatopancreases of vitellogenic and nonvitellogenic females and males. In situ hybridization. A) Stage 3 vitellogenic female antisense probe. B) Stage 3 vitellogenic female sense probe. C) Stage 1 vitellogenic female antisense probe. D) Mature male antisense probe. Immunohistochemistry. E) Stage 3 vitellogenic female. F) Mature male. The arrows point to a representative positive signal. T, Hepatopancreatic tubule; L, lumen of the tubule. Bars ¼ 50 lm(A–E) and 200 lm(F). 142 ZMORA ET AL.

FIG. 4. VTG and VT detection in the female blue crab hepatopancreas, hemolymph, and ovary. A) Coomassie blue-stained 7% SDS-PAGE. B) Western blot analysis using anti-VT serum. Lanes 1 and 2, HDL fractions of hepatopancreas stages 1 and 3, respectively; lanes 3 and 4, HDL fractions of hemolymph stages 1 and 3, respectively; lanes 5 and 6, total protein extracts from ovary stages 1 and 3, respectively. Molecular mass markers (kDa) are indicated on the left. C) Coomassie blue-stained PVDF membrane transferred from SDS-PAGE at 6% for the hepatopancreas and hemolymph and 7% for the ovary. All samples were obtained from one female at ovarian stage 3. a, Hepatopancreas; b, hemolymph; c, ovary. The bands numbered 1 to 8 were subjected to N-terminal sequencing and the results are listed in Table 1.

Figure 4C and a band of ;250 kDa in the hemolymph). As first six amino acids of cryptocyanin from the swimming crab stated earlier, hepatopancreatic and hemolymph samples were Portunus pelagicus (GenBank accession no. ABM54471). resolved in 6% SDS-PAGE, while the ovarian proteins were electrophoresed on a 7% gel. The resulting amino acid VTG and VT levels sequences are listed in Table 1. The initial amino acid sequence Late-prepubertal females approaching their final molt were of band 4 of the hepatopancreas was XPYGG, which was divided randomly into two groups. After the final molt, the consistent with the sequences of bands 6 and 8 of the females in one group were allowed to mate and the others hemolymph and ovary samples, respectively (Fig. 4C, lanes remained as virgins. Using a competitive ELISA, the VTG a–c). Band 5 of the hemolymph and ovarian band 7 were levels in the hemolymph were determined in samples collected sequenced as SVD(X)AA and SVDYAA, respectively. The semiweekly over 10 wk. SVDYAA sequence agrees with the predicted amino acid As shown in Figure 5, the hemolymph VTG levels in both residues at positions 732–737 that immediately follow the groups were low (10–20 lg/ml) during the first 2.5 wk, then putative RERR cleavage site. Surprisingly, ovarian band 7 also increased gradually until they peaked at 200 lg/ml by Week 5. yielded the sequence MEYTRSS, which corresponded to