Ovarian inhibition of juvenile hormone synthesis in the viviparous cockroach, Diploptera punctata

The mature ovary can actively prevent increases in rates of juvenile hormone (JH) synthesis by corpora allata (CA). This was unequivocally shown in two ways. (1) By combining in a single host female one ovary known to be stimulatory and one known to be nonstimulatory. Since the combination of a stimulatory and nonstimulatory mature ovary resulted in rates of JH synthesis intermediate between those found following implantation of either of these ovaries alone, it is concluded that the nonstimulatory mature ovary is indeed inhibitory (a nonstimulatory immature ovary had no such inhibitory effect). (2) By implanting nearly mature ovarioles and female CA into males. The ovarioles prevented the increase in rates of JH synthesis by the implanted CA that occurs in the absence of implanted ovarioles. This ovarian-elicited difference in rates of JH synthesis by CA implanted into males was not accompanied by differences in hemolymph ecdysteroid titers. Also, following removal of nearly mature ovaries from normal females the rates of JH synthesis remained above those of control females for only 24 hr and then declined to rates of controls. This suggests that there is an additional factor regulating the decline in JH synthesis at the end of the gonadotrophic cycle.     

Juvenile hormone synthesis as related to egg development in neotenic reproductives of the termite Reticulitermes flavipes, with observations on urates in the fat body

The relationship between juvenile hormone (JH) synthesis and egg development, which is well documented in cockroaches, is much less studied in their close relatives, the termites. In this study of neotenic reproductives of the subterranean termite Reticulitermes flavipes, in vitro rates of juvenile hormone (JH) synthesis by corpora allata (CA) are related to vitellogenic egg development and the size of CA. The first study compared brachypterous and apterous neotenics in their first cycle of egg development and a second study compared physogastric and non-physogastric brachypterous and apterous neotenics. In both studies, rates of JH synthesis correlated with the size of CA as indicated by their length. Unlike the cockroach in which all basal oocytes are in the same stage of development, those in termites are in various stages. In brachypterous and apterous in the first cycle of egg development, CA with high rates of JH synthesis were from females with early vitellogenic basal oocytes, whereas CA with low rates of JH synthesis were from females with either pre-vitellogenic or mature basal oocytes. This pattern of JH synthesis is similar to the cycle of JH synthesis correlated with oocyte development in several cockroach species. In later oocyte maturations, CA from physogastric apterous females with ovaries containing mature, as well as growing oocytes, showed a wide range of JH production; the CA with the highest rates of JH synthesis were from females with the highest proportion of early vitellogenic oocytes suggesting that both mature and early vitellogenic oocytes interact to regulate JH synthesis. Rates of JH synthesis were related to the number of vitellogenic ovarioles. Physogastric brachypterous neotenics, compared to the other classes of neotenic females, had CA with 2- to 4-fold higher rates of JH synthesis and ovaries with 2.5- to 8-fold greater number of vitellogenic ovarioles. However, both physogastric brachypterous and apterous neotenics had more vitellogenic basal oocytes and less urate in their fat bodies than the respective non-physogastric neotenics. These results demonstrate the similarities and differences between the classes of neotenic termites and between reproductive females in cockroaches and termites.     

Ovarian stimulation of juvenile hormone biosynthesis in the viviparous cockroach, Diploptera punctata

A direct radiochemical assay for juvenile hormone (JH) biosynthesis by the corpora allata (CA) showed that the cyclic changes in biosynthesis associated with the gonadotrophic cycle were abolished in the absence of ovaries whether or not the CA were innervated. In ovariectomized animals denervated CA synthesized JH at slightly higher rates than did innervated CA. However, the low rates of JH synthesis in ovariectomized females were sufficient to result in accumulation of vitellogenin in the hemolymph to levels twice those of normal females. Implantation of one ovary 1 week after ovariectomy resulted in a cycle of JH biosynthesis qualitatively similar to that observed during a normal gonadotrophic cycle. Implantation of one ovariole did not result in a detectable cycle of JH biosynthesis whereas normal rates of JH synthesis were observed after implantation of three to six ovarioles. Implantation of four ovaries resulted in a cycle of CA activity more attenuated than that observed in the presence of one ovary. Nonetheless all four ovaries sequestered a normal quantity of vitellin.     

