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Immunorecognition and distribution of progesterone receptors in the Chinese mitten crab Eriocheir sinensis during ovarian development.

ABSTRACT Progesterone is an important sex steroid that plays a vital role during ovarian development in crustaceans. In vertebrates, progesterone mediates reproduction via the progesterone receptor (PR). Previous studies have shown that PR is present in the ovary, hepatopancreas, and nerve tissues of some crustacean species. The Chinese mitten crab, Eriocheir sinensis, is an important aquaculture species in China and has become an invasive species in Europe and North America. A better understanding of the relevant reproductive mechanisms could potentially benefit artificial propagation and production of E. sinensis. Our intention was to immunorecognize and immunolocalize PR in the ovary, hepatopancreas, optic ganglion, brain ganglion, and thoracic ganglion of female E. sinensis using Western blot and immunohistochemistry. Changes in the distribution of PR were also investigated in these tissues during ovarian development. With an apparent molecular weight of 70 kDa, PR was identified in the optic, brain, and thoracic ganglion of female E. sinensis. During ovarian development, follicle cells were stained with positive PR at each ovarian stage. In germinal cells, positive PR was found in the cytoplasm only during the early ovarian development stages (I-III), whereas positive PR stained in the nucleus of germinal cells from stage III-stage V. In the hepatopancreas, PR was localized in the nucleus of resorptive cells as well as in the cytoplasm and nucleus of fibrillar cells for all stages of ovarian development. On the contrary, no PR-like substance was found in the other types of hepatopancreatic cells, such as blisterlike cells and embryonic cells, during ovarian development. However, a PR-like substance was detected in the nerve tissues of female E. sinensis. In the optic ganglion. PR was localized in the nucleus only of nerve cells. In the thoracic ganglion, PR was detected in the cytoplasm and nuclei of nerve cells during all ovarian development stages, with stronger detection during late ovarian development (stages III-V) rather than early (stages I and II). In addition, PR was localized in the brain ganglion, which is supported by evidence that the nuclei of nerve cells stained positively for PR antibody during all ovarian development stages. We suggest that progesterone not only regulates vitellogenesis and ovarian development directly by binding PR in the ovary and hepatopancreas, but also modulates indirectly ovarian development through nerve tissue.

KEY WORDS: Chinese mitten crab, Eriocheir sinensis, progesterone receptor. Western blot, immunohistochemistry, ovarian development

INTRODUCTION

In vertebrates, sex steroid hormones, such as progestins, estrogens, and androgens, are involved in sex-specific regulation of molecular processes (Kohler et al. 2007, Lafont & Mathieu 2007). It has been reported that most vertebrate sex steroids also occur in invertebrate tissues (Janer & Porte 2007). Progesterone is one of the important progestins, and it plays an important role in mediating female reproductive physiology and behavior in all vertebrates. In mammalian species, progesterone is indispensable for ovulation to implantation, maintenance of pregnancy, and breast development (Arck et al. 2007, Gellersen et al. 2009). The effects of progesterone can be mediated by genomic and nongenomic actions, and nuclear and membrane progesterone receptors (PRs) are thought to be involved in these female reproductive events (Thomas 2003, Arck et al. 2007, Thomas et al. 2007, Zhu et al. 2008, Dressing et al. 2011).