At 9.5 wk, the VTG levels in the unmated females began to amino acid residues 1775–1782 of the deduced amino acid decline and returned to the basal level, while in the mated sequence of VTG (Fig. 4C, lane c). females, the extent of decrease was moderate, stabilizing at a Electrophoresis in 6% SDS-PAGE was chosen for the significantly higher level of ;100 lg/ml in the final 2 wk, hepatopancreatic and hemolymph samples, since band 7 in the which corresponded to ovarian stage 4 (Fig. 5). ovarian tissue samples gave two sequences (Fig. 4C, lane c). The The VTG levels in the hepatopancreas and ovary during vitellogenesis were measured. As shown in Figure 6, the VTG sequence of band 1 of the hepatopancreas (Fig. 4C, lane a) started content of the hepatopancreas increased from 1.0 6 0.3 (stage with SSSGQ, which corresponded to amino acids 1781–1785 of 1) to 7.0 6 3.0 (stage 3) lg/mg of total protein, and then VTG, while band 2 sequenced as LYGPQY, which correspond- dropped to ;3 lg/mg of total protein at stage 4. ed to amino acids 745–751 of VTG. Both proteins begin 6 and 15 As expected, the VT content of the ovary increased amino acids after the expected MEYTRSS and SVDYAA gradually during vitellogenesis from 128 6 19 lg/mg at stage motifs, respectively. The ;250-kDa band did not produce a 1 to 478 6 4 lg/mg at stage 4, which was equivalent to ;10% sequencing result (Fig. 4C, lane b), whereas band 3 started with to ;50% of the total proteins (Fig. 6). the sequence DEPDGV (Fig. 4C, lane a), which is identical to the VTG Gene Expression TABLE 1. N-terminal sequencing results of the bands (1–8, shown in Fig. During the course of vitellogenesis, the VTG transcript 4C) obtained from SDS-PAGEs (6% for hepatopancreas and hemolymph levels in the hepatopancreas and ovary of the same animals % and 7 for ovary). were measured using QRT-PCR. VTG expression in the VTG band no. Source N-terminal sequence hepatopancreas, which was initially very low at stage 1 (1174 copies/20 ng total RNA), increased ;900-fold at stages 1 Hepatopancreas SSSGQ 2 and 3, followed by an additional dramatic increase of 73 000- 2 Hepatopancreas LYGPQY 7 3 Hepatopancreas DEPDGV* fold (8.6 3 10 copies/20 ng RNA) at stage 4 (Fig. 7A). The 4 Hepatopancreas (X)PYGG VTG expression levels in the ovary (although not detected by 5 Hemolymph SVD(X)AA Northern blot analysis, Fig. 2) were generally ;3000-times 6 Hemolymph APYGGT lower than those in the hepatopancreas, with the exception of 7 Ovary SVDYAA:MEYTRSS stage 1, when the expression levels were similar. The general 8 Ovary APYGGTTQ expression pattern in the ovary resembled that in the * Non-VTG. hepatopancreas (Fig. 7B). VITELLOGENESIS IN THE BLUE CRAB 143

