Physiological role of apyrene spermatozoa ofBombyx mori

Summary Observation by electron and phase-contrast microscopy demonstrated that inBombyx mori the dissociation of eupyrene bundles, apparently through digestion of the prostatic secretion or an endopeptidase, was promoted by the vigorously flagellating movement of apyrene spermatozoa in the spermatophore.     

Disruption of sperm release from insect testes by cytochalasin and β-actin mRNA mediated interference

Release of sperm bundles from moth testes is controlled by the local circadian oscillator. The mechanism which restricts migration of sperm bundles to a few hours each day is not understood. We demonstrate that a daily cycle of sperm release is initiated by the migration of folded apyrene sperm bundles through a cellular barrier at the testis base. These bundles have conspicuous concentrations of actin filaments at their proximal end. Inhibition of actin polymerization by cytochalasin at a specific time of day inhibited sperm release from the testis. Likewise, application of double-stranded actin RNA specifically inhibited sperm release. This RNA-mediated interference (RNAi) lowered the pool of actin mRNA in tissues involved in sperm release. The decline in mRNA levels resulted in the selective depletion of F-actin from the tip of apyrene sperm bundles, suggesting that this actin may be involved in the initiation of sperm release. Combined results of RNAi experiments at physiological, cellular and molecular levels identified unique cells that are critically involved in the mechanism of sperm release.Received 11 April 2003; received after revision 2 May 2003; accepted 27 May 2003     

Occurrence of retinal and 3-hydroxyretinal in a possible photoreceptor of the silkworm brain involved in photoperiodism

Summary Deficiency of dietary carotenoid and vitamin A caused an absence of photoperiodic response of diapause induction in the silkworm,Bombyx mori, and an addition of vitamin A to the diet restored the response. By high pressure liquid chromatography (HPLC) a possible photoperiodic receptor of the silkworm brain was found to contain both retinal and 3-hydroxyretinal which are chromophores of insect visual pigments. These pieces of evidence suggest that a retinoid protein might function in the photoperiodic response of the silkworm.     

Two-step acquisition of motility by insect spermatozoa

High motility of eupyrene sperm of a grasshopper (Omocestus ventralis) was induced by cAMP. Both trypsin and cAMP were necessary for high motility of eupyrene sperm of three other species of grasshoppers. The same was found for apyrene sperm of the silkmoth,Bombyx mori, when they had been wahsed free from seminal plasma. On the other hand, in locusts (Locusta migratoria), in which the yellow gland has high Arg-C endopeptidase activity, sperm motility was induced by trypsin, like that of apyrene sperm of Lepidoptera. Thus in these two orders of Insecta, sperm motility appears to be induced by the same two-step process: the first step by Arg-C endopeptidase, and the second by cAMP.     

DNA synthesis in the nuclei of differentiating muscle fibers of the silkworm,Bombyx mori L.

Summary Incorporation of³H-thymidine (³HTdr) into the nuclei of myofibril-containing myofibers of larvae of the silkworm,Bombyx mori, was shown by means of light microscope (LM) and electron-microscope (EM) autoradiography. The number of DNA-synthesizing myonuclei attains 42% 12–18 h after each molt. Thus in the developing silkworm DNA replication and myofibrillogenesis are coexisting and not mutually exclusive processes as is the rule in vertebrate somatic myogenesis.     

7-Dehydrosterols in prothoracic glands of the silkworm,Bombyx mori

Summary Sterol analysis of prothoracic glands of the silkworm,Bombyx mori revealed the presence of 7-dehydrocampesterol and 7-dehydrositosterol together with 7-dehydrocholesterol. It was also found that the amounts of these 7-dehydrosterols were increased in proportion to the ecdysone titer.     

Neuropeptides of the silkworm,Bombyx mori

Six neuropeptides of the silkworm,Bombyx mori, have been isolated and chemically characterized during the past 10 years. They are bombyxin, prothoracicotropic hormone, pheromone-biosynthesis-activating neuropeptide/melanization-and-reddish-coloration hormone, diapause hormone, eclosion hormone, and adipokinetic hormone. Recent progress in research on these neuropeptides is described.     