In invertebrates, progesterone was first identified in starfish (Pisatser ochraceus) as early as 1960 by Botticelli et al. Subsequently, progesterone has also been detected in some crustacean species, such as the American lobster, Homarus americanas (Couch et al. 1987); the mud crab Scylla serrata (Warrier et al. 2001); the mole crab Emerita asiatica (Gunamalai et al. 2006); the swimming crab Portunus trituberculatus (Feng et al. 2009); the Chinese mitten crab, Eriocheir sinensis (Lu et al. 2010); and the mud crab Scylla paramamosain (Ye et al. 2010b). The concentrations of progesterone fluctuate during ovarian development in crustaceans. In E. sinensis, the progesterone concentrations of ovary and hepatopancreas increase markedly during the early and middle vitellogenic stages, and then decrease significantly during the late vitellogenic stage (Lu et al. 2010). Other investigations indicate that a certain amount of exogenous progesterone has positive effects on oocyte development, vitellogenesis, vitellogenin messenger RNA levels, and spawning of female crustaceans (Yano 1985, Ramachandra Reddy et al. 2006, Meunpol et al. 2007). Unfortunately, the mechanisms of progesterone activity in crustaceans are unclear. Therefore, the regulation of progesterone during ovarian development in crustaceans has become of key interest in the study of invertebrate reproductive biology. To date, PR has been identified by immunological methods in some tissues of crustaceans, including ovary, hepatopancreas, and in different nerve tissues (Paolucci et al. 2002, Song et al. 2010, Yeet al. 2010a, Ye et al. 2010b). Therefore, it is proposed that the regulation of progesterone is mediated mainly by the binding of PR during ovarian development in crustaceans (Preechaphol et al. 2010).

The Chinese mitten crab, Eriocheir sinensis, originally distributed in East Asian countries, has become an invasive species in Europe and North America (Dittel & Epifanio 2009). This crab has become a commercially important aquaculture species in China, with annual production reaching ~600,000 t (Qiu et al. 2012). A better understanding of the reproductive mechanisms of E. sinensis would potentially benefit its artificial propagation and fisheries management. In this study, we intended to immunorecognize and immunolocalize PR in the ovary, hepatopancreas, optic ganglion, brain ganglion, and thoracic ganglion of female E. sinensis via Western blot assay and immunohistochemistry. Changes in and distribution of PR were also investigated in these tissues during ovarian development. Our investigation facilitates further understanding of reproductive physiology and modulation of sex steroid hormones during ovarian development in E. sinensis.

MATERIALS AND METHODS

Crab Maintenance

Female Eriocheir sinensis (body weight, 60-150 g) were obtained from a Chongming mitten crab farm near Shanghai, China, May 2011 to March 2012. Fifteen to twenty female crabs were taken randomly each month. The crabs were transported alive to the laboratory at Shanghai Ocean University, Shanghai, China, where they were maintained in aquarium tanks (length x width x depth, 75 cm x 53 m x 45 cm) filled with a recirculating water system. The temperature ranged from 18-25[degrees]C. The pH, dissolved oxygen, ammonia, and nitrite concentration were monitored regularly and maintained at pH 7.0-9.0; dissolved oxygen, more than 5 mg/L; ammonia, less than 0.5 mg/L; and nitrite, less than 0.2 mg/L. All crabs were fed daily at 17:00 with fresh fish at a ration of 3%-10% total biomass, adjustable based on daily observation of residual feed in each tank. Before daily feeding, feces and uneaten food were removed by siphoning. The crabs were acclimated for at least 7 days before being sacrificed.

Crab Dissection and Ovarian Staging

Animals were anesthetized on ice. Ovaries, hepatopancreas, and other nerve tissue were removed and treated for immunocytochemistry and Western blot analysis. Ovarian staging of Eriocheir sinensis was based on the ovarian morphology and histology modified from Xue et al. (1987). There are 5 ovarian stages during the ovarian development cycle: stage I, before puberty molting and with a triangular pleonal flap, with the ovary mainly include oogonium; stage II, the ovary is milky white and comprised mainly of previtellogenic oocytes and endogenous vitellogenic oocytes; stage III, the ovary is light yellow or tan and comprised primarily of exogenous vitellogenic oocytes and endogenous vitellogenic oocytes; stage IV, the ovary is deep purple and comprised mainly of nearly mature oocytes and exogenous vitellogenic oocytes; and stage V: the ovary is chocolate brown and comprised mainly of mature oocytes. Four to 6 females were dissected and staging for further study.