FIG. 6. VTG levels in the hepatopancreas (gray bars) and VT levels in the ovary (black bars) during vitellogenesis, as determined by ELISA. Results are presented as the mean 6 SEM (n ¼ 5). *P 0.05; ** P 0.01.

FIG. 5. VTG levels in the hemolymph of mature females from final molt immunohistochemistry (Fig. 3) indicate that the hepatopancre- to 10 wk after molting. The VTG levels were measured by ELISA and are presented as mean 6 SEM. *P 0.05; **P 0.01. Gray circles, mated as is the site of expression and translation of VTG. This females (n ¼ 5); black circles, unmated females (n ¼ 6). conclusion contradicts the results of previous studies conducted at the protein level, which concluded that the ovary was the sole site of VTG production in the blue crab, while ruling out DISCUSSION expression in the hepatopancreas [31, 32]. The ICC pattern, indicated by granular signals in the epithelial cells of the In order to gain a better understanding of the reproductive hepatopancreatic tubules (Fig. 3, E and F), is similar to those of physiology of the female blue crab, C. sapidus, we utilized a C. quadricarinatus [46] and M. rosenbergii [53] and implies combined molecular and biochemical approach to study that VTG is present in a packaged form. Although the above vitellogenesis. This complex process involves a network of methods suggest that the ovary is unlikely to express VTG, the tissues (hepatopancreas, hemolymph, and ovary) and includes highly sensitive method of QRT-PCR revealed the presence of the synthesis, transport, and processing of VTG, as well as the VTG mRNA in this tissue, albeit at a level 3000-times lower accumulation of VT during oocyte growth. The full-length than in the hepatopancreas. To ascertain that the QRT-PCR cDNA of VTG was sequenced and a quantitative assay (QRT- result was not an artifact, a 50-RACE amplicon of 1.5 kb was PCR) to measure gene expression was developed. In addition, amplified from the ovary, sequenced, and found to be identical ovarian VT was purified, antibodies were generated, and a to that prepared from the hepatopancreas (data not shown). competitive ELISA was established. Although the 30 portion may differ in length or sequence from The analysis of the VTG cDNA revealed that the ORF the hepatopancreas form, these results suggest that vitellogen- encodes a 2563-amino acid precursor with a predicted esis in the suborder Pleocyamata, as in Dendrobranchiata, molecular mass of 282 kDa. This protein is most homologous originates in both the hepatopancreas and ovary. As alluded to (80%) to the VTG of another brachyuran species, the Japanese earlier, the difference may lie in the relative VTG contribution blue crab Portunus trituberculatus [38, 39]. Similar to other of each tissue, which appears to be significantly lower in the VTGs, the blue crab VTG contains multiple potential N- ovary of Pleocyamata, at levels undetectable by most glycosylation sites, of which seven out of the nine are located procedures. downstream of amino acid 728 [36, 50]. Unlike vertebrates, It is widely accepted that the VT primary precursor (prepro- crustacean VTGs, which included the blue crab VTG, lack the VTG) undergoes several proteolytic cleavages, to generate the prominent motif characterized by a polyserine domain. subunits that comprise ovarian VT. Using immunoblotting and Polyserine domains bind bivalent cations [36] and have immunoprecipitation of HDL fractions, we were able to previously been implicated in receptor binding [51, 52]. In concentrate approximately 2000-fold the VTG/VT in the crustaceans, the lower serine residue content (5% to 10%) is hepatopancreas and hemolymph (ovarian stage 3) for N- typically attributed to the general scarcity of polyserine terminal sequencing. For example, 25 mg of fresh hepatopan- domains (relative to most vertebrate VTGs) [36]. The serine creas yielded 10–15 lg of VTG, whereas ;20 lg of VTG was content of blue crab VTG (8.5%) falls within this range. obtained from 100 ll of hemolymph. While the immunopre- A phylogenetic tree was constructed using the 11 crustacean cipitation procedure provided in pure form for N-terminal VTGs currently available in GenBank, including the blue crab sequencing the VTG/VT subunits that are involved in the sequence, and the rainbow trout VTG as an outgroup. The tree processing of VTG, it also yielded an unexpected result for the showed two main clusters, Penaeids and Brachyurans, and hepatopancreas sample. We anticipated fewer bands in the clearly demonstrated that the blue crab VTG belongs to the hepatopancreas, as the site of VTG synthesis, than in the family of crustacean VTGs. In addition, our data indicate hemolymph. To our surprise, four bands were evident in the specific lineage modifications for Brachyurans vs. Penaeids hepatopancreas (Fig. 4C, lane a), while only two bands were (Fig. 1). It is anticipated that additional crab VTGs will cluster noted in the hemolymph (Fig. 4C, lane b). Moreover, as listed with the three existing crab VTGs, allowing the construction of in Table 1, the sequencing results for bands 1 and 2, compared a more representative evolutionary distribution of crustacean with those of the ovarian samples, are missing 6 and 13 amino VTGs. acids at the N-terminus, which may reflect aminopeptidase The VTG cDNA was initially isolated from the hepatopan- activity. It is possible that these differences resulted from the creas of a vitellogenic female. The results obtained in the immunoprecipitation steps, since the hepatopancreas is the Northern blot analysis (Fig. 2), in situ hybridization, and major digestive tissue with a variety of metabolic enzymes. 144 ZMORA ET AL.

FIG. 7. VTG transcript levels in the hepa- topancreas (A) and ovary (B), as determined by QRT-PCR during ovarian development. The results are presented as copy number per 20 ng total RNA (n ¼ 5). **P 0.01.

