Molecular cloning and sequence of retinoid X receptor in the green crab Carcinus maenas: a possible role in female reproduction.

Retinoid X receptor (RXR) belongs to an ancient superfamily of nuclear hormone receptors, and plays an important role in reproduction of vertebrates. However, the reproductive role of RXR has not been clarified in crustaceans. In this investigation, we first report the cloning of two alternative splice variants of RXR cDNA from green crab ovarian RNA. RXR mRNA levels were quantified in different vitellogenic stages of the crab hepatopancreas (HP) and ovary. The expression of RXR mRNA relative to the arginine kinase mRNA was significantly increased in the HP of vitellogenic crabs in a stage-dependent manner. The relative levels of RXR mRNA in the ovary were significantly lower in vitellogenic stage III crabs than in crabs in the other three stages. These data indicate that the HP and ovary of the crab are capable of expressing RXR, which may regulate, in part, vitellogenesis in the crab. We also examined the effects of methyl farnesoate (MF) and RXR-dsRNA treatments on vitellogenin and RXR gene expression. Vitellogenin and RXR mRNA levels in HP and ovarian fragments incubated in MF were significantly (P<0.001) higher than in control tissue fragments prepared from the same animal. Treatment of crabs with RXR-dsRNA significantly (P<0.001) reduced mRNA levels for RXR and for vitellogenin as well as MF levels in hemolymph. These results indicate that, MF and RXR form a complex (MF-RXR) directly and together stimulate ovarian development in these green crabs. This interaction of RXR, MF, and ovary development axis is a novel finding and is the first report to the best of our knowledge.

Methyl farnesoate synthesis in the lobster mandibular organ: the roles of HMG-CoA reductase and farnesoic acid O-methyltransferase.

Eyestalk ablation (ESA) increases crustacean production of methyl farnesoate (MF), a juvenile hormone-like compound, but the biochemical steps involved are not completely understood. We measured the activity of 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR) and farnesoic acid O-methyl transferase (FAOMeT), an early step and the last step in MF synthesis. ESA elevated hemolymph levels of MF in male lobsters. Enzyme activity suggested that increased MF production on day one was due largely to elevated HMGR activity while changes in FAOMeT activity closely paralleled changes in MF levels on day 14. Transcript levels for HMGR and FAOMeT changed little on day one, but both increased substantially on day 14. We treated ESA males with a partially purified mandibular organ-inhibiting hormone (MOIH) and observed a significant decline in MF levels, FAOMeT activity, and FAOMeT-mRNA levels after 5h. However, no effect was observed on HMGR activity or its mRNA indicating that they must be regulated by a separate sinus gland peptide. We confirmed that lobster HMGR was not a phosphoprotein and was not regulated by reversible phosphorylation, an important mechanism for regulating other HMGRs. Nevertheless, molecular modeling indicated that the catalytic mechanisms of lobster and mammalian HMGR were similar.

Purification and characterization of a mandibular organ protein from the American lobster, Homarus americanus: a putative farnesoic acid O-methyltransferase.

Methyl farnesoate (MF) appears to have important roles in the development, morphogenesis, and reproduction of crustaceans. To better understand the regulation of MF synthesis, we studied farnesoic acid O-methyltransferase (FAOMeT, the final enzyme in the MF biosynthetic pathway) in the American lobster (Homarus americanus). FAOMeT purified from mandibular organ (MO) homogenates had a MW of approximately 38,000. The sequences of trypsin fragments of purified FAOMeT were used to design PCR primers to amplify a cDNA fragment, which was used to isolate a full-length cDNA containing a single open reading frame (ORF) of 828 bp encoding a protein of 276 amino acids. The deduced amino acid sequence of this putative FAOMeT protein contained two copies of a conserved approximately 135 amino acid domain we term the CF (CPAMD8/FAOMeT) domain; single copies of this domain also occur in the human CPAMD8 protein (a member of the alpha-2 macroglobulin family) and an uncharacterized Drosophila protein. The recombinant protein had no FAOMeT activity. However, its addition to MO homogenates from eyestalk ablated (ESA) lobsters increased enzyme activity by up to 75%, suggesting that FAOMeT may require an additional factor or modification (e.g., phosphorylation) for its activation. The mRNA for the putative FAOMeT was primarily found in the proximal region of the MO, the predominant site of MF synthesis. FAOMeT transcripts were found in muscle tissue from ESA animals, but not in green gland, hepatopancreas, or in muscle tissue from intact animals. FAOMeT mRNA was also detected in embryos and larval stages. This is the first comprehensive report of this protein in the lobster, and is an important step in elucidating the functions of MF in these animals.

The lobster mandibular organ produces soluble and membrane-bound forms of 3-hydroxy-3-methylglutaryl-CoA reductase.

