William J. Biggers

Unifying Concepts Learned from Methyl Farnesoate for Invertebrate Reproduction and Post-Embryonic Development1

SYNOPSIS. Since the discovery that methyl farnesoate (MF), the unepoxidated form of the insect juvenile hormone (JHIII), is produced by mandibular organs of numerous crustaceans, extensive evidence has accumulated that this compound appears to perform similar functions in the Crustacea as JH performs in insects. A major function of MF appears to be in enhancing reproductive maturation. This was first shown by indirect experimentation with eyestalk ablation, which augmented MF production. Subsequently, direct treatments of several species of crustacea with MF showed that reproductive maturation was enhanced. A second function of MF, similar to that of the JH of insects, is in the maintenance of juvenile morphology. This is especially true in the late larval transformations into juveniles, where MF plays an inhibitory role, as well as during the transformation of juveniles into adults. These results were inferred from eyestalk removal experiments. In the case of the larval-juvenile transition, inhibitory results were also obtained with MF by direct hormone treatments. However, the transition from very early larval stages, such as one nauplius stage proceeding to the next, which in many cases also involves morphogenetic changes, may be occurring in the presence of MF. Indeed, MF appears to be stimulatory to early postembryonic larval stages of Crustacea. Again, this function of MF in Crustacea appears to be similar to functions of JH in early postembryonic insects. However, it should be pointed out that there are many more ‘‘early’’ stages in Crustacea than there are in insects, and very few of these cases have been investigated. When considering the animal kingdom and larval metamorphosis, the question may be raised whether there are other members of the JH family regulating metamorphosis and reproduction. One plausible example appears to be among certain annelids. The trochophores of Capitella respond to various juvenoids, but are most responsive, within one hour, to MF and eicosatrienoic acid. This latter compound is present also in adult annelids, where it has been named ‘‘Sperm Maturation Factor,’’ since it seems to function in the maturation of sperm in Arenicola. Therefore, eicosanoids perform in annelids two functions performed in insects by JHs. In conclusion, it seems that there are morphogenesis promoting responses to JHs in early larval development in crustaceans, annelids, and possibly other forms, which differ from those MF effects in later larvae of Crustacea where MF retards morphogenesis. Such early responses as noted here have recently also been described for insects. Furthermore, it is clear that the polyunsaturated 8,11,14-eicosatrienoic and aracidonic acids seem to be juvenoids, and appear to function as such in annelids, and may also be functionally active in insects and crustaceans. It seems reasonable to conclude therefore that new and novel juvenoids exist, while others still await discovery.

Settlement and Metamorphosis of Capitella Larvae Induced by Juvenile Hormone-Active Compounds Is Mediated by Protein Kinase C and Ion Channels

The signal transduction pathway by which juvenile hormone-active compounds induce settlement and metamorphosis of metatrochophore larvae of the polychaete annelid Capitella sp. 1 was investigated. The known protein kinase C (PKC) activator phorbol-12, 13-dibutyrate was an active inducer of settlement and metamorphosis, whereas H-7, an inhibitor of PKC, inhibited settlement and metamorphosis in response to juvenile hormone III (JH III). JH III and methyl farnesoate (MF) also directly activated, in vitro, both a PKC-like enzyme present in Capitella homogenates and PKC purified from rat brain. In addition, binding studies using the fluorescent PKC inhibitor RIM-1 revealed the presence of a PKC-like enzyme in intact Capitella larvae and juveniles. Settlement and metamorphosis of the larvae was also stimulated by membrane-depolarizing concentrations of KCI. This response to KCl was inhibited by tetraethylammonium. The potassium channel blocker 4-aminopyridine induced settlement and metamorphosis, whereas settlement and metamorphosis in response to JH III was inhibited by the potassium channel ionophore nigericin. Settlement and metamorphosis induced by JH III was inhibited by the calcium channel blockers Ni2+, Zn2+, and verapamil, whereas settlement and metamorphosis was induced by the calcium ionophore A23187. These results suggest that in mediating this response, juvenile hormones may cause activation of PKC, leading to subsequent modulation of potassium and calcium channels.