Ecdysteroid conjugates in the ovaries of the silkworm,Bombyx mori: 3-phosphates of 2,22-dideoxy-20-hydroxyecdysone and of bombycosterol

Summary Two novel ecdysteroid conjugates, 2,22-dideoxy-20-hydroxyecdysone 3-phosphate (1) and bombycosterol 3-phosphate (2), as well as four known ecdysteroid 22-phosphate esters, have been isolated and characterized from the ovaries of the silkworm,Bombyx mori.     

Sex pheromone conversion and degradation in antennae of the silkworm mothBombyx mori L.

Summary In living antennae of the silkworm mothBombyx mori L. the pheromone compound (E, Z)-10,12-hexadecadienol and hexadecanol are enzymatically converted to their corresponding fatty aldehydes, acids and long-chain fatty acid esters. The pheromone is completely degraded at high rates in the antennae of freshly hatched moths. The polar volatile [³H]metabolites exclusively consist of tritiated water. The half-life of the pheromone is about 2.5 min in males and 0.5 min in females. Drying inactivates the enzymes responsible for pheromone oxidation.Dedicated to Professor Adolf Butenandt on the occasion of his 85th birthday     

Injection of a soluble sperm fraction into sea-urchin eggs triggers the cortical reaction

Summary Fertilization membranes from around unfertilized sea-urchin eggs after microinjection of a soluble spermatozoa fraction isosmotic with seawater. This demonstrates that the spermatozoon contains a chemical that triggers an increase in cytosolic calcium, leading to exocytosis of cortical granules. It also demonstrates that the triggering mechanism does not require an externally-activated egg-membrane process. Further experiments show that the chemical trigger is not calcium.     

Studies on some enzymes of the toad (Bufo melanostictus) testis and their probable role at the time of fertilization

Summary Glycosidases like sialidase,-galactosidase, -L-fucosidase, N-acetyl hexosaminidase and proteases were detected in toad testis. Neuraminic acid aldolase activity was also detected. The enzyme activities were found to vary as production of spermatozoa varied. All enzymes, except N-acetyl glucosaminidase, were shown to decrease after injection of toad pituitary extract and they were also found to be absent from testis containing no spermatozoa. The glycosidases were found to act on toad oviduct jelly and they may therefore be involved in the degradation of the jelly after fertilization, into smaller bits, which may be utilized as nutrients by the fertilized zygote.Acknowledgment. We thank Prof. T.R. Ramaiah, Head of the Department of Biochemistry, University of Mysore, for his help. We also acknowledge the financial assistance of University Grants Commission to one of us (MS) and CSIR through a grant No. 9 (165)83/EMR-II to HSS. Please address all correspondence to H.S. Seshadri.     

Spermatogenesis is extraordinarily accelerated in metamorphosis-arrested larvae of a salamander,Hynobius retardatus

Laboratory experiments were conducted to induce neoteny inHynobius retardatus, which had been reported to propagate in larval forms like axolotl. A large number of newly hatched larvae were reared in an aqueous solution of thiourea (TU) and sodium perchlorate (SPC) in order to arrest the metamorphosis. Gonadal development in the metamophosis-arrested larvae was compared with that in normally metamorphosing and metamorphosed controls. Metamorphosis-arrested male larvae produced morphologically mature spermatozoa approximately 4 months after hatching, when the gonads in the controls began to differentiate into testes, or to show the premeiotic proliferation of germ cells. Possible endocrine controls of these phenomena are discussed.     

Vitamin A in human semen

Summary Vitamin A concentration has been determined in human semen with and without hydrolysis by fluorometry and high performance liquid chromatography (HPLC). The vitamin A content of human spermatozoa is much lower than that reported for rabbit spermatozoa but approximately equal to that of bull spermatozoa.     

