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Effects of estradiol and progesterone on the reproduction of the freshwater crayfish Cherax albidus.

Introduction

Reproductive physiology in crustaceans is highly controlled and regulated by the nervous and endocrine systems (Engelmann, 1994). Endocrine control of female reproduction is governed by a variety of hormonal and neuronal factors that involve neuropeptide hormones, such as gonad-stimulating hormone (GSH) and vitellogenesis-inhibiting hormone (VIH); terpenoids, such as methyl farnesoate, a stimulator of vitellogenesis; ketosteroids, such as ecdysteroids; and finally sex steroids such as estradiol and progesterone (Huberman, 2000; Zapata et al., 2003). Ecdysteroids are the primary hormonal factors of molting and positively affect vitellogenesis also (Subramoniam, 2000). Crustacean ecdysteroids are very polar molecules, and there is no evidence for carrier proteins in the hemolymph. However, nucleotide sequences responsive to DNA binding domain (DBD) of steroid receptors have been found in the DNA of Metapenaeus ensis, providing evidence that steroids, upon the binding to specific receptors, activate the transcription of specific genes (Chan, 1998).

Vertebrate-type steroids have been reported to be present in the hepatopancreas, ovary, and hemolymph of crustaceans, their levels changing in correlation with the oocyte maturation cycle (Lafont and Mathieu, 2007 for review). Indeed, a positive correlation between vitellogenin (VTG) circulating levels and hemolymph levels of progesterone and 17[beta]-estradiol have been reported for crabs (Shih, 1997; Warrier et al., 2001; Zapata et al., 2003) and shrimps (Quinitio et al., 1994; Yano, 2000). Moreover, the stimulatory effects of some vertebrate-type steroids such as 17[beta]-estradiol and progesterone on ovarian growth in decapods have been reported by several authors. In the crayfish Macrobrachium rosenbergii, 17[beta]-estradiol behaved as a metabolic activator at the cellular level, causing an increase in mitochondrial ATP-ase, cytosolic malate dehydrogenase, and glucose-6-phosphate dehydrogenase in the hepatopancreas (Ghosh and Ray, 1993a). In Procambarus clarkii, 17[beta]-estradiol and 17[alpha]-hydroxyprogesterone produced a significant increase in the gonadosomatic index, while only the latter brought about a significant increase in oocyte diameter (Rodriguez et al., 2002b). On the other hand, 17alpha]-hydroxyprogesterone, when administered in combination with methyl farnesoate, inhibited oocyte growth by suppressing the stimulatory action of the methyl farnesoate on the ovary of Procambarus clarkii (Rodriguez et al., 2002a); and in Cherax quadricarinatus 17[alpha]-hydroxypro-gesterone administration failed to increase the number of spawns during the reproductive period (Cahansky et al., 2003). An endogenous origin for vertebrate-type steroid hormones has been investigated through the presence of steroidogenic enzymes. The activity of 17[beta]-hydroxysteroid dehydrogenase, a key enzyme in steroid metabolism, has been determined in the hepatopancreas and the ovary of Macrobrachium rosenbergii. The enzyme activity was upregulated by 17[beta]-estradiol and thus was higher in the hepatopancreas of maturing females (Ghosh and Ray, 1993b).

Despite numerous reports on the occurrence of vertebrate-type steroid hormones in crustaceans, their exact mode of action remains to be elucidated. The evidence points to a physiological role for the vertebrate-type steroids in crustaceans, which implies the presence of specific receptors. Immunological evidence has recently been reported for progesterone receptors in the ovary and for both progesterone and estradiol receptors in the hepatopancreas of the crayfish Austropotamobius pallipes (Paolucci et al., 2002), suggesting dichotomous roles for these hormones in vitellogenesis.

Among crustaceans there is evidence that vitellogenin is synthesized in several tissues including the hepatopancreas and the ovary itself (Shafir et al., 1992; Khayat et al., 1994; Lee and Chang, 1999). In these animals, secondary vitellogenesis is accompanied by the accumulation of yolk, composed of lipids, carbohydrates, and proteins (Adiyodi and Subramoniam, 1983; Charniaux-Cotton and Payen, 1988), circulating in the hemolymph as VTG, a high-density lipoprotein (HDL) (Lee and Puppione, 1988; Komatsu et al., 1993). The HDL called LPII in Cherax quadricarinatus is specific for secondary vitellogenic females and contains four polypeptides with masses between 80 and 208 kDa (Abdu et al., 2000; Yehezkel et al., 2000). The lack of LPII in the hemolymph of spawning females and in females that are not in their reproductive season indicates that LPII may be a useful marker of secondary vitellogenesis.