Changes in the sensitivity of adult cockroach corpora allata to a brain allatostatin

There are major changes in the sensitivity of corpora allata from the cockroach Diploptera punctata toward the allatostatic tridecapeptide APSGAQRLYGFGL-amide (ASAL) during the female reproductive cycle, as revealed by measurement of juvenile hormone (JH) biosynthesis in vitro. Glands from recently molted adult females show only 30-40% inhibition at 10 nM ASAL, falling to a minimum of less than 10% on day 5 at the peak of spontaneous JH synthesis in vitro. The decline in JH synthesis observed in post-vitellogenic females is accompanied by a dramatic increase to ca. 90% ASAL sensitivity at 10 nM by day 6. Then sensitivity slowly wanes during subsequent ovulation and pregnancy to the levels typical of previtellogenic and virgin females. Full dose/response studies reveal a second level of response at ca. 1 microM, which resembles the pattern obtained from whole brain extracts. We conclude that physiological sensitivity to ASAL (IC50 ca. 0.1 nM) is correlated with the preparation for choriogenesis, and we suggest that 1000 times higher doses give a cross-reaction with related allatostatic receptor(s) that confer important sensitivity at other development stages.     

The activity and stability of injected juvenile hormones (JH I, JH II, and JH III) in last-instar larvae and adult females of the cockroach Nauphoeta cinerea.

The relative activities of the three known juvenile hormones (JHs) injected in olive oil are investigated in a larva test, a vitellogenin test, and an ovary growth test. The degree and character of juvenilization obtained after injection of JHs is dependent on the developmental stage at the time of injection, on the dose, and on the kind of JH used. In all developmental stages investigated JH I and JH II have similar and up to 10-fold higher juvenilizing activity than JH III. Measurement of the exogenous JH I and JH III titers at different developmental stages reveals that until at least 2–3 days after injection, the titer of JH III in hemolymph is higher than that of JH I and that only 1% or less of the injected JH circulates in hemolymph. This indicates that the difference in juvenilizing activity between JH I and JH III cannot be due to different elimination rates and suggests that JH I and JH III have different modes of action in larval Nauphoeta. For the vitellogenin and the ovary growth test decapitated adult females are used. Injection of small doses of each JH induces the synthesis of vitellogenin while high doses of each JH have to be injected to stimulate oocyte growth. Thus in adult females the three JHs have similar relative activities.     

Regulation of Y-organ ecdysteroidogenesis by molt-inhibiting hormone in crabs: involvement of cyclic AMP-mediated protein synthesis

The crustacean neuropeptide, molt-inhibiting hormone (MIH), directly inhibits Y-organ ecdysteroidogenesis, an effect mediated by cyclic AMP (cAMP) and antagonized by calcium-calmodulin. We investigated regulation of Y-organ protein. RNA, and DNA syntheses by MIH, cAMP, and calcium in relation to steroidogenesis in vitro. Ecdysteroid production and [3H]leucine incorporation into protein were inhibited 50-60 and 80-90%, respectively, by MIH activity in eyestalk extracts (4 eyestalk equivalents), 10(-6) M forskolin, or a combination of 10(-2) M dibutyryl cAMP and 10(-4) M 3-isobutyl-1-methylxanthine (dbcAMP-IBMX). Calcium ionophore A23187 (10(-4) M) stimulated ecdysteroidogenesis two-fold, did not affect the relatively high basal (control) rate of protein synthesis, and reduced the inhibitory effects of forskolin on steroidogenesis and protein synthesis. Incorporation of [3H]uridine into RNA was unaffected by MIH, forskolin, or A23187 but was reduced 50% by dbcAMP-IBMX. Basal rates of [3H]thymidine incorporation into DNA were low and were not affected by treatments. The effects of MIH were specific; extracts of brain or muscle did not alter Y-organ steroidogenesis or protein synthesis, while muscle extract increased precursor incorporation into RNA. Eyestalk extract did not affect [3H]leucine incorporation into protein of brain, muscle, or gill. Cycloheximide (5 micrograms/ml) depressed protein synthesis 90% and steroidogenesis 60%, enhanced the inhibition induced by MIH, and blocked the stimulation of steroidogenesis induced by A23187; effects on basal steroidogenesis were evident after 1 hr. Actinomycin D (1 microgram/ml) depressed RNA synthesis 86% but did not alter basal, MIH-inhibited, or A23187-stimulated ecdysteroidogenesis during incubations. These results indicate that MIH suppresses Y-organ steroidogenesis in part by inhibiting protein synthesis at the translational level; the effect is mediated by cAMP. The stimulation of steroidogenesis by calcium, mediated by lowering cAMP, also appears to depend in part upon protein synthesis.     