Role of low ecdysteroid levels in the early last larval instar ofBombyx mori

The relationship between juvenile hormone and ecdysteroid titers during the early stage of the last larval instar of the silkworm,Bombyx mori, was examined in this study. When larvae were fed 20-hydroxyecdysone-supplemented mulberry leaves throughout the last larval instar, 100% underwent supernumerary larval molting instead of metamorphosis. The application of juvenile hormone mimic during the early last instar did not induce supernumerary larval molting, but did delay metamorphosis. Temporal and quantitative ecdysteroid titer measurements revealed that in normal larvae the titers maintained very low levels (3–12 ng/ml) during the early stage of the last instar; however, in 20-hydroxyecdysone-treated larvae, levels ranging from 24 to 45 ng/ml were detected, and a major peak (246 ng/ml) was observed on day 6. These results show that very low ecdysteroid titer levels during the early stage of the last larval instar may play an important role in initiating decreases in juvenile hormone titers as well as in directing metamorphosis.     

Isoenzymes of glutamate-oxalacetate transaminase in the larvae of silkwormsBombyx mori infected with nuclear polyhedrosis virus

Summary Nuclear polyhedrosis virus infection induced changes in the activity and isoenzyme pattern of glutamate-oxalacetate transaminase in the larvae of silkworm,Bombyx mori.The author wish to thank the director of CSRTI, Mysore for providing the virus preparation and the silkworm larvae.     

Occurrence of 3-epi-22-deoxy-20-hydroxyecdysone and its phosphoric ester in diapause eggs of the silkworm,Bombyx mori

Ecdysteroids in diapause eggs of the silkworm,Bombyx mori, were analyzed using high-performance liquid chromatography (HPLC) combined with radioimmunoassay (RIA). A relatively large amount of an unidentified free ecdysteroid and its phosphoric ester (conjugated form) were detected. These two compounds were isolated by a combination of column chromatography on silicic acid, thin-layer chromatography (TLC), and HPLC using a reverse-phase (RP) column. The purified compounds were identified as 3-epi-22-deoxy-20-hydroxyecdysone (22d20E) and 3-epi-22-deoxy-20-hydroxyecdysone 2-phosphate (22d20E2P) by means of mass spectrometry and nuclear magnetic resonance spectroscopy. to our knowledge, this is the first report of 22d20E and 22d20E2P.     

Toxic effects of methylmercury on spermatozoa in vitro

In an in vitro investigation, methylmercury (MeHg) reduced the motility of rat spermatozoa probably by the inhibition of succinate dehydrogenase and ATPase activities. Concomitant morphological changes observed in the spermatozoa were coiled tails and kinks in midpiece and tail regions.     

Toxic effects of methylmercury on spermatozoa in vitro

Summary In an in vitro investigation, methylmercury (MeHg) reduced the motility of rat spermatozoa probably by the inhibition of succinate dehydrogenase and ATPase activities. Concomitant morphological changes observed in the spermatozoa were coiled tails and kinks in midpiece and tail regions.     

Metabolism of amphibian spermatozoa in relation to their motility.

Bufo arenarum spermatozoa were able to sustain motility both under aerobic and anaerobic conditions. In aerobiosis, the oxygen consumption varies between 2.6 and 4.2 microliter O2/10(8) cells/h at 30 degree C. The synthesis of lactic acid by anaerobic spermatozoa demonstrated the existence of an active glycolytic pathway.     

Uptake of glusose-C14 and fructose-C14 by buffalo spermatozoa in extenders during cold storage

Buffalo spermatozoa exhibit insignificant uptake of glucose-C14 and fructose-C14 when semen samples were preserved in cold for 96 h in 2 extenders. Incorporation of C14 in spermatozoa, TCA precipitable proteins and soluble sugar phosphates was either equal or less in semen samples preserved in cold. CAW appeared to be a better extender than SKMEY as revealed by sugar uptake in cold.