A complete VTG cDNA has been cloned in Cherax quadricarinatus. The gene is expressed as a single transcript and is present in the hepatopancreas of females during secondary vitellogenesis (Abdu et al., 2002; Serrano-Pinto et al., 2004); in intersex individuals it is negatively regulated by the androgenic gland, as demonstrated by the fact that removal of these glands results in VTG transcription (Abdu et al., 2002). However, the investigation of VTG expression in both female and intersex crayfish reveals that the gene is under a multifactorial regulation (Khalaila et al., 2001). The X-organ-sinus gland (XO-SG) inhibits the VTG gene, and its removal results in a partial recovery of VTG synthesis in males and a total recovery in intersex individuals (Shechter et al., 2005). On the basis of these data, VTG in crustaceans seems to be under a multifactorial regulation, including vertebrate-like steroids, although with different features according to species.

In this study we investigate the effect of the sex steroids 17[beta]-estradiol and progesterone on crayfish reproduction, using VTG as a marker. We employed the genus Cherax (spp.) as a crayfish model because of the advanced status of knowledge about its VTG structure and synthesis.

Materials and Methods

Animals

Males and females of the crayfish Cherax albidus (Clark, 1936) were obtained from the "Pilot Aquaculture Laboratory for Cherax spp. intensive farming," located in Siculiana (Sicily, Italy), and transferred to our laboratory. Crayfish were originally imported from Mulataga Aquaculture (Perth, Western Australia, P.O. Box 343 Gosnells 6110), as Cherax albidus, in April 2005, and were, according to the European Community Law, in good health and disease-free (Health Certificate n. 4436915). For the present study, animals were kept under natural photoperiod and the water temperature was set at 20 [degrees]C. Animals were fed a natural diet ad libitum every second day. We utilized adult females that had already spawned once and that were in different stages of the reproductive cycle at the time of the study. The stage of the reproductive cycle was defined on the basis of the time of the year the animals were analyzed and on the gonadosomatic index (GSI) and the oocyte diameter according to Sagi et al. (1996, 1999). Mean oocyte diameter ([+ or -]SD) was calculated from a sample of 15 oocytes per ovary. Females were defined as non-vitellogenic (NV) with a GSI = 0.21 [+ or -] 0.05 and whitish oocytes with a diameter = 0.5 [+ or -] 0.1 mm; early-vitellogenic (EV) with a GSI = 3.0 [+ or -] 0.5 and yellow oocytes with a diameter--2.0 [+ or -] 0.2 mm; and full-vitellogenic (FV) with a GSI = 5.0 [+ or -] 0.5 and blue oocytes with a diameter = 2.5 [+ or -] 0.3 mm. Adult males were employed as negative controls. Crayfish body weight ranged from 25 to 40 g.

Experimental design

On the basis of the stage of their reproductive cycle, females were divided into three groups of 24 animals each: Group one = NV females; Group two = EV females; Group three = FV females. Each group was separated into the following treatments, with 6 crayfish in each treatment: Control, injected with saline solution for freshwater crayfish (Van Harreveld, 1936); E treatment, injected with 17[beta]-estradiol; P treatment, injected with progesterone; E+P treatment, injected with both steroid hormones.

Steroids were dissolved in ethanol and the obtained solutions were diluted in the saline solution to reach the final concentration of [10.sup.7] mol [1.sup.1]/crayfish, according to previous studies on crayfish (Rodriguez et al., 2002b). The steroids were injected in the abdominal muscle, in proximity to the fifth pleopod, two times a week for 4 weeks. The injection volume was 100 [macro]. The same experimental design was carried out on males, with only two animals per group. At the end of the treatment, the animals were anesthetized on ice, weighed, and sacrificed. First, the hemolymph was extracted by a syringe from each animal, then transferred into a glass tube with EDTA. The samples were centrifuged at 10000 X g for 1 h and were preserved at -80 [degree]C. Subsequently, gonads and hepatopancreas was divided into three pieces: one treated for histological and immunohistochemical analysis; one placed in a solution of phosphate buffer and protease inhibitor to evaluate the lipidic profile; and one used for total RNA extraction. Ovaries were treated for histological analysis. The following variables were measured: gonadosomatic index (GSI = fresh weight of ovary/whole crayfish X 100); hepatosomatic index (HIS = fresh weight of hepatopancreas/whole crayfish X 100); mean oocyte diameter (MOD = major diameter + minor diameter/2).

Histological analysis

A piece of hepatopancreas and a piece of ovary for each animal were fixed in Bouin's solution for about 10 h, dehydrated with ascending alcoholic series, cleared in xylene, and then embedded in paraffin wax. The sections were cut to a thickness of 7 [micro]m and stained with hematoxylineosin.