Chemical and Reagents

Polyclonal rabbit antihuman PR antibody (category code sc538; recognizes the C terminus of human) was from Santa Cruz Biotechnology USA (Dallas, TX). The prediluted goat antirabbit immunoglobulin (Ig) G peroxidase conjugate (category code PV-6001) was from Zhongshan Biological Technology, Ltd (Beijing, China). The goat antirabbit IgG peroxidase conjugate (category code BA1054), prestained protein molecular weight markers (category code ARI 113), N-N'-methylene-bis-acrylamide (category code AR1161), PMSF (AR1178), sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) protein loading buffer (AR1112), TBS (AR0193), TBST (AR0195), phosphate-buffered saline (PBS; 10 mM, pH 7.2-7.6; AR0030), and citrate acid buffer (10 mM, pH 6.0; AR0024) were from Boster Biological Technology, Ltd. (Wuhan, Hubei Province, China). Diaminobenzidine (DAB; category code BY11057) was from Sigma Chemical Co. (St. Louis., MO). The buffer used, radio immunoprecipitation assay lysis buffer (category code WB0103), was from Weiao Biological Technology, Ltd. (Shanghai, China). Western buffers consisted of SDS-PAGE protein loading buffer, TBS, and TBST with 0.05% Tween-20. Immunocytochemistry buffers included PBS and citrate acid buffer.

Western Blot

Optic ganglia, brain ganglia, thoracic ganglion, and muscle from stage III were dissected, weighed, minced, and homogenized in the radio immunoprecipitation assay lysis buffer according to the manufacturer's instruction. Briefly, samples were homogenized on ice by glass potter sand and centrifuged at 10,000gfor 15 min at 4[degrees]C. The supernatants were then collected and protein concentrations were determined by micro-UV spectrophotometer (Q5000; Quawell). The protein extracts were separated by on 5%-10% gradient SDS-PAGE. After electrophoresis, proteins were transferred to the nitrocellulose membrane using a Transblot chamber (BG-VER-Mini; Baygene, Beijing, China) for 80 min at 150 mA. The nitrocellulose was rinsed 3 times in TBS for 10 min and then blocked for 1 h at room temperature with skim milk. After rinsing 3 times with TBST for 10 min, the nitrocellulose was incubated with PR antibodies (dilution, 1:200 dilution) for 12 h at 4[degrees]C. The blot was rinsed 3 times in TBST for 10 min and then incubated with goat antirabbit IgG peroxidase conjugate (dilution, 1:500) at room temperature for 2 h. After washing, immunoreactive bands were revealed by incubation with DAB. The Western blot image was captured by digital camera (EOS D60; Canon, Tokyo, Japan).

Immunohistochemistry

The ovaries, hepatopancreas, and nerve tissues in different stages were fixed in Bouin's solution for 24 h before being dehydrated. The samples were then dehydrated progressively in ascending concentrations of ethanol solution, and the dehydrated specimens were cleared with xylene before being embedded in paraffin wax (melting point, 56-58[degrees]C). Serial sections of 5-7 pm were cut using a rotary microtome (Leica RM 2016; Leica Microsystems Inc., Bannockburn, IL) and attached to glass slides with egg white liquid and glycerin solution (vol./vol., 1:1). The glass slides were placed in 10 mM citrate acid buffer (pH 6.0) and then were heated to 94[degrees]C for 15 min of thermal-induced antigen retrieval by an electromagnetic oven. After cooling the slides to room temperature, they were rinsed 3 times in PBS for 5 min. Slides were then incubated in 0.3% [H.sub.2][O.sub.2] for 20 min to block the activity of endogenous peroxidase. After rinsing 3 times with PBS for 5min, sections were incubated at 4[degrees]C with antihuman PR antibody (dilution in PBS, 1:50) overnight. The sections were then rinsed 3 times in PBS for 5 min and incubated with prediluted goat antirabbit IgG peroxidase conjugate for 30 min at room temperature. The immunostaining signal was visualized by using DAB as the chromogen. Sections were then dehydrated and mounted in Permount. All sections were observed with a Leica DM2500 microscope equipped with a computer-controlled display system (Leica Microsystems Inc., Wetzlar, Germany) while microphotographs were taken with a digital camera (Leica DFC295; Leica Microsystems Inc.).