Overall, seven out of the eight proteins detected by the anti-VT and c), it seems that the second cleavage takes place in the serum turned out to be subunits of VTG/VT, with the ovary, splitting band 5 to band 7, which contains two subunits. exceptions of an ;80-kDa protein (Fig. 4C, band 3) in the Based on our N-terminal sequencing results (Table 1), this hepatopancreas, which was identified as cryptocyanin, and an second cleavage was expected to produce two bands with unknown ;250-kDa protein in the hemolymph. calculated molecular masses of ;119.4 kDa and ;87.9 kDa. Consensus proteolytic motifs (RXXR) specifically recog- However, in SDS-PAGE (Fig. 4, A and C), bands 2 nized by the subtilisin-like endopeptidase or convertase are (hepatopancreas) and 7 (ovary) migrated below the 100-kDa distributed throughout the VTG protein sequences. However, marker, indicating the molecular masses of less than 100 kDa only one of these motifs has been proven to be utilized and is for both subunits. This may be due to glycosylation and conserved among crustacean VTGs. N-terminal amino acid conjugation of lipid to this subunit, which contains many sequencing of the different VT subunits illustrated that this putative glycosylation sites, thereby affecting its mobility in functional RXXR motif is positioned at 710–728 of other SDS-PAGE. Interestingly, while Lee and Walker [29] observed crustacean VTGs [15, 33, 34, 37, 38] Cleavage gives rise to a an additional ;107-kDa lipoprotein in the hemolymph, only 78–90-kDa protein, which is homologous to invertebrate and the subunits of VTG (;207.3 kDa and ;78.5 kDa) and a vertebrate lipoproteins [54, 55] and fish VTG [56, 57]. minor band (;250 kDa) of unknown sequence were found in The C. sapidus VTG possesses a functional RERR motif at the current study. In , the cleavage of the larger amino acids 728–731, which results in a calculated subunit M. rosenbergii mass of ;78.5 kDa for the subunit beginning with VTG subunit occurs in the hemolymph only at advanced APYGGTTQ (http://bioinformatics.org/sms2/protein_mw. vitellogenic stages [37]. Likewise, it is possible that cleavage html) and a calculated molecular mass of ;207.3 kDa for of the ;207.3-kDa subunit (band 5) in the blue crab the remainder of the VTG, starting at SVDYAA (Table 1). As hemolymph occurs after stage 4, although this motion was presented in Table 1, this result is expected and is in agreement not tested in the present study. Based on the N-terminal with the bands seen for the hemolymph (Fig. 4C, band 5) and sequencing results, a putative model for the processing of C. ovary (Fig. 4C, band 7). Since only two bands were observed sapidus VTG is proposed in Figure 8. Since the same cleavage in the hemolymph, it appears that the first cleavage of VTG sites have been reported in the Penaeus semisulcatus VTG occurs in the hepatopancreas immediately after synthesis, prior subunits [15], it can be concluded that this is a common to its secretion into the hemolymph. From a comparison of the processing pattern for crustacean VTGs. banding patterns of hemolymph and ovary (Fig. 4C, lanes b The prepro-VTG, with a predicted molecular mass of 282 kDa, was not detected in the hepatopancreas (the established site of synthesis) by staining or immunoblotting (Fig. 4, A-C). This may be due to rapid processing of this protein immediately after synthesis. Alternatively, since the anti-VT serum was generated against VT, the targeted epitopes on this precursor may be unavailable for interaction with the anti-VT serum. Cleavages, glycosylations, conjugation of lipids, and consequent refolding are involved in the conversion of prepro- VTG to VT, as well as in the general formation of epitopes [58, 59]. The significance of the tertiary structure of the VTG subunits has been demonstrated in the turtle Chinemys reevesii [60], in which four different monoclonal antibodies did not recognize the denatured forms of the VTG subunits. Similar to the hemolymph VTG (Fig. 5), the hepatopancre- atic VTG levels increased significantly in stage 3 and waned in FIG. 8. Proposed model for the VTG cleavage pattern in the female blue stage 4 (Fig. 6). Concomitantly, VT accumulated in the ovary crab. The hepatopancreas and hemolymph VTGs, which lack the first 18 aa of the signal peptide, are composed of 78.5-kDa and 207.3-kDa (Fig. 6). However, the VTG transcripts in the hepatopancreas subunits. VT in the ovary is composed of 78.5-kDa, 119.4-kDa, and 87.9- showed a different pattern, with a sharp increase at stage 4 (Fig. kDa subunits. The mature VT is derived by two consecutive cleavages: the 7A) after relatively moderate increases at stages 2 and 3 of first takes place in the hepatopancreas and the second occurs in the ovary. vitellogenesis. This discrepancy between the transcript level and The different subunits with calculated molecular masses and N-terminal aa sequences are indicated. The amino acid sequence shown in gray was protein content has not been observed previously in crustaceans. observed in the hepatopancreas but it is unlikely to occur under normal Instead, in P. japonicus hepatopancreas, the VTG mRNA levels conditions. decreased at the equivalent stage of vitellogenesis [34, 61]. VITELLOGENESIS IN THE BLUE CRAB 145