Immunohistochemistry

For each animal, a piece of hepatopancreas was fixed in formalin, dehydrated in ethanol, cleared in xylene, and embedded in paraffin wax. Serial sections (7 [micro]m) were cut and placed on silane-coated slides. The sections were processed by the immunoperoxidase method. Tissues were deparaffinized, rehydrated, and then washed in 0.1 mol (1.sup.1) phosphate buffer solution (PBS) at pH 7.4 for 15 min. Endogenous peroxidase activity was blocked by incubation for 30 min in a 0.3% hydrogen peroxide solution diluted in methanol. After incubation with nonfat dry milk (Bio-Rad) to reduce background staining, the sections were placed overnight in a moist chamber at 4 [degree]C with anti-VTG primary antibody made against Cherax quadricarinatus (a generous gift of Prof. Sagi, Ben Gurion University, Beer Sheva, Israel) at an optimal dilution of 1:500. Afterward, the sections were rinsed in several baths of PBS and incubated for 1 h at room temperature with secondary antibody--antirabbit IgG conjugated with horseradish peroxidase (Pierce), diluted 1:250. The peroxidase reaction was developed in a solution of 3,3'-diaminobenzidine tetrahydrochloride (Sigma Chemical Co., St. Louis, MO) 0.015% w/v in Tris-HCl 0.01 mol (1.sup.1)pH 7.5, containing 0.03% hydrogen peroxide. Slides were then dehydrated and mounted in Canada balsam and examined using a Nikon Eclipse E600 microscope. Controls were treated by the same methods except that the primary antibody was omitted.

Fatty acid analysis

The hepatopancreas was homogenized in PBS and centrifuged at 14,000 X g. The pellet was employed for fatty acid analysis. Lipids were extracted following the two-step method of Bligh and Dyer (1959). The fatty acid transesterification was accomplished using the protocol suggested by Kramer et al. (1997). Lipid analyses were performed at the Lipidomic laboratory of Lipinutragen srl (Bologna, Italy), a spin-off company of the Consiglio Nazionale delle Ricerche, Bologna (Italy). Fatty acid methyl ester analysis was carried out by gas chromatography on a Varian CP-3800 gas chromatograph equipped with a flame ionization detector and a Rtx-2330 column (90% biscyanopropyl-10% phenylcyanopropyl polysiloxane capillary column; 60 m, 0.25 mm i.d., 0.20[micro]m film thickness). Helium was the carrier gas at the constant pressure of 29 psi. Oven temperature started from 160 [degree]C held for 55 min, followed by an increase of 5 [degrees]C/min up to 195 [degrees]C, held for 10 min, followed by a second increase of 10 [degrees]C/min up to 250 [degrees]C. Fatty acid methyl ester values were identified by comparison with the retention times of authentic samples (Ferreri et al., 2001, 2002).

RNA extraction and cDNA synthesis

Total RNA was isolated from the hepatopancreas using SV Total RNA Isolation System (Promega). Reverse transcription was performed using 4[micro]g of total RNA, oligo dT primers and the ImProm-II Inverse Transcription System (Promega).

PCR amplification

Oligonucleotide primers (VgForward-5'AACGAGGAA-GACGCTGTGG 3'; VgReverse-5'GGGTATCGCCGAA-TAAAGG 3') were designed on the cDNA sequence of the Cherax quadricarinatus VTG reported by Abdu et al. (2002) (GenBank Accession no. AF306784). PCR amplification was carried out in a Helix Thermal Cycler (DiaTech), in a 20-[micro]1 reaction using 1.25 units of Taq DNA Poly, 1 X Taq DNA Poly Buffer, 1.5 mmol(1.sup.1) MgCl2, 0.2 mmol (1.sup.1) of each dNTP, 0.1[miceo]mol[1.sup 1] of each primer, and 2-5 [micro]l of template cDNA. PCR conditions consisted of denaturation at 95 [degrees]C for 5 min, followed by 35 cycles of denaturation at 94 [degrees]C for 30 s, annealing at 58 [degrees]C for 30 s, and extension at 72 [degrees]C for 1 min. A final elongation step was performed at 72 [degrees]C for 10 min. The PCR product was separated by 1% agarose gel electrophoresis with ethidium bromide and visualized with Chemidoc UV transilluminator (BioRad).

cDNA cloning and nucleotide sequencing

The PCR fragment was purified using a QIAquick gel extraction (Qiagen). The PCR fragment (900-bp long) was cloned into a pGEM-T Easy Vector (Promega) to transform Escherichia coli (strain DH-5[alpha]) using standard methods. Clones containing the PCR insert were isolated and the plasmid DNA was purified using a QIAprep Spin Miniprep kit (Qiagen). The nucleotide sequence was carried out by PRIMM srl.

Real-Time RT-PCR

The amount of VTG mRNA was determined with realtime RT-PCR to estimate the effects of the hormonal treatments on the VTG expression in the hepatopancreas. Preliminary cDNA synthesis was performed as previously described. The VTG transcript was quantitatively analyzed and normalized using both VTG sense (5'-TTTTGGT-GAAGGCTACGC-3') and antisense (5'-TCTTGCAGCTGTTCCAGT-3') primers and adding to the PCR reaction an additional pair of primers amplifying a fragment of [beta]-actin cDNA as a housekeeping gene. The additional primers (sense: 5'-GGTCGGTATGGGTCAGAAG-3'; antisense: 5'-GTGGTGGTGAAGGAGTAGCC-3') were designed based on the Cherax quadricarinatus [beta]-actin cDNA sequence reported in the GenBank database (Martinez-Perez et al., 2005) (GenBank Accession no. AY430093). Realtime reactions were carried out on an ABI 7300 Real-Time PCR System (Applied Biosystem, Foster City, CA) using SYBR Green I dye. The real-time PCR mix contained 12.5 [micro]1 of 2X Brilliant SYBR Green QPCR Master Mix (Stratagene), 0.1 [micro]mol (1.sup.1) downstream primers, and 50 ng of template DNA in a 25-[micro]1 final volume. The system was initially incubated at 95 [degrees]C for 10 min for the initial AmpliTaq enzyme activation, followed by 40 cycles of denaturation at 95 [degrees]C for 30 s, annealing at 58 [degrees]C for 1 min, and extension at 72 [degrees]C for 1 min. A final elongation step was performed at 72 [degrees]C for 10 min.