Identification of Juvenile Hormone-Active Alkylphenols in the Lobster Homarus americanus and in Marine Sediments

We have identified, by gas chromatography/mass spectrometry, four alkylphenols that are present in the hemolymph and tissues of the American lobster Homarus americanus and in marine sediments. These alkylphenols are used industrially in antioxidant formulations for plastic and rubber polymer manufacturing, and are similar in structure to a known endocrine disruptor, bisphenol A. The compound 2-t-butyl-4-(dimethylbenzyl)phenol was present at concentrations of 0.02 to 1.15 μg/ml in hemolymph and 8.95 to 21.58 μg/g in sediments. A second compound, 2,4-bis-(dimethylbenzyl)phenol, was present at concentrations between 0.07 and 19.78 μg/ml in hemolymph and 138.94 to 224.89 μg/g in sediment, while a third compound, 2,6-bis-(t-butyl)-4-(dimethylbenzyl)phenol, was found at concentrations between 0.01 and 13.00 μg/ml in hemolymph, 2.55 and 6.11 μg/g in hepatopancreas, and 47.85 and 74.66 μg/g in sediment. A fourth compound, 2,4-bis-(dimethylbenzyl)-6-t-butylphenol, was found at concentrations of 0.20 to 70.71 μg/ml in hemolymph, 23.56 to 26.89 μg/g in hepatopancreas, and 90.68 to 125.58 μg/g in sediment. These compounds, along with bisphenol A, 4-dimethylbenzylphenol, and nonylphenol, display high juvenile hormone activity in bioassays. Alkylphenols at high concentrations are toxic to crustaceans and may contribute significantly to lobster mortality; at lower concentrations, they are likely to have endocrine-disrupting effects.

METHYL FARNESOATE INDUCED OVARIAN MATURATION IN THE SPIDER CRAB, LIBINIA EMARGINATA

Summary To overcome the problem of getting crustaceans to reproduce in captivity, eyestalk ablation or X-organ sinus gland removal is commonly utilized in commercially important species such as shrimp. We have investigated the effect of unilateral and bilateral eyestalk ablation on methyl farnesoate (MF) production by mandibular organs (MOs) and on ovarian maturation in female spider crabs Libinia emarginata, a useful model since these animals are in a terminal molt and are devoid of a functional Y-organ. Non-reproductive, over-wintering female L. emarginata were induced to be reproductive by feeding and increasing the holding temperature to stimulate the endocrine system. In addition, we removed X-organ sinus glands by eyestalk ablation either unilaterally (UEA) or bilaterally (BEA) to further stimulate MF synthesis by MOs. Endogenous MF in the hemolymph was extracted and quantified by means of HPLC and in some cases by GC/MS. Oocyte growth and egg quality were studied simultaneously to determine how the...

Augmentation of polynuclear aromatic hydrocarbon metabolism of human placental tissues of first-trimester pregnancy by cigarette smoke exposure

OBJECTIVE Polynuclear aromatic hydrocarbons are important components of cigarette smoke. The toxicity of polynuclear hydrocarbons depends on their metabolic biotransformation by tissues. This study was performed to assess the effects of cigarette smoke exposure on polynuclear aromatic hydrocarbon metabolism in the human placental tissues of first-trimester pregnancy. STUDY DESIGN The expression of essential enzymes that metabolize polynuclear aromatic hydrocarbons and regulate toxic metabolism, aryl hydrocarbon hydroxylase, epoxide hydrolase, and glutathione S-transferase, were determined by immunocytochemical staining of the specific enzymes in first-trimester placental samples from both smoker and nonsmoker donors. The overall polynuclear aromatic hydrocarbon metabolism by such tissues was quantitated by a radiometric assay with benzo[a]pyrene substrate in placental villi tissues and also in isolated trophoblast cells or in cultured trophoblast cells in the presence of polynuclear aromatic hydrocarbon agent. RESULTS Immunocytochemical staining revealed that aryl hydrocarbon hydroxylase was localized on trophoblast cells of first-trimester placentas from smoker donors. Epoxide hydrolase was present in stromal and trophoblast cells, and glutathione S-transferase (pi) was present in trophoblast cells of both nonsmoker and smoker subjects. In addition, the overall metabolism of polynuclear aromatic hydrocarbon xenobiotics in such tissues (8 to 11 weeks) of donors who smoked cigarettes was observed to be increased compared with that of nonsmokers by radiometric assay of metabolic products. The increased polynuclear aromatic hydrocarbon metabolism from such exposure was also shown in isolated and purified trophoblast cells of first-trimester placental villi and in culture of such trophoblast cells of nonsmoker donors with polynuclear aromatic hydrocarbon by the same assay procedure. CONCLUSIONS Therefore, contrary to previous assumptions, these data demonstrate that cigarette smoke exposure increases the polynuclear aromatic hydrocarbon metabolism of placentas even during the early stages of pregnancy. Augmented polynuclear aromatic hydrocarbon metabolism may produce genotoxic metabolites deleterious to conceptus development.