Monitoring VTG levels in the hemolymph allowed us to 4. Valle D. Vitellogenesis in insects and other groups—a review. Mem Inst determine the time required to reach each stage of ovarian Oswaldo Cruz 1993; 88:1–26. 5. Wallace RA. Vitellogenesis and oocyte growth in nonmammalian development under our experimental conditions. By comparing vertebrates. Dev Biol 1985; 1:127–177. the levels of VTG in the hemolymph samples of mated and 6. Browdy C. A review of the reproductive biology of penaeus species: unmated females, we observed some differences in the patterns perspectives on controlled shrimp maturation systems for high quality of vitellogenesis. The effect of semen on ovarian development is nauplii production. In: Wyban J (ed.), Proceedings of the Special Session of interest, since the female blue crab mates only once in her on Shrimp Farming. Baton Rouge, Florida; 1992:22–51. lifetime, stores the sperm in spermathechae, and uses it for 7. Byard EH, Aiken DE. The relationship between molting, reproduction and a hemolymph female specific protein in the lobster, Homarus americanus. several rounds of fertilization [26]. Since the regulation of Comp Biochem Physiol 1984; 77:749–757. vitellogenesis by peptides in semen, which simultaneously 8. Fyffe WE, O’Connor JD. Characterization and quantification of a decrease female receptivity to mating and enhance vitellogen- crustacean lipovitellin. Comp Biochem Physiol B 1974; 47:851–867. esis, has been reported in Drosophila [62], the regulation of 9. Lee FY, Shin TW, Chang CF. Isolation and characterization of female vitellogenesis in the blue crab by factors in the semen is plausible. specific protein (vitellogenin) in mature female hemolymph of the freshwater prawn, Macrobrachium rosenbergii: comparison with ovarian In the present study, we have shown that: 1) blue crab VTG vitellin. Gen Comp Endocrinol 1997; 108:406–415. is expressed mainly in the hepatopancreas, 2) prepro-VTG 10. Meusy JJ, Payen GG. Female reproduction in relation to molting and undergoes two cleavages, one in the hepatopancreas and a growth in malacostracan crustacea. Zool Sci 1988; 5:217–265. second one primarily in the ovary, 3) VT may be serologically 11. Tsukimura B. Crustacean vitellogenesis: its role in oocyte development. different from its hepatopancreatic primary precursor, 4) the Am Zool 2001; 41:465–467. VTG mRNA levels in the hepatopancreas do not correlate with 12. Vasquez-Boucard C, Ceccaldi HJ, Benyamin Y, Roustan C. Identification, purification, et characterisation de la lipovitelline chez un crustacean VTG protein levels at stage 4 in the hepatopancreas and decapode Natania Penaeus japonicus (Bate). J Exp Mar Biol Ecol 1986; hemolymph, and 5) the onset and advancement of vitellogen- 97:37–50. esis in the female blue crab are not dependent upon mating. 13. Kerr MS. The hemolymph proteins of the blue crab, Callinectes sapidus. However, the fate of the ovary at stage 4 is probably dependent II. A lipoprotein serologically identical to oocyte lipovitellin. Dev Biol upon semen-borne factors. 1969; 20:1–17. 14. Warrier S, Subramoniam T. Receptor mediated yolk protein uptake in the Vitellogenesis in most crustaceans of the Pleocyamata and crab Scylla serrata: crustacean vitellogenin receptor recognizes related Dendrobranchiata suborders is co-ordinated with the molting mammalian serum lipoproteins. Mol Reprod Dev 2002; 61:536–548. cycles, although the pattern varies across species [63]. In some 15. Avarre JC, Michelis R, Tietz A, Lubzens E. Relationship between cases, the two processes are tightly linked [10] and can affect vitellogenin and vitellin in a marine shrimp (Penaeus semisulcatus) and each others duration [64, 65]. Therefore, it is believed that molecular characterization of vitellogenin complementary DNAs. Biol vitellogenesis and molting are controlled by the same Reprod 2003; 69:355–364. 16. Wallace RA, Selman K. Cellular and dynamic aspects of oocyte growth in regulatory factors, which include neuropeptides of the XO- teleost. Amer Zool 1981; 197:325–343. SG complex [31, 66, 67], ecdysteroids [68–70], and juvenoid 17. Gerber-Huber S, Nardelli D, Haefliger JA, Cooper DN, Givel F, Germond hormones [38, 71]. Of great interest are the few species in JE, Engel J, Green NM, Wahli W. Precursor-product relationship between which the molting cycle is halted upon puberty, the female blue vitellogenin and the yolk proteins as derived from the complete sequence crab being among them. On the one hand, we have found that of a Xenopus vitellogenin gene. Nucleic Acids Res 1987; 15:4737–4760. 