Each reaction was run in triplicate. Accurate amplification of the target amplicon was checked by performing a melting curve. Data were analyzed according to the relative quantification method. All the statistical analyses were performed using GraphPad.

SDS-PAGE and Western blotting

Electrophoretic analysis was performed according to the Laemmli method (Laemmli, 1970) using a marker of known molecular weight (Color Burst, Sigma). Samples were re-suspended in 0.125 mol [1.sup.1] Tris-HCl (pH 6.8) containing 2% SDS, 10% glycerol, 0.02% bromophenol blue, and 5% [beta]-mercaptoethanol; boiled for 2 min; and loaded into the wells of a 7.5% denaturing SDS-polyacrylamide gel. After the run the gel was electroblotted onto nitrocellulose filter (Millipore). The filter was blocked for 1 h with a solution containing 0.1% Tween-20 in PBS and 5% BSA and then incubated for 1 h with anti-VTG primary antibody (kindly provided by Prof. Sagi) diluted 1:5000 in 1% PBS-Tween-BSA. Subsequently, the filter was washed five times with PBS-Tween and incubated for 1 h with the secondary antibody, anti-rabbit IgG conjugated with horseradish peroxidase (Pierce), diluted 1:10000 in 1% PBS-Tween-BSA. After five washings with PBS-Tween, the filter was developed using Super Signal West-Pico Chemiluminescent Substrate kit (Pierce) reagents, and the bands were visualized with Chemidoc (Biorad).

Total protein concentration

Total protein concentration was determined with the Bradford method (Sigma) using bovine serum albumin (BSA) as standard.

Statistical analysis

Values were expressed as mean [+.or.-] standard error (S.E.). Data were analyzed by one-way analysis of variance (ANOVA), and any significant difference was determined at the 0.05 level by Duncan's multiple range test. The analyses were carried out with the Statistica statistical package, ver. 7.0 (Statsoft Inc., Tulsa, OK).

Results

Histology of hepatopancreas

In decapods, the hepatopancreas is a bilobed brown and yellowish organ that occupies much of the cephalothoracic cavity. It is formed of a mass of blind tubules. Tubules consist of a cylindrical epithelial layer, which constitutes the glandular epithelium, surrounded by a basal lamina of connective tissue. Each tubule is differentiated into three zones: the distal and medial zones of the tubules delimit an irregular and narrow lumen and constitute the cortical region of the gland, while the proximal zones form the medullar region and delimit an ample tubular (Vogt, 1994). In Cheraxalbidus, as in other decapods, the hepatopancreas was composed by tubules lined by a single-layered epithelium. Figure 1 shows the histological section of a typical mid-region of hepatopancreatic tubules of NV, EV, and FV females. In untreated NV female the tubules were characterized by cylindrical epithelial cells with the nucleus basally located and numerous vacuoles in the apical zone. The lumen of the tubules was narrow. The hepatopancreas histology of females injected with 17[beta]-estradiol (E), progesterone (P), and both hormones (E+P) did not show any changes when compared to the control. In untreated EV females the tubules appeared similar to those of untreated NV females. The treatment with E caused an enlargement of the lumen of the tubules with a consequent compression of the epithelial cells, which formed a thin layer. The lumen of the tubules was occupied by abundant secretions. The treatment with P and E+P caused an increase of the vacuoles present in the cylindrical epithelial cells, while the tubule lumen size was unaffected (Fig. 1A-C). In untreated FV females the cylindrical epithelial cells appeared swollen and were occupied by voluminous vacuoles. After steroid treatment the epithelial cells appeared engorged with large vacuoles, the tubular walls appeared brittle, and the whole tissue appeared to be rather loose (Fig.1D--F).

[FIGURE 1 OMITTED]

Vitellogenin immunolocalization in the hepatopancreas

To determine the possible relationship between the stages of females and the presence of VTG in the vacuoles of epithelial cells of the hepatopancreas, we employed an antibody generated against Cherax quadricarinatus vitellogenin. No VTG immunoreactivity was present in NV females or in males (not shown). In EV females (Fig. 2A) VTG immunoreactivity was present in the vacuoles of some epithelial cells.