[FIGURE 1 OMITTED]

RESULTS

Western Blot Analysis of PR

Western blot analysis was carried out on protein samples from the optic ganglion, brain ganglion, thoracic ganglion, and muscle. As previous studies have demonstrated, there was a strong immunoreaction of antihuman PR antibody against PR in crustaceans based on a conservative region of the PR protein chain (Coccia et al. 2010, Ye et al. 2010a, Ye et al. 2010b). Rabbit antihuman PR antibody was used to detect the PR homolog of Eriocheir sinensis in this experiment. As shown in Figure 1, there was only 1 band detected in the Western blot of nerve tissue, with an apparent molecular weight of 70 kDa. Muscle of E. sinensis and bovine serum albumin were used in this experiment as negative controls and they did not display any immunoreactive bands in the Western blot.

Distribution and Change of PR in the Ovary

During ovarian development of female Eriocheir sinensis, PR-positive substances were detected mainly in follicle cells, germinal cytoplasm of early ovarian stages (stages I-III), and oocyte nuclei of later ovarian stages (stages III-V). The distribution and immunohistological intensity of PR changed during ovarian development (Table 1). During stage I, the ovary contained mainly oogonium, and many follicle cells were distributed among germinal zones. At this stage, PRs were localized primarily in follicle cells (strongly positive) and the cytoplasm of oogonium (moderately positive; Fig. 2A). Stage II ovaries consisted mainly of previtellogenic oocytes and endogenous vitellogenic oocytes. During this stage, follicle cells moved gradually to oocytes nearby, and PR substances were localized mainly in follicle cells (strongly positive) and the cytoplasm of oocytes (moderately positive; Fig. 2B, C). During stage III, the ovary contained primarily exogenous vitellogenic oocytes, and a small amount of endogenous vitellogenic oocytes was also observed in the center of germinal zones. During this stage, the oocytes were enclosed by a monolayer of follicle cells, and PR immunoreactivity was found in the cytoplasm of endogenous vitellogenic oocytes (strongly positive) and the nucleus of exogenous vitellogenic oocytes (moderately positive; Fig. 2D). The follicle cells were also strongly positive for immunoreactivity with PR antibody (Fig. 2E). In stage IV ovaries, the majority of oocytes were nearly mature oocytes, and follicle cells were merged into the cell membrane of nearly mature oocytes. Both the nucleus of nearly mature oocytes and follicle cells showed moderate PR immunoreactivity at this stage (Fig. 2F). During the final stage (stage V), mature oocytes were the typical oocytes found in the ovary, whereas follicle cells were hardly recognized. Progesterone receptor immunoreactivity existed in the nucleus of mature oocytes (moderately positive; Fig. 2G), whereas the immunoreactivity of follicle cells decreased to weakly positive during this stage. In addition, no immunoreactivity was detected in the muscle of E. sinensis used as a negative control (Fig. 2M).

Distribution and Change of PR in the Hepatopancreas

The hepatopancreas of Eriocheir sinensis is composed of multiple blind-end tubules, and the tubule walls consist of 4 kinds of epithelial cells, embryonic cells, resorptive cells, fibrillar cells, and blisterlike cells. Progesterone receptor-positive substances were localized in the nucleus of resorptive cells as well as in the nucleus and cytoplasm of fibrillar cells in the hepatopancreas for all ovarian development stages (stages I-V; Fig. 2H-L, Table 1), and the degree of staining of the immune response was strongly positive. On the contrary, no positive PR-like substance was found in the other types of hepatopancreatic cells.