Juvenile hormone synthesis in vitro by larval and pupal corpora allata of Manduca sexta

The synthesis of juvenile hormone I (JH I) and juvenile hormone III (JH III) in vitro by isolated corpora allata (CA) and brain-corpora cardiaca-corpora allata complexes (BR-CC-CA) from fourth and fifth instar larvae and pupae of Manduca sexta was assessed by JH I and JH III radioimmunoassays of incubation medium. Fluctuations in synthesis occurred at specific stages during larval-pupal development. These fluctuations temporally approximated those of previously determined hemolymph titers of JH I and III for this developmental period, except for the synthesis of JH III during the prepupal period, when the JH III hemolymph titer was negligible but JH HI synthesis in vitro increased to the highest rate observed. The overall agreement between CA activity and the JH hemolymph titers suggests that the in vitro system supports physiological gland activity and that changes in biosynthesis contribute significantly to changes in the titers. A comparison of JH I and JH III synthesis by isolated CA with that by BR-CC-CA suggests that the BR-CC can differentially modulate the synthesis of these two homologs. The significance of these findings with regard to the possible endocrine function of JH III and the proposed neuroendocrine control of the CA is discussed.     

Juvenile hormone inhibits ecdysone secretion and responsiveness to prothoracicotropic hormone in prothoracic glands of Bombyx mori

The role of juvenile hormone (JH) in the regulation of prothoracic gland activity was investigated during the early days of the last (fifth) larval instar of Bombyx mori. Allatectomy on the day of larval ecdysis into the fifth instar or 1 day before ecdysis shortened the time between larval ecdysis and gut purge. Prothoracic glands of the freshly ecdysed fifth instar larvae were inactive and did not respond to the prothoracicotropic hormone (PTTH), whereas those larvae that were allatectomized 1 day before ecdysis exhibited secretory activity in vitro and were capable of responding to PTTH. When corpora allata were removed from freshly ecdysed fifth instar larvae, the prothoracic glands became competent to respond to PTTH in 6 hr and exhibited secretory activity in vitro 9 hr after the allatectomy. Treatment of allatectomized larvae with a JH analog resulted in the recovery of the normal inactive state of the glands. These data suggest that JH acts during the early stages of the instar to suppress both the secretory activity of prothoracic glands and also the acquisition of competence to respond to PTTH.     

Immunosuppressive serum factors in viral hepatitis. I. Characterization of serum inhibition factor(s) as lymphocyte antiactivator(s)

Sera from patients with acute viral hepatitis B were found to inhibit the in vitro proliferation of normal lymphocytes induced by different mitogens and antigens. In addition, an effect on concanavalin A-induced T suppressor cell activity and pokeweed mitogen-stimulated IgG and IgM synthesis was demonstrated. Studies concerning the kinetics of serum immunosuppressive effects indicated that serum immunosuppressive factor (SIF) interfered with the intermediate phase of mitogen-induced lymphocyte activation which was defined by protein and RNA synthesis. Thus, when SIF-positive sera were added to lymphocytes, which were already activated by phytohemagglutinin, for 8, 12, or 18 hr, the inhibitory effect decreased in relation to the duration of lymphocyte activation. No inhibition could be demonstrated when SIF-positive sera were added 24 hr after initiation of mitogen stimulation. Furthermore, similar inhibitory effects were found measuring either uptake of [3H]uridine (RNA synthesis) or [3H]leucine (protein synthesis) in a 24 hr culture of phytohemagglutinin-stimulated lymphocytes or [3H]thymidine uptake (DNA synthesis) after 48 hr. These results indicate that SIF act(s) like an antiactivator and may belong to immunoregulatory physiologic serum factors.     