Chemical induction of settlement and metamorphosis of Capitella capitata Sp. I (Polychaeta) larvae by juvenile hormone-active compounds

Summary The influence of juvenile hormone (JH)-active chemicals on the settlement and metamorphosis of metatrochophore larvae of the polychaete annelid Capitella sp. I of the Capitella complex has been investigated. These studies demonstrate that JH-active chemicals are able to induce settlement and metamorphosis of Capitella larvae, and that these effects may possibly be mediated by protein kinase C induction. Evidence for the presence of JH-active compounds in marine sediments is also presented, suggesting that these chemicals may serve a natural role as chemical cues for settlement and metamorphosis for Capitella larvae in the marine environment.

Detection of juvenile hormone-active compounds by larvae of the marine annelid Capitella sp. I

Several bioassay systems have been previously developed to detect the presence of juvenile hormones or compounds with juvenile hormone-activity (JH activity). While investigating the effects of juvenile hormones on polychaete annelid development, we have previously found that metatrochophore larvae of the polychaete Capitella sp. I are induced to settle and metamorphose in response to compounds with JH-activity in less than 1 h. Investigating further the settlement-inducing effects of chemicals with varying degrees of JH-activity, we have found that there is a positive relationship between the JH-activity of chemicals in the Galleria insect cuticle bioassay and their ability to induce settlement and metamorphosis of the Capitella larvae. The juvenoids JH I, JH III, and methyl farnesoate induced 50% of the larvae to settle and metamorphose in 1 h at concentrations of 25, 12, and 1 μM dissolved in the seawater (EC50s). The JH-active fatty acid eicosatrienoic acid also was very active having an EC50 of 10 μM, whereas non-JH-active chemicals such as cholesterol and alkane hydrocarbons were not effective in inducing settlement and metamorphosis. These results therefore suggest that the Capitella settlement and metamorphosis assay may aid in the detection and purification of chemicals with JH-activity.

Lessons learned from inshore and deep-sea lobsters concerning alkylphenols

Summary In earlier studies it was found that metamorphosis-inducing activity brings about the settlement of swimming trochophore larvae of the marine worm Capitella capitata. Such activity was attributable to alkylphenols, which are present in marine sediments as well as in hemolymph and embryos of the lobster Homarus americanus. These samples were taken from Long Island Sound and Vineyard Sound. Little or none of this activity was found in offshore lobsters taken from the edge of the continental shelf far from human influence. A comprehensive analytical method based on organic solvent microwave-assisted extraction, followed by solid phase extraction and gas chromatography-mass spectrometry was developed for the simultaneous identification and quantification of bioactive compounds. These compounds include the following four alkylphenols: #1: 2-t-butyl-4-(dimethylbenzyl)phenol; #2: 2,6-bis-(t-butyl)-4-(dimethylbenzyl)phenol; #3: 2,4-bis-(dimethylbenzyl)phenol; and #4: 2,4-bis-(dimethlybenzyl)-6-t-butylphenol. It is reported that 95 (36%) of 262 inshore lobsters contained one or more alkylphenols with average values as follows: compound #1, 11.2 ± 55.9 ng/mL; #2, 23.3 ± 355.9 ng/mL; #3, 116.6 ± 434.2 ng/mL; and #4, 107.1±1167.9 ng/mL, respectively. Only one of 15 offshore lobsters (6.7%), had detectable alkylphenols. Three of five (60%) of deep-sea lobsters had embryos that were carrying higher levels of alkylphenols. These results suggest that inshore lobsters at higher water temperatures are exposed to alkylphenol contamination. Offshore lobsters at lower temperatures appear to be remediated and have lower alkylphenol levels in their blood by having remained offshore, presumably in less contaminated waters. The embryos were most likely formed during inshore reproductive maturation because offshore temperatures are too low for ovarian and egg maturation. These embryos remained contaminated because of their isolation within a relatively impervious shell.

Insulin modulates neuronal plasma membrane development in human fetal spinal cord neurons in culture

The number of intramembrane protein particles (IMP) in the protoplasmic face of the perikaryal plasma membrane was evaluated in neurons from 9 week-old and 12 week-old human fetal spinal cord in culture. An increased number of IMP was observed in membranes from 12 week-old fetal neurons when compared to membranes from 9 week-old fetal neurons. The addition of insulin (100-2500 microU/ml) to the culture media resulted in a significantly increased number of IMP in neuronal membranes. Incubation with glucose (1.5-6 mg/ml) did not modify the number of IMP and glucose did not potentiate the effect of insulin when both glucose (3 mg/ml) and insulin (500 microU/ml) were added to the culture media. The results suggest that insulin may modulate the development of neuronal membranes and that this effect is not mediated by an increased glucose utilization.