Fatty acid composition of hepatopancreas membranes

To investigate the cause of the crumbling of the tubule walls of the hepatopancreas of the FV females treated with hormones, we analyzed the fatty acid composition of the cell membranes. Results are shown in Table 1. Data represent the relative percentages of peaks observed in the GC analysis. Peaks correspond to the more representative 13 fatty acids, and their sum was set to 100. The relative percentages indicate approximately the weight of every fatty acid that constitutes the phospholipids of the cellular membrane. The comparison of the data highlights possible changes in the metabolism and, consequently, in the type and percentages of the fatty acids in the cellular membrane.

Palmitic was the most abundant among saturated fatty acids (SFA); oleic was the most abundant among the mono-unsaturated fatty acids (MUFA); and linoleic, eicosapentanoic acid (EPA), and docosahexanoic acid (DHA) were the most abundant among polyunsaturated fatty acids (PUFA). When treated FV females were compared with untreated FV controls, palmitic acid showed a statistically significant increase with E treatment, oleic showed a statistically significant decrease with P and E P treatment, linoleic acid showed a statistically significant increase with P treatment, EPA showed a statistically significant increase with E+P treatment, and DHA showed a statistically significant decrease with E treatment. Total SFA were higher in E- and E+P-treated females, while total PUFA and MUFA were lower in E-treated females.

[FIGURE 2 OMITTED]

VTG cDNA sequencing

The nucleotide sequence of the PCR fragment obtained by amplification of the cDNA derived from the hepatopancreas of the vitellogenic females was compared to VTG sequences deposited in GenBank. The result reported in Figure 3 shows that our cDNA fragment (GenBank Accession no. GQ420689) shared 97% identity with the Cherax quadricarinatus vitellogenin mRNA (GenBank Accession no. AF306784).

VTG levels in the hepatopancreas

To ascertain the effect of steroids on the expression of VTG, RNA from hepatopancreas of both untreated and treated animals was employed as a template in real-time PCR experiments. Samples were always run in triplicate, and the analysis of the dissociation curves from both experimental and [beta]-actin control samples revealed a single melting peak, indicating a specific signal for both transcripts. In all negative control samples, no amplification of the fluorescent signal was detected. The quantitative analysis of VTG gene expression was relative to the VTG mRNA level in controls that was set as a reference value of one. The VTG mRNA results for EV females treated with hormones are as follows: when injected with E, the average increase was 1.8-fold, corresponding to a percentage increase of 80% [+ or -] 5.2%, which was statistically significant; when injected with P, the average increase was 1.67-fold, corresponding to an increase of 68% [+ or -] 3.4%, which was statistically significant; when injected with both steroids, the average increase was 1.15-fold, corresponding to an increase of 10% [+ or -] 2.3%, which was not statistically significant. For FV females, treatment with E produced an average VTG mRNA increase of 1.32-fold, corresponding to a percentage increase of 35% [+ or -] 4.2%; treatment with P induced an average increase of 1.25-fold, corresponding to an increase of 22% [+ or -] 1.8%; injection with both steroids showed an average increase of 1.22-fold, corresponding to an increase of 24% [+ or -] 2% (Fig. 4). The VTG increase in FV females was thus not statistically significant with respect to the control. Finally, in NV females and males, hormonal treatment did not cause any increase in VTG mRNA (data not shown).

VTG in the hemolymph

The antibody generated against the Cherax quadricarinatus yolk polypeptide of 106 kDa crossreacted with an immunoreactive band of about 80 kDa in the hemolymph of Cherax albidus EV (Fig. 5) and FV (Fig. 6) females. In both cases, treatment with steroids increased the intensity of the immunoreactive band. This increase was mainly evident in females injected with progesterone. The immunoreactive band was not present in the hemolymph of NV females and untreated males, which were used as a negative control (data not shown).

Discussion

In this study the effects of 17[beta]-estradiol and progesterone on the reproduction of the freshwater crayfish Cherax albidus are reported. Vitellogenin (VTG) as a marker of reproduction is widely employed in both vertebrates and invertebrates (Sumpter and Jobling, 1995; Marin and Matozzo, 2004). The use of VTG as a marker of endocrine disruption has revealed itself to be particularly useful (Hutchinson and Pickford, 2002; Rotchell and Ostrander, 2003; Porte et al., 2006, for review). Moreover, progesterone or its metabolites seems to induce VTG synthesis in some invertebrates, although the effects are not entirely clear. A direct stimulatory effect of 17 [alpha]-hydroxyprogesterone on VTG production has been suggested in the red swamp crayfish Procam-barus clarkii (Rodriguez et al., 2002b), and Reddy et al.(2006) demonstrated that this hormone induced ovarian growth and ovarian VTG synthesis in the freshwater crab Oziotelphusa senex senex. This study shows that in vivo treatment with 17[beta]-estradiol and progesterone, alone or in combination, brought about an increase in VTG mRNA in early-vitellogenic (EV) females and, although to a lesser extent, in full-vitellogenic (FV) females of the crayfish Cherax albidus. 17[beta]-estradiol seemed to be more effective than progesterone on VTG mRNA synthesis in the hepatopancreas, in agreement with Yano (2000) and Yano and Hoshino (2006) who suggest that in penaeids 17[beta]-estradiol could be the actual hormone that stimulated VTG production, using progesterone as a precursor. Hepatopancreas explants of the shrimp Metapenaeusensis incubated in vitro with steroid hormones demonstrated that both 17[beta]-estradiol and progesterone stimulated VTG gene expression, although 17[beta]-estradiol was more effective (Tiu et al., 2006). In our study both 17[beta]-estradiol and progesterone stimulated VTG mRNA synthesis, although at different rates; and the treatment with 17[beta]-estradiol plus progesterone did not show any additive effect on mRNA VTG transcription, proving that these steroids do not act in synergy, at least in the doses employed here. Although hemolymph levels of 17[beta]-estradiol and progesterone have not been detected in crayfish, the dose employed here falls within the physiological concentrations reported for both hormones in the hemolymph of the mud crab Scylla serrata (Warrier et al., 2001) and the prawn Marsupenaeus japonicus (Okumura and Sakiyama, 2004). It seems that in Cherax albidus 17[beta]-estradiol and progesterone act differently on VTG regulation. Moreover, dichotomous roles for these hormones in vitellogenesis have been hypothesized for the red mud crab Scylla serrata, in which maximum levels of 17[beta]-estradiol were found in the hepatopancreas but the highest concentration of progesterone was detected in the ovary (Warrier et al., 2001).