In a previous study [Li, Wagner, Friesen and Borst (2003) Gen. Comp. Endocrinol. 134, 147-155], we showed that the MO (mandibular organ) of the lobster Homarus americanus has high levels of HMGR (3-hydroxy-3-methylglutaryl-CoA reductase) and that most (approx. 75%) of the enzyme activity is soluble. In the present study, we report the biochemical and molecular characteristics of this enzyme. HMGR had two forms in the MO: a more abundant soluble form (66 kDa) and a less abundant membrane-bound form (72 kDa). Two cDNAs for HMGR were isolated from the MO. A 2.6-kb cDNA encoded HMGR1, a 599-amino-acid protein (63 kDa), and a 3.2-kb cDNA encoded HMGR2, a 655-amino-acid protein (69 kDa). These two cDNAs had identical 3'-ends and appeared to be products of a single gene. The deduced amino acid sequences of these two proteins revealed a high degree of similarity to other class I HMGRs. Hydropathy plots indicated that the N-terminus of HMGR1 lacked a transmembrane region and HMGR2 had a single transmembrane segment. Recombinant HMGR1 expressed in Sf9 insect cells was soluble and had kinetic characteristics similar to native HMGR from the MO. Treatment with phosphatase did not affect HMGR activity, consistent with the observation that neither HMGR1 nor HMGR2 has a serine at position 490 or 546, the position of a conserved phosphorylation site found in class I HMGR from higher eukaryotes. Other lobster tissues (i.e. midgut, brain and muscles) had low HMGR activities and mRNA levels. MO with higher HMGR activities had higher HMGR mRNA levels, implying that HMGR is regulated, in part, at the transcription level.

3-hydroxy-3-methylglutaryl-coenzyme A reductase in the lobster mandibular organ: regulation by the eyestalk.

The mandibular organ (MO) of the lobster, Homarus americanus, produces the isoprenoid methyl farnesoate (MF), a compound related to insect juvenile hormone (JH). To better understand the synthesis and regulation of MF, we studied 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase (HMGR), the rate-limiting enzyme in isoprenoid biosynthesis. Lobster HMGR had a Km of 11.4 microM for HMG-CoA, a Km of 14.8 microM for NADPH, and was at least 2000-fold more selective for this cofactor than for NADH. Lovastatin and mevalonic acid inhibited HMGR, with KI values of 1.3 nM and 25.3 microM, respectively, whereas MF, farnesoic acid, cholesterol, 20-hydroxyecdysone, and progesterone had no effect. Approximately 75% of the HMGR activity in lobster MO was soluble. Similar levels of HMGR activity were observed in all regions of the MO. Eyestalk removal increased MF synthesis and the activity of farnesoic acid O-methyltransferase (FAOMeT, the final step in MF synthesis) in the MO by 10.7- and 5.7-fold, respectively, and caused a 3.1-fold increase of HMGR activity. Injection of the eyestalk ablated lobsters with an extract of two sinus glands (SG), a neuroendocrine organ in the eyestalk, decreased MF synthesis, FAOMeT activity and HMGR activity to 3, 8, and 20%, respectively, of the levels observed in saline-treated animals. The regulation of crustacean HMGR by the SG suggests that the lobster MO is a useful model system for investigating the cellular regulation of HMGR activity.

Characterization and quantification of yolk proteins in the lobster, Homarus americanus.

Yolk protein (vitellin, Vn) and its precursor (vitellogenin, Vg) were isolated and characterized in the ovary and hemolymph, respectively, of the adult female lobster, Homarus americanus. Vn had a molecular mass of 360 kDa when analyzed by gel filtration. When analyzed by SDS-PAGE, Vn had six bands (110, 105, 94, 90, 81, and 78 kDa). An anti-Vn antiserum was developed using purified Vn, and the antiserum was used to detect Vn and Vg by ELISA and western blot techniques. ELISA analysis of hemolymph proteins separated by gel filtration indicated that Vg was similar in mass to Vn (360 kDa). However, western blots of hemolymph proteins separated by SDS-PAGE indicated that Vg contained a pair of protein subunits, 194 kDa and 179 kDa. Furthermore, the elution profiles of Vn and Vg from anion exchange chromatography indicated that Vg had a more negative charge. Thus, Vg appears to be processed after its uptake by the ovary to form Vn. Vg was undetectable in hemolymph from adult males by either ELISA or by western blot analysis. However, hemolymph levels of Vg in adult females increased 40-fold during the reproductive cycle, rising from 18 microg/mL in ovarian stage II to 789 microg/mL at stage V. This increase correlates well with oocyte growth during the cycle. Hence, this method may be useful for studying the regulation of lobster vitellogenesis.