Immunolocalization of a lipase-like protein in the reproductive apparatus of female Phlebotomus papatasi, at various stages of the gonotrophic cycle

In female phlebotomine sandflies, little is known about the reproductive accessory glands that presumably contribute to egg production and/or oviposition. The main protein secreted in the accessory glands of female Phlebotomus papatasi was recently characterised as a lipase-like protein, the first to be found in the female accessory glands of any insect. This protein, named PhpaLIP (for Phlebotomus papatasi lipase), has now been detected and localized in the reproductive tissues of female P. papatasi, at different stages of the gonotrophic cycle, using a polyclonal anti-PhpaLIP serum and both confocal scanning laser and immuno-electron microscopy. PhpaLIP appears to be always present in the accessory glands (with a secretory peak shortly before oviposition) but was also detected in the follicle cells of the ovarioles, within the developing vitelline envelope, and in the oviducts. The results are discussed in relation to the functions that PhpaLIP could have during the gonotrophic cycle, in the various reproductive structures of female P. papatasi.     

A factor that initiates myocardial hypertrophy in hypertension

A lack of correlation between blood pressure and myocardial hypertrophy was established in spontaneously hypertensive rats, suggesting that factors other than blood pressure control might be responsible for the modulation of myocardial hypertrophy. An in vitro system that is independent of blood pressure and hemodynamic effects was developed by use of isolated myocytes to study myocardial protein synthesis. The validity of this system was determined by means of morphology, by receptor integrity, and by studying the incorporation of tritiated leucine into myocyte protein (dpm/mg/hr). Addition of a supernatant of spontaneously hypertensive rat myocardial homogenate (centrifuged at 1500 g) to the myocyte system resulted in a significant increase in tritiated leucine incorporation into myocyte protein when compared with the addition of homogenates from normal controls. The protein from the homogenate was partially purified by high performance liquid chromatography. The resultant purified protein also stimulated protein synthesis by 70%. Furthermore, a significant increase in the specific activity of the transfer RNA and the rate of protein synthesis was observed after addition of homogenate from hypertrophied heart (4.02 +/- 0.3 vs 7.0 +/- 0.2 pmol leucine/microgram protein/hr; p less than 0.05). These data demonstrate the existence of a soluble factor in the hypertrophied myocardium that stimulated protein synthesis. This factor may play a key role in modulation of myocardial structure during development or regression of myocardial hypertrophy in hypertension.     

Corpus allatum activity in vitro during the reproductive cycle of the viviparous cockroach, Diploptera punctata (eschscholtz)

Isolated pairs of corpora allata (CA) from the viviparous cockroach, Diploptera punctata have been shown to synthesize and release C₁₆JH at a linear rate for at least 5 hr. No storage of C₁₆JH has been observed at any time during the first oviposition cycle. It is suggested that rate limitation in JH biosynthesis does not occur at the terminal enzymic stage because the immediate precursor, methyl farnesoate, does not accumulate at any level of CA activity. It is concluded that the short-term incubation procedures employed represent an accurate assessment of CA activity in vivo.The synthesis and release of C₁₆JH by CA has been followed during the first oviposition cycle. High rates of release of JH were observed during rapid oocyte growth—CA became highly active over a 24 hr period as the oocytes were entering vitellogenesis. An analysis of CA activity relative to oocyte length revealed that the release rate of C₁₆JH was highest when oocyte length was in the range 1.0–1.6 mm. However, it was not possible to ascertain if vitellogenesis was initiated before or after the increase in the JH release rate. C₁₆JH synthesis of 40 pmol hr^?1 is the highest mean rate yet reported for an insect.     