A strong correlation between estrogens and expression of the heat shock protein HSP90 in the shrimp Metapenaeus ensis indicates that the expression of VTG may be under the regulation of estrogen hormones through a mechanism similar to that in vertebrates (Wu and Chu, 2008). However, the presence of an estrogen receptor gene in crustaceans is still controversial. Estrogen receptors have not been reported in crustaceans, although nuclear receptors sharing high similarity with estrogen receptors have been identified in Drosophila (Maglich et al., 2001); and specific androgen binding sites--but no estrogen binding sites--have been found in the amphipod Hyalella azteca (reviewed in Koheler et al., 2007).

In this study, the effect of steroid treatment was blunted during the full vitellogenesis period in comparison to the early vitellogenesis period. This phenomenon may be ascribable to the higher rate of VTG expression in FV females that were therefore less responsive to steroid stimulation than NV females. In contrast, neither VTG mRNA nor circulating VTG in the hemolymph were detected in NV females of Cherax albidus treated with sex steroids. These results are consistent with those reported by Tsukimura (2001) for the ridgeback shrimp Sicyonia ingentis, in which sexually quiescent females treated with progesterone, 17[alpha]-hydroxyprogesterone, and 17 [beta]-estradiol did not show increased levels of yolk protein precursor in the hemolymph. One possible explanation the author advanced for these results is that the endocrine environment of NV females probably involves high levels of gonadotropin inhibiting harmone, causing the animals to be unresponsive to vertebrate-like steriods. According to our data we can extend and deepen these observations by suggesting that neither progesterone nor 17[beta]-estradiol affects VTG synthesis in Cherax albidus females in a sexual quiescent phase.
Table 1

Fatty acid composition of the hepatopancreas membranes of
full-vitellogenic Cherax albidus females injected with steroids 17
[beta]-estradiol (E[sub.2.]), progesterone (P). 17[beta]-estradiol
plus progesterone (E[.sub.2]+P), and females injected with saline
solution (control)

      Fatty acids           Control            E2

16:0(palmitic acid         20.37 [+ or -] 1.15a   23.37 [+ or -] 1.18b

16:1(palmitoleic acid)      6.96 [+ or -] 0.60     7.09 [+ or -] 1.09

18:0(stearic acid)          4.96 [+ or -] 0.52     6.00 [+ or -] 0.63

9-trans 18:1(elaidic        0.18 [+ or -] 0.02     0.09 [+ or -] 0.02
acid)
9-cis 18:1(oleic acid)     27.69 [+ or -] 1.10a   27.07 [+ or -] 1.14a

11-cis 18:1 (vaccenic       5.03 [+ or             4.62 [+ or -] 0.49
acid                       -] 0.53

18:2(linoleic acid)        19.21 [+ or            18.53 [+ or -] 1.15a
                           -] 1.12a

20:2(eicosadienoic acid)   0.85 [+ or              0.88 [+ or -] 0.03
                           -] 0.08

20:3(dihomo-*-linolenic    0.17 [+ or              0.13 [+ or -] 0.01
acid)                      -] 0.10

20:4n-6(arachidonic acid)  1.17 [+ or              1.30 [+ or -] 0.09
                           -] 0.10

Trans 20:4                 0.18 [+ or
                            -] 0.03                0.17 [+ or -] 0.02
(trans-arachidonic acid)

20:5n-3(EPA)               7.14 [+ or
                            -] 0.80a               6.25 [+ or -] 0.79a

22:6n-3(DHA)               5.48[+ or               3.79 [+ or -] 0.38b
                           -]0.52a

Total PUFA                 34.02[+ or             30.88 [+ or -] 0.35b
                           -]0.38a

Total MUFA+PUFA            21.99[+ or             20.29 [+ or -] 0.51b
                           -]047a