18. Engelman F. Insect vitellogenin: identification, biosynthesis and role of vitellogenesis, although not directly related to molting, shares vitellogenesis. Adv Insect Physiol 1979; 14:49–108. some features with other crustacean species, such as the site of 19. Yang W-J, Ohira T, Tsutsui N, Subramonian T, Huong DTT, Aida K, vitellogenesis and processing of VTG and ovarian develop- Wilder MN. Determination of amino acid sequence and site of expression ment. On the other hand, we have also observed a unique of four vitellins in the giant freshwater prawn, Macrobrachium rose- increase in VTG transcript levels in the hepatopancreas and nbergii. J Exp Zool 2000; 287:413–422. 20. Wolin EM, Laufer H, Albertini DF. Uptake of the yolk protein, ovary at stage 4 and a dependency on mating for the completion lipovitellin, by developing crustacean oocytes. Dev Biol 1973; 35:160– of egg maturation. These differences may be related to the halt 170. in molting and a different mode of regulation. Nevertheless, the 21. Charniaux-Cotton H. Vitellogenesis and its control in malacostracan present study establishes the basis for studying the mechanism crustacean. Am Zool 1985; 25:197–206. of regulation of vitellogenesis in this species. 22. Rankin SM, Bradfield JY, Keeley LL. Ovarian protein synthesis in the South American white shrimp, Penaeus vannamei, during the reproductive cycle. Invertebr Reprod Dev 1989; 15:27–33. ACKNOWLEDGMENTS 23. Yano I, Chinzei Y. Ovary is the site of vitellogenin synthesis in Kuruma prawn, Penaeus japonicus. Comp Biochem Physiol 1987; 86:213–218. We thank Oded Zmora for advice and help in the execution of the 24. Browdy CL, Fainzilber M, Tom M, Loya Y, Lubzens E. Vitellin synthesis project, John Stubblefield for reading the manuscript, and Sarah Bembe in relation to oogenesis in in vitro incubated ovaries of Penaeus and Bridget Bystry for technical assistance with the animals. We also semisulcatus (Crustacea, Decapoda, Penaeidae). J Exp Zool 1990; 255: thank Professors Richard Lee (Skidaway Institute of Oceanography, 205–215. Savannah, Georgia) for sharing the N-terminal sequences of VTG, Allen 25. Lui CW, Sage BA, O’Connor JD. Biosynthesis of lipovitellin by the Place (COMB) for advice on VT purification, and Dr. Zeev Pancer for crustacean ovary. J Exp Zool 1974; 188:289–296. guidance with the phylogenetic analysis. This article is contribution #06– 26. Churchill EP. Life history of the blue crab. Bull Bureau Fish 1919; 36:95– 142 from the Center of Marine Biotechnology (University of Maryland 128. Biotechnology Institute, Baltimore, MD). 27. Tagatz ME. Biology of the blue crab, Callinectes sapidus Rathbum, in the St. Johns River, Florida. Fish Bull 1968; 67:17–33. REFERENCES 28. Hines AH, Lipcius RN, Haddon AM. Population dynamics and habitat partitioning by size, sex, and molt stages of blue crab (Callinectes sapidus) 1. Charniaux-Cotton H, Payen G. Crustacean reproduction. In: Laufer H, in a subestuary of central Chesapeake Bay. Mar Ecol Prog Ser 1987; 36: Downer RGH (eds.), Endocrinology of Selected Invertebrate Types. New 55–64. York: Liss; 1988:279–303. 29. Lee RF, Puppione DL. 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Moult cycle-related changes in biological activity 70. Fyhn UE, Fyhn HJ, Costlow JD. Cirriped vitellogenesis: effect of of moult-inhibiting hormone (MIH) and crustacean hyperglycaemic ecdysterone in vitro. Gen Comp Endocrinol 1977; 32:266–271. hormone (CHH) in the crab, Carcinus maenas. Eur J Biochem 2003; 71. Rodriguez EM, Lopez Greco LS, Medesani DA, Laufer H, Fingerman M. 270:3280–3288. Effect of methyl farnesoate, alone and in combination with other 50. Khalaila I, Peter-Katalinic J, Tsang C, Radcliffe CM, Aflalo ED, Harvey hormones, on ovarian growth of the red swamp crayfish, Procambarus JH, Dwek RA, Rudd PM, Sagi A. Structural characterization of the N- clarkii, during vitellogenesis. Gen Comp Endocrinol 2002; 125:34–40. בקרה הורמונלית של רביה בנקבת הסרטן הכחול Callinectes sapidus על ידי נוירופפטידים בקומפלקס ה-XOSG בגבעול העין