Okadaic acid regulation of the retinoblastoma gene product is correlated with the inhibition of growth factor-induced cell proliferation in mouse fibroblasts.

Okadaic acid, a specific inhibitor of protein phosphatases 1 and 2A, was used to study the mechanism of action of transforming growth factor beta (TGF-beta) on cell cycle progression in C3H/10T1/2 mouse embryonic fibroblasts, where TGF-beta exerts a growth-stimulatory effect. Concentrations of okadaic acid as low as 5 nM inhibited TGF-beta (5 ng/ml)- or 10% serum-induced [3H]thymidine incorporation into postconfluent, quiescent cells. Further, these inhibitory effects were observed when okadaic acid was added as late as 10 hr after TGF-beta or serum stimulation. Since C3H/10T1/2 fibroblasts undergo the G1/S transition at 10-14 hr after TGF-beta and 8-12 hr after serum stimulation, these observations indicate that a phosphatase activity may be required for S-phase entry. In a parallel experiment, okadaic acid partially inhibited TGF-beta-induced [14C]leucine incorporation by 20-65%, depending upon the okadaic acid concentration. In conjunction with the effect of okadaic acid on DNA and protein synthesis, Western blot analysis indicated that okadaic acid inhibited phosphorylation of the retinoblastoma gene product and decreased its protein level, even when added 10 hr after TGF-beta or 8 hr after serum stimulation. These findings strongly suggest that protein phosphatases play a pivotal role for S-phase entry in mouse fibroblasts. Moreover, protein phosphatases may be required in the intermediate steps of TGF-beta or serum growth factor signal-transduction pathways for the stimulation of phosphorylation of the retinoblastoma protein, especially in late G1.     

A Novel DNA Polymerase Activity Found in Association with Intracisternal A-Type Particles

A DNA polymerase activity that promotes the synthesis of poly(dT) has been found in association with intracisternal A-type particles isolated from several mouse tumors. The poly(dT) synthesis activity requires a DNA or RNA primer, is optimal at high salt concentration, prefers magnesium over manganese, and is stimulated by poly(rA). No significant incorporation of dAMP, dGMP, or dCMP was detected in the presence of several RNA and DNA template-primers. The enzyme activity differs in several of its properties from the poly(rA)-directed DNA polymerase activity associated with Rauscher murine leukemia virus.     

Characterization and regulation of HMG-CoA reductase during a cycle of juvenile hormone synthesis

The enzyme 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase was characterized in cockroach corpora allata which produce insect juvenile hormone III (methyl-(10R)10,11-epoxy-3,7,11-tri-methyl-2E,6E-dodecadienoate ). HMG-CoA reductase is a microsomal enzyme dependent on NADPH and dithiothreitol (or glutathione) for activity. The enzyme selectively reduced (3S)-HMG-CoA to (3R)-mevalonate with an apparent KM of 7.6 microM. Mevinolin was a competitive inhibitor of HMG-CoA reductase with a KI of 2.4 nM. No evidence for a modulation of enzyme activity by phosphorylation was obtained. Levels of HMG-CoA reductase were not altered after incubation of the corpora allata with either mevinolin (to decrease isoprenoid flux) or with mevalonate or farnesol (to increase isoprenoid flux). Split pairs of corpora allata were used to compare JH III synthetic activity with HMG-CoA reductase activity during the cycle of JH III synthesis that controls vitellogenesis and oocyte growth in adult females. Both activities changed over 10-fold and peaked on day 5 after emergence/mating, but JH III synthesis did not parallel HMG-CoA reductase activity precisely thereafter. The half-life of HMG-CoA reductase measured in the presence of cycloheximide was significantly different between low and high activity glands and was not related to the half-life of JH III synthesis. The results suggest that HMG-CoA reductase should not be considered 'the rate-limiting enzyme' in juvenile hormone synthesis by Diploptera punctata corpora allata.     