Total SFA                  12 .67[+ or            14.68 [+ or -] 0.90b
                           -]0.83a

    Fatty acids                 P                 E2+P
16:0(palmitic acid         20.71.[+ or -].1.14a  20.29 [+ or
                                                 -] 1.16a

16:1(palmitoleic acid)      6.99.[+ or -].1.07    5.63 [+ or
                                                 -] 0.70

18:0(stearic acid)          4.93.[+ or -].0.52    6.06 [+ or
                                                 -] 0.79

9-trans 18:1(elaidic        0.13.[+ or -].0.01    0.07 [+ or
acid)                                            -] 0.03

9-cis 18:1(oleic acid)     25.83.[+ or -].1.18b  25.15 [+ or
                                                 -] 1.15b

11-cis 18:1 (vaccenic       4.11.[+ or -].0.43    4.15 [+ or
acid                                             -] 0.47

18:2(linoleic acid)        20.98.[+ or -].1.21b  19.95 [+ or
                                                 -] 1.18a

20:2(eicosadienoic acid)    0.79.[+ or -].0.01    0.86 [+ or
                                                 -] 0.02

20:3(dihomo-*-linolenic     0.15.[+ or -].0.2     0.10 [+ or
acid)                                            -] 0.01

20:4n-6(arachidonic acid)   1.01.[+ or -].0.2     1.63 [+ or
                                                 -] 0.95
Trans 20:4                  0.16.[+ or -].0.01    0.14 [+ or
(trans-arachidonic acid)                         -] 0.02

20:5n-3(EPA)                8.16.[+ or -].0.01    9.31+0 .88b

22:6n-3(DHA)                5.47.[+ or -].0.60a   6.15[+ or
                                                 -] 0.61a

Total PUFA                 36.56.[+ or -].0.60a  38.00[+ or
                                                 -] 053c

Total MUFA+PUFA            22.91.[+ or -].057c   23.37 [+ or
                                                 -] 056c

Total SFA                  12.82.[+ or -].0.83a  13.37 [+ or
                                                 -] 0.98a
Vaues are reported as relative percentage of peaks with the
total sum of peaks set at 100%. Eace examination was carried
out on three animals table values are the man +- standard error.
Values were analyzed by factorial ANOVA. Values in the same row
with different letters are significantly different (P[less than]0.05).
EPA-eicosapentanoic acid; DHA. docosahexanoic acid; SFA=saturated
fatty acids; MUFA=monounsaturated
fatty acids; PUFA=polyunsaturated fatty acids.


The localization of the VTG mRNA in the hepatopancreas of Cherax albidus confirms what has already been shown in other crayfish, that exogenous VTG synthesis occurs in the hepatopancreas. The site of VTG synthesis in crustaceans has been established in many species. The ovary (Lui et al, 1974; Lui and O'Connor, 1976, 1977; Eastman-Reks and Fingerman, 1985; Yano and Chinzei, 1987; Rankin et al, 1989; Browdy et al, 1990), hepatopancreas (Paulus and Laufer, 1987; Lee and Watson, 1995), or adipose tissue (Tom et al., 1987) have been proposed as organs of VTG synthesis in decapod crustaceans. Hepatopancreas was suggested as the main synthetic site of this protein in the freshwater crayfish Macrobrachium rosenbergii (Lee and Chang, 1999; Chen et al., 1999) and Macrobrachium nipponense (Han et al., 1994). The presence of the VTG mRNA in the hepatopancreas of Cherax albidus represents further evidence that this organ is the synthetic site of VTG.

[FIGURE 3 OMITTED]