מחקר לשם מילוי חלקי של הדרישות לקבלת תואר "דוקטור " לפילוסופיה"

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טבת תש "ע ינואר 2010 באר שבע בקרה הורמונלית של רביה בנקבת הסרטן הכחול Callinectes sapidus על ידי נוירופפטידים בקומפלקס ה-XOSG בגבעול העין

מחקר לשם מילוי חלקי של הדרישות לקבלת תואר "דוקטור " לפילוסופיה"

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העבודה נעשתה בהדרכת פרופ' אמיר שגיא ד"ר סוק צ'ונג, אוניברסיטת מרילנד, ארה"ב פרופ' יונתן זוהר, אוניברסיטת מרילנד, ארה"ב במחלקה למדעי החיים, הפקולטה למדעי הטבע, אוניברסיטת בן גוריון באר שבע

ובמכון לביוטכנולוגיה ימית, המכון הביוטכנולוגי של אוניברסיטת מרילנד, בולטימור, ארה"ב

אישור המנחים: פרופסור אמיר שגיא: ______ד"ר סוק צ'ונג: ______(Dr. J. Sook Chung) פרופסור יונתן זוהר:______אישור דיקן בית הספר ללימודי מחקר מתקדמים ע"ש קרייטמן- ______תקציר

רביה מהווה שלב חיוני ובלתי נפרד ממהלך החיים של כל יצור חי. להבנה ושליטה על תהליכי רביה קיים ערך

ישומי בממשק של יצורים בעלי ערך מסחרי. התפתחות גונדלית, ובעיקר תהליך הויטלוגניזה, הם תהליכים צורכי

אנרגיה ולפיכך מבוקרים על ידי גורמים אנדוקרינים. נכון לעכשיו, הגורמים המעורבים בבקרה של ויטלוגניזה

בסרטנים ומנגנוני פעולתם אינם מובנים בשלמותם. יתרה מזאת, למרות הבדלי צורות ופיזולוגיות הרביה של מיני

הסרטנים השונים, רק כמה מודלים כללים של בקרה הורמונלית הוצעו עד כה.

הסרטן הכחול, Callinectes sapidus הוא סרטן בעל ערך כלכלי-סביבתי התומך בענף דייג גדול בחוף המזרחי

האטלנטי של ארצות הברית ומפרץ מקסיקו, אשר נמצא בסכנת הכחדה בשל ירידה משמעותית באוכלוסיה הבוגרת . .

לנקבת הסרטן הכחול התנהגות ופיזיולגיה רבייתית יחודית, ובשל כך, עבודה זו התמקדה בלמידת ההתפתחות

גונדלית ובקרתה על-ידי נוירופפטידים של הקומפלקס אשר נמצא בגבעול העין -X organ sinus gland)

(XOSG. במחקר זה, נמדדה ההתפתחות הגונדלית ע"י מדידת ויטלוגניזה – תהליך יצירת חלבון החלמון וספיגתו

בביצית המתפתחת. בחלקה הראשון של העבודה, הונח הבסיס ללמידת הבקרה ההורמונלית ע"י הגדרת תהליך

הויטלוגניזה הפרופילים ושלו הן ברמת החלבון ורמת ביטוי הגן לאורך ההתפתחות הגונדלית. כתוצאה מכך, הדנ "א "

המשלים של גן הויטלוגנין של הסרטן הכחול שובט במלואו מההפטופנקריאס , ובכך תוקנה ההנחה השגויה שבוססה

על ידי דיווחים קודמים, שהויטלוגנין בסרטן הכחול מיוצר בשחלות. יתרה מכך, תוצאות מחקר זה מראות שלא כרוב

החלבונים, חלבון הויטלוגנין אינו מצטבר ונשמר בהפטופנקריאס אלא מופרש מיד לאחר התרגום. תופעה זו מצביעה

על כך ששעתוק ותרגום גן הויטלוגנין מבוקרים באופן נפרד.