Temporal variations of hemolymph esterase activity and juvenile hormone titers during ovocyte maturation in Acheta domesticus (Orthoptera)

Using in vitro methods, juvenile hormone (JH) esterase activity and alpha-naphthylacetate esterase activity were determined in the hemolymph during the first reproductive cycle of the house cricket, Acheta domesticus. Biochemical properties of the hemolymph JH esterase were studied. alpha-Naphthylacetate esterases increased during the first gonotrophic cycle: peaks of their activity could be observed concomitant with peaks of JH esterase activity. The fluctuations in JH esterase activity correlated with those of hemolymph JH titers. The results are discussed.     

The changing effect of the ovary on rates of juvenile hormone synthesis in Diploptera punctata

The ovary is necessary for the cycle of juvenile hormone (JH) synthesis by the corpora allata which normally accompanies oocyte development in mated adult Diploptera punctata (B. Stay and S. S. Tobe, Gen. Comp Endocrinol.34, 276–286, 1978; B. Stay, S. S. Tobe, E. C. Mundall, and S. Rankin, Gen. Comp. Endocrinol.52, 341–349, 1983). Implanting parts of the ovary into ovariectomized females showed that only the basal oocytes were capable of stimulating an increase in rates of JH synthesis (measured by an in vitro radiochemical assay) within 4 days of implantation; apical oocytes and the oviduct were ineffective. Ovariectomized 2-day-old, mated females were used to test the capacity of ovaries of various developmental stages to stimulate increases in rates of JH synthesis within 24 or 48 hr. Previtellogenic ovaries, with basal oocytes <0.8 mm long, and those near the end of vitellogenesis, with basal oocytes >1.5 mm long, did not result in an increase in rates of JH synthesis above Ringer-injected controls. Vitellogenic ovaries within this size range did elicit an increase in rates of JH synthesis. Furthermore the degree of increase was dependent upon the size of the oocytes; the ovaries with largest oocytes showed the greatest capacity to increase rates of JH synthesis.     

Spontaneous synthesis and release of C16 juvenile hormone by isolated corpora allata of female locust Schistocerca gregaria and female cockroach Periplaneta americana.

Isolated pairs of corpora allata (CA) from adult females of both Schistocerca gregaria and Periplaneta americana incorporate radioactivity from [methyl-¹⁴C] methionine into C₁₆ juvenile hormone (JH) when incubated for up to several hours, such that the rate of JH synthesis can be accurately determined. Analysis of the medium shows that CA from P. americana continue to release newly synthesised JH at constant rates for at least 5 hr, whereas those from S. gregaria may show a marked decrease in rate of JH release after 3 hr, particularly in the case of glands having high initial rates of synthesis and release. In both cases the rates of release of JH are strictly proportional to the rates of JH synthesis when measured over a period of 3 hr incubation, independent of the rate of synthesis. It is concluded that the spontaneous level of endocrine activity in glands from both these species can be faithfully quantified by precise radiochemical methods using the short-term incubation procedures described.     

Regulation of the biosynthesis of insulin in isolated brockmann bodies of the carp (Cyprinus carpio)

The biosynthesis of proinsulin and insulin was followed by the incorporation of radiolabeled leucine and phenylalanine into carp islet protein. Even in the absence of glucose or amino acids in the incubation medium the share of proinsulin in the total protein biosynthesis amounted to ～ 40% of the incorporated [³H]leucine during 2 hr of incubation at 17°. Glucose increased only the rate of total protein synthesis by approximately 20% but did not stimulate the (pro)insulin synthesis. Furthermore, leucine did not stimulate the [³H]-phenylalanine incorporation into proinsulin. Cyproheptadine, a specific inhibitor of proinsulin synthesis in mammals, had no effect on carp islets. The rate of [³H]leucine incorporation into carp islet protein was strongly dependent on the temperature. The share of proinsulin in the total protein synthesis decreased to about 20% at 4°. The effect of temperature on the enzyme system converting proinsulin to insulin was most dramatic. Whereas the half-time of conversion was between 3 and 4 hr between 25° and 17°, it was 58 hr at 9° and undetectably slow at 4°. The results indicate that, in contrast to mammals, the regulation of insulin biosynthesis in islets of the poikilotherm carp is not determined by the supply of metabolic substrates, but by the environmental temperature.