In addition to the effect of steroids on VTG transcription, we also analyzed VTG presence in the hemolymph. We employed antibodies generated against a Cherax quadricarinatus egg yolk polypeptide of about 106 kDa. In the hemolymph of Cherax albidus the antibodies against the 106-kDa polypeptide crossreacted with a polypeptide that appeared as a band of about 80 kDa (present data). The difference in molecular weight may reflect the high variability of polypeptides generated by the proteolytic cleavage of VTG when analyzed under denaturing conditions. Although it appears that the VTG gene organization and expression pattern in decapods is highly conserved (Tiu et al., 2009), a vast array of VTG subunits of different molecular weight has been reported (Tsang et al., 2003; Kung et al., 2004; Serrano-Pinto et al., 2004; Market al., 2005; Tiu et al., 2006). The 80-kDa band of Cherax albidus crossreacted with the antibodies against the 106-kDa polypeptide corresponding to the molecular weight of one of the four VTG proteins that Yehezkel et al. (2000) considered specific to secondary-vitellogenic females. The absence of the 80-kDa band in both the male and inter sex (data not shown) of Cherax albidus strongly sustains its validity as a marker of vitellogenesis in this species. The treatment with sex steroids caused an increase in the intensity of the 80-kDa band that was particularly evident in the hemolymph of vitellogenic females treated with progesterone. In progesterone-treated females in early vitellogenesis, progesterone brought about a 2-fold increase in the optical density of the 80-kDa band with respect to the control, while in the FV females there was a 3-fold increase. This result is in conflict with the effect of sex steroids on VTG mRNA levels in the hepatopancreas, where 17[beta]-estradiol was more effective than progesterone. This result may indicate different VTG regulation at the level of gene transcription and protein translation. RNA metabolism regulation involves an emerging class of proteins--the RNA binding proteins (Burd and Dreyfuss, 1994; Mattaj, 1993)--that appear to play critical roles in mRNA splicing (Hodgkin, 1989: Dreyfuss et al., 1993), nuclear export (Maquat, 1991), translation (Kozak, 1992; Melefors and Hentze, 1993), stabilization and degradation (Nielsen and Shapiro, 1990a; Sachs, 1993). The control of mRNA stability and degradation represents a crucial step in the coupling (or uncoupling) of gene transcription and protein production. An increasing number of molecules have been shown to regulate RNA stability, including steroid hormones (Brock and Shapiro, 1983; Note-born et al., 1986; Paek and Axel, 1987; Nielsen and shapiro, 1990b). Dodson et al. (1995) identified, in Xenopus laevis liver, a protein that binds, in a specific manner, a segment of the 3'-untransiated region (3'-UTR) of VTG mRNA. This protein is induced by 17[beta]-estradiol and mediates stabilization of VTG transcript. It can be hypothesized that, in Cherax albidus, progesterone induces an increase of VTG synthesis while 17[beta]-estradiol induces an increase of gene transcription and/or stabilizes immature mRNA, regulating its translation. In this way 17[beta]-estradiol may avoid a sudden impoverishment of the whole VTG mRNA pool.

[FIGURE 4 OMITTED]

[FIGURE 5 OMITTED]

[FIGURE 6 OMITTED]

In this study, 17[beta]-estradiol and progesterone treatment modified the hepatopancreas morphology of EV and FV females. In contrast, NV females did not show any difference between the control and steroid-treated females, a result consistent with the unresponsiveness of NV females to steroid stimulation in this phase of the reproductive cycle. In EV and FV females the histological analysis of the hepatopancreas highlighted that an increase in the size of the cells in females treated with steroids was mainly due to large vacuoles probably occupied in vivo by lipids. The immunohistochemistry of vitellogenic females showed that the content of some, but not all, vacuoles crossreacted with antibodies against Cherax quadricarinatus VTG.

In FV females treated with steroids the hepatopancreas tubular walls were fragmented and brittle in appearance. Since SFA reduce the permeability and fluidity of membranes and promote their stiffening while PUFA maintain permeability and fluidity (Vance and Vance, 2002), we analyzed hepatopancreas membrane fatty acid composition in control and treated females to get an insight into the possible causes of membrance fragility. We found that 17[beta]-estradiol treatment caused an increase in SFA and a decrease in PUFA and MUFA, which might explain the membrane fragility. One the other hand, progesterone treatment increased both MUFA and PUFA, which does not provide any satisfactory explanation for the membrane fragility. However, we should keep in mind that the paucity of data available in the literature does not allow the formulation of any hypothesis, and that although the effects of 17[beta]-estradiol on lipid biosynthesis have been reported, these are not directly related to reproduction (Smith et al., 1978; Canesi et al., 2007; Sharpe and MacLatchy, 2007).

In conclusion, in this study we have monitored how the administration of 17[beta]-estradiol and progesterone affects the reproduction of the crayfish Cherax albidus. We evaluated the effects mainly in terms of changes in VTG expression in the hepatopancreas and VTG concentration in the hemo-lymph in relation to the phase of the reproductive cycle. Both sex steroids caused an increase in VTG expression and concentration, although their effects were not cumulative and depended on the phase of the reproductive cycle. The emerging picture is one of great complexity due to the high variability of actions ascribable to the so-called "vertebrate sex steroids." In support of this view, the ample literature available along with this study requires a deep investigation into the molecular mechanisms underlying sex steroid regulation of VTG in invertebrates.

Acknowledgments

This research was supported by the "Camera di Commercio di Benevento" and the "Regione Siciliana, Assessorato Agricoltura e Foreste" grants to Professoressa Marina Paolucci. We thank Sig. Dario D'Argenio for his technical assistance and Dr. Carla Ferreri for assistance in lipid analysis.

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Received 4 August 2009; accepted 17 November 2009.

* To whom correspondence should be addressed. E-mail: paolucci@unisannio.it

Abbreviations: EV, early-vitellogenic; FV, full-vitellogenic; GSI, gonadosomatic index; NV, non-vitellogenic; VTG, vitellogenin.

E. COCCIA(1), E. DE LISA(1),C. DI CRISTO(1), A. DI COSMO(2), AND M. PAOLUCCI(1),*

(1) Department of Biological and Environmental Sciences, Faculty of Sciences, University of Sannio, Via Port'Arsa, 11-82100 Benevento, Italy; and (2) Department of Structural and Functional Biology, University of Naples "Federico II," Via Cinthia, 80126 Napoli, Italy
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