Minoru Osanai

Control mechanism of diapause of the pharate first-instar larvae of the silkmoth Antheraea yamamai.

Abstract The application of an imidazole derivative (KK-42), body ligation and extirpation of specific ganglia suggest a new endocrine mechanism responsible for the regulation of diapause in pharate first-instar larvae of insects. The present results indicate that a humoral repressive factor originates in the region of the mesothorax and that a humoral maturation factor is released from the region of the 2nd to 5th abdominal segments. Our data suggest that the former inhibits the action of the latter during diapause.

Energy metabolism in the spermatophore of the silkmoth, Bombyx mori, associated with accumulation of alanine derived from arginine

In the female silkmoth, Bombyx mori, labeled glucose-1-phosphate and pyruvate were found to be metabolized with the formation of alanine in the spermatophore, which contains various secretions from some of the glands and the seminal fluid of the male reproductive system. Moreover, in the spermatophore 14CO2 was released not only from these radioactive glycolytic intermediates and end products, but also from 2-oxoglutarate and ornithine. The ratio of the rates of metabolic formation of carbon dioxide from pyruvate and alanine was about 1:2 at 150 min after the beginning of mating. Oxidative decarboxylation of pyruvate in the TCA-cycle was accelerated by added 2-oxoglutarate. Thus, there is a pathway of cascade reactions from arginine to 2-oxoglutarate coupled with glycolysis in the spermatophore.

A specific endopeptidase, BAEE esterase, in the glandula prostatica of the male reproductive system of the silkworm, Bombyx mori

Abstract A specific endopeptidase, arginine ester-hydrolyzing enzyme (BAEEase), was demonstrated in the glandula (g.) prostatica, the lower region of the ejaculatory duct of the silkworm, Bombyx mori . Of the male reproductive glands, only the g. prostatica was found to contain this enzyme. The enzyme was partially purified by ammonium sulfate precipitation and characterized enzymologically. The BAEEase activity was maximal at pH 9.0–9.5. The enzyme was very labile above pH 5.0 at 25°C, but was still stabilized at pH 4.0 by the presence of 0.1 M KCl. Unlike bovine trypsin, prostatic BAEEase showed marked hydrolytic activity on BAEE, and slight activity on TAME, but none on TLME. The trypsin inhibitors, p- NPGB , leupeptin and antipain, markedly inhibited the enzyme activity, but other serine protease inhibitors, thiol protease inhibitors and a carboxyl protease inhibitor were not inhibitory. The results indicate that prostatic BAEEase is probably a new endopeptidase of the serine protease group that specifically cleaves protein on the C-side of arginine residues.

Role of arginase transferred from the vesicula seminalis during mating and changes in amino acid pools of the spermatophore after ejaculation in the silkworm, Bombyx mori

Abstract . 1. 1. Ejaculation of seminal fluid into the spermatophore formed inside the female bursa copulatrix terminates 20 min after the beginning of copulation of Bombyx mori . The amounts of amino acids transferred are small, but the amounts of amino acids in the spermatophore continue to increase after this time due to protein degradation and amino acid interconversions. 2. 2. Arginase, which has high activity in the vesicula seminalis, is transferred to the spermatophore without loss of activity during ejaculation. During mating, ornithine and much urea are formed in the spermatophore, indicating activity of the transferred arginase. 3. 3. In the spermatophore, marked increase of alanine with low concentrations of ornithine and glutamate suggests the presence of a pathway for the active formation of 2-oxoglutarate with pyruvate via glutamate from arginine. During mating, proline and glutamine also increase, but at low rates. 4. 4. Of the exocrine glands in the male reproductive system, the vesicula seminalis secretes the highest concentration of glutamate (30% of the total amino acids); serine is the amino acid present at highest concentration in secretions of other glands (20–30%). No urea was found in the secretions of any of the glands.

The spermatophore and its structural changes with time in the bursa copulatrix of the silkworm, Bombyx mori

The bursa copulatrix of the silkmoth is filled with various secretions and seminal fluid transferred from the male reproductive system during mating. The contents of the bursa include a pearly body, spermatophragma, and spermatophore. The latter consists of a four‐layered wall, an inner and outer matrix, and a soft plug. The components of the spermatophore are all heterogeneous, since they are formed by the partially mixed, viscous streams of the male secretions. Apyrene spermatozoa and eupyrene bundles are present only in the inner matrix, but both the matrices are important sites of sperm maturation. After spermatophore formation, the basophilic regions and periodic acid‐Schiff (PAS)‐granules in both matrices gradually decrease with time. The PAS‐granules were identified as glycogen by α‐amylase treatment. The inner matrix containing sperm and the outer matrix decrease in volume and become concentrated near the neck of the spermatophore. Apyrene and eupyrene spermatozoa can move toward the ductus seminalis after their maturation. A large, beltlike space which does not stain remains at the periphery of the spermatophore. These structural changes of spermatophore contents seem to reflect metabolic reactions in the spermatophore as the reactor for sperm maturation.