[FIGURE 2 OMITTED]

Distribution and Change of PR in Neural Tissues

The optic ganglion of Eriocheir sinensis consists of 4 parts: the lamina ganglionaris, medulla externa, medulla interna, and medulla terminalis. The X-organ (XO) is located in the basal lateral medulla terminalis, and the sinus gland is between the medulla interna and the medulla terminalis. Progesterone receptor immunoreactivity was found exclusively in the nucleus of neurons for all ovarian development stages (moderately positive; Table 2). Briefly, PR-positive substances were observed not only in the nerve cells of the sinus gland and X-organ, but also in the outer margin of the lamina ganglionaris, medulla externa, and medulla interna (Fig. 3A-F). The thoracic ganglion is a part of the thoracic ganglion mass, which also includes the subesophageal ganglion and abdominal ganglion. In the thoracic ganglion, PR immunoreactivity was detected in the nucleus and cytoplasm of nerve cells. During ovarian stages I and II, nerve cells of the thoracic ganglion had weakly positive PR immunoreactivity whereas PR immunoreactivity increased to moderately positive in nerve cells during the mid to late ovarian stages (stages, III-V; Fig. 3G-J, Table 2). The brain ganglion of E. sinensis is composed of protocerebrum, deutocerebrum, and tritocerebrum. Strongly positive PR immunoreactivity existed only in the nucleus of nerve cells for each ovarian stage, except for stage IV, when it was moderately positive (Fig. 3K-0, Table 2).

DISCUSSION

Isoforms and Molecular Weight of PR

Progesterone receptor protein has been identified in several crustacean species, including the mud crab Scylla paramamosain (Ye et al. 2010b), the swimming crab Portunus trituberculatus (Chen et al. 2013), and the freshwater crayfish Austropotamobius pallipes (Coccia et al. 2010), using immunological methods. To explore the presence of PR in Eriocheir sinensis, Western blot analysis with a polyclonal PR antibody was used to confirm the PR homolog of E. sinensis in the nerve tissues of this species. Only 1 immunoreactive band of 70 kDa was detected in these tissues. No positive band was observed in the control group with the same antibody. This finding is similar to previous results obtained for other invertebrates, including Octopus vulgaris, A. pallipes and S. paramamosain (Di Cosmo et al. 1998, Paolucci et al. 2002, Ye et al. 2010b). However, unlike invertebrates, more than one PR isoform has been identified in vertebrates (Sasaki et al. 2001, Merlino et al. 2007). Most vertebrates have 2 isoforms, PR-A and PR-B, which may originate from alternative splicing of a single gene and act as ligand-activated transcription factors to regulate the expression of reproductive target genes (Sasaki et al. 2001, Custodia-Lora & Callard 2002, Conneely et al. 2000). Recent studies have also revealed differences in molecular weight, expression distribution, and physiological function between PR-A and PR-B (Wang et al. 1998, Polzonetti-Magni et al. 2004, Merlino et al. 2007, Hammouche et al. 2012). Therefore, the PR differences between invertebrates and vertebrates may indicate they have different modulating mechanisms.

Relationship Between the Distribution of PR and Ovarian Development

The concentrations of progesterone in the hepatopancreas, ovary, and hemolymph of Eriocheir sinensis have been measured at different vitellogenesis stages by radioimmunoassay (Lu et al. 2010). Results showed that the change in progesterone levels correlated with the progress of vitellogenesis, suggesting progesterone might be involved in this process. The immunological evidence of PR has also been reported in some tissues of decapod crustaceans, including the ovary and hepatopancreas in the Austropotamobiuspallipes (Paolucci et al. 2002), as well as the optic ganglion, thoracic ganglion, and brain ganglion in Scylla paramamosain (Song et al. 2010, Ye et al. 2010a). In vertebrates, previous studies have demonstrated that the major physiological regulation functions of progesterone are mediated by the binding of PR (Arck et al. 2007, Thomas et al. 2007, Ye et al. 2010b). In view of these situations, it is suggested that PR may be present in the tissues of E. sinensis to bind progesterone for the regulation of ovarian development. With Western blot, the PR homolog was also identified in tissues of E. sinensis during ovarian development.