בשלב הבא, זוהה הנוירופפטיד הגבעול עיני אשר מבקר ויטלוגניזה בנקבת הסרטן הכחול. מערכת הדגרה של

הפטופנקריאס פותחה וזיהתה את ההורמון מעכב ההתנשלות (Molt-inhibiting hormone- MIH) כבעל תפקיד

בבקרת הויטלוגניזה. במחקר זה התגלה של-MIH יש תפקיד מזרז של תהליכי הויטלוגניזה בשלבים המתקדמים ולא

הראשוניים של ההתפתחות הגונדלית, הן ברמת השעתוק והן ברמת התרגום וההפרשה של הויטלוגנין. בנוסף , לא ,

זוהה גורם מעכב ויטלוגינזה בנקבת הסרטן הכחול כפי שתואר במיני סרטנים אחרים. ממצאים אלה מנוגדים להנחה המקובלת של נוכחות הורמון מעכב התפתחות גונדלית וויטלוגניזה בכל הסרטנים. מציאותו של גורם מזרז ויטלוגניזה

קשור כנראה לפיזיולוגית הרביה היחודית של נקבת הסרטן אשר עוצרת את תהליכי ההתנשלות בשלב הבגרות

המינית.

במחקר זה, תוארה נוכחותם של אתרי קישור ספציפיים ל MIH בהפטופנקריאס של נקבת סרטן כחול

ויטלוגנית, אשר צפיפותם עולה בהתאם להתקדמות ההתפתחות הגונדלית. השוואת קינטיקת הקישור של MIH

לממברנות ההפטופנקריאס לזו של הקישור לממברנות בלוטת ההתנשלות (Y organ- YO) הראתה שני סוגי

קישור: קישור בעל זיקה נמוכה וצפיפות גבוהה לעומת זיקה גבוהה וצפיפות נמוכה, בהתאם. יתרה מכך , בעוד MIH

מפעיל cGMP כשליח משני ב - YO , הוא מפעיל cAMP בהפטופנקריאס. בנוסף לחדוש בממצא זה , נוכחותו של ,

קולטן ממברנלי בהפטופנקריאס, בנוסף ל - YO , מפריך פרדיגמה שניה הטוענת שה - YO הוא איבר המטרה היחיד

של MIH .

הממצא שהקולטן ל MIH בהפטופנקריאס של נקבת הסרטן הכחול שונה מזה של ה YO בתזמון הופעתו,

בפעילותו, בקינטיקת הקישור ובהעברת הסיגנל מציע מודל לתופעה רווחת ולא פתורה של פעילות הנוירופפטידים

של גבעול העין המתבטאת בריבוי תפקידים של הורמון אחד (pleiotropicity). מודל זה מציע שריבוי התפקידים

של הנוירופפטידים הללו מתווכים על-ידי קולטנים שונים לאותו הורמון, כאשר כל פונקציה מבוצעת על -ידי קולטן -

שונה .

לסיכום, מחקר זה מהווה הבנה מקיפה של תהליך הויטלוגניזה בנקבת הסרטן הכחול, אשר בו מוצג ה-MIH כבעל

תפקיד מרכזי בבקרת התהליך. ה-MIH מוצג כנושא ומבצע שני תפקידים מקבילים בנקבת הסרטן הכחול , בשני ,

התהליכים המשמעותיים ביותר בחיי הסרטנים : התנשלות (המקבילה לגדילה) ורביה. ובכך, מוצע שהאנטגוניזם בין

תהליכי התנשלות והרביה המתואר בסרטנים באופן כללי , יכול להיות מתווך על ידי הורמון אחד , ה MIH , אשר ,

מזרז התפתחות גונדלית מצד אחד ובו בעת מעכב התנשלות.