Isolations of eupyrene sperm bundles and apyrene spermatozoa from seminal fluid of the silkmoth, Bombyx mori

Abstract Fractions of eupyrene sperm bundles and apyrene spermatozoa were separated by centrifugation of the seminal fluid of the vesicula seminalis of normal 1-day-old male moths of the silkworm, Bombyx mori , on a discontinuous gradient of Percoll in silkworm Ringer at 25°C. On similar centrifugation of the seminal fluid of 1-day-old males that had been kept at 32°C only during the pharate-pupal period, eupyrene bundles were obtained in lower yield, and apyrene spermatozoa were recovered in poor yield. Thus, maintenance at 32°C during the pharate-pupal stage seemed to cause not only marked decrease in production of apyrene spermatozoa, but also some decrease in production of eupyrene spermatozoa. Apyrene spermatozoa separated from the seminal plasma were not activated by treatment with only a serine protease such as trypsin or initiatorin, which is present in the prostatic secretion. Both an endopeptidase and a cyclic nucleotide, cAMP or cGMP were required for their activation, and the further addition of 2-oxoglutarate enhanced their mortality. No glycogen phosphorylase activity was detected in either the isolated apyrene spermatozoa or the eupyrene sperm bundles.

Changes in amino acid pools in the silkworm, Bombyx mori during embryonic life: Alanine accumulation and its conversion to proline during diapause

Abstract Alanine accumulated in silkworm eggs at the onset of diapause. When the eggs were kept at 4°C during diapause, this alanine was converted to glutamate, glutamine and especially proline. On resumption of development at 25°C after diapause, proline was used as an energy source for protein synthesis. In HCl-treated diapause eggs, which develop like non-diapause eggs, most amino acids showed similar developmental changes to those in eggs in resumption of embryogenesis after diapause. However, the proline level increased until the middle of embryonic development and then decreased. Continuous incubation of diapause eggs at 25°C after day 10 of oviposition caused a decrease in alanine with increases in glutamine and proline, while the levels of most other amino acids either decreased slightly or remained unchanged until day 80, when most eggs died. These results show that diapause eggs have a metabolic complex coupled with carbohydrate and amino acid metabolism inclusive of the 2-oxoglutarate-glutamate shuttle. Under conditions when embryogenesis proceeded, the level of phosphoethanolamine decreased rapidly.

Induction of motility of apyrene spermatozoa and dissociation of Eupyrene sperm bundles of the silkmoth, Bombyx mori, by initiatorin and trypsin

Summary The activator, or inducer of motility, of apyrene spermatozoa of the silkmoth, Bombyx mori, was shown to be present in the posterior glandula (g.) prostatica. The activity of this factor in this gland was lost by boiling at 100°C for 10 min. Bundles of eupyrene sperm were dissociated by treatment with either a g. prostatica homogenate or trypsin, and their dissociation was concentration-dependent. On the contrary, for activation of apyrene sperm, the optimal trypsin concentration was 0.45–1.80 μg/ml and activation decreased at higher and lower trypsin concentrations. Microscopic observation showed that the dissociation rate of eupyrene sperm bundles by the g. prostatica homogenate corresponded to that by 0.45–9.0 μg/ml of trypsin. An autolysate of the g. prostatica and digests of seminal fluid with the g. prostatica homogenate or trypsin did not activate apyrene sperm. Of 11 endopeptidase inhibitors tested, antipain, leupeptin, TLCK, TPCK and PMSF strongly inhibited sperm activation by the g. pros...

System for supply of free arginine in the spermatophore of Bombyx mori: Arginine-liberating activities of contents of male reproductive glands

Abstract The mechanism of supply of free arginine in the Bombyx mori spermatophore was analyzed by examining the arginine-liberating activities of the contents of male reproductive glands, which are constituents of the spermatophore formed during ejaculation. On autolysis, only the contents of glandula (g.) prostatica showed significant production of only free arginine. On incubation in combination with the contents of the g. prostatica, arginine liberation from the vesicula (v.) seminalis contents was highest. Similarly, the contents of the g. spermatophorae also showed arginine liberation, but those of the g. lacteola showed generalized liberation of various amino acids. Following digestion of these preparations with bovine trypsin, the v. seminalis, g. spermatophorae and g. prostatica liberated free lysine as well as arginine, while those of the g. lacteola liberated various amino acids. These findings strongly suggest that a specific proteolytic system for production of free arginine is active in the spermatophore. That is, an endopeptidase derived from the g. prostatica specifically cleaves protein at the C-terminal side of arginine residues, and then exopeptidases derived from the v. seminalis, g. spermatophorae and g. prostatica liberate free arginine from the peptides produced by the endopeptidase.

13C and 31P NMR studies on sugar metabolism in Bombyx mori and Philosamia cynthia ricini larvae

Abstract Studies were made on 13 C and 31 P NMR in larvae of two species of silkworm, Bombyx mori and Philosamia cynthia ricini, in vivo as well as in vitro to determine the pathways of glucose utilization, especially those to amino acids as components of silk fibroin. Results showed that the 13 C of [1- 13 C]glucose administered orally into 5th instar larvae of both species was incorporated into glucose-1-phosphate, glucose-6-phosphate and trehalose. Serine, glutamate, glutamine, citrate, malate, trehalose and sorbitol-6-phosphate were detected in the hemolymphs of these larvae as metabolites of [1- 13 C]glucose. Two days after [1- 13 C]glucose administration, labeled alanine, glycine, serine, urea, glycogen, trehalose and glycerol were clearly detected in Bombyx larvae. Starvation caused rapid consumption of administered [1- 13 C]glucose with very little accumulation of 13 C in glycogen or trehalose. In the in vivo 31 P NMR spectra of Bombyx larvae, ATP, arginine phosphate, sorbitol-6-phosphate, uridine diphosphoglucose, phosphoenolpyruvate and inorganic phosphate were detected with some sugar phosphates, such as glucose-1-phosphate and glucose-6-phosphate. During starvation, the intensity of the signal of inorganic phosphate increased and those of sugar phosphate other than sorbitol-6-phosphate decreased, but these changes were reversed by oral administration of glucose.