To explore the possible mechanisms of progesterone function in ovarian development of female Eriocheir sinensis, it is necessary to detect further the change and distribution of PR in the tissues associated with vitellogenesis. Our immunochemical results showed that the follicle cells were stained with strongly positive PR for each ovarian stage in female E. sinensis, whereas PR immunoreactivity weakened from stage III to stage V. As for the germinal cells, positive PR existed in the cytoplasm during the early ovary development stages (stages I-III), including oogonium and endogenous vitellogenic oocytes, whereas positive PR stained in the nucleus of oocyte cells during stage III to stage V. Previous studies have shown that progesterone can promote the differentiation of oogonium to oocytes and the synthesis of vitellogenin during early ovarian developmental stages (Warrier et al. 2001, Paolucci et al. 2002). The existence of PR in oogonium and endogenous vitellogenic oocytes suggests that progesterone may mediate these processes via the binding of PR during early ovary development. With the development of oocytes, immunological reaction of PR transferred from cytoplasm into the nucleus. Similar results have been reported in Scylla paramamosain (Ye et al. 2010b). Although Lu et al. (2010) found that progesterone levels decreased significantly during late vitellogenesis (ovarian stages IV and V), the role of progesterone still cannot be ignored during these stages, and may be involved in facilitating the final maturation of oocytes and breakdown of the germinal vesicle (Yano 1985, Warrier et al. 2001, Gunamalai et al. 2006). The occurrence of PR immunoreactivity in the nucleus of exogenous vitellogenic oocytes, nearly mature oocytes, and mature oocytes confirmed the effect of progesterone existing during the late stages of ovarian development. A previous study has shown that the cytosol receptor of the steroid complex occurred earlier than the nuclear receptor of the steroid complex (Paolucci et al. 2002); therefore, the transformation of PR immunoreactivity from cytoplasm into nucleus may indicate the trafficking of PR between the nucleus and the cytoplasm in E. sinensis (Pinter & Thomas, 1995, McKenna & O'Malley 2002, Thomas 2003, Thomas et al. 2007). Actually, oocyte maturation of E. sinensis consists of growth maturity and physiological maturity (Gu & He 1997). At the end of ovarian growth maturity, the germ vesicle breakdown of mature oocytes occurs, and then the oocytes reach final physiological maturity and are ready to spawn (Meusy & Payen 1988). However, the duration of growth maturity to physiological maturity is relatively short, and it is difficult to observe in female E. sinensis broodstock (Gu & He 1997). In the current study, the final ovarian stage (stage V) is defined as the growth of mature oocytes, not physiological maturity for female E. sinensis. Therefore, another study needs to investigate the distribution and function of PRs during the process of oocyte physiological maturity and spawning for female E. sinensis.

[FIGURE 3 OMITTED]

The follicle cell is a key cell in ovarian development of crustaceans, and the migration of follicle cells could be used as an important indicator for the start of exogenous vitellogenesis in decapod crustaceans (Cheng et al. 2002). In vertebrates, such as fish and amphibians, the follicle cell is the major site of synthesis of maturation-inducing steroids (Patino & Purkiss 1993). Whether the follicle cell has similar capabilities in crustaceans is still unclear. It has been observed that follicle cells play an important role in the transportation of yolk precursors (vitellogenin) into oocytes directly or indirectly during ovarian development of Eriocheir sinensis (Du et al. 1999). Furthermore, previous research has also reconfirmed progesterone, as a derivative of cholesterol, possibly bound the vitellogenin at its lipid binding sites during vitellogenin transportation (Warrier et al. 2001). Here, PR immunoreactivity was observed in the follicle cells of the ovary during ovarian development. Similar results have been found in other invertebrate species, such as Octopus vulgaris (Di Cosmo et al. 1998), and Scylla paramamosain (Ye et al. 2010b). These results imply a possibility that PR may mediate progesterone activities in follicle cells for the transportation of vitellogenin.

In crustaceans, the hepatopancreas is not only the organ of nutrient digestion, absorption, and storage, but also it plays an extremely important role in reproductive endocrinology (Holland & Cabbott 1971, Rosa & Numes 2002). It has been shown that high titers of progesterone were observed in the hepatopancreas during early vitellogenesis of Eriocheir sinensis (Lu et al. 2010). In addition, Li et al. (2006) found both the ovaries and hepatopancreas were the sites of vitellogenin synthesis whereas the hepatopancreas was the extraovarian site of exogenous vitellogenesis for female E. sinensis. As observed in the current study, during ovarian development, the nucleus of hepatopancreatic resorptive cells as well as the nucleus and cytoplasm of hepatopancreatic fibrillar cells were stained with strongly positive PR. These observations suggest a possibility that hepatopancreas may be a site of PG biosynthesis, which can then bind with PR to promote the synthesis of vitellogenin in hepatopancreas (Swevers et al. 1991, Warrier et al. 2001).

The optic ganglion, thoracic ganglion, and brain ganglion have been identified as important neuroendocrine organs in crustaceans, and some hormones excreted from these neuroendocrine organs play an important role in the regulation of gonadal maturation (Mazurova et al. 2008, Raviv et al. 2008, Nagaraju 2011). It is well known that gonadal maturation of crustaceans is regulated principally by two antagonistic neural hormones: gonad-inhibiting hormone synthesized and secreted from the X-organ-sinus gland complex of the eyestalk, and gonad-stimulating hormone thought to be produced by the brain and thoracic ganglion (Subramoniam 2011, Swetha et al. 2011). Recently, immunological evidence has been reported that PR existed in the optic, thoracic, and brain ganglion of the mud crab Scylla paramamosain (Song et al. 2010, Ye et al. 2010a). However, whether PR is mediating progesterone activities in the neural tissues has not been reported for crustaceans. In vertebrates, sex steroid hormones are involved in the feedback regulation of neuroendocrine systems by combining with steroid hormone receptors in nerve tissues, such as the hypophysis (Moguilewsky & Raynaud 1980), hypothalamus (Guerriero & Ciarcia 2001), and locus coeruleus (Helena et al. 2006).

The presence of receptor which specifically bind to steroids is a prerequisite for hormone action (Ye et al. 2010a, Swetha et al. 2011). Consequently, the existence of PR immunoreactivity in the optic, thoracic, and brain ganglions of Eriocheir sinensis indicates the potential ability of PR to bind progesterone and act as a mediator in progesterone activation of neuroendocrine regulation for female ovarian development. However, the exact mechanism of progesterone activities in the nerve tissue remains unknown for crustaceans. Clearly, further research is needed in this area in the future.

Mechanism of Progesterone Action for Ovarian Development

Progesterone plays an important role across all vertebrates in mediating female reproductive physiology and behavior. Besides these functions, progesterone is also the precursor of some other steroid hormones, suggesting a key role in the regulation of steroid metabolism in vertebrates (Janer & Porte 2007, Swetha et al. 2011). Therefore, this area has drawn much attention from reproductive biologists to investigate progesterone function and its mechanism for animal reproduction (Katsu et al. 2008, Stout et al. 2010, Hammouche et al. 2012). Progesterone is considered to bind its corresponding receptor (PR) to generate physiological actions (Yong-Feng, 2002, Thomas et al. 2007). In vertebrates, previous studies have indicated there are two major actions of progesterone during ovarian development and vitellogenesis: the classic genomic mechanism and a rapid nongenomic mechanism. The classic genomic mechanism involves the passage of progesterone through the plasma membranes of target cells, where they bind to specific intracellular receptors called nuclear progesterone receptors (nPRs). The activated PR complex then binds to response elements on target genes to alter their transcriptional activity (Thomas et al. 2007, O'Connell et al. 2011). However, many actions of progesterone are too rapid to be mediated by the classic genomic mechanism, which is relatively slow and typically occurs over a timescale of hours to days. Recent research has shown that progesterone also acts at the cell surface of target tissues and cells to initiate rapid activation of intracellular signaling pathways, alterations in calcium concentrations, and second messengers occurring within a few minutes or even a few seconds (Thomas et al. 2007, Gellersen et al. 2009). These rapid progesterone actions are mediated by binding to the receptors on the cell surface, called membrane progesterone receptors (Bramley 2003, Thomas 2003, Dressing et al. 2011).

Although it is clear for the manner of progesterone mechanism in vertebrate reproduction, particularly in mammals, the precise action mechanism of progesterone remains unclear for crustaceans throughout the reproductive cycle (Kohler et al. 2007, Coccia et al. 2010). However, the general scheme seems remarkably similar in all organisms. To date, research in progesterone regulation in crustaceans has concentrated mainly on the effects of exogenous progesterone on ovarian development and vitellogenesis, with no available publications found in the action mechanism of progesterone (Kulkami et al. 1979, Meunpol et al. 2007, Coccia et al. 2010). Recent studies have shown that nPR is present in the ovary, hepatopancreas, and nerve tissues of Austropotamobius pallipes as well as Scylla paramamosain by immunohistochemical methods with anti-nPR antibody (Paolucci et al. 2002, Ye et al. 2010b). We also found nPR was widely distributed in these tissues in female Eriocheir sinensis, and suggest that the action of nPR may be present during ovarian development in crustaceans. Recently, progestin membrane receptor component 1, a kind of membrane PR, has been identified in the giant tiger shrimp Penaeus monodon, and played an important role during ovarian development of this species (Preechaphol et al. 2010). Therefore, we propose that genomic and nongenomic mechanisms of progesterone action are also involved in the progesterone regulation of ovarian development in crustaceans. The presence of PR-positive substances in the nerve tissue of crustaceans suggests that progesterone may modulate ovarian development indirectly through the feedback of the neuroendocrine system.

ACKNOWLEDGMENTS

This study was supported by grants from the National High Technology Research R&D Program of China (2012AA10A40905), Shanghai Municipal Natural Science Foundation project (12ZR1413000), and the Outstanding Academic Leader Project (12XD1402700) from Shanghai Municipal Science and Technology Commission. Infrastructure costs were supported by the second batch innovation research group developing project in the universities of Shanghai (no. 2009-026) and Shanghai Universities First-Class Disciplines Project of Fisheries (no. 2012-62-0908) from Shanghai Municipal Education Committee.

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XUGAN WU, (1) ([dagger]) HAO CHEN, (1) ([dagger]) ZHIJUN LIU (1) AND YONGXU CHENG (1,2) *

(1) Key Laboratory of Exploration and Utilization of Aquatic Genetic Resources, Shanghai Ocean University, No 999 Huchenghuan Road, Lingang New District, Shanghai 201306, China; (2) Shanghai Universities Knowledge Service Platform, Shanghai Ocean University, No 999 Huchenghuan Road, Lingang New District, Shanghai 201306, China

* Corresponding author. E-mail: chengyongxucrablab@hotmail.com

([dagger]) These authors contributed equally to this work.

DOI: 10.2983/035.033.0105
TABLE 1. The distribution and change of progesterone receptor in the
ovary and hepatopancreas during ovarian development of Eriocheir
sinensis.

                                    Cellular type
Ovarian              Cytoplasm of    Nucleus of       Nucleus of
stage     Follicle    germ cell       germ cell     resorptive cell

I           +++           ++              -               +++
II          +++           ++              -               +++
III         +++          +++             ++               +++
IV           ++           -              ++               +++
V            +            -              ++               +++

          Cytoplasm and
Ovarian     nucleus of
stage     fibrillar cell

I              +++
II             +++
III            +++
IV             +++
V              +++

+++, strongly positive; ++, moderately positive; +, weakly
positive; -, negative.

TABLE 2. The distribution and change of progesterone receptor in the
optic, thoracic, and brain ganglion during ovarian development of
Eriocheir sinensis.

                                  Tissue type

                                                     Brain ganglion,
Ovarian    Optic ganglion,    Thoracic ganglion,         nucleus
stage        nucleus of          cytoplasm and        of nerve cell
             nerve cell      nucleus of nerve cell

I                ++                    +                   +++
II               ++                    +                   +++
III              ++                   ++                   +++
IV               ++                   ++                   ++
V                ++                   ++                   +++

+++, strongly positive; ++, moderately positive; +, weakly positive.
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Author:Wu, Xugan; Chen, Hao; Liu, Zhijun; Cheng, Yongxu
Publication:Journal of Shellfish Research
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Geographic Code:9CHIN
Date:Apr 